platessa) caught by gill netting at different sites in the Hvaler. Archipelago. Indices of biochemical effects in liver S9- fractions were studied by measuring ...
Arch. Environ. Contam. Toxicol. 21,486--496 (1991)
Archives
of
E novni rt oa mn mi neantti aoln c Tox c o 9 1991 Springer-Verlag New York Inc.
Environmental Contaminants and Biochemical Responses in Flatfish from the Hvaler Archipelago in Norway Anders G o k s C y r *], Astrid-Mette HusCy*, Hfivard E. Larsen*, Jarle KlungsCyr**, Svein Wilhelmsen**, A m u n d Maage***, Einar M. Brevik #, T o m m y A n d e r s s o n e#, Malin Celander e#, Maija P e s o n e n e e , and L a r s F6rlin # e *Department of Biochemistry, University of Bergen, Arstadveien 19, N-5009 Bergen, Norway, **Institute of Marine Research, N-5024 Bergen, Norway, ***Institute of Nutrition, Directorate of Fisheries, N-5024 Bergen, Norway, ~Norwegian Institute for Air Research, N-2000 LillestrCm, Norway, and ##Department of Zoophysiology, University of G6teborg, S-400 31 G6teborg, Sweden Abstract. The levels of several environmental contaminants, including selected polyaromatic hydrocarbons (PAH), organochlorines (DDT/DDE, hexachlorobenzene), 15 polychlorinated biphenyl (PCB) congeners, and polychlorinated dibenzofnrans and dibenzo-p-dioxins, PCDF/PCDD), and heavy metals (Cd, Hg, Pb, and As) were analyzed in muscle and liver of three different flatfish species (dab, Limanda limanda; flounder, Platichthys flesus; plaice, PIeuronectes platessa) caught by gill netting at different sites in the Hvaler Archipelago. Indices of biochemical effects in liver S9fractions were studied by measuring cytochrome P450dependent monooxygenase and UDP-glucuronyl transferase activities, and by immunoquantitating cytochrome P450 1A 1 using an indirect enzyme-linked immunosorbent assay (ELISA). Only low levels of PCDD/PCDF, Cd, and Pb were observed, whereas PCB levels were significantly elevated in fish from the inner sites of the Archipelago compared to a reference site. The contaminant gradient toward the Glomma estuary was correlated with increased cytochrome P450 1A1 activity, measured as 7-ethoxyresorufin Odeethylase (EROD), and with immunoquantitated P450 1A 1. In contrast, fish from the site at Idefjorden, although containing elevated contaminant levels, did not show elevated EROD activity, but apparently elevated P450 1A1 protein. These findings may reflect different pollution histories of the sites, and indicate the applicability of biochemical effect indices (i.e., EROD and P450 IAI immunoquantitation) to monitoring studies. The integrated chemical-biochemical approach employed in this study can obviously be expanded to give fruitful information about cause-effect relationships in other contaminant situations.
1 To whom correspondence should be addressed.
The increasing pollution of the marine environment is a threat to the health of organisms inhabiting the seas (Murchelano 1990), as well as to human beings as predators or consumers of such organisms (Dawe 1990). In highly polluted areas, fish populations have high incidences of liver lesions and neoplasms (Malins et al. 1988; Murchelano and Wolke 1985). These incidences are often associated with elevated levels of toxic contaminants in the sediments, such as aromatic hydrocarbons, chlorinated hydrocarbons, and metals. At lower levels of pollution, when gross pathologies are not apparent, biochemical and physiological disturbances may be present in fish inhabiting such waters. Studies of such biological effects of contaminants at lower levels of organization can provide rapid and sensitive indications of stress, and have been recommended in monitoring strategies of marine pollution (Stebbing 1989). The biochemical responses of the cytochrome P450 system to organic toxicants, and of metallothioneins to heavy metals, are regarded as especially promising candidates (Haux and Frrlin 1988). For such studies, simplified immunochemical methods have been developed (GoksCyr 1991; GoksCyr et al. 1991a; Hogstrand et al. 1989). The Hvaler Archipelago is situated close to the Swedish border in southeastern Norway, and is the recipient of water from Norway's largest river, the Glomma, 550 km long running through rich agricultural areas and with a dumping site, pulp mills, and metallurgic as well as other industrial activities in the cities of Fredrikstad and Sarpsborg in the delta area. This situation creates a north-south gradient of pollution through the archipelago (Skei 1984). In addition, the fjord Idefjorden which enters the area from east has a long pollution history derived from a pulp mill in the city of Halden (see map of the area, Figure 1). Sediments in the archipelago contain elevated levels of chlorinated hydrocarbons and metals (Knutzen et al. 1986). In order to study the levels of contaminants in flatfish in
Contaminants and Responses in Hvaler Fish
487
V 00'
Oi
P , fl
G 6
O0
0~ 0
O
O -,lJ
"
Fig. 1. Map of the Hvaler area with the locations of the sampling sites indicated. For more precise information on positions, see Materials and Methods. Site II and III are influenced by water masses running southwards from the Glomma estuary (arrows), while site IV is receiving water from Idet]orden. Site I is located on the Swedish side of the border, and is considered a reference site for this study. Inserted is a map of southern Norway indicating the position of the Hvaler area (open square) in the estuary of the 550 km long Glomma fiver
/
o
-[,O(I
the H v a l e r area, and the e f f e c t s o f t h e s e on s e l e c t e d bioc h e m i c a l p a r a m e t e r s , an i n t e g r a t e d a p p r o a c h i n c l u d i n g c h e m i c a l a n d b i o c h e m i c a l a n a l y s e s w a s e s t a b l i s h e d . Organic c h e m i c a l s , m e t a l s , and b i o c h e m i c a l p a r a m e t e r s w e r e investigated in fish c a u g h t at d i f f e r e n t sites in the area.
Materials and Methods
Table 1. Biological data on flatfish caught at four different locations in the Hvaler Archipelago, April 1988
Site a
Species (N) b
m:ff
Cond. factor d
LSI ~
I
L. limanda (2) P. flesus (4) P. platessa (4) L. limanda (6) P. flesus (5) P. platessa (4) P. flesus (9) P. platessa (3) P. flesus (7) P. platessa (9)
1:i 4:0 1:3 1:5 5:0 0:4 2:7 1:2 5:2 4:5
0.94 0.91 0.86 0.94 0.98 0.97 0.97 1.01 0.87 0.88
1.22 1.16 1.04 2.10 1.28 1.98 1.67 1.26 1.25 1.28
II
Sampling ofFish Flatfish were caught by gill netting at four different locations (see map, Figure 1) in late April 1988. The positions of the sites were: Site I, 11~ 5' E, 58~ 57' N; site II, 11~ 1' E, 59~ 8' N; site III, 10~ 59' E, 59~ 1'30" N; site IV, 11~ 12' E, 59~ 5' N. The total catches consisted of 8 dab (Limanda limanda), 25 flounder (Platichthys flesus), and 20 plaice (Pleuronectes platessa), ranging between 130 and 300 g. Most of the fish had maturing gonads, and fish of both sexes were caught. The biological data of the fish are presented in Table 1. The samples were processed immediately after collecting the net. Livers were excised and cut in smaller pieces. For biochemical analyses, approximately 0.5 g of liver was homogenized in 4 vol. 0.1 M Na-phosphate buffer pH 7.4 with 10% glycerol, and S9-fractions
III IV
-+ 0.12 -+ 0.05 -+ 0.05 -+ 0.07 +- 0.11 -+ 0.11 +- 0.13 +- 0.08 +- 0.10 -+ 0.11
-+ 0.47 - 0.30 -+ 0.42 +- 0.57 -+ 0.31 +- 0.27 + 0.24 --- 0.19 -+ 0.18 -+ 0.51
a Site number refers to the map in Figure 1 b Species: dab, L. limanda; flounder, P. flesus; plaice, P. platessa. (N) total number of individuals caught at the site c m:f = no. of males:no, of females d Condition factor, body weight (g) x 100/(length (cm))3 (Bagenal and Tesch 1978) Liver somatic index, liver weight as percent of body weight (All values are presented as mean +_ SD)
A. GoksCyr et al.
488
Table 2. Percent lipid and levels (ng/g lipid) o f aromatic h y d r o c a r b o n s (PAH), h e x a c h l o r o b e n z e n e (HCB), D D E , and PCB in m u s c l e of flatfish from the H v a l e r Archipelago Site
Species (N) a
% lipid
PAH b
HCB
I
L1 (2) Pp(2) L1 (2) Pp (2) Pf(2) Pp (1) Pf (1) Pp (4)
2.3 -+ 1.7 0.7-+0.2 1.2 --- 0.6 0.7 --- 0.5 1.1 - 0.6 0.7 0.9 0.8 -+ 0.2
410 --- 120 280--- 140 390 --_ 60 1190 -+ 770 302 -+ 20 600 756 1030 -+ 360
18 -+ 30-+ 60 -+ 40 190 + 100 133 70 -
II III IV
13 10 50 40 210
40
DDE ~
S u m PCB d
110 --- 90 1 1 0 - 20 500 _+ 550 210 -+ 220 730 - 360 243 633 340 -+ 130
1170 -+ 450 1460--- 140 7000 -+ 3840 4370 --- 520 5630 _-x-1130 4257 7700 4950 --- 1770
a L1 = dab; Pf = flounder; Pp = plaice; (N) = n u m b e r o f individuals analyzed b S u m of selected polyaromatic h y d r o c a r b o n s (see Materials and Methods) r Includes D D T t r a n s f o r m e d into D D E during saponification d S u m of 15 selected PCB c o n g e n e r s (see Materials and Methods)
PAH, m u s c l e 8
DDE, m u s c l e 10
A
7"
9
6-
8-
C
7
5-
m
6 4-
5
~
3"
4
3
2-
2 l: 1 o
II
III
IV
I
II
Site
IV
Site
HCB, muscle 7
III
PCB,
10
B
9
6-
muscle
D
8-
5'
7
6
4"
[ I
3' 2" 1' 0 I
II
Ill
IV
Site
Fig. 2. Relative levels of organic c o n t a m i n a n t s in m u s c l e of flatfish from the H v a l e r Archipelago. Site no. refer to Figure 1, and levels are e x p r e s s e d relative to the levels at site I. (A) s u m of selected polyaromatic h y d r o c a r b o n s , P A H , ( B ) h e x a c h l o r o b e n z e n e , H C B , (C) D D E , (D) s u m o f selected chlorobiphenyl congeners, PCB (see
5 4
3 2 1 I
/ / i
II
IIl
IV
Site text for more details). O p e n bars r e p r e s e n t levels in dab (Limanda limanda); grey bars r e p r e s e n t plaice (Pleuronectes platessa), presented as m e a n --- SD. Since no site I data were available for flounder (Platichthys flesus), this species in not included in the figure
Contaminants and Responses in Hvaler Fish
489
Table 3. Percent lipid and levels (ng/g lipid) of hexachlorobenzene (HCB), DDE, and PCB in liver of flatfish from the Hvaler Archipelago
Site a
Species (N)
% lipid
I
L1 (2) Pp (2) L1 (2) Pp (2) Pf (2) Pp (2) Pf (1) Pp (4)
14.3 17.6 9.9 6.3 5.0 17.7 4.8 7.1
II III IV
-+ --+-+ -+
3.4 4.7 4.0 5.2 2.4 11.9
-
2.3
HCB
DDE
18 (1) n.d. 75 -+ 27 44 + 47 n.d. 77 (1) n.d. 32 (1)
275 n.d. 840 230 n.d. 1142 n.d. 295
Sum PCB (1)
4660 1740 6990 9280 5980 9260 2708 7590
-+ 260 -+ 160 (1) (1)
-+ -+ -+ -+ -+ -+
4140 10 1580 10425 360 4250
+- 4710
a For explanations, see Table 2 were prepared by centrifugation in Eppendorf tubes at 9000 x g for 15 rain. S9-fractions were frozen in liquid nitrogen for transport to the laboratory. For chemical analyses, muscle and liver samples were frozen on dry ice~
HCB, liver 6"
A
5" I
4"
Biochemical Analyses ~
All analyses were performed on S9-fractions. 7-ethoxyresorufin Odeethylase (EROD) activity was measured fluorometrically as described by Prough et al. (1978), using a Perkin-Elmer LS-5 Spectrofluorometer and resorufin (Pierce) as internal standard in each series. The concentration of the standard was assessed, using an extinction coefficient of 73 mM-lcm-1 (Klotz e t al. 1984). 7ethoxycoumarin O-deethylase (ECOD) activity was determined as p r e v i o u s l y d e s c r i b e d ( G o k s C y r e t a l . 1987), a n d U D P glucuronyltransferase (UDP-GT) activity with the aglycon substrate p-nitrophenol as described by Andersson et al. (1985). Immunochemical analyses were performed with polyclonal antibodies against cod cytochrome P450 1A1 ( = c o d P-450c, GoksCyr 1985), in Western blotting (GoksCyr et al. 1991b), and in an indirect enzyme-linked immunosorbent assay, ELISA (GoksCyr 1991). Protein was determined by the method of Lowry et al. (1951), using bovine serum albumin as the standard.
Chemical Analyses Selected polyaromatic hydrocarbons (PAH) were analyzed by gas c h r o m a t o g r a p h y / m a s s s p e c t r o m e t r y (GC/MS): N a p h t h a l e n e , phenanthrene, dibenzothiophene, and their C1 to C3 (C2 for phenanthrene) alkyl homologues; fluoranthene; pyrene; benz(a)anthracene; chrysene; benzo(b+j+k)fluoranthene; benzo(e)pyrene; benzo(a)pyrene; perylene; indeno(1,2,3-c,d)pyrene; benzo(ghi)perylene; dibenzo(a,a+c,h)anthracene. Selected organochlorines, i.e., polychlorinated biphenyls (PCBs; IUPAC No. 28, 52, 101, 99, 110, 118, 153, 105, 138, 183, 128, 156, 180, 170, 194), hexachlorobenzene (HCB) and DDT/DDE, were analyzed by capillary gas chromatography (GC/ECD). The methods have been described by KlungsCyr et al. (1988). Samples of muscle (approx. 5 g) and liver (0.5-1 g) were saponified under reflux for 2 h with methanolic KOH (0.5 N) and the resulting methanol-water phase extracted with 2 • 30 ml pentane. The two portions were combined and reduced in volume to 500 ~xlby a rotary evaporator and a gentle stream of nitrogen. A 50 • 6 mm ID silica gel column (70-230 mesh, 5% deactivated) slurry packed in pentane:dichloromethane (90:5) was used for clean up of extracts for GC and GC/MS analysis. Compounds were eluted from the column with 10 ml pentane: dichloromethane (90:5). After evaporation of the solvent, the extracts were redissolved in 100 t~1 hexane. Fully deuterated biphenyl, anthracene and pyrene were used as internal standards for the analysis of aromatic hydrocarbons and 2,2',6,6'-tetrachlorobiphenyl (CB No. 53) for the analysis of chlori-
3" 2"
1" 0
II
III
IV
III
IV
III
IV
Site DDE, 5
liver
B m
4-
__m
3'
2"
"I o
II Site
PCB, liver 12
C
10" 8" 6" 4"
2",
-
0 I
I1
Fig. 3. Relative levels of organic contaminants in liver of flatfish from the Hvaler Archipelago. (A) HCB, (B) DDE, and (C) sum PCB as explained in Figure 2 nated hydrocarbons. These were added to samples prior to extraction/saponification. Lack of material made it necessary to determine fat content from saponified tissue. After the saponification process mentioned above,
A. GoksCyr et al.
490
Scatter Plot Matrix
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Correlations 1 'Variable PAH M
PAH M HCB M DDEM CB-52 M CB-101 M CB-153 M :~PCB M CB-52 L CB-101 L CB-153 L ~t~CB L
,
1,0000 -0,3125 -0,2247 -0,1055 -0,0637 0,0433 -0,0003 0,5343 0,6693 0,6379 0,7623
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123456780,51,52,53,51234567
HCB M -0,3125 1,0000 0,6927 0,5186 0,3015 0,2286 0,2916 0,3092 0,0605 -0,0402 -0,0535
DDE M -0,2247 0,6927 1,0000 0,4912 0,4109 0,7090 0,6670 0,0044 -0,0649 -0,1340 -0,1757
CB-52 M -0,1055 0,5186 0,4912 1,0000 0,9623 0,7441 0,8529 -0,0124 -0,1105 0,5109 0,4107
[
I
5 10 15 1 0 3 0 5 0 7 0
CB-101 M -0,0637 0,3015 0,4109 0,9623 1,0000 0,8110 0,9035 -0,1641 -0,1991 0,5753 0,4449
CB-153 M 0,0433 0.2286 0,7090 0,7441 0,8110 1,0000 0,9815 -0,2421 -0,1755 0,3516 0,2487
i
i
i
i
20 60 100
.~,PCB M -0,0003 0,2916 0,6670 0,8529 0,9035 0,9815 1,0000 -0,1955 -0,1622 0,4196 0,3123
CB-52 L 0,5343 0,3092 0,0044 -0,0124 -0,1641 -0,2421 -0,1955 1,0000 0,9302 0,2450 0,4633
i i
i i i
i [
i
50100 200
CB-101 L 0,6693 0,0605 -0,0649 -0,1105 -0,1991 -0,1755 -0,1622 0,9302 1,0000 0,2804 0,5160
i
100300500
i
I
I
1
i
i
5001500 30s
CB-153 L 0,6379 -0,0402 -0,1340 0,5109 0,5753 0,3516 0,4196 0,2450 0,2804 1,0000 0,9639
]~PCB L 0,7623 -0,0535 -0,1757 0,4107 0,4449 0,2487 0,3123 0,4633 0,5160 0,9639 1,0000
Fig. 4. Scatter plot analysis and correlation coefficients for levels of PAH, HCB, DDE, PCB congeners 52, 101, 153, and sum of 15 PCBs in muscle (M), and (only PCBs) liver (L). (A) (above) Results from individual fish in gradient I-III.
1 ml water-methanol phase was transferred to a 10 ml Sovirel tube and acidified with 3 ml 1 N HC1. The acidified solution was extracted with 2 • 4 ml petroleum benzine (40-60). Gel occurring after shaking of the tubes was removed by centrifugation. The petroleum benzine extract was evaporated under a gentle stream of nitrogen and redissolved in 1 ml hexane. An aliquot (500 t~l) was determined after evaporation of the solvent by gravimetry on a Chan 29 electrobalance. Chlorinated dibenzofurans and dibenzo-p-dioxins (PCDF/PCDD) were determined in pooled samples of 4-5 fish from each station. In this case, only flounder (Platichthysflesus) were analyzed. Samples were treated and GC/MS analysis was performed as described by Oehme et al. (1989). 13C-labelled 2,3,7,8-chlorine-substituted PCDDs and PCDFs were added to the samples to monitor the quality of sample extraction and recovery. The recovery rates were within the quality assurance limits (>50 and
