Carp Blood after Zinc Exposure Short Original Communications The Changes in Common Carp Blood after Short-term Zinc Exposure Matgorzata Witeska* and Barbara Ko~ciuk Department of Animal Physiology, University of Podlasie, Prusa 12, 08-110 Siedlce, Poland * Correspondingauthor (wites@ap.siedlce.pl) DOh httD://dx.doi.orq/10.1065/espr2003.07.161 Abstract Blood zinc level, hematological parameters and blood cell morphology were evaluated in common carp immediately after 3 h exposure to 20 mg dm-3 of zinc (Zn0), and in 24, 48 and 96 hours after the end of it (Zn24, Zn48, Zn96). Blood zinc level in the non-exposed fish was 8 mg dm-3, reached a maximum of 20 mg dm-3 in Zn48, while it dropped to 9 mg dm-3 in Zn96. Zinc caused a stress reaction in fish indicated by an increase in hematocrit value in Zn0, and elevated plasma glucose level and trombocytosis which persisted until the end of the experiment. Zinc-exposed fish showed an increased frequency of abnormal erythrocytes, and a compensatory release of immature erythrocytes to the blood stream. In zinc-treated fish, leukocyte count initially increased and subsequently decreased significantly below the control level due to a drop in lymphocyte number. Lymphocyte viability was reduced, and abnormal lymphocytes appeared. A decreased count of juvenile neutrophiles, and reduced phagocyte activity also occurred. The results indicate possible zinc-induced disturbances in both specific and non-specific immune mechanisms. Keywords: Carp blood; erythrocytes; fish; hematology; leukocytes; metal; thrombocytes; toxicity; zinc exposure Introduction Zinc is an important micronutrient, present in natural waters at levels from 0.002 to 0.2 mg dm -2, whereas its concentrations may reach up to 43 mg dm -3 [1] in polluted ones near effluent discharge. Fish take up waterborne zinc mainly via the gills [2]. Median lethal concentrations of zinc (96LC50) for fish range from 3.6 pg dm -3 [3] to 87.9 mg dm -3 [4], and depend on various abiotic and biotic factors [5]. Sublethal zinc intoxication may change hematological parameters in fish, although no data are available on zinc deposition and dynamics in blood. Various changes in red blood cell count and hemoglobin content were observed [6,7,8,9] in fish intoxicated with 70 lag dm -3 - 100 mg dm -3 of zinc, while the same treatments always reduced white blood cell counts, and the share of lymphocytes. Zinc is also known to suppress specific and non-specific immune response in fish [10,11]. 284 The present study was undertaken to show zinc dynamics in fish blood, and the changes in the counts and morphology of common carp blood cells after short-term exposure to high concentrations of zinc. 1 Materials and Methods Common carps of an average body weight of 75 g harvested in October 1999 from the rearing pond were acclimated for a month to the laboratory conditions in the holding tank with water recirculation system, at 16-18~ Then, the fish were immersed for 3 hours in the ZnSO 4 solution in tap water (17~ pH 8.2, hardness 227 mg dm -3 as CaCO3) of nominal Zn concentration 20 mg dm -3, in groups of 10 fish per 20 dm 3 aquarium. After the exposure, the fish were transferred to clean tap water. Control fish were kept for 3 hours in tap water at the same density as experimental groups, and then also transferred to another tank. No mortalities were observed during the experiment. Blood was sampled from 10 fish of each group: control (K0), and zinc treatment immediately after the end of exposure (Zn0), and after 24, 48 and 96 h post exposure (Zn24, Zn48, and Zn96) by heart puncture. For blood zinc level measurements, 0.5-1.5 cm 3 of blood was subjected to hot wet digestion with H N O 3 and H202. Zinc concentrations were measured using the atomic absorption spectrophotometry technique (AAS). For each fish hematocrit value (Ht), red and white blood cell counts (RBC and WBC) were determined. Phagocytic ability was measured using the spectrophotometric NBT method. Lymphocyte viability was evaluated using trypan blue exclusion. Glucose level was measured using a spectrophotometric method with trichloracetic acid and o-toluidine reagent. Blood smears were stained according to Pappenheim, and viewed under 12.5 x 100 magnification. In each smear, 300 erythrocytes and 100 leukocytes were classified. Thrombocyte counts were estimated from the number of these cells in the smears per 100 leukocytes, and WBC value. The significance of differences of all measured parameters among the groups (at p<0.05) was tested with the least significant difference (LSD) test, using a Statistica package. 2 Results The blood zinc concentration in the non-exposed fish was equal to 8 mg dm -3. In Zn0, a significant increase up to ESPR - Environ Sci & Pollut Res 10 (5) 284 - 286 (2003) 9 ecomed publishers, D-86899 Landsberg, Germany and Ft. Worth/TX 9Tokyo 9Mumbai 9Seoul ~ Melbourne * Paris Short Original Communications Carp Blood after Zinc Exposure 14 mg dm -3 occurred, and the maximum value of 20 mg dm -3 was noted in Zn48. In Zn96, the blood zinc level was 9 mg dm -3 and did not significantly differ from the control. Hematocrit of the zinc-exposed fish was transiently elevated in the Zn0 group (Table 1). RBC values did not significantly differ among the groups. An increased frequency of abnormal erythrocytes occurred only in the Zn0 group. The abnormalities included irregular cell shape, indented nucleus, amitosis or swelling. The number of juvenile erythrocytes temporarily increased in the zinc-exposed fish, showing a maximum in Zn24. White blood cell count was significantly elevated in the Zn0 group, and reduced in the Zn48 group, as compared to the control. Lymphocyte number initially increased, and subsequently dropped signficantly below the control level (Zn24, Zn48). Viability of lymphocytes was reduced in Zn0 and Zn48. In Zn24 and Zn48, a number of damaged leukocytes increased significantly. They included mainly lymphocytes of irregular shape or divided nucleus. Neutrophile count increased immediately after zinc exposure, and then dropped to a level similar to the controls. Among the neutrophiles, juvenile stages distinctly predominated in the control, and just after the end of zinc exposure (Zn0). In Zn24Zn96, the count of juvenile neutrophiles decreased, and a shift towards PMN occurred. Monocyte number significantly increased in Zn24 and remained elevated until the end of the experiment. Phagocytic ability was reduced in Zn96. Thrombocyte counts were significantly higher in zinc-exposed fish as compared to the controls (except for Zn48). Glucose level was elevated in Zn24 and Zn48 (Table 1). 3 Discussion No data were found in the literature on the zinc concentrations and dynamics in full blood of fish. In the present study, the blood zinc level increased after the end of exposure and reached a maximum in 48 hours after the end of exposure, which is in accordance with the data obtained by [12] for mercury dynamics in rainbow trout. This is also consistent with the general pattern of metal transport in the fish body [5]. Waterborne metal is initially bound to the gills, subsequently taken up by blood, and then may be deposited in other tissues, even if toxic agent is removed from the water. Temporary Ht increase, together with elevated thrombocyte counts and glucose levels in zinc-exposed fish, as compared to the control, may indicate that zinc induced a typical stress reaction in fish. No significant differences in the RBC suggest little effect of zinc on the carp erythrocytes. However, temporary increases in abnormal and juvenile cell numbers indicate some cellular disturbances, quickly compensated by a supply of new cells. Zinc might alter the properties of erythrocyte cell mem- Table 1: The changes in blood parameters in common carps exposed for 3 hours to 20 mg dm-3 of zinc 26.5-+3.5 30.3+4.7 28.9• .98+_.32 1.06_+.56 1.06+_.28 .09+-.05* .02-+.01 .02+_.02 .01+.01 .21+_.09" .40• .25+_.15" .09+_.06 76.4+_11.8 58.3-+12.2" 99.3+_16.0 43.3• 28.1+_5.8" 81.3+_14.4 8.6-+3.0 31.6• Ht[%] hematocrit RBC [106 mm-3] red blood cell count .92-+.20 .95+-.27 EAbn [106 mm-3] abnormal erythrocytes .01• EJuv [106 mm-3] juvenile .10-+.05 24.6• erythrocytes WBC [103 dm-3] white blood cell count 83.6+_14.2 LYM [103 dm-3] lymphocytes 67.1• 112.2• 86.9• NEU [103 dm~3] neutrophiles 13.8• 21.1-+16.6" 10.4-+2.0 10.0-+4.3 NJuv [103 dm -3] myelocytes + metamyelocytes 11.1• 18.6+_15.6* 7.0+_1.9 3.7+_1.9" 4.8+_1.7 PMN [103 dm -3] neutrophiles with elongated nucleus + neutrophiles with lobed nucleus, 2.7-+1.4 2.5-+1.5 3.4-+0.8 6.3• 3.9+_2.3 MONO [103 dm-3] monocytes 2.0• 2.2+_1.2 5.7+_2.1" 7.5• 6.1 +2.3* 14.9+_5.1" 11.6+_3.6* LDam [103 dm-3] damaged leukocytes THRO [103 dm-3] thrombocytes NBT [g dm4] phagocytic ability LymVia [%] viability of lymphocytes Glucose [mg dm-3] 1.7_+0.7 32.9• .98• 95• 538• 3.0+2.0 64.3• 62.7• 1.05+_.07 .88+_.14 81 +3* n.d. 721 +_178 799+_295* 37.5• 1.07+_.09 90• 935• 3.3+_1.0 60.7+_9.6* .79-+.13" 92+_4 n.d. mean for 10 fish • S.D. * different from the control at p<0.05 n.d.: no data ESPR - Environ Sci & Pollut Res 10 (5) 2003 285 Carp Blood after Zinc Exposure branes rendering them more fragile and permeable, which probably resulted in cell swelling (observed in the smears, and indicated by an increase of Ht value), deformation and damage. According to [13], zinc causes damage to the membrane lipid layer. A significant drop in WBC and lymphocyte counts, reduced lymphocyte viability, and the presence of abnormal cells indicate cytotoxic effect of zinc upon carp lymphocytes. Zincinduced decrease in white blood cell count was reported by [6,7,8,14], and a reduction in the blood lymphocyte levels was noted by [6,7,8]. According to [15], in vitro zinc treatment reduced kidney lymphocyte count and their cytotoxic activity in zebrafish, while [16] observed that zinc reduced proliferation of common carp lymphocytes, both in vitro and in vivo. That was confirmed by the results obtained by [11] who observed total suppression of in vitro lymphocyte proliferation at 0.75-200 mg dm -3 of zinc. An increase in plasma zinc level in humans resulted in a significant impairment of lymphocyte functions [17]. The increase in neutrophile count in Zn0 was related to a raise in the juvenile cell number. In the subsequent samples - Zn24 and Zn48 - the numbers of these cells gradually decreased. That suggests a transient inhibition of granulopoiesis. A significant reduction of phagocyte activity observed in Zn96 indicates an impairment of their function. Monocyte count started to increase later than granulocyte number, and remained significantly elevated until the end of the experiment, which might have been related to zincinduced damage of blood cells or other fish tissues. The data on the effect of zinc on non-specific immune mechanisms in fish are scarce. According to [6,7], an increase in P M N cell count occurred after acute zinc exposure. According to [15], zinc enhanced the macrophage response, while [11] revealed that the effect of zinc on kidney macrophages was dose-dependent (stimulation at 50-100 mg dm -3, and suppression at 6-12 and over 200 mg dm-3). Zinc may also cause a reduction of chemotactic migration and bacteria ingestion by P M N cells [17]. The results of the present study show that even a short-term exposure of fish to high concentrations of zinc may cause considerable changes in their blood due to metal absorption and deposition in the organism. Such changes may develop and persist after removal of toxic agent from the water. It seems that red blood cell count is little sensitive to intoxication, and may be easily compensated, while the white blood cell system is particularly susceptible to zinc intoxication, which results in disturbances in both specific and non-specific immune mechanisms. Such disturbances may result in impaired resistance of fish to diseases. Short Original Communications [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] Andros JD, Garton RR (1980): Acute lethality of copper, cadmium and zinc to northern squawfish. 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Wydawnictwo Ekonomia i Srodowisko, 84-87 [In Polish] Kock G, Bucher F (1997) Accumulation of zinc inrainbow trout (Oncorhynchus mykiss) after waterborne and dietary exposure. Bull Environ Contam Toxicol 58, 305-310 Received: March 31 st, 2003 Accepted: July 18th, 2003 Onlinefirst: July 19th, 2003 ESPR - Environ Sci & Pollut Res 10 (5) 2003