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The authors examined the potential of pulp mill effluent from pulp-producing countries (Canada, Brazil, New Zealand) to affect fish reproduction. Specifically, the estrogenic effects in juvenile rainbow trout (Oncorhynchus mykiss) pulse-exposed to 11 different mill effluent extracts (intraperitoneal injections of solid-phase extraction-dichloromethane nonpolar fraction). The results indicated that effluent extracts were estrogenic in juvenile trout irrespective of the gender, as reflected by increasing level of plasma vitellogenin (VTG; Brazil > New Zealand > Canada). Despite the high variability observed among mills, differences in VTG levels were related to the type of mill process (kraft > elementary chlorine-free kraft > thermomechanical pulping). Moreover, effluent treatments did not appear to significantly decrease VTG induction. A consistent estrogenic effect was observed in those mills that process a combination of feedstocks (softwood and hardwood), with the highest increase in VTG related to eucalyptus feedstock. The results demonstrate significant estrogenic effects of pulp mill effluents on chronically exposed juvenile trout, suggesting that in vivo metabolic activation of precursors is necessary to cause the observed increases in VTG levels. This molecular estrogenic response provides a useful starting point for predicting population-level impacts through the adverse outcome pathway methodology. Environ Toxicol Chem 2017;36:1547-1555. © 2016 SETAC., (© 2016 SETAC.)

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.

This study investigates factors affecting reproduction in fish exposed to pulp and paper mill effluents by comparing effluents from countries with varying levels of documented effects. To explore the hypothesis of wood as a common source of endocrine disrupting compounds, feedstocks from each country were analyzed. Analyses included in vitro assays for androgenic activity (binding to goldfish testis androgen receptors), estrogenic activity (yeast estrogen screen), and neurotransmitter enzyme inhibition (monoamine oxidase and glutamic acid decarboxylase). Chemical analyses included conventional extractives, known androgens, and gas chromatograph index (GCI) profiles. All effluents and wood contained androgenic activity, particularly in nonpolar fractions, although known androgens were undetected. Effluents with low suspended solids, having undergone conventional biotreatment had lower androgenic activities. Estrogenic activity was only associated with Brazilian effluents and undetected in wood. All effluents and wood inhibited neurotransmitter enzymes, predominantly in polar fractions. Kraft elemental chlorine free mills were associated with the greatest neurotransmitter inhibition. Effluent and wood GCI profiles were correlated with androgenic activity and neurotransmitter enzyme inhibition. Differences in feedstock bioactivities were not reflected in effluents, implying mill factors mitigate bioactive wood components. No differences in bioactivities could be discerned on the basis of country of origin, thus we predict effluents in regions lacking monitoring would affect fish reproduction and therefore recommend implementing such programs.

Pulp and paper mill effluents have been linked to effects on fish reproduction for more than 25 years. To date, the causes of these effects and remedial strategies have eluded investigators. Recent work has shown that the degree of reproductive effect caused by a mill effluent is related to the overall organic content. If verified, this could lead to breakthroughs for best management practices (BMPs). For this study, the effluents from seven kraft mills were assessed for their ability to reduce egg production in fathead minnow (Pimephales promelas) in the laboratory. At the same time, the effluents were analyzed for three parameters thought to be good indicators of organic losses: biochemical oxygen demand (BOD), methyl-substituted 2-cyclopentenones (cellulose degradation products), and a gas chromatographic (GC) profile index, which integrates the total area of the chromatographic peaks of solvent-extracted effluents using low-resolution mass spectrometry. The results showed that the degree to which the effluents reduced egg production increased with increased organic losses as characterized by BOD and the GC profile index. Therefore, these parameters could be used to guide BMPs at kraft mills according to specific targets: BOD < 20 mg/L and GC profile index equivalent to effluent with BOD of 20 mg/L. Such targets should be achievable by good in-plant control of organic losses and optimized effluent biotreatment systems., (Copyright © 2011 SETAC.)

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.

Here we show for the first time that components of pulp and paper mill effluents contain neuroactive substances that may impair fish reproduction. Grab samples of primary and secondary effluent were obtained from a representative pulp and paper mill in Eastern Canada. Effluents were fractionated using classic polarity and polyphenolic extraction methods into solvents of selected polarities (water, ethanol, ethyl acetate, and hexane). By means of in vitro, competitive assays on goldfish (Carassius auratus) brain tissues, the extracts were screened for their ability to interact with enzymes and receptors involved in gamma-aminobutyric acid (GABA), dopamine, glutamate, and acetylcholine-dependent neurotransmission. These neurotransmission pathways have essential regulatory roles in fish reproduction. Radioligand binding to the following neurotransmitter receptors were significantly impacted following in vitro incubations with extracts (percentage change from controls indicated in brackets): dopamine-2 (D2; 21-48% increase), GABA(A) receptor binding (65-67% decrease and 189% increase), N-methyl-o-aspartic acid (NMDA; 26-75% decrease), and muscarinic cholinergic (mACh; 42% increase). Activities of the following neurotransmitter-related enzymeswere significantly impacted: monoamine oxidase (MAO; 14-48% decrease), GABA-transaminase activity (33% decrease and 21-69% increase), and acetylcholinesterase (AChE; 21-50% decrease). No changes in glutamic acid decarboxylase (GAD) activity were detected. These findings provide a novel and plausible mechanism by which pulp and paper mills effluents impair fish reproduction by interacting with neurotransmitter systems. Furtherwork is required to identify the active compounds and explore whether these changes occur in vivo.

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.

Reproductive effects have been recorded in wild fish in waters receiving pulp mill effluent (PME) since the mid to late 1980s. Laboratory assays with fathead minnow (FHM; Pimephales promelas) have been developed to better understand fish responses to PME. However, observations from laboratory studies have been variable, making it difficult to establish cause/effect relationships. A lack of environmental relevance in these laboratory studies may have contributed to the variability observed. The objectives of the present study were, first, to determine the effects of bleached kraft PME (BKME) on FHM under environmentally realistic conditions (i.e., ambient water and effluent quality) and, second, to determine the suitability of pair-breeding FHM to better link BKME-induced changes in indicators at the biochemical, individual, and population levels. A mobile bioassay trailer was situated on-site at a bleached kraft mill for 60 d, allowing supply of both ambient water (Lake Superior, Canada) and final BKME. The reproductive output of FHM was initially assessed for 21 d to obtain baseline data pre-exposure. At the end of the pre-exposure period, selected breeding pairs were exposed to final BKME (100% v/v and 1% v/v) for 21 d. Results demonstrated a stimulatory response pattern at 1% BKME (e.g., increased egg production) compared to control. In the 100% treatment, spawning events were reduced and fewer eggs were produced during the first two weeks of exposure. Exposure to 100% (v/v) BKME also resulted in ovipositor development in males and development of male secondary sex characteristics in females. Obtaining pre-exposure data and use of pair-breeding FHM in this assay gave a sensitive indication of effluent effects and allowed accurate comparisons of endpoints to be made.

Reproductive effects of pulp mill effluents on fish continue to be reported in Canadian waters. Spawning delays, reduced gonad size, and altered levels of sex steroid hormones have been found in both sexes of various fish species exposed to effluents. We initiated a project to identify the source/cause of such effects. In part 1 of this two-part series, we exposed mature adult fathead minnow (FHM; Pimephales promelas) for 21 d to final treated effluent (1% and 100% v/v) from a bleached kraft pulp mill in Terrace Bay (ON, Canada). Results suggested pulp mill effluent from this mill affected reproductive indicators in FHM and effects were dependent on effluent concentration, duration of exposure, and method of data analysis. The main objective of this paper was to use the FHM assay to identify waste stream sources within the mill that affect reproductive indicators. Various process streams were selected, characterized with respect to effluent chemistry and acute toxicity, and a subset was tested on-site with the 21-d FHM bioassay. Results showed that both the combined mill effluent (before secondary treatment) and the combined alkaline stream (CALK) caused both decreased spawning events (approximately 55% for both streams) and decreased egg production (28 and 74%, respectively), and the CALK stream resulted in significant male ovipositor development. By comparing response patterns we were able to identify the CALK stream as a source of compounds at this mill affecting reproductive indicators in FHM and highlight this stream for further toxicity identification evaluation.

As part of a continuing survey, effluents from five mills in Canada were tested in the laboratory for their potential to affect fish reproduction. The study included effluents from two thermomechanical pulp (TMP) mills, two kraft pulp mills, and one mill that used both chemical and mechanical pulping. The laboratory test used adult fathead minnows and involved a 21-day exposure to each effluent. All the effluents were tested at 2 and 20% concentration. The effluent from 1 of the kraft mills was also tested at 40% concentration. The endpoints of the test included, egg production, gonad size, sex steroids, secondary sexual characteristics, and vitellogenin concentration in males, considered to be an indicator of estrogenicity. The results of this study were similar to the results of our previous survey. None of the effluents produced noteworthy changes at 2% concentration. At 20% concentration, only the effluent from the multiprocess mill produced a significant reduction in eggs, which was considered to be the most important indicator of reproductive performance. Some effluents did produce an increase and/or a decrease in a variety of endpoints other than egg production, but the most consistent response was an induction of vitellogenin in males exposed to three of the five effluents tested. In summary, these results indicate that most mill effluents up to 20 or 40% concentration do not affect the overall reproductive capacity of minnows in the laboratory. However, the mill effluents do seem to contain substances that cause vitellogenin induction.

Resin acids, a class of wood extractives, are potential toxic constituents in many pulp and paper mill effluents. In the present investigation, the effects of two predominant resin acids, dehydroabietic acid (DHA) and abietic acid (ABA), on survival, reproduction, and growth of the freshwater crustacean Daphnia magna were assessed over its life cycle. Based on the experimentally determined acute toxicity data (48-h EC(50)'s) for DHA (7.48 mg/L) and ABA (7.98 mg/L), D. magna was treated chronically with each resin acid at nominal concentrations of 0, 0.25, 0.5, 1.0, 2.0, 4.0, and 8.0mg/L for 21 days. Both DHA and ABA at concentrations as high as 4.0mg/L did not affect physiological and reproductive parameters such as time to maturation, number of molting, number of broods, and number of offspring produced from surviving daphnids, while significant mortality was observed only at 8.0mg/L in both cases. However, a small but statistically significant decrease in Daphnia growth (body length) at the end of exposure was detected at concentrations as low as 0.5mg/L for DHA and 1.0mg/L for ABA, respectively. These results indicated that both DHA and ABA had the potential to exhibit weak growth inhibition without apparent negative effects on reproduction to D. magna at nonlethal concentration levels. This slight effect is not expected to be ecologically significant because the concentrations of DHA and ABA in biologically treated pulp and paper mill effluents are well below the effective levels observed in the present study.

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.

This study evaluated the effects of bleached and unbleached kraft mill effluent on reproductive success of largemouth bass (Micropterus salmoides). Bass were exposed to effluent concentrations (0, 10, 20, 40, or 80%) for 28 and 56 d. Parameters measured included hepatosomatic index (HSI) and gonadosomatic index (GSI) and plasma concentrations of 17beta-estradiol (E2), 11-ketotestosterone (11-KT), and vitellogenin (VTG). At the end of the 56-d period, bass were moved to hatchery ponds to evaluate spawning success. Spawning mats with eggs either were brought indoors for evaluation of fecundities, hatchabilities, and egg and fry size (measured at age 3 d), or were left in ponds and fry number and size recorded (average age of 14 d). Effluent exposure was verified by measuring resin acids (isopimaric, abietic. and dehydroabietic acids) in bile. Compared to controls, exposed bass had greater concentrations of resin acids in bile. In general, exposed females had lower concentrations of E2 and VTG (> or = 20% effluent), whereas males had lower concentrations of 11-KT (> or = 20% effluent) and increased E2 (> or = 20% effluent). The HSI values increased in females (> or = 10% effluent), and GSI values decreased in both sexes (> or = 40% effluent). Fecundity, egg size, and hatchability did not differ across treatments, but an increase in the frequency of fry abnormalities and a decrease in fry weights was observed at effluent exposures of 40% and higher. However, results from the pond study, revealed a significant reduction in fry growth and survival (> or = 10%). This decline may have been caused by an increased frequency of deformities, in conjunction with alterations of growth. These changes could have resulted from alterations in egg quality because of failure of parental reproductive systems, from acute embryo toxicity after translocation of contaminants from the mother to the developing embryo, or from both.

Rainbow trout were exposed to a secondary treated, thermomechanical/bleached kraft pulp and paper effluent in 12,000-L, flow-through exposure tanks at an environmental research facility located at a pulp and paper mill in Kawerau, New Zealand. Trout (age, 2+ years) were obtained from a local hatchery and exposed either to upstream river water or a nominal concentration of 12% (v/v) effluent diluted in upstream river water. Three treatment groups were used: Effluent exposure that started approximately three months before gonadal growth (eight-month total exposure), effluent exposure that started approximately halfway through gonadal development (two-month total exposure), and trout exposed to reference water alone for the total duration of the experiment. Trout were sacrificed just before spawning; exposure, growth, and reproductive endpoints were assessed during and at the termination of the experiment. Reduction in growth was observed in both sexes in the eight-month treatment group relative to the river water reference treatment group. No differences were observed in condition factor or liver size in either treatment. Females in the eight-month exposure group also had significantly lower ovary weight. The two-month exposure group showed no differences from the reference group in growth or somatic indices. Estradiol and testosterone were reduced in blood samples taken from the eight-month exposure group by four months into the experiment as compared to the reference treatment. Steroid and vitellogenin levels in individual female trout from this treatment were significantly correlated with gonadosomatic indices (GSI) measured at the termination of the experiment. The GSI was not correlated strongly or consistently with pregnenolone, nor were any treatment-related pregnenolone differences observed, indicating that the steroid hormone reductions likely were not related to cholesterol side-chain cleavage. Male trout showed significant induction of vitellogenin and lower 11-ketotestosterone during the experiment (only the eight-month group was examined), but this did not result in any significant differences in testes development. Thus, this study has shown an impact of pulp mill effluent exposure on the reproductive physiology of female trout that appeared to be hormonally mediated. Furthermore, the effect could only be manifest when the exposure was initiated before the start of gonad development.

The objective of this study was to describe and compare several reproductive parameters for Florida largemouth bass (Micropterus salmoides floridanus) inhabiting the St. Johns River and exposed to different types and/or degrees of contamination. Welaka was selected as the reference site in this study because of its low urban and agricultural development, Palatka is in close proximity to a paper mill plant, the Green Cove site is influenced by marine shipping activities and Julington Creek site receives discharges of domestic wastewater and storm water runoff from recreational boating marinas. For this study, bass were sampled both prior to (September 1996) and during the spawning season (February 1997). In order to characterize chemical exposure, bass livers were analyzed for up to 90 trace organics and 11 trace metal contaminants. Reproductive parameters measured included gonadosomatic index (GSI), histological evaluation of gonads and plasma concentrations of vitellogenin (VTG), 17beta-estradiol (E2) and 11-ketotestosterone (11-KT). In general, the sum of organic chemicals was highest in livers from Palatka bass and bass from Green Cove and Julington Creek had higher hepatic concentrations of low molecular polycyclic aromatic hydrocarbons and polychlorinated biphenyls when compared to fish from Welaka. Metals were more variable across sites, with highest mean concentrations found in bass from either Julington Creek (Ag, As, Cr, Cu, Zn) or Welaka (Cd, Hg, Pb, Se, Tn). Female bass from Palatka and Green Cove had lower concentrations of E2, VTG and lower GSI in relation to Welaka. Males from Palatka and Green Cove showed comparable declines in 11-KT in relation to males from Julington Creek and GSI were decreased only in Palatka males. These results indicate a geographical trend in reproductive effects, with changes being most pronounced at the site closest to the paper mill (Palatka) and decreasing as the St. Johns River flows downstream. Since reproductive alterations were most evident in bass sampled from the site closest to the paper mill discharge, it is possible that exposure to these effluents might explain at least some of the results reported here. However, the presence of reproductive alterations in fish sampled at a considerable distance from the mill discharge (Green Cove, 40 km) would suggest exposure to chemicals released from sources other than the paper mill plant. It is clear that additional studies are needed to evaluate the potential impact of these reproductive changes in populations of Florida largemouth bass inhabiting the St. Johns River.

Long-term studies on the reproductive fitness of fish under controlled exposure conditions are necessary to address some of the controversy surrounding the field-based studies of pulp and paper effluent effects. This study undertook effluent exposures of 2+ age rainbow trout that were approximately halfway through gonadal growth. Trout were exposed to a mixed thermomechanical/bleached kraft effluent in 12,000-L flow-through exposure tanks at an environmental research facility located at a pulp and paper mill in Kawerau, New Zealand. Trout were exposed to either upstream river water or 10% effluent in upstream river water and were maintained at a ration of 0.7% of body wet weight during the experiment. Results of the 2-month study indicated that trout survival was not significantly different between effluent-exposed tanks and reference tanks. There was extensive growth during the exposure but no differences were found due to effluent exposure. Gonadal development was not significantly different between treatments. Steroid hormone concentrations in males and females were not affected by effluent exposure. The effluent showed no potential to be estrogenic as indicated by a lack of vitellogenin induction in male trout. Other physiological indicators of energy storage and utilization also showed no significant differences. Modest induction of hepatic 7-ethoxyresorufin-O-deethylase (2.5-fold) was the only detectable biological effect of the exposure. Biliary concentration of effluent-related compounds were typical of pulp mill effluent exposure and further suggested that the source of phytosterols was in fact dietary and not effluent-derived.

This study evaluated the potential effects of different concentrations of bleached/unbleached kraft mill effluent (B/UKME) on several reproductive endpoints in adult largemouth bass (Micropterus salmoides). The kraft mill studied produces a 50/50 mix of bleached/unbleached market pulp with an estimated release of 36 million gal of effluent/day. Bleaching sequences were C90d10EopHDp and CEHD for softwood (pines) and hardwoods (mainly tupelo, gums, magnolia, and water oaks), respectively. Bass were exposed to different effluent concentrations (0 [controls, exposed to well water], 10, 20, 40, or 80%) for either 28 or 56 days. At the end of each exposure period, fish were euthanized, gonads collected for histological evaluation and determination of gonadosomatic index (GSI), and plasma was analyzed for 17beta-estradiol, 11-ketotestosterone, and vitellogenin (VTG). Largemouth bass exposed to B/UKME responded with changes at the biochemical level (decline in sex steroids in both sexes and VTG in females) that were usually translated into tissue/organ-level responses (declines in GSI in both sexes and in ovarian development in females). Although most of these responses occurred after exposing fish to 40% B/UKME concentrations or greater, some were observed after exposures to 20% B/UKME. These threshold concentrations fall within the 60% average yearly concentration of effluent that exists in the stream near the point of discharge (Rice Creek), but are above the <10% effluent concentration present in the St. Johns River. The chemical(s) responsible for such changes as well as their mode(s) of action remain unknown at this time.

Perch (Perca fluviatilis L.) and roach (Rutilus rutilus L.) populations in a state of late vitellogenesis were studied downstream of two pulp and paper mills and at upstream references in southern Lake Saimaa, Finland. The mills used elemental chlorine-free bleaching and activated sludge effluent treatment technologies. The exposure of fish to pulp mill effluents, as measured by concentrations of chlorophenolics in the bile and liver ethoxyresorufin-O-deethylase (EROD) activity, was low and almost similar to the references. Nevertheless, bile resin acid concentrations in exposed perch and roach (260-320 micrograms/ml) and bile beta-sitosterol concentrations in exposed roach (1.5-3.5 micrograms/ml) were, respectively, 10 to 30 times and 2 to 5 times higher compared with the references. Reproductive parameters like plasma 17 beta-estradiol and testosterone levels were lower in exposed male and female perch but the same in roach. Vitellogenin concentrations in plasma and liver cytosol of roach were similar. Gonad weight and fecundity were lower in exposed female perch but similar in roach. The relative liver weight was higher in exposed roach but not in perch. The body condition and immunologic parameters in fish were the same. The growth and age at maturity of exposed perch and roach were similar to the references. The size and age distribution of perch in the recipient of one of the mills was shifted toward smaller and younger fish. Our results show that several reproductive variables were altered in late vitellogenic perch in the vicinity of the mills, but they were not altered in roach.

Two different materials-aspen wood-wool and paper towel-were compared as nesting material for three inbred mouse strains (BALB/c, C57BL/6J and DBA/2) housed in barrier conditions. In addition, the effect of varying the number of females per cage (one to three per cage) of these three strains and with NIH/S outbred mouse stock was studied. The number of litters, litter size and neonatal mortality were determined, as well as age, sex and weight of weanlings. The type of nesting material did not affect the characteristics monitored. In all strains, the number of weanlings per female was greatest in singly-housed females. In terms of the number of weanlings per cage, two females per cage gave the best result. In DBA/2 mice, neonatal mortality increased when several females were caged together.

In the early 1990s, many Canadian pulp and paper mills implemented process changes to comply with new regulations that came into effect in 1993. These regulations placed stricter guidelines on a number of parameters in effluent discharges, including limits on acute toxicity, on the discharges of suspended solids, and on biochemical oxygen demand. To meet these new regulations, many of the older Canadian pulp and paper mills had to install secondary treatment systems. The investment by the Canadian pulp and paper industry was in excess of $5 billion, and the implementation of the new regulations and the process changes took several years. The new regulations were an extension of regulations designed in the early 1970s and were not designed specifically to address the reproductive responses recently reported in fish collected downstream of mills in Scandinavia and North America. This report describes a series of projects conducted between 1991 and 1996 to evaluate the effectiveness of the new regulations to address the issue of reproductive responses in fish associated with exposure to pulp-mill effluents. These studies have shown that the existing short-term bioassays do not adequately predict the potential of effluents to affect reproduction in wild fish. Laboratory testing using fathead minnows exposed over a full life cycle confirmed depression in sex steroid production, delay in sexual maturity, reduced egg production, and changes in secondary sex characteristics documented at some sites. Our studies demonstrated that both steroid hormone changes and induction of liver detoxification enzymes take place quickly. While short-term exposures can predict the potential of some effluents to impact steroid hormone production, there is no readily available assay that can be widely applied. In the absence of a usable and transferable laboratory bioassay, field collections were conducted at a number of sites. Generalizations are not possible at this time, but impacts have been seen at a variety of sites, and partial recovery has been documented at five sites in North America following various process and waste treatment changes. Data gaps and critical research areas are identified.

The results and interpretations of published Canadian field studies on the reproductive status of fish in waters receiving pulp and paper mill effluent discharges were reviewed. Most of the information was obtained from indicator measurements such as gonad size, fecundity, and serum steroid levels in wild fish sampled at reference and effluent-exposed sites. Difficulties in selecting appropriate sampling sites, natural variability, and the ecological relevance of the indicator measurements were identified as major complicating factors for the interpretation of the field data. Consequently, it was not possible to conclude to what extent, if any, widespread effects on fish reproduction are being caused by pulp and paper mill effluents or that specific manufacturing processes are causing such effects. Further research on the normal variability and predictive capability of reproductive indicators, for example, using an integrated approach (i.e., laboratory testing, mesocosm studies, and field work), is recommended.

Fish populations residing in a river receiving bleached kraft mill effluents (BKME) and in an uncontaminated river were investigated to evaluate causal relationships between exposure to BKME and various indicators of fish health. The Index of Biotic Integrity demonstrated that species richness and composition were much lower in the contaminated river with an obvious imbalance in the trophic structure of the fish community. Biomolecular and biochemical responses such as DNA damage and elevated activity of detoxification enzymes indicated that fish in the contaminated river had been exposed to toxicants. The status of various condition indices such as the liver and visceral somatic indices suggested metabolic and nutritional imbalances in sunfish as a result of exposure to pulp mill effluents. Fish populations in the contaminated river also demonstrated an abnormal size distribution and age structure. Female redbreast sunfish from the BKME-impacted river contained a large number of atretic oocytes and had lower serum levels of estradiol than fish from the reference site. These observations plus data from previous developmental toxicity studies suggest that the primary mechanism by which BKME may affect fish populations in this river is through reproductive dysfunction and recruitment failure. Decreased recruitment may have caused a reduction in population size and resulted in decreased competition and increased resource availability. With increased food and habitat availability, more energy may have been available for growth and lipid storage of survivors in the contaminated river.