ent 378 (2007) 376–402www.elsevier.com/locate/scitotenv

Science of the Total Environm

Chemical contaminants, health indicators, and reproductivebiomarker responses in fish from the Colorado

River and its tributaries

Jo Ellen Hinck a,⁎, Vicki S. Blazer b, Nancy D. Denslow c, Kathy R. Echols a,Timothy S. Gross d, Tom W. May a, Patrick J. Anderson e,

James J. Coyle e, Donald E. Tillitt a

a U.S. Geological Survey (USGS), Columbia Environmental Research Center (CERC), 4200 New Haven Rd., Columbia, MO 65201, USAb USGS Leetown Science Center, 11649 Leetown Rd., Kearneysville, WV 24530, USA

c Center for Environmental and Human Toxicology, P.O. Box 100185, University of Florida, Gainesville, FL 32611, USAd USGS, Florida Integrated Science Center, 7920 NW 71st St., Gainesville, FL 32653, USA

e USGS, BEST Program, 2150 Centre Ave, Building C, Fort Collins, CO 80526, USA

Received 20 December 2006; received in revised form 14 February 2007; accepted 19 February 2007Available online 5 April 2007

Abstract

Common carp (Cyprinus carpio), black bass (Micropterus spp.), and channel catfish (Ictalurus punctatus) were collected from14 sites in the Colorado River Basin (CRB) to document spatial trends in accumulative contaminants, health indicators, andreproductive biomarkers. Organochlorine residues, 2,3,7,8-tetrachlorodibenzo-p-dioxin-like activity (TCDD-EQ), and elementalcontaminants were measured in composite samples of whole fish, grouped by species and gender, from each site. Selenium (Se)and mercury (Hg) concentrations in fish were elevated throughout the CRB, and pesticide concentrations were greatest in fish fromagricultural areas in the Lower Colorado River and Gila River. Selenium concentrations exceeded toxicity thresholds for fish(N1.0 μg/g ww) at all CRB sites except the Gila River at Hayden, Arizona. Mercury concentrations were elevated (N0.1 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 the Gila River at Arlington,Arizona (N1.0 μg/g ww) and Phoenix, Arizona (N0.5 μg/g ww). Concentrations of other formerly used pesticides includingtoxaphene, total chlordanes, and dieldrin were also greatest at these two sites but did not exceed toxicity thresholds. Currently usedpesticides such as Dacthal, endosulfan, γ-HCH, and methoxychlor were also greatest in fish from the Gila River downstream ofPhoenix. Total polychlorinated biphenyls (PCBs; N0.11 μg/g ww) and TCDD-EQs (N5 pg/g ww) exceeded wildlife guidelines infish from the Gila River at Phoenix. Hepatic ethoxyresorufin O-deethylase (EROD) activity was also relatively high in carp fromthe Gila River at Phoenix and in bass from the Green River at Ouray NWR. Fish from some sites showed evidence of contaminantexposure as indicated by fish health indicators and reproductive biomarker results. Multiple health indicators including alteredbody and organ weights and high health assessment index scores may be associated with elevated Se concentrations in fish fromthe Colorado River at Loma, Colorado and Needles. Although grossly visible external or internal lesions were found on most fishfrom 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. Intersex fish were found at

⁎ Corresponding author. Tel.: +1 573 876 1808; fax: +1 573 876 1896.E-mail address: jhinck@usgs.gov (J.E. Hinck).

0048-9697/$ - see front matter. Published by Elsevier B.V.doi:10.1016/j.scitotenv.2007.02.032

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seven of 14 sites and included smallmouth bass (M. dolomieu), largemouth bass (M. salmoides), catfish, and carp and may indicateexposure to endocrine disrupting compounds. A high proportion of smallmouth bass from the Yampa River at Lay (70%) wasintersex but the cause of this condition is unknown. Male carp, bass, and catfish with low concentrations of vitellogenin werecommon in the CRB. Comparatively high vitellogenin concentrations (N0.2 mg/mL) were measured in male bass from the GreenRiver at Ouray NWR and the Colorado River at Imperial Dam and indicate exposure to estrogenic or anti-androgenic chemicals.Anomalous reproductive biomarkers including low GSI and gonadal abnormalities (calcifications, edema, and parasites) observedin fish downstream of Phoenix are likely related to the poor water-quality of the Gila River in this area.Published by Elsevier B.V.

Keywords: Selenium; Mercury; Organochlorine pesticides; PCBs; Ethoxyresorufin O-deethylase (EROD) activity; Health assessment index (HAI);Biomarkers; Intersex; Vitellogenin

1. Introduction

The Colorado River (CR) is the largest river of thesouthwest U.S. and one of the world's most regulatedrivers. Numerous dams and diversion on the CR and itstributaries provide water for municipal and industrialusage, agriculture, hydroelectric power, and the tourismindustry but have also altered the ecological, morpho-logical, and hydrological characteristics of the river.Dams have changed water temperature, turbidity, waterchemistry, and aquatic habitat; disrupted fish migrationand spawning; increased competition and predation bynon-native species; and extirpated native species (Stan-ford and Ward, 1986; Minckley, 1991). Dams also divertmost CRwater for irrigation, which has severely reducedthe large riparian and wetlands areas of CR delta inMexico and eliminated important habitat for endangeredspecies and migratory birds (Mora et al., 2003).

The Colorado River Basin (CRB) is generally arid andhighly mineralized, and much of the basin is underlain bypetroleum-rich geologic formations. Salts, oil, and traceelements such as arsenic (As), selenium (Se), and heavymetals are consequently available for potential release tothe environment through environmental weathering andresource extraction. Irrigation and mining (e.g., copper,coal, uranium) occur throughout the CRB and acceleratethe rates of the processes controlling the release anddistribution of these naturally occurring elements.Elevated concentrations of chemical contaminants inCR tributaries result from agriculture, mining, andenergy-related activities and from the human populationsthey support. A variety of chemicals including arsenic-based defoliants, pesticides, metals, and salts have beenused in agricultural areas along the Gila River (GR) and inthe Mexicali Valley of the CR delta (García-Hernándezet al., 2001, 2006; Mora et al., 2003).

Increasing water demands and drought have impactedwater-quality in the CR and its tributaries. Annual flows

from snow melt and thunderstorms fluctuated greatlybefore the construction of major dams and reservoirs, butincreased water supply demands have resulted inephemeral flows in many streams and rivers. Flows inthe GR Basin consist primarily of irrigation return water,storm water, and effluent from wastewater treatmentplants, which may negatively affect aquatic wildlife(Gebler, 1998; Anning, 2003). Exploitation of naturalresources, weathering of mineralized geologic forma-tions, wastewater effluents, urban runoff, and agricul-tural practices has contributed to declines in water-quality and has impacted habitat quality for biota in theCRB. As a result, many waters are listed as impaired, andstates have made efforts to restore native aquatic speciesand to characterize contaminants. Elevated concentra-tions of metals and metalloids have been reported inwater, sediment, and biota throughout the CRB, andelevated concentrations of organochlorine pesticidessuch as DDT and its metabolites have been found inwater, sediments, and biota in agricultural areas (Bakeret al., 1992; Stephens et al., 1992; King et al., 1993,1997; Bevans et al., 1996; Schmitt et al., 1999; Gebler,2000; García-Hernández et al., 2001, 2006; Gellenbeckand Anning, 2002). These studies have concluded thatCRB biota are at risk from exposure to elevatedcontaminant concentrations, and human fish consump-tion advisories for DDT metabolites, dieldrin, toxa-phene, and chlordane have been issued for the GRdownstream of Phoenix (USEPA, 2004). However, littleinformation is available on the effects of chemical conta-minants in fish (individual and populations) in the CRB.

Our primary objective was to document the occur-rence and distribution of chemical contaminants, healthindicators, and reproductive biomarkers in fish from theCR and several of its largest tributaries. Secondaryobjectives were to compare results from our study toprevious CRB studies and other U.S. river systems andto further refine benchmarks for quantification of long-

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term trends and interpretation of biomarker results.These latter objectives were achieved by building on theresults of similar investigations in the Mississippi RiverBasin (Schmitt, 2002), Rio Grande Basin (Schmitt et al.,2005), Columbia River Basin (Hinck et al., 2006a), andYukon River Basin (Hinck et al., 2006b, in press). Thispaper summarizes the most pertinent findings of theCRB study, which are reported in greater detail byHinck et al. (2006c). Data from this and relatedinvestigations are available atbhttp://www.cerc.usgs.gov/data/best/search/index.htmN.

2. Materials and methods

An overview of the methods is presented here. Moredetail is provided by Hinck et al. (2006c).

2.1. Sampling and field procedures

Common carp (Cyprinus carpio, henceforth carp;n=260), smallmouth bass (Stations 311 and 312 only)or largemouth bass (henceforth, bass; Micropterus spp.;n=117), and channel catfish (Ictalurus punctatus;n=89) were collected between late August and late

Fig. 1. Map of the Colorado River Basin illustrating waterways,

October 2003 by electrofishing (Fig. 1; Table 1). Thesenon-native species were targeted because of theirwidespread distribution and the abundant contaminant,health indicator, and reproductive biomarker dataavailable. Fish were collected from 14 sites located onthe CR and its large tributaries. Sites were chosen torepresent a range of contaminant sources (e.g., mining,agricultural, and urban areas) and were often locatednear river confluences. Detailed site descriptions wereprovided by Hinck et al. (2006c). Adult fish of similarsize were targeted at each site to reduce variation due toage; however, the age of fish varied (Table 2). Fish wereheld in aerated live-wells until processed (usually lessthan 3 h). All collection, handling, and euthanasiaprocedures followed animal care and use guidelines(American Fisheries Society et al., 2004). A bloodsample was obtained from the posterior caudal artery andvein using a heparinized needle and syringe and waschilled on wet ice. The fish was then weighed, measured,and killed with a blow to the head. Observations ofexternal features were recorded, and grossly visibletissue anomalies were dissected and preserved in 10%neutral buffered formalin (NBF) for histopathologicalanalysis. The liver (bass and catfish only; carp have a

state and international boundaries, and locations sampled.

Table 1Location and collection dates (2003) in the Colorado River Basin (CRB)

Station information Collection dates Latitude, longitude

Upper CRB311 — Yampa R. near Lay, CO 10/21–10/23 40°25′38.00″N, 107°51′24.00″W312 — Green R. at Ouray National Wildlife Refuge (NWR), UT 9/8–9/9 40°08′31.00″N, 109°39′17.00″W313 — Green R. near San Rafael, UT 9/5–9/6 38°45′56.00″N, 110°05′16.00″W314 — Gunnison R. at Delta, CO 9/6–9/7 38°45′59.58″N, 108°02′30.30″W315 — Colorado R. near Loma, CO 9/9–9/11 39°09′39.00″N, 108°48′28.56″W316 — Colorado R. at Gold Bar Canyon, UT 9/3–9/4 38°34′02.00″N, 109°38′51.00″W317— San Juan R. near Hogback Diversion, NM 9/3–9/4 36°44′41.00″N, 108°41′28.00″W

Lower CRB319 — Colorado R. at South Cove, AZ 10/1 36°05′23.70″N, 114°06′12.30″W320 — Colorado R. at Willow Beach, AZ 9/29–9/30 35°52′33.12″N, 114°39′53.10″W321 — Colorado R. near Needles, CA 9/23–9/24 34°43′44.64″N, 114°20′12.96″W322 — Colorado R. near Imperial Dam, AZ 9/25–9/26 32°54′05.94″N, 114°28′09.48″W323 — Gila R. near Hayden, AZ 8/22–8/23 33°01′22.14″N, 110°44′16.32″W324 — Gila R. at Phoenix, AZ at 115th Avenue 8/25–8/27 33°22′33.42″N, 112°18′19.20″W325 — Gila R. near Arlington, AZ 8/18–8/20 33°19′06.92″N, 112°40′26.46″W

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dispersed liver), spleen, and gonads were removed andweighed. The liver, gall bladder, posterior and anteriorkidneys, gonads, and spleen were examined forabnormalities. Pieces of liver were collected andimmediately flash-frozen in liquid nitrogen for ethoxyr-esorufinO-deethylase (EROD) analysis. Samples (b5 g)of gill, gonad, kidney, spleen, and additional pieces ofliver were collected and preserved for histopathologicalexamination, gender confirmation (gonad), and macro-phage aggregate analysis (spleen). Otoliths, scales, orspines were collected for age determination (Berg andGrimaldi, 1967; Casselman, 1990; Cowan et al., 1995).All remaining tissues (those not frozen or fixed) werewrapped in aluminum foil and frozen for analysis oforganochlorine chemical and elemental contaminantsand TCDD-EQ. Work surfaces and contact instrumentswere cleaned with ethanol and acetone (contact instru-ments only) between fish to prevent cross-contamina-tion. Blood samples were centrifuged, and the plasmawas aspirated and frozen in liquid nitrogen forvitellogenin (vtg) and steroid hormone analysis. Cryo-genically frozen liver and plasma samples were shippedto the laboratory on dry ice and stored at −80 °C. Afternecropsy, whole fish were grouped by gender and site,frozen, and shipped to the analytical laboratory.

2.2. Laboratory analyses

Individual fish were partly thawed, cut into pieces,and ground to a fine texture. Fifteen percent of the totalbody weight was sub-sampled (14–1040 g) to maintainthe proportional size representation of each fish in acomposite sample. The ground sub-samples were then

grouped to create a single homogenous composite sam-ple for each site, species, and gender combination. Thecomposite sample was then sub-sampled (200 g) and re-frozen (−20 °C). All equipment was disassembled andchemically cleaned between composite samples toprevent cross-contamination. All fish collected wereincluded in one of the 53 composite samples, which hadfrom 1 to 13 fish in each sample. Both bass and catfish(both predator species) were collected from Stations 312,315, and 324; five composite samples (female and malebass from Station 312; male bass from Station 324 (nofemale bass collected); female and male catfish fromStation 315) were excluded from the chemical analysis inorder to reduce analytical costs.

Dichloromethane extracts (0.1 g) of a 10-g sub-samplewere analyzed gravimetrically for lipid content. Sampleswere analyzed by high-resolution capillary gas chroma-tography with electron capture detection (GC-ECD) for29 organochlorine pesticide residues and total polychlori-nated biphenyls (PCBs) after size exclusion and adsorp-tion column cleanup procedures (Hinck et al., 2006b,c).Total PCBs were reported as the sum of 139 congeners.Toxaphene residues were quantified on the basis of 20component peaks of a technical toxaphene standard.Quality assurance (QA) measures for the organochlorinepesticide and PCB analyses included the analysis ofblanks, triplicate analyses, and matrix spikes. In addition,recovery standards were added to each sample to correctfor analytical losses. Pesticides were identified by dual-column GC-ECD. Recoveries were 91±7% (mean±SD)for organochlorine pesticides and 77±7% to 91±7% forPCBs. The limit-of-detection (LOD) for each compoundwas calculated by adding the average procedural blank

Table 2Mean±standard error of total length, weight, and age of carp, bass, and catfish from the Colorado River Basin

Taxon, station Female Male

n Length (mm) Weight (g) Age (years) n Length (mm) Weight (g) Age (years)

Carp312 — Ouray NWR, UT 11 456±9 1167±73 7.7±0.3 9 442±16 1052±92 15.8±4.4313 — San Rafael, UT 10 467±12 1237±92 22.7±4.0 11 452±9 1026±65 34.4±2.6314 — Delta, CO 13 575±19 2076±222 19.8±1.8 8 562±24 1633±207 18.1±3.8315 — Loma, CO 10 a 543±22 1901±211 8.3±0.9 10 b 499±21 1497±133 7.5±0.3316 — Gold Bar Canyon, UT 10 466±13 1105±89 31.5±3.5 10 440±6 918±33 26.2±2.0317 — Hogback Diversion, NM 7 c 520±8 1811±80 25.0±4.0 13 511±10 1622±71 18.2±2.5319 — South Cove, AZ 11 550±18 2039±189 23.9±4.4 6 482±24 1352±208 34.7±5.2320 — Willow Beach, AZ 9 a 517±14 1735±128 41.9±2.8 11 518±13 1608±118 44.2±2.0321 — Needles, CA 12 506±16 1517±110 26.8±2.3 8 489±19 1384±118 22.0±2.1322 — Imperial Dam, AZ 12 540±10 1972±177 14.3±2.3 8 518±9 1580±72 14.3±2.5323 — Hayden, AZ 7 462±49 1486±385 9.9±3.8 13 419±30 1024±183 6.4±1.4324 — Phoenix, AZ 8 d 444±33 1163±181 6.1±1.2 10 357±17 566±87 3.7±0.5325 — Arlington, AZ 13 370±20 690±114 5.2±0.5 7 331±19 418±81 5.0±0.7

Bass311 — Lay, CO 9 a 354±16 532±90 6.4±0.8 10 a 309±16 532±90 5.3±0.4312 — Ouray NWR, UT 6 e 312±7 349±36 2.2±0.2 8 281±9 349±36 1.9±0.1315 — Loma, CO 5 404±26 404±58 5.0±0.4 7 b 354±16 404±58 4.5±0.2321 — Needles, CA 11 451±17 636±58 5.3±1.0 9 394±17 636±58 4.8±0.9322 — Imperial Dam, AZ 10 382±28 609±99 2.3±0.7 10 348±18 609±99 1.9±0.3323 — Hayden, AZ 4 267±12 311±60 1.5±0.3 5 280±20 311±60 1.2±0.2324 — Phoenix, AZ 0 – – – 2 376±24 895±325 2.5±0.5325 — Arlington, AZ 11 317±15 895±325 1.8±0.3 9 341±16 626±77 2.1±0.4

Catfish312 — Ouray NWR, UT 5 323±22 255±58 8.8±0.9 6 308±10 209±27 6.7±0.7313 — San Rafael, UT 4 320±14 210±26 5.0±0.6 14 338±17 360±59 5.7±0.5315 — Loma, CO 2 f 485±21 713±93 15.0 1 g 488 1230 –316 — Gold Bar Canyon, UT 10 332±18 272±51 6.3±0.5 10 328±12 256±36 6.2±0.8317 — Hogback Diversion, NM 11 456±14 876±107 10.0±0.8 8 456±25 885±142 8.1±0.7324 — Phoenix, AZ 12 450±36 1079±270 2.8±0.4 3 h 493±84 1855±420 2.7±0.3

Note. –, Not applicable.a n=8 for age.b n=6 for age.c n=6 for weight.d n=7 for length.e n=5 for age.f n=1 for age.g n=0 for age.h n=2 for age.

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concentration to three times the procedural blank standarddeviation (Keith, 1991). The nominal LODs were0.0014 μg/g wet-weight (ww) for individual compounds,0.048 μg/g ww for total PCBs, and 0.024 μg/g ww fortoxaphene. Polybrominated diphenyl ethers (PBDEs)present in samples from Stations 323, 324, and 325interfered with o,p′-DDT and p,p′-DDD concentrationsin these samples; therefore, samples from Stations 323,324, and 325 were subjected to an additional basicalumina column cleanup step and re-evaluated by gaschromatography with mass spectrometry (GC–MS) toaccurately quantify the DDTs with PBDE interferences.

Sub-samples for elemental analyses (100 g) werefreeze-dried. Percent moisture was determined as weight

lost during lyophilization.One portion of the driedmaterialwas digested in nitric acid and analyzed by inductivelycoupled plasmamass spectroscopy (ICP-MS) for cadmium(Cd), copper (Cu), and zinc (Zn). A second portion wasdry-ashed (magnesium nitrate–nitric acid–HCl) andanalyzed by hydride generation atomic absorption spec-troscopy for arsenic (As) and selenium (Se). A thirdportion was analyzed directly for total mercury (Hg) usingthermal combustion, amalgamation, and atomic absorptionspectroscopy. QA measures for elemental determinationsincluded 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/gdw for Hg, 0.014 μg/g dw for Se, 0.4 μg/g dw for Cd and

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Cu, and 1.0 μg/g dw for Zn. Elemental concentrations(including LODs) were converted from dry-weight (dw) toww for statistical analysis and reported using the moisturecontent of each sample (range: 66–76%).

A third sub-sample (10 g) was solvent-extracted andsubjected to reactive cleanup for use in theH4IIE bioassay(Tillitt et al., 1991; Whyte et al., 2004). Concentrations of2,3,7,8-tetrachlorodibenzo-p-dioxin equivalent doses(TCDD-EQ; pg/g ww) were determined by slope ratioassay as modified by Ankley et al. (1991). QA measuresfor the H4IIE bioassay included analysis of duplicatesamples and reference materials. Limits-of-quantification(LOQs; 0.1–0.8 pg/g) and LODs (0.5–1.7 pg/g) werecomputed separately for each set of samples.

Hepatic EROD activity was determined on micro-somal fractions, and protein content was quantified usingthe fluorescamine protein assay (Hinck et al., 2006c).EROD activity was reported as the mean of triplicatedeterminations. The LOD was calculated by adding theaverage basal EROD rate to three times the standarddeviation of that rate for each set of samples analyzed(n=19). QA measures included LODs (0.01–0.23 pmol/min/mg), LOQ (0.12–0.87 pmol/min/mg), and theanalysis of reference materials and duplicate samples.Hepatic EROD activity was bLOQ in 18 of 517 samples.

Body and organ weights were used to compute con-dition factor (CF) and organosomatic indices accordingto the following formulae: CF=body weight in g/(lengthin cm)3; hepatosomatic index (HSI)= liver weight / (totalbody weight−gonad weight) × 100; splenosomaticindex (SSI)=spleen weight / (total body weight−gonadweight) × 100; gonadosomatic index (GSI) =gonadweight / total body weight×100. The weight of thegonads was subtracted from the bodyweight to minimizethe effect of the reproductive cycle on these indices.

The occurrence of gross external and internal patho-logical disorders was determined during field proces-sing. Tomaintain consistencywith previous studies (e.g.,Fournie et al., 2001; Schmitt, 2002; Schmitt et al., 2005;Hinck et al., 2006a), only grossly visible disorders ofthe eye, opercles, body surface, fins, and skeleton wereincluded. A necropsy-based health assessment index(HAI) score was calculated for each fish by assigningnumerical values to gross lesions (Adams et al., 1993;Schmitt, 2002), then summing the values for all organsobserved. An HAI score, which can range from 0 to 220,was computed for a fish only if observations were pres-ent for all components.

Preserved gill, liver, gonad, spleen, anterior kidney,and posterior kidney tissues were prepared for histo-pathological analysis (Schmitt, 2002). Tissue sampleswere dehydrated, embedded in paraffin, sectioned at 6-

μm, and stained with hematoxylin and eosin (H & E) formicroscopic examination. In general, two to fivesections of each tissue sample were examined forabnormalities in each fish. Transverse ovary sectionswere assigned to developmental stages 0 (immature), 1(previtellogenic), 2 (early vitellogenic) 3 (mid-vitello-genic), 4 (late vitellogenic), and 5 (spent) based on thepredominant size and appearance of oocytes, andtransverse testes sections were similarly classified intodevelopmental stages 0 (immature), 1 (early spermato-genic), 2 (mid-spermatogenic), 3 (late spermatogenic),and 4 (spent; Blazer, 2002). Gonad tissue was alsoexamined microscopically for abnormalities such asintersex and oocyte atresia (reabsorbed or degeneratingeggs). Atresia was quantified by counting one hundredoocytes in each sample and reported as a percent. Fishwere identified as intersex (i.e., when an ovotestiscondition was detected) when individual or small foci ofundeveloped oocytes were observed within testiculartissue or when spermatocytes were observed withinovarian tissue. Macrophage aggregates (MA) in spleensections were stained using Perl's method (Luna, 1992).MA parameters included the number of aggregates in2 mm2 of tissue (MA-#) and the mean size (area) ofaggregates within those 2 mm2 (MA-A). The percentageof tissue occupied by aggregates (MA-%) was computedfrom these measurements. MA parameters were quan-tified using computer-based image analysis in spleensections from Stations 311, 313, 314, 316, 317, 321,322, 323, and 325 and manually digitized microscopicanalysis in spleen tissues from Stations 312, 315, 319,320, and 324. MAs in bass and catfish from Stations 312and 315 were quantified using both methods. Measure-ment differences in the methods were not consistentlysignificant among species or MA parameters; therefore,data from bothmethods were combined for data analysis.

Concentrations of vtg in carp (n=251) and bass(n=114) were determined by direct enzyme-linkedimmunosorbent assay and were reported as the meanof triplicate measurements (Denslow et al., 1999).Quality assurance measurements included the LOD(0.0005 mg/mL for carp; 0.001 mg/mL for bass),coefficient of variation (b10% for all samples), andinter-assay variability (b10%). Vitellogenin concentra-tions were bLOD in 91 of 365 (25%) plasma samples,of which 62 (68%) were from male fish. Concentrationsof 17β estradiol (E2) and 11-ketotestosterone (KT) inplasma samples were measured by radioimmunoassay(Hinck et al., 2006c). QA measurements included theLOD (10.7 pg/mL for E2; 14.3 pg/mL for KT),extraction efficiency (86–90%), coefficient of variation(b10%), and inter-assay variability (b9%). Cross-

Fig. 2. Concentrations of total mercury and selenium (all μg/g wet-weight) in whole-body composite samples of fish in the Upper(Stations 311–317) and Lower (Stations 319–325) Colorado RiverBasin. Reference lines on mercury graph include protective thresholdsfor piscivorous mammals (0.1 μg/g ww; Yeardley et al., 1998),juvenile and adult fish (0.2 μg/g ww; Beckvar et al., 2005), andpiscivorous birds (0.3 μg/g ww; Barr, 1986). Reference lines on theselenium graph include protective thresholds for piscivorous wildlife(0.75 μg/g ww) and larval fish (1.0 μg/g ww; Lemly, 1996, 2002).

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reactivities of the E2 antiserum with estrone (1.32%),estriol (2.46%), 17α-estradiol (1.32%), and othersteroids (b0.2%) and the KTantiserum with testosterone(9.65%), dihydrotestosterone (3.7%), androstenedione(b1.0%), and other steroids (b0.1%) were low.

2.3. Data set composition and statistical analyses

All results for analytes in whole-body compositesamples were converted to, reported as, and analyzedstatistically as ww concentrations. Avalue of one-half theLOD was substituted for censored values in all statisticalanalyses and graphs. Concentrations of many contami-nants were bLOD, which limited the extent and rigor ofstatistical analyses that could be performed. Spatialdifferences in concentrations of Hg, Se, and p,p′-DDEwere tested with analysis-of-variance (ANOVA) usingFisher's unrestricted least significant difference (LSD;Saville, 1990). Log-transformed concentrations of thesecontaminants were analyzedwith a series of t-tests using apooled error mean-square (MSe) representing differencesbetween samples of the same species. A conservative α-level of 0.01 was used in these comparisons to protectagainst experiment-wise error.

Biomarker results were analyzed using ANOVA totest for differences among sites and to examine var-iation due to gender, age, and reproductive stage. Fishfor which age could not be determined (see Table 2)were excluded from all analyses that included age as afactor. Fish for which the gender (and subsequentlygonadal stage) could not be verified histologically(carp from Station 324 (n=2); bass from Station 311(n=1); and catfish from Stations 315 (n=2) and 317(n=1)) were also excluded from analyses that includedgender as a factor. Hepatic EROD activity, MA-A,MA-%, and vtg concentrations were log10-transformedto approximate normality and homogeneity-of-vari-ance. A value of one-half the LOQ was substituted forcensored (bLOQ) EROD activities and vtg concentra-tions in all statistical analyses. Differences in hepaticEROD activity among sites were tested with ANOVAusing Fisher's unrestricted LSD (Saville, 1990). Toaccount for unequal sample sizes, the pooled errormean-square was used to determine significant differ-ences among sites. Males and females were analyzedand presented separately if ANOVA indicated thatgender was a significant factor for a biomarker.Histological descriptions of tissues were qualitativeand not included in the statistical analyses. All com-putations and statistical analyses were performed withVersion 9.1 of the Statistical Analysis System (SASInstitute, Cary, NC).

3. Results

3.1. Lipid and moisture content (data not shown)

Lipid content of whole-body composite sam-ples differed among sites and species but was typically2–10%. Samples contained 66–76% water.

3.2. Exposure indicators

3.2.1. Elemental contaminantsElemental contaminants were measured in carp, bass,

and catfish. Spatial differences were analyzed for Hg andSe because concentrations exceeded literature-basedtoxicity thresholds. Total Hg was detected in all samples(0.01–0.37 μg/g), and the greatest Hg concentrations(N0.20 μg/g) were in catfish from Stations 312 and 313and bass from Station 311 (Fig. 2). Concentrations were

Table 3Concentrations a (all μg/g wet-weight) of total mercury (Hg), selenium (Se), and p,p′-DDE (DDE) that exceeded minimum toxicity thresholds in atleast one composite sample b

Taxon, station Hg Se DDE

Carp312 — Ouray NWR, UT 0.15±0.01 gh 1.43±0.04 b 0.012±0.004 a313 — San Rafael, UT 0.17±0.01 h 1.69±0.06 bcd 0.011±0.001 a314 — Delta, CO 0.06±0.01 de 2.92±0.03 h 0.235±0.015 gh315 — Loma, CO 0.07±0.01 ef 2.15±0.11 efg 0.140±0.030 fg316 — Gold Bar Canyon, UT 0.14±0.01 gh 2.02±0.01 cdef 0.044±0.011 cd317 — Hogback Diversion, NM 0.10±0.00 fg 1.50±0.22 b 0.017±0.002 ab319 — South Cove, AZ 0.04±0.00 c 2.13±0.05 defg 0.009±0.003 a320 — Willow Beach, AZ 0.02±0.00 ab 1.63±0.07 bcd 0.105±0.006 ef321 — Needles, CA 0.03±0.00 bc 2.57±0.05 gh 0.031±0.004 bcd322 — Imperial Dam, AZ 0.02±0.01 a 2.47±0.14 fgh 0.058±0.020 de323 — Hayden, AZ 0.15±0.01 gh 0.76±0.04 a 0.042 ± 0.005 cd324 — Phoenix, AZ 0.04±0.00 c 1.69±0.04 bcd 0.310±0.030 h325 — Arlington, AZ 0.03±0.01 b 2.38±0.32 fgh 1.500±0.200 I

Bass311 — Lay, CO 0.25±0.03 d 0.95±0.09 b 0.002±0.000 b315 — Loma, CO 0.09±0.02 bc 2.03±0.06 d 0.137±0.053 b321 — Needles, CA 0.10±0.03 c 1.86±0.08 d 0.015±0.001 a322 — Imperial Dam, AZ 0.04±0.00 a 2.67±0.05 e 0.036±0.008 b323 — Hayden, AZ 0.10±0.01 c 0.53±0.01 a 0.016±0.000 a325 — Arlington, AZ 0.06±0.01 ab 1.47±0.06 c 2.150±0.550 b

Catfish312 — Ouray NWR, UT 0.21±0.00 c 0.97±0.03 b 0.016±0.002 a313 — San Rafael, UT 0.34±0.03 d 0.81±0.01 b 0.008±0.001 a316 — Gold Bar Canyon, UT 0.18±0.00 bc 1.02±0.09 b 0.057±0.005 b317 — Hogback Diversion, NM 0.12±0.01 b 0.52±0.01 a 0.016±0.001 a324 — Phoenix, AZ 0.05±0.00 a 0.64±0.07 a 0.712±0.069 c

ANOVA F25,26 47.71⁎ 73.66⁎ 81.74⁎

a Shown are arithmetic means (±standard errors). Also shown are results of one-way analysis-of-variance (ANOVA) as F-values and degrees-of-freedom for differences among sites (⁎P≤0.01). Within each taxon-station group, means followed by the same letter are not significantly different(Pb0.01). Concentrations were log-transformed for statistical anlaysis.b n=2 composite samples for all taxon-station group.

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greater in piscivores than benthivores at most sites.Differences in total Hg among stations were significant incarp, bass, and catfish (Table 3). Seleniumwas detected inall samples (0.51–2.95 μg/g), and the greatest concentra-tions (≥2.0 μg/g) were in carp from Stations 314, 315,316, 319, 321, 322, and 325 and bass from Stations 315and 322 (Fig. 2). Selenium concentrations were generallyb1.0 μg/g in catfish. Among station differences in Seconcentrations were significant in carp, bass, and catfish(Table 3). Arsenic was detected in all samples, but con-centrations were low (0.01–0.19 μg/g). The greatest Asconcentrations (N0.14 μg/g) were in carp samples fromStations 319, 320, 321, 322, and 323 (data not shown).Cadmium concentrations were NLOD (0.03 μg/g) in 22of 48 samples (46%) representing Stations 312, 313,314, 315, 316, 317, 319, 320, 322, and 323. Cadmiumconcentrations were greater in carp (bLOD-0.24 μg/g)than in bass (bLOD) and catfish (0.03–0.04 μg/g) andhighest (N0.20 μg/g) in carp from Stations 313 and 316(data not shown). Copper was detected in all samples, and

concentrations were generally greater in carp (0.71–1.39 μg/g) than in bass (0.34–1.16 μg/g) and catfish(0.36–0.68 μg/g; data not shown). Zinc (Zn) was detectedin all samples at concentrations of 12.8–99.6 μg/g, andconcentrations were greater in carp (N52 μg/g) than inother taxa (b36 μg/g; data not shown).

3.2.2. Organochlorine pesticidesConcentrations of p,p′-DDTwere NLOD (0.0014μg/g)

in 19 of 48 samples from seven stations (data notshown). p,p′-DDE, the most persistent metabolite of p,p′-DDT, was detected in all samples, and p,p′-DDEconcentrations were greatest (N0.5 μg/g) in carp andbass from Station 325 and catfish from Station 324(Fig. 3). Among station differences in p,p′-DDE con-centrations were significant in all taxa (Table 3). Con-centrations of p,p′-DDD were detected in 44 of 48samples. Relatively high concentrations (N0.025 μg/g)were measured in samples from Stations 324 and325 (data not shown). Concentrations of o,p′-DDE

384 J.E. Hinck et al. / Science of the Total Environment 378 (2007) 376–402

(b0.00008–0.017 μg/g), o,p′-DDD (b0.00055–0.017 μg/g), and o,p′-DDT (b0.00053–0.11 μg/g)were greatest in male largemouth bass from Station

325 (data not shown). Concentrations of o,p′-DDTweregreater in fish from the GR (0.05–0.11 μg/g) than thosefrom other sites in the CRB (b0.003 μg/g).

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Seven chlordane-related compounds (cis-chlordane,trans-chlordane, cis-nonachlor, trans-nonachlor, oxy-chlordane, heptachlor, and heptachlor epoxide) weremeasured. Trans-nonachlor was detected in all samples,and concentrations were N0.01 μg/g in carp from Stations315, 316, and 324, bass fromStation 325, and catfish fromStations 316 and 324 (data not shown). Cis-nonachlorwas detected (N0.0001 μg/g) in most (98%) samples, butconcentrations N0.01 μg/g were measured only in carpfrom Station 315 and catfish from Station 324. Cis-chlordane was detected (N0.00004 μg/g) in 47 of 48samples, and concentrationswereN0.01μg/g in carp fromStations 315, 324, and 325 and catfish from Station 324.Trans-chlordane was detected (N0.00023 μg/g) in 39 of48 samples. Concentrations were N0.01 μg/g in carp fromStations 315 and 324 and catfish from Station 324.Concentrations of oxychlordane, heptachlor, and hepta-chlor epoxide were low (b0.01 μg/g) in all samples (datanot shown). Total chlordane concentrations (sum of sevencompounds) ranged from 0.002 to 0.12 μg/g and wereN0.05 μg/g in carp from Stations 315, 324, and 325 andcatfish from Station 324 (Fig. 3).

Concentrations of other formerly used organochlo-rine pesticides including dieldrin, endrin, mirex, toxa-phene, and hexachlorobenzene were also relatively highin samples from Stations 324 and 325. Concentrationsof dieldrin were ≥LOD (0.00005 μg/g) in 43 of 48samples and were N0.01 μg/g in carp from Stations 315and 324, bass from Station 325, and catfish from Station324 (Fig. 3). Endrin was detected (N0.0001 μg/g) in 47of 48 samples, and concentrations were N0.05 μg/g incarp and catfish from Station 324 and carp and bassfrom Station 325 (data not shown). Mirex was detected(N0.00005 μg/g) in 44 of 48 samples, but concentrationswere ≤0.001 μg/g in all samples (data not shown).Toxaphene was NLOD (0.024 μg/g) in samples fromStations 323 (0.049 μg/g), 324 (0.072–0.50 μg/g), and325 (0.50–0.87 μg/g; Fig. 3). Hexachlorobenzene wasdetected at trace concentrations in most samples (94%),and the greatest concentrations (N0.002 μg/g) weremeasured in carp from Stations 314 and 324 and catfishfrom Station 324 (Fig. 3).

Concentrations of currently used organochlorineresidues and their metabolites followed patterns similar

Fig. 3. Concentrations of formerly used (first column) and currently used (swet-weight) in whole-body composite samples of fish in the Upper (Stationschlordanes are the sum of cis- and trans-chlordanes and nonachlors, oxychloof endosulfan I and II and endosulfan sulfate. Censored values are plottedconcentrations in one or more CRB samples exceeded the threshold; toxicReference lines on the p,p′-DDE graph include toxicity thresholds for fish (0The reference line on the toxaphene graph represents the threshold where rep1975).

to those of formerly used pesticides. Pentachloroben-zene was detected (N0.00007 μg/g) in 32 of 48 samples,but concentrations were b0.0002 μg/g in all samplesexcept those from Stations 324 and 325 (Fig. 3).Pentachloroanisole, a metabolite of pentachlorophenol,was detected (N0.00013 μg/g) in 46 of 48 samples, andconcentrations were N0.01 μg/g in carp from Stations312 and 316 and catfish from Station 324 (Fig. 3).Lindane (γ-hexachlorocyclohexane) was detected(N0.00051 μg/g) in 18 of 48 samples, and concentra-tions were N0.004 μg/g in all samples from Station 324(Fig. 3). Dacthal was detected (N0.0005 μg/g) in 24 of48 samples, and concentrations were greatest (0.005–0.009 μg/g) in fish from Station 324 (Fig. 3). Mostendosulfan concentrations (sum of endosulfan I, endo-sulfan II, and endosulfan sulfate) were b0.02 μg/g, butthe greatest concentrations (N0.07 μg/g) were in carpand bass from Station 325 (Fig. 3). Methoxychlor wasdetected (N0.00035 μg/g) in 11 of 48 samples, andconcentrations were greatest (N0.002 μg/g) in fish fromStations 324 and 325 (data not shown).

3.2.3. Total PCBs and TCDD-EQConcentrations of total PCBswere NLOD (0.048μg/g)

in 22 of 48 samples from nine stations (data not shown).Concentrations ranged from 0.05 to 2.1 μg/g, with themaximum measured in female catfish from Station 324.Other samples with relatively high concentrations(≥0.8 μg/g) included carp from Stations 320 and 324and catfish from Station 324.

TCDD-EQ concentrations were NLOD (0.10–0.25 pg/g) in 6 of 48 samples, and concentrationsranged from 2 to 6 pg/g (data not shown). TCDD-EQswere N2 pg/g in male carp from Station 320 and malecatfish from 316 and were N5 pg/g in all samples fromStations 324 and 325.

3.2.4. Hepatic ethoxyresorufin O-deethylase (EROD)activity

Hepatic EROD activity was analyzed statistically incarp, bass, and catfish. In carp, a significant ANOVAmodel containing the factors station, gender, gonadalstage, and their interactions explained 41% of the totalvariation in EROD activity (F41, 207=3.45, Pb0.01).

econd column) organochlorine residues and their metabolites (all μg/g311–317) and Lower (Stations 319–325) Colorado River Basin. Totalrdane, heptachlor, and heptachlor epoxide. Total endosulfan is the sumas 50% of LOD. Literature-based toxicity thresholds were plotted ifity threshold were not available for all organochlorine contaminants..6 μg/g; Beckvar et al., 2005) and avian wildlife (1 μg/g; Blus, 1996).roductive effects have been documented in fish (0.4 μg/g; Mayer et al.,

Table 4Hepatic ethoxyresorufin O-deethylase (EROD) activity a (pmol/min/mg protein) by taxon, station, and gender

Taxon, station Female Male

n EROD(pmol/min/mg)

n EROD(pmol/min/mg)

Carp312 — Ouray NWR, UT 11 0.93+0.38 a 9 1.85+0.68 ab313 — San Rafael, UT 10 3.44+1.00 bc 11 8.79+2.33 cd314 — Delta, CO 13 2.01+0.37 ab 8 4.01+0.46 bcd315 — Loma, CO 10 1.46+0.40 ab 10 4.20+1.01 bcd316 — Gold Bar Canyon, UT 10 0.98+0.45 a 10 3.80+0.85 bc317 — Hogback Diversion, NM 7 0.81+0.21 a 13 3.15+1.14 ab319 — South Cove, AZ 11 3.37+2.04 bc 6 3.42+0.73 abcd320 — Willow Beach, AZ 9 4.37+1.15 bc 11 4.49+1.07 bcd321 — Needles, CA 12 2.03+0.37 ab 8 3.73+1.10 abcd322 — Imperial Dam, AZ 12 0.95+0.12 a 8 1.22+0.59 a323 — Hayden, AZ 6 0.85+0.52 a 13 1.97+0.64 ab324 — Phoenix, AZ 8 9.05+3.55 c 10 10.7+2.42 d325 — Arlington, AZ 13 2.40+0.75 ab 7 2.54+1.73 abANOVA F12, 120=5.70⁎ F12, 111=4.56⁎

Bass311 — Lay, CO 9 6.95+0.77 ab 10 23.4+1.77 c312 — Ouray NWR, UT 6 18.6+27.8 b 8 61.2+7.12 d315 — Loma, CO 5 11.6+3.67 ab 7 15.1+5.43 bc321 — Needles, CA 11 7.27+1.05 ab 9 13.1+3.52 bc322 — Imperial Dam, AZ 10 4.47+1.08 a 10 3.51+0.88323 — Hayden, AZ 4 2.50+1.49 a 5 6.29+2.27 ab324 — Phoenix, AZ 0 – 2 4.36+2.34 ab325 — Arlington, AZ 11 9.01+1.85 ab 9 9.36+3.40 bANOVA F6, 48=2.76⁎ F7, 52=14.84⁎

Catfish312 — Ouray NWR, UT 5 6.30+1.49

1.49.14 a6 10.8+1.76 a

313 — San Rafael, UT 4 8.90+3.74 a 14 6.89+1.06 a315 — Loma, CO 2 11.2+6.14 a 1 4.59 a316 — Gold Bar Canyon, UT 10 5.08+1.19 a 10 6.44+1.79 a317 — Hogback Diversion, NM 11 7.52+1.16 a 8 6.84+1.09 a324 — Phoenix, AZ 13 4.91+1.29 a 3 3.71+1.77 aANOVA F5, 38=1.39 F5, 36=1.62

a Shown are geometric means (+standard error). Also shown are results of one-way analysis-of-variance (ANOVA) as F-values and degrees-of-freedom for differences among sites (⁎P≤0.05). Within each taxon-station group, means followed by the same letter are not significantly different(Pb0.01). Censored values were represented by 50% of the limit-of-quantification in all computations. Data were log-transformed for statisticalanlaysis.

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Hepatic EROD activity was generally greater in malecarp than in female carp. Mean EROD activity differedamong sites, with mean activity N5 pmol/min/mg infemales from Station 324 and males from Stations 313and 324 (Table 4). Hepatic EROD activity in individualcarp was N15 pmol/min/mg in some females fromStations 319, 320, and 324 and males from Stations 313,315, 317, 320, 324, and 325. In bass, a significantANOVA model explained 49% of the total variation inEROD activity (F23, 92=3.78, Pb0.01). Similar to carp,EROD activity was greater in male bass than in femalebass. Mean EROD activity differed among sites and wasgreatest in bass from Station 312 (Table 4). Mean EROD

activity was N10 pmol/min/mg in females from Stations312 and 315 and N20 pmol/min/mg in males fromStations 311 and 312. In catfish, mean EROD activitydid not differ significantly among sites (F21, 63=1.17,PN0.05). Mean EROD activity was N10 pmol/min/mgin female catfish from Station 315 and male catfish fromStation 312 (Table 4).

3.3. Fish health indicators

3.3.1. Health assessment index (HAI) and histologyFish with high HAI scores were generally considered

to be in poorer health than those with low HAI scores.

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HAI scores for most carp (91%) were 0–70 and were≥100 in individual fish from Stations 313, 314, 315,319, 320, 321, and 322. Mean HAI scores were b40 incarp in the Upper CRB and the GR and N47 in carp fromthe Lower CR (Fig. 4). Liver discoloration, granularspleen and kidney, and pale gills accounted for mosthigh HAI scores at Stations 319, 320, 321, and 322. HAIscores for most bass (87%) ranged from 0 to 100. Mean

Fig. 4. Mean health assessment index (HAI) scores by lesion locationin carp, bass, and catfish in the Upper (Stations 311–317) and Lower(Stations 319–325) Colorado River Basin.

HAI scores ranged from 50 to 103 and were uniformlygreater (N90) in bass from Stations 315, 321, and 322(Fig. 4). Liver discoloration; granular liver, kidney, andspleen; and body surface lesions elevated HAI scores infish from Stations 315, 321, and 322. HAI scores inmost catfish (85%) were 0–70, and mean HAI scoreswere b43 except in fish from Stations 315 and 317(Fig. 4). High HAI scores in fish from Stations 315 and317 were attributed to head and eye lesions, frayed anderoded fins, liver discoloration, and granular spleen(Fig. 4).

Histopathological examinations were conducted ongill, liver, gonad, kidney, and spleen tissues of fish fromStations 312, 315, 319, 320, and 324. Mild basalhyperplasia of the lamellar epithelium was noted in thegill tissue of carp from Stations 312 and 320 and catfishfrom Station 312. Edema, parasites, and fused lamellaewere also found in carp from Station 320. Cystic andproliferated bile ducts, hypertrophy, and MAs wereidentified in carp liver tissues from Station 320. A largegranuloma with calcification that was likely parasite-related was also found in liver tissue of a catfish fromStation 312. Extensive parasitic infestations of trema-todes and some myxosporeans were identified in liverand gonad tissues of many largemouth bass. Degener-ation and calcification in some liver samples wereparasite-related, and associated granulomas were occa-sionally observed in these same tissues. Macrophageaggregates were present in the gonadal tissue of multiplecarp from Station 320, and ova containing fluid werepresent in several female carp from Station 319.Anomalies in gonadal tissues were observed in manycarp and catfish from Station 324; calcification, edema,and parasites were the most common abnormalitiespresent. In splenic tissue, parasitic infestations inlargemouth bass were similar to, but less prevalentthan, those found in liver and gonad tissues. A largehematoma was present in spleen tissue of a carp fromStation 312, and an edema was found in individual carpfrom Stations 315 and 319. MAs were common inkidney tissues of all species. Ectopic thyroid follicles(TFs) were also common in the head and hind kidney ofcarp from all stations; however, TFs were not found inbass or catfish. Thyroid follicles differed in size, werefilled with eosinophilic-staining colloid, and weregenerally surrounded by flattened squamous epithelium.Hind kidney TFs were generally smaller but morenumerous than those in the head kidney. Similar to liverand spleen tissues, parasites and associated lesions wereprevalent in head and hind kidney of largemouth bass.Crystallization, calcification, or both were present inhind kidney tubules of multiple carp from Station 320.

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Hyaline droplet degeneration was also present in thehind kidney of five female catfish from Station 324.

Histopathological examination of external lesionsrevealed that most nodules on the body surface and finswere inflammatory responses from parasitic infesta-tions. Two raised fin lesions on carp from Station 320were diagnosed as fibromas with inflammation andincreased numbers of melanocytes, and a hematoma wasidentified on the opercle of a carp from Station 324. Afibroma and papilloma were observed on catfish fromStation 313, and a papilloma was observed on one bassand one carp from Station 321.

3.3.2. Condition factor and organosomatic indices(data not shown)

Condition factor, HSI, and SSI computed from bodyand organ weights were used as general indicators ofoverall fish health. Condition factor values of 1.0–2.0are common in many fish species. In carp, a significantANOVA model containing the factors station, gender,gonadal stage, and their interactions explained 27% ofthe total variation in condition factor (F41, 205=1.87,Pb0.01). Mean CF values ranged from 1.0 at Station314 to 1.2 at Station 317 in carp, but the CF value was1.9 in one carp from Station 311. CF values wererelatively high (N1.6) in carp from Stations 311 and 320and low (b0.8) in carp from Stations 314, 315, and 321.In bass, a significant ANOVA model explained 60% ofthe total variation in condition factor (F23, 92=5.76,Pb0.01). Mean CF values ranged from 1.0 at Stations315 and 321 to 1.7 at Station 311. Condition factorvalues N1.5 were calculated for fish from all stationsexcept Station 321, and CF values b1.0 were calculatedfor individual fish from Stations 315 and 321, where lowCF values were also observed in carp. The ANOVAmodel for CF in catfish was statistically significant andexplained 67% of the total variation (F21, 62=6.11,Pb0.01). Differences among stations (F5, 62=7.45,Pb0.01) and between genders (F1, 62=6.41, Pb0.05)were significant. Mean CF values ranged from 0.7 atStations 313 and 315 to 0.9 at Station 324 in females and0.7 at Station 316 to 1.1 at Station 315 (n=1) in males.CF values in most catfish (91%) were b1.0.

In bass, theANOVAmodel forHSIwas significant andexplained 57% of the total variation (F14, 101=9.72,Pb0.01). Differences among stations were also signifi-cant (F7, 101=18.83,Pb0.01). Station means ranged from0.7% at Station 312 to 1.7% at Station 311, and mean HSIvalues were low (b1.0%) in bass from Stations 312, 321,and 322. Hepatosomatic index values were N2.0% inseveral fish fromStation 311 only. TheANOVAmodel forHSI in catfish was statistically significant and explained

24% of the total variation (F11, 73=2.06, Pb0.05),and differences among stations were also significant(F5, 73=2.69, Pb0.05). Station means ranged from 1.3%at Station 316 to 1.7% at Station 313. Individual HSIvalues were N2.0% in fish from Stations 312, 313, and317 and b1.0% in fish from Stations 316 and 317.

Splenosomatic index values in carp differed signif-icantly among stations (F12, 212=2.96, Pb0.01) andbetween genders (F1, 212=14.86, Pb0.01). Mean SSIvalues ranged from 0.16% at Stations 324 and 325 to0.30% at Station 317 in female carp and 0.15% atStation 324 to 0.46% at Station 315 in male carp.Individual SSI values were ≥0.30% in females fromStations 312, 314, 315, 316, 317, and 320 and ≤0.10%in females from Stations 319 and 324. Individual SSIvalues were N0.50% in males from Stations 312, 315,and 320. A high SSI value (1.74%) was calculated for amale carp from Station 315. SSI values in bass differedsignificantly among stations (F7, 101=3.56, Pb0.01).Mean SSI values ranged from 0.03% at Station 324(n=2) to 0.16% at Station 315, and most individual bass(85%) had SSI values between 0.05% and 0.20%. Incatfish, SSI values differed significantly among stations(F5, 73=3.53, Pb0.01) but not between genders (F1, 73=0.00, PN0.05). Station means ranged from 0.08% atStation 324 to 0.20% at Station 315, and SSI valueswere 0.08–0.20% in most individuals (80%). In general,spleens were larger in catfish from Stations 315 and 317and smaller in those from Station 324.

3.3.3. Macrophage aggregatesMacrophage aggregates were quantified and analyzed

statistically in splenic tissues of carp, bass, and catfish. Incarp, an ANOVA model containing the factors station,gender, age, and their interactions was significant forMA-A (F51, 199=1.97, Pb0.01), MA-# (F51, 199=3.51,Pb0.01), and MA-% (F51, 199=3.31, Pb0.01). Stationwas not a significant factor in any of these models(PN0.05), but age was a significant factor in the MA-A(F1, 199=5.32, Pb0.05) and MA-% (F1, 199=16.75,Pb0.01) models. Mean MA-A and MA-% values weregreatest in carp from Stations 319 and 320 (Fig. 5), wheremean age was also the greatest. The high mean MA-Avalues (N9000 μm2) in carp from Stations 319 and 320were the greatest documented by the BEST-LRMNprogram. Station means were 4.1–10.9 MA/mm2 forMA-#, 2349–11463 μm2 for MA-A, and 1.8–6.6% forMA-% in carp (excluding Station 311 where n=1).

In bass, an ANOVA model containing the factorsstation, gender, age, and their interactions was signif-icant for MA-A (F29, 81=2.38, Pb0.01), MA-# (F29, 81=3.97, Pb0.01), and MA-% (F29, 81=4.96, Pb0.01).

Fig. 5. Mean (±standard error) splenic macrophage aggregateparameters in fish from the Upper (Stations 311–317) and Lower(Stations 319–325) Colorado River Basin. Parameters include macro-phage density (MA-#), macrophage aggregate area (MA-A), and percentsplenic tissue occupied by macrophage aggregates (MA-%).

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MA parameters did not differ significantly (PN0.05)among stations, but MA parameters were generallylower in fish from the Upper CRB than the Lower CRB(Fig. 5). Age was a significant factor in the MA-# model(F1, 81 =4.45, Pb0.05). Station means were 0.7–5.5 MA/mm2 for MA-#, 612–9389 μm2 for MA-A,and 0.1–3.5% for MA-% in bass.

In catfish, an ANOVA model containing the factorsstation, gender, age, and their interactions was signif-icant for MA-A (F20, 63=5.26, Pb0.01) but not MA-#(F20, 63=0.85, PN0.05) or MA-% (F20, 63=1.53,PN0.05). The MA-A values differed significantlyamong stations for catfish (F4, 63=7.90, Pb0.01) and

were greater in fish from Stations 313, 316, and 317 thanStations 312, 315, and 324 (Fig. 5). Station means were0.9–2.3 MA/mm2 for MA-#, 932–4508 μm2 for MA-A,and 0.3–0.7% for MA-% in catfish.

3.4. Reproductive biomarkers

3.4.1. Gonadal histopathologyGonadal stages 0 (1%), 1 (1%), 2 (10%), and 3 (88%)

were present in female carp (n=132) from 13 sites. Onestage-0 fish was present at Station 315, and stage-1 fishwere present at Stations 320 and 324. One female carpfrom Station 320 had evidence of intersex as identifiedby the presence of spermatozoa with previtellogenicoocytes (Fig. 6). Gonadal stages 1 (50%), 2 (30%), 3(18%), and 5 (2%) were present in female bass (n=56)from seven stations. Fifty percent of female bass werestage-1. Female smallmouth bass from Stations 311 and312 were more advanced (stages 2 and 3) thanlargemouth bass from other sites in the Lower CR(most stage-1). Gonadal stages 0 (7%), 1 (48%), 2 (25%),3 (11%), and 4 (9%) were present in female catfish(n=44) from six sites. Stage-1, -2 and -3 fish werepredominant at most stations, but stage-0 fish werepresent only at Station 324. Female carp, bass, andcatfish had varying degrees of oocyte atresia. Atresia wastypically b20% in carp, b5% in bass, and b6% in catfish.An ANOVA model containing the factors station, age,gonadal stage, and their interactions was significant foratresia only in bass. The model explained 68% of thetotal variation in oocyte atresia (F12, 31=5.46, Pb0.05).Oocyte atresia in bass differed significantly amongages (F1, 31=15.29, Pb0.05) and reproductive stages(F1, 31=15.84, Pb0.05). Mean atresia was greatest inbass from Station 321 (5.5%) compared to all other sites(b3%) and was generally greater in older bass.

Gonadal stages 0 (2%), 1 (2%), 2 (46%), 3 (49%), and4 (1%) were present in male carp (n=117) from 13 sites.All stage-0 and -1 male carp were from Station 324, andthe stage-4 male carp was from Station 323. Gonadalstages 0 (16%), 1 (44%), 2 (33%), 3 (5%), and 4 (2%)were present in male bass (n=60) from eight sites.Smallmouth bass from Stations 311 and 312 were moreadvanced (stage-2 and -3) than largemouth bass fromStations 322, 323, 324, and 325 (stage-0 and -1). Stage-0male bass were from Stations 324 and 325, and the stage-4 male bass was from Station 324. Intersex gonads werefound inmale bass from Stations 311, 322, and 323.Mildto moderate numbers of oocytes in testicular tissue werefound in seven of ten male smallmouth bass from Station311 (Fig. 6). Mild occurrence of oocytes in testiculartissue was identified in four of ten largemouth bass from

Fig. 6. Selected gonadal histological observations in CRB fish.A. Spermatozoa (s) and previtellogenic oocytes (o) in ovarian tissue offemale carp fromWillowBeach, Arizona (Station 320). B. Intersex or thepresence of previtellogenic oocytes (arrows) in testicular tissue of a malebass from Lay, Colorado (Station 311). Hematoxylin and eosin stain.

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Station 322 and two of five largemouth bass from Station323. All intersex largemouth bass were stage-1;however, intersex smallmouth bass were stages 2 and3. Gonadal stages 0 (19%), 1 (2%), 2 (14%), 3 (21%),and 4 (43%) were present in male catfish (n=45) fromsix sites. Multiple stages were found in fish from moststations, but all males from Station 324 (n=3) werestage-4 (spent). Intersex gonads were observed in arelatively small proportion of male catfish from Stations312, 317, and 324. One male fish from each of thesestations had evidence of intersex. Intersex males fromStations 312 and 317 were stage-0, but the intersexcatfish from Station 324 was stage-4.

3.4.2. Gonadosomatic index (GSI)The GSI can provide information about gonadal

health and can be influenced by gender and gonadalstage. ANOVA models containing the factors station,

gender, gonadal stage, and their interactions weresignificant for GSI in carp (F42, 195=19.37, Pb0.01),bass (F23, 91=60.61, Pb0.01), and catfish (F21, 60=7.90,Pb0.01). Themodel explained 81% of the total variationin carp. The GSI differed among stations (F11, 195=2.20,Pb0.05) and gonadal stage (F1, 195=23.66, Pb0.01),but was not influenced by gender (F1, 195=2.48,PN0.05). Mean GSI values in female carp ranged from5.0% at Station 316 to 17.6% at Station 325 (Fig. 7).Ovaries were proportionately larger in female carp fromStations 314, 317, 321, and 325 (N14%). The GSI valueswere lowest (b2.0%) in less advanced (stage-0 and -1)fish from Stations 315, 320, and 324. The GSI in malecarp differed among CRB stations, but GSI data were notavailable for male carp from Station 317. Mean GSIvalues in male carp ranged from 0.9% at Station 324 to9.6% at Station 321 (Fig. 7). Low GSI values (b1.0%)were calculated for individual male carp from Stations320 and 324; these fish were in gonadal stages 0–4.Histopathological analyses revealed that most male carpwith GSI values b1.0% also had inflammation, calcifieddeposits, macrophage aggregates, and edema in theirtesticular tissue.

The ANOVA model accounted for 94% of the totalGSI variation in bass; GSI values differed significantlyamong stations (F5, 91=5.91, Pb0.01) and betweengenders (F1, 91=14.60, Pb0.01). Overall, GSI valueswere generally greater in fish from the Upper CRBcompared to the Lower CRB (Fig. 7). Mean GSI valuesranged from 0.3% at Station 322 to 3.0% at Station 311 infemale bass (Fig. 7). The GSI values were uniformlygreater in smallmouth bass from Station 311 (1.9–3.5%)than in bass from other stations, which may reflect thelater collection date (Table 1). Mean GSI values in malebass were 0.01–0.05% (Fig. 7), and GSI values wereN0.6% in individual fish from Stations 311, 312, and 323.The GSI values were generally greater in more advanced(stage-2 and -3) male bass from the Upper CRB than inless advanced (stage-0 and -1) fish from the Lower CRB.

The ANOVA model explained 73% of the total GSIvariation in catfish. The GSI values differed signif-icantly among stations (F5, 60=4.34, Pb0.01), gender(F1, 60 = 5.05, Pb0.05), and gonadal stages (F1,

60=22.37, Pb0.01). Mean GSI values in female catfishranged from 0.3% at Station 324 to 1.2% at Stations 312and 315 (Fig. 7). In general, female catfish with GSIvalues N1.0% were in advanced reproductive stages(stages 3 or 4). Mean GSI values in male catfish were0.2–0.4% (Fig. 7) and were not correlated with stage.Low GSI values (b0.1%) were calculated for male fishat Stations 312, 313, 316, and 317 that were primarily instages 0 and 4.

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3.4.3. Vitellogenin (vtg)Vitellogenin concentrations were measured only in

carp and bass. Concentrations were NLOD in 84% ofmale carp (0.0005 mg/mL) but only 27% of male bass(0.001 mg/mL). Vitellogenin concentrations in male basswere not analyzed statistically because of the manycensored (bLOD) values. In male carp, an ANOVAmodel containing the factors station, gonadal stage, andtheir interactions explained 41% of the total variation invtg (F19, 91=3.53, Pb0.01). Station differences were notsignificant (F6, 91=0.85, PN0.05), but gonadal stage wasa significant factor (F1, 91=5.77, Pb0.05). Mean con-centrations in male carp ranged from b0.0005 mg/mL atStation 320 to 0.06 mg/mL at Stations 322 and 323(Fig. 7). Concentrations were NLOD in at least one fishfrom all sites except Station 320 and were generallygreatest in fish from the GR (Fig. 7). The highest vtgconcentrations were in stage-2 male carp from Stations323 (n=3; 0.11–0.17 mg/mL) and Station 322 (n=1;0.30mg/mL) and may represent an estrogenic response toenvironmental contaminants. Mean vtg concentrations inmale bass ranged from b0.001 mg/mL at Station 311 to0.075 mg/mL at Station 312 (Fig. 7). Concentrationswere NLOD in at least one male bass from all sites exceptStation 311 and were relatively high (N0.28 mg/mL) intwo stage-2 males from Station 312.

In female carp and bass, ANOVAmodels that includedthe factors station, gonadal stage, and their interactionswere significant for vtg. The model explained 37% of thetotal vtg variation in carp (F20, 109=3.16, Pb0.01) and67% in bass (F10, 43=8.89, Pb0.01). In carp, stationdifferences were not significant (F7, 109=1.73, PN0.05),but gonadal stage was a significant factor (F1, 109=11.14,Pb0.01). In contrast, vtg concentrations in bass differedamong stations (F3, 43=2.88, Pb0.05) but not gonadalstage (F1, 43=0.03, PN0.05). Mean vtg concentrations infemale carp ranged from 0.57 mg/mL at Station 324 to5.95 mg/mL at Station 320 (Fig. 7). Vitellogeninconcentrations were uniformly low (b1.8 mg/mL) infemales at Station 324, most of which were stage-3 fish.Mean concentrations in female bass ranged from0.005 mg/mL at Station 321 to 8.4 mg/mL at Station311 (Fig. 7). Concentrations were N5.0 mg/mL inmultiple female bass from Station 311 but wereb0.37 mg/mL in female bass from all other sites. Thegreatest vtg concentrations were found in stage-2 and -3female bass from Stations 311 and 312.

3.4.4. Steroid hormones

3.4.4.1. 17β estradiol (E2). ANOVA models contain-ing the factors station, gender, gonadal stage, and their

interactionswere significant for E2 in carp (F41, 206=30.56,Pb0.01), bass (F23, 88=8.65, Pb0.01), and catfish(F21, 61=3.07, Pb0.01). E2 concentrations in carp dif-fered among stations (F12, 206=2.52, Pb0.01) andbetween genders (F1, 206=6.37, Pb0.01), but was notinfluenced by gonadal stage (F1, 206=1.73, PN0.05).Mean E2 concentrations in female carp ranged from275 pg/mL at Station 323 to 1334 pg/mL at Station 314and were generally greatest in fish from the Upper CRB(Fig. 7). Relatively low concentrations (400 pg/mL)were measured in female carp from the GR (Stations323, 324, and 325; Fig. 7). Mean E2 concentrations inmale carp ranged from 29 pg/mL at Station 319 to313 pg/mL at Station 312 and were uniformly low atStation 319 (Fig. 7).

E2 concentrations in bass did not differ among stations(F5, 88=0.34, PN0.05), gonadal stages (F1, 88=0.08,PN0.05), or between genders (F1, 88=0.36,PN0.05). Thelack of statistical differences between genders may be dueto variation in site sample sizes (n=4–11 for females andn=2–10 for males) or the immature gonadal development(stage-1) of both males and females at many sites. MeanE2 concentrations in female bass ranged from 305 pg/mLat Station 325 to 1007 pg/mL at Station 322 (Fig. 7), andconcentrations were uniformly high (N755 pg/mL) atStation 322. In male bass, mean concentrations rangedfrom 164 pg/mL at Station 322 to 434 pg/mL at Station311 (Fig. 7). E2 concentrationswere relatively high (340–602 pg/mL) in intersex smallmouth bass from Station 311but were lower (108–324 pg/mL) in intersex largemouthbass from Stations 322 and 323. Fish from the LowerCRB were less advanced in their reproductive stage,which may explain the lower hormone concentrations inthese fish.

Like bass, E2 concentrations in catfish did not differamong stations (F5, 61=0.64, PN0.05), gonadal stages(F1, 61=1.13, PN0.05), or between genders (F1, 61=1.10,PN0.05). Mean E2 concentrations in female catfishranged from 296 pg/mL at Station 317 to 827 pg/mL atStation 312 (Fig. 7). Relatively low concentrations(b200 pg/mL) were measured in individual fish atStations 317 and 324. Mean E2 concentrations in malecatfish ranged from62 pg/mL at Station 324 to 239 pg/mLat Station 313; E2 concentrations in males were relativelylow (b200 pg/mL) in at Stations 316 and 324 and high atStation 317 (Fig. 7).

3.4.4.2. 11-ketotestosterone (KT). ANOVA modelscontaining the factors station, gender, gonadal stage,and their interactions were significant for KT in carp(F41, 206=6.94, Pb0.01), bass (F23, 88=15.63, Pb0.01),and catfish (F21, 61=7.73, Pb0.01). KT concentrations in

Fig. 7. Mean (±standard error) reproductive biomarkers including gonadosomatic index (GSI), vitellogenin (vtg), 17β estradiol (E2), and 11-ketotestosterone (KT) in fish from the Upper (Stations 311–317) and Lower (Stations 319–325) Colorado River Basin. (Note: different scales areused for female and male plots, and censored vtg concentrations are plotted as 50% of LOD).

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carp differed between genders (F1, 206=11.48, Pb0.01),but were not influenced by station (F12, 206=1.38,PN0.05) or gonadal stage (F1, 206=1.57, PN0.05).Mean KT concentrations in female carp ranged from158 pg/mL at Station 323 to 473 pg/mL at Station 315(Fig. 7). Mean KT concentrations in male carp rangedfrom 121 pg/mL at Station 319 to 1141 pg/mL at Station312 and were generally greater in the Upper CRB than theLower CRB (Fig. 7). Like E2 concentrations, KTconcen-trations were uniformly low in male carp at Station 319.

KT concentrations in bass differed among stations(F5, 88 =2.24, Pb0.05) and gender (F1, 88=8.08,Pb0.01), but not gonadal stage (F1, 88 = 1.85,PN0.05). Mean KT concentrations in female bassranged from 143 pg/mL at Station 325 to 581 pg/mLat Station 322 (Fig. 7); concentrations were uniformlylow (b264 pg/mL) in individual fish from Stations 323and 325. Mean KT concentrations in male bass rangedfrom 398 pg/mL at Station 323 to 1501 pg/mL at Station315 (Fig. 7). Concentrations were low (b500 pg/mL) infish from Stations 323 and 324. KT concentrations weregreater than E2 concentrations in six female bass atStation 321.

KT concentrations in catfish were influenced bystation (F5, 61=3.52, Pb0.01) and gender (F1, 61=5.19,Pb0.05) but not gonadal stage (F1, 61=0.06, PN0.05).Mean KT concentrations in female catfish rangedfrom 92 pg/mL at Station 317 to 733 pg/mL at Station312 (Fig. 7). Concentrations were uniformly high(N650 pg/mL) in fish at Station 312 and low(b305 pg/mL) in those at Stations 315, 316, and 317.Mean KT concentrations in male catfish ranged from87 pg/mL at Station 317 to 865 pg/mL at Station 312(Fig. 7). KT concentrations were uniformly low(b400 pg/mL) in males from Stations 316, 317, and324 (Fig. 7). E2 concentrations (N200 pg/mL) weregreater than KT concentrations (b85 pg/mL) in multiplemale catfish at Station 317.

4. Discussion

4.1. Exposure indicators

Concentrations of most elemental contaminants,including As, Cd, and Zn, were relatively low and didnot exceed toxicity thresolds. In contrast, concentrationsof Hg and Se exceeded toxicity thresholds at one ormore sites (Table 5) and have been previously identifiedas contaminants of concern in the CRB (Baker et al.,1992; King et al., 1993; USEPA, 2004).

Methylmercury, which is the most toxic form of Hg,represents N90% of the Hg that occurs in fish (Wiener

et al., 2002). Total Hg concentrations in fish from ourstudy were less than historical concentrations measuredin carp, bass, and catfish from National ContaminantBiomonitoring Program (NCBP) sites in the CRB(Schmitt et al., 1999) and previous LRMN investigations(Schmitt, 2002; Schmitt et al., 2005; Hinck et al., 2006a).Overall, Hg concentrations in our study were similar tothose measured previously in the Upper CRB and LowerCR (Radtke et al., 1988; Rowland et al., 2002) but lowerthan historical concentrations in the GR (King et al.,1997). Consumption advisories for Hg have been issuedfor sport fish from reservoirs in the San Juan River andDolores River Basins and lakes in southern Arizona(USEPA, 2004). Potential Hg sources in these areasinclude historical ore milling (Cu, gold, silver, Pb, andZn) and gold amalgamation, naturally mineralized soils,and atmospheric deposition from coal-fired power plants(Tewalt et al., 2001). Total Hg concentrations in CRBsamples exceeded toxicity thresholds for fish andwildlife. Mercury concentrations in at least one samplefrom Stations 311, 312, 313, 315, 316, 317, 321, and 323exceeded 0.1 μg/g, which may be a threat to piscivorousmammals (Table 5; Yeardley et al., 1998). Mercuryconcentrations N0.2 μg/g in bass from Station 311 andcatfish from Stations 312 and 313 exceed protectivethresholds for juvenile and adult fish (Beckvar et al.,2005), and concentrations N0.3 μg/g in catfish samplesfrom Station 312 may represent a threat to piscivorousbirds (Barr, 1986).

High Se concentrations have been measured histor-ically in CRB fish as a result of natural weathering ofseleniferous shales, irrigation practices, uranium ore andcoal extraction, and combustion of coal at hydroelectricgenerating stations (Radtke et al., 1988). Elevated Seconcentrations in the Lower CR are the result oftransportation from the Upper CR rather than localagricultural practices (Welsh and Maughan, 1994).Concentrations of Se were high in all fish samples andconfirmed previous CRB findings (Table 5). HistoricalNCBP concentrations were also relatively high in carp(0.36–3.65 μg/g), bass (0.23–3.00 μg/g), and catfish(b0.05–2.50 μg/g) near Stations 312, 319, 320, 321,322, and 323 (Schmitt et al., 1999), and Se concentra-tions in CRB fish were generally greater than thosereported in previous LRMN studies (Schmitt, 2002;Schmitt et al., 2005; Hinck et al., 2006a,b). Historically,Se concentrations were generally low and below toxicitythresholds in fish from the GR (Baker et al., 1992; Kinget al., 1997) but threatened fish reproduction andpiscivorous wildlife in the Lower CR (King et al.,1993; Andrews et al., 1997). Selenium concentrations incarp from Stations 321 and 322 were similar to those

Table 5Summary of chemical contaminant concentrations and biological endpoints that exceeded protective thresholds or were anomalous in fish from theColorado River Basin

Station Chemical contaminants and EROD activity Health indicators Reproductive biomarkers

311 — Lay, CO a b Hg (b); Se (b) (None observed) Int (mb); vtg (fb); E2 (mb)312 — Ouray NWR, UT Hg (c, ct); Se (c, ct); EROD (b) HSI (−b) Int (mct); vtg (mb)313 — San Rafael, UTc Hg (c, ct); Se (c) T (ct) (None observed)314 — Delta, COb c Se (c) CF (−c) (None observed)315 — Loma, CO Hg (b); Se (c, b) HAI (b, ct); CF (−c,−b); SSI (mc,−b) (None observed)316 — Gold Bar Canyon, UTc Hg (c, ct); Se (c, ct) (None observed) (None observed)317 — Hogback Diversion, NMc Hg (c, ct); Se (c) HAI (ct) Int (mct); E/KT (mct)319 — South Cove, AZ b c Se (c) HAI (c); MA (c) E2 (−mc); KT (−mc)320 — Willow Beach, AZ b c Se (c); PCB (c) HAI (c); MA (c); T (c) Int (fc)321 — Needles, CAb Hg (b); Se (c, b) HAI (c, b); CF (−c,−b); HSI (−b);

T (c, b)(None observed)

322 — Imperial Dam, AZb Se (c, b) HAI (c, b); HSI (−b) Int (mb); vtg (mc)323 — Hayden, AZ b Hg (c, b) (None observed) Int (mb); vtg (mc); E2 (−fc);

KT (−fc)324 — Phoenix, AZd Se (c); DDE (c, ct); Tox (ct); PCB (c, ct);

TCDD (c, ct); EROD (c)SSI (−b,−ct) Int (mct); GSI (−mc);

vtg (−fc)325 — Arlington, AZ b Se (c, b); DDE (c, b); Tox (c, b) (None observed) (None observed)

Male (m) and female (f) carp (c; Cyprinus carpio), bass (b,Micropterus spp.), and catfish (ct; Ictalurus punctatus) were collected from all sites unlessotherwise indicated. If gender is not specified, then the indicated condition was present in both. Additional abbreviations. DDE, p,p′-DDE; Tox,toxaphene; PCB, total PCBs; TCDD, dioxin-like activity as determined by H4IIE bioassay; Hg, total mercury; Se, selenium; EROD, hepaticethoxyresorufin O-deethylase activity; CF, condition factor; HAI, health assessment index; SSI, splenosomatic index; HSI, hepatosomatic index; T,tumor; MA, macrophage aggregates (one or more parameters); GSI, gonadosomatic index; vtg, vitellogenin; Int, intersex; E2, 17β estradiol; KT, 11-ketotestosterone; − indicates that the response or condition was smaller or lower than most; all others larger or greater.a No carp collected.b No catfish collected.c No bass collected.d No female bass collected.

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reported previously (Radtke et al., 1988) but were lowerin fish from Station 312, where historical concentrationswere high (Rowland et al., 2002). A consumptionadvisory for all freshwater fish was issued in the LakeStewart Wildlife Management area (near Station 312)after high Se concentrations negatively affected wildlife(Stephens et al., 1992; USEPA, 2004).

Diet is the primary route of Se exposure and toxicityin aquatic vertebrates (Lemly, 2002; Hamilton, 2004),and toxicity thresholds associated with Se tissueconcentrations are relatively low because of Se's hightoxicity and potential to bioaccumulate. Whole-bodyconcentrations of 8–16 μg/g dw (2–4 μg/g wwassuming 75% moisture) have been associated withreproductive failure in fathead minnows (Schultz andHermanutz, 1990) and bluegill (Lepomis macrochirus;Gillespie and Baumann, 1986; Hermanutz et al., 1992;Coyle et al., 1993), and concentrations of 1.2 μg/g wwreduced growth in larval rainbow trout (Vidal et al.,2005). Whole-body concentrations of Se should notexceed 4 μg/g dw (1.0 μg/g ww assuming 75%moisture) to avoid toxicity to larval fish and 3 μg/gdw (0.75 μg/g ww assuming 75% moisture) to avoid

toxicity to piscivorous wildlife (Lemly, 1996, 2002). Atleast one sample from all CRB stations exceeded one ofthese thresholds (Table 5).

Concentrations of formerly used organochlorinepesticides were elevated in fish from the GR down-stream of Phoenix (Stations 324 and 325), where lowannual precipitation and high pesticide (includingorganochlorine, organophosphate, and organocarba-mate) application rates have contributed to declines inaquatic habitat quality (Gebler, 1998; Gellenbeck andAnning, 2002). Concentrations of p,p′-DDE weregreatest (N0.5 μg/g) in samples from Stations 324 and325. Our findings were consistent with the relativelyhigh historical concentrations of total DDT (primarily asp,p′-DDE) in fish from the intensively farmed valleys ofthe Lower CR and GR (Clark and Krynitsky, 1983;Baker et al., 1992; King et al., 1997; Schmitt et al.,1999; García-Hernández et al., 2001). Concentrations ofp,p′-DDE in fish from Stations 324 and 325 weregenerally greater than those from previous LRMNinvestigations (Schmitt, 2002; Schmitt et al., 2005;Hinck et al., 2006a,b). Nevertheless, little or no p,p′-DDT was detected at these stations, indicating the

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continued weathering of residual DDT rather than theinput of new material. Concentrations of p,p′-DDE in atleast one sample from Stations 324 and 325 exceededtoxicity thresholds for fish (0.6–0.7 μg/g; Beckvar et al.,2005) or avian wildlife (1–3 μg/g; Blus, 1996). Whole-body p,p′-DDE concentrations of 1.0 μg/g altered sexsteroid hormone (E2 and KT) concentrations in bloodplasma and gene expression involved in the endocrinepathway in adult largemouth bass (Garcia-Reyero et al.,2006). Although total DDT and p,p′-DDE concentra-tions in the GR have declined over the past decade, fishand wildlife, including migratory birds, are still at riskfrom current concentrations in fish downstream ofPhoenix (Stations 324 and 325; Table 5).

Technical DDTcontains o,p′-DDT as an impurity (upto approximately 15%), and residues of this compoundand its metabolites also remain widespread (Schmittet al., 1999). After comparing sample chromatographicpeaks with PBDE standards, we determined that severalPBDE congeners were present in the GR samples andwere interfering with o,p′-DDT and p,p′-DDD insamples from Stations 323, 324, and 325. An additionalcleanup step and GC–MS were used to accurately quan-tify o,p′- and p,p′-homologs in the GR samples. ThePBDE congeners were not quantified for this study.Little is currently known about the effects of PBDEon fish, but PBDEs may affect thyroid homeostasis(Tomy et al., 2004) and reproductive development infish (Patiño et al., 2003). Concentrations of o,p′-DDTwere greatest in fish from Stations 324 and 325, ando,p′-DDD concentrations (0.006–0.017 μg/g) wererelatively high compared to p,p′-DDD concentrations(0.003–0.037 μg/g) in samples from Stations 324 and325. Concentrations of o,p′-homologs were generallynot detected or were low in bass and carp from previousLRMN studies (Schmitt et al., 2005; Hinck et al., 2006a).The o,p′-homologs were historically considered relativelybenign, but studies have found that these compounds areestrogenic (Donohoe and Curtis, 1996; Metcalfe et al.,2000; Papoulias et al., 2003). The total risk to CRB fishand wildlife represented by concentrations of o,p′-DDTand its homologs is unknown.

Concentrations of other formerly used pesticides andtheir metabolites including total chlordanes, dieldrin,endrin, and hexachlorobenzene were greatest in samplesfrom the Lower GR but did not exceed toxicitythresholds for fish or wildlife. Many of these pesticideshave been reported historically in fish from the LowerGR (Baker et al., 1992; King et al., 1997; Schmitt et al.,1999). The greatest toxaphene concentrations weremeasured in fish from cotton-producing areas of theLower GR (Stations 324 and 325), where concentrations

in fish have been high historically (King et al., 1997).Toxaphene concentrations in fish from Stations 324 and325 exceeded concentrations (0.4 μg/g) associated withreproductive effects in fish (Table 5; Mayer et al., 1975).

Concentrations of currently used organochlorineresidues and their metabolites including pentachloro-benzene, pentachloroanisole, γ-HCH, Dacthal, endo-sulfan, and methoxychlor were also relatively high inthe GR downstream of Phoenix, although aquatictoxicity thresholds for most of these chemicals werenot available. These residues have not been reported infish from previous CRB studies but have beenassociated with reproductive and developmental effectsin fish and wildlife (Shukla and Pandey, 1986;McDonald, 1991; USEPA, 2002; Ortiz et al., 2003;Versonnen et al., 2004) and should continue to bemonitored in the CRB.

Total PCBs concentrations were generally lowthroughout the CRB except in fish from Stations 320and 324 (N0.8 μg/g), which were located downstream ofmetropolitan areas. High PCB concentrations in fishdownstream of the urban and industrial areas of Phoenixhave been reported previously (King et al., 1997). TotalPCB concentrations in fish from Stations 320 and 324exceeded a concentration (0.48 μg/g) known to causeinferior reproductive performance and offspring surviv-al in piscivorous wildlife (Table 5; Hornshaw et al.,1983), but all concentrations were less than thoseassociated with reproductive and developmental effectsin fish (5 μg/g; Monosson, 2000).

Most TCDD-EQ concentrations in CRB fish weresimilar to those reported in fish from reference sites inprevious studies (Giesy et al., 1995; van den Heuvel et al.,1995) and did not exceed toxicity thresholds for fish(30 pg/g; Walker et al., 1996;Whyte et al., 2004). TCDD-EQs concentrations N4 pg/g were measured in carp andcatfish from Station 324, which in conjunction with thegreater PCB concentrations at this station, indicated thatthe dioxin-like activity in these fish was likely due toPCBs. The TCDD-EQs in fish from Station 324 exceededtoxicity thresholds for mammalian and avian wildlife(4.4–5 pg/g; Nosek et al., 1992; Tillitt et al., 1996);therefore, piscivorous wildlife from this area may be atrisk from dioxin-like compounds (Table 5).

We considered the normal ranges of hepatic ERODactivity to be 0–16 pmol/min/mg in female bass, 0–22 pmol/min/mg in male bass, 0–4 pmol/min/mg infemale carp, and 0–6 pmol/min/mg in male carp (Whyteet al., 2000; Schmitt, 2002). Mean EROD activityexceeded these criteria in male bass from Stations 311and 312, female carp from Stations 320 and 324, andmale carp from Stations 312 and 324 and indicates

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exposure to aryl hydrocarbon receptor (AhR) agonists atthese sites (Table 5). The normal range of hepatic ERODactivity in catfish was unknown, but EROD activity inCRB catfish was similar to those reported in catfishfrom previous LRMN studies (Schmitt, 2002; Schmittet al., 2005). High EROD activities in male and femalecarp from Station 324 were correlated with the elevatedTCDD-EQ and PCB concentrations in these samples.TCDD-EQs and PCB concentrations were low in basssamples from Station 312, which indicated that bass atthis site were exposed to some other type of AhR agonist(such as polycyclic aromatic hydrocarbons (PAHs)).Large bituminous coal beds in northeastern Utah couldbe a potential PAH source in this area and may becontributing to the elevated EROD activities in fish.

4.2. Health indicators

The quantitative fish health indicators incorporatedinto this study have been widely used and discussed inthe literature but have had limited use in CRB studies.Relatively low CF values for carp from Stations 314,315, and 321 (b0.8) and bass from Stations 315 and 321(b1.0) were lower than typical values for these taxa(Table 5; Carlander, 1969, 1977; Sepúlveda et al., 2001;Schmitt, 2002; Schmitt et al., 2005; Hinck et al., 2006a).Selenium concentrations in carp and bass from Stations314, 315, and 321 were among the highest measured inour study (N2.0 μg/g) andmay be responsible for the lowCF values in fish from these sites (Hamilton, 2004;Muscatello et al., 2006). The liver normally constitutes1–2% of the body in most fish (Gingerich, 1982). MeanHSI values were b1.0% in bass from Stations 312, 321,and 322 (Table 5), although similar HSI values werereported in bass from previous LRMN studies (Schmitt,2002; Schmitt et al., 2005; Hinck et al., 2006a).Histopathological examination determined that the liversof bass from Stations 321 and 322 had high parasite loadsand fatty vacuolization. Decreased liver size has beenreported in various fish species after exposure tocontaminants including metals and bleached kraft milleffluent (McMaster et al., 1991; Adams et al., 1992).

The SSI values in carp and catfish were similar tothose measured in previous LRMN studies, but values inbass were considered low (Schmitt, 2002; Schmitt et al.,2005; Hinck et al., 2006a). Relatively low SSI valueswere calculated for bass and catfish from Station 324,but histopathological examination determined thatspleen tissue was normal in these fish. Contracted(i.e., smaller) spleens in fish have been associated withexposure to organic contaminants including PCBs,PAHs, and bleach kraft mill effluent (Payne et al.,

1978; Kiceniuk and Khan, 1987; Pulsford et al., 1995).Relatively high PCB concentrations (N1.0 μg/g) weremeasured in fish from Station 324, and wastewatertreatment plant effluent and urban runoff from thePhoenix area comprises much of the water in the GRnear Station 324 (Gebler, 1998; Anning, 2003; Arnoldet al., 2004). These factors may have affected SSI valuesin fish from Station 324 (Table 5).

The HAI in carp and bass has been reported inprevious studies (Adams et al., 1993; Coughlan et al.,1996; Schmitt, 2002; Schmitt et al., 2005; Hinck et al.,2006a) but has had limited use in catfish (BEST-LRMNprogram, unpublished data). Health assessment indexscores were elevated in carp from Stations 319, 320,321, and 322, bass from Stations 315, 321, and 322, andcatfish from Stations 315 and 317 (Table 5). The HAIscores in bass were greater than those reported in bassfrom other LRMN studies (Schmitt, 2002; Schmitt et al.,2005; Hinck et al., 2006a) and would be consideredunhealthy or contaminated by criteria from other studies(Adams et al., 1993; Coughlan et al., 1996). Therelatively high HAI scores for carp and bass in theLower CR indicate that these fish were in generallypoorer health than those from other CRB stations. Fewincidences of confirmed tumors or other grossly visibleindications of exposure to toxic chemicals were found inCRB fish. Tumors (fibroma, papilloma) were found on atotal of five CRB fish (0.01%) representing Stations313, 320, and 321 (Table 5).

Increases in MA parameters in fish from specificcontaminated sites relative to reference sites have beendocumented in both laboratory and field studies (Blazeret al., 1987; Wolke, 1992). Fournie et al. (2001)suggested a value of N40 splenicMA/mm2 as a thresholdindicative of possible effects due to hypoxia or sedimentcontamination in marine and estuarine fishes. Althoughmean MA-# values in CRB fish did not exceed 40 MA/mm2, more information is needed to interpret values infreshwater fishes. Splenic MAs were larger and morenumerous in carp than in bass or catfish. MAs wereN15,000 μm2 in multiple carp from Stations 319 and 320and were the largest measured in any LRMN study(Schmitt, 2002; Schmitt et al., 2005; Hinck et al., 2006a).Increases in MA parameters have been associated withcontaminants in laboratory and field studies (see reviewby Blazer et al., 1987; Wolke, 1992), but can vary withfish size, age, and nutritional status (Wolke et al., 1985;Blazer et al., 1987; Couillard and Hodson, 1996).Pigmented cell accumulations, which are similar toMAs, were also found in gonadal tissue of several maleand female carp from Stations 319 and 320. Highincidences of testicular MAs were correlated with lower

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GSI values in male carp from Lake Mead and may becaused by exposure to environmental contaminants(Patiño et al., 2003); however, infectious diseases canalso increase MAs in fish tissues (Wolke, 1992).Histopathological examination also revealed TFs werecommon in head and hind kidney tissues of carp, butmore information is needed to determine the function ofTFs in the head and hind kidney of carp.

4.3. Reproductive biomarkers

The reproductive biomarkers used in this study arethe key measures of reproductive function and areroutinely used to help evaluate contaminant effects orsimply assess general reproductive health in fish. Allfish samples were collected post-spawn and within an 8-week time period (late August to late October) tominimize the variation of reproductive biomarkers fromtemperature, photoperiod, and annual reproductivecycle. However, natural changes or fluctuations inreproductive biomarkers may have occurred during thiscollection period. Reproductive stage was more variablein carp from Station 324 (stages 0–4) than those fromother CRB sites (most stages 2–3). Water temperature ofthe GR downstream of Phoenix exceeds 24 °C much ofthe year (Anning, 2003) and likely affects the spawningcycle of fish in the GR.

Most GSI values corresponded with gonadal stage(i.e., GSI values increased as gonadal stage advanced),but male carp from Station 324 had abnormally low GSIvalues (b1.0%) and were at different reproductivestages than carp from other sites (Table 5). Histopath-ological analyses determined that most male carp withGSI values b1.0% also had inflammation, calcifieddeposits, pigmented cell accumulations, and edemaobserved in their testicular tissue. Some of theseobservations have been previously associated withexposure to treated municipal sewage effluent in carp(Lavado et al., 2004; Diniz et al., 2005). Patiño et al.(2003) suggested that environmental contaminants suchas PCBs, dioxins, furans, and PBDEs can affectreproductive development in male carp.

Intersex was identified in carp, bass, and catfish fromseven of 14 CRB stations sampled (Table 5). Gonadaltissue of all intersex bass and catfish was primarilytesticular tissue with an apparent invasion of mild tomoderate numbers of immature oocytes. In contrast, thegonads of the intersex carp from Station 320 containedprimarily ovarian tissue with some spermatozoa.Previous LRMN studies have observed intersex onlyin male bass (Schmitt, 2002; Schmitt et al., 2005; Hincket al., 2006a), but an intersex carp was previously

reported in a Lake Mead study (Snyder et al., 2004). Toour knowledge, the background occurrence of intersexfish has not been established for any of these species, butthe high proportion of intersex smallmouth bass fromStation 311 (70%) is cause for concern. These resultsindicate that the fish from Station 311 may have beenexposed to endocrine modulating chemicals althoughthe cause of intersex is unknown and warrants furtherinvestigation.

Mean plasma vtg concentrations were higher in fe-male smallmouth bass from Station 311 (8.41 mg/mL)compared to other stations (b1.27 mg/mL). Otherreproductive biomarkers were normal in female bassalthough the incidence of intersex in male bass from thissite was high (70%). Mean vtg concentrations were lower(0.57 mg/mL) in female carp from Station 324 comparedto other stations (N3.03 mg/mL). Other reproductivebiomarkers and organochlorine concentrations wereanomalous in fish from this station (Table 5). Vitellogeninconcentrations were N0.1 mg/mL in male carp fromStations 322 and 323 and male bass from Station 312 andmay indicate exposure to estrogen mimics (Table 5;Denslow et al., 1999). Some organochlorine pesticidesare estrogen mimics, but concentrations of the organo-chlorine pesticides measured in this study were low inmale fish from Stations 312, 322, and 323. Detectableconcentrations of vtg have been documented in male fishas a result of low assay LODs, but concentrationsN0.001 mg/mL are generally considered anomalous inmale fish. However, vtg concentrations in male CRB fishdid not fall within the range of early or mid-vitellogenicfemales (N0.8 mg/mL) considered to be high in previousstudies (Schmitt, 2002; Schmitt et al., 2005; Hinck et al.,2006a). Relatively low E2 and KT concentrations weremeasured in female carp from Station 323 and male carpfrom Station 319, but high E2 concentrations weremeasured in male bass from Station 311. Sex steroidhormones can vary due to various factors including age,season, and geographical location.

5. Conclusions

The agricultural industry in the Lower CRB is one ofthe most productive in the U.S. and relies heavily onirrigation canals and pesticide applications for high cropyields. Residential applications of pesticides in the GRhave also been high (Gellenbeck and Anning, 2002).Although concentrations were generally less than thecurrent established water-quality limits, pre-emergentpesticides such as simazine, trifluralin, and Dacthal havebeen frequently detected in waters of the GR fromDecember to April (Gellenbeck and Anning, 2002),

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which indicates that fish may be exposed to these contam-inants prior to spawning. Previous studies have reportedthat fish and wildlife may be at risk from p,p′-DDT andother pesticides in agricultural areas along the GR and inthe Lower CR (Baker et al., 1992; King et al., 1993, 1997;Schmitt et al., 1999; Gebler, 2000; García-Hernándezet al., 2001, 2006).Our findings support these conclusionsand also indicate that currently used organochlorineresidues and their metabolites including pentachloroani-sole, Dacthal, and endosulfan were elevated in fish fromthe GR and should be monitored. Although concentra-tions were generally low in CRB fish, currently usedorganochlorine residues have been associated withhistological, developmental, and reproductive effects infish and other wildlife (Shukla and Pandey, 1986;McDonald, 1991; ATSDR, 2002; USEPA, 2002; Ortizet al., 2003; Versonnen et al., 2004).

Anomalous reproductive biomarkers including re-duced gonad size, less advanced gonadal development,and histological gonad abnormalities were present in fishdownstream of Phoenix (Station 324) but not fartherdownstream in the GR (Station 325). We conclude thatmunicipal inputs including wastewater treatment planteffluent and urban runoff from the Phoenix metropolitanarea are likely affecting reproductive health in fish fromStation 324. Other studies have documented pesticide-contaminated fish and sediment and decreased taxarichness in aquatic invertebrate communities in effluent-dependent reaches downstream of Phoenix (Gebler, 1998;Gellenbeck and Anning, 2002), and fish consumptionadvisories have been issued because of DDT metabolites,toxaphene, and chlordane for the Gila, Lower Has-sayampa, Salt, and Aqua Fria Rivers in this area (USEPA,2004). Arnold et al. (2004) reported that effluent-dominated waters near Phoenix contain among thehighest concentrations of known estrogenic compoundsand have the potential to negatively affect aquatic life.Estrogenic chemicals have been associated with repro-ductive effects in fish (Donohoe and Curtis, 1996;Ungerer and Thomas, 1996; Metcalfe et al., 2000;Hassanin et al., 2002; Papoulias et al., 2003; Lavadoet al., 2004; Diniz et al., 2005), and the reproductivebiomarker responses in fish from Station 324 may berelated to those chemicals. The PBDEs present in fishfrom Stations 324 and 325 may also affect reproduc-tive biomarkers (Patiño et al., 2003). More studies, par-ticularly downstream of Las Vegas and Phoenix, arewarranted to describe the distribution and potential effectsof new and emerging contaminants such as personal careproducts and pharmaceuticals in aquatic systems.

Substantial irrigation is required for agricultural cropproduction in the Upper CRB, and water in irrigation

return flows may be highly contaminated with dissolvedSe salts leached from soils (Lemly, 1996). Previousinvestigations determined that Se concentrations wereelevated in sediment, water, and biota in the Upper CRB(Stephens et al., 1992; Engberg, 1999; Schmitt et al.,1999; Seiler et al., 1999). Higher Se concentrations infish have also been reported in the Lower CR as a resultof transport from the Upper CRB rather than from localagricultural activities (Radtke et al., 1988; Baker et al.,1992; King et al., 1993; Andrews et al., 1997). Ourfindings support previous conclusions that Se concen-trations pose a risk to fish and piscivorous wildlife in theCRB. Selenium concentrations exceeded protectivecriteria for fish, piscivorous wildlife, or both at allCRB stations, but teratogenic defects (e.g., spine, head,and mouth deformities) associated with seleniumtoxicosis in CRB fish were rare (Lemly, 1997). Thelack of teratogenic defects in CRB fish does not implythat the risk of Se is minimal. Studies have documentedreproductive effects in fish of multiple life stagesexposed to high Se concentrations (Gillespie andBaumann, 1986; Schultz and Hermanutz, 1990; Herma-nutz et al., 1992; Hamilton et al., 2005b,c). Studiesexamining the effects of elevated Se concentrations inmultiple life stages of CRB fish are limited to theendangered razorback sucker (Xyrauchen texanus;Hamilton et al., 2005a,b,c), but studies with otherspecies are warranted. The effects of Se on reproductionof the razorback sucker in the Lower CRB, where Seconcentrations were highest in our study, are unknownbut could provide relevant information on the species'limited population in the CRB. Further investigationsare also needed to determine the effect of Se onmolecular and reproductive pathways in fish andwildlife in Se-contaminated areas of the CRB.

Mercury sources in the CRB include historical oremilling and gold amalgamation, naturally mineralizedsoils, and atmospheric deposition. Large coal beds in theGreen River, Yampa River, and San Juan River Basinsalso contain Hg, which can be released during coalcleaning (crushing, sifting, froth flotation) and fromcoal-fired power plants (Tewalt et al., 2001). Fish fromthese areas in our study had among the highest Hgconcentrations and exceeded protective criteria for fishand wildlife. Acid deposition from coal-fired powerplants in the Yampa River Basin at Craig, Colorado(near Station 311) has been associated with reproductiveproblems in amphibians from nearby waters (Corn andVertucci, 1992). Abnormal reproductive biomarkerswere found in the GR. Evidence of intersex was foundin 70% of male bass from Station 311. Liney et al.(2005) concluded that intersex is age-related and occurs

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at higher incidences in adult fish exposed to estrogeniccompounds during early development. The cause ofintersex in male smallmouth bass in the Yampa River isunknown and should be investigated further.

Acknowledgements

This study was conducted jointly by the USGS, the U.S. Fish and Wildlife Service (USFWS), University ofFlorida (UF), California Department of Fish and Game,and the Colorado Division ofWildlife as part of the LargeRiver Monitoring Network of the Biomonitoring ofEnvironmental Status and Trends (BEST) Program.Many individuals representing USGS, USFWS, UF, andother organizations contributed to this study. R. Lipkinprovided themap, andM. Ellersieck provided informationfor the statistical analyses. S. Goodbred,K.Koch, C.Marr,and R. Patiño reviewed earlier versions of this paper.

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