function (Davis 1976; Walden and Howard 1981). Histopathological changes were studied in rainbow trout exposed to an untreated bleached kraft pulp milt ...
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Arch. Environ. Contam. Toxicol. 17,319-323 (1988)
9 1988 Springer-Verlag New York Inc.
Histopathology of Rainbow Trout Exposed to a Bleached Kraft Pulp Mill Effluent C. M. Couillard i, R. A. B e r m a n 2, and J. C. Panisset 3 Occupational and Environmental Health, University of Montreal, C.R 6128, Montreal, Quebec, Canada, H3C 3J7 Abstract. T h e toxicity of an u n t r e a t e d b l e a c h e d kraft pulp mill effluent was evaluated histopathologically, using rainbow trout. Gills, liver, kidney, spleen, digestive tract, and skin were examined in a 96-hr LC50 acute test. Fish e x p o s e d to lethal concentrations of effluent had an extensive fusion of gill lamellae. This lesion was also present in fish that s u r v i v e d the 96-hr LC50 acute test but to a lesser extent. Trout e x p o s e d for 20, 40, and 60 days to sublethal effluent concentrations were also examined. N o lesions that could be attributed specifically to the effluent were observed, but e x p o s e d fish had a higher incidence of fin necrosis and damaged gills than the u n e x p o s e d fish after 40 and 60 days. L o s s of resistance to bacterial pathogens m a y be a significant stress-related effect of this effluent in fish e x p o s e d for long periods to low concentrations.
et at. (1978) d e m o n s t r a t e d deterioration of the fine ridged structure on the s e c o n d a r y lameltae of gills in fish e x p o s e d to dehydroabietic acid, an important toxic c o m p o n e n t of pulp and p a p e r effluent. A variety of sublethal tests of fish exposed to pulp and p a p e r effluent have revealed respiratory dysfunction (Davis 1976; Walden and H o w a r d 1981). H i s t o p a t h o l o g i c a l c h a n g e s w e r e s t u d i e d in r a i n b o w t r o u t e x p o s e d to an u n t r e a t e d b l e a c h e d kraft pulp milt effluent. Tissues were examined in fish e x p o s e d to high effluent c o n c e n t r a t i o n s in a 96-hr LC50 test, and in fish e x p o s e d to sublethal effluent concentrations (0.10 and 0.25 x LC50) for 20, 40, and 60 days. The objective of this investigation was to identify the target tissues, if any, affected by the effluent at lethal and sublethal concentrations. Material and Methods
F e w studies h a v e e x a m i n e d the histopathological effects of pulp and p a p e r mill effluent in fish. Fujiya (1961) described extensive histological damage in fish, Sparus macrocephalus e x p o s e d in situ to w a t e r c o n t a m i n a t e d b y a J a p a n e s e kraft mill. Lesions were found in the liver, kidney, spleen, and intestine. N o histological d a m a g e has b e e n reported in fish e x p o s e d to N o r t h A m e r i c a n pulp and p a p e r mill effluent ( M c L e a y and B r o w n 1979). Monteith
Effluent Collection and Handling Tests were conducted on combined wastewater from all the bleaching stages and from the chemical plant of an eastern Canadian bleached kraft mill. The effluent was collected by batch sampling in 20-L tightly closed plastic containers. Samples were sent on the day of collection to the testing laboratory where they were stored at 4~ for up to 4 days before commencing bioassays (Walden et al. 1975).
Dilution Water 1 Department of Veterinary Pathology, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Canada, S7N 0W0. 2 Pulp and Paper Research Institute of Canada, 570 St John's Blvd, Pointe Claire, Quebec, Canada, HgR 3J9. 3 Occupational and Environmental Health, University of Montreal, C.R 6128, Montreal, Quebec, Canada, H3C 3J7.
Dechlorinated tapwater from the city of Longueuil, Quebec, (pH 7.8 -+ 0.1, hardness 173 ppm), which was filtered on three columns of activated charcoal and exposed to ultraviolet light, was used for effluent dilution and fish holding water. A temperature of 15~ and a 12-hr photoperiod were maintained during the tests and the dissolved oxygen was kept above 9 mg/L with aeration.
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C. M. Couillard et al.
Table 1. Gill lesions in rainbow trout exposed to a bleached kraft pulp mill effluent in a 96-hr LC50 test Fish exposed to % of death after 96 hr Number of fish examined Gill Lesions --Hyperplasia a
--Cellular Hypertrophyb - - C l u b b e d lamellaeb -
-
0% V/V 0 9
10% V/V 0 9
2.1 (-+0.4)
1.9 (-+0.8) ns d
9/27 10/27
13.5% V/V 70 5
18% V/V 100 3
1.8 (-+0.6) ns d
ND
11/27 ns e
ND
ND
11/27 ns e
ND
ND
6 7 7 4 24/27 s~ 3/9 ns f
0 1 2 12 15/15 s e ND
0 2 2 5 9/9 se ND
Lamellar fusionb:
+ ++ +++ ++++ Total --Telangiectasisc
0 0 0 0 0/27 4/9
V/V Volume/Volume a number of cellular layers, mean and standard deviation, measured on microphotographs b prevalence, measured on microphotographs c prevalence, measured by microscopic examination d Mann-Whitney, p ~< 0.05 Chi-squared, p ~< 0.05 f Fisher, p ~< 0.05 ns not statistically significant compared to control s statistically significant compared to control ND not determined
Lethal Test Toxicity tests were performed on juvenile rainbow trout, Salmo gairdneri (2.0 _+ 0.8 g), which were acclimated to laboratory conditions for two weeks prior to evaluation. A static 96-hr LC50 test was completed according to the method of Sprague (1969, 1973) as modified by Environment Canada (1980). Four groups of 10 trout each were exposed in 60-L polyethylene tanks to 4 different effluent dilutions, ranging from 7.5 to 24%. The pH of each dilution was adjusted to 7.0 -+ 0.1 with NaOH. Volume to fish ratios in the tanks were 1 L/g. The 96-hr LC50 was estimated from mortality observed at each concentration.
Sublethal Test In the sublethal test, fifteen groups of 10 trout each were randomly distributed in 11-L polystyrene tanks with plastic-netting covers. Three fish per tank were immediately removed and weighed. The remaining fish were kept in dilution water for 24-hr before the beginning of exposure. After 20 days and 40 days of exposure, 2 fish per tank were removed, weighed and fixed for histopathological examination. The same procedure was repeated after 60 days with all the surviving fish. Experimental tanks were divided in 3 groups of 5 tanks each. The control group (C) contained dilution water, group A contained an effluent concentration of 0.25 96-hr LC50 (4% Volume/Volume)
and group B contained 0.10 LC50 (about 1.6% Volume/Volume). The mean LC50 of the effluent was estimated in preliminary experiments from the 96-hr LC50s of four different samples of effluent (Couillard et al., unpublished). The test was conducted with a flow-through system. The effluent was delivered to each tank through a peristaltic pump (Carlo Erba with PVC Technicon tubes, 0.56 mm inner diameter). The effluent was pre-diluted to 50% (for group A) and 25% (for group B) and was constantly agitated in two 20-L polystyrene tanks placed before the pump. The effluent was mixed with dilution water, by air agitation, in a plastic beaker fixed in each tank, before overflowing into the tank. The dilution water reached the tanks by gravity, its flow being determined by the diameter and the length of the delivering tubes (Technicon 025116-0536-06). In each tank, a volume of 10-L of water was maintained by an overflow mechanism and the flow (water + effluent) was set at 20 ml/min. Flows were monitored daily and adjusted as required. Volume to fish ratios varied from 1.2 to 2.0 L/g/day during exposure. The lowest ratios were found after 40 days of exposure. Ammonium nitrogen concentrations which were measured in the tanks at this time were slightly lower than 0.5 mg/L. The pH, which was measured daily in each tank, varied from 7.2 to 8.4. Throughout the test period, fish were fed a dry pelleted commercial trout food (Ewos II | grower diet) twice daily. In each tank, fish received an individually weighed quantity of food, corresponding to 2% of their wet weight during the first 40 days and 1% thereafter. The bottom of each tank was cleaned with a manual vacuum cleaner before each meal.
321
Effects of Pulp Mill Effluent on Rainbow Trout Table 2. Mortality rate and fin rot occurrence in rainbow trout during the 60 days sublethal test
--Total number of fish --Number of dead fish --Percent mortality --Number of finrot cases --Day of observable onset of fin rot
Group C (0% V/V)~
Group A Group B (2% V/V) (4% V/V)
35 3 8 1
35 5nsb 17ns 4ns
35 6ns 14ns 8s~
53
40
20
v / v Volume/Volume b ns non significant, Fisher, p ~< 0.05 c s significant, Fisher, p ~< 0.05
Pathological Examination Fish were fixed in phosphate buffered formalin (Roberts 1978). Tissues were embedded in paraffin blocks and 5 ~ sections were prepared and stained with hematoxylin-eosin. Gill, liver, kidney, spleen, alimentary canal, and skin were examined in each fish. Lesions that were uniformly distributed on gills were quantified by microphotography, using a Zeiss Axiomat microscope with a 16X planapochromat objective. For each fish, 3 fields depicting gill lamellae were selected arbitrarily and photographed. The photographs were taken blindly, without knowledge of the treatment regime each specimen had been exposed to~ The number of photographs showing cellular hypertrophy and clubbed lamellae per fish (0-3) was used as an index of lesion prevalence and severity for comparison among treatment groups. The prevalence and severity of lamellar fusion was determined using the same technique and was also graded from 1+ (0 to 25% of the lamellae affected) to 4+ (75 to 100% of the lamellae affected) based on number of fused lameltae in each photograph. One measurement of the number of cellular layers at the base of the gill lamellae was made on each photograph. The mean and the standard error of the number of cellular layers for the total number of photographs was calculated for each group and was used as an index of cellular hyperplasia. The prevalence of telangiectasia (lamellar aneurism) and of lamellar fusion, in the sublethal test, was determined by microscopic examination and not by microphotography since the number of affected lamellae was small.
Results
Lethal Test T h e 96-hr L C 5 0 o f the e f f l u e n t was b e t w e e n 10 a n d 13.5% V o l u m e / V o l u m e . M o r t a l i t y v a r i e d a b r u p t l y f r o m o n e c o n c e n t r a t i o n to a n o t h e r . F u s i o n o f the gill l a m e l l a e was f o u n d o n l y in e x p o s e d fish. T h e e x t e n t o f the l e s i o n i n c r e a s e d with the e f f l u e n t c o n c e n t r a t i o n . I n fish e x p o s e d to the h i g h e s t c o n c e n t r a t i o n s , 13.5% a n d 18% V o l u m e / V o l u m e , m o s t of t h e gill l a m e l l a e w e r e f u s e d (4 + ). All t r o u t e x p o s e d to 10% V o l u m e / V o l u m e for 96 hr s u r v i v e d , b u t t h e i r gills w e r e d a m a g e d . C e l l u l a r h y p e r p l a s i a a n d by-
Table 3. Gill lesions in rainbow trout exposed for 20 days to a bleached kraft pulp mill effluent Group C (0% V/V) Number of fish examined Gill Lesions --Hyperplasiaa
--Cellular Hypertrophyb
10 2.5 (+0.5)
Group B (2% V,~v') 10
Group A (4% V/V) 10
2.4 2.7 (_+0.4)nsd (-+0.5)nsd
17/30
23/30 ns~
I7/30ns~
--Clubbed tamellaeb
8/30
9/30 ns~
13/30ns~
--Lameltar fusionc
2/10 nsr
4/10 nsf
5/10
--Telangiectasis~
5/10 nsf
5/10 nsf
5/10
v / v Volume/Volume number of cellular layers, mean and standard deviation, measured on microphotographs b prevalence, measured on microphotographs r prevalence, measured by microscopic examination d Mann-Whitney, p ~< 0.05 r Chi-squared, p ~< 0.05 f Fisher, p ~< 0.05 ns not statistically significant compared to control s statistically significant compared to control pertrophy, clubbed lamellae and telangiectasia were s e e n in gills o f b o t h c o n t r o l a n d e x p o s e d fish with e q u a l f r e q u e n c y (Table 1). T h e s e c o n d i t i o n s w e r e n o t d e t e r m i n e d in s p e c i m e n s f r o m the two higher e x p o s u r e g r o u p s , b e c a u s e the c e l l u l a r o u t l i n e s w e r e i n d i s t i n c t in s e v e r e l y f u s e d gill l a m e l l a e .
Sublethal Test M o r t a l i t y w a s o b s e r v e d i n all g r o u p s in t h e s u b lethal test (Table 2). M o s t o f the d e a d fish had skin l e s i o n s c o n s i s t e n t with a d i a g n o s i s o f fin rot: necrosis, fibrosis, epithelial hyperplasia, melanop h o r e a g g r e g a t i o n or i n f i l t r a t i o n with i n f l a m m a t o r y cells a n d the d i s i n t e g r a t i o n o f the fin rays. In m a n y i n s t a n c e s , b a c t e r i a l c o l o n i e s (long f i l a m e n t o u s rods a n d s h o r t rods) w e r e p r e s e n t in the l e s i o n s ( B u l l o c k et al. 1971). O n l y o n e case o f fin rot w a s identified in the c o n t r o l g r o u p a n d it a p p e a r e d t o w a r d the e n d o f the test (day 56). I n the e x p o s e d g r o u p , the dise a s e w a s o b s e r v e d s o o n e r (days 20 a n d 40) a n d in m o r e fish. E x c e p t for the fin rot c a s e s , the skin o f the fish was i n t a c t . N o h i s t o p a t h o l o g i c a l c h a n g e s t h a t c o u l d be att r i b u t e d s p e c i f i c a l l y to the e f f l u e n t were s e e n in the e x p o s e d fish. V a r i o u s d e g r e e s o f c e l l u l a r h y p e r p l a s i a a n d h y p e r t r o p h y , c l u b b e d Iameltae a n d telan-
322
C.M. Couillard et at.
Table 4. Gill lesions in rainbow trout exposed for 40 days to a bleached kraft pulp mill effluent Group C (0% V/V) Number of fish examined
Gill Lesions - - Hyperplasiaa
10
10
Group A (4% V/V) 10
Group C (0% V/V) Number of fish examined
Group B (2% V/V)
11
Gill Lesions --Hyperplasiaa
9
Group A (4% V/V) 10
2.9 (-+0.8) s~
2.8 (-+0.6) sd
2/30
17/30 s~
12/30 s~
--Cellular Hypertrophyb
18/33
12/27 nse
14/30ns~
27/30
17/30 ns ~
21/30ns c
--Clubbed lamellaeb
19/33
16/27 ns ~
18/30ns~
--Lamellar fusionc
2/10
5/10 nsf
3/10 ns f
--Lamellar fusionc
3/11
4/9 ns f
3/10 ns f
--Telangiectasis~
2/10
2/t0 nse
4.10 nsf
--Telangiectasisc
4/11
3/9 nsf
3/10 ns f
--Cellular Hypertrophyb --Clubbed lamellaeb
2.1 (--_0.6)
Group B (2% V/V)
Table 5. Gill lesions in rainbow trout exposed for 60 days to a bleached kraft pulp mill effluent
V/V Volume/Volume a number of cellular layers, mean and standard deviation, measured on microphotographs b prevalence, measured on microphotographs prevalence, measured by microscopic examination d Mann-Whitney, p ~< 0.05 Chi-squared, p ~< 0.05 f Fisher, p ~< 0.05 ns not statistically significant compared to control s statistically significant compared to control
g i e c t a s i a w e r e f o u n d in gills o f c o n t r o l a n d e x p o s e d f i s h ( T a b l e 3). L a m e l l a r f u s i o n w a s o b s e r v e d b u t o n l y 3 o r 4 l a m e l l a e p e r gill f i l a m e n t w e r e a f f e c t e d . A f t e r 40 d a y s o f e x p o s u r e , t h e r e w a s m o r e e v i d e n c e o f c e l l u l a r h y p e r t r o p h y a n d h y p e r p l a s i a in e x p o s e d f i s h t h a n in c o n t r o l s (Table 4). A f t e r 60 d a y s , gills o f e x p o s e d fish e x h i b i t e d a s i m i l a r s p e c t r u m o f l e s i o n s a s gills f r o m t h e u n e x p o s e d fish e x c e p t for c e l l u l a r h y p e r p l a s i a w h i c h w a s m o r e s e v e r e in t h e g r o u p e x p o s e d to 2% V / V (Table 5). Liver, spleen, kidney, stomach, intestine and pancreas from fish of each treatment group contained no histological lesions. The mean body w e i g h t s w e r e n o t a f f e c t e d b y 20, 40, o r 60 d a y s o f e f f l u e n t ( F i g u r e 1).
2.0 (-+0.8)
2.5 (-+0.8) sd
V/V Volume/Volume a number of cellular layers, mean and standard deviation, measured on microphotographs b prevalence, measured on microphotographs c prevalence, measured by microscopic examination d Mann-Whitney, p ~< 0.05 Chi-squared, p
