Comparative Effects of Embryonic Metformin Exposure on Wild and Laboratory-Spawned Fathead Minnow (Pimephales promelas) Populations.

Metformin is routinely detected in aquatic ecosystems because of its widespread use as a treatment for Type 2 diabetes. Laboratory studies have shown that exposure to environmentally relevant concentrations of metformin can alter metabolic pathways and impact the growth of early life stage (ELS) fish; however, it is unknown whether these effects occur in wild populations. Herein, we evaluate whether findings from laboratory studies are representative and describe the relative sensitivities of both populations. Duplicate exposures (0, 5, or 50 µg/L metformin) were conducted using wild- and lab-spawned fathead minnow (Pimephales promelas) embryos. Apart from the water source, exposure conditions remained constant. Wild embryos were exposed to previously dosed lake water to account for changes in bioavailability, while reconstituted freshwater was used for the laboratory study. Developmental metformin exposure differentially impacted the growth and morphology of both cohorts, with energy dyshomeostasis and visual effects indicated. The fitness of wild-spawned larvae was impacted to a greater extent relative to lab-spawned fish. Moreover, baseline data reveal important morphological differences between wild- and lab-spawned ELS fatheads that may diminish representativeness of lab studies. Findings also confirm the bioavailability of metformin in naturally occurring systems and suggest current exposure scenarios may be sufficient to negatively impact developing fish.

Lethal and sublethal toxicity of perfluorooctanoic acid (PFOA) in chronic tests with Hyalella azteca (amphipod) and early-life stage tests with Pimephales promelas (fathead minnow).

Perfluoroalkyl substances (PFAS), including perfluorooctanoic acid (PFOA), are industrial chemicals that are of concern due to their environmental presence, persistence, bioaccumulative potential, toxicity, and capacity for long-range transport. Despite a large body of research on environmental exposure, insufficient chronic aquatic toxicity data exist to develop water quality targets for clean-up of federal contaminated sites in Canada. Thus, our objective was to assess the aqueous toxicity of PFOA in chronic tests with Hyalella azteca (amphipod) and early-life stage tests with Pimephales promelas (fathead minnow). Toxicity data were analyzed based on measured PFOA concentrations. Amphipod exposures were 42 d (0.84-97 mg/L) and examined survival, growth, and reproduction. Fathead minnow exposures were 21 d (0.010-76 mg/L), which encompassed hatching (5 d) and larval stages until 16 d post-hatch; endpoints included hatching success, deformities at hatch, and larval survival and growth. Amphipod survival was significantly reduced at 97 mg/L (42-d LC50 = 51 mg/L), but growth and reproduction were more sensitive endpoints (42-d EC50 for both endpoints = 2.3 mg/L). Fathead minnows were less sensitive than amphipods, exhibiting no significant effects in all endpoints with the exception of uninflated swim bladder, which was significantly higher at 76 mg/L (15%) than controls (0%). Maximum concentrations of PFOA are generally in the ng/L range in global surface waters, but can reach the µg/L range in close proximity to major source inputs; therefore, environmental concentrations are well below those that caused toxicity in the current study. Our data will provide valuable information with which to assess the risk of PFOA at contaminated sites, and to set a target for site remediation.

Increased Temperature and Turbulence Alter the Effects of Leachates from Tire Particles on Fathead Minnow (Pimephales promelas).

Tire particles are of concern as a stressor due to the combination of their chemical constituents, high emission rates, and global distribution. Once in the environment, they will interact with physical parameters (e.g., UV, temperature). The interaction of chemical pollution with changing physical environmental parameters is often underestimated in ecotoxicology. Here, we investigate the role of temperature, mechanical stress (i.e., turbulence), UV, and CO2 on the effects of tire leachates on fish. Two samples of tire particles were exposed to four different levels of each physical stressor. A toxicological assessment was performed with fathead minnow embryos assessing five end points (hatching success, time to hatch, length, deformities, and heart rate). Results showed that variations of temperature and mechanical stress affect the toxicological impact of tire leachates. Zn and/or polycyclic aromatic hydrocarbons (pyrene, phenanthrene, chrysene, benzo[a]pyrene, anthracene, naphthalene, fluoranthene, and benzo[ghi]perylene) were identified in the leachate and tire samples by Raman/surface-enhanced Raman spectroscopy and gas chromatography with mass spectroscopy, respectively.

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

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

Development of environmental effects monitoring protocol in Brazil: a fish guide study of three river estuaries.

In Brazil, there are no unified and effective environmental monitoring models for bodies of water. Thus, several methodologies are used that result in information that is often difficult to compare, especially for stakeholders involved in regional water management. Studies in some countries such as Australia, Chile, the USA, and Sweden use the monitoring model implemented in Canada that was developed in the early 1990s. This model was designed to evaluate whether the current environmental regulations are sufficiently protective for pulp and paper effluents and for metal mining effluents. In this study, the Canadian Environmental Effects Monitoring methodologies were applied to three different Brazilian river basins, with the goal of constructing a framework for monitoring environmental effects. Pilot studies were carried out in the estuarine regions of the Benevente, Jucu, and Santa Maria da Vitória river basins, which are important rivers in the state of Espírito Santo. Evaluations included fish health, bioaccumulation studies, benthic invertebrate survey, and physical-chemical analyses of water and sediment. The quality of the environments was evaluated by means of seasonal samplings and comparisons between discharge, upstream, and downstream areas. This study made it possible to identify appropriate fish species to be used in environmental effects monitoring and the environmental quality of the rivers themselves as well as knowledge and policy gaps to implement such monitoring programs in Brazil. The study raises questions about the adequacy of Brazilian environmental legislation concerning tidal rivers.

An evaluation of germline mutations and reproductive impacts in fathead minnow (Pimephales promelas) exposed to contaminated sediment.

Polycyclic aromatic hydrocarbons (PAHs) have become ubiquitous in the aquatic environment. Some PAHs are mutagenic, potentially causing germline mutations in fish that inhabit PAH contaminated waters. We evaluated the effect of exposure to sediment-borne PAHs on reproduction and germline mutation rates in fathead minnows (Pimephales promelas). Exposure to the contaminated sediment had no significant impact on the reproductive endpoints measured in this study. Germline mutations rates at three microsatellite DNA loci were 1.69 × 10-3 in fish exposed to PAH-contaminated sediment and 0.55 × 10-3 in control fish, with zero mutations being observed in fish exposed to sediment from a reference site. While the difference in mutation rates between treatments was not statistically significant for the sample size used (15-19 families per treatment), the observed mutations rates enabled us to estimate the sample size required to detect a significant effect. To our knowledge, this is the first report of germline mutation rates in fathead minnow exposed to an environmental contaminant, providing baseline data for use in the design of future experiments.

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.

Profiling oil sands mixtures from industrial developments and natural groundwaters for source identification.

The objective of this study was to identify chemical components that could distinguish chemical mixtures in oil sands process-affected water (OSPW) that had potentially migrated to groundwater in the oil sands development area of northern Alberta, Canada. In the first part of the study, OSPW samples from two different tailings ponds and a broad range of natural groundwater samples were assessed with historically employed techniques as Level-1 analyses, including geochemistry, total concentrations of naphthenic acids (NAs) and synchronous fluorescence spectroscopy (SFS). While these analyses did not allow for reliable source differentiation, they did identify samples containing significant concentrations of oil sands acid-extractable organics (AEOs). In applying Level-2 profiling analyses using electrospray ionization high resolution mass spectrometry (ESI-HRMS) and comprehensive multidimensional gas chromatography time-of-flight mass spectrometry (GC × GC-TOF/MS) to samples containing appreciable AEO concentrations, differentiation of natural from OSPW sources was apparent through measurements of O2:O4 ion class ratios (ESI-HRMS) and diagnostic ions for two families of suspected monoaromatic acids (GC × GC-TOF/MS). The resemblance between the AEO profiles from OSPW and from 6 groundwater samples adjacent to two tailings ponds implies a common source, supporting the use of these complimentary analyses for source identification. These samples included two of upward flowing groundwater collected <1 m beneath the Athabasca River, suggesting OSPW-affected groundwater is reaching the river system.

A subchronic in situ exposure method for evaluating effects in small-bodied fish at contaminated sites.

In situ fish-caging studies at contaminated sites can provide information that is more realistic compared to traditional laboratory-based studies. However, few methods have been developed for exposing sentinel fish species for subchronic durations, and fewer still are optimized for exposing small-bodied fish while maintaining fish health and growth throughout the caging trial. Those methods typically lack a feeding regimen during the fish caging trial. While that may be acceptable or even appropriate for typical short-term toxicity testing, it does limit the duration of the exposure, and may not be suitable when post-caging trials or observations are necessary. Returning healthy fish to the lab following the in situ exposure would be important, for example, in studies designed to examine long-term or multigenerational effects following an in situ exposure. In this article we describe a subchronic method for caging small fish at contaminated sites while maintaining growth and reproductive development. Fathead minnow (Pimephales promelas) were caged in situ for 6 weeks, after which time they were returned to the lab where they were evaluated for health and reproductive performance. Growth and reproductive endpoints revealed no adverse effect on fish due to fish caging and related handling, demonstrating the suitability of our caging and feeding method for long-term caging studies.

Selecting optimal eggs and embryonic developmental stages of fathead minnow (Pimephales promelas) for early life-stage toxicity tests.

Aquaculture research has indicated that fish embryo hatching success and larval survival can sometimes be predicted by embryo characteristics, such as blastomere cleavage patterns. An analogous strategy of individual assessment of spawned eggs could also be used to improve the quality of toxicity tests using early life-stages of fish where control-group survival determines experimental validity. Here we explored whether a simple method of assessing fathead minnow eggs and embryos for abnormalities could predict hatch success, and larval size at hatch, as indicators of embryo larval quality. Embryos were classified according to both their developmental stage and the presence of any abnormalities: uneven blastomere cleavage, atypical embryo size or shape, and the presence of inclusions in the yolk. Clutch size and fertilization rate did not predict embryo larval quality. Fewer abnormalities in embryos with ≤32 cells correlated with longer larvae at hatch. Normal embryos were more likely to hatch successfully than abnormal embryos of the same clutch, but because abnormality rates were generally low, much of the variation in hatch success could not be attributed to visible embryo malformations. Blastomere symmetry may be a useful selection criterion in embryos <3 h postfertilization. Where toxicant exposures early in embryonic development are not required or possible, hatch success could be increased by using older embryos that have survived gastrulation. Purposeful selection of embryos with at least two blastomeres, blastomere symmetry, and few inclusions can improve control survival and improve the quality of any generated (sub)lethality data. In our laboratory, application of the egg-selection criteria significantly improved control group hatch success increasing it from a mean of 84.4 to 94.2%.

Effects of metformin on wild fathead minnows (Pimephales promelas) using in-lake mesocosms in a boreal lake ecosystem.

Due to its widespread use for the treatment of Type-2 diabetes, metformin is routinely detected in surface waters globally. Laboratory studies have shown that environmentally relevant concentrations of metformin can adversely affect the health of adult fish, with effects observed more frequently in males. However, the potential risk to wild fish populations has yet to be fully elucidated and remains a topic of debate. To explore whether environmentally relevant metformin exposure poses a risk to wild fish populations, the present study exposed wild fathead minnows (Pimephales promelas) to 5 or 50 µg/L metformin via 2 m diameter in-lake mesocosms deployed in a natural boreal lake in Northern Ontario at the International Institute for Sustainable Development - Experimental Lakes Area (IISD-ELA). Environmental monitoring was performed at regular intervals for 8-weeks, with fish length, weight (body, liver and gonad), condition factor, gonadosomatic index, liver-somatic index, body composition (water and biomolecules) and hematocrit levels evaluated at test termination. Metabolic endpoints were also evaluated using liver, brain and muscle tissue, and gonads were evaluated histologically. Results indicate that current environmental exposure scenarios may be sufficient to adversely impact the health of wild fish populations. Adult male fish exposed to metformin had significantly reduced whole body weight and condition factor and several male fish from the high-dose metformin had oocytes in their testes. Metformin-exposed fish had altered moisture and lipid (decrease) content in their tissues. Further, brain (increase) and liver (decrease) glycogen were altered in fish exposed to high-dose metformin. To our knowledge, this study constitutes the first effort to understand metformin's effects on a wild small-bodied fish population under environmentally relevant field exposure conditions.

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.

Chronic Embryo-Larval Exposure of Fathead Minnows to the Pharmaceutical Drug Metformin: Survival, Growth, and Microbiome Responses.

Metformin is a glucose-lowering drug commonly found in municipal wastewater effluents (MWWEs). The present study investigated the chronic effects of metformin in early-life stages of the fathead minnow (Pimephales promelas). Endpoints assessed were growth, survival, and deformities. The larval gut microbiome was also examined using 16 S ribosomal RNA gene amplicon sequencing to determine microbial community composition and alpha and beta diversity. Eggs and larvae were exposed to metformin measured concentrations (mean [standard deviation]) of 0.020 (0.017) µg/L (for controls) and 3.44 (0.23), 33.6 (1.6), and 269 (11) µg/L in a daily static-renewal setup, with 20 embryos per beaker. The low and middle metformin exposure concentrations represent river and MWWE concentrations of metformin. To detect small changes in growth, we used 18 replicate beakers for controls and 9 replicates for each metformin treatment. Over the 21-d exposure (5 d as embryos and 16 d posthatch [dph]), metformin did not affect survival or growth of larval fish. Hatch success, time to hatch, deformities in hatched fry, and survival were similar across all treatments. Growth (wet wt, length, and condition factor) assessed at 9 and 16 dph was also unaffected by metformin. Assessment of the microbiome showed that the larvae microbiome was dominant in Proteobacteria and Firmicutes, with small increases in Proteobacteria and decreases in Firmicutes with increasing exposure to metformin. No treatment effects were found for microbiome diversity measures. Control fish euthanized with the anesthetic tricaine methane sulfonate had decreased alpha diversity compared to those sampled by spinal severance. This experiment demonstrates that metformin at environmentally relevant concentrations (3.44 and 33.6 µg/L) and at 10 times MWWE concentrations (269 µg/L) does not adversely affect larval growth or gut microbiome in this ubiquitous freshwater fish species. Environ Toxicol Chem 2022;41:635-647. © 2021 Her Majesty the Queen in Right of Canada. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC. Reproduced with the permission of the Minister of Environment and Climate Change Canada.

Temperature modulates the impacts of wastewater exposure on the physiology and behaviour of fathead minnow.

Municipal wastewater treatment plant (WWTP) effluent is a substantial source of pollution in aquatic habitats that can impact organisms across multiple levels of biological organization. Even though wastewater effluent is discharged continuously all year long, its impacts across seasons, specifically during winter, have largely been neglected in ecotoxicological research. Seasonal differences are of particular interest, as temperature-driven metabolic changes in aquatic organisms can significantly alter their ability to respond to chemical stressors. In this study, we examined the effects of multiple levels of wastewater effluent exposure (0, 25, or 50% treated effluent) on the physiological and behavioural responses of adult fathead minnow (Pimephales promelas) at temperatures simulating either summer (20 °C) or winter (4 °C) conditions. At 20 °C, wastewater exposure posed a metabolic cost to fish, demonstrated by higher standard metabolic rate and was associated with increased haematocrit and a reduction in boldness. In contrast, fish exposed to wastewater at 4 °C experienced no change in metabolic rate but performed fewer social interactions with their conspecifics. Taken together, our results demonstrate that wastewater exposure can lead to metabolic and behavioural disruptions, and such disruptions vary in magnitude and direction depending on temperature. Our findings highlight the importance of studying the interactions between stressors, while also underscoring the importance of research during colder periods of the year to broaden and deepen our understanding of the impacts of wastewater contamination in aquatic ecosystems.

Transformation of bitumen-derived naphthenic acid fraction compounds across surface waters of wetlands in the Athabasca Oil Sands region.

Bitumen is extracted from oil sands in the Athabasca Oil Sands region (AOSR) of Alberta, Canada. Much of the bitumen-derived toxicity in mine waste is attributable to naphthenic acid fraction compounds (NAFCs). Mines in the AOSR are required to be returned to a natural state after closure; thus, cost-effective strategies are needed to reduce toxicity from NAFCs. Previous studies have demonstrated the capability of constructed wetlands to attenuate NAFCs. However, the capacity of wetlands in the natural environment to degrade and transform NAFCs to less toxic components is poorly understood. To better understand the spatial distribution and fate of NAFCs in natural wetlands, samples were collected across the surfaces of two mature opportunistic wetlands near active oil sands mines. The first wetland has a well-defined surface flow pathway and inflows affected by overburden containing lean bitumen ore. The second wetland, in contrast, is a stagnant water body with raw bitumen visible along its edges. For the wetland with a well defined flow path, NAFCs decreased in concentration down gradient, while oxidized NAFCs constituted a greater proportion of NAFCs with increase in flow path. Likewise there was a decrease in the molecular weights of NAFCs, similar to trends observed in constructed wetland treatment systems. In comparison, NAFCs were more uniformly distributed across the relatively stagnant wetland. Overall, these data provide new evidence that mature opportunistic wetlands in the AOSR can promote the degradation and oxidation of bitumen-derived naphthenic acids into less toxic compounds.

A Deep Dive into the Complex Chemical Mixture and Toxicity of Tire Wear Particle Leachate in Fathead Minnow.

The ecological impact of tire wear particles in aquatic ecosystems is a growing environmental concern. We combined toxicity testing, using fathead minnow (Pimephales promelas) embryos, with nontarget high-resolution liquid chromatography Orbitrap mass spectrometry to characterize the toxicity and chemical mixture of organic chemicals associated with tire particle leachates. We assessed: 1) exposure to tire particle leachates after leaching for 1-, 3-, and 10-d; and 2) the effect of the presence and absence of small tire particulates in the leachates. We observed a decrease in embryonic heart rates, hatching success, and lengths, as well as an increase in the number of embryos with severe deformities and diminished eye and body pigmentation, after exposure to the leachates. Overall, there was a pattern whereby we observed more toxicity in the 10-d leachates, and greater toxicity in unfiltered leachates. Redundancy analysis showed that several benzothiazoles and aryl-amines were correlated with the toxic effects observed in the embryos. These included benzothiazole, 2-aminobenzothiazole, 2-mercaptobenzothiazole, N,N'-diphenylguanidine, and N,N'-diphenylurea. However, many other chemicals characterized as unknowns are likely to also play a key role in the adverse effects observed. Our study provides insight into the types of chemicals likely to be important toxicological drivers in tire leachates, and improves our understanding of the ecotoxicological impacts of tire wear particles. Environ Toxicol Chem 2022;41:1144-1153. © 2021 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

The acute toxicity of bitumen-influenced groundwaters from the oil sands region to aquatic organisms.

The extraction of surface mined bitumen from oil sands deposits in northern Alberta, Canada produces large quantities of liquid tailings waste, termed oil sands process-affected water (OSPW), which are stored in large tailings ponds. OSPW-derived chemicals from several tailings ponds migrating past containment structures and through groundwater systems pose a concern for surface water contamination. The present study investigated the toxicity of groundwater from near-field sites adjacent to a tailings pond with OPSW influence and far-field sites with only natural oil sands bitumen influence. The acute toxicity of unfractionated groundwater and isolated organic fractions was assessed using a suite of aquatic organisms (Pimephales promelas, Oryzias latipes, Daphnia magna, Hyalella azteca, Lampsilis spp., Ceriodaphnia dubia, Hexagenia spp., and Vibrio fischeri). Assessment of unfractionated groundwater demonstrated toxicity towards all invertebrates in at least one far-field sample, with both near-field and far-field samples with bitumen influence toxic towards P. promelas, while no toxicity was observed for O. latipes. When assessing the unfractionated groundwater and isolated organic fractions from near-field and far-field groundwater sites, P. promelas and H. azteca were the most sensitive to organic components, while D. magna and L. cardium were most sensitive to the inorganic components. Groundwater containing appreciable amounts of dissolved organics exhibited similar toxicities to sensitive species regardless of an OSPW or natural bitumen source. The lack of a clear distinction in relative acute toxicities between near-field and far-field samples indicates that the water-soluble chemicals associated with bitumen are acutely toxic to several aquatic organisms. This result, combined with the similarities in chemical profiles between bitumen-influenced groundwater originating from OSPW and/or natural sources, suggests that the industrial bitumen extraction processes corresponding to the tailings pond in this study are not contributing unique toxic substances to groundwater, relative to natural bitumen compounds present in groundwater flow systems.

In vivo and in vitro mixed-function oxygenase activity and vitellogenin induction in fish and in fish and rat liver cells by stilbenes isolated from scotch pine (Pinus sylvestris).

Many types of pulp and paper mill effluents have the ability to induce mixed-function oxygenase (MFO) activity and vitellogenin (VTG) protein in exposed male fish. The search for the compounds responsible for MFO induction has led to several classes of compounds, among them retene and stilbenes. The objective of this study was to investigate the biological activities of candidate stilbene compounds. Three stilbenes, 3,5-dihydroxystilbene (pinosylvin; P1), 3-hydroxy-5-methoxystilbene (P2), and 3,5-dimethoxystilbene (P3), were extracted from Scotch pine (Pinus sylvestris) and purified to evaluate their ability to induce MFO activity in vitro using ethoxyresorufin-O-deethylase (EROD) activity in a rat hepatoma cell line (H4IIE). As these compounds may be chlorinated during pulp bleaching, chlorination of P2 was undertaken, producing di- and trichlorinated isomers (Cl-P2), which were also tested. Compounds were tested for EROD-inducing ability in vivo by exposing juvenile rainbow tout (Oncorhynchus mykiss) to waterborne concentrations (0.010 to 1.0 mg/L) for 4 days. Compounds were also tested for their ability to induce VTG in trout primary liver cells in vitro. The stilbenes were weak inducers of EROD and VTG. H4IIE EROD was induced by all four compounds, with the most potent induction by P3, followed by P1, the Cl-P2 mixture, and then P2. Induction for all four stilbenes was from 3.13 × 10⁻³ to 3.57 × 10⁻4 as potent as retene and about 1.11 × 10⁻5 to 1.20 × 10⁻6 as potent as TCDD. Juvenile rainbow trout did not show EROD induction after exposures to P1, P2, or the Cl-P2 mixture, whereas P3 caused activity fourfold above that of controls. P1, P3, and Cl-P2 all weakly induced VTG in rainbow trout hepatocytes. The most potent inducer of VTG was Cl-P2, followed by P3 and P1. The results show the ability of wood-derived stilbenes to cause weak MFO induction in fish and in rat liver cells and to weakly induce vitellogenin in fish liver cells.

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.