The effect of body size on digestive chemistry and absorption efficiencies of food and...

Journal of Experimental Marine Biology and EcologyŽ .263 2001 185–209

www.elsevier.comrlocaterjembe

The effect of body size on digestive chemistryand absorption efficiencies of food and

sediment-bound organic contaminants in Nereisž /succinea Polychaeta

Michael J. Ahrens), Jonathan Hertz, Elizabeth M. Lamoureux,Glenn R. Lopez, Anne E. McElroy, Bruce J. Brownawell

Marine Sciences Research Center, State UniÕersity of New York, Stony Brook, NY 11794-5000, USA

Received 22 February 2001; received in revised form 25 May 2001; accepted 5 June 2001

Abstract

Ž .We investigated the hypothesis that absorption efficiencies for organic matter OM andŽ .hydrophobic organic contaminants HOC correlate with body size in the deposit feeding

Ž . Ž .polychaete Nereis succinea Frey and Leuckart . Gut passage time GPT in N. succinea isapproximately five to ten times shorter in juveniles than in adults. Since shorter GPT is likely todiminish the efficiency of intestinal digestion and solubilization, one would expect juvenile wormsto have significantly diminished absorption efficiencies compared to adults. To test this hypothe-

Ž . Žsis, we measured absorption efficiencies AE for radioactively labeled phytoplankton Pseudo-. Žnitzschia sp. , sediment OM, and three sediment-bound hydrophobic organic contaminants tetra-

w x w x Ž . w x.chlorobiphenyl TCBP , hexachlorobenzene HCB and benzo a pyrene BaP over a gradient ofbody size spanning 10–110 mm. We furthermore measured gut pH and gut fluid surface tensionŽ .surfactancy in relation to body size, to examine whether small worms might compensate forshorter GPT by having comparatively more aggressive gut conditions. Absorption efficiencieswere measured in pulse-chase feeding experiments over 5–48 h. Live phytoplankton was absorbedwith AEs of 55–95%, while bulk sediment OM was absorbed with AEs of only 5–18%.Sediment-bound TCBP, HCB and BaP were absorbed with AEs of 55–92%. AEs were commonlyhigher in larger worms, and linear and Ivlev-type regressions of AE onto body size explainedmore than 59% of the AE variance in any treatment. AEs for phytoplankton and OM correlatedstrongly with depuration time, i.e. the time until first detection of non-radioactive AchaserB-feces.In contrast, no time dependency of AE was detected for HOCs, albeit over a narrower range of

) Ž .Corresponding author. National Institute of Water and Atmospheric Research NIWA , Gate 10 SilverdaleRoad, P.O. Box 11-115, Hamilton, New Zealand. Tel.: q64-7-856-1730; fax: q64-7-856-0151.

Ž .E-mail address: [email protected] M.J. Ahrens .

0022-0981r01r$ - see front matter q2001 Elsevier Science B.V. All rights reserved.Ž .PII: S0022-0981 01 00305-7

( )M.J. Ahrens et al.rJ. Exp. Mar. Biol. Ecol. 263 2001 185–209186

depuration times. Gut fluid pH of N. succinea ranged between pH 5.8 and 7.7, and was onŽ .average slightly higher closer to seawater pH in larger individuals. Surface tension, measured as

drop contact angle, was greatly diminished relative to seawater in all worms, with greatestdifferences found in large worms. In contrast to AE measurements, only 10–20% of the variancein gut surfactancy and pH data was explained by body size, suggesting that differences in gut

Ž .chemistry pH and surfactancy play a subordinate role in explaining the higher AEs in adultworms. We conclude that gut chemistry is likely to set upper and lower limits on absorptionefficiency of food and sediment-bound HOCs in N. succinea, while body size, and in the case offood uptake, differences in GPT, probably account for most of the variance within this range.q 2001 Elsevier Science B.V. All rights reserved.

Keywords: Absorption; Deposit feeding; Digestion; Nereis succinea; Nutrition; Organic contaminants; Poly-chaetes

1. Introduction

Studies on the digestive physiology of deposit feeding organisms have establishedŽ . Žthat gut passage time GPT tends to increase with body size Cammen, 1980a; Forbes

.and Lopez, 1987; Penry and Jumars, 1990 . It has furthermore been shown, boththeoretically and experimentally, that longer GPT tends to increase absorption efficiencyŽCalow, 1975a,b, 1977; Klump et al., 1987; Penry and Jumars, 1987; Kofoed et al.,

.1989; Wang and Fisher, 1996; Penry and Weston, 1998 . The intuitive explanation forŽ .this is that digestion and absorption are time-dependent processes Kofoed et al., 1989

that are slow in relation to GPT so that longer digestion time should result in greaterŽ .hydrolysis andror solubilization, and subsequently greater absorption efficiencies AE

Ž .of ingested food. Following this reasoning and assuming similar digestive physiology ,one may therefore expect juvenile animals, which possess shorter GPT than adults, tohave lower AEs if fed the same food. The same prediction should apply to otheringested substances whose digestion and absorption are time-dependent, e.g. hydropho-bic contaminants. Despite its simplicity, this hypothesis has not been rigorously testedfor animals with simple one-compartmental guts, such as the majority of deposit feedingpolychaetes.

Models of optimal foraging predict that organisms adjust their feeding strategy so asŽ .to maximize the rate of food absorption Sibly, 1981; Dade et al., 1990 . Organisms

have two options to maximize gain: either by modifying the rate of delivery—i.e. byhigh ingestion rates andror particle selection—or by modifying the intensity of thedigestive environment. While numerous theoretical and experimental studies havedemonstrated the role of ingestive processes in controlling absorption in deposit feedersŽ .Penry and Jumars, 1987; Lopez and Levinton, 1987 , the importance of digestive

Žchemistry as a covariate has only been addressed recently Mayer et al., 1997; Weston.and Mayer, 1998a,b; Bock and Mayer, 1999 and is the subject of this paper.

In addition to food, deposit feeders often ingest considerable amounts of primarilynon-nutritive, yet nonetheless digestively accessible material. Prominent examples are

Ž .hydrophobic organic contaminants HOCs , such as polycyclic aromatic hydrocarbonsŽ . Ž .PAHs , polychlorinated biphenyls PCBs and other chlorinated compounds. Benthic

( )M.J. Ahrens et al.rJ. Exp. Mar. Biol. Ecol. 263 2001 185–209 187

deposit feeders are particularly prone to high dietary HOC exposure because of theirhigh ingestion rates, and because concentrations of HOCs tend to be greatly elevated insediments in relation to seawater. The extent to which deposit feeding behavior increases

Ž .the bioaccumulation of organic chemicals has been reviewed by Leppanen 1995 .¨One of the great uncertainties in models of food and contaminant uptake is the

efficiency with which food or contaminants are absorbed during gut passage. AbsorptionŽ .efficiency AE , or bioavailability, depends on a multitude of abiotic and biotic factors,

Žranging from the chemical characteristics of the toxicant e.g. hydrophobicity,. Žhydrogen-bonding capacity and chemical reactivity , properties of the sediment e.g.

organic carbon content, particle size, mineralogy, cation exchange capacity, redox and. ŽpH , to characteristics of the organism’s feeding physiology e.g. ingestion rate, gut

passage time and digestive chemistry; Leppanen, 1995; Standley, 1997; Mayer et al.,¨. Ž1997 . Furthermore, AEs vary not only among species but also intraspecifically Weston

and Mayer, 1998a,b; Forbes and Forbes, 1997; Kukkonen and Landrum, 1995; Lydy and.Landrum, 1993; Lee et al., 1990; but see Calow, 1975a . As a consequence, bioener-

getic–kinetic models commonly assume a range of AEs for a given animal species andŽ .substance e.g. Thomann et al., 1995; Wang et al., 1998 . The variance in AE

measurements can be greatly diminished by pre-selecting animals of uniform age, sizeŽ .and condition, as shown for trace metal uptake in mussels Wang and Fisher, 1997 .

While this is practical for comparisons and monitoring purposes, it does so at the cost ofgeneralizability and a mechanistic understanding of the digestive and absorptive process.We currently lack theory and data to help explain the high degree of intraspecificvariability observed in AE measurements.

If we assume that intestinal absorption of food and HOCs are kinetically controlled,although the type of relationship and time scale may differ, AEs should be a function of

Ž .gut passage time GPT and digestive intensity. Evidence demonstrating the strongŽ .influence of GPT on absorption has been presented by Klump et al. 1987 , Weston

Ž . Ž .1990 and Penry and Weston 1998 , who observed AEs for PAHs and chlorinatedŽ .biphenyls to correlate inversely with weight-specific ingestion or egestion rates. Since

Žingestion rate is inversely proportional to GPT normalized to gut fullness in one-com-.partment, plug-flow guts , this is identical to stating that AEs correlate directly with

ŽGPT it may be noted that gut fullness should formally be expressed as a mass, rather.than a volume, for units to match . Some readers might note that previous research on

Ž .mollusks and crustaceans has led to contrary conclusions. For example, Calow 1975a,bfound AEs of organic carbon in freshwater gastropods to be invariant to body sizeŽ .although AEs did increase with GPT . This finding may be attributable to the generallymore complicated digestive systems of gastropods: many mollusks have two-compart-ment guts, in which material of higher food value is selectively retained and processedby the digestive glands, resulting in long GPTs of selected components even for smallanimals. The present paper focuses on a polychaete species with a one-compartment gut,in which sediment particle processing may be assumed to be plug-flow.

We chose to work with Nereis succinea, a cosmopolitan deposit feeding polychaeteŽ .of temperate salt marshes Fauchald and Jumars, 1979 , and one of the dominant

intertidal deposit feeding species in Long Island Sound. GPT of N. succinea rangesbetween 30 min for 10 mm juveniles and more than 6 h for 100 mm adults in the


Ž . Žlaboratory Ts24 8C . A similar range in GPT has been observed in the field 45–200.min at 24 8C; recalculated from Cammen, 1980b . In addition to GPT, it is possible that

digestive intensity and selectivity could vary between juvenile and adult worms. Largedeposit feeding polychaetes and holothuroids have high digestive enzyme activities and

Ž .surfactant concentrations Mayer et al., 1997 , which have been shown to facilitatesolubilization and subsequent absorption of sediment-bound polynuclear aromatic hydro-

Ž . Ž . Žcarbons PAHs , polychlorinated biphenyls PCBs and other HOCs Mayer et al., 1996;.Weston and Mayer, 1998a,b; Penry and Weston, 1998; Ahrens et al., 2001 . No

comparable data currently exists for very small deposit feeders, such as juveniles. Theunexplained intraspecific variability in absorption efficiencies could, thus, in part be dueto differences in digestive intensity.

Another digestive variable of great physiological importance is pH. Gut pH of mostŽ .deposit feeders is commonly slightly acidic Ahrens and Lopez, in press , yet small

Žvariations in pH can greatly affect enzyme activity and the relative sorption partition-. Ž .ing of HOCs to dissolved organic carbon DOC , ultimately affecting bioavailability

Ž .Standley, 1997; Jota and Hassett, 1991 . Possible changes of gut pH over ontogenycould, thus, alter the bioavailability of ingested food and HOCs.

The goal of this study was to compare absorption efficiencies for food carbon andsediment-bound HOCs between large and small N. succinea, to test the hypothesis thatbody size is a crucial determinant of absorption efficiencies within a species. Wehypothesized that AEs should differ between adult and juvenile worms, and reflect

Žbody-size dependent differences in GPT and gut fluid chemical properties i.e. surface.tension and pH . The prevailing route for uptake of organic carbon, trace metals and

Žorganic contaminants in N. succinea is via ingested sediment Cammen, 1980c; Wang et.al., 1999; Fowler et al., 1978; Rubinstein et al., 1983 . Due to its high ingestion rates

Žadult worms of 6 mg dry weight ingest about 20 mg of sediment per day at 15 8C;. Ž y2Cammen, 1980a and its high abundance 1500 individuals m in Flax Pond, personal

.observation , N. succinea may play an influential role in the remobilization and trophictransfer of surficial organic matter and sediment-associated HOCs in coastal ecosystems.

2. Materials and methods

2.1. Animal collection

Ž .Juvenile and adult N. succinea body length 10–110 mm were collected from FlaxŽ .Pond, a temperate salt marsh bordering on Long Island Sound, NY USA , on several

Ž .occasions during July–December 1998. Small individuals -25 mm length wereŽstrained from sediments using a 2-mm sieve, while larger organisms 25–110 mm

.length were collected by hand and shovel. Following collection, worms were promptlytransferred to the laboratory and placed into individual containers to prevent aggressiveinteractions. Adult worms were maintained in customized feeding chambers, constructedfrom two 100-ml polypropylene vials connected by a U-shaped piece of Tygon tubingŽ .i.d. 3.2 mm , and a second piece of Tygon tubing serving to equilibrate water level

Ž .differences Wang et al., 1999; Ahrens et al., 2001 . Juvenile worms were maintained in


Ž .pieces of glass capillary tubing VitroCom , placed in individual chambers of 12-wellŽ .culture plates Falcon, B 2.2 cm, volume is approximately 3 ml per well filled with

Žseawater. Worms were acclimated to experimental conditions Ss28 ppt, Ts24 8C,.approximately 12:12 darkrlight cycle for several days, while being fed on surface

sediment.

2.2. Sediment collection

Sediment for AE experiments was collected during the winters of 1997 and 1998from the same location as worms. Sediment was collected at low tide, using polypropy-lene core tubes to retain the loose surface layer. A 0.5-cm thick layer was carefullyscooped off the surface and rinsed through a 300-mm screen using seawater, to remove

Ž .macrofauna and coarse debris pebbles and Spartina alterniflora stalks . Only theŽ .-300 mm fraction was saved for ensuing experiments and refrigerated q1 8C until

Ž .use. Sediment bulk characteristics grain size and C and N content were determined forone sediment batch, as summarized in Table 1. We assume that differences among

Ž .sediment batches were negligible; Montlucon 1997 , who conducted a 20-monthsedimentological survey of Flax Pond from 1995 to 1997 at the same location assampled by us, found no significant seasonally related variability in sediment grain size,carbon content, or surface area. His measurements revealed a general predominance ofsilt- and clay-sized particles and an organic carbon content of 2.1–3.1%, similar to our

Ž .findings. Surface area, not determined by us but measured by Montlucon 1997 , ranged2 Ž .between 6.8 and 10.0 m rg see also Yamamoto and Lopez, 1985 . Water content of

surface sediment was approximately 52%, based on our own measurements.

2.3. Absorption efficiency experiments

We used radiotracer methodology to determine absorption efficiencies of 14C-labeledphytoplankton, 14C-formaldehyde-labeled sedimentary organic matter and three hy-

Ž14 X X 14drophobic organic contaminants C-2,2 ,4,4 tetrachlorobiphenyl, C-hexachloroben-3 Ž . .zene, and H-benzo a pyrene .

2.3.1. Algal food labelingŽ .The absorption of fresh organic matter OM was determined by feeding worms

cultures of a uniformly 14C-labeled planktonic diatom, Pseudonitzschia sp., native to

Table 1ŽFlax Pond sediment characteristics Org Csorganic carbon, Nsnitrogen, layersdepth in sediment core,

.n.d.snot determined

Layer Org C N Sand Silt Clay Surface area Reference2Ž . Ž . Ž . Ž . Ž . Ž .% % % % % m rg

Ž .Surface 0–0.5 cm 2.6 0.27 34 55 11 n.d. this studyŽ .Deep 10–10.5 cm 3.0 0.23 59 34 7 n.d. this study

Ž . Ž .Yearly range 0–10 cm 2.1–3.1 n.d. 25–44 41–61 11–24 6.8–10.0 Montlucon 1997


Ž .Flax Pond courtesy of I. Stupakoff . Algae were cultured in fr2 medium made of 300ml-0.2 mm filtered and autoclaved Flax Pond seawater at 15 8C on a 14:10 lightrdark

14 Žcycle. 0.370 MBq of C Irvine Radiochemicals, Irvine, CA; specific activity, 321.9. 14MBqrmmol was added as NaH CO , and pH was adjusted to pH 8.0. Algae were3

grown for 5 days and harvested at a cell density of 4=104 cellsrml, at which 83% ofthe added 14C activity was associated with algal cells. Algae were concentrated and

Žrinsed by centrifugation 10,000=g for 5 min; and rinsed five times with 40 ml.seawater .

2.3.2. Sediment organic matter labelingŽ . 14Sediment organic matter OM was non-specifically radiolabeled with C-formal-

Ž .dehyde, following Lopez and Crenshaw 1982 . Five milliliter of settled surfacesediment were centrifuged at 1600=g for 6 min, after which overlying water was

Ž .replaced with 30 ml of 30% NaCl solution by weight , to reversibly inhibit microbialŽ . 14 Žactivity Lopez and Elmgren, 1989 . A total of 0.366 MBq of C formaldehyde NEN,

.Boston, MA; specific activity 1.9 MBqrmmol were added to the vial and shakenŽvigorously, resulting in a spike of approximately 0.073 MBqrml sediment 0.15

.MBqrg dry weight . This addition corresponded to a maximum calculated formal-Ž .dehyde-sediment concentration of approximately 38 nmolrml 79 nmolrg . The sedi-

Ž . Žment was left to equilibrate at room temperature 24 8C for 5 days shaken once per. Ž . Žday after which it was centrifuged 8 min at 1100=g and rinsed five times each

.cycle with 30 ml Flax Pond sea water to remove unbound formaldehyde. Formaldehydeactivity associated with the particulate phase at the end of the labeling period was 12%of the activity originally added, corresponding to a formaldehyde concentration ofapproximately 10 nmolrg. The maximal water concentration resulting from feedingapproximately 100–500 mg of this material to worms was several orders of magnitudelower than the concentration at which formaldehyde exerts toxic effects to marine

Ž .invertebrates Office of Pesticide Programs, 1995 .

2.3.3. Sediment organic contaminant labelingLabeling of sediments with hydrophobic organic contaminants followed the slurry

Ž .method of McElroy and Means 1988 , in which radioactive tracers were first added toglass jars and then adsorbed onto sediments. Compounds used were 14C-2,2X,4,4X

Ž . 14tetrachlorobiphenyl TCBP , specific activity 0.385 MBqrmmol; C-hexachloroben-Ž . 3 Ž . Ž .zene HCB , specific activity 0.392 MBqrmmol; and H-benzo a pyrene BaP , specific

activity 0.330 MBqrmmol.ŽFor TCBP and HCB sediment labeling, two 5-ml batches of settled -300 mm

. 14sieved sediment were spiked with 0.062 MBq of C-TCBP, and 0.044 MBq of14 14 Ž . Ž .C-HCB, to render final C activities of 0.012 TCBP and 0.009 MBqrml HCB ,respectively. This corresponded to sediment concentrations of approximately 32 nmolrmlŽ . Ž .68 nmolrg for TCBP and 22 nmolrml 47 nmolrg for HCB on a volume or dryweight basis, respectively. After spiking, jars were placed on an orbital shaker at room

Ž .temperature 24 8C for 1 day to thoroughly homogenize the labeled sediment.Another 5-ml batch of sediment was dual-labeled with 3H-BaP and 14C-TCBP to

simultaneously compare the absorption efficiencies of two different hydrophobic organic


contaminants. TCBP spiking procedure and activity added were identical to describedŽ 14 .above i.e. 0.062 MBq of C-TCBP for 5-ml sediment . Sediment was then additionally

3 3 Ž .spiked with 0.063 MBq of H-BaP, for a H activity of 0.013 MBqrml 0.026 MBqrg .ŽThis corresponded to sediment concentrations of approximately 32 nmolrml 68

. Ž .nmolrg of TCBP and 38 nmolrml 80 nmolrg of BaP. After spiking, sediment washomogenized and left to stand for a minimum of 1 day.

2.3.4. Feeding and depurationFeeding experiments followed a pulse-chase protocol as described in Ahrens et al.

Ž .2001 . To improve feeding success, worms were starved 2 days prior to conductingexperiments. Worms were fed on radiolabeled algae or sediment for approximately 1Ž . Ž .small worms or 3 h large worms , whereupon the radiolabeled sediment was removed,and animals given unlabeled sediment to purge their digestive tracts of all unabsorbedradioactive material. Fecal matter was collected for each animal in separate vials. Toease identification, non-radioactive sediment was optically labeled with fluorescent

Ž . Žpigment Radiant Color, Magruder Color, Elizabeth, NJ . Only healthy i.e. uninjured.and actively feeding and defecating worms were used. Temperature during feeding

experiments was 24 8C.To facilitate feeding and removal of radiolabeled food material, food cohesiveness

was augmented in phytoplankton and sediment OM treatments by congealing foodŽparticles with gelatin. For this purpose, a 20% gelatin solution 1 g KNOX unflavored

. Ž . Žgelatin in 5 ml seawater was mixed with sediment or algae in a 1:1 ratio i.e. 1 ml.gelatin solution per 1 ml sediment or algal suspension . The gelatin solution was

Ž .warmed in a water bath just above its melting point approximately 30 8C beforemixing with sediment or algae, and then left to harden in a refrigerator. Gelatinized foodwas cut into small cubes prior to feeding. Gelatin additions were only performed for14C-formaldehyde labeled sediment and 14C-labeled algae, whereas HOC-labeled sedi-ments were fed in loose form to avoid potential gelatin–HOC interaction effects.

ŽWorms were fed by placing a small amount of radiolabeled food less than one gut.filling in front of an animal’s mouth. Worms usually initiated feeding within 1–30 min

Žof food presentation. After approximately 1–3 h of feeding a time period shorter than.the minimum gut residence time in the laboratory , chambers were pipetted clear of all

radiolabeled sediment, and unlabeled, fluorescently tagged sediment was added. SmallŽ .worms -25 mm were transferred to clean culture plates. Worms were then left to

clear their guts of unabsorbed food for 3–48 h, depending on individual performance,and fecal material was removed on occurrence. Since N. succinea possess straight,

Žcylindrical guts with negligible axial mixing of solid gut contents Cammen, 1980b and.personal observation , depuration was considered complete when several strings of

Ž .fluorescent non-radioactive feces were defecated. In order to minimize disturbance,depuration progress was monitored only every few hours. GPT could therefore only be

Žestimated from our irregularly spaced time records of depuration progress i.e. the time.until first appearance of non-radioactive AchaserB feces . We conducted three sets of

absorption efficiency experiments: one experiment determined AEs for phytoplanktonand sediment organic matter, another experiment measured AEs for TCBP and HCB,and the third experiment compared AEs of TCBP and BaP.


2.3.5. Sample preparationUpon appearance of fluorescently labeled feces indicating the complete defecation of

all radiolabeled sediment, worms were removed from their feeding chambers, rinsed,blotted, weighed andror measured, and frozen at y20 8C until further analysis. Tocheck for leaching or excretion of radiotracer into chamber water, 1 ml aliquots weretaken from each chamber at the end of the depuration period and found to containnegligible amounts of radioactivity. No water samples were taken at the end of the

Ž .feeding period, but since feeding times were kept short 1–3 h , indirect uptake ofleachates from the dissolved phase was assumed to be negligible. Feces and worm

Žsamples were solubilized with 1–2 ml of an alkaline tissue solubilizer Solvable,.Packard and digested at 60 8C for at least 4 h. Turbid samples were divided among

Žseveral vials. In preparation for scintillation counting, 10–15 ml cocktail Ultima Gold.XR, Packard were added to each vial. Blanks received 10 ml cocktail and 1 ml

Solvable. Samples were left standing under low light at room temperature for at least 1 hprior to scintillation counting to decrease chemiluminescence.

2.3.6. Counting and calculationsSamples were counted for 300 s on a Wallac LKB 1217 Rackbeta liquid scintillation

Ž3 14 .counter, employing dual-channel H and C counting mode, automatic quenchŽ .correction and background blank subtraction. Samples with less than four times

Ž .background activity i.e. -100 dpm or from obviously non-feeding worms wereŽ .excluded from further analysis. Absorption efficiencies AE were calculated according

Ž .to Eq. 1 , assuming a mass balance,

A Aretained wormAEs100P s100P 1Ž .

A A qAingested worm feces

Ž . 3 14 Ž .in which AE is the absorption efficiency in % , A is the H or C activity in dpmworm3 14 Ž .of the organism tissue, and A is the H or C activity in dpm of the feces. Thefeces

sum of A qA is assumed to approximate the activity ingested, if feces areworm feces

collected quantitatively, and respiration and leaching or excretion into surrounding waterŽ .can be neglected as was the case in our experiments . For a discussion of the merits and

Ž . Ž .drawbacks of the mass balance method, refer to Penry 1998 and Ahrens et al. 2001 .

2.4. Gut chemistry characterization

In addition to AE measurements, we determined gut fluid surfactancy and gut pH aschemical proxies for digestive intensity. We focused on animals with body lengths )25mm, due to the much greater ease of dissection. However, we were able to obtain asmall data set on gut pH for juveniles of 10–25 mm length, by employing a novel in

Ž .vivo microfluorimetric technique Ahrens and Lopez, in press . All measurements wereperformed on animals that had been starved for a minimum of 2 days.

2.4.1. Surfactancy measurementsPrior to dissection, adult worms were anaesthetized in 10% MgCl solution, and their2

length was measured to the nearest mm. Animals were dissected dorsally, taking care


not to rupture the main blood vessel. After placing a small incision into the gut lining,the emerging brownish gut fluid was collected with a glass capillary. Gut fluid wascollected from three different regions of the digestive tract between stomach and rectumŽ .operationally termed fore, mid and hind gut . Only brownish fluid, uncontaminated by

Ž .blood identifiable by a red hue was used for measurements. Surface tension ofŽ .digestive fluids was measured by drop contact angle Mayer et al., 1997 . For this, a

Ž1–2-mL droplet of gut fluid was placed onto a Parafilm American National Can,.Neenah, WI wrapped glass slide, and viewed sideways using a 458 inclined first surface

mirror, projecting toward a dissecting microscope with an attached video camera. Theinner angle between the drop and the hydrophobic Parafilm surface was measured using

Ž .an image analysis program NIH Image . This angle was termed the fluid’s contactŽ .angle. Contact angle values smaller than seawater sapproximately 908 signified

Ž .reduced surface tension and consequently higher surfactancy Fig. 1 . Care was taken toŽ .measure the contact angle immediately i.e. within seconds after placing the drop onto

the Parafilm surface, to minimize evaporation of gut fluid. For each drop, the contactŽ .angle was determined twice on each side of the droplet and averaged. Surfactancy

Ž .measurements were restricted to worms of a dissectible body size i.e. )25 mm only.

2.4.2. Gut pH measurementsGut pH was measured using two techniques, depending on the size of the worm.

Ž . ŽLarger worms )25 mm length were measured using a microelectrode MI-413,.Microelecrodes, tip diameter 1.3 mm , while small worms were measured microfluori-

metrically. For large worms, depending on the volume of gut fluid collected, pH wasŽ .either measured in the collection vial also used for contact angle measurements , or the

microelectrode was directly inserted into the gut perforation and pushed slightly into thegut lumen to immerse its tip. Readings were taken after approximately 20–30 s ofequilibration time. After each reading, the electrode was rinsed in seawater and blotteddry. Each measurement session was preceded by calibration with low ionic strength

Ž .buffers pH 4, 7, 10; VWR Scientific , and electrode performance was checked

Fig. 1. Drop contact angles of seawater and N. succinea gut fluid, showing greatly reduced surface tension forŽ .gut fluid. Dark spots are due to camera oversaturation light reflection .


repeatedly between measurements to assure comparability. The pH readout by theelectrode sometimes jumped erratically, which was assumed to be due to coating of theelectrode tip by the highly DOC-rich gut fluids. Regular rinsing minimized this artifact.

To measure gut pH in worms smaller than 25 mm length, we used an in vivoŽmicrofluorimetric technique, recently developed in our laboratory Ahrens and Lopez, in

. Žpress . Worms were fed fluorescein-labeled yeast cells Saccharomyces cereÕisiae,.Bioparticles, Molecular Probes . The fluorescein served as a pH sensitive indicator

Ž .sensitivity range approximately pH 5–8 . Gut pH was measured in live worms byratioing the fluorescence intensity at 495 and 450 nm excitation wavelength, andcalibrating the ratio to buffers of known pH. In order to minimize quenching effectsfrom gut contents, worms were pre-fed glass beads.

2.5. Statistical procedures

Since absorption efficiencies are expressed as percentages, statistical tests wereŽ . Ž .generally performed after arcsine angular transformation Sokal and Rohlf, 1995 , in

order to normalize the tails of the distribution and remove unwanted dependence of thevariance on the mean. For greater power, comparisons were conducted using parametric

Ž . Ž .statistics e.g. t-test . However, since sample size was small for AE data , caution isadvised when interpreting significance. Significance levels in multiple comparisons wereadjusted to the number of comparisons made, to assure an experiment-wise error rate of

ŽpF0.05. Regressions onto body size were performed using Kaleidagraph Abelbeck.Software .

For allometric comparisons, we used body length as the preferred independentvariable, since this parameter proved to be a considerably more accurate measure ofbody size than wet weight, which tended to fluctuate greatly due to variable amounts ofadhering water. This required transformation of wet weight to body length for a number

Žof worms whose length we did not measure i.e. for the TCBP and BaP absorption.efficiency comparison . To transform weight to length, we used the allometric relation-

Ž Ž ..ship Eq. 2

Ls5.40PW 0.44 ; r 2s0.92 2Ž .Ž .determined for an ns40 samples of worms, in which L signifies body length in mm

Ž .and W is wet weight in mg .

3. Results

3.1. Body size effects on absorption efficiencies

3.1.1.1. Absorption efficiencies for algal and sediment organic matter14 Ž .N. succinea absorbed C-labeled algae with 55–95% efficiency Fig. 2 . Linear

Ž .regression of absorption efficiency AE onto body length explained 88% of the varianceŽ .p-0.01 , if we excluded one uncharacteristically low AE value of 56% for a 53 mm


Ž .Fig. 2. N. succinea absorption efficiencies for Pseudonitzschia sp. phytoplankton PN-algae , sedimentŽ . Ž . Ž . Ž .organic matter Corg , tetrachlorobiphenyl TCBP and benzo a pyrene BaP , plotted against body length

Ž .mm . Regression lines are for linear and Ivlev fits of AE vs. body length. Regression equations andcoefficients are given in the legend next to the corresponding food treatment. One exceptionally low data point

Ž .in the Pseudonitzschia data set 56%, labeled AoutlierB was excluded from regressions. With the exception ofthe Pseudonitzschia absorption data, linear and Ivlev fits plotted very close to each other. Length data for BaP

Ž Ž ..and TCBP were estimated from weight using the allometric equation given in the text Eq. 2 . HCB AE datawere not plotted for lack of accurate individual body-length measurements.

long individual. According to this regression, a 10-mm increase in body length resultedin an absolute increase of AE by approximately 4% within the size range investigated.

Ž .An Ivlev fit i.e. exponential rise to the maximum of the data did not improve fit, butmay be a more appropriate model, considering that AE cannot exceed 100%. However,to compare trends of AE vs. body size among different food types by analysis ofcovariance the linear model was more convenient.

14 Ž .C-labeled sediment organic matter OM was absorbed with AEs of only 5–18%,Ž .i.e. to a much lesser degree than fresh algal organic carbon. Again, smaller worms

generally had lower AEs than adults. Linear regression of AE onto body length againrevealed a significant increase of AE with body size, explaining 62% of the variance,while an Ivlev fit of the data explained 59% of the variance. Employing the linearmodel, a 10-mm increase in body length increased AE by roughly 2%. Nonetheless, the


slopes for Pseudonitzschia phytoplankton and sediment OM absorption were notŽ .significantly different from each other analysis of covariance, p)0.05 .

Respiration and excretion of ingested 14C were assumed to be negligible, since the14C radioactivity determined in chamber water at the end of the depuration period wasminor compared to tissue or feces activity. When recalculating AEs and including the14 ŽC activity of the chamber water as originally assimilated material assuming solubiliza-

14 y.tion of respired CO as dissolved HCO , AEs increased, on average, by 1% for2 314C-labeled algae, and 2% for 14C-labeled organic matter. However, since it is equallypossible that a fraction of the dissolved 14C activity in the chamber water was derived

Ž .from post-ingestive leaching of feces therefore constituting a negative bias , we decidedto ignore chamber water 14C activity in AE calculations altogether.

While we did not determine individual gut passage time in AE experiments, wenoticed that worms that cleared their guts faster usually had lower AEs for both livePseudonitzschia phytoplankton and sediment OM. We therefore used our irregularlyrecorded observational logs of depuration progress, i.e. the time interval until firstdetection of non-radioactive AchaserB feces, as a first estimate of GPT. Fig. 3 shows that

Ž .shorter depuration times corresponding simultaneously to shorter worm length indeed

Ž . Ž .Fig. 3. N. succinea absorption efficiencies for phytoplankton PN-algae and sediment organic matter Corg ,Ž .plotted vs. depuration time i.e. time until first record of non-radioactive AchaserB feces . Each data point

represents one worm. Model fits plotted are linear and Ivlev regressions of AE vs. depuration time ofindividual worms. Regression equations are given in the legend next to the symbol for the respectivetreatment.


correlated with lower AEs for Pseudonitzschia phytoplankton and sediment OM. Oneshould note, however, that worms in these AE experiments tended to retain food forseveral hours longer than they typically do in the field, and that intervals betweenobservations were significantly longer than typical GPT. It is conceivable that wormsincreased GPT in response to the augmented food quality due to the added gelatin inthese treatments. Ivlev fits of AE vs. GPT seemed to reflect the observed trends betterthan linear fits, and were capable of explaining approximately 80% and 64% of thePseudonitzschia and OM AE variance, respectively.

3.1.2. Absorption efficiencies for hydrophobic organic contaminants

Absorption efficiencies for the three hydrophobic organic contaminants studiedŽ .ranged between 55% and 92% for tetrachlorobiphenyl TCBP , between 63% and 92%

Ž . Ž . Žfor hexachlorobenzene HCB , and between 64% and 80% for benzo a pyrene BaP;.Table 2; Fig. 2 . Because of incomplete length data for small worms, AEs for the HCB

Žand TCBP experiments were only compared between two size categories )25 and.-25 mm; Table 2 . AEs for TCBP and HCB were, on average, significantly higher in

Žlarge worms than in small worms p-0.05, one-tailed t-test of arcsine transformed.data . However, body size-related variance was also present within the operationally

Ždefined size classes. When comparing across treatments separately for juveniles and. Ž .adults , TCBP and HCB AEs did not differ significantly t-test; p)0.05 .

A side-by-side comparison of 3H-BaP and 14C-TCBP absorption efficiencies fromŽ .dual-labeled sediment Fig. 2 and Table 2 revealed TCBP to be absorbed with about

Ž .12% higher efficiency than BaP paired t-test, p-0.001 . Furthermore, as in theŽ .previous experiments, AEs increased with increasing body size Fig. 2 , although the

Ždata set is limited to larger worms with body sizes greater than 60 mm as noted above,.length was estimated from weight . The regression slopes of TCBP and BaP AEs vs.

Žbody length were significantly different from zero ps0.01 for TCBP and ps0.018.for BaP and explained 64–70% of the variance. While slopes did not differ significantly

Ž Ž . .between each other, the y-intercepts did analysis of covariance ANCOVA , p-0.01 ,suggesting that TCBP was absorbed with greater efficiency than BaP by N. succinea.

Table 2Ž . ŽAverage absorption efficiencies % of N. succinea for two body size classes )25 mm body length, -25

. 14 14mm body length and five food treatments: C-labeled Pseudonitzschia-phytoplankton, C-formaldehydeŽ . 14 Ž . 14 Ž .labeled sediment organic matter Org C , C-hexachlorbenzene HCB , C-tetrachlorobiphenyl TCBP , and

3 Ž . Ž .H-benzo a pyrene BaPa aŽ .Length mm Pseudonitzschia Org C HCB TCBP TCBP BaP

Large 87"13 13"4 87"7 87"4 81"6 69"8Ž . Ž . Ž . Ž . Ž . Ž . Ž .)25 mm ns8 ns6 ns4 ns4 ns8 ns8Small 64"8 7"1 69"6 74"12 n.d. n.d.Ž . Ž . Ž . Ž . Ž .-25 mm ns7 ns3 ns3 ns5

Ž .Data shown are averages"1 SD. Number of individuals is given in parentheses n.d.snot determined .aResults from dual-labeled sediment.


Similarly, analysis of covariance for the BaP, TCBP, Pseudonitzschia and sediment OMŽ .AE data sets using body length as a covariate revealed no difference in slopes

Ž .ANCOVA, ps0.27 , but significant differences among pooled means. This suggeststhat although different types of organic matter were absorbed with significantly different

Ž .AEs by N. succinea ranging from 5% to 92% similar size-dependent digestiveconstraints seemed to apply, resulting in similar slopes.

Depuration time, chosen as a semi-quantitative proxy for GPT, was a poor predictorof HOC absorption efficiencies, in marked contrast to results from phytoplankton andsediment OM experiments. Instead of increasing with longer depuration time, AEs for

Ž .TCBP and BaP did not reveal any significant time-related trend Fig. 4 . ExploratoryŽanalysis of AE data from other HOC absorption experiments conducted by us results

.not shown also indicated AE to be unaffected by depuration time. The range ofŽ .depuration times in TCBP and BaP treatments 2.5–4.5 h was conspicuously shorter

Žthan in phytoplankton and organic matter feeding experiments 12–45 h; compare Figs..3 and 4 . It is conceivable that AEs for TCBP and BaP would also have shown greater

variation and time-dependence if worms of a wider range of body size and consequentlya wider range of depuration times had been used. However, measured depuration times

Ž . Ž . Ž .Fig. 4. N. succinea absorption efficiencies for tetrachlorobiphenyl TCBP and benzo a pyrene BaP forŽ .dual-labeled sediment, plotted vs. depuration time h of eight individual worms. No significant time-depend-

ency was detected. Depuration time was defined as the time until detection of non-radioactive AchaserB feces.Model fits shown are linear and Ivlev regressions of AE vs. depuration time. The regression equation for eachfit is given in the legend.


for worms in HOC treatments were concordant with typical gut passage times for N.Ž .succinea in the field approximately 1–4 h; Cammen, 1980b , which suggests that

worms in HOC treatments did not behave unusually. It is more probable that worms inphytoplankton and sediment OM experiments took exceptionally long to digest theirfood, perhaps in response to its higher nutritional quality by the added gelatin. The lackof a time-dependency for TCBP and BaP absorption may be due to differences insolubilization rates for food and HOC carbon, and the fact that HOCs, once solubilized,are probably directly absorbed by the gut epithelium, without any further digestive steps.

3.2. Body size effects on gut chemistry

3.2.1. Surfactancy

Gut fluids of N. succinea were amber to mahogany-colored and often of syrup-likeconsistency, making it difficult to collect more than a few microliters of fluid per worm.Contact angles of gut fluids ranged between 258 and 508, and were significantly lower

Ž . Ž . Ž .Fig. 5. Surface tension drop contact angle of N. succinea fore gut stomach fluid vs. body length mm ,Ž .fitted by linear regression equation given in plot, with x signifying body length in mm . Each data point

Ž .represents one worm. The decrease of contact angle with body length was significant p-0.01 .


Ž . Ž .than seawater approximately of 90–958 , or gut fluid diluted by blood )608 . Contactangles were measured in 94 gut fluid samples, for worms of 25–100 mm body length.

Ž .Mean contact angle of all gut fluid samples was 398 SDs6 . No significant differenceŽin contact angles was detectable among the three separate gut regions fore, mid, hind;

t-test, pX40.017; with significance level adjusted for an experiment-wise error rate of

.pF0.05 for three comparisons . Variability of contact angles along the digestive tract ofan individual worm was commonly less than 108. Regressions of contact angle ontobody length, performed separately for the three gut regions, revealed a common trend of

Ž .decreasing contact angles i.e. stronger surfactancy with increasing body length.Ž .However, this trend was only significant in the case of fore gut fluid Fig. 5, p-0.001 ,

where body length explained 26% of the variance. Furthermore, absolute differencesbetween contact angles of large and small worms, if present, were only moderate. Forexample, a 25-mm worm had an average fore gut contact angle of 448, whereas a

Ž .100-mm worm had an angle of 318 averages based on linear regression . When poolingall contact angle data from the three gut regions, a significant correlation with body

Ž .length p-0.001 was still detectable, although it explained only 13% of the varianceof the combined data set.

Ž .Fig. 6. Fore and hind gut pH of N. succinea vs. body length mm . Linear regressions of pH onto body lengthŽ .were significant for both gut regions p-0.01 . No differences in slopes or intercept were detectable by

ANCOVA. Regression equations for the fore and hind gut data are given at the bottom of the figure. Mid gutŽ .pH was not significantly affected by body size data not shown .


3.2.2. Gut pH

Gut pH of N. succinea was close to neutrality, ranging between pH 5.8 and 7.7. Of234 pH measurements taken, 16 values were obtained fluorimetrically and 218 by

Ž .microelectrode. Overall mean gut pH was pH 6.75 SDs0.35 , and pH variations alongan individual’s digestive tract were 0.3 pH units on average. Multiple paired t-tests

Žamong the three gut regions revealed mid gut pH to be significantly lower pH 6.67; SD. Ž . Ž .0.38 than fore gut pH pH 6.80; SD 0.36 , and hind gut pH pH 6.79; SD 0.31 . Fore

Ž Xand hind gut pH did not differ significantly between each other paired t-test, p )0.017;with significance level adjusted for an experiment-wise error rate of pF0.05 for three

.comparisons . This finding may be indicative of slightly greater water exchange betweenthe anterior and posterior gut regions with the outside environment. Regression of pHvs. body length gave no significant relationship for mid gut pH, while a significant

Ž .relationship was found for both fore and hind gut pH Fig. 6 , explaining 10% and 21%of the variance, respectively. Differences between slopes, tested by analysis of covari-ance, were not significant. Thus, a 25-mm long worm had a predicted fore or hind gutpH of pH 6.6–6.7, while a 100-mm individual had a pH of approximately 7.1. Plotting

Fig. 7. N. succinea gut fluid contact angle vs. pH, for pooled data from fore, mid and hind gut. Note theconspicuous absence of contact angles -358 for pH-6.7.


all pooled pH values vs. all pooled contact angle values indicated a significant inverseŽ .relationship Fig. 7; p-0.01 , but explained only 8% of the variance. We noticed a

Ž .conspicuous absence of very low surface tension values -358 for acidic pH valuesŽ .pH-6.7 .

4. Discussion

A large proportion of the intraspecific variance in absorption efficiencies of N.Ž .succinea was explainable by body size. Absorption efficiencies AE for live phyto-

Ž .plankton, sediment organic matter OM and three hydrophobic organic contaminantsŽ .TCBP, HCB and BaP were on average 10–20% higher in larger worms. While these

Ž .trends, in large, probably reflect differences in gut passage time GPT , chemicalproperties of the digestive tract, represented by gut pH and gut surface tension, alsoco-varied with body size. Average gut pH tended to increase and gut fluid surfacetension tended to decrease with body length, although this size-relationship was rathersubtle for both parameters and explained only about 10–20% of the observed variance.

While linear and Ivlev regressions of AE onto body size explained similar propor-tions of the variance, the Ivlev fit is certainly the more appropriate functional model forfitting absorption efficiency, as it constrains AE to values -100%, and forces AEthrough the origin. Nonetheless, linear regression offered statistical convenience and wasimperative for using analysis of covariance.

Live Pseudonitzschia phytoplankton was absorbed very efficiently by both juvenileŽ .and adult worms AEs55–95% , whereas sediment OM was absorbed with very low

Ž .efficiencies by all worm sizes AEs5–18% . These findings suggest that although AEsŽ .tended to increase with body size and presumably GPT , the overall magnitude of

Žabsorption efficiency i.e. whether organic carbon was absorbed with roughly 10% or. Ž .60% AE was largely body-size and GPT independent. The general similarity of AEs

of juvenile and adult N. succinea suggest an overall similarity in their digestiveenvironment. This is supported by our measurement of uniformly low surface tension,and a rather narrow gut pH range of pH 5.8–7.7. The findings of AEs of similarmagnitude for both large and small worms could signify two things: either the digestiblecarbon fraction is removed rapidly from food, or small worms slowed their GPT tosimilar values as adults during feeding experiments. A third explanation, namely, thatsmall worms have comparatively more aggressive gut chemistries, is not supported byour measurements of pH and surfactancy.

The AEs determined for N. succinea ingesting phytoplankton and bulk organic matterwere similar to values published for other benthic invertebrates. Absorption efficienciesfor bulk sedimentary organic matter by deposit feeders commonly range between 0%

Žand 40% see reviews by Lopez and Levinton, 1987 and Lee et al., 1990; furthermore.Lopez and Elmgren, 1989 and usually average less than 20%. In contrast, AEs for

Ž .microalgae are often as high as 80–90%. For instance, Nielsen and Kofoed 1982Ž .measured AEs of 80–92% for Corophium Õolutator, and Bricelj et al. 1984 measured

AEs of up to 82% for the suspension-feeding bivalve Mercenaria mercenaria ingesting


Ž .Pseudoisochrysis paradoxa. However, Bricelj et al. 1982 also reported much lowerŽ . Ž .AEs 13–28% for three other algal species. Kofoed 1975 determined AEs of 60–71%

Ž .for Hydrobia Õentrosa gastropoda ingesting microalgae.The roughly 10 times higher absorption efficiencies of juveniles for phytoplankton

Ž . Ž .approximately 60% compared to sediment OM 5% should make ingestion ofphytoplankton and phytodetritus greatly more preferable to small worms than sedimen-

Ž .tary OM. In fact, a simple comparison of weight-specific absorption rates sgain vs.Ž .weight-specific metabolic rates scost between a 10-mm and a 100-m individual

Ž Ž .using our AE measurements and ingestion rates IR and respiration rates from.Cammen, 1980b reveals a 10-mm worm to have roughly five times higher metabolic

Ž .requirements, but only a 2.5 times higher absorption rate absorption ratesAEP IRwhen feeding on the same food as adults. If sediment OM constitutes the majority ofavailable carbon, a juvenile N. succinea can greatly improve its energy budget byselecting for algal carbon instead. This could be accomplished by feeding primarily at

Žthe sediment surface. By ingesting half of all carbon as phytoplankton i.e. fresh.phytodetritus , and maintaining its approximately 3.5= higher ingestion rate, a juvenile

N. succinea’s weight-specific absorption would be able to keep up with its metabolicrequirements. Since absorption efficiencies in adult worms differ by only a factor of 6

Ž . Ž .between sediment OM 15% and phytoplankton 90% , selective ingestion of algae,though preferable, has less drastic consequences for maximizing the rate of absorptivegain, and can be more easily achieved simply by having a high ingestion rate. However,ingestion of algae may nonetheless be consequential for the adequate supply of nitrogen,phosphorus or other nutritionally important elements.

Ž w xDespite significant differences in hydrophobicity log K HCB s5.50; logoww x .K TCBP s6.31 , HCB and TCBP were absorbed with very similar efficiencies by N.ow

Ž w x .succinea. BaP, the most hydrophobic of the three HOCs tested log K BaP s6.50 ,ow

was absorbed with slightly lower—but nonetheless high—AEs. Even though AEs for agiven organic compound tended to diminish with decreasing body length, the y-inter-

Ž .cepts of the regression slopes were generally high i.e. )35% , indicative of highbioavailability and an efficiently solubilizing digestive chemical environment in N.succinea.

Ž .Kukkonen and Landrum 1995 measured AEs of 56% for the freshwater amphipodDiporeia spp. and 10–26% for the oligochaete Lumbriculus Õariegatus, for animals fed

Ž .sediment-sorbed BaP. Weston and Mayer 1998a,b , also investigating BaP, determinedAEs of 21% for Abarenicola pacifica and 29–35% for A. brasiliensis. AEs for other

Ž .PAHs fall within a similar range see review by Wang and Fisher, 1999 : fluoranthenewas absorbed with AEs of 36–42% by the gastropod Potamopyrgus antipodarumŽ . ŽForbes and Forbes, 1997 , and up to 56% by the polychaete Capitella sp. 1 Forbes et

.al., 1996 . The polychaetes A. pacifica and A. brasiliensis absorbed phenanthrene withŽAEs of 33% and 12–50%, respectively Penry and Weston, 1998; Weston and Mayer,

. Ž .1998b . Klump et al. 1987 measured AEs of 15–36% for Limnodrilus hoffmeisteriŽ .oligochaeta ingesting the PCB congener hexachlorobiphenyl. The absorption efficien-

Žcies reported by us for N. succinea for BaP, TCBP and HCB ranging between 55% and.92% were, thus, higher than the majority of previously published values. This greater

bioavailability could have a number of explanations. For one, the specific composition


of Flax Pond sediment or the artificial spiking procedure with radioactively labeledcompounds could have resulted in a weaker adsorption of the respective contaminant tothe sediment matrix. Furthermore, the short labeling times used by us may have resultedin exceptionally high bioavailability. When re-feeding the dual-labeled, TCBP and BaP

Žamended sediment to large worms 29 days later i.e. after contaminant-sediment contact. Ž .times of nearly 1 month , we found AEs to have decreased to 66% SDs10 for TCBP

Ž . Ž .and 33% SDs12 for BaP unpublished data . This supports the notion that increasedsediment-contact time renders organic contaminants progressively less bioavailableŽ .Alexander, 2000 . However, continued aging of the same sediment did not appear todecrease bioavailability any further, since AEs for TCBP were still around 73% in an

Ž .experiment conducted 430 days later Ahrens et al., 2001 . Alternatively, selectiveŽ .retention and therefore non-plug flow conditions , or a generally more desorptive

digestive environment by N. succinea could have resulted in the observed greaterbioavailability. Since our AEs for algal and sediment OM fell within the range ofprevious studies, we believe selective retention, incomplete depuration or poor physio-logical state of the organism to be unlikely explanations. Subsequent experiments by usŽ .Ahrens et al., 2001 have confirmed the high AEs for TCBP and HCB for N. succinea,

Žwhile demonstrating lower AEs for other species e.g. 37% for HCB by the polychaete)Pectinaria gouldii , providing further evidence for a strongly desorptive digestive

environment in N. succinea.Recent studies have shown that solubilization and absorption of sediment-bound

ŽHOCs are greatly enhanced in the presence of digestive surfactants Weston and Mayer,.1998a,b; Voparil and Mayer, 2000; Ahrens et al., 2001 . N. succinea has high

concentrations of digestive surfactants, as indicated by low contact angles of digestivefluids. Although the molecular identities of these digestive surfactants are currentlyunknown, desorption efficiencies determined by in vitro gut fluid extractions haveproven to be excellent predictors of HOC absorption efficiencies, and the majority of

Žbioavailable HOC fraction is solubilized within the first minute of contact Weston and.Mayer, 1998b; Ahrens et al., 2001 . If digestive desorption is the primary requisite for

absorption, then a high HOC desorption rate within the first hour of gut fluid contactŽmay explain why juvenile worms can attain very high absorption efficiencies e.g.

.69–74% for TCBP and HCB despite short gut passage times of 30–60 min. Analo-gously, enzymatic hydrolysis of a given food bolus is likely to proceed fastest in theperiod immediately following ingestion, since digestion rates commonly follow

ŽMichaelis–Menten-like hyperbolic kinetics Kofoed et al., 1989; Jumars and Penry,. Ž1989 . Enzyme activities in deposit feeder gut fluids are known to be high Mayer et al.,.1997 and are capable of depleting hyrolyzable substrate concentrations within minutes

Ž .Ahrens and Lopez, in press .Ž .Penry and Weston 1998 observed a decrease of AEs for phenanthrene in A.

pacifica when increasing ingestion rates by manipulating food quality. Since ingestionŽ . Žrate normalized to body weight relates inversely to gut passage time assuming gut

.fullness does not change , this finding supports the conclusion that AEs increase withŽ .longer GPTs. However, Penry and Weston 1998 noted that in five out of six of their

treatments, correlations of AE with ingestion rate were non-significant. This could beexplained if the bioavailable PAH fraction desorbed quickly inside the gut, analogous to


Ž .our findings for PCB and hexachlorobenzene desorption Ahrens et al., 2001 . It wouldŽ . Žbe interesting to reanalyze Penry and Weston’s 1998 data using body length or

.weight as a covariate. If correlations with body length also proved to be non-significant,Ž .then differences in digestive chemistry between smaller and larger worms if present

could be assumed to be unimportant for PAH absorption.While the near neutral conditions in N. succinea’s digestive tract suggest that gut pH

Žhas little influence on HOC bioavailability similar conclusions were also reached by.Wang et al., 1998, 1999 for trace metals , we cannot rule out its importance entirely.

Ž .Many enzymatic reactions are strongly pH-sensitive, and Standley 1997 and Jota andŽ .Hassett 1991 have demonstrated that slight changes in sediment pH can measurably

affect HOC sorption and subsequently bioavailability, presumably by altering the tertiarystructure of humic substances. If we assume that various organic carbon pools in the

Žworm gut compete for HOC sorption e.g. sediment organic carbon, gut surfactant DOC,.and non-surfactant gut DOC, such as proteins , changes in bioavailability may result if

the sorption properties of one or more of the carbon pools were disproportionatelyaltered by pH changes.

In light of the recent evidence that surfactant micelles are one of the chief agents forŽ .HOC solubilization Voparil and Mayer, 2000 , it would have been informative to have

Ž .determined critical micelle dilution CMD as a measure of solubilizing potential. CMDis operationally defined as the threshold at which further dilution of a surfactant-contain-ing solution leads to a complete disaggregation of surfactant micelles into singlemolecules, resulting in a rapid rise in contact angle. Subsequent work by us has shownthat N. succinea’s CMD is reached when its gut fluid is diluted to approximately 10% of

Ž .its original strength Ahrens et al., 2001 , and we have confirmed micelle presence inevery N. succinea gut fluid sample measured so far. In light of these results, the highvariability of contact angles among individuals is surprising. We suggest two explana-

Ž . Ž .tions, a measurement errors and b presence of possibly more than one surfactanttype, whose relative abundance andror properties may vary depending on feedinghistory, body size or some other physiological parameter. Measurement errors associated

Žwith the contact angle procedure were indeed considerable, differing up to 68 usually.2–38 between paired measurements of the same drop. However, pair-averaging greatly

reduced variance between subsequent measurements, and it may be safely assumed thatthe same error applied to all measurements, regardless of the worm size of a particulargut fluid sample. Additional errors could have been incurred from slightly varying dropvolumes, since we dispensed drops directly from the capillary with which they were

Ž .collected we have since upgraded to a micropipette . As an alternative explanation, theŽ .observed differences in surfactant properties could be real: Bock and Mayer 1999 have

presented evidence that gut surfactancy can increase or diminish in response to diet,especially the presencerabsence of sediment. It is therefore conceivable that the

Ž .observed body size dependency of contact angles and perhaps of AEs as well wasrelated to allometric starvation effects. If gut surfactancy in juveniles and adults changedat different rates in response to the 2-day starvation period, this differential change insurfactancy could have lead to differences in desorption efficiency and, ultimately, inabsorption efficiency. Furthermore, our present data indicates that lower pH valuesŽ .pH-6.7 were conspicuously associated with contact angles )358. While this inverse


Ž .pH-surface tension relationship was weak Fig. 7 , it may indicate a slight negativeŽinfluence of gut pH on micelle formation perhaps by slightly altering electronegativity

.within a surfactant molecule , which in turn could result in slightly lower HOCsolubilization and AEs for individuals with lower gut pH.

Since the chemical identity of deposit feeder digestive surfactants is currentlyunknown, one can only speculate about their structure, chemical properties and numberin N. succinea. Is there more than one surface-active compound? We do not knowwhether these compounds are synthesized by the organism itself or constitute byproductsof sediment digestion. Efforts to identify the chemical structure of N. succinea’sdigestive surfactants are currently underway in our laboratory, yet additional work onthe gut fluids of other species and taxa is clearly needed. Knowledge of the compositionand relative size of the various dissolved and particulate organic carbon fractions in N.succinea’s and other deposit feeders’ gut contents would be most useful for formulatingexpanded equilibrium partition models that describe partitioning of organic contaminantsamong more than just two phases. It would be interesting to compare the sorptivestrengths of the organic carbon pools in response to changing physicochemical condi-

Ž .tions e.g. pH or redox .An estimation of the degree to which different digestive parameters contribute to the

observed variance in AEs would be enticing, but cannot be conducted with our limiteddata. Gut chemistry was determined for different worms than those used for AEexperiments, and we have no accurate individual GPT measurements to use as anindependent covariate other than body size. GPT was merely inferred from our recordsof depuration progress, and since the interval between feces collection was longer than10 h in some experiments, our statistical analysis based on these estimates is likely to beweak. While a direct linear relationship between GPT with body length is well

Ž .established for N. succinea Cammen, 1980b , calculating GPT from size and using thisdata to estimate its variance contribution would be tautological. GPT is not the onlydigestive parameter that changes with body size, as demonstrated by gut surfactancy andpH, so that we cannot conclude that lower GPTs are the single explanation for the lowerAEs observed in smaller worms. Particularly for organic contaminant absorption, whichseems to proceed much faster than food uptake, GPT measurements with high temporal

Ž .resolution on the order of minutes would be desirable. A full multivariate experimentaldesign that simultaneously determined body size, gut passage time, digestive parametersŽ .such as pH and surfactancy and absorption efficiency for individual worms would bethe proper experimental approach for estimating the contribution of various digestiveparameters to AE variance.

We conclude that differences in body size strongly affect absorption efficiencies oflive phytoplankton, bulk sediment organic matter, and hydrophobic organic contami-nants by the polychaete N. succinea. While differences in gut passage time appear to bethe most likely explanation for the observed variability in the absorption of foodŽ .phytoplankton and sediment OM , juvenile and adult N. succinea also differ slightly intheir respective digestive chemistry, i.e. pH and surfactancy. Particularly the trend of

Ž .elevated surfactancy s lower surface tension in larger animals may act synergisticallywith prolonged GPT to facilitate desorption and subsequently increased uptake ofsediment-bound organic matter and hydrocarbons.


Acknowledgements

We would like to thank I. Stupakoff for the technical assistance with algal culturingand feeding methodology, and L. Mayer, R. Aller, N. Fisher and two anonymousreviewers for helpful comments to the manuscript. Grant 003-96-A from Hudson RiverFoundation and Grant OCE 9711793 from NSF funded this work. This is contribution

[ ]1223 from the Marine Sciences Research Center. RW

References

Ahrens, M.J., Lopez, G.R., 2001. In vivo characterization of the gut chemistry of small deposit feedingŽ .polychaetes. In: Woodin, S. Ed. , Organism–Sediment Interactions Symposium, Belle Baruch Institute for

Marine Biology and Coastal Research, University of South Carolina, Georgetown, SC, Oct. 23–25, 1998.In press.

Ahrens, M.J., Hertz, J., Lamoureux, E.M., Lopez, G.R., McElroy, A.E., Brownawell, B.J., 2001. The role ofdigestive surfactants in determining bioavailability of sediment-bound hydrophobic organic contaminantsto 2 deposit-feeding polychaetes. Mar. Ecol. Prog. Ser. 212, 145–157.

Alexander, M., 2000. Aging, bioavailability and overestimation of risk from environmental pollutants.Ž .Environ. Sci. Technol. 34 20 , 4259–4265.

Bock, M.J., Mayer, L.M., 1999. Digestive plasticity of the marine benthic omnivore Nereis Õirens. J. Exp.Ž .Mar. Biol. Ecol. 240 1 , 77–92.

Bricelj, V.M., Bass, A.E., Lopez, G.R., 1984. Absorption and gut passage time of microalgae in a suspensionfeeder: an evaluation of the 51Cr:14C twin tracer technique. Mar. Ecol. Prog. Ser. 17, 57–63.

Calow, P., 1975a. The feeding strategies of two freshwater gastropods, Ancylus fluÕiatilis Mull. and¨Ž .Planorbis contortus Linn. Pulmonata in terms of ingestion rates and absorption efficiencies. Oecologia

20, 33–49.Calow, P., 1975b. Defecation strategies of two freshwater gastropods, Ancylus fluÕiatilis Mull. and Planorbis¨

Ž .contortus Linn. Pulmonata with a comparison of field and laboratory estimates of food absorption rate.Oecologia 20, 51–63.

Calow, P., 1977. Ecology, evolution and energetics: a study in metabolic adaptation. Adv. Ecol. Res. 10,1–62.

Cammen, L.M., 1980a. Ingestion rate: an empirical model for aquatic deposit feeders and detritivores.Oecologia 44, 303–310.

Cammen, L.M., 1980b. A method for measuring ingestion rate of deposit feeders and its use with theŽ .polychaete Nereis succinea. Estuaries 3 1 , 55–60.

Cammen, L.M., 1980c. The significance of microbial carbon in the nutrition of the deposit-feeding polychaeteNereis succinea. Mar. Biol. 61, 9–20.

Dade, W.B., Jumars, P.A., Penry, D.L., 1990. Supply-side optimization: maximizing absorptive rates. In:Ž .Hughes, R.N. Ed. , Behavioural Mechanisms of Food Selection. NATO ASI Series, vol. G20. Springer-

Verlag, Berlin, pp. 531–556.Fauchald, K., Jumars, P.A., 1979. The diet of worms: a study of polychaete feeding guilds. Oceanogr. Mar.

Biol. Annu. Rev. 17, 193–284.Forbes, V.E., Forbes, T.L., 1997. Dietary absorption of sediment-bound fluoranthene by a deposit-feeding

Ž .gastropod using the C-14:Cr-51 dual-labeling method. Environ. Toxicol. Chem. 16 5 , 1002–1009.ŽForbes, T.L., Lopez, G.R., 1987. The allometry of deposit feeding in Capitella species I polychaeta:

.capitellidae : the role of temperature and pellet weight in the control of egestion. Biol. Bull. 172, 187–201.Forbes, V.E., Forbes, T.L., Holmer, M.H., 1996. Inducible metabolism of fluoranthene by the opportunistic

polychaete, Capitella sp. I. Mar. Ecol. Prog. Ser. 132, 63–70.Fowler, S.W., Polikarpov, G.G., Elder, D.L., Parsi, P., Villeneuve, J.P., 1978. Polychlorinated biphenyls:


accumulation from contaminated sediments and water by the polychaete Nereis diÕersicolor. Mar. Biol.48, 303–309.

Jota, M.A.T., Hassett, J.P., 1991. Effects of environmental variables on binding of a PCB congener byŽ .dissolved humic substances. Environ. Toxicol. Chem. 10 4 , 483–491.

Jumars, P.A., Penry, D.L., 1989. Digestion theory applied to deposit feeding. In: Lopez, G.R., Taghon, G.,Ž .Levinton, J. Eds. , Ecology of Marine Deposit Feeders. Springer-Verlag, New York, pp. 114–128.

Klump, J.V., Krezoski, J.R., Smith, M.E., Kaster, J.L., 1987. Dual tracer studies of the assimilation of anorganic contaminant from sediments by deposit feeding oligochaetes. Can. J. Fish. Aquat. Sci. 44,1574–1583.

Ž .Kofoed, L.H., 1975. The feeding biology of Hydrobia Õentrosa Montague : I. The assimilation of differentcomponents of food. J. Exp. Mar. Biol. Ecol. 19, 233–241.

Kofoed, L.H., Forbes, V.E., Lopez, G.R., 1989. Time dependent absorption in deposit feeders. In: Lopez,Ž .G.R., Taghon, G., Levinton, J. Eds. , Ecology of Marine Deposit Feeders. Springer-Verlag, New York,

pp. 129–148.Kukkonen, J., Landrum, P.F., 1995. Measuring assimilation efficiencies for sediment-bound PAH and PCB

congeners by benthic organisms. Aquat. Toxicol. 32, 75–92.Lee, H. II, Boese, B.L., Randall, R.C., Pelletier, J., 1990. A method for determining gut uptake efficiencies of

hydrophobic pollutants in a deposit-feeding clam. Environ. Tox. Chem. 9, 215–219.Leppanen, M., 1995. The role of feeding behavior in bioaccumulation of organic chemicals in benthic¨

organisms. Ann. Zool. Fenn. 32, 247–255.Lopez, G.R., Crenshaw, M.A., 1982. Radiolabeling of sedimentary organic matter with 14-C-formaldehyde:

preliminary evaluation of a new technique for use in deposit feeding studies. Mar. Ecol. Prog. Ser. 8,283–289.

Lopez, G.R., Elmgren, R., 1989. Feeding depths and organic absorption for the deposit feeding benthicŽ .amphipods Pontoporeia affinis and Pontoporeia femorata. Limnol. Oceanogr. 34 6 , 982–991.

Lopez, G.R., Levinton, J.S., 1987. Ecology of deposit-feeding animals in marine sediments. Q. Rev. Biol. 62Ž .3 , 235–260.

Ž .Lydy, M.J., Landrum, P.F., 1993. Assimilation efficiency for sediment-sorbed benzo a pyrene by Diporeiaspp. Aquat. Toxicol. 26, 209–224.

Mayer, L.M., Chen, Z., Findlay, R.H., Fang, J., Sampson, S., Self, R.F.L., Jumars, P.A., Quetel, C., Donard,O.F.X., 1996. Bioavailability of sedimentary contaminants subject to deposit-feeder digestion. Environ.

Ž .Sci. Technol. 30 8 , 2641–2645.Mayer, L.M., Schick, L.L., Self, R.F.L., Jumars, P.A., Findlay, R.H., Chen, Z., Sampson, S., 1997. Digestive

environments of benthic macroinvertebrate guts: enzymes, surfactants and dissolved organic matter. J. Mar.Res. 55, 785–812.

McElroy, A.E., Means, J.C., 1988. Factors affecting the bioavailability of hexachlorobiphenyls to benthicŽ .organisms. In: Adams, W.J., Chapman, G.A., Landis, W.G. Eds. , Aquatic Toxicology and Hazard

Assessment, vol. 10. ASTM STP 971, American Society for Testing and Materials, Philadelphia, pp.149–158.

Montlucon, D., 1997. Mechanisms controlling adsorption capacity of Flax Pond Sediments. MS Thesis,Marine Sciences Research Center, State University of New York, Stony Brook, 112 pp.

Nielsen, M.V., Kofoed, L.H., 1982. Selective feeding and epipsammic browsing by the deposit-feedingamphipod Corophium volutator. Mar. Ecol. Prog. Ser. 10, 81–88.

Ž .Office of Pesticide Programs, 1995. Environmental Effects Database EEDB . Environmental Fate and EffectsDivision, US EPA, Washington, DC.

Penry, D.L., 1998. Applications of efficiency measurements in bioaccumulation studies: definitions, clarifica-Ž .tions and a critique of methods. Environ. Toxicol. Chem. 17 8 , 1633–1639.

Penry, D.L., Jumars, P.A., 1987. Modeling animal guts as chemical reactors. Am. Nat. 129, 69–96.Penry, D.L., Jumars, P.A., 1990. Gut architecture, digestive constraints and feeding ecology of deposit-feeding

and carnivorous polychaetes. Oecologia 82, 1–11.Ž .Penry, D.L., Weston, D.P., 1998. Digestive determinants of benzo a pyrene and phenanthrene bioaccumulationŽ .by a deposit-feeding polychaete. Environ. Toxicol. Chem. 17 11 , 2254–2265.

Rubinstein, N.I., Lores, E., Gregory, N.R., 1983. Accumulation of PCBs, mercury and cadmium by Nereis


Õirens, Mercenaria mercenaria and Palaemonetes pugio from contaminated harbor sediments. Aquat.Toxicol. 3, 249–260.

Ž .Sibly, R.M., 1981. Strategies of digestion and defecation. In: Townsend, C.R., Calow, P. Eds. , PhysiologicalEcology: An Evolutionary Approach to Resource Use. Sinauer Associates, Sunderland, pp. 109–139.

Sokal, R.R., Rohlf, F.J., 1995. Biometry. Freeman, NY, 887 pp.Standley, L.J., 1997. Effect of sedimentary organic matter composition on the partitioning and bioavailability

Ž .of dieldrin to the oligochaete Lumbriculus Õariegatus. Environ. Sci. Technol. 31 9 , 2577–2583.Thomann, R.V., Mahony, J.D., Mueller, R., 1995. Steady-state model of biota sediment accumulation factor

for metals in two marine bivalves. Environ. Toxicol. Chem. 14, 1989–1998.Ž .Voparil, I.M., Mayer, L.M., 2000. Dissolution of sedimentary polycyclic aromatic hydrocarbons PAHs into

Ž . Ž .the lugworm’s Arenicola marina digestive fluid. Environ. Sci. Technol. 34 7 , 1221–1228.Wang, W.-X., Fisher, N.S., 1996. Assimilation of trace elements and carbon by the mussel Mytilus edulis.

Ž .Effects of food composition. Limnol. Oceanogr. 4 2 , 197–207.Wang, W.-X., Fisher, N.S., 1997. Modeling the influence of body size on trace element accumulation in the

mussel Mytilus edulis. Mar. Ecol. Prog. Ser. 161, 103–115.Wang, W.-X., Fisher, N.S., 1999. Assimilation efficiencies of chemical contaminants in aquatic invertebrates:

Ž .a synthesis. Environ. Toxicol. Chem. 18 9 , 2034–2045.Wang, W.-X., Stupakoff, I., Gagnon, C., Fisher, N.S., 1998. Bioavailability of inorganic and methylmercury to

a marine deposit-feeding polychaete. Environ. Sci. Technol. 32, 2564–2571.Wang, W.-X., Stupakoff, I., Fisher, N.S., 1999. Bioavailability of dissolved and sediment-bound metals to a

marine deposit-feeding polychaete. Mar. Ecol. Prog. Ser. 178, 281–293.Weston, D.P., 1990. Hydrocarbon bioaccumulation from contaminated sediment by the deposit-feeding

polychaete Abarenicola pacifica. Mar. Biol. 107, 159–169.Weston, D.P., Mayer, L.M., 1998a. In vitro digestive fluid extraction as a measure of the bioavailability of

sediment-associated polycyclic aromatic hydrocarbons: sources of variation and implications for partition-Ž .ing models. Environ. Toxicol. Chem. 17 5 , 820–829.

Weston, D.P., Mayer, L.M., 1998b. Comparison of in vitro digestive fluid extraction and traditional in vivoapproaches as measures of polycyclic aromatic hydrocarbon bioavailability from sediments. Environ.

Ž .Toxicol. Chem. 17 5 , 830–840.Yamamoto, N., Lopez, G., 1985. Bacterial abundance in relation to surface area and organic content in marine

sediments. J. Exp. Mar. Biol. Ecol. 90, 209–220.

The effect of body size on digestive chemistry and absorption efficiencies of food and...

Documents

Transcript of The effect of body size on digestive chemistry and absorption efficiencies of food and...