Low Salinity Decreases the Tolerance to Two Pesticides, Beta-cypermethrin and Acephate, of White-leg Shrimp, Litopenaeus vannamei

With the rapid development of green revolution in agriculture, pesticides are widely used in pest control. However, it could lead to high concentrations of these pesticides in surface water especially in intensive agricultural areas [1], which are hazards for aquatic species [2]. Most insecticides used in agriculture are organophosphate, Carbamate and synthetic Pyrethroid compounds [3] because of their relatively non-persistent characteristics in the environment. Though these pesticides can rapidly degrade in natural environment, their high acute toxicity to some non-target species has been demonstrated in many laboratory tests [4-7].


Introduction
With the rapid development of green revolution in agriculture, pesticides are widely used in pest control. However, it could lead to high concentrations of these pesticides in surface water especially in intensive agricultural areas [1], which are hazards for aquatic species [2]. Most insecticides used in agriculture are organophosphate, Carbamate and synthetic Pyrethroid compounds [3] because of their relatively non-persistent characteristics in the environment. Though these pesticides can rapidly degrade in natural environment, their high acute toxicity to some non-target species has been demonstrated in many laboratory tests [4][5][6][7].
Beta-cypermethrin and Acephate are two commonly used pesticides worldwide. Beta-cypermethrin is a synthetic analog of Pyrethrins, extracts from the ornamental Chrysanthemum Cinerariaefolium, and widely used in field pest control [8]. Due to the Lipophilicity, Pyrethroids have a high rate of gill absorption. So they have been reported to be extremely toxic to fish [8][9][10][11][12], but information in crustacean species is still limited. Acephate is a water soluble foliar spray insecticide of moderate persistence and is used on fruit and vegetable crops grown across the world [13]. A number of studies have been conducted on the toxicity of Acephate on different organisms and indicated as a potent Neurotoxicant [14]. It is also found to be mutagenic [15], carcinogenic [16], and Cytotoxic [17]. However, this compound is considered relatively non-toxic to fish with a median lethal concentration (LC 50 ) for goldfish of 9,550 mg/l and rainbow trout >1,000 mg/l over 96 h [13]. But potential hazard to aquatic crustaceans has been speculated for the heavy use of these compounds and deficiency in the immune system, since lower LC 50 values have been found in Americanmysis bahia, Homarus americanus, Penaeus aztecus and P. duprarum [18][19].
The white-leg shrimp, Litopenaeus vannamei, is a typical tropical crustacean that can adapt to a wide range of salinities ranging from 1 to 50.0% [20], and with the development of inland saline water farming, it has become one of the most popular shrimp for aquaculture in the Central and South American countries [21][22], Thailand [23] and China [24]. Toxicology of ambient pesticides on this species mainly focused on diazinon [25], Propiconazole [25], parathion [26], pyrazosulfuron-ethyl [27], permethrin [27], chlordane [28], DDT [28], lorsban [28] and lindane [28], while there is no report on the toxicology of beta-cypermethrin and Acephate to this species at either salinity, though environment characteristics may affect the toxicity of toxicants to aquatic organisms [29].
L. vannamei were obtained from a farm with pond salinity of 20.0% in Haikou, Hainan, China. Shrimps were cultured in fiberglass tanks (60×50×50 cm) at a salinity of 20.0% for 1 week, then divided randomly into two groups and acclimated to the target salinities (5.0% and 20.0%) by changing 2.0% per day. After reaching the targeted salinities, each group was acclimated an additional week. Individual shrimp, weighing 4.54 ± 0.33 g, were selected for the toxicology experiments. During the pre-culture and acclimation period, shrimps were fed with a commercial feed containing 41.1% crude protein, 9.6% crude lipid, 12.0% ash, and 10.0% moisture. Seawater was pumped from the Xiuying Coast in Haikou City, and filtered through an activated carbon cartridge for at least 3 days before entering the culture system. The tap water was aerated before being added to the tank to adjust the salinity levels. The water quality parameters during the experiment were 27.7-29.2°C for temperature, 5.58-6.79 mg/L or dissolved oxygen, 8.3 ± 0.2 for pH value and <0.01 mg/L for total ammonia-nitrogen.
Beta-cypermethrin and Acephate (Yujing Bio-Tech Co. Ltd., Beijing City, China) stock solutions were prepared at the day of trial starting. As beta-cypermethrin was only slightly dissolved in water, it was prepared by diluting it in acetone to give the stock material. The control group received a same acetone volume used in the dilution of the dosing concentrations. For both pesticides, six concentrations with a control were used with triplicates for each treatment. The beta-cypermethrin stock solution was diluted to the following concentrations: 0 (control), 0.05, 0.1, 0.2, 0.4, 0.8, 1.6 μg/L at 5.0%, and 0 (control), 0.1, 0.2, 0.4, 0.8, 1.6, 3.2 μg/L at 20.0%. And the Acephate stock solution was diluted to the following concentrations: 0 (control), 13.3, 20, 30, 45, 67.5, 101.25 mg/L at 5.0%, and 0 (control), 20, 30, 45, 67.5, 101.25, 151.88 mg/L at 20.0%. Ten shrimp were randomly assigned to each treatment and all the tanks were aerated continuously to keep oxygen at adequate levels. Test solutions were renewed totally every 24 h. The experiment was conducted for 96 h and no feed was supplied to the shrimps during the test period. Observations on mortality and abnormal behavior of the shrimps were conducted at least two times each day. Death was assumed when juvenile white shrimps were non-motile and showed no response when touched with a glass rod [30]. Dead shrimps were removed immediately when they were found. All treatments were run in triplicate with similar conditions. Median lethal concentrations (LC 50 ) with 95% confidence limits were calculated by Trimmed Spearman-Karber Method [31]. The standard error of the difference was used to determine statistical differences in LC 50 values and the effect of salinity to the acute toxicity of beta-cypermethrin and Acephate at different exposure time was analyzed by the t-test.

Results and Discussion
Signs of toxicity in fish poisoned with beta-cypermethrin generally include the changes of loss equilibrium, color darkening and staying motionless [10]. Signs of toxicity in terrestrial animals poisoned with Acephate include muscular weakness, tremors, reduced activity [32], but reports on signs of toxicity in aquatic animals were limited. In this study, less toxic symptom can be observed for L. vannamei poisoned with both beta-cypermethrin and Acephate. Dying shrimp showed pronounced whiter body color compared with the alive, and the shrimp dying, when turned over, was nearly immobile on the bottom or showed little movement when touched with a glass stick.
The percentages of mortality recorded for each treatment are shown in Table 1. No mortality was observed in the controls at either salinity or in the treatments of 0.10 μg/L at 20.0% for beta-cypermethrin during the 96 h. However, 100% mortality occurred in the treatments of 101.25 mg/L at 5.0% and 151.88 mg/L at 20.0% for Acephate and 3.20 μg/L at 20.0% for beta-cypermethrin after 24 h of exposure. The mean LC 50

Salinity
Beta-cypermethrin Acephate    (Figure 1 and Table 2). The results indicated that L. vannamei at low salinity is more sensitive to the ambient beta-cypermethrin and Acephate toxicity. Meanwhile, beta-cypermethrin had higher toxicity than Acephate to L. vananmei at either salinity.
The LC 50 values for beta-cypermethrin and Acephate to aquatic animals reported in the existing literatures are shown in Table 3, which support the finding of higher toxicity of beta-cypermethrin to white shrimp in this study, though the LC 50 values of both pesticides varied in different aquatic organisms. When compared with the toxicity of other pesticides reported to L. vannamei (Table 4), except for permethrin, beta-cypermethrin is more highly toxic to L. vannamei than some other commonly used pesticides, such as diazinon [25], propiconazole [25], parathion [26], pyazosulfuron-ethyl [27], chlordane [27], DDT [28], lorsban [28], and lindane [28]. While the toxicity of Acephate to L. vannamei is less than that of other pesticides except for pyrazosulfuronethyl (Table 4). According to the determination of toxicity for different pesticides [33], beta-cypermethrin is extremely toxic to L. vannamei (96 h LC 50 <0.1 mg/L), while Acephate is low toxic (96 h LC 50 is 10-100 mg/L).
L. vannamei is more susceptible to ambient beta-cypermethrin and Acephate at 5.0% than at 20.0%, and significant differences were both found at 24 h, 72 h and 96 h (P<0.05). Similar findings were reported in the toxic effects of ambient ammonia, boron and nickel to L. vannamei. The 96 h LC 50 with 95% confidence limit of L. vannamei at 3.0% to ambient ammonia-N was 9.33 mg/L [36], which was significantly lower than the previous results [37], which reported that the LC 50  and acephate (down) to L. vannamei exposed from 24 to 96 h at 5% (∆) and 20.0% (•). "*" and "**"mean p<0.05 and p<0.01 of t-test, respectively.

Scientific name LC 50 Source
Beta-cypermethrin (μg/L)  Chlordane 0.0632 mg/L-48 h 2.44 g [28] DDT 0.0087 mg/L-48 h 2.84 g [28] Lorsban 0.0048 mg/L-48 h 2.49 g [28] Lindance 0.0039 mg/L-48 h 2.76 g [28]  219.52, 147.8, 80.06 mg/L at 20.0% [38]. The 96 h LC 50 values were 41 μmol/L and 362 μmol/L for L. vannamei at 5% and 25%, respectively [39] many previous studies have demonstrated that the optimal salinity for growth of L. vannamei should be around 20% [36,40]. When L. vannamei is exposed to dilute medium or low salinity, many problems such as the passive loss of Na + and Cl − will occur [41], and though L. vannamei could cope with these problems in different physiological ways [42], extremely low salinity is still a serious stress for L. vannamei. Therefore, lower mean LC 50 values of beta-cypermethrin and Acephate to white shrimp were observed at 5.0% when compared with those at 20.0% in the present study.
Overall, ambient beta-cypermethrin and Acephate can pose an acute toxic effect on L. vannamei. Beta-cypermethrin is extremely toxic to L. vannamei, while Acephate is low toxic. L. vannamei at low salinity was more sensitive to the toxicity of these two toxicants.