U.S. Department of the Interior

U.S. Geological Survey

Scientific Investigations Report 2006—5163

Biomonitoring of Environmental Status and Trends (BEST) 

Program: Environmental Contaminants, Health Indicators, 

and Reproductive Biomarkers in Fish from the Colorado River 

Basin
Cover: The U.S. map shows the Colorado River Basin (green) and stations sampled during this study (orange). Shown in gray are 

major river basins and stations in the conterminous U.S. and Alaska sampled during Biomonitoring of Environmental Status and Trends 

Program (BEST) investigations.
Biomonitoring of Environmental Status and 

Trends (BEST) Program: Environmental 

Contaminants, Health Indicators, and 

Reproductive Biomarkers in Fish from the 

Colorado River Basin

By Jo Ellen Hinck, Vicki S. Blazer, Nancy D. Denslow, Timothy S. Gross, Kathy R. 

Echols, Anne P. Davis, Tom W. May, Carl E. Orazio, James J. Coyle, and Donald E. 

Tillitt

Scientific Investigations Report 2006–5163

U.S. Department of the Interior

U.S. Geological Survey
U.S. Department of the Interior

Dirk Kempthorne, Secretary

U.S. Geological Survey

P. Patrick Leahy, Acting Director

U.S. Geological Survey, Reston, Virginia: 2006

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Suggested citation:

Hinck, J.E., Blazer, V.S., Denslow, N.D., Gross, T.S., Echols, K.R., Davis, A.P., May, T.W., Orazio, C.E., Coyle, J.J., and 

Tillitt, D.E., 2006, Biomonitoring of Environmental Status and Trends (BEST) Program: environmental contaminants, 

health indicators, and reproductive biomarkers in fish from the Colorado River Basin: U.S. Geological Survey, Scientific 

Investigations Report 2006-5163, 119 p. 
Preface    iii

Preface

This study was conducted as part of the U.S. Geological Survey (USGS) Biomonitoring of 

Environmental Status and Trends (BEST) Program’s Large River Monitoring Network (LRMN). 

BEST evolved from previous Federal monitoring programs including the National Pesticide Monitoring Program (NPMP) of the 1960s, renamed the National Contaminant Biomonitoring Program 

(NCBP) in the early 1970s, which also screened for elemental contaminants. The U.S. Fish and 

Wildlife Service (USFWS) participated in the NPMP and maintained the NCBP by monitoring 

concentrations of persistent contaminants in freshwater fish and avian wildlife through 1986 

(Schmitt and others, 1999). The BEST Program was initiated in the 1990s to build on information produced by these earlier programs and to provide more biologically relevant information 

regarding potential contaminant effects on lands and species under USFWS management. The 

program was transferred to the National Biological Survey in 1993 and ultimately to USGS in 

1996. The LRMN has principal emphasis to identify, monitor, and assess the effects of chemical contaminants on the fish health in the nation’s large rivers. The 2003 Colorado River Basin 

(CDRB) study is one in a series of BEST-LRMN Program monitoring investigations. Previous 

studies include the Mississippi River Basin in 1995, the Columbia River and Rio Grande Basins in 

1997, and the Yukon River Basin in 2002. 

Acknowledgements

This study was conducted jointly by the USGS through its research centers in Columbia, 

Missouri (Columbia Environmental Research Center, CERC), Gainesville, Florida (Florida Integrated Science Center, FISC), and Kearneysville, West Virginia (Leetown Science Center, LSC). 

The study was facilitated through a Research Work Order with the USGS-Florida Cooperative 

Fish and Wildlife Research Unit at the University of Florida (UF), Gainesville, Florida. The U.S. 

Fish and Wildlife Service (USFWS) offices in Salt Lake City, Utah; Vernal, Utah; Grand Junction, 

Colorado; Albuquerque, New Mexico; Las Vegas, Nevada; Phoenix, Arizona; Parker, Arizona; and 

Willow Beach, Arizona participated in fish collections. California Department of Fish and Game 

in Blythe, California and the Colorado Division of Wildlife in Fort Collins, Colorado also collected 

fish. Many individuals representing USGS, USFWS, UF, and other organizations contributed 

substantially during the conduct of the study. M. Annis (CERC), P. Anderson [BEST Program, 

(BEST)], D. Baker (USFWS), T. Bartish (BEST), J. Candrl (CERC), J. Coyle (BEST), L.R. DeWeese 

(USGS National Water Quality Assessment (NAWQA) Program), S. Goodbred (NAWQA), D. Nicks 

(CERC), C. Marr (USFWS), E. Orsak (USFWS), and D. Papoulias (CERC) participated in field dissections. Chemical analyses were conducted by CERC Environmental Chemistry laboratories (B. 

Brumbaugh, K. Feltz, J. Meadows, M. Tanner, G. Tegerdine, and M. Walther). Laboratory analyses for biomarkers were performed by D. Nicks, A. Donahue, C. Wieser (FISC), D. Bowling (LSC), 

and K. Kroll (UF). D. Bowling (LSC) and K. Spring (LSC) assisted in the histology slide preparation, and B. Martin (LSC) assisted with the macrophage aggregate evaluation for samples from 

Stations 311, 313, 314, 316, 317, 321, 322, 323, and 325. C. Smith (Colorado Histo-Prep, Fort 

Collins, Colorado) analyzed the histology samples from Stations 312, 315, 319, 320, and 324. L. 

Stanley (Tottenham, Ontario) and J. Isely (USGS Coop Unit, Clemson University) aged the fish. R. 

Lipkin (CERC) prepared the report for publication. R. Claunch (CERC) and M. Keuss (CERC) provided quality assurance for tables and figures. S. Goodbred, K. Koch (Bureau of Land Management), C. Marr, and R. Patiño (USGS Coop Unit, Texas Tech University) reviewed the report and 

provided additional information.
Contents

Preface ...........................................................................................................................................................iii

Acknowledgements......................................................................................................................................iii

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

Introduction.....................................................................................................................................................2

Colorado River Basin Overview..........................................................................................................2

Hydrology and Environmental Setting......................................................................................2

Geology..........................................................................................................................................4

Landownership and Urban Areas .............................................................................................4

Dams..............................................................................................................................................5

Water Quality Impairments and Fish Consumption Advisories............................................5

Major Sources of Contaminants to the Colorado River Basin ......................................................7

Agriculture ....................................................................................................................................7

Mining and Extractive Industries ..............................................................................................7

Industrial and Municipal Sources.............................................................................................9

Perchlorate .................................................................................................................................10

Extant Sources of Information on Contaminants in the Colorado River Basin.........................10

Materials and Methods...............................................................................................................................12

Collection Sites....................................................................................................................................12

Target Species and Sampling Strategy...........................................................................................12

Monitoring Methods Overview.........................................................................................................12

Fish Collection .....................................................................................................................................17

Sample Processing.............................................................................................................................17

Laboratory Analysis............................................................................................................................18

Composite Sample Preparation...............................................................................................18

Elemental Contaminants and Moisture Content...................................................................18

Organochlorine Contaminants and Lipid Content ................................................................19

H4IIE Rat Hepatoma Cell Bioassay .........................................................................................19

EROD Activity..............................................................................................................................19

Fish Health Indicators ........................................................................................................................20

General Histopathological Analyses ......................................................................................20

Quantitative Organism-Level Indicators ................................................................................20

Macrophage Aggregates ........................................................................................................20

Reproductive Biomarkers..................................................................................................................20

Gonadal Histopathology ..............................................................................................20

Vitellogenin .................................................................................................................................21

Sex Steroid Hormones ..............................................................................................................21

Data Set Composition and Statistical Analyses ............................................................................22

Results and Discussion...............................................................................................................................22

Geographic Distribution and Demographic Characteristics of the Fish Collected..................22

Accumulative contaminants, H4IIE Bioassay, and EROD Activity..............................................24

Elemental Contaminants...........................................................................................................24

Arsenic................................................................................................................................24

Selenium ............................................................................................................................28

Mercury .............................................................................................................................29

Cadmium.............................................................................................................................34

iv    Contaminants, Health Indicators, and Reproductive Biomarkers in Fish from the Colorado River Basin
Lead.....................................................................................................................................35

Zinc......................................................................................................................................35

Copper.................................................................................................................................35

Chromium ...........................................................................................................................36

Nickel..................................................................................................................................37

Organochlorine Contaminants.................................................................................................37

DDT and its primary metabolites....................................................................................37

Chlordane and heptachlor...............................................................................................41

Dieldrin................................................................................................................................42

Endrin..................................................................................................................................44

Mirex...................................................................................................................................44

Toxaphene..........................................................................................................................44

Hexachlorobenzene (HCB)..............................................................................................44

Pentachlorobenzene ........................................................................................................45

Pentachloroanisole (PCA) ...............................................................................................45

Hexachlorocyclohexane (HCH) ......................................................................................45

Dacthal................................................................................................................................45

Endosulfan .........................................................................................................................45

Methoxychlor ....................................................................................................................45

Total PCBs, H4IIE-Derived Dioxin Equivalents and Ethoxyresorufin O-Deethylase 

(EROD) Activity..............................................................................................................47

Total PCBs ..........................................................................................................................47

H4IIE Bioassay ..................................................................................................................47

Ethoxyresorufin O-Deethylase (EROD) Activity ...........................................................48

EROD in Carp ............................................................................................................48

EROD in bass ............................................................................................................48

EROD in channel catfish .........................................................................................49

EROD in other species ...........................................................................................49

Accumulative Contaminants, H4IIE, and EROD Activity: Summary...................................51

Fish Health Indicators ........................................................................................................................53

Organism-Level Indicators.......................................................................................................53

External Gross Lesions ....................................................................................................53

Health Assessment Index................................................................................................53

Condition factor and Organosomatic Indices .......................................................................53

Condition factor and Organosomatic Indices in Carp ................................................53

Condition factor and Organosomatic Indices in Bass................................................55

Condition factor and Organosomatic Indices in Channel catfish.............................57

Cellular and Histopathological Indicators .............................................................................59

MA measurements in carp..............................................................................................60

MA measurements in bass .............................................................................................61

MA measurements in channel catfish ..........................................................................62

General Histopathology ...................................................................................................63

Fish Health Indicators: Summary ............................................................................................65

Reproductive Biomarkers..................................................................................................................67

Reproductive Biomarkers in Carp...........................................................................................67

Reproductive Biomarkers in Bass ..........................................................................................71

Reproductive Biomarkers in Channel catfish .......................................................................77

Reproductive Biomarkers: Summary......................................................................................78

Contents    v
Figures

Figure 1. Map of the Colorado River Basin ..........................................................................................3

Figure 2. Map of land ownership in the Colorado River Basin .........................................................6

Figure 3. Map of coal deposits and mining operations in the Colorado River Basin....................8

Figure 4. Concentrations arsenic (As) and selenium (Se) in whole-body fish composite 

samples from the Colorado River Basin.............................................................................28

Figure 5. Concentrations total mercury (Hg) in whole-body fish composite samples 

from the Colorado River Basin.............................................................................................34

Figure 6. Concentrations cadmium (Cd) and lead (Pb) in whole-body fish composite samples 

from the Colorado River Basin.............................................................................................35

Figure 7. Concentrations of zinc (Zn), copper (Cu), chromium (Cr), and nickel (Ni) in whole-

body fish composite samples from the Colorado River Basin........................................36

Figure 8. Concentrations of banned pesticides or pesticide products in whole-body fish 

composite samples from the Colorado River Basin.........................................................39

Figure 9. Unweighted geometric mean concentrations of total DDT and chlordane-

related compounds in whole-body fish composite samples from the Colorado 

 River Basin..............................................................................................................................42

Figure 10. Concentrations of unlisted or restricted use pesticides or pesticide products 

in whole-body fish composite samples from the Colorado River Basin.......................46

Figure 11. Concentrations of total PCBs and H4IIE bioassay-derived TCDD-EQ in whole-

body fish composite samples from the Colorado River Basin........................................48

Figure 12. Hepatic microsomal EROD activity in female and male carp, bass, and channel

catfish from the Colorado River Basin ...............................................................................49

Figure 13. Mean HAI scores by lesion location in carp, bass, and channel catfish from 

the Colorado River Basin......................................................................................................55

Spatial patterns in contaminant concentrations and biomarker responses ............................80

Correlations between Chemical and Biological Endpoints ................................................80

Geographic Summaries ............................................................................................................80

Upper Colorado River Basin (Upper CDRB)..................................................................80

Yampa River, Lay, CO (Station 311)........................................................................83

Green River, Ouray NWR, UT (Station 312)..........................................................83

Green River, San Rafael, UT (Station 313)............................................................83

Gunnison River, Delta, CO (Station 314)................................................................84

Colorado River, Loma, CO (Station 315)................................................................84

Colorado River, Gold Bar Canyon, UT (Station 316)............................................85

San Juan River, Hogback Diversion, NM (Station 317) .....................................85

Lower Colorado River Basin (Lower CDRB) .................................................................86

Colorado River, South Cove, AZ (Station 319) .....................................................86

Colorado River, Willow Beach, AZ (Station 320).................................................86

Colorado River, Needles, CA (Station 321)...........................................................87

Colorado River, Imperial Dam, AZ (Station 322)..................................................87

Gila River, Hayden, AZ (Station 323)......................................................................87

Gila River, Phoenix, AZ (Station 324).....................................................................87

Gila River, Arlington, AZ (Station 325)...................................................................88

Conclusions...................................................................................................................................................88

References....................................................................................................................................................91

Appendices.................................................................................................................................................107

vi    Contaminants, Health Indicators, and Reproductive Biomarkers in Fish from the Colorado River Basin
Figure 14. Fish health indicators by station in female and male carp from the Colorado 

River Basin..............................................................................................................................57

Figure 15. Fish health indicators by station in female and male bass from the Colorado River 

 Basin ........................................................................................................................................59

Figure 16. Fish health indicators by station in female and male channel catfish from the 

Colorado River Basin.............................................................................................................61

Figure 17. Splenic macrophage aggregate parameters by station in female and male carp 

from the Colorado River Basin.............................................................................................63

Figure 18. Splenic macrophage aggregate parameters by station in female and male bass 

from the Colorado River Basin............................................................................................64

Figure 19. Splenic macrophage aggregate parameters by station in female and male 

channel catfish from the Colorado River Basin................................................................64

Figure 20. Gonadal stage proportions by station in female and male carp, bass, and 

channel catfish from the Colorado River Basin................................................................68

Figure 21. Selected gonadal histological observations in CDRB fish..............................................69

Figure 22. Reproductive health indicators by station in female carp from the Colorado 

River Basin..............................................................................................................................71

Figure 23. Reproductive health indicators by station in male carp from the Colorado 

River Basin..............................................................................................................................72

Figure 24. Reproductive health indicators by station in female bass from the Colorado 

River Basin..............................................................................................................................75

Figure 25. Reproductive health indicators by station in male bass from the Colorado 

River Basin..............................................................................................................................76

Figure 26. Reproductive health indicators by station in female channel catfish from the 

Colorado River Basin.............................................................................................................77

Figure 27. Reproductive health indicators by station in male channel catfish from the 

Colorado River Basin.............................................................................................................78

Figure 28. Maximum concentrations of selenium (Se) and mercury (Hg) in composite 

samples of whole fish ...........................................................................................................84

Figure 29. Plasma vitellogenin (vtg) concentrations in male fish and occurrence of 

intersex fish in the Colorado River Basin...........................................................................85

Tables

Table 1. National Wildlife Refuges located within the Colorado River Basin ...............................5

Table 2. Stations sampled in 2003 and collection date in the Colorado River Basin..................13

Table 3. Methods incorporated into the Colorado River Basin......................................................14

Table 4. Organochlorine chemical and elemental contaminants measured in whole-

body fish composite samples ...............................................................................................15

Table 5. Monitoring and assessment strategy for polycyclic aromatic and 

polyhalogenated hydrocarbons ...........................................................................................17

Table 6. Number of fish collected in the Colorado River Basin......................................................23

Table 7. Lengths, weights, and age of carp collected in the Colorado River Basin....................25

Table 8. Lengths, weights, and ages of bass collected in the Colorado River Basin..................26

Table 9. Lengths, weights, and ages of channel catfish collected in the Colorado 

River Basin................................................................................................................................27

Table 10. Percent of samples and stations that exceeded limit of detection 

concentrations for elemental contaminants in composite samples of whole 

 fish from the Colorado River Basin .......................................................................................29

Contents    vii
Table 11. Unweighted geometric mean, minimum, and maximum concentrations of 

elemental contaminants in fish collected in the Colorado River Basin.........................30

Table 12. Spatial trends of chemical contaminants in fish collected in the Colorado 

River Basin...............................................................................................................................32

Table 13. Unweighted geometric mean, minimum, and maximum concentrations of 

select elemental contaminants in fish from BEST-LRMN studies..................................33

Table 14. Percent of samples and stations with concentrations exceeding the limit 

of detection for organochlorine chemical residues in composite samples 

 of whole fish in the Colorado River Basin...........................................................................38

Table 15. Unweighted geometric mean, minimum, and maximum concentrations 

of organochlorine chemical contaminants in fish from stations in the 

 Colorado River Basin..............................................................................................................40

Table 15. Unweighted geometric mean, minimum, and maximum concentrations 

of organochlorine chemical contaminants in fish from stations in the 

 Colorado River Basin...............................................................................................................41

Table 16. Unweighted geometric mean, minimum, and maximum concentrations of select

organochlorine chemical contaminants fish from BEST-LRMN studies.......................43

Table 17. Geometric mean, sample size, minimum, and maximum of microsomal EROD 

activity in fish collected in the Colorado River Basin.......................................................50

Table 18. Number and location of external lesions identified on fish collected in the 

Colorado River Basin...............................................................................................................52

Table 19. Distribution of health assessment index scores among carp, bass, catfish, 

brown trout, and white sucker collected in the Colorado River Basin...........................54

Table 20. Arithmetic mean of condition factor by species and station in carp, bass, 

and channel catfish collected in the Colorado River Basin .............................................56

Table 21. Arithmetic mean of splenosomatic index by species and station in carp, bass, 

and channel catfish collected in the Colorado River Basin .............................................58

Table 22. Arithmetic mean of the hepatosomatic index in bass and channel catfish 

collected in the Colorado River Basin..................................................................................60

Table 23. Arithmetic mean of macrophage aggregate density, area, and percent tissue 

occupied in carp, bass, and channel catfish from the Colorado River Basin ...............62

Table 24. Arithmetic mean of gonadosomatic index and vitellogenin in carp, bass, 

and channel catfish collected in the Colorado River Basin in 2003 ................................70

Table 25. Arithmetic mean of 17β-estradiol, 11-ketotestosterone, and the ratio of 

17β-estradiol to 11-ketotestosterone in carp, bass, and channel catfish 

 from the Colorado River Basin..............................................................................................73

Table 25. Arithmetic mean of 17β-estradiol, 11-ketotestosterone, and the ratio of 

17β-estradiol to 11-ketotestosterone in carp, bass, and channel catfish 

 from the Colorado River Basin..............................................................................................74

Table 26. Statistically significant correlations between biological and chemical 

endpoints in carp, bass, and channel catfish ....................................................................81

Table 27. Summary of chemical and biological indicator results by CDRB station.......................82

viii    Contaminants, Health Indicators, and Reproductive Biomarkers in Fish from the Colorado River Basin
Appendix 1. Selected species within the Colorado River Basin identified as 

having special status by the USFWS. ..........................................................................108

Appendix 2. Results of quality assurance and limits of detection for elemental 

contaminants in whole-body fish composites from the Colorado River Basin .....109

Appendix 3. Results of quality assurance, method detection limit, and method 

quantitative limit for organochlorine compounds in whole-body fish 

 composites from the Colorado River Basin..................................................................110

Appendix 4. Lengths, weights, and ages of non-target species collected in the 

Colorado River Basin .......................................................................................................113

Appendix 5. DDT homolog concentrations in composite samples from Stations 

      324 and 325........................................................................................................................114

Appendix 6. Results of preliminary analysis-of-variance investigating the effects 

of various factors on biomarker responses in carp, bass, and 

 channel catfish in the Colorado River Basin................................................................115

Appendix 7. Fish health indicators for non-target species collected in the 

      Colorado River Basin......................................................................................................118

Conversion Factors

Multiply By To obtain

Length

centimeter (cm) 0.3937 inch (in)

millimeter (mm) 0.03937 inch (in)

meter (m) 3.281 foot (ft)

kilometer (km) 0.6214 mile (mi)

Area

square kilometer (km2) 0.3861 square mile (mi)

cubic kilometer (km3) 0.2399 cubic mile (mi3)

Volume

milliliter (mL) 0.03381 fluid ounce (fl. oz)

liter (L) 1.057 quart (qt)

liter (L) 0.2642 gallon (gal)

cubic meter (cm3) 0.0002642 million gallons (Mgal)

Mass

kilogram (kg) 2.205 pound, avoirdupois (lb)

Concentration

microgram per gram (μg/g) = part per million (ppm; 106)

nanogram per gram (ng/g) = part per billion (ppb; 109)

picogram per gram (pg/g) = part per trillion (pptr; 1012)

milligram per millimeter (mg/mL) = part per thousand (ppt; 103)

milligram per liter (mg/L) = part per million (ppm; 106)

microgram per liter (μg/L) = part per billion (ppb; 109)

Temperature in degrees Celsius (°C) may be converted to degrees Fahrenheit (°F) as follows:

 °F=(1.8×°C)+32

Temperature in degrees Fahrenheit (°F) may be converted to degrees Celsius (°C) as follows:

 °C=(°F-32)/1.8

Contents    ix
x    Contaminants, Health Indicators, and Reproductive Biomarkers in Fish from the Colorado River Basin
Abstract

Seven fish species were collected from 14 sites on rivers 

in the Colorado River Basin (CDRB) from August to October 

2003. Spatial trends in the concentrations of accumulative 

contaminants were documented and contaminant effects on 

the fish were assessed. Sites were located on the mainstem of 

the Colorado River and on the Yampa, Green, Gunnison, San 

Juan, and Gila Rivers. Common carp (Cyprinus carpio), black 

bass (Micropterus sp.), and channel catfish (Ictalurus punctatus) were the targeted species. Fish were field-examined for 

external and internal anomalies, selected organs were weighed 

to compute somatic indices, and tissue and fluid samples were 

preserved for fish health and reproductive biomarker analyses. 

Composite samples of whole fish, grouped by species and 

gender, from each site were analyzed for organochlorine and 

elemental contaminants using performance-based and instrumental methods. 2,3,7,8-tetrachlorodibenzo-p-dioxin-like 

activity (TCDD-EQ) was measured using the H4IIE rat hepatoma cell bioassay. Selenium (Se) and mercury (Hg) concentrations were elevated throughout the CDRB, and pesticides 

concentrations were greatest in fish from agricultural areas in 

the Lower Colorado River and Gila River. Selenium concentrations exceeded toxicity thresholds for fish (>1.0 μg/g ww) 

at all sites except from the Gila River at Hayden, Arizona. 

Mercury concentrations were elevated (>0.1 μg/g ww) in fish 

from the Yampa River at Lay, Colorado; the Green River at 

Ouray National Wildlife Refuge (NWR), Utah and San Rafael, 

Utah; the San Juan River at Hogback Diversion, New Mexico; 

and the Colorado River at Gold Bar Canyon, Utah, Needles, 

California, and Imperial Dam, Arizona. Concentrations of 

p,p’-DDE were relatively high in fish from Arlington, Arizona 

(>1.0 μg/g ww) and Phoenix, Arizona (>0.5 μg/g ww). Concentrations of other banned pesticides including toxaphene, 

total chlordanes, and dieldrin were also greatest at these two 

sites but did not exceed toxicity thresholds. Current-use or 

unlisted pesticides such as dacthal, endosulfan, γ-HCH, and 

methoxychlor were also greatest in fish from Gila River. Total 

polychlorinated biphenyls (PCBs; >0.11 μg/g ww) and TCDDEQs (>5 pg/g ww) exceeded wildlife guidelines in fish from 

the Gila River at Phoenix, Arizona. Hepatic ethoxyresorufin 

O-deethylase (EROD) activity was also relatively high in carp 

from the Gila River at Phoenix, Arizona and in bass from the 

Green River at Ouray NWR, Utah. Altered biomarkers were 

noted in fish throughout the CDRB. Fish from some stations 

responded to chronic contaminant exposure as indicated by 

fish health and reproductive biomarker results. Multiple fish 

health indicators including altered body and organ weights 

and high health assessment index scores may be associated 

with elevated Se concentrations in fish from the Colorado 

River at Loma, Colorado and Needles, California. Although 

grossly visible external or internal lesions were found on 

most fish from some sites, histopathological analysis determined many of these to be inflammatory responses associated with parasites. Edema, exophthalmos, and cataracts 

were noted in fish from sites with elevated Se concentrations. 

Reproductive biomarkers including gonad development and 

maturation, vitellogenin concentrations, and steroid hormone 

concentrations were anomalous in fish from the Gila River 

at Hayden and Phoenix, Arizona. In addition, intersex fish 

were found at seven of 14 sites. The intersex condition was 

identified in smallmouth bass (M. dolomieu), largemouth bass 

(M. salmoides), channel catfish, and carp and may indicate 

exposure to endocrine disrupting compounds. Seven of ten 

male smallmouth bass from the Yampa River at Lay, ColoBiomonitoring of Environmental Status and Trends (BEST)

Program: Environmental Contaminants and their Effects

on Fish in the Colorado River Basin

Jo Ellen Hinck1, Vicki S. Blazer2, Nancy D. Denslow3, Timothy S. Gross4*, Kathy R. Echols1, Anne P. Davis1†,Tom 

W. May1, Carl E. Orazio1, James J. Coyle5, and Donald E. Tillitt1

1

U.S. Geological Survey (USGS), Columbia Environmental Research Center (CERC), 4200 New Haven Rd., Columbia, MO 65201

2

USGS Leetown Science Center, 11649 Leetown Rd., Kearneysville, WV 

24530

3

Center for Environmental and Human Toxicology, PO Box 110885, University of Florida, Gainesville, FL 32611

4

USGS Florida Integrated Science Center, 7920 NW 71st St, Gainesville, 

FL 32653

5

USGS, BEST Program, Fort Collins Science Center, 2150 Centre Ave., 

Building C, Fort Collins, CO 80526

*

Current address Center for Environmental and Human Toxicology, PO Box 

110885, University of Florida, Gainesville, FL 32611

†

Current address New Mexico Environment Department, 1190 St. Francis 

Dr., Santa Fe, NM 87502
rado were intersex. Male carp, bass, and channel catfish 

with low concentrations of vitellogenin were common in the 

CDRB. Comparatively high vitellogenin concentrations (>0.2 

mg/mL) were measured in male fish from the Green River at 

Ouray NWR, Utah and the Colorado River at Imperial Dam, 

Arizona and may indicate exposure to estrogenic chemicals. 

Biomarker responses may be associated with industrial and 

municipal discharges, mining operations, irrigated agriculture, 

and atmospheric deposition. Continued biological monitoring 

is warranted to identify consistently degraded sites and those 

with emerging problems in the CDRB. 

Introduction

The Colorado River (CDR) is the most important river of 

the arid southwest U.S. The CDR provides water for municipal and industrial water supplies, agriculture, hydroelectric 

power, and the tourism industry including sport fisheries in 

seven states from its headwaters in the Rocky Mountains to the 

Gulf of California (fig. 1). Increasing water demands and a 

recent drought have impacted water quality in the CDR and its 

major tributaries. Exploitation of natural resources, weathering of mineralized geologic formations, wastewater effluents, 

urban runoff, and agricultural practices have contributed to 

declines in water quality and have impacted habitat quality 

for biota in the Colorado River Basin (CDRB). As a result, 

many CDRB waters are listed as impaired. Multiple CDRB 

states have made efforts to restore native aquatic species 

and to characterize contaminants in CDRB waters. Elevated 

concentrations of metals and metalloids have been reported in 

water, sediment, and biota throughout the CDRB, and elevated 

concentrations of organochlorine pesticides such as DDT and 

its metabolites have been found in water, sediments, and biota 

in the agricultural areas of the Lower CDRB (Baker and others, 1992; Stephens and others, 1992; King and others, 1993, 

1997; Abell, 1994; Bevans and others, 1996; Schmitt and 

others, 1999; Gebler, 2000; Spahr and others, 2000; GarcíaHernández and others, 2001, 2006; Gellenbeck and Anning, 

2002). These studies have concluded that CDRB biota are at 

risk from exposure to elevated contaminant concentrations.

The Biomonitoring of Environmental Status and Trends 

(BEST) Program’s Large River Monitoring Network (LRMN) 

studied the CDR and several of its largest tributaries from 

August to October 2003. The LRMN is unique among 

national monitoring programs with its emphasis on characterizing the effects of environmental contaminants on fish health. 

The LRMN accomplishes this by measuring chemical concentrations within an organism and evaluating the physiological, 

morphological, and histopathological responses of contaminant exposure by the organism. Our primary objective was to 

document the occurrence and distribution of contaminants and 

their effects on CDRB fish and to evaluate the potential risk 

from these contaminants to other wildlife. Secondary objectives were to compare biomonitoring results to other major 

rivers systems in the U.S. and to further refine benchmarks for 

quantification and interpretation of biomarker results. These 

latter objectives were achieved by building on the results of 

similar LRMN investigations in the Mississippi River Basin 

(MRB; Schmitt, 2002), Rio Grande Basin (RGB; Schmitt and 

others, 2004), Columbia River Basin (CRB; Hinck and others, 2004a), and Yukon River Basin (YRB; Hinck and others, 

2004b). Contaminant concentrations in fish from our study 

were also compared to historical and contemporaneous CDRB 

data sets (Baker and others, 1992; García-Hernández and others, 2001; King and others, 1993, 1997; Schmitt and others, 

1999).

Findings of the 2003 CDRB study are reported in this 

document. Data from this study have been incorporated into 

an interactive national database at: <http://www.cerc.usgs.gov/

data/best/search/index.htm>. Results from this study, together 

with those from other LRMN investigations, will help resource 

managers and scientists to assess contaminant effects on fish 

and wildlife and to identify CDRB areas that warrant further 

investigation of contaminant risks.

Colorado River Basin Overview

Hydrology and Environmental Setting

The CDR is the largest river in the southwestern U.S. and 

is the sixth largest river in the U.S. in terms of flow. The CDR 

flows 2,250 km (kilometer) through seven states, from the 

Rocky Mountains of Northern Colorado to the Gulf of California in Mexico (Patrick, 2000) and drains 630,000 km2

 (U.S. 

Department of the Interior (USDOI), 2001). Major tributaries 

include the Green, Gunnison, White, Yampa, San Juan, and 

Gila Rivers (fig. 1). The San Juan River converges with the 

CDR near Lake Powell in northern Arizona and is the largest 

tributary to the CDR with an annual discharge of 3.1 million 

m3 (Patrick, 2000). The Gila River (GR) in southern Arizona 

flows through arid and desert areas to connect with the CDR 

near Yuma, Arizona. The CDR then flows 50 km in Mexico 

although much of the water is consumed or diverted before it 

reaches the Gulf of California.

The CDR hydrology has been altered to accommodate 

water demands, control flooding, and generate hydroelectric 

power. The CDRB was legally divided into the Upper CDRB 

and Lower CDRB at Lee’s Ferry upstream of the Grand Canyon by the Colorado Compact of 1922 (Brown, 1927; Patrick, 

2000; Stanford and Ward, 1986; USDOI, 2001). A series of 

dams, reservoirs, and diversion canals were constructed to 

deliver water to each CDRB state, Native American Indian 

Tribes, and Mexico (Stanford and Ward, 1986). The compact 

allocated water amounts for each state in the CDRB based 

on estimated average flow at Lee’s Ferry. Federal legislation 

apportioned 7.5 million acre-ft (9.25 million m3) of water 

annually to each the Upper and Lower CDRB. Distribution of 

the annual flow in the Upper CDRB is as follows: Colorado, 

2    Contaminants, Health Indicators, and Reproductive Biomarkers in Fish from the Colorado River Basin
51%; New Mexico, 11%; Utah, 23%; Wyoming, 14% (Patrick, 

2000). Arizona, California, and Nevada receive 37%, 59%, 

and 4%, respectively, of the annual flow in the Lower CDRB 

(Patrick, 2000). The Mexican Treaty of 1944 guarantees 1.5 

million acre-ft (1.85 million m3) of water delivered to Mexico. 

The flow of the CDR at Lee’s Ferry was over-estimated when 

the allocations were calculated, and as a result, water demands 

will soon exceed water supplies (Spofford, 1980; Stanford and 

Ward, 1986).

Impoundments, channelized reaches, and irrigation diversions affect water flow in the CDRB (Gloss and others, 2005; 

Starnes, 1995). Annual flows from snow melt and thunderstorms fluctuated greatly before the construction of major 

dams and reservoirs. Today, some reaches only receive flow 

during the monsoon season (July-September), or flows consist 

entirely of irrigation return, wastewater effluent, or urban 

runoff (Anning, 2003; Arnold and others, 2004; Gebler, 1998; 

Starnes, 1995). Hydrology in the Upper CDR is characterized 

by rapids created from steep slopes in topography, lower sediment and dissolved-solids concentrations, and cooler temperatures (Gloss and others, 2005; Patrick, 2000; Spahr and others, 

2000). The Lower CDR has reduced flow from dams and reservoirs, little slope in the topography, increased sediment load, 

diversions from channelization and extensive irrigation, and 

high evaporation rates from the arid climate. Mean monthly 

flows in the mainstem CDR (>10,000 ft3/s; >283 m3/s) were 

greater than those from large tributaries (<1,000 ft3/s; <28 

m3/s; U.S. Geological Survey (USGS), 2006). Some of the 

lowest flows (<100 ft3/s; <3 m3/s) occurred in the GR (Anning, 

2003; USGS, 2006). More than 40% of the CDRB annual 

flow derives from the Upper CDRB, where the elevation is 

>3,000 m (Stanford and Ward, 1986) and heavy precipitation 

from snow and rainfall (>100 cm per year) provides 75% of 

the surface water. The semiarid and arid regions of the CDRB 

receive < 25 cm of precipitation annually and provide little 

additional flow to the mainstem river (Brown, 1927; Patrick, 

2000). The amount of precipitation decreases to <15 cm 

annually in the Lower CDRB at elevations <1500 m. Flow in 

many of the lower basin tributaries, such as the Lower GR, is 

slow and intermittent due to dams and diversions upstream. 

Tributaries in the Lower CDRB rely on infrequent rainstorms 

for surface water recharge.

Figure 1. Map of the Colorado River Basin (CDRB) including state boundaries, cities, major rivers and tributaries, dams, National 

Wildlife Refuges, and sites sampled in 2003. See Table 2 for station descriptions.

Introduction    3
The CDRB has a diverse landscape that reflects the 

climate in the region and ranges from needleleaf forest in 

mountain regions to shrub and grassland in the arid desert 

regions. Six ecoregion divisions are found in the CDRB 

(USDOI, 1997). Temperate Steppe Mountains are found west 

of the Green River in northeastern Utah and at high altitudes 

in Colorado. Temperate Desert Mountains are located in south 

central Utah and along the Yampa River in Colorado, and 

Temperate Deserts characterize western Colorado and eastern Utah. Northern Arizona and southern Utah have Tropical/Subtropical Steppe, and Tropical/Subtropical Mountains 

are found in these same areas at higher altitudes. Most of the 

Lower CDRB including southern Arizona and California is 

Tropical/Subtropical Desert. 

Climate in the Upper CDRB varies with altitude and 

ranges from alpine to arid conditions. The climate in the 

Lower CDRB is influenced by air masses from the Gulf of 

California, Gulf of Mexico, and the Eastern Pacific Ocean 

which often produce a subtropical high pressure ridge that 

results in low amounts of precipitation, clear skies, warm 

weather, and low humidity. This region is mostly arid with 

high rates of evapotranspiration and has little surface water 

recharge. Water temperature are typically <20°C in the mainstem CDRB and larger tributaries, although temperature in 

the GR can exceed 25°C during the late summer and early fall 

(Anning, 2003; USGS, 2006). 

Geology

Crystalline and sedimentary rocks and alluvium comprised of stream, landslide and glacial deposits are present in 

the CDRB. Formations are also of marine origin and contain 

sodium chloride (halite) and calcium sulfate (gypsum) salts 

(Moody and Mueller, 1984). The CDR is high in dissolved 

solids, carrying >9 million tons of salt downstream annually (Patrick, 2000; USDOI, 2001). Natural mineral springs, 

irrigation, weathering of the soils, reservoir evaporation, and 

municipal and industrial sources contribute to high dissolved 

solids in the CDRB (Patrick, 2000). Federal legislation authorized the U.S. Bureau of Reclamation, Department of Interior, 

and Department of Agriculture to implement cost-effective 

measures for managing salinity in the CDRB (Trueman, 

1998). Salinity control projects include erosion control and 

lined irrigation ditches and sprinkler systems. Desalination 

plants, which have been developed to reduce salt loads, have 

had limited success in the Lower CDRB (Stanford and Ward, 

1986). 

Total dissolved solids (TDS) in the CDR and its tributaries reflect the geology of the CDRB. Weathering of the 

Mancos shale formation, composed of gypsum and limestone, 

increases dissolved mineral concentrations in CDRB waters 

(Moody and Mueller, 1984; Stanford and Ward, 1986; Patrick, 

2000; USDOI, 2001). The Mancos formation in the Upper 

CDRB is extensively drained, which has increased sulfate concentrations two to four fold as water flows through the formation (Stanford and Ward, 1986). Residual soils derived from 

sandstone or shale are generally shallow and contain large 

amounts of soluble minerals including selenium (Se; Presser 

and others, 1994). Irrigation, natural springs, and man-made 

wells intercept water high in TDS associated with these 

formations and discharge into the CDR. As a result, TDS 

increase from approximately 50 mg/L at CDR headwaters to 

800 mg/L at Imperial Dam (Moody and Mueller, 1984). The 

current TDS load is attributed to irrigation and impoundments, 

diversions of headwaters low in salinity, flow depletion from 

evapotranspiration on irrigated land, and reservoir evaporation 

(Gloss and others, 2005; Moody and Mueller, 1984; Stanford 

and Ward, 1986).

Landownership and Urban Areas

Landownership in the CDRB consists of Federal (64%), 

Native American (19%), military (2%), and private or state 

lands (15%; fig. 2). The U.S. National Park Service, U.S. 

Bureau of Reclamation, and U.S. Fish and Wildlife Service 

(USFWS) own much of the land along the CDR and its major 

tributaries. Many of these areas are designated as national 

parks, monuments, and recreation areas. Private lands used 

for grazing and agriculture are typically located within the 

flood plains of these rivers. Large Native American reservations in the CDRB include the Unitah and Ouray Tribes along 

the Green River, the Southern Ute Tribe and Navajo Nation 

along the San Juan River, the Hualapai and Havasupai Tribes 

along the Grand Canyon, Fort Apache and San Carlos Apache 

Tribes in the Upper GR, and the Colorado Indian Tribe along 

the Lower CDR. Large military facilities include the Barry 

M. Goldwater Air Force Range, the Yuma Marine Corps Air 

Station, and the Yuma Proving Ground in the Lower CDRB. 

Land use in the CDRB has changed in the past 50 years with 

decreases in forests and pastures and increases in cropland, 

highways, airports, roads, wilderness areas and parks, and 

industrial and urban areas (O’Donnell, 1997).

Threatened and Endangered species are found on Federal 

lands of the CDRB (Appendix 1). Several areas have been 

designated National Wildlife Refuges (NWRs) that provide 

important nesting habitat for waterfowl and wintering areas 

and stopover points for migratory birds (table 1; fig. 1). Several regional conservation programs have been established to 

monitor and protect endangered large river fishes [bonytail 

chub (Gila elegans), humpback chub (G. cypha), razorback 

sucker (Xyrauchen texanus), and Colorado pikeminnow 

(Ptychocheilus lucius)] in CDRB waterways designated as 

critical habitat (Holden, 2000; Lower Colorado River MultiSpecies Conservation Program, 2005; Upper Colorado River 

Endangered Fish Recovery Program (UFRP), 2005). Recovery strategies include raising endangered fish in hatcheries for 

stocking, managing non-native fish, improving river habitat, 

and conducting research (Hamilton and others, 2005a; UFRP, 

2005). 

The CDRB is sparsely populated due to climate and 

topography, but population centers are growing at a rapid 

pace. The two largest urban areas are Phoenix, Arizona and 

4    Contaminants, Health Indicators, and Reproductive Biomarkers in Fish from the Colorado River Basin
Las Vegas, Nevada. Phoenix, the sixth largest city in the U.S., 

has a population >1,300,000 (Perry and Mackun, 2001). Las 

Vegas, one of the fastest growing metropolitan areas from 

1990 to 2000, has a population >1,500,000. Other cities in the 

basin with populations >100,000 include Tucson and Yuma, 

Arizona. The population in the CDRB increased >13% from 

1990 to 2000 and populations growth has increased >50% 

in some areas in western Colorado, southern Nevada, southwestern Utah, and northwestern Arizona (Perry and Mackun, 

2001). In addition, resort mountain communities have been 

steadily growing at a rate of 10% a year. As the CDRB population continues to grow, demands on water and other natural 

resources will increase.

Dams

The CDR is one of the most highly regulated river 

systems in the world. Multiple dams have been built for 

agricultural irrigation, hydroelectric power generation, river 

regulation and flood control, recreation, municipal and 

industrial supplies, and wastewater disposal (fig. 1; Gloss and 

others, 2005; Paulson and Baker, 1983; USDOI, 1997). More 

than 117 reservoirs have been created (Stanford and Ward, 

1986). The dams and reservoirs have altered the ecological, 

morphological, and hydrological characteristics of the CDR. 

Effects of regulation include changes in water temperature, 

turbidity, water chemistry, aquatic habitat, disruption of fish 

migration and spawning, increased predation and competition 

by non-native species, and extinction of native species (Gloss 

and others, 2005; Holden, 1979; Holden and Stalnaker, 1975; 

Minckley, 1991; Standford and Ward, 1986). 

Lake Mead, the nation’s largest reservoir, extends from 

the mouth of the Grand Canyon at South Cove to the Black 

Canyon at Hoover Dam. Hoover Dam is used to generate 

hydroelectric power for Nevada, Arizona, and California (Harbeck and others, 1958; Paulson and Baker, 1983). Approximately 90% of Lake Mead’s 28 million acre-feet (34.5 million 

m3) volume is from the CDR (Paulson and Baker, 1983), and 

Lake Mead supplies most of the water storage and regulation 

for the Lower CDRB (Patrick, 2000). Glen Canyon Dam, the 

only major dam located on the CDR mainstem in the Upper 

CDRB, forms Lake Powell and controls most of the water 

from the Upper CDRB (Patrick, 2000). The Davis Dam which 

forms Lake Mohave and Parker Dam which creates Lake 

Havasu are major municipal diversions of the Colorado River 

Aqueduct and Central Arizona Project, respectively. Other 

important dams in the CDRB include the Navajo Dam on the 

San Juan River, and the Headgate Rock, Palo Verde Diversion, 

and Imperial Dams located on the Lower CDR. The Morelos 

Dam, located south of the Northerly International Boundary 

with Mexico, is a diversion dam that assists in rerouting water 

to Mexico via the Alamo Canal (Patrick, 2000; USDOI, 1997). 

Construction of dams has diverted most of water for irrigation 

and has severely reduced the large riparian and wetlands areas 

in the Colorado River Delta Region in Mexico (Mora and 

others, 2003). As a result, important habitat for endangered 

species and migratory birds has been lost.

Water Quality Impairments and Fish 

Consumption Advisories

Impairments are issued for rivers, streams, and lakes that 

do not meet state defined water quality standards as required 

by Section 303(d) of the Clean Water Act. Many CDRB water 

bodies are listed as impaired for turbidity, TDS, fecal coliform, 

temperature, metals, and pesticides. The review of 303(d) 

listed waters for this report focused only on those associated 

with rivers and reservoirs related to the CDR and its major 

tributaries.

Impairments in the Green River Basin were primarily 

in small streams, and common impairments included fecal 

coliform, TDS, and temperature (Utah Department of Environmental Quality (UDEQ), 2000; Wyoming Department of 

Environmental Quality (WDEQ), 2002). Impairments for 

TDS and chlorine were identified for the Lower Green River 

(WDEQ, 2002). Impairments for copper (Cu), silver (Ag), 

and cadmium (Cd) were associated with the Ferris-Haggarty 

Mine located on Haggarty Creek in the Little Snake River 

Basin of the Upper CDRB (WDEQ, 2002). A Se impairment 

for Ashley Creek is associated with irrigation flow to the 

Stewart Lake Management Area in Utah (UDEQ, 2000). In 

Colorado, Cd, Cu, lead (Pb), manganese (Mn), zinc (Zn), and 

iron (Fe) impairments in rivers are associated with mining 

activities (Colorado Department of Public Health and Environment (CDPHE), 1998). The entire length of the Eagle River, 

a tributary of the CDR in Colorado, is listed as impaired for 

Cd, Mn, and Zn from Eagle Mine (CDPHE, 1998). Streams in 

the Gunnison and Lower Dolores River Basins have impairments for Se and ammonia, and Cd, Mn, and Zn impairments 

have been issued near the historical mining area of Idarado 

(CDPHE, 1998). The Upper Animas River in the San Juan 

River Basin (SJB) has aluminum (Al), Cd, Cu, Fe, Pb, and Zn 

impairments associated with abandoned mines in the San Juan 

Mountains, and impairments in the Lower SJB are primarily 

for stream bottom deposits possibly from agriculture, erosion, or urban runoff (New Mexico Environment Department 

(NMED), 2000).

Table 1. Sampling stations located within approximately 75 km 

of a National Wildlife Refuge (NWR) in the Colorado River Basin. 

CDRB station NWR name

Lay, CO (311) Browns Park

Ouray NWR, UT (312) Ouray

Willow Beach, AZ (320) Desert

Needles, CA (321) Bill Williams River, Havasu

Imperial Dam, AZ (322) Cibola, Imperial, Kofa

Introduction    5
Most water quality impairments in the Lower CDRB are 

associated with metropolitan areas and agricultural practices 

rather than mining. Rivers near Las Vegas have impairments 

for Fe, boron (B), phosphorus (P), and temperature (Nevada 

Division of Environmental Protection, 2002). The Imperial 

Valley of California, which uses the Lower CDR for irrigation, 

has impairments for siltation, pesticides, and Se associated 

with agricultural return flows (California Environmental Protection Agency (CEPA), 2003). Most impairments in streams 

and rivers in the Upper GRB, including turbidity, conductivity, 

and metals (Al, Cu, Cd, Pb, and Zn) are associated with agriculture, erosion, and resource extraction (Arizona Department 

of Environmental Quality (ADEQ), 2004; NMED, 2000). Turbidity and fecal coliform impairments have also been issued 

for many tributaries in the Lower GRB, and impairments for 

Cu and Zn are likely from extensive mining in the region.

Fish consumption advisories are designed to protect 

human health and are also good indicators of water quality 

because most are issued after assessing contaminant concentrations in fish tissue. More than 50 fish consumption 

advisories have been issued for reservoirs and rivers in the 

CDRB (USEPA, 2004a). Advisories for mercury (Hg), DDT, 

dieldrin, chlordane, and toxaphene were the most common.

Fish consumption advisories have not been issued for the 

mainstem of the CDR. A Se advisory for all freshwater fish 

located from the confluence of Ashley Creek with the Green 

River to the Lake Stewart Wildlife Management area did not 

extend into the mainstem of the Green River (USEPA, 2004a). 

Lake Stewart was studied extensively after Se contaminated 

the area and negatively impacted wildlife (Stephens and others, 1992). A Hg advisory for the consumption of common 

carp (Cyprinus carpio) and channel catfish (Ictalurus punctatus) was issued on the San Juan River from the Hammond 

Diversion to the mouth of the Mancos River (USEPA, 2004a). 

Potential Hg sources in the area include two coal-fired power 

plants, the San Juan Power Plant and the Four Corners Power 

Plant (Abell, 1994). Multiple consumption advisories in 

the GRB have been associated with historic use of banned 

pesticides. Consumption advisories for chlordane, p,p’-DDD,

p,p’-DDE, p,p’-DDT, dieldrin, and toxaphene were issued for 

all fish in the Hassayampa River from the lower portion of 

Buckeye Canal to the GR (USEPA, 2004a). In addition, the 

Salt River west of 59th Avenue in Phoenix to the GR and the 

Figure 2. Map of land ownership in the Colorado River Basin (CDRB) including government and private lands and sites sampled in 

2003.

6    Contaminants, Health Indicators, and Reproductive Biomarkers in Fish from the Colorado River Basin
GR from it’s confluence with the Salt River to near Gila Bend, 

Arizona have p,p’-DDT, dieldrin, chlordane, and toxaphene 

advisories for all fish (USEPA, 2004a).

Reservoirs have more fish consumption advisories than 

rivers in the CDRB. Sweitzer Reservoir, constructed on the 

alluvial plain of the Gunnison and Uncompahgre Rivers in 

Colorado, has a Se advisory for all fish (USEPA, 2004a). 

Selenium has leached from underlying shales. Stewart Lake 

Management Area near Vernal, Utah also has a Se advisory for 

all fish due to contaminated irrigation water (USEPA, 2004a). 

Waters from this area flow into the Green River. The Navajo 

Reservoir on the San Juan and Piedra Rivers and the McPhee 

Reservoir along the Dolores River and nearby Narraguinnep 

Reservoir have Hg advisories for multiple sport fish including 

largemouth bass (Micropterus salmoides), smallmouth bass 

(M. dolomieu), channel catfish, black crappie (Pomoxia nigromaculatus), yellow perch (Perca flavescens), rainbow trout 

(Oncorhynchus mykiss), kokanee salmon (O. nerka), northern 

pike (Esox lucius), and walleye (Stizostedion vitreum; USEPA, 

2004a). The Hg source in these reservoirs has been attributed 

to mining and atmospheric deposition from coal-fired power 

plants in the area (Abell, 1994; Melancon and others, 1979). 

Nearby Lake Farmington in New Mexico also has Hg advisories issued for largemouth bass and channel catfish, and Bear 

Canyon Reservoir in the Upper GRB has Hg advisories for 

largemouth bass, channel catfish, white crappie (P. annularis), 

and bluegill (Lepomis macrochirus; USEPA, 2004a). Several 

lakes in southern Arizona including Alamo Lake on the Bill 

Williams River have Hg advisories for sport fish, and potential 

Hg sources include historic ore milling and amalgamation processes, naturally mineralized soils, and atmospheric deposition 

(USEPA, 2004a).

Major Sources of Contaminants to the Colorado

River Basin

The CDRB is generally arid and highly mineralized, and 

petroleum-rich geologic formations underlay much of the 

basin. These conditions allow salts, oil, and trace elements 

such as As, Se, and heavy metals to be available for potential 

release into the environment through natural weathering and 

resource extraction. Irrigation and mining occur throughout 

the CDRB and accelerate the rates of the processes controlling 

the release and distribution of these naturally occurring compounds (Schmitt, 1998). Therefore, elevated concentrations 

of contaminants in CDRB tributaries result from agriculture, 

mining, and energy-related activities and from the populations 

they support. Dams and diversions alter the flux of water and 

sediment and their associated contaminants, and irrigation 

practices leach contaminants from underlying soil and rock 

formations.

Agriculture

Historically, sediment transported from the Upper CDRB 

created marshlands and fertile floodplains in the Lower CDRB 

during the monsoon season (Minckley, 1991; Mueller and 

Marsh, 2002). Crops flourished and diverse wildlife communities were supported (Mueller and Marsh, 2002). Today, 

agriculture in the Lower CDRB is important for the economies 

of California and Arizona. Most farms are located in the 

GRB in southern Arizona and the Imperial Valley in southern 

California which have mild climates and fertile soils. Major 

crops include cotton, wheat, alfalfa, and vegetables, including broccoli, carrots, and lettuce, and farms in the region have 

some of the highest yields per acre in the U.S. (USDOI, 1997). 

Agriculture is more limited in the Upper CDRB, although 

hay, corn, small grains, dry beans, onions, melons, fruit, and 

grapes are important in some areas (Spahr and others, 2000). 

Agricultural demands and low annual precipitation have led 

to extensive irrigation systems throughout the CDRB. Nearly 

four million acres of farmland are irrigated in the CDRB 

(USDOI, 2001). Some counties in southern Arizona and 

California rely on water from irrigation canals for their crops. 

Pesticide applications in this area are high. Active ingredients in larger applications (>1,100 kg or 9,500 liters per acre) 

included atrazine, 1,3 dichloropropene, metam-sodium, trifluralin, chloropicrin, aldicarb, S-Ethyl dipropylthiocarbamate, 

benefin, bensulate, dimethyl tetrachloroterephthalate, and 

pronamide in 2002 (ADEQ, 2002). Total pesticides applied 

to Arizona soils in 1988 exceeded 816,500 kg with most 

(56%) being insecticides (Artiola and Dubois, 1995). Wildlife 

in these areas may have been exposed to various chemicals 

including arsenic-based defoliants, stable organic herbicides and insecticides, metals, and salts (Aritola and Dubois, 

1995). DDT and toxaphene remain present in GRB soils and 

sediments including those in the Phoenix metropolitan area 

(ADEQ, 1992). Many contaminants such as p,p’-DDE and 

Se are also a concern in the Mexicali Valley of the CDR delta 

(García-Hernández and others, 2001, 2006; Mora and others, 

2003).

Mining and Extractive Industries

Oil and natural gas extraction and mining provide a 

substantial proportion of several state economies in the CDRB, 

but exploitation of these natural resources have negatively 

affected aquatic habitat and biota. Abandoned and active 

mines eliminated >19 km of fisheries in the Upper CDRB 

(Abell, 1994). One of the most heavily polluted areas is the 

Animas River where surface water was severely polluted by 

abandoned mine runoff near Silverton, Colorado, in the San 

Juan Mountains (Abell, 1994). Minerals such as Cu, gold 

(Au), Ag, Pb, and Zn have been heavily mined throughout the 

CDRB (fig. 3). Major oil and natural gas deposits occur in the 

Upper CDRB from northwestern New Mexico to Wyoming. 

Oil shale and tar sands located in the Upper Green River Basin 

are not considered major commercial deposits (USDOI, 1997). 

Introduction    7
Coal mining in Colorado, Utah, and New Mexico has grown in 

the past several decades, and a large uranium deposit occurs in 

southeastern Utah and western Colorado.

Copper is used for electrical and electronic products, 

industrial machinery and equipment, building construction, 

and transportation equipment. Copper deposits in Arizona 

have been exploited, and mining districts in Arizona produce 

67% of the U.S. supply (Niemuth, 2004). The Morenci, Ray, 

and Sierrita mines in the GRB and the Bagdad mine in west 

central Arizona produce >90% (635,000 kg) of the copper 

in Arizona (fig. 3). Additional copper mines in Arizona are 

being considered. Utah and New Mexico are ranked second 

and third in U.S. copper production. Copper mining operations are also found in the Upper GRB in southwest New 

Mexico (Kramer and others, 1997; New Mexico Energy, 

Minerals, and Natural Resources Department (NMRD), 2003). 

Although emissions are monitored, contaminants from 15 

copper smelters in Arizona and New Mexico are a growing 

concern in the region. Smelters are point sources for sulfur 

oxides, toxic metals (for example, As, Pb, Hg), fine particulates, and acid precipitation and can contaminate surrounding areas (World Bank Group, 1998). Biota near Hayden, 

Arizona, where a copper smelter is in operation, may be at 

risk (Arizona Department of Health Services, 2002; King 

and Baker, 1995). The Hayden smelter is located directly on 

the GR, one of the principal sources of irrigation water for 

southern Arizona (USEPA, 1977). In the 1970s, biota including algae, caddis fly and black fly larvae, and fish were absent 

below the Hayden smelter (USEPA, 1977). Runoff from 

storm events, erosion of mine tailings, and emissions from 

the smelters may adversely impact threatened and endangered 

species in this area (G. Beatty, oral communication). Other 

mineral deposits in the CDRB include Au, Ag, Pb, and Zn 

in the Uncompahgre and San Juan Mountains in southwestern Colorado (Colorado Mining Association (CMA), 2004). 

These metals are also mined in the Green River and Colorado 

River placers in Utah although they do not produce a significant amount of ore. Rising gold prices and funding availability have renewed interest in precious metal exploration in 

southern Arizona (Niemuth, 2004).

One of the largest U.S. natural gas deposits is located 

in the SJB, resulting in New Mexico being a major U.S. 

natural gas producer (NMRD, 2003). Smaller deposits in 

northeastern Utah and northwestern Colorado are associated 

with coalbeds. A portion of the natural gas in New Mexico is 

coalbed methane (CBM) in which New Mexico ranks first in 

U.S. production and reserves (NMRD, 2003). The Fruitland 

Coal formation in the SJB is the largest CBM producer in the 

Figure 3. Map of coal deposits and mining operations in the Colorado River Basin (CDRB). Mineral extraction facilities include ferrous 

and nonferrous mines and construction and agricultural mineral operations. Ferrous mineral and nonferrous metal processing plants 

are also shown.

8    Contaminants, Health Indicators, and Reproductive Biomarkers in Fish from the Colorado River Basin
U.S. Natural gas extraction and processing have been associated with PAH contamination that can affect water quality 

(Abell, 1994). Several large surface coal mines including the 

San Juan, Navajo, and La Plata are also active in the SJB and 

produce bituminous coal for electrical power. Two coalfired power plants, the San Juan and Four Corners, located 

west of Farmington, New Mexico, may contaminate the San 

Juan River with trace elements and PAHs through wet or dry 

deposition (Abell, 1994; NMRD, 2003). Gernard and others (1983) suggested that the Cd and Hg from power plant 

effluent pose the largest threat to aquatic ecosystems, but Se 

has also been measured at high concentrations in power plant 

disposal waters. Colorado and Utah have active coal mines, 

both surface and underground, that excavate bituminous coal 

for electricity production (Bon and Wakefield, 1999; CMA, 

2004). Coal mining operations can contaminate surface water 

by increasing sediment load and TDS concentrations, increasing erosion from strip mining, and altering pH, which may 

enhance the transport of other trace elements (Abell, 1994). 

Acid deposition from coal-fired power plants in the Yampa 

River Basin near Craig and Hayden, Colorado have caused 

reproduction problems in amphibians in nearby waters, and 

problems may persist if emissions remain high (Turk and 

Campbell, 1997).

Other non-metallic mineral deposits with the potential 

to affect water quality in the CDRB include gypsum, sulfur, 

fluorospar, barite, and asbestos. The Henderson mill located 

in Grand County, Colorado, processes molybdenum to make 

alloy steels, stainless steel, and cast iron and is the largest 

molybdenum mine in the world (CMA, 2004). Large uranium 

deposits are located in Colorado, Utah, and New Mexico 

although significant production is limited to the Colorado 

Plateau in western Colorado and southeastern Utah (Bon 

and Wakefield, 1999; CMA, 2004; NMRD, 2003). Uranium 

mine and mill tailings may also contain large quantities of 

TDS, Se, molybdenum, and vanadium as well as radioactive 

materials (Abell, 1994). One of the largest uranium mining 

operations near Moab, Utah ceased production in 1984, but the 

mill tailings site is still located nearby. The unlined tailings 

site, located along the CDR, covers more than 53 hectares and 

leaches radioactive wastes and ammonia into the CDR near 

potential habitat for endangered aquatic species (Fairchild and 

others, 2002). Radioactive isotopes released from the site can 

be transported downstream towards Lake Mead by sediment 

and water, and groundwater used for irrigation and drinking 

water is also at risk to become contaminated. Consequently, 

the U.S. Department of Energy will remove 11.9 million 

tons of radioactive tailings from the banks of the CDR (U.S. 

Department of Energy, 2005). A study at the uranium mine on 

the Animas River at Durango, Colorado found bottom fauna 

and invertebrate populations were eliminated from mill wastes 

for 48 km downstream of the site, but radioactive chemical 

concentrations declined after the mill closed in 1963 (Anderson and others, 1963).

Industrial and Municipal Sources

Heavy demands have been placed on the CDR to meet 

municipal supply and wastewater disposal needs of large 

population centers. The CDRB provides municipal and industrial water to over 27 million people (USDOI, 2001). Much of 

the CDRB is sparsely populated with many towns and cities 

located along river corridors. Main population centers including Las Vegas, Nevada and Phoenix and Yuma, Arizona are 

located in the Lower CDRB, which already has a limited water 

supply due to recent drought conditions, heavy irrigation, and 

mandated state water allocations. Such demands on the water 

supply result in ephemeral flows in many streams and rivers. Las Vegas uses the CDR at Lake Mead as its major water 

resource, but the city obtains 10% of its water from groundwater. The CDR is also the primary source of drinking water in 

Yuma. In the early 1970s, the Central Arizona Project (CAP) 

was initiated to divert water via a concrete aqueduct from the 

CDR at Lake Havasu to Phoenix and Tucson. The surface 

water from CAP and the Verde and Salt Rivers provide 95% of 

the water to Phoenix. Flows in the GRB consist primarily of 

irrigation return water, storm water, and effluent from wastewater treatment plants (Anning, 2003; Cordy and others, 1998; 

Gebler, 1998). Effluents from seven wastewater treatment 

plants dominate the water in the GR from its confluence with 

the Salt River at Phoenix to Gillespie Dam (Cordy and others, 

1998). Several studies concluded that chemicals in wastewater 

effluent from the Las Vegas and Phoenix areas may negatively 

impact aquatic wildlife (Arnold and others, 2004; Bevans 

and others, 1996; Gross and others, 2004; Patiño and others, 

2003b); therefore, water quality from municipal wastewater is 

a concern in these areas.

Federal law requires that permits be issued for companies 

to discharge wastewater into rivers. Industries that manufacture, process, or use toxic chemicals are required to annually 

report releases of these chemicals. There is little industrial 

development and associated industrial permitted discharges 

to the CDR or its tributaries; most are near Las Vegas and 

Phoenix. Most permitted point sources are municipal wastewater treatment facilities, and discharges include biological 

oxygen demand (BOD) and TDS (USEPA, 2004b). The 

Phoenix area has multiple wastewater treatment facilities that 

discharge inorganic compounds (Se, As, Zn, Cr, Cu, Mn, and 

Hg) into the Salt River and GR, and an aerospace manufacturer discharges Pb, Ag, and Cu into the Salt River. Permitted 

discharges from the Las Vegas area were limited to BOD and 

TDS from a wastewater treatment facility (USEPA, 2004b). 

Power plants and mining industries reported most of the 2002 

toxic releases (non-permit releases) into the CDRB (USEPA, 

2004e). Releases from power plants were mostly dioxins and 

dioxin-like compounds and were generally <500 g. Mining operations in Colorado and Arizona commonly reported 

small releases of Cu and Mn. The San Juan Coal Company 

(San Juan Mine) reported one time releases (>5,000 kg) of 

antimony (Sb), As, barium (Ba), Cr, and Cu, but no other 

large releases were reported in the CDRB. Areas with large 

Introduction    9
quantities of air emissions include Phoenix, Las Vegas, Grand 

Junction, and near Farmington. The most common compound 

released into the air from fugitive and point source emissions 

were sulfuric acid, hydrochloric acid, hydrogen fluoride, 

ammonia, Pb, styrene, and Cu (USEPA, 2004e). The amount 

of unreported contaminant releases in the CDRB is unknown.

Several CDRB locations are designated as Superfund 

sites (USEPA, 2004c). A zinc mine and a uranium and vanadium processing plant in the Upper CDRB are associated with 

mine and mill tailings; As, Cd, Cr, Cu, Pb, Hg, Zn, polychlorinated biphenyls (PCBs), uranium, radium, and vanadium 

have contaminated surface water, groundwater, and soils near 

these sites. The Monticello uranium mill in Utah is listed for 

radioactive materials (radium, uranium, and vanadium) and 

some metals (As, Cd, Cr, Cu, Pb, Se, and Zn) in the surface 

water and groundwater. The United Nuclear Corporation near 

Gallup, New Mexico in the SJB began mining uranium in the 

late 1960s and operating a mill in 1977, and contaminants 

from tailing fluids containing As, Cd, Cobalt (Co), Nickel 

(Ni), and Se were released after a dam broke and infiltrated 

the alluvial aquifers near the mill (USEPA, 2004c). A landfill 

near Farmington, New Mexico in the SJB has been associated 

with groundwater contamination by Mn and volatile organic 

compounds. Superfund sites in Arizona are located in the 

GRB and associated with groundwater and soil contamination (USEPA, 2004c). Most facilities were landfills, airports, 

and military operations near Phoenix and Yuma, and common contaminants included volatile organic compounds, Cr, 

As, Cd, Cu, petroleum hydrocarbons, PAHs, pesticides, and 

nitroaromatics.

Perchlorate

Perchlorate is associated with releases of ammonium 

perchlorate by military operations, aerospace programs, and 

defense contractors and has been measured in surface and 

groundwater in Arizona, Nevada, and California (Urbansky, 

2002). Manufacturing facilities near Henderson, Nevada in 

the Las Vegas Valley, currently owned by the Kerr-McGee 

Corporation, are the primary source of perchlorate in the 

Lower CDR (Owens and others, 2004). The aquifer located 

beneath the facility was contaminated with perchlorate that 

leached from unlined evaporation ponds. As a result, significant amounts of perchlorate, as much as 408 kg/day (900 lbs/

day), entered the Las Vegas Wash in the late 1990s (USEPA, 

2004d). Kerr-McGee, in conjunction with the Nevada Division of Environmental Protection and the USEPA, initiated a 

control strategy to capture and treat the perchlorate in 1997 

after the facility was identified as the source of contamination. 

Perchlorate concentrations in the surface water in Las Vegas 

Bay, Lake Mead, and the Lower CDR have declined since 

1999, although decreased dilution of the contaminated water 

in Lake Mead is likely if recent drought conditions persist 

(USEPA, 2004d). Perchlorate contamination is also being 

monitored in Arizona. Concentrations in the Lower CDR are 

attributed to the Henderson facility although other facilities 

including military areas near Yuma and manufacturing plants 

in Phoenix have introduced perchlorate to the GRB (Owens 

and others, 2004). 

Perchlorate can disrupt thyroid function by interfering 

with iodide accumulation, which blocks hormone production 

(Urbansky, 2002; Wolff, 1998). Thyroid-related studies in 

mammals (National Research Council, 2005), fish (Brown 

and others, 2004a; Mukhi and others, 2005; Patiño and others, 

2003a), and amphibians (Goleman and others, 2002) indicate 

that further research is needed to characterize the potential 

deleterious effects of perchlorate.

Extant Sources of Information on Contaminants

in the Colorado River Basin

Water quality in the CDRB has been studied due to 

concerns for threatened and endangered species, elevated Se 

concentrations in irrigation return flows, and decreasing water 

availability. Regional-scale contaminant studies in the CDRB 

are limited. The CDR drains an extensive landscape, and 

many water quality studies have focused on particular areas 

or regions within the basin. Several large Federal programs 

including the National Contaminant Biomonitoring Program 

(NCBP), the National Water Quality Assessment (NAWQA) 

Program, and the National Irrigation Water Quality Program 

(NIWQP) have conducted contaminant studies in various 

regions of the CDRB. The USFWS has also conducted multiple contaminant studies in the basin.

The main objective of the NCBP was to document temporal and spatial trends of organochlorine and inorganic concentrations in fish (Schmitt and others, 1999). The program 

reported concentrations of many persistent contaminants such 

as organochlorine pesticides, PCBs, and Hg were decreasing in whole-body fish samples from 107 locations across the 

U.S. by the mid-1980s. Historical NCBP concentration data 

from 1970 to 1986 are available for fish including carp, bass 

(Micropterus sp.), and channel catfish from seven locations 

(Lake Powell and Lake Mead; San Carlos Reservoir; Lake 

Martinez; Lake Havasu; Yuma, Arizona; and Vernal, Utah) 

in the CDRB (Schmitt and others, 1999). The fourth highest 

mean Cd concentration in the U.S. (>0.05 μg/g) was in fish 

from Vernal. Mining activities in Colorado and Utah are likely 

sources of Cd in this area (May and McKinney, 1981). Mean 

concentrations of arsenic (As) in fish from Lake Havasu and 

Yuma were high (>0.2 μg/g) and could be from arsenical agricultural chemicals in intensively farmed regions of the Lower 

CDRB (Schmitt and others, 1999). The highest mean Se 

concentrations (>1.0 μg/g) reported by the NCBP were in fish 

from the CDRB, and these concentrations could be a threat to 

piscivorous wildlife (Schmitt and others, 1999). Conversely, 

organochlorine pesticides were generally low in the CDRB, 

although high concentrations of total DDT (>0.75 μg/g) were 

documented in agricultural areas of the Lower CDRB. 

10    Contaminants, Health Indicators, and Reproductive Biomarkers in Fish from the Colorado River Basin
The NAWQA Program of the USGS examines water 

quality in river basins and groundwater systems in the U.S. 

Three NAWQA study units including the Upper Colorado 

River Basin (UCOL), Nevada Basin and Range (NVBR), and 

Central Arizona Basins (CAZB) are located in the CDRB. 

The NAWQA Program studied water quality in the UCOL 

of western Colorado from 1996 to 1998 and reported that 

elemental concentrations exceeded water quality guidelines 

(Spahr and others, 2000). Specifically, concentrations of 

Cd, Zn, Cu, and Pb in streambed sediments from historical 

mining districts could be harmful to aquatic wildlife (Mize 

and Deacon, 2002; Spahr and others, 2000). In addition, Se 

was leached out off the irrigation drainage soils in agricultural areas of the Colorado Plateau, causing this area to be the 

major Se source for Lake Powell (Engberg, 1999). Pesticide 

concentrations in this area were low and below protective 

criteria for aquatic wildlife (Spahr and others, 2000). Few biological samples are available, but Deacon and Stephens (1998) 

measured Cd, Se, Cu, and Zn concentrations in fish liver from 

the UCOL. Concentrations of Cd and Se were highest in 

fish from mining and agricultural areas, respectively, while 

concentrations of Cu and Zn were highest in areas of mixed 

land-use (Deacon and Stephens, 1998).

The NVBR study unit, which includes the Las Vegas Valley in southern Nevada, was sampled in 1992-1996 (Bevans 

and others, 1996). The NAWQA Program determined that 

urban activities in the Las Vegas Valley have contributed 

nutrients (ammonia, nitrate), synthetic organic compounds 

(herbicides, insecticides, trihalomethanes, methyl tert-butyl 

ether), organochlorines (polycyclic aromatic hydrocarbons 

(PAHs), phenols, phthalate esters), and trace elements (Cd, 

Cu, chromium (Cr), Pb, and Zn) to surface water and sediments in the Las Vegas Wash (Bevans and others, 1998). The 

Las Vegas Wash receives large quantities of sewage effluent 

from Las Vegas and surrounding areas. Chemicals in the 

effluent are eventually discharged into Las Vegas Bay of Lake 

Mead via the Las Vegas Wash and may be transported further 

downstream in the CDR. Several studies have found evidence 

of endocrine disruption in carp in the Las Vegas Wash and Las 

Vegas Bay of Lake Mead (Bevans and others, 1996; Gross and 

others, 2004; Patiño and others, 2003b).

The NAWQA Program initiated water quality studies 

in the CAZB in 1994. Gebler (2000) reported on the occurrence and distribution of organochlorine compounds in tissues 

of aquatic biota in CAZB streams. Organic and inorganic 

contaminants also entered the CAZB from industrial, mining, 

agricultural, and municipal activities in Mexico (Cordy and 

others, 1998; Gellenbeck and Anning, 2002). Water use in 

this area is dominated by agriculture, primarily cotton, which 

introduces organochlorines into multiple streams and rivers. 

Water quality in CAZB streams in urbanized areas is poor due 

to flow being composed primarily of sewage effluent. Concentrations of total DDT and toxaphene exceeded criteria to 

protect piscivorous wildlife in fish collected near a wastewater 

treatment facility in western Phoenix and in Buckeye Canal 

near Hassayampa (Gebler, 2000). Concentrations of PCBs 

also exceeded protective wildlife criteria in fish tissues from 

Buckeye Canal. 

The NIWQP was initiated in the mid-1980s to identify and address water quality and contamination problems, 

specifically Se contamination from irrigation, in the western 

U.S. Reconnaissance investigations included five areas in the 

CDRB: the San Juan River area in New Mexico, the Dolores-Ute Mountain area and Gunnison-Grand Valley (GGV) 

in Colorado, the Middle Green River Basin in Utah, and the 

Lower Colorado River Valley in Arizona and California (Seiler 

and others, 1999). Remediation planning, based upon results 

of reconnaissance and detailed studies, began in the GGV and 

MGR areas to reverse adverse impacts to fish and wildlife 

from irrigation projects.

NIWQP activities in the GGV focused on degraded fish 

habitat and Se loading in the Gunnison River Basin (Butler 

and Leib, 2002). NIWQP and other groups working in the 

GGV proposed flushing channels and alternative uses of 

irrigation drainage as remediation processes for backwater and 

bottomland sites used by endangered fish in the Grand Valley. 

Most Se is the result of irrigation drainage in the Uncompahgre River Basin near Montrose and Delta, Colorado. Remediation efforts include reducing irrigation seepage from ponds 

and irrigation ditches, phytoremediating to remove Se from 

top soil, and developing management practices for Se sources 

unrelated to agriculture. The MGR area is located near Stewart Lake Wildlife Management Area near Vernal, Utah, and 

Stewart Lake has received irrigation drainage since the early 

1980s as a stable water supply. Selenium has contaminated 

water, sediments, and biota. Deformed embryos and reduced 

hatch success has been documented in migratory birds in 

the lake and outfall to the Green River (Stephens and others, 1992). Diversions were constructed to redirect irrigation 

drainage from the Lake Stewart to the Green River. Proposed 

remediation efforts include flushing contaminated sediments, 

mechanical (tilling) and chemical options, and bioremediation. 

However, NIWQP became inactive in 2004, and remediation 

efforts ended.

The San Juan River Seven-Year Research Program 

(SJRP), under the auspices of the San Juan River Basin Recovery Implementation Program, was initiated in the early 1990s. 

A primary goal of the program was to review water quality and 

contaminant information in the SJB (Abell, 1994). Irrigation 

and mineral extraction and processing were identified as the 

major sources of contaminants in the SJB. The SJRP recommended that future research should include the effects of Se on 

rare fish, the sources and effects of PAHs, and correlation of 

degraded fish health with contaminants (Abell, 1994).

The USFWS has conducted multiple studies on environmental contaminants in fish associated with agricultural drainage canals in the Lower CDRB. The GRB provides important 

habitat for Threatened and Endangered species, migratory 

birds, and waterfowl. Agricultural canals have transported 

pesticides to the GR and contributed to elevated concentrations of organochlorines and metals in fish (Baker and others, 

1992; García-Hernández and others, 2001; King and others, 

Introduction    11
1997). Numerous migratory waterfowl use the delta system of 

the Lower CDRB and may be exposed to agricultural pesticides in this area (Baker and others, 1992; García-Hernández 

and others, 2001, 2006). Copper mining operations instead 

of agricultural or municipal discharges are the primary source 

of contaminants in the Middle GR from Coolidge Dam to the 

Ashurst-Hayden Dam (King and Baker, 1995). The Ray copper mine contaminated Mineral Creek, a tributary to the GR, 

which resulted in high concentrations of Cu, Pb, and Zn in 

fish; however, the overall ecology of the creek improved after 

renovations at the mine (Andrews and King, 1997).

Irrigation drainage waters in the Yuma Valley of southern 

Arizona are potentially harmful to fish and wildlife (Baker 

and others, 1992). Concentrations of toxaphene, p,p’-DDE, 

dieldrin, and total chlordane in fish tissue were consistent with 

historical NCBP concentrations, but p,p’-DDD and p,p’-DDT 

concentrations have decreased in fish tissue (Baker and others, 

1992). In addition, concentrations of As, Cu, and Se exceeded 

protective criteria for fish, and concentrations of As and Cu 

in fish have increased in the Yuma Valley since the late 1970s 

(Baker and others, 1992).

King and others (1997) measured pesticides and metals concentrations in fish and wildlife in the Lower GRB. 

The greatest mean concentration of p,p’-DDE (11.17 μg/g) 

documented in the U.S. from areas associated with agriculturally applied pesticides was measured in common carp from 

Buckeye Canal (King and others, 1997). Concentrations of 

p,p’-DDE have declined even though current levels remain 

extremely high in fish collected from GR tributaries and 

canals. Concentrations of other pesticides and PCBs were less 

than protective criteria for fish and wildlife (King and others, 

1997). Copper was detected at high concentrations (>5 μg/g) 

in carp and channel catfish and remains a concern due to its 

tendency to interact with other compounds and elements (King 

and others, 1997). 

The USFWS measured Se and other trace elements in 

sediment and fish from Imperial, Cibola, and Havasu NWRs 

along the Lower CDR in the late 1980s (King and others, 

1993). Concentrations of As and Hg in fish were below protective criteria for piscivorous wildlife, and only one sample 

exceeded criteria for Cd. Concentrations of Se (0.89-4.39 

μg/g) exceeded the minimum threshold level shown to cause 

reproductive impairment in fish but remained stable from 

1984 to 1989 (King and others, 1993). The study recommended that each NWR consider issuing fish consumption 

advisories in selected areas with known high Se concentrations. A subsequent study by Andrews and others (1997) 

measured organochlorines and inorganic contaminants in fish 

and wildlife of the Havasu NWR in Arizona. Arsenic, Cd, Cr, 

Cu, Pb, and Se concentrations were potentially hazardous to 

fish and wildlife in the area, but organochlorine concentrations 

were below protective wildlife criteria (Andrews and others, 

1997). Concentrations of Se in largemouth bass and common 

carp ranged from 0.79 to 2.60 μg/g, which were considered 

potentially lethal for piscivorous wildlife. Andrews and others 

(1997) concluded that further investigations on the effects of 

Se in fish and piscivorous birds should be a priority in this area 

of the Lower CDRB.

Materials and Methods

Collection Sites

Fish were collected at fourteen sites in the CDRB (fig. 

1; table 2). Six of the 14 sites were located on the CDR, two 

were on the Green River, three were on the Gila River, and 

one each was on the Yampa, Gunnison, and San Juan Rivers. 

Stations were grouped into two sub-basins, the Upper CDRB 

and Lower CDRB. Fish were collected post-spawn between 

August and October 2003 (table 2). Sampling at each site was 

completed during one visit spanning 1-3 days.

Target Species and Sampling Strategy

This study was designed to retain comparability with previous investigations which measured chemical concentrations 

in whole fish composite samples, while also accommodating 

biological measurements, many of which are gender-specific 

and require live or freshly killed individual fish, incorporated 

into the BEST Program’s Large River Monitoring Network 

(LRMN; Schmitt and Dethloff, 2000). The same species were 

targeted at each site to standardize fish health and reproductive 

biomarker results. The most prevalent bottom-dwelling species and predator species were common carp (henceforth carp) 

and largemouth bass (Micropterus salmoide), respectively, in 

previous NCBP collections (Schmitt and others, 1999). These 

were also the targeted species in other BEST-LRMN investigations (Hinck and others, 2004a; Schmitt, 2002; Schmitt and 

others, 2004). These species have a widespread distribution, 

abundant extant contaminant data, and biological endpoint 

data and were the preferred taxa at all sites. Alternate species 

were permitted if these taxa could not be obtained. Preferred alternate species included white sucker (Catostomus 

commersoni) as alternate benthivores, and smallmouth bass 

(henceforth Micropterus sp., bass), channel catfish, brown 

trout (Salmo trutta) and flathead catfish (Pylodictis olivaris) 

as piscivores. The collection target at each site was 10 (each) 

adult male and female of each taxon for a total of 40 fish per 

site. Adult carp and bass of a size representative of those 

believed to be present based on extant information were preferred and extremely large or small fish were avoided. More 

than two species were collected at sites if the quota for the 

target taxa could not be obtained.

Monitoring Methods Overview

A suite of chemical and biological methods including reproductive biomarkers, measures of cytochrome P450 

12    Contaminants, Health Indicators, and Reproductive Biomarkers in Fish from the Colorado River Basin
enzyme induction, fish health assessments, and chemical analyses of fish carcasses was used to characterize the exposure 

of fish to contaminants and the effects of exposure (table 3; 

Schmitt and Dethloff, 2000). Concurrent determination of tissue residue concentrations along with the suite of fish health, 

immune system responses, and reproductive assessments supports the interpretation of relationships between exposure and 

biological responses.

Multiple organochlorine chemical residues and elemental 

contaminants were measured in the whole-body fish composite samples (table 4). These analytes were selected to provide 

the maximum amount of information on accumulative contaminants of interest at minimal cost and to maintain continuity with the historical NCBP database. Instrumental analyses 

of specific planar halogenated hydrocarbons (PHHs), such 

as polychlorinated dibenzo-p-dioxin (PCDD) and polychlorinated dibenzofuran (PCDF) congeners, were excluded due 

to their high analysis cost. Instead, extracts of the composite samples were screened with the H4IIE rat hepatoma cell 

bioassay (Tillitt and others, 1991; Whyte and others, 2004), 

which responds to planar PHHs (table 5). Ethoxyresorufin Odeethylase (EROD) activity, which indicates recent exposure 

to exogenous aryl hydrocarbon receptor (AhR) ligands including PHHs and PAHs, were measured in the livers of individual 

fish (Kennedy and Jones, 1994; Pohl and Fouts, 1980; Whyte 

and others, 2000). Together these assays and analyses allowed 

for the estimation of the relative concentrations of potential 

biological effects of PHHs and PAHs without the expense of 

instrumental analyses for these compounds (table 5).

Measurements of fish health, immune system responses, 

and reproductive status were included in the suite of indicators 

to examine potential impacts from non-accumulative contaminants and contaminant mixtures (table 3). Fish health indicators included gross observations for abnormalities, condition 

and organosomatic indices, and histopathological examination 

(Goodbred and others, 1997; Hinton, 1993; Hinton and others, 

1992). Gross observations and determination of indices based 

on relative fish and organ sizes such as condition factor (CF), 

hepatosomatic index (HSI), and splenosomatic index (SSI) are 

relatively simple and indicative of cumulative, organism-level 

changes (Grady and others, 1992). However, these indicators are non-specific in terms of causal mechanisms and may 

reflect early, subtle alterations and foreshadow subsequent 

effects at the individual- or population-level.

The SSI is an indicator of overall organism health as 

well as a measure of immune system stress. Other immune 

system indicators included the measurement of macrophage 

aggregates (MA) in preserved spleen tissue samples (table 

3). Macrophage aggregates, also known as melanomacrophage centers, are discrete aggregations of pigment-bearing 

macrophages found in spleen, kidney, and sometimes liver of 

advanced teleosts (Agius, 1980). Pigmented cell accumulations can also occasionally found in the gonad and other tissues. These specialized cells are thought to be responsible for 

centralizing foreign material and debris for destruction, detoxification or reuse, storing waste products, contributing to the 

immune response, and storing/recycling iron (Ellis and others, 

1976; Ferguson, 1976). MA measurements have responded to 

Table 2. Stations sampled in 2003 and collection date (first-last) in the Colorado River Basin. Stations are grouped by 

sub-basin and ordered upstream to downstream.

Sub-basin and river Station

number Nearby landmark Collection dates Latitude, Longitude

Upper Colorado River Basin

Yampa 311 Lay, CO 10/21/03-10/23/03 40°25’38.00’’N, 107°51’24.00’’W

Green 312 Ouray NWR, UT 9/8/03-9/9/03 40°08’31.00’’N, 109°39’17.00’’W

Green 313 San Rafael, UT 9/5/03-9/6/03 38°45’56.00’’N, 110°05’16.00’’W

Gunnison 314 Delta, CO 9/6/03-9/7/03 38°45’59.58’’N, 108°02’30.30’’W

Colorado 315 Loma, CO 9/9/03-9/11/03 39°09’39.00’’N, 108°48’28.56’’W

Colorado 316 Gold Bar Canyon, UT 9/3/03-9/4/03 38°34’02.00’’N, 109°38’51.00’’W

San Juan 317 Hogback Diversion, NM 9/3/03-9/4/03 36°44’41.00’’N, 108°41’28.00’’W

Lower Colorado River Basin

Colorado 319 South Cove, AZ 10/1/03 36°05’23.70’’N, 114°06’12.30’’W

Colorado 320 Willow Beach, AZ 9/29/03-9/30/03 35°52’33.12’’N, 114°39’53.10’’W

Colorado 321 Needles, CA 9/23/03-9/24/03 34°43’44.64’’N, 114°20’12.96’’W

Colorado 322 Imperial Dam, AZ 9/25/03-9/26/03 32°54’05.94’’N, 114°28’09.48’’W

Gila 323 Hayden, AZ 8/22/03-8/23/03 33°01’22.14’’N, 110°44’16.32’’W

Gila 324 Phoenix, AZ 8/25/03-8/27/03 33°22’33.42’’N, 112°18’19.20’’W

Gila 325 Arlington, AZ 8/18/03-8/20/03 33°19’06.92’’N, 112°40’26.46’’W

Materials and Methods    13
Table 3. Methods incorporated into the Colorado River Basin in 2003.

Method Description Tissue(s) examined Sensitivity Primary reference(s)

Histopathology Microscopic examination for the presence of lesions; can provide early 

indication of chemical exposure

Liver, gill, gonads, 

spleen, and kidney

Overall organism health 

and contaminants

Hinton and others (1992); Hinton (1993); 

Goodbred and others (1997)

Ethoxyresorufin 

O-deethylase 

(EROD) activity

Enzyme induction by planar hydrocarbons Liver PCBs; chlorinated, diox- ins and furans; PAHs Pohl and Fouts (1980); Kennedy and Jones (1994); Whyte and others (2000)

Macrophage aggregate analysis

Macrophages are important in the immune system, serving as a first line 

of defense for the organism and as an antigen processing cell

Spleen Multiple contaminants 

including PAHs and metals

Blazer and others (1994, 1997)

H4IIE bioassay A screening tool to determine the 

presence of certain classes of planar halogenated compounds

Whole fish (composite 

samples)

PCBs; chlorinated dioxins 

and furans

Tillitt and others (1991); Whyte and others 

(2004)

Vitellogenin A precursor of egg yolk, normally synthesized in the liver of female fish Blood plasma Endocrine-modulating substances Denslow and others (1999)Steroid hormones (17β-estradiol and 

11-ketotestosterone)

Determine reproductive health and 

status

Blood plasma Endorcrine-modulating 

substances

Guillette and others (1994); Goodbred and 

others (1997)

Chemical analyses Organochlorine chemical residues and 

elemental contaminants

Whole fish (composite 

samples)

Specific analytes Schmitt and others 

(1999)

Somatic indices The relative mass of some organs is often indicative of chemical exposure Gonads, spleen, liver Overall organism health Grady and others (1992)Necropsy-based fish health assessment

Visual assessment of external/internal 

anomalies (for example, lesions, parasites, tumors), which may indicate contaminant-related stress

All Overall organism health Goede (1988, 1996); Adams and others (1993); 

Adams (1990)

14    Contaminants, Health Indicators, and Reproductive Biomarkers in Fish from the Colorado River Basin
Table 4. Organochlorine chemical and elemental contaminants measured in whole-body fish composite samples.—Continued

Contaminant class and analyte Chemical name(s) or atomic symbol Principal uses and sources to aquatic ecosystems

Organochlorine chemicals

p,p’-DDE 2,2-bis (p-chlorophenyl)-1,1-dichloroethylene

DDT-metabolite

p,p’-DDD (TDE) 2,2-bis (p-chlorophenyl)-1,1-dichloroethane

Insecticide; DDT-metabolite

p,p’-DDT 2,2-bis (p-chlorophenyl)-1,1,1-trichloroethane

Insecticide

o,p’-DDE 2-(o-chlorophenyl)-2-(p-chlorophenyl)-1,1-

dichloroethylene

o,p’-DDT metabolite

o,p’-DDD (TDE) 2-(o-chlorophenyl)-2-(p-chlorophenyl)-1,1-

dichloroethane

o,p’-DDT metabolite

o,p’-DDT 2-(o-chlorophenyl)-2-(p-chlorophenyl)-

1,1,1-trichloroethane

p,p’-DDT impurity

Total polychlorinated biphenyls (PCBs) Mixture containing as many as 209 monothrough octa-chloro-substituted biphenyl 

congeners.

Dielectric, hydraulic, and transformer fluids; lubricants; extenders; de-dusting agents; carbonless 

copy paper

Aldrin 1,2,3,4,10,10-hexachloro-1,4,4a,5,8,8ahexahydro-1,4:5,8-dimethanonaphthaleneInsecticide

Dieldrin 1,2,3,4,10,10-hexachloro-6,7-epoxy1,4,4a,5,8,8a-hexahydro-1,4-endnnnoexo-5,8-dimethanonaphthaleneInsecticide; aldrin metabolite

Endrin 1,2,3,4,10,10-hexachloro-6,7-epoxy1,4,4a,5,6,7,8,8a-octahdyro-1,4- endoendo-5,8-dimethanonaphthaleneInsecticide; isodrin metabolite

Heptachlor epoxide 1,4,5,6,7,8,8-heptachloro-2,3-epoxy3a,4,7,7a-tetrahydro-4,7-methano-1HindeneHeptachlor metabolite; technical chlordane constituent/metabolite

cis-Chlordane 1,2,4,5,6,7,8,8-octachloro-2,3,3a,4,7,7ahexahydro-4,7-methano-1H-indene 

(1-α,2-α,3a-α,4-β,7-β,7a-α)

Insecticide; technical chlordane constituent

trans-Chlordane 1,2,4,5,6,7,8,8-octachloro-2,3,3a,4,7,7ahexahydro-4,7-methano-1H-indene(1-

α,2-β,3a-α,4-β, 7-β,7a-α)

Technical chlordane constituent

cis-Nonachlor 1,2,3,4,5,6,7,8,8-nonachloro-2,3,3a,4,7,7ahexahydro-4,7-methano-1H-indene 

(1-α,2-α, 3-α, 3a-α, 4-β, 7-β, 7a-α)

Technical chlordane constituent

trans-Nonachlor 1,2,3,4,5,6,7,8,8-nonachloro-2,3,3a,4,7,7ahexahydro-4,7-methano-1H-indene(1-

α,2-β,3-α, 3a-α,4-β,7-β,7a-α)

Technical chlordane constituent

Oxychlordane (octachlor epoxide) 2,3,4,5,6,6a,7,7-octachloro-1a,1b,5,5a,6, 

6a-hexahydro-2,5-methano-2H-indeno(1,2-b)oxirene (1a-α,1b-β,2-α,5-α,5aβ,6-β,6a-α)

cis-Chlordane metabolite

Heptachlor 1H-1,4,5,6,7,8-8-heptachloro-3a,4,7,7a-tetrahydro-4,7-methanoindeneInsecticide

Methoxychlor 1,1’-(2,2,2-trichloroethylidene)-bis[4-methoxybenzene]Insecticide

Toxaphene Chlorinated camphene mixture averaging 

62% chlorine by weight

Insecticide; herbicide

Materials and Methods    15
Table 4. Organochlorine chemical and elemental contaminants measured in whole-body fish composite samples.—Continued

Contaminant class and analyte Chemical name(s) or atomic symbol Principal uses and sources to aquatic ecosystems

α-Hexachlorocyclohexane (HCH) 1,2,3,4,5,6-hexachlorocyclohexane Constituent of insecticide mixture containing various 

HCH isomers; also know as α-benzene hexachloride (BHC)

β-HCH 1,2,3,4,5,6-hexachlorocyclohexane Technical HCH (BHC) constituent

δ-HCH 1,2,3,4,5,6-hexachlorocyclohexane Technical HCH (BHC) constituent

γ-HCH (Lindane) 1,2,3,4,5,6-hexachlorocyclohexane Insecticide; technical HCH (BHC) constituent

Hexachlorobenzene (HCB) Perchlorobenzene Fungicide; industrial intermediate

Pentachlorobenzene Chlorinated benzene Fungicide; fire retardant

Pentachloroanisole Chlorinated benzene Fungicide; metabolite of chlorinated aromatic chemicals

Endosulfin I (α-Endosulfan) 6,9-methano-2,4,3-benzodioxathiepin,6,7,8,9,10,10-hexachloro1,5,5a,6,9,9aPesticide

Endosulfin II (β-Endosulfan) 6,7,9,10,10-hexachloro-1,5,5a,6,9,9a-hexahydro-6,9-methano-2,4,3-benzodiozathiepin-3-oxide, (3α, 5aα, 6β, 9β, 9aα)Pesticide

Endosulfan sulfate 6,7,8,9,10,10-hexachloro-1,5,5a,6,9,9ahexahydro-6,9-methano,2,4,3-benodiatiepin 3,3-dioxideEndosulfan byproduct

Dacthal dimethyl-2,3,5,6-tetrachlorobenzene-1,4-

dicarboxylic acid

Herbicide; may contain dioxin (2,3,7,8-TCDD) and 

HCB as impurities

Mirex 1,1a,2,2,3,3a,4,5,5,5a,5b,6-dodecachloro-octahydro-1,3,4-metheno-1Hcyclobuta(cd)pentaleneInsecticide; fire retardant

Elemental contaminants

Arsenic As Industrial sources; herbicides; defoliants

Cadmium Cd Mining, smelting and other industrial sources; urban 

runoff; sewage discharges

Chromium Cr Mining, tanning, and other refractory and chemical 

industrial sources

Copper Cu Mining, smelting and other industrial sources

Lead Pb Mining, smelting and other industrial sources; urban 

runoff; atmospheric pollution; fishing sinkers; lead 

shot

Mercury Hg Herbicides; fungicides; pulp, paper, and textile effluents; open-cycle chloralkali cells; landfills; mining; 

atmospheric pollution

Nickel Ni Mining, smelting, and other industrial sources

Selenium Se Coal-fired powerplants; irrigation return flows

Zinc Zn Mining, smelting and other industrial sources; urban 

runoff

16    Contaminants, Health Indicators, and Reproductive Biomarkers in Fish from the Colorado River Basin
contaminant exposure in both field and laboratory studies 

although they may be affected by a variety of factors (reviews 

by Wolke, 1992 and Blazer and others, 1997).

Measures of reproductive condition included plasma 

vitellogenin (vtg) and sex steroid hormone concentrations, 

gonadosomatic index (GSI), and gonadal histopathology (table 

3). Contaminants, particularly estrogen mimics, have been 

shown to impact reproduction in laboratory and field studies, 

although the reproductive condition in fish can be influenced 

by many factors including gender, age, reproductive stage, season, and water temperature (Allen and others, 1999; Gimeno 

and others, 1998). Estrogen mimics are capable of stimulating 

the production of vtg, a precursor of yolk protein, in the livers 

of oviparous vertebrates, and a number of endocrine disrupting compounds have been shown to induce abnormal vitellogenesis (Servos, 1999; Tyler and others, 1998). Vitellogenin 

production is normally associated with female fish, but vtg can 

be produced in males if estrogen or an estrogen-like chemical 

is present. The detection of concentrations typical of early- to 

mid-vitellogenic females in male fish has been associated with 

exposure to exogenous estrogens (Bowman and others, 2002; 

Denslow and others, 1999; Folmar and others, 1996, 2000, 

2001), but low vtg concentrations in males may be normal for 

certain species (Gross and others, 2002). Vitellogenin was 

measured in both male and female CDRB fish to document 

these and other possible alterations. The GSI and gonadal 

histopathology [reproductive stage, presence of atretic oocytes, 

and intersex condition (presence of female reproductive tissue in males or vice-versa)] were also assessed as measures 

of reproductive health and status. The GSI relates the proportional size of the gonad to the body size and may reflect 

changes resulting from a variety of physiological factors such 

as reproductive stage and environmental factors, including 

exposure to contaminants. Elevated occurrence of atretic 

(unfertilized, reabsorbed, or both) eggs has been noted in fish 

exposed to contaminants (Cross and Hose, 1988; Johnson and 

others, 1988), although other factors may also be involved. 

Feminization of male fish (that is, intersex condition) has been 

reported in laboratory and field studies of contaminants (Allen 

and others, 1999; Gimeno and others, 1997, 1998; Jobling 

and others, 1998) and in previous LRMN studies (Blazer and 

others, 2002; Hinck and others, 2004a; Schmitt and others, 

2004). Reproductive biomarkers can vary with temperature, 

photoperiod, and annual reproductive cycle. To minimize the 

effect of these factors on the reproductive biomarkers, all fish 

were collected post-spawn. Carp, bass, and channel catfish 

are annual spawners that typically spawn from April to June in 

the CDRB. All fish in our study were collected within eight 

weeks of one another from August 18 to October 23.

Fish Collection

Fish were collected by boat electrofishing along the 

shoreline or backwater areas of the river being sampled. All 

specimens of the target species were collected, irrespective of 

size, although electrofishing tends to be biased toward larger 

fish (Reynolds, 1983). More than 10 fish of a given species 

and gender were collected at some sites. GPS coordinates 

were obtained for the upstream and downstream locations at 

the sampling site in which fish were collected. Fish were held 

in on-board live wells and transported to shore for processing, 

usually within a few hours of collection. Fish at certain stations were held alive overnight in tanks or net pens containing 

ambient river water following night collections.

Sample Processing

A live fish was selected and identified to species. A 

blood sample was collected from the posterior caudal artery 

and vein with a heparinized needle and syringe and placed on 

wet ice. Plasma from this blood sample was later obtained 

for determination of vtg and steroid hormone concentrations. 

The fish was weighed, measured, and killed with a blow to the 

head. Observations of external features were recorded, and 

tissue anomalies were removed by dissection and preserved 

in 10% neutral buffered formalin (NBF) for histopathological 

analysis. The abdominal cavity was dissected open and the 

liver, spleen, and gonads were removed and weighed. Liver 

weights for carp and sucker were not determined because 

these species have a dispersed hepatopancreas. The liver, gall 

bladder, posterior and anterior kidneys, gonads, mesenteric 

fat (in certain species), and spleen were examined, and the 

gender of the fish was determined. Pieces of liver collected 

for EROD analysis were immediately flash-frozen in a dry 

ice-ethanol slurry, then transferred to dry ice for storage and 

shipment. Samples of gonad, kidney, spleen, and additional 

pieces of liver were collected and preserved in 10% NBF for 

histopathological examination, gender confirmation (gonad), 

and macrophage aggregate analysis (spleen). Upon compleTable 5. Monitoring and assessment strategy for polycyclic 

aromatic and polyhalogenated hydrocarbons (PAHs and PHHs). 

a

Total PCBs were determined by gas chromatography with 

electron-capture detection. b7-ethoxyresorufin O-deethylase. c

H4IIE bioassay was performed after reactive cleanup to 

remove aryl hydrocarbon receptor (AhR)-active PAHs. dAnd 

other planar organic compounds. + responds; – does not 

respond; *AhR-active isomers and congeners only.

Endpoint

Contaminants

PCBs

PCDDs

&

PCDFs

PAHsd

GC-ECDa (carcass) + - -

EROD activityb (liver) * * *

H4IIE bioassayc (carcass) * * -

Materials and Methods    17
tion of the internal examination and dissection, otoliths 

(asterisci in carp, sagitta in bass), scales, and/or spines were 

collected for age determination. Remaining tissues (those not 

frozen or fixed) were placed back into the body cavity and 

the entire fish was wrapped in aluminum foil. The wrapped 

carcass was labeled and placed in a bag with other carcasses 

of the same species and gender. These samples were chilled 

and later frozen for analysis of organochlorine chemical and 

elemental contaminants and dioxin-like activity. The entire 

field procedure was typically conducted in 15-20 min (per 

fish), and tissue samples, especially liver for EROD analysis, were collected and frozen as rapidly as possible. Blood 

samples were centrifuged, and the plasma was drawn off with 

a transfer pipette into a cryovial and frozen in dry ice following the processing of the fish.

Laboratory Analysis

Fish samples were shipped frozen on dry ice to the 

Columbia Environmental Research Center (CERC) of the 

USGS and stored frozen (-20°C) until analyzed. CERC 

performed analyses of composite fish samples for organic 

and elemental contaminants and completed quality assurance 

(QA) and quality control (QC) procedures described in Hinck 

and others (2004b). Fish were homogenized and lyophilized 

by LET, Inc. (Columbia, Missouri) according to protocols 

provided by CERC. Cryogenically frozen liver samples for 

EROD analysis were also shipped on dry ice to CERC for 

analysis. Cryogenically frozen plasma samples were similarly 

shipped to the Center for Environmental and Human Toxicology of the University of Florida for analysis of vtg and the 

Florida-Caribbean Science Center of the USGS for analysis of 

sex steroid hormones. Preserved tissue samples were shipped 

to the National Fish Health Laboratory of the USGS Leetown Science Center and Colorado Histo-Prep (Fort Collins, 

Colorado) for histopathological analysis. Information on 

these latter procedures are given by Blazer and others (2002) 

and McDonald and others (2002). Age determination was 

conducted by L. Stanley (Tottenham, Ontario) and confirmed 

by the USGS South Carolina Cooperative Fish and Wildlife Research Unit (Clemson, South Carolina). Scales were 

processed for age determination when otoliths were unavailable by estimating age (years) from the number of completed 

annuli (Berg and Grimaldi, 1967; Hesthagen, 1985). Transverse section of otoliths (asterisci in carp, sagitta in bass) 

and spines were processed for age determination following 

modified procedures from Cowan and others (1995) and 

Casselman (1987, 1990). Annual growth increments (annuli) 

were designated at the distal edge of an opaque zone imbedded 

between two translucent zones. As collection period varied 

and samples were examined in the blind, ages were reported as 

the number of increments regardless of time of year. 

Composite Sample Preparation

Individual fish carcasses were composited by gender 

and species and prepared by band-sawing and grinding with 

a commercial meat grinder. All equipment was disassembled 

and chemically cleaned between composite samples to prevent 

cross contamination. One sub-sample (~100 g) of the composite was refrozen (-20°C) for analysis of moisture content 

and lyophilized prior to elemental contaminant determination; a second (~10 g) sample was retained for organochlorine 

chemical residues by GC-ECD and gravimetric determination 

of lipid content; and a third (~10 g) sample was extracted with 

methylene chloride, subjected to the reactive cleanup procedure and ampulated for use in the H4IIE bioassay. These 

methods have been described previously (Hinck and others, 

2004b). 

Elemental Contaminants and Moisture Content

Sub-samples for elemental analyses (~100 g) were freezedried, and percent moisture was determined as weight lost 

during lyophilization. One portion of the dried material was 

digested in nitric acid and analyzed by inductively coupled 

plasma mass spectroscopy (ICP-MS) for the determination 

of Cd, Cr, Cu, Fe, Pb, Ni, and Zn. A homogenized aliquot of 

each dried sample (~0.25 g) was heated with 6 mL of nitric 

acid in a sealed low-pressure Teflon® vessel in a microwave 

oven. The cooled digestate liquid was transferred to a 125 mL 

polyethylene bottle and ultrapure H2O (>10 megOhm/cm) was 

added, yielding a final weight of 101.5 g (100 mL). The final 

acid matrix was 6% HNO3. All samples were diluted 10X 

by a CETAC ASD-500 auto-diluter as part of the analytical 

sequence. Internal standards were germanium (50 ng/g), rhodium (10 ng/g), and thorium (10 ng/g). The external standard 

consisted of a NIST traceable reference solution to which five 

elements (praseodymium, terbium, thulium, tantalum, and 

gold) were added for improved calibration in the rare earth 

region of the mass spectral range. A second portion (~0.5 

g) was dry-ashed (magnesium nitrate-nitric acid-HCl) and 

analyzed by hydride generation atomic absorption spectroscopy for As and Se. The dry ashing procedure consisted of 

three steps: 1) boiling with nitric acid for solubilization and 

partial oxidation; 2) 500°C ashing with magnesium nitrate 

to complete the oxidation and decompose remaining organic 

matter; and 3) heating with HCl to dissolve the ash and reduce 

Se+6 to the Se+4 oxidation state required for hydride generation. Digestates were diluted following the HCl reduction to 

~100 mL with de-ionized water that yielded a final acid matrix 

of 10% HCl. The digestates were mixed with HCl carrier 

solution and reduced by sodium tetrahydridoborate which 

was stabilized with sodium hydroxide. The resulting volatile 

hydrogen selenide or arsenide was transferred with argon 

carrier gas into a heated quartz cell mounted on an atomic 

absorption spectrophotometer for decomposition and measurement. A third portion was analyzed directly for total Hg using 

18    Contaminants, Health Indicators, and Reproductive Biomarkers in Fish from the Colorado River Basin
thermal combustion, amalgamation, and atomic absorption 

spectroscopy. 

Quality assurance (QA) measures for elemental determinations included the analysis of reagent blanks, replicate 

samples, certified reference materials, and fortified samples. 

Nominal LODs were 0.018 μg/g dw for As, 0.0007 μg/g dw 

for Hg, 0.014 μg/g dw for Se, 0.1 μg/g dw for Cd, and 1.0 μg/g 

dw for Cr, Ni, and Pb (Appendix 2). Elemental concentrations (including LODs) were converted from dry-weight (dw) 

to wet-weight (ww) for statistical analysis and reporting using 

the moisture content of each sample, which ranged from 66 to 

76%. 

Organochlorine Contaminants and Lipid Content

One sub-sample (~10 g) was solvent-extracted and analyzed gravimetrically for lipid content (range: 2-12%) and by 

high-resolution capillary gas chromatography with electron 

capture detection (GC-ECD) for 29 organochlorine pesticide 

residues and total polychlorinated biphenyls (PCBs; Appendix 

3). Total PCBs were calculated and reported as the sum of 

139 congeners. The analytical procedure began with blending anhydrous sodium sulfate with the composite sub-sample. 

Targeted chemicals were then extracted from the dried sample 

with dichloromethane. The extract was quantitatively split 

into portions for H4IIE bioassay (80%), OCP/PCB/toxaphene 

analyses (8%), percent-lipid determination (2%), and archive 

(10%). The analytical portion was spiked with the following chemical standards to track method recoveries: PCB 029 

(2,4,6-trichlorobiphenyl) for early-eluting PCBs (Cl1 - Cl3); 

PCB 155 (2,2’,4,4’,6,6’-hexachlorobiphenyl) for mid-range 

eluting congeners (Cl4 - Cl6), PCB 204 (2,2’,3,4,4’,5,6,6’-octachlorobiphenyl) for later-eluting PCBs (Cl7

 - Cl10), tetrachloro-m-xylene, and dibutylchlorendate. Quality assurance 

measures for the organochlorine analyses included the analysis 

of triplicate and fortified samples, use of internal standards to 

monitor recoveries of each sample, and the confirmation of 

residue identities by dual-column gas chromatography-electron capture detection. 

The analytical portion was purified by removing interfering co-extracted lipids and biogenic materials prior to 

the gas chromatographic quantification. Most interferences 

were removed by low-pressure size-exclusion chromatography (LPSEC). High performance SEC (HPSEC) was used 

to remove residual interferences. A two-layered octadecyl 

silica/activated silica gel column was used to separate the 

organochlorine pesticide residues from the PCBs prior to gas 

chromatography (GC) analysis. Dual-column GC-ECD data 

were used to confirm organochlorine pesticide identities. 

Total toxaphene concentrations were determined by quantifying 20 component peaks in a technical toxaphene standard. 

Nominal LODs were ≤1.4 ng/g for individual compounds, 

48 ng/g for total PCBs, and 24 ng/g for toxaphene. Polybrominated diphenyl ethers (PBDEs) present in samples from 

Stations 324 and 325 interfered with o,p’-DDT and p,p’-DDD. 

These samples were subjected to an additional cleanup step 

and re-evaluated by gas chromatography with mass spectrometry (GC-MS) to accurately quantify the DDTs with PBDE 

interferences. 

H4IIE Rat Hepatoma Cell Bioassay

Sub-samples for H4IIE analysis were kept frozen at 

CERC until the initiation of sample processing. Details of 

processing have been previously described (Schmitt and others, 2002; Whyte and others, 2004). Briefly, samples were 

thawed, homogenized, and extracted from a column with 

methylene chloride. Percent lipid was determined on a 1% 

portion of the extract. The remainder was concentrated and 

cleaned up by two-stage column chromatography. Extracts 

were evaporated, re-dissolved with isooctane and ampulated. 

Matrix QC of prepared samples (blanks and spikes) included 

ground tissues from laboratory-raised bluegill (Lepomis macrochirus) and samples of a CERC standard positive control 

tissue (carp from Saginaw Bay, Michigan).

The H4IIE rat hepatoma cells were seeded at 23,000 

cells/well in 300 μL of D-MEM culture media (Tillitt and others, 1991) and allowed to proliferate for 24 h. The cells were 

then dosed with sample extracts or standards in isooctane and 

incubated for 72 h to allow for maximal EROD induction. A 

standardized TCDD solution was used to generate an analytical dose-response curve, and a total of six dose-response 

curves were analyzed on each assay date. A linear regression 

was performed on each sample well to obtain the slope and 

estimate the rate of the reaction (pmol/min). The amount of 

protein in each well was determined by the fluorescamine 

assay (Lorenzen and Kennedy, 1993), and the protein values 

were used to normalize dose to each well and EROD activity. The reaction rate observed in each well was normalized 

according to the measured protein content, generating a value 

of specific activity (pmols resorufin formed/minute/mg of 

protein). The reaction rate in each well was then divided by 

the measured dose given to each well (gram equivalents/mg) to 

result in specific activity per min per gram equivalent (g.eq.) 

dosed. Reported results are the average of four replicate 

concentrate doses. The mean EROD reaction rate (pmol/min/

g.eq.) was divided by the average initial slope obtained for the 

TCDD standard curves, resulting in a measure of an equivalent 

dose of TCDD (TCDD-EQ; pg/g) for each sample. Assay 

LODs ranged from 0.1 to 0.8 pg/g, and the limits of quantification (LOQs) ranged from 0.5 to 1.7 pg/g. 

EROD Activity

Cryogenically frozen liver samples were stored at –80°C 

by CERC until the preparation of microsomal fractions, which 

were used the same day they were prepared. The kinetic 

microsomal assays were conducted in 96-well microtiter 

plates (Whyte and others, 2000). Briefly, triplicate determinations of EROD activity were performed on 10 μL portions of 

each microsomal preparation, and mean EROD activity was 

Materials and Methods    19
reported. Protein content was determined using the fluorescamine protein assay (Lorenzen and Kennedy, 1993) in 

the same 96-well microtiter plate as the EROD analyses. A 

positive control material, liver microsomes of male Spague 

Dawley rats injected with 500 mg/kg of Aroclor 1254, was 

also analyzed. A linear regression was performed on the data 

from each well to determine an EROD rate (pmol/min) along 

with its associated estimate of variance. Protein content was 

used to normalize EROD activity (pmol/min/mg) in each well. 

The LOD was calculated by adding the average basal EROD 

rate to three times the standard deviation of that rate, and the 

LOQ was calculated by adding the average basal EROD rate 

to ten times the standard deviation of that rate. LODs ranged 

from 0.01 to 0.23 pmol/min/mg, and the LOQs ranged from 

0.12 to 0.87 pmol/min/mg. 

Fish Health Indicators

General Histopathological Analyses

Tissues preserved in 10% NBF (liver, kidney, spleen, 

gill, gonad, and grossly visible lesions) were shipped to the 

LSC (Leetown, West Virginia) and Colorado Histo-Prep (Fort 

Collins, Colorado) and prepared for routine histopathological 

analysis (Blazer and others, 2002). Tissue sections (5 to 6-μm, 

on glass slides) were stained with hematoxylin and eosin (H & 

E) for light microscopic examination.

Quantitative Organism-Level Indicators

The prevalence of gross external pathological disorders 

was determined with a rating of present (1) or absent (0) 

deduced from field data. Gross pathologies were selected 

for consistency with other monitoring programs that have 

used this type of assessment (Fournie and others, 1996). 

Gross abnormalities included grossly visible disorders of the 

eye (exophthalmia, hemorrhage, opacity, emboli, missing), 

opercles (shortening, deformities, parasites), and body surface 

(ulcers, parasites, discolored areas or raised growths). In addition, disorders of the fins and skeleton were included. Numerical values were assigned to internal and external observations 

of lesions recorded in the field, and a necropsy-based fish 

health assessment index (HAI) score was calculated for each 

fish by summing these values for all organs (Blazer and others, 

2002). An index was only computed for a fish if there was a 

complete assessment.

Body and organ weights measured in the field were used 

to calculate condition and organosomatic indices. Condition 

factor was computed as body weight (g)/length3 (cm). The 

HSI was calculated as liver weight/(total body weight – gonad 

weight) X 100. Similarly, the SSI was calculated as spleen 

weight/(total body weight – gonad weight) X 100. Subtracting 

the weight of the gonads from the body weight in the computation of HSI and SSI minimizes the effect of the reproductive 

cycle on these indices (Dethloff and Schmitt, 2000). GSI was 

calculated as gonad weight/total body weight X 100. Additional information on these indices is given by Blazer and others (2002) and Dethloff and Schmitt (2000).

Macrophage Aggregates 

Macrophage aggregates were quantified using two 

methods, manual digitized analysis and computer-based image 

analysis, and then the data were compared. For the manual 

digitized analysis, MAs in splenic tissues from Stations 312, 

315, 319, 320, and 324 were visualized with H & E and quantified by viewing multiple fields (n = 5) using a 10X eyepiece 

and a 20X objective. The total viewed area was 3.2 mm2 per 

spleen section. For the computer-based image analysis, MAs 

in splenic tissues from Stations 311, 313, 314, 316, 317, 321, 

322, 323, and 325 were visualized through the Perl’s method 

staining procedure, which enhance the pigments in the MAs 

(Luna, 1992). The total viewed area was 2 mm2 per spleen 

section. Bass and channel catfish from Stations 312 and 

315 were analyzed using both methods. MA measurements 

included the number of aggregates in a mm2 of tissue (MA-#) 

and the area of each aggregate (MA-A). The percentage of 

tissue occupied by MA (MA-%) was computed from these 

measurements (Blazer and others, 2002). 

Reproductive Biomarkers

Gonadal Histopathology

Tissue pieces representing the entire gonad length were 

preserved in 10% NBF. Transverse sections were processed 

for routine light microscopy (embedded in paraffin, sectioned 

at 5 to 6 μm, and stained with H & E).

Female gonadal tissue was staged using developmental 

stages (designated 0-5) to classify each section (Blazer, 2002; 

McDonald and others, 2002; Nagahama, 1983; Rodriguez 

and others, 1995; Treasurer and Holliday, 1981). Fish ovaries 

typically contain oocytes in several developmental stages and 

were classified according to the maturity of the predominant 

stage of oogenesis in each tissue sample. Ovaries containing 

only undeveloped, previtellogenic oocytes were assigned to 

stage 0 (immature). Samples containing only previtellogenic 

chromatin nucleoli and perinuclear oocytes, identified by 

cytoplasm that stained basophilic with H & E, were assigned 

to stage 1 (previtellogenic). Ovaries containing previtellogenic oocytes as in stage 1 plus some cortical alveoli oocytes 

were classified as stage 2 (early vitellogenic). Those containing larger oocytes in which the cortical alveoli were pushed to 

the periphery of the cell, yolk globules filled the center, and 

the chorion of the developing oocytes were thicker than in 

earlier stages were designated as stage 3 (mid-vitellogenic). 

Ovaries containing oocytes with fused yolk globules were 

designated as stage 4 (late vitellogenic). Ovaries containing 

20    Contaminants, Health Indicators, and Reproductive Biomarkers in Fish from the Colorado River Basin
post-ovulatory follicles, which can be observed for some time 

after ovulation, are typically assigned to stage 5 (spent). After 

the ovarian tissues were staged they were further examined 

by light microscopy for atresia and other abnormalities. One 

hundred oocytes in each fish sample were counted when possible. Those showing morphological evidence of resorption 

or necrosis were quantified, and the percent of atretic oocytes 

were calculated. Oocyte atresia data in fish from Stations 

312, 315, 319, 320, and 324 were categorized and were not 

included in the statistical analyses or figures but are presented 

in the tables. 

Analogous to the procedure used to stage ovaries, male 

gonadal tissue was classified into developmental stages (0-4) 

according to the maturity of the predominant stage of spermatogenesis of each tissue sample (Blazer, 2002; Nagahama, 

1983). Immature, undeveloped, or regressed testes containing only spermatogonia were classified as stage 0 (immature) where as those containing primarily spermatocytes and 

spermatids were designated as stage 1 (early spermatogenic). 

Stage-2 (mid-spermatogenic) testes contain approximately 

equal proportions of spermatocytes, spermatids, and spermatozoa, and testes containing primarily mature spermatozoa were 

identified as stage 3 (late spermatogenic). Stage-4 testes were 

identified as post-spawning or spent. Testicular tissue was 

also examined microscopically for any abnormalities such as 

intersex. Male fish were classified as intersex when individual 

or small foci of undeveloped oocytes were observed within 

testicular tissue (that is, when an ovotestis condition was 

detected).

Vitellogenin

Concentrations of plasma vtg were determined by 

direct Enzyme-Link Immunosorbent Assay (ELISA) using 

the monoclonal antibodies (mAb), 3G2, 2D4, 2C11, and 

1C8 for the bass, carp, sucker, and trout, respectively. The 

plasma samples were diluted 1:200 (1:100 for carp), 1:10,000, 

1:100,000 and 1:1,000,000 with 10mM phosphate, 150mM 

NaCl, 0.02% azide, 10 KIU/mL Aprotinin, pH 7.6 (PBSZAP). Species-specific vtg standards (0, 0.005, 0.01, 0.02, 

0.04, 0.06, 0.08, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0 μg/mL) containing 

1:200, 1:10,000, 1:100,000, and 1:1,000,000 male plasma (in 

PBSZ-AP) were added to account for matrix effect (Denslow 

and others, 1999). Samples and standards were loaded onto a 

96-well ELISA plate in triplicate and stored overnight at 4oC 

in a humidified container. The following day the plates were 

washed four times with PBSZ and then blocked with 1% BSA 

in 10mM tris, 150mM NaCl, 0.05% tween, 0.02% azide, 10 

KIU/mL Aprotinin, pH 7.6 (1% BSA/TBSTZ-AP) for 2 h at 

room temperature. The plates were rewashed with PBSZ (4 

times) and the monoclonal antibody was loaded into wells on 

each plate. The lowest dilution (1:200) was probed with 1-3 

μg/mL of the mAb (depending on species) and dilutions of 

1:10k and higher with 0.1 μg/mL. After the addition of the 

mAb, the plates were stored at 4oC overnight in the humidified 

container. The following day the plates were washed and the 

biotinylated secondary antibody (goat anti mouse IgG-biotin) 

was added to each well at 1:1000 dilution in 1% BSA/TBSTZAP and incubated at room temperature for 2 h. The plates 

were washed, and strepavidin-alkaline phosphatase was added 

at 1:1,000 dilution in 1% BSA/TBSTZ-AP and incubated 

for 2 h at room temperature. After a final wash of the plates, 

the color was developed by adding 1 mg/mL p-nitro-phenyl phosphate in carbonate buffer (0.03M carbonate, 2mM 

MgCl2, pH 9.6) and measuring the color using an ELISA plate 

reader (SpectraMax Plus384, Applied Biosystems) at 405 nm. 

Concentrations of the unknowns were determined from the 

standard curves.

The LOD for the vtg direct ELISA was 0.001 mg/mL for 

bass, 0.0005 mg/mL for carp and sucker, and 0.002 mg/mL for 

trout. All assays were performed in triplicate and reported as 

the mean of the three measurements. The coefficient of variation was <10% for all samples analyzed. Inter and intra-assay 

variability was routinely measured by analyzing controls on 

several plates and different runs was found to be <10%, and 

<5%, respectively.

Sex Steroid Hormones

Concentrations of 17β-estradiol (E2) and 11-ketotestosterone (KT) in plasma samples collected from carp, bass, 

channel catfish, brown trout, white sucker, and flathead catfish 

were measured by radioimmunoassay (RIA). For analysis, 

samples were thawed and split. Duplicate plasma samples 

(50 μL) were extracted twice by adding 4 mL of ethyl ether, 

vortexing for 1 min, freezing the aqueous layer in a methanol-dry ice bath, and decanting the ether layer containing the 

lipophilic sex steroids. Standard curves were prepared in 

phosphate buffered saline with gelatin and azide (PBSGA) 

buffer using variable amounts of unlabeled E2 or KT (1, 5, 10, 

25, 50, 100, 250, 500 and 1,000 pg) and a constant concentration of radiolabeled hormone. Cross-reactivities of the E2 and 

KT antiserums with other steroids were low. Reactions were 

comprised of plasma extract (50 μL), radiolabeled sex steroid 

hormone (100 μL), and corresponding sex steroid hormonespecific antibody (100 μL) in PBSGA buffer (250 μL). The 

reaction solutions were allowed to equilibrate overnight, during which time the unlabeled hormone from the extract and 

a constant concentration of the corresponding radiolabeled 

sex steroid hormone competed for the same antibody binding 

sites. Following incubation, non-antibody bound radiolabeled 

hormone was removed by adding 250 μL of charcoal dextran and centrifuging at 3,000 X g for 10 min. Supernatant 

aliquots (0.4 mL) containing bound radiolabeled hormone 

were removed and placed in a vial with 4 mL of scintillation 

fluid. Radioactivity was measured using scintillation spectrophotometry. Sex steroid concentrations in plasma extracts 

were determined using a four-parameter logistics regression 

analysis of standard curves, which was then used to calculate 

concentrations for plasma extracts.

Pooled samples in triplicate were assayed serially in 10-, 

20-, 30-, 40-, and 50-μl volumes (final volume of 50 μl with 

Materials and Methods    21
charcoal-stripped plasma). The resulting inhibition curves 

were parallel to the respective standard curve, with the tests 

for homogeneity of regression indicating that the curves did 

not differ. Further characterization of the assays involved 

measurement of known amounts (1, 2, 5, 10, 25, 50, 100, 250 

and 500 pg) of E2 or KT in 50 μl charcoal-stripped plasma. 

Hormone concentrations in the plasma samples were corrected 

for extraction efficiency of 90±5.2% for E2 and 86±7.0% for 

KT. The LOD was 10.7 pg/mL for E2 and 14.3 pg/mL for KT. 

Inter- and intra-assay coefficients of variation were 9.4% and 

11.2%, respectively, for E2 and 9.4% and 12.1%, respectively, 

for KT.

The ratio of E2 to KT (E/KT) is an additional variable 

used to analyze sex steroid hormones (Folmar and others, 

1996; Goodbred and others, 1997; Hileman, 1994). Typically, E/KT ratio is >1.0 in female fish and <1.0 in male fish, 

but exact ranges of normality and seasonal fluctuations in this 

variable have not been established. 

Data Set Composition and Statistical Analyses

Carp, bass, and channel catfish were included in the statistical analyses. For indicators based on individual fish (that 

is, biomarkers and demographic endpoints), data were discussed in terms of the magnitude of the means within a gender 

for different species. Data were summarized graphically with 

box plots presenting the mean, median, range, and 25th and 

75th percentiles. Some biomarkers were also analyzed more 

rigorously (see following paragraphs).

Descriptive statistics were calculated for length, weight, 

and age data for species at individual stations. Fish for which 

otoliths, spines, or scales were unreadable for age determination (7 carp; 4 bass; 3 channel catfish; 3 white sucker; 1 flathead catfish) were not included in the presentation or analysis 

of age data. Gender was reported as NG (no gonad) for fish in 

which no gonad tissue was collected (3 carp; 3 channel catfish; 

1 bass; 1 flathead catfish; 1 brown trout). Other biological 

data for carp, bass, and channel catfish are presented in tabular 

form and are discussed. Length and weights of carp, bass, and 

channel catfish were also analyzed statistically because of the 

influence of fish size on concentrations of Hg. 

A total of 52 composite samples from 14 stations were 

analyzed for chemical contaminants in fish tissue. Twenty six 

samples (50%) from 13 stations were carp, 12 samples (23%) 

from six stations were bass, 10 samples (19%) from five stations were channel catfish, two samples (4%) from one station 

were brown trout, and two samples (4%) from one station 

were white sucker. All results for composite samples were 

converted to, analyzed statistically as, and reported as ww 

concentrations. A value of one-half the LOD was substituted 

for censored values (that is, values <LOD) in the computation 

of un-weighted geometric station means and for statistical 

analyses and censored values in all graphs (Schmitt and others, 

1999). 

Concentrations of many contaminants were <LOD, which 

precluded rigorous statistical analysis. Tissue concentration data are presented graphically and as tabular summaries. 

Geographic differences in contaminant concentrations were 

also examined statistically using ANOVA. Log-transformed 

concentrations of these analytes in carp, bass, and channel 

catfish were analyzed with series of t-tests using a pooled 

error mean-square (MSe) representing differences between- or 

among-samples of the same species. A nominal α-level of 

0.01 was used in these comparisons to protect against experiment-wise error. Because concentrations of Hg in predatory 

fish increase with size, age, or both (for example, Wiener and 

others, 2002), log-transformed length-adjusted (HgL) and 

weight-adjusted (HgW) concentrations were also tested using 

this procedure. Following the method of Brumbaugh and others (2001), the adjusted Hg values were computed by dividing 

the measured concentration in each composite sample by the 

mean length (m) and weight (kg) of the individual fish comprised by the sample. Unadjusted Hg, HgW, and HgL were 

also analyzed separately using the one-way analysis, which 

resulted in a more conservative test with fewer degrees-offreedom and MSe

-values based on only one species. Toxicity 

thresholds and contaminant concentration data from other 

studies were converted to ww concentrations, assuming 75% 

moisture, if the original study documented concentrations in 

dw and percent moisture was not reported.

Biomarkers can differ among species, gender, age, and 

reproductive stage (Schmitt, 2002; Schmitt and Dethloff, 

2000). Accordingly, a series of linear ANOVA models were 

fit to the individual fish data for carp, bass, and channel catfish 

to determine which factors influenced biomarker responses 

in these taxa. Analyses conducted on larger, previous LRMN 

datasets were used as a guide. The results of these analyses 

were reported as F-values and significance levels and were 

used to guide the graphical presentation and discussion of the 

findings. Because of the complexity of the models and the 

small size of the data set, the means could not be adjusted for 

the factors in the models. Data are presented and discussed in 

terms of the magnitudes of the means for combined genders or 

each gender within a species and at different stations within a 

species. Some individual points are also discussed. Data are 

presented graphically and summarized as described for the 

demographic data.

Results and Discussion

Geographic Distribution and Demographic

Characteristics of the Fish Collected

A total of 517 fish representing seven species were collected (table 6). Carp, bass, and channel catfish accounted 

for 90% of the fish obtained. Carp were collected from all 14 

stations, largemouth bass and channel catfish from six stations, 

22    Contaminants, Health Indicators, and Reproductive Biomarkers in Fish from the Colorado River Basin
Table 6. Number of fish collected, organized by station, species, and gender, in the 

Colorado River Basin in 2003. Stations are grouped by sub-basin and listed upstream 

to downstream. Station numbers are given in parentheses. Species totals that include 

individual(s) of unknown gender are designated by an asterisk (*).

Sub-basin, station and species Male Female Species total Station total

Upper Colorado River Basin

Lay, CO (311) 40

Smallmouth bass 10 9 20*

White sucker 8 11 19

Carp 1*

Ouray NWR, UT (312) 45

Carp 9 11 20

Channel catfish 6 5 11

Smallmouth bass 8 6 14

San Rafael, UT (313) 39

Carp 11 10 21

Channel catfish 14 4 18

Delta, CO (314) 42

Brown trout 9 11 21*

Carp 8 13 21

Loma, CO (315) 37

Carp 10 10 20

Channel catfish 1 2 5*

Largemouth bass 7 5 12

Gold Bar Canyon, UT (316) 40

Carp 10 10 20

Channel catfish 10 10 20

Hogback Diversion, NM (317) 40

Carp 13 7 20

Channel catfish 8 11 20*

Lower Colorado River Basin

South Cove, AZ (319) 17

Carp 6 11 17

Willow Beach, AZ (320) 20

Carp 11 8 20*

Needles, CA (321) 40

Carp 8 12 20

Largemouth bass 9 11 20

Imperial Dam, AZ (322) 40

Carp 8 12 20

Largemouth bass 10 10 20

Hayden, AZ (323) 40

Carp 13 7 20

Flathead catfish 7 3 11*

Largemouth bass 5 4 9

Phoenix, AZ (324) 37

Carp 10 8 20*

Channel catfish 2 12 15*

Largemouth bass 2 2

Arlington, AZ (325) 40

Carp 7 13 20

Largemouth bass 9 11 20

Results and Discussion    23
smallmouth bass from two stations, and white sucker, brown 

trout, and flathead catfish from one station each (table 6). 

Only carp were captured from Stations 319 and 320 although 

attempts were made to collect predatory species at these stations. Bass or channel catfish were collected from all stations 

except Station 314 where brown trout was the predator species.

Total length, weight, and age were examined in CDRB 

carp. The mean total length (TL) of carp was 477 mm, the 

mean weight was 1,365 g, and the mean age was 18.9 y (table 

7). Female and male carp were similar in length, weight, 

and age (table 7). Carp were generally smallest (mean TL 

and weight) from Station 325 and largest from Station 314 

(table 7). Age varied among stations with carp from the GRB 

(Stations 323, 324, and 325) being younger (<10 y) than 

those from other stations (>20 y). CDRB carp were slightly 

smaller but older than carp collected in previous LRMN 

studies (Hinck and others, 2004a; Schmitt, 2002; Schmitt 

and others, 2004). Carp from previous LRMN investigations 

were younger (<15 y) than CDRB carp although previous 

age estimates were based on scales (Hinck and others, 2004a; 

Schmitt, 2002; Schmitt and others, 2004). Age was estimated 

from otoliths (asterisci) in CDRB carp and verified by two 

independent laboratories. Otoliths have been used to age 

carp in other studies, but the use of otoliths to estimate age 

in carp has not been validated for fish >14 years old (Brown 

and others, 2004b; Vilizzi and Walker, 1999; Wilde and Pope, 

2002). A previous study in Lake Mead using whole otoliths 

to age fish estimated carp to be 1-3 y (Wilde and Pope, 2002), 

but conditions in the CDRB are favorable for carp to be >20 

y (D. Scarnecchio, oral communication). Carp, which were 

introduced to the CDRB prior to the 1880s, have no natural 

predators in the basin and can successfully spawn in vegetated 

areas with slow current. Carp may be younger in the GRB 

due to intermittent water flows during the year. The age of 

some CDRB carp may have been overestimated if otolith rings 

represented changes in water temperature, periods of rapid 

growth, or lunar cycles rather than annual growth. More information is needed to determine if otoliths are the appropriate 

structures to estimate age in carp. 

Total length, weight, and age varied in CDRB bass. The 

mean TL of bass was 348 mm, and mean weight was 597 g 

(table 8). The mean age bass was 3.3 y. Female bass outweighed male bass (676 g vs. 529 g) and were longer (365 mm 

vs. 334 mm), but the average age for both females and males 

was similar (3.6 y vs. 3.1 y). The largest female and male bass 

(mean TL and weight) were from Station 321, and the smallest 

bass were from Station 323 (table 8). Bass were older from 

Stations 311, 315, and 321 than from other CDRB stations. 

Smallmouth bass from Stations 311 and 312 were similar in 

size to largemouth bass collected from other CDRB stations 

(table 8). The average lengths, weights, and ages of CDRB 

bass were similar to bass collected in previous LRMN studies 

(Hinck and others, 2004a; Schmitt, 2002; Schmitt and others, 

2004).

Total length, weight, and age varied in female and male 

CDRB channel catfish. In channel catfish, mean TL was 389 

mm in TL and mean weight was 593 g (table 9). Female channel catfish were slightly larger than males (table 9). The mean 

age for channel catfish was 6.5 y (range 1-15 y), and female 

and male ages were similar (6.6 y vs. 6.2 y). Channel catfish 

from Stations 312, 313, and 316 were smaller (mean TL and 

weight) than those from Stations 315, 317, and 324 (table 

9). The youngest channel catfish were collected from Station 

324 (<3 y) while those from Station 315 were the oldest (>9 

y). Length, weight, and age data for the other species are in 

Appendix 4.

Accumulative contaminants, H4IIE Bioassay,

and EROD Activity

Elemental Contaminants

Arsenic

Arsenic was detected in all CDRB samples, and the greatest concentrations (>0.14 μg/g) were measured in carp from 

Stations 319, 320, 321, 322, and 323 (fig. 4; table 10). The 

greatest concentration (0.19 μg/g) was measured in male carp 

from Station 320, and the greatest mean concentration (0.17 

μg/g) was also measured at this site (table 11). Concentrations of As in other species were generally <0.10 μg/g (fig. 4). 

Arsenic concentrations differed significantly among CDRB 

stations in carp but not in bass or channel catfish (table 12). 

Arsenic concentrations were significantly greater in carp from 

Stations 319, 320, 321, 322, and 323 in the Lower CDRB than 

carp from Stations 314, 316, and 317 in the Upper CDRB.

Concentrations of As in 2003 samples were similar to 

those documented in other CDRB studies. The maximum 

NCBP concentrations of As in the CDRB were 0.33 μg/g in 

carp, 0.49 μg/g in bass, and 0.29 μg/g in channel catfish from 

1971 to 1986 (Schmitt and others, 1999). Historical NCBP 

concentrations in carp and channel catfish were similar to 

those measured in 2003. Concentrations in bass near Stations 

319, 320, 321, and 322 were generally greater (>0.15 μg/g) 

in NCBP samples than in 2003 samples. Arsenic concentrations were lower (<0.08 μg/g) in whole-body carp collected 

near Stations 321 and 322 in conjunction with a 1986 NIWQP 

investigation (Radtke and others, 1988) than those in 2003 

carp samples. Butler and others (1996) reported that most As 

concentrations in fish were <0.27 μg/g in western Colorado, 

and concentrations were 0.04-0.16 μg/g in carp and 0.07-0.14 

μg/g in white sucker from the Ouray NWR (Station 312) 

in 1991-1993 (Rowland and others, 2002). Relatively high 

As concentrations were also measured in liver of bluehead 

sucker (Catostomus discobolus) from the CDR near Cameo 

and Loma, Colorado (near Station 315; Deacon and Stephens, 

1998).

24    Contaminants, Health Indicators, and Reproductive Biomarkers in Fish from the Colorado River Basin
Table 7. Lengths, weights, and age of carp (Cyprinus carpio) collected in the Colorado River Basin in 2003. Stations are listed upstream to downstream. Station 

numbers are given in parentheses. Sample size (n), arithmetic mean, standard deviation (SD), and range are also given. Fish in which gender could not be determined are identified as having no gonad (NG).

Sub-basin, station Gender Length (mm) Weight (g) Age (years)

n Mean SD Range n Mean SD Range n Mean SD Range

Basin total All 259 477 85 265-705 259 1365 627 215-3735 253 18.9 13.8 1-54

M 124 463 81 275-635 124 1213 527 260-2475 120 19.6 14.5 1-54

F 132 495 82 270-705 132 1528 667 250-3735 131 18.6 13.2 1-49

NG 3 301 58 265-368 3 458 409 215-930 2 3.0 0.0 3

Upper Colorado River BasinLay, CO (311) NG 1 368 -- -- 1 930 -- -- 0 -- -- --Ouray NWR, UT (312) M 9 442 47 333-480 9 1052 277 450-1380 9 15.8 13.1 4-39

F 11 456 31 413-504 11 1167 242 845-1505 11 7.7 0.9 6-9

San Rafael, UT (313) M 11 452 29 410-495 11 1026 215 695-1460 11 34.4 8.6 10-41

F 10 467 37 433-549 10 1237 289 945-1770 10 22.7 12.6 7-40

Delta, CO (314) M 8 562 69 436-635 8 1633 585 495-2475 8 18.1 10.8 4-34

F 13 575 69 495-705 13 2076 802 945-3735 13 19.8 6.4 11-39

Loma, CO (315) M 10 499 65 398-595 10 1497 419 770-2045 6 7.5 0.8 6-8

F 10 543 71 413-623 10 1901 669 880-3065 8 8.3 2.6 6-13

Gold Bar Canyon, UT (316) M 10 440 20 407-463 10 918 106 760-1075 10 26.2 6.3 15-33

F 10 466 41 417-525 10 1105 280 805-1565 10 31.5 11.0 9-41

Hogback Diversion, NM (317) M 13 511 35 450-565 13 1622 254 1300-2060 13 18.2 8.8 10-40

F 7 520 21 495-560 6 1811 195 1605-2035 7 25.0 10.5 11-40

Lower Colorado River BasinSouth Cove, AZ (319) M 6 482 58 420-577 6 1352 510 880-2150 6 34.7 12.7 16-45

F 11 550 61 438-615 11 2039 627 915-3095 11 23.9 14.5 10-48

Willow Beach, AZ (320) M 11 518 43 465-590 11 1608 392 1110-2240 11 44.3 6.6 35-54

F 9 517 41 457-576 9 1735 383 1225-2370 9 40.9 7.9 25-49

Needles, CA (321) M 8 489 53 438-595 8 1384 333 935-1860 8 22.0 5.8 13-34

F 12 506 54 434-615 12 1517 380 1080-2245 12 26.8 8.0 8-42

Imperial Dam, AZ (322) M 8 518 25 483-563 8 1580 205 1390-2035 8 14.3 7.1 6-23

F 12 540 36 465-600 12 1972 404 1285-2825 12 14.3 7.9 6-25

Hayden, AZ (323) M 13 419 107 275-559 13 1024 661 260-2110 13 6.4 4.9 1-14

F 7 462 130 290-615 7 1486 1018 310-3025 7 9.9 10.0 1-30

Phoenix, AZ (324) M 10 357 53 288-441 10 566 275 270-1080 10 3.7 1.5 3-7

F 7 444 86 304-550 8 1163 513 335-1830 8 6.1 3.5 3-11

NG 2 268 4 265-270 2 223 11 215-230 2 3.0 0.0 3

Arlington, AZ (325) M 7 331 51 293-415 7 418 214 280-815 7 5.0 1.8 4-9

F 13 370 72 270-523 13 690 410 250-1730 13 5.2 2.0 3-9

Results and Discussion    25
Table 8. Lengths, weights, and ages of bass (Micropterus sp.) collected in the Colorado River Basin in 2003. Stations are listed upstream to downstream. 

Station numbers are given in parentheses. Sample size (n), arithmetic mean, standard deviation (SD), and range are also given. Fish in which gender could not be determined are identified as having no gonad (NG). 

Sub-basin, station Gender Length (mm) Weight (g) Age (years)

n Mean SD Range n Mean SD Range n Mean SD Range

Basin total All 117 348 70 212-567 117 597 374 130-2705 112 3.3 2.4 1-12

M 60 334 58 212-465 60 529 258 130-1220 57 3.1 1.9 1-9

F 56 365 78 243-567 56 676 458 190-2705 54 3.6 2.7 1-12

NG 1 265 -- -- 1 295 -- -- 1 5.0 -- --

Upper Colorado River Basin

Lay, CO (311)

M 10 309 50 249-391 10 532 285 235-1120 8 5.3 1.2 4-7

F 9 354 47 305-428 9 784 400 450-1620 8 6.4 2.3 5-12

NG 1 265 -- -- 1 295 -- -- 1 5.0 -- --

Ouray NWR, UT (312)

M 8 281 26 232-324 8 349 101 180-530 8 1.9 0.4 1-2

F 6 312 16 292-336 6 448 95 330-575 5 2.2 0.4 2-3

Loma, CO (315)

M 7 354 43 296-415 7 404 153 240-665 6 4.5 0.5 4-5

F 5 404 57 345-475 5 755 503 215-1510 5 5.0 1.0 4-6

Lower Colorado River Basin

Needles, CA (321)

M 9 394 50 322-465 9 636 175 390-885 9 4.8 2.6 2-9

F 11 451 58 338-555 11 857 407 365-1715 11 5.3 3.4 2-11

Imperial Dam, AZ (322)

M 10 348 56 265-430 10 609 313 235-1140 10 1.9 1.0 1-4

F 10 382 88 280-567 10 815 746 250-2705 10 2.3 2.2 1-8

Hayden, AZ (323)

M 5 280 44 212-320 5 311 135 130-460 5 1.2 0.4 1-2

F 4 267 24 243-300 4 268 101 190-415 4 1.5 0.6 1-2

Phoenix, AZ (324)

M 2 376 48 342-410 2 895 460 570-1220 2 2.5 0.7 2-3

Arlington, AZ (325)

M 9 341 47 257-383 9 626 232 245-860 9 2.1 1.1 1-3

F 11 317 50 250-396 11 515 240 240-980 11 1.8 0.9 1-3

26    Contaminants, Health Indicators, and Reproductive Biomarkers in Fish from the Colorado River Basin
Multiple USFWS studies have documented As concentrations in CDRB fish. In 1994 and 1995, King and others 

(1997) measured As in whole-body carp (<0.05-0.89 μg/g), 

largemouth bass (0.39-0.85 μg/g), and channel catfish (0.06-

0.46 μg/g) in the cotton growing regions of the Lower GRB 

near Stations 324 and 325. Concentrations were 0.07-0.13 

μg/g in channel catfish from the Lower GRB and Yuma 

Valley (Baker and others, 1992). Arsenic in fish from Imperial, Cibola, and Havasu NWRs did not represent a threat to 

piscivorous wildlife in the late 1980s (King and others, 1993), 

but Andrews and others (1997) concluded that concentrations 

(<0.10-0.20 μg/g) in fish from the Havasu NWR may represent a risk to fish and wildlife. Concentrations of As in fish 

collected in the Middle GR from Coolidge Dam to AshurstHayden Dam (near Station 323) ranged from <0.05 μg/g to 

1.11 μg/g and have the potential to harm wildlife in some 

areas (Andrews and King, 1997).

Concentrations of As in carp, bass, and channel catfish 

were measured in previous LRMN studies. Concentrations 

in carp ranged from 0.12 to 0.32 μg/g in the MRB (Schmitt 

and others, 2002), 0.05 to 0.55 μg/g in the RGB (Schmitt and 

others, 2004), and 0.24 to 0.56 μg/g in the CRB (Hinck and 

others, 2004a). In bass, As concentrations were 0.10-0.57 

μg/g in the MRB (Schmitt and others, 2002), 0.04-0.25 μg/g 

in the RGB (Schmitt and others, 2004), and 0.22-0.53 μg/g 

in the CRB (Hinck and others, 2004a). Concentrations of As 

in channel catfish ranged from 0.05 to 0.23 μg/g in the RGB 

(Schmitt and others, 2004). Overall, As concentrations in 

CDRB fish (0.01-0.19 μg/g) were less than those from other 

LRMN studies (table 13).

Arsenic concentrations in CDRB fish (0.01–0.19 μg/g) 

were not considered a hazard to the fish or piscivorous wildlife 

(USEPA, 1984). A review by Jarvinen and Ankley (1999) 

included several laboratory studies in which As effects were 

evaluated relative to whole-body concentrations. Concentrations of 8.1-13.5 μg/g were associated with loss of equilibrium 

and 5.4 μg/g caused increased mortality in rainbow trout fingerlings (McGreachy and Dixon, 1990, 1992). Adult bluegill 

experienced reduced survival and growth at 11.6 μg/g (Gilderhus, 1966). Concentrations of As in all CDRB fish were 

Table 9. Lengths, weights, and ages of channel catfish (Ictalurus punctatus) collected in the Colorado River Basin in 2003. Stations are 

listed upstream to downstream. Station numbers are given in parentheses. Sample size (n), arithmetic mean, standard deviation (SD), 

and range are also given. Fish in which gender could not be determined are identified as having no gonad (NG).

Sub-basin, station Gender Length (mm) Weight (g) Age (years)

n Mean SD Range n Mean SD Range n Mean SD Range

Basin total All 88 389 96 234-685 88 593 553 90-3210 86 6.5 3.0 1-15

F 44 399 98 234-685 44 655 627 90-3210 43 6.6 3.6 1-15

M 42 370 90 247-620 41 510 475 110-2275 41 6.2 2.3 2-12

NG 3 501 72 440-580 3 807 163 665-985 2 8.5 0.7 8-9

Upper Colorado River Basin

Ouray NWR, UT (312) M 6 308 26 279-345 6 209 65 140-290 6 6.7 1.6 5-9

F 5 323 49 279-390 5 255 130 155-400 5 8.8 1.9 6-11

San Rafael, UT (313) M 14 338 64 247-430 14 360 220 110-690 14 5.7 1.9 3-10

F 4 320 27 289-346 4 210 51 175-285 4 5.0 1.2 4-6

Loma, CO (315) M 1 488 -- -- 1 1230 -- -- 0 -- -- --

F 2 485 29 464-505 2 713 131 620-805 1 15.0 -- --

NG 2 531 69 482-580 2 878 152 770-985 1 9.0 -- --

Gold Bar Canyon, UT (316) M 10 328 39 270-399 10 256 114 135-520 10 6.2 2.5 3-11

F 10 332 58 234-420 10 272 162 90-590 10 6.3 1.6 4-10

Hogback Diversion, NM (317) M 8 460 69 375-600 8 888 402 480-1760 8 8.1 2.1 6-12

F 11 453 47 400-555 11 875 354 565-1790 11 10.0 2.5 7-15

NG 1 440 -- -- 1 665 -- -- 1 8.0 -- --

Lower Colorado River Basin

Phoenix, AZ (324) M 3 493 84 335-620 2 1855 594 1435-2275 3 2.7 0.6 2-3

F 12 450 125 290-685 12 1079 934 205-3210 12 2.8 1.4 1-5

Results and Discussion    27
below these thresholds and were not expected to adversely 

affect fish or wildlife in the basin.

Selenium 

Selenium was detected in all samples, and the greatest 

concentration (2.95 μg/g) was measured in male carp from 

Station 314 (table 10). Concentrations were >1.0 μg/g in most 

samples except those from Stations 311 and 323 (fig. 4). The 

greatest station mean (2.57 μg/g) was measured at Station 322 

(table 11). Concentrations of Se were generally greatest in 

carp and bass and lowest in channel catfish (fig. 4). Selenium 

concentrations differed significantly among sites in carp, bass, 

and channel catfish (table 12). Concentrations in carp from 

Stations 314, 315, and 316 in the Upper CDR, Station 319 

near Lake Mead, Stations 321 and 322 in the Lower CDR, 

and Station 325 in the Lower GR were significantly greater 

than those from other stations. Similarly, concentrations in 

bass from Stations 315, 321, 322, and 325 were significantly 

greater than those from Stations 311 and 323. Concentrations 

were significantly greater in channel catfish from Stations 312 

and 316 than those from Station 317.

Relatively high Se concentrations have been measured 

historically in CDRB fish as a result of natural weathering of 

seleniferous shales, irrigation practices, uranium ore and coal 

extraction, and combustion of coal at hydroelectric generating 

stations (Radtke and others, 1988). Elevated concentrations 

in the Lower CDR are the result of transportation from the 

Upper CDR rather than local agricultural practices (Welsh and 

Maughan, 1994). Relatively high concentrations in carp (0.36-

3.65 μg/g), bass (0.23-3.00 μg/g), and channel catfish (<0.05-

2.50 μg/g) near Stations 312, 319, 320, 321, 322, and 323 

were measured by the NCBP from 1971 to 1986 (Schmitt and 

others, 1999). Historical concentrations were generally lowest 

in carp and bass in the GR near San Carlos Reservoir (Station 

323; Schmitt and others, 1999). Selenium concentrations in 

carp were 1.6 μg/g near Station 321 and 2.5 μg/g upstream 

Station 322 (Radtke and others, 1988), which are similar to 

2003 concentrations at these stations. Butler and others (1996) 

reported concentrations >0.73 μg/g (assuming 75% moisture) 

in fish from western Colorado, and concentrations in carp 

(1.52-22.0 μg/g) and white sucker (0.71-16.0 μg/g) were high 

in the Ouray NWR where selenium remediation efforts had 

been initiated (Rowland and others, 2002). Concentrations 

of Se were generally lower and below toxicity thresholds in 

most fish from the GRB (Andrews and King, 1997; Baker and 

others, 1992; King and others, 1997) compared to other CDRB 

fish. However, Se concentrations in fish from NWRs in the 

Lower CDR were elevated and may threaten fish reproduction 

(King and others, 1993) and piscivorous wildlife (Andrews 

and others, 1997).

Selenium concentrations in carp, bass, and channel 

catfish were measured in previous LRMN studies. Concentrations in carp were <0.1-4.66 μg/g in the MRB (Schmitt 

and others, 2002), 0.23-1.54 μg/g in the RGB (Schmitt and 

others, 2004), and 0.32-1.1 μg/g in the CRB (Hinck and others, 2004a). In bass, concentrations were 0.21-4.46 μg/g in 

the MRB (Schmitt and others, 2002), 0.47-1.26 μg/g in the 

RGB (Schmitt and others, 2004), and <0.02-0.81 μg/g in the 

CRB (Hinck and others, 2004a). Selenium concentrations in 

channel catfish were 0.32-0.46 μg/g in the RGB (Schmitt and 

others, 2004). Selenium concentrations in CDRB fish were 

greater than those measured in previous LRMN studies (table 

13).

Several Se studies were included in a review by Jarvinen 

and Ankley (1999) on the effects of inorganic chemicals to 

aquatic organisms. Various studies from this review reported 

that whole-body concentrations of Se between 8 and 16 

μg/g dw (2-4 μg/g ww assuming 75% moisture) have led to 

reproductive failure in fathead minnows (Pimephales promelas; Schultz and Hermanutz, 1990) and bluegill (Gillespie 

and Baumann, 1986; Hermanutz and others, 1992; Coyle 

and others, 1993). In addition, concentrations of Se present 

in the egg stage or at hatch affected larval survival (Coyle 

and others, 1993; Hamilton and others 2005b, 2005c), which 

As (

μg/g)

0.00

0.05

0.10

0.15

0.20

Station

 311 312 313 314 315 316 317 319 320 321 322 323 324 325

Se (

μg/g)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Upper CDRB Lower CDRB

Common carp

Bass

Channel catfish

White sucker

Brown trout

Figure 4. Concentrations (μg/g ww) of arsenic (As) and 

selenium (Se) by station and taxon in whole-body fish composite 

samples from the Colorado River Basin in 2003. Censored values 

are plotted as one half the LOD. Stations are ordered from 

upstream to downstream and are grouped by sub-basin. See 

Table 2 for station descriptions.

28    Contaminants, Health Indicators, and Reproductive Biomarkers in Fish from the Colorado River Basin
emphasizes that multiple life stages need to be examined to 

correctly assess toxicity and tissue concentration relationships 

(Jarvinen and Ankley, 1999). Selenium accumulation varies 

among tissues in fish. Relatively high Se concentrations are 

found in spleen, liver, kidney, and gonads (Hamilton and others, 2005a). Selenium contamination has also been associated 

with histopathological effects in the gill, liver, kidney, and 

ovary of freshwater fish (Sorenson, 1988; Sorenson and others, 

1984). Whole-body concentrations of Se should not exceed 4 

μg/g dw (1.0 μg/g ww assuming 75% moisture) to avoid toxicity to the fish and should not exceed 3 μg/g dw (0.75 μg/g ww 

assuming 75% moisture) to avoid toxicity to piscivorous wildlife (Hamilton, 2004; Lemly, 1996). At least one sample from 

all CDRB stations exceeded one or both of these thresholds. 

Selenium continues to represent a risk to aquatic and piscivorous wildlife in the CDRB. 

Mercury 

Mercury was detected in all CDRB samples, and the 

maximum concentration (0.37 μg/g) was measured in female 

channel catfish from Station 313 (table 10). Concentrations 

were >0.1 μg/g in samples from Stations 311, 312, 313, 315, 

316, 317, 321, and 323 (fig. 5). Mean station concentrations 

were greatest from Stations 313 (0.24 μg/g) and 311 (0.23 μg/

g) in the Upper CDRB (table 11). Predatory fish (bass, channel catfish) accumulate greater Hg concentrations than bottom 

feeding fish (carp, sucker; Hinck and others, 2004a, 2004b; 

Schmitt and others, 1999, 2002, 2004), although carp and bass 

concentrations were similar at many CDRB stations (fig. 5). 

Mercury concentrations in predatory fish increase with size 

(that is, heavier and longer fish have greater concentrations; 

Brumbaugh and others, 2001); therefore, Hg concentrations 

of Hg adjusted for weight and length were examined. Relating concentrations of Hg in composite samples to individual 

length and weight measurements is difficult although overall 

trends or patterns can be identified. The length- or weightadjusted Hg concentrations (HgL and HgW, respectively) were 

greater (some >0.4 μg/g) than the unadjusted concentrations 

(none >0.5 μg/g). The greatest HgL and HgW concentrations 

were measured in channel catfish from Stations 312, 313, and 

316 and white sucker from Station 311 (fig. 5). The current 

study design (that is, measuring concentrations in composite 

samples) cannot definitively account for the contribution of 

size to concentrations of Hg. 

Concentrations of Hg in carp, bass, and channel catfish 

differed significantly among CDRB stations (table 12). Concentrations of unadjusted Hg in carp were significantly greater 

from Stations 312, 313, and 316 in the Upper CDRB and 

Station 323 in the Middle GR than from most other stations. 

Significantly greater concentrations of Hg were measured in 

bass from Station 311 (all smallmouth bass) than those from 

other stations (all largemouth bass; table 12). Similar to carp, 

concentrations of Hg were significantly greater in channel catfish from Stations 312 and 313 on the Green River than from 

other sites (table 12). The relative rankings of the stations for 

Hg, HgL, and HgW were similar for carp, bass, and channel 

catfish (table 12). Most relative differences remained after 

adjusting for fish size, which suggests that spatial differences 

were not entirely artifacts of fish size.

Mercury concentrations in CDRB fish were similar 

to those from other CDRB studies. The maximum historical NCBP concentrations were 0.23 μg/g in carp, 0.41 μg/g 

in bass, and 0.2 μg/g in channel catfish from 1971 to 1986 

(Schmitt and others, 1999), which were greater than most 

Table 10. Percent of samples and stations that exceeded limit of detection (LOD) concentrations for elemental contaminants in 

composite samples of whole fish from the Colorado River Basin in 2003. The maximum concentrations and associated sample 

information (station, gender, and species) from this study are also given. NA, not applicable.

Analyte Samples

(% of 52)

Stations

(% of 14) LOD range (μg/g)

Maximum concentration

(μg/g) Station Gender Species

Arsenic 100 100 NA 0.19 Willow Beach, AZ (320) M Common carp

Cadmium 46 79 0.024-0.034 0.24 San Rafael, UT (313) M Common carp

Chromium 81 100 0.24-0.35 2.38 Arlington, AZ (325) F Largemouth bass

Copper 100 100 NA 2.80 Delta, CO (314) F Brown trout

Lead 2 7 0.24-0.35 0.29 Gold Bar Canyon, UT (316) M Common carp

Mercury 100 100 NA 0.37 San Rafael, UT (313) F Channel catfish

Nickel 6 21 0.24-0.35 1.73 Loma, CO (315) F Common carp

Selenium 100 100 NA 2.95 Delta, CO (314) M Common carp

Zinc 100 100 NA 99.6 Arlington, AZ (325) F Common carp

Results and Discussion    29
Table 11. Unweighted geometric mean, minimum (Min), and maximum (Max) concentrations (μg/g, wet-weight) of 

elemental contaminants in fish collected in the Colorado River Basin in 2003. Censored values were replaced by one-half 

the value for the LOD for the computation of station means, but only if at least one value exceeded the LOD. The maximum 

geometric station mean is shown in bold for each contaminant. Stations are listed upstream to downstream.—Continued

Sub-basin, station Element

As Cd Cr Cu Hg Ni Pb Se Zn

Upper Colorado River Basin

Lay, CO (311) Mean 0.06 0.03 0.50 0.59 0.23 <0.34 <0.34 0.88 14.0

n = 4 Min 0.05 <0.03 0.37 0.40 0.18 <0.31 <0.31 0.81 12.8

Max 0.07 0.05 0.85 0.85 0.27 <0.34 <0.34 1.03 16.3

Ouray NWR, UT (312) Mean 0.07 0.05 0.50 0.66 0.18 <0.30 <0.30 1.18 39.1

n = 4 Min 0.06 0.03 0.39 0.47 0.14 <0.24 <0.24 0.95 17.6

Max 0.09 0.10 0.62 0.82 0.21 <0.30 <0.30 1.48 93.8

San Rafael, UT (313) Mean 0.05 0.05 0.27 0.62 0.24 <0.29 <0.29 1.17 40.8

n = 4 Min 0.02 <0.03 <0.24 0.41 0.16 <0.24 <0.24 0.80 18.6

Max 0.13 0.24 0.73 0.95 0.37 <0.29 <0.29 1.75 89.3

Delta, CO (314) Mean 0.05 0.02 0.23 1.58 0.07 <0.33 <0.33 2.28 53.4

n = 4 Min 0.04 <0.03 <0.29 1.05 0.05 <0.28 <0.28 1.63 34.3

Max 0.10 0.04 0.43 2.80 0.09 <0.33 <0.33 2.95 95.1

Loma, CO (315) Mean 0.05 0.03 0.39 0.68 0.08 0.27 <0.34 2.08 31.6

n = 4 Min 0.03 <0.03 0.31 0.34 0.06 <0.27 <0.27 1.97 13.5

Max 0.08 0.09 0.56 1.20 0.10 1.73 <0.34 2.25 81.9

Gold Bar Canyon, UT (316) Mean 0.02 0.04 0.47 0.64 0.16 <0.29 0.15 1.43 39.8

n = 4 Min 0.02 <0.02 0.31 0.36 0.13 <0.24 <0.24 0.93 19.9

Max 0.03 0.21 0.86 0.91 0.18 <0.29 0.29 2.03 88.3

Hogback Diversion, NM (317) Mean 0.03 0.03 0.32 0.73 0.11 <0.31 <0.31 0.88 38.2

n = 4 Min 0.03 <0.03 <0.31 0.46 0.10 <0.29 <0.29 0.51 17.7

Max 0.04 0.09 0.34 1.16 0.12 <0.31 <0.31 1.72 82.5

Lower Colorado River Basin

South Cove, AZ (319) Mean 0.14 0.08 0.28 1.23 0.04 <0.30 <0.30 2.13 73.5

n = 2 Min 0.11 0.06 <0.30 1.09 0.04 <0.30 <0.30 2.08 72.4

Max 0.17 0.12 0.54 1.39 0.04 <0.30 <0.30 2.19 75.4

Willow Beach, AZ (320) Mean 0.17 0.04 0.48 1.21 0.02 <0.31 <0.31 1.63 76.1

n = 2 Min 0.15 0.04 0.40 1.20 0.02 <0.31 <0.31 1.56 64.6

Max 0.19 0.05 0.58 1.23 0.02 <0.31 <0.31 1.70 89.0

Needles, CA (321) Mean 0.08 0.01 0.30 0.85 0.05 0.27 <0.34 2.19 35.4

n = 4 Min 0.05 <0.03 <0.34 0.52 0.03 <0.26 <0.24 1.79 15.9

Max 0.13 <0.03 0.68 1.12 0.12 1.53 <0.34 2.62 89.4

Imperial Dam, AZ (322) Mean 0.10 0.02 0.29 0.58 0.02 <0.35 <0.35 2.57 31.8

n = 4 Min 0.05 <0.03 <0.34 0.34 0.01 <0.29 <0.29 2.34 15.6

Max 0.14 0.04 0.72 0.92 0.04 <0.35 <0.35 2.72 64.7

30    Contaminants, Health Indicators, and Reproductive Biomarkers in Fish from the Colorado River Basin
concentrations in 2003 samples from Stations 312, 319, 320, 

321, 322, and 323. Concentrations in carp from Stations 321 

and 322 were similar to those reported by Radtke and others (1988), and Hg concentrations were <LOD (0.05 μg/g) in 

whole-body carp near Station 321 and upstream Station 322. 

Mercury concentrations in carp and white sucker from Station 

312 were 0.01-0.22 μg/g and 0.01-0.02 μg/g, respectively, in 

1991-1993 (Rowland and others, 2002).

Multiple USFWS studies have documented Hg concentrations in fish from the GRB. In 1994 and 1995, concentrations of Hg were measured in whole-body carp (0.04-0.41 

μg/g), largemouth bass (0.07-0.09 μg/g), and channel catfish 

(0.03-0.11 μg/g) from the GRB near Stations 324 and 325 

(King and others, 1997). Concentrations were low in channel catfish (0.02 μg/g) from the Lower GRB and Yuma Valley 

(Baker and others, 1992). Mercury concentrations in fish from 

Imperial, Cibola, and Havasu NWRs (<0.08 μg/g) did not represent a threat to piscivorous wildlife in 1988-1989 (Andrews 

and others, 1997; King and others, 1993), and concentrations 

in fish from the Middle GR near Station 323 (<0.01-0.08 μg/g) 

were below protective criteria for wildlife (Andrews and King, 

1997). 

Concentrations of Hg in carp, bass, and channel catfish 

were measured in previous LRMN studies. In carp, concentrations were 0.04-0.34 μg/g in the MRB (Schmitt and 

others, 2002), 0.03-0.20 μg/g in the RGB (Schmitt and others, 

2004), and <0.05-0.20 μg/g in the CRB (Hinck and others, 

2004a). Concentrations in bass were 0.05-0.45 μg/g in the 

MRB (Schmitt and others, 2002), 0.07-0.45 μg/g in the RGB 

(Schmitt and others, 2004), and 0.06-0.31 μg/g in the CRB 

(Hinck and others, 2004a). Mercury concentrations in channel 

catfish ranged from 0.05 to 0.09 μg/g in the RGB (Schmitt and 

others, 2004). Overall, Hg concentrations in CDRB fish were 

less than those measured in previous LRMN studies (table 13).

Fish populations are at greatest risk from Hg during 

embryonic and larval stages partially due to maternal transfer 

(Wiener and Spry, 1996). Behavioral effects in laboratory 

studies have been documented in fish containing whole-body 

concentrations of 0.7-5.4 μg/g (Kania and O’Hara, 1974; 

Wiener and Spry, 1996). Permanent impairment of grayling 

(Thymallus thymallus) fry feeding efficiency and competitive ability occurred at Hg concentrations of 0.27 μg/g (Fjeld 

and others, 1998). Dietary Hg exposure increased mortality 

(0.20-0.47 μg/g) and altered sex ratios (0.44-1.1 μg/g) of adult 

mummichog (Fundulus heteroclitus) and reduced fertilization 

success (0.01-0.63 μg/g) of eggs (Matta and others, 2001). 

Jarvinen and Ankley (1999) reviewed various laboratory studies evaluating the effects of Hg on reproduction in freshwater 

fish. Included were studies that found reduced reproduction at 

whole-body concentrations of 4.47 μg/g in fathead minnows 

(Snarski and Olson, 1982) and 9.4 μg/g in second-generation 

brook trout (Salvelinus fontinalis) (McKim and others, 1976). 

In fathead minnows, dietary Hg concentrations of 0.87 μg/g 

dw (0.22 μg/g ww assuming 75% moisture) increased wholebody concentrations over 10-fold, suppressed hormone levels, 

and inhibited gonadal development in females (Drevnick and 

Sandheinrich, 2003). Whole-body concentrations associated 

with behavioral and reproductive effects were approximately 

5 μg/g for brook trout and 10 μg/g for rainbow trout (Wiener 

and Spry, 1996; Wiener and others, 2002). However, caution 

should be used with these thresholds because many factors 

can contribute uncertainty to these critical tissue concentration 

estimates (Wiener and others, 2002).

Dietary concentrations of Hg in wildlife as low as 0.3 

μg/g have been associated with reproductive impairment in 

common loons (Gavia immer; Barr, 1986), and reproduction in 

mallards (Anas platyrhynchos) was affected at concentrations 

as low as 0.1 μg/g (Heinz, 1979). Dietary concentrations 

of 0.25-1.0 μg/g may also be toxic to piscivorous mammals 

Table 11. Unweighted geometric mean, minimum (Min), and maximum (Max) concentrations (μg/g, wet-weight) of 

elemental contaminants in fish collected in the Colorado River Basin in 2003. Censored values were replaced by one-half 

the value for the LOD for the computation of station means, but only if at least one value exceeded the LOD. The maximum 

geometric station mean is shown in bold for each contaminant. Stations are listed upstream to downstream.—Continued

Sub-basin, station Element

As Cd Cr Cu Hg Ni Pb Se Zn

Hayden, AZ (323) Mean 0.12 0.03 0.47 1.11 0.12 0.22 <0.28 0.63 33.6

n = 4 Min 0.08 <0.03 0.33 0.89 0.09 <0.26 <0.26 0.52 15.5

Max 0.16 0.11 0.87 1.27 0.16 0.87 <0.28 0.80 73.3

Phoenix, AZ (324) Mean 0.03 0.02 0.42 0.64 0.04 <0.33 <0.33 1.03 42.1

n = 4 Min 0.01 <0.03 0.36 0.43 0.04 <0.30 <0.30 0.57 24.1

Max 0.08 <0.03 0.46 0.90 0.05 <0.33 <0.33 1.73 75.2

Arlington, AZ (325 Mean 0.06 0.01 0.58 0.61 0.04 <0.29 <0.29 1.86 32.1

n = 4 Min 0.03 <0.03 0.32 0.38 0.02 <0.28 <0.28 1.42 13.2

Max 0.08 <0.03 2.38 0.88 0.06 <0.29 <0.29 2.69 99.6

Results and Discussion    31
Table 12. Spatial trends of chemical contaminants in fish collected in the Colorado River Basin in 2003. Least-squares mean concentrations (all in μg/g ww 

unless otherwise noted) of arsenic (As), cadmium (Cd), copper (Cu), total mercury (Hg), length-adjusted Hg (HgL; μg/g/m ww), weight-adjusted Hg (HgW; μg/g/kg ww), selenium (Se), zinc (Zn), p,p’-DDE, toxaphene, and total PCBs. Values within each group of taxon-station means followed by the same letter are not significantly different (

P < 0.01 Fisher’s protected LSD). Also shown are ANOVA F-values, degrees-of-freedom (df), and significance levels (*

P

≤ 0.01). Stations 

are listed upstream to downstream within each taxon grouping.

Taxon and station Contaminant

As Cd Cu Hg HgL HgW Se Zn p,p’-DDE Toxaphene PCB

CarpOuray NWR, UT (312) 0.08abc 0.09cd 0.77a 0.15gh 0.34fg 0.13fg 1.43b 85.5b 0.01a 0.01a 0.02aSan Rafael, UT (313) 0.11bcd 0.18f 0.92a 0.16h 0.35g 0.14g 1.69bc 87.5b 0.01a 0.01a 0.02aDelta, CO (314) 0.05a 0.04abc 1.18a 0.05de 0.09cd 0.03bcde 2.92h 82.5ab 0.24a 0.01a 0.08a

Loma, CO (315) 0.08abc 0.08bcd 1.14a 0.07ef 0.13de 0.04cde 2.15def 72.0ab 0.14a 0.01a 0.06a

Gold Bar Canyon, UT (316) 0.03a 0.17ef 0.90a 0.13gh 0.30fg 0.13fg 2.02cde 79.5ab 0.04a 0.01a 0.02a

Hogback Diversion, NM (317) 0.04a 0.09cd 1.10a 0.10fg 0.19ef 0.06ef 1.50b 81.0ab 0.02a 0.01a 0.04a

South Cove, AZ (319) 0.14de 0.09cd 1.24a 0.04c 0.07bcd 0.02bcd 2.13def 73.5ab 0.01a 0.01a 0.02a

Willow Beach, AZ (320) 0.17e 0.05abc 1.21a 0.02ab 0.05ab 0.01ab 1.63bc 77.0ab 0.10a 0.01a 1.24b

Needles, CA (321) 0.13cde 0.02ab 1.09a 0.03bc 0.06bc 0.02abc 2.57gh 75.0ab 0.03a 0.01a 0.06a

Imperial Dam, AZ (322) 0.13cde 0.03abc 0.89a 0.01a 0.03a 0.01a 2.47fg 58.5a 0.06a 0.01a 0.06a

Hayden, AZ (323) 0.15de 0.11de 1.20a 0.15gh 0.34fg 0.13fg 0.76a 69.0ab 0.04a 0.03a 0.05a

Phoenix, AZ (324) 0.07ab 0.02ab 0.88a 0.04c 0.10cd 0.05de 1.69bc 71.0ab 0.31a 0.10a 1.10b

Arlington, AZ (325) 0.06ab 0.01a 0.88a 0.02b 0.07bcd 0.05de 2.38efg 81.5ab 1.50b 0.62b 0.12a

Bass

Lay, CO (311) 0.07a 0.02a 0.44a 0.24d 0.73d 0.37d 0.95b 13.5a 0.00a 0.01a 0.02a

Loma, CO (315) 0.04a 0.01a 0.41a 0.08bc 0.23bc 0.16bc 2.03d 14.0a 0.14a 0.01a 0.04a

Needles, CA (321) 0.06a 0.01a 0.68ab 0.09c 0.21bc 0.12ab 1.86d 17.0a 0.01a 0.01a 0.02a

Imperial Dam, AZ (322) 0.09a 0.02a 0.37a 0.04a 0.10a 0.05a 2.67e 17.5a 0.04a 0.01a 0.02a

Hayden, AZ (323) 0.09a 0.01a 1.03b 0.09c 0.35c 0.33c 0.53a 16.5a 0.02a 0.01a 0.02a

Arlington, AZ (325) 0.08a 0.01a 0.42a 0.05ab 0.16ab 0.09ab 1.47c 13.0a 2.15b 0.72b 0.15a

Channel Catfish

Ouray NWR, UT (312) 0.06a 0.04a 0.58a 0.20c 0.65cd 0.89c 0.97c 18.0a 0.02a 0.01a 0.02a

San Rafael, UT (313) 0.03a 0.02a 0.42a 0.34d 1.02d 1.23c 0.81abc 19.0a 0.01a 0.01a 0.02a

Gold Bar Canyon, UT (316) 0.02a 0.01a 0.48a 0.18bc 0.54c 0.67c 1.02bc 20.0a 0.06a 0.01a 0.02a

Hogback Diversion, NM (317) 0.04a 0.01a 0.49a 0.11b 0.25b 0.13b 0.52a 18.0a 0.02a 0.01a 0.04a

Phoenix, AZ (324) 0.01a 0.02a 0.47a 0.05a 0.10a 0.03a 0.64ab 25.0a 0.71b 0.26b 1.60b

F25,26 8.28* 8.97* 8.56* 47.71* 36.93* 32.22* 50.56* 20.28* 18.66* 8.66* 11.65*

32    Contaminants, Health Indicators, and Reproductive Biomarkers in Fish from the Colorado River Basin
Table 13. Unweighted geometric mean, minimum (Min), and maximum (Max) concentrations (μg/g ww) of select elemental contaminants in piscivorous (Pisc) 

and benthivorous (Ben) fish from BEST-LRMN studies including the Colorado River Basin (CDRB), Columbia River Basin (CRB; Hinck and others, 2004a), Mississippi River Basin (MRB; Schmitt, 2002a), Rio Grande Basin (RGB; Schmitt and others, 2004), and Yukon River Basin (YRB; Hinck and others, 2004b). Censored values were replaced by one-half of the value for the LOD for the computations of basin means. 1 Piscivorous fish samples sizes for all elemental contaminants were n = 12 for the CDRB, n = 14 for the CRB, n = 34 for the MRB, n = 9 for the RGB, and n = 10 for the YRB. 2 Benthivorous fish samples sizes for all elemental contaminants were n = 14 for the CDRB, n = 16 for the CRB, n = 46 for the MRB, n = 10 for the RGB, and n = 5 for the YRB.

Arsenic Cadmium Chromium Copper Mercury Lead Selenium Zinc

Basin Pisc 1 Ben 2 Pisc Ben Pisc Ben Pisc Ben Pisc Ben Pisc Ben Pisc Ben Pisc Ben

CDRB 

Min 0.01 0.02 <0.02 <0.03 <0.24 <0.34 0.34 0.71 0.04 0.01 <0.24 <0.25 0.51 0.72 12.8 13.1

Max 0.12 0.19 0.04 0.24 2.38 0.73 2.80 1.39 0.37 0.25 0.33 0.35 2.72 2.95 36.1 99.6

Mean 0.05 0.08 0.02 0.05 0.36 0.41 0.56 1.00 0.11 0.06 0.16 0.14 1.11 1.75 18.1 67.1

CRB

Min <0.22 <0.21 <0.04 <0.04 0.30 0.50 0.34 0.81 <0.05 <0.05 <0.09 <0.09 <0.22 <0.22 11.6 17.7

Max 0.53 0.56 0.14 0.51 3.70 11.2 2.32 3.92 0.61 0.22 0.29 9.29 0.84 1.10 22.9 105.6

Mean 0.16 0.17 0.03 0.09 0.80 1.69 0.70 1.42 0.19 0.11 0.06 0.12 0.36 0.34 16.3 43.0

MRB

Min <0.11 <0.12 <0.02 <0.03 <0.16 0.25 0.35 0.47 0.05 <0.04 <0.01 <0.01 0.20 <0.12 13.7 16.5Max 0.57 0.33 0.22 0.51 2.29 7.48 3.84 2.68 0.45 0.34 0.49 0.69 4.46 4.66 41.6 150.0

Mean 0.16 0.11 0.02 0.07 0.51 0.91 0.55 1.09 0.17 0.09 0.02 0.09 0.49 0.62 19.7 70.1

RGB

Min <0.05 <0.10 <0.02 <0.02 0.28 0.38 0.29 0.56 <0.02 0.03 <0.03 <0.07 0.17 0.23 11.1 16.6

Max 0.44 0.55 0.04 0.12 70.2 71.8 1.01 1.80 0.46 0.20 0.83 0.43 1.87 1.73 55.9 83.6

Mean 0.12 0.16 0.01 0.03 4.23 6.31 0.57 1.03 0.10 0.10 0.07 0.10 0.54 0.63 18.3 54.2

YRB

Min 0.04 0.03 <0.01 0.04 <0.22 0.26 0.41 0.63 0.11 0.08 <0.27 <0.27 0.23 0.45 10.5 15.6Max 1.95 0.17 <0.01 0.12 1.26 1.64 0.94 1.49 0.65 0.19 <0.27 <0.27 0.85 0.62 56.4 30.8

Mean 0.12 0.07 <0.01 0.07 0.30 0.52 0.53 0.99 0.27 0.13 <0.27 <0.27 0.46 0.55 39.6 19.3

Results and Discussion    33
(studies reviewed by Wolfe and others, 1998). Neurotoxicity 

and mortality occurred in adult minks (Mustela vison) after 

chronic exposure to dietary Hg concentrations >1 μg/g (Dansereau and others, 1999; Wobeser and others, 1976; Wren and 

others, 1987). Consequently, guidelines for the protection of 

piscivorous wildlife range from 0.5 to 1.0 μg/g (Eisler, 1987; 

Thompson, 1996), and values as low as 0.1 μg/g for mammals 

and 0.02 μg/g for birds have been derived from water quality criteria and bioaccumulation factors (Yeardley and others, 

1998). Selenium affords a degree of protection against Hg 

toxicity in wildlife by demethylation to inorganic mercury 

when Se and Hg are in molar ratio of 1:1 (Dietz and others, 

1990; Heinz and Hoffman, 1998; Scheuhammer and others, 

1998; Wiener and others, 2002). However, studies have shown 

Se-enhanced Hg embryo toxicity in birds (Heinz and Hoffman, 1998). Thus, although the significant amounts of Se may 

protect adult birds from the toxic effects of Hg, reproductive 

effects may be exacerbated. Mercury concentrations in channel catfish from Station 313 exceeded 0.3 μg/g, and at least 

one sample from Stations 311, 312, 313, 315, 316, 317, 321, 

and 323 exceeded 0.1 μg/g (fig. 5). Therefore, CDRB wildlife 

may be at risk from exposure to Hg.

Cadmium

Concentrations of Cd were >LOD (0.024-0.034 μg/g) 

in 24 samples (46%) from 11 stations (table 10). Concentrations were >0.2 μg/g in two carp samples only (fig. 6), and 

the maximum concentration (0.24 μg/g) was measured in 

male carp from Station 313. The greatest station mean (0.08 

μg/g) was at Station 319 (table 11). Cadmium concentrations 

were greater in carp compared to other species, and concentrations were greater in carp than predator species collected 

concomitantly in previous LRMN studies (Hinck and others, 

2004a; Schmitt and others, 2002, 2004). Concentrations of Cd 

differed significantly among stations in carp but not in bass or 

channel catfish (table 12). Concentrations were significantly 

greater in carp from Stations 313 and 316 than at all others 

except Station 323 (table 12; fig. 6).

Cadmium concentrations in 2003 samples were low as 

reported by previous CDRB investigations (Andrews and 

King, 1997; Baker and others, 1992; Butler and others, 1996; 

King and others, 1997; Rowland and others, 2002; Stephens 

and others, 1988). Historical NCBP concentrations of Cd 

were generally low in carp (≤0.18 μg/g), bass (≤0.05 μg/g), 

and channel catfish (≤0.12 μg/g) from 1971 to 1986, but 

relatively high concentrations (>0.15 μg/g) were measured 

in carp near Stations 312, 319, and 320 (Schmitt and others, 

1999). In previous LRMN studies, Cd concentrations in carp 

were <0.02-0.51 μg/g in the MRB (Schmitt and others, 2002), 

<0.02-0.12 μg/g in the RGB (Schmitt and others, 2004), 

and <0.04-0.51 μg/g in the CRB (Hinck and others, 2004a). 

In bass, concentrations were <0.02-0.22 μg/g in the MRB 

(Schmitt and others, 2002) and <LOD (0.02 μg/g) in the RGB 

and CRB (Hinck and others, 2004a; Schmitt and others, 2004). 

Concentrations in channel catfish were <LOD (~0.03 μg/g) 

in the RGB (Schmitt and others, 2004). Cadmium concentrations in CDRB fish were similar to those measured in previous 

LRMN studies (table 13).

Birds and mammals are comparatively resistant to Cd. 

Dietary toxicity thresholds were >100 μg/g in the studies 

reviewed by Eisler (1985). Eisler (1985) suggested that a Cd 

concentration of 2 μg/g in fish is evidence of contamination, 

that 5 μg/g is potentially life-threatening to the fish, and that 

13-15 μg/g is a threat to higher trophic levels. A review by 

Jarvinen and Ankley (1999) included several laboratory studHg (

μg/g)

0.0

0.1

0.2

0.3

0.4

Common carp

Bass

Channel catfish

White sucker

Brown trout

HgL (

μg/g/m)

0.0

0.5

1.0

Station

 311 312 313 314 315 316 317 319 320 321 322 323 324 325

HgW (

μg/g/kg)

0.0

0.5

1.0

1.5

Upper CDRB Lower CDRB

Figure 5. Concentrations (μg/g ww) of total mercury (Hg) by 

station and taxon in whole-body fish composite samples from the 

Colorado River Basin in 2003. Unadjusted (Hg), length-adjusted 

(HgL), and weight-adjusted (HgW) concentrations are shown. 

Censored values are plotted as one half the LOD. Stations are 

ordered from upstream to downstream and are grouped by 

sub-basin. See text for computations and Table 2 for station 

descriptions

34    Contaminants, Health Indicators, and Reproductive Biomarkers in Fish from the Colorado River Basin
ies in which whole-body concentrations of Cd in freshwater 

fish of 0.12-15.6 μg/g resulted in reduced survival, growth, or 

both, and concentrations of 2-8 μg/g caused decreased spawning and embryo production. Concentrations of Cd in CDRB 

fish were below these benchmarks.

Lead

Concentrations of Pb were <LOD in all samples except 

male carp (0.29 μg/g) from Station 316 (fig. 6; table 10). 

These low concentrations of Pb are similar to those reported 

by previous CDRB studies (Andrews and others, 1997; Baker 

and others, 1992; Radtke and others, 1988; Roland and others, 

2002; Schmitt and others, 1999; Stephens and others, 1988). 

Andrews and King (1997) reported that Pb concentrations 

(0.54-2.34 μg/g) in fish from the Middle GR, an area with 

mine drainage, may threaten fish survival and reproduction; 

however, concentrations of most samples from the study 

were <LOD. Concentrations of Pb in carp, bass, and channel catfish from previous LRMN studies were generally less 

than those measured in the CDRB (table 13; Hinck and others, 

2004a; Schmitt and others, 2002, 2004).

The effects threshold of Pb in fish is ≥0.4 μg/g based on 

whole-body concentrations [(Holcombe and others, 1976) as 

reviewed in Jarvinen and Ankley (1999)]. Whole-body Pb 

concentrations of 0.4 μg/g reduced hatchability and 4.0-8.8 

μg/g reduced growth in third generation brook trout at various 

life stages (Holcombe and others, 1976). Concentrations in 

CDRB fish did not approach these values. CDRB wildlife are 

not at risk from Pb.

Zinc

Zinc was detected in all samples (12.8-99.6 μg/g), and 

the maximum concentration was measured in female carp 

from Station 325 (fig. 7; table 10). Concentrations were >52 

μg/g in carp samples while all other samples were <36 μg/g. 

Station means were >70 μg/g at Stations 319 and 320, but carp 

was the only species from these sites (table 11). Carp were 

not collected from Station 311, the station with the lowest 

geometric station mean (14.0 μg/g). Concentrations of Zn 

differed significantly among stations in carp but not in bass or 

channel catfish (table 12). Concentrations were significantly 

greater in carp from Stations 312 and 313 on the Green River 

than from Station 322 in the Lower CDRB.

Zinc concentrations were similar to historical NCBP 

concentrations in carp (42-110 μg/g), bass (11-27 μg/g), and 

channel catfish (17-23 μg/g; Schmitt and others, 1999). Some 

of the greatest historical Zn concentrations were in carp from 

the Green River (near Station 312) and the CDR at Lake Mead 

(near Stations 319 and 320), Lake Martinez (near Station 322), 

and Lake Havasu (near Station 321). Historical concentrations 

in carp, bass, and channel catfish in the Lower CDR (Andrews 

and others, 1997; King and others, 1993; Radtke and others, 

1988) and the GRB (Baker and others, 1992; King and others, 

1997) were similar to 2003 concentrations. However, Zn concentrations in carp from Station 312 (>75 μg/g) were greater 

than those from a previous study (27-54 μg/g; Rowland and 

others, 2002). Sun and Jeng (1998) reported that concentrations of Zn in carp commonly exceed 100 μg/g and determined 

that carp partition Zn in their digestive tissue and generally 

had greater concentrations than other species. Zinc concentrations in CDRB fish were similar to those reported in the MRB 

(Schmitt and others, 2002), RGB (Schmitt and others, 2004), 

and CRB (Hinck and others, 2004a; table 13). Because Zn is 

an essential nutrient, it is unlikely to be potentially harmful to 

CDRB fish or piscivorous wildlife (Eisler, 1993).

Copper

Copper was detected in all samples, and the maximum 

concentrations (1.61-2.80 μg/g) were in brown trout from Station 314 (fig. 7; table 10). All other concentrations were <1.39 

μg/g. Stations means were >1.0 μg/g at Stations 314, 319, 

320, and 323 (table 11). Concentrations of Cu differed significantly among CDRB stations in bass but not in carp or channel 

catfish (table 12). Concentrations of Cu were significantly 

Cd (

μg/g)

0.00

0.05

0.10

0.15

0.20

0.25

Common carp

Bass

Channel catfish

White sucker

Brown trout

Station

 311 312 313 314 315 316 317 319 320 321 322 323 324 325

Pb (

μg/g)

0.10

0.15

0.20

0.25

Upper CDRB Lower CDRB

Figure 6. Concentrations (μg/g ww) of cadmium (Cd) and lead 

(Pb) by station and taxon in whole-body fish composite samples 

from the Colorado River Basin in 2003. Censored values are 

plotted as one half the LOD. Stations are ordered from upstream 

to downstream and are grouped by sub-basin. See Table 2 for 

station descriptions.

Results and Discussion    35
greater in bass from Station 323 than all other sites except 

Station 321 (table 12). Station 323 is located near Cu mining 

operations in the GRB.

In general, Cu concentrations in CDRB fish were similar 

to those from the NCBP (Schmitt and others, 1999) and previous LRMN investigations (table 13; Hinck and others, 2004a; 

Schmitt and others, 2002, 2004). Copper was identified as 

a contaminant of concern in the GRB where sources include 

mining operations and urban runoff (Andrews and King, 1997; 

Baker and others, 1992; King and others, 1997) and in several 

NWRs in the Lower CDR (Andrews and others, 1997; King 

and others, 1993).

The ecological relevance of Cu in CDRB fish is 

unknown, and tissue-based criteria for Cu are not available 

for the protection of avian and mammalian wildlife (Eisler, 

1997). Cyprinids appear to be less sensitive to Cu toxicity 

than salmonids although high concentrations can cause more 

severe gill damage and epithelial swelling in carp (De Boeck 

and others, 2004). Chronic Cu exposure has been associated 

with physiological effects including changes in oxygen consumption, ionic regulation, and cell types as well as endocrine 

disrupting effects such as adrenergic response and cortisol 

release (Handy, 2003). Copper accumulates in the kidney during chronic exposure, and fish exposed to dietary Cu may also 

have increased MA activity in the kidney (Handy, 2003). 

Chromium 

Concentrations of Cr were >LOD (0.24-0.35 μg/g) in 

42 of 52 samples (81%) representing all sites (table 10). The 

maximum concentration was measured in female bass (2.38 

μg/g) from Station 325, and all other concentrations were 

<0.87 μg/g (fig. 7; table 10). The greatest mean concentration 

(0.58 μg/g) was from Station 325 on the GR (table 11). 

Previous studies have measured Cr in fish from the 

CDRB. Chromium concentrations were 0.22-0.75 μg/g in 

carp, 0.41-0.52 μg/g in bass, and 0.28-0.62 μg/g channel 

catfish in the GR southwest of Phoenix near Station 325 (King 

and others, 1997). Concentrations were relatively low (<0.06 

μg/g) in channel catfish from agricultural areas of the Lower 

GR (Baker and others, 1992). Concentrations from Station 

323 were similar to those measured in fish from the AshurstHayden Dam (Andrews and King, 1997). Chromium concentrations were >2.4 μg/g in fish from the Lower CDR near Stations 321 and 322 (King and others, 1993), and Cr was named 

a contaminant of concern in fish from Havasu NWR (Andrews 

and others, 1997). Concentrations were 0.14-1.13 μg/g in carp 

and 0.30-4.37 μg/g in white sucker from the Green River near 

the Ouray NWR (Rowland and others, 2002). Chromium concentrations in carp were 0.38-71.8 μg/g in the RGB (Schmitt 

and others, 2004) and 0.76-3.96 μg/g in the CRB (Hinck and 

others, 2004a), and concentrations in bass were 0.71-70.1 μg/g 

in the RGB (Schmitt and others, 2004) and 0.30-3.70 μg/g in 

the CRB (Hinck and others, 2004a). Chromium concentrations were generally lower than those from previous LRMN 

studies (table 13).

Eisler (1986) suggested that concentrations of Cr >4.0 

μg/g dw (1.0 μg/g ww assuming 75% moisture) in the tissues and organs of fish and wildlife indicate environmental 

contamination, but the significance of such a value is unclear. 

Studies linking whole-body Cr concentrations to survival or 

Zn (

μg/g)

0

50

100

150

200

Cu (

μg/g)

0.0

0.5

1.0

1.5

2.0

2.5

Common carp

Bass

Channel catfish

White sucker

Brown trout

Cr (

μg/g)

0.0

0.5

1.0

1.5

2.0

 311 312 313 314 315 316 317 319 320 321 322 323 324 325

Ni (

μg/g)

0.0

0.5

1.0

1.5

Upper CDRB Lower CDRB

Station

Figure 7. Concentrations (μg/g ww) of zinc (Zn), copper (Cu), 

chromium (Cr), and nickel (Ni) by station and taxon in whole-body 

fish composite samples from the Colorado River Basin in 2003. 

Censored values are plotted as one half the LOD. Stations are 

ordered from upstream to downstream and are grouped by subbasin. See Table 2 for station descriptions.

36    Contaminants, Health Indicators, and Reproductive Biomarkers in Fish from the Colorado River Basin
growth effects in freshwater fishes were not found (Jarvinen 

and Ankley, 1999).

Nickel

Concentrations of Ni were >LOD (0.24-0.35 μg/g) in 

only 3 of 52 samples (6%) from three stations (table 10). Concentrations were 0.87-1.73 μg/g, and the maximum concentration was in female carp from Station 315 (fig. 7; table 10). 

Mean concentrations were >0.2 μg/g at Stations 315, 321, and 

323 (table 11). 

Nickel was not a contaminant of concern in previous 

CDRB investigations (Andrews and King, 1997; Andrews and 

others, 1997; King and others, 1993, 1997; Rowland and others, 2002; Stephens and others, 1988). Nickel concentrations 

in carp were 0.18-4.21 μg/g in the RGB (Schmitt and others, 

2004) and <0.25-0.75 μg/g in the CRB (Hinck and others, 

2004a), and concentrations in bass were 0.23-3.29 μg/g in the 

RGB (Schmitt and others, 2004) and <0.22-0.31 in the CRB 

(Hinck and others, 2004a). Studies are lacking for linkages 

of whole-body concentrations to effects for Ni (Jarvinen and 

Ankley, 1999), and tissue-based criteria for the protection 

of fishes and piscivorous wildlife are not available. Data for 

additional elements from this study are available at <http://

www.cerc.usgs.gov/data/search.htm>.

Organochlorine Contaminants

DDT and its primary metabolites

The U.S. banned the use of DDT in 1972 although concentrations of this persistent organochlorine insecticide and its 

metabolites remain present in the environment from historical 

use and as a consequence of atmospheric transport (Fernadez 

and Grimalt, 2003). Elevated concentrations of DDT residues 

are most common in cotton-growing areas of the U.S., near 

former sites of production and formulation, and through atmospheric transport from sites where DDT is still used (Schmitt 

and others, 2002). We found the parent compound, p,p’-DDT, 

exceeded the LOD (>0.0014 μg/g) in 21 of 52 fish samples 

(40%) from seven stations (table 14), but all concentrations 

of p,p’-DDT were low. The maximum p,p’-DDT concentration (0.031 μg/g) was measured in male bass at Station 325 

(table 14). The major metabolite of p,p’-DDT, p,p’-DDE, was 

detected in all samples, and the maximum concentration (2.7 

μg/g) was measured in male bass from Station 325 (table 14; 

fig. 8). Samples with concentrations >0.5 μg/g included carp 

from Station 325 (1.3 and 1.7 μg/g), bass from Station 325 

(1.6 and 2.7 μg/g), and channel catfish from Station 324 (0.64 

and 0.78 μg/g; fig. 8), and station means were also greatest at 

Stations 324 and 325 (table 15). Concentrations of p,p’-DDE 

differed significantly among stations in carp, bass, and channel 

catfish (table 12). Concentrations of p,p’-DDE were significantly greater in bass and carp from Station 325 and channel 

catfish from Station 324. Mean concentrations of total DDT 

(p,p’- homologs) were greatest at Stations 324 and 325 (fig. 9). 

Concentrations of p,p’-DDD from p,p’-DDT breakdown and 

use as an insecticide were detected in 48 of 52 (92%) samples 

representing all stations (table 14). 

Our findings are consistent with the relatively high historical concentrations of total DDT (primarily as p,p’-DDE) 

in fish from the intensively farmed valleys of the Lower CDR 

and GR (Baker and others, 1992; Clark and Krynitsky, 1983; 

García-Hernández and others, 2001; Kepner, 1987; King and 

others, 1997; Schmitt and others, 1999). Conversely, Stephens 

and others (1988) reported concentrations were <LOD in fish 

from the Upper CDRB. Massive amounts of DDT (>25 kg/ha) 

have been applied to cotton growing regions of the Lower GR 

from the late 1950s to the early 1970s that have resulted in 

some of the highest reported concentrations in the U.S. (King 

and others, 1997). Concentrations of total DDT were <0.05 

μg/g in fish from historical NCBP sites except in fish from 

Yuma, Arizona (Schmitt and others, 1999), but p,p’-DDE concentrations were high in carp (0.03-0.43 μg/g) and largemouth 

bass (0.79-2.06 μg/g) from 1976 to 1986 at this site. Concentrations of p,p’-DDE in carp were 11.17 μg/g from Buckeye 

Canal, an agricultural drain, near Station 325 (King and others, 

1997). Concentrations of p,p’-DDE may also be hazardous to 

fish in Lake Mead (Bevans and others, 1996; Tuttle and Orsak, 

2002).

Concentrations of p,p’-DDE in carp, bass, and channel 

catfish measured in previous LRMN were greater than those 

measured in CDRB fish (table 16). In carp, concentrations 

were <0.01-8.3 μg/g in the MRB (Schmitt and others, 2002), 

0.01-0.67 μg/g in the RGB (Schmitt and others, 2004), and 

0.01-1.1 μg/g in the CRB (Hinck and others, 2004a). Concentrations in bass were <0.01-0.53 μg/g in the MRB (Schmitt 

and others, 2002), <0.01-0.40 μg/g in the RGB (Schmitt and 

others, 2004), and <0.01-1.2 μg/g in the CRB (Hinck and others, 2004a), and concentrations in channel catfish ranged from 

0.10 to 1.4 μg/g in the RGB (Schmitt and others, 2004). 

Concentrations of total DDT in fish >0.15 μg/g are potentially harmful to the brown pelican (Pelicanus occidentalis), a 

sensitive avian species (Anderson and others, 1975). Protective wildlife criteria as low as 0.20 μg/g have been suggested 

by Newell and others (1987). Concentrations of 1-3 μg/g are 

potentially hazardous to most piscivorous birds (see review 

by Blus, 1996), and whole-body concentrations as low as 0.5 

μg/g have been associated with toxic effects to fish (see review 

by Jarvinen and Ankley, 1999). Whole-body concentrations of 

total DDT <5.0 μg/g have reduced survival of fry or fingerlings in multiple freshwater fish species (Burdick and others, 

1964; Cuerrier and others, 1967; Hopkins and others, 1969; 

Johnson and Pecor, 1969; Macek, 1968). Total DDT concentrations were >1.0 μg/g in fish from Station 325 only (fig. 9), 

and concentrations were >0.15 μg/g in individual samples 

from Stations 314, 315, 324, and 325. Concentrations of total 

DDT and p,p’-DDE in the GR have declined over the past 

decade, although fish and wildlife, including migratory birds, 

are still at risk from current concentrations. 

Results and Discussion    37
Table 14. Percent of samples and stations with concentrations exceeding the limit of detection (LOD) for organochlorine 

chemical residues in composite samples of whole fish in the Colorado River Basin in 2003. The maximum concentrations 

and associated sample information (station, species, and gender) from this study are also given. ND, not detected. a Sum of 

p,p’-DDT, p,p’-DDE, and p,p’-DDD, with censored values represented by one half the LOD. b Sum of cis- and trans-chlordanes 

and nonachlors; oxychlordane; heptachlor; and heptachlor epoxide, with censored values represented by one half the LOD. 

C

 1,1a,2,2,3,3a,4,5,5,5a,5b,6-dodecachloro-octahydro-1,3,4-metheno-1H-cyclobuta(cd)pentalene. d Sum of α-, β-, γ-, and δhexachlorocyclohexane with censored represented by one half the LOD. NA – not applicable.

Analyte(s) Samples

(% of 52)

Stations

(% of 14)

Maximum 2003 concentration

μg/g Station Gender Species

p,p’-DDT 40 50 0.031 Arlington, AZ (325) M Largemouth bass

p,p’-DDD 92 93 0.037 Arlington, AZ (325) M Largemouth bass

p,p’-DDE 100 100 2.7 Arlington, AZ (325) M Largemouth bass

Total p,p’-homologs a NA NA 2.8 Arlington, AZ (325) M Largemouth bass

o,p’-DDT 38 71 0.003 Willow Beach, AZ (320) M Common carp

o,p’-DDD 38 50 0.017 Arlington, AZ (325) M Common carp

o,p’-DDE 83 100 0.017 Arlington, AZ (325) M Largemouth bass

Aldrin 0 0 NA NA NA NA

Dieldrin 90 100 0.022 Phoenix, AZ (324) M Channel catfish

Endrin 98 100 0.10 Arlington, AZ (325) M Largemouth bass

cis-Chlordane 96 100 0.043 Arlington, AZ (325) M Common carp

trans-Chlordane 79 93 0.027 Phoenix, AZ (324) M Channel catfish

cis-Nonachlor 98 100 0.017 Loma, CO (315) M Common carp

trans-Nonachlor 100 100 0.045 Phoenix, AZ (324) M Channel catfish

Oxychlordane 77 86 0.011 Phoenix, AZ (324) M Channel catfish

Heptachlor epoxide 85 100 0.004 Loma, CO (315) M Common carp

Heptachlor 15 43 0.001 Imperial Dam, AZ (322) M Largemouth bass

Total chlordane-related residues b NA NA 0.121 Phoenix, AZ (324) M Channel catfish

Toxaphene 15 21 0.87 Arlington, AZ (325) M Largemouth bass

Mirex c 92 100 0.001 San Rafael, UT (313) M Channel catfish

Hexachlorobenzene (HCB) 94 100 0.007 Phoenix, AZ (324) M Channel catfish

Pentachlorobenzene 65 86 0.001 Phoenix, AZ (324) M Channel catfish

Pentachloroanisole 94 100 0.021 Gold Bar Canyon, UT (316) M Common carp

alpha-HCH 69 100 0.001 South Cove, AZ (319) F Common carp

beta-HCH 62 79 0.004 San Rafael, UT (313) F Common carp

gamma-HCH (Lindane) 35 64 0.008 Phoenix, AZ (324) M Channel catfish

delta-HCH 52 71 0.003 Phoenix, AZ (324) M Channel catfish

Hexachlorocyclohexane (HCH) d 94 100 0.015 Phoenix, AZ (324) M Channel catfish

Dacthal 46 79 0.009 Phoenix, AZ (324) M Common carp

Endosulfan I 21 50 0.004 Hayden, AZ (323) F Common carp

Endosulfan II 46 64 0.054 Arlington, AZ (325) M Common carp

Endosulfan sulfate 96 100 0.079 Arlington, AZ (325) M Common carp

Methoxychlor 21 36 0.010 Arlington, AZ (325) M Largemouth bass

Total PCBs 42 64 2.1 Phoenix, AZ (324) F Channel catfish

38    Contaminants, Health Indicators, and Reproductive Biomarkers in Fish from the Colorado River Basin
p,p'-DDE (

μg/g)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Common carp

Bass

Channel catfish

White sucker

Brown trout

Total chlordanes (

μg/g)

0.00

0.03

0.06

0.09

0.12

Dieldrin (

μg/g)

0.000

0.005

0.010

0.015

0.020

Station

 311 312 313 314 315 316 317 319 320 321 322 323 324 325

Endrin (

μg/g)

0.00

0.03

0.06

0.09

Upper CDRB Lower CDRB

Mirex (

μg/g)

0.0000

0.0002

0.0004

0.0006

0.0008

0.0010

Toxaphene (

μg/g)

0.0

0.2

0.4

0.6

0.8

Station

 311 312 313 314 315 316 317 319 320 321 322 323 324 325

Hexachlorobenzene (

μg/g)

0.000

0.002

0.004

0.006

Upper CDRB Lower CDRB

Figure 8. Concentrations (μg/g ww) of banned pesticides or pesticide products including p,p’-DDE, total chlordanes, dieldrin, endrin, 

mirex, toxaphene, and hexachlorobenzene by station and taxon in whole-body fish composite samples from the Colorado River Basin 

in 2003. Total chlordanes are the sum of cis- and trans-chlordanes and nonachlors, heptachlor, heptachlor epoxide, and oxychlordane. 

Censored values are plotted as one half the LOD. Stations are ordered from upstream to downstream and are grouped by sub-basin. 

See Table 2 for station descriptions.

Results and Discussion    39
Table 15. Unweighted geometric mean, minimum (Min), and maximum (Max) concentrations (μg/g ww) of organochlorine 

chemical contaminants in fish from the Colorado River Basin in 2003. Censored values were replaced by one-half of the 

value for the LOD for the computation of station means, but only if at least one value exceeded LOD. Total PCBs is the sum 

of all congeners. Total chlordane is the sum of cis-chlordane, trans-chlordane, cis-nonachlor, trans-nonachlor, heptachlor, 

heptachlor epoxide and oxychlordane. The maximum geometric station mean is shown in bold for each contaminant. Stations 

are listed from upstream to downstream.

Station p,p’-DDE Dieldrin Total PCBs Toxaphene Total chlordane

Upper Colorado River Basin

Lay, CO (311) Mean 0.003 0.0002 <0.048 <0.024 0.007

n = 4 Min 0.001 <0.0002 <0.048 <0.024 0.002

Max 0.006 0.0008 <0.048 <0.024 0.014

Ouray NWR, UT (312) Mean 0.014 0.0005 <0.048 <0.024 0.006

n = 4 Min 0.009 0.0002 <0.048 <0.024 0.004

Max 0.018 0.0010 <0.048 <0.024 0.008

San Rafael, UT (313) Mean 0.010 0.0007 <0.048 <0.024 0.007

n = 4 Min 0.007 0.0004 <0.048 <0.024 0.003

Max 0.013 0.0009 <0.048 <0.024 0.011

Delta, CO (314) Mean 0.116 0.0022 0.044 <0.024 0.010

n = 4 Min 0.057 0.0006 <0.048 <0.024 0.004

Max 0.250 0.0089 0.081 <0.024 0.021

Loma, CO (315) Mean 0.131 0.0055 0.045 <0.024 0.031

n = 4 Min 0.083 0.0022 <0.048 <0.024 0.016

Max 0.190 0.0192 0.065 <0.024 0.121

Gold Bar Canyon, UT (316) Mean 0.049 0.0029 <0.048 <0.024 0.023

n = 4 Min 0.033 0.0015 <0.048 <0.024 0.011

Max 0.061 0.0041 <0.048 <0.024 0.032

Hogback Diversion, NM (317) Mean 0.016 0.0008 0.035 <0.024 0.010

n = 4 Min 0.015 0.0006 <0.048 <0.024 0.007

Max 0.019 0.0009 0.052 <0.024 0.011

Lower Colorado River Basin

South Cove, AZ (319) Mean 0.009 0.0004 <0.048 <0.024 0.004

n = 2 Min 0.007 0.0004 <0.048 <0.024 0.003

Max 0.012 0.0004 <0.048 <0.024 0.005

Willow Beach, AZ (320) Mean 0.104 0.0015 1.20 <0.024 0.020

n = 2 Min 0.099 0.0013 0.87 <0.024 0.017

Max 0.110 0.0018 1.60 <0.024 0.023

Needles, CA (321) Mean 0.021 0.0005 0.039 <0.024 0.004

n = 4 Min 0.014 0.0002 <0.028 <0.024 0.002

Max 0.034 0.0012 0.067 <0.024 0.008

40    Contaminants, Health Indicators, and Reproductive Biomarkers in Fish from the Colorado River Basin
Technical DDT contains o,p’-DDT as an impurity (up to 

approximately 15%), and residues of this compound and its 

metabolites also remain widespread (Schmitt and others, 1999, 

2002). Concentrations of o,p’-DDE (<0.00008-0.017 μg/g), 

o,p’-DDD (<0.00055-0.017 μg/g), and o,p’-DDT (<0.00053-

0.11 μg/g) were greatest in male largemouth bass from Station 

325 (table 14). Concentrations of o,p’-DDT were generally 

greater in fish from the GR (0.05-0.11 μg/g) than all others 

from the CDRB (<0.003 μg/g). Concentrations of o,p’-DDD 

were relatively high compared to p,p’-DDD concentrations in 

samples from the Station 324 and 325 (Appendix 5); o,p’-

DDD concentrations were expected to be 10 times lower than 

p,p’-DDD concentrations Concentrations of o,p’-homologs 

were generally not detected or low (≤0.01 μg/g) in bass or 

carp from the RGB and CRB (Hinck and others, 2004a; 

Schmitt and others, 2004). The o,p’ homologs were historically considered relatively benign, but multiple studies have 

found that these compounds are estrogenic (Ackerman and 

others, 2002; Donohoe and Curtis, 1996; Guillette and others, 

1996; Metcalfe and others, 2000; Papoulias and others, 2003; 

Toppari and others, 1996). Dietary exposure to estrogenic 

chemicals including o,p’-DDT and o,p’-DDE produced hepatotoxicity in rainbow trout, potentially causing decreased HSI 

values, plasma vtg concentrations, and lipid levels (Donohoe 

and Curtis, 1996). Papoulias and others (2003) reported that 

low concentrations of o,p’-DDT and o,p’-DDE may interfere 

with the binding of natural ligands to steroid binding receptors and proteins resulting in endocrine-disrupting effects such 

as decreased GSI values. Conversely, Ungerer and Thomas 

(1996) found that increases in o,p’-DDT concentrations were 

associated with increased GSI values in female but not male 

Atlantic croaker (Micropogonias undulatus). The study 

concluded that o,p’-DDT binds to different lipoproteins in the 

plasma of female fish and compartmentalizes in the oocyte 

(Ungerer and Thomas, 1996). However, Metcalfe and others (2000) suggested that continuous exposure to estrogenic 

compounds such as o,p’-DDT must begin in ovo and continue 

throughout early development to affect reproductive endpoints 

in fish. The total risk to fish and wildlife represented by concentrations of o,p’-DDT and its homologs is unknown.

Chlordane and heptachlor

Chlordane is a mixture of cyclopentadiene-derived 

compounds that was widely used as a soil insecticide. Concentrations of these compounds are typically greatest in fish 

from corn-growing regions, urban areas in the “termite belt” 

or southeastern U.S., and near production and formulation 

facilities (Schmitt and others, 1999; Schmitt, 2002). Seven 

chlordane-related components and metabolites were measured: 

cis-chlordane, trans-chlordane, cis-nonachlor, trans-nonachlor, 

oxychlordane, heptachlor, and heptachlor epoxide (table 14). 

Heptachlor epoxide, a metabolite of heptachlor, is a minor 

constituent of chlordane and was also used historically as an 

insecticide, and environmental concentrations result from 

both sources. Oxychlordane is a metabolite of cis-chlordane. 

Concentrations of oxychlordane, heptachlor, and heptachlor 

epoxide were low (table 14). Concentrations of cis-chlordane 

were >LOD in 52 of 54 (96%) samples representing all staTable 15. Unweighted geometric mean, minimum (Min), and maximum (Max) concentrations (μg/g ww) of organochlorine 

chemical contaminants in fish from the Colorado River Basin in 2003. Censored values were replaced by one-half of the 

value for the LOD for the computation of station means, but only if at least one value exceeded LOD. Total PCBs is the sum 

of all congeners. Total chlordane is the sum of cis-chlordane, trans-chlordane, cis-nonachlor, trans-nonachlor, heptachlor, 

heptachlor epoxide and oxychlordane. The maximum geometric station mean is shown in bold for each contaminant. 

Stations are listed from upstream to downstream.—Continued

Station p,p’-DDE Dieldrin Total PCBs Toxaphene Total chlordane

Imperial Dam, AZ (322) Mean 0.043 0.0002 0.038 <0.024 0.004

n = 4 Min 0.028 <0.0002 <0.048 <0.024 0.003

Max 0.078 0.0005 0.067 <0.024 0.004

Hayden, AZ (323) Mean 0.026 0.0002 0.031 0.017 0.003

n = 4 Min 0.016 <0.0002 <0.048 <0.024 0.002

Max 0.047 0.0003 0.070 0.049 0.006

Phoenix, AZ (324) Mean 0.467 0.0152 1.30 0.09 0.070

n = 4 Min 0.280 0.0109 1.00 <0.024 0.046

Max 0.780 0.0220 2.10 0.50 0.122

Arlington, AZ (325) Mean 1.758 0.0082 0.13 0.65 0.040

n = 4 Min 1.300 0.0061 0.12 0.50 0.026

Max 2.700 0.0102 0.18 0.87 0.062

Results and Discussion    41
tions (table 14), and concentrations were >0.01 μg/g in carp 

from Stations 315, 324, and 325 and channel catfish from 

Station 324. Trans-chlordane was detected in 41 of 52 (79%), 

and concentrations were >0.01 μg/g in carp from Stations 315 

and 324 and channel catfish from Station 324 (table 14). Cisnonachlor was detected in most (98%) samples, but concentrations >0.01 μg/g were only measured in carp from Station 315 

and channel catfish from Station 324. Trans-nonachlor was 

detected in all samples, and concentrations were >0.01 μg/g 

in carp from Stations 315, 316, and 324, bass from Station 

325, and channel catfish from Stations 316 and 324 (table 14). 

Concentrations of total chlordanes (sum of seven compounds) 

ranged from 0.002 to 0.12 μg/g, and concentrations were 

greatest in fish from Stations 315, 316, 320, 324, and 325 

(figs. 8 & 9). Mean total chlordane concentrations were greatest at Stations 324 (0.07 μg/g) and 325 (0.04 μg/g; table 15). 

Trans-nonachlor, trans-chlordane, and cis-chlordane were the 

primary constituents of total chlordanes at these sites (fig. 9).

Historical NCBP concentrations of total chlordane were 

<0.1 μg/g in fish from 1976 to 1986, and most concentrations 

for individual constituents were <LOD (Schmitt and others, 

1999). Other studies reported that total chlordane concentrations were relatively low in agricultural areas of the Lower GR 

(Kepner, 1987; King and others, 1997) and in irrigation drainages of the Yuma Valley (Baker and others, 1992) and Middle 

Green River Basin (Stephens and others, 1988). Most total 

chlordane concentrations were <0.1 μg/g in carp, bass, and 

channel catfish from previous LRMN investigations (Hinck 

and others, 2004a; Schmitt and others, 2002, 2004), but mean 

basin concentrations were generally lower in the CDRB than 

the CRB, MRB, and RGB (table 16). Total chlordane concentrations >0.3 μg/g are of concern for the health of predatory 

fish and fish-eating birds (Eisler, 1990). Fish and wildlife are 

not at risk from chlordane in the CDRB.

Dieldrin

Most environmental dieldrin is present due to the breakdown of aldrin, which has not been used in the U.S. since 1974 

and was not detected in CDRB samples (table 14). Concentrations of dieldrin were detected in 47 of 52 (90%) samples 

representing all stations (table 14). Concentrations were >0.01 

μg/g in carp from Stations 315 and 324, bass from Station 

325, and channel catfish from Station 324 (fig. 8). Trace 

concentrations (0.001–0.01 μg/g) were detected in samples 

from Stations 314, 315, 316, 320, and 321 (fig. 8). All mean 

concentrations were ≤0.008 μg/g except for Station 324 (0.015 

μg/g; table 15).

Most concentrations of dieldrin were ≤LOD (0.1 μg/g) 

in carp, bass, and channel catfish from NCBP stations in the 

CDRB from 1976 to 1986 (Schmitt and others, 1999), and 

dieldrin concentrations were ≤0.03 μg/g in fish from the Yuma 

Valley (Baker and others, 1992). Dieldrin concentrations were 

<LOD (~0.1 μg/g) in fish from irrigation drainages in the 

Middle Green River Basin (Stephens and others, 1988). Dieldrin was also rarely detected in carp, bass, and channel catfish 

in the Lower GR although concentrations were 0.01-0.09 μg/g 

in bass collected downstream of Phoenix (King and others, 

1997). Previous LRMN studies from the MRB (Schmitt and 

others, 2002), RGB (Schmitt and others, 2004), and CRB 

(Hinck and others, 2004a) reported low concentrations of 

dieldrin in most carp (<0.01-0.25 μg/g), bass (<0.01-0.08 

μg/g), and channel catfish (<0.01-0.05 μg/g). Whole-body 

concentrations of 0.36-2.13 μg/g in juvenile rainbow trout 

did not effect survival or growth, but concentrations of 5.65 

μg/g reduced survival (Shubat and Curtis, 1986; Macek and 

others, 1970 as cited in Jarvinen and Ankley, 1999). Dieldrin 

concentrations in CDRB fish samples are unlikely to represent a significant threat to either fish or piscivorous wildlife 

(Jarvinen and Ankley, 1999; Peakall, 1996).

Station

311312313314315316317319320321322323324325

Total chlordane (

μg/g)

0.00

0.02

0.04

0.06

heptachlor 

heptaclor epoxide 

oxychlordane 

cis-chlordane 

trans-chlordane

cis-nonachlor 

trans-nonachlor 

Total DDT (

μg/g)

0.0

0.4

0.8

1.2

1.6

2.0

p,p'-DDE 

p,p'-DDD 

p,p'-DDT 

Upper CDRB Lower CDRB

Figure 9. Unweighted geometric mean concentrations (μg/g 

ww) of total DDT (p,p’-DDT, DDE, and DDD) and chlordanerelated compounds (cis- and trans-chlordanes and nonachlors, 

heptachlor, heptachlor epoxide, and oxychlordane) by station 

in whole-body fish composite samples from the Colorado River 

Basin in 2003. Censored values are represented by one half the 

LOD in the computation of means and totals but are not shown in 

the figure. Stations are ordered from upstream to downstream 

and are grouped by sub-basin. See Table 2 for station 

descriptions.

42    Contaminants, Health Indicators, and Reproductive Biomarkers in Fish from the Colorado River Basin
Table 16. Unweighted geometric mean, minimum (Min), and maximum (Max) concentrations [all μg/g ww except TCDD-EQ (pg/g)] of select organochlorine 

chemical contaminants in piscivorous (Pisc) and benthivorous (Ben) fish from BEST-LRMN studies including the Colorado River Basin (CDRB), Columbia River Basin (CRB; Hinck and others, 2004a), Mississippi River Basin (MRB; Schmitt, 2002a), Rio Grande Basin (RGB; Schmitt and others, 2004), and Yukon River Basin (YRB; Hinck and others, 2004b). Censored values were replaced by one-half the LOD for the computations of basin means. Total DDT is the sum of p,p’-DDD, p,p’-DDE, and p,p’-DDT. Total chlordane is the sum of cis-chlordane, trans-chlordane, cis-nonachlor, trans-nonachlor, heptachlor epoxide, and oxychlordane. Total PCBs is the sum of all congeners. 1 Piscivorous fish samples sizes for all organochlorine chemical contaminants were n = 12 for the CDRB, n = 14 for the CRB, n = 34 for the MRB, n = 9 for the RGB, and n = 10 for the YRB. 2 Benthivorous fish samples sizes for all organochlorine chemical contaminants were n = 14 for the CDRB, n = 16 for the CRB, n = 46 for the MRB, n = 10 for the RGB, and n = 5 for the YRB.

p,p’-DDE Total DDT Total Chlordane Total PCBs Toxaphene TCDD-EQ

Basin Pisc 1 Ben 2 Pisc Ben Pisc Ben Pisc Ben Pisc Ben Pisc Ben

CDRB 

Min 0.001 0.004 0.003 0.008 0.002 0.003 <0.024 <0.024 <0.050 <0.050 <0.01 <0.01Max 2.700 1.700 1.740 2.770 0.121 0.121 2.100 1.600 0.870 0.740 6.00 6.00Mean 0.040 0.049 0.047 0.060 0.009 0.012 0.042 0.066 0.020 0.019 0.15 0.13

CRB

Min <0.010 <0.010 0.015 0.015 0.030 0.030 <0.030 <0.030 <0.030 <0.030 0.00 0.00Max 1.200 1.100 1.390 1.310 0.072 0.125 1.300 0.750 <0.030 0.050 8.00 43.00Mean 0.144 0.167 0.185 0.220 0.033 0.034 0.076 0.080 <0.030 0.016 2.40 1.97

MRB

Min <0.010 <0.010 0.015 0.015 0.030 0.030 <0.050 <0.050 <0.050 <0.050 <0.21 <0.40Max 0.530 8.300 0.840 11.110 0.363 0.527 2.000 3.300 0.740 8.300 62.00 68.00Mean 0.037 0.079 0.061 0.120 0.042 0.054 0.138 0.129 0.028 0.041 3.00 3.40

RGB

Min <0.010 0.012 0.015 0.022 0.030 0.030 <0.050 <0.050 <0.050 <0.050 <0.40 <0.35Max 1.600 0.670 1.686 0.700 0.211 0.051 <0.050 <0.050 2.400 0.670 6.00 3.00Mean 0.075 0.100 0.103 0.121 0.041 0.033 <0.050 <0.050 0.044 0.034 1.70 1.20

YRB

Min <0.001 <0.001 0.001 0.001 <0.001 <0.001 <0.010 0.024 <0.020 0.020 <1.70 <1.70Max 0.004 0.005 0.014 0.013 0.007 0.005 0.063 0.087 0.034 0.019 <1.70 <1.70Mean <0.0010 <0.001 0.002 0.002 0.002 0.003 0.027 0.037 0.010 0.008 <1.70 <1.70

Results and Discussion    43
Endrin

Endrin, one of the most toxic organochlorine pesticides 

to fish (Johnson and Finley, 1980), was used on comparatively 

few crops historically. Endrin was detected in 51 of 52 (98%) 

samples (table 14), and concentrations were >0.05 μg/g in carp 

and channel catfish from Station 324 and carp and bass from 

Station 325 (fig. 8). Only trace endrin concentrations (<0.01 

μg/g) were measured at historical NCBP sites in the CDRB 

(Schmitt and others, 1999), and concentrations in fish from the 

Middle Green River were <LOD (Stephens and others, 1988). 

Previous LRMN investigations reported non-detected or trace 

concentrations (0.01 μg/g) of endrin in carp, bass, and channel 

catfish (Hinck and others, 2004a; Schmitt and others, 2002, 

2004). Toxicity studies documenting whole-body concentrations of endrin in fish were not found; therefore, the total risk 

to fish and wildlife represented by concentrations of endrin is 

unknown.

Mirex

Mirex was used as an insecticide to combat red imported 

fire ants (Solenopsis wagneri) in the southern U.S. Elsewhere, 

mirex was also used as a flame retardant and as a polymerizing 

agent (Kaiser, 1987). Mirex was detected in 48 of 52 (92%) 

samples (table 14). All concentrations were ≤0.001 μg/g, and 

the greatest concentrations were in fish from Stations 313 and 

314 (fig. 8). Concentrations were not detected in NCBP samples in the CDRB (Schmitt and others, 1999). Other LRMN 

studies also reported low concentrations of mirex (most <0.01 

μg/g; Hinck and others, 2004a, 2004b; Schmitt and others, 

2002, 2004). Toxicity studies reporting whole-body concentrations of mirex in fish were not found; therefore, the total 

risk to fish and wildlife represented from mirex is unknown.

Toxaphene

Toxaphene was the most heavily used insecticide in the 

U.S. following the ban on DDT (Schmitt and Winger, 1980). 

Use of toxaphene in the U.S. peaked in the late 1970s, and 

the pesticide was subsequently banned. Historical NCBP 

concentrations of toxaphene in fish samples reflected use, 

which peaked in the mid-1970s (Schmitt and others, 1999). 

Although toxaphene was used mostly on cotton, this pesticide 

has been atmospherically transported to remote locations 

and residues have been detected in fish from the Arctic and 

the Great Lakes (Muir and others, 1999; Schmitt and others, 

1999). Toxaphene was detected (>0.024 μg/g) in samples 

from Stations 323, 324, and 325 (fig. 8; table 15).

Previous studies have documented toxaphene concentrations in fish from agricultural areas of the Lower CDRB. The 

historical NCBP concentrations of toxaphene were generally 

low (≤0.1 μg/g) in carp, bass, and channel catfish although 

relatively high concentrations were measured in carp from 

the GR near Station 323 (<0.01-0.8 μg/g) and largemouth 

bass from the Lower CDR near Station 322 (<0.01-1.0 μg/g; 

Schmitt and others, 1999). Concentrations were <LOD in 

carp from the Middle Green River Basin (Stephens and others, 1988). Relatively high toxaphene concentrations (>5.0 

μg/g) were reported in agricultural areas of the Lower GR in 

the mid-1980s (Kepner, 1987). A subsequent study reported 

lower concentrations in much of this area, but concentrations 

remained relatively high (>1.5 μg/g) in channel catfish (King 

and others, 1997). Toxaphene concentrations (≤0.38 μg/g) in 

fish from irrigation drainages in the Yuma Valley were less 

than protective criteria for fish (0.40 μg/g; Baker and others, 

1992). The greatest toxaphene concentrations in the CDRB 

were found in cotton producing areas of the Lower GR, which 

is similar to the relatively high concentrations (>2.0 μg/g) 

reported in carp from cotton producing regions of the MRB 

(Schmitt and others, 2004). Total toxaphene concentrations 

were generally greater in CDRB fish than those from the RGB 

and YRB (table 16).

Acute and chronic effects of toxaphene on freshwater 

fish have been reported at whole-body concentrations ≥0.4 

μg/g (Eisler and Jacknow, 1985; Jarvinen and Ankley, 1999). 

Jarvinen and Ankley (1999) cited a number of laboratory 

studies on the acute and chronic effects of toxaphene. Adult 

brook trout containing whole-body concentrations of 0.4 μg/g 

produced eggs with reduced viability, and lake trout (Salvelinus namaycush) and white sucker containing 0.035-0.203 μg/g 

also produced eggs with reduced viability (Mayer and others, 

1975). Survival and growth of several freshwater fish species 

at various life stages were reduced at concentrations >0.90 

μg/g (Mayer and others, 1975, 1978). Concentrations of toxaphene were >0.4 μg/g in fish from Stations 324 and 325. Fish 

and wildlife from these sites may be at risk from toxaphene.

Hexachlorobenzene (HCB)

HCB was produced for use as a fungicide and was a byproduct of the production of other chlorinated hydrocarbons. 

This compound is less toxic to fish than many other persistent 

organochlorines but contains toxic impurities including polychlorinated dibenzo-p-dioxins and polychlorinated dibenzop-furans (Schmitt and others, 1999; Villanueva and others, 

1974). HCB was detected at trace concentrations in most 

samples (94%; table 14). The greatest concentrations (>0.002 

μg/g) were measured in carp from Stations 314 and 324 and 

channel catfish from Station 324 (fig. 8). Historical NCBP 

concentrations of HCB were <0.01 μg/g (Schmitt and others, 

1999), and previous LRMN investigations generally reported 

concentrations ≤0.01 μg/g in carp, bass, and channel catfish 

(Hinck and others, 2004a; Schmitt and others, 2002, 2004). 

Concentrations of HCB were ≤27 μg/g in whole fish from 

monitoring studies across the U.S. (Nowell and others, 1999). 

Protective criteria for HCB are limited. Concentrations as 

low as 0.33 μg/g in whole fish have been suggested to protect 

piscivorous wildlife (Newell and others, 1987), although HCB 

concentrations in CDRB fish were well below this benchmark.

44    Contaminants, Health Indicators, and Reproductive Biomarkers in Fish from the Colorado River Basin
Pentachlorobenzene

Pentachlorobenzene is used as a precursor in the synthesis of the fungicide pentachloronitrobenzene and as a fire 

retardant. Pentachlorobenzene can enter aquatic systems 

through industrial discharge and as a degradation product of 

other organochlorine compounds such as hexachlorobenzene 

(Barber and others, 1997). Pentachlorobenzene was detected 

in 34 of 52 samples (65%; table 14), but concentrations were 

<0.0002 μg/g in most samples (fig. 10). Concentrations 

were 0.00022-0.00041 μg/g in samples from Station 325 and 

0.00041-0.00089 μg/g in samples from Station 324 (fig. 10). 

Histological lesions in the kidney, liver, and thyroid have been 

associated with pentachlorobenzene exposure in mice and rats 

(McDonald, 1991). The risk of pentachlorobenzene to fish 

and wildlife in the CDRB is unknown.

Pentachloroanisole (PCA)

PCA, a metabolite of the wood preservative pentachlorophenol (PCP), is toxic and potentially carcinogenic (National 

Research Council of Canada (NRCC), 1982; Schmitt and others, 1999). Early formulations of PCP contained chlorinated 

dioxins and other toxic impurities (Schmitt and others, 1999). 

PCA was detected in 49 of 52 samples (94%; table 14), and 

concentrations were >0.01 μg/g in carp from Stations 312 and 

316 and channel catfish from Station 324 (fig. 10). Concentrations of PCA were ≤LOD (0.005 μg/g) in all NCBP samples 

from the CDRB (Schmitt and others, 1999). The risk of PCA 

to fish and wildlife in the CDRB is unknown.

Hexachlorocyclohexane (HCH)

Four HCH isomers (α-, β-, γ-, δ- HCH) were measured 

in the CDRB samples. Although a mixture of isomers was 

historically used on cotton and other crops in the U.S., only γHCH (lindane) is still used in North America for some agricultural and domestic applications. However, lindane is not heavily used in the CDRB (USGS, 2003). HCH isomers, which 

are relatively short-lived, were detected at trace concentrations 

in most samples (94%; table 14). γ-HCH concentrations were 

>0.004 μg/g in all samples from Station 324 (fig. 10). Concentrations of α- and γ- HCH were ≤LOD (0.01 μg/g) in most 

samples collected by the NCBP (Schmitt and others, 1999) 

and previous LRMN studies (Hinck and others, 2004a, 2004b; 

Schmitt and others, 2002, 2004). γ-HCH concentrations were 

≤0.12 μg/g in whole fish from monitoring studies across the 

U.S. (Nowell and others, 1999). Histopathological alterations 

in the gill, liver, and kidney of freshwater fish have been associated with γ-HCH contamination (Ortiz and others, 2003). 

Concentrations of γ-HCH <0.10 μg/g in whole fish have been 

suggested to protect piscivorous wildlife (Newell and others, 

1987). Concentrations in CDRB fish were well below this 

benchmark. 

Dacthal

Dacthal remains registered as a broad-spectrum herbicide 

for use on ornamental plants, turf, and vegetable and field 

crops (Cox, 1991; Schmitt and others, 1999) and is heavily 

used in the GRB (USGS, 2003). The technical product of 

the dacthal can contain 2,3,7,8-TCDD and HCB as impurities (Cox, 1991). Dacthal was detected in 24 of 52 samples 

(46%; table 14), and similar to γ- HCH, dacthal concentrations 

were greatest (0.005-0.009 μg/g) in fish from Station 324 (fig. 

10). Dacthal concentrations were ≤0.01-0.06 μg/g in NCBP 

samples from the CDRB (Schmitt and others, 1999). The risk 

of dacthal to fish and wildlife in the CDRB is unknown.

Endosulfan

Endosulfan is a broad spectrum insecticide used on a 

wide variety of vegetables, fruits, cereal grains, cotton, and 

ornamental plants and is heavily used in the Lower CDRB 

(USGS, 2003). Technical-grade endosulfan contains two pure 

isomers, endosulfan I and II. Endosulfan sulfate is a reaction 

product of technical endosulfan and can be found in organisms as a result of oxidation of endosulfan I and II (Agency 

for Toxic Substances and Disease Registry (ATSDR), 2000). 

Most endosulfan I concentrations were <0.001 μg/g, and the 

greatest concentrations were in carp from Stations 316 and 

323 (fig. 10). Endosulfan II was detected in 24 of 52 samples 

(46%; table 14), and concentrations were <0.01 μg/g in all 

samples except those from Station 325 (fig. 10). Endosulfan 

sulfate was detected in most (96%) samples, and like endosulfan II, the greatest concentrations (>0.02 μg/g) were in fish 

from Station 325 (fig. 10). Endosulfan concentrations were 

≤0.17 μg/g in whole fish from monitoring studies across the 

U.S. (Nowell and others, 1999). Amphibians, fish, birds, and 

mammals treated with endosulfan exhibited developmental 

and reproductive effects typically associated with endocrine 

disrupting chemicals in previous studies (Dutta and others, 

2006; USEPA, 2002). Dutta and others (2006) reported that 

significant damage to testicular tissue in adult bluegill after 

exposure to endosulfan may have deleterious effects on spermatogenesis and male fertility. Endosulfan exposure impaired 

genital tract development in birds (30-120 μg/g) and reduced 

hormone levels and sperm production in mammals (15-75 

μg/g; USEPA, 2002). Effects in fish were observed at much 

lower concentrations, and growth and survival were the most 

sensitive endpoints (USEPA, 2002). Reproductive effects 

including decreased GSI, reduced oocyte size, and increased 

oocyte atresia were documented in tilapia (Sarotherodon mossambicus) after exposure to endosulfan (0.001 μg/g; Shukla 

and Pandey, 1986). Endosulfan concentrations in fish from 

Stations 316, 323, 324, and 325 exceeded this threshold. 

Methoxychlor

Methoxychlor, a derivative of DDT, is an insecticide used 

on field crops, vegetables, fruits, stored grain, livestock, and 

domestic pets (ATSDR, 2002). Historical pesticide applicaResults and Discussion    45
Pentachloroanisole (

μg/g)

0.000

0.005

0.010

0.015

0.020

γ-HCH (

μg/g)

0.000

0.002

0.004

0.006

0.008

Station

 311 312 313 314 315 316 317 319 320 321 322 323 324 325

Dacthal (

μg/g)

0.000

0.002

0.004

0.006

0.008

0.010

Pentachlorobenzene (

μg/g)

0.0000

0.0002

0.0004

0.0006

0.0008

0.0010

Common carp

Bass

Channel catfish

White sucker

Brown trout

Upper CDRB Lower CDRB

Station

0.000

0.001

0.002

0.003

0.004

0.005

0.00

0.02

0.04

0.06

0.00

0.02

0.04

0.06

0.08

Station

 311 312 313 314 315 316 317 319 320 321 322 323 324 325

0.000

0.002

0.004

0.006

0.008

0.010

Upper CDRB Lower CDRB

Endosulfan I (

μg/g) Endosulfan II (

μg/g) Endosulfan sulfate (

μg/g) Methoxychlor (

μg/g)

Figure 10. Concentrations (μg/g ww) of unlisted or restricted use pesticides or pesticide products including pentachlorobenzene, 

pentachloroanisole, λ-BHC (lindane), dacthal, endosulfan I, endosulfan II, endosulfan sulfate, and methoxychlor by station and taxon in 

whole-body fish composite samples from the Colorado River Basin in 2003. Censored values are plotted as one half the LOD. Stations 

are ordered from upstream to downstream and are grouped by sub-basin. See Table 2 for station descriptions.

46    Contaminants, Health Indicators, and Reproductive Biomarkers in Fish from the Colorado River Basin
tion records indicate that methoxychlor is not heavily used in 

the CDRB (USGS, 2003). Methoxychlor can bioaccumulate 

in fish, insects, and mammals although it is not as persistent 

as DDT. Methoxychlor was detected in 11 of 52 samples 

(21%; table 14), and concentrations were <0.001 μg/g in most 

samples (fig. 10). Like many of the other pesticides measured in this study, methoxychlor concentrations were greatest (>0.002 μg/g) in fish from Stations 324 and 325 (fig. 10). 

Methoxychlor can be weakly estrogenic in fish although not 

all the effects of methoxychlor in aquatic wildlife are mediated through the estrogen receptor (ATSDR, 2002; Versonnen 

and others, 2004). Methoxychlor concentrations were low in 

CDRB fish, but the risk of methoxychlor exposure to fish and 

wildlife in the CDRB is unknown.

Total PCBs, H4IIE-Derived Dioxin Equivalents and 

Ethoxyresorufin O-Deethylase (EROD) Activity

Total PCBs

PCBs, mixtures of 209 chlorinated compounds, were 

used as coolants and lubricants in electrical capacitors and 

transformers, for pressure treating lumber, and paper manufacturing until banned in the U.S. in 1977. Total PCBs concentrations were >LOD (0.048 μg/g) in 22 of 52 samples (42%) 

from nine stations (table 14). Concentrations were 0.05-2.1 

μg/g, with the maximum concentration in female channel 

catfish from Station 324 (fig. 11; table 14). Concentrations 

were relatively high (0.8 μg/g) in carp from Stations 320 

and 324 and channel catfish from Station 324 (fig. 11), and 

mean concentrations were greatest at Stations 320 (1.2 μg/g) 

and 324 (1.3 μg/g; table 15). Concentrations of PCBs were 

significantly greater in carp from Stations 320 and 324 and 

channel catfish from Station 324; concentrations did not differ 

significantly among stations in bass (table 12).

Previous studies reported low PCB concentrations in 

the CDRB. Concentrations (as Aroclor mixtures 1248, 1254, 

1260) at NCBP sites were historically <LOD (0.1 μg/g) in the 

CDRB (Schmitt and others, 1999). Aroclor concentrations 

<0.1 μg/g were reported in carp from the CDR near Stations 

312, 321, and 322 in previous studies (Radtke and others, 

1988; Stephens and others, 1988). King and others (1997) 

measured total PCBs in carp (<0.05-0.65 μg/g), bass (<0.05-

0.70 μg/g), and channel catfish (<0.05-0.17 μg/g) in the GR 

and concluded that concentrations were generally greatest 

downstream of the urban and industrial areas of Phoenix. 

Concentrations of PCBs were <0.05-3.3 μg/g in carp and 

0.05-2.0 μg/g in bass in the MRB (Schmitt and others, 2002), 

<0.03-0.45 μg/g in carp and <0.03-0.64 in bass in the CRB 

(Hinck and others, 2004a), and were <LOD (<0.05 μg/g ) in 

the RGB (Schmitt and others, 2004). Total PCBs concentrations in CDRB fish were similar to those from other LRMN 

studies (table 16).

The New York State Department of Environmental 

Conservation (NYSDEC) wildlife guideline for total PCBs 

in fish is 0.11 μg/g (Newell and others, 1987), a concentration exceeded by all samples from Stations 320, 324, and 325 

(fig. 11). The toxicity of individual PCB congeners ranges 

over several orders of magnitude (Ahlborg and others, 1994; 

van den Berg and others, 1998) and varies with the endpoint 

being considered (Hansen, 1998). Survival of fry decreased 

at approximately 5 μg/g of Aroclor 1254 in several laboratory 

studies (Hansen and others, 1973; Schimmel and others, 1974 

as cited in Monosson, 2000). Concentrations of PCBs exceeding 100 μg/g in fish tissues can affect reproduction in females 

or be lethal, and concentrations of 50 μg/g can reduce growth 

and survival in offspring (Niimi (1996) as cited in Beyer and 

others, 1996). These concentrations may be lower in more 

sensitive fish species (Niimi, 1996). Inferior reproductive performance and offspring survival were found in mink fed Great 

Lakes fish or fish products with PCB concentrations of 0.48 

μg/g (Hornshaw and others, 1983). Concentrations of PCBs 

in CDRB fish were generally low, but concentrations in fish 

from Stations 320 and 324 were elevated and could be a risk to 

piscivorous wildlife.

H4IIE Bioassay

TCDD-EQs were generally low and detected in only 6 

of 52 samples (12%) from three stations (fig. 11). Concentrations of TCDD-EQs were >2 pg/g in male channel catfish 

from Station 316, male carp from Station 320, and carp and 

channel catfish from Station 324. Relatively high PCB concentrations were also measured in samples from Station 324 

(fig. 11). 

Previous studies have examined TCDD-EQ concentrations in fish, but data for CDRB fish is limited. The dietary 

toxicity threshold for TCDD is 4.4 pg/g in mammals (Heaton 

and others, 1995; Tillitt and others, 1996) and 5 pg/g in avian 

wildlife (Nosek and others, 1992). Most concentrations in 

CDRB fish were similar to those reported in fish from reference sites in previous studies (Giesy and others, 1995; van 

den Heuvel and others, 1995). As a comparison, TCDD-EQ 

concentrations ≥60 pg/g were reported in the MRB, and in that 

basin those samples with concentrations ≥20 pg/g were widespread (Schmitt and others, 2002). TCDD-EQ concentrations 

were generally low (<8 pg/g) in carp and bass from the RGB 

and CRB (Hinck and others, 2004a; Schmitt and others, 2004). 

No CDRB sample approached 30 pg/g, which is the threshold 

for toxic effects in fish (Walker and others, 1996; Whyte and 

others, 2004). TCDD and octochlorodibenzo-p-dioxin were 

frequently detected in sediments from Lake Mead, and maximum concentrations (>20 pg/g) were measured in Las Vegas 

Bay (Covay and Beck, 2001). TCDD-EQ concentrations were 

relatively low in fish near Lake Mead (Station 319 and 320) in 

2003 samples. The TCDD-EQs in fish from Station 324 were 

exceeded toxicity thresholds; therefore, piscivorous wildlife 

from this area may be at risk from dioxin-like compounds.

Results and Discussion    47
Ethoxyresorufin O-Deethylase (EROD) Activity

Many factors including species, gender, and gonadal 

stage affect hepatic EROD activity (Whyte and others, 2000). 

An ANOVA model which included station (location), gender, 

gonadal stage, and their interactions was significant in carp 

(F41, 207 = 3.45, P < 0.01) and bass (F23, 92 = 3.78, P <0.01), 

but not channel catfish (F21, 63 = 1.17, P > 0.05; Appendix 6). 

Genders were analyzed and reported separately in all three 

taxa to maintain comparability with data from previous LRMN 

studies and the scientific literature.

EROD in Carp

Mean EROD activity was greatest (9.05 pmol/min/mg) 

in female carp from Station 324, with individual activities 

ranging from 1.67 to 44.4 pmol/min/mg (fig. 12; table 17). 

Other station means were ≤4.37 pmol/min/mg. Schlenk and 

others (1996) determined basal EROD activity in carp from 

uncontaminated sites to be 0-5 pmol/min/mg, which is similar 

to basal activity (0-4 pmol/min/mg) determined for carp by 

Schmitt and others (2002). Hepatic EROD activities were 

>5 pmol/min/mg in individual female carp from all stations 

except Stations 315, 317, 322, and 323, but activities >10 

pmol/min/mg in individual females were rare (fig. 12).

Mean EROD activity was greatest (10.7 pmol/min/mg) in 

male carp from Station 324, with individual activities ranging 

from 5.04 to 41.7 pmol/min/mg (fig. 12). Other station means 

ranged from 1.22 pmol/min/mg at Station 322 to 8.79 pmol/

min/mg at Station 313. Individual activities exceeded basal 

levels (25 pmol/min/mg; Schlenk and others, 1996; Schmitt 

and others, 2002) in male carp from several stations, and activity in males from Stations 313, 315, 316, 317, 320, 324, and 

325 was >10 pmol/min/mg (fig. 12).

Hepatic EROD activity in carp was measured in other 

studies including previous LRMN investigations. However, 

EROD studies for carp in the CDRB were not found. Mean 

EROD activity in female carp from the CDRB (0.67-9.05 

pmol/min/mg) were similar to those from the RGB (0.25-16.8 

pmol/min/mg; Schmitt and others, 2004) and CRB (0.27-

10.3 pmol/min/mg; Hinck and others, 2004a). Mean EROD 

activity in male carp was 0.34-32.6 pmol/min/mg in the RGB 

(Schmitt and others, 2004) and 0.89-10.6 pmol/min/mg in the 

CRB (Hinck and others, 2004a), which were similar to those 

in the CDRB (1.22-10.7 pmol/min/mg). In laboratory studies, EROD activities were 7.1-25 pmol/min/mg in reference 

juvenile carp (Kosmala and others, 1998; Taysee and others, 

1998; Marionnet and others, 1997, 1998) and 2.7-41.9 pmol/

min/mg in reference adult carp (Deér and others, 1996; Solé 

and others, 2000). Overall, mean hepatic EROD activity was 

generally highest in carp from Station 324. Relatively high 

PCB and TCDD-EQ concentrations, which are known AhR 

agonists, were also measured in carp from Station 324.

EROD in bass

Hepatic EROD activity was generally greater in bass than 

carp. Mean EROD activity was greatest (13.2 pmol/min/mg) 

in female bass from Station 312, with individual activities 

ranging from 0.03 to 66.1 pmol/min/mg (fig. 12; table 17). 

Other station means ranged from 2.50 pmol/min/mg at Station 

323 to 11.6 pmol/min/mg at Station 315 (table 17). Adams 

and others (1994) determined basal EROD activity for female 

bass to be 0-5 pmol/min/mg, which is less than basal EROD 

activity (0-16 pmol/min/mg) determined for female bass from 

the MRB (Schmitt and others, 2002). Activities >16 pmol/

min/mg were measured in fish from Stations 312, 315, 321, 

and 325.

Mean EROD activity was greatest (61.2 pmol/min/mg) in 

male bass from Station 312, and individual activities ranged 

from 39.8 to 104.5 pmol/min/mg (fig. 12; table 17). Other station means were ≤23.4 pmol/min/mg (table 17). EROD activities in male bass were greater in bass from the Upper CDRB 

than those from the Lower CDRB (fig. 12).

Hepatic EROD activity in bass has been reported by 

previous LRMN investigations and other studies, but EROD 

data specific to the CDRB were not found. Adams and others 

Total PCBs (

μg/g)

0.0

0.5

1.0

1.5

2.0

Station

 311 312 313 314 315 316 317 319 320 321 322 323 324 325

TCDD-EQ (pg/g)

0

2

4

6

Common carp

Bass

Channel catfish

White sucker

Brown trout

Upper CDRB Lower CDRB

Figure 11. Concentrations of total PCB (μg/g ww) and H4IIE 

bioassay-derived TCDD-EQ (pg/g) by station and taxon in wholebody fish composite samples from the Colorado River Basin in 

2003. Stations are ordered from upstream to downstream and are 

grouped by sub-basin. See Table 2 for station descriptions.

48    Contaminants, Health Indicators, and Reproductive Biomarkers in Fish from the Colorado River Basin
(1994) determined basal EROD activity for male bass to be 

0-17 pmol/min/mg, which is slightly less than basal EROD 

activity (0-22 pmol/min/mg) determined for male bass by 

Schmitt and others (2002). Mean EROD activities in female 

bass from the CDRB (2.50-13.2 pmol/min/mg) were lower 

than those from the RGB (21.3-108 pmol/min/mg; Schmitt 

and others, 2004) and CRB (5.21-40.4 pmol/min/mg; Hinck 

and others, 2004a). In male bass, mean EROD activities 

were 17.0-75.9 pmol/min/mg in the RGB (Schmitt and others, 2004) and 6.9-68.3 pmol/min/mg in the CRB (Hinck and 

others, 2004a), which were also similar to those in the CDRB 

(6.70-61.2 pmol/min/mg). Activity was >22 pmol/min/mg 

in bass from Stations 311, 312, 315, 321, and 325. Overall, 

mean hepatic EROD activity was generally highest in bass 

from Station 312. PCB and TCDD-EQ concentrations were 

low in bass from Station 312, which indicates induced hepatic 

EROD activity may have been caused by another AhR agonist 

(for example, PAH).

EROD in channel catfish

Hepatic EROD activity in channel catfish was similar to 

those in bass. Mean activity was greatest (11.2 pmol/min/mg) 

in female channel catfish (n = 2) from Station 315 (fig. 12; 

table 17). Other station means ranged from 5.08 pmol/min/mg 

at Station 316 to 8.90 pmol/min/mg at Station 313 (table 17). 

Mean EROD activity in male channel catfish was comparable 

to females with the greatest activity (10.8 pmol/min/mg) in 

males from Station 312, and other station means were <6.9 

pmol/min/mg (fig. 12; table 17).

Hepatic EROD activity was measured in channel catfish 

from the MRB and RGB. Mean EROD activities in RGB 

channel catfish were 12.2-40.6 pmol/min/mg in females, 17.5-

20.3 pmol/min/mg in males, and 14.1-38.9 pmol/min/mg in 

juveniles (Schmitt and others, 2004). Activities were generally lower (0.4-14.2 pmol/min/mg) in channel catfish from the 

MRB (Schmitt and others, 2002) than those from the CDRB. 

More data is needed to determine whether EROD activity was 

elevated or within the normal range in channel catfish from the 

CDRB.

EROD in other species 

Hepatic EROD activity was also measured in flathead 

catfish, brown trout, and white sucker. Activity differed 

among fishes, and few studies have described basal activity in 

Figure 12. Hepatic microsomal EROD activity (pmol/min/mg) 

by station in female and male carp, bass (Micropterus sp.), and 

channel catfish from the Colorado River Basin in 2003. Shown for 

each group are points representing individual fish and the mean 

(red horizontal line), median (black horizontal line), interquartile 

range (box), and the 10th and 90th percentiles (whiskers). 

Stations are ordered from upstream to downstream and are 

grouped by sub-basin. See Table 2 for station descriptions.

Female carp

pmol/min/mg

0.001

0.01

0.1

1

10

100

1000

Male carp

pmol/min/mg

0.01

0.1

1

10

100

Female bass

pmol/min/mg

0.01

0.1

1

10

100

Male Bass

pmol/min/mg

1

10

100

Female channel catfish

pmol/min/mg

2

4

10

20

Male channel catfish

Station

 311 312 313 314 315 316 317 319 320 321 322 323 324 325

pmol/min/mg

2

4

10

20

Upper CDRB Lower CDRB

Results and Discussion    49
Table 17. Geometric mean, sample size (n), minimum (Min), and maximum (Max) of microsomal EROD activity (pmol/mg/min protein) 

in fish collected in the Colorado River Basin in 2003. Censored values were represented by one half the LOD in the computation of 

geometric means. Fish in which gender was undetermined or no gonad was obtained are listed as NG. The maximum geometric 

station mean is shown in bold for each taxon. Stations are ordered upstream to downstream. --, not applicable.

Taxon and station

Female Male NG

n Mean Min Max n Mean Min Max n Mean Min Max

Carp

Lay, CO (311) 0 -- -- -- 0 -- -- -- 1 0.9 -- --

Ouray NWR, UT (312) 11 0.7 0.03 5.3 9 1.5 0.03 5.0 0 -- -- --

San Rafael, UT (313) 10 3.4 0.9 13.9 11 8.8 2.7 31.6 0 -- -- --

Delta, CO (314) 13 2.0 0.9 5.1 8 4.0 2.4 5.3 0 -- -- --

Loma, CO (315) 10 1.5 0.5 4.9 10 4.2 1.6 17.4 0 -- -- --

Gold Bar Canyon, UT (316) 10 1.0 0.2 8.1 10 3.8 1.2 10.2 0 -- -- --

Hogback Diversion, NM (317) 7 0.8 0.4 1.7 13 3.2 0.4 39.9 0 -- -- --

South Cove, AZ (319) 11 3.4 0.5 136.2 6 3.4 1.4 5.4 0 -- -- --

Willow Beach, AZ (320) 9 4.4 1.4 18.4 11 4.5 1.6 22.7 0 -- -- --

Needles, CA (321) 12 2.0 1.1 6.3 8 3.7 0.6 6.0 0 -- -- --

Imperial Dam, AZ (322) 12 1.0 0.4 1.8 8 1.2 0.2 7.4 0 -- -- --

Hayden, AZ (323) 6 1.3 0.9 3.2 13 2.0 0.3 6.9 0 -- -- --

Phoenix, AZ (324) 8 9.1 1.7 44.4 10 10.7 5.0 41.7 2 0.1 0.01 1.5

Arlington, AZ (325) 13 1.9 0.1 12.9 7 2.1 0.1 15.8 0 -- -- --

Bass

Lay, CO (311) 9 7.0 3.8 11.6 10 23.4 14.5 30.1 1 7.8 -- --

Ouray NWR, UT (312) 6 13.2 0.03 66.1 8 61.2 39.8 104.5 0 -- -- --

Loma, CO (315) 5 11.6 4.8 20.3 7 15.1 3.8 41.4 0 -- -- --

Needles, CA (321) 11 7.3 3.8 16.5 9 13.1 2.6 28.6 0 -- -- --

Imperial Dam, AZ (322) 10 4.5 1.4 12.5 10 3.5 1.2 9.8 0 -- -- --

Hayden, AZ (323) 4 2.5 1.4 10.1 5 6.3 2.6 14.7 0 -- -- --

Phoenix, AZ (324) 0 -- -- -- 2 4.4 2.8 6.7 0 -- -- --

Arlington, AZ (325) 11 9.0 4.2 27.6 9 9.4 2.2 27.8 0 -- -- --

Channel catfish

Ouray NWR, UT (312) 5 6.3 3.1 10.2 6 10.8 5.7 14.9 0 -- -- --

San Rafael, UT (313) 4 8.9 4.3 18.2 14 6.9 3.1 18.0 0 -- -- --

Loma, CO (315) 2 11.2 7.3 17.4 1 4.6 -- -- 2 4.8 3.2 7.3

Gold Bar Canyon, UT (316) 10 5.1 2.0 11.0 10 6.4 1.6 20.5 0 -- -- --

Hogback Diversion, NM (317) 11 7.5 3.8 17.3 8 6.8 4.0 13.2 1 5.7 -- --

Phoenix, AZ (324) 13 5.3 1.4 14.1 3 3.7 1.8 6.4 0 -- -- --

Flathead catfish

Hayden, AZ (323) 3 5.0 3.6 6.4 7 5.3 3.0 8.5 1 4.6 -- --

Brown trout

Delta, CO (314) 11 1.4 0.2 9.6 9 8.0 2.3 19.3 1 7.7 -- --

White sucker

Lay, CO (311) 11 3.4 1.2 12.9 8 27.4 11.5 111.4 0 -- -- --

50    Contaminants, Health Indicators, and Reproductive Biomarkers in Fish from the Colorado River Basin
these species (Whyte and others, 2000). Those for which such 

comparisons can be made are summarized below.

White sucker were collected from Station 311, and mean 

EROD activity was greater in males (27.4 pmol/min/mg) than 

in females (3.41 pmol/min/mg; table 17). Hepatic EROD 

activity was 5-15 pmol/min/mg in white sucker from reference 

sites in previous studies (Couillard and Hodson, 1996; Schrank 

and others, 1997; Whyte and others, 2000), and gender differences were not significant in any of these studies. Mean 

EROD activity was 35.2 pmol/min/mg in white sucker from a 

site contaminated by PCBs, PAHs, and heavy metals (Schrank 

and others, 1997). Mean EROD activity in brown trout was 

1.38 pmol/min/mg in females, 7.98 pmol/min/mg in males, 

and 7.66 pmol/min/mg in a juvenile from Station 314 (table 

17). These activities were similar to basal activities reported 

in brown trout from previous studies (Whyte and others, 

2000). Mean EROD activity in flathead catfish from Station 

323 was 5.02 pmol/min/mg in females, 5.25 pmol/min/mg in 

males, and 4.54 pmol/min/mg in a juvenile (table 17) and were 

similar to those measured in channel catfish. Other studies 

measuring EROD activity in flathead catfish were not found.

Accumulative Contaminants, H4IIE, and EROD 

Activity: Summary

Concentrations of most organochlorine residues and 

elemental contaminants measured in CDRB fish were low. 

Concentrations of Se and Hg exceeded toxicity thresholds in 

samples from multiple stations. Se naturally occurs in the 

Mancos shale of the Upper CDRB, and Se contamination has 

received much attention in the CDRB. Although the main 

source of Se is leaching in irrigation canals, Se is transported 

to the Lower CDR with the high silt and sediment load of the 

Upper CDR (Welsh and Maughan, 1994). Selenium concentrations were >1.0 μg/g in at least one sample from all stations 

except Station 323, which could be harmful to piscivorous 

wildlife (Lemly, 1996, 2002). Probable Hg sources in the 

CDRB include historical mining areas and associated processes, coal-fired power plants, and naturally mineralized 

soils. Mercury concentrations were >0.3 μg/g in channel 

catfish at Station 313 and may represent a threat to piscivorous 

birds (Barr, 1986). Mercury concentrations were >0.1 μg/g 

in at least one sample from Stations 311, 312, 313, 315, 316, 

317, 321, and 323 and may be hazardous to piscivorous mammals at these sites (Yeardley and others, 1998). 

Organochlorine concentrations of banned pesticides in 

fish from the Lower GRB exceeded wildlife threshold criteria, 

which confirmed previous findings. Concentrations of p,p’-

DDE in all samples from Station 325 (>1.0 μg/g) and channel 

catfish from Station 324 (>0.5 μg/g) may pose a risk to fish 

and wildlife (Blus, 1996; Jarvinen and Ankley, 1999). Little 

or no p,p’-DDT was detected at these stations, indicating the 

continued weathering of residual DDT rather than the input 

of new material. However, relatively high concentrations of 

o,p’-homologs were initially reported in samples from Stations 324 and 325. After comparing sample chromatographic 

peaks with PBDE standards, we determined that several PBDE 

congeners were interfering with o,p’-DDT and p,p’-DDD in 

these samples. An additional cleanup step and GC-MS were 

needed to accurately quantify o,p’- and p,p’-homologs in 

samples from Stations 324 and 325 only. The PBDE congeners were not quantified for this report. Similar to p,p’-DDE, 

toxaphene concentrations were relatively high (>0.4 μg/g) in 

those same samples. The greatest concentrations of dieldrin 

and total chlordanes were also measured at Stations 324 and 

325 as well as Stations 314, 315, and 316. Concentrations 

of these contaminants were less than the effects thresholds. 

Concentrations of unlisted or restricted use pesticides including pentachlorobenzene, pentachloroanisole, γ-HCH, dacthal, 

endosulfan, and methoxychlor were also relatively high in 

the Lower GR although toxicity thresholds for most of these 

chemicals were unavailable. Previous CDRB studies have 

either not measured or reported these unlisted or restricted use 

pesticides in fish, and comparison of 2003 concentrations to 

other studies is difficult. Nevertheless, many of these current use organochlorine pesticides can cause reproductive and 

developmental effects in fish and wildlife (McDonald, 1991; 

Ortiz and others, 2003; Shukla and Pandey, 1986; USEPA, 

2002; Versonnen and others, 2004) and should continued to be 

monitored in the CDRB. 

Concentrations of total PCBs were also generally low 

throughout the CDRB. The exceptions were fish collected 

from Stations 320 and 324, where concentrations of PCBs 

were >0.5 μg/g. Both of these stations were located downstream of major metropolitan areas. TCDD-EQs concentrations >4 pg/g were measured in carp and channel catfish from 

Station 324, which in conjunction with the greater PCB concentrations at this station indicated that the dioxin-like activity 

in fish from that site was likely due to PCBs. Concentrations 

of TCDD-EQs were >2 pg/g in channel catfish from Station 

316 and carp from Station 320. Hepatic EROD activity was 

similar among stations, with a few exceptions. In carp, the 

greatest mean EROD activity was in fish from Station 324, 

which correlates with the elevated TCDD-EQ and PCB concentrations in these samples. In bass, mean EROD activities 

were greatest at Station 312; TCDD-EQs and PCB concentrations were low in these samples, which indicated that bass 

at this site were exposed to some other type of AhR agonist, 

such as a PAH. Large bituminous coal beds in northeastern 

Utah could be a potential PAH source in this area and may be 

contributing to the elevated EROD activities in fish.

Results and Discussion    51
Table 18. Number and location of external lesions identified on fish collected in the Colorado River Basin in 2003. Body, 

eyes, opercles, and fins of each fish were examined for the presence of lesions, and the proportion of fish with lesions was 

calculated. Stations are listed upstream to downstream. 

Lesion location

Station and species n Body Eyes Opercles Fins Total no.

w/lesions Proportion

Lay, CO (311)

Smallmouth bass 20 0 0 1 0 1 0.05

White sucker 19 2 0 0 7 7 0.37

Carp 1 0 0 0 0 0 0.00

Ouray NWR, UT (312)

Carp 20 8 1 1 5 14 0.70

Channel catfish 11 1 0 0 2 3 0.27

Smallmouth bass 14 2 1 0 1 3 0.21

San Rafael, UT (313)

Carp 21 2 1 0 5 7 0.33

Channel catfish 18 7 0 1 7 10 0.56

Delta, CO (314)

Brown trout 21 0 1 0 15 16 0.76

Carp 21 6 3 2 3 9 0.43

Loma, CO (315)

Carp 20 2 1 1 5 8 0.40

Channel catfish 5 3 2 0 3 4 0.80

Largemouth bass 12 4 2 1 7 9 0.75

Gold Bar Canyon, UT (316)

Carp 20 2 0 0 1 3 0.15

Channel catfish 20 5 1 0 5 8 0.40

Hogback Diversion, NM (317)

Carp 20 0 0 1 3 4 0.20

Channel catfish 20 6 1 0 12 12 0.60

South Cove, AZ (319)

Carp 17 2 0 1 0 3 0.18

Willow Beach, AZ (320)

Carp 20 2 4 0 6 9 0.45

Needles, CA (321)

Carp 20 3 0 2 2 5 0.25

Largemouth bass 20 6 0 3 0 9 0.45

Imperial Dam, AZ (322)

Carp 20 1 0 1 3 5 0.25

Largemouth bass 20 0 2 2 0 4 0.20

Hayden, AZ (323)

Carp 20 0 0 1 4 4 0.20

Flathead catfish 11 1 0 0 0 1 0.09

Largemouth bass 9 0 0 0 1 1 0.11

Phoenix, AZ (324)

Carp 20 2 0 1 5 7 0.35

Channel catfish 15 1 0 0 6 7 0.47

Largemouth bass 2 0 0 0 0 0 0.00

Arlington, AZ (325)

Carp 20 2 0 1 8 9 0.45

Largemouth bass 20 2 0 1 6 7 0.35

Basin Totals

All 517 72 20 21 122 189 0.37

Carp 260 32 10 12 50 87 0.34

Bass 117 14 5 8 15 34 0.58

Channel catfish 89 23 4 1 35 44 0.49

White sucker 19 2 0 0 7 7 0.37

Brown trout 21 0 1 0 15 16 0.76

Flathead catfish 11 1 0 0 0 1 0.09

52    Contaminants, Health Indicators, and Reproductive Biomarkers in Fish from the Colorado River Basin
Fish Health Indicators

Organism-Level Indicators

External Gross Lesions

External gross lesions (abnormalities) were identified on 

37% of CDRB fish during the fish health examination in the 

field. These lesions were categorized by location including 

lesions on the body surface, eyes, opercles, and fins. More 

lesions were generally found on Upper CDRB fish than Lower 

CDRB fish (table 18). High proportions of lesions (≥70%) 

occurred in carp from Station 312, brown trout from Station 

314, and bass and channel catfish from Station 315.

Overall, lesions were identified on 34% of carp. External 

lesions were identified on <50% of carp from most sites (table 

18). Lesions on carp were primarily attributed to frayed fins 

and nodules on the body surface and fins. Eye lesions on carp 

from Station 314 (n = 3) were identified as black spots or 

exophthalmic, and those from Station 320 (n = 4) were 

opaque. External lesions were found on 58% of bass, and the 

greatest proportion (75%) was found on bass from Station 315 

(table 18). Most bass lesions were described as nodules on 

the fins or mouth and frayed fins. External lesions were found 

on 49% of channel catfish, and the greatest lesion occurrence 

(>50%) was observed in fish from Stations 313, 315, and 

317 (table 18). Most external lesions in channel catfish were 

parasites on the body or fins. In general, the proportion of 

CDRB fish with external lesions was lower than the proportion identified in previous LRMN investigations (Blazer and 

others, 2002; Hinck and others, 2004a; Schmitt and others, 

2004). Statistical comparisons among basins are not part of 

this report.

External lesions were collected during field necropsy for 

histopathological examination. Most nodules on the body 

surface and fins were inflammatory responses from parasitic 

infestations. Two raised fin lesions on carp from Station 320 

were diagnosed as fibrotic lesions with inflammation and 

increased numbers of melanocytes, and a hematoma was identified on the opercle of a carp from Station 324. A fibroma 

and papilloma were observed on catfish from Station 313, and 

a papilloma was observed on a bass and a carp from Station 

321. 

Health Assessment Index

The health assessment index (HAI) is a systematic 

method to identify external and internal lesions for each fish 

during field necropsy. A higher HAI score indicates that a 

greater number of lesions were identified on the fish. The HAI 

may vary depending on gender and gonadal stage (Schmitt, 

2002). The gender and gonadal stage did not influence HAI in 

carp, bass, or channel catfish (Appendix 7). Therefore, gender 

and gonadal stage data were combined for data analysis.

Most HAI scores in carp (91%) ranged from 0 to 70, indicating that most fish had zero to three lesions (table 19). Mean 

HAI scores were <40 in carp in the Upper CDRB and the 

GRB and >47 in carp from the Lower CDR (table 19; fig. 13). 

Liver discoloration, granular spleen and kidney, and pale gills 

accounted for most elevated HAI scores at Stations 319, 320, 

321, and 322 (fig. 13). These data are similar to HAI scores in 

carp from the MRB (Blazer and others, 2002), RGB (Schmitt 

and others, 2004), and CRB (Hinck and others, 2004a). 

HAI scores in bass were greater than those in carp. Most 

HAI scores in bass (87%) ranged from 0 to 100 (table 19). 

Mean HAI scores ranged from 50 to 103 from all stations 

except Stations 311 and 312, where all smallmouth bass were 

collected (fig. 13). HAI scores in bass from Stations 315, 

321, and 322 were greater (>90) than those from other stations (table 19). Liver discoloration, granular liver, kidney, 

and spleen, and body surface lesions elevated HAI scores in 

fish from Station 315, 321, and 322. In general, HAI scores 

in bass from these three stations were greater than those 

measured in bass from the MRB (Blazer and others, 2002), 

RGB (Schmitt and others, 2004), and CRB (Hinck and others, 

2004a).

Most HAI scores in channel catfish (85%) ranged from 0 

to 70 (table 19), and mean HAI scores were <43 except in fish 

from Stations 315 (70; n = 5) and 317 (81; fig. 13). Elevated 

HAI scores in fish from Stations 315 and 317 were attributed 

to head and eye lesions, frayed and eroded fins, liver discoloration, and granular spleen (fig. 13). HAI scores in channel 

catfish were 0-30 in the MRB and 10-70 in the RGB (BESTLRMN program, unpublished data).

Condition factor and Organosomatic Indices

These indices were calculated from body and organ 

weights in individual fish and considered general indicators 

of the overall fish health. Alterations of these indices may be 

indicative of effects resulting from exposure to contaminants 

(Schmitt and Dethloff, 2000). These indices can vary among 

species, gender, and gonadal stage. ANOVA models containing the factors station, gender, and gonadal stage were tested 

to determine if these factors influenced condition factor, HSI, 

and SSI in carp, bass, and channel catfish (Appendix 7).

Condition factor and Organosomatic Indices in Carp

Condition factor values did not differ among stations 

(F12, 205 = 1.22, P > 0.05) or between genders (F1, 205 = 0.01, P 

> 0.05) in carp. The basin-wide mean CF value was 1.17 in 

carp. Means were similar among stations and ranged from 

1.00 at Station 314 to 1.24 at Station 317 (table 20). The CF 

value was 1.87 in one carp from Station 311. Relatively high 

CF values (>1.60) were calculated for carp from Stations 311 

and 320, and low CF values (<0.80) characterized multiple 

carp from Stations 314, 315, and 321 (fig. 14). Mean CF 

values in carp were 1.1-1.5 in the MRB (Blazer and others, 

2002), 1.2-1.5 in the RGB (Schmitt and others, 2004), and 

Results and Discussion    53
Table 19. Distribution of health assessment index (HAI) scores among carp, bass, catfish, brown trout, and white sucker collected in the Colorado River Basin 

in 2003. Sample size (n), arithmetic mean, and standard error (SE) are also given. Station totals where HAI scores were not available for all individual fish are noted with an asterisk (*). --, not applicable.

Species and station Health assessment index (HAI) score

0 10 30 40 50 60 70 80 90 100 110 120 130 150 160 n Mean SE

Carp Lay, CO (311) 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 -- Ouray NWR, UT (312) 1 5 2 7 0 0 4 0 0 0 0 0 0 0 0 19* 35.6 5.26

 San Rafael, UT (313) 6 6 4 4 0 0 0 0 0 0 0 0 1 0 0 21 22.4 6.36

 Delta, CO (314) 5 1 2 7 0 0 4 0 0 2 0 0 0 0 0 21 39.5 6.89

 Loma, CO (315) 1 4 3 7 0 1 1 0 2 1 0 0 0 0 0 20 41.0 6.36

 Gold Bar Canyon, UT (316) 3 6 4 6 0 1 0 0 0 0 0 0 0 0 0 20 22.1 2.04

 Hogback Diversion, NM (317) 2 7 2 7 0 2 0 0 0 0 0 0 0 0 0 20 26.5 4.25

 South Cove, AZ (319) 1 0 7 2 0 3 2 0 1 1 0 0 0 0 0 17 47.1 6.23

 Willow Beach, AZ (320) 1 2 5 1 0 1 3 0 3 4 0 0 1 0 0 20 57.5 7.74

 Needles, CA (321) 1 1 1 2 0 2 6 0 0 6 0 0 0 0 0 20 69.5 7.45

 Imperial Dam, AZ (322) 0 0 5 7 0 5 2 0 0 1 0 0 0 0 0 20 48.5 4.12

 Hayden, AZ (323) 12 5 2 1 0 0 0 0 0 0 0 0 0 0 0 20 7.5 2.70

 Phoenix, AZ (324) 9 4 2 4 0 0 1 0 0 0 0 0 0 0 0 20 16.5 4.60

 Arlington, AZ (325) 9 6 3 0 0 0 2 0 0 0 0 0 0 0 0 20 14.5 4.84

 Basin total 52 47 42 55 0 15 25 0 6 15 0 0 2 0 0 259 34.3 1.88

Bass

 Lay, CO (311) 0 0 15 0 0 5 0 0 0 0 0 0 0 0 0 20 37.5 2.98

 Ouray NWR, UT (312) 5 3 3 2 0 0 1 0 0 0 0 0 0 0 0 14 19.3 5.69

 Loma, CO (315) 0 1 1 0 0 1 3 0 0 1 0 1 2 1 1 12 91.6 13.7

 Needles, CA (321) 0 0 0 1 0 1 3 0 0 10 0 0 5 0 0 20 98.0 5.69

 Imperial Dam, AZ (322) 0 0 0 0 0 1 0 1 3 10 1 1 2 0 1 20 103.0 4.59

 Hayden, AZ (323) 0 0 0 0 0 1 0 0 7 1 0 0 0 0 0 9 87.8 3.64

 Phoenix, AZ (324) 0 0 1 0 0 0 1 0 0 0 0 0 0 0 0 2 50.0 20.0

 Arlington, AZ (325) 0 1 2 1 0 8 2 0 2 4 0 0 0 0 0 20 65.5 5.73

 Basin total 5 5 22 4 0 17 10 1 12 26 1 2 9 1 2 117 71.3 3.57

Channel catfish

 Ouray NWR, UT (312) 6 2 1 0 0 0 0 0 1 0 0 0 0 0 0 10* 14.0 8.97

 San Rafael, UT (313) 2 3 2 3 0 3 4 0 0 1 0 0 0 0 0 18 42.8 6.85

 Loma, CO (315) 0 0 0 1 0 0 3 0 0 1 0 0 0 0 0 5 70.0 9.49

 Gold Bar Canyon, UT (316) 5 3 7 3 0 0 2 0 0 0 0 0 0 0 0 20 25.0 4.78

 Hogback Diversion, NM (317) 0 2 0 2 0 1 5 0 1 7 0 0 0 0 2 20 81.0 8.82

 Phoenix, AZ (324) 3 2 3 2 0 2 3 0 0 0 0 0 0 0 0 15 34.7 6.89

 Basin total 16 12 13 11 0 6 17 0 2 9 0 0 0 0 2 88 41.4 3.73

White sucker

 Lay, CO (311) 4 2 4 3 0 5 1 0 0 0 0 0 0 0 0 19 33.2 5.57Flathead catfish

 Hayden, AZ (323) 6 0 3 0 0 0 1 0 0 0 0 0 0 0 0 10* 16.0 7.48Brown trout

 Delta, CO (314) 0 3 3 6 0 2 6 0 1 0 0 0 0 0 0 21 47.1 5.03

54    Contaminants, Health Indicators, and Reproductive Biomarkers in Fish from the Colorado River Basin
1.2-1.9 in the CRB (Hinck and others, 2004a), and CF values 

<0.80 were rare in carp from these studies. Mean CF values 

were approximately 1-2 in carp from the Las Vegas Bay of 

Lake Mead (Patiño and others, 2003b). The CF values in 

CDRB carp were generally less than those reported for carp 

(1.2 to >2.0) in a national survey (Carlander, 1969). 

Relative spleen sizes in carp differed among stations (F12, 

212 = 2.96, P < 0.01) and between genders (F1, 212 = 14.86, P < 

0.01); therefore, males and females were analyzed separately. 

The basin-wide mean SSI value in female carp was 0.21% 

(table 21). Station means ranged from 0.16% at Stations 324 

and 325 to 0.30% at Station 317. Values were ≥0.30% in fish 

from Stations 312, 314, 315, 316, 317, and 320 and ≤0.10% 

in fish from Stations 319 and 324 (fig. 14). Mean SSI values 

in female carp from the CDRB were similar to those from the 

MRB (0.09-0.87%; Blazer and others, 2002), the RGB (0.10-

0.30%; Schmitt and others, 2004), and the CRB (0.15-0.39%; 

Hinck and others, 2004a). 

The SSI was generally greater in male carp than female 

carp. The basin-wide mean SSI value in male carp was 0.27% 

(table 21). Station means ranged from 0.15% at Station 324 to 

0.46% at Station 315. Individual SSI values were >0.50% in 

fish from Stations 312, 315, and 320 (fig. 14). The SSI value 

was 1.74% in one fish from Station 315, but the spleen tissue 

was histologically described as normal. This is the greatest 

SSI value calculated for male carp from any LRMN investigation, but SSI values >2.0% were measured in several female 

carp from the MRB (Blazer and others, 2002). Mean SSI 

values in male carp from the CDRB were similar to those from 

the MRB (0.04-0.50%; Blazer and others, 2002), the RGB 

(0.10-0.40%; Schmitt and others, 2004), and the CRB (0.22-

0.44%; Hinck and others, 2004a).

Condition factor and Organosomatic Indices in Bass

Condition factor values in bass did not differ among stations (F5, 92 = 1.47, P > 0.05) or between genders (F1, 92 = 1.43, 

P > 0.05). The basin-wide mean CF value in bass was 1.34, 

and station means ranged from 0.99 at Stations 315 and 321 to 

1.65 at Station 311 (table 20). Values >1.5 were calculated for 

fish from all stations except Station 321, and values <1.0 were 

calculated for fish from Stations 315 and 321 only (fig. 15). 

As previously mentioned, CF values were also low in carp 

from Stations 315 and 321. Multiple studies have reported 

that CF values of 1.0-2.0 are typical in healthy largemouth and 

smallmouth bass (Blazer and others, 2002; Carlander, 1977; 

Hinck and others, 2004a; Schmitt and others, 2004; Sepúlveda 

and others, 2001). Some internal lesions in bass at Station 321 

may be associated with the low CF values. A number of bass 

exhibited fatty infiltration and degeneration of hepatocytes, 

large MAs, and a very high helminth parasite load in the liver, 

kidney, and spleen. Some bass also had myxosporidian parasites in the kidney.

HSI values in bass differed among stations (F7, 101 = 

18.83, P < 0.01) but not between genders (F1, 101 = 0.50, P > 

0.05). The basin-wide mean HSI value in bass was 1.15%, 

Carp

HAI score

0

20

40

60

80

Bass

HAI score

0

30

60

90

Channel catfish

Station

 311 312 313 314 315 316 317 319 320 321 322 323 324 325

HAI score

0

20

40

60

80

Upper CDRB Lower CDRB

Fin

Body surface

Eye

Opercle

Gill

Liver

Spleen 

Kidney 

Figure 13. Mean HAI scores by lesion location in carp, bass, 

and channel catfish from the Colorado River Basin in 2003. 

Stations are ordered from upstream to downstream and are 

grouped by sub-basin. See Table 2 for station descriptions.

Results and Discussion    55
Table 20. Arithmetic mean of condition factor (CF) by species and station in carp, bass, and channel 

catfish collected in the Colorado River Basin in 2003. Female and male fish were separated when 

analysis-of-variance modeling determined that gender was a significant factor. Number of samples (n), 

minimum (Min), maximum (Max), and standard error (SE) are also given. Stations are ordered upstream to 

downstream. The maximum station mean for each taxon is shown in bold. --, not applicable.

Taxon and station

Condition factor (CF)

n Mean Min Max SE

Carp

Basin Total 258 1.17 0.60 1.87 0.01

Lay, CO (311) 1 1.87 -- -- --

Ouray NWR, UT (312) 20 1.21 1.02 1.34 0.02

San Rafael, UT (313) 21 1.15 0.97 1.44 0.03

Delta, CO (314) 21 1.00 0.60 1.49 0.05

Loma, CO (315) 20 1.19 0.72 1.50 0.06

Gold Bar Canyon, UT (316) 20 1.08 0.96 1.23 0.02

Hogback Diversion, NM (317) 19 1.24 0.94 1.54 0.03

South Cove, AZ (319) 17 1.18 1.06 1.33 0.02

Willow Beach, AZ (320) 20 1.20 0.63 1.79 0.05

Needles, CA (321) 20 1.19 0.71 1.54 0.05

Imperial Dam, AZ (322) 20 1.20 1.00 1.31 0.02

Hayden, AZ (323) 20 1.22 1.10 1.38 0.02

Phoenix, AZ (324) 19 1.17 0.98 1.38 0.02

Arlington, AZ (325) 20 1.19 1.01 1.45 0.02

Bass

Basin Total 117 1.34 0.52 2.07 0.03

Lay, CO (311) 20 1.65 1.26 2.07 0.04

Ouray NWR, UT (312) 14 1.49 1.33 1.71 0.03

Loma, CO (315) 12 0.99 0.52 1.77 0.13

Needles, CA (321) 20 0.99 0.55 1.36 0.07

Imperial Dam, AZ (322) 20 1.27 0.69 1.54 0.04

Hayden, AZ (323) 9 1.35 1.26 1.54 0.03

Phoenix, AZ (324) 2 1.60 1.42 1.77 0.18

Arlington, AZ (325) 20 1.51 1.31 1.71 0.03

Female Channel catfish

Basin Total 44 0.79 0.45 1.10 0.02

Ouray NWR, UT (312) 5 0.70 0.59 0.85 0.04

San Rafael, UT (313) 4 0.65 0.45 0.75 0.07

Loma, CO (315) 2 0.65 0.48 0.81 0.17

Gold Bar Canyon, UT (316) 10 0.67 0.56 0.85 0.03

Hogback Diversion, NM (317) 11 0.89 0.75 1.05 0.03

Phoenix, AZ (324) 12 0.92 0.76 1.1 0.03

Male Channel catfish

Basin Total 41 0.79 0.59 1.09 0.02

Ouray NWR, UT (312) 6 0.70 0.59 0.78 0.03

San Rafael, UT (313) 14 0.81 0.68 1.09 0.03

Loma, CO (315) 1 1.06 -- -- --

Gold Bar Canyon, UT (316) 10 0.68 0.59 0.82 0.02

Hogback Diversion, NM (317) 8 0.89 0.81 0.97 0.02

Phoenix, AZ (324) 2 0.97 0.95 0.99 0.02

56    Contaminants, Health Indicators, and Reproductive Biomarkers in Fish from the Colorado River Basin
and station means ranged from 0.66% at Station 312 to 1.73% 

at Station 311 (table 22). Hepatosomatic index values were 

>2.0% in fish from Station 311 only. Relatively low HSI 

values (<1.0%) were found in fish from all stations except Station 315, and station means were <1.0% at Stations 312, 321, 

and 322 (fig. 15). At Station 312, liver tissue was described 

as normal during both the field necropsy and histopathological examination. The livers of bass from Stations 321 and 322 

were identified as abnormal (that is, tan and nodular) during 

field examination, but most livers from other stations were 

normal. The pale or tan appearance was likely due to the fatty 

infiltration or vacuolization of the hepatocytes, and the nodular 

appearance may be from helminth parasites and associated 

inflammation. Mean HSI values in bass were 0.6-2.0% in 

the MRB (Blazer and others, 2002), 0.7-1.0% in the RGB 

(Schmitt and others, 2004), and 0.9-2.3% in the CRB (Hinck 

and others, 2004a). Gingerich (1982) determined a comparative range for normal liver weight in fish to be 1-3% of the 

total body weight. Liver weights in many CDRB bass were 

below this benchmark but were similar to those reported in 

other LRMN studies.

SSI values in bass differed among stations (F7, 101 = 3.56, 

P < 0.01) but not between genders (F1, 101 = 0.08, P > 0.05). 

The basin-wide mean SSI value in bass was 0.11%, and station 

means ranged from 0.03% at Station 324 (n = 2) to 0.16% at 

Station 315 (table 21). Individual SSI values were >0.2% in 

fish from Stations 311, 315, and 321. SSI values were ≤0.03% 

in fish from Stations 311, 315, and 324 (fig. 15), and splenic 

tissues in these fish appeared histologically normal except for 

the presence of parasites. Mean SSI values in bass were 0.09-

0.24% in the MRB (Blazer and others, 2002), 0.1-0.2% in the 

RGB (Schmitt and others, 2004), and 0.11-0.25% in the CRB 

(Hinck and others, 2004a). The SSI values in CDRB bass 

were generally lower than those measured in previous studies.

Condition factor and Organosomatic Indices in Channel 

catfish

Condition factor values in channel catfish differed among 

stations (F5, 62 = 7.45, P < 0.01), gonadal stages (F1, 62 = 12.25, 

P < 0.01), and between genders (F1, 62 = 6.41, P < 0.05); 

therefore, females and males were analyzed separately. The 

basin-wide mean CF value in female channel catfish was 0.79, 

and station means ranged from 0.65 at Stations 313 and 315 to 

0.92 at Station 324 (table 20). The CF values were 0.60-1.00 

in most females (80%) and were generally greatest (>0.80) 

in fish from Stations 317 and 324 (fig. 16). CF values were 

low (<0.60) in females from Stations 312, 313, 315, and 316, 

and most of these fish were stage 1. The basin-wide mean 

CF value in male channel catfish was 0.79 (table 20). Station 

means ranged from 0.68 at Station 316 to 1.06 at Station 315 

(n = 1), and most CF values (95%) were <1.0 (fig. 16). Similar to female channel catfish, CF values were low (<0.60) in 

males from Stations 312, 313, 315, and 316. These fish were 

in stages 0-4. The CF values in CDRB channel catfish were 

CF

0.4

0.8

1.2

1.6

2.0

Female

SSI (%)

0.0

0.1

0.2

0.3

0.4

Male

Station

 311 312 313 314 315 316 317 319 320 321 322 323 324 325

SSI (%)

0.0

0.5

1.0

1.5

Upper CDRB Lower CDRB

Figure 14. Fish health indicators by station in female and male 

carp from the Colorado River Basin in 2003. Indicators include 

condition factor (CF) and splenosomatic index (SSI). Females 

and males were plotted separately when analysis-of-variance 

modeling determined gender was a significant factor. Shown for 

each group are points representing individual fish and the mean 

(red horizontal line), median (black horizontal line), interquartile 

range (box), and the 10th and 90th percentiles (whiskers). 

Stations are ordered from upstream to downstream and are 

grouped by sub-basin. See Table 2 for station descriptions.

Results and Discussion    57
Table 21. Arithmetic mean of splenosomatic index (SSI; %) by species and station in carp, bass, 

and channel catfish collected in the Colorado River Basin in 2003. Female and male fish were 

separated when analysis-of-variance modeling determined gender was a significant factor. Number 

of samples (n), minimum (Min), maximum (Max), and standard error (SE) are also given. Stations are 

ordered upstream to downstream. The maximum station mean for each taxon is shown in bold. --, 

not applicable.

Taxon and station Splenosomatic index (SSI)

n Mean Min Max SE

Carp

 Female

Basin Total 132 0.21 0.09 0.45 0.01

Ouray NWR, UT (312) 11 0.25 0.16 0.39 0.02

San Rafael, UT (313) 10 0.18 0.13 0.25 0.01

Delta, CO (314) 13 0.23 0.16 0.32 0.01

Loma, CO (315) 10 0.24 0.15 0.35 0.02

Gold Bar Canyon, UT (316) 10 0.21 0.11 0.31 0.02

Hogback Diversion, UT (317) 6 0.30 0.16 0.45 0.05

South Cove, AZ (319) 11 0.17 0.09 0.24 0.01

Willow Beach, AZ (320) 9 0.20 0.11 0.35 0.03

Needles, CA (321) 12 0.22 0.17 0.28 0.01

Imperial Dam, AZ (322) 12 0.19 0.12 0.29 0.01

Hayden, AZ (323) 7 0.21 0.13 0.28 0.02

Phoenix, AZ (324) 8 0.16 0.10 0.30 0.02

Arlington, AZ (325) 13 0.16 0.11 0.22 0.01

 Male

Basin Total 105 0.27 0.09 1.74 0.02

Ouray NWR, UT (312) 9 0.28 0.17 0.70 0.06

San Rafael, UT (313) 11 0.25 0.18 0.32 0.01

Delta, CO (314) 3 0.31 0.25 0.39 0.04

Loma, CO (315) 10 0.46 0.19 1.74 0.15

Gold Bar Canyon, UT (316) 10 0.26 0.17 0.44 0.03

Hogback Diversion, UT (317) 0 -- -- -- --

South Cove, AZ (319) 6 0.26 0.17 0.41 0.04

Willow Beach, AZ (320) 11 0.26 0.14 0.56 0.04

Needles, CA (321) 8 0.25 0.13 0.43 0.03

Imperial Dam, AZ (322) 8 0.25 0.13 0.32 0.02

Hayden, AZ (323) 12 0.26 0.12 0.41 0.02

Phoenix, AZ (324) 10 0.15 0.09 0.26 0.01

Arlington, AZ (325) 7 0.20 0.13 0.31 0.02

Bass

Basin Total 117 0.11 0.02 0.47 0.01

Lay, CO (311) 20 0.14 0.03 0.39 0.02

Ouray NWR, UT (312) 14 0.07 0.05 0.12 0.01

Loma, CO (315) 12 0.16 0.02 0.47 0.04

Needles, CA (321) 20 0.14 0.05 0.28 0.02

Imperial Dam, AZ (322) 20 0.09 0.04 0.14 0.01

Hayden, AZ (323) 9 0.08 0.05 0.11 0.01

Phoenix, AZ (324) 2 0.03 0.02 0.03 0.01

Arlington, AZ (325) 20 0.09 0.05 0.20 0.01

Channel Catfish

Basin Total 88 0.15 0.05 0.51 0.01

Ouray NWR, UT (312) 11 0.14 0.07 0.20 0.01

San Rafael, UT (313) 18 0.16 0.10 0.25 0.01

Loma, CO (315) 5 0.20 0.10 0.51 0.08

Gold Bar Canyon, UT (316) 20 0.14 0.08 0.25 0.01

Hogback Diversion, UT (317) 20 0.19 0.07 0.45 0.02

Phoenix, AZ (324) 14 0.08 0.05 0.11 0.01

58    Contaminants, Health Indicators, and Reproductive Biomarkers in Fish from the Colorado River Basin
similar to those reported in catfish from the MRB (0.9-1.1) 

and RGB (0.7-1.8; BEST-LRMN program, unpublished data).

HSI values in channel catfish differed among stations (F5, 

73 = 2.69, P < 0.05) but not between genders (F1, 73 = 0.20, P > 

0.05). The basin-wide mean HSI value was 1.45% in channel 

catfish, and station means ranged from 1.31% at Station 316 to 

1.73% at Station 313 (table 22). Individual HSI values were 

>2.0% in fish from Stations 312, 313, and 317 and <1.0% in 

fish from Stations 316 and 317 (fig. 16). The HSI values in 

channel catfish were 0.8-1.7% in the MRB and 1.9-2.2% in 

the RGB (BEST-LRMN Program, unpublished data). The HSI 

values in CDRB channel catfish were normal (1-3%) according to Gingerich (1982).

SSI values in channel catfish differed among stations (F5, 

73 = 2.69, P < 0.05) but not between genders (F1, 73 = 0.00, P > 

0.05). The basin-wide mean SSI value in channel catfish was 

0.15%, and station means ranged from 0.08% at Station 324 

to 0.20% at Station 315 (table 21). SSI values were >0.4% 

in fish from Stations 315 and 317. Relatively low SSI values 

(<0.07%) characterized fish from Station 324 (fig. 16), but 

spleen tissue in these fish were histologically normal. The SSI 

values in CDRB channel catfish were similar to those measured in previous LRMN investigations. SSI values in channel 

catfish were 0.14-0.17% in the MRB and 0.03-0.77% in the 

RGB (BEST-LRMN program, unpublished data). Fish health 

indicators for non-target species are located in Appendix 7.

Cellular and Histopathological Indicators

Macrophage aggregates (MAs) contain endogenous 

and exogenous waste products and are active in the immune 

response to these materials (Schmitt and Dethloff, 2000). 

Three MA parameters, density or number of aggregates per 

mm2 (MA-#), mean size of aggregates in μm2 (MA-A), and 

percent of tissue occupied by macrophage aggregates (MA-%) 

were analyzed for carp, bass, and channel catfish.

MA parameters were quantified using computer-based 

image analysis in spleen tissues from Stations 311, 313, 314, 

316, 317, 321, 322, 323, and 325 and manually digitized 

microscopic analysis in spleen tissues from Stations 312, 315, 

319, 320, and 324. MAs in bass and channel catfish from Stations 312 and 315 were quantified using both methods. Manually digitized measurements were significantly lower than 

computer-based image measurements for MA-# in bass from 

Station 312 (t

7 = 4.06, P = 0.005) and channel catfish from Station 315 (t

4 = 3.20, P = 0.03) and MA-A in bass from Station 

315 (t

11 = 2.80, P = 0.02) and channel catfish from Station 312 

(t

10 = 4.04, P = 0.002). However, MA-% calculations did not 

differ significantly between methods. Differences in the methods were not consistent among species or MA parameter. In 

general, manually digitized measurements provided estimates 

that were less than the estimates obtained with computer-based 

image measurements, but differences were not always significant. Therefore, MA data from Stations 312, 315, 319, 320, 

and 324 were excluded from statistical modeling.

CF

0.5

1.0

1.5

2.0

2.5

Station

 311 312 313 314 315 316 317 319 320 321 322 323 324 325

SSI (%)

0.0

0.1

0.2

0.3

0.4

0.5

HSI (%)

1

2

3

Upper CDRB Lower CDRB

Figure 15. Fish health indicators by station in female and male 

bass (Micropterus sp.) from the Colorado River Basin in 2003. 

Indicators include condition factor (CF), hepatosomatic index 

(HSI), and splenosomatic index (SSI). Shown for each group are 

points representing individual fish and the mean (red horizontal 

line), median (black horizontal line), interquartile range (box), and 

the 10th and 90th percentiles (whiskers). Stations are ordered 

from upstream to downstream and are grouped by sub-basin. 

See Table 2 for station descriptions.

Results and Discussion    59
The MA parameters varied significantly in both bass and 

carp based on the collection station and age of the fish (and 

the interaction of these factors; ANOVA, Appendix 6). Station, gender, and age of the fish were not significant factors 

influencing MA parameters in channel catfish. Female and 

male fish were analyzed together in all three taxa because MA 

parameters did not differ between genders. Age was a significant factor in some species and is discussed where appropriate. MA parameters from all stations were included in the 

tables and figures because of the differences in methodology 

reported previously. 

MA measurements in carp

The basin-wide mean MA-# value was 7.0 MA/mm2 in 

CDRB carp, and station means ranged from 4.1 MA/mm2 at 

Stations 314 and 315 to 10.9 MA/mm2 at Station 316 (table 

23). The mean MA-# was also >10 MA/mm2 at Stations 313 

and 322, but the MA-# in a carp from Station 311 was low 

(0.6 MA/mm2). Relatively high MA-# values (>15 MA/mm2) 

were measured in fish from Stations 313, 316, 322, and 325 

(fig. 17). Overall, MA-# values in CDRB carp were similar 

to those from previous LRMN studies. Mean MA-# values in 

carp were 5.1-18.3 MA/mm2 in the MRB (Blazer and others, 

2002), 1-16 MA/mm2 in the RGB (Schmitt and others, 2004), 

and 3.0-10.2 MA/mm2 in the CRB (Hinck and others, 2004a).

The size of MAs in carp differed among CDRB stations. 

Stations means were 2,349-5,596 μm2 except at Stations 319 

(9,764 μm2) and 320 (11,463 μm2; table 23). The MA-A values were >15,000 μm2

 in multiple fish from these two stations 

(fig. 17), and these fish were also among the oldest collected 

(table 7). The MA-As in carp from Stations 319 and 320 were 

the greatest documented by the LRMN. Mean MA-A values 

in carp were 1,670-4,684 μm2 in the MRB (Blazer and others, 2002), 1,500-8,000 μm2

 in the RGB (Schmitt and others, 

2004), and 2,690-5,850 μm2 in the CRB (Hinck and others, 

2004a).

Similar to MA-A values, mean MA-% values were greatest (>6%) in carp from Stations 319 and 320 (table 23). Other 

station means ranged from 1.8% at Stations 312 and 314 to 

4.7% at Station 316 (table 23), and an MA-% value of 0.1% 

was calculated for carp from Station 311. The MA-% values were >10% in numerous fish from Stations 319 and 320, 

which is consistent with the high MA-A values from these 

stations (fig. 17). Mean MA-% values were 1.2-6.4% in the 

MRB (Blazer and others, 2002), 1-13% in the RGB (Schmitt 

and others, 2004), and 0.9-4.7% in the CRB (Hinck and others, 2004a).

Table 22. Arithmetic mean of the hepatosomatic index (HSI; %) by species and 

station in bass and channel catfish collected in the Colorado River Basin in 2003. 

Number of samples (n), minimum (Min), maximum (Max), and standard error (SE) are 

also given. Stations are ordered upstream to downstream. The maximum station mean 

for each taxon is shown in bold.

Taxon and station Hepatosomatic index (HSI)

n Mean Min Max SE

Bass

Basin Total 117 1.15 0.36 3.00 0.04

Lay, CO (311) 20 1.73 0.69 3.00 0.11

Ouray NWR, UT (312) 14 0.66 0.36 0.93 0.04

Loma, CO (315) 12 1.43 1.03 1.84 0.07

Needles, CA (321) 20 0.85 0.59 1.30 0.04

Imperial Dam, AZ (322) 20 0.96 0.48 1.68 0.07

Hayden, AZ (323) 9 1.23 0.80 1.65 0.10

Phoenix, AZ (324) 2 1.39 0.84 1.94 0.55

Arlington, AZ (325) 20 1.14 0.64 1.58 0.06

Channel Catfish

Basin Total 88 1.45 0.87 2.79 0.04

Ouray NWR, UT (312) 11 1.53 1.08 2.79 0.15

San Rafael, UT (313) 18 1.73 1.29 2.32 0.07

Loma, CO (315) 5 1.33 1.15 1.64 0.09

Gold Bar Canyon, UT (316) 20 1.31 0.97 1.78 0.05

Hogback Diversion, NM (317) 20 1.36 0.87 2.06 0.07

Phoenix, AZ (324) 14 1.42 1.07 1.85 0.06

60    Contaminants, Health Indicators, and Reproductive Biomarkers in Fish from the Colorado River Basin
The size of MAs in carp from Stations 319 and 320 were 

larger, but not more numerous, than those from other CDRB 

stations. MA parameters have been positively correlated with 

age in previous studies, and carp from Stations 319 and 320 

were relatively old (>30 y). Therefore, age is an important 

factor to consider when interpreting these data. However, MA 

parameters did not appear anomalous in relatively old carp 

(>20 y) from Stations 313, 316, and 321.

MA measurements in bass

The basin-wide mean density of MAs in bass (2.3 MA/

mm2) was less than in carp (7.0 MA/mm2). Station means 

ranged from 0.7 MA/mm2 at Station 312 to 5.5 MA/mm2 at 

Station 324 and were generally less in fish from the Upper 

CDRB compared to those from the Lower CDRB (table 23). 

Mean MA-# values were relatively low (<0.9 MA/mm2) at 

Stations 311, 312, and 315, and values were ≥6 MA/mm2 in 

fish from Stations 321, 322, 324, 325 (fig. 18). Age was a 

significant factor in the ANOVA model, but large MAs (≥6 

MA/mm2) were measured in fish from various age classes (1 

to 11 y). Overall, MA-# values in the CDRB were generally 

less than those from previous LRMN studies. Mean MA-# 

values in bass were 2.2-11.2 MA/mm2 in the MRB (Blazer and 

others, 2002), 4-8 MA/mm2 in the RGB (Schmitt and others, 

2004), and 4.1-9.5 MA/mm2 in the CRB (Hinck and others, 

2004a).

Bass from the Upper CDRB not only had fewer numbers 

of MAs but also smaller MAs (table 23). Station means were 

612-2,317 μm2 in the Upper CDRB compared to 3,994-9,389 

μm2 in the Lower CDRB (table 23). The MA-A values were 

>15,000 μm2 in bass from Stations 321, 323, and 325 (fig. 18). 

MA-A values in bass from the Lower CDRB (Stations 323, 

324, and 325) were generally greater than those from previous 

LRMN studies. Mean MA-A values in bass were 1,049-4,440 

μm2 in the MRB (Blazer and others, 2002), 3,000-5,000 μm2 in 

the RGB (Schmitt and others, 2004), and 2,118-5,095 μm2 in 

the CRB (Hinck and others, 2004a).

Mean MA-% values were lower (<0.5%) in bass from 

Stations 311, 312, and 315 and greater (>1.6%) in bass from 

Stations 321, 322, 323, 324, and 325 (table 23). Individual 

MA-% values were >6% in bass from Stations 321, 322, and 

323 (fig. 18). Age was a significant factor in the statistical 

model, but large MAs were measured in fish from various 

age classes (2-11 y). The MA-% values in CDRB bass were 

generally less than those measured in bass from previous 

LRMN studies. Mean MA-% values were 0.3-3.8% in the 

MRB (Blazer and others, 2002), 2-3% in the RGB (Schmitt 

and others, 2004), and 1.1-4.0% in the CRB (Hinck and others, 2004a).

In general, all MA parameters were lower in bass from 

Stations 311, 312, and 315 in the Upper CDRB than those 

from Stations 321, 322, 323, 324, and 325 in the Lower 

CDRB. However, mean MA measurements in bass were 

lower than those from the MRB, RGB, and CRB. Age was a 

significant factor in the statistical model, but relatively high 

Figure 16. Fish health indicators by station in female and male 

channel catfish from the Colorado River Basin in 2003. Indicators 

include condition factor (CF), hepatosomatic index (HSI), and 

splenosomatic index (SSI). Shown for each group are points 

representing individual fish and the mean (red horizontal line), 

median (black horizontal line), interquartile range (box), and the 

10th and 90th percentiles (whiskers). Stations are ordered from 

upstream to downstream and are grouped by sub-basin. See 

Table 2 for station descriptions. CF

0.6

0.8

1.0

Station

 311 312 313 314 315 316 317 319 320 321 322 323 324 325

SSI (%)

0.0

0.1

0.2

0.3

0.4

0.5

0.6

HSI (%)

1.0

1.5

2.0

2.5

3.0

Upper CDRB Lower CDRB

CF

0.4

0.6

0.8

1.0

1.2

Female

Male

Results and Discussion    61
MA parameters were measured in fish of various age classes 

(1-11 y).

MA measurements in channel catfish

The MA measurements in channel catfish were the lowest 

of the target species. The basin-wide mean for MA-# was 

1.4 MA/mm2, and station means ranged from 0.9 MA/mm2 at 

Station 313 to 2.3 MA/mm2 at Station 316 (table 23). Values 

were ≥6 MA/mm2 only in fish from Station 316 (fig. 19). MA 

size was generally small. Mean MA-A values ranged from 

932 μm2 at Station 315 to 4,508 μm2 at Station 313 (table 23). 

Fish from Station 313 had a few, very large MAs. The MA-A 

values were relatively high (>5,000 μm2) in fish from Stations 

313, 316, and 317 (fig. 19). The MA-% values in channel 

catfish were similar among stations. Mean MA-% values were 

Table 23. Arithmetic mean of macrophage aggregate (MA) density (MA-#), MA area (MA-A), and percent tissue occupied by 

MA (MA-%) by taxon and station in carp, bass, and channel catfish from the Colorado River Basin in 2003. Number of samples (n), 

minimum (Min), maximum (Max), and standard error (SE) are also given. Stations are grouped by taxon and ordered upstream to 

downstream. The maximum station mean for each taxon is shown in bold. Measurements from Stations 312, 315, 319, 320, and 324 

were made using manual microscopic analysis; measurements from other stations were made using computer-based image analysis. 

--, not applicable.

Taxon and station

MA-# (MA/mm2) MA-A (μm2) MA-% (%)

n Mean Min Max SE Mean Min Max SE Mean Min Max SE

Carp

Basin Total 260 7.0 0.0 25.3 0.27 4847 0 34549 271 3.4 0.0 23.9 0.20

Lay, CO (311) 1 0.6 -- -- -- 2349 -- -- -- 0.1 -- -- --

Ouray NWR, UT (312) 20 5.3 0.0 9.4 0.54 2842 0 8101 433 1.8 0.0 6.4 0.34

San Rafael, UT (313) 21 10.5 1.2 25.3 1.36 4244 650 8147 459 4.5 0.1 9.2 0.59

Delta, CO (314) 21 4.1 1.2 8.8 0.47 4369 1663 14532 593 1.8 0.5 8.8 0.40

Loma, CO (315) 20 4.1 0.0 10.9 0.65 3526 0 7881 538 2.0 0.0 6.2 0.41

Gold Bar Canyon, UT (316) 20 10.9 6.5 19.4 0.79 4414 1749 10518 461 4.7 1.3 9.6 0.46

Hogback Diversion, UT (317) 20 6.3 2.9 11.8 0.52 4517 1247 10346 551 3.0 0.4 7.5 0.44

South Cove, AZ (319) 17 5.8 0.0 8.8 0.51 9764 0 34549 2307 6.6 0.0 23.9 1.67

Willow Beach, AZ (320) 20 5.5 2.8 8.1 0.36 11463 3902 29113 1503 6.1 1.4 14.4 0.89

Needles, CA (321) 20 7.7 2.4 14.7 0.72 5596 2121 10909 474 4.0 1.7 7.0 0.31

Imperial Dam, AZ (322) 20 10.5 2.4 23.5 1.21 4002 1974 11570 565 4.2 0.7 16.3 0.76

Hayden, AZ (323) 20 6.6 0.0 14.1 0.93 2926 0 10236 486 2.1 0.0 6.2 0.37

Phoenix, AZ (324) 20 5.0 0.0 7.5 0.43 3487 0 6439 345 2.0 0.2 3.1 0.18

Arlington, AZ (325) 20 8.9 1.2 19.4 1.11 2779 795 5194 295 2.5 0.2 9.5 0.45

Bass

Basin Total 117 2.3 0 10.6 0.20 4513 0 19762 402 1.4 0.0 10.3 0.17

Lay, CO (311) 20 0.9 0.0 2.9 0.18 2317 0 6994 427 0.3 0.0 1.5 0.08

Ouray NWR, UT (312) 14 0.7 0.0 1.3 0.11 612 0 1570 117 0.1 0.0 0.3 0.02

Loma, CO (315) 12 0.8 0.0 5.3 0.45 1948 0 13843 1168 0.5 0.0 2.9 0.29

Needles, CA (321) 20 3.7 0.6 7.7 0.40 7196 2243 18628 969 2.9 0.2 7.0 0.47

Imperial Dam, AZ (322) 20 3.3 0.6 10.6 0.73 3994 1338 11756 564 1.6 0.1 7.1 0.42

Hayden, AZ (323) 9 2.7 0.6 5.3 0.66 9389 2803 19762 2067 2.7 0.3 10.3 1.06

Phoenix, AZ (324) 2 5.5 5.0 5.9 0.47 6992 6415 7568 577 3.5 3.5 3.6 0.08

Arlington, AZ (325) 20 2.8 0.6 5.9 0.35 6371 869 17354 916 1.7 0.1 4.6 0.27

Channel catfish

Basin Total 89 1.4 0.0 10.0 0.17 2741 0 25823 327 0.4 0.0 2.2 0.05

Ouray NWR, UT (312) 11 1.4 0.0 5.9 0.54 1236 0 3995 427 0.4 0.0 2.0 0.17

San Rafael, UT (313) 18 0.9 0.6 1.8 0.10 4508 976 25823 1356 0.4 0.1 1.6 0.10

Loma, CO (315) 5 1.3 0.0 2.8 0.49 932 0 1618 284 0.3 0.0 0.5 0.08

Gold Bar Canyon, UT (316) 20 2.3 0.6 10.0 0.58 3213 1056 5774 317 0.7 0.1 2.2 0.14

Hogback Diversion, UT (317) 20 1.0 0.6 2.4 0.14 3153 643 6439 386 0.3 0.0 1.1 0.06

Phoenix, AZ (324) 15 1.4 0.0 4.7 0.34 1342 0 4776 351 0.4 0.0 1.3 0.12

62    Contaminants, Health Indicators, and Reproductive Biomarkers in Fish from the Colorado River Basin
≤0.4% at all stations except Station 316 (table 23), and MA-% 

values were >1.5% in fish from Stations 312, 313, and 316. 

MAs were generally larger but less numerous in CDRB channel catfish compared to MAs in channel catfish from a previous study (Matsche and Grizzle, 1999); however, fish from the 

Matsche and Grizzle study were younger (<5 months) than 

CDRB fish.

General Histopathology

Gill, liver, gonad, spleen, head kidney, hind kidney, and 

lesion tissues were collected for histopathological examination 

during the field examination. Results were limited to Stations 

312, 315, 319, 320, and 324 and summarized by tissue.

Gill tissues were typically taken from the second or third 

gill arch and were normal in most fish with a few exceptions. Mild basal hyperplasia of the lamellar epithelium was 

noted in carp from Stations 312 and 320 and channel catfish 

from Station 312. Edema, parasites, and fused lamellae were 

also found in carp from Station 320. Most aneurysms in the 

gills were likely trauma induced from collection and processing, but aneurysms in several fish from Stations 315 and 319 

showed thrombosis, which were considered pathological 

anomalies.

Liver tissues were normal in most CDRB fish evaluated. A large granuloma with calcification was found in a 

channel catfish from Station 312 and was likely parasitic in 

origin. Extensive parasitic infestations of helminth parasites 

were identified in the livers of largemouth bass from all sites. 

Lesions from trematodes were typically mild to severe, and 

myxosporean were minimal to mild. Parasites were degenerating surrounding tissues and creating calcified areas in some 

tissues. Occasionally, associated granulomas were observed 

in these same liver tissues. Cystic and proliferated bile ducts, 

hypertrophy, and MAs were found in carp liver tissues from 

Station 320.

Gonadal tissues were examined for the presence of ovotestes and quantification of oocyte atresia. Encysted parasites, associated lesions, or both were common in female and 

male gonads of largemouth bass from Stations 312, 315, 319, 

320, and 324. Pigmented cell accumulations were found in 

multiple carp from Station 320, and ova containing fluid were 

described for several female carp from Station 319. Anomalies in gonadal tissues were described in many carp and channel catfish from Station 324; calcification, poorly developed 

testes, edema, and parasites were the most common abnormalities observed in the gonadal tissues of fish from Station 324.

Histopathological conditions in splenic tissue (other than 

MAs) were rare in CDRB fish. Splenic tissues were analyzed 

for MAs, as discussed previously (see previous section). 

Parasitic infestations in largemouth bass were similar but less 

prevalent to those described in liver tissues. A large hematoma was observed in a carp from Station 312, and edema was 

observed in two carp (Stations 315 and 319).

Anomalies were observed in head and hind kidney tissues 

of all species collected in the CDRB. MAs were common in 

MA-# (No. MA/mm

2)

0

5

10

15

20

25

30

MA-A (

μ

m

2)

0

10000

20000

30000

40000

Station

 311 312 313 314 315 316 317 319 320 321 322 323 324 325

MA-% (%)

0

5

10

15

20

25

Upper CDRB Lower CDRB

Figure 17. Splenic macrophage aggregate parameters by 

station in female and male carp from the Colorado River Basin in 

2003. Parameters include macrophage aggregate density (MA-#), 

macrophage aggregate area (MA-A), and percent of splenic 

tissues occupied by macrophage aggregates (MA-%). Shown for 

each group are points representing individual fish and the mean 

(red horizontal line), median (black horizontal line), interquartile 

range (box), and the 10th and 90th percentiles (whiskers). 

Stations are ordered from upstream to downstream and are 

grouped by sub-basin. See Table 2 for station descriptions.

Results and Discussion    63
MA-# (No. MA/mm

2)

0

2

4

6

8

10

12

MA-A (

μ

m

2)

0

5000

10000

15000

20000

Station

 311 312 313 314 315 316 317 319 320 321 322 323 324 325

MA-% (%)

0

2

4

6

8

10

Upper CDRB Lower CDRB

Figure 18. Splenic macrophage aggregate parameters by 

station in female and male bass (Micropterus sp.) from the 

Colorado River Basin in 2003. Parameters include macrophage 

aggregate density (MA-#), macrophage aggregate area (MAA), and percent of splenic tissues occupied by macrophage 

aggregates (MA-%). Shown for each group are points 

representing individual fish and the mean (red horizontal line), 

median (black horizontal line), interquartile range (box), and the 

10th and 90th percentiles (whiskers). Stations are ordered from 

upstream to downstream and are grouped by sub-basin. See 

Table 2 for station descriptions.

MA-# (No. MA/mm

2)

0

2

4

6

8

10

12

MA-A (

μ

m

2)

100

300

1000

3000

10000

30000

Station

 311 312 313 314 315 316 317 319 320 321 322 323 324 325

MA-% (%)

0.0

0.5

1.0

1.5

2.0

Upper CDRB Lower CDRB

Figure 19. Splenic macrophage aggregate parameters by 

station in female and male channel catfish from the Colorado 

River Basin in 2003. Parameters include macrophage aggregate 

density (MA-#), macrophage aggregate area (MA-A), and percent 

of splenic tissues occupied by macrophage aggregates (MA-

%). Shown for each group are points representing individual 

fish and the mean (red horizontal line), median (black horizontal 

line), interquartile range (box), and the 10th and 90th percentiles 

(whiskers). Stations are ordered from upstream to downstream 

and are grouped by sub-basin. See Table 2 for station 

descriptions.

64    Contaminants, Health Indicators, and Reproductive Biomarkers in Fish from the Colorado River Basin
kidney tissues of all species. Ectopic thyroid follicles (TFs) 

were also common in the head and hind kidney of carp from 

all stations evaluated, but TFs were not found in bass or channel catfish. TFs differed in size, were filled with eosinophilicstaining colloid (caused by a reserve of thyroglobulin), and 

were generally surrounded by flattened squamous epithelium. 

Active TFs are usually surrounded by tall cuboidal or columnar epithelial cells and may contain less colloid (Fournie and 

others, 2005). The TFs in the hind kidney were generally 

smaller but more numerous than those found in the head kidney. Similar to liver and spleen tissues, parasites and associated lesions were prevalent in head and hind kidney tissues of 

largemouth bass. Crystallization, calcification, or both were 

observed in hind kidney tubules of multiple carp from Station 

320. Hyaline droplet degeneration was also found in the hind 

kidney of five female channel catfish from Station 324. 

Fish Health Indicators: Summary

The fish health indicators, which can be affected by 

various factors including age, gender, reproductive status, 

geographic location, and contaminants, were selected to reflect 

overall organismal health of the fish and their populations. 

Most of the endpoints measured in CDRB fish were limited to 

the target species (that is, carp, bass, and channel catfish) to 

remain consistent with other portions of the study. However, 

some endpoints were measured in all species.

The occurrence of external lesions has been used to 

evaluate fish health in freshwater and estuarine systems (Fournie and others, 1996; Sanderson and van den Berg, 1999). Fin 

erosion (Cross, 1985; Lindesjoo and Thulin, 1990; Murchelano and Ziskowski, 1982; Reash and Berra, 1989), skin and 

liver tumors (Baumann and others, 1991; Malins and others, 

1988; Vogelbein and others, 1990), and skeletal deformities 

(Bengtsson, 1979; Bengtsson and others, 1985; Mehrle and 

others, 1982) are commonly associated with degraded environments. Errors in proportions of anomalous fish can result 

from biased or differential examination of fish, species composition, habitat, and other factors unrelated to environmental 

degradation (Leonard and Orth, 1986). This study evaluated 

abnormalities of the body surface, eyes, opercles, and fins, 

including deformities and parasites. Thus, comparisons of this 

data to other studies must also consider the type of anomaly 

considered in each examination.

In general, the proportion of CDRB fish with external lesions was lower compared to previous LRMN studies 

(Blazer and others, 2002; Hinck and others, 2004a; Schmitt 

and others, 2004). Overall, more lesions were identified on 

fish from the Upper CDRB than those from the Lower CDRB, 

and high proportions of lesion occurrence (≥70%) were 

reported in carp from Station 312 and bass and channel catfish 

from Station 315. Body surface nodules and frayed fins 

were the most common external lesions identified in CDRB 

fish. Most nodules were inflammatory responses to parasitic 

infestations, and frayed fins may result from normal wear as 

a fish ages. Tumors were found in several fish. A hematoma 

on an opercle was identified on a carp from Station 324, and 

fibrotic fin lesions with inflammation and increased numbers 

of melanocytes were identified on two carp from Station 320. 

A fibroma and papilloma were observed on catfish from Station 313, and a papilloma was observed on a bass and a carp 

from Station 321. 

The HAI, which is also an assessment of grossly visible 

lesions, is more comprehensive than the incidence of external lesions because it accounts for both external and internal 

abnormalities. The HAI in carp and bass has been used in 

previous studies (Adams and others, 1993; Coughlan and 

others, 1996; Blazer and others, 2002; Schmitt and others, 

2004; Hinck and others, 2004a) but has had limited use in 

channel catfish (BEST-LRMN program, unpublished data). 

Mean HAI scores from the CDRB were 15-70 in carp, 19-103 

in bass, 14-81 in channel catfish, and relatively high HAI 

scores were calculated for carp and bass from the Lower CDR. 

Liver discoloration, granular spleen and kidney, and pale gills 

accounted for most elevated HAI scores in carp from Stations 

319, 320, 321, and 322. Liver discoloration, granular liver, 

kidney, and spleen, and body surface lesions elevated HAI 

scores in bass from Station 315, 321, and 322. The HAI scores 

in bass were greater than those measured previously in bass 

from other LRMN studies (Blazer and others, 2002; Hinck 

and others, 2004a; Schmitt and others, 2004) and would be 

considered unhealthy or contaminated by criteria from other 

studies (Adams and others, 1993; Coughlan and others, 1996). 

The relatively high HAI scores for carp and bass in the Lower 

CDR indicate that these fish were in generally poorer health 

than those from other CDRB stations.

Condition factor may indicate changes at the organism level and is directly affected by nutrition, season, sexual 

maturation, and disease (Adams and others, 1982; Denton and 

Yousef, 1976; Möller, 1985; Tyler and Dunn, 1976). High CF 

values have been associated with contaminant exposure such 

as pulp mill effluent (Adams and others, 1992a; McMaster and 

others, 1991; Oakes and others, 2004) and municipal sewage 

effluent (Oakes and others, 2004), whereas lower CF values 

have been observed after exposure to contaminants such as 

metals and petroleum (Kiceniuk and Khan, 1987; Miller and 

others, 1992; Munkittrick and Dixon, 1988). Condition factor 

can also vary among locations within a species (Doyon and 

others, 1988; Fisher and others, 1996). Relatively low CF 

values were calculated for carp from Stations 314, 315, and 

321 (<0.8) and bass from Stations 315 and 321 (<1.0), and 

these values were also lower than those measured in fish from 

other studies (Blazer and others, 2002; Carlander, 1969, 1977; 

Hinck and others, 2004a; Schmitt and others, 2004; Sepúlveda 

and others, 2001). Selenium concentrations in carp and bass 

from Stations 314, 315, and 321 were among the highest 

measured in our study (>2.0 μg/g), and may be associated with 

low CF values in fish from these sites (Hamilton, 2004).

The HSI can vary with season (Beamish and others, 

1996; Delahunty and de Vlaming, 1980), temperature (Fine 

and others, 1996), nutrition (Daniels and Robinson, 1986; 

Foster and others, 1993), gender, and reproductive status 

Results and Discussion    65
(Fabacher and Baumann, 1985; Förlin and Haux, 1990; 

Grady and others, 1992). Changes in HSI have often been 

associated with contaminant exposure (Adams and McLean, 

1985). Increased HSI values have been reported with exposure of wild fish to organic contaminants, most often PAHs 

and PCBs, whereas laboratory exposures of fish to metals, 

crude oil, certain pesticides, and bleached kraft mill effluent 

have resulted in HSI decreases (Schmitt and Dethloff, 2000). 

Other studies have reported that HSI increased in largemouth 

bass after exposure to bleached/unbleached kraft mill effluent 

(Sepúlveda and others, 2001, 2003). A comparative range for 

normal liver weight in fish is 1-3% of body weight, with relative weights >2% being uncommon (Gingerich, 1982). Mean 

HSI values were <1.0% in bass from Stations 312, 321, and 

322 but were not related to gonadal stage. Similar HSI values 

in bass were reported in previous LRMN studies (Blazer and 

others, 2002; Hinck and others, 2004a; Schmitt and others, 

2004). Histopathological examination determined liver tissue 

from Stations 321 and 322 has high parasites loads and fatty 

vacuolization.

The SSI can differ among species, genders, and locations 

and change over age, size, gonadal development, and season 

(Krykhtin, 1976; Ruklov, 1979; White and Fletcher, 1985). 

Fish exposed to organic contaminants alone or in combination 

with metals had decreased SSI values (Kiceniuk and Khan, 

1987; Payne and others, 1978; Pulsford and others, 1995), but 

increased SSI values have rarely been documented with contaminant exposure (Adams and others, 1992b). An increase 

in relative spleen size is considered indicative of disease or 

immune problems (Goede and Barton, 1990). Capture and 

holding stress during field studies can also alter SSI and HSI 

(Schmitt and Dethloff, 2000). The SSI values in carp and 

channel catfish were similar to those measured in previous 

LRMN studies, but values in CDRB bass were generally lower 

than those from other studies (Blazer and others, 2002; Hinck 

and others, 2004a; Schmitt and others, 2004). Relatively low 

SSI values were observed in bass and channel catfish from 

Station 324, but histopathology determined spleen tissue was 

normal in these fish. Decreased spleen sizes have been associated with hypoxia as well as exposure to PCBs, PAHs, and 

metals (see Schmitt and Dethloff, 2000). Relatively high PCB 

concentrations (>1.0 μg/g) were measured in fish from Station 

324, and wastewater treatment plant effluent and urban runoff 

from the Phoenix area comprises much of the water in the GR 

near Station 324 (Anning, 2003; Arnold and others, 2004; 

Gebler, 1998). These factors may have affected SSI values in 

fish from Station 324.

Various factors including size, nutritional status (Agius, 

1979, 1980; Agius and Roberts, 1981; Wolke and others, 

1985), age (Blazer and others, 1987; Brown and George, 1985; 

Couillard and Hodson, 1996), and exposure time (Schmitt and 

Dethloff, 2000) can affect MAs in different species. Increases 

in MA parameters in fish from specific contaminated sites 

relative to reference sites have been documented in both laboratory and field studies (Blazer and others, 1994; Blazer and 

others, 1997; Wolke, 1992). The USEPA Environmental Monitoring and Assessment Program (EMAP) Estuaries Program 

(Fournie and others, 2001; Summers and others, 1993) and 

National Oceanic and Atmospheric Administration’s Status 

and Trends Program (Chang and others, 1998) have also used 

MA parameters as bioindicators. MAs have been identified 

in various tissues including spleen, liver, gonad, and kidney, 

and many studies using splenic MAs have been performed 

in marine or estuarine environments but not in freshwater 

environments. Splenic MA-# >40 MA/mm2 were correlated 

with hypoxic stress or high levels of sediment contamination 

in estuarine fishes of varying age (Fournie and others, 2001). 

More information is needed to interpret values in freshwater 

fishes.

MAs were generally larger and more numerous in carp 

than in bass or channel catfish. MAs were >15,000 μm2 in 

multiple carp from Stations 319 and 320, and these were the 

largest MAs measured in any LRMN study (Blazer and others, 

2002; Hinck and others, 2004a; Schmitt and others, 2004). 

Carp from Stations 319 and 320 were relatively old (>35 y). 

However, smaller MAs (<7,000 μm2) were measured in other 

CDRB carp >35 y. MA parameters were generally lower in 

bass from Stations 311, 312, and 315 in the Upper CDRB than 

those from Stations 321, 322, 323, 324, and 325 in the Lower 

CDRB. Mean MA measurements were lower than those from 

the MRB, RGB, and CRB studies (Blazer and others, 2002; 

Hinck and others, 2004a; Schmitt and others, 2004).

MAs were examined but not quantified in other tissues 

from Stations 312, 315, 319, 320, and 324. MAs were common in the head and hind kidney of carp, bass, and channel 

catfish from all stations. Pigmented cell accumulations were 

also found in gonadal tissue of several male and female carp 

from Stations 319 and 320. MAs in carp gonads have been 

reported previously in Lake Mead (Patiño and others, 2003b). 

Patiño and others (2003b) suggested that high incidences of 

testicular MAs were correlated with lower GSI values in males 

and may be caused by exposure to environmental contaminants; however, infectious diseases can also increase MAs in 

fish tissues (Wolke, 1992). Histopathological examination 

also revealed TFs were common in head and hind kidney tissues of carp, but the size and number of TFs differed between 

tissues and among sites. TFs in the hind kidney of carp have 

described in previous LRMN investigations (Blazer and others, 2002; Schmitt and others, 2004). Perchlorate, a known 

contaminant in the Lower CDR, can disrupt thyroid function 

in mammals (National Research Council, 2005), fish (Brown 

and others, 2004a; Patiño and others, 2003a), and amphibians (Goleman and others, 2002). In our study, TF presence, 

number, or size in carp were not related to an area with known 

perchlorate contamination (Lower CDR). However, further 

research is needed to determine the function of TFs in the head 

and hind kidney of carp and TF occurrence in carp but not 

bass or channel catfish.

66    Contaminants, Health Indicators, and Reproductive Biomarkers in Fish from the Colorado River Basin
Reproductive Biomarkers

Reproductive biomarkers including gonadosomatic index 

(GSI), gonadal histopathology (for example, oocyte atresia 

and intersex condition), vitellogenin concentrations, and steroid hormone concentration were examined. These endpoints 

provide information on reproductive health of the fish and 

are quantifiable measures of biochemical, physiological, and 

histological changes that occur throughout the reproductive 

cycle. To minimize the effects of temperature, photoperiod, 

and annual reproductive cycle, all fish were collected postspawn within an eight week time period (August to October). 

Reproductive biomarkers can also be influenced by gender, 

maturational stage, age, and contaminants (Barry and others, 

1990; Bromage and others, 1982; Chang and Chen, 1990; 

Denslow and others, 1999; Down and others, 1990; Goodbred and others, 1997; So and others, 1989). Reproductive 

biomarkers were tested using ANOVA models that contained 

the factors station (location), gender, gonadal stage, age, and 

their interactions (Appendix 6). Although gender was not a 

significant factor in all ANOVA models, females and males 

were analyzed separately because of the known gender influence on the reproductive biomarkers measured in this study. 

In this section, gonadal stage distribution, gonadal histopathology, GSI, vitellogenin concentrations, oocyte atresia (females 

only), and steroid hormone concentrations are described 

for each species and gender. Gonadal stage was a significant factor in many ANOVA models and is discussed where 

appropriate for each reproductive biomarker. Oocyte atresia 

data in fish from Stations 312, 315, 319, 320, and 324 were 

categorized and were not included in the statistical analyses or 

figures but are described where appropriate. 

Reproductive Biomarkers in Carp

Gonadal stage in female carp was similar among CDRB 

stations. Most females (88%) were stage 3, and the remaining 

fish were stage 0 (1%), 1 (1%), and 2 (10%; fig. 20). A single 

stage-0 fish was from Station 315; stage-1 fish were found at 

Stations 320 and 324; and stage-2 fish were found at Stations 

313, 316, 319, 320, 322, and 323. Ovarian tissue from a single 

female carp from Station 320 contained primarily previtellogenic oocytes with a few spermatocytes, and this fish was 

considered to be intersex (fig. 21A).

In general, male carp were less advanced in gonadal stage 

than female carp from the same station (fig. 20). Most male 

carp were stage 2 (46%) and 3 (49%), and the remaining fish 

were stage 0 (2%), 1 (2%), and 4 (1%; fig. 20). Stage-0 and 

-1 fish were from Station 324 only, and the single stage-4 fish 

was from Station 323. Collection date did not explain the less 

advanced stage of fish from Station 324, because fish from 

Stations 323 and 325 were collected within several days of 

those from Station 324 (table 2).

GSI values in carp differed among stations (F11, 195 = 

2.20, P < 0.05) but not between genders (F1, 195 = 2.48, P > 

0.05). Station means ranged from 5.0% at Station 316 to 

17.6% at Station 325 (table 24) and were <10% at Stations 

312, 313, 316, 319, 323, and 324 (fig. 22). Proportionately 

larger ovaries were found in female carp from Stations 314, 

317, 321, and 325 compared to those from most other stations. 

GSI values were >20% only in female carp from Station 325 

(fig. 22); these females were stage 3. The GSI values were 

lowest (<2.0%) in female carp that were less advanced, stage-0 

female from Station 315 (0.6%) and the stage-1 fish from 

Stations 320 and 324 (1.5% and 2.0%, respectively). One 

stage-2 fish from Station 323 was the only other female carp 

to have a GSI <2.0%. The GSI values in CDRB female carp 

were similar to those from previous LRMN and CDRB studies. Mean GSI values in female carp were 1-18% in the MRB 

(McDonald and others, 2002), 5-20% in the RGB (Schmitt and 

others, 2004), 2-14% (Hinck and others, 2004a), and 5-20% in 

the Las Vegas Bay of Lake Mead (Patiño and others, 2003b).

GSI in male carp differed among CDRB stations. GSI 

data was not available for male carp from Station 317. Station 

means were lowest (0.9%) at Station 324, and other means 

ranged from 3.6% at Stations 320 and 325 to 9.6% at Station 

321 (table 24). Proportionately smaller testes (GSI <1.0%) 

were found in carp from Stations 320 and 324 compared to 

those from other stations (fig. 23), and these fish were in 

gonadal stages 0-4. Histopathological analyses determined 

that most male carp with GSI values <1.0% had poor testicular 

development with inflammation, calcified deposits, pigmented 

cell accumulations, and edema. Some of these observations 

have been previously associated with exposure to treated 

municipal sewage effluent in carp (Diniz and others, 2005; 

Lavado and others, 2004). The GSI values were >12% in fish 

from Stations 314 and 321. In previous LRMN and CDRB 

studies, mean GSI values in male carp were approximately 

2-12% in the MRB (McDonald and others, 2002), 1-13% in 

the RGB (Schmitt and others, 2004), <0.1-11.0% in the CRB 

(Hinck and others, 2004a), and 4-8% in the Las Vegas Bay of 

Lake Mead (Patiño and others, 2003b).

Vitellogenin concentrations in carp differed among stations (F12, 199 = 2.29, P < 0.01) but not between genders (F1, 195 

= 0.60, P > 0.05). Gender differences were likely not statistically significant as the result of the analytical model, which 

computes the F-statistic after accounting for all other factors 

(station, stage) in the model (Appendix 6). When stage was 

removed from the model, vtg concentrations differed between 

genders (F1, 223 = 759.8, P < 0.05). Concentrations of vtg were 

1-10 mg/mL in most female carp (73%), and station means 

ranged from 0.57 mg/mL at Station 324 to 5.95 mg/mL at Station 320 (table 24). Concentrations were low (≤1.81 mg/mL) 

in females from Station 324, most of which were stage-3 fish 

(figs. 20 & 22). In addition to a stage-0 fish from Station 315 

and a stage-1 fish from Station 324, concentrations were <0.1 

mg/mL in stage-2 and -3 females from Stations 316 and 325. 

Concentrations >8 mg/mL were measured in fish from all stations except Station 324, and relatively high vtg concentrations 

(>15 mg/mL) were measured in females from Stations 316 

and 320 (fig. 22). Concentrations in female carp were generResults and Discussion    67
ally greater than concentrations measured in previous LRMN 

investigations. Mean vtg concentrations in female carp were 

≤2.9 mg/mL in the MRB (McDonald and others, 2002), ≤2.5 

mg/mL in the RGB (Schmitt and others, 2004), and ≤3.9% 

mg/mL in the CRB (Hinck and others, 2004a).

Vitellogenin concentrations were detected (>0.0005 mg/

mL) in male carp from all stations except Station 320 (fig. 23). 

Station means ranged from <0.0005 mg/mL at Station 320 to 

0.058 mg/mL at Station 322 and were generally greatest in fish 

from the GRB (table 24; fig. 23). Concentrations were >0.01 

mg/mL in 57% of male carp that were in stages 0-4 (table 24). 

A. Common carp

B. Bass (Micropterus sp.)

C. Channel catfish

0%

20%

40%

60%

80%

100% Proportion

0%

20%

40%

60%

80%

100%

Proportion

0%

20%

40%

60%

80%

100%

F M F M F M F M F M F M F M F M F M F M F M F M F M F M

311 312 313 314 315 316 317 319 320 321 322 323 324 325

Station

Proportion

Stage 0 Stage 1 Stage 2 Stage 3 Stage 4 Stage 5

Figure 20. Gonadal stage proportions by station in female (F) and male (M) carp (A), bass (B; Micropterus sp.), 

and channel catfish (C) from the Colorado River Basin in 2003. Stations are ordered from upstream to downstream. 

See Table 2 for station descriptions and collection dates.

68    Contaminants, Health Indicators, and Reproductive Biomarkers in Fish from the Colorado River Basin
Relatively high concentrations in three males from Station 

323 (0.11-0.17 mg/mL) and one male from Station 322 (0.30 

mg/mL) may indicate an estrogenic response in these fish (fig. 

23). Concentrations of vtg were more frequently detected in 

male CDRB carp compared those from previous LRMN investigations, but lower method LOD may be a factor in increased 

detection frequency. Most mean concentrations in male carp 

were near LOD (0.001 mg/mL) in the MRB (McDonald and 

others, 2002), RGB (Schmitt and others, 2004), and CRB 

(Hinck and others, 2004a), and vtg concentrations >0.01 mg/

mL in male carp were rare in these studies.

An ANOVA model for oocyte atresia in female carp containing the factors station, age, gonadal stage, and their interactions was not significant (F23, 59 = 1.62, P > 0.05; Appendix 

6). Station means ranged from 6.8% at Station 325 to 17.6% 

at Station 316. Percent atresia was <20% in most carp (89%) 

but was >25% in individual fish from Stations 313, 314, and 

316 (fig. 21B). Percent oocyte atresia was also <20% in most 

fish (89%) from Stations 312, 315, 319, 320, and 324, where 

atresia data was categorized, but was 21-30% in fish from 

Stations 312 (n = 1), 319 (n = 3), and 324 (n = 1). In previous LRMN studies, mean percent atresia was approximately 

0-25% in the MRB (McDonald and others, 2002), 1-13% in 

the RGB (Schmitt and others, 2004), and 0-18% in the CRB 

(Hinck and others, 2004a).

Concentrations of E2 differed among stations (F12, 206 

= 2.52, P < 0.01) and between genders (F1, 206 = 26.37, P < 

0.01), and KT concentrations differed between genders (F1, 

206 = 11.48, P < 0.01) but not among stations (F12, 206 = 1.38, 

P > 0.05). Both E2 and KT concentrations were generally 

greater in carp from the Upper CDRB than those from the 

Lower CDRB. Mean concentrations of E2 for females ranged 

from 275 pg/mL at Station 323 to 1,334 pg/mL at Station 314 

(table 25). Mean E2 concentrations were >1,200 pg/mL in 

females from Stations 313, 314, and 315 but were relatively 

low (400 pg/mL) in fish from Stations 323, 324, and 325 

(fig. 22). These differences did not reflect stage differences 

among female carp. Mean E2 concentrations in female carp 

were slightly lower than those from the MRB (357-2,410 

pg/mL; McDonald and others, 2002). Concentrations of KT in 

females were similar among CDRB stations, and KT concentrations were 100-500 pg/mL in most female carp (83%). Station means for females ranged from 158 pg/mL at Station 323 

to 473 pg/mL at Station 315 (table 25), and differences in KT 

concentrations did not reflect stage differences among female 

carp. Concentrations were >600 pg/mL in fish from Stations 

312, 313, 314, 315, and 321 (fig. 22). Mean KT concentrations in female carp from the MRB (109-987 pg/mL) were 

slightly greater than CDRB concentrations (McDonald and 

others, 2002). Concentrations of E2 were generally greater 

than KT concentrations in female carp as reflected by E/KT 

ratios >1.0 (fig. 22). The E/KT ratios were <1.0 in individual 

fish from Stations 312, 320, 323, 324, and 325 (table 25). The 

E/KT ratio was 0.76 in the intersex carp from Station 320. 

The two lowest E/KT ratios (0.34 and 0.51) were in female 

carp from Station 325. 

Figure 21. Selected gonadal histological observations in CDRB 

fish. A. Spermatocytes (s) and previtellogenic oocytes (o) in 

ovarian tissue of female carp from Willow Beach, Arizona (Station 

320). B. High porportion of atretic oocytes (a) among healthyappearing oocytes (b) in carp from Delta, Colorado (Station 314). 

C. Intersex or the presence of previtellogenic oocytes (arrows) in 

testicular tissue of a male bass from Lay, Colorado (Station 311). 

Hematoxylin and eosin stain.

Results and Discussion    69
Table 24. Arithmetic mean of gonadosomatic index (GSI; %) and vitellogenin (Vtg; mg/mL) by species and station in carp, bass, and channel catfish collected in the Colorado 

River Basin in 2003. Number of samples (n), minimum (Min), maximum (Max), and standard error (SE) are also given. Censored values for vtg were represented by one half the LOD in the computation of means. The percent males with concentrations of vtg >0.01 mg/mL are also presented. Stations are grouped by gender and taxon and ordered upstream to downstream. The maximum station mean for each taxon is shown in bold. ND – not determined. --, not applicable.

Taxon and station

GSI (%) Vtg (mg/mL)

Female Male Female Male

n Mean Min Max SE n Mean Min Max SE n Mean Min Max SE n % Mean Min Max SE

Carp

Basin Total 132 10.7 0.6 23.0 0.4 104 5.6 0.2 12.0 0.3 131 3.80 0.0003 17.32 0.28 118 57 0.022 0.0003 0.308 0.003Ouray NWR, UT (312) 11 8.5 4.0 15.5 0.9 9 5.9 4.1 7.1 0.3 11 4.95 2.20 9.29 0.73 9 56 0.018 0.0050 0.039 0.004San Rafael, UT (313) 10 6.6 3.1 9.8 0.9 11 6.3 3.3 8.4 0.4 10 3.73 2.14 9.22 0.76 11 36 0.009 0.0003 0.023 0.003Delta, CO (314) 13 14.3 9.4 19.3 0.7 3 9.1 7.3 12.0 1.5 13 3.20 2.41 3.88 0.11 8 75 0.024 0.0040 0.075 0.008Loma, CO (315) 10 10.7 0.6 14.3 1.3 10 7.6 3.4 10.4 0.7 10 4.43 0.04 9.22 0.86 10 20 0.007 0.0003 0.041 0.004Gold Bar Canyon, UT (316) 10 5.0 2.2 8.8 0.7 9 6.4 3.5 9.9 0.6 10 4.78 0.0003 15.54 1.64 10 30 0.009 0.0003 0.024 0.002Hogback Diversion, NM (317) 6 14.4 8.6 18.7 1.4 0 -- -- -- -- 7 3.43 2.12 5.17 0.37 13 46 0.012 0.0003 0.039 0.003South Cove, AZ (319) 11 7.6 2.4 16.0 1.3 6 5.5 4.6 7.0 0.4 11 4.42 0.14 10.94 1.08 6 67 0.015 0.0003 0.026 0.004Willow Beach, AZ (320) 9 10.6 1.5 16.3 1.7 11 3.6 0.7 6.0 0.5 8 5.95 0.03 17.32 2.09 5 0 0.0003 0.0003 0.0003 0.000Needles, CA (321) 12 14.8 11.8 19.4 0.7 8 9.6 7.3 12.0 0.6 12 3.46 0.49 8.14 0.59 8 63 0.016 0.0003 0.030 0.004Imperial Dam, AZ (322) 12 10.5 7.2 14.0 0.6 8 6.7 5.4 8.0 0.3 12 3.46 0.38 9.63 1.06 8 63 0.058 0.0020 0.308 0.036Hayden, AZ (323) 7 8.1 1.5 15.4 2.0 12 5.5 3.0 7.8 0.4 7 4.02 0.19 9.69 1.37 13 85 0.057 0.0020 0.174 0.015Phoenix, AZ (324) 8 6.8 2.0 11.7 1.2 10 0.9 0.2 5.1 0.5 8 0.57 0.02 1.81 0.21 10 80 0.028 0.0003 0.070 0.008Arlington, AZ (325) 13 17.6 11.4 23.0 1.0 7 3.6 3.1 4.9 0.2 12 3.03 0.01 10.80 0.94 7 57 0.009 0.0003 0.022 0.003

Bass

Basin Total 56 0.9 0.1 3.5 0.1 59 0.3 0.0 1.0 0.0 54 1.27 0.001 20.94 0.520 59 14 0.013 0.001 0.293 0.007Lay, CO (311) 9 3.0 1.9 3.5 0.2 10 0.5 0.3 0.9 0.1 8 8.41 0.001 20.94 2.300 10 0 0.001 0.001 0.001 0.000Ouray NWR, UT (312) 6 0.8 0.6 1.0 0.1 8 0.5 0.3 0.6 0.1 6 0.184 0.001 0.37 0.075 8 38 0.075 0.001 0.293 0.046Loma, CO (315) 5 1.1 0.9 1.3 0.1 7 0.3 0.1 0.5 0.1 4 0.008 0.001 0.03 0.007 7 14 0.004 0.001 0.017 0.002Needles, CA (321) 11 0.7 0.4 1.2 0.1 9 0.2 0.1 0.4 0.0 11 0.005 0.001 0.02 0.002 9 11 0.004 0.001 0.018 0.002Imperial Dam, AZ (322) 10 0.3 0.1 0.4 0.0 10 0.1 0.0 0.4 0.0 10 0.008 0.001 0.04 0.004 10 0 0.002 0.001 0.008 0.001Hayden, AZ (323) 4 0.4 0.4 0.4 0.0 5 0.2 0.1 1.0 0.2 4 0.009 0.001 0.02 0.004 5 0 0.002 0.001 0.006 0.001Phoenix, AZ (324) 0 -- -- -- -- 2 0.1 0.1 0.1 0.0 0 -- -- -- -- 2 50 0.007 0.001 0.012 0.006Arlington, AZ (325) 11 0.4 0.2 0.6 0.0 8 0.1 0.0 0.1 0.0 11 0.007 0.001 0.023 0.002 8 25 0.007 0.001 0.015 0.002

Channel Catfish

Basin Total 44 0.7 0.0 3.2 0.1 39 0.2 0.0 0.7 0.0 ND -- -- -- -- ND -- -- -- -- --Ouray NWR, UT (312) 5 1.2 0.5 3.2 0.5 6 0.2 0.0 0.4 0.1 ND -- -- -- -- ND -- -- -- -- --San Rafael, UT (313) 4 0.7 0.6 0.8 0.0 14 0.3 0.0 0.7 0.1 ND -- -- -- -- ND -- -- -- -- --Loma, CO (315) 2 1.2 0.5 1.9 0.7 1 0.4 -- -- -- ND -- -- -- -- ND -- -- -- -- --

Gold Bar Canyon, UT (316) 10 0.6 0.3 1.8 0.1 8 0.2 0.0 0.5 0.1 ND -- -- -- -- ND -- -- -- -- --Hogback Diversion, NM (317) 11 0.9 0.2 3.1 0.3 8 0.3 0.1 0.5 0.1 ND -- -- -- -- ND -- -- -- -- --Phoenix, AZ (324) 12 0.3 0.0 0.6 0.0 2 0.2 0.2 0.2 0.0 ND -- -- -- -- ND -- -- -- -- --

70    Contaminants, Health Indicators, and Reproductive Biomarkers in Fish from the Colorado River Basin
E2 concentrations in most male carp (87%) were 50-400 

pg/mL. Station means ranged from 29 pg/mL at Station 319 

to 313 pg/mL at Station 312 (table 25) and were >200 pg/mL 

in males from Stations 312, 313, 314, and 315. Concentrations were uniformly low (≤46 pg/mL) in male carp from Station 319 (fig. 23), and these fish were stage-3 like most male 

carp from this study. Mean concentrations in male carp from 

the MRB (203-1,209 pg/mL) were greater than those from the 

CDRB (McDonald and others, 2002). Concentrations of KT 

were 100-1,100 pg/mL in most male CDRB carp (77%), and 

station means ranged from 121 pg/mL at Station 319 to 1,141 

pg/mL at Station 312 (table 25). Concentrations were >1,500 

pg/mL in fish from Stations 312, 313, 315, 320, and 325 (fig. 

23). Mean concentrations of KT in male carp from the MRB 

were 215-3,663 pg/mL (McDonald and others, 2002). Concentrations of KT were generally greater than E2 concentrations in male CDRB carp as reflected by E/KT ratios <1.0 (fig. 

23). Ratios were >1.0 in fish from Stations 314, 316, 317, 

321, 323, and 324 (table 25), and E/KT ratios were >1.2 in 

three fish from Station 316 (fig. 23).

Reproductive Biomarkers in Bass

Female bass were generally less advanced in gonadal 

stage than female carp at the same station (fig. 20). Bass 

from Stations 311 and 312 were exclusively smallmouth bass 

whereas bass from all other stations were largemouth bass. 

Most female bass were stage 1 (50%), and the remaining fish 

were stage 2 (30%), 3 (18%), and 5 (2%; fig. 20). Stage-2 fish 

were found at Stations 311, 312, 315, and 321, and stage-3 

fish were at Stations 311 and 312. One fish from Station 321 

was stage 1. In general, fish from Imperial Dam in the Lower 

CDR and the GR were less advanced (stage-0 and -1) than 

those from the Upper CDRB.

Male bass were generally less advanced in gonadal stage 

than male carp at the same station, but in similar stages as 

female bass. Most male bass were stage 1 (44%) and 2 (33%), 

and the remaining fish were stage 0 (16%), 3 (5%), and 4 

(2%; fig. 20). Similar to female bass, smallmouth bass from 

Stations 311 and 312 were more advanced (stage-2 and -3) and 

largemouth bass from Stations 322, 323, 324, and 325 were 

less advanced (stage-0 and -1). Most male bass from Stations 

324 and 325 were stage 0 (immature), and the stage-4 (spent) 

male bass was from Station 324. Intersex gonads were found 

Figure 22. Reproductive health indicators by station in female 

carp from the Colorado River Basin in 2003. Indicators include 

gonadosomatic index (GSI), vitellogenin (vtg), atresia, 17βestradiol (E2), 11-ketotestosterone (KT), and the ratio of estradiol 

and II-ketotestosterone (E/KT). Shown for each group are points 

representing individual fish and the mean (red horizontal line), 

median (black horizontal line), interquartile range (box), and the 

10th and 90th percentiles (whiskers). Stations are ordered from 

upstream to downstream and are grouped by sub-basin. See 

Table 2 for station descriptions.

GSI (%)

0

5

10

15

20

25

Vtg (mg/mL)

0

4

8

12

16

atresia (%)

0

10

20

30

40

E2 (pg/mL)

0

400

800

1200

1600

KT (pg/mL)

0

200

400

600

800

Station

 311 312 313 314 315 316 317 319 320 321 322 323 324 325

E/KT ratio

0

2

4

6

8

10

Upper CDRB Lower CDRB

Results and Discussion    71
in male bass from Stations 311, 322, and 323. A relatively 

high proportion of male bass from Station 311 were intersex. 

Mild to moderate numbers of oocytes in testicular tissue were 

found in seven of ten male smallmouth bass from Station 311 

(fig. 21C). Mild occurrence of oocytes in testicular tissue was 

identified in four of ten largemouth bass from Station 322 and 

two of five largemouth bass from Station 323. All intersex 

largemouth bass were stage 1, but intersex smallmouth bass 

(Station 311) were stage 2 and 3.

GSI values in bass differed among stations (F5, 91 = 5.91, P 

< 0.01) and between genders (F1, 91 = 14.60, P < 0.01). Lower 

GSI values were calculated for females from the Lower CDRB 

than those from the Upper CDRB. Station means ranged from 

0.3% at Station 322 to 3.0% at Station 311 and were >1.0% at 

Stations 311 and 315 (table 24). The GSI values were greater 

in female smallmouth bass from Station 311 (1.9-3.5%) than 

females from other stations (fig. 24). Most female bass from 

Station 311 were stage 3, but GSI values were <1.0% in stage3 fish collected from Station 312. The greater GSI values in 

the bass may reflect the later collection from Station 311 (table 

2). The GSI values were <0.6% in female bass from Stations 

322, 323, and 325 (table 24), which reflected the less advanced 

stage (stage-1) of these fish (fig. 20). Mean GSI values were 

<2% in the MRB (McDonald and others, 2002), 0.6-0.9% in 

the RGB (Schmitt and others, 2004), and 0.5-2.9% in the CRB 

(Hinck and others, 2004a).

GSI values in male bass were generally low (<1.0%). 

The GSI values were generally greater in more advanced 

(stage-2 and -3) male bass from the Upper CDRB than in less 

advanced (stage-0 and -1) fish from the Lower CDRB. Station 

means ranged from 0.1% to 0.5% (table 24), and GSI values 

were >0.6% in fish from Stations 311, 312, and 323 (fig. 25). 

Mean GSI values in male bass were approximately 0.1-0.7% 

in the MRB (McDonald and others, 2002), 0.2-0.4% in the 

RGB (Schmitt and others, 2004), and 0.2-0.9% in the CRB 

(Hinck and others, 2004a).

Vitellogenin concentrations in bass did not differ among 

stations (F5, 89 = 1.73, P > 0.05) or between genders (F1, 89 = 

1.75, P > 0.05). However, concentrations of vtg were greater 

in female smallmouth bass from Station 311 than those from 

other stations (fig. 24). Station means ranged from 0.005 

mg/mL at Station 321 to 8.4 mg/mL at Station 311 (table 24). 

Concentrations were >5.0 mg/mL in multiple females from 

Station 311 but were <0.37 mg/mL in all other female bass 

GSI (%)

0

3

6

9

12

Vtg (mg/mL)

0.0001

0.001

0.01

0.1

E2 (pg/mL)

0

100

200

300

400

500

KT (pg/mL)

0

500

1000

1500

2000

Station

 311 312 313 314 315 316 317 319 320 321 322 323 324 325

E/KT ratio

0.0

0.4

0.8

1.2

1.6

Upper CDRB Lower CDRB

Figure 23. Reproductive health indicators by station in male 

carp from the Colorado River Basin in 2003. Indicators include 

gonadosomatic index (GSI), vitellogenin (vtg), 17β-estradiol 

(E2), 11-ketotestosterone (KT), and the ratio of estradiol and 

II-ketotestosterone (E/KT). Shown for each group are points 

representing individual fish and the mean (red horizontal line), 

median (black horizontal line), interquartile range (box), and the 

10th and 90th percentiles (whiskers). Stations are ordered from 

upstream to downstream and are grouped by sub-basin. See 

Table 2 for station descriptions.

72    Contaminants, Health Indicators, and Reproductive Biomarkers in Fish from the Colorado River Basin
Table 25. Arithmetic mean of 17β-estradiol (E2; pg/mL), 11-ketotestosterone (KT; pg/mL), and the ratio of 17β-estradiol to 11-ketotestosterone (E/KT) 

by taxon and station in carp, bass, and channel catfish from the Colorado River Basin in 2003. Number of samples (n), minimum (Min), maximum (Max), and standard error (SE) are also given. Stations are grouped by gender and taxon and ordered upstream to downstream. The maximum station mean for each taxon is shown in bold. --, not applicable.—Continued

Taxon and station E2 (pg/mL) KT (pg/mL) E/KT

n Mean Min Max SE Mean Min Max SE Mean Min Max SE

Female Carp

Basin Total 132 848 25 1605 35 308 15 880 16 3.3 0.3 9.7 0.2Ouray NWR, UT (312) 11 844 372 1275 88 364 113 807 66 3.2 0.5 8.0 0.7San Rafael, UT (313) 10 1328 1152 1600 46 399 136 738 68 4.3 1.6 9.1 0.7Delta, CO (314) 13 1334 1142 1605 42 377 136 880 62 4.8 1.7 9.7 0.7Loma, CO (315) 10 1201 531 1573 91 473 133 829 70 3.2 1.4 8.3 0.7Gold Bar Canyon, UT (316) 10 954 502 1159 78 270 113 431 34 3.9 2.7 8.0 0.5Hogback Diversion, NM (317) 7 834 508 1140 96 285 177 420 34 2.9 2.6 3.2 0.1South Cove, AZ (319) 11 850 568 1154 74 253 100 403 30 3.9 1.9 5.8 0.5Willow Beach, AZ (320) 9 657 162 991 93 244 108 371 29 3.2 0.8 7.8 0.7Needles, CA (321) 12 961 603 1350 73 369 120 802 61 3.3 1.7 7.1 0.5Imperial Dam, AZ (322) 12 674 111 1161 102 254 62 590 39 3.0 1.0 6.0 0.5Hayden, AZ (323) 7 275 169 501 43 158 77 288 27 2.1 0.7 4.3 0.5Phoenix, AZ (324) 8 407 93 951 103 188 84 539 55 2.6 0.5 5.4 0.6Arlington, AZ (325) 12 384 25 772 69 274 15 598 53 1.8 0.3 3.9 0.3

Male Carp

Basin Total 124 181 20 511 9 605 21 2160 40 0.4 0.1 1.6 0.0Ouray NWR, UT (312) 9 313 235 414 19 1141 473 2160 174 0.3 0.1 0.5 0.0San Rafael, UT (313) 11 295 169 511 32 1074 316 1564 112 0.3 0.1 0.7 0.1Delta, CO (314) 8 221 103 370 28 648 200 1177 126 0.5 0.1 1.3 0.1Loma, CO (315) 10 210 106 402 30 1121 569 1846 111 0.2 0.1 0.3 0.0Gold Bar Canyon, UT (316) 10 125 33 300 25 488 21 935 108 0.6 0.1 1.6 0.2Hogback Diversion, NM (317) 13 129 53 271 17 322 99 831 59 0.5 0.1 1.3 0.1South Cove, AZ (319) 6 29 20 46 4 121 30 306 41 0.4 0.1 1.0 0.1Willow Beach, AZ (320) 11 185 85 413 28 727 133 1571 144 0.3 0.1 0.6 0.0Needles, CA (321) 8 166 69 268 27 387 177 685 73 0.5 0.2 1.2 0.1Imperial Dam, AZ (322) 8 113 20 279 29 359 53 529 67 0.4 0.0 1.0 0.1Hayden, AZ (323) 13 200 33 380 28 334 103 661 46 0.6 0.3 1.1 0.1Phoenix, AZ (324) 10 161 87 262 22 443 176 1070 91 0.4 0.2 1.2 0.1Arlington, AZ (325) 7 127 26 254 33 572 113 1553 181 0.3 0.1 0.5 0.1

Female Bass

Basin Total 54 530 171 1225 44 346 97 1055 33 1.9 0.4 6.6 0.2Lay, CO (311) 8 549 465 675 24 271 103 521 54 2.7 1.1 6.6 0.6Ouray NWR, UT (312) 6 418 247 535 43 353 106 844 113 2.0 0.5 3.7 0.6Loma, CO (315) 4 566 196 1175 219 370 141 541 94 1.7 0.4 2.2 0.4Needles, CA (321) 11 384 174 888 66 442 104 1055 89 1.2 0.5 3.4 0.3Imperial Dam, AZ (322) 10 1007 755 1225 49 581 419 880 50 1.9 0.9 2.9 0.2Hayden, AZ (323) 4 455 211 1161 235 164 97 264 39 2.5 1.2 4.4 0.7Arlington, AZ (325) 11 305 171 772 51 143 107 203 11 2.1 1.2 3.8 0.2

Results and Discussion    73
Table 25. Arithmetic mean of 17β-estradiol (E2; pg/mL), 11-ketotestosterone (KT; pg/mL), and the ratio of 17β-estradiol to 11-ketotestosterone (E/KT) 

by taxon and station in carp, bass, and channel catfish from the Colorado River Basin in 2003. Number of samples (n), minimum (Min), maximum (Max), and standard error (SE) are also given. Stations are grouped by gender and taxon and ordered upstream to downstream. The maximum station mean for each taxon is shown in bold. --, not applicable.—Continued

Taxon and station E2 (pg/mL) KT (pg/mL) E/KT

n Mean Min Max SE Mean Min Max SE Mean Min Max SE

Male Bass

Basin Total 58 270 88 602 15 1216 148 2056 59 0.3 0.1 1.7 0.0Lay, CO (311) 10 434 347 602 27 1432 926 1754 100 0.3 0.2 0.5 0.0Ouray NWR, UT (312) 8 322 239 374 18 1347 1006 1531 63 0.2 0.2 0.3 0.0Loma, CO (315) 7 234 107 444 43 1501 1083 1871 103 0.2 0.1 0.3 0.0Imperial Dam, AZ (322) 9 286 175 376 22 1291 582 1828 123 0.2 0.1 0.5 0.0Yuma, AZ (322) 10 164 108 234 14 1102 645 1419 79 0.2 0.1 0.3 0.0Hayden, AZ (323) 5 187 88 340 45 398 303 542 41 0.5 0.3 1.0 0.1Phoenix, AZ (324) 2 277 179 375 98 456 406 506 50 0.6 0.4 0.7 0.2Arlington, AZ (325) 7 201 99 244 19 1338 148 2056 235 0.3 0.1 1.7 0.2

Female Channel Catfish

Basin Total 42 521 94 1357 52 286 30 882 38 2.7 0.7 14.0 0.4Ouray NWR, UT (312) 4 827 521 1357 199 733 678 782 21 1.1 0.7 1.8 0.3San Rafael, UT (313) 4 688 326 1190 184 455 109 729 148 1.9 1.0 3.0 0.4Loma, CO (315) 2 414 223 604 191 275 245 305 30 1.4 0.9 2.0 0.5Gold Bar Canyon, UT (316) 10 561 231 1134 91 195 50 294 23 3.2 1.2 6.5 0.5Hogback Diversion, NM (317) 10 296 94 572 53 92 41 190 14 3.8 1.1 14.0 1.2Phoenix, AZ (324) 12 547 151 1205 109 307 30 882 77 2.5 0.7 6.9 0.6

Male Channel Catfish

Basin Total 42 209 38 662 18 488 20 1411 65 1.5 0.1 10.1 0.4Ouray NWR, UT (312) 6 227 58 333 39 865 283 1411 151 0.3 0.2 0.4 0.0San Rafael, UT (313) 14 239 78 363 24 815 249 1383 88 0.3 0.1 0.9 0.1Loma, CO (315) 1 201 -- -- -- 834 -- -- -- 0.2 -- -- --Gold Bar Canyon, UT (316) 10 119 49 183 13 183 31 411 45 1.2 0.3 3.5 0.3Hogback Diversion, NM (317) 8 279 102 662 61 87 20 216 21 4.7 0.9 10.1 1.2Phoenix, AZ (324) 2 62 38 86 24 185 169 201 16 0.4 0.2 0.5 0.2

74    Contaminants, Health Indicators, and Reproductive Biomarkers in Fish from the Colorado River Basin
(table 24; fig. 24). The greatest vtg concentrations (>0.3 mg/

mL) were in the more advanced stage (most stage-3) fish from 

Stations 311 and 312. The mean vtg concentration in female 

bass from Station 311 was among the greatest measured in any 

LRMN investigation. Mean vtg concentrations in female bass 

were ≤3.7 mg/mL in the MRB (McDonald and others, 2002), 

0.01-3.1 mg/mL in the RGB (Schmitt and others, 2004), and 

0.06-14.3 mg/mL in the CRB (Hinck and others, 2004a).

Vitellogenin concentrations were detected (>0.001 

mg/mL) in male bass from all stations except Station 311 

(fig. 24). Concentrations of vtg were ≤0.02 mg/mL in most 

(97%) males, and station means ranged from <0.001 mg/mL at 

Station 311 to 0.075 mg/mL at Station 312 (table 24). Males 

from Station 311 were generally in more advanced stages 

(stages 2 and 3) than those from other stations, and seven of 

10 male bass from Station 311 were intersex. Concentrations 

were relatively high (≥0.28 mg/mL) in two stage-2 males from 

Station 312 and may indicate an estrogenic response in these 

two fish. These concentrations are similar to those reported 

in male bass from the MRB (McDonald and others, 2002), 

RGB (Schmitt and others, 2004), and CRB (Hinck and others, 

2004a), but vtg concentrations >0.1 mg/mL in male bass were 

rare in these previous studies. 

Oocyte atresia in female bass differed among gonadal 

stage (F1, 31 = 15.84, P < 0.01) and was generally lower in 

female bass (<5%) than in carp (>10%). Station means ranged 

from 0.0% at Station 323 to 5.5% at Station 321 (fig. 24). Percent atresia was <5% in bass from all stations except Station 

321, where oocyte atresia was >5% in six fish. Oocyte atresia 

was generally greater in stage-2 fish (>4%) than those in other 

gonadal stages (<2%). Percent oocyte atresia was also <10% 

in fish from Stations 312 and 315 where atresia data was 

categorized. Percent atresia was generally less in CDRB bass 

than those from previous LRMN studies. Mean percent atresia 

in bass was approximately 0-6% in the MRB (McDonald and 

others, 2002), 0-30% in the RGB (Schmitt and others, 2004), 

and 1-12% in the CRB (Hinck and others, 2004a).

Concentrations of E2 in bass did not differ among stations (F5, 88 = 0.34, P > 0.05), gonadal stage (F1, 88 = 0.08, P 

> 0.05), or between genders (F1, 88 = 0.36, P > 0.05). KT 

concentrations differed among stations (F5, 88 = 2.24, P < 0.05) 

and between genders (F1, 88 = 8.08, P < 0.01) but not among 

gonadal stage (F1, 88 = 1.85, P > 0.05). Most E2 concentrations (89%) in female bass were 200-1,200 pg/mL, and station 

Figure 24. Reproductive health indicators by station in female 

bass (Micropterus sp.) from the Colorado River Basin in 2003. 

Indicators include gonadosomatic index (GSI), vitellogenin (vtg), 

atresia, 17β-estradiol (E2), 11-ketotestosterone (KT), and the 

ratio of estradiol and II-ketotestosterone (E/KT). Shown for each 

group are points representing individual fish and the mean (red 

horizontal line), median (black horizontal line), interquartile range 

(box), and the 10th and 90th percentiles (whiskers). Stations are 

ordered from upstream to downstream and are grouped by subbasin. See Table 2 for station descriptions.

GSI (%)

0

1

2

3

4

Vtg (mg/mL)

0.0001

0.001

0.01

0.1

1

10

atresia (%)

0

5

10

15

E2 (pg/mL)

0

300

600

900

1200

KT (pg/mL)

0

300

600

900

Station

 311 312 313 314 315 316 317 319 320 321 322 323 324 325

E/KT ratio

0

2

4

6

Upper CDRB Lower CDRB

Results and Discussion    75
means ranged from 305 pg/mL at Station 325 to 1,007 pg/mL 

at Station 322 (table 25). Concentrations were relatively high 

(>900 pg/mL) in females from Station 322 (fig. 24). Concentrations of KT were 100-625 pg/mL in most female bass 

(89%). Station means ranged from 143 pg/mL at Station 325 

to 581 pg/mL at Station 322 (table 25). KT concentrations in 

females were greatest at Station 322 and were relatively low 

(<300 pg/mL) at Stations 323 and 325 (fig. 24). Mean concentrations of E2 and KT in female bass from the MRB were 

238-1,726 pg/mL and 62-1,019 pg/mL, respectively (McDonald and others, 2002). Concentrations of E2 were generally 

greater than KT concentrations in female bass as reflected 

by E/KT ratios >1.0 (fig. 24). Ratios <1.0 were reported in 

females from Stations 312, 315, 321, and 322 (table 25; fig. 

24), and ratios were <1.0 in six female bass from Station 321 

due to low E2 concentrations (≤300 pg/mL) in these fish. 

Most E2 concentrations (90%) were 100-450 pg/mL 

in male bass, and station means ranged from 164 pg/mL at 

Station 322 to 434 pg/mL at Station 311 (table 25). Relatively low concentrations (≤100 pg/mL) were measured in 

males from Stations 323 and 325 (fig. 25); GSI values and 

vtg concentrations were also low in these male bass. Concentrations of E2 were generally greatest (>350 pg/mL) in 

males from Station 311, which was the only station where vtg 

concentrations were not detected in male bass. Concentrations 

of E2 were relatively high (340-602 pg/mL) in intersex male 

smallmouth bass from Station 311 but were lower (108-324 

pg/mL) in intersex male largemouth bass from Stations 322 

and 323. However, bass from the Lower CDRB were less 

advanced in their gonadal stage, which may explain the lower 

sex steroid hormone concentrations in these fish (fig. 20). The 

KT concentrations were 400-1,800 pg/mL in most male bass 

(88%), and station means ranged from 398 pg/mL at Station 

323 to 1,501 pg/mL at Station 315 (table 25). Concentrations 

of KT were much lower in male bass from Stations 323 and 

324 than all other stations (fig. 25) and were not related to 

gonadal stage. Mean concentrations of E2 and KT were 201-

855 pg/mL and 167-2,502 pg/mL, respectively, in male bass 

from the MRB (McDonald and others, 2002). The E/KT ratio 

was generally <1.0 in male bass (fig. 25), and ratios were >1.0 

in only two fish from Stations 323 and 325 (table 25).

GSI (%)

0.0

0.3

0.6

0.9

Vtg (mg/mL)

0.0001

0.001

0.01

0.1

E2 (pg/mL)

0

200

400

600

KT (pg/mL)

0

500

1000

1500

2000

Station

 311 312 313 314 315 316 317 319 320 321 322 323 324 325

E/KT ratio

0.0

0.4

0.8

1.2

1.6

Upper CDRB Lower CDRB

Figure 25. Reproductive health indicators by station in male 

bass (Micropterus sp.) from the Colorado River Basin in 2003. 

Indicators include gonadosomatic index (GSI), vitellogenin 

(vtg), 17β-estradiol (E2), 11-ketotestosterone (KT), and the ratio 

of estradiol and II-ketotestosterone (E/KT). Shown for each 

group are points representing individual fish and the mean (red 

horizontal line), median (black horizontal line), interquartile range 

(box), and the 10th and 90th percentiles (whiskers). Stations are 

ordered from upstream to downstream and are grouped by subbasin. See Table 2 for station descriptions.

76    Contaminants, Health Indicators, and Reproductive Biomarkers in Fish from the Colorado River Basin
Reproductive Biomarkers in Channel catfish

Gonadal stage was more variable in female channel 

catfish than in female carp or bass at the same stations. Most 

fish were stage 1 (48%), and the remaining were stage 0 (7%), 

2 (25%), 3 (11%), and 4 (9%; fig. 20). Stage-0 (immature) 

females were found at Station 324 only. Stage-1 fish were 

from Stations 313, 316, 317, and 324, and stage-2 females 

were from Stations 312, 313, and 317. Female channel catfish 

identified as stage-3 were from Stations 312, 315, 316, and 

317, and stage-4 females were from Stations 312, 315, and 

317.

Similar to female channel catfish, gonadal stage was 

more variable in male channel catfish than in male carp or bass 

from the same station. Most male channel catfish were stage 

4 (43%) and 3 (21%), and the remaining males were stage 0 

(17%), 1 (2%), and 2 (17%; fig. 20). Stage-0 males were from 

Stations 312, 316, and 317. The one stage-1 fish was from 

Station 313, and stage-2 fish were from Stations 312, 313, and 

317. Males identified as stage-3 were from Stations 312, 313, 

315, 316, and 317, and stage-4 males were from Stations 313, 

316, 317, and 324. All male channel catfish from Station 324 

(n = 3) were spent (stage-4). Intersex gonads were observed 

in a relatively small proportion of male channel catfish from 

Stations 312, 317, and 324. One male fish from each of these 

stations had evidence of ovotestes. Intersex males from Stations 312 and 317 were stage 0, but the intersex male channel 

catfish from Station 324 was stage 4.

GSI values differed among stations (F6, 60 = 4.34, P < 

0.01), gonadal stage (F1, 60 = 22.37, P < 0.01), and between 

genders (F1, 60 = 5.05, P < 0.05). Most GSI values were 

<3.0%, and station means ranged from 0.3% at Station 324 

to 1.2% at Stations 312 and 315 in female channel catfish 

(table 24). Relatively low GSI values (<0.4%) were calculated 

for most females from Station 324 (fig. 26), and all of these 

females were stage 0 and 1. Fish with GSI values >1.0% 

were in advanced reproductive stages (stage 3 or 4), although 

these stages were also observed in females with GSI values 

<0.8%. In male channel catfish, station means for GSI values 

ranged from 0.2% to 0.4% (table 24; fig. 27). Values <0.1% 

were only observed in stage-0, -1, and -4 males, and GSI 

values were generally greater in stage-2 and -3 males. These 

GSI values are similar to those in female (0.6-2.3%) and male 

(0.02-0.10%) channel catfish from the RGB (BEST-LRMN 

Program, unpublished data). 

GSI (%)

0

1

2

3

atresia (%)

0

3

6

9

12

E2 (pg/mL)

0

300

600

900

1200

KT (pg/mL)

0

200

400

600

800

Station

 311 312 313 314 315 316 317 319 320 321 322 323 324 325

E/KT ratio

0

4

8

12

Upper CDRB Lower CDRB

Figure 26. Reproductive health indicators by station in female 

channel catfish from the Colorado River Basin in 2003. Indicators 

include gonadosomatic index (GSI), vitellogenin (vtg), atresia, 

17β-estradiol (E2), 11-ketotestosterone (KT), and the ratio 

of estradiol and II-ketotestosterone (E/KT). Shown for each 

group are points representing individual fish and the mean (red 

horizontal line), median (black horizontal line), interquartile range 

(box), and the 10th and 90th percentiles (whiskers). Stations are 

ordered from upstream to downstream and are grouped by subbasin. See Table 2 for station descriptions.

Results and Discussion    77
An ANOVA model for oocyte atresia in female channel 

catfish containing the factors station, age, gonadal stage, and 

their interactions was not significant (F10, 13 = 1.53, P > 0.05; 

Appendix 6). Oocyte atresia was ≤6% in most female channel 

catfish (fig. 26) and was not related to gonadal stage. Oocyte 

atresia in females was similar among stations; station means 

ranged from 1.5% at Station 317 to 3.6% at Station 316. In 

addition, oocyte atresia was <10% in fish from Stations 312 

and 324 where the data were categorized. Other studies measuring atretic eggs in channel catfish were not found.

E2 concentrations in channel catfish did not differ among 

stations (F5, 61 = 0.64, P > 0.05), gonadal stage (F1, 61 = 1.13, 

P > 0.05), or between genders (F1, 61 = 1.10, P > 0.05). KT 

concentrations differed among stations (F5, 61 = 3.52, P < 0.01) 

and between genders (F1, 61 = 5.19, P < 0.05) but not among 

gonadal stage (F1, 61 = 0.06, P > 0.05). Most E2 concentrations 

(83%) in female channel catfish were 100-900 pg/mL (table 

25), and station means ranged from 296 pg/mL at Station 317 

to 827 pg/mL at Station 312. Concentrations were relatively 

low (<200 pg/mL) in individual females from Stations 317 

and 324 (fig. 26). The KT concentrations were 50-420 pg/mL 

in most female channel catfish (74%). Station means ranged 

from 92 pg/mL at Station 317 to 733 pg/mL at Station 312 

(table 25). Concentrations were uniformly high (>650 pg/mL) 

in females from Station 312 and low (<305 pg/mL) in those 

from Stations 315, 316, and 317 (fig. 26). Few E/KT ratios 

were <1.0 in female channel catfish. However, ratios >5.0 

were observed in fish from Stations 316, 317, and 324 as a 

result of low KT concentrations in these males (fig. 26). 

In male channel catfish, most E2 concentrations (86%) 

were 80-400 pg/mL (table 25), and station means ranged 

from 62 pg/mL at Station 324 to 239 pg/mL at Station 313. 

Concentrations were relatively low (<200 pg/mL) in fish from 

Stations 316 and 324 (fig. 27). Most (76%) KT concentrations 

were 60-1,000 pg/mL in male channel catfish, and station 

means ranged from 87 pg/mL at Station 317 to 865 pg/mL at 

Station 312 (table 25). Concentrations were uniformly low 

(<400 pg/mL) in males from Stations 316, 317, and 324 (fig. 

27). The E/KT ratio was >5.0 in several males from Station 

317 as a result of low KT concentrations (<81 pg/mL) in these 

fish (fig. 27), and low KT concentrations were also measured 

in female channel catfish from this station. Steroid hormone 

concentrations in channel catfish from other studies were not 

found.

Reproductive Biomarkers: Summary

The reproductive biomarkers used in this study are the 

key measures of reproductive function and are routinely used 

to help evaluate contaminant effects or simply assess general 

reproductive health in fish. Age, species, water temperature, 

photoperiod, and other biotic and abiotic factors can influence these biomarkers over the course of the reproductive 

cycle. Therefore, care must be taken when interpreting these 

reproductive biomarkers and these ancillary factors considered 

when possible. Our evaluations consider the age, species, 

GSI (%)

0.0

0.2

0.4

0.6

0.8

E2 (pg/mL)

0

200

400

600

KT (pg/mL)

0

400

800

1200

Station

 311 312 313 314 315 316 317 319 320 321 322 323 324 325

E/KT ratio

0

3

6

9

Upper CDRB Lower CDRB

Figure 27. Reproductive health indicators by station 

in male channel catfish from the Colorado River Basin 

in 2003. Indicators include gonadosomatic index (GSI), 

vitellogenin (vtg), 17β-estradiol (E2), 11-ketotestosterone 

(KT), and the ratio of estradiol and II-ketotestosterone 

(E/KT). Shown for each group are points representing 

individual fish and the mean (red horizontal line), median 

(black horizontal line), interquartile range (box), and the 

10th and 90th percentiles (whiskers). Stations are ordered 

from upstream to downstream and are grouped by subbasin. See Table 2 for station descriptions.

78    Contaminants, Health Indicators, and Reproductive Biomarkers in Fish from the Colorado River Basin
and gonadal stage directly; while photoperiod and temperature may be considered through indirect information about 

each station (collection date and available gauging station 

information, respectively). All fish samples were collected 

post-spawn and within an eight week time period (August to 

October) to minimize the variation of reproductive biomarkers 

from temperature, photoperiod, and annual reproductive cycle. 

However, natural changes or fluctuations in reproductive biomarkers may have occurred during this collection period. 

The GSI is often used to evaluate reproductive status and 

health, although interpretations of GSI values rely on understanding natural variations among fish of similar age, gender, 

and species. Environmental influences and behavioral patterns 

may also confound the interpretation of the data. Considerable variations in gonad size have been reported throughout 

the reproductive cycle of most species of fish (de Vlaming and 

others, 1981). The gonads can constitute substantially differing proportions of the total body weight of a fish (that is, GSI). 

For example, gonads made up a greater proportion of the total 

body mass in carp than in bass or channel catfish in this study. 

Proportionately larger gonads were found in female carp from 

Stations 314, 317, 321, and 325, male carp from Stations 314 

and 321, female bass from Station 311, and relatively small 

gonads were identified in male carp from Stations 320, 324, 

and 325, female and male bass from Stations 322, 323, and 

325, and female channel catfish from Station 324. Attempts 

were made to minimize the differences in gonad size due to 

collection period by collection the fish post-spawn and within 

an eight week time period. Most GSI values corresponded 

with gonadal stage (that is, GSI values increased as gonadal 

stage advanced). Abnormally low GSI values were measured 

in multiple male carp from Station 324. Exposure to wastewater effluent has been correlated with decrease GSI values in 

fish (Diniz and others, 2005; Gross and others, 2004; Lavado 

and others, 2004). Patiño and others (2003b) reported that 

GSI values in male carp from Las Vegas Bay were consistently 

lower (<7.5%) than males from the Overton Arm of Lake 

Mead regardless of season and suggested that environmental 

contaminants such as PCBs, dioxins, furans, and PBDEs may 

be affecting the reproductive development in male carp. However, reproductive development was not impaired in female 

carp from Las Vegas Bay (Patiño and others, 2003b).

Gonadal histopathology was used to confirm gender, 

assign reproductive stage, and detect anatomical abnormalities 

such as the presence of ovotestes and excessive oocyte atresia. 

Collection date did not appear to affect gonadal stage. Carp 

were in similar stages of gonadal maturity except for male 

carp from Station 324, which were less advanced (stage-0 

and -1) and had low GSI values. These differences were not 

related to age or size of carp from this station. In bass, most 

females and males in the Lower CDRB were less advanced 

than those from the Upper CDRB. Gonadal stage was variable 

in channel catfish.

Oocyte atresia is defined as the involution or resorption of 

oocytes by the ovaries and has been validated as a histopathological biomarker. Although oocyte atresia is a normal physiological event in all fish, it can become a pathological condition following exposure to certain environmental contaminants 

(Cross and Hose, 1988, 1989; Johnson and others, 1988; 

Kirubagaran and Joy, 1988). Other factors such as water temperature can also influence pathological oocyte atresia (June, 

1970, 1977). Oocyte atresia ≥25% in female carp and >10% 

in female bass were defined as high in a previous LRMN study 

(McDonald and others, 2002). Atresia was low in most CDRB 

fish, but individual fish with relatively high atresia were noted 

in carp from Stations 313, 314, 316, and 319 and bass from 

Station 321.

Intersex was identified in carp, bass, and channel catfish 

from seven of 14 CDRB stations. Evidence of ovotestes was 

found in seven of ten male smallmouth bass from Station 311, 

four of ten largemouth bass from Station 322, two of five 

largemouth bass from Station 323, and one of eight carp from 

Station 320. Stations 312, 317, and 324 each had one male 

channel catfish with ovotestes. Gonadal tissue of all intersex 

bass and channel catfish was primarily testicular tissue with 

an apparent invasion of mild to moderate amounts of ovarian 

follicles or oocytes. In contrast, the intersex carp collected 

at Station 320 had gonads which contained mainly ovarian 

tissue with some spermatocytes. During field necropsy of 

this female carp, the gonads were described as dark red lax 

tissue with pouches of clear fluid; other females from this site 

appeared normal with gonads full of eggs. A thin abdominal 

wall and fluid-filled body cavity were also noted for the intersex female carp during the field examination. This is the first 

occurrence of intersex carp and channel catfish in the BESTLRMN Program. Previous LRMN studies have only observed 

ovotestes in bass, which have been predominantly found to 

contain small numbers of oocytes in otherwise normal testicular tissue (Hinck and others, 2004a; McDonald and others, 

2002; Schmitt and others, 2004). To our knowledge, the background occurrence of intersex fish has not been established 

for any of these species, but the high proportion of intersex 

smallmouth bass from Station 311 (70%) is cause for concern. 

Ovotestes were not found in any male smallmouth bass from 

Station 312, located several hundred kilometers downstream 

of Station 311 in the Upper CDRB. These results indicate that 

the fish from Station 311 may have had an estrogenic response 

although the cause is unknown and warrants further investigation. Other reproductive biomarkers in bass from Station 311 

were anomalous. Station 311 was the only station where vtg 

concentrations were not detected in male bass, but concentrations in female bass were the greatest measured (>5.0 mg/mL) 

in CDRB fish. Concentrations of E2 were also relatively high 

(>300 pg/mL) in all male smallmouth bass (including intersex bass) from Station 311; high E2 concentrations in these 

fish could be a result of these fish being in advanced gonadal 

stages (stage 2) and later collection date (late October). 

Vitellogenin concentrations were measured in carp and 

bass but not in channel catfish. In female bass, concentrations were >5 mg/mL in smallmouth bass from Station 311 

but <0.37 mg/mL in smallmouth bass from other stations. 

Concentrations were relatively low (<1.8 mg/mL) in stage-2 

Results and Discussion    79
female carp from Station 324. Other biomarkers were normal 

in carp from this station, but high concentrations of organochlorines have been reported historically in this area. Vitellogenin was detected in male carp and bass from most CDRB 

stations, but concentrations were <LOD in all males from 

Station 320. Concentrations were >0.1 mg/mL in male carp 

from Stations 322 and 323 and male bass from Station 312 

and may indicate that these fish have been exposed to estrogen 

mimics. Some organochlorine pesticides can act like estrogen 

mimics, but concentrations of the organochlorine pesticides 

measured in this study were low in male fish from Stations 

312, 322, and 323. Other estrogen mimics including nonylphenol and ethynylestradiol may affect vtg concentrations 

in male fish but were not measured in this study. Previous 

studies have localized vtg receptors to the testes, muscle, and 

spermatocytes (Bidwell and Carlson, 1995; Tao and others, 

1996), but the function and importance of these receptors in 

male fish are unknown. Detectable concentrations of vtg have 

been documented in male fish as a result of lower assay LODs, 

but concentrations >0.001 mg/mL are generally considered 

anomalous in male fish.

Like many other reproductive biomarkers, concentrations 

of sex steroid hormones can vary by gender, age, geographical 

locations, species, and season (Barry and others, 1990; Bromage and others, 1982; Chang and Chen, 1990; Denslow and 

others, 1999; Down and others, 1990; Goodbred and others, 

1997; So and others, 1989). Variation in sex steroid hormones 

concentrations within each species is expected (McDonald 

and others, 2002), but E/KT ratios >1.0 in females and <1.0 

in males are generally considered normal (Folmar and others, 

1996; Hileman, 1994). 

Sex steroid hormone concentrations differed by species, 

station, and gender in CDRB fish. Steroid hormone concentrations in carp were generally greatest in fish from the Upper 

CDRB. Relatively low E2 and KT concentrations were measured in females from Station 323, 324, and 325 and males 

from Station 319. Gross and others (2004) also reported that 

KT concentrations were low in male fish from Las Vegas Bay 

of Lake Mead and suggested that wastewater effluent may be 

affecting reproductive biomarkers in the fish. Concentrations 

of KT were also relatively low in male bass from Stations 

323 and 324. In contrast, steroid hormone concentrations in 

female bass were greater at Station 322 than those from other 

stations, and E2 concentrations were greatest in male bass 

from Station 311, which included seven intersex fish. Concentrations of KT in female channel catfish were relatively high 

at Station 312 and low at Stations 315, 316, and 317; E/KT 

ratios were >5.0 in multiple female catfish from Station 316. 

In male channel catfish, E2 and KT concentrations were low in 

fish from Stations 316 and 324. The KT concentrations were 

low and E2 concentrations were high in male channel catfish 

from Station 317, resulting in E/KT ratios >5 in many fish.

Spatial patterns in contaminant concentrations

and biomarker responses

Correlations between Chemical and Biological 

Endpoints

Spearman Rank correlations were examined to determine 

if chemical concentrations were related to biomarker responses 

in the CDRB (table 26). Significant correlations (P < 0.05) 

were determined for each gender and species. Few biomarker 

responses were found to be correlated with contaminants and 

present in more than one species, gender, or both (table 26). 

Interpretation of these data may be limited by the range of 

contaminant concentrations and sample sizes.

Geographic Summaries

Geographic station summaries were made to emphasize 

relatively high contaminant concentrations, consistent biomarker responses, or both (table 27). The highlighted findings indicate contaminant concentrations or EROD activities 

that exceeded known thresholds or were outside expected 

ranges relative to other CDRB stations. The colors for the 

fish health indicators and reproductive biomarkers are relative 

and indicate the number, magnitude, or both of the anomalies 

at a station. The summaries are intended to draw attention to 

particular stations discussed in the text, possibly for further 

investigation. Increased frequencies of external lesions or 

elevated HAI scores, which represent the cumulative total 

number of grossly visible internal and external lesions, do not 

necessarily indicate direct contaminant effects. Many factors 

other than contaminants can indirectly influence fish health 

indicators and reproductive biomarkers, including nutrients, 

organic matter, and water temperature. Considerably more is 

known about risk to fish and piscivorous wildlife associated 

with bioaccumulative contaminants and EROD activities than 

about long- and short-term risks represented by the other biomarkers. Therefore, greater relative risk has been associated 

with elevated contaminant concentrations and EROD activities 

than with anomalous fish health indicators or reproductive 

biomarkers (table 27).

Upper Colorado River Basin (Upper CDRB)

The Upper CDRB includes Station 311 on the Yampa 

River, Stations 312 and 313 on the Green River, Station 314 

on the Gunnison River, Stations 315 and 316 on the CDR, and 

Station 317 on the San Juan River. Carp and bass or channel catfish were the target species, but white suckers (Station 

311) and brown trout (Station 314) were collected when target 

species were not found. Certain areas in the Upper Colorado 

including NWRs near Station 311 and 312 provide important nesting sites for large populations of ducks and geese 

and resting and feeding areas for migratory birds. Certain 

80    Contaminants, Health Indicators, and Reproductive Biomarkers in Fish from the Colorado River Basin
Table 26. Statistically significant Spearman rank correlations (P < 0.05) between biological and chemical endpoints in carp (c), bass (b; Micropterus sp.), and channel 

catfish (h). Positive correlations are regular text, and negative correlations are italicized text. Genders were not combined in this analysis because many biomarkers differed between males (m) and females (f). aHSI in bass and channel catfish only. bPercent oocyte atresia was measured in females only. -- no significant correlations.

Biological endpoint

Chemical endpoint

p,p’-DDE Toxaphene TCDD-EQ As Cd Cr Cu Hg Ni Pb Se Zn

Age mc fc, mc fb -- -- -- mc -- -- -- -- --Total Length

-- fc, mc -- fb -- -- fc fh fb, fc, mc, mh fc, mh -- --

Weight

-- fc, mc -- fb -- -- fc fh mb, fc, mc, fh, mh fc, fh, mh -- --

Condition factor

-- -- -- -- -- -- -- -- fh, mh fh, mh mh --

Splenosomatic Index (SSI) fh fc mb, fc mb -- -- -- fb -- -- -- --Hepatosomatic Index (HSI)a mh -- mh -- -- -- fh mh -- -- -- --Gonadosomatic Index (GSI)

-- mc -- fh fc, mh -- -- fb, mb -- mc mc fh

MA-A

-- mc fb, mb -- -- -- mc -- -- -- -- --

Atresiab

-- -- -- -- -- -- -- -- -- -- fh fh

Health Assessment Index (HAI)

-- mc -- -- -- fb -- -- fc, mc fc -- mb

17β-estradiol (E2)

-- -- fc -- fh -- -- -- -- -- -- --

11-ketotestosterone (KT)

-- -- fc -- fh mc -- -- -- -- -- --

E/KT

-- -- fc fb fh -- -- -- fb -- mb --

Vitellogenin (vtg)

-- fc -- -- -- -- -- -- fb -- -- mb

Hepatic EROD activity fh -- -- mb -- mc -- -- mh mh -- --

Results and Discussion    81
Table 27. Summary of chemical and biological indicator results by CDRB station. Within each column, colors indicate the severity, prevalence, or both of the indicated 

condition or conditions at each station (green<yellow<red). These designations are relative; see text for explanations. Species collected included carp (c), bass (b), channel catfish (ct), white sucker (s), brown trout (t), and flathead catfish (ft). See Table 2 and Figure 2 for station and sub-basin locations. Hg, mercury; Se, selenium; DDE, p,p’-DDE; Tox, toxaphene; PCB, total polychlorinated biphenyls; TCDD-EQ, dioxin-like activity as determined by H4IIE bioassay; EROD, ethoxyresorufin 

O-deethylase; EL, 

external lesions; HSI, hepatosomatic index; T, tumor; CF, condition factor; SSI, splenosomatic index; HAI, health assessment index; MA, macrophage aggregates (one or more parameters); GSI, gonadosomatic index; ovt, ovotestes; vtg, vitellogenin; E2, 17β-estradiol; KT, 11-ketotestosterone; E/KT, estradiol/11-ketotestosterone ratio; stage, gonadal stage of reproduction; m, male; f, female. - indicates smaller; all others larger.

Station Species collected Contaminants and EROD Fish health indicators Reproductive biomarkers

Upper Colorado River BasinYampa R. at Lay, CO (311) carp, bass, sucker Hg (b, s); Se (b); EROD (ms) None ovt (mb); vtg (fb); E2 (mb)Green R. at Ouray NWR, UT (312) carp, bass, channel catfish Hg (c, ct); Se (c, ct); EROD (b) EL (c); HSI (-b) ovt (mct); vtg (mb)Green R. at San Rafael, UT (313) carp, channel catfish Hg (c, ct); Se (c) T (ct) NoneGunnison R. at Delta, CO (314) carp, brown trout Se (c, t) CF (-c) NoneColorado R. at Loma, CO (315) carp, bass, channel catfish Hg (b); Se (c, b) EL (b, ct); HAI (b, ct); CF (-c, 

-b); SSI (mc, -b)

None

Colorado R. at Gold Bar Canyon, UT (316) carp, channel catfish Hg (c, ct); Se (c, ct) None NoneSan Juan R. at Hogback Diversion, NM (317) carp, channel catfish Hg (c, ct); Se (c) HAI (ct) ovt (mct); E/KT (mct)Lower Colorado River BasinColorado R. at South Cove, AZ (319) carp only Se (c) HAI (c); MA (c) E2 (-mc); KT (-mc)Colorado R. at Willow Beach, AZ (320) carp only Se (c); PCB (c) HAI (c); MA (c); T (c) ovt (fc)Colorado R. at Needles, CA (321) carp, bass Hg (b); Se (c, b) HAI (c, b); CF (-c, -b); HSI (-b); 

T (c, b)

E/KT (-fb)

Colorado R. at Imperial Dam, AZ (322) carp, bass Se (c, b) HAI (c, b); HSI (-b) stage (-b); ovt (mb); vtg (mc)Gila R. at Hayden, AZ (323) carp, bass, flathead catfish Hg (c, b) None stage (-b); ovt (mb); vtg (mc); E2 

(-fc); KT (-fc)

Gila R. at Phoenix, AZ (324) carp, bass, channel catfish Se (c); DDE (c, ct); Tox (ct); 

PCB (c, ct); TCDD-EQ (c, ct); EROD (c)

SSI (-b, -ct); T (c) stage (-mc, -mb, -ct); ovt (mct); 

GSI (-mc); vtg (-fc)

Gila R. at Arlington, AZ (325) carp, bass Se (c, b); DDE (c, b); Tox (c, b) None stage (-b)

82    Contaminants, Health Indicators, and Reproductive Biomarkers in Fish from the Colorado River Basin
piscivorous species including the bald eagle (Haliaeetus 

leucocephalus) overwinter in the Upper CDRB at Ouray NWR 

(Station 312) and western Colorado (Station 315). Chemical 

contaminants can enter the waters of the Upper CDRB through 

a variety of basin activities and processes. Mining activities have introduced heavy metals including Cd, Cu, Pb, and 

Zn and degraded water quality in many Upper CDRB rivers 

and streams (CDPHE, 1998; UDEQ, 2000). Major commercial deposits of oil shale, tar sands, and coal are located 

in the Upper Green River Basin. Irrigation canals transport 

Se leached from the underlying shales and have contaminated 

many Upper CDRB waters that have resulted in multiple fish 

consumption advisories in rivers and reservoirs (USEPA, 

2004a). Mercury advisories for sport fish in reservoirs of the 

San Juan River and Dolores River Basins are potentially from 

mining and atmospheric deposition from coal-fired power 

plants (Abell, 1994; Melancon and others, 1979). Colorado’s 

largest coal-fired power plants located along the Yampa River 

near Craig have high Hg emissions and acid deposition, which 

has been linked to reproductive impairments in amphibians in 

north central Colorado (Turk and Campbell, 1997). 

Yampa River, Lay, CO (Station 311)

Station 311 was located on the Yampa River near Lay, 

Colorado. Smallmouth bass and white sucker were collected 

in late October 2003. Several contaminants and biomarkers 

exceeded threshold criteria or appeared anomalous in fish 

(table 27). Selenium concentrations were >0.75 μg/g in all 

samples and >1.0 μg/g in female smallmouth bass (fig. 28); 

these concentrations are potentially hazardous to fish and 

piscivorous wildlife (Lemly, 1996). Mercury concentrations 

ranged from 0.18 μg/g to 0.27 μg/g and were among the greatest measured in this study (fig. 28). Mercury concentrations 

>0.1 μg/g may be harmful to piscivorous mammals (Yeardley 

and others, 1998). Seven of 10 male bass from Station 311 

were intersex (fig. 29), which is one of the greatest percentages of intersex fish found at a site by the BEST-LRMN Program. Hepatic EROD activity was relatively high (>25 pmol.

min/mg) in male white sucker. Altered sex ratios have been 

documented in the offspring of fish with whole-body Hg concentrations of 0.44-1.1 μg/g (Matta and others, 2001), but it is 

unknown whether the relatively high Hg concentrations and 

intersex condition in male bass from Station 311 were related. 

The estrogenic effects of metals and metal mixtures (for 

example, Le Guével and others, 2000) are largely unknown but 

should be considered when interpreting reproductive biomarkers. Concentrations of E2 were elevated in all male bass (>400 

pg/mL) from this station as compared to other stations, yet vtg 

concentrations in these same fish were within normal ranges. 

Relatively high sex steroid hormone concentrations and low 

vtg concentrations can occur when circulating steroid hormones are bound to steroid hormone binding proteins in the 

plasma and are not free to enter into target tissues such as the 

liver (Tollefsen, 2002). Further investigations are required to 

determine whether these concentrations are a result of contaminant exposure. GSI values were normal in the intersex fish, 

which is consistent with the lack of induction of vtg found 

in males from this station. Concentrations of vtg in female 

bass from Station 311 were also greater than observed at other 

stations, and the mean vtg concentration in female bass (8.4 

mg/mL) was among the greatest measured in bass from any 

LRMN investigation. Station 311 was sampled last (late October) in our study (table 2); this later collection may partially 

explain the higher vtg concentrations. Overall, reproductive 

biomarkers were anomalous at this site. The high incidence of 

intersex smallmouth bass has rarely been documented at this 

magnitude in previous studies. Facilities commonly associated with estrogenic contaminants (for example, wastewater 

treatment plants) are not located near Station 311, but water 

from the Yampa River is diverted near Station 311 to irrigate 

fields of alfalfa or grass hay along the river (R. Krueger, written communication). Further research is needed to determine 

the cause of intersex fish in the Yampa River. 

Green River, Ouray NWR, UT (Station 312)

Station 312 was located in the Ouray NWR along the 

Green River in Utah, an area known for its historical Se contamination. Carp, smallmouth bass, and channel catfish were 

collected in September 2003. Several elemental contaminants 

and biomarkers exceeded threshold criteria or appeared anomalous in fish (table 27). Selenium concentrations were >0.9 

μg/g in channel catfish and >1.4 μg/g in carp (fig. 28); these 

concentrations are potentially harmful to fish and piscivorous 

wildlife (Lemly, 1996). Mercury concentrations exceeded 

0.1 μg/g, which may be hazardous to piscivorous mammals 

(fig. 28; Yeardley and others, 1998). Hepatic EROD activities in female and male bass were uniformly greater at Station 

312 compared to other CDRB stations but were generally 

lower than those from previous LRMN investigations (Hinck 

and others, 2004a; Schmitt and others, 2004). Decreased 

liver sizes, as reflected by the low HSI values (most <1.0%), 

were also found in bass although histopathological examination determined liver tissue to be normal in these fish. The 

highest vtg concentrations (>0.28 mg/mL) in male bass were 

measured from this site (fig. 29) and may indicate isolated 

estrogenic responses to environmental conditions at Station 

312. A stage-0 male channel catfish was intersex, containing 

primarily testicular tissue with previtellogenic oocytes (fig. 

29). External lesions were identified on 74% of carp from 

Station 312 and were related to parasitic infestations which 

caused frayed fins and nodules on the body surface and fins. 

Further studies are needed to determine the cause of anomalous biological endpoints in fish from this site.

Green River, San Rafael, UT (Station 313)

Station 313 was located on the Green River near the 

confluence of the San Rafael River. Carp and channel catfish 

were collected in September 2003. Mercury and Se were the 

only environmental contaminants that exceeded threshold 

criteria in fish (table 27). The highest Hg concentrations (>0.3 

Results and Discussion    83
μg/g) of this study were measured in channel catfish from 

Station 313 (fig. 28) and may cause reproductive impairment 

in sensitive avian species (Wiener and Spry, 1996; Wiener and 

others, 2002). In addition, Hg concentrations in carp (>0.1 μg/

g) may be a threat to piscivorous mammals (Yeardley and others, 1998). Selenium concentrations in carp (>1.6 μg/g) may 

be hazardous to fish and piscivorous wildlife (fig. 28; Lemly, 

1996). Tumors, a fibroma and a papilloma, were observed 

on catfish from Station 313. Other fish health indicators and 

reproductive biomarkers were not anomalous in fish from this 

site. 

Gunnison River, Delta, CO (Station 314)

Station 314 was located on the Gunnison River near 

Delta, Colorado. Carp and brown trout were collected in 

September 2003. Few contaminants and biomarkers exceeded 

threshold criteria or appeared anomalous in fish (table 27). 

Selenium was the only contaminant to exceed protective 

criteria for fish and piscivorous wildlife (Lemly, 1996), and 

concentrations were >2.9 μg/g in carp, the highest measured in 

this study, and 1.6 μg/g in trout (fig. 28). Natural weathering 

of Mancos shale is the primary source of Se in the Gunnison 

River Basin (Osmundson and others, 2000). Many carp also 

had relatively low body mass, as reflected by CF values <0.8 

in these fish. Several studies have reported that lower CF values are associated with Se accumulation in fish (see review by 

Hamilton, 2004). Other fish health indicators and reproductive 

biomarkers were not anomalous in fish from this site.

Colorado River, Loma, CO (Station 315)

Station 315 was located on the CDR at the Loma Boat 

Launch downstream of Grand Junction, Colorado. Carp, 

largemouth bass, and channel catfish were collected in September 2003. Several elemental contaminants and biomarkers 

exceeded effects thresholds or appeared anomalous in fish 

(table 27). Selenium concentrations in carp and channel catfish (>2.0 μg/g) were potentially harmful to fish and piscivorous wildlife (fig. 28; Lemly, 1996). The Gunnison River 

confluence with the CDR, upstream of Station 315, contributes 

large amounts of Se from the marine shales of western Colorado to the CDR. Mercury concentrations were generally low, 

but concentrations in female bass (0.1 μg/g) were potentially 

hazardous to piscivorous mammals (Yeardley and others, 

Figure 28. Maximum concentrations (μg/g ww) of selenium 

(Se) and mercury (Hg) in composite samples of whole fish. For 

Se, concentrations should be <0.75 μg/g ww to avoid toxicity 

to piscivorous wildlife and <1.0 μg/g ww to avoid toxicity to fish 

(Lemly, 1996). A Hg concentration of 0.1 μg/g ww in fish has 

been suggested as a guideline for the protection of piscivorous 

mammals (Yeardley and others, 1998), and concentrations of 0.3 

μg/g ww can cause reproductive impairments in the common 

loon (Gavia immer; Wiener and Spry, 1996; Wiener and others, 

2002). See Table 2 for station descriptions.

84    Contaminants, Health Indicators, and Reproductive Biomarkers in Fish from the Colorado River Basin
1998). External lesions were found on 75% of bass and 80% 

of channel catfish, which contributed to the elevated HAI 

scores in these taxa. Liver discoloration, granular liver, kidney, and spleen, and body surface lesions in bass and head and 

eye lesions and liver discoloration in channel catfish elevated 

HAI scores. Histopathological examination determined that 

liver, gonad, spleen, head kidney, and hind kidney tissues in 

most bass were infested with parasites, and external lesions 

collected for histopathology contained hemorrhages, inflammation, and repair from parasitic infestations. Similar to 

carp from Station 314, low CF values (<0.8) were calculated 

for several carp and bass and may be associated with high 

Se concentrations (Hamilton, 2004). A SSI value of 1.74% 

was the highest calculated in male carp from this study and 

previous LRMN investigations. In contrast, SSI values in bass 

were 0.02-0.47%; values <0.03% have rarely been reported in 

previous LRMN studies. Reproductive biomarkers were not 

anomalous in fish from Station 315.

Colorado River, Gold Bar Canyon, UT (Station 316)

Station 316 was located in Gold Bar Canyon of the CDR 

downstream of Moab, Utah. Carp and channel catfish were 

collected in September 2003. Mercury and Se concentrations 

exceeded protective criteria for fish (table 27). Concentrations 

of Hg in carp (0.13-0.14 μg/g) and channel catfish (0.18 μg/g) 

may pose a threat to piscivorous mammals (fig. 28; Yeardley 

and others, 1998). Selenium concentrations were >2.0 μg/g 

in carp and >1.0 μg/g in channel catfish and may be hazardous to fish and piscivorous wildlife (fig. 28; Lemly, 1996). 

Fish health indicators and reproductive biomarkers were not 

anomalous in fish from this station.

San Juan River, Hogback Diversion, NM (Station 317)

Station 317 was located near the Hogback Diversion 

along the San Juan River downstream of Farmington, New 

Mexico. Carp and channel catfish were collected in September 2003. Several elemental contaminants and biomarkers 

exceeded threshold criteria or appeared anomalous in fish 

(table 27). Concentrations of Hg in carp and channel catfish 

(≥0.1 μg/g) may be harmful to piscivorous mammals (fig. 28; 

Yeardley and others, 1998). Selenium concentrations in carp 

(>1.2 μg/g) may pose a threat to fish and piscivorous wildlife 

(fig. 28; Lemly, 1996). Several biological endpoints were 

anomalous in channel catfish from this site. The greatest mean 

Figure 29. Plasma vitellogenin (vtg) concentrations in male 

fish and occurrence of intersex fish. For vtg, thresholds indicate 

stations where at least one male had a detectable concentration 

of vtg (>0.01 mg/mL). Concentrations >0.1 mg/mL indicate an 

estrogenic response to environmental contaminants. Percentage 

of CDRB fish with intersex (that is, gonads containing ovarian and 

testicular tissue). See Table 2 for station descriptions. M, male; 

F, female; SMBS, smallmouth bass; CNCF, channel catfish; LMBS, 

largemouth bass; CARP, common carp.

Results and Discussion    85
HAI score (81) in this study was calculated for channel catfish 

from Station 317. Head and eye lesions, discolored livers, and 

granular spleens contributed to the elevated HAI scores. Liver 

tissue from fish at Station 317 had focal areas of inflammation and necrosis, and microsporidan cysts were occasionally 

observed within the hepatic parenchyma. A stage-0 male 

channel catfish was intersex, containing primarily testicular 

tissue with previtellogenic oocytes (fig. 29). The E/KT ratios 

were high (>5.0) in multiple male channel catfish as a result of 

uniformly low KT concentrations (<81 pg/mL). 

Lower Colorado River Basin (Lower CDRB)

The Lower CDRB includes Stations 319, 320, 

321, and 322 on the CDR and Stations 323, 324, and 

325 on the GR. Carp and bass or channel catfish were 

the target species, but flathead catfish were also collected from Station 323. The many NWRs in the Lower 

CDRB provide wintering areas and stopover points for migratory birds and are the primary source of water in some of the 

more arid areas. Water quality impairments in the Lower 

CDRB are primarily associated with agriculture and Cu mining. Agricultural drainage canals have transported pesticides 

and contributed to elevated concentrations of organochlorines 

and metals in fish from the GR, and these areas along the GR 

are important to Threatened and Endangered species, migratory birds, and waterfowl (Baker and others, 1992; King and 

others, 1997). Consequently, pesticides and Se associated 

with agricultural return flows could be harmful to fish and 

wildlife in the Lower CDR (Baker and others, 1992; CEPA, 

2003). Selenium is transported in silt and sediment from the 

Upper CDRB to the Lower CDR, where high concentrations 

have been measured in fish since the 1980s (King and others, 

1993). Other elemental contaminants including As, Cd, Cr, 

Cu, and Pb exceeded protective criteria in fish from the Lower 

CDR in previous studies (Andrews and others, 1997). Copper and Zn impairments in the Upper GR are likely the result 

of extensive mining (ADEQ, 2004; King and Baker, 1995; 

NMED, 2000), and relatively high concentrations of Cu, Pb, 

and Zn have been measured in fish near these mines (Andrews 

and King, 1997). The GR is unique in that arid conditions 

cause ephemeral flows in some portions of the river, while 

the flow downstream of Phoenix is controlled by releases of 

wastewater treatment plant effluent and storm runoff (Anning, 

2003; Arnold and others, 2004; Gebler, 1998). In addition, 

several lakes in southern Arizona including Alamo Lake on 

the Bill Williams River have Hg advisories for sport fish, and 

potential sources include historic ore milling and amalgamation processes, naturally mineralized soils, and atmospheric 

deposition (USEPA, 2004a). Studies in Las Vegas Bay have 

documented relatively high occurrences of environmental 

contaminants including PCBs, dioxins, furans, and PBDEs in 

sediments (Covey and Beck, 2001) and disruptions in reproductive fish health (Bevans and others, 1996; Gross and others, 

2004; Patiño and others, 2003b).

Colorado River, South Cove, AZ (Station 319)

Station 319 was located at South Cove, Arizona, in the 

Gregg Basin of Lake Mead. Carp were collected in early 

October 2003. Selenium and several biomarkers exceeded 

threshold criteria or appeared anomalous in fish (table 27). 

Selenium concentrations in carp (>2.1 μg/g) were potentially 

hazardous to fish and piscivorous wildlife (fig. 28; Lemly, 

1996). Liver discoloration, granular spleen and kidney tissues, and pale gills accounted for most of the elevated HAI 

scores. Macrophage aggregates in carp were >15,000 μm2, 

the largest measured in any LRMN study (Blazer and others, 

2002; Hinck and others, 2004a; Schmitt and others, 2004), and 

carp from Station 319 were also among the oldest in our study. 

Steroid hormone concentrations were uniformly low in male 

carp, although they were in similar gonadal stages as carp 

from other stations. These low hormone concentrations may 

indicate an endocrine response in male carp from this site. A 

study in Las Vegas Bay of Lake Mead, downstream of South 

Cove, also found decreased KT concentrations in fish and concluded that exposure to wastewater effluent may have altered 

reproductive biomarkers in fish (Gross and others, 2004). 

Colorado River, Willow Beach, AZ (Station 320)

Station 320 was located on the CDR downstream of 

Hoover Dam near the Willow Beach Fish Hatchery in Arizona. 

Carp were collected in late September 2003. Several contaminants and biomarkers exceeded threshold criteria or appeared 

anomalous in fish (table 27). Selenium concentrations in carp 

(>1.6 μg/g) may be hazardous to fish and piscivorous wildlife 

(fig. 28; Lemly, 1996). Concentrations of PCBs in male carp 

(0.9 μg/g) and female carp (1.6 μg/g) exceeded the NYSDEC 

guideline (0.11 μg/g) to protect wildlife (Newell and others, 

1987). Relatively high HAI scores were attributed to discolored livers, granular spleen and kidney, and pale gills. Fibrotic 

fin lesions with inflammation and increased numbers of melanocytes were identified on two carp from Station 320. Similar 

to Station 319, MAs were >15,000 μm2 in several carp, and 

carp were older than those from other CDRB stations. One 

female carp was intersex, containing primarily ovarian tissue 

with a few spermatocytes (fig. 29). The lowest GSI value 

(1.5%) and E2 concentration (162 pg/mL) from this station 

were mesured in the intersex female, but vtg data was unavailable for this fish. Intersex carp have not been found in previous LRMN studies; however, one intersex carp was reported in 

a recent Lake Mead study (Snyder and others, 2004). Patiño 

and others (2003b) suggested that low GSI values in male 

carp from Las Vegas Bay may be associated with exposure to 

environmental contaminants such as PCBs, dioxins, furans, 

and PBDEs. Aquatic biota in Las Vegas Bay and the Lower 

CDR below Hoover Dam may be at risk from exposure to 

these and other contaminants (for example, pharmaceuticals 

and personal care products) as the population of the Las Vegas 

area grows. 

86    Contaminants, Health Indicators, and Reproductive Biomarkers in Fish from the Colorado River Basin
Colorado River, Needles, CA (Station 321)

Station 321 was located on the CDR downstream of 

Needles, California near the Park Moabi Recreation Area. 

Carp and largemouth bass were collected in September 2003. 

Mercury, Se, and several biomarkers exceeded threshold criteria or were anomalous in fish (table 27). Mercury concentrations in female bass (0.12 μg/g) may be a threat to piscivorous 

mammals (fig. 28; Yeardley and others, 1998). Selenium concentrations in bass (>1.8 μg/g) and carp (>2.5 μg/g) exceeded 

protective criteria for fish and piscivorous wildlife (fig. 28; 

Lemly, 1996). Liver discoloration, granular spleen and kidney, 

and pale gills in carp and liver discoloration, granular liver, 

kidney, and spleen, and body surface lesions in bass accounted 

for most elevated HAI scores. A number of fish from this site 

exhibited fatty infiltration and degeneration of hepatocytes, 

large MAs, and a very high helminth parasite load in the liver, 

kidney, and spleen. Papillomas were also found on a carp and 

bass from Station 321. Similar to fish from Station 315, low 

CF values (<0.8) in several carp and bass may be related to 

high Se concentrations in these fish (Hamilton, 2004). The 

HSI values in some bass were relatively low (<1.0%). The 

E/KT ratios were <1.0 in six female bass. Overall, E2 concentrations were low (<300 pg/mL) and KT concentrations were 

high (>500 pg/mL) compared to other CDRB female bass. 

Further studies are warranted to determine the cause of the 

multiple anomalous fish health indicators from this site.

Colorado River, Imperial Dam, AZ (Station 322)

Station 322 was located on the CDR downstream of the 

Imperial Dam. Carp and largemouth bass were collected in 

September 2003. Selenium and several biomarkers exceeded 

threshold criteria or appeared anomalous in fish (table 27). 

Selenium concentrations in bass (>2.7 μg/g) and carp (>2.3 

μg/g) exceeded protective criteria for fish and piscivorous 

wildlife (fig. 28; Lemly, 1996). Mean HAI scores were relatively high in bass (103) and carp (49). Anomalies were similar to those found in fish from Station 321 and included liver 

discoloration, granular liver, kidney, and spleen, pale gills, 

and body surface lesions. The HSI values were low (<1.0%) 

in multiple bass. Female and male bass were generally less 

advanced (all stage 1) than bass from other stations. Four of 

10 male largemouth bass were intersex, containing primarily testicular tissue with few previtellogenic oocytes (fig. 29); 

other reproductive biomarkers including GSI, vtg, and steroid 

hormones appeared normal in the intersex fish. The highest 

vtg concentration in male carp (>0.31 mg/mL) was measured 

at this station (fig. 29) and was greater than concentrations in 

some CDRB female carp. Very low concentrations of E2 (31 

pg/mL) and KT (53 pg/mL) were also measured in this fish. 

Further investigation is needed to understand the anomalous 

reproductive biomarker responses in fish from Station 322.

Gila River, Hayden, AZ (Station 323)

Station 323 was located on the Upper GR near Hayden, 

Arizona. Carp, largemouth bass, and flathead catfish were collected in late August 2003. Mercury and several reproductive 

biomarkers exceeded threshold criteria or appeared anomalous 

(table 27). Mercury concentrations in carp (0.14-0.16 μg/g) 

and male bass (0.1 μg/g) may be hazardous to piscivorous 

mammals (fig. 28; Yeardley and others, 1998). Gill tissue 

was histologically normal for most bass and flathead catfish 

at Station 323, but focal areas of hyperplasia leading to fused 

secondary lamellae were observed in some fish. Gill tissue from most carp at Station 323 had some hyperplasia and 

fusion of secondary lamellae, which may relate to contaminant 

exposure, but monogenetic trematodes were also present in the 

gill tissue. Like Station 322, female and male bass were generally less advanced (all stage 1) and were younger (<2 y) than 

bass from other stations. Two of five male largemouth bass 

were intersex. KT concentrations were relatively low in the 

intersex bass as reflected in the E/KT ratios >1.0. Intersex fish 

also had greater GSI values (0.96%) compared to female bass 

from this station. Concentrations of vtg were >0.1 mg/mL in 

three male carp and may indicate an estrogenic response in 

these fish. Relatively low concentrations of E2 (<400 pg/mL) 

and KT (<200 pg/mL) were measured in female carp, which 

did not appear to be related to differences in gonadal stage. 

More studies are needed to determine the cause of anomalous 

reproductive biomarkers in fish from this site.

Gila River, Phoenix, AZ (Station 324)

Station 324, located on the GR downstream of its confluence with the Salt River near Estrella Park, has a history of 

pesticide contamination in fish. Carp, largemouth bass, and 

channel catfish were collected in late August 2003. Several 

contaminants and biomarkers exceeded threshold criteria or 

appeared anomalous in fish (table 27). Selenium concentrations in carp (>1.7 μg/g) and channel catfish (>0.6 μg/g) 

exceeded protective criteria for fish and piscivorous wildlife 

(fig. 28; Lemly, 1996). The greatest concentrations of banned 

organochlorine pesticides or pesticide products including 

p,p’-DDE, toxaphene, total chlordanes, dieldrin, endrin, and 

hexachlorobenzene were in fish from Station 324. Concentrations of pentachlorobenzene, PCA, γ-HCH, dacthal, and 

methoxychlor, all currently registered pesticides or pesticide 

products, were also relatively high in fish from Stations 324. 

Concentrations of p,p’-DDE and toxaphene exceeded available protective criteria. Concentrations of p,p’-DDE in carp 

(>0.3 μg/g) and channel catfish (>0.6 μg/g) may be harmful to 

sensitive avian species (Anderson and others, 1975) and other 

wildlife (Newell and others, 1987). Concentrations of o,p’-

DDD (0.006-0.010 μg/g) were also relatively high. Toxaphene 

concentrations in male channel catfish (0.50 μg/g) exceeded 

toxicity thresholds to protect fish (Eisler and Jacknow, 1985; 

Jarvinen and Ankley, 1999). Concentrations of PCBs in all 

samples exceeded the NYSDEC guideline (0.11 μg/g) to 

protect wildlife (Newell and others, 1987), and TCDD-EQs 

Results and Discussion    87
concentrations in carp and channel catfish also exceeded the 

toxicity threshold for avian wildlife (5 pg/g; Nosek and others, 1992). Hepatic EROD activities were generally greater 

in female and male carp from Station 324 compared to other 

stations and exceeded basal EROD activity in carp, which 

may indicate exposure to PAHs, PCBs, and dioxins (Schmitt 

and others, 2002). SSI values were relatively low in bass 

(≤0.03%; n = 2) and channel catfish (≤0.11%), but histopathological examination determined spleen tissue to be normal 

in most fish. Decreased spleen sizes have been associated 

with exposure to PCBs, PAHs, and metals (see Schmitt and 

Dethloff, 2000). A hematoma on an opercle was identified on 

a carp from Station 324. The gonadal development of male 

carp, male bass, and channel catfish at Station 324 were less 

advanced compared to male carp, male bass, and channel 

catfish from other CDRB sites. Histopathological examination revealed that the testes of these male fish were abnormal, 

containing calcified, poorly developed, edemic, or parasitic 

tissues; these abnormalities were not found in fish from other 

stations. Some of these observations have been previously 

associated with exposure to wastewater effluent in carp (Diniz 

and others, 2005; Lavado and others, 2004). Moreover, one 

male channel catfish was intersex (fig. 29). The GSI values in 

male carp were low (<1.0%), which may be associated with 

estrogenic chemicals in the water (Diniz and others, 2005; 

Hassanin and others, 2002; Lavado and others, 2004). Relatively low vtg concentrations (<2.0 mg/mL) were measured 

in female carp. These data indicate that contaminants may be 

interfering with steroidogenesis. Many of the organochlorine 

pesticides measured at this site have been associated with 

reproductive effects and histological changes in gill, liver, and 

kidney tissues in fish (ATSDR, 2002; McDonald, 1991; Ortiz 

and others, 2003; Shukla and Pandey, 1986; USEPA, 2002; 

Versonnen and others, 2004). Studies have reported that o,p’ 

homologs were estrogenic and caused delayed gonad development and altered plasma vtg concentrations (Ackerman and 

others, 2002; Donohoe and Curtis, 1996; Guillette and others, 

1996; Metcalfe and others, 2000; Papoulias and others, 2003; 

Toppari and others, 1996). Wastewater effluent dominates 

the water in the GR downstream of Phoenix, and reproductive effects in fish from Station 324 may be associated with 

contaminants in the effluent. Slow, intermittent flows and 

high water temperatures (>25°C) also need to be considered 

as they may also influence reproductive biomarker responses. 

Further investigations are warranted to determine the cause(s) 

of altered reproductive biomarkers in multiple taxa from this 

site and to continue to monitor pesticide contamination in fish 

from the GR.

Gila River, Arlington, AZ (Station 325)

Station 325 was located on the GR near Arlington, 

Arizona. Carp and largemouth bass were collected in August 

2003. Several environmental contaminants exceeded threshold 

criteria (table 27). Selenium concentrations potentially hazardous to fish and piscivorous wildlife were measured in carp 

(>2.1 μg/g) and bass (>1.4 μg/g; Lemly, 1996). Like Station 

324, fish from Station 325 had high concentrations of banned 

and currently registered pesticides or pesticide products. 

Specifically, concentrations of p,p’-DDE, toxaphene, endosulfan II, endosulfan sulfate, and methoxychlor were greatest 

in fish from Station 325. Concentrations of pentachlorobenzene, PCA, γ-HCH, dacthal, and methoxychlor, all currently 

registered pesticides or pesticide products, were also relatively 

high in fish from Stations 325. Like Station 324, p,p’-DDE 

and toxaphene exceeded protective criteria. Concentrations of 

p,p’-DDE in carp >1.3 μg/g) and channel catfish (>1.6 μg/g) 

may be harmful to sensitive avian species (Anderson and others, 1975) and other wildlife (Newell and others, 1987). Concentrations of o,p’-DDD (0.024-0.037 μg/g) were the greatest 

measured in the study. Toxaphene concentrations in carp (0.50 

μg/g) and bass (0.60 μg/g) exceeded toxicity thresholds to 

protect fish (Eisler and Jacknow, 1985; Jarvinen and Ankley, 

1999). Although fish health indicators and reproductive biomarkers were not anomalous at this site, many of the organochlorine pesticides measured at this site have been associated 

with reproductive, developmental, and histological effects in 

fish (Ackerman and others, 2002; ATSDR, 2002; Donohoe 

and Curtis, 1996; Guillette and others, 1996; McDonald, 1991; 

Metcalfe and others, 2000; Ortiz and others, 2003; Papoulias and others, 2003; Shukla and Pandey, 1986; Toppari and 

others, 1996; USEPA, 2002; Versonnen and others, 2004). 

Similar to bass from Stations 322 and 323, female and male 

bass were generally less advanced (stage 1 and 0, respectively) 

and were younger (<3 y) at Station 325 compared to bass from 

most other sites. Pesticide contamination in fish should be 

continued to be monitored at this site. 

Conclusions

Few CDRB fish had evidence of being exposed to 

extremely high concentrations of toxic chemicals, but concentrations of some organochlorine pesticides and elemental 

contaminants were elevated and may pose a risk to aquatic 

systems and fish-eating wildlife. Fish from several CDRB stations may have responded to chronic contaminant exposure as 

indicated by fish health indicator and reproductive biomarker 

results.

The agricultural industry in the Lower CDRB is one of 

the most productive in the U.S. and relies heavily on irrigation 

canals and pesticide applications for high crop yields. Pesticides have also been heavily applied in the GR for residential 

use (Gellenbeck and Anning, 2002). Chemicals such as arsenic-based defoliants, stable organic herbicides and insecticides, metals, and salts are potentially accessible to wildlife 

(Aritola and Dubois, 1995). Pre-emergent pesticides (for 

example, simazine, trifluralin, and dacthal) are most frequently 

detected in the GR from December to April (Gellenbeck and 

Anning, 2002), which indicates that fish may be exposed 

to the contaminants prior to spawning. Concentrations of 

88    Contaminants, Health Indicators, and Reproductive Biomarkers in Fish from the Colorado River Basin
pesticides were generally less than the current established 

water-quality limits, however. Previous studies have reported 

that fish and wildlife may be at risk from p,p’-DDT and other 

pesticides in agricultural areas of the GRB and Lower CDRB 

(ADEQ, 1992; Baker and others, 1992; García-Hernández and 

others, 2001, 2006; Gebler, 2000; King and others, 1993;1997; 

Schmitt and others, 1999), and multiple fish consumption 

advisories are listed for these waters (USEPA, 2004a). Our 

findings support these conclusions and also indicate that 

unlisted and restricted use organochlorine pesticide concentrations (for example, PCA, dacthal, endosulfan) are elevated in 

the GR and should be monitored. Although concentrations 

were generally low in CDRB fish, restricted use and unlisted 

organochlorine pesticides have been associated with histological, developmental, and reproductive effects in fish and other 

wildlife (ATSDR, 2002; McDonald, 1991; Ortiz and others, 

2003; Shukla and Pandey, 1986; USEPA, 2002; Versonnen and 

others, 2004).

Concentrations of p,p’-DDE and toxaphene in the Lower 

GRB may be hazardous to fish and piscivorous wildlife; 

o,p’-DDD, PCBs, TCDD-EQs, and EROD activity were also 

elevated in fish downstream of Phoenix, Arizona. PBDEs 

were also detected in fish samples from Stations 324 and 325 

in the GR. Fish from Station 324 (Phoenix, Arizona) also had 

several reproductive biomarkers that were noteworthy. The 

gonadal development of male carp, male bass, and channel 

catfish at Station 324 were less advanced compared to male 

carp, male bass, and channel catfish from other CDRB sites, 

and reduced gonad size (that is, low GSI values) was also 

noted in male carp and bass from Station 324. In addition, vtg 

concentrations were relatively low in female carp from Station 

324. These anomalous biomarkers were observed in fish from 

Station 324 but not Station 325 further downstream on the 

GR, where fish were collected several days prior to those from 

Station 324. We conclude that municipal inputs including 

wastewater treatment plant effluent and urban runoff from the 

Phoenix metropolitan area are likely involved in the reproductive biomarker responses in fish from Station 324. 

The GR and its tributaries have ephemeral or intermittent 

flows and are primarily recharged by irrigation return water, 

storm water, and wastewater treatment plant effluent (Anning, 

2003; Gebler, 1998). Aquatic invertebrate community data 

indicated that water quality in effluent-dependent reaches near 

Phoenix was poor (Gebler, 1998). Arnold and others (2004) 

reported that effluent-dominated waters near Tucson and Phoenix contained the highest concentrations of known estrogenic 

compounds in a nationwide survey and have the potential to 

negatively impact aquatic life. Moreover, estrogenic chemicals have been correlated with inhibited testes growth and 

delayed gonadal maturation (Diniz and others, 2005; Hassanin and others, 2002; Lavado and others, 2004). Exposure 

to estrogenic chemicals including o,p’-DDT and o,p’-DDE in 

fish have been associated with decreased HSI values, plasma 

vtg concentrations, and lipid levels (Donohoe and Curtis, 

1996) and altered GSI values (Papoulias and others, 2003; 

Ungerer and Thomas, 1996). Continuous exposure to estrogenic compounds such as o,p’-DDT from in ovo and throughout early development can affect reproductive endpoints in 

fish (Metcalfe and others, 2000). Anomalous reproductive 

biomarkers in fish from Station 324 are likely associated with 

contaminants in the wastewater-dominated effluent in the GR 

downstream of Phoenix. Altered reproductive biomarkers 

were also noted in fish from Stations 319 and 320 near Lake 

Mead. Several studies concluded that exposure to wastewater 

effluent and municipal runoff in Lake Mead may be altering reproductive biomarkers in fish (Gross and others, 2004; 

Patiño and others, 2003b). Studies are needed to determine 

the effects and ecological significance of wastewater effluent 

and agricultural runoff on fish from these areas. As found in 

our study, new or emerging contaminants such as PBDEs are 

present in effluent downstream of urban areas but their effects 

on fish are largely unknown. More studies, particularly downstream of Las Vegas and Phoenix, are warranted to describe 

the distribution and potential effects of these new contaminant 

concentrations in aquatic systems. Other known estrogenic 

compounds including nonlyphenol and ethoxylates commonly 

found in effluents should also be monitored. Furthermore, 

investigations are needed that describe how contaminant 

effects in fish may be altered in arid environments like the 

GRB. 

Selenium-rich soils are produced by the weathering of 

marine shales and are widespread in the arid and semiarid 

regions of the U.S. including the Upper CDRB. Substantial 

irrigation is required for agricultural crop production in the 

Upper CDRB, and water in irrigation return flows may be 

highly contaminated with dissolved selenium salts that have 

been leached from the soil (Lemly, 1996). Our study addresses 

recommendations of previous monitoring efforts to examine 

the effects of Se in fish and correlate degraded fish health 

with contaminants (Abell, 1994). Previous investigations 

have determined Se concentrations were elevated in sediment, 

water, and biota in the Upper CDRB (Abell, 1994; Butler 

and Leib, 2002; Deacon and Stephens, 1998; Engberg, 1999; 

Schmitt and others, 1999; Seiler and others, 1999; Spahr and 

others, 2000; Stephens and others, 1992). However, some of 

the highest Se concentrations in fish have been reported in the 

Lower CDR (Andrews and others, 1997; Baker and others, 

1992; King and others, 1993; Schmitt and others, 1999). Elevated Se concentrations in the Lower CDR are likely the result 

of transporting Se from the Upper CDRB rather than sources 

from local agricultural practices (Welsh and Maughan, 1994). 

Our findings support conclusions from previous CDRB studies 

that Se concentrations may pose a risk to fish and piscivorous 

wildlife. Concentrations of Se exceeded protective criteria for 

fish, piscivorous wildlife, or both at all CDRB stations. Concentrations were greatest in carp from Stations 314, 321, 322, 

and 325 and bass from Station 322, but teratogenic defects (for 

example, spine, head, and mouth deformities) associated with 

selenium toxicosis were rare in fish from these sites (Lemly, 

1997). Edema, exopthalmus, and cataracts, which were noted 

in fish from Stations 314, 315, 320 and 322, can also be selenium-induced (Lemly, 1997). However, the lack of teratoConclusions    89
genic defects in CDRB fish does not preclude that the risk to 

Se is minimal. Multiple studies have documented reproductive effects in fish (egg to adult) exposed to high Se concentrations (Gillespie and Baumann, 1986; Schultz and Hermanutz, 

1990; Hermanutz and others, 1992; Coyle and others, 1993; 

Hamilton and others 2005b, 2005c). Studies examining the 

effects of elevated Se concentrations in multiple life stages of 

CDRB fish are limited to the razorback sucker (Hamilton and 

others, 2005a, 2005b, 2005c), but studies with other species 

are warranted. The effects of Se on reproduction of the endangered razorback sucker in the Lower CDRB, where Se concentrations were highest in our study, are unknown but could 

provide relevant information on their limited populations in 

the Lower CDR. High Se concentrations have been associated 

with low CF values from fish being underweight (Hamilton, 

2004). Low CF values were noted in fish from Stations 314, 

315, and 321 where Se concentrations were the highest. Our 

results indicate that Se concentrations may be associated with 

biomarker responses in CDRB from several stations. Further 

investigations are warranted to determine cause-effect relationships between Se and biomarkers (reproductive, morphological, and molecular) in CDRB fish.

In addition to the extensive agriculture in the CDRB, 

mining is an important industry. Minerals including Cu, 

Au, Pb, and Zn have been heavily mined in much of the 

CDRB, and uranium, coal, oil, and natural gas have also been 

exploited. Many Cu mines are located along the GR in New 

Mexico and Arizona and are a primary source of contaminants 

in the Middle GR (King and Baker, 1995). Fifteen Cu smelters in this area are point sources for sulfur oxides, toxic metals 

(for example, As, Pb, Hg), fine particulates, and acid precipitation. One smelter in Hayden, Arizona (Station 323) is located 

near the GR, and storm water runoff, erosion of mine tailings, 

and emissions from the smelters may adversely impact threatened and endangered species in this area (G. Beatty, oral communication). Several studies have reported relatively high Cu 

concentrations in fish from the GR (Baker and others, 1992; 

King and others, 1997); our results are similar. Concentrations 

of Cu were significantly higher in bass from Station 323 than 

other CDRB stations. Although tissue-based criteria for Cu 

are unavailable for piscivorous wildlife (Eisler, 1997), several 

studies have correlated physiological effects in fish with Cu 

exposure (see review by Handy, 2003). Monitoring histological changes in fish, especially those from the GR, are likely 

necessary to understand the effects of Cu exposure to fish. 

Large natural gas and coal deposits are located in the Upper 

CDRB, and New Mexico has the largest reserves and production of CBM (NMRD, 2003). Extraction and processing of 

these natural resources have caused degraded water quality 

in the SJB and the Green River. Continued monitoring of 

environmental contaminants and their effects in aquatic biota 

is needed in these areas as mining operations expand to meet 

consumer demands.

Mercury sources in the CDRB include historic ore milling and amalgamation processes, naturally mineralized soils, 

and atmospheric deposition (USEPA, 2004a). King and others 

(1993) concluded that Hg concentrations did not pose a risk 

to piscivorous wildlife in the Lower CDRB; a conclusion 

that was also supported by our data. Large coal beds found 

in the Green River, Yampa River, and San Juan River Basins 

also contain Hg, which can be extracted during coal cleaning 

and released from coal-fired power plants (Tewalt and others, 2001). Fish from these areas in our study had among the 

highest Hg concentrations and exceeded protective criteria for 

fish and wildlife. Mercury deposition from coal-fired power 

plants in New Mexico (near Station 317) may have contributed 

to the fish consumption advisories for nearby reservoirs and 

rivers (Abell, 1994; Melancon and others, 1979). In addition, 

acid deposition from power plants in the Yampa River Basin 

at Craig, Colorado (near Station 311) has been associated 

with reproductive problems in amphibians from nearby waters 

(Turk and Campbell, 1997). Plasma E2 concentrations were 

relatively high in male smallmouth bass from Station 311, and 

evidence of ovotestis was found in 70% of these males, which 

indicates reproductive biomarker responses in these fish. This 

occurrence of intersex is one the most severe reported by 

the BEST-LRMN Program. The cause of intersex in these 

fish is unknown, although the condition has been reported in 

similarly high proportions in smallmouth bass in other U.S. 

river basins (Blazer and others, 2002; Blazer, 2006). Liney 

and others (2005) concluded that intersex is age-related and 

occurs at higher incidences in adults exposed to estrogenic 

compounds during early development. Blazer (2006) suggests 

that intersex in smallmouth bass from the Potomac River were 

caused by a mixture of estrogenic chemicals from various 

sources including human wastewater, agricultural runoff, 

industrial discharge, and atmospheric deposition. These types 

of endocrine disrupting sources are not found near Station 311. 

However, the high occurrence of intersex and high E2 concentrations in male smallmouth bass along with the relatively high 

vtg concentrations in female smallmouth bass from this site 

indicate that the reproduction pathway in these fish has been 

affected and needs to be investigated further. 

The demand for water continues to increase as the human 

population expands and drought conditions persist throughout 

the CDRB. The magnitude and type of chemical contaminants 

released in the CDRB are likely to expand, and water quality 

will decline as industrial and municipal discharges increase. 

Toxic trace elements will likely continue to leach into river 

systems as a result of mining operations and irrigated agriculture. Densely populated areas including Las Vegas and Phoenix will continue to introduce emerging or new generation 

chemicals such as pharmaceuticals and person care products 

to CDRB waters. The effects of these contaminants to aquatic 

biota are largely unknown. Biological responses would be 

expected to increase in magnitude as chemical concentrations increase, which may ultimately place fish populations at 

risk. Results from this study and other investigations indicate 

that continued monitoring is needed to identify consistently 

degraded sites and those with emerging problems, specifically 

those in the GRB. Focused investigations are also needed in 

90    Contaminants, Health Indicators, and Reproductive Biomarkers in Fish from the Colorado River Basin
the CDRB to document chemical sources, interactions with 

other factors, and cause-effect relationships.

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Wyoming Department of Environmental Quality (WDEQ), 

2002, Wyoming’s 2002 303(d) list of waters requiring 

TMDLs: Wyoming Department of Environmental Quality, 

at URL (http://deq.state.wy.us/wqd/watershed/downloads/

TMDL/2-2227.doc.pdf ), 13 p.

Yeardley, R.B., Jr., Lazorchak, J.M., and Paulsen, S.G., 1998, 

Elemental fish tissue contamination in northeastern U.S. 

lakes: evaluation of an approach to regional assessment: 

Environmental Toxicology and Chemistry, v. 17, p. 1875-

1884.

106    Contaminants, Health Indicators, and Reproductive Biomarkers in Fish from the Colorado River Basin
Appendices

Appendices    107
Appendix 1. Selected species within the Colorado River Basin identified as having special status by the USFWS. aUSFWS 

Threatened and Endangered Species System (TESS) webpage (https://ecos/fws/gov/ecos/sec/species.do) accessed 8/23/04.

Common name Scientific name Designationa

American bittern Botarurus lentiginosus Sensitive 

Colorado River toad Bufo alvarius Sensitive 

Western toad Bufo boreas Sensitive 

Southwestern toad Bufo microscaphus Sensitive 

June sucker Chasmistes liorus Endangered

Trumpeter swan Cygnus buccinator Sensitive

Beautiful shiner Cyprinella formosa Threatened 

Desert pupfish Cyprinodon macularius Endangered 

Humpback chub Gila cypha Endangered 

Sonora chub Gila ditaenia Threatened 

Bonytail chub Gila elegans Endangered 

Chihuahua chub Gila nigrescenas Threatened 

Yaqui chub Gila purpurea Endangered

Virgin River chub Gila seminuda Endangered

Whooping crane Grus americana Endangered

Bald eagle Haliaeetus leucocephalus Threatened

Harlequin duck Histrionicus histrionicus Sensitive

Yaqui catfish Ictaturus pricel Endangered

Little Colorado spinedace Lepidomeda vittata Threatened

River otter Lontra canadensis Sensitive

Spikedace Meda fulgida Threatened

Hornyhead chub Nocomis biguttatus Sensitive

Apache trout Oncorhynchus apache Threatened

Gila trout Oncorhynchus gilae Endangered

Kanab ambersnail Oxyfoma haydeni kanabensis Endangered

Woundfin Plagopterus argentissimus Endangered

Gila topminnow Poeciliopsis occidentalis Endangered

Colorado pikeminnow Ptychocheilus lucius Endangered 

Yuma clapper rail Rallus longirostris yumanensis Endangered 

Chiricahua leopard frog Rana chiricahuensis Threatened 

Mountain yellow-legged frog Rana muscosa Endangered 

Wood frog Rana sylvatica Sensitive 

Kendall Warm Springs speckled dace Rhinichthys osculus thermalis Endangered

Least tern Sterna antillarum Endangered 

Mexican spotted owl Strix occidentalis lucida Threatened 

Loach minnow Tiaroga cobitis Threatened 

Razorback sucker Xyrauchen texanus Endangered 

108    Contaminants, Health Indicators, and Reproductive Biomarkers in Fish from the Colorado River Basin
Appendix 2. Results of quality assurance (QA) and limits of detection (LOD) for elemental contaminants (μg/g dry weight) in whole-body fish composites from the Colorado River 

Basin using a quantitative analysis for arsenic, selenium, and total mercury and an ICP-MS semi-quantitative scan of other elements. aSample sizes for Hg were n = 18 for blank 

equivalent concentrations; n = 19 for triplicate sample analyses; n = 3 for NRCC DORM-2; n = 6 for CERC striped bass; n = 12 for spiked samples. Samples sizes for As and Se were n= 6 for spiked samples. bDogfish muscle. cColumbia Environmental Research Center whole-body striped bass research material.

QA component

Element

Arsenic Cadmium Copper Chromium Mercury Nickel Lead Selenium Zinc

Blank equivalent concentrations (μg/g dw); n = 6a

Min. 0.027 <0.1 <1 <1 0.0001 <1 <1 0.009 <1

Max. 0.118 <0.1 <1 <1 0.0006 <1 <1 0.049 <1

Triplicate sample analyses (% relative standard deviation); n = 3

Min. 2.8 0.0 4.3 0.0 2.9 0.0 0.0 2.4 2.1

Max. 54.0 5.8 12.0 3.3 30.0 0.0 0.0 4.8 5.4

Reference materials (% recovery) NRCC DORM-2b; n = 2

Min. 87 100 100 89 100 83 100 100 90Max. 100 100 100 100 100 100 100 105 99Cert. conc. range 

(μg/g dw) 18.0 ± 1.1 0.043 ± 0.008 2.34 ± 0.16 34.7 ± 5.5 4.64 ± 0.26 19.4 ± 3.1 0.065 ± 0.007 1.40 ± 0.09 25.6 ± 2.3

CERC striped bassc; n = 2

Min. 100 100 100 152 100 100 100 103 98

Max. 100 100 102 154 100 100 100 105 100Recommended conc. 

range (μg/g dw) 3.20 ± 0.5 0.33 ± 0.12 4.20 ± 0.5 1.40 ± 1.2 2.26 ± 0.51 1.80 ±1.4 3.62 ± 1.2 2.26 ± 0.2 43.9 ± 2.0

Pre-digestion spiked samples (% recovery); n = 4

Min. 92 84 97 99 90 95 102 94 86

Max. 107 108 103 103 131 103 112 115 104

Nominal LOD (μg/g dw) 0.018 --- --- --- 0.0007 --- --- 0.014 ---Reporting Limit (μg/g 

dw; semi-quantitative scan)

--- 0.1 0.1 0.1 --- 0.1 1.0 --- 1.0

Appendices    109
Appendix 3. Results of quality assurance (QA), method detection limit (MDL), and method quantitative limit (MQL) for organochlorine compounds (ng/g wet weight) in wholebody fish composites from the Colorado River Basin. Sample sizes were n = 5 for all contaminants. aPercent relative standard deviation. bReference material was a Columbia 

Environmental Research Center positive control fish tissue, carp from Saginaw Bay (n = 16). cToxaphene has not been evaluated historically in this fish material. PCB, polychlorinated biphenyl; QCB, pentachlorobenzene; HCB, hexachlorobenzene; PCA, pentachloroanisole; HCH, hexachlorocyclohexane.

QA component

Compound

Total PCB Total toxaphene QCB HCB PCA

α-HCH β-HCH

σ-HCH γ-HCH Heptachlor Heptachlor epoxide Aldrin

Procedure blanks(ng/g ww)

Min. 7.20 1.00 0.005 0.02 0.02 0.00 0.00 0.00 0.000 0.00 0.000 0.0Max. 34.5 12.6 0.020 0.10 0.05 0.03 0.25 0.30 0.005 0.05 0.002 0.0Mean 16.5 8.00 0.010 0.04 0.04 0.02 0.06 0.11 0.002 0.01 0.000 0.0

Matrix blanks(ng/g ww)

Min. 44.4 5.30 0.04 0.21 0.19 0.33 0.00 0.00 0.00 0.00 0.09 0.0Max. 93.2 14.5 0.09 0.29 0.46 0.58 0.86 1.06 0.50 0.10 0.31 0.0Mean 60.0 10.6 0.07 0.26 0.27 0.43 0.31 0.28 0.11 0.04 0.25 0.0

Triplicate sample analyses (% RSD)a

Min. 2.8 7.1 3.8 3.7 6.9 26 5.0 7.1 12 20 6.9 0.0Max. 4.8 76 21 8.1 28 83 41 25 136 33 173 0.0Mean 3.8 31 13 5.8 14 49 23 16 48 26 79 n/a

Reference material b(ng/g ww)

Min. 5900 160 4.7 13 2.7 3.5 1.2 1.8 0.7 0.3 2.9 0.6Max. 7100 280 6.3 14 3.9 7.5 3.1 6.4 0.7 3.8 3.8 0.9Mean 6500 220 5.5 14 3.1 5.5 2.3 3.4 0.7 1.5 3.3 0.8Long Term. conc. (ng/g ww) 6700 ---c 5.4 12 3.0 6.1 1.6 2.5 0.4 0.1 3.9 <MDL

Fortified spiked sample (% recovery)

Min. 93 88 72 75 89 78 69 74 73 67 74 65Max. 102 109 85 88 101 102 84 90 105 86 86 90Mean 98 95 79 83 95 92 77 83 88 79 79 83

Nominal MDL (ng/g ww) 48 24 0.07 0.26 0.13 0.08 0.19 0.51 0.05 0.19 0.01 0.27Nominal MQL (ng/g ww) 121 67 0.18 0.70 0.34 0.23 0.54 1.50 0.14 0.57 0.02 0.83

110    Contaminants, Health Indicators, and Reproductive Biomarkers in Fish from the Colorado River Basin
Appendix 3. Results of quality assurance (QA), method detection limit (MDL), and method quantitative limit (MQL) for organochlorine compounds (ng/g wet weight) in whole-body 

fish composites from the Colorado River Basin. Sample sizes were n = 5 for all contaminants. aPercent relative standard deviation. bReference material was a Columbia Environmental Research Center positive control fish tissue, carp from Saginaw Bay (n = 16). cToxaphene has not been evaluated historically in this fish material. PCB, polychlorinated biphenyl; QCB, pentachlorobenzene; HCB, hexachlorobenzene; PCA, pentachloroanisole; HCH, hexachlorocyclohexane.—Continued

QA component Compound Dacthal Dieldrin Endrin Oxychlordane cis-Chlordane trans-Chlordane cis-Nonachlor trans-Nonachlor o,p’-DDE o,p’-DDD o,p’-DDT

Procedure blanks(ng/g ww)Min. 0.010 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000Max. 1.017 0.221 0.054 0.114 0.049 0.005 0.079 0.025 0.044 0.005 0.010Mean 0.242 0.048 0.022 0.036 0.014 0.002 0.028 0.008 0.011 0.001 0.002Matrix blanks(ng/g ww)Min. 0.12 1.8 0.0 1.1 0.1 0.0 1.0 3.3 0.0 0.00 0.0Max. 2.01 2.6 4.9 7.8 0.3 1.2 1.4 5.4 1.0 0.05 1.6Mean 0.83 2.3 1.7 3.2 0.2 0.4 1.1 3.9 0.4 0.02 0.8Triplicate sample analyses (% RSD)aMin. 1.3 13 17 10 3.8 7.7 1.2 2.8 21 9.3 29Max. 33 89 138 95 87 34 15 22 23 9.3 91Mean 14 39 61 39 27 19 7.3 9.4 22 9.3 69Reference material b(ng/g ww)Min. 2.1 18 0.12 5.2 21 13 12 27 6.8 1.7 23Max. 3.0 26 7.90 9.2 49 18 19 39 9.3 70 23Mean 2.5 21 4.20 7.1 32 15 15 31 7.9 51 23Long Term. conc. (ng/g ww) 5.0 19 4.40 4.0 23 11 12 25 6.1 52 2.0Fortified spiked sample (% recovery)Min. 69 73 66 84 74 76 72 77 68 72 88Max. 85 88 101 100 93 91 99 92 85 92 112Mean 78 83 79 94 85 84 89 85 79 81 101Nominal MDL (ng/g ww) 0.5 0.15 0.1 0.08 0.04 0.23 0.10 0.03 0.08 0.6 0.53Nominal MQL (ng/g ww) 1.5 0.44 0.3 0.25 0.12 0.68 0.27 0.09 0.26 1.7 1.70

Appendices    111
Appendix 3. Results of quality assurance (QA), method detection limit (MDL), and method quantitative limit (MQL) for organochlorine compounds (ng/g wet 

weight) in whole-body fish composites from the Colorado River Basin. Sample sizes were n = 5 for all contaminants. aPercent relative standard deviation. bReference material was a Columbia Environmental Research Center positive control fish tissue, carp from Saginaw Bay (n = 16). cToxaphene has not been evaluated historically in this fish material. PCB, polychlorinated biphenyl; QCB, pentachlorobenzene; HCB, hexachlorobenzene; PCA, pentachloroanisole; HCH, hexachlorocyclohexane.—Continued

QA component Compound p,p’-DDE p,p’-DDD p,p’-DDT Endosulfan I Endosulfan II Endosulfan sulfate Methoxychlor Mirex

Procedure blanks(ng/g ww)Min. 0.04 0.00 0.000 0.00 0.000 0.000 0.000 0.000

Max. 1.20 0.40 0.025 0.00 0.059 0.050 0.020 0.010

Mean 0.36 0.08 0.005 0.00 0.012 0.011 0.007 0.002Matrix blanks(ng/g ww)Min. 2.1 0.0 0.00 0.00 0.0 0.2 0.00 0.00

Max. 5.0 1.0 0.34 0.00 0.5 0.5 0.13 0.44

Mean 2.9 0.4 0.23 0.00 0.2 0.3 0.03 0.14Triplicate sample analyses (% RSD)aMin. 1.8 6.3 6.1 0.0 14 3 10 6

Max. 12 87 6.3 0.0 92 59 15 63Mean 6.5 25 6.2 n/a 53 25 13 28

Reference material b(ng/g ww)Min. 340 240 2.3 0.0 0.6 2.8 0.5 2.4

Max. 660 360 3.8 0.0 0.6 8.6 7.8 3.9Mean 510 310 3.1 0.0 0.6 4.4 4.5 3.0

Long Term. conc. (ng/g ww) 440 260 3.3 5.8 3.1 3.2 9.7 3.7Fortified spiked sample (% recovery)Min. 72 65 91 61 84 82 90 71

Max. 95 93 133 76 90 106 125 90

Mean 82 86 107 66 86 98 110 84Nominal MDL (ng/g ww) 0.86 0.67 1.4 0.21 0.17 0.05 0.35 0.05

Nominal MQL (ng/g ww) 2.40 2.00 4.5 0.64 0.53 0.15 1.10 0.15

112    Contaminants, Health Indicators, and Reproductive Biomarkers in Fish from the Colorado River Basin
Appendix 4. Lengths, weights, and ages of non-target species including white sucker (Catostomus commersoni), flathead catfish (Pylodictis olivaris), 

and brown trout (Salmo trutta) collected in the Colorado River Basin in 2003. Stations are listed upstream to downstream. Station numbers are given in parentheses. Sample size (n), arithmetic mean, standard deviation (SD), and range area also given. Fish in which gender could not be determined are identified as having no gonads (NG). --, not applicable.

Species and station Gender

Length (mm) Weight (g) Age (years)

n Mean SD Range n Mean SD Range n Mean SD Range

White suckerLay, CO (311) M 8 448 63 340-516 8 1261 432 470-1595 7 8.9 4.2 4-15

F 11 463 70 312-546 11 1323 522 395-2095 9 9.3 3.0 5-14

Brown troutDelta, CO (314) M 9 387 63 258-472 9 529 265 175-885 9 2.6 0.7 2-4

F 11 371 78 269-498 11 575 353 160-1230 11 2.5 1.0 2-5

NG 1 380 -- -- 1 220 -- -- 1 2.0 -- --

Flathead catfishHayden, AZ (323) M 7 507 272 265-845 7 2534 3016 155-6935 6 2.7 2.1 1-6

F 3 258 45 225-310 3 182 59 115-300 3 1.3 0.6 1-2

NG 1 321 -- -- 1 305 -- -- 1 2.0 -- --

Appendices    113
Appendix 5. DDT homolog concentrations (μg/g) in composite samples from Stations 324 and 325 compared to other CDRB samples. Total 

DDT concentrations are the sum of p,p’- and o,p’-DDT, DDE, and DDD.

Station and species

% lipid p,p’-DDT o,p’-DDT p,p’-DDE o,p’-DDE p,p’-DDD o,p’-DDD Total DDT

Phoenix, AZ (324) Carp, female 5.57 0.003 <0.0005 0.280 0.002 0.003 0.006 0.295 Carp, male 7.17 0.006 <0.0005 0.340 0.004 0.005 0.010 0.365 Channel catfish, female 8.03 0.010 <0.0005 0.643 0.002 0.005 0.006 0.666 Channel catfish, male 11.2 0.015 <0.0005 0.780 0.003 0.009 0.007 0.814Arlington, AZ (325) Carp, female 4.80 0.008 0.0006 1.30 0.008 0.024 0.016 1.357 Carp, male 5.72 0.015 <0.0005 1.70 0.012 0.028 0.017 1.773 Bass, female 4.83 0.019 <0.0005 1.60 0.010 0.025 0.012 1.666 Bass, male 4.95 0.031 <0.0005 2.70 0.017 0.037 0.015 2.800All other samples (range) 2.28-

12.40

<0.001-

0.008

<0.0005-

0.0029

0.001-

0.250

<0.00008-

0.005

<0.0007-

0.016

<0.0006-

0.004

0.004-

0.277

114    Contaminants, Health Indicators, and Reproductive Biomarkers in Fish from the Colorado River Basin
Appendix 6. Results of preliminary analysis-of-variance investigating the effects of various factors on biomarker responses in carp, 

bass, and channel catfish in the Colorado River Basin in 2003. Degrees-of-freedom (df), F-values with levels of significance (*0.01 < 

P ≤ 0.05; **P ≤ 0.01), and coefficients of determination (R2) are presented. ND, not determined. a Data from Stations 312, 315, 319, 320, 

and 324 excluded from analysis.—Continued

Variable, source, and

(transformation)

Carp Bass Channel Catfish

df F R2 df F R2 df F R2

EROD (log)

Model 41 3.45** 0.41 23 3.78** 0.49 21 1.17 0.28

Station 12 1.81* 5 0.60 5 1.28

Gender 1 1.09 1 1.98 1 0.66

Station*Gender 2 1.47 3 1.90 3 0.59

Stage 1 0.57 1 1.00 1 3.88

Stage*Station 12 1.40 4 0.85 5 1.35

Stage*Gender 1 0.42 1 0.15 1 1.27

Stage*Station*Gender 2 1.20 3 1.36 3 0.62

Error 207 92 63

Condition Factor

Model 41 1.87** 0.27 23 5.76** 0.60 21 6.11** 0.67

Station 12 1.22 5 1.47 5 7.45**

Gender 1 0.01 1 1.43 1 6.41*

Station*Gender 2 0.00 3 2.03 3 0.32

Stage 1 0.01 1 0.07 1 12.25**

Stage*Station 12 0.88 4 1.54 5 1.29

Stage*Gender 1 0.03 1 2.02 1 4.95*

Stage*Station*Gender 2 0.02 3 1.73 3 0.40

Error 205 92 62

Splenosomatic Index

Model 24 2.55** 0.22 14 1.86* 0.21 11 3.21** 0.33

Station 12 2.96** 7 3.56** 5 3.53**

Gender 1 14.86** 1 0.08 1 0.00

Station*Gender 11 0.98 6 0.15 5 0.94

Error 212 101 73

Hepatosomatic Index

Model ND ND ND 14 9.72** 0.57 11 2.06* 0.24

Station ND 7 18.83** 5 2.69*

Gender ND 1 0.50 1 0.20

Station*Gender ND 6 0.87 5 0.59

Error ND 101 73

HAI (rank)

Model 41 4.05** 0.45 23 10.62** 0.73 21 3.07** 0.51

Station 12 1.34 5 4.00** 5 1.48

Gender 1 0.27 1 1.21 1 0.20

Station*Gender 2 0.31 3 2.34 3 0.04

Stage 1 1.40 1 0.17 1 3.29

Stage*Station 12 1.07 4 1.51 5 0.78

Stage*Gender 1 0.03 1 0.71 1 0.02

Stage*Station*Gender 2 0.49 3 2.56 3 0.24

Error 206 92 62

Appendices    115
Appendix 6. Results of preliminary analysis-of-variance investigating the effects of various factors on biomarker responses in carp, 

bass, and channel catfish in the Colorado River Basin in 2003. Degrees-of-freedom (df), F-values with levels of significance (*0.01 < 

P ≤ 0.05; **P ≤ 0.01), and coefficients of determination (R2) are presented. ND, not determined. a Data from Stations 312, 315, 319, 320, 

and 324 excluded from analysis.—Continued

Variable, source, and

(transformation)

Carp Bass Channel Catfish

df F R2 df F R2 df F R2

MA-% (log) a

Model 11 3.19** 0.43 19 4.55** 0.57 11 0.63 0.13

Station 7 1.14 4 2.27 2 0.34

Gender 1 0.13 1 1.07 1 0.08

Station*Gender 7 2.63* 4 1.18 2 0.28

Age 1 8.23** 1 6.54* 1 0.32

Age*Station 7 0.47 4 1.02 2 0.03

Age*Gender 1 1.18 1 2.33 1 0.04

Age*Station*Gender 7 1.84 4 0.34 2 0.25

Error 130 65 45

MA-A (log) a

Model 11 1.92** 0.31 19 1.76* 0.34 11 0.43 0.09

Station 7 1.54 4 1.27 2 0.28

Gender 1 1.85 1 0.10 1 0.81

Station*Gender 7 1.02 4 0.11 2 0.04

Age 1 2.81 1 0.50 1 0.06

Age*Station 7 1.58 4 0.21 2 0.17

Age*Gender 1 0.11 1 0.62 1 0.38

Age*Station*Gender 7 0.41 4 0.12 2 0.03

Error 130 65 45

MA-# a

Model 11 2.98** 0.42 19 3.75** 0.53 11 1.06 0.21

Station 7 1.09 4 1.53 2 0.02

Gender 1 3.78 1 0.06 1 0.09

Station*Gender 7 1.98 4 1.34 2 0.07

Age 1 1.84 1 5.84* 1 0.59

Age*Station 7 0.11 4 2.68 2 0.19

Age*Gender 1 2.94 1 0.03 1 0.05

Age*Station*Gender 7 1.16 4 0.39 2 0.08

Error 130 65 45

Gonadosomatic Index

Model 42 19.37** 0.81 23 60.61** 0.94 21 7.90** 0.73

Station 11 2.20* 5 5.91** 5 4.34**

Gender 1 2.48 1 14.60** 1 5.05*

Station*Gender 2 3.06* 3 4.50** 3 3.13*

Stage 1 23.66** 1 0.01 1 22.37**

Stage*Station 11 2.55** 4 1.47 5 3.98**

Stage*Gender 1 7.27** 1 0.01 1 16.92**

Stage*Station*Gender 1 2.32 3 0.29 3 2.83*

Error 195 91 60

116    Contaminants, Health Indicators, and Reproductive Biomarkers in Fish from the Colorado River Basin
Appendix 6. Results of preliminary analysis-of-variance investigating the effects of various factors on biomarker responses in carp, 

bass, and channel catfish in the Colorado River Basin in 2003. Degrees-of-freedom (df), F-values with levels of significance (*0.01 < 

P ≤ 0.05; **P ≤ 0.01), and coefficients of determination (R2) are presented. ND, not determined. a Data from Stations 312, 315, 319, 320, 

and 324 excluded from analysis.—Continued

Variable, source, and

(transformation)

Carp Bass Channel Catfish

df F R2 df F R2 df F R2

Vitellogenin (log)

Model 41 23.33** 0.83 23 7.65** 0.66 ND ND ND

Station 12 2.29** 5 1.73 ND

Gender 1 0.60 1 1.75 ND

Station*Gender 2 0.01 3 3.30* ND

Stage 1 0.01 1 0.07 ND

Stage*Station 12 1.78 4 1.48 ND

Stage*Gender 1 8.68* 1 0.11 ND

Stage*Station*Gender 2 0.50 3 2.31 ND

Error 199 89 ND

Estradiol

Model 41 30.56** 0.86 23 8.65** 0.69 21 3.07** 0.51

Station 12 2.52** 5 0.34 5 0.64

Gender 1 6.37** 1 0.36 1 1.10

Station*Gender 2 2.77* 3 0.53 3 1.40

Stage 1 1.73 1 0.08 1 1.13

Stage*Station 12 2.40** 4 0.16 5 0.90

Stage*Gender 1 0.88 1 0.00 1 0.49

Stage*Station*Gender 2 2.12 3 0.46 3 1.45

Error 206 88 61

11-ketotestosterone

Model 41 6.94** 0.58 23 15.63** 0.80 21 7.73** 0.73

Station 12 1.38 5 2.24* 5 3.52**

Gender 1 11.48** 1 8.08** 1 5.19*

Station*Gender 2 5.18* 3 0.12 3 2.55

Stage 1 1.57 1 1.85 1 0.06

Stage*Station 12 0.84 4 0.53 5 0.35

Stage*Gender 1 6.24* 1 0.01 1 2.78

Stage*Station*Gender 2 3.85* 3 0.22 3 0.90

Error 206 88 61

Estradiol/11-ketotestosterone

Model 41 7.65** 0.63 23 6.08** 0.61 21 2.39** 0.45

Station 12 0.58 5 0.15 5 0.59

Gender 1 2.15 1 3.61 1 0.22

Station*Gender 2 0.36 3 0.11 3 0.52

Stage 1 0.37 1 0.12 1 0.25

Stage*Station 12 0.56 4 0.06 5 0.57

Stage*Gender 1 0.18 1 0.14 1 0.01

Stage*Station*Gender 2 0.17 3 0.08 3 0.15

Error 206 88 61

Appendices    117
Appendix 6. Results of preliminary analysis-of-variance investigating the effects of various factors on biomarker responses in carp, 

bass, and channel catfish in the Colorado River Basin in 2003. Degrees-of-freedom (df), F-values with levels of significance (*0.01 < 

P ≤ 0.05; **P ≤ 0.01), and coefficients of determination (R2) are presented. ND, not determined. a Data from Stations 312, 315, 319, 320, 

and 324 excluded from analysis.—Continued

Variable, source, and

(transformation)

Carp Bass Channel Catfish

df F R2 df F R2 df F R2

Atresia (females only) a

Model 23 1.62 0.39 12 5.46** 0.68 10 1.53 0.54

Station 3 0.12 0 ND 1 0.12

Stage 1 0.12 1 15.84** 1 0.66

Stage*Station 3 0.09 0 ND 1 0.29

Age 1 0.00 1 10.25** 1 0.22

Age*Station 3 0.11 0 ND 1 0.10

Stage*Age 1 0.01 1 15.29** 1 0.50

Stage*Age*Station 3 0.11 0 ND 1 0.19

Error 59 31 13

118    Contaminants, Health Indicators, and Reproductive Biomarkers in Fish from the Colorado River Basin
Appendix 7. Fish health indicators for non-target species collected in the Colorado River Basin in 2003. Arithmetic mean, number of samples (n), minimum (Min), maximum (Max), 

and standard error (SE) are given for gonadosomatic index (GSI; %), splenosomatic index (SSI; %), condition factor (CF), and health assessment index (HAI). Fish with undetermined gender from which no gonad tissue was obtained are listed as NG. --, not applicable.

Species and station

Female Male NG

n Mean Min Max SE n Mean Min Max SE n Mean Min Max SE

Brown Trout

Delta, CO (314)

GSI 11 6.86 0.13 14.65 1.70 9 0.72 0.02 3.44 0.42 1 0.05 0.05 0.05 --SSI 11 0.19 0.08 0.96 0.08 9 0.08 0.03 0.13 0.01 1 0.27 0.27 0.27 --CF 11 1.00 0.63 1.34 0.06 9 0.88 0.42 1.10 0.09 1 0.40 0.40 0.40 --HAI 11 47.3 10 90 8.6 9 44.4 30 70 5.0 1 70 70 70 --

Flathead Catfish

Hayden, AZ (323)

GSI 3 0.50 0.17 0.92 0.22 7 0.14 0.06 0.21 0.02 1 0.10 0.10 0.10 --SSI 3 0.19 0.06 0.44 0.13 7 0.06 0.00 0.09 0.01 1 0.07 0.07 0.07 --CF 3 0.99 0.83 1.14 0.09 7 0.97 0.78 1.26 0.06 1 0.92 0.92 0.92 --HAI 3 10.0 0 30 10.0 7 18.6 0 70 10.1 0 -- -- -- --

White Sucker

Lay, CO (311)

GSI 11 6.63 0.38 10.37 0.81 8 2.00 0.07 6.05 0.91 0 -- -- -- --

SSI 11 0.18 0.08 0.42 0.03 8 0.18 0.05 0.28 0.02 0 -- -- -- --CF 11 1.26 1.14 1.36 0.02 8 1.35 1.16 1.56 0.05 0 -- -- -- --HAI 11 30.9 0 60 7.8 8 36.3 0 70 8.2 0 -- -- -- --

Appendices    119
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Hinck, J.E., Blazer, V.S., Denslow, N.D., Gross, T.S., Echols, K.R., Davis, A.P., May, T.W., Orazio, C. E., Coyle, J.J., and Tillitt, D.E. —Biomonitoring of Environmental Status and Trends (BEST) Program: Environmental Contaminants, Health Indicators, and Reproductive Biomarkers in Fish from the Colorado River Basin—U.S.Geological Survey–Scientific Investigations Report 2006—5163