Sources and Effects of Contaminants of Emerging Concern in Southern California Coastal Waters
INTEGRATED COASTAL EFFECTS STUDY: SYNTHESIS OF FINDINGS
STEVEN M. BAY,*y DORIS E. VIDAL-DORSCH,y DANIEL SCHLENK,z KEVIN M. KELLEY,§
KEITH A. MARUYA,y and JOSEPH R. GULLYk
ySouthern California Coastal Water Research Project, Costa Mesa, California, USA
zUniversity of California, Riverside, California, USA
§California State University Long Beach, Long Beach, California, USA
kSanitation Districts of Los Angeles County, Whittier, California, USA
(Submitted 10 February 2012; Returned for Revision 2 April 2012; Accepted 23 July 2012)
Abstract—Municipal wastewater discharges constitute a major source of contaminants of emerging concern (CECs) to coastal waters,
yet uncertainty exists regarding their linkage to adverse biological effects such as endocrine disruption. Limited information is available
concerning the types and fate of CECs discharged to the Southern California Bight (SCB) from municipal wastewater and their potential
for ecological impacts. The present study investigated the impacts of CECs from ocean wastewater discharges on SCB fish.
Concentrations of CECs were measured in effluents from four major municipal wastewater dischargers. Seawater, sediment, and
hornyhead turbot (Pleuronichthys verticalis) from the discharge sites and a reference area were collected and analyzed for chemical and
biological indicators. Low concentrations of pharmaceuticals, personal care products, and industrial and commercial compounds were
measured in effluent. Some CECs were also detected in sediment, seawater, and fish livers near the outfalls, confirming exposure to
CECs. Fish plasma hormone analyses suggested the presence of physiological effects, including a reduced stress response, altered
estrogen synthesis or estrogenic exposure, and reduced thyroxine. Most fish responses were found at all sites and could not be directly
associated with effluent discharges. However, concentrations of thyroxine were lower at all discharge sites relative to the reference, and
estradiol concentrations were lower at three of the four outfall sites. The physiological responses found were not associated with adverse
such as survival and reproduction. Environ. Toxicol. Chem. 2012;31:2711–2722. # 2012 SETAC
Keywords—Contaminants of emerging concernSeawaterEffluentFish Biological responses
Three decades of monitoring by southern California water
quality agencies have provided a great deal of information
regarding legacy priority pollutants such as dichlorodiphenyl-
trichloroethane (DDT), polychlorinated biphenyls (PCBs), Hg,
and Pb. In contrast, little is known about the sources, fate, and
effects of thousands of other chemicals in current use. Some of
these are recently developed compounds, many of which are
designed to affect biological systems. These compounds have a
widespread use and are continuously discharged into aquatic
habitats [1–3]. These so-called contaminants of emerging con-
cern (CECs) can be classified into four major categories:
pharmaceuticals and personal care products (PPCPs); cur-
rent-use pesticides; natural and synthetic hormones; and indus-
trial and commercial compounds (ICCs).
The occurrence and effects of most CECs have not been
extensively studied, perhaps because of the lack of available
analytical methods, yet they may represent a risk to aquatic life
after being released to the environment [3–5]. For example,
current-use pesticides such as pyrethroids have been identified
as a cause of sediment toxicity throughout California [6,7].
Some CECs disrupt the endocrine systems of animals by
interfering with the action of hormones involved with repro-
duction or growth [8–10]. Research in regions outside southern
California suggests that environmental concentrations of some
CECs may be sufficient to produce endocrine disruption in fish
living in coastal waters [11–14].
Recent studies have detected multiple CECs in sediments
collected near wastewater effluent discharges in coastal waters
of the Southern California Bight (SCB) [15,16]. Previous
studies have also reported the presence of indicators of endo-
crine disruption in local fish, such as the production of vitello-
genin (VTG) (an egg-yolk protein) by male flatfish [16–19]. A
toxicity identification evaluation (TIE) of sediment extracts
inducing VTG synthesis failed to identify causative agents
. The significance of these results is uncertain because
the previous studies were limited in scope and interpretation
and PCBs) that may produce similar effects. As a result, the
nature and magnitude of endocrine disruption in southern
California fish is not known. Additional information is needed
ing fish health, and whether current CEC inputs from municipal
wastewater discharges are responsible. Without such informa-
decisions regarding the need to monitor and regulate CECs.
The present study, the 2006 CEC Coastal Effects Study, was
designed to address data gaps regarding CECs in southern
California coastal waters. The goal of the project was to help
answer the following questions: (1) What types of CECs are
discharged into the SCB from municipal wastewater outfalls?
(2) Are SCB marine life exposed to CECs from municipal
wastewater discharges? (3) Is evidence present of endocrine
disruption or other physiological effects in SCB fish? (4) Are
Environmental Toxicology and Chemistry, Vol. 31, No. 12, pp. 2711–2722, 2012
# 2012 SETAC
Printed in the USA
All Supplemental Data may be found in the online version of this article.
* To whom correspondence may be addressed
Published online 15 September 2012 in Wiley Online Library
effects on fish related to historical or current municipal waste-
water discharges? (5) Are specific chemicals responsible for the
effects? (6) Are the biological effects adversely impacting fish
Sample collection, analysis, and data interpretation was a
collaborative effort among the Southern California Coastal
Water Research Project, southern California’s four largest
municipal wastewater treatment agencies, and major univer-
sities. The present study provides a synthesis of the key findings
of the study and is intended to complement other papers in this
series that describe the results in greater detail.
southern California that found physiological changes sugges-
tive of endocrine disruption in the hornyhead turbot (Pleuro-
nichthys verticalis), a common species of flatfish that lives on
soft bottom sediments along the coast of the SCB. Flatfish such
as hornyhead turbot are used for environmental monitoring
because they live in contact with the sediment, have high site
fidelity, feed on sediment-dwelling animals, and are monitored
locally for tissue contamination, thus providing a model organ-
ism for studying exposure to environmental contaminants in
specific areas [20,21].
A comprehensive and integrated chemical/biological inves-
tigation was conducted that focused on the four largest ocean
discharges of municipal wastewater to the SCB (Fig. 1). A total
these outfalls, which are located at depths ranging from approx-
imately 60 to 100m . Samples of final effluent from each of
the treatment plants, as well as near-bottom seawater, sediment,
and hornyhead turbot from the study areas, were collected to
characterize the fate of a diverse suite of CECs. The effluents
investigated have been described by Vidal et al. .
Three different sampling designs were used to address the
study questions. The first sampling design consisted of collec-
tion of quarterly samples of wastewater effluent and near
bottom water from each of the five sites. Chemical analysis
of these samples was used to describe the occurrence and water
exposure concentrations of CECs. Samples were collected
between May 2006 and February 2007.
Thesecond samplingdesignwas intendedtodocumentspatial
patterns of sediment contamination, fish exposure, and biological
fish were collected from the five study sites (Santa Monica Bay
and San Diego [SD]) during a single sampling event between the
was a composite of surface sediment (top 5cm) from three
separate grabs. Fifty hornyhead turbot were collected by otter
for chemical analysis (liver) and biological analysis (blood, liver,
and gonad).The liver samples were composited by gender before
analyses were conducted on individual fish samples.
The third sampling design was intended to investigate
temporal and smaller-scale spatial variations in biological
indicators such as reproduction. This sampling was focused
on three sites that represented a wide range of expected con-
taminant exposure: PV, OC, and DP. Spatial variation was
investigated by collecting fish from two far-field sites used in
monitoring programs as a local reference for Palos Verdes
(PVF) and Orange County (OCF). Thirty hornyhead turbot
were collected from each site at quarterly intervals between
May 2006 and February 2007 (samples from the first quarter
were the same as those collected for the spatial study design).
Fig. 1. Project study sites. Four sites were located near large discharges of municipal wastewater (Santa Monica Bay [LA], Palos Verdes [PV], Orange County
[OC], and San Diego [SD]. In addition, samples were collected from one reference station, Dana Point (DP), and two far-field stations Palos Verdes and Orange
County (PVF and OCF, respectively).
Environ. Toxicol. Chem. 31, 2012S.M. Bay et al.
Effluents and study sites
Each of the effluent types and associated sampling sites
represented a different combination of effluent treatment and
historical contamination, providing an opportunity to examine
the relative impacts of current CEC inputs and legacy contam-
ination on the fish. For example, effluents discharged at the LA
and PV locations received 100% secondary treatment, and the
sediments of these areas contained relatively high legacy con-
tamination from DDT and PCBs when compared with the other
stations investigated in the present study. The OC site received
effluent with partial secondary treatment and contained little
legacy sediment contamination. The SD site received chemi-
cally enhanced advanced-primary treated effluent and also had
little legacy sediment contamination. The DP site—used as a
reference site for the study—was located in an area away from
been used as a reference site by previous studies . The DP
site was located 35km away from the nearest major municipal
wastewater discharge (OC), although a much smaller discharge
of secondary treated municipal wastewater effluent was located
The gradient of sediment contamination among the study
sites is well known, and it is characterized by the highest
concentrations of DDTs, PCBs, and polycyclic aromatic hydro-
carbons at PV, intermediate contaminant concentrations at LA,
and relatively low and similar concentrations at the remaining
sites . Overall contaminant exposure at these sites was
quantified using the mean effects range-median quotient
(ERMq)  calculated for sediment data from 2003 to 2007
monitoring and showed a similar pattern. Mean effects range-
median quotients for PV and LA were 16 and 0.1, respectively,
whereas the quotients for OC, SD, and DP were lower and
study conducted by Brown et al.  showed lower polycyclic
aromatic hydrocarbon levels in fish collected at DP, when
compared with fish collected near wastewater outfalls and other
coastal areas with contaminant inputs.
A diverse suite of CECs and legacy contaminants were
measured in effluent, seawater, sediment, and fish liver. The
analytes included pharmaceuticals, personal care products,
industrial and commercial compounds, and current-use pesti-
cides. A complete description of these measurements can be
found in Vidal et al.  and in Maruya et al. . Not all
analytes were measured in all types of samples because of
limitations in sample size, analytical methods, and low prob-
ability of occurrence in a given matrix. Legacy contaminants
(PCBs and chlorinated pesticides) were only measured in sedi-
ment and liver samples, because the effluents are currently not a
significant source of these compounds.
Effluent and seawater. A total of 56 analytes, including
pharmaceuticals, personal care products, hormones, current
use pesticides, and industrial and commercial compounds, were
measured in effluent and seawater samples. Fifty of those
analytes were analyzed on a quantitative basis, and six on a
semiquantitative basis . Each wastewater sample consisted
of a 2-L peak flow grab. Effluent samples were collected in two
1-L glass, silanized, amber bottles following procedures
described in Vidal et al. . The seawater samples were
collected from areas near the publicly owned treatment work
(POTW) effluent discharges. A Teflon-coated Niskin bottle was
used to collect seawater, then the samples were placed into two
1-L glass, silanized, amber bottles. Seawater samples were
taken at site-specific depths: 61m at LA, 59m at PV, 54m
at OC, 53m at DP, and 95m at SD.
Samples were extracted using 500mg hydrophilic–lipophilic
solid-phase extraction (SPE) cartridges. Extractions were per-
formed using an Autotrace automated SPE system (Zymark
concentrated with a gentle stream of nitrogen (N), part of it was
placed into a vial for liquid chromatography tandem mass
spectrometry analysis, and another part was retained for
solution was extracted twice; the resulting extracts were com-
and internal solutions of standards were added. Complete details
for sample extraction methods have been previously published
A Varian CP-3800 Gas Chromatograph with a CP-8400
autosampler and Varian 2200 mass spectrometer was used for
all gas chromatography tandem mass spectrometry analyses.
Mass spectrometry was performed using multiple-reaction
monitoring in positive electron impact mode for all analytes.
Liquid chromatography–tandem mass spectrometry was used
to analyze nonsteroid compounds  and steroid compounds
. Six compounds were analyzed on a semiquantitative
basis, which included acetaminophen, erythromycin, hydro-
codone, oxybenzone, ibuprofen, and iopromide . Liquid
chromatography–tandem mass spectrometry was performed
using an API 4000 triple quadrupole mass spectrometer
(ABSCIEX) equipped with an Agilent 1100 LC and HTC-
PAL autosampler (CTC Analytics).
For quality control, labeled surrogates were added before
SPE to monitor analyte recoveries and matrix interference. The
isotope surrogates used represented a variety of compound
classes. Matrix spikes were applied to effluent and seawater
samples, with percentage of recoveries ranging from 80 to
120%. Reporting limits were designated based on a value of
three to five times the method detection limit and set as the
lowest calibration point for each analyte. The reporting limits
ranged from 0.0002 to 0.05mg/L. For gas chromatography–
tandem mass spectrometry, analyte quantitation was conducted
with internal standard calibrations using linear or quadratic
regression with 1/x2weighting. Liquid chromatography tandem
mass spectrometry quantitation was conducted with isotope
dilution with linear or quadratic regression with 1/x2weighting.
The calibration points for gas chromatography–tandem mass
spectrometry were at 10, 25, 50, 100, 250, and 500mg/L,
whereas liquid chromatography–tandem mass spectrometry
calibration points were at 1.0, 2.5, 5.0, 10, 25, 50, 100, and
200mg/L. Correlation coefficients were consistently 0.99 or
greater. Additional quality assurance and quality control meth-
ods are described elsewhere [29,30].
Sediment and tissue. Three sediment grabs were collected
within 100m of the original station coordinates to a depth of
2cm using a Van-Veen–type grab deployed from a support
research vessel. Sediment from each grab was removed with a
precleaned stainless scoop and placed into a stainless steel bowl
for homogenization into a composite sample. An aliquot of the
composite sample was then transferred to a 500-ml I-Chem
Integrated coastal effects study
Environ. Toxicol. Chem. 31, 20122713
glass jar and immediately placed in an ice-filled cooler.
Between grabs, all stainless steel implements were carefully
cleaned with site water, deionized water, and rinsed with
pesticide grade methanol. On delivery to the laboratory, each
sediment composite sample was immediately frozen at ?208C.
Pleuronichthys verticalis livers were excised from individ-
ual fish collected with a 7.6-m-wide semi-balloon otter trawl
and segregated by size (retaining those conforming to the 75%
size rule), sex, and station for a total of 10 composite samples
(five female/five male; 5g minimum wet tissue each). A differ-
ential Global Positioning System was used to locate the sam-
pling sites and to control the trawling speed at 50 to 60m/min.
Each trawl lasted 10min, covering distances ranging from 500
to 600m. A methanol-rinsed stainless steel scalpel was used to
of 15 and 23 individual livers were composited for each female
and male sample, respectively. Composite samples were stored
in precleaned 500-ml glass I-Chem jars and kept frozen at
?208C during shipping and before analysis.
A suite of 98 compounds were analyzed in sediment and 79
in tissue . Analytes measured included 48 legacy organo-
chlorines (such as PCB congeners, chlordanes, DDTs), four
butyltin compounds (only in sediment), 21 current-use pesti-
17 industrial and commercial compounds (PBDEs, phthalates,
and 4-nonylphenol), five pharmaceutical or personal care prod-
ucts (carbamazepine, diazepam, oxybenzone, simvastatin, and
triclosan), and three hormones (estrone, 17b-estradiol and 17b-
ethinyl estradiol). In tissue samples, only the synthetic hormone
17b-ethinylestradiol was analyzed.
Different methods were employed to extract and analyze
the sediment and tissue samples. For example, legacy organo-
chlorines, urea herbicides (including diuron), 4-nonylphenol,
and PBDEs were extracted from samples by accelerated or
sequential solvent extraction and analyzed by gas chromato-
graphy with electron capture or mass spectrometric detection
or by liquid chromatography–mass spectrometry. Compounds
such as organophosphate and triazine pesticides were extracted
and isolated by SPE and analyzed with gas chromatography
with electron capture or with thermionic-specific detection.
Analytical methods for the PPCPs are described in detail else-
where . An Alliance 2695 Quattro Micro liquid chromato-
graphy tandem mass spectrometry operating in the electrospray
ionization (ESI) negative ion or multiple reaction monitoring
ESI positive ion modes was used to analyze hormones and
ance analysis was conducted and included blanks and matrix
spikes for both sample types. Method detection limits ranged
from 0.1 to 50mg/kg. Mean recoveries of analytes spiked into
sediment and tissue ranged from 62 to 99%. Further details
regarding the methods and procedures used for the analysis of
sediment and tissue samples can be found in Maruya et al. .
Multiple biological indicators representing different levels
fish. The parameters were selected to link highly sensitive
molecular responses to more ecologically relevant measures
such as reproduction and survival.
After the fish were collected, they were each placed in
individual buckets with seawater. Then each fish was measured
and weighed. Subsequently, a heparinized syringe was used to
obtain blood from the caudal artery. The blood was placed in
vials and centrifuged for plasma isolation. The next step was
humane sacrifice via cervical dislocation, and then dissection to
remove the tissues needed for subsequent analysis. After
plasma, liver, and gonad were removed, the samples were
preserved. The gonad was fixed in Dietrich’s solution for
histopathology analysis. The blood plasma and liver samples
at ?208C until further analysis.
Blood plasma samples were used to determine the concen-
ketotestosterone [11-KT]), development (thyroxine), and stress
response (cortisol). The concentration of VTG was also meas-
ured in plasma. Reproductive hormones and VTG were meas-
ured in all fish, whereas thyroxine and cortisol were only
measured in samples from the spatial study component. Sam-
pling and preservation methods for biological samples can be
found in Vidal et al.  and Forsgren et al. .
One half of the gonad from each fish was preserved for
histological analysis of sex, maturity state (based on the degree
of gonad development ), and abnormalities in sexual devel-
opment (e.g., presence of eggs in male gonad). The remaining
half of the gonad was weighed to calculate the half gonadal
somatic index (0.5 GSI), a measure of reproductive status. The
0.5 GSI was calculated as the ratio of the sampled gonad weight
divided by the total body weight of the fish.
Concentrations of estradiol and testosterone were measured
by radioimmunoassay using labeled estradiol or cortisol. These
analyses were conducted with a Perkin-Elmer Cobra II gamma
counter(Packard Instruments).Plasmaconcentrations of11-KT
and thyroxine were measured by enzyme immunoassay using a
microplate spectrophotometer (Spectramax R250, Molecular
Devices). The intra-assay precision ranged between 7.4 and
11.2%, whereas the inter-assay precision ranged from 8.1 to
8.6%. Further information regarding plasma hormone analysis
can be found in Reyes et al . Plasma VTG was measured by
enzyme-linked immunosorbent assay as described in Rempel
et al.  and Forsgren et al. . Plasma VTG concentration
was normalized to the total protein concentration in the plasma
(ng/ml protein). This normalization was used to reduce varia-
bility among samples and the number of false positives.
Trawl-caught fish diversity and abundance data were used to
calculate the fish response index, which measures pollution
stress in demersal fish communities. The fish response index
values indicate whether the species composition is similar to
that characteristic of reference conditions. Fish community data
were obtained from outfall site monitoring datasets and from
regional monitoring studies conducted between 1998 and 2008
for fish collected near DP. Further information about fish
response index methods can be found in Allen et al., 2001
and 2011 [35,36].
Summary statistics and analysis of variance were used to
analyze the data. Chemical measurements in effluent and sea-
water samples were summed by chemical group (e.g., hor-
mones, PPCPs, ICCs; group members listed in Supplemental
Data, Table S2), averaged by station, and analyzed using one-
way analysis of variance (p<0.05). Half of the detection limit
was substituted for constituent concentrations reported by the
laboratory as not detected. Station averages of hormones and
VTG concentrations were also compared using analysis of
variance. Samples with significant differences by analysis of
variance were subsequently analyzed using a Tukey test to
identify differences among specific sites.
Environ. Toxicol. Chem. 31, 2012 S.M. Bay et al.
RESULTS AND DISCUSSION
The present study represents the most comprehensive inves-
tigation to date of CECs and their effects in coastal offshore
waters. Thousands of chemical and biological measurements
were made during the present study; detailed results can be
found in related publications [23,25,33,34,37]. The key findings
are described in the present study, organized with respect to the
study questions. The data created by the present study are
available in a relational database (www.sccwrp.org).
What types of CECs are discharged into the SCB from municipal
Diverse types of CECs were discharged into the SCB from
municipal wastewater outfalls. Most of the target PPCPs, ICCs,
and hormone analytes were frequently detected in effluent
samples from each of the four wastewater treatment facilities.
Of the 31 PPCPs measured, 11 were detected in every sample
analyzed, regardless of treatment level. Five ICCs and one of
the hormones were also detected in 100% of the effluent
Effluent CEC concentrations were low, with values less than
compounds were lower than available toxicity thresholds .
Concentrations of individual constituents were variable among
effluent types in some cases, and showed no consistent trend
between sampling times or effluent types. However, the total
concentration of PPCPs and ICCs varied significantly among
effluent types (Fig. 2), with the lowest concentrations in the
effluents that received full secondary treatment (LA, PV). This
trend is consistent with the results of other studies that show
greater removal of CECs with longer treatment plant residence
times [39,40]. Influent CEC concentrations were not measured
in the present study; trends in effluent CEC concentrations
could therefore reflect differences in waste input characteristics
among geographic regions.
Are SCB marine life exposed to CECs from municipal wastewater
The results indicated that fish are likely exposed to CECs
from effluent discharges through multiple pathways. Some
PPCPs and ICCs were detected in seawater samples collected
near the ocean floor at the fish sampling locations (Table 1).
Only a small proportion of the target analytes were detected in
seawater, and the concentrations were 400 to 1,000 times lower
than those present in the effluent, which is consistent with the
expected dilution of the effluent on discharge. These seawater
concentrations (usually less than one part per trillion or nano-
grams per liter) were generally near the analytical detection
limits for the compounds .
Fewer compounds were detected at DP (n¼7) than at the
effluentdischargeareas(n¼17).These CECsoccurred atlower
concentrations at DP than at the effluent discharge areas (con-
centrations ranged from 0.0004 to 0.1mg/L). The detection of
Fig. 2. Average total concentration of pharmaceutical and personal care
(PPC) products (þstandard error) in quarterly wastewater effluent samples.
The percentage of secondarytreatmentis shownfor each effluent typeat the
each other. LA¼Santa Monica Bay; PV¼Palos Verdes; OC¼Orange
County; SD¼San Diego. [Color figure can be seen in the online version of
this article, available at wileyonlinelibrary.com]
Table 1. Effluent and seawater occurrence and median concentrations (mg/L) for chemicals detected in seawater samplesa
Chemical groupChemical useChemical name
Median Occurrence (%)Median Occurrence (%)
aA list of all the contaminants of emerging concernanalyzed in effluentand seawater is shown in Supplemental Data, Table S2. Only those compounds detected
in seawater are included in the table. The compounds have been organized by chemical group: current-use pesticides, hormones, industrial and commercial
compounds, and pharmaceutical and personal care products. This table presents modified data from Vidal et al .
bMedian was not calculated because of low frequency of detection.
PPCP¼pharmaceutical and personal care products; ICC¼ industrial and commercial compounds.
Integrated coastal effects study
Environ. Toxicol. Chem. 31, 20122715
some CECs at DP could reflect transport from effluent dis-
charges (e.g., transport by the eddies formed by the California
undercurrent of the California Current System ). Other
contaminant sources could also be responsible for the presence
the site or urban runoff.
those expected to produce short-term toxic effects. For exam-
ple, the U.S. EPA seawater aquatic life water toxicity threshold
for nonylphenol is 1.7mg/L , and the maximum concen-
tration found in the seawater samples was 0.23mg/L. However,
evaluation of potential chronic effects for CECs is uncertain
because aquatic life toxicity thresholds have only been devel-
oped for a few of these compounds, and little research has been
done to determine the effects of mixtures of CECs in environ-
Sediment contamination is a likely pathway of fish exposure
to some CECs. Sediment samples from all locations, including
the DP reference site, contained triclosan (antimicrobial) and
nonylphenol (surfactant), and the LA, PV, and OC sediments
from all of the sites also contained PBDEs and nonylphenol
 (Table 2). Some chemicals that were not detected in the
sediment were found in the livers of fish from the same site,
such as PCBs (SD and DP stations) and diazepam (all stations;
). This finding highlights the fact that even if a contaminant
is not found at detectable levels in sediment or seawater, the
contaminant may still be present in the environment and able to
be accumulatedby organisms inthat area. Onlyapartial suite of
PPCPs were analyzed in the liver and sediment samples, so no
conclusion can be made regarding the accumulation of other
PPCPs in fish.
The sediment and tissue data also illustrate that hornyhead
turbot were exposed to multiple legacy contaminants at con-
pesticides). This pattern of exposure is the result of historical
discharges that produced widespread contamination in the SCB
. Because chlorinated pesticides and PCB compounds have
the potential to cause endocrine disruption and other effects
often associated with some CECs, these legacy contaminants
must be considered when evaluating the biological effects of
CECs in the SCB. Sediment and tissue levels of legacy con-
taminants for the DP site were consistently among the lowest
concentrations measured, indicating the suitability of this
station as a low contamination reference in the present study.
Is there evidence of endocrine disruption or other physiological
effects in SCB fish?
The presentstudy detected severalmolecular-level
responses associated with physiological changes in hornyhead
turbot. The relationship of these changes to contaminant expo-
sure and endocrine disruption cannot be established without
further study. However, no adverse impacts on fish condition or
reproduction were found.
Hormones and VTG. Potentially abnormal variations were
observed in some blood plasma indicators. Yet, in most cases
these responses were found at all sites, including the reference
site, and could not be directly associated with effluent dis-
charges. These widespread responses included the frequent
detection of low levels of VTG in male fish, and high concen-
trations of estradiol in male fish relative to females (Fig. 3).
More than half of the male hornyhead turbot sampled
contained detectable concentrations of VTG in plasma. Male
VTG concentrations were generally 1,000-fold lower than
females and unlikely to disrupt reproduction, but they may
be indicative of fish exposure to estrogens in the environment.
Male fish from the SD site had significantly higher concen-
trationsofVTGrelativetoPVand LAfish,butnodifferences in
female VTG were present. Male hornyhead turbot caught at DP
also contained similar concentrations of VTG when compared
with males collected near the effluent discharge areas. These
results suggested that the presence of plasma VTG in male fish
was not associated with current outfall discharges.
Unexpectedly high concentrations of plasma estradiol in
male hornyhead turbot were observed at all study sites. Estra-
diol concentration in males is lower than that in females for
most fish species; yet, male hornyhead turbot estradiol concen-
trations were similar to or greater than those of females (Fig. 3).
The high relative estradiol levels in males could have been
attributable to low female estradiol levelsresulting from diurnal
fluctuations related to post-spawning conditions because this
species is an asynchronous spawner . However, diurnal
fluctuations are an unlikely explanation, because even pre-
spawning females (which had the highest estradiol concentra-
tions) had estradiol levels similar to or lower than the concen-
trations observed in males. In addition, this pattern of relatively
high male estradiol concentrations was observed during all
sampling events, regardless of the level of spawning activity.
Elevated estradiol in males did not appear to be associated
with outfall discharges, as a similar pattern was detected at the
DP reference site. Measurements of estradiol in other species of
southern California flatfish do not show this unusual pattern
, although flatfish species from other areas have shown a
similar pattern of relative estradiol concentration . This
pattern may either represent a widespread response to environ-
mental factors or be a normal characteristic of the species.
Statistically significant differences in estradiol concentra-
tions among stations were observed. Estradiol concentrations in
fish (either males or females) from LA, PV, and OC were
approximately half of those in fish from SD and DP (Fig. 3)
. This trend may represent a response to historical outfall
Table 2. Concentrationsofselectedlegacycontaminantsandcontaminants
of emerging concern in sediment and liver tissue (mg/g)a
Stations OC pesticidesPCBs PBDEs Nonylphenol
oethane (DDD), dichlorodiphenyldichloroethylene (DDE), dichlorodiphe-
nyltrichloroethane (DDT), dichlorodiphenylmonochloroethylene (DDMU),
dieldrin, endrin, heptachlor, heptachlor epoxide, lindane, methoxychlor,
oxychlordane, nonachlor, and toxaphene (OC Pesticides); PCBs¼sum of
tissue (triclosan was not detected in tissue; diazepam was not detected in
sediments). Table modified from Maruya et al .
polybrominated diphenyl ethers; LA¼Santa Monica Bay; PV¼Palos
Verdes; SD¼San Diego; DP¼Dana Point; ND¼Not detected.
Environ. Toxicol. Chem. 31, 2012S.M. Bay et al.
discharges, as fish from LA, PV, and OC also have higher
concentrations of legacy chlorinated hydrocarbon contaminants
in their tissues, which can have antiestrogenic effects, as has
been observed in fish from other areas .
No consistent pattern in the plasma concentration of 11-KT,
the principal form of testosterone in fish, was present among the
outfall sites (Fig. 4). This hormone regulates reproduction and
growth in fish. As expected, males had significantly higher
concentrations of the androgen as compared with females.
Although the concentration of 11-KT varied approximately
threefold among stations in both sexes, a significant difference
was only present in PV females. The observed variation in
11-KT concentrations may represent differences in reproduc-
tive stages of the fish among sites. Legacy contamination is
unlikely to be responsible for the variations in 11-KT, because
the hormone concentrations do not correspond to trends in fish
tissue chlorinated hydrocarbon exposure among the sites.
Evidence of a regionwide inhibition in the stress response
system of hornyhead turbot was observed in the present study.
The hormone cortisol is normally produced in response to
stress, such as that resulting from the fish capture and handling
methods used in the present study. Cortisol concentrations in
hornyhead turbot from all sites were less than half the concen-
tration observed in other fish species subjected to a similar
degree of stress (Fig. 4). These results may be indicative of
chronic stress, which is known to diminish the ability of fish to
produce cortisol in response to stress, or could be attributable to
contaminant impacts on the cortisol-producing endocrine tissue
The average concentration of thyroxine was reduced in fish
from each of the outfall sites relative to the reference site
(Fig. 4), particularly at the PV site, where the greatest legacy
contamination exists. Recent studies in San Francisco Bay have
also observed reduced thyroxine levels in two species of fish
from contaminated locations and associated with the changes
with PCB bioaccumulation . Thyroid hormones have
important roles in regulating growth, early development, and
metabolism. Reduced levels of thyroid hormones could lead to
impairment of physiological functions essential to the well-
being and survival of the organism. This is the first report of
thyroxine concentrations in hornyhead turbot; whether this
pattern is present at other times of the year or in other southern
California species is unknown.
Gonad histopathology and feminization. No evidence of
feminization or abnormal sexual differentiation was observed
in the present study. Histological analysis of the gonads found
no instances of feminization (e.g., presence of developing eggs
in male gonad) out of 373 male fish examined in both the spatial
and temporal components of the present study.
Atresia (oocyte degeneration) was observed in females from
all stations. The incidence of atresia was low (20% or less) at all
sites and did not show any apparent relationship with effluent
discharges or legacy contamination. The presence and severity
of atresia seemed to correspond to the fish reproductive stage,
as a normal feature of the reproductive cycle. Atresia can be
caused by a disruption in steroidal signaling .
Wide variations in male to female sex ratios were observed,
Fig. 3. Averagevitellogenin(VTG)andestradiolconcentrationsinmaleandfemalehornyheadturbot.Barswithsameletterarenotstatisticallydifferentfromeach
other. The numbers on top of the bars indicate the percentage of males with detectable VTG. Samplescollected in May and June 2006. LA¼Santa MonicaBay;
PV¼Palos Verdes; OC¼Orange County; SD¼San Diego; DP¼Dana Point. [Color figure can be seen in the online version of this article, available at
Integrated coastal effects study
Environ. Toxicol. Chem. 31, 2012 2717
as sampling variability and sex-specific aggregation behavior.
Reproductive cycle. Analyses of quarterly fish collections at
selected sites were used to compare temporal changes in
hormones and gonad condition as an indicator of subtle effects
on reproduction among sites. Female fish tended to be sexually
mature (larger gonads) during the May to August sampling
periods, as indicated by their maturity grade (Fig. 5). This trend
developing eggs in the gonad. During the May and August
sampling periods, the females had the greatest percentage of
percentage of vitellogenic oocytes ranged between 6 and 18%,
from 0 to 9% in August, between 0 and 3% in November and
from 0 to 14% in January and February. The reproductive cycle
of males was similar to that of the females, in general. Females
from OCF (farfield site for OC discharge site) did not exhibit
this general reproductive cycle. Little variation wasfound in the
GSI or gonad maturity of OCF females throughout the year.
Male and female plasma VTG concentrations also varied
temporally. The VTG variation generally corresponded to
variations in GSI and maturity grade, especially in females.
Little temporal variation in female VTG concentrations was
observed at OCF,a finding consistent with the GSI and maturity
state data, and perhaps evidence of an impaired reproductive
cycle at this station.
In males, variation in 11-KT concentrations corresponded to
the other measures of the male reproductive cycle (e.g., matur-
ity grade), with elevated levels in May and June at all sites
(Fig. 5). In females, the androgen was present at very low levels
and did not correspond with other measures of the female
reproductive cycle (as expected for females).
In contrast to 11-KT, the concentrations of estradiol showed
little similarity to other measures of the reproductive cycle.
Estradiol was high in both sexes, at levels expected in repro-
differences. These results cannot be directly associated with
exposure of hornyhead turbot to effluent contaminants, because
they were evident at both discharge and reference sites. How-
ever, the duration and sampling frequency for the assessment of
reproduction cycles was limited (e.g., one year and four sam-
pling events) and may not have been sufficient to detect subtle
changes in reproductive cycles between sites. Additional
research needs to be conducted to better understand estradiol
concentration changes that are responding to diurnal fluctua-
tions as batches of eggs are spawned.
The present study and another recent study seem to indicate
an extended reproductive season for hornyhead turbot. Deng
et al.  reported early signs of spawning activities in
February and higher percentages of spawning/post-spawning
individuals through August, suggesting an extended (spring
through summer) spawning period. Forsgren et al. 
reported that during the spring season, female ovarian devel-
opmental stages were characterized by a high percentage of
spawning and post-spawning stage follicles when compared
with females collected in other seasons. Additional work is
needed beyond the scope of the present study to further
understand reproductive seasonality.
Fig. 4. Average11-ketotestosterone,cortisol,andthyroxineconcentrationsinmaleandfemalehornyheadturbot.Cortisolandthyroxineconcentrationsrepresent
combined data for males and females. Bars with same letter are not statistically different from each other. Samples collected in May and June 2006. LA¼Santa
Environ. Toxicol. Chem. 31, 2012 S.M. Bay et al.
Fish condition. Overall measures of fish condition, the
condition factor (CF) and liver somatic index (LSI), generally
corresponded with the reproductive cycle of hornyhead turbot.
Variations among sites in specific parameters were observed,
however. Fish from OC, OCF, and DP (males) had the highest
CF during the period of higher reproductive activity, whereas
PV and PVF did not. Relative liver size (LSI) varied among
sites, with the highest LSI values in PV fish at all time periods
(Fig. 5). Elevated LSI values at PV may be related to increased
exposure to chlorinated hydrocarbons, which has been associ-
ated with liver enlargement in fish .
Are these effects associated with either historical or current
municipal wastewater discharges?
The association of the molecular responses observed in the
present study with municipal wastewater discharge is uncertain
for most parameters. Biological responses such as reduced
cortisol response, VTG production in males, and comparable
estradiol levels in males and females were found at both the
discharge and reference sites, indicating little relationship to the
presence of effluent discharge or historical sediment contam-
ination patterns. If these responses are attributable to chemical
exposure, then this exposure must be widely distributed
throughout the southern California Bight ecosystem and may
have multiple sources. Bight-wide chemical exposure at low
levels doesoccur, as shown bysediment and tissue analyses. An
alternative explanation for the cortisol, VTG, and estradiol
results is that they represent normal, but unusual, characteristics
of hornyhead turbot. Additional analyses of hornyhead turbot
from reference areas and laboratory studies are needed to
determine the normal range of variation for the molecular
indicators used in the present study.
Two molecular indicators did show an apparent association
with multiple municipal wastewater discharge sites: thyroxine
and estradiol. Hornyhead turbot thyroxine concentrations at all
four discharge sites were less than in fish from DP. Fish thyroid
hormone production is known to be reduced as a result of
exposure to several types of contaminants that are more prev-
alent near outfall sites, such as PCBs and polybrominated
diphenyl ethers (PBDEs) [52,53]. The thyroxine results need
confirmation, because the results are based on a single collec-
tion event in May and June 2006. Whether this reduction at
outfall discharge sites persists over time or occurs at other
locations is not known.
Reduced plasma estradiol concentrations were observed in
fish from those sites, with the highest concentrations of con-
taminants in the sediment: LA, PV, and OC. Quarterly samples
for LA and OC confirmed the trend for estradiol (Fig. 5),
suggesting that this response may be related to contaminant
exposure. Legacy contamination is a potential cause of the
estradiol response, because the reduced concentrations were
only present at discharge sites with substantial legacy contam-
ination and higher quality effluents (LA, PV, OC), and not
present at SD (lower legacy contamination).
In contrast, increased male plasma estradiol concentrations
were present at only the SD outfall site where both legacy
contamination and level of treatment are relatively lower. The
association of the SD estradiol response with municipal waste-
observed at other discharge sites, and there were no repeated
measurements over time at SD. Additional samples of SD fish
need to be analyzed to determine whether the plasma estradiol
results represent a site-specific response, as opposed to normal
variations in the physiology of hornyhead turbot.
Are specific chemicals responsible for the effects?
No specific associations between individual chemicals and
biological effects can be determined from the present study.
The responses observed for estradiol, cortisol, and thyroxine
are not diagnostic for a single chemical type. The ability to
evaluate chemical-specific associations in the present study
was limited because tissue chemical analyses were conducted
on composites rather than on individual fish. Without chem-
ical data on individuals, a robust statistical evaluation of
possible cause–effect relationships cannot be conducted. Stat-
istical associations with chemicals also need to be confirmed
by controlled laboratory exposure studies, because the stat-
istical associations may be attributable to correlations with
unmeasured chemicals or environmental factors. The mixture
of exposure from legacy and current discharges also compli-
cates determination of chemical linkages. Similar impacts
on hormone concentration can be caused by both legacy
contaminants (e.g., DDTs, PCBs) and CECs (e.g., PBDEs,
The important role of legacy contaminants in some of these
responses is suggested by the plasma estradiol results. This
molecular indicator showed patterns of response associated with
the LA, PV, and OC sites, where legacy sediment contamination
and effluent quality is greatest. A role in the biological responses
Fig. 5. Temporaltrendsinthepercentageofmaturity,estradiol,and11-keto
testosterone (11-KT) concentrations, and liver somatic index (LSI) of male
and female hornyhead turbot. PV¼Palos Verdes; PVF¼Palos Verdes far-
field station; OC¼Orange County; OCF¼Orange County far-field station;
DP¼Dana Point. [Color figure can be seen in the online version of this
article, available at wileyonlinelibrary.com]
Integrated coastal effects study
Environ. Toxicol. Chem. 31, 20122719
observed is possible, because legacy contaminants such as
PCB and DDTs were detected in tissue (Table 2); these legacy
contaminants are known for altering estradiol levels in fish
Are the biological effects adversely impacting fish populations?
The biological responses observed in the present study did
not appear to be associated with reduced hornyhead turbot
reproduction or survival. The gender ratio (relative proportion
of male and female fish) of hornyhead turbot varied among
sampling events and sites, but did not show a consistent trend
indicative of altered sexual differentiation. In general, gender
ratios were skewed in some samples of fish from the outfall
sites, but this type of pattern was also observed in fish collected
at Dana Point. In addition, no gender ratio patterns were seen
that were consistent over time . Furthermore, pathology
studies showed no feminization incidence in male fish; of the
373 testes analyzed in the present study none had testis-ova
(presence of early oocytes in testis).
Lack of evidence for fish feminization is consistent with the
revised results from the Bight 2003 regional survey. The 2003
study examined 42 hornyhead turbot males collected through-
out the Southern California Bight. The findings of the present
study initially reported a seemingly high incidence (12%) of
intersex (presence of developing eggs in male gonad) and noted
that all five males with presumed intersex were collected near
POTW discharges. In addition, intersex was also reported to be
present in 8% of English sole (Pleuronectes vetulus). However,
reanalysis of the tissue samples determined that the presence of
oocytes in all but two of hornyhead turbot and all of the English
sole classified as having intersex was the result of sample
contamination introduced in the field or laboratory.
Analysis of long-term monitoring data for the study sites
indicated that hornyhead turbot populations are stable or
increasing throughout the region . Statistically significant
differences in the average abundance of hornyhead turbot were
present among each of the discharge sites, but in all cases, the
abundance in the 2000s was greater than in previous decades.
Analysis of long-term monitoring data from PV and OC indi-
cates that annual variability in hornyhead turbot abundance
appears to be related to variations in ocean temperature, as the
relative abundance of hornyhead turbot tends to increase when
coastal water temperature is lower (M.J. Allen, Southern
California Coastal Water Research Project, Costa Mesa,
California, USA, personal communication).
Fish communities were healthy at the study sites, as
indicated by the fish response index values. The species com-
position and abundance of demersal (bottom-associated) fish
measured in recent monitoring surveys was typical of that
expected in unimpacted reference areas of the SCB (Fig. 6)
. These results are consistent with Bight 2008 regional
monitoring data, which indicate that the condition of offshore
fish communities throughout the SCB is equivalent to that of
reference areas .
Most of our knowledge regarding CEC exposure and effects
in southern California is limited to offshore coastal habitats and
two species of flatfish. Similar studies are needed for other
habitats with high potential for CEC exposure (e.g., estuaries
and effluent-dominated rivers) and other species to provide
better context to determine constituents, areas, and responses of
Tables S1-S2 (33 KB DOC).
Acknowledgement—The present study could not have been accomplished
without the collaboration of local water quality management agencies and
universities, and the existence of adaptive National Pollutant Discharge
Elimination System monitoring programs in the region that provide the
flexibility to conduct special studies. The authors acknowledge funding and
field support from the City of Los Angeles (Environmental Monitoring
Division), Los Angeles County Sanitation Districts, Orange County
Sanitation District, and the City of San Diego (Public Utilities Department,
Environmental Monitoring and Technical Services Division). Financial
Program/California Ocean Protection Council (grant #06-043 to California
Armstrong, S. Johnson, T. Stebbins, and M. Baker for their assistance with
projectdesign, implementation, anddata interpretation.M. Myers (National
samples. We thank the following individuals for assistance with laboratory
analyses: D. Young, D. Greenstein, M. Mays, and B. Layton (Southern
California Coastal Water Research Project); J.Reyes (California State
University Long Beach); S. Snyder, B. Vanderford, J. Zeigler, and R.
Trenholm (Southern Nevada Water Authority); K. Armburst and K. Xia
(Mississippi State Chemistry Laboratory); and J. Wolf (Experimental
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