Int. J. Aquat. Biol. (2015) 3(5): 339-345 E-ISSN: 2322-5270; P-ISSN: 2383-0956 Journal homepage: www.ij-aquaticbiology.com © 2015 Iranian Society of Ichthyology Original Article Determination of lethal concentrations of indoxacarb in fingerling Cyprinus carpio at two temperatures Ali Taheri Mirghaed, Melika Ghelichpour*1 Department of Aquatic Animal Health, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran. Article history: Received 8 September 2015 Accepted 3 October 2015 Available online 2 5 November 2015 Keywords: Common carp Insecticide Toxicity LC50 Abstract: Acute (24-96 hrs) toxicity of indoxacarb, a new insecticide generation, was determined in Cyprinus carpio under the semi-static condition. Fish (~5 g) were exposed to increasing concentrations of indoxacarb over 24-96 hrs at 17ºC, and over 24 hrs at 22ºC, and mortality was recorded every 24 hrs. Indoxacarb-LC50 values at 17ºC were found to be 37.55, 20.92, 18.77 and 16.85 ppm after 24, 48, 72 and 96 hrs, respectively. LC50 after 24 hrs at 22ºC was 21.55 ppm, which was significantly lower than that obtained at 17ºC. The lowest observed effect concentration (LOEC) values at 17ºC were 14 ppm for 24, 48, 72 hrs, and 11 ppm for 96 hrs. No observed effect concentration (NOEC) values at 17ºC were 11 ppm for 24, 48, 72 hrs, and 8 ppm for 96 hrs. NOEC and LOEC values after 24 hrs at 22ºC were 8 and 11 ppm, respectively. The results indicated that indoxacarb is classified as "harmful" substance in common carp and, the higher temperature, the more toxicity of indoxacarb. Introduction Chemical pesticides are considered as an economic access to control pests; however, these chemicals may be highly toxic to non-target species in the environment. Nowadays, the indiscriminate use of pesticides and their excretion to the aquatic ecosystem threaten the health of organisms (Rao, 2006). Indoxacarb is an oxadiazin pesticide used to control sucking pests of crops, especially acts against lepidopteran larvae. The chemical name of indoxacarb is (S)-methyl 7- chloro-2, 5-dihydro-2- [[(methoxycarbonyl) [4-(trifluoromethoxy) phenyl] amino] carbonyl] indeno[1,2-e][1,3,4] oxadiazine- 4a(3H)-carboxylate (C22H17ClF3N3O7). It is marketed under the names Indoxacarb Technical Insecticide, Steward Insecticide and Avaunt Insecticide. Avant is more common name for this substance in Iran. This relatively new insecticide was registered in California, January 2001 (Monkada, 2003). As soon as indoxacarb is absorbed or ingested * Corresponding author: Melika Ghelichpour E-mail address: ml.ghelichpour@gmail.com by insects, feeding cessation occurs. It kills pests by binding to a site on sodium channels and blocking the flow of sodium ions into nerve cells. The result of exposure is impaired nerve function, feeding cessation, paralysis and death of pests. It may take days for insects to die (Brugger, 1997). Indoxacarb is used on a range of crops, which include fruits (apples, pears and tomatoes), vegetables (broccoli, Brussels sprouts, cabbage, cauliflower, eggplant, potato, and lettuce), soybeans, alfalfa and peanuts. It is used to control or suppress many insects, including Beet armyworm, Cabbage looper, Corn earworm, Diamondback moth, Fall armyworm, Imported cabbageworm, Southern armyworm, Tomato pinworm and Tomato fruitworm (Dupont, 2002). This pesticide is somehow a hydrophobic pesticide and its penetration to aquatic system is slow, so it is considered as a safe insecticide in environmental standpoint. It provides a much safer alternative to some pesticides such as organophosphates and 340 Int. J. Aquat. Biol. (2015) 3(5): 339-345 pyrethroids. The relatively low mammalian toxicity of this insecticide provides improved safety to workers, as well as terrestrial mammals and birds when compared to competitive organophosphates and carbamates. Lack of cross resistance to existing insect control products, environmental suitability and its safety to non-target organisms makes indoxacarb an excellent candidate for controlling some pests (Wing et al., 2000). Since pH elevation and sunlight speed up indoxacarb hydrolysis and photolysis, it seems that this insecticide has low persistence in aquatic system. However, it is classified as moderately to very highly toxic to freshwater and estuarine/marine fish on an acute basis (Moncada, 2003). There are very limited studies about the effect of this insecticide on fish. Veeraiah et al. (2013) investigated the biochemical parameters of Indian major carp Labeo rohita exposed to lethal and sub- lethal concentrations of indoxacarb showing that indoxacarb is highly toxic to fish with 96 hrs LC50 of 0.053 ppm. Also, indoxacarb exposure resulted in decrease in protein, glycogen and nucleic acids content in different organs. As, there is no information available about the toxicity of this pesticide to common carp, Cyprinus carpio, hence, this study aimed to determine the lethal concentrations of indoxacarb in common carp at two different temperatures. Materials and methods Fish and maintenance conditions: In summer 2015, a total of 700 fingerlings were obtained from Fishery Research Station of Gharahsoo (Bandar Turkman, Iran) and brought to the laboratory. Fish were stocked in a 2000 L fiberglass tank for adaptation to the laboratory conditions. The fish were maintained under continuously-aerated condition in the tank for seven days. They were fed commercial pellets (Energy Co., 4EF3000) at 2% body weight per day, twice a day. Water exchange was about fifty percent daily (ground water of Gharahsoo). The fish total length and weight were 5.83 ± 0.87 cm and 5.64 ± 0.74 g, respectively. Water quality parameters were as following: temperature = 17ºC, pH = 8.23 ± 0.02, dissolved oxygen = 8.68 ± 0.5 ppm, oxygen saturation = 97.05 ± 2.3 %, electro conductivity = 4.8 ± 0.15 μs m-1, salinity = 2.8 ± 0.08 ppt. These parameters were measured by Hach HQ40d portable pH, conductivity, dissolve oxygen and salinity meter (Loveland, Colorado, USA). Total hardness 300 ± 17 ppm (as CaCO3), alkalinity 350.75 ± 20 ppm (as CaCO3), and calcium 110.8 ± 11 ppm were measured by a Portable photometer (Wagtech 7100, Berkshire, UK). No mortality was observed during adaption period. Toxicity test: Lethal concentration was determined at two temperatures, 17 ± 1 and 22 ± 1°C, according to OECD (1992). Based on the preliminary tests, the fish were exposed to concentrations of 0 (control), 8, 11, 14, 17, 20, 23, 26, 35, 45 and 60 ppm indoxacarb (Hefiran Co., 15%, Tehran, Iran). At 22°C, mortality was recorded after 24 hrs exposure, whereas, at 17°C mortality was recorded at 24, 48, 72 and 96 hrs. Eleven tanks were assigned for each temperature. A 300 L, white and cylindrical tank was used for each concentration. Each tank stocked with 30 fish (150 g biomass) and 160 L water. The fish were allowed to adapt to these conditions for seven days under aerated condition, during which they were fed (2% of body weight, twice a day) with commercial pellets (Energy Co., 4EF3000). Tanks' water was exchanged fifty percent daily. Water quality parameters were monitored during experiment and they were similar to those of adaptation period. No mortality was observed during this period. Feeding was ceased 24 hrs before dosing. Indoxacarb solution was added to each tank to set concentrations. Seventy five percent of the exposed solution was renewed each day to maintain water quality and the appropriate concentration of indoxacarb. During the experiment, the fish behavior and the number of dead fish were recorded at 24 hrs intervals. Statistical analysis: LC50 values were calculated using probit regression in SPSS v.22. The lowest observed effect concentration (LOEC) was 341 Taheri Mirghaed and Ghelichpour/ Effect of indoxacarb on Common carp determined as the minimum concentration which caused mortalities at each time point (Rand, 1995). No observed effect concentration (NOEC) was determined as maximum concentration at which no mortality was occurred (Rand, 1995). Difference between 24 hrs-LC50 of the temperatures was considered significance if LC50 of one temperature was outside the confidence interval of the other temperature (Marking and Bills, 1975; Hoseini and Jafar Nodeh, 2011). Significant difference between mortality percentages was investigated using t-test. Results No mortality was observed in the control group during the exposure. The control and 8 ppm indoxacarb treatment showed normal swimming and natural body coloration during the experiment. Abnormal behavioral changes such as dark coloration, unilateral or bilateral exophthalmia, loss of equilibrium, lateral swimming near the surface, motionless on the bottom of tank, hyper excitement and lethargy were observed in the fish exposed to higher indoxacarb concentrations (20-60 ppm) during 24 hrs of exposure period. These behaviors were observed in 11 ppm of indoxacarb 72 hrs after the start of the experiment, and in 14 ppm indoxacarb 48 hrs after exposure. LC5-95, NOEC and LOEC of indoxacarb are presented in Tables 1-4. LC50 after 24, 48, 72 and 96 hrs at 17ºC were 37.55 (32.26-42.44), 20.92 (19.12- 23.08), 18.77 (17.87-19.70) and 16.85 (15.97-17.73) ppm, respectively. LC50 after 24 hrs at 22ºC was 21.55 (18.25-26.70). There was a significant difference between 24 hrs-LC50 at the tested 95% Confidence Limits Time Temperature LC5 Lower Upper Slope ± S.E Intercept ± S.E 24 hrs 22ºC 14.98 7.86 17.83 10.42 ± 1.25 -13.89 ± 1.65 24 hrs 17ºC 19.57 13.76 23.61 5.81 ± 0.68 -9.15 ± 0.96 48 hrs 17ºC 14.80 11.41 16.70 10.94 ± 1.37 -14.45 ± 1.80 72 hrs 17ºC 13.70 12.17 14.80 12.03 ± 1.50 -15.31 ± 1.93 96 hrs 17ºC 12.10 10.67 13.15 11.44 ± 1.39 -14.03 ± 1.73 Table 1. LC5 (ppm) of indoxacarb after 24, 48, 72 and 96 hrs at 17 and 22ºC. 95% Confidence Limits Time Temperatu re LC50 Lower Upper Slope±S.E Intercept±S.E 24 hrs 22ºC 21.55* 18.25 26.70 10.42±1.25 -13.89±1.65 24 hrs 17ºC 37.55 32.26 46.44 5.81±0.68 -9.15±0.96 48 hrs 17ºC 20.92 19.12 23.08 10.94±1.37 -14.45±1.80 72 hrs 17ºC 18.77 17.87 19.70 12.03±1.50 -15.31±1.93 96 hrs 17ºC 16.85 15.97 17.73 11.44±1.39 -14.03±1.73 Table 2. LC50 (ppm) of indoxacarb after 24, 48, 72 and 96 hrs at 17 and 22ºC. Asterisk in front of 24 hrs LC50 at 22ºC means significant difference compared to 24 hrs LC50 at 17ºC. 95% Confidence Limits Time Temperature LC95 Lower Upper Slope ± S.E Intercept ± S.E 24 hrs 22ºC 31.00 55.33 124.9 10.42 ± 1.25 -13.89 ± 1.65 24 hrs 17ºC 72.05 25.54 66.26 5.81 ± 0.68 -9.15 ± 0.96 48 hrs 17ºC 29.57 25.95 39.38 10.94 ± 1.37 -14.45 ± 1.80 72 hrs 17ºC 25.72 23.90 28.87 12.03 ± 1.50 -15.31 ± 1.93 96 hrs 17ºC 23.48 21.79 26.22 11.44 ± 1.39 -14.03 ± 1.73 Table 3. LC95 (ppm) of indoxacarb after 24, 48, 72 and 96 hrs at 17 and 22ºC. 342 Int. J. Aquat. Biol. (2015) 3(5): 339-345 temperatures. NOEC after 24, 48, 72 and 96 hrs at 17ºC were 11, 11, 11 and 8 ppm, respectively. LOEC after 24, 48, 72 and 96 hrs at 17ºC were 14, 14, 14 and 11 ppm, respectively. NOEC and LOEC after 24 hrs at 22ºC were 8 and 11 ppm, respectively. Mortality of fish exposed to different concentrations of indoxacarb, at 22ºC was significantly higher than 17ºC (Fig. 1). Discussion The results revealed that the severity of the behavioral responses was dependent to the indoxacarb concentration and exposure time. There were no studies about fish behavioral alterations which are exposed to indoxacarb. Environmental pollutants such as metals, pesticides, and other organic pollutants cause serious risks to aquatic organisms. Accordingly, there is a great deal of researches about physiological mechanisms in animals exposed to contaminants. Behavioral signs of toxicity appear to be ideal tools to assess the effects of aquatic pollutants on fish, because behavioral indicators reflex the physiological states. Toxicant exposure may completely alter behaviors that are essential for fish survival in natural ecosystems, particularly in toxicant concentrations lower than lethal concentration. Behavioral changes are mainly related to cholinesterase inhibition, alternation of brain neurotransmitter levels, sensory deficiency, and impaired gonadal or thyroid hormone levels. Scott and Sloman (2004) investigated interrelationships between behavioral and physiological indicators of toxicity in fish. Also, a positive correlation was observed between brain acetyl cholinesterase activity and swimming speed of mosquito fish, Gambusia affinis after lethal exposure of an organophosphate pesticide, monocrotophos (Kavitha and Rao, 2007). According to 96 hrs LC50, indoxacarb is classified as "harmful" substance in common carp (Commission Directive, 2001). The results showed that LC50 of indoxacarb in the present study was higher than other studies which reported 96 hrs LC50 of 0.024-0.053 in different fish species (Veeraiah et al., 2013; Hoke, 1997). Such differences may be species specific. Also, water physico-chemical properties are the most important factors involved in the different results of indoxacarb toxicity. Hydrolysis rates of indoxacarb elevate with increasing pH. The half-life at pH 5, 7 and 9 were considered to be approximately 500, 38 and 1 day, respectively (Ferraro and McEuen, 1996). In this study, pH was 8.23 ± 0.02; therefore, it can cause faster hydrolysis of indoxacarb and continuously lower toxicity in comparison with other studies; although, those studies did not mention water pH. Other water physic-chemical factors may affect toxicity of pesticide. For instance, increasing total alkalinity from 19 to 90-120 ppm is resulted in decreasing mortality of Oncorhynchus mykiss exposed to methiocarb and endosulfan (Altinok et al., 2006; Capkin et al., 2006). Water alkalinity in the present study was relatively high (~350 ppm) and this may be another reason for higher LC50 compared to the previous studies that needs a further study. Cyprinus carpio is an ectothermic organism, hence, Time Temperature NOEC LOE C 24 hrs 22ºC 8 11 24 hrs 17ºC 11 14 48 hrs 17ºC 11 14 72 hrs 17ºC 11 14 96 hrs 17ºC 8 11 Table 4. NOEC and LOEC of indoxacarb after 24, 48, 72 and 96 hrs at 17 and 22ºC. Figure 1. Mortality percentages at the final time point of observation (17 and 22ºC: 24 hrs after exposure). Columns represent means and standard deviation. Gray bars show mortalities at 22ºC and black bars show mortalities at 17ºC. Asterisks above the bars show significant difference in mortality percentages between the temperatures. * P<0.05; *** P<0.001. 343 Taheri Mirghaed and Ghelichpour/ Effect of indoxacarb on Common carp temperature is a fundamental factor influencing all its physiological processes. Many authors affirmed the assumption that temperature elevation increases toxicity of harmful substances in certain species (Fisher and Wadleigh, 1985; Persoone et al., 1989; Song et al., 1997; Van Wezel and Jonker, 1998; Heugens et al., 2001). Exposure to a toxic substance would increase metabolism and oxygen demand of fish as an ectothermic organism; with elevated temperature, the oxygen solubility in water is decreased, therefore, temperature elevation may worsen the toxic effects of the toxic substance (Osterauer and Kohler, 2008). In addition, bioavailability of toxic materials like any substances is dependent on temperature. At high temperatures, the absorption of substances by aquatic animals is elevated due to better solubility of the substance and an intensified distribution or active uptake rate of the substance by gill or skin (Heugens et al., 2001; Osterauer and Kohler, 2008). According to this, toxic effect of a substance on an aquatic organism may be potentiated with increasing temperature. Similarly, Altinok et al. (2006) and Capkin et al. (2006) reported increase in methiocarb and endosulfan toxicity in O. mykiss as a result of temperature elevation. As conclusion, the results suggest that indoxacarb is categorized as "harmful" pesticide in common carp with 96 hrs LC50 value of 16.85 ppm. Also, the pesticide toxicity increases along with temperature elevation. References Altinok I., Capkin E., Karahan S., Boran, M. (2006). Effects of water quality and fish size on toxicity of methiocarb, a carbamate pesticide, to rainbow trout. Environmental Toxicology and Pharmacology, 22: 20-26. Brugger K.E. (1997). DPX-MP062: Prospective tier I ecological effects assessment for non-target organisms. DuPont Agricultural Products Document No. AMR 4782-97. Capkin E., Altinok I., Karahan S. (2006). Water quality and fish size affect toxicity of endosulfan, an organochlorine pesticide, to rainbow trout. Chemosphere, 64: 1793-1800. Directive C. (1967). Council Directive 67/548/EEC of 27 June 1967 on the approximation of laws, regulations and administrative provisions relating to the classification, packaging and labeling of dangerous substances. Official Journal of the European Communities, 196 (16.8): 1. Dupont Product Profile. (2002). Indoxacarb. Available Online: http://www.avaunt.dupont.com Ferraro P., McEuen S.F. (1996). Hydrolysis of DPX- JW062 (A Racemic Mixture of DPX-KN128 and DPX-KN127) in buffer solutions of pH 5, 7, and 9. DuPont Agricultural Products Document No. AMR 2789-93. Fisher S.W., Wadleigh R.W. (1985). Effects of temperature on the acute toxicity and uptake of lindane by Chironomus riparius (Meigen) (Diptera: Chironomidae). Journal of Economic Entomology, 78: 1222-1226. Hoke R.A. (1997). IN-JT333-20: Flow-through, acute, 96-hour LC50 to rainbow trout, Oncorhynchus mykiss, DuPont Haskell Laboratory, Elkton Road, P.O. Box 50, Newark, DE 19714 USA, Report No. HL-1997- 00180, 19 June 1997. Hoseini S.M., Jafar Nodeh A. (2011). Acute toxicity of potassium permanganate to Caspian roach Rutilus rutilus caspicus in two size classes and under different aeration conditions. Toxicological Environmental Chemistry, 93: 996-1001 Kavitha P., Rao J.V. (2007). Oxidative stress and locomotor behavior response as biomarkers for assessing recovery status of mosquito fish, Gambusia affinis after lethal effect of an organophosphate pesticide, monocrotophos. Pesticide Biochemistry and Physiology, 87: 182-188. Marking L.L., Bills T.D. (1975). Toxicity of potassium permanganate to fish and its effectiveness for detoxifying antimycin. Transactions of the American Fisheries Society, 104: 579-83. Moncada A. (2003). Environmental fate of indoxacarb. Environmental Monitoring Branch, Dept. of Pesticide Regulation, California Environmental Protection Agency, Sacramento, CA. OECD. (1992). Guideline for testing of chemicals. Fish, acute toxicity test. pp. 1-9. Osterauer R., Köhler H.R. (2008). Temperature- dependent effects of the pesticides thiacloprid and diazinon on the embryonic development of zebrafish 344 Int. J. Aquat. Biol. (2015) 3(5): 339-345 (Danio rerio). Aquatic Toxicology, 86: 485-494. Persoone G., Van De Vel A., Van Steertegem M., De Nayer B. (1989). Predictive value of laboratory tests with aquatic invertebrates: influence of experimental conditions. Aquatic Toxicology, 14: 149-166. Rand G.M. (1995). Fundamental of aquatic toxicology effects, environmental fate and risk assessment. 2nd ed. Washington, DC: Taylor & Francis. pp: 3-67. Rao J.V. (2006). Toxic effects of novel organophosphorus insecticide (RPR-V) on certain biochemical parameters of euryhaline fish, Oreochromis mossambicus. Pesticide Biochemistry and Physiology, 86: 78-84. Scott G.R., Sloman K.A. (2004). The effects of environmental pollutants on complex fish behavior: integrating behavioural and physiological indicators of toxicity. Aquatic Toxicology, 68: 369-392. Song M.Y., Stark J.D., Brown J.J. (1997). Comparative toxicity of four insecticides, including imidacloprid and tebufenozide, to four aquatic arthropods. Environmental Toxicology and Chemistry, 16: 2494- 2500. Van Wezel A.P., Jonker M.T.O. (1998). Use of the lethal body burden in the risk quantification of field sediments; influence of temperature and salinity. Aquatic Toxicology, 42: 287-300. Veeraiah K., Rao S. (2013). Changes in biochemical parameters of freshwater fish Labeo rohita exposed to lethal and sub-lethal concentrations of indoxacarb. International Journal of Bioassays, 2: 1282-1387. Wing K.D., Sacher M., Kagaya Y., Tsurubuchi Y., Mulberig L., Connair M., Schnee, M. (2000). Bioactivation and mode of action of the oxadiazine indoxacarb in insects. Crop Protection, 19: 537-545. Int. J. Aquat. Biol. (2015) 3(5): 339-345 E-ISSN: 2322-5270; P-ISSN: 2383-0956 Journal homepage: www.ij-aquaticbiology.com © 2015 Iranian Society of Ichthyology چکیده فارسی مختلف دمای دو در قدانگشت کپورماهیان بر ایندوکساکارب سم کشندة هایغلظت تعیین *پور قلیچ ملیکا میرقائد، طاهری علی .ایران تهران، تهران، دانشگاه دامپزشکی، دانشکده آبزیان، بهداشت گروه چکیده: . شد تعیین ایستا نیمه شرایط در( Cyprinus carpio) کپورمعمولی ماهی در( ساعت 69 -42) ایندوکساکارب جدید کشحشره حاد سمیت در ساعت 42 مدت به گرادسانتی درجة 44 حرارتدرجه در و ساعت 69 تا 42 مدت به گرادسانتی درجة 71 دمای در گرم 5 حدود هایماهی مقادیر گرادسانتی درجة 71 دمای در. شد گزارش ساعت 42 هر میرومرگ درصد و گرفتند قرار ایندوکساکارب مختلف هایغلظت معرض 50LC 45/79 و 11/74 ،55/64،71/42 ترتیببه ساعت 69 و 14 ،24 ،42 از پس ایندوکساکارب سم برای ppm درجه در. آمد دستبه در آن معادل مقدار از کمتر داریمعنی طوربه که شد محاسبه ppm 55/47 ساعت 42 از پس 50LC مقدار گراد،سانتی درجة 44 حرارت 14 و 24 ،42 هایزمان طی گرادسانتی درجة 71 ترحرادرجه در( LOEC) مؤثر غلظت کمترین مقدار. بود گرادسانتی درجة 71 دمای 24 ،42 از پس گرادسانتی درجة 71 دمای در سم این( NOEC) اثربی غلظت. شد محاسبه 77ppm ساعت 69 از پس و ppm 72 ساعت درجة 44 دمای در ایندوکساکارب سم مؤثر غلظت کمترین و اثربی غلظت. بود ppm 4 ساعت 69 گذشت از پس و ppm 77 ساعت 14 و دما افزایش و داشته قرار مضر مواد دستة در ایندوکساکارب سم که داد نشان نتایج. بود ppm 77 و 4 ترتیببه ساعت 42 از پس و گرادسانتی .گرددمی کشحشره این سمیت افزایش سبب .50LC سمیت، کش،حشره کپورمعمولی،: کلمات کلیدی