Int. J. Aquat. Biol. (2017) 5(1): 1-6; DOI: ISSN: 2322-5270; P-ISSN: 2383-0956 Journal homepage: www.ij-aquaticbiology.com © 2016 Iranian Society of Ichthyology Original Article Acute toxicity of nonylphenol ethoxylate-6 to whiteleg shrimp, Penaeus vannamei (Boone, 1931) (Decapoda, Penaeidae) Azin Ahmadi1, Ahmad Noori1*, Mahdi Banaee2 1Department of Fisheries Science, Faculty of Marine Science and Technology, University of Hormozgan, Bandar Abbas, Iran. 2Department of Aquaculture, Faculty of Natural Resources and Environment, Behbahan Khatam Al-Anbia University of Technology, Iran. Article history: Received 24 April 2016 Accepted 15 December 2016 Available online 2 5 February 2017 Keywords: Nonylphenol ethoxylate-6 Shrimp Penaeus vannamei Acute toxicity Abstract: Nonylphenol ethoxylate-6 (NP6EO) is widely used in industrial and domestic products and easily detected in the environment. The toxicity and estrogenic potency of alkylphenols have been investigated in several studies. However, to the best of our knowledge available, acute toxicity data about the effects of NP6EO on decapod and aquatic ecosystem in literature is yet scarce. Therefore, in this study the adult male and female whiteleg shrimp, Penaeus vannamei, were exposed to various concentrations of NP6EO (0.04, 1, 5, 25, 125, 625 µL L-1) for four days. Acute toxicity potential of NP6EO on adult P. vannamei was assessed by calculating LC50 for different times. Median lethal concentration (LC50) of NP6EO at 96 hours was 7.017 μL L-1. The LC50 of this compound revealed a positive correlation between shrimp mortality and exposure periods. The data exhibited that NP6EO was considered as "toxic" to P. vannamei and further toxicity assessment to other species is strongly recommended. Introduction Man-made chemicals are an important part of the modern life. Human beings as well as wildlife populations cannot avoid coming into contact with many of chemicals employed in variety of industries like food production (plants and meat), pathogen control (insecticides), production of modern materials (plastics), or in the built environment (insulations and fire retardants) (Bergman et al., 2012). Considering the importance of these compounds and their widespread presence in the environment, it is important that comprehensive strategies are developed to preclude widespread environmental contamination with endocrine disruptors (EDs) and protect environment (David et al., 2009; Bergman et al., 2012). Nonylphenol ethoxylate-6 (NP6EO) is used in countless number of applications and because of its extensive use, discharged to the sewer system and make their way into wastewater and aquatic systems * Corresponding author: Ahmad Noori E-mail address: nooryahmad@gmail.com (Ying et al., 2002). NP6EO is a nonionic surfactant that is used in a wide range of industrial applications and consumer products, such as laundry detergents, dust-control agents and deicers, industrial liquid soaps and cleaners, cosmetics, paints, and as the dispersing agents in pesticides and herbicides (Jobling and Sumpter, 1993). Concern has recently increased about the use of alkylphenol ethoxylates (APEs) because of the relative stability of their metabolites such as nonylphenol (NP), octylphenol (OP) and nonylphenol ethoxylate-1-3 (NP1-3EO) in the environment (Giger et al., 1984) and their estrogenic effects on organisms (Ying et al., 2002) which is considered as EDs. Testing strategies employed acute toxicity studies to evaluate and measure the effect(s) of one or more pollutants on one or more species. This implies that tests at high doses will inform us about low-dose exposures (Reish and Oshida, 1986; Bergman et al., 2012). The lethality of the EDs was used as the 2 Ahmadi et al./ Acute toxicity of nonylphenol ethoxylate-6 to whiteleg shrimp endpoint in an aquatic acute toxicity testing system (Faheem and Lone, 2013). In general, determination of lethal concentrations, such as the median lethal concentration (LC50), is recognized as the first step for risk assessment of synthetic and natural chemicals (Johnson and Finley, 1980; Ura et al., 2002). These data assist in the development and application of water quality criteria for the protection of the aquatic environment. In spite of evidence that proved the toxicity of NPnEO on many aquatic animals (Dorn et al., 1993; Lussier et al., 2000; Hirano et al., 2004; Oliveira- Filho et al., 2005; Ricciardi et al., 2008; Liu et al., 2011), there is no attempts have been made to determine the impacts of NP6EO on Penaeus vannamei. Hence, this study aims to determine and compare the acute toxicity of NP6EO to P. vannamei upon modification of the exposure conditions. Materials and Methods Chemicals: NP6EO (CAS No.9016-45-9) was obtained from Kimiagaran Emroz Company (Tehran, Iran). Stock solution of the NP6EO was prepared by dissolving appropriate concentration in 96% ethanol as solvent. Required concentrations were obtained by serial dilution and stored in dark at 4°C until usage. Solvent concentration was kept at 0.01% (v/v) for all treatments. Animal maintenance and exposure to NP6EO: Adult P. vannamei (both sexes; body weight: 25.89±0.79 g, total length: 14.26±0.16 cm) were obtained from shrimp farms located at southern coast lines of Iran and were transferred to Kolahi Aquatic Restock Center (KARC). Adult shrimps were acclimated in 300L fiberglass tanks containing ultraviolet-filtered recirculating water (pH=7.77±0.017) for 2 weeks prior to the experiment. Feeding was done on daily basis in four times at the rate of 2.5% of the body weight by commercial feed until 24 hrs prior to the initiation of the test. After the acclimation period, 240 adult shrimp were randomly distributed among 24 tanks, comprising control, vehicle control (ethanol with a final concentration of 1:1,000 v/v water), 0.04, 1, 5, 25, 125, 625 µL L-1 of NP6EO. The experiment was run in triplicate, without feeding and water exchange during the experiment. The mortality of shrimps in each treatments were counted and recorded over the exposure period at 24, 48, 72 and 96 hrs. Dead shrimps were removed from treatments immediately. The study was approved by the Iranian Society for the Animal Welfare. Data analysis and statistics: Data are expressed as mean with the corresponding standard error (SE). LC values and 95% confidence intervals (95% CI) were calculated using probit analysis. Shapiro-Wilk and Levene’s tests were used to check the normality of data distribution and the homogeneity of variances, respectively (Zar, 2010). If data support the prerequisites for parametric analysis, one-way analysis of variance (ANOVA) followed by Tukey’s multiple range test was applied. Otherwise, Kruskal- Wallis and Mann-Whitney U test were applied to determine the statistical significance (Zar, 2010). All the analysis was performed by SPSS 16.0. The significant level in all analysis was set at P≤0.05. Results The percent mortality of P. vannamei after exposure to various concentrations of NP6EO for 24, 48, 72 and 96 hrs has been depicted in Figure 1. Mortality increased with increasing concentrations and exposure time (Fig. 1). The LC50 values were 437.052 ± 326.250 µL L-1 for 24 hrs, 33.627±15.443 µL L-1 for 48 hrs, 10.816±3.936 µL L-1 for 72 hrs and 7.017±2.391 µL L-1 for 96 hrs. The LC values, their upper and lower confidence limits and slope functions for NP6EO have been given in Table 1. Discussion Acute toxicity tests provide a measure of the toxicity of the given compounds to experimental species under specific environmental conditions (Reish and Oshida, 1986). They also reflect the severe and rapid damage caused by sudden exposure to lethal concentrations of contaminants (Alam and Maughan, 1993). In the present study, calculated LC50-96 hrs value https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=2&cad=rja&uact=8&ved=0ahUKEwiYstvgg4rNAhVHbRQKHRiECPEQFggkMAE&url=https%3A%2F%2Fen.wikipedia.org%2Fwiki%2FUltraviolet_germicidal_irradiation&usg=AFQjCNHP4CeJi4utFBNFUpPqESvaxLwjQw&sig2=KgVHTTNjloxvOHxTAoMR3Q 3 Int. J. Aquat. Biol. (2017) 5(1): 1-6 of NP6EO for P. vannamei was assessed as 7.017 μL L-1, which was in agreement with the results reported for other species have been tested with NP and NPnEO (Dorn et al., 1993; Mann and Bidwell, 2000; Oliveira-Filho et al., 2005; Ricciardi et al., 2008). The acute and chronic toxicity of alkylphenol ethoxylates and their metabolites have been investigated for several freshwater and marine species (Servos, 1999; Staples et al., 2004). Previous studies showed that species sensitivity varies from 17 μg L-1 of para-nonylphenol (PNP) for winter flounder (Pleuronectes americanus) (Lussier et al., 2000) to 9.2 mg L-1 of NP8EO for Litoria adelaidensis (Mann and Bidwell, 2000). Median lethal concentrations of 4 to 6.6 mg L-1 of NP9EO was reported for fathead minnow, Pimephales promelas (Dorn et al., 1993; Staples et al., 1998). Also in the other similar studies, LC50-96 hrs for NP, NP1EO and NP2EO for fathead minnow were 136, 218, and 323 μg L-1, respectively (TenEyck and Markee, 2007). The varying data of available toxicity tests resulted as a function of ethoxy chain Table 1. Effective dose, confidence limits, and slope function for nonylphenol ethoxylate-6 (NP6EO) at different intervals for the whiteleg shrimp, Penaeus vannamei. Exposure periods Effective dose (µL/L) SE limits Slope function 't' ratio Heterogeneity LCL UCL 24 hrs LC1=0.006 LC5=0.161 LC10=0.922 LC20=7.642 LC50=437.052 LC80=* LC90=* LC95=* LC99=* 0.009 0.150 0.647 3.976 326.250 * * * * 0.000 0.012 0.144 2.346 131.656 3340.90 * * * 0.052 0.683 2.895 20.537 3241.25 * * * * 0.479±0.081 5.920 1.135 48 hrs LC1=0.001 LC5=0.026 LC10=0.128 LC20=0.866 LC50=33.627 LC80=1305.031 LC90=8834.585 LC95=* LC99=* 0.002 0.023 0.089 0.447 15.443 1032.11 8962.67 * * 0.000 0.003 0.022 0.255 14.568 362.622 1755.602 6344.154 * 0.010 0.107 0.400 2.124 95.011 9758.932 * * * 0.530±0.070 7.548 1.540 72 hrs LC1=0.002 LC5=0.028 LC10=0.104 LC20=0.512 LC50=10.816 LC80=228.489 LC90=1125.541 LC95=4199.877 LC99=* 0.002 0.020 0.063 0.237 3.936 127.492 802.962 3578.53 * 0.000 0.000 0.001 0.022 2.243 39.623 133.766 348.322 1984.653 0.042 0.247 0.664 2.438 74.971 * * * * 0.635±0.075 8.436 2.903 96 hrs LC1=0.003 LC5=0.027 LC10=0.091 LC20= 0.406 LC50=7.017 LC80=121.319 LC90=538.201 LC95=1841.874 LC99=* 0.002 0.018 0.052 0.182 2.391 59.905 338.229 1382.53 * 0.000 0.000 0.002 0.028 1.708 26.836 87.163 219.975 1182.462 0.035 0.193 0.495 1.673 33.301 2593.424 * * * 0.680±0.078 8.755 2.538 * Values more than 10000 are not shown 4 Ahmadi et al./ Acute toxicity of nonylphenol ethoxylate-6 to whiteleg shrimp length, the type of test used, and the species tested. Considering the relative toxicity values, or NP toxic equivalency factors (TEFs) which calculated for nonylphenol compounds, toxic concentrations for different nonylphenolic compounds such as NPnEO with various EO chain length could be matched with similar endpoints for NP for the same species. If TEF for NP be considered as 1, for example, toxic equivalency factors for NPnEO (1