Long-term effects of bleached kraft mill effluents on carbohydrate metabolism and hepatic xenobiotic biotransformation enzymes in fish

ECOTOXICOLOGY

AND

ENVIRONMENTAL

SAFETY

13,53-60 (1987)

Long-Term Effects of Bleached Kraft Mill Effluents on Carbohydrate Metabolism and Hepatic Xenobiotic Biotransformation Enzymes in Fish

*Department of Zoophysiology, University of Gtiteborg, Box 25059, S-400 31 Gijteborg, Sweden; TSwedfsh Environmental Protection Board, Brackish Water Toxicology Laboratory, Studsvik. S-61 182 Nykiiping, Sweden; and *Department of Zoophysiology. Uppsala University, Box 560,4 75122 Uppsala, Sweden Received May 6, 1986 In a laboratory investigation, fourhorn sculpin (Myoxocephalus quadricornis) were exposed for 5-9 months to waste water from pine and birch lines from a bleached kraft pulp plant. The bleached krati mill effluents (BKME) affected both the carbohydrate metabolism and the xenobiotic metabolism. Thus, an elevated muscle glycogen content in fish exposed to effluent from th.e pine pulp line suggestsa metabolic imbalance. A liver enlargement and a strong elevation of the hepatic cytochrome P-450dependent ethoxyresorufin-O-deethylase activity after exposure to the bleached pine pulp effluents indicate the presence of cytochrome P-450-inducing agents in the BKME. The results also demonstrate that many physiological test parameters may be used1 as good indicators of sublethal disturbances in fish to BKME exposure. Q 1987 Academic Press, Inc.

INTRODUCTION In the process of pulping wood, large volumes of waste water containing organic compounds are released. Although efforts have been made to reduce the outlets, 90% of the total effluent of soluble organic materials in Sweden originates from the forest industry. In Sweden the kraft process dominates the pulp capacity and to produce white paper the pulp must be bleached. The predominant method used comprises multiple stages, several of which employ chlorine-containing agents. In Sweden about 15,000 tons per year of organically bound chlorine (TOCL) are released into the aquatic environment (Anonymous, 1984). Knowledge of the impact of pulp and paper mill discharges on aquatic life is therefore of paramount importance. Much data are available concerning acute toxicity of pulp and paper mill effluents to fish. The reported 96-hr LC~O on fish to bleached kraft mill effluents (BKME) are between 0.6 and 30 ~01% (Seppovaara, 1973; Walden, 1976). Meaningful comparison between test results are difficult to make because the various pulp mill effluents used are qualitatively and quantitatively very different, even when effluents from the same industry are taken at different times. Furthermore, acute toxicity responses (e.g., death) am insufficient to assessthe impact of various pollutants on aquatic ecosystems. In this context, various sublethal toxicity tests are more relevant and make it possible to detect early arising effects on fish and to measure threshold concentrations below which deleterious effects do not occur. Among such tests, physiological and biochemical methods have been found in previous studies to be very useful for detect53

0147-6513/87$3.00 Copyright 0 1987 by Academic Press, Inc. All rights of reproduction m any form reserved

54

ANDERSSON

ET AL.

ing and diagnosing sublethal disturbances in fish induced by toxic substances or industrial waste effluents (for reviews see Payne, 1984; Larsson et al., 1985; Forlin et al., 1986). This report describes the first part of an extensive trial to use physiological methods as health indicators in laboratory and field investigations on fish exposed to BKME. In the present investigation, fourhorn sculpin, Myoxocephalus quadricornis, were exposed for 5-9 months to different qualities of BKME from Husum kraft pulp industry, situated at the coast of Gulf of Bothnia in northern Sweden. A large number of sublethal responses were measured in the fourhorn sculpin after the long-term exposure. The present report describes effects of BKME on carbohydrate metabolism and hepatic xenobiotic biotransformation enzymes. The effects of BKME on the red and white blood cell status, the ion balance and vertebral structure of the fourhorn sculpin, and residue levels of chlorinated organic compounds are presented by Hardig et al. ( 1986). A third report deals with the influence of BKME on the mechanical and biochemical characteristics of the vertebrae (Mayer et al., 1986). MATERIALS

AND

METHODS

Animals. Fourhorn sculpin (M. quadricornis) were obtained by artificial fertilization with eggs and milt from three females and three males caught by gill nets in the vicinity of the Husum kraft plant. At the start of the experiment, the fish were about 10 months old and had an average weight of 1 g. Exposure conditions. During the experiment the fish were fed with live Tubifex sp., Neomysis sp., and Lumbricus sp. Thirty to 35 fish were kept in each of 70-liter aquaria with flowthrough brackish water (40-80 ml/min) from Tvaren bay outside Studsvik. The water temperature in the aquaria, which was measured daily, followed the natural fluctuations in Tvaren bay. The salinity, pH, and dissolved oxygen content were measured once weekly. The mean values * SD of the test water were temperature 8.8 ? 0.7, salinity 7.4 f 0.3, pH 7.5 + 0.1. The oxygen content was close to saturation level (i.e., >90% saturation) from the start of the exposure (March 11, 1983) until May 6, 1983, when it dropped to 70% saturation and after which the water flows were doubled (from 40 to 80 ml/min). On September 29, the oxygen levels dropped again to 70% saturation and airstones were placed in all aquaria and remained in use until the end of the experiment (December 12, 1983). Fish were exposed for 9 months to BKME from the Husum industry. Samples of waste water from the pine pulp and birch pulp lines were taken from the industry in January, June, and August 1983. Physicochemical characteristics and some acute toxicity of the different batches of waste water from pine and birch lines are shown in Table 1. The waste water was kept frozen (-20 to -25°C) before use. The stock solutions of BKME were continuously pumped by a peristaltic pump (SPlA-20, Stalprodukter, Sweden) which delivered a flow of 15 + 1 ml/hr during the first 2 months and 30 _t 2 ml/hr during the rest of the experimental period, depending on doubled water flow (see above). The BKME flow was mixed with the incoming natural brackish water. The intended test concentrations were 0.12% (pine “low” dose, PL) and 0.6% (pine and birch “high” dose, PH and BH, respectively) of the BKME batches presented in Table 1. A combination of pine and birch BKME was obtained by mixing PH BH, thus giving pine/birch medium dose (PBM) containing 0.3% of each BKME category.

EFFECTS OF KRAFI-

MILL

EFFLUENTS

TABLE

55

ON FISH

1

PHYSIOCHEMICAL CHARKTERISTICS AND SOME ACUTE TOXICITY DATA OF THE THREE BATCHES OF WASTE WATER FROM PINE PULP AND BIRCH PULP LINES OF THE INVESTIGATED

KRAfl

“Pine-batch” I Jan. 1983 Washing loss Na2S04 b&o pulp” BOD, (mg/liter) COD (mg/liter) Kappa number Suspended matter (mg/liter) PH TOCL (mmol/liter) Waste water volume m31b0 ~ulpl 96-hr L&,, Nitocra spinipes, 90 w/w 5-min EC&,, Microtox, % w/w

16 270 1130 20-22

II June 1983

13 330 1200-1400 13-16

PULP

INDUSTRY

No.

“Birch-batch” III Aug.

1983

15 350 1200 20

I Jan. 1983

II June 1983

16 250 1060 17-18

15 235 960 14-17

No.

III Aug.

1983

15 270 1050 18

25 2.5 3.3

13 2.1 2.4

11 2.1 1.05

42 3.9 0.4

42 4.3 0.4

34 2.5 0.94

70

31

45

60

60

60

8.7

7.4

8.8

28.0

19.4

19.4

18.0

12.4

10.6

11.5

16.7

8.7

a fw. metric tons of 90% dryness.

Since batches II and III from June and August, respectively, of the pine pulp effluents were more concentrated due to a lower waste water flow per ton produced pulp (Table I), these effluents were diluted before use by a factor of 2 to obtain comparable testing conditions throughout the test. By this adjustment the concentration of pine BKME was 0.3% (high dose) and 0.06% (low dose) during the last 5 months of the experilment. Sampling and analyses. After 9 months of exposure the fish were rapidly removed one at a time from the water. The samplings of blood, liver, and muscle tissue and analytical techniques for determination of blood glucose and lactate, as well as liver and muscle glycogen content were performed as previously described (Dave et al., 1975). Ascorbic acid content in the liver was measured according to the method of Zannoni et al. (1974). Liver tissue for assay of the enzymes involved in xenobiotic metabolism was quickly frozen in liquid nitrogen according to Fiirlin and Andersson ( 1985). 7-Ethoxyresorufin-O-deethylase and UDP glucuronosyltransferase activities were measured as described by Andersson et af. (1985). All enzyme activities were measured at 20°C. The pH levels used for the enzyme reactions were found to be optimal in all cases and enzyme activities showed linear correlations with time and protein concentration at the incubation temperature used. The liver somatic index (LSI) was calculated according to the formula liver weight (g) X loo/body weight (g), and the condition factor was calculated according to the formula body weight (g) X lOO/[length (cm)13. Statistical methods. The statistical treatment of the data was performed by simple variance analysis followed by Duncan’s new multiple-range test (Winer, 197 1) using the data program MULREG 800 on ABC Microcomputer-system (Luxor, Sweden).

56

ANDERSSON

TABLE

ET AL

2

EFFECTSOF VARIOUS PINE AND BIRCH PULP LINE BKME

ON PHYSIOLOGICAL

PARAMETERS IN FOURHORN SCULPIN Parameter

Control

Body weight(g) Length (cm) Condition factor

12.3 k 5.0 11.1 f 1.3 0.84 + 0.10 2.7? 1.0

LSI Muscle glycogen mgigf UDPGT activity’ nmol/mg prot X min

PL (24) (24) (24) (15)

3.8 + I .7 (14)

0.14+0.07(11)

10.9 10.7 0.83 3.2

PH

f 4.5 I? 1.3 k 0.08 k 0.6

(24) (24) (24) (16)

3.2 f 1.5

(16)

0.16+0.07(14)

7.1 f 3.5 9.3 * 1.3 0.82 + 0.09 3.5kO.6

BH

PBM

(30) (30) (30) (16).

8.9 f 2.4 10.1 * 0.9 0.83 + 0.07 3.2k0.9

(30) (30) (30) (16)

5.5 k 1.8 (14)*

3.5 k 1.6

(14)

0.13~0.07(10)

No&. The results are means f SD; number of animals in parentheses. birch “high” dose (BH); pine/birch “medium” dose (PBM). ’ Hepatic microsomal UDP glucuronosyltiansferase activity. l p
0.11~0.09(10) Pine

“low”

dose (PL);

7.0 * 3.1 9.4 2 1.2 0.77 f 0.06 2.5kO.6

(33) (33) (33)* (15)

3.6 2 1.8 (14)

0.17kO.l0(8) pine

“high”

dose (PH);

Statistically significant differences between exposed and control fish have been indicated at P < 0.05 and P < 0.0 1. RESULTS

AND

DISCUSSION

Due to an accidental stop in the waterflow in the BH aquarium all fish died in that group on June 26 and were replaced with new fish, which were then treated identically to the control fish. This latter group of fish was thus exposed for 5 months instead of 9 months. At sampling, the body weight and length were significantly lower in fourhom sculpin exposed to PH, BH, and PBM effluents compared to the control fish (Table 2). No attempts to evaluate the effects of BKME on growth are made since the mortality was suspected to be selective; some bigger fish swallowed smaller fish, which might affect the fish size differently in the various experimental groups. The muscle glycogen level was significantly higher in sculpins exposed to PH effluent, but was not affected in the other BKME groups (Table 2). Blood glucose and lactate, as well as the liver content of glycogen, was measured only in fourhom sculpin from control and PL groups due to the small amount of material. No significant effect of the PL dose on these parameters was observed (Table 3).

TABLE EFFECTS

Control PL Note.The

OF

3

BKME FROM THE PINE LINE ON PHYSIOLOGICAL PARAMETERS FOURHORN SCULPIN (Myoxocephalus quadricornis)

IN

Blood glucose (mg/lOO ml)

Blood lactate (mg/lOO ml)

Liver ascorbic acid k/g)

Liver glycogen (mg/ 100 ms)

14.4 + 3.7 (16) 14.5 k 2.4 (17)

13.8 -t 1.8 (14) 13.4 f 2.0(10)

77.8 zt 12.1 (16) 84.7 i- 14.7 (19)

7.6f2.8(15) 7.3 * 3.1 (13)

results are expressed

as means k SD, number

ofanimals

in parentheses.

Pine “low”

dose (PL).

64

LIU

AND

HE

TABLE MERCURY

CONCENTRATIONS

IN FISHES

1976 ______ Species Ctenopharyngodon idellus (Cuvier et Valenciennes) Xenocypris macrolepis Gunther Acanthobrama simoni Bleeker Hemibarbus maculatus Bleeker Cyprinus carpio haematopterus Tern. et Schl. Carassius auratus gibelio (Bloch) Hypophthalmichthys molitrix (Cuvier et Valenciennes) Aristichthys nobilis (Richardson) Parabramis pekinensis (Basilewsky) Culter erythropterus (Basilewsky) Erythroculter mongolicus (Basilewsky) Eryfhroculter ilishaeformis (Bleeker) Parasilurus asotus (Linnaeus) Siniperca chuatsi (Basilewsky)

“From

II SONGHUA

1978

6 0.371 51 0.850

1980 .r

54 0.346

49

0.697

n

1981 x

5 0.410 8 0.150

66 0.258

97

0.279

0.405

338

0.545

33 0.191

46

0.181

I1 0.263

8 0.421

9 0.360

18 0.258

5 0.230

8 0.109

100

5 0.325

5 0.325

7 0.159

9 0.168

12 0.637

14 0.581

8 0.538

67 0.408

2 0.200 1 0.350 49

1 0.350 5 1.120

68 0.915

0.887

202

1 0.135

0.366

0.494

10 0.510

3 0.437

5 0.423

3 0.323

79

0.532

65 0.540

185

0.441

1 0.200 6 0.608 32 0.323 105

.G

0.063 0.311 0.483 0.280

2 0.038

0.397

n

6 123 4 19

17 0.350

74

.r

total

0.063 0.269 0.483 0.300

6 0.295

0.697

n

6 years’

6 43 4 1

3 0.097

46

(mg/kg)”

RIVER

1979

nxnxnxn

119 Yu ad.

OFTHE

1977

30 0.626 Total

1

0.324

17 17 11 116

0.193 0.359 0.263 0.351

446

0.328

19 104 11 260 1122

0.202 0.779 0.263 0.434 0.448

(1983)

survive for long in the river water. The gravel bed and bank at the upper reaches of the river were covered with mine tailings, and the diversity of biotic community was lessened; apparently, the environmental conditions for spawning and for feeding of the juvenile was ruined. Besides, the salmon manifests a distinct avoidance reflex against the pulp mill wastewater even at low concentration, therefore it cannot pass through the heavily polluted section of the river on its upstream migration toward the spawning ground. The anadromous spawning migration was thus impaired. The Jiyun River of Hebei Province has a length of about 300 km. and was noted for its high production of “ice fish” (Salangidae) and a sort of “purple crab.” Because of the untreated wastewater from chemical works, the river was polluted with yellow phosphorus, mercury, HCH, and DDT (Department of Biology, Nankai Univ. and Beitang Commune, 1977; Department of Chemistry, Nankai Univ.. 1977; Institute of Zoology, Academia Sinica, 1977). Surveys revealed that in the heavily polluted sections of the river, death of fish occurred frequently, and the only fish that survived was the pollution-tolerant crucian carp (Curussius aurutus). Further downstream, the number of fish species and the size of fish population increased with the distance from the source of pollution, but the fish had an odor of pesticide, thus rendering them unfit for consumption. The death of the fish was due to the fact that the bottom sediments in the river contained yellow phosphorus at the level of 1.8-2.2 mg dry weight/kg of sediment. This pollutant under the action of water current was dissolved or suspended in the water mass and the fish were poisoned. The poisoned fish had

58

ANDERSSON

mg prot

ET AL.

min

0.6 1

0.4.

02

o-

-

FIG. 1. Effects of various pine and birch line BKME on 7-ethoxyresorufin-O-deethylase activity in liver microsomes from fourhom sculpin (Myoxocephalus quadricornis). Control (C); pine “low” dose (PL): pine “high” dose (PH); birch “high” dose (BH); pine/birch “medium” dose (PBM). The columns represent means + SD of(n) animals. **Indicates statistically significant differences between exposed fish and control fish(P
(Bend and James, 1978). The initial biotransformation of lipophilic compounds is usually associated with an enzyme system known as cytochrome P-450 monooxygenase or mixed function oxidase (MFO) system. The cytochrome P-450 enzyme system introduces functional groups into the substrate which facilitates their further metabolism by conjugating enzymes. The ability of many chemicals to induce hepatic cytochrome P-450-dependent activities is a well-documented feature. In fish, polyaromatic hydrocarbon (PAH) type inducers increase the activities of several cytochrome P-450-mediated enzymes (Stegeman, 1981). Induction of hepatic cytochrome P-450 activities in fish exposed to petroleum contaminations is a very sensitive biological response and has been used in several biological monitoring programs (Payne, 1984). In the present study, fish exposed to bleached pine pulp effluent or combined bleached pine/birch pulp effluent showed markedly higher hepatic cytochrome P-450-dependent ethoxyresorufin-Odeethylase (EROD) activities (5 and 3-4 times higher, respectively) than control fish (Fig. 1). These results strongly suggest the presence of potent cytochrome P-450 inducers, such as aromatic chlorinated compounds, in the bleached pulp effluents and the potential use of this response parameter as a sensitive indicator for BKME exposure. In previous laboratory studies, cytochrome P-450 inducing agents have also been shown to induce conjugation reactions, e.g., the UDP glucuronosyltransferase activities, in rainbow trout (Sulmo gairdneri) (Andersson et al., 1985; Forlin and Haux, 1985). The hepatic UDP glucuronosyltransferase activity in fourhom sculpin was, however, not affected by pine or birch pulp effluents (Table 2). This may be due to a low sensitivity of UDP glucuronosyltransferase to the cytochrome P-450-inducing agent(s) in BKME. Another explanation may be that the UDP glucuronosyltransfer-

EFFECTS OF KRAFT

MILL

EFFLUENTS

ON FISH

59

ase is inhibited by certain chemicals in the BKME. Thus, Oikari and Nakari (1982) have found decreased UDP glucuronosyltransferase activities in fish exposed to BKME. Furthermore, it has been demonstrated that one of the major toxicants in BKME, dehydroabietic acid, is a potent inhibitor of UDP glucuronosyltransferase in rainbow trout (Oikari et al., 1983). It is obvious that the birch line BKME caused weaker effects in the fourhorn sculpins than corresponding pine line BKME (Fig. 1 and Table 2). This difference may be due to less potent toxic compounds in the BKME from the birch pulp line, but it can also be explained by shorter exposure time (about 5 months) for this group of fish. CONCLUSIONS The pmsent, long-term, laboratory exposure of fish to bleached kraft mill effluents, affects important physiological functions, such as carbohydrate metabolism and hepatic xenolbiotic metabolism. These results also indicate that many physiological test parameters may be good indicators of sublethal responses in fish exposed to BKME. There is a need to develop monitoring techniques and sublethal bioassays procedures to detect ;and diagnose disturbances on fish populations in polluted areas. This is particularly important for the very complex character of BKME, which consists of several hundred compounds, many of which have not yet been chemically characterized and fi3r which analytical methods are not at hand. Further studies are in progress with the purpose to further elucidate the effects of BKME on fish and to evaluate the physiological parameters used in the present investigation. These studies are performed as both laboratory and field experiments, which should facilitate the interpretation of results and the value of the response parameters as monitoring indices.

ACKNOWLEDGMENTS We are grateful to Eva Nilsson, Inger Petterson, and Birgitta Samuelsson for excellent technical assistance. The investigation was financed by the Swedish Environmental Protection Board.

REFERENCES ANDERSSON, T., F~RLIN, L., H;~RDIG, J., AND LARSSON, A. (I 986). Physiological disturbances in fish living in the receiving body ofwater of a kraft pulp mill industry (manuscript in preparation). ANDERSSOP~,T., PESONEN,M., AND JOHANSSON.C. (1985). Differential induction of cytochrome P-450dependent monooxygenase. epoxide hydrolase, glutathione transferase and UDP glucuronosyltransferase activities in the liver of the rainbow trout by P-naphthoflavone or Clophen A50. Biochem. Pharmacol.34,3309-3314. Anonymous (1984). Environment/Celltr/ose. National Swedish Environment Protection Board. Bulletin 1793. BEND, J. R., AND JAMES, M. 0. (1978). Xenobiotic metabolism in marine and fresh water species. In Biochemical and Biophysical Perspectives in Marine Bio1og.v(D. C. Malins and J. R. Sargent, eds.), Vol. 4, pp. 125- 187. Academic Press, New York. DAVE, G., JOHANSSON-SJ~BECK, M.-L., LARSSON. A., LEWANDER, K.. AND LIDMAN, U. (1975). Metabolic and hematological effectsof starvation in the European eel, AnguiNa anguilla L. I. Carbohydrate, lipid, protein and inorganic ion metabolism. Comp. Biochem. Physiol. 52A, 423-430. F~RLIN, L., AND ANDERSSON,T. (1985). Storage conditions of rainbow trout liver cytochrome P-450 and conjugatmg enzymes. Comp. Biochem Physiol. SOB, 569-512.

60

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ET AL.

F~~RLIN,L., AND HAUX, C. (1985). Increased excretion in the bile of 178.(‘H)estradiol-derived radioactivity in rainbow trout treated with B-naphthoflavone. .4quat. To,yico/. 6, 197-208. F~RLIN, L., HAUX, C.. ANDERSSON. T., OLSSON. P.-E., AND LARSSON, A. (1986). Physiological methods in fish toxicology: Laboratory and field studies. In Fish Physiology: Recent ,4dvances (S. Nilsson and S. Holmgren, eds.). Croom Helm, London. in press. HARDIG, J., ANDERSSON. T.. F~~RLIN,L., AND LARSSON, A. (1986). Long-term effects on bleached kraft mill effluents on red and white cell status, ion balance, and vertebral structure in fish (manuscript in preparation). KLAUNIG, J. E.. LIPSKY, M. M., AND TRUMP, B. F. (1979). Biochemical and ultrastructural changes in teleost liver following subacute exposure to PCB. J. Environ. Pathol. To..uicol. 2,953-963. LARSSON, A.. HAUX, C.. AND SJ~~BECK.M.-L. (1985). Fish physiology and metal pollution: Results and experiences from laboratory and field studies. Exotoxicol. Environ. Suf 9,250-28 1. LIDMAN, U., F~RLIN, L., MOLANDER, O., AND AXELSON. G. (1976). Induction of the drug metabolizing system in rainbow trout (Sulmo guirdneri) liver by polychlorinated biphenyls (PCBs). Acta Pharmacol. et Toxicol. 39,262-272. MAYER, F. L., BENGTSSON, B.-E., HAMILTON, S.. AND BENGTSSON, A. (1986). Influence of the pulp mill and ore smelter effluents on vertebral composition of fourhom sculpins (M.voxocephahu quadrocornis) in the laboratory and Gulf of Bothnia, Sweden. In Proceedingfrom the 9th .4STMSymposium on Aquatic Toxicology and Environmental Fate, Philadelphia. Pa., in press. MCLEAY, D. J., AND BROWN, D. A. (1974). Growth stimulation and biochemical changes in juvenile coho salmon (Oncorhynchus kiszctch) exposed to bleached kraft pulpmill effluent for 200 days. J. Fish. Res. Board Canad. 31,1043-1949. MCLEAY, D. J., AND BROWN, D. A. (1975). Effects of acute exposure to bleached kraft pulpmill effluent on carbohydrate metabolism of juvenile coho salmon (Oncorhynchus kisutch) during rest and exercise. J. Fish. Res. Board Canad. 32,753-760. OIKARI, A.. LYNN, B.-E., CASTR~N. M.. NAKARI, T., SNICKARS-NIKINMAA. BISTER, H.. AND VIRTANEN, E. (1983). Toxicological effects of dehydroabietic acid (DHHA) on the trout, Salmo guirdneri, in fresh water. Water Res. 17,8 l-89. OIKARI, A. 0. J., AND NAKARI, T. (1982). Kraft pulp mill effluent components cause liver dysfunction in trout. Bull. Environ. Contamin. Toxicol. 28,266-270. OKARI, A.. NAKARI, T., AND HOLMBOM B. (I 984). Sublethal actions ofsimulated kraft pulp mill effluents (KME) in Salmo gairdneri: Residues of toxicants, and effects on blood and liver. Ann. Zool. Fenn. 21, 45-53.

PAYNE, J. F. (1984). Mixed-function oxygenase in biological monitoring programs: Review of potential usage in different phyla ofaquatic animals. In Ecotoxicological Testing For theMarine Environment (G. Persoone, E. Jaspers, and C. Claus, eds.). Vol. 1, pp. 625-650. State Univ. Ghent and Inst. Mar. Scient. Res., Bredene, Belgium. SEPPOVAARA,0. (I 973). The toxicity of the sulphate pulp bleaching effluents. Puperi Ja Puu 55,7 13-720. STEGEMAN, J. J. (198 1). Polynuclear aromatic hydrocarbons and their metabolism in the marine environment. In Pol.vcyclicHydrocarbons and Cancer (H. V. Gelboin and P. 0. P. Ts’o, eds.), Vol. 3, pp. l-60. Academic Press, Orlando, Fla. THOMAS. P., BALLY, M. B., AND NEFF, J. M. (1982). Ascorbic acid status of mullet, Mugil wpha!us L., exposed to cadmium. J. Fish. Biol. 20, I83- 196. WALDEN, C. C. (1976). The toxicity of pulp and paper mill ellhrents and corresponding measurement procedure. Water Res. 20,639-664. WINER, B. J. (197 1). Statistical Principals in Experimental Design. McGraw-Hill, New York. ZANNONI, V., LYNCH. M., GOLDSTEIN. S., AND SATO. P. (1974). A rapid micromethod for determination of ascorbic acid in plasma and tissues. Biochem. Med. 11,4 l-48.