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The kinetics of zinc accumulation by the marine prosobranch gastropod Littorina littorea
Marine Environmental Research 24 (1988) 135-139
The Kinetics of Zinc Accumulation by the Marine Prosobranch Gastropod Littorina littorea
A. Z. Mason Molecular EcologyInstitute, California State University, Long Beach, California 90840, USA
The kinetics of accumulation of~SZn by L. iittorea from seawater containing low (2"5 x 10-~o M) and high (150 x 10-~°M) concentrations of Zn has been studied. The accumulation of~SZn by the animals was essentially linear with time at both external concentrations of the metal. Analyses of the individual tissues show that at low ambient concentrations, ~SZn is accumulated primarily by the kidney while at elevated concentrations, the stomach and visceral complex also play a prominent role in accumulating the metal. Comparative analyses of the isotopic and total concentrations of Zn in the animals exposed to high concentrations of Zn show, however, that the rates of turnover andflux of the metal in these tissues are different. Thus, although the initial rates of Zn accumulation are highest in the kidney when normalized for weight, there is no net accumulation of the Zn in the tissue after 1 week of exposure since the biological half-life is short and there is rapid bidirectional flux of the metal. This is in marked contrast to the stomach and visceral complex which have long biological half-lives for the metal and have been identoqed as major sites of Zn accumulation in Zn contaminated environments.
Previous studies on L. littorea collected from pristine and Zn polluted field sites have provided evidence for environmentally induced shifts in metal metabolism and have shown that certain tissues, such as the kidney and visceral complex, can assume a prominent role in accumulating Zn when the ambient concentrations of the metal are high. L2 These studies provide no temporal information, however, on the rates of accumulation of the metal in the tissues in response to changes in the environmental concentrations of Zn. The present study aims to supplement the field data by using 65Zn to study 135 Marine Environ. Res. 0141-1136/88/$03"50 © 1988 ElsevierApplied SciencePublishers Ltd, England. Printed in Great Britain
136
•
A. Z. Mason
quantitatively the flux of the metal through various tissues of L. littorea at low and high external concentrations of the metal. The two concentrations of metal chosen for the present study were approximately equivalent to those recorded for the pristine and Zn contaminated field sites monitored in earlier studies. 1'2 A population of L. littorea of approximately the same size and weight were collected from a pristine coastal field site. The concentration of Zn in the seawater at this site was approximately 1.8-2.0 × 10-lo M. The animals were acclimated for 1 week to laboratory conditions in large plastic holding tanks containing 50 litres of 2 ~tm filtered, UV treated seawater. After acclimation, the animals were divided into two groups. One group was transferred to tanks containing seawater with a Zn concentration of 2.5 × 10-1°M. The other group was maintained in seawater with a Zn concentration of 150 × 10-x°M. Radiolabelled 6SEn (17/~Ci 65Zn/liter) was present in all the tanks and was added 48 h before the beginning of the experiment to allow equilibration with non-isotopic Zn in the water. Groups of 6-10 animals were removed periodically from the flasks during the 42 day experimental period, killed and dissected into a number of component tissues. The tissues were dried, weighed and the accumulation of radiotracer in each tissue type measured. Linear equations, fitted to the data by the method of least squares, describing the kinetics of accumulation of 6SEn in the various tissues in the two experimental groups are shown in Table 1. The data are expressed as #g 65Zn/g dry wt/day and are derived from the specific activity of the isotope in the water. The fitted equations shown in Table 1 highlight a number of points. Firstly, the accumulation of 6SEn by both groups of intact animals is linear with time. Similarly, with the exception of the gills and the kidney tissue in the animals exposed to high external concentrations of Zn, the tissues show relatively high r 2 values indicating a constant rate of accumulation of the metal. The low r 2 values in the gills and kidney appear to be attributable to equilibration between the 6SEn in these tissues and the media. Thus, the accumulation of Zn in these tissues tends to reach an asymptotic value after 1-2 weeks of exposure. Secondly, the equations show high positive intercept values for the kidney and gills. Previous studies indicate there is rapid accumulation of metal within the first few hours of exposure. 3 These earlier studies also indicate that these kinetics cannot be solely explained by non-specific adsorption which implies that these tissues have particularly efficient biological mechanisms for rapidly taking up and accumulating Zn. Thirdly, at low external concentrations, Zn is accumulated primarily by the kidney but at higher concentrations the majority of the metal is vectored to the stomach and visceral complex.
Zinc accumulation by Littorina littorea
137
TABLE 1 E q u a t i o n s Describing the Rates o f Accumulation o f 65Zn in the Different Tissues o f Littorina littorea under Different E n v i r o n m e n t a l C o n d i t i o n s (expressed as/~g 65Zn/g dry wt/day) G r o u p 1: Animals Exposed to 2.5 × 10-1°M Z n Tissue
Equation
r2
Whole animal
y = 0.14 + 0.83
0-95
Mantle Gills R e c t u m / h y p o b r a n c h i a l gland Head/foot Columellar muscle Stomach Visceral complex Kidney
y= y= y= y= y= y= y= y=
0.80 0-54 0'70 0"77 0.87 0"95 0-95 0"96
0.10 + 0.15 + 0'07 + 0"05 + 0.05 + 0.28 + 0.28 + 1.64 +
1-09 2-41 0.65 0.14 0-14 0.87 0.31 4.05
G r o u p 2: Animals Exposed to 150 × 10-1°M Z n Tissue
Equation
rz
Whole animal
y = 13"6 + 6-4
0'96
Mantle Gills R e c t u m / h y p o b r a n c h i a l gland Head/foot Columellar muscle Stomach Visceral complex Kidney
y= y= y= y= y= y= y= y=
0-93 0-85 0"92 0.96 0"93 0.98 0-92 0"58
1.6 + 6.4 2.2 + 18-4 5"5 - 9"6 1.4 + 3.2 1.5 - 9"6 40.0 - 82.4 47.2 - 97.6 24-0 + 486'0
Finally, comparisons of the rate constants for Zn accumulation in the different tissues at low and high external concentrations show evidence of saturation of Zn accumulation at higher external concentrations in the mantle, head/foot, muscle, gills and kidney. An approximate 60-fold increase in the external concentration of Zn results in only a 19-30-fold increase in the rates of accumulation of the metal in these tissues. In complete contrast, there is a 143-169-fold increase in accumulation of Zn in the stomach and visceral complex indicating enhanced accumulation and retention of the metal at higher external concentrations. These differences in the dose response relationship shown by the tissues may result from tissue specific alterations in metal turnover or transportation. An indication of the relative rates of turnover of the Zn in the different tissues of the animals
A. Z. Mason
138
TABLE 2 Variations in Total Concentrations of Zn a in the Intact Organism and Various Component Tissues of Littorina littorea Exposed for 42 days to 150 x 10- lo M Zn (expressed as pg Zn/g dry wt tissue)
Tissue
Day 0 Day 7 Day 14 Day 21 Day 28 Day 35 Day 42
Whole animal
113"8
242.7
292"8 405'0
612'6
618"5 605.3
Gills Rectum/hypobranchial gland Head/foot Columellar muscle Stomach Visceral complex Kidney
185-1 1 8 7 . 5 1 9 1 . 6 193.9 1 4 6 . 2 1 8 8 . 0 255.0 111.5 128.6 73.5 156,5 284.1 324.1 267.0 81.5 98.8 78.4 99.0 101.5 102-9 120-2 118.9 118.0 80.2 1 3 2 . 4 1 4 6 . 4 124.5 195.0 142.7 410.8 546.0 835.7 1 396-7 1 355"5 1 918.1 101-6 253.7 480'8 800.6 l 533-4 1 664.5 1 321.7 372'1 2976.0 2493.0 2149.0 1946-0 1481.0 2t53.0
a Analyses of the total levels of zinc in the tissues of the animals during the experimental period were performed by atomic absorption spectroscopy.
exposed to 150 x 10-~°M Zn can be obtained by comparing the rates of accumulation of isotopic metal with the changes in the total concentration of metal in the tissues over time (Table 2). On the basis of the characteristics of Zn accumulation and turnover which emerge from the data given in Tables 1 and 2, the tissues can be divided into three groups. Group 1 includes the columellar muscle, head/foot, mantle edge, and rectum/hypobranchial gland. These tissues appear to have low flux rates and balanced rates of uptake and excretion. Collectively, they account for approximately 20% of the total Zn in individuals chronically exposed to high levels of Zn and are not important in metabolizing or storing the metal. Group 2 includes the visceral complex and stomach. These tissues show a high rate of accumulation of Zn and a low rate of loss. The tissues have a large biomass and a high capacity to accumulate the metal. Consequently, the tissues are an important site of storage of the metal and may contain more than 70% of the total quantity of metal in animals collected from Zn contaminated areas. G r o u p 3 includes the kidney and gills. In the experiment designed to mimic heavily contaminated waters, the concentration of 65Zn in the kidney rises rapidly during the first 14 days of exposure. U p o n further exposure, the rate of 6 5 Z n accumulation decreases. This decrease coincides with a fall in the total concentration of metal in the tissue, indicating a finite capacity to accumulate the metal. It can be proposed that these characteristics are due principally to increases in the rate of efflux of metal which are accompanied by a shift in the direction of accumulation of the metal away from the kidney
Z&c accumulation by Littorina littorea
139
towards the group 2 tissues. Thus, although the kidney is actively involved in metabolizing Zn and has a high rate of influx, the rate of efflux is comparable and there is no net accumulation of metal. A similar, but less pronounced, pattern of Zn metabolism is observed for the gills. Due to their small biomass, the residual amount of metal in these tissues is relatively small and they rarely contain more than 10% of the total body dose of Zn in animals from Zn polluted environments.
REFERENCES 1. Mason, A. Z. & Simkiss, K. J. mar. biol. Ass. UK, 63, 661-72 (1983). 2. Mason, A. Z., Simkiss, K. & Ryan, K. J. mar. biol. Ass. UK, 64, 699-720 (1984). 3. Mason, A. Z. Ph.D Thesis, University of Wales (1983).
The Kinetics of Zinc Accumulation by the Marine Prosobranch Gastropod Littorina littorea
A. Z. Mason Molecular EcologyInstitute, California State University, Long Beach, California 90840, USA
The kinetics of accumulation of~SZn by L. iittorea from seawater containing low (2"5 x 10-~o M) and high (150 x 10-~°M) concentrations of Zn has been studied. The accumulation of~SZn by the animals was essentially linear with time at both external concentrations of the metal. Analyses of the individual tissues show that at low ambient concentrations, ~SZn is accumulated primarily by the kidney while at elevated concentrations, the stomach and visceral complex also play a prominent role in accumulating the metal. Comparative analyses of the isotopic and total concentrations of Zn in the animals exposed to high concentrations of Zn show, however, that the rates of turnover andflux of the metal in these tissues are different. Thus, although the initial rates of Zn accumulation are highest in the kidney when normalized for weight, there is no net accumulation of the Zn in the tissue after 1 week of exposure since the biological half-life is short and there is rapid bidirectional flux of the metal. This is in marked contrast to the stomach and visceral complex which have long biological half-lives for the metal and have been identoqed as major sites of Zn accumulation in Zn contaminated environments.
Previous studies on L. littorea collected from pristine and Zn polluted field sites have provided evidence for environmentally induced shifts in metal metabolism and have shown that certain tissues, such as the kidney and visceral complex, can assume a prominent role in accumulating Zn when the ambient concentrations of the metal are high. L2 These studies provide no temporal information, however, on the rates of accumulation of the metal in the tissues in response to changes in the environmental concentrations of Zn. The present study aims to supplement the field data by using 65Zn to study 135 Marine Environ. Res. 0141-1136/88/$03"50 © 1988 ElsevierApplied SciencePublishers Ltd, England. Printed in Great Britain
136
•
A. Z. Mason
quantitatively the flux of the metal through various tissues of L. littorea at low and high external concentrations of the metal. The two concentrations of metal chosen for the present study were approximately equivalent to those recorded for the pristine and Zn contaminated field sites monitored in earlier studies. 1'2 A population of L. littorea of approximately the same size and weight were collected from a pristine coastal field site. The concentration of Zn in the seawater at this site was approximately 1.8-2.0 × 10-lo M. The animals were acclimated for 1 week to laboratory conditions in large plastic holding tanks containing 50 litres of 2 ~tm filtered, UV treated seawater. After acclimation, the animals were divided into two groups. One group was transferred to tanks containing seawater with a Zn concentration of 2.5 × 10-1°M. The other group was maintained in seawater with a Zn concentration of 150 × 10-x°M. Radiolabelled 6SEn (17/~Ci 65Zn/liter) was present in all the tanks and was added 48 h before the beginning of the experiment to allow equilibration with non-isotopic Zn in the water. Groups of 6-10 animals were removed periodically from the flasks during the 42 day experimental period, killed and dissected into a number of component tissues. The tissues were dried, weighed and the accumulation of radiotracer in each tissue type measured. Linear equations, fitted to the data by the method of least squares, describing the kinetics of accumulation of 6SEn in the various tissues in the two experimental groups are shown in Table 1. The data are expressed as #g 65Zn/g dry wt/day and are derived from the specific activity of the isotope in the water. The fitted equations shown in Table 1 highlight a number of points. Firstly, the accumulation of 6SEn by both groups of intact animals is linear with time. Similarly, with the exception of the gills and the kidney tissue in the animals exposed to high external concentrations of Zn, the tissues show relatively high r 2 values indicating a constant rate of accumulation of the metal. The low r 2 values in the gills and kidney appear to be attributable to equilibration between the 6SEn in these tissues and the media. Thus, the accumulation of Zn in these tissues tends to reach an asymptotic value after 1-2 weeks of exposure. Secondly, the equations show high positive intercept values for the kidney and gills. Previous studies indicate there is rapid accumulation of metal within the first few hours of exposure. 3 These earlier studies also indicate that these kinetics cannot be solely explained by non-specific adsorption which implies that these tissues have particularly efficient biological mechanisms for rapidly taking up and accumulating Zn. Thirdly, at low external concentrations, Zn is accumulated primarily by the kidney but at higher concentrations the majority of the metal is vectored to the stomach and visceral complex.
Zinc accumulation by Littorina littorea
137
TABLE 1 E q u a t i o n s Describing the Rates o f Accumulation o f 65Zn in the Different Tissues o f Littorina littorea under Different E n v i r o n m e n t a l C o n d i t i o n s (expressed as/~g 65Zn/g dry wt/day) G r o u p 1: Animals Exposed to 2.5 × 10-1°M Z n Tissue
Equation
r2
Whole animal
y = 0.14 + 0.83
0-95
Mantle Gills R e c t u m / h y p o b r a n c h i a l gland Head/foot Columellar muscle Stomach Visceral complex Kidney
y= y= y= y= y= y= y= y=
0.80 0-54 0'70 0"77 0.87 0"95 0-95 0"96
0.10 + 0.15 + 0'07 + 0"05 + 0.05 + 0.28 + 0.28 + 1.64 +
1-09 2-41 0.65 0.14 0-14 0.87 0.31 4.05
G r o u p 2: Animals Exposed to 150 × 10-1°M Z n Tissue
Equation
rz
Whole animal
y = 13"6 + 6-4
0'96
Mantle Gills R e c t u m / h y p o b r a n c h i a l gland Head/foot Columellar muscle Stomach Visceral complex Kidney
y= y= y= y= y= y= y= y=
0-93 0-85 0"92 0.96 0"93 0.98 0-92 0"58
1.6 + 6.4 2.2 + 18-4 5"5 - 9"6 1.4 + 3.2 1.5 - 9"6 40.0 - 82.4 47.2 - 97.6 24-0 + 486'0
Finally, comparisons of the rate constants for Zn accumulation in the different tissues at low and high external concentrations show evidence of saturation of Zn accumulation at higher external concentrations in the mantle, head/foot, muscle, gills and kidney. An approximate 60-fold increase in the external concentration of Zn results in only a 19-30-fold increase in the rates of accumulation of the metal in these tissues. In complete contrast, there is a 143-169-fold increase in accumulation of Zn in the stomach and visceral complex indicating enhanced accumulation and retention of the metal at higher external concentrations. These differences in the dose response relationship shown by the tissues may result from tissue specific alterations in metal turnover or transportation. An indication of the relative rates of turnover of the Zn in the different tissues of the animals
A. Z. Mason
138
TABLE 2 Variations in Total Concentrations of Zn a in the Intact Organism and Various Component Tissues of Littorina littorea Exposed for 42 days to 150 x 10- lo M Zn (expressed as pg Zn/g dry wt tissue)
Tissue
Day 0 Day 7 Day 14 Day 21 Day 28 Day 35 Day 42
Whole animal
113"8
242.7
292"8 405'0
612'6
618"5 605.3
Gills Rectum/hypobranchial gland Head/foot Columellar muscle Stomach Visceral complex Kidney
185-1 1 8 7 . 5 1 9 1 . 6 193.9 1 4 6 . 2 1 8 8 . 0 255.0 111.5 128.6 73.5 156,5 284.1 324.1 267.0 81.5 98.8 78.4 99.0 101.5 102-9 120-2 118.9 118.0 80.2 1 3 2 . 4 1 4 6 . 4 124.5 195.0 142.7 410.8 546.0 835.7 1 396-7 1 355"5 1 918.1 101-6 253.7 480'8 800.6 l 533-4 1 664.5 1 321.7 372'1 2976.0 2493.0 2149.0 1946-0 1481.0 2t53.0
a Analyses of the total levels of zinc in the tissues of the animals during the experimental period were performed by atomic absorption spectroscopy.
exposed to 150 x 10-~°M Zn can be obtained by comparing the rates of accumulation of isotopic metal with the changes in the total concentration of metal in the tissues over time (Table 2). On the basis of the characteristics of Zn accumulation and turnover which emerge from the data given in Tables 1 and 2, the tissues can be divided into three groups. Group 1 includes the columellar muscle, head/foot, mantle edge, and rectum/hypobranchial gland. These tissues appear to have low flux rates and balanced rates of uptake and excretion. Collectively, they account for approximately 20% of the total Zn in individuals chronically exposed to high levels of Zn and are not important in metabolizing or storing the metal. Group 2 includes the visceral complex and stomach. These tissues show a high rate of accumulation of Zn and a low rate of loss. The tissues have a large biomass and a high capacity to accumulate the metal. Consequently, the tissues are an important site of storage of the metal and may contain more than 70% of the total quantity of metal in animals collected from Zn contaminated areas. G r o u p 3 includes the kidney and gills. In the experiment designed to mimic heavily contaminated waters, the concentration of 65Zn in the kidney rises rapidly during the first 14 days of exposure. U p o n further exposure, the rate of 6 5 Z n accumulation decreases. This decrease coincides with a fall in the total concentration of metal in the tissue, indicating a finite capacity to accumulate the metal. It can be proposed that these characteristics are due principally to increases in the rate of efflux of metal which are accompanied by a shift in the direction of accumulation of the metal away from the kidney
Z&c accumulation by Littorina littorea
139
towards the group 2 tissues. Thus, although the kidney is actively involved in metabolizing Zn and has a high rate of influx, the rate of efflux is comparable and there is no net accumulation of metal. A similar, but less pronounced, pattern of Zn metabolism is observed for the gills. Due to their small biomass, the residual amount of metal in these tissues is relatively small and they rarely contain more than 10% of the total body dose of Zn in animals from Zn polluted environments.
REFERENCES 1. Mason, A. Z. & Simkiss, K. J. mar. biol. Ass. UK, 63, 661-72 (1983). 2. Mason, A. Z., Simkiss, K. & Ryan, K. J. mar. biol. Ass. UK, 64, 699-720 (1984). 3. Mason, A. Z. Ph.D Thesis, University of Wales (1983).