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Accumulation and depuration of tetramethyllead by rainbow trout
P,'ater Re.search Vol. 15. pp. 621 to 625, 1981 Printed in Great Britain. All rights reserved
00,43-1354/81/050621-06502.00/0 Copyright © 1981 Pergamon Press Lid
A C C U M U L A T I O N A N D D E P U R A T I O N OF TETRAMETHYLLEAD BY RAINBOW TROUT P. T. S. WONG, Y. K. CHAU, O. KRAMAR and G. A. BENGERT Canada Centre for Inland Waters, P.O. Box 5050, Burlington. Ontario, Canada L7R 4A6 (Received December 1980) Absiract--Tetramethyllead was found very toxic especially to juvenile rainbow trout (Salmo gairdneri). It was accumulated rapidly from the water by the fish and the highest concentration was found in the lipid layer of the intestine. Deputation of Me4Pb from the fish organs was initially quite rapid, followed by a slower decrease and eventually reaching residual levels. Environmental concentrations of Me,LPb detected in the fishery products thus represent a balance between uptake and deputation of this compound in fish. In conjunction with this study, a dosing system for exposing rainbow trout to Me,,Pb was developed.
INTRODUCTION
Environmental concern has stimulated much public a t t e n t i o n to the global impacts of pollutants on the biosphere. Lead is one of the pollutants that attract m u c h interest because of its widespread uses and high toxicity. Organolead c o m p o u n d s are generally more toxic t h a n inorganic lead c o m p o u n d s with the tetraalkyl derivatives being the most toxic form (Wong et al., 1978). Tetramethyllead and tetraethyllead have. been used as antiknock additives to gasoline since 1923. Recently tetramethyllead was reported to be produced from biological and chemical methylation of several inorganic and organic lead c o m p o u n d s in the aquatic e n v i r o n m e n t (Wong et al., 1975; Jarvie et al., 1975: Schmidt & Huber, 1976; D u m a s et al., 1977; Harrison & Laxen, 1978). Tetraalkylleads were subsequently detected in muscles (Mor & Becaria, 1977), lobster and cod (Sirota & Uthe. 1977) and trout (Chau et al.. 1979). In this report we present expertmental data to indicate that rainbow trout are able to accumulate M e 4 P b from water. The concentration of M e 4 P b found in fish represents an equilibrium between the uptake of M e ~ P b from water and the d e p u t a t i o n of the c o m p o u n d from the fish. MATERIALS AND METHODS The dosiml .~ystem Since tetrameth.xllead (Mc,~Pb) is volatile, relatively xvaler-insoluble and is commercially available only in :oluene solution (80°,, MeePb in toluene. Alfa Chemicals. MAt, dosing of Me~Pb at a constant level to fish necessitates the design of a nex~ dosing system (Fig. lk Excess Me~Pb was added to a 20-1. dark bottle (to avoid photodegradation of MeaPb) mixed vigorously with a magnetic stirrer. A second reservoir (4-1. dark bottle) was used to ensure that any undissohed Me~Pb from the first bottle would settle to the bottom. The supernate in the second bottle contained a relatixeh constant and saturated level of Me~Pb at 12.Stag 1 ~ as Pb. The desired dosing level of Me~Pb was achiexed by mixing the appropriate amount of the Mc:Pb-saturated water (regulated with peristaltic 621
pump) with dechlorinated water in the mixing chamber. To avoid exposure of personnel to volatile Me~Pb, the dosing system was set up in a fume-hood. Water The dilution water from Lake Ontario was filtered through a charcoal filter and its flow rate at 500ml m i n - t to the mixing chamber was regulated with a head tank. The means and standard deviations of water temperature. dissolved oxygen, pH, conductivity and hardness were 14.9 _+ 0.9°C, 8.2 _+ 1.2mgl-l, 7.86 _+0.19, 273 _+ 8 #mhos cm-~ and 135 _+ 2.5 mg l -~ as CaCO3 respectively (Hodson et al., 1980). The flow rate of 500 ml min-~ to an 8-1. test tank would provide a 99°o replacement time of approx. 2 h (Sprague. 1973). Accumulation and deputation of Me, Pb Three separate experiments were performed to determine the accumulation of Me4Pb by rainbow trout (Salmo gairdneri). In the first experiment, 30 fish each weighing approx. l g were exposed to Me4Pb at an average concentration of 3.5/~gl-1. The control containing the same number of fish was not treated with Me4Pb but was exposed to an equivalent amount of toluene. Fish were fed daily with 5°0 fish food per g of fish. Ration levels and pellet sizes were adjusted for fish size according to the recommendations of a commercial feed manufacturer (Martins Feed Mills, Elmira. Ontario, Canada). The test tanks were cleaned prior to feeding to eliminate feces and excess food from the previous day. Fish (alive or dead) were removed from the tanks daily, rinsed briefly with distilled water, dried with blotting paper, weighed, dissected and extracted with 5 ml hexane and 5 ml of 4°0 EDTA (Chau et al., 1979). Tetramethyllead in fish and water was analyzed by a gas chromatograph-atomic absorption spectrometer system. The detection limits for fish and water were 0.025pgg -~ and 0.50,ugl -~ respectively as Pb. In the second experiment, l0 larger fish each weighing more than 20g were used in each of the control and exposed tanks. One fish was removed from the control and the exposure tanks after the third, eighth and tenth days of the experiment. Fish tissues and organs were dissected and extracted. The remaining 7 fish were used for Me4Pb depuration study. The fish from the exposed tank were immediately transferred to the clean tank circulated with dechlorinated water. One of the 7 exposed fish was immediately removed for analysis (0-h depuration) and 1 of the remaining 6 fish was removed after 8. 26, 56. 72. 80 and
.20 L ,
_
Water
7
Pump
,ib
r--,---~
~
~__ _ _ :
Ch~c-oal Filter
Head Tank
Overflow Port
Toxin Reservoir . (4L)
---Stirrer
~-~:- ....
__,.
~_
Mixing Chamber
~
-Stirrer
"
~~'*~
_ To D r a i n
Dosing System Fig. 1. A dosing system for exposing Me,LPb to fish. 170 h of depuration. Fish tissues and organs were analyzed for Me,Pb. In the third experiment, the concentration of Me4Pb in the exposed tank was increased to 51 #g 1-~. Three fish, each weighing an average of 20 8, were exposed to Me4Pb for 14 days. Fish were disse~ed, organs and tissues were pooled and analyzed for Me, Pb. RESULTS
Accumulation of Me,Pb by rainbow trout In the first experiment in which 30 small fish (approx. 1 g) were exposed to a low level of Me4Pb
(3.5 + 0.9 #g 1- 1), 1 fish died in the first day of exposure. Two fish were dead in each of the second and third day. The remaining fish survived even after 7 days exposure. Me4Pb levels in the fish were observed to steadily increase from 0.43 #g g-1 (wet wt) in the first day to 1.54 (+_0.65), 1.70 (_+0.54) and 2.54 (_+0.67) gg g - x (wet wt) in second, third and seventh day treatment respectively (Fig. 2). No further increase in Me4Pb concentrations in fish was observed in the 2-week experiment. Neither mortality nor Me, Pb was found in controlled fish exposed to an equivalent amount of toluene.
3
-r 2 u,.. .,o 13,.
_/ 0
1
2
3
4
5
Days of Exposure Fig. 2. Accumulation of Me4Pb by fish.
6
7
Accumulation and depuration of tetramethyilead by rainbow trout
623
Table 1. Accumulation of tetramethyllead in organs of rainbow trout
Organs Intestine (a) Lipid (b) Pyloric caeca (c) Small intestine (d) Large intestine Gill Skin/head Air bladder Liver Fillet Heart Kidney Stomach Gallbladder Spleen Fish alive or dead Wt of fish (g) Conc. of Me~Pb in water (#g l-1)
Concentrations of Me4Pb in organs (/ag g- ~ wet wt) Expt. II (Exposure day) Expt. I11 3 8 10 14 63.7 --* ---27.4 17.8 11.3 9.1 3.2 -----Alive 30.0 23
140.4 ----47.5 43.7 26.1 20.0 16.6 -11.7 5.4 -3.4 Dead 40.0 25
-415.7 183.9 91.9 33.9 54.8 61.4 44.2 16.6 20.4 10.4 9.0 6.7 5.9 3.5 Dead 62.8 25
300.6 106.1 52.7 -21.1 48.7 -17.9 9.0 8.5 18.2 24.5 -36.2 Dead 20.4 51
* Not analyzed.
The initial rate of uptake of Me4Pb by fish can be calculated by the equation: dCf KtC,.~- K2Cf dt where -
Cf = C,o = K 1C,., = K:Cf = K1 = K2 = t =
-
=
cone. of Me4Pb in fish cone. of Me,,Pb in water uptake rate depuration rate uptake rate constant depuration rate constant time.
After integration:
K1C,., Cf -
(1 - e-r2'), K2
At t = ~ . Cf - K IC,, = Ccqum
K2
where C~qum = equilibrium conc. of Me,LPb in fish. K1 and K2 were calculated by plotting In [C,~u,, - Cf] against t with various estimates of C¢qu,, to give the best fit to a straight line. The K1 and K., were found to be 300 and 0.37 d a y - 1 respectively, The initial uptake rate in Fig. 2 was 1 gig Me,LPb g - t fish d a y - 1. In the second experiment, in which 10 larger fish 1 > 2 0 g l were exposed to a higher concentration of MeaPb in water 124 ___ 1 l t g l - l l , mortality was not
observed until the eighth day of exposure (Table I). Since the fish were larger, tissues instead of whole fish were analyzed for M e , Pb. The intestine contained the highest level (63-140ggg-1), followed by gill, skin/ head and air bladder. Fillet also contained 3.2-20.4 #g g - ]. Analyses of intestine revealed that the lipid layer (mesentery fat) accumulated very high amounts of Me4Pb ( 4 1 5 g g g - ] ) . Based on 25gg Me4Pb l - 1 of water, the concentration factor of Me4Pb in the lipid layer of the intestine is 16,000 times. Only 2 out of l0 fish died when exposed to 24 _ 1/~g Me4Pb l - 1 for 10 days. When Me4Pb concentration was further increased to 5 1 / z g l - l , 2 out of 3 fish died during the 2 weeks experiment (Experiment 3). Since the dead fish were of smaller size (approx. 20 g), their tissues were pooled for Me4Pb analyses. Similar to the results in Experiment 2, the lipid layer and pyloric caeca of the intestine again contained the highest concentration of Me4Pb. Other tissues also accumulated appreciable quantities of the compound. Increased Me4Pb concentration from 25 to 51/~g 1- l in the water, however, did not increase Me4Pb in most of the fish tissues.
Depurationof Me4Pb from rainbow trout Seven fish exposed to 24 + 1/ag Me4Pb 1- ] for 10 days in experiment 2 were used for the depuration study. As shown in Fig. 3, fish had accumulated Me4Pb to various levels in the tissues, again with the lipid layer of the intestine containing the highest amount (400/1g Me4Pb g - I I . When fish were placed in clean-water, concentrations of Me,~Pb in the tissues decreased quite rapidly, followed by a slower decrease and eventually reached residual levels. The order of initial Me,~Pb levels in the tissues seemed to corre-
624
P T' S Woxc~~' ,,l -
lO00n
I
I
1°° 1 O
10 IntestinalFat
5
Skin and Head c Gill
\
Stomach L~ver ~
1 o
io
Kidney
Fillet 9o
oo
12o
15o
18o
HOURS
Fig.-3. Depuration of Me4Pb from fish organs
spond well with that of the residual levels at the end of the deputation experiment. The depuration rate was calculated from C, = C,,e -r-'' where Co was initial concentration of Me, Pb in fish. The K2 for intestinal fat and skin/head estimated from Fig. 3 were 0.58 and 0.29 day-t respectively,
stability of Me,Pb in freshwater than in sea-water. Juvenile rainbow trout has also been shown to be more sensitive to chemicals than adult animals (McKim et ul., 1975). In our experiments, more mature fish (approx. 30 g fish-') could tolerate Me,Pb as high as 24/ag l-~. It will be of interest to compare the sensitivity of the life cycle of fish (from egg to adult stages) to Me,Pb toxicity. Tetraalkyllead DISCUSSION compounds are more readily absorbed through the skin and the gastro intestinal tract than inorganic There have been few studies on the effects of lead compounds (WHO, 1977). Once in the animal. tetraalkyllead compounds to aquatic biota primarily tetraalkyllead compounds are metabolized to trialkylbecause of the difficulties in maintaining a relatively lead compounds which exert the toxic effect by obliconstant level of the volatile compounds during the terating the normal "'pH gradient" across the mitoexperiment and in measuring the compounds. The chondrial membrane and thus uncoupling oxidative design in our dosing system ensures a continuous sup- phosphorylation (Skilleter, 1975). ply of the chemicals and at a relatively constant conMe, Pb may be accumulated by fish either through centration, water or food. In marine biota, the water route has Results in our experiments confirm that Me, Pb is been demonstrated to be more important (Maddock one of the more toxic forms of lead compounds & Taylor, 1978). In our results. Me,Pb was taken up (Wong et at., 1978). Exposure of rainbow trout to by fish at a steady rate from 0.4 #g g -~ in thefirst day tetramethyllead at a concentration as low as to 2.5/agg-~ in the seventh day of exposure (Fig. 2). 3.5/ag l-~ caused immediate mortality to several fish. Since the concentration of Me,Pb in the water was The 96 h LC50 value was 50/,~g1- t for marine fish 3.5/~gI- 1 the accumulation factor for Me4Pb in fish (Maddock & Taylor, 1978). The higher value for these was approx. 100 x and 700 × in first and seventh days marine fish could be due to the differences in fish respectively on a whole fish basis. species (trout vs plaice) size (1 vs 3 g) and the high The order of distribution of Me4Pb ira tish organs
Accumulation and depuration of tetramethyllead by rainbow trout
625
and tissues is shown in Table 1. Intestine especially
REFERENCES
the fatty tissue of the intestine, contained the highest level of Me4Pb. Since Me4Pb is lipophilic, it is not too surprising that the compound was concentrated in the lipid layer. Other organs such as gills, air bladder and liver also accumulated much Me4Pb. No
Botr~ C., Malizia E., Melchiorri P., Stacchini E., Tirayanti G. & Zorsi C. D. (19771 Study and evaluation of organic lead levels in fishes and phytoplankton near Otranto. European Society for Toxicology 19th Meeting, Copenhagen. Chau Y. K., Wong P. ,T. S., Bengert G. A. & Kramar O. (1979) Determination of tetraalkyllead compounds in water, sediment and fish samples. Analyt. Chem. 51, 186-188. Chau Y. K., Wong P. T. S., Kramar O., Bengert G. A.. Cruz R. B.. Kinrade J. O., Lye J. & Van Loon J. C. (1980) Occurrence of tetraalkyllead compounds in the aquatic environment. Bull. envir, contain. Toxic'. 24, 265-269. Dumas J. P., Pazdernik L., Belloncik S., Bouchard D. & Vaillancourt G. (1977) Methylation du plomb en milieu aquatique. Proc. 12th Can. Syrup. War. Pollut. Res. 12, , 91-100. Grandjean P.&NielsenT.(19791Organolead compounds: environmental health aspects. Residue Rer. 72, 97-148. Harrison R. M. & Laxen D. P. H. {1978) Natural source of tetraalkyllead in air. Nature 275, 738-740. Hodson P. V.. Hilton J. W., Blunt B. R. & Slinger S. J. {1980) Effects of dietary ascorbic acid on chronic lead toxicity to young rainbow trout {Salmo #airdneri). Can. J. Fish. Aquat. Sci. 37, 170-176. Jarvie A. W. P., Markall R. N. & Potter H. R. (1975~ Chemical alkylation of lead. Nature 255, 217-218. Maddock B. G. & Taylor D. (1977) The acute toxicity and bioaccumulation of some lead alkyl compounds in marine animals. Presented at the Internat. Experts Discussion Meeting on: Lead-occurrence, fate and pollution in the marine environment, Rovinz, Yugoslavia, 18-22 October, 1977. McKim J. M., Benoit D. A., Biesinger K. E., Brungs W. A. & Siefert 11975) Effects of pollution on freshwater fish. J. War. Pollut. Control Fed. 47, 1711-1768. Mor E. D. & Beccaria A. M. (1977) A dehydration method to avoid loss Of trace elements in biological samples. Presented at the lnternat. Experts Discussion Meeting on: Lead-occurrence. fate and pollution in the marine environment, Rovinz. Yugoslavia. 18-22 October, 1977. Schmidt U. & Huber F. (1976l Methylation of organolead and lead(Ill compounds to (CH314Pb by micro-organisms. Nature 259, 157-158. Silverberg B. A., Wong P. T. S. & Chau Y. K. 11977) Effect of tetramethyl lead on freshwater green algae. Archs envir, contam. Toxic'. 5, 305-313. Sirota G. R. & Uthe J. F. [1977) Determination of tetraalkyllead compounds in biological materials. Analyt. Chem. 49, 823-825. Skilleter D. N. I1975) The decrease of mitochondrial substrate uptake caused by trialkyltin and trialkyllead cornpounds in chloride media and its relevance to inhibition of oxidative phosphorylation. Biochem. J. 146, 465-471. Sprague J. B. (19731 The ABC's of pollutant bioassay using fish. In: Biological Methods for the Assessment of Water Quality, ASTM STP 528. American Society for ,Testing and Materials, 6-30. WHO (19771 World Health Organization: environmental health criteria. Lead p. 3. Geneva. Wong P. "1-.S.. Chau Y. K. & Luxon P. L. 11975) Methylation of lead in the environment. Nature 253, 263-264. Wong P. T. S., Silverberg B. A., Chau Y. K. & Hodson P. V. t1978) Lead and the aquatic biota. In Biogeochemistry of Lead (Edited by Nriagu J.). pp. 279-342. Elsevier Press. New York.
other similar experimental data in fish tissues can be found to contrast with the present study. Various marine tissues (cod, lobster and mackerel) were found to contain from 0.1 to 4.79 ppm of tetraalkyllead cornpounds. In the case of lobster digestive gland, the level of tetraalkyllead compounds represented 81% of the total lead content (Sirota & Uthe, 1977). In algae, MeaPb was accumulated in the concretion bodies of the cytoplasm (Silverberg e t a / . , 1977). Mussels " tMytilus edulisl exposed to trimethyllead chloride resulted in the lead distribution in the following order: gill > foot > gonad IMaddock & Taylor, 1978). When fish are allowed to accumulate Me,LPb and then placed in Me,~Pb-free water, the compound was released from the organs at different rates (Fig. 3). For example, the half lives (time required for Me4Pb to decrease to 507~oof its original level)for Me4Pb in the intestinal fat and skin/head were estimated to be 30 and 45 h respectively. This demonstrates that Me4Pb could be rapidly depurated from the organs when fish were placed in clean water. However, because of the extreme dilutions of Me,,Pb and its metabolic products in water, we were not able to follow their concentrations in water. The depuration rate was established by analyzing the Me4Pb in fish. Several species of fish .and animals have been reported to metabolize tetraalkyllead to trialkyllead compounds by the enzymes, mixed function oxidases (Botr6 et al., 1977). The trialkyllead compounds were responsible for the toxic actions in animals {Grandjean & Nielson, 1979). The guideline for total lead level in fish permissible for human consumption in the United Kingdom is 2 mg kg- ~. There is no similar guideline for lead in Canada. The results in this study indicate that the concentrations of tetraalkyllead compounds in fish represent an equilibrium between the accumulation and the depuration of the compound in fish. The equilibrium levels must be significant in the aquatic environment as shown by the frequent detection of a variety of fisherx products containing these compounds tSirota & Uthe. 1977: Chau et al.. 19801. Since tetraalkyllead compounds are much more toxic than other lead compounds (Wong et al., 1978: Grandjean & Nielson, 1979). special considerations should be given in formulating guidelines for total lead and tetraalkvllead compounds in fishery products. .4ckllowled~lemem--We v¢ish to thank Dr Uwe Borgmann for advice in the kinetic analyses.
00,43-1354/81/050621-06502.00/0 Copyright © 1981 Pergamon Press Lid
A C C U M U L A T I O N A N D D E P U R A T I O N OF TETRAMETHYLLEAD BY RAINBOW TROUT P. T. S. WONG, Y. K. CHAU, O. KRAMAR and G. A. BENGERT Canada Centre for Inland Waters, P.O. Box 5050, Burlington. Ontario, Canada L7R 4A6 (Received December 1980) Absiract--Tetramethyllead was found very toxic especially to juvenile rainbow trout (Salmo gairdneri). It was accumulated rapidly from the water by the fish and the highest concentration was found in the lipid layer of the intestine. Deputation of Me4Pb from the fish organs was initially quite rapid, followed by a slower decrease and eventually reaching residual levels. Environmental concentrations of Me,LPb detected in the fishery products thus represent a balance between uptake and deputation of this compound in fish. In conjunction with this study, a dosing system for exposing rainbow trout to Me,,Pb was developed.
INTRODUCTION
Environmental concern has stimulated much public a t t e n t i o n to the global impacts of pollutants on the biosphere. Lead is one of the pollutants that attract m u c h interest because of its widespread uses and high toxicity. Organolead c o m p o u n d s are generally more toxic t h a n inorganic lead c o m p o u n d s with the tetraalkyl derivatives being the most toxic form (Wong et al., 1978). Tetramethyllead and tetraethyllead have. been used as antiknock additives to gasoline since 1923. Recently tetramethyllead was reported to be produced from biological and chemical methylation of several inorganic and organic lead c o m p o u n d s in the aquatic e n v i r o n m e n t (Wong et al., 1975; Jarvie et al., 1975: Schmidt & Huber, 1976; D u m a s et al., 1977; Harrison & Laxen, 1978). Tetraalkylleads were subsequently detected in muscles (Mor & Becaria, 1977), lobster and cod (Sirota & Uthe. 1977) and trout (Chau et al.. 1979). In this report we present expertmental data to indicate that rainbow trout are able to accumulate M e 4 P b from water. The concentration of M e 4 P b found in fish represents an equilibrium between the uptake of M e ~ P b from water and the d e p u t a t i o n of the c o m p o u n d from the fish. MATERIALS AND METHODS The dosiml .~ystem Since tetrameth.xllead (Mc,~Pb) is volatile, relatively xvaler-insoluble and is commercially available only in :oluene solution (80°,, MeePb in toluene. Alfa Chemicals. MAt, dosing of Me~Pb at a constant level to fish necessitates the design of a nex~ dosing system (Fig. lk Excess Me~Pb was added to a 20-1. dark bottle (to avoid photodegradation of MeaPb) mixed vigorously with a magnetic stirrer. A second reservoir (4-1. dark bottle) was used to ensure that any undissohed Me~Pb from the first bottle would settle to the bottom. The supernate in the second bottle contained a relatixeh constant and saturated level of Me~Pb at 12.Stag 1 ~ as Pb. The desired dosing level of Me~Pb was achiexed by mixing the appropriate amount of the Mc:Pb-saturated water (regulated with peristaltic 621
pump) with dechlorinated water in the mixing chamber. To avoid exposure of personnel to volatile Me~Pb, the dosing system was set up in a fume-hood. Water The dilution water from Lake Ontario was filtered through a charcoal filter and its flow rate at 500ml m i n - t to the mixing chamber was regulated with a head tank. The means and standard deviations of water temperature. dissolved oxygen, pH, conductivity and hardness were 14.9 _+ 0.9°C, 8.2 _+ 1.2mgl-l, 7.86 _+0.19, 273 _+ 8 #mhos cm-~ and 135 _+ 2.5 mg l -~ as CaCO3 respectively (Hodson et al., 1980). The flow rate of 500 ml min-~ to an 8-1. test tank would provide a 99°o replacement time of approx. 2 h (Sprague. 1973). Accumulation and deputation of Me, Pb Three separate experiments were performed to determine the accumulation of Me4Pb by rainbow trout (Salmo gairdneri). In the first experiment, 30 fish each weighing approx. l g were exposed to Me4Pb at an average concentration of 3.5/~gl-1. The control containing the same number of fish was not treated with Me4Pb but was exposed to an equivalent amount of toluene. Fish were fed daily with 5°0 fish food per g of fish. Ration levels and pellet sizes were adjusted for fish size according to the recommendations of a commercial feed manufacturer (Martins Feed Mills, Elmira. Ontario, Canada). The test tanks were cleaned prior to feeding to eliminate feces and excess food from the previous day. Fish (alive or dead) were removed from the tanks daily, rinsed briefly with distilled water, dried with blotting paper, weighed, dissected and extracted with 5 ml hexane and 5 ml of 4°0 EDTA (Chau et al., 1979). Tetramethyllead in fish and water was analyzed by a gas chromatograph-atomic absorption spectrometer system. The detection limits for fish and water were 0.025pgg -~ and 0.50,ugl -~ respectively as Pb. In the second experiment, l0 larger fish each weighing more than 20g were used in each of the control and exposed tanks. One fish was removed from the control and the exposure tanks after the third, eighth and tenth days of the experiment. Fish tissues and organs were dissected and extracted. The remaining 7 fish were used for Me4Pb depuration study. The fish from the exposed tank were immediately transferred to the clean tank circulated with dechlorinated water. One of the 7 exposed fish was immediately removed for analysis (0-h depuration) and 1 of the remaining 6 fish was removed after 8. 26, 56. 72. 80 and
.20 L ,
_
Water
7
Pump
,ib
r--,---~
~
~__ _ _ :
Ch~c-oal Filter
Head Tank
Overflow Port
Toxin Reservoir . (4L)
---Stirrer
~-~:- ....
__,.
~_
Mixing Chamber
~
-Stirrer
"
~~'*~
_ To D r a i n
Dosing System Fig. 1. A dosing system for exposing Me,LPb to fish. 170 h of depuration. Fish tissues and organs were analyzed for Me,Pb. In the third experiment, the concentration of Me4Pb in the exposed tank was increased to 51 #g 1-~. Three fish, each weighing an average of 20 8, were exposed to Me4Pb for 14 days. Fish were disse~ed, organs and tissues were pooled and analyzed for Me, Pb. RESULTS
Accumulation of Me,Pb by rainbow trout In the first experiment in which 30 small fish (approx. 1 g) were exposed to a low level of Me4Pb
(3.5 + 0.9 #g 1- 1), 1 fish died in the first day of exposure. Two fish were dead in each of the second and third day. The remaining fish survived even after 7 days exposure. Me4Pb levels in the fish were observed to steadily increase from 0.43 #g g-1 (wet wt) in the first day to 1.54 (+_0.65), 1.70 (_+0.54) and 2.54 (_+0.67) gg g - x (wet wt) in second, third and seventh day treatment respectively (Fig. 2). No further increase in Me4Pb concentrations in fish was observed in the 2-week experiment. Neither mortality nor Me, Pb was found in controlled fish exposed to an equivalent amount of toluene.
3
-r 2 u,.. .,o 13,.
_/ 0
1
2
3
4
5
Days of Exposure Fig. 2. Accumulation of Me4Pb by fish.
6
7
Accumulation and depuration of tetramethyilead by rainbow trout
623
Table 1. Accumulation of tetramethyllead in organs of rainbow trout
Organs Intestine (a) Lipid (b) Pyloric caeca (c) Small intestine (d) Large intestine Gill Skin/head Air bladder Liver Fillet Heart Kidney Stomach Gallbladder Spleen Fish alive or dead Wt of fish (g) Conc. of Me~Pb in water (#g l-1)
Concentrations of Me4Pb in organs (/ag g- ~ wet wt) Expt. II (Exposure day) Expt. I11 3 8 10 14 63.7 --* ---27.4 17.8 11.3 9.1 3.2 -----Alive 30.0 23
140.4 ----47.5 43.7 26.1 20.0 16.6 -11.7 5.4 -3.4 Dead 40.0 25
-415.7 183.9 91.9 33.9 54.8 61.4 44.2 16.6 20.4 10.4 9.0 6.7 5.9 3.5 Dead 62.8 25
300.6 106.1 52.7 -21.1 48.7 -17.9 9.0 8.5 18.2 24.5 -36.2 Dead 20.4 51
* Not analyzed.
The initial rate of uptake of Me4Pb by fish can be calculated by the equation: dCf KtC,.~- K2Cf dt where -
Cf = C,o = K 1C,., = K:Cf = K1 = K2 = t =
-
=
cone. of Me4Pb in fish cone. of Me,,Pb in water uptake rate depuration rate uptake rate constant depuration rate constant time.
After integration:
K1C,., Cf -
(1 - e-r2'), K2
At t = ~ . Cf - K IC,, = Ccqum
K2
where C~qum = equilibrium conc. of Me,LPb in fish. K1 and K2 were calculated by plotting In [C,~u,, - Cf] against t with various estimates of C¢qu,, to give the best fit to a straight line. The K1 and K., were found to be 300 and 0.37 d a y - 1 respectively, The initial uptake rate in Fig. 2 was 1 gig Me,LPb g - t fish d a y - 1. In the second experiment, in which 10 larger fish 1 > 2 0 g l were exposed to a higher concentration of MeaPb in water 124 ___ 1 l t g l - l l , mortality was not
observed until the eighth day of exposure (Table I). Since the fish were larger, tissues instead of whole fish were analyzed for M e , Pb. The intestine contained the highest level (63-140ggg-1), followed by gill, skin/ head and air bladder. Fillet also contained 3.2-20.4 #g g - ]. Analyses of intestine revealed that the lipid layer (mesentery fat) accumulated very high amounts of Me4Pb ( 4 1 5 g g g - ] ) . Based on 25gg Me4Pb l - 1 of water, the concentration factor of Me4Pb in the lipid layer of the intestine is 16,000 times. Only 2 out of l0 fish died when exposed to 24 _ 1/~g Me4Pb l - 1 for 10 days. When Me4Pb concentration was further increased to 5 1 / z g l - l , 2 out of 3 fish died during the 2 weeks experiment (Experiment 3). Since the dead fish were of smaller size (approx. 20 g), their tissues were pooled for Me4Pb analyses. Similar to the results in Experiment 2, the lipid layer and pyloric caeca of the intestine again contained the highest concentration of Me4Pb. Other tissues also accumulated appreciable quantities of the compound. Increased Me4Pb concentration from 25 to 51/~g 1- l in the water, however, did not increase Me4Pb in most of the fish tissues.
Depurationof Me4Pb from rainbow trout Seven fish exposed to 24 + 1/ag Me4Pb 1- ] for 10 days in experiment 2 were used for the depuration study. As shown in Fig. 3, fish had accumulated Me4Pb to various levels in the tissues, again with the lipid layer of the intestine containing the highest amount (400/1g Me4Pb g - I I . When fish were placed in clean-water, concentrations of Me,~Pb in the tissues decreased quite rapidly, followed by a slower decrease and eventually reached residual levels. The order of initial Me,~Pb levels in the tissues seemed to corre-
624
P T' S Woxc~~' ,,l -
lO00n
I
I
1°° 1 O
10 IntestinalFat
5
Skin and Head c Gill
\
Stomach L~ver ~
1 o
io
Kidney
Fillet 9o
oo
12o
15o
18o
HOURS
Fig.-3. Depuration of Me4Pb from fish organs
spond well with that of the residual levels at the end of the deputation experiment. The depuration rate was calculated from C, = C,,e -r-'' where Co was initial concentration of Me, Pb in fish. The K2 for intestinal fat and skin/head estimated from Fig. 3 were 0.58 and 0.29 day-t respectively,
stability of Me,Pb in freshwater than in sea-water. Juvenile rainbow trout has also been shown to be more sensitive to chemicals than adult animals (McKim et ul., 1975). In our experiments, more mature fish (approx. 30 g fish-') could tolerate Me,Pb as high as 24/ag l-~. It will be of interest to compare the sensitivity of the life cycle of fish (from egg to adult stages) to Me,Pb toxicity. Tetraalkyllead DISCUSSION compounds are more readily absorbed through the skin and the gastro intestinal tract than inorganic There have been few studies on the effects of lead compounds (WHO, 1977). Once in the animal. tetraalkyllead compounds to aquatic biota primarily tetraalkyllead compounds are metabolized to trialkylbecause of the difficulties in maintaining a relatively lead compounds which exert the toxic effect by obliconstant level of the volatile compounds during the terating the normal "'pH gradient" across the mitoexperiment and in measuring the compounds. The chondrial membrane and thus uncoupling oxidative design in our dosing system ensures a continuous sup- phosphorylation (Skilleter, 1975). ply of the chemicals and at a relatively constant conMe, Pb may be accumulated by fish either through centration, water or food. In marine biota, the water route has Results in our experiments confirm that Me, Pb is been demonstrated to be more important (Maddock one of the more toxic forms of lead compounds & Taylor, 1978). In our results. Me,Pb was taken up (Wong et at., 1978). Exposure of rainbow trout to by fish at a steady rate from 0.4 #g g -~ in thefirst day tetramethyllead at a concentration as low as to 2.5/agg-~ in the seventh day of exposure (Fig. 2). 3.5/ag l-~ caused immediate mortality to several fish. Since the concentration of Me,Pb in the water was The 96 h LC50 value was 50/,~g1- t for marine fish 3.5/~gI- 1 the accumulation factor for Me4Pb in fish (Maddock & Taylor, 1978). The higher value for these was approx. 100 x and 700 × in first and seventh days marine fish could be due to the differences in fish respectively on a whole fish basis. species (trout vs plaice) size (1 vs 3 g) and the high The order of distribution of Me4Pb ira tish organs
Accumulation and depuration of tetramethyllead by rainbow trout
625
and tissues is shown in Table 1. Intestine especially
REFERENCES
the fatty tissue of the intestine, contained the highest level of Me4Pb. Since Me4Pb is lipophilic, it is not too surprising that the compound was concentrated in the lipid layer. Other organs such as gills, air bladder and liver also accumulated much Me4Pb. No
Botr~ C., Malizia E., Melchiorri P., Stacchini E., Tirayanti G. & Zorsi C. D. (19771 Study and evaluation of organic lead levels in fishes and phytoplankton near Otranto. European Society for Toxicology 19th Meeting, Copenhagen. Chau Y. K., Wong P. ,T. S., Bengert G. A. & Kramar O. (1979) Determination of tetraalkyllead compounds in water, sediment and fish samples. Analyt. Chem. 51, 186-188. Chau Y. K., Wong P. T. S., Kramar O., Bengert G. A.. Cruz R. B.. Kinrade J. O., Lye J. & Van Loon J. C. (1980) Occurrence of tetraalkyllead compounds in the aquatic environment. Bull. envir, contain. Toxic'. 24, 265-269. Dumas J. P., Pazdernik L., Belloncik S., Bouchard D. & Vaillancourt G. (1977) Methylation du plomb en milieu aquatique. Proc. 12th Can. Syrup. War. Pollut. Res. 12, , 91-100. Grandjean P.&NielsenT.(19791Organolead compounds: environmental health aspects. Residue Rer. 72, 97-148. Harrison R. M. & Laxen D. P. H. {1978) Natural source of tetraalkyllead in air. Nature 275, 738-740. Hodson P. V.. Hilton J. W., Blunt B. R. & Slinger S. J. {1980) Effects of dietary ascorbic acid on chronic lead toxicity to young rainbow trout {Salmo #airdneri). Can. J. Fish. Aquat. Sci. 37, 170-176. Jarvie A. W. P., Markall R. N. & Potter H. R. (1975~ Chemical alkylation of lead. Nature 255, 217-218. Maddock B. G. & Taylor D. (1977) The acute toxicity and bioaccumulation of some lead alkyl compounds in marine animals. Presented at the Internat. Experts Discussion Meeting on: Lead-occurrence, fate and pollution in the marine environment, Rovinz, Yugoslavia, 18-22 October, 1977. McKim J. M., Benoit D. A., Biesinger K. E., Brungs W. A. & Siefert 11975) Effects of pollution on freshwater fish. J. War. Pollut. Control Fed. 47, 1711-1768. Mor E. D. & Beccaria A. M. (1977) A dehydration method to avoid loss Of trace elements in biological samples. Presented at the lnternat. Experts Discussion Meeting on: Lead-occurrence. fate and pollution in the marine environment, Rovinz. Yugoslavia. 18-22 October, 1977. Schmidt U. & Huber F. (1976l Methylation of organolead and lead(Ill compounds to (CH314Pb by micro-organisms. Nature 259, 157-158. Silverberg B. A., Wong P. T. S. & Chau Y. K. 11977) Effect of tetramethyl lead on freshwater green algae. Archs envir, contam. Toxic'. 5, 305-313. Sirota G. R. & Uthe J. F. [1977) Determination of tetraalkyllead compounds in biological materials. Analyt. Chem. 49, 823-825. Skilleter D. N. I1975) The decrease of mitochondrial substrate uptake caused by trialkyltin and trialkyllead cornpounds in chloride media and its relevance to inhibition of oxidative phosphorylation. Biochem. J. 146, 465-471. Sprague J. B. (19731 The ABC's of pollutant bioassay using fish. In: Biological Methods for the Assessment of Water Quality, ASTM STP 528. American Society for ,Testing and Materials, 6-30. WHO (19771 World Health Organization: environmental health criteria. Lead p. 3. Geneva. Wong P. "1-.S.. Chau Y. K. & Luxon P. L. 11975) Methylation of lead in the environment. Nature 253, 263-264. Wong P. T. S., Silverberg B. A., Chau Y. K. & Hodson P. V. t1978) Lead and the aquatic biota. In Biogeochemistry of Lead (Edited by Nriagu J.). pp. 279-342. Elsevier Press. New York.
other similar experimental data in fish tissues can be found to contrast with the present study. Various marine tissues (cod, lobster and mackerel) were found to contain from 0.1 to 4.79 ppm of tetraalkyllead cornpounds. In the case of lobster digestive gland, the level of tetraalkyllead compounds represented 81% of the total lead content (Sirota & Uthe, 1977). In algae, MeaPb was accumulated in the concretion bodies of the cytoplasm (Silverberg e t a / . , 1977). Mussels " tMytilus edulisl exposed to trimethyllead chloride resulted in the lead distribution in the following order: gill > foot > gonad IMaddock & Taylor, 1978). When fish are allowed to accumulate Me,LPb and then placed in Me,~Pb-free water, the compound was released from the organs at different rates (Fig. 3). For example, the half lives (time required for Me4Pb to decrease to 507~oof its original level)for Me4Pb in the intestinal fat and skin/head were estimated to be 30 and 45 h respectively. This demonstrates that Me4Pb could be rapidly depurated from the organs when fish were placed in clean water. However, because of the extreme dilutions of Me,,Pb and its metabolic products in water, we were not able to follow their concentrations in water. The depuration rate was established by analyzing the Me4Pb in fish. Several species of fish .and animals have been reported to metabolize tetraalkyllead to trialkyllead compounds by the enzymes, mixed function oxidases (Botr6 et al., 1977). The trialkyllead compounds were responsible for the toxic actions in animals {Grandjean & Nielson, 1979). The guideline for total lead level in fish permissible for human consumption in the United Kingdom is 2 mg kg- ~. There is no similar guideline for lead in Canada. The results in this study indicate that the concentrations of tetraalkyllead compounds in fish represent an equilibrium between the accumulation and the depuration of the compound in fish. The equilibrium levels must be significant in the aquatic environment as shown by the frequent detection of a variety of fisherx products containing these compounds tSirota & Uthe. 1977: Chau et al.. 19801. Since tetraalkyllead compounds are much more toxic than other lead compounds (Wong et al., 1978: Grandjean & Nielson, 1979). special considerations should be given in formulating guidelines for total lead and tetraalkvllead compounds in fishery products. .4ckllowled~lemem--We v¢ish to thank Dr Uwe Borgmann for advice in the kinetic analyses.