Metabolites of Chlorinated Syringaldehydes in Fish Bile as Biomarkers of Exposure to Bleached Eucalypt Pulp Effluents

Metabolites of chlorinated phenolic compounds in fish bile have been found to be sensitive biomarkers of bleached pulp mill effluent exposure. Chlorinated syringaldehydes are largely unstudied chlorophenolics found in bleached hardwood effluent. Sand flathead (Platycephalus bassensis), Australian marine fish, were exposed to 100% chlorine dioxide-bleached eucalypt pulp effluent at concentrations of 0.5, 2, and 8% (v/v) for 4 days. Metabolites of 2-chlorosyringaldehyde (2-CSA), the predominant chlorophenolic in this effluent, were measured in the bile. The major metabolite was the conjugate of 2-chloro-4-hydroxy-3,5-dimethoxy-benzylalcohol (2-CB-OH), the reduced product of 2-CSA. 2-CB-OH was found in all fish exposed to diluted effluent and was concentrated in the bile over 1000 times above 2-CSA levels in the effluent. A separate experiment examined the metabolic fate of 2,6-dichlorosyringaldehyde (2,6-DCSA), which is one of the major chlorophenolics in chlorine-bleached eucalypt pulp effluent. Sand flathead were exposed to 2,6-DCSA by intraperitoneal injection at 15 mg/kg or through the water to 0.5, 2, or 8 μg/liter for 4 days. Analysis of the bile revealed the major metabolite of 2,6-DCSA to be the conjugate of 2,6-dichloro-4-hydroxy-3,5-dimethoxybenzylalcohol, which was found in all exposed fish and was concentrated in the bile over 20,000 times above 2,6-DCSA exposure levels. Results reveal that the analysis of metabolites of chlorinated syringaldehydes in fish bile can provide a biomarker of bleached hardwood effluent exposure that is sensitive to low levels of exposure, specific to certain bleaching sequences, and correlates well with exposure concentrations.     

Expression of the vitellogenin gene in the liver of juvenile whitefish (Coregonus lavaretus L. s.l.) exposed to effluents from pulp and paper mills.

Juvenile whitefish (Coregonus lavaretus L. s.l.) were exposed by caging in the field to diluted effluents from three operating pulp, paper, and paperboard mills in Southern Lake Saimaa, Finland. The expression of the vitellogenin gene, used as a biomarker of estrogenic contamination of effluents, was measured using a Northern blotting method. Increased mRNA levels, the most specific and reliable evidence for estrogen receptor-mediated actions in vivo, were found in fish caged in the vicinity of one of three mills studied. This mill was found to discharge wood-derived compounds, such as sterols and resin acids, into Lake Saimaa in amounts considerably exceeding those from the other two mills. The increased vitellogenin gene expression suggests that the effluent is a source of estrogenic contaminants.     

Biomarker responses and resin acids in fish chronically exposed to effluents from a total chlorine-free pulp mill during regular production

This study used rainbow trout (Oncorhynchus mykiss) to investigate the biological effects of long-term exposure to a total chlorine-free (TCF) pulp mill effluent. Fish tanks were set up on the premises of the pulp mill, and fresh effluent water was led through a pipe directly from the pulp mill to the tanks. The fish were exposed to effluent for up to 50 days and kept for up to 70 days afterward to study the recovery process. Two independent experiments were carried out. Ethoxyresorufin-O-deethylase (EROD), glutathione-S-transferase (GST), and glutathione reductase (GR) activity were elevated in fish exposed to a 2% concentration of pulp mill effluent, as were levels of DNA adducts. These effects could be detected during exposure and also after a period of recovery. The results demonstrate that the pulp mill effluent contains substances that affect the detoxification process and also have genotoxic potential. The continued occurrence of effects after a 70-day recovery period was demonstrated in both experiments, and may indicate that the effluent contained compounds with persistent properties. The content of free and conjugated resin acids in the bile of the fish was found to be a useful indicator of exposure to pulp mill effluent.     

Persistent Organochlorines in the Effluents from a Chlorine-Bleached Kraft Integrated Pulp and Paper Mill in Southeast Asia

A bleached sulfate integrated pulp and paper mill producing printing and writing paper from mixed tropical hardwood and bamboo was studied. The mill uses a “conventional bleaching sequence,” C-E-H1-H2, with an average molecular chlorine consumption of 50 kg per ton of air-dried pulp (ADP). The content of polychlorinated dibenzofurans (PCDFs) and dibenzo-p-dioxins (PCDDs) in the bleaching filtrate in terms of the nordic toxicity equivalent (N-TEQ) was 33.5, 1.15, 0.56, and 0.014 pg/L for the E, C, H1, and H2 bleaching stages, respectively. The corresponding PCDFs and PCDDs loads in ng/t ADP were in the same ranking, i.e., 670, 69, 11.2, and 0.28, respectively. The congener and isomeric pattern of PCDFs and PCDDs of the bleaching filtrate and the bleached pulp was found to be typical for the chlorine bleaching plant effluent. The obtained dioxin load formed in the mill is in agreement with Western studies for the given multiple chlorine of 0.21–0.23. The load is, however, lower than reported discharges from Scandinavian mills using 1980s bleaching technologies, but substantially higher than the discharges from mills with modern bleaching technologies. Modifications in the bleaching plant to reduce molecular chlorine use are necessary to reduce dioxin formation. Received: 15 July 1998/Accepted: 17 March 1999     

Juvenile sea bass liver biotransformation and erythrocytic genotoxic responses to pulp mill contaminants

This research represents the first study concerning liver phase I biotransformation induction, measured as cytochrome P-450 (P450) and ethoxyresorufin-O-deethylase (EROD), and genotoxic responses, measured as erythrocytic micronuclei (EMN) and nuclear abnormalities (ENA), in Dicentrarchus labrax L. (sea bass) exposed to pulp and paper mill contaminants. Juvenile sea bass were exposed for 6 h to 0 (control) and 0.0125 microM concentrations of the resin acids (RAs) abietic acid (AA) and dehydroabietic acid (DHAA) or 7-isopropyl-1-methylphenanthrene (retene) (Experiment 1). Sea bass were exposed for 6 h to 0 (control), 0.78, 1.56, 3.12, 6.25, and 12.5% bleached kraft pulp mill environmental contaminated water (BKPMECW) collected at the old sewage outlet of a pulp and paper industry (Experiment 2). The time-dependent response was studied in sea bass at 0, 8, 16, 24, 48, and 72 h exposure to 0.78 and 12.5% BKPMECW (Experiment 3). The experimental results demonstrated the presence of genotoxic compounds in BKPMECW. AA, DHAA, and retene may be the constituents responsible for high BKPMECW genotoxicity, since they induced similar sea bass EMN and ENA frequency increases. The BKPMECW, the RAs, and retene failed to significantly increase liver EROD activity and P450 content at 6 h. Furthermore, 3.12% BKPMECW, 0.0125 microM AA, and 0.0125 microM retene significantly decreased liver EROD activity. However, P450 was significantly increased from 8 up to 72 h exposure to BKPMECW. Therefore, the low or inhibited EROD levels could be a consequence of a general membrane disturbance by BKPMECW, RAs, and retene. However, liver ALT results indicate significant liver damage or enzyme inhibition only at 8, 16, 48, and 72 h exposure to BKPMECW.     

Chronic Effects of <Emphasis Type="Italic">Pinus radiata</Emphasis> and <Emphasis Type="Italic">Eucalyptus globulus</Emphasis> Kraft Mill Effluents and Phytosterols on <Emphasis Type="Italic">Daphnia magna</Emphasis>

Two kraft pulp mill effluents were compared in terms of their chronic toxicity to Daphnia magna. One resulted from pulping Pinus radiata and the other came from a parallel processing of Pinus radiata and Eucalyptus globulus (mixed kraft pulp mill effluent). The concentration of phytosterols found in the mixed kraft pulp mill effluent was higher than in the effluent from Pinus radiata, with values of 0.1082 and 0.02 μg/L, respectively. The phytosterols per se are responsible for 12.9% and 8.1% of the deviation from the natural shape, while the kraft pulp mill effluents account for 25.6%–27.8% of shape deviation. The role of β-sitosterol and stigmasterol is discussed in relation to endocrine disruption.     

Measures of Resting Immune Function and Related Physiology in Juvenile Rainbow Trout Exposed to a Pulp Mill Effluent

Juvenile rainbow trout (Oncorhynchus mykiss) were exposed to 0, 10, 30, and 70% (v/v) of a secondary-treated, integrated thermomechanical-bleached kraft pulp and paper mill effluent. The immunological parameters oxidative burst and phagocytosis of head kidney macrophages and total and differential circulating leukocytes were measured after 21 d of exposure. General parameters of stress and exposure including erythrocyte counts, numbers of degenerating erythrocytes, splenic pigmented macrophage aggregates (PMAs), spleen size, bile chemistry, and hepatic EROD activity were also assessed. Contrary to parallel chronic studies on the same effluent, EROD induction did not occur. Analyses of bile indicated uptake and accumulation of resin acids and some sterols. There was no measurable macrophage-related immunological dysfunction. However, circulating leukocytes, specifically lymphocytes, were reduced. The density of splenic PMAs increased over the exposure period, possibly in association with degenerating blood cells. There were statistical differences between staggered days of sampling in head kidney oxidative burst, white and mature red blood cell counts, and spleen size, indicating that relatively minor capture and handling stress could result in rapid changes in some parameters. Overall, it was concluded that the observed minor, indirect alterations in the immune response were likely the result predominantly of a nonspecific mechanism such as a cortisol-mediated stress response.     

Effects of Acute Handling Stress on Whitefish Coregonus lavaretus After Prolonged Exposure to Biologically Treated and Untreated Bleached Kraft Mill Effluent

Exposure of fish to water of impaired quality has been shown to disrupt the function of the hypothalamo-pituitary-interrenal (HPI) axis and alter the interpretation of data from field studies due to the varying effects of handling and delayed sampling on exposed and reference animals. In the present study, juvenile whitefish, Coregonus lavaretus, were exposed for 6 weeks to diluted (4–8%) untreated and biologically treated bleached kraft mill effluent (BKME) and their response to acute handling was investigated. Liver microsomal EROD activity and glycogen phosphorylase (GPase) activity, in addition to gill Na⁺-K⁺-ATPase activity, and blood hemoglobin and hematocrit levels were increased in whitefish exposed for 6 weeks to untreated BKME, whereas those exposed to treated BKME exhibited increased blood hemoglobin and red blood cell K⁺ concentrations. Both handling procedures, exposure to a shallow water (10 cm, 5 min) and to an air challenge (10 s air/10 s water/30 s air/10 s water/10 s air), resulted in acute physiological stress, as recorded after 5-, 60-, and 120-min recovery periods. Following air exposure, the levels of plasma cortisol, blood glucose, hemoglobin, and hematocrit as well as the liver GPase activity were increased, and liver glycogen concentration decreased in control fish. These responses were attenuated in fish exposed to untreated or treated BKME. Plasma estradiol and testosterone levels were not affected by the BKME exposures or by the air challenge. Handling also resulted in attenuated EROD induction in fish exposed to untreated BKME. According to the present findings, the sensitivity of some widely used cellular and physiological variables may be improved by time-dependent standardization when interpreting data obtained following delayed sampling. Received: 3 July 2000/Accepted: 26 January 2001     

Biotransformation and other physiological responses in whitefish caged in a lake receiving pulp and paper mill effluents

Hepatic monooxygenase (MO) and conjugation enzyme activities, metabolites of chlorinated phenolics in the bile, and blood ionoregulatory parameters were studied in juvenile whitefish (Coregonus muksun Pallas and C. muksun X Coregonus peled Gmelin hybrid) held in cages downstream from a mill producing chlorine-bleached kraft pulp and printing paper. MO activities, measured as benzo[a]pyrene hydroxylase, 7-ethoxycoumarin O-deethylase, and 7-ethoxyresorufin O-deethylase, were significantly induced in whitefish caged about 5 km from the effluent outlet. The highest mean increase detected was 17 times the control value. In the nearest caging station (3 km) the induction was lower, indicating inhibition or toxicity caused by the effluent. The levels of bile metabolites of chlorinated phenolics showed highest concentrations at the nearest station and decreased levels at more distant locations over the whole water area studied (15 km). Bile metabolites in whitefish exposed in control areas confirmed low-level background pollution of the lake system due to chlorinated phenolics. Observations on blood ionic concentrations suggest that whitefish were able to regulate their hydromineral balance despite the environmental pollution affecting physiology of biotransformation.     

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 (<Emphasis Type="Italic">Pinus sylvestris</Emphasis>)

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.     

Cytochrome P450-Dependent Enzymes and Oxidant-Mediated Responses in Rainbow Trout Exposed to Contaminated Sediments

Hatchery-reared immature rainbow trout (Oncorhynchus mykiss) were exposed to different concentrations (2 and 4 liters) of contaminated sediment taken from a site receiving unbleached pulp mill effluents. The fish were held in aquaria and sampled three times during an experimental period of 21 days. The monooxygenase activity, measured as the deethylation of 7-ethoxyresorufin (EROD activity), increased three- to fourfold in the exposed fish relative to controls. The increase was not dependent on exposure concentration. Cytochrome P450IA1, the EROD catalyst, demonstrated proportional induction in the 2-liter exposed fish. However, exposure to 4 liters sediment strongly induced P450IA1 and did not reflect EROD activity. This may suggest inhibition of P450IA1 activity by the amount of chemicals discharged from pulp mills. UDPglucuronosyl-transferase increased at one stage of the experimental period, while glutathione S-transferase remained unchanged. Amounts of total glutathione in blood, liver, and muscle were slightly increased by exposure to contaminated sediments, but hepatic enzyme activities of superoxide dismutase and catalase were not affected. In conclusion, monooxygenase activities appear to be a sensitive tool in the monitoring of sediment toxicity.     

Paper and Pulp Mill Effluent-Induced Immunotoxicity in Freshwater Fish Channa punctatus (Bloch)

The influence of temperature variation on the humoral immune response using the plaque-forming cell (PFC) assay in freshwater fish Channa punctatus (Bloch) was investigated. The fish was exposed to 1% concentration (v/v in water) of paper and pulp mill effluent under standard laboratory conditions in glass aquaria. Effect of effluent exposure on the organ weight and organ cellularity was also evaluated. In general, lower temperature in winter produced a subdued response in control as well as experimental group. At higher temperature of summer and rainy seasons, an elevated response was observed. It was revealed that though suppressive effect of effluent was evident during all the seasons, in summer, extent of suppression was greater as compared to winter. Paper and pulp mill effluent exposure resulted in a decrease in the splenic and pronephric (head kidney) cellularity, with more pronounced effect in the summer season. We also studied the effects of the length of exposure on various parameters. Fish were exposed for 15, 30, 60, and 90 days to effluent. Short-term exposure for 15 days induced an elevated PFC response, but change was not statistically significant. Conversely, the exposure for 30, 60, and 90 days significantly reduced (p < 0.05–0.001) the PFC response. Long-term exposure also caused significant reduction (p < 0.05–0.01) in the weights of lymphoid organs (spleen, head kidney, and total kidney). These results show a suppressive effect of chemical constituents of paper and pulp mill effluent on the immune functions. Furthermore, results demonstrated that though modulatory effect of the temperature on immune functions is recognized, it was not a major contributory factor to the elevated immunotoxicity of paper and pulp mill effluent in the seasons of high temperature. Received: 14 March 2000/Accepted: 28 July 2000     

Environmental Assessment of Benthic Impacts Associated with Pulp Mill Discharges. II. Distribution of Sediment EOX in Relation to Environmental Factors

Between 1991 and 1992, comprehensive toxicological, chemical, and benthic community structure surveys were conducted at Jackfish Bay, Lake Superior, Ontario, to assess sediment quality below a bleached kraft mill effluent (BKME) outfall. In this report, we describe the spatial distribution and concentration of extractable organic chlorines (EOX) in sediments below the outfall in relation to effluent discharge patterns, sediment deposition, and bioaccumulation potential. Triplicate sediment samples were collected by Ekman grab from 29 and 44 stations in 1991 and 1992, respectively, and from 15 stations in three reference areas not exposed to the effluent. For each sample, organic carbon content, dry weight, and sediment-associated EOX were determined. In addition, sediment traps were used to determine sediment deposition along two tangents transecting the effluent plume and at a reference station located approximately 4,200 m from the outfall. The spatial distribution of sediment EOX consisted of two distinct concentration gradients, each linked to effluent discharge patterns from Blackbird Creek and dominant circulatory patterns in Jackfish Bay: a north–south gradient, extending along the west shore of Jackfish Bay, and a east–west gradient. Sediment EOX ranged from 7,000 mg/kg organic carbon (oc) (2,050 mg/kg dry solids (ds)) in depositional sediments of Moberley Bay to nondetectable at some stations in eastern and outer Jackfish Bay. Reference (background) concentrations ranged from nondetectable to 3,000 mg/kg oc (1–15 mg/kg ds). The attenuation of EOX was significantly correlated with the rate of sediment deposition (r² = 0.57; p ≤ 0.001). Although the concentration of sediment EOX was strongly correlated with toxicity to some invertebrates (Sibley et al.[1997] Arch Environ Contam Toxicol 32:274–284), bioaccumulation factors were typically <2 based on analysis of oligochaete tissues. The results of this study demonstrate that the spatial distribution of sediment-associated contaminants adjacent to pulp mill effluent outfalls are determined primarily by hydrodynamic and depositional characteristics of the receiving waters and that EOX can be used to accurately delineate areas of contamination. Received: 7 May 1997/Accepted: 29 September 1997     

Studies of Transformation and Particle-Binding of Resin Acids During Oxidative Treatment of Effluent from Two New Zealand Pulp Mills

Reactor studies of aerobic degradation of effluent from the first and last ponds of the treatment system of two New Zealand pulp and paper mills indicated that filterable BOD₅, resin acids and transformed resin acids, free and bound, degraded at similar rates. During oxidative treatment the resin acids of untreated effluent became increasingly bound to particulate material and a sediment high in abiet-13-enoic acid was formed.     

Sublethal Effects of Chlorine-Free Kraft Mill Effluents on <Emphasis Type="Italic">Daphnia magna</Emphasis>

The implementation of elemental chlorine-free (ECF) bleaching methods has drastically reduced the aquatic toxicity of Kraft mill effluents during the last decade. However, the residual toxicity of Kraft mill effluents is still a potential concern for the environment, even when subjected to secondary wastewater treatment. The aim of this study is characterize potential sublethal effects of ECF Kraft mill effluents using Daphnia magna as model species. D. magna exposed towards increasing concentration of ECF Kraft mill effluent showed a significant, dose-dependent reduction in feeding. Conversely, post-feeding assay, life history, and allometric growth analyses showed stimulatory, rather than inhibitory effects in exposed animals at low concentrations, while high concentrations of ECF Kraft mill effluents reduced their reproductive output. These results suggest a hormetic effect in which moderate concentrations of the effluent had a stimulatory effect with higher concentrations causing inhibition in some variables.     

Contaminants in Ospreys from the Pacific Northwest: I. Trends and Patterns in Polychlorinated Dibenzo-p-Dioxins and -Dibenzofurans in Eggs and Plasma

Osprey (Pandion haliaetus) eggs were collected from 1991 to 1997 at nests (n = 121) upstream and downstream of bleached kraft pulp mills and at reference sites in the Fraser and Columbia River drainage systems of British Columbia, Washington, and Oregon. Blood samples were collected from nestling ospreys during the 1992 breeding season on the Thompson River. Samples were analyzed for polychlorinated dibenzo-p-dioxins (PCDDs) and -dibenzofurans (PCDFs). Mean concentrations of 2,3,7,8-TCDD were significantly higher in eggs collected in 1991 at downstream compared to upstream nests near pulp mills at Kamloops and Castlegar, British Columbia. There were no significant temporal trends in 2,3,7,8-TCDD, -TCDF or other measured compounds at a sample of nests monitored between 1991 and 1994 downstream of the Castlegar pulp mill, despite changes in bleaching technology (CIO₂ substitution). However, by 1997 concentrations of 2,3,7,8-TCDD and -TCDF were significantly lower than previous years in nests sampled downstream at both Castlegar and Kamloops. An unusual pattern of higher chlorinated PCDDs and PCDFs was found in many of the osprey eggs collected in this study, and considerable individual variation in the pattern existed among eggs from the same site. For example, eggs from four different nests at one study area (Quesnel) on the Fraser River had concentrations of 1,2,3,4,6,7,8-HpCDD ranging from <1 to 1,100 ng/kg and OCDD from <1 to 7,000 ng/kg wet weight. Higher mean concentrations of HpCDD and OCDD were found in eggs from the Thompson River, a tributary of the Fraser, compared to the Columbia River, and concentrations were generally higher at nests upstream of pulp mills. In plasma samples, 1,2,3,4,6,7,8-HpCDD and OCDD were the main compounds detected, with no significant differences measured between samples upstream versus downstream or earlier versus later in the breeding season. Use of chlorophenolic wood preservatives by lumber processors was considered the main source of higher chlorinated PCDD/Fs throughout the systems, based on patterns of trace PCDFs in eggs and significant correlations between egg concentrations of pentachlorophenol and both HpCDD (r = 0.891, p < 0.01) and OCDD (r = 0.870, p < 0.01). Received: 17 February 1998/Accepted: 11 June 1998     

A New Developmental Toxicity Test for Pelagic Fish Using Anchoveta (<Emphasis Type="Italic">Engraulis ringens</Emphasis> J.)

A series of six 96-h static bioassays were performed to validate the use of anchoveta (Engraulis ringens) embryos as test organisms for ecotoxicological studies. The standardization protocol utilized potassium dichromate (K₂Cr₂O₇) as a reference toxicant and egg mortality as the endpoint. The results indicated that the mean sensitivity of anchoveta embryos to potassium dichromate was 156.1 mg L⁻¹ (range: 131–185 mg L⁻¹). The statistical data analysis showed high homogeneity in LC50 values among bioassays (variation coefficient = 11.02%). These results demonstrated that the protocol and handling procedures implemented for the anchoveta embryo bioassays comply with international standards for intra-laboratory precision. After secondary treatment, an effluent from a modern Kraft pulp mill was tested for E. ringens embryo toxicity, finding no significant differences from the controls.     

Male-Biased Sex Ratios and Vitellogenin Induction in Zebrafish Exposed to Effluent Water from a Swedish Pulp Mill

Juvenile zebrafish (Danio rerio) were exposed to different dilutions (0, 0.67, 2.5, 10, and 50%) of effluent water from a Swedish pulp mill that previously has been reported to be androgenic to fish. Exposure was performed between days 10–38 days post-hatch. Fish were sampled for whole-body vitellogenin concentrations at day 38 post-hatch and for histological examination of gonads at day 60 post-hatch. In fish exposed to the highest concentration of pulp mill effluent, elevated concentrations of vitellogenin were measured. The androgenicity of the pulp mill water was confirmed by the increased number of males recorded at 60 days post-hatch. Image analysis of testes indicated stimulation of spennato genesis. Intersex fish were observed in all exposure groups. An androgenic activity equivalent to 5.6 ng/L dihydroxytestosterone was measured using the yeast androgen screen (YAS) assay. The present study demonstrates that both androgenic and estrogenic effects can be detected when exposing zebrafish during the juvenile period to complex mixtures of chemicals.     

Constituents within pulp mill effluent deplete retinoid stores in white sucker and bind to rainbow trout retinoic acid receptors and retinoid X receptors

Wild female and male white sucker (Catostomus commersoni) inhabiting an area receiving pulp mill effluent had reduced hepatic levels of retinol, didehydroretinol, retinyl esters, and didehydroretinyl esters, while vitamin E levels were unaffected. This disruption of the retinoid system led us to test methanol and dichloromethane extracts from the effluent of 11 pulp mills from across Canada for their ability to bind to rainbow trout (Oncorhynchus mykiss) retinoic acid receptors (RARs) from the gill and retinoid X receptors (RXRs) from the liver. Concentrated extracts of the final effluent from 6 of the 11 pulp mills were able to displace greater than 25% of the receptor-bound [3H]all-trans retinoic acid (RA) or [3H]9-cis RA from trout RARs and RXRs, respectively. The ability of the extracts to displace retinoic acid did not appear to be linked to the pulping or treatment processes. Moreover, extracts with the greatest activity came from thermomechanical mills, suggesting the compounds may originate from the wood furnish. In addition, extracts prepared from wood furnish (wood chips: white spruce [50%], lodgepole pine [47%], and balsam fir [3%]) from one mill were able to displace [3H]RA from the RARs and RXRs. The 4-hydroxy RA, a metabolite of RA that has been shown to be generated in greater quantities in fish exposed to P450-inducing xenobiotics, was able to displace [3H]all-trans RA from trout RARs as effectively as unlabeled all-trans RA. These results suggest that pulp mill effluent may impact the retinoid system of fish at multiple sites, either by decreasing hepatic retinoid stores or through contributing additional ligands (from the wood furnish) that can bind to RA receptors.