- Email: [email protected]
Response of hematological and biochemical parameters to heavy metal exposure: Implications in environmental monitoring
Marine Environmental Research 24 (1988) 219-222
Response of Hematological and Biochemical Parameters to Heavy Metal Exposure: Implications in Environmental Monitoring William H. Benson, Carl F. Watson, Kevin N. Baer & R. Allan S t a c k h o u s e Division of Pharmacologyand Toxicology,School of Pharmacy, Northeast Louisiana University,Monroe, Louisiana 71209-0470, USA
The use of selected hematological and biochemical parameters as indicators of metal exposure in aquatic organisms was evaluated. The hematological and biochemical parameters examined include glucose, hematocrit and aminotransferase h'vels in golden shiners exposed to cadmium. Cadmium exposure produced significant alterations in the levels of glucose, aspartate aminotran.~ferase and alanine aminotransferase; however, hematocrit was not altered by exposure to cadmium. In addition, the comparative activity of Na/K adenosine triphosphatase (ATPase) was evaluated in the fathead minnow, golden shiner and bluegill sunfish. Basal Na/K A TPase activity was lowest in golden shiner (1"01 pmol Pi/mg protein~h) and highest in bluegill sunfish ( 1.45 #mol Pi/mg protein~h). While a stimulation of Na/K A TPase activi O' was observed at an exposure concentration of 1 pg Cd/liter in the fathead minnow and bluegill sunfish, inhibition of enzymatic activity was observed at higher exposure concentrations (10 and lO0 #g Cd/liter). Gill Na/K A TPase activity in golden shiner was not significantly influenced by cadmium exposure. The observed insensitivity of A TPase in shiner may, in part, be related to higher background and accumulated concentrations of cadmium in gill tissue.
Chemically-induced alterations in the biochemistry and physiology of aquatic organisms are being investigated as potential diagnostic tools in environmental monitoring. Hematological and biochemical parameters are attractive as indicators of environmental health because they offer a rapid and sensitive means of monitoring the impact of chemicals on aquatic organisms. Various industrial processes, agricultural practices and fossil fuel 219 Marine Environ. Res. 0141-1136/88/$03-50 © 1988 ElsevierApplied SciencePublishers Ltd, England. Printed in Great Britain
220
William H. Bensonet al.
combustion provide a multiplicity of point and diffuse sources of heavy metal contamination. 1 Exposure to heavy metals, such as cadmium, results in hematological changes such as reduction in hematocrit, hemoglobin and red blood cell count. 2 Alterations in N a / K adenosine triphosphatase (ATPase) enzymatic activity also have been shown in response to a wide variety of environmental contaminants. 3'4 In the present investigation, we evaluated the use of selected hematological and biochemical parameters as biological indicators of metal exposure to aquatic organisms. Fathead minnows (Pimephales promelas), golden shiners (Notemigonus crysoleucas) and bluegill sunfish (Lepomis macrochirus) were exposed in soft water to cadmium using 48 or 96 h static renewal bioassays. Blood was withdrawn by caudal transection and hematocrit was analyzed using a micro-capillary reader. The plasma, obtained by centrifugation, was analyzed for glucose, aspartate aminotransferase (AST) and alanine aminotransferase (ALT). Gill Na/K ATPase activity was analyzed by a modified procedure of Zaugg 5 and inorganic phosphate was determined by a modification of the method of Peterson. 6 Treatment effects on all parameters were evaluated using analysis of variance and comparison of means were determined using Duncan's multiple-range test. 7 Homogeneity of variance was determined by the Fmax test. 8 A non-parametric method based on ranks was used for data sets with heterogeneous variances. 7 Although impaired respiratory function was observed in golden shiners exposed to cadmium, as evidenced by excess mucus secretion from the gills, no significant differences were observed in hematocrit values. Peak glucose levels occurred after 12 h of exposure to 1.35 and 2.40 mg Cd/liter. Although a decline in glucose was observed, the 96 h glucose values for golden shiners exposed to 2-40 mg Cd/liter remained significantly different from control. Significant alterations in AST were observed during the entire 96 h exposure period for both 1.36 and 2.40 mg Cd/liter exposure concentrations. On the other hand, ALT values progressively increased with time of exposure. Although exposure of golden shiners to cadmium influenced plasma glucose and aminotransferase levels, it is unclear whether these parameters would be useful as diagnostic tools in environmental monitoring because they can be influenced by many unknown stressors (handling, parasites, undiagnosed diseases, etc.). 9 If the nature of the applied stressor is known then hematological and biochemical parameters, such as AST and ALT, may suggest cellular injury and/or disturbed metabolic status, If, however, the applied stressor is unknown, then hematological and biochemical parameters are unlikely to permit identification. Exposure of fish to sublethal cadmium concentrations for 48 h resulted in a concentration-related increase of cadmium in gill tissue (Table 1). A
Hematological and biological responses to metal exposure
TABLE 1 Gill Cadmium Content of Freshwater Teleost Species Exposed to Sublethal Concentrations of Cadmium for 48 h a
Species
Bluegill sunfish h Fathead minnowc Golden shiner h
Cadmium exposure concentrations (pg/liter) 0
1
10
100
0.21 (0-01) 0"10 (0"00) 0.52 (0.07)
0.37 (0.01) 0'47 (0'09) 1.21 (0.08)
0.54* (0.o4) 1'89" (0'28) 2.15" (0.10)
1-76" (0.10) 2-58* (090) 4-72* (0"85)
a Values represent mean gill cadmium content (pg Cd/g wet weight tissue). Standard error presented in parentheses; n = 3. h Variances are homogeneous. c Variances are heterogeneous. *Significantly different, p<0.05, from the simultaneous control (Duncan's multiple-range test).
TABLE 2 Gill Na/K ATPase Activity in Freshwater Teleost Species Exposed to Sublethal Concentration of Cadmium for 48 h a
Cadmium exposure concentration (pg/liter)
Bluegill sunfish
0
1.46 (009) 2-13" (0-13) 1-22 (0-09) 123 (014)
1 10 100
Fathead minnow
1"29 (0'06) 142 (009) 087* (0'10) 093* (O'O8)
Golden shiner
1.01 (0-07) 0'80 (0"10) 094 (004) 070 (0-15)
a Values represent mean Na/K ATPase activity (#mol Pi/mg protein/h). Standard error presented in parentheses; n = 3. Variances are homogeneous. *Significantlydifferent, p < 0-05, from the simultaneous control (Duncan's multiple-range test).
221
222
William H. Benson et al.
differential response in gill N a / K ATPase activity to sublethal cadmium exposure was observed in the three species examined (Table 2). There was stimulation of N a / K ATPase in bluegill sunfish and fathead minnow at an exposure concentration of !/~g/liter cadmium, while inhibition was observed at 10 and 100 pg Cd/liter. Although variability in Na/K ATPase activity in golden shiners was exhibited as a result of cadmium exposure, enzymatic activity was not significantly different from control. Basal Na/K ATPase activity was demonstrated to be highest in bluegill sunfish (1-46/~mol Pi/mg protein/h) and lowest in golden shiners (1.01/lmol Pi//mg protein/h). The observed insensitivity of ATPase in shiners appears related to higher background and accumulated cadmium concentrations in gill tissue. The elevated background gill cadmium content in golden shiners suggests that these fish received prior exposure to cadmium. The apparent down-regulation of golden shiner ATPase, resulting from prior exposure and/or acquired tolerance to cadmium, may confound the use of biochemical parameters, such as N a / K ATPase, in environmental monitoring. This work forms part of the Toxicology Program of the School of Pharmacy, an operational unit of the College of Pharmacy and Health Sciences, Northeast Louisiana University.
REFERENCES 1. Beijer, K. & Jernelov, A. In Handbook on the Toxicology of Metals (L. Friberg, G. F. Nordberg & V. B. Vouks, eds), Elsevier/North Holland, New York, pp. 47-63, 1979. 2. Houston, A. H. & Keen, J. E. Canad. J. Fish. Aquat. Sci., 41, 1829-34 (1984). 3. Leadem, T. P., Campbell, R. D. & Johnson, D. W. Comp. Biochem. Physiol., 49A, 197-205 (1974). 4. Boese, B. L., Johnson, V. G., Chapman, D. E. & Ridlington, J. W. Comp. Biochem. Physiol., 71C, 63-7 (1982). 5. Zaugg, W. S. Canad. J. Fish. Aquat. Sci., 39, 215-17 (1982). 6. Peterson, G. L. A. Anal. Biochem., 84, 164-72 (1978). 7. SAS Institute, Inc. S A S User's Guide: Statistics, 1982 Edition, SAS Institute, Inc., Cary, NC, USA, 1982. 8. Sokal, R. R. & Rohlf, F. J. Biometry, W. H. Freeman and Co., San Francisco, 1981. 9. Lockhart, W. L. & Metner, D. A. In Contaminant Effects on Fisheries (V. M. Cairns, P. V. Hodson & J. O. Nriagu, eds), John Wiley, New York, pp. 73-85, 1984.
Response of Hematological and Biochemical Parameters to Heavy Metal Exposure: Implications in Environmental Monitoring William H. Benson, Carl F. Watson, Kevin N. Baer & R. Allan S t a c k h o u s e Division of Pharmacologyand Toxicology,School of Pharmacy, Northeast Louisiana University,Monroe, Louisiana 71209-0470, USA
The use of selected hematological and biochemical parameters as indicators of metal exposure in aquatic organisms was evaluated. The hematological and biochemical parameters examined include glucose, hematocrit and aminotransferase h'vels in golden shiners exposed to cadmium. Cadmium exposure produced significant alterations in the levels of glucose, aspartate aminotran.~ferase and alanine aminotransferase; however, hematocrit was not altered by exposure to cadmium. In addition, the comparative activity of Na/K adenosine triphosphatase (ATPase) was evaluated in the fathead minnow, golden shiner and bluegill sunfish. Basal Na/K A TPase activity was lowest in golden shiner (1"01 pmol Pi/mg protein~h) and highest in bluegill sunfish ( 1.45 #mol Pi/mg protein~h). While a stimulation of Na/K A TPase activi O' was observed at an exposure concentration of 1 pg Cd/liter in the fathead minnow and bluegill sunfish, inhibition of enzymatic activity was observed at higher exposure concentrations (10 and lO0 #g Cd/liter). Gill Na/K A TPase activity in golden shiner was not significantly influenced by cadmium exposure. The observed insensitivity of A TPase in shiner may, in part, be related to higher background and accumulated concentrations of cadmium in gill tissue.
Chemically-induced alterations in the biochemistry and physiology of aquatic organisms are being investigated as potential diagnostic tools in environmental monitoring. Hematological and biochemical parameters are attractive as indicators of environmental health because they offer a rapid and sensitive means of monitoring the impact of chemicals on aquatic organisms. Various industrial processes, agricultural practices and fossil fuel 219 Marine Environ. Res. 0141-1136/88/$03-50 © 1988 ElsevierApplied SciencePublishers Ltd, England. Printed in Great Britain
220
William H. Bensonet al.
combustion provide a multiplicity of point and diffuse sources of heavy metal contamination. 1 Exposure to heavy metals, such as cadmium, results in hematological changes such as reduction in hematocrit, hemoglobin and red blood cell count. 2 Alterations in N a / K adenosine triphosphatase (ATPase) enzymatic activity also have been shown in response to a wide variety of environmental contaminants. 3'4 In the present investigation, we evaluated the use of selected hematological and biochemical parameters as biological indicators of metal exposure to aquatic organisms. Fathead minnows (Pimephales promelas), golden shiners (Notemigonus crysoleucas) and bluegill sunfish (Lepomis macrochirus) were exposed in soft water to cadmium using 48 or 96 h static renewal bioassays. Blood was withdrawn by caudal transection and hematocrit was analyzed using a micro-capillary reader. The plasma, obtained by centrifugation, was analyzed for glucose, aspartate aminotransferase (AST) and alanine aminotransferase (ALT). Gill Na/K ATPase activity was analyzed by a modified procedure of Zaugg 5 and inorganic phosphate was determined by a modification of the method of Peterson. 6 Treatment effects on all parameters were evaluated using analysis of variance and comparison of means were determined using Duncan's multiple-range test. 7 Homogeneity of variance was determined by the Fmax test. 8 A non-parametric method based on ranks was used for data sets with heterogeneous variances. 7 Although impaired respiratory function was observed in golden shiners exposed to cadmium, as evidenced by excess mucus secretion from the gills, no significant differences were observed in hematocrit values. Peak glucose levels occurred after 12 h of exposure to 1.35 and 2.40 mg Cd/liter. Although a decline in glucose was observed, the 96 h glucose values for golden shiners exposed to 2-40 mg Cd/liter remained significantly different from control. Significant alterations in AST were observed during the entire 96 h exposure period for both 1.36 and 2.40 mg Cd/liter exposure concentrations. On the other hand, ALT values progressively increased with time of exposure. Although exposure of golden shiners to cadmium influenced plasma glucose and aminotransferase levels, it is unclear whether these parameters would be useful as diagnostic tools in environmental monitoring because they can be influenced by many unknown stressors (handling, parasites, undiagnosed diseases, etc.). 9 If the nature of the applied stressor is known then hematological and biochemical parameters, such as AST and ALT, may suggest cellular injury and/or disturbed metabolic status, If, however, the applied stressor is unknown, then hematological and biochemical parameters are unlikely to permit identification. Exposure of fish to sublethal cadmium concentrations for 48 h resulted in a concentration-related increase of cadmium in gill tissue (Table 1). A
Hematological and biological responses to metal exposure
TABLE 1 Gill Cadmium Content of Freshwater Teleost Species Exposed to Sublethal Concentrations of Cadmium for 48 h a
Species
Bluegill sunfish h Fathead minnowc Golden shiner h
Cadmium exposure concentrations (pg/liter) 0
1
10
100
0.21 (0-01) 0"10 (0"00) 0.52 (0.07)
0.37 (0.01) 0'47 (0'09) 1.21 (0.08)
0.54* (0.o4) 1'89" (0'28) 2.15" (0.10)
1-76" (0.10) 2-58* (090) 4-72* (0"85)
a Values represent mean gill cadmium content (pg Cd/g wet weight tissue). Standard error presented in parentheses; n = 3. h Variances are homogeneous. c Variances are heterogeneous. *Significantly different, p<0.05, from the simultaneous control (Duncan's multiple-range test).
TABLE 2 Gill Na/K ATPase Activity in Freshwater Teleost Species Exposed to Sublethal Concentration of Cadmium for 48 h a
Cadmium exposure concentration (pg/liter)
Bluegill sunfish
0
1.46 (009) 2-13" (0-13) 1-22 (0-09) 123 (014)
1 10 100
Fathead minnow
1"29 (0'06) 142 (009) 087* (0'10) 093* (O'O8)
Golden shiner
1.01 (0-07) 0'80 (0"10) 094 (004) 070 (0-15)
a Values represent mean Na/K ATPase activity (#mol Pi/mg protein/h). Standard error presented in parentheses; n = 3. Variances are homogeneous. *Significantlydifferent, p < 0-05, from the simultaneous control (Duncan's multiple-range test).
221
222
William H. Benson et al.
differential response in gill N a / K ATPase activity to sublethal cadmium exposure was observed in the three species examined (Table 2). There was stimulation of N a / K ATPase in bluegill sunfish and fathead minnow at an exposure concentration of !/~g/liter cadmium, while inhibition was observed at 10 and 100 pg Cd/liter. Although variability in Na/K ATPase activity in golden shiners was exhibited as a result of cadmium exposure, enzymatic activity was not significantly different from control. Basal Na/K ATPase activity was demonstrated to be highest in bluegill sunfish (1-46/~mol Pi/mg protein/h) and lowest in golden shiners (1.01/lmol Pi//mg protein/h). The observed insensitivity of ATPase in shiners appears related to higher background and accumulated cadmium concentrations in gill tissue. The elevated background gill cadmium content in golden shiners suggests that these fish received prior exposure to cadmium. The apparent down-regulation of golden shiner ATPase, resulting from prior exposure and/or acquired tolerance to cadmium, may confound the use of biochemical parameters, such as N a / K ATPase, in environmental monitoring. This work forms part of the Toxicology Program of the School of Pharmacy, an operational unit of the College of Pharmacy and Health Sciences, Northeast Louisiana University.
REFERENCES 1. Beijer, K. & Jernelov, A. In Handbook on the Toxicology of Metals (L. Friberg, G. F. Nordberg & V. B. Vouks, eds), Elsevier/North Holland, New York, pp. 47-63, 1979. 2. Houston, A. H. & Keen, J. E. Canad. J. Fish. Aquat. Sci., 41, 1829-34 (1984). 3. Leadem, T. P., Campbell, R. D. & Johnson, D. W. Comp. Biochem. Physiol., 49A, 197-205 (1974). 4. Boese, B. L., Johnson, V. G., Chapman, D. E. & Ridlington, J. W. Comp. Biochem. Physiol., 71C, 63-7 (1982). 5. Zaugg, W. S. Canad. J. Fish. Aquat. Sci., 39, 215-17 (1982). 6. Peterson, G. L. A. Anal. Biochem., 84, 164-72 (1978). 7. SAS Institute, Inc. S A S User's Guide: Statistics, 1982 Edition, SAS Institute, Inc., Cary, NC, USA, 1982. 8. Sokal, R. R. & Rohlf, F. J. Biometry, W. H. Freeman and Co., San Francisco, 1981. 9. Lockhart, W. L. & Metner, D. A. In Contaminant Effects on Fisheries (V. M. Cairns, P. V. Hodson & J. O. Nriagu, eds), John Wiley, New York, pp. 73-85, 1984.