ECOTOXICOLOGY
AND
ENVIRONMENTAL
SAFETY
19,99- 105 (1990)
In Vivo Effect of the Organophosphorus Insecticide Trichlorphon on Immune Response of Carp (Cyprinus carpio) II. Effect of High Doses of Trichlorphon on Nonspecific Immune Response ANDRZEJ K. SIWICKI,* MURIEL COSSARINI-DUNIER,~ MARIA STUDNICKA,* AND ANDREE DEMAEL$ *Inland Fisheries Institute in Olsztyn, Departments in Zabieniec, 05-500 Piaseczno, Poland; TLaboratoire d’Ecotoxicologie, INRA. Ecole Nationale V&%naire de Lyon, 69280 Marcy L ‘Etoile, France; and SLaboratoire de Physiologie G&&ale et Cornparke, Universith Claude Bernard, Lyon, 69622 Villeurbanne Cedex, France Received May 2. I989 The effect of trichlorphon, one of the most widely used organophosphorus insecticides, on the nonspecific immune response in carp (Cyprinus carpio) was studied. The effect of 20,000 ppm trichlorphon on the immune response was followed for 3 and 56 days after intoxication. The effect of 10,000 ppm trichlorphon on the nonspecific immune response of carp experimentally infected by Pseudomonas alcaligenes and Aeromonaspunctata was also examined. Leucocyte number, phagocytic ability of neutrophils, percentage NBT-positive PMN cells, phagocytic index, lysozyme level in serum, and ceruloplasmin activity in plasma were examined on Days 2, 4, 6, 8, 10, 14, 18, 22, and 26 after carp were exposed. After intoxication leukopenia was observed as decreases in phagocytic ability of neutrophils and in phagocytic index. Lysozyme level in serum was also decreased compared to that of control. The percentage of NBT-positive PMN cells decreased when the ceruloplasmin activity in plasma increased in intoxicated fish. 0 1990 Academic
Press. Inc.
INTRODUCTION The organophosphorus insecticides are the most widely used substances employed in agriculture and in fish culture in ponds (Cossarini-Dunier et al., 1990). Studies concerning the effects of organophosphorus insecticides on fish health have concerned such aspects as toxicity for fish, kinetics of elimination from the fish organism after treatment, and determination of the necessary latency period (Studnicka, 1970; Hoffman and Mayer, 1974; Studnicka and Sopinska, 1983). Also examined were the effects of organophosphorus insecticides on different biochemical and physiological parameters of carp (Sakaguchi, 1972; Jeney and Jeney, 1986). Organophosphorus compounds are known to produce a specific inhibition of acetylcholinesterase, which in some cases is accompanied by the inhibition of neuro target esterase (Studnicka, 1970; Repetto et al., 1988; Jeney and Jeney, 1986). No study has been made on the effects of organophosphorus insecticides on the nonspecific immune response in carp. Nonspecific immunity in fish depends on a complex of factors and mechanisms. Phagocytosis is the primary mechanism of the nonspecific immune response of fish to pathogenic factors such as bacteria, viruses, and parasites. The phagocytic cells constitute important homeostatic and immunological factors in fish. Among them many cells have phagocytic activity, the most important being macrophages and polymorphonuclear (PMN) leucocytes. Two of the methods used to assessthe phago99
0147-6513/90 $3.00 Copyright 0 1990 by Academic Press, Inc. All rights ofreproduction in any form reserved.
100
SIWICKI
ET
AL.
cytic activity of leucocytes are the nitroblue tetrazolium (NBT) index (Siwicki et al., 1985) and the phagocytic index (Avtalion and Shahrabani, 1975; Siwicki, 1989). Lysozyme is an element of the nonspecific humoral response in fish. Lysozyme, a hydrolytic enzyme exerting bacteriolytic properties, is present in mucus, serum and, phagocytic cells (Fletcher and White, 1973; Studnicka et al., 1986). Ceruloplasmin is an (Yeglobulin of plasma which contains ferroxidase and functions in copper binding as well as affecting genetic variation. The physiological function of plasma ceruloplasmin is unknown, but it is an indicator of the acuteness of a disease, an “acute-phase reactant” (Rice, 1960; Siwicki and Studnicka, 1987). This study was performed to examine the effect of the organophosphorus insecticide trichlorphon on the nonspecific immune response in carp with and without an experimental infection. MATERIALS Animals and Experimental
AND
METHODS
Design
First experiment. Carp (Cyprinus carpio), each weighing about 100 g, were the same as those used by Cossarini-Dunier et al. (1990). Blood samples were collected from control and experimental groups of carp on Days 3 and 56 after fish were contaminated in 20,000 ppm trichlorphon solution for 15 min. Second experiment. Two hundred and seventy healthy carp (Cyprinus carpio), each weighing loo-150 g, were examined. Fish were distributed into two groups of 135 carps each and were kept at 20°C. They were maintained in 500-liter tanks. Fish from group I were kept in 10,000 ppm trichlorphon solution for 30 min and fish from group II were control. On Day 7 after intoxication, blood samples were collected from 10 fish from each group and all fish were injected intraperitoneally with Pseudomonas alcaligenes and Aeromonaspunctata ( 1: 1,400 X lo6 in 0.2 ml-‘). Blood samples were collected from the dorsal aortas of 10 fish of each group 2,4,6,8, 10, 14, 18,22, and 26 days after the injection of bacteria. Methods. Total leucocyte numbers, phagocytic ability of neutrophils (NBT index), percentage of nitroblue tetrazolium-positive PMN cells, phagocytic index, lysozyme level in serum, and ceruloplasmin activity in plasma were determined. The NBT index was performed according to the spectrophotometric method (Siwicki et al., 1985). Extinction was determined in l-cm cuvettes, at a wave length of 545 nm (spectrophotometer Specol 10, Carl Zeiss Jena, GDR). Simultaneously, the total number of leucocytes was assessed using a hemocytometer, and stained blood smears were prepared and examined in order to calculate the absolute numbers of neutrophils in the blood. Percentage of NBT-positive PMN cells was determined by cytochemistry methods (Szczylik et al., 1979; Siwicki, 1989). The percentage of NBT-positive cells was determined by treating slide smears. The phagocytic index was determined by a cytochemistry method (Avtalion and Shahrabani, 1975; Siwicki, 1989). The lysozyme level in serum was measured using the turbidimetric assay (Studnicka et al., 1986). The standard used was hen egg white lysozyme (Sigma). Lysozyme activities were measured at 450 nm using an Eskalab spectrophotometer (Smith Kline Instruments Co., U.S.A.). The ceruloplasmin activity in plasma was determined according to the method of Siwicki and Studnicka (1987). The freshly prepared 0.1% p-phenylenediamine (PPD)
EFFECT OF TRICHLORPHON
ON CARP, II
101
TABLE 1 PERIPHERAL BLEND LEUUXYTE NUMBERS, PHAG~CYTIC ABILITY OF NEUTROPHILS (NBT INDEX), LYSOZYME LEVEL IN SERUM, AND CERULOPLASMIN ACTIVITY IN PLASMA OF CARP AFTER A 2%TRICHLORFON BATH FOR 15 min
NBT index
Lysozyme in serum (rkcml-‘)
Ceruloplasmin in plasma (mg. 100ml-‘)
Group
Day
Leucocytes (x10-3)
Control Assay
3 3
40.5 f 3.4 34.2 + 3.3”
0.41 f 0.07 0.28 t 0.04”
0.73 f 0.23 0.41 + 0.12”
17.6f 0.62 20.1 + 0.86”
Control Assay
56 56
35.7 f 3.5 31.6k2.5”
0.39 e 0.09 0.38 t 0.04
0.55 + 0.07 0.32 k 0.08”
16.9f 0.9 18.4f 1.07
Note. Mean f SE. ’ P < 0.01 in Scheffe’stest.
in acetate buffer at pH 5.2 and 0.02% sodium azide were used. After 15 min of incuba-
tion (plasma with a buffered solution of PPD), the ceruloplasmin activity was measured spectrophotometrically (525 nm) using an Eskalab spectrophotometer-alpha (Smith Kline Instrument Co.). Statistical analysis. An analysis of variance was done on the areas under the kinetic curves. Each individual kinetic was integrated and the Fvalues of Snedecor and Bartlett’s tests were used to determine whether the difference observed between groups was statistically significant. RESULTS
First experiment. The values for number of leucocytes, NBT index, lysozyme activity level in serum, and ceruloplasmin activity in plasma after intoxication with 20,000 ppm trichlorphon are presented in Table 1. On Day 3 after intoxication the total leucocyte number, NBT index, and lysozyme level in serum were significantly diminished (P < 0.05) when the ceruloplasmin activity in plasma increased. On Day 56 the total leucocyte number and lysozyme level in serum diminished, compared to those of the control group (statistically significant, P < 0.05), but the NBT index and ceruloplasmin activity in plasma were equivalent. Second experiment. On Day 6 after intraperitoneal injection of bacteria some fish from experimental group I developed skin lesions like ecchymosis, shallow ulceration, and anal redness. These symptoms had increased by Day 18 (65% fish). Twenty percent of the fish from the control group (group II) presented these symptoms after 14 days. On Days 18,22, and 26 after infection, mortality of fish from the experimental group (25%) was observed, whereas in fish from the control group the ulceration tended to cicatrize and no mortality occurred. In intoxicated fish immune response was signincantly reduced after injection of live bacteria, compared to that of controls. The kinetics of alteration in total leucocyte number are presented in Fig. l., the phagocytic ability of neutrophils (NBT index) in Fig 2., the percentage of NBT-positive PMN cells in Fig. 3., the phagocytic index in Fig 4., lysozyme level in serum in Fig. 5., and the ceruloplasmin activity in plasma in Fig 6. From the time of experimental infection, the leucocyte number, NBT index, percentage NBT-positive PMN cells, phagocytic index, and lysozyme level in serum were
102
SIWICKI
20’
0
10
ET AL.
Days
20
30
FIG. 1. Kinetics of leucocyte number in blood after injection of bacteria on Day 0 to Day 26 (mean f SE, n = 10). (0) Control; (+) after intoxication with 1% trichlorphon.
significantly diminished (P < 0.05) in fish from group I compared to those of the control group; there was a significant increase (P < 0.05) in the ceruloplasmin activity in plasma, compared to that of control. DISCUSSION In the present work, the immunotoxic effect of trichlorphon baths (20,000 ppm and 10,000 ppm) on the nonspecific immune response of carp was studied. After in vivo contamination, leukopenia was observed and a decrease in phagocytic ability of neutrophils in parallel with a decrease in lysozyme level in serum. In intoxicated fish (10,000 ppm trichlorphon), nonspecific immune response was reduced after experimental infection with live bacteria and a prolonged immunosuppressive effect was
10
Days
20
30
FIG. 2. Kinetics of NBT index in blood after injection of bacteria on Days 0 to Day 26 (mean + SE, n= 10). (El) Control; (+) after intoxication with 1% trichlorphon.
EFFECT OF TRICHLORPHON
10
Days
103
ON CARP, II
20
4 30
FIG.3. Kinetics of percentage NBT-positive PMN cells in blood after injection of bacteria on Day 0 to Day 26 (mean + SE, n = 10). (0) Control; (+) after intoxication with 1% trichlorphon.
observed. Results similar to those observed in the control group in phagocytic ability of neutrophils, level of lysozyme in serum, and leucocyte numbers have been observed in carp infected with P. alcaligenes and A. punctata by Siwicki and Studnicka ( 1987). The highest activity of ceruloplasmin in plasma was found in correlation with disease conditions in the fish. Enhanced activity of ceruloplasmin has been observed in carp infected with bacteria (Siwicki and Studnicka, 1987). Cossarini-Dunier et al. (1990) studied the effect on carp of high doses (20,000 ppm) of trichlorphon in viva; no effect on antibody production was observed. The lymphoid organs were not significantly contaminated as analyzed by acetylcholinesterase activity.
FIG.4. Kinetics of phagocytic index in blood after injection of bacteria on Day 0 to Day 26 (mean + SE, n = 10). (El) Control; (+) after intoxication with 1% trichlorphon.
104
SIWICKI
10
ET AL.
Days
20
30
FIG.5. Kinetics of lysozyme level in serum after injection of bacteria on Day 0 to Day 26 (mean f SE, n = 10). (Cl) Control; (+) after intoxication with 1% trichlorphon.
After intoxication with high doses of trichlorphon, chemical stress was observed in carp, due to hypoglycemia and an increase in the level of adrenalin and noradrenalin in brain and heart (Jeney and Jeney, 1986). Trichlorphon specifically inhibited acetylcholinesterase (ACHE) (Holland et al., 1967; Studnicka, 1970; Jeney and Jeney, 1986) and decreased ACHE in brain and plasma of carp-proof of the very toxic effect of trichlorphon on the central nervous system. The immune system is important for defense against a variety of pathogenic agents. It is a highly evolved system and is distributed throughout the body. The cellular units of this system, primarily lymphocytes, neutrophils, and macrophages, are found in
L E
40-
T
8r . g 5 B
30.
*;
20.
2 5t 2 s
1010
’ o
Days
2 ’
7 30
FIG.6. Kinetics ofceruloplasmin activity in plasma after injection of bacteria on Day 0 to Day 26 (mean + SE, n = 10). (0) Control: (+) after intoxication with 1% trichlorphon.
EFFECT OF TRICHLORPHON
ON CARP, II
105
most organs and are regulated by a variety of multistep control processes. The system is also sensitive to homeostatic adjustments via endocrine regulation and is influenced by the biochemical status of the nervous system. Cellular cooperations and interactions are necessary for the ultimate response of this system. In the present study we observed the cytotoxic effect of trichlorphon on the immunocompetent cells in blood. The immunosuppressive effect was found in correlation with disease conditions in the carp. On Day 6 after experimental infection the disease appeared and on Days 18 to 26 mortality of fish was observed, whereas no mortality was observed in the control group and the ulceration tended to cicatrize. Intoxication with trichlorphon in carp leads to an immunosuppressive effect due to chemical toxicity. This work has investigated the toxicity of high doses of trichlorphon on fish, and it is hoped that, as a consequence, stricter control of the use of trichlorphon in the treatment of parasitic diseases in fish will be implemented. REFERENCES AVTALION, R. R., AND SHAHRABANI, R. (197.5). Studies on phagocytosis in fish. Immunology 29,118 1. COSSARINI-DUNIER, M., DEMAEL, A., AND SIWICKI, A. (1990). In vivo effect of the organophosphorus
insecticide trichlorphon on the immune response of carp (Cyprinus carpio). I. Effect of contamination on antibody production in relation to residue level in organs. Ecotoxicol. Environ. Suf, (Part I) 19. FLETCHER, T. C., AND WHITE, A. (1973). Lysozyme activity in the plaice (Pleuronectesplutessu L.). Experimentia 29, 1283-1285. HOFFMAN, G. L.. AND MAYER, F. P. (1974). Parasites of Freshwater Fishes: A Review of Their Control and Treatment. TFH Publications. Neptune, NJ. HOLLAND, H. T., COPPAGE, D. L., AND BUTLER, P. A. (1967). Use of fish brain acetylcholinesterase to monitor pollution by organophosphorus pesticides. Bull. Environ. Contam. Toxicol. 2, 156-162. JENEY, Z., AND JENEY, G. (1986). Studies on the effect oftrichlorphon on different biochemical and physiological parameters of common carp (Cyprinus carpio L.). Aquacultura Hungarica (Szarvas) 5,79-89. REPETTO, G., SANZ, P., AND REPETTO, M. (I 988). In vivo and in vitro effect of trichlorfon on esterasesof the red crayfish Procambarus clarkii. Bull. Environ. Contam. Toxicol. 41,597-603. RICE, E. W. (1960). Correlation between serum copper, ceruloplasmin activity and C-reactive protein. Clin. Chim. Acta 5,632-636. SAKAGUCHI. H. (1972). On the effects of agricultural chemicals upon fish. I. Changes of chemical components in serum and liver of carp exposed to organophosphate compounds. BUN. Japan. Sot. Sci. Fish. 38,555-560. SIWICKI. A., STUDNICKA,
M., AND RYKA, B. (1985). Phagocytic ability of neutrophils in carp (Cyprinus carpio L.). Bamidgeh 37, 123-l 28. SIWICKI, A.. STUDNICKA, M., AND RYKA, B. (1986). Ceruloplasmin activity in carp (Cyprinus carpio L.). Bamidgeh 38, 126- 129. SIWICKI, A., AND STUDNICKA, M. (1987). The phagocytic ability ofneutrophils and serum lysozyme activity in experimentally infected carp, Cyprinus carpio L. J. Fish Biol. 31,57-60. SIWICKI, A. (1989). Immunostimulating influence of levamisole on non-specific immunity in carp (Cyprinus carpio). Dev. Comp. Immunol. 13(I), 87-92. STUDNICKA, M. (1970). Investigation on the remanants of the phospho-organic compound Foschlor in tissues of fishes subjected to the treatment with this preparation. Pol. Arch. Weter. 13, 107- 12 I. STUDNICKA, M., AND SOPINSKA. A. (1983). Toxicity of technical Foschlor for fish. Rocz. Nauk Roln. 2, 111-116. STUDNICKA, M., SIWICKI, A., AND RYKA, B. (1986). Lysozyme level in carp (Cyprinus carpio L.). Bamidgeh 1,22-25. SZCZYLIK, C., GORNAS, P., ANDCAREWICZ, R. (1979). Test redukcji NBT-metodyka i praktyczne zastosowanie. Diagn. Lab. 1,35-42.
AND
ENVIRONMENTAL
SAFETY
19,99- 105 (1990)
In Vivo Effect of the Organophosphorus Insecticide Trichlorphon on Immune Response of Carp (Cyprinus carpio) II. Effect of High Doses of Trichlorphon on Nonspecific Immune Response ANDRZEJ K. SIWICKI,* MURIEL COSSARINI-DUNIER,~ MARIA STUDNICKA,* AND ANDREE DEMAEL$ *Inland Fisheries Institute in Olsztyn, Departments in Zabieniec, 05-500 Piaseczno, Poland; TLaboratoire d’Ecotoxicologie, INRA. Ecole Nationale V&%naire de Lyon, 69280 Marcy L ‘Etoile, France; and SLaboratoire de Physiologie G&&ale et Cornparke, Universith Claude Bernard, Lyon, 69622 Villeurbanne Cedex, France Received May 2. I989 The effect of trichlorphon, one of the most widely used organophosphorus insecticides, on the nonspecific immune response in carp (Cyprinus carpio) was studied. The effect of 20,000 ppm trichlorphon on the immune response was followed for 3 and 56 days after intoxication. The effect of 10,000 ppm trichlorphon on the nonspecific immune response of carp experimentally infected by Pseudomonas alcaligenes and Aeromonaspunctata was also examined. Leucocyte number, phagocytic ability of neutrophils, percentage NBT-positive PMN cells, phagocytic index, lysozyme level in serum, and ceruloplasmin activity in plasma were examined on Days 2, 4, 6, 8, 10, 14, 18, 22, and 26 after carp were exposed. After intoxication leukopenia was observed as decreases in phagocytic ability of neutrophils and in phagocytic index. Lysozyme level in serum was also decreased compared to that of control. The percentage of NBT-positive PMN cells decreased when the ceruloplasmin activity in plasma increased in intoxicated fish. 0 1990 Academic
Press. Inc.
INTRODUCTION The organophosphorus insecticides are the most widely used substances employed in agriculture and in fish culture in ponds (Cossarini-Dunier et al., 1990). Studies concerning the effects of organophosphorus insecticides on fish health have concerned such aspects as toxicity for fish, kinetics of elimination from the fish organism after treatment, and determination of the necessary latency period (Studnicka, 1970; Hoffman and Mayer, 1974; Studnicka and Sopinska, 1983). Also examined were the effects of organophosphorus insecticides on different biochemical and physiological parameters of carp (Sakaguchi, 1972; Jeney and Jeney, 1986). Organophosphorus compounds are known to produce a specific inhibition of acetylcholinesterase, which in some cases is accompanied by the inhibition of neuro target esterase (Studnicka, 1970; Repetto et al., 1988; Jeney and Jeney, 1986). No study has been made on the effects of organophosphorus insecticides on the nonspecific immune response in carp. Nonspecific immunity in fish depends on a complex of factors and mechanisms. Phagocytosis is the primary mechanism of the nonspecific immune response of fish to pathogenic factors such as bacteria, viruses, and parasites. The phagocytic cells constitute important homeostatic and immunological factors in fish. Among them many cells have phagocytic activity, the most important being macrophages and polymorphonuclear (PMN) leucocytes. Two of the methods used to assessthe phago99
0147-6513/90 $3.00 Copyright 0 1990 by Academic Press, Inc. All rights ofreproduction in any form reserved.
100
SIWICKI
ET
AL.
cytic activity of leucocytes are the nitroblue tetrazolium (NBT) index (Siwicki et al., 1985) and the phagocytic index (Avtalion and Shahrabani, 1975; Siwicki, 1989). Lysozyme is an element of the nonspecific humoral response in fish. Lysozyme, a hydrolytic enzyme exerting bacteriolytic properties, is present in mucus, serum and, phagocytic cells (Fletcher and White, 1973; Studnicka et al., 1986). Ceruloplasmin is an (Yeglobulin of plasma which contains ferroxidase and functions in copper binding as well as affecting genetic variation. The physiological function of plasma ceruloplasmin is unknown, but it is an indicator of the acuteness of a disease, an “acute-phase reactant” (Rice, 1960; Siwicki and Studnicka, 1987). This study was performed to examine the effect of the organophosphorus insecticide trichlorphon on the nonspecific immune response in carp with and without an experimental infection. MATERIALS Animals and Experimental
AND
METHODS
Design
First experiment. Carp (Cyprinus carpio), each weighing about 100 g, were the same as those used by Cossarini-Dunier et al. (1990). Blood samples were collected from control and experimental groups of carp on Days 3 and 56 after fish were contaminated in 20,000 ppm trichlorphon solution for 15 min. Second experiment. Two hundred and seventy healthy carp (Cyprinus carpio), each weighing loo-150 g, were examined. Fish were distributed into two groups of 135 carps each and were kept at 20°C. They were maintained in 500-liter tanks. Fish from group I were kept in 10,000 ppm trichlorphon solution for 30 min and fish from group II were control. On Day 7 after intoxication, blood samples were collected from 10 fish from each group and all fish were injected intraperitoneally with Pseudomonas alcaligenes and Aeromonaspunctata ( 1: 1,400 X lo6 in 0.2 ml-‘). Blood samples were collected from the dorsal aortas of 10 fish of each group 2,4,6,8, 10, 14, 18,22, and 26 days after the injection of bacteria. Methods. Total leucocyte numbers, phagocytic ability of neutrophils (NBT index), percentage of nitroblue tetrazolium-positive PMN cells, phagocytic index, lysozyme level in serum, and ceruloplasmin activity in plasma were determined. The NBT index was performed according to the spectrophotometric method (Siwicki et al., 1985). Extinction was determined in l-cm cuvettes, at a wave length of 545 nm (spectrophotometer Specol 10, Carl Zeiss Jena, GDR). Simultaneously, the total number of leucocytes was assessed using a hemocytometer, and stained blood smears were prepared and examined in order to calculate the absolute numbers of neutrophils in the blood. Percentage of NBT-positive PMN cells was determined by cytochemistry methods (Szczylik et al., 1979; Siwicki, 1989). The percentage of NBT-positive cells was determined by treating slide smears. The phagocytic index was determined by a cytochemistry method (Avtalion and Shahrabani, 1975; Siwicki, 1989). The lysozyme level in serum was measured using the turbidimetric assay (Studnicka et al., 1986). The standard used was hen egg white lysozyme (Sigma). Lysozyme activities were measured at 450 nm using an Eskalab spectrophotometer (Smith Kline Instruments Co., U.S.A.). The ceruloplasmin activity in plasma was determined according to the method of Siwicki and Studnicka (1987). The freshly prepared 0.1% p-phenylenediamine (PPD)
EFFECT OF TRICHLORPHON
ON CARP, II
101
TABLE 1 PERIPHERAL BLEND LEUUXYTE NUMBERS, PHAG~CYTIC ABILITY OF NEUTROPHILS (NBT INDEX), LYSOZYME LEVEL IN SERUM, AND CERULOPLASMIN ACTIVITY IN PLASMA OF CARP AFTER A 2%TRICHLORFON BATH FOR 15 min
NBT index
Lysozyme in serum (rkcml-‘)
Ceruloplasmin in plasma (mg. 100ml-‘)
Group
Day
Leucocytes (x10-3)
Control Assay
3 3
40.5 f 3.4 34.2 + 3.3”
0.41 f 0.07 0.28 t 0.04”
0.73 f 0.23 0.41 + 0.12”
17.6f 0.62 20.1 + 0.86”
Control Assay
56 56
35.7 f 3.5 31.6k2.5”
0.39 e 0.09 0.38 t 0.04
0.55 + 0.07 0.32 k 0.08”
16.9f 0.9 18.4f 1.07
Note. Mean f SE. ’ P < 0.01 in Scheffe’stest.
in acetate buffer at pH 5.2 and 0.02% sodium azide were used. After 15 min of incuba-
tion (plasma with a buffered solution of PPD), the ceruloplasmin activity was measured spectrophotometrically (525 nm) using an Eskalab spectrophotometer-alpha (Smith Kline Instrument Co.). Statistical analysis. An analysis of variance was done on the areas under the kinetic curves. Each individual kinetic was integrated and the Fvalues of Snedecor and Bartlett’s tests were used to determine whether the difference observed between groups was statistically significant. RESULTS
First experiment. The values for number of leucocytes, NBT index, lysozyme activity level in serum, and ceruloplasmin activity in plasma after intoxication with 20,000 ppm trichlorphon are presented in Table 1. On Day 3 after intoxication the total leucocyte number, NBT index, and lysozyme level in serum were significantly diminished (P < 0.05) when the ceruloplasmin activity in plasma increased. On Day 56 the total leucocyte number and lysozyme level in serum diminished, compared to those of the control group (statistically significant, P < 0.05), but the NBT index and ceruloplasmin activity in plasma were equivalent. Second experiment. On Day 6 after intraperitoneal injection of bacteria some fish from experimental group I developed skin lesions like ecchymosis, shallow ulceration, and anal redness. These symptoms had increased by Day 18 (65% fish). Twenty percent of the fish from the control group (group II) presented these symptoms after 14 days. On Days 18,22, and 26 after infection, mortality of fish from the experimental group (25%) was observed, whereas in fish from the control group the ulceration tended to cicatrize and no mortality occurred. In intoxicated fish immune response was signincantly reduced after injection of live bacteria, compared to that of controls. The kinetics of alteration in total leucocyte number are presented in Fig. l., the phagocytic ability of neutrophils (NBT index) in Fig 2., the percentage of NBT-positive PMN cells in Fig. 3., the phagocytic index in Fig 4., lysozyme level in serum in Fig. 5., and the ceruloplasmin activity in plasma in Fig 6. From the time of experimental infection, the leucocyte number, NBT index, percentage NBT-positive PMN cells, phagocytic index, and lysozyme level in serum were
102
SIWICKI
20’
0
10
ET AL.
Days
20
30
FIG. 1. Kinetics of leucocyte number in blood after injection of bacteria on Day 0 to Day 26 (mean f SE, n = 10). (0) Control; (+) after intoxication with 1% trichlorphon.
significantly diminished (P < 0.05) in fish from group I compared to those of the control group; there was a significant increase (P < 0.05) in the ceruloplasmin activity in plasma, compared to that of control. DISCUSSION In the present work, the immunotoxic effect of trichlorphon baths (20,000 ppm and 10,000 ppm) on the nonspecific immune response of carp was studied. After in vivo contamination, leukopenia was observed and a decrease in phagocytic ability of neutrophils in parallel with a decrease in lysozyme level in serum. In intoxicated fish (10,000 ppm trichlorphon), nonspecific immune response was reduced after experimental infection with live bacteria and a prolonged immunosuppressive effect was
10
Days
20
30
FIG. 2. Kinetics of NBT index in blood after injection of bacteria on Days 0 to Day 26 (mean + SE, n= 10). (El) Control; (+) after intoxication with 1% trichlorphon.
EFFECT OF TRICHLORPHON
10
Days
103
ON CARP, II
20
4 30
FIG.3. Kinetics of percentage NBT-positive PMN cells in blood after injection of bacteria on Day 0 to Day 26 (mean + SE, n = 10). (0) Control; (+) after intoxication with 1% trichlorphon.
observed. Results similar to those observed in the control group in phagocytic ability of neutrophils, level of lysozyme in serum, and leucocyte numbers have been observed in carp infected with P. alcaligenes and A. punctata by Siwicki and Studnicka ( 1987). The highest activity of ceruloplasmin in plasma was found in correlation with disease conditions in the fish. Enhanced activity of ceruloplasmin has been observed in carp infected with bacteria (Siwicki and Studnicka, 1987). Cossarini-Dunier et al. (1990) studied the effect on carp of high doses (20,000 ppm) of trichlorphon in viva; no effect on antibody production was observed. The lymphoid organs were not significantly contaminated as analyzed by acetylcholinesterase activity.
FIG.4. Kinetics of phagocytic index in blood after injection of bacteria on Day 0 to Day 26 (mean + SE, n = 10). (El) Control; (+) after intoxication with 1% trichlorphon.
104
SIWICKI
10
ET AL.
Days
20
30
FIG.5. Kinetics of lysozyme level in serum after injection of bacteria on Day 0 to Day 26 (mean f SE, n = 10). (Cl) Control; (+) after intoxication with 1% trichlorphon.
After intoxication with high doses of trichlorphon, chemical stress was observed in carp, due to hypoglycemia and an increase in the level of adrenalin and noradrenalin in brain and heart (Jeney and Jeney, 1986). Trichlorphon specifically inhibited acetylcholinesterase (ACHE) (Holland et al., 1967; Studnicka, 1970; Jeney and Jeney, 1986) and decreased ACHE in brain and plasma of carp-proof of the very toxic effect of trichlorphon on the central nervous system. The immune system is important for defense against a variety of pathogenic agents. It is a highly evolved system and is distributed throughout the body. The cellular units of this system, primarily lymphocytes, neutrophils, and macrophages, are found in
L E
40-
T
8r . g 5 B
30.
*;
20.
2 5t 2 s
1010
’ o
Days
2 ’
7 30
FIG.6. Kinetics ofceruloplasmin activity in plasma after injection of bacteria on Day 0 to Day 26 (mean + SE, n = 10). (0) Control: (+) after intoxication with 1% trichlorphon.
EFFECT OF TRICHLORPHON
ON CARP, II
105
most organs and are regulated by a variety of multistep control processes. The system is also sensitive to homeostatic adjustments via endocrine regulation and is influenced by the biochemical status of the nervous system. Cellular cooperations and interactions are necessary for the ultimate response of this system. In the present study we observed the cytotoxic effect of trichlorphon on the immunocompetent cells in blood. The immunosuppressive effect was found in correlation with disease conditions in the carp. On Day 6 after experimental infection the disease appeared and on Days 18 to 26 mortality of fish was observed, whereas no mortality was observed in the control group and the ulceration tended to cicatrize. Intoxication with trichlorphon in carp leads to an immunosuppressive effect due to chemical toxicity. This work has investigated the toxicity of high doses of trichlorphon on fish, and it is hoped that, as a consequence, stricter control of the use of trichlorphon in the treatment of parasitic diseases in fish will be implemented. REFERENCES AVTALION, R. R., AND SHAHRABANI, R. (197.5). Studies on phagocytosis in fish. Immunology 29,118 1. COSSARINI-DUNIER, M., DEMAEL, A., AND SIWICKI, A. (1990). In vivo effect of the organophosphorus
insecticide trichlorphon on the immune response of carp (Cyprinus carpio). I. Effect of contamination on antibody production in relation to residue level in organs. Ecotoxicol. Environ. Suf, (Part I) 19. FLETCHER, T. C., AND WHITE, A. (1973). Lysozyme activity in the plaice (Pleuronectesplutessu L.). Experimentia 29, 1283-1285. HOFFMAN, G. L.. AND MAYER, F. P. (1974). Parasites of Freshwater Fishes: A Review of Their Control and Treatment. TFH Publications. Neptune, NJ. HOLLAND, H. T., COPPAGE, D. L., AND BUTLER, P. A. (1967). Use of fish brain acetylcholinesterase to monitor pollution by organophosphorus pesticides. Bull. Environ. Contam. Toxicol. 2, 156-162. JENEY, Z., AND JENEY, G. (1986). Studies on the effect oftrichlorphon on different biochemical and physiological parameters of common carp (Cyprinus carpio L.). Aquacultura Hungarica (Szarvas) 5,79-89. REPETTO, G., SANZ, P., AND REPETTO, M. (I 988). In vivo and in vitro effect of trichlorfon on esterasesof the red crayfish Procambarus clarkii. Bull. Environ. Contam. Toxicol. 41,597-603. RICE, E. W. (1960). Correlation between serum copper, ceruloplasmin activity and C-reactive protein. Clin. Chim. Acta 5,632-636. SAKAGUCHI. H. (1972). On the effects of agricultural chemicals upon fish. I. Changes of chemical components in serum and liver of carp exposed to organophosphate compounds. BUN. Japan. Sot. Sci. Fish. 38,555-560. SIWICKI. A., STUDNICKA,
M., AND RYKA, B. (1985). Phagocytic ability of neutrophils in carp (Cyprinus carpio L.). Bamidgeh 37, 123-l 28. SIWICKI, A.. STUDNICKA, M., AND RYKA, B. (1986). Ceruloplasmin activity in carp (Cyprinus carpio L.). Bamidgeh 38, 126- 129. SIWICKI, A., AND STUDNICKA, M. (1987). The phagocytic ability ofneutrophils and serum lysozyme activity in experimentally infected carp, Cyprinus carpio L. J. Fish Biol. 31,57-60. SIWICKI, A. (1989). Immunostimulating influence of levamisole on non-specific immunity in carp (Cyprinus carpio). Dev. Comp. Immunol. 13(I), 87-92. STUDNICKA, M. (1970). Investigation on the remanants of the phospho-organic compound Foschlor in tissues of fishes subjected to the treatment with this preparation. Pol. Arch. Weter. 13, 107- 12 I. STUDNICKA, M., AND SOPINSKA. A. (1983). Toxicity of technical Foschlor for fish. Rocz. Nauk Roln. 2, 111-116. STUDNICKA, M., SIWICKI, A., AND RYKA, B. (1986). Lysozyme level in carp (Cyprinus carpio L.). Bamidgeh 1,22-25. SZCZYLIK, C., GORNAS, P., ANDCAREWICZ, R. (1979). Test redukcji NBT-metodyka i praktyczne zastosowanie. Diagn. Lab. 1,35-42.
