metabolic effects of technical pentachlorophenol (pcp) on the eel anguilla anguilla l
TRANSCRIPT
Comp. Biochem. PhysioL, 1972, Vol. 43B, pp. 171 to 183. Pergamon Press. Printed in Great Britain
METABOLIC EFFECTS OF TECHNICAL PENTACHLOROPHENOL (PCP) ON THE EEL
ANGUILLA ANGUILLA L.
BO H O L M B E R G , *t SOREN JEN S EN , * A K E LARSSON, 1 K E R S T I N L E W A N D E R t and M A T S O L S S O N s
tDepartment of Zoophysiology, University of Goteborg, Fack, S-400 33 G6teborg 33, Sweden; ~The Swedish Environmental Protection Board, University of Stockholm, Wallen- berg Laboratory, S-104 05 Stockholm 50, Sweden; and 3Swedish Museum of Natural
History, S-104 05 Stockholm 50, Sweden
(Received 7 January 1972)
Abstract--1. Eels (Anguilla anguilla L.) were exposed to sea water and fresh water containing 0"1 ppm pentachlorophenol (PCP). The accumulation of the pesticide as well as the effects on different metabolites in blood, muscle and liver were studied.
2. PCP exposure caused changes, which indicated a hypermetabolic state with accelerated utilization of tissue energy reserves.
3. An altered cholesterol metabolism, a decreased activity of liver glutamate pyruvate transaminase (GPT) and an enlargement of the liver suggested a disturbed liver function.
4. The effects of PCP seemed to persist in spite of a recovery period in clean water for about 2 months.
INTRODUCTION
P~NTACHLOROPH~OL (PCP) has been used as a fungicide and a molluscicide and is described as a strong uncoupling agent of oxidative phosphorylation (for review, see Bevenue & Beckman, 1967). In Sweden PCP is used widely in different branches of industry. In pulp-mills PCP has in some cases up to 1971 replaced phenylmercury as a fungicide preventing slime production. PCP is also used as a lumber preservative and an impregnating agent in paints and in saw mills.
The extensive use of PCP has resulted in a serious pollution problem in the water environment. Careless handling of PCP has caused a number of severe fish kills in recent years.
In work on snails Weinbach (1956) postulated that uncoupling of the oxidative phosphorylation provides a biochemical mechanism of PCP's molluscicidal action. Subsequent in vitro studies on isolated rat mitochondria (Weinbach & Garbus, 1965) have shown that PCP is very tightly bound to mitochondrial proteins. T h e authors suggested that this protein-pentachlorophenol interaction could be an important factor in the uncoupling phenomenon. As a consequence of its effect on the oxidative phosphorylation PCP has been commonly used as an uncoupling agent in studies on biochemical processes in animal tissues (Garbus & Weinbach, 1963; Nicholls et al., 1967).
* Present address: National Board of Fisheries, Fack, S-402 20 G6teborg 5, Sweden.
171
172 B. HOLMBERG, S. JENSEN, A. LARSSON, K. LEWANDEa AND M. OLSSON
S o m e in vivo s tud ies of the effect of P C P on fishes have also been r epo r t ed . G o o d n i g h t (1942) showed tha t P C P in le thal concen t ra t ions caused inc reased r e s p i r a t o r y m o v e m e n t s and b l eed ing resu l t ing f rom capi l la ry rup tu re . W e b e r (1965) o b s e r v e d fo l lowing acute effects of N a - P C P (8 p p m ) on carp (Cyprinus carpio) and r a inbow t rou t (Salmo gairdneri): i l l -ba lanced move me n t s , gasp ing for air at the wa te r surface, i r regu la r and r a p i d r e sp i r a to ry m o v e m e n t s and a s l imy cover ing of the gills. I n work on a c ichl id (Cichlasoma bimaculatum) K r u e g e r et al. (1966) showed tha t P C P had obv ious effects on growth , food c o n s u m p t i o n and s w i m m i n g pe r fo rmance . T h e y also d e m o n s t r a t e d tha t P C P exposu re caused lower adenos ine t r i p h o s p h a t e levels and inh ib i t i on of d i f ferent enzyme sys tems.
T h i s p a p e r r epo r t s on l abo ra to ry e x p e r i m e n t s c o n d u c t e d to show the m e t a - bol ic d i s t u rbances wh ich occur when eels (Anguilla anguilla L.) are exposed to P C P at a low concen t r a t i on (0.1 p p m in the water) . T h e me tabo l i c effects of P C P on eels in sea wa te r and f resh wate r are p re sen ted , as well as the up t ake of the pes t i - c ide in b lood , l iver and musc le t issues. T h e p a p e r also desc r ibes the m e t a b o l i c changes d u r i n g a r ecovery pe r iod in clean water .
M A T E R I A L S A N D M E T H O D S Fish
Yellow eels (Anguilla anguilla L.) weighing from 65 to 130 g were used in these investi- gations. The fish were caught in October-November from the relatively clean coastal area, Sm6gen, on the Swedish west coast. Fish for the sea-water test were caught in 1969 and fish for the fresh-water test in 1970. After capture and transport to the laboratory the fish were acclimated to laboratory conditions in aquaria with filtered, recirculating sea water with a salinity of 30-32~o. These conditions were maintained at least 10 days before the experiments took place. In the fresh-water experiment the sea-water captured eels were transferred to fresh-water aquaria, where they were held for at least 2 weeks. The temper- ature in both sea-water and fresh-water systems was about 12°C. The eels were neither fed during the acclimatization period nor during the experiments.
Test substance Sodium pentachlorophenate, technical grade (Fluka AG) was dissolved in distilled
water to the PCP concentration of 250 ppm. * The test medium was prepared by adding 10ml of this stock solution to 25 I. water in each experimental jar, to obtain a final concentration of 0"1 ppm.
Sea-water experiment Test containers were 30 I. glass jars containing 25 1. of aerated sea water with a salinity of
32~o. The studies were conducted at about 12°C. In each jar three eels were exposed to the test medium or to clean sea water (controls). The pH was 8'1 at the beginning of the experiment. Every 24 hr the test medium or the control medium was replaced with a new solution of the same concentration. No significant decrease of the PCP level could be
*Recently it has been shown that technical PCP preparations sometimes contain con- siderable amounts of chlorinated dibenzo-p-dioxins with 7 and 8 chlorines per molecule (D7 and D8) and the corresponding chlorinated hydroxy phenylethers (pre-dioxins, PD7 and PD8) (Jensen & Renberg, 1972). Analysis of the pentachlorophenol used in this experi- ment gave the following values in ppm D8 : 620, PD8 : 3600 and D7 + PD7 : 460.
M E T A B O L I C EFFECTS OF T E C H N I C A L PCP O N T H E EEL 173
observed in the water in an experiment without eels and water exchange lasting for a period of 2 weeks. After 8 days of exposure to the pesticide medium the treated animals were transferred to clean sea water for a recovery period for a further 8 days without water exchange.
For determination of pesticide content in muscle, liver and blood tissue, two eels were removed after 0, 1, 2, 4, 6, 8, 9, 10, 12 and 16 days.
For metabolic analyses nine eels from the test group were taken out after 8 days of ex- posure to PCP and nine eels after the recovery period (a further 8 days). At the same time eels were removed from the control group.
Fresh-water experiment The same arrangement as mentioned above was used in the fresh-water experiment
using dechlorinated tap water with pH 7"1. In a pre-investigation all eels died after 5 days of exposure to 0"1 ppm of PCP, therefore an exposure time of only 4 days was used in the experiment. The eels were then transferred to clean fresh-water aquaria with running dechlorinated tap water for 55 days of recovery.
For PCP analyses three eels were taken at 0, 1, 2, 3, 4, 5, 6, 8, 16, 42 and 59 days after the beginning of the experiment. For metabolic analyses nine eels from the test group were taken after 4 days of exposure in PCP and after 4 and 55 days of recovery. From the control group eels were taken out only after 4 and 8 days, because eels in the control group died of a disease before 55 days of recovery.
Tissue collection Each fish was removed from the water and immediately stunned with a blow on the head.
Individual body weight was recorded. Blood was obtained from caudal vessels by a heparin- ized syringe. Portions of whole blood were taken for hematocrit, hemoglobin, glucose and lactate determinations. The rest of the blood sample was centrifuged, and the plasma was stored in sealed plastic tubes at - 2 0 ° C until analyzed for chloride, inorganic phosphate, total protein, cholesterol, triglycerides and free fatty acids.
After blood collection the fish was killed by decapitation. Samples of muscular tissue were excised from the epaxial muscle just behind the level of the anus. Weighed samples of muscle for glycogen, triglyceride and transaminase determinations were immediately deep frozen and stored at -20°C.
The liver was removed from the animal and weighed to the nearest 0"01 g. The liver was then divided in weighed samples for glycogen, triglyceride and transaminase deter- minations. These liver samples were deep frozen immediately and stored at - 2 0 ° C until analyzed.
Analytical methods Estimation of pentachlorophenol in tissue. The tissue samples (about 0.2 g) were homo-
genized in methanol and the lipids were extracted by chloroform according to Carlsson (1963). The chloroform phase was then evaporated. The solid residue was dissolved in 2 ml of toluene and this solution was transferred to a 1 2 - m l centrifugation tube. Five ml of 1 M potassium hydroxide was added and after being shaken for 2 min the tube was centri- fuged and then chilled in dry ice in ethanol and the toluene phase was decanted off. Three ml of 1 M phosphoric acid and 2 ml of toluene was added to the tube containing the potas- sium hydroxide phase. The tube was then shaken for 2 min and centrifuged. After chilling the tube in dry ice in ethanol the toluene phase was decanted to a 3-ml test-tube and 0-3 ml of diazomethane in ether (Kirkland, Dow Chemical 33:1521) was added.
After 1 hr the ether was evaporated and 10/zl of the toluene extract was injected into the gas chromatograph (the Hewlett-Packard 7500 with electron capture detector (rSNi) was
174 B. HOLMBERG, S. JENSEN, A. LARSSON, K. LEWANDER AND M. OLSSON"
used). The all glass column (160 cm x 0"20 cm) treated with HMDS (hexarnethyldisil- azane) was filled with 100-120 mesh HMDS-treated and acid washed Chromosorb W, covered with 2"66% QF 1 + 1 ~o SF 96 (w/w). The carrier gas was nitrogen purified by means of a molecular sieve. The gas speed was about 30 ml/min. Detector and injector temperatures were 180 and 190°C respectively. The column temperature was chosen to give PCP a retention time of 8 min (about 130°C). The standard PCP solution used was treated in the same way as the samples.
Metabolic analyses. Routine clinical methods used in an earlier investigation on eels (Larsson & F~inge, 1969) were employed for determinations of hematocrit, hemoglobin, chloride, inorganic phosphate, total plasma protein, cholesterol, triglycerides, free fatty acids (FFA) and lactate.
Glucose was determined enzymatically using a premixed glucose oxidase-dye-buffer reagent (Kabi, Sweden). Glycogen in liver and muscle tissue was digested in potassium hydroxide and precipitated with ethanol according to a modification of the method of Van Handel (1965). After acid hydrolysis the formed glucose was estimated by the glucose oxidase method mentioned above. Muscle and liver triglycerides were estimated according to Carlsson (1963).
Glutamate pyruvate transaminase (GPT) was measured by a modification (Biochernica Test Combination, Boehringer) of the method of Reitman & Frankel (1957). The measure- ments were made on a 1 : 100 homogenate of liver and a 1 : 10 homogenate of muscle in 0"15 M potassium chloride solution. Liver somatic index (LSI) was derived by multiplying the ratio of liver weight to body weight by 100.
Statistical treatment. All data in Tables 3 and 4 were analyzed by Student's t-test. Significant differences between the PCP treated eels and corresponding control eels were established at the 0.05 level.
RESULTS
The pesticide accumulation
T h e course of accumulat ion in liver, muscle and blood tissues is shown in Fig. 1 and in Tables 1 and 2. In both experiments the highest levels were found in the liver. In blood and muscle the levels of PCP were of the same magni tude of size, and about one-quarter of the level in the liver. In the fresh-water test the PCP content in blood was higher than in muscle while in the sea-water test the values were inverted.
Dur ing the exposition t ime there was a rapid increase of the pesticide content in the tissues and this rise was accompanied by a slight sign of poisoning in the fish. T h e visible symptoms of poisoning which were increased respiratory movements , hypersensit ivity to external stimuli and loss of equilibrium, appeared in the fresh- water test during the third and fourth day and in the sea-water case at a higher level between the sixth and eighth day.
The re was a good correlation between the exposure t ime and the m a x i m u m level of PCP in the tissues (4 and 8 days in the fresh-water and sea-water test respectively).
Dur ing the recovery period the level of PCP decreased, but in the fresh-water exper iment the treated eels still hold about 1-2 p p m in the liver and 0.08 p p m in muscle tissue after 55 days of recovery. We failed to show any measurable amount of PCP in the protein fraction of blood, liver or muscle tissues.
tvmr~oLic m,~-~CTS OF ~Cm~ICAL PCP ON Tm~ EEL 175
3°t-t~ /Jl l-(b)
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Time, days
FIo. 1. Levels of PCP in various tissues of eel from the sea-water test (a) and the fresh-water test (b). The amount in ppm in fresh tissue, logarithmic scale.
O, Liver; I , Muscle; ©, blood.
Metabolic effects The effects of PCP in vivo on different blood, liver and muscle metabolites in
the eels are summarized in Tables 3 and 4. Average values for hematocrit, liver- somatic index, GPT activity in liver and muscle, changes in body weight are also presented in the same tables.
Body weights. The fish exposed to PCP lost considerably more weight than corresponding control fish.
Hematocrit and hemoglobin. Both hematocrit and hemoglobin values increased significantly when fish were exposed to PCP. These increased values persisted during the recovery in the sea-water study. In the fresh-water test only the increased hematocrit value persisted after transfer to clean water.
Chloride. There were no significant difference between the control eels and the PCP treated eels in the plasma concentration of chloride. In the long-term fresh- water study the chloride level after 55 days of recovery was about 10 per cent lower than the initial level.
Inorganic phosphate. PCP caused initially an increase in the content of in- organic phosphate in eel plasma. During 4 or 8 days of recovery the level had
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METABOLIC EFFECTS OF TECHNICAL PCP ON THE EEL 177
returned to normal. After 55 days of recovery a low level of plasma inorganic phosphate was observed.
Total plasma protein. In the fresh-water study no changes were observed in the protein content in blood plasma after exposure to PCP. On the contrary the sea- water experiment resulted in a difference in total protein concentration between the control and the PCP treated eels after the first 8 days of exposure to PCP, but after the recovery period there was no difference between the two groups.
Plasma cholesterol. There was a tendency towards increased cholesterol levels due to PCP treatment. After exposure to PCP the content of total and free choles- terol in plasma were significantly elevated after 8 days of recovery in sea water. In fresh water total and esterified cholesterol were increased after 4 days of exposure, but after 4 days of recovery there was no significant difference.
Plasma triglycerides. The PCP treatment caused a significant increase in the amount of triglycerides in blood plasma only in the sea water eels after 8 days of exposure.
Plasma free fatty acids (FFA). There was a tendency towards increased plasma FFA levels after PCP tretament. This was most obvious after the recovery periods.
Blood glucose. Blood glucose was markedly increased after PCP treatment. The hyperglycemia still persisted after 55 days of recovery in clean fresh water.
Blood lactate. In the sea-water experiment there was an elevation in blood lactate after PCP treatment, which remained after 8 days of recovery. In the fresh-water study there was no significant difference in the level of blood lactate.
Liver-somatic index (LSI). During the exposure to PCP the LSI was un- affected. But after 8 days of recovery in sea water and 55 days of recovery in fresh water, respectively, the PCP treated eels seemed to have increased their LSI values.
Liver and muscle triglycerides. PCP exposure caused no significant decrease in the triglyceride content in the liver or skeletal muscle.
Liver glycogen. As regards the glycogen levels in liver tissue we have only values from the sea-water experiment because of some fault in the freezing equip- ment. The PCP treated eels showed no significant change in liver glycogen after 8 days of exposure or after the recovery period.
Muscle glycogen. The pattern of muscle glycogen changes was somewhat different in the two experiments. In the sea-water study there was a significant decrease initially, but in fresh water there was no change in the muscle glycogen level. After 55 days of recovery in fresh water the eels had a high level of muscle glycogen.
Glutamate pyruvate transaminase (GPT). The activity of GPT was only deter- mined in the sea-water study. The GPT activity was significantly lower in both liver and muscle tissues after 8 days of recovery in clean sea water. The controls showed no significant changes in muscle GPT activity during the experiment, but in the liver there was an obvious rise in the activity of this enzyme between 8 and 16 days.
178 B. HOLMBERG, S. JENSEN, A. LARSSON, K. LEWANDER AND M. OLSSON
DISCUSSION*
The eels in this investigation were not fed, so the uptake of PCP from the water probably took place through the gills and/or the skin. Such an uptake of pesti- cides is common in fish (Johnson, 1968). Fish in sea water in contrast to fish in fresh water drink large amounts of water (Evans, 1968), so it cannot be excluded that PCP in this case can enter the body through the intestine. PCP is probably distributed by the blood to different organs, where the main part is stored in fat tissue (Johnson, 1968). In our experiments the highest level of PCP in eel tissues was found in the liver. The content of PCP in muscle tissue was only one-quarter of that in liver in spite of higher lipid content. This might be of interest in view of the importance of the liver in the vital process of detoxification.
The relatively slow uptake of PCP initially was followed by an increased up- take rate, which coincided with the increased respiratory movements. This change in respiration may increase the amount of water passing through the gills. It cannot be excluded, however, that some resistance mechanisms (excretion or detoxification processes) may be disturbed at the same moment as the impaired condition and poisoning signs appear. This study gives no information about the fish's ability to eliminate or metabolize PCP. We can only show that PCP dis- appeared during the recovery period. Simultaneously the outer signs of poisoning, except the increased respiratory movements, became less marked. In studies on shellfish, Kobayashi et al. (1970a, b) observed that PCP could be transformed to some bound form by detoxification mechanisms. They found that a possible mode of detoxification was by conjugation with sulphate.
The difference in pH between sea water and fresh water media (8.1 and 7.1 respectively) might be responsible for the more rapid action in fresh water. The pH affects the ionization of the phenol and probably the uptake rate (Chapman, 1969). Unfortunately the number of PCP analyses are too few to ascertain if the uptake rate in our study was influenced by the water media. Whitley & Sikora (1970) have shown that pH of the media is a very important factor at PCP ex- posure. In a work on tubificid worms they found that the effect of PCP was more pronounced at lower pH values. Goodnight (1942) showed that fish exposed to PCP survived longer at pH 7.6 than 6.6 or lower.
According to the in vitro studies by Weinbach & Garbus (1965) PCP is very tightly bound to mitochondrial proteins. We found that the main part of stored PCP appeared in the lipid fraction of the analyzed tissues. Probably there is only a very small amount of adsorbed PCP which is bound to mitochondrial proteins. However, the mitochondria are very rich in lipid. Thus the lipid soluble pesticide may be concentrated in this organelle and therefore may be particularly available for binding to mitochondrial proteins. It cannot be excluded that this binding can persist even after recovery in clean water. The decreased PCP level in lipid de- posits does not necessarily mean that the protein bound PCP if any, in mitochondria
* I t c a n n o t be exc luded t ha t some of the observed effects of t echnica l P C P on the metabolism of eels are due to the presence of chlorinated dioxins and pre-dioxins recently found in the chemical used in this experiment (see Materials and Methods).
M E T A B O L I C EFFECTS OF T E C H N I C A L PCP O N T H E EEL 179
has diminished. After 55 days of recovery there still remained measurable amounts of PCP in the lipid deposits. During this long recovery period the starving eels probably used their lipid stores in muscle and liver tissues, and this may lead to a redistribution of accumulated PCP within the organism.
From the results of the metabolic analyses (Tables 3 and 4) it is apparent that pronounced metabolic differences existed between PCP exposed and control fishes. On the whole the metabolic effects of PCP were the same in the sea-water and fresh-water experiment. PCP treatment seems to cause a hypermetabolic state characterized by the accelerated utilization of tissue energy reserves with accompanying loss of body weight. The increase in respiratory movements, which was observed during the exposure, and the rise in hematocrit and hemoglobin are also in accordance with this hypermetabolic state. The increased ventilation rate might be attributed to the accumulation of metabolites.
As shown in other works (Weinbach, 1956; Garbus & Weinbach, 1963) the action of PCP appears to be an uncoupling of the oxidative phosphorylation. It is likely that most of the metabolic effects mentioned above might be a result of such an uncoupling action. As a consequence of the uncoupling effect different sub- strates certainly are oxidized, but the transfer of this oxidation energy to formation of high-energy phosphate compounds probably occurs with reduced efficiency. Thus, to meet basal energy requirements the exposed fishes probably had to increase the metabolic rate and are forced to a hypermetabolic state.
The trend in the changes of cholesterol, triglycerides and FFA in blood plasma and liver and muscle triglycerides may indicate an increased utilization of lipids in PCP exposed eels. This is in agreement with another work on fish (Hanes et al., 1968). They found that young coho salmon (Oncorhynchus kisutch) catabolized 47 per cent of their total lipids during 14 days' exposure to 0.1 ppm potassium pen- tachlorophenol, compared to 25 per cent in control fish. Our observed effects of PCP on blood glucose and plasma FFA agree well with findings from studies on another uncoupling agent, 2,4-dinitrophenol (2,4-DNP) (Snarr & Lein, 1968). They showed that 2,4-DNP injections in dogs caused a rise in blood glucose and plasma FFA.
In a study parallel to ours Bostr6m & Johansson (1972) concluded that PCP exposure increased the aerobic metabolism and reduced the anaerobic breakdown of carbohydrates in the liver. They found a decreased activity of lactate dehy- drogenase (LDH) in the liver of PCP exposed eels. However, we found in the sea-water study a marked increase in the accumulation of blood lactate. According to Dando (1969) it is possible that there is no relationship between the glycolysis in the muscle tissue and the activities of lactate dehydrogenases in the liver.
Most whole blood and plasma constituents increased after PCP exposure. Therefore it cannot be excluded that a dehydration might be partly responsible for these elevated concentrations of different blood metabolites. Such a loss of water from blood to other tissues has been reported as a response of severe stress or exercise (Love, 1970). However, the unchanged levels of chloride and total protein in the blood plasma in PCP treated eels argue against such a water loss.
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glo
bin
(ra
g/10
0 m
l)
Pla
sma
chlo
ride
(m
M)
Pla
sma
inor
gani
c ph
osph
ate
(rag
/100
ml)
T
ota
l pl
asm
a pr
otei
n (g
/100
ml)
T
ota
l pl
asm
a ch
oles
tero
l (m
g/10
0 m
l)
Est
erif
ied
plas
ma
chol
este
rol
(mg/
100
ml)
F
ree
plas
ma
chol
este
rol
(mg/
100
ml)
P
lasm
a tr
igly
ceri
des
(mM
) P
lasm
a fr
ee f
atty
aci
ds (
FF
A)
(/~M
) B
lood
glu
cose
(m
g/10
0 m
l)
Blo
od l
acta
te (
mg/
100
ml)
L
iver
-so
mat
ic i
ndex
[(L
W
x 10
0)/B
W]
Liv
er t
rigl
ycer
ides
(m
g/10
0 m
g w
et w
t.)
Mus
cle
trig
lyce
ride
s (m
g/10
0 m
g w
et w
t.)
Mu
scle
gly
coge
n (m
g/10
0 m
g w
et w
t.)
2'4
+ 1
'0
6"0
_+ 1
"3 *
3"
9 _+
0"8
7"
9 +
1 "5
*
28"3
_+
1"5
37"7
+ 2
"3 t
31"0
_+
1"6
37"5
+ 0
"7t
9'2_
+0'
3 11
"1 +
_0"4
t 10
"1 _
+0"4
10
"6_+
0"5
11
4+
3
111
_+ 1
11
3 _+
3 10
3 +
4
7"0
+_ 0
"7
10"3
+ 1
"3 *
6"
9 -+
0"6
7"
0 +_
0"3
5"
1 +_
0"4
5-2_
+0"
2 5'
3 _+
0"2
5"1
_+0"
2 25
2 _+
23
339
_+ 1
6"
270
_+ 1
8 30
4 _+
17
166
_+ 1
2 22
5 +
lOt
174
-+ 1
3 19
5 _+
13
86 -
+ 14
11
1 +_
14
99 _
+ 7
109
+_ 1
2 3"
5 +
0"7
4'0
_+0"
9 4"
1 +
0"7
4"8
+_ 1
'5
459
_+43
51
7 +
33
437
_+ 3
4 51
3 _+
37
53"8
-+6"
7 15
1'6
+ 17
"9t
67"5
+14
"7
123"
5_+
20-1
" 1
1"5
+2
"2
16'7
-+2"
8 7'
1 -+
0"6
6"9-
+1"
3 1"
20 +
0"1
0 1"
23 _
+ 0"0
8 1"
30 -+
0'0
6 1"
26 _
+ 0'0
8 5'
1 +-
1'4
4"
3 _+
0"8
3'
4 -+
1"7
3"
2 -+
1"3
9"
1 -+
1"3
6"
5 _+
1'1
7"
1 _+
0"4
5"
5 +
0'7
0"13
+0"
01
0"13
-+0'
01
0"1
4+
0'0
2
0't
2_
+0
'03
16-2
+ 1
.8
35.7
+ 1
.8
9-4
+0
.6
96
+4
5.
4 +
0.3
5-2_
+0.
3 3O
6 _+
22
201
+ 19
10
5 _+
9
4.5
_+ 0
.6
552
+ 42
10
9.0
+ 29
.0
12.3
+ 1
.6
1-51
+ 0
.05
3"1
-+ 1
-2
4.9_
+O
.6
0-24
_+ 0
"02
Z
Z
O
z ~J
t~
~J
©
Res
ults
are
exp
ress
ed a
s m
ean
_+
S.E
. *
P <
0"05
. ?P
< 0
'005
. N
um
ber
of
fis
hes
in e
ach
grou
p in
par
enth
eses
.
TA
BL
E
4 M
ET
AB
OL
IC E
FF
EC
TS
OF
PE
NT
AC
HL
OR
OP
ItE
NO
L (
0"1
pp
m)
oN
E
EL
S I
N S
EA
WA
TE
R
8 d
ays'
ex
po
sure
8
day
s' e
xp
osu
re
+
8 d
ays'
rec
ov
ery
Co
ntr
ol
PC
P-t
reat
ed
Co
ntr
ol
PC
P-t
reat
ed
eels
(8)
ee
ls (
9)
eels
(8)
ee
ls (
9)
Dec
reas
e in
in
itia
l b
od
y w
eig
ht
(%)
Hem
ato
crit
(%
) H
emo
glo
bin
(ra
g/10
0 m
l)
Pla
sma
chlo
rid
e (m
M)
Pla
sma
ino
rgan
ic p
ho
sph
ate
(mg
/10
0 m
l)
To
tal
pla
sma
pro
tein
(g/
100
ml)
T
ota
l pl
asnm
ch
ole
ster
ol
(mg
/10
0 m
l)
Est
erif
ied
pla
sma
cho
lest
ero
l (m
g/1
00
ml)
F
ree
pla
sma
cho
lest
ero
l (m
g/1
00
ml)
P
lasm
a tr
igly
ceri
des
(m
M)
Pla
sma
free
fat
ty a
cids
(F
FA
) (/
zM)
Blo
od g
luco
se (
mg
/10
0 m
l)
Blo
od
lac
tate
(m
g/1
00
ml)
L
iver
--so
mat
ic i
nd
ex [
(LW
x
100)
/BW
] L
iver
tri
gly
ceri
des
(m
g/1
00
mg
wet
wt.
) M
usc
le t
rig
lyce
rid
es (
mg
/10
0 m
g w
et w
t.)
Liv
er g
lyco
gen
(ra
g/10
0 m
g w
et w
t.)
Mu
scle
gly
cog
en (
mg
/10
0 m
g w
et w
t.)
Liv
er G
PT
(m
U/m
g w
et w
t.)
Mu
scle
GP
T (
mU
/mg
wet
wt.
)
1.3
+ 0.
5 1.
7 +
0.6
38.4
+ 0
.5
41.1
+ 0
-8*
10.5
_+ 0
.5
12.3
+ 0
.5 *
14
1 +-
5
141
+ 3
7.
2 +_
0.4
8-
3 +-
0.3
*
4.0
+ 0
.1
4.5
+- 0
.1 t
37
3 _+
40
378
+ 3
9 22
7 +
23
253
+ 1
9 14
7 +
27
125
+ 2
3 3"
6+-0
.9
6.2
+0
.8*
39
9 +
16
42
4+
17
45.1
+ 2
.7
116-
3 +
5"5
t 7"
5 +
-0"8
12
"0+
- l'
lt
1"29
+ 0
"09
1"25
+ 0
"11
6"0
+ 1
"5
4"2
+ 1
"5
11"3
+ 2
"4
8"2
+ 1
"7
3"2
+ 0
"5
2"5
+ 0
"4
0"16
+0"
01
0"11
+0
"02
*
30"7
+ 7
"6
27"3
+4
"3
0"65
+ 0
"12
0"47
_+ 0
"07
2"2
+0
-7
6"2
+ 0
-8t
35"3
+0
"4
38"3
+0
"3t
10"0
+__
0"3
11"6
__+
0"4t
1
44
+2
1
45
+1
7"
0 +
0"2
7"
1 +
0"2
4"
4 __+
0"2
4"6
+- 0
"2
336
+ 2
2 43
3 +
31
* 23
4 +_
_ 19
260
+ 2
4 10
1 +
15
172
+ 1
1 t
3"7
+ 0
"4
5-0
+ 0
"7
369
+ 1
3 56
5 +
51
t 40
.4 +
2"1
85
"7 +
2"9
t 7-
4+-1
"1
12.1
+1
"3"
1"21
+ 0
"04
1-44
+ 0
-08
* 5"
4 +
1"9
5"
3 +
1"5
9"
0 +
2"1
6"
6 +
1-4
3"
3 +
0"3
2"
6 +
0"6
0"
11 +
-0"0
2 0"
14+
-0"0
3 42
"6 +
6"9
24
.9 +
- 3"7
* 0"
59 +
0"0
7 0"
39 +
_ 0"0
4*
O
O
t"
~0
m
t~
Res
ult
s ar
e ex
pre
ssed
as
mea
n
+
S.E
. *
P <
0"0
5.
tP <
0"0
05.
Nu
mb
er
of
fish
es i
n ea
ch g
rou
p i
n p
aren
thes
es.
182 B. HOLMBERG, S. JENSEN, A. LARSSON, K. LEWANDER AND M. OLSSON
In both sea-water and fresh-water experiments there was a significant elevation of plasma inorganic phosphate initially after the PCP exposure. It is possible that this rise in plasma phosphate reflects the breakdown of high-energy phosphates, which probably takes place due to PCP.
The action of PCP on the metabolism has striking similarities with an exercise or stress situation. Severe exercise in fish results, as summarized by Love (1970), in a rise of several blood constituents (e.g. glucose, lactate and inorganic phosphate). A similar response was observed in a study of the metabolic effects of adrenalin injections to eels (A. Larsson, unpublished observations). In that study there also was a rise in hemoglobin and the plasma content of FFA, triglycerides and cho- lesterol, resembling the PCP response. In another work in our laboratory (M. Johansson, personal communication) it was observed that an intraperitoneal in- jection of adrenalin and a PCP exposure had the same effect on the spleen of the eel. Both treatments gave the result that the spleen was contracted and emptied on the supply of blood cells, thus causing a polycythemia in the blood.
The effect of PCP on plasma cholesterol might partly be explained by the hypermetabolic state. It cannot be excluded, however, that the altered content of esterified and free cholesterol in the plasma from PCP exposed eels also might be a result of an impaired liver function. Another effect of PCP, which may indicate a disturbed liver function is the lowered activity of liver GPT. In the sea-water study the effect on GPT activity was most pronounced after the recovery period. At the same time there was a significant increase in the liver-somatic index (LSI). This increase cannot only be explained by the decrease in body weight but must also and be mainly due to an enlargement of the liver. A fish liver enlargement has been reported as a common result of pesticide action (Johnson, 1968).
Despite the recovery period the PCP treated eels seem to retain the hyper- metabolism. These remaining effects may depend on the high binding capacity of PCP to mitochondrial proteins. Another possible explanation is that there is a gradual release of accumulated PCP from lipid stores when the eels had to use lipids during the long starvation period. Then PCP would be redistributed and could exert its action on other tissues. However, further study is necessary to throw light on this persisting effect of PCP.
Acknowledgements--We are grateful to Mr. Gurmar Lind, Mrs. Eva Nilsson, Mrs. Waltraud Persson and Mrs. Siv Ostling for excellent technical assistance during the course of this study. We are also greatly indebted to Dr. R. L. Saunders, St. Andrews, Canada; and Dr. S. BostrSm and Dr. R. Johansson, GStenborg, for constructive criticism during the preparation of this manuscript.
This work was supported by grants from the Swedish Environmental Protection Board (Contract No. 7-91/69. Dnr. 1168-7-69), National Board of Fisheries and Stiftelsen Wilhelm och Martina Lundgrens Ventenskapsfond.
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its occurrence as a residue in human and animal tissues. Residue Rev. 19, 83-134. BOSTR/SM S-L. 86 JOHANSSON R. G. (1972) Effects of pentachlorophenol on enzymes in-
volved in energy metabolism in the liver of the eel. Comp. Biochem. Physiol. 41B, 359-369.
METABOLIC EFFECTS OF TECHNICAL PCP ON THE EEL 183
CARLSSON L. A. (1963) Determination of serum triglycerides. J . Atheroscler. Res. 3, 334-336. CHAPMAN G. g . (1969) Toxicity of pentachlorophenol to trout alevins. Ph.D. thesis,
Corvallis, Oregon State University. DANDO P. R. (1969) Lactate metabolism in fish..7, mar. biol. Ass. U.K. 49, 209-223. EvANs D. H. (1968) Measurement of drinking rates in fish. Comp. Biochem. Physiol. 25,
751-753. GARBUS J. & WEINBACH E. C. (1963) Restoration of oxidative phosphorylation and related
reactions in uncoupled mitochondria by albumin. Fedn Proc. Fedn Am Socs. exp. Biol. 22, 405.
GOODNIGHT C. J. (1942) Toxicity of sodium pentachlorophenate and pentachlorophenol to fish. Ind. Eng. Chem. 34, 868-872.
HANES D., KRUEGER H., TINSLEY I. & BOND C. (1968) Influence of pentachlorophenol on fatty acids of coho salmon. Proc. west. Pharmacol. Soc. 11, 121-125.
JENSEN S. & RSNBERC L. (1972) Polychlorinated dibenzo-p-dioxins and polychlorinated hydroxy phenylethers (pre-dioxins) in technical pentachlorophenol (PCP)..4mbio (In press.)
JOHNSON D. W. (1968) Pesticides and fishes--a review of selected literature. Trans. Am. Fish. Soc. 97, 398-424.
KOBAYASHI K., AKIT^KS H. & TOMIYAMA T. (1970a) Studies on the metabolism of penta- chlorophenate, aherbicide, in aquatic organisms--II. Biochemical change of PCP in sea water by detoxification mechanisms of Tapes philippinarum. Bull. yap. Soc. Sd. Fish. 36, 96-102.
KOBAYASHI K., AKITAK~ H. & TOMIYAMA T. (1970b) Studies on the metabolism of pen- tachlorophenate, herbicide, in aquatic organisms--III . Isolation and identification of a conjugated PCP yielded by a shell-fish, Tapes philippinarum. Bull. yap. Soc. Sci. Fish. 36, 103-108.
KRUEGER H., Lu S. D., CHAPMAN G. & CHENO J. T. (1966) Effects of pentachlorophenol on the fish, Cichlasoma bimaculatum. Abstract from the 3rd International Pharmacological Congress, S. Paulo, Brazil, 24-30 July 1966.
L~mssoN A. & FgNOE R. (1969) Chemical differences in the blood of yellow and silver phase of the European eel (Anguilla anguilla L.). Archs. intern. Physiol. Biochem. 77, 701-709.
Love R. M. (1970) The Chemical Biology of Fishes, pp. 39-57. Academic Press, New York. NICHOLLS D. G., SHEPI~RD D. & GARLAND P. B. (1967) A continuous recording technique
for the measurement of carbon dioxide, and its application to mitochondrial oxidation and decarboxylation reactions. Biochem. ~. 103, 677-691.
RSITMAN S. & FRA}aCSL S. (1957) A colorimetric method for determination of serum G O T and GPT. Am.ft. clin. Pathol. 28, 56-63.
SNARR J. F. & LEIN A. (1968) Response of plasma glucose and free fatty acids to hyper- metabolism. Proc. Soc. exp. Biol. Med. 127, 694-697.
VAN HANDEL E. (1965) Estimation of glycogen in small amounts of tissue. Analyt. Bio- chem. 11, 256-265.
WEBER E. ( 1965) Einwirkung yon Pentachlorophenolnatrium auf Fische und Fischniihrtiere. Biol. Zentralbl. 84, 81-93.
WEINBACH E. C. (1956) The influence of pentachlorophenol on oxidative and glycolytic phosphorylation in snail tissue. Archs Biochem. Biophys. 64, 129-143.
WEINBACH E. C. & GARBUS J. (1965) The interaction of uncoupling phenols with mito- chondria and mitochondrial protein. ~. biol. Chem. 240, 1811-1819.
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Key Word Index---Pentachlorophenol (PCP); pesticide; chlorinated hydrocarbons; pesticide accumulation; Anguilla anguilla L.; fish; metabolism; metabolic disturbances; hypermetabolism; liver disturbances; chlorinated dioxins.