Critical Review ALTERED REPRODUCTION IN FISH EXPOSED TO PULP AND PAPER MILL EFFLUENTS: ROLES OF INDIVIDUAL COMPOUNDS AND MILL OPERATING CONDITIONS

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1 Environmental Toxicology and Chemistry, Vol. 27, No. 3, pp , SETAC Printed in the USA /08 $ Critical Review ALTERED REPRODUCTION IN FISH EXPOSED TO PULP AND PAPER MILL EFFLUENTS: ROLES OF INDIVIDUAL COMPOUNDS AND MILL OPERATING CONDITIONS L. MARK HEWITT,* TIBOR G. KOVACS, MONIQUE G. DUBÉ, DEBORAH L. MACLATCHY, PIERRE H. MARTEL, MARK E. MCMASTER, MICHAEL G. PAICE, JOANNE L. PARROTT, MICHAEL R. VAN DEN HEUVEL,# and GLEN J. VAN DER KRAAK Aquatic Ecosystem Protection Research Division, Environment Canada, Burlington, Ontario L7R 4A6 FP Innovations-Paprican Division, Pointe-Claire, Quebec H9R 3J9, Canada Toxicology Centre, University of Saskatchewan, Saskatoon, Saskatoon S7N 5B3, Canada Canadian Rivers Institute and Department of Biology, Wilfrid Laurier University, Waterloo, Ontario N2L 3C5 #Canadian Rivers Institute and Department of Biology, University of Prince Edward Island, Charlottetown, Prince Edward Island C1A 4P3 Department of Integrative Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada (Received 11 March 2007; Accepted 14 September 2007) Abstract 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. Keywords Pulp/paper mill effluents Endocrine disruption Biotreatment Fish reproduction INTRODUCTION For more than 25 years, there have been reports that effluents from pulp and paper mills affect fish reproduction. Studies involving wild fish, in situ experiments, and laboratory in vivo tests conducted in Sweden, Canada, Finland, the United States, and New Zealand have documented reductions in sex steroid hormone levels, gonad size and fecundity, alterations in secondary sex characteristics, and delayed sexual maturity associated with exposure to mill effluents. Since the late 1980s, the global industry has made significant changes in mill operating conditions; these changes were designed, in part, to mitigate environmental impacts and to comply with regulatory requirements. The elimination of polychlorinated dioxins and furans from mill effluents through the reduced use of molecular chlorine in bleach plants, the installation of biotreatment systems, and the implementation of color-removal strategies have combined to produce effluents with significantly reduced loadings of organochlorines, biological oxygen demand (BOD), acute lethal toxicity, suspended solids, and color [1 4]. Despite these changes, effects on fish reproduction continue to be reported in Canada [5], the United States [6], Sweden * To whom correspondence may be addressed (mark.hewitt@ec.gc.ca). Published on the Web 10/31/2007. [7,8] Finland [9], and New Zealand [10], with effects also noted recently in countries with emerging pulp and paper industries, such as Chile [11,12]. Perhaps the most up-to-date trends are evident from the regulatory Environmental Effects Monitoring (EEM) program implemented in Canada during the mid-1990s. As part of the EEM program, mills are required to monitor fish and benthos in their receiving environments in three-year cycles. This information is then used to assess the adequacy of the effluent regulations in protecting the environment on a site-specific basis [13]. Within the EEM, an effect on the fish population and on the benthic invertebrate community is defined as a statistically significant difference in a measured parameter between an area exposed to effluent and a reference area or as a statistically significant gradient within the exposure area [14]. If effects are observed above a critical effect size, mills are required to undertake an investigation of cause [15] and, pending regulatory approval, an investigation of solutions to remedy the situation. Information from the first three cycles of the EEM in Canada has revealed two national average response patterns in fishes namely, enhanced growth and enhanced condition that can be attributed to nutrient enrichment and metabolic disruption [14]. Metabolic disruption is characterized by increased body condition, increased liver size, and decreased gonad size (all relative to reference), and metabolic disruption has been documented in mills employing a range of pulp pro- 682

2 Altered reproduction in fish exposed to pulp mill effluents Environ. Toxicol. Chem. 27, Table 1. Summary of effects of pulp mill effluents on wild fish Location and time of studies Mill process type a Effluent dilution (%) Species Main effects noted References Florida (USA) rivers, late 1970s to present Baltic Sea (Sweden), 1980s to 1990s Canadian freshwaters ( 15 sites), late 1980s to present Baltic Sea (Sweden), late 1990s to 2002 North Carolina/Tennessee (USA) rivers, 1989 and 1990 Florida (USA) rivers, late 1990s Waikato River (New Zealand), 2002 Lake Saimaa (Finland), 1995 and 1996 Bleached kraft 80 Mosquito fish and other Poicillidae Bleached and unbleached kraft Bleached kraft, mechanical/sulfite, sulfite TMP, BCTMP (overall average. 1) Mainly perch (Perca fluviatilis) and roach (Rutilus rutilus) 10 species, mainly white sucker (Catostomus commersoni) Bleached kraft 1 5 Eelpout (Zoarces vivparous) Bleached kraft 75 Redbreast sunfish (Lepomis auritus) Bleached kraft Largemouth bass (Micropterus salmoides floridanus) Bleached kraft 50 Brown bullhead (Ameiurus nebulosis) Bleached kraft 1 4 European perch (Perca fluviatilis L.) and roach (Rutilus rutilus L.) Masculinization of females based on secondary sex characteristics Reduced gonad size; larval mortality Reduced gonad size, circulating sex [5] hormones, and fecundity; delayed maturity; changes in secondary sex characteristics Greater proportion of male embryos [5] Lower serum levels of estradiol and increased incidence of atretic vitellogenic oocytes in females Reduced gonad size, lower plasma sex hormones, and reduced vitellogenin in females Lower serum levels of steroid hormones, no change in gonad size Decreased gonad size and plasma sex steroid hormones in perch only [5,57,117,121] [16,20,135,136] [137] [5] [138] [9,139] a TMP thermomechanical pulping; BCTMP bleached chemithermomechanical pulping. duction and effluent treatment types discharging into a variety of environments (e.g., riverine, lake, and estuarine). The consistency of this response pattern as well as the evidence from other studies worldwide has highlighted the necessity for mitigation. Because the sources, identities, and biological mechanisms associated with fish reproductive impairment, however, remain unresolved, little information is available about what mills can do to improve effluent quality in relation to fish reproduction. The purpose of this review is to provide an overview of the primary reproductive effects observed in fish exposed to pulp and paper mill effluents, the current state of knowledge regarding the sources/causes of reproductive effects, and the consequences of changes in mill operating conditions in relation to fish reproductive impacts. It is hoped that this information will facilitate research leading to the formulation of cost-effective strategies for eliminating reproductive effects associated with the effluent discharges, thereby ensuring the sustainability of both fish populations and the pulp and paper industry. REPRODUCTIVE RESPONSES IN FISH EXPOSED TO PULP MILL EFFLUENTS The first indications linking mill effluents to reproductive effects were noted in wild fish (summarized in Table 1). Subsequently, fish exposures in laboratory, mesocosm, and in situ studies also demonstrated that mill effluents can influence a variety of reproductive endpoints (Table 2). Whereas some results vary by species and from study to study, effluents can affect fish reproduction in multiple ways, with the most notable and consistent responses including decreased gonad size, decreased production and/or levels of gonadal sex steroids, hormone receptor interactions, altered expression of secondary sex characteristics, and decreased egg production. Decreased gonad size Numerous studies have shown that gonad size is decreased in wild fish exposed to pulp and paper mill effluents (Table 1). It is one of the most consistent responses observed, and it has been reported in Sweden [16], Finland [17], Canada [5], and New Zealand [18]. Decreased gonad size has coincided with delayed sexual maturation of fish in Canada [18,19] and Sweden [20]. In Canada, reductions in gonad size have been consistently measured in white sucker (Catostomus commersoni) exposed to effluent from a bleached kraft mill located at Jackfish Bay (ON, Canada) since the late 1980s [21]. Decreased gonad size also has been consistently found in perch (Perca fluviatilis) collected near the bleached kraft mill located at Norrsundet (Sweden) [7]. Reductions of gonad size have been observed in fish at other Canadian mills as well [22,23]. Furthermore, three successive cycles of Canadian EEM studies from 1992 to 2003 evaluated multiple fish species and determined an average decrease in gonad size of both sexes of fish at approximately 60 mills [14]. Decreased gonad size, combined with increased energy use and storage, had been interpreted as a form of metabolic disruption [24]. Although observed primarily in wild fish, gonad size reductions have been observed in laboratory-based, life-cycle studies with fathead minnow (Pimephales promelas) [25] and in mesocosm (i.e., artificial stream) studies with mummichog (Fundulus heteroclitus) [26]. Decreased production of gonadal sex steroids In many studies, decreases in circulating levels of gonadal sex steroids (testosterone, 17 -estradiol, and 11-ketotestosterone) coincide with decreased gonad size. Studies with white sucker exposed to bleached kraft mill effluent (BKME) at Jack-

3 684 Environ. Toxicol. Chem. 27, 2008 L.M. Hewitt et al. Table 2. Summary of effects of pulp mill effluents from in vivo laboratory, in situ, and mesocosm fish studies Species Effluents (location of studies) a Test description and exposure duration End points affected at effluent eoncentration References Fathead minnow (Pimphelas promelas) Mosquito fish (Gambusia affinis) Shortfin eel (Anguilla australis) Guppy (Poecilia promelas) Mummichog (Fundulus heteroclitus) Goldfish (Carassius auratus) Rainbow trout (Oncorhynchus mykiss) Largemouth bass (Micropterus salmoides) Three-spined stickleback (Gasterosteus aculeatus) BKME, BSME, and TMP (Canada and United States) Life cycle (egg to sexual maturity); 6 months Egg production at %; also changes in secondary sex characteristics, including masculinization and feminization, changes in sex hormones, delayed sexual maturity Egg production and VTG induction at 20% BKME, TMP, and MP (Canada) Adult reproduction; 21 d [1,54] BKME and TMP (New Adult exposure; 21 d Masculinization at 100% [10] Zealand) BKME, TMP, and Juvenile in situ exposure; Increased plasma estradiol and tes- [140] CTMP (New Zealand) 21 d tosterone at 10% BKME (Sweden) Adult exposure; 42 d Masculinization at 5 25% [134] BKME (Canada) Adult exposure; 7 57 d Reduced plasma testosterone at 1 and 5% BSME, BKME, and Adult exposure; 8, 16, Reduced circulating sex hormones TMP (Canada) and 21 d and gonadal hormone production BKME and TMP (New Zealand) BKME and unbleached KME (USA) Primary BKME (Sweden) Maturing (two years or older); 8 months Adult (1.5 years); d Adult females; 42 d at % Reduced gonads, testosterone, and estradiol in females at 12%; reduced larval size in progeny of exposed adults. Reduced sex hormones and gonad size at 20% Masculinization (increased spiggin and kidney epithelial cell height) at 10% [61 66, ] [30,31] [96,123] [48,124] [50,141] [142] a BKME bleached kraft mill effluent; BSME bleached sulfite mill effluent; MP multiprocess mill (both chemical and mechanical pulping); TMP thermomechanical mill effluent. fish Bay showed both sexes had lower levels of sex hormones in effluent-exposed zones [27], and a number of sites within the pituitary gonadal axis were affected. This included reduced levels of gonadotropin II (i.e., luteinizing hormone), reduced ovarian steroid biosynthetic capacity, and altered peripheral steroid metabolism [28]. Interestingly, ovarian prostaglandin production was not affected in these fish, suggesting that the effects were specific to steroid biosynthesis and not a general response of the ovary to effluent exposure. More recent tests with Canadian bleached sulfite mill effluent (BSME) showed inhibition of steroidogenesis, as evidenced by decreases in pregnenolone concentrations in female rainbow trout (Oncorhynchus mykiss) [29]. Decreases in circulating and gonadal production of testosterone also have been observed in the estuarine mummichog after exposure to primary-treated BKME [30] and in-mill process streams [31]. Other work has focused on potential mechanisms underlying the altered steroid biosynthetic capacities of effluentexposed fish. Studies with white sucker identified a number of sites within the steroid biosynthetic pathway effected by effluent exposure [32]. By measuring the synthesis of the major steroid intermediates in the biosynthetic pathway, it was possible to identify sites where the activities of specific enzymes were altered. Unfortunately, these sites changed with the reproductive state of the fish and did not account for all the reductions. It was more probable that reductions in the availability of the primary substrate cholesterol could account for the major reductions in the production of steroids in exposed fish [32]. Ovarian tissues from these fish also have reduced mrna expression of the steroid acute regulatory protein (StAR) that is responsible for mobilization of cholesterol across the mitochondrial membrane (M. McMaster, Environment Canada, Burlington, ON, unpublished data). Other studies have shown that sitosterol, the dominant plant sterol found in mill effluents, affects steroid biosynthesis in gonadal tissues by interfering with cholesterol mobilization and its conversion to pregnenolone [33]. In the gonad, the StAR protein normally facilitates cholesterol mobilization to the mitochondria, where it is converted into pregnenolone by the cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc). Emerging evidence suggests that expression of StAR and P450scc is reduced in fish exposed to aryl hydrocarbon receptor ligands [34], which are present in mill effluents. Another potential pathway may involve the reduced levels of luteinizing hormone observed in white sucker exposed to BKME [28] and the finding that StAR protein levels are regulated by gonadotropins in zebrafish [28,35]. Recently, StAR mrna transcript levels and plasma testosterone levels have been shown to be reduced in male goldfish (Carassius auratus) after five months of exposure to sitosterol [36]. Hormone receptor interactions Pulp mill effluents contain ligands for nuclear sex steroid receptors and the plasma sex steroid binding protein, thereby having the potential to affect steroid hormone signaling and transport in fish. Ligands for estrogen and androgen receptors and the sex steroid binding protein have been detected in hepatic tissues of fish exposed to effluent for 4 d at the Jackfish Bay mill [37], at a Canadian bleached sulfite mill [38], and in wild fish collected during the spring spawning migration below an additional Canadian bleached kraft mill [39]. Effluents from each of these mills have been shown to cause reproductive

4 Altered reproduction in fish exposed to pulp mill effluents Environ. Toxicol. Chem. 27, Table 3. Summary of studies investigating the effects of pulp mill effluents on vitellogenin (VTG) in fish Mill type a Species b Study type (location) Length of exposure Sex c response d VTG References BKME White sucker Field (Canada) Life M [44,142] F 0 MP White sucker Field (Canada) Life M 0 [44] F 0 BKME Largemouth bass Field (USA) Life M 0 [143] F BKME Largemouth bass Mesocosm (USA) d M 0 [51] F BKME Bluegill Mesocosm (USA) 8 months M and F 0 [52] BKME Perch Field (Finland) Life M 0 [9] F BKME Perch Field (Finland) Life M 0 [9] F 0 BKME Roach Field (Finland) Life M 0 [9] F 0 BKME Roach Field (Finland) Life M 0 [9] F 0 BKME Whitefish Field (Finland) 30 d I [144] BKME Whitefish Field (Finland) 30 d I 0 [144] BKME Whitefish Field; Finland 30 d I 0 [144] TMP and kraft Rainbow trout Caged (Sweden) 21 d I 0 [46] BSME Rainbow trout Laboratory (Canada) 21 d I [53] BKME Rainbow trout Laboratory (Canada) 21 d I [53] BSME Rainbow trout Laboratory (Canada) 21 d I [29] BKME Rainbow trout Laboratory (Canada) 21 d I 0 [29] BKME and TMP Rainbow trout Laboratory (New Zealand) 60 d M 0 [48] BKME and TMP Rainbow trout Laboratory (New Zealand) 60 d M [48] F BKME and TMP Rainbow trout Mesocosm and laboratory 7, 14, 21, 28, and 320 d I 0 [47] (New Zealand) BKME Brown trout Laboratory (Canada) 15 d I 0 [45] BKME Brown trout Laboratory (Canada) 28 d I 0 [45] TMP Fathead minnow Laboratory (Canada) 21 d I [54] BKME Fathead minnow Laboratory (Canada) 21 d I [54] MP Fathead minnow Laboratory (Canada) 21 d I [54] TMP Fathead minnow Laboratory (Canada) 21 d I 0 [54] BKME Fathead minnow Laboratory (Canada) 21 d I 0 [54] MP Fathead minnow Laboratory (Canada) 21 d I 0 [54] TMP Fathead minnow Laboratory (Canada) 21 d I [1] BKME Fathead minnow Laboratory (Canada) 21 d I [1] MP Fathead minnow Laboratory (Canada) 21 d I [1] TMP Fathead minnow Laboratory (Canada) 21 d I 0 [1] BKME Fathead minnow Laboratory (Canada) 21 d I 0 [1] BKME Fathead minnow Laboratory (Canada) 5 months M and F [145] River sediment Rainbow trout Laboratory (Chile) 29 d I [11] BKME Rainbow trout In situ (Chile) 11, 21, and 30 d F [12] BKME, TMP, BCTMP, and BSME Rainbow trout hepatocytes Laboratory (Canada) 4 d (extract incubation), 0, [55] a BCTMP bleached chemithermomechanical mill effluent; BKME bleached kraft mill effluent; BSME bleached sulfite mill effluent; MP multiprocess mill (both chemical and mechanical pulping); TMP thermomechanical mill effluent. b Bluegill, Lepomis macrochirus; brown trout, Salmo trutta; fathead minnow, Pimephales promelas; largemouth bass, Micropterus salmoides; perch, Perca fluviatilis; rainbow trout, Oncorhynchus mykiss; roach, Rutilus rutilus; white sucker, Catostomus commersoni; whitefish, Coregonus lavaretus. c F female; I immature; M male. d 0 no change; increase; decrease. alterations in wild fish. Ligands for the androgen receptors have been detected in Swedish kraft mill effluent [8] that causes male-biased sex ratios of eelpout (Zoarces viviparous) [40] and have been linked to masculinization of mosquito fish (Gambusia affinis) in Florida (USA) [6,41] and New Zealand [10]. Hormone receptor interactions also are evident in the expression of estrogenic responses. Increased amounts of the yolk precursor vitellogenin (VTG) are detected in males following exposure to estrogen agonists as well as to androgenic substances that are metabolized to estrogens [42]. In the case of pulp mill effluents, the available information concerning effluent effects on VTG induction is highly variable and may be related, in part, to exposure concentrations and differences in species sensitivities (Table 3). Several studies have found no effects of effluent exposure on VTG production, and other studies have shown that circulating levels of VTG in females is depressed [43]. For example, in the Moose River basin of Canada, circulating levels of VTG were reduced in white sucker females downstream from a bleached kraft mill, corresponding to reduced circulating levels of 17 -estradiol [43,44]. No VTG induction was observed in immature brown trout (Salmo

5 686 Environ. Toxicol. Chem. 27, 2008 L.M. Hewitt et al. trutta) exposed to two Canadian kraft effluents, one at 25 to 100% for 15 d and the other at 100% for 28 d. [45]. Elevated VTG was not seen in roach (Rutilus rutilus) caged downstream of two elemental chlorine free (ECF) mills in Finland, and VTG in females was decreased in perch downstream from one of the two mills [9]. Similarly, no VTG induction has been observed in sexually mature rainbow trout exposed to mill effluents in Sweden [46]. In New Zealand, a BKME/thermomechanical (TMP) effluent was shown to induce VTG production in only one of four experiments [47 49], suggesting temporal fluctuations in effluent compositions. In the United States, largemouth bass (Micropterus salmoides; mesocosm and wild) exposed to BKME in Florida [50,51] and bluegill (Lepomis macrochirus) exposed in artificial streams for eight months to 12 and 30% BKME from a mill in North Carolina (USA) [52] also showed no evidence of estrogenic effects as measured by VTG induction. Conversely, examples also exist in which the induction of VTG was associated with effluent exposure (Table 3). Sexually immature rainbow trout exposed in the laboratory to effluents from Canadian bleached sulfite and bleached kraft mills exhibited significant VTG induction [53]. In tests with 11 Canadian mill effluents, male fathead minnows exposed to 20 or 40% effluent from TMP, kraft, and multiprocess (both chemical and mechanical pulping) mills showed VTG induction as the most frequent response [1,54]. Emerging information regarding the potential of effluents from South American mills to affect fish reproduction showed a twofold induction of plasma VTG in rainbow trout exposed for 29 d in the laboratory to sediments collected below the discharge of four Chilean pulp and paper mills in the Biobio River [11]. The increased level of VTG also was associated with increased gonad size and the presence of more mature ovarian follicles in females. Immature female trout caged for 21 d at the same locations where the sediments were collected exhibited four- to fivefold increases in VTG levels that coincided with enhanced gonadal maturation (presence of vitellogenic oocytes) [12]. Other studies have shown that different pulp mill effluents may contain compounds that exhibit estrogenic or antiestrogenic activity. Biotreated effluent extracts from Canadian mills of several process types were shown to increase VTG production in primary cultures of rainbow trout hepatocytes, whereas others antagonized the actions of 17 -estradiol on VTG induction [55]. These observations, in combination with exposure concentrations and species sensitivities, may account for the discrepancies noted in the literature. Altered expression of secondary sex characteristics The earliest evidence of pulp mill effluent induced changes in secondary sex characteristics comes from the 1980s: Female mosquito fish in the Fenholloway River of Florida were found to be masculinized [56,57]. One of the most conspicuous indicators for masculinization of female mosquito fish is the formation of a gonopodium, which involves elongation of the anal fin that typifies sexual development in males. This has been observed in mosquito fish exposed in the laboratory to the kraft effluent discharged to the Fenholloway River [57,58] and, more recently, to ECF kraft/tmp effluent from New Zealand, where effluent filtration eliminated the masculinization effect in laboratory exposures [10]. Follow-up work at the New Zealand mill showed no masculinization in wild mosquito fish and that effluent-associated suppression of in vitro production of ovarian sex steroids was not related to masculinization [59]. A significant amount of work has been directed at the role of microbial transformation of phytosterols contributing to masculinization and is discussed below (see Experiments with individual compounds and Toxicity identification evaluation). An example of reduced expression of secondary sex characteristics are white sucker collected from Jackfish Bay that showed reduced numbers of tubercles in males exposed to primary-treated effluent [27]. Interestingly, after installation of secondary treatment, tubercles were noted in some females, and some recovery, although not complete, was found in males [3]. Effects on secondary sex characteristics also have been noted in fathead minnow and represent one of the most sensitive responses of this species to effluent exposure in the laboratory [60]. Responses that have been observed in laboratory studies include delayed development of sex characteristics, demasculinization of male fish, feminization of male fish (e.g., ovipositor development), and masculinization of female fish (tubercles and dorsal fin dot) [25,61 63]. Decreased egg production Controlled laboratory studies have consistently found that mill effluents have the capacity to affect egg production. Lifecycle studies involving fathead minnows exposed to effluents from U.S. and Canadian bleached kraft mills [63 66], unbleached kraft mills [64,65], and one bleached sulfite mill [25] have found reductions in the number of eggs produced. Effects on egg production commonly have been assessed with laboratory studies using fathead minnows in life-cycle assays (from eggs to reproductively mature adults; approximately five months) and in abbreviated life-cycle tests of 28 d. Abbreviated tests involve a pre-exposure period, from which spawning pairs of fish are selected, followed by an exposure phase [67]. Egg production was negatively affected at concentrations of effluent lower than approximately half the effluent concentrations that negatively impacted in vitro steroid production [65]. Employing a 28-d adult reproductive test [67], egg production recently has been used to survey the effects of 11 Canadian effluents [1,54] as well as to track sources of endocrine-disrupting substances at the bleached kraft mill in Jackfish Bay [68,69]. In the 11-mill survey, in which effluent concentrations did not exceed 40%, egg production was halted completely by one of the effluents. For the Jackfish Bay mill, on-site exposures subsequently were used to test the effects of various process streams within the mill. This showed that both the combined mill effluent (before secondary treatment) and the combined alkaline stream caused decreased spawning events and decreased egg production. SOURCE AND NATURE OF SUSPECTED CAUSATIVE AGENTS Pulp and paper mill effluents are complex matrices containing material from wood (e.g., extractives, phytosterols, and trace metals), process derivatives or compounds formed during pulping/bleaching (e.g., dimethyl disulfide formed during kraft pulping), additives (e.g., polymeric formulations used as retention aids in papermaking), and if the effluent is biotreated, (partially) biodegraded products of the above. Much of the chemical characterization of mill effluents was performed during the 1980s and 1990s [70 73], with no reports of detailed chemical characterization of effluents appearing since the mid- 1990s. Following process modifications over the last decade,

6 Altered reproduction in fish exposed to pulp mill effluents Environ. Toxicol. Chem. 27, however, effluent compositions have changed markedly since the previous characterizations, and this lack of information impedes efforts to establish cause-and-effect relationships with present-day effluents. Insight regarding the identities and sources of chemicals affecting fish reproductive homeostasis has arisen indirectly from experiments with individual effluent extractives and longterm field studies. Several attempts also have been made to identify directly the source and nature of substances in mill effluents impacting fish reproduction, classified as toxicity source evaluation (TSE) and toxicity identification evaluation (TIE) studies. The TSE studies involve determinations of individual process wastewaters within the mill that are causing the effect (see Toxicity source evaluation), and the TIE studies describe effluent (or individual waste stream) manipulations that lead to the isolation, identification, and confirmation of the suspected causative agents (see Toxicity identification evaluation). Experiments with individual compounds Many studies have examined the reproductive effects of individual effluent extractives, and much of this work has been reviewed [74] ( documents/mrc report.pdf). These extractives include resin acids (e.g., abietic acid), isoflavonoids (genistein), and phytosterols (sitosterol and stigmastanol). All originate from wood and may end up in the final discharge either unaltered or as a biotransformation product of microbial activity. Several researchers have hypothesized that because the chemical structures of these compounds are similar to those of endogenous steroids, these wood components may be causing the reproductive effects via interference with receptor-mediated pathways. It is important to recognize that although these studies have tested the effects of selected compounds on an individual basis, the effects of mixture interactions that could occur in actual exposures have not been considered. The greatest body of work to date involves phytosterols, mainly sitosterol and, to a lesser extent, stigmastanol. Various formulations of sitosterol have been found to affect various aspects of fish reproduction (e.g., steroid production and VTG induction) in goldfish and rainbow trout [53,75 77]. Detailed follow-up studies have found that the actions of sitosterol leading to depressions in hormone biosynthesis are mediated through effects within the steroid biosynthetic pathway [33,78]. When sitosterol exposures were performed at typical North American mill effluent concentrations [79] in parallel with effluent from a Canadian bleached kraft and a bleached sulfite mill, however, plasma cholesterol was reduced by sitosterol but not by the mill effluents [53], suggesting that sitosterol was not the direct source of the effects. Several long-term fish exposures with sitosterol have been conducted. A phytosterol preparation from wood, containing mainly sitosterol, was used in a multigenerational test with zebrafish (Danio rerio) [80]. Exposure to sitosterol at 10 g/l in the mixture did not affect spawning success. Induction of VTG was noted, however, and the sex ratio of the F 1 and F 2 generations was altered, with evidence for masculinization of the former and feminization of the latter. This suggested that the phytosterol preparation had both estrogenic and androgenic effects. In another experiment, maturing brown trout (Salmo trutta lacustris) were exposed to 10 and 20 g/l solutions of phytosterol (mainly sitosterol) [81]. After artificial fertilization, the development of the next generation was followed. Exposure to phytosterols affected the next generation, as evidenced by higher prevalence of disease, deformities, and mortality. Work on understanding the causes of masculinization associated with sterol exposure dates back to the mid-1980s, with conflicting reports concerning the role of microbial transformation in the effects. During an initial study attempting to explain the cause of masculinization, mosquito fish were exposed in the laboratory to predominantly sitosterol or stigmastanol with associated sterol impurities [82]. Fish also were exposed to the phytosterols mixed with Mycobacterium smegmatis as well as to the bacteria alone. No evidence was found of females being masculinized when exposed to the bacteria alone or to the phytosterols alone. Conversely, masculinization was observed in females exposed to the phytosterol/bacterial mixtures, with slightly greater potency observed for the stigmastanol/bacteria mixture [82]. This indicated that the active compounds are not the phytosterols themselves but, rather, their bacterial degradation products. The actual degradation products were not identified, which has led to much speculation regarding their identities. Other studies with stigmastanol using fathead minnow life-cycle exposures have shown that concentrations an order of magnitude higher than those typically found in final effluents ( 73 g/l) caused no effect, and no correlations have been found between threshold effects on egg production and other phytosterols, including sitosterol, campesterol, stigmasterol, and total phytosterols [83]. Recent work from New Zealand demonstrates that sterols in effluents and their oxidation products resulting from chlorine dioxide bleaching are not androgenic [84]. Other studies have determined that multiple compounds are functioning as ligands for the androgen receptor and that these likely are involved in the responses (see Toxicity identification evaluation). Resin acids have been tested for their ability to affect fish reproduction because of their acute toxicity and ubiquitous nature in effluents from softwood mills. Abietic acid was reported to have weak estrogenic activity [84,85], although the purity of the product tested was low. Because resin acids typically are reduced by more than 90% in biotreatment systems [86], their probable role in causing reproductive effects with fish exposure to biotreated effluent generally is regarded as minor. Genistein was found at concentrations of approximately 13 and 11 g/l at a Canadian kraft mill before and after biotreatment, respectively [87]. In tests with the Japanese medaka (Oryzias latipes), genistein as well as equol, another isoflavonoid, were found to cause feminization of secondary sex characteristics in the males as well as a variety of ovarian alterations in the females [88]. Evidence of intersex (testisova) also was found in males. These effects, however, occurred at genistein concentrations (e.g., 1 mg/l) 100-fold higher than those found in the effluent. Pulp mill effluents also may contain additives; however, to date, only nonylphenol has been examined for its potential to affect fish reproduction. Nonylphenol is a biodegradation product of alkyl phenol ethoxylates present in formulations used for pitch dispersants, felt washers, cleaners, defoamers, and deinking agents, and it has been shown to possess estrogenic properties [89]. The pulp and paper industry did use alkylphenol ethoxylate products, but the use of such products was voluntarily discontinued [90]. As such, nonylphenols likely are not the cause of the effects in the effluents discharged by present-day mills.

7 688 Environ. Toxicol. Chem. 27, 2008 L.M. Hewitt et al. Indirect evidence suggesting cause Based on field studies that monitored fish as mills implemented process changes (e.g., before and after the installation of effluent biotreatment; see Role of effluent biotreatment), several inferences were drawn about the nature of the effluent components associated with changes in circulating steroid hormones [5]. These include the following: Biotreatment does not completely eliminate the causative agents, the causative substances affect fish through waterborne exposure, and continuous effluent exposure was necessary to maintain the effects. Finally, effects on steroid levels are not long lasting that is, once removed from effluent, fish appear to recover, and the effects caused by discharges from modernized or current mills are unlikely to be caused by highly substituted, chlorine-containing compounds. Attempts to correlate water chemistry with biological effects have been largely unsuccessful and underscore the complexity of establishing cause and effect. For example, water samples from sites on three rivers in Florida receiving effluents from three bleached kraft mills were analyzed for chlorinated phenolics, resin/fatty acids, phytosterols, and various group parameters, such as polyphenolics and condensable tannins [91]. The masculinization response in female mosquito fish was greatest at sites where resin/fatty acids and sitosterol peaked in concentration; however, the opposite was found at another site, suggesting that these compounds are not involved with the responses. An additional problem interfering with establishing cause and effect relates to quantifying exposures in field and laboratory studies. To establish dose response relationships and to compare the potencies of different effluents, effluent exposures typically have been quantified using percentage effluent. This measure of exposure alone does not take into account several factors that would influence the toxicological potency of an effluent. The types of wood species used, process type, water use, and efficiency of biotreatment all influence the potency of a given effluent, but these are not consistently reported. Bulk measures, such as BOD [53], total suspended solids [92], conductivity [52], polyphenolics [91], and measures of specific ions (Ca and K ) [49], frequently are used as supporting information to provide an estimate of effluent concentrations. Commonly measured effluent extractives, such as resin acids [50], plant sterols [93], and chlorophenolics [9], also have been used. Several studies do not report any measures or estimates of effluent concentrations [6,94,95]. These inconsistent measures of effluent quality throughout the literature have contributed to difficulties when interpreting bioassay responses between studies and needs to be addressed in future work aimed at finding solutions. Toxicity source evaluation To circumvent problems related to working with extremely complex final effluent matrices, several studies have adopted an approach that investigates individual in-plant process wastes as potential sources of reproductive effects. This TSE approach does not consider chemical modifications that can occur during biotreatment (see Role of effluent biotreatment) or from metabolic alterations, but it offers the advantage of eliminating wastes not involved in the effects as well as simplifying effluent matrices for potential TIE experiments. In one such study, goldfish were exposed to 13 wastewater streams from a bleached sulfite and a bleached kraft mill, and both circulating sex steroids and in vitro gonadal steroid production were measured [96]. Except for the final biotreated BSME tested at 100%, none of the other wastewater streams caused statistically significant reductions. Similarly, only 100% effluent from the secondary clarifier of the biotreatment system elicited steroid responses with the wastewaters in the bleached kraft mill. Using mummichog, a series of experiments have linked depressed steroids to condensates generated from weak black liquor evaporation during chemical recovery as well as from bleachery effluents [30,31,96,97]. This work has continued onsite at another Canadian kraft mill but with a different fish species, because the mill in question discharged into a freshwater recipient [68,69]. Bioassays of 21-d duration were conducted with fathead minnows exposed to 1 and 100% secondary-treated effluent and four process streams. Exposure to the final effluent caused ovipositor development and induction of VTG in males, whereas females developed male secondary sex characteristics. Two process streams were the dominant contributors to the effects measured in the final effluent. Exposure to the acid stream (24% of the total final effluent volume) produced an increase in egg production in females and in VTG induction in males. The alkaline stream (67% of the final effluent flow) decreased egg production and caused VTG induction and ovipositor development in males. Condensates at this mill also contained chemicals associated with the steroid depressions in mummichog [98]. Additional studies are required to further delineate these sources. Toxicity identification evaluation In addition to TSE studies, attempts have been made to identify directly the substances responsible for reproductive effects. The chief method employed is bioassay-directed fractionation. One of the critical aspects in this approach is the choice of endpoint used to drive the chemical manipulations. The development of mechanistic-based bioassays with different pathways of fish reproductive perturbations and shorter turnaround times has enabled their use as investigative tools. Most of the work conducted to date has employed in vitro binding assays to characterize ligands for sex steroid receptors, chemicals associated with in vivo sex steroid depressions, and a combination of in vivo and in vitro techniques to evaluate androgenic and estrogenic substances. A collection of international studies since 2001 has pursued the identities of compounds causing masculinization effects. Using in vitro tests, several attempts have been made to determine the cause of effluent-induced masculinization in Florida. Fenholloway River water receiving BKME was found to contain androgen-receptor agonists that were characterized as nonpolar organic material [6,99]. In one case, further fractionation and TIE work indicated androstenedione as the causative agent [99]. An earlier study in which solid-phase extraction was followed by high-pressure liquid chromatographic fractionation, however, identified a fraction without androstenedione that caused androgenic activity [100]. Further studies have highlighted the possibility that phytosterols can be converted to progesterone, and then to androstenedione and androstadienedione, by microbial populations in the water and sediment downstream from mills [41]. Recent work from New Zealand, however, has shown that relatively high waterborne concentrations ( g/l) of androstenedione and androstadienedione are required to elicit masculinization in mosquito fish [59]. This is consistent with other evidence from New Zealand that sterols present in effluents, as well as their

8 Altered reproduction in fish exposed to pulp mill effluents Environ. Toxicol. Chem. 27, oxidation products resulting from chlorine dioxide bleaching, are not involved in the masculinization of mosquito fish and did not show activity in rainbow trout brain androgen receptor binding assays. Multitiered bioassay directed fractionation experiments of primary and biotreated effluents from a chlorinefree Swedish kraft mill directed by androgen receptors isolated from Atlantic croaker (Micropogonias undulates) ovaries have shown that unidentified compounds possessing diterpenoid skeletons are present in multiple fractions exhibiting binding affinities and that a receptor-mediated pathway is the primary route by which masculinization effects seem to be occurring [8,84]. Using controlled fish exposures, a series of laboratory and field experiments have determined that Canadian BKME and BSME contain multiple ligands for fish sex steroid receptors and the aryl hydrocarbon receptor [37 39], demonstrating bioaccumulation of compounds capable of affecting steroid signaling after brief waterborne exposures. At the Canadian bleached kraft mill where condensates have been identified as a primary source of chemicals affecting steroid levels in mummichog, investigations also have shown that substances reducing testosterone are readily bioavailable, are polar, and can cause an effect within days [101]. In-depth chemical characterization (solid-phase extraction, reverse-phase high-pressure liquid chromatography, and gas chromatography mass spectrometry) of condensates have identified various lignin degradation products and terpenoids to be associated with the effects and to be present in condensates at another bleached kraft mill with documented effects on wild fish [98]. The relevance of these findings to the broader industry (kraft and nonkraft processes as well as biotreated effluents) is the focus of current efforts. To summarize, bioactive substances have been characterized as being polar as well as nonpolar, being water soluble as well as associated with solids, having the potential to act as ligands for a variety of receptors (including estrogen and androgen receptors), and mainly being nonbioaccumulative, requiring a sustained exposure to cause the effects. These findings further highlight the complexity of the presence of multiple bioactive substances functioning by multiple mechanisms. Questions remain about the possibility of common causative agents, particularly those related to wood furnish, that are independent of mill process type. MILL PROCESS/OPERATING CONDITIONS AND EFFECTS ON FISH REPRODUCTION Understanding how manufacturing processes, mill furnish, effluent biotreatment, and actual mill operating conditions (e.g., spill control) can influence effluent quality are important pieces of information for understanding the sources of the reproductive effects and developing corrective strategies. Throughout the course of studies regarding effluent effects on fish reproduction, the industry has undergone substantial changes in operating conditions. These include changes in bleaching practices to eliminate the formation of 2,3,7,8-tetrachlorodibenzodioxin, the increasing use of TMP and bleached chemithermomechanical pulping processes, and the installation and/or upgrading of secondary-treatment facilities. This provides a good opportunity to track the influence of these changes on effluent quality, but it is difficult to form generalizations about the mills of today based on studies conducted 10 years ago. Today, for example, virtually all mills have effluent biotreatment for meeting regulatory BOD and acute lethal toxicity limits. In the 1980s and early 1990s, however, only approximately 60% of the mills in Canada had effluent biotreatment [102]. Nevertheless, reviewing the literature on how mill processes/operating conditions may affect fish reproduction could provide clues as to the origin of the fish responses. Manufacturing processes and effluent quality Mills produce pulp mainly by chemical (e.g., kraft and sulfite) or mechanical (e.g., TMP) means; multiprocess mills employ both processes. Because the effluent components from mills using different manufacturing processes can vary substantially [71], questions exist regarding whether manufacturing processes themselves influence the ability of an effluent to affect fish reproduction. Swedish field studies during the 1980s indicated that effects on fish in the vicinity of bleached kraft mills were greater than effects near unbleached kraft mills [ ]. Coupled with the discovery around the same time of 2,3,7,8-tetrachlorodibenzodioxin in effluents discharged by mills employing chlorine for bleaching as well as a small number of studies at mill sites using other kinds of processes, this led to the speculation that effects on fish were caused exclusively by chlorine-containing compounds [107]. As additional studies were conducted, however, a different picture began to emerge. The initial studies at Jackfish Bay involved an examination of effects associated with BKME. To investigate the health of fish exposed to effluents from other types of mills, fish were examined in the recipients at seven Canadian mills in Ontario during the early 1990s [23,108]. Of the seven study sites, four received inputs from bleached kraft mills, one from a mainly unbleached kraft mill, one from a sulfite mill, and one from a multiprocess mill. At that time, only three of the effluents were biotreated. In general, gonad size and plasma hormone concentrations in effluent-exposed fish were decreased irrespective of mill process, suggesting a uniform response to differing production types. Laboratory tests with fathead minnow have since confirmed that fish reproduction can be affected by effluents from mills using different manufacturing processes. Thus far, tests have been conducted in North America with bleached and unbleached kraft mill effluent [54,63,65,66, ], BSME [62,113,114], effluents from TMP mills [1,54,61], and effluents from multiprocess mills [1,54]. One series of experiments used the same protocol (adult fathead minnows and effluent exposure for 21 d) and allowed a direct comparison of 11 effluents [1,54]. Effluents from four TMP, four bleached kraft, and two multiprocess mills were tested at concentrations of 2 and 20% (v/v); one BKME also was tested at 40% (v/v). Except for the effluent from one multiprocess mill, the remaining 10 effluents affected at least one reproductive endpoint. With the objective of comparing effects from bleached mills employing kraft and sulfite digestion, 21-d laboratory tests with rainbow trout showed that both BSME and BKME decreased pregnenolone concentrations in immature rainbow trout and increased VTG [53]. Only the kraft effluent, however, caused a reduction in circulating testosterone concentrations. Additional comparisons included studies of oxidative stress indicators and circulating sex steroids [29]. In this case, both effluents caused similar oxidative stress responses, but substantial differences were observed in endocrine-disrupting potential. The BSME reduced pregnenolone concentrations in females and caused VTG induction. The BKME caused in-