Bioaccumulation in Fish of Chlorinated Phenols from Kraft Pulp Mill Bleachery Effluents L. Landner', K. Lindstr6m 2, M. Karlsson ~, J. Nordin ~ and L. S6rensen' 'Swedish Water and Air Pollution Research Laboratory (IVL) Box 21060, S-100 31 Stockholm, Sweden ~Swedish Forest Products Research Laboratory Box 5604, S-114 86 Stockholm, Sweden
During the last ten years the gross water p o l l u t i o n caused by oxygen consuming organic substances in the effluents from the pulp and paper industry has been considerably reduced. The greatest r e m a i n i n g pollution problem in the pulp industry is c o n n e c t e d with the b l e a c h i n g process. In this process the pulp is treated first w i t h chlorine or a mixture of chlorine and chlorine dioxide in several steps and thereafter with strong alkali in order to extract the colored b r e a k d o w n products of lignin from the pulp. The effluents from this m u l t i - s t e p bleaching procedure partly possess a strong ~ r o w n i s h colour and generally constitute about half of the total effluent volume from a kraft pulp mill. This means that only in Sweden the kraft pulp bleacheries discharge about 300 m i l l i o n m 3 of waste water per annum. To get a detailed d e s c r i p t i o n of the chemical composition of the very complex mixture of d i f f e r e n t organic compounds discharged from the cellulose pulp mills is a formidable task, still to a great extent refractory to fulfilment. An interesting approach to this p r o b l e m has recently been used by the Canadian workers LEACH and THAKORE (1973, 1975), who c o n c e n t r a t e d their identification work on those compounds that could be shown to cause clear-cut biological effects on aquatic organisms. In their studies on effluents from the first extraction step in kraft pulp bleacheries, LEACH and THAKORE (1975) were able to show that almost the total acute toxic effect to rainbow trout was exerted by a limited number of resin acid derivatives and c h l o r i n a t e d lignin breakdown products. The compounds responsible for 90 percent of the acute toxic action to fish were mono- and d i c h l o r o d e h y d r o a b i e t i c acid, tri- and t e t r a c h l o r o g u a i a c o l and epoxystearic acid. This result relates of course only
663 Bulletin o| Environmental Contamination & Toxicology, VoL 18, No. 6 9 1977 by Springer-Verlag New York, Inc.
to that p a r t i c u l a r raw m a t e r i a l u s e d by the m i l l s in w e s t e r n C a n a d a t h a t w e r e i n c l u d e d in the i n v e s t i g a t i o n and c a n n o t be d i r e c t l y g e n e r a l i z e d to E u r o p e a n c o n d i tions. No s t u d i e s h a v e so far - to our k n o w l e d g e - b e e n r e p o r t e d on the a c c u m u l a t i o n or long t e r m e f f e c t s of t h e s e c o m p o u n d s on fish or o t h e r a q u a t i c animals. The first c h a r a c t e r i z a t i o n of the a c i d - p h e n o l i c f r a c t i o n of E u r o p e a n , p i n e - w o o d p u l p b l e a c h e r y e f f l u e n t s , w i t h r e s p e c t to l o w - m o l e c u l a r c h l o r f n a t e d c o m p o u n d s was c a r r i e d out by L I N D S T R O M and N O R D I N (1976). They i d e n t i fied t r i c h l o r o p h e n o l , i s o m e r i c t r i c h l o r o c a t e c h o l s and t e t r a c h l o r o c a t e c h o l in the e f f l u e n t s f r o m the first c h l o r i n a tion step and t r i c h l o r o p h e n o l , i s o m e r i c t r i c h l o r o g u a i a c o l s and t e t r a c h l o r o g u a i a c o l in the e f f l u e n t s from the f i r s t a l k a l i n e e x t r a c t i o n step. H o w e v e r , the n e u t r a l f r a c t i o n of these e f f l u e n t s still r e m a i n s to be c h a r a c t e r i z e d for low-molecular chlorinated compounds. The p r e s e n t w o r k is an a t t e m p t to find out if the l o w - m o l e c u l a r c h l o r i n a t e d c o m p o u n d s , i d e n t i f i e d to be p r e s e n t in low c o n c e n t r a t i o n in the e f f l u e n t s f r o m S w e d i s h k r a f t p u l p b l e a c h e r i e s and some of w h i c h h a v e b e e n shown to be a c u t e - t o x i c to fish, f u r t h e r m o r e show t e n d e n c i e s to a c c u m u l a t e in fish. If such a b i o a c c u m u l a tion is d e m o n s t r a t e d to occur, this w o u l d be a s t r o n g r a t i o n a l e for f u r t h e r s t u d i e s on p o s s i b l e long t e r m or c h r o n i c e f f e c t s of this g r o u p of c o m p o u n d s .
MATERIAL AND METHODS Accumulation
experiments
R a i n b o w t r o u t y e a r l i n g s (Salmo ~ a i r d i n e r i ) w e r e e x p o s e d to e f f l u e n t s f r o m the c h l o r i n a t i o n (C-step) and e x t r a c tion steps (E-step), r e s p e c t i v e l y , in a d i l u t i o n of a b o u t 40 times in B a l t i c Sea w a t e r (salinity 7 ppt) in flowt h r o u g h w h o l e g l a s s aquaria. F r e s h e f f l u e n t s a m p l e s w e r e t a k e n e v e r y w e e k at the mills, p r o d u c i n g full b l e a c h p i n e k r a f t pulp. The s a m p l e s w e r e s t o r e d in c o m p l e t e l y f i l l e d and c l o s e d p o l y e t e n c o n t a i n e r s at 3~ b e f o r e use. Two d i f f e r e n t E - s t e p e f f l u e n t s w e r e tested; one after a C - s t e p u s i n g i00 per c e n t c h l o r i n e and the other a f t e r a C - s t e p w h e r e 5 per c e n t of the c h l o r i n e was r e p l a c e d by c h l o r i n e d i o x i d e (CD-step). In b o t h cases, the c h l o r i n e a d d i t i o n in the C - s t e p was 70-90 k g / t o n p u l p and the a d d i t i o n of caustic in the E - s t e p a b o u t 60 k g / t o n pulp.
664
The e f f l u e n t v o l u m e s were: m 3 / t o n p u l p and from the E - s t e p
from the C - s t e p ~40 ~20 m 3 / t o n pulp.
All e x p o s u r e s w e r e c a r r i e d out at a t e m p e r a t u r e of 8-10~ The pH of the s o l u t i o n s was 7 ~ 0.5. W a t e r flow was a d j u s t e d to about i0 i/h and the fish d e n s i t y was about i0 g/l. In one of the e x p e r i m e n t s , e x p o s u r e was d i s c o n t i n u e d after 7 w e e k s and the fish w e r e a l l o w e d to e x c r e t e the a c c u m u l a t e d c o m p o u n d s d u r i n g 8 f u r t h e r w e e k s in n o n p o l l u t e d sea water. Liver s a m p l e s w e r e c o m p o s i t e samples from 4 to 6 i n d i v i d u a l s , w h e r e a s m u s c l e s a m p l e s were from i n d i v i d u a l fish.
Anal~tical Sample
preparation
P r e p a r a t i o n of fish t i s s u e s and i s o l a t i o n of the c h l o r o p h e n o l s in a b e n z e n e e x t r a c t was b a s e d on the procedure by R E N B E R G (1974). The b e n z e n e e x t r a c t was then e v a p o r a t e d to about 0.5 ml, d e r i v a t i z e d w i t h d i a z o e t h a n e L I N D S T R O M and N O R D I N (1976) and f i n a l l y p u r i f i e d on a c o l u m n (2 cm x 0.5 cm i.d.) p a c k e d w i t h s i l i c i c a c i d (Mallinchrodt, i00 m e s h a n a l y t i c a l grade) w h i c h was prew a s h e d w i t h benzene. The e l u a t e (about 0.5 ml) was taken to gas c h r o m a t o g r a p h i c analysis.
Gas c h r o m a t o g r a p h y A P e r k i n - E l m e r gas c h r o m a t o g r a p h m o d e l 900 e q u i p p e d w i t h an e l e c t r o n c a p t u r e d e t e c t o r (ECD) was used. The c h r o m a t o g r a p h was r e c o n s t r u c t e d to suit glass o p e n - t u b u lar c o l u m n s w i t h a G r o b type i n j e c t o r and a p u r g e gas system. The c o l u m n used was a SE-30 glass o p e n - t u b u l a r c o l u m n (25 m x 0.25 mm i.d.) k i n d l y given to us by J o h a n R o r a a d e at the S w e d i s h T o b a c c o Company. The c o l u m n temp e r a t u r e was 170~ i n j e c t o r t e m p e r a t u r e 250~ and m a n i fold and ECD t e m p e r a t u r e s 200~ t h r o u g h o u t the e x p e r i m e n t s . C a r r i e r gas was a r g o n - m e t h a n e 95:5 w i t h a c o l u m n f l o w of 0.35 m l / m i n u t e . Split ratio was 1:20. The p u r g e gas f l o w was 90 m l / m i n u t e .
665
GC-MID A F i n n i g a n gas c h r o m a t o g r a p h (9500)- mass spectrom e t e r (3200 F) w i t h an o n - l i n e c o m p u t e r (6000) was used. To o b t a i n i n t e r p r e t a b l e mass spectra, the small sample size is a limitation. However, the s e n s i t i v i t y can be i n c r e a s e d by m o n i t o r i n g on specific ions (multiple ion d e t e c t i o n , MID). Gas c h r o m a t o g r a p h i c d a t a w e r e as d e s c r i b e d above e x c e p t for c a r r i e r gas flow (0.60 m l / m i n u t e , He). The p l a t i n u m c a p i l l a r y i n t e r f a c e p e r a t u r e s w e r e 250~ respectively.
and ion source
tem-
The i n s t r u m e n t was c a l i b r a t e d b e f o r e each run to 0.1 a.m.u. D e t e c t i o n of ions was p e r f o r m e d at m/e 195.9, 197.9, 251.9 and 253.9 in the first cluster, 210.9, 212.9, 225.9 and 227.9 in the second, 244.9, 246.9, 259.9 and 261.9 in the third. M a x i m u m run time for each c l u s t e r was 6, 3.5 and 2.5 minutes, r e s p e c t i v e l y . So that the total run was 12 m i n u t e s . The first c l u s t e r h a d ions of e t h y l a t e d 2 . 4 . 6 - t r i c h l o r o p h e n o l and 2.6-dib r o m o p h e n o l , the second 4 . 5 . 6 - t r i c h l o r o g u a i a c o l and the third t e t r a c h l o r o g u a i a c o l . Each ion was m o n i t o r e d for 64 m s e c every 200 msec. Ion source
controls
were
set as follows:
E m i s s i o n current: 0.5 mA E l e c t r o n energy: 70 eV Ion energy: ~i0 V E l e c t r o n m u l t i p l i e r : 2.5 kV -8 P r e a m p l i f i e r s e n s i t i v i t y : 10 Amps/V
Reference
compounds
2.4.6=trichlorophenol (Fluka AG, pract, grade). 4.5.6-trichloroguaiacol (m.p. 112.6 ~ - i13.3~ and tetrachloroguaiacol (m.p. 1 2 2 . 3 ~ - 122.6~ were synthetized by Lars S t r 6 m b e r g et al. at the S w e d i s h F o r e s t P r o d u c t s R e s e a r c h L a b o r a t o r y and k i n d l y given to us.
Determination I n t e r n a l s t a n d a r d (i0 ~I of 260 ~g 2 . 6 - d i b r o m o p h e n o l per ml ethanol) was added to the b e n z e n e e x t r a c t b e f o r e the ion e x c h a n g e p r o c e d u r e R E N B E R G (1974). Q u a n t i t a t i v e a n a l y s i s was p e r f o r m e d on the gas c h r o m a t o g r a p h (ECD) as p r e v i o u s l y d e s c r i b e d L I N D S T R O M and N O R D I N (1976). All samples w e r e also q u a n t i f i e d by m e a n s of MID as a d o u b l e check. The v a r i o u s p r o c e d u r e s a g r e e d f a i r l y well.
666
The p r e c i s i o n of the GC analysis on replicate injections was around ~ 1.5 % (calculated on 4.5.6-trichloroguaiacol). The overall accuracy had a c o e f f i c i e n t of v a r i a t i o n of about 12 % based on a triplicate analysis of various fat solutions (25 mg fat/ml benzene) spiked w i t h the reference substances (about 1 ~g/ml). All peaks quantified were w i t h i n the linearity of the detector.
RESULTS AND DISCUSSION As can be seen from table i, a several week long exposure of rainbow trout for caustic extraction effluents diluted in Baltic Sea water to 2.5 % resulted in a significant uptake of at least three d i f f e r e n t chlorinated phenols into the fish body. Both the E-step effluent after a pure C-step and that after a C~-step supported U the uptake of the three chlorinated phenols. However, the first m e n t i o n e d E-step effluent resulted in the highest equilibrium concentrations in the fish liver. A somewhat lower uptake of chlorinated phenols followed the exposure to c h l o r i n a t i o n effluents of similar concentration. In particular, t e t r a c h l o r o g u a i a c o l was taken up only to a small extent after exposure to C - s t e p effluents. This difference might be due to the fact that the C-step effluents contain only low c o n c e n t r a t i o n s of c h l o r i n a t e d guaiacols, but instead relatively high amounts of the corresponding c a t e c h o l s (LINDSTR~M and NORDIN, 1976). These latter compounds have a more polar c h a r a c t e r than the guaiacols and may be m o r e readily cleared from the fish body. In table 1 it is also indicated that the uptake in the liver was more efficient than that in the muscle. This was p a r t i c u l a r l y evident for trichloroguaiacol. Figure 1 summarizes some p r e l i m i n a r y data showing the rate of uptake of chlorinated phenols into fish liver during exposure to bleachery effluents. It also gives a p r e l i m i n a r y idea of the rate of clearance after discontinuation of exposure. A steady state, where uptake is balanced by excretion and/or metabolism, seems to be achieved already after two weeks, at least for trichloroguaiacol. The here obtained rate of clearance for 2 . 4 . 6 - t r i c h l o r o phenol and t r i c h l o r o g u a i a c o l gives a tentative b i o l o g i c a l half life in the liver of less than i0 days. For tetrachloroguaiacol the picture is still somewhat obscure.
667
c%
1
-
6 6 ii ii ii ii Ii 60 70
63 62 59 38 86 50 34
130
120 130
150 150
on
Liver Muscle
Liver Liver Liver Liver Muscle Muscle Muscle
Liver
Liver Liver
Liver Liver
Fish Tissue mean weight (g)
ECD Response factor (calculated w e i g h t of p u r e phenol)
Control "
E-step after C D - s t e p " " " " "
-
2 5
E-step "
Control
2 5
Exposure time (weeks)
C-step "
Effluent
2.8 2.5
2.5 2.9 2.8 3.4 1.5 2.6 1.6
2.3
2.9 2.5
2.6 2.3
Fat content (%)
0.64
0.75
During the last ten years the gross water p o l l u t i o n caused by oxygen consuming organic substances in the effluents from the pulp and paper industry has been considerably reduced. The greatest r e m a i n i n g pollution problem in the pulp industry is c o n n e c t e d with the b l e a c h i n g process. In this process the pulp is treated first w i t h chlorine or a mixture of chlorine and chlorine dioxide in several steps and thereafter with strong alkali in order to extract the colored b r e a k d o w n products of lignin from the pulp. The effluents from this m u l t i - s t e p bleaching procedure partly possess a strong ~ r o w n i s h colour and generally constitute about half of the total effluent volume from a kraft pulp mill. This means that only in Sweden the kraft pulp bleacheries discharge about 300 m i l l i o n m 3 of waste water per annum. To get a detailed d e s c r i p t i o n of the chemical composition of the very complex mixture of d i f f e r e n t organic compounds discharged from the cellulose pulp mills is a formidable task, still to a great extent refractory to fulfilment. An interesting approach to this p r o b l e m has recently been used by the Canadian workers LEACH and THAKORE (1973, 1975), who c o n c e n t r a t e d their identification work on those compounds that could be shown to cause clear-cut biological effects on aquatic organisms. In their studies on effluents from the first extraction step in kraft pulp bleacheries, LEACH and THAKORE (1975) were able to show that almost the total acute toxic effect to rainbow trout was exerted by a limited number of resin acid derivatives and c h l o r i n a t e d lignin breakdown products. The compounds responsible for 90 percent of the acute toxic action to fish were mono- and d i c h l o r o d e h y d r o a b i e t i c acid, tri- and t e t r a c h l o r o g u a i a c o l and epoxystearic acid. This result relates of course only
663 Bulletin o| Environmental Contamination & Toxicology, VoL 18, No. 6 9 1977 by Springer-Verlag New York, Inc.
to that p a r t i c u l a r raw m a t e r i a l u s e d by the m i l l s in w e s t e r n C a n a d a t h a t w e r e i n c l u d e d in the i n v e s t i g a t i o n and c a n n o t be d i r e c t l y g e n e r a l i z e d to E u r o p e a n c o n d i tions. No s t u d i e s h a v e so far - to our k n o w l e d g e - b e e n r e p o r t e d on the a c c u m u l a t i o n or long t e r m e f f e c t s of t h e s e c o m p o u n d s on fish or o t h e r a q u a t i c animals. The first c h a r a c t e r i z a t i o n of the a c i d - p h e n o l i c f r a c t i o n of E u r o p e a n , p i n e - w o o d p u l p b l e a c h e r y e f f l u e n t s , w i t h r e s p e c t to l o w - m o l e c u l a r c h l o r f n a t e d c o m p o u n d s was c a r r i e d out by L I N D S T R O M and N O R D I N (1976). They i d e n t i fied t r i c h l o r o p h e n o l , i s o m e r i c t r i c h l o r o c a t e c h o l s and t e t r a c h l o r o c a t e c h o l in the e f f l u e n t s f r o m the first c h l o r i n a tion step and t r i c h l o r o p h e n o l , i s o m e r i c t r i c h l o r o g u a i a c o l s and t e t r a c h l o r o g u a i a c o l in the e f f l u e n t s from the f i r s t a l k a l i n e e x t r a c t i o n step. H o w e v e r , the n e u t r a l f r a c t i o n of these e f f l u e n t s still r e m a i n s to be c h a r a c t e r i z e d for low-molecular chlorinated compounds. The p r e s e n t w o r k is an a t t e m p t to find out if the l o w - m o l e c u l a r c h l o r i n a t e d c o m p o u n d s , i d e n t i f i e d to be p r e s e n t in low c o n c e n t r a t i o n in the e f f l u e n t s f r o m S w e d i s h k r a f t p u l p b l e a c h e r i e s and some of w h i c h h a v e b e e n shown to be a c u t e - t o x i c to fish, f u r t h e r m o r e show t e n d e n c i e s to a c c u m u l a t e in fish. If such a b i o a c c u m u l a tion is d e m o n s t r a t e d to occur, this w o u l d be a s t r o n g r a t i o n a l e for f u r t h e r s t u d i e s on p o s s i b l e long t e r m or c h r o n i c e f f e c t s of this g r o u p of c o m p o u n d s .
MATERIAL AND METHODS Accumulation
experiments
R a i n b o w t r o u t y e a r l i n g s (Salmo ~ a i r d i n e r i ) w e r e e x p o s e d to e f f l u e n t s f r o m the c h l o r i n a t i o n (C-step) and e x t r a c tion steps (E-step), r e s p e c t i v e l y , in a d i l u t i o n of a b o u t 40 times in B a l t i c Sea w a t e r (salinity 7 ppt) in flowt h r o u g h w h o l e g l a s s aquaria. F r e s h e f f l u e n t s a m p l e s w e r e t a k e n e v e r y w e e k at the mills, p r o d u c i n g full b l e a c h p i n e k r a f t pulp. The s a m p l e s w e r e s t o r e d in c o m p l e t e l y f i l l e d and c l o s e d p o l y e t e n c o n t a i n e r s at 3~ b e f o r e use. Two d i f f e r e n t E - s t e p e f f l u e n t s w e r e tested; one after a C - s t e p u s i n g i00 per c e n t c h l o r i n e and the other a f t e r a C - s t e p w h e r e 5 per c e n t of the c h l o r i n e was r e p l a c e d by c h l o r i n e d i o x i d e (CD-step). In b o t h cases, the c h l o r i n e a d d i t i o n in the C - s t e p was 70-90 k g / t o n p u l p and the a d d i t i o n of caustic in the E - s t e p a b o u t 60 k g / t o n pulp.
664
The e f f l u e n t v o l u m e s were: m 3 / t o n p u l p and from the E - s t e p
from the C - s t e p ~40 ~20 m 3 / t o n pulp.
All e x p o s u r e s w e r e c a r r i e d out at a t e m p e r a t u r e of 8-10~ The pH of the s o l u t i o n s was 7 ~ 0.5. W a t e r flow was a d j u s t e d to about i0 i/h and the fish d e n s i t y was about i0 g/l. In one of the e x p e r i m e n t s , e x p o s u r e was d i s c o n t i n u e d after 7 w e e k s and the fish w e r e a l l o w e d to e x c r e t e the a c c u m u l a t e d c o m p o u n d s d u r i n g 8 f u r t h e r w e e k s in n o n p o l l u t e d sea water. Liver s a m p l e s w e r e c o m p o s i t e samples from 4 to 6 i n d i v i d u a l s , w h e r e a s m u s c l e s a m p l e s were from i n d i v i d u a l fish.
Anal~tical Sample
preparation
P r e p a r a t i o n of fish t i s s u e s and i s o l a t i o n of the c h l o r o p h e n o l s in a b e n z e n e e x t r a c t was b a s e d on the procedure by R E N B E R G (1974). The b e n z e n e e x t r a c t was then e v a p o r a t e d to about 0.5 ml, d e r i v a t i z e d w i t h d i a z o e t h a n e L I N D S T R O M and N O R D I N (1976) and f i n a l l y p u r i f i e d on a c o l u m n (2 cm x 0.5 cm i.d.) p a c k e d w i t h s i l i c i c a c i d (Mallinchrodt, i00 m e s h a n a l y t i c a l grade) w h i c h was prew a s h e d w i t h benzene. The e l u a t e (about 0.5 ml) was taken to gas c h r o m a t o g r a p h i c analysis.
Gas c h r o m a t o g r a p h y A P e r k i n - E l m e r gas c h r o m a t o g r a p h m o d e l 900 e q u i p p e d w i t h an e l e c t r o n c a p t u r e d e t e c t o r (ECD) was used. The c h r o m a t o g r a p h was r e c o n s t r u c t e d to suit glass o p e n - t u b u lar c o l u m n s w i t h a G r o b type i n j e c t o r and a p u r g e gas system. The c o l u m n used was a SE-30 glass o p e n - t u b u l a r c o l u m n (25 m x 0.25 mm i.d.) k i n d l y given to us by J o h a n R o r a a d e at the S w e d i s h T o b a c c o Company. The c o l u m n temp e r a t u r e was 170~ i n j e c t o r t e m p e r a t u r e 250~ and m a n i fold and ECD t e m p e r a t u r e s 200~ t h r o u g h o u t the e x p e r i m e n t s . C a r r i e r gas was a r g o n - m e t h a n e 95:5 w i t h a c o l u m n f l o w of 0.35 m l / m i n u t e . Split ratio was 1:20. The p u r g e gas f l o w was 90 m l / m i n u t e .
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GC-MID A F i n n i g a n gas c h r o m a t o g r a p h (9500)- mass spectrom e t e r (3200 F) w i t h an o n - l i n e c o m p u t e r (6000) was used. To o b t a i n i n t e r p r e t a b l e mass spectra, the small sample size is a limitation. However, the s e n s i t i v i t y can be i n c r e a s e d by m o n i t o r i n g on specific ions (multiple ion d e t e c t i o n , MID). Gas c h r o m a t o g r a p h i c d a t a w e r e as d e s c r i b e d above e x c e p t for c a r r i e r gas flow (0.60 m l / m i n u t e , He). The p l a t i n u m c a p i l l a r y i n t e r f a c e p e r a t u r e s w e r e 250~ respectively.
and ion source
tem-
The i n s t r u m e n t was c a l i b r a t e d b e f o r e each run to 0.1 a.m.u. D e t e c t i o n of ions was p e r f o r m e d at m/e 195.9, 197.9, 251.9 and 253.9 in the first cluster, 210.9, 212.9, 225.9 and 227.9 in the second, 244.9, 246.9, 259.9 and 261.9 in the third. M a x i m u m run time for each c l u s t e r was 6, 3.5 and 2.5 minutes, r e s p e c t i v e l y . So that the total run was 12 m i n u t e s . The first c l u s t e r h a d ions of e t h y l a t e d 2 . 4 . 6 - t r i c h l o r o p h e n o l and 2.6-dib r o m o p h e n o l , the second 4 . 5 . 6 - t r i c h l o r o g u a i a c o l and the third t e t r a c h l o r o g u a i a c o l . Each ion was m o n i t o r e d for 64 m s e c every 200 msec. Ion source
controls
were
set as follows:
E m i s s i o n current: 0.5 mA E l e c t r o n energy: 70 eV Ion energy: ~i0 V E l e c t r o n m u l t i p l i e r : 2.5 kV -8 P r e a m p l i f i e r s e n s i t i v i t y : 10 Amps/V
Reference
compounds
2.4.6=trichlorophenol (Fluka AG, pract, grade). 4.5.6-trichloroguaiacol (m.p. 112.6 ~ - i13.3~ and tetrachloroguaiacol (m.p. 1 2 2 . 3 ~ - 122.6~ were synthetized by Lars S t r 6 m b e r g et al. at the S w e d i s h F o r e s t P r o d u c t s R e s e a r c h L a b o r a t o r y and k i n d l y given to us.
Determination I n t e r n a l s t a n d a r d (i0 ~I of 260 ~g 2 . 6 - d i b r o m o p h e n o l per ml ethanol) was added to the b e n z e n e e x t r a c t b e f o r e the ion e x c h a n g e p r o c e d u r e R E N B E R G (1974). Q u a n t i t a t i v e a n a l y s i s was p e r f o r m e d on the gas c h r o m a t o g r a p h (ECD) as p r e v i o u s l y d e s c r i b e d L I N D S T R O M and N O R D I N (1976). All samples w e r e also q u a n t i f i e d by m e a n s of MID as a d o u b l e check. The v a r i o u s p r o c e d u r e s a g r e e d f a i r l y well.
666
The p r e c i s i o n of the GC analysis on replicate injections was around ~ 1.5 % (calculated on 4.5.6-trichloroguaiacol). The overall accuracy had a c o e f f i c i e n t of v a r i a t i o n of about 12 % based on a triplicate analysis of various fat solutions (25 mg fat/ml benzene) spiked w i t h the reference substances (about 1 ~g/ml). All peaks quantified were w i t h i n the linearity of the detector.
RESULTS AND DISCUSSION As can be seen from table i, a several week long exposure of rainbow trout for caustic extraction effluents diluted in Baltic Sea water to 2.5 % resulted in a significant uptake of at least three d i f f e r e n t chlorinated phenols into the fish body. Both the E-step effluent after a pure C-step and that after a C~-step supported U the uptake of the three chlorinated phenols. However, the first m e n t i o n e d E-step effluent resulted in the highest equilibrium concentrations in the fish liver. A somewhat lower uptake of chlorinated phenols followed the exposure to c h l o r i n a t i o n effluents of similar concentration. In particular, t e t r a c h l o r o g u a i a c o l was taken up only to a small extent after exposure to C - s t e p effluents. This difference might be due to the fact that the C-step effluents contain only low c o n c e n t r a t i o n s of c h l o r i n a t e d guaiacols, but instead relatively high amounts of the corresponding c a t e c h o l s (LINDSTR~M and NORDIN, 1976). These latter compounds have a more polar c h a r a c t e r than the guaiacols and may be m o r e readily cleared from the fish body. In table 1 it is also indicated that the uptake in the liver was more efficient than that in the muscle. This was p a r t i c u l a r l y evident for trichloroguaiacol. Figure 1 summarizes some p r e l i m i n a r y data showing the rate of uptake of chlorinated phenols into fish liver during exposure to bleachery effluents. It also gives a p r e l i m i n a r y idea of the rate of clearance after discontinuation of exposure. A steady state, where uptake is balanced by excretion and/or metabolism, seems to be achieved already after two weeks, at least for trichloroguaiacol. The here obtained rate of clearance for 2 . 4 . 6 - t r i c h l o r o phenol and t r i c h l o r o g u a i a c o l gives a tentative b i o l o g i c a l half life in the liver of less than i0 days. For tetrachloroguaiacol the picture is still somewhat obscure.
667
c%
1
-
6 6 ii ii ii ii Ii 60 70
63 62 59 38 86 50 34
130
120 130
150 150
on
Liver Muscle
Liver Liver Liver Liver Muscle Muscle Muscle
Liver
Liver Liver
Liver Liver
Fish Tissue mean weight (g)
ECD Response factor (calculated w e i g h t of p u r e phenol)
Control "
E-step after C D - s t e p " " " " "
-
2 5
E-step "
Control
2 5
Exposure time (weeks)
C-step "
Effluent
2.8 2.5
2.5 2.9 2.8 3.4 1.5 2.6 1.6
2.3
2.9 2.5
2.6 2.3
Fat content (%)
0.64
0.75
