Keywords: diazinon, acute toxicity, Barbonymus gonionotus, Cyprinus carpio

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1 Acute toxicity of diazinon on Silver barb (Barbonymus gonionotus) and Common carp (Cyprinus carpio) fingerlings Banthita Sawasdee a*, Leklai Chantubut a, Chanawan Thowanna a, Chonlada Yodying b a Program in Aquaculture, Faculty of Agricultural Technology, Rajabhat Maha Sarakham, Thailand b Program in Agricultural Machinery Technology, Faculty of Agricultural Technology, Rajabhat Maha Sarakham, Thailand * Corresponding author. Tel.: address: banthitas@yahoo.com 1. Abstract This study examined the toxicity of diazinon on Barbonymus gonionotus and Cyprinus carpio fingerlings. The fingerlings were treated with diazinon at the concentrations of 0, 0.001, 0.005, 0.01 and 0.1 mg/l, respectively. The results showed that the mortality rate (%) increased from 16.67±4.44, 90.00±6.67 to 100 in B. ginionotus and 3.33±4.44, 6.70±8.89 to 100 in C. carpio at 96 hr. The fingerlings exposed to diazinon, the external abnormalities including curved spine and discoloration of skin were also found. At the same concentration, the 100% mortality was observed in B. ginionotus fingerlings within one hour, whereas the 100% mortality of C. carpio was found within two hours after the exposure. In addition, the results revealed that fingerlings of B. ginionotus to be more sensitive to diazinon than C. carpio. Keywords: diazinon, acute toxicity, Barbonymus gonionotus, Cyprinus carpio 2. Introduction Thailand uses large quantities of pesticides in the Southeast Asia region for the control of crop pests, vectors of disease and increase yields. In order to boost agricultural production and efficiency there has been a marked increase in the use of more powerful agricultural chemicals. Based on data from the Office of Agriculture Regulation of the Department of Agriculture, about 70,000 tons of pesticides comprised of 265 individual active ingredients were imported into Thailand in Diazinon is one of the most pesticides use in Thailand (WHO,1996). Diazinon is organophosphorus compound with an anticholinesterase which causes loss of functional coordination that result in immobilization of organisms (Brooks, 1976; Tinoco-

2 Ojanguren and Halperin, 1998). It is used extensively to control flies, lice, insect pests of ornamental plants and food crops, as well as nematodes and soil insects in lawns and croplands. Diazinon degrades rapidly in the environment, with half-time persistence usually less than 14 days. Diazinon may remain biologically active in soils for 6 months or longer under conditions of low temperature, low moisture, high alkalinity and lack of suitable microbial degraders (Eisler, 1986). Because of its aquatic distribution, diazinon affects a wide range of non-target organisms, like invertebrates, mammals, birds and fishes, especially those inhabiting aquatic environment (Burkepile et al., 2000; Hamm and Hilton, 2000) Fish are widely used to evaluate the health of aquatic ecosystem and physiological changes serve as biomarkers of environmental pollution (Kock et al., 1996). The fish B. gonionotus and C. carpio have been selected as the test organism because of its wide availability in local tanks and ponds. They are quite sturdy and ideally suited for experimentation in laboratory for longer periods. These fish are cultured in ponds and they have a very good commercial value, because of their nutritive value and taste. The both species are the majority of production consumed domestically in Asia (FAO, 2004). The purpose of this research was to determine the acute toxicity of pesticides diazinon on B. gonionotus and C. carpio fingerlings. 3. Material and methods Adult B. gonionotus and C. carpio strain used for egg production originated from Maha Sarakham Inland Fisheries Research and Development Center, Maha Sarakham, Thailand. Fish were maintained in cement tank with flow-through system at ambient temperature. They were fed twice a day with commercial fish food. The physicochemical characterization of the water used for fish bioassay was carried out using standard methods (APHA, 1998) and the following values were obtained: Temperature 27ºC, ph , dissolved oxygen mg/l, alkalinity mg/l and water hardness mg/l. Effects of pesticides were investigated by exposing B. gonionotus and C. carpio fingerlings to a range of diazinon (0, 0.001, 0.005, 0.01 and 0.1 mg/l). Solutions were prepared with double-distrilled water. For the test with diazinon, glass Petri dishes were used and the stock solutions were kept in glass bottles. Base on previous studies, each of 10 B. gonionotus and C. carpio fingerlings (newly hatched) were exposed for 96 h to the nominal concentrations of 0, 0.001, 0.005, 0.01 and 0.1 mg/l diazinon. The entire experiment was conducted in with a total of 90 fingerlings per treatment group (10 per petri dish, 9 replicates). The replacement of the medium were done daily to ensure even distribution of the chemical. The fingerlings

3 were not fed because they are not able to feed themselves and carry a yolk-sac which provides their nutrition. Treatment effects on the acute toxicity were determined using one-way analysis of variance (ANOVA). ANOVA was followed by Duncan s new multiple range test to compare the treatment with respective control. Significant difference occurs for a given parameter when p < Results At 24 h to 96 h, fingerlings treated with a concentration of mg/l diazinon or higher showed a trend to increased mortality. This finding, however, was significant only at the high concentration tested (0.1 mg/l for 24 h, 0.01 mg/l for 48 h, mg/l for 72 h and 96 h, respectively) (Table1). Table 1 The mortality rate (%) of B. gonionotus exposed to diazinon Concentration % mortality (mg/l) 24 h 48 h 72 h 96 h 0 0±0 b 0±0 b 3.33±4.44 b 16.67±4.44 c ±4.44 b 3.33±4.44 b 10.00±6.67 b 73.33±15.56 b ±4.44 b 13.33±11.11 b 86.67±17.78 a 100±0 a ±6.67 b 83.00±22.22 a 100±0 a 100±0 a ±0 a 100±0 a 100±0 a 100±0 a Note: Groups with different letters are significantly different (p < 0.05) The mortality of C. carpio was observed during the exposure times at 24, 48, 72 and 96 h with the range concentrations of 0, 0.001, 0.005, 0.01 and 0.1 mg/l diazinon (Table 2). At 24 hr to 48 hr, fishes treated with diazinon showed 100% mortality. This finding, however, was significant only at the highest concentration tested (0.1 mg/l). At 72 h, the results showed a significant difference of mortality at 0.1 mg/l, whereas, at 96 h of exposure, a significant difference of mortality was found at the concentrations of 0.01 mg/l or higher.

4 Table 2 The mortality rate (%) of C. carpio exposed to diazinon Concentration % mortality (mg/l) 24 h 48 h 72 h 96 h 0 0±0 b 0±0 b 0±0 c 0±0 c ±0 b 0±0 b 0±0 c 3.33±4.44 c ±0 b 0±0 b 0±0 c 6.67±8.89 c ±0 b 0±0 b 6.67±4.44 b 66.67±26.67 b ±0 a 100±0 a 100±0 a 100±0 a Note: Groups with different letters are significantly different (p < 0.05) According to the results, at the same concentration, the 100% mortality was observed in B. ginionotus fingerlings within one hour, whereas the 100% mortality of C. carpio was found within two hours after the exposure. In addition, the results revealed that fingerlings of B. ginionotus to be more sensitive to diazinon than C. carpio. Furthermore, the external abnormalities including curved spine and skin discoloration of treatment group were found. However, no significant malformations were observed in B. gonionotus and C. carpio fingerlings exposed to the different treatment groups. 5. Discussion Our study showed clearly that diazinon is increased mortality rates in B. ginionotus and C. carpio fingerlings. In previous studies, several fish were sensitive to diazinon: according to Aydin and Köprücü (2005), common carp embryos exposed to diazinon had significant effects on mortality and hatching success. In addition, with increasing diazinon concentrations, the larvae exposed duration h significantly increased the number of dead larvae, similar to our results. Hossain et al. (2000) reported that LC50 of diazinon to three Indian major carp (Catla catla, Labeo rohita and Cirrhinus mrigala) fingerlings were 1.399, 1.308, and mg/l at 24, 48, 72 and 96 h, respectively. There are differences in the acute toxicity of diazinon for various fish species. In European eel (Anguilla anguilla), the 96 h LC50 values range even in hundredths of milligrams per liters (Sancho et al., 1993). The 96 h LC50 values of diazinon was reported as mg/l for fry bluegill (Lepomis macrochirus), 1.47 mg/l for sheepshead minnow (Cyprinodon variegates), 1,65 mg/l for fry rainbow trout (Oncorhynchus mykiss) and 7.80 mg/l for fathead minnow, Pimephales minnow (US EPA, 2000). The different toxicity of diazinon may be

5 demonstrated on the example of two fish species used for ecotoxicological assessment of chemical substances. The 96 h LC50 values of diazinon for guppy (Poecilia reticulate) was found to be 0.8 mg/l but for zebrafish (Brachydanio rerio) it was found to be 8 mg/l (Keizer et al., 1991). Similarly, the LC50 96 h of diazinon for fingerlings and larvae of spirlin (Alburnoides bipunctatus) were 7.88 and 0.69, respectively (Vajargah et al., 2013). Different factors have been suggested to cause selective toxicity of diazinon for various fish species: different inhibition of acetylcholinesterase, different detoxification and absorption (Oh et al., 1991). 6. Conclusions This study demonstrates that diazinon has a detrimental impact on mortality and the abnormalities of fish fingerlings. The results also revealed that B. ginionotus is more sensitive to diazinon than C. carpio. The results indicate that low level of diazinon in the aquatic environment may have a significant effect on fish population. Considering fish as experimental biological models to measure the environmental impact of different concentration substances, these results indicate that tested pesticide could produce potential environmental contamination. Acknowledgement Thanks are due to Maha Sarakham Inland Fisheries Research and Development Center, for providing animals from which our fish stock has originated. References American Public Health Association (APHA), Standard Methods for the Evaluation of Water and Waste Water. 20th Edn. American Public Health. Washington D.C. pp: Aydin R, Köprücü K Acute toxicity of diazinon on the common carp (Cyprinus carpio L.) embryos and larvae. Pest. Biochem. Phys. 82: Brooks GT. 1976, Penetration and Distribution of Insecticides. In: Wilkinson, C.F. (Ed.), Insecticide Biochemistry and Physiology, (Heyden, London, 1976) 3-60

6 Burkepile DE, Moore MT, Holland MM The susceptibility of five nontarget organisms to aqueous diazinon exposure. Bull. Environ.Contam. Toxicol. 64: Eisler R Diazinon hazards to Fish, Wildlife and Invertebrates: A synoptic review. Patuxent Wildlife Research Center. U.S. Fish and Wildlife Service, Biol. Rep. 85(1.9): 25 Hamm JT, Hinton DE The role of development and duration of exposure to the embryotoxicity of diazinon. Aquat. Toxicol. 48: Hossin Z, Haldar GC, Mollah MFA Acute toxicity of chlorpyrifos, cadusafos and diazinon to three Indian major carp (Catla catla, Labeo rohita and Cirrhinus mrigala) fingerlings. Bangladesh J. Fish. Res. 4(2): Kock, G., Triendl, M. and Hofer, R Seasonal patterns of metal accumulation in Arctic char (Salvelinus alpinus) from an oligitrophic Alpine lake related to temperature. Can. J. Fish. Aquat. Sci. 53: Tinoco-Ojanguren R, Halperin DC Poverty, production health: inhibition of erythrocyte cholinesterase via occupational exposure to organophosphate insecticides in Chiapas, Mexico. Arch. Environ. Health. 53: FAO Pesticide residues in food Evaluation Part I- Residues. FAO plant production and protection paper 142 FAO FAO statistical yearbook Information Division FAO. Viale delle Terme di Caracalla, Rome; Italy Sancho E, Ferrando MD, Andreau E, Gamon M Bioconcentration and excretion of diazinon by eel, Bull. Environ. Contam. Toxicol. 50: Keizer J, De Agostino G, Vittozzi I The importance of biotransformation in the toxicity of xenobiotics to fish. 1: toxicity and bioaccumulation of diazinon in guppy (Poecilia reticulata) and zebra fish (Brachydanio rerio), Aquat. Toxicol. 21: Oh HS, Lee SK, Kim YH, Roh JK Mechanism of selective toxicity of diazinon to killifish (Oryzias latipes) and loach (Misgurnus anguillicaudatus), Aquat. Toxicol. Risk Assess. 14: Office of Pesticide Programs, Pesticide Ecotoxicity Database (Formerly: Environmental Effects Database), Environmental Fate and Effects Division, U.S. EPA, Washington, DC, 2000.

7 Vajargah MF, Hossaini SA, Hedayati A Acute toxicity test of two pesticides diazinon and deltamethrin on spirlin (Alburnoides bipunctatus) larvae and fingerlings. J. Toxicol. Environ: Health. Sci. 5(6):