Proceedings of International Symposium on Environmental Pollution Control and Waste Management 7- January 02, Tunis (EPCOWM 02), p.38-42. Sensitivity of some freshwater fishes for water pollutants Abbas N. BALASEM, Abdul-Sahib K. ALI and Amal J. MUTAR Fish Research Center, P.O. Box 765, Tuwaitha, Baghdad, Iraq INTRODUCTION Ecosystems are vulnerable to various kinds of pollutants such as organic materials heavy metals, radioactive substances petroleum and its products, thermal pollution and domestic wastes. It is estimated that abut 0 000 organic materials, including pesticides, are available in the nature and more substances are introduced continuously. Millions of tons of these substances have been produced every years and a large quantity of these substances finds it way to the surface water eventually. Most of these materials are foreign to the nature and emerge deleterious effects to various kinds of organisms. The phenomenon of accumulation of these materials, leading to long term effects, makes the situation even more complicated. The second most dangerous group of pollutants are heavy metals which some of them exceeded their maximum permissible limits in the surface water in some locations. Heavy metals such as mercury, copper, lead, cadmium and zenic are regarded highly toxic to man and living systems. On other hand radioactive materials are another source of contamination to the environment. These dangerous substances may be resulted from nuclear accidents, atomic bomb tests, radioactive waste and other related activities. Oil may reach the surface as well as the underground water during exploration, extraction, transport, storage and refining of crude oil. Once it reaches the water it is almost impossible to recover the oil compleldy. Finally, domestic wastes and thermal pollution are among the important pollutants to the environment. There are many physical, chemical and biological methods to detect water pollutants and their effects on the biological systems. Some fish species were used as biological indicators for water pollution because of many reasons i.e. 1. Fishes are utilized a relatively large quantity of water during respiration as well as feeding, 2. Many pollutants could be accumulated in the fish organs, 3. They are inexpensive and easily handled, 4. Large number of fish can be used experimentally (Kligerman 1981). In the present report, the sensitivity of five species of fishes to copper was observed. Hematological and cytogenetic investigations were made on the most sensitive species of these fishes. MATERIALS AND METHODS Fishes Five species of freshwater fishes were employed in the present experiment namely: Liza abu, Barbus luteus, Cyprinus carpio, Carassius carassinus and Gambusia affinis. More emphasis was made on L. abu based on a previous findings in this laboratory showing the sensitivity of this species for some water pollutants. The Median Lethal Concentration (LC 50 ) Six groups of L. abu were exposed to various concentrations of copper i.e.,, 0.3, 0.4, 0.5 and 0.6 ppm, 38
Balasem et al. respectively. The exposure time was hours at 23-24 o C, ph 6.5-7.5. An additional group of L.abu was used as a control. After the determination of LC 50 of copper to L.abu, the other above-mentioned four species of fishes were exposed to that concentration of the metal at similar conditions of exposure. The Micronucleus Assay The frequencies of micronuclei (MN) in red blood cells (RBCs) obtained from various groups of fishes were conducted according to the methods described by Al-Sabti (1991) with some modification. In brief, blood samples were withdrawn from the caudal veins by heparinized sterile 1-ml plastic syringes. Blood smears were made, dried and fixed with 99.8% methanol for five minutes. The fixed preparations then stained with Giemsa for minutes. Microscopy was made at 00X and at least 00 RBCs were observed for the presence of MN. Haematological examinations Red blood cell (RBC) counts, white blood cell (WBC) counts, packed cell volumes (PCV) and haemoglobin (Hb) concentrations were measured. The methods described by ( Blaxhall and Daislly, 1973) were used to carry out these tests. RESULTS AND DISCUSSION After exposing of L. abu to various concentrations of copper ranged from zero to 0.6 ppm for hrs at 23-24 C. The LC 50 was found to 0.4 ppm (Table 1 ). Four species of fishes namely: Barbus luteus, Cyprinus carpio, Carassius carassinus and Gambusia affinis were exposed to 0.4 ppm of copper at similar conditions to those mentioned above. None of these fishes were died during the hours of exposure (Table 2). It may be concluded that at the conditions of the present experiment, L. abu is more sensitive to copper than any of the species described above. In a previous study in this laboratory (Ali et al., 1999) L. abu was found to be more sensitive to arsenic than any of eight species of freshwater fishes namely Barbus sharpeyi, B.xanthopterus,B.grypus,B.luteus,Cyprinuscarpio,Ctenopharyngodon idella, Carassius carassinus and Gambusia affinis. A follow-up study was made on two groups of fishes (L.abu) exposed to the lowest concentrations i.e. and ppm of copper. In this part of the study micronucleus assay was made on the above-mentioned groups and apparently healthy group. The assay was conduced on days th, th and 30 th after the end of exposure to reveal the presence of micronuclei (MN) in RBCs of the exposed fishes and controls. Table 3 shows that the frequencies of MN in RBCs were elevated significantly above the corresponding values in the control group. This elevation was persisted at least 30 days after the end of exposure. Micronucleus is though to be a whole chromosome or part of it lagged behind in the cytoplasm during mitosis.therefore micronucleus assay may be used as replacement for or in adjacent to chromosomal aberration analysis. Micronucleus assay is simpler and less labourous and needs less experience to perform in a comparison with chromosomal aberration test. However, chromosome complement of L. abu was found to be 50 chromosome which suggesting it as a suitable species to study the genotoxic effects of water pollutants (Balasem et al. 1999). Exposure of L. abu to concentrations of mercury (5, 0.005 and 0.01 ppm, respectively) led to increase the number of MN in RBC for at least eight weeks after the end exposure (Al-Tamimi, et al. 1999). 39
Sensitivity of fresh water fishes Table 1 : Mortality of L. abu exposed to Copper for hr at 23-24 C. Concentration (ppm) No. of Fish % of mortality 0.00 6 0% 0 6 0% 0. 6 0% 0.30 6 33.3% 0.40 6 50.0% 0.50 6 0.0% 0.60 6 0.0% Table 2 : Sensitivity of L. abu exposed to 0.4 ppm copper for hrs in a comparison with some other freshwater fishes. Species of Fish Total No. % of mortality Liza abu 6 50% Barbus luteus 6 0% Cyprinus carpio 6 0% Carassius carassius 6 0% Gambosia affinis 6 0% Table 3 : The frequency of MN in RBCs of L. abu exposes to copper. Days after the end of exposure Group 1 (Control) Micronuclei Per cell Group II (Exposed to ppm) Group III (Exposed to ppm) days 0 7 days 0.0013 0.0033 0.0037 30 days 0.0017 0.0037 0.0050 In previous studies in this laboratory it was found that the exposure of fish to chemical agents led to elevate the frequencies of micronuclei in RBC for various periods after the and of exposure ( Table 4). Regarding the haematological effects of copper on L. abu, table 5 shows no significant difference between the exposed groups and controls as red blood cell counts, packed cell volumes and haemoglobin concentration concerned. However fishes exposed to ppm of copper showed an elevation of white blood cell counts, in a comparison with non-exposed fishes, at th days after the end of exposure(table 3 ). White blood cells were reported to be increased in fishes exposed to some insecticides (Ibraheem et al., 00). The increase in the number of WBCs may be as a defense mechanism of the organism. However, some authors observed reductions in most blood parameters, including WBCs, in fish exposed to heavy metals (Al-Tamimi, 1999) or insecticides (Abdul-Ahad, 1999). The explanation for this discrepancy between these responses is unknown. 40
Balasem et al. Table 4: The Induction of micronuclei in RBC of fish after exposure to some chemical agents Agent Conc. (ppm) Duration of Exposure (hrs) Time of Test (Days after exposure) Species of Fishes MN/ cell MN/cell (Normal Value) Reference. As L. abu L. adu 0.0055 5 5 Ali, et al., 1999 Pb 48 5.0 48 0.5 48 CN - 1.0 48 C. carassius 0.0068 0.0078 0.0095 0.0060 C. carassius 0.0090 0.01 0.0058 0.0065 C. carassius 0.0054 0.0073 0.05 0.0058 C. carassius 0.00 0.0078 0.01 0.00 Balasem et al., 01a 0.0040 Balasem et al., 0l b 7 7 0.001 Cr 43 43 C. carpio 0.005 0.003 0.005 0.001 nom published Cu 60 60 30 30 L. abu 7 0.0033 0.0037 0.0037 0.0050 The present report Table 5 : Haematological values in groups of L. abu after days from the end of exposure to copper. (Hb : Haemoglobin concentration, PCV: Packed cell volume, WBCs: White blood cell counts, RBCs: Red blood cell counts). Tests Group 1 (Control) Group II (Exposed to ppm) Group III (Exposed to ppm) Hb (gm %) 3.4 3.3 PCV(%) 11.3 11.0.0 WBCs (Cell/mm 3 ) 74 100 RBCs(Cell/mm 3 ) 1.13X 6 1.X 6 1.23X 6 41
Sensitivity of fresh water fishes REFERENCES Al-Sabti, K. (1991). Handbook of Genotoxic Effects and Fish Chromosomes, Lujubljana pp: 221. Al-Tamimi, M.T.; A.N. Balasem and H.S. Ammash (1999). Cytogenetic, haematological and molecular study on Liza abu as a biological indicator for mercury pollution. Iraqi J. Agric. 4 (5): 142-150. Ali, A.S.K.; A.N. Balasem and A.J. Mutar (1999). A preliminary study on the sensitivity of Liza abu (Heckel) to arsenic in a comparison with eight species. of local and breeding fishes. The Veterinarian 9 (3): 35-42. Balasem, A.N.; A.S.K. Ali and A.J. Mutar (01a). The sensitivity of Carassius carassius to heavy metals pollution. II. Lead. Proceeding of the Forth Scientific. Conference, University of Al-Qadisiya, 28-29 th March, 01. Balasem, A.N.; A.J. Mutar and A.S.K. Ali.(01b). The frequencies of micronuclei in red blood cells of Carassius carassius exposed to cyanide. The 2 nd Symposium for Environmental Pollution, College of Science. Babylon University 14-15 th March, 01. Blaxhall, P. C. and Daislly, K. W. (1973). Routine haematological methods for use with fish blood. J. Fish Biology, 5:771-781. International Labor Office (19). Encyclopaedia of Occupational Heath and Safety Vol. I Geneva, Switzerland. (Copper pp 546-548). Kinne, O. (ed.) Marine Ecology Vol. V part3: pollution and protection of seas- Radioactive materials, Heavy Metals and Oil. John Wiley and sons. 1984, pp1618. Kligerman, A.D. (1981). The use of cytogenetics to study genotoxic agents in fishes. In Cytogenetic Assays of Environmental Mutagen, (Hsu, T.C and Totowa, N.J., eds.) Allanheld osmun, pp: 161-181. Mutar, A.J. and A.N. Balasem Cytogenetic and haematological effects of chromium on Common carp (nompublished). 42