RESEARCH IN FISHERIES...1967

COLLEGE OF FISHERIES

FISHERIES RESEARCH INSTITUTE

UNIVERSITY OF WASHINGTON

SEATTLE, WASHINGTON 98105

CONTRIBUTION NO. 280

MARCH 1968

■

CONTENTS

High Seas Salmon Studies ........5

Sampling and Tagging. Allan C. Hartt, Michael B. Dell, Forrest E. Carvey, and Wariboko Q. B. West . . . 5

1967 Operations ....................................................................... 5

Tag Returns ............................................................................ 6

Scale Study of Juvenile Coho Salmon. Thomas H. Peck 7

Food, Feeding Habits, and Growth of .0 Age Pacific Salmon. Rollin David Andrews, III ................................... 8

Racial Analysis of Juvenile Pink Salmon. Gary T Sakagawa ................................................................. 8

Physiological Studies of Juvenile Salmon. Lynwood S. Smith, James B. Saddler, Harry M. Miles, Rick D. Cardwell, Timothy W. Newcomb, and Peter B. Swierkowski ............................................................ 8

Distribution and Survival of Juvenile Salmon in Belling-ham Bay. Wayne G. Williams, Danny E. Sjolseth, Rob- ert A. Bishop, and Ernest 0. Salo ................................... 24

Estuarine Ecology Studies. Denny M. Miller, Jerry A. Wet herall, Steven Zebold, William H. Lenarz, Gary D. Stauffer, Jeffrey Fujioka, Michael Halstead, Ernest 0. Salo, and T Saunders English ............................................. 24

Studies of Water Quality ........................................................ 24

Studies of Juvenile Chinook Salmon ................................... 26

Studies of Adult Chinook Salmon ................................... 28

Studies of Demersal Fishes ................................................... 30

Food and Feeding Habits of Juvenile Coho Salmon and Steelhead Trout in Worthy Creek, Washington. James Johnston and Ernest 0. Salo .............................................

32

Compilation of Pacific Salmon Literature. Ernest 0. Salo and Edwin K. Holmberg ........................................................

32

Invertebrate Fisheries

Alaskan Salmon Research ...................................................................................... 9

Kodiak Island Salmon Studies. Richard W. Taylor and Donald E. Bevan ............................................................ 9

Forecast for 1968 9

Reliability of Sampling Gear and Technique . . 10

Chignik Lakes Sockeye Salmon Studies. Duane E. Phinney and Michael L. Dahlberg ...................................

Chignik Lagoon Studies ........................................................

Distribution of Maturing Chignik Sockeye Salmon in the North Pacific Ocean ..............................................

Nushagak District Salmon Studies. Donald E. Rogers and Robert L. Burgner ........................................ 12

Pink Salmon in the Nushagak District ..............................

Some Results of a Survey of the Bottom Fauna in Nushagak District Lakes. Jerry E. Reeves

Magnitudes of Sockeye Spawning Colonies. Kenneth Roberson ................................................................. 16

Epizootics in Experimental Marine Shellfish Populations. Albert K. Sparks, Kenneth K. Chew, Donald Weitkamp, Evelyn J. Jones, Lieselotte Schwartz ................................... 33

10 Oyster Pathology. Albert K. Sparks, Michael C. Mix, Don- ald Weitkamp, David Des Voigne, Evelyn J. Jones, Liese-

10 lotte Schwartz ........................................................................... 34

33

Postembryonic Development of Laboratory-reared Spot

12 Shrimp, Pandalus platyceros Brandt. Vincent A. Price and Kenneth K. Chew ............................................................

Shellfish Toxicity Study. John Dupuy, Louisa Norris, and

12 Albert K. Sparks ......................................................................

Chromosomes of Oysters and Mussels. Muzammil Ahmed . . 14 and Albert K. Sparks ............................................................

35

36

36

21

Washington Fish Studies .....................................................................

Big Beef Creek Salmon Studies. Kenneth Williams, K Victor Koski, and Ernest 0. Salo ................................... 22

Coho Salmon Studies ............................................................. 22

Chum Salmon Studies ............................................................. 23

Radioecology ................................................................................................................. 37

Johnston Atoll Program. Edward E. Held and Allyn H 18 Seymour .................................................................................... 37

18 Bikini Atoll Resurvey. Edward E. Held .............................. 37

Fern Lake Program. Lauren R. Donaldson, Sigurd M. 20 Olsen, Paul R. Olson, Zella Short, James C. Olsen, Har-

old E. Klaassen, and Rufus W. Kiser ................................... 38

Columbia River Studies. Allyn H. Seymour ......................... 39

22 Uptake and Loss of Zinc Oysters ......................................... 39

Effects of Radionuclides on Oyster Larvae .................... 39

The Combined Effects of Irradiation and Temperature and Other Environmental Factors on Salmonid Embryos. Arthur D. WeZander, Lorna J. Matson, Gerald Wadley, and Dorothy Treadwell ....................................................... 40

Kvichak Sockeye Salmon Studies. Ole A. Mat hisen, Orra E. Kerns, Jr., John W. Anderson, Stephen H. Hoag, Richard A. Baxter, T B. Gunnerod, and Patrick H. Poe

Variability in Survival Rates of Sockeye Salmon in the Kvichak River System .........................................

Relationship Between Food Supply and Salmon Production in Iliamna Lake ..............................

Tagging Studies in Newhalen River ...................................

Ecology and Water Utilization .........................................................................

Washington Cooperative Fishery Unit. Richard R. Whit-new, Edward S. Marvich, Aven M. Andersen, and Brian J. Allee .................................................................................... 41

Water Resources Research Needs in the State of Washing- ton, Milo C. Bell ..................................................................... 42

41 Study of the Basic Microbiological and Biochemical Fac- tors Involved in the Irradiation of Marine Products. John Liston, Jack R. Matches, Bohdan M. Slabyj, Jong R. Chung, Gary A. Houghtby, and Susan J. Westcott . 56

Anaerobic Bacteria from the Marine Environment. John Liston, Jack R. Matches, Donald D. Curran, and Mary L. Holman ............................................................................... 59

An Initial Study of the Water Resources of the State of Washington: Volume 1. A First Estimate of Future Demands for Water in the State of Washington. Milo C. Bell .................................................................................... 43

A Compendium on the Success of Passage of Small Fish Through Turbines. Milo C. Bell ........................................ 43

Estimates of the Acute Toxicity of Ammonia-urea Plant Wastes to Coho Salmon, Oncorhynchus kisutch. Max Katz and Robert A. Pierro .................................................. 44

The Transport of Endrin from the Yolk Sac to the Em-bryo in Developing Steelhead Fry. Ken Kimura, Dov Grajcer, and Max Katz ....................................................... 45

The Uptake of Endrin by the Steelhead Trout Embryo from the Yolk Sac and from the Environment and Its Effect on Lipid Metabolism. Dov Grajcer and Max Katz 46

Electrolytes in Developing Steelhead Trout Embryos and Yolk Sacs. Bruce Fowler, Dov Grajcer, and Max Katz . 47

Enzymes of Intermediary Nitrogen Metabolism in Fishes. George W. Brown, Jr ................................................................ 47

Research Vessel, M. V. Commando. Allan C. DeLacy . 47

Marine Microbiology John Liston, John G. Chan, and John A. Baross .....................................................................

60

Vibrio parahaemolyticus Studies 62

A Biological Model of a Freshwater Community: A Gno-tobiotic Ecosystem. Frieda B. Taub, Ruth Hung, and Richard Tomlinson ................................................................. 63

Viral Accumulation and Elimination by Shellfish and Crustacea of the Pacific Coast. John Liston, Jack R. Matches, Rudolph G. DiGrolamo, and Albert K. Sparks 65

Pelleting of Dry Feed for Hatchery Salmon. Car/ D. Decker and George M. Pigott .............................................

A Method for Determining Protein Thiol Groups in Foods. Glen R. Limb ............................................................

Faculty and Staff, January 1, 1968

Graduate Students, Spring 1967—Winter 1968 .................................... 68

Students Receiving Fellowships during the Year ................................. 69

Students Receiving Scholarships during the Year ................................. 69

Students Receiving B.S. Degrees from the College of Fisheries from July 1966 to July 1967 ............................................................................. 70

Students Receiving Advanced Degrees in Fisheries during 1967 and Their Thesis Titles ...................................................................................... 70

Contributions by Members of the College of Fisheries, University of Washington, during 1967 .................................................................. 71

Food Science 48

Introduction

48

65

66

67

Survival of Bacteria on Seafoods. John Liston, Jack R. Matches, Gary A. Houghtby, David Bannerjee, Donald D. Curran, and Susan J. Westcott ........................................ 48

Inhibitory Studies 49

Growth Studies 49

Food-Poisoning Bacteria 50

Effect of Cold Shock on Biosynthesis 52

Oxidative Metabolism of Nonprotein Nitrogen Compo-nents by Fish Spoilage Bacteria and Their Physiology of Psychrotropic Growth During Storage of Fish . . . 53

Mechanism of the Chemistry of the Spoilage Process in Fish. John Listron, Flor B. Irlandez, Jong R. Chung, and David Bannerjee ............................................................ 55 Abstracts of Ph.D. Theses 74

Longli. UNITED STATES r4 Puree seine gear gear

1./Intlexing abundance a. age connotatien of Seestura .1 and .1 age sockeye salmon south of Ad.

2. Sampling and tagging juven la sale. in eastern Bering Sea 1- 8.1PlAng fad tagging juvenile salmon in Gulf of Alaska T

18

CASABA Longline gear

High Seas Salmon Studies'

SAMPLING AND TAGGING

The Fisheries Research Institute sampled and tagged salmon on the high seas in 1967 in continuing a pro-gram begun in 1955 to study the oceanic life history and ecology of the Pacific salmon (genus Oncorhyn-chus) and steelhead trout (Salmo gairdneri). The studies are part of a large program of research that the U.S. Fish and Wildlife Service, the Fisheries Re-search Board of Canada, and the Fisheries Agency of Japan are conducting jointly.

1967 Operations There were three major sampling and tagging oper-

ations in 1967. Fig. 1 shows the areas of these oper-ations and also the areas in which tagging was carried out by Canada and Japan. The numbers of salmon caught and tagged in the operations were:

Steel-head

Pink

Chinook trout Sockeye (0. Coho (0. (S.

(0. Chum gorbu- (0. tshawy- gaird-

nerka) (0. keta) scha) kisutch tscha neri) Total

Total caught 11,014 14,397 18,441 1,799 243 3 45,897 Total tagged 3,150 889 2,345 701 85 0 7,170

Fig. 1.—Areas of salmon tagging operations by Canada, Japan, and the United States on the high seas in 1967.

The primary objective in the work south of Adak was to obtain an index to the abundance and age composition of immature sockeye salmon upon which to base the forecast of the 1968 run to Bristol Bay. A secondary objective was to compare the efficiency of purse seines and gill nets as sampling tools for immature salmon by fishing the two types of gear simultaneously at a number of stations. Seining was done from the Fisheries Research Institute chartered vessel Commander, and gillnetting from the Bureau of Commercial Fisheries vessel G. B. Kelez. The re-sults will be published by the Bureau of Commercial Fisheries. Our purse seine operations south of Adak extended from June 24 through August 13. A total of 3,801 immature sockeye salmon was caught, of which approximately 78 per cent were .1 age. Thus, it appears that .2 age fish will predominate in the run to Bristol Bay in 1968. A forecast of the 1968 run to Bristol Bay will be published by the Alaska Depart-ment of Fish and Game together with forecasts of runs to the individual river systems, which will be derived from other data. Chum salmon were also relatively abundant in the sampling, but since the vast majority of immature chum salmon south of Adak Island are of Asian origin the data are not of direct interest to North American fishermen.

Juvenile2 salmon (mainly sockeye) were sampled and tagged in the eastern Bering Sea from August 17 through September 15, one month later than in 1966. Objectives were to study the distribution, abundance, size, and feeding habits of juvenile salmon in this area during late summer. The catch distribution indicated that juvenile salmon were present mainly to the east of 164°W. They occurred farther north

1 The studies are part of a research program for the purposes of the International North Pacific Fisheries Commission (Can-ada, Japan, United States) and are subsidized under a contract with the U.S. Fish and Wildlife Service, Bureau of Commercial Fisheries, No. 14-17-0007-869.

2 The term "juvenile" as used herein refers to salmon of .0 age.

5

than in 1966 when they were found only in a narrow belt close to the north side of the Alaska Peninsula. Juvenile salmon apparently disperse seaward from Bristol Bay rivers at a leisurely pace; they had ad-vanced a maximum of 300 miles by September 15. Such a limited distribution suggests that feeding con-ditions were favorable in the eastern Bering Sea. The migratory pace was in sharp contrast to that observed in the Gulf of Alaska, which will be discussed later. The average length of juvenile sockeye salmon in the Bering Sea samples was about 17 cm.

A sockeye that had been spray-dyed as a smolt in the Ugashik River, a Bristol Bay tributary, between May 29 and June 3 by biologists of the Auke Bay laboratory of the U.S. Bureau of Commercial Fisheries was recaptured in a set at 55°36'N x 162°57'W on September 7. It was found among 970 specimens from one seine set that were examined under ultraviolet light. Approximately 600,000 sockeye smolts from Bristol Bay rivers were spray-dyed by federal biologists in 1967 in a study of estuarine and early ocean mi-grations.

Returns from the 778 juveniles tagged in 1967 are not expected until 1969 and 1970. Approximately 900 of the Bering Sea juveniles were preserved for lab-oratory studies of stomach contents, parasites and racial features.

Sampling and tagging of juvenile salmon in the Gulf of Alaska have been gradually expanded since 1964. Objectives in 1967 were to study the migration, distribution, abundance, growth, and feeding habits of juvenile salmon of all species during their early ocean residence period. The chartered purse seine

Fig. 2.—Dennison tag being applied to a juvenile salmon with Dennison Swiftacher applicator.

Fig. 3—Dennison tag after application.

vessel Storm was used for the field work, which lasted from July 1 through November 1, in an area extend-ing from Cape Flattery, Washington to Yakutat, Alaska (Fig. 1). Juvenile salmon of all species were found, as in past years, migrating generally northward throughout the area of sampling. Data on migrations are derived from the direction of set of the purse seine, tag returns, and morphometric, meristic, scale, and parasite studies of specimens. A record catch of 4,551 juveniles (mainly pink salmon) was made in Dixon Entrance on July 14.

Most of the tags used on juvenile salmon this year in the Bering Sea and in the Gulf of Alaska were of a new type of molded plastic internal anchor tag that is applied by means of an injection tool (Dennison Swiftacher), which dispenses the tag from a cartridge of 25 tags (Figs. 2 and 3). The tag and applicator, manufactured by the Dennison Mfg. Co., Framing-ham, Massachusetts, are described in detail by Michael B. Dell in a report soon to be published in the Transactions of the American Fisheries Society. The new tags appeared satisfactory on the juveniles at sea at time of release, but we must await returns in 1968, 1969, and 1970 to compare them with the tags used on juveniles in previous years. In preliminary tests of the new tag on trout in the Seattle area, the rate of return over a 5-month period was comparable to that of disk tags.

Tag Returns

Tag returns in 1967 most worthy of mention are those from four sockeye that were tagged as juveniles in 1965 in the Gulf of Alaska (Fig. 4). All were re-leased in the northern part of the Gulf of Alaska and

6

MI 1,.

4. 6

1

ii VIL

5ot4111F11.1'1IRLRLPL

NORTON

I*

$. 1101111k - S'-`-11160° • ' F V

1 ' KODIAK

ALASK .z...1

PRIBILOF BRISTO 111-- I: IS. BAY 1.0 .4111.

VA U.3 55° IILAND 2

- ifANALASK

50°

4/1855 = 0.2$

45°

W 170 °W I65°W I60° W I55°W I50°W 145'W I40°W 135° W

Fig. 4.—Locations of release and recovery of 4 sockeye salmon that were released as juveniles in 1965 and recovered in 1967.

were recovered southeast of the release points. The returns demonstrate that juvenile sockeye salmon mi-grate rapidly northwestward along the coast as pink and coho salmon do. This migratory pattern prob-ably applies to most sockeye in the eastern Gulf of Alaska since the seine catch data each year have shown that the center of abundance of sockeye is in the northern Gulf by mid-August. Dates of release ranged from August 9 through August 26, 1965. Thus, juve-niles from as far south as the Fraser River had mi-grated approximately 1,000 miles by mid-August of their first summer at sea. In contrast is the slow migratory pace of juvenile sockeye in Bristol Bay (described earlier). Lengths at release ranged from 19 to 21 cm. Additional sockeye from the same tag-

ging operations may be recovered in 1968 as .3 age fish.

Two pink salmon tagged as juveniles in 1966 were recovered in 1967. One of them is particularly note-worthy since it provided the first evidence that Fraser River pink salmon also migrate as far north as the northern Gulf of Alaska during their early ocean feeding period. The specimen was tagged near 581/2 0N x 138°W on August 12, 1966 and recovered September 17, 1967 in the San Juan Island area.

Sixteen coho salmon tagged as juveniles in 1966 were recovered in 1967. Their early ocean migration pattern was similar to that discussed for sockeye and pink salmon and agreed with findings in earlier years. As in 1965 and 1966, we received no returns from Puget Sound and vicinity. This lack suggests that these stocks may not migrate as extensively as most of the other stocks that originate between California and southeastern Alaska. The rate of return from the 1966 tagging was poorer than that from either the 1964 or 1965 operations (shown below).

Rates of return of juvenile cohos tagged in the Gulf of Alaska in 1964, 1965, and 1966

Number Number

Year Number recovered recovered Total Rate of tagged tagged year N year N + I recovered return

1964 554 8 8 16 2.9%

1965 1,062 1 46 47 4.4%

1966 1,562 16 16 32 2.0%

Allan C. Hartt, Michael B. Dell, Forrest E. Carvey, and Wariboko Q. B. West

SCALE STUDY OF JUVENILE COHO SALMON

In recent years we have placed increased emphasis on determination of the freshwater age of the salmon sampled and tagged in our high seas operations, par-ticularly the juvenile salmon. Our age interpretations are generally believed to have been accurate and con-sistent except in the case of coho salmon. It appears that a much higher percentage of coho salmon had spent two and three years in freshwater than one would expect from the known freshwater life history of coho salmon in the coastal areas adjacent to the locations of our high seas sampling. The discrepancy is believed to have been due to improper identifica-tion of marks as annuli. It is suspected that feeding

and handling techniques in hatcheries are causing marks with the characteristics of annuli to appear on the scales of coho salmon. The recent increase in hatchery production of cohos may have caused the high incidence of aberrant scale patterns in our sam-ples. Therefore, a study of scale patterns in juvenile hatchery-reared and stream coho salmon has been undertaken.

Monthly samples of about 25 juvenile coho salmon, composed of all available age groups, are being col-lected from selected hatcheries and streams: the Nook-sack, Issaquah, Green River, and Minter Creek hatch-eries in Washington and their associated streams and

7

lus formation on scales and otoliths are being deter-mined. Comparisons will be made between the hatch-ery-reared and stream fish.

Thomas H. Peck

the Klaskanine and Big Creek hatcheries in Oregon and their associated streams. Sampling began in early October of 1967 and will continue through the spring migration of 1968. Scale patterns and times of annu-

FOOD, FEEDING HABITS, AND GROWTH OF .0 AGE PACIFIC SALMON

In the past several years, we have collected young Pacific salmon in the Bering Sea and outer coastal waters of the Northeast Pacific. These fish are being examined in a study of the following features of the biology of salmon during the first few months of ocean residence: (1) food and feeding habits for each species and (2) the influence of these habits on the migrations and growth patterns of each species.

The pattern of feeding activity must be taken into consideration for each species before comparisons can be made of the amount of food consumed and the extent of competition for food between species. The

work to date has shown that red salmon consume food at varying rates within a 24-hr period. It is not known whether this variation results from differences in avail-ability of food items or from other causes, e.g., innate physiological demands.

The diet of sockeye salmon ranging in length from 10-28 cm included euphausiids, amphipods, copepods, shrimp and crab larvae, fish larvae (some were identi-fied as herring), and pteropods. The importance of these items in the diet is being evaluated in conjunc-tion with investigations of feeding activity.

Rollin David Andrews III

RACIAL ANALYSIS OF JUVENILE PINK SALMON

A preliminary analysis of small samples from our 1965 sampling and tagging operations has indicated that certain meristic, morphometric, parasitic, and growth characters can be used to determine the coastal origin of juvenile pink salmon collected in the Gulf of Alaska. Work was begun in 1967 on a more corn-

prehensive racial analysis of the 1966 and 1967 collec-tions by using larger samples and more refined meth-ods. The findings are expected to supplement the tagging results and to contribute to the biology of salmon at sea.

Gary T Sakagawa

PHYSIOLOGICAL STUDIES

The primary effort was devoted this year to super-vision of the design and construction of the floating laboratory. Upon its completion sometime during the early spring, it will be used for the study of the physiological responses of salmon to the stresses of tagging and various environmental changes. Instru-ments that will be installed aboard the floating lab-oratory after construction is completed were set up temporarily in a basement laboratory in the College of Fisheries and tested. These instruments measure

Supported by the Federal Water Pollution Control Admin-istration (Department of the Interior), No. WP 01056-02 and by the U.S. Fish and Wildlife Service, Bureau of Commercial Fish-eries, No. 14-17-0007-869.

OF JUVENILE SALMON'

heart rate and blood pressure, several aspects of blood chemistry, and certain details of lipid metabolism.

Secondary activities included research on the natural stresses experienced by coho salmon as they migrate in Big Beef Creek. Mr. Harry Miles described for his M.S. thesis the transitory changes that occur in plasma ions of juvenile coho salmon as they enter and adapt to the marine environment. He found that from 30 to 40 hr are required for the adjustment. He then began similar observations on adult coho salmon as they entered freshwater. Other studies of changes occur-ring in the serum proteins and lipids of these fish are being conducted by Mr. Rick Cardwell and Dr. James Saddler.

8

Dr. L. S. Smith completed preliminary experiments on the effects of endotoxin and histamine injected into the blood stream of rainbow trout. Histamine produced symptoms similar to those that occur in immature salmon after they have suffered severe scale loss. This work was done in collaboration with Dr.

Gary Weidemeyer, of the Western Fish Disease Lab-oratory.

Lynwood S. Smith, James B. Saddler, Harry M. Miles, Rick D. Cardwell, Timothy W. Newcomb,

Peter B. Swierkowski

Alaskan Salmon Research

KODIAK ISLAND SALMON STUDIES

The 1967 pink salmon run to Kodiak Island was one of the smallest in the 50-year history of the fish-ery; by season's end the pack amounted to only 18,000 cases. So poor was the run that virtually the entire island was closed to commercial fishing for the dura-tion of the fishing season. Several years ago a run failure of this magnitude would have caused severe losses to the fishing industry and jeopardized the chances of the succeeding run. However, since the run was forecast one year ahead by the Alaska Depart-ment of Fish and Game and the Fisheries Research Institute, the industry was prepared for a low level of production and good spawning escapements were achieved.

Since 1962 the odd-year runs have been considerably smaller than the even-year runs. It appears from the size and distribution of the 1967 escapement that the situation may improve in 1969, when returns may be considerable.

Forecast for 1968'

Sampling was conducted by trawl net during June and July of 1967 in five bays for the purpose of obtaining indices to the abundance of juvenile salm-on: Alitak, Uyak, Uganik, Ugak, and Kaiugnak. This year, as in years past, a disproportionate amount of our effort was spent in determining the distribution and relative abundance of the dense concentration of fingerling pink salmon in Portage Bay, an arm of Alitak Bay. Catches near the center of this concentra-tion were considerably more than during any previous year of sampling; they ranged up to 9,240 pink salmon

per 10-min haul; but the concentration occupied less area than in previous years; therefore, the total abun-dance was not substantially greater.

Our method of indexing is not applicable to the runs from Karluk and Red Rivers—two of the major pink salmon-producing streams on Kodiak Island. These streams are unique in that they both drain into open ocean rather than the head of a bay, or fiord. Consequently, the juveniles do not spend time in an estuary and are unavailable to our trawl net. The Alaska Department of Fish and Game conducts fry sampling annually in each stream, as well as in most of the major streams on Kodiak Island, to obtain indices to the abundance of return runs.

Fingerling abundance varied widely among the five bays surveyed (Table 1). Within Alitak Bay, large numbers of fingerlings were found in the Deadman Bay and Portage Bay arms and a few were found in the Olga Bay arm. We forecast good returns to Dead-man River and Humpy River, tributaries of Alitak

TABLE 1.

Indices of fingerling pink salmon abundance at Kodiak Islanda

Year Bay 1963 196-1 1965 1966 1967

Alitak 215.2 372.0 207.0 135.9 332.6 Olga 177.4 29.3 4.0 2.8 5.9 Uganik 30.8 3.1 30.9 20.2 2.9 Uyak 58.5 74.1 16.2 17.3 Ugak 6.2 20.9 Kaiugnak 52.7

a Average fingerling catch per tow. Supported by the Salmon Packers of Kodiak Island through

the Association of Pacific Fisheries.

9

cc I

Chignik Boy

Mount Veniominof

Bay, and a poor return to Dog Salmon River of Olga Bay. Our surveys of Uyak and Uganik Bays yielded very few fingerlings; therefore, we forecast poor re-turns to Uyak River and Uganik River. Fingerling abundance in Ugak Bay was several times greater than in 1966; however, because the 1967 return was very poor, we forecast a fair return for 1968. Since Kaiug-nak Bay was surveyed this year for the first time, we have no basis for comparison with previous years and therefore no basis for a forecast.

Reliability of Sampling Gear and Technique2

In July the Fisheries Research Institute received a one-year grant from the U.S. Bureau of Commercial Fisheries under The Anadromous Fish Act of 1967 to determine the variability of Kodiak trawl catches and the efficiency of the Kodiak trawl in catching available fingerlings.

To accomplish the first objective, we made a total of 125 paired trawl hauls in Alitak and Uganik Bays during late July. We fished duplicate nets simultane-ously, using the research vessel Malka and two seine skiffs to tow the nets (Fig. 1). An outrider pram was attached to the cod end of each net, in which a man rode to examine and note the contents of the net at 5-min intervals during continuous hauls of an hour or more. Unfortunately, most of the fish had migrated out of the bays, and catches were representative only of low levels of abundance. Analysis of the data showed a good correlation between paired catches

I Supported by a grant from the U.S. Fish and Wildlife Serv-ice, Bureau of Commercial Fisheries, under the Anadromous Fish Act of 1967.

Fig. 1.—Sketch showing simultaneous towing of two surface trawls.

during the darkest hours of night (rxy = 0.98 between 2300 and 0100 hours). Catches of less than 20 fish per haul were not significantly correlated.

In an attempt to achieve the second objective, we made a comparison between catches of a 150-fathom small mesh purse seine and a trawl net. While each of 14 round hauls was made with the purse seine in Ali-tak Bay, several trawl hauls were made around the perimeter of the seine. The purse seine provided in-formation on actual fish densities for comparison with catches by the trawl.

Because of low fish densities at the time of the ex-periment the data thus collected were insufficient to permit an accurate evaluation of the efficiency of the Kodiak trawl.

We plan to continue work towards the attainment of both objectives in 1968.

Richard W. Tyler, Donald E. Bevan

CHIGNIK LAKES SOCKEYE SALMON STUDIES1

The Fisheries Research Institute has studied the sockeye salmon of the Chignik River system, Alaska (Fig. 1), for thirteen years. The scope of the research and the importance of the run were detailed in past issues of Research in Fisheries. This report concerns studies of postsmolt sockeye in the Chignik estuary and distribution of maturing fish in the North Pacific Ocean.

Chignik Lagoon Studies Early in our program of studies, it became apparent

that Chignik Lagoon, a shallow, nearly enclosed estu-

'Supported by the Alaska Packers Association, Chignik Fish-eries Co., and the U.S. Fish and Wildlife Service, Bureau of Commercial Fisheries, under the Anadromous Fish Act of 1967.

Fig. 1.—Map of the Chignik River watershed with inset of western Alaska.

10

TABLE I.

Fishes of Chignik Lagoon, Alaska, and their relative frequency of occurrence

-icipenser sp. .-I gonus acipenserinus Tilesius Antmodytes hexapterus Pallas Blepsias cirrhosus (Pallas) Citharichthys sp. Ciupea harengus pallasi Valenciennes Gasterosteus aculeatus Linnaeus Hexagrammos superciliosus (Pallas) Hexagrammos stelleri Tilesius Hip poglossus stenolepis Schmidt Hypomesus pretiosus pretionts (Girard) al Hypomesus olidus (Pallas) Lepidopsetta bilineata (Ayres) Leptocottus armatus Girard Lumpenus sagitta Wilimovsky Mallotus villosus (Muller) Microgadus proximus (Girard) Myoxocephalus polyacanthocephalus (Pallas) Occa dodecaedron (Tilesius) Oncorhynchus gorbuscha (Walbaum) Oncorhynchus keta (Walbaum) Oncorhynchus kisutch (Walbaum) Oncorhynchus nerka (Walbaum) Oncorhynchus tschwytscha (Walbaum) Pallasina barbata (Steindachner) Pholis laeta (Cope) Platichthys stellatus (Pallas) Psettichthys melanostictus Girard Pungitius pungitius (Linnaeus) Salmo gairdneri Richardson Salvelinus malma (Walbaum) Sebastodes 4p. Thaleichthys pacificus (Richardson) Trichodon trichodon (Tilesius)

Sturgeon Rare Sturgeon poacher Rare Pacific sand lance Common Silverspotted sculpin Occasional Sanddab Rare Pacific herring Common Threespine stickleback Common Rock greenling Occasional Whitespotted greenling Occasional Pacific halibut Occasional Surf smelt Rare Pond smelt Occasional Rock sole Rare Pacific staghorn sculpin Common Pacific snakeblenny Occasional Capelin Rare Pacific tomcod Occasional Great sculpin Occasional Bering poacher Rare Pink salmon Common Chum salmon Common Coho salmon Common Sockeye salmon Common Chinook salmon Common Tubenose poacher Occasional Crescent gunnel Occasional Starry flounder Common Sand sole Rare Ninespine stickleback Occasional Rainbow trout Rare Dolly Varden Common Rockfish Rare Eulachon Occasional Pacific sandfish Rare

a Previously recorded only as far west as Sitkalidak Strait, Kodiak Island.

ary at the mouth of the Chignik River, is a secondary nursery area for Chignik sockeye salmon smolts. The smolts leave the lakes at the relatively small size of about 70 mm and remain in the lagoon until they reach about 100 mm, when they continue to the open ocean. Annually since 1962, we have sampled the smolts in the lagoon with a 200-ft beach seine and a tow net with a 6-ft-square opening. A list of the 34 species of fish captured incidentally in Chignik La-goon is given in Table 1.

Since the young sockeye salmon are a part of the community of fauna in the lagoon during their resi-dency, we sought to determine the species of fish with which they normally associated and relationships that might influence their distribution and the length of their residency. We could not observe these relation-ships directly; therefore, we assumed that the samples taken by beach seine and tow net reflected the actual distribution, abundance, and associations of fishes. The equation Nab /(N.NO-1/2(Nb)% was used with the aid of a computer program to calculate an index

of affinity from the sample data for all possible com-binations of two species. Nab is the number of joint occurrences of species a and b, Na is the number of occurrences of species a, and Nb is the number of occurrences of species b in the samples. The species were assigned the letters a and b in such a manner that Nb > Na. When the index of affinity was greater than the pre-assigned level, the two species were con-sidered to have significant affinity.

Each species with any significant affinity values was placed in only one group if it had significant affinity with each member, or was determined to have affinity with one or more groups if it had affinity with only some members. When a pair of species in different groups had an affinity index greater than the assigned value, we considered that the groups were associated. In this manner we determined the hierarchy of associ-ations within the community (Fig. 2).

Sockeye salmon smolts were found to have the greatest affinity with adult threespine sticklebacks. Sockeye salmon smolts, adult threespine sticklebacks,

11

Dolly Varden, Pacific herring, and pink salmon fry were found to make up a group with mutual affinity. Within this group might be found relationships of interest to our study of the ecology of sockeye smolts in Chignik Lagoon.

Ninespine stickleback (adult) Pond smelt

Threespine stickleback (fry) Sockeye salmon (fry)

Sockeye salmon ( smolt) Threespine stickleback ( adult)

Dolly Varden Pacific herring Chum salmon

Pacific sand lance

Pacific tomcod Snakeblenny

Staghorn sculpin Starry flounder

Chinook salmon

Coho salmon Isopod Shrimp Crescent gunnel Whitespotted greenling Pink salmon

Eulachon

Cape h o Tubenose poacher Great sculpin

Fig. 2.—Diagram of the hierarchy of associations of the common fauna of Chignik Lagoon, Alaska.

Distribution of Maturing Chignik Sockeye Salmon in the North Pacific Ocean

Within the past 12 years we have learned a great deal about the distribution of salmon in the North Pacific. Since the Chignik stocks form a small segment of the ocean population of sockeye in any given year, it has been only recently that we have accumulated enough tagging data to accurately determine distribu-tion of the stocks. Both U.S. and Canadian tagging data were used in the analysis; Mr. Allan C. Hartt of the Fisheries Research Institute kindly made available the raw data for analysis.

Table 2 presents the limits of distribution for 100, 97, and 77 per cent of tags recovered. Fig. 3 shows the number of Chignik recoveries by area of tagging and the limits of distribution of 97 and 77 per cent of

.

...

... ... ....,___

IN - ....

ft

•

' ,

11"

_ _;._ _,_

L

7" J

...

_ ■0

##-

\ \

r"

i

.....r.. ,

40111,11V0 0 #

0#1. i

_

• -

ffl

175 I ISO 1.■3 W W

Fig. 3.—Numbers of tags recovered at Chignik, Alaska, summarized by INPFC tagging areas, 1956-1967.

TABLE 2.

Limits of ocean distribution of sockeye tagged in their last summer at sea and later recovered at Chignik, Alaska. Tags

returned from 1956 through October 6, 1967

Recoveries

Latitude (N) Longitude (W)

Number Percentage

91 100 58°03' 47 °50' 168°06' 137°45' 88 97 58°00' 50°00' 155°00' 135°00' 70 77 58°00' 52°00' 150°00' 140°00'

recoveries. Nearly all maturing sockeye from Chignik were found east of the longitude of Chignik. Although 40 per cent of the U.S. and Canadian high seas tagging effort since 1956 was expended west of Chignik, only 2 per cent of the tags were recovered from tagging west of the watershed. The center of distribution of maturing sockeye from Chignik appears to be in the central Gulf of Alaska near the northern limits of the Alaska gyre.

Duane E. Phinney and Michael L. Dahlberg

NUSHAGAK DISTRICT SALMON STUDIES1

The 1967 season marked the twenty-second year in our long-range study of salmon populations in the Nushagak District of Bristol Bay, Alaska. The pro-gram of research was reviewed in the 1965 Research in Fisheries, and recent studies of sockeye salmon

Supported jointly by the Bristol Bay canning industry through the Association of Pacific Fisheries, the U.S. Fish and Wildlife Service, Bureau of Commercial Fisheries, and the Uni-versity of Washington.

escapement and young fish production were sum-marized in the 1966 Research in Fisheries. The re-sults of some specialized studies are presented in this report.

Pink Salmon in the Nushagak District The even-year run of pink salmon in the Nushagak

District of Bristol Bay has become a significant re-source for the commercial fishery of the district. From

12

Geo

met

ric m

ean c

atch

1922 to 1956, the annual pink salmon catch averaged less than 250,000, and little interest was shown in this resource by either the fishermen or biologists. Since 1958, the average even-year catch (1.2 million) has been comparable to that in the early period of the fishery (1904-1920) and has nearly equalled the average annual sockeye catch in recent years. A good pink salmon catch in the Nushagak District in 1968 will be particularly welcome in view of the forecasted low sockeye runs to Bristol Bay.

The majority of pink salmon enter Bristol Bay in late July and are bound for the Nushagak system, where spawning is particularly concentrated in the Nuyakuk River. A smaller fraction of the run enters the Wood River (Fig. 1). Most of these fish spawn in the Agulowak and Agulukpak Rivers, two of the inter-connecting rivers in the lake system. They share these spawning grounds with larger populations of sockeye salmon. Another fraction of the Nushagak run spawns in the lower part of Tikchik River, which is tributary to Tikchik Lake, in the Nuyakuk River headwaters.

Although our freshwater research in the Nushagak District has been primarily concerned with sockeye salmon production, we have collected some data on abundance and growth of young pink salmon in Lake Aleknagik and Tikchik Lake. All indicate differences in the early life history of pink salmon that spawn in a lake system from those that spawn in coastal rivers.

In coastal rivers, the fry migrate to salt water upon emergence from the gravel. They are then about 30 mm long. The young fry obtain little food from the freshwater environment and subsist largely on the yolk. In the Wood River lakes and Tikchik Lake, the fry must travel some distance to reach the outlet rivers (60 miles in the case of Agulukpak River fry); and it is quite apparent that they feed actively during the course of their travel.

We do not know whether a proportion of the pink

Fig. 1.—A pair of pink salmon from the Wood River spawning grounds.

4.0

3.5

3.0

2.5

2.0

1.5

1.0

0.5

30 10 20 30 10 July August

Date

Fig. 2.—Geometric mean catches of pink salmon fry in sampling Lake Aleknagik in 1965 and 1967. Each point is the

geometric mean from 10 seine hauls.

salmon fry in these lakes move out directly after emergence; however, we have found substantial num-bers of pink salmon fry in the lakes long after emer-gence. Pink salmon fry have been caught by tow net in Lake Aleknagik as late as September 10. Results of sampling by beach seine in Lake Aleknagik in 1965 and 1967 are shown in Fig. 2. The maximum catch was taken between June 20 and July 10, and a decline in catch was observed during the later part of July, presumably because of mortality, migration out of the lake, and a movement to the pelagial area of the lake. Offshore movement was evident during August 5-10, when tow net catches were large and inshore seine catches were very small or nil (Table 1).

On July 28, 1967, small schools of pink fry were observed migrating upstream alongshore through the narrows between Tikchik Lake and Nuyakuk Lake in company with larger numbers of sockeye fry and year-lings. This behavior is unusual for young pink salmon The young pinks were intermediate in size between the two year classes of young sockeye and were more active than the young sockeye.

The average growth rate of pink fry in Lake Alek-nagik from June 23 to August 7 in 1965 and 1967 was

13

0.6 mm/day, nearly 21/2 times greater than the rate for sockeye fry in the same lake. The mean lengths of pink and sockeye fry (preserved specimens) from sam-pling in the lake on August 7 in odd years from 1959-1967 were inversely correlated with sockeye pop-ulation density (Table 1). The mean length of pink fry caught in early September was 89 mm and was comparable to the length of yearling sockeye salmon.

TABLE 1

Geometric mean catches and lengths of pink salmon fry and sockeye salmon fry from tow net sampling in Lake Aleknagik

in odd years, 1959-1967

Pink salmon fry Sockeye salmon fry

Mean Mean

length length Number of

Geometric mean catch (mm), (mm), parent Year Aug.5-10 Sept.1-10 Aug. 7 Aug. 7 spawners

1959 0.23 0.00 78 53 63,000 1961 - 0.00 85,400

1963 0.36 0.00 77 51 48,000

1965 0.79 0.08 67 42 155,000 1967 0.05 0.03 60 40 287,500

In the years when there was relatively good growth among salmon in Lake Aleknagik (1959 and 1963), the mean lengths of pink salmon fry in catches were nearly comparable to those of pink salmon smolts caught in Chignik Lagoon (a salt-water estuary) on August 7, 1965 and 1967 (77 and 83 mm) (personal communication from Mr. Duane Phinney, Fisheries Research Institute). However, young pink salmon usually leave the salt-water estuaries by early August in most areas of Alaska, and at this time their average length is considerably greater than that of pink salmon in Lake Aleknagik. For example, the average length of pink salmon caught outside of Uganik Bay (Kodiak Island) in early August, 1963, was 117 mm (personal communication, Mr. Richard Tyler, Fisher-ies Research Institute), and juvenile pink salmon

TABLE 2

Percentage composition of food in stomachs of pink salmon fry, sockeye salmon, and threespine sticklebacks from Lake Aleknagik,

July 1-8, 1967

Pink salmon

fry

Sockeye salmon,

age 0

Sockeye salmon, age!

Threespine stickleback

Number of stomachs 50 45 20 40 Mean length of fish (mm) 39 31 55 52 Percentage composition

by numbers Zooplankton

Bosmina 5.7 4.6 37.3 16.4 Daphnia 6.5 20.3 4.4 1.8 Holopedium 0.8 8.9 3.6 0.4 Cyclopoida 83.8 44.8 39.5 73.4 Calanoida 2.0 0 0 5.3

Insects (by number) 1.1 21.4 15.2 2.7 Percentage of zooplankton

by volumeb 66 21 27 73

a;Numbers of each item expressed as percentage of total items in stomach.

b The average estimated percentage of zooplankton in indi-vidual stomach contents.

caught in the Bering Sea on September 10, 1967 aver-aged 166 mm (data provided by Mr. Allan Hartt, Fisheries Research Institute), nearly twice the length of pink salmon in Lake Aleknagik in early September.

The stomach contents of pink salmon fry taken in four seine hauls from Lake Aleknagik during July 1-8, 1967 were examined, and the food was compared with that of juvenile sockeye and threespine sticklebacks, which constituted the majority of fish in the catches (Table 2). Food items were common to all species; however, insects (primarily midge pupae and adults, Chironomidae) made up the bulk of the food in sock-eye stomachs, whereas, zooplankton made up the bulk of the food of pink fry and stickleback. Although the pink fry were caught in the lake littoral zone (in-shore), their stomach contents indicated that they had foraged mainly in the pelagial zone of the lake.

Donald E. Rogers and Robert L. Burgner

SOME RESULTS OF A SURVEY OF THE BOTTOM FAUNA IN NUSHAGAK DISTRICT LAKES

The emphasis in our research on Nushagak District salmon is on a comparative ecological study of lake systems in the district to determine the factors respon-sible for differences in sockeye salmon production. One aspect concerns the populations of resident fishes, primarily Arctic char and whitefish, and their relation-ships to salmon populations and production. The bottom fauna of each lake was sampled in an effort

to determine abundance of food organisms available to resident fishes in the different lake systems. This investigation and its results will be detailed in a later paper, and only a general description of the results of the survey of benthic macrofauna of the Nushagak District lakes in June and July of 1965 is presented here.

Over 300 samples were collected in eight lakes of

14

Fig. 1.—Sampling in Lake Aleknagik, summer 1965

the Wood River, Snake River, Igushik River, and Tikchik lake systems. Sampling effort was allocated among systems in proportion to the percentage of lake bottom less than 40 m in depth. Stations were located along transects perpendicular to shore. Two samples of bottom sediments were taken at each sta-tion with a one-foot-square Petersen dredge (Fig. 1) and washed to remove the mud (Fig. 2). The remain-ing organic matter was preserved and examined later in the laboratory.

The relative composition of the macrofauna by major groups in the four lake systems is shown in Table 1. Percentages were calculated from geometric means of samples taken between 3 and 15 m, where most of the samples were taken. This method is valid since all lake systems exhibited a similar decline in numbers of organisms (by major categories) with in-

Fig. 2.—Washing sample.

creasing depth. There were two noticeable differences among the systems: all differed in the ratio of tendi-pedid larvae to annelids (oligochaetes and leeches), and the Tikchik system had a greater abundance of molluscs than the other systems. However, the simi-larity of composition among the systems becomes apparent when the data are compared with those for five other large North American lakes (Table 2). Al-though sampling was conducted over greater depths and longer timespans in these five lakes, the contrast in relative abundance of organisms, particularly am-phipods, is sufficiently marked to indicate striking differences in composition of bottom fauna. Amphi-pods were rarely encountered in the Nushagak Dis-trict samples, but were the most abundant organisms in samples of bottom fauna from the other lakes.

Densities of total bottom fauna to a depth of 20 m

TABLE 1.

Percentage numerical composition of bottom fauna of four major lake systems of the Nushagak District, Alaska, in June and July, 1965

Tendipeclid

Av. sampling System

larvae Annelids Pe lecypods

Gastropods Nematodes Misc. depth (m)

Wood River lakes 53 32 8 4 2 1 9.3 Snake River lakes 43 44 8 4 1 0 9.2 Igushik lakes 65 20 8 4 3 0 7.8 Tikchik lakes 32 44 10 11 1 2 9.2

TABLE 2.

Approximate percentage numerical composition of bottom fauna of five large North American lakesa

Lake Tendipedids Oligochaetes Pelecy pods Gastropods Amphipods Misc.

Great Slave 8 10 11 3 62 6 Athabaska 11 13 9 2 61 4 Reindeer 4 5 16 1 72 2 Winnipeg 0.4 9 1 64 15 Michigan 19 5 64 10

a Taken from Rawson, D. S. 1953. The bottom fauna of Great Slave Lake. J. Fish. Res. Bd. Canada 10(8):486-520.

15

TABLE 4

in the four lake systems are presented in Table 3. Densities were determined from weighted average densities over depth strata, and each stratum average was weighted by the relative area of that stratum. Data from several other lake regions are presented in Table 4 for comparison. Again, qualifications must be made for the reasons mentioned above (depth and timespan differences). In addition, mollusc weights were not excluded from the calculations for Table 3 as they were for some of the lakes given in Table 4. The Alaskan data were derived from geometric means and the data for the European and North American lakes, from arithmetic means.

TABLE 3.

Densities of bottom fauna to 20 m in the four major lake systems of the Nushagak District, Alaska, in summer, 1965

Wet weight Number of Total number in kg per

Lake system samples per m2 hectare

Densities of total bottom fauna in selected lake regionsa

Region Number of lakes

Wet weight in kg per hectare

Alpine 43 76.1 N. Germany 64 115.0 N. Canada 13 88.9 Eastern U.S.A. 38 87.2 Finland 75 23.6 Sweden 5 31.1 Russia 10 41.3 N. Brunswick 3 25.2

a Taken from Haynes, F. R. 1957. On the variation in bottom fauna and fish yeild in relation to trophic levels and lake dimension. J. Fish. Res. Bd. Canada 14(1):1-32.

Benthic Macrofauna /wet wt. in kg. x I 0-1/ho)

l0

5

114

726

74.9

20

1,198

76.0

19

393

41.1

64

1,598

85.5

Wood River lakes Snake River Lake Igushik lakes Tikchik lakes

Zooplankton ( Total organisms 10-3/m3)

■atig#

Whitefish (Numbers x 10/net. hr.)

Tikchik

The trend in macrofaunal density among systems is compared with the trends in densities of whitefish, zooplankton, and sockeye salmon spawners in Fig. 3. Points for whitefish represent a three-year average for the Tikchiks and two-year averages in the other sys-tems. Points for zooplankton represent two to five-year averages, depending on the lake system, and points for salmon spawner density represent a seven-year average. The trends for salmon and zooplankton are similar, whereas the trends for whitefish and mac-rofauna tend to be just the opposite. The trends for zooplankton and macrofauna differ, possibly because lake nutrients not used by small zooplankton popula-tions are utilized by benthic animal populations or available nutrients differ in quality. If it is assumed that the fish populations in these lake systems are

Sockeye Salmon Spawners

(Numbers x I0-3/km2 of lake surface)

Snake Wood lgushik River River River

Lake System

Fig. 3.—Trends in benthic macrofauna, zooplankton, sockeye salmon, and whitefish densities among four lake systems of

the Nushagak District, Alaska.

limited by the food supply, then it appears from Fig. 3 that the salmon populations, which feed largely on zooplankton, are limited by zooplankton abundance, and that whitefish, which feed primarily on benthos, are limited by bottom macrofaunal density.

Jerry E. Reeves

MAGNITUDES OF SOCKEYE SPAWNING COLONIES

Numerical estimates of spawning populations are essential statistics in our study of salmon production and survival in the Wood River system. Tagging experiments have been conducted annually since 1965 for the purpose of evaluating aerial and ground sur-vey procedures used in estimating the sockeye popula-tion in each of three types of spawning grounds:

creeks, rivers, and beaches. In certain of the Wood River lakes, the majority of spawners utilize the beaches, whereas in others, the majority spawn in tributary creeks and rivers. Should there be a ten-dency to overestimate the abundance of spawners in one type of spawning ground in relation to another, then there would be a bias in estimating the popula-

16

tions of isolated spawning areas and also of individual lakes in the system. Thus, there is a need for deter-mining the reliability of estimates obtained through mark-and-recovery techniques before they can be used for evaluating population estimates obtained by other means.

In the 1965 pilot study, the population of a short connecting river was estimated in a tagging experi-ment. Petersen disc tags were used. The results indi-cated that an accurate estimate was obtained, but that considerable time and personnel would be necessitated by tag application in a large-scale operation.

In 1966, the spawning populations of a creek, a river, and a beach were estimated. The dorsal fin of each fish was perforated with a paper punch and the adipose fish removed with a linoleum knife. Two workers were able to seine and tag from 500 to 1,000 adult sockeye per day depending on the availability of fish. Thus, the requirements for speed and person-nel were met. The results showed that removal of the adipose fin was a reliable mark, but perforation of the dorsal fin was not.

The populations of areas studied in 1966 and an additional spawning area of each type were estimated in 1967. Each fish was tagged with a Dennison tag and its adipose fin was removed. The tag was inserted with a Dennison Swiftacher tag applicator either into the flesh below the dorsal fin or through the fish at the same location. Two workers were able to seine, tag, and remove the adipose fin from 400 to 700 fish per day depending on availability. Tag losses ranged from 22.3 per cent to 73.7 per cent; therefore, mark-and-recovery population estimates were based on the adipose fin mark.

The time and location of marking greatly influ-enced estimates, especially in the case of creeks and rivers. Marking too early in the season may result in

the marking of transient fish, whereas marking too late will result in the marking of members of the population that are unlikely to be recovered until most spawners have died. In the creeks, marking is best conducted at the mouths, where spawners school before entering. Most spawning rivers in the Wood River system are connecting rivers between lakes, and spawners school at both the head and mouth of these streams. Marking operations were conducted at each end of the rivers, but operations at the heads of these streams yielded better mark distributions and popula-tion estimates.

Recovery effort requires proper timing. On creeks and rivers only dead fish were examined for marks, whereas on beaches both live and dead were exam-ined. Dead fish from creeks were collected until nearly all fish had died. A comparison of the esti-mated spawning populations of Hansen Creek with the peak ground counts in 1966 and 1967 (Table 1) shows the results of proper timing and marking and recovery effort. It is assumed here that the peak ground count does not include salmon removed by predators or those schooled at the mouth; thus, the ground count should be lower than the actual popu-lation. Frequent tag recovery efforts are precluded in large rivers by the amount of time required to cover the river, hence recovery should be conducted at se-lected periods, and the last effort should be made when nearly all spawners have died. This procedure was followed in the Agulukpak River in 1966 and 1967, and reliable population estimates were obtained (Table 1). On the other hand, during the last recovery effort in the Agulowak River in 1967, there were many live fish, mark distribution was poor, and the two marked units gave dissimilar estimates; thus, a biased estimate was obtained.

In nearly all the estimates, sex ratios calculated

TABLE I.

Estimated magnitudes of isolated population units in the Wood River system from ground and aerial survey counts and the results of marking experiments

Area Date Marking estimate

95% confidence interval

From To

Peak ground count

Aerial survey estimates Peak Adjusted

estimate estimate

Bear Creek 1967 6,190 4,178 8,798 2,123 1,000 1,993a Hansen Creek 1966 14,630 9,328 19,932 14,658 110,000 I I9,948a Hansen Creek 1967 4,565 3,989 5,141 3,332 1,200 1,993a Agulowak River 1967 123,598 99,136 148,060 63,000 70,000b Agulowak River 1966 115,893 86,029 145,757 100,000 110,000b Agulukpak River 1967 31,610 27,835 35,385 25,000 25,000b Little Togiak River 1965 8,946 6,165 11,727 8,000 14,000b Anvil Bay 1966 29,350 24,000 34,700 35,200 107,994a Anvil Bay 1967 18,752 16,876 20,628 17,400 51,191a Silver Horn 1967 26,994 24,777 29,211 15,210 36,109a

a,b - Only connecting rivers and lakes are considered as units for the adjusted aerial estimates, thus creeks and beach areas are adjusted as one for each lake. The method of adjustment is explained in the tet.

17

from the marking estimates were not in agreement with sex ratios determined directly from the recov-eries. Unequal predation and differences in the num-bers that drifted, floated, or wandered may have been the cause. Moreover, male spawners schooled at the river mouths did not enter the recovery areas in pro-portion to males marked at the river heads, females marked at the river heads, or females marked at the river mouths. Females were consistently more abun-dant than males among dead fish recovered in sections near the river head. The percentages of males in four nearly equal sections of the Agulowak River, from head to mouth, were 28.5, 32.7, 47.5, and 62.5 per cent.

Recovery of marked fish from beach spawning areas was carried out in two ways: dead fish were collected or speared, and live fish were seined. Because of the considerable length of occupancy of beaches by spawn-ers, recovery effort of dead fish only would necessitate sampling over a period of several weeks. At Anvil Bay, where both live and dead fish were recovered, separ-ately based estimates were 24,451 and 13,720 spawners, respectively. Estimates based on live fish recovered from three separate tagged units at Silver Horn before any had died were: 27,349, 27,092, and 26,209 spawn-ers. Although there was a 22.3 per cent tag loss at this area, fish bearing only the adipose mark were assigned to the three tagged units on a prorated basis as the ratios of tags applied to tags recovered for all groups were similar. In beach spawning areas, marking is best accomplished when most fish are schooled, and recovered, by systematically seining along the entire area before any fish have died.

Estimated populations of the areas studied are shown in Table 3, together with aerial survey esti-mates and adjusted estimates from the Alaska Depart-ment of Fish and Game. Adjustment is done to ac-count for escapement into the system that was not accounted for by aerial survey and is based on the relationship between peak aerial counts and adjusted estimates from the previous year. When the mark and recapture estimates are used as a basis for judg-ment, it appears that the weighting factors used to adjust aerial estimates in the different types of spawn-ing areas may need modification. Adjusted estimates for beach areas appear to be too high; those for rivers and perhaps creeks, too low.

Our effort to estimate the spawning populations of different spawning areas met with varied success. Creeks were not difficult areas to survey. The least expensive and most consistent results were gained from ground counts at the peak of spawning, supple-mented with distribution information from aerial sur-veys where creeks could not be covered in their entir-ety. Estimates of the populations of rivers and beaches made by the mark-and-recapture method were reliable when proper technique was used.

The procedures for collecting fish for marking and the recovery techniques used in these studies have been satisfactory. Removal of the adipose fin, although satisfactory as a secondary mark, does not provide a means of identifying components of the population. We need a tag that is simple and easy to apply and that will remain in the fish from the time of tagging until considerable deterioration has taken place.

Kenneth Roberson

KVICHAK SOCKEYE

Variability in Survival Rates of Sockeye Salmon in the Kvichak River System

Cyclic changes occur in the magnitude of the sock-eye salmon run to the Kvichak River system. During the last cycle, the run increased from 652,000 (1963) to 46,566,000 fish (1965) and fell to 4,914,000 fish (1967). Such abrupt changes in abundance create numerous problems for the fishermen, industry, and management authorities alike. It appears that they persist because of a lower survival rate among the progeny in some of the off years of the cycle than in

1 This work is supported by the U.S. Fish and Wildlife Service, Bureau of Commercial Fisheries, and the Association of Pacific Fisheries.

SALMON STUDIES'

the peak years. Therefore, it is important to establish where and when these changes in survival rate occur. Thereby, a rational basis will be provided for con-sideration of different strategies for management of cyclic salmon runs.

The variability in total survival rates for 1952-1961 is given in Table 1. In the three peak brood years 1952, 1956, and 1960, the total return was almost four times as great as the parent escapement, but in the other years except 1954 and 1955, the total return was less than twice as much as the parent escapement and sometimes even smaller than it. The cyclic pat-terns have not always been consistent, as may be seen from the high returns for the 1954 and 1955 brood

18

TABLE 1

Variability in total survival rates of the Kvichak sockeye salmon runs, 1952-1961

Year of escapement

Escapement, in millions

Total return, in millions

Ratio of parent

escapement to return

1952 5.970 23.041 3.86 1953 .321 .592 1.84 1954 .241 .755 3.13 1955 .250 2.080 8.32 1956 9.443 37.763 4.00 1957 2.964 4.016 1.35 1958 .535 .289 .54 1959 .680 .552 .81 1960 14.630 54.019 3.69 1961 3.706 3.362 .91

years, but they have become more pronounced in recent years; thus changes in survival rate have also become more pronounced.

Variability in survival rate in the ocean. Yearly changes in total survival rate may take place either in freshwater or in the sea or in both environments. A measure of variability in ocean survival is afforded by the total return per smolt unit that migrates to sea. Because of the size of the Kvichak River and the large number of smolts involved, no estimate exists of the absolute magnitude of the smolt migration. A relative index is obtained annually by the Alaska Department of Fish and Game. This is the catch obtained by a fyke net fished in a standard pattern. The index is not a precise one and is greatly influ-enced by environmental factors, especially by ice drift-ing in the river at the time of smolt migration. Hence, the available data give only indications of smolt mag-nitude. From these data, it appears that there has been great variability in survival rate, and not all large smolt migrations provided a high return per migrating smolt unit.

Factors other than total number may contribute to the final survival rate, such as the size of the smolts, the timing of their migration, and the degree of infec-tion from parasites. Much work on these and other factors remains to be done on the Kvichak sockeye salmon before returns to this district can be accurately forecasted.

Variability in survival rate in freshwater. We know more of the variability in survival rate during the freshwater phase of life than the marine phase. Our studies in freshwater were greatly strengthened last summer, when our facilities were augmented with a new boat provided by the salmon industry (Fig. 1).

The first life stage for which we have data covers the time from spawning until the fry have moved

Fig. 1.-The new research vessel, the Sa-yak.

into the pelagic area of the lake in the following sum-mer. At this time the fry can be sampled quantita-tively by tow nets, and the mean catch per tow net haul is used as a measure of relative abundance. For four of the six year classes sampled to date, the ratios of parent escapement (in millions) to fry abundance index (mean catch per tow in the following year) varied from 1:3 to 1:6 (Table 2). However, for the 1962 and 1966 year classes, the ratios were 1:10.7 and 1:17.8, respectively. These estimates do not indicate a higher survival rate from spawning to the fry stage for the peak cycle year class (1965), and the two high survival rates (1962 and 1966 year classes) are not in syncrony with the cycle. It appears that the survival advantage of large year classes (as indicated in escape-ment-return data) may not show until later life stages or the peak cycle spawning population in 1965 was excessive.

Tow net catches in 1967 indicated a compar-atively high survival rate for the 1966 year class. Its abundance was estimated to be nearly half that of the

TABLE 2.

Parent escapements of sockeye salmon and mean tow net catches of fry in Iliamna Lake

Ratio of escapement

Kvichak escapement (in millions) Number Mean catch offry to mean

Year (x105) Year Number fry catch

1961 3.7 1962 21.8 5.9 1962 2.6 1963 27.8 10.7 1963 0.3 1964 0.9 3.0 1964 1.0 1965 4.4 4.4 1965 24.3 1966 151.7 6.2 1966 3.8 1967 67.6 17.8

19

Age I Smolt Index - - - -Age 1.2 Return - in Mi lions

1962 1964 r —Ow

1967 Escapement

in Millions FryCatch/Tow 1960 E315/h1 1965 1966

1 5 6 4 2 a

1970 Age 2.3 Return 1966

961---1

Yearling trol■Elndex

1962 1963 V93A —OM 1967

196

1969 Age 1.3 Return 1965

Age 2.2 Return 1965

.-1971

1970

1966 1967

Newhalen Village Section 4

01'

Fig. 3.—Map of Iliamna Lake, the Newhalen River, and Lake Clark, showing divisions of Iliamna Lake and tagging and

recovery sites on Newhalen River.

Iliamna Lake

we

100-

Perc

ent C

yclo

ps so

lder

80-

60-

40-

20- \•

80-

60-

40-

20-

Per

cent

Bosm

ino

1 2 3 4 Lake Sections

1965 year class, even though its parent escapement was only about one-seventh of the latter's (the largest es-capement on record). If a normal survival rate should continue, substantial numbers of this class would re-turn in 1970, 1971, and 1972.

Part of the 1965 year class migrated seaward in the spring of 1967, and part still remains in the lake. Relative data on abundance for the 1960, 1965, and 1966 year classes are summarized in Fig. 2 for com-parison. Fish from the 1960 year class made up almost the entire 1965 run. The yearling smolt production

in 1967 (1965 year class) was over 21/2 times that in

1962 (1960 year class), yet the parent escapement of the year class was about 1.7 times larger than the other's. Returns from this part of the year class are expected in 1967 and 1970. The relative abundance of yearlings remaining in Iliamna Lake in 1967 was 80.9 per tow, somewhat less than in 1962 (93.3 per tow). These fish will migrate seaward in the spring of 1968. Returns are expected in 1970 and 1971.

Year of Spawning) Years after Spawning

Fig. 2.—Sockeye salmon production from the 1960 and 1965 peak brood years and the 1966 subpeak brood year,

Kvichak River system.

Relationship Between Food Supply and Salmon Production in !Hamm Lake

We have conducted investigations in Iliamna Lake since 1962 to determine the relationship between food supply and growth and survival of salmon. Cyclops scutifer and Bosmina coregoni, the two planktonic crustaceans that predominate in the lake, are the prin-cipal food species for both fry and yearlings. In 1967, we conducted sampling of zooplankton and juvenile salmon in certain parts of the lake. We used a No. 6 mesh net for plankton and a tow net. Cyclops scutifer dominated in the zooplankton fauna in the eastern sections of the lake (3 and 4, Fig. 3) and Bosmina coregoni in the western sections. The stomach con-tents of fish captured reflected the nature of the food supply in all four sections, but yearlings had fed heavier on the larger form, Cyclops scutifer, whereas

Lake Clark

0 10 20 30 40 Nondalton I I 1 1 Fish Camp

KILOMETERS

Noma

fry had fed more intensively on the smaller organism, Bosmina coregoni (Fig. 4). However, the difference in feeding habits between the two age groups of fish does not preclude competition for food since it is related to the total availability of food in the nursery areas. We have ascertained the standing crop of zooplanktoh each year since 1962, and will soon describe its rela-tionship to fish production.

The standing crops of zooplankton in the last three summers are shown in Table 3. The standing crop was less in the summer of 1967 than in the two pre-vious summers, except July, 1965. The large biomass

—•—# 6 Mesh Net Samples --•--- Sockeye Fingerling Stomachs

• Sockeye Fry Stomachs

O r 1 2 3 4

Lake Sections Fig. 4.—Percentages (by numbers) of Cyclops scutifer and

Bosmina coregoni in food samples from August, 1967 sampling, Iliamna Lake.

20

15- _

10- _

of juvenile salmon in Iliamna Lake may be expected to reduce the standing crop of food organisms during the winter of 1967-68. However, the standing crop may not reflect directly the total food available since it does not measure the reproductive rate of the food organisms. The reproductive rate involves birth and death rates (from natural causes and grazing by the young salmon). These factors are under study.

TABLE 3.

Average numbers of zooplankton organisms per cubic meter of Iliamna Lake in the upper 100 m in the last three summers a

Year June July August

30

20- _

"ts

lb

5- _

I l l l i t l i t t i l l l i l t I t t t t I l i t I I

5 10 15 20 25 30 4 July August

Date of Tagging

Fig. 5—Migration time of fish tagged at Newhalen Village and recovered at Nondalton fishing camp.

Total

Cyclops scutifer

Bosmina coregoni

1965 1966 1967

1965 1966 1967

1965 1966 1967

5,528 6,363 4,450

3,755 4,585 3,643

329 496 147

5,774 8,829 9,770 10,174 6,182 7,232

3,637 3,743 6,252 4,389 2,858 1,883

944 3,037 2,220 3,473 1,261 2,384

a Calculated from geometric means weighted according to volume.

Availability of zooplankton food is related to the production of phytoplankton, which forms the first trophic level. Studies of phytoplankton production through the 14C technique have been conducted in Iliamna Lake since 1962. Relationships cannot be expressed as yet in quantitative terms because certain aspects of the environmental variability are not clear. For example, the variability in photosynthetic rate observed at one station in Iliamna Lake during a 6-day period in 1967 approached the variability some-times observed among sections of the lake within the same year or between years (Table 4).

Tagging Studies in Newhalen River The Kvichak salmon investigations have largely

been devoted to Iliamna Lake. Lake Clark, the next largest nursery lake in the Kvichak system (Fig. 3) is

TABLE 4.

Variability in photosynthetic rate in Knutson Bay, Iliamna Lake, from July 14-19, 1967

12C assimilated Date Per m2 Per m3

July 14 75.3 1.67 July 15 60.5 1.51 July 18 49.4 1.40 July 19 64.2 1.82

143 square miles in area and provides 12 per cent of the total nursery area. The size of the escapement to Lake Clark has not been determined exactly because the glaciated water in the trunk stream, the lake, and some of the tributaries does not permit visual observa-tion.

In an effort to develop a method for escapement enumeration, a tagging experiment was conducted in 1967. Salmon were tagged at the mouth of Newhalen River at Newhalen Village and recovered at the upper end of the river at the Nondalton fishing camps lo-cated 25 miles upstream. The Dennison tag, a new type of tag, was used (see Figs. 2 and 3, High Seas Salmon Studies, in this issue). Dates of tagging and mean times in days before recovery are given in Fig. 5. Salmon tagged late in the season completed ascent of the Newhalen River in 20 days, whereas those tagged early in the season covered the distance in 30 days. The average period was 23 days. The water level has varied from year to year, and in some seasons it may have delayed or prevented migration. A similar situ-ation has been observed in the Fraser River, B.C., and found to limit success of spawning. Perhaps a similar effect is the cause of the comparatively small salmon production in Lake Clark. We plan to continue in-vestigations in this regard.

Ole A. Mathisen, Orra E. Kerns, Jr., John W. Anderson, Stephen H. Hoag, Richard A. Baxter,

Tor B. Gunnerod, and Patrick H. Poe

21

Washington Fish Studies

BIG BEEF CREEK SALMON STUDIES'

Research facilities at the University's field station at Big Beef Creek have been augmented, and some minor changes have been made in the natural environ-ment. Laboratory and storage space, bathroom facil-ities, and a fireplace were added to the cabin, and a telephone was installed. Log jams were cleared in the lower stream and some of the bends riprapped to re-pair damage from the spring floods.

During the year the facilities were utilized also by independent investigators and research units, includ-ing Big Beef Creek Research II (Initiative 171), Salm-on Physiology (Bureau of Commercial Fisheries and Public Health Service), the Cooperative Fisheries Unit (Trout and Salmon Behavior), and Chum Spawning Channel Research (Anadromous Fish Act, P.L. 89-304). The last project contributed the most significant addi-tions to facilities (Figs. 1 and 2).

Coho Salmon Studies The extensive urban development that is taking

place in the Puget Sound area will undoubtedly alter ecological conditions in many salmon-producing

Fig. 1.—Chum salmon spawing channel, during construction, showing placement of gravel in spawning pens.

Fig. 2.—Lower pond, showing trap for adult chum salmon. Pond can also be used for rearing—either in freshwater or

salt water—of fry produced in channel.

streams. Some of the studies at Big Beef Creek are concerned with the effects of the Wm. Symington real estate development. A feature of the development is an artificial lake with a surface area of approximately 80 acres and a storage capacity of about 800 acre-feet, which was created by damming the creek. It is antici-pated that, in the next eight years, from 700 to 800 homes will be constructed along its shores.

Since the spring of 1966 we have been collecting information on environmental conditions, the magni-tudes of populations of adult and smolt coho salmon, and the growth rates of juvenile coho in the lake and in the stream above and below the lake. Temperature, water transparency, concentrations of nitrates, phos-phates, total dissolved solids, and abundance and com-position of zooplankton are monitored.

The adult run in the fall of 1968 was estimated by a tagging program. The fish were trapped and tagged

Supported by Graduate School Research Fund—Initiative 171.

22

near the mouth of the stream and were recovered by means of a temporary trap at the dam. The estimated

spawning population was 5,536 fish (N = 6,791 and N = 4,429 — 95 per cent confidence limits). Two thousand and one fish were passed over the dam to spawn in the stream above the lake, and the remain-der spawned in the stream below the dam. The majority of these fish spawned within a mile of the lake, and it is thought that some of them had been spawned in the upper part of the stream prior to the construction of the dam.

The populations of smolts in the lake and the stream above and below the lake were also estimated. Traps were located at the lake inlet and outlet and near the stream mouth. Smolts in the area above the upper trap were marked with fluorescent pigment and re-leased. The ratios of marked fish to unmarked in catches at the three traps were noted and used as indices of abundance. The results follow:

'I ABLE I.

Estimated smolt populations of the lake and stream above and below the lake

.1c1

95% Confidence limits

Stream above lake 26,349 30,292 23,115 Lake proper 4,456 5,011 3,989 Stream below lake 33,998 37,058 31,303

Total 64,803 70,011 60,147

Growth in length was measured at monthly inter-vals, except during the winter when high flows pre-vented sampling. By September, the mean length of lake inhabitants was 90.6 mm, whereas the mean lengths of fish at sites in the upper stream were 55.6 and 51.9 mm. Immediately below the lake, the fish averaged 60.3 mm; and the fish near the stream mouth had a mean length of 77.5 mm. Winter growth was greater among fish in the upper stream than among fish in the lower stream; thus, by spring, the fish in both streams were of nearly equal size. Growth in the lake was good all winter, and by spring (1967), the fish were from 30 to 40 mm larger than the stream fish on the average.

Smolts of 0 age were captured in a trap at the lake outlet during the outmigration. These fish were larger than the average yearling coho salmon from the upper stream.

Condition factors were determined for coho salmon collected in the upper stream, the lake, and at five sites along the lower stream. As juvenile coho salmon are socially organized, condition factors vary accord-

ing to social status within a particular microhabitat; the dominant individuals have larger condition fac-tors than subordinate members of the organization. In this study, fish in a complete sample were grouped into three size groups: large, average, and small. Those fish in the first group were considered to have been dominant, whereas those in the last group were considered subordinate. The farther upstream the samples had been taken (excluding lake samples) the greater the difference between condition factors of dominants and subordinates was and the smaller the condition factors of both dominants and subordinates were. It is believed that these phenomena are typical of natural streams with high population densities and intensive intraspecific competition. In the lake, where competition was essentially absent, condition factors were significantly greater.

The mean summer temperature in the lower creek was only 3°F warmer than that of the upper stream, whereas the mean lake temperature was 12°F warmer. It appears that the stream below the lake recovers rapidly from the high temperatures of the outflow; temperatures less than one mile below the dam were virtually the same as those at the mouth of the stream. Finally, the lake caused increased variance in the diurnal fluctuation of temperatures in the stream be-low the lake during the summer months.

Chum Salmon Studies'

Experiments to determine the effects of various qualities of gravel on the survival of eggs and quality of fry were begun in a spawning facility that was constructed in an existing side channel of Big Beef Creek in the fall of this year. The facility was com-pleted by mid-December, in time to accommodate the last spawners, It (Fig. 1) is 600 ft long and 10 ft wide and has 24 50-ft x 5-ft spawning pens. Four different grades of spawning gravels, each with a different per-centage of fines, are being tested. The objective is to determine the effects of various qualities of gravel on the survival of eggs and quality of fry. Standpipes are inserted into the gravel at 96 locations for the sam-pling of intragravel water.

At the lower end of the channel, three ponds were excavated, diked, and equipped with tide gates (Fig. 2). They will be used in studies of the influence of tidal action on the lower spawning channel and rear-ing of salmon in freshwater and salt water.

Kenneth R. Williams, K Victor Koski, and Ernest 0. Salo

Supported by the Anadromous Fish Program, Bureau of Commercial Fisheries, Contract No. 14-17-0007-877.

23

Bellingham Bay

South Bellingham

Bellingham

45,

NAUTICAL MU/ -Silver • Chinook

DISTRIBUTION AND SURVIVAL OF JUVENILE SALMON IN BELLINGHAM BAY'

Research was directed towards a description of the life of juvenile salmon in Bellingham Bay, and inves-tigations, begun in 1966, into the abundance and natural mortality and growth rates of juvenile coho salmon in three streams tributary to the Nooksack River were continued (1966 Research in Fisheries).

A total of 271,362 chinook salmon and 176,640 coho salmon were taken from the Nooksack River State Salmon Hatchery, marked with fluorescent pigment, and released at the hatchery. Townetting was begun in the bay on April 27 and continued until the out-ward migration of salmon was nearly complete. Tow-ing was conducted with a 10-ft tow net along selected standard transects. Water temperature, turbidity, and tide were determined and water samples were taken in conjunction with fish sampling. Samples were analyzed later for salinity and content of sulphite waste and dissolved oxygen. Salinity will be plotted and analyzed by the EXTAB-2 computer program in an effort to determine whether the migration routes are related to these variables, especially SSL.

The first catch of salmon was taken on May 3, and the outward migration of salmon was nearly complete on June 20. A total of 2,002 chinook and 769 coho salmon and numbers of chum salmon, herring, smelt, sticklebacks, and other fishes common to Puget Sound were captured. Coho and chinook were observed under ultraviolet light to detect those that had been

'Supported by the Georgia-Pacific Corporation and the State of Washington Water Research Center.

Fig. 1.—Distribution of marked salmon in Bellingham Bay.

marked earlier at the Nooksack Hatchery. Forty-one marked coho salmon and 128 marked chinook salmon were captured. The distribution of marked fish in the bay is shown in Fig. 1. Catch and environmental data will be plotted and analyzed by the EXTAB-2 computer program for the purpose of determining whether migration routes were related to water qual-ity.

Wayne G. Williams, Danny E. Sjolseth, Robert A. Bishop, and Ernest 0. Salo

ESTUARINE ECOLOGY STUDIES'

A program of research was initiated by the Fisheries Research Institute in 1965 to assess the effects of pollutional loads on the water quality and ecology of pelagic and demersal fishes in Elliott Bay and to forecast through simulation techniques the effects of pollution abatement. The program of research and studies in 1966 were reviewed in the 1966 Research in Fisheries, and the present report is restricted to prog-ress in studies of water quality, juvenile and adult chinook salmon, and demersal fishes.

Studies of Water Quality Man's ability to change and monitor the estuarine

environment has resulted in the creation of numerous

'Supported by the U.S. Public Health Service, WP-00642-03.

agencies for preventing its impairment and alleviating damage. Much data on water quality have been col-lected by these agencies, but often they are not fully utilized because of the lack of data processing and analytical techniques.

We developed techniques for processing and ana-lyzing data obtained from four automatic water qual-ity monitoring stations maintained in the Duwamish Estuary by the Municipality of Metropolitan Seattle (METRO) and United States Geological Survey. The stations measure dissolved oxygen, conductivity, tem-perature, pH, and solar radiation intensity and are located at Renton (station 1), East Marginal Way (station 3), 16th Avenue South (station 5), and Spo-kane Street (station 7) (Fig. 1). Considerable effort was

24

• Observed DO. content

--- -12.32893+0.18558 x tidal prism +0.41500x station 70.0. +4.1007 x log Istreamtlowl

— -13.86181+0.20941 x tidal prism + 5.56699v log (streamflord

Fig. 1.—Elliott Bay, Duwamish Estuary, and Duwamish-Green River.

required to convert the data from an unreliable and not readily usable form to an easily read summary table. Eight computer programs were written and used in this task. Although our data processing system is designed for the Duwamish Estuary monitors and the IBM 7040-94 DCS at the University of Washington Computer Center, the techniques used are general and would aid agencies undertaking similar projects.

There are four major steps in data processing. The data are screened to remove observations that fall outside probable limits, show too rapid a change, or are known to be invalid from monitor failures. Dis-solved oxygen trends that are shown by field calibra-tions to be invalid are removed. Occasional missing observations are estimated by an interpolation pro-gram that assumes a quadratic relationship between time and the variable to be interpolated. Summary tables of daily and weekly means, maxima, and min-ima and standard deviations are prepared.

Preliminary analysis has indicated that simple mul-tiple regression models may be used to provide de-scriptions of the relationships among the measured variables and external conditions. The models allow

14 28 14 28 II 25 9 23 6 20 4 18 1 15 29 2 26 February March April May June July August September

Date

Fig. 2.—Observed and predicted daily average dissolved oxygen values 0.9m above the bottom at station 5 during the period

February through September, 1966.

estimation of daily average dissolved oxygen values at the 16th Avenue South Station, the critical area of the estuary, from streamflow, tidal prism, and dis-solved oxygen values from adjacent stations. Less precise estimates are obtained when the dissolved oxygen values from adjacent stations are deleted from the model. Fig. 2 illustrates the predicted and ob-served values. It is apparent that adjacent deviations between predicted and observed values tend to be similar. Thus, the assumption of homogeneous and independent error terms was probably violated and probability statements concerning the significance of the models and coefficients cannot be made without further manipulation of the data.

The results of the regressions agree with intuitive expectations. The positive correlation between stream-flow, tidal prism, and dissolved oxygen content could be due to several factors. High streamflow is associated with a high aeration rate in the Duwamish River and high stream influence on the estuary. High stream-flow also causes great turbulence in the estuary. Thus, the highly saline bottom water of the estuary is mixed with the surface water and replaced by new, highly saline water from Puget Sound at a rapid rate during periods of high streamflow. A large tidal prism also results in increased turbulence and replacement of the bottom water. Welch2 demonstrated that organic matter, including dead algae, which have a high BOD, settles to the bottom in the area of the 16th Avenue South Station. The dissolved oxygen content of the bottom water is a function of the amount of time

2 Welch, E. 1967. Factors initiating phytoplankton blooms and resulting effects on dissolved oxygen in an enriched estuary. Ph.D. Thesis, Univ. Washington, Seattle. 101 p.

25

— Dissolved oxygen --- Tidal level

that BOD is high. Thus, low streamflows and tidal prism would be expected to be associated with low dissolved oxygen content.

The strong correlation between tidal prism and dissolved oxygen content at the bottom of the 16th Avenue South Station is not caused solely by the relationship between circulation and tidal prism. Tidal action pushes the salt wedge back and forth in the estuary. Correlation between tidal stage and dissolved oxygen content is direct until the salt wedge passes the station, when it becomes indirect (Fig. 3). Thus, the correlation between tidal prism and dis-solved oxygen content is a measure of both the effect of flushing and the body of water whose dissolved oxygen content the probe is measuring.

Our future work will include the construction of more complex regression models and spectral analysis. A simulation model of the estuary will be developed in which some of the results of the analysis will be incorporated.

8 16 24 8 16 24 8 16 24 8 16 24 8 16 24 8 16 24 8 16 24 8 16 24 8 16 24 17 18 19 20 21 22 23 24 25 26

September Date

Fig. 3.—Comparison of hourly dissolved oxygen concentrations 0.9m above the bottom at station 5 and predicted tidal heights

at Elliott Bay, September 17-26, 1966.

Studies of Juvenile Chinook Salmon

The effective management of a salmon stock de-pends on a thorough knowledge of every phase in its life cycle. Thus far, studies of chinook salmon have focused primarily on the ecology of reproduction and on the period of life encompassing ocean feeding, maturity, and spawning migration. Although the per-iod of downstream migration and early marine life is probably the most critical phase in the life of the chinook salmon, it has been largely ignored. Fishery biologists and resource managers are becoming increas-ingly more aware of the need for information in this area.

In the spring and summer of 1966, and again in 1967, we investigated the ecology of juvenile chinook salmon in the Duwamish Estuary. All aspects of the studies focused on the general objective of understand-ing the dynamics of young chinook salmon in an estuary receiving domestic and industrial wastes. Spe-cifically, our studies were designed to determine the survival rate of fingerling chinook salmon during the downstream migration, and their growth and distri-bution in the estuary.

The survival of downstream migrants. Assessing mortalities among the migrating salmon is a difficult problem. Except in cases of heavy loss, i.e., fish kills, deaths of juvenile salmon are generally unobservable, so we cannot account for all mortalities. We can esti-mate the total loss and compute a mortality rate in some general area and interval of time. This was the procedure followed in our Duwamish Estuary studies.

In 1967 we conducted a mark-and-recapture experi-ment designed to estimate survival of downstream migrants at two different levels of streamflow. The U.S. Army Corps of Engineers controlled the water discharges from Howard Hanson Dam, on the upper Green River, to provide us with the necessary flow differential. Juvenile chinook salmon were provided by the Washington Department of Fisheries.

Two groups of fish were released at the Soos Creek Hatchery, one group of 150,000 fluorescent pigment-marked fingerlings when streamflow was about 1,000 cfs, and another group of comparable size ten days later when streamflow was approximately 3,000 cfs. Each flow regime extended for a period of about ten days. As each marked population migrated seaward and arrived in the estuary, two additional marked groups of 150,000 fish were released at this point. Sub-sequent sampling of survivors of the four groups of marked fish with a tow net in the estuary enabled us to estimate survival rates. Unfortunately, many of the fish were not ready to migrate when released be-cause of poor growth in the hatchery. They delayed in the river and gradually moved seaward. Thus, the two groups did not migrate under greatly different flows.

The survival rate of the first group, /SI, which mi-grated under an average flow of about 1,470 cfs, was estimated preliminarily as 0.513. The survival rate of the second group, t, which migrated under a flow of approximately 1,550 cfs, was estimated as 0.678. It is likely that, after we have obtained estimated travel times, the values for average flow will change and differ greatly. In any case, estimates will be examined for bias.

If it turns out, after corrections are made, that t

26

Upper Duwamish River

Lower Duwamish River

Inner Elliott Bay

Outer Elliott Bay

• • • •

• •

I • MN

50

Mean c

atc

h p

er

standard

tow

40

30

20

10

0

• • • •

June 1-15 June 16-30 July 1-15

Fig. 4.—Changes in distribution and average abundance of juvenile chinook salmon in the Duwamish Estuary during the

1966 sampling season.

• I •

• •

•

• • . •

---0

is significantly greater than /„ then we might as-sume that the success, i.e., percentage return, of chi-nook stocks will be greater in general if the seaward migrants are released under high flow conditions.

In these studies, we viewed mortality as a function of some stress per unit of time and length of exposure to the stress. For example, we postulated that higher streamflows would result in a greater stream velocity, which, in turn, would hasten the downstream migra-tion and reduce exposure to the stresses that the migrants encountered.

Timing the downstream migration. Once we have estimated the survival rate of downstream migrants in a certain area without bias, we will have to define the time interval (travel time) to which it applies. This would be a simple matter if the migrants, released in one group, traveled down the river together and the survivors passed the downstream boundary of the particular interval at once. Although the fingerlings were released in single groups, some fish traveled faster than others, and the survivors entered the estu-ary at different times. Moreover, we assume that mor-talities were not strictly random with respect to swim-ming speed since we feel that the slow-migrating individuals were exposed to stresses for a longer pe-riod of time than faster-migrating individuals and fewer of them survived.

Taking account of these facts, we will compute average travel times from immigration curves, which we can generate from data on catch and effort in the estuary. Although these curves will at first underesti-mate average travel time (show negative bias), we can possibly correct for the bias by assuming that, in the original group of marked fish, swimming speeds were distributed symmetrically.

Distribution of juvenile chinook salmon. We used the townet to obtain samples of juvenile chinook sal-mon from the surface waters of the entire estuary over most of the summer. We made about 1,100 five-minute tows in 1966 and approximately 1,800 five-minute tows in 1967. Catch data from the 1966 and 1967 op-erations indicate that the fingerlings spent at least two months in the estuary, but after the downstream mi-gration was completed, their numbers decreased stead-ily (Fig. 4). They appeared to congregate in clusters or schools and were most abundant along the periphery of Elliott Bay and in the Duwamish Waterway.

Water conditions in the Duwamish Estuary during the summer months. Surface water samples were taken routinely in conjunction with the townetting. Tem-perature, dissolved oxygen concentration and salinity were determined. Throughout June and July, dis-solved oxygen concentrations were probably above the

Region of sampling area

minimum requirements for young salmonids, although these requirements are poorly defined under field con-ditions.

In 1967, temperatures in the low 70's (°F) were recorded in the lower Duwamish River, as Seattle had one of its warmest summers on record. The relatively warm water and sunny conditions prevailing in July and August helped to promote large blooms of phyto-plankton in the estuary. The surface waters of the Duwamish Waterway were supersaturated with dis-solved oxygen during at least two days, even in the early morning hours (Fig. 5).

0 saturation values July 14 (1500-1700 hours)

• observed values July 14 11500-1700 hours)

• saturation values July 16 (0230-0400 hours)

°observed values July 16 (0230-0400 hours)

1st Avenue 16 th Avenue —

2 3 4

Miles upstream from river mouth

Fig. 5.—Observed concentrations of dissolved oxygen in the sur-face waters of the Duwamish River on July 14 and July 16, 1967. Points on the lower curves represent concentration at 100 per cent saturation and computed from data on water temperature

and salinity.

25

20

;13

15

Z,‘

10

(z Spokane Street

27

Visibility (secchi disc transparency) was much lower in the waterway than in the bay, and salinity was not only much lower but also more variable. The mixing of freshwater and salt water was much greater in the river than in the bay, except perhaps along the eastern periphery of the bay, where most of the river water disperses. Until evidence is found to the contrary, in view of catch data and information on water condi-tion, it is reasonable to believe that the fingerling salmon reside largely near the surface of the estuary. This is the region were nutrients, which come from the river and various sewage outfalls, are most con-centrated and is probably the area of greatest primary productivity. Thus, this region may be the best feeding area for all pelagic organisms. The food and feeding habits of the young chinook salmon have not been investigated.

Growth of juvenile chinook salmon. In 1966 and 1967, samples of juvenile chinook salmon were re-moved from catches and preserved in formalin for future studies of growth and scale development. A preliminary analysis of length measurements indicated disparities in growth rates, or at least average size, between salmon in different areas of the estuary. The mean length of 126 fingerlings (measured live) taken above 16th Avenue on June 22, 1967, was 93 mm, and the mean weight was 8.4 g. A sample of 126 finger-lings from the East Waterway had a mean length of 85 mm and a mean weight of 6.2 g. In fact, young chinooks taken from the upper part of the estuary seemed always larger than those taken elsewhere, al-though there was a fair degree of variability. The reason for this is not known and should be a subject of future study.

Predation by lampreys on juvenile chinook salmon. One of the common fishes appearing in the Duwam-ish Estuary tow net catches is the river lamprey, Lam petra ayresi. Little is known of its life history. It spawns in freshwater and the young migrate to sea after several years and grow to a length of about 300 mm. The lamprey becomes predatory after emergence from the stream bottom. In the Duwamish Estuary we observed lamprey predation on numerous occasions. Many specimens of chinook salmon taken in the es-tuary bore either fresh lamprey wounds or scars, and several were taken with lampreys firmly attached to the dorsolateral surface. In a typical case, a 204-mm lamprey was attached to a 100-nun fingerling. In series of tows, as many as 7 per cent of chinooks captured had been wounded by lampreys. In general the lampreys displayed a distribution similar to that of the young salmon, but were especially abundant in the upper portion of the Duwamish Waterway. For the estuary

as a whole, the abundance of lampreys throughout the summer followed the same pattern of decline observed for juvenile chinook salmon (Table 1).

TABLE 1.

Statistics on catch per standard tow of river lamprey and juvenile chinook salmon in the Duwamish Estuary, June and July, 1966

Juvenile chinook

River lamprey Standard

Standard Date

Mean deviation

Mean deviation

June 1-15 31.95 48.54 0.5715 1.548 June 16-30 16.05 23.79 0.2459 0.595 July 1-15 7.28 12.99 0.1915 0.522

Studies of Adult Chinook Salmon

Studies were conducted in the months of August, September, and October in 1967 and were designed to determine the distribution and migration patterns of adult chinook salmon in the Duwamish Estuary and to assess the effect of water quality on distribu-tion, timing of migration, and behavior. Previous studies had shown that chinook salmon reside in the Duwamish Waterway in these months, a period when water temperatures range sometimes from 70-75°F, microbial activity is high, and streamflow and dis-solved oxygen concentration are low.

Distribution in the estuary. Forty-seven fish that were captured by purse seine at the mouth of the Duwamish River were equipped with internal sonic tags and released (group 1). Their movements were monitored by project personnel at high tide three days a week between August 27 and October 12 in a skiff equipped with a sonic-receiving unit.

The data from this operation have not been ana-lyzed in detail; the results of a preliminary analysis are shown in Fig. 6. It is evident that there was a gradual movement of fish up the estuary as the season progressed. Prior to the week of September 10, sonic-tagged fish were concentrated in the Spokane Street basin, and only one fish was noted in the oxygen-depleted section. After this week and until the fish migrated into freshwater, some of them were noted in the 16th Avenue Bridge section. A marked decline in temperature and dissolved-oxygen concentration occurred in this section during the week of September 10-16 that may have been correlated with fish move-ment.

Timing of Migration. The movements of sonic-tagged fish were followed by means of two stationary, recording sonic tag monitors, one located 4.5 miles from the mouth of the river (at the 16th Avenue Bridge) and the other 5 miles farther upstream (at

28

15 10 5

........................n ...... I 5 10- 5- 0

15 10 5 o nn ..............................,,,„

15 101 5 0 rin r 1 ......

rLrr

f

rl I n

Nu

mb

er

of d

etec

tions

TI

II

---- Bottom — Surface

75°-

70°

65°

60°

55°

50°

20.0

-r

8/20

-8/2

6

L\I

8/15

-8/1

9

9/1

0-9

/16

-

9/24

-9/3

0-

9//7

-9/2

3-

/0/1

-10/

12-

Ran

ges

of

tem

per

atu

re °F

R

ange

s of

dis

solv

ed o

xyge

n m

g/I

10.0

5.0

0.0

15.0-

-L

.LZ 16th

Ave

. Br

Aug. 27- Sept. 2

Sept. 3 -Sept. 9

Sept. 10-Sept. 16

Sept. I?- Sept. 23

Sept. 24 - Sept. 30

Oct. 1 - Oct. 12

Fig. 6.—Locations of detections of sonic-tagged salmon in group 1, Fall 1967.

Tukwila). Visual counts of migrating fish were taken immediately above tidal influence, and date and time of arrival of fish were noted at the Green River State Salmon Hatchery.

Records from the monitors have not been examined to date. They will show the periods of migration activity in their vicinities. Approximately 140 of 305 tagged fish have been recovered at the time of writing at the Green River Hatchery. The majority of the run arrived between September 25 and October 23. Recovery data have not been analyzed; they will show the times of arrival of various groups of tagged fish.

The effect of dissolved oxygen sag in the vicinity of the 16th Avenue Bridge on migration was determined by releasing a group of tagged fish (group 2) above the area and another group of tagged fish below (group 1) and tracking their movements. A compar-ison of estuarine distribution and dates of arrival at the hatchery of group 1 and group 2 may allow us to determine to what extent high temperatures and low dissolved oxygen content of the water affected migra-tion. During the week of August 27 - September 3,

Fig. 7.—Weekly ranges of temperature and dissolved oxygen at the surface and bottom waters of the 16th Ave. S. Bridge,

Duwamish River, August 15-October 12, 1967.

dissolved oxygen concentration at the bottom dropped to 1.0 mg/1 and surface water temperature reached 74°F (Fig. 7).

Relationship between water quality and fish move-ment and distribution. To determine the effect of water quality on fish distribution and migration in the estuary, intensive water sampling was conducted at high and low tides at the surface and bottom of each of 12 stations three times a week. Temperature was taken and dissolved oxygen content and salinity were determined. Analysis of the data is under way. Changes in temperature and dissolved oxygen con-centration during the sampling period at the 16th Avenue South Bridge are shown in Fig. 7. It is appar-ent that water quality shifted during the period of residency of chinook salmon in the estuary. Data on riverflow, tidal prism, and barometric pressure are

29

Perc

ent

fulln

ess

of s

tom

ach

also available and will be examined for correlation with migration.

Studies of Demersal Fishes

Demersal fishes were sampled with a beam trawl at Duwamish Head (Elliott Bay), Port Gardner Bay, Port Susan, Holmes Harbor, and Skagit Bay. Fishing was conducted over a broad range of depths seasonally in 1965, 1966, and 1967.

Intensive sampling was conducted at Duwamish Head. Samples were collected by trawl over 24-hr periods in the months of March, May, and September of 1966. The sampling design was essentially a 42-haul grid, wherein each of seven depths (10, 15, 20, 30, 40, 60, and 80 m) was sampled at 4-hr intervals during the diel period. Sampling was also conducted with a 120-ft beach seine over a 24-hr period at Duwamish Head on the same dates in May that trawling was conducted.

The demersal fish trawl sampling was conducted from the Department of Oceanography's 65-ft R/V Hoh. The Hoh is equipped with a fathometer and a Simrad echo sounder, both of which were used to monitor and record trawling depths. An electronic telemetering system was used to record, and modify when necessary, sampling in progress.

The net, which was attached to a 3-m beam devel-oped by the Department of Oceanography, was a 16-ft semiballoon shrimp trawl. It had a stretched mesh size of 11/2 inches in the body and 11/4 inches in the codend and a liner of 1/4-inch woven mesh. Mounted on the beam were "pacers," to provide an estimate of the fishing distance, a bathykymograph, to give a reading of depth versus time, and a flowmeter, to indicate the rate of trawling.

All fish within a sample were examined to deter-mine the species, length, weight, sex, approximate state of sexual maturity, stomach content, and the presence of any abnormalities, such as nematode in-festation or tumorous growths. After one year of this detailed processing, only the species, length, sex, and presence of abnormalities were noted for all fish in a sample.

At Duwamish Head fish of the numerically impor-tant demersal species, Parophrys vetulus, Lepidopsetta bilineata, Chitonotus pugetensis, and Citharichthys stigmaeus, and the uncommon species Citharichthys sordidus were segregated from other fish within a sample and injected with formalin for the purpose of preserving the contents of the digestive tract. Each fish was examined in detail, as outlined above, and the estimated per cent fullness of stomach and the nature of stomach contents were noted.

( 0113- 440 mm (b) 11 3-167 mm

(d)213-267 mm 60

40

20

0

60

40

20-

0 e3

Time,

Fig. 8.—Did l feeding patterns of all fish of the species Parophrys (a), length classes (b,c,d,e), and sexes of length class 213-440mm

(f). Numbers on the abscissa denote time when fish were captured.

The diel migrations and the food and feeding habits of the five numerically important species and of selected length classes within the species have been analyzed for the most part. The data from past inten-sive sampling at Duwamish Head and quarterly samp-ling at the other locations are being transferred to IBM cards for statistical analysis. Length-frequency distributions will be studied by species, sex, depth, and season. Length-weight relationships and relative growth rates can then be compared between polluted and nonpolluted areas. Further, estimates of variabil-ity can be used to design more efficient sampling and monitoring programs.

Results of trawling at Duwamish Head. An analysis of the diel migration patterns of the five species at Duwamish Head suggests diel migrations in one or more months. The fishes appear to collect at the 40-m depth during the day and move into the 10-m to 20-m depths at night. Nocturnal onshore migration is con-firmed somewhat by the larger numbers of fish (ex-cluding C. sordidus) caught in the beach seine by night as compared to daytime catches. However, the sizable decrease in abundance of the common species in the later months of the year, particularly at night, combined to limit the magnitude of migra-tional trends.

80-

60-

40-

20-

0 Fi nn (c) 168-212 mm

(e) 268-440 mm (f)213-440 mm

to •e- 03 C\J (\j ez, r\I

hr.

30

Duwamish Head

N • 563 Total volumetric displacement . 106.817 ml

Duwamish River

N = 65 Total volumetric displacement = 70.418 ml

100

90

80

70

60

50

40

30

20

10

II

S.

The diel feeding habits of four of the five species have been analyzed. The mean estimated per cent fullness of stomach and the mean volume of food eaten were determined for each fish. Fig. 8 shows the diel feeding habits from the estimated per cent fullness of stomach of all fish of the species Parophrys, four length classes, and the sexes within the two largest length classes. The following characteristics are apparent:

1. The maxima in feeding intensity for all fish (Fig. 8a) occurred at approximately dawn (0600) and dusk (1800) in apparently a rather great and sustained feeding activity throughout the diel period.

2. Fish within the size range 113-167 mm ate almost exclusively during the nocturnal period (2200-0600) (Fig. 8b).

3. Fish within the size range 213-267 mm (Fig. 8d) ate principally during the diurnal period (1000-1800).

4. An intermediate feeding pattern (between those described in 1 and 2 above) was exhibited by fish within the size range 168-212 mm (Fig. 8c).

5. The maximum intensity of feeding of fish in the size range 268-440 mm occurred in the late after-noon (1400-1800). A minor peak occurred at dawn (Fig. 8e).

6. There appears to have been a difference in the feeding habits of the sexes in the length class 213-440 mm (Fig. 8f). Somewhat obscure feeding maxima in the early morning and late afternoon are apparent for the males and one peak at dusk for the females.

Results of trawl sampling in the Duwamish Estuary. The sampling locations in the Duwamish Estuary were chosen for their relative freedom from debris. The sampling method and gear were the same as those used at the other areas except that the trawl was stripped to essentials. The common fishes of the estuary may be divided into three categories: "shallow water" fish - Leptocottus and Platichthys; Eurybenthic fish - Parophrys; and "deep water" fish - Microgadus, Lumpenus, and Artedius (?).

Fig. 9 shows the food habits by major food groups of fish of the species Parophrys at Duwamish Head in May of 1966 and in the Duwamish Estuary in May

Fig. 9.—Per cent volumetric displacement of major food groups in fish of the species Parophrys from Duwamish Head and

Duwamish River.

of 1967. The fish in the estuary eat slightly larger crustaceans and a greater proportion of polychaetes than the fish on the outside do. Also, they eat differ-ent types of polychaetes, principally Arenicola and Capitella. Furthermore Parophrys in the river con-sume a smaller volume of molluscs and no echino-derms or fish.

The incidence of hemangiomas in fish of the species Parophrys during the three years of sampling at Du-wamish Head was less than 1 per cent. The smaller fish were the most commonly infected (the 31 tumor-ous fish were all less than 250 mm; 25 were less than 200 mm and 20 were less than 150 mm). The inci-dence of this disease in fish of this species in the estuary appeared to be approximately the same; one fish in 113 was infected. It should be noted that small fish of this species were relatively rare in the estuary.

A high proportion of fish of the species Platichtys taken in the estuary were tumorous. Of 105 individu-als analyzed, nine were infected (8.6 per cent). Again, the small fish (between 94 mm and 150 mm) were the most commonly infected. Denny M. Miller, Jerry A. Wetherall, Stephen Zebold,

William H. Lenarz, Gary D. Stauffer, Jeffrey Fujioka, Michael Halstead, Ernest 0. Salo, and

T. Saunders English

31

FOOD AND FEEDING HABITS OF JUVENILE COHO SALMON AND

STEELHEAD TROUT IN WORTHY CREEK, WASHINGTON'

A study of the food and feeding habits of juvenile coho salmon (Oncorhynchus kisutch) and steelhead trout (Salmo gairdneri) in the macrohabitats of Worthy Creek, a stream in Western Washington, was conducted from April 1966 to March 1967. The follow-ing were determined and used as indices for compari-son of food organisms in the macrohabitat and diet: microhabitats occupied by coho salmon and steelhead trout and their influence on diet, seasonal variation in the diets of coho and steelhead and abundance of surface and subsurface food organisms, food selection from organic stream drift by coho and steelhead, and the total volumes of food organisms consumed by coho and steelhead.

Analysis of the data indicated a minor influence of macrohabitat on diet. With the exception of Ephe-

meroptera nymphs, no significant difference (95 per cent confidence level) was found in food items among

Supported by the Everett Technical Committee, composed of the Scott Paper Company, Simpson-Lee Paper Company and the Weyerhaeuser Company of Everett, Washington.

coho taken from four areas. F tests were conducted in an attempt to determine significant differences in the diet of coho salmon in time as well as space during the sampling period. Diptera larvae, Diptera adults, Ephemeroptera nymphs, Tricoptera larvae, Homoptera adults, and aquatic annelids differed sig-nificantly in abundance with time. There was no significant difference between the diets of steelhead trout from two of the study areas for any food organ-ism. Only Diptera adults, Plecoptera nymphs, and aquatic annelids showed a statistical significance for any monthly differences. Conclusions were not reached concerning utilization of drift by coho and steelhead because of inadequate sampling. Coho sal-mon were found near the water surface in areas of low stream velocity, whereas steelhead trout were associated with the substrate regardless of the water velocity. In both species the greatest volume of the diet was comprised of subsurface food organisms (immature forms) but coho salmon had fed more extensively than steelhead trout on surface foods.

James Johnston and Ernest 0. Salo

COMPILATION OF PACIFIC SALMON LITERATURE'

The Pacific Salmon Interagency Council, through its technical committee (TECOM), has contracted with the Fisheries Research Institute for continuation of the compilation of the literature on salmon. The present Salmon Compendium, as it is commonly called, is a coded file of the literature on Pacific sal-

Supported by the Anadromous Fish Act, P.L. 89-304, Con-tract No. 14-17-0007-763; Project No. AFCS 20-1.

mon that was published during the period 1900 through 1959. The compilation was made in 1963 by members of the Seattle Laboratory of the Bureau of Commercial Fisheries in consultation with the late Dr. W. F. Thompson. Five copies of the compendium have been distributed to subscribers in Alaska, Seattle (2), Portland, and Washington, D.C.

Ernest 0. Salo and Edwin K. Holmberg

32

Invertebrate Fisheries

EPIZOOTICS IN EXPERIMENTAL MARINE SHELLFISH POPULATIONS'

This investigation is a continuation of the study of Mytilicola orientalis in oysters and this was the fifth and final year of support.1 Five experimental stations, consisting of a float and a rack at the +2-ft tide level of the oyster beds, were established in Hood Canal, Oyster Bay, and Willapa Bay in Washington and in Humboldt Bay, California. A float station only was established in Yaquina Bay, Oregon. Each station was stocked with experimental populations of the Pacific oyster (Crassostrea gigas), the native oyster (Ostrea lurida), and the bay mussel (Mytilus edulis). The populations were regularly checked for growth and survival and sampled for fatness, gross examina-tion for infection by M. orientalis (an endoparasitic copepod found in the digestive tract), and histological determination of the presence of other pathogens and pathological effects of infection.

The seasonal patterns of shell growth of several year classes of Pacific oysters in Humboldt Bay and Ya-quina Bay have been determined from the beginning of 1963 to the present time. Shell growth occurred largely during spring, summer, and autumn when temperatures were approximately 12°C or above. During winter when the temperatures were 10°C or below, minimal growth or cessation of growth oc-curred. For the past five years, Pacific oysters in Humboldt Bay grew continuously throughout the year, but the most significant growth occurred be-tween April and November. The growth of Pacific oysters in Yaquina Bay occurred principally between the months of May through October and definitely ceased during the winter months when temperatures fell below 10°C.

Again, the focus of attention was on Pacific oyster populations at Humboldt Bay, where decisive mortal-ities of imported Japanese seed occurred during the first three summers of this investigation and virtually none during the winters. Since the beginning of the study, we have introduced new populations each year and are now monitoring the survival of five different

Supported by the U.S. Public Health Service, National Insti-tutes of Health, Grant No. UI-00122-05.

year classes of Pacific oysters in Humboldt Bay. As shown in Table 1, mortalities from 1963 through 1965 became progressively greater from one year class to the next, especially during their second summer of existence in Humboldt Bay. Since 1965, mortalities have decreased markedly to less than 7.1 per cent recorded in 1967 for any given year class of oysters monitored.

TABLE 1.

Summary of mortality rates from May through October among different year classes of Pacific oysters imported as seed from

Japan into Humboldt Bay

1963a

1964 1965 1966 1967

1962 Seed planting Float Float 12.6% 10.9% Loss - Intertidal 13.4 5.6 8.5% 7.7% 0.0%

1963 Seed planting Float 22.6% 26.0% 4.7% 7.1% Intertidal 13.9 17.9 3.3 0.0

1964 Seed planting Float 39.3% 0.0% 3.0% Intertidal 23.3 4.6 0.0

1965 Seed planting Float 16.0% 3.2% Intertidal 9.7 3.2

1966 Seed planting (transported after one summer in Willapa Bay, Washington)

Float 3.0% Intertidal 1.0

a Second year for the 1962 plantings.

Pacific oyster seed of the 1965 set in Willapa Bay and Hood Canal, Washington, and Pendrille Sound, British Columbia, were introduced into the Hum-boldt Bay station in early 1966 in search for a strain of Pacific oysters resistant to the disease organism causing the mortalities in Humboldt Bay. Miyagi Pacific oyster seed of the 1965 year class from Japan were in short supply and were only planted in a few areas, such as Willapa Bay and southern Puget Sound, Washington. No seed was shipped to Humboldt Bay.

33

Therefore, imported seed was shipped via Willapa Bay to Humboldt Bay to add another group of oysters from the same year class for comparison of survival and growth. Survival of these four populations dur-ing their second summer was excellent, as shown in Table 2. At no time during their second summer in Humboldt Bay did any group exceed 4.2 per cent mortality.

TABLE 2.

Summary of mortality rates in the second summer, May through October 1967, among Pacific oysters of the 1965 year class

transplanted into Humboldt Bay from selected areas.

1967 May. October

3.0% 1.0%

3.9% 0.0%

2.0% 1.0%

4.2% 2.0%

Japanese Miyagi seed Float Intertidal

Pendrille Sound, Canada, seed Float Intertidal

Hood Canal, Washington, seed Float Intertidal

Willapa Bay, Washington, seed Float Intertidal

The Mytilicola infection rates were computed and analyzed further since last year's report, and the over-all average number of copepods per infected host was again found to be highest for Pacific oysters, lowest for native oysters, and intermediate for bay mussels. This finding was not related to the percentage of shellfish infected since, in all areas, the total number infected by Mytilicola was highest in bay mussels, followed by Pacific oysters and native oysters.

The Mytilicola dissected from the gut of Pacific oysters were examined further and categorized into mature of immature forms. The results demonstrated that this copepod does breed throughout the year.

When examining the three areas (Humboldt, Yaquina, and Willapa Bays) most heavily infected with Mytili-cola, we continue to almost always find immature or copepodid female stages and mature females with or without egg cases throughout the year.

Studies on the early life history of Mytilicola are being continuel. There appears to be two meta-nauphius stages before the parasite reaches the first copepodid stage. This will be confirmed with further culture work.

As indicated earlier, with few exceptions, we can now state that Pacific oysters with below-average con-dition index values generally had significantly higher percentages of infestation by Mytilicola. Also, in those areas where the numbers of Mytilicola were high enough to warrant computation of the average number per infected oyster, the average number of parasites per infected oyster was significantly higher in the group with below-average condition than in the group with above-average condition.

There were heavy mortalities among native oysters in Yaquina Bay, Oregon, and Willapa Bay, Washing-ton, and replacement populations were generally nec-essary within 1 to 11/2 years. Mortalities in the two areas were usually heaviest during the fall, winter, and early spring months, in contrast to the mortality patterns (1963-1965) among Pacific oysters in Hum-boldt Bay, where heaviest mortalities occurred during the summer. The mortalities of native oysters during the fall, winter, and early spring of 1966-67, however, were much lower than in previous years.

Although heavy sets of bay mussels in the baskets during each summer caused serious problems in the collection of survival data, general observations of mussel mortalities for all stations were made and the highest mortalities usually occurred during the sum-mer months; mortalities were comparatively low dur-ing the fall and winter months.

Albert K. Sparks, Kenneth K. Chew, Donald E. Weitkamp, Evelyn J. Jones, and Lieselotte Schwartz

OYSTER PATHOLOGY'

This study was initiated in 1961 by a grant from the Bureau of Commercial Fisheries, entitled "Studies in Oyster Pathology." Primarily, the reactions to injuries of various types were studied. Some of the studies undertaken were the normal post-mortem

1 Supported by the U.S. Bureau of Commercial Fisheries, Grant No. 14-17-0001-1736.

changes in the Pacific oyster (Crassostrea gigas), the acute inflammatory response, pathological effects of low salinity on oysters, wound healing, tumors, bac-terial and certain parasites, such as Mytilicola orien-talis and the larval tapeworm Tylocephalum.

Approximately 21/2 years ago, the Bureau of Com-mercial Fisheries began a collaborative program of

34

investigations of oyster mortalities on the west coast by awarding contracts to the state fishery departments of Washington, Oregon, and California to study mor-tality rates and causes of mortalities. The Oregon Fish Commission and the Washington Department of Fisheries established and check the stations, mon-itoring the mortality rates, collecting hydrographic data, and fixing representative normal and all dying oysters encountered. Our responsibility in the new program is to process all tissues and examine them for the presence of microorganisms and pathological conditions. Three slides are prepared of tissues from each oyster; one is retained by our laboratory, one is returned to the originating laboratory, and one is sent to the Bureau of Commercial Fisheries at Oxford, Maryland, for confirmatory study. A report of the results of the histological examination of each oyster accompanies the slide.

At the present time we are making an intensive study of all unidentified organisms, using special staining techniques, which we hope will aid in identi-fying these possible pathogens.

In addition to providing diagnostic service to the state fishery departments of Washington and Oregon, this project has continued to support, on a greatly reduced scale, research in experimental pathology of oysters.

One such study completed during 1967 was of the wound repair process and reaction to intraspecific tissue implants in the Pacific oyster. Oysters were wounded with a sterile blade and one-half of the wounded group received an implant of living mantle tissue. Although the rate of wound repair varied considerably among the experimental oysters, the pattern was remarkably consistent. Leucocytes infil-trated the lesion; those along the margins of the

wound elongated to form a parallel band, and those in the center of the wound channel grouped into whorls of fusiform cells histologically resembling scar tissue. These cells were gradually replaced by Leydig cells, probably by differentiation of the cells already present, but possibly by infiltration of Leydig cells outside the periphery of the wound. The leucocytes filling the wound at the body surface differentiated into a "pseudoepithelium" that was indistinguishable from normal epithelium adjacent to the wound. The totipotency of the oyster leucocyte was further demon-strated in the repair of digestive diverticulae or in-testinal epithelium damaged in the wounding. Leu-cocytes infiltrated the damaged area, phagocytosed the necrotic tissue debris, then replaced the destroyed epithelium with tissue that was indistinguishable from undamaged digestive epithelium.

Homologous tissue implant studies were not as successful because of a high percentage of rejection of the implant and ejection via the wound channel. Since mantle edge was used as the tissue for trans-plantation, it is now realized that the activity of the mantle folds and fimbria may have resulted in the implants crawling out of the host tissue rather than being expelled by the host.

Successful homologous implants fused with the host tissue and became continuous with it. Fusion was brought about by fusiform leucocytes, which formed a connecting tissue at the host-implant interface. The tissues became continuous, fusiform leucocytes differ-entiated into Leydig tissue or pseudoepithelium, and cross-circulation appeared to occur.

Albert K. Sparks, Michael C. Mix, Donald E. Weitkamp, David M. Des Voigne, Evelyn J. Jones,

and Lieselotte Schwartz

POSTEMBRYONIC DEVELOPMENT OF LABORATORY-REARED SPOT SHRIMP, PANDALUS PLATYCEROS BRAN Di

Spot shrimp were reared successfully in the labora-tory from eggs stripped from females caught in Dabob Bay, Washington, on January 20, 1967. The eggs were incubated in circulating bubbling seawater at 53° F. Hatching occurred after 41 days, but ovigerous females at the same temperature took 22 days longer. Larvae were cultured in floating 800-ml beakers and were fed newly hatched brine shrimp nauplii. Five stages were recorded before the postlarval stage, and

larvae survived for 83 days. They reached 19 mm at the ninth stage. All six stages were distinguished by the telson. Larvae were also kept at 51°, 53° and 55° F. Low temperatures were optimum for survival in the early stages, but larvae survived the longest at comparatively higher temperatures. At 55° larvae moulted 4.5 days ahead of larvae kept at 51°. The highest mortalities occurred during the moulting process.

Vincent A. Price and Kenneth K. Chew

35

SHELLFISH TOXICITY STUDY'

Several aspects of the relationship of Mytilus cal-if ornianus and Crassostrea gigas to the toxic dino-flagellate Gonyau lax washingtonensis (Hsu) from Sequim Bay on the Strait of Juan de Fuca were studied by using unialgal mass cultures for feeding experiments.

Five feeding experiments were conducted in which the mussels (M. californianus) received G. washington-ensis at a concentration of 50 cells/ml. From 75 to 92 percent of the toxin was recovered from the shellfish meats by standard extraction and mouse bioassay.

• This relatively vigorous uptake of toxin by the mussel is similar to the uptake of toxin by the mussel under natural conditions.

In contrast, feeding and water pumping rate experi-ments with the oyster, Crassostrea gigas, demonstrated no measurable uptake of toxin after a period of three months of experimentation. Two types of reaction were observed when the oyster was fed Gonyau lax washingtonensis. The most common reaction was drastic curtailment of the volume of water filtered together with vigorous clapping of the valves or com-plete cessation of pumping activity. The second reac-tion recorded was a 30-per cent reduction in pumping rates together with the production of large amounts of pseudofeces. Concentrations of 20, 40, 80, and 120 cells/ml curtailed or caused cessation of the pumping activity. Culture medium, with cells removed, added

'Supported by the U.S. Public Health Service, National Insti-tutes of Health. Shellfish Training Grant TO1 UI-01039-03.

to filtered seawater did not appear to affect the oys-ter's pumping rate. This general physiological reac-tion would tend to lend evidence to the hypothesis that oysters under field conditions may sometimes refuse to accept G. washingtonensis initially, but accept the dinoflagellate after a period of acclimation. Results from sampling C. gigas at Sequim Bay, Wash-ington, showed a 2-week delay in its uptake of para-lytic shellfish poison in comparison to the California mussel. Furthermore, results from bioassay of field samples showed that the levels of toxin in C. gigas were three to four times lower than in M. calif or-nianus.

Culture work with Gonyaulax washingtonensis has been continued. Emphasis in the current study is on nutritional requirements of this organism. The pre-liminary work of devising a defined medium to re-place the soil extract medium previously used is in progress.

Field collections made during the summer of 1967 showed that, contrary to a commonly held belief, G. washingtonensis can occur in Puget Sound. This species was found in fair abundance off Richmond Beach in June and July and at a series of points from West Point, Seattle, to Bainbridge Island in July. Cultures isolated from these locations, as well as a culture received from Friday Harbor, are being main-tained and will be tested for their capacity to pro-duce toxin.

John Dupuy, Louisa A. Norris, and Albert K. Sparks

CHROMOSOMES OF OYSTERS AND MUSSELS'

Studies of the chromosomes of bivalves from the Pacific Coast of the U.S. were continued. The native Olympia oyster, Ostrea lurida, and the European flat oyster, 0. edulis, have a diploid number of 20. The diplotene meiotic bivalents of 0. lurida resemble "lampbrush" chromosomes and are being further ana-lyzed for chiasmal frequency and will be compared with those of 0. edulis.

Both Miyagi and Kumamoto varieties of the Pacific oyster, Crassostrea gigas, also have a diploid number of 20. Preliminary studies have shown differences in

'Supported by the U.S. Public Health Service, National Insti-tutes of Health. Shellfish Training Grant TO1 UI-01039-03.

the chiasmal frequency of meiotic bivalents and in the morphology of mitotic chromosomes. The Kuma-moto is a stunted oyster compared to the Miyagi variety; the latter grows almost three times as large in the same time under similar conditions. It cannot be said whether C. gigas Kumamoto is a variety, sub-species, or a full species. Both Kumamoto and Miyagi forms are known to successfully hybridize.

The California mussel, Mytilus californianus, and Bay mussel, M. edulis, have a common diploid chro-mosome number of 28. The 14 meiotic metaphase-1 bivalents, however, show considerable differences in their morphology. In reciprocal crosses, only few eggs were seen to cleave and it is not known whether

'36

hybridization can be induced in the laboratory or occurs in nature.

Karyotype analyses showed that all the oyster spe-cies have stable chromosome sets; the majority of chromosomes are metacentric and few are submeta-

centric. Both species of mussels, however, also have acrocentric chromosomes besides metacentric and sub-metacentric. Conservatism of chromosome numbers on generic and species levels is noteworthy.

Muzammil Ahmed and Albert K. Sparks

Radioecology

JOHNSTON ATOLL PROGRAM'

The Johnston Atoll program was begun in 1966 as a continuing program to investigate the conse-quences of a potential introduction of radionuclides into the ocean near the Johnston Atoll located about 700 miles west-southwest of Hawaii. The objectives of the program are 1) to maintain a capability for radiological surveys of the biota and environment of the area, 2) to document current levels of radionu-clides in tuna from the central Pacific fishery and in the organisms at Johnston Atoll, and 3) to provide basic information for predicting and evaluating the biological consequences of the introduction of radio-nuclides into the ocean in the Johnston Atoll area.

During the period January-March, 1967, fallout from the Chinese nuclear detonations of December 1966 was collected at the program's portable labora-tory in Seattle and analyzed by gamma ray spec-trometry. The primary objective was to test the ef-ficacy of the counting systems in an actual fallout situation. Neodymium-147, cerium-141, cerium-144, tellurium-132, iodine-131, barium-140-lanthanum-140, ruthenium-103-rhodium-130, ruthenium-106-rhodium-106, and zirconium-95-niobium-95 were detected in precipitation and grass samples collected in January 1967.

'Supported by the U.S. Atomic Energy Commission, Contract No. AT(26-1)-269.

A second annual field trip was made to Johnston Atoll in May 1967 and invertebrates, fish, algae, sea-birds, guano and sediments were collected. The ra-dionuclides manganese-54 and zinc-65 were found in birds and guano and zirconium-95 was found in sea urchins. The amount of each radionuclide, presum-ably from worldwide fallout, was less than that of naturally occurring potassium-40. Plutonium-239, which accidentally contaminated a small portion of the reef at Johnston Atoll in 1962, was still present in the sediments in 1967.

Tuna samples are being obtained monthly from the Japanese tuna fishery in the central Pacific in cooperation with the Japanese National Institute of Health in Tokyo. Thus far, only cesium-137 has been found in muscle tissue and in amounts less than potassium-40.

Laboratory studies with tritium have been con-cerned mainly with the development of techniques for liquid scintillation counting of seawater. Up to 5-ml samples can be counted with efficiencies of 20 per cent. Studies of the possible discrimination be-tween tritium and hydrogen by intertidal organisms have been initiated, but there are as yet no conclusive results. Mr. Robert C. Erickson has undertaken a doctoral thesis problem on the effects of tritium on the behavior of fish.

Edward E. Held and Allyn H. Seymour

BIKINI ATOLL RESURVEY'

The Laboratory of Radiation Ecology participated in an environmental survey of Bikini Atoll from April 23 to May 7, 1967 at the request of the Division of Biology and Medicine of the AEC. The survey team consisted of four Marshallese natives and ten scientists

'Supported by the U.S. Atomic Energy Commission, Contract No. AT(45-1)1385, Letter Agreement 67-2.

and technicians, representing the AEC, the U.S. Naval Radiological Defense Laboratory, the Trust Territory of the Pacific, and the University of Washington.

The major objective of the survey was to obtain as precise a measure as possible of gamma-dose rates at the atoll and to compare the results obtained with measurements by radiation survey instruments in gen-

37

FERN LAKE 1965 1966

- Surface wafer ----- Bottom water

' J.. F65 Aor May Jun Jul Au p Sep Oct Nov Dec Jan Feb Mar ' I I ' I ' Mar ' Apr May Jun Jul Aug Sep Oct 'Noy Dec

1 1' 120

era! use and with more sophisticated instruments. Synoptic measurements were taken with an ANPDR-27F beta-gamma meter, a high-pressure ionization chamber, a portable gamma-ray spectrometer, thermo-luminescent dosimeters and film badges. Preliminary results indicate that the ANPDR-27F gave readings as much as 50 per cent higher than the true values because of energy dependence and the varying pro-portions of nuclides at the atoll.

Radiation measurements and observations were made at seventeen of the eighteen islets of Bikini Atoll, and a few biological and environmental samples were taken to supplement the laboratory's studies at Bikini in 1964. The results of the survey will be used as part of an evaluation of the habitability of Bikini Atoll.

Edward E. Held

FERN LAKE

A detailed ecological investigation of the Fern Lake watershed is being conducted jointly by the College of Fisheries of the University of Washington, the Wash-ington State Department of Game, and the U.S. Atomic Energy Commission. Among the 1967 studies of the Fern Lake system, we should like to cite especi-ally the examination of the zooplankton and the ef-fects of the 1965 mineral fertilization on the physical and chemical properties of the water, the bottom animals, and the steelhead trout population.

The littoral zooplankton have been studied for a seven-year period (1958-1964). Forty-seven species of Cladocerans and ten species of Copepoda were identi-fied from more than 2,000 samples. The dominant Cladocerans were Ceriodaphnia reticulata, Scaphole-beris kingi, Diaphanosoma brachyurum, Acroperus harpae, Sida crystallina, and Chydorus sphaericus. In-dications of autecological differentiation were found.

The limnetic zone of Fern Lake is characterized by the presence of the Cladoceran Holopedium gibberum and the Copepoda Epischura nevadensis and Diapto-mus oregonensis. The rare occurrence of Daphnia species is also significant. However, after the fertiliza-tion in August, 1965, Daphnia did appear in signifi-cant numbers, primarily in the littoral area. The genus is recognized as preferring waters with a cal-cium concentration above the levels normally occur-ring in Fern Lake. The standing crop of limnetic zooplankton is generally greatly reduced during the winter period; however, there was a significant in-crease in the zooplankters the first winter after the fertilization. Eight years' observations preceding the fertilization showed an average dry weight of 30 mg/m3 from October to December (range 8 to 43

'Work performed under Contract Nos. AT(45-1)1385 and AT(45-1)2050 with the U.S. Atomic Energy Commission and in cooperation with the Washington State Department of Game.

PROGRAM'

mg/m3). After the fertilization, the figure was 128 mg/m3.

We found from three years' observations at Fern Lake that the filterability index is a useful tool for characterizing a given water body. Regional and seasonal trends are apparent and are dependent, in part, on the colloid content of the water.

The lake was fertilized during the summer of 1965 by adding 81/2 tons of inorganic fertilizer. Applications were performed from the middle of August, with four-day intervals, through the first week of September. An example of the effect of the mineral fertilization on the water quality is shown in Fig. 1. Specific con-ductance rose sharply and then decreased gradually until December. Around Christmas snow fell, and heavy rain followed. Runoff was excessive as a con-sequence of this condition. The flushing of the lake caused a loss of roughly 50 per cent of the salts in

Fig. 1.—Variation of specific conductance.

solution at the onset of the flushing. Conductance soon dropped to prefertilization levels. For some time, the annual flushing of the lake has been re-garded as a major cause of its low productivity; the fertilization experiment proved this premise to be true.

An examination of the response of the bottom animals to the fertilization showed that Diptera re-sponded most noticeably. In these organisms the response was rapid and resulted in an increase in the number and total weight of organisms and in

38

an acceleration of the life cycle of the more slowly maturing groups. However, the effect was brief. In about one year after the fertilization, the number of organisms was approximately the same as that ob-served in the previous years.

Studies were continued on the population dynamics of steelhead trout (Salmo gairdneri) to determine the effects of the 1965 fertilization. Net production estimates of the juvenile fish in the lake before, dur-ing, and after fertilization show definite differences. Production for the 9-month period (July 15 to April 15) was double that of the previous and following years (Table 1). Monthly production estimates indi-cate that the increase occurred in the fall of 1965 and coincided with an increase in zooplankton and aquatic insect production. Net fish production in the following summer and fall was less than the pre-

TABLE 1. Total net production of age 0 and age I and older fish from

July 15 to April 15

Age 0

Age land older

Total Time

production

production

production (kg)

(kg)

(kg)

1964-1965 192 16 208 1965-1966 416 32 448 1966-1967 167 20 187

fertilization levels. A simulation study of the relation-ship between fish production and food production is now under way and should lead to a better under-standing of the interaction between trophic levels.

Lauren R. Donaldson, Sigurd M. Olsen, Paul R. Olson, Zella F. Short, James C. Olsen, Harold E. Klaassen,

and Rufus W. Kiser

COLUMBIA RIVER STUDIES'

Uptake and Loss of Zinc in Oysters

A 2-year experiment to determine the rate of uptake and rate of loss of radioactive zinc (65Zn) by oysters in a natural environment was completed recently. Oysters were transferred from Willapa Bay, where 65Zn is present, to Puget Sound-Hood Canal, where there is essentially no 65Zn. Also, a reverse transfer of oysters was made—from Puget Sound-Hood Canal to Willapa Bay.

Although all of the waters of the world contain a barely detectable quantity of 65Zn from fallout, Will-apa Bay has an additional very small quantity of this radionuclide as a consequence of the operations of the Hanford reactors on the Columbia River. (Zinc-65 from worldwide fallout accounts for about one per cent of this radionuclide in Willapa Bay.) Periodic sampling and counting of oysters exchanged between the two environments provided data from which rates of uptake and loss were calculated. Since oysters do not distinguish between the stable and radioactive isotopes of zinc, the observations for 65Zn rates are equally applicable to the rates for total zinc.

Uptake undoubtedly is by a two-or-more compart-mental system; hence, complete equilibrium is not reached until all systems are at equilibrium. These experiments indicate that at the concentration of 65Zn present in Willapa Bay, a year is required for

'Supported by the U.S. Atomic Energy Commission, Contract No. AT(45-l)2047.

the system to reach complete equilibrium, which is a considerably longer period than had been expected from reports of laboratory experiments. The differ-ence may be accounted for by the lower concentration of 65Zn in Willapa Bay than in the laboratory experi-ment, or a difference in the chemical form of 65Zn in the two experiments, or the fact that complete equi-librium may not have been reached in the laboratory experiment.

The biological half-life of zinc in oysters was deter-mined from the rate of loss and was found to be approximately the same as the physical half-life for 65Zn, 245 days. Hence, the effective half-life is about 125 days; in other words, if Willapa Bay oysters were transferred to Puget Sound, one-half of the 65Zn present at the time of transfer would be lost in four months.

Effects of Radionuclides on Oyster Larvae

An investigation of the effects of radionuclides on oyster larvae has been undertaken by use of a method developed by the Washington State Department of Fisheries to determine the effects of waste sulfite liquor on oyster larvae. The only difference in the experi-ments is the substitution of various radionuclides for waste sulfite liquor as the stress factor. The investiga-tion is being conducted by Victor Nelson as thesis research for the M.S. degree.

Three radionuclides have been used: 65Zn and 51Cr

39

(chromium-51), because of their presence in Willapa Bay, and 90Sr-60Y (strontium-90, yttrium-90), because of the controversy between Russian and English re-search scientists over the effects of these radionuclides on the pelagic eggs and larvae of certain marine fishes. Inspection of the data, without mathematical analyses, indicates that effects are not present at concentrations of less than 10-4 to 10-5 cil for any of the three radionuclides. Since the concentration of 65Zn in

Willapa Bay is about 10-11 cp, this means that the concentration of 65Zn in Willapa Bay would have to be increased by a factor of a million, before effects on oyster larvae would be expected. Also, preliminary inspection of the data indicates good agreement with the findings of the English scientists, although it is recognized that pelagic fish larvae are perhaps more sensitive than oyster larvae.

Allyn H. Seymour

THE COMBINED EFFECTS OF IRRADIATION AND TEMPERATURE AND OTHER ENVIRONMENTAL FACTORS ON SALMONID EMBRYOS

This project, initiated during October, 1967, is de-signed to study the effects of X-rays administered to salmonids at various embryological stages and at nor-mal and above normal temperatures. Salmonid fishes have been artificially propagated for many years, and much is known about their life history, habits, and physiology. The development of most of the species is well known and can be controlled within the range of temperatures normal for the species. The objective of the experiment is to relate the effects of different doses of X-rays on different developmental stages of the embryo. Of special interest are the effects of low doses on the early cleavage stages and the effects on certain characters, such as numbers of vertebrae, scale rows, fin rays, growth rate, and color.

The salmon eggs that will be used for the experi-ment (Oncorhynchus tshawytscha) are to be taken from members of a run to the University of Washing-ton hatchery, which was developed by Dr. L. R. Don-aldson. The eggs of one female (5,000 or more) will be divided into lots of about 100 each and irradiated with 5, 10, 20, 50, 100, and 200 r at about 100 r/min, all during one developing stage. A control lot of 100 eggs will be kept for every irradiated lot. The objec-tive in the initial experiments will be to attempt to determine the combined influence of X-irradiation and temperature on the cleavage, gastrular, and blas-tular stages of the embryo.

Some of the eggs, embryos, and larvae will be sacri-ficed for study of chromosome preparation; the re-mainder will be allowed to live through the feeding stages, when most will be sacrificed for study of growth, body proportions, number of vertebrae, fin rays, scales (and other meristic characters), and parr marks. A check on mortalities will be made twice a day, and dead embryos will be preserved as soon as noted. Temperatures and light will be controlled as closely as possible.

Chromosome studies will be made on cleavage stages for study of early embryonic material, and on epithelial cells and gills for later embryonic and larval material. Usually, there is an alteration in number of structures in embryos developing at different tem-peratures; average values occur close to normal tem-perature range, and extreme values at the extreme temperature for the species.

In this experiment, we hope to determine the inter-relationship of X-ray dose, stage of development, and chromosome damage and to compare the latter with subsequent anomalies appearing in the developing embryos and larvae. The number of aberrations ap-pears to be dependent on the developmental stage at the moment of irradiation. Development of the chi-nook salmon is slow enough at normal temperatures to permit an X-ray of the embryo several times be-tween fertilization and the two-cell stage (about 7 hr). The four-cell stage is complete 5 hr later; thus, there are several opportunities to X-ray the two cells at different stages of division. The remaining cleavage stages occur at increasing rates but still offer oppor-tunities for irradiation at different mitotic stages. During these cleavage stages the chromosomes are reduced in size; thus, we can compare radiosensitivity with chromosome volume. In addition, the egg is semitransparent and allows fairly accurate identifica-tion of the developmental stages.

Should irradiation dose and developmental stage be determined for these experiments, we would learn much about the embryology of salmonid fishes, espe-cially about the organizers of various characteristics of the adult fish. Further, they might present a way of controlling development to some degree by the use of ionizing irradiation, as we now attempt to do with plants.

Arthur D. Welander, Lorna J. Matson, Gerald W. Wadley, and Dorothy Treadwell

40

Ecology and Water Utilization

WASHINGTON COOPERATIVE FISHERY UNIT'

The Washington Cooperative Fishery Unit, located at the College of Fisheries, University of Washington, began operations in the fall of this year. This unit is one of twenty-three nationwide units and is a part of the Cooperative Program of the Bureau of Sport Fish-eries and Wildlife, as provided for in P.L. 86-686 (74 Stat. 733). The purpose of the unit, as stated in the Act, is "to facilitate cooperation between the Federal Government, colleges and universities, the States, and private organizations for cooperative unit programs of research and education relating to fish and wildlife and for other purposes."

Cooperating agencies are the Washington Depart-ment of Game, Washington Department of Fisheries, University of Washington, and the Bureau of Sport Fisheries and Wildlife of the Fish and Wildlife Ser-vice. A coordinating committee with a representative from each of the cooperating agencies, provides gen-eral guidance to the unit, including a review of pro-posals for graduate studies and annual budget. The Bureau provides the unit leader and assistant unit leader, limited funds for support of graduate studies, and facilities, such as fish hatcheries and laboratories, for project use. The State Departments of Game and Fisheries provide funds for support of graduate re-search projects and the use of their facilities. The University provides office and laboratory space, special facilities, stenographic services and other assistance in support of the research and teaching effort. Thus, the unit benefits from the combined skills, profes-sional competence, facilities, and services of all the cooperating agencies.

Training fishery biologists is the single most impor-tant function of the unit. Increasing demands for public recreational fishing require more and better-trained personnel to carry out research and manage-ment responsibilities to resolve today's increasingly complex problems in fisheries. The unit's program,

1 Supported jointly by the Washington Game Department, the Washington Fisheries Department, Bureau of Sport Fisheries and Wildlife, and the University of Washington.

therefore, contributes to the pool of trained biologists necessary to meet critical federal, state, and private employment requirements. Research by unit person-nel and graduate students is also a major objective of the program. Research projects are chosen to pro-vide maximum public recreation in response to state, regional, and national needs. Extension-type activities are included as a part of the unit's effort, depending on the teaching workload and the desires of the coop-erators. The unit contributes information designed to achieve better public understanding of fisheries research and management. Special assignments may also be undertaken by unit personnel, such as partici-pating in national surveys, inventory of aquatic re-sources, design of research projects and programs, and others.

The unit program of the University offers a wide selection of courses and research projects for students interested in advanced training. Considerable latitude is granted each student in the selection of a research subject. Students are encouraged to exercise indepen-dent initiative in pursuing their selected projects. The cooperating agencies' interests and needs are considered by the students in selecting their subjects for investigation; however, study can be undertaken by a student in such broad areas as fishery manage-ment and ecology, population dynamics, taxonomy, fish culture, diseases, nutrition, and the closely related fields of limnology, oceanography, statistics, econom-ics, and biometrics.

Four research assistantships are provided for in the budget for fiscal year 1968. Each carries a $4,000 annual stipend and provides the necessary funds for equipment, supplies, temporary help, and miscellane-ous expenses. Two research projects being pursued at the time of this writing are:

(a) A study of the life history of Washington's larg-est clam, the geoduck, Panope generosa, and its contribution to the sport fishery of the state, in partial fulfillment of requirements for a Ph.D. degree, by Aven M. Andersen. This clam, which

41

commonly reaches a weight of 5 lb, lives in the lower intertidal and subtidal zones on protected beaches from California to Alaska. It is avidly sought by sportsmen. Commercial fishing for this species is prohibited in the State of Wash-ington. There are no scientific publications on the life history of the geoduck clam. The rate of growth, natural and fishing mortality, life expectancy, time of spawning, larval develop-ment, distribution, rate of recruitment, relative population densities in specific areas, among others, are deserving of special scientific inquiry. These essential facts are needed for the conserva-tion and wise use of this valuable resource. Among the areas selected for field studies are the following: (1) private property adjacent to the College of Fisheries Research Station at Big Beef Creek, Hood Canal; (2) Battelle North-west's property in Sequim Bay; and (3) Sylopash Point (Dosewallips River Flats).

(b) A study of the behavioral interactions between juvenile coho salmon and steelhead trout, in

partial fulfillment of requirements for a M.S. degree, by Brian J. Allee. This study is designed to compare the different ecological requirements for juvenile coho salmon and steelhead trout, to determine how the various forces within the habitat influence segregation of the two species, and to determine the influences each species exerts in establishing territorial rights. Big Beef Creek has been selected for making direct ob-servations of the coho-steelhead interrelation-ships. Two plastic rearing ponds, 15 ft in dia-meter, have been set up alongside Big Beef Creek to be used in this study. These pools enable the researcher to manipulate environ-mental factors under specifically controlled con-ditions and to assess how these changes influence the behavioral patterns of the two species in-volved.

The remaining two research assistantships have not been formalized at this time.

Richard R. Whitney, Edward S. Marvich, Aven M. Andersen, and Brian J. Allee

WATER RESOURCES RESEARCH NEEDS IN THE STATE OF WASHINGTON

An interdisciplinary study of current research needs in water development and utilization in the State of Washington has been made by faculty members of the University of Washington and Washington State University and has been reported in a single report, Report No. 1, of the Water Research Center. This report is a compilation of ideas and suggestions ex-pressed in reports written by several professors in the water resources field at the University of Washington and Washington State University on water resources research needs and prepared as a part of the "Plan-ning and Evaluation Project" funded by the Office of Water Resources Research, Department of the Interior, under Public Law 88-379.

The section on fish and wildlife was prepared at the College of Fisheries and summarized study areas in which knowledge is needed immediately for expan-sion of the resources to keep pace with the expanding use of water. Forty-five subtopics for study are sug-gested, which are covered by eleven general topics, as follows:

Determination of the quantity and quality of water required locally for recreational fishing and wildlife for the years 1980 and 2020.

Determination of the quantity and quality of water required locally for maintenance of the com-mercially important anadromous fish stocks for the years 1980 and 2020.

Development of practical techniques of enhanc-ing fish production of natural lakes, streams, and man-made impoundments.

Summary of knowledge on the efficiency of pres-ent fish passage facilities and establishment of more precise criteria for future facilities.

Determination of the effects of reservoir regula-tion on fish and wildlife stocks.

Study of the effects of man-made changes in streams and lakes on environmental ecology and community dynamics, succession, and changes in species dominance.

Studies of lakes and artificial impoundments. Research on fish and wildlife in streams. Research into facilities as related to fisheries and

wildlife production. Determination of water values required for the

future maintenance of fish and game populations. Research into means of reducing fish and wildlife

losses at water projects caused by project operations. Milo C. Bell

42

AN INITIAL STUDY OF THE WATER RESOURCES OF THE STATE OF WASHINGTON:

VOLUME 1. A FIRST ESTIMATE OF FUTURE DEMANDS FOR WATER IN THE STATE

OF WASHINGTON'

A chapter covering water requirements for fish, wildlife, and recreation was prepared at the College of Fisheries and is included in the above report. The total study is an interdisciplinary publication prepared by the faculties of the University of Wash-ington and Washington State University and pub-lished as a part of the Water Research Center activi-ties of the State of Washington. The Water Research Center is a joint operation of the two universities and has its headquarters at Pullman.

The purpose of the report is to present an initial analysis of the availability of water to supply the present needs of the entire state and to estimate the growing demands on such supplies to the year 2020. Factual information is presented and made available to departments and individuals responsible for the planning and management of the water resources of this state. The section on fish, wildlife, and recreation is concerned with the quantity of flow or volume as related to maintenance of environment. It is recog-nized that volume per se, without consideration of quality and its ramifications, would be ineffective. Flow or volume, however, is a controlling factor in the maintenance of quality levels as associated with environmental needs. The report, by necessity, is held to the defining of flow levels to permit the maximum production of fish as may be controlled

1 Supported by the State of Washington Department of Con-servation, the Legislative Interim Committee on Water Resources and matching funds from the University of Washington and Washington State University.

by spawning conditions. Flow requirements needed to provide maximum food production are acknowl-edged but are not developed in this first report.

The State of Washington is divided into fifty basins. The choice was made by those working generally in fields of runoff or supplies. While this is not the most convenient method for determining fish, wildlife and recreation needs, the use of these divisions made it possible for flow requirements for fish, wildlife, and recreation to be kept parallel with other requirements within the basins and within the projected water yields of such basins. The period from July to Octo-ber is critical for both volume of runoff and flow needs for maintenance of natural fish environment. This critical period has been investigated for the general fish, wildlife, and recreation needs as a special part of the total annual requirements. The demands are compared to the yield from the basins in an at-tempt to determine whether surpluses or shortages exist at present and how conditions will be altered to the year 2020. Basic formulae, based on fish spawn-ing requirements, were determined and used to obtain the optimum wetted areas in streams. It was found that the elevation of the stream's source and its geo-graphic position in the storm paths from the Pacific produce different bed width and depth characteristics and water volume requirements. Spawning times and requirements are indicated for typical streams. Evapo-ration losses and precipitation ranges are included as an aid in the study of lakes.

Milo C. Bell

A COMPENDIUM ON THE SUCCESS OF PASSAGE OF SMALL FISH THROUGH TURBINES

Faculty members Milo C. Bell, Allan C. DeLacy, and Gerald J. Paulik co-authored the compendium for the U.S. Army Corps of Engineers. The results of tests conducted in the United States, Canada, Eur-ope, and the British Isles for success of fish passage at both Kaplan and Francis-type runners were com-pared and analyzed. Pertinent comments from the literature were included.

The results of the tests were studied under three general headings: types of injuries sustained by test and control fish, factors relating to or causing losses

or injuries to test and control fish, and survival rates of test and control fish. These were subcategorized as follows: kills by mechanical or pressure causes, effects of sizes of fish, species differences, types of turbine, heads on the plants, sigma values, diameters of runners, volumes of flow and other critical design criteria for turbines, and hydroplant operations. Be-cause of the interrelationship of many of the operat-ing and design features of turbines, computer tech-niques were used in the analyses. Scarcity of data during the test periods for many of the machines,

43

such as temperature conditions, condition of the test specimens, and method of recovery, precluded com-plete analysis of many tests.

Kaplan and Francis wheels were considered separ-ately. The principal causes for losses were given for each test. The combined data indicate that for Fran-cis wheels, per cent of wicket gate opening is the most important variable, and sigma and fish length are next in importance. For the Kaplan turbines, im-portant variables are square root of head and sigma; wicket gate opening is not important. The determin-ation of the importance of different variables was based on a regression analysis of data points collected during tests conducted under normal operation when several factors were being varied simultaneously. Thus, undetected confounding of apparent causes may have occurred. This could be eliminated by designing future tests according to the principles outlined in the section on future tests. The findings, however, logically follow the engineering design of the turbines. Kaplans are run at the most efficient

level for a given head and load by blade angle adjust-ments. The fish survival rate for both Kaplans and Francis wheels follows the general efficiency curve. The highest survival occurred at the point of highest total efficiency in both types of turbines. The Kaplans have a flatter survival curve for load changes because of inherent design. The machines tested appear to have had a minimum of shock waves and subsequent pressure drops, which could account for a part of the unexplained losses. There appears to be general agreement that the reported mechanical losses in Francis turbines are related to the clearance between the wicket gates and the runners and to runner size and speed. The relationship between clearance and fish mortalities is not apparent for Kaplans in proto-type dimensions.

The test data used are shown also on y vs. x plots; they indicate the many factors that must be consid-ered in the conduct of such experiments.

Milo C. Bell

ESTIMATES OF THE ACUTE TOXICITY OF AMMONIA-UREA PLANT WASTES TO COHO SALMON, ONCORHYNCHUS KISUTCH

In order to properly plan the waste disposal system of an ammonia-urea fertilizer plant to be constructed on an estuary, it was necessary to determine the me-dian tolerance level (TLm) and the 10-per cent toler-ance level (TLio) of the effluent to salmon. Young coho salmon were chosen as test animals because of their availability at the time that the experiments were conducted. A synthetic waste that contained all of the ions and had the important physical properties of the waste was prepared. The toxicity of the syn-thetic waste was determined by a series of bioassays during July and August, 1967. The study was con-ducted at the Bowman's Bay Experimental Laboratory near Anacortes, Washington.

Experimental Fish

The fish used were fingerling coho salmon obtained from the Samish Hatchery of the Washington State Department of Fisheries. They were between 6.2 and 8.4 cm in total length and had a mean total length of 7.5 cm. With the cooperation of the personnel of the Bowman's Bay station, the fish were acclimated to the salinity of the seawater supply, 29 parts per thousand (ppth). The salinity of the water in the tank holding the salmon was increased to 7 ppth on

the first day, and in the following days to 14 ppth, 18 ppth, 25 ppth, and 29 ppth. No mortalities were observed during the procedure. The fish were fed a diet of Oregon pellets identical to their diet in the hatchery.

Experimental Procedures

Several series of bioassays were conducted. The first was a static bioassay, in which the range of con-centrations to be used in the definitive, continuous-flow bioassay was determined. Ten fish were tested in 100 1 of water in each experiment. The tests were conducted in the large, concrete and glass aquaria of the laboratory. Ten fish were used at each concentra-tion of simulated waste and in the controls. Water temperature was kept between 15.5° and 16°C, and the salinity was kept at 29 ppth.

The remainder of the bioassays were continuous-flow bioassays, in which the exchange rate was about 1.5 1 per hr and continuous volume of 100 1 was maintained. The synthetic waste was metered by means of a Marriott bottle into a mixing chamber.

It was our intention to conduct the bioassays for 96 hr, but we reduced the time to 48 hr after the first few bioassays because almost all mortalities were

44

observed within the first 24 hr. In some experiments, fish that were moribund at the end of 24 hr died within the next 24-hr period. The same observation was made by English workers who studied the toler-ance of salmonid fish to ammonium chloride in estu-arine waters.

There were three series of running-water bioassays. The first series was conducted at the ambient salinity of 29 ppth, the second series at about 25 ppth (23.8 to 25.6 ppth), and the third series at 20 ppth (19.4 to 21.8 ppth). The salinity was reduced by the intro-duction of water from Pass Lake, the source of the hatchery freshwater supply.

The final two experiments were standing-water bio-assays conducted with NH4C1 in water of 29 ppth salinity to determine whether the toxicity of the sim-ulated waste was due solely to NH3 - NH4 + alone or to the combination of substances in the simulated waste.

Results The results of the bioassay experiments are sum-

marized in Table 1. It is known that the toxicity of an ammonium-

ammonia mixture is due to the undissociated am-monia (NH3) present in the solution. The toxicity of the mixed effluent was greater than that of NH4C1. This may have been because the amount of undis-sociated ammonia was greater in the simulated waste; calculated amounts of NH3 were 0.60 ppm dissolved in water of the simulated waste and 0.50 ppm in the

NH4C1 solution. The toxicity of the waste decreased as the salinity

decreased, within the range of the experimental con-ditions. At a salinity of 29 ppth, the TLm was about

47 ppm NH3-NH4 (0.60 ppm NH3); at 25 ppth, the

TABLE 1.

A summary of 48-hr TLma and TLiob of fingerling coho salmon subjected to the simulated waste of an ammonia-urea fertilizer plant and of NH4C1. The data were obtained in static and continuous-flow bioassays in seawater of 29, 25, and 20 ppth salinity. Temperatures were 15.5° and 16°C. Concentrations of NH3-NH4-11 were obtained experimentally and NH3 concentra-

tions were calculated.

Type of bioassay Salinity, in ppth

TLm, in ppm of

NH3-NH4-1-4 NH3

TTio, in ppm of

NH3-NH4̀ P NH3

Static bioassay 1. Simulated

waste 29 47 0.60 35.5 0.45

2. NH4C1 29 58 0.50 51.0 0.43

Continuous-flow bioassays

Replicate 1 29 49 0.62 45 0.57 2 29 46 0.58 43 0.56

3 29 47 0.60 45 0.57

Replicate 1 25 52 0.66 48 0.61 2 25 53 0.69 49 0.62

Replicate 1 20 55 0.88 49 0.79

2 20 53 0.85 48 0.77

a TLm - median tolerance limit. b TLio - 10-per cent tolerance limit.

TLm was about 52.5 ppth NH3-NH4 (0.68 ppm NH3); and at 20 ppth, the TLm was about 54 ppm NH3-NH4 (0.87 ppm NH3).

This observation is of some practical importance, for it indicates that if the waste effluent is regulated so that it does not affect fish at the maximum salinity, an added safety factor will be obtained during the period of spring freshets, when young salmon might be migrating through the area on their way to the ocean.

Max Katz and Robert A. Pierro

THE TRANSPORT OF ENDRIN FROM THE YOLK SAC

TO THE EMBRYO IN DEVELOPING STEELHEAD FRY1

Great interest has been shown in recent years re-garding the use and abuse of chlorinated hydrocarbon pesticides. Yet, the fundamental and theoretical as-pects of accumulation and mode of deleterious action of those toxicants on beneficial aquatic species have not been studied.

A study was undertaken of the effects of endrin, a representative chlorinated hydrocarbon insecticide, on lipid (phospholipids, neutral lipids, and choles-

1 Supported by the Federal Water Pollution Control Admin-

istration, Grant No. WP00956-02.

terol) and electrolyte metabolism in developing steel-head eggs and larvae (embryo and yolk sac). Lipids were selected for intensive study because it was known that the chlorinated hydrocarbons dissolve and are stored in the fats. Any effect on fish physiology would be readily observed in the mechanisms that dealt with the transportation and metabolism of the lipids.

Answers to the following questions were sought:

1. Will prolonged exposure of the egg to endrin permit the concentration of the insecticide in the egg? If so, then (a) is the transport active or passive;

45

(b) will the prolonged exposure of the egg to endrin affect the lipid metabolism of the newly hatched embryo; and (c) does the egg integument give any protection from endrin?

2. Will the embryo have any endrin at hatching or will it take it up gradually from the yolk sac? How is the endrin (which may be absorbed by the egg) distributed in the larvae and in the yolk sac?

3. Will the embryo take up additional endrin from the water?

4. Will the embryo metabolize or excrete any of the endrin?

5. Will the endrin affect the uptake and metabo-lism of lipids and electrolytes in the newly hatched embryo?

Before these questions could be answered, it was necessary to establish the normal metabolism of lipids and electrolytes in steelhead eggs and larvae. A series of investigations was undertaken and brief summaries are given below.

The Uptake of Endrin by Fertilized and Unfertilized Steelhead Trout Eggs

Fertilized and unfertilized trout eggs were exposed to 150 mg/1 of endrin in a constantly flowing stream of water at 12°C. The assumption was that the un-fertilized eggs would be less active metabolically and therefore would have a different uptake of endrin than the fertilized egg. Samples of 10 eggs each were taken each day, and the quantity of endrin was assayed.

The fertilized eggs took up progressively more en-drin than the unfertilized eggs, although the total uptake in the first 12 days was so small that it seemed unlikely to affect the hatched fish. However, during hatching (day 13), the concentration of endrin in-creased seventyfold in the yolk sac fry. This finding suggests the extreme importance of preventing the contamination of water in the hatchery from the day of hatching on.

Kenneth S. Kimura, Dov Grajcer, and Max Katz

THE UPTAKE OF ENDRIN BY THE STEELHEAD TROUT EMBRYO FROM THE

YOLK SAC AND FROM THE ENVIRONMENT

Three groups of fertilized steelhead trout eggs were used: One group of eggs was exposed to 150 ppm of endrin throughout the experiment. One group of eggs was exposed to 150 ppm of endrin up to and including the day of hatching. One group of eggs was not exposed to endrin.

Samples were taken at short intervals during the period of development from hatching until the com-plete absorption of the yolk sac. Separate insecticide and lipid assays were done for the embryo and yolk sac. Assays were conducted for phospholipids, neutral lipids, cholesterol, and endrin.

These were the findings:

1. There was no endrin in freshly hatched embryos.

2. There was progressive absorption of endrin from the yolk sac when the egg had been exposed to endrin prior to hatching.

3. There was direct uptake of endrin from the water by the developing embryo.

4. There was a breakdown of endrin by the embryo, but not by the yolk sac.

5. The quantity of neutral lipids in the embryo

Supported by the Federal Water Pollution Administration, Grant No. WP00956-02.

AND ITS EFFECT ON LIPID METABOLISM'

increased, while the concentration of neutral lipids was first reduced and then gradually increased.

6. Endrin-treated eggs produced embryos with a higher concentration of neutral lipids. This con-centration diminished gradually to a level only slightly higher than the normal.

7. Neutral lipids in the normal yolk sac were re-duced proportionally to the uptake in the embryo, whereas they remained unchanged in the endrin-treated yolk sac. 8. The phospholipid concentration in the normal embryo increased from .43 mg/embryo to 3.04 mg/embryo, whereas, in the endrin-treated samples, phospholipid uptake was impaired to a large degree. The greatest impairment was observed in embryos that were maintained continuously in the endrin solution.

9. The cholesterol content was similar for treated and untreated samples. There was a gradual in-crease of cholesterol levels in all embryos (from 200 mg per cent at hatching to 350 mg per cent at absorption of yolk sac) and a smaller increase in the yolk sac (from 390 mg per cent to 525 mg per cent).

Dov Grajcer and Max Katz

46

ELECTROLYTES IN DEVELOPING STEELHEAD TROUT EMBRYOS AND YOLK SACS

The electrolyte balance of normally developing steelhead larvae was assayed as a preliminary investi-gation to a study of changes due to endrin exposure. The Perkin-Elmer atomic absorption spectrophoto-meter was used. The embryos and yolk sacs were separated and assayed throughout the period of yolk sac absorption. It was found that the calcium con-centration of the embryo increased, whereas the cal-cium level in the yolk sac remained constant. Potas-sium levels increased greatly in the embryo and stayed

constant in the yolk sac. The sodium concentration increased slightly in the embryo and remained nearly constant in the yolk sac. Magnesium concentration was constant in the embryo but fluctuated greatly in the yolk sac. On the basis of the above results, it was concluded that calcium, potassium, and sodium are taken by the fry from the water and that mag-nesium needed by the embryo is taken from the yolk sac.

Bruce Fowler, Dov Grajcer, and Max Katz

ENZYMES OF INTERMEDIARY NITROGEN METABOLISM IN FISHES

Basic studies on the occurrence and levels of various enzymes of intermediary nitrogen metabolism in fishes is being undertaken in the College of Fisheries. Ex-pansion of our knowledge of the interconversions of nitrogenous substances in fishes is a prerequisite to a clear understanding of their development, growth, nutrition, resistance to disease, and the biological effects of chemicals and radioactive materials pollut-ing the aquatic environment. A study of the enzymic

patterns and metabolic machinery of elasmobranchs and primitive fishes, such as the lungfishes and the coelacanth, provides information on the evolution of the progenitors of the higher vertebrates, including man. Projected studies on the enzymes of intermed-iary nitrogen metabolism in salmon will provide valu-able information for correlating nutritional require-ments, growth rate, and productivity of these impor-tant food fishes.

George W Brown, Jr.

RESEARCH VESSEL, M. V. COMMANDO

The 67-ft M. V. Commando served the needs of classes in several departments on the University cam-pus and was used as well by various research projects and agencies.

University of Washington classes that made use of the Commando included:

Biology 473 (Limnology) Mechanical Engineering 492 (Naval Architecture) Oceanography 412 (Ocean Currents) Fish. 314, 405, 406, 425, 426, 427, 453 Food Science

Class trips by the College of Fisheries frequently serve multiple needs. Representatives of a federal or state agency may accompany a Commando trip in order to secure fish for special studies. Many times fish samples are secured for processing into fish food

for the resident salmon and trout in the college's experimental hatchery.

One trip in December, 1966 and another in Febru-ary, 1967 was made to Juneau under charter to the University of Alaska in the interest of gear training for University of Alaska students.

Other agencies and departments that chartered the Commando included the Washington State Depart-ment of Fisheries; Undersea Gardens, Seattle; Depart-ment of Oceanography, University of Washington; and Bureau of Commercial Fisheries, U.S. Fish and Wildlife Service.

A combination of academic and research activity was accomplished on several training cruises in Puget Sound. These trips generally proceed from the Seattle area to Everett, to Bellingham, to the San Juan Is-lands and back to Seattle. Five or six students and

47

and Pickering of Bristol University, England. On the same trip a student from the University of British Columbia obtained shark specimens for use in a de-gree program.

Allan C. DeLacy

professors participate in a training cruise at any one time.

In September, 1967, three days were devoted to catching several thousand ratfish (Hydrolagus). These were used for excision of the pituitary by Drs. Heller

Food Science

INTRODUCTION

The year 1967 was a productive one for the Food Science Department. Research projects ranged from fundamental studies connected with the chemistry and microbiology of foods to the application of engi-neering and technological principles to processing, handling, and storing of foods. Work was expanded in production of fish protein concentrate, pelleting of dry feeds for hatchery fish, investigation of effects of radiation on microorganisms, marine and fresh-water microbiology, and extension service activities. One Ph.D. degree was awarded during the year. The registration of freshman and sophomore classes indi-cates a considerably increased interest in the field of food science. Members of the faculty and staff pre-sented papers at several national meetings, including the American Society for Microbiology, the American Association of Agricultural Engineers, the Institute of Food Technology, and the American Institute of Biological Sciences.

A major development during the year was the estab-lishment of a program sponsored by the Rockefeller Foundation to develop and expand the general pro-gram of studies in fisheries technology at the Catholic University of Valparaiso, Chile. The original pro-gram was for a two-year period. During 1967, the first year, five faculty members of the Valparaiso Fish-eries Department took advanced courses in the Uni-versity of Washington Food Science Department. Dr. John Liston left on December 15 to assist in the development of the over-all school curriculum. A second member of the faculty will spend some time in Valparaiso during the forthcoming year.

That the Food Science Department will continue to expand is indicated by the increased enrollment and the growing interest by commercial firms in work-ing on cooperative research programs. Summaries of some of the research programs are presented in this section.

SURVIVAL OF BACTERIA ON SEAFOODS1

This is the final report of three years' work. Much of the data were reported briefly in earlier reports, but most of the studies will be summarized here.

Two general trends in the U.S. food industry have caused new problems in food protection: the increas-ing production of prepared food items and the in-creasing use of nonsterilizing processes to extend shelf storage of foodstuffs. Most of these products are usu-ally stored frozen or chilled. Potential hazards have been recognized in the case of precooked frozen foods

I Supported by the U.S. Public Health Service, No. 5 ROI EF 00558-03.

and much work has been done in this area. Not as much attention has been paid to seafoods held in chill storage.

When low-acid foods, such as seafoods, are held at chill temperatures (ca.0-10°C) without special packag-ing, the naturally associated bacterial population will rapidly cause spoilage of the products. There is much indirect evidence that the abundant psychrophilic growth will inhibit development of potentially dan-gerous bacteria. In the case of pasteurized and speci-ally packaged foods, the growth of the indigenous flora is greatly depressed, spoilage is delayed, and the

48

competitive position of potential food pathogens is greatly enhanced. At the same time, the lengthened storage period provides an opportunity for the devel-opment of the potentially dangerous bacterial popu-lation and perhaps for the elaboration of significant amounts of toxin. In order to assess the possible hazard, it was necessary to know the effect of storage conditions on the bacteria normally present on the food, the effect on organisms that are likely to con-stitute a health hazard, and the capabilities for growth by such organisms in the presence of varying degrees of competition from the spoilage bacteria.

Bacteria were isolated from the skin and underlying flesh of fish held in ice for 3, 9, and 16 days. A heter-ogeneous group of organisms was noted in 3-day-iced fish, and a homogeneous group in 16-day-iced fish. Of the 3-day isolates, 28 per cent were Pseudomonas and the remainder were Achromobacter, Flavobacterium, Alcaligenes. micrococci, and yeasts. After 9 and 16 days, the flora was composed mainly of Pseudomonas and Achromobacter.

Inhibitory Studies

Since there was much indirect evidence that the psychrophilic growth would inhibit the development of potentially dangerous bacteria, on foods, the effect of mixed culture growth was determined. A pure culture of a spoilage organism was grown in direct competition with Staphylococcus aureus or Salmonella and inhibition of the pathogen by the spoilage organ-ism was established by comparison of the viable count of control cultures of the test pathogen with the viable count of the pathogen when grown in mixed culture with a spoilage organism. When the viable count differed by a factor of 1 log or 90 per cent, i.e., the test pathogen count was 1 log lower (90 per cent lower) than the control count, the spoilage organism was con-sidered to have inhibited the pathogen. Of the 332 spoilage organisms tested, 25 were able to significantly inhibit the growth of S. aureus. Twenty of these inhibitors were isolated from fish held in iced storage for 16 days and the remaining five were isolated from fish held in ice for nine days. The composition of this spoilage flora was Pseudomonas, 18; Achromo-bacter, 4; and gram-positive rods, 3. Three hundred and eighteen spoilage organisms were tested with Salmonella. Again, the inhibitors were predominantly from the fish held in ice for 16 days (25 of 36); 10 were isolated from 9-day-iced fish, and 1 from 3-day-iced fish. Even more Pseudomonas were found in this group of inhibitors than in the other group inhibiting S. aureus. There was also very little correlation be-tween inhibitors of S. aureus and inhibitors of Sal-

monella, and only four spoilage organisms were able to effectively control both food pathogens.

Of the over 300 spoilage organisms tested, only 61 were able to inhibit either S. aureus or Salmonella. Also, the majority of these inhibitors were Pseudo-monas. Since Pseudomonas organisms are known to grow rapidly at low temperatures, it was not known whether the inhibition was due to competitive growth or the production of inhibitory compounds. There-fore, selected spoilage organisms were grown in a broth medium. After 18-48 hours' growth, the spoil-age organisms were removed by centrifugation and filtration. The resulting sterile supernatant was inoc-ulated with the pathogen and a nutrient supplement, and a medium was obtained that was nutritionally comparable to fresh medium. Control samples were also kept so that the degree of inhibition could be determined. The supernatants from 76 spoilage or-ganisms were tested for inhibition of S. aureus. Only 18 samples of supernatants were inhibitory and pre-vented or markedly inhibited (90 per cent less than the control samples) the growth of S. aureus. The or-ganisms that produced a supernatant inhibitory to staphylococci were not in every case the same organ-isms inhibiting staphylococci in mixed culture tests. It was apparent that some organisms produced inhibi-tory material in the medium in sufficient quantity to prevent maximum growth of staphylococci. An at-tempt was made to isolate and characterize the inhibi-tory material produced by two spoilage organisms. Although the characterization was not complete, the inhibitory material was not protein in nature since it was filterable, possessing a molecular weight less than 10,000, and was relatively heat-stable. Also, little activity was lost when the supernatant was passed through both anionic and cationic exchange resins.

There was little correlation between inhibition in mixed culture and culture supernatant, and only a few organisms produced inhibitory supernatants. These data indicated that some inhibition was due to production of inhibitory compounds, but the large part of inhibition was due to the rapid competitive growth of the spoilage organisms.

Growth Studies

To better understand the competitive effects of the spoilage organisms, it was necessary to determine the lengths of their lag and growth rates. Over 300 spoil-age organisms were grown at 2, 12, and 22°C and the change in growth was followed by viable count and/ or measurement of the turbidity of the culture me-dium. The results are shown in Table 1. It can be

49

seen that the rate of growth (generation time) de-creased as the incubation temperature was decreased. Also, organisms isolated at 3 days had shorter genera-tion times than those isolated after 9 or 16 days' iced storage. The length of lag phase did not correlate well with generation time. Organisms that showed rapid growth (short generation time) did not neces-sarily have short lag phases.

TABLE I

Generation times and lag phases for bacteria isolated from spoiling fish

Number Generation tj,me, hr Lag, hr Incubation Day of of temperature isolation organisms Mean Range Mean Range

2°C 16 164 9.6 3.9-20.5 24 2-72 9 35 11.6 8.7-16.0 15 4-45 3 3 21 16.6-43

12°C 16 156 2.1 0.8- 7.5 7.7 0-18 9 125 2.3 1.0-11.0 7.2 0-18 3 38 5.8 2.1-13 21 0-76

22°C 16 145 1.1 0.7- 4.0 2.6 0-11 9 120 1.3 0.8- 5.0 2.2 0-6 3 39 4.5 1.2-18 2.4 0-6

Studies with two groups of selected organisms, one with extended lag phase at 2°C and the other with short lag periods at 2°C, showed that the generation times were temperature-dependent. However, very little difference in generation times was obtained be-tween the two groups of organisms with long and short lags at different incubation temperatures. These results suggest that a fundamental reorganization may have been taking place in the organisms with long lags at 2°C, perhaps involving the synthesis of new psychrophilic enzyme systems.

Food-poisoning Bacteria

Growth on Seafoods. Salmonella is a potential health hazard in many foods; however, little infor-mation was available on the growth of this organism in seafoods and conditions influencing growth.

The growth of Salmonella was tested in untreated and irradiated crabmeat. S. give grew competitively in crabmeat at 22°C, but not at 11, 8, or 5°C. The cells grew rapidly and reached high numbers in all samples at 22°C, but they decreased in numbers at all lower temperatures. In English sole tissue, S. hei-delberg, S. typhimurium, and S. derby all grew ra-pidly at temperatures as low as 8°C from inocula as low as 101 cells/g and even in the presence of 10- to 100-fold higher numbers of competing saprophytes. Ionizing radiation at relatively low levels enhanced the growth of salmonellae on fish fillets by reducing

the numbers of competing saprophytes. No growth was obtained under any condition when the temper-ature was held below 6°C.

Low Temperature Growth. With the increasing numbers of food products held under refrigerated storage, the problems of low temperature growth of food pathogens have become more acute. It is nor-mally assumed that refrigerated storage will prevent the growth of these food pathogens during storage. The growth of salmonellae and staphylococci have been reported at temperatures as low as 6.8°C. It is not inconceivable that some refrigerated foods reach temperatures above this, especially during defrosting. Since foods may be subjected to warm temperatures for short periods of time, food-poisoning bacteria may be able to grow and increase in numbers. Since low temperature is used to control the growth of bacteria, the low temperature growth of salmonellae was in-vestigated. Salmonellae were grown on agar in a tem-perature-gradient incubator. The results showed that salmonellae normally grow at high temperatures, but can also grow at much lower temperatures. Minimum temperatures for growth for 7 serotypes, as determined by visible growth, ranged from 5.5 to 6.8°C.

The pattern of survival and growth of Salmonella derby, S. heidelberg, and S. typhimurium was followed by inoculating the organisms into broth and incubat-ing the cultures in a polythermostat at temperatures ranging from 1.1 to 12.3°C. Minimum growth tem-perature obtained for S. heidelberg after 19 days' incubation was 5.3°C. The minimum growth temper-atures for the same length of time for S. typhimurium and S. derby were 6.2 and 6.9°C, respectively.

The results indicate a growth temperature shift during extended incubation of Salmonella at low temperatures. This phenomenon and the capability of Salmonella for growth at low temperatures could be significant in foods stored for long periods of time at temperatures above 5°C.

Effect of Sodium Chloride on the Growth of Sal-monella. In addition to temperature, the effects of other environmental factors on the growth of salmon-ellae were tested. Sodium chloride is used in many products as a preserving agent. The concentration used may be either lethal or inhibitory depending on the concentration. Little data was available in the literature on the growth of salmonellae in the pre-sense of sublethal salt concentrations at optimum and suboptimum temperatures.

Experiments were designed to determine the effects of low levels of salt (1-8 per cent) on the growth of salmonellae at 37, 22, 12, and 8°C in nutrient broth and fish tissue homogenate. For these studies, nutrient

50

6 7 3 4

Time, days

S. heidelberg

0.9 12°C

0.1

Abso

rban

ce

0.8

0.7

0.6

0.5

0.4

0.3

0.2

„a 1.0

2.0

S. heidelberg 37°C

0.9

4.0 0 50

,

6.0

/

..A 7.0

/ /

/ :

• 8.0

I! .

/ / / I I /* / /1

/ / / /

/ II ,

/.

cr .

Abs

orba

nce

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

1 5 21 25 30

Time, hours

40 45

i/ 0

0 3 4.5

broth was prepared containing sodium chloride at the desired levels. Fish tissue homogenate was prepared and 10 ml was placed in 25-ml Erlenmeyer flasks. Sufficient sodium chloride was added to give the de-sired concentration of 1-8 per cent.

The rate of growth of Salmonella serotypes in nutrient broth decreases with increasing salt concen-trations. When incubated at 8°C, S. heidelberg in-creased throughout the 8 days' incubation period in 1 and 2 per cent salt, but increased only 4 days in 3 per cent salt. At the higher salt concentrations, the numbers of cells either did not change or de-clined. When the incubation temperature was raised, the organism was able to increase in numbers over a wider range of salt concentrations. At 12°C, increases were obtained up to 5 per cent salt (Fig. 1). The lower levels of salt, 1 and 2 per cent, also appear to be stimulatory. When the temperature was raised to 22°C, the growth of the organisms in 1 to 3 per cent salt was very rapid, and then the rate of growth decreased as the salt concentration increased. The maximum number of cells reached was lower at the higher salt concentrations. When growth at 37°C in salt concentrations between 4.0 and 8.0 per cent were

Fig. 1.-Growth of Salmonella heidelberg at 12°C in nutrient broth containing 0-6 per cent salt.

tested, the organisms grew well (Fig. 2). Although the salt concentrations affect the rates of growth of these organisms, the cells also grow at different tem-peratures, several of which are suboptimal. These two factors, salt and temperature, interact to produce differences in the rates of growth. The rates of growth at 8°C in 1 and 2 per cent salt cannot be compared with those at 22° or 37°C in 1 and 2 per cent salt. These data show that the rate of growth is directly proportional to temperature and inversely propor-tional to salt concentration.

Similar experiments were carried out on S. typhi-mui ium and S. derby, and the results were almost identical. Both nutrient broth and fish homogenate were tried as the growth medium. The results ob-tained with fish homogenate paralleled very closely the results obtained with nutrient broth.

Effect of pH on the Growth of Salmonella. Another environmental factor that has been shown to affect the growth of bacteria is the pH of the growth me-

/.

....••• ............ 3 0

-64.0

Fig. 2.-Growth of Salmonella heidelberg at 37°C in nutrient broth containing 4-8 per cent salt.

51

dium. Experiments were performed to determine the combined effects of pH and temperature. S. heidel-berg was grown on a temperature gradient incubator. The medium in each channel of the incubator was adjusted to a different pH value; values ranged from 4.0 to 9.0. Temperatures in each channel ranged from -1.0°C to 15.5°C. The data obtained are shown in Fig. 3. After 7 days' incubation at pH 4.0, no growth

40 5.0 6.I0 7.0 8.0 91.0 pH of medium

Fig. 3.—Effect of the pH of the medium on the minimum growth temperature of Salmonella heidelberg.

of Salmonella was obtained. At pH 5.0, the organism was able to grow, but the minimum growth tempera-ture was 9.5°C. At pH values 6.0, 7.0, and 8.0, the minimum growth temperatures were 5.3, 5.2, and 5.3°C. At pH 9.0, the minimum growth temperature again increased to 6.7°C.

Similar results were obtained when organisms of this species were grown in broth in a polythermostat. At pH 4.0, the cells slowly decreased in numbers during the incubation period. The results obtained at higher pH values were similar to those obtained when S. heidelberg was grown on agar.

Vibrio parahaemolyticus. Vibrio parahaemolyticus is a major cause of food poisoning in Japan, especially in the summer months. While it is usually associated with dishes prepared from raw seafoods, other dishes have been implicated. The distribution in nature appears to be limited to coastal sea waters and bot-toms surrounding Japan. Since the early Japanese

reports, the organisms have been reported in Puget Sound (see V parahaemolyticus Studies in this issue).

Investigations on some parameters of growth showed that the ability of V parahaemolyticus to grow is highly dependent on salt concentration and growth temperature. Growth studies were carried out with strains responsible for food poisoning in Japan. These strains were able to grow at 22°C at salt con-centrations that ranged from 0.1 to 7.0 per cent, but not at 8.9 or 10 per cent. The optimum salt concen-tration was found to be about 3 per cent.

Before the growth potential of V. parahaemolyticus on seafoods could be examined, it was necessary to develop a medium that would enable us to quanti-tatively enumerate and differentiate organisms of this species from the normal flora of seafoods. This me-dium was developed and contained trypticase soy broth supplemented with 0.4 per cent starch, 4 per cent NaCl and a pH of 7.2. A number of inhibitors and antibiotics were added in an effort to inhibit the normal spoilage flora. Penicillin at 2 units/ml was the only inhibitor that proved effective. It was, however, not as effective with crabmeat as with English sole fillets and oysters. When used, the medium permitted the growth of organisms of this species but inhibited many of the spoilage organisms. The organisms, which will hydrolyze starch, were detected by flooding the surface of the agar medium with iodine. Areas of starch hydrolysis surrounding colonies of V parahae-molyticus were evident by clear zones in the medium stained purple as a result of the starch-iodine reaction.

Inoculated pack studies were carried out on Eng-lish sole, crabmeat, and oysters. When added to Eng-lish sole fillets, the organisms failed to grow and actually declined in numbers at 1, 4, and 8°C. In crabmeat, growth was obtained at 22.°C and I3°C, but not at lower temperatures. In preliminary studies on oysters, the organisms failed to grow at 13 and 8°C, the only temperatures tested.

These studies, although not complete, showed that V. parahaemolyticus can grow in seafoods provided adequate growth conditions are met. Further studies are necessary before we can fully understand the health hazard of V. parahaemolyticus in seafoods.

Effect of Cold Shock on Biosynthesis

The effects of cold shock on biosynthetic processes of Escherichia coli K12 were examined by use of radioactively labeled precursors of protein, RNA and DNA. The incorporation of C1-4-aspartic acid into protein, H3-uracil into RNA, and H3-thymidine into DNA was followed in suspensions reincubated at 22 and 35°C after cold shock.

10.0-

Z) 9.0-

E

7.0-

E 6.0-

5.0-

52

Protein and RNA synthesis were depressed by chil-ling, and the degree of depression increased with time in the cold. DNA synthesis was very severely de-pressed after 10 min of chilling and completely halted after 30 min. Extended incubation of chilled suspen-sions at 22 and 35°C did not result in incorporation of thymidine and growth of cells even though the num-ber of viable cells, which was determined by plating on a complete medium, remained constant although growth is possible in this dilute medium.

Because of the lack of growth in dilute medium, cells were harvested by filtration after chilling, resus-pended in full-strength medium, and incubated at 35° C in the presence of labeled thymidine. Cold-shocked cells incorporated thymidine after 4 hours, whereas control cells immediately began incorporation. Cells chilled in 0.3 per cent phenethyl alcohol or Ringer's solution, two protective diluents, were able to imme-diately incorporate thymidine. Thus, it appears that they protected the DNA replication system from cold shock damage.

Electron micrographs of thin sections of chilled cells showed that the nuclear material had condensed into the center of the cells, whereas nuclear material was normally dispersed throughout control cells. The proportion of cells with condensed nuclei agreed very well with the proportion of cells inactivated by cold shock. Therefore, it appears that there might have been a physical effect on DNA that resulted in the in-ability to duplicate the chromosome.

The effects of metabolic inhibitors on cold shock, which were reported in the 1966 Research in Fisheries, can be interpreted in view of these results. Events prior to assembly of the DNA strands can be elimi-nated since hydroxyurea, which inhibits DNA synthe-sis by halting synthesis of deoxyribonucleotide triphos-phate pools, has no effect on cold shock lethality. The protective effect of phenethyl alcohol is unre-lated to its previously reported activities. Addition to growing cultures had no effect on lethality; thus, completion of the chromosome, which occurred with growth inhibition by phenethyl alcohol, did not con-fer stability, and initiation of replication was probably not involved since cold shock sensitivity would have decreased prior to initiation of DNA replication.

The protection provided by phenethyl alcohol when present in the chilling diluent further proves that permeability control, proposed by other workers as a key area for cold shock, is not a significant factor in cold shock lethality since phenethyl alcohol is known to alter permeability control in E. coli. This protection then appears to be due to a stabilizing influence on the DNA replication system of some

unknown manner as phenethyl alcohol has been shown to have no effect on the physicochemical prop-erties of DNA.

The protection from divalent cations (Ca ++ in Ring-er's solution; also Mg, Mn++) would also indicate a stabilizing effect on the replication system since these ions are well known for stabilizing structures (mem-branes, ribosomes) and in enzyme function; DNA polymerase, for instance, requires Mg + for activity. DNA polymerase is not inactivated by low temper-ature, so the divalent cations could be stabilizing polymerase.

Thus, cold shock appears to interfere with DNA synthesis at the replication point. Whether interfer-ence is due to an effect of chilling on DNA polymer-ase, to a physical impairment caused by irreversible condensation of the nucleus, or to another effect is subject to further exploration.

Oxidative Metabolism of Nonprotein Nitrogen Com-ponents by Fish Spoilage Bacteria and Their Physiology of Psychrotrophic Growth During Storage of Fish

There is general agreement among investigators that gram-negative asporogenous rods that mainly belong to the genera Pseudomonas, Achromobacter, and Flavo bacterium comprise the microflora of the majority of newly caught fish. Reports by many in-vestigators indicate that Pseudomonas becomes the predominant genus as fish reach advanced stages of spoilage. Most studies have been concerned with enumeration and partial identification of the bacteria found on fresh and spoiling fish, and little is known about the mechanisms by which Pseudomonas be-comes the predominant flora in spoiled fish.

In our studies, the microflora of English sole during spoilage was shown to be dominated by Pseudomonas species. The percentage of these organisms increased from less than 30 per cent in the initial flora to over 90 per cent as spoilage proceeded. Pseudomonas were the principal organisms that invaded the fish flesh during storage in ice (Table 2). The metabolically active Pseudomonas, which are present in small num-bers on the fish skin initially, overgrow all the other types of bacteria during storage of fish in ice. The organisms invaded the fish through the skin in about 7 days. Overgrowth of all the other types of bacteria by Pseudomonas is due to ability to utilize a wide range of amino compounds, high rate of oxidative capability, and ability to adaptively utilize creatine, and possibly lysine, at ice temperatures.

Since all the spoilage bacteria were active oxidizers of many of the nonprotein nitrogen (NPN) compo-nents, particularly amino acids, they produced total

53

Pseudomonas Achromobacter Flavo bacterium Alcaligenes Cocci (Gram+) Rods (Gram+) Yeasts

28 0 72 83 98 18 50 5 3 1 3 25 3 2 0 3 0 7 2 1

33 0 0 0 0 10 25 13 3 0 5 0 0 0 0

87 1 0 4 0 7

alanine

glutamic acid

glycine

lysine

60

creatine, taurine

50

40

4O 30

20

1 0

Cells grown at 8°C

Cells grown at 22°C

4.

"'•

TABLE 2.

Generic composition of the microbial flora of English sole stored in ice

Days in ice

Type of bacteria

3 days 9 days . 16 days Skin Flesh Skin Flesh Skin Flesh

`70 % % % %

Total % 100 100 100 100 100 100

Total number of isolates 39 4 64 68 85 82

volatile base (TVB) at a similar rate up to a certain period—about 10-12 days in fish stored in ice. After this period, proteolytic Pseudomonas types hydrolyzed protein and released amino acids that were probably utilized by both the proteolytic and nonproteolytic types of bacteria, and spoilage of the fish muscle ensued. All amino acids tested except lysine were oxidized rapidly; lysine was oxidized slowly and in-completely. Alanine, among all the amino acids pre-sent in the fish, was most rapidly utilized by all groups of bacteria (Fig. 4). Taurine was not oxidized. Very active and complete oxidation of creatine and lysine was shown by cells cultured in media containing these substances in high concentration.

0 10 20 30 40 50 60 Time, in minutes

Fig. 4.—Rate of oxidation of alanine, glutamic acid, glycine, lysine, creatine and taurine by unadapted Pseudomonas #2.

A detailed study of the oxidation of alanine was carried out since this amino acid was used preferen-tially by the spoilage bacteria. The bacteria isolated from spoiling fish could be classified into three groups on the basis of the rate of alanine oxidation: viz., group 1, 1.260 02 uptake/min; group 2, 0.260 02 uptake/min, and group 3, 0.009p,1 02 uptake/min. Organisms from actively spoiling fish (stored in ice for 9 and 16 days) all fell into group 1. Groups 1 and 2 consisted entirely of Pseudomonas; all other bacteria isolated from fish fell into group 3. The bacteria invading the flesh of the fish all fell into group 1. It was postulated that the rapid rate of amino substrate oxidation by the Pseudomonas and their ability to utilize creatine effectively, and possibly lysine, was the main cause of their effectiveness. Group 1 bacteria produced greater amounts of vola-tile base (an indicator of fish spoilage) in fish tissue preparations than either group 2 or group 3 bacteria. However, proteinase production by proteolytic group 1 Pseudomonas was inhibited at ice storage temper-atures, and the inhibition accounts in part for the small amount of proteolysis seen in spoiling fish.

The spoilage Pseudomonas were found to be psychrotrophic. Growth occurred at temperatures ranging from 0-35°C. Cells grown at low temperatures (e.g., 8°C) produced an alanine oxidase system that was active at tempreatures below 12°C. This appears to be a temperature-induced psychrophilic oxidase that is produced in addition to the normal mesophilic oxidase system present in Pseudomonas grown at any temperature in the growth range. The psychrophilic system was not produced by cells grown at about 22°C (Fig. 5). It was heat-labile and was inactivated by exposure to 35°C, at which temperature the meso-philic system was stable.

10 20 30 40 50 60 70 Time in minutes

Fig. 5.—Effect of growth temperature on the oxidation of alanine at 4°C by Pseudomonas cells.

54

Experiments with 2-4 dinitrophenol seemed to rule out a permease system, and it was assumed that a psychrophilic isozyme was involved. The general distribution of such inducible systems in psychrotro-phic bacteria would explain their ability to shift to high activity at low temperatures after a suitable lag phase period.

These studies showed that there is a quantitative and qualitative change in bacterial flora on the skin and in the flesh of fish during storage in ice. Pseudo-

monas species become the dominant species because of their rapid growth at ice temperatures and their efficient utilization of the N-containing substances in the water-soluble components of fish tissue. They apparently also possess a temperature-induced psy-chrophilic enzyme system, which enables them to be active at any temperature in their growth range.

John Liston, Jack R. Matches, Gary A. Houghtby, David Bannerjee, Donald D. Curran, and

Susan J. Wescott

MECHANISM OF THE CHEMISTRY OF THE SPOILAGE PROCESS IN FISH1

The work under this contract was devoted to an investigation of the biochemical mechanisms involved in the spoilage of iced fish and how these mechanisms are affected by the use of ice containing chlortetra-cycline. In general, the investigation was centered on the oxidative production of volatile base from fish extracts by spoilage bacteria and the influence of various conditions on this process, including the effect of chlortetracycline (CTC). The activity of proteolytic spoilage bacteria was investigated also.

Much of the work was done on bacteria isolated originally from stored iced fish, and they were repre-sentative of the types that most commonly occur in spoiling fish. Two types of fish were used in these studies, Pacific rockfish (Sebastodes spp.) and English sole (Parophrys vetulus), both of which are major food fishes.

The major chemical changes that occur in iced fish during spoilage are due to bacterial degradation of soluble, small-molecule components of fish tissue. This source was established by experiments in which chemical and organoleptic changes in aseptically ex-cised fish tissues held at 0-5°C were compared with changes in similarly treated inoculated or septic fish muscle. The dominant measurable products of fish spoilage seem to be volatile basic substances, trimethy-lamine, and volatile acids. Trimethylamine (TMA) is generally assumed to be produced by bacterial re-duction of trimethylamine oxide (TMAO) . Since there is no evidence that appreciable protein break-down occurs during the earlier stages of spoilage, it is assumed that the volatile bases and acids are pro-duced by degradation of the soluble materials present in fish juice.

Supported by the Federal Food and Drug Authority, Con-tract No. 66-10.

Pseudomonas bacteria dominate the microbial popu-lation of spoiling fish. These organisms comprise 60-95 per cent of the measurable microflora, are aerobic and oxidative, and characteristically utilize amino compounds as a source of C and energy by oxidizing them to pyruvic acid. The acid is passed into the citric acid cycle via acetyl CoA. The soluble (extractive) material in fish muscle tissues is largely composed of N-containing compounds, such as creatine, taurine, and amino acids, as well as trimethylamine oxide. Spoilage bacteria have been shown in our laboratories to be capable of using creatine and the amino acids normally present in fish nonprotein nitrogen (NPN) compounds as a sole source of C and energy. The first step in the utilization is apparently an oxidative deamination of the amino compound. Experiments indicate that the NH3 produced in fish during spoil-age could be accounted for solely by such oxidative deamination processes.

In this study, it was initially assumed that micro-organisms representative of the spoilage types could serve as useful analytical tools in evaluating the spoil-age process in fish and the effect of CTC on this process. It was hoped that by fractionation of NPN extracts from fish, the "spoilage" substrates could be separated and indicated by the action of spoilage bacteria on them. Moreover, it was expected that the concentration and distribution of "spoilage" sub-strates would change as fish from which extracts were taken themselves underwent spoilage.

Chlortetracycline, besides selectively slowing down cell multiplication, has a direct effect on metabolism; it reduces the oxidative activity of spoilage bacteria. It also seems to have a depressant effect on proteinase synthesis in proteolytic bacteria, although the evi-dence for this is not clear-cut.

55

Respirometric studies of oxygen uptake were com-pared with total volatile base (TVB) production. In these studies, the molar ration of 02 to NH3 lay be-tween 1.33 and 1.70 for cells tested at 25°C. These values are within the expected range for the oxidation of amino acids. Ratios in the presence of CTC were higher and indicated less efficient oxidation of amino substrates. Molar ratios higher than 1.33 to 1.70 were also encountered, particularly from extracts of fresh, iced rockfish. It seems likely that in such cases oxi-dizable substrates other than amino compounds (e.g., ribose) may have been available. No definite pattern was observable in production of TVB from extracts of fish stored for different periods in ice. In the final run, there was a slight increase in TVB production as extracts from fish stored for longer periods in ice were tested, except that a minimum value occurred with extracts from fish stored for 7 days in normal and CTC ice. This result provides somewhat tenuous support for the hypothesis that oxidizable amino com-pounds are reduced to a minimum at some stage of the spoilage process and permit derepression of pro-teinase production by proteolytic bacteria. A mini-mum concentration of TVB was found in tissues after 7 days in normal ice but not until 10 days in CTC ice.

The results obtained in experiments in which pro-teolytic and nonproteolytic spoilage pseudomonads were grown in sterile fish tissue certainly indicate a

cycle of oxidation of amino compounds that leads to derepression of proteinase production, which leads, in turn, to accelerated NH3 production as oxidizable amino acids are released by hydrolysis of fish protein. Tests on individual components of NPN clearly showed that certain amino acids are most effective in repressing proteinase production. Arginine, glutamic acid, alanine, and methionine seem most effective. Unfortunately, the data obtained by thin-layer chro-matography analysis of the amino acids in fish extracts during spoilage are too fragmentary to indicate con-clusively the actual occurrence and quantitative vari-ation in amount of these acids with time. Most other published analyses indicate a decline in all amino compounds except taurine and, to a certain extent, lysine during normal spoilage. Neither of these com-pounds is repressive for proteinase synthesis.

An intriguing observation made during inoculated tissue experiments was that TMA production was higher in the case of proteolytic than nonproteolytic organisms. Moreover, the production of TMA by combined cultures at 22°C was low, whereas that from combined cultures at 2°C was relatively high. This raises the possibility that TMAO reduction is coupled in some way with the oxidative processes involved in TVB production or possibly with proteolysis.

John Liston, Flor B. Irlandez, Jong R. Chung, and David Bennerjee

STUDY OF THE BASIC MICROBIOLOGICAL AND BIOCHEMICAL FACTORS INVOLVED IN THE IRRADIATION OF MARINE PRODUCTS'

During the period covered by this report, work was devoted mainly to microbiological and technological studies of the effects of low-dose and multiple-dose irradiation on seafoods and the continued investiga-tion of the factors involved in radiation damage sus-tained by Staphylococcus aureus when exposed to radiation. The project on survival and growth of Salmonella in irradiated seafoods was completed and additional work was done on the effect of radiation of proteolytic activities of bacteria in spoiling fish.

Salmonella Studies Salmonellae grew competitively in English sole ti-

sue at temperatures as low as 8°C from inocula as low as 101 cells/g, but they increased in numbers in non-sterile crab tissue only at higher temperatures (>11°

Supported by the U.S. Atomic Energy Commission, Contract No. AT(45-1)1730.

C). Irradiation facilitated Salmonella growth at low temperature in English sole and at 22°C in crab meat by reducing the competitive microflora. Irradiation had no effect on minimum growth temperatures and Salmonella growth was not obtained in seafood under any condition at temperatures below 6°C.

Low Dose and Double Dose Studies Previous investigations showed that two treatments

of 50 Krad each followed at a few days' interval by a treatment of 100 Krad yielded extension of storage life of English sole fillets equivalent to that obtained by single exposure to about 300 Krad. It was shown that double irradiation is most effective when the primary dose is given to very fresh fish. Little exten-tion was obtained by double treatment of fish stored 8 days or longer in ice prior to primary treatment. The bacteriological quality of spoiled fish with counts

56

in excess of 106 bacteria/g could not be falsified by a double treatment of 50 and 100 Krad. To test the applicability of the technique to shipboard irradia-tion, whole fish in polyethylene bags were irradiated at 50 Krad, stored at 1°C for 7 days, filleted, and a portion of the fillets packaged in polymylar pouches exposed to a second dose of 100 Krad and stored at 1°C. Fillets were prepared also from the fresh fish and irradiated at 50 Krad or irradiated at 50 and then at 100 Krad. Fillets from the non-irradiated fish had a total shelf life (measured bacteriologically) of about 12 days, fillets from whole fish irradiated at 50 Krad about 18 days, fresh fillets exposed to a single dose about 18 days, fillets from fish exposed to a double dose about 18-22 days, and fillets exposed to a double dose (50+100 Krad) greater than 28 days. Thus, a single dose of 50 Krad on whole fresh fish is effective in prolonging the shelf life of fillets significantly. A preliminary experiment in which fillets were taken at intervals from whole fish exposed to 50 Krad and stored at 1°C indicated diminishing effectiveness of a single, low dose in extending the shelf life of fillets as the treated whole fish remain in ice storage.

Microbiology and Chemistry

Volatile base, trimethylamine, and tyrosine N (for proteolysis) assays were conducted on twice-irradiated fish and indicated, as expected, that the double dose further depressed TMA and TVB production, which is, in any case, greatly delayed in irradiated fish. There was a marked depression of tyrosine N increase in twice-irradiated fish as compared with nonirradi-ated or once-irradiated fish (about 30, 20, and 15 mg tyrosine N/100 g of fish for 0, 50 Krad, and 50 + 100 Krad fish, respectively, after 20-25 days' storage). This finding indicates suppression of bacterial pro-teolysis, which occurs during late spoilage in un-treated fish. Direct counts of proteolytic bacteria confirmed that 50 Krad reduced proteolytic bacteria to very low levels, and 50 + 100 Krad virtually elim-inated them. Analysis of 494 microorganisms isolated from untreated, once- and twice-irradiated fillets dur-ing storage is proceeding. The untreated fillets devel-oped a spoilage flora of Pseudomonas, fillets irradiated at 50 Krad carried mainly Achromobacter organisms, with a few yeasts, and fillets exposed to a double dose of radiation (50 + 100 Krad) consistently showed Achromobacter organisms, but developed yeasts and, ultimately, lactobacilli during storage. Biochemical analysis of the populations revealed rapid develop-ment of an active TVB-producing population on un-treated fillets and a slower development on irradiated fillets. Proteolytic bacteria developed in untreated fish

late in storage and occurred sporadically in low num-bers on fillets irradiated at 50 Krad. Several attempts have been made to follow changes in amino acid con-tent of fillets during storage since this is believed to be closely related to the spoilage process. However, no significant results have been obtained by thin-layer chromatography.

Radiation Damage Studies

In this work, effort has been concentrated on re-covery processes in S. aureus cells suffering sublethal radiation damage by using specific inhibitors of DNA, RNA, and protein synthesis and tracer studies of uracil and thymidine uptake. Repair has been shown to occur optimally at 37°C and a pH of about 5.5 and appears to involve DNA synthesis, protein and RNA synthesis with a consequent energy requirement. The evidence suggests that the primary radiation lesion is in the DNA molecule. Recovery depends on com-pletion of the DNA repair process before normal DNA replication is completed. Cells recovering from radiation damage are hypersensitive to a second ex-posure to radiation (and to heat shock) during the repair period. This finding provides a partial ex-planation of the effectiveness of double irradiation.

Role of Pseudomonas Bacteria in Post-mortem Degrada-tion of Fish Muscle Proteins at Chill Temperatures

Degradation of nitrogenous components by bacteria represents most of the complex deteriorative events occurring in spoiling fish muscle. Bacterial spoilage becomes apparent soon after the resolution of rigor in dead fish, and bacteria increase in numbers by using the nonprotein nitrogenous (NPN) components of fish muscle during the early stages of spoilage. It is commonly accepted that very little or none of the protein is attacked by bacteria until fish enter into advanced stages of spoilage.

During spoilage, Pseudomonas organisms increase in numbers and become predominant among the microflora. Since this event coincides with the gross alteration or organoleptic qualities of fish and is accompanied by the onset of extensive protein break-down in the fish muscle, the nature and factors affect-ing the formation of proteinases by fish spoilage bac-teria have been the subject of investigations for a number of years.

Proteoly tic Pseudomonas. Low dose irradiation (50 Krad) was effective in reducing the numbers of pro-teolytic bacteria in English sole fillets. The radiation-sensitive bacteria were responsible for much of the proteolysis that occurred in irradiated fish stored at 0-2°C (Tables 1 and 2), and the extent of this activity

57

was dependent on the numbers of proteolytic bacteria that survived irradiation. Pseudomonas bacteria, which are known to be responsible for much of the proteolysis in spoiling fish, were greatly reduced in numbers by low dose irradiation.

TABLE 1.

Proteolytic bacteria in irradiated and nonirradiated English sole fillets stored at 0-2°C

Dose Days of storage

( Krad ) 0 4 7 II 14 19 22

(Per cent of total number) 0 31.3 17.6 58.3 76.0 80.0 81.8 85.5

50 <I <I <I <I <1 <I <1

TABLE 2.

Proteolysis in irradiated and nonirradiated English sole fillets stored at 0-2°C

Dose

( Krad)

Days of storage

0 4 7 11 14 19 22 36

0 50

8.20 8.75

(mg of tyrosine N per 100 g of muscle)

10.17 12.25 14.18 19.20 24.42 30.05

9.95 12.35 13.20 14.42 17.78 19.88 29.50

An analysis of 303 organisms isolated from English sole during storage in ice revealed that the proportion of Pseudomonas organisms increased as storage pro-gressed and the percentage of proteolytic Pseudomo-nas bacteria also increased.

Even though proteolytic Pseudomonas bacteria were present in fresh fish and increased in numbers during spoilage, protein breakdown was delayed until the total numbers of bacteria reached >107 cellsig of tissue, which corresponded to the late logarithmic phase of growth. Similar results were obtained when cells of a proteolytic strain of Pseudomonas were cultured in sterile fish homogenate at 22° and 2°C. This delay in fish protein breakdown was due to regulation of proteinase formation by the proteolytic bacteria by controlling mechanisms. These control-ing mechanisms were studied by inoculating cells of a proteolytic strain of Pseudomonas (D-62) isolated from spoiling fish into media containing different fish muscle components, i.e., proteins, NPN.

Control Mechanisms of Proteinase Production by D-62. Cells of D-62 produced proteinases in a medium containing only soluble fish proteins. Enzyme pro-duction decreased gradually after the viable cell pop-ulation reached its maximum level. Also, the amount of proteinases produced by D-62 cells was temperature-dependent; the greatest amount was produced at 22°C and comparatively less was produced at 12 and 2°C. These results indicate that the proteinases are extra-

cellular enzymes that are liberated into the growth medium through physiological processes rather than by cell lysis. The enzymes produced, whether at 22° or at 2°C, had an optimum growth temperature be-tween 40 and 50°C, well above the maximum growth temperature (30°C). The enzymes also displayed activ-ity over a broad pH range; maximum activity oc-curred at pH 9.0.

Enzyme production did not begin in the medium that contained fish proteins and NPN (9:1 v/v) until the viable population reached 103 cells/ml, which corresponded to the late exponential phase of growth. Also, the amount of enzyme produced was much less than in the medium that contained only fish proteins. Enzyme production was further delayed in the me-dium that contained NPN and the medium that con-tained a mixture of amino acids with no proteins. The amount produced in the fish protein medium was fivefold to tenfold less than that produced in the medium that contained only NPN and the medium that contained fish proteins and NPN. These results suggest that the enzymes are inducible and that the NPN fraction has some repressive effect on the pro-duction of enzymes by D-62.

The repressive effect of each individual NPN com-ponent on enzyme production was tested by incorpor-ating each component at 10-3 to 10-4 M concentration in a diazotized fish protein medium. The repressive effect appeared to be confined to amino acids; argi-nine, niethionine, alanine, glutamic acid, aspartic acid, hydroxyproline, threonine, and glycine were the principal components among those tested that showed a pronounced repressive effect.

It appeared that proteinase production was induced during spoilage in the following manner. During early spoilage, bacterial proteinase production was repressed by the NPN fraction. The spoilage bac-teria, both proteolytic and nonproteolytic, utilized NPN in place of protein until the NPN fraction reached a critical level. At this point, the NPN con-tent was decreased, the repression was overcome, and proteinase production was induced.

The hypothesis was tested by inoculating D-62 cells into fish homogenates prepared from fish that had been stored 0, 7, 14, and 22 days in ice. Although growth of the cells was not significantly different from one homogenate to another, the proteinase production in the 14-day-iced fish homogenate was greatest, fol-lowed, in decreasing order, by the 22-, 7-, and 0-day-iced fish homogenates. Thus, the results confirmed the hypothesis. John Liston, Jack R. Matches, Bohdan M. Slabyj, Jong R. Chung, Gary A. Houghtby, and Susan J. Wescott

58

ANAEROBIC BACTERIA FROM THE MARINE ENVIRONMENT'

The occurrence of anaerobic bacteria in the marine environment was investigated during the past year. Samples of marine sediment were taken in Puget Sound from locations representing polluted and clean areas, areas with great tidal flushing, and areas with relatively little tidal flushing. Bacterial samples were collected from the stomach and gut contents of fish caught just off the West Point (greater Seattle) dis-posal plant outfall and in areas at some distance from the outfall. Sediment samples were taken above and below the Ballard Locks in the Lake Washington Ship Canal.

The following procedure was followed in isolation of bacteria from sediments or fish stomach and gut contents. The mud-water slurries taken from the surface of sediment cores or the fish stomach and gut contents were diluted and 0.1-ml quantities spread over a surface of blood agar (blood agar base, seawater and 5 per cent human blood) and incubated anaerob-ically at 30°C. One-milliliter quantities of each slurry were also added to tubes of a cooked meat medium and incubated at 30°C. At intervals of 3, 7, 15, and 45 days, samples were removed from the tubes of cooked medium and spread on the surface of blood agar. Col-onies developing on blood agar plates were picked, purified, and tested for oxygen requirement. The numbers of organisms isolated from sampling stations in Puget Sound and the dates and depths of sampling are shown in Table I. Samples were always taken at each station from approximately the same area, and the variation in total numbers between samplings was found to be approximately 1 log except at station G (Bellingham Bay), where the variation was found to be 2 logs. The highest counts were obtained at this station during July. During this time of year many fish and agricultural products are processed at

Supported by the U.S. Public Health Service, No. EF 00882- 02.

Bellingham, and much of the processing water is dumped into the bay, which may account for the higher counts.

The numbers of organisms isolated from sediment samples taken from other areas in Puget Sound (Table 2) compare favorably with the numbers found at the other Puget Sound locations.

Obligate anaerobic organisms were subjected to morphological and biochemical tests for identification. Two hundred fifty-nine obligate anaerobic bacteria were isolated from sediment samples and 99 of these organisms have been identified and named (Table 3).

The increase in population and number of sewage disposal plants with outfalls into the marine environ-ment has created an interest in the effects of the sew-age, which contains pathogenic anaerobic organisms, on the anaerobic flora of fish. An investigation of the problem is under way. Bottom fish were taken by otter trawl immediately off the end of the West Point disposal plant outfall and at other stations at increasing distances south and west of this outfall. The stations south of the outfall were in the direction

TABLE 2. Numbers of anaerobic and facultative bacteria isolated from

marine sediment samples taken in Puget Sound

Location Depth, m 1967

Apr. 26 May 5 June 12 Oct. 3 Oct. 31

Number x 104/m1 Murden Cove 31 1.95 0.41 Above Ballard

Locks 15 30.0 4.9 Below Ballard

Locks 15 1.7 1.3 Oyster Bay

Sample I 28.0 Sample 2 13.0

West Pointa Position 1 152 0.12

2 236 2.0 3 155 2.5 5 248 0.7

aSee Table 4 for locations of positions.

TABLE 1. Numbers of anaerobic and facultative bacteria isolated from marine sediments taken in Puget Sound

Location

West Point West Point Everett Bay No. Saratoga Pass Penn Cove Skagit Flats Bellingham Bay East Sound-Orcas Is. Lopez Sound

Depth, m 1967

Nov. 15 Apr. 26 May 6 May 18 June 12 July 21 Nov.7

30 236 100 135 63 25 27 30 40

0.95 0.50 1.4 6.3

3.5 9.0 7.0 1.1

0.56 0.40 Number x 104 /MI

6.3 0.60 3.4 6.0

2.3 7.5

2.1 1.5

1.3 5.6 9.7

11.7 6.3 1.9

103.0 1.7

0.42 4.6 4.9 1.2 0.65 4.4

35.0 3.8 2.7

59

TABLE 4. Average numbers of aerobic and anerobic bacteria isolated from

fish gut samples taken at various stations off West Point

TABLE 3. Clostridia species isolated from marine sediment samples taken

in Puget Sound

Organism Number

Clostridium botulinum 49 Clostridium novyi 38 Clostridium perfringens 5 Clostridium bifermentans 4 Clostridium sordelli 2 Clostridium sporo genes

of the tidal flow and the stations west of the outfall were at right angles to tidal flow. The station loca-tions, depths, and average numbers of bacteria iso-lated from fish stomach and gut content at the stations are shown in Table 4. The species of fish sampled at the six stations on July 25, 1967 and the average num-bers of bacteria isolated from the fish are shown in Table 5. Fish were also caught and sampled on three other occasions. Numbers of bacteria varied within samples, between stations, and even 'between sam-plings. The numbers of bacteria in the stomach and gut contents of fish taken from the various stations varied by no more than 1 log. As expected, feeding fish contained a larger number of both aerobic and anaerobic bacteria in their stomach and gut contents than nonfeeding fish. A greater variation was found, however, between species of fish.

The bacteria isolated from fish gut and stomach content were tested for their oxygen requirements and 499 obligate anaerobes were selected. These or-ganisms are being tested for morphological and bio-chemical capabilities as an aid to their identification. Identification of these isolates will give some insight into the distribution of pathogenic anaerobic bacteria

Aerobic Anaerobic bacteria bacteria

Distance from Number Mean Mean Station outfall Depth, m offish number number

Number x 104Ig 153 4 0.25 0.1 216 4 0.16 0.063 141 4 0.50 0.2 72 4 0.39 0.063

216 4 1.0 0.32 29 4 0.39 0.08

TABLE 5. Average numbers of aerobic and anaerobic bacteria isolated from

fish taken from Puget Sound, by species

Number q f Aerobic Anaerobic Fish fish samples bacteria bacteria

Number x 104/g

Cod 2 0.25 0.063 Dogfish 2 0.63 0.40 Dover sole 4 0.79 0.25 English sole 3 0.20 0.063 Hake 2 10.0 0.63 Rat fish 6 0.13 0.05 Rockfish 5 0.50 0.063

in fish at different locations and will also indicate whether fish caught near the sewage outfall contain larger numbers of pathogenic anaerobes than fish caught at some distance from the outfall. These data will have public health significance especially in the case of fish caught and utilized as food.

John Liston, Jack R. Matches, Donald D. Curran, and Mary L. Holman

1 0.25 mile south 2 1.1 mile south 2 2.1 mile south 4 0.5 mile west 5 1.2 mile west 6 2.0 mile west

MARINE MICROBIOLOGY'

Studies in the past year on marine microorganisms in Puget Sound waters and sediments centered on chitinoclastic bacteria. Samples of Puget Sound water, sediment, fishes, and invertebrates were obtained on three extensive and four one-day cruises with the MV Commando. Various intertidal areas in Purdy, Oyster Bay, Willapa Bay and Elliott Bay were also sampled for bacterial studies. Sampling stations in Puget Sound were selected for differences in water depth, hydrographic conditions, and proximity to populated areas and anticipated pollution (Table 1).

Supported by the Office of Naval Research, No. NONR 477(41).

Intertidal areas were generally sampled near oyster beds, which contribute organic material continually to the sediments through biodeposition. It was of interest to determine whether differences existed among stations in bacterial numbers, types, and bio-chemical activities.

Total heterotrophic bacterial counts varied from station to station in Puget Sound, but generally the counts in sediments ranged from 103 to 106/g of sediments. On rare occasions, counts of 106 bac-teria/g of sediments were encountered. Chitinoclastic bacteria made up 1 to 20 per cent of the total bacteria. Samples of intertidal sediments had consistently

60

TABLE I.

Puget Sound and intertidal sampling stations

Code Location Depth,

Salinity range, °/00

Temperature range °

G Sediment

type Samples a

A West Point 30 25-29 9-12 Sand-mud S,W,F,I West Point 236 26-29 9-12 Brown mud S,W,F,I Everett Bay 100 26-29 10-13 Brown mud S.W.F.I N. Saratoga Passage 135 25-29 8-12 Brown mud S,W,F,I

Penn Cove 63 24-27 7-12 Grey-green mud S,W Skagit Flats 25 25-30 8-12 Sand-gravel S,W Bellingham Bay 27 26-30 9-14 Brown mud S,W,F,I East Sound 30 26-30 9-15 Grey-green mud S,W,F,I

Orcas Island Lopez Sound 40 26-30 9-14 Grey-green mud S,W Marrowstone Pt. 97 30 10-12 Gravel S,W

OB Oyster Bay Intertidal NA Wide Sand-shell S,W,1 P91 Elliott Bay Intertidal NA Wide Brown-green clay S,F

Purdy Intertidal NA 10-20 Sand-gravel S,W,1 SB Shils hole Bay Intertidal NA Wide Sand WB Willapa Bay Intertidal NA Wide Sand-shell S,W,I

aS-sediment, W-water, F-fishes, I-invertebrates.

higher total and chitinoclastic counts than samples of Puget Sound sediments. The total heterotrophic counts of intertidal sediments ranged from 106 to 108 bacteria/g, and chitinoclastic bacteria accounted for 13 to 50 per cent of the total numbers. Bacterial numbers in water samples were consistently lower than those in sediments from the same area by at least 10 to 1,000 times. Agar digesters usually made up less than 0.1 per cent of the total bacterial popu-lation.

Total biochemical activity measurements, which were determined by multiple-tube-enrichment tech-niques, showed different levels of activity for a num-ber of organic compounds. The utilization of glucose, galactose, and alanine and the reduction of nitrate occurred at high levels, whereas cellulose digestion and gelatin liquification occurred at much lower fre-quencies in the same samples.

During the past year, 87 chitinoclastic bacteria iso-lated from Puget Sound sediments, waters, and fauna were studied in detail. All isolates were gram-nega-tive, motile, asporogenous rods and predominantly of Vibrio types. All isolates grew well and showed chitin-digesting activity between 8° and 22°C. At 35°C, 13 of 87 cultures grew, and none grew at 42°C. It appears that the majority of chitinoclastic bacteria isolated from Puget Sound were psychrophilic. The chitino-clastic isolates from both Puget Sound and intertidal areas showed distinct salt requirements for growth (Table 2). Whereas all the chitinoclastic isolates grew well in seawater agar, only 6 per cent of the Puget Sound isolates and none of the intertidal isolates grew in freshwater agar. Growth was usually well sustained

in nutrient agar supplemented with 5 per cent and 7 per cent NaC1, but not when supplemented with 0.5 per cent NaCl. At 10.0 per cent NaC1, a large majority of isolates was unable to grow. Freshwater agar supplemented with 3.0 per cent Rilamix, an artificial seawater-salts mixture, supported growth very well.

TABLE 2.

Effect of salt concentration on growth of chitinoclastic isolates at 13°C

Per cent of isolates that grew

Puget Sound Intertidal Growth medium

isolates isolates

Seawater agar

100 100 Freshwater agar

6 Freshwater agar ± 0.5% NaCI

72 Not tested

Freshwater agar ± 5.0% NaCl

98 100 Freshwater agar + 7.0% NaCI

93 81

Freshwater agar + 10.0% NaCI

23 5 Freshwater agar -I- 3.0% Rilamix

95 Not tested

The examination of additional chitinoclastic iso-lates from intertidal sediments, waters, and shellfish was also initiated during the last year. Preliminary results suggest again the predominance of Vibrios. A number of chitinoclastic species of Cytophaga, which are rarely seen in Puget Sound samples, and a chitinoclastic Spirillum have been isolated from intertidal areas. In addition, a Vibrio showing simul-taneous chitin and agar digestion was isolated.

Various factors affecting chitinoclastic activity were investigated. It was found that chitinoclastic activity

61

was dependent on aerobiosis. Under anaerobic growth conditions, chitinoclastic activity was either poor or absent. The depressed activity may have been related to the decreased growth under anaerobiosis. Cultures, which showed no activity under anaerobi-iosis, did show activity when exposed to aerobic con-ditions. The effect of various organic compounds on chitinoclastic activity was also examined. N-acetyl-glucosamine, an end product of chitin hydrolysis, stimulated chitinoclastic activity in a majority of the isolates. Glucosamine, acetate, and ribose stimulated a lesser number of isolates. Starch, cellobiose, acetate, citrate and ribose generally had little effect on the activity of a majority of chitinoclastic isolates. Glu-cose was distinct in its inhibition of chitinoclastic activity in most of the isolates. The effect of glucose may have been due to enzyme repression or to lowered pH resulting from glucose metabolism.

Efforts to develop a method for routinely assaying chitinase activity in cell-free culture medium have not been successful thus far. Concentrated chitinase preparations obtained by ammonium sulfate precipi-tation are now being tested. When a suitable enzyme assay method is developed, it will be possible to study the physical parameters that influence the production and activity of chitinase.

The assimilatory capabilities of Puget Sound sedi-ments was investigated with the model seabed system. Analysis of test sediments after contact with a large volume (ca. 17,000 ml) of flowing seawater showed no significant increase in organic carbon, as compared to control sediments. This finding is in contrast to previous observations. Further runs with the seabed system in an attempt to study assimilatory processes, as well as degradative processes, are planned.

Vibrio parahaemolyticus Studies

In the United States and other countries, food-borne illnesses with unrecognized causative agents constitute an important group of diseases. In this country, for example, it is estimated that over 50 per cent of the cases of food poisoning were due to agents of unknown etiology. This is understandable since little or no effort has been made by Public Health Laboratories to determine causative agents other than Clostridium botulinum, staphylococci, Salmonella, and very recently, Clostridium perfringens.

In 1951, Japanese investigators made the first report of an outbreak of gastroenteritis caused by a faculta-tively halophilic bacterium, which was recently named Vibrio parahaemolyticus. This report did not attract much attention until 1956, when a similar organism was isolated during an outbreak of food poisoning

in Yokohama. This isolation was significant since these investigators used a selective medium for Staph-ylococcus aureus that contained 4 per cent salt; thus, it appeared that salt was a requirement for growth of the Vibrio. Subsequently, many public health bac-teriologists throughout Japan reported the isolation of organisms of this species from the stools of patients suffering from gastroenteritis. Today, it is estimated that over 50 per cent of the outbreaks of gastroenter-itis in Japan are caused by Vibrio parahaemolyticus.

V. parahaemolyticus food poisoning is associated primarily with fish and fish products. In Japan, this vibrio is found in abundance in marine sediments, coastal waters, and various marine animals during the summer months, and total numbers decline mark-edly during the winter season.

The first reported isolation of V. parahaemolyticus-like organisms in the United States was from Puget Sound and Washington coast sediments. Approxi-mately 10 per cent of the vibrios isolated from these sources showed characteristics similar to those de-scribed for V parahaemolyticus. This discovery prompted a more extensive survey. In the spring and summer of 1967, samples of water and sediment were taken from various inshore and offshore locations in Puget Sound. In addition, oysters were obtained from Purdy Lagoon, Oyster Bay, and Willapa Bay. Organ-isms were isolated from these sources by using a selec-tive technique based on the invariant ability of V parahaemolyticus to utilize starch. The starch medium contained 5 per cent NaC1, a salt concentration which is most favorable for the growth of vibrios, but too high to permit growth of many terrestrial and other contaminants, including the coliforms. Further selec-tion was imposed by incubation of the samples under anaerobic conditions, a technique that prevents the growth of aerobic gram-negative Pseudomonas, which occurred in large numbers in all marine samples.

V. parahaemolyticus was found in relatively large numbers during the summer months from all Puget Sound water, sediment, and oyster samples. Numbers of this vibrio ranged from 30 to 350/m1 in the water samples, 100 to 2,000/g in the sediment samples, and 65 to 4,000/g in the oyster samples. During the early spring and early fall, however, the counts of V. para-haemolyticus fell drastically. The situation paralleled that described by the Japanese investigators. From all the samples tested, over 500 organisms that met the physiological characteristics of V. parahaemolyti-cus, as described by the Japanese investigators, were isolated. Two hundred of these isolates were studied extensively by standard taxonomic procedures. Simul-taneously, 40 strains of V. parahaemolyticus and 6

62

strains of two related vibrios obtained from Japan were tested. From these investigations, it was shown that the Puget Sound isolates composed two groups: a group of the same strains as the Japanese strains, and a group that differed from the Japanese strains only in their ability to rapidly ferment sucrose. Slow sucrose fermentation was shown by some of the Japan-ese strains of V. parahaemolyticus; thus, the group 2 vibrios from Puget Sound were probably biotypes of this species.

The Japanese investigators showed that serological identification of V. parahaemolyticus is not reliable; therefore, an attempt was made to isolate bacterio-phages specific for V parahaemolyticus by enrich-ment of water, sediment, and homogenized oyster preparations with the organism. Enrichment of 35 samples yielded two bacteriophages specific for V. parahaemolyticus, both from Pacific oyster homogen-ates. So far, one Puget Sound isolate has proved susceptible to both phages, but there is some evidence that other isolates tested may be lysogenic. At pre-sent, investigations on the physiological and morpho-logical properties of these bacteriophages are being

carried out. In addition, attempts are being made to isolate a sufficient number of bacteriophages for the purpose of greatly facilitating the rapid identifi-cation of V. parahaemolyticus.

The occurrence of V parahaemolyticus in U.S. in-shore waters and its apparent association with oysters is of considerable significance, particularly in view of food poisoning outbreaks in Japan from this organ-ism. Preliminary observations in our laboratory indi-cated that strains of V parahaemolyticus are very sensitive to freezing and die out fairly rapidly when held at temperatures below 5°C. The widespread use of refrigeration by the U.S. food industry may ameli-orate the potential hazard to the public. What role, if any, the organism plays in the marine environment, where it forms part of a large population of polysac-charide-degrading, halophilic vibrios, is not clear at this time, but Japanese reports of seasonable abun-dance of the organisms when water temperatures are relatively high suggest that it may be an active com-ponent of the microflora which are degrading chitin at those times.

John Liston, John G. Chan, and John A. Baross

A BIOLOGICAL MODEL OF A FRESHWATER COMMUNITY: A GNOTOBIOTIC ECOSYSTEM'

Ecologists are presently seeking unified theories to relate community structure aspects, such as species succession and abundance, to element and energy flux. Portions of natural communities (microcosms) were brought into the laboratory by some investiga-tors to facilitate measurements, but these still consti-tuted very complex biological communities. Like natural communities, they were not very amenable to description or analysis of population structure, primarily because the nonalgal microorganisms, which must be responsible for major portions of the com-munity metabolism, remained refractory to analysis. Most ecologists are not trained in microbiology, and even the aquatic microbiologists stress the limitations of present enumeration techniques for effective mea-surement of the active microbial community. Because of these difficulties, most ecologists either ignore the nonalgal microorganisms or attribute to them all sys-tem functions not shown by the limited segment of the community that they do measure. Thus, there has been no critical evaluation of their role.

Supported by the Department of Interior, FWPCA Grants WP 00982-01 and -02.

In answer to these problems, gnotobiotic ecosystems, mixed but defined cultures of organisms selected to simulate natural communities, are being tested as ecological tools. Because species composition and di-versity can be experimental variables, and because total enumeration of the composite species can be made, these systems offer greater opportunities for the study of community structure than undefined laboratory microcosms do.

A preliminary ecological study of a simple gnotobi-otic ecosystem involving the alga Chlamydomonas re-inhardtii, the protozoan Tetrahymena vorax, and the bacteria Pseudomonas fluorescens, Aerobacter aero-genes, and Cytophaga hutchinsonii has recently been undertaken. The alga was the only organism capable of growing on the inorganic medium and was there-fore the primary producer. The protozoan was in-tended as the primary consumer. The bacteria, all common freshwater bacteria, were selected as reducers to recycle the organic waste materials back to the alga. This preliminary study was concerned primarily with population enumeration as a means of evaluating the degree to which such a simple community could function.

63

Chl rinyora s

DILUTION

DILUTION

0.1

0.01 ' DILUTION+

SYSTEM SEALED

Total bacteria

Absorbance

Pseudomonas i,Den city )

0.001

ion

A ::°1"°1 "" a 2 2.12 7

102

Pseudomonas +Cytophago inoculated r,--Tetrohymena inoculated (1.6/m1)

10' , . . , , , , , , . , . 0 10 20 30 40 50 60 /0 80 90 IOU 110 120 130 140 I 501 160 170

Days

—1.0

10

iDenryl

1 / r174173na' I

1 Y

1 1

1 1

1 Tetrahymena 1 ( Density )

170 • • 110 120 130 140 150 0 10 20 30 40 50 60 70 80 90 1

Days

DILUTION DILUTION DILUTION

110 120 130 140 150 160 170 0 10 20 30 40 50 60 70 80 90 100

Days

107 B1 Chlomydomonos

y

to5

GNOTOBIOTIC ECOSYSTEMS 01 and B,

Chlomydornanas ( only )

Absorbance

Densoy

DILUTION. SYSTEM SEALED DILUTION DILUTION

60 70 80 90 100 110 120 130 140 150 160 170

Density

Absorbance

Total bacteria

DILUTION DILUTION

1 1106

0.01

001

F,72°.'2" i actS eyes

Pseudomonas + Cytophaga inoculated Tetrohymena no doted

1 03

10

Fig. 2.—Density of organisms and absorbance in gnotobiotic ecosystem A2.

DILUTION

The algal cells grew logarithmically until they reached a plateau at x 105 cells/ml, after which they slowly declined both in the presence and absence of protozoa and bacteria (Figs. 1, 2, and 3). The Pseudomonas maintained a population of x 105 cells/ml, but the Cytophaga failed to grow. The pro-tozoans grew logarithmically for several days, during which many, but not most, of the cells contained algal cells. When logarithmic growth ceased at x 103 cells/ml, few of them contained algal cells and the animals appeared food-limited although there were no significant reductions in the potential prey organisms. At no time was there effective cropping of the algal cells. Since neither the Pseudomonas nor Tetrahymena can grow in the medium without the alga, the alga must have supported them by extra-cellular products. Although the population numbers remained relatively stable, the state of the cells changed; the ratio of carotinoid to chlorophyll in algal cells and the size of algal and protozoan cells shifted.

Dilution with fresh mineral nutrient resulted, with one exception, in a short-term decrease in density, followed by population increases to levels slightly higher than the previous stabilization level, and a gradual decline to the stabilization level. One excep-tion was noted; in the first dilution of one system (Fig. 1), the alga showed rapid recovery, but the Pseudomonas population failed to recover immedi-ately, and the Tetrahymena population did not re-cover until after the Pseudomonas population in-creased. Thus, it appears that the algal products became available to the protozoans only through the

GNOTOBIOTIC ECOSYSTEM A, Chlamydomonas, Tetrohymeno, Pseudomonas. C > la g1 _a, and Aerobacter.

Fig. 1.—Density of organisms and absorbance in gnotobiotic ecosystem AI.

GNOTOBIOT1C ECOSYSTEM Ay Chlarnydomonas,Tetr nena. Pseudomonas, Cytophaga, and Aerobocter

Fig. 3.—Density of Chlamydomonas and absorbance of control ecosystems l32 and 132.

mediation of the bacteria, and the small biomass of the bacteria, estimated as approximately 1/60 of the weight of the algal cells, gave little measure of their importance to the functioning of the system.

As a model of a dynamic aquatic community, the system was a very limited success, but it resembled an algal bloom to a remarkable degree. The grazing chain was ineffective because of a change in the feed-ing pattern of the protozoan, and recycling appeared nil. Future work is being directed at finding other combinations of organisms that may permit a more adequate grazing chain and input-yield systems.

FRIEDA B. TAUB, RUTH HUNG, AND RICHARD TOMLINSON

64

VIRAL ACCUMULATION AND AND CRUSTACEA OF

ELIMINATION BY SHELLFISH THE PACIFIC COAST'

An investigation into the accumulation and elimi-nation of viruses by shellfish and crustacea of the Pacific Coast was initiated during the past year by the Food Science Department in collaboration with the Invertebrate Fisheries Group. To date, a labora-tory has been equipped for viral studies and work is under way. Preliminary cultures of monkey kidney tissue have been started and a study of enteroviruses in the marine environment will soon be initiated.

In recent years, there have been a number of reports of outbreaks of infectious hepatitis that were directly traceable to the consumption of raw, polluted shell-fish. These findings resulted in the initiation of a series of studies on the East Coast into the role of pathogenic viruses in shellfish and the marine environ-ment. Because the nature of the infectious hepatitis virus has not been determined, researchers used pri-marily the pathogenic enteroviruses as "model sys-tems." Data obtained indicate an ability of these viruses to survive in the marine and estuarine environ-ment for varying lengths of time. It was confirmed that shellfish can accumulate high levels of viruses readily. It was also noted that shellfish will depurate themselves readily, if afforded an opportunity to do so.

'Supported by U.S. Public Health Service, National Institutes of Health. Shellfish Training Grant T01-U1-01039-03.

Data of this nature are lacking for the West Coast. Fortunately, there have been no known cases to date of viral infection after consumption of shellfish. How-ever, the population is increasing on this coast and is placing a strain on sewage disposal facilities; it is possible that the situation will change. Therefore, a multiphase study of enteroviruses in the marine environment was initiated. The objectives are:

I. To determine whether there are, at present, path-ogenic viruses in the marine environment.

2. To determine whether West Coast shellfish are capable of accumulating viruses, the quantity ab-sorbed, the sites of absorption, the ability to depurate, and the possibility of viral replication. The last will likewise be investigated in crustacea.

3. To determine whether viruses are capable of surviving processing or cooking in seafoods.

Crassostrea gigas, Tapes japomea, Pandalus platy-cerus and Cancer magister have been selected as test animals. Virus titers will be determined by the plaque-forming technique of Hsuing and Melnick. Irradiation and cooking will be the primary process-ing procedures employed.

John Liston, Jack R. Matches, Rudolph G. DiGirolamo, and Albert K. Sparks

PELLETING OF DRY FEED

The development of dry pelleted feeds for animal feeding has paralled the development of compounded diets. The conventional methods of compacting di-etary ingredients into homogeneous particles produce a pellet that is dense, has a polished, shiny surface, and disintegrates rather slowly when exposed to moist-ure. This type of pellet, although adequate for most animal diets, has not proven satisfactory for diets of hatchery-raised fish, particularly salmon. The pellets sink immediately and force the fish to eat much of their food off the contaminated bottom. Moreover, important, heat-sensitive dietary components are de-stroyed during the process of pelleting. Future feed-ing programs, however, will depend on dry diets since they will not necessitate freezing, frozen storage, and

'Supported by the Washington State Department of Fisheries, Contract No. 1-33-D, under P.L. 88-309.

FOR HATCHERY SALMON'

transportation in a frozen condition and can be fed throughout the year. Therefore, a project was initi-ated to develop an improved technique for producing pellets that would retain the nutritional components available in the original dietary ingredients.

It was discovered earlier in the year that dry fish feed will form pellets when sprayed with a water mist on an oscillating table. The pellets were physi-cally better than those prepared by the conventional means of manufacture. It was also discovered that the technique had been used in the fertilizer and chemical industries and that equipment was com-mercially available. Therefore, a Dravo 14-inch-dia-meter Pelletizing Disc was ordered. The machine has been successfully used in the manufacture of fish feed pellets. The process involves feeding a dry mixture into a disc that rotates at an angle of approximately

65

450. As water is sprayed into the disc, pellets form

and come off the disc at the same rate at which feed is introduced. Pellet size is controlled by manipula-tion of disc speed, feed rate, spray rate, and angle of disc rotation. The pellets float for a short time and are relatively soft. Neither heat nor pressure is used in the pelleting process, so maintenance costs are low and there is no damage to heat-sensitive components.

Since water must be removed after the pelleting operation, research effort is being directed towards finding processing conditions that will permit the best

retention of heat-sensitive components during drying. Since thiamine is an important heat-sensitive vitamin, its retention under various drying conditions is being used as an index of thermal destruction during the drying operation. It has been found that retention of thiamine during drying can be improved in fish meals by the addition of soy oil. Additional research will be conducted so that this information can be used and a nutritionally as well as physically adequate pellet can be prepared commercially.

Carl D. Decker and George M. Pigott

A METHOD FOR DETERMINING PROTEIN THIOL GROUPS IN FOODS

During the past year a new analytical method for determination of protein thiol (sulfhydryl) groups in foods was developed. The thiol groups form ferrous ion when reacted with an excess ferric ion. The fer-rous ion produces an orange-red color with o-phenan-throline, and the absorbance of the solution is directly proportional to the thiol content. Experimental work indicates that the reaction is stoichiometric; 2 moles of protein thiol react with 2 moles of ferric ion to pro-duce 2 moles of ferrous ion and I mole of disulfide.

2 RSH +2 Fe+3 2 Fe+2 + RSSR.

The reaction medium is sodium metaphosphate buffer (pH 7.2-7.5), saturated in cystine and 0.3 per cent in o-phenanthroline.

The method has proven to be useful in the deter-mination of thiol content of purified protein from English sole muscle tissue and compares favorably with other methods in simplicity, sensitivity, selectiv-ity, and reproducibility. Preliminary analyses of thiol groups in protein from fresh English sole muscle tissue indicate that the ratio of thiol to protein is relatively consistent at 70+5 micromoles thiol/g of protein. In irradiated muscle tissue (300 KR), the ratio of thiol to protein is reduced to 55+5 micro-

moles thiol/g of protein. Work is continuing on the study of irradiation effects on protein thiols.

This method also has utility in the quantitative determination of other nonprotein biological reducing substances that occur naturally in tissues. The reac-tion is quantitative for small thiol compounds, such as cysteine and glutathione, in the pH range 7.2-7.5. It is also quantitative for ascorbic acid in the pH range 2.0-2.5. One mole of ascorbic acid reacts stoi-chiometrically with 2 moles of ferric ion in this pH range to form 2 moles of ferrous ion and one mole of dehydroascorbic acid.

AA + 2 Fe+3 —> DHAA + 2 Fe+3

This reaction appears to be particularly useful in determination of ascorbic acid in tissues in which the ascorbic acid content is low, as in muscle tissue.

The extent of interference from other substances in muscle tissue has not been completely determined. Potential reducing substances, such as glucose, tyro-sine, ribose, and hypoxanthine, do not reduce ferric ion in these metaphosphate-buffered solutions. How-ever, further work is being done on this method to improve its usefulness in determining sulfhydryl groups present in foods and other biological systems.

Glen R. Limb

66

UNIVERSITY OF WASHINGTON COLLEGE OF FISHERIES RICHARD VAN CLEVE, Dean

FACULTY AND STAFF, JANUARY 1, 1968

FACULTY

Alverson, Dayton L., Affiliate Assistant Professor Jones, G. Ivor, Affiliate Associate Professor Royce, William F., Associate Dean Bell, Milo C., Professor Katz, Max, Research Associate Professor Rucker, Robert, Affiliate Professor Bevan, Donald E., Associate Dean Klontz, G. W., Affiliate Associate Professor Saddler, James B., Research Assistant Professor Bonham, Kelshaw, Research Associate Prof essorListon, John, Professor Salo, Ernest 0., Associate Professor Brown, George W., Jr., Associate Professor Lynch, James E., Professor Emeritus Seymour, Allyn H., Professor Burgner, Robert L., Professor Marvich, Edward S., Affiliate Assistant Pro fessorSmith, Lynwood S., Associate Professor Chew, Kenneth K., Associate Professor Matches, Jack, Research Assistant Professor Sparks, Albert K., Professor Collins, Gerald B., Affiliate Professor Mathisen, Ole A., Associate Professor Taub, Frieda B., Research Associate Professor DeLacy, Allan C., Professor Nakatani, Roy E., Affiliate Associate Professor Thompson, Richard B., Affiliate Assistant Donaldson, Lauren R., Professor Paulik, Gerald J., Professor Professor Hagen, Donald W., Assistant Professor Pigott, George, Assistant Professor Welander, Arthur D., Professor Held, Edward E., Research Professor Whitney, Richard R., Associate Professor

Teaching Assistants

Ahmed, Muzammil Hoag, Stephen H. Limb, Glen R. McKenzie, Daniel H. Tillman, Michael E.

Agarwala, Om, Engineer II Allee, J. Brian, Research Assistant Andersen, Aven M., Research Assistant Baross, John A., Research Assistant Chan, John G., Research Assistant Cheney, Daniel P., Research Assistant Chung, Jong R., Research Assistant Daneault, Louis P., Research Assistant Daniels, Roy J., Hatchery Caretaker Decker, Carl D., Research Assistant Dupuy, John L., Predoctoral Research Associate

III Dupuy, Monica, Chemist DiGirolamo, Rudolph, Research Assistant Eagleton, James G., Hatchery Supervisor Erickson, Robert C., Predoctoral Research

Associate I

Oswold, Tom, Jr., Master Commando

Andersen, Shirley J., Administrative Secretary Anderson, Vivian J., Secretary II Chase, Marion L., Editor I Cooprider, Christina, Secretary II Granger, Camille, Secretary II

Research Staff

Grajcer, Dov, Research Assistant Holman, Mary L., Laboratory Technician II Houghtby, Gary A., Assistant Microbiologist Hung, Ruth, Laboratory Technician III Johnson, Irene, Laboratory Helper I Kines, Dorothy, Laboratory Assistant II Kynard, Boyd, Graduate Staff Assistant Leon, Kenneth A., Research Assistant Lueders, Louis H., Laboratory Technician II Lusk, Raymond T, Instrumentation Technician McAlpin, Marguerite E., Laboratory

Technician II Matson, Lorna J., Laboratory Technician III Mearns, Alan J., Research Assistant Meldrim, John W., Research Assistant Nelson, Victor A., Research Assistant Norris, Louisa A., Research Assistant

Technical Staff

Rockness, Olaf, Marine Engineer, Commando

Administrative Staff

Hurley, Agnes, Secretary II Kohlwes, Mae, Secretary II Lewarch, Ruth, Secretary II Linstedt, Grace, Secretary II Loomis, Nel L., Secretary III Mannhalt, Evelyne, Secretary II

Olsen, James C., Predoctoral Research Associate I Olsen„ Sigurd M., Biologist Olson, Paul R., Senior Fisheries Biologist Pederson, Gary L., Predoctoral Research

Associate I Ruddell, Craig L., Research Assistant Savage, Lura Mae, Stockroom Attendant I Schneider, Ronald L., Research Assistant Short, Zella F., Botanist Sutton, William R., Chemist Thorne, Richard, Research Assistant Vick, Charles E., Statistician I Wadley, Gerald W., Research Assistant Wescott, Susan J., Laboratory Technician II WoeIke, Charles E., Research Assistant Woodey, James C., Predoctoral Research

Associate I

Rowlands, Fred, Developmental Machinist

Martin, Jane, Secretary III Nelson, Karen S., Office Assistant III Pautzke, Maxine M., Secretary IV Peterson, Eileen M., Secretary III Pierre, Ophelia J., Secretary II

Lecturers 1967-68 Academic Year

Anderson, Andrew W. Dassow, John A. Greenough, Joseph W. Loosanoff, Victor L. Ruthford, Eugene A. Beck, William Ellis, Cadwalader H. Groninger, Herman Malins, Donald Stansby, Maurice M. Bell, F. H. Erkins, Robert A. Hall, W. B. Millenback, Clifford Steinberg, Maynard Brett, John R. Germain, Louis G • Halver, John E. Nelson, Richard, Jr. Suomela, Arne J. Burchard, George W. Foster, Richard F. Hartt, Allan C. Olsen, Sigurd M. Tollefson, Roger Chakravarti, Diptiman Fredin, R. A. Hodgins, Harold Olson, Paul Wolfe, John N. Conte, Frank Gibbs, Charles V. Isaac, Gary Ordal, Erling J. Yonker, Walter V.

Glude, John B. Lokken, Harold Pautzke, Clarence F.

LIBRARY (Fisheries-Oceanography) STAFF

Durrance, Raymond E., Librarian Andreassen, Janet, Library Assistant Sandell, Kerstin, Library Assistant Mercado, Heidi, Assistant Librarian Buell, Sally, Library Assistant

67

FISHERIES RESEARCH INSTITUTE Robert L. Burgner, Director

Bevan, Donald E., Professor Chew, Kenneth K., Associate Professor Katz, Max, Research Associate Professor

Fisheries Research Institute Research Faculty

Mathisen, Ole A., Associate Professor Salo, Ernest 0., Associate Professor Paulik, Gerald J., Professor Smith, Lynwood S., Associate Professor Royce, William F., Associate Dean Sparks, Albert K., Professor Saddler, James B., Research Assistant Professor

Anderson, John W., Fisheries Biologist I Andrews, Rollin David III, Fisheries Biologist Barnes, Richard N., Research Assistant Baxter, Richard A., Research Assistant Byrnes, George C., Research Assistant Cardwell, Rick D., Research Assistant Dahlberg, Michael L., Predoctoral Research

Associate II Dell, Michael B., Fisheries Biologist III Eldridge, Peter J., Predoctoral Research

Associate I Friend, Richard C., Marine Technician I Gunnerod, Tor B., Fisheries Biologist I Hartt, Allan C., Fisheries Biologist IV Hoag, Stephen H., Research Assistant Holmberg, Edwin K., Fisheries Biologist IV Isakson, John S., Fisheries Biologist I

Allison, Martha R., Administrative Secretary Bailey, Helen I., Key Punch Operator III Battey, Mary M., Key Punch Operator II Beall, Dorothy D., Office Assistant IV Conner, Alice M., Secretary II

Research Staff

Jones, Evelyn, Laboratory Technician II II Kerns, Orra E., Jr., Fisheries Biologist IV

Kimura, Kenneth S., Research Assistant Koski, K Victor, Predoctoral Research

Associate I Lenarz, William H., Predoctoral Research

Associate I Miles, Harry M., Predoctoral Research

Associate I Miller, Denny M., Research Assistant Mix, Michael C., Research Assistant Newcomb, Timothy W., Research Assistant Peck, Mary McQuaid, Research Assistant Peck, Thomas H. Research Assistant Phinney, Duane E., Research Assistant Reeves, Jerry E., Predoctoral Research

Associate II

Administrative Staff

Fisher, Janet L., Clerk II Gregory, Diane, Office Assistant I Johnston, Mary M., Secretary I Leonard, Alice P., Office Assistant III Loomis, Nel L., Secretary III

Roberson, Kenneth, Fisheries Biologist I Rogers, Donald E., Fisheries Biologist III Sakagawa, Gary T., Predoctoral Research

Associate I Schwartz, Lieselotte, Laboratory Technician II Sjolseth, Danny E., Research Assistant Stauffer, Gary D., Research Assistant Swierkowski, Peter B., Machinist' Mechanic Tyler, Richard W., Fisheries Biologist III Vick, Charles E., Statistician I Weinmann, Fredrick C., Research Assistant Weitkamp, Donald E., Research Assistant West, Wariboko, Q. B. Fisheries Biologist I Wetherall, Jerry A., Research Assistant Williams, Kenneth R., Research Assistant Yoshinaka, Marvin S., Research Assistant Zebold, Stephen L., Marine Technician I

Moore, Remedios W., Editor I Pederson, Carol J., Key Punch Operator I Robson, Alice M., Magnetic Tape Typist I Sievanen, Judith M., Magnetic Tape Typist II Steele-Shaw, Dorothy M., Office Assistant II

STUDENTS, SPRING 1966-WINTER 1967 GRADUATE

AHMED, MUZAMMIL (P)

ALLEE, BRIAN j. (M)

ALTON, MILES S. (M)

ALVERSON, DAYTON L. (P)

ANDERSEN, AVEN M. (M)

ANDERSON, SANDRA (P) ARMAH, MARQUAYE (M)

BANNERJEE, DAVID (P)

BAROSS, JOHN A. (P) BAXTER, RICHARD A. (M)

BERGMAN, PETER K. (P) BEST, EDGAR A. (M)

BOATMAN, EDWIN S. (P) BRANNON, ERNEST L. (M)

BROWNE, EDWARD F. (M)

CHAN, JOHN G. (P)

CHENEY, DANIEL P. (P)

Cu, CHUNG-LING (P) CHUNG, JONG R. (P) CLEAVER, FREDERICK C. (P)

CURRAN, DONALD D. (M)

DAHLBERG, MICHAEL L. (P) DECKER, CARL D. (M)

DESVOIGNE, DAVID M. (M)

DIGIROLAMO, RUDOLPH G. (P)

DOMENOWSKE, RALPH S. (M) DONALDSON, JOHN R. (P)

DUPUY, JOHN L. (P)

ELLIOTT, ERIC 0. (M)

EMERICK, JOHN C. (M)

ERICKSON, ROBERT C. (P) FOWLER, CHARLES F. (M)

FREEL, MARVIN E. (M)

FUKUHARA, FRANCIS M. (P) GADAU, EUGENE L. (M)

GALLAGHER, EDWARD C. (P) GILBERTSON, LARRY G. (M)

GRAJCER, Dov (P) GREENFIELD, DAVID W. (P) GREENOUGH, JOSEPH W. (M)

68

GRONLUND, WILLIAM D. (M)

GUNSTROM, GARY K. (M)

HEINZ, WAYNE A. (P) HIRSCHHORN, GEORGE (P) HOAG, STEPHEN H. (M)

HOUGHTBY, GARY A. (P)

Hsu, BENNY (M)

IKUSEMIJU, KOLAWOLE (M)

ISAKSON, JOHN S. (M) JOHNSON, HOWARD E. (P) JOHNSTON, JAMES M. (M)

JONES, RONALD L. (M)

JOSEPH, JAMES (P) .KATKANSKY, STANLEY C. (P) KILAMBI, VARAD R. (P) KLAASSEN, HAROLD E. (P) Kosiu, K VICTOR (P) LANDER, ROBERT H. (M) LARSON, GARY L. (M) LEE, YOUNG-J00 (M)

LENARZ, WILLIAM H. (P) LEON, KENNETH A. (P) LEONG, CHOON C. (M) LICHTENHELD, RICHARD W. (P) LIMB, GLEN R. (P) MANICKAM, BALASINGAM (M) MATHEWS, STEPHEN B. (P) MCKENZIE, DANIEL H. (P) MELDRIM, JOHN W. (M) MILES, HARRY M. (M) MILLER, BRUCE S. (P) MILLIKAN, ALAN E. (M) MIX, MICHAEL C. (M) MOORE, JAMES L. (P) MYHRE, RICHARD J. (P) NASSRALLA, MOUNIR G. (M) NEAL, RICHARD A. (P) NELSON, MARTIN 0. (M) NELSON, VICTOR A. (M)

NEWMAN, H. WILLIAM (M) NICOLA, STEPHEN H. (M) NORRIS, LOUISA A. (P) OLSEN, JAMES C. (P) PARISOT, THOMAS J. (P) PECK, THOMAS H. (M) PEDERSON, GARY L. (P) PELLA, JEROME J. (P) PEREYRA, WALTER T. (P) PHINNEY, DUANE E. (M) REEVES, JERRY E. (P) ROGERS, DONALD E. (P) RONHOLT, LAEL L. (M) RUDDELL, CRAIG L. (M) SAISITHI, PRASERT (P) SALCEDO, LYDIA G. (M) SCHNEIDER, RONALD L. (M) SILLIMAN, RALPH P. (P) SLABYJ, BOHDAN M. (P) SOUTHWARD, GLEN M. (P)

STAUFFER, GARY D. (M) TAN, ENG-CHOW (P) THOMPSON, RICHARD B. (P) TILLMAN, MICHAEL F. (M) UBOMA, BERTRAM, M. (M) VATHANAPRIDA, CHARE (P) VIDAL-JUNEMANN, JULIO (M) WADLEY, GERALD W. (M) WATERS, BRIAN F. (M) WEITKAMP, DONALD E. (M) WELCH, EUGENE B. (P) WEST, WARIKOBO Q. B. (M) WETHERALL, JERRY A. (P) WHITMORE, CECIL M. (P) WHITESIDE, BOGGY G. (P) WILLIAMS, KENNETH R. (M) WOELKE, CHARLES E. (P) WOODEY, JAMES C. (P) ZEBOLD, STEPHEN L. (M)

STUDENTS RECEIVING FELLOWSHIPS DURING THE YEAR

Fellowship

Fish and Wildlife Service Shellfish Sanitation Public Health Service Office of Education National Science Foundation Office of Education Public Health Service Shellfish Sanitation Public Health Service Department of Interior

Recipient

JOHN G. CHAN RUDOLPH G. DIGIROLAMO JOHN L. DUPUY LARRY G. GILBERTSON GARY L. LARSON DANIEL H. MCKENZIE ALAN J. MEARNS TIMOTHY D. SCHINK CHARLES E. WOELKE GLEN R. LIMB

STUDENTS RECEIVING SCHOLARSHIPS DURING THE YEAR

Scholarship Recipient

Northern Commercial Company Scholarship CARL D. DECKER Tacoma Sportsmen's Club Auxiliary Scholarship KENNETH A. LEON W. F. Thompson Award LARRY G. GILBERTSON Olympia Salmon Club, Inc. Scholarship Fund DANIEL P. CHENEY Northwest Salmon Canners Association JAMES C. WOODEY National Canners Scholarship CHUNG-LING CHU Faculty Merit Awards DANIEL H. MCKENZIE

MARIE SCHMIDT Crown Zellerbach Foundation Scholarship ....................... discontinued Goldstein Memorial Scholarship ............................................. discontinued Pacific Fishery Biologists Scholarship ................................... only awarded every 4 years

69

STUDENTS RECEIVING B.S. DEGREES FROM THE COLLEGE OF FISHERIES FROM JULY 1966 TO JULY 1967

BERNHARDT, JOHN C. BESSEY, ROBERT F. BISHOP, ROBERT A. BLOFELD, ROBERT L. BRUNSON, WAYNE D. CASTLE, PETER T. CARVEY, FORREST E. CHRIST, ROBERTA E. DOBLE, BRUCE D. DONNELLY, ROBERT F.

EMERICK, JOHN C. HARTSOCK, FRANKLIN B. HUNTER, CARL H. ISAKSSON, ARM KIMURA, KENNETH S. LEGG, GORDON H. MODES, VERNON E. NUTLEY, LINDA J. ORACH, FAUSTINO PARENTE, WILLIAM D.

PITCHFORD, LOREN C. SHEARER, JOHN D. SCHINK, TIMOTHY D. S JOLSETH, DANNY E. TORMALA, CARL P. TUTMARK, GREGORY J. WILLIAMS, WAYNE G. WINNOR, RICHARD A. YOUNG, WILLIAM C.

STUDENTS RECEIVING ADVANCED DEGREES IN FISHERIES DURING 1967 AND THEIR THESIS TITLES

ALVERSON, DAYTON L. "A study of demersal fishes and fisheries of the Northeastern Pacific Ocean." Ph.D.

BANNER JEE, DAVID "Oxidative metabolism of nonprotein nitrogen com-ponents by fish spoilage bacteria and their physiol-ogy of psychrotrophic growth during storage of fish (English sole) ." Ph.D.

BOATMAN, EDWIN S. "The effects of hydrostatic pressure on the structure of terrestrial and marine bacteria." Ph.D.

GUNSTROM, GARY K. "A comparison of the occurrence of gross physical anomalies between F1 progeny of normal and irradi-ated coho salmon." M.S.

IKUSEMI JU, KOLAWOLE "The life history and ecology of Cottus sp in Lake Washington." M.S.

JOHNSON, HOWARD E. "Effects of endrin on the reproduction of a fresh-water fish (Oryzias latipes)." Ph.D.

KATKANSKY, STANLEY C. "Some effects of the parasitic copepod, Mytilicola orientalis Mori, on three species of bivalve molluscs in selected sites in Washington, Oregon and Cali-fornia." Ph.D.

KLAASSEN, HAROLD E. "An evaluation of the bottom fauna and its role as fish food in Fern Lake, Washington." Ph.D.

NEAL, RICHARD A. "Fluctuations in the levels of paralytic shellfish toxin in four species of lamellibranch molluscs near Ketchikan, Alaska, 1963-1965." Ph.D.

ROGERS, DONALD E. "Estimation of pelagic fish populations in the Wood River lakes, Alaska, from tow net catches and echogram marks." Ph.D.

SAISITHI, PRASERT "Studies on the origins and development of the typical flavor and aroma of Thai fish sauce." Ph.D.

VIDAL-JUNEMANN, JULIO "Some population dynamics of the Spanish sardine (Sardinops sagax musica) in Antofagasta, Chile." M.S.

WELCH, EUGENE B. "Factors initiating phytoplankton blooms and re-sulting effects on dissolved oxygen in an enriched estuary." Ph.D.

WEST, WARIBOKO Q. B. "The use of the Bergman-Jefferts tag on the "spot" shrimp, Pandalus platyceros Brandt." M.S.

70

CONTRIBUTIONS BY MEMBERS OF THE COLLEGE OF FISHERIES, UNIVERSITY OF WASHINGTON, DURING 1967

(Including those that were published in 1966 but failed to appear in previous year's listing)

I. Publications

Contribution No.

200 TAUB, FRIEDA B., From wastes to resources. Activities Report, 17 (1) :72-78. 1965.

208 THOMPSON, W. F., Fishing treaties and sal-mon of the North Pacific. Science, 150 (3705) :1786-1789. December 1965.

218 ROYCE, WILLIAM F., Population dynamics and marine ecology: Research needs and strategy, p. 53-65. In Theodore A. Olson and Fredrick J. Burgess [eds.], Pollution and marine ecology. Proceedings of the Conference on Ecology and Pollution PIroblems held at Texas A & M Univer-sity Marine Laboratory, Galveston, Tex-as, March 24-26, 1966. Interscience Pub-lishers, John Wiley 8c Sons, New York. 1967. [Abstract only available.]

220 KATKANSKY, STANLEY C., and ALBERT K. SPARKS, Seasonal sexual pattern in Pacif-ic oyster Crassostrea gigas in Washington State. Washington [State] Department of Fisheries, Fisheries Research Papers, 2 (4) :80-89. December 1966.

222 CHEW, KENNETH K., ALBERT K. SPARKS, STANLEY C. KATKANSKY, and DAVID HUGHES, Preliminary observations on the seasonal-size distribution of Mytilicola orientalis Mori in the Pacific oyster Crass-ostrea gigas (Thunberg) at Humboldt Bay, California and Yaquina Bay, Ore-gon. Proceedings, National Shellfisheries Association, 55, 1964:1-8.

224 BURGNER, ROBERT L., Food production in two lake chains of Southwestern Alaska. Verh. Internat. Verein. Limnol., 16:1036- 1043. December 1966.

225 MATHISEN, OLE A., Some adaptations of sockeye salmon races to limnological fea-tures of Iliamna Lake, Alaska. Verh. In-ternat. Verein. Limnol., 16:1025-1035. December 1966.

228 VAN CLEVE, R., W. F. THOMPSON, Obitu-ary. Journal of the Fisheries Research Board of Canada, 23(11):1790-1793. 1966.

230 KILAMBI, R. V., F. M. UrrER, and A. C DELACY, Differentiation of spawning pop-ulations of the surf smelt Hypomesus pre-tiosus (Girard) by serological methods. Journal of the Marine Biological Associ-ation of India, 7 (2) :364-368. 1965.

231 KATZ, MAX, J. C. WOODEY, C. D. BECKER, P. T. K. Woo, and J. R. ADAMS, Records of Cry ptobia salmositica from sockeye sal-mon from the Fraser River drainage and from the state of Washington. Journal of the Fisheries Research Board of Canada, 23(12) :1965-1966. 1966.

232 PHINNEY, DUANE E., and MICHAEL L. DAHLBERG, The use of adipose fin pig-mentation for distinguishing between ju-venile chinook and coho salmon in Alas-ka. Journal of the Fisheries Research Board of Canada, 24(1) :209-211. 1967.

233 PAULEY, GILBERT B., and ALBERT K. SPARKS, The acute inflamatory reaction in two different tissues of the Pacific oys-ter, Crassostrea gigas. Journal of the Fisheries Research Board of Canada, 23 (12) :1913-1921. 1966.

234 RUDDELL, CRAIG, Hydroxyethyl methacry-late (glycol methacrylate) combined with polyethylene glycol 400 and water; an embedding medium for routine 1-2 mi-cron sectioning. Stain Technology, 42 (3) :119-123. 1967.

235 Koo, TED S. Y., and ANDHI ISARANKURA, Ob-jective studies of scales of Columbia Riv-er chinook salmon, Oncorhynchus tshaw-ytscha (Walbaum) . U.S. Fish and Wild-life Service, Fishery Bulletin 66 (2) :165-180. April 1967.

236 NARVER, DAVID W., A prototype kokanee spawning channel. Progressive Fish-Cul-turist, 28 (3) :183-184. July 1966.

237 BECKER, CLARENCE D., Try panosoma occi-dentalis sp. n. from freshwater teleosts in Washington State. Journal of Protozo-ology. 14 (1) :153-156. 1967.

238 McPHAIL, J. D., Distribution of fresh water fishes in Western Washington. North-west Science, 41(1) :1-11. 1967.

239 PAULIK, G. J., and WILLIAM H. BAYLIFF, A generalized computer program for the Ricker Model of equilibrium yield per recruitment. Journal of the Fisheries Re-search Board of Canada, 24 (2) :249-259. 1967.

240 Washington [State], University of, College of Fisheries, Fisheries Research Institute. Research in Fisheries-1966. 72 p. 1967.

71

241 GUNSTROM, GARY K., The occurrence of a Katadidymus chinook salmon Alevin. Transactions of the American Fisheries Society, 96 (2) :214-216. April 1967.

242 PHINNEY, DUANE E., DENNY M. MILLER, and MICHAEL L. DAHLBERG, Mass-mark-ing young salmonids with fluorescent pig-ment. Transactions of the American Fisheries Society, 96 (2) :157-162. April 1967.

243 MILLER, DENNY M., JERRY A. WETHERALL, and WILLIAM H. LENARZ. A modern ap-proach to the study of estuaries, with specific reference to the Duwamish River, Washington. Transactions of the Thirty-Second North American Wildlife and Natural Resources Conference. p. 165-173. 1967.

244 KILAMBI, RAJ V., and ALLAN C. DELACY, Heterogeneity of surf smelt Hypomesus pretiosus (Girard) in the Sate of Wash-ington, as judged by incidence of larval Anisakis (Nematoda) . Journal of the Fisheries Research Board of Canada, 24 (3):629-633. 1967.

247 CHEW, KENNETH K., and GILBERT B. PAULEY, Experimental infection of oys-ters (Crassostrea gigas) with Thig-motri-chid Ciliates. Journal of Invertebrate Pathology, 9(2):230-234. June 1967.

248 PAULEY, GILBERT B., and ALBERT K. SPARKS, Observations on experimental wound re-pair in the adductor muscle and leydig cells of the oyster Crassostrea gigas. Jour-nal of Invertebrate Pathology, 9 (3) :298-309. September 1967.

249 CHEW, KENNETH K., GREGORY J. TUTMARK, and WARIBOKO Q. B. WEST, Preliminary study on the use of Bergman-Jefferts co-ded tags on crabs. 1966 Proceedings of the National Shellfisheries Association, 57:24-26. June 1967.

250 KATKANSKY, STANLEY C., ALBERT K. SPARKS, and KENNETH CHEW, Distribution and some effects of the endo parasitic cope-pod, Mytilicola orientalis, on the Pacific oyster, Crassostrea gigas, on the Pacific Coast. 1966 Proceedings of the National Shellfisheries Association, 57:50-58. June 1967.

251 OLSON, PAUL R., and SIGURD OLSEN, Mem-brane filtration of freshwater. Nature, 214 (5094) :1217-1218. June 1967.

252 OLSEN, SIGURD, DIPTIMAN CHAKRAVARTI, and PAUL R. OLSON, Water, bottom depo-sits, and zooplankton of Fern Lake, Wash-ington. Limnology and Oceanography, 12 (3) :392-404. July 1967.

253 SMITH, L. S., J. R. BRETT, and J. C. DAVIS, Cardiovascular dynamics in swimming adult sockeye salmon. Journal of the Fisheries Research Board of Canada, 24 (8) :1775-1790. 1967.

256 ROBERSON, KENNETH, An occurrence of chi-nook salmon beach spawning in Lake Washington. Transactions of the Amer-ican Fisheries Society, 96 (4) :423-424. Oc-tober 1967.

257 COLWELL, R. R., and ALBERT K. SPARKS, Properties of Pseudomonas enalis, a ma-rine bacterium pathogenic for the inver-tebrate Crassostrea gigas (Thunberg) . Applied Microbiology, 15 (5) :980-986. September 1967.

259 ROYCE, WILLIAM F., and EDWARD D. HAN-SEN, Food fishery policies in the western United States. Washington Law Review, 43(1):231-267. 1967.

260 ROYCE, WILLIAM F., Fish and fishing. Bul-letin of the Atomic Scientists, 23(7):26-27. September 1967.

261 BECKER, C. DALE, and WAYNE D. BRUNSON. Diphyllobothrium (Cestoda) infections in salmonids from three Washington lakes. Journal of Wildlife Management, 31(4):813-824. 1967.

262 AHMED, MUZAMMIL, and ALBERT K. SPARKS, A preliminary study of chromosomes of two species of oysters (Ostrea lurida and Crassostrea gigas). Journal of the Fisher-ies Research Board of Canada, 24 (10) : 2155-2159. 1967.

264 SALO, ERNEST 0., Study of the effects of logging on pink salmon in Alaska. Pro-ceedings, Society of American Foresters, Seattle, Washington, 1966:59-62. 1967.

267 BECKER, C. DALE, The parasitic fauna of teleosts in six Washington lakes. North-west Science, 41(4) :160-168. 1967.

270 SMITH, LYNWOOD S., The Pacific salmon's long voyage. Pacific Search, 2 (3) :67-22. December 1967.

DUPLICATED REPORTS FOR LIMITED SPECIAL CIRCULATION

Fisheries Research Institute Circulars

Circular No.

67-1 The Green River hatchery, Washington: A historical and statistical review. By Clar-ence D. Becker. January 1, 1967. 3'7p.

67-2 Abundance, distribution, and species com-position of zooplankton in the lakes of the

72

Nushagak District, Alaska, 1961-1965. By Brian F. Waters. January 23, 1967. 27 p.

*67-3 Detailed release data for all United States high seas tagging in 1966. By the high seas tagging staff. March 20, 1967. IBM listing.

67-4 Study of the migration and spawning distri-bution of the runs of chinook and coho in the Green-Duwamish River system in the fall of 1965. By Denny M. Miller and Gary D. Stauffer. April 10, 1967. 21 p.

67-5 Report on age composition and abundance of sockeye salmon sampled from spawning grounds in the Wood River lakes, 1966. By Kenneth Roberson. April 20, 1967. 24 p.

67-6 Thermal studies of Iliamna Lake, 1966. By Richard A. Baxter. April 25, 1967. 15 p.

67-7 Studies of mature sockeye salmon at Chig-nik, 1966. By Michael L. Dahlberg and Duane E. Phinney. April 21, 1967. 41 p.

*67-8 High seas salmon tagging - table of fresh-water and ocean ages of red salmon tagged or saved for examination in 1966. By the high seas tagging staff. May 2, 1967. IBM listing.

*67-9 High seas salmon tagging - 1966 totals of salmon tagged or saved for examination by

*Not available for distribution

location, date, species, age and maturity. By the high seas tagging staff. May 2, 1967. IBM listing.

67-10 Historical data on the sockeye salmon runs to the Nushagak District, Bristol Bay, Alas-ka. Compiled by Billy S. Batts and Ken-neth J. Fischler. May 19, 1967. 63 p.

67-11 Climatological observations, water level and water temperatures, Iliamna Lake, Alaska, 1966. By Tor B. Gunnerod. June 20, 1967. 31 p.

67-12 Fisheries Research Institute presentation at the 29th Annual Canned Salmon Cutting Demonstration and Technical Conference held at the Olympic Hotel, Seattle, March 7, 1967. July 17, 1967. 29 p.

67-13 Timing, escapement distribution, and catch, Kodiak Island salmon, 1964. By Don-ald E. Bevan and Dexter F. La11. August 31, 1967. 60 p.

*6744 Tag returns in 1966 - United States high seas tagging. (Final report of 1966 returns.) By the high seas tagging staff. October 6, 1967. IBM listing [Supersedes Circ. 66-19.]

*6745 Tag returns in 1967 - United States high seas tagging. Received through October 10, 1967. (Preliminary report.) By the high seas tagging staff. October 12, 1967. IBM listing.

LABORATORY OF RADIATION ECOLOGY

UWFL-93 Bikini-Eniwetok Studies, 1964. Part I. Ecological Studies. By A. D. Welander, K. Bonham, L. R. Donaldson, R. F. Palumbo, S. P. Gessel, F. G. Lowman and W. B. Jackson. September 15, 1966. 277 p. Part II. Radiobiological Studies. By A. D. Welander, K. Bonham, R. F. Palumbo, S. P. Gessell, F. G. Lowman, W. B. Jackson, R. McClin, and G. B. Lewis. Spetember 8, 1967. 233 p.

73

ABSTRACTS OF Ph.D. THESES

A STUDY OF DEMERSAL FISHES AND FISHERIES OF THE NORTHEASTERN PACIFIC OCEAN

by

DAYTON LEE ALVERSON

Otter trawling for demersal fishes commenced in the eastern Pacific about 1875 in the waters adjacent to San Francisco, California. For a number of years the fishery remained relatively small, conducted by small boats generally operating out of major popula-tion centers. During World War II, increased protein needs accelerated the growth of trawl fisheries in both the United States and Canada. The fishery, however, remained small in relation to major world trawl fish-eries. In postwar years production of bottomfish-exclusive of Pacific halibut—by United States and Canadian vessels ranged between approximately 100 million and 140 million pounds.

Bottom fisheries conducted by Asian countries in the eastern North Pacific were initiated in 1929 by Japan. In 1959 Soviet vessels entered the eastern Bering Sea and began heavy exploitation of the yel-lowfin flounder. The entrance of the Soviet Union into the eastern Pacific led to a rapid expansion of both Soviet and Japanese fisheries in these waters. By 1960 trawlers were exploiting the waters south of the Aleutians, and by the middle of this decade trawl fisheries expanded throughout the Gulf of Alaska and south to northern California.

This study involves investigation of data collected during a series of exploratory fishing surveys during the years 1940 through 1962. The purpose of this study was to (1) review the development and sizes of demersal fisheries in the northeastern Pacific Ocean; (2) describe the methods and techniques used to har-vest the resources; (3) summarize results of United States exploratory fishing surveys in these waters; (4) investigate the demersal fish communities, and the distribution, relative abundance, and sizes of impor-tant demersal fish stocks inhabiting the continental shelf and slope off North America from Oregon to the Bering Sea; (5) examine •the species complexes and distribution patterns of its dominant elements in relation to known hydrographic features; and (6) consider the magnitude and uses of these resources.

Catch records from 1755 exploratory drags with a standard otter trawl were analyzed to provide infor-mation on average catch rates for individual species, percentage of total drags in which species occurred, percentage each species contributed to the group cate-

gory, percentage contribution of each group to the aggregate fish catch, and percentage that each species constituted in the aggregate groundfish catch.

The flounders formed the most important single group taken by the standard otter trawl on the con-tinental shelf in all regions except the Arctic, and also dominated catches on the continental slope in the Gulf of Alaska and Alaska Peninsula regions. In the Bering Sea the flounder group constituted 80 per cent of the aggregate fish catch, with one species-yellowfin sole—accounting for 53 percent of the floun-der catch. In any one regional depth zone, a relatively few species of flatfish dominated the flounder catch.

There was a pronounced decrease in the number of rockfish species encountered progressing north and west from southern Oregon to Unimak Pass. The Pacific ocean perch (Sebastodes alutus) was by far the most important species encountered, dominating the catch of rockfish in almost all depth zones and regions. Many of the shallower water forms of rock fishes which were relatively abundant in waters south of Cape Spencer were either absent or occurred in only trace quantities in the more northern regions.

Three cod forms (Pacific cod, pollock, and hake) are important constituents of the demersal fish com-munity of the eastern Pacific. The pollock appears to be the dominant roundfish form in the Bering Sea and was also abundant in waters from Unimak Pass to southeastern Alaska. The true cod co-exists in this same geographic range, but appears to reach its max-imum density in somewhat shallower waters than does pollock. Pacific hake is the dominant roundfish in the more southern part of the geographic range in-vestigated; that is from southern Oregon to Vancouver Island, B.C.

Of the elasmobranchs, the dogfish shark, ratfish, and a variety of skates were the most important forms taken during exploratory surveys. Dogfish shark is a dominant form in waters off Oregon and Washington and appears to be particularly important in the de-mersal fish complex inside Puget Sound and in the Strait of Juan de Fuca.

Investigation of the distribution and abundance of species by geographic areas as related to environ-mental features showed a marked change in the fauna

74

north and south of the westwind drift which has its eastern terminus in the region of Vancouver Island, B. C. Another marked faunal boundary appeared to be the Aleutian Chain. The author suggests that the current systems are major factors influencing the dis-tribution and kinds of fauna encountered, and that oceanographic features in the Gulf of Alaska may

markedly affect the general productivity of this area. A minimum standing crop of approximately 19

billion pounds in the waters of the eastern Pacific from the Bering Sea to southern Oregon is forecasted. It is expected that the eastern Pacific could produce 3.2 billion pounds of demersal fishes annually, having a value of approximately $150,000,000.

OXIDATIVE METABOLISM OF NONPROTEIN NITROGEN COMPONENTS BY FISH. SPOILAGE BACTERIA AND THEIR PHYSIOLOGY

OF PSYCHROTROPHIC GROWTH DURING STORAGE OF FISH (ENGLISH SOLE)

by

DAVID BANNERJEE

The microflora of English sole during spoilage was shown to be dominated by Pseudomonas species which increased from less than 30 per cent in the initial flora to over 90 per cent as spoilage proceeded. Pseudomo-nas were the principal organisms invading the fish flesh after 9 days storage in ice.

Dominance of the Pseudomonas was shown to be due to their rapid growth at ice temperatures and their efficient utilization of the N-containing sub-stances in the water soluble fraction of fish tissue. Nonprotein nitrogen material (NPN) such as amino acids and creatine were oxidized by these organisms yielding ammonia and other substances characteristic of spoiling fish. All amino acids tested except lysine were oxidized rapidly, lysine was oxidized slowly and incompletely. Taurine was not oxidized. Very active and complete oxidation of creatine and lysine was shown by cells induced by culture in media containing high concentration of these substances.

A detailed study of the oxidation of alanine was carried out since this amino acid was used preferen-tially by the spoilage bacteria. The bacteria isolated from spoiling fish could be classified into three groups on the basis of the rate of alanine oxidation: viz., Group 1, 1.26 p litre 02 uptake/min., Group 2, 0.26 p,

litre 02 uptake/min. and Group 3, 0.09 p, litre 02 up-take/min. Organisms from actively spoiling fish (9 and 16 days ice storage) all fell into Group 1. Groups 1 and 2 consisted entirely of Pseudomonas species, all other bacteria isolated from fish fell into Group 3. The bacteria invading the flesh of the fish all fell into Group 1. It was postulated that the rapid rate of amino substrate oxidation by these Pseudomonas and

their ability to adapt for effective utilization of crea-tine and possible lysine was the main reason for their effectiveness as spoilage bacteria in fish. Group 1 bacteria were shown to produce greater amounts of volatile base (an indicator of fish spoilage) in fish tissue preparations than Group 2 or Group 3 bacteria. However, proteinase production by proteolytic Group 1 Pseudomonas was inhibited at ice storage temper-atures and this provides a partial explanation of the small amount of proteolysis seen in spoiling fish.

The spoilage Pseudomonas were found to be psy-chrotrophic growing over a range from 0 - 35°C. A partial explanation of the mechanism for growth over this wide temperature range was provided by the observation that cells grown at low temperature (eg 8°C) produced an alanine oxidase system which was active at temperatures below 12°C. This appears to be a temperature induced psychrophilic oxidase which was produced in addition to the normal mesophilic oxidase system present in Pseudomonas grown at any temperature in the growth range. The psychrophilic system was not produced by cells grown at about 22°C. It was heat labile and may be inactivated by exposure to 35°C at which temperature the meso-philic system was stable.

Experiments with 2,4 dinitrophenol seemed to rule out a permease system and it was assumed that a psychrophilic isozyme was involved. The general dis-tribution of such inducible systems in psychrotophic bacteria would explain their ability to shift to high activity at low temperature after a suitable lag phase period.

75

THE EFFECTS OF HYDROSTATIC PRESSURE ON THE STRUCTURE OF TERRESTRIAL AND MARINE BACTERIA

by

EDWIN SPENCER BOATMAN

An investigation was carried out to determine the effects of hydrostatic pressure on the in vitro growth, viability and structure of species of marine and terres-trial bacteria.

The marine group of bacteria comprised species of Pseudomonas, V ibrio, Coryne bacteria and Bacillus and were isolated from sediments obtained at a depth of 1700 m. off the coast of Washington. The terres-trial group of bacteria consisted of species of Escher-ichia, Aerobacter and Bacillus.

All experiments were conducted within a pressure range of 15 psi to 10,000 psi and at incubation tem-peratures of either 4°C or 21°C.

In order to inquire into the possible intracellular sites affected by pressure parallel investigations were carried out using acriflavine, because in low concen-trations it appeared to have similar functional and morphological effects on bacteria as pressure.

All of the organisms grew at 21°C at 15 psi in media with salinities of 20 per cent and with the exception of the mesophilic terrestrial organism E. coli all repro-duced at 4°C at this salinity.

Under the influence of increased hydrostatic pres-sure a pronounced variability in cell growth and morphology was found. The level of nutrients used or oxygen concentration did not account for this vari-ability. The highest pressure affording some degree of growth at 21°C was 8000 psi. Of the four organisms that showed growth at this pressure two were marine (Pseudomonas sp. and Bacillus sp.) and two were terrestrial in origin (E. coli and Aerobacter sp.). At 4°C growth rates were considerably reduced at all pres-sures. However, some cultures contained viable cells at 10,000 psi at 21°C or at 4°C after 4 days or 14 days respectively. It was concluded from the growth stud-ies that pressures up to 4,000 psi at 4°C were not un-duly detrimental to the growth of the marine bacteria or to most of the terrestrial bacteria for the time peri-ods instituted.

Structural differences between the organisms were elucidated by both light and electron microscopy. For the latter, a procedure was devised to fix the bacteria during growth under pressure. At pressures of 4,000 psi and higher the Gram-negative organisms as a whole, with the exception of the marine Pseudomonas, grew in the form of long filaments. Upon removal of the stress, most of the filaments reverted to form single cells. Similar results were obtained in the case of cells grown in the presence of acriflavine. Glucose (1 per cent) incorporated into growth media repressed filament formation in the presence of pressure or acri-flavine.

By electron microscopy the filamentous organisms were largely of uniform width and compared to con-trols possessed similar amounts of DNA and RNA. Since cell wall and cytoplasmic membrane growth was unimpaired, it was postulated that cell division was specifically inhibited.

Because the marine Pseudomonas was virtually un-affected in terms of growth and morphology by pres-sure, this resistance may be of significance in view of the abundance of this organism in offshore environ-ments.

Of the Gram-positive group of bacteria (marine and terrestrial) filament formation was less marked but, nevertheless, cell division in all except the marine Bacillus 871 N, appeared affected. Inhibition of cell division appeared related to the trapping of meso-somes between the cell wall and cytoplasmic mem-brane and to other cell wall damage.

On the basis of the acriflavine experiments it is hypothesized that cell division at least in the Gram-negative forms is activated by an episome-like cyto-plasmic DNA fragment which, at certain levels, is rendered inoperative by pressure. In the Gram-positive species the site of action appears to involve the meso-some cell-wall complex. Resistance to pressure or acriflavine may be related to the base composition of the cell genome.

76

THE EFFECTS OF ENDRIN ON THE REPRODUCTION OF A FRESHWATER FISH (Oryzias latipes)

by

HOWARD ERNEST JOHNSON

A series of laboratory experiments were conducted in 1965 and 1966 to determine the effects of endrin, a chlorinated hydrocarbon insecticide, on the repro-ductive success of a freshwater fish, the medaka (Oryz-ias latipes). This species is easily reared in the labora-tory and mature individuals spawn nearly every day when they are provided with proper light and temper-ature conditions. The experiments were designed to determine if exposure to endrin affected spawning be-havior and egg production of adult fish, and the devel-opment and survival of their progeny.

A series of adult exposure studies were conducted in which sexually mature medaka were exposed to different endrin concentrations for periods of 23 to 45 days. Endrin solutions in the test tanks were con-tinuously renewed by a special apparatus. The high-est concentration tested was 1.3 ug/1 and the lowest was was about 0.04 ug/l. Actual concentrations in the test tanks were periodically determined by gas chromatography.

Endrin concentrations of about 0.6 ug/1 or greater were lethal to most adult fish during the exposure periods. Prior to death, acute endrin poisoning in-duced abnormal behavior which disrupted spawning activity and in turn prevented fertilization of the eggs. Food consumption was reduced and emaciated fe-males ceased producing eggs. Fish exposed to endrin concentrations of 0.3 ug/1 or less did not suffer any observable adverse effects, and their egg production was not different from control fish.

Eggs from exposed fish, collected shortly after spawning, were incubated in endrin-free water. No effects on the developing embryos were observed until just prior to or at the time of hatching, when the

progeny of all exposed groups exhibited symptoms of endrin toxicity which ranged from slight behavior changes to severe muscle tetanus. The appearance and severity of these responses in the hatching fry were related to both the concentration and length of time the parent fish had been exposed. These results, sup-ported by gas chromatography analysis of fish and egg tissues, demonstrate the rapid rate at which fish absorb endrin from solution and transfer it to their eggs. The survival of the fry was reduced in propor-tion to the length of time adult fish were exposed.

Fish which survived lethal concentrations recovered rapidly when they were removed from exposure. The number of affected fry produced by fish during post-exposure decreased at a rate inverse to the concentra-tion to which the parent fish had been exposed but all fish eventually produced normal-appearing fry.

Eggs from non-exposed medaka and stickleback were incubated in various concentrations of endrin until just prior to hatching. Fry hatching from these eggs exhibited identical effects as those from exposed parents. Endrin concentrations of 15.0 ug/1 or greater in the incubating solution caused immobilization of the hatching medaka fry. Stickleback eggs incubated in concentrations of 20 and 10 ug/1 accumulated con-centrations of 14.3 and 7.8 mg/kg endrin respectively, and the hatching fry suffered severe muscle tetanus and immobilization; those incubated in 5 and 2 ug/1 accumulated 1.8 and 0.8 mg/kg respectively and the fry suffered slight to marked behavioral changes.

These investigations demonstrate that even very low concentrations of endrin in water may signifi-cantly reduce fish populations by affecting their pro-geny.

77

SOME EFFECTS OF THE PARASITIC COPEPOD, MYTILICOLA ORIENTALIS MORI, ON THREE SPECIES OF BIVALVE

MOLLUSCS IN SELECTED SITES IN WASHINGTON, OREGON, AND CALIFORNIA

by

STANLEY CHARLES KATKANSKY

The endoparasitic copepod, Mytilicola orientalis, was studied from February, 1963 to March, 1965, at experimental stations in Humboldt Bay, California; Yaquina Bay, Oregon; Hood Canal, Oyster and Wil-lapa Bays in Washington. The incidence of and inten-sity of infestation by this parasite was described for three host species (Pacific and native oysters and blue mussels) in all areas. The effect of M. orientalis on the condition, growth and survival of the Pacific oys-ter was investigated.

No short term cyclic variations in the incidence or intensity of infestation of shellfish by M. orientalis were observed in the experimental populations in this study.

Blue mussels in the bed baskets generally exhibited significantly heavier infestation rates than those in float baskets, but these differences were not noted for Pacific and native oysters.

Although no effect on the survival of Pacific oysters that could be attributed to infestations by Mytilicola was noted, the infested oysters exhibited a significantly lower Condition Index. There was little evidence that shell growth was adversely affected.

A high degree of correlation was found between

the incidence and intensity of infestation. Pacific oysters were the most heavily infested host species in this study followed by blue mussels and native oysters. However, when considering the number of parasites per gram of host tissue, the native oysters were the most heavily infested, followed by the blue mussel and Pacific oysters.

The growth pattern of Pacific oysters at Humboldt Bay was continuous with no obvious cessations. A definite growth cessation was noted at Yaquina Bay each year between October and March.

Larval Mytilicola were cultured and nauplius stages were noted 22 hours after the experiment began. Two copeodid stages were noted; the first being evident at 96 hours and the second at 150 hours.

On the basis of the results of the study, M. orien-talis cannot be considered a serious threat to commer-cial shellfish populations on the Pacific Coast. How-ever, other workers have described mortalities of shell-fish attributable to high levels of infestation by Mytili-cola; therefore, it is suggested that any future investi-gations be concerned with the distribution and early life history of the parasite which may be used in its control in the event that problems arise in the future.

AN EVALUATION OF THE BOTTOM FAUNA AND ITS ROLE AS FISH FOOD IN FERN LAKE, WASHINGTON

by

HAROLD EUGENE KLAASSEN

This study is primarily a qualitative and quanti-tative evaluation of the bottom fauna in Fern Lake, and secondarily a description of the role of the bottom fauna in the diet of the fish.

Fern Lake is an unproductive lake located in Kitsap County, Washington. The lake area is 9.65 hectares; its maximum depth, 7.6 meters. The lake is managed for production of steelhead trout.

The lake bottom was surveyed and divided into zones based on the composition of the bottom mate-rial. The zones and their relative areas were: Ooze 47 per cent, Fine Debris 20 per cent, Coarse Debris 23

per cent, Sand 5 per cent, and Gravel, 5 per cent. These zones were sampled by optimum allocation of subsamples.

The organisms form two distinct communities, each occupying half of the bottom area. The ooze was inhabited primarily by large midge (Tendipes sp.) larvae and oligochaetes with smaller amounts of Chao-borus larvae. The other zones were inhabited by a heterogeneous sublittoral community where midge larvae were dominant. Other organisms were present in small amounts; e.g. Sialis larvae, Ephemeroptera nymphs, amphipods, and oligochaetes.

78

Numbers and weights of the organisms in the two bottom communities were distinctly different in addi-tion to their pattern of fluctuation. The weight of midge larvae in the ooze area was much higher than in the other areas and did not form a distinct pattern. Most of these larvae had a two year life cycle. The larvae of the non-ooze zones exhibited an annual pattern with maximum occurring in late winter and early spring and the low during late summer. The Chaoborus larvae were in the ooze area mainly. Their fluctuation was irregular, but showed annual cycles. The oligochaetes were most abundant in the ooze zone, and their fluctuation did not exhibit any dis-cernible pattern.

The dry weight of the average standing crop of total benthic macrofauna was 5.61 g/m2 in the pro-fundal zone and 0.91 g/m2 in the sublittoral zone. The average for the whole lake was 3.26 g/m2.

Insect emergence was determined with submerged emergence traps. Tendipedidae were the most abun-dant collected. Distinctly different emergence pat-terns were found in the ooze and the debris areas. The emergence pattern of the midges in the ooze area had two peaks, one in early spring and another after the fall turnover. The magnitude of the peaks was different between 1963 and 1964. The midge emergence pattern in the debris areas was similar in

1963 and 1964 and exhibited three peaks, one mid-spring, one in July, and one in September.

Chaoborus emerged from May to October and formed two peaks, one in July and the other in Sep-tember.

The annual dry weight of midge emerging per unit area in the different communities was similar, about 0.5 g/m2. The total weight of midges emerging in 1963 and 1964 was 38.2 and 39.9 kg. The total calculated Chaoborus emergence was 3.0 kg for 1963 and 3.4 kg for 1964.

The food habits of the young trout were deter-mined through content analysis of 430 stomachs. A slight shift in diet to larger organisms was detected as the fish grew. The diet showed a definite seasonal change. During spring the predominant food was bottom insects most of which were midge pupae. As these became scarce, the terrestrial insects increased in the diet; by late summer they were the main part of the diet.

The comparison of the Fern Lake benthic biomass with literature data, revealed that the Fern Lake values were higher than most lakes and were exceeded only by more productive lakes. Though the standing crop of bottom fauna was high, the production was low due to a slow rate of turnover of the bottom fauna.

FLUCTUATIONS IN THE LEVELS OF PARALYTIC SHELLFISH IN FOUR SPECIES OF LAMELLIBRANCH MOLLUSCS

NEAR KETCHIKAN, ALASKA, 1963-1965

by

RICHARD ALLAN NEAL

TOXIN

Samples of butter clams, Sazidomus giganteus (De-shayes), little-neck clams, Protothaca staminea (Con-rad) and bay mussels, Mytilus edulis Linnaeus, were taken at regular intervals from five locations near Ketchikan, Alaska between May, 1963 and Febraury, 1965. Samples of Pacific oysters, Crassostrea gigas (Thunberg), were taken from populations held in baskets at four of the stations. Standard extraction and mouse-bioassay methods were used to determine concentrations of paralytic shellfish toxin in the shell-fish. Separate tests of toxicity were run on whole butter clams, butter clam siphons and butter clam bodies.

Water samples were collected at each station along with the shellfish samples, and chemical analyses were made to determine levels of salinity, inorganic silicon,

inorganic nitrogen and inorganic phosphorous. Con-tinuous water temperature recordings were made at four of the sampling sites and local meteorological data were collected by the U.S. Weather Bureau. Samples of the phytoplankton were collected regularly from all stations and analyzed qualitatively and quan-titatively.

Mussels accumulated and released toxin rapidly while little-neck clams and Pacific oysters accumulated only small amounts of the toxin. Butter clams, how-ever, accumulated toxin rapidly but released it very slowly. As a result butter clams were toxic throughout the year and apparently accumulated toxin from year to year, old clams being more toxic than young ones. Marked differences were observed between stations in the levels of toxicity of butter clams, but only

79

slight differences in the toxicities of mussels were noted. The tidal level at which Pacific oysters and mussels were held did not influence the amounts of toxin present.

The periods of uptake of toxin by butter clams did not always coincide with periods of uptake by the other species examined. The uptake of toxin by mus-sels was very closely correlated with the presence of Gonyaulax spp. at three stations. The uptake of toxin by butter clams was not consistently related to the environmental conditions or populations of phyto-plankton observed.

Blooms of G. catenella occurred following diatom blooms under conditions of high solar radiation, low salinity and low nutrient concentrations. Conditions of lowered salinity were caused by heavy run-off due to melting snow.

Toxic butter clams transplanted to an area of low

toxicity lost half of their toxicity during an eight-month period, while clams of low toxicity increased only slightly in toxicity when transplanted to a beach of high toxicity for a similar period. Highly toxic butter clams apparently lost toxin more rapidly than clams of low toxicity. Butter calms released toxin more rapidly at warmer temperatures.

An average of 63 per cent of the toxin present in butter clams is held in the siphon.

Several experiments were conducted to investigate the possibility of reducing the toxicity of shellfish by destruction of the toxin at an intermediate pH or by extracting toxin at a low pH.

It is suggested that butter clams obtain toxin from sources other than the active cells of G. catenella. Possible sources are the bottom sediments, organic debris or encysted cells of G. catenella.

ESTIMATION OF PELAGIC FISH POPULATIONS IN THE WOOD RIVER LAKES, ALASKA, FROM TOW NET CATCHES AND ECHOGRAM MARKS

by

DONALD EUGENE ROGERS

A method was developed for estimating pelagic populations of juvenile sockeye salmon and three-spine stickleback in the Wood River lake system of southwestern Alaska from tow net catches and echo-gram marks.

The fish ranged in length from 25 to 120 mm. They occupied the upper 20 m of the lake at night and tended to concentrate between 0-9 m. The fish in the offshore regions of the lakes occurred as indi-viduals as opposed to schools; however, there was frequent clustering near shore.

The echosounder provided a measure of fish abun-dance below a depth of 3 m. Echogram marks were counted by 3-m depth intervals, and the relationship between catch and marks was calculated from 287 observations at 3-6 m and 117 at 6-9 m. An effective sound beam was estimated, and echogram counts were adjusted to a common sampling unit.

The tow net sampling provided an estimate of fish density in the upper 6 m in water deeper than 20 m. The echosounder provided a means of estimating the availability of fish to the tow net, defined as the percentage of fishable area of total area occupied by

the fish. Vertical availability was estimated from echograms

obtained while towing, and horizontal availability was estimated from transects with the echosounder across the width of the lake. Population size was calculated from catch per unit of effort, volume of water within the tow net sampling region, and availability.

Estimates of population were calculated for six lakes for five years. Populations of sockeye fry were of primary interest. They ranged from .91 to 43.46 million. Approximate 95 per cent confidence intervals on the estimates ranged from +40 per cent to +88 per cent.

An optimum sampling scheme for future years was calculated from variances and total sampling effort in this study. Lake populations of about 10 million could be estimated within approximately +50 per cent with 95 per cent confidence by making 30 tows and 15 transects per lake. This could be accomplished for the lake system with one tow net and two echo-sounders during a three-week period.

Sources of error in the estimates and modification of the method are discussed.

80

STUDIES ON THE ORIGINS AND DEVELOPMENT OF THE TYPICAL FLAVOR AND AROMA OF THAI FISH SAUCE

by

PRASERT SAISITHI

The microbiology and chemistry of Thai fish sauce was investigated in relation to the development of color, flavors and aromas. Total viable counts of bac-teria from fish sauce samples decreased with the in-crease of fermentation time. Aerobic and anaerobic counts at 37°C. were higher on media containing 10 per cent salt than 0.5 per cent or 20 per cent salt. The number of viable bacteria was too low to have signifi-cant role in protein degradation. Furthermore, the bacteria apparently lacked the necessary enzymes to utilize fish muscle protein homogenate. It was con-cluded that protein degradation was due to free prote-olytic enzymes present in fish digestive systems or de-rived from spoilage bacteria normally present on fish prior to salting.

Total volatile acid values of fish sauce samples were higher than the acceptable values for either fresh or salted fish. However, the total volatile base values at one half month and at the end of fermentation pro-cess were considerably lower than the standard accept-able value for fresh fish. The maximum values for both total volatile acids and bases were found at 9-month fermentation time. On milli-equivalent basis, total volatile acids were higher than total volatile bases at one half month and at the end of the fermen-tation process (12 months). The increase in volatile acids at the end of fermentation suggested that the bouquet of Thai fish sauce came from volatile acids. Substances responsible for the typical aroma of Thai fish sauce, separated by steam distillation and identi-fication by paper chromatography, were formic, ace-tic, propionic and isobutyric acids.

Qualitative determination of amino acids from sam-

pies of commercial fish sauce obtained at intervals during the fermentation process revealed no changes in amino acid patterns. The principal amino acids found were lysine, aspartic acid, glutamic acid, gly-cine, alanine, histidine, threonine, valine, leucine or isoleucine and phenylalanine. These amino acids were assumed to be responsible for the flavor of fish sauce. Taurine, B-alanine, 1-methyl histidine, sulfur amino acids (except taurine), aromatic amino acids (except phenylalanine), proline and hydroxy proline which are usually present in fresh fish tissues were not detected in fish sauce samples. The first three amino acids probably disappeared much earlier dur-ing fermentation process through non-enzymic brown-ing reactions whereas the remainders were metabolized by bacteria found in fish sauce samples.

The contribution of Maillard reaction products to the development of color, flavors and aromas of fish sauce was also investigated using model systems con-sisting of individual amino acid in combination with ribose in phosphate buffer pH 6.5. None of the aro-mas obtained from 21 ribose-amino acid systems was similar to that of the Thai fish sauce. Non-enzymic browning was considered to be mainly responsible for the color production in fish sauce. Various aspects of ribose-amino acid browning reactions in relation to the browning intensity were discussed.

Bacteriological studies of the bacteria isolated from fish sauce samples showed that the typical fish sauce aroma was produced by the homofermentative lactic acid bacterium, Pediococcus halo philus. The possible mechanisms by which the bacterium produced vola-tile acids from amino acid catabolism were discussed.

81

FACTORS INITIATING PHYTOPLANKTON BLOOMS AND RESULTING EFFECTS ON DISSOLVED OXYGEN IN

AN ENRICHED ESTUARY

by

EUGENE BRUMMER WELCH

Phytoplankton productivity, standing stock, and related environmental factors were studied during 1964 to 1966 in the Duwamish River estuary, at Se-attle, Washington. The study was prompted by the introduction of the effluent of a secondary sewage treatment plant into the Duwamish River at Renton, Washington, 20.4 km upstream from the mouth. The Renton Treatment Plant began operation in June 1965. The purpose of the study was to understand the factors affecting phytoplankton in the estuary in order to detect and evaluate the possibility of a direct effect of the effluent nutrients on the production of phytoplankton. The indirect effects of phytoplankton blooms on dissolved-oxygen concentrations were also considered because of the possible consequences of reduced oxygen content upon important salmonoid populations which migrate through the estuary.

Ammonia and phosphate concentrations were found to increase significantly downstream from the treatment plant outfall after it began operating. Phy-toplankton blooms, primarily of diatoms, occurred in the lower estuary during August 1965 and 1966, fol-lowing the introduction of effluent. No bloom oc-curred during 1964 before the effluent was introduced, but in August 1963 the surface waters were found to be supersaturated with oxygen which indicated that a bloom had occurred. Nutrients were probably not the primary factor controlling the timing of phyto-plankton blooms because concentrations of phosphor-us and nitrogen were relatively high before the RTP began operating and during periods of low phyto-plankton production. Rather, the consistent occur-rence of blooms coincident with minimum fresh water discharge and tidal exchange conditions during Aug-ust throughout the study period indicate that the timing of blooms is probably controlled primarily by hydrographic factors which determine retention time and stability of the surface-water layer. This has been demonstrated in part by a highly significant correla-tion between gross productivity and the factors which indicate retention time (fresh water discharge) and vertical stability (difference between mean surface and

mean bottom temperatures). The failure of a bloom to develop in 1964 is related to a freshwater discharge during that summer that was much greater than normal. The high discharge in 1964 probably resulted in greater turbulence and mixing, and a shorter reten-tion time of the surface water during that year than is common during the summers when the discharge is less. This conclusion is supported by lower mean surface-water temperatures in 1964. Hydrographic factors are apparently important, because as shown in other estuarine environments by other workers, phytoplankton production increases when the zone of vertical turbulent mixing is not markedly deeper than the compensation depth.

Phytoplankton cells produced in the surface waters sink, contributing oxidizable organic matter to the saline-water wedge. The maximum BOD in this bot-tom-wedge occurs in the same section of the estuary at the same time as the maximum phytoplankton biomass (as indicated by chlorophyll a) and minimum dissolved-oxygen concentrations. Although there are other sources of BOD in the estuary, and minimum discharge and tidal exchange conditions assist in re-ducing dissolved oxygen, the highly significant corre-lation of chlorophyll a with BOD throughout the summer indicates that respiration of live phytoplank-ton and decomposition of dead phytoplankton cells are clearly important contributors of BOD.

Increase in the biomass of blooms caused by in-creased effluent nutrients would probably decrease the dissolved-oxygen co-ncentrations further as a result of the increased BOD of the larger mass of phyto-plankton. This possible effect of effluent nutrients was evaluated by laboratory bioassays and a compar-ison between years of the mean annual biomass in the estuary. A green algal population in vitro in-creased in response to added effluent nutrients. How-ever, the available field data suggest that a 46 percent increase in effluent discharge during 1966, as com-pared to 1965, did not increase the phytoplankton biomass in the estuary significantly.

82