Biological Effects of Hydrocarbon Degradation Intermediates: Is the Total Petroleum Hydrocarbon Analytical Method Adequate for Risk Assessment?

In crude oil contaminant plumes, the dissolved organic carbon (DOC) is mainly hydrocarbon degradation intermediates only partly quantified by the diesel range total petroleum hydrocarbon (TPHd) method. To understand potential biological effects of degradation intermediates, we tested three fractions of DOC: (1) solid-phase extract (HLB); (2) dichloromethane (DCM-total) extract used in TPHd; and (3) DCM extract with hydrocarbons isolated by silica gel cleanup (DCM-SGC). Bioactivity of extracts from five wells spanning a range of DOC was tested using an in vitro multiplex reporter system that evaluates modulation of the activity of 46 transcription factors; extracts were evaluated at concentrations equivalent to the well water samples. The aryl hydrocarbon receptor (AhR) and pregnane X receptor (PXR) transcription factors showed the greatest upregulation, with HLB exceeding DCM-total, and no upregulation in the hydrocarbon fraction (DCM-SGC). The HLB extracts were further studied with HepG2 chemically activated luciferase expression (CALUX) in vitro assays at nine concentrations ranging from 40 to 0.01 times the well water concentrations. Responses decreased with distance from the source but were still present at two wells without detectable hydrocarbons. Thus, our in vitro assay results indicate that risks associated with degradation intermediates of hydrocarbons in groundwater will be underestimated when protocols that remove these chemicals are employed.

Intramolecular [2 + 2] Photocycloaddition of Altrenogest: Confirmation of Product Structure, Theoretical Mechanistic Insight, and Bioactivity Assessment.

While studying the environmental fate of potent endocrine-active steroid hormones, we observed the formation of an intramolecular [2 + 2] photocycloaddition product (2) with a novel hexacyclic ring system following the photolysis of altrenogest (1). The structure and absolute configuration were established by X-ray diffraction analysis. Theoretical computations identified a barrierless two-step cyclization mechanism for the formation of 2 upon photoexcitation. 2 exhibited progesterone, estrogen, androgen, and pregnane X receptor activity, albeit generally with reduced potency relative to 1.

Toxicity Assessment of Groundwater Contaminated by Petroleum Hydrocarbons at a Well-Characterized, Aged, Crude Oil Release Site.

Management of petroleum-impacted waters by monitored natural attenuation requires an understanding of the toxicology of both the original compounds released and the transformation products formed during natural breakdown. Here, we report data from a groundwater plume consisting of a mixture of crude oil compounds and transformation products in an effort to bridge the gap between groundwater quality information and potential biological effects of human exposures. Groundwater samples were characterized for redox processes, concentrations of nonvolatile dissolved organic carbon (NVDOC) and total petroleum hydrocarbons in the diesel range, as well as for activation of human nuclear receptors (hNR) and toxicologically relevant transcriptional pathways. Results show upregulation of several biological pathways, including peroxisome proliferator-activated receptor gamma and alpha, estrogen receptor alpha, and pregnane X receptor (PXR) with higher levels of hNR activity observed in more contaminated samples. Our study of affected groundwater contaminated by a crude-oil release 39 years ago shows these types of waters may have the potential to cause adverse impacts on development, endocrine, and liver functioning in exposed populations. Additionally, positive trends in activation of some of the molecular targets (e.g., PXR) with increasing NVDOC concentrations (including polar transformation products) demonstrate the importance of improving our understanding of the toxicity associated with the unknown transformation products present in hydrocarbon-impacted waters. Our results begin to provide insight into the potential toxicity of petroleum-impacted waters, which is particularly timely given the ubiquitous nature of waters impacted by petroleum contamination not only recently but also in the past and the need to protect drinking-water quality.

Examining Natural Attenuation and Acute Toxicity of Petroleum-Derived Dissolved Organic Matter with Optical Spectroscopy.

Groundwater samples containing petroleum-derived dissolved organic matter (DOMHC) originating from the north oil body within the National Crude Oil Spill Fate and Natural Attenuation Research Site near Bemidji, MN, USA were analyzed by optical spectroscopic techniques (i.e., absorbance and fluorescence) to assess relationships that can be used to examine natural attenuation and toxicity of DOMHC in contaminated groundwater. A strong correlation between the concentration of dissolved organic carbon (DOC) and absorbance at 254 nm ( a254) along a transect of the DOMHC plume indicates that a254 can be used to quantitatively assess natural attenuation of DOMHC. Fluorescence components, identified by parallel factor (PARAFAC) analysis, show that the composition of the DOMHC beneath and adjacent to the oil body is dominated by aliphatic, low O/C compounds ("protein-like" fluorescence) and that the composition gradually evolves to aromatic, high O/C compounds ("humic-/fulvic-like" fluorescence) as a function of distance downgradient from the oil body. Finally, a direct, positive correlation between optical properties and Microtox acute toxicity assays demonstrates the utility of these combined techniques in assessing the spatial and temporal natural attenuation and toxicity of the DOMHC in petroleum-impacted groundwater systems.

Rapid effects of the aromatase inhibitor fadrozole on steroid production and gene expression in the ovary of female fathead minnows (Pimephales promelas).

Cytochrome P450 aromatase catalyzes conversion of C19 androgens to C18 estrogens and is critical for normal reproduction in female vertebrates. Fadrozole is a model aromatase inhibitor that has been shown to suppress estrogen production in the ovaries of fish. However, little is known about the early impacts of aromatase inhibition on steroid production and gene expression in fish. Adult female fathead minnows (Pimephales promelas) were exposed via water to 0, 5, or 50µg fadrozole/L for a time-course of 0.5, 1, 2, 4, and 6h, or 0 or 50µg fadrozole/L for a time-course of 6, 12, and 24h. We examined ex vivo ovarian 17ß-estradiol (E2) and testosterone (T) production, and plasma E2 concentrations from each study. Expression profiles of genes known or hypothesized to be impacted by fadrozole including aromatase (cytochrome P450 [cyp] 19a1a), steriodogenic acute regulatory protein (star), cytochrome P450 side-chain cleavage (cyp11a), cytochrome P450 17 alpha hydroxylase/17,20 lyase (cyp17), and follicle stimulating hormone receptor (fshr) were measured in the ovaries by quantitative real-time polymerase chain reaction (QPCR). In addition, broader ovarian gene expression was examined using a 15k fathead minnow microarray. The 5µg/L exposure significantly reduced ex vivo E2 production by 6h. In the 50µg/L treatment, ex vivo E2 production was significantly reduced after just 2h of exposure and remained depressed at all time-points examined through 24h. Plasma E2 concentrations were significantly reduced as early as 4h after initiation of exposure to either 5 or 50µg fadrozole/L and remained depressed throughout 24h in the 50µg/L exposure. Ex vivo T concentrations remained unchanged throughout the time-course. Expression of transcripts involved in steroidogenesis increased within the first 24h suggesting rapid induction of a mechanism to compensate for fadrozole inhibition of aromatase. Microarray results also showed fadrozole exposure caused concentration- and time-dependent changes in gene expression profiles in many HPG-axis pathways as early as 4h. This study provides insights into the very rapid effects of aromatase inhibition on steroidogenic processes in fish.

Environmental Photochemistry of Altrenogest: Photoisomerization to a Bioactive Product with Increased Environmental Persistence via Reversible Photohydration.

Despite its wide use as a veterinary pharmaceutical, environmental fate data is lacking for altrenogest, a potent synthetic progestin. Here, it is reported that direct photolysis of altrenogest under environmentally relevant conditions was extremely efficient and rapid (half-life ∼25 s). Photolysis rates (observed rate constant kobs = 2.7 ± 0.2 × 10(-2) s(-1)) were unaffected by changes in pH or temperature but were sensitive to oxygen concentrations (N2-saturated kobs = 9.10 ± 0.32 × 10(-2) s(-1); O2-saturated kobs = 1.38 ± 0.11 × 10(-2) s(-1)). The primary photoproduct was identified as an isomer formed via an internal 2 + 2 cycloaddition reaction; the triplet lifetime (8.4 ± 0.2 µs) and rate constant (8 × 10(4) s(-1)) of this reaction were measured using transient absorption spectroscopy. Subsequent characterization determined that this primary cycloaddition photoproduct undergoes photohydration. The resultant photostable secondary photoproducts are subject to thermal dehydration in dark conditions, leading to reversion to the primary cycloaddition photoproduct on a time scale of hours to days, with the photohydration and dehydration repeatable over several light/dark cycles. This dehydration reaction occurs more rapidly at higher temperatures and is also accelerated at both high and low pH values. In vitro androgen receptor (AR)-dependent gene transcriptional activation cell assays and in silico nuclear hormone receptor screening revealed that certain photoproducts retain significant androgenic activity, which has implications for exposure risks associated with the presence and cycling of altrenogest and its photoproducts in the environment.

Using transcriptomic tools to evaluate biological effects across effluent gradients at a diverse set of study sites in Minnesota, USA.

The aim of this study was to explore the utility of "omics" approaches in monitoring aquatic environments where complex, often unknown stressors make chemical-specific risk assessment untenable. We examined changes in the fathead minnow (Pimephales promelas) ovarian transcriptome following 4-day exposures conducted at three sites in Minnesota (MN, USA). Within each site, fish were exposed to water from three locations along a spatial gradient relative to a wastewater treatment plant (WWTP) discharge. After exposure, site-specific impacts on gene expression in ovaries were assessed. Using an intragradient point of comparison, biological responses specifically associated with the WWTP effluent were identified using functional enrichment analyses. Fish exposed to water from locations downstream of the effluent discharges exhibited many transcriptomic responses in common with those exposed to the effluent, indicating that effects of the discharge do not fully dissipate downstream. Functional analyses showed a range of biological pathways impacted through effluent exposure at all three sites. Several of those impacted pathways at each site could be linked to potential adverse reproductive outcomes associated with the hypothalamic-pituitary-gonadal (HPG) axis in female fathead minnows, specifically signaling pathways associated with oocyte meiosis, TGF-beta signaling, gonadotropin-releasing hormone (GnRH) and epidermal growth factor receptor family (ErbB), and gene sets associated with cyclin B-1 and metalloproteinase. The utility of this approach comes from the ability to identify biological responses to pollutant exposure, particularly those that can be tied to adverse outcomes at the population level and those that identify molecular targets for future studies.

Transcriptomic effects-based monitoring for endocrine active chemicals: assessing relative contribution of treated wastewater to downstream pollution.

The present study investigated whether a combination of targeted analytical chemistry information with unsupervised, data-rich biological methodology (i.e., transcriptomics) could be utilized to evaluate relative contributions of wastewater treatment plant (WWTP) effluents to biological effects. The effects of WWTP effluents on fish exposed to ambient, receiving waters were studied at three locations with distinct WWTP and watershed characteristics. At each location, 4 d exposures of male fathead minnows to the WWTP effluent and upstream and downstream ambient waters were conducted. Transcriptomic analyses were performed on livers using 15,000 feature microarrays, followed by a canonical pathway and gene set enrichment analyses. Enrichment of gene sets indicative of teleost brain-pituitary-gonadal-hepatic (BPGH) axis function indicated that WWTPs serve as an important source of endocrine active chemicals (EACs) that affect the BPGH axis (e.g., cholesterol and steroid metabolism were altered). The results indicated that transcriptomics may even pinpoint pertinent adverse outcomes (i.e., liver vacuolization) and groups of chemicals that preselected chemical analytes may miss. Transcriptomic Effects-Based monitoring was capable of distinguishing sites, and it reflected chemical pollution gradients, thus holding promise for assessment of relative contributions of point sources to pollution and the efficacy of pollution remediation.

Identifying non-point sources of endocrine active compounds and their biological impacts in freshwater lakes.

Contaminants of emerging concern, particularly endocrine active compounds (EACs), have been identified as a threat to aquatic wildlife. However, little is known about the impact of EACs on lakes through groundwater from onsite wastewater treatment systems (OWTS). This study aims to identify specific contributions of OWTS to Sullivan Lake, Minnesota, USA. Lake hydrology, water chemistry, caged bluegill sunfish (Lepomis macrochirus), and larval fathead minnow (Pimephales promelas) exposures were used to assess whether EACs entered the lake through OWTS inflow and the resultant biological impact on fish. Study areas included two OWTS-influenced near-shore sites with native bluegill spawning habitats and two in-lake control sites without nearby EAC sources. Caged bluegill sunfish were analyzed for plasma vitellogenin concentrations, organosomatic indices, and histological pathologies. Surface and porewater was collected from each site and analyzed for EACs. Porewater was also collected for laboratory exposure of larval fathead minnow, before analysis of predator escape performance and gene expression profiles. Chemical analysis showed EACs present at low concentrations at each study site, whereas discrete variations were reported between sites and between summer and fall samplings. Body condition index and liver vacuolization of sunfish were found to differ among study sites as did gene expression in exposed larval fathead minnows. Interestingly, biological exposure data and water chemistry did not match. Therefore, although results highlight the potential impacts of seepage from OWTS, further investigation of mixture effects and life history factor as well as chemical fate is warranted.

Assessment of Mitochondrial Function in the AmE-711 Honey Bee Cell Line: Boscalid and Pyraclostrobin Effects.

There is a growing concern that chronic exposure to fungicides contributes to negative effects on honey bee development, life span, and behavior. Field and caged-bee studies have helped to characterize the adverse outcomes (AOs) of environmentally relevant exposures, but linking AOs to molecular/cellular mechanisms of toxicity would benefit from the use of readily controllable, simplified host platforms like cell lines. Our objective was to develop and optimize an in vitro-based mitochondrial toxicity assay suite using the honey bee as a model pollinator, and the electron transport chain (ETC) modulators boscalid and pyraclostrobin as model fungicides. We measured the effects of short (~30 min) and extended exposures (16-24 h) to boscalid and pyraclostrobin on AmE-711 honey bee cell viability and mitochondrial function. Short exposure to pyraclostrobin did not affect cell viability, but extended exposure reduced viability in a concentration-dependent manner (median lethal concentration = 4175 µg/L; ppb). Mitochondrial membrane potential (MMP) was affected by pyraclostrobin in both short (median effect concentration [EC50] = 515 µg/L) and extended exposure (EC50 = 982 µg/L) scenarios. Short exposure to 10 and 1000 µg/L pyraclostrobin resulted in a rapid decrease in the oxygen consumption rate (OCR), approximately 24% reduction by 10 µg/L relative to the baseline OCR, and 64% by 1000 µg/L. Extended exposure to 1000 µg/L pyraclostrobin reduced all respiratory parameters (e.g., spare capacity, coupling efficiency), whereas 1- and 10-µg/L treatments had no significant effects. The viability of AmE-711 cells, as well as the MMP and cellular respiration were unaffected by short and extended exposures to boscalid. The present study demonstrates that the AmE-711-based assessment of viability, MMP, and ETC functionality can provide a time- and cost-effective platform for mitochondrial toxicity screening relevant to bees. Environ Toxicol Chem 2024;43:976-987. © 2024 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Photochemical mobilization of dissolved hydrocarbon oxidation products from petroleum contaminated soil into a shallow aquifer activate human nuclear receptors.

Elevated non-volatile dissolved organic carbon (NVDOC) concentrations in groundwater (GW) monitoring wells under oil-contaminated hydrophobic soils originating from a pipeline rupture at the National Crude Oil Spill & Natural Attenuation Research Site near Bemidji, MN are documented. We hypothesized the elevated NVDOC is comprised of water-soluble photooxidation products transported from the surface to the aquifer. We use field and laboratory samples in combination with complementary analytical methods to test this hypothesis and determine the biological response to these products. Observations from optical spectroscopy and ultrahigh-resolution mass spectrometry reveal a significant correlation between the chemical composition of NVDOC leached from photochemically weathered soils and GW monitoring wells with high NVDOC concentrations measured in the aquifer beneath the contaminated soil. Conversely, the chemical composition from the uncontaminated soil photoleachate matches the NVDOC observed in the uncontaminated wells. Contaminated GW and photodissolution leachates from contaminated soil activated biological targets indicative of xenobiotic metabolism and exhibited potential for adverse effects. Newly formed hydrocarbon oxidation products (HOPs) from fresh oil could be distinguished from those downgradient. This study illustrates another pathway for dissolved HOPs to infiltrate GW and potentially affect human health and the environment.

A graphical systems model and tissue-specific functional gene sets to aid transcriptomic analysis of chemical impacts on the female teleost reproductive axis.

Oligonucleotide microarrays and other 'omics' approaches are powerful tools for unsupervised analysis of chemical impacts on biological systems. However, the lack of well annotated biological pathways for many aquatic organisms, including fish, and the limited power of microarray-based analyses to detect low level differential expression of individual genes can hinder the ability to infer and understand chemical effects based on transcriptomic data. Here we report on the supervised assembly of a series of tissue-specific functional gene sets intended to aid transcriptomic analysis of chemical impacts on the female teleost reproductive axis. Gene sets were defined based on an updated graphical systems model of the teleost brain-pituitary-gonadal-hepatic axis. Features depicted in the model were organized into gene sets and mapped to specific probes on three zebrafish (Danio rerio) and two fathead minnow (Pimephales promelas) microarray platforms. Coverage of target genes on the microarrays ranged from 48% for the fathead minnow arrays to 88% for the most current zebrafish platform. Additionally, extended fathead minnow gene sets, incorporating first degree neighbors identified from a Spearman correlation network derived from a large compendium of fathead minnow microarray data, were constructed. Overall, only 14% of the 78 genes queried were connected in the network. Among those, over half had less than five neighbors, while two genes, cyclin b1 and zona pellucida glycoprotein 3, had over 100 first degree neighbors, and were neighbors to one another. Gene set enrichment analyses were conducted using microarray data from a zebrafish hypoxia experiment and fathead minnow time-course experiments conducted with three different endocrine-active chemicals. Results of these analyses demonstrate the utility of the approach for supporting biological inference from ecotoxicogenomic data and comparisons across multiple toxicogenomic experiments. The graphical model, gene mapping, and gene sets described are now available to the scientific community as tools to support ecotoxicogenomic research.

The Eco-Exposome Concept: Supporting an Integrated Assessment of Mixtures of Environmental Chemicals.

Organisms are exposed to ever-changing complex mixtures of chemicals over the course of their lifetime. The need to more comprehensively describe this exposure and relate it to adverse health effects has led to formulation of the exposome concept in human toxicology. Whether this concept has utility in the context of environmental hazard and risk assessment has not been discussed in detail. In this Critical Perspective, we propose-by analogy to the human exposome-to define the eco-exposome as the totality of the internal exposure (anthropogenic and natural chemicals, their biotransformation products or adducts, and endogenous signaling molecules that may be sensitive to an anthropogenic chemical exposure) over the lifetime of an ecologically relevant organism. We describe how targeted and nontargeted chemical analyses and bioassays can be employed to characterize this exposure and discuss how the adverse outcome pathway concept could be used to link this exposure to adverse effects. Available methods, their limitations, and/or requirement for improvements for practical application of the eco-exposome concept are discussed. Even though analysis of the eco-exposome can be resource-intensive and challenging, new approaches and technologies make this assessment increasingly feasible. Furthermore, an improved understanding of mechanistic relationships between external chemical exposure(s), internal chemical exposure(s), and biological effects could result in the development of proxies, that is, relatively simple chemical and biological measurements that could be used to complement internal exposure assessment or infer the internal exposure when it is difficult to measure. Environ Toxicol Chem 2022;41:30-45. © 2021 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Impacts of an anti-androgen and an androgen/anti-androgen mixture on the metabolite profile of male fathead minnow urine.

Male and female fathead minnows (Pimephales promelas, FHM) were exposed via water to 20 or 200 microg/L of cyproterone acetate (CA), a model androgen receptor (AR) antagonist. FHM were also exposed to 500 ng/L of 17beta-trenbolone (TB), a model AR agonist, and to mixtures of TB with both concentrations of CA. The urine metabolite profile (as measured by 1H NMR spectroscopy) of male FHM exposed to the high concentration of CA was markedly different from that of controls, and this difference was less for males coexposed to the associated TB+CA mixture. The exposure to TB alone had almost no impact on the male urine profile. These results suggest that male FHM urinary metabolite profiling may be useful for directly detecting effects of anti-androgens. In contrast, the urinary profile of male FHM exposed to the lower concentration of CA was not very different from that of controls, but, unexpectedly, this difference was increased when coexposed to the associated TB+CA mixture. This suggests that TB with CA at the lower concentration impacts male FHM through an interactive effect possibly unrelated, or in addition, to AR antagonism. The relative occurrence of male-like nuptial tubercles in female FHM exposed to TB and to the mixtures of TB and CA supported the metabolomics data.

Toxicity prediction and effect characterization of 90 pharmaceuticals and illicit drugs measured in plasma of fish from a major European river (Sava, Croatia).

Chemical analysis of plasma samples of wild fish from the Sava River (Croatia) revealed the presence of 90 different pharmaceuticals/illicit drugs and their metabolites (PhACs/IDrgs). The concentrations of these PhACs/IDrgs in plasma were 10 to 1000 times higher than their concentrations in river water. Antibiotics, allergy/cold medications and analgesics were categories with the highest plasma concentrations. Fifty PhACs/IDrgs were identified as chemicals of concern based on the fish plasma model (FPM) effect ratios (ER) and their potential to activate evolutionary conserved biological targets. Chemicals of concern were also prioritized by calculating exposure-activity ratios (EARs) where plasma concentrations of chemicals were compared to their bioactivities in comprehensive ToxCast suite of in vitro assays. Overall, the applied prioritization methods indicated stimulants (nicotine, cotinine) and allergy/cold medications (prednisolone, dexamethasone) as having the highest potential biological impact on fish. The FPM model pointed to psychoactive substances (hallucinogens/stimulants and opioids) and psychotropic substances in the cannabinoids category (i.e. CBD and THC). EAR confirmed above and singled out additional chemicals of concern - anticholesteremic simvastatin and antiepileptic haloperidol. Present study demonstrates how the use of a combination of chemical analyses, and bio-effects based risk predictions with multiple criteria can help identify priority contaminants in freshwaters. The results reveal a widespread exposure of fish to complex mixtures of PhACs/IDrgs, which may target common molecular targets. While many of the prioritized chemicals occurred at low concentrations, their adverse effect on aquatic communities, due to continuous chronic exposure and additive effects, should not be neglected.

Bioactive Rearrangement Products from Aqueous Photolysis of Pharmaceutical Steroids.

In an ongoing effort to study the environmental fate of endocrine-active steroid hormones, we report the formation of phenolic rearrangement products (3 and 4) with a novel 6,5,8,5-ring system following aqueous photolysis of dienogest (1) and methyldienolone (2). The structures were established by analysis of 2D NMR and HRMS data, and that of 3 was confirmed by X-ray diffraction analysis. These photoproducts exhibit progestogenic and androgenic activity, albeit with less potency than their parent compounds.

Assessment of river sediment toxicity: Combining empirical zebrafish embryotoxicity testing with in silico toxicity characterization.

Quantitative chemical analyses of 428 organic contaminants (OCs) indicated the presence of 313 OCs in the sediment extracts from Sava River, Croatia. Pharmaceuticals were present in higher concentrations than pesticides thus confirming their increasing threat to freshwater ecosystems. Toxicity evaluation of the sediment extracts from four locations (Jesenice, Rugvica, Galdovo and Lukavec) using zebrafish embryotoxicity test (ZET) accompanied with semi-quantitative histopathological analyses exhibited correlation with cumulative number and concentrations of OCs at the investigated sites (10.05, 15.22, 1.25, and 9.13 µg/g respectively). Toxicity of sediment extracts and sediment was predicted using toxic unit (TU) approach and persistence, bioaccumulation and toxicity (PBT) ranking. Additionally, influential OCs and genes were identified by graph mining of the prior knowledge informed, site-specific chemical-gene interaction models. Predicted toxicity of sediment extracts (TUext) was similar to the results obtained by ZET and associated histopathology with Rugvica sediment being the most toxic, followed by Jesenice, Lukavec and Galdovo. Sediment TU (TUsed) favoured OCs with low octanol-water partition coefficients like herbicide glyphosate and antibiotics ciprofloxacin and sulfamethazine thus indicating locations containing higher concentrations of these OCs (Galdovo and Rugvica) as the most toxic. Results suggest that comprehensive in silico sediment toxicity predictions advocate providing equal attention to organic contaminants with either very low or very high log Kow.

Prior knowledge-based approach for associating contaminants with biological effects: A case study in the St. Croix River basin, MN, WI, USA.

Evaluating potential adverse effects of complex chemical mixtures in the environment is challenging. One way to address that challenge is through more integrated analysis of chemical monitoring and biological effects data. In the present study, water samples from five locations near two municipal wastewater treatment plants in the St. Croix River basin, on the border of MN and WI, USA, were analyzed for 127 organic contaminants. Known chemical-gene interactions were used to develop site-specific knowledge assembly models (KAMs) and formulate hypotheses concerning possible biological effects associated with chemicals detected in water samples from each location. Additionally, hepatic gene expression data were collected for fathead minnows (Pimephales promelas) exposed in situ, for 12 d, at each location. Expression data from oligonucleotide microarrays were analyzed to identify functional annotation terms enriched among the differentially-expressed probes. The general nature of many of the terms made hypothesis formulation on the basis of the transcriptome-level response alone difficult. However, integrated analysis of the transcriptome data in the context of the site-specific KAMs allowed for evaluation of the likelihood of specific chemicals contributing to observed biological responses. Thirteen chemicals (atrazine, carbamazepine, metformin, thiabendazole, diazepam, cholesterol, p-cresol, phenytoin, omeprazole, ethyromycin, 17ß-estradiol, cimetidine, and estrone), for which there was statistically significant concordance between occurrence at a site and expected biological response as represented in the KAM, were identified. While not definitive, the approach provides a line of evidence for evaluating potential cause-effect relationships between components of a complex mixture of contaminants and biological effects data, which can inform subsequent monitoring and investigation.