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Assessment of toxic effects of triclosan on the terrestrial snail (Achatina fulica)
Chemosphere xxx (2014) xxx–xxx
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Assessment of toxic effects of triclosan on the terrestrial snail (Achatina fulica) Xiaonan Wang a,b, Zhengtao Liu a,⇑, Wanhua Wang a, Zhenguang Yan a, Cong Zhang c, Weili Wang a, Lihong Chen a a State Key Laboratory of Environmental Criteria and Risk Assessment, State Environmental Protection Key Laboratory of Ecological Effect and Risk Assessment of Chemicals, Chinese Research Academy of Environmental Sciences, Beijing 100012, China b College of Water Sciences, Beijing Normal University, Beijing 100875, China c China Offshore Environmental Services Co. Ltd., Tianjin 300452, China
h i g h l i g h t s Toxic effects of triclosan on Achatina fulica were assessed via various approaches. NOECs of TCS for the biomass, shell diameter, and food intake were 24 mg kg
1
TCS caused significant oxidative stress even at low concentration of 24 mg kg
1
a r t i c l e
i n f o
Article history: Received 7 July 2013 Received in revised form 6 December 2013 Accepted 5 January 2014 Available online xxxx Keywords: Triclosan Terrestrial environment Snail (Achatina fulica) Chronic toxicity test Risk assessment Antioxidant enzymes
a b s t r a c t Triclosan (TCS) is a broad-spectrum antimicrobial agent used in personal care products, and as a result, is widespread in the environment. Toxicity tests of TCS on aquatic organisms have been reported, but limited toxicity data on terrestrial species are available. In this study, the 28-d chronic toxicity of TCS on the biomass, shell diameter growth, and total food intake of the terrestrial snail Achatina fulica were tested. Moreover, biochemical responses, including changes in the activity of catalase (CAT), superoxide dismutase (SOD), peroxidase (POD), and the content of malondialdehyde (MDA), were examined after 14-d and 28-d exposure. Results showed that TCS had toxic effects on the biomass, shell diameter growth, and total food intake of A. fulica with no observed effect concentration (NOEC) values of 24 mg kg 1. As for the antioxidant enzymes, TCS caused significant oxidative stress even at the low concentration of 24 mg kg 1. The CAT and POD activities at the high concentrations of 200 and 340 mg kg 1, respectively, were significantly inhibited. The SOD and CAT activity in treatments below 118 mg kg 1 and the MDA content in all treatments showed dose–effect relationships. This study demonstrated that TCS caused adverse effects on terrestrial invertebrates, and provided valuable information for the risk assessment imposed by TCS in the terrestrial environment. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Personal care products (PCPs) represent an important type of emerging organic pollutants and can be found in all environmental media (Ternes et al., 2004; Amorim et al., 2010). The presence of PCPs in the environment and their possible effects on nontarget organisms are of great concern worldwide (Daughton and Ternes, 1999; Kümmerer, 2004; Veldhoen et al., 2006). Among the PCPs, triclosan (5-chloro-2(2,4-dichlorophenoxy) phenol, or TCS) is a broad-spectrum antimicrobial agent used in a variety of personal ⇑ Corresponding author. Tel.: +86 10 84915175; fax: +86 10 84915173. E-mail address: [email protected] (Z. Liu).
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care products including soaps, deodorant, and toothpaste (McAvoy et al., 2009; Price et al., 2010), as well as in household cleaners and even in textiles (sportswear, bed clothes, shoes, and carpets) and children’s toys (Singer et al., 2002). TCS has already been the subject of various scientific fields, and has been detected in surface water and agricultural soil worldwide in recent years (Benotti et al., 2008; Kasprzyk-Hordern et al., 2008; Cha and Cupples, 2009; Zhao et al., 2010; Ramaswamy et al., 2011). TCS is relatively persistent in the environment with a half-life of at least 11 d in surface water (Bester, 2005) and 18 d in aerobic soil (Ying et al., 2007). As for the terrestrial environment, TCS is known to be adsorbed to soil and may enter soil primarily through agricultural application of sewage sludge. Once in the soil, it mostly remains in the upper
http://dx.doi.org/10.1016/j.chemosphere.2014.01.044 0045-6535/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Wang, X., et al. Assessment of toxic effects of triclosan on the terrestrial snail (Achatina fulica). Chemosphere (2014), http://dx.doi.org/10.1016/j.chemosphere.2014.01.044
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10–20 cm incorporation layer (Reiss et al., 2009) and the final soil concentration depends on the initial TCS biosolid concentration, the method of biosolid application, soil types, and other factors Heidler and Halden (2007) reported that the concentration of TCS in sewage sludge was 20–55 mg kg 1, which could cause toxicological risk to terrestrial organisms. The adverse effects of TCS on terrestrial organisms have been reported Liu et al. (2009) reported the effect of TCS on soil microbial processes, and Amorim et al. (2010) provided the chronic toxicity data of TCS on plants (Triticum aestivum, Brassica rapa), worms (Eisenia andrei, Enchytraeus albidus), and collembolans (Folsomia candida). However, the toxicity data, especially the chronic toxicity data of TCS on terrestrial species, are very limited or unavailable. As for the biochemical effect of TCS, very few studies have been conducted on terrestrial organisms, as confirmed by Lin et al. (2010), who examined the biochemical and genetic toxicity of TCS on Eisenia fetida. Therefore, further studies need to be performed on TCS toxicity. Antioxidant enzymes, including catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD), play a major role in removing and detoxifying reactive oxygen species (ROS) and other free radicals caused by environmental contamination (Tripathi et al., 2006; Asagba et al., 2008). In addition, the level of malondialdehyde (MDA) is an important indicator of lipid peroxidation and it is a sensitive diagnostic index of oxidative injury in cells (Avci et al., 2005; Chen et al., 2012). In the present study, the toxic effects of TCS on a terrestrial snail (Achatina fulica) were tested. The A. fulica was often used to determine the biochemical and other toxic effects of chemicals (Rao and Singh, 2002; Chandran et al., 2005; Salway et al., 2010). The aims of this study were to (1) determine the toxic effect of TCS on the biomass, shell diameter growth, and total food intake of A. fulica; (2) determine the toxic effect of TCS on the activity of SOD, CAT, and POD, and the content of MDA. This work could provide valuable information for the environmental risk assessment and pollution management imposed by TCS in the terrestrial environment. 2. Materials and methods 2.1. Chemicals and reagents TCS, C12H7Cl3O2, P97% purity (HPLC), was purchased from Sigma Aldrich. All reagents and standards used in enzyme assays were purchased from the Nanjing Jiancheng Bioengineering Institute (Nanjing, China). All other reagents used in the tests were purchased from the Beijing Guoyao Chemical Co., Ltd. (Beijing, China) and were of analytical grade with chemical purity of 97–99%. 2.2. Test organism The juvenile snails were purchased from a snail culturing farm in Beijing, China and were selected from synchronous breeding to ensure the same size, mass, and age. The snails were bred with lettuce for 2 weeks in the same conditions as the toxicity test: temperature (20 ± 0.5 °C), moisture content (50% of the water holding capacity). Two days before starting the test, the snails were woken by spraying water, and the proportion of snails not woken was less than 2%. The juvenile snails (4 weeks old) with a mean fresh mass of 1.03 ± 0.10 g and a shell diameter of 16.50 ± 0.20 mm were selected for all experiments. 2.3. Test soil All tests were performed with natural soil. Surface (5–20 cm) soil was collected from an agricultural field in Henan, China. For
more than 5 years, wastewater or sewage sludge had not been applied to the agricultural field. The main characteristics of the test soil were: organic matter content of 23.19 ± 0.47 g kg 1, pH of 8.05 ± 0.05, cation exchange capacity of 17.10 cmol kg 1, 3.91% clay, 32.04% silt, and 64.05% sand. Before the test, the soil was air-dried and then passed through a 4-mm sieve. Appropriate amounts of TCS dissolved in acetone were spiked into air-dried soil to obtain the desired treatment concentrations. The total amount was carefully mixed and the acetone evaporated under darkness in a fume cupboard for 24 h. The sub-samples of each batch were introduced into the test replicates.
2.4. Toxicity test The test was performed according to the standard guideline ISO 15952:2006 (E) (ISO, 2006). The final treatment concentrations were 14, 24, 40, 69, 118, 200, and 340 mg TCS kg 1 soil DW (dry weight) with a common ratio of 1.7. In addition, a control and a solvent control (acetone) were tested in parallel. Three replicates per treatment were used. Six juvenile snails were randomly selected for each container containing a 1-cm layer of 140 g soil. The snails were fed randomly with pieces of fresh lettuce put in a petri dish. The test duration was 28 d. The test ran at 20.0 ± 0.5 °C under a 16:8 (light:dark) photoperiod. Soil moisture content was maintained at its initial level by regular spraying with ultrapure water. The containers were cleaned and the food was renewed three times a week. At days 14 and 28, the organisms were counted and weighed, and shell diameter and total food intake were measured.
2.5. Biochemical assays The test procedures were the same as above. The snail extracts were prepared at days 14 and 28. All procedures were carried out at 4 °C. The whole soft body was removed from the shell, and thereafter the foot and the viscera were separated (Vaufleury and Pihan, 2002). The viscera were analyzed in this study because they contain the digestive gland. The method used for enzyme extraction was adopted from a previous study (Liang et al., 2013). In brief, the viscera part was homogenized in an ice-cold Tris–HCl buffer (0.25 M sucrose, 0.1 M Tris–HCl, 1 mM EDTA, pH 7.4) with a ratio of 1:9 (w/v). The homogenates were centrifuged at 10 000 g for 30 min at 4 °C. The supernatant was used for the analysis of enzymes and protein determination. The activity of SOD, CAT, and POD was determined as described by McCord and Fridovich (1969), Lyttle and DeSombre (1977), and Aebi (1984), respectively. Protein content was determined according to Bradford (1976) using bovine serum albumin as the standard. Lipid peroxidation was measured using the thiobarbituric acid test for MDA according to Ohkawa et al. (1979).
2.6. Statistical analysis Data were expressed as mean ± standard deviation (SD) and checked for homogeneity of variance by using Levene’s test. Statistical analyses were performed with one-way analysis of variance (ANOVA). The least significant differences (LSD) test was used for comparing the significance of differences between blank and treatment groups. A p value less than 0.05 or 0.01 was considered to be statistically significant. The no observed effect concentration (NOEC) was defined as the highest concentration that did not result in a significant effect compared with the control. SPSS 20.0 and OriginLab 8.0 were used in this study.
Please cite this article in press as: Wang, X., et al. Assessment of toxic effects of triclosan on the terrestrial snail (Achatina fulica). Chemosphere (2014), http://dx.doi.org/10.1016/j.chemosphere.2014.01.044
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3. Results 3.1. Toxicity of TCS The toxicity effects of TCS on gains in biomass and shell diameter (days 14 and 28) as well as effect on total food intake of A. fulica are shown in Figs. 1–3. All NOEC values are presented in Table 1. After 14-d and 28-d exposure, the biomass and shell diameter growth of A. fulica in the 40 mg kg 1 and above treatments were significantly reduced (p < 0.05; ANOVA; Figs. 1 and 2). After a 28d exposure, the total food intake in the 40 mg kg 1 and above treatments were significantly reduced (p < 0.05; ANOVA; Fig. 3). Based on the statistical analysis, the NOECs of TCS for the biomass, shell diameter growth, and total food intake were 24 mg kg 1. The NOEC of TCS for 28-d survival was 200 mg kg 1. After a 14-d exposure, there was no mortality in any treatment. Therefore, the 14-d acute EC50 for survival of A. fulica was much larger than 340 mg kg 1.
Fig. 2. Effect of TCS on gains in diameter after 14-d and 28-d exposure. Data are presented as mean ± standard deviation (SD). And represent significant differences compared with the control at p < 0.05 and p < 0.01, respectively.
3.2. Biochemical assays The biochemical effects of TCS on the activity of SOD, CAT, POD, and MDA in the viscera of A. fulica are depicted in Fig. 4. Compared with the control, the SOD activity in the treatments above 14 mg kg 1 was significantly induced with increasing TCS concentration after a 14-d exposure (p < 0.05; ANOVA; Fig. 4A). The activity of SOD in the treatments above 24 mg kg 1 was significantly enhanced after a 28-d exposure (p < 0.01; ANOVA; Fig. 4A). The activity of CAT in A. fulica was significantly induced in the treatments of 40, 69, and 118 mg kg 1, but was significantly inhibited in high concentrations of 200 and 340 mg kg 1 after a 14-d exposure (p < 0.05; ANOVA; Fig. 4B). Similarly, after a 28-d exposure, the CAT activity in the treatments between 24 and 69 mg kg 1 was significantly induced, but was significantly inhibited in the treatments above 118 mg kg 1. After a 14-d exposure, there was no significant change in the activity of POD below 118 mg kg 1. In contrast, the POD activity decreased with increasing TCS concentration above 118 mg kg 1, and was significantly inhibited in the 340 mg kg 1 group. After a 28-d exposure, a slight increase was observed in the concentrations below 69 mg kg 1, but it was not significantly higher than the control. The activity of POD decreased as TCS concentration
Fig. 3. Effect of TCS on food intake after 28-d exposure. Data are presented as mean ± standard deviation (SD). Represents significant difference compared with the control at p < 0.01.
Table 1 Effect concentrations of TCS on different endpoints after 14-d and 28-d exposure. The p value was performed with one-way analysis of variance (ANOVA).
Fig. 1. Effect of TCS on gains in biomass after 14-d and 28-d exposure. Data are presented as mean ± standard deviation (SD). And represent significant differences compared with the control at p < 0.05 and p < 0.01, respectively.
1
Endpoint
Time (d)
NOEC (mg kg
Growth of biomass
14 28
24 24
)
p Value p < 0.05 p < 0.05
Growth of shell diameter
14 28
24 24
p < 0.05 p < 0.05
Inhibition of food intake Survival
28 28 14
24 200 >340 (EC50)
p < 0.05 p < 0.05 –
increased above 69 mg kg 1, and reached the minimum level in the 340 mg kg 1 group (p < 0.05; ANOVA; Fig. 4C). After a 14-d exposure, there was a noticeable dose-dependent toxic effect in terms of MDA content. The content of MDA increased noticeably as TCS concentration increased after 14-d and 28-d exposure (p < 0.05; ANOVA; Fig. 4D). The peak was observed in the 340 mg kg 1 group on day 14, reaching 188% of that in the control.
Please cite this article in press as: Wang, X., et al. Assessment of toxic effects of triclosan on the terrestrial snail (Achatina fulica). Chemosphere (2014), http://dx.doi.org/10.1016/j.chemosphere.2014.01.044
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Fig. 4. Effect of TCS on the activity of SOD (A), CAT (B), POD (C), and content of MDA (D) of A. fulica after 14-d and 28-d exposure. Data are presented as mean ± standard deviation (SD). And represent significant differences compared with the control at p < 0.05 and p < 0.01, respectively.
4. Discussion 4.1. Toxicity effect and risk assessment of TCS As presented in Table 1, the NOECs of TCS for the biomass, shell diameter growth, and total food intake were 24 mg kg 1, which showed that these three test endpoints for A. fulica had a similar level of sensitivity to TCS. For survival of A. fulica, the 28-d NOEC was 200 mg kg 1 Amorim et al. (2010) reported that the NOECs of TCS on the reproduction of terrestrial invertebrates Enchytraeus albidus, Eisenia andrei, and Folsomia candida were 3.2, <10.0, and 3.2 mg kg 1, respectively, whereas the NOEC values for survival of the three invertebrates were 3.2, 320.0, and P320.0 mg kg 1, respectively. This was consistent with our study, that survival was less sensitive than growth, reproduction, and total food intake for TCS exposure on terrestrial invertebrates. Additionally, the 21-d NOECs of TCS on the emergence of plants Brassica rapa and Triticum aestivum were reported to be 32 and 180 mg kg 1, respectively (Amorim et al., 2010), which were larger than the NOECs for terrestrial invertebrates. This indicated that plants B. rapa and T. aestivum were less sensitive than invertebrates A. fulica, E. albidus, E. andrei, and F. candida when exposed to TCS in the terrestrial environment. In the aquatic environment, Brausch and Rand (2011), and Wang et al. (2013) reported that plants (especially algae) were
more sensitive than invertebrates. Reasons for this difference need further investigation. In the present study, the 14-d EC50 for survival (acute test) of A. fulica was much larger than 340 mg kg 1, while the 28-d NOEC for growth (chronic test) was 24 mg kg 1, which showed a difference larger than a factor of 10 (in risk assessment, it is common to use a factor of 10 in extrapolating chronic responses from acute) (EC, 2003). The result indicated that this extrapolation might underestimate the risk for growth of A. fulica, as confirmed by Amorim et al. (2010), who provided a factor much larger than 10 for the earthworm E. andrei. The predicted no effect concentration (PNEC) estimation in European Union (EU) risk assessment usually uses either the Assessment Factor of 10-1000 on the lowest NOEC or the species sensitivity distribution (SSD) method (EU, 2006). The SSD method is considered to be more robust, but data from a series of species are required (always at least 10 toxicity data). In this study, an Assessment Factor of 10 was used, since there were chronic NOEC values for the biomass, shell diameter growth, total food intake, and survival of A. fulica. A PNEC value of 2.4 mg kg 1 was derived, which was larger than in the previous study (Amorim et al., 2010) of 0.32 mg kg 1 using the lowest NOEC values for the reproduction and survival of the three terrestrial invertebrates. The difference was mainly because only one species was tested in our work.
Please cite this article in press as: Wang, X., et al. Assessment of toxic effects of triclosan on the terrestrial snail (Achatina fulica). Chemosphere (2014), http://dx.doi.org/10.1016/j.chemosphere.2014.01.044
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Therefore, species of various trophic levels, as well as endpoints of different levels, should be included in the toxicity test and environmental risk assessment.
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damage the antioxidant defense system and peroxide the lipid membranes in organisms. 5. Conclusion
4.2. Biochemical assays of TCS Antioxidant enzymes and their oxidation products are important indicators of oxidative stress (Koivula and Eeva, 2010). They are of great importance to remove and detoxify ROS and other free radicals (Tripathi et al., 2006; Asagba et al., 2008), which can increase oxidative stress and oxidize proteins, lipids, and nucleic acids, leading to damage in cell structure and even cell death (Atli and Canli, 2010; Cao et al., 2010). As a primary remover of oxygen free radicals O2 , SOD plays an important role in defending against the accumulation of toxic ROS. It can catalyze the conversion of reactive O2 to produce hydrogen peroxide (H2O2), resulting in an increase in the activity of enzymes and high level of H2O2 (Verma and Dubey, 2003). In the present study, the activity of SOD in A. fulica was stimulated significantly in response to TCS above 40 mg kg 1 after 14-d and 28-d exposure (p < 0.05; ANOVA; Fig. 4A). Similarly, Lin et al. (2012) found a significant increase in the activity of SOD in E. fetida at treatments above 50 mg kg 1 TCS after 28-d exposure. After conversion of O2 to H2O2 by SOD, H2O2 and other free radicals are then scavenged in the presence of important enzymes such as CAT and POD in the antioxidant defense system. CAT in peroxisomes and mitochondria can be induced by H2O2 and decompose H2O2 to H2O and O2 (Tang et al., 2005). In this work, the activity of CAT in the snail A. fulica treated by TCS of 24, 40, and 69 mg kg 1 was significantly higher than that in the control after 14-d and 28-d exposure, but it returned to the levels of the control at 118 mg kg 1 after a 28-d exposure (p < 0.05; ANOVA; Fig. 4B). Finally, CAT activity was significantly inhibited in the high concentration treatments (200 and 340 mg kg 1). Previous studies (Lin et al., 2010) reported that the activity of CAT in E. fetida was significantly induced in low concentrations but inhibited in high concentrations of TCS, and this strongly supported our work. The increase in CAT activity in low TCS concentrations can be explained by the large synthesis of the enzyme as a result of ROS production. In contrast, when the generation of ROS exceeds the enzyme synthesis, the CAT activity is significantly inhibited in a higher level of TCS, resulting in damage to the antioxidant defense system (Geret et al., 2002; Atli and Canli, 2010). POD also acts on H2O2 and plays an important role in eliminating H2O2 in organisms. In our study, TCS exhibited a different impact on the activity of POD from that of SOD and CAT in A. fulica. There was no significant change in the activity of POD in low TCS concentrations. However, the POD activity was significantly inhibited in the 340 mg kg 1 group (p < 0.05; ANOVA; Fig. 4C). This could be caused by decreased de novo synthesis of enzyme proteins or irreversible inactivation of enzyme proteins resulting from excess ROS (Jafari, 2007; Atli and Canli, 2010). In our work, CAT was more sensitive to TCS than POD, since its activity significantly changed in almost all concentration groups. Liu et al. (2011) reported that CAT played a more primary role than POD in eliminating H2O2 caused by polycyclic musk in E. fetida. The level of MDA has been proven to be an important indicator of lipid peroxidation. In this study, the oxidative stress induced by TCS was demonstrated by MDA content. Moreover, the content of MDA displayed a dose-dependent increase after 14-d and 28-d exposure, indicating that most products of peroxidized unsaturated fatty acids had accumulated in A. fulica with increasing TCS concentration. Similar findings were reported by previous studies (Xue et al., 2009; Lin et al., 2010). When the generation of ROS exceeded the scavenging capacity of SOD, CAT, and POD, they could
This study was a contribution to the assessment of adverse effects of TCS in the terrestrial environment, where limited data exist. Our results indicated that TCS caused different levels of toxicity to various tested endpoints and biochemical responses of A. fulica. The NOECs of TCS for the biomass, shell diameter growth, and total food intake of A. fulica were 24 mg kg 1. As for biochemical responses, environmental exposure to TCS was significantly harmful even at the low concentration of 24 mg kg 1. The significant changes of antioxidant enzymes indicated wide oxidative stress in A. fulica caused by TCS pollution, and finally, adverse effects of TCS on total food intake and growth were presented. Biochemical responses of SOD, CAT, and MDA were considered suitable biomarkers to improve our understanding of toxicological mechanisms of TCS to terrestrial invertebrates. However, the response of POD was not so sensitive to low TCS concentrations in the present study. Acknowledgments This work was financially supported by the Program of Environmental Protection Commonweal Research (Grant Nos. 2011467054, 201109052-1) and the National Science and Technology Project of Water Pollution Control and Abatement of China (Grant No. 2012ZX07501-003-06). References Aebi, H., 1984. Catalase in vitro. Method. Enzymol. 105, 121–126. Amorim, M.J., Oliveira, E., Soares, A.M., Scott-Fordsmand, J.J., 2010. Predicted no effect concentration (PNEC) for triclosan to terrestrial species (invertebrates and plants). Environ. Int. 36, 338–343. Asagba, S.O., Eriyamremu, G.E., Igberaese, M.E., 2008. Bioaccumulation of cadmium and its biochemical effect on selected tissues of the catfish (Clarias gariepinus). Fish Physiol. Biochem. 34, 61–69. Atli, G., Canli, M., 2010. Response of antioxidant system of freshwater fish Oreochromis niloticus to acute and chronic metal (Cd, Cu, Cr, Zn, Fe) exposures. Ecotoxicol. Environ. Saf. 73, 1884–1889. _ 2005. Peroxidation in muscle and liver tissues from Avci, A., Kaçmaz, M., Durak, I., fish in a contaminated river due to a petroleum refinery industry. Ecotoxicol. Environ. Saf. 60, 101–105. Benotti, M.J., Trenholm, R.A., Vanderford, B.J., Holady, J.C., Stanford, B.D., Snyder, S.A., 2008. Pharmaceuticals and endocrine disrupting compounds in US drinking water. Environ. Sci. Technol. 43, 597–603. Bester, K., 2005. Fate of triclosan and triclosan-methyl in sewage treatment plants and surface waters. Arch. Environ. Contam. Toxicol. 49, 9–17. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Brausch, J.M., Rand, G.M., 2011. A review of personal care products in the aquatic environment: environmental concentrations and toxicity. Chemosphere 82, 1518–1532. Cao, L., Huang, W., Liu, J.H., Yin, X.B., Dou, S.Z., 2010. Accumulation and oxidative stress biomarkers in Japanese flounder larvae and juveniles under chronic cadmium exposure. Comp. Biochem. Physiol. C 151, 386–392. Cha, J., Cupples, A.M., 2009. Detection of the antimicrobials triclocarban and triclosan in agricultural soils following land application of municipal biosolids. Water Res. 43, 2522–2530. Chandran, R., Sivakumar, A.A., Mohandass, S., Aruchami, M., 2005. Effect of cadmium and zinc on antioxidant enzyme activity in the gastropod Achatina fulica. Comp. Biochem. Physiol. C 140, 422–426. Chen, F., Gao, J., Zhou, Q.X., 2012. Toxicity assessment of simulated urban runoff containing polycyclic musks and cadmium in Carassius auratus using oxidative stress biomarkers. Environ. Pollut. 162, 91–97. Daughton, C.G., Ternes, T.A., 1999. Pharmaceuticals and personal care products in the environment: agents of subtle change? Environ. Health Perspect. 107, 907. EC, 2003. Technical Guidance Document (TGD) on risk assessment in support of Commission Directive 93/67/EEC on risk assessment for new notified substances and Commission Regulation (EC) No. 1488/94 on risk assessment for existing substances and Directive 98/8/EC of the European parliament and of the council concerning the placing of biocidal products on the market, Part 11, Technical report, European Commission, Brussels, Belgium.
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Assessment of toxic effects of triclosan on the terrestrial snail (Achatina fulica) Xiaonan Wang a,b, Zhengtao Liu a,⇑, Wanhua Wang a, Zhenguang Yan a, Cong Zhang c, Weili Wang a, Lihong Chen a a State Key Laboratory of Environmental Criteria and Risk Assessment, State Environmental Protection Key Laboratory of Ecological Effect and Risk Assessment of Chemicals, Chinese Research Academy of Environmental Sciences, Beijing 100012, China b College of Water Sciences, Beijing Normal University, Beijing 100875, China c China Offshore Environmental Services Co. Ltd., Tianjin 300452, China
h i g h l i g h t s Toxic effects of triclosan on Achatina fulica were assessed via various approaches. NOECs of TCS for the biomass, shell diameter, and food intake were 24 mg kg
1
TCS caused significant oxidative stress even at low concentration of 24 mg kg
1
a r t i c l e
i n f o
Article history: Received 7 July 2013 Received in revised form 6 December 2013 Accepted 5 January 2014 Available online xxxx Keywords: Triclosan Terrestrial environment Snail (Achatina fulica) Chronic toxicity test Risk assessment Antioxidant enzymes
a b s t r a c t Triclosan (TCS) is a broad-spectrum antimicrobial agent used in personal care products, and as a result, is widespread in the environment. Toxicity tests of TCS on aquatic organisms have been reported, but limited toxicity data on terrestrial species are available. In this study, the 28-d chronic toxicity of TCS on the biomass, shell diameter growth, and total food intake of the terrestrial snail Achatina fulica were tested. Moreover, biochemical responses, including changes in the activity of catalase (CAT), superoxide dismutase (SOD), peroxidase (POD), and the content of malondialdehyde (MDA), were examined after 14-d and 28-d exposure. Results showed that TCS had toxic effects on the biomass, shell diameter growth, and total food intake of A. fulica with no observed effect concentration (NOEC) values of 24 mg kg 1. As for the antioxidant enzymes, TCS caused significant oxidative stress even at the low concentration of 24 mg kg 1. The CAT and POD activities at the high concentrations of 200 and 340 mg kg 1, respectively, were significantly inhibited. The SOD and CAT activity in treatments below 118 mg kg 1 and the MDA content in all treatments showed dose–effect relationships. This study demonstrated that TCS caused adverse effects on terrestrial invertebrates, and provided valuable information for the risk assessment imposed by TCS in the terrestrial environment. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Personal care products (PCPs) represent an important type of emerging organic pollutants and can be found in all environmental media (Ternes et al., 2004; Amorim et al., 2010). The presence of PCPs in the environment and their possible effects on nontarget organisms are of great concern worldwide (Daughton and Ternes, 1999; Kümmerer, 2004; Veldhoen et al., 2006). Among the PCPs, triclosan (5-chloro-2(2,4-dichlorophenoxy) phenol, or TCS) is a broad-spectrum antimicrobial agent used in a variety of personal ⇑ Corresponding author. Tel.: +86 10 84915175; fax: +86 10 84915173. E-mail address: [email protected] (Z. Liu).
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care products including soaps, deodorant, and toothpaste (McAvoy et al., 2009; Price et al., 2010), as well as in household cleaners and even in textiles (sportswear, bed clothes, shoes, and carpets) and children’s toys (Singer et al., 2002). TCS has already been the subject of various scientific fields, and has been detected in surface water and agricultural soil worldwide in recent years (Benotti et al., 2008; Kasprzyk-Hordern et al., 2008; Cha and Cupples, 2009; Zhao et al., 2010; Ramaswamy et al., 2011). TCS is relatively persistent in the environment with a half-life of at least 11 d in surface water (Bester, 2005) and 18 d in aerobic soil (Ying et al., 2007). As for the terrestrial environment, TCS is known to be adsorbed to soil and may enter soil primarily through agricultural application of sewage sludge. Once in the soil, it mostly remains in the upper
http://dx.doi.org/10.1016/j.chemosphere.2014.01.044 0045-6535/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Wang, X., et al. Assessment of toxic effects of triclosan on the terrestrial snail (Achatina fulica). Chemosphere (2014), http://dx.doi.org/10.1016/j.chemosphere.2014.01.044
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10–20 cm incorporation layer (Reiss et al., 2009) and the final soil concentration depends on the initial TCS biosolid concentration, the method of biosolid application, soil types, and other factors Heidler and Halden (2007) reported that the concentration of TCS in sewage sludge was 20–55 mg kg 1, which could cause toxicological risk to terrestrial organisms. The adverse effects of TCS on terrestrial organisms have been reported Liu et al. (2009) reported the effect of TCS on soil microbial processes, and Amorim et al. (2010) provided the chronic toxicity data of TCS on plants (Triticum aestivum, Brassica rapa), worms (Eisenia andrei, Enchytraeus albidus), and collembolans (Folsomia candida). However, the toxicity data, especially the chronic toxicity data of TCS on terrestrial species, are very limited or unavailable. As for the biochemical effect of TCS, very few studies have been conducted on terrestrial organisms, as confirmed by Lin et al. (2010), who examined the biochemical and genetic toxicity of TCS on Eisenia fetida. Therefore, further studies need to be performed on TCS toxicity. Antioxidant enzymes, including catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD), play a major role in removing and detoxifying reactive oxygen species (ROS) and other free radicals caused by environmental contamination (Tripathi et al., 2006; Asagba et al., 2008). In addition, the level of malondialdehyde (MDA) is an important indicator of lipid peroxidation and it is a sensitive diagnostic index of oxidative injury in cells (Avci et al., 2005; Chen et al., 2012). In the present study, the toxic effects of TCS on a terrestrial snail (Achatina fulica) were tested. The A. fulica was often used to determine the biochemical and other toxic effects of chemicals (Rao and Singh, 2002; Chandran et al., 2005; Salway et al., 2010). The aims of this study were to (1) determine the toxic effect of TCS on the biomass, shell diameter growth, and total food intake of A. fulica; (2) determine the toxic effect of TCS on the activity of SOD, CAT, and POD, and the content of MDA. This work could provide valuable information for the environmental risk assessment and pollution management imposed by TCS in the terrestrial environment. 2. Materials and methods 2.1. Chemicals and reagents TCS, C12H7Cl3O2, P97% purity (HPLC), was purchased from Sigma Aldrich. All reagents and standards used in enzyme assays were purchased from the Nanjing Jiancheng Bioengineering Institute (Nanjing, China). All other reagents used in the tests were purchased from the Beijing Guoyao Chemical Co., Ltd. (Beijing, China) and were of analytical grade with chemical purity of 97–99%. 2.2. Test organism The juvenile snails were purchased from a snail culturing farm in Beijing, China and were selected from synchronous breeding to ensure the same size, mass, and age. The snails were bred with lettuce for 2 weeks in the same conditions as the toxicity test: temperature (20 ± 0.5 °C), moisture content (50% of the water holding capacity). Two days before starting the test, the snails were woken by spraying water, and the proportion of snails not woken was less than 2%. The juvenile snails (4 weeks old) with a mean fresh mass of 1.03 ± 0.10 g and a shell diameter of 16.50 ± 0.20 mm were selected for all experiments. 2.3. Test soil All tests were performed with natural soil. Surface (5–20 cm) soil was collected from an agricultural field in Henan, China. For
more than 5 years, wastewater or sewage sludge had not been applied to the agricultural field. The main characteristics of the test soil were: organic matter content of 23.19 ± 0.47 g kg 1, pH of 8.05 ± 0.05, cation exchange capacity of 17.10 cmol kg 1, 3.91% clay, 32.04% silt, and 64.05% sand. Before the test, the soil was air-dried and then passed through a 4-mm sieve. Appropriate amounts of TCS dissolved in acetone were spiked into air-dried soil to obtain the desired treatment concentrations. The total amount was carefully mixed and the acetone evaporated under darkness in a fume cupboard for 24 h. The sub-samples of each batch were introduced into the test replicates.
2.4. Toxicity test The test was performed according to the standard guideline ISO 15952:2006 (E) (ISO, 2006). The final treatment concentrations were 14, 24, 40, 69, 118, 200, and 340 mg TCS kg 1 soil DW (dry weight) with a common ratio of 1.7. In addition, a control and a solvent control (acetone) were tested in parallel. Three replicates per treatment were used. Six juvenile snails were randomly selected for each container containing a 1-cm layer of 140 g soil. The snails were fed randomly with pieces of fresh lettuce put in a petri dish. The test duration was 28 d. The test ran at 20.0 ± 0.5 °C under a 16:8 (light:dark) photoperiod. Soil moisture content was maintained at its initial level by regular spraying with ultrapure water. The containers were cleaned and the food was renewed three times a week. At days 14 and 28, the organisms were counted and weighed, and shell diameter and total food intake were measured.
2.5. Biochemical assays The test procedures were the same as above. The snail extracts were prepared at days 14 and 28. All procedures were carried out at 4 °C. The whole soft body was removed from the shell, and thereafter the foot and the viscera were separated (Vaufleury and Pihan, 2002). The viscera were analyzed in this study because they contain the digestive gland. The method used for enzyme extraction was adopted from a previous study (Liang et al., 2013). In brief, the viscera part was homogenized in an ice-cold Tris–HCl buffer (0.25 M sucrose, 0.1 M Tris–HCl, 1 mM EDTA, pH 7.4) with a ratio of 1:9 (w/v). The homogenates were centrifuged at 10 000 g for 30 min at 4 °C. The supernatant was used for the analysis of enzymes and protein determination. The activity of SOD, CAT, and POD was determined as described by McCord and Fridovich (1969), Lyttle and DeSombre (1977), and Aebi (1984), respectively. Protein content was determined according to Bradford (1976) using bovine serum albumin as the standard. Lipid peroxidation was measured using the thiobarbituric acid test for MDA according to Ohkawa et al. (1979).
2.6. Statistical analysis Data were expressed as mean ± standard deviation (SD) and checked for homogeneity of variance by using Levene’s test. Statistical analyses were performed with one-way analysis of variance (ANOVA). The least significant differences (LSD) test was used for comparing the significance of differences between blank and treatment groups. A p value less than 0.05 or 0.01 was considered to be statistically significant. The no observed effect concentration (NOEC) was defined as the highest concentration that did not result in a significant effect compared with the control. SPSS 20.0 and OriginLab 8.0 were used in this study.
Please cite this article in press as: Wang, X., et al. Assessment of toxic effects of triclosan on the terrestrial snail (Achatina fulica). Chemosphere (2014), http://dx.doi.org/10.1016/j.chemosphere.2014.01.044
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3. Results 3.1. Toxicity of TCS The toxicity effects of TCS on gains in biomass and shell diameter (days 14 and 28) as well as effect on total food intake of A. fulica are shown in Figs. 1–3. All NOEC values are presented in Table 1. After 14-d and 28-d exposure, the biomass and shell diameter growth of A. fulica in the 40 mg kg 1 and above treatments were significantly reduced (p < 0.05; ANOVA; Figs. 1 and 2). After a 28d exposure, the total food intake in the 40 mg kg 1 and above treatments were significantly reduced (p < 0.05; ANOVA; Fig. 3). Based on the statistical analysis, the NOECs of TCS for the biomass, shell diameter growth, and total food intake were 24 mg kg 1. The NOEC of TCS for 28-d survival was 200 mg kg 1. After a 14-d exposure, there was no mortality in any treatment. Therefore, the 14-d acute EC50 for survival of A. fulica was much larger than 340 mg kg 1.
Fig. 2. Effect of TCS on gains in diameter after 14-d and 28-d exposure. Data are presented as mean ± standard deviation (SD). And represent significant differences compared with the control at p < 0.05 and p < 0.01, respectively.
3.2. Biochemical assays The biochemical effects of TCS on the activity of SOD, CAT, POD, and MDA in the viscera of A. fulica are depicted in Fig. 4. Compared with the control, the SOD activity in the treatments above 14 mg kg 1 was significantly induced with increasing TCS concentration after a 14-d exposure (p < 0.05; ANOVA; Fig. 4A). The activity of SOD in the treatments above 24 mg kg 1 was significantly enhanced after a 28-d exposure (p < 0.01; ANOVA; Fig. 4A). The activity of CAT in A. fulica was significantly induced in the treatments of 40, 69, and 118 mg kg 1, but was significantly inhibited in high concentrations of 200 and 340 mg kg 1 after a 14-d exposure (p < 0.05; ANOVA; Fig. 4B). Similarly, after a 28-d exposure, the CAT activity in the treatments between 24 and 69 mg kg 1 was significantly induced, but was significantly inhibited in the treatments above 118 mg kg 1. After a 14-d exposure, there was no significant change in the activity of POD below 118 mg kg 1. In contrast, the POD activity decreased with increasing TCS concentration above 118 mg kg 1, and was significantly inhibited in the 340 mg kg 1 group. After a 28-d exposure, a slight increase was observed in the concentrations below 69 mg kg 1, but it was not significantly higher than the control. The activity of POD decreased as TCS concentration
Fig. 3. Effect of TCS on food intake after 28-d exposure. Data are presented as mean ± standard deviation (SD). Represents significant difference compared with the control at p < 0.01.
Table 1 Effect concentrations of TCS on different endpoints after 14-d and 28-d exposure. The p value was performed with one-way analysis of variance (ANOVA).
Fig. 1. Effect of TCS on gains in biomass after 14-d and 28-d exposure. Data are presented as mean ± standard deviation (SD). And represent significant differences compared with the control at p < 0.05 and p < 0.01, respectively.
1
Endpoint
Time (d)
NOEC (mg kg
Growth of biomass
14 28
24 24
)
p Value p < 0.05 p < 0.05
Growth of shell diameter
14 28
24 24
p < 0.05 p < 0.05
Inhibition of food intake Survival
28 28 14
24 200 >340 (EC50)
p < 0.05 p < 0.05 –
increased above 69 mg kg 1, and reached the minimum level in the 340 mg kg 1 group (p < 0.05; ANOVA; Fig. 4C). After a 14-d exposure, there was a noticeable dose-dependent toxic effect in terms of MDA content. The content of MDA increased noticeably as TCS concentration increased after 14-d and 28-d exposure (p < 0.05; ANOVA; Fig. 4D). The peak was observed in the 340 mg kg 1 group on day 14, reaching 188% of that in the control.
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Fig. 4. Effect of TCS on the activity of SOD (A), CAT (B), POD (C), and content of MDA (D) of A. fulica after 14-d and 28-d exposure. Data are presented as mean ± standard deviation (SD). And represent significant differences compared with the control at p < 0.05 and p < 0.01, respectively.
4. Discussion 4.1. Toxicity effect and risk assessment of TCS As presented in Table 1, the NOECs of TCS for the biomass, shell diameter growth, and total food intake were 24 mg kg 1, which showed that these three test endpoints for A. fulica had a similar level of sensitivity to TCS. For survival of A. fulica, the 28-d NOEC was 200 mg kg 1 Amorim et al. (2010) reported that the NOECs of TCS on the reproduction of terrestrial invertebrates Enchytraeus albidus, Eisenia andrei, and Folsomia candida were 3.2, <10.0, and 3.2 mg kg 1, respectively, whereas the NOEC values for survival of the three invertebrates were 3.2, 320.0, and P320.0 mg kg 1, respectively. This was consistent with our study, that survival was less sensitive than growth, reproduction, and total food intake for TCS exposure on terrestrial invertebrates. Additionally, the 21-d NOECs of TCS on the emergence of plants Brassica rapa and Triticum aestivum were reported to be 32 and 180 mg kg 1, respectively (Amorim et al., 2010), which were larger than the NOECs for terrestrial invertebrates. This indicated that plants B. rapa and T. aestivum were less sensitive than invertebrates A. fulica, E. albidus, E. andrei, and F. candida when exposed to TCS in the terrestrial environment. In the aquatic environment, Brausch and Rand (2011), and Wang et al. (2013) reported that plants (especially algae) were
more sensitive than invertebrates. Reasons for this difference need further investigation. In the present study, the 14-d EC50 for survival (acute test) of A. fulica was much larger than 340 mg kg 1, while the 28-d NOEC for growth (chronic test) was 24 mg kg 1, which showed a difference larger than a factor of 10 (in risk assessment, it is common to use a factor of 10 in extrapolating chronic responses from acute) (EC, 2003). The result indicated that this extrapolation might underestimate the risk for growth of A. fulica, as confirmed by Amorim et al. (2010), who provided a factor much larger than 10 for the earthworm E. andrei. The predicted no effect concentration (PNEC) estimation in European Union (EU) risk assessment usually uses either the Assessment Factor of 10-1000 on the lowest NOEC or the species sensitivity distribution (SSD) method (EU, 2006). The SSD method is considered to be more robust, but data from a series of species are required (always at least 10 toxicity data). In this study, an Assessment Factor of 10 was used, since there were chronic NOEC values for the biomass, shell diameter growth, total food intake, and survival of A. fulica. A PNEC value of 2.4 mg kg 1 was derived, which was larger than in the previous study (Amorim et al., 2010) of 0.32 mg kg 1 using the lowest NOEC values for the reproduction and survival of the three terrestrial invertebrates. The difference was mainly because only one species was tested in our work.
Please cite this article in press as: Wang, X., et al. Assessment of toxic effects of triclosan on the terrestrial snail (Achatina fulica). Chemosphere (2014), http://dx.doi.org/10.1016/j.chemosphere.2014.01.044
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Therefore, species of various trophic levels, as well as endpoints of different levels, should be included in the toxicity test and environmental risk assessment.
5
damage the antioxidant defense system and peroxide the lipid membranes in organisms. 5. Conclusion
4.2. Biochemical assays of TCS Antioxidant enzymes and their oxidation products are important indicators of oxidative stress (Koivula and Eeva, 2010). They are of great importance to remove and detoxify ROS and other free radicals (Tripathi et al., 2006; Asagba et al., 2008), which can increase oxidative stress and oxidize proteins, lipids, and nucleic acids, leading to damage in cell structure and even cell death (Atli and Canli, 2010; Cao et al., 2010). As a primary remover of oxygen free radicals O2 , SOD plays an important role in defending against the accumulation of toxic ROS. It can catalyze the conversion of reactive O2 to produce hydrogen peroxide (H2O2), resulting in an increase in the activity of enzymes and high level of H2O2 (Verma and Dubey, 2003). In the present study, the activity of SOD in A. fulica was stimulated significantly in response to TCS above 40 mg kg 1 after 14-d and 28-d exposure (p < 0.05; ANOVA; Fig. 4A). Similarly, Lin et al. (2012) found a significant increase in the activity of SOD in E. fetida at treatments above 50 mg kg 1 TCS after 28-d exposure. After conversion of O2 to H2O2 by SOD, H2O2 and other free radicals are then scavenged in the presence of important enzymes such as CAT and POD in the antioxidant defense system. CAT in peroxisomes and mitochondria can be induced by H2O2 and decompose H2O2 to H2O and O2 (Tang et al., 2005). In this work, the activity of CAT in the snail A. fulica treated by TCS of 24, 40, and 69 mg kg 1 was significantly higher than that in the control after 14-d and 28-d exposure, but it returned to the levels of the control at 118 mg kg 1 after a 28-d exposure (p < 0.05; ANOVA; Fig. 4B). Finally, CAT activity was significantly inhibited in the high concentration treatments (200 and 340 mg kg 1). Previous studies (Lin et al., 2010) reported that the activity of CAT in E. fetida was significantly induced in low concentrations but inhibited in high concentrations of TCS, and this strongly supported our work. The increase in CAT activity in low TCS concentrations can be explained by the large synthesis of the enzyme as a result of ROS production. In contrast, when the generation of ROS exceeds the enzyme synthesis, the CAT activity is significantly inhibited in a higher level of TCS, resulting in damage to the antioxidant defense system (Geret et al., 2002; Atli and Canli, 2010). POD also acts on H2O2 and plays an important role in eliminating H2O2 in organisms. In our study, TCS exhibited a different impact on the activity of POD from that of SOD and CAT in A. fulica. There was no significant change in the activity of POD in low TCS concentrations. However, the POD activity was significantly inhibited in the 340 mg kg 1 group (p < 0.05; ANOVA; Fig. 4C). This could be caused by decreased de novo synthesis of enzyme proteins or irreversible inactivation of enzyme proteins resulting from excess ROS (Jafari, 2007; Atli and Canli, 2010). In our work, CAT was more sensitive to TCS than POD, since its activity significantly changed in almost all concentration groups. Liu et al. (2011) reported that CAT played a more primary role than POD in eliminating H2O2 caused by polycyclic musk in E. fetida. The level of MDA has been proven to be an important indicator of lipid peroxidation. In this study, the oxidative stress induced by TCS was demonstrated by MDA content. Moreover, the content of MDA displayed a dose-dependent increase after 14-d and 28-d exposure, indicating that most products of peroxidized unsaturated fatty acids had accumulated in A. fulica with increasing TCS concentration. Similar findings were reported by previous studies (Xue et al., 2009; Lin et al., 2010). When the generation of ROS exceeded the scavenging capacity of SOD, CAT, and POD, they could
This study was a contribution to the assessment of adverse effects of TCS in the terrestrial environment, where limited data exist. Our results indicated that TCS caused different levels of toxicity to various tested endpoints and biochemical responses of A. fulica. The NOECs of TCS for the biomass, shell diameter growth, and total food intake of A. fulica were 24 mg kg 1. As for biochemical responses, environmental exposure to TCS was significantly harmful even at the low concentration of 24 mg kg 1. The significant changes of antioxidant enzymes indicated wide oxidative stress in A. fulica caused by TCS pollution, and finally, adverse effects of TCS on total food intake and growth were presented. Biochemical responses of SOD, CAT, and MDA were considered suitable biomarkers to improve our understanding of toxicological mechanisms of TCS to terrestrial invertebrates. However, the response of POD was not so sensitive to low TCS concentrations in the present study. Acknowledgments This work was financially supported by the Program of Environmental Protection Commonweal Research (Grant Nos. 2011467054, 201109052-1) and the National Science and Technology Project of Water Pollution Control and Abatement of China (Grant No. 2012ZX07501-003-06). References Aebi, H., 1984. Catalase in vitro. Method. Enzymol. 105, 121–126. Amorim, M.J., Oliveira, E., Soares, A.M., Scott-Fordsmand, J.J., 2010. Predicted no effect concentration (PNEC) for triclosan to terrestrial species (invertebrates and plants). Environ. Int. 36, 338–343. Asagba, S.O., Eriyamremu, G.E., Igberaese, M.E., 2008. Bioaccumulation of cadmium and its biochemical effect on selected tissues of the catfish (Clarias gariepinus). Fish Physiol. Biochem. 34, 61–69. Atli, G., Canli, M., 2010. Response of antioxidant system of freshwater fish Oreochromis niloticus to acute and chronic metal (Cd, Cu, Cr, Zn, Fe) exposures. Ecotoxicol. Environ. Saf. 73, 1884–1889. _ 2005. Peroxidation in muscle and liver tissues from Avci, A., Kaçmaz, M., Durak, I., fish in a contaminated river due to a petroleum refinery industry. Ecotoxicol. Environ. Saf. 60, 101–105. Benotti, M.J., Trenholm, R.A., Vanderford, B.J., Holady, J.C., Stanford, B.D., Snyder, S.A., 2008. Pharmaceuticals and endocrine disrupting compounds in US drinking water. Environ. Sci. Technol. 43, 597–603. Bester, K., 2005. Fate of triclosan and triclosan-methyl in sewage treatment plants and surface waters. Arch. Environ. Contam. Toxicol. 49, 9–17. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Brausch, J.M., Rand, G.M., 2011. A review of personal care products in the aquatic environment: environmental concentrations and toxicity. Chemosphere 82, 1518–1532. Cao, L., Huang, W., Liu, J.H., Yin, X.B., Dou, S.Z., 2010. Accumulation and oxidative stress biomarkers in Japanese flounder larvae and juveniles under chronic cadmium exposure. Comp. Biochem. Physiol. C 151, 386–392. Cha, J., Cupples, A.M., 2009. Detection of the antimicrobials triclocarban and triclosan in agricultural soils following land application of municipal biosolids. Water Res. 43, 2522–2530. Chandran, R., Sivakumar, A.A., Mohandass, S., Aruchami, M., 2005. Effect of cadmium and zinc on antioxidant enzyme activity in the gastropod Achatina fulica. Comp. Biochem. Physiol. C 140, 422–426. Chen, F., Gao, J., Zhou, Q.X., 2012. Toxicity assessment of simulated urban runoff containing polycyclic musks and cadmium in Carassius auratus using oxidative stress biomarkers. Environ. Pollut. 162, 91–97. Daughton, C.G., Ternes, T.A., 1999. Pharmaceuticals and personal care products in the environment: agents of subtle change? Environ. Health Perspect. 107, 907. EC, 2003. Technical Guidance Document (TGD) on risk assessment in support of Commission Directive 93/67/EEC on risk assessment for new notified substances and Commission Regulation (EC) No. 1488/94 on risk assessment for existing substances and Directive 98/8/EC of the European parliament and of the council concerning the placing of biocidal products on the market, Part 11, Technical report, European Commission, Brussels, Belgium.
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Please cite this article in press as: Wang, X., et al. Assessment of toxic effects of triclosan on the terrestrial snail (Achatina fulica). Chemosphere (2014), http://dx.doi.org/10.1016/j.chemosphere.2014.01.044