Effects of pulp and paper mill effluents on reproduction, bile conjugates and liver MFO (mixed function oxygenase) activity in fish at Southern Lake Saimaa, Finland

Effects of pulp and paper mill effluents on reproduction, bile conjugates and liver MFO (mixed function oxygenase) activity in fish at Southern Lake Saimaa, Finland

~ Pergamon Wal Sci Tech Vol 40. No. 11-12, pp. 109-114. 1999 '01999 Published by ElsevIer SCIence LId on behalfofille IAWQ Prlnced In Grear Britain...

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Pergamon

Wal Sci Tech Vol 40. No. 11-12, pp. 109-114. 1999 '01999

Published by ElsevIer SCIence LId on behalfofille IAWQ Prlnced In Grear Britain All nghls reserved

PH: S0273-1223(99)00707-6

0273-1223/99 $20.00 + 0.00

EFFECTS OF PULP AND PAPER MILL EFFLUENTS ON REPRODUCTION, BILE CONJUGATES AND LIVER MFO (MIXED FUNCTION OXYGENASE) ACTIVITY IN FISH AT SOUTHERN LAKE SAIMAA, FINLAND Aamo Karels*, Markus Soimasuo** and Aimo Oikari* • Department ofBiological and Environmental &iences. University ofJyvliskylii. P.D. Box 35, FIN-4035l Jyvtiskylti. Finland •• Biomark. EnVIronmental Research, Y/iopistonkatu 42. FIN-40100. Jyvtiskylii. Finland

ABSTRACT Reproductive mdlces like gonad SIZe. fecWldity, egg sIZe and sex sterOid levels of estradiol-170 and testosterone, vitellogernn 10 the blood as well as bJle conjugates and liver 7-ethoxyresorufin O-deethylase (EROD) actIVIty were studied in populattons of perch (Percaf/uvlatilis L.) and roach (Ruti/us rutilus L.) and expenmentally exposed Juvenile whitefish (Coregonus lavarelUs L. s.l.) at the Southern Lake SalD13a (S.E. Fmland). Our studies showed that the introduction of elemental chlonne free (ECF) bleaching and secondary treatment of effiuents in a modem activated sludge plant at the tnIl1 m 1992 have substantially reduced the exposure of feral and caged fish to organochlonnes. The Itver EROD activity was noticeably lower in feral and caged fish near the mill indicating lesser impacts on the liver ID1xed function oxygenase (MFO) syslem However, at the mill site, liver EROD activles ID feral and caged fish still tend to be one to four limes higher than al the reference slles. Significantly decreased plasDl3 estradiol-I?ll and lestasterone concentrations in perch and roach ID the period of development of the gonads (autumn and winter) mdieate that there were endocrine disrupting compounds present in the lake receivmg ECF pulp and paper 1D111 effiuents. Causeeffect relationships, however, are difficult to establish. ~ 1999 Published by Elsevier Science Ltd on behalf of the IAWQ. All rights reserved

KEYWORDS Bile conjugates; EROD; fish; pulp mill effiuent; reproduction. INTRODUCTION It has been demonstrated that fish exposed to pulp mill effiuents evoke a variety of sublethal effects (Owens, 1991; Sandstr6m, 1996) which can lead to fish population and community changes (Neuman and KarAs. 1987; Hakkari. 1992; Karels and Niemi. in preperation). For example. salmoniform fishes like lake trout, whitefish and vendace have disappeared from Finnish waters receiving bleached kraft mill effiuent (BKME) whereas certain others, like perch and roach have survived (Hakkari, 1992; Karels and Niemi, in preperation). While the most consistent biomarkers of exposure to pulp mill effiuents are bile chlorophcnolic conjugates and liver mixed function oxygenase (MFO) activity, the translation of these responses to higher levels of organisation are still not wen understood (Hodson, 1996; Kloepper-Sams, 1996). More recent Canadian and Scandinavian studies have also demonstrated effects of BKME on reproduction in feral fish 109

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populations as well as in fish exposed to BKME in the laboratory. Decreased steroid hornlOne levels, gonad size and fecundity were demonstrated in fish exposcd to efflucnts of pulp mills with different processes and effluent treatments (Munkittrick et tli.. 199 J. 1992. 1994: McMaster et tI/., 1991, 1992; Gagnon et al., 1994; Sandstrom, 1996; Karels et ai.. 1998. in prep.; Van Ocr Kraak et al. 1998). While some studies did not show any significant reproductive effects (Hodson et tli., 1992; Kloepper-Sams et £/1. 1(94), the interpretation of results often set:ms to be hindt:rcd by many site, seasonal and lish population speci fie factors. In the Southern Lake Saimaa (Finland) a series of studies have been conductt:d before and after the substitution of elemental chlorine by chlorine dioxide in the bleaching of kraft pulp and replacement of aerated lagoons by activated sludge treatmenl of effluents in 1992. Results showed dramatic reductions in chlorinated phenolics and other organochlorincs, both in the lakc water and fish exposed to it. (Oikari and Holmbom. 1996; Soimasuo et til, 1998: Karels et tI/.. 1998. submitted, in prep.; Leppanen et ai, 1998). In the light of decreased biomarker responses in experimentally exposed fish (Oikari and Holmbom, 1996; Soimasuo e/ al., 1998), the recovery of thc ecotoxicological status of a lake system after m;~or technical alterations was suggested to be relatively fast. We theret()fc hypothesized that the improved water quality should lead to recovery of fish populations and fauna at the lake area polluted by BKME for several decades. More specifically, as the first sign, the reproductive and physiological status of feral {ish populations downstream of effluent sources was considered to give important information in respect to prerequisites of their normalisation and recovery. To lest this hypothesis. we investigated possible eflects of elemental chlorine free (ECF) BKME on hiomarkers of exposure and reproduction of feral perch and roach and experimentally exposed juvenile whitefish .



Caging ..ttes Sampling sites

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Figure I Sampling sites of feral perch and roadl <..Inti caging sites of juvenile whitcfi",h al the Southern Lake Saima:J

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MATERIALS AND METIIODS Study sites and research area The study area received ahout 200 000 m 'Id biologically treated effluents, discharged from an integrated elemental chlorine free (EeF) bleached kraft pulp and paper mill with a modernized activated sludge treatment (mill A), and an Eer pulp mill (mill H) with hiologieallreatmenl of eflluenls. Samples of the fish populations were collected 1-2 km downstream of the mills (Fig. I). Caging sites of the juvenile whitefish were located downstream and upstream of the discharge point of the mills (Fig. I). The mills are the prinwry source of chemical contamination in the research area (chemicals, nutrients and log-Iloating) and there is no contamination by munieip;d effluents.

Pulp and paper mill effluents

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Collection of feral perch and roach and exposure of whitefish Perch and roach were caught in spring 1995 and 1996 during the spawning period, autumn 1995, during early vitellogenesis, and in winter 1997, during advanced vitellogenesis. Fish were sampled as described in Karels et al. (1998). In May 1995,1996 and 1997, 1+-year-old whitefish (Coregonus lavaretus L. s.1.) were exposed by caging at the experimental area in Southern Lake Saimaa. After one month of exposure, fish were sampled on the research vessel ''Muikku'' as described earlier in Soimasuo et al. (1998). Analytical methods Conjugated and free chlorophenolics in the bile, the 7-ethoxyresorufin O-deethylase (EROD) activity of liver microsome fractions and plasma steroid hormone concentrations were measured as described earlier in Soimasuo et al. (1998) and Karels et al. (1998). Data were compared by one-way ANOVA or by the nonparametric Kruskall-Wallis test when necessary. Data were log-transformed where appropriate. The significance was set at p<0.05. The data were analysed using SPSS computer software. RESULTS AND DISCUSSION Exposure of fish to pulp and paper mill effiuents Bile chlorophenolics (CPs) and the liver EROD activity are commonly used as biomarkers of exposure to pulp and paper mill effiuents (SOderstrom and Wachtmeister, 1992; Oikari and Holmbom, 1996; Hodson, 1996). The process alterations at the mills caused that bile CPs of caged whitefish in 1995, 1996 and 1997 were only 0.2-0.5% of those measured in 1991 (Soimasuo et al., 1998, Lepplinen et aI., 1998), and bile CPs in perch and roach in 1995, 1996 and 1997 only 0.01-0.02% of those in 1983 (Oikari, 1986; Karels et al., 1998, submitted, in prep.). These results indicate a dramatically released exposure of fish to chlorophenolic compounds. The liver EROD activity in caged and feral fish near the mill was decreased by 50 to 75% after the process changes, indicating lesser impacts on the liver MFO system (Soimasuo et al., 1998; Karels et al., 1998, submitted, in prep.). However, at the mill site, liver EROD activies in whitefish and roach are still one to four times higher than at the reference sites, indicating a decreased but still existing exposure to pulp mill effiuent. Although MFO induction is apparently a useful biomarker, the ecological or even physiological significance of MFO induction in fish exposed to BKME still remains a point of discussion (Munkittrick et al., 1994; Kloepper-Sams, 1996). Reproductive indices

In contrast to caged juvenile whitefish, plasma concentrations of 17Ll-estradiol and tetosterone in feral perch and roach near the mill in autumn 1995 and winter 1997 tended to be lower from the reference sites (Figs. 2 and 3). Lower plasma hormone concentrations were only found in autumn and winter, during the period of development of the gonads, when hormone levels are more stable than during the spawning period. These results correspond with earlier studies in Canada on white sucker populations living in waters contaminated by pulp and paper mill effiuents (Munkittrick et al., 1994). Several studies have shown that there is substantial evidence that exposure to BKME leads to alterations in endocrine and reproductive function in fish (Van Der Kraak et aI., 1998). Fish showed e.g. reduced levels of the dominant sex steroid hormones (Munkittrick et al., 1991, 1992, 1994; McMaster et al., 1991, 1992; Gagnon et al., 1994; Karels et aI., 1998, submitted, in prep.; Soimasuo et al., 1998b; Van der Kraak et al., 1998). Other studies, on the contrary, did not observe any differences in plasma steroid hormone concentrations of fish exposed to BKME (Hodson et al., 1992; Kloepper-Sams et al., 1994; Soimasuo et al., 1998a). However, phytosterols of BKME, like Ll-sitosterol, have demonstrated alterations on the reproductive status in a number of fish species (MacLatchy et al., 1998; Mellanen et al., 1996, VanDer Kraak et al., 1998; Tremblay and Van Der Kraak, 1999). A broad range of compounds, however, including sterols, lignans, stilbenes and resin acids that are present in BKME are also known to have weak estrogenic activity (Van Oer Kraak et al., 1998).

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Pulp and paper milI effiuents

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Plasma vitellogenin concentrations in feral roach in winter 1997 and juvenile whitefish in spring 1997, however, were similar compared to the reference sites. In female fishes, VTG is the egg yolk precursor protein synthesized in the liver in response to circulating estrogens like estradiol-17B. It is transported in the blood to the ovary. The relative gonad size (GSI), fecundity and egg size of feral perch and roach downstream of the mills in spring 1996 (pre-spawning fish) and winter 1997 (figure 3) Were not significantly different from the reference sites (Karels et aI., submitted, in prep.). A decrease in gonad size was found in several Canadian and Swedish studies (review Sandstrllm, 1996). The relatively few studies on effects of BKME on fecundity of fish show contradictory results. Fecundity in BKME exposed fish was decreased, increased or similar to the references (Sandstrllm, 1996). CONCLUSIONS Our studies have shown that the introduction of ECF bleaching and secondary treatment of effiuent in a modern activated sludge plant have substantially reduced the exposure of fish to pulp mill effiuent compounds. However, liver EROD activies in caged and feral fish at the mill site still are one to four times higher than on the reference sites, indicating a lower but still existing exposure to BKME. Significantly lower plasma steroid hormone concentrations in feral fish downstream of the mill could be indicative of the presence of endocrine disrupting compounds in the lake area affected by BKME. Cause and effect relationships, however, are difficult to establish because of many natural environmental and biological factors affecting the endocrine regulation of spawning fishes. ACKNOWLEDGEMENTS The authors wish to thank the crew ofRV "Muikku" and the personnel of the South-Carelia Fisheries Centre (Etelli-Karjalan Kalatalouskeskus), Lappeenranta for their help in the field work. We also thank all our colleagues and collaborators who participated in these studies. The studies with caged whitefish (ECOBALANCE project) received grants from the Maj and Tor Nessling Foundation, UPM KymmenelKaukas Inc. and the Academy of Finland. Fundings for the feral fish studies were provided by the Raija and Ossi Tuuliainen Foundation, Lappeenranta and the Rural Business District of KymilFisheries Unit, Kouvola. REFERENCES Gagnon. M. M., Dodson, J. J., Hodson, P. V., Van Der Kraak, G. and Carey, J. H. (1994). Seasonal effects of bleached kraft milI effiuent on reproductive parameters of white sucker (Catostomus commersoni) populations of the St. Maurice River, Quebec, Canada. Can. J. Fish. Aquatic. Sci. 51,337-347. Hallari, L. (1992). Effects ofpulp and paper milt effiuents on fish populations in Finland. Finnish Fisheries Research 13,93-106. Hodson, P. V., McWhirther, M., Ralph, K., Gray, B., Thivierge, D., Carey, J. H., Van Der Kraale, G., Whittle, D. M. and Levesque, M.-C. (1992). Effects of bleached kraft milt effiuent on fish in the S1. Maurice River, Quebec. Environ. Toxicol. Chem. 11, 1635-I65 1. Hodson, P. V. (1996). Mixed function oxygenase induction by pulp mill effluents· advances since 1991. In: Environmental/ale and effecis ofpulp and paper mill efJIuenls. M.R. Servos, K.R. MunkiUricle, J.H. Carey and GJ. Van Ocr Kraak (eds), S1. Lucie press, Delray Beach, Florida: 349-358. Karels, A. E., Soimasuo, M., Lappivaara, J., leppAnen, H., Aaltonen, T., Mellanen, P. and Oikari, A. O. J. (1998). Effects ofECFbleached kraft null effiuent on reproductIve steroids and liver MFO activity in populations of perch and roach. ECOloxicology 7, 123-132. Karels, A., Soimasuo, M. and Olkari, A. (in press). Biomarker responses in experimentally exposed fish and feral fISh in • lake receiving pulp mill effiuents. In: Environmenlal fate and effects ofpulp and paper mill effluents, Sluthridge tl al. (eds). SETAC press, leWIS Publishers, Boca Raton (in press). Karels, A. E. and Oikari. A. O. J. (subnutled). Effects of pulp and paper mill effiuents on bile conjugales, liver EROD activity and plasma sex steroids in spawning perch and roach populations. Karels, A., SOImasuo, M., Suutan, R. and Odeari, A. (submitted). Monitoring the recovery of a lake with aid of fish biomarkers: Responses of whitefish (Coregonus IQvarelUS L. s.l.) experimentally exposed in a large lake receiving pulp and paper mill effluents. Karels, A. and Niemi, A. (in preparation). Fish community responses to changes in pulp milt processes and treatment of effiuents. Karels, A., Soimasuo, M., LeppAnen, H., Markkula, E., Paranko, J. and Oikari, A. (in preparation). Fish bile metabolites, liver EROD activity, reproductive steroids and vitellogenin levels in caged juvenile whitefISh in a large take receiving pulp mill effluents.

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Karels, A. E., Jokinen, I. and Oikari, A. O. J. (in preparahon). Effects of pulp null effluents on biomarkers of exposure and reproduction in feral perch and roach populatIons In the Southern Lake Saimaa, Fmland. Kloepper-Sams, P. J., Swanson, S. M., Marchant, T., Schryer, R. and Owens, J. W. (1994). Exposure offish to biologically treated bleached-kraft effluent. 1. BlOchenucal, physiological and pathologIcal assesment of Rocky Mountain whitefish (Prosopium wllilamsom) and longnose sucker (Catostomus catostomus). Environ. Toxicol Chem. 13(9): 1469·1482. Kloepper-Sams, P. J. (1996). FIeld and laboratory studIes of blochenucal responses associated WIth pulp null effluents: status in 1991, 1994 and beyond. In: Environmental fate and effects of pulp and paper mill effluents, M. R. Servos, K. R. Munkittrick, J. H. Carey and G. J. Van Der Kraak (eds), St. Lucie press, Delray Beach, Florida: 439-445. Leppanen, H., Mantmen, S. and Oikan, A. (1998). The use of fish bile metabolite analyses as exposure biomarkers to pulp and paper null effluents. Chemosphere 36( 12), 2621-2634. MacLatchy, D., Peters, L., NIckle, J. and Van Der Kraak, G. J. (1997). Exposure to Il-sitosterol alters the endocrine status of goldfish dIfferently than 1711-estradiol. Environ Toxieol Chem 16, 1895-1904, 1998. McMaster, M. E., Van Der Kraak, G. J., Pont, C. B., Munklttrick, K. R., Sibley, P. K., Snuth, I. R and Dixon, D. G. (1991). Changes in hepaliC mixed-function oxygenase (MFa) activity, plasma steroid levels and age at maturIty of a white sucker (Catostomus commersom) populatIOn exposed to bleached kraft pulp null effluent. Aquat Toxicol. 21, 199-218. McMaster, M. E., Portt, C. B., Munkittnck, K. R. and Dixon, D. G. (1992) Milt charactenstics, reproduchve performance and larval survival and development of wlute sucker exposed to bleached kraft null effluent. Ecotoxieol. EnVIron. Sa! 23, 103-117. Mellanen, P., Pellinen, T., Lehtimliki, J., Makela, S., Bylund, G., Holmbom, B., Mannila, E., Otkan, A. and Santti, R. (1996). Wood-{jerived estrogens: Studies 1D VItro WIth breast cancer cell lines and In VIVO m trout. Toxlcol. Appl Pharmacol. 136,381-388. Munklttnck, K. R., Pont, C. B., Van Der Kraak, G. 1., Smith, I. R. and Rokosh, D. A. (1991). Impact of bleached kraft mill effluent on population charaetenstlcs, liver MFO actlvity, and serum steroid levels of a lake Superior white sucker (Catostomus commersoni) population. Can. J FISh. Aquat. &i. 48, 1371-1380. Munklttnck, K. R., Van Der Kraak, G. J., McMaster, M. E. and Portt, C. B. (1992). Response of hepatic MFa activity and plasma sex sterOIds to secondary treatment of bleached kraft pulp null effluent and null shutdown. Envrron. Toxieol. Chem. t I, 1427-1439. Munkittnck, K. R., Van Der Kraak, G. J., McMaster, M. E., Portt, C. B., van den Heuvel, M. R. and Servos, M. R. (1994). Survey of receivIng-water envIronmental Impacts assocIated WIth dIscharges from pulp nulls. 2. Gonad size, liver size, hepatic EROD activity and plasma sex steroid levels in white sucker. Environ Toxicol Chem. 13(7), 1098-1101. Neuman, E. and Karb, P. (1988). Effects of pulp null effluent on a Baltic coastal fish community. Wat.Sci Tech. 20,95-106. Oikari, A. O. J. (1986). Metabohtes of xenobiotlcs m the btle of fish 1D waterways polluted by pulpmill effluents. Bull. Environ. Contamin. Tox/col. 36, 429-436. Olkari, A. and Holmbom, B. (1996). Ecotoxlcologlcal effects of process changes implemented 1D a pulp and paper mill: A Nordic case study. In: Environmental fate and effect.· of pulp and paper mill effluents, M.R. Servos, K.R. Munkittnck, J.H. Carey and GJ. Van Der Kraak (eds), Delray Beach, Florida, St. Lucie press: 613-625 Owens, J. W. (1991). The hazard assesrnent of pulp and paper effluents in the aquatic environment. A review. Environ. Toxicol. Chem 10(12),1511-1540. SandstrOm, O. (1996). In sItu assesments of the impact of pulp mill effluent on hfe-Iustory variables in fish. In: Environmental fate and effeets ofpulp and paper mIll effluents, M.R. Servos, K.R. Munklttrick, J.H. Carey and GJ. Van Der Kraak (eds) Delray Beach, Florida, St. Lucie press: 449-457. Soimasuo, R., Jokinen, I., Kukkonen, J., Pelllnen, T, Rlstola, T. and Oikari, A. (1995). Biomarker responses along a pollution gradient: Effects of pulp and paper mill effluents on caged whitefish. Aquat Toxieol 31,329-345. Soimasuo, M. R., Karels, A. E., Leppanen, H., Santh, R. and Olkari, A. O. J. (I 998a). Biomarker responses in whitefish (Coregenus lavaretus L. 5.1.) experimentally exposed in a large lake receIving effluents from pulp and paper industry. Arch. Environ. Contam Toxicol. 34, 69-80. Soimasuo, M. R.• Lappivaara, J. and Oikan, A. 0 ..1. (1998b). Confirmation of in situ exposure of fish to secondary treated bleached-kraft null effluent using a laboratory simulation Environ Toxicol. Chern. 17, 1371-1379. SllderstrOm, M. and Wachtmeister, C. A. (1992). Fish bIle as a monitoring inslrument for phenolics and other substances related to bleached kraft mill effluent and a study of chlorophenolic metabolic transformation products in a periphyton community. In: EnVironmental Fate and Effects ofBleached Pulp Mill Effluents, A. Slldergren (ed). Swedish EPA Report 4031: 203206. Tremblay, L. and Van Der Kraak, G. (1999). Comparison between the effects of the phytosterol II-sitosterol and pulp and paper mill effluents on sexually immature rainbow trout Environ. Toxico/. Che", 18(2),329-336. Van Der Kraak, GJ., Munkittnck K.R., McMaster M.E. and MacLatchey D.L. (1998). A comparison of bleached kraft null effluent, I 78-estradiol, and Il-s.toslerol effects on reproductive function in fish. In: Principles and processes for evaluatmg endocrine disruptIon in WIldlife, R. Kendall, R. DIckerson, J. Giesy and W. Suk (eds). SETAC Press, pp. 249-265.