Ecotoxicology and Environmental Safety 116 (2015) 137–142

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Toxicological effects and oxidative stress responses in freshwater snail, Lanistes carinatus, following exposure to chlorpyrifos Abdelmonem M. Khalil Department of Zoology, Faculty of Science, Zagazig University, Zagazig, Egypt

art ic l e i nf o

a b s t r a c t

Article history: Received 18 November 2014 Received in revised form 9 March 2015 Accepted 10 March 2015

Chlorpyrifos is a widely used organophosphorous pesticide in agriculture and environmental health. Laboratory studies of chlorpyrifos have revealed acute lethal toxicity at very low concentration (96-h LC50) of 0.39 μg L  1 to the freshwater snail, Lanistes carinatus. Acetylcholinesterase (AChE) inhibition progressed and reached 52% and 78% of the control after 28-d exposure to 0.09 and 0.29 μg L  1 chlorpyrifos, respectively. Catalase (CAT), glutathione peroxidase (GPx) and glutathione-S-transferase (GST) activities increased in comparison to control group in the first period of exposure (7–21 d), then decreased relative to the control in the second period of exposure (21–28 d). A significant (po 0.05) glutathione (GSH) depletion was observed in snails exposed to 0.09 and 0.29 μg L  1 in comparison to the control, whereas malondialdehyde (MDA) content gradually increased in a dose-dependent manner. This study showed that alterations in antioxidant enzyme activities along with depletion of GSH content and elevation of MDA content can be used as biomarkers in environmental assessment programs. & 2015 Elsevier Inc. All rights reserved.

Keywords: Chlorpyrifos Antioxidant enzymes Glutathione Lipid peroxidation Lanistes carinatus Acetylcholinesterase

1. Introduction Human activities release hundreds of chemicals, which contaminate water bodies such as ponds, lakes, irrigation and drainage canals. Aquatic pollution has become an important global problem during the last several decades. Toxicological properties of synthetic insecticides have been improved over the past sixty years (Nauen and Bretschneider, 2002). Chlorpyrifos is a crystalline organophosphate pesticide that is widely used throughout the world in agriculture and non-agriculture applications (Farahat et al., 2011; Ramzy, et al., 2014). WHO (1997) classified chlorpyrifos as a moderately dangerous, Class II insecticide. This insecticide has been reported to induce oxidative stress (Gupta et al., 2010) and DNA damage (Jett and Navoa, 2000). As with other organophosphorus, chlorpyrifos inhibits nervous system function by binding with acetylcholinestrase (AChE), the enzyme responsible for the hydrolysis of the neurotransmitter acetylcholine at cholinergic synapses (Yu et al., 2008). Binding AChE with chlorpyrifos allows the accumulation of acetylcholine (ACh) at the nerve junction and this leads to overstimulation of nicotinic and muscarinic receptors, and disrupted neurotransmission (Reigart and Roberts, 1999). Ma et al. (2015) determined the acute toxicity of chlorpyrifos on Carassius auratus goldfish. They also evaluated the enzymatic activities of AChE, SOD and CAT and the contents of MDA in the liver and brain of the goldfish exposed to 1/10 and 1/3 E-mail address: [email protected] http://dx.doi.org/10.1016/j.ecoenv.2015.03.010 0147-6513/& 2015 Elsevier Inc. All rights reserved.

96-h LC50 of chlorpyrifos. Ishii et al. (2004) reported that chlorpyrifos could inhibit mitochondrial ATP production through the uncoupling of oxidative phosphorylation, which could lead to ROS production. Oxidative stress is believed to occur when there is an imbalance in the biological pro-oxidant-antioxidant ratio. Thus, it can be defined as a disturbance in the balance between the production of reactive oxygen species (ROS) and antioxidant defenses (Barzilai and Yamamoto, 2004). This production of ROS can lead to damage in lipids, proteins, DNA, and cell structure. Living organisms have unparalleled systems for protecting themselves against the damage effects of activated ROS. For instance, superoxide (O−2 ), the parental form of intracellular ROS, is a reactive molecule, but it can be converted to H2O2 by GST and then to oxygen and water by several enzymes including CAT and GPx. Therefore, monitoring the change in activity of antioxidant enzymes such as GST, CAT and GPx is considered as an effective method of denoting oxidative stress. Although there is a battery of antioxidant defense enzymes to protect against ROS, excessive ROS produced as a result of exposure to contaminants such as pesticides can overwhelm defense mechanisms and cellular damage may supervene. It seems that oxidation of lipids is the most commonly used approach in free radical research field because many organisms, especially aquatic ones contain high amount of lipids with polyunsaturated fatty acid residues, a substrate for oxidation (Huang et al., 2003). The most frequently used methods to examine lipid peroxidation are based on measuring of the end product MDA (Lushchak, 2011). L. carinatus (Olivier, 1804) is one of the most common

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freshwater snails in Egypt. This snail is found in numerous water body types with preference for standing water. As a typical freshwater snail, it is used as sentinel species for monitoring aquatic pollution by heavy metals (Abd El Gawad, 2009). In the present study, this animal was selected for study because it is locally abundant, easily collected and easily maintained under laboratory conditions. The survey of literature indicated that the work is scanty in dealing with the antioxidant and oxidative stress related responses in the freshwater snails exposed to chlorpyrifos. Therefore, the present study had three main aims: (1) to examine the lethal and sublethal effects of chlorpyrifos on L. carinatus snail, (2) to examine the relevance of AChE activities as early warning tools of neurotoxic stress in freshwater snail, (3) to observe the antioxidant activity alterations and the oxidative damage in the hepatopancreas after 28 days exposure to 25% and 75% 96-h LC50 chlorpyrifos.

Chlorpyrifos was tested at 0.1, 0.5, 1.0, 5.0 and 10.0 μg L  1 distilled water. After spiking the dosing solution directly into the test medium, containers were carefully covered with plastic mesh to prevent snails from escaping and kept at 237 2 °C under 12 h:12 h dark/light regime. Live/dead snails were counted after 24, 48, 72 and 96 h by gently prodding and monitoring the movement of head–foot region. Snails were counted as dead if they failed to withdraw their head–foot regions into their shells after prodding for three times. Dead snails were removed from test containers and the test solutions and control water were renewed daily. LC50 of the test pesticide was calculated from the data obtained in acute toxicity bioassays following the probit analysis method as described by Finney (1971). The probit analysis was carried out using Statview v.5 (SAS software Inc., USA). During bioassay, the mortality in treated groups was corrected by using the formula of Abbott (1987) for control mortality. Abbott's formula: corrected % mortality ¼(% alive control  % alive treated)  100%/(% alive control).

2. Materials and methods 2.4. Sublethal toxicity assay 2.1. Chemicals The insecticide used in this study was the commercial grade formulation of chlorpyrifos [0,0-diethyl O-(3,5,6-trichloro-2-pyridyl) phosphorothioate]. Dursbans 48 EC (active ingredient chlorpyrifos, 480 g L  1) was provided by Syngenta Co., Giza, Egypt. All other chemicals were of highest purity and were purchased from Sigma-Aldrich, Cairo, Egypt. 2.2. Snail collection and acclimatization Adult freshwater L. carinatus snails were collected from uncontaminated irrigation and drainage channels near Belbeis city (30° 25′N, 31° 34′E), Sharkia Governorate, Egypt in the summer of 2013. The mean shell size of snails was 33.671.3 mm length and 19.2 71.1 mm width. After their transfer to the laboratory, they were acclimatized in plastic containers for a minimum of one week to the experimental conditions at 23 72 °C and a 12 h:12 h light/dark photoperiod cycle, in aerated and dechlorinated tap water. Dissolved oxygen and pH were determined by a digital oxygen meter and pH meter. The mean values for test water quality were as follows: dissolved oxygen 7.19 70.32 mg L  1, pH 8.5 70.06 and hardness 184 712 mg L  1. Snails were fed on fresh lettuce leaves and all containers were covered by plastic mesh to prevent snails from escaping. 2.3. Acute toxicity assay A semi-static renewal acute toxicity bioassay was conducted according to the American Public Health Association standard methods (APHA, 1998) to determine the median lethal concentration causing 50% mortality (LC50). Experiments were carried out in 1 L plastic containers, each containing 500 ml of pre-aerated, dechlorinated tap water and 15 adult snails at the start of the experiment. Stock solution was made by diluting 1 mL Dursbans 4E in 1 L distilled water. The specimens were not fed a day prior to and during toxicity tests to reduce fecal and excess food contaminating the test solution. Preliminary experiments were carried out to establish the effective range of the tested insecticide. Concentrations of the test compound used in short-term definitive tests were between the highest concentration at which there was 0% mortality and the lowest concentration at which there was 100% mortality. A geometric dilution series of five concentrations was used in each toxicity test, with three replicate containers per concentration (i.e. 45 snails per concentration) and one blank control container.

Sublethal toxicity of chlorpyrifos was determined using biomarkers of oxidative stress (GST, CAT and GPx activities and MDA and GSH contents) and AChE inhibition as a neurotoxic biomarker. The same methodology as the acute toxicity–exposure was used for the subchronic study, except there were 10 snails per treatment in triplicates. Two chlorpyrifos sublethal concentrations corresponding to two 25% and 75% of the 96-h LC50 value (0.39 μg L  1) were used. Snails were exposed for 28 days and were fed every other day on fresh lettuce leaves. The test solutions and control water were renewed on alternate day. The snails were divided into three groups, 30 specimens per group (each container contained 10 snails). Group I (control): the snails of this group received no treatment; group II: the snails of this group were treated with 0.09 μg L  1 (the lowest observed adverse effect level, LOAEL), which is nearly equal to 25% 96-h LC50 and group III: the snails of this group were treated with a concentration of 0.29 μg L  1 (3-fold higher than LOAEL), which is nearly equal to 75% 96-h LC50. 2.5. Tissue preparation The sublethal effects of chlorpyrifos on some of the biochemical parameters of snails were monitored at 7, 14, 21 and 28 d after treatment. At the end of each time interval, the head–foot region from 6 randomly selected live snails of each experimental group (two animals from each container) was excised with sharp scissors and the cerebral ganglia were dissected and removed under stereomicroscope (Radwan and Mohamed, 2013). Cerebral ganglia were pooled and homogenized in 10 volumes (w/v) of 0.1 M phosphate buffer (pH 7.5) for 1 min. The homogenates were then centrifuged at 4000g for 30 min at 4 °C and the supernatant was used to analyze AChE activity. The digestive gland of each snail was dissected, taken out, washed with ice-cold physiological saline (0.9%) and weighed. Digestive glands were pooled and homogenized in 10 volume of ice-cold saline solution for 1 min (Radwan and Mohamed, 2013). The homogenates were centrifuged at 4000g for 30 min at 4 °C and the supernatant was used to assay the oxidative stress biomarkers. 2.6. Biomarkers assay 2.6.1. Enzymatic measurements Acetylcholinesterase (AChE; EC 3.1.1.7) activity was determined by the method of Ellman et al. (1961) using 5,5′-dithio-bis-(2-nitrobenzoic acid (DTNB) and acetylthiocholine iodide as substrate.

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AChE activity was expressed as nmol mg  1 protein min  1. Glutathion-S-transferase (GST; EC 2.5.1.18) activity was evaluated using the method described by Habig et al. (1974) using 1-chloro2,4-dinitrobenzene (CDNB) as substrate. Enzyme activity was expressed as nmol mg  1 protein min  1. Catalase (CAT; EC 1.11.1.6) activity was assayed by the method of Sinha (1972). Hydrogen peroxide (0.2 M) was used as substrate. Dichromate/ acetic acid reagents were added to start and terminate the reaction, respectively. Enzyme activity was expressed as specific activity in Unit mg  1 protein. Glutathione peroxidase (GPx; EC 1.11.1.9) activity was measured in an assay linking the reaction to that of glutathione reductase (Paglia and Valentine, 1967) and following the decrease in nicotinamide adenine dinucleotide (NADPH) with tert-butyl hydroxide as substrate. The activity was expressed as specific activity in Unit mg  1 protein. 2.6.2. Malondialdehyde and glutathione assay The malondialdehyde (MDA) assay was based on the reaction of chromogenic reagent N-methyl-2-phenylindole with MDA at 45 °C, giving rise to a chromophore with absorbance at 586 nm (Esterbauer et al., 1991). MDA content was expresses as nmol mg  1 protein. Glutathione (GSH) as a non-enzymatic antioxidant was quantified using Ellman's reagent (Ellman, 1959). The amount of GSH was expressed in terms of nmol mg  1 protein. 2.6.3. Determination of total protein Total soluble protein contents were determined according to the method of Lowry et al. (1951), using bovine serum albumin as standard. 2.7. Statistical analysis

Fig. 1. Activity of acetylcholinesterase (AChE) in the adult freshwater snail L. carinatus exposed to chlorpyrifos contaminated water. Data is expressed as mean 7 S.D. Different letters indicate significant differences among treatments one-way ANOVA, followed by Tukey's multible comparison test (po 0.05).

in Table 1. Snail mortality increased significantly when the concentration and the time of exposure were increased showing that chlorpyrifos is highly toxic to L. carinatus. A 100% mortality rate of the L. carinatus was observed after exposure to 5 μg L  1 for 48 h and 10 μg L  1 for 1 h. The LC50 values (95% confidence limit, CL) for 1, 24, 48, 72 and 96 h were calculated as 5.89 (4.13–7.74), 3.02 (2.18–3.89), 0.84 (0.59–1.11), 0.56 (0.32–0.81) and 0.39 (0.24– 0.58) μg L  1, respectively. 3.2. Acetylcholinestrase inhibition

Data are presented as means 7S.D. All data were tested and exhibited a normal distribution according to Shapiro–Wilk test (at α ¼0.05) and a homogeneity of variances according to Levene's test (at α ¼0.05). One-way analysis of variances (ANOVA) was used to analyze overall differences among treatments, time and replicates. Tukey's post-hoc test was carried out when each group of the experimental data was compared with the control. All data were analyzed statistically using the software package SPSS, version 21 (SPSS Inc. Chicago, IL, USA).

Fig. 1 illustrates the effect of chlorpyrifos on AChE activity in L. carinatus snails during sublethal exposure for 28 days. Exposure to 0.09 and 0.29 μg L  1 of chlorpyrifos caused significant (Tukey's test, po 0.05) inhibition in AChE activity for all tested durations of exposure compared to the control. About 32% and 51% inhibition in AChE activities of treated snails were recorded after 7-d exposure to 0.09 and 0.29 μg L  1, respectively. This inhibition progressed and reached 61 and 82% at 21 d, then slightly reduced to 52% and 78% at 28 d in 0.09 and 0.29 μg L  1 treated snails. After 21-d exposure, the lowest AChE activities 18.33 73.61 and 8.37 71.71 nmol mg  1 protein min  1 were observed in treated snails exposed to 0.09 and 0.29 μg L  1, respectively.

3. Results

3.3. Antioxidant enzyme activities

3.1. LC50 determination

The antioxidant enzyme activities in the digestive gland tissue of L. carinatus snails as induced by two sublethal concentrations of chlorpyrifos (0.09 and 0.29 μg L  1) are illustrated in Figs. 2, 3 and 4. GST activity increased significantly (Tukey's test, po 0.05) in comparison to the control (Fig. 2). After 7-d exposure, GST activity in snails exposed to 0.09 μg L  1 was insignificantly higher (63.33 nmol mg  1 protein min  1) than that of the control (53.36 nmol mg  1 protein min  1), whereas it sharply increased to reach 115.69 nmol mg  1 protein min  1 in the snails treated with 0.29 μg L  1. GST induction were 118%,147%, 138% and 123% of the control in snails treated with 0.09 μg L  1 chlorpyrifos at 7, 14, 21 and 28-d exposure, respectively. The corresponding values as percent of the control for snails treated with 0.29 μg L  1 were 216, 181, 162 and 123 (Fig. 2). As shown in Fig. 3, during the initial exposure period (7 d), the CAT activity of 0.09 and 0.29 μg L  1 chlorpyrifos treated snails increased significantly (Tukey's test, po 0.05) to 151% and 120% of the control, respectively. CAT activity of snails treated with 0.09 and 0.29 μg L  1 dropped to the control level after 21-d exposure and suppressed to 73% and 68%, respectively after 28-d exposure.

The number of dead snails at different concentrations of chlorpyrifos after exposure for 1, 24, 48, 72 and 96 h is presented

Table 1 Cumulative mortality values of chlorpyrifos after application to L. Carinatus snails. For each concentration, average based on three replicates7 S.D. n ¼45). Chlorpyrifos (mg L  1)

Control 0.1 0.5 1.0 5.0 10.0

Average percent mortality 7 S.D. 1h

24 h

48 h

72 h

96 h

þ þ 6.6 72.1 15.5 7 4.7 40.0 75.8 100.0 7 0.0

þ þ 17.8 7 5.2 28.9 7 6.1 57.6 79.8 –

þ 4.5 71.1 31.1 78.8 60.0 79.4 100.0 7 0.0 –

2.2 7 0.7 13.3 7 4.7 55.6 7 11.1 77.8 7 13.6 – –

4.4 7 1.9 31.17 5.3 71.17 13.4 91.3 714.7 – –

( þ )¼ no mortality. (  ) ¼ no data, because of 100% mortality.

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Fig. 5. Activity of malondialdehyde (MDA) content in the adult freshwater snail L. carinatus exposed to chlorpyrifos contaminated water. Data is expressed as mean7 S.D. Different letters indicate significant differences among treatments one-way ANOVA, followed by Tukey's multible comparison test (p o 0.05). Fig. 2. Activity of glutathione-S-transferase (GST) in the adult freshwater snail L. carinatus exposed to chlorpyrifos contaminated water. Data is expressed as mean 7S.D. Different letters indicate significant differences among treatments one-way ANOVA, followed by Tukey's multible comparison test (p o 0.05).

The effects of chlorpyrifos on the GPx activity of L. carinatus snails are presented in Fig. 4. After 7-d exposure, the GPx activity increased 2.3 and 3.4-fold that of the control at 0.09 and 0.29 μg L  1, respectively. The corresponding values of GPx activity after 14-d exposure were 1.8 and 1.5-fold that of the control. GPx activity dropped to the level of control after 28-d exposure at 0.09 and 0.29 μg L  1. 3.4. MDA elevation and GSH depletion

Fig. 3. Activity of catalase (CAT) in the adult freshwater snail L. carinatus exposed to chlorpyrifos contaminated water. Data is expressed as mean 7 S.D. Different letters indicate significant differences among treatments one-way ANOVA, followed by Tukey's multible comparison test (p o 0.05).

The MDA content of snails was used to evaluate lipid peroxidation by chlorpyrifos and the results are illustrated in Fig. 5. After 7-d exposure, MDA content was significantly (p o0.05) greater (1.75 fold) than that of the control at the low treatment concentration (0.09 μg L  1). However, MDA content of the snails exposed to the high treatment concentration (0.29 μg L  1) was kept at the control level. After 14-d exposure, MDA content values increased significantly (p o0.05) to 1.6 and 1.7-fold that of the control at sublethal concentrations 0.09 and o.29 μg L  1, respectively. MDA content dropped to control level at 0.09 μg L  1 after 21-d exposure and rose to a significantly high level again (2-fold that of the control) after 28-d exposure. A significant (Tukey’s test, po 0.05) decrease of GSH levels was observed in snails exposed to 0.09 and 0.29 μg L  1 chlorpyrifos in comparison to the control. GSH depletion was 20%, 30%, 58% and 69% of the control in snails treated with 0.09 μg L  1 at 7, 14, 21 and 28-d exposure, respectively. The corresponding values as percent of the control of snails treated with 0.29 μg L  1 were 31, 58, 60 and 89. In general, it was shown that this reduction was clearly dose- and time-dependent except for 21-d duration of exposure (Fig. 6).

4. Discussion

Fig. 4. Activity of glutathione peroxidase (GPx) in the adult freshwater snail L. carinatus exposed to chlorpyrifos contaminated water. Data is expressed as mean 7S.D. Different letters indicate significant differences among treatments one-way ANOVA, followed by Tukey's multible comparison test (p o 0.05).

Current study shows that chlorpyrifos has a considerable killing effect against the non-target freshwater snail L. carinatus with 96h LC50 0.39 μg L  1. The data of the present study are consistent with that of the other studies. The 24-h LC50 for the intermediate host snail Bulinus truncates was 1.32 μg L  1 (Hasheesh and Mohamed, 2011). However, Odenkirchen and Eisler (1988) reported that L. carinatus snail was not affected by chlorpyrifos. Chlorpyrifos has several toxic properties, the most prominent effect of which is AChE inhibition. AChE activity is therefore widely used in biomonitoring studies as a biomarker of organophosphate pesticide exposure (Rendón-von Osten et al., 2005). Evaluation of AChE activity in the nervous tissues (cerebral ganglia) of L. carinatus after treatment with chlorpyrifos, revealed an inhibition of AChE. In this

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Fig. 6. Activity of glutathione (GSH) content in the adult freshwater snail L. carinatus exposed to chlorpyrifos contaminated water. Data is expressed as mean 7 S.D. Different letters indicate significant differences among treatments one-way ANOVA, followed by Tukey's multible comparison test (p o0.05).

study, the reduction of AChE activity is assumed to result from the direct action of chlorpyrifos exposure on the active site of this enzyme (Blodgett, 2006). A positive correlation with chlorpyrifos concentration and the time of exposure associated with the degree of AChE inhibition of L. carinatus was recorded in the successive durations of exposure (7, 14 and 21 d) however, insignificant reduction in the inhibitory effect of AChE was detected at 28-d exposure. This result could be explained on the basis of the findings of Ahammad et al. (1980) who reported that in the presence of high amount of accumulated acetylcholine in the fish tissues (resulting from AChE inhibition under the pesticide stress), the inhibitory effect produced by malathion is reduced with the time of exposure. The data of the present study are in agreement with literature data. Cacciatore et al. (2013) showed that chlorpyrifos produced a concentration-dependent inhibition of cholinesterase (ChE) activity in the tissues of the gastropod Planorbarius conrneus. Antioxidant enzymes (e.g., CAT, GPx and GST) play an important role in the adaptation of living organisms to stressful conditions through the elimination of ROS (Barata et al., 2005). GST is best known for its ability to catalyze the conjugation of the reduced form of glutathione (GSH) to xenobiotic substrates for the purpose of detoxification (Jifa et al., 2006). In the present study, GST activity at the low sublethal concentration 0.09 μg L  1 chlorpyrifos was significant after 14 days of exposure compared with the insignificant enhancement of GST after the 7-d exposure. At the high concentration 0.29 μg L  1, GST activity progressively decreased after 14 and 21 days of exposure compared with that after 7-d exposure indicating that more ROS were produced and the GST activity was more effectively enhanced at the high sublethal concentration. This enhancement suggested that detoxification against pro-oxidation powers, mediated by GST was induced (Elia et al., 2007). The extra generated ROS inactivated GST activity by interacting with the enzyme (Cunha et al., 2007). The insignificant alteration in GST activity at the 0.09 μg L  1 chlorpyrifos within the exposure duration may have been caused by the low level of oxidative stress which could not effectively induce GST activity in the snails. GST activity of the snails exposed to the two sublethal concentrations decreased with the increase of exposure time. Thus, GST activity displayed both dose- and time-response relationship. A similar relationship of GST activity was observed by Zhang et al. (2005) who reported that GST activity showed a statistically significant response to oxidative stress caused by exposure to 2,4-dichlorophenol at the beginning of experiment and subsequently dropped to the control level at the end of the experiment. CAT activities play an important role in the antioxidant

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protection of invertebrates (Livingstone, 2003). In the current study, it was observed that CAT activities were significantly increased. The data suggest that increases in antioxidant defense would be due to enhanced oxygen free radicals generation, which could stimulate antioxidant activities (Torres et al., 2002) to cope with this increased oxidative stress and protect the cells from damage. Although CAT is inducible under condition of oxidative stress (Di Giulio et al., 1989), CAT activity dropped to the control level following exposure to chlorpyrifos for 21 days and significantly suppressed under the control level after 28-d exposure. This observation suggests that GST plays a major role in the protection of L. carinatus snails against ROS whereas CAT is of minor importance. In this study, a positive correlation between chlorpyrifos and the antioxidant enzymes CAT and GST levels has been observed. Similar correlation has been found between same enzymes and the physiological conditions of a control population of terrestrial isopod Porcellio scaber (Jemec et al., 2012). GPx is the most important peroxidase for the detoxification of hydroperoxide (Orbea et al., 2000). This catalyzes H2O2 and organic hydroperoxides to water and alcohol using GSH as a cofactor. However, the decreased activity of GPx might be due to the over production of ROS, especially O−2 by chlorpyrifos and depletion of its substrate (GSH) level. In the present study, chlorpyrifos at two tested sublethal concentrations significantly increased the activity of GPx in a dose- and time-dependent manner until the 21-d exposure. GPx activity of snails exposed to 0.29 μg L  1 was significantly suppressed after the 21-d exposure. Both tested sublethal concentrations dropped to the control level after 28 days of exposure. The obtained results are in accordance with the finding of Escobar et al. (1996) who reported that under a high free-radical formation rate, the enzyme inactivation prevails leading to reduced antioxidant enzyme activities and to autocatalysis of oxidative damage process. Generally, the aforementioned antioxidant enzymes play an important role in the efficient destruction of O−2 and H2O2. The activity of these enzymes increased monotonically under chlorpyrifos-induced conditions after 14 days of exposure. Based on the observed alterations in antioxidant enzyme activities, exposure can be divided into two main periods. In the first period of exposure (7–21 d), the induction of activity was observed in all enzymes, which revealed that CAT, GPx and GST act in a cooperative or synergistic way for the protection of membrane lipids against oxidative stress caused by chlorpyrifos. In the second period (21– 28 d), the activities of the enzymes decreased to the levels observed in the control. This may be due to the fact that antioxidants appear to be overwhelmed by ROS and the antioxidant enzyme activities show a gradual decrease up to a severe depletion. Elevation of lipid peroxidation in snail exposed to sublethal concentrations of chlorpyrifos, as evidenced by increased MDA production in this study, proposes participation of free radicals-induced oxidative cell injury in mediating the toxicity of chlorpyrifos. In the present study, it is clearly evident that the increased trend of MDA content was contrary to that of GSH. GSH is a tripeptide synthesized from the amino acids glycine, glutamate and cysteine. It is one of the most important factors protecting against oxidative attacks by reactive oxygen species such as lipid peroxidation because GSH acts as a reducing agent and free radical scavenger (Verma et al., 2007). Compared with GST, CAT and GPx activities, the positive and linear relationship between GSH concentration and exposure concentration suggests that GSH is an excellent biomarker by which oxidative stress in snails exposed to chlorpyrifos could be estimated. As the exposure time increased, the trend of GSH concentration was in accordance with the decrease of anioxidant enzymes activity.

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5. Conclusion The present study represents, to my best knowledge, the first work to evaluate the ecotoxicological effects on the freshwater snail, L. carinatus exposed to chlorpyrifos. The results of the present study, which involved an integrated examination of the neurotoxic effects and possible alterations in lipid peroxidation and antioxidant defenses, demonstrated that sublethal exposure of chlorpyrifos to L. carinatus stimulated oxidative stress leading to elevated MDA level, depleted GSH content, induced antioxidant enzymes and inhibited AChE in the subject snail species. Further ecotoxicological studies with L. carinatus is necessary to prove that this organism is good model organism, where there is little information about the response of this species to different stressors. The tested biomarkers linked with other endpoints such as the behavior of chlorpyrifos-exposed snails should be investigated.

Acknowledgments The author is grateful to Department of Zoology, Faculty of Science, Zagazig University, Egypt for providing the research facilities. The author also would like to thank Prof. Fagr Khamis from National Research Center in Cairo, Egypt for her continuous interest and support of this study.

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Toxicological effects and oxidative stress responses in freshwater snail, Lanistes carinatus, following exposure to chlorpyrifos.

Chlorpyrifos is a widely used organophosphorous pesticide in agriculture and environmental health. Laboratory studies of chlorpyrifos have revealed ac...
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