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Bioconcentration and excretion of benthiocarb and simetryne by carp
Wat. Res. Vol. 23, No. 4, pp. 529-531, 1989 Printed in Great Britain. All rights reserved
0043-1354/89 $3.00+0.00 Copyright © 1989 Pergamon Press ple
RESEARCH NOTE BIOCONCENTRATION A N D EXCRETION OF BENTHIOCARB A N D SIMETRYNE BY CARP TAIZO TSUDA, SHIGERUAOKI, MIHOKO KOJIMA and HIROYUKI HARADA Shiga Prefectural Institute of Public Health and Environmental Science, 13-45, Gotenhama, Ohtsu, Shiga 520, Japan
(First received April 1988; accepted in revised form November 1988) Abstract--Bioconcentration and excretion of benthiocarb(S-p-chlorobenzyl N,N-diethylthiocarbamate) and simetryne [2,4-bis(ethylamino)-6-methylthio- 1,3,5-triazine)] were studied for carp (Cyprinus carpio L.). The concentrations of both chemicals in muscle and viscera of carp reached plateaus after 12 h exposure. Bioconcentration factors (BCF) of benthiocarb were 25.5 in muscle, 62.7 in liver, 72.7 in kidney and 63.3 (mean, n = 4) in gallbladder over the 168 h exposure period. Similarly, BCF of simetryne were 2.4 in muscle, 13.5 in liver, 8.1 in kidney and 10.9 in gallbladder over the 72 h exposure period. The excretion rate constants of benthiocarb were 0. I 0 h - ~for muscle, 0.09 h - t for liver, 0.13 h - ~for kidney and 0.09 hfor gallbladder assuming their processes to be first-order kinetics. Similarly, those of simetryne were 0.44 h -I for muscle, 0.37 h -l for liver, 0.29 h -l for kidney and 0.23 h -I for gallbladder.
Key words--bioconcentration, excretion, benthiocarb, simetryne, carp, Cyprinus carpio L., muscle, liver, kidney, gallbladder
INTRODUCTION In recent years, a large number of pesticides such as herbicides or insecticides have been produced and discharged into the environment. The data on their bioconcentration and excretion by fish are useful for the evaluation of their safety to man together with their toxicity and metabolism, and further for the evaluation of the contamination of fish by pesticides in lakes or rivers. Bioconcentrations of various pesticides in fish have been reported by several investigators (Neely et al., 1974; Veith et al., 1979; Kanazawa, 1981), but there are still many pesticides that have not been studied in relation to their bioconcentration in fish. We have already studied the bioconcentration and excretion of benthiocarb and simetryne (both herbicides) by the freshwater fish, willow shiner (Gnathopogon caerulescens) (Tsuda et al., 1988). In this study, we researched in detail the bioconcentration and excretion of these herbicides for muscle and viscera (liver, kidney and gallbladder) in carp. In the present study we report the results of these experiments compared with those of the willow shiner (Tsuda et al., 1988). MATERIALS AND METHODS
Chemicals Benthiocarb(S-p-chlorobenzyl N,N-diethylthiocarbamate) and simetryne [2,4-bis(ethylamino)-6-methylthio-l,3,5triazine] were purchased from Wako Pure Chemical Industries Ltd (Osaka, Japan). These chemicals, both reagent grade (>98.0 and 99.0%), were used without further purification.
Test fish Carp (Cyprinus carpio L.) were purchased from Nango Suisan Center (Shiga Prefecture, Japan). They were 9.0ll.0cm in body length and weighed 19.0-28.7g. Benthiocarb and simetryne were not detected in carp before exposure to these chemicals. Commercial assorted feed (10g/15 fish) was given once a day throughout the experiment.
Test system The study was carried out under the continuous flowthrough system. Peristaltic pumps (Cole--Parmer Master Flex PA-21A type ) were used to obtain continuous flow of test solutions. Aquaria (about 501.) made of acrylic plastic were used. For bioconcentration of the test chemicals (benthiocarb 168 h, simetryne 72 h), stock solutions (101.) of benthiocarb (2000 [t g 1-t) and simetryne (1600 #g 1-m) were respectively diluted 150 times continuously with dechlorinated city water and supplied to each of the two aquaria containing 15 fish. These stock solutions were prepared by diluting ethanol solutions of benthiocarb (4000/zg ml-~, 5 ml) and simetryne (4000/zgml -t, 4ml) with water to I01., respectively. The experimental arrangements were the same as our previous report (Tsuda et aL, 1987). During the tests, the flow rate and temperature of each test water were maintained 181 h -m and 19+ I°C, respectively. The concentrations of the chemicals in each test tank water were 4.0-5.0/~gl -m for benthiocarb and 8.0-8.9 ~ug1-~ for simetryne. The low concentrations of these chemicals are probably because they are adsorbed to the tubes of the experimental arrangement or the walls of the aquaria. Measurements were carried out at 0, 24, 72 and 168 h for benthiocarb, and at 0, 24, 48 and 72 h for simetryne. Three fish were taken at 12, 24, 72 and 168 h for benthiocarb, and at 12, 24, 48 and 72 h for simetryne. Excretion experiments of benthiocarb (72 h) and simetryne (24h) from carp were separately carried out in dechlorinated city water after bioconcentration of the test chemicals, in the same manner as the above bioconcentration experiments. In each excretion experiment, 15 fish 529
530
Research Note Table 1. BCF of bentbiocarb and simetryne in carp BCF* of benthiocarb
BCF* of simetryne
Organ
t2h
24h
72h
168h
12h
24h
48h
72h
Muscle Liver Kidney Gallbladder
26.8 82.5 94.4 72.4
25.9 84.5 69.0 79.8
21.3 50.5 54.3 61.1
28.1 33.3 73.2 39.8
2.4 t4.6 9.2 11.9
2.8 16.5 10.0 11.3
2.6 I 1.8 0.7 13.5
1.9 11 I 6.4 6.8
*Mean value (n = 3) for muscle and single determination value for liver, kidney and gallbladder.
were exposed to benthiocarb for 168 h and to simetryne for 72h, 12 fish were transferred to each aquaria and dechlorinated city water was supplied to them. During the experiment, the flow rate and temperature of the test water were maintained at 901 h t and 19 + I°C, respectively. The concentrations of the test chemicals excreted from the fish in the test tanks were <0.2 and 0.1 ltgl t for benthiocarb and simetryne, respectively, after 8 h in these excretion experiments. Three fish were taken at 0, 12, 24, 48 and 72 h for benthiocarb, and at 0, 7, 12 and 24 h for simetryne. Under these conditions, none of the fish showed signs of tiredness or agitation during the bioconcentration and excretion experiments.
Analysis The concentration of benthiocarb or simetryne in the water was determined by the following procedure. A measured volume (100 ml) of water was shaken with 100 ml of hexane after addition of 5 g of NaCI. The organic layer was injected into a gas chromatograph equipped with a flame thermionic detector ( F T D ~ C ) after rotary-vacuum evaporation to 5 ml. Average recoveries (n = 3) were 91% for benthiocarb and 89% for simetryne at 10/~gl -t spiked levels. Detection limits were 0.2/~g 1 ~ for benthiocarb and 0.1 #g 1-t for simetryne. Determination of benthiocarb or simetryne in fish samples was carried out by the following method (Goto and Kato, 1980). In brief, about 5 g of fish sample were homogenized with 30 ml of acetonitrile by high-speed homogenizer (Ultra-Turrax) and the organic layer was filtrated. The residue was rinsed with 10 rnl of acetonitrile and the rinse was again filtrated. The combined filtrate was shaken twice with 50 ml of dichloromethane after addition of 100 ml of 20% NaC1. The combined dichloromethane layer was dehydrated with anhydrous Na 2SO4 and rotary-vacuum evaporated just to dryness at 40°C. The residue was dissolved with 5 ml of acetone and passed through a column (a mixture of activated charcoal 0.3 g and microcrystalline cellulose 2.7 g). Benthiocarb or simetryne was eluted with 100 ml of acetone. The herbicide concentrations of the eluate were measured by FTD-GC after rotary-vacuum evaporation to 1-5 ml. Analyses of the herbicides were carried out as three separate samples for muscle and as a mixture of three samples for liver, kidney and gallbladder at each sampling time of bioconcentration and excretion experiments. Average recoveries (muscle, n = 3) were 80% for benthiocarb and 85% for simetryne at 200ngg -t spiked levels. Detection limits were 0.2 ngg -~ in muscle and 2 n g g -~ in viscera for benthiocarb, and similarly 0.1 and I ng g-~ for simetryne.
where C = chemical concentration in each part of fish (ng g ~) at time, t C0 = chemical concentration in each part offish (ng g~) initially k = excretion rate constant (h ~) t = time (h). RESULTS AND DISCUSSION
Bioconcentration of benthiocarb and simetryne The experimental results are shown in Table 1. The concentrations o f b o t h chemicals in all four parts o f the fish reached plateaus after 12 h exposure. This was similar to the results o f the same experiment on willow shiner in our study (Tsuda et al., 1988). BCF values o f benthiocarb were 25.5 in muscle, 62.7 in liver, 72.7 in kidney and 63.3 (mean, n = 4 ) in gallbladder over the ! 68 h exposure period. Similarly, those o f simetryne were 2.4 in muscle, 13.5 in liver, 8.1 in kidney and 10.9 in gallbladder over the 72 h exposure period. The order o f the BCF values in the four parts o f the carp for benthiocarp was slightly different to that o f simetryne. But for both chemicals, the values o f B C F in the viscera were higher than those in muscle.
Excretion of benthiocarb and simetryne The experimental results are s h o w n in Figs 1 and 2. The concentrations o f both chemicals in the fish rapidly decreased, so could not be measured by F T D - G C after 72 h for benthiocarb in the viscera o f the fish and after 24 h for simetryne in all four parts o f the fish. The rate constants (k) and biological half-lives o f all four parts o f the fish are shown in 1 0 3 ....
A
,102 ~
o
MuscLe
*
Liver
•
GaLLbLadder
Calculation of BCF BCF was calculated by the following equation
"6
chemical concentration in each part of fish BCF = • chemical concentration in water
I
The concentration of chemical in the water at each sampling time was used for the calculation of BCF.
Calculation of excretion rate constants The following equation was used for the calculation of excretion rate constants of chemicals from fish C = C0e-k'
10 -~ 0
I
I
12
24
l
I
48
"/'2
(hi
Fig. l. Excretion of benthiocarb from carp.
Research Note
531
Table 2. Excretion rate constants and biological half-lives of benthiocarb and simetryne by carp Benthiocarb k (h t) Half-life (h)
Organ Muscle Liver Kidney Gallbladder
10 2 r -
o •
MuscLe Liver
•
GaLLbtodder
0.10 0.09 0.13 0.09
6.9 7.7 5.3 7.7
0.44 0.37 0.29 0.23
Simetryne Half-life (h) 1.6 1.9 2.4 3.0
pared with the rate for the whole body of the willow shiner (k = 0.073 h -]) in our study (Tsuda et al., 1988). Similarly for simetryne, the rate for the carp (k =0.23~).44h -l) was considerably faster compared with that for the willow shiner (k = 0.13 h-~).
Kidney
c
k (h i)
10
REFERENCES
o~
U
lo'
0
I
6
12
(h)
Fig. 2. Excretion of simetryne from carp.
Table 2. For benthiocarb, there was no difference in the excretion rate for three parts (all parts except kidney) of the fish. For simetryne, the rate for kidney and gallbladder was slower than for muscle and liver. The excretion rate of benthiocarb for all four parts of the carp (k = 0.09-0.13 h-~) was slightly faster com-
Goto S. and Kato S. (1980) Analytical Methods of Pesticide Residues. Soft Science, Tokyo. Kanazawa J. (1981) Measurement o f the bioconcentration factors of pesticides by freshwater fish and their correlation with physiochemical properties or acute toxicities. Pestic. Sci. 12, 417-424. Neely W. B., Branson D. R. and Blau G. E. (1974) Partition coefficient to measure bioconcentration potential o f organic chemicals in fish. Envir. Sci. Technol. 8, 1113-1115. Tsuda T., Aoki S., Kojima M. and Harada H. (1988) Bioconcentration and excretion o f benthiocarb and simetryne by willow shiner. Toxic. envir. Chem. 18, 31-36. Tsuda T., Nakanishi H., Aoki S. and Takebayashi J. (1987) Bioconcentration and metabolism of phenyltin chlorides in carp. Wat. Res. 21, 949-953. Veith G. D., DeFoe D. L. and Bergstedt B. V. (1979) Measuring and estimating the bioconcentration factor of chemicals in fish. J. Fish. Res. Bd Can. 36, 1040-1048.
0043-1354/89 $3.00+0.00 Copyright © 1989 Pergamon Press ple
RESEARCH NOTE BIOCONCENTRATION A N D EXCRETION OF BENTHIOCARB A N D SIMETRYNE BY CARP TAIZO TSUDA, SHIGERUAOKI, MIHOKO KOJIMA and HIROYUKI HARADA Shiga Prefectural Institute of Public Health and Environmental Science, 13-45, Gotenhama, Ohtsu, Shiga 520, Japan
(First received April 1988; accepted in revised form November 1988) Abstract--Bioconcentration and excretion of benthiocarb(S-p-chlorobenzyl N,N-diethylthiocarbamate) and simetryne [2,4-bis(ethylamino)-6-methylthio- 1,3,5-triazine)] were studied for carp (Cyprinus carpio L.). The concentrations of both chemicals in muscle and viscera of carp reached plateaus after 12 h exposure. Bioconcentration factors (BCF) of benthiocarb were 25.5 in muscle, 62.7 in liver, 72.7 in kidney and 63.3 (mean, n = 4) in gallbladder over the 168 h exposure period. Similarly, BCF of simetryne were 2.4 in muscle, 13.5 in liver, 8.1 in kidney and 10.9 in gallbladder over the 72 h exposure period. The excretion rate constants of benthiocarb were 0. I 0 h - ~for muscle, 0.09 h - t for liver, 0.13 h - ~for kidney and 0.09 hfor gallbladder assuming their processes to be first-order kinetics. Similarly, those of simetryne were 0.44 h -I for muscle, 0.37 h -l for liver, 0.29 h -l for kidney and 0.23 h -I for gallbladder.
Key words--bioconcentration, excretion, benthiocarb, simetryne, carp, Cyprinus carpio L., muscle, liver, kidney, gallbladder
INTRODUCTION In recent years, a large number of pesticides such as herbicides or insecticides have been produced and discharged into the environment. The data on their bioconcentration and excretion by fish are useful for the evaluation of their safety to man together with their toxicity and metabolism, and further for the evaluation of the contamination of fish by pesticides in lakes or rivers. Bioconcentrations of various pesticides in fish have been reported by several investigators (Neely et al., 1974; Veith et al., 1979; Kanazawa, 1981), but there are still many pesticides that have not been studied in relation to their bioconcentration in fish. We have already studied the bioconcentration and excretion of benthiocarb and simetryne (both herbicides) by the freshwater fish, willow shiner (Gnathopogon caerulescens) (Tsuda et al., 1988). In this study, we researched in detail the bioconcentration and excretion of these herbicides for muscle and viscera (liver, kidney and gallbladder) in carp. In the present study we report the results of these experiments compared with those of the willow shiner (Tsuda et al., 1988). MATERIALS AND METHODS
Chemicals Benthiocarb(S-p-chlorobenzyl N,N-diethylthiocarbamate) and simetryne [2,4-bis(ethylamino)-6-methylthio-l,3,5triazine] were purchased from Wako Pure Chemical Industries Ltd (Osaka, Japan). These chemicals, both reagent grade (>98.0 and 99.0%), were used without further purification.
Test fish Carp (Cyprinus carpio L.) were purchased from Nango Suisan Center (Shiga Prefecture, Japan). They were 9.0ll.0cm in body length and weighed 19.0-28.7g. Benthiocarb and simetryne were not detected in carp before exposure to these chemicals. Commercial assorted feed (10g/15 fish) was given once a day throughout the experiment.
Test system The study was carried out under the continuous flowthrough system. Peristaltic pumps (Cole--Parmer Master Flex PA-21A type ) were used to obtain continuous flow of test solutions. Aquaria (about 501.) made of acrylic plastic were used. For bioconcentration of the test chemicals (benthiocarb 168 h, simetryne 72 h), stock solutions (101.) of benthiocarb (2000 [t g 1-t) and simetryne (1600 #g 1-m) were respectively diluted 150 times continuously with dechlorinated city water and supplied to each of the two aquaria containing 15 fish. These stock solutions were prepared by diluting ethanol solutions of benthiocarb (4000/zg ml-~, 5 ml) and simetryne (4000/zgml -t, 4ml) with water to I01., respectively. The experimental arrangements were the same as our previous report (Tsuda et aL, 1987). During the tests, the flow rate and temperature of each test water were maintained 181 h -m and 19+ I°C, respectively. The concentrations of the chemicals in each test tank water were 4.0-5.0/~gl -m for benthiocarb and 8.0-8.9 ~ug1-~ for simetryne. The low concentrations of these chemicals are probably because they are adsorbed to the tubes of the experimental arrangement or the walls of the aquaria. Measurements were carried out at 0, 24, 72 and 168 h for benthiocarb, and at 0, 24, 48 and 72 h for simetryne. Three fish were taken at 12, 24, 72 and 168 h for benthiocarb, and at 12, 24, 48 and 72 h for simetryne. Excretion experiments of benthiocarb (72 h) and simetryne (24h) from carp were separately carried out in dechlorinated city water after bioconcentration of the test chemicals, in the same manner as the above bioconcentration experiments. In each excretion experiment, 15 fish 529
530
Research Note Table 1. BCF of bentbiocarb and simetryne in carp BCF* of benthiocarb
BCF* of simetryne
Organ
t2h
24h
72h
168h
12h
24h
48h
72h
Muscle Liver Kidney Gallbladder
26.8 82.5 94.4 72.4
25.9 84.5 69.0 79.8
21.3 50.5 54.3 61.1
28.1 33.3 73.2 39.8
2.4 t4.6 9.2 11.9
2.8 16.5 10.0 11.3
2.6 I 1.8 0.7 13.5
1.9 11 I 6.4 6.8
*Mean value (n = 3) for muscle and single determination value for liver, kidney and gallbladder.
were exposed to benthiocarb for 168 h and to simetryne for 72h, 12 fish were transferred to each aquaria and dechlorinated city water was supplied to them. During the experiment, the flow rate and temperature of the test water were maintained at 901 h t and 19 + I°C, respectively. The concentrations of the test chemicals excreted from the fish in the test tanks were <0.2 and 0.1 ltgl t for benthiocarb and simetryne, respectively, after 8 h in these excretion experiments. Three fish were taken at 0, 12, 24, 48 and 72 h for benthiocarb, and at 0, 7, 12 and 24 h for simetryne. Under these conditions, none of the fish showed signs of tiredness or agitation during the bioconcentration and excretion experiments.
Analysis The concentration of benthiocarb or simetryne in the water was determined by the following procedure. A measured volume (100 ml) of water was shaken with 100 ml of hexane after addition of 5 g of NaCI. The organic layer was injected into a gas chromatograph equipped with a flame thermionic detector ( F T D ~ C ) after rotary-vacuum evaporation to 5 ml. Average recoveries (n = 3) were 91% for benthiocarb and 89% for simetryne at 10/~gl -t spiked levels. Detection limits were 0.2/~g 1 ~ for benthiocarb and 0.1 #g 1-t for simetryne. Determination of benthiocarb or simetryne in fish samples was carried out by the following method (Goto and Kato, 1980). In brief, about 5 g of fish sample were homogenized with 30 ml of acetonitrile by high-speed homogenizer (Ultra-Turrax) and the organic layer was filtrated. The residue was rinsed with 10 rnl of acetonitrile and the rinse was again filtrated. The combined filtrate was shaken twice with 50 ml of dichloromethane after addition of 100 ml of 20% NaC1. The combined dichloromethane layer was dehydrated with anhydrous Na 2SO4 and rotary-vacuum evaporated just to dryness at 40°C. The residue was dissolved with 5 ml of acetone and passed through a column (a mixture of activated charcoal 0.3 g and microcrystalline cellulose 2.7 g). Benthiocarb or simetryne was eluted with 100 ml of acetone. The herbicide concentrations of the eluate were measured by FTD-GC after rotary-vacuum evaporation to 1-5 ml. Analyses of the herbicides were carried out as three separate samples for muscle and as a mixture of three samples for liver, kidney and gallbladder at each sampling time of bioconcentration and excretion experiments. Average recoveries (muscle, n = 3) were 80% for benthiocarb and 85% for simetryne at 200ngg -t spiked levels. Detection limits were 0.2 ngg -~ in muscle and 2 n g g -~ in viscera for benthiocarb, and similarly 0.1 and I ng g-~ for simetryne.
where C = chemical concentration in each part of fish (ng g ~) at time, t C0 = chemical concentration in each part offish (ng g~) initially k = excretion rate constant (h ~) t = time (h). RESULTS AND DISCUSSION
Bioconcentration of benthiocarb and simetryne The experimental results are shown in Table 1. The concentrations o f b o t h chemicals in all four parts o f the fish reached plateaus after 12 h exposure. This was similar to the results o f the same experiment on willow shiner in our study (Tsuda et al., 1988). BCF values o f benthiocarb were 25.5 in muscle, 62.7 in liver, 72.7 in kidney and 63.3 (mean, n = 4 ) in gallbladder over the ! 68 h exposure period. Similarly, those o f simetryne were 2.4 in muscle, 13.5 in liver, 8.1 in kidney and 10.9 in gallbladder over the 72 h exposure period. The order o f the BCF values in the four parts o f the carp for benthiocarp was slightly different to that o f simetryne. But for both chemicals, the values o f B C F in the viscera were higher than those in muscle.
Excretion of benthiocarb and simetryne The experimental results are s h o w n in Figs 1 and 2. The concentrations o f both chemicals in the fish rapidly decreased, so could not be measured by F T D - G C after 72 h for benthiocarb in the viscera o f the fish and after 24 h for simetryne in all four parts o f the fish. The rate constants (k) and biological half-lives o f all four parts o f the fish are shown in 1 0 3 ....
A
,102 ~
o
MuscLe
*
Liver
•
GaLLbLadder
Calculation of BCF BCF was calculated by the following equation
"6
chemical concentration in each part of fish BCF = • chemical concentration in water
I
The concentration of chemical in the water at each sampling time was used for the calculation of BCF.
Calculation of excretion rate constants The following equation was used for the calculation of excretion rate constants of chemicals from fish C = C0e-k'
10 -~ 0
I
I
12
24
l
I
48
"/'2
(hi
Fig. l. Excretion of benthiocarb from carp.
Research Note
531
Table 2. Excretion rate constants and biological half-lives of benthiocarb and simetryne by carp Benthiocarb k (h t) Half-life (h)
Organ Muscle Liver Kidney Gallbladder
10 2 r -
o •
MuscLe Liver
•
GaLLbtodder
0.10 0.09 0.13 0.09
6.9 7.7 5.3 7.7
0.44 0.37 0.29 0.23
Simetryne Half-life (h) 1.6 1.9 2.4 3.0
pared with the rate for the whole body of the willow shiner (k = 0.073 h -]) in our study (Tsuda et al., 1988). Similarly for simetryne, the rate for the carp (k =0.23~).44h -l) was considerably faster compared with that for the willow shiner (k = 0.13 h-~).
Kidney
c
k (h i)
10
REFERENCES
o~
U
lo'
0
I
6
12
(h)
Fig. 2. Excretion of simetryne from carp.
Table 2. For benthiocarb, there was no difference in the excretion rate for three parts (all parts except kidney) of the fish. For simetryne, the rate for kidney and gallbladder was slower than for muscle and liver. The excretion rate of benthiocarb for all four parts of the carp (k = 0.09-0.13 h-~) was slightly faster com-
Goto S. and Kato S. (1980) Analytical Methods of Pesticide Residues. Soft Science, Tokyo. Kanazawa J. (1981) Measurement o f the bioconcentration factors of pesticides by freshwater fish and their correlation with physiochemical properties or acute toxicities. Pestic. Sci. 12, 417-424. Neely W. B., Branson D. R. and Blau G. E. (1974) Partition coefficient to measure bioconcentration potential o f organic chemicals in fish. Envir. Sci. Technol. 8, 1113-1115. Tsuda T., Aoki S., Kojima M. and Harada H. (1988) Bioconcentration and excretion o f benthiocarb and simetryne by willow shiner. Toxic. envir. Chem. 18, 31-36. Tsuda T., Nakanishi H., Aoki S. and Takebayashi J. (1987) Bioconcentration and metabolism of phenyltin chlorides in carp. Wat. Res. 21, 949-953. Veith G. D., DeFoe D. L. and Bergstedt B. V. (1979) Measuring and estimating the bioconcentration factor of chemicals in fish. J. Fish. Res. Bd Can. 36, 1040-1048.