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Review of concepts useful for maintaining quality of male reproductive field samples for laboratory study

Investigations into cellular and molecular characteristics of male gametes obtained from fish in natural ecosystems require careful sample handling and shipping in order to minimize artifacts. Maintaining sample integrity engenders confident assessments of ecosystem health, whereby animal condition is often reflected by gamete biomarkers - indicators that respond in measurable ways to changes. A number of our investigations have addressed the hypothesis that biomarkers from fish along a pollution gradient are reflective of site location. Species biology and the selected biological endpoints direct choice of parameters such as: temperature, buffer osmolality, time in transit, fixation, cryoprotectants, protease inhibition, and antibiotic inclusion in extender. This paper will highlight case studies, and outline parameters and thoughts on approaches for use by field and laboratory researchers.

Conference Paper

Assessment of general health of fishes collected at selected sites in the Great Lakes Basin In 2012

During the past decade, there has been a substantive increase in the detection of “emerging contaminants”, defined as a new substance, chemical, or metabolite in the environment; or a legacy substance with a newly expanded distribution, altered release, or a newly recognized effect (such as endocrine disruption). Emerging contaminants include substances such as biogenic hormones (human and animal), brominated flame retardants, pharmaceuticals, personal care products, plasticizers, current use pesticides, detergents, and nanoparticles. These contaminants are frequently not regulated or inadequately regulated by state or Federal water quality programs. Information about the toxicity of these substances to fish and wildlife resources is generally limited, compared to more highly regulated contaminants, and some classes have been shown to cause affects (for example feminization of male fish, immunomodulation) that are not evaluated via traditional toxicity testing protocols. As a result, these compounds may pose a substantial, but currently poorly documented threat to aquatic ecosystems. Failure to identify and understand the impacts of these emerging contaminants on fish and wildlife resources may result in deleterious impacts to Great Lakes resources that can result in adverse ecological, economic and recreational consequences. The U. S. Fish and Wildlife Service received funding through the Great Lakes Restoration Initiative (GLRI) for an Early Warning Program to detect and identify emerging contaminants and to evaluate the effects of these contaminants on fish and wildlife. The U.S. Geological Survey (WV Cooperative Fish and Wildlife Research Unit and National Fish Health Research Laboratory, Leetown Science Center) developed and implemented a biological effects monitoring protocol to assist in this program. Fish collections and measurements of biomarkers of exposure in Fall 2010 and Spring 2011 occurred at individual sites within select Areas of Concern (AOCs). They provided an assessment of the utility of the suite of biomarkers and also identified sites for more in-depth analyses. Selected areas are characterized as areas with known emerging contaminants, sensitive or listed species, areas downstream from municipal wastewater discharges or receiving waters for industrial facilities, and/or areas susceptible to agricultural or urban contamination, or harbors or ports. The results of the 2010- 2011 studies were summarized in Blazer et al. 2014 a, b, c; Braham et al. in review and Blazer et al. in review.

Great Lakes

The ecological condition of estuaries in the Gulf of Mexico

The Gulf of Mexico is a vast natural resource encompassing the coastal areas of western Florida, Alabama, Mississippi, Louisiana, and Texas, as well as a portion of Mexico. Many estuaries flow into the Gulf of Mexico and serve as nursery grounds for fish, habitat for a wide variety of wildlife, shipping routes, and a source of recreation. Estuarine-dependent species constitute more than 95 percent of the commercial fishery harvests from the Gulf of Mexico, and many important recreational fishery species depend on estuaries during some part of their life cycle. Gulf estuaries are diverse and productive ecosystems that provide a variety of valuable resources, including fish and shellfish, recreation, transportation, and water supply. Assessing the overall condition of Gulf of Mexico estuaries required incorporating data from other federal, state, and local monitoring programs to augment the information on ecological indicators collected by the U.S. Environmental Protection Agency’s (USEPA) Environmental Monitoring and Assessment Program (EMAP). The resulting document would provide a synthesis of the available knowledge about the condition of Gulf of Mexico estuaries. This document is intended for use by scientists and other citizens concerned with the ecological condition of estuaries, as well as by managers and lawmakers interested in the sustained use of estuaries for commercial and recreational purposes. It also addresses public concerns about the aesthetic quality of coastal areas vital to tourism and recreation. By producing this report on the ecological condition of estuaries in the Gulf of Mexico, we have taken one step in assessing the health of this environmental resource. We have produced an environmental “report card” to be used as a guide in the evaluation of management decisions and research directions. This report is organized in three parts: (1) an introduction that gives background information on the Gulf of Mexico, estuarine ecology, and the factors that impact estuaries in the gulf, (2) the main section on priority ecological indicators used to measure the condition of estuaries in the gulf and (3) an ecological report card that summarizes the data on ecological indicators and provides a rating of the condition of estuaries in each gulf state and for gulf estuaries overall. Many of the ratings were based on the percent area of estuaries in each state exhibiting degraded or adverse levels of an indicator. Eutrophication, a condition of high nutrients often resulting in low oxygen levels and other adverse effects, is an important water quality concern for estuaries along the gulf coast. The National Oceanic and Atmospheric Administration (NOAA) has compared the Gulf of Mexico to other coastal regions like the middle Atlantic and has ranked the Gulf of Mexico as having the highest number of point sources of nutrients and the highest percentage of land use devoted to agriculture. We evaluated monitoring data for nitrogen, chlorophyll, and dissolved oxygen as indicators of eutrophication. Although most of the estuaries exhibited high nitrogen or chlorophyll or low dissolved oxygen concentrations at least once during a survey, many times these conditions were observed in small rivers or bayous rather than in the entire estuary. Often, the percent area affected was low. The gulf estuaries had moderate conditions overall for nutrients and dissolved oxygen. Definite nutrient problems were observed in >25% of the estuarine area in Louisiana and Texas and definite dissolved oxygen problems were observed in Alabama. Contaminants in estuarine sediments provide evidence of the accumulation of chemicals from anthropogenic sources. We compared the concentrations of sediment contaminants to established guideline values to determine the proportion of estuarine area that could have potential adverse effects on living organisms. Although detectable levels of contaminants were measured in almost every estuary in the Gulf of Mexico, <25% of the estuarine area in all states had contaminant concentrations that exceeded these guidelines. Wetlands are integral parts of estuarine systems. Declining acreage means habitat loss that may be the result of commercial and residential development, hydrologic alterations, or dredge and fill operations. The Gulf of Mexico region contains more than 50% of the coastal wetland acreage in the U.S. and yet it also has the highest rate of coastal wetland loss. Nine of the top ten estuarine drainage areas ranked by total wetland area are in the Gulf of Mexico region. The most current estimates of total wetland loss over the past 200 years range from 41% to 54% for the gulf states. Although coastal wetlands continue to be altered or destroyed, some estimates indicate that the rate of loss has slowed. All gulf states were rated as having severe problems with wetland loss. The condition of benthic (bottom-dwelling) invertebrates, fish and shellfish, birds, and threatened and endangered species was used to evaluate the health of estuarine fauna. Degraded benthic communities inhabited <25% of the estuarine area in all gulf states except for Texas. Commercial fish and shellfish landings may be used as an indicator of population stability while fish biomarkers are used to measure the health of individuals in the population. Commercial landings of the top four fisheries (shrimp, menhaden, blue crab, and oyster) are stable in the gulf states while fish biomarkers indicate fair to poor fish health in Alabama and Texas and good fish health elsewhere. Coastal and marine bird populations appear to be in good condition throughout the gulf. Four threatened or endangered species inhabit coastal areas in the Gulf of Mexico: brown pelican, Gulf sturgeon, manatee, and Kemp’s ridley sea turtle. In general, populations of these species are in good to fair condition in the gulf states. Public health indicators include shellfish bed closures and chemicals found in edible fish tissue. Harvest of shellfish (primarily oyster in the Gulf of Mexico) is restricted or prohibited when concentrations of bacteria or other pathogens reach levels that could impair human health. The gulf states contain the most acreage of shellfish-growing waters in the U.S. but also have the most acreage restricted for harvest. All gulf states except Mississippi have >25% of their shellfish-growing waters restricted for harvest, mostly due to pollution from wastewater treatment plants or other upstream sources. Advisories may be issued that limit consumption when the concentrations of chemicals in fish tissue exceed levels known to be harmful to humans. Although seafood consumption advisories have been issued in all gulf states, the percent of the fish population with high concentrations of contaminants is relatively low in the gulf overall.

Alabama, Florida, Louisiana, Mississippi, Texas

Hydroclimate and fire paleorecords across the southern Rockies and Colorado Plateau over the common era

The southwestern US has been experiencing a severe drought and increased fire activity over the past two decades, affecting people’s health, homes, and businesses. Many individual fires occurring in the Southwest are the most severe in recorded history both in terms of dollars of damages as well as in fire extent. It is essential to be able to place the present drought and fires into the context of the current changing climate as well as in the framework of climate variability and human activity over centennial to millennial timescales. Dendrochronology can determine both the seasonality of fires as well as changes in precipitation. Charcoal, pollen, and the biomarkers levoglucosan, mannosan, and galactosan can help determine fire activity and the type of burned vegetation. People have controlled and utilized fire in the southwestern US for thousands of years. While proxies cannot yet determine if people ignited a specific fire, fecal sterols can determine if people were within an individual watershed. Here, we examine the strengths and weaknesses of using high-resolution tree-ring data in conjunction with biomarkers in ice and lake cores from the southwestern US to study interactions between changes in hydroclimate, fires, and human activity.

Conference Paper

Wind River Subbasin Restoration Annual Report of USGS Activities January 2021 through December 2022

We sampled juvenile wild Steelhead Trout Oncorhynchus mykiss in headwater streams of the Wind River, WA, to characterize population attributes and investigate life-history metrics, particularly migratory patterns, and early life-stage survival. We used passive integrated transponder (PIT) tagging and a series of instream PIT-tag interrogation systems (PTISs) to track juveniles and adults. The Wind River subbasin is considered a wild Steelhead refuge by Washington Department of Fish and Wildlife (WDFW). No hatchery Steelhead Trout have been released in the Wind River subbasin since 1997, and hatchery adults are estimated at less than one percent of spawners in most years. Over twenty years of Steelhead Trout status and trend monitoring and research in the subbasin is contributing to understanding of population response to numerous restoration actions in the subbasin, including removal of Hemlock Dam from Trout Creek in 2009, which had an outdated adult ladder and contributed to increased water temperatures reducing performance of juvenile Steelhead Trout. Data from our study, and companion work by Washington Department of Fish and Wildlife, are contributing to Bonneville Power Administrations (BPA) Research, Monitoring, and Evaluation (RM&E) Program Strategy of Fish Population Status Monitoring (https://www.cbfish.org/ProgramStrategy.mvc/Index). Specifically, this work addresses the sub-strategies of 1) Assessing the Status and Trends of Diversity of Natural Origin Fish Populations and Uncertainties Research regarding differing life histories of a wild Steelhead Trout population, 2) Assessing the Status and Trend of Adult Natural Origin Fish Populations, and 3) Monitoring and Evaluating the Effectiveness of Tributary Habitat Actions Relative to Environmental, Physical, or Biological Performance Objectives. During summer and fall 2021 and 2022, we PIT-tagged 1,889 and 1,391 Steelhead parr (age-0 and age-1), respectively, in the Trout Creek and upper Wind River watersheds. Age-0 parr were at lower densities in 2022 than many years due to a poor return of adult Steelhead spawners in 2022. Steelhead Trout parr were recaptured and detected through repeat headwater sampling, smolt trap operations, and instream PTISs and Columbia River PIT-tag detection infrastructure. We maintained, and upgraded in 2022, a series of six instream PTISs to monitor movement of tagged Steelhead Trout parr, smolts, and adults, providing data to population assessments, and life-cycle research and modeling. We continue to improve our PTISs in the Wind River subbasin. The improvements in siting and addition of grid power to the upper Wind River PTIS (site code WRU, rkm 27.6) during 2016 and 2017, and the addition of the Mine Reach site (site code MIN, rkm 36.0) have much improved PIT-tagged fish monitoring in the upper Wind River watershed. The paired PTIS design in the upper Wind River watershed (sites WRU and MIN) matches that in the Trout Creek watershed (sites TRC, rkm 2.0; and TC4, rkm 11.5) and will allow comparisons of Steelhead Trout population metrics between the two watersheds as response to Hemlock Dam removal continues and future restoration efforts occur in Trout Creek. During summer 2022, we upgraded three PTISs with new transceivers and new or reconfigured antennas. We replaced the Biomark 1001 Multiplexing Transceivers with Biomark MTS IS1001 Master Controller and individual IS1001 Transceivers at WRU, TRC (Trout Creek, rkm 2.0), and TC4 (Trout Creek at 43 Road Bridge, rkm 11.5). These new transceivers and antennas will improve detection performance due to increased read range and decreased susceptibility to noise. We also installed an additional IS1001 Transceiver and 11-foot antenna at WRA in summer 2021 to increase cross-channel and water column coverage. Detection data from PIT-tagged adult Steelhead Trout at PTISs allow assessment of adult escapement to tributary watersheds within the Wind River subbasin. Adult Steelhead Trout detection efficiency estimates at our primary PTIS in Trout Creek have been greater than 99 percent during six of the past eight years and have exceeded 97% at our primary PTIS in the Wind River during seven of the past eight years. Adult escapement estimates to tributary watersheds are helping us evaluate the efficacy of the 2009 removal of Hemlock Dam from rkm 2.0 of Trout Creek. The dam had potential negative effects on Steelhead Trout populations in Trout Creek due to hydrologic impairment, increased temperatures, and adult passage issues. Hemlock Dam was laddered for adult passage, but not to modern standards, which likely resulted in avoidance by some adult Steelhead Trout. Detections at the instream PTISs have demonstrated trends of age-0 and age-1 parr emigration from natal areas during summer and fall, in addition to the expected movement of parr and smolts in spring. We have estimated that from 15 to 51% of parr tagged as age-0 fish in headwater areas make downstream migrations at age 1 for additional rearing. Downstream movement occurs primarily during spring but also in fall. We have estimated that up to 27% of Steelhead Trout parr, tagged as age-1 fish, make downstream migrations during fall. Fall migration of age-1 parr has been more common in the upper Wind River watershed than the Trout Creek watershed. These findings raise questions about where parr most successfully rear and whether migrations are density- or habitat-quality driven. Broader monitoring programs would give a more comprehensive understanding of juvenile Steelhead Trout production and rearing and contributions to adult recruitment from varied rearing strategies. Repeat sampling at consistent locations in the subbasin has enabled assessment of juvenile Steelhead Trout growth patterns. Growth rates (relative change in weight) of age-0 PIT-tagged parr during summer were similar across the subbasin, though slightly lower in the Trout Creek watershed. The greatest summer growth rate was in the mainstem of the Wind River (rkm 37 and 41). Summer growth rates were lower for age-1 parr in the Trout Creek watershed than the upper Wind River watershed. Yearly relative growth was similar across the subbasin for both age-0 and age-1 tagged parr. Lower Layout Creek had the highest yearly growth rate of parr from age-0 to age-1. Mainstem Wind River (rkm 37) had the highest yearly growth rate of parr from age-1 to age-2. Non-native Brook Trout Salvelinus fontinalis are present in the subbasin, chiefly the Trout Creek watershed, and repeat sampling provides an index of their prevalence. Mean percent-of-catch that is Brook Trout, at four sample sites in Trout Creek, has declined from the period 1998 2003 to the period 2011 2022. Percent-of-catch and number of Brook Trout at the Trout Creek sites from 2011 through 2022 has generally declined, though both metrics have been somewhat variable. Evaluation and planning of habitat restoration efforts are critical to ensure efficient use of money and resources. Assessing Steelhead Trout life history variation in the Wind River subbasin will inform research and tracking of many populations and help inform habitat restoration and water allocation planning. Movement of Steelhead Trout parr from natal areas to other rearing areas raises questions regarding juvenile abundance, origin, and habitat use within watersheds. Improved PTISs and focused PIT-tagging of age-0 and age-1 Steelhead Trout parr allow investigation of such questions. Increasingly detailed viable salmonid population information, such as that provided by PIT-tagging and instream PTIS networks like those in the Wind River, can provide data to inform fisheries policy and management and understand life-history strategies and limiting factors. Such efforts also enable assessment of long-term effects of habitat restoration actions such as the removal of Hemlock Dam on Trout Creek, and the proposed Stage-0 restoration effort for upper Trout Creek, which would be a large-scale effort to reset sections of stream within their floodplain, restoring connectivity and interaction with surrounding landscape.

Report

Biomonitoring of Environmental Status and Trends (BEST) Program: Selected methods for monitoring chemical contaminants and their effects in aquatic ecosystems

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 steroid hormones (17β-estradiol 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 endpoint(s) and methods, and discuss the indicator?s value and limitations for contaminant monitoring and assessment.

Information and Technology Report

GDGT and alkenone flux in the northern Gulf of Mexico: Implications for the TEX 86 and U K 1 37 paleothermometers

The TEX 86 and molecular biomarker proxies have been broadly applied in down-core marine sediments to reconstruct past sea surface temperature (SST). Although both TEX 86 and have been interpreted as proxies for mean annual SST throughout the global ocean, regional studies of GDGTs and alkenones in sinking particles are required to understand the influence of seasonality, depth distribution and diagenesis on downcore variability. We measure GDGT and alkenone flux, as well as the TEX 86 and indices in a 4-year sediment trap time series (2010-2014) in the northern Gulf of Mexico (nGoM), and compare these data with core-top sediments at the same location. GDGT and alkenone fluxes do not show a consistent seasonal cycle, however the largest flux peaks for both occurs in winter. co-varies with SST over the 4-year sampling interval, but the -SST relationship in this data set implies a smaller slope or non-linearity at high temperatures when compared with existing calibrations. Furthermore, the flux-weighted value from sinking particles is significantly lower than that of underlying core-top sediments, suggesting preferential diagenetic loss of the tri-unsaturated alkenone in sediments. TEX 86 does not co-vary with SST, suggesting production in the subsurface upper water column. The flux-weighted mean TEX 86 matches that of core-top sediments, confirming that TEX 86 in the nGoM reflects local planktonic production rather than allochthonous or in-situ sedimentary production. We explore potential sources of uncertainty in both proxies in the nGoM, but demonstrate that they show nearly identical trends in 20 th century SST, despite these factors.

Paleoceanography

Comparisons of stereological and other approaches for quantifying macrophage aggregates in piscine spleens

Macrophage aggregates (MA s) are focal accumulations of pigmented macrophages in the spleen and other tissues of fish. A central role of MA s is the clearance and destruction of degenerating cells and recycling of some cellular components. Macrophage aggregates also respond to chemical contaminants and infectious agents and may play a role in the adaptive immune response. Tissue damage or physiological stress can result in increased MA accumulation. As a result, MA s may be sensitive biomarkers of environmental stress in fish. Abundance of MA s in tissues has been reported in a variety of ways—most commonly as density, mean size, and relative area—but the utility of these estimates has not been compared. In this study, four different types of splenic MA abundance estimates (abundance score, density, relative area, and total volume) were compared in two fish populations (Striped Bass Morone saxatilis and White Perch M. americana ) with a wide range in ages. Stereological estimates of total volume indicated an increase in MA abundance with spleen volume, which generally corresponded to fish age, and with splenic infections (mycobacteria or trematode parasites). Abundance scores were generally limited in the ability to detect changes in MA abundance by these factors, whereas density estimates were greatly influenced by changes in spleen volume. In some instances, densities declined while the total volume of MA s and spleen volume increased. Experimentally induced acute stress resulted in a decrease in spleen volume and an increase in MA density, although the total volume of MA s remained unchanged. Relative area estimates accounted for the size and number of MA s but not for changes in organ volume. Total volume is an absolute measure of MA abundance irrespective of changes in organ volume or patterns of accumulation and may provide an improved means of quantifying MA s in the spleens of fish.

Maryland

Detect and exploit hidden structure in fatty acid signature data

Estimates of predator diet composition are essential to our understanding of their ecology. Although several methods of estimating diet are practiced, methods based on biomarkers have become increasingly common. Quantitative fatty acid signature analysis (QFASA) is a popular method that continues to be refined and extended. Quantitative fatty acid signature analysis is based on differences in the signatures of prey types, often species, which are recognized and designated by investigators. Similarly, predator signatures may be structured by known factors such as sex or age class, and the season or region of sample collection. The recognized structure in signature data inherently influences QFASA results in important and typically beneficial ways. However, predator and prey signatures may contain additional, hidden structure that investigators either choose not to incorporate into an analysis or of which they are unaware, being caused by unknown ecological mechanisms. Hidden structure also influences QFASA results, most often negatively. We developed a new method to explore signature data for hidden structure, called divisive magnetic clustering (DIMAC). Our DIMAC approach is based on the same distance measure used in diet estimation, closely linking methods of data exploration and parameter estimation, and it does not require data transformation or distributional assumptions, as do many multivariate ordination methods in common use. We investigated the potential benefits of the DIMAC method to detect and subsequently exploit hidden structure in signature data using two prey signature libraries with quite different characteristics. We found that the existence of hidden structure in prey signatures can increase the confusion between prey types and thereby reduce the accuracy and precision of QFASA diet estimates. Conversely, the detection and exploitation of hidden structure represent a potential opportunity to improve predator diet estimates and may lead to new insights into the ecology of either predator or prey. The DIMAC algorithm is implemented in the R diet estimation package qfasar.

Ecosphere

Micronuclei and other erythrocyte nuclear abnormalities in fishes from the Great Lakes Basin, USA

Biological markers (biomarkers) sensitive to genotoxic and mutagenic contamination in fishes are widely used to identify exposure effects in aquatic environments. The micronucleus assay was incorporated into a suite of indicators to assess exposure to genotoxic and mutagenic contamination at five Great Lakes Areas of Concern (AOCs), as well as one non-AOC (reference) site. The assay allowed enumeration of micronuclei as well as other nuclear abnormalities for both site and species comparisons. Erythrocyte abnormality data was also compared to skin and liver tumor prevalence and hepatic transcript abundance. Erythrocyte abnormalities were observed at all sites with variable occurrence and severity among sites and species. Benthic-oriented brown bullhead ( Ameiurus nebulosus ) and white sucker ( Catostomus commersonii ) expressed lower rates of erythrocyte abnormalities, but higher rates of skin and liver neoplasms, when compared to pelagic-oriented largemouth bass ( Micropterus salmoides ) or smallmouth bass ( Micropterus dolomieu ) at the same site. The reduced erythrocyte abnormalities, increased transcript abundance associated with Phase I and II toxicant responsive pathways, and increased neoplastic lesions among benthic-oriented taxa may indicate the development of contaminant resistance of these species to more acute effects.

Great Lakes

Pathway-based approaches for assessment of real-time exposure to an estrogenic wastewater treatment plant effluent on fathead minnow reproduction

Wastewater treatment plant (WWTP) effluents are known contributors of chemical mixtures into the environment. Of particular concern are endocrine-disrupting compounds, such as estrogens, which can affect the hypothalamic-pituitary-gonadal axis function in exposed organisms. The present study examined reproductive effects in fathead minnows exposed for 21 d to a historically estrogenic WWTP effluent. Fathead minnow breeding pairs were held in control water or 1 of 3 effluent concentrations (5%, 20%, and 100%) in a novel onsite, flow-through system providing real-time exposure. The authors examined molecular and biochemical endpoints representing key events along adverse outcome pathways linking estrogen receptor activation and other molecular initiating events to reproductive impairment. In addition, the authors used chemical analysis of the effluent to construct a chemical-gene interaction network to aid in targeted gene expression analyses and identifying potentially impacted biological pathways. Cumulative fecundity was significantly reduced in fish exposed to 100% effluent but increased in those exposed to 20% effluent, the approximate dilution factor in the receiving waters. Plasma vitellogenin concentrations in males increased in a dose-dependent manner with effluent concentration; however, male fertility was not impacted. Although in vitro analyses, analytical chemistry, and biomarker responses confirmed the effluent was estrogenic, estrogen receptor agonists were unlikely the primary driver of impaired reproduction. The results provide insights into the significance of pathway-based effects with regard to predicting adverse reproductive outcomes.

Environmental Toxicology and Chemistry

Gene expression changes in female zebrafish (Danio rerio) brain in response to acute exposure to methylmercury

Methylmercury (MeHg) is a potent neurotoxicant and endocrine disruptor that accumulates in aquatic systems. Previous studies have shown suppression of hormone levels in both male and female fish, suggesting effects on gonadotropin regulation in the brain. The gene expression profile in adult female zebrafish whole brain induced by acute (96 h) MeHg exposure was investigated. Fish were exposed by injection to 0 or 0.5 µg MeHg/g. Gene expression changes in the brain were examined using a 22,000‐feature zebrafish microarray. At a significance level of p < 0.01, 79 genes were up‐regulated and 76 genes were down‐regulated in response to MeHg exposure. Individual genes exhibiting altered expression in response to MeHg exposure implicate effects on glutathione metabolism in the mechanism of MeHg neurotoxicity. Gene ontology (GO) terms significantly enriched among altered genes included protein folding, cell redox homeostasis, and steroid biosynthetic process. The most affected biological functions were related to nervous system development and function, as well as lipid metabolism and molecular transport. These results support the involvement of oxidative stress and effects on protein structure in the mechanism of action of MeHg in the female brain. Future studies will compare the gene expression profile induced in response to MeHg with that induced by other toxicants and will investigate responsive genes as potential biomarkers of MeHg exposure.

Environmental Toxicology and Chemistry

Biomonitoring environmental contamination with pipping black-crowned night heron embryos: Induction of cytochrome P450

Cytochrome P450-associated monooxygenase activities and cytochrome P450 proteins were measured in pipping black-crowned night heron ( Nycticorax nycticorax ) embryos collected from a reference site (next to the Chincoteague National Wildlife Refuge, VA) and three polluted sites (Cat Island, Green Bay, Lake Michigan, WI; Bair Island, San Francisco Bay, CA; West Marin Island, San Francisco Bay, CA). In a laboratory study, artificially incubated night heron embryos from the reference site were treated with 3-methylcholanthrene (200 μg administered into the air cell 2 d before pipping) or phenobarbital (2 mg daily for 2 d before pipping). Compared to controls (untreated + vehicle-treated embryos), 3-methylcholanthrene induced a greater than fivefold increase in activities of several monooxygenases (arylhydrocarbon hydroxylase, AHH; benzyloxyresorufin- O -dealkylase, BROD; ethoxyresorufin- O -dealkylase, EROD; pentoxyresorufin- O -dealkylase, PROD) and a greater than 100-fold increase in the concentration of immunodetected cytochrome P450 1A (CYP1A). Phenobarbital treatment resulted in only a slight increase in BROD activity but induced proteins recognized by antibodies to cytochrome P450 2B (CYP2B) by 2,000-fold. In a field study, activities of AHH, BROD, EROD, and ethoxycoumarin- O -dealkylase (ECOD) were up to 85-fold higher in pipping black-crowned night herons collected from Cat Island compared to other sites. Hepatic CYP1A and CYP2B cross-reactive proteins were detected in significantly more individuals from Cat Island than from the reference site. Greatest burdens of total PCBs and p,p ′-DDE were detected in embryos from Cat Island. Cytochrome P450-associated monooxygenase activities and cytochrome P450 proteins (AHH, BROD, EROD, ECOD, CYP1A, CYP2B) were significantly associated with total PCB burdens ( r = 0.50-0.72). These data indicate that cytochrome P450 may be a useful biomarker of exposure to some PCB mixtures in black-crowned night heron embryos.

Environmental Toxicology and Chemistry

Relation among cytochrome P450, AH-active PCB congeners and dioxin equivalents in pipping black-crowned night-heron embryos

Pipping black-crowned night-heron (Nycticorax nycticorax) embryos were collected from a relatively uncontaminated site (next to Chincoteague National Wildlife Refuge, VA) and three polluted sites (Cat Island, Green Bay, Lake Michigan, WI; Bair Island, San Francisco Bay, CA; West Marin Island, San Francisco Bay, CA). Hepatic cytochrome P-450-associated monooxygenases and cytochrome P-450 proteins, induced up to 85-fold relative to the reference site, were associated with concentrations of total polychlorinated biphenyls (PCBs) and 11 PCB congeners that are presumed to express toxicity through the arylhydrocarbon (Ah) receptor. Multiple regression revealed that up to 86% of the variation of cytochrome P450 measurements was accounted for by variation in the concentration of these PCB congeners. Toxic equivalents (TEQs) of sample extracts, predicted mathematically (summed product of PCB congener concentrations and toxic equivalency factors), and dioxin equivalents (TCDD-EQs), derived by bioassay (ethoxyresorufin-O-dealkylase activity of treated H4IIE rat hepatoma cells), were greatest in Cat Island samples. Cytochrome P450-associated monooxygenases and cytochrome P450 proteins were related to TEQs and TCDD-EQs; adjusted r-2 often exceeded 0.5 for the relation among mathematically predicted TEQs and cytochrome P450 measurements. These data extend previous observations in heron embryos of an association between P450 and total PCB burdens to include Ah-active PCB congeners, and presumably other compounds, which interact similarly with the Ah receptor. Benzyloxyresorufin O-dealkylase, ethoxyresorufin O-dealkylase, and cytochrome P450 1A appear to be the most reliable measures of exposure to Ah-active PCB congeners in black-crowned night-heron embryos. These findings provide further evidence that cytochrome P450-associated parameters have considerable value as a biomarker for assessing environmental contamination of wetlands.

Environmental Toxicology and Chemistry

Cytochrome P450 and contaminant concentrations in nestling black-crowned night-herons and their interrelation with sibling embryos

Hepatic cytochrome P450-associated monooxygenase activities were measured in 11-d-old nestling black-crowned night-herons ( Nycticorax nycticorax ) collected from a reference site (next to Chincoteague National Wildlife Refuge, VA, USA) and three contaminated sites (Cat Island, Green Bay, WI, USA; Bair Island, San Francisco Bay, CA, USA; and West Marin Island, San Francisco Bay, CA, USA). Arylhydrocarbon hydroxylase and benzyloxyresorufin- O -dealkylase activities of nestlings from contaminated sites were only slightly elevated (less than threefold) compared with the reference site. Organochlorine pesticide and total polychlorinated biphenyl (PCB) concentrations in nestlings were greatest at contaminated sites, although much lower than found in concurrently collected eggs and pipping embryos. Pollutant concentrations of nestlings were rarely associated with monooxygenase activity. In contrast, concurrently collected pipping heron embryos (often siblings of the nestlings) exhibited pronounced monooxygenase induction (means at contaminated sites were elevated up to sevenfold and values of some embryos exceeded 25-fold induction). Furthermore, monooxygenase activity of pipping embryos was significantly correlated with total PCBs, arylhydrocarbon receptor-active PCB congeners, and toxic equivalents. The modest monooxygenase responses of heron nestlings suggest that this biomarker may have only limited value during this rapid-growth life stage.

California, Virginia, Wisconsin

Cytochrome P450 and organochlorine contaminants in black-crowned night-herons from the Chesapeake Bay region, USA

Black-crowned night-heron ( Nycticorax nycticorax ) offspring were collected from a relatively uncontaminated coastal reference site (next to Chincoteague National Wildlife Refuge, VA, USA) and two sites in the Chesapeake Bay watershed (Baltimore Harbor, MD and Rock Creek Park, Washington, DC, USA). Hepatic microsomal activities of benzyloxyresorufin- O -dealkylase and ethoxyresorufin- O -dealkylase were significantly elevated (up to sixfold and ninefold induction, respectively) in pipping embryos from the Baltimore Harbor colony compared to the reference site, whereas values in embryos from the Rock Creek Park colony were intermediate. Concentrations of organochlorine pesticides and metabolites in pipping embryos from both sites in the Chesapeake watershed were greater than at the reference site but below the known threshold for reproductive impairment. However, concentrations of 10 arylhydrocarbon receptor-active polychlorinated biphenyl (PCB) congeners and estimated toxic equivalents were up to 37-fold greater in embryos collected from these two sites in the Chesapeake Bay region, with values for toxic congeners 77 and 126 exceeding those observed in pipping heron embryos from the Great Lakes. Monooxygenase activity of pipping embryos was associated with concentrations of several organochlorine pesticides, total PCBs, arylhydrocarbon receptor-active PCB congeners, and toxic equivalents ( r = 0.30–0.59), providing further evidence of the value of cytochrome P450 as a biomarker of organic contaminant exposure. Organochlorine contaminant levels were greater in 10-d-old nestlings from Baltimore Harbor than the reference site but had no apparent effect on monooxygenase activity or growth. These findings demonstrate induction of cytochrome P450 in pipping black-crowned night-heron embryos in the Chesapeake Bay region, probably by exposure to PCB congeners of local origin, and the accumulation of organochlorine pesticides and metabolites in nestling herons from Baltimore Harbor. Bio-monitoring with additional waterbird species (e.g., bald eagle, common tern, great blue heron) that appear to be more sensitive to PCBs than black-crowned night-herons is recommended to document health of waterbirds and remediation of the Chesapeake Bay.

Chesapeake Bay region

Induction of cytochrome P450-associated monooxygenases in northern leopard frogs, Rana pipiens, by 3,3′,4,4′,5-Pentachlorobiphenyl

Northern leopard frogs ( Rana pipiens ) were injected intraperitoneally either with a solution of polychlorinated biphenyl (PCB) 126 in corn oil at a concentration of 0.2, 0.7, 2.3, or 7.8 mg/kg body weight or with corn oil alone. Appropriate assay conditions with hepatic microsomes were determined for four cytochrome P450-associated monooxygenases: ethoxyresorufin- O -dealkylase (EROD), methoxy-ROD (MROD), benzyloxy-ROD (BROD), and pentoxy-ROD (PROD). One week after PCB administration, the specific activities of EROD, MROD, BROD, and PROD were not elevated at doses ≤0.7 mg/kg ( p > 0.05) but were significantly increased at doses ≥2.3 mg/kg compared to the control groups ( p < 0.05). The increased activities of these four enzymes were 3 to 6.4 times those in the control groups. The increased activities were maintained for at least 4 weeks. Because of a lack of induction at low doses of PCB 126, which were still relatively high compared to currently known environmental concentrations, we suspect that EROD, MROD, BROD, and PROD activities are not sensitive biomarkers for coplanar PCB exposure in leopard frogs.

Environmental Toxicology and Chemistry

Blood changes in mallards exposed to white phosphorus

White phosphorus (P 4 ) has been extensively used by the military for various purposes, including marking artillery impacts and as an obscurant. Target practice in an Alaskan tidal marsh during the last 4 decades has deposited large amounts of P 4 particles in sediments and water, which have resulted in die-offs of several waterfowl species. Because the toxicity of P 4 in birds has not been well documented and because it is quickly excreted or metabolized in living animals, we sought to determine the effects of experimental dosing on blood characteristics in game farm mallards ( Anas platyrhynchos ). In two experiments, one employing single doses of 5.4 mg P 4 per kilogram body weight in corn oil and the other using daily repeated doses of pelletized P 4 at either 0.5 or 1.0 mg/kg, there were significant changes in aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase (LDH), inorganic P, hematocrit, and hemoglobin. Other indications of exposure included changes in uric acid, creatinine, and total protein, which were consistent with reported liver and kidney damage due to this contaminant. Changes in white blood cells included a greater frequency of thrombocytes and fewer lymphocytes in dosed birds compared to controls. A biomarker of exposure based on LDH activity and hemoglobin is proposed.

Environmental Toxicology and Chemistry