Long-Term Studies of Fish Health before and after the Closure of a Bleached Kraft Pulp Mill in Northern Ontario, Canada.

Changes in ecosystems after the removal of stress provide a rich source of information for conservation science. We used a long-term regional data set on the performance of white sucker (Catostomus commersonii) collected before and after the closure of a pulp mill to explore recovery in fish. Physiological indicators, including liver enzymes and plasma steroids, showed some compelling changes after the closure of the mill consistent with reduced exposure to pulp mill effluent but did not unequivocally demonstrate recovery. However, persistent signals in these fish may indicate effects of impoundment or discharge of sewage. We also used quantile regression with environmental covariates and bootstrap iteration to determine if systematic variation remained in relative body weight, liver weight, and gonad weight. In fish formerly exposed to pulp mill effluent, we found evidence of improvements (male gonad weight and liver weight of males and females), degradation (gonad weight of females), and no change (body weight). Although the observed patterns may be associated with closure of the mill, some differences were also found at regional locations, suggesting roles of additional stressors and challenging the clear association of change at the Mattagami River exposure site with the closure of the mill. However, fish captured at this location show responses consistent with regional locations, suggesting no residual impacts and highlights the challenges of identifying changes in fish even after large and known interventions. Environ Toxicol Chem 2021;40:162-176. © 2020 SETAC.

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.

Chronic toxicity of oil sands tailings pond sediments to early life stages of fathead minnow (Pimephales promelas).

In this study fathead minnow (Pimephales promelas) embryo-larval stages were exposed to two oil sands tailings pond sediments which had previously been shown to decrease the survival of embryo-larval larval stages of walleye (Sander vitreus) and northern pike (Esox lucius). Fathead minnow are standard test species and we wanted to compare their sensitivity to the other two species. Fathead minnow larvae were exposed for 20 days (5 days in the egg stage and 15 days in the larval stage) with daily renewal of sediments and waters. Sediments contained polycyclic aromatic hydrocarbons (PAHs) and alkylated PAHs (APAHs). Results from an earlier study showed that Sediment 1 contained 173 µg/g total PAHs + APAHs (97 % alkylated), and sediment 2 contained 401 µg/g total PAHs + APAHs (95 % alkylated). Fathead minnow larvae exposed to oil sands tailings pond sediments had decreased survival, decreased weight, and increased deformities. Fathead minnow survival was unaffected at the embryo stage and at hatch. Most deaths occurred at the larval stages 1-8 days after hatching, showing the importance of exposing the fish for at least a week after hatch. Toxicity was seen at 0.2 g/L of sediment, which was equivalent to the addition of 35 and 80 µg total PAHs + APAHs to 1 L of overlying water for sediment 1 and 2, respectively. When compared to embryo-larval northern pike and walleye results from previous studies, all three species of fish responded more strongly to sediment 2 compared to sediment 1. For effects on lethality, fathead minnow were equally sensitive to pike, but walleye were 5-28 times more sensitive to the lethal effects of the sediments compared to both fathead minnow and pike. The study (and comparisons to our previous studies) shows the difference in sensitivity between a model laboratory species (fathead minnow) and some species of wild fish that are highly relevant to the oil sands area of Alberta.

Long-term effects of an early-life exposure of fathead minnows to sediments containing bitumen. Part I: Survival, deformities, and growth.

The aim of this study was to investigate the long-term effects of a short exposure to natural sediments within the Athabasca oil sand formation to critical stages of embryo-larval development in fathead minnows (Pimephales promelas). Three different sediments were used: Ref sediment from the upper Steepbank River tested at 3 g/L (containing 12.2 ng/g ∑PAHs), and two bitumen-rich sediments tested at 1 and 3 g/L; one from the Ells River (Ells downstream, 6480 ng/g ∑PAHs) and one from the Steepbank River (Stp downstream, 4660 ng/g ∑PAHs). Eggs and larvae were exposed to sediments for 21 days, then transferred to clean water for a 5-month grow-out and recovery period. Larval fish had significantly decreased survival after exposure to 3 g/L sediment from Stp downstream, and decreased growth (length and weight at 16 days post hatch) in Ells and Stp downstream sediments at both 1 and 3 g/L. Decreased tail length was a sensitive endpoint in larval fish exposed to Ells and Stp downstream sediments for 21 days compared to Ref sediment. After the grow-out in clean water, all growth effects from the bitumen-containing sediments recovered, but adult fish from Stp downstream 3 g/L sediment had significant increases in jaw deformities. The study shows the potential for fish to recover from the decreased growth effects caused by sediments containing oil sands-related compounds, but that some effects of the early-life sediment exposure occur later on in adult fish.

Putative Rodlet Cell Neoplasms in the Livers of Two White Suckers (Catostomus commersonii).

Although discovered more than a century ago, piscine rodlet cells (RCs) remain somewhat of a mystery to scientists in terms of their origin and function. Initially described as parasites, and later as potential secretory cells, the prevailing theory is that RCs are leucocyte-like cells that possess pathogen defence capabilities. The current case report involves a novel type of neoplasm discovered in the livers of two adult female white suckers (Catostomus commersonii) that were collected as part of a survey of fish from the St. Mary's River Area of Concern, in which sediment contaminated by polyaromatic hydrocarbons has been associated historically with a high prevalence of liver neoplasms in white suckers. The two tumours in this study were investigated by light microscopy, histochemical staining, immunohistochemical labelling for S100 protein and transmission electron microscopy. The evidence from these investigations suggests that these neoplasms may be derived from de-differentiated RCs or RC precursors. The unanticipated existence of these solid mesenchymal-like tumours may prompt a reassessment of the current dogma regarding the physiological function of RCs.

Meltwater from snow contaminated by oil sands emissions is toxic to larval fish, but not spring river water.

To assess the toxicity of winter-time atmospheric deposition in the oil sands mining area of Northern Alberta, embryo-larval fathead minnow (Pimephales promelas) were exposed to snowmelt samples. Snow was collected in 2011-2014 near (<7km) oil sands open pit mining operations in the Athabasca River watershed and at sites far from (>25km) oil sands mining. Snow was shipped frozen back to the laboratory, melted, and amended with essential ions prior to testing. Fertilized fathead minnow eggs were exposed (<24h post-fertilization to 7-16days post-hatch) to a range of 25%-100% snowmelt. Snow samples far from (25-277km away) surface mining operations and upgrading facilities did not affect larval fathead minnow survival at 100%. Snow samples from sites near surface mining and refining activities (<7km) showed reduced larval minnow survival. There was some variability in the potencies of snow year-to-year from 2011 to 2014, and there were increases in deformities in minnows exposed to snow from 1 site on the Steepbank River. Although exposure to snowmelt from sites near oil sands surface mining operations caused effects in larval fish, spring melt water from these same sites in late March-May of 2010, 2013 and 2014 showed no effects on larval survival when tested at 100%. Snow was analyzed for metals, total naphthenic acid concentrations, parent PAHs and alkylated PAHs. Naphthenic acid concentrations in snow were below those known to affect fish larvae. Concentrations of metals in ion-amended snow were below published water quality guideline concentrations. Compared to other sites, the snowmelt samples collected close to mining and upgrading activities had higher concentrations of PAHs and alkylated PAHs associated with airborne deposition of fugitive dusts from mining and coke piles, and in aerosols and particles from stack emissions. CAPSULE: Snow collected close to oil sands surface mining sites is toxic to larval fathead minnows in the lab; however spring melt water samples from the same sites do not reduce larval fish survival.

Molecular pathways associated with the intersex condition in rainbow darter (Etheostoma caeruleum) following exposures to municipal wastewater in the Grand River basin, ON, Canada. Part B.

Rainbow darter (Etheostoma caeruleum; RBD) is a small benthic fish found in North America. This species is sensitive to sewage effluent, and intersex is found in up to 80% of males in near-field areas in the Grand River, Ontario, Canada. To learn more about the molecular signaling cascades associated with intersex, a developed customized oligonucleotide microarray (4×180 K) using next generation sequencing was developed to characterize the transcriptome in the gonad of male and female RBD. Gene expression profiling was performed in males and females from both a reference site and a polluted site. Males with and without intersex condition from the areas closest to effluent outfalls were compared to males and females from a reference site. Microarray analysis revealed that there was increased mRNA abundance for genes associated with oogenesis in intersex males (i.e. the presence of eggs within the testis), and a decrease in mRNA abundance for genes associated with spermatid development. In females exposed to effluent, cell processes related with hatching and ovulation were down-regulated, and genes involved in immune responses were increased in abundance. In the non-intersex males exposed to effluent, cell processes such as sperm cell adhesion were decreased at the transcript level relative to males from the reference site. Microarray analysis revealed that heat shock proteins (HSP) were significantly increased in non-intersex males exposed to effluent; however, HSPs were not differentially expressed in intersex males exposed to the effluent. Genes involved in sex differentiation (sox9, foxl2 and dmrt1) and reproduction (esr1, esrb, ar, vtg, cyp19a1 and cyp11a) were measured in males, females, and intersex individuals. Consistent with the intersex condition, many transcripts showed an intermediate expression level in intersex males when compared to phenotypic males and females. This study improves our knowledge regarding the molecular pathways that underlie the intersex condition and develops a suite of qPCR bioassays in RBD that are able to discriminate pollutant-exposed males without intersex from those males with intersex. Part A of this study reports on the effects of municipal wastewater effluents (MWWEs) on RBD in the Grand River and demonstrates that there are disruptions in higher level endpoints that include altered steroid levels. Here we develop a new tool for assessing and monitoring the intersex condition in RBD in polluted natural environments and begin to characterize gene networks that are associated with the condition.

Whole organism responses and intersex severity in rainbow darter (Etheostoma caeruleum) following exposures to municipal wastewater in the Grand River basin, ON, Canada. Part A.

Municipal wastewater effluents (MWWEs) contain anthropogenic substances that can exhibit endocrine-disrupting activity. These complex mixtures have been observed to exert adverse effects on fish. Rainbow darter (Etheostoma caeruleum, RBD) is a small benthic fish that is widespread throughout the Grand River, Ontario, Canada, and has been previously shown to be adversely affected by MWWE exposure in this watershed. The objectives of this study were to quantify biological responses in this sentinel species and intersex severity in male fish, in relation to the area of urbanization. It focused on RBD populations adjacent to wastewater outfalls in the Grand River watershed. In May 2011, nine sites across the urban gradient were selected to evaluate the impact of MWWEs. Endpoints for energy storage (i.e. condition factor, k; liversomatic index, LSI) as well as reproductive endpoints (i.e. gonadosomatic index, GSI; gonad development, hormone production), and intersex were assessed in the fish. Rainbow darter showed a high incidence of intersex downstream of the wastewater outfalls, especially below the largest treatment plant outfall at Kitchener (∼85%). We applied an intersex index (score from 0 to 7) that considers the number of eggs within the testis and the stage of maturation of the egg. RBD exposed downstream of the largest wastewater outfall at Kitchener had a score of 3.81±0.37 compared to upstream to the urban areas where there were no intersex males found other than a single individual with a score 1 (average intersex score of site 0.06±0.06). In addition, several fish associated with the Kitchener outfall had macroscopic vitellogenic eggs in the testes (intersex scores 5 and 6). The sub-population of fish located at the wastewater outfall also showed a tendency towards skewed sex ratios (greater proportion of females to male fish) compared to the population at the reference sites. Male fish inhabiting the urbanized area of the Grand River also showed reduced levels of testosterone (T) and 11-ketotestosterone (11KT). Intersex males had the lower levels of 11KT relative to the upstream reference fish but could not be distinguished from normal males collected at the exposed sites. Despite the high levels of intersex at these sites, no relationships were evident among intersex severity and other measured endpoints such as GSI, LSI or in vitro steroid production. The effects observed appear to be associated with urbanization and exposure to treated MWWEs in the watershed. Although intersex incidence and severity was a very good indicator of wastewater exposure, intersex could not be directly linked to other effects in this wild population. The effects of MWWEs on transcriptional changes in adult RBD exposed to the effluents are reported in the corresponding report (Part B).

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.

Molecular signatures in rainbow darter (Etheostoma caeruleum) inhabiting an urbanized river reach receiving wastewater effluents.

Rainbow darter (Etheostoma caeruleum) is a small benthic fish species found in North America that are abundant and distributed throughout the Grand River watershed, ON, Canada. Rainbow darter exhibit intersex in males at sites adjacent to municipal wastewater effluents (MWWE). In October 2010, female and male rainbow darter were collected at 3 sites (1 upstream reference and 2 downstream exposed sites) in the Grand River near the cities of Kitchener and Waterloo. The primary objectives of this research were (1) to characterize the responses of whole organism endpoints (i.e. condition factor (K), liversomatic (LSI) and gonadosomatic index (GSI), histopathology) to MWWEs and (2) to identify transcripts showing altered steady state abundance with exposure to MWWE in fish inhabiting municipal wastewater effluent-exposed areas. Genes measured in this study included vitellogenin, Sry-box containing protein 9 (sox9), forkhead box L2 (foxl2), doublesex and mab-3 related transcription factor 1 (dmrt1), cytochrome P450, family 11, subfamily A, polypeptide 1 (cyp11a) as well as estrogen (esr1, esrb) and androgen (ar) receptors. There were no changes in condition factor; however, there was a significant increase in LSI and a decrease in GSI in fish inhabiting downstream environments when compared with fish collected from the reference site. Males had a high incidence (∼ 70%) of intersex in downstream sites; characterized by the presence of oocytes within the testis. In the gonad, there were sex specific differences for genes related to sexual differentiation; dmrt1 was only expressed in males whereas foxl2 and sox9 were highly expressed in females compared to males. Expression levels of ar and esr1 were higher in females than males. Conversely, esrb was not differentially expressed between sexes or among sites. There were no differences detected for the genes investigated within sex among sites. This study is the first to report on gene expression changes in the rainbow darter, with emphasis on the differences in transcript abundance between sexes and how these changes relate to exposures to MWWEs. Molecular approaches are being investigated for their potential application to field ecotoxicology, and molecular bioassays for relevant, sentinel species in environmental monitoring programs are required to better understand the impact of anthropogenic impacts on species at risk in river systems.

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.

Differential changes in gene expression in rainbow trout hepatocytes exposed to extracts of oil sands process-affected water and the Athabasca River.

The oil sands region of northern Alberta represents the world's largest reserves of bitumen, and the accelerated pace of industrial extraction activity has raised concern about the possible impacts on the Athabasca River and its tributaries. An ecotoxicogenomic study was undertaken on Oncorhynchus mykiss trout hepatocytes exposed to extracts of water samples near the oil sand development area, as well as to oil sands process-affected water (OSPW) extracts using the quantitative reverse transcriptase polymerase chain reaction technique. The expression of the following genes (mRNA) was monitored to track changes in xenobiotic biotransformation (CYP1A1, CYP3A4, glutathione S-transferase, multi-drug resistance transporter), estrogenicity (estrogen receptor and vitellogenin), oxidative stress (superoxide dismutase and metallothionein) and DNA repair activity (DNA ligase). The extent of DNA-aromatic hydrocarbon adducts was also determined in cells by immuno-staining. A comparative analysis of gene expression between the river/lake and OSPW samples revealed that CYP3A4, metallothioneins, DNA ligase and GST genes, were specifically expressed by OSPW. Cells exposed to OSPW, commercial naphthenic acids, and benzo(a)pyrene showed increased polyaromatic hydrocarbon DNA-adducts, as determined by cell immunofluorescence analysis. Other genes were induced by all types of water samples, although the induction potential was stronger in OSPW most of the time (e.g., VTG gene was expressed nearly 15-fold by surface waters from the lake and river samples but increased to a maximum of 31-fold in OSPW). A multivariate discriminant function analysis revealed that the lake and river water samples were well discriminated from the OSPW. The CYP3A4 gene was the most highly expressed gene in cells exposed to OSPW and responded less to the lake or river water in the Athabasca River area. This study identified a suite of gene targets that responded specifically to OSPW extracts, which could serve as toxicogenomic fingerprints of OSPW contamination.

Preliminary fingerprinting of Athabasca oil sands polar organics in environmental samples using electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry.

There is a growing need to develop analytical methods that can distinguish compounds found within industrially derived oil sands process water (OSPW) from those derived from natural weathering of oil sands deposits. This is a difficult challenge as possible leakage beyond tailings pond containments will probably be in the form of mixtures of water-soluble organics that may be similar to those leaching naturally into aquatic environments. We have evaluated the potential of negative ion electrospray ionization high-resolution Fourier transform ion cyclotron resonance mass spectrometry (FTICRMS) for comparing oil sands polar organics from tailing ponds, interceptor wells, groundwater, river and lake surface waters. Principal component analysis was performed for all species observed. which included the O(2) class (often assumed to be monocarbxoylic naphthenic acids) along with a wide range of other species including humic substances in the river and lake samples: O(n) where n=1-16; NO(n) and N(2)O(n) where n=1-13; and O(n)S and O(n)S(2) where n=1-10 and 1-8, respectively. A broad range of species was investigated because classical naphthenic acids can be a small fraction of the 'organics' detected in the polar fraction of OSPW, river water and groundwater. Aquatic toxicity and environmental chemistry are attributed to the total organics (not only the classical naphthenic acids). The distributions of the oil sands polar organics, particularly the sulfur-containing species, O(n)S and O(n)S(2), may have potential for distinguishing sources of OSPW. The ratios of species containing O(n) along with nitrogen-containing species: NO(n), and N(2)O(n), were useful for differentiating organic components derived from OSPW from those found in river and lake waters. Further application of the FTICRMS technique for a diverse range of OSPW of varying ages and composition, as well as the surrounding groundwater wells, may be critical in assessing whether leakage from industrial sources to natural waters is occurring.

Responses of white sucker (Catostomus commersoni) to 20 years of process and waste treatment changes at a bleached kraft pulp mill, and to mill shutdown.

The impacts of pulp mill effluents on white sucker (Catostomus commersoni) have been studied at Jackfish Bay, ON, Canada since the late 1980s. The site receives effluent from a large bleached kraft pulp mill which is the only source of chemical contamination in the area. Many laboratory studies have looked at the toxicological consequences of pulping process changes, but the benefit of these changes have not been looked at in wild fish. Jackfish Bay white sucker showed impacts on sexual maturity, gonad size, secondary sexual characteristics and circulating steroids hormone levels in the early years of the studies, and impacts were evaluated after installation of secondary treatment (1989), major pulping process changes (1995) and after the mill ceased pulp production and effluent release (2006). The addition of secondary treatment resulted in minor improvements in wild fish health, and the conversion to elemental chlorine free (ECF) bleaching at the mill was associated with more recovery in liver and gonad size. While some impacts persist at the exposure site, reproductive parameters showed further improvement during the mill shutdown period demonstrating that biologically active chemicals are still being discharged from modernized mills.

Androgenic effects of a Canadian bleached kraft pulp and paper effluent as assessed using threespine stickleback (Gasterosteus aculeatus).

The presence of unidentified estrogens and androgens in effluents from pulp and paper mills is well documented. However, their role in effluent effects on fish reproduction remains unclear. The objective of this study was to investigate the hypothesis that reproductive impacts of a modern pulp mill effluent are mediated by androgens and/or estrogens in the effluent. Male and female threespine stickleback were exposed to biologically treated Canadian bleached kraft mill effluent under flow-through conditions in the laboratory at 0, 1, 10 and 100% (v/v) dilutions. After 7 and 21 d of exposure, steroidogenesis was assessed using in vitro incubations of gonadal tissue in both males and females. mRNA expression of the estrogen-regulated gene vitellogenin, and the androgen-responsive gene spiggin were assessed using quantitative RT-PCR in the livers of male and posterior kidneys of female stickleback, respectively. Hepatic 7-ethoxyresorufin-O-deethylase (EROD) activity was assessed in both sexes. Effluent extracts were examined for estrogenic and androgenic bioactivity using receptor binding bioassays, and were screened for pulp and paper related extractives and steroidal androgens using GC-MS. This effluent up-regulated spiggin mRNA in the kidney of female stickleback at 10% and 100% (v/v) effluent at 21 d, but not at 7 d of exposure. This change at the mRNA expression of the gene was associated with an increase in cell height in kidney proximal tubule epithelial cells at 100% effluent after both 7 and 21 d. Liver vitellogenin mRNA in male stickleback was not induced at either 7 or 21 d. EROD was induced at 10 and 100% after 21 d of exposure in both sexes, but not after 7 d of exposure. Despite evidence of exposure to androgens, there was no reduction in steroidogenic capacity at any effluent dilution. Effluent extracts were capable of eliciting the displacement of androgens and estrogens from receptors, but androgenic potency was 4-fold greater. A screen of more than 30 androgenic androstane steroids showed no detections. Hence, the androgenic constituents in this particular effluent remain unknown.

Northern Rivers Ecosystem Initiative: distribution and effects of contaminants.

In response to a number of recommendations following the Northern Rivers Basin Studies (NRBS) contaminant program, the Northern Rivers Ecosystem Initiative (NREI) focused considerable attention on assessing contaminants from specific sources including pulp mill effluents, atmospheric transport of mercury and the Alberta oil sands operations. NRBS identified a number of major contaminants of concern including polychlorinated biphenyls, dioxins and furans, mercury and various hydrocarbons. Together, the NRBS and the NREI studies have demonstrated major declines in the levels of dioxins and furans over the last decade as pulp and paper mills have changed their process and treatment strategies in response to new Federal regulations. Polychlorinated biphenyls however, continue to be a concern for the region as their levels have not declined in fish and sediments over the course of these studies. Higher levels in sediments downstream of Grande Prairie and Hinton were identified, but the source of these contaminants remains unknown. Chlorinated pesticides were also investigated, and although toxaphene, DDT and other chlorinated organic pesticides were detected in fish tissue, they were present at very low levels. Studies on the oil sands industry in northern Alberta demonstrated limited impacts on the Athabasca River to date, although studies did identify slight to moderate impacts of natural oil seeps on fish and benthic communities in tributary streams. NREI studies also identified endocrine active compounds in the three pulp and paper mill effluents tested, but endocrine disruptive effects in wild fish were minimal. Municipal sewage effluents also contain endocrine active compounds and it is recommended that monitoring continue around these point sources.

The effects of pulp mill effluent on the sex steroid binding protein in white sucker (Catostomus commersoni) and longnose sucker (C catostomus).

The objective of this study was to characterize the effects of pulp mill effluent on the properties of the sex steroid binding protein (SBP) in the plasma of white sucker (Catostomus commersoni) and longnose sucker (C. catostomus). SBPs which specifically bind estradiol and testosterone with high affinity (k(D) approximately 3 nM) and low capacity (B(max) approximately 73-81 nM) were identified in both species. Subsequent studies determined if the properties of the SBP in white sucker exposed to bleached kraft mill effluent (BKME) at Terrace Bay, ON, and in longnose sucker exposed to BKME at Grande Prairie, AB. differed from appropriate reference fish. There were no effects of BKME exposure on the binding affinity (k(D)) of the SBP in either species, but there was a significant increase in the binding capacity (B(max)) of longnose sucker SBP exposed to BKME. The livers of nai;ve white sucker exposed to effluent at Terrace Bay or a bleached sulfite/groundwood mill in Edmundston, NB, rapidly accumulated compounds of differing hydrophobicity that bound to both the white sucker and goldfish (Carassius auratus) SBP. Conversely, there was reduced accumulation of SBP ligands in the bile of effluent-exposed fish. We have demonstrated that constituents present within pulp mill effluent bind to both the white sucker and goldfish SBP, and that native species residing downstream of pulp mill effluents may experience modifications in the properties of their SBP.

An interlaboratory study on the use of steroid hormones in examining endocrine disruption.

In recent years, there has been an increased use of the measurement of sex steroid hormone levels in the blood of animals exposed to chemicals as an indicator of reproductive impairment or an alteration in endocrine function. Although levels of hormones are often compared among animals and laboratories, there has been no study to examine the between-laboratory variability in actual steroid measurements. Therefore, we initiated a study with white sucker collected from a site receiving pulp mill effluent, previously documented as having reduced steroid levels, to address this issue. Samples of plasma and media from in vitro gonadal incubations were delivered to eight outside laboratories with the ability to measure steroid hormones. These laboratories ranged from well-established fish endocrine laboratories to wildlife toxicology laboratories, which have recently implemented the methods to measure steroid hormones. In this study, we have considered both the absolute measure of steroid content between laboratories as well as the ability to discriminate between reference and exposed populations as important criteria when evaluating the utility of these measures. Of the eight outside laboratories conducting the analyses, six detected identical site differences in circulating levels of testosterone and 17beta-estradiol to those documented by our Burlington laboratory (ON, Canada). However, the absolute value of the steroid hormones measured in the plasma varied significantly (plasma testosterone 0.6-23.1 ng/ml, 17beta-estradiol 77.6-1782.7 pg/ml) with coefficients of variation of 70.4% and 60.3% respectively. Similar results were demonstrated for the measurement of steroid hormones in media following in vitro gonadal incubation. Although there was a fair amount of variability in the absolute measure of steroid hormone levels, we would predict a far greater coherence of interlaboratory results through the sharing of reagents and the use of a common methodology between laboratories. These results are very promising, providing evidence for the inclusion of steroid hormones in monitoring endocrine disruption in wildlife species.