Effects of atrazine on fish, amphibians, and reptiles: update of the analysis based on quantitative weight of evidence.

Quantitative weight of evidence (QWoE) provides a framework and process for evaluating different toxicological studies based on quality and relevance of the results. This framework allows for data from these studies to be combined in separate lines of evidence to address causality, and relevance to environmental risks. In 2014, such a QWoE that examined the body of available company reports and peer reviewed literature regarding the effects of the herbicide atrazine on fish, amphibians, and reptiles was published. Since that time, new studies have been conducted and/or published. One of the advantages of the QWoE framework is that additional information can be added as it becomes available. Thus, these new studies were evaluated in the same manner as previously and the new data incorporated into the existing QWoE. As before, the new updated QWoE was based on the same process of objective scoring of individual studies with respect to the quality of the methods and the relevance of individual responses to the apical endpoints of survival, growth, development, and reproduction. These new data did not identify new responses or indicate any relevant effects of atrazine. The new updated QWoE analysis concluded that atrazine does not adversely affect fish, amphibians, and reptiles, at environmentally relevant concentrations (<100 µg atrazine/L), which is consistent with the previous conclusions. These new studies and data are discussed in this paper and the accompanying supplement information provides detailed and transparent information to support these conclusions.

The Relationship between Organic Loading and Effects on Fish Reproduction for Pulp Mill Effluents across Canada.

This study builds upon the work of a multiagency consortium tasked with determining cost-effective solutions for the effects of pulp mill effluents on fish reproduction. A laboratory fathead minnow egg production test and chemical characterization tools were used to benchmark 81 effluents from 20 mills across Canada, representing the major pulping, bleaching, and effluent treatment technologies. For Kraft and mechanical pulp mills, effluents containing less than 20 mg/L BOD5 were found to have the greatest probability of having no effects. Organic loading, expressed as the total detected solvent-extractable components by gas chromatography/mass spectrometry (GC/MS), also correlated with decreased egg laying. Exceptions were found for specific Kraft, mechanical, and sulfite mills, suggesting yet unidentified causative agents are involved. Recycled fiber mill effluents, tested for the first time, were found to have little potential for reproductive effects despite large variations in BOD5 and GC/MS profiles. Effluent treatment systems across all production types were generally efficient, achieving a combined 82-98% BOD5 removal. Further reductions of final effluent organic loadings toward the target of less than 20 mg/L are recommended and can be realized through biotreatment optimization, the reduction of organic losses associated with production upsets and selecting best available technologies that reduce organic loadings to biotreatment.

Effects of atrazine in fish, amphibians, and reptiles: an analysis based on quantitative weight of evidence.

A quantitative weight of evidence (WoE) approach was developed to evaluate studies used for regulatory purposes, as well as those in the open literature, that report the effects of the herbicide atrazine on fish, amphibians, and reptiles. The methodology for WoE analysis incorporated a detailed assessment of the relevance of the responses observed to apical endpoints directly related to survival, growth, development, and reproduction, as well as the strength and appropriateness of the experimental methods employed. Numerical scores were assigned for strength and relevance. The means of the scores for relevance and strength were then used to summarize and weigh the evidence for atrazine contributing to ecologically significant responses in the organisms of interest. The summary was presented graphically in a two-dimensional graph which showed the distributions of all the reports for a response. Over 1290 individual responses from studies in 31 species of fish, 32 amphibians, and 8 reptiles were evaluated. Overall, the WoE showed that atrazine might affect biomarker-type responses, such as expression of genes and/or associated proteins, concentrations of hormones, and biochemical processes (e.g. induction of detoxification responses), at concentrations sometimes found in the environment. However, these effects were not translated to adverse outcomes in terms of apical endpoints. The WoE approach provided a quantitative, transparent, reproducible, and robust framework that can be used to assist the decision-making process when assessing environmental chemicals. In addition, the process allowed easy identification of uncertainty and inconsistency in observations, and thus clearly identified areas where future investigations can be best directed.

A survey of Canadian mechanical pulp and paper mill effluents: insights concerning the potential to affect fish reproduction.

Building on breakthroughs recently made at kraft mills, a survey of mechanical pulp and paper mill effluents was undertaken to gain insights concerning potential effects on fish reproduction. Effluents from seven Canadian mills were characterized chemically for conventional parameters such as biochemical oxygen demand (BOD) and total suspended solids (TSS). Each sample was further subjected to solvent extraction followed by gas chromatographic separation for the determination of resin/fatty acids and for the estimation of a gas chromatography (GC) profile index. Each mill effluent was assessed for the potential to affect fish reproduction in the laboratory using a five day adult fathead minnow (Pimephales promelas) egg production bioassay with exposures to 100% effluent. The seven effluents were found to have substantial variation both in terms of chemical characterization and effects on fish reproduction. Temporal variations were also noted in effluent quality at mills sampled on different occasions. Similar to what has been observed for kraft mills, a general trend of greater reductions in egg production caused by effluents with greater BOD concentrations and GC profile indices was noted. Effluents with BOD > 25 mg/L and GC Profile indices >5.0 caused a complete cessation of egg production. At the same time, about half of the total effluents sampled had BOD < 25 mg/L and GC profile indices <5.0 and caused no significant effects on egg production, suggesting these values may be useful as effluent quality targets for mechanical pulp and paper mills. However, 3 out of 14 effluents sampled had BOD < 25 mg/L and GC profile indices <5.0 and caused significant reductions in egg production. The reason(s) for reproductive effects caused by such effluents is presently unclear. The effluent quality parameters considered in this study may require further refinement to address their utility in predicting the adverse reproductive effects induced by effluents from mechanical pulp and paper mills.

Effluent monitoring at a bleached kraft mill: directions for best management practices for eliminating effects on fish reproduction.

A long-term monitoring study was conducted on effluents from a bleached kraft pulp and paper mill located in Eastern Canada. The study was designed to gain insights into temporal effluent variability with respect to fish reproduction as it related to production upsets, mill restarts and conditions affecting biological treatment performance. Final effluent quality was monitored between February 2007 and May 2009 using biochemical and chemical oxygen demand, total suspended solids, resin and fatty acids, a gas chromatographic profiling index, and the presence of methyl substituted 2-cyclopentenones. Selected effluent samples were evaluated for effects on fish reproduction (egg production) using a shortened version of the adult fathead minnow reproductive test. The events relating to negative effects on fish reproduction were upsets of the pulping liquor recovery system resulting in black liquor losses, operational upsets of the hardwood line resulting in the loss of oxygen delignification filtrates, and conditions that reduced the performance of biological treatment (e.g., mill shutdown and low ambient temperatures). The reductions in egg production observed in fathead minnow were associated with biochemical oxygen demand values > 20 mg/L, GC profiling indices > 1.2 and the presence of methyl-substituted 2-cyclopentenones at concentrations > 100 µg/L. This study demonstrated the importance of both in-plant measures for controlling the loss of organics as well as the optimum operation of biological effluent treatment for eliminating effluent-related effects on fish reproduction (egg production) in the laboratory.

Regulation of prostaglandin synthesis in ovaries of sexually-mature zebrafish (Danio rerio).

This study investigates the regulation of prostaglandin (PG) synthesis in the ovaries of sexually-mature zebrafish (Danio rerio). We examined the ovarian expression of genes within the arachidonic acid (AA) pathway, and the ovarian levels of 17alpha,20beta-dihydroxy-4-pregnen-3-one (17alpha,20beta-P), 17beta-estradiol (E(2)), and PGF(2alpha) in spawning and nonspawning fish during the ovulatory cycle. Real-time RT-PCR analysis revealed that the expression levels of cytosolic phospholipase A(2) (cpla2) and cyclooxygenases (COX)-2 (ptgs2) in ovarian fragments and in isolated full-grown follicles of spawning fish were highest at 6:00 when ovulation was expected to occur. In nonspawning fish, cpla2 expression levels declined over time while ptgs2 expression displayed the same temporal pattern as in spawning fish. Elevated levels of 17alpha,20beta-P in the spawning fish occurred at 3:30, but there were no changes in the nonspawning fish. In other studies conducted to investigate the hormonal regulation of AA pathway genes, fish exposed via the water for 24 or 96 hr to 17alpha,20beta-P or E(2) exhibited reduced ovarian expression levels of COX-1 (ptgs1) and PG E synthase-2 (ptgsl), and E(2) reduced the expression of cpla2. Injection of human chorionic gonadotropin (hCG) (100 IU) led to increased expression levels of cpla2 and ptgs2 at 2 and 18 hr post-treatment, but consistently reduced ptgs1 and ptgsl expression. In these fish, ovarian levels of 17alpha,20beta-P were elevated at all time points and PGF(2alpha) levels in the hCG-treated group were significantly higher than the control fish at 18 hr. Collectively, these in vivo results suggest that gonadotropins and steroids are involved in the regulation of the AA pathway in ovarian follicles of zebrafish.

Effects of atrazine on fish, amphibians, and aquatic reptiles: a critical review.

The herbicide atrazine is widely used in agriculture for the production of corn and other crops. Because of its physical and chemical properties, atrazine is found in small concentrations in surface waters--habitats for some species. A number of reports on the effects of atrazine on aquatic vertebrates, mostly amphibians, have been published, yet there is inconsistency in the effects reported, and inconsistency between studies in different laboratories. We have brought the results and conclusions of all of the relevant laboratory and field studies together in this critical review and assessed causality using procedures for the identification of causative agents of disease and ecoepidemiology derived from Koch's postulates and the Bradford-Hill guidelines. Based on a weight of evidence analysis of all of the data, the central theory that environmentally relevant concentrations of atrazine affect reproduction and/or reproductive development in fish, amphibians, and reptiles is not supported by the vast majority of observations. The same conclusions also hold for the supporting theories such as induction of aromatase, the enzyme that converts testosterone to estradiol. For other responses, such as immune function, stress endocrinology, parasitism, or population-level effects, there are no indications of effects or there is such a paucity of good data that definitive conclusions cannot be made.

Reproduction, larval growth, and reproductive development in African clawed frogs (Xenopus laevis) exposed to atrazine.

Reproductive success and development of F2 offspring from F1 adult African clawed frogs (Xenopus laevis) exposed to atrazine throughout larval development and as sexually mature adults was examined. Larval X. laevis were exposed to one of four nominal concentrations of atrazine (0, 1, 10, 25 microg atrazine/l) beginning 96 hr after fertilization and continuing through two years post-metamorphosis. Clutch size and survival of offspring were used as measurement endpoints to gauge reproductive success of the F1 frogs. Larval survivorship and time to metamorphosis were used to gauge developmental success of the F2 offspring from atrazine-exposed frogs. Testes in F1 and F2 frogs were examined for incidence of anomalies, such as testicular ovarian follicles, and sex ratios in F2 offspring were investigated to determine if exposure to atrazine caused trans-generational effects (effects on F2 individuals due to exposure of F1 individuals). There were no effects of any of the studied concentrations of atrazine on clutch size of F1 frogs. There were also no effects on hatching success or time to metamorphosis. Sex ratios did not differ between F2 offspring among treatments. There was no evidence to suggest a transgenerational effect of atrazine on spawning success or reproductive development of X. laevis. This is consistent with the presence of robust populations of X. laevis in areas where they are exposed to atrazine that has been used for several decades for weed control in production of corn. Our observations also are consistent with the results of most other studies of frogs where no effects were found to be associated with exposure to atrazine. Our data do not support the hypothesis that atrazine significantly affects reproductive fitness and development of frogs.

Altered reproduction in fish exposed to pulp and paper mill effluents: roles of individual compounds and mill operating conditions.

For the last 20 years, studies conducted in North America, Scandinavia, and New Zealand have shown that pulp and paper mill effluents affect fish reproduction. Despite the level of effort applied, few leads are available regarding the factors responsible. Effluents affect reproduction in multiple fish species, as evidenced by decreased gonad size, decreased circulating and gonadal production of reproductive steroids, altered expression of secondary sex characteristics, and decreased egg production. Several studies also have shown that effluent constituents are capable of accumulating in fish and binding to sex steroid receptors/ binding proteins. Studies aimed at isolating biologically active substances within the pulping and papermaking process have provided clues about their source, and work has progressed in identifying opportunities for in-mill treatment technologies. Following comparisons of manufacturing processes and fish responses before and after process changes, it can be concluded that effluent from all types of mill processes are capable of affecting fish reproduction and that any improvements could not be attributed to a specific process modification (because mills normally performed multiple modifications simultaneously). Improved reproductive performance in fish generally was associated with reduced use of molecular chlorine, improved condensate handling, and liquor spill control. Effluent biotreatment has been effective in reducing some effects, but biotreated effluents also have shown no difference or an exacerbation of effects. The role of biotreatment in relation to effects on fish reproduction remains unclear and needs to be resolved.

Assessing recovery of in vitro steroid production in male rainbow darter (Etheostoma caeruleum) in response to municipal wastewater treatment plant infrastructure changes.

The present study examined in vitro 11-ketotestosterone and testosterone production by the testes of rainbow darter (Etheostoma caeruleum) collected from selected reference sites and downstream of 2 municipal wastewater treatment plants (MWWTPs; Waterloo and Kitchener) on the central Grand River (Ontario, Canada), over a 6-yr period (2011-2016). The main objective was to investigate if infrastructure upgrades at the Kitchener MWWTP in 2012 resulted in a recovery of this response in the post-upgrade period (2013-2016). Two supporting studies showed that the fall season is appropriate for measuring in vitro sex steroid production because it provides stable detection of steroid patterns, and that the sample handling practiced in the present study did not introduce a bias. Infrastructure upgrades of the Kitchener MWWTP resulted in significant reductions in ammonia and estrogenicity. After the upgrades, 11-ketotestosterone production by MWWTP-exposed fish increased in 2013 and it continued to recover throughout the study period of 2014 through 2016, returning to levels measured in reference fish. Testosterone production was less sensitive and it lacked consistency. The Waterloo MWWTP underwent some minor upgrades but the level of ammonia and estrogenicity remained variable over time. The production of 11-ketotestosterone and testosterone in rainbow darter below the Waterloo MWWTP was variable and without a clear recovery pattern over the course of the present study. The results of the present study demonstrated that measuring production of sex steroids (especially 11-ketotestosterone) over multiple years can be relevant for assessing responses in fish to environmental changes such as those resulting from major infrastructure upgrades. Environ Toxicol Chem 2018;37:501-514. © 2017 SETAC.

Population impacts in white sucker (Catostomus commersonii) exposed to oil sands-derived contaminants in the Athabasca River.

Biological and chemical endpoints were measured in white sucker collected downstream of Athabasca oil sands developments (AB, Canada) and compared with those at Calling Lake (AB, Canada), a reference location upstream of the Athabasca oil sands deposit. Naphthenic acid concentrations were also measured at 14 sites in the Athabasca River watershed. Concentrations of naphthenic acids were elevated in tributaries adjacent to oil sands mining developments. Tributary naphthenic acid profiles were more similar to aged oil sands process water than samples from the Athabasca River, suggesting an influence of tailings in the tributaries. White sucker showed higher energy storage in the Athabasca River as indicated by significantly higher condition and liver size. White sucker were not investing that energy into reproductive effort as measured by gonad size and fecundity, which were significantly reduced relative to the reference location. White sucker showed increased exposure to polycyclic aromatic hydrocarbons as indicated by hepatic cytochrome P4501A (CYP1A) activity and fluorescent bile metabolites, as well as higher concentrations of naphthenic acids in bile. Cadmium, copper, nickel, and selenium were also elevated in white sucker liver tissue compared with the reference location. Based on the exposure profile and response pattern observed, effects on energy storage and utilization in white sucker from the Athabasca River most likely resulted from exposure to polycyclic aromatic hydrocarbons derived from petrogenic and pyrolytic sources. Environ Toxicol Chem 2017;36:2058-2067. © 2017 SETAC.

Oxidative stress and sex steroid levels in fish following short-term exposure to pulp-mill effluents.

This study investigates the relationship between oxidative stress and reproductive dysfunction in wild white sucker (Catostomus commersoni) with short-term exposures to pulp-mill effluent. Hepatic oxidative damage, as quantified using 2-thiobarbituric acid-reactive substances (TBARS), was often increased with effluent exposure within 4-8 d, but responses varied by species, sex, and effluent. Fatty acyl-coenzyme A (CoA) oxidase (FAO) and ethoxyresorufin O-deethylase (EROD) activities were also significantly induced between 4 and 8 d of exposure. There were marked species differences in oxidative stress, as TBARS, FAO, and EROD responses in white sucker differed dramatically from those of longnose sucker (Catostomus catostomus) exposed under identical conditions. Exposure for 8 d to pulp-mill effluent delayed ovulation in white sucker, and these delays were independent of changes in circulating testosterone and 17alpha,20beta-dihydroxy-4-pregnen-3-one titers. Evaluations of the effects of pulp mill effluent on in vivo plasma steroid levels and in vitro steroidogenic capacities were compromised due to caging stress. In vivo preexposure to pulp-mill effluent did not reduce in vitro ovarian follicle steroidogenic capacities when exposed to additional reactive oxygen species (ROS) generators. Endocrine and oxidative stress parameters may be interrelated, as the in vivo administration of ferric nitrilotriacetate (Fe(3+)NTA) significantly reduced circulating sex steroids. Administration of a superactive GnRH analog containing a dopamine inhibitor significantly increased TBARS within 24 h, indicating endocrine status is capable of modifying oxidative stress responses. This study provides new knowledge regarding the onset of oxidative stress and changes in reproductive endpoints in fish following pulp-mill effluent exposure.

Morphological abnormalities during early-life development of the estuarine mummichog, Fundulus heteroclitus, as an indicator of androgenic and anti-androgenic endocrine disruption.

We tested the hypothesis that gross morphological abnormalities are a sensitive indicator of exposure to waterborne androgenic and anti-androgenic compounds during embryonic, larval and juvenile stages of development in the common estuarine killifish, the mummichog (Fundulus heteroclitus; Pisces: Cyprinodontidae). Static exposures with daily renewal were carried out with 10-100,000 ng/L of the androgen agonist, 17alpha-methyltestosterone (MT), or the androgen antagonist, cyproterone acetate (CA), for 60 days post-fertilization (PF) in duplicate exposures. Measured concentrations were 78.4-155.8% of nominal concentrations for MT and 13.5-168.1% for CA. No dose-related or consistent effects of MT or CA were observed before hatch. In 60 days PF juveniles, incidence of skeletal abnormalities (scoliosis, lordosis, head, facial and fin), soft tissue abnormality (anal swelling) and hemorrhaging were significantly increased by MT but only at high concentrations (> or =1000 ng/L). The 10,000 and 100,000 ng/L concentrations of MT produced a wider range of abnormalities than 1000ng/L. Over 90% of fish exposed to 10,000 or 100,000 ng/L were abnormal with an average of over 3.5 abnormalities per fish. CA did not increase the incidence of any type of abnormality. Survival of juveniles to the end of the exposure was reduced by MT at concentrations of 1000 ng/L and greater in the first experiment and at concentrations of 10,000 ng/L and greater in the second experiment. Juvenile length was reduced by high concentrations of MT (> or =10,000 ng/L) in the first experiment and by most concentrations in the second experiment. We conclude that morphological abnormalities in early-life stages of mummichogs are not a sensitive indicator of exposure to androgenic or anti-androgenic waterborne EDSs at environmentally relevant concentrations.

Short-term lab exposures of immature rainbow trout (Oncorhynchus mykiss) to sulfite and kraft pulp-mill effluents: effects on oxidative stress and circulating sex steroids.

This study investigates the temporal onset of reactive oxygen species (ROS) damage and changes in circulating sex steroids in immature rainbow trout exposed over 21 d to two pulp-mill effluents. Exposure to effluent from a bleached sulfite mill produced increases in 2-thiobarbituric acid reactive substances (TBARS), ethoxyresorufin O-deethylase (EROD) activity, hepatic free iron, and significant depressions in hepatic ascorbic acid. Impairments in pregnenolone production relative to cholesterol availability suggest an effect of sulfite-mill effluent early in the steroidogenic pathway. Induction of vitellogenin in immature fish exposed to effluent from this mill, relative to waterborne 17 beta-estradiol treatments, indicated sulfite-mill effluent contained constituents capable of binding the estrogen receptor. Exposure to a kraft-mill effluent also elevated hepatic TBARS, tissue normalized fatty acyl-coenzyme A oxidase (FAO) activity, and hepatic free iron while producing commensurate declines in hepatic ascorbic acid. Plasma testosterone, 11-ketotestosterone, and 17 beta-estradiol were elevated with kraft-mill effluent exposure, but no changes in vitellogenin induction were observed. In summary, effluent from bleached sulfite and bleached kraft mills yielded similar oxidative stress responses, but marked differences were observed in the endocrine-disrupting potential of each effluent.

Short-term exposures of fish to perfluorooctane sulfonate: acute effects on fatty acyl-coa oxidase activity, oxidative stress, and circulating sex steroids.

This study investigated the effects of exposure to waterborne perfluorooctane sulfonate (PFOS) on oxidative stress and reproductive endpoints in fish. Exposures utilized species commonly used in toxicological testing, including the fathead minnow (Pimephales promelas) and rainbow trout (Oncorhynchus mykiss), as well as relatively insensitive taxa such as creek chub (Semotilus atromaculatus), spottail shiner (Notropis hudsonius), and white sucker (Catostomus commersoni). In all fish species, short-term (14-28 d) exposure to PFOS produced only modest mortality at concentrations consistent with environmental spill scenarios. However, PFOS consistently increased hepatic fatty acyl-CoA oxidase activity and increased oxidative damage, as quantified using the 2-thiobarbituric acid-reactive substances assay. Plasma testosterone, 11-ketotestosterone, and 17beta-estradiol titers were often elevated with PFOS exposure. Vitellogenin, the egg yolk precursor protein, was occasionally altered in the plasma with PFOS exposure, but responses varied with maturity. Oviposition frequency and egg deposition in fathead minnow were not significantly impaired with PFOS exposure, despite a trend toward progressive impairment with increasing exposure concentrations. Although short-term PFOS exposure produced significant impacts on biochemical and reproductive endpoints in fish at concentrations consistent with environmental spills, the impact of long-term exposure to environmentally relevant concentrations of PFOS is unclear.

Assessment of biomarkers for contaminants of emerging concern on aquatic organisms downstream of a municipal wastewater discharge.

Contaminants of emerging concern (CECs), including pharmaceuticals, personal care products and estrogens, are detected in wastewater treatment plant (WWTP) discharges. However, analytical monitoring of wastewater and surface water does not indicate whether CECs are affecting the organisms downstream. In this study, fathead minnows (Pimephales promelas) and freshwater mussels Pyganodon grandis Say, 1829 (synonym: Anodonta grandis Say, 1829) were caged for 4 weeks in the North Saskatchewan River, upstream and downstream of the discharge from the WWTP that serves the Edmonton, AB, Canada. Passive samplers deployed indicated that concentrations of pharmaceuticals, personal care products, an estrogen (estrone) and an androgen (androstenedione) were elevated at sites downstream of the WWTP discharge. Several biomarkers of exposure were significantly altered in the tissues of caged fathead minnows and freshwater mussels relative to the upstream reference sites. Biomarkers altered in fish included induction of CYP3A metabolism, an increase in vitellogenin (Vtg) gene expression in male minnows, elevated ratios of oxidized to total glutathione (i.e. GSSG/TGSH), and an increase in the activity of antioxidant enzymes (i.e. glutathione reductase, glutathione-S-transferase). In mussels, there were no significant changes in biomarkers of oxidative stress and the levels of Vtg-like proteins were reduced, not elevated, indicating a generalized stress response. Immune function was altered in mussels, as indicated by elevated lysosomal activity per hemocyte in P. grandis caged closest to the wastewater discharge. This immune response may be due to exposure to bacterial pathogens in the wastewater. Multivariate analysis indicated a response to the CECs Carbamazepine (CBZ) and Trimethoprim (TPM). Overall, these data indicate that there is a 1 km zone of impact for aquatic organisms downstream of WWTP discharge. However, multiple stressors in municipal wastewater make measurement and interpretation of impact of CECs difficult since water temperature, conductivity and bacteria are also inducing biomarker responses in both fish and mussels.

Oxidative stress and bioindicators of reproductive function in pulp and paper mill effluent exposed white sucker.

This study investigates oxidative stress and bioindicators of reproductive function in wild white sucker (Catostomus commersoni) collected from environments receiving pulp and paper mill effluent discharges in northern Ontario. Samples were collected over an eight-year period adjacent to three pulp and paper mills using a variety of processing and bleaching techniques. Fish collected downstream of pulp and paper mills within the Moose River basin exhibited elevated hepatic and gonadal 2-thiobarbituric acid reactive substances (TBARS), the presence of which is indicative of oxidative stress in these tissues. Within the Jackfish Bay system, exposure to pulp and paper mill effluent did not elevate hepatic or gonadal TBARS. Hepatic cytochrome P4501A activity (CYP1A) and fatty acyl-CoA oxidase (FAO) activities were frequently increased in livers of Moose River basin fish exposed to pulp and paper mill effluent, while lower activities of both enzymes were found within fish from the Jackfish Bay system. This suggests that oxidative stress may be related to CYP1A and FAO activities. Within the Moose River system, increases in measures of oxidative stress (TBARS, FAO) were generally coincident with decreased levels of 17 beta-estradiol; however, testosterone was often lower in Jackfish Bay system fish without any commensurate changes in oxidative stress. The suite of reproductive and oxidative stress parameters measured in this study varied between seasons and mills suggesting responses to effluent are dynamic and effects are complicated by different receiving environments. The relationship between gonad size, gonadal oxidative stress, and circulating plasma steroids remains unclear.

Hormonal, morphological, and physiological responses of yellow perch (Perca flavescens) to chronic environmental metal exposures.

The effects of a chronic environmental exposure to metals on the hormonal, physiological, and reproductive status were assessed in yellow perch (Perca flavescens) sampled in six lakes situated along a contamination gradient of Cd, Zn, Cu, Pb, and Ni in the mining region of Rouyn-Noranda, Québec. Fish were captured in the summer and fall, and sampled before or after a confinement of one hour. Metal concentrations in the kidneys and the interrenal tissues (homologous to mammalian adrenals) were measured to compare tissue-specific metal accumulation. An exposure-related decrease of condition factor, gonadosomatic index (GSI), branchial Na+/K(+)-ATPase activity, plasma thyroxine (T4), triiodothyronine (T3), and 17 beta-estradiol and an impaired capacity to enhance cortisol levels after confinement were observed. Fish from the metal-contaminated lakes possessed gonads at less mature stages and exhibited structural alterations of their gills, interrenal cells, and thyroid follicle epithelium. A comparison of the morphological, biochemical, and physiological endpoints measured in the present study revealed that plasma concentrations of hormones and parameters of gill function were the most affected by metal contamination. The results of this study indicate that lifelong exposures to sublethal concentrations of metals alter the physiological functions of fish and delay reproduction.

Utility of the TBARS assay in detecting oxidative stress in white sucker (Catostomus commersoni) populations exposed to pulp mill effluent.

Recent evidence indicates that contaminant-stimulated free radical production and resulting oxidative damage may be an important mechanism of toxicity in organisms exposed to water-borne contaminants. This study tested the hypothesis that increases in oxidative stress and associated biochemical alterations would be present in white sucker (Catostomus commersoni) living in an environment receiving pulp mill effluent. Consistent increases in liver TBARS and frequent increases in gonadal TBARS were observed immediately downstream of the pulp mill effluent discharge; observable effects were attenuated with distance until they were not significantly different from reference values. Increases detected with the TBARS assay were commensurate with increases detected using the lipid hydroperoxides assay, a mechanistically independent technique. Fish exposed to pulp mill effluent also exhibited significant increases in hepatic free iron and ascorbic acid and a reduced free radical scavenging capacity in the livers of fish downstream of the effluent discharge relative to reference fish. Increases in oxidative stress are not necessarily dependent on increases in lipid substrate or related to reductions in ascorbic acid. TBARS values similar to those observed in fish resident below pulp mill effluent discharges were observed in white sucker 2 h after intraperitoneal injection using 15 mg/kg body weight ferric nitrilotriacetate (Fe(3+) NTA) as a positive control. This study indicates oxidative stress could be a mechanism of toxicity in fish exposed to pulp mill effluent and demonstrates the utility of TBARS in delineating zones of exposure to pulp mill effluent.

Oxidative stress responses in longnose sucker (Catostomus catostomus) exposed to pulp and paper mill and municipal sewage effluents.

While recent evidence indicates that the generation of reactive oxygen species (ROS) and associated oxidative damage are frequently observed in fish populations with exposure to pulp and paper mill effluents, the potential for ROS generation from municipal sewage effluents has not been addressed. This study investigates the utility of measures of oxidative stress in delineating the effects of both pulp and paper mill and municipal sewage discharges. Longnose sucker (Catostomus catostomus) were collected below three pulp and paper mill and two municipal sewage effluent discharges over a 3-year period within the Wapiti and Athabasca River systems in northern Alberta. Biochemical responses in longnose sucker varied between the two rivers systems, with more pronounced changes occurring within the Wapiti River. Of the suite of biochemical parameters examined, fatty acyl-CoA oxidase (FAO) activity was the most sensitive indicator of pulp and paper mill exposure, but was only infrequently induced with exposure to municipal sewage effluent. Hepatic and gonadal 2-thiobarbituric acid reactive substances, lipid hydroperoxides, and hepatic free iron were less consistently elevated with exposure to pulp and paper mill effluent than FAO activity, and were also only infrequently altered with sewage effluent exposure. Hepatic ascorbic acid, liver somatic index, and condition factor were consistently altered with exposure to both sewage and pulp and paper mill effluents. While specific biochemical and organismal responses varied with effluents and time, the collective suite of oxidative stress endpoints proved to be useful tools in identifying relative influences of municipal sewage and pulp and paper mill effluent on fish populations in adjacent receiving waters.