Assessing the biological status of fish in a river receiving pulp and paper mill effluents

Assessing the biological status of fish in a river receiving pulp and paper mill effluents

Environmental Pollution 118 (2002) 123–140 www.elsevier.com/locate/envpol Assessing the biological status of fish in a river receiving pulp and paper ...

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Environmental Pollution 118 (2002) 123–140 www.elsevier.com/locate/envpol

Assessing the biological status of fish in a river receiving pulp and paper mill effluents T.G. Kovacs*, P.H. Martel, R.H. Voss Paprican, 570 boul. St-Jean, Pointe-Claire, Que´bec, Canada H9R 3J9 Received 6 April 2001; accepted 13 July 2001

‘‘Capsule’’: Unlike sentinel fish species, fish community structure analysis tracked improved effluent quality resulting from changes in mill operating conditions. Abstract This study compared the use of sentinel species- and community-based field approaches for assessing the biological status of fish living in a river receiving pulp and paper mill effluents. Three approaches were compared. Two approaches used sentinel species. One of these involved an internal/external examination of the fish that leads to the calculation of a fish health assessment index (HAI) and the other involved biochemical measurements of hepatic mixed function oxidase (MFO) activity and plasma steroid levels. The third approach characterized the fish community structure according to an index of biotic integrity (IBI). The comparison focused on how the methods respond to the hypothesis that recent process modifications/effluent treatment changes, resulting in demonstrable improvements in effluent quality, have beneficial effects on fish. Neither of the approaches using sentinel fish indicated clear mill-related influences either before or after the process modifications/effluent treatment changes. There was no evidence of depressed plasma steroids and increased MFO activity in fish frequently associated with mill effluent exposure in previous studies. While the HAI was higher at stations downstream from two mills, this could not be linked to effluent exposure alone. In contrast, the study of community structure showed a substantial improvement in fish assemblages at all the mill sites. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: Pulp mills; Paper mills; Effluents; Fishes; Biological tests; Effluent treatment; Sampling

1. Introduction The pulp and paper industry in Canada and worldwide has undergone major modernization in the 1990s to meet new effluent regulations and to operate what have been termed minimum impact mills (Axega˚rd et al., 1997). The improved effluent quality resulting from this modernization program, as determined by laboratory biotests, has been well documented (Kovacs and Megraw, 1996; Kovacs et al., 1997b). However, it is also important to assess whether the modernization steps resulted in benefit for the aquatic organisms inhabiting the waterways receiving mill discharges. In fact, field studies have increasingly gained importance as a means of judging the role of mill effluents in receiving waters * Corresponding author. Tel.: +1-514-630-4101, ext. 2363; fax: +1-514-630-4134. E-mail address: [email protected] (T.G. Kovacs).

both from a regulatory and a research perspective (Environment Canada, 1992; Dell et al., 1996; Hall and Miner, 1997; Keough and Mapstone, 1997; Sandstro¨m et al., 1997). In Canada, the regulatory environmental effects monitoring (EEM) program is meant to assess the adequacy of current regulations pertaining to mill effluents (Environment Canada, 1992). The EEM program requires mills to test the sublethal toxicity of discharges twice a year and to study the condition of benthic invertebrates and fish in the receiving waters every 3 years. In the case of fish, the regulation calls for the examination of morphological and life history characteristics of two sentinel species. The assumption of the sentinel species approach is that the morphological/life history characteristics of two species provide a good indication of the condition of all the fish in the receiving environment (Courtney et al., 1998). The first cycle of the EEM program was completed in 1995. At the end of

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this cycle, several difficulties were identified regarding the fish component of the EEM. Some of these included fish mobility, natural variability as well a lack of knowledge concerning the link between physiological/ morphological endpoints and responses at the whole population or community level (Munkittrick et al., 1997b). There were also issues related to capture success, reference site selection and discriminating the influence of one discharge from another when there were numerous inputs to a particular receiving water. These difficulties are not unique to the regulatory EEM program and have been known to affect the ability to interpret the results of all field studies (US EPA, 1991), especially the establishment of causality when upstream–downstream type differences are observed (Kovacs et al., 1997a). While overall, the fish component of the first cycle EEM program provided useful information, there was general consensus that, for the future, some refinements to the regulatory protocols were needed. There was also consensus that, in some situations, consideration needs to be given to alternative approaches to account for sitespecific differences (Courtney et al., 1998). Various alternative approaches ranging from mesocosm experiments to caged bivalves were recommended (Munkittrick et al., 1997b; Courtney et al., 1998). However, these alternatives either did not involve fish or were more like on-site bioassays than real fieldwork studying wild fish. Because of this, some work evaluating the usefulness of alternate field approaches examining the status of wild fish was warranted. The objective of this study was to assess the biological condition of fish in the vicinity of three pulp and paper mills situated on the St. Franc¸ois River in Quebec, Canada by three different approaches other than what is currently required for the regulatory EEM program in Canada. The study focused on how the three approaches responded to the hypothesis that process and effluent treatment changes, implemented by the mills in 1995 to improve effluent quality and meet regulatory limits, should lead to demonstrable improvements for wild fish in the river. Two of the three approaches used sentinel species captured upstream and downstream of the mills in 1994/1995 and 1998. One of these involved an internal/external examination of the fish that leads to the calculation of a fish health assessment index or HAI (Adams et al., 1993) and the second involved the measurement of two biochemical parameters, hepatic mixed function oxygenase (MFO) activity and plasma steroid levels. The third approach involved the characterization of the fish community structure, according to an Index of Biotic Integrity (IBI) developed by Karr (1981). The latter was conducted at locations along a 75-km stretch of river in 1998 following the protocols used by the Quebec Ministry of the Environment (Richard, 1996) for a study in 1991 on the same river.

2. Study area 2.1. River Description The St. Franc¸ois River (Fig. 1a) in Quebec, Canada originates from Lake St. Franc¸ois and then flows west and north into the St. Lawrence River draining a 10,230-km2 region (Richard, 1996). This region is comprised of the Appalachian plateau and St. Lawrence lowlands. The sampling stations for this study were located between the town of Bishopton and the town of Richmond representing a 75-km stretch (Fig. 1b) in the Appalachian plateau region of the river. This portion of the river, characterized by relatively steep slopes, rocky bottoms and rapidly fluctuating water levels, receives discharges from three pulp and paper mills as well as from the municipality of Sherbrooke. This municipality subjects its wastewater to primary and secondary treatment. Between 1991 and 1998, there were several changes made in the treatment process that included improvements in the biofiltration system, reduced chemical usage and disinfection by ultraviolet (UV) irradiation. Other potential inputs to this stretch of the river include tributaries, runoff from agricultural activities as well as smaller municipalities and industries, including a small (47 t/day) tissue paper mill at Lennoxville on a tributary river (Massawippi). There are dams (Fig. 1b) just above each mill to control water levels and produce electricity. There is also a hydroelectric dam just above the town of East Angus. The dams restrict fish movement, particularly between regions upstream and downstream of mill discharges. The 13 fish community (IBI stations 1–13) and six sentinel species (SS stations 1–6) sampling locations shown in Fig. 1b were strategically selected to take advantage of the restriction of fish mobility caused by the dams thereby minimizing the interaction between upstream and downstream populations. 2.2. Mill descriptions Mill A, located at East Angus, is a manufacturing complex comprised of two plants, one producing about 145 t/day unbleached kraft pulp from softwood chips (95% spruce and 5% pine) and the other producing about 180 t/day boxboard from recycled fiber. The kraft pulp, together with recycled fiber, is used to produce about 250 t/day of a variety of kraft papers. A biological treatment system consisting of a three-cell aerated lagoon with a total residence time of about 10 days became operational in August 1995. The final mill effluents are treated concurrently with municipal effluent from the city of East Angus. The BOD and TSS from the city of East Angus contributes about 7 and 6%, respectively, of the BOD and TSS load to the treatment system. The average effluent flow in 1998 was

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m3/day. The biological treatment plant, a sequential batch reactor with a retention time of approximately 5 hours, began operation in August 1995. Mill C, located at Windsor, pulps hardwood (58% maple, 18% birch, 15% poplar, 4% beech and 5% unknown species) using a Kamyr modified continuous cooking digester followed by a DoEoD(P) bleaching sequence to produce a variety of bleached kraft specialty papers. The daily production capacity is approximately 1600 t/day and the effluent discharge is between 66,000 and 79,000 m3/day. The mill effluent has been treated since 1987 in an aerated lagoon with a 5-day retention time. In 1995, a flotation system was added to the secondary clarifier to reduce suspended solids losses. As well, during that year, the efficiency of pulp washing was improved and bleaching changed from a 55% ClO2 sequence, C/DEoHD(P), to the present sequence. 2.3. Effluent dilution

Fig. 1. (a) Geographical position of the study area, and (b) location of sentinel species and index of biotic integrity (IBI) sampling stations.

13,575 m3/day. The mill contribution was an average of 10,105 m3/day and represents about 80% of the total treated effluent. Mill B, located at Bromptonville, is a thermomechanical pulp (TMP) mill producing about 700 t/day of newsprint from softwood chips composed of approximately 50% black spruce and 50% balsam fir. The mill also operates a 160 t/day deinking plant, established in 1992, and, as a result, incorporates about 25 to 30% recycled paper into its pulp stock. The pulp is brightened using sodium hydrosulphite. The average water usage for the mill is between 28,818 and 38,650

The concentration of effluent from the three mills in the St. Franc¸ois River was estimated on the basis of average effluent flows and average river flows nearest the mill as well as on the basis of 7Q10 (i.e. the period of lowest stream flow during a 7-day interval that is expected to occur once every 10 years) river flows in the fish sampling area of this study using the CORMIX model (Ame´natech, 1999). On the basis of average river flows, the minimum and maximum dilutions occur in April and August/September, respectively. At East Angus, the concentrations range between 0.07% in April and 0.3% in September and the yearly average is 0.14%. At Bromptonville, the concentrations are 0.08% in April and 0.42% in September. The yearly average concentration is 0.22%. At Windsor, the concentration of effluent in the river is 0.2% in April and 0.9% in August. The yearly average is 0.48%. On the basis of 7Q10 river flow using the CORMIX model, the effluent concentrations are 9% at East Angus (Ame´natech, 1999), 1% at Bromptonville, also confirmed by a rhodamine dye study (Harvey, 2000), and the maximum effluent dilution downstream from the Windsor mill all the way to the confluence with the St. Lawrence River is 1.9% (GDG, 2000).

3. Methods 3.1. Habitat assessment In 1998, the habitat was assessed at each fish community and sentinel species sampling station according to the characteristics established by Richard (1996) for his study of the river in 1991. The habitat characterization included substrate, width, depth, current, vegetation and general description of the environment.

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Depth measurement was made by a depth sounder (Humminbird) from a boat that was driven diagonally from one shore to the other along the 0.5-km sampling stretch. Velocity measurements at all fish community sampling stations were made with a portable flow meter (Marsh-McBirney). At sentinel species sampling stations, velocity was estimated visually as slow, moderate or fast. Temperature was recorded by an Onset StowAway TidBit temperature logger for the period between May and October. Water dissolved oxygen levels were also measured during the sampling. Prior to the fisheries work, water samples were collected during July and analyzed for several parameters such as pH, conductivity and nutrients according to standard methods (APHA, AWWA, WPCF, 1985). 3.2. Sentinel species assessment The sentinel species were captured at six locations, upstream and downstream from the three mills, between East Angus and Windsor. The sampling in 1998 occurred between 28 September and 19 October at the East Angus mill, 14 September and 23 October at the Bromptonville mill and 21 September to 24 October at the Windsor mill. The upstream and downstream sampling zones in 1998 (Fig. 1b) were the same as those used for the first cycle of the regulatory EEM requirements (Environment Canada, 1998) that must be completed once every 3 years. The cycle 1 EEM studies at Windsor (September 1994), Bromptonville (September 1995) and East Angus (September 1995) were completed by consultants (Ame´natech, 1996a, 1996b, 1996c). During the first cycle of EEM, Paprican staff used the fish captured at Windsor and Bromptonville for health assessment and biochemical measurements according to the same procedures as used during the 1998 study described later. The sentinel species for this study were white sucker (Catostomus commersoni) at all three mill site locations both for work related to cycle 1 EEM and in 1998, smallmouth bass (Micropterus dolomieui) at Windsor in 1998 and tesselated darter (Etheostoma olmstedi) at Bromptonville and East Angus in 1998. The white sucker was the most common species sampled for the first cycle freshwater EEM studies in Canada (Munkittrick et al., 1997b). The smallmouth bass is a popular sport fish in the St. Franc¸ois River basin. The tesselated darter is considered to be a small-bodied fish and such fish have been recommended for cycle 2 EEM work (Courtenay et al., 1998; Environment Canada, 1998). It is presumed that such fish are easier to capture in large numbers and their mobility is more restricted. In 1994 and 1995 (cycle 1 EEM), the objective was to collect 20 adult (mature) specimens of each sex for two species. Sampling was by gill nets and electrofishing (Ame´natech, 1996a, 1996b, 1996c). In 1998, the objective at

each mill site was to collect 30 adult specimens for each sex of two species. For the 1998 work, the fish were captured either by electrofishing (as described for community assessment, below) in water about 0.5–2.0 m deep or by hoopnets. The hoopnets were 6 m long, 1.4m in diameter and were equipped with side wings of 6-m length and a central panel 31.6-m long. They were installed parallel to the shore at intervals within the fishing zones in about 1.5–6.0 m of water and with their opening facing downstream. The hoopnets were left overnight and lifted each morning. After capture, the fish were maintained alive in the river in holding cages until processing. Typically, fish were kept in these cages from a few hours to overnight, as it was not possible to process all the fish simultaneously. The fish were transported from the cages to a mobile laboratory in plastic tubs filled with river water. The holding times in these tubs never exceeded 20 min. The fish were sampled for biochemical analyses (determination of liver MFO activity and plasma sex steroids) as well as being subjected to an external/ internal examination for calculation of a HAI. 3.2.1. HAI approach The HAI assessment followed a procedure described by Adams et al. (1993). The procedure involves an external and internal examination of the fish (anesthetized in 100 mg/l MS-222) including eyes, skin, gills, pseudobranchs, thymus, spleen, hindgut, liver, kidney and opercules (the latter not listed by Adams et al., 1993). The presence or absence of parasites and fin erosion is also recorded. In addition, a blood sample was taken from the caudal vein of white suckers and smallmouth bass with a heparinized vacuum tube. The blood sample was used for determining packed cell volumes of erythrocytes and leukocytes, as well as plasma protein. A blood sample of sufficient volume could not be obtained from the darters. Thus, tesselated darters were examined for 12 parameters rather than 15. Each of the 15 variables is assigned a numeric value where the normal condition is rated as zero. Any abnormalities in the spleen, kidney, liver, eyes, gills and pseudobranchs are given values of 30. For the other variables, such as the thymus, abnormalities are rated as 10, 20 or 30 depending on the degree of abnormality, with the greatest abnormality ranked as 30. For each fish, the variable rankings are summed and a HAI for the population is calculated by summing the individual HAIs and dividing by the number of fish in the population. Thus, a low HAI value represents healthy fish while a high HAI value suggests poorer health. Except for blood parameters in suckers, the anomalies were judged according to the descriptions outlined by Adams et al. (1993). In the case of the blood parameters for suckers, the normal range was established on the basis of fish sampled in the uncontaminated tributaries

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of the St. Maurice River in Quebec. A total of 192 suckers were captured in the fall of 1997 and the mean hematocrit, leucocrit and plasma protein levels for these fish were determined. The normal range was set as  2 S.D. units from the mean and was assigned a value of zero. For example, in the case of hematocrit, the normal range, given a zero score, was 24–50%. Scores of 30, 20 and 10 were assigned to fish with hematocrit < 14%, between 14 and 24% and >50%, respectively. Fish with leucocrit 52 and < 2 were assigned scores of 30 and 0, respectively, and fish with protein levels < 4.3 mg/ml, between 4.3 and 8.0 mg/ml and >8.0 mg/ml were scored as 30, 0 and 10, respectively. In addition to the variables used for the HAI calculations, bile color and mesenteric fat content, as recommended by Adams et al. (1993), the condition of scales and the occurrence of skeletal as well as fin deformities were recorded. 3.2.2. Biochemical measurement approach The liver and blood of individual white suckers were used to measure hepatic MFO activity and plasma steroid levels, respectively. The liver samples were preserved on dry ice in the field and were assayed in the laboratory for ethoxyresorufin-O-deethylase (EROD) activity. Briefly, all livers were processed at 4  C and all reagents were kept chilled. From each liver, a subsample of 0.5 g was removed. The liver piece was rinsed with 50 mM TRIS–HCl, 0.15 M KCl, pH 7.4 buffer, dried on absorbent paper and homogenized in buffer for 6 s. The homogenate volume was adjusted to 20% weight/ volume with buffer and then spun at 2000 g for 5 min and 10,000 g for 20 min. The post mitochondrial supernatant was removed and analyzed for EROD activity according to the fluorometric method described by Hodson et al. (1991). The whole procedure is described in detail elsewhere (Martel et al., 1995). The results of the assay were expressed as pmoles of resorufin produced per minute per mg of total protein in the post mitochondrial supernatant (pmol/min/mg). The blood was kept on ice and centrifuged (1700 g for 10 min) within 1 h of sampling. The resulting plasma was stored at 80  C until analysis. After ether extraction, analysis for sex steroids by radioimmunoassay was conducted according to the procedure described by McMaster et al. (1992). Samples taken from females were analyzed for estradiol and testosterone while samples taken from males were analyzed for testosterone only. The results were expressed in picograms per ml. All plasma samples were assayed in duplicate and the interassay variation was < 15%. 3.2.3. Statistical analysis The HAI values, liver MFO activity and plasma steroid levels of the fish captured at the various locations (e.g. upstream–downstream) as well as in different years

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(e.g. 1995 and 1998) were compared for statistically significant differences by the Mann–Whitney test (P < 0.05). The Kruskall–Wallis one-way non-parametric ANOVA (P < 0.05) and Dunn’s multiple comparison test (P < 0.05) were also used to determine statistically significant differences when all the stations were considered together for any one year. 3.3. Fish community assessment Sampling for the purpose of assessing the status of fish communities occurred between 5 August and 19 August 1998. Sampling was done at 13 locations from just above the town of Bishopton to Richmond 10 km below the mill discharge at Windsor. The sampling locations (Fig. 1b) corresponded to those selected by Richard (1996) for a 1991 study on the same stretch of the river and included stations both above and below the three mills as well as the city of Sherbrooke. Each of the 13 stations was sampled along 500 m of shoreline on both sides of the river by using a Smith Root electric shocker (model 2.5 GPP) mounted on a 14-foot inflatable boat. The anode was a 1-m diameter aluminum ring supporting 12 strands of stainless steel wire. The cathode, made from stainless steel wire strands, was installed on the opposite side to the anode. Two persons using insulated dip nets collected the stunned fish while a third person steered the boat and controlled the shocking unit. The captured fish were kept alive in a tub filled with aerated river water. All fish were identified, measured and weighed. A maximum of 20 fish of each species was examined for external anomalies such as skeletal deformation, fin erosion, lesions, tumors and the presence of parasites. Fish that could not be positively identified in the field, mostly juvenile cyprinids, were preserved in formalin for later identification by using standard texts such as Legendre (1954, 1960), Scott and Crossman (1974), and Brisebois et al. (1998). In addition, all the fish from station IBI-8 were subjected to verification by an independent ichtyologist (Huguette Masse´). The greatest numbers of species were captured at station IBI-8. For each sampling location, an IBI, developed by Karr (1981, 1987, 1991) and modified by Richard (1996), was calculated. Of the 12 metrics proposed by Karr to calculate an IBI, Richard (1996) retained six. These included the number of Catostomidae species, the number of pollution intolerant species, the percent omnivores, the percent insectivores, the percent piscivores and the proportion of fish with visible anomalies. In addition to the six metrics, Richard (1996) added a metric utilizing the index of well being (IWB) as described by Gammon (1980): IWB ¼ 0:5 lnN þ 0:5 lnB þ H0 n þ H0 b

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where: N=total catch per unit effort; B=total biomass per unit effort; H0 n=2.303[log n(1/n ni log ni)]; H0 b=2.303[log b(1/n bi log bi)]; n=total number of individuals per catch per unit effort (CUE) at each station; ni=number captured per CUE of species i; b=total biomass per CUE at each station; and bi=biomass per CUE for species i. This index incorporates two abundance and two diversity estimates and, hence, is counted as two metrics. For the IBI calculation, Richard (1996) calculated the difference between the IWB and the modified IWBm. In the latter, the contribution in terms of catch per unit effort and biomass per unit effort of pollution tolerant species is removed from the biomass component of the IWB without changing the Shannon’s diversity indices (H0 n and H0 b). A detailed description of the scoring method for each metric is shown in Table 1. The score of 1, 3 or 5 assigned to each metric was added and multiplied by 1.5 to calculate the final IBI value (Richard, 1996). The 1.5 multiplication factor allows for the comparison of an IBI score based on eight metrics (i.e. six metrics and the modified IWBm that counts for two metrics) as used by Richard (1996) to the original IBI score proposed by Karr (1981) based on 12 metrics. Hence, an IBI value of 12 is the lowest rating possible and the highest possible value is 60. Based on the IBI values, the fish assemblages at various locations could be classified (Richard, 1996) as excellent (54–60), good (45–54), average (36–45), poor (24–36) and very poor (12–24).

4. Results 4.1. Habitat assessment 4.1.1. Sentinel species sampling stations There were some differences in habitat between the sampling stations upstream (SS-1, SS-5) and downstream (SS-2, SS-6) from the effluent discharge of two mills on the St. Franc¸ois River (Table 2). At East Angus, where the river was wider and deeper, the current was Table 1 Metric scores used for IBI calculation (Richard, 1996) Metric description

No. of Catostomidae species No. of pollution intolerant species Percent omnivores Percent insectivores Percent piscivores Percent fish with anomalies IWB-IWBma a

Metric Score 1

3

5

0 0 >45% <20% <1% >5% >1.0

1 1–2 20–45% 20–45% 1–5% 2–5% 0.5–1.0

2+ 3+ <20% >45% >5% <2% <0.5

IWB, index of well being. IWBm, modified index of well-being.

slower upstream from the mill (SS-1). At the sampling station upstream of the mill at Windsor (SS-5), the current was slower and the dominant substrate was more sandy and gravel-like. The habitats of the two sampling stations at Bromptonville (SS-3 and SS-4) were fairly similar. 4.1.2. Fish community sampling stations The habitat characteristics of the 13 fish community sampling stations are shown in Table 2. There were also some differences in the habitats between the sampling stations, most notably with respect to maximum width/ depth, flow and dominant substrate. This was definitely the case between stations upstream and downstream from the mills (that is, IBI stations 3/4, 9/10 and 11/12), probably reflecting the influence of dams above the effluent discharge. Differences in shore and aquatic vegetation were less evident. 4.1.3. River water physical/chemical measurements Between 14 September and 19 October, the average 6.21 a.m. temperature at the sentinel species stations SS1, SS-2, SS-3, SS-4, SS-5 and SS-6 was 12.7  C (2.9), 13.1  C (3.1), 14.2  C (3.1), 14.3  C (3.1), 14.5  C (3.1) and 14.9  C (3.2), respectively (numbers in parentheses represent standard deviations). At 6.21 p.m. the average temperature ranged between 14.4–15.2  C (standard deviations 3.0–3.5) at the same sampling stations. During the IBI sampling period, the temperature at IBI stations one to 10 ranged between 23.1 and 24  C (standard deviation 1.6–2.6). At the stations further downstream (IBI-11 to IBI-13), the temperature was 26  C. The dissolved oxygen levels were above 8 mg/l at IBI sampling stations one to 10 and just greater than 7 mg/l at stations further downstream (IBI-11 to IBI-13). There was a general increase of alkalinity (18–29 mg/l as CaCO3), hardness (41–61 mg/l as CaCO3) and conductivity (83–160 mmhos) in going from upstream to downstream that likely reflects physical/geological conditions rather than anthropomorphic influence (Richard, 1996). Several parameters such as color (27– 50 color units), total suspended solids (2–8.5 mg/l), ammonia (0.01–0.17 mg/l), turbidity (0.75–3.25 N.T.U.), total nitrogen (0.7–1.7 mg/l), phosphorous (0.02–0.09 mg/l) and total organic carbon (18–28 mg/l) varied to different degrees between stations, but, without any apparent trend. In particular, there was no increase in these parameters resulting from the discharge of the mill effluents. 4.2. Sentinel apecies assessment 4.2.1. HAI approach There was no sex-related difference for the HAI score of any of the three species. Hence, the data for both sexes were combined for interpretation. The HAI scores

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T.G. Kovacs et al. / Environmental Pollution 118 (2002) 123–140 Table 2 Habitat description of sentinel-species and fish-community sampling stations Station number

Maximum widtha (m)

Sentinel species SS-1 200 SS-2 120 SS-3 280 SS-4 SS-5 SS-6

Velocityb (cm/s)

Dominant substratec

Shore vegetationd

15 5.0 20

Moderate Fast Moderate

Bedrock Bedrock Boulder, alluvium, bedrock Boulder, alluvium Sand, gravel

–-e – –

<20 <20 <20

Wilderness Industry, wilderness Wilderness, residential

– – –

<20 <20 <20

Wilderness Wilderness, cottages Wilderness, residential

Trees, shrubs, grass

<20

Wilderness, cottages

5.0 5.0 4.0

Slow to moderate Slow Moderate

Fish community IBI-1 96

3.0

600

IBI-2 IBI-3

170 70

7.0 8.0

IBI-4 IBI-5 IBI-6 IBI-7

100 60 98 57

IBI-8 IBI-9 IBI-10 IBI-11 IBI-12 IBI-13 a b c d e f

240 200 200

Maximum deptha (m)

Aquatic vegetation (%)

80 20

Boulder, bedrock, alluvium, gravel, Clay, sand, gravel Clay, sand

Trees Trees

1.5 1.3 1.3 2.0

810 800 720 550

Alluvium, gravel Alluvium Alluvium Alluvium, gravel, rock

Trees, shrubs, grass Trees, shrubs, Grass, shrubs Trees, grass

<20 <20 <20 <20

98

3.0

350

Clay

Trees, grass

<20

85 110 130 125 179

3.5 3.5 5.0 8.0 2.0

300 370 190 400 200

Clay Sand, gravel Sand, gravel Alluvium, sand, bedrock Sand, gravel, alluvium

Grass, shrubs Grass, trees Grass, trees Trees, grass Trees, grass, shrubs

20–50 20–50

<20 <20 <20 None <20

General shore description

Wilderness, cottages Wilderness, cottages, agriculture Agriculture Wilderness Cottages, agriculture Residential, cottages, industryf Residential, cottages, industryf Agriculture, residential Wilderness Wilderness, cottages Wilderness, agriculture Wilderness, residential

Within 0.5 km sampling zone. At one location considered most typical within 0.5 km sampling zone. All substrates shown represent at least 15% of the total; decreasing importance from left to right. For a distance of 5 m from the shoreline. Not determined. Non pulp mill industry.

for tesselated darters and smallmouth bass in 1998 are shown in Fig. 2. The score for darters from all locations above and below mills at East Angus and Bromptonville was 11 or less. When comparing the darters from upstream and downstream of each mill site, the scores downstream from mills at East Angus and Bromptonville were significantly lower than scores from upstream. The few observations of abnormalities were related to mild parasite (black spot) infestations, whereas, other parameters were found to be normal in 97% of the cases. There was no significant HAI difference between the smallmouth bass upstream (HAI: 28) and downstream (HAI: 26) from the mill at Windsor (Fig. 2). These fish had slightly higher overall scores than darters and the abnormalities included mainly liver discoloration, mild to severe black spot infestation and frayed fins (at the upstream station only). All plasma protein and packed red cell volumes were found to be above the normal range reported by Adams et al. (1993). The HAI scores for white sucker in 1995 and 1998 are shown in Fig. 3. The scores ranged between 38 and 79

depending on location and time of capture. In 1995, the HAI scores of the fish upstream (SS-3) and downstream (SS-4) of the Bromptonville mill were not significantly different from each other. When considering the fish from all three of the sampling locations, the HAI of fish downstream (SS-4) from the mill at Bromptonville was significantly different from the HAI score upstream (SS-1) from the mill at East Angus. The fish from the two upstream stations (SS-1 and SS-3) had HAI scores that were not significantly different. The HAI values did not change significantly between 1995 and 1998 at two of the stations (SS-1 and SS-4). However, the HAI declined significantly upstream (SS-3) from the mill at Bromptonville. Simple upstream–downstream comparisons (e.g. SS-1 and SS-2) or multiple comparisons (i.e. all the stations) of the 1998 fish indicated significant increases in the HAI values downstream from mills at East Angus and Bromptonville. The abnormalities contributing to the HAI scores in white suckers were observed mostly on the eyes, gills, livers as well as in the percent hematocrit/plasma protein

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Fig. 2. The health assessment index (HAI) score for tesselated darters (white) and smallmouth bass (black) at the six sampling stations of the study in 1998. The numbers in parentheses represent standard deviation values. * Denotes that the HAI of the downstream darters is significantly lower than the HAI of the upstream darters (Mann– Whitney, P<0.05).

concentration and presence of anomalies (Table 3). The eye abnormalities included whiteness of the cornea and lens (possibly indicating parasitic infestation) or missing eyes. For the gills, the most frequent abnormalities were clubbed (mostly swelling of lamellae) and marginated (whiteness at the margin) gills and paleness (possibly indicating anemia). The most frequent liver discoloration observed was a coffee-cream color instead of the normal reddish/brown. Fish from some locations also had external and/or internal parasites that increased the HAI values. For all of the other HAI parameters (e.g. kidney, hindgut) the percent of fish with a normal condition ranged between 80 and 100%. The parameters examined but not included in the HAI calculation for white sucker included bile coloration and percent fat content as recommended by Adams et al. (1993) and the condition of the scales, fin deformation and skeletal abnormalities. Bile coloration was almost always yellow and the fat content was highly

Fig. 3. The health assessment index (HAI) score for white suckers at the six sampling stations in 1995 (white) and 1998 (black). The numbers in parentheses represent standard deviation values. Bars not sharing the same letter (upper case for 1995; lower case for 1998) are significantly different from each other (Dunn’s multiple comparisons, P <0.05). * Denotes that the HAI of the downstream suckers is significantly higher than the HAI of the upstream suckers (Mann– Whitney, P<0.05).

variable lacking any consistent trend (data not shown). Skeletal abnormalities, such as lordosis, scoliosis, deformation of the head, jaws or operculum, were extremely rare, and never found in more than 1.8% of the fish at any of the stations. Scale anomalies, such as disruption of the normal pattern, regeneration (rosettes) and missing or healing scales were found in 6–37% of the fish (Table 3). These anomalies generally increased in going from the most upstream to downstream stations. Fin deformities, such as abnormal shapes, missing and reoriented fins were not found at the station upstream from the mill at Windsor (SS-5), but were observed in 11–35% of the fish at other stations (Table 3). Finally, of the suckers used for the HAI approach, nine individuals downstream from the Windsor mill were found with tissue growth on the lips and five individuals at various locations were found to be in

Table 3 The percent of white sucker in 1995 and 1998 with abnormal scores for various HAI parameters Sentinel species sampling stations Mill A

Mill B

SS-1

SS-2

SS-3

Mill C SS-4

SS-5

SS-6

1995 (n=20)

1998 (n=64)

1998 (n=65)

1995 (n=21)

1998 (n=72)

1995 (n=47)

1998 (n=73)

1998 (n=60)

1998 (n=55)

45 55 10 0 10 50

19 48 19 42 48 33

40 66 43 58 17 66

71 71 48 5 33 29

28 24 39 10 28 53

98 70 47 11 26 26

85 71 45 8 34 29

48 48 35 0 27 42

67 62 18 4 36 47

Non-HAI parameters Scales –a Fins –

6 19

12 35

– –

13 11

– –

16 12

37 0

35 15

HAI parameters Eyes Gills Liver Parasites Hematocrit Plasma protein

a

Not determined in 1995.

131 Values not sharing the same letter (upper case 1995; lower case 1998) are statistically significant from each other (Dunn’s multiple comparisons, P< 0.05). Numbers in parentheses represent standard deviations. b For any one year, downstream fish significantly different from upstream fish (Mann–Whitney test, P <0.05). c PMS, post mitochondrial supernatant.

a

1710a (1333) 1549ab (963) 3.5a (2) 1618b (846) 1428b (962) 5.8b (3) 1597a (967) 1225abc (740) 3.6a (2) 963 (628) 897 (501) 9.0 (3) 1157ab (742) 1358abcb (755) 3.4a (2) 2719B (1194) 2708B (1279) 3.1Ab (2) 986b (791) 1052c (950) 3.3a (2) 2468B (747) 2415B (1012) 7.9B (3) 1878ab (1255) 1845ab (1057) 4.8a (3) 660A (276) 913A (602) 2.8A (2)

1009b (763) 1065bc (628) 6.8a (9)

1089 (664) 14 (8) 1656a (790) 15a (8) 1388 (832) 15 (7) 1261a (548) 13a (8) 2268 A (1078) 7.9Ab (3) 1498a (887) 12a (6) 2423A (832) 18B (8) 1965ab (1156) 15a (8)

Females Testosterone, pg/ml Estradiol, pg/ml EROD activity, pmol/min/mg PMSc

1998

1416a (800) 17a (22) 1562A (664) 5.2A (2) Males Testosterone, pg/ml EROD activity, pmol/min/mg PMSc

1994 1995 1998 1995

1995

SS-3 SS-2 SS-1

1998

SS-4

1998

1994

1998

SS-6 SS-5

Mill C Mill B Mill A

Sentinel species sampling stationsa

Table 4 Hepatic EROD activity and plasma steroid levels in white sucker captured at the various sentinel species sampling stations, 1994–1998

4.2.2. Biochemical measurement approach The hepatic EROD activity and plasma steroid levels of male and female white suckers captured at the various sentinel species sampling stations are shown in Table 4. In 1994, the testosterone and EROD activity in males captured upstream (SS-5) and downstream (SS6) from the Windsor mill was not significantly different. In contrast, for the females, both hepatic MFO activity as well as plasma steroid levels were significantly different. Enzyme activity was lower and steroid levels were higher in fish captured downstream from this mill. In 1995, comparison of the fish upstream (SS-3) and downstream (SS-4) from the mill at Bromptonville indicated reduced EROD activity in the downstream fish. When fish from all three mill locations were considered together, the levels of testosterone in males upstream from mill A as well as both upstream and downstream from mill B were not statistically different. A significant increase of enzyme activity in males upstream from mill B when compared with males from the other two locations was evident. In the case of females, testosterone and estradiol levels were significantly lower for individuals sampled upstream from mill A. As for males, liver MFO activity was higher in females captured upstream from mill B. In 1998, comparison of the fish upstream and downstream from the three mills indicated increased testosterone in males downstream (SS-2) from the mill at East Angus (Mill A) and increased testosterone and estradiol in females downstream from this mill. When all the sampling stations were considered together, testosterone levels and EROD activity in males were not significantly different. In females, EROD activity was also not site dependent. Plasma testosterone levels were similar for fish captured upstream from the East Angus mill (SS-1) and upstream/downstream (SS-3/SS-4) of the mill at Bromptonville. The plasma testosterone levels were higher in females from the two sampling locations (SS-5 and SS-6) around the Windsor mill as well as downstream (SS-2) from the East Angus mill than in females

1998

an emaciated condition. Because the overall HAI score of the downstream fish was not higher, the tissue growth on the lips was not considered to have great significance concerning overall health. Tissue growth was also observed on the lips of suckers captured at various locations along the river during the sampling for the IBI approach indicating that this condition was not unique at the furthest downstream sentinel species sampling site. In the two other species, smallmouth bass and tesselated darter, there were no significant observations worthy of note concerning the parameters not used for HAI calculation. Only one bass at station SS-5 and one bass at station SS-6 were judged to have both male and female gonads suggesting that these fish were hermaphrodites.

1888a (1043) 12a (9)

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upstream (SS-1 and SS-3) from the mills at East Angus and Bromptonville. In general, estradiol levels were higher in females sampled at the locations downstream from the three mills and the lowest level was found in fish upstream from the Bromptonville mill. There was substantial variability in the plasma testosterone levels of female white suckers sampled from the reference stations in 1998. Levels ranged from 986 to 1597 pg/ml, and the plasma concentration in fish from upstream of the Windsor mill was significantly higher than in fish from the other two sites. In contrast, estradiol levels and hepatic EROD activity in the same fish and EROD activity and testosterone levels in males from the same reference stations were not significantly different. 4.3. Fish community assessment Information about the fish captured at the 13 stations studied for fish community structure is summarized in Table 5. A total of 5003 individuals were sampled representing 28 species, several trophic levels and different degrees of pollution tolerance. Overall, the total number of species and total number of individuals at the 13 sampling stations varied between 11–19 and 82– 758, respectively. Three species of fish (M. dolomieui, S. corporalis and P. caprodes) were captured at all 13 sampling locations. Six other species (I. unicuspis, S. vitreum, E. olmstedi, C. commersoni, L. cornutus and A. rupestris) were found at least at 10 of the 13 stations. In general, the species captured at the most stations were also the most abundant in quantity. Except for the lack of cyprinids at the uppermost station (IBI-1), there was no general trend in species distribution or species richness in going from upstream to downstream. The data for the various metrics and the corresponding scores as well as IBI values are shown in Table 6. The metrics with the greatest variation between stations included the percent omnivores and the percent insectivores. The percent of fish with anomalies, the number of pollution intolerant species and the number of Catostomidae exhibited intermediate variability between the 13 study stations. The scores for percent piscivores and the modified index of well-being metrics were the maximum possible at all the sampling stations. In the case of the piscivores, the high percentages were the result of large numbers of young of the year fish. There were only small differences in the individual metric scores of the fish communities immediately above and below mill effluent outlets. At East Angus, there was an increase in the percent of fish with anomalies downstream from the mill. At Bromptonville, there were more species of Catostomidae upstream from the mill whereas at Windsor, the percent insectivores, the percent of fish with anomalies, the number of intolerant species and the number of Catostomidae were all greater downstream from the effluent.

While there were obvious differences in species composition and abundance resulting in different metric scores for the fish inhabiting different regions of the river, the overall IBI values ranged from 45 to 57 and such values according to Richard (1996) represent fish communities that can be rated as average to excellent. The fish community in the two stations immediately above East Angus mill (IBI-2 and IBI-3) with a score of 57 fell in the excellent category. The metrics for the fish from these two locations were found to have the highest possible score, except for the presence of pollution intolerant species. The other fish communities were classified as either average (e.g. the uppermost location, IBI-1; IBI-8, downstream from Bromptonvile, IBI-10; upstream from Windsor, IBI-11; and the lowermost location IBI-13) with a score of 45 or good (all other stations) with scores of 48 and 54. The major factors contributing to the lower IBI values that resulted in the average or good classification were greater number of omnivorous species, fewer insectivorous species and greater incidences of anomalies. Fore et al. (1994) suggested that IBI values that differ by 48 units should not be considered to be significantly different. On the basis of this criterion, there was no degradation of the fish communities immediately downstream from mill discharges, when compared with fish communities immediately upstream from the three mills. On the contrary, the fish community below the Windsor mill was found to have an IBI value nine points higher than the fish community just above the mill. At this location, the percent of fish with anomalies was 4.9, but this was counterbalanced by the presence of 53% insectivores, two Catostomidae species and one pollution-intolerant species.

5. Discussion The main objective of pollution abatement programs is to protect the ecological integrity of our waterways, for example, fish populations and communities. More and more, the success of such efforts is gauged by the study of fish in the wild. There are numerous approaches to assess the biological status of wild fish (Cash, 1995), but, essentially, they can be broadly classified into sentinel species and community-based approaches. The assumption of the sentinel species approaches is that one or two species can be considered to be indicators of the condition of all the fish inhabiting a particular waterway (Gibbons and Munkittrick, 1994). Community-based approaches do not focus on individual species but, rather, assess the fish community structure directly in terms of various metrics such as proportion of pollution intolerant species (Karr, 1981). The interpretability of the results from the various approaches, that is our ability to establish causality,

Table 5 Taxonomic classification, trophic level (PIS, piscivore; INS, insectivore; OMN, omnivore), tolerance rating (TOL, pollution tolerant; INR, intermediate; INT, pollution intolerant) and abundance of fish captured at 13 different stations in the St. Franc¸ois River in 1998 Family

Catostomidae (Suckers)

Species

a

INS OMN INS INS INS PIS INS PIS OMN HERa INS OMN INS INS OMN INS OMN OMN PIS PIS INS INS INS PIS INS INS PIS PARa

INR TOL INR INR INT INR INR INR TOL INR INR TOL INR INT TOL INR TOL INR INR INR INR INT INR INR INR INT INR INR

2 2 3

1 1

12 4 7

3

4 2 8

23 26 15

13

3 9

178

467

37

IBI-4

IBI-5

5 5

2 63 6 1

1

43

25 3

IBI-7

5

14 5

1

22

68

1 132

19

24

23

17

4 6 11 3 213

IBI-8

IBI-9

2 17

9 34

IBI-10 IBI-11 IBI-12 IBI-13 1 1

2

5

4 103 17

IBI-6

45

3

1 2 5 42

5 4 64 1 4 1

1 5 7 142

5

3 33

6 3 144

5 3 92

2 2

1 11

7 8

1 49 1

1 76 2

2 69

69 2

24

23

14 3 380 2 2

2 1 3

1

4 3 1

22 1

2 13 1 102 1

1 2

3

2 47 16 2 75 49 4 22 2 2 2 9 3 17 18 63 49 7 37 5 3 72 80 3 6 6 3 8 2 1 11 44 9 250 307 36 87 13 78 8 2 1 8 10 2 4 2 11 1 1 1 1641 2301 3326 2066 2553 2320 2213 3047 2832 2596 3156 2635 3561 86 274 758 243 500 338 220 545 683 227 236 185 708 12 12 16 12 18 12 14 19 15 12 11 14 16 3.1 7.1 13.7 7.1 11.8 8.7 6.0 10.7 14.5 5.2 4.5 4.2 11.9 5

3 14 5

9 119 34

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Catostomus catostomus Catostomus commersoni Moxostoma anisurum Moxostoma macrolepidotum Moxostoma valenciennesi Centrarchidae (Bass, Sunfish) Ambloplites rupestris Lepomis gibbosus Micropterus dolomieui Cyprinidae (Minnows, Shiners) Cyprinus carpio Hybognatus regius Luxilus cornutus Notemigonus crysoleucas Notropis atherionides Notropis volucellus Pimephales notatus Rhinichthys cataractae Semotilus atromaculatus Semotilus corporalis Esocidae (Pike) Esox lucius Gadidae (Burbot) Lota lota Gasterosteidae (Stickleback) Culaea inconstans Hiodontidae (Goldeye) Hiodon tergisus Percidae (Darters, Perch, Walleye) Etheostoma olmstedi Perca flavescens Percina caprodes Percina copelandi Stizostedion vitreum Petromyzontida (Lamprey) Ichthyomyzon unicuspis Electrofishing time Total number of fish Total number of species Catch per unit effort

Trophic Pollution Sampling stations level tolerance IBI-1 IBI-2 IBI-3

Not included in trophic metrics; HER, herbivore; PAR, parasitic.

133

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T.G. Kovacs et al. / Environmental Pollution 118 (2002) 123–140

Table 6 Metrics and values used to calculate a modified index of biotic integrity (IBI) for the St. Franc¸ois River in 1998a Station

[1] Percent omnivores

[2] Percent insectivores

[3] Percent piscivores

[4] Percent of fish with anomalies

[5] No. of pollution intolerant species

[6] No. of Catostomidae

[7] Modified index of well-being (IWB-IWBm)

Modified IBI score P ( 1+2+3+4+5+6+7)1.5

IBI-1 IBI-2 IBI-3 IBI-4 IBI-5 IBI-6 IBI-7 IBI-8 IBI-9 IBI-10 IBI-11 IBI-12 IBI-13

22 16 6 11 57 14 41 23 15 12 14 12 17

19 74 73 77 24 33 31 18 16 6 3 53 16

50 10 21 9 17 52 26 59 67 82 82 34 67

4.7 0.7 0.3 2.6 0.2 2.8 2.4 2.1 3.3 4.1 0 4.9 6.8

1 1 1 1 2 1 2 1 1 2 0 1 1

3 2 3 3 4 1 2 3 3 1 1 2 3

0.04 0.11 0.13 0.02 0.17 0.18 0.07 0.11 0.03 0.05 0.03 0.06 0.12

45 57 57 54 48 48 48 45 48 45 45 54 45

a

(3) (5) (5) (5) (1) (5) (3) (3) (5) (5) (5) (5) (5)

(1) (5) (5) (5) (3) (3) (3) (1) (1) (1) (1) (5) (1)

(5) (5) (5) (5) (5) (5) (5) (5) (5) (5) (5) (5) (5)

(3) (5) (5) (3) (5) (3) (3) (3) (3) (3) (5) (3) (1)

(3) (3) (3) (3) (3) (3) (3) (3) (3) (3) (1) (3) (3)

(5) (5) (5) (5) (5) (3) (5) (5) (5) (3) (3) (5) (5)

(10) (10) (10) (10) (10) (10) (10) (10) (10) (10) (10) (10) (10)

The metric scores in parentheses were assigned according to the criteria in Table 2.

measure the degree of environmental degradation if it exists, or, convincingly prove absence of harm and even improvements in relation to effluent discharges, depends on a variety of factors. Most field study designs involve comparison of areas receiving discharges to reference areas. Such ‘‘upstream/downstream’’ designs introduce problems of pseudoreplication (Hurlbert, 1984) or the lack of appropriate replicates that would allow statistical inferences concerning impact caused by an effluent. This hampers our ability to establish causality. The assumption in such studies is that except for the effluent in question all other conditions are identical between the two stations. The validity of this assumption is open to debate and touches on issues related to habitat, biotic–abiotic interactions and natural variability. In the case of fish, mobility and lack of knowledge concerning life history have the potential to further influence our ability to establish causality. There are other factors that tend to be more specific for a particular approach. For sentinel fish, especially when measurements are made at the biochemical or organ level, there is a lack of evidence that changes can be related to the population/ community level (Kloepper-Sams, 1996). For community studies, such as the IBI, concerns have been raised about lack of sensitivity to ‘‘chemicals’’ and the long time needed to see changes thereby reducing the possibility of remedial action (Haux and Fo¨rlin, 1988; Cash, 1995). As all field approaches have advantages and disadvantages (Cash, 1995), it was felt that the simultaneous use of three approaches in this study could provide valuable leads as to which methods provide potential for gauging the outcome of industry strategies aimed at environmental protection. The comparison of the approaches in this study focused on the study design

and the ability of the three approaches to track changes in effluent quality. 5.1. Study design The St. Franc¸ois River situation provided a unique opportunity to assess the status of fish by using three different approaches both on a spatial (upstream/ downstream comparisons in any one year) and temporal (comparisons between different years) basis. The sampling locations in 1998 were chosen to match the sites of previous sentinel species and community studies. While the sampling locations for sentinel species and community assessment were not identical, they were sufficiently close to allow for the comparisons of the different approaches. Spatial comparisons for any one-year could verify if responses (e.g. with respect to plasma steroids) previously reported for fish exposed to mill effluents existed in this system. The temporal comparison, or the beforeafter-control-impact (BACI) study design suggested by Stewart-Oaten et al. (1986), could overcome concerns about the virtually impossible task of selecting ideal reference stations and provide the best opportunity to assess the outcome of process/treatment changes implemented by three mills in 1995. The description of habitats in Table 2 illustrates the difficulty of matching reference and exposed stations, for example, regarding differences in habitats caused by dams. On the other hand, the existence of dams restricting fish movement between stations upstream and downstream from mills in this study minimized concerns about fish mobility and life history that plague all studies done in the field. In terms of process/treatment changes, two of the three mills installed secondary treatment facilities in

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1995 that resulted in meeting new regulations regarding BOD, suspended solids and acute lethal toxicity. The third mill was already in compliance as it had a biological treatment system since the time of the mill startup in 1987, but made further improvements through process changes and fine-tuning of the treatment system. The benefit of the changes at the three mills with respect to effluent quality, including sublethal toxicity, is summarized in Table 7. The new treatment facilities at mills A (East Angus) and B (Bromptonville) reduced BOD and TSS by 89–97% and 70–87%, respectively. At mill C (Windsor), the fine-tuning of the existing treatment system resulted in a further reduction of BOD and TSS discharges by 59 and 55%, respectively, for the secondary-treated effluent. As a result of the installation of secondary treatment, effluent acute lethal toxicity to rainbow trout was eliminated at mills A and B. The effluent from mill C already had 96-h LC50>100%. The sublethal toxicity of the effluent from the three mills, particularly for mills A and B after the implementation of secondary treatment, was also substantially reduced (Table 7). For example, the average IC25 for mill B effluent prior to secondary treatment in fathead minnow, Cerioaphnia and algae tests was 6, 0.4 and 9%, respectively. These IC25s increased, indicating reduced sublethal toxicity, to >100, 91 and 70% after secondary treatment of the effluent.

5.2. Comparison of sentinel-species and communitybased field approaches The biological status of fish in relation to pulp and paper mill effluent exposure has been the focus of many field investigations in the past. Some of the work was reviewed by Owens (1991), Carey et al. (1993) and Kovacs et al. (1997a). At the biochemical level, reduced plasma steroids and increased hepatic EROD activity were frequently observed (Munkittrick et al., 1992). At the organ/whole organism level, increased condition factor/liver size, decreased gonad size and fecundity (Munkittrick et al., 1991, 1994; Parker and Smith, 1997; Langlois and Dubuc, 1999) as well as increased HAI (Adams et al., 1993), mostly due to liver, gill and kidney anomalies, were reported. Mill modernizations have been found to reduce effluent effects on these parameters although in some cases they have not completely eliminated them (Sandstro¨m, 1994, 1996; Munkittrick et al., 1997a; Seegert et al., 1997). Studies directly assessing the potential effects of mill effluents on the whole fish community have been less common. In one case (Adams et al., 1992), deleterious influences on the whole community were related to effects at lower levels of biological organization (e.g. biochemical, organ). In another study involving a community-based approach, mill modernization was shown to improve the fish community structure (Seegert et al., 1997).

Table 7 The quality of effluents discharged by the three mills in this study before and after process/treatment changes in 1995 as determined by regulatory physical/chemical and toxicity tests Mill A

Physical/chemical tests BOD, kg/ta TSS, kg/ta

a

Mill C

Pre 1995

Post 1995

Pre 1995

Post 1995

Pre 1995

Post 1995

7.1 (5.8–8.3) 5 (4.8–5.2)

0.8 (0.6–1.0) 1.5 (1.0–1.8)

15.4 (14.8–16.8) 6 (5.5–6.5)

0.4 (0.3–0.6) 0.8 (0.5–1.0)

3.2 (1.2–6.3) 6.6 (0.7–11.5)

1.3 (1.1–2.0) 3.0 (2.7–3.5)

>100c n=12

21d (8–35)

>100c n=12

>100e n=12

>1003 n=12

20g (9–32) n=4 >100 n=3

6h (5–7) n=3

>100 n=4

74 (40–99) n=3 84 (37–>100) n=4

9g (0.5–26) n=4 58 (10–100) n=3

0.4h (0.1–0.6) n=3 91 (65–>100) n=4 16 (8–22) n=3

Acute toxicity tests Rainbow trout, 96-h LC50 (%) 16–72b Sublethal toxicity tests Fathead minnow growth, 7-day IC25 (%)f Ceriodaphnia reproduction, 7-day IC25 (%)f Selenastrum growth, 7-day IC25 (%)f

Mill B

5g (3–10) n=4

77 (32–>100) n=3 9h (7–10) n=3

70 (35–>100) n=4 9 (5–11) n=3

57 (27–>100) n=4 45 (15–>100) n=4

1992–1994 (pre 1995) and 1996–1998 (post 1995) averages. Prior to the installation of secondary treatment in 1995, the mill complex discharged four separate wastewaters. The LC50 values shown here represent the range of average values for the four discharges (Ame´natech, 1996a). c The average LC50 for 1998. d This mill had two discharges prior to the installation of secondary treatment in 1995. In 1994, the average LC50 for the principal sewer, which accounted for about 85% of the total flow, was 21% (Ame´natech, 1996b). e Represents the toxicity data for 1994. f Values represent averages of number of tests (n) indicated. The numbers in parentheses represent the range of IC25 values. g A composite effluent sample was prepared in proportion to the flow of the four individual wastewater stream discharges to the river from the mill complex prior to the installation of secondary treatment (Ame´natech, 1996a) h A composite sample was prepared from the two discharges prior to the installation of secondary efflluent treatment in 1995 (Ame´natech, 1996b) b

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The most frequently observed responses previously attributed to mill effluents at the biochemical level in wild fish (i.e. increased EROD activity and lower steroids) were not evident in this study either before or after the process/treatment changes. To the contrary, when differences between fish above and below mills were observed, they involved higher steroid levels and lower MFO activity. However, no clear or consistent trends were evident in upstream and downstream comparisons at the downstream stations throughout the 75-km stretch study site or in terms of temporal comparisons between 1995 and 1998. Hence, any biochemical differences were judged more to be the result of natural variability than an effluent-related phenomenon. It appears that compounds (e.g. wood extractives, phytosterols) previously shown or hypothesized to be capable of causing such responses (MacLatchy and Van Der Kraak, 1995; Martel et al., 1997) were not present in the river or were at levels below those able to cause effects. For biochemical measurements, the normal conditions in fish are largely unknown (Sprague, 1990; Thomas, 1990; Schlenk, 1999) making it difficult to draw inferences about biological significance. For example, increased MFO activity in fish from some locations was initially thought to be associated with adverse effects on other biological functions such as reproduction (Hodson, 1996). However, with time, it was realized that increased enzyme activity was only indicative of exposure to mill effluent components, such as extractives from wood, and is not an indication of serious health problems (Hodson, 1996; Martel et al., 1997). In the case of serum steroids, there is no clear guidance as to what reductions constitute reproductive problems. In the field, links of reduced hormones to lower gonad size and fecundity have been made in some studies (Adams et al., 1992; McMaster et al., 1991; Munkittrik et al., 1991) but not in others (Gagnon et al., 1994; KloepperSams et al., 1994). As stated earlier, there is no evidence for links to population and ecosystem effects (KloepperSams, 1996). Finally, it appears that measurements of circulating sex hormones at a single point in time are not good indicators of reproductive capacity of fish even in controlled laboratory studies (Kramer et al., 1998). As such, biochemical measurements appear to be most useful as a tool for explaining effects at higher levels of biological organization (Sprague, 1990), if such effects are found. The HAI assessment in this study was completed for three sentinel species at six locations in 1998, but was done only for white sucker from three locations in 1995. Hence, only a limited temporal comparison could be made on the basis of the HAI approach. In the case of white sucker, there were significant increases in the HAI of fish downstream from mills A and B in 1998 indicating possible mill-related influences. However, a clear

link of increased HAI to mill effluents was complicated by comparison with the situation in 1995. At that time, there was no difference in the HAI of fish from above and below mill B. Between 1995 and 1998, the HAI for fish upstream from mill B decreased significantly, but stayed the same for the fish downstream from the mill. As the significant difference between the upstream and downstream fish in 1998 is the result of seemingly improved HAI for the upstream fish, it is difficult to relate the higher HAI score of the downstream fish in 1998 only to the presence of the mill effluent. Also, the type of abnormalities that contributed to the increased HAI, such as eye and liver anomalies, were more of the type that can be found in fish from virtually all locations, that is with or without man-made influences (Fournie et al., 1996). This is in contrast to the types of anomalies previously reported for fish from mill impacted stations (Adams et al., 1993). Finally, for two of the other three sentinel species, namely, tesselated darters and smallmouth bass, no mill-related influences could be established in 1998. In fact, the HAI score of darters was lower downstream from two of the mills. As for biochemical measurements, there is also no documentation what HAI value represents healthy or sick fish. Rather, the HAI of fish from exposed areas are compared with the HAI of reference fish for statistical differences (Adams et al., 1993). For example, the HAI of largemouth bass (Micropterus salmoides) from uncontaminated areas ranged between 17 and 42, whereas, the HAI of fish from stations considered impacted ranged from 64 to 79 (Adams et al., 1993). In the St. Franc¸ois River study, the HAI of the three sentinel species was found to be different. The HAI was the lowest for tesselated darters, followed by smallmouth bass and white sucker. This HAI trend probably reflects differences in the age of the fish, older fish are expected to have more abnormalities, and lifestyle, as, for example, the incidence of gross pathological abnormalities are higher in demersal species than in pelagic species (Fournie et al., 1996). The mean age of the suckers (a bottom-dwelling species) in this study ranged from 4.3 to 7.6 years, while the mean age of darters (inhabiting more quiet shoreline waters) and bass (pelagic species) ranged from 1.9 to 2.8 years and 3.5 to 6.0 years, respectively. It is also worth noting that the age of suckers downstream from mill B, with a higher HAI score than fish from upstream, was about 2 years greater than the age of suckers upstream (Tables 4 and 5). Overall, the darters in this study had relatively few abnormalities and their HAI scores were less than 12. Such scores can probably be considered representative of healthy fish considering the maximum possible score of 360 for this species. The HAI score for bass ranged between 25 and 35 and these values fell in the range of what was considered normal for largemouth bass (a phylogenetically similar species) by Adams et al.

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(1993). The scores for suckers ranged between 37 and 78 representing larger percentages of fish with anomalies, although, even in this case, the scores were relatively low in comparison with the maximum possible score of 450. Suckers sampled in 1997 from four locations along non-impacted tributaries to the St. Maurice River in Quebec had HAI values between 30 and 34 (Paprican unpublished results). In view of this, overall, a HAI assessment on its own is unlikely to provide sufficient information for environmental decision making purposes unless consistent and dramatic differences are observed. Rather, as appeared to be the case with biochemical measurements in sentinel species, the HAI approach seems to have potential as a supportive tool used in conjunction with other approaches. In contrast to the sentinel species approaches, Richard (1996) concluded from his fish community assessment in 1991 that on the basis of IBI values, fish assemblages were considered to be impaired below the two mills (A and B) which at that time were discharging non-secondary-treated effluents (Fig. 4). The overall condition of the fish community was reflected by the percentages of fish with anomalies (sometimes reaching close to 20%), suggesting that man-made influences were at least partially responsible. This was not the case below the mill (C) discharging secondary-treated effluent where the fish community was found to have a slightly higher IBI score relative to the upstream station. However, 3 years after the installation of secondary treatment at the two mills, the fish communities downstream from mill A and B effluent discharges improved from a poor classification to a level considered good and average, respectively. By 1998, there was little, if any difference relative to the immediate upstream station(s). One reason for the improved IBI score was the much lower percentage of fish with anomalies. Other metrics that contributed to the improvement included the percent piscivores and the modified index of well being. Downstream from mill C, the fish community improved from an average to a good classification

Fig. 4. A comparison of the condition of the fish communities along the St. Franc¸ois River in 1991 and 1998 according to index of biotic integrity (IBI) assessments. IBI results from 1991 were taken from Richard (1996).

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probably reflecting better water quality throughout the river as well as process modifications and fine-tuning of the treatment system at this mill. The improvements that were evident on the basis of the IBI approach mirrored the improvements in effluent quality at the three mill sites as determined by regulatory tests for BOD, TSS, acute lethal and sublethal toxicity. In the case of the IBI approach, the condensation of information from several parameters into a single number, while providing directions as to what is acceptable (excellent, good) and what is not (poor, very poor), is sometimes considered to be an oversimplification (Landis and Yu, 1995). However, there is nothing preventing the use of individual metrics in the interpretation of the IBI value (James Karr, personal communication), should it be deemed of value. More importantly, for the IBI, the scores assigned for each metric as well as the selection of specific metrics can influence the overall interpretation of the available data. For example, in 1998, the score for the modified index of well being was identical at all sites suggesting this metric was not sufficiently sensitive for detecting significant differences between community characteristics. Due to the high percentage of piscivores at some sites in 1998, the maximum score of five for this metric was assigned for assemblages with nine to 82% piscivores. Such a broad range in percent of fish representing one trophic level may seem unreasonable. However, for the 1998 portion of this study, we followed the metrics and assigned scores used by Richard (1996) for his study in 1991. This is what allowed a direct comparison between the situation in 1991 and 1998. On the basis of such a temporal comparison the shear changes observed in the modified index of well being as well as percent piscivores at some locations indicates that substantial improvements were evident. Because Richard’s (1996) metrics and scores were based on the conditions existing in the early 1990s and because the situation in the river changed substantially since 1991, there may be some justification for changing the original metrics and scores for a spatial comparison later in the decade. We made some calculations of the IBI on the basis of changes to the metric selection (e.g. dropping the index of well being, including total number of taxa) as well as the assignment of scores for a particular metric (e.g. changes assigned to the percent piscivore metric). While such changes obviously changed the IBI scores, it did not change the general trend for 1998 presented in Fig. 4. Just as importantly, it did not change the evidence for substantial improvements since 1991, particularly when the same changes were applied for the calculation of the IBI values in 1991. This suggests a certain robustness of this approach that can be considered to be useful for monitoring studies. In summary, there were differences in the trends of the status of the St. Franc¸ois River fish based on

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sentinel-species and community-based field approaches. Most importantly, in this river system, the communitybased approach appeared to be the most sensitive in responding to mill discharges as well as in tracking the benefits of mill process/treatment changes that resulted in improved effluent quality. This finding confirms the potential value of this approach for monitoring the status of fish in relation to effluents from pulp and paper mills. Previous investigators have reported that sentinel species approaches, not corresponding to effects seen at the community level in this study, did detect responses in fish exposed to mill effluents as well as improvements in effluent quality at other sites (Sandstro¨m, 1996; Munkittrick et al., 1997a). This illustrates the possibility that the nature and concentration of effluent components responsible for the effects may vary from site to site. As such, while there appear to be several approaches that can be used for impact assessment work, the selection of the appropriate method may need to be site specific depending on what is likely to be the agent or endpoint of concern.

6. Summary and conclusions 1. Comparisons of three approaches used to assess the biological condition of fish in the St. Franc¸ois River showed different capabilities to detect both impacts related to mill effluents and improvements related to process changes/secondary effluent treatment. 2. The biochemical measurements (plasma steroid levels, hepatic MFO activity) in white sucker did not appear to be deleteriously affected by the mill effluent and were not indicative of effects at the community level. This suggests the possibility that, in this case, effluent components causing effects at the lower level of biological organization were different than what may cause effects at the higher level of biological organization or that the concentrations of causative agents were below what can cause biochemical changes. 3. The HAI assessment of sentinel fish indicated differences in fish at different locations, but these could not be exclusively linked to mill effluents. 4. In contrast to the sentinel species approaches, the IBI approach in the St. Franc¸ois River system showed evidence of both mill-related effects on fish assemblages as well as improvements in fish communities. From a study conducted in 1991, Richard (1996) concluded that the fish communities were deleteriously impacted below two mills not having secondary effluent treatment. In 1998, significant improvements in the fish communities were evident after the installation of secondary

treatment at two mills and process modifications/ improved effluent treatment at a third mill. The improvements reflected enhancements in effluent quality with respect to BOD, suspended solids and toxicity.

Acknowledgements We thank Maria Ricci, Patrice Delisle, David McMullen, Serge Nielly, Jim Larocque, Anne Corriveau, Dany Boudrias and Brian Berra for their invaluable technical support. The assistance of Bob Poole with the statistical analyses is appreciated. Dr. James Karr, University of Washington, kindly reviewed the manuscript and provided many useful comments for improvements. This work was supported by the maintaining members of the Pulp and Paper Research Institute of Canada. Partial financial support for the work was provided by the Technology Partnerships Canada (TPC) program of Industry Canada.

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