Environ Sci Pollut Res (2015) 22:4660–4669 DOI 10.1007/s11356-014-3716-8

RESEARCH ARTICLE

Biomarker responses in earthworms (Eisenia fetida) to soils contaminated with di-n-butyl phthalates Li Du & Guangde Li & Mingming Liu & Yanqiang Li & Suzhen Yin & Jie Zhao

Received: 6 May 2014 / Accepted: 10 October 2014 / Published online: 21 October 2014 # Springer-Verlag Berlin Heidelberg 2014

Abstract Di-n-butyl phthalates (DBP) are recognized as ubiquitous contaminants in soil and adversely impact the health of organisms. Changes in the activity of antioxidant enzymes and levels of glutathione-S-transferase (GST), glutathione (GSH), and malondialdehyde (MDA) were used as biomarkers to evaluate the impact of DBP on earthworms (Eisenia fetida) after exposure to DBP for 28 days. DBP was added to artificial soil in the amounts of 0, 5, 10, 50, and 100 mg kg−1 of soil. Earthworm tissues exposed to each treatment were collected on the 7th, 14th, 21st, and 28th day of the treatment. We found that superoxide dismutase (SOD) and catalase (CAT) levels were significantly inhibited in the 100 mg kg−1 treatment group on day 28. After 21 days of treatment, GST activity in 10–50 mg kg−1 treatment groups was markedly stimulated compared to the control group. MDA content in treatment groups was higher than in the control group throughout the exposure time, suggesting that DBP may lead to lipid peroxidation (LPO) in cells. GSH content increased in the treatment group that received 50 mg kg−1 DBP from 7 days of exposure to 28 days. These results suggest that DBP induces serious oxidative damage on earthworms and induce the formation of reactive oxygen species (ROS) in earthworms. However, DBP concentration in current agricultural soil in China will not constitute any threat to the earthworm or other animals in the soil. Responsible editor: Philippe Garrigues Electronic supplementary material The online version of this article (doi:10.1007/s11356-014-3716-8) contains supplementary material, which is available to authorized users. L. Du : G. Li (*) : M. Liu : Y. Li : S. Yin : J. Zhao College of Resources and Environment, Key Laboratory of Colleges and Universities in Shandong Province Agricultural Environment, National Engineering Laboratory for Efficient Utilization of Soil and Fertilizer, Shandong Agricultural University, 61 Daizong Road, 271018 Taian, China e-mail: [email protected]

Keyword Di-n-butyl phthalate . Earthworm (Eisenia fetida) . Antioxidant enzyme . Lipid peroxidation . Glutathione-S-transferase

Introduction Phthalic acid esters (PAEs) are organic substances that are used in plastic matter industries because they can increase the plasticity of many materials. Phthalates are typically applied in the production process of plastics and cosmetics. During the manufacture, usage, and waste disposal of plastics, phthalates are released and distributed in sediments, natural water, wastewater, and soil (IPCS 1992; Jianlong et al. 1996; Willie and Peijnenburg 2006). Exposure to these harmful compounds by inhalation or dermal contact may interfere with reproduction in humans and wildlife (Jobling et al. 1995; Lee et al. 2004; Tyl et al. 2004; Borch et al. 2006). In addition, some phthalates are suspected teratogens, mutagens, and carcinogens (Huff and Kluwe 1984). The US Environmental Protection Agency (US EPA 1991), and the China National Environmental Monitoring Center (Wang et al. 1995) have already classified the most common PAEs as priority environmental pollutants. Di-n-butyl phthalate (DBP) is widely used in the manufacture of phthalates and is classified as a type of PAEs. DBP may induce lesions in the reproductive system of rabbits, especially during the intrauterine period (Higuchi et al. 2003). Exposure to low concentrations of DBP impairs spermatogenesis in frogs (Xenopus laevis) (Lee and Veeramachaneni 2005) and it is reported that in utero exposure to DBP resulted in reduced testosterone levels, Leydig cell aggregates, and multinucleated gonocytes in fetal testes (Kleymenova et al. 2005). DBP has been reported to reduce steroidogenesis by fetal-type Leydig cells in primates and rodents (Hallmark et al. 2007). DBP can also cause oxidative stress in organisms. Qin et al. (2011a) found that superoxide dismutase (SOD) and catalase

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(CAT) activities in the mantle were induced significantly and the lipid peroxidation (LPO) level was also obviously induced in Perna viridis during chronic DBP exposure. Gill SOD activity in Lutjanus erythropterus increased significantly as the concentration of DBP increased. Gill and liver malondialdehyde (MDA) content increased then decreased after being exposed to DBP (Qin et al. 2011b). Chen et al. (2010) also found that SOD activity in stem and leaves increased first and then decreased with the increase of DBP concentrations and that DBP caused increase in MDA content in Arabidopsis seedlings. DBP has relatively high residual levels in the soils, ranging from 3.18 to 29.37 mg kg−1 in aquic soils of the Handan District (average 14.06 mg kg−1) and 2.75–14.62 mg kg−1 in black soils of the Harbin District (average 7.60 mg kg−1). The total content of four kinds of phthalates (DMP, DEP, DBP, and DEHP) is 0.89– 10.03 mg kg−1 in agricultural soil in China (Hu et al. 2003). Sludges contained DBP of 4.2 to approximately 5.7 mg kg−1 dried solids in Shanghai (Zheng et al. 2008). All noncultivated soils contain the lowest contents of phthalates, suggesting that the kinds of pollutants are largely derived from human agricultural activities (Xu et al. 2008). The half-life period of DBP is about 20 years. Many studies have focused on the deleterious effects of DBP on oxidative stress in animals. Superoxide dismutase (SOD) and catalase (CAT) are regarded as antioxidant enzymes that prevent oxidative stress and their levels which reflect change on biological cells may be used to monitor oxidative stress (Yu et al. 2008). Peroxidase (POD) is considered molecular bioindicators for contaminant-mediated oxidative stress to reflect the magnitude of responses in different populations exposed to xenobiotics (Li 2003). Malondialdehyde (MDA), another indicator of oxidative stress, shows the degree of lipid peroxidation (LPO) in the body. It has been reported that antioxidant enzyme activities and LPO levels in fishes and bivalve animals may be used as biomarkers of oxidative stress and reflections of the extent to the organisms (Di Giulio et al. 1993; Solé et al. 1995). Farombi et al. (2006) indicated that the reduction in the activity of CAT observed in rats may reflect the inability of the testes to eliminate hydrogen peroxide possibly produced by activation of DBP and its metabolites or inactivation of the enzymes caused by excess generation of reactive oxygen species (ROS) in the testes, and that the overwhelming generation of free radicals in the testicular milieu may therefore inactivate SOD as observed in rats. Glutathione-S-transferase (GST) enzymes have been shown to respond to toxins. Most earthworm GST studies have been based on alterations in biochemical activity after exposure to industrial metals and pesticide contaminated soils (Aly and Schröder 2008; Maity et al. 2008; Lqaszczyca et al. 2004; Lukkari et al. 2004; SaintDenis et al. 2001; Booth et al. 2000). Glutathione (GSH) is an important water-soluble antioxidant. It can directly combine

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cellular electrophilic reagents with GST for antioxidation. GSH also plays a key role in cellular detoxification metabolism (Mather-Mihaich and Di Giulio 1993; Cossu et al. 1997; Li et al. 2010). Earthworms play a critical role in soil structure and function (Saint-Denis et al. 1999). Furthermore, they contribute significantly to organic matter decomposition and nutrient cycling (Coleman and Ingham 1988). Since they are ecologically important and plentiful, earthworms have been one of the most commonly used organisms to examine biological effects of potentially harmful materials. The earthworm Eisenia fetida has been regarded as a model biomonitor for measuring soil environmental pollution since 1984 (OECD 1984). Zeng et al. (2010) found that in Esisenia foelide DEHP induced oxidative damage to cells and changed the activity of enzymes. Because DBP, as a kind of PAE, has a similar structural formula and properties to DEHP (Charles et al. 1997), we supposed that E. fetida may be sensitive to DBP and can cause damage in E. fetida. Therefore, E. fetida was chosen for the evaluation of toxicity of DBP in the present study. Although the effects of DBP have been investigated in several vertebrate species, there is no data about effects of DBP on earthworms. The present study focuses on the effects of DBP on the activity of antioxidant enzymes and antioxidant content under artificial soil conditions. The study permits the exploration of possible protective mechanisms of E. fetida after exposure to DBP and the screening of sensitive early-warning molecular biomarkers for DBP contamination.

Material and methods Chemicals DBP (98 % pure), was purchased from Shandong Jingbo Agricultural Chemical Co., Ltd. (Beijing). Other chemicals used in this study were also of analytical grade and were purchased from local commercial sources. Glassware was meticulously cleaned to reduce any background contamination of phthalates. All chromic acid washed glassware was placed in a 300 °C oven overnight. After cooling, the glassware was air-dried until use. Earthworms E. fetida between 300 and 400 mg each were obtained from laboratory cultures at Shandong Agricultural University. The cultures are maintained with cow dung as a substrate and food. Earthworms were removed from culture, rinsed with tap water, and stored in Petri dishes on damp filter paper for 24 h (in the dark at 20±2 °C) to void the gut contents. The earthworms

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chosen for this assay had all reached adulthood and exhibited well-developed clitellum.

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in a climate chamber with a stable temperature of 20±2 °C, moisture of 75±2 % with a 12:12-h light:dark regimen.

Filter paper contact test DBP exposure The acute toxicity of DBP to earthworms was conducted according to the filter paper contact test to the OECD normal method (OECD 1984). Briefly, the sides of culture dishes were lined with filter paper of 63 cm2 without overlapping. DBP dissolved in acetone solvent (1 mL) was added into the filter paper, and the culture dish was rotated for uniform distribution of DBP. Acetone solvent was dried under a stream of compressed air, and then distilled water (1 mL) was added to each culture dish. Seven concentration levels of 1, 2, 10, 25, 50, 75, 100 mg cm−2 were selected for the test. Each culture dish has 10 depurated earthworms in it. The culture dishes were capped firmly with a small ventilation hole to prevent loss of DBP and incubated in an environmental chamber under 20±2 °C and 12/12-h photoperiod. Earthworms were exposed to seven different concentrations of DBP for a period of 24 and 48 h. From the number of organisms that died during the exposure, the values of lethal concentrations 50 % (LC50) were calculated. Every treatment group and the control group had three replicates each.

DBP concentrations of 0, 5, 10, 50, and 100 mg DBP kg−1 artificial soil (dry weight) were used for the toxicity tests. A single earthworm was collected from each replicate bowl on the 7th, 14th, 21st, and 28th day after application of DBP. Toxicity tests were conducted according to the OECD (1984) guidelines in artificial soil and similar to the acute toxicity tests described in detail above.

Sample preparation Earthworms were placed into a prechilled mortar and pestled under ice-cold conditions homogenized in Tris-HCl buffer (250 mM sucrose, 50 mM Tris (pH 7.5), 1 mM DTT, and 1 mM EDTA) in a 1/4 w/v ratio for 1 min using a XHF-D homoge niz er. Homogen ates w ere centrifuged at 10000 r min−1 for 15 min. After centrifugation, the supernatants were collected and stored at −20 °C until analysis. The samples were carried out at 4 °C.

Acute toxicological tests The acute toxic effects of DBP on E. fetida were investigated by artificial soil tests according to the OECD normal method (OECD 1984). The composition of artificial soil samples (dry weight) was 70 % fine sand, 20 % kaolin clay, and 10 % sphagnum peat. Calcium carbonate (about 1 mg per 100 mg −1 of artificial soil) was added in artificial soil to adjust the pH to a value of 7.0±0.2. According to the preexperimental results, five concentrations, i.e., 500, 1000, 2000, 5000, and 10,000 mg DBP kg−1 artificial soil (dry weight), were set for the acute toxicity tests. DBP was dissolved in acetone and thoroughly mixed into the artificial soil at the various concentrations. The soils were placed in a well-ventilated fume hood and turned daily for 7 days in order to evaporate acetone and age the spiked soil. Following acetone evaporation, all soils were rehydrated to 35 % moisture and left 1 day to equilibrate. The control was mixed with the same volume of acetone, distilled water, and pH as the treatment groups. Mortality was determined at 14 days. The earthworms were cultivated for 24 h in untreated artificial soil and then they were put in the DBP treated artificial soil. Each 1000 mL container was filled with 500 g of artificial soil (wet weight). Ten worms with uniform body lengths and weights were randomly divided into the five treatment groups with three replicates of each treatment. The containers were sealed with plastic film to reduce evaporation and punched with holes for ventilation. Tests were conducted

SOD activity Superoxide dismutase (SOD) activity was determined according to a modification of the method of Marklund and Marklund (1974). This assay is based on the ability of SOD to inhibit the autoxidation of pyrogallol (50 mM) in a 50 mM Tris-HCl buffer (pH 8.3). The reaction mixture contained 4.5 mL Tris-HCl buffer, 100 mL supernatant, and 10 mL pyrogallol. Oxidation of pyrogallol was monitored by measuring absorbance at 325 nm. One unit of SOD activity was defined as 50 % inhibition of the oxidation process. Enzymatic activity was expressed as units per milligram of protein.

CAT activity Catalase (CAT) activity was determined according to a modified method of Greanwald (1985). A 3 mL solution contained 0.67 M substrate was prepared by adding 0.16 mL 30 % H2O2 to 100 mL phosphate buffer, pH 7.0, and 100 mL sample or blank (digestive gland and gills, respectively). Kinetics were recorded at 240 nm. The concentration of H2O2 was determined at 3–4 min intervals after the initiation of the reaction by addition of supernatant. One unit of CAT activity was defined as 50 % H2O2 consumption at 1 min. The results are expressed as enzyme unit per milligram protein.

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POD activity Peroxidase (POD) activity was determined according to the method established by Kochba et al. (1977) with slight modifications. The supernatant was added to a 5 mL reaction mixture containing 100 mM potassium phosphate buffer (pH 6.0), 20 mM guaiacol, and 0.2 % (w/v) H2O2. Changes in absorbance were recorded at 470 nm at 25 °C. One activity unit of POD was defined as the amount of enzyme that caused an increase of 0.01 absorbance unit per minute. The results were expressed as △OD470 min−1 g−1 protein. GST activity Glutathione peroxidase (GST) activity was determined according to the method of Habig et al. (1974). Various substrates were used in assays to further characterize GSTs using 1-chloro-2,4-dinitrobenzene (CDNB) as a substrate. The assay was carried out by monitoring the appearance of the conjugated complex of CDNB and GSH at 340 nm. The mixture contained 190 mL of 0.1 M Tris buffer pH 7.0, 0.5 mL of 1 M MGSH, 1 mL of 1 M MCDNB, and 10 mL enzyme extract. Specific activities were expressed as nanomole per minute per milligram protein. GSH content Glutathione (GSH) content was measured by a modified method of Sen et al. (1992). The tissue extract sample was mixed with 3 volumes of 5 % TCA and the mixture was centrifuged at 10000 rev at 4 °C for 10 min. A 100 mL aliquot of supernatant was mixed with 1.4 mL of 0.4 M Tris-HCl buffer (pH 8.9) and 100 mL of 0.01 M DTNB. After 5 min of incubation, GSH content was assessed by absorbance at 412 nm. GSH content was calculated from the calibration curve and expressed in microgram per 100 mg−1 of tissue. MDA content Malondialdehyde (MDA) content was used as an indicator of lipid peroxidation (LPO) level and was quantified by measuring the formation of thiobarbituric acid reactive substances according to the methods described by Ohkawa et al. (1979) with some modifications. The concentration of MDA formed was calculated using the absorbance coefficient 1.56×105 M−1 cm−1 (Flecha et al. 1991) and results are expressed as nanomole MDA per milligram protein. Statistical analysis The data were analyzed using SPSS (Standard Version 13.0, SPSS Inc.). The relationships between DBP concentration and SOD, CAT, POD, and GST activities; MDA content; and GSH

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content were tested by analysis of variance (ANOVA). When significant differences were considered at level P