INTRODUCTION IJT 2015;

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1 Volume 8, No 7, Winter 5 Acute Toxicity Behavioral Changes Gold Fish (Carassius Auratus) Exposed to Malathion Hinosan Saeid Shahbazi Naserabad *, Alireza Mirvaghefi, Mohammad Hasan Gerami, Hamed Ghafari Farsani Received: 5.7. Accepted:.8. Downloaded from ijt.arakmu.ac.ir at : +33 on Thursday January 3rd 9 ABSTRACT Background: Pesticides are widely used in agriculture. Excessive use pesticides has health risk for human threatens non-target organisms. This research aimed to determine lethal concentrations malathion Hinosan for Carassius auratus (5± gr) [mean ± SD]. Methods: Experiments were performed according to O.E.C.D for days (96 h) concentrations,,,, 8 mg L- Hinosan,,,, 6 mg L- malathion with three replicates. LC, LC, LC3, LC5, LC7, LC9 LC99 for, 8, 7 96 h were determined using a probit analysis. Results: The results indicated that 96 h LC5 value Hinosan malathion for Gold fish was..7 mg/l, respectively. Fishes exhibited irregular, erratic darting swimming movements, hyper excitability, bruise in caudal section, loss equilibrium sinking to bottom. Conclusion: Malathion Hinosan have medium toxicity for C. auratus could cause irreversible harm behavioral changes. Keywords: Carassius auratus, Hinosan, LC5, Malathion. IJT 5; 3-8 INTRODUCTION Aquatic ecosystems are largest natural environments which are constantly faced with threats such as deterioration in genetic biological diversity. Although, se environments are not targets for pesticides, previous studies sighted presence pesticides ir metabolites in surface waters [3]. The most commonly used organophosphates (OPs) are parathion, malathion, methyl parathion, chlorpyrifos, diazinon, dichlorvos phosmet. Malathion is an organophosphate pesticide that is widely used in agriculture, residential lscaping, public recreation areas, in public health pest control programs such as mosquito eradication []. Hinosan, anor organophosphate pesticide (Edifenphos; -ethyl S, S-diphenylphosphorodithioate) was marketed by Bayer AG in 968 to control Pyricularia oryzae on rice [5]. OPs are alkylating agents that chemically reaction with main cell macromolecules such as proteins, nucleic acids lipids disturb ir performance [6,7]. The main mechanism OPs is enzyme inhibition acetylcholinesterase which can cause disruption in central nervous system (CNS) peripheral nervous system. Goldfish (Carassius auratus) is a freshwater, benthopelagic fish family Cyprinidae order Cypriniformes which is one most commonly kept aquarium fish with a wide distribution in central Asia, China Japan.In Iran, it has been reported in Atrak, Gorgan, Gharasu, Tajan, Babol, Haraz, Sardab, Aras (including middle aras lower reaches its tributary in Qareh Chai), Tonekabon, Pol-e Rud, Safid rivers, Anzali Mordab where it is now largest population, Gorgan Bay, Boojagh Wetl Alma- Ala-Gol. The trade ornamental fish is a significant worldwide economic activity for aquaculture industry with wild caught freshwater fish being most commercialized species [8]. C.auratus is considered as most popular favorable aquarium fish in Iran approximately 5 million goldfish are reproduced during newyear's holidays (Nowruz) [9]. This study aimed to investigate lethal concentration (LC5 96-h) malathion. Department Fisheries, University Tehran, Karaj, Iran.. Department Fisheries, University Gonbad Kavous, Golestan, Iran. *Corresponding Author: saeid.shahbazi@ut.ac.ir

2 Downloaded from ijt.arakmu.ac.ir at : +33 on Thursday January 3rd 9 MATERIAL AND METHODS Two hundred fifty Carassius auratus with an average weight 5± g were acclimatized to laboratory condition in 8 3 cm aquarium for 5 days. In order to measure biological capability determine survival, specimens were kept in natural toxin-free environment to determine natural mortality. Physicochemical properties water used for se experiments were as follows: temperature ± ºC, dissolved oxygen 7 to 7.5 ppm, ph 7.5 to 8 CaCO3 5 mg/l. During main experiment, water was not changed. The specimens were fed twice a day with Biomar at % body weight, before test. All Experiments were performed on 6-h light 8 hours darkness. Fish behavior clinical signs were recorded. Experiments were performed according to OECD stard method [], to determine 96 h LC5 malathion Hinosan to Carassius auratus. To determine lethal concentration Hinosan fishes were divided to 5 treatments ( toxin control treatment) were exposed to concentrations,,,, 8 mg L-. For malathion specimens were exposed to 6 treatments (5 toxin control treatment) i.e.,,,, 6 mg L-. Mortality rates were recorded after, 8, 7 9 hours dead specimens were removed from aquarium. The nominal concentration toxin causing mortality (LC LC LC3 LC5 LC7 LC9 LC99) within, 8, 7 9 hours for each toxin were computed on basis probit analysis version 6/ with SPSS 8 []. Eventually, maximum allowable concentration (MAC), lowest observable effect concentration (LOEC) no observable effect concentration (NOEC) were also determined. RESULTS No mortality was observed in control group in main experiment during acclimatization. Hours after poison exposure absolute mortality (hundred percent) with Hinosan occurred in 8 ppm concentration; while, this value was 6 ppm for malathion (Table ). The values LC5 for malathion Hinosan in Carassius auratus were.7. mg L-, respectively (Table -). Behavioral changes clinical symptoms in fishes that were exposed to malathion started at 8 mg L- after 5 hours, but at 6 mg L- symptoms were observed after hours. These changes occurred for Hinosan toxicity in 8 mg L- after hours. The control group treatments with concentrations mg L- had normal behavior during experiment. Fishes with acute toxicity had asnia, imbalance, spiral swimming collapse to bottom aquarium. Abnormal swimming, increasing respiratory motions, bruise in caudal fin was symptoms Hinosan acute toxicity in fishes. No Observed Effect Concentration (NOEC) Lower Observed Effect Concentration (LOEC) are shown in Figure. Based on computed LC5 for every species, contaminants MAC in natural environments is. LC5. In fact, MAC is amount concentration in range concentrations a toxic substance in an environment that is between NOEC LOEC level. Therefore, calculated values were..7 mg/l for Hinosan malathion respectively, in ± C. In addition, LOEC which is lowest concentration or amount a toxic substance found by experiment or observation that causes an adverse effect on function, capacity, growth, development, or lifespan a target species under defined conditions exposure was calculated to be mg/l for both toxins. NOEC, which represent toxicant consecration that has no effect on organisms exposed to yields no statistically significant deviation from a control group; was determined to be mg/l for both toxins. 6 5 Hinosan(ppm) Hinosan in gold fish Carassius auratus. Due to proximity breeding places gold fish to rivers, rice paddies orchards, this study can to be useful for breeding gold fish. Saeid Shahbazi Naserabad et al 3 NOEC LOEC Acute Toxicity Test LC5 Figure. Acute toxicity test Hinosan on Carassius auratus. Volume 8, No 7, Winter 5;

3 Acute Toxicity Behavioral Changes Gold Malathion(ppm) 8 Table. Cumulative mortality goldfish (n=, each concentration) exposed acutely to Hinosan. 6 Downloaded from ijt.arakmu.ac.ir at : +33 on Thursday January 3rd 9 NOEC LOEC LC5 Acute Toxicity Test Figure. Acute toxicity test malathion on Carassius auratus. Mortality (No.) Concentration (ppm) control 8 Table. Lethal Concentrations (LC-99) Hinosan depending on time (-) for goldfish (mean ± SE). Point LC LC LC3 LC5 LC7 LC9 LC99 Concentration (ppm) (95 % confidence limits).95 ±.5.68 ±.8.68 ±.8.6 ± ± ± ± ±.7.5 ± ± ± ±.7.88 ±.5.9 ±.8.9 ±.8. ± ±.5. ±.8. ±.8.33 ± ±.5.87 ±.8.87 ±.8.78 ± ± ± ± ±.7 Table 3. Cumulative mortality goldfish (n=, each concentration) exposed acutely to malathion. Mortality (No.) 9 Concentration (ppm) control 8 6 Table. Lethal Concentrations (LC-99) malathion depending on time (-) for Goldfish (mean ± SE). Point LC LC LC3 LC5 LC7 LC9 LC99 Concentration (ppm) (95 % confidence limits) 5.9 ±.8 5. ± ± ± ± ±.7.5 ± ± ± ±.7 5. ±.36.9 ± ± ± ±.36.7 ± ± ±.7 6. ± ± ± ± ± ±.36.8 ± ± ± ± Volume 8, No 7, Winter 5

4 Downloaded from ijt.arakmu.ac.ir at : +33 on Thursday January 3rd 9 DISCUSSION Result LC5 for both toxins showed that it decreases with increasing toxin concentration duration exposure. It means that by increasing duration, lower concentrations toxins were required to kill 5% fish LC5 values in first hours experiment were more than end 96-hour LC5. Exposure time is one effective factors in OPs toxicity []. When fish are exposed to a constant concentration toxins, ir tolerance diminishes over time toxin has more time to affect. When toxin accumulates in fish tissues, adverse effects increase which reby causes a decrease in LC5. Overall, LC5 for both toxins used in C. auratus, showed a decreasing trend over 96 hours in listed physicochemical conditions. Previous studies on acute toxicity Diazinon Deltamethrin in Cyprinus carpio [3,] also reported similar decreasing trends in LC5. Contrasting results are limited on acute toxicity malathion Hinosan for Carassius auratus. Sailatha et al. observed LC5 for technical commercial grades malathion on Tilapia mossambica as ppm, respectively [5]. They declared that commercial grade malathion was more toxic than technical one. Forget et al. reported mortality LC5 values for several stages marine copepod Tigriopus brevicornis (Müller) exposed to malathion as.3 µg liter [6]. They indicated that dichlorvos was most toxic substance for T. brevicornis, followed by malathion. Pey et al. determined LC5 value for malathion in Channa punctatus to be 6.6 ppm [7]. They declared that LC5 value for malathion was higher than value reported (.8 mg/l) by Tsuda et al. at in killifish (Oryzias latipes) [8] Khangarot Ray reported a lower LC5 value (.738, ppm) at in C. punctatus [9]. Also acute toxicity malathion for some fishes like, Labeo rohita, Ptychocheilus Lucius, Colisa fasciatus Ctenopharyngodon idella were found to be.9, 3.7,..38 mgl/l, respectively [- 3]. Data on Hinosan toxicity in fish are limited too. Shamoushaki determined LC5 values Hinosan for Acipenser nudiventris as 8 ppb reported that A. nudiventris are Saeid Shahbazi Naserabad et al severely irritated by Hinosan []. Also Alinezhad determined LC5 values Hinosan for A. persicus A. stellatus as 37 ppb.6 ppb, respectively [5]. Behavior is considered a promising tool in ecotoxicology [6, 7]. In toxic environments, fish exhibited irregular, erratic darting swimming movements loss equilibrium which is due to inhibition acetylcholinesterase (AChE) activity leading to accumulation acetylcholine in cholinergic synapses causing hyperstimulation [8]. Pesticides are lipophilic are rapidly absorbed in fish gills which cause respiratory limitations [9]. They also attack neural organs resulting in imbalance, spiral swimming collapse to bottom aquarium. Our observed behavioral changes were in acordance with previous researches on organophosphorus pesticides [3-33] malathion on CtenoPharyngodon idella[6]. Change in respiration rate is one common physiological responses to toxicants []. Gulping air at surface swimming at water surface were also observed in acute toxicity by malathion in Labeo rohita [3]. Abnormal swimming pattern, intense opercular movements loss coordinated movement with fish lying on ir side in acute toxicity diazinon malathion was reported by Banaee et al. in Oncorhynchus mykiss [3], Uner et al. in Oreochromis niloticus [3], Pey et al. in Channa punctatus [7] Salwa El Ella in Ctenopharyngodon idella[6]. Maximum Allowable Concentration (MAC) in natural environments according to LC5 is. LC5 level. The value MAC for malathion Hinosan in mean temperature ± Cº were calculated as.7. mg/l, respectively. The lowest observable effect concentration (LOEC) is analogous to limit detection conventional methods analysis. LOEC represents initial toxicity threshold a chemical while NOEC represent concentration toxicant that will not cause any effect. Sensitivity bioassays, toxicity evaluation comparative evaluation effects pesticides was evaluated using LOEC values [35]. The acute toxicity response, LOEC NOEC are shown in Figure. Also, according to Table 5 (determination toxicity in different pesticides) both malathion Hinosan have medium toxicity for Carassius 6 Volume 8, No 7, Winter 5;

5 Acute Toxicity Behavioral Changes Gold auratus. Malathion Hinosan are abundant in many ecosystems are used extensively in farmls. Employing biological methods to reduce pests instead chemical pesticides could help to improve environment [36]. Downloaded from ijt.arakmu.ac.ir at : +33 on Thursday January 3rd 9 Table5. Determination toxicity in different pesticides. LC5 (mg/l) Up to Less to. Degree toxicity Nearly no poison Toxicity Low Toxicity Medium Toxicity High Toxicity Very high CONCLUSION Acute toxicity studies are first step in toxicological reaserch. Our results demonstrated that malathion Hinosan have harmful effects on behavior fish. This study showed that se toxicants at relatively low concentrations cause mortality in fishes even at short periods exposure. Thus, it can be concluded that Gold fishes are sensitive to Hinosan malathion, ir mortality rate is dose dependent, as well. ACKNOWLEDGEMENT The authors would like to sincerely thank Department Fisheries, Faculty Natural Resources, University Tehran, for supporting this research. REFERENCES. Tsuda T, Inoue T, Kojima M, Aoki S. Pesticides in water fish from rivers flowing into Lake Biwa. Bulletin environmental contamination toxicology. 996;57(3):-9.. Mansingh A, Wilson A. Insecticide contamination Jamaican environment III. Baseline studies on status insecticidal pollution Kingston Harbour. Marine pollution bulletin. 995;3(): Van Der Geest H, Stuijfz S, Kraak M, Admiraal W. Impact a diazinon calamity in 996 on aquatic macroinvertebrates in River Meuse, The Nerls. Nerl Journal Aquatic Ecology. 997;3(): Bonner MR, Coble J, Blair A, Freeman LEB, Hoppin JA, Sler DP, et al. Malathion exposure incidence cancer in agricultural health study. American journal epidemiology. 7;66(9): Scheinpflug H, Jung H. Use organophosphates for control fungal diseases crops. Bayer PflSchutz-Nachr. 968;: Kuroda K, Yamaguchi Y, Endo G. Mitotic toxicity, sister chromatid exchange, rec assay pesticides. Archives environmental contamination toxicology. 99;3(): Evenson D, Jost L. Sperm chromatin structure assay for fertility assessment. Current protocols in cytometry. : Andrews C. The ornamental fish trade fish conservation. Journal fish Biology. 99;37(sA): Coad BW, Abdoli A. Exotic fish species in fresh waters Iran. Zoology in Middle East. 993;9(): OECD. OECD Guidelines for Testing Chemicals: Organization for Economic; Finney DJ. Probit analysis; a statistical treatment sigmoid response curve Larkin DJ, Tjeerdema RS. Fate effects diazinon. Reviews environmental contamination toxicology. ;66: Svoboda M, Luskova V, Drastichova J, Žlabek V. The effect diazinon on haematological indices common carp (Cyprinus carpio L.). Acta Veterinaria Brno. ;7(): Svobodova Z, Luskova V, Drastichova J, Svoboda M, Žlábek V. Effect deltamethrin on haematological indices common carp (Cyprinus carpio L.). Acta Veterinaria Brno. 3;7(): Sailatha D, Sahib I, Rao K. Toxicity technical commercial grade malathion to fish, Tilapia mossambica (Peters). Proc Indian Acad Sci (Anim Sci). 98;9: Forget J, Pavillon J, Menasria M, Bocquene G. Mortality LC5 Values for Several Stages Marine Copepod Tigriopus brevicornis Müller) Exposed to Metals Arsenic Cadmium Pesticides Atrazine, Carburan, Dichlorvos, Malathion. Ecotoxicology environmental safety. 998;(3): Pey S, Kumar R, Sharma S, Nagpure N, Srivastava SK, Verma M. Acute toxicity bioassays mercuric chloride malathion on air-breathing fish Channa punctatus (Bloch). Ecotoxicology environmental safety. 5;6():-. 8. Tsuda T, Kojima M, Harada H, Nakajima A, Aoki S. Acute toxicity, accumulation excretion organophosphorous insecticides ir oxidation products in killifish. Chemosphere. 997;35(5): Khangarot B, Ray P. Studies on toxicity malathion to freshwater teleosts, Channa 7 Volume 8, No 7, Winter 5

6 . Downloaded from ijt.arakmu.ac.ir at : +33 on Thursday January 3rd punctatus (Bloch) Puntius sophore (Hamilton). Archiv für Hydrobiologie. 988;3(3):65-9. Beyers DW, Sikoski PJ. Acetylcholinesterase inhibition in federally endangered Colorado squawfish exposed to carbaryl malathion. toxicology chemistry. 99;3(6): Singh S, Tripathi P, Yadav R, Singh D, Singh A. Toxicity malathion carbaryl pesticides: effects on some biochemical priles freshwater fish Colisa fasciatus. Bulletin environmental contamination toxicology. ;7(3):59-9. Patil VK, David M. Behaviour respiratory dysfunction as an index malathion toxicity in freshwater fish, Labeo rohita (Hamilton). Turkish Journal fisheries aquatic sciences. 8;8:33-7. Salwa M, El Ella A. Toxicity malathion its effect on activity acetylcholinesterase in varioustissues grass carp, Ctenopharyngodon idella Val. Egyptian Journal Aquatic Biology Fisheries. 8;:9 7. Shamooshaki MM, Nezami SA, Sari AE, Khara H, Pazh Z, Garagorakei MY. Determination LC5 96 h Diazinon, Hinosan Tilt for Acipenser nudiventris fingerlings. Iranian Scientific Fisheries Journal. 8;:3.[Persian] Alinezhad R. Determining LC5 in Regent Insecticide, Hinosan Fungicide, Roundup Herbicide to A. Stellatus A. persicus. [MSc sis]. Lahijan Azad University.. Drummond RA, Russom CL. Behavioral toxicity syndromes: A promising tool for assessing toxicity mechanisms in juvenile faad minnows. toxicology chemistry. 99;9():37-6. Cohn J, MacPhail RC. Ethological experimental approaches to behavior analysis: implications for ecotoxicology. health perspectives. 996;():99-3. Saeid Shahbazi Naserabad et al 8. Mushigeri S, David M. Fenvalerate induced changes in Ach associated AchE activity in different tissues fish Cirrhinus mrigala (Hamilton) under lethal sub-lethal exposure period. toxicology pharmacology. 5;(): Masud S, Singh IJ. Temperature dependent toxicity behavioural responses in freshwater fish Cyprinus carpio exposed to pyrethroid pesticide, cypermethrin. Journal Science Water Resources. 3;(): Üner N, Oruç EÖ, Sevgiler Y, Şahin N, Durmaz H, Usta D. Effects diazinon on acetylcholinesterase activity lipid peroxidation in brain Oreochromis niloticus. toxicology pharmacology. 6;(3): Banaee M, Sureda A, Mirvaghefi A, Ahmadi K. Effects diazinon on biochemical parameters blood in rainbow trout (Oncorhynchus mykiss). Pesticide biochemistry physiology. ;99(): Rauf A, Arain N. Acute toxicity diazinon its effects on hematological parameters in Indian carp, Cirrhinus mrigala (Hamilton). Turkish Journal Veterinary Animal Sciences. 3;37(5): Shamoushaki M. Acute toxicity insecticide, Diazinon fungicide, Tilt (Propiconazole) on Pacific white Shrimp, Litopenaeus vannamei postlarva e Palaemon adspersus juveniles. Journal Research in Biology. ;: Ural M, Koprucu SS. Acute toxicity dichlorvos on fingerling European catfish, Silurus glanis. Bulletin environmental contamination toxicology. 6;76(5): Fernández-Alba A, Herno M, Piedra L, Chisti Y. Toxicity evaluation single mixed antifouling biocides measured with acute toxicity bioassays. Analytica chimica acta. ;56(): Moore A, Waring CP. The effects a syntic pyrethroid pesticide on some aspects reproduction in Atlantic salmon (Salmo salar L.). Aquatic toxicology. ;5():-. 8 Volume 8, No 7, Winter 5;