Pathway-Based Approaches for Assessing Biological Hazards of Complex Mixtures of Contaminants: A Case Study in the Maumee River.

Assessment of ecological risks of chemicals in the field usually involves complex mixtures of known and unknown compounds. We describe the use of pathway-based chemical and biological approaches to assess the risk of chemical mixtures in the Maumee River (OH, USA), which receives a variety of agricultural and urban inputs. Fathead minnows (Pimephales promelas) were deployed in cages for 4 d at a gradient of sites along the river and adjoining tributaries in 2012 and during 2 periods (April and June) in 2016, in conjunction with an automated system to collect composite water samples. More than 100 industrial chemicals, pharmaceuticals, and pesticides were detected in water at some of the study sites, with the greatest number typically found near domestic wastewater treatment plants. In 2016, there was an increase in concentrations of several herbicides from April to June at upstream agricultural sites. A comparison of chemical concentrations in site water with single chemical data from vitro high-throughput screening (HTS) assays suggested the potential for perturbation of multiple biological pathways, including several associated with induction or inhibition of different cytochrome P450 (CYP) isozymes. This was consistent with direct effects of water extracts in an HTS assay and induction of hepatic CYPs in caged fish. Targeted in vitro assays and measurements in the caged fish suggested minimal effects on endocrine function (e.g., estrogenicity). A nontargeted mass spectroscopy-based analysis suggested that hepatic endogenous metabolite profiles in caged fish covaried strongly with the occurrence of pesticides and pesticide degradates. These studies demonstrate the application of an integrated suite of measurements to help understand the effects of complex chemical mixtures in the field. Environ Toxicol Chem 2021;40:1098-1122. © 2020 SETAC. This article has been contributed to by US Government employees and their work is in the public domain in the USA.

Metabolomics for informing adverse outcome pathways: Androgen receptor activation and the pharmaceutical spironolactone.

One objective in developing adverse outcome pathways (AOPs) is to connect biological changes that are relevant to risk assessors (i.e., fecundity) to molecular and cellular-level alterations that might be detectable at earlier stages of a chemical exposure. Here, we examined biochemical responses of fathead minnows (Pimephales promelas) to inform an AOP relevant to spironolactone's activation of the androgen receptor, as well as explore other biological impacts possibly unrelated to this receptor. Liquid chromatography with high resolution mass spectrometry (LC-MS) was used to measure changes in endogenous polar metabolites in livers of male and female fish that were exposed to five water concentrations of spironolactone (0, 0.05, 0.5, 5, or 50µgL-1) for 21days. Metabolite profiles were affected at the two highest concentrations (5 and 50µgL-1), but not in the lower-level exposures, which agreed with earlier reported results of reduced female fecundity and plasma vitellogenin (VTG) levels. We then applied partial least squares regression to assess whether metabolite alterations covaried with changes in fecundity, VTG gene expression and protein concentrations, and plasma 17ß-estradiol and testosterone concentrations. Metabolite profiles significantly covaried with all measured endpoints in females, but only with plasma testosterone in males. Fecundity reductions occurred in parallel with changes in metabolites important in osmoregulation (e.g., betaine), membrane transport (e.g., l-carnitine), and biosynthesis of carnitine (e.g., methionine) and VTG (e.g., glutamate). Based on a network analysis program (i.e., mummichog), spironolactone also affected amino acid, tryptophan, and fatty acid metabolism. Thus, by identifying possible key events related to changes in biochemical pathways, this approach built upon an established AOP describing spironolactone's androgenic properties and highlighted broader implications potentially unrelated to androgen receptor activation, which could form a basis for the development of an AOP network.

Evaluation of whole-mount in situ hybridization as a tool for pathway-based toxicological research with early-life stage fathead minnows.

Early-life stage fish can be more sensitive to toxicants than adults, so delineating mechanisms of perturbation of biological pathways by chemicals during this life stage is crucial. Whole-mount in situ hybridization (WISH) paired with quantitative real-time polymerase chain reaction (QPCR) assays can enhance pathway-based analyses through determination of specific tissues where changes in gene expression are occurring. While WISH has frequently been used in zebrafish (Danio rerio), this technology has not previously been applied to fathead minnows (Pimephales promelas), another well-established small fish model species. The objective of the present study was to adapt WISH to fathead minnow embryos and larvae, and use the approach to evaluate the effects of estrone, an environmentally-relevant estrogen receptor (ER) agonist. Embryos were exposed via the water to 0, 18 or 1800 ng estrone/L (0, 0.067 and 6.7nM) for 3 or 6 days in a solvent-free, flow-through test system. Relative transcript abundance of three estrogen-responsive genes, estrogen receptor-α (esr1), cytochrome P450-aromatase B (cyp19b), and vitellogenin (vtg) was examined in pooled whole embryos using QPCR, and the spatial distribution of up-regulated gene transcripts was examined in individual fish using WISH. After 3 days of exposure to 1800 ng estrone/L, esr1 and cyp19b were significantly up-regulated, while vtg mRNA expression was not affected. After 6 days of exposure to 1800 ng estrone/L, transcripts for all three genes were significantly up-regulated. Corresponding WISH assays revealed spatial distribution of esr1 and vtg in the liver region, an observation consistent with activation of the hepatic ER. This study clearly demonstrates the potential utility of WISH, in conjunction with QPCR, to examine the mechanistic basis of the effects of toxicants on early-life stage fathead minnows.

Metabolite profiling of fish skin mucus: a novel approach for minimally-invasive environmental exposure monitoring and surveillance.

The application of 'omics tools to biologically based monitoring and surveillance of aquatic environments shows considerable promise for complementing chemical monitoring in ecological risk assessments. However, few of the current approaches offer the ability to sample ecologically relevant species (e.g., fish) in a way that produces minimal impact on the health of the organism(s) under study. In the current study we employ liquid chromatography tandem mass spectrometry (LC-MS/MS) to assess the potential for skin mucus-based metabolomics for minimally invasive sampling of the fathead minnow (FHM; Pimephales promelas). Using this approach we were able to detect 204 distinct metabolites in the FHM skin mucus metabolome representing a large number of metabolite classes. An analysis of the sex specificity of the skin mucus metabolome showed it to be highly sexually dimorphic with 72 of the detected metabolites showing a statistically significant bias with regard to sex. Finally, in a proof-of-concept fashion we report on the use of skin mucus-based metabolomics to assess exposures in male and female fathead minnows to an environmentally relevant concentration of bisphenol A, a nearly ubiquitous environmental contaminant and an established endocrine active chemical.

Metabolomics for in situ environmental monitoring of surface waters impacted by contaminants from both point and nonpoint sources.

We investigated the efficacy of metabolomics for field-monitoring of fish exposed to wastewater treatment plant (WWTP) effluents and nonpoint sources of chemical contamination. Lab-reared male fathead minnows (Pimephales promelas, FHM) were held in mobile monitoring units and exposed on-location to surface waters upstream and downstream of the effluent point source, as well as to the actual effluent at three different WWTP sites in Minnesota. After four days of exposure, livers were collected, extracted, and analyzed by (1)H NMR spectroscopy and GC-MS to characterize responses of the hepatic metabolome. Multivariate statistical analysis revealed distinct metabolite profile changes in response to effluent exposure from each of the three WWTPs. Differences among locations (i.e., upstream, downstream, and effluent) within each of the three sites were also identified. These observed differences comport with land-use and WWTP characteristics at the study sites. For example, at one of the sites, the metabolomic analyses suggested a positive interactive response from exposure to WWTP effluent and nearby nonpoint (likely agricultural related) contamination. These findings demonstrate the utility of metabolomics as a field-based technique for monitoring the exposure of fish to impacted surface waters.

Field-based approach for assessing the impact of treated pulp and paper mill effluent on endogenous metabolites of fathead minnows (Pimephales promelas).

A field-based metabolomic study was conducted during a shutdown of a pulp and paper mill (PPM) to assess the impacts of treated PPM effluent on endogenous polar metabolites in fathead minnow (FHM; Pimephales promelas) livers. Caged male and female FHMs were deployed at a Great Lakes area of concern during multiple periods (pre-, during, and post-shutdown) near the outflow for a wastewater treatment plant. Influent to this plant is typically 40% PPM effluent by volume. Additional FHMs were exposed to reference lake water under laboratory conditions. A bioassay using T47D-KBluc cells showed that estrogenic activity of receiving water near the outflow declined by 46% during the shutdown. We then used (1)H NMR spectroscopy and principal component analysis to profile abundances of hepatic endogenous metabolites for FHMs. Profiles for males deployed pre-shutdown in receiving water were significantly different from those for laboratory-control males. Profiles were not significantly different for males deployed during the shutdown, but they were significant again for those deployed post-shutdown. Impacts of treated effluent from this PPM were sex-specific, as differences among profiles of females were largely nonsignificant. Thus, we demonstrate the potential utility of field-based metabolomics for performing biologically based exposure monitoring and evaluating remediation efforts occurring throughout the Great Lakes and other ecosystems.

Alternatives to in vivo tests to detect endocrine disrupting chemicals (EDCs) in fish and amphibians--screening for estrogen, androgen and thyroid hormone disruption.

Endocrine disruption is considered a highly relevant hazard for environmental risk assessment of chemicals, plant protection products, biocides and pharmaceuticals. Therefore, screening tests with a focus on interference with estrogen, androgen, and thyroid hormone pathways in fish and amphibians have been developed. However, they use a large number of animals and short-term alternatives to animal tests would be advantageous. Therefore, the status of alternative assays for endocrine disruption in fish and frogs was assessed by a detailed literature analysis. The aim was to (i) determine the strengths and limitations of alternative assays and (ii) present conclusions regarding chemical specificity, sensitivity, and correlation with in vivo data. Data from 1995 to present were collected related to the detection/testing of estrogen-, androgen-, and thyroid-active chemicals in the following test systems: cell lines, primary cells, fish/frog embryos, yeast and cell-free systems. The review shows that the majority of alternative assays measure effects directly mediated by receptor binding or resulting from interference with hormone synthesis. Other mechanisms were rarely analysed. A database was established and used for a quantitative and comparative analysis. For example, a high correlation was observed between cell-free ligand binding and cell-based reporter cell assays, between fish and frog estrogenic data and between fish embryo tests and in vivo reproductive effects. It was concluded that there is a need for a more systematic study of the predictive capacity of alternative tests and ways to reduce inter- and intra-assay variability.

Metabolite profiling and a transcriptional activation assay provide direct evidence of androgen receptor antagonism by bisphenol A in fish.

Widespread environmental contamination by bisphenol A (BPA) has created the need to fully define its potential toxic mechanisms of action (MOA) to properly assess human health and ecological risks from exposure. Although long recognized as an estrogen receptor (ER) agonist, some data suggest that BPA may also behave as an androgen receptor (AR) antagonist. However, direct evidence of this activity is deficient. To address this knowledge gap, we employed a metabolomic approach using in vivo exposures of fathead minnows (FHM; Pimephales promelas ) to BPA either alone or in a binary mixture with 17ß-trenbolone (TB), a strong AR agonist. Changes in liver metabolite profiles in female FHM in response to these exposures were determined using high resolution (1)H NMR spectroscopy and multivariate and univariate statistics. Using this approach, we observed clear evidence of the ability of BPA to mitigate the impact of TB, consistent with an antiandrogenic MOA. In addition, a transcriptional activation assay with the FHM AR was used to confirm the AR antagonistic activity of BPA in vitro. The results of these in vivo and in vitro analyses provide strong and direct evidence for ascribing an antiandrogenic MOA to BPA in vertebrates.

Mechanism-based categorization of aromatase inhibitors: a potential discovery and screening tool.

Cytochrome P450 aromatase is a key steroidogenic enzyme that converts androgens to estrogens in vertebrates. There is much interest in aromatase inhibitors (AIs) both because of their use as pharmaceuticals in the treatment of estrogen-sensitive breast cancers, and because a number of environmental contaminants can act as AIs, thereby disrupting endocrine function in humans and wildlife through suppression of circulating estrogen levels. The goal of the current work was to develop a mechanism-based structure-activity relationship (SAR) categorization framework highlighting the most important chemical structural features responsible for inhibition of aromatase activity. Two main interaction mechanisms were discerned: steroidal and non-steroidal. The steroid scaffold is most prominent when the structure of the target chemical is similar to the natural substrates of aromatase - androstenedione and testosterone. Chemicals acting by non-steroidal mechanism(s) possess a heteroatom (N, O, S) able to coordinate the heme iron of the cytochrome P450, and thus interfere with steroid hydroxylation. The specific structural boundaries controlling AI for both analyzed mechanisms were defined, and a software tool was developed that allowed a decision tree (profile) to be built discriminating AIs by mechanism and potency. An input chemical follows a profiling path and the structure is examined at each step to decide whether it conforms with the structural boundaries implemented in the decision tree node. Such a system would aid drug discovery efforts, as well as provide a screening tool to detect environmental contaminants that could act as AIs.

Investigating compensation and recovery of fathead minnow (Pimephales promelas) exposed to 17alpha-ethynylestradiol with metabolite profiling.

1H NMR spectroscopy was used to profile metabolite changes in the livers of fathead minnows (Pimephales promelas) exposed to the synthetic estrogen 17alpha-ethynylestradiol (EE2) via a continuous flow water exposure. Fish were exposed to either 10 or 100 ng EE2/L for 8 days, followed by an 8 day depuration phase. Livers were collected after days 1, 4, and 8 of the exposure, and at the end of the depuration phase. Analysis of polar extracts of the liver revealed a greater impact of EE2 on males than females, with metabolite profiles of the former assuming similarities with those of the females (i.e., feminization) early in the exposure. Biochemical effects observed in the males included changes in metabolites relating to energetics (e.g., glycogen, glucose, and lactate) and liver toxicity (creatine and bile acids). In addition, amino acids associated with vitellogenin (VTG) synthesis increased in livers of EE2-exposed males, a finding consistent with increased plasma concentrations of the lipoprotein in the fish. Using partial least-squares discriminant analysis (PLS-DA), the response trajectories of the males at both exposure concentrations were compared. This revealed an apparent ability of the fish to compensate for the presence of EE2 later in the exposure, and to partially recover from its effects after the chemical was removed.

NMR analysis of male fathead minnow urinary metabolites: a potential approach for studying impacts of chemical exposures.

The potential for profiling metabolites in urine from male fathead minnows (Pimephales promelas) to assess chemical exposures was explored using nuclear magnetic resonance (NMR) spectroscopy. Both one-dimensional (1D) and two-dimensional (2D) NMR spectroscopy was used for the assignment of metabolites in urine from unexposed fish. Because fathead minnow urine is dilute, we lyophilized these samples prior to analysis. Furthermore, 1D 1H NMR spectra of unlyophilized urine from unexposed male fathead minnow and Sprague-Dawley rat were acquired to qualitatively compare rat and fish metabolite profiles and to provide an estimate of the total urinary metabolite pool concentration difference. As a small proof-of-concept study, lyophilized urine samples from male fathead minnows exposed to three different concentrations of the antiandrogen vinclozolin were analyzed by 1D 1H NMR to assess exposure-induced changes. Through a combination of principal components analysis (PCA) and measurements of 1H NMR peak intensities, several metabolites were identified as changing with statistical significance in response to exposure. Among those changes occurring in response to exposure to the highest concentration (450 microg/L) of vinclozolin were large increases in taurine, lactate, acetate, and formate. These increases coincided with a marked decrease in hippurate, a combination potentially indicative of hepatotoxicity. The results of these investigations clearly demonstrate the potential utility of an NMR-based approach for assessing chemical exposures in male fathead minnow, using urine collected from individual fish.

Polychlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs), biphenyls (PCBs), and polycyclic aromatic hydrocarbons (PAHs) and 2,3,7,8-TCDD equivalents (TEQs) in sediment from the Hyeongsan River, Korea.

Sediment, pore water and water samples from the Hyeongsan River, Korea were analyzed for several classes of halogenated aromatic hydrocarbons (HAHs) and their dioxin-like activities were evaluated using the in vitro H4IIE-luc bioassay. Polychlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs), and biphenyls (PCBs) were detected in sediments from all six sampling locations with mean concentrations of 2.8 x 10(2) pg/g, 190 pg/g, and 61.4 ng/g, dw, respectively. Polycyclic aromatic hydrocarbons (PAHs) were predominated by 4-6 ring compounds with concentrations in the range of 5.30-7680 ng/g, dw. Chemical profiles of target analytes in sediment and water samples revealed that there was a gradient of concentrations along the river from upstream to downstream, which suggested that the primary source was a wastewater reservoir adjacent to a sewage treatment plant (STP). TEQs derived by summing the product of concentrations of individual congeners by their respective relative potencies (REPs or TEFs) ranged from 4.3 x 10(-1) to 1.1 x 10(3) pg/g, dw. Raw Soxhlet extracts from all six sampling locations induced significant dioxin-like responses in the H4IIE-luc bioassay. TCDD-EQs derived from H4IIE bioassay ranged from 7 x 10(-3) to 1.5 x 10(3) pg/g, dw, which were significantly correlated with TEQs (r2 = 0.994, p < 0.05). Among the three Florisil fractions tested, PCDD/Fs in fraction (F2) induced the greatest magnitude of response (range: 24-83%-TCDD-max.) in the H4IIE-luc assay. Comparison of the TEQ and TCDD-EQ suggested little non-additive interaction between fractions and AhR-active and inactive compounds. Concentrations of individual congeners as well as TEQs and TCDD-EQs suggest inputs from the industrial center waste stream in the Hyeongsan River.

Trace organic contaminants, including toxaphene and trifluralin, in cotton field soils from Georgia and South Carolina, USA.

Residues of organic contaminants--including toxaphene, DDT, trifluralin, hexachlorocyclohexanes, polychlorinated biphenyls, polycyclic aromatic hydrocarbons (PAHs) and nonylphenol--were measured in 32 cotton field soils collected from South Carolina and Georgia in 1999. Toxaphene, trifluralin, DDT and PAHs were the major contaminants found in these soils. The maximum concentration of toxaphene measured was 2,500 ng/g dry weight. Trifluralin was detected in all the soils at concentrations ranging from 1 to 548 ng/g dry weight. Pesticide residues were not proportional to soil organic carbon content, indicating that their concentrations were a reflection of application history and dissipation rates rather than air-soil equilibrium. Soil extracts were also subjected to in vitro bioassays to assess dioxinlike, estrogenic, and androgenic/glucocorticoid potencies. Relatively more polar fractions of the soils elicited estrogenic and androgenic/glucocorticoid activities, but the magnitude of response was much less than those found in coastal marine sediments from industrialized locations.

Effects of waterborne exposure to 4-nonylphenol on plasma sex steroid and vitellogenin concentrations in sexually mature male carp (Cyprinus carpio).

4-Nonylphenol (NP) has been shown to elicit estrogenic responses both in vivo and in vitro. The mechanism by which NP exerts estrogenic and other endocrine-modulating effects in vivo remains unclear, however. The goal of this study was to evaluate the ability of NP to elicit estrogenic responses through indirect mechanisms of action involving the modulation of endogenous steroid hormone concentrations. Sexually mature male common carp (Cyprinus carpio) were exposed to aqueous NP concentrations ranging from <0.05 to 5.4 microg NP/l for 28-31 d. Approximately 0.5-3.5 ppm of NP was detected in pooled plasma samples or tissue samples from the carp studied. NP exposure did not significantly increase plasma concentrations of 17beta-estradiol (E2), testosterone (T) or vitellogenin (VTG). Excluding outliers, plasma E2 concentrations ranged from <175 to 700 pg E2/ml. T concentrations ranged from 940 to 24,700 pg T/ml plasma. The greatest VTG concentration detected was 52 microg/ml. One-third of the plasma samples tested contained <1 microg VTG/ml. Overall, the results of this study did not support the hypothesis that exposure to waterborne NP can modulate concentrations of steroid hormones in the plasma of sexually mature male carp. The results did, however, raise a number of questions regarding the utility of estradiol equivalent (EEQ) estimates as a means of predicting in vivo effects of estrogenic substances. Furthermore, they provide information regarding the concentrations and variability of E2, T, and VTG in the plasma of sexually mature male carp, which may aid in design and interpretation of future studies.

Relative potencies of individual polycyclic aromatic hydrocarbons to induce dioxinlike and estrogenic responses in three cell lines.

The dioxinlike and estrogenic relative potencies (REPs) of 16 priority polycyclic aromatic hydrocarbons (PAHs), seven methylated PAHs, and two hydroxylated PAHs were examined using three in vitro cell bioassays. An in vitro ethoxyresorufin-O-deethylase assay with PLHC-1 fish hepatoma cells and in vitro luciferase assay with H4IIE-luc recombinant rat hepatoma cells were used to evaluate dioxinlike potency. An in vitro luciferase assay with MVLN, recombinant human breast carcinoma cells, was used to evaluate estrogenic potency. Seven of the 16 priority PAHs tested induced significant dioxinlike responses. Excluding outliers with large ranges of uncertainty, the dioxinlike REPs for the PAHs ranged from 10(-6) to 10(-3). This is similar to the REPs reported for other xenobiotics of concern including polychlorinated naphthalenes (PCNs) and some polychlorinated biphenyls (PCBs). In general, REP estimates generated in this study were similar to those reported previously. However, a comparison of the estimates of total 2,3,7,8-tetrachlorodibenzo-p-dioxin equivalents derived using assay-specific REPs with REPs reported in other studies indicated that the use of nonspecific REPs could lead to significant error in mass-balance (potency-balance) analyses. A 10-h acid treatment completely destroyed the dioxinlike activity of a PAH mixture. Among the compounds tested, only benzo[a]anthracene and dibenz[a,h]anthracene induced significant responses in the MVLN bioassay. Relative estrogenic potencies were estimated to be approximately 10(-7). Overall, this research contributes to the growing consensus regarding the dioxinlike potency of priority PAHs and PAH derivatives and provides some additional evidence about potentially estrogenic PAHs.

In vitro response of fish and mammalian cells to complex mixtures of polychlorinated naphthalenes, polychlorinated biphenyls, and polycyclic aromatic hydrocarbons.

In vitro characterization and comparison of responses to different classes of biologically active compounds can increase the utility of bioassays. In this study, the relative potencies (REPs) of mixtures of polychlorinated naphthalenes (PCNs), polychlorinated biphenyls (PCBs), and polycyclic aromatic hydrocarbons (PAHs), to induce in vitro ethoxyresorufin-O-deethylase (EROD) in PLHC-1 fish hepatoma cells, H4IIE wild type (H4IIE-wt) rat hepatoma cells, and recombinant H4IIE cells (H4IIE-EROD) were determined. The mixtures were also analyzed by in vitro luciferase assay with recombinant H4IIE cells (H4IIE-luc). Halowaxes 1051, 1014, and 1013 caused significant induction in all three H4IIE assays at concentrations less than 10 mg/l, but did not elicit a significant response in the PLHC-1 assay. Based on H4IIE results, the Halowaxes were estimated to have relative potencies (REPs) of approximately 10(-6)-10(-8) relative to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Less than 5 mg/l of Aroclors 1242, 1248, 1254; Clophens A60, T64; and Chlorofen induced significant responses in the H4IIE assays, while only Clophens A60 and T64 caused a significant response in the PLHC-1 assay. The efficacy of the Aroclor mixtures was generally insufficient to allow for quantitative REP estimates, but, based on their responses in the H4IIE assays, Clophen A60 and Chlorofen were estimated to have REPs of approximately 10(-6) and 10(-7), respectively. A mixture of 16 priority PAHs caused significant induction in all four cell types and was estimated to have a REP of approximately 10(-4). Overall, the results of this study add to a growing database on the dioxin-like potency of complex mixtures of xenobiotics, and suggested that H4IIE-based in vitro bioassays were more sensitive than PLHC-1 cells for detecting dioxin-like activity in complex mixtures.

Organochlorine insecticides, polychlorinated biphenyls, and metals in water, sediment, and green frogs from southwestern Michigan.

In an attempt to explain the etiology of frog deformities and population declines, many possible causative factors have been examined, including the input of synthetic chemicals into aquatic systems, where frogs spend much of their lives, including their entire developmental stages. Deformities in populations of green frogs in wetlands of southwestern Michigan that are influenced by agricultural, urban, or industrial inputs were assessed in this study. Of the 1445 green frogs (Rana clamitans) examined, only four (0.3%) exhibited morphological deformities. This deformity rate is less than the recognized background level of deformities for this species, which is approximately 1%. Concentrations of organochlorine insecticides, polychlorinated biphenyls (PCBs), and metals were determined in water, sediment, frog eggs, tadpoles, and adult green frog tissues. Concentrations of all individual organochlorine insecticides in tissue were less than 6 ng/g, wet wt. Concentrations of sigmaPCBs in tissue did not exceed 100 ng/g, wet wt. Concentrations of toxic metals were less than the limits of detection. Because no significant numbers of green frog deformities were observed in this region, it can be assumed that at these low concentrations, physical malformations in green frogs should not be observed. Significance of study. This study provides information on the incidence of deformities in green frog populations in southwestern Michigan and offers background data on chemical residues in green frogs and their environment.

Trace organic contaminants in sediment and water from Ulsan Bay and its vicinity, Korea.

Sediment and water samples collected from 32 locations in Ulsan Bay and adjacent inland areas were analyzed for polycyclic aromatic hydrocabons (PAHs), nonylphenol (NP), octylphenol (OP), bisphenol A (BPA), organochlorine (OC) pesticides (HCB, HCHs, CHLs, and DDTs), and polychlorinated biphenyls (PCBs) to characterize their spatial distribution and contamination status. PAHs were detected in nearly all sediment and water extracts from Ulsan Bay and its inland locations. The sedimentary PAH concentrations ranged from 17 to 3,100 ng/g on a dry weight basis (DW), which were predominated by two- and three-ring aromatic hydrocarbons in river and/or stream, and four- to six-ring compounds in Ulsan Bay sediment. Concentrations of PAHs in pore water samples were generally two or three orders magnitude less than those of corresponding sediment samples. Maximum concentrations of NP, OP, and BPA in sediments were 1,040, 120, and 54 ng/g DW, respectively. Concentrations of OP and BPA were, on average, 5- to 13-fold less than those of NP. PCB concentrations in sediment ranged from 1.4 to 77 ng/g DW, which were predominated by lower chlorinated congeners such as di- through pentachlorinated biphenyls. Among different OC pesticides analyzed, concentrations of DDTs were the greatest, ranging from 0.02 to 41.9 ng/g DW. NP concentrations were greater at inner locations proximal to municipal wastewater discharges into rivers and/or streams, whereas the concentrations of PCBs and PAHs were great near the sites of high industrial activities. Sediment-pore water partitioning coefficients correlated with those of reported Koc or Kow values for selected PAHs in Ulsan Bay, but these varied by an order of magnitude for stream and/or river sediments.

In vitro bioassay determination of dioxin-like and estrogenic activity in sediment and water from Ulsan Bay and its vicinity, Korea.

Extracts of sediment and water samples collected from Ulsan Bay, Korea, were screened for their ability to induce dioxin-like and estrogenic gene expression in vitro. Each sample was tested as raw extract (RE) and fractionated extract (FE). Based on the initial screening of RE, 23 of 31 sediment samples showed significant dioxin-like activity in H4IIE-luc bioassay, whereas most sediment samples did not elicit estrogenic response in MVLN bioassay. Most of the activities associated with FE samples revealed that mid-polar (F2) and most polar (F3) fractions were responsible for the significant reporter gene expression in H4IIE-luc bioassay. The results suggest that complex interactions may have depressed the activities of the known arylhydrocarbon receptor (AhR) agonists present in F1 samples. The F2 samples were the most active fraction. All F2 samples except one induced significant dioxin-like activity, and over half of the F2 samples induced significant estrogenic activity. Ten of the F2 samples produced magnitudes of response in H4IIE-luc bioassay similar to those induced by a 2,3,7,8-tetrachlorodibenzo-p-dioxin standard. Sediment associated with F2 samples was estimated to contain 24.9-826 pg TCDD-EQ/g DW. Based on a qualitative mass balance analysis, polycyclic aromatic hydrocarbons (PAHs) appeared to account for both the estrogenic and dioxin-like responses observed. Over half of the F3 samples were either cytotoxic or caused morphological changes in both H4IIE-luc and MVLN cells. Known concentrations of alkylphenols and bisphenol A were not great enough to account for both the estrogenic response and cytotoxicity observed for F3 samples. Despite the apparent toxic or stressful effects, most of F3 samples induced significant dioxin-like activity in vitro, adding to a growing body of evidence that suggests the presence of unidentified, relatively polar, AhR agonists in sediment from some areas.

Identification and quantification of estrogen receptor agonists in wastewater effluents.

Total concentrations of several known xenobiotic estrogen receptor (ER) agonists and natural and synthetic estrogen were measured in water by use of a combination of instrumental and bioanalytical approaches. Samples from 3 municipal wastewater treatment plants (WWTPs) in south central Michigan (upstream and effluent); 4 point source locations on the Trenton Channel of the Detroit River, MI; and 5 locations in Lake Mead, NV were analyzed. Organic compounds were extracted from 5 L water samples using solid-phase extraction disks and separated into three fractions based on polarity. Whole extracts and fractions were tested for ER agonist potency using the MVLN in vitro bioassay. ER agonist potency was characterized by comparing the magnitude of induction elicited by the extract or fraction to the maximum induction caused by 17beta-estradiol (E2). The greatest concentrations of ER agonists were associated with the most polar fraction (F3). Instrumental analyses and further fractionation were used to identify specific ER agonists associated with bioassay responses. Bioassay data were compared to extract concentrations in order minimize variability associated with the extraction procedure. Concentrations of endogenous estrogen, E2, and the synthetic estrogen ethynylestradiol (EE2) ranged from nondetectable to 14.6 ng/mL extract (nondetectable to 3.66 ng/L water) and represented from 88 to 99.5% of the total estrogen equivalents in the water samples analyzed. Concentrations of alkylphenols (APs) ranged from nondetectable to 148 microg/mL extract (nondetectable to 37,000 ng/L water). In general, alkylphenols contributed less than 0.5% of the total estrogen equivalents in the water samples. Both bioassay-directed fractionation results and comparison of ER agonist concentrations, adjusted for their known relative potencies, support the conclusion that E2 and EE2 were the dominant environmental estrogens in water samples from mid-Michigan and Lake Mead, NV.