Environ. Sci. Technol. 2004, 38, 6385-6395
Reproductive Responses of Common
Carp (Cyprinus carpio) Exposed in
Cages to Influent of the Las Vegas
Wash in Lake Mead, Nevada, from
Late Winter to Early Spring
E R I N M . S N Y D E R , * ,†
S H A N E A . S N Y D E R , †,‡ K E V I N L . K E L L Y , §
TIMOTHY S. GROSS,O
D A N I E L L . V I L L E N E U V E , †,|
SCOTT D. FITZGERALD,⊥
S E R G I O A . V I L L A L O B O S , †,# A N D
JOHN P. GIESY†
Department of Zoology,
National Food Safety and Toxicology Center, and
Center for Integrative Toxicology, Michigan State University,
218C Food Safety and Toxicology Building,
East Lansing, Michigan 48824, U.S. Bureau of Reclamation,
Ecological Research and Investigations (D-8220),
Denver Federal Center, P.O. Box 25007,
Denver, Colorado 80225, U.S. Geological SurveysBiological
Resources Division, Florida Caribbean Science Center,
7920 NW 71st Street, Gainesville, Florida 32653, and
Department of Pathobiology and Diagnostic Investigation and
Diagnostic Center for Population and Animal Health,
P.O. Box 30076, Lansing, Michigan 48909-7576
in LW influent. Male carp caged at LW had slightly
greater concentrations of plasma VTG than those at other
sites, and a modest elevation in plasma E2 was observed
in male carp at LX and LW, but causes other than contaminant
exposure cannot be ruled out. Water temperature
differences among sites complicated interpretation of the
results. Variation in some end points among carp at
two different reference sites supports the use of multiple
reference sites in field studies of the effects of endocrinedisrupting chemicals.
Introduction
Lake Mead is a large reservoir formed by impoundment of
the Colorado River behind the Hoover Dam. The reservoir
serves as a source of domestic and agricultural water for
more than 22 million users (1) and is a popular recreation
area. The Las Vegas Wash (LW) delivers tertiary-treated
municipal wastewater effluent (87-88%), nonpotable shallow
groundwater (6%), and urban runoff (6%) from the urbanized
Las Vegas Valley (1, 2) to the Boulder Basin of Lake Mead via
the Las Vegas Bay (LX) (Figure 1). LW serves as the sole
drainage of the Las Vegas Valley hydrographic basin and
contributes approximately 1.5% of the flow into Lake Mead,
while the Colorado River provides approximately 97% of the
flow, and the Virgin and Muddy Rivers contribute an
additional 1.5%.
The Las Vegas Wash (LW) delivers tertiary-treated
municipal wastewater effluent, nonpotable shallow
groundwater seepage, and runoff from the urbanized Las
Vegas Valley to Las Vegas Bay (LX) of Lake Mead. To
investigate the potential for contaminants in LW influent
to produce effects indicative of endocrine disruption in vivo,
adult male and female common carp (Cyprinus carpio)
were exposed in cages for 42-48 d at four sites in Lake
Mead: LW, LX, and two reference locations in the lake. End
points examined included gonadosomatic index; gonad
histology; concentrations of plasma vitellogenin (VTG) and
plasma sex steroids (17β-estradiol (E2), testosterone (T), 11ketotestosterone (11-KT)); plasma estrogen:androgen ratios
(E2:T, E2:11-KT), in vitro production of T by gonad tissue,
and hepatopancreas ethoxyresorufin O-deethylase activity.
Few differences among fish caged at different sites
were potentially attributable to exposure to contaminants
In 1996, the U. S. Geological Survey (USGS) reported that
endocrine disruption was occurring in feral adult male and
female common carp (Cyprinus carpio) collected in the
vicinity of LW and LX (3). Evidence included alterations in
plasma sex steroid concentrations in male and female carp
relative to those in carp at a reference site and increased
concentrations of the estrogen-inducible plasma protein
vitellogenin (VTG) in the blood of male carp in the absence
of a concomitant increase in plasma 17β-estradiol (E2). The
latter indicates that the fish were exposed to an unidentified
exogenous estrogenic chemical (4). Previous research has
revealed increased concentrations of VTG in the blood of
carp (4-10) and other cyprinids (11, 12) exposed to municipal
wastewater treatment plant (WWTP) effluent in enclosures
or captured from riverine sites influenced by WWTP effluent.
Estrogenic chemicals found in municipal WWTP effluent and
implicated as potential causative agents for VTG induction
in male fish exposed to these effluents include the animal
steroids E2 and estrone, the oral contraceptive medication
component ethinylestradiol (EE2), and the alkylphenol
polyethoxylates (APE) and their degradation products, the
alkylphenols (AP) (5, 13-15).
* Corresponding author present address: Black & Veatch Corporation, 4040 S. Eastern Ave., Suite 330, Las Vegas, NV 89119;
phone: (702)732-0448; fax: (702)732-7578; e-mail: SnyderEM@
bv.com.
† Michigan State University.
‡ Present address: Southern Nevada Water Authority, 1350 Richard
Bunker Ave., Henderson, NV 89015.
§ U.S. Bureau of Reclamation.
O U.S. Geological Survey.
| Present address: U.S. EPA Mid-Continent Ecology Division, 6201
Congdon Blvd., Duluth, MN 55804-2595.
⊥ Department of Pathobiology and Diagnostic Investigation and
Diagnostic Center for Population and Animal Health, Michigan State
University, East Lansing, MI 48824.
# Present address: Syngenta Crop Protection, Inc., Ecological
Toxicology & Environmental Safety, P.O. Box 18300, Greensboro, NC
27419.
Extracts from water samples collected from LW and LX
in April 1997 were subjected to a bioassay-directed fractionation and analysis scheme designed to identify the bioactive
estrogenic constituents of the LW influent (16). Results
indicated that the relatively polar compounds E2 and EE2
were largely responsible for the observed in vitro bioactivity,
while relatively nonpolar compounds (polycyclic aromatic
hydrocarbons (PAHs), polychlorinated biphenyls, organochlorine pesticides) and moderately polar compounds (octylphenol, nonylphenol) were not likely to have contributed
significantly to the estrogenic activity (16). The current study
was conducted in mid-February through late March to early
April of 1999 to determine whether exposure to LW influent,
and particularly to estrogenic contaminants found therein,
causes reproductive endocrine disruption in adult carp.
10.1021/es049690n CCC: $27.50
Published on Web 11/02/2004
 2004 American Chemical Society
VOL. 38, NO. 23, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 1. Map of fish cage sites in the Boulder Basin of Lake Mead, NV.
TABLE 1. Cage Deployment and Retrieval Dates and Mean
Weights of Carp at Initiation and Completion of the In Situ
Exposure Perioda
site
sex
HF
HF
LW
LW
LX
LX
MC
MC
WB
WB
male
female
male
female
male
female
male
female
male
female
final mean
date
date
initial mean
deployed retrieved wt per fish (g)b wt per fish (g)c
na
na
02/17
02/17
02/18
02/18
02/16
02/16
02/15
02/15
na
na
04/05
04/05
04/07
04/07
04/01
04/01
03/29
03/29
na
na
163
123
187
153
184
141
173
136
160
107
159
114
178
115
189
123
171
124
sites. Two cages were placed directly at the point (site LW)
where LW enters LX such that the carp received maximum
exposure to the influent. Two cages were placed further out
in LX at a point (site LX) where the LW influent was more
dilute but still readily detected by its greater conductivity
and turbidity than main body lake water. Cages (each 2.1 m
× 2.1 m × 1.4 m deep) were suspended approximately 1.41.7 m below the surface of the water to prevent sunburn and
to avoid boat traffic and vandalism. Fish were fed approximately twice weekly by dropping a sinking pellet food
(Silver Cup trout pellets; Nelson & Sons, Inc.; Murray, UT)
through the top of each cage. Fish were observed feeding on
pellets at the reference sites, but greater turbidity prevented
observation of fish at LW and LX.
Materials and Methods
Water Quality. Water quality was monitored periodically
at each exposure site. Water temperature, pH, dissolved oxygen (DO), conductivity, and turbidity were measured using
a Hydrolab Surveyor 4 data recorder equipped with a H2O
Sonde water quality multiprobe (Hydrolab Corporation,
Austin, TX). Measurements taken at 1, 2, and 3 m depths at
each site were averaged on each sampling day to cover the
entire span of the fish cages. Water samples for measurement
of alkalinity, hardness, total ammonia, and nitrite were collected with a horizontal water sampler (Wildlife Supply
Company, Saginaw, MI) at the approximate mid-depth of the
cages at each site, and the water quality parameters were
measured with Hach test kits (Hach Company, Loveland, CO).
Caged Fish. Sexually mature, adult male and female common
carp (2-3 yr of age) were purchased from J&J Aquafarms
(Sanger, CA). Males and females were held in separate cement
holding ponds at the Lake Mead Fish Hatchery, Boulder City,
NV, for 4-7 d prior to placing them into cages in Lake Mead.
While fish were held at the hatchery, they were fed daily ad
libitum with a floating dense culture pellet food for pond
fish (Aquatic Ecosystems, Apopka, FL). Some carp were
retained at the hatchery as hatchery references (HF) to
determine initial concentrations of plasma VTG and stage of
sexual maturation of the fish at the beginning of the study.
In situ exposures began in mid-February and lasted through
late March or early April (Table 1). Two fish cages (Aquatic
Ecosystems, Apopka, FL), one containing 30 male carp and
one containing 30 female carp, were deployed at each of
four sites in Lake Mead (Figure 1). Two sites, Water Barge
Cove (WB) and Moon Cove (MC), are not influenced
significantly by the flow of LW and were considered reference
Fish Retrieval and Sample Collection. The exposure
durations for pairs of cages at different sites were: LW, 47
d; LX, 48 d; WB, 42 d; and MC, 44 d. At the end of the exposure
period, the carp were transported in aerated livewells a short
distance to shore, where they were dispatched by immersion
in 200 mg/L tricaine methanesulfonate (Tricaine-S or MS222; Aquatic Ecosystems, Apopka, FL). Blood was collected
immediately from the caudal vasculature of each fish using
a chilled, heparinized needle and syringe. For each carp, up
to 3 mL of blood was placed into a chilled polyethylene
centrifuge tube pretreated with a protease inhibitor (aprotinin; Sigma, St. Louis, MO) for a final concentration of 1 TIU
(trypsin inhibitor unit) per mL of blood. To minimize variation
in plasma sex steroid concentrations caused by diel cycling,
blood samples were collected from fish of the same sex at
different sites within the same 2-3-h time period (17). Gentle
pressure was applied to the abdomen of male fish to
determine whether milt was running. Each fish was weighed
a
wt, weight; na, not applicable; LW, Las Vegas Wash; LX, Las Vegas
Bay; MC, Moon Cove; WB, Water Barge Cove; HF, Hatchery Fish
(hatchery controls). b Calculated by dividing initial total weight of fish
(in kg) by the number of fish weighed (30 per cage) and converting to
mean weight in grams per fish. Initial total weight of carp placed in
each cage was determined to the nearest 0.01 kg by weighing a bucket
of water on a scale, then adding the fish to the bucket and re-weighing.
c Calculated by dividing final total weight of fish (in kg) by number of
fish present when the cage was retrieved and converting to mean weight
in grams per fish. Final total weight was determined by summing the
individual fish weights recorded after cage retrieval.
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ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 38, NO. 23, 2004
to the nearest 1 g and measured for standard length to the
nearest 0.1 cm.
Fish carcasses and blood samples were transported on
ice to the laboratory. The sex of each fish was confirmed by
gross observation of the gonads. A random subsample of 16
fish from each cage was selected for measurement of gonad
weights, collection of hepatopancreas samples and tissues
for histological examination, and in vitro gonad incubations.
Gonads were removed and weighed to the nearest 0.001 g
with a portable analytical balance. The caudal half of one
gonad (right side) from each fish was preserved in 10%
neutral-buffered formalin. Samples of hepatopancreas tissue
were frozen immediately in liquid nitrogen for subsequent
measurement of ethoxyresorufin O-deethylase (EROD) activity. In vitro incubations of gonad tissue for measurement
of basal testosterone (T) production (in vitro T production)
were conducted using methods similar to those described
previously (18). For each female carp, 12 × 75 mm test tubes,
each containing 20 vitellogenic follicles (0.7-1.1 mm diameter) in 0.5 mL of medium 199 without phenol red (Sigma,
St. Louis, MO), were incubated in triplicate at 16-17 °C for
18 h. For male carp, a preweighed portion of testicular tissue
(18-25 mg) was incubated in the same manner as the ovarian
follicles. Blood samples were centrifuged at 3000g for 10 min
at 4 °C. Plasma was collected, divided into several aliquots
per sample, and frozen at -80 °C. Reference carp maintained
at the fish hatchery (HF) were sampled in the same manner
14 d after the last fish cages were deployed in the lake.
Gonad Histology. From each preserved gonad sample, a
section that represented the centermost portion of the organ
was embedded in paraffin, sectioned at 5 µm, and stained
with haematoxylin and eosin. Gonad samples from 12 to 16
male carp per site and from 12 to 16 female carp per site
were examined histologically, as described previously (19,
20), and testes were designated as spermatogenically active
or inactive.
Plasma VTG. Plasma samples were analyzed for VTG with
a competitive enzyme-linked immunosorbent assay (ELISA)
described elsewhere (21). Inter-assay percent coefficient of
variation was 14.2% for a single blood plasma sample
analyzed in each ELISA run.
Sex Steroids and EROD Activity. Concentrations of the
sex steroid hormones E2, T, and 11-ketotestosterone (11KT) were measured in blood plasma and concentrations of
T were measured in gonad incubation medium by competitive radioimmunoassay as described elsewhere (22). EROD
activity in postmitochondrial supernatant prepared from
hepatopancreas tissue samples was measured using a method
adapted from previous methods (21, 23, 24).
Calculation of Indices and Statistical Analyses. Gonadosomatic index (GSI) was calculated for each fish as GSI )
[gonad weight/(body weight - gonad weight)], where all
weights are in grams. Fulton-type condition factor (K) was
calculated for each fish as K ) (W/L3) × 10 000, where W)
weight in g and L ) standard length in mm. Data were
analyzed with the aid of SYSTAT Version 9 for Windows (SPSS
Science, Chicago, IL; 1998), the SAS System for Windows
Release 7.00 (SAS Institute, Cary, NC; 1998), and SAS JMP
Version 4.0.4 (SAS Institute, Cary, NC; 2001). When data met
the assumptions of parametric statistical methods, differences
among sites and between sexes were examined by one-way
or two-way analysis of variance (ANOVA) followed by Tukey’s
HSD post-hoc comparison of means. When log transformation failed to cause data to meet the assumptions of
parametric methods, a Kruskal-Wallis test was approximated
by examining the ranks of the data by ANOVA, followed by
a Tukey-like test conducted on the ranks of the data. A
Student’s t-test or Mann-Whitney test was used to explore
differences in some end points between male and female
carp held in cages at the same site. Effects were considered
to be statistically significant at the 0.05 level of type I error
(R) for all analyses. In the few cases when plasma samples
contained VTG concentrations less than the method detection
limit (MDL) of 0.267 µg of VTG/mL, the value of one-half the
MDL was substituted for statistical analyses.
As in other fish species, gonadal sex steroid synthesis,
concentrations of plasma sex steroids, and EROD activity
vary with the stage of sexual maturation in carp (22, 25, 26).
Therefore, comparisons of plasma sex steroid concentrations,
in vitro T production, plasma VTG (because it is dependent
on plasma E2, at least in females), and EROD activity among
fish held in cages at different sites were restricted to those
carp determined to be in a similar stage of sexual maturation
within each sex. Spermiating males with running milt and
testes determined by histological examination to be spermatogenically active were considered to be in the same stage
of sexual maturation. Females with ovaries containing more
than 50% primary follicles and containing some intact tertiary
follicles were considered to be in the same stage of sexual
maturation.
Multivariate profile analysis (27) conducted with SAS was
used to examine patterns of ovarian follicles in different stages
of development (primary, secondary, or tertiary). The analysis
was conducted on arcsine-transformed proportions of follicles (parametric) and also on the ranks of these proportions
(nonparametric). The arcsine transformation was executed
to cause proportions to assume a nearly normal distribution
(28). Because this transformation does not perform well at
the extreme ends of the range of possible values, for the
proportions X/n (where X ) the number of follicles in a
particular condition and n ) 50 follicles counted), the arcsine
transformation was improved by replacing 0/n with 1/4n
and n/n with 1-1/4n, as suggested by Zar (28).
Results and Discussion
One of the major challenges in determining whether animals
are experiencing endocrine disruption in the environment
is deciding whether statistically significant changes constitute
biologically significant changes, with negative consequences
at the organism or population levels. It is particularly difficult
to assess whether changes in plasma sex steroid or VTG levels
can be considered to be adverse impacts because normal
ranges have not been established for most fish species,
particularly for wild fish. Factors such as temperature,
photoperiod, nutritional status, age, season, reproductive
cycles, and diel cycles can affect reproductive end points,
including circulating sex steroid concentrations. In this study,
the effects of these factors were minimized, and they were
considered in the interpretation of the results. Results of this
study are compared to previously reported baseline information on reproductive end points in common carp.
However, data from different studies were not collected under
a single standardized protocol, so caution should be used in
making comparisons. Variation in reproductive end points
can occur among cyprinid fish at different sites regardless
of potential impacts due to pollution (22, 29, 30); therefore,
two reference sites were included in this study.
Water Quality. Water quality parameters measured at
each site were generally within tolerance guidelines for
optimal health and growth of cyprinid fish (Table 2) (31).
Water hardness at all sites reached levels greater than the
optimum range for carp, but they can tolerate hardness
exceeding 250 mg/L (31). Hardness was greater at sites LX
and LW than at the reference sites. There were consistent
water temperature differences among sites (LW > LX > WB
> MC), with average site temperatures ranging from 17.1 °C
at LW to 13.3 °C at MC. Conductivity measurements were
greatest at LW, less at LX, and least at WB and MC, indicating
that carp caged at LW were receiving the greatest exposure
and those caged at LX were receiving less exposure to Las
VOL. 38, NO. 23, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 2. Water Quality Parameters Measured at Exposure Sites for Carp Held in Cages in Lake Mead, NVa
water quality
parameter
temperatureb
(°C)
pHb
DOb
(mg/L)
conductivityb
(µS)
turbidityb
(NTU)
alkalinityb
(mg/L)
hardnessc,d
(mg/L)
tot ammoniac
(mg/L)
site
LW
LX
MC
WB
17.1
(16.2-18.7)
8.38
(8.10-8.53)
12.02
(9.58-13.31)
1561
(1423-1636)
3.4
(2.8-4.1)
108
(107-108)
348
(336-360)
0.1
(0.0-0.2)
16.0
(15.3-16.9)
8.40
(8.08-8.61)
11.66
(9.57-12.83)
1250
(1088-1464)
2.0
(0.8-3.7)
107
(105-108)
333
(306-360)
0.2
(0.0-0.3)
13.3
(12.6-14.2)
8.14
(8.05-8.23)
9.79
(9.58-10.13)
903
(893-922)
1.6
(0.0-4.6)
109
(107-110)
270
(268-272)
0.2
(0.0-0.3)
14.5
(14.0-14.9)
8.06
(8.00-8.11)
9.70
(9.57-9.83)
889
(883-895)
0.4
(0.4-0.4)
110
(110-110)
202
(134-270)
0.1
(0.0-0.2)
a All measurements were made between March 5 and March 26, and sampling days were spread throughout the carp exposure period. Reported
values are means, with ranges in parentheses. DO, dissolved oxygen; tot ammonia, total ammonia; LW, Las Vegas Wash; LX, Las Vegas Bay; MC,
Moon Cove; WB, Water Barge Cove. b Measurements were taken at 1, 2, and 3 m depths at each site to span the depths of the cages, and a mean
was calculated for each sampling day. Reported values are grand means for three sampling days per site. c Reported values are means for two
measurements conducted on different sampling days. d As CaCO3 equivalents.
Vegas Wash influent. Conductivity measurements at the
reference sites were consistent with that of main body lake
water. Nitrite (MDL ) 2 mg/L) was not detected in any of
the water samples.
Survival and Condition. Four carp died during the
exposure: two males at WB, one male at LX, and one female
at LX. The number of fish that died was small in comparison
with other studies in which fish were held in municipal WWTP
effluents (5, 32, 33) or in rivers receiving municipal WWTP
effluents (15, 29, 34). With the exception of male carp held
at MC, the mean weight per fish in each cage was less at the
end of the exposure than it was at the beginning (Table 1),
possibly indicating that the fish were stressed by the exposure
conditions. However, this might be an artifact of the different
methods used to collect initial and final mean weight data
(see legend for Table 1). Mean K for female carp at LX was
less than that for females at both reference sites (Table 3).
The reason for this difference is not clear, but the difference
is small, and the fish at LX did not appear to be unhealthy.
Male carp at LW and LX had K values similar to those for
males from HF and MC (Table 3). Results were not different
when statistical analysis of K values was restricted to carp in
the same stage of sexual maturation.
Gonad Development and Histology. Previous studies
have demonstrated decreased GSI in male, female, and
intersex fish exposed to municipal WWTP effluent (12, 14).
After repeated seasonal sampling of feral common carp from
Las Vegas Bay and from the Overton Arm of Lake Mead, a
location not influenced by the influent of LW and thus
considered a reference site, Patiño et al. found that males
from Las Vegas Bay consistently had smaller GSI (35). In the
current study, no statistically significant differences in mean
GSI occurred among female carp held in cages at the lake
sites, but mean GSI of female carp at HF was significantly
less than that of females at each of the lake sites (Table 3).
No statistically significant differences in mean GSI were
observed among male carp held at different sites, including
HF (Table 3). Results were similar when statistical analyses
were restricted to females or males in the same stage of sexual
maturation.
Adult fathead minnows exposed to waterborne E2 showed
changes in the proportions of ovarian follicles in different
stages of development, with greater exposure concentrations
resulting in the appearance of atretic follicles and the absence
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of mature (tertiary) follicles (20). In the current study, there
were no statistically significant differences in ovarian follicle
development profile among carp held at different lake sites
(Figure 2), indicating that they were in the same stage of
ovarian development. Ovaries of all caged carp contained a
preponderance of primary follicles and more tertiary than
secondary follicles. Incidence and severity of oocyte atresia
were low. Only five of the 63 caged carp subsampled for
histological examination demonstrated atretic ovarian follicles, and in each case, only one follicle per 50 examined in
one fish was atretic. Carp with atretic ovarian follicles were
evenly distributed among the exposure sites.
Fish undergoing active gonadal growth and development,
particularly those in the middle stages of gonadal recrudescence, provide the most useful information regarding the
end points examined in this study. Sex steroid synthesis and
gonadal growth occur during that time, making it possible
to detect decreases in circulating sex steroid concentrations
or in GSI caused by exposure to endocrine-disrupting
chemicals (EDCs), while variability and sensitivity to siterelated differences in reproductive timing unrelated to
contaminant exposure are minimized (17). The carp held at
HF were sampled earlier than the carp held at the lake sites
and initially were assumed to be representative of the
condition of carp held in cages prior to their deployment in
the lake with respect to GSI and plasma VTG concentrations.
The lesser GSI in female carp held at HF relative to those in
females held in the lake suggests that the latter were
experiencing gonadal growth and development during the
exposure. However, proportions of primary follicles were less
and proportions of secondary follicles were greater in ovaries
of female carp held at HF. This difference was not anticipated
and warrants caution in making comparisons between HF
females and those caged in the lake.
Mean GSI for female carp held in cages at all sites in the
lake (Table 3) indicate that the majority of female carp were
in the middle to late stages of gonadal recrudescence and
probably not ripe for spawning, when GSI could be expected
to be approximately 17-28% (36-40), although lesser GSI
have been reported for ripe female carp (41, 42). The average
water temperatures measured at the exposure sites were less
than the optimal range of 19-24 °C that typically induces
carp to spawn in temperate regions (42-44). No indications
of ovulation (36, 37) were apparent during gross examination,
TABLE 3. Condition Factor (K), Gonadosomatic Index (GSI), In Vitro Testosterone (T) Production, and Hepatopancreas
Ethoxyresorufin O-Deethylase (EROD) Activity in Adult Female and Male Carp Held in Cages in Lake Meada
females
males
mean
Tukey
group
0.21-0.28
0.22-0.33
0.22-0.28
0.23-0.36
0.23-0.29
0.25
0.24
0.24
0.26
0.26
Condition Factor (K)
abc
HF
ab
LW
a
LX
c
MC
bc
WB
12
29
29
28
28
12
16
16
15
15
0.022-0.150
0.065-0.207
0.081-0.190
0.060-0.218
0.030-0.179
0.085
0.134
0.141
0.154
0.130
Gonadosomatic Index (GSI)
a
HF
b
LW
b
LX
b
MC
b
WB
LW
LX
MC
WB
15
16
13
14
65-119
137-387
81-194
84-160
82
239
121
120
In Vitro T Production
a
LW
c
LX
b
MC
b
WB
HF
LW
LX
MC
WB
10
15
16
13
14
5.3-15
4.2-37
4.7-25
2.6-13
4.1-14
Hepatopancreas EROD Activity (pmol mg-1 min-1)
9.5
a
HF
12
18
b
LW
15
11
a
LX
16
7.8
a
MC
16
8.8
a
WB
15
site
n
HF
LW
LX
MC
WB
12
30
27
28
29
HF
LW
LX
MC
WB
range
site
mean
Tukey
group
0.22-0.25
0.19-0.28
0.19-0.26
0.18-0.27
0.23-0.30
0.24
0.23
0.23
0.24
0.26
a
a
a
ab
b
12
16
16
16
15
0.025-0.062
0.016-0.069
0.018-0.096
0.025-0.099
0.012-0.086
0.046
0.050
0.054
0.056
0.062
a
a
a
a
a
15
16
16
15
3.8-5.9
6.8-16
4.0-9.7
3.8-8.8
5.0
12
5.8
5.9
a
b
a
a
8.5-22
7.7-50
10-29
1.2-25
1.5-27
12
24
16
8.8
12
abc
c
bc
a
ab
n
range
a Fulton-type condition factors (K) are reported for all caged carp, regardless of stage of sexual maturation. K values were calculated as K )
(W/L3) × 10 000, where W ) weight in g and L ) standard length in mm. Statistical analyses were conducted on log-transformed K values for both
sexes. GSI are reported for all fish for which gonad weight was measured. GSI ) [gonad weight/(body weight - gonad weight)], where all weights
are in g. Statistical analyses were conducted on untransformed GSI values for both sexes. In vitro T production is reported only for those carp
in the same stage of sexual maturation within each sex. For female carp, in vitro T production per 20 ovarian follicles (pg of T/20 follicles) is reported.
For male carp, in vitro T production per mg of testicular tissue (pg of T/mg of tissue) is reported. Statistical analyses were conducted on log
transformed in vitro T production values for both sexes. Statistical analyses were conducted on log transformed hepatopancreas EROD activity
values (pmol mg-1 min-1) for both sexes. Within each combination of end point and sex, fish at sites marked with common letters (Tukey groups)
were not significantly different. Differences were considered to be significant at p < 0.05. LW, Las Vegas Wash; LX, Las Vegas Bay; MC, Moon
Cove; WB, Water Barge Cove; HF, Hatchery Fish (hatchery controls).
stages of gonadal recrudescence at the end of the exposure
period.
FIGURE 2. Profiles of percentages of follicles in each of three
stages (primary, secondary, and tertiary) in the ovaries of adult
female carp held in cages in Lake Mead. Each stacked bar represents
the mean percentages of follicles in the corresponding stages.
Numbers above bars are sample sizes. LW, Las Vegas Wash; LX,
Las Vegas Bay; MC, Moon Cove; WB, Water Barge Cove.
and no post-ovulatory follicles were observed during histological examination of the ovaries.
Although the difference was not statistically significant,
the lesser mean GSI for male carp held at HF suggests that
the male carp caged in the lake were undergoing active
gonadal growth during the exposure period. On the basis of
GSI reported in male common carp or mirror carp in different
stages of sexual maturation (36-40, 45, 46), most of the male
carp used in this study appeared to be in the middle to late
All of the male carp used in this study had spermatogenically active testes, and no degenerative changes were
observed during histological examination of the testes. Only
two male carp did not produce running milt (one at MC and
one at LW) at the end of the exposure period. Sertoli cell
proliferation was observed previously in testes of fathead
minnows exposed to the natural estrogen E2 or to the
estrogenic chemical 4-nonylphenol (NP) (19, 20). Median
Sertoli cell proliferation scores were not significantly different
among male carp held in cages at different sites in the lake
or at HF. Only two male carp, one caged at LX and one at
LW, exhibited Sertoli cell proliferation. Both of these males
had only mild lesions, but the one caged at LW failed to
produce running milt. Although it is possible that these lesions
were related to contaminants in LW influent, the low
incidence and mild presentation provide little support for
that conclusion.
Four male carp were incorrectly presumed to be females
at the beginning of the study due to their failure to release
milt and lack of any other indication of male gender (nuptial
tubercles, male coloration) and were placed in cages
designated for females. None of these fish produced milt at
the end of the exposure period, and three (one at each of the
following sites: LX, MC, and WB) were later examined
histologically and found to contain spermatogenically inactive testes. The fourth fish, placed with the females at LX,
was not examined histologically. Because some of the carp
used in this study were only 2 years old, it is possible that
a few had not reached sexual maturity prior to being placed
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FIGURE 3. Concentrations of plasma vitellogenin (VTG, µg/mL) in
adult female carp held in cages in Lake Mead. Data are reported
only for carp in the same stage of sexual maturation. Closed circles
represent individual data points, and bar heights represent site
medians. Sample sizes are as follows: HF ) 10, LW ) 15, LX )
16, MC )13, WB ) 14. Nonparametric statistical analyses were
conducted, and letters above bars represent Tukey-like groupings
(p < 0.05); i.e., fish at sites with bars marked with common letters
were not significantly different with regard to plasma VTG
concentration. LW, Las Vegas Wash; LX, Las Vegas Bay; MC, Moon
Cove; WB, Water Barge Cove; HF, Hatchery Fish (hatchery controls).
in the cages. All of the other male carp examined were actively
spermatogenic, and most were spermiating (producing
running milt). It is unlikely that the presence of the
undeveloped males affected the reproductive condition of
the females with which they were held.
Intersex Carp. One carp placed with the females at
reference site MC was determined to be intersex upon gross
and histological examination, with approximately half testicular tissue and half ovarian tissue in one gonad. It is unlikely
that this single occurrence of intersex gonad resulted from
exposure to chemicals in the environment during the study
period. The fish were sexually differentiated adults prior to
commencement of the study, and concentrations of EDCs
sufficient to produce intersex gonads in adult fish were not
likely to have been encountered at the reference site.
Occasionally, intersexuality appears to occur spontaneously
under natural conditions in cyprinid fish (47).
Plasma VTG in Females. In male fish or in sexually
immature female fish, elevated plasma VTG concentration
is an indicator of exposure to estrogenic chemicals. Reduced
plasma VTG, possibly indicating endocrine disruption, has
been reported in sexually mature female carp collected
downstream from municipal WWTP effluent discharges (26).
Concentrations of VTG in blood plasma of all female carp
used in this study ranged from 0.024 to 29.7 mg of VTG/mL.
There were no statistically significant differences in plasma
VTG concentration among female carp held in cages at
different sites in Lake Mead (Figure 3), but only females at
LX had elevated plasma VTG concentrations relative to those
in females held at HF, which might be representative of initial
plasma VTG concentrations in the caged carp. Conclusions
regarding site-to-site differences were the same regardless
of whether the analysis was restricted to carp in the same
stage of sexual maturation. In a study of feral Lake Mead
carp, females caught in LX had greater plasma VTG concentrations than those caught in LW or at a reference site (3).
If greater plasma VTG concentrations in female carp at LX
are due to waterborne contaminants, it is not clear why the
more dilute influent of the Las Vegas Wash at LX might
produce greater plasma VTG concentrations than the more
concentrated influent at LW.
Excluding reference site WB, plasma E2 (Figure 4) and
plasma VTG concentrations in female carp varied together
among the Lake Mead sites, although differences among
females held at different sites in the lake were not statistically
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ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 38, NO. 23, 2004
FIGURE 4. Concentrations of plasma sex steroids 17β-estradiol
(E2), testosterone (T), and 11-ketotestosterone (11-KT) in adult female
carp held in cages in Lake Mead. All concentrations are in pg/mL.
Data are reported only for those carp in the same stage of sexual
maturation. Bar heights represent site means, and error bars are
2 SEM. Sample sizes are as follows: LW ) 15, LX ) 16, MC ) 13,
WB ) 14. Nonparametric statistical analyses were conducted on
E2 and 11-KT data. Parametric statistical analyses were conducted
on log transformed T data. Letters of the same style above bars
represent Tukey or Tukey-like groupings (p < 0.05) for a single
steroid; i.e., fish at sites with bars marked with common letters of
the same style were not significantly different with regard to the
steroid of interest. LW, Las Vegas Wash; LX, Las Vegas Bay; MC,
Moon Cove; WB, Water Barge Cove.
significant for either E2 or VTG. Concentrations of plasma
VTG in female carp were generally within the range of
concentrations previously reported for female carp collected
in a national reconnaissance study in the United States (22)
or collected from reference sites in rivers in Spain (8) and
were similar to those previously reported for female carp
collected in the spring from a reference site in Lake Mead
(less than 10 mg/mL) (3). Overall, these findings indicate
that plasma VTG concentrations in female carp held in cages
in Lake Mead were not significantly affected by LW influent.
Plasma VTG in Males. Plasma VTG concentrations in male
carp ranged from less than the MDL (0.267 µg of VTG/mL)
to 51.8 µg of VTG/mL for all male carp used in this study and
for males in the same stage of sexual maturation. The median
plasma VTG concentration in male carp at LW was significantly greater (3-10-fold) than that of males at the other
sites. The number of nondetects was small at each site: HF
(1/12), MC (1/28), WB (2/28), LX (1/29), and LW (0/29). When
the analysis was restricted to carp in the same stage of sexual
maturation, conclusions regarding site-to-site differences
were the same, and the number of nondetects per site
remained small: HF (1/12), MC (1/15), WB (1/16), LX (1/16),
and LW (0/15).
Greater plasma VTG concentrations in male carp at LW
were accompanied by greater plasma E2 concentrations
(Figure 5 and 6) relative to those in males at both reference
sites. However, male carp held at LX or at HF had similar or
greater mean concentrations of plasma E2 but did not exhibit
an increase in plasma VTG. This finding implies that male
carp held at LW were exposed to an exogenous estrogenic
substance that produced an increase in plasma VTG independent of endogenous E2. Alternatively, plasma E2 in the
fish at LX and HF might have been bound to a greater extent
by plasma binding proteins and less available to hepatocytes
(48). Water temperature also must be considered as a factor
potentially influencing VTG induction. In a study by Hernandez et al., plasma VTG was induced in male carp given
i.p. injections of E2 when they were held at 18-20 °C, but
there was no detectable response at 9-10 °C (49). The water
temperature at LW was slightly greater than the temperature
at LX (average 17.1 and 16.0 °C, respectively). It is not known
FIGURE 5. Concentrations of plasma vitellogenin (VTG, µg/mL) in
adult male carp held in cages in Lake Mead. Data are reported only
for carp in the same stage of sexual maturation. Closed circles
represent individual data points, and bar heights represent site
medians. Sample sizes are as follows: HF ) 12, LW ) 15, LX )
16, MC ) 16, WB ) 15. Nonparametric statistical analyses were
conducted, and letters above bars represent Tukey-like groupings
(p < 0.05); i.e., fish at sites with bars marked with common letters
were not significantly different with regard to plasma VTG
concentration. LW, Las Vegas Wash; LX, Las Vegas Bay; MC, Moon
Cove; WB, Water Barge Cove; HF, Hatchery Fish (hatchery controls).
whether the slight differences in temperature were sufficient
to explain differences in plasma VTG among male carp at
different sites.
The elevated plasma VTG concentrations in male carp
held at LW are not as great as those reported for feral male
carp caught at LX and LW in 1995 (3). Low milligrams per
milliliter concentrations of plasma VTG were reported in
male carp exposed to the influent of LW, as opposed to
concentrations less than the MDL of 1 mg of VTG/mL in
male carp caught at a reference site (3). Elevated concentrations and relative induction of plasma VTG in male carp held
at LW were not dramatic when compared to results from
some other studies of male carp exposed to municipal WWTP
effluent or to other potential sources of contamination
elsewhere in the United States (4) and in Japan (10). However,
in research conducted in Spain with wild adult carp collected
from rivers, more modest increases in plasma VTG concentration were associated with exposure to municipal WWTP
effluents (7-9). Plasma VTG concentrations in male carp in
the current study fall within the range of concentrations
previously reported for male carp collected upstream of
WWTP discharges (25), for apparently normal male carp
collected from rivers (6), and for male carp sampled as
laboratory controls (40, 50-52). In a recent study in Spain,
plasma VTG reached approximately 4-10 µg of VTG/mL in
adult male carp collected from reference sites upstream of
municipal WWTP discharges in rivers in March and May,
when water temperatures spanned those observed in our
study (8).
Median plasma VTG concentration for female carp was
significantly greater than that for males within each site.
Concentrations of VTG in the plasma of male and female
carp held in cages at the same site overlapped slightly and
only within reference site WB. In contrast, concentrations of
plasma VTG in feral male carp collected from LX and LW in
1995 overlapped substantially with concentrations in females
(3). Likewise, Folmar et al. reported overlapping plasma VTG
concentrations in male and female common carp collected
from a municipal WWTP effluent canal where the fish also
demonstrated alterations in sex steroid concentrations that
were indicative of endocrine disruption (4). That VTG in males
was induced to levels seen in females might indicate a more
severe effect on male carp than was observed in the current
study.
FIGURE 6. Concentrations of plasma sex steroids 17β-estradiol
(E2), testosterone (T), and 11-ketotestosterone (11-KT) in adult male
carp held in cages in Lake Mead. All concentrations are in pg/mL.
Data are reported only for those carp in the same stage of sexual
maturation. Bar heights represent site means, and error bars are
2 SEM. Samples sizes are as follows: LW ) 15, LX ) 16, MC )
16, WB ) 15. Parametric statistical analyses were conducted on
untransformed E2 data and on log transformed T and 11-KT data.
Letters of the same style above boxes represent Tukey groupings
(p < 0.05) for a single steroid; i.e., fish at sites with bars marked
with common letters of the same style were not significantly different
with regard to the steroid of interest. LW, Las Vegas Wash; LX, Las
Vegas Bay; MC, Moon Cove; WB, Water Barge Cove.
Plasma Sex Steroids in Males. The androgens 11-KT and
T are dominant sex steroids in male fish including carp (53),
and 11-KT is often considered to be the principal androgen
in carp (53-55). E2 also occurs in the blood plasma of male
fish. Changes in circulating sex steroid concentrations have
been documented in male fish exposed to municipal WWTP
effluents, but the effects are not consistent. Male walleye
collected from a natural side channel of the Mississippi River
receiving municipal WWTP effluent had elevated serum E2
concentrations (56), but E2 concentrations were not affected
in male carp collected from the same area (4). In those same
two studies, male walleye and carp exposed to municipal
WWTP effluent had depressed serum T concentrations. In
May 1995, feral male carp collected at LX, but not those
collected at LW, had depressed plasma E2 and plasma 11-KT
concentrations relative to males from a reference site (3);
plasma T concentrations were not measured.
Plasma E2 concentrations in male carp ranged from 134
to 649 pg of E2/mL for all male carp used in the current study
and for males in the same stage of sexual maturation. Male
carp held in cages at LW and LX had plasma E2 concentrations
greater than those in males held at the reference sites (Figure
6). Plasma E2 concentrations in male carp held at the
reference sites were similar to or slightly greater than those
reported previously for male carp outside the spawning
season (4, 22, 25, 57-60). Even the greater concentrations of
plasma E2 in male carp at LW and LX in the current study
are comparable to, and generally less than, concentrations
measured in feral male carp captured in May 1995 from a
reference site in Lake Mead (3). The elevation in E2
concentrations in males at LW and LX relative to the reference
sites is modest as compared to the larger changes in plasma
E2 that may take place over the reproductive cycle of male
carp. Basal concentrations of plasma E2 less than 0.3 ng/mL,
and significant peaks at 1-2 ng/mL during the spawning
season have been reported (57).
Concentrations of T in plasma of male carp ranged from
213 to 3462 pg of T/mL for all male carp used in the study
and from 241 to 3462 pg of T/mL among male carp in the
same stage of sexual maturation. There were no statistically
significant differences in plasma T concentrations among
males held at different sites in the lake (Figure 6), and site
VOL. 38, NO. 23, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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6391
means were on the lower end of the range of normal
concentrations of plasma T previously reported for male carp
outside the spawning season (4, 57-62).
Concentrations of 11-KT in plasma of male carp ranged
from 277 to 4364 pg of 11-KT/mL for all male carp used in
the current study and for male carp in the same stage of
sexual maturation. There were no statistically significant
differences in plasma 11-KT concentrations among male carp
held in cages at LW, LX, and reference site WB (Figure 6), and
concentrations were similar to those reported for feral male
carp captured from LW and from a reference site in Lake
Mead (3). Plasma 11-KT concentrations in male carp in the
current study also were greater than or similar to concentrations previously reported for male carp outside the spawning
period (25, 58, 62).
Relative proportions of plasma E2, T and 11-KT concentrations in male carp were similar among sites (Figure 6), so
absolute steroid concentrations might reflect site-to-site
differences in steroidogenic capacity related to water temperature. There were no differences in E2:11-KT ratio or E2:T
ratio among sites that would indicate an imbalance of the
male sex steroid milieu (see below).
Plasma Sex Steroids in Females. E2 and T are important
sex steroids in female teleosts (18). During gonadal recrudescence, T serves as a precursor for E2, which in turn induces
vitellogenesis (18). Although 11-KT has been detected in the
plasma or serum of the females of a wide variety of teleost
fish species, including koi carp (63) and common carp (3, 22,
25, 58), the functional significance of this hormone in female
fish is unknown. Elevated plasma 11-KT concentrations in
female common carp have been cited previously as evidence
of endocrine disruption (3), and plasma 11-KT concentrations
in female carp collected from streams in the United States
were reported to be positively associated with exposure to
dissolved pesticides (22).
Plasma E2 concentrations ranged from 92 to 1289 pg of
E2/mL for all females used in this study and for females in
the same stage of sexual maturation. Plasma E2 concentrations in female carp held in cages at LW and LX were not
significantly different from those in females held at the MC
reference site (Figure 4). Plasma T concentrations in female
carp ranged from 183 to 2660 pg of T/mL for all female carp
used in the study and from 198 to 2660 pg of T/mL for females
in the same stage of sexual maturation. Although there were
statistically significant differences in plasma T among females
held in cages at different sites (WB and LW < LX < MC)
(Figure 4), the differences do not appear to be related to LW
influent. Plasma E2 and T concentrations in female carp held
in cages at all sites fell within the range of concentrations
reported elsewhere for female carp captured from reference
sites in rivers (25) or held in aquaria under simulated natural
photoperiod (58), in outdoor tanks (62), or in ponds (64).
Plasma E2 concentrations in female carp also were similar
to those reported for feral female carp collected in May 1995
from a reference site in Lake Mead (3).
Plasma 11-KT concentrations ranged from 64 to 3632 pg
of 11-KT/mL for all female carp used in the study and from
64 to 667 pg of 11-KT/mL among female carp in the same
stage of sexual maturation. Female carp held at LW and LX
had plasma 11-KT concentrations similar to those in females
at both reference sites (Figure 4), and concentrations in most
female carp were less than 0.7 ng/mL. Site median plasma
11-KT concentrations for female carp were less than concentrations reported for a captive stock of Japanese koi carp
(0.61 ng/mL, SEM ) 0.20, n ) 4) (63) or for feral female carp
captured from Lake Mead (3) and were within the range of
plasma 11-KT concentrations reported for female carp
captured in the fall in the Western United States (22), held
in aquaria under simulated natural photoperiod in spring
(58), or captured from reference sites in streams (25).
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FIGURE 7. Ratio of plasma 17β-estradiol to plasma 11-ketotestosterone (E2:11-KT) in adult female and male carp. Data are reported
only for those carp in the same stage of sexual maturation. Bar
heights represent site means, error bars are 2 SEM, and numbers
on bars are sample sizes. Log transformed data were subjected to
parametric statistical analyses for both sexes. Letters above bars
represent Tukey groupings (p < 0.05) within each sex; i.e., fish at
sites with bars marked with common letters were not significantly
different with regard to E2:11-KT ratio. LW, Las Vegas Wash; LX, Las
Vegas Bay; MC, Moon Cove; WB, Water Barge Cove; HF, Hatchery
Fish (hatchery controls).
Plasma Estrogen to Androgen (E:A) Ratios. Plasma
concentrations of androgens are typically greater than
concentrations of estrogens in male carp, while the opposite
is true for female carp (3, 22), so estrogen to androgen (E:A)
ratios typically are less than 1 for males and greater than 1
for females (with the exception of E2:T in females during the
spawning periodssee below). E:A ratios (E2:T ratios or
E2:11-KT ratios) appear to be sensitive indicators of abnormal
sex steroid milieu and to be better indicators of the
reproductive health of fish than the concentrations of
individual sex steroids (29), since the ratios tend to exhibit
differences between male and female fish regardless of
absolute steroid concentrations.
Plasma E2:11-KT ratios ranged from 0.14 to 1.13 for all
male carp used in this study and for males in the same stage
of sexual maturation. There were no statistically significant
differences in E2:11-KT ratio among males held at different
sites (Figure 7). All males in the same stage of sexual
maturation had E2:11-KT ratios less than 1, except for one
at LW (1.13). Mean E2:11-KT ratio for females exceeded that
for males within each site by more than 2-9-fold. Plasma
E2:11-KT ratios ranged from 0.17 to 5.16 in all female carp
used in the study and from 0.31 to 5.16 for females in the
same stage of sexual maturation. Female carp caged at LW
and LX did not differ significantly from those at MC with
regard to plasma E2:11-KT ratio (Figure 7). E2:11-KT ratios
were greater than 1 for most female carp in the same stage
of sexual maturation, with the exception of those at site WB,
where most females (13/14) had E2:11-KT ratios less than
1.0. Only three other female carp in the same stage of sexual
maturation, one at LW (0.87) and two at MC (0.95 and 0.99),
had E2:11-KT ratios less than 1.0, indicating a normal state
for most female carp in the current study.
E2:T ratios ranged from 0.15 to 1.77 for all male carp used
in this study and for males in the same stage of sexual
maturation. There were no significant differences in E2:T
ratio among male carp held at different sites in Lake Mead
(Figure 8). Plasma E2:T ratios ranged from 0.17 to 3.86 for
all female carp used in the current study and for female carp
in the same stage of sexual maturation. Females held at LW
and HF had significantly greater plasma E2:T ratios than
females held at both reference sites (Figure 8). E2:T ratios
were significantly different between males and females at all
sites but reference site WB.
of 15-20 °C, it is possible that the small site-to-site differences
in water temperature might have affected sex steroid
production.
FIGURE 8. Ratio of plasma 17β-estradiol to plasma testosterone
(E2:T) in adult female and male carp. Data are reported only for
those carp in the same stage of sexual maturation. Bar heights
represent site means, error bars are 2 SEM, and numbers on bars
are sample sizes. Parametric statistical analyses were conducted
on log transformed and untransformed data for females and males,
respectively. Letters above bars represent Tukey groupings within
each sex (p < 0.05); i.e., fish at sites with bars marked with common
letters were not significantly different with regard to E2:T ratio. LW,
Las Vegas Wash; LX, Las Vegas Bay; MC, Moon Cove; WB, Water
Barge Cove, HF, Hatchery Fish (hatchery controls).
Changes in E2:T ratio can be difficult to interpret in female
carp because the ratio can change over the course of gonadal
recrudescence. Also, changes in sex steroid profiles and E2:T
ratio in relation to the reproductive cycle appear to vary for
female carp of different strains and geographic locations, so
the general assertion that E:A ratios are greater than 1 for
females does not always hold. In a recent assessment of
seasonal changes in plasma sex steroid concentrations in
female carp, plasma T concentrations were generally greater
than plasma E2 concentrations except during fall gonadal
recrudescence (62). In the U.K., Manning and Kime found
that plasma E2 was greater in female carp with late vitellogenic ovaries, whereas plasma T was greater in the most
sexually mature female carp with post-vitellogenic ovaries
(42). Likewise, Colombo et al. reported that aromatase activity
decreases in the post-vitellogenic ovary in carp (65). E2:T
ratios, then, shift from greater than 1 to less than 1 as female
carp reach more advanced stages of sexual development.
Thus, greater E2:T ratio in female carp held at LW (and
perhaps at LX, although the increase is not significant relative
to both reference sites) might indicate that they were in a
less advanced stage of sexual development, possibly due to
adverse effects of LW influent. There were no statistically
significant differences in GSI or ovarian follicle development
profiles among females at different sites to support that
hypothesis. However, because carp have asynchronous
ovaries, it is difficult to determine their precise stage of
reproductive development, so it is possible that there were
slight differences in reproductive timing among fish at
different sites that would result in differences in sex steroid
profiles that are unrelated to contaminants.
Average water temperatures at the exposure sites exhibited
a pattern (MC < WB < LX < LW) that paralleled the pattern
of median E2:T ratios among female carp held in cages at
these sites. Because T is the precursor for synthesis of E2 in
the teleost ovary (18), conversion of T to E2 might have
increased with water temperature if females were still
undergoing vitellogenesis. In female carp in the earlier stages
of vitellogenesis, with high circulating concentrations of E2,
in vitro E2 production by ovarian fragments increases very
significantly from 15 to 20 °C, while for those in the later
stages of vitellogenesis, with greater circulating concentrations of T, in vitro T production increases very significantly
over the same temperature range (42). Because water
temperatures at sites used in this study fall within the range
The post-vitellogenic shift from production of E2 to T
was not observed in female carp in a study in China (66) or
in a study in Israel (64). In the latter study, plasma E2 and
T varied together throughout the reproductive cycle until
the post-vitellogenic phase of ovarian development, when
plasma E2 concentrations remained at an elevated level
during and for several weeks after spawning, while plasma
T declined just prior to spawning (64). Likewise, female carp
held in aquaria under simulated natural photoperiod had
elevated plasma E2 in spring relative to summer and fall
(58). If this same pattern of steroidogenesis occurred in female
carp caged in Lake Mead and the fish were in a later stage
of gonadal recrudescence, the observed steroid concentrations and E2:T ratios might indicate that females at the
warmer LW and LX sites were closer to spawning than the
females at the reference sites, with plasma E2 concentrations
remaining at elevated levels and plasma T beginning to
decline. This explanation appears to roughly fit the steroid
patterns observed for female carp in this study, with the
exception of the generally low steroid concentrations in
females at WB. Clearly, a thorough understanding of the
pattern of steroidogenesis and its relationship to the reproductive cycle for the specific fish population under study
would improve the ability to interpret plasma sex steroid
profiles for female carp undergoing gonadal recrudescence.
In Vitro T Production by Gonad Tissue. Both male and
female carp held in cages at LX had greater basal capacity
to produce T in gonad tissue than did fish held at other sites,
and ovarian tissue in female carp held at LW had less capacity
to produce T than that in females at other sites (Table 3).
Since reduced in vitro steroid production is the expected
finding in fish with impaired steroidogenic capacity due to
exposure to contaminants (18), the reason for increased in
vitro T production in fish at LX relative to those at LW, where
contaminants are presumably present in greater concentrations, is not clear. However, in vitro T production values for
all carp in this study were similar to basal in vitro T production
values reported previously for other cyprinids (goldfish and
fathead minnows) (18). Likewise, basal in vitro T production
for male carp was similar to that reported for adult male carp
in a different study (55). The lesser in vitro T production in
females at LW did not result in significantly reduced
circulating sex steroid concentrations in LW females relative
to females at both reference sites, and greater in vitro T
production in male and female carp caged at LX did not
produce a corresponding increase in plasma T relative to
fish at both reference sites.
EROD Activity. EROD activity is a measure of the mixed
function oxygenase (MFO) activity catalyzed by hydrocarboninducible cytochrome P450 1A proteins (67). EROD activity
in fish is commonly used as a biomarker of exposure to certain
planar halogenated hydrocarbons, PAHs, and structurally
related compounds that bind to the aryl hydrocarbon receptor
(68) (i.e., the “dioxin-like” compounds). EROD activity in
common carp hepatopancreas is induced by exposure to a
variety of dioxin-like compounds and is particularly useful
as a biomarker in this species because of the wide range
between basal and induced activities (68). Because the MFO
system metabolizes both xenobiotics and steroids (69), it
has been hypothesized that exposure to chemicals that induce
MFO activity might interfere with steroid metabolism in fish
(17, 26) such that MFO activity should be considered as a
potential modifying factor in field studies where circulating
steroid concentrations are end points. However, EROD
induction is not consistently related with decreased circulating sex steroid concentrations in fish (68).
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Female and male carp held at LW had significantly greater
hepatopancreas EROD activity than those held at all other
sites or at both reference sites, respectively (Table 3). In fish,
EROD activity and other monooxygenase activities are
modulated by many factors other than contaminant exposure, including season, temperature, photoperiod, nutritional
status, sex, and reproductive cycles (70, 71), so interpretation
of small differences in EROD activity in field studies should
be done with caution. The elevation in EROD activity in LW
carp is slight since EROD induction less than 10-fold is
considered to be weak (68). Basal EROD activity in carp from
different studies varies greatly (0-4600 pmol min-1 mg-1)
(68), and EROD activities for all carp used in this study fall
within that range. EROD activity for male carp held at HF,
where they were not exposed to LW influent, was not
significantly different from that for males held at LW or LX.
Water temperature was greater at LW than at the other sites,
and others have noted that EROD activity in carp increased
with water temperature (26). Thus, available evidence
suggests that dioxin-like contaminants did not have a
substantial effect on fish used in this study and that site
characteristics such as water temperature might be responsible for observed differences.
Biological Significance of Observed Effects. Although
plasma VTG was greater in male carp held in cages at LW
than in males at the other sites, the difference was small.
Exposure to estrogenic contaminants might have caused VTG
induction in LW males, but greater water temperature at
that site was a potential confounding factor. Furthermore,
a causal link between low-level VTG induction and reproductive dysfunction in male fish has not been established,
so this cannot be considered a detrimental effect. Plasma E2
was moderately greater in male carp caged at LW and LX
than in males at the reference sites, but temperature was a
potential confounding factor, and E:A ratios did not indicate
an imbalance in the male sex steroid milieu. Other differences
among fish caged at different sites are small, potentially
explained by temperature differences among sites, and/or
not clearly related to concentration of LW influent.
Comparison among Studies of Caged Carp and Feral
Carp in Lake Mead. Lower plasma E2 and 11-KT were
observed in feral male carp captured from LX but not in
those captured from LW (3), while increased plasma E2 was
observed in male carp caged in LW and LX in the current
study. The reason for this difference is not apparent. VTG
was detected in the plasma of feral male carp caught from
LW and LX but was not detected in males from a reference
site (3), and increased VTG also was observed in male carp
held in cages in LW in current study. Increased plasma 11KT and decreased plasma E2:11-KT ratio were observed in
feral femal carp caught in LW, and increased plasma VTG
was observed in feral female carp caught from LX (3), but
none of these effects were seen in caged female carp in the
current study. No effects of LW influent on GSI or ovarian
histology were observed in feral (3, 35) or caged female carp,
indicating that any effects were not severe enough to damage
ovarian tissue structure or to impair gonadal recrudescence.
In the 1995 study of feral male carp (3) and in the current
study of caged male carp, no effects on GSI or testicular
histology were observed, but another study determined that
feral male carp captured from LX had consistently lower GSI
and, on one sampling date, a lower proportion of sperm
relative to other germ cell stages (35).
There are many potential reasons for the inconsistencies
between results of the current study and previous studies of
feral carp caught from Lake Mead (3, 35). First, the fish used
in the current study were exposed only for a limited duration
and as sexually differentiated adults, whereas feral fish might
have been exposed to contaminants associated with LW
during the more sensitive period of sexual differentiation
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and for a longer period of time that allowed for bioaccumulation and delayed effects. Results from the few laboratory
studies (50, 60) that sought to characterize the reproductive
system responses of adult carp to waterborne EDCs suggest
that they are not as sensitive as the adults of some other fish
species. Feral carp had access to sediment and natural forage
and might have accumulated contaminants through those
routes of exposure, which were not available to the caged
carp. Contaminants present in LW might have varied
qualitatively and quantitively during the different time
periods studied. The 1995 study of feral carp (3) was
conducted in May, when water temperatures probably were
slightly greater than those experienced by carp in the current
study and might have allowed for greater production of VTG
in response to exposure to estrogenic contaminants. Patiño
et al. (35) sampled feral carp repeatedly over the course of
two spawning seasons, increasing the likelihood of identifying
effects that might be apparent only seasonally. Finally, the
caged carp might have lost weight during the exposure period,
possibly indicating that the exposure conditions produced
some stress that might have affected the responses of the
fish. The variation in some end points among carp held in
cages at two different reference sites emphasizes the
importance of using multiple reference sites whenever it is
feasible in field studies where unknown and/or uncontrolled
factors might modulate endocrine responses under study.
Acknowledgments
The Southern Nevada Water Authority (SNWA) and the U.S.
Bureau of Reclamation provided funding, field and laboratory
support, and assistance in study design. Peggy Roefer, Kim
Zikmund, and Alan Sims of SNWA were particularly helpful.
Michigan State University student Peter Horvath assisted
with the field work. The National Institute of Environmental
Health Sciences provided a Research Service Award (NIH
Training Grant ES07255) to E.M.S. The National Park Service
(NPS) at Lake Mead National Recreational Area and the
Nevada Division of Wildlife (NDOW) provided advice on study
design, field support, and facilities for holding fish. Bill Burke
(NPS), John Hutchings (NDOW), and the employees of the
Lake Mead Fish Hatchery (NDOW) deserve special mention.
The NPS volunteers, particularly Fred DeSilva, generously
donated their time to operate boats. Dr. Nancy Denslow and
Kevin Kroll, University of Florida, provided advice on
adaptation of the VTG ELISA.
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Received for review February 27, 2004. Revised manuscript
received July 15, 2004. Accepted August 4, 2004.
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