Ecotoxicology and Environmental Safety 101 (2014) 31–35
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Biochemical responses to the toxicity of the biocide abamectin on the freshwater snail Physa acuta Junguo Ma, Chune Zhou, Yao Li, Xiaoyu Li n College of Life Science, Henan Normal University, Xinxiang, Henan 453007, China
art ic l e i nf o
a b s t r a c t
Article history: Received 14 September 2013 Received in revised form 3 December 2013 Accepted 11 December 2013 Available online 8 January 2014
The toxic effects of abamectin (ABM), an anthelmintic drug, on the snail, Physa Acuta, and the biochemical responses to the exposure stress were evaluated. The activities of superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST), acetylcholinesterase (AChE), and nitric oxide synthase (NOS), and the contents of malondialdehyde (MDA) were determined in snail soft tissues (head, foot, visceral mass, and the mantle) for up to 96 h of exposure to 3.4, 9.6, 19.2, or 27.4 μg L 1 of ABM. The results showed that SOD and GST activities were promoted by ABM-exposure at the earlier periods of treatment (12–48 h) while these activites were inhibited at the end of test. The tendency of CAT activity was similar to that of SOD, but it increased at the end of test. MDA levels of the snail soft tissues increased in all treatment groups, including the recovery group, indicating that lipid peroxidation occurred in snail soft tissues. ABMexposure inhibited AChE activity. However, NOS activities increased by ABM-exposure. In addition, activities of antioxidant enzymes and AChE from the snail soft tissues resumed the normal levels after 96 h of recovery period, but MDA level did not attain the original level. This study provides information on the biochemical mechanism of ABM toxicity on the snail. & 2014 Published by Elsevier Inc.
Keywords: Abamectin Physa acuta Acetylcholinesterase Nitric oxide synthase Antioxidant enzymes Lipid peroxidation
1. Introduction Abamectin (ABM) is a kind of large ring lactone disaccharide compounds and the natural fermentation product of the soildwelling actinomycete Streptomyces avermitilis (Roth et al., 1993; Luo et al., 2013). It is highly lipophilic and used both as a biocide and as an anthelminthic drug. According to the classification standard by WHO, ABM belongs to highly toxic chemicals with neurological and developmental toxicity. The toxicological mechanism of ABM is believed to affect the γ-aminobutyric acid (GABA) system and Cl channels of animal cells (Maioli et al., 2013), in which the GABA receptor is responsible for regulating the neural basal tone (Turner and Schaeffer, 1989). Since twenty years ago, ABM had been widely used for preventing parasitic diseases from fish (Lorio et al., 1992; Johnson and Margolis, 1993). Therefore it may enter aquatic environment and pose environmental risks to aquatic animals and ecosystems. Toxicologically, ABM usually has only slight toxicity to earthworms and birds (Halley et al., 1993), but it is highly toxic to fish (Jenčič et al., 2006), representing a potential threat to aquatic animals. Aquatic snail Physa acuta is an invasive species and now world widely distributed in freshwater body (Albrecht et al., 2009; Guo
n
Corresponding author. Fax: þ 86 373 3329390. E-mail address: [email protected] (X. Li).
0147-6513/$ - see front matter & 2014 Published by Elsevier Inc. http://dx.doi.org/10.1016/j.ecoenv.2013.12.009
et al., 2009). Moreover, the snail is very sensitive to toxicants and thus appropriate for toxicity testing (Evans-White and Lamberti, 2009; Sánchez-Argüello et al., 2009; Lance et al., 2010; Musee et al., 2010; De Castro-Català et al., 2013; Hossain and Aditya, 2013; Seeland et al., 2013). Therefore it was adopted as experimental animal to determine ABM toxicity and biochemical alterations in the snail P. acuta by conducting an acute exposure of ABM in the present study.
2. Materials and methods 2.1. Chemicals and reagents ABM as two percent microemulsions was obtained from Biogen Crop Science Limited, China. It was first dissolved in distilled water for stock solutions and then diluted to obtain the experimental concentrations. The Superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST), acetylcholinesterase (AChE), and nitric oxide synthase (NOS) Diagnostic Reagent Kit were purchased from the Nanjing Jiancheng Bioengineering Institute (China). Bovine serum albumin and thiobarbituric acid (TBA) were obtained from Sigma (USA). 2.2. Snails The adult P. acuta with an average wet weight of 0.136 7 0.035 g and an average shell length of 6.853 7 1.438 mm were collected from a fishery pond in China and cultured in glass jars (3 L in volume) with aerated tap water (total hardness of
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The acute toxicity test was conducted according to the Spearman-Kärber method (Kärber, 1931) and the exposure concentrations were 3.4 μg L 1 (1/10 96 h LC50), 9.6 μg L 1 (1/10 48 h LC50), 19.2 μg L 1 (1/5 48 h LC50), and 27.4 μg L 1 (2/7 48 h LC50). These concentrations were designed based on the results of our previous report, in which the 96 h and 48 h LC50 of ABM to P. acuta was 34 and 96 μg L 1, respectively (Ma and Li, 2011). Two hundred snails were randomly divided into five groups (40 snails in each group), out of which four groups were ABM-treated groups (3.4, 9.6, 19.2, and 27.4 μg L 1 of ABM) and exposed for 12, 24, 48, and 96 h. Another group was maintained as a control. The animals in each test group were placed in a 1000 mL beaker containing 800 mL of ABM solution, and the control snails were bred temporarily in a beaker with aerated tap water. The beaker was covered with cotton gauze to prevent snails from escaping. The cotton gauze must be on the surface of water in the beaker so that the snails cannot choke due to dryness when they attach to the cotton gauze. Each test was conducted in triplicate. Except snails were not fed, ABM exposure conditions were maintained as to those used for the breeding of the snails. At the end of each exposure period (12, 24, 48, and 96 h), ten snails from each group were taken and placed in Eppendorf tube. After their shells were removed, all the soft tissues (head, foot, visceral mass, and the mantle) were homogenized (10 percent w/v) in 0.1 M phosphate buffer (pH 7.5). The homogenate was centrifuged at 12,000 g for 10 min at 4 1C and the supernatant obtained was stored at –20 1C for biochemical assays.
2.4. Recovery test The recovery test was carried out to evaluate the surviving capacity of snails from the acute exposure of ABM. Sixty snails were randomly divided into three groups, in which two groups were the ABM-treatment groups (19.2 and 27.4 μg L 1 of ABM) and another as a control group. After 96 h of treatment, the snails were removed to the aerated tap water immediately and recovered in the ABM-free water for another 96 h. During the recovery test, snails were fed on commercial food at a rate of 0.5–1 percent of body weight/day. At 48 or 96 h time intervals, ten snails were collected and homogenized as described above for biochemical assays. Each test was conducted in triplicate.
2.5. Biochemical analysis The activities of SOD, CAT, GST, AChE, and NOS in snail soft tissues were determined by using the Diagnostic Reagent Kit purchased from the Nanjing Jiancheng Bioengineering Institute (China) according to the manufacturer0 s instructions. The assay results were given in units of enzymatic activity per milligram of protein (U/mg protein), in which one unit was defined as the amount of enzyme producing 50 percent inhibition of the enzyme, as the amount of enzyme decomposing 1 μM H2O2 per second, as the amount of enzyme causing GSH concentration down 1 μM L 1 in 1 min, as the amount of enzyme decomposing 1 nM NO per minute, and as the amount of enzyme capable of converting 1 μM of acetylthiocholine iodide as a substrate per minute for SOD, CAT, GST, NOS, and AChE, respectively. The total protein contents of samples were measured by the method of Lowry et al. (1951) using bovine serum albumin as a standard. Lipid peroxidation was measured by the TBA method (Ohkawa et al., 1979). The assay was monitored for the appearance of the conjugated complex of TBA and malondialdehyde (MDA) at 532 nm. The concentration of MDA was expressed as nM MDA per mg protein.
3.1. Antioxidant enzymes 3.1.1. SOD activity No change was found in SOD activity of the snail soft tissues between the treatment and control groups after 12 h of ABMexposure, except for an increase in the group of 19.2 μg L 1 of ABM when compared to the control (Fig. 1A). However, at 24 h exposure, SOD activities in the groups of 9.6 and 19.2 μg L 1 of ABM were significantly higher than that of control, while it was remarkably inhibited in the highest concentration group (27.4 μg L 1). Its activity was maintained unchanged in the lowest concentration group (3.4 μg L 1). Then (48 h later), SOD activity in 19.2 μg L 1 of ABM group also decreased while still increased in 9.6 μg L 1 group. At the end of test (96 h), SOD activities in all treated groups (except for 3.4 μg L 1) were lower than that of control (Fig. 1A).
3.1.2. CAT activity The average change tendency of CAT activity was similar to that of SOD before 48 h of ABM-exposure, but it increased in all treated groups (except for 3.4 μg L 1) at the end of test (Fig. 1B).
60 SOD activities (U/mg protein)
2.3. ABM-exposure
3. Results
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50
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40
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30 20
10 0 12
24
48
96
Exposure times (h)
80 CAT activites (U/mg protein)
water 340 mg L 1, pH 7.6, turbidity 1.5 nephelometric turbidity units, and total dissolved solid content 660 mg L 1, which were detected according to the Standards for Drinking Water Quality, China, GB5479-2006) at a constant temperature (22 7 1 1C) and a 16 h: 8 h light/darkness photoperiod under laboratory conditions for several generations prior to the experiment. Snails were fed on a mixture of commercial goldfish food (Wannong Fishery Company, China) at a rate of 0.5–1 percent of body weight/day and lettuce leaves. The water was changed weekly. The snail was handled according to the guidelines in the China Law for Animal Health Protection and Instructions for Granting Permits for Animal Experimentation for Scientific Purposes (Ethics approval No. SCXK (YU) 20050001).
**
70
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60
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50
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40 30 20 10 0
12
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Exposure times (h)
70 GST activities (U /mg protein)
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60 **
50
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40
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30 20
10 0
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48
96
Exposure times (h)
2.6. Statistical analysis Statistical analysis was performed using the SPSS 13.0 software (SPSS Inc.). All data were analyzed by one-way ANOVA and post-hoc pairwise comparisons among all groups using the Duncan0 s test (po 0.05).
Fig. 1. Effects of ABM on activities of SOD (A), CAT (B), and GST (C) in the snail soft tissues after 12, 24, 48, or 96 h of exposure. Values represent the means and vertical bars indicate the standard deviation of three separate experiments. Asterisks denote a response that is significantly different from the control (npo 0.05, nn p o0.01).
J. Ma et al. / Ecotoxicology and Environmental Safety 101 (2014) 31–35 8
2 0μg/L 3.4μg/L 9.6μg/L 19.2μg/L 27.4μg/L
1.5 1 0.5
12
24
48
NOS activities U / mg protein)
MDA concentration (n mol/ mg protein)
2.5
0
7 6 5 4 3 2 1 0
96
12
24
Exposure times (h)
AChE activites (U/mg protein)
48
96
Exprosure tims (h)
Fig. 2. MDA contents in the snail soft tissues after 12, 24, 48, or 96 h of ABM-exposure. Values represent the means and vertical bars indicate the standard deviation of three separate experiments. Asterisks denote a response that is significantly different from the control (npo0.05,nnpo0.01).
Fig. 4. NOS activities of the snail soft tissues after 12, 24, 48, or 96 h of ABMexposure. Values represent the means and vertical bars indicate the standard deviation of three separate experiments. Asterisks denote a response that is significantly different from the control (npo 0.05,nnp o 0.01).
Table 1 Activities of SOD, CAT, GST, AChE, and NOS, and MDA level in the snail soft tissues after 48 or 96 h of recovery from ABM-exposure for 96 h.
0.6 0.5 0.4 0.3 0.2 0.1 0
33
12
24
48
96
Exprosur times (h) Fig. 3. AChE activities of the snail soft tissues after 12, 24, 48, or 96 h of ABMexposure. Values represent the means and vertical bars indicate the standard deviation of three separate experiments. Asterisks denote a response that is significantly different from the control (npo0.05,nnpo0.01).
3.1.3. GST activity GST activities of the snail soft tissues increased after 24 or 48 h of ABM-exposure in comparison with that of control while they were inhibited at the end of test, which was similar to SOD, except for an increase in 9.6 μg L 1 of ABM group (Fig. 1C).
Antioxidant system
Revert time (h)
Control
19.2 mg L 1
27.4 mg L 1
SOD (U/mg protein) CAT (U/mg protein) GST (U/mg protein) AChE (U/mg protein) NOS (U/mg protein) MDA (nM/mg protein)
48 96 48 96 48 96 48 96 48 96 48 96
48.5 71.24 48.5 70.966 51.6 71.03 52.1 71.43 36.8 70.585 36.4 70.889 0.486 70.0138 0.50770.0165 4.88 70.110 4.85 70.210 0.995 70.089 0.97770.026
47.5 7 0.453 41.5 7 1.22n 49.27 1.23 45.8 7 1.55 55.4 7 1.62n 59.17 1.24n 53.17 1.23 52.0 7 1.16 36.3 7 0.757 34.3 7 1.01 36.0 7 0.460 38.8 7 0.978 0.4927 0.022 0.4887 0.008 0.5157 0.020 0.502 7 0.010 4.8317 0.268 5.46 7 0.278nn 4.889 7 0.201 4.977 0.166 1.4317 0.083n 1.767 0.137nn n 1.463 7 0.084 1.63 7 0.048nn
Values represent the means 7SD of three separate experiments. Asterisks denote a response that is significantly different from the control. n
p o 0.05. po 0.01.
nn
4. Discussion 3.2. MDA content MDA levels in the snail soft tissues from all treated groups (except for 3.4 μg L 1) were significantly higher than that of the control group during the periods of ABM-exposure (Fig. 2).
3.3. AChE activity AChE activities of the snail soft tissues were demonstrated in Fig. 3, in which an obvious tendency of activity inhibition was observed.
3.4. NOS activity NOS activities in the snail soft tissues were generally promoted after 24, 48, or 96 h of ABM-exposure (Fig. 4).
3.5. Recovery test The results of recovery test showed that the activities of SOD, CAT, GST, and AChE of the snail viscera usually could resume the normal levels after 96 h of recovery (Table 1). However, MDA level and NOS activity failed to recover.
Recently antioxidant enzymes and MDA have been used as biomarkers of pollutants that generate oxidative stress in aquatic animals (Ait Alla et al., 2006; Zapata-Vivenes and Nusetti, 2007; Siwela et al., 2010). In this study, it was found that acute ABMexposure caused significant changes in the activities of the antioxidant enzymes (SOD, CAT, and GST) and the levels of MDA in the snail P. acuta, especially persistent increase of MDA content, suggesting that MDA level of the snail soft tissues might be a biomarker of ABM-toxicity. SOD and CAT are two key antioxidant enzymes responsible for elimination of cellular reactive oxygen species (ROS) induced by toxicants, in which SOD firstly disproportionates the highly reactive and potentially toxic superoxide radicals (O2 ) to hydrogen peroxide (H2O2) (Reddy and Sreenivasula, 1997; Kumar et al., 2003). Then H2O2 was converted to molecular oxygen and water by CAT catalyzing. Therefore SOD-CAT system provides the first line of defense against ROS. Generally SOD activity tends to increase when mild external pressure acts on organisms, but then its activity will be inhibited after severe injury by toxicants, resulting in excess ROS accumulation that leads to a decrease in antioxidant defenses or causes oxidative damage in organisms (Liesivuori and Savolainen, 1991). In the present study, SOD activities were promoted by ABM-exposure at the earlier periods of exposure (12 and 48 h) at lower concentrations of ABM (3.4, 9.6, and even 19.2 μg L 1), while they were inhibited almost in all treated groups at the end of test. This result indicates that ABM
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induces excess ROS in snail soft tissues and SOD fails to timely eliminate the ROS, which in turn results in activity inhibition of SOD. Notably, it was found that SOD activity in the lowest concentration group (3.4 μg L 1 maintained unchanged while it always increased in highest one (27.4 μg L 1) during the ABMexposure. The tendency of CAT activity change was similar to that of SOD, but it increased in almost all treated groups at the end of test. Some researchers believed that CAT activity showed a positive relationship with SOD activity (Porte et al., 1991; Wu et al., 2011; Richardson et al., 2008). However, our results are consistent with this viewpoint at the earlier periods of ABM- exposure, while an opposite trend was observed at the later time. This result could be due to the disproportionation reaction of O2, which is not the only source of H2O2 that could also be generated by amino acids or cytochrome P450 oxidation activated (Livingstone et al., 1992). GST belongs to the phase II detoxification enzyme of animal liver and plays an important role in metabolizing and detoxification of chemicals (Dallinger, 1993; Storey, 1996). Moreover, GST also can transform a variety of hydrophobic pollutants to prevent cellular membrane from lipid peroxidation (Hayes and Pulford, 1995; Marrs, 1996; van der Oost et al., 2003; Lushchak et al., 2005). Li et al.(2008) proposed that GST could be a promising biomarker of aquatic pollutants to the freshwater snail, Bellamya purificata. In this study, all treatments promoted GST activity in snail soft tissues at the earlier stage of exposure but inhibited it at the end of test, showing a basically similar tendency to SOD and CAT. MDA is the product of the reaction between ROS and unsaturated fatty acids in cellular membrane and its content alternation in tissue indirectly reflects the damage to cellular membrane caused by excess ROS (Papadimitriou and Loumbourdis, 2002; Li et al., 2013). Therefore it has been considered to be an indicator of cellular oxidative damage. In the present study, ABM-exposure increased MDA levels of the snail soft tissues for all treated groups, including the recovery group, indicating that lipid peroxidation occurred in snail soft tissues. Furthermore, unrestored MDA level in the recovery group suggests that the excess ROS may has not been completely eliminated by the antioxidant system of the snail. This result also indicates that ROS and the antioxidant system are probably involved in the toxicity mechanism of ABM in the snail. AChE is an important enzyme that plays a crucial role in catalyzes the hydrolysis of the acetylcholine (ACh) neurotransmitter to maintain the normal conduct of nerve impulses (Chen et al., 2012). Now a lot of reports have approved that AChE is the main target of organophosphate and carbamate pesticides, and it can be used as an important biomarker of pesticide toxicity to snails (Singh and Agarwal, 1983, Radwan et al., 1992; Essawy et al., 2009; Laguerre et al., 2009). However, there has been no report regarding the possible effects of ABM on snail AChE till now (Lu et al., 2009; Ucán-Marín et al., 2012). In the present study, ABM-exposure inhibited AChE activity in most treatment groups. NOS is a key enzyme to catalyze L-arginine and molecular oxygen to generate of NO that plays an important role in sensory and motor systems (Funakoshi et al., 1999), learning and memory (Susswein et al., 2004), neurogenesis (Estrada and MurilloCarretero, 2005), and autonomic nervous activities (Guo and Longhurst, 2003). Additionally, NOS also plays an important role in resisting to disease and immune regulation in the immune system of aquatic animals. In this study, NOS activities of the snail soft tissues were increased by ABM-exposure, indicating that ABM promoted NOS activity and more NO production to resist the stress from ABMexposure. To the best of our knowledge, it is the first report on the effects of biocide on mollusk NOS. Toxicologically, recovery test is very important for evaluation and determination of chemical toxicity on organisms. The results of our recovery tests showed that the activities of antioxidant enzymes (SOD, CAT, and GST) and AChE, except for MDA level, in the snail soft tissues could recover to the normal levels after
96 h of recovery period. This result suggests that ABM toxicity on the snail might be not irreversible and snail antioxidant enzymes and AChE could restore from the stress. However, after recovery time, MDA contents were still significantly higher than that of control, indicating ROS induced by ABM and the damage to the cellular membrane by ROS were persistent and non-eliminated. Much more long term of recovery may be needed for the snail to resume due to serious injury caused by ABM. In summary, the results of the present study indicate that ABMexposure alters the activities of antioxidant enzymes, AChE, and NOS, and causes lipid peroxidation in snail viscera. This result suggests that ROS and the antioxidant system may be involved in the toxicity mechanism of ABM on the snail.
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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Biochemical responses to the toxicity of the biocide abamectin on the freshwater snail Physa acuta Junguo Ma, Chune Zhou, Yao Li, Xiaoyu Li n College of Life Science, Henan Normal University, Xinxiang, Henan 453007, China
art ic l e i nf o
a b s t r a c t
Article history: Received 14 September 2013 Received in revised form 3 December 2013 Accepted 11 December 2013 Available online 8 January 2014
The toxic effects of abamectin (ABM), an anthelmintic drug, on the snail, Physa Acuta, and the biochemical responses to the exposure stress were evaluated. The activities of superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST), acetylcholinesterase (AChE), and nitric oxide synthase (NOS), and the contents of malondialdehyde (MDA) were determined in snail soft tissues (head, foot, visceral mass, and the mantle) for up to 96 h of exposure to 3.4, 9.6, 19.2, or 27.4 μg L 1 of ABM. The results showed that SOD and GST activities were promoted by ABM-exposure at the earlier periods of treatment (12–48 h) while these activites were inhibited at the end of test. The tendency of CAT activity was similar to that of SOD, but it increased at the end of test. MDA levels of the snail soft tissues increased in all treatment groups, including the recovery group, indicating that lipid peroxidation occurred in snail soft tissues. ABMexposure inhibited AChE activity. However, NOS activities increased by ABM-exposure. In addition, activities of antioxidant enzymes and AChE from the snail soft tissues resumed the normal levels after 96 h of recovery period, but MDA level did not attain the original level. This study provides information on the biochemical mechanism of ABM toxicity on the snail. & 2014 Published by Elsevier Inc.
Keywords: Abamectin Physa acuta Acetylcholinesterase Nitric oxide synthase Antioxidant enzymes Lipid peroxidation
1. Introduction Abamectin (ABM) is a kind of large ring lactone disaccharide compounds and the natural fermentation product of the soildwelling actinomycete Streptomyces avermitilis (Roth et al., 1993; Luo et al., 2013). It is highly lipophilic and used both as a biocide and as an anthelminthic drug. According to the classification standard by WHO, ABM belongs to highly toxic chemicals with neurological and developmental toxicity. The toxicological mechanism of ABM is believed to affect the γ-aminobutyric acid (GABA) system and Cl channels of animal cells (Maioli et al., 2013), in which the GABA receptor is responsible for regulating the neural basal tone (Turner and Schaeffer, 1989). Since twenty years ago, ABM had been widely used for preventing parasitic diseases from fish (Lorio et al., 1992; Johnson and Margolis, 1993). Therefore it may enter aquatic environment and pose environmental risks to aquatic animals and ecosystems. Toxicologically, ABM usually has only slight toxicity to earthworms and birds (Halley et al., 1993), but it is highly toxic to fish (Jenčič et al., 2006), representing a potential threat to aquatic animals. Aquatic snail Physa acuta is an invasive species and now world widely distributed in freshwater body (Albrecht et al., 2009; Guo
n
Corresponding author. Fax: þ 86 373 3329390. E-mail address: [email protected] (X. Li).
0147-6513/$ - see front matter & 2014 Published by Elsevier Inc. http://dx.doi.org/10.1016/j.ecoenv.2013.12.009
et al., 2009). Moreover, the snail is very sensitive to toxicants and thus appropriate for toxicity testing (Evans-White and Lamberti, 2009; Sánchez-Argüello et al., 2009; Lance et al., 2010; Musee et al., 2010; De Castro-Català et al., 2013; Hossain and Aditya, 2013; Seeland et al., 2013). Therefore it was adopted as experimental animal to determine ABM toxicity and biochemical alterations in the snail P. acuta by conducting an acute exposure of ABM in the present study.
2. Materials and methods 2.1. Chemicals and reagents ABM as two percent microemulsions was obtained from Biogen Crop Science Limited, China. It was first dissolved in distilled water for stock solutions and then diluted to obtain the experimental concentrations. The Superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST), acetylcholinesterase (AChE), and nitric oxide synthase (NOS) Diagnostic Reagent Kit were purchased from the Nanjing Jiancheng Bioengineering Institute (China). Bovine serum albumin and thiobarbituric acid (TBA) were obtained from Sigma (USA). 2.2. Snails The adult P. acuta with an average wet weight of 0.136 7 0.035 g and an average shell length of 6.853 7 1.438 mm were collected from a fishery pond in China and cultured in glass jars (3 L in volume) with aerated tap water (total hardness of
J. Ma et al. / Ecotoxicology and Environmental Safety 101 (2014) 31–35
The acute toxicity test was conducted according to the Spearman-Kärber method (Kärber, 1931) and the exposure concentrations were 3.4 μg L 1 (1/10 96 h LC50), 9.6 μg L 1 (1/10 48 h LC50), 19.2 μg L 1 (1/5 48 h LC50), and 27.4 μg L 1 (2/7 48 h LC50). These concentrations were designed based on the results of our previous report, in which the 96 h and 48 h LC50 of ABM to P. acuta was 34 and 96 μg L 1, respectively (Ma and Li, 2011). Two hundred snails were randomly divided into five groups (40 snails in each group), out of which four groups were ABM-treated groups (3.4, 9.6, 19.2, and 27.4 μg L 1 of ABM) and exposed for 12, 24, 48, and 96 h. Another group was maintained as a control. The animals in each test group were placed in a 1000 mL beaker containing 800 mL of ABM solution, and the control snails were bred temporarily in a beaker with aerated tap water. The beaker was covered with cotton gauze to prevent snails from escaping. The cotton gauze must be on the surface of water in the beaker so that the snails cannot choke due to dryness when they attach to the cotton gauze. Each test was conducted in triplicate. Except snails were not fed, ABM exposure conditions were maintained as to those used for the breeding of the snails. At the end of each exposure period (12, 24, 48, and 96 h), ten snails from each group were taken and placed in Eppendorf tube. After their shells were removed, all the soft tissues (head, foot, visceral mass, and the mantle) were homogenized (10 percent w/v) in 0.1 M phosphate buffer (pH 7.5). The homogenate was centrifuged at 12,000 g for 10 min at 4 1C and the supernatant obtained was stored at –20 1C for biochemical assays.
2.4. Recovery test The recovery test was carried out to evaluate the surviving capacity of snails from the acute exposure of ABM. Sixty snails were randomly divided into three groups, in which two groups were the ABM-treatment groups (19.2 and 27.4 μg L 1 of ABM) and another as a control group. After 96 h of treatment, the snails were removed to the aerated tap water immediately and recovered in the ABM-free water for another 96 h. During the recovery test, snails were fed on commercial food at a rate of 0.5–1 percent of body weight/day. At 48 or 96 h time intervals, ten snails were collected and homogenized as described above for biochemical assays. Each test was conducted in triplicate.
2.5. Biochemical analysis The activities of SOD, CAT, GST, AChE, and NOS in snail soft tissues were determined by using the Diagnostic Reagent Kit purchased from the Nanjing Jiancheng Bioengineering Institute (China) according to the manufacturer0 s instructions. The assay results were given in units of enzymatic activity per milligram of protein (U/mg protein), in which one unit was defined as the amount of enzyme producing 50 percent inhibition of the enzyme, as the amount of enzyme decomposing 1 μM H2O2 per second, as the amount of enzyme causing GSH concentration down 1 μM L 1 in 1 min, as the amount of enzyme decomposing 1 nM NO per minute, and as the amount of enzyme capable of converting 1 μM of acetylthiocholine iodide as a substrate per minute for SOD, CAT, GST, NOS, and AChE, respectively. The total protein contents of samples were measured by the method of Lowry et al. (1951) using bovine serum albumin as a standard. Lipid peroxidation was measured by the TBA method (Ohkawa et al., 1979). The assay was monitored for the appearance of the conjugated complex of TBA and malondialdehyde (MDA) at 532 nm. The concentration of MDA was expressed as nM MDA per mg protein.
3.1. Antioxidant enzymes 3.1.1. SOD activity No change was found in SOD activity of the snail soft tissues between the treatment and control groups after 12 h of ABMexposure, except for an increase in the group of 19.2 μg L 1 of ABM when compared to the control (Fig. 1A). However, at 24 h exposure, SOD activities in the groups of 9.6 and 19.2 μg L 1 of ABM were significantly higher than that of control, while it was remarkably inhibited in the highest concentration group (27.4 μg L 1). Its activity was maintained unchanged in the lowest concentration group (3.4 μg L 1). Then (48 h later), SOD activity in 19.2 μg L 1 of ABM group also decreased while still increased in 9.6 μg L 1 group. At the end of test (96 h), SOD activities in all treated groups (except for 3.4 μg L 1) were lower than that of control (Fig. 1A).
3.1.2. CAT activity The average change tendency of CAT activity was similar to that of SOD before 48 h of ABM-exposure, but it increased in all treated groups (except for 3.4 μg L 1) at the end of test (Fig. 1B).
60 SOD activities (U/mg protein)
2.3. ABM-exposure
3. Results
*
** **
50
** **
**
40
** **
** **
30 20
10 0 12
24
48
96
Exposure times (h)
80 CAT activites (U/mg protein)
water 340 mg L 1, pH 7.6, turbidity 1.5 nephelometric turbidity units, and total dissolved solid content 660 mg L 1, which were detected according to the Standards for Drinking Water Quality, China, GB5479-2006) at a constant temperature (22 7 1 1C) and a 16 h: 8 h light/darkness photoperiod under laboratory conditions for several generations prior to the experiment. Snails were fed on a mixture of commercial goldfish food (Wannong Fishery Company, China) at a rate of 0.5–1 percent of body weight/day and lettuce leaves. The water was changed weekly. The snail was handled according to the guidelines in the China Law for Animal Health Protection and Instructions for Granting Permits for Animal Experimentation for Scientific Purposes (Ethics approval No. SCXK (YU) 20050001).
**
70
*
*
60
**
** *
50
* *
*
40 30 20 10 0
12
24
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96
Exposure times (h)
70 GST activities (U /mg protein)
32
**
60 **
50
** **
**
**
40
** **
30 20
10 0
12
24
48
96
Exposure times (h)
2.6. Statistical analysis Statistical analysis was performed using the SPSS 13.0 software (SPSS Inc.). All data were analyzed by one-way ANOVA and post-hoc pairwise comparisons among all groups using the Duncan0 s test (po 0.05).
Fig. 1. Effects of ABM on activities of SOD (A), CAT (B), and GST (C) in the snail soft tissues after 12, 24, 48, or 96 h of exposure. Values represent the means and vertical bars indicate the standard deviation of three separate experiments. Asterisks denote a response that is significantly different from the control (npo 0.05, nn p o0.01).
J. Ma et al. / Ecotoxicology and Environmental Safety 101 (2014) 31–35 8
2 0μg/L 3.4μg/L 9.6μg/L 19.2μg/L 27.4μg/L
1.5 1 0.5
12
24
48
NOS activities U / mg protein)
MDA concentration (n mol/ mg protein)
2.5
0
7 6 5 4 3 2 1 0
96
12
24
Exposure times (h)
AChE activites (U/mg protein)
48
96
Exprosure tims (h)
Fig. 2. MDA contents in the snail soft tissues after 12, 24, 48, or 96 h of ABM-exposure. Values represent the means and vertical bars indicate the standard deviation of three separate experiments. Asterisks denote a response that is significantly different from the control (npo0.05,nnpo0.01).
Fig. 4. NOS activities of the snail soft tissues after 12, 24, 48, or 96 h of ABMexposure. Values represent the means and vertical bars indicate the standard deviation of three separate experiments. Asterisks denote a response that is significantly different from the control (npo 0.05,nnp o 0.01).
Table 1 Activities of SOD, CAT, GST, AChE, and NOS, and MDA level in the snail soft tissues after 48 or 96 h of recovery from ABM-exposure for 96 h.
0.6 0.5 0.4 0.3 0.2 0.1 0
33
12
24
48
96
Exprosur times (h) Fig. 3. AChE activities of the snail soft tissues after 12, 24, 48, or 96 h of ABMexposure. Values represent the means and vertical bars indicate the standard deviation of three separate experiments. Asterisks denote a response that is significantly different from the control (npo0.05,nnpo0.01).
3.1.3. GST activity GST activities of the snail soft tissues increased after 24 or 48 h of ABM-exposure in comparison with that of control while they were inhibited at the end of test, which was similar to SOD, except for an increase in 9.6 μg L 1 of ABM group (Fig. 1C).
Antioxidant system
Revert time (h)
Control
19.2 mg L 1
27.4 mg L 1
SOD (U/mg protein) CAT (U/mg protein) GST (U/mg protein) AChE (U/mg protein) NOS (U/mg protein) MDA (nM/mg protein)
48 96 48 96 48 96 48 96 48 96 48 96
48.5 71.24 48.5 70.966 51.6 71.03 52.1 71.43 36.8 70.585 36.4 70.889 0.486 70.0138 0.50770.0165 4.88 70.110 4.85 70.210 0.995 70.089 0.97770.026
47.5 7 0.453 41.5 7 1.22n 49.27 1.23 45.8 7 1.55 55.4 7 1.62n 59.17 1.24n 53.17 1.23 52.0 7 1.16 36.3 7 0.757 34.3 7 1.01 36.0 7 0.460 38.8 7 0.978 0.4927 0.022 0.4887 0.008 0.5157 0.020 0.502 7 0.010 4.8317 0.268 5.46 7 0.278nn 4.889 7 0.201 4.977 0.166 1.4317 0.083n 1.767 0.137nn n 1.463 7 0.084 1.63 7 0.048nn
Values represent the means 7SD of three separate experiments. Asterisks denote a response that is significantly different from the control. n
p o 0.05. po 0.01.
nn
4. Discussion 3.2. MDA content MDA levels in the snail soft tissues from all treated groups (except for 3.4 μg L 1) were significantly higher than that of the control group during the periods of ABM-exposure (Fig. 2).
3.3. AChE activity AChE activities of the snail soft tissues were demonstrated in Fig. 3, in which an obvious tendency of activity inhibition was observed.
3.4. NOS activity NOS activities in the snail soft tissues were generally promoted after 24, 48, or 96 h of ABM-exposure (Fig. 4).
3.5. Recovery test The results of recovery test showed that the activities of SOD, CAT, GST, and AChE of the snail viscera usually could resume the normal levels after 96 h of recovery (Table 1). However, MDA level and NOS activity failed to recover.
Recently antioxidant enzymes and MDA have been used as biomarkers of pollutants that generate oxidative stress in aquatic animals (Ait Alla et al., 2006; Zapata-Vivenes and Nusetti, 2007; Siwela et al., 2010). In this study, it was found that acute ABMexposure caused significant changes in the activities of the antioxidant enzymes (SOD, CAT, and GST) and the levels of MDA in the snail P. acuta, especially persistent increase of MDA content, suggesting that MDA level of the snail soft tissues might be a biomarker of ABM-toxicity. SOD and CAT are two key antioxidant enzymes responsible for elimination of cellular reactive oxygen species (ROS) induced by toxicants, in which SOD firstly disproportionates the highly reactive and potentially toxic superoxide radicals (O2 ) to hydrogen peroxide (H2O2) (Reddy and Sreenivasula, 1997; Kumar et al., 2003). Then H2O2 was converted to molecular oxygen and water by CAT catalyzing. Therefore SOD-CAT system provides the first line of defense against ROS. Generally SOD activity tends to increase when mild external pressure acts on organisms, but then its activity will be inhibited after severe injury by toxicants, resulting in excess ROS accumulation that leads to a decrease in antioxidant defenses or causes oxidative damage in organisms (Liesivuori and Savolainen, 1991). In the present study, SOD activities were promoted by ABM-exposure at the earlier periods of exposure (12 and 48 h) at lower concentrations of ABM (3.4, 9.6, and even 19.2 μg L 1), while they were inhibited almost in all treated groups at the end of test. This result indicates that ABM
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induces excess ROS in snail soft tissues and SOD fails to timely eliminate the ROS, which in turn results in activity inhibition of SOD. Notably, it was found that SOD activity in the lowest concentration group (3.4 μg L 1 maintained unchanged while it always increased in highest one (27.4 μg L 1) during the ABMexposure. The tendency of CAT activity change was similar to that of SOD, but it increased in almost all treated groups at the end of test. Some researchers believed that CAT activity showed a positive relationship with SOD activity (Porte et al., 1991; Wu et al., 2011; Richardson et al., 2008). However, our results are consistent with this viewpoint at the earlier periods of ABM- exposure, while an opposite trend was observed at the later time. This result could be due to the disproportionation reaction of O2, which is not the only source of H2O2 that could also be generated by amino acids or cytochrome P450 oxidation activated (Livingstone et al., 1992). GST belongs to the phase II detoxification enzyme of animal liver and plays an important role in metabolizing and detoxification of chemicals (Dallinger, 1993; Storey, 1996). Moreover, GST also can transform a variety of hydrophobic pollutants to prevent cellular membrane from lipid peroxidation (Hayes and Pulford, 1995; Marrs, 1996; van der Oost et al., 2003; Lushchak et al., 2005). Li et al.(2008) proposed that GST could be a promising biomarker of aquatic pollutants to the freshwater snail, Bellamya purificata. In this study, all treatments promoted GST activity in snail soft tissues at the earlier stage of exposure but inhibited it at the end of test, showing a basically similar tendency to SOD and CAT. MDA is the product of the reaction between ROS and unsaturated fatty acids in cellular membrane and its content alternation in tissue indirectly reflects the damage to cellular membrane caused by excess ROS (Papadimitriou and Loumbourdis, 2002; Li et al., 2013). Therefore it has been considered to be an indicator of cellular oxidative damage. In the present study, ABM-exposure increased MDA levels of the snail soft tissues for all treated groups, including the recovery group, indicating that lipid peroxidation occurred in snail soft tissues. Furthermore, unrestored MDA level in the recovery group suggests that the excess ROS may has not been completely eliminated by the antioxidant system of the snail. This result also indicates that ROS and the antioxidant system are probably involved in the toxicity mechanism of ABM in the snail. AChE is an important enzyme that plays a crucial role in catalyzes the hydrolysis of the acetylcholine (ACh) neurotransmitter to maintain the normal conduct of nerve impulses (Chen et al., 2012). Now a lot of reports have approved that AChE is the main target of organophosphate and carbamate pesticides, and it can be used as an important biomarker of pesticide toxicity to snails (Singh and Agarwal, 1983, Radwan et al., 1992; Essawy et al., 2009; Laguerre et al., 2009). However, there has been no report regarding the possible effects of ABM on snail AChE till now (Lu et al., 2009; Ucán-Marín et al., 2012). In the present study, ABM-exposure inhibited AChE activity in most treatment groups. NOS is a key enzyme to catalyze L-arginine and molecular oxygen to generate of NO that plays an important role in sensory and motor systems (Funakoshi et al., 1999), learning and memory (Susswein et al., 2004), neurogenesis (Estrada and MurilloCarretero, 2005), and autonomic nervous activities (Guo and Longhurst, 2003). Additionally, NOS also plays an important role in resisting to disease and immune regulation in the immune system of aquatic animals. In this study, NOS activities of the snail soft tissues were increased by ABM-exposure, indicating that ABM promoted NOS activity and more NO production to resist the stress from ABMexposure. To the best of our knowledge, it is the first report on the effects of biocide on mollusk NOS. Toxicologically, recovery test is very important for evaluation and determination of chemical toxicity on organisms. The results of our recovery tests showed that the activities of antioxidant enzymes (SOD, CAT, and GST) and AChE, except for MDA level, in the snail soft tissues could recover to the normal levels after
96 h of recovery period. This result suggests that ABM toxicity on the snail might be not irreversible and snail antioxidant enzymes and AChE could restore from the stress. However, after recovery time, MDA contents were still significantly higher than that of control, indicating ROS induced by ABM and the damage to the cellular membrane by ROS were persistent and non-eliminated. Much more long term of recovery may be needed for the snail to resume due to serious injury caused by ABM. In summary, the results of the present study indicate that ABMexposure alters the activities of antioxidant enzymes, AChE, and NOS, and causes lipid peroxidation in snail viscera. This result suggests that ROS and the antioxidant system may be involved in the toxicity mechanism of ABM on the snail.
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