Acclimatization of roach, Rutilus rutilus (L.), to toxic components of kraft pulp mill effluents

ECOTOXICOLOCY

AND

ENVIRONMENTAL

SAFETY

15,282-288

(1988)

Acclimatization of Roach, Rutilus rutilus (L.), to Toxic Components of Kraft Pulp Mill Effluents A. OIKARI AND J. KUKKONEN Department ofBioIogy, University ofJoensuu, PL 111, SF-80101 Joensuu, Finland Received April 14, I98 7 Roach (Rutilus rutihs) were exposed, along with their appropriate controls, to a simulated bleached kraft mill effluent (WE-Sa+CP) first for 38 days at a concentration of 0.035 X LC& and then for 14 days at 0.07 X LCro. During the experiment, their tolerances to KME-Sa+CP were tested five times by measuring the 48-hr LCsO values. In addition, the growth of roach was monitored. At the end ofthe exposure, accumulation of [‘4C]pentachlorophenol in various parts of the fish (total PCP in water 15.6 &liter) was measured. When the fish were preexposed to KME-Sa+CP, the acute tolerance of this mixture in roach increased by 30-39%, but the response was abolished in 3 1 days. Fish growth remained unchanged during the experiment. Measurement of PCP accumulation revealed no difference in the absorption rate, but under steadystate conditions the degree of bioconcentration was 16% lower (P < 0.02) in preexposed roach than in their unexposed controls. This difference was entirely accounted for in the head and visceral parts of the fish. Even when no final changes were noted in tolerance and growth rate of the fish, the authors suggest that the significantly lowered body burden implied acclimatizatory compensation under subchronic exposure ofthis xenobiotic. 0 1988 Academic PKSS, hc.

INTRODUCTION A usual result of nonlethal exposure in a challenging environment is an increase in tolerance or resistance of the given factor. Reorganization of the metabolism during acclimatization will eventually increase the fitness of an individual, i.e., its probable genetic contribution to succeeding generations (Prosser, 1973; Keeton, 1980). The physiological acclimatization to environmental chemicals, or lack of it, may thus be used as a rational endpoint in assessing the potential harm the chemicals possibly cause to animal populations. Therefore, knowledge of the limits of compensatory acclimatization must be considered as an important determinant when interpreting the ecotoxicological significance of the low-level chemical contamination of the environment. The tolerance acclimatization of fish exposed to organic toxicants has received little attention. Preexposure of fish to parathion did not change the acute parathion toxic ity (see Bradley et al., 1985). On the other hand, acclimatory processes were suggested in liver protein metabolism of rainbow trout exposed to low concentrations of bleached kraft mill effluent (BKME) (Oikari and Niittyki, 1985). Similarly, several of the observed effects of chronic exposure of coho salmon to BKME (McIeay and Brown, 1979) can be considered adaptive in their nature. Biotransformation of xenobiotic chemicals may occur in fish at inherent rates which are sufficient to affect their toxicity and residue dynamics (Lech, 1974; Southworth et al., 198 1; Melanchon et al., 1985). Furthermore, induction of biotransformation enzymes in fish tissues 0147-6513/88 $3.00 Copyright 8 1988 by Academic Press, Inc. AU rigbt.7 of reproduction in any form reSeNed.

282

ACCLIMATIZATION

OF

ROACH

TO

KRAFIY

MILL

EFFLUENT

283

can lead to an enhanced disposition rate of xenobiotics from the body (Lech and Bend, 1980). In the present study roach were exposed to the low levels of toxicants normally occurring in inland water bodies receiving BKME. The possible acclimatization to the new chemical environment was assessed by measuring the growth of fish, changes in the tolerance of the same toxicant, and bioaccumulation of one essential substance present in water under the preexposure period. In this study we used roach because of their occurrence even in waters heavily polluted by BKME; i.e., their capacity to acclimatize in this respect might be better than that generally found in fish. MATERIALS

AND

METHODS

Roach came from a natural fish pond (&la, near Joensuu in eastern Finland) where they were reared over one summer season. The weight of fish varied from 0.75 to 0.95 g (length from 4.8 to 5.4 cm), and all were of the age group O+. Roach were transported to a laboratory aquarium supplied with unchlorinated tap water, originally ground water, of the town of Joensuu. Fish were allowed to acclimatize to this water (pH 7.1, CaCO,-hardness 50 mg/liter, Na 2.2, Ca 14.3, Mg 3.0, Fe 0.01, and Mn 0.0 1 mg/liter) for about 7 weeks before the experiments were started in late October. Water temperature was kept between 12.5 and 13.5”C, and a L:D rhythm of 14: 10 h (L 06-20) was maintained in the aquarium room. Water oxygen concentration stayed >8 mg/liter. Roach were fed daily with salmon fodder (Evos-Vaasa, Suomen Rehu Inc., Finland) to the ration of ca. 0.5% of body weight per day. About 1200 roach were randomly divided into two groups of the same size, weighed in water, and placed into two identical 400-liter all-steel aquaria. A water flow of 1.5 liters/min (ca. 4.5 liters/g fish/day) was provided to both aquaria; all other conditions were similar to those maintained before. One group was exposed for 52 days to a simulated bleached kraft mill effluent (KME-Sa+CP; cf. Oikari et al., 1984) containing the two major classes of acutely toxic substances, resin acids and chlorophenolics, normally present in this type of effluent. During the first 38 days the KMESa+CP concentration corresponded, as determined with 0+-year-old brown trout (Salvo trutta m. lacustris), to 0.035 X LC~O (96 hr) but was increased to the level of 0.07 X LCso for the last 14 days (see Fig. 2). During the experiment fish were fed, using the same lot of salmon fodder as before, with the daily ration of 1.5% of body weight, divided into two equal parts. The second group of fish was treated identically but no KME-Sa+CP was added to the water. The mortality of roach was negligible in both groups (14 fish in the group exposed to KME-Sa+CP and 4 in the controls). One liter of KME-Sa+CP stock contained 1.O g (dry wt) sulfate soap (A. Ahlstrom, Inc., Finland; 25% dry wt total resin acids and 40% dry wt total fatty acids; for the composition, see Oikari et al., 1984) 100 mg 2,4,6-trichlorophenol (CP-3), 143 mg 2,3,4,6-tetrachlorophenol (CP-4), and 57 mg pentachlorophenol (PCP; all of grade “purissimum”). The mixture was further diluted with distilled water (1:2000) and pumped to the test aquarium, yielding the concentration as desired. At the start of experiment, therefore, the following concentrations (&liter) prevailed. total resin acids, 4.4; total fatty acids, 7.0; CP-3, 1.75; CP-4,2.5; and PCP, 1.O. Tolerance of roach to KME-Sa+CP was measured as LCsO (48 hr) values, which were determined according to the guidelines recommended by the OECD (198 1). The dilution water used was the same as that supplied to the fish throughout the

284

OIKARI

AND KUKKONEN

RG. 1. The six parts of roach (RutiLus rutilus,ca. 1 g) used in counting [U-‘4C]pentachlorophenol activity. I, head, including most of the gills and the heart; II, visceral organs, including some of the gills but excluding the kidneys; III, anterior part of the dorsal musculature; IV, middle part of the dorsal musculature; V, ventrolateral musculatures; and VI, the tail. Fins and skin were shared as shown.

experiment. The test water pH was 7.0 and its temperature was 12.5 f 0.5”C (mean + range). For each test about 100 roach were randomly netted from the shoal and 70 of them were further divided between seven all-glass jars (fish density, 0.25 g/]&r water). For one day before the test no food was given to the fish. The I&, values with their 95% confidence intervals were calculated according to probit analysis (SAS, 1982). The growth of fish was estimated by weighing 12 randomly selected subgroups, 3 or 4 roach in each, from both study groups. No food was given for the last 24 hr before weighing. In order to measure the absorption rate and accumulation of PCP from water to roach, a bath (4.0 liters, 12.5”C) containing KME-Sa+CP at the concentration of 0.07 X L& was prepared. Radioactive PCP ([U-‘4C]pentachlorophenol, CEA/CMM438, France; sp act, 40 mCi/mmol) was added to the bath, yielding 4200 dpm/ml. The total concentration of PCP was 15.6 &liter. At the end of the experiment (Day 52) 13 roach preexposed to KME-Sa+CP and 13 unexposed control fish were transferred to the bath. A fence, made of PVC net (4 2 mm), kept the two fish groups apart. Water in the bath was gently aerated through glass pipets to ensure identical conditions on both sides of the fence. Because tracer concentrations at each moment of time were identical for all fish, the technique allowed direct comparison of the two study groups. After 1,3, and 24 hr, 4 or 5 fish were removed from the bath, rinsed in nonradioactive water for 5 set, placed on ice-cold petri dishes, and killed by spinal transsection by means of a fine pair of scissors. Each roach was divided into six parts (ca. 100-200 mg each; Fig. 1) for radioactivity measurements. The tissue pieces were solubilized in 1 ml Lumasolve (Packard, The Netherlands) at 50°C for 24 hr before counting. The samples were then bleached by 2-3 additions of hydrogen peroxide (to prevent excess foaming isopropanol also was added) and allowed to stand for a few hours before 10 ml scintillation fluid (Lumagel, Packard) was added to each vial. The well-shaken samples were left to age for 24 hr before counting by LSC (Rackbeta, Wallac-LKB, Finland) using automated external standard correction. The quenching curve was prepared by using solubilized fish blood as a colored quenching agent. The radioactivity of the bath was measured from I- to 1.5-ml samples directly mixed with the LSC fluid. Tissue radioactivities, calculated on a wet weight basis, were compared by two-tailed t tests (SAS, 1982). RESULTS

Tolerance Acclimatization and Growth ofRoach Preexposure to low concentrations of KME-Sa+CP initially increased the tolerance in roach (after 9 days by 39% and after 16 days by 30% Fig. 2) to identical

ACCLIMATIZATION

OF ROACH TO KRAFT

MILL

EFFLUENT

285

FIG. 2. Effects of roach preexposure to a simulated bleached kraft mill efluent (KME-Sa+CP) on its acute tolerance (=48 h LC,a) to KME-Sa+CP. One toxic unit refers to the 96-hr LC50 value of KMESa+CP in 0+-year-old lake trout (Safmo trutta m. lucustris) under identical test conditions. Asterisk refers to statistically significant difference between the two groups; NS = not significant.

toxicant mixtures. After 3 1 days, however, the enhanced tolerance was abolished and this condition was maintained until the end of the experiment despite doubling of the KME-Sa+CP concentration. During the experiment the average weight gain of roach was 16.2%, but no difference (P > 0.4) between the groups could be demonstrated. No consistent differences were noted in fish weights after 9, 16, 31, 38, or 52 days of exposure, or in survivors (N = 19-37) of LCsO tests. In summary, our measurements did not reveal any net assimilatory or energetic changes in roach during this subchronic exposure to toxicants.

Accumulation of [‘4C]Pentachlorophenol in PreexposedRoach This was studied at the end (52 days) of KME-Sa+CP exposure. During the 24-hr accumulation period, water radioactivity exponentially decreased from 4200 dpm/ ml (= 15.6 pg PCP/liter) to 1500 dpm/ml(=5.6 pg PCP/liter), but a steady state was attained already by 20 hr. This time scale is in accordance with previous observations (Lech et al., 1977; Saarikoski and Viluksela, 1982). No differences, either in the total body basis or in any of the six parts of roach (Fig. l), were found in the initial absorption rate of [r4C]PCP from the water to fish (Fig. 3). On the other hand, after 24 hr zoooHEAWISCERA

1 parts I+H 1

(51 Cmtrols

IMO-

(5) Pm-exposed 6

9

12

15

18

21

TIME Ihl FIG. 3. Effects of 52&y preexposure of roach to a simulated bleached kraft mill effluent (KME-Sa+CP) on the accumulation of radioactive pentachlorophenol ([U-“CJPCP) from water to fish. Total PCP concentration in water bath at the start was 15.6 &liter. For the test concentrations of IWE-Sa+CP, see Fig. 2.

286

OIKARI

AND

KUKKONEN

the radioactivity found in roach preexposed to KME-Sa+CP was 16.1% (P < 0.02) lessthan that in the control fish, and the head and visceral organs entirely accounted for this difference. The other parts, predominantly composed of musculature, bones, and skin (parts III-VI of Fig. l), revealed no significant differences in [‘4C]PCP accumulation (Fig. 3). At the end of the uptake period, the highest [‘4C]PCP activity was found in body part II (control, 2570 dpm/mg; preexposed,2 140 dpm/mg), possiblybecausethe liver and gall bladder were included. Radioactivity in the other body parts (Fig. 1) followed the order I g V = VI > III = IV. In part II, however, great variation led to a statistically insignificant difference, which might have been causedby the difficulties in dividing each fish in identica1 ways. If the head of fish (control, 1480 dpm/mg; preexposed, 1025 dpm/mg, P c 0.02) was combined with the visceral region, these parts of the preexposedroach contained 24% less(P < 0.0 1) [‘4C]PCP activity than their controls. During the initial phaseof [‘4C]PCP absorption, the radioactivity in head and visceral parts was equal, which points to the dominant role of gills in the uptake of PCP. In terms of radioactivity, bioconcentration (dpm in tissue/dpm in water) results are parallel to those of direct tissue activities. Accordingly, statistically significant differences between preexposed and unexposed roach were noted only under steadystate conditions. In roach kept in KME-Sa+CP for 52 days, both the head (control, 965; preexposed, 670) and the head+viscera (controls, 1180; preexposed, 895) displayed significantly lower (P < 0.01) bioconcentration. On the total body basis the corresponding bioconcentration factors were 520 and 440 (P < O.Ol), the order of magnitude as suchbeing similar to that of some other teleosts(Saarikoski and Viluksela, 1982; Niimi and Cho, 1983; Huckins and Petty, 1983). Similar to the direct tissue activities, no differences in the bioconcentration of PCP in those body parts with dominating musculature were found (parts III-VI of Fig. 1; control, 265; preexposed, 250). The percentage distribution of [ “C]PCP activity in the six parts of the roach body was significantly increased in the visceral organs-from 13% during the first 3 hr to 27% in 24 hr (P < O.Ol)-but the two study groups were similar in this respect. In contrast, the percentageradioactivity content in the headwas higher during the initial absorption phase (41%) than it was in a steady-statefish (31%). In other body parts (III-VI, Fig. l), no differences were seenin relative radioactivity contents during the exposure. DISCUSSION An essentialfinding of this study is the significantly decreased[‘4C]PCP accumulation in roach preexposedto a low concentration of a BKME-type mixture containing several chlorophenols (Fig. 3). The lower body burden of a xenobiotic chemical should be advantageous to a fish living in a contaminated environment. Rather unexpectedly, however, theseroach revealed unchanged acute tolerance of the samechemical mixture when they were preexposed,Becausethis might have been due to sensitization of target tissues,no long-term tolerance enhancement, although’initially evident (Fig. 2), was followed due to the exposure.To testthe hypothesis of sensitization, tolerances to pure PCP should have been measured. We may hypothesize, however, that not only an increased tolerance but also an unchanged tolerance (vs decreasedtolerance) could indicate adjustment to an envi-

ACCLIMATIZATION

OF ROACH TO KRAFl-

MILL

EFFLUENT

287

ronment which is potentially injurious to an organism. Therefore, the ability of the roach population to live in waters filled with BKME may be not only genetically determined but also affected by physiological acclimatization. It is important to realize that in our study the growth and the tolerance of roach did not decrease due to a subchronic exposure; i.e., no responses which could be interpreted as an uncompensation in respect to a toxicologically challenging environment were found. The hypothesis that the unchanged tolerance and growth actually could be compensatory outcomes in a severe environment need further verification by measurements of the fitness (Keeton, 1980) of fish under controlled conditions. On the other hand, several published studies with salmonids, which, in contrast to roach, do not occur in receiving waters close to puIp mills, have shown reduced production or impaired reproduction success due to exposures to low concentrations of unbleached or bleached KME (Webb and Brett, 1972; Stoner and Livingston, 1978; Seim et al., 1977; Landner et al., 1985, Vuorinen and Vuorinen, 1985). In conclusion, whether unequivocally measurable, determination of the limit of the compensatory acclimatization could serve as a biologically meaningful endpoint in ecotoxicological surveys of chronic contamination of the aquatic environment. Our observations on roach should also be combined with the cellular mechanisms responsible for the decreased tissue content of [‘4C]PCP. In teleost fishes, the major pathway of PCP metabolism is its direct conjugation with glucuronic acid or with sulfate in hepatocytes followed by its excretion to the surrounding water (Lech et al., 1977; Kobayashi, 1977; Huckins and Petty, 1983; Oikati and &is, 1985). The tissue activity of PCP thus consisted of both the free and the bound parent compounds together with its metabolites. Because no difference in absorption rates between preexposed and nonexposed roach was found, it is suggested that the lower body burden of [‘4C]PCP in preexposed fish was due mainly to enhanced elimination, probably via the liver-intestine route. As suggested by Niimi and Cho (1983), the development of this response would need several weeks at a sufficiently low level. On the hepatocellular level the compensatory response might be caused by the induction of conjugate ing enzymes, in particular, UDP-glucuronosyltransferase (UDP-GT). However, effects of chlorophenols and resin acids, the two major classes of toxicants in KMESa+CP, on the liver UDP-GT activity are not similar. At concentration levels of micrograms per liter, the former substances seem to have primarily an inductive and the latter an inhibitory effect (Oikari et al., 1983; J. Tana, 1986 unpublished). In all, the net response of UDP-GT seems to depend on such factors as the chlorophenol/ resin acid ratio in water. CONCLUSION The present results on roach, a species having permanent populations even in lake areas close to pulp mill sewers, showed a significant ability for reduced body burden of pentachlorophenol under long-term low-level exposure. The response, however, was not associated with enhanced tolerance toward the total effluent containing pentachlorophenol only as one toxic ingredient. On the other hand, the unchanged growth rate of roach can also be considered as an expression of compensatory acclimatization under environmental conditions potentially toxic to fishes. ACKNOWLEDGMENTS This study was supported by the Academy of Finland/Research Council for the Environmental Sciences (Project 06/085).

288

OIKARI

AND KUKKONEN

REFERENCES BRADLEY, R. W., DUQUESNAY, C., AND SPRAGUE, J. B. (1985). Acclimation of rainbow trout, S&no gairdneri Richardson, to zinc: Kinetics and mechanism of enhanced tolerance induction. J. Fj.r/r Bjo/. 27,367-379.

HUCKINS, J. N., AND PETTY, J. D. (1983). Dynamics of purified and industrial pentachlorophenol in fathead minnows. Arch. Environ. Contam. Toxicol. 12,667-672. KEETON, W. T. (1980). BiologicalScience, 3rd ed. Norton, New York, KOBAYASHI, K. ( 1977). Metabolism of pentachlorophenol in fishes. In Penrac/rlorophenol (K. Ranga Rao, Ed.), pp. 89-105. Plenum, New York. LANDNER, L., NEILSON, A. H., WRENSEN, L., T~~RNHOLM, A., AND VIKTOR, T. (1985). Short-term test for predicting the potential of xenobiotics to impair reproductive success in fish. Ecoroxicol. Envjrort SaJ: 9,282-293. LECH, J. J. (1974). Glucuronide formation in rainbow trout, effect of salicylamide on the acute toxicity, conjugation and excretion of 3-trilluoromethyl nitrophenol. Biochem. Pharmacol. 23,2403-24 10. LECH, J. J., AND BEND, J. R. (1980). Relationship between biotransformation and the toxicity and fate of xenobiotic chemicals in fish. Environ. Health Perspect. 34, 115-l 3 1. LECH, J. J., GLICKMAN, A. H., AND STATHAM, C. N. (1977). Studies on the uptake, disposition and metabolism of pentachlorophenol and pentachloroanisole in rainbow trout (Salmogairdnert). In Pentachlorophenol (K. Ranga Rao, Ed.), pp. lO7- 113. Plenum, New York. MCLEAY, D. J., AND BROWN, D. A. (1979). Stress and chronic effects of untreated and treated bleached krat? pulpmill effluent on the biochemistry and stamina of juvenile coho salmon (Oncorhynchus kisutch). J. Fish. Res. Board Canad. 36, 1049-1059. MELANCON, M. J., WILLIAMS, D. E., BUHLER, D. R., AND LECH, J. J. (1985). Metabolism of 2-methylnaphthalene by rat and rainbow trout hepatic microsomes and purified cytochromes P-450. DrugMetab. Dispos. 13,542-547. NIIMI, A. J., AND CHO, C. Y. (1983). Laboratory and field analysis ofpentachlorophenol (PCP) accumulation by salmonids. Water Res. 17, 179 l-l 795. OECD (198 1). OECD (Organization for Economical Cooperation and Development) Guidelines for Testing of Chemicals, 203. OECD Paris. OIKARI, A., AND NIITIYL& J. (1985). Subacute physiological effects of bleached kraft mill effluent (BKME) on the liver of trout, Salmo gairdneri. Ecotoxicol. Environ. SaJ: 10, 159-I 72. OIKARI, A., AND AN.&, E. (1985). Chlorinated phenolics and their conjugates in the bile of trout (Salmo gairdnert) exposed to contaminated waters. Bull. Environ. Contam. Toxicol. 35,802-809. OIKARI, A., LYNN, B-E., CASTREN, M., NAKARI, T., SNICKARS-NIKINMAA, B., BISTER, H., AN? VIRTANEN, E. (1983). Toxicological effects of dehydroabietic acid (DHAA) in the trout, Salmo gairdneri Richardson, in fresh water. WaterRes. 17,81-89. OIKARI, A,, NAKARI, T., AND HOLMBOM B. (1984). Sublethal actions of simulated kraft pulp mill effluents (KME): residues oftoxicants, and effects on blood and liver. Ann. 2001. Fennici 21,45-53. PROSSER,C. L. (1973). Comparative Animal Physiology. Saunders, Philadelphia. SAARIKOSKI, J., AND VILUKSELA, M. (1982). Relation between physicochemical properties of phenols and their toxicity and accumulation in fish. Ecotoxicol. Environ. Saj 6,501-5 12. SAS (1982). Users Guide: Statistics. SAS Institute Inc., Cary, NC. SEIM, W. K., LICHATOWICH, J. A., ELLIS, R. H., AND DAVIS, G. E. (1977). Effects of kraft mill effluents on juvenile salmon production in laboratory streams. Water Res. 11, 189-I 96. SOUTHWORTH, G. R., KEFFER, C. C., AND BEAUCHAMP, J. J. (1981). The accumulation and disposition of benz[a]acridine in the fathead minnow, Pimephales promelas. Arch. Environ. Contam. Toxicol. 10, 561-569. STONER, A. W., AND LIVINGSTON, R. J. (1978). Respiration, growth and food conversion efficiency of pi&h (Ladogon rhomboides) exposed to sublethal concentrations of bleached kraft mill effluent. Environ. Pollut. 17,207-2 17. WEBB, P. W., AND BRETT, J. R. (1972). The effects of sublethal concentrations of whole bleached kraft mill effluent on the growth and food conversion efficiency of underyearling sockeye salmon (Oncorhynthus nerka). J. Fish. Res. Board Canad. 29, 1555- 1563. VUORINEN, P., AND VUORINEN, M. (1985). Effects ofbleached kraft mill effluent on reproduction ofbrown trout (Salmo trutta L.) on a restricted diet. Finnish Fish. Res. 6,92-105.