Oxidative stress responses in longnose sucker (Catostomus catostomus) exposed to pulp and paper mill and municipal sewage effluents

Oxidative stress responses in longnose sucker (Catostomus catostomus) exposed to pulp and paper mill and municipal sewage effluents

Aquatic Toxicology 67 (2004) 255–271 Oxidative stress responses in longnose sucker (Catostomus catostomus) exposed to pulp and paper mill and municip...

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Aquatic Toxicology 67 (2004) 255–271

Oxidative stress responses in longnose sucker (Catostomus catostomus) exposed to pulp and paper mill and municipal sewage effluents Ken D. Oakes a , Mark E. McMaster b , Glen J. Van Der Kraak a,∗ a

b

Department of Zoology, University of Guelph, Guelph, Ont., Canada N1G 2W1 National Water Research Institute, Environment Canada, Burlington, Ont., Canada L7R 4A6

Received 11 September 2003; received in revised form 14 January 2004; accepted 16 January 2004

Abstract While recent evidence indicates that the generation of reactive oxygen species (ROS) and associated oxidative damage are frequently observed in fish populations with exposure to pulp and paper mill effluents, the potential for ROS generation from municipal sewage effluents has not been addressed. This study investigates the utility of measures of oxidative stress in delineating the effects of both pulp and paper mill and municipal sewage discharges. Longnose sucker (Catostomus catostomus) were collected below three pulp and paper mill and two municipal sewage effluent discharges over a 3-year period within the Wapiti and Athabasca River systems in northern Alberta. Biochemical responses in longnose sucker varied between the two rivers systems, with more pronounced changes occurring within the Wapiti River. Of the suite of biochemical parameters examined, fatty acyl-CoA oxidase (FAO) activity was the most sensitive indicator of pulp and paper mill exposure, but was only infrequently induced with exposure to municipal sewage effluent. Hepatic and gonadal 2-thiobarbituric acid reactive substances, lipid hydroperoxides, and hepatic free iron were less consistently elevated with exposure to pulp and paper mill effluent than FAO activity, and were also only infrequently altered with sewage effluent exposure. Hepatic ascorbic acid, liver somatic index, and condition factor were consistently altered with exposure to both sewage and pulp and paper mill effluents. While specific biochemical and organismal responses varied with effluents and time, the collective suite of oxidative stress endpoints proved to be useful tools in identifying relative influences of municipal sewage and pulp and paper mill effluent on fish populations in adjacent receiving waters. © 2004 Elsevier B.V. All rights reserved. Keywords: Pulp and paper mill effluent; Sewage; Fish; Oxidative stress; Multiple point sources

1. Introduction

∗ Corresponding author. Tel.: +1-519-824-4120x53598; fax: +1-519-767-1656. E-mail address: [email protected] (G.J. Van Der Kraak).

It has been well established that pulp and paper mill (Karels et al., 1999; Sepúlveda et al., 2003) and sewage treatment plant (Folmar et al., 1996; Routledge et al., 1998) effluents contain constituents with the capacity to disrupt physiological function in exposed

0166-445X/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.aquatox.2004.01.007

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organisms. As the chemical composition and toxicity of both types of effluents are complex and variable (Suntio et al., 1988; Ahtiainen et al., 1996; Woodworth et al., 1999), the bioactive constituents responsible for disruptions in physiological function are often unknown (Hewitt et al., 2000; McArdle et al., 2000; Sheahan et al., 2002). Recent evidence suggests that waterborne contaminants which generate reactive oxygen species (ROS) may be a significant source of toxicity for aquatic organisms living in polluted environments and may be partially responsible for disruptions in physiological function (Livingstone, 2001; Oakes et al., 2003, in press). Excessive ROS production in response to xenobiotic inducing compounds can overwhelm endogenous detoxifying mechanisms producing the cumulative damage to cellular constituents termed oxidative stress. Biochemical changes consistent with oxidative stress have been demonstrated in fish below numerous pulp and paper mills utilizing differing furnish, bleaching techniques, and effluent treatment strategies (Mather-Mihaich and Di Giulio, 1991; Fatima et al., 2000; Oakes et al., 2003). However, there has been only limited research investigating the occurrence of oxidative stress in organisms exposed to municipal sewage effluent (Avery et al., 1996; Isamah et al., 2000). Estrogens and estrogenic compounds, which are frequently found at elevated concentrations in fish exposed to sewage treatment plant effluent (Folmar et al., 1996; Routledge et al., 1998; McArdle et al., 2000) have been shown to increase concentrations and alter composition of lipids in fish (Mercure et al., 2001). As lipids, especially polyunsaturated fatty acids are readily peroxidized by ROS, endocrine-induced changes in lipids can directly affect oxidative stress susceptibility. Changes in endocrine status produced by sewage treatment plant effluent exposure may also indirectly affect oxidative stress susceptibility through hormonal modification of antioxidant levels, as has been demonstrated in mammals (Aten et al., 1992; Peltola et al., 1996). Elevated estrogens may further contribute to oxidative stress by their production of ROS through redox-cycling (Liehr and Roy, 1990). The objectives of the present study were to evaluate biochemical and organismal responses in fish exposed to municipal sewage and pulp and paper mill effluent, and to determine if these responses were associated with oxidative stress. Measurement end-

points included hepatic and gonadal 2-thiobarbituric acid reactive substances (TBARS) as indicators of oxidative stress associated with lipid peroxidation, fatty acyl-CoA oxidase (FAO) activity and hepatic free iron as possible sources of ROS generation, and ascorbic acid as a measure of antioxidant status. Collectively, the assessment of these endpoints in two river systems receiving both municipal sewage and pulp and paper mill effluent discharges over a 3-year period will help to determine the efficacy of oxidative stress endpoints in delineating the relative impacts of multiple effluents on resident fish populations.

2. Materials and methods 2.1. Study sites Longnose sucker (Catostomus catostomus) were collected from pre-selected reference, municipal sewage effluent exposed, and pulp and paper mill effluent exposed locations over a 3-year period (fall 1999–2001) in the Wapiti and Athabasca Rivers in northern Alberta, Canada, as described in detail by McMaster et al. (in press). All three mills and both municipal sewage treatment plants examined in this study employed primary treatment to remove settleable solids and secondary treatment to reduce biochemical oxygen demand before effluent release. The sewage treatment plant on the Wapiti River system had tertiary treatment installed to reduce the nutrient content of the treated effluent following the 1999 collections, but prior to the fall 2001 collections. Discharge from both sewage treatment plants fluctuated throughout the year with greater discharges throughout the summer months. The pulp and paper mills examined in this study employed three different process types, utilized differing bleaching techniques and mill furnish, and released fairly constant volumes of effluent throughout the year (Table 1). 2.1.1. Fish collection and processing Within the Wapiti River system, fish were collected from four locations. The most upstream is a reference station within the Wapiti River adjacent to its confluence with Pipestone Creek (55◦ 02 50 N; 119◦ 06 10 W). The Pipestone Creek station is approximately 21 km upstream of the City of Grande Prairie,

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Table 1 Description of pulp and paper mills and municipal sewage facilities discharging to the Wapiti and Athabasca River systems Pulp and paper mill

Identifying code for multiple mills

Bleaching sequencea

Process type

Productionb

Wapiti River system (Grande Prairie, AB) Athabasca River system (Whitecourt, AB)

Single mill

DEop D EDc

Kraft (Pulp)

Mill A

Nonee

TMP (Paper)

Mill B

Unknownf

bcTMPg (Paper)

Municipal sewage plant

Plant capacity (m3 per day)

Discharge minimum (m3 per day)

Discharge maximum (m3 per day)

Discharge daily average (m3 per day)

Tertiary treatment

Sampling occasions

Wapiti River system (Grande Prairie AB) Athabasca River system (Whitecourt, AB)

Not known

345100 (winter)

618500 (spring)

439450

ASBNRh

Fall 1999, 2001

17272

3300 (winter)

7000 (spring)

No

Fall 2000

a b c d e f g h

Effluent discharge (m3 per day)

Sampling occasions

885

48236d

Fall 1999, 2001

698

Unknown

Fall 2000

767

Unknown

3800

D: chlorine dioxide; P: hydrogen peroxide; E: alkaline extraction; O: molecular oxygen. Air dried tonnes per day. Mill furnish is 35% lodgepole pine, 62% white spruce, 3% balsam fir and black spruce. Based on 1999 data. Mill furnish dominantly white spruce, but includes lodgepole pine and balsam fir. Mill furnish is 40% softwood chips (50% lodgepole pine, 30% white spruce, 20% black spruce); 60% hardwood chips (100% aspen). Bleached-chemi thermomechanical. Activated sludge biological nutrient removal (removes solids, phosphorus and nitrogen). Installed May 2001.

AB and its municipal sewage outfall (55◦ 04 30 N; 118◦ 41 45 W). The second station was immediately downstream of the sewage outfall, with the third immediately below the effluent discharge of the pulp mill. The mill releases its effluent into the Wapiti River a further 10 km downstream of the municipal sewage discharge. Finally, a second reference station, located 22 km further east within the limnologically similar Little Smoky River, was utilized in the fall 1999 collection only. Fish were collected from this cleanwater tributary about 15 km upstream of its discharge into the Smoky River (55◦ 02 50 N; 119◦ 06 10 W). More detailed descriptions of sampling locations and maps of the study area are provided in McMaster et al. (in press). Within the Athabasca River system, fish were also collected from four locations. The most upstream location is a reference station located adjacent the confluence of the tributary Windfall Creek with the Athabasca River (54◦ 13 35 N; 116◦ 10 15 W) approximately 31 km upstream of the Town of Whitecourt, AB. The first paper mill, referred to hereafter as Mill A, is approximately 21 km downstream of Windfall Creek and 10 km upstream of the town of Whitecourt.

A second paper mill, Mill B, is located at the confluence of the McLeod and Athabasca Rivers, inside the Town of Whitecourt (54◦ 08 25 N; 115◦ 41 45 W). Fish were sampled immediately downstream of both mills. Finally, fish were also collected below the municipal sewage outfall, 4 km further downstream of the effluent discharge of Mill B. Collections below the sewage outfall represent the furthest downstream station sampled in the Athabasca River system. Although the present study only considers the effects of municipal sewage and paper mill effluent discharges at the Town of Whitecourt, influences of both effluent types (sewage and pulp mill) entering the Athabasca River at Hinton, AB (located 185 km upstream to the southwest), cannot be dismissed and may contribute to background responses at all stations. More detailed descriptions of sampling locations and maps of the study area are provided in McMaster et al. (in press). All fish collected within the Wapiti and Athabasca River systems were captured using a Smith Root® electrofishing boat. Captured fish were placed in water-filled holding tanks (<1.5 h) prior to transport to shore for processing. Fish were initially sexed using external morphology with definitive sex and

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maturity determined by visual inspection after the animal was sacrificed. Only sexually mature fish were used in the present study. Fish were sacrificed by a sharp blow to the head followed by severance of the spinal cord in accordance with protocols approved by local animal care committees. Measurements of fork length, total weight, and liver weight were taken and liver-somatic indices (LSI = liver weight/ (total body weight − liver weight)) and condition factor (K = (weight/length3 )) were calculated. Portions of liver and gonad were snap frozen in liquid nitrogen, followed by laboratory storage at −80 ◦ C prior to analysis. Care was taken to expedite the sampling process to minimize time from the death of the fish to immersion of tissues in liquid nitrogen. Tissues remained frozen until analysis and all analyses were performed on samples within 18 months of collection.

the equimolar production of a methylene blue product which was quantified spectrophotometrically using a Biochrom Ultrospec 3100 Pro UV/Visible spectrophotometer. Further assay details are provided in Oakes and Van Der Kraak (2003). 2.2.3. FAO activity The FAO activity assay quantifies production of the ROS H2 O2 specifically generated by an enzyme unique to peroxisomal ␤-oxidation, fatty acyl-CoA oxidase (EC 1.3.99.3). Production of H2 O2 , via the direct transfer of electrons to oxygen by fatty acyl-CoA oxidase, is quantified using lauroyl-CoA as the enzymatic substrate with concurrent measurement of the oxidation of 4-hydroxyphenylacetic acid to a fluorescent product in a horseradish peroxidase coupled reaction (Poosch and Yamazaki, 1986). Further assay details and assay validation are provide in Oakes and Van Der Kraak (2003).

2.2. Biochemical measures 2.3. Iron content 2.2.1. TBARS assay The TBARS assay was used to quantify oxidative stress (lipid peroxidation) in fish liver and gonad samples as described previously (Ohkawa et al., 1979; Oakes and Van Der Kraak, 2003). Briefly, this method involves the reaction of malondialdehyde (MDA), a degradation product of lipid peroxidation, with 2-thiobarbituric acid (TBA) under conditions of high temperature and acidity to generate a fluorescent adduct that was measured spectrofluorometrically using a Perkin-Elmer Luminescence Spectrometer LS50. TBARS were determined on 10% (w/v) whole tissue homogenates containing butylated hydroxytoluene (BHT) in the homogenizing buffer according to the assay optimization for white sucker tissue described in Oakes and Van Der Kraak (2003). 2.2.2. Lipid hydroperoxides (LPO) A mechanistically independent commercially available kit (K-Assay LPO-CC kit; Kamiya Biomedical Company, Seattle, WA) was used to validate oxidative stress responses detected using the TBARS assay. The LPO test was used for verification purposes only on a subset of representative samples. Briefly, the principle of the LPO test is that in the presence of hemoglobin, lipid hydroperoxides produced by lipid peroxidation are reduced to hydroxyl derivatives with

Hepatic iron content was determined by the method described by Chrichton et al. (1980). Briefly, this involves reacting reduced forms of iron within a 10% (w/v) liver homogenate with 2,2 -bipyridyl to produce a complex which absorbs maximally at 520 nm using a Biochrom Ultrospec 3100 Pro UV/Visible spectrophotometer. Further assay details are provided in Oakes and Van Der Kraak (2003). 2.3.1. Ascorbic acid Hepatic vitamin C content was measured according to the protocol described by Jagota and Dani (1982) which involves the reaction the Folin-Ciocalteu reagent with strong reducing agents, dominantly ascorbic acid, to produce a quantifiable blue product measured against an ascorbic acid standard using a Biochrom Ultrospec 3100 Pro UV/Visible spectrophotometer. 2.3.2. Statistics TBARS, FAO, LPO, hepatic ascorbic acid and iron data were checked for homogeneity of variance and normality prior to two-way analysis of variance (ANOVA) for each year with sampling site and sex as the main factors (SPSS version 11.0 for Windows). Differences between sites were detected by

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Tukey’s HSD. Where interactions between the main factors were significant (Psex×site < 0.05), data were re-analyzed by sex using one-way ANOVA followed by Tukey’s HSD and responses (by sex) were plotted on separate axes; otherwise analysis was by two-way ANOVA as indicated by both sexes plotted on the same axis. When differences between the sexes at each station were significant (Psex < 0.05), results of Tukey post-hoc analysis were presented with female differences indicated by different lower case letters, and males with different upper case letters. Where interaction (Psex×site > 0.05) and sex (Psex > 0.05) factors were not significant, parameter differences with exposure (Psite < 0.05) determined by Tukey’s HSD are indicated by different lower case letters above a bar encompassing both sexes. LSI and K were evaluated using analysis of covariance (ANCOVA). Multiple regression analysis was performed by sex on hepatic TBARS using hepatic iron and ascorbic acid as independent variables (SPSS version 11.0 for Windows). Correlation analyses among measured endpoints were performed using both sexes if each endpoint responded similarly with exposure to sewage and pulp and paper mill effluents (Psex×site ≥ 0.05), but by sex if interactions in either parameter were significant (Psex×site < 0.05).

3. Results 3.1. TBARS In all stations examined, male and female hepatic TBARS responded similarly to municipal sewage effluent and pulp and paper mill effluent exposure (Psex×site ≥ 0.469). In the Wapiti River system in fall 1999, hepatic TBARS values did not differ between the sexes while male TBARS were higher than those of females in the Athabasca River system in fall 2000 and the Wapiti River system in fall 2001. Within the Wapiti River system, hepatic TBARS were increased with exposure to pulp mill effluent only in the fall of 1999 and then only relative to the Little Smoky reference station (Fig. 1a and c). Within the Athabasca River system, hepatic TBARS were increased only below Mill B (Fig. 1b). No increases in hepatic TBARS were detected below municipal sewage outfalls in either river system.

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TBARS values in ovarian tissue were always higher than in testicular tissue (Psex < 0.001). Overall, male and female gonadal tissue responded differently to sewage and pulp and paper mill effluent exposure (Psex×site < 0.001) although in a single case both sexes responded similarly. Within the Wapiti River system in fall 1999, exposure to pulp mill effluent increased gonadal TBARS relative to the Pipestone reference station in both sexes, while sewage effluent increased only ovarian, and not testiscular TBARS (Fig. 2a and b). Ovarian TBARS concentrations were also higher in the Little Smoky station relative to the upstream reference site. In the Athabasca River in fall 2000, there were no changes in gonadal TBARS in either sex within the river system (Fig. 2c and d). Within the Wapiti River in fall 2001, ovarian TBARS were significantly increased with exposure to pulp mill effluent with no other changes observed in the river system (Fig. 2e and f). Testis TBARS from the Wapiti River system in 1999 were significantly lower than concentrations observed in other collections. 3.2. LPO Within the Athabasca River system in fall 2000, male longnose sucker had significantly higher LPO concentrations than females (Psex < 0.001). Both sexes responded to paper mill effluent similarly (Psex×site = 0.263) with significant increases in fish collected below mill B relative to the Windfall reference site (Fig. 3). No assessment was made on the impact of municipal sewage discharge on LPO concentrations. 3.3. Hepatic FAO activity In both river systems and in all sample years, male and female FAO activities did not differ between the sexes, and both sexes responded similarly with exposure to municipal sewage effluent and pulp and paper mill effluent (Psex×site ≥ 0.056). There were pronounced differences in apparent FAO responses between activities expressed as nmoles H2 O2 /g liver and those protein normalized to nmoles H2 O2 /min/mg protein (Fig. 4). FAO activities in fish from the Wapiti River system in fall 1999 were significantly increased with pulp mill effluent exposure when expressed as

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Fig. 1. Hepatic TBARS in longnose sucker collected (a) from the Wapiti River system in the fall of 1999, (b) the Athabasca River system in the fall of 2000, and (c) from the Wapiti River system in the fall of 2001. Females (open bars) and males (solid bars) are presented as means (and S.E.) with sample size in parenthesis. Statistical differences (P < 0.05) were detected by two-way ANOVA and Tukey’s HSD as indicated by different letters.

nmoles H2 O2 /g liver (Fig. 4a), but were significantly increased with exposure to municipal sewage effluent when expressed as nmoles H2 O2 /min/mg protein (Fig. 4b). Within the Athabasca River system in fall 2000, significant increases in FAO activity with exposure to paper mill effluent were detected when activities were expressed both as nmoles H2 O2 /g liver (Fig. 4c) and as nmoles H2 O2 /min/mg protein (Fig. 4d), although enzyme activities below mill B were significantly lower than those below Mill A when protein normalized. Finally, in the Wapiti River

in fall 2001, FAO activities were significantly increased regardless of how activity was expressed, although FAO activity was only increased in fish collected below the municipal sewage outfall when enzyme activities were expressed as nmoles H2 O2 /g liver (Fig. 4e and f). Hepatic FAO activities were positively correlated with hepatic TBARS when FAO activity was expressed as nmoles H2 O2 /g liver (R = 0.132, P = 0.030, n = 267) but not when expressed as nmoles H2 O2 /min/mg protein (R = 0.031, P = 0.608, n = 267).

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Fig. 2. Gonadal TBARS in longnose sucker (a) females and (b) males collected from the Wapiti River system in the fall of 1999; in (c) females and (d) males from the Athabasca River system in the fall of 2000; and in (e) females and (f) males from the Wapiti River system in the fall of 2001. Females (open bars) and males (solid bars) are presented as means (and S.E.) with sample size in parenthesis. Statistical differences (P < 0.05) were assessed within each sex by one-way ANOVA and Tukey’s HSD as indicated by different lower case letters.

3.4. Ascorbic acid Hepatic ascorbic acid concentrations in female and male fish responded differently to municipal sewage and pulp mill effluent in the Wapiti River system in the fall of 1999 (Psex×site = 0.039). In this year, both sexes had significantly reduced hepatic ascorbic acid with exposure to pulp mill effluent relative to the Pipestone reference station (Fig. 5a and b). However, only females had significant increases in ascorbic acid with exposure to the municipal sewage discharge. Additionally, male fish from the Little Smoky reference site

had significantly lower hepatic ascorbic acid than the upstream reference site. Within the Athabasca River system in fall 2000 and in the Wapiti River system in fall 2001, fish of both sexes responded similarly with exposure to sewage and pulp and paper mill effluent (Psex×site ≥ 0.311). Within the Athabasca River system, hepatic ascorbic acid was significantly increased below both mills A and B, as well as the municipal sewage discharge (Fig. 5c). Within the Wapiti River system in fall 2001, ascorbic acid was significantly higher in the livers of fish exposed to municipal sewage effluent, but unchanged with exposure to

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Fig. 3. Hepatic LPO in longnose sucker collected from the Athabasca River system near Whitecourt AB in the fall of 2000. Females (open bars) and males (solid bars) are presented as means (and S.E.) with sample size in parenthesis. Statistical differences (P < 0.05) were detected by two-way ANOVA as indicated by different letters.

pulp mill effluent (Fig. 5d). While hepatic concentrations of ascorbic acid differed between the sexes in the Wapiti River system in fall 1999 (especially at the Little Smoky reference station), ascorbic acid concentrations did not differ between female and male fish in the Athabasca River system (fall 2000) nor in the Wapiti River system in fall 2001. Hepatic ascorbic acid concentrations were positively correlated with hepatic TBARS in females (R = 0.456, P < 0.001, n = 135) and males (R = 0.387, P < 0.001, n = 131) but not with FAO activity expressed as nmoles H2 O2 /g liver in females (R = 0.057, P = 0.508, n = 135) or males (R = 0.117, P = 0.181, n = 131). However, FAO activity expressed as nmoles H2 O2 /min/mg protein was positively correlated with ascorbic acid in females (R = 0.296, P < 0.001, n = 135) and males (R = 0.375, P < 0.001, n = 131). 3.5. Iron content In the Wapiti River system in fall 1999 and the Athabasca River system in fall 2000, changes in male and female hepatic iron content responded similarly with exposure to both municipal sewage effluent and pulp and paper mill effluent (Psex×site ≥ 0.110). Within the Wapiti River system in 1999, there were no changes in hepatic iron with exposure to either municipal sewage or pulp mill effluent (Fig. 6a).

Within the Athabasca River system, significant increases in hepatic iron were detected in fish collected downstream of both paper mills, while fish resident below the sewage treatment plant outfall had hepatic iron levels unchanged from the reference condition (Fig. 5b). Within the Wapiti River system in fall 2001, hepatic iron content responded differently in female than male fish with exposure to sewage and pulp mill effluent (Psex×site = 0.001). Female fish in 2001 did not differ in hepatic iron content with exposure to effluents, while male hepatic iron was significantly increased in fish collected downstream of the pulp mill (Fig. 5c and d). Hepatic iron content was positively correlated with hepatic TBARS in females (R = 0.265, P < 0.001, n = 135) and males (R = 0.239, P = 0.005, n = 131). While hepatic iron was correlated to hepatic ascorbic acid in females (R = −0.242, P = 0.004, n = 135) and males (R = −0.147, P = 0.009, n = 131), the correlation was negative in both sexes. Multiple regression analysis was used to determine the effect of hepatic ascorbic acid and iron on hepatic TBARS. In females, both parameters were positively correlated with TBARS (R2model = 0.234, Piron = 0.038, Pascorbic acid < 0.001) but with the slope of ascorbic acid relatively steep compared with iron (Biron = 5.3E−3, Bascorbic acid = 1.364). Similarly, both iron and ascorbic acid were positively correlated with TBARS in

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Fig. 4. Hepatic FAO activity expressed as (a) nmoles H2 O2 /g liver and (b) nmoles H2 O2 /min/mg protein in longnose sucker collected from the Wapiti River system in the fall of 1999; (c) nmoles H2 O2 /g liver and (d) nmoles H2 O2 /min/mg protein in fish from the Athabasca River system in the fall of 2000; (e) nmoles H2 O2 /g liver and (f) nmoles H2 O2 /min/mg protein in fish from the Wapiti River system in the fall of 2001. Females (open bars) and males (solid bars) are presented as means (and S.E.) with sample size in parenthesis. Statistical differences (P < 0.05) were detected by two-way ANOVA and Tukey’s HSD as indicated by different lower case letters.

males (R2model = 0.184, Piron = 0.022, Pascorbic acid < 0.001) again with a shallow iron slope and a relatively steep ascorbic acid slope (Biron = 1.4E−3, Bascorbic acid = 0.426). From the multiple regression coefficients of determination, 23.4% of the female and 18.4% of the variation in male hepatic TBARS are explained by ascorbic acid and iron. 3.6. LSI Within the Wapiti River system in fall 1999, both sexes captured below the sewage and pulp mill out-

falls had significantly larger livers than reference fish, while female livers were significantly higher at the Pipestone Creek than the Little Smoky Reference (Table 2). In the Athabasca River system in fall 2000, female fish captured below the sewage outfall had significantly smaller livers than reference or paper mill effluent exposed fish. Within the Wapiti River system in fall 2001, both sexes had significantly larger livers with sewage and pulp mill effluent exposure, with increases below the pulp mill discharge greater than those below the sewage discharge (Table 2).

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Fig. 5. Hepatic ascorbic acid in (a) female and (b) male longnose sucker collected from the Wapiti River system in the fall of 1999; (c) the Athabasca River system in the fall of 2000; and (d) the Wapiti River system in the fall of 2001. Females (open bars) and males (solid bars) are presented as means (and S.E.) with sample size in parenthesis. Statistical differences (P < 0.05) were detected within each sex by one-way ANOVA and Tukey’s HSD in the Wapiti River system in the fall of 1999, and by two-way ANOVA and Tukey’s HSD in the Athabasca River system in fall 2000 and the Wapiti River system in fall 2001 as indicated by different lower case letters.

3.7. Condition factor Within the Wapiti River system in fall 1999 and 2001, condition factor was significantly increased in fish exposed to municipal sewage and pulp mill effluent (Table 2). Within the Athabasca River system in fall 2000, there were no changes in condition factor with exposure to either sewage or paper mill effluent (Table 2).

4. Discussion To our knowledge, this study is unique in investigating the utility of oxidative stress endpoints in delineating the relative impacts of multiple point-source

effluents on resident fish populations. Of the many measures of oxidative stress currently available, the TBARS assay is the most widely used (Liu et al., 1997). Longnose sucker captured below pulp and paper mill effluent discharges in the two river systems in northern Alberta sometimes had increased levels of oxidative stress, as quantified by hepatic and gonadal TBARS, while no changes in TBARS with sewage exposure were observed. Although increased oxidative stress with contaminant exposure has not been previously demonstrated in this species, our earlier studies have demonstrated increased hepatic and gonadal TBARS in white sucker (Catostomus commersoni) with pulp and paper mill effluent exposure in a northern Ontario river system (Oakes and Van Der Kraak, 2003; Oakes et al., 2003). In the present study,

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Fig. 6. Hepatic iron content in longnose sucker collected from (a) the Wapiti River system in the fall of 1999; (b) the Athabasca River system in the fall of 2000; and in (c) female and (d) male fish from the Wapiti River system in the fall of 2001. Females (open bars) and males (solid bars) are presented as means (and S.E.) with sample size in parenthesis. Statistical differences (P < 0.05) were detected by two-way ANOVA and Tukey’s HSD in fish from Grande Prairie AB in fall 1999 and Whitecourt AB in fall 2000 and by one-way ANOVA and Tukey’s HSD in the Wapiti River system in fall 2001 as indicated by different lower case letters.

changes in hepatic and gonadal TBARS in resident fish with pulp and paper mill effluent exposure were not as consistent as those observed in previous studies. These responses differences may be attributable to inherent differences in species sensitivity, or to the fact that the three mills examined in northern Alberta are among the most modern in Canada. Within the Wapiti River system, no consistent changes in hepatic or gonadal TBARS were observed with pulp mill effluent exposure, and municipal sewage effluent alone did not significantly increase TBARS values. However, hepatic and gonadal TBARS concentrations at sewage exposed sites were often intermediate between those of the reference and pulp mill effluent exposed locations suggesting sewage effluents may play a minor role in promoting oxidative

stress. Such evaluations, however, should consider both the volume and composition of all effluent contributions. On the Wapiti River, the pulp mill effluent discharge accounts for approximately 2.5% of the average annual flow of the Wapiti River; by comparison, the sewage discharge contributes a seasonal average of only 0.77% (0.60–1.09%). The advent of tertiary treatment to the Wapiti River sewage treatment facility in the summer of 2001 appears to have reduced effluent constituents capable of producing the TBARS increases (relative to the Pipestone reference location) seen in the fall of 1999. As tertiary treatment removes nutrients known to increase primary productivity, and consequently secondary productivity within the benthic invertebrate community, reductions in oxidative stress responses

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Table 2 Liver-somatic index (LSI) and condition factor (K) in longnose sucker collected from the Wapiti River system in the fall of 1999 and 2001 and from the Athabasca River system in the fall of 2000 Sex

Pipestone creek (Ref)

Wapiti River system (fall 1999) LSI F 1.89 ± 0.07 M 1.51 ± 0.05 K F 1.01 ± 0.04 M 1.03 ± 0.03 Wapiti River system (fall 2001) LSI F 1.26 ± 0.04 M 0.90 ± 0.06 K F 1.03 ± 0.02 M 1.05 ± 0.01 Sex

Little smoky (Ref)

Below sewage

Below mill

(15) a (22) a

1.55 ± 0.04 (25) b 1.37 ± 0.04 (21) a

2.45 ± 0.06 (20) c 2.70 ± 0.11 (20) b

2.13 ± 0.16 (20) c 2.40 ± 0.07 (20) b

(16) a (22) a

1.07 ± 0.02 (25) a 1.06 ± 0.02 (21) a

1.24 ± 0.02 (20) b 1.24 ± 0.02 (20) b

1.32 ± 0.08 (20) b 1.25 ± 0.02 (20) b

(20) a (20) a

– –

1.56 ± 0.05 (20) b 1.37 ± 0.04 (21) b

1.97 ± 0.06 (20) c 1.83 ± 0.07 (23) c

(20) a (20) a

– –

1.12 ± 0.01 (20) b 1.16 ± 0.01 (21) b

1.14 ± 0.02 (20) b 1.18 ± 0.01 (23) b

Below Mill A

Below Mill B

Below sewage

1.92 ± 0.14 (18) a 1.91 ± 0.04 (20) a

1.95 ± 0.04 (19) ab 1.66 ± 0.05 (19) a

1.69 ± 0.13 (21) b 1.72 ± 0.07 (21) a

1.21 ± 0.02 (18) a 1.20 ± 0.01 (20) a

1.17 ± 0.02 (19) a 1.20 ± 0.03 (20) a

1.20 ± 0.02 (21) a 1.21 ± 0.01 (21) a

Windfall (Ref)

Athabasca River system (fall 2000) LSI F 2.07 ± 0.06 (20) a M 1.64 ± 0.09 (21) a K F 1.18 ± 0.03 (20) a M 1.22 ± 0.02 (21) a

Values are presented as means (and S.E.) with sample size in parenthesis. No fish were collected from the Little Smoky station in 2001. Statistical differences (P < 0.05) were detected by ANCOVA as indicated by different lower case letters. Data is adapted from McMaster et al. (in press).

may be a function of modified food and vitamin availability. Enrichment or impoverishment of benthic invertebrate communities below effluent outfalls is dependent on the relative toxicity and nutrient content of the specific effluent, but changes in community structure are common (Culp et al., 2000; Archambault et al., 2001; Soltan et al., 2001) and may contribute to altered hepatic monounsaturated and polyunsaturated lipid concentrations in higher trophic levels (Avery et al., 1998). While overall lipid concentrations themselves do not appear to affect TBARS (Oakes and Van Der Kraak, 2003), changes in diet could result in changes in hepatic mono- and polyunsaturated fatty acids which vary in susceptibility to peroxidation and TBARS formation (Pryor et al., 1976; Avery et al., 1998). While tertiary treatment may modify fish diets, it is also possible that heavy metals which can increase oxidative stress (Oakes and Van Der Kraak, 2003) have been taken up by tertiary treatment organisms prior

to effluent release, consequently reducing the ROS generating capacity of the effluent (Avery et al., 1996; Miranda and Ilangovan, 1996; Dellarossa et al., 2001). In addition to hepatic TBARS, hepatic lipid hydroperoxides (LPO) were also evaluated within the Athabasca River system. Both the TBARS and LPO assays were able to resolve similar increases in hepatic oxidative damage within the Athabasca River system confirming that, as in white sucker, both assays detecting different lipid peroxidation products can be used to evaluate oxidative stress responses in longnose sucker (Oakes and Van Der Kraak, 2003). While TBARS and LPO were significantly increased in hepatic tissue with paper mill effluent exposure in the Athabasca River system, gonadal indices of oxidative stress were unaltered with paper mill or sewage effluent exposure. The sewage discharge for the Town of Whitecourt on the Athabasca River is located 4 km downstream of both paper mill discharges, which provides an

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opportunity to evaluate if sewage effluent produced cumulative oxidative stress effects in combination with the paper mill discharges. No increases in TBARS below the sewage discharge were observed which, although there is no tertiary treatment, was not surprising given the relatively small discharge of the sewage treatment plant (only 0.01–0.05% of the Athabasca River volume). What was surprising was the anticipated contribution of ROS-generating materials from both Mills A and B, just 4 km upstream of the sewage outfall, was not evident. Previous studies have demonstrated increases in TBARS and alterations in other biochemical parameters are not attenuated until distances greater than 40 km downstream of the pulp and paper mill effluent discharge (Gibbons et al., 1998; Oakes and Van Der Kraak, 2003). The only explanation we can offer for the apparent lack of paper mill effluent contribution is the current patterns produced by the Athabasca’s confluence with the McLeod River may have altered effluent mixing so that the chemical composition across the width of the river may not be homogeneous. Such a scenario has recently been well described in the St. Lawrence River (deBruyn et al., 2003) where the addition of sewage effluent was confined to a narrow ribbon between two distinct lotic streams (water discharged from the Ottawa River displayed no lateral mixing with Great Lakes water) for some distance below their confluence. Our own studies have previously described a similar scenario in a river system where TBARS in fish immediately below a discharge were not as high as at a station 14 km further downstream (Oakes et al., 2003). Elevations in FAO activity have been associated with exposure to constituents of pulp and paper mill effluents (Mather-Mihaich and Di Giulio, 1991; Haasch et al., 1998; Oakes et al., 2003), but may also be induced by elevated plasma lipids (Crockett and Sidell, 1993). Plasma hyperlipidemia, in turn, may be a direct response to estrogenic compounds in sewage and pulp mill effluents (Routledge et al., 1998; Mercure et al., 2001) or produced indirectly by an enriched diet resulting from pulp mill or sewage effluent nutrient additions (Culp et al., 2000; Chambers et al., 2000). Changes in peroxisomal FAO activity were measured to determine if oxidative stress increases (quantified using the TBARS and LPO assays) were associated with the obligatory production of H2 O2

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by FAO-catalyzed hepatic ␤-oxidation. Although catalase, an enzyme which detoxifies H2 O2 is co-induced with FAO activity, it often cannot detoxify all of the ROS produced which may result in oxidative damage (Mather-Mihaich and Di Giulio, 1991). In the present study, FAO activity, while always increased with pulp and paper mill effluent exposure when expressed as nmoles H2 O2 /g liver, was only intermediate in activity or occasionally increased with sewage exposure. Interestingly, the FAO assay was able to resolve activity changes with exposure to pulp and paper mill effluent in the Athabasca River in the fall of 2000 and in the Wapiti River in the fall of 2001 that were not detected with the TBARS assay. Consistent with our previous studies in white sucker tissue (Oakes et al., 2003), FAO activity in longnose sucker did not correspond well with determinations of oxidative stress, especially when FAO activity was protein normalized. While most, but not all (Palace et al., 1996), measures of enzyme activities related to oxidative stress in the primary literature are presented protein normalized, previous studies (Scarano et al., 1994; Oakes et al., 2003) have found that expressing FAO activity as nmoles H2 O2 /g liver provides different information than protein normalized FAO activity. The lack of correspondence between oxidative stress and protein normalized FAO activity, especially with exposure to complex effluents, may be due in part to the production of many proteins in hepatic tissue, such as cytochrome P4501As (CYP1As), which do not necessarily generate oxidative stress (Oakes et al., 2003). Significant increases in CYP1A activity, and hence increased CYP1A protein production, are associated with exposure to pulp and paper mill effluent in the present study (McMaster et al., in press) and serve to artificially reduce the apparent FAO activity relative to exposure scenarios where single-compound inducers increase FAO proteins only (Oakes et al., 2003). A good example of this artificial reduction in apparent activity in the present study can be seen by comparing responses to pulp mill effluent exposure in the Wapiti River system in the fall of 1999 between activities expressed as nmoles H2 O2 /g liver and nmoles H2 O2 /min/mg protein. In deference to the influences which may complicate interpretation of FAO activity and oxidative stress, we and others report FAO activity both as nmoles H2 O2 /g liver to most accurately reflect ROS tissue concentrations, as well as nmoles

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H2 O2 /min/mg protein to primarily reflect changes in enzymatic activity (Scarano et al., 1994; Oakes et al., 2003). Hepatic ascorbic acid, an important dietary antioxidant, was significantly altered with exposure to pulp and paper mill and municipal sewage effluent on several sampling occasions. As teleost fishes cannot synthesize ascorbic acid (Chatterjee, 1973), any increases in hepatic ascorbic acid content below sewage outfalls and pulp and paper mill effluent discharges must be due to dietary enrichment contributed by the allochthonous inputs, likely through alterations in benthic invertebrate communities (Culp et al., 2000). However, reduced levels of ascorbic acid may result from either a dietary deficiency, or by depletion through ascorbic acids’ antioxidant properties in response to elevated ROS generation. Within the Wapiti River system, there were pronounced decreases in hepatic ascorbic acid associated with exposure to pulp mill effluent, while elevated levels of ascorbic acid were found in the livers of fish below the paper mill discharge on the Athabasca River as well as below the municipal sewage discharges in both river systems. Increases in ascorbic acid are likely attributable to the addition of nutrients by municipal sewage and paper mill discharges on the Athabasca River (6–16% of phosphorus and 4–10% of the nitrogen load, Chambers et al., 2000), and also within the Wapiti River system (22% of phosphorus and 20% of the nitrogen load, Chambers et al., 2000). In contrast, the depletion of hepatic ascorbic acid below the pulp mill discharge in the Wapiti River system likely corresponds to increases in ROS generation, which may offset any enrichment effects associated with this effluent. Although the sewage treatment plant on the Wapiti River utilizes tertiary treatment to reduce nutrient inputs, the treatment plant on the Athabasca River does not. These treatment differences suggest potentially greater enrichment, and hence potentially higher availability of an ascorbic acid enriched diet downstream of the sewage outfall on the Athabasca River relative to the outfall on the Wapiti River. Our data, however, suggest the volume of effluent discharged into the Wapiti River from the City of Grande Prairie (population 37 000) relative to that discharged to the Athabasca River from the Town of Whitecourt (population 8300) masks any effects of the presence of tertiary treatment.

The hepatic ascorbic acid and TBARS findings in the present study differ from our previous work in white sucker which demonstrated significant increases in hepatic TBARS whenever hepatic ascorbic acid levels were increased (Oakes and Van Der Kraak, 2003). Ascorbic acid in the present study may be behaving as an antioxidant or may be functioning as a pro-oxidant in the presence of redox-active metal species such as iron (Vreman et al., 1998; Griffiths and Lunec, 2001) where it can undergo Haber–Weiss type reactions to generate the very reactive ROS hydroxyl radical (Livingstone, 2001). The strong negative correlation between ascorbic acid and iron in the present study may be a function of ascorbic acid depletion concomitant with the reduction of iron to facilitate the Haber–Weiss reaction (Livingstone, 2001). Multiple regression analysis demonstrates the plausibility of Haber–Weiss processes in producing oxidative stress as approximately 20% of the change in TBARS could be explained by the predictors iron and ascorbic acid. It should be noted that the ascorbic acid response represents only the hydrophilic antioxidant potential in the liver. Lipophilic antioxidants, such as retinoids, are often dramatically decreased in livers of fish exposed to pulp and paper mill effluent by ROS dependent or independent mechanisms (Schoff and Ankley, 2002; Alsop et al., 2003). Hepatic iron concentrations were often elevated with pulp and paper mill effluent exposure, but rarely with exposure to sewage effluent alone. However, fish collected below sewage outfalls occasionally had hepatic iron concentrations intermediate between those observed at reference stations and pulp and paper mill effluent exposed collections suggesting there may be influences of the sewage outfalls on hepatic iron content as well. It is unclear if increases in hepatic iron with effluent exposure represent uptake from an iron-enriched environment, or are the result of the liberation of endogenous iron stores by ROS (Matés, 2000; Agrawal et al., 2001). While little information is known about iron concentrations in pulp and paper mill effluents, previous studies have demonstrated chelated iron is a potent source of ROS (Oakes and Van Der Kraak, 2003). It is reasonable to assume metals in pulp and paper mill effluent may be complexed with organic constituents, which may alter the iron reactivity towards that of a chelated species (Hamm et al., 1986).

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Detection of biochemical changes associated with pollutant discharges at the cellular level are important as even the most subtle change in morphology or organismal function must be preceded by biochemical change. However, it is important to evaluate if effects of environmental pollutants can also be detected at an organismal level. Enlarged livers, as indicated by LSI, are often a result of altered lipid concentrations (Oakes and Van Der Kraak, 2003), hyperplasia (increase in cell number) or hypertrophy (increase in cell size) as adaptive responses of the liver to foreign compounds (Goede and Barton, 1990). Although hepatic lipids were not evaluated in this study, a subjective internal fat index identified increased lipid stores in fish collected downstream of the two effluent discharges on the Wapiti River (McMaster et al., in press). LSI measures in each river system were also closely paralleled by the induction of CYP1A detoxifying enzymes; with significant induction in the Wapiti River system in both years and no elevation of CYP1A activity with exposure to either effluent within the Athabasca River system (McMaster et al., in press). CYP1A enzymes, which are a plausible source of oxidative stress (Oakes et al., 2003), further illustrate the linkages between biochemical and organismal endpoints. Condition factor (K) was measured as an index of overall health and somatic fitness as K reflects changes in food intake, fat deposition, and muscle development (Goede and Barton, 1990). As the northern rivers examined in the present study are nutrient-limited, longnose sucker appear to respond to the allochthonous nutrient addition by showing signs of increased K. Similar to LSI, K was increased below municipal sewage and pulp mill effluent discharges in the Wapiti River system. No such differences were found for K in the Athabasca River system, however, as previous studies have demonstrated that the reference site selected for these studies (Windfall) is nutrient saturated from a pulp mill and sewage effluent discharge located upstream in the town of Hinton. The additional nutrient input from the three discharges examined in these studies do not result in increased K as the system is already saturated (McMaster et al., in press). Alterations in LSI and K were frequently associated with biochemical measures of oxidative stress (particularly FAO), suggesting biochemical endpoints are not only useful assessment tools, but indicative of organismal impacts of pollutant exposure.

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5. Conclusion One of the challenges of assessing environmental impacts under field-type conditions is attributing biochemical and organismal effects to specific pollution sources within a context of environmental and temporal variability. In this study, specific parameters varied in their ability to resolve biochemical and ROS-induced changes in fish exposed to pulp and paper mill and sewage discharges. Although most oxidative stress measures in longnose sucker were altered downstream of pulp and paper mill effluent and municipal sewage discharges, only FAO activity appears to be a consistent indicator of exposure. While individual biochemical responses may be influenced by a myriad of variables that change over time in response to varying effluent constituents, the suite of parameters examined were collectively able to resolve and, to some extent quantify, the ability of each effluent to produce changes in exposed fish. Oxidative stress endpoints, applied with organismal assessments of fitness and pollution exposure, are useful tools in identifying relative influences of municipal sewage and pulp and paper mill effluent on fish in adjacent receiving waters. Acknowledgements The authors gratefully acknowledge the technical assistance of K. Daynes, M. Edwards, B. Gray, M. Hewitt, A. Lister, N. Jones, J. Kraft, L. Peters, C. Portt, G. Tetreault, and K. Wells. We thank the anonymous reviewers for their constructive comments, which greatly strengthened the article. This work was sponsored by the Canadian Network of Toxicology Centers (CNTC) and Natural Sciences and Engineering Research Council (NSERC) of Canada grants to GVDK. MEM received support from Environment Canada, the Northern Rivers Ecosystem Initiative, and the Toxic Substances Research Initiative. KDO received financial support from CNTC, NSERC, and the Ontario Graduate Scholarship program.

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