Oxidative Damage of Naphthenic Acids on the Eisenia fetida Earthworm Jie Wang, Xiaofeng Cao, Liwei Chai, Jingqiu Liao, Yi Huang, Xiaoyan Tang State Key Joint Laboratory of Environmental Simulation and Pollution Control, College of Environmental Sciences and Engineering, Peking University, Beijing 100871, China

Received 21 October 2014; revised 16 February 2015; accepted 7 March 2015 Abstract: Naphthenic acids (NAs) have been gaining recognition in recent years as potentially harmful environmental contaminants. Few studies have focused on the potential ecotoxicity of NAs to terrestrial environment. In this study, the responses of antioxidant system and lipid peroxidation and DNA damage were investigated after exposing Eisenia fetida to soil contaminated with NAs. The results indicated that NAs induced a significant increase (p < 0.05) in superoxide dismutase and catalase enzyme activities. The glutathione peroxidase enzyme activities were significantly inhibited (p < 0.05) in the medium and high dose treatments. An increase in malondialidehyde indicated that NAs could cause cellular lipid peroxidation in the tested earthworms. The percentage of DNA in the tail of comet assay of coelomocytes as an indication of DNA damage increased after treatment with different doses of NAs, and a dosedependent DNA damage of coelomocytes was found. In conclusion, oxidative stress caused by NAs C 2015 Wiley Periodicals, Inc. exposure induces physiological responses and genotoxicity on earthworms. V Environ Toxicol 00: 000–000, 2015.

Keywords: naphthenic acids; oxidative stress; genotoxicity; antioxidant enzyme; lipid peroxidation

INTRODUCTION Naphthenic acids (NAs) are a mixture of aliphatic or alicyclic carboxylic acids that are described by the general formula CnH2n 1 ZO2, where n represents the number of carbon atoms in the molecule and Z specifies hydrogen deficiency in the case of cyclic NAs (Headley and McMartin, 2004; Scott et al., 2008). NAs are naturally occurring low molecular weight constituents of petroleum, and widely used in a variety of commercial and industrial applications such as tire fabrication and wood preservatives (Grewer et al., 2010; Melvin and Trudeau, 2012; Clemente and Fedorak, 2005). NAs have been gaining recognition in recent years as potentially harmful environmental contaminants, and research Correspondence to: Y. Huang; e-mail: [email protected] Contract grant sponsor: Public Welfare Project of Ministry of Environmental Protection. Contract grant number: 201309034. Published online 00 Month 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/tox.22139

has been carried out to determine the toxicological risks from expose to NAs (Clemente and Fedorak, 2005; Kannel and Gan, 2012). These organic acids have significant detrimental effects in aquatic environments and are toxic to variety of aquatic organisms (fish, algae, microorganisms) with some evidence that the mechanism is related to their surfactant activity (Colavecchia et al., 2004; Jones et al., 2011; Gagne et al., 2012; Woodworth et al., 2012; Melvin et al., 2013). NAs tend to penetrate the cell wall of a biological membrane and produce narcosis-like effects in aquatic species (Kannel and Gan, 2012). These compounds are also reported to be toxic to mammals, which have effects on the formation of red and white blood cells, inhibition of cellular respiration, and toxic to the liver and can be lethal after acute, high dose exposures (Rogers et al., 2002). Furthermore, several studies using fish hepatocytes and embryos revealed that exposure to oil sand process-affects water (OSPW) resulted in greater expression of genes related to the biotransformation of xenobiotics and oxidative stress (Gagne et al., 2012, 2013; He et al., 2012), and NAs have been considered as the primary candidate contaminants in OSPW.

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However, very few studies have investigated toxicity of NAs to terrestrial organism, despite high concentrations of NAs entered into soil from crude oil exploration and production processes (Wang et al., 2015). The toxicity of contaminants varies between soil and water, and knowledge of this variance is essential to develop soil environmental quality guidelines. Earthworms plays a major role in the soil ecosystem for their favorable effects on soil structure and function (Groenigen et al., 2014). They may expose to contaminants through their intestine and skin via alimentary and dermal uptake routes. Hence, earthworms are suitable indicator species for ecotoxicological assessment of soil pollution (Schreck et al., 2008; Xie et al., 2012). Moreover, the use of biomarkers in earthworms for the evaluation of the effects of soil contaminants has received increased attention, and can also be used to monitor soil pollution and provide an ecotoxicological diagnosis as an early warning system (Hankard et al., 2004; Liu et al., 2012; Velki and Hackenberger, 2013). In this article, Eisenia fetida (the most commonly used species) was used as a model organism to study the effects of NAs on the mortality, antioxidant defense system and DNA damage. Specifically, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and malondialdehyde (MDA) contents in earthworms were measured, and a comet assay was conducted to detect DNA damage in coelomocytes. The purpose was to get a comprehensive understanding of the effects of NAs on earthworms, and provide more information about the potential ecological risks of NAs on terrestrial ecosystems.

MATERIALS AND METHODS Chemicals NAs were purchased from Sigma (USA). Stock solution of NAs was prepared in dichloromethane (HPLC grade) at a concentration of 100 mg/L. All other reagents used in the following tests were purchased from Sinopharm Chemical (Beijing, China) and were of analytical grade with a chemical purity  97%.

Earthworm Adult E. fetida with well-developed clitellum (wet weight of 350–500 mg) were purchased from the Lutai Earthworm Breeding Farm (Tianjin, China). They were acclimatized for two weeks under laboratory conditions in culture pots containing soil and cattle manure. Water was added as necessary to maintain moisture content. Twenty-four hours prior to exposures, the earthworms were removed from the culture, rinsed in water, and kept on damp filter paper in the dark at 20 C to void the gut contents.

Test Soil The toxic effects of NAs on E. fetida were investigated by natural soil tests. Soil samples were taken from the upper

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layer (0–20 cm) beside Weiming Lake in Peking University, Beijing, China. The soil sample were air dried at room temperature, and sieved with a 2 mm mesh. The main physical and chemical properties of the soil were as follows: pH 6.37, organic matter 1.67%, cation exchange capacity (CEC), 15.6 cmol kg21, total N 0.94 g kg21, total P 2.67 g kg21, clay 35.7%, silt 46.8%, sand 17.5%.

Experimental Design Experiments were performed according to the OECD methods (OECD, 2004). The NAs concentrations were set as 0, 5, 10, 50, 100, 150, 250, 500, 750, and 1000 mg/kg dry weight, and six replicates (each container representing one replicate) for each exposure treatment and control. There were ten adult earthworms and 500 g (dry weight) of soil in each container. All exposures were conducted in an artificial climate incubator at 20 C in 70% relative humidity with a 16:8 h light: dark regime. After 7 days and 14 days exposure, the living earthworms were collected, observed and counted to evaluate the acute 7-days LC50 and 14-days LC50. Based on the acute toxicity, concentrations with no death observed were chose to evaluate the biomarkers in earthworms. Therefore, 0 mg/kg NAs was set as blank control, and 10, 50, and 100 mg/kg were designed as low, medium, and high exposure concentrations, respectively. Four exposure periods were employed: 1, 3, 7, and 14 days. After exposure, the earthworms were collected and left for 1 day to void their gut contents, and then transferred into liquid nitrogen. The entire earthworm samples were prepared for biomarker analysis. For the Comet assay, the earthworms were removed from each container and prepared for earthworm coelomocytes individually.

Biochemical Assays All procedures were conducted at 4 C. Earthworms were homogenized for 1 min in Tris buffer (100 mM with 250 mM sucrose and 1 mM EDTA, pH 7.5, w/v 5 1:4) using a PRO200 homogenizer at 10,000 rpm. The homogenate was centrifuged at 9000 rpm for 15 min and the supernatants were collected and stored at 280 C for the enzyme determinations. The contents of total protein, SOD, CAT, and GPx were determined using kits obtained from CELL BIOLABS INC according to the manufacturer’s instructions. For each sample, enzyme activity was normalized to its own protein concentration, and then the relative fold-changes were determined. MDA concentration was used as an indicator of lipid peroxidation. The amount of MDA formed was calculated by measuring the absorbance at 532 nm using a molar extinction coefficient of 1.56 3 105 M21cm21 (GonzalezFlecha et al.). The final results are expressed as nmol MDA/mg protein, and then the relative fold-changes were determined.

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Alkaline Comet Assay DNA damage was tested on earthworm immune cells, the coelomocytes, which are present in the coelomic fluid. The coelomocytes were obtained as described by Eyambe et al. (1991). Briefly, individual earthworm was rinsed in 1 mL cold extrusion medium (5% ethanol, 95% saline, 2.5 mg/mL EDTA, 10 mg/mL guaiacol glyceryl ether, pH 7.3) for 3 min. Coelomocytes were spontaneously secreted in the medium, and then the extrusion were centrifuged (4 C, 9000 rpm, 10 min) and discarded the supernatant. The cells were washed three time with phosphate-buffered saline (PBS) and final cell density was adjusted to about 1 3 106 cells/ mL with PBS. The alkaline comet assay was performed as described originally by Singh with slight modifications (Singh et al., 1988). The cell suspension (10 lL) was mixed with 90 lL of 0.75% (w/v in PBS) low-melting agarose at 37 C, and pipette onto a frosted slide precoated with a layer of 100 lL 0.75% (w/v in PBS) normal melting agarose. After solidification in 4 C for 15 min, the slides were immerse into a lysis solution (2.5 M NaCl, 10 mM Tris, 100 mM Na2EDTA, 1% Na-sarcosinate, 10% dimethyl sulfoxide, and 1% Triton X-100) for 16 h in the dark at 4 C to remove cellular proteins and release damaged DNA. The slides were transferred to a horizontal electrophoresis tank containing alkaline buffer (300 mM NaOH, 1 mM EDTA, pH > 13) for 20 min to unwind DNA. Next, electrophoresis was run for 30 min at 18 V and 300 mA at 4 C. The slides were then neutralized (TE buffer, pH 7.5) thrice at 5 min intervals and stained with ethidium bromide (2 lg/mL) for 5 min. The slides were analyzed using a Nikon 80i fluorescent microscope.

Statistical Analysis Each treatment was conduct in quadruplicate. SPSS 20.0 statistical software was used to analyze the experimental, and the results were expressed in the form of mean 6 SD. The relative fold-changes were determined for SOD, CAT, GPx, and MDA. Significant differences between treatment groups were determined using the post hoc LSD test (p < 0.05). Images of comets were recorded with a digital camera and analyzed with the software program Lucia Comet Assay 4.X. At least 50 cells per slide were analyzed and tail DNA% and Olive tail moment (OTM) were used to quantify the extent of DNA damage.

RESULTS Effect of NAs on Mortality of Earthworms Deaths were observed when NAs concentrations in soil were higher than 100 mg/kg, and 100% of mortality was noted in soil with concentrations higher than 750 and 500 mg/kg after 7 days and 14 days exposure, respectively (Fig. 1). In addi-

Fig. 1. Acute lethal response of E. fetida exposed to NAs after 7-days and 14-days exposures in nature soil toxicity test.

tion, earthworms drilled into soils with great reluctance when NAs concentrations were higher than 250 mg/kg, for more earthworms stayed on the top of soils. The LC50 of NAs to E. fetida for 7 days and 14 days was 299.15 and 180.26 mg/kg using Probit Analysis in SPSS.

Effects of NAs on the Activities of Antioxidant-Related Enzymes and Lipid Peroxidation in E. fetida The change in SOD activity between control and NAstreated E. fetida is shown in Figure 2. In comparison with control, significant increases in SOD activity were observed on day 1 after expose to 50–100 mg/kg NAs. This response continued with the exposure period, although activity decreased on day 14, it remained higher than that of the control. The same trend was found for CAT activity (Fig. 3). Compared with that of control, a significantly higher level in the activity of CAT was observed when the concentration of NAs in soil was 50 and 100 mg/kg during 14 days exposure. After 14 days of exposure, CAT activity returned to control levels at low doses of NAs, and slightly higher activity was observed at medium and high doses without significant differences. The effect of NAs on GPx activity in earthworms is displayed in Figure 4. No significant changes could be found in the initial exposures except for the high doses on day 1 (1.37 fold changes, p < 0.05). However, as the exposure continued, the activity of GPx decreased (p < 0.05) for the medium and high dose, falling to 0.82 and 0.67 folds of the control, respectively. The effect of NAs on lipid peroxidation was determined by evaluating the MDA content in earthworms (Fig. 5). On day 1, the MDA contents were significantly elevated by the

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Fig. 2. The relative levels of SOD enzymatic activity in E. fetida exposed to different treatments of NAs. Data are described as means 6 SD (n 5 6). Different small letters indicated significant (p < 0.05) difference between different treatments.

three levels of NAs and the maximum content was about 1.60 fold changes compared with that in the control. The similar changes were observed during the whole exposure period, and there was a noticeable dose-dependent toxicity effect in terms of MDA contents.

Fig. 3. The relative levels of CAT enzymatic activity in E. fetida exposed to different treatments of NAs. Data are described as means 6 SD (n 5 6). Different small letters indicated significant (p < 0.05) difference between different treatments.

Environmental Toxicology DOI 10.1002/tox

Fig. 4. The relative levels of GPx enzymatic activity in E. fetida exposed to different treatments of NAs. Data are described as means 6 SD (n 5 6). Different small letters indicated significant (p < 0.05) difference between different treatments.

DNA Damage Induced by NAs The DNA damage of earthworm coelomocytes was performed with control and different doses treatments after 14 days exposure. Figure 6 shows a type comet image of undamaged DNA and three comet images of damaged DNA

Fig. 5. The relative levels of MDA concentrations in E. fetida exposed to different treatments of NAs. Data are described as means 6 SD (n 5 6). Different small letters indicated significant (p < 0.05) difference between different treatments.

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Fig. 6. Typical comet figures. (a) 100, (b) 50, (c) 10, and (d) 0 mg/kg NAs. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

of low, medium, and high doses exposure, respectively. Significant DNA damage could be observed from the comet shape changes. The values for tail DNA and OTM in treatment groups (10, 50, and 100 mg/kg) had statistically significant (p < 0.05) increases versus the control (Table I). There was a strong linear correlation (y 5 0.573x 1 21.443; R2 5 0.9396) between the tail DNA% and the concentrations of NAs. And the same strong linear correlation was confirmed by the OTM (y 5 0.7942x – 0.6043; R2 5 0.9901).

DISCUSSION The goal of this study was to obtain a comprehensive understanding of the effects of NAs on earthworms, and to evaluate ecological risk of NAs in terrestrial environment. NAs have been gaining recognition as potentially harmful environmental contaminants, and this is growing concern for its adverse effects on biological health. Moreover, according to our knowledge, no study about NAs toxicity was performed with E. fetida as a model sentinel species. Thus, this study TABLE I. Effect of NAs on DNA damage evaluated by the alkaline comet assay in coelomocytes of E. fetida exposed for 14 days Concentrations (mg/kg) 0 10 50 100

Tail DNA (%) a

1.14 6 0.87 22.95 6 9.98b 57.64 6 12.56c 78.29 6 14.26d

OTM 0.41 6 0.31a 9.63 6 4.23b 34.98 6 13.67c 65.87 6 17.34d

Different small letters indicated significant (p < 0.05) difference between different treatments according to one-way ANOVA.

might be the first attempt to investigate the effects of NAs exposure on its disturbance of earthworms. Oxidative stress is a plausible explanation for the toxicity of NAs to the earthworms (He et al., 2012). Oxidative stress results when production of reactive oxidative species (ROS) exceeds the capacity of cellular antioxidant defenses to remove these toxic species (Limon-Pacheco and Gonsebatt, 2009). SOD, CAT, and GPx play key functions in clearance of ROS, which belong to the enzymatic component of cellular antioxidant defense systems. Changes of activity of these enzyme suggested that there was production of ROS in organisms. In our study, significant increases in the activity of SOD were observed in exposure treatments, suggesting the formation of superoxide anion radical (•O22), which indicated that the tested earthworms suffered from oxidative stress. After transformation of •O22 to H2O2 by SOD, H2O2, and other free radicals are then removed by in the presence of important enzymes such as CAT and GPx in antioxidant defense systems (Lin et al., 2012; Liu et al., 2012; Velki and Hackenberger, 2013). CAT is present in peroxisomes and mitochondria where it decomposed H2O2 to water and oxygen. Our study showed that the activity trend of CAT was in general according with the activity of SOD. NAs caused decrease of SOD and CAT activity at the end of exposure period. The reason for SOD might be that the natural antioxidant defenses were saturated, and too great ROS accumulated in earthworms, which became CAT inhibitor (Wu et al., 2011). Unlike CAT, GPx remove H2O2 by coupling its reduction with the oxidation of glutathione (GSH). NAs induced the activity of GPx with no significant differences in the low and medium doses groups in the initial exposure. At the end of exposure period, NAs inhibited the activity of GPx in the medium and high doses treatments, the reasons

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might be that excessive ROS in cell inhibited the GPx activity, or large amount of GSH was consumed to remove ROS. Our group is working with this problem. In these results, CAT potentially played a more primary role in eliminating H2O2 caused by NAs, since its activity was still stimulated when the activity of GPx declined to the control level. There are few studies to investigate the changes of activity of antioxidant enzymes in organisms exposed to NAs. He et al. (2012) demonstrated greater abundances of transcripts of SOD and CAT genes in fathead minnow embryos exposed to OSPW than those in control, which suggested there was greater production of ROS in OSPW exposure groups. This observation is consistent with the results of other research groups (Gagne et al., 2012, 2013), and NAs in OSPW were considered to be most relevant with oxidative stress. Although the reasons for the changes described NAs effects are not fully understood, it is conceivable that NAs could interact with the tissues and result in fluctuated enzyme activities. Moreover, these changes are mainly caused by increased ROS in E. fetida under oxidative stress. MDA is the product of the reaction between free radicals and unsaturated fatty acids in cellular membranes. The oxidative stress induced by NAs can be demonstrated by MDA formation. The increased concentration of MDA could be detected significantly in all three levels of exposure. Shalata and Tal (1998) suggested that one of the most damage effects of ROS is the peroxidation of membrane lipids. The results of this study showed that the MDA concentrations in earthworms were strongly enhanced in response to acute exposure. Thus, it is concluded that the increased MDA concentrations in E. fetida was due to oxidative stress caused by NAs exposure. The Comet Assay is a rapid, sensitive and relatively simple method for detecting DNA damage at the level of individual cells (Kumaravel et al., 2009; Hu et al., 2014). In this study, significant DNA damages were observed in the coelomocytes of E. fetida in soil tests. Moreover, a dose-related response of DNA damage in cells exposed to NAs was clearly observed in our study. A previous study conducted by Lacaze et al. (2014) showed that NAs mixture either industrial (Merichem) or commercial (Sigma) caused primary DNA damage in rainbow trout hepatocytes. And what’s more interesting is that the same dose of different NAs resulted in a different pattern of response, which implied that the genotoxicity of NAs is highly dependent on their structure and complexity (Frank et al., 2008; Grewer et al., 2010; West et al., 2011). Future research is needed to explore the relationships between the chemical compounds and structure of NAs and their genotoxicity.

180.26 mg/kg. The activities of SOD and CAT and the MDA content showed significant responses (p < 0.05) to NAs exposure. Significant DNA damage was observed through the comet assay. All these results implied oxidative stress might be involved in the mechanism of NAs-induced toxicity in animals. Further studies are needed to elucidate the gene expression and the oxidative mechanisms of NAs acting on the organisms.

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CONCLUSION

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Environmental Toxicology DOI 10.1002/tox

Oxidative damage of naphthenic acids on the Eisenia fetida earthworm.

Naphthenic acids (NAs) have been gaining recognition in recent years as potentially harmful environmental contaminants. Few studies have focused on th...
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