19 l-203 (1989)
Biotransformation and Other Physiological Responses in Whitefish Caged in a Lake Receiving Pulp and Paper Mill Effluents PIRJO LINDSTR~~M-SEPP.X*
*University of Kuopio, Department ofPhysiology, POB 6. SF-7021 I Kuopio, Finland, and t university of Joensuu, Department of Biology, POB I1 1, SF-80101 Joensuu, Finland Received December 4, I988 Hepatic monooxygenase (MO) and conjugation enzyme activities, metabolites of chlorinated phenolics in the bile, and blood ionoregulatory parameters were studied in juvenile whitefish (Coregonus m&sun Pallas and C. muksun X Coregonus peled Gmelin hybrid) held in cages downstream from a mill producing chlorine-bleached kraft pulp and printing paper. MO activities, measured as benzo[a]pyrene hydroxylase, ‘l-ethoxycoumarin Odeethylase, and 7-ethoxym sorufin Odeethylase, were significantly induced in whitefish caged about 5 km from the effluent outlet. The highest mean increase detected was 17 times the control value. In the nearest caging station (3 km) the induction was lower, indicating inhibition or toxicity caused by the effluent. The levels of bile metabolites of chlorinated phenolics showed highest concentrations at the nearest station and decreased levels at more distant locations over the whole water area studied ( 15 km). Bile metabolites in whitefish exposed in control areas confirmed low-level background pollution of the lake system due to chlorinated phenolics. Observations on blood ionic concentrations suggest that whitefish were able to regulate their hydromineral balance despite the environmental pollution affecting physiology of biotransformation. 0 1989 Academic PCS, IIIC.
INTRODUCTION It is a commonly known fact that fish populations in downstream water areas extensively polluted by effluents discharged from pulp and paper industry are different from those in upstream waters. This dissimilarity also exists farther downstream where the effluents have been diluted and many of their organic components have been degraded or transformed. In addition to direct toxic effects caused by variably persistent chemicals, indirect consequences like decreased water oxygen concentration, color, and suspended material are reasons for alterations of fish population structure. Most typically, in Scandinavian inland waters, salmonid species have disappeared from waters polluted by pulp and paper industry effluents and have been replaced by such fishes as pike, perch, and roach, species which have less value. Lately much attention has been directed toward development of biological markers suitable for early and sensitive detection of potentially harmful risks caused by environmental chemicals. One direction has been the physiological/biochemical responses, including variables describing biotransformation and ionic/osmotic regulation of fishes (Oikari et al., 1985; Evans, 1987; Payne et al., 1987; LindstriimSeppl, 1988). In autumn 1986 an extensive caging study was carried out in the southern part of Lake Saimaa (SE Finland), the receiving water body of a kraft pulp mill with conventional chlorine-based bleachery integrated with a paper mill producing high-quality printing paper. This process represents the most typical case of toxic pollution due to pulp and paper industry. The effects of the bleached kraft pulp (BICME) and paper 191
0147-6513/89 $3.00 Copyright Q 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.
mill effluent were studied using two fish-e.g., whitefish (Coregonus m&sun Pallas) reported here-and one mussel species caged at 10 field stations, representing different effluent concentrations, including two control areas. The aim of the present study was to investigate the effects of BKME and paper mill effluent using xenobiotic biotransformation enzyme activities, bile metabolites of selected BKME toxicants, and ionoregulatory parameters of C. muksun Pallas. Previously this species was living in many of the inland water areas polluted today by waste waters from pulp and paper industry. By using whitefish, an authentic species suffering from these effluents, as an experimental model, responses in physiological/ biochemical markers can be directly associated with other biological responses leading to altered population structure. Furthermore, because whitefish is a plankton feeder it is not necessary to feed the fish by fodder even during extended field exposure. This also makes the exposure pattern very realistic, because whitefish are ingesting pollutants through both water and food. EXPERIMENTAL
Fish and Their Caging Juvenile 1+-year-old, hatchery-reared whitefish (C. muksun, Kontiolahti Fish Farm) were transported to five field stations, 3-l 5 km downstream from a pulp and paper mill in the southern part of Lake Saimaa, Finland. Before transportation from the hatchery (water temperature 16-17”C), whitefish had not been fed for 3 days. PVC bags were partially filled with oxygen and surrounded with ice to prevent the water from warming during the 6-hr transportation time. When transferred to cages, all the fish were visually examined and found to be in good condition and no late mortality developed during the first days of the experiment. The fish were exposed for 3 weeks to lake water contaminated with BKME. The controls were treated in a similar manner at two upstream reference locations. The fish were enclosed in 50liter cages, lo- 15 fish in each, and placed at 3-m depth. When sampled, each animal (plankton feeders) had food remains in the intestine indicating normal feeding behavior. The weight of the fish varied from 16 to 103 g (standard length, 12.7-23.0 cm). It is well-known that different species of the genus Coregonus have not been described taxonomically in a clear-cut manner. Different species are often difficult to determine on the basis of their general morphology. Therefore, the genetic identity of the fish used was determined by electrophoretic separation of proteins followed by demonstration of specific enzymes. This identification was carried out by Dr. Jukka Vuorinen (Vuorinen, 1984). The majority of the whitefish sampled were, as expected, C. muksun (n = 3 1) but there were also hybrids of C. muksun X C. pled (Gmelin) (n = 25). Both types were divided into all caging stations (see Table 3). The results were also calculated after separating the two species into their own categories. The mill produced chlorine-bleached kraft pulp (approximately 3 1,000 tons/ month, September 1986) and high-quality printing paper (approximately 2 1,400 tons/month). Both pine ( f ) and birch ($) were used as raw materials. During the 3week exposure period there were two short breaks in the normal operation of the mill. The volumes of the biologically treated effluents averaged 220,000 m3/day. The detailed description of the mill, the study area, and the experimental approach applied has been given in previous papers (Oikari et al., 1985; Oikari and KunnamoOjala, 1987; Lindstriim-Seppl and Oikari, 1989).
Collecting of the Fish Samples Fish were taken one by one from the cages and stunned by a blow to the head, and the total weight (to the nearest 10 mg) was recorded. Blood was taken from the tail vessels; plasma and red blood cells were immediately separated by centrifugation. Bile was aspirated from the gall bladder, and the liver was removed and weighed (accuracy of 1 mg). A 50-mg piece was taken for species identification by electrophoresis and the rest was used for enzyme determinations. Condition factor (CF) was calculated from the total weight of the fish and the standard length by the formula CF = [ W(g)/(L(cm)3] X 100 (Table 1). Liver somatic index (LSI, the percentage weight of detached liver of the total fish weight) was also calculated. The entire procedure for each fish required 8-10 min. All the samples were stored in liquid nitrogen for further processing. Xenobiotic
The frozen tissue samples were thawed at +4”C, put in ice-cold 0.25 M sucrose, and homogenized in a Potter-Elvehjem-type glass-Teflon homogenizer. The postmitochondrial supernatant solution ( 10,OOOg for 20 min) was spun further at 105,OOOg for 60 min in a Kontron TGA-65 ultracentrifuge to obtain the microsomal fraction. Microsomes were resuspended in 0.25 Msucrose containing 20% glycerol, 1 ml corresponding to 1 g liver wet wt, and stored at -80°C for further analysis. The control and experimental samples were analyzed after a similar storage time (in the same day or after 2 weeks depending on the parameter). The content of cytochrome P450 was determined from freshly prepared microsomes according to the method of Johannesen and DePierre (1978). The difference between the spectrum of dithionite reduced and that of nonreduced microsomes was recorded with a Carry 118 spectrophotometer. The method was selected because of its minimal disturbing effect on hemoglobin and methemoglobin. The monooxygenase activities were measured with three different substrates. The deethylation of 7-ethoxyresort&n (EROD) was measured immediately after preparation of microsomes by Pet-kin-Elmer MPF-43A fluorescence spectrophotometer in a kinetic reaction with resorufin as reference (Burke and Mayer, 1974). Also 7-ethoxycoumarin O-deethylation (ECOD) (Aitio, 1978) and arylhydrocarbon hydroxylase activity (AHH) with benzolalpyrene as substrate (Nebert and Gelboin, 1968) were determined ffuorometrically. The NADPH-cytochrome c reductase activity was measured by the method of Dallner et al. ( 1966). UDPglucuronosyltransferase activity (UDP-GT) was measured spectrophotometrically with pnitrophenol as aglycone (Hanninen, 1968) and glutathione S-transferase with 1-chloro-2,4-dinitrobenzene as substrate (Habig et al., 1974). All the determinations were carried out at 18°C. The amount of protein was measured using the method of Lowry et al. (195 1). Other Analyses Concentrations of free and conjugated chlorinated phenolics in the bile (sample size 50-200 ~1) of the whitefish were measured by capillary gas chromatography (Hewlet-Packard 5800; Oikari and Kunnamo-Ojala, 1987). Free chlorophenolics (CPs) were extracted first from diluted and acidified (pH 3-3.5) bile, after addition of
an internal standard (2,4-dibromophenol), with three 2-ml lots of n-hexane:acetone (3: 1). Organic phases were combined and evaporated to a small volume, and CPs were acetylated according to the method described in Voss et al. (198 1). The aqueous fraction was treated with concentrated HCl(1: 1) for 2.5 hr at 7o”C, neutralized with NaOH, and processed further as described for the free CPs. The concentrations of plasma sodium and magnesium were determined by atomic absorption spectrometry and chloride concentration by electrometric titration (Radiometer CTMlO). The sodium concentration of erythrocytes was measured as in Nikinmaa et al. ( 1987) without extracellular space correction.
Statistical Data Processing Because the studied material consisted of groups that varied in size, the assumption of equal variances was tested with Cochran’s C test. The degree of heterogeneity in many variables was significant (P < 0.05). The data were thereafter tested with a nonparametric Kruskall-Wallis one-way analysis of variances using the data programs SPSS-X release 2.2+ and 3.0 for VAX/VMS in VAX-l l/780 VMS V4.3 of the Computing Centre of the University of Kuopio. The data were also tested with analysis of variance (SPSS-X, subprogram ONE-WAY). Differences between groups were screened by Duncan’s multiple range test (a = 0.05). RESULTS
Water PhysicochemicalFeatures during Exposure Water temperature during the 3-week study period varied between 13.8 and 9.8”C (from the water surface to the caging depth) at different caging sites. Oxygen concentrations determined at the control (11.4 and 10.7 mg/liter) and experimental stations (6.0 mg/liter in El) just before fish sampling showed a considerable oxygen demand of the discharges. Determination of total chlorinated phenolics as well as resin acids from the lake water indicated highest toxicant concentrations at station El, 3 km from the effluent pipe, and decreasing values at more remote areas so that low levels were detectable even at control sites. A gradient was also seen in other water parameters such as pH, conductivity, color, and sodium content, indicating the presence as well as dilution and transformations of the effluent throughout the study area. A more detailed description of the study area and water quality has been reported by Lindstriim-SeppZ and Oikari (1989).
General Condition of White$sh It was not possible to demonstrate any statistical differences between the two control stations in any of the variables measured. Control groups were therefore handled as one entity and in this manner compared to experimental groups. Condition factor of the whitefish did not vary between groups; neither did liver somatic index in most cases (Table 1). This is in accordance with the observation in rainbow trout, which were exposed in a similar manner (Lindstriim-Seppg and Oikari, 1989). In general, LSI and CF did not seem to be sensitive indicators of subacute exposure of fish to diluted BKME. However, at the caging station 5.6 km from the sewer, liver size was increased by 19% (P < 0.05) on an average. Liver size in rainbow trout in a corresponding study was slightly increased (22%) 3 km from the effluent
IN WHITEFISH TABLE
UNDER PULP MILL
CONDITIONFACTORANDLIVERSOMATIC INDEX(%)OF~HITERSH EXPOSED TOPULPANDPAPERMILLEFJFTSJENTSINCAGESFOR 3 WEEKS ATFIVEFIELDSTATIONSBELOWAMILLSEWER IN THESOUTHERNPARTOFLAKESAIMAA
Station (km) Cl El E2 E3 E4 E5
+ C2 (-3-12)b (3.1) (5.6) (6.8) (8.9) (15.0)
CF” 0.894 0.984 0.870 0.918 0.891 0.949
f + + + + f
0.026’ 0.020 0.03 1 0.033 0.041 0.018
0.524 0.568 0.622 0.563 0.544 0.586
+ iI f + f +
14 6 5 12 7 12
0.023 0.025 0.026* 0.019 0.03 1 0.032
Note. The control fish were caged upstream from the effluent outlet (see Fig. 1). o CF, condition factor [ W(g)/L (cm)3] X 100; LSI, liver somatic index (percentage weight of liver of total fish weight). b Distance from the mill sewer (cf. Fig. 1). c Mean f SEM; N, number of fish; statistics (compared to controls Cl + C2): *P < 0.05 (Duncan).
outlet, but was not statistically significant (Lindstriim-Seppl and Oikari, 1989). Increased LSI has also occurred in perch collected from waters affected by pulp and paper mill effluents (Andersson et al., 1988). Exposure of juvenile rainbow trout to a sulfate soap preparation (mainly resin and fatty acids) has been demonstrated to lead to significantly increased LSI (Oikari and Nakari, 1982). This was due to the increased liver water concentration.
Biotransformation EnzymeActivities The amount of cytochrome P450 was similar at all sampling locations (Table 2). At station E3, however, there was a tendency of elevated cytochrome P450 concentration (36%), but the variation was high and the increase was not significant. Also in rainbow trout caged under corresponding conditions there were slight, but statistically insignificant, increases in cytochrome P450 content (up to 27%) (LindstriimSeppl and Oikari, 1989). There were no differences in NADPH-cytochrome c reductase activity among the studied whitefish groups (Table 2). In rainbow trout NADPH-cytochrome c reductase activity was unresponsive except in fish collected at the station 15 km from the sewer, which showed decreased values (- 17%). On the other hand, cytochrome P450-mediated polysubstrate monooxygenase (PSMO) enzyme activities in whitefish were affected by the exposure and, when compared to control values, there were significant inductions (Fig. I) of AHH and EROD activities. A 3.5-fold induction of AHH activity was observed at the closest exposure station, 3 km from the effluent pipe. However, farther away (6.8 km, E3), the induction was up to 16.9 times the control values. Induction indicated by EROD was also highest at the E3 station (11.5-fold), i.e., more intense than at El (6.8-fold), which is closer to the mill. The lower induction detected at the caging station E 1 compared to that at station E3 may be due to an inhibitory and/or direct hepatotoxic effect of the effluent appearing in the vicinity of the mill. Water analyses in the study area in September 1986
TABLE 2 BIOTRANSFORMATION ENZYME ACTIVITIES IN THE LIVER OF WHITEFISH EXPOSED TO PULP AND PAPER MILL EFFLUENTS FOR 3 WEEKIS AT LAKE SAIMAA
Station (km) Cl + C2’(-3-12) El (3.1) E2 (5.6) E3 (6.8) E4 (8.9) E5 (15.0)
CYT P450” 117.3r 11.1’ 118.9f 10.2 128.9+ 11.4 158.6+ 16.7 131.2t- 16.8 128.9+ 22.0
Kruskall-Wallis analysisof variances Pd 0.501
CYT C-RED 11.5f 12.6f 14.2f 12.0It 13.1f 13.3f
1.1 1.4 0.7 0.7 1.2 0.8
UDP-GT 56.6 f 47.7 f 46.5 f 64.6 f 31.9* 44.1 +
6.7 19.4 3.8 14.8 2.0 17.1
GST 19.2f 5.4 16.2f 4.0 23.7 f 3.0 33.2 + 4.5* 12.3f 3.4 22.1 + 4.9 0.094
Note. For details see Table 1. ’ CYT P450, cytochrome P450 content (pmol/mg protein), CYT C-RED, cytochrome c reductase (pmol/min X mg protein), UDP-GT, UDPglucuronosyltransferase (pmol/min X mg protein), GST, glutathione S-transferase (nmol/min X mg protein). b Cl + C2 = controls. ’ Mean values HEM; statistics (compared to controls Cl + C2, n = 6 pooled from 14 fish, eachother n = 3-6 pooled from 6-12 fish): *P < 0.05. d Significance, corrected for ties.
showed that the concentrations of BKME toxicants were higher at station El than at E3; e.g., total resin acids were 14.0 and 3.6 &liter (Lindstrom-Seppi and Oikari, 1989). The corresponding values for chloroquaiacols were 0.35 and 0.22 &liter, respectively. Ahokas et al. (1976) detected decreased AHH and aminopyrine N-demethylase activities in pike (Esox lucius L.) caught from a lake heavily polluted with effluents from a pulp mill using sulfate and sulfite processes and bleaching. These were thought to be due to the hepatotoxic effects of the effluent. In a subchronic laboratory experiment with lake trout (Sulmo trutta lacustris), where the fish were kept in simulated BKME consisting of sulfate soap and chlorophenols, the induction of liver PSMO activities was partially abolished at highest exposure concentrations (Oikari et al., 1988). On the other hand, resin acids have been demonstrated to decrease PSMO activities in fish hepatocyte culture (M. Pesonen, personal communication). In rainbow trout, in contrast to whitefish, field studies conducted on the same water area as the present studies revealed no inhibition: the activities of PSMO were highest at the nearest caging station and gradually decreased at more distant locations (Lindstriim-Seppl and Oikari, 1989). Other stations, farther away, were found to have only slightly increased or statistically unaltered PSMO enzyme activities (Fig. 1). The average increase in EROD activity at station E2 was 3.5fold, but this was statistically insignificant (P > 0.05). Similarly, caging station E5, 15 km from the effluent pipe, indicated some induction of AHH and EROD activities but the results indicated a high variation. In rainbow trout caged at the same locations as whitefish, the induction was seen at all sampling sites with all PSMO activities assayed [AHH, ECOD, EROD, and PROD (pentoxyresorufin Odealkylase); Lindstriim-Seppa and Oikari, 19891. In rainbow trout, however, the degree of maximal induction (7-fold in EROD) did not rise to values as high as those in whitefish. This could be due partially to different exposure patterns;
FIG. 1. Influence of bleached krah pulp and paper mill effluents on hepatic monooxygenase activities ECOD, 74hoxycoumarin O-deethylase; m EROD, 7-ethoxyr(0 AHH, benzo[a]pyrene hydroxylase; esorufin O-deethylase) in juvenile whitefish caged for 3 weeks at five field stations below the sewer (Lake Saimaa, September 1986). The control fish (Cl + C2 = 1) were caged in the waters upstream from the effluent outlet. (Mean f SEM; (*) P < 0.05 relative to the controls; Duncan).
whitefish did receive pollutants both from water and from food. Therefore the body load of inducing xenobiotics from BKME in whitefish was probably higher than that in rainbow trout fed only occasionally with pellet fodder. It has been demonstrated that pulp and paper mill effluents cause a polycyclic aromatic hydrocarbon (PAH) type of induction in fish (Fiirlin et al., 1985). EROD and AHH are known to be specific substrates for the appropriate Ah locus-coded cytochrome P450 isoenzyme (P450LM4,, in rainbow trout, Williams and Buhler, 1983; P450, in cod, Goksoyr, 1987; P45Or in scup, Klotz et al., 1983), which is inducible with PAH-type inducers like 3-methylcholanthrene (Nebert and Atlas, 1978). Increased deethylation of 7-ethoxucoumarin is not as specific an indicator of PAHtype induction as EROD and AHH. Accordingly, as seen also in this study, ECOD appeared to be induced less than the two other monooxygenase enzyme activities analyzed (Fig. 1). Simple linear correlation coefficients were calculated between the three hepatic PSMO activities and the amount of cytochrome P450. A rather strong positive correlation was seen between AHH and EROD, AHH and ECOD, and ECOD and EROD, the respective r values being 0.88,0.65, and 0.62 (P < 0.001). These correlation coefficients are equal to or somewhat smaller than those observed in a laboratory study on simulated BKME with lake trout (Oikari et al., 1988). Therefore, for practical purposes of environmental management, determination of one of these-preferably
AND OIKARI 3
MON~~XYGENASE ENZYME ACTIVITIES IN Two WHITEFISH POPULATIONS (C. m&sun AND HYBRID OF C. m&sun X C. peled) AT DIFFERENT CAGING STATIONS IN LAKE SAIMAA ACT.
2.17” 0.37 3.70 7
3.80 N.D.d 11.90 1
1.65 0.25 10.90 4
16.93* N.D. 34.47 * 9
0.60 N.D. 3.90 3
0.80 0.15 2.90 7
0.164 0.757 0.088
0.27 0.47 2.90 7
3.25* 1.40 24.05 * 5
2.90* 0.80 10.00 1
35.40* 3.40* 42.60* 3
0.90 0.25 8.45 4
2.90* 0.90 14.0 5
0.121 0.157 0.159
C. m&sun AHHb ECOD EROD Ne
C. muksunX C. peled AHH ECOD EROD N
Note. For location of caging areas see Fig. 1. ’ Kruskall-Wallis analysis of variances, significance corrected for ties. b AHH, benzo[a]pyrene hydroxylase; ECOD, 7-ethoxycoumarin O-deethylase; EROD, 7-ethoxyresorufin Odeethylase; pmol/min X mg protein. ’ Mean; *statistics, ranges, Duncan (0.05) compared to controls (Cl + C2). d N.D., not detectable. ’ Number of fish.
EROD-is generally sufficient. On the other hand, there were no significant correlations between the PSMO activities and the amount of cytochrome P450. The effect of different whitefish species, C. muksun and C. muksun X C. peled hybrid, on the results obtained is noteworthy, although the overall patterns of responses were much alike (Table 3). First, C. muksun had higher control AHH activity and, therefore, a decree of induction lower than that of more responsive hybrids. C. muksun from station E3 (site of maximal increase in PSMO) had lower AHH and EROD activities than the hybrids in the same cage. The hybrids also had high ECOD activities. AHH activity in hybrids was elevated again at stations El, E2, and E5 as was EROD at station E 1. UDPglucuronosyltransferase activity in whitefish liver (Table 2) did not change, in contrast to the tendency to higher activities in rainbow trout studied at the same time and in the same area (Lindstriim-Seppa and Oikari, 1989). In whitefish, especially at station E4, a slight tendency of inhibition (-44%, not significant) of UDP-GT was observed. Earlier reports on the effects of pulp and paper mill effluents or effluent constituents have given quite contradictory results of hepatic UDP-GT activities. According to Oikari and Kunnamo-Ojala ( 1987) UDP-GT activity in trout, in the same study area as that of the present study, was increased by 31-57% in areas 4-6 km from the mill sewer. Perch (Percafluviatilis) captured in the vicinity of a Baltic pulp bleach plant had induced UDP-GT activities (Andersson et al., 1988). Fourhom sculpin (Myxocephalus quadricornis), much like whitefish in the present study, was not affected during a long-term exposure (Andersson et al., 1987). On the other hand, inhibition of liver UDP-GT activity in rainbow trout caged for 5 days, like that in whitefish at station E4, has been reported (Oikari et al., 1985). Furthermore, laboratory studies demonstrated that resin acids, as well as chlorophenols at high sublethal concentrations, significantly decrease liver UDP-GT activity in rainbow trout (Oikari
UNDER PULP MILL
TABLE 4 THECONCENTRATIONSOFFREECHLORINATEDPHENOLICS (&ml) INTHEBILEOFWHITEFISHCAGEDUPSTREAMANDDOWNSTREAM FROM THE PULP AND PAPER MILL SEWER
Cl +C2(n = 7p El(n=2) E2(n=2) E3(n=3) E4(n = 2) E5 (n = 4)
0.7 -+0.2’ 1.OkO.l 6.3 + 1.2 1.7a0.5 1.5kO.O 0.9 f 0.3
0.4fO.l 2.5 + 0.6 2.1 + 0.7 4.0 + 3.1 5.0 -+0.7 l.OkO.4
0.3 + 0.1 9.5 + 8.8 1.220.1 0.6 + 0.2 2.2 + 1.9 1.0+ 0.3
Note. For details see Table 1. ” CGs, chloroguaiacols (3,4,5trichloroguaiacol, 3,5,6-trichloroguaiacol, tetrachloroguaiacol); CPs, chlorophenols (2,4,6-trichlorophenol, tetrachlorophenol, pentachlorophenol); CS, trichlorosyringol. b n = number of pools from l-5 biles. ’ Mean f SEM.
and Nakari, 1982; Oikari et al., 1983; Castren and Oikari, 1987; Mattsoff and Oikari, 1987). Inhibition of hepatic UDP-GT activity by sulfate soap preparation and resin acids has also been found. In all, response of liver UDP-GT seems to depend on the length of exposure, the concentration of toxicants, and the fish species studied. Glutathione S-transferase (GST) activity of whitefish was induced at station E3, in contrast to the parallel study on rainbow trout (Lindstriim-Seppa and Oikari, 1989), where no changes were observed. Cytoplasmic GSTs catalyze a multitude of conjugation reactions of reactive electrophilic compounds, e.g., those formed in PSMO reactions, utilizing glutathione as a cosubstrate (Mannervik, 1987). In whitefish, PSMO induction was much higher than that in rainbow trout and this may have produced more substrates to GST, which could be counteracted by GST induction, On the other hand, in lake trout exposed to simulated pulp mill effluent in the laboratory, a decrease in liver GST activity was detected (Oikari et al., 1988). The induction of PSMO in lake trout was much lower than the induction in rainbow trout and, furthermore, an increase in kidney GST was detected.
Bile Conjugates of Chlorophenolics In whitefish about 94% of total chlorophenolics in the bile were conjugated, possibly, as in other salmonids, primarily with glucuronic acid (Oikari and Ank, 1985). Compared to those in rainbow trout (Lindstrbm-Seppa and Oikari, 1989), there were higher concentrations of free chlorinated phenolics in the bile of whitefish (Table 4). However, the levels of free chlorophenolics in the bile were lower than those in lake trout (Oikari ef al., 1988). Most distinctly, the concentrations of hydrophilic metabolites of chlorinated phenolics (chloroquaiacols, chlorophenols, and trichlorosyringol), measured from the bile of whitefish, indicate a clear distance-related response (Fig. 2). The levels were highest at station E 1 (3 km) and decreased at more distant locations. Since low levels could also be detected at the control sites, it seems that the whole southern part of
FIG. 2. The concentrations (&ml) of conjugated chlorinated phenolics (0 CGs, chloroquaiacols: 3,4,5trichloroquaiacol, 4,5,6-trichloroquaiacol, tetrachloroquaiacol; CPs, chlorophenols: 2,4,6-trichlorophenol, tetrachlorophenol, pentachlorophenol; t!~ CS, trichlorosyringol) in the bile of whitefish caged up stream and downstream from the pulp and paper mill. (Mean + SEM; (*) P < 0.05 relative to the controls; Duncan).
Lake Saimaa is contaminated by BKME. At station E3 (6.8 km), a location having the highest induction of PSMO system, the levels of conjugated chloroguaiacols were lower than those in three other experimental areas. Therefore, if the level of chloroguaiacol conjugates in the bile is considered an index of environmental exposure to BKME, it is concluded that induction of hepatic PSMO system was partially abolished not only at El (3.1 km), but also at E2, in an area 2.5 km farther away from the source of BKME. In all, the results indicate that bile metabolites of conjugated pollutants serve, as stated earlier (Oikari and Holmbom, 1986), as highly sensitive indicators of aquatic exposure of fishes to pulp and paper industry effluents.
Blood Electrolytic Balance In previous and accompanying studies with rainbow trout, plasma Na+ concentration has been noted to be increased throughout the study area (Oikari et al., 1985, Lindstriim-Seppa and Oikari, 1989). Similarly, whitefish also displayed a tendency toward elevated plasma Na level, most distinctly at station E2, but the increase was not statistically significant (Table 5). This response has been thought to be due to increased uptake rate of Na in an environment where sodium concentration is about three times higher, because of BKME, than that in the background situation of Lake Saimaa. On the other hand, plasma chloride and magnesium concentrations in whitefish were largely unchanged, in contrast to rainbow trout (Lindstriim-Seppl and Oi-
TABLE 5 IONIC CONCENTRATIONS IN F&D BLOOD CELLS AND PLASMA FROM WHITEFISH CAGED FOR 3 WEEILS AT DIFFERENT DISTANCES (E 1-E5) DOWNSTREAM FROM THE SEWER OF A PULP AND PAPER MILL AND IN Two CONTROL AREAS (C 1+ C2) UPSTREAM
Station (km) Cl + C2 (-3-12) El (3.1)
E2 (5.6) E3 (6.8) E4 (8.9) ES(15.0)
RBC-Na’ 25.2 f 2.46 28.6 + 1.9 25.5 _+0.5 28.1 -t 4.8 18.8+- 1.8 23.6 f 2.1
105 + 4
0.95 -t 0.10
118? 5 126+ 2 118k 5 109f 17 115+- 7
1.06 f 0.04 1.04 f 0.02
1.06+ 0.03 0.97 + 0.08 0.98 + 0.05
124 rt 10 139+ 6
136+ 125+ 138k 135-t
3 11 1 1
6 3 10 6 11
analysis of variance 0.120
u RBC-Na, red blood cell sodium (mmol/liter); PLNa, plasma sodium (mmol/liter); PL-Mg, plasma magnesium (mmol/liter); PL-Cl, plasma chloride (mmol/liter). ’ Mean ? SEM; N = number of fish, statistics compared to controls C 1 + C2, *P < 0.05 (Duncan). ’ Significance, corrected for ties.
kari, 1989), indicating good regulation of ionic and osmotic balance in whitefish exposed to BKME (Table 5). Compared to those in control animals, concentrations of sodium in red blood cells (RBC) of whitefish remained unchanged (Table 5). This is also in contrast to the observations on rainbow trout, showing increased sodium level in RBC at caging sites closest to the pulpmill sewer. CONCLUSIONS The plankton-feeding whitefish (C. muksun) proved to be a suitable species for field caging experiments. The main advantage is its ability to feed even in cages. Therefore, xenobiotic chemicals in the true solution are absorbed not only from the ambient water by the fish, but also from particles filtered and eaten. This trait also makes possible long-term experiments in the field. The physiological and toxicological responses of whitefish were pronounced. Induction of monooxygenase enzyme activities indicated clear physiological effects of BKME, although fish were able to maintain their hydromineral balance. At the same time toxic and inhibitory effects were observed. Bile analysis of conjugated chlorinated phenolics indicated clear distancerelated responses. ACKNOWLEDGMENTS This study was supported by grants from The Academy of Finland/Research Council of the Environmental Sciences (Project 06/085) and the Kymenlaakso Fund of the Finnish Cultural Foundation. We thank Dr. Jukka Vuorinen for the species identification of whitefish. We are indebted to M. SC. Jussi Kukkonen, Ms. Riitta Pietarinen, Ms. Eeva-Liisa Palkisp&, and Ms. Sirkku Hartikainen for skillful technical assistance. We also thank Professor Dsmo Hanninen, M.D., Ph.D., for comments made on the manuscript. The whitefish studied were donated by Kontiolahti Fish Farm (Mr. Ossi Puhakka, director).
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