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A Decade of Research on the Environmental Impacts of Pulp and Paper Mill Effluents in Canada (1992-2002)

4.2 History

4.2.1 Acute bioassays in EEM

Controlled laboratory exposures of fish and invertebrates to pulp mill effluent (PMEs) have been part of the EEM program for the past decade (Figure 3). The purpose of the short-term assays was to assess potential acute effects of exposure to final effluent, and to monitor changes in effluent quality as the mills implemented treatment and process changes over time. Acute bioassays were conducted twice a year.

The fish short-term assays measure survival and growth in native species exposed to effluent for a week. Fish species used include fathead minnows (Pimphales promelas) or rainbow trout (Oncorhynchus mykiss) at pulp and paper mills discharging into freshwater environments, and inland silverside (Menidia beryllina) or topsmelt (Atherinops affinis) at pulp and paper mills discharging into marine environments. Invertebrate assays examined survival, growth and reproduction/fertilization success in invertebrates exposed to PME for up to 3 weeks. Test organisms used in the invertebrate assays were Ceriodaphnia dubia at pulp and paper mills discharging into freshwater environments, and gametes of sea urchin (Strongylocentrotus purpuratus) or sand dollars (Dendraster excentricus ) at pulp and paper mills discharging into marine environments (Environment Canada, 1992a, b, c, 1998b).

Results of these acute studies showed that implementation of secondary treatment greatly improved the quality of Canadian PME: There were fewer impacts on fish and invertebrate survival and growth as mills installed secondary treatment (Environment Canada, 1997a, b; Scroggins & Miller, 2000; Lowell et al., 2003).

Short-term laboratory tests were assessed for their ability to predict the impact of final effluents on invertebrates and fish in receiving environments. Borgmann et al. (2001) showed that in data collected during the EEM program, invertebrate tests agreed with results from benthic field surveys in three case studies involving seven Ontario pulp mills. As well, fish acute test results agreed well with impacts on wild fish at most of the 16 Ontario mills (Borgmann et al., 2001).

4.2.2 Short-term exposures assessing MFO and steroids

Although acute lethality and growth bioassays could assess short-term effects of pulp and paper mill effluents, it was obvious that better tests were needed, as wild fish health was affected in receiving environments even though the mill's final effluent routinely passed all acute toxicity tests (Figure 3). In an early review of to PME toxicity test data, Kovacs and Megraw (1996) found no consistent patterns in laboratory toxicity test results conducted at several pulp mills utilizing different processes and effluent treatment. Robinson et al. (1994) saw no effects on survival or growth of fathead minnow larvae exposed for 7 d to 14 different pulp and paper mill receiving waters, even though wild fish showed changes in growth and reproductive indicators at several mill sites (Munkittrick et al., 1994a). It should be emphasized that Robinson et al. (1994) tested receiving water, not final mill effluent. Performance of laboratory tests is maximized when final effluents are tested in a series of dilutions, so that a dose-response may be assessed and threshold effluent concentrations may be estimated.

The development of short-term sublethal tests to assess the impacts of pulp and paper mill effluents was based on the observations in wild fish that were common to most pulp and paper mill sites: MFO induction and steroid depressions. Some of the earliest controlled exposures of fish to pulp and paper mill effluent involved caging wild fish in pulp mill effluent streams. These caging studies were useful in determining the short-term responses to pulp mill effluent (MFO induction, steroid depression), and were the first step in progressing from field observations to short-term predictive laboratory tests.

Measurement of MFO enzymes was one of the first biomarkers to be used in laboratory bioassays of fish exposed to pulp mill effluents. To assess the MFO-inducing potential of PMEs, ethoxyresorufin-O-deethylase (EROD) activity in fish liver homogenates was measured after exposures as short as 4 days (Hodson et al., 1996; Parrott et al., 1999).

White sucker captured from a reference site and exposed to bleached kraft mill effluent (BKME) in cages or 80-L containers had dramatic MFO induction (Munkittrick et al., 1999; Parrott et al., 1999). Immature white sucker and juvenile rainbow trout exposed to BKME had dramatic MFO induction (Munkittrick et al., 1999). However, goldfish caged in 50 % BKME showed no MFO response (McMaster et al., 1996a). This may be due to the recalcitrant nature of goldfish to MFO inducers. Kidd et al. (1993) had to intraperitoneally dose goldfish with 50 µg/kg PCB126 to see even moderate MFO induction.

In-situ caging experiments demonstrated some characteristics of the MFO induction which have provided clues to the properties of the inducing substances (See Characterization of Bioactive Chemicals). Studies in 1989 showed that white sucker captured from the effluent plume and held in clean water for 4 d had a marked decrease in liver MFO activity (McMaster, 1991). Exposures of juvenile white sucker to BKME showed EROD induction peaked in 8 d, and remained elevated until 14 d, whether or not the fish were removed to depurate in clean water. EROD activities returned to pre-exposure levels after 24 days (Munkittrick et al., 1994b, 1999).

Laboratory exposures of fish confirmed that EROD activity was induced by effluent from pulp and paper mills of various production types. In a survey using 31 samples of secondary-treated PMEs from eight different mills, Martel et al. (1994) found that rainbow trout MFO was most consistently induced after exposure to kraft mill effluent (both bleached and unbleached). In a broader examination of MFO induction by various PMEs, rainbow trout exposed for 4 d to 10 % final effluent showed significant MFO induction in 29 of 46 effluent samples tested (Martel et al., 1996). Induction was seen in effluent from several types of pulp and paper mills: unbleached and bleached kraft mills as well as thermomechanical and chemi-thermomechanical mills (Martel et al., 1996). MFO induction was seen in trout exposed to primary treated effluent, as well as secondary treated effluent. Ten of the 29 effluent samples caused less than 2-fold induction over control fish MFO. Nineteen of the 29 effluent samples caused higher induction (up to 15 fold) (Martel et al., 1996). Laboratory exposures of juvenile rainbow trout showed induction by final effluent from four bleached kraft mills and one unbleached kraft mill (Williams et al., 1996). Thresholds for significant MFO induction ranged from 0.3 to 9 % final effluent (Williams et al., 1996). Wood condensates in black liquors also induced MFO in laboratory exposures of rainbow trout (Martel et al., 1994; Hodson et al. 1996; Coakley et al., 2001). Four-day exposures to spent bleaching liquors from two Ontario BKMEs (both using ClO 2 bleaching) caused MFO induction (Coakley et al., 2001). Filtrates from softwood bleaching were about 3-fold more potent than filtrates from hardwood bleaching, and filtrates from the last stage of bleaching were more potent than filtrates from the first stage of bleaching (Coakley et al., 2001).

Because the MFO induction in fish is a very rapid response, studies have examined which mill process streams cause induction, as well as which effluent treatments successfully reduce inducing potency of final effluent. Treatment for the removal of hard-to-degrade organics and an activated sludge treatment process consistently removed more than 85 % of effluent-induced MFO activity from a BKME with 60 % ClO 2 bleaching (Schnell et al., 2000). Characterization of various in-plant process waters sampled at the mill indicated that the softwood-line bleach plant was a major contributor (>70 %) to the MFO induction potential of untreated and biologically-treated BKME (Schnell et al., 2000).

Retene (7-isopropyl-1-methylphenanthrene) in pulp mill effluent and sediments has been investigated as one of the potential causes of MFO induction in fish downstream of pulp mill effluents. This substituted phenanthrene is a derivative of resin acids, and can be produced in anaerobic sediments by bacterial metabolism of resin acids (Wakeham et al., 1980). However, resin acids were not able to induce MFO in fish (Ferguson et al., 1992). Retene can be found at concentrations over 3,000 m g/g dry wt in sediments of rivers receiving PME (Oikari et al., 2002). Increased ethoxyresorufin-O-deethylase (EROD) activity was observed in juvenile trout exposed for 4 d to waterborne retene at 100 m g/L (Parrott et al., 1994; Fragoso et al., 1998, 1999). Induction peaked at 4 days, and moving exposed fish to clean water showed elimination of retene as indicated by EROD activities returning to pre-exposure levels (Fragoso et al., 1998, 1999).

Although much research has focused on the identity of the MFO inducer in PMEs, the conclusions are still elusive. Compounds in mill effluents such as retene, chlorinated and unchlorinated diarylethanes (stilbenes; Burnison et al., 1996, 1999), and compounds from wood extracts such as juvabione and dehydrojuvabione (Martel et al., 1997) can all induce EROD, but there is no definitive list of MFO inducers at various mills. It appears that the MFO inducing compounds are a diverse group, and vary with mill process, wood type, and effluent treatment (See Characterization of Bioactive Chemicals).

With the search for the MFO inducer came considerable debate over the biological relevance of MFO induction (Martel et al., 1994). MFO induction was associated with but not mechanistically-linked to repro-ductive effects in wild fish (Hodson, 1996). Evidence mounted that the compounds causing MFO induction and the compounds causing steroid depression in PME may be different groups of chemicals (Munkittrick et al., 1994a). The focus of the laboratory bioassays then shifted to steroid depression and reproductive effects.

Short-term fish exposures were used to assess the steroid-reducing capacity of PMEs. Field-captured fish could be caged in PME and assessed for changes in circulating steroids. Laboratory fish transported to the field and exposed in situ were used to assess the steroid-reducing potential of PMEs. Laboratory exposures of small fish species were also used to assess PMEs ability to depress steroids. Laboratory exposures for steroid assessments were usually static-renewal assays, lasting from a week to a month. The steroids measured included testosterone, 11-ketotestosterone and estradiol.

A variety of pulp and paper mill effluents have been shown to decrease fish reproductive indicators (such as GSI, fecundity) and alter circulating steroid hormones (See Field Studies and Mechanistic Research). Alterations in reproduction and circulating sex steroid hormones also have been seen in fish exposed to pulp mill effluent in laboratory or mesocosm studies. In general, fish exposed to a variety of pulp mill effluents under controlled conditions in the laboratory have depressed sex hormone levels.

Caging of laboratory test species in BKME streams were the first assays that were able to mimic the reproductive changes and steroid depression seen in wild fish exposed to BKME (McMaster et al., 1996a). Effects on goldfish were most dramatic when exposures were conducted on site, with fish caged in effluent streams. A decrease in potency was noted in laboratory exposures of goldfish to effluent under static, daily renewal conditions. However, McMaster et al. (1996a) propose that this could be due to fish loading density in laboratory exposures, as well as to biological factors (testing of goldfish during reproductive senescence). As well, mill process changes and upgrades make it difficult to assess test sensitivity over time, as effluent quality is continually changing.

Development of techniques for measurement of steroid production by excised gonads of fish (McMaster et al., 1995b) allowed the production of steroids to be assessed in testes and ovaries, under either basal or stimulated conditions (with added human chorionic gonadotropin (hCG), to stimulate the gonad to produce hormones). Techniques for measurement of testicular and ovarian in vitro steroid production paved the way for investigations into the effects of PMEs on the reproductive functions of smaller-bodied fish species (too small to sample blood for sex steroid measurement). Fathead minnows and marine mummichogs exposed to PMEs showed decreases in production of sex steroids by excised testes and ovaries (Robinson, 1994; Dubé & MacLatchy, 2000a). In general, measurements of steroids produced by excised gonads correlate well with levels of circulating steroids.

Controlled exposures using laboratory fish have been used to address whether current PME discharges have potential to cause steroid disruptions, or whether historical input may be associated with the effects. Early studies at Jackfish Bay demonstrated depressions of sex steroid hormones in white sucker (Munkittrick et al., 1994a; McMaster et al., 1996b). Goldfish that were transported to this site and exposed to BKME also showed changes in sex steroids after 4-8 d exposures (McMaster et al., 1996a), indicating current discharges were sufficient to cause the reproductive effects. These findings were confirmed at a bleached sulphite mill (BSM) where goldfish exposed for 21 d on-site in 1997 to final effluent also had reduced circulating sex steroids (Parrott et al., 1999, 2000a). At a bleached kraft mill located in Saint John NB , the receiver is a tidal environment with inputs from other industries and it is not possible to conduct wild fish assessments. In this case, mesocosm studies using mummichog were necessary to evaluate the potential of this effluent to affect fish reproduction. Mummichog exposed for a month to 1 % BKME had depressed circulating sex steroid concentrations (Dubé & MacLatchy, 2000a).

The development of short-term laboratory exposures of fish that could detect the steroid-reducing potential of PMEs allowed researchers to search for bioactive compounds in effluents. The search for the compounds responsible for reproductive impairment in fish exposed to PMEs has examined several plant-based compounds, such as ß-sitosterol and genistein. ß-sitosterol was detected in a survey of 22 US pulp and paper mill effluents at concentrations ranging from 71 to 535 µ g/L (Cook et al., 1996). Goldfish (Carassius auratus) exposed to ß-sitosterol (intraperitoneally injected or waterborne) had decreased plasma steroid concentrations (MacLatchy & Van Der Kraak, 1995; MacLatchy et al., 1995, 1997). ß-sitosterol (= 75 µg/L) reduced the concentrations of plasma testosterone, pregnenolone and cholesterol in immature rainbow trout after 21 d laboratory exposures (Tremblay & Van Der Kraak, 1998). ß-sitosterol also binds weakly to trout hepatic estrogen receptor (ER) and weakly induces vitellogenin in trout hepatocytes and in fish exposed for 21 d to concentrations of 25-100 µg/L (Tremblay & Van Der Kraak, 1998). However, Tremblay and Van Der Kraak (1998) propose that the steroid reducing and ER-agonist activities represent two different modes of action of ß-sitosterol, as it acts differently than traditional ER-agonists such as estradiol or nonylphenol. MacLatchy et al. (2002) have shown that brook trout (Salvelinus fontinalis) and goldfish implanted with ß-sitosterol had a decrease in the available gonadal mitochondrial cholesterol pool for the P450 enzyme responsible for side chain cleavage (P450scc), but had no change in the activity of this enzyme (that converts cholesterol to pregnenolone). The proposed mechanism of action for ß-sitosterol is the impairment of cholesterol transfer across mitochondrial membranes, thereby decreasing pregnenolone and sex steroid levels (MacLatchy et al., 2002).

Another plant-based compound, genistein, was found to alter gonadal development in fish. Genistein is a plant flavanoid, found in the heartwood of many tree species. Concentrations of genistein in final BKME from Ontario were 10 m g/L (Kiparissis et al., 2000). Newly-hatched Japanese medaka (Oryzias latipes) exposed to genistein for 3 months had altered secondary sex characteristics and abnormal gonadal development. Histological examination of the gonads showed that male medaka testes contained eggs (a condition called "testis-ova"), and that male fish also showed a feminization of secondary sex characteristics at concentrations of 1,000 m g/L genistein. Female medaka showed masculinization (male-like dorsal and anal fins) and histological examination of the ovaries showed oocyte atresia (Kiparissis et al., 2000).

Recently, short exposures of laboratory fish have shed light on the genes altered by PME exposure that may be related to decreased hormones. Advances in genetic techniques have determined that the gene expression response pattern of fish to a paper mill effluent was different from the effects of estradiol. Largemouth bass exposed for 7 d to 2 months to 10-80 % bleached paper mill effluent had decreased circulating steroids and vitellogenin. Male fish held for 7 d in the effluent showed a different gene expression pattern than male fish exposed to estradiol (Denslow et al., 2000). The authors suggest that the mechanism for the steroid reductions is not through interference with the estrogenic cascade, but through some other pathway (Denslow et al., 2000). As molecular and genetic techniques advance, laboratory bioassays will evolve to assess the reproductive and steroid-reducing capacity of PMEs with efficient and biologically relevant endpoints.

Short-term exposure bioassays have also been used to assess the potential mutagenicity of compounds in PMEs. Easton et al. (1997) exposed Chinook salmon to PME, and found increases in size variability of red blood cell (RBC) nuclei. There was considerable debate over the findings, and the potential link to the health of BC Chinook salmon populations. Exposure to the PME caused fragmentation of the RBC nuclei but not heritable mutagenic and carcinogenic changes. Laboratory studies assessing the ability of PME to cause genetic mutations have shown that solvent fraction of some PMEs can induce back-mutation of Salmonella bacteria in the Ames assays (Rao et al., 1994, 1995a, b), and can induce micronuclei formation in rainbow trout (Rao et al., 1995b).

In general, field studies have rarely found increases in neoplasms in wild fish exposed to PMEs. However, one field study has shown increases in DNA adducts in fish exposed to BKME. In the Fraser River, BC, a study of wild Chinook salmon exposed to PME detected increased DNA adducts in liver microsomes, although there were no increases in liver lesions as detected by histology (Wilson et al., 2000).

Laboratory exposures of fish to PME have been able to duplicate the DNA adducts seen in wild fish. Wilson et al., (2001) exposed juvenile Chinook salmon for 28 d to up to 2-16 % final effluent from an elemental chlorine-free (ECF) BKME mill. Hepatic microsomal DNA adducts were elevated in fish exposed to 8 and 16 % effluent, and EROD was induced in fish exposed to effluent concentrations of 2 % and higher. The magnitude of DNA adducts correlated well with the EROD activity, and may suggest a mechanistic link: An increase in biological oxidation (increased MFO) within liver cells may generate reactive metabolites. These compounds may then bind to proteins, lipids and other components of cells (DNA and RNA), and may alter their function. Such studies provide insight into the potential biological significance of MFO induction.

Laboratory studies of mussels exposed to PME have shown similar reproductive changes to fish: suppression of sex steroids and decreases in fecundity. Freshwater mussels (Elliptio buckleyi) exposed for 56 d to 40 and 80 % paper mill effluent (from the Georgia Pacific Mill in Palatka , Florida) had decreases in mantle concentrations of estradiol in females and testosterone in males (Gross et al., 2000). None of the female mussels from the 40 and 80 % PME treatment were gravid, compared to 50 % gravid females in control mussels, and 42-46 % gravid females in the 10 and 20 % PME treatments, respectively (Gross et al., 2000). Gross et al. (2000) suggested that mussels may be a sensitive organism to study the reproductive potency of PMEs. Similar decreases in fecundity were seen in fish exposed to this effluent as largemouth bass (Micropterus salmoides) showed a decrease in the number of eggs spawned per female at concentrations of 10 % and above (Sepulveda et al., 2000).

Martel et al. (2003) have investigated the use of caged freshwater mussels (Elliptio complanata) at three pulp mills. Growth of mussels caged for 60 d at 10 stations along a river in Quebec was found to be a sensitive endpoint. The unbleached kraft mill showed increased mussel growth, the thermomechanical pulp mill showed no difference in mussel growth, and the bleached kraft mill showed decreased growth. The decrease in mussel growth was reflective of the decreased benthic density, richness and diversity downstream of this mill (Martel et al., 2003).

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