This document describes the suite of biological methods of the U.S. Geological Survey Biomonitoring of Environmental Status and Trends program for monitoring chemical contaminants and their effects on fish. The methods, which were selected by panels of experts, are being field-tested in rivers of the Mississippi River, Columbia River, and Rio Grande basins. General health biomarkers include a health assessment index based on gross observation; histopathological examination of selected organs and tissues; condition factor; and the heptosomatic and splenosomatic indices. Immune system indicators are plasma lysozyme activity and measures of splenic macrophage aggregates. Reproductive biomarkers include plasma concentrations of sex steroids hormones (17beta-estradiaol and 11-ketotestosterone) and vitellogenin, gonadal histopathology (including reproductive stage and, in females, gonadal atresia), and the gonadosomatic index. Indicators of exposure to polycyclic aromatic and polyhalogenated hydrocarbons are the H4IIE rat hepatoma cell bioassay (performed on solvent extracts of composite fish samples) and hepatic ethoxyresorufin-O-deethylase activity. Stable nitrogen isotope ratios are used to assess the trophic position of the fish and their exposure to sewage and other animal wastes. For each indicator we describe endpoints(s) and methods, and discuss the indicator's value and limitations for contaminant monitoring and assessment.

We collected examined and analyzed 1378 fish of 99 species from 47 sites in the Mississippi River basin (MRB) during 1995 and from a reference site in 1996. The sampling sites in the MRB represented National Contaminant Biomonitoring Program (NCBP) stations situated at key points on major rivers and National Water Quality Assessment Program (NAWQA) stations located on lower-order rivers and streams in the Eastern Iowa Basins (FIB) and Mississippi Embayment (MSE) Study Units. The reference site was the water supply system of the USGS-Leetown Science Center in rural Jefferson County WV Common carp (Cyprinus carpio; carp) and black basses (Micropterus spp., bass) the targeted species together represented 82% of the fish collected. Each fish was examined in the field for externally and internally visible gross lesions selected organs were weighed to compute various ponderal and organo-somatic indices and selected tissues and fluids were obtained and preserved for analysis of biomarkers. Fish health indicators included splenic macrophage aggregates lysozyme activity and hispathological anlysis of liver kidney and of liver tissues. Reproductive biomarkers included analysis of plasma concentrations of vitellogenin (vtg) and the sex steroid hormones 17beta-estradiol (E2) and 11-ketotestosterone (11- kt), and the histological determination of percent oocyte atresia (in female fish) and gonadal stage. Hepatic ethoxyresorufin O-deethylase (EROD) activity was also measured. Composite samples of whole fish from each station were grouped by species and gender and analyzed for persistent organochlorine and elemental contaminants and for dioxin-like activity (TCDD-EQ) using the H411E rat hepatoma cell bioassay.

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