Setting the stage to advance oil toxicity testing: Overview of knowledge gaps, and recommendations.

The Chemical Response to Oil Spills: Ecological Effects Research Forum created a standardized protocol for comparing the in vivo toxicity of physically dispersed oil to chemically dispersed oil to support science-based decision making on the use of dispersants in the early 2000s. Since then, the protocol has been frequently modified to incorporate advances in technology; enable the study of unconventional and heavier oils; and provide data for use in a more diverse manner to cover the growing needs of the oil spill science community. Unfortunately, for many of these lab-based oil toxicity studies consideration was not given to the influence of modifications to the protocol on media chemistry, resulting toxicity and limitations for the use of resulting data in other contexts (e.g., risk assessments, models). To address these issues, a working group of international oil spill experts from academia, industry, government, and private organizations was convened under the Multi-Partner Research Initiative of Canada's Oceans Protection Plan to review publications using the CROSERF protocol since its inception to support their goal of coming to consensus on the key elements required within a "modernized CROSERF protocol".

Behavioral and hypothalamic transcriptome analyses reveal sex-specific responses to phenanthrene exposure in the fathead minnow (Pimephales promelas).

Environmental concentrations of the polycyclic aromatic hydrocarbon phenanthrene can become elevated with petroleum processing, industrial activities, and urban run-off into waterbodies. However, mechanisms related to its neurotoxicity in fish are not fully described. Here, we exposed adult fathead minnows (FHM) to an average measured concentration of 202 µg phenanthrene/L over a 47-d period. Behaviors of male and female FHM were assessed using a novel aquarium test. Phenanthrene exposed females displayed equilibrium loss, while phenanthrene exposed males spent less time in the aquarium bottom, suggesting phenanthrene reduced anxiety-related behavior. To elucidate putative mechanisms underlying behaviors, we determined the hypothalamic transcriptome profile, a critical integration centre for the regulation of behaviors. There were 1075 hypothalamic transcripts differentially expressed between males and females (sex-specific) while 15 transcripts were phenanthrene-specific. Thus, sex of the animal was more pervasive at influencing the transcriptome compared to phenanthrene and this may partially explain the divergent behavioral responses between sexes. Transcripts altered by phenanthrene included palmitoylated 3 membrane protein, plectin 1,ATP synthase membrane subunit c, and mitochondrial ribosomal protein S11. Gene set enrichment analysis revealed less than 5% of the gene networks perturbed by phenanthrene were shared between males and females, thus phenanthrene altered the hypothalamic transcriptome in a sex-specific manner. Gene networks shared between both sexes and associated with phenanthrene-induced neurotoxicity included processes related to mitochondrial respiratory chain dysfunction, epinephrine/norepinephrine release, and glutamate biosynthesis pathways. Such energy deficits and neurotransmitter disruptions are hypothesized to lead to behavioral deficits in fish. This study provides mechanistic insights into phenanthrene-induced neurotoxicity and how it may relate to changes in fish behaviors.

Transcriptome network data in larval zebrafish (Danio rerio) following exposure to the phenylpyrazole fipronil.

Fipronil is a phenylpyrazole pesticide that is used in both residential and agricultural applications. Fipronil is detected in run-off and water systems that are near areas in which the pesticide has been applied. The pesticide acts to antagonize gamma aminobutyric acid receptors, leading to over-excitation in the central nervous system. Fipronil has relatively high toxicity to fish, but the mechanisms underlying the toxicity are not well understood in embryonic stages. Zebrafish embryos were exposed to a single concentration of fipronil for 48 h at ∼3-4 h-post-fertilization. Following a 7-day depuration phase, transcriptome and behavioral analyses were conducted. Transcriptomics identified neural processes as those differentially expressed with different doses of fipronil (0.2 µg, 200 µg and 2 mg fipronil/L). Gene networks associated with astrocyte differentiation, myelination, neural tube development, brain stem response, innervation, nerve regeneration, astrocyte differentiation, among other pathways were altered with exposure. In addition, miRNA-related events are disrupted by fipronil exposure and genes associated with primary or pri-miRNA processing were increased in larval fish exposed to the pesticide. These data present putative mechanisms associated with neurological impacts at later ages of zebrafish. This is important because it is not clear how early exposure to pesticides like fipronil affect central nervous system function and organisms later in life.

Residual molecular and behavioral effects of the phenylpyrazole pesticide fipronil in larval zebrafish (Danio rerio) following a pulse embryonic exposure.

Pesticides are typically applied to crops as acute applications, and residual effects of such intermittent exposures are not often characterized in developing fish. Fipronil is an agricultural pesticide that inhibits γ-amino-butyric acid (GABA) gated chloride channels. In this study, zebrafish (Danio rerio) embryos were exposed for 48 h (starting at ~3 h post fertilization, hpf) to various concentrations of fipronil (0.02 µg/L up to 4000 µg/L). Following this acute exposure, a subset of fish was transferred to clean water for a 7-day depuration phase. We hypothesized that a pulse exposure to fipronil during critical periods of central nervous system development would adversely affect fish later in life. After a 48 hour pulse exposure, survival was reduced in embryos exposed to 2 µg fipronil/L or greater. However, there was no further mortality during the depuration phase, nor were there changes in body length nor notochord length in larvae 9 dpf (days post-fertilization) compared to controls. Additional experiments were carried out at higher concentrations over 96 h (up to 4 dpf) to also elucidate developmental effects and teratogenicity of fipronil (43.7 µg/L up to 4370 µg/L). Fipronil at these higher concentrations significantly impacted the development of zebrafish, and the following morphometric and teratogenic effects were observed in 4 dpf fish; reduced body length, yolk sac and pericardial edema, reduced midbrain length, reduced optic and otic diameter, and truncation of the lower jaw. In depurated fish, we hypothesized that there would exist residual effects of exposure at the molecular level. Transcriptome profiling was therefore conducted on 9 dpf depurated larvae exposed initially for 48 h to one dose of either 0.2 µg/L, 200 µg/L or 2000 µg/L fipronil. The expression of gene networks associated with glycogen and omega-3-fatty acid metabolism were decreased in larvae exposed to each of the three concentrations of fipronil, suggesting metabolic disruption. Moreover, transcriptomics revealed that fipronil suppressed gene networks related to light-dark adaptation, photoperiod sensing, and circadian rhythm. Based on these data, we tested fish for altered behavioral responses in a Light-Dark preference test. Larvae exposed to >200 µg fipronil/L as embryos showed fewer number of visits (20-30% less) to the dark zone compared to controls. Larvae also spent a lower amount of time in the dark zone compared to controls, suggesting that fipronil strengthened dark avoidance behavior which is indicative of anxiety. This study demonstrates that a short pulse exposure to fipronil can affect transcriptome networks for metabolism, circadian rhythm, and response to light in fish after depuration, and these molecular responses are hypothesized to be related to aberrant behavioral effects observed in the light-dark preference test.

Transcriptome Profiling in Larval Fathead Minnow Exposed to Commercial Naphthenic Acids and Extracts from Fresh and Aged Oil Sands Process-Affected Water.

Surface mining and extraction of oil sands results in the generation of and need for storage of large volumes of oil sands process-affected water (OSPW). More structurally complex than classical naphthenic acids (NAs), naphthenic acid fraction components (NAFCs) are key toxic constituents of OSPW, and changes in the NAFC profile in OSPW over time have been linked to mitigation of OSPW toxicity. Molecular studies targeting individual genes have indicated that NAFC toxicity is likely mediated via oxidative stress, altered cell cycles, ontogenetic differentiation, endocrine disruption, and immunotoxicity. However, the individual-gene approach results in a limited picture of molecular responses. This study shows that NAFCs, from aged or fresh OSPW, have a unique effect on the larval fathead minnow transcriptome and provides initial data to construct adverse outcome pathways for skeletal deformities. All three types of processed NAs (fresh, aged, and commercial) affected the immunome of developing fish. These gene networks included immunity, inflammatory response, B-cell response, platelet adhesion, and T-helper lymphocyte activity. Larvae exposed to both NAFCs and commercial NA developed cardiovascular and bone deformities, and transcriptomic networks reflected these developmental abnormalities. Gene networks found only in NAFC-exposed fish suggest NAFCs may alter fish cardiovascular health through altered calcium ion regulation. This study improves understanding regarding the molecular perturbations underlying developmental deformities following exposure to NAFCs.

Part B: Morphometric and transcriptomic responses to sub-chronic exposure to the polycyclic aromatic hydrocarbon phenanthrene in the fathead minnow (Pimephales promelas).

Phenanthrene is a tricyclic polycyclic aromatic hydrocarbon and environmental contaminant found in high concentrations around urban catchments and in the vicinity of oil extraction activities. Fish exposed to phenanthrene can exhibit altered reproductive hormone profiles and/or differences within gonadosomatic index and altered gamete proportions, but the mechanisms underlying these changes are not fully understood. In this study, we conducted a sub-chronic bioassay and measured transcriptional responses in the liver, the major tissue involved in generating lipids for oocyte growth. Adult male and female fathead minnow (Pimephales promelas) were exposed to an average measured concentration of 202 µg phenanthrene/L for a 7 week period. Condition factor was reduced in both males and females, while female fish also showed decreased gonadosomatic index relative to control females. In females exposed to phenanthrene, perinucleolar proportions were increased ∼1.9-fold relative to the control group whereas the proportions of vitellogenic oocytes decreased ∼8.8 fold. In males exposed to phenanthrene, spermatogonia proportions were increased ∼2.3 fold in testicular tissues compared to control fish. Thus, gametes were at an earlier stage of maturation in phenanthrene-treated fish compared to controls. However, no differences were detected in the production of 17ß-estradiol or testosterone from the gonad in either sex. Catalase activity was also assessed in the liver as a measure of oxidative stress and this biomarker did not change in activity in either sex. In addition to endpoints in the ovary, the female hepatic transcriptome was measured, as this tissue produces lipids for oocyte maturation. Transcriptomic responses to phenanthrene exposure suggested a reduction in vitellogenin mRNA, and lipid metabolism and immune system pathways. Comparisons of hepatic transcriptome responses with Part A (72 h phenanthrene exposure) showed that energy homeostasis pathways were consistently altered following phenanthrene exposure over multiple durations and concentrations. We suggest that altered energy homeostasis may be adversely affecting reproductive efforts, as impaired reproduction has been observed in other studies investigating polycyclic aromatic hydrocarbons.

Part A: Temporal and dose-dependent transcriptional responses in the liver of fathead minnows following short term exposure to the polycyclic aromatic hydrocarbon phenanthrene.

Phenanthrene is a low molecular weight polycyclic aromatic hydrocarbon (PAH) that is composed of three fused benzene rings. PAHs are formed naturally through incomplete combustion of organic materials, and are environmental contaminants due to anthropogenic activities (e.g. oil extraction and refining, industrial and municipal effluents, fossil fuel burning). Fish exposed to PAHs such as phenanthrene have been reported to exhibit altered reproductive axis endpoints, however the mechanisms that underlie these responses are not fully characterized. To better understand effects at the mechanistic level, we applied transcriptomics to identify molecular pathways altered after acute exposure to phenanthrene on both a dose and temporal scale. Female fathead minnow (Pimephales promelas) were exposed to an average measured concentration of either 0, 29.8, 389 or 943 µg phenanthrene/L for 24, 48, and 72 h in a static-renewal bioassay. Ovaries were assessed for oocyte distribution as well as in vitro 17ß-estradiol production and gene expression for transcripts related to steroidogenesis and estrogen signalling. In addition, the liver transcriptome was measured as this tissue is the primary source of the egg yolk precursor protein vitellogenin. Exposure to 29.8 µg phenanthrene/L increased proportions of the cortical alveolar stage in the ovaries after 48 h while the proportion of cortical alveolar oocyte were decreased in fish exposed to 943 µg phenanthrene/L for 48 h. Phenanthrene did not affect 17ß-estradiol production at any time or dose, and did not affect transcripts associated with hormone synthesis nor signalling pathways. In the liver, the transcriptome showed fewer genes in common across time when compared to those transcripts affected by concentration at a single time point. Cholesterol metabolism was the only pathway perturbed in the liver following all comparisons in both the dose and time course experiments. Our data suggest that transcriptome networks associated with hepatic lipid metabolism are rapidly affected by phenanthrene, and this may indirectly reduce resources available for reproductive efforts.

Physiological and molecular responses of juvenile shortnose sturgeon (Acipenser brevirostrum) to thermal stress.

The shortnose sturgeon (Acipenser brevirostrum LeSueur, 1818) is a vulnerable species that is found along the eastern coast of North America. Little is known about temperature tolerance in this species and with a rapidly changing global climate, it becomes increasingly important to define the thermal tolerance of this species to better predict population distribution. Using a modified critical thermal maximum test (CTMax), the objectives of this study were to determine the impact of heating rate (0.1, 0.2 and 0.25°Cmin-1) on the thermal tolerance, associated hematological responses, and oxygen consumption in juvenile sturgeon. In addition, transcripts associated with physiological stress and heat shock (i.e., heat shock proteins) were also measured. Heating rate did not alter the CTMax values of shortnose sturgeon. Neither heating rate nor thermal stress affected plasma sodium and chloride levels, nor the expression of transcripts that included catalase, glucocorticoid receptor, heat shock protein70 (hsp70), heat shock protein 90α (hsp90α) and cytochrome P450 1a (cyp1a). However, regardless of heating rate, thermal stress increased both plasma potassium and lactate concentrations. Glucose levels were increased at heating rates of 0.2 and 0.25°Cmin-1, but not at 0.1°Cmin-1. Overall, oxygen consumption rates increased with thermal stress, but the response patterns were not affected by heating rate. These data support the hypothesis that shortnose sturgeon can tolerate acute heat stress, as many physiological and molecular parameters measured here were non-responsive to the thermal stress.

Molecular initiating events of the intersex phenotype: Low-dose exposure to 17α-ethinylestradiol rapidly regulates molecular networks associated with gonad differentiation in the adult fathead minnow testis.

Intersex, or the presence of oocytes in the testes, has been documented in fish following exposure to wastewater effluent and estrogenic compounds. However, the molecular networks underlying the intersex condition are not completely known. To address this, we exposed male fathead minnows to a low, environmentally-relevant concentration of 17alpha-ethinylestradiol (EE2) (15ng/L) and measured the transcriptome response in the testis after 96h to identify early molecular initiating events that may proceed the intersex condition. The short-term exposure to EE2 did not affect gonadosomatic index and proportion of gametes within the testes. However, the production of 11-ketotestosterone and testosterone from the testis in vitro was decreased relative to controls. Expression profiling using a 8×60K fathead minnow microarray identified 10 transcripts that were differentially expressed in the testes, the most dramatic change being that of coagulation factor XIII A chain (20-fold increase). Transcripts that included guanine nucleotide binding protein (Beta Polypeptide 2), peroxisome proliferator-activated receptor delta, and WNK lysine deficient protein kinase 1a, were down-regulated by EE2. Subnetwork enrichment analysis revealed that EE2 suppressed transcriptional networks associated with steroid metabolism, hormone biosynthesis, and sperm mobility. Most interesting was that gene networks associated with doublesex and mab-3 related transcription factor 1 (dmrt1) were suppressed in the adult testis, despite the fact that dmrt1 itself was not different in expression from control males. Transcriptional networks involving forkhead box L2 (foxl2) (transcript involved in ovarian follicle development) were increased in expression in the testis. Noteworthy was that a gene network associated to granulosa cell development was increased over 100%, suggesting that this transcriptome network may be important for monitoring estrogenic exposures. Other cell processes rapidly downregulated by EE2 at the transcript level included glucose homeostasis, response to heavy metal, amino acid catabolism, and the cyclooxygenase pathway. Conversely, lymphocyte chemotaxis, intermediate filament polymerization, glucocorticoid metabolism, carbohydrate utilization, and anterior/posterior axis specification were increased. These data provide new insight into the transcriptional responses that are perturbed prior to gonadal remodeling and intersex following exposure to estrogens. These data demonstrate that low concentrations of EE2 (1) rapidly suppresses male hormone production, (2) down-regulate molecular networks related to male sex differentiation, and (3) induce transcriptional networks related to granulosa cell development in the adult testis. These responses are hypothesized to be key molecular initiating events that occur prior to the development of the intersex phenotype following estrogenic exposures.

The influence of breeding strategy, reproductive stage, and tissue type on transcript variability in fish.

Characterizing factors that contribute to transcript variability is necessary before molecular endpoints are widely adopted as biomarkers for environmental monitoring programs and risk assessment. Here, we employed a meta-analysis approach to understand how reproductive stage, breeding strategy, and tissue type influence transcript variability in multiple fish species. Transcript abundance from the scientific literature was examined by method of quantification (qPCR or microarray), and the extracted data were used to calculate the coefficient of variation (CoV) for each transcript. Based on qPCR data, variability in the abundance of estrogen receptor 1 and hydroxysteroid dehydrogenase 3b was dependent upon reproductive stage and/or breeding strategy in the female ovaries. The variability of other transcripts in the steroid biosynthesis pathway as well as other steroid receptors did not depend upon sex, breeding strategy, or reproductive stage. Variability estimates were used to determine sample size requirements for detecting specific critical effects in molecular endpoints. It was estimated that only 37.8% of published studies used in the qPCR meta-analysis had sufficient experimental power (0.8) to detect a 2-fold expression difference in a transcript. To build upon these analyses, microarray data were used to measure overall variability of the transcriptome, and it was determined that the vitellogenic reproductive stage had the lowest transcriptomic variability compared to other reproductive stages. This variability was lower in a single-spawning species (largemouth bass) compared to a multiple-spawner (fathead minnow). Following this, a meta-analysis of 777 microarrays for multiple fish species was performed to determine the influence of breeding strategy and tissue type on transcriptomic variability. In this analysis, single-spawning fish showed lower gonadal and hepatic transcriptome variability compared to multiple-spawning species. Thus, these species may be more appropriate for sampling molecular endpoints in monitoring programs. Transcript variability was lowest in the brain, followed by the gonads and liver, which may reflect fewer morphological changes relative to these tissues. The results of this study should be used in conjunction with other experimental and sampling recommendations to optimize the use of molecular endpoints in regulatory ecotoxicology and environmental monitoring programs.

Lethal and sublethal effects of phthalate diesters in Silurana tropicalis larvae.

Phthalates are compounds used in polymers to increase their flexibility and are now ubiquitous in the environment as a result of widespread use. Because few studies have focused on the adverse effects of these chemicals in aquatic species, the present study aimed to determine the effects of phthalate diesters in amphibians. Western clawed frog (Silurana tropicalis) tadpoles were acutely exposed to water spiked with monomethyl phthalate (MMP; 1.3-1595.5 mg/L), dimethyl phthalate (DMP; 0.03-924.0 mg/L), or dicyclohexyl phthalate (DCHP; 0.3-99.3 mg/L). Because few studies have addressed the toxicity of these specific phthalates in most organisms, the present study used higher concentrations of these chemicals to determine their toxicity pathways in amphibians and at the same time investigate a suite of genes known to be altered by the well-studied phthalates. Both DMP and DCHP increased larval mortality (9.1-924.0 mg/L DMP and 4.1-99.3 mg/L DCHP), increased frequency of malformations in tadpoles (0.1-34.1 mg/L DMP and 4.1-19.0 mg/L DCHP), and up-regulated cellular stress-related messenger-RNA (mRNA) levels (4.1 mg/L DCHP). To characterize the molecular toxicity pathway of these phthalates in tadpoles, transcriptome analysis was conducted using a custom microarray. Parametric analysis of gene set enrichment revealed important changes in the expression of genes related to drug metabolism and transport, liver metabolism, xenobiotic clearance, and xenobiotic metabolism after DMP and DCHP treatments, although these responses were less pronounced with MMP (the metabolite of DMP). The present study is one of the few studies that demonstrated complementarity between gene expression analysis and organismal effects. Environ Toxicol Chem 2016;35:2511-2522. © 2016 SETAC.

Transcriptional networks associated with 5-alpha-dihydrotestosterone in the fathead minnow (Pimephales promelas) ovary.

Androgens play a significant role in regulating oogenesis in teleost fishes. The androgen dihydrotestosterone (DHT) is a potent non-aromatizable androgen involved in sexual differentiation in mammals; however, its actions are not well understood in teleost fish. To better characterize the physiological role of DHT in the fathead minnow (FHM) ovary on a temporal scale, in vitro assays for 17ß-estradiol (E2) production were conducted in parallel with microarray analysis. Ovarian explants were incubated at different concentrations of DHT (10(-6), 10(-7), and 10(-8)M DHT) in three separate experiments conducted at 6, 9, and 12h. DHT treatment resulted in a rapid and consistent increase in E2 production from the ovary at all three time points. Therefore, DHT may act to shift the balance of metabolites in the steroidogenic pathway within the ovary. Major biological themes affected by DHT in the ovary in one or more of the time points included those related to blood (e.g. vasodilation, blood vessel contraction, clotting), lipids (e.g. lipid storage, cholesterol metabolism, lipid degradation) and reproduction (e.g. hormone and steroid metabolism). Gene networks related to immune responses and calcium signaling were also affected by DHT, suggesting that this androgen may play a role in regulating these processes in the ovary. This study detected no change in mRNA levels of steroidogenic enzymes (cyp19a1, star, 11ßhsd, 17ßhsd, srd5a isoforms), suggesting that the observed increase in E2 production is likely more dependent on the pre-existing gene or protein complement in the ovary rather than the de novo expression of transcripts. This study increases knowledge regarding the roles of DHT and androgens in general in the teleost ovary and identifies molecular signaling pathways that may be associated with increased E2 production.

Transcriptomic Responses During Early Development Following Arsenic Exposure in Western Clawed Frogs, Silurana tropicalis.

Arsenic compounds are widespread environmental contaminants and exposure elicits serious health issues, including early developmental anomalies. Depending on the oxidation state, the intermediates of arsenic metabolism interfere with a range of subcellular events, but the fundamental molecular events that lead to speciation-dependent arsenic toxicity are not fully elucidated. This study therefore assesses the impact of arsenic exposure on early development by measuring speciation and gene expression profiles in the developing Western clawed frog (Silurana tropicalis) larvae following the environmental relevant 0.5 and 1 ppm arsenate exposure. Using HPLC-ICP-MS, arsenate, dimethylarsenic acid, arsenobetaine, arsenocholine, and tetramethylarsonium ion were detected. Microarray and pathway analyses were utilized to characterize the comprehensive transcriptomic responses to arsenic exposure. Clustering analysis of expression data showed distinct gene expression patterns in arsenate treated groups when compared with the control. Pathway enrichment revealed common biological themes enriched in both treatments, including cell signal transduction, cell survival, and developmental pathways. Moreover, the 0.5 ppm exposure led to the enrichment of pathways and biological processes involved in arsenic intake or efflux, as well as histone remodeling. These compensatory responses are hypothesized to be responsible for maintaining an in-body arsenic level comparable to control animals. With no appreciable changes observed in malformation and mortality between control and exposed larvae, this is the first study to suggest that the underlying transcriptomic regulations related to signal transduction, cell survival, developmental pathways, and histone remodeling may contribute to maintaining ongoing development while coping with the potential arsenic toxicity in S. tropicalis during early development.

Transcript variability and physiological correlates in the fathead minnow ovary: Implications for sample size, and experimental power.

Fundamental studies characterizing transcript variability in teleost tissues are needed if molecular endpoints are to be useful for regulatory ecotoxicology. The objectives of this study were to (1) measure transcript variability of steroidogenic enzymes and steroid receptors in the fathead minnow (FHM; Pimephales promelas) ovary to better determine normal variability and the sample sizes needed to detect specific effect sizes and to (2) determine how expression patterns related to higher level endpoints used in some regulatory ecotoxicology programs (e.g. relative gonad size). Estrogen receptor 2b (esr2b) and 5α-reductase a3 (srd5a3) showed high variability in the ovary (CV>1.0) while progesterone receptor (pgr), androgen receptor (ar), and esr2a showed comparatively low variability (CV=~0.5--0.7). Using these estimates, a power analysis revealed that sample sizes for real-time PCR experiments would need to be>20 to detect a 2-fold change for 7 of the transcripts examined; thus many molecular studies conducted in the fish ovary may have insufficient power to detect smaller effects. Two transcripts were correlated to steroid production in the ovary; cyp19a1 levels were positively correlated to in vitro E2 production, while ar levels were negatively correlated to in vitro T production. Thus, these transcripts may be informative molecular surrogates for ovarian steroid production. No transcript investigated showed any correlation to GSI, condition, or body weight/length. Molecular approaches in fish are increasingly used to assess biological impacts of chemical stressors; however additional studies are required that determine how molecular variability relates to higher level biological endpoints.

Understanding the chronic impacts of oil refinery wastewater requires consideration of sediment contributions to toxicity.

Previous studies at an oil refinery in Saint John, New Brunswick, Canada, found a diminished fish community downstream of the effluent outfall that appeared to be associated with periodic low dissolved oxygen concentrations due to episodic discharges of contaminated transport vessel ballast water. This study was initiated after the ballast water was removed from the effluent to further investigate the potential causes of residual effects in the study stream, Little River. We used field caging of fish, laboratory bioassays, and chemical analysis of effluents and sediments from the field site to determine if the effluent or contaminated sediments were affecting the recovery of the fish community in Little River. The field studies suggested that exposed, caged fish were affected, displaying >40 % increases in liver sizes and increased liver detoxification enzyme activity (cytochrome P450 1A, CYP1A); however, similar responses were absent in laboratory exposures that used effluent only. Adding sediments collected from the vicinity of the refinery's outfall to the laboratory bioassays reproduced some of the field responses. Chemical analyses showed high concentrations of PAHs in sediments but low concentrations in the effluent, suggesting that the PAHs in the sediment were contributing more to the impacts than the effluent. Application of effects-based monitoring is suggested as beneficial to identify impacts to fisheries where refinery effluents of this type are involved.