Brown bullhead at the St. Lawrence River (Cornwall) Area of Concern: health and endocrine status in the context of tissue concentrations of PCBs and mercury.

The St. Lawrence River, at Cornwall Ontario, has accumulated sediment contaminants, mainly mercury (Hg) and polychlorinated biphenyls (PCBs), from industrial point sources over many years. Although those sources are past, the river at Cornwall remains an Area of Concern (AOC). Because of remediation and other changes in the AOC, improved knowledge of contaminants in wild-fish and their putative links to health effects could help decision makers to better assess the AOC's state. Thus, we compared tissue concentrations of Hg, PCBs, morphometric measures of health, and biomarkers of exposure, metabolic-, and reproductive health in native brown bullhead (Ameiurus nebulosus) from the AOC to those of upstream reference fish. Linear discriminant analysis separated the adult fish of both sexes among upstream and downstream sites without misclassification. Burdens of total-Hg (all sites) and PCB toxic equivalents (downstream sites) exceeded the guidance for the protection of wildlife consumers. There were subtle effects of site on physiological variables, particularly in female fish. Total-Hg in tissue correlated negatively to plasma testosterone and 17ß-estradiol in female fish at Cornwall: moreover, concentrations of both hormones were lower within the AOC compared to reference site fish. A similar effect on vitellogenin, which was uncorrelated to E2/T at the downstream sites, indicated the potential for reproductive effects. Downstream fish also had altered thyroidal status (T3, TSH, and ratio of thyroid epithelial cell area to colloid area). Despite spatial and temporal variability of the endocrine-related responses, these subtle effects on fish health within the AOC warrant further study.

Tissue contaminants and wild fish health in the St. Clair River Area of Concern - Part 2: Spatial trends and temporal declines in organics.

We explored tissue concentrations of polychlorinated biphenyls (PCBs), chlorinated pesticides, and relevant organochlorines and fish health in the following adult wild fish in the St. Clair River Area of Concern (Ontario, Canada): shorthead redhorse (Moxostoma macrolepidotum), yellow perch (Perca flavescens), and emerald shiner (Notropis atherinoides). We collected adult fish from sites within the river's industrial zone (Stag Island), a downstream site adjacent to Walpole Island (Chenal Écarte), and an upstream reference site in Lake Huron in 2002/2003 and 2014. We tested for trends in tissue concentrations of organic contaminants across sites and over time; we assessed the potential effects of contaminants on morphological indicators of fish health across sites by year. Over the 12-year period, the tissue concentrations of most PCBs declined at the river sites, except for some non-legacy PCBs (PCB11 and 185), which increased in yellow perch at Stag Island, a new observation for fish in the St. Clair River AOC. There was little difference between the concentrations of calculated toxic equivalents (TEQs) of the Lake Huron and the St. Clair River fish in 2014, except for emerald shiners from Stag Island which had elevated ΣPCB and TEQs. Each fish species at all sites exceeded the Canadian tissue residue guideline for PCBs for the protection of mammalian wildlife consumers of aquatic biota, but fish-derived TEQs indicated little potential health risk to fish. Over time, hexachlorobutadiene and hexachlorobenzene concentrations increased in some fish at Stag Island by about 8- and 4-fold, respectively, whereas they decreased at other sampling locations. Principal Component Analysis followed by Linear Discriminant Analysis of the 2014 SHRH data suggested that although the fish separated by site, tissue concentrations of PCB and organochlorine contaminants did not have consistent relationships to the morphological health indicators, including egg production in females, which implied the absence of causative relationships.

Altered expression of metabolites and proteins in wild and caged fish exposed to wastewater effluents in situ.

Population growth has led to increased global discharges of wastewater. Contaminants that are not fully removed during wastewater treatment, such as pharmaceuticals and personal care products (PPCPs), may negatively affect aquatic ecosystems. PPCPs can bioaccumulate causing adverse health effects and behavioural changes in exposed fish. To assess the impact of PPCPs on wild fish, and to assess whether caged fish could be used as a surrogate for resident wild fish in future monitoring, we caged goldfish in a marsh affected by discharges of wastewater effluents (Cootes Paradise, Lake Ontario, Canada). We collected plasma from resident wild goldfish, and from goldfish that we caged in the marsh for three weeks. We analyzed the plasma proteome and metabolome of both wild and caged fish. We also compared proteomic and metabolic responses in caged and wild fish from the marsh to fish caged at a reference site (Jordan Harbour Conservation Area). We identified significant changes in expression of over 250 molecules that were related to liver necrosis, accumulation and synthesis of lipids, synthesis of cyclic AMP, and the quantity of intracellular calcium in fish from the wastewater affected marsh. Our results suggest that PPCPs could be affecting the health of wild fish populations.

Reduced anxiety is associated with the accumulation of six serotonin reuptake inhibitors in wastewater treatment effluent exposed goldfish Carassius auratus.

Pharmaceuticals and personal care products (PPCPs) have been found in wastewater treatment plant (WWTP) effluents and their recipient watersheds. To assess the potential of WWTP effluents to alter fish behaviour, we caged male goldfish (Carassius auratus) for 21-days at three sites along a contamination gradient downstream from a WWTP which discharges into Cootes Paradise Marsh, on the western tip of Lake Ontario. We also included a fourth caging site as an external reference site within Lake Ontario at the Jordan Harbour Conservation Area. We then measured concentrations of PPCPs and monoamine neurotransmitters in caged goldfish plasma, and conducted behavioural assays measuring activity, startle response, and feeding. We detected fifteen different PPCPs in goldfish plasma including six serotonin reuptake inhibitors (amitriptyline, citalopram, fluoxetine/norfluoxetine, sertraline, venlafaxine, and diphenhydramine). Plasma concentrations of serotonin were significantly greater in plasma of fish caged closer to the WWTP effluent outfall site. The fish caged near and downstream of the WWTP effluent were bolder, more exploratory, and more active overall than fish caged at the reference site. Taken together, our results suggest that fish downstream of WWTPs are accumulating PPCPs at levels sufficient to alter neurotransmitter concentrations and to also impair ecologically-relevant behaviours.

Wild fish from the Bay of Quinte Area of Concern contain elevated tissue concentrations of PCBs and exhibit evidence of endocrine-related health effects.

The Bay of Quinte (BOQ) is an Area of Concern listed under the Great Lakes Water Quality Agreement. The presence of dioxins and dioxin-like PCBs in fish in the BOQ AOC has led to restrictions on fish consumption by humans, which is a beneficial use impairment. Adult yellow perch (Perca flavescens) and brown bullhead (Ameiurus nebulosus) were sampled from Trenton, Belleville, and Deseronto (reference site) in the BOQ. A suite of hormone assays and various measures of exposure and/or sublethal health effects were used to assess the health status of fish of both species and sex. Condition factor, hepatosomatic index, ethoxyresorufin-O-deethylase activity, circulating steroid and thyroid hormones, thyroid activation, oocyte size distribution, spermatogenic cell stages, and plasma vitellogenin were among the endpoints that were significantly (p < 0.05) affected by location. Many of those effects corresponded with significantly (p < 0.05) greater tissue concentrations of polychlorinated biphenyls (PCBs) at Belleville and Trenton. Hepatic extracts from brown bullhead sampled from Trenton had significantly (p < 0.05) greater binding activity to the androgen receptor and sex steroid binding protein. Taken together, these data and preliminary data from a concomitant study suggest that PCBs are likely being hydroxylated in vivo, resulting in enhanced bioactivity at endocrine receptors and measurable health responses. The present study supports the growing body of evidence that PCBs and their metabolites can affect fish thyroid and steroid hormone systems.

Interaction of Galaxolide® with the human and trout estrogen receptor-α.

Synthetic musks have been detected in sewage effluents, surface waters, and fish tissues where the polycyclic musk compound, HHCB (Galaxolide®) is the dominant compound in those matrices. In the present study, the Galaxolide® formulation was tested in the yeast estrogenicity screening (YES) assay, and also tested in in vitro and in vivo teleost systems to determine whether it interacts with the estrogen receptor as either an agonist or antagonist. In those tests, Galaxolide® did not act as an estrogen agonist, however there was strong evidence of antagonistic activity as Galaxolide® inhibited the estrogenic activity of 17ß-estradiol (E2). In the YES assay based on a recombinant strain of yeast containing the human estrogen receptor (i.e. hERα), Galaxolide® inhibited the effects of E2 in a dose-dependent manner (IC50=1.63×10(-5)M). In a luciferase reporter gene assay based on the rainbow trout estrogen receptor (i.e. rtER) transfected into a rainbow trout gonadal (RTG-2) cell line, the IC50 for the antagonistic effect of Galaxolide® was 2.79×10(-9)M. In an in vivo assay based on modulation of vitellogenin in rainbow trout, Galaxolide® i.p. injected into trout at a dose of 3.64mg/kg caused inhibition of E2-induced vitellogenin production. That dose is within the range of concentrations of Galaxolide® that have been detected in tissues of fish from contaminated locations.

The effect of creosote on vitellogenin production in rainbow trout (Oncorhynchus mykiss).

As part of a broader investigation into the effects of creosote treatments on the aquatic biota in pond microcosms, we examined the possible implications for vitellogenin (Vtg) production in Oncorhynchus mykiss [rainbow trout (RT)]. Vtg is the precursor of egg yolk protein and has emerged as a useful biomarker of exposure to estrogenic substances. Our a priori intent was to assess the ability of the creosote treatments (nominal cresoste concentrations were 0, 3, and 10 microl/L immediately after the last subsurface addition) to induce estrogenic responses in RT. The data showed no evidence of an estrogenic response in the treated fish. During the course of the experiment, however, the fish matured and began to produce Vtg, probably in response to endogenous estrogen. A posteriori analysis of the Vtg data from the maturing fish showed that after 28 days, the plasma Vtg concentrations were about 15-fold lower in fish from the creosote-treated microcosms compared with fish from the reference microcosm. Although the experiment design does not permit mechanistic insights, our observation suggests that exposure of female fish to PAH mixtures such as creosote can impair the production of Vtg with possible health implications for embryos and larvae.

Ability of fractionated petroleum refinery effluent to elicit cyto- and photocytotoxic responses and to induce 7-ethoxyresorufin-O-deethylase activity in fish cell lines.

The ability of fractionated petroleum refinery effluent to cause cellular responses in fish cell lines was evaluated. The cellular responses, which included direct and indirect cytotoxicity, photocytotoxicity and induction of 7-ethoxyresorufin-O-deethylase (EROD) activity, may potentially be linked to sublethal effects observed in effluent-exposed fish and fish larvae. In order to be able to quantify cellular responses rapidly, microtitre plates were used along with fluorescent probes. For the quantification of cyto- and photocytotoxicity, the fluorescent probes were alamar Blue and carboxyfluorescein diacetate acetoxymethyl ester (CFDA-AM), which monitor metabolic activity and cell membrane integrity, respectively. EROD activity was measured as the rate of conversion by EROD of the substrate 7-ethoxyresorufin to its fluorescent product, resorufin. Effluent from an Ontario refinery was fractionated into aqueous and particulate phase. As well, a solid phase extract (SPE) was used to prepare concentrated effluent for testing in the cell lines. The effluent was able to elicit all of the responses of interest although significant cyto- and photocytotoxicity required effluent equivalent concentrations above 100% effluent and could only be revealed upon exposure of cells to the SPE concentrated effluent. Based on their retention on C18, the cytotoxicants are likely to be non-polar to moderately polar chemicals. The presence of polar compounds affecting cellular metabolism was indicated by the responses of exposed cells to a 90% aqueous phase effluent. In contrast to cyto- and photocytotoxicity, EROD induction occurred at effluent equivalent concentrations well below 100% effluent and was elicited by the SPE and the particulate fraction thereby suggesting that most EROD-inducers were particle-bound. Among other applications, the described techniques could help to determine the source of causative agents of sublethal effects in the refining process.

The fate and impact of oil and oil-dispersant mixtures in freshwater pond ecosystems: introduction.

Oil and oil-dispersant mixtures were added to the surface waters of a series of man-made ponds. The fate of the oil and dispersant (Norman Wells crude and Corexit 9527 respectively) were studied as well as the impact of the added chemicals on the ponds' ecosystems. Elements of the ecosystems studied include bacteria, fungi, phytoplankton, periphyton , proto- and mesozooplankton , zoobenthos and surface insects. In addition a number of water quality parameters were regularly monitored. Comparisons were made between oil-treated and control ponds, as well as oil-dispersant treated and oil and/or control ponds. This paper describes the experimental set up and provides a summary of the findings reported in the following five papers.

The impact of oil and oil-dispersant mixtures on fungi in freshwater ponds.

The impact of oil and oil-dispersant mixtures on the mycoflora of a freshwater ecosystem was assessed using man made ponds. Data indicated that the ponds were well stabilized before pond treatment. Sufficient Norman Wells crude oil to give 100 ppm was added to each treated pond. The dispersant, Corexit 9527, was initially one-fifth of the oil concentration in the oil-dispersant-treated ponds. After pond treatment, an immediate increase occurred in the number of geoaquatic fungi in the oil-treated pond whereas an increase, followed by a sharp decrease, which was in turn followed by a recovery occurred in the oil-dispersant-treated ponds. The fungal enhancement effects, which were slight, were largely short term in duration (7-83 days). No clear cut medium or long term geoaquatic fungal enhancement effects were observed. The pond treatments had no detectable effect on the vertical distribution of fungi in the ponds. No significant treatment effects were observed on the water mould levels in the treated ponds. No obvious treatment effect was observed on the percentage of viable fungi capable of growth on non-degraded or biodegraded oil as a sole carbon source. Fifteen of 39 selected pond water fungi were able to grow on non-degraded Norman Wells oil. Corexit 9527 incorporated into agar plates inhibited the radial growth of the oil utilizing fungal isolates; EC50 values were determined.

The occurrence of Candida Albicans in Lake Ontario bathing beaches.

There are inherent weaknesses associated with currently used bacterial fecal pollution indicator systems. Fecal pollution indicator data would be more meaningful if supplemented with information relating to the occurrence of pathogens in recreational water. Through surveys of four bathing beaches on Lake Ontario, it was established that the opportunistically pathogenic yeast Candida albicans occurs in near shore waters. The beaches surveyed could be differentiated on the basis of bacterial fecal pollution indicator levels and numbers of the pathogens C. albicans and Pseudomonas aeruginosa. The occurrence of C. albicans and P. aeruginosa appeared to be related to elevated fecal pollution indicator levels. Maximum numbers of all parameters were observed in July and August in association with peak bather loads at the beaches. In only one instance does the data suggest that a beach was subjected to human fecal contamination.