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Abstract.................................................................................................................................................................................................... 1

Introduction ............................................................................................................................................................................................. 1

Brief description of fish endocrine systems ..................................................................................................................................... 2

Acknowledgments............................................................................................................................................................................ 3

Approach and methods ............................................................................................................................................................................ 3

Study design and site selection......................................................................................................................................................... 3

Fish collection and sample processing ............................................................................................................................................. 9

Laboratory analyses and quality control .......................................................................................................................................... 9

Data analyses.................................................................................................................................................................................. 10

Selected factors affecting biological characteristics.............................................................................................................................. 12

Gonad maturation and fish age....................................................................................................................................................... 12

Streamflow and wastewater discharge ........................................................................................................................................... 14

Biological characteristics as indicators of hormonally active agents.................................................................................................... 17

Vitellogenin concentrations in common carp plasma .................................................................................................................... 17

Sex steroid hormone concentrations in common carp plasma ....................................................................................................... 17

Gonado-Somatic index values for common carp ........................................................................................................................... 19

Gonad histopathology of common carp ......................................................................................................................................... 23

Relations of biological characteristics to wastewater treatment plant effluent and land use ................................................................ 23

Summary................................................................................................................................................................................................ 26

References ............................................................................................................................................................................................. 27

Supplemental information ..................................................................................................................................................................... 33

#'88#?'

Figure1. Schematic showing endocrine regulation of selected sex steroid hormones and vitellogenin synthesis in female fish........... 3

Figure 2. Map showing location of stream sites sampled in Minnesota during August-September 1999.............................................. 7

Figures 3a-5b. Photomicrographs showing: 

3a. Female common carp ovary containing perinucleolar oocytes; classified as stage-0, undeveloped and immature .................11

3b. Female common carp ovary containing a mixture of perinucleolar and cortical alveoli oocytes; classified as stage-1, 

previtellogenic..........................................................................................................................................................................11

3c. Female common carp ovary containing some vitellogenic oocytes with moderate numbers of vitelline granules and 

a few perinucleolar and cortical alveoli oocytes; classified as stage-2, early vitellogenic......................................................11

3d. Male common carp testes containing a mix of spermatocytes, spermatids, and spermatozoa in approximately equal 

proportions; classified as stage-2, mid spermatogenic.............................................................................................................11

3e. Male common carp testes containing a thin germinal epithelium with scattered spermatogenic activity characteristic 

of full-grown testes; classified as stage-3, late spermatogenic and sexually mature...............................................................11

4. Examples of degenerating atretic oocytes in female common carp ........................................................................................... 12

5a. Ceroid/lipofuscin tissue in female common carp gonads......................................................................................................... 12

5b. Ceroid/lipofuscin tissue in male common carp gonads............................................................................................................ 12

Figures 6a-7 Graphs showing:

6a. Concentrations of vitellogenin, 17β-estradiol, and 11-ketotestosterone in plasma and gonado-somatic index values 

of stage-0, female carp collected during 1999 ........................................................................................................................ 18

6b. Concentrations of vitellogenin, 17β-estradiol, and 11-ketotestosterone in plasma and gonado-somatic index values 

of stage-1 and-2 female carp collected during 1999 ............................................................................................................... 19

7. Concentrations of vitellogenin, 17β-estradiol, and 11-ketotestosterone in plasma and gonado-somatic index values 

of stage-2 and -3 male carp collected during 1999 ................................................................................................................. 20

8 $'

1. Concentrations of vitellogenin, 17β-estradiol, 11-ketotestosterone, and testosterone in female and male common carp plasma 

reported in other studies ................................................................................................................................................................... 4

2. Description of the biological characteristics used in this study .......................................................................................................... 6

3. Study sites and basin land-use characteristics ..................................................................................................................................... 8

4. Histological characteristics used to classify gonad maturation stages for male and female common carp in this study.................. 10
iv

8 $'@?8#$

5. Comparisons of biological characteristics between gonad stages, and between fish ages for female common carp ....................... 13

6. Comparisons of biological characteristics between gonad stages, and between fish ages for male common carp .......................... 13

7. Mean and range of female fish age by gonadal stage of maturation ................................................................................................. 15

8. Mean and range of male fish age by gonadal stage of maturation .................................................................................................... 15

9. Hydrologic conditions at streamflow-gaging stations located near sampling sites during May - August, 1999 .............................. 16

10. Discharges from wastewater treatment plants upstream of sampling sites, streamflow, and percent streamflow consisting 

of wastewater treatment plant discharges....................................................................................................................................... 16

11. Spearman correlation coefficients between biological characteristics and percent streamflow consisting of wastewater 

for female and male common carp................................................................................................................................................. 17

12. Comparisons of biological characteristics for female fish between upstream and downstream site groups for each gonad 

maturation stage, and between upstream and downstream paired sites ......................................................................................... 21

13. Comparisons of biological characteristics for male fish between upstream and downstream site groupsfor each gonad 

maturation stage, and between upstream and downstream paired sites ......................................................................................... 21

14. Summary of biological indicators of hormonally active agents for stage-0, -1 and -2 female and stage-2 and 3 male 

common carp at individual sites collected during 1999 from Minnesota streams ......................................................................... 22

15. Values for length, weight, age, biological characteristics and water temperature for individual female fish 

collected during 1999 ..................................................................................................................................................................... 34

16. Values for length, weight, age, biological characteristics and water temperature for individual male fish 

collected during 1999 ..................................................................................................................................................................... 41

?3$'#?:8?' $3#8#?'

Multiply metric unit By To obtain inch-pound unit

micrometers (μm) 0.0000393 inch (in.)

centimeter (cm) 0.393 inch (in.)

millimeters(mm) 0.0393 inch (in.)

meter (m) 3.281 foot (ft)

gram (g) 0.035 ounce avoirdupois (oz)

liter (L) 0.264 gallon (gal)

square kilometer (km2) 0.3861 square mile (mi2

)

degrees Celsius (°C) 1.8 (Temp. oC) + 32 degrees Fahrenheit (°F)

cubic meter per second (m3/s) 35.31 cubic foot per second (ft3

/s)

cubic meter per second (m3/s) 22.82 million gallons per day(Mgal/day)

Concentrations of substances in fish plasma are given milligrams per milliliter (mg/mL), micrograms per milliliter (μg/

mL), nanograms per milliliter (ng/mL), or picograms per milliliter (pg/mL). A milligram is one thousandth of a gram, a 

microgram is one millionth of a gram, a nanogram is one billionth of a gram, a picogram is one trillionth of a gram, and a 

milliliter is one thousandth of a liter. 
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The presence of hormonally active agents (HAAs) was 

determined in selected Minnesota streams using biological 

characteristics (measures of endocrine disruption) of common carp (Cyprinus carpio) exposed to wastewater treatment plant (WWTP) effluent and runoff from agricultural 

and forested land. Four biological characteristics of common carp were used as indicators of HAAs in the streams 

selected for this study: (1) high concentrations of vitellogenin in male fish and low concentrations in female fish, (2) 

high or low plasma concentrations of the sex steroid hormones (17β-estradiol and 11-ketotestosterone), (3) low 

gonado-somatic index (GSI) (gonad weight divided by total 

body weight multiplied by 100) values, and (4) abnormal 

gonad histopathology (high percent of atretic oocytes in 

female ovaries and high percent ceroid/lipofuscin tissue in 

male or female gonads). The study design was a paired site 

approach targeting sites downstream and upstream of 

WWTP discharges on different streams. Male (221 individuals) and female (201 individuals) common carp were collected using electrofishing techniques from seven streams 

with sites at two locations (upstream and downstream of 

WWTPs), and eight sites located downstream of WWTPs 

with no upstream-paired sites. Samples were collected 

between August 3 and September 13, 1999.

The presence of HAAs in selected Minnesota streams 

was indicated by biological characteristics in common 

carp. Biological characteristics used in this study identified 

WWTP effluent as a potential source of HAAs. Additionally, fish located at sites upstream of WWTP effluent primarily draining agricultural land show indications of 

HAAs, which may be the result of agricultural runoff or 

other sources of HAAs. There was variability among all 

sites and among sites within each site group. Differences 

among sites may be due to differences in water chemistry 

or fish exposure time. Natural variation in the biological 

characteristics may account for some of the differences 

observed in this study. This study and others indicate the 

presence of HAAs in surface water and the potential signs 

of endocrine disruption in resident fish populations. 

Detailed controlled studies could confirm the effects of particular chemicals such as pesticides or components of 

WWTPs on fish reproduction and population structure.

#8?8#?

Concern about the effects of 

chemicals in the environment that act 

as hormonally active agents (HAAs) 

is widespread (Colburn and Clement, 

1992; Guillette and others, 1995; 

Goodbred and others, 1997). HAAs 

may interfere with natural regulation 

of the endocrine system of animals by 

either mimicking or blocking the 

function of natural hormones (U.S. 

Environmental Protection Agency, 

1997; Kime, 1998). A variety of 

anthroprogenic chemicals (xenobiotics) including organochlorine compounds, surfactants (alkylphenols), 

plasticizers, coatings, detergent breakdown products (nonylphenols (NP)), 

human excreted estrogens (17β-estradiol and 17α-ethynylestradiol), agricultural pesticides, and some trace 

elements may act as HAAs and interfere with normal endocrine function 

in animals including humans (U.S. 

Environmental Protection Agency, 

1997). It has also been suggested that 

HAAs may be the cause of an 

increased incidence of breast and ovarian cancer, and endometriosis in 

females (Davis and others, 1993), 

cryptorchidism, testicular and prostate 

cancer, and diminished sperm counts 

in males (Sharpe and Skakkebaek, 

1993) although these hypotheses have 

not been substantiated (National 

Research Council, 1999). 

Two potential sources of HAAs to 

streams in Minnesota are treated sewage (domestic and industrial) and runoff from agricultural or forested land. 

Domestic and industrial sewage may 

contain a variety of chemicals including natural and synthetic estrogens 

(Tabak and others 1981; Desbrow and 

others, 1998), alkylphenolic compounds (Ahel and others, 1994a and 

1994b; Barber and others, 2000), and 

bisphenol-A that are known to interact 

with the estrogen receptors in fish 

(Routledge and Sumpter, 1996; Nagel 
2

and others, 1997; Desbrow and others, 

1998). Runoff from agricultural land 

and atmospheric deposition of pesticides has resulted in the presence of 

pesticides in streams. Herbicides 

including atrazine, cyanazine, 

alachlor and metolachlor are commonly detected in streams draining 

agricultural areas in Minnesota (Minnesota Department of Agriculture, 

1996; Fallon and others, 1997). 

Fish are important organisms for 

indicating the extent of HAAs in surface water because they are directly 

exposed to contaminants, and their 

endocrine systems have many physiological similarities to mammals, 

including humans. Evidence of endocrine disruption in fish was found in 

response to a variety of potential 

HAAs including wastewater treatment 

discharge (Purdom and others, 1994; 

Sumpter and Jobling, 1995; and Folmar and others, 1996), pulp and paper 

mill discharges (Van der Kraak, 

1992), and agricultural chemicals 

(Goodbred and others, 1997; Grady 

and others, 1998; Kime, 1998).

 Potential effects of HAAs on fish 

include disruption of the endocrine 

system, specifically reproductive 

function. Effects of HAAs on fish 

reproductive systems include changes 

in sex steroid hormone synthesis, 

degenerative changes in the ovaries 

and testes including intersex conditions (oocytes in testes tissue), abnormal oogenesis and spermatogenesis, 

reduced sperm viability and motility, 

reduced gonad size, reduced fertility, 

reduced viability of young, and 

changes in the timing of sexual reproduction (U.S. Environmental Protection Agency, 1997; and Kime, 1998). 

 Endocrine disruption in common 

carp (Cyprinus carpio) and walleye 

(Stizostedion vitreum) was documented in Minnesota during 1995–96. 

Several male carp collected in the 

effluent channel from the Minneapolis 

and St. Paul Metropolitan Wastewater 

Treatment Plant effluent channel had 

elevated concentrations of vitellogenin and were considered to be estrogenized by the effluent (Folmar and 

others, 1996). 

Although evidence of HAAs has 

been documented in Minnesota by the 

presence of endocrine disruption in 

fish within a wastewater treatment 

plant (WWTP) channel consisting of 

100 percent effluent from a major 

metropolitan area, little is known 

about the presence of HAAs in 

streams receiving less WWTP effluent or from streams that drain agricultural and forested land. A study was 

conducted to determine the presence 

of HAAs in selected Minnesota 

streams using biological characteristics (measures of endocrine disruption) of common carp exposed to 

WWTP effluent and runoff from agricultural and forested land. The study 

was conducted cooperatively among 

the U.S Geological Survey (USGS), 

the Minnesota Pollution Control 

Agency (MPCA), the Minnesota 

Department of Natural Resources 

(MDNR), and the Legislative Commission on Minnesota Resources 

(LCMR).

The purpose of this report is to 

present the results of this study. Specifically, this report describes: (1) the 

influence of gonad maturation and age 

on biological characteristics (2) biological characteristics as indicators of 

HAAs, and (3) relations of biological 

characteristics to WWTP discharges 

and land use. Results presented in this 

report were based on common carp 

collected at 22 sites from 15 streams 

in Minnesota during August and September 1999.

	-	
	/	


8	


Fish reproduction involves a complex chain of hormonal and developmental events. Gonadal recrudescence 

is the growth of the gonad from the 

regressed to the fully mature state 

(Matty, 1985; Kime, 1998). After 

reaching the fully mature state, 

spawning occurs and recrudescence 

begins again. Common carp spawning 

activity is the greatest during May and 

June when water temperatures are 

between 16 and 23.9o

C (Becker, 

1983; Billard, 1999). Common carp 

begin recrudescence immediately 

after spawning and by autumn they 

are ready for spawning the next spring 

when conditions are optimal (Down 

and others, 1990). External signals 

such as photoperiod and/or water temperature initiate gonadal recrudescence, which is modulated by the 

endocrine system. These external signals stimulate the hypothalamus gland 

to produce gonadatrophin-releasing 

hormone (GnRH) (fig. 1). GnRH 

stimulates the pituitary gland to produce gonadatrophins (GtH), which 

stimulate the synthesis of sex steroid 

hormones in the ovaries and testes. 

Testosterone is present in both male 

and female fish (Matty, 1985; Chang 

and Chen, 1990; Kime, 1998;). Testosterone is converted to 17β-estradiol 

through aromatase action or to 11-

ketotestosterone via 11β-hydroxytestosterone (Kime, 1998). The function 

of 11-ketotestosterone in male fish is 

for gonadal maturation and for the 

development of secondary sex characteristics (Matty, 1985, Mylonas and 

others, 1997; Cavaco and others; 

1998, Kime, 1998; Todo and others, 

1999), while its function in female 

fish is not clear. In females, 17βestradiol serves as a major hormone 

responsible for oocyte development 

(Matty, 1985). A major role of 17βestradiol in females is to stimulate the 

liver to produce vitellogenin. Vitellogenin is a protein that is the precursor 

to egg yolk proteins and is responsible 

for increased gonad weight in females 

during gonadal recrudescence 

(Specker and Sullivan, 1994). In male 

fish, vitellogenesis is stimulated in the 

liver cells (hepatocytes) when they are 

exposed to various natural and synthetic estrogens and xenobiotics such 

as NP (Jobling and Sumpter, 1993). 

Concentrations of measurable 

amounts of plasma vitellogenin in 

male fish are usually very low (ng/

mL) or undetectable, thus making the 

presence of vitellogenin in male fish 

an indicator of the presence of estrogen or estrogenic chemicals in the 

environment (Purdom and others, 

1994, Sumpter and Jobling, 1995, 

Folmar and others, 1996). Although 

the presence of vitellogenin concentrations in male common carp under 
3

natural conditions is not considered 

normal, Jobling and others (1998) 

measured concentrations of 0.0001 

mg/mL, and Sumpter and Jobling 

(1995) measured 0.01 mg/mL for 

male fish at control sites. This suggests that some low concentrations of 

vitellogenin may be present in male 

fish. The effect of vitellogenin in male 

fish is largely unknown; however, elevated vitellogenin concentrations in 

flounder (Herman and Kincaid, 1988), 

and in trout (Hammer and others, in 

press) caused renal failure and subsequent death. Normal ranges in vitellogenin, 17β-estradiol, and 11-

ketotestosterone concentrations in 

female and male common carp have 

not been established. Means or ranges 

of 17β-estradiol, 11-ketotestosterone, 

and testosterone concentrations 

reported in the literature are listed in

table 1.

39"


 The authors express appreciation 

to Judy Helgen, Linda Brooks, Gerald 

Blaha, and Gary Kimball of the Minnesota Pollution Control Agency; 

David Wright and Jack Enblom of the 

Minnesota Department of Natural 

Resources; Leroy Folmar of the U.S. 

Environmental Protection Agency; 

Hilary Carpenter of the Minnesota 

Department of Health; Deborah 

Swackhammer, Ira Adelman, and 

Peter Sorensen of the University of 

Minnesota; and Robert Polta of the 

Metropolitan Council Environmental 

Services for their assistance in study 

design, site selection, field collection, 

and ancillary data retrieval.

?&$8&?'

Four biological characteristics of 

common carp used as indicators of 

HAAs in the streams selected for this 

study are: (1) High concentrations of 

plasma vitellogenin in male fish and 

low concentrations in female fish, (2) 

high or low plasma concentrations of 

the sex steroid hormones (17β-estradiol and 11-ketotestosterone), (3) low 

gonado-somatic index (GSI) (gonad 

weight divided by total body weight, 

multiplied by 100) values, and (4) 

abnormal gonad histopathology (high 

percent of atretic oocytes in female 

ovaries and high percent ceroid/lipofuscin tissue in male or female 

gonads). 

 These selected biological characteristics are commonly used in other 

studies and are a subset of the measures used by the USGS Biomonitoring of Environmental Status and 

Trends Program (Schmitt and Dethloff, 2000) to identify the influence of 

contaminants on fish throughout the 

United States. Features of each biological characteristic as described by 

Schmitt and Dethloff (2000) and 

Kime (1998) are listed in table 2. The 

selected biological characteristic can 

be influenced by age and gonad maturation; therefore, the influence of 

these factors on each biological characteristic was determined.


-	"

	

	

 The study design was a paired 

site approach targeting sites downstream and upstream of WWTP discharges on selected streams. Sites 

downstream of WWTPs are influenced primarily by effluent discharge 

and to a lesser extent by runoff from 

local urban and upstream land. Sites 

upstream of WWTP effluent were primarily influenced by runoff from agricultural land. The criteria for site 

selection included streams that 

receive a continuous effluent discharge from a WWTP and had a dam 

(fish barrier) upstream of that discharge. Additionally, streams with no 

additional WWTPs upstream of the 

selected site and those with the greatest amount of WWTP discharge per 

streamflow during summer low-flow 

conditions had top priority for selection. Given these constraints, 22 sites 

GnRH GtH

17 -estradiol

Hypothalamus

Pituitary

Testosterone

Vitellogenin

Liver

Ovaries

Figure 1. Endocrine regulation of selected sex steroid hormones and vitellogenin synthesis in female fish

(modified from Kime, 1998; GnRH, gonadotrophin-releasing hormone; GtH, gonadotrophin).
4

Table 1. Concentrations of vitellogenin, 17β-estradiol, 11-ketotestosterone, and testosterone in female and male common carp pla

[mg/mL, milligrams per milliliter; pg/mL, picograms per milliliter; na, not available; oC, degrees centigrade; >, greater than]

Sex Vitellogenin (mg/mL) 17β-estradiol (pg/mL) 11-ketotestosterone (pg/mL) Testosterone (pg/mL) Number of samples Study Geographic location Date

Female

128.4 ± 1.80 12110 ± 150 11074 ± 110 11116 ± 117 69 Goodbred and others, 1997 Northeastern United States Septem

Novem

126.5 ± 1.5 11260 ± 68 1707 ± 42 11008 ± 65 97 Goodbred and others, 1997 Mississippi River Basin, United States August 

Septem

128.3 ± 1.3 12463 ± 231 1617 ± 75 1862 ± 110 72 Goodbred and others, 1997 Northern- midcontinental United States Septem110.8 ± 1.1 11045 ± 283 1282 ± 64 1369 ± 75 10 Goodbred and others, 1997 Southern-midcontinental United States Decem127.9 ± 1.5 11767 ± 158 11359 ± 158 11288 ± 148 53 Goodbred and others, 1997 Western United States Octobe11.7 ± 0.9 1216.7 ± 51.5 na 1720.8 ± 100 7 Folmar and others, 1996 Minneapolis and St. Paul Metropolitan Wastewater 

Treatment Plant effluent channel

August 

na 1750.5 ± 89.6 na 11250 ± 100 10 Folmar and others, 1996 Mississippi River near St. Paul. Minnesota Septem

ber 199

na 2490 2180 2800 5 Down and others, 1990 Indoor flow-through aquaria under a simulated natural 

photoperiod

Spring ing and spawnilasting 

na 1140 1140 1250 9 Down and others, 1990 Indoor flow-through aquaria under a simulated natural 

photoperiod

Summpostspaperiod (water tures >

na 1140 1130 1250 10 Down and others, 1990 Indoor flow-through aquaria under a simulated natural 

photoperiodd

Fall peSeptemNovem
5

1 mean, plus or minus one standard error of the mean2 estimated mean based on evaluation of graphs3 estimated range based on evaluation of graphs

Male

10 1480 ± 37 11439 ± 111 11917 ± 159 79 Goodbred and others, 1997 Northeastern United States Septem

Novem

10 + 0.1 1311 ± 25 11148 ± 92 11488 ± 110 77 Goodbred and others, 1997 Mississippi River Basin, United States August 

ber, 19

10 1716 ± 49 1805 ± 69 11084 ± 111 66 Goodbred and others, 1997 Northern- midcontinental United States Septem 10 1258 ± 130 1627 ± 106 1838 ± 154 2 Goodbred and others, 1997 Southern-midcontinental United States Decem10 + 0.1 1598 ± 77 11561 ± 158 12141 ± 351 53 Goodbred and others, 1997 Western United States Octobe11.13 ± 0.9 1125.4 ± 19.2 na 12970 ± 1500 10 Folmar and others, 1996 Minneapolis and St. Paul Metropolitan Wastewater Treatment Plant effluent 

channel

August 

na 1125.9 ± 7.3 na 14160 ± 800 10 Folmar and others, 1996 Mississippi River near St. Paul, Minnesota Septem

ber 199

na 1147.3 ± 14.3 na 15130 ± 700 11 Folmar and others, 1996 Mississippi River near Hast- ings, Minnesota Septem

ber 199

na 1133.6 ± 13.7 na 12920 ± 400 10 Folmar and others, 1996 Minnesota River in Minne- sota Septem

ber 199

na 1105.4 ± 22.0 na 16410 ± 200 5 Folmar and others, 1996 St. Croix River upstream of St. Croix Falls, Wisconsin Septem

ber 199

na 260 2300 2900 5 Down and others, 1990 Indoor flow-through aquaria under a simulated natural 

photoperiod

Spring ing and spawnilasting 

na 250 2150 2200 9 Down and others, 1990 Indoor flow-through aquaria under a simulated natural 

photoperiod

Summpostspaperiod peratur

na 270 2300 21200 9 Down and others, 1990 Indoor flow-through aquaria under a simulated natural 

photoperiod

Fall peSeptemNovem

na 3100 - 1300 na 31000 - 3500 50-100 Chang and Chen, 1990 Laboratory aquaria na na 3100 - 2000 na 31000 - 3300 8 Chang and Chen, 1990 Laboratory aquaria Novem

through 1989

Table 1. Concentrations of vitellogenin, 17β-estradiol, 11-ketotestosterone, and testosterone in female and male common carp plasma re

[mg/mL, milligrams per milliliter; pg/mL, picograms per milliliter; na, not available; oC, degrees centigrade; >, greater than]

Sex Vitellogenin (mg/mL) 17β-estradiol (pg/mL) 11-ketotestosterone (pg/mL) Testosterone (pg/mL) Number of samples Study Geographic location Date
6

Table 2. Description of the biological characteristics used in this study

 [modified from Schmitt and Dethloff (2000) and Kime, (1998)].

Biological characteristic Description Use 

Vitellogenin A precursor of egg yolk proteins, synthesized in the 

liver of female fish upon stimulation by 17β-estradiol 

Primarily controlled by estrogen so is a valuable indicator of exogenous estrogens and estrogen mimics. Because it is not normally present in male fish plasma, it is a good indicator of hormonally active agents that act as estrogenic substances. 

Can vary tion, specfish at oncompariso

Sex steroid hormones (17β-estradiol and 11-ketotestosterone) Chemical messengers that stimulate secondary sex characteristics, development of gametes, and spawning. Indicators of reproductive health and status. Can vary 

tion, specHigh variThese fac

Gonado-somatic index Gonad weight divided by total body weight, multiplied by 100. Indicator of reproductive status and chemical exposure. Indicator is related to reproductive success. Changes w

tion, and ated with 

Gonad histopathology Microscopic examination for the presence of abnormalities (ceroid/lipofuscin deposits in males and 

females, and atretic eggs in females).

Cellular level abnormalities often occur prior to macroscopic abnormalities. Good early warning system of sublethal health affects. 

Standardicompariso
7

Lower

Red Lake

Upper

Red Lake

Lake Winnibigoshish

Leech

Lake

Mille

Lacs

Lake

Lake

of the

Woods

94°

92°

90°

96°

48°

46°

44°

NORTH DAKOTA SOUTH DAKOTA

WISCONSIN

MINNESOTA

IOWA

CANADA

0 20 40 60 80 100 MILES

0 20 40 60 80 100 KILOMETERS

EXPLANATION

Lake Superior

River

Minnesota

River

Red

River

of

the

North

St.

Croix

Base from U.S. Geological Survey

Digital data, 1:2,000,000, 1983

U.S. Albers projection.

DSite location upstream D

of wastewater treatment

plant (number is site

location number)

Figure 2. Location of stream sites sampled in Minnesota during August-September 1999.

9

2 Site location downstream

of wastewater treatment

plant (number is site

location number)

(Sites are listed in table 3)

N. Dak. S. Dak.

Iowa

Wisc.

CANADA

Minnesota

Location Map

Rock

Valley Shell

Rock

Study

Area

D

DD

D D

D

D

D

D D D

D

D

D

D

D

DD DD

D

D

15

16

4

9 10

3

17

18

6

14

13 5

11

12

21

2

22 19

20

7 1

8

Minneapolis

Saint Paul Saint Paul

Duluth

Rochester

Mankato

Moorhead

St. Cloud

Royalton

Winona

Albert Lea Albert Lea Austin

Faribault

Hastings

Hibbing

Owatonna

Red Wing Red Wing

Willmar

Alexandria

Crookston

East

Grand

Forks

Fairmont

Garden City Garden City

Fergus Falls

Grand Rapids Grand Rapids

International Falls International Falls

Little Falls

Marshall

New Ulm New Ulm

Stillwater

St. Croix Falls St. Croix Falls

Worthington

Jackson

Bemidji

Thief River Falls River Falls

Brainerd

Cloverdale

Danbury

Hutchinson

Rockford

Windom

Heron Lake

Saint James Saint James

Luverne

Hardwick

Melrose

West Union

Sauk Center

La Salle La Salle Madelia
8

Table 3. Study sites and basin land-use characteristics 

[D, site downstream of wastewater treatment plant; U site located upstream of wastewater treatment plant; shaded cells are downstream sites; na, not available; km

Site 

location 

number 

(fig. 2)

Sampling 

date Site name Latitude Longitude Site group Drainage area (km2) Drainage basin land 

 Urban Agricultural Wetland 

1 8/31/99 Cedar River downstream of Austin. Minn. 43°38′58′′ 92°58′24′′ D 633 4.0 89.1 2.7 2 8/6/99 St. James Creek downstream of St. James, 

Minn.

44°00′20′′ 94°35′21′′ D 118 5.9 85.8 3.0 

3 8/13/99 Minneapolis and St. Paul Metropolitan 

Wastewater Treatment Plant effluent channel

44°55′08′′ 93°02′50′′ D na 100 0.00 0.00 

4 8/24/99 Mississippi River downstream of Little 

Falls, Minn.

45°58′00′′ 94°22′30′′ D 29,505 1.0 22.1 25.1 

5 8/4/99 Okabena Creek near Worthington, Minn. 43°38′51′′ 95°31′57′′ D 18 36.6 59.1 0.8 6 8/3/99 Redwood River near Marshall, Minn. 44°29′12′′ 95°45′56′′ D 694 2.5 87.1 4.8 7 8/18/99 Shell Rock River near Albert Lea, Minn. 43°36′02′′ 93°17′30′′ D 388 7.6 81.7 2.6 8 8/30/99 Zumbro River downstream of Rochester, 

Minn.

44°03′55′′ 92°27′57′′ D 785 8.0 79.4 3.5 

9 8/16/99 South Fork of the Crow River upstream of 

Hutchinson, Minn.

44°52′20′′ 94°21′20′′ U 1,059 1.3 79.6 8.7 

10 8/9/99 South Fork of the Crow River downstream 

of Hutchinson, Minn.

44°52′39′′ 94°26′45′′ D 1,171 2.1 79.4 8.5 

11 8/5/99 Des Moines River upstream of Windom, 

Minn.

43°51′54′′ 95°18′47′′ U 1,432 1.1 89.5 3.9 

12 8/5/99 Des Moines River downstream of Windom, 

Minn.

43°51′27′′ 95°06′28′′ D 2,944 1.7 90.4 3.3 

13 8/17/99 Rock River upstream of Luverne, Minn. 43°43′04′′ 96°09′49′′ U 792 1.3 94.9 2.2 

14 8/17/99 Rock River downstream of Luverne, Minn. 43°39′02′′ 96°11′47′′ D 1,092 1.5 94.9 2.0 

15 8/23/99 Sauk River upstream of Sauk Center, Minn. 45°49′51′′ 95°02′16′′ U 416 1.0 67.2 14.5 

16 8/19/99 Sauk River downstream of Melrose, Minn. 45°40′37′′ 94°48′13′′ D 1,110 1.5 71.8 12.6 

17 9/13/99 St. Croix near Cloverdale, Minn. 45°57′05′′ 92°33′20′′ U 7,119 0.4 13.5 10.2 

18 8/26/99 St. Croix downstream of Stillwater, Minn. 45°02′10′′ 92°46′50′′ D 18,351 0.8 28.9 16.8 

19 9/1/99 Straight River upstream of Owatona, Minn. 44°03′10′′ 93°15′03′′ U 505 1.8 90.2 2.9 

20 9/1/99 Straight River downstream of Owatona, 

Minn.

44°05′55′′ 93°13′47′′ D 629 3.4 87.4 3.4 

21 8/11/99 Watonwan River upstream of Madelia, 

Minn.

44°03′53′′ 94°35′22′′ U 529 0.9 93.2 2.8 

22 8/11/99 Watonwan River downstream of Madelia, 

Minn.

44°02′41′′ 94°24′44′′ D 1,660 1.8 91.7 2.3 
9

on 15 streams in Minnesota were 

selected for study (fig. 2 and table 3). 

Although paired sites on each stream 

were preferred, this was not always 

possible due to the inability to collect 

desired fish species. Seven streams 

had sites at two locations (upstream 

and downstream of WWTPs), and 

eight sites were downstream of 

WWTPs with no upstream-paired 

sample.

The study area includes streams 

draining land in Minnesota and Wisconsin. Sites are located primarily in 

the southern part of Minnesota in 

basins that drain agricultural land. 

Exceptions are the St. Croix River 

(sites 17 and 18) and the Mississippi 

River downstream of Little Falls, 

Minnesota (site 4), that drain primarily forested land. Environmental factors such as land use, hydrologic 

conditions, and WWTP characteristics were described for each site. The 

data set used to characterize land use 

in the study basins was produced by a 

cooperative effort of the USGS and 

the U.S. Environmental Protection 

Agency (USEPA) (U.S. Geological 

Survey, 2000). The classified data are 

based on 30-m-resolution Landsat satellite data. Land use for the drainage 

basin upstream of each site was determined with geographic-informationssystem (GIS) analyses of these data. 

/	

	

&	"

Each fish species may have 

unique responses to HAAs because of 

differences in reproductive strategies. 

For example, the reproductive strategy of common carp includes the 

release of numerous gametes during 

several spawning periods. In contrast, 

smallmouth bass (Micropterus dolomieui) produce few gametes during a 

more discrete spawning period. Common carp was the only species 

selected for collection among all sites 

because of the potential confounding 

factors associated with the comparison among different fish species. 

Common carp were also selected 

because they are common species in 

Minnesota streams and have been 

studied previously in Minnesota (Folmar and others, 1996, Goodbred and 

others, 1997; and Schmitt and Dethloff, 2000). Male and female common 

carp were collected using electrofishing techniques with pulsed DC current. Fish were sampled between 

August 3 and September 13, 1999. 

This time period was selected because 

it was reported to be the post-spawn 

period when gonadal recrudescence 

occurs in carp (Down and others, 

1990; Becker, 1983). At this time sex 

steroid hormones should be relatively 

stable. Paired sites were sampled 

within a 1-week period, to reduce 

potential variability in biological 

characteristics due to differences in 

hydrologic and chemical conditions, 

with the exception of sites 17 and 18, 

which were sampled within a 30-day 

period. The downstream sample was 

collected within an area of the stream 

where the effluent was well mixed 

(within 400 m downstream of the discharge). Fish were kept alive in a 

holding container with native water 

and processed within a few hours of 

capture. Each fish was weighed, 

length was measured, and scales were 

collected for age determination. At 

least 3 scales from the same area of 

each fish were examined under a 

microscope and scale annuli counted 

to determine fish age.

Approximately 3–5 mL of blood 

was drawn with a syringe from the 

caudal vein and transferred to a 5-mL 

heparinized vacutainer. The vacutainer was stored on wet ice prior to 

being centrifuged for 10 minutes at 

3,500 revolutions per minute to separate plasma. Plasma from each fish 

was transferred to two cryovials and 

stored on dry ice or liquid nitrogen 

prior to transfer to laboratories. One 

plasma sample was sent to the University of Florida’s Molecular Biomarkers Core Facility in Gainesville, 

Florida, for analysis of vitellogenin, 

and the remaining plasma sample was 

sent to the USGS laboratory in 

Gainesville, Florida, for 17β-estradiol 

and 11-ketotestosterone analyses. All 

plasma samples were stored at – 80o

C 

until analyzed. Gonads were removed 

and weighed to the nearest gram for 

GSI calculation. Subsamples of 

gonad, spleen, and liver tissue were 

fixed in 10 percent formalin and sent 

to the USGS laboratory in Leetown, 

West Virginia, for histopathological 

examination. 

(
3

:	



Plasma samples from common 

carp were analyzed for vitellogenin, 

17β-estradiol, and 11-ketotestosterone 

concentrations. Vitellogenin concentrations in plasma were analyzed 

using a direct Enzyme-Linked Immunosorbent Assay (ELISA) as previously described (Denslow and others, 

2000; Folmar and others, 1996; Goodbred and others,1997; and Schmitt 

and Dethloff, 2000). A monoclonal 

antibody, HL 1638 (2D3), specifically 

raised against common carp vitellogenin, was used for these assays. This 

antibody reacts with high affinity and 

specificity with vitellogenin and not 

with other plasma proteins. The 

ELISA used in this study can detect 

10–100 ng of vitellogenin per sample, 

resulting in a sensitivity of 0.001 mg/

mL, as plasma must be diluted onehundred times to eliminate interferences. Each ELISA plate had a positive control (plasma with a known 

vitellogenin concentration) to test for 

interassay and intra-assay variation. 

The assay was performed in triplicate 

and if the coefficient of variation 

exceeded 10 percent, the samples 

were rerun. Correlations between 

standards and field samples yielded 

Pearson correlation coefficients

between 0.95 and 0.99.

Radioimmunoassay procedures 

were used for 17β-estradiol and 11-

ketotestosterone determination as previously described (Goodbred and others, 1997; and Schmitt and Dethloff 

2000). Each sample was analyzed in 

duplicate. Extraction efficiencies of 

92 ± 2.8 percent and 86 ± 3.3 percent 

were observed for 17β-estradiol and 

11-ketotestosterone, respectively. The 

minimum concentration detectable 

was 6.4 pg/mL for 17β-estradiol and 

8.1 pg/mL for 11-ketotestosterone. 

The assays are subject to cross reac-
10

tivity with other steroids. Cross reactivity of 17β-estradiol with other 

steroids was: 11.2 percent for estrone, 

1.7 percent for estriol, and less than 

1.0 percent for 17α-estradiol and 

androstendione. The cross reactivity 

of 11-ketotestosterone with other steroids was 9.7 percent for testosterone, 

3.7 percent for α-dihydrotestosterone, and less than 1 percent for 

androstendione. 

 Samples of female and male 

gonad tissues were classified into 

stages of sexual maturation and examined for histopathological abnormalities. Methods used for laboratory 

preparations and characterizations are 

described in Goodbred and others 

(1997), and Schmitt and Dethloff 

(2000), and briefly here. Gonad tissue 

was processed for light microscopy by 

dehydration and embedding in paraffin. Blocks of paraffin were cut at 6-

μm thickness and stained with hematoxylin and eosin.

 Ovaries and testes were examined for stage of sexual maturation 

and gonadal abnormalities (percent 

atretic oocytes in females, and presence of ceroid/lipofuscin in both 

females and males). Determination of 

gonad maturation stage was based on 

the maturity of the predominant stage 

of oogenesis and spermatogenesis for 

female and male gonads, respectively 

(table 4). Stage- 0, -1,and -2 females 

were observed in this study and are 

shown in figures 3a, 3b, and 3c. 

Although stage-3 is typical of females 

approaching spawning, stage-1, -2, 

and -3 are all sexually mature fish. 

Stages 1–3 are characteristic of sexually mature male fish. Stages 2 and 3 

were common among male common 

carp in this study and are shown in 

figures 3d and 3e.

Atresia in the female gonadal tissue is presented as the percent of 

oocytes that are atretic (fig. 4). When 

possible, 100 oocytes were counted. A 

semiquantitative method (Reimschuessel and others, 1992) was used 

to classify ceroid/lipofuscin tissue in 

female and male gonads (figs 5a and 

5b). The presence of ceroid/lipofuscin 

tissue in the female and male gonads 

was ranked on a scale of 0–4, with 0 

being none observed, 1 rarely 

observed, 2 mild, 3 moderate, and 4 

severe. 

-
3

Because the selected biological 

characteristics can be influenced by 

physiological factors such as gonad 

maturation and fish age, a one-way 

analysis of variance (ANOVA) was 

used to determine if any biological 

characteristic was different among 

gonad maturation stages or fish ages. 

Comparisons of biological characteristics between site groups (7 upstream 

and 15 downstream sites) were 

accomplished with a two-sided student’s t-test for unequal sample sizes. 

Comparisons for the paired sites (7 

upstream and downstream pairs) were 

accomplished with a paired t-test. 

This test reduces the variability 

between streams so that differences 

between groups can be detected 

(Helsel and Hirsch, 1992). ANOVA 

was used to make comparisons of biological characteristics among all sites. 

All variables were tested for normality through visual inspection of normal probability plots prior to analyses 

and transformed when necessary to 

achieve normality. A significance 

level of 0.05 was used as the criterion 

for statistical significance. The statistical-test result is represented by 

p-values, the attained level of significance.

 Because baseline values for biological characteristics have not been 

established, the values for each characteristic were ranked relative to one 

another. Mean values of the selected 

Table 4. Histological characteristics used to classify gonad maturation stages for female and male common carp in this study

 (modified from Schmitt and Dethloff, 2000; and Goodbred and others, 1997)

Gonad 

maturation 

stage

 Female ovary characteristics Male testes characteristics

0 Undeveloped (immature):

Predominantly contained early perinucleolar oocytes that are previtellogenic and no developing ova. 

Undeveloped (immature):

Testicular tissue contained exclusively immature

stages of spermatogenesis (spermatocytes) with no

spermatozoa observed.

1 Previtellogenic:

Ovaries contained greater than 90 percent previtellogenic oocytes, 

and few cortical alveoli.Ova are slightly to moderately enlarged 

and contain vacuoles or lipid droplets but few vitelline granules.

Early spermatogenic: 

Testicular tissues predominantly contained immature stages (spermatocytes to spermatids), with some spermatozoa observed. 

2 Early vitellogenic:

Ovaries contained a majority of oocytes that were early to midvitellogenic with few to moderate numbers of vitelline granules 

and very few fully developed oocytes.

Mid-spermatogenic:

Testicular tissue contained a mix of spermatocytes,

spermatids and spermatozoa in roughly equal proportions. 

3 Late vitellogenic:

Ovaries contained a majority of oocytes that were late vitellogenic 

with eosinophillic yolk globules distributed throughout the cytoplasm.

Late-spermatogenic:

Testicular tissues contained mature sperm. 
11

Figure 3a. Female common carp ovary containing

perinucleolar oocytes; classified as stage-0,

undeveloped and immature (Magnification 365x).

Figure 3b. Female common carp ovary containing a

mixture of perinucleolar and cortical alveoli oocytes;

classified as stage-1, previtellogenic (Magnification 365x).

Figure 3c. Female common carp ovary containing some

vitellogenic oocytes with moderate numbers of vitelline

granules and a few perinucleolar and cortical alveoli

oocytes; classified as stage-2, early vitellogenic

(Magnification 365x).

Figure 3d. Male common carp testes containing a mix

of spermatocytes, spermatids, and spermatozoa in

approximately equal portions; classified as stage-2,

mid spermatogenic (Magnification 412x).

Figure 3e. Male common carp testes containing a thin

germinal epithelium with scattered spermatogenic

activity characteristic of full-grown testes; classified

as stage-3, late spermatogenic, sexually mature

(Magnification 412x).
12

biological characteristics at each site 

were ranked and classified into one of 

three categories: high, medium, or 

low. The high class consisted of sites 

with mean values of a biological characteristic greater than or equal to the 

75th percentile (the highest 25 percent). The medium class consisted of 

sites with mean values of a biological 

characteristic between the 25th and 

75th percentiles, and the low class 

consisted of sites with mean values of 

a biological characteristic less than or 

equal to the 25th percentile (the lowest 25 percent). Samples in the high 

and low classes were of the greatest 

interest because fish in these two 

classes represent the samples that are 

distinct among all sites and thus may 

indicate potential HAA exposure and 

endocrine disruption. 

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/	
3"

The selected biological characteristics can be influenced by physiological factors such as gonad maturation 

and fish age (Goodbred and others, 

1997). Both gonad maturation stage 

and fish age varied among sites. 

Among all fish collected (201 females 

and 221 males), female common carp 

age ranged from 1 to 6 years with 

gonad maturation stages from 0 to 3 

(table 15, at the back of the report). 

Male common carp age ranged from 1 

to 5 years with gonad maturation 

stages from 1 to 4 (table 16, at the 

back of the report). There were small 

sample sizes in select gonad stages 

(one female in gonad stage-3, and two 

males each in gonad stages-1 and 4) 

that did not allow for analyses of the 

influence of those gonad stages on 

biological characteristics; therefore, 

these five samples were eliminated 

from further analyses.

 Selected biological characteristics were different among gonad maturation stages and fish ages for both 

female and male fish (tables 5 and 6, 

respectively). In female fish, vitellogenin, 11-ketotestosterone, GSI valFigure 4. Examples of atretic oocytes in female common carp (Magnification 412x).

Figure 5a. Ceroid/lipofuscin tissue in female common

carp gonads (Magnification 824x).

Figure 5b. Ceroid/lipofuscin tissue in male common

carp gonads (Magnification 412x).
13

Table 5. Comparisons of biological characteristics between gonad stages, and between fish ages for female com

[SE, one standard error; p-values shown in bold indicate statistically significant differences; n, number of samples; GSI, gonado-soma

1 Female common carp in gonad stages-0 through -2 and ranging in age from 1- to 6- years old were used in these analyses.

Biological 

characteristic Gonad stage Fish age

Gonad stage-0

Mean ± SE

Gonad stage-1

Mean ± SE

Gonad stage-2

Mean ± SE

p-value 

among gonad 

stages

1 year

Mean ± SE

2 years

Mean ± SE

3 years

Mean ± SE

4 years

Mean ± SE

M

n= 21 n=49 n=130 n=16 n=71 n=65 n=33 

Vitellogenin (mg/mL) 0.217 ± 0.048 3.58 ± 0.67 3.44 ± 0.30 <0.001 3.65 ± 1.25 2.85 ± 0.48 3.34 ± 0.49 2.88 ± 0.44 3

17β-estradiol

(pg/mL) 428 ± 40 548 ± 46 560 ± 28 0.3 486 ± 70 544 ± 37 544 ± 37 598 ± 56 

11-ketotestosterone (pg/mL) 299 ± 38 263 ± 28 361 ± 21 0.02 271 ± 48 332 ± 23 278 ± 26 352 ± 35 

GSI (percent) 1.1 ± 0.1 4.2 ± 0.4 9.9 ± 0.3 <0.001 3.41 ± 0.68 7.0± 0.6 7.9 ± 0.5 9.1 ± 0.9 

Atretic oocytes (percent) 2.5 ± 1.2 11.4 ± 1.6 10.2± 0.7 <0.001 4.4 ± 1.6 8.6 ± 0.9 10.6 ± 1.3 12.0 ± 1.7 

Ceroid/lipofuscin ranking 0.57 ± 0.16 1.1 ± 0.11 1.0 ± 0.04 0.001 0.69 ± 0.12 0.76 ± 0.06 1.1± 0.08 1.1 ± 0.08 

1 Male common carp in gonad stages-2 and -3 and ranging in age from 1- to 5- years old were used in these analyses.

Table 6. Comparisons of biological characteristics between gonad stages, and between fish ages for male com

[SE, standard error; p-values shown in bold indicate statistically significant differences; n, number of samples; GSI, gonado-somatic 

Biological 

characteristic Gonad stage Fish age

Gonad stage-2

Mean ± SE

Gonad stage-3

Mean ± SE

p-value 

among gonad 

stages

1 year

Mean ± SE

2 years

Mean ± SE

3 years

Mean ± SE

4 years

Mean ± SE

n = 35 n = 182 n = 14 n = 75 n = 79 n = 31 

Vitellogenin (mg/mL) 0.02 ± 0.009 0.01 ± 0.001 0.02 0.009 ± 0.0002 0.018 ± 0.002 0.01 ± 0.001 0.02 ± 0.01 

17β-estradiol

(pg/mL) 256 ± 22 264 ± 10 0.8 157 ± 20 263 ± 17 283 ± 13 264 ± 30 

11-ketotestosterone (pg/mL) 774 ± 70 620 ± 32 0.04 900 ± 159 522 ± 42 671 ± 50 804 ± 87 

GSI (percent) 4.5 ± 0.4 5.3 ± 0.2 0.05 3.5 ± 0.4 4.3 ± 0.24 5.1 0.25 6.6 ± 0.4 

Ceroid/lipofuscin ranking 0.89 ± 0.11 1.01 ± 0.05 0.2 0.78 ± 0.15 0.56 ± 0.06 1.1 ± 0.07 1.3 ± 0.11 
14

ues, percent atretic oocytes, and 

ceroid/lipofuscin rankings were significantly different among gonad maturation stages (table 5). Stage-0 

females had lower mean vitellogenin 

concentrations, GSI values, percent 

atretic oocytes, and ceroid/lipofuscin 

rankings than stage-1 and -2 females. 

The 11-ketotestosterone concentrations, GSI values, percent atretic 

oocytes, and ceroid/lipofuscin rankings were significantly different 

among females of different ages(table 

5). The 11-ketotestosterone concentrations were greatest in 6-year-old 

females. GSI values and percent 

atretic oocytes were lowest in 1- and 

6-year-old females.

In male fish, vitellogenin, 11-

ketotestosterone, and GSI values differed significantly by gonad maturation stage (table 6). Vitellogenin and 

11-ketotestosterone concentrations 

were greater in stage-2 than in stage-3 

males. GSI values were greater in 

stage-3 than in stage-2 males. All biological characteristics in male fish differed significantly by age (table 6). 

Vitellogenin concentrations were lowest in 1- and 5-year old males. Oneyear-old males had lower concentrations of 17β-estradiol, greater concentrations of 11-ketotestosterone and 

lower GSI values than older males 

(table 6). 

To account for age and gonad 

maturation stage as potential confounding factors, analyses of biological characteristics were limited to a 

subset of the original data set. Analyses of female characteristics were limited to 181 females in gonad 

maturation stages-0, -1, and -2 and 

ranging from 2- to 5-years old (table 

15). Analyses of male characteristics 

were limited to 185 males in gonad 

maturation stages-2 and -3, and ranging from 2- to 4-years old (table 16). 

Analyses were completed for each 

gonad stage individually (stage-2 

males were only compared to other 

stage-2 males). Even after removal of 

1- and 5-year-old male fish, GSI values increased with increasing fish age 

within each stage of maturation. The 

GSI should normalize the relation of 

gonad weight to body weight. However, this was not observed for male 

common carp in this study. This relation should not affect the comparison 

between the upstream and downstream group because the percents of 

2-, 3-, and 4-year old fish were similar 

within each site group. Likewise, fish 

age was similar between the paired 

sites. Comparisons of GSI values for 

male fish among sites may be biased 

because female and male ages were 

not the same at each site (tables 7 and 

8, respectively). For example, the 

mean age for gonad stage-3 males at 

sites 4 and 17 was 4 years old. In contrast, gonad stage-3 males were on 

average 2–3 years old among other 

sites.


#



-	
"

 Physiological responses of fish to 

contaminants are often dose dependent (the response increases as the 

concentration of a contaminant 

increases). Therefore, fish in streams 

with more concentrated WWTP effluent may show a greater response than 

fish in streams with less concentrated 

WWTP effluent. 

The flow of the receiving stream 

and the amount of WWTP effluent 

determine the dilution of the effluent 

in the stream. Streamflows on the day 

of sampling ranged from 0.10 to 248 

m3/s among all sites. Streamflows the 

three months prior to and during sampling in August 1999 were not all at 

low-flow conditions that would have 

been ideal to assure that the WWTP 

effluent was maximally concentrated. 

USGS streamflow data from gaging 

stations located near sampling locations were used to estimate streamflow conditions. Antecedent 

streamflows generally were greater 

than normal during May, June, and 

July (table 9), but returned to approximately normal flow during August. 

Exceptions to these observations were 

the Cedar River near Austin, Minnesota (site 1), the Mississippi River 

near Royalton, Minnesota (site 4), the 

South Fork of the Zumbro River at 

Rochester, Minnesota (site 8), and the 

St. Croix River near St. Croix Falls, 

Wisconsin (site 18). The streamflow 

at these four sites was greater than 

average during August by a factor of 

two or more. 

 There was also variability in the 

amount of discharge that each WWTP 

contributed to the receiving stream 

(table 10). To account for differences 

in WWTP dilution among sites, the 

percent of streamflow consisting of 

wastewater (sum of discharges from 

all WWTPs upstream of the sampling 

site, divided by streamflow at the 

sampling site, multiplied by 100) was 

estimated for each site. The discharge 

data for each WWTP was obtained 

from the Permit Compliance System 

(Linda Brooks, Minnesota Pollution 

Control Agency, written commun., 

1999). The average daily WWTP 

effluent discharges for August 1998 

(August 1999 data were not readily 

available) were used to represent the 

discharge data from each WWTP. 

This value is considered an estimate 

because of the potential temporal variability in WWTP effluent amounts.

The characteristics of the influent 

and waste processing at each WWTP 

also affect the chemistry of the effluent. While the primary influent to 

each plant was domestic waste, there 

were likely variable amounts and 

types of industrial waste influent. The 

amount and types of industrial waste 

entering each WWTP was not quantified in this report. 

To determine if the percent of 

streamflow consisting of wastewater 

influenced the biological characteristics, Spearman rank correlations were 

determined for each biological characteristic and the percent of streamflow consisting of wastewater (table 

11). Most Spearman correlation coefficients were low (< 0.5) between biological characteristics and the percent 

of streamflow consisting of wastewater. Vitellogenin concentrations in 

female and male fish of selected 

gonad stages were related to the percent of streamflow consisting of 

wastewater. Vitellogenin concentrations in stage-0 and-1 females were 

negatively correlated with the percent 

of streamflow consisting of wastewater (-0.41 and -0.30, respectively). 
15

Table 7. Mean and range of female fish age by gonad stage of maturation 

[shaded cells are downstream sites]

Site location 

number

(fig. 2)

Gonad stage–0 Gonad stage–1 Gonad stage–2

Number of fish Age (years) Number of fish Age (years) Number of fish Age (years)

Downstream

Mean Range Mean Range Mean Range

1 10 3.9 3 - 5

2 6 2 2 - 2

3 2 3.5 3 - 4 5 3.2 3 - 4 7 3.2 3 - 4

4 3 5 5 1 5 5

5 5 2.8 2 - 4

6 1 3 3 

7 2 3 3 - 3 8 2.6 2 - 3

8 4 3.5 2 - 5

Paired upstream-downstream

9 2 3.5 3 - 4 6 3.1 2 - 4

10 2 3.5 3 - 4 10 3 2 - 4

11 2 2.5 2 - 3 9 2.4 2 - 3

12 1 3 3 1 2 2 7 2 2 - 2

13 4 2.2 2 - 3 7 2.7 2 - 4

14 6 2.6 2 - 3 5 2.6 2 - 4

15 7 2.7 2 - 4

16 1 2 2 4 2.25 2 - 3

17 5 5 5 - 5

18 2 4.5 4 - 5 8 3.25 2 - 4

19 5 3.4 2 - 4

20 1 2 2 3 3 2 - 4 4 3.7 3 - 4

21 2 2 2 - 2 5 2 2 

22 11 2.1 2 - 3 7 2.4 2 - 3

Summary 14 47 120

Table 8. Mean and range of male fish age by gonad stage of maturation 

[shaded cells are downstream sites]

Site location 

number 

(fig. 2)

Gonad stage-2 Gonad stage-3

Number of fish Age (years) Number of fish Age (years)

Downstream

Mean Range Mean Range

1 2 3 3 - 3 9 3.6 3 - 4

2 12 2.1 2 - 3

3 5 3.2 3 - 4 1 2 2 

4 3 4 4 - 4

5 1 2 2 1 3 3 

6 11 2.8 2 - 3

7 5 2.4 2 - 3 7 3 2 - 4

8 2 3 2 - 4

Paired upstream-downstream

9 10 3.1 3 - 4

10 11 2.8 2 - 4

11 8 2.2 2 - 3

12 1 2 2 7 2.1 2 - 3

13 13 2.2 2 - 3

14 1 3 3 12 2.3 2 - 3

15 3 2.3 2 - 3 1 2 2 

16 3 2.3 2 - 3 9 2.5 2 - 4

17 2 4 4 - 4 4 4 4 

18 3 3.3 3 - 4 6 3.5 3 - 4

19 7 3.1 2 - 4

20 12 3.0 2 - 4

21 1 3 3 - 3 6 2.1 2 - 3

22 6 2.5 2 - 3

Summary 27 158
16

Table 9. Hydrologic conditions at streamflow-gaging stations located near sampling sites during May-August 1999. 

U.S. 

Geological 

Survey site 

identifier

Corresponding site 

location number 

(fig. 2)

U.S. Geological Survey station name 

(period of record)

Percent difference between 1999 mean monthly streamflow 

and the mean monthly streamflow for the period of record1

1

Negative numbers indicate that the 1999 stream flow was less than the mean streamflow for the period of record.

May June July August

05457000 1 Cedar River near Austin, Minn. (1909-99) 336 85 238 107

05267000 4 Mississippi River near Royalton, Minn. (1924-99) 89 42 77 135

05315000 6 Redwood River near Marshall, Minn. (1940-99) 62 55 59 -43

05462000 7 Shell Rock River at Shell Rock, Iowa (1953-99) 225 130 293 63

05372995 8 South Fork of the Zumbro River at Rochester, Minn. 

(1981-99) 116 1 42 102

05280000 9 and 10 Crow River at Rockford, Minn. (1906-99) 56 43 0 -26

05476000 11 and 12 Des Moines River at Jackson, Minn. (1930-99) 92 14 8 -63

06483500 13 and 14 Rock River near Rock Valley, Iowa (1948-99) 66 18 8 -41

05270500 15 and 16 Sauk River near St. Cloud, Minn. (1909-99) 105 71 -21 -6

05333500 17 St. Croix River near Danbury, Wisc. (1914-99) -26 -6 -63 -1

05340500 18 St. Croix River near St. Croix Falls, Wisc. (1902-99) -6 -18 20 146

05353800 19 and 20 Straight River near Faribault, Minn. (1966-99) 192 74 93 35

05319500 21 and 22 Watonwan River near Garden City, Minn. (1940-99) 38 -24 84 -54

1Sum of the average daily discharges for August 1998 from all wastewater treatment plants upstream of the sampling site.

Table 10. Discharges from wastewater treatment plants upstream of sampling sites, streamflow, and percent of streamflow 

consisting of wastewater treatment plant discharges 

[na, not available; WWTP, wastewater treatment plant; m3/sec, cubic meters per second; shaded cells are downstream sites].

Site location number 

(fig. 2)

Average discharge from 

WWTP (m3/sec)

Streamflow 

(m3/sec) 

Percent of streamflow consisting 

of WWTP discharge

Downstream 

1 0.242 3.85 6

2 0.048 0.10 48

3 11.0 11.0 100

4 0.765 229 0.3

5 0.093 0.14 66

6 0.121 1.43 8

7 0.198 2.41 8

8 0.599 7.92 8

9 0 2.08 0

Paired upstream-downstream

10 0.111 3.34 3.3

11 0 1.14 0

12 0.039 3.82 1

13 0.002 0.71 0.3

14 0.034 1.05 3.2

15 0 na 0

16 0.105 4.56 2.3

17 0 na 0

18 0.280 248 0.1

19 0 2.29 0

20 0.153 3.05 5

21 0.001 0.68 0.1

22 0.031 2.02 1.5
17

Vitellogenin concentrations in stage-2 

males were positively correlated with 

percent of streamflow consisting of 

wastewater (Spearman correlation 

coefficient = 0.53). In female fish, 

there were low to moderate negative 

correlations between 17β-estradiol 

and 11-ketotestosterone concentrations and the percent of streamflow 

consisting of wastewater. In male fish, 

correlations with both 17β-estradiol 

and 11-ketotestosterone concentrations and the percent of streamflow 

consisting of wastewater were low. 

GSI values for females and males 

were weakly related to the percent of 

streamflow that wastewater contributed. 

 #??1#&8$#'+

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Concentrations of vitellogenin 

and sex steroid hormones, GSI values, 

and gonad histopathology were used 

as biological indicators of HAAs for 

purposes of this report. These characteristics are described for both female 

and male common carp. 

	
"	

	

	&

Mean vitellogenin concentrations 

in females were approximately 300 

times greater than those in males (fig. 

6a, 6b, and 7; tables 12 and 13). A 

comparison of vitellogenin concentrations in female fish between the site 

groups (7 upstream and 15 downstream sites) revealed no significant 

differences. Additionally, there were 

no differences between the paired 

sites (7 upstream–downstream pairs) 

for female fish. 

Vitellogenin concentrations in 

females were significantly different 

among all sites (p <0.001) (figs. 6a 

and 6b). Mean vitellogenin concentrations were low (less than the 25th percentile) for females in at least one 

gonad stage at downstream sites 2, 3, 

12, 16, and 20 and upstream sites 11, 

13, and 21 (figs. 6a and 6b, and table 

14).

In male fish, vitellogenin concentrations were observed at all sites. 

Because vitellogenin has been 

observed in male fish under controlled 

conditions at concentrations of 

0.0001 mg/mL (Jobling and others, 

1998), and 0.01 mg/mL (Sumpter and 

Jobling, 1995), concentrations of 

vitellogenin less than 0.01 mg/mL are 

considered as background concentrations for this study. Vitellogenin concentrations above background 

concentrations were detected in 0 to 

92 percent (mean = 36 percent) of the 

males at each site. Vitellogenin concentrations above background were 

observed in males at five downstream 

sites (3, 5, 6, 7 and 16) and four 

upstream sites (9, 13, 15, and 

21).Vitellogenin concentrations in 

stage-3 males were significantly 

greater in the upstream than in the 

downstream site group (table 13). At 

paired sites, vitellogenin concentrations in males were greater at 

upstream than downstream sites 

(p = 0.01). Vitellogenin concentrations in males were significantly different among all sites (p <0.001) (fig. 

7). Vitellogenin concentrations in 

males in at least one gonad stage were 

greatest (greater than or equal to the 

75th percentile) at downstream sites 

1, 3, and 7; and upstream sites 9, 13, 

15, 21 (table 14). 

.
	%



		

&

The 17β-estradiol concentrations 

in females were on average 2.7 times 

greater than 11-ketotestosterone concentrations. In contrast, the 11-

ketotestosterone concentrations in 

male fish were on average 2.3 times 

greater than 17β-estradiol concentrations. Normal ranges of 17β-estradiol 

and 11-ketotestosterone in female and 

Table 11. Spearman correlation coefficients between biological characteristics and the percent of streamflow consisting of wastewater 

for female and male common carp 

[mg/mL, milligrams per milliliter; pg/mL, picograms per milliliter; na, not applicable].

Biological characteristic Spearman correlation coefficient

Female common carp Male common carp

Gonad stage-0 Gonad stage-1 Gonad stage-2 Gonad stage-2 Gonad stage-3

Vitellogenin (mg/mL) -0.41 -0.30 0.014 0.53 -0.003

17β-estradiol (pg/mL) -0.35 -0.54 -0.14 0.20 0.004

11-ketotestosterone (pg/mL) -0.71 -0.22 -0.19 -0.23 0.11

Gonado-somatic index (percent) 0.34 -0.19 0.03 -0.12 -0.20

Atretic oocytes (percent) 0.31 -0.16 0.04 na na
18

0.0

0

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

00 2000 0 0 0 0 2 1 0 0 7 1 0 0 0 1 0 0

400

800

1,200

1,600

STAGE-0

Downstream Paired upsteam-downstream

0.2

0.4

0.6

0.8

1.0

17 -ESTRADIOL AND

11-KETOTESTOSTERONE

CONCENTRATIONS,

IN PICOGRAMS PER MILLILITER

VITELLOGENIN CONCENTRATIONS,

IN MILLIGRAMS PER MILLILITER

GONADO-SOMATIC INDEX,

IN PERCENT

0.0

0.5

1.0

1.5

2.0

2.5

3.0

SITE LOCATION NUMBER (FIG.2 AND TABLE 3)

Figure 6a. Concentrations of 11-ketotestosterone

and gonado-somatic index values of stage-0 female common carp

collected during 1999.

vitellogenin, 17 -estradiol, and

in plasma

17 - estradiol

11-ketotestosterone

2

EXPLANATION

Number of samples

+1 Standard error

Mean

-1 Standard error







male fish have not been established. 

However, Down and others (1990) 

reported that 17β-estradiol concentrations were approximately two times 

greater than 11-ketotestosterone concentrations in female common carp and that 

11-ketotestosterone concentrations were 

approximately four times greater than 

17β-estradiol concentrations in male 

common carp. Goodbred and others 

(1997) observed that 17β-estradiol concentrations were on average 2.5 times 

greater than 11-ketotestosterone concentrations in female common carp and that 

11-ketotestosterone concentrations were 

approximately 2.5 times greater than 

17β-estradiol concentrations in male 

common carp. This reoccurring pattern 

suggests that although both female and 

male fish have both 17β-estradiol and 

11-ketotestosterone concentrations in 

their plasma, 17β-estradiol concentrations generally are expected to be greater 

than 11-ketotestosterone concentrations 

in females. Conversely, 11-ketotestosterone concentrations are expected to be 

greater than 17β-estradiol concentrations 

in males. 

Steroid hormone concentrations in 

female fish tended to be greater in the 

upstream than in the downstream site 

group although there were some gonad 

maturation stages for which there were 

no significant differences (fig. 6a, 6b, 

and 7; tables 12 and 13). 

At the paired sites, 17β-estradiol 

concentrations tended to be greater at 

upstream than downstream sites. However there were no statistically significant differences between upstream and 

downstream sites (table 13). The paired 

sites 17–18 have a large influence on this 

result. Site 17 is unusual because the 

17β-estradiol concentrations in female 

fish were low and 11-ketotestosterone 

concentrations were high (in the range of 

male concentrations). Concentrations of 

11-ketotestosterone in females were 

greater at upstream than downstream 

paired sites (p = 0.04). 

Both 17β-estradiol and 11-ketotestosterone concentrations in females differed among sites (p <0.001) (figs. 6a 

and 6b). Mean concentrations of 17βestradiol in female fish were low in at 

least one gonad stage at downstream 

sites 2, 4, 5, 6, 10, 12, 16, and 20; and 

upstream sites 17 and 21 (table 14). 

Mean concentrations of 11-ketotestosterone in female fish of at least 

one gonad stage were high at downstream sites 1, 8, 12, 20, and 22; and 

upstream sites 11, 15, 17, 19, and 21 

(table 14). Mean concentrations of 

11-ketotestosterone were greater 

than 17β-estradiol concentrations in 

female fish within at least one gonad 

stage at downstream sites 2, 12, and 

20; and upstream site 17 (table 14).

 In male fish, there were some 

significant differences in 11-ketotestosterone, but not 17β-estradiol concentrations between the upstream 

and downstream site groups (table 

13). In stage-2 males, 11-ketotestosterone concentrations were greater 

at the upstream than at the downstream site groups (p = 0.02); however, concentrations in stage-3 males 

were not different between site 

groups. 
19

0

400

800

1,200

1,600

0

0

4

4

8

8

12

12

16

16

20

20

Figure 6b. Concentrations of 11-ketotestosterone in plasma and gonado-somatic

index values of stage-1 and-2 female common carp collected during 1999.

vitellogenin, 17 -estradiol, and

VITELLOGENIN CONCENTRATIONS,

IN MILLIGRAMS PER MILLILITER

GONADO-SOMATIC INDEX,

IN PERCENT

0 0 5 3 0 0 2 0 2 2 0 1 4 6 0 4 0 2 0 3 2 11 10 6 7 1 5 1 8 4 6 10 9 7 7 5 0 0 5 8 5 4 5 7

STAGE-1 STAGE-2

Downstream Downstream Paired upsteam-downstream Paired upsteam-downstream

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

SITE LOCATION NUMBER (FIG. 2 AND TABLE 3) SITE LOCATION NUMBER (FIG. 2 AND TABLE 3)

17 -ESTRADIOL

11- KETOTESTOSTERONE

AND

CONCENTRATIONS,

IN PICOGRAMS PER MILLILITER

17 - estradiol

11-ketotestosterone

2

EXPLANATION

Number of samples

+1 Standard error

Mean

-1 Standard error







At the paired sites, there were no 

significant differences in 11-ketotestosterone or 17β-estradiol concentrations in male fish between upstream 

and downstream. Although there were 

no significant differences at the 0.05 

significance level, 11-ketotestosterone 

concentrations tended to be greater at 

upstream than downstream sites 

(p = 0.07) at most of the paired sites. 

 Both 11-ketotestosterone and 

17β-estradiol concentrations in male 

fish were significantly different 

among sites (p <0.001). Mean concentrations of 17β-estradiol in male 

fish were high in at least one gonad 

stage at downstream sites 2, 3, 4, 5, 6, 

14, and 18; and upstream sites 13 and 

19 (fig. 7, table 14). Mean concentrations of 11-ketotestosterone in male 

fish were low in at least one gonad 

stage at downstream sites 5, 12, 14, 

and 16, and upstream sites 9, and 13. 

Additionally, 17β-estradiol concentrations were greater than 11-ketotestosterone concentrations at upstream 

site 13 and downstream site 14. 

1
	!.



The gonads of female and male 

fish are considered to be fully sexually mature when GSI values reach 

approximately 20–30 percent of 

female body weight, and 5–10 percent 
20

0

400

800

1,200

1,600

VITELLOGENIN CONCENTRATIONS,

IN MILLIGRAMS PER MILLILITER

GONADO-SOMATIC INDEX,

IN PERCENT

2 0 5 0 1 0 5 0 0 0 0 1 0 1 3 3 2 3 0 0 1 0 9 12 1 3 1 11 6 2 10 11 8 7 13 12 1 9 4 6 7 12 6 6

STAGE-2 STAGE-3

Downstream Downstream Paired upsteam-downstream Paired upsteam-downstream

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

SITE LOCATION NUMBER (FIG. 2 AND TABLE 3) SITE LOCATION NUMBER (FIG. 2 AND TABLE 3)

Figure 7. Concentrations of 11-ketotestosterone in plasma and gonado-somatic

index values of stage-2 and-3 male common carp collected during 1999.

vitellogenin, 17 -estradiol, and

0.00

0

0.02

2

0.04

4

0.06

6

0.08

8

0.10

10

17 -ESTRADIOL AND

11- KETOTESTOSTERONE

CONCENTRATIONS,

IN PICOGRAMS PER MILLILITER

17 - estradiol

11-ketotestosterone

2

EXPLANATION

Number of samples

+1 Standard error

Mean

-1 Standard error







of male body weight (Kime, 1998). 

Contaminants can result in reduced 

gonad weights and reduced reproductive success (Kime, 1998). 

There were some differences in 

female GSI values between upstream 

and downstream site groups. GSI values in stage-1 females were greater in 

the upstream than the downstream site 

group (p = 0.03) (table 12). At the 

paired sites, there were no significant 

differences in GSI values for females 

between upstream and downstream. 

GSI values in female fish were 

significantly different among all sites 

(p <0.001) (figs. 6a and 6b). Mean 

GSI values were low for female fish 

in at least one gonad stage at downstream sites 3, 5, 7, 10, 16, 18, and 20; 

and upstream sites 11, 15, and 21 

(table 14).

Male GSI values in stage-3 fish 

were greater in the upstream than the 

downstream site group (p = 0.009) 

(table 13). At the paired sites, GSI 

values were significantly greater in 

males from upstream than downstream sites (p = 0.04). 

Male GSI values were significantly different among all sites 

(p <0.001) (fig. 7). Mean GSI values 

were low in male fish in at least one 

gonad stage at downstream sites 3, 5, 
21

1 Female common carp in gonad stages-0 through -2, and ages ranging from 2 to 5 years old were used in these analyses 2 Two sided t-test results3 Paired t-test result

Table 12. Comparisons of biological characteristics for female fish1 between upstream and downstream site groups for each gonad matu

and downstream paired sites 

[SE, standard error; n, number of fish sampled; p-values shown in bold indicate statistically significant differences]

Biological 

characteristic Comparison of upstream and downstream site groups 

 Gonad stage–0 Gonad stage–1 Gonad stage–2

Upstream

Mean ± SE

Downstream 

Mean ± SE p-value 2 Upstream Mean ± SE Downstream Mean ± SE p-value 2 Upstream Mean ± SE Downstream Mean ± SE p-value 2 U

M

n = 9 n = 5 n = 8 n = 39 n = 37 n = 83 

Vitellogenin (mg/mL) 0.27 ± 0.07 0.24 ± 0.14 0.6 6.03 ± 2.29 2.89 ± 0.63 0.2 2.87 ± 0.52 3.46 ± 0.37 0.26 3

17β-estradiol

(pg/mL)

531 ± 75 357 ± 42 0.08 802 ± 147 499 ± 46 0.04 567 ± 49 559 ± 36 0.84 

11-ketotestosterone (pg/mL) 428 ± 27 191 ± 53 0.02 242 ± 78 277 ± 31 0.63 379 ± 46 337 ±24 0.71 

GSI (percent) 0.9 ± 0.2 1.5 ± 0.3 0.18 6.3 ± 0.9 3.8 ± 0.4 0.03 9.9 ± 0.6 10.1 ± 0.4 0.69 7

Atretic oocytes (percent) 3.2 ± 2.3 5.0 ± 5.4 0.68 15.0 ± 4.0 10.8 ± 1.8 0.09 10.9 ± 0.9 9.5 ± 1.3 0.93 

Ceroid/lipofuscin ranking 0.82 ± 0.68 0.75 ± 0.48 0.33 0.92 ± 0.22 1.15 ± 0.48 0.94 1.06 ± 0.06 1.03 ± 0.10 0.05 0

Table 13. Comparisons of biological characteristics for male fish1 between upstream and downstream site groups for each g

and between upstream and downstream paired sites

 [SE, standard error; n, number of fish sampled; p-values shown in bold indicate statistically significant differences] 

1 Male common carp in gonad stages-2 and -3, and ages ranging from 2 to 4 years old were used in these analyses2 Two sided t-test results 3 Paired t-test result

Biological Characteristic Comparison of upstream and downstream site groups 

 Gonad Stage–2 Gonad Stage–3

Upstream

Mean ± SE

Downstream 

Mean ± SE p-value 2 Upstream Mean ± SE Downstream Mean ± SE p-value 2 Upstre

Mean ± 

n = 6 n =21 n = 49 n =109 n = 

Vitellogenin (mg/mL) 0.01 ± 0.0045 0.02 ± 0.01 0.44 0.016 ± 0.0028 0.0089 ± 0.0012 0.01 0.018 ± 

17β-estradiol (pg/mL) 254 ± 54 292 ± 30 0.57 287 ± 19 264 ± 14 0.28 260 ± 

11-ketotestosterone (pg/mL) 1022 ± 115 705 ± 96 0.02 561 ± 51 620 ± 42 0.44 709 ± 

GSI (percent) 4.7 ± 1.2 3.7 ± 0.3 0.49 6.0 ± 0.3 4.9 ± 0.2 0.009 5.8 ± 

Ceroid/lipofuscin ranking 0.83 ± 0.44 0.88 ± 0.12 0.43 0.90 ± 0.24 1.06 ± 0.08 0.21 0.86 ± 
22

Table 14. Summary of biological indicators of hormonally active agents for stage-0, -1 and -2 female, and stage-2 and 3 male co

collected during 1999 from Minnesota streams 

[Low, lowest 25th percentile within each gonad stage; High, 75th percentile (highest 25th percentile); pg/mL, picogram per milliliter; mg/mL, milligrams per milliliter; ≤, 

to; shading shows downstream sites; X indicates that the mean value for at least one gonad stage met the criteria listed in the biological ind

Biological indicators

Site location number (shown on fig. 2)

Downstream sites Paired upstream-d

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 

Female common carp

Low vitellogenin concentrations (≤ 0.07, ≤ 0.89 and ≤ 2.22 mg/mL for stage-0, -1 and -2, respectively)

X

X

X X X

Low 17β-estradiol concentrations (≤ 326, ≤ 316 and ≤

350 pg/mL for stage-0, -1 and -2, respectively)

X

X

X

X

X

X 

High 11-ketotestosterone (≥ 390, ≥356 and ≥ 445 pg/

mL for stage -0, -1, and -2, respectively)

X

X X

X X

11-ketotestosterone > 17β-estradiol 

X

X

Low gonado-somatic index values (≤ 0.7, ≤ 2.9 and ≤ 8.8 percent for stage-0, -1 and -2, respectively)

X

X

X X X X

High percent atretic oocytes (≥ 10.0, ≥ 15.3 and 

≥ 13.8 for stage-0, -1 and-2, respectively)

X

X

X

X

X

X

X

High ceroid/lipofuscin ranking (≥ 1.4, ≥ 1.6 and ≥ 1.1 

for stage-0, -1 and -2, respectively)

X

X

X

X

X

X

Male common carp

High vitellogenin concentrations (≥ 0.015 and ≥

0.016 mg/mL for stage-2 and -3, respectively)

X

X

X

X

X

X

High 17β-estradiol concentrations (≥ 366 and ≥ 339 

pg/mL for stage-2 and 3, respectively)

X

X

X

X

X

X

X 

Low 11-ketotestosterone (≤ 529 and ≤ 422 pg/mL for 

stage-2 and -3, respectively)

X

X X X

X

17β-estradiol ≥ 11-ketotestosterone

X

X

Low gonado-somatic index values (≤ 2.8 and ≤ 3.7 

percent for stage-2 and 3, respectively)

X

X X X

X

X

High ceroid/lipfuscin ranking (≥ 1.2 and ≥ 1.3 for 

stage-2 and-3, respectively)

X

X

X

X

X

Total number of biological indicators 3 4 8 4 5 2 2 4 3 4 6 7 7 5 4 
23

10, 12, and 22, and upstream sites 11 

and 15 (table 14). 

Water temperature is a stimulus 

for spawning and recrudescence and 

can influence GSI values (Kime, 

1998). Water temperatures ranged 

from 16 to 29 o

C among all sites. 

Female GSI values were not consistently related to water temperature 

(Spearman rank correlation coefficients of 0.08, -0.01, -0.34, for stage0, -1, and -2, respectively). In contrast, GSI values in male fish declined 

as water temperature increased 

(Spearman rank correlation coefficients of -0.52 for males in gonad 

stages-2 and -3). Male fish at three of 

the sites (5, 10, and 11) with the greatest water temperatures had low GSI 

values; however, it is not possible 

with data collected during this study 

to determine if lower GSI values at 

sites 5, 10, and 11 are due to greater 

temperatures or other factors. 

%	


"



An average of 86 percent of the 

female fish per site had atretic 

oocytes. The amount of atretic 

oocytes per female ranged from 0 to 

57 percent (mean = 10.25 percent). 

There was no difference in percent 

atretic oocytes between site groups 

(table 12). At the paired sites, there 

were no significant differences in percent atretic oocytes between upstream 

and downstream. 

 Percent atretic oocytes in females 

were significantly different between 

sites (p <0.001) (figs. 6a and 6b). The 

mean percent atretic oocytes in female 

fish within at least one gonad stage 

were high at downstream sites 3, 4, 8, 

14, and 18; and upstream sites 9, 11, 

and 13) (table 14).

 Ceroid/lipofuscin tissue rankings 

in female gonad tissue ranged from 0 

to 3 among all females (table 15). 

Most females were ranked 1 in both 

upstream and downstream site groups 

(75 and 66 percent respectively). 

Ceroid/lipofuscin tissue rankings in 

females were not different between 

site groups for stage-0 and -1 fish; 

however, ceroid/lipofuscin rankings 

were greater in the upstream than 

downstream site group for stage-2 fish 

(p = 0.05) (table 12). Mean ceroid/

lipofuscin rankings in female fish varied among sites (p <0.001). Mean 

ceroid/lipofuscin tissue rankings were 

high for females in at least one gonad 

stage at downstream sites 3, 4, 8, 14 

and 20 and at upstream sites 11, 13 

and 17 (table 14). 

Ceroid/lipofuscin tissue rankings 

ranged from 0 to 3 among all male 

fish (table 16). On average, 82 percent 

of males at each site had ceroid/lipofuscin tissue. There was no difference 

in ceroid/lipofuscin rankings between 

the upstream and downstream site 

groups or for the upstream–downstream paired sites. Ceroid/lipofuscin 

rankings varied among sites (p = 

0.006). Ceroid/lipofuscin rankings 

were high for male fish in at least one 

gonad stage at sites 1, 3, 8, 10, 12, and 

18; and upstream sites 17 and 19 

(table 14).

$8#?'?: #??1#

&8$#'8#'8?

*'8$*8$8$8$8

8$::$8

'$

The presence of HAAs in selected 

Minnesota streams was indicated by 

biological characteristics in common 

carp. Biological characteristics used 

in this study identified WWTP effluent as a potential source of HAAs. 

Fish located at sites upstream of 

WWTP effluent draining agricultural 

and forested land also show indications of HAAs. 

 The paired sites offered the best 

opportunity to compare biological 

characteristics between sites upstream 

and downstream of WWTPs. Because 

the dominant chemicals at upstream 

sites likely influence downstream 

sites, the effect at downstream sites 

was expected to be greater due to the 

combination of dominant upstream 

factors and WWTP effluent. This pattern was not observed for vitellogenin 

concentrations in male fish.Vitellogenin concentrations in male fish tended 

to be greater in upstream than downstream paired sites. Two of the 

upstream sites where males had elevated vitellogenin concentrations 

(sites 13 and 21) had minimal percents of streamflow that consisted of 

WWTP discharge (0.3 and 0.1 percent, respectively). It is possible that 

males at these two sites were exposed 

to effluent, which resulted in elevated 

vitellogenin concentrations. However, males with elevated vitellogenin 

concentrations were also observed at 

upstream sites with no known continuous-flow WWTPs upstream (sites 9 

and 15). This may suggest that HAAs 

that are not associated with WWTP 

effluent are present at upstream sites. 

 Sex steroid concentrations and 

GSI values at the paired sites indicated a greater effect downstream 

than upstream of WWTPs. The 17βestradiol concentrations in females 

and 11-ketotestosterone concentrations in males tended to be lower at 

sites downstream of WWTPs. Similarly, Folmar and others (1996) found 

decreases in testosterone in male common carp collected from WWTP 

effluent. GSI values in male fish were 

lower at downstream than upstream 

sites. A similar result was reported by 

Jobling and others (1996), for rainbow trout exposed to NP and 17αethynylestradiol, which are compounds observed in WWTP effluent.

Although there were some patterns in biological characteristics 

between upstream and downstream 

sites, there was a great amount of 

variability among sites within each 

group. For example, high vitellogenin 

concentrations in males were 

observed at three downstream sites (1, 

3, and 7) and four upstream sites (9, 

13, 15, 21). Female fish had low 

vitellogenin concentrations at five 

downstream and three upstream sites. 

The presence of vitellogenin in male 

fish and low concentrations in females 

may be due to a variety of chemicals, 

including agricultural pesticides and 

chemicals in WWTP effluent, that are 

reported to induce vitellogenin production in male fish. Vitellogenin 

induction has been observed in male 
24

fish from streams draining agricultural areas. Grady and others (1998) 

observed that male largemouth bass 

treated with 100 μg/L of atrazine for 

20 days had increased vitellogenin 

concentrations. Atrazine at that high 

concentration is not typically 

observed in larger streams. However, 

it may be an environmentally relevant 

concentration for some periods of the 

year, especially for common carp, 

because they move upstream into very 

small tributaries where the concentrations tend to be greater than in larger 

streams.

Vitellogenin induction has also 

been observed in male fish exposed to 

WWTP effluent. Purdom and others 

(1994) measured vitellogenin concentrations of 0.00002–0.015 mg/mL in 

caged common carp after a 3-week 

exposure to WWTP effluent. Similarly, Folmar and others (1996) 

reported the presence of vitellogenin 

in male common carp downstream of 

the Minneapolis and St. Paul Metropolitan WWTP effluent channel (site 

3). NP concentrations of 2.1 to 2.3 μg/

L during the fall of 1997 and spring of 

1998, measured by Barber and others 

(2000) in the Minneapolis and St. 

Paul Metropolitan WWTP effluent 

channel (site 3), are below those concentrations of NP (10–20 μg/L)

reported by Sumpter and Jobling 

(1995) and Hammer and others (in 

press) to induce vitellogenin in male 

fish. The Minneapolis and St. Paul 

Metropolitan WWTP effluent channel 

contained 43–150 μg/L of total combined alkylphenols (AP), alkylphenolpolyethoxylates (APEO), and 

alkylphenolethoxycarboxylates 

(APEC), which in combination may 

have resulted in vitellogenin induction 

(Barber and others, 2000). 

Both 17β-estradiol and 11 

ketotestosterone concentrations were 

variable among sites. Most of the sites 

where female fish had low 17β-estradiol concentrations, and had 11-

ketotestosterone concentrations that 

were greater than 17β-estradiol concentrations were located downstream 

of WWTPs. In male fish, 11-ketotestosterone concentrations were low at 

more sites located downstream than 

upstream of WWTPs. The tendency 

for 17β-estradiol concentrations to be 

low in female fish, and 11-ketotestosterone concentrations to be low in 

male fish and at sites downstream of 

WWTPs could be due to water chemistry. Folmar and others (1996) found 

reduced testosterone concentrations in 

male common carp exposed to 

WWTP effluent. HAAs associated 

with WWTP effluent may inhibit testosterone production and its conversion to both 17β-estradiol and 11-

ketotestosterone. 

GSI values were low for female 

and male fish at sites located 

upstream and downstream of 

WWTPs. Fish with low GSI values 

may have limited reproduction potential (Kime, 1998). Low GSI values for 

both male and female fish were 

observed at sites 3 and 5. Reduced 

GSI values have been observed by 

others in male fish below kraft mills 

(McMaster and others, 1992) and in 

male fish exposed to pesticides (Arora 

and Kulshrestha, 1984). 

The percent atretic oocytes and 

ceroid/lipofuscin tissue rankings in 

females and males were not clearly 

related to just one site group, but were 

predominant at specific sites and may 

indicate that some contaminant or 

environmental factor has reduced the 

potential for spawning or has induced 

egg reabsorption. Kumar and Pant 

(1998) and Shukla and others (1984) 

found that atresia was increased with 

pesticide exposures. 

The number of biological characteristics that indicated HAAs varied 

among sites. All sites had at least one 

biological characteristic that indicated 

the presence of HAAs in female and 

male fish (table 14). Downstream 

sites 3, 12, and 20 and upstream sites 

11 and 13 had the greatest number of 

HAA indications while downstream 

sites 1, 6, 7, and 22 and upstream sites 

9 and 19 had few indications of 

HAAs. 

At downstream sites, the number 

of indications of HAAs does not seem 

to be strongly related to the dilution of 

the effluent in the receiving stream. 

The downstream site with the greatest 

number of HAA indicators was site 3, 

which is a 100 percent effluent stream 

from the Minneapolis and St. Paul 

Metropolitan WWTP. Sites 12 and 20 

also had a high number of biological 

characteristics that indicate HAAs, 

yet had lower estimated percentages 

of wastewater (1 and 5 percent, 

respectively). In addition, sites 2 and 

5 have relatively higher estimated percentages of wastewater (48 and 66 

percent respectively) and have relatively fewer indications of HAAs. 

Differences in the types and numbers 

of biological characteristics that indicate HAAs among downstream sites 

may be due to differences in WWTP 

effluent chemistry rather than the percent streamflow consisting of WWTP 

discharge. 

At the upstream sites, there is not 

a clear factor that explains the lack of 

a consistent number of biological 

indicators. Upstream sites 11 and 13 

had the greatest number of indications 

of HAAs, while sites 9 and 19 had 

few indications of HAAs. Potential 

reasons may include differences in the 

types of pesticides used and amount 

of pesticide runoff into streams, 

unknown discharges of WWTP effluent, and runoff from animal feedlots 

that may contain HAAs. The lack of 

consistency at upstream and downstream sites may also be due to differences in the exposure time of fish 

sampled or natural variability in the 

biological characteristics.

The presence of biological indicators of HAAs at sites may be due to a 

variety of chemicals including agricultural pesticides and chemicals in 

WWTP effluent. Although water 

chemistry at each sampling site is 

unknown, observations from other 

water quality monitoring studies in 

Minnesota provide some evidence of 

chemicals that may be present at sites 

in this study that may act as HAAs. 

While this ancillary information does 

not provide the type of detailed chemical information needed to establish 

cause and effect relations, it can provide insight into the presence of 

potential HAAs in streams sampled 

during this study. 

Determination of evidence of 

HAAs is complicated by the presence 
25

of natural variability in biological 

characteristics. Natural variation in 

the biological characteristics may 

account for some of the differences 

observed in this study. Analyses were 

completed on separate gonad stages to 

remove potential natural variability in 

reproductive stage; however, there is 

likely still some variability in the biological characteristics due to natural 

reproductive status. 

This study and others indicate the 

presence of HAAs in surface water 

and the potential signs of endocrine 

disruption in resident fish populations in Minnesota streams. Detailed 

controlled studies could confirm that 

endocrine disruption has occurred and 

establish the effects of particular 

chemicals such as pesticides or components of WWTPs on fish reproduction and population structure.
26

'A

Concern about the effects of chemicals in the environment that act as hormonally active agents (HAAs) is widespread. HAAs may interfere with natural regulation of the 

endocrine system of animals by either mimicking or blocking the function of natural hormones. Potential effects of 

HAAs on fish include disruption of the endocrine system, 

specifically reproductive function. Potential sources of 

HAAs to streams in Minnesota are treated sewage (domestic and industrial) and runoff from agricultural or forested 

land. Fish are important organisms for indicating the extent 

of HAAs in surface water because they are directly exposed 

to contaminants and their endocrine systems have many 

physiological similarities to mammals, including humans. 

Male (221 individuals) and female (201 individuals) 

common carp were collected using electrofishing techniques from seven streams with sites at two locations 

(upstream and downstream of WWTPs), and eight sites 

located downstream of WWTPs with no upstream–paired 

sites. Samples were collected between August 3 and September 13, 1999. Four biological characteristics of common carp used as indicators of HAAs in the streams 

selected for this study are: (1) high concentrations of vitellogenin in male fish and low concentrations in female fish, 

(2) high or low plasma concentrations of the sex steroid 

hormones (17β-estradiol and 11-ketotestosterone), (3) low 

gonado-somatic index (GSI) (gonad weight divided by total 

body, weight multiplied by 100) values, and (4) abnormal 

gonad histopathology (high percent of atretic oocytes in 

female ovaries and high percent ceroid/lipofuscin tissue in 

male or female gonads). 

The presence of HAAs in select Minnesota streams 

was indicated by biological characteristics in common 

carp. Biological characteristics used in this study identified 

WWTP effluent as a potential source of HAAs. Additionally, fish located at sites upstream of WWTP effluent draining agricultural and forested land show indications of 

HAAs. 

Because the dominant chemicals at upstream sites 

likely influence downstream sites, the effect at downstream 

sites was expected to be greater due to the combination of 

dominant upstream factors and WWTP effluent. This pattern was not observed for vitellogenin concentrations in 

male fish.Vitellogenin concentrations in male fish tended to 

be greater in upstream than downstream paired sites. Sex 

steroid concentrations and GSI values at the paired sites 

indicated a greater effect downstream than upstream of 

WWTPs. The 17β-estradiol concentrations in females and 

11-ketotestosterone concentrations in males were lower at 

sites downstream of WWTPs. GSI values in males were 

also lower at sites downstream of WWTPs. 

Although there were some patterns in biological characteristics between upstream and downstream paired sites, 

there was a great amount of variability among all sites. All 

sites had at least one biological characteristic that indicated 

the presence of HAAs in female and male fish. Downstream sites 3, 12, and 20 and upstream sites 11 and 13 had 

the greatest number of HAA indications while downstream 

sites 1, 6, 7, and 22 and upstream sites 9 and 19 had few 

indications of HAAs. At downstream sites, the number of 

indications of HAAs does not seem to be strongly related to 

the dilution of the effluent in the receiving stream. 

The presence of biological indicators of HAAs located 

at all sites may be due to a variety of chemicals including 

agricultural pesticides and chemicals in WWTP effluent. 

Determination of evidence of HAAs is complicated by the 

presence of natural variability in biological characteristics. 

Natural variation in the biological characteristics may 

account for some of the differences observed in this study. 

This study and others indicate the presence of HAAs in 

surface water and the potential signs of endocrine disruption in resident fish populations in Minnesota streams. 

Detailed controlled studies could confirm that endocrine 

disruption has occurred and establish the effects of particular chemicals such as pesticides or components of WWTPs 

on fish reproduction and population structure.
27

$:$$$'

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Munkittrick, K.R., Van der Kraak, G.J., 

McMaster, M.E., and Portt, C.B., 

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K.A., Dahr, M.G., Boechler, M., and 

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Chang, J.P., 1992, Exposure to 

bleached kraft pulp mill effluent disrupts the pituitary—Gonadal axis of 

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Miltenburg, J.K., and Opperhuizen, 

A., 1996, Membrane burdens of 

chlorinated benzenes lower the main 

phase transition temperature in 

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vesicles—Implications for toxicity 

by narcotic chemicals: Environmental Toxicology and Chemistry, v. 15, 

no. 2, p. 203–212.

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and Janes, A.B., 1997, Mass spectral 

characterization of p-nonylphenol 

isomers using high-resolution capillary GC-MS: Journal of Chromatographic Science, v. 35, p. 19–30.

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1994, Environmentally persistent 

alkylphenolic compounds are estrogenic: Endocrinology, v. 135, no. 1, 

p. 175–187.
32
33

'$$8#:?8#?
34

 Table 15. Water temperature and values for length, weight, age, and biological characteristics of individual female fish c

[--, no data; mm, millimeter; oC, degrees in centigrade; mg/mL, milligrams per milliliter; pg/mL, picograms per milliliter; D, downstream; U, up

Site 

location 

number

Site 

group

Length 

(mm)

Weight 

(grams)

Gonad 

stage

Age 

(years)

Water 

temperature 

(oC)

Vitellogenin 

(mg/mL)

17β-estradiol 

(pg/mL)

11-

ketotestosterone 

(pg/mL)

Gonadsomatic index 

(percent)

A

oo

(pe

1 D 416 998 2 3 19 3.028 1,341 701 16.3 1 D 416 1089 2 3 19 3.111 1,078 731 15.9 

1 D 485 1588 2 4 19 2.007 866 366 7.3 

1 D 428 1315 2 4 19 3.036 941 533 10.7 

1 D 454 1406 2 4 19 2.164 432 488 9.7 

1 D 533 2359 2 4 19 2.349 497 537 15.1 

1 D 410 862 2 4 19 3.402 747 623 13.3 

1 D 450 1452 2 4 19 3.158 972 831 14.1 

1 D 465 1361 2 4 19 1.785 801 595 10.9 

1 D 456 1452 2 5 19 2.692 1,134 760 14.3 

1 D 552 2132 2 6 19 2.415 383 698 6.5 

2 D 474 1225 2 2 23 1.974 305 618 6.5 2 D 477 1542 2 2 23 1.86 307 349 17.9 

2 D 490 1769 2 2 23 1.868 502 551 8.5 

2 D 483 1678 2 2 23 1.585 379 416 15.1 

2 D 442 1315 2 2 23 1.15 303 316 8.8 

2 D 481 1542 2 2 23 1.537 118 353 14.2 3 D 551 2087 0 3 22 0.053 437 238 1.9 

3 D 571 2087 0 4 22 0.34 364 46 1.5 

3 D 520 2041 1 3 22 0.097 620 558 3.9 

3 D 511 1905 1 3 22 0.165 303 54 1.9 

3 D 517 1633 1 3 22 0.089 1,326 507 3.2 

3 D 522 1950 1 3 22 0.021 420 136 3. 

3 D 555 2268 1 4 22 0.446 453 125 3.4 

3 D 515 1996 2 3 22 0.015 343 502 6.5 

3 D 514 2177 2 3 22 1.342 747 284 8.4 

3 D 532 1814 2 3 22 0.078 283 43 4.0 

3 D 512 2087 2 3 22 0.75 1,035 258 3.1 

3 D 500 1633 2 3 22 0.252 678 237 5.5 

3 D 570 2812 2 4 22 1.003 247 256 9.3 

3 D 554 2495 2 4 22 1.329 884 111 5.1 

4 D 710 4536 1 5 -- 1.337 716 533 4.0 
35

4 D 685 3856 1 5 -- 1.72 619 186 3.9 

4 D 690 4672 1 5 -- 1.68 477 202 3.6 

4 D 638 4763 2 5 -- 4.104 296 193 8.9 5 D 319 499 0 1 28 0.138 297 63 1.0 

5 D 281 318 0 1 28 0.006 386 103 0.9 

5 D 321 499 0 1 28 0.364 168 558 0.8 

5 D 318 454 0 1 28 0.261 375 88 1.5 

5 D 331 544 1 1 28 13.047 442 75 2.6 

5 D 403 1497 2 2 28 11.597 402 113 3.5 

5 D 367 771 2 2 28 17.029 337 74 4.7 

5 D 505 1864 2 3 28 1.282 105 36 8.3 

5 D 425 1315 2 3 28 6.847 321 113 9.7 

5 D 493 1724 2 4 28 4.744 557 313 6.6 

6 D 429 1089 2 3 25 16.815 158 87 -- 7 D 415 907 1 1 23 0.989 532 58 2.5 7 D 418 998 1 3 23 5.909 319 97 2.6 

7 D 418 1043 1 3 23 0.047 419 184 1.8 

7 D 470 1406 2 2 23 5.752 374 106 11.0 

7 D 424 1089 2 2 23 1.791 342 127 12.0 

7 D 491 1678 2 2 23 6.454 385 123 10.3 

7 D 519 1860 2 3 23 5.553 243 91 11.3 

7 D 465 1542 2 3 23 3.146 365 52 11.9 

7 D 452 1225 2 3 23 4.834 343 201 11.5 

7 D 444 1361 2 3 23 8.415 449 129 11.6 

7 D 414 1043 2 3 23 4.255 556 154 10.7 

8 D 365 771 2 1 -- 1.441 133 112 6.5 8 D 430 1043 2 2 -- 1.781 114 141 8.8 

8 D 500 1905 2 3 -- 1.733 955 947 11.8 

8 D 623 3402 2 4 -- 3.309 1,014 647 7.5 

8 D 640 3901 2 5 -- 2.832 1,120 880 9.2 

--

9 U 570 2132 1 3 24 15.395 639 191 9.3 

 Table 15. Water temperature and values for length, weight, age, and biological characteristics of individual female fish c

[--, no data; mm, millimeter; oC, degrees in centigrade; mg/mL, milligrams per milliliter; pg/mL, picograms per milliliter; D, downstream; U, up

Site 

location 

number

Site 

group

Length 

(mm)

Weight 

(grams)

Gonad 

stage

Age 

(years)

Water 

temperature 

(oC)

Vitellogenin 

(mg/mL)

17β-estradiol 

(pg/mL)

11-

ketotestosterone 

(pg/mL)

Gonadsomatic index 

(percent)

A

oo

(pe
36

9 U 560 2404 1 4 24 0.861 667 107 6.3 

9 U 500 1678 2 2 24 2.596 783 145 12.1 

9 U 550 1905 2 3 24 0.686 653 152 8.1 

9 U 538 2087 2 3 24 4.061 259 177 10.5 

9 U 587 2586 2 3 24 3.251 396 107 10.4 

9 U 602 2631 2 4 24 1.768 1,090 139 7.8 

9 U 579 2313 2 4 24 1.888 791 140 6.4 

10 D 511 1860 1 3 26 0.759 394 53 2.7 

10 D 501 1814 1 4 26 10.024 38 135 3.0 

10 D 485 1452 2 2 26 0.461 319 191 5.2 

10 D 465 1361 2 3 26 2.223 235 84 7.2 

10 D 441 1270 2 3 26 4.959 1184 401 11.3 

10 D 490 1588 2 3 26 1.206 301 39 9.7 

10 D 525 1950 2 3 26 5.41 578 94 7.3 

10 D 505 1814 2 3 26 6.495 1,107 46 8.9 

10 D 442 1270 2 3 26 3.035 864 37 10.0 

10 D 499 1814 2 3 26 0.424 228 324 13.5 

10 D 500 1588 2 3 26 3.103 528 650 3.8 

10 D 515 1633 2 4 26 0.662 290 544 9.9 

11 U 392 998 0 2 29 0.59 722 489 1.8 11 U 465 1452 0 3 29 0.664 416 552 2.4 

11 U 389 998 2 2 29 2.557 301 376 4.0 

11 U 388 1089 2 2 29 0.295 797 348 4.6 

11 U 421 1315 2 2 29 1.512 634 487 6.5 

11 U 379 998 2 2 29 0.173 401 415 4.4 

11 U 381 998 2 2 29 0.599 923 373 11.0 

11 U 548 1905 2 3 29 0.458 433 401 12.4 

11 U 520 2041 2 3 29 0.35 677 542 7.3 

11 U 403 1134 2 3 29 0.715 766 504 5.1 

11 U 520 1996 2 3 29 1.102 801 427 7.1 

12 D 462 1225 0 3 24 0.749 454 205 2.6 12 D 407 680 1 2 24 0.408 161 356 3.2 

12 D 365 680 2 1 24 2.473 762 429 6.8 

 Table 15. Water temperature and values for length, weight, age, and biological characteristics of individual female fish c

[--, no data; mm, millimeter; oC, degrees in centigrade; mg/mL, milligrams per milliliter; pg/mL, picograms per milliliter; D, downstream; U, up

Site 

location 

number

Site 

group

Length 

(mm)

Weight 

(grams)

Gonad 

stage

Age 

(years)

Water 

temperature 

(oC)

Vitellogenin 

(mg/mL)

17β-estradiol 

(pg/mL)

11-

ketotestosterone 

(pg/mL)

Gonadsomatic index 

(percent)

A

oo

(pe
37

12 D 389 816 2 2 24 0.467 399 141 8.9 

12 D 380 816 2 2 24 1.139 833 341 14.3 

12 D 442 953 2 2 24 0.799 602 243 6.6 

12 D 375 771 2 2 24 0.131 122 517 11.8 

12 D 350 590 2 2 24 1.425 511 550 9.8 

12 D 441 1089 2 2 24 0.681 210 242 13.4 

12 D 404 862 2 2 24 0.496 274 390 11.0 13 U 455 1089 1 2 27 12.883 638 96 5.7 

13 U 496 1361 1 2 27 13.066 697 121 8.8 

13 U 460 1315 1 2 27 2.385 588 151 6.8 

13 U 487 1542 1 3 27 1.878 301 84 8.4 

13 U 445 1270 2 2 27 1.766 333 120 13.4 

13 U 452 1134 2 2 27 1.987 582 98 15.0 

13 U 492 1406 2 2 27 2.126 828 155 13.7 

13 U 552 2631 2 3 27 2.094 246 100 14.7 

13 U 510 1814 2 3 27 0.149 368 127 11.4 

13 U 488 1497 2 3 27 2.116 287 61 10.8 

13 U 616 2812 2 4 27 2.252 301 41 6.6 14 D 447 1089 1 2 22 1.066 397 100 2.9 

14 D 464 1270 1 2 22 1.301 449 99 2.9 

14 D 485 1406 1 3 22 2.232 335 203 8.8 

14 D 535 1860 1 3 22 2.34 365 107 9.8 

14 D 555 2268 1 3 22 16.996 628 159 6.7 

14 D 515 1814 1 3 22 2.335 433 89 6.6 

14 D 420 907 2 2 22 0.751 397 85 12.8 

14 D 405 862 2 2 22 2.499 311 86 13.0 

14 D 435 1179 2 2 22 2.121 561 104 10.9 

14 D 488 1497 2 3 22 2.007 359 147 7.6 

14 D 564 2495 2 4 22 11.683 381 108 15.6 15 U 330 816 0 1 24 0.067 542 442 0.2 

15 U 404 1179 0 2 24 0.201 911 355 0.68 

15 U 378 907 0 2 24 0.108 653 408 0.66 

15 U 420 1270 0 2 24 0.176 328 425 0.24 

 Table 15. Water temperature and values for length, weight, age, and biological characteristics of individual female fish c

[--, no data; mm, millimeter; oC, degrees in centigrade; mg/mL, milligrams per milliliter; pg/mL, picograms per milliliter; D, downstream; U, up

Site 

location 

number

Site 

group

Length 

(mm)

Weight 

(grams)

Gonad 

stage

Age 

(years)

Water 

temperature 

(oC)

Vitellogenin 

(mg/mL)

17β-estradiol 

(pg/mL)

11-

ketotestosterone 

(pg/mL)

Gonadsomatic index 

(percent)

A

oo

(pe
38

15 U 388 1043 0 2 24 0.076 702 531 0.38 

15 U 420 1315 0 3 24 0.068 228 374 0.84 

15 U 450 1406 0 4 24 0.305 432 305 1.0 

15 U 455 1588 0 4 24 0.288 385 412 0.82 

16 D 360 816 0 1 22 0.033 331 128 1.1 16 D 323 635 0 1 22 0.022 327 87 0.95 

16 D 415 1089 0 2 22 0.032 302 112 0.92 

16 D 424 1225 1 2 22 0.008 110 49 1.3 

16 D 446 1497 1 2 22 0.043 133 92 0.9 

16 D 410 1270 1 2 22 0.015 609 151 0.78 

16 D 448 14062 1 3 22 0.758 413 33 0.16 

17 U 770 7348 2 5 16 7.013 306 363 9.4 17 U 698 4853 2 5 16 5.015 69 1594 14.4 

17 U 732 5670 2 5 16 6.139 199 546 11.5 

17 U 751 7484 2 5 16 3.953 105 220 13.6 

17 U 745 5715 2 5 16 4.411 132 515 6.9 

17 U 750 6396 2 6 16 3.326 61 565 12.8 

17 U 785 6804 2 6 16 3.83 629 932 12.7 

17 U 734 5216 3 5 16 0.094 158 445 9.6 18 D 610 3221 1 4 23 1.831 911 399 4.3 

18 D 602 3039 1 5 23 2.7 857 288 5.4 

18 D 485 1814 2 2 23 2.583 453 356 10.0 

18 D 542 2223 2 3 23 2.916 1223 515 9.3 

18 D 515 2041 2 3 23 2.759 394 507 7.3 

18 D 530 2223 2 3 23 3.449 823 420 8.3 

18 D 536 2268 2 3 23 2.226 923 400 12.1 

18 D 555 2404 2 4 23 1.606 1483 446 7.5 

18 D 605 3402 2 4 23 2.153 741 534 6.1 

18 D 614 3402 2 4 23 3.712 648 334 8.1 

19 U 407 907 2 2 21 2.275 381 385 4.1 19 U 531 2132 2 3 21 3.715 738 497 8.0 

19 U 551 2767 2 4 21 2.168 685 427 13.6 

 Table 15. Water temperature and values for length, weight, age, and biological characteristics of individual female fish c

[--, no data; mm, millimeter; oC, degrees in centigrade; mg/mL, milligrams per milliliter; pg/mL, picograms per milliliter; D, downstream; U, up

Site 

location 

number

Site 

group

Length 

(mm)

Weight 

(grams)

Gonad 

stage

Age 

(years)

Water 

temperature 

(oC)

Vitellogenin 

(mg/mL)

17β-estradiol 

(pg/mL)

11-

ketotestosterone 

(pg/mL)

Gonadsomatic index 

(percent)

A

oo

(pe
39

19 U 670 4309 2 4 21 3.951 591 557 15.1 

19 U 662 5534 2 4 21 3.262 629 445 18.7 

20 D 367 726 0 2 19 0.027 228 356 0.68 20 D 426 771 1 2 19 1.188 129 737 3.2 

20 D 500 1860 1 3 19 3.323 105 251 14.0 

20 D 519 1542 1 4 19 3.726 386 308 6.0 

20 D 505 1542 2 3 19 3.533 886 428 12.2 

20 D 550 2359 2 4 19 2.898 287 252 18.2 

20 D 578 2449 2 4 19 4.485 122 183 15.3 

20 D 518 1905 2 4 19 6.518 113 331 17.0 21 U 411 816 1 2 22 0.207 1505 645 2.0 

21 U 435 907 1 2 22 1.582 1382 541 2.8 

21 U 375 635 2 1 22 8.451 1303 503 6.0 

21 U 350 544 2 1 22 9.35 433 489 7.7 

21 U 394 771 2 1 22 14.563 482 383 3.1 

21 U 354 544 2 1 22 5.703 472 382 6.1 

21 U 380 499 2 1 22 1.507 791 431 6.8 

21 U 425 771 2 2 22 0.069 1185 636 6.0 

21 U 378 726 2 2 22 17.297 1275 689 9.1 

21 U 369 635 2 2 22 1.696 571 689 12.6 

21 U 440 1179 2 2 22 0.838 902 595 8.1 

21 U 395 862 2 2 22 9.956 556 435 10.7 

22 D 402 907 1 2 24 13.358 763 722 2.4 22 D 405 1043 1 2 24 8.231 334 126 4.0 

22 D 434 1225 1 2 24 5.775 391 562 5.5 

22 D 410 862 1 2 24 1.364 733 352 2.4 

22 D 394 816 1 2 24 0.075 431 315 1.8 

22 D 358 635 1 2 24 4.335 422 331 2.4 

22 D 402 953 1 2 24 0.692 1244 513 1.6 

22 D 428 998 1 2 24 -- 691 415 4.7 

22 D 394 862 1 2 24 4.155 851 282 2.7 

22 D 472 1225 1 3 24 0.635 501 459 3.0 

22 D 444 1179 1 3 24 8.71 575 536 5.3 

 Table 15. Water temperature and values for length, weight, age, and biological characteristics of individual female fish c

[--, no data; mm, millimeter; oC, degrees in centigrade; mg/mL, milligrams per milliliter; pg/mL, picograms per milliliter; D, downstream; U, up

Site 

location 

number

Site 

group

Length 

(mm)

Weight 

(grams)

Gonad 

stage

Age 

(years)

Water 

temperature 

(oC)

Vitellogenin 

(mg/mL)

17β-estradiol 

(pg/mL)

11-

ketotestosterone 

(pg/mL)

Gonadsomatic index 

(percent)

A

oo

(pe
40

1Female common carp in gonad stages-0, 1, and 2 and ages ranging from 1 to 6 (200 samples) were used in the inital analyses to determine the in

age on biological characteristics. Because sample size for females in gonad stage-3 (1 sample) was too small for further analyses, this sample was remples (females 1- and 6-years old ) were excluded from further analyses because biological characteristics were found to vary with age. Samples markeanalyses. Samples marked with a Y were used in subsequent analyses of biological characteristics between site groups, among paired sites, and among 

22 D 411 953 2 2 24 1.352 741 345 7.0 

22 D 405 953 2 2 24 6.858 410 361 7.1 

22 D 375 816 2 2 24 1.396 691 315 14.2 

22 D 431 1043 2 2 24 3.146 993 662 9.7 

22 D 468 1497 2 3 24 5.58 442 342 4.3 

22 D 472 1588 2 3 24 11.295 622 382 11.3 

22 D 452 1270 2 3 24 13.216 401 544 12.7 

 Table 15. Water temperature and values for length, weight, age, and biological characteristics of individual female fish c

[--, no data; mm, millimeter; oC, degrees in centigrade; mg/mL, milligrams per milliliter; pg/mL, picograms per milliliter; D, downstream; U, up

Site 

location 

number

Site 

group

Length 

(mm)

Weight 

(grams)

Gonad 

stage

Age 

(years)

Water 

temperature 

(oC)

Vitellogenin 

(mg/mL)

17β-estradiol 

(pg/mL)

11-

ketotestosterone 

(pg/mL)

Gonadsomatic index 

(percent)

A

oo

(pe
41

 Table 16. Water temperature and values for length, weight, age, and biological characteristics of individual male fish c

[--, no data; mm, millimeter; oC, degrees in centigrade; mg/mL, milligrams per milliliter; pg/mL, picograms per milliliter; D, downstream; U, ups

Site location 

number

Site 

group

Length 

(mm)

Weight 

(grams)

Gonad 

stage

Age 

(years)

Water 

temperature 

(oC)

Vitellogenin 

(mg/mL)

17β-estradiol 

(pg/mL)

11-

ketotestosterone 

(pg/mL)

Gonad

somatic i

(percen

1 D 394 726 2 3 19 0.0011 158 1,421 5.9 1 D 394 726 2 3 19 0.013 172 1,189 4.7 

1 D 550 2132 2 5 19 0.024 206 1,362 4.5 

1 D 518 2132 3 3 19 0.006 212 1,111 6.9 

1 D 381 816 3 3 19 0.007 319 1,740 5.1 

1 D 395 1134 3 3 19 0.007 165 1,507 7.5 

1 D 516 1860 3 4 19 <0.001 178 787 5.2 

1 D 436 1043 3 4 19 0.05 169 1,187 7.4 

1 D 526 1950 3 4 19 0.009 334 2,211 5.9 

1 D 547 2177 3 4 19 0.006 247 914 6.2 

1 D 396 1361 3 4 19 0.106 90 823 4.3 

1 D 419 953 3 4 19 0.005 185 862 5.3 

1 D 529 1996 3 5 19 0.005 226 779 6.5 

2 D 336 771 3 1 23 0.02 181 514 3.1 2 D 442 1134 3 2 23 0.001 456 671 3.6 

2 D 404 907 3 2 23 0.002 361 592 2.2 

2 D 440 1225 3 2 23 0.003 478 642 4.3 

2 D 443 1179 3 2 23 0.013 223 476 4.2 

2 D 439 1270 3 2 23 0.03 222 550 5.5 

2 D 480 1542 3 2 23 0.003 298 431 3.4 

2 D 488 1633 3 2 23 <0.001 965 1,709 5.8 

2 D 442 1179 3 2 23 <0.001 438 1,006 3.1 

2 D 436 1179 3 2 23 <0.001 131 889 3.0 

2 D 445 1089 3 2 23 0.008 264 602 2.9 

2 D 482 1406 3 3 23 0.009 125 385 2.8 

2 D 276 1452 3 3 23 <0.001 297 679 3.5 

3 D 465 1361 1 3 22 0.014 252 712 1.5 3 D 505 1814 2 3 22 0.028 527 892 3.9 

3 D 532 2087 2 3 22 0.005 309 590 3.1 

3 D 500 1724 2 3 22 0.026 320 811 1.6 

3 D 480 1633 2 3 22 0.03 256 851 3.1 

3 D 550 2087 2 4 22 0.32 424 109 3.2 

3 D 460 1270 3 2 22 0.019 277 522 3.5 
42

3 D 510 1225 4 3 22 0.016 171 752 0.74 D 614 2994 3 4 -- <0.001 594 650 5.3 

4 D 625 3629 3 4 -- 0.004 281 329 5.1 

4 D 580 3357 3 4 -- 0.004 421 529 9.4 

4 D 627 4264 3 5 -- 0.00073 374 901 6.0 

4 D 733 5035 3 5 -- <0.001 383 618 8.3 

4 D 675 4400 3 5 -- <0.001 421 435 6.3 

5 D 374 771 2 2 28 0.009 434 821 2.2 5 D 485 1406 3 3 28 0.01 224 422 3.1 6 D 388 1089 3 1 25 0.007 214 561 3.9 

6 D 378 771 3 1 25 0.021 321 844 3.6 

6 D 467 1195 3 2 25 0.004 267 350 5.1 

6 D 412 1134 3 2 25 0.029 409 533 2.5 

6 D 609 2631 3 3 25 0.00012 336 464 5.1 

6 D 531 2359 3 3 25 0.016 254 389 3.5 

6 D 541 1901 3 3 25 0.015 265 281 4.3 

6 D 551 2299 3 3 25 0.012 208 822 4.7 

6 D 482 1678 3 3 25 0.021 368 502 2.9 

6 D 502 2223 3 3 25 0.009 553 1,221 3.0 

6 D 546 2586 3 3 25 0.008 346 311 4.6 

6 D 465 1452 3 3 25 0.006 245 832 4.3 

6 D 527 2132 3 3 25 0.02 424 437 3.9 

6 D 629 3447 3 5 25 <0.001 248 259 7.9 

7 D 365 635 1 2 23 0.042 132 598 1.1 7 D 463 1270 2 2 23 0.026 147 155 4.4 

7 D 383 726 2 2 23 0.067 185 995 3.4 

7 D 408 907 2 2 23 0.013 386 712 4.5 

7 D 394 816 2 3 23 0.019 283 322 2.8 

7 D 440 1225 2 3 23 0.027 347 988 7.4 

7 D 427 1089 3 2 23 0.017 185 177 6.4 

7 D 495 1497 3 3 23 0.013 238 581 3.6 

7 D 482 1633 3 3 23 0.019 298 481 6.2 

 Table 16. Water temperature and values for length, weight, age, and biological characteristics of individual male fish c

[--, no data; mm, millimeter; oC, degrees in centigrade; mg/mL, milligrams per milliliter; pg/mL, picograms per milliliter; D, downstream; U, ups

Site location 

number

Site 

group

Length 

(mm)

Weight 

(grams)

Gonad 

stage

Age 

(years)

Water 

temperature 

(oC)

Vitellogenin 

(mg/mL)

17β-estradiol 

(pg/mL)

11-

ketotestosterone 

(pg/mL)

Gonad

somatic i

(percen
43

7 D 483 1588 3 3 23 0.006 287 322 6.1 

7 D 392 907 3 3 23 0.037 224 447 4.6 

7 D 468 1315 3 3 23 0.042 339 529 5.9 

7 D 574 2313 3 4 23 0.015 -- -- 4.1 

8 D 464 1361 3 2 -- 0.005 102 306 6.0 8 D 575 2586 3 4 -- 0.003 230 1,498 8.1 9 U 394 953 3 3 24 0.019 326 333 3.6 

9 U 524 1905 3 3 24 0.021 281 270 4.2 

9 U 522 1678 3 3 24 0.008 339 234 4.2 

9 U 545 2041 3 3 24 0.02 408 228 7.6 

9 U 515 1905 3 3 24 0.023 276 197 6.7 

9 U 554 1905 3 3 24 0.017 204 229 8.1 

9 U 489 1497 3 3 24 0.011 246 369 7.9 

9 U 546 2087 3 3 24 0.012 298 385 6.3 

9 U 562 2404 3 3 24 0.007 342 423 7.7 

9 U 557 2404 3 4 24 0.035 378 562 9.9 

10 D 488 1452 3 2 26 0.005 399 162 3.0 10 D 469 1406 3 2 26 0.009 268 534 3.1 

10 D 468 1588 3 2 26 0.004 81 34 2.5 

10 D 501 1860 3 2 26 0.002 291 607 3.6 

10 D 480 1497 3 3 26 0.004 533 408 3.9 

10 D 490 1542 3 3 26 0.002 483 867 3.2 

10 D 501 1542 3 3 26 0.003 302 265 2.6 

10 D 501 1542 3 3 26 <0.001 281 125 

10 D 476 1542 3 3 26 0.003 321 415 3.9 

10 D 489 1542 3 4 26 0.002 188 752 3. 

10 D 496 1588 3 4 26 0.003 129 1,869 2.0 

10 D 529 2041 3 5 26 0.005 299 583 4.2 

11 U 378 953 3 1 29 0.007 58 396 2.3 

11 U 365 862 3 1 29 0 105 571 1.4 

11 U 383 953 3 2 29 <0.001 108 404 1.2 

11 U 382 998 3 2 29 0.012 104 524 2.4 

 Table 16. Water temperature and values for length, weight, age, and biological characteristics of individual male fish c

[--, no data; mm, millimeter; oC, degrees in centigrade; mg/mL, milligrams per milliliter; pg/mL, picograms per milliliter; D, downstream; U, ups

Site location 

number

Site 

group

Length 

(mm)

Weight 

(grams)

Gonad 

stage

Age 

(years)

Water 

temperature 

(oC)

Vitellogenin 

(mg/mL)

17β-estradiol 

(pg/mL)

11-

ketotestosterone 

(pg/mL)

Gonad

somatic i

(percen
44

11 U 382 1043 3 2 29 0.003 120 708 2.9 

11 U 370 907 3 2 29 0.005 232 739 3.4 

11 U 400 1134 3 2 29 0.009 298 752 1.7 

11 U 372 953 3 2 29 0.001 111 456 2.2 

11 U 564 1406 3 3 29 0.005 161 641 2.7 

11 U 515 1996 3 3 29 <0.001 33 625 2.2 

12 D 385 680 2 2 24 0.005 132 238 2.2 12 D 367 635 3 1 24 <0.001 106 178 2.7 

12 D 373 590 3 1 24 0.006 65 849 3.6 

12 D 355 635 3 1 24 0.011 143 413 2.2 

12 D 409 771 3 2 24 0.006 205 186 2.1 

12 D 373 635 3 2 24 0.003 102 535 1.6 

12 D 417 680 3 2 24 0.002 106 125 1.6 

12 D 385 680 3 2 24 0.006 55 412 2.9 

12 D 426 771 3 2 24 0.002 95 534 2.7 

12 D 402 862 3 2 24 <0.001 167 588 3.0 

12 D 445 1089 3 3 24 <0.001 112 420 1.6 

12 D 415 816 4 2 24 0.002 109 351 1.1 13 U 430 1043 3 2 27 0.023 185 154 4.8 

13 U 450 1134 3 2 27 0.012 256 94 5.6 

13 U 434 1089 3 2 27 0.009 298 551 5.1 

13 U 409 816 3 2 27 0.064 636 358 6.9 

13 U 460 1270 3 2 27 0.012 395 313 6.3 

13 U 445 1043 3 2 27 0.029 299 441 7.6 

13 U 455 1134 3 2 27 0.043 391 367 7.6 

13 U 397 816 3 2 27 0.02 338 197 8.5 

13 U 433 1043 3 2 27 0.04 190 82 6.9 

13 U 453 1225 3 2 27 0.094 316 148 6.9 

13 U 467 1270 3 3 27 0.024 391 227 4.3 

13 U 495 1542 3 3 27 0.007 396 482 6.3 

13 U 507 1633 3 3 27 0.015 348 308 5.0 14 D 530 1814 2 3 22 0.01 319 222 4.3 

14 D 442 1089 3 2 22 0.02 362 137 6.0 

 Table 16. Water temperature and values for length, weight, age, and biological characteristics of individual male fish c

[--, no data; mm, millimeter; oC, degrees in centigrade; mg/mL, milligrams per milliliter; pg/mL, picograms per milliliter; D, downstream; U, ups

Site location 

number

Site 

group

Length 

(mm)

Weight 

(grams)

Gonad 

stage

Age 

(years)

Water 

temperature 

(oC)

Vitellogenin 

(mg/mL)

17β-estradiol 

(pg/mL)

11-

ketotestosterone 

(pg/mL)

Gonad

somatic i

(percen
45

14 D 440 953 3 2 22 0.008 477 206 4.9 

14 D 480 1270 3 2 22 <0.001 301 115 

14 D 533 1361 3 2 22 0.004 335 201 2.6 

14 D 455 998 3 2 22 0.007 407 187 4.6 

14 D 591 2223 3 2 22 0.005 421 381 6.5 

14 D 405 726 3 2 22 0.023 246 279 6.3 

14 D 455 1043 3 2 22 <0.001 488 558 4.1 

14 D 535 1724 3 3 22 0.006 332 209 3.7 

14 D 523 1588 3 3 22 0.005 324 363 5.6 

14 D 561 2087 3 3 22 0.005 320 292 5.3 

14 D 505 1588 3 3 22 <0.001 525 451 6.4 15 U 362 907 2 1 24 0.01 61 519 1.3 

15 U 417 1406 2 2 24 0.002 421 759 3.3 

15 U 386 1089 2 2 24 0.008 292 814 2.0 

15 U 417 1179 2 3 24 0.033 383 1,158 2.5 

15 U 404 1270 3 2 24 0.08 182 729 4.0 

15 U 590 2948 3 5 24 <0.001 244 626 7.1 

15 U 588 2994 3 5 24 <0.001 493 1,152 7.8 16 D 397 1134 2 2 22 0.017 95 992 3.9 

16 D 398 1089 2 2 22 0.003 113 102 1.9 

16 D 596 2177 2 3 22 0.003 153 178 3.5 

16 D 430 1134 3 2 22 0.039 99 178 5.8 

16 D 421 1089 3 2 22 0.008 127 469 4.6 

16 D 424 1315 3 2 22 0.006 174 175 4.9 

16 D 401 953 3 2 22 0.003 117 1,135 2.5 

16 D 461 1542 3 2 22 0.013 349 352 10.0 

16 D 437 1179 3 2 22 0.015 132 271 4.5 

16 D 552 2268 3 3 22 0.02 301 148 5.7 

16 D 542 1678 3 4 22 0.005 158 368 5.2 

16 D 507 1996 3 4 22 0.015 234 598 7.4 

17 U 619 3493 2 4 16 0.007 111 1,231 8.7 17 U 655 4082 2 4 16 0.008 121 755 8.1 

17 U 675 4536 2 5 16 <0.001 139 826 7.1 

 Table 16. Water temperature and values for length, weight, age, and biological characteristics of individual male fish c

[--, no data; mm, millimeter; oC, degrees in centigrade; mg/mL, milligrams per milliliter; pg/mL, picograms per milliliter; D, downstream; U, ups

Site location 

number

Site 

group

Length 

(mm)

Weight 

(grams)

Gonad 

stage

Age 

(years)

Water 

temperature 

(oC)

Vitellogenin 

(mg/mL)

17β-estradiol 

(pg/mL)

11-

ketotestosterone 

(pg/mL)

Gonad

somatic i

(percen
46

17 U 670 4309 2 5 16 <0.001 175 261 9.8 

17 U 700 4717 2 5 16 0.005 271 401 7.1 

17 U 700 5126 2 5 16 <0.001 78 636 10.6 

17 U 680 4264 3 4 16 <0.001 165 791 9.2 

17 U 624 3674 3 4 16 <0.001 679 536 8.2 

17 U 627 3810 3 4 16 0.002 104 223 8.7 

17 U 667 4309 3 4 16 0.011 75 613 10.5 

17 U 610 3357 3 5 16 <0.001 131 758 8.0 

17 U 665 4672 3 5 16 <0.001 363 268 11.4 

17 U 760 5987 3 5 16 <0.001 113 574 10.1 

17 U 728 5625 3 5 16 <0.001 102 303 13.8 

17 U 660 4445 3 5 16 0.009 31 621 10.6 

18 D 530 1769 2 3 23 <0.001 518 924 2.9 18 D 515 1905 2 3 23 0.003 482 1,596 4.7 

18 D 566 2676 2 4 23 <0.001 372 699 3.4 

18 D 500 1678 3 3 23 <0.001 287 618 6.2 

18 D 486 1633 3 3 23 0.00039 203 473 5.8 

18 D 480 1724 3 3 23 0.003 367 739 5.5 

18 D 595 3130 3 4 23 0.003 344 605 4.2 

18 D 604 3039 3 4 23 07 496 564 4.9 

18 D 554 2449 3 4 23 <0.001 285 501 6.5 

19 U 449 1089 3 2 21 <0.001 388 579 10.4 19 U 580 2404 3 3 21 0.006 370 585 9.2 

19 U 621 3629 3 3 21 0.003 487 761 8.3 

19 U 594 2767 3 3 21 <0.001 236 950 7.8 

19 U 577 2812 3 3 21 <0.001 344 662 8.0 

19 U 718 4717 3 4 21 <0.001 389 850 6.4 

19 U 628 3130 3 4 21 <0.001 556 899 7.2 

20 D 423 907 3 2 19 <0.001 181 582 5.4 20 D 516 1814 3 3 19 <0.001 421 1,080 8.6 

20 D 511 1724 3 3 19 0.003 101 5,13 8.3 

20 D 438 907 3 3 19 <0.001 20 624 7.0 

20 D 502 1406 3 3 19 0.002 95 1,897 10.0 

 Table 16. Water temperature and values for length, weight, age, and biological characteristics of individual male fish c

[--, no data; mm, millimeter; oC, degrees in centigrade; mg/mL, milligrams per milliliter; pg/mL, picograms per milliliter; D, downstream; U, ups

Site location 

number

Site 

group

Length 

(mm)

Weight 

(grams)

Gonad 

stage

Age 

(years)

Water 

temperature 

(oC)

Vitellogenin 

(mg/mL)

17β-estradiol 

(pg/mL)

11-

ketotestosterone 

(pg/mL)

Gonad

somatic i

(percen
47

1 Male common carp in gonad stages 2 and 3 and ages ranging from 1- to 5- years old(217 samples) were used in the inital analyses to determine 

and age on biological characteristics. Because sample size for males in gonad stage-1 (2 samples) and gonad stage-2 (2 samples) were too small for furemoved from further analyses. Some samples (males 1- and 5-years old) were excluded from further analyses because biological characteristics were fwith a N were excluded from further analyses. Samples marked with a Y were used in subsequent analyses of biological characteristics between site grosites.

20 D 463 1452 3 3 19 0.005 211 1,846 11.6 

20 D 502 1678 3 3 19 <0.001 167 1,195 8.8 

20 D 536 1860 3 3 19 0.004 229 1,826 7.1 

20 D 429 953 3 3 19 <0.001 77 531 4.8 

20 D 441 998 3 3 19 <0.001 172 546 7.0 

20 D 567 2132 3 4 19 0.005 107 221 9.0 

20 D 557 2268 3 4 19 0.01 125 622 8.8 21 U 383 726 2 1 22 0.021 238 1,301 4.3 

21 U 343 499 2 1 22 0.009 131 844 6.2 

21 U 446 1179 2 3 22 0.016 193 1,413 3.9 

21 U 374 726 3 1 22 <0.001 221 1,867 5.8 

21 U 364 680 3 1 22 0.011 178 2,065 4.3 

21 U 350 680 3 1 22 0.013 177 1,679 3.7 

21 U 398 726 3 2 22 0.021 226 951 5.4 

21 U 393 862 3 2 22 0.01 320 1,162 7.7 

21 U 392 816 3 2 22 0.007 185 901 5.8 

21 U 420 1043 3 2 22 0.02 249 1,834 3.8 

21 U 442 1225 3 2 22 0.018 197 1,537 5.6 

21 U 447 1270 3 3 22 0.012 212 1,100 3.6 

22 D 427 998 3 2 24 0.017 107 444 4.6 22 D 447 998 3 2 24 0.013 122 505 4.7 

22 D 322 680 3 2 24 0.006 217 252 3.8 

22 D 522 1633 3 3 24 0.032 149 491 2.0 

22 D 484 1270 3 3 24 0.002 58 419 2.1 

22 D 397 953 3 3 24 0.01 106 1,187 4.6 

 Table 16. Water temperature and values for length, weight, age, and biological characteristics of individual male fish c

[--, no data; mm, millimeter; oC, degrees in centigrade; mg/mL, milligrams per milliliter; pg/mL, picograms per milliliter; D, downstream; U, ups

Site location 

number

Site 

group

Length 

(mm)

Weight 

(grams)

Gonad 

stage

Age 

(years)

Water 

temperature 

(oC)

Vitellogenin 

(mg/mL)

17β-estradiol 

(pg/mL)

11-

ketotestosterone 

(pg/mL)

Gonad

somatic i

(percen