Seasonal Evaluation of Reproductive Statusand Exposure to Environmental Estrogens inHornyhead Turbot at the Municipal WastewaterOutfall of Orange County, CA

Xin Deng,1 Mary Ann Rempel,1 Jeff Armstrong,2 Daniel Schlenk1

1Department of Environmental Sciences, University of California, Riverside, Riverside,California 92521, USA

2Orange County Sanitation District, Fountain Valley, California 92708-7018, USA

Received 9 May 2007; accepted 12 May 2007

ABSTRACT: Seasonal changes in developmental stages, condition factor (CF), gonadosomatic index, andplasma vitellogenin (Vtg) concentrations in male and female hornyhead turbot were examined at the waste-water outfall (T1) of the Orange County Sanitation District, and two farfield sites T11 (7.7 km northwest of theoutfall) and Dana Point (35 km south of the outfall) between February 2005 and May 2006. Fish collectedfrom the three sites exhibited male-oriented sex ratios. With few exceptions, developmental stages, CF, andGSI of both genders and plasma Vtg concentrations of females were not significantly different in samples col-lected from different sites at the same sampling period. More advanced gonad developmental stages andhigher plasma Vtg concentrations in females were observed in August, indicating the seasonality of the repro-ductive cycle for this species. Plasma Vtg concentrations in males were observed in all of the sampling siteswith the highest prevalence at T11 relative to T1 and Dana Point. The Vtg expression in males from the threesampling sites indicated widespread exposure to estrogenic compounds in waters of coastal California.However, the histopathological and reproductive relevance of the responses appeared to be insignificant andmay not affect the population in these locations. # 2007 Wiley Periodicals, Inc. Environ Toxicol 22: 464–471, 2007.

Keywords: hornyhead turbot; reproductive status; wastewater; estrogens; vitellogenin

INTRODUCTION

Concerns of contamination by environmental estrogens and

their adverse impacts on reproductive health of aquatic ani-

mals have been the subject of several studies near the

wastewater outfall of the Orange County Sanitation District

(OCSD) (Roy et al., 2003; Schlenk et al., 2005; Rempel

et al., 2006). This area receives �240 million gallons per

day of 50% blended primary and secondary treated waste-

water effluent. Bioindicators in flatfish have been used to

evaluate the potential effects of the discharge on fish pop-

ulations. In the sediments near the outfall, estrogen and its

mimicking compounds, such as 17b-estradiol, several

alkylphenol ethoxylates, and alkylphenols, were observed

(Schlenk et al., 2005). Further investigations at the site indi-

cated a skewed sex ratio towards male fish, presence of

plasma vitellogenin (Vtg) concentrations in males, and

sperm DNA damage (Rempel et al., 2006). However, bio-

logical endpoints regarding the sexual maturity, gonadoso-

matic index (GSI), and species abundance for fish at the

outfall appeared to be unaffected compared with those at a

farfield reference site (Roy et al., 2003; Rempel et al.,

2006). Although reproductive maturity was estimated from

the fish size, the effects of environmental estrogen exposure

on reproductive development were not assessed.

Correspondence to: X. Deng; e-mail: xin.deng@ucr.edu

Contract grant sponsor: Orange County Sanitation District (OCSD)

Published online in Wiley InterScience (www.interscience.wiley.com).

DOI 10.1002/tox.20287

�C 2007 Wiley Periodicals, Inc.

464

Hornyhead turbot (Pleuronichthys verticalis), one of thesentinel species used in previous studies, was selected as

the target species for this study for it is a common, rela-

tively stationary, resident species in the Southern California

Bight (Allen et al., 2002). Despite its use as a sentinel spe-

cies for biomonitoring projects, its reproductive biology as

well as life history is not well understood. Reports have

been rather fragmentary and somewhat contrary regarding

the reproductive cycle. Based on different regional datasets

in southern California, hornyhead turbot have been reported

to spawn seasonally (Budd, 1940) or year-round (Goldberg,

1982; Cooper, 1994). As an ongoing biomonitoring project,

the aim of the present study was to evaluate effects of envi-

ronmental estrogenic exposure in hornyhead turbot

throughout the seasonal reproductive status of this species.

MATERIALS AND METHODS

Study Sites and Fish Collection

Hornyhead turbot were collected using a 7.6-m wide semi-

balloon otter trawl in February, August, and November of

2005 from two locations—the OCSD outfall (T1) and a far-

field site (T11), and February and May of 2006 from T1,

T11, and Dana Point. The outfall (Lat 33 34.6410; Long 118

00.5670) is located 7 km offshore on the San Pedro Shelf

within the Southern California Bight. Site T11 (Lat 33

36.0550; Long 118 05.1990) is 7.7 km northwest of the out-

fall. Male California halibut (Parlichthys californicus)either directly exposed to sediments or injected with sedi-

ment extracts demonstrated significantly induced Vtg levels

when exposed to T1 sediments but not T11 sediments

(Schlenk et al., 2005). Thus, T11 is likely a cleaner site as a

reference for evaluating the response of environmental

estrogens in flatfish at the wastewater outfall. In addition,

Dana Point (Lat 33 24.9980; Long 117 41.0010), 35 km

south of T1, was also sampled at a similar depth as a poten-

tial reference. To minimize variation of each sampling

event, a differential Global Positioning System was used to

accurately locate the sampling sites and to control the

trawling path in a similar direction towards northwest fol-

lowing the surface current. The trawling was controlled at a

speed of 50–60 m/min for 10 min, which converted to a dis-

tance of 500–600 m. As the sampling sites were relatively

accurate, the sampled fish were presumably from the same

population of each site. Their standard length ranged from

92 to 190 mm for males and 110–230 mm for females.

As a separate control, six adult male hornyhead turbot

collected at T11 in August 2005 were transported to the

laboratory and held in a living steam aquarium with

bioassay-grade artificial seawater for 3 weeks. The water

was changed and the fish were fed live earthworms daily.

The plasma from the purged animals was then collected,

and the Vtg concentration was measured.

Sampling Procedures

Upon retrieval of the trawl net, hornyhead turbot were blot-

ted on paper towels, individually measured by standard

length to the nearest 1.0 mm, and weighed to the nearest

1 g for condition factor (CF) calculation. Blood was drawn

by a heparinized 22-G syringe from the dorsal aorta and im-

mediately centrifuged with a portable centrifuge for 2 min

at 500 rpm. Plasma was removed and kept in a 1.5-mL cen-

trifuge tube on dry ice until transport to a 2808C freezer

for further analysis of Vtg concentrations. Exsanguinated

fish were then sacrificed by transecting the spinal cord pos-

terior to the head. One-half of the gonads were removed

and weighed to calculate GSI as the other half was used for

histology. For histological analysis, ovarian tissue was

sliced into 5-mm thicknesses with a razor blade and half of

one-side of the testis was placed into a tissue cartridge, and

fixed in 10% phosphate buffered formalin for developmen-

tal and histopathologic examinations. CF and [1/2] GSI

were calculated according to the following formulae:

CF 5 100 3 body weight (g)/[standard length (cm)]3

[1/2] GSI5 1003 half of gonad weight (g)/body weight (g)

Gonadal Development and Histopathology

To prepare the gonad tissue for developmental and histo-

pathological examinations, the fixed gonads were dehy-

drated in increasing concentrations of ethanol, embedded in

paraffin, sectioned into 5-lm thicknesses, stained with he-

matoxylin and eosin, and examined under light microscope.

The morphologic criterion for staging ovaries and testes

were adapted from previous studies (Htun-Han, 1978a,b;

Johnson et al., 1991; Sol et al., 1998). Ovarian stages were

determined by oocytes of the most advanced maturation in

examined sections. Briefly, ovarian developmental stages

were characterized as follows: stage 1—regressed stage,

ovaries with primary and secondary oocytes; stage 2—pre-

vitellogenic stage, oocytes with cortical alveoli and zonal

radiata; stage 3—vitellogenic stage, oocytes with yolk

globules; stage 4—ovaries with hydrated oocytes; stage

5—spawning stage, ovaries with hydrated oocytes and post-

ovulatory follicles; stage 6—postspawning stage, ovaries

with many postovulatory follicles and atretic oocytes. Tes-

ticular stages were classified based on the proportion of var-

ious germ cells, which include spermatogonia, spermato-

cytes (primary and secondary), spermatids and spermato-

zoa. Criteria for testicular developmental stages were: stage

1—regressed stage, testes with only spermatogonia; stage

2—gonial proliferation, testes with primary and secondary

spermatocytes; stage 3—onset of meiosis, testes with sper-

matids but predominated by primary and secondary sper-

matocytes; stage 4—spermatogenesis, testes with sperm

and all cell types. Stage 5—spermiating stage, tubules par-

tially filled with sperm; stage 6—postspermiating stage,

465EXPOSURE TO ENVIRONMENTAL ESTROGENS IN HORNYHEAD TURBOT AT WASTEWATER OUTFALL

Environmental Toxicology DOI 10.1002/tox

tubules largely empty with few remaining sperm. Ovaries

and testes were also examined for incidences of ovarian

atresia and ova-testes, respectively.

Vitellogenin Assay in Plasma

The vitellogenin assay followed the procedure developed

by Rempel et al. (2006). Briefly, the wells of a 96-well

plate were coated with either 1% non-fat milk for nonspe-

cific binding wells or 100 lL of 0.8 lg/mL California hali-

but Vtg in 50 mM carbonate buffer, and incubated at 378Cfor 2 h. The plate was then washed three times with 10 mM

Tris-phosphate buffer saline (TPBS), blocked with 2% non-

fat milk in TPBS, incubated 378C for 45 min, and washed

with TPBS again after the incubation. Diluted standard

(purified halibut Vtg) or plasma samples and primary anti-

body (rabbit antiturbot Vtg purchased from Cayman Chem-

ical, Ann Arbor, MI) in TPBS were mixed at 1:1 ratio in a

final concentration of antibody of 1:1000. The mixture was

incubated, added in triplicate into each of the treated wells,

and incubated before the secondary antibody (goat anti-

rabbit labeled with alkaline phosphatase purchased from

Biorad, Hercules, CA) diluted to 1:2000 in TPBS was

added. p-Nitrophenylphosphate was used as the detection

substrate with absorbance measurements at 405 nm. Values

were normalized to total plasma protein, which was meas-

ured by the method of Bradford (1976), using bovine serum

albumin as a standard. The detection limit of Vtg concen-

tration was at 0.1 ng/lg plasma protein. Vtg values below

the detection limit were set at 0.1 for the purposes of com-

parison among different seasons and sites.

Data Analysis

Data were analyzed using STATISTICA Version 6.0 (Stat-

Soft, Tulsa, OK). All datasets were checked for normality

of distribution and homogeneity of variance by Shapiro-

Wilk and Levene tests prior to further analyses. Logarith-

mical transformation was necessary for GSI values to meet

the normality of distribution. CF and log-transformed GSI

were subjected to ANOVA analysis for site differences

and seasonal changes of each gender. A nonparametric

Kruskal–Wallis test was performed for plasma Vtg concen-

trations because Vtg datasets could not be remedied to meet

the normality of distribution. When significant differences

(p\ 0.05) were identified, differences among means were

compared by the Tukey’s multiple comparison test (CF and

GSI) or post hoc test (Vtg concentrations). In addition, dif-

ferences in sex ratios and developmental stages between

sites or seasons were tested by the likelihood ratio test.

RESULTS

Gender Ratios

Gender ratios of hornyhead turbot were significantly skewed

toward males (p \ 0.05) in most of the sampling events

except fish collected from T1 in August 2005, and from T1

and T11 in May 2006, where gender ratios were not signifi-

cantly deviated from one (Table I). When all the fish in

2005–2006 were summed up for each site, the male ratio at

T11 was significantly higher than that at T1 but not at Dana

TABLE I. Sex ratio and incidence of Vtg induction in hornyhead turbot collected at T1, T11, and Dana Point (DP)in 2005–2006

Month Site

Number of Fish Collected Percent of Fish with Vtg Induction (%)

Sex Ratio Male:FemaleMale Female Male Female

February-2005 T1 16 10 0 70.0 1.6:1

T11 20 5 15.0 80.0 4:1a

August-2005 T1 16 14 6.25 92.9 1.1:1

T11 10 5 80.0 100.0 2:1

November-2005 T1 18 11 38.9 54.5 1.6:1

T11 19 1 0 0 19:1a

February-2006 T1 20 10 15.0 20.0 2:1

T11 18 12 33.3 25.0 1.5:1

DP 19 11 0 54.5 1.7:1

May-2006 T1 24 26 4.2 84.6 0.9:1

T11 5 9 0 11.1 0.6:1

DP 23 12 17.4 66.7 1.9:1

2005–2006 T1 94 71 12.8 70.4 1.3:1

T11 72 32 23.6 40.6 2.3:1a

DP 42 23 9.5 60.9 1.8:1

1988–2006 T1 308 185 N/A N/A 1.7:1

T11 136 83 1.6:1

a Indicates a significant higher values in fish collected at site T11 relative to T1 or DP.

466 DENG ET AL.

Environmental Toxicology DOI 10.1002/tox

Point. Totaling the annual records, a total of 493 and 219

fish were collected at T1 and T11, respectively, since 1988.

The gender ratios were significantly male-oriented with a

similar male: female ratio in T1 and T11, which was also

similar to the gender ratio at Dana Point (Table I).

CF and GSI

Male and female fish collected in the same season exhibited

similar mean values of CF with two exceptions where signif-

icantly lower CF values were observed in female fish collect

at T11 in February 2005 and at Dana Point in May 2006

(Fig. 1). Seasonal change in CF was observed in males at

site T11, but not site T1 and Dana Point. In females, the

change was observed at site T1 and T11 but not at Dana

Point. It appears that fish in August tend to have lower CF,

whereas fish in November tends to have higher CF.

Similar GSI values were observed in male and female

fish collected in the same season from different sites with

three exceptions where significantly lower GSI values were

observed in males collected at T11 in August 2005, and

females at T11 in February 2005 and May 2006. Seasonal

changes in GSI were observed in males at T1 and T11 with

a lower value in November but not at Dana Point. GSI of

females at T1 and Dana Point exhibited significant seasonal

changes with a tendency of higher values in August and

May. Only one female fish was collected at site T11 in No-

vember, which constrained the statistical evaluations

between seasons and sites.

Plasma Vtg Concentrations

The prevalence of Vtg induction among sampling events

varied from 0–80% with the highest in fish collected at T11

in August 2005. However, no site or seasonal specific

trends were observed (Table I). Fish in 6 out of 12 sampling

events exhibited significantly elevated Vtg concentrations

relative to the purged control (PC) animals [Fig. 3(a)]. The

pooled yearly data indicated that male fish collected at T11

had a significantly higher prevalence of Vtg induction com-

pared with fish from T1 and Dana Point (Table I). Overall,

male fish had low Vtg concentrations ranging from 0.1 to

0.38 ng/lg plasma protein. Significant differences of Vtg

concentrations among sampling sites were observed only in

fish collected in February and August 2005 [Fig. 3(a)].

Vtg concentrations in female hornyhead turbot collected

at the same site and season did not differ statistically [Fig.

3(b)]. Seasonal changes were only observed in fish from

site T11 that had a significantly higher value in August than

in the rest of the seasons [Fig. 3(b)]. The pattern of seasonal

changes of plasma Vtg concentrations appears to corre-

spond with that observed in GSI of female animals [Fig.

2(b)]. The percentages of females in vitellogenesis were the

highest in August (Table I).

Developmental Stages and Histopathology

Hornyhead turbot gonads collected in August and Novem-

ber 2005 at T1 and T11, and in February and May 2006 at

T1, T11, and Dana Point were examined for developmental

stages and histopathology. Ovaries and testes in hornyhead

turbot exhibited an asynchrous developmental pattern. De-

velopmental stages in animals of different seasons gener-

ally overlapped. However, seasonal changes in profiles of

developmental stages were significantly pronounced in

Fig. 1. Condition factors (CF) for male (a) and female (b)hornyhead turbot collected at sites T1, T11, and Dana Point(DP) in February, August, November 2005 and February,May 2006. For all the Box & Whisker graphs (Figs. 1–3),small squares represent means, large rectangles representthe range of standard errors and whiskers indicate the rangeof standard deviations. Different letters (capital for T11 orDP, and lowercase for T1) represent significant differences(p\ 0.05) among sampling seasons of the same site. * indi-cates the significant difference between sampling sites atthe same sampling season.

467EXPOSURE TO ENVIRONMENTAL ESTROGENS IN HORNYHEAD TURBOT AT WASTEWATER OUTFALL

Environmental Toxicology DOI 10.1002/tox

male and female fish from both sites (Fig. 4). In August,

over 70% of males from both sites were spent and �80%

females reached either the late spawning or postspawning

stage. In November, 89% and 67% of males were immature

from site T11 and T1, respectively, and none of the females

were mature. Early signs of spawning were observed in

February when each of the two females at T1 and Dana

Point presented hyaline oocytes (stage 4), and four males

from T1, T11, and Dana Point had testes with spermatozoa

(stage 4 or 5). Differences in profiles of developmental

stages were not significant in male fish among the three

sites in all of the sampling seasons (Fig. 4). Significantly

higher percentages of immature females were observed at

DP in February 2006 and at T11 in May 2006.

Histopathological examinations revealed incidences of

ovarian atresia in 4 of 5 fish collected at T11 and 1 of 14

fish at T1 in August 2005. Ova-testis was found in one fish

collected at T1 in February 2006 with 4 oogonia present in

the testicular cysts.

Figure 5 a and b showed the plasma Vtg concentrations

as a function of developmental stages for all the male and

females collected in 2005–2006. No correlation (R2 50.0435) was evident in male fish [Fig. 5(a)]. However,

plasma Vtg concentrations significantly corresponded with

developmental stages (R2 5 0.757) in female fish whose

plasma Vtg concentrations were significantly elevated

beyond the vitellogenic stage (stage 3) [Fig. 5(b)].

DISCUSSION

The current study examined the reproductive status of hor-

nyhead turbot and potential exposures to environmental

estrogens at the wastewater outfall of the OCSD relative to

two farfield sites in the southern California bight.

Fig. 3. Vitellogenin (Vtg) concentrations for male (a) andfemale (b) hornyhead turbot collected at sites T1 and T11 inFebruary, August, November, February 2006, and DanaPoint (DP) in February, May 2006. # indicates a significantelevation of the Vtg concentration relative to purged controlanimals (PC).

Fig. 2. 1/2 Gonadosomatic index for male (a) and female(b) hornyhead turbot collected at sites T1, T11, and DP inFebruary, August, November 2005 and February, May 2006.

468 DENG ET AL.

Environmental Toxicology DOI 10.1002/tox

Evaluation of hornyhead turbot populations from 1988 indi-

cated a skewed sex ratio toward male fish at sites T1 and

T11 (Rempel et al., 2006) as well as Dana Point. In labora-

tory conditions, skewed sex ratios towards females were

demonstrated in several fish species exposed to estrogens

(Nimrod and Benson, 1998; Orn et al., 2006; Seki et al.,

2005). However, the phenomenon was rarely observed in

field investigations. Examination of the sex ratios of Euro-

pean flounder (Platichthys flesus) in 11 estuarine and ma-

rine waters in the UK revealed significant estrogenic

responses in male Vtg inductions with 20% male fish pre-

senting ova-testis at contaminated waters, but sex ratios in

flounder from each site appeared to be unaffected (Allen

et al., 1999a,b). While the male-oriented sex ratios of hor-

nyhead turbot populations could imply masculinization of

the population at the Southern California Bight, lack of

knowledge on spawning behavior, and seasonal distribution

of this species has been an impediment to validating the

skewness as a natural or anthropogenically influenced life

history attribute. In winter flounder (Pseudopleuronectesamericanus), several males were commonly involved in the

spawning activity of a single female without agonistic

behavior among those males (Stoner et al., 1999). Field sur-

veys on greenback founder (Rhombosolea tapirina) indi-cated that females appeared to be more abundant in shal-

low water whereas males were more abundant in deep

water (Gibson, 2005). Hornyhead turbot appeared to be

most abundant between depths of 20–60 m along the south-

ern California bight (Allen et al., 2002; Allen, 2006). Our

trawling was conducted at a depth of 55–60 m at all the

sampling sites, which was near the distribution boundary of

this species. If hornyhead turbot had similar spawning

behavior or segregation patterns as the winter flounder or

greenback flounder, it was then not surprising to have

higher catches of males at this depth and location.

Consistent seasonal changes of CF and GSI were not

observed in either sites or gender of hornyhead turbot.

Nevertheless, reproductive seasonality in males and

females was well supported by the high percentage of

mature fish and significantly elevated plasma Vtg concen-

trations in females in May and August [Figs. 3(b) and 4(b)].

The reproductive seasonality observed in this study was in

agreement with an early observation by Budd (1940), who

Fig. 4. Developmental stages for male (a) and female (b)hornyhead turbot collected at sites T1, T11, and DP in Au-gust, November 2005 and February, May 2006. Numbers inbars indicate the sample size.

Fig. 5. Correlations between plasma Vtg concentrationsand developmental stages in male (a) and female (b) horny-head turbot in 2005–2006.

469EXPOSURE TO ENVIRONMENTAL ESTROGENS IN HORNYHEAD TURBOT AT WASTEWATER OUTFALL

Environmental Toxicology DOI 10.1002/tox

reported that hornyhead turbot at Monterey Bay, CA

spawned from March to August. A more recent study by

Cooper (1994) observed potential seasonal changes of GSI

and maximum oocyte diameter, but failed to conclude sea-

sonality of spawning due to a lack of statistical analysis. In

examining 140 hornyhead turbot randomly collected

through the year from San Clemente to Santa Monica Bay,

CA in 5 years, Goldberg (1982) reported that 76–100% of

fish collected in each month were in spawning status, and

concluded that this species spawned year-round. Since cri-

teria of developmental stages was not well characterized in

the article, it was impossible to compare the results with the

current study. The presence of postovulatory follicles in

ovaries usually indicates recent spawning in fish. The

author observed only 2.6% of the spawning fish possessing

postovulatory follicles, which is not consistent with high

percentages of spawning individuals. Since temperature

plays a vital role in triggering spawning in fish, the discrep-

ancy of the spawning period in previous studies might have

resulted from the differences of temperature regimes in dif-

ferent geological locations. In fact, flatfish species generally

have a single or a bimodal spawning period (Rijnsdorp and

Witthames, 2005). The early signs of spawning activities

observed in February and the high percentage of post-

spawning individuals of both genders in August implies an

extended spawning period for hornyhead turbot. Whether

hornyhead turbot possess a single or bimodal reproductive

pattern remains to be a subject of future studies.

The results in the present study indicated that low concen-

trations of plasma Vtg were continuously evident in male

hornyhead turbot collected at sites T1 and T11 in 2005–

2006. The plasma Vtg concentrations were significantly ele-

vated relative to the purged animals in 6 out of 12 sampling

events from all of the three sampling sites. In the previous

study of 2003–2004, prevalences of Vtg induction were 73

and 83% in the sampled males at T1 and T11, respectively

(Rempel et al., 2006). The present study revealed signifi-

cantly decreased prevalences at both sites. The plasma Vtg

concentrations among sampling sites were statistically differ-

ent in 2 out of 5 sampling seasons. However, considering the

low Vtg concentrations, the differences may not be biologi-

cally significant in relation to estrogenic effects (Rempel

et al., 2006). It has been recognized that both estrogen and

testosterone present in male and female animals work in con-

cert to regulate sex differentiation and development (Hess

and Carnes, 2004). Previous studies demonstrated the pres-

ence of low plasma Vtg levels in male fish not exposed to

estrogens in laboratory conditions (Hotta et al., 2003; Seki

et al., 2003; Van den Belt et al., 2003). This phenomenon

suggests that endogenous estrogen may partially contribute

to the Vtg induction. However, compared with the purged

animals, the plasma Vtg concentrations in male hornyhead

turbot from T1, T11, and Dana Point were significantly

elevated, which implies exposure to either exogenous estro-

gens or compounds that elicit estrogenic activity in vivo (i.e.,

anti-androgens). This hypothesis may be indirectly supported

by the detectable levels of estrogenic compounds in sedi-

ments (Schlenk et al., 2005) or due to exposure in water or

prey. Hornyhead turbot primarily feed on benthic dwelling

polychaetes that spent their whole life in the sediments and

may accumulate significant levels of estrogen mimics via

sediments. Thus, trophic transfer from sediments to benthic

dietary items may provide a better source of exposure.

The farfield site T11 has been used as a reference site to

evaluate biological endpoints of hornyhead turbot for envi-

ronmental estrogenic activity at the outfall T1 since 2000.

Fish collected from T1 and T11 in multiple years exhibited

similar values in all the measured endpoints. Plasma Vtg

and estradiol concentrations in males, GSI, gonadal devel-

opment, sexual maturity, sperm DNA damage, and popula-

tion abundance rarely showed significant differences

between the two sites. Since T11 is 7.7 km down-

current of the outfall, it is possible that environmental

chemicals coming from the outfall may eventually be trans-

ported to T11. Thus, it may be necessary to investigate

other locations for reference comparisons. Male fish from

Dana Point showing the lowest prevalence of the plasma

Vtg concentrations suggested that Dana Point is a better ref-

erence site for future investigations. Studies are currently

underway to compare seasonal concentrations of Vtg and

gonadal development of hornyhead turbot at other waste-

water outfalls in the Southern California Bight.

In summary, male fish from the OCSD wastewater out-

fall T1 and the farfield site T11 consistently expressed Vtg

throughout the year. However, evidence of developmental

impairments or alteration of gender ratios was not

observed. Thus, the relevance of Vtg expression in horny-

head turbot to reproduction or development may be limited.

The authors would like to thank OCSD Ocean Monitoring

Crew, Jesus Reyes, and Gabriela Rodriguez-Fuentes for help in

collecting samples. We also wish to acknowledge the Southern

California Coast Water Research Group for providing histology

equipment.

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471EXPOSURE TO ENVIRONMENTAL ESTROGENS IN HORNYHEAD TURBOT AT WASTEWATER OUTFALL

Environmental Toxicology DOI 10.1002/tox