ECOTOXICOLOGY
Effects
AND
ENVIRONMENTAL
SAFETY
of Chromium
ANIL Department
on the Blood
K. SRIVASTAVA, of Zoology,
3, 321-324 (1979)
S. J. AGRAWAL,
University Received
of Gorakhpur, February
of a Freshwater
Teleost
AND H. S. CHAUDHRY Gorakhpur
273 001, U.P.,
India
26, 1979
Exposure to a sublethal concentration of 35 ppm chromium induced pronounced increases in the population of circulating erythrocytes, hematocrit, clotting time, and hepatosomatic index at 90 hr post-treatment in a freshwater teleost, Colisa fasciatus. Leukopenia due to reduction in the number of small lymphocytes, thrombocytopenia, and a significant increase in the erythrocyte sedimentation rate were also noted in fish subjected to exposure to the metal; hemoglobin and the number of circulating immature erythrocytes and large lymphocytes were not affected. The data suggest that hematology can serve as an economical model for the study of toxic effects caused by chromium.
Chromium is a common pollutant of natural waters primarily from the discharge of industrial wastes. While C1-3+is considered a minor problem, CP is very toxic (Krenkel, 1974). In general, studies using fish have been confined to the determination of lethal concentrations (L& values) of hexavalent chromium (Adelman et al., 1976) with little information available on the effects of sublethal doses of this metal (Stokes and Fromm, 1965). This study was undertaken to determine if some hematological characteristics of a freshwater teleost, Colisa fasciatus, would change following acute exposure to sublethal levels of hexavalent chromium in the form of chromium trioxide (CrO,). Hematology of this fish has proven valuable in monitoring stress responses (Srivastava and Agrawal, 1977), including those due to exposure to sublethal levels of heavy metals (Srivastava and Agrawal, 1979; Srivastava and Mishra, 1979). McLeay and Gordon (1977) have also suggested that changes in the blood may well represent the most definitive test in determining physiological responses of fish to toxic substances. MATERIALS
AND METHODS
Adult female specimens (weight, 5.12 + 0.42 g) of Colisa fasciatus were used in the study. The fish, captured locally, were kept for 10 days under natural photoperiod in glass aquaria containing tap water. They were fed powdered dried shrimp daily, but food was withheld during the experimental period. The properties of the tap water were as follows: temperature, 25 +- 1°C; pH, 7.3; electrical conductivity, 1433.33 -t 33.33 @U/cm; dissolved oxygen content, 6.8 ppm; hardness, 120 mg/liter as CaCO,. Six groups of 12 fish each were exposed for 90 hr to chromium as chromium trioxide at a sublethal concentration of 35 ppm. A sample of 6 fish from each group of survivals (approximately 70%) was selected for blood analysis. The 96-hr LC,, value, determined concurrently by graphical interpolation in plotting percentage survival vs log CrO, concentration (Greenberg et al., 1975), was 40 ppm. Each fish was anesthetized with MS 222, blotted dry with absorbant
322
SRIVASTAVA, AGRAWAL, AND CHAUDHRY
paper, and weighed. Fresh blood samples were collected from the caudal artery by transection of the caudal peduncle of the fish. The hematological techniques have been described previously (Srivastava, 1968a,b,1969; Srivastava and Mishra, 1979). In one of the treated groups of fish, body and liver weights were recorded for calculating the hepatosomatic index (liver wt/body wt x 100). RESULTS The effects of the administration of an acutely sublethal concentration of chromium on the blood of Colisa are presented in Table 1. The total erythrocyte count, number of red blood cells per 1000 cells, and hematocrit value for fish were elevated significantly from control values after 90 hr exposure to 35 ppm of chromium trioxide. Erythrocyte sedimentation rate (ESR), total leukocyte count, and number of small lymphocytes per 1000 cells were significantly lower in the treated fish than corresponding values for the untreated group. The number of thrombocytes per 1000 cells was also significantly depressed following acute exposure to the metal; the total thrombocyte count was not altered significantly, but it showed a trend toward decreasing (P < 0.1) at 90 hr subsequent to exposure to the toxicant. The values for number of immature erythrocytes and of large lymphocytes per 1000 cells and the hemoglobin were not altered appreciably by metal exposure. A marked increase in hepatosomatic index after exposure of the fish to the metal was also observed. DISCUSSION The increases in the number of circulating red blood cells and the hematocrit in Colisa are comparable with unpublished observations made by us on this fish TABLE
1
EFFECT OF CHROMIUM ON HEMATOLOGICAL OF A FRESHWATER TELEOST~
Parameter Erythrocytes (X 106/mm3) Leukocytes (X lo3/mm3) Thrombocytes (X 103/mm3) Total differential cell counts0 Mature erythrocytes Immature erythrocytes Small lymphocytes Large lymphocytes Thrombocytes Hematocrit (%) ESR (mm/hr) Hemoglobin (g%) Clotting time (set) Hepatosomatic index
Control (water)
VALUES
Experimental (chromium)
5.24 r 0.21 59.17 4 7.24 35.00 2 7.07
6.44 r 0.40’ 33.29 _t 6.08d 19.00 2 2.45
962.43 k 1.38 1.86 _’ 0.34 31.00 !I 1.51 1.71 _c 0.42 3.00 r 0.00 47.20 ‘- 2.01 1.57 +- 0.17 11.61 IC_0.20 27.94 4 0.89 0.95 -c-0.05
980.57 ” 1.33e 1.00 * 0.22 16.00 & 1.18’ 1.00 t 0.22 1.43 k 0.20e 54.94 5 1.9Od 0.86 +- 0.14e 11.50 ? 0.27 34.25 f 1.40” 1.13 -c 0.06”
a Fish (Colisafasciatus) were exposed to 35 ppmof chromium trioxide for 90 hr. All values in the table represent the mean t- SE (IV = 6). * Per thousand cells of all types. c P i 0.05.
d P -=c0.01. e P < 0.001, Student’s t test.
EFFECTS
OF CHROMIUM
Oh’ FISH BLOOD
323
exposed to acutely sublethal concentrations of nickel and copper. Similar increases in the circulating erythrocytes were reported in teleosts exposed to cobalt (Frovola, 1960) or on exposure to pulpmill effluent (McLeay, 1975). The increased red blood cell count probably reflects a hypoxic stress exposure resulting in secondary polycythemia. This increase in the total erythrocyte count also explains the retardation of erythrocyte sedimentation rate. The leukopenia in Co&a following exposure to chromium is mainly due to a decrease in small lymphocytes; lymphopenia has also been reported in this fish exposed to stressful conditions (Srivastava and Agrawal, 1977) or on acute exposure to sublethal concentrations of cobalt (Srivastava and Agrawal, 1979). Occurrence of leukopenia (predominantly lymphopenia) has been reported in other teleosts exposed to toxicants (McLeay and Gordon, 1977). The retarded blood clotting time in Colisa after exposure to chromium may have resulted mainly from a decrease in the number of circulating thrombocytes which function in the fish blood clotting process (Doolittle and Surgenor, 1962). Srivastava (1969) reported that clotting rate in teleosts was an apparent function of the number of thrombocytes present. He demonstrated that the higher the number of thrombocytes present, the shorter the blood clotting in several teleosts. A retardation in clotting time and thrombocytopenic response has also been reported in salmonids subjected to chemicals arising from industrial effluents (Iwama et al., 1976; McLeay, 1973). The increased hepatosomatic index due to stress is a generalized feature of fish and is related to the internal detoxification processes (Eisler and Kiss& 1971). The results of the study suggest that besides leukopenia, an indicator of generalized stress response, erythrocytosis and hypocoagulability of whole blood may be used in monitoring chromium toxicity in fish. REFERENCES ADELMAN, I. R., SMITH, L. L., AND SIESENNOP, G. D. (1976). Acute toxicity of sodium chloride, pentachlorophenol, Guthion, and hexavalent chromium to fathead minnows (Pimephales promelas) and goldfish (Curassius auratus). .I. Fish. Res. Bd. Canad. 33, 203-208. DOOLITTLE, R. F., AND SURGENOR, D. M. (1962). Blood coagulation in fish. Amer. J. Physiol. 203, 964-970. EISLER, R., AND KISSIL, G. W. (1975). Toxicities of crude oils and oil dispersant mixtures to juvenile rabbitfish, Siganus rivulatus. Trans. Amer. Fish. Sot. 104, 571-578. FROVOLA, L. K. (1960). The effect of inorganic cobalt on the morphological picture of the blood of Cyprinus
carpio.
Dokl.
Akad.
Nauk
SSSR
131, 983-984.
GREENBERG, A., et al. (eds.) (1975). Standard Methods for the Examination of Water and Wastewater, 14th ed. Amer. Pub. Health Ass., Washington, D.C. IWAMA, G. K., GREER, G. L., AND LARKIN, P. A. (1976). Changes in some hematological characteristics of coho salmon (Oncorhynchus kisutch) in response to acute exposure to dehydroabietic acid (DHAA) at different exercise levles. J. Fish. Res. Bd. Cunad. 33, 285-289. KRENKEL, P. A. (1974). Sources and classification of water pollutants. In Industrial Pollution (N. I. Sax, ed.), pp. 197-219. Van Nostrand Reinhold, New York. MCLEAY, D. J. (1973). Effects of cortisol and dexamethasone on the pituitary-interrenal axis and abundance of white blood cell types in juvenile coho salmon, Oncorhynchus kisutch. Gen. Camp. Endocrinol. 21, 441-450. MCLEAY, D. J. (1975). Sensitivity of blood cell counts in juvenile coho salmon (Oncorhynchus kisutch) to stressors including sublethal concentration of pulpmill effluent and zinc. J. Fish. Res. Bd. Cunad. 32, 2357-2364. MCLEAY, D. J., AND GORDON, M. R. (1977). Leucocrit: A simple hematological techique for measuring acute stress in salmonid fish, including stressful concentrations of pulpmill effluent. J. Fish. Res. Bd. Cunud. 34, 2164-2175.
324
SRIVASTAVA,
AGRAWAL,
AND CHAUDHRY
SRIVASTAVA, A. K. (1968a). Studies on the hematology of certain fresh water teleosts. I. Erythrocytes. Anat. Anz. 123, 233-249. SRIVASTAVA, A. K. (1968b). Studies on the hematology of certain fresh water teleosts. IV. Hemoglobin. Folia Haematol. (Leipzig) 90, 411-418. SRIVASTAVA, A. K. (1%9). Studies on the hematology of certain fresh water teleosts. V. Thrombocytes and the clotting of blood. Anat. Anz. 124, 368-374. SRIVASTAVA, A. K., AND AGRAWAL, U. (1977). Involvement of pituitary-interrenal axis and cholinergic mechanism during the cold-shock leucocyte sequence in a fresh water tropical teleost, Colisa fascia&s. Arch. Anat. Microsc. Morphol. Exp. 66, 97-108. SRIVASTAVA, A. K., AND AGRAWAL, S. J. (1979). Hematological anomalies in a fresh water teleost, Colisa fasciatus, on acute exposure to cobalt. Acta Pharmacol. Toxicol. 44, 197-199. SRIVASTAVA, A. K., AND MISHRA, S. (1979). Blood dyscrasia in a teleost (Colisa fasciatus) following acute exposure to sublethal concentration of lead. J. Fish. Biol. 14, 199-203. STOKES, R. M., AND FROMM, P. 0. (1965). Effects of chromate on glucose transport by the gut of rainbow trout. Physiol. 2001. 38, 202-205.
Effects
AND
ENVIRONMENTAL
SAFETY
of Chromium
ANIL Department
on the Blood
K. SRIVASTAVA, of Zoology,
3, 321-324 (1979)
S. J. AGRAWAL,
University Received
of Gorakhpur, February
of a Freshwater
Teleost
AND H. S. CHAUDHRY Gorakhpur
273 001, U.P.,
India
26, 1979
Exposure to a sublethal concentration of 35 ppm chromium induced pronounced increases in the population of circulating erythrocytes, hematocrit, clotting time, and hepatosomatic index at 90 hr post-treatment in a freshwater teleost, Colisa fasciatus. Leukopenia due to reduction in the number of small lymphocytes, thrombocytopenia, and a significant increase in the erythrocyte sedimentation rate were also noted in fish subjected to exposure to the metal; hemoglobin and the number of circulating immature erythrocytes and large lymphocytes were not affected. The data suggest that hematology can serve as an economical model for the study of toxic effects caused by chromium.
Chromium is a common pollutant of natural waters primarily from the discharge of industrial wastes. While C1-3+is considered a minor problem, CP is very toxic (Krenkel, 1974). In general, studies using fish have been confined to the determination of lethal concentrations (L& values) of hexavalent chromium (Adelman et al., 1976) with little information available on the effects of sublethal doses of this metal (Stokes and Fromm, 1965). This study was undertaken to determine if some hematological characteristics of a freshwater teleost, Colisa fasciatus, would change following acute exposure to sublethal levels of hexavalent chromium in the form of chromium trioxide (CrO,). Hematology of this fish has proven valuable in monitoring stress responses (Srivastava and Agrawal, 1977), including those due to exposure to sublethal levels of heavy metals (Srivastava and Agrawal, 1979; Srivastava and Mishra, 1979). McLeay and Gordon (1977) have also suggested that changes in the blood may well represent the most definitive test in determining physiological responses of fish to toxic substances. MATERIALS
AND METHODS
Adult female specimens (weight, 5.12 + 0.42 g) of Colisa fasciatus were used in the study. The fish, captured locally, were kept for 10 days under natural photoperiod in glass aquaria containing tap water. They were fed powdered dried shrimp daily, but food was withheld during the experimental period. The properties of the tap water were as follows: temperature, 25 +- 1°C; pH, 7.3; electrical conductivity, 1433.33 -t 33.33 @U/cm; dissolved oxygen content, 6.8 ppm; hardness, 120 mg/liter as CaCO,. Six groups of 12 fish each were exposed for 90 hr to chromium as chromium trioxide at a sublethal concentration of 35 ppm. A sample of 6 fish from each group of survivals (approximately 70%) was selected for blood analysis. The 96-hr LC,, value, determined concurrently by graphical interpolation in plotting percentage survival vs log CrO, concentration (Greenberg et al., 1975), was 40 ppm. Each fish was anesthetized with MS 222, blotted dry with absorbant
322
SRIVASTAVA, AGRAWAL, AND CHAUDHRY
paper, and weighed. Fresh blood samples were collected from the caudal artery by transection of the caudal peduncle of the fish. The hematological techniques have been described previously (Srivastava, 1968a,b,1969; Srivastava and Mishra, 1979). In one of the treated groups of fish, body and liver weights were recorded for calculating the hepatosomatic index (liver wt/body wt x 100). RESULTS The effects of the administration of an acutely sublethal concentration of chromium on the blood of Colisa are presented in Table 1. The total erythrocyte count, number of red blood cells per 1000 cells, and hematocrit value for fish were elevated significantly from control values after 90 hr exposure to 35 ppm of chromium trioxide. Erythrocyte sedimentation rate (ESR), total leukocyte count, and number of small lymphocytes per 1000 cells were significantly lower in the treated fish than corresponding values for the untreated group. The number of thrombocytes per 1000 cells was also significantly depressed following acute exposure to the metal; the total thrombocyte count was not altered significantly, but it showed a trend toward decreasing (P < 0.1) at 90 hr subsequent to exposure to the toxicant. The values for number of immature erythrocytes and of large lymphocytes per 1000 cells and the hemoglobin were not altered appreciably by metal exposure. A marked increase in hepatosomatic index after exposure of the fish to the metal was also observed. DISCUSSION The increases in the number of circulating red blood cells and the hematocrit in Colisa are comparable with unpublished observations made by us on this fish TABLE
1
EFFECT OF CHROMIUM ON HEMATOLOGICAL OF A FRESHWATER TELEOST~
Parameter Erythrocytes (X 106/mm3) Leukocytes (X lo3/mm3) Thrombocytes (X 103/mm3) Total differential cell counts0 Mature erythrocytes Immature erythrocytes Small lymphocytes Large lymphocytes Thrombocytes Hematocrit (%) ESR (mm/hr) Hemoglobin (g%) Clotting time (set) Hepatosomatic index
Control (water)
VALUES
Experimental (chromium)
5.24 r 0.21 59.17 4 7.24 35.00 2 7.07
6.44 r 0.40’ 33.29 _t 6.08d 19.00 2 2.45
962.43 k 1.38 1.86 _’ 0.34 31.00 !I 1.51 1.71 _c 0.42 3.00 r 0.00 47.20 ‘- 2.01 1.57 +- 0.17 11.61 IC_0.20 27.94 4 0.89 0.95 -c-0.05
980.57 ” 1.33e 1.00 * 0.22 16.00 & 1.18’ 1.00 t 0.22 1.43 k 0.20e 54.94 5 1.9Od 0.86 +- 0.14e 11.50 ? 0.27 34.25 f 1.40” 1.13 -c 0.06”
a Fish (Colisafasciatus) were exposed to 35 ppmof chromium trioxide for 90 hr. All values in the table represent the mean t- SE (IV = 6). * Per thousand cells of all types. c P i 0.05.
d P -=c0.01. e P < 0.001, Student’s t test.
EFFECTS
OF CHROMIUM
Oh’ FISH BLOOD
323
exposed to acutely sublethal concentrations of nickel and copper. Similar increases in the circulating erythrocytes were reported in teleosts exposed to cobalt (Frovola, 1960) or on exposure to pulpmill effluent (McLeay, 1975). The increased red blood cell count probably reflects a hypoxic stress exposure resulting in secondary polycythemia. This increase in the total erythrocyte count also explains the retardation of erythrocyte sedimentation rate. The leukopenia in Co&a following exposure to chromium is mainly due to a decrease in small lymphocytes; lymphopenia has also been reported in this fish exposed to stressful conditions (Srivastava and Agrawal, 1977) or on acute exposure to sublethal concentrations of cobalt (Srivastava and Agrawal, 1979). Occurrence of leukopenia (predominantly lymphopenia) has been reported in other teleosts exposed to toxicants (McLeay and Gordon, 1977). The retarded blood clotting time in Colisa after exposure to chromium may have resulted mainly from a decrease in the number of circulating thrombocytes which function in the fish blood clotting process (Doolittle and Surgenor, 1962). Srivastava (1969) reported that clotting rate in teleosts was an apparent function of the number of thrombocytes present. He demonstrated that the higher the number of thrombocytes present, the shorter the blood clotting in several teleosts. A retardation in clotting time and thrombocytopenic response has also been reported in salmonids subjected to chemicals arising from industrial effluents (Iwama et al., 1976; McLeay, 1973). The increased hepatosomatic index due to stress is a generalized feature of fish and is related to the internal detoxification processes (Eisler and Kiss& 1971). The results of the study suggest that besides leukopenia, an indicator of generalized stress response, erythrocytosis and hypocoagulability of whole blood may be used in monitoring chromium toxicity in fish. REFERENCES ADELMAN, I. R., SMITH, L. L., AND SIESENNOP, G. D. (1976). Acute toxicity of sodium chloride, pentachlorophenol, Guthion, and hexavalent chromium to fathead minnows (Pimephales promelas) and goldfish (Curassius auratus). .I. Fish. Res. Bd. Canad. 33, 203-208. DOOLITTLE, R. F., AND SURGENOR, D. M. (1962). Blood coagulation in fish. Amer. J. Physiol. 203, 964-970. EISLER, R., AND KISSIL, G. W. (1975). Toxicities of crude oils and oil dispersant mixtures to juvenile rabbitfish, Siganus rivulatus. Trans. Amer. Fish. Sot. 104, 571-578. FROVOLA, L. K. (1960). The effect of inorganic cobalt on the morphological picture of the blood of Cyprinus
carpio.
Dokl.
Akad.
Nauk
SSSR
131, 983-984.
GREENBERG, A., et al. (eds.) (1975). Standard Methods for the Examination of Water and Wastewater, 14th ed. Amer. Pub. Health Ass., Washington, D.C. IWAMA, G. K., GREER, G. L., AND LARKIN, P. A. (1976). Changes in some hematological characteristics of coho salmon (Oncorhynchus kisutch) in response to acute exposure to dehydroabietic acid (DHAA) at different exercise levles. J. Fish. Res. Bd. Cunad. 33, 285-289. KRENKEL, P. A. (1974). Sources and classification of water pollutants. In Industrial Pollution (N. I. Sax, ed.), pp. 197-219. Van Nostrand Reinhold, New York. MCLEAY, D. J. (1973). Effects of cortisol and dexamethasone on the pituitary-interrenal axis and abundance of white blood cell types in juvenile coho salmon, Oncorhynchus kisutch. Gen. Camp. Endocrinol. 21, 441-450. MCLEAY, D. J. (1975). Sensitivity of blood cell counts in juvenile coho salmon (Oncorhynchus kisutch) to stressors including sublethal concentration of pulpmill effluent and zinc. J. Fish. Res. Bd. Cunad. 32, 2357-2364. MCLEAY, D. J., AND GORDON, M. R. (1977). Leucocrit: A simple hematological techique for measuring acute stress in salmonid fish, including stressful concentrations of pulpmill effluent. J. Fish. Res. Bd. Cunud. 34, 2164-2175.
324
SRIVASTAVA,
AGRAWAL,
AND CHAUDHRY
SRIVASTAVA, A. K. (1968a). Studies on the hematology of certain fresh water teleosts. I. Erythrocytes. Anat. Anz. 123, 233-249. SRIVASTAVA, A. K. (1968b). Studies on the hematology of certain fresh water teleosts. IV. Hemoglobin. Folia Haematol. (Leipzig) 90, 411-418. SRIVASTAVA, A. K. (1%9). Studies on the hematology of certain fresh water teleosts. V. Thrombocytes and the clotting of blood. Anat. Anz. 124, 368-374. SRIVASTAVA, A. K., AND AGRAWAL, U. (1977). Involvement of pituitary-interrenal axis and cholinergic mechanism during the cold-shock leucocyte sequence in a fresh water tropical teleost, Colisa fascia&s. Arch. Anat. Microsc. Morphol. Exp. 66, 97-108. SRIVASTAVA, A. K., AND AGRAWAL, S. J. (1979). Hematological anomalies in a fresh water teleost, Colisa fasciatus, on acute exposure to cobalt. Acta Pharmacol. Toxicol. 44, 197-199. SRIVASTAVA, A. K., AND MISHRA, S. (1979). Blood dyscrasia in a teleost (Colisa fasciatus) following acute exposure to sublethal concentration of lead. J. Fish. Biol. 14, 199-203. STOKES, R. M., AND FROMM, P. 0. (1965). Effects of chromate on glucose transport by the gut of rainbow trout. Physiol. 2001. 38, 202-205.
