Life-cycle studies with 2 marine species and bisphenol A: The mysid shrimp (Americamysis bahia) and sheepshead minnow (Cyprinodon variegatus).

Bisphenol A (BPA) is a high production volume compound primarily used to produce epoxy resins and polycarbonate plastic. Exposure to low concentrations of BPA occurs in freshwater and marine systems, primarily from wastewater treatment plant discharges. The dataset for chronic toxicity of BPA to freshwater organisms includes studies on fish, amphibians, invertebrates, algae, and aquatic plants. To broaden the dataset, a 1.5-generation test with sheepshead minnow (Cyprinodon variegatus) and a full life-cycle test with mysid shrimp (Americamysis bahia) were conducted. Testing focused on apical endpoints of survival, growth and development, and reproduction. The respective no-observed-effect concentration (NOEC) and lowest-observed-effect concentration (LOEC) values of 170 and 370 µg/L for mysid and 66 and 130 µg/L for sheepshead were based on reduced fecundity. The hazardous concentrations for 5% of the species (HC5) values of 18 µg/L were calculated from species sensitivity distributions (SSDs) with freshwater-only data and combined freshwater and marine data. Inclusion of marine data resulted in no apparent difference in SSD shape, R2 values for the distributions, or HC5 values. Upper-bound 95th percentile concentrations of BPA measured in marine waters of North America and Europe (0.024 and 0.15 µg/L, respectively) are below the HC5 value of 18 µg/L. These results suggest that marine and freshwater species are of generally similar sensitivity and that chronic studies using a diverse set of species can be combined to assess the aquatic toxicity of BPA. Environ Toxicol Chem 2018;37:398-410. © 2017 The Authors. Environmental Toxicology and Chemistry Published by Wiley Periodicals, Inc. on behalf of SETAC.

Characterizing the effects of bisphenol A on sediment-dwelling benthic organisms.

Bisphenol A (BPA) is a high production volume chemical intermediate used primarily in the production of polycarbonate plastics and epoxy resins. It primarily enters surface water and sediment via effluent discharges during its manufacture and use. The physical properties of BPA suggest that sediment is a potential sink and may result in exposure to benthic organisms. Currently there are no studies measuring the chronic toxicity of BPA to benthic organisms via direct sediment exposure. The present study examined the chronic toxicity of BPA to 3 commonly used test organisms that are generally representative of invertebrates occupying the base of the benthic food web and for which standardized testing protocols are available: the oligochaete Lumbriculus variegatus (mean numbers and biomass), the midge Chironomus riparius (emergence and development rate), and the estuarine amphipod Leptocheirus plumulosus (survival, growth, and reproduction). No-observed-effect concentrations (NOECs) for the 3 species ranged from 12 mg/kg to 54 mg/kg dry weight. All NOEC values were higher than all measured concentrations of BPA in freshwater and marine sediments reported in reliable, fully reported studies from North America and Europe from the 1990s to the present. For the first time, there are studies with BPA measuring the chronic toxicity to 3 taxa of sediment dwelling invertebrates, which are suitable to support region-specific risk assessments.

Relevance of drinking water as a source of human exposure to bisphenol A.

A comprehensive search of studies describing bisphenol A (BPA) concentrations in drinking water and source waters (i.e., surface water and groundwater) was conducted to evaluate the relevance of drinking water as a source of human exposure and risk. Data from 65 papers were evaluated from North America (31), Europe (17), and Asia (17). The fraction of drinking water measurements reported as less than the detection limit is high; 95%, 48%, and 41%, for North America, Europe, and Asia, respectively. The maximum quantified (in excess of the detection limit) BPA concentrations from North America, Europe, and Asia are 0.099 µg/l, 0.014 µg/l, and 0.317 µg/l. The highest quantified median and 95th percentile concentrations of BPA in Asian drinking water are 0.026 µg/l and 0.19 µg/l, while high detection limits restricted the determination of representative median and 95th percentile concentrations in North America and Europe. BPA in drinking water represents a minor component of overall human exposure, and compared with the lowest available oral toxicity benchmark of 16 µg/kg-bw/day (includes an uncertainty factor of 300) gives margins of safety >1100. Human biomonitoring data indicate that ingestion of drinking water represents <2.8% of the total intake of BPA.

Treatment of wastewaters containing bisphenol A: state of the science review.

The presence of microconstituents in effluents from municipal wastewater treatment plants (WWTPs) and their receiving waters has attracted considerable recent attention. This review summarizes the state of the science on the removal of bisphenol A (BPA) by WWTPs and presents evidence that the compound should be effectively removed in well-operated activated sludge systems. The biological treatment of BPA has been extensively studied in laboratory, pilot, and full-scale municipal WWTPs. Although removal efficiencies varied, the average of the reported removal efficiencies for BPA in full-scale facilities was 84%. Mass balance studies conducted in the laboratory with 14C-labeled BPA and studies of full-scale facilities show that biodegradation is the dominant removal process, consistent with the fact that BPA is readily degradable and able to support microbial growth. Many of the plants were able to reduce BPA concentrations in the wastewater effluent to significantly less than 1 microg/L. This review examines operating parameters important for optimizing and sustaining the performance of wastewater treatment systems including solids residence time (SRT), which proved to be the most critical. The weight of evidence suggests the optimum SRT needs to be approximately 10 days to ensure high treatment efficiencies. Other optimum operating conditions include maintaining dissolved oxygen concentrations of greater than or equal to 2.0 mg/L, elevating the SRT during periods of low temperature, and implementing step-feed control during storm-induced high flow conditions to avoid the washout of biomass.

Early life-stage and multigeneration toxicity study with bisphenol A and fathead minnows (Pimephales promelas).

Regulatory guidelines for long term testing to assess the toxicity of xenobiotic compounds such as bisphenol A (BPA) with fish have focused on survival, growth, and development in early life stages. Early life stages are critical windows of exposure, but do not address later phases in the life cycle, such as reproduction, that are equally important for the continued survival of the organisms. Residual amounts of BPA are released to surface water. BPA has, therefore, been the subject of considerable toxicity testing with fish and other aquatic organisms. A long term multigeneration test with fish has been conducted to better interpret the environmental relevance of detectable levels of BPA. Fathead minnow (Pimephales promelas) were exposed for 444 days over the course of three generations that included F0 reproducing adults, F1 eggs grown to be reproducing adults, and F2 eggs. Endpoints included survival, growth, reproduction, and vitellogenin concentrations. Concentrations tested ranged from 1 to 1,280 µg/L. No observed effect concentrations (NOEC) of 640 µg/L and higher for growth parameters show few differences between age or generation. Reproductive NOEC in F0 and F1 breeding pairs were 640 and 160 µg/L, respectively. The lowest NOEC related to survival, growth and development or reproduction was 16 µg/L for F2 hatching success. This long term study covered both early life and adult reproduction stages that allowed examination of all critical windows of exposure. Overall, NOEC ranging from 16 to 1,280 µg/L were found, which are well above median and upper 95th percentile concentrations of BPA in fresh waters in North America and Europe (0.081 and 0.47 µg/L and 0.01 and 0.035 µg/L, respectively). The likelihood is low that measured concentrations of BPA in surface water would affect fish, even if exposed over more than one generation.

Estimating potential risks to terrestrial invertebrates and plants exposed to bisphenol A in soil amended with activated sludge biosolids.

Bisphenol A (BPA) is a high production volume substance primarily used to produce polycarbonate plastic and epoxy resins. During manufacture and use, BPA may enter wastewater treatment plants. During treatment, BPA may become adsorbed to activated sludge biosolids, which may expose soil organisms to BPA if added to soil as an amendment. To evaluate potential risks to organisms that make up the base of the terrestrial food web (i.e., invertebrates and plants) in accordance with international regulatory practice, toxicity tests were conducted with potworms (Enchytraeids) and springtails (Collembolans) in artificial soil, and six plant types using natural soil. No-observed-effect concentrations (NOEC) for potworms and springtails were equal to or greater than 100 and equal to or greater than 500 mg/kg (dry wt), respectively. The lowest organic matter-normalized NOEC among all tests (dry shoot weight of tomatoes) was 37 mg/kg-dry weight. Dividing by an assessment factor of 10, a predicted-no-effect concentration in soil (PNEC(soil)) of 3.7 mg/kg-dry weight was calculated. Following international regulatory guidance, BPA concentrations in soil hypothetically amended with biosolids were calculated using published BPA concentrations in biosolids. The upper 95th percentile BPA biosolids concentration in North America is 14.2 mg/kg-dry weight, and in Europe is 95 mg/kg-dry weight. Based on recommended biosolids application rates, predicted BPA concentrations in soil (PEC(soil)) would be 0.021 mg/kg-dry weight for North America and 0.14 mg/kg-dry weight for Europe. Hazard quotients (ratio of PEC(soil) and PNEC(soil)) for BPA were all equal to or less than 0.04. This indicates that risks to representative invertebrates and plants at the base of the terrestrial food web are low if exposed to BPA in soil amended with activated sludge biosolids.

Evaluation of the amphibian metamorphosis assay: exposure to the goitrogen methimazole and the endogenous thyroid hormone L-thyroxine.

The U.S. Environmental Protection Agency (U.S. EPA) has included an amphibian metamorphosis assay (AMA) to detect thyroid active chemicals in Tier 1 testing of their endocrine screening program. To understand the variability, specificity, and reliability of the key endpoints of this assay, two exposure studies with Xenopus laevis tadpoles were conducted with two known thyroid-active compounds, namely, methimazole or L-thyroxine, for a total of 21 d. In addition, various increased-flow-rate treatments were included in the exposures to evaluate the effects of physical stress on metamorphic development. The endpoints examined in the exposures were wet weight, snout-vent length, hind-limb length, developmental stage, and thyroid and gonadal histopathology. As expected, the results indicated that both methimazole and L-thyroxine were thyroid active in the AMA, hind-limb length and thyroid histopathology being the most sensitive endpoints of thyroid activity. Tadpoles that were exposed to the various physical stressors in these experiments showed no signs of altered metamorphic development, and exposure to the thyroid-active compounds had no effect on the developing gonad of X. laevis. Taken together, these results support the use of the AMA as a Tier 1 endocrine screen for detection of potential thyroid pathway activity; however, the lack of a true negative response (no-effect) during the validation process prevents a full evaluation of this assay's specificity at this time.

Chemicals of emerging concern in the Great Lakes Basin: an analysis of environmental exposures.

This review and statistical analysis was conducted to better understand the nature and significance of environmental exposures in the Great Lakes Basin and watershed to a variety of environmental contaminants. These contaminants of interest included current-use pesticides, pharmaceuticals, organic wastewater contaminants, alkylphenol ethoxylates, perfluorinated surfactants, flame retardants, and chlorinated paraffins. The available literature was critically reviewed and used to develop a database containing 19,611 residue values for 326 substances. In many papers, sampling locations were characterized as being downstream from municipal wastewater discharges, receiving waters for industrial facilities, areas susceptible to agricultural or urban contamination, or harbors and ports. To develop an initial assessment of their potential ecological significance, the contamination levels found were compared with currently available regulatory standards, guidelines, or criteria. This review was prepared for the IJC multi-board work group, and served as background material for an expert consultation, held in March, 2009, in which the significance of the contaminants found was discussed. Moreover, the consultation attempted to identify and assess opportunities for strengthening future actions that will protect the Great Lakes. Based on the findings and conclusions of the expert consultation, it is apparent that a wide variety of chemicals of emerging concern have been detected in environmental media (air, water, sediment, biota) from the Great Lakes Basin, although many are present at only trace levels. Although the presence of these contaminants raises concerns in the public and among the scientific community, the findings must be placed in context. Significant scientific interpretation is required to understand the extent to which these chemicals may pose a threat to the ecosystem and to human health. The ability to detect chemicals in environmental media greatly surpasses our ability to understand the implications of such findings. As advances in analytical technologies occur, it is probable that substances previously found to be non-detectable will be detected. However, their presence in environmental media should not be construed to mean that they are necessarily toxic or hazardous. Current-use pesticides are tightly regulated and extensive efforts have been made to analyze for their presence in surface waters from the Great Lakes Basin. The concentrations found in surface waters for many of the pesticides are below current regulatory criteria. However, the concentrations of certain pesticides exceeded current criteria in 6-32% of the samples analyzed. Detectable concentrations of pharmaceutical compounds were present in 34% of the surface water samples. Various prescription and non-prescription drugs were detected, most frequently at locations that were proximate to the point of discharge from wastewater treatment plants or agricultural operations. At present, there are no standards, guidelines, or criteria with which to compare these contaminant concentrations. Concentrations of alkylphenol ethoxylates and their metabolites have been well studied. All surface water nonylphenol concentrations were below US ambient water quality criteria. However, the concentrations reported for some locations exceeded Canadian guidelines for water or sediment. Only limited data were available for a wide variety of organic wastewater contaminants. Measured concentrations in Great Lakes waters were generally low. Where criteria exist for comparison, the concentrations found were generally below the associated regulatory standards. However, exceedences were noted for some classes of compounds, including phthalates and polycyclic aromatic hydrocarbons. The highest environmental concentrations were reported in biota for a number of persistent, bioaccumulative, and toxic compounds (e.g., polybrominated diphenyl ethers, perfluorinated surfactants). Various stewardship as well as government risk assessment and risk management programs have been implemented over the past years for many of these compounds. Because risk management strategies for some of these contaminants have been implemented only recently, their impact on environmental concentrations, to date, remains unclear. Current evidence suggests that the concentrations of some brominated flame retardants are trending downward, while the concentrations of others appear to be increasing. Regulatory criteria are not available for many of the chemicals of emerging concern that were detected in the Great Lakes Basin. When criteria do exist, it is important to recognize that they were developed based on the best available science at the time. As the science evolves, regulatory criteria must be reassessed in light of new findings (e.g., consideration of new endpoints and mechanisms of action). Further, there are significant scientific gaps in our ability to interpret environmental monitoring data, including the need for: (a) improving the understanding of the effects of mixtures, (b) information on use of, and the commercial life cycle of chemicals and products that contain them, (c) information on source contributions and exposure pathways, and (d) the need for thoughtful additional regulatory,environmental, and health criteria. Discharges from wastewater treatment plants were identified as an important source of contaminants to surface waters in the Great Lakes Basin. Combined sewer overflows and agricultural operations were also found to be important contributors to concentrations in surface waters. Concentrations of many of the chemicals were generally the highest in the vicinity of these sources, decline with increasing distance from sources, and were generally low or non-detectable in the open waters of the Great Lakes.

Occurrence of nonylphenol ethoxylates and their metabolites in municipal wastewater treatment plants and receiving waters.

Nonylphenol ethoxylates (NPEs) are surfactants often used in applications that result in their disposal and treatment in wastewater treatment plants (WWTPs). In this study, three municipal activated sludge WWTPs, receiving primarily residential wastewater, were monitored, with their receiving waters, to determine the occurrence of NPEs and their biodegradation metabolites, including nonylphenol (NP), low-mole and higher-mole nonylphenol ethoxylates (NPE1, NPE2-8, and NPE > or =9), and nonylphenol ether carboxylates (NPECs). The facilities were moderately sized and operating normally when influent and effluent samples were taken. Treatment efficiencies, taking into account concentrations of the parent ethoxylate and metabolites, ranged from 97.2 to 99.8%. Samples of receiving stream water, sediment, and pore water were collected upstream, downstream at the end of the mixing zones, and farther downstream from the WWTP discharges, to determine the occurrence of NPE and their metabolites and to assess the potential effect on the receiving stream ecosystems. Concentrations of nonylphenol or total nonylphenol equivalents measured upstream (<0.02 to 1.29 microg/L), at the end of the mixing zone (0.2 to 3.15 microg/L), and farther downstream (<0.02 to 1.84 microg/L) were compared with the recently established national ambient water quality chronic criteria of 6.6 mcirog/L. On the basis of this analysis, the likelihood of adverse effects on aquatic species within the three receiving streams is low.

Simultaneous quantitation of testosterone, estradiol, ethinyl estradiol, and 11-ketotestosterone in fathead minnow fish plasma by liquid chromatography/positive atmospheric pressure photoionization tandem mass spectrometry.

In the present work, for the first time, a rapid and sensitive liquid chromatography/positive atmospheric pressure photoionization tandem mass spectrometry (LC/APPI-MS/MS) method has been developed and validated for the simultaneous quantitation of testosterone, estradiol, ethinyl estradiol, and 11-ketotestosterone in fathead minnow fish plasma using no more than 10 microL of plasma. Compounds present in plasma were directly derivatized with dansyl chloride and 25 microL of the derivatized mixture was injected into the LC/APPI-MS/MS system. The gradient chromatographic elution was achieved on an Agilent Zorbax SB-C18 analytical column (2.1 mm x 50 mm, 1.8 microm particle size) with mobile phases consisting of acetonitrile, water and acetic acid. The flow rate was 0.5 to 0.7 mL/min and the total run time was 11.5 min. The lower limits of quantitation for testosterone, estradiol, ethinyl estradiol, and 11-ketotestosterone and were 1, 1, 1, and 2.5 ng/mL, respectively. Intra-batch precision was less than 19.4% and inter-batch precision was less than 11.7% for all four analytes. Accuracy was within 83.5-115.4% of nominal concentrations. This method is used for quantitation of sex steroid levels in fathead minnow tested in endocrine disruptor screening experiments.

Exposure analysis of bisphenol A in surface water systems in North America and Europe.

This study was conducted to develop a statistical understanding of exposures to bisphenol A (BPA) in aquatic environments in North America and Europe. Concentrations of BPA have been reported by 89 investigations published between 1997 and 2007. On the basis of an analysis of weighted observations (n = 1068 and 848 for North America and Europe, respectively), BPA was reported at concentrations above the detection limit in 20-51% of freshwater samples. Median BPA concentrations for fresh surface waters for North America and Europe were 0.081 and 0.01 microg/L, respectively, while 95th percentiles were 0.47 and 0.35 microg/L, respectively. In contrast to fresh surface waters, only limited data are available for sediments and less for marine ecosystems. For freshwater sediments in North America (n = 71), the median and 90th percentile concentration (the 95th percentile was not calculable) were 0.6 and 3.4 ng/ g-dw, respectively, while the median and 95th percentile concentration in Europe (n = 249) were 16 and 256 ng/g-dw, respectively. To assess the potential ecological significance, we compared exposure concentrations with available regulatory criteria. The results suggest the frequency of locations in which concentrations are likely to cause adverse effects on aquatic ecosystems is low, with the exception of sediments collected from some highly urbanized and industrial locations.

Environmental persistence of organic pollutants: guidance for development and review of POP risk profiles.

Environmental persistence is an important property that can enhance the potential of a chemical substance to exert adverse effects and be transported to remote environments. The persistence of organic compounds is governed by the rates at which they are removed by biological and chemical processes, such as biodegradation, hydrolysis, atmospheric oxidation, and photolysis. The persistence workgroup in a recent Society of Environmental Toxicology and Chemistry (SETAC) Pellston workshop (Pensacola, FL, USA, January 2008) focused on evaluating persistence of organic compounds in environmental media (air, water, soil, sediment) in terms of their single-medium degradation half-lives. The primary aim was to provide guidance to authors and reviewers of chemical dossiers for persistent organic pollutants (POPs) and persistent, bioaccumulative, and toxic substances (PBTs) proposed for action. A second objective was to provide a summary of the current state of the science with respect to POP fate assessment. Assessing the persistence of chemical substances in the environment is not straightforward. A common misconception is that, like many chemical properties, environmental persistence is an inherent property of the substance and can be readily measured. In fact, rates of degradation of a substance in the environment are determined by a combination of substance-specific properties and environmental conditions. This article addresses how persistence can be evaluated based on an assortment of supporting information. Special attention is given to several critical issues, including transformation products, nonextractable residues, and treatment of uncertainty and conflicting data as part of a weight-of-evidence assessment.

Introduction to special series: science-based guidance and framework for the evaluation and identification of PBTs and POPs.

There is a growing sense of urgency among scientists and environmental policy-makers concerning the need for improving the scientific foundation supporting international regulations for identifying and evaluating persistent, bioaccumulative, and toxic (PBT) substances and persistent organic pollutants (POPs) in the environment. The current national and international regulations define PBTs and POPs in terms of fairly strict criteria that are based on the state of the science in the late 1970s and early 1980s. Since then, an evolution in the state of the science has produced new insights into PBT substances and an array of new methods to identify PBT chemicals. The development of regulatory criteria has not kept up with the rapid development in environmental chemistry and toxicology, and as a result, scientists often find themselves in the situation where guidance on PBT and POPs criteria is limited and, in some respects, out of date. With this background, a Society of Environmental Toxicology and Chemistry (SETAC) Pellston Workshop brought together experts from academia, government, and industry to reach consensus on the significance of advancements in our understanding of the behavior and potential impact of POPs and PBTs in the environment, the current understanding of the state of the science, as well as recommendations for policy-makers to improve and coordinate national and international regulations on this issue. The workshop builds on the outcome of a previous Pellston workshop, held in 1998, which focused on the evaluation of persistence and long-range transport of organic chemicals in the environment, and is linked to other recent Pellston workshops, among them the Tissue Residue Approach for Toxicity Assessment workshop held in 2007. The results of this workshop are conveyed in a series of 9 articles, published in this issue of Integrated Environmental Assessment and Management, and describe the coordination of science, regulation, and management needed to more effectively achieve a common goal of managing chemicals on our planet.

Acute and chronic toxicity testing of bisphenol A with aquatic invertebrates and plants.

Bisphenol A (BPA, 4,4'-isopropylidine diphenol) is a commercially important chemical used primarily as an intermediate in the production of polycarbonate plastic and epoxy resins. Extensive effect data are currently available, including long-term studies with BPA on fish, amphibians, crustaceans, and mollusks. The aim of this study was to perform additional tests with a number of aquatic invertebrates and an aquatic plant. These studies include acute tests with the midge (Chironomus tentans) and the snail (Marisa cornuarietis), and chronic studies with rotifers (Brachionus calyciflorus), amphipods (Hyalella azteca), and plants (Lemna gibba). The effect data on different aquatic invertebrate and plant species presented in this paper correspond well with the effect and no-effect concentrations (NOECs) available from invertebrate studies in the published literature and are within the range found for other aquatic species tested with BPA.

Plasma sampling and freezing procedures influence vitellogenin measurements by enzyme-linked immunoassay in the fathead minnow (Pimephales promelas).

The present study compared three different methods for measuring plasma vitellogenin (VTG) in fathead minnow (FHM; Pimephales promelas): A procedure using liquid chromatography with electrospray ionization combined with tandem mass spectrometry (LC/ESI-MS/MS), and two commercial enzyme-linked immunoassay (ELISA) kits using either anti-carp or anti-FHM antibodies. The influence on plasma VTG measurements of using the protease-inhibitor aprotinin during blood sampling and of submitting the plasma samples to a freeze-thaw cycle before analysis also was evaluated. The addition of aprotinin to the blood during sampling significantly reduced the plasma VTG concentrations measured by ELISA, whereas the VTG values measured after plasma samples were submitted to a freeze-thaw cycle were significantly higher than those measured before freezing. This inflating effect of freezing on VTG measurements made by ELISA could be prevented if plasma samples were frozen diluted in citrate buffer containing 16 mg/ml of polyethylene glycol (PEG). In contrast, measurements of VTG made by LC/ESI-MS/MS were unaffected by freezing and, conceptually, are independent from enzymatic degradation. Although the use of aprotinin and PEG effectively reduced the influence of enzymatic and physical degradation caused by freezing and thawing on VTG measurements made by ELISA, it did not improve agreement between the three analytical techniques evaluated. More information is needed regarding the molecular structure and the existence of possible multiple forms of VTG before this protein can be measured adequately in FHM.

Potentiation of the vitellogenic response to 17alpha-ethinylestradiol by cortisol in the fathead minnow Pimephales promelas.

The effects of elevated plasma cortisol levels on vitellogenin (VTG) induction were examined in the fathead minnow (Pimephales promelas) in an attempt to evaluate the potential influence of stress on this commonly used biomarker of estrogenicity. Two separate experiments were conducted in which fish plasma cortisol was elevated to various levels for 14 d by noninvasive additions of cortisol to the aquaria water. Fathead minnows were exposed to either cortisol alone, 17alpha-ethinylestradiol (EE2) alone, or a combination of the two hormones, and plasma levels of VTG as well as liver expression of VTG mRNA were measured. Both experiments gave similar results, with an exposure to 4 ng/L of EE2 resulting in significantly greater levels of plasma VTG in the presence of, compared to that in absence of, cortisol, whereas exposure to cortisol alone at concentrations between 144 and 800 microg/L had no effect on plasma VTG levels. This potentiation of the EE2-induced vitellogenesis by cortisol was dose-dependent, with plasma VTG reaching 125, 167, and 295% of the values obtained with EE2 alone when 144, 360, and 800 microg/L of cortisol, respectively, were added to the water. Liver mRNA results were consistent with plasma VTG, although they generally were more variable. The present study demonstrates that cortisol does not independently induce vitellogenesis but can potentiate estrogen-induced VTG synthesis in fathead minnow. The implications of these findings for the use of VTG as a biomarker of estrogenicity are discussed.

Natural attenuation of chlorinated solvents at Area 6, Dover Air Force Base: characterization of microbial community structure.

A polyphasic approach based on cultivation and direct recovery of 16S rRNA gene sequences was utilized for microbial characterization of an aquifer contaminated with chlorinated ethenes. This work was conducted in order to support the evaluation of natural attenuation of chlorinated ethenes in groundwater at Area 6 at Dover Air Force Base (Dover, DE). Results from these studies demonstrated the aquifer contained relatively low biomass (e.g. direct microscopic counts of < 10(7) bacteria/g of sediment) comprised of a physiologically diverse group of microorganisms including iron reducers, acetogens, sulfate reducers, denitrifiers, aerobic and anaerobic heterotrophs. Laboratory microcosms prepared with authentic sediment and groundwater provided direct microbiological evidence that the mineralization of vinyl chloride and cis-dichloroethene as well as each step in the complete reductive dechlorination of tetracloroethene to ethene can occur in the Area 6 aquifer. Enrichment cultures capable of the oxidative degradation of cis-1,2-dichloroethene (cis-DCE) and vinyl chloride (VC) were obtained from groundwater across the aquifer demonstrating the possible importance of direct, non-cometabolic oxidation of cis-DCE and VC in natural attenuation. Culture-independent analyses based upon recovery of 16S rRNA gene sequences revealed the presence of anaerobic organisms distributed primarily between two major bacterial divisions: the delta subdivision of the Proteobacteria and low-G + C gram positive. Recovery of sequences affiliated with phylogenetic groups containing known anaerobic-halorespiring organisms such as Desulfitobacterium, Dehalobacter, and certain groups of iron reducers provided qualitative support for a role of reductive dechlorination processes in the aquifer. This molecular data is suggestive of a functional linkage between the microbiology of the site and the apparent natural attenuation process. The presence and distribution of microorganisms were found to be consistent with a microbially driven attenuation of chlorinated ethenes within the aquifer and in accord with a conceptual model of aquifer geochemistry which suggest that both reductive and oxidative mechanisms are involved in heterogeneous, spatially distributed processes across the aquifer.

Natural attenuation of chlorinated solvents at Area 6, Dover Air Force Base: groundwater biogeochemistry.

Monitored natural attenuation (MNA) has recently emerged as a viable groundwater remediation technology in the United States. Area 6 at Dover Air Force Base (Dover, DE) was chosen as a test site to examine the potential for MNA of tetrachloroethene (PCE) and trichloroethene (TCE) in groundwater and aquifer sediments. A "lines of evidence" approach was used to document the occurrence of natural attenuation. Chlorinated hydrocarbon and biogeochemical data were used to develop a site-specific conceptual model where both anaerobic and aerobic biological processes are responsible for the destruction of PCE, TCE, and daughter metabolites. An examination of groundwater biogeochemical data showed a region of depleted dissolved oxygen with elevated dissolved methane and hydrogen concentrations. Reductive dechlorination likely dominated in the anaerobic portion of the aquifer where PCE and TCE levels were observed to decrease with a simultaneous increase in cis-1,2-dichloroethene (cis-DCE), vinyl chloride (VC), ethene, and dissolved chloride. Near the anaerobic/ aerobic interface, concentrations of cis-DCE and VC decreased to below detection limits, presumably due to aerobic biotransformation processes. Therefore, the contaminant and daughter product plumes present at the site appear to have been naturally atteuated by a combination of active anaerobic and aerobic biotransformation processes.