Int. J. Aquat. Biol. (2015) 3(5): 346-351 E-ISSN: 2322-5270; P-ISSN: 2383-0956 Journal homepage: www.ij-aquaticbiology.com © 2015 Iranian Society of Ichthyology Original Article Acute toxicity of Euphorbia turcomanica on Aphanius dispar Homa Zare1, Ahmad Noori*1, Morteza Yusefzadi2, Mahdi Banaee3 1Department of Fisheries Science, Faculty of Marine Science and Technology, Hormozgan University, Bandar Abbas, Iran. 2Department of Marine Biology, Faculty of Marine Science and Technology, Hormozgan University, Bandar Abbas, Iran. 3Department of Aquaculture, Faculty of Natural Resources and Environment, Behbahan Khatam Alanbia University of Technology, Behbahan, Iran. Article history: Received 10 July 2015 Accepted 25 O c t o b e r 2015 Available online 2 5 N o v e m b e r 2015 Keywords: Euphorbia turcomanica Dried powder Piscicidal Killifish Abstract: Piscicidal and molluscicidal activities of aqueous extracts of many members of the family Euphorbiaceae are well-known, but the toxicity potential of Euphorbia turcomanica was not yet studied on any aquatic animals. An acute toxicity test was performed by using a four-day static renewal test to determine the LC50 value of dried powder of E. turcomanica for the euryhaline fish, Aphanius dispar. The LC50 values at various exposure periods are 0.177±0.039 g/L for 24 hrs, 0.131±0.030 g/L for 48 hrs, 0.073±0.018 g/L for 72 hrs, and 0.052±0.013 g/L for 96 hrs. The toxicity of dried powder of E. turcomanica exhibits a positive correlation between fish mortality and exposure periods. As this is the first report about toxicity of E. turcomanica on A. dispar, the results could be only compared to that of other Euphorbiaceae as well as other fishes. It is concluded that the toxicity potential of E. turcomanica is comparable and close to that of well-documented Euphorbiaceae. It has been suggested that E. turcomanica products cannot be used directly in fish-inhabiting water reservoirs. Introduction Increased awareness of the negative effects caused by overexposure to synthetic organo-piscicides (Reidinger and Russell, 1976) has led to efforts for finding products from plant origin to substitute. Being the products of biosynthesis, they are potentially biodegradable (Marston and Hostettmann, 1985). The Euphorbiaceae is a large families with about 300 genera and 7500 species (Vasas and Hohmann, 2014). Of the member of this family, the genus Euphorbia with about 2000 species (Frodin, 2004), is one of the five most species-rich genera of flowering plants (Govaerts et al., 2000). They have a poisonous milky white latex-like sap and unique kind of floral structures. The chemical constituents of these plants, include triterpenoids and related compounds (sterols, alcohols and hydrocarbons), phenolic compounds (flavonoids, lignans, coumarins, tannins, phenanthrenes, * Corresponding author: Ahmad Noori E-mail address: nooryahmad@gmail.com quinones, phenolic acids, etc.), alkaloids, cyanogenic glucosides, and glucosinolates (Abdel‐ Fattah, 1987; Neuwinger, 2004; Kumar et al., 2010) that are poisonous to target and non-target aquatic organisms (Singh et al., 1996; Ebenso, 2004; Prasad et al., 2010). Many of these plants are cosmopolitan distributing in subtropics and temperate regions (Horn et al., 2012). In the flora of Iran, this genus comprises 70 species, of which 17 species are endemic (Mozaffarian, 1996). Euphorbia turcomanica Boiss, is an annual herb, which grows wild in plains of Iran (Mozaffarian, 1996; Pahlevani and Riina, 2011). Different species of the genus Euphorbia are used as insecticide, piscicide, and molluscicide (Sastry and Siddiqui, 1983; Oliveira-Filho and Paumgartten, 2000; Tiwari and Singh, 2004; Tiwari et al., 2004; Singh et al., 2005; Oliveira-Filho et al., 2010; Hassan et al., 2011). Since, there is no information available 347 Zare et al./ Acute toxicity of E. turcomanica on A. dispar regarding the effect of E. turcomanica on fishes as piscicid. Therefore, due to wide use of various parts of the members of the family Euphorbiacea (Bani et al., 1997; Abdel-Hamid, 2003; Srivastava et al., 2004; Tiwari et al., 2004), this study was conducted to assess the acute toxicity of the lethal concentrations of aqueous extracts of E. turcomanica’s aerial parts on Aphanius dispar, an euryhaline fish of Iranian inland water. Materials and Methods A total of 240 individuals of A. dispar (both sexes: with mean body weight and total length of 2.03±0.5 g, and 47.7±0.45 mm, respectively) were collected from seasonal rivers in Bandar Abbas and transported to the Hormozgan University Fisheries laboratory. In the laboratory, healthy fish were introduced into a 100 L tank with continuous aeration, where they were acclimatize for 14 days to the laboratory conditions. The fish were considered fully acclimatized when no death was observed for seven successive days. The fish were fed 2-3 times a day during this period with commercial pellets, containing protein >28%, lipid >3%, fiber <4% and moisture <10%. The stem, branches and leaves of E. turcomanica were dried in room temperature away from direct sunlight. Then, they were powdered and mixed with water to obtain the required plant concentrations. For the determination of LC50 of E. turcomanica on A. dispar, the four-day static renewal acute toxicity test was performed based on Clesceri et al. (1998). Fish were exposed to 0.001, 0.01, 0.1, 1, 2, 4 and 6 g/L of the dried powder of E. turcomanica with three replicates each containing 10 fish (kept in 4 L plastic aquaria). In addition, control group with three replicates in similar stocking density and aquaria were considered for this experiment. The water was renewed every day and required dried plant was added after water renewal to keep experimental concentrations. The fish were not feed 24 hrs before and during the experiment. Dead fish were counted and removed from the treatments immediately. A toxic effect was determined by a statistically significant decrease in the survival of fish exposed to the plant relative to the survival of Figure 1. Percent mortality of the fish Aphanius dispar after 24, 48, 72 and 96 hrs exposures to different concentrations of Euphorbia turcomanica powder (g/L). 348 Int. J. Aquat. Biol. (2015) 3(5): 346-351 fish in a control. The physico-chemical parameters of the water during experiment, including temperature (23.5±1ºC), dissolved oxygen (4.17±0.1 mg/L), electrical conductivity (840.41±2.46 µS/cm), and pH (8.12±0.03) were measured daily. At different exposure periods (24, 48, 72 and 96 hrs), the mortality of the fish was subjected to probit analysis using Minitab software (Minitab®16.2.4) to calculate the LC values, their slope functions, and confidence limits. Results The percent mortality of the exposed A. dispar to Exposure periods Effective dose (g/L) SE limits Slope function 't' ratio LCL UCL 24 hrs LC1=0.010 LC5=0.024 LC10=0.037 LC20=0.063 LC50=0.177 LC80=0.496 LC90=0.851 LC95=1.327 LC99=3.056 0.004 0.008 0.011 0.017 0.039 0.117 0.228 0.401 1.155 0.004 0.012 0.020 0.038 0.115 0.313 0.503 0.734 1.457 0.024 0.047 0.067 0.106 0.272 0.789 1.439 2.400 6.411 1.414±0.229 6.183 48 hrs LC1=0.005 LC5=0.014 LC10=0.023 LC20=0.041 LC50=0.131 LC80=0.414 LC90=0.757 LC95=1.246 LC99=3.172 0.002 0.005 0.007 0.011 0.030 0.104 0.219 0.409 1.317 0.002 0.007 0.012 0.024 0.084 0.253 0.430 0.654 1.406 0.013 0.028 0.043 0.071 0.204 0.678 1.334 2.373 7.156 1.485±0.226 6.556 72 hrs LC1=0.001 LC5=0.005 LC10=0.009 LC20=0.018 LC50=0.073 LC80=0.296 LC90=0.618 LC95=1.133 LC99=3.540 0.001 0.002 0.003 0.006 0.018 0.083 0.202 0.427 1.714 0.001 0.002 0.004 0.010 0.045 0.171 0.325 0.541 1.371 0.004 0.011 0.018 0.033 0.118 0.512 1.173 2.373 9.144 1.570±0.219 7.163 96 hrs LC1=0.001 LC5=0.003 LC10=0.005 LC20=0.012 LC50=0.052 LC80=0.230 LC90=0.500 LC95=0.949 LC99=3.155 0.000 0.001 0.002 0.004 0.013 0.067 0.172 0.378 1.621 0.000 0.001 0.003 0.006 0.032 0.130 0.255 0.435 1.153 0.003 0.007 0.012 0.023 0.087 0.407 0.981 2.070 8.635 1.675±0.228 7.332 Table 1. Effective dose, confidence limits, and slope function for aqueous extract of Euphorbia turcomanica at different intervals on Aphanius dispar. 349 Zare et al./ Acute toxicity of E. turcomanica on A. dispar various concentrations of the plant extract of E. turcomanica for 24, 48, 72 and 96 hrs are depicted in Figure 1. The LC50 values at various exposure periods were 0.177 g/L for 24 hrs, 0.131 g/L for 48 hrs, 0.073 g/L for 72 hrs, and 0.052 g/L for 96 hrs. The LC values, their upper and lower confidence limits, and slope functions are given in Table 1. The toxicity of dried powder of E. turcomanica was found to be time and dose-dependent (P<0.05). The regression coefficient demonstrated a significant positive correlation (P<0.05) between mortality percent and concentration of E. turcomanica. Also a significant negative correlation (P<0.05) was found between the exposure time and different LC values. Discussion The results revealed that the dried powder of E. turcomanica has a high piscicidal activity causing more mortality with increasing its concentration. The toxicity was both time and dose dependent. A significant negative correlation demonstrated between different LC values and exposure time. LC50 value of E. turcomanica decrease with increasing exposure time in A. dispar from 0.177 g/L after 24 hrs to 0.052 g/L after 96 hrs. To my best knowledge, there is no report on the toxicity effects of E. turcomanica on aquatic animals. Although, some reports are present on the toxicity effects of the aqueous and latex extracts of other members of the family Euphorbiaceae on some animals, including fish and molluscs (Singh and Singh, 2002; Singh and Singh, 2005; Tiwari and Singh, 2006; dos Santos et al., 2007; Oliveira-Filho et al., 2010). The LC50 value for 24 hrs of dried powder of E. tirucalli stem bark latex for Colisa fasciatus was 8.14 mg/L, whereas this value for Channa punctatus was 9.01 mg/L (Tiwari et al., 2003). In another experiment, the toxicity of four plants belonging to members of Euphorbiaceae and Apocynaceae on C. punctatus evaluated (Singh and Singh, 2005). In this study, 96 hrs LC50 values of E. royleana, Nerium indicum, Jatropha gossypifolia, and Thevetia peruviana were 0.020, 0.041, 4.34, and 3.17 g/L, respectively (Singh and Singh, 2005). This value for E. turcomanica was 0.052 g/L on A. dispar. In general, juicy and latex-bearing Euphorbiaceae are more potent in their toxic effects than rotenone- yielding plants (Neuwinger, 2004). In the present study, the dried powder of whole plant is used directly and nevertheless, the LC50 values of E. turcomanica is comparable in potent toxicity to the other studies. 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(2015) 3(5): 346-351 E-ISSN: 2322-5270; P-ISSN: 2383-0956 Journal homepage: www.ij-aquaticbiology.com © 2015 Iranian Society of Ichthyology چکیده فارسی ( Euphorbia turcomanica) ترکمنی فرفیون گیاه کشنده های غلظت تعیین (Aphanius dispar) گورخری آفانیوس ماهی روی بر 3بنایی مهدی ،2زادی یوسف مرتضی ،*1نوری احمد ،1زارع هما .ایران بندرعباس، هرمزگان، دانشگاه دریایی، فنون و علوم دانشکده شیالت، گروه1 .ایران بندرعباس، هرمزگان، دانشگاه دریایی، فنون و علوم دانشکده دریا، زیست گروه2 .ایران بهبهان، االنبیاء، خاتم دانشگاه طبیعی، منابع و زیست محیط دانشکده شیالت، گروه3 چکیده: مطالعه مورد تناننرم و ماهی کشنده سم عنوانبه Euphorbiaceae خانواده به متعلق هایگونه از بسیاری از حاصل آبی هایعصاره تاثیر تاکنون در آبزی موجودات روی بر E. turcomanica ترکمنی فرفیون گیاه کشندگی و سمیت توان مورد در اطالعاتی وجود این با است، گرفته قرار هایغلظتمنظور تعیین به محصور محیط در روزه چهار آزمایش روش از استفاده با گیاه این کشنده هایغلظت تعیین آزمون. باشدمین دسترس 05LC گیاه شده خشک پودر E. turcomanica گورخری آفانیوس ماهی روی بر A. dispar، اجرایک گونه با دامنه تحمل باالی شوری، به لیتر بر گرم 131/5±535/5 ساعت، 22 برای لیتر بر گرم 111/5±530/5 ترتیب به در معرض قرارگیری مختلف هایزمان در 05LC مقادیر. درآمد شده خشک پودر سمیت. آمد دست به ساعت 09 برای لیتر بر گرم 502/5±513/5 و ساعت 12 برای لیتر بر گرم 513/5±514/5 ساعت، 24 برای گزارش یناول این تحقیق نتایج اینکه به توجه با. داد نشان تاثیرگذاری زمان مدت و ماهی تلفات بین را مثبتی همبستگی E. turcomanica گیاه Euphorbiaceae های گونه سایر نتایج با مقایسه قابلتنها نتایج این باشد،می A. dispar ماهی روی بر E. turcomanica گیاه سمیت مورد در هایگونه سایر به نزدیک و مقایسه قابل E. turcomanica گیاه سمیت توان که نمود استنباط توانمی حاصل نتایج از. است دیگر ماهیان روی بر Euphorbiaceae گیاه از تولیدی ترکیبات که شودمی پیشنهاد همچنین. اندگرفته قرار بررسی مورد تاکنون که است E. turcomanica توجه با .دگردن استفاده است، مختلف ماهیان زیست محل که آبی هایمحیط در مستقیم طوربه آن باالی سمیت به .ماهی گورخری ماهی، کشنده سم خشک، پودر ترکمنی، فرفیون :کلمات کلیدی