748

Environmental Toxicology and Chemistry, Vol. 23, No. 3, pp. 748–758, 2004Printed in the USA

0730-7268/04 $12.00 1 .00

SURVEYS OF PLASMA VITELLOGENIN AND INTERSEX IN MALE FLOUNDER(PLATICHTHYS FLESUS) AS MEASURES OF ENDOCRINE DISRUPTION BY

ESTROGENIC CONTAMINATION IN UNITED KINGDOM ESTUARIES: TEMPORALTRENDS, 1996 TO 2001

MARK F. KIRBY,*† YVONNE T. ALLEN,† ROBERT A. DYER,† STEVE W. FEIST,‡ IOANNA KATSIADAKI,‡PETER MATTHIESSEN,§ ALEX P. SCOTT,‡ ANDREW SMITH,† GRANT D. STENTIFORD,‡ JOHN E. THAIN,†

KEVIN V. THOMAS,† LAURA TOLHURST,† and MICHAEL J. WALDOCK‡†Centre for Environment, Fisheries and Aquaculture Science, Burnham Laboratory, Remembrance Avenue, Burnham-on-Crouch,

Essex CM0 8HA, United Kingdom‡Centre for Environment, Fisheries and Aquaculture Science, Weymouth Laboratory, The Nothe, Barrack Road, Weymouth,

Dorset DT4 9UB, United Kingdom§Centre for Ecology and Hydrology, The Ferry House, Far Sawrey, Ambleside, Cumbria LA22 0LP, United Kingdom

(Received 20 March 2003; Accepted 6 August 2003)

Abstract—Plasma vitellogenin (VTG) concentrations and the presence of the ovo-testis (intersex) condition have been recordedin male flounder (Platichthys flesus) captured from several United Kingdom (UK) estuaries since 1996 as part of the endocrinedisruption in the Marine Environment (EDMAR) project and earlier programs. It has been confirmed that plasma VTG concentrationsin male flounder have remained elevated in several UK estuaries (e.g., Tees, Mersey, and Tyne) throughout the period covered bythis study. However, the time-series data indicate that plasma VTG, a measure of environmental estrogen contamination, hasdecreased in fish captured from several estuaries, especially those of the Tyne and Mersey. Shorter time-series data sets from theForth and Clyde estuaries also suggest a decrease in estrogen contamination at these sites. Trends associated with specific pointsources of estrogenic contamination show site-specific patterns. For instance, plasma VTG levels in male flounder captured nearthe Howdon sewage treatment outfall (Tyne) have shown a steady decline to near baseline levels in 2001, while the plasma ofmale fish captured at a site adjacent to the Dabholm Gut discharge in the Tees estuary have shown little evidence of a sustaineddecline. The occurrence of the intersex condition was detected at a low but consistent prevalence through the study period, withthe majority of cases recorded in fish captured from the Tyne and Mersey estuaries. The data set does not allow conclusions to bedrawn about any temporal trends associated with this condition. The significance of the findings and possible mitigating influencesare discussed in terms of the impacts on wild fish and the role of effluent treatment in reducing these.

Keywords—Vitellogenin Platichthys flesus Estrogen Intersex Endocrine disruption

INTRODUCTION

In recent years, evidence of the exposure of fish to endo-crine disrupters has been found in freshwater, estuarine, andmarine ecosystems [1–3] in many parts of the world. Thesystematic focus on fish reflects their importance as a resource,as a pivotal component of the ecosystem, and as sentinel or-ganisms for environmental perturbations in most aquatic en-vironments. While more recent concern has been raised as tothe potential effects of androgenic, antiandrogenic, and anti-estrogenic [4–6] endocrine disrupters (including effects at-tributable to paper pulp mill discharges), the most widespreadevidence of environmental effects is associated with the fem-inization of males through their exposure to substances withestrogenic activity.

The perceived dominance of estrogenic endocrine disrup-tion in the environment is indicative of the many classes ofubiquitous contaminants that have been shown to demonstrateestrogenic activity. Such compounds include the alkylphenols,the phthalates, some pesticides, the polychlorinated biphenyls,certain brominated flame retardants, and, of course, the naturaland synthetic estrogens being released via sewage outfalls asa consequence of normal human excretion and the use of the

* To whom correspondence may be addressed(m.f.kirby@cefas.co.uk).

contraceptive pill and hormone replacement therapies [7]. Anumber of feminizing effects have been noted in male fish asa potential consequence of exposure to these substances. Sucheffects include the production of the female-specific yolk pre-cursor protein vitellogenin (VTG) [2,3,7–10], the presence ofthe ovo-testis intersex condition, and other morphologicalchanges [11]. The most widely used of these feminizationbiomarkers is the presence of VTG in male blood plasma.Vitellogenin has been shown to be a sensitive and robust bio-marker that can be measured relatively easily using enzyme-linked immunosorbent assays (ELISA) or radioimmunoassay(RIA) techniques [3].

In the United Kingdom, the earliest evidence of estrogenicendocrine disruption, in terms of VTG induction, came fromstudies of sewage treatment effluent exposure of rainbow trout(Oncorhynchus mykiss) [12] and the presence of ovo-testis inthe freshwater fish species the roach (Rutilus rutilus) [1]. Laterstudies investigating potential estrogenic effects in estuarineenvironments showed that these biomarkers could also be de-tected in the flounder (Platichthys flesus) [2,3,7,13]. Further-more, the levels of plasma VTG and of intersex in male floun-der were highest in those estuaries most associated with in-dustrial activity or high inputs of domestic sewage effluent[3].

In response to the growing evidence that species inhabiting

VTG and intersex in UK flounder Environ. Toxicol. Chem. 23, 2004 749

Fig. 1. Sampling locations in the United Kingdom estuaries.

estuarine and coastal waters were being exposed to highenough concentrations of estrogenic substances to elicit sig-nificant responses, the endocrine disruption in the Marine En-vironment (EDMAR) program was initiated in 1998. The ED-MAR program had a wide remit to investigate the effects ofendocrine-disrupting chemicals in the United Kingdom (UK)marine environment and to develop new techniques and bio-markers for fish and invertebrates. A significant component ofthe program involved the regular sampling of flounder fromseveral UK estuaries to follow up on the data reported inrelated studies. Concentrations of plasma VTG in male floun-der and the occurrence of ovo-testis were investigated overthe period from 1999 to 2001 and, in conjunction with datafrom 1996 and 1997, formed a short time series that is col-lectively presented here.

MATERIALS AND METHODS

Field surveys

Six estuaries were selected in which to conduct floundersampling surveys. These were a relatively uncontaminated ref-erence estuary, the Alde; three English estuaries, the Mersey,Tees, and Tyne; and two Scottish estuaries, the Clyde andForth. The location of these estuaries and the sampling sites

therein are shown in Figure 1. The frequency and time of yearof each survey (sampling trip) are shown in Table 1.

Surveys were conducted using a 2-m beam trawl fitted withan 8-mm mesh liner towed at 2.5 to 3 knots for approximately10 to 15 min. The catch from each tow was sorted by hand,and flounder were transferred immediately to tanks of flowingestuarine water.

Fish processing/tissue sampling

The length of all fish was measured to the nearest 1 mm.While whole fish, liver, and gonad weights were recorded dur-ing the September–December 2001 surveys, a suitable balancewas not available on board the small survey vessels used dur-ing the period from 1999 to early 2001. In this period, theweights of the fish, their livers, and gonads, could not be taken.As a result, the hepatosomatic index (HSI 5 liver weight/totalweight 3 100) could be determined only for the former sam-ples.

Approximately 1 to 2 ml of blood were taken from thecaudal vein of each fish (unanesthetized) using a heparinizedsyringe, transferred to an Eppendorf tube, and placed on ice.Whole blood samples were centrifuged at 10,000 rpm for 5min, and the plasma supernatant was drawn off, frozen in liquid

750 Environ. Toxicol. Chem. 23, 2004 M.F. Kirby et al.

Table 1. Numbers, mean lengths, and mean plasma vitellogenin (VTG) concentrations of male floundersampled from each site between 1996 and 2001 in United Kingdom estuaries

Estuary Site Date No.

Mean plasmaVTG concn.

(mg/ml)

Mean length,cm

(standard error)

Alde North of Orford 26/11/9628/10/9708/10/9927/03/0019/12/0017/09/01

102310132515

0.20.20.20.80.20.2

24.7 (2.5)25.9 (2.0)17.0 (3.8)18.9 (2.5)24.7 (3.4)23.4 (3.0)

Clyde Rosneath

Pillar Bank

15/03/0028/11/0011/09/0102/10/9730/09/99

225

51916

0.20.20.20.20.2

21.6 (1.8)21.3 (2.5)21.1 (2.8)22.1 (1.2)20.4 (1.3)

Bowling

14/03/0028/11/0010/09/0102/10/9729/09/9914/03/0027/11/0010/09/01

115

181417

52920

15.33.60.20.2

53.9275.9

1.00.2

17.5 (2.5)20.0 (0.9)20.9 (1.5)20.6 (1.1)22.1 (1.4)16.9 (1.9)19.9 (1.8)21.5 (2.1)

Tees Dabholm Gut 14/10/9709/11/9920/03/00

722

4,265.51,318.2

12,987.5

23.7 (5.8)17.0 (3.6)17.1 (3.7)

20/11/0019/11/01

2520

3,355.2597.0

22.6 (1.7)22.3 (2.7)

Tyne St. Anthonys 16/10/9621/10/9710/11/9923/03/0023/11/0013/02/0109/10/01

3101316141213

1.5286.3

26.4166.7

1.866.3

0.2

19.8 (0.9)23.4 (5.0)23.7 (4.5)22.2 (1.6)23.1 (2.6)23.3 (1.6)23.1 (3.5)

Hebburn 16/10/9616/10/9710/11/9922/03/0022/11/0013/02/0109/10/01

99

1412182121

113.0448.3

39.76.96.30.90.5

23.4 (4.9)24.6 (5.2)21.1 (4.3)20.6 (1.9)24.4 (1.3)23.3 (1.8)23.4 (2.9)

Howdon 16/10/9616/10/9711/11/9922/03/0022/11/0012/02/0109/10/01

11151820232324

116.290.3

150.7238.8

2.12.81.9

23.4 (4.6)19.7 (2.7)24.7 (4.7)20.2 (1.7)24.6 (1.3)24.0 (2.8)24.5 (2.4)

Mersey Eastham Sands 02/12/9602/09/9706/09/9901/11/0020/02/0125/09/01

1428

6201718

19,226.21,239.5

307.5194.0

1,268.53.5

26.3 (3.1)26.1 (5.0)18.5 (3.4)23.8 (3.9)22.8 (2.8)20.8 (3.2)

Forth Port Edgar 16/03/0001/12/0028/02/0122/10/01

7151010

55.60.22.00.2

20.6 (2.3)22.6 (3.4)22.6 (1.8)17.9 (2.2)

nitrogen, and stored at 2208C until analysis for VTG usingthe ELISA/RIA methods described in the following.

Following collection of each blood sample, fish were eu-thanized by a blow to the head followed by severing of thespinal column. The testes were carefully removed, placed intohistological cassettes, and fixed in 10% neutral-buffered for-malin for 24 h. The cassettes were gently agitated throughoutthis period to ensure thorough fixation. Tissues were trans-

ferred to industrial methylated spirit for storage prior to his-tological processing.

VTG analysis

Up to the year 2000, flounder plasma VTG levels weremeasured by RIA as described by Allen et al. [2] and Scottand Hylland [14]. After this, plasma VTG has been measuredby ELISA. The equipment, plate-coating buffer, plate-washing

VTG and intersex in UK flounder Environ. Toxicol. Chem. 23, 2004 751

Table 2. Mean length and somatic data for male flounder in UnitedKingdom estuaries sampled between September and November 2001

Estu-aries Sites No.

Length(cm)

Weight(g)

Liverweight

(g) HSIa

Alde North of Orford 15 23.4 143.9 1.47 0.99

Clyde RosneathPillar BankBowling

51820

21.120.921.5

108.4105.9120.9

0.941.261.48

0.871.161.16

Forth Port Edgar 10 17.9 62.5 0.84 1.28

Mersey Eastham Sands 18 20.8 102.6 1.37 1.29

Tees Dabholm Gut 20 22.3 127 — —

Tyne St. AnthonysHebburnHowdon

132124

23.123.424.5

161.4156.4194.2

2.92.883.9

1.841.931.95

a HSI 5 hepatosomatic index.

buffer, assay buffer, anti-rabbit IgG alkaline phosphatase con-jugate, revelation solution, and pipetting procedures were thesame as those described for ELISA measurement of spiggin[15]. A new batch of standard, prepared exactly as describedby Scott and Hylland [14], was introduced in 1999 and anotherin 2001. The concentrated standard solution was made by dis-solving 1 mg VTG in 500 ml distilled water followed by 500ml of plate-coating buffer. For coating of the polystyrene plates,a 100-ml aliquot was added to 100 ml of coating buffer, and100 ml of this dilute solution were then added to all wells ofan appropriate number of plates (one for every 20 samples).The initial standards in the polypropylene plates were madeby diluting the concentrated standard solution 1:20 (50 mg/ml) and 1:100 (10 mg/ml) in assay buffer. The flounder VTGantiserum (the same as that used for RIA) was diluted 1:10,000in assay buffer, and 65 ml were added to all wells of thepolypropylene plates (to give a final volume per well of 200ml). At the end of the assay, VTG concentrations were cal-culated from the dilution of plasma that gave the reading thatwas closest to the middle (and most accurate) part of the stan-dard curve (plotted as a sigmoid).

After the results of this procedure had been calculated, allplasma samples that registered VTG concentrations of ,0.5mg/ml were reassayed with a more sensitive ELISA procedure.This was exactly the same as described previously except thatthe concentration of coating buffer was fivefold lower, theantiserum concentration was sevenfold lower, and the startingconcentration of the two rows of standards were 10 and 2 mg/ml. When run under these conditions, color development ofplasma samples with concentrations of 20 ng/ml fell withinthe steep and most accurate range of the standard curve, whichwas roughly the same sensitivity as the RIA. However, sincemany, but not all, published ELISA procedures have reportedfalse-positive (matrix) effects of plasma at low dilutions (seediscussion in Scott and Hylland [14]), we set the level ofdetection at 0.2 mg/ml, equivalent to a dilution of plasma inbuffer of 1:100 (v/v) below which matrix effects would beunlikely to show.

In order to ensure that no problems occurred in the change-over from one method of assay to another during the courseof the study, 104 plasma samples (with a wide range of VTGconcentrations) were assayed using both RIA and ELISA. Theresults were compared by linear regression. In order to estab-lish whether plasma VTG analysis results prior to 2001 werecomparable to those in 2001, a calibration study was under-taken in February 2001. All but a few of the samples that hadbeen collected from the Mersey from 1996 to 2001 and thathad been stored frozen at 2208C were thawed out and reas-sayed (all at the same time) with the ELISA. The three prep-arations of standard that were used during the course of thestudy were also compared with other in a single RIA in 1997.

Histology

Fixed samples of testis were processed to wax in a vacuuminfiltration processor using standard protocols. Sections werecut at 3 to 5 mm on a rotary microtome, mounted onto glassslides, and stained with haematoxylin and eosin. Stained sec-tions were examined using a Nikon Eclipse E800 light micro-scope, and digital images were obtained using the Luciay

Screen Measurement System (Nikon, Kingston-upon-Thames,Surrey, UK). Assessments were made as to the presence ofthe intersex (ovo-testis) condition.

Statistics

The range of plasma VTG concentrations measured in maleflounder varied enormously during the study, even on occasionin fish captured from the same site on the same sampling date.Concentrations ranged across five orders of magnitude, frombelow the nominal limit of detection of 0.2 mg/ml to .100,000mg/ml. Even when logarithmically transformed, the data didnot follow a Gaussian distribution, and on many occasions alldata were below the limit of detection (yielding zero variance),and therefore comparisons between sites by analysis of vari-ance were not appropriate. However, for the purposes of com-parison with previous studies, mean VTG concentrations foreach sampling time were calculated, and values below the limitof detection (,0.2 mg/ml) were assigned the value of 0.2.

As an alternative, data for each sampling period were sortedinto ranges denoted by differing levels of plasma VTG in-duction, and the proportions of each data set falling into eachrange were compared. The ranges were ,0.2, 0.2 to 100, 100to 1,000, 1,000 to 10,000, and .10,000 mg/ml. To examinefor a significant difference in mean plasma VTG expressionacross sampling periods, binomial logistic models were fittedto values falling within a particular range at each time point.This was done only for the ranges where sufficient readingsfor the models to be estimated fell within the particular rangeover the time periods. Statistical significance values were ob-tained from comparing the reductions in deviance and corre-sponding degrees of freedom from the model containing timeas a covariate as compared to the null model with no covariate.

Where available, size and gonadosomatic and HSI datawere analyzed for significant (p , 0.05) intersite differencesusing single-factor analysis of variance.

RESULTS

Somatic index and length data

The numbers and mean lengths of male flounder sampledper site are summarized in Table 1. Fish selected for analysiswere generally between 20 and 22 cm in length. However,because of the scarcity of fish at some sites, mean lengthsranged from 16.9 to 26.3 cm. A full suite of somatic indexdata could be obtained only for the final set of surveys towardthe end of 2001. Even then, the immature nature of the malegonads meant that the gonadosomatic index was unobtainable.Hepatosomatic indices and other data from these final surveys

752 Environ. Toxicol. Chem. 23, 2004 M.F. Kirby et al.

Fig. 2. Comparison of vitellogenin concentrations (mg/ml) in the sameplasma samples as measured by radioimmunoassay (RIA) and en-zyme-linked immunosorbent assays (ELISA).

Fig. 3. Radiolabel displacement curves (within a single assay) of thethree batches of standard used over the course of the present study:m 5 1997 to 1999; m 5 1999 to 2001 v 5 2001 on.

Table 3. Assay sensitivity comparison data

Yearset No.

Mean vitellogenin(mg/ml)

OriginalReassay

2001 Ratio

199619972000

59114

32

28,7782,0671,572

18,3561,5491,519

0.60.71

are shown in Table 2. For most sites, HSIs were significantly(p , 0.05) greater than at the Alde, except for Rosneath onthe Clyde. No apparent association between HSI and VTGconcentration was observed.

Plasma VTG data

In the 104 plasma samples that were measured by bothELISA and RIA, VTG concentrations were highly comparable(Fig. 2). The correlation coefficient (r2) was 0.94, the slopewas 0.99, and the intercept was 46 mg/ml. When all plasmasamples from the Mersey estuary were thawed out and reas-sayed by ELISA in 2001, it was found that average plasmaconcentrations in the 1996 and 1997 samples were 60 and70%, respectively, of those recorded in the original assays,while those for the year 2000 were the same (Table 3). Thethree batches of standard used during the course of the studygave displacement curves that were not overtly different fromeach other (Fig. 3).

Mean plasma VTG concentrations for all sampling sitesand times are shown in Table 1. Trends in mean plasma VTGconcentration for male flounder at certain estuarine sites be-tween 1996 and 2001 are illustrated in Figure 4.

Evidence for year-by-year changes in average VTGconcentrations

The results from the statistical analyses are summarized inTable 4. Most of the changes in plasma VTG concentrationover time are unlikely to have happened by chance as the pvalues are generally low. Confirming that differences occurredover time cannot differentiate between clear temporal trends

and more general fluctuations in the data. In order to expandfurther on the statistical evidence of intersampling differences,we have looked at the data for each station in more detail andcommented appropriately.

Alde

Plasma VTG concentrations in male flounder from the Alderemained at consistently low levels (,0.2 mg/ml) since thetime series was initiated in 1996. An exception to this wasseen in one male flounder captured during the March 2000sampling trip that showed an elevated but low level of VTGinduction (8.2 mg/ml).

Mersey

Plasma VTG concentrations from male flounder capturedat Eastham Sands on the Mersey showed a precipitate declinebetween 1996 and 1997 and a slower decline thereafter. In1996, mean concentrations were the highest ever detected inmale flounder from a UK estuary (.104 mg/ml) with nearly60% of males showing very high levels of induction. However,by September 2001, mean plasma VTG levels had reduced to,10 mg/ml. Although this reduction is a substantial drop, theplasma VTG levels in males remain significantly elevated, andin the early 2001 round of surveys, this estuary had the second-highest plasma VTG concentrations. With this said, it is per-haps noteworthy that 70% of the male flounder sampled inSeptember 2001 showed no evidence of elevated plasma VTG.

Tyne

The three sampling sites visited on the Tyne estuary haveshown different trend characteristics over the time course ofthis study. In particular, the site farthest upstream (St. An-thonys) has been the most inconsistent. While the mean plasmaVTG concentration in males captured on the most recent sam-pling occasion (0.24 mg/ml), in September 2001, was muchlower than that reported previously and approximately 80%of the male specimens captured showed no induction, it isdifficult to discern any unequivocal downward trend in thedata. Conversely, plasma VTG data from the Hebburn siteshowed a very clear and consistent downward trend from apeak of .102 mg/ml in 1997 to ,10 mg/ml in September 2001.In 1996 at the Hebburn site, all male fish showed evidence ofVTG induction, with over 30% of specimens moderately in-

VTG and intersex in UK flounder Environ. Toxicol. Chem. 23, 2004 753

Fig. 4. Proportions of plasma vitellogenin concentrations at a range of induction levels in male flounder from United Kingdom estuaries, 1996–2001 trends. Estuaries/sites: (a) Alde (North of Orford); (b) Clyde (Bowling); (c) Clyde (Pillar Bank); (d) Clyde (Rosneath); (e) Tees (DabholmGut); (f) Tyne (St. Anthonys); (g) Tyne (Hebburn); (h) Tyne (Howdon); (i) Mersey (Eastham Sands); (j) Forth (Port Edgar).

754 Environ. Toxicol. Chem. 23, 2004 M.F. Kirby et al.

Table 4. Statistical analysis showing p values from testing sites inthe United Kingdom if proportions of induced fish, falling withincertain plasma vitellogenin induction ranges, differ as a function of

sampling time; all ranges in mg/ml

Sites ,0.2 0.2–100 100–1,0001,000–10,000 .10,000

AldeRosneathPillar BankBowlingDabholm GutSt. AnthonysHebburnHowdonMerseyPort Edgar

0.540.78

,0.01,0.01

—0.01

,0.01,0.01,0.01,0.01

—a

—

0.010.430.250.010.540.13—

——

————

,0.01,0.01

—

——

—0.03———

0.03—

——

———————

a — 5 insufficient data for analysis.

Fig. 5. Mean plasma vitellogenin (VTG) concentrations (mg/ml) inmale flounder for all samples taken during 2000 and 2001.duced. However, by October 2001, .80% of males contained

,0.2 mg/ml plasma VTG. At the Howdon site, VTG concen-trations remained high (.102 mg/ml) between 1996 and March2000. From November 2000, plasma VTG concentrationsshowed a pronounced decline of two orders of magnitude tojust above 10 mg/ml and appeared to remain at this reduced(though still elevated) level at subsequent samplings. This re-duction is reflected in the proportions of fish showing VTGinduction between November 2000 and October 2001, withbetween 40 and 60% showing no evidence of moderate or highinduction, unlike on previous sampling occasions where 80 to100% of male fish showed induction.

Tees

Plasma VTG concentrations in male flounder sampled fromthe Dabholm Gut site were routinely high through the studyperiod. In all samples taken between 1997 and November 2000,mean concentrations were consistently greater than 103 mg/mlwith a peak at mean concentrations .104 mg/ml in male fishfrom the March 2000 sample. The most recent data set (Sep-tember 2001) showed a small reduction to ,103 mg/ml, butthis is still two orders of magnitude higher than that of anyother sites sampled during this period. Essentially, the meanplasma VTG concentration time series shows consistently highlevels with little evidence of any major reduction over time.However, it is notable that when one considers the proportionof male fish showing plasma VTG induction, the first evidenceof noninduced specimens was found during the most recentsampling periods (in November 2000 and in 2001).

Clyde

Male flounder captured from the Rosneath site showed near-baseline levels of plasma VTG induction on all three samplingoccasions. Male flounder captured farther upstream at the PillarBank site showed no evidence of VTG induction during the1997, 1999, and 2001 sampling periods. However, some ev-idence of plasma VTG induction was observed on both sam-pling occasions in 2000. Similarly, at the Bowling site (evenfarther upstream), low to moderate plasma VTG induction wasobserved in 1997 and 2001 but considerably more inductionin the September 1999 and March 2000 sampling periods. InMarch 2000, definite elevations were observed in plasma VTGconcentrations at both upstream sites, with mean concentra-tions of .10 and .102 mg/ml at the Pillar Bank and Bowlingsites, respectively. At this time, between 40 and 60% of the

fish at these sites showed evidence of plasma VTG induction,including a few highly induced specimens from the Bowlingsite. Subsequent surveys have shown a reduction in this con-centration of plasma VTG at this site, reaching baseline levelsin September 2001.

Forth

The Port Edgar site on the Forth estuary has been sampledonly since September 1999. Although clear evidence of plasmaVTG induction was observed in over 70% of male floundercaptured during the March 2000 sampling period (with meanconcentrations at .10 mg/ml), it was low to absent at othertimes. Taking the period from September 1999 as a whole, noevidence of change exists.

Evidence for seasonal changes in VTG concentrations

Sampling was too infrequent between 1996 and 1999 todemonstrate conclusive short-term variations in plasma VTGconcentrations in any of the estuaries sampled. However, theincreased frequency of sampling during 2000 and 2001 enableda seasonal comparison of plasma VTG concentrations. Com-bining means of plasma VTG concentration in all male floun-der sampled between September and December and betweenJanuary and March in 2000 and in 2001 demonstrates a clearupward trend for all areas (except for the Rosneath site on theClyde estuary, at which concentrations were consistently ,0.2mg/ml, and at the Hebburn site within the Tyne estuary, atwhich plasma VTG concentrations appeared to oscillate overthe sampling period) (Fig. 5).

Intersex analysis

The prevalence of the intersex (ovo-testis) condition inflounder sampled between 1999 and 2001 is shown in Table5. A typical example of ovo-testis is shown in Figure 6. Asshown from the data from 1996 to 1997 and from 1999 to2001, flounder from the Mersey (5/58 5 8.6%) and the Tyne(5/236 5 2.1%) constituted the bulk of the incidences of thiscondition, though isolated examples have also been recordedin flounder captured from the Clyde and Thames estuaries (notreported here). Such data suggest that intersex may be a wide-spread phenomenon in flounder inhabiting contaminated es-tuaries.

VTG and intersex in UK flounder Environ. Toxicol. Chem. 23, 2004 755

Table 5. Occurrence of ovo-testis in male flounder from estuarine surveys in the United Kingdom,1999–2001

Estuaries Location DateTotalno.

Intersexno. %

Alde North of OrfordNorth of OrfordNorth of Orforda

North of Orforda

North of Orford

08/10/9927/03/00May 00September/

October 0019/12/00

101212

8

26

0000

0

0000

0

Mersey EasthamEasthama

Easthama

EasthamEastham

07/09/9925/05/0011/10/0001/11/0020/02/01

6131220

7

01121

088

1014

Forth Port EdgarPort EdgarPort EdgarPort Edgar

16/09/9916/03/0001/12/0028/02/01

57

1510

0000

0000

Clyde RosneathPillar BankPillar BankBowlingBowlingBowling

28/11/0030/09/9914/03/0029/09/9914/03/0027/11/00

25171117

529

011000

069000

Tees Dabholm GutDabholm Guta

Dabholm GutDabholm Gut

09/11/9929/09/0020/03/0020/11/00

213

225

0000

0000

Tyne St. AnthonysSt. AnthonysSt. AnthonysSt. AnthonysHebburnHebburnHebburnHebburnHowdonHowdonHowdona

Howdona

HowdonHowdon

10/11/9923/03/0023/11/0013/02/0110/11/9922/03/0023/11/0013/02/0111/11/9922/03/0009/05/0028/09/0022/11/0012/02/01

1315141514121821182010132330

00020010100001

000

130060600003

a Additional data from Stentiford et al. [23].

DISCUSSION

Surveys of flounder that have been carried out since thestudies of Allen et al. [2,7] have shown that male fish capturedfrom certain UK estuaries still have elevated concentrationsof VTG in their plasma. However, in most estuaries, the degreeof elevation does not appear to be as high as when large-scaleestuarine sampling programs were first established in 1996.This is particularly apparent at some sites on the Mersey andTyne estuaries, the temporal trend for VTG in fish from Eas-tham Sands on the Mersey being a good example, with areduction in the mean concentration from approximately 104

to 10 mg/ml over the five-year study period.The apparent downward trend in mean plasma VTG con-

centrations is supported by a general increase in the propor-tions of fish showing no induction of plasma VTG and a re-duction in the number of fish showing highly elevated VTGinduction. Of particular significance are the changes seen infish captured from the Tyne and Clyde estuaries during 2000that showed large reductions in plasma VTG concentrationsand also the latest surveys in 2001 where male fish from thesesites showed little evidence of any significant plasma VTG

response. Furthermore, while most of the male fish capturedfrom the Howdon site on the Tyne showed very high plasmaVTG induction in early studies, 60% of the fish showed noevidence of induction in 2001. Such reductions need to betreated with some caution, as they are based on limited sam-pling occasions and could be influenced by other factors, suchas significant migration of fish into the area or altered feedinghabits of resident fish. The low plasma VTG concentrationsin male flounder from the Alde estuary support the continueduse of this area as a suitable reference site. Mean VTG levelsfrom Alde fish remain a relevant baseline against which tocompare results from other estuaries.

The sites that demonstrated the smoothest downward VTGtrend over time are the Eastham Sands site on the Merseyestuary and the Hebburn site on the Tyne estuary. These sitesare associated with contamination from several diffuse sourcesand therefore may act as a monitor of general estrogenic con-tamination of the estuary. As such, the downward trend mayreflect an overall improvement in contaminant levels in theserivers.

An unexpected finding from the current study has been the

756 Environ. Toxicol. Chem. 23, 2004 M.F. Kirby et al.

Fig. 6. Ovo-testis in a male flounder. Note the large zonal accumulationof vitellogenic and previtellogenic oocytes surrounded by normal tes-ticular tissue. Mature testis (Mt) containing spermatozoa, * (vitello-genic oocytes), arrow (previtellogenic oocytes and putative oogonia).

Table 6. Natural and synthetic steroid concentrations, measured bygas chromatography–mass spectrometry (GC-MS), and in vitroestrogen receptor (ER) agonist potency, using the yeast estrogenscreen (YES), in Howdon sewage treatment works effluent. Data

reproduced from Thomas et al. [19,20]

Samplingdate

Instrumental data(GC-MS)

Estradiol(ng/L)

Estrone(ng/L)

Ethynyl-estradiol(ng/L)

ER agonistpotency(YES)

ngestradiol/La

06/09/0014/09/0021/09/0028/09/0002/10/0012/10/0015/09/0121/09/0127/09/0104/10/0106/10/0110/10/01

218.047NDNDND4.75.78.8NDND,1

961554NDNDND8.08.25.1NDND5.0

151.61.5NDNDND2.51.43.0NDND,1

130717665

10525

,4,4,4,4,4,4

a All YES data are blank subtracted. Concentrations represent 17b-estradiol equivalents in the original sample before analytical manip-ulations. ND 5 no data, where sample has failed strict quality as-surance procedures.

discovery of short-term variation in VTG concentrations inmost of the estuaries sampled (including the control estuary,the Alde). Such variations have precluded conclusive diagnosisof downward trends in plasma VTG concentrations at thesesites. Examination of the 2000 and 2001 data indicates thatmale flounder caught in September have generally lower con-centrations of VTG than in the following months. With theprogress of autumn and then winter, mean plasma concentra-tions increase, reaching their peak around March. Such a pat-tern is consistent with fish entering the contaminated estuariesby the summer and leaving again for spawning at sea in March/April [16]. The assumption follows that, in September fishhave been resident only since the summer, and therefore con-taminant exposure has been for a relatively short period. Thespawning migration generally takes place in the winter [17]but may start as early as October and continues through thewinter [18] so that by February/March those late or nonmi-grators still caught in estuaries have had over six months ofexposure to estrogenic compounds (whether in the water, thesediment, or the diet). One outcome of this discovery of month-to-month variation is that future annual surveys should, when-ever possible, be conducted at the same time of the year inany particular estuary in order to provide interpretable long-term trend data.

At least four methodological problems could give rise toan apparent downward or upward trend in VTG concentrationsover the course of the study. These are the change from RIAto ELISA in 2000, degradation of VTG in plasma samplesduring storage, degradation of the standard during storage, andthe introductions of new batches of standard in 1999 and 2001.We have shown that results from RIA are ELISA are highlycomparable and also that no substantial differences existedbetween the standard preparations. Reassay of all plasma sam-ples from fish captured in the Mersey estuary between 1996and 2000 showed that VTG concentrations in the 1996 and1997 samples were 40 and 30% lower, respectively, than thosethat had been originally measured. This could possibly be dueto degradation of VTG in the plasma samples during long-term frozen storage. However, the differences are very smalland insignificant in relation to the 10- to 10,000-fold interyearand intermonth differences noted elsewhere in this study. Some

of the difference can also be ascribed to interassay error. It isunlikely that degradation of the standard during long-term stor-age would have occurred over the course of the study, as thiswould actually have led to an apparent increase in VTG con-centrations with time.

The two sites (Howdon on the Tyne and Dabholm Gut onthe Tees) specifically associated with previously identified [19]point sources of estrogenic contamination show different trendcharacteristics. The Howdon site on the Tyne is associatedwith a major sewage effluent outfall. The plasma VTG con-centrations in male flounder from this site remained high andconsistent from 1996 to March 2000 and then showed a clearreduction, remaining consistently low from November 2000to September 2001. Around the end of 2000, secondary treat-ment processes were brought on line at the Howdon sewagetreatment plant (D. Waites, Northumbrian Water, Durham,County Durham, UK, personal communication). Work com-missioned by Northumbrian Water [20], the owners of thetreatment plant, measured the levels of the main estrogen re-ceptor agonists (17b-estradiol [E2], estrone [E1], and 17a-ethynylestradiol [EE2]) in the Howdon sewage treatmentworks effluent during 2000–2001 by gas chromatography cou-pled to mass spectrometry using the method of Kelly [21]. Aspart of the same study, estrogenic potency (as E2 equivalents)of the effluent was also determined using the in vitro yeastestrogen screen [19]. The analytical data show that the sec-ondary treatment of the effluent successfully reduced meanconcentrations of E2 from 25 to 5 ng/L, E1 from 55 to 7 ng/L, and EE2 from 6 to 2 ng/L. Furthermore, the estrogenicpotency (E2 equivalents) was greatly reduced from a meanactivity of approximately 80 ng/L E2 to ,4 ng/L E2 (Table6). The close relationship between the introduction of sec-ondary treatment, the reduction in estrogen receptor agonistconcentrations, and the reduction in plasma VTG in maleflounder from the Howdon site as reported in the current studyis striking.

The survey site at Dabholm Gut on the Tees estuary is

VTG and intersex in UK flounder Environ. Toxicol. Chem. 23, 2004 757

Table 7. Total annual discharges (kg) of nonylphenol (NP),nonylphenol ethoxylates, and octylphenol into the Tees, via DabholmGut (United Kingdom), between 1997 and 2001 (Environment Agency

of England and Wales, unpublished data)

Year Nonylphenol Octylphenola NP ethoxylates

19971998199920002001

23,2006,0001,360

660805

NDb

300300300300

ND6,0005,7806,0002,300

a Estimated data only.b ND 5 no data.

associated with multiple discharges containing significantquantities of both industrial and domestic effluents. Estrogen-icity of the Dabholm Gut effluent has been associated withhigh levels of industrial contaminants, such as nonylphenol[19], as well as steroidal agents, such as ethynylestradiol, es-trone, and estradiol, that have also been shown to be the primecause of estrogenicity in domestic effluents at the Howdon site[19]. A consistently high and relatively unabated plasma VTGinduction has been observed in male flounder from the Dab-holm Gut site between 1997 and 2000. In the most recentsampling period (November 2001), the mean plasma VTGconcentration was the lowest so far recorded at more than 20times below values seen during peak VTG induction. However,no firm evidence yet exists for a sustained reduction in theVTG levels in male fish from this site. This is rather surprising,as it is known that the loads of nonylphenol and its ethoxylatesin the main industrial effluent at this site have reduced overthe study period (see Table 7) (D. Tarttelin, Environment Agen-cy of England and Wales, Washington, Tyne and Wear, UK,personal communication). Furthermore, the large volume ofmunicipal sewage effluent discharging to Dabholm Gut has,like the effluent at the Howdon site on the Tyne, undergonesecondary treatment since late 2000 (D. Waites, personal com-munication). The fact that a more significant reduction in maleplasma VTG, such as that observed at Howdon, has not oc-curred at the Dabholm Gut site may be due to inputs of un-known estrogenic chemicals with the potential of inducingVTG synthesis or the fact that effluent treatment regimes arenot reducing the load of estrogenic chemicals sufficiently. Al-ternatively, historical estrogenic contamination at this site, per-haps associated with the sediments, may be more persistentthan at other sites.

Further analysis of the somatic data in Table 2 reveals nocorrelation of male plasma VTG concentrations with fish sizeor with HSI. The HSIs are significantly higher (p , 0.05) thanthose from the reference site at the Alde for all locations otherthan at Rosneath on the Clyde. This is not unexpected in fishfrom estrogen-contaminated estuaries because the liver growsin response to estrogen exposure in order to synthesize VTG.However, fish from these sites may also be expected to havetheir hepatic mixed-function oxygenase detoxification systemsstimulated by other contaminants, such as polycyclic aromatichydrocarbons. This can also result in increased HSI values, afinding previously associated with fish captured from sites inthe Tyne, Tees, and Mersey estuaries [22].

Earlier studies of plasma VTG induction and the occurrenceof ovo-testis in flounder revealed no close relationship betweenthe two. Studies from 1996 [2] and later in 2000 [9] demon-strated that the intersex condition appeared to occur solely in

flounder from the Mersey (5/30 5 17% prevalence), whilesurveys from 1997 [7] revealed the condition in fish from boththe Mersey (6/62 5 9.2%) and the Tyne (5/65 5 7.5%) es-tuaries. The results presented in this paper show that theseestuaries continue to be the hot spots of estrogenic contami-nation, even though in some areas VTG induction in male fishappears to be decreasing. It remains difficult to draw any firmconclusions with respect to the presence of ovo-testis and con-tamination by endocrine-disrupting chemicals. However, theconsistent, if relatively low, prevalence of the ovo-testis con-dition in the Tyne and Mersey estuaries coincides with areasof known historical estrogenic contamination as shown bychemical analysis of sediments, water, and biota and by theplasma VTG data presented here. What is more difficult toexplain is the apparent lack of ovo-testis in fish taken fromthe Tees estuary. This suggests that, if the condition is causedby exposure to endocrine-disrupting chemicals (a highly likelysupposition), it may not simply be due to estrogenically activecompounds in general but rather to specific classes of sub-stances that have been more prevalent at sites in the Tyne andMersey estuaries. It might be significant that the discharge atDabholm Gut has been more readily associated with the es-trogenicity of alkyphenolic compounds than at the other sites.It is also recognized that because the numbers of specimenscaught in the Tees is lower than in the other estuaries, theapparent lack of ovo-testis may be sampling artifact.

The studies reported here provide important evidence thatplasma VTG concentrations in male flounder remain elevatedin certain UK estuaries. Furthermore, a low but consistentprevalence of the intersex (ovo-testis) condition has continuedto be recorded in the Tyne and Mersey estuaries. While thelong-term impacts of estrogenic endocrine disruption such asthis remain uncertain, continued monitoring of the situation isrecommended. Furthermore, the potential for effluent treat-ment programs and reduced discharges of estrogenic contam-inants to improve water quality and reduce environmental im-pacts, as has been alluded to in this study, requires furtherinvestigation.

CONCLUSION

This study has demonstrated that plasma VTG concentra-tions in male flounder have remained elevated at some stationsin several industrialized estuaries in the United Kingdom (e.g.,Tees, Mersey, and Tyne) over the period 1996–2001. However,when considered as a time series, these data strongly suggestthat a long-term reduction had occurred in estrogenic contam-ination, measured by VTG induction in male flounder, at mostsites. These reductions were particularly apparent in the Mer-sey and Tyne, but the most recent data also suggest near-baseline levels in some other estuaries (e.g., Clyde and Forth).Analysis of data from 2000 and 2001 indicated that, for mostestuaries, VTG concentrations from fish caught in the winterand spring months are higher than those from fish caught inthe autumn months, presumably because of the migratory be-havior of the flounder.

The reduction in VTG concentrations in male fish caughtnear Howdon sewage treatment works is associated with re-duced levels of estrogenic contamination in the Howdon dis-charge that are probably related to the introduction of sec-ondary treatment at the plant. No major change in mean plasmaVTG concentrations in male fish captured near the DabholmGut discharges was noted over the study period.

Finally, the occurrence of ovo-testis in male flounder con-

758 Environ. Toxicol. Chem. 23, 2004 M.F. Kirby et al.

tinues to be detected at a relatively low but consistent level.Fish from the Tyne and Mersey estuaries continue to providethe bulk of cases in this species, although a few examples havebeen observed elsewhere (such as the Clyde).

Acknowledgement—The authors wish to acknowledge all the crewsof the survey vessels used: research vessel Coastal Guardian, WaterGuardian, and Sea Vigil (Environment Agency); research vessel ForthRanger and Endrick II (Scottish Environment Protection Agency);and the MV Nicola Dawn. We greatly appreciate the advice we havereceived from Mike Nicholson and Jon Barry concerning appropriatestatistical analysis and representation of the data. Finally, we are alsoindebted to the Endocrine Disruption in the Marine Environment (ED-MAR) Program funded by the UK Department for Environment, Foodand Rural Affairs, the Environment Agency of England and Wales,the Scotland and Northern Ireland Forum for Environmental Research,and the European Chemical Industry Council.

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