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Evaluation of the toxicity of 1-butyl-3-methyl imidazolium tetrafluoroborate using earthworms (Eisenia fetida) in two soils
Science of the Total Environment 686 (2019) 946–958
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Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv
Evaluation of the toxicity of 1-butyl-3-methyl imidazolium tetrafluoroborate using earthworms (Eisenia fetida) in two soils Yuting Shao a,1, Kaixuan Hou a,1, Zhongkun Du a, Bing Li a, Jun Wang a, Albert Juhasz b, Jinhua Wang a, Lusheng Zhu a,⁎ a College of Resources and Environment, Shandong Agricultural University, Key Laboratory of Agricultural Environment in Universities of Shandong, National Engineering Laboratory for Efficient Utilization of Soil and Fertilizer Resources, 61 Daizong Road, Taian 271018, PR China b Future Industries Institute, Centre for Environmental Risk Assessment and Remediation, University of South Australia, Mawson Lakes, SA 5095, Australia
H I G H L I G H T S
G R A P H I C A L
A B S T R A C T
• Ecotoxicity of [Bmim]BF4 to earthworms in artificial and nature soil was compared. • The toxicity of [Bmim]BF4 in different soils were compared by computing IBRv2 index. • [Bmim]BF4 can influence the enzyme activities and cause DNA damage in earthworms. • [Bmim]BF4 showed greater toxicity in nature (fluvo-aquic) soil.
a r t i c l e
i n f o
Article history: Received 2 April 2019 Received in revised form 30 May 2019 Accepted 1 June 2019 Available online 06 June 2019 Editor: Jay Gan Keywords: [Bmim]BF4 Earthworm Artificial soil Fluvo-aquic soil IBR
a b s t r a c t Herein, to research the toxic effect of ionic liquids (ILs) on earthworms and compare their different toxicities in different soils, 1-butyl-3-methyl imidazolium tetrafluoroborate ([Bmim]BF4) was selected as a test substance, Eisenia fetida was selected as the experimental indicator organism, and artificial and fluvo-aquic soils were selected as the media. The acute toxicity, reactive oxygen species (ROS) content, detoxification enzyme (GST) activity, anti-oxidant enzyme activities, lipid peroxidation oxidative and DNA damage in earthworms were all measured to evaluate the toxicity of [Bmim]BF4. The results showed that either in fluvo-aquic soil or artificial soil, [Bmim]BF4 can stimulate the accumulation of ROS in earthworms, inducing activities of antioxidant enzymes and detoxification enzymes, inevitably causing lipid peroxidation and DNA damage in earthworms. The integrated biomarker response (IBR) indicated that the toxicity of [Bmim]BF4 in fluvo-aquic soil was greater than that in artificial soil. This experiment is relevant to the reliability of artificial soil toxicity research, and maybe this paper can provide a more authentic understanding of traditional toxicity experiments. © 2019 Elsevier B.V. All rights reserved.
⁎ Corresponding author at: Shandong Agricultural University, 61 Daizong Road, Taian 271018, PR China. E-mail addresses: [email protected] (Z. Du), [email protected] (J. Wang), [email protected] (A. Juhasz), [email protected] (J. Wang), [email protected] (L. Zhu). 1 Yuting Shao and Kaixuan Hou contributed equally to this work.
https://doi.org/10.1016/j.scitotenv.2019.06.010 0048-9697/© 2019 Elsevier B.V. All rights reserved.
Y. Shao et al. / Science of the Total Environment 686 (2019) 946–958
1. Introduction With the increasing requirements of environmental quality for human life, green chemistry is a key factor in achieving economic and socially sustainable development. Ionic liquids (ILs) have excellent chemical and thermodynamic stability and are largely applied in organic synthesis, isolation and purification, electro-chemistry and other fields because of the lower vapor pressure, higher thermal stability and adjustable dissolution (Studzinska et al., 2009; Made et al., 2015; Mehrkesh and Karunanithi, 2016). It is unlikely that ILs become air pollutants because of their low vapor pressure, but they can inevitably be released into water and soil through accidental spills, effluents or irrigation (Liwarska-Bizukojc, 2011). Besides, common ILs have been proven to remain stable in water and soil medium (Shao et al., 2017). Therefore, it is especially important to study the toxicity of ILs in water and soil. Nowadays, with the extensive use of ILs, there are a large number of studies on their toxicity. For aquatic organisms, Ma et al. (2010) demonstrated the toxicity of [Cnmim]Br (n = 4, 6, 8, 10, 12) to green algae, after S. obliquus was exposed to [Cnmim]Br, the 96 h-EC50 values ranged from 22.24 to 0.02 mg/L, while Ruokonen et al. (2016) highlighted the viability effect and behavioral toxicity of 11 kinds of amidinium, imidazolium, and phosphonium based ILs to Danio rerio. At present, the study on the toxicity of ILs to aquatic organisms is comprehensive relatively, while the toxicity research on terrestrial organisms is relatively rare. For terrestrial impact, earthworms (Eisenia fetida) are commonly utilized to assess the impact of soil contamination (Zhao et al., 2014; Li et al., 2015; Li et al., 2017) using Organization of Economic and Cooperation Development (OECD) (1984) methods using artificial soil. Many studies have highlighted the toxic effects of ILs on earthworms and confirmed that ILs would cause enzyme induction (Li et al., 2010; Shao et al., 2018a; Shao et al., 2019). However, the toxicity research of ILs in artificial soil may not represent the true toxicity. But few studies have been performed to compare the toxic effects of ILs between artificial soil and natural soil. In the present study, 1-butyl-3-methyl imidazolium tetrafluoroborate ([Bmim]BF4), a commonly used imidazole-based ILs, which has good electrical conductivity and biocompatibility, was used to assess its impact on Eisenia fetida in both artificial soil and natural (fluvo-aquic) soil. A variety of toxicological biomarkers were utilized including acute toxicity, the content of reactive oxygen species (ROS), anti-oxidant enzyme systems, de-toxification enzyme glutathione-S-transferase (GST), lipid peroxidation (LPO) product malondialdehyde (MDA), and DNA damage (olive tail moment (OTM) in the comet essay) to evaluate the effect of [Bmim]BF 4 on earthworms. The integrated biomarker response (IBR) calculated with biomarker responses could be used to evaluate the ecological risk of the polluted area (Wang et al., 2011). Because of the shortcomings of IBR, IBR was improved to IBR version 2 (IBRv2) (Sanchez et al., 2013). Subsequently, IBRv2 was widely used to assess the environmental quality (Estefanía et al., 2018; Wan et al., 2018). In this study, the IBR index was computed to evaluate the toxicity to earthworms of [Bmim]BF 4 in artificial soil and fluvo-aquic soil. It was hypothesized that Eisenia fetida's toxicological response to [Bmim]BF4 would vary between artificial and natural soil due to the influence of soil properties on IL bioavailability.
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Healthy adult earthworms (Eisenia fetida), weighing approximately 300 to 600 mg, were sourced from Shandong Agricultural University breeding base. Eisenia fetida were adapted to lab conditions for 14 days prior to toxicity testing. The artificial soil used for this study was prepared according to the OECD (1984) guidelines. Briefly, the soil contained plant ash (10%), kaolin clay (20%) and quartz sand (70%), and the pH was adjusted to 6.0 ± 0.5 by CaCO3. The natural soil used in this study was a fluvo-aquic soil, collected from alluvial sediments of the Yellow River, Shandong Province, China. The basic information of the two soils are detailed in supplementary material. (Table S1). 2.2. Methods 2.2.1. IL soil preparation [Bmim]BF4 was added to artificial and fluvo-aquic soils to achieve concentrations ranging from 5 to 1000 mg/kg. Briefly, 500 g of airdried soil was thoroughly mixed with aqueous solutions of [Bmim]BF4 to achieve the desired concentration and a water holding capacity of 60%. Earthworms were pre-cultured for 1 d in non-toxic soils before experiments. 2.2.2. Acute toxicity test For acute toxicity tests, [Bmim]BF4 in artificial and fluvo-aquic soil was assessed at 0, 200, 400, 500, 600, 800, and 1000 mg/kg. Assays were conducted in triplicate (3 × 500 g) with each soil containing ten earthworms. All treatments were maintained in a climate chamber (20 °C, 12 h Light: 12 h Dark) with survival monitored over a 14-day exposure period. In this study, the earthworms' mortality of controls was 0, and the earthworms' mortality of the highest concentrations was N90%. 2.2.3. Subchronic toxicity test In the subchronic toxicity test, the concentrations of [Bmim]BF4 were set to 5, 10, 20 and 40 mg/kg. As with acute toxicity tests, assays were conducted in triplicate, however, fifteen earthworms were included in each treatment. All treatments were maintained in a climate chamber (20 °C, 12 h Light: 12 h Dark) and on days 7, 14, 21 and 28, earthworms were randomly selected (3 per treatment) for the assessment of subchronic toxicity indicators (ROS, antioxidant enzymes, DNA damage). During the 28 d, the earthworms' mortality of controls was 0. 2.2.4. Assessment of protein concentration Protein content of earthworms was determined in the light of Bradford (1976), with bovine serum albumin (BSA) used to construct standard curves. 2.2.5. Assessment of ROS The 2′,7′- dichlorohydro fluorescein diacetate (DCFH-DA) method used in this study is commonly utilized for the determination of ROS content (Zhang et al., 2013). DCFH-DA, an oxygen-sensitive fluorescent probe, can react with ROS thereby allowing its quantification. Earthworms were previously depurated for 12 h, after which they were washed and homogenized in 0.1 mol/L phosphate buffer saline (PBS).
2. Materials and methods 2.1. Materials [Bmim]BF4 (CAS: no. 174501-65-6, purity above 99.0%) was purchased from the Cheng Jie Chemical Co., Ltd. (Shanghai, China). All other reagents used in this study were analytical grade constituents and bought from Sigma Chemical Co. (St. Louis, MO, USA).
Table 1 Acute toxicity of [Bmim]BF4 on earthworm (Eisenia fetida) in artificial soil and fluvo-aquic soil.
Artificial soil Fluvo-aquic soil a
7 d-LC50 (mg/kg)
14 d-LC50 (mg/kg)
870 (750–1089)a 744 (678–835)a
678 (617–746)a 489 (439–541)a
Values in parenthesis represent the 95% confidence intervals of the mean.
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ROS level (Fluorescence Intensity)
c
c
c
c
1000 b
b
b
b a
a 800
0
5
10
20
1200
40
0
5
10
20
40
d c
c 1000
b
b
ab
b a
a
800
600
C
Fluvo-aquic soil
1200
600
B
Artificial soil
1400 ROS level (Fluorescence Intensity)
A
0
5
10
20
40
0
ab
5
10
20
40
ROS level (Fluorescence Intensity)
1200 1100 1000
b a
b
d
a c
900
bc
800 a
ab
700 0
D
b
5
10
20
40
0
5
10
20
40
ROS level (Fluorescence Intensity)
1500 d
1400 1300
c
1200 1100
c b
1000 900
a
b
ab
a
a
800
a
700 600
0
5
10
20
40
0
5
10
20
40
Concentration (mg/kg) Fig. 1. The ROS level of earthworms effected by [Bmim]BF4 after 7 (A), 14 (B), 21 (C), 28 (D) days in artificial and fluvo-aquic soil at diverse concentrations. The colors (black, red, blue, pink and green) mean the concentration of [Bmim]BF4 (0, 5, 10, 20 and 40 mg/kg on the X axis). The line and little box within the box plot indicate the median and mean values. The boundaries of the box indicate the means ± standard error (SE). The whiskers indicate minimum and maximum. Least significant difference test was adopted with a significance of p b 0.05 between control and exposure treatments shown by a, b, c etc. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
The homogenate was centrifuged (20,000 ×g, 20 min, 4 °C) and resuspended to extract mitochondria for ROS assessment. Mitochondrial extracts were mixed with DCFH-DA at 37 °C for 20 min, then the reaction was terminated by 1 mol/L HCl and the OD value was read at 538 nm under an excitation wavelength of 485 nm by a fluorescence spectrophotometer.
2.2.6. Assessment of antioxidant enzymes, GST activities and MDA content Following depuration, earthworms were washed and then homogenized in PBS (10 times the body weight). The homogenate was centrifuged (10,000 rpm, 15 min, 4 °C) and resuspended prior to the assessment of antioxidant enzyme and de-toxification enzyme activities and MDA content.
Y. Shao et al. / Science of the Total Environment 686 (2019) 946–958
Artificial soil
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Fluvo-aquic soil
A 4.5
SOD activity(U/mg pr)
b 4.0
a
ab
b
d e
ab c b
3.5 a 3.0
2.5
0
5
10
20
40
0
5
10
20
40
b
b
20
40
3.6
B SOD activity(U/mg pr)
3.4 3.2
b
ab ab
a
a
a
3.0
b
c 2.8 2.6 0
5
10
20
40
0
5
10
4.5
C SOD activity(U/mg pr)
c b
4.0
b
b
10
20
40
a
ab
20
40
b c c
a a
3.5 a 3.0
2.5
0
5
10
20
40
0
5
4.0
D SOD activity(U/mg pr)
c 3.5 b
d 3.0
b
e
ab a
a 2.5
2.0
0
5
10
20
40
0
5
10
Concentration (mg/kg) Fig. 2. The SOD activity of Eisenia fetida exposed to [Bmim]BF4 after 7 (A), 14 (B), 21 (C), 28 (D) days in artificial soil and fluvo-aquic soil at diverse concentrations. The colors (black, red, blue, pink and green) mean the concentration of [Bmim]BF4 (0, 5, 10, 20 and 40 mg/kg). The line and little box within the box plot indicate the median and mean values. The boundaries of the box indicate the means ± standard error (SE). The whiskers indicate minimum and maximum. Least significant difference test was adopted with a significance of p b 0.05 between control and exposure treatments shown by a, b, c etc. Pr, protein. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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A
Artificial soil
CAT activity(U/mg pr)
7
B
6
a
a
0
5
a
Fluvo-aquic soil b
b
a
20
40
0
a
a
a
a
5
10
20
40
a
a
20
40
5
4
10
11 a
CAT activity(U/mg pr)
10 9 a
8 a
7 6
a
a
0
5
10
a
a
a
a
a a
5 4 3 2
CAT activity(U/mg pr)
C 6
20
40
0
a
a
a
5
10
a
a
a a
4
2 0
5
10
20
40
0
5
10
a
a
20
40
8
D
a
CAT activity(U/mg pr)
ab 7 a 6
ab
ab
ab
b b
5
4 0
5
10
20 40 0 Concentration (mg/kg)
5
10
20
40
Fig. 3. The CAT activity of earthworms exposed to [Bmim]BF4 after 7 (A), 14 (B), 21 (C), 28 (D) days in artificial soil and fluvo-aquic soil at diverse concentrations. The colors (black, red, blue, pink and green) mean the concentration of [Bmim]BF4 (0, 5, 10, 20 and 40 mg/kg). The line and little box within the box plot indicate the median and mean values. The boundaries of the box indicate the means ± standard error (SE). The whiskers indicate minimum and maximum. Least significant difference test was adopted with a significance of p b 0.05 between control and exposure treatments shown by a, b, c etc. Pr, protein. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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A
Fluvo-aquic soil
Artificial soil
1.0
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POD activity (U/mg pr)
0.9 c
0.8 0.7 0.6
b
ab
a
a
10
20
40
ab
ab
ab
10
20
40
bc
c
20
40
a
a
a
0
5
ab
a
0.5 0.4 0.3 0.2
B
0
5
10
20
40
POD activity (U/mg pr)
0.8 0.7
b
a
a
a a
0.6
a
a
0.5 0.4 0.3
0
5
10
20
40
0
5
C POD activity (U/mg pr)
0.8 b
a
a
ab ab
c
0.6
c c
0.4
0
POD activity (U/mg pr)
D
5
10
20
40
0
5
a
ab
10
0.8 0.7
a a
ab
a a
ab
a
b
0.6 0.5 0.4 0.3
0
5
10
20 40 0 Concentration (mg/kg)
5
10
20
40
Fig. 4. The POD activity of Eisenia fetida exposed to [Bmim]BF4 after 7 (A), 14 (B), 21 (C), 28 (D) days in artificial soil and fluvo-aquic soil at diverse concentrations. The colors (black, red, blue, pink and green) mean the concentration of [Bmim]BF4 (0, 5, 10, 20 and 40 mg/kg). The line and little box within the box plot indicate the median and mean values. The boundaries of the box indicate the means ± standard error (SE). The whiskers indicate minimum and maximum. Least significant difference test was adopted with a significance of p b 0.05 between control and exposure treatments shown by a, b, c etc. Each trial contained three replicates. Pr, protein. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Superoxide dismutase (SOD) activity was assessed according to Han et al. (2014), where an enzyme activity unit (U) expresses the amount of SOD required to inhibit the chemical reduction of 1/2 added nitrogen blue tetrazole (NBT). Enzyme extracts (50 μL, PBS for control) were
mixed with PBS, methionine, NBT, Na2-EDTA, riboflavin and deionized water (totally 3 mL) and after 30 min, the UV absorption was measured at 560 nm. One control tube was selected randomly and placed in the dark, while the others were reacted under a 4000 lx fluorescent lamp.
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Artificial soil
Fluvo-aquic soil
210
A GST activity(nmol/min/pr)
200 190
b
c
180 170 160
ab
a
bc
bc
b
ab
c
a
150 140 130 120 110 100 90
0
5
10
20
40
0
5
a
a
a
10
20
40
180
B GST activity(nmol/min/pr)
170 a
160 150
a
a
a
a
b c
140 130 120 110 100 90 80
C
0
5
10
20
40
0
10
20
40
GST activity(nmol/min/pr)
170 a 160
a ab
150
ab
ab ab
b
b
c
140
d
130 120 110 0
5
10
20
40
0
5
10
20
40
170
D
a
GST activity(nmol/min/pr)
160 150
a
b b
140
bc
b bc
bc
c
130 120
c
110 100 90 80
0
5
10
20
40
0
5
10
20
40
Concentration (mg/kg) Fig. 5. The GST activities of earthworms effected by [Bmim]BF4 after 7 (A), 14 (B), 21 (C), 28 (D) days exposure in artificial soil and fluvo-aquic soil at diverse concentrations. The colors (black, red, blue, pink and green) mean the concentration of [Bmim]BF4 (0, 5, 10, 20 and 40 mg/kg). The line and little box within the box plot indicate the median and mean values. The boundaries of the box indicate the means ± standard error (SE). The whiskers indicate minimum and maximum. Least significant difference test was adopted with a significance of p b 0.05 between control and exposure treatments shown by a, b, c etc. Pr, protein. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Catalase (CAT) activity was also assessed in the light of Han et al. (2014), where an enzyme activity unit (U) expresses the amount of enzyme required to decompose 50% hydrogen peroxide (H2O2). Enzyme extracts (10 μL) were mixed with PBS (3 mL) as the reference, or mixed with PBS and H2O2 (3 mL) as experimental group, after which the dynamics of UV (250 nm) was monitored every 5 s for 1 min. In the light of Han et al. (2014), POD (guaiacol peroxidase) activity was assessed. In this study, reaction solution (3 mL, including PBS, guaiacol, and hydrogen H2O2) was mixed with PBS (20 μL) as the reference or with enzyme extracts (20 μL) as experimental group, after which the dynamics of UV (470 nm) was monitored every 30 s for 3 min. According to Han et al. (2014), GST activity was assessed, as reduced glutathione (GSH) can bind with 1-chloro-2,4-dinitro-benzene. The homogenization buffer contained Na2HPO4, NAH2PO4, glycerin, phenylmethylsulfonyl fluoride, EDTA, and dithiothreitol. Enzyme extraction was combined with CNDB, glutathione and buffer solution and the dynamics of UV (340 nm) was monitored every 30 s for 3 min. Determination of MDA content was performed according to Zhang et al. (2013). Enzyme extracts (PBS for control), sodium dodecyl sulfonate, acetic acid, TBA and water were combined sequentially, and incubated at 90 °C (water bath). After 1 h, the absorbance (532 nm) was monitored. 2.2.7. Assessment of DNA damage DNA damage was assessed by the single cell gel electrophoresis (SCGE) experiment according to Song et al. (2009). Following depuration, earthworms were soaked in physiological saline, and then immersed in extracting solution (containing anhydrous ethanol, normal saline, ethylenediaminetetraacetic acid (EDTA), guaiacol glyceryl ether) to extract coelomocytes. Extracts were centrifuged (3000 ×g, 15 min, 4 °C) and resuspended in PBS prior to use. Electrophoretic sheets were made by preparing three gel layers, composed of normal melting agar (NMA), a mixture of extraction and low melting agar (LMA), and LMA, whereby gel layers were attached to the surface of glass slides. Slides were put into cell-lysis solution for 60 min, placed in electrophoretic solution (NaOH and Na2EDTA) for 30 min. After which slides were electrophoresed (300 mA, 25 V) and neutralized. Finally, slides were dehydrated in ethanol and stored in a humid environment prior to use. Slides were stained with EB and viewed under the fluorescence inverted microscope (Olympus BX51). At least one hundred cells at each concentration were analyzed by the comet assay software project (CASP) to obtain the OTM, which reflects cell damage.
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toxicity of [Bmim]BF4 to earthworms in fluvo-aquic soil is more severely than that in artificial soil. When earthworms were exposed to low-dose ILs (taking the possible concentrations in the soil into account, the dose was set as: 5, 10, 20, 40 mg/kg) for a long time (28 days), the results of the comparison of toxicity between different soils were shown below. 3.2. ROS level A substance that promotes oxidative stress in earthworms is ROS (Dayem et al., 2017), which influences the generation, cross-linking, single-strand breaks, base alkylation and oxidation of pyridine dimers and the deletions of DNA bases (Tuteja et al., 2001). ROS content in earthworms following exposure to [Bmim]BF4 over a 28-day period in different soils was presented in Fig. 1. After the initial exposure of earthworms to [Bmim]BF4 in artificial soil (7 days), ROS levels were significantly higher compared to earthworms exposed to non-amended soil. On day 7, there appeared to be a linear increase in fluorescence intensity with increasing [Bmim]BF4 up to 20 mg/kg. At higher concentrations (i.e. 40 mg/kg), there was no significant difference in ROS level compared to an exposure dose of 20 mg/kg. A similar result was observed for the fluvo-aquic soil spiked with [Bmim]BF4. However, on day 28, the disparity between ROS response in the artificial control (unexposed) group and [Bmim]BF4 spiked soil group decreased. The 10 mg/kg and 40 mg/kg group resulted in a ROS response that was significantly higher than unexposed earthworms; the remaining [Bmim]BF4 exposure concentrations (5 and 20 mg/kg) produced a ROS response that was similar to the control group. Within contrast, for earthworms exposed to [Bmim]BF4 in the fluvo-aquic soil, ROS levels at day 28 were significantly higher compared to those in the unexposed group. Xu et al. (2018) researched the toxic impacts of ILs [C8mim]R (R = Cl−, Br−, BF−4) on wheat seedings in brown soil, then the ROS levels accumulated and formed concentration dependent. Guo et al. (2015) researched the toxic impacts of ILs [C8mim]Cl on Eisenia fetida (the same concentrations as this study) and in their study, the ROS levels in earthworms also showed the concentration dependent. Shao et al. (2019) researched the toxicity of CnBr to Eisenia fetida and a similar conclusion was obtained. They all proved that ILs can affect the biobalance and stimulate the accumulation of ROS in organisms. Although the ROS accumulation was observed in both soils, the change is not exactly the same, and the ROS levels in earthworms in fluvo-aquic soil were higher than that in artificial soil especially in the second half of the experiment.
2.3. Statistical analyses
3.3. Antioxidant enzyme activities
All experiments were performed in triplicate with data in this study analyzed by SPSS 20.0 and plotted by Origin 8.5. In the figures, each bar depicts the mean of replicates and error bars depict the standard deviations (SD). Different letters in the figures reflect significant difference (p b 0.05) between the treatments. The figures of treatment effect ratio of all biomarkers are presented in SI (Figs. S1–S7). Data of all biomarker responses of earthworms (Eisenia fetida) after 28 days exposure to [Bmim]BF4 under 40 mg/kg was selected to calculate IBR index according to the method of Sanchez et al. (2013). Detail of calculation process was listed in supplementary material.
The enzymes that remove ROS include SOD, CAT, and POD. SOD eliminates superoxide radicals, by converting O−2 to H2O2 while CAT and POD are anti-oxidant enzymes that eliminate H2O2 (Qu et al., 2010); CAT catalyzes H2O2 to produce H2O (Wu et al., 2011) while POD oxidizes cosubstrates (such as guaiacol or ascorbate) to decompose H2O2. The above enzymes can interact and then reflect the degree of oxidative stress and thus the toxicity of [Bmim]BF4 to earthworms. SOD activity in earthworms following exposure to [Bmim]BF4 over a 28-day period was displayed in Fig. 2. After the initial exposure of earthworms to [Bmim]BF4 in artificial soil (7 days), the SOD activities of all treated groups were significantly higher than that of the control. On day 14, all treatment groups returned to the control level (except the 20 mg/kg treated group). On day 21, the SOD activities were re-activated in all treated groups and higher than that of control, but the SOD activities in the high concentration treated groups (20, 40 mg/kg) were lower than others. On day 28, the SOD activities for all concentrations were still higher than those in the control, but they showed a tendency to first be activated and then inhibited as the concentration increased. Meanwhile, after the earthworms
3. Results and discussion 3.1. Acute toxicity Eisenia fetida acute toxicity test results are shown in Table 1. In artificial soil, the 7 d-LC50 was 870 mg/kg compared to 744 mg/kg in fluvoaquic soil. Similarly, the 14 d-LC50 was obviously higher in artificial soil (678 mg/kg) compared to the in fluvo-aquic soil (489 mg/kg). The acute
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Artificial soil
MDA content (nmol/mg pr)
A
Fluvo-aquic soil
3.0 c
a
bc
2.8 ab ab
2.6 a a
a a
a
2.4
2.2
MDA content (nmol/mg pr)
B
0
5
10
20
40
0
5
10
20
40
3.0 2.8
d
2.6
c
c
d
2.4 2.2
a
b
ab
b a
ab
0
5
2.0 1.8 1.6 0
MDA content (nmol/mg pr)
C
5
10
20
40
10
20
40
3.4 c
3.2 3.0
b
b
2.8 2.6 2.4 2.2
b
ab
5
10
c
c
20
40
a
a
0
5
a
2.0 0
MDA content (nmol/mg pr)
D
10
20
2.8
40
d d c
2.6 b
ab
c
b
b
5
10
2.4 a
a 2.2
2.0
0
5
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Fig. 6. The MDA contents of earthworms exposed to [Bmim]BF4 after 7 (A), 14 (B), 21 (C), 28 (D) days in artificial soil and fluvo-aquic soil at diverse concentrations. The colors (black, red, blue, pink and green) mean the concentration of [Bmim]BF4 (0, 5, 10, 20 and 40 mg/kg). The line and little box within the box plot indicate the median and mean values. The boundaries of the box indicate the means ± standard error (SE). The whiskers indicate minimum and maximum. Least significant difference test was adopted with a significance of p b 0.05 between control and exposure treatments shown by a, b, c etc. Pr, protein. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
contacted [Bmim]BF4 in the fluvo-aquic soil, the SOD activity of all treatment groups was activated on day 7. After that (on day 14), only the 10 mg/kg treatment group maintained a level equal to control, and the SOD activity of the remaining three concentrations treatment
groups was inhibited. On day 21, similar with the artificial soil, the SOD activities were re-activated in all treatment groups. On day 28, no treatment groups differed significantly from the control except the 10 mg/kg treatment group.
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When Xiong et al. (2013) studied the toxic impacts of copper sulfate on Eisenia fetida, and Duan et al. (2015) studied the toxic impacts of benzo[a]pyrene on Eisenia fetida, they all observed that the SOD activities in poisoned earthworms were first activated and then inhibited. The SOD activities were activated first, because excessive ROS induced the synthesis of anti-oxidase or activated SOD as a mechanism to resist oxidative stress. Then, the SOD activity decreased because the removal of ROS consumed some SOD. However, as the exposure time prolonged, increasing amounts of ROS forced the earthworms to activate more SOD to counter the impact, and SOD activity thus increased again. Ultimately, the SOD activity showed inhibition at higher concentrations, possibly because earthworms had higher ROS levels at higher concentrations (and more SOD was thus consumed) or because the ROS accumulated beyond the catalytic ability of SOD and inhibited the SOD activity. For these two soils, the SOD activity in earthworms in fluvo-aquic soil had a more obvious response. CAT activity in earthworms following exposure to [Bmim]BF4 over a 28-day period was presented in Fig. 3. After 7 d exposure to [Bmim]BF4 in artificial soil, the CAT activity in all earthworms was activated, and significant difference was observed only in the 20 and 40 mg/kg treatment groups. However, on day 14 and day 21, the activation effect disappeared, and the levels in all the treatment groups returned to control level. On day 28, all treatment groups showed a significant effect in artificial soil and an inapparent inhibitory effect in fluvo-aquic soil. POD activity following exposure to [Bmim]BF4 over a 28-day period was presented in Fig. 4. After 7 d exposure to [Bmim]BF4 in artificial soil, the POD activity in earthworms of all treatment groups was significantly higher than control, and the highest concentration treatment group had the biggest growth. On day 14, the POD activities were no longer activated. On day 21, the POD activities were decreased, while a significant activation was observed in the lowest concentration treatment group. On day 28, all the treatment groups were slightly inhibited. Meanwhile, after earthworms contacted [Bmim]BF4 in the fluvo-aquic soil, the POD activity was unchanged on day 7. On day 14, the POD activities were little activated. On day 21, the POD activity of all the treated groups was significantly inhibited, which lasted until day 28. CAT and POD, two enzymes with the same function, collectively remove excess H2O2 in earthworms. In previous studies, Du et al. (2014) researched the toxic effect of ILs [C8mim]Br in Danio rerio; under the induction of ILs, the CAT activity in zebrafish was first activated and then gradually inhibited to reduce the damage. Duan et al. (2015) researched the toxic impacts of benzo[a]pyrene on Eisenia fetida and confirmed that
the POD activities in earthworms were first activated and then gradually inhibited as the earthworms were poisoned. Similarly, in this study, H2O2 accumulated under the action of SOD, and both CAT and POD were thus activated to eliminate the effects of H2O2. After that, due to the different catalytic effects of CAT and POD, CAT disproportionated H2O2 directly, while POD catalyzed the oxidation of H2O2 with another substrate (Vidossich et al., 2012). Thus, CAT dominated the reaction and still maintained the activated state, but POD was gradually returned to the control's level on day 14. Beginning on day 21, with the prolonged exposure time, oxidative stress increased, and more O−2 and H2O2 accumulated in the earthworms. The adaptability and health of the earthworms were thus reduced, leading to toxic reactions that decreased the activities of CAT and POD; these changes were more obvious in the higher concentration groups. The inhibitory effect of enzymes in fluvo-aquic soil was more obvious. 3.4. Detoxification enzyme activities GST is an important metabolic detoxification enzyme which catalyzes the activity of reduced glutathione (GSH) to react with contaminants and increases the water solubility of toxicants which facilitates their elimination/excretion. GST also catalyzes the binding of GSH to electrophilic intermediate metabolites and reduces the possibility of such compounds to react with intracellular biological macromolecules, thus maintaining genomic integrity of cells (LaCourse et al., 2009). GST activity following exposure to [Bmim]BF4 over a 28-day period in earthworms was presented in Fig. 5. After 7 d exposure to [Bmim]BF4 in artificial soil, GST activities in earthworms were activated in all treatment groups, and the higher concentration treated groups showed the greater differences. On day 14, the activation was diminished, and the levels restored to the level of control. On days 21 and 28, the GST activity began to be inhibited, and all treatment groups showed significant differences on day 21. Meanwhile, when earthworms contacted [Bmim]BF4 in the fluvo-aquic soil, on day 7, GST activity was also activated and showed a concentrationeffect relationship. On day 14, the GST activity began to be inhibited in the 20 and 40 mg/kg treatment groups. After days 21 and 28, the inhibition was strengthened, all treatment groups were inhibited, and more obvious inhibitory effects were observed with increasing concentrations. In this study, the GST activities in earthworms increased first and then decreased. Lin et al. (2012) studied the physiological and molecular responses of Eisenia fetida to chlortetracycline, and Shao et al. (2012) studied the oxidative stress induced by endosulfan in zebrafish. Though
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the organisms were exposed to different contaminants and concentrations, they both observed that the GST activity in organisms increased first and then decreased. The GST activity increased first, possibly as an adaptive response of earthworms, and the enhanced GST detoxification effect helped to reduce the damage caused by ILs. After that, the detoxification process produced large amounts of intermediate metabolites, perhaps altering the composition of the GST subunit and reducing the GST activity. At the same time, GST was consumed as the substrate (Shao et al., 2018b,). Shao et al. (2019) studied oxidative stress and genotoxic effects in earthworms induced by five imidazolium bromide ILs with different alkyl chains. They find that, GST activity was stimulated at lower concentrations but inhibited at higher concentrations particularly after 28 days exposure to [C10mim]Br. Therefore, the GST activity was significantly inhibited in the second half of the experiment. In these two soils, the inhibitory effect of GST in the fluvo-aquic soil occurred earlier. This means that earthworms in fluvo-aquic soil were more seriously damaged. 3.5. Oxidative damage Exposure to [Bmim]BF4may result in the generation of ROS, enzyme stress and oxidative damage (such as LPO and DNA damage) (Shi and Zhou, 2010). [Bmim]BF4 exposure may lead to the accumulation of free radicals, which may oxidize unsaturated fatty acids, damaging the structure and function of biological membranes. As LPO may result in the formation of MDA, the intensity of LPO damage and the extent of oxidative damage can be inferred from changes in MDA content (Gill and Tuteja, 2010). MDA content in earthworms was shown in Fig. 6. After 7 d exposure to [Bmim]BF4 in the artificial soil, there was no significant change in the MDA content in any of the treatment groups. On day 14, the MDA content was significantly increased. On days 21 and 28, the MDA content in all treated groups was significantly increased, and the degree of the effect gradually increased. Which may be due to the combined effects of ROS and GST. As IL toxicity increased, ROS content increased and antioxidant enzymes were inhibited, causing significant increase in MDA content. Guo et al. (2015) researched the toxic effect of [C8mim]Cl on Eisenia fetida, Shao et al. (2018a) researched the toxicity of ILs ([C8mim]Br and [C8mim]BF4) on Eisenia fetida. The concentration they set was same as this study, and their experiments showed that ILs can induce serious LPO in earthworms, same as this study. The MDA content increased as the exposure time and dose increased. It can be speculated that the degree of LPO intensified. The LPO in fluvo-aquic soil was more obvious. [Bmim]BF4 may also cause DNA damage whereby ROS reacts with DNA molecules directly, resulting in DNA spiral structure damage. In addition, ROS reacts with LPO products and intermediate alkyl radicals, indirectly exacerbating further damage to DNA (Possalmai et al., 2007). OTM value in earthworms was shown in Fig. 7. After earthworms contacted [Bmim]BF4 in the artificial and the fluvo-aquic soil, the OTM value of nearly all these treatment groups was higher than control, and this value increased gradually as the dose and exposure time increasing. Dong et al. (2013) studied the DNA damage by [C10mim]Br in zebrafish and showed that ILs can continuously induce DNA damage. Shao et al. (2018a) also proved that [Omim]BF4 and [Omim]Br can cause DNA damage in Eisenia fetida and observed nearly a linear increase in OTM value with increasing concentration. In the present study, the DNA in earthworms was damaged, and the OTM value increased as the increasing dose and exposure times. Zhang et al. (2018) and Ma et al. (2019) verified the same results when studied the DNA damage by fluoxastrobin and pyraclostrobin respectively and they even find that the comet assay was the most sensitive biomarkers. Besides, DNA damage in the fluvo-aquic soil was more obvious than that in the artificial soil.
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3.6. Toxicity between the two soils As an integrated index, IBR stands for toxicity level of [Bmim]BF4. The IBR index histogram and a star plot under 40 mg/kg concentration on day 28 were shown in Fig. 8. The star plot including all biomarkers, the blue circle can be seen as the base line. Indices inside base line mean inhibited, while indices outside the base line mean activated. On day 28, under 40 mg/kg concentration, the IBR index in fluvoaquic (21.61) soil was higher than that in artificial soil (19.90), which indicates that [Bmim]BF4 can cause severer toxicity in fluvo-aquic soil. The responses of biomarkers of the star plot indicate that the toxicity effects were the increase of ROS and MDA contents, the inhibition of CAT, POD and GST activity, and the damage of DNA. Among all selected indicators, the bigger contribution indicators to IL toxicity were ROS and OTM. Combining the acute and subchronic experimental results, we can predict that [Bmim]BF4 showed greater toxicity in fluvo-aquic soil. Artificial soil experiments cannot fully represent the toxicity of ILs. This may be due to the fact that natural soils can introduce all sorts of variables to the tests, such as the soil organic matter, pH, cation exchange capacity (CEC) and ratio of organic carbon to total nitrogen (C:N), resulting in toxic differences, the most possibility is that increasing organic matter leads to decreasing toxicity (Amorim et al., 2005; Stepnowski et al., 2007). Schnug et al. (2014) also highlighted that the toxic effects of pesticide in the field are greater than laboratory. Specific impacts, such as whether all ILs exhibit these effects, still require further study. In the future, related experiments should be designed and ecotoxicity tests should be performed more in real environmental condition (other natural soils) to help understand the real danger of IL under natural conditions. At the same time, experiments should be designed to investigate what is the possible factor for the difference. 4. Conclusions The present study measured biochemical toxicity and genotoxicity in earthworms (Eisenia fetida) after exposure to [Bmim]BF4 in artificial soil and fluvo-aquic soil, and the primary conclusions were as follows: (1) [Bmim]BF4 can increase the ROS contents and cause the increase of MDA contents. (2) [Bmim]BF4 can stimulate the SOD activity and inhibit the activities of CAT, POD and GST. (3) [Bmim]BF4 above 5 mg/kg can cause DNA damage in earthworms. (4) IBR index indicated that the toxicity of [Bmim]BF4 in fluvo-aquic soil was higher than that in artificial soil when exposure to 40 mg/kg [Bmim]BF4 on day 28. (5) Combining all results, [Bmim]BF4 showed more toxicity in fluvoaquic soil to earthworms (Eisenia fetida) than that in artificial soil. (6) The authors declare no competing financial interest. Acknowledgments Funding: The present study was supported by “the National Key R&D Program of China [grant numbers: 2016YFD0800202], the National Natural Science Foundation of China under grants: 41771282 and 41701279, the Natural Science Foundation of Shandong Province, China under grants: ZR2017MD005 and ZR2017BB075, and the Special Funds of Taishan Scholar of Shandong Province, China”. All applicable international, national, and/or institutional guidelines for the care and use of earthworms were followed. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.scitotenv.2019.06.010.
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Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv
Evaluation of the toxicity of 1-butyl-3-methyl imidazolium tetrafluoroborate using earthworms (Eisenia fetida) in two soils Yuting Shao a,1, Kaixuan Hou a,1, Zhongkun Du a, Bing Li a, Jun Wang a, Albert Juhasz b, Jinhua Wang a, Lusheng Zhu a,⁎ a College of Resources and Environment, Shandong Agricultural University, Key Laboratory of Agricultural Environment in Universities of Shandong, National Engineering Laboratory for Efficient Utilization of Soil and Fertilizer Resources, 61 Daizong Road, Taian 271018, PR China b Future Industries Institute, Centre for Environmental Risk Assessment and Remediation, University of South Australia, Mawson Lakes, SA 5095, Australia
H I G H L I G H T S
G R A P H I C A L
A B S T R A C T
• Ecotoxicity of [Bmim]BF4 to earthworms in artificial and nature soil was compared. • The toxicity of [Bmim]BF4 in different soils were compared by computing IBRv2 index. • [Bmim]BF4 can influence the enzyme activities and cause DNA damage in earthworms. • [Bmim]BF4 showed greater toxicity in nature (fluvo-aquic) soil.
a r t i c l e
i n f o
Article history: Received 2 April 2019 Received in revised form 30 May 2019 Accepted 1 June 2019 Available online 06 June 2019 Editor: Jay Gan Keywords: [Bmim]BF4 Earthworm Artificial soil Fluvo-aquic soil IBR
a b s t r a c t Herein, to research the toxic effect of ionic liquids (ILs) on earthworms and compare their different toxicities in different soils, 1-butyl-3-methyl imidazolium tetrafluoroborate ([Bmim]BF4) was selected as a test substance, Eisenia fetida was selected as the experimental indicator organism, and artificial and fluvo-aquic soils were selected as the media. The acute toxicity, reactive oxygen species (ROS) content, detoxification enzyme (GST) activity, anti-oxidant enzyme activities, lipid peroxidation oxidative and DNA damage in earthworms were all measured to evaluate the toxicity of [Bmim]BF4. The results showed that either in fluvo-aquic soil or artificial soil, [Bmim]BF4 can stimulate the accumulation of ROS in earthworms, inducing activities of antioxidant enzymes and detoxification enzymes, inevitably causing lipid peroxidation and DNA damage in earthworms. The integrated biomarker response (IBR) indicated that the toxicity of [Bmim]BF4 in fluvo-aquic soil was greater than that in artificial soil. This experiment is relevant to the reliability of artificial soil toxicity research, and maybe this paper can provide a more authentic understanding of traditional toxicity experiments. © 2019 Elsevier B.V. All rights reserved.
⁎ Corresponding author at: Shandong Agricultural University, 61 Daizong Road, Taian 271018, PR China. E-mail addresses: [email protected] (Z. Du), [email protected] (J. Wang), [email protected] (A. Juhasz), [email protected] (J. Wang), [email protected] (L. Zhu). 1 Yuting Shao and Kaixuan Hou contributed equally to this work.
https://doi.org/10.1016/j.scitotenv.2019.06.010 0048-9697/© 2019 Elsevier B.V. All rights reserved.
Y. Shao et al. / Science of the Total Environment 686 (2019) 946–958
1. Introduction With the increasing requirements of environmental quality for human life, green chemistry is a key factor in achieving economic and socially sustainable development. Ionic liquids (ILs) have excellent chemical and thermodynamic stability and are largely applied in organic synthesis, isolation and purification, electro-chemistry and other fields because of the lower vapor pressure, higher thermal stability and adjustable dissolution (Studzinska et al., 2009; Made et al., 2015; Mehrkesh and Karunanithi, 2016). It is unlikely that ILs become air pollutants because of their low vapor pressure, but they can inevitably be released into water and soil through accidental spills, effluents or irrigation (Liwarska-Bizukojc, 2011). Besides, common ILs have been proven to remain stable in water and soil medium (Shao et al., 2017). Therefore, it is especially important to study the toxicity of ILs in water and soil. Nowadays, with the extensive use of ILs, there are a large number of studies on their toxicity. For aquatic organisms, Ma et al. (2010) demonstrated the toxicity of [Cnmim]Br (n = 4, 6, 8, 10, 12) to green algae, after S. obliquus was exposed to [Cnmim]Br, the 96 h-EC50 values ranged from 22.24 to 0.02 mg/L, while Ruokonen et al. (2016) highlighted the viability effect and behavioral toxicity of 11 kinds of amidinium, imidazolium, and phosphonium based ILs to Danio rerio. At present, the study on the toxicity of ILs to aquatic organisms is comprehensive relatively, while the toxicity research on terrestrial organisms is relatively rare. For terrestrial impact, earthworms (Eisenia fetida) are commonly utilized to assess the impact of soil contamination (Zhao et al., 2014; Li et al., 2015; Li et al., 2017) using Organization of Economic and Cooperation Development (OECD) (1984) methods using artificial soil. Many studies have highlighted the toxic effects of ILs on earthworms and confirmed that ILs would cause enzyme induction (Li et al., 2010; Shao et al., 2018a; Shao et al., 2019). However, the toxicity research of ILs in artificial soil may not represent the true toxicity. But few studies have been performed to compare the toxic effects of ILs between artificial soil and natural soil. In the present study, 1-butyl-3-methyl imidazolium tetrafluoroborate ([Bmim]BF4), a commonly used imidazole-based ILs, which has good electrical conductivity and biocompatibility, was used to assess its impact on Eisenia fetida in both artificial soil and natural (fluvo-aquic) soil. A variety of toxicological biomarkers were utilized including acute toxicity, the content of reactive oxygen species (ROS), anti-oxidant enzyme systems, de-toxification enzyme glutathione-S-transferase (GST), lipid peroxidation (LPO) product malondialdehyde (MDA), and DNA damage (olive tail moment (OTM) in the comet essay) to evaluate the effect of [Bmim]BF 4 on earthworms. The integrated biomarker response (IBR) calculated with biomarker responses could be used to evaluate the ecological risk of the polluted area (Wang et al., 2011). Because of the shortcomings of IBR, IBR was improved to IBR version 2 (IBRv2) (Sanchez et al., 2013). Subsequently, IBRv2 was widely used to assess the environmental quality (Estefanía et al., 2018; Wan et al., 2018). In this study, the IBR index was computed to evaluate the toxicity to earthworms of [Bmim]BF 4 in artificial soil and fluvo-aquic soil. It was hypothesized that Eisenia fetida's toxicological response to [Bmim]BF4 would vary between artificial and natural soil due to the influence of soil properties on IL bioavailability.
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Healthy adult earthworms (Eisenia fetida), weighing approximately 300 to 600 mg, were sourced from Shandong Agricultural University breeding base. Eisenia fetida were adapted to lab conditions for 14 days prior to toxicity testing. The artificial soil used for this study was prepared according to the OECD (1984) guidelines. Briefly, the soil contained plant ash (10%), kaolin clay (20%) and quartz sand (70%), and the pH was adjusted to 6.0 ± 0.5 by CaCO3. The natural soil used in this study was a fluvo-aquic soil, collected from alluvial sediments of the Yellow River, Shandong Province, China. The basic information of the two soils are detailed in supplementary material. (Table S1). 2.2. Methods 2.2.1. IL soil preparation [Bmim]BF4 was added to artificial and fluvo-aquic soils to achieve concentrations ranging from 5 to 1000 mg/kg. Briefly, 500 g of airdried soil was thoroughly mixed with aqueous solutions of [Bmim]BF4 to achieve the desired concentration and a water holding capacity of 60%. Earthworms were pre-cultured for 1 d in non-toxic soils before experiments. 2.2.2. Acute toxicity test For acute toxicity tests, [Bmim]BF4 in artificial and fluvo-aquic soil was assessed at 0, 200, 400, 500, 600, 800, and 1000 mg/kg. Assays were conducted in triplicate (3 × 500 g) with each soil containing ten earthworms. All treatments were maintained in a climate chamber (20 °C, 12 h Light: 12 h Dark) with survival monitored over a 14-day exposure period. In this study, the earthworms' mortality of controls was 0, and the earthworms' mortality of the highest concentrations was N90%. 2.2.3. Subchronic toxicity test In the subchronic toxicity test, the concentrations of [Bmim]BF4 were set to 5, 10, 20 and 40 mg/kg. As with acute toxicity tests, assays were conducted in triplicate, however, fifteen earthworms were included in each treatment. All treatments were maintained in a climate chamber (20 °C, 12 h Light: 12 h Dark) and on days 7, 14, 21 and 28, earthworms were randomly selected (3 per treatment) for the assessment of subchronic toxicity indicators (ROS, antioxidant enzymes, DNA damage). During the 28 d, the earthworms' mortality of controls was 0. 2.2.4. Assessment of protein concentration Protein content of earthworms was determined in the light of Bradford (1976), with bovine serum albumin (BSA) used to construct standard curves. 2.2.5. Assessment of ROS The 2′,7′- dichlorohydro fluorescein diacetate (DCFH-DA) method used in this study is commonly utilized for the determination of ROS content (Zhang et al., 2013). DCFH-DA, an oxygen-sensitive fluorescent probe, can react with ROS thereby allowing its quantification. Earthworms were previously depurated for 12 h, after which they were washed and homogenized in 0.1 mol/L phosphate buffer saline (PBS).
2. Materials and methods 2.1. Materials [Bmim]BF4 (CAS: no. 174501-65-6, purity above 99.0%) was purchased from the Cheng Jie Chemical Co., Ltd. (Shanghai, China). All other reagents used in this study were analytical grade constituents and bought from Sigma Chemical Co. (St. Louis, MO, USA).
Table 1 Acute toxicity of [Bmim]BF4 on earthworm (Eisenia fetida) in artificial soil and fluvo-aquic soil.
Artificial soil Fluvo-aquic soil a
7 d-LC50 (mg/kg)
14 d-LC50 (mg/kg)
870 (750–1089)a 744 (678–835)a
678 (617–746)a 489 (439–541)a
Values in parenthesis represent the 95% confidence intervals of the mean.
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ROS level (Fluorescence Intensity)
c
c
c
c
1000 b
b
b
b a
a 800
0
5
10
20
1200
40
0
5
10
20
40
d c
c 1000
b
b
ab
b a
a
800
600
C
Fluvo-aquic soil
1200
600
B
Artificial soil
1400 ROS level (Fluorescence Intensity)
A
0
5
10
20
40
0
ab
5
10
20
40
ROS level (Fluorescence Intensity)
1200 1100 1000
b a
b
d
a c
900
bc
800 a
ab
700 0
D
b
5
10
20
40
0
5
10
20
40
ROS level (Fluorescence Intensity)
1500 d
1400 1300
c
1200 1100
c b
1000 900
a
b
ab
a
a
800
a
700 600
0
5
10
20
40
0
5
10
20
40
Concentration (mg/kg) Fig. 1. The ROS level of earthworms effected by [Bmim]BF4 after 7 (A), 14 (B), 21 (C), 28 (D) days in artificial and fluvo-aquic soil at diverse concentrations. The colors (black, red, blue, pink and green) mean the concentration of [Bmim]BF4 (0, 5, 10, 20 and 40 mg/kg on the X axis). The line and little box within the box plot indicate the median and mean values. The boundaries of the box indicate the means ± standard error (SE). The whiskers indicate minimum and maximum. Least significant difference test was adopted with a significance of p b 0.05 between control and exposure treatments shown by a, b, c etc. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
The homogenate was centrifuged (20,000 ×g, 20 min, 4 °C) and resuspended to extract mitochondria for ROS assessment. Mitochondrial extracts were mixed with DCFH-DA at 37 °C for 20 min, then the reaction was terminated by 1 mol/L HCl and the OD value was read at 538 nm under an excitation wavelength of 485 nm by a fluorescence spectrophotometer.
2.2.6. Assessment of antioxidant enzymes, GST activities and MDA content Following depuration, earthworms were washed and then homogenized in PBS (10 times the body weight). The homogenate was centrifuged (10,000 rpm, 15 min, 4 °C) and resuspended prior to the assessment of antioxidant enzyme and de-toxification enzyme activities and MDA content.
Y. Shao et al. / Science of the Total Environment 686 (2019) 946–958
Artificial soil
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Fluvo-aquic soil
A 4.5
SOD activity(U/mg pr)
b 4.0
a
ab
b
d e
ab c b
3.5 a 3.0
2.5
0
5
10
20
40
0
5
10
20
40
b
b
20
40
3.6
B SOD activity(U/mg pr)
3.4 3.2
b
ab ab
a
a
a
3.0
b
c 2.8 2.6 0
5
10
20
40
0
5
10
4.5
C SOD activity(U/mg pr)
c b
4.0
b
b
10
20
40
a
ab
20
40
b c c
a a
3.5 a 3.0
2.5
0
5
10
20
40
0
5
4.0
D SOD activity(U/mg pr)
c 3.5 b
d 3.0
b
e
ab a
a 2.5
2.0
0
5
10
20
40
0
5
10
Concentration (mg/kg) Fig. 2. The SOD activity of Eisenia fetida exposed to [Bmim]BF4 after 7 (A), 14 (B), 21 (C), 28 (D) days in artificial soil and fluvo-aquic soil at diverse concentrations. The colors (black, red, blue, pink and green) mean the concentration of [Bmim]BF4 (0, 5, 10, 20 and 40 mg/kg). The line and little box within the box plot indicate the median and mean values. The boundaries of the box indicate the means ± standard error (SE). The whiskers indicate minimum and maximum. Least significant difference test was adopted with a significance of p b 0.05 between control and exposure treatments shown by a, b, c etc. Pr, protein. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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A
Artificial soil
CAT activity(U/mg pr)
7
B
6
a
a
0
5
a
Fluvo-aquic soil b
b
a
20
40
0
a
a
a
a
5
10
20
40
a
a
20
40
5
4
10
11 a
CAT activity(U/mg pr)
10 9 a
8 a
7 6
a
a
0
5
10
a
a
a
a
a a
5 4 3 2
CAT activity(U/mg pr)
C 6
20
40
0
a
a
a
5
10
a
a
a a
4
2 0
5
10
20
40
0
5
10
a
a
20
40
8
D
a
CAT activity(U/mg pr)
ab 7 a 6
ab
ab
ab
b b
5
4 0
5
10
20 40 0 Concentration (mg/kg)
5
10
20
40
Fig. 3. The CAT activity of earthworms exposed to [Bmim]BF4 after 7 (A), 14 (B), 21 (C), 28 (D) days in artificial soil and fluvo-aquic soil at diverse concentrations. The colors (black, red, blue, pink and green) mean the concentration of [Bmim]BF4 (0, 5, 10, 20 and 40 mg/kg). The line and little box within the box plot indicate the median and mean values. The boundaries of the box indicate the means ± standard error (SE). The whiskers indicate minimum and maximum. Least significant difference test was adopted with a significance of p b 0.05 between control and exposure treatments shown by a, b, c etc. Pr, protein. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Y. Shao et al. / Science of the Total Environment 686 (2019) 946–958
A
Fluvo-aquic soil
Artificial soil
1.0
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POD activity (U/mg pr)
0.9 c
0.8 0.7 0.6
b
ab
a
a
10
20
40
ab
ab
ab
10
20
40
bc
c
20
40
a
a
a
0
5
ab
a
0.5 0.4 0.3 0.2
B
0
5
10
20
40
POD activity (U/mg pr)
0.8 0.7
b
a
a
a a
0.6
a
a
0.5 0.4 0.3
0
5
10
20
40
0
5
C POD activity (U/mg pr)
0.8 b
a
a
ab ab
c
0.6
c c
0.4
0
POD activity (U/mg pr)
D
5
10
20
40
0
5
a
ab
10
0.8 0.7
a a
ab
a a
ab
a
b
0.6 0.5 0.4 0.3
0
5
10
20 40 0 Concentration (mg/kg)
5
10
20
40
Fig. 4. The POD activity of Eisenia fetida exposed to [Bmim]BF4 after 7 (A), 14 (B), 21 (C), 28 (D) days in artificial soil and fluvo-aquic soil at diverse concentrations. The colors (black, red, blue, pink and green) mean the concentration of [Bmim]BF4 (0, 5, 10, 20 and 40 mg/kg). The line and little box within the box plot indicate the median and mean values. The boundaries of the box indicate the means ± standard error (SE). The whiskers indicate minimum and maximum. Least significant difference test was adopted with a significance of p b 0.05 between control and exposure treatments shown by a, b, c etc. Each trial contained three replicates. Pr, protein. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Superoxide dismutase (SOD) activity was assessed according to Han et al. (2014), where an enzyme activity unit (U) expresses the amount of SOD required to inhibit the chemical reduction of 1/2 added nitrogen blue tetrazole (NBT). Enzyme extracts (50 μL, PBS for control) were
mixed with PBS, methionine, NBT, Na2-EDTA, riboflavin and deionized water (totally 3 mL) and after 30 min, the UV absorption was measured at 560 nm. One control tube was selected randomly and placed in the dark, while the others were reacted under a 4000 lx fluorescent lamp.
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Artificial soil
Fluvo-aquic soil
210
A GST activity(nmol/min/pr)
200 190
b
c
180 170 160
ab
a
bc
bc
b
ab
c
a
150 140 130 120 110 100 90
0
5
10
20
40
0
5
a
a
a
10
20
40
180
B GST activity(nmol/min/pr)
170 a
160 150
a
a
a
a
b c
140 130 120 110 100 90 80
C
0
5
10
20
40
0
10
20
40
GST activity(nmol/min/pr)
170 a 160
a ab
150
ab
ab ab
b
b
c
140
d
130 120 110 0
5
10
20
40
0
5
10
20
40
170
D
a
GST activity(nmol/min/pr)
160 150
a
b b
140
bc
b bc
bc
c
130 120
c
110 100 90 80
0
5
10
20
40
0
5
10
20
40
Concentration (mg/kg) Fig. 5. The GST activities of earthworms effected by [Bmim]BF4 after 7 (A), 14 (B), 21 (C), 28 (D) days exposure in artificial soil and fluvo-aquic soil at diverse concentrations. The colors (black, red, blue, pink and green) mean the concentration of [Bmim]BF4 (0, 5, 10, 20 and 40 mg/kg). The line and little box within the box plot indicate the median and mean values. The boundaries of the box indicate the means ± standard error (SE). The whiskers indicate minimum and maximum. Least significant difference test was adopted with a significance of p b 0.05 between control and exposure treatments shown by a, b, c etc. Pr, protein. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Catalase (CAT) activity was also assessed in the light of Han et al. (2014), where an enzyme activity unit (U) expresses the amount of enzyme required to decompose 50% hydrogen peroxide (H2O2). Enzyme extracts (10 μL) were mixed with PBS (3 mL) as the reference, or mixed with PBS and H2O2 (3 mL) as experimental group, after which the dynamics of UV (250 nm) was monitored every 5 s for 1 min. In the light of Han et al. (2014), POD (guaiacol peroxidase) activity was assessed. In this study, reaction solution (3 mL, including PBS, guaiacol, and hydrogen H2O2) was mixed with PBS (20 μL) as the reference or with enzyme extracts (20 μL) as experimental group, after which the dynamics of UV (470 nm) was monitored every 30 s for 3 min. According to Han et al. (2014), GST activity was assessed, as reduced glutathione (GSH) can bind with 1-chloro-2,4-dinitro-benzene. The homogenization buffer contained Na2HPO4, NAH2PO4, glycerin, phenylmethylsulfonyl fluoride, EDTA, and dithiothreitol. Enzyme extraction was combined with CNDB, glutathione and buffer solution and the dynamics of UV (340 nm) was monitored every 30 s for 3 min. Determination of MDA content was performed according to Zhang et al. (2013). Enzyme extracts (PBS for control), sodium dodecyl sulfonate, acetic acid, TBA and water were combined sequentially, and incubated at 90 °C (water bath). After 1 h, the absorbance (532 nm) was monitored. 2.2.7. Assessment of DNA damage DNA damage was assessed by the single cell gel electrophoresis (SCGE) experiment according to Song et al. (2009). Following depuration, earthworms were soaked in physiological saline, and then immersed in extracting solution (containing anhydrous ethanol, normal saline, ethylenediaminetetraacetic acid (EDTA), guaiacol glyceryl ether) to extract coelomocytes. Extracts were centrifuged (3000 ×g, 15 min, 4 °C) and resuspended in PBS prior to use. Electrophoretic sheets were made by preparing three gel layers, composed of normal melting agar (NMA), a mixture of extraction and low melting agar (LMA), and LMA, whereby gel layers were attached to the surface of glass slides. Slides were put into cell-lysis solution for 60 min, placed in electrophoretic solution (NaOH and Na2EDTA) for 30 min. After which slides were electrophoresed (300 mA, 25 V) and neutralized. Finally, slides were dehydrated in ethanol and stored in a humid environment prior to use. Slides were stained with EB and viewed under the fluorescence inverted microscope (Olympus BX51). At least one hundred cells at each concentration were analyzed by the comet assay software project (CASP) to obtain the OTM, which reflects cell damage.
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toxicity of [Bmim]BF4 to earthworms in fluvo-aquic soil is more severely than that in artificial soil. When earthworms were exposed to low-dose ILs (taking the possible concentrations in the soil into account, the dose was set as: 5, 10, 20, 40 mg/kg) for a long time (28 days), the results of the comparison of toxicity between different soils were shown below. 3.2. ROS level A substance that promotes oxidative stress in earthworms is ROS (Dayem et al., 2017), which influences the generation, cross-linking, single-strand breaks, base alkylation and oxidation of pyridine dimers and the deletions of DNA bases (Tuteja et al., 2001). ROS content in earthworms following exposure to [Bmim]BF4 over a 28-day period in different soils was presented in Fig. 1. After the initial exposure of earthworms to [Bmim]BF4 in artificial soil (7 days), ROS levels were significantly higher compared to earthworms exposed to non-amended soil. On day 7, there appeared to be a linear increase in fluorescence intensity with increasing [Bmim]BF4 up to 20 mg/kg. At higher concentrations (i.e. 40 mg/kg), there was no significant difference in ROS level compared to an exposure dose of 20 mg/kg. A similar result was observed for the fluvo-aquic soil spiked with [Bmim]BF4. However, on day 28, the disparity between ROS response in the artificial control (unexposed) group and [Bmim]BF4 spiked soil group decreased. The 10 mg/kg and 40 mg/kg group resulted in a ROS response that was significantly higher than unexposed earthworms; the remaining [Bmim]BF4 exposure concentrations (5 and 20 mg/kg) produced a ROS response that was similar to the control group. Within contrast, for earthworms exposed to [Bmim]BF4 in the fluvo-aquic soil, ROS levels at day 28 were significantly higher compared to those in the unexposed group. Xu et al. (2018) researched the toxic impacts of ILs [C8mim]R (R = Cl−, Br−, BF−4) on wheat seedings in brown soil, then the ROS levels accumulated and formed concentration dependent. Guo et al. (2015) researched the toxic impacts of ILs [C8mim]Cl on Eisenia fetida (the same concentrations as this study) and in their study, the ROS levels in earthworms also showed the concentration dependent. Shao et al. (2019) researched the toxicity of CnBr to Eisenia fetida and a similar conclusion was obtained. They all proved that ILs can affect the biobalance and stimulate the accumulation of ROS in organisms. Although the ROS accumulation was observed in both soils, the change is not exactly the same, and the ROS levels in earthworms in fluvo-aquic soil were higher than that in artificial soil especially in the second half of the experiment.
2.3. Statistical analyses
3.3. Antioxidant enzyme activities
All experiments were performed in triplicate with data in this study analyzed by SPSS 20.0 and plotted by Origin 8.5. In the figures, each bar depicts the mean of replicates and error bars depict the standard deviations (SD). Different letters in the figures reflect significant difference (p b 0.05) between the treatments. The figures of treatment effect ratio of all biomarkers are presented in SI (Figs. S1–S7). Data of all biomarker responses of earthworms (Eisenia fetida) after 28 days exposure to [Bmim]BF4 under 40 mg/kg was selected to calculate IBR index according to the method of Sanchez et al. (2013). Detail of calculation process was listed in supplementary material.
The enzymes that remove ROS include SOD, CAT, and POD. SOD eliminates superoxide radicals, by converting O−2 to H2O2 while CAT and POD are anti-oxidant enzymes that eliminate H2O2 (Qu et al., 2010); CAT catalyzes H2O2 to produce H2O (Wu et al., 2011) while POD oxidizes cosubstrates (such as guaiacol or ascorbate) to decompose H2O2. The above enzymes can interact and then reflect the degree of oxidative stress and thus the toxicity of [Bmim]BF4 to earthworms. SOD activity in earthworms following exposure to [Bmim]BF4 over a 28-day period was displayed in Fig. 2. After the initial exposure of earthworms to [Bmim]BF4 in artificial soil (7 days), the SOD activities of all treated groups were significantly higher than that of the control. On day 14, all treatment groups returned to the control level (except the 20 mg/kg treated group). On day 21, the SOD activities were re-activated in all treated groups and higher than that of control, but the SOD activities in the high concentration treated groups (20, 40 mg/kg) were lower than others. On day 28, the SOD activities for all concentrations were still higher than those in the control, but they showed a tendency to first be activated and then inhibited as the concentration increased. Meanwhile, after the earthworms
3. Results and discussion 3.1. Acute toxicity Eisenia fetida acute toxicity test results are shown in Table 1. In artificial soil, the 7 d-LC50 was 870 mg/kg compared to 744 mg/kg in fluvoaquic soil. Similarly, the 14 d-LC50 was obviously higher in artificial soil (678 mg/kg) compared to the in fluvo-aquic soil (489 mg/kg). The acute
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Artificial soil
MDA content (nmol/mg pr)
A
Fluvo-aquic soil
3.0 c
a
bc
2.8 ab ab
2.6 a a
a a
a
2.4
2.2
MDA content (nmol/mg pr)
B
0
5
10
20
40
0
5
10
20
40
3.0 2.8
d
2.6
c
c
d
2.4 2.2
a
b
ab
b a
ab
0
5
2.0 1.8 1.6 0
MDA content (nmol/mg pr)
C
5
10
20
40
10
20
40
3.4 c
3.2 3.0
b
b
2.8 2.6 2.4 2.2
b
ab
5
10
c
c
20
40
a
a
0
5
a
2.0 0
MDA content (nmol/mg pr)
D
10
20
2.8
40
d d c
2.6 b
ab
c
b
b
5
10
2.4 a
a 2.2
2.0
0
5
10
20 40 0 Concentration (mg/kg)
20
40
Fig. 6. The MDA contents of earthworms exposed to [Bmim]BF4 after 7 (A), 14 (B), 21 (C), 28 (D) days in artificial soil and fluvo-aquic soil at diverse concentrations. The colors (black, red, blue, pink and green) mean the concentration of [Bmim]BF4 (0, 5, 10, 20 and 40 mg/kg). The line and little box within the box plot indicate the median and mean values. The boundaries of the box indicate the means ± standard error (SE). The whiskers indicate minimum and maximum. Least significant difference test was adopted with a significance of p b 0.05 between control and exposure treatments shown by a, b, c etc. Pr, protein. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
contacted [Bmim]BF4 in the fluvo-aquic soil, the SOD activity of all treatment groups was activated on day 7. After that (on day 14), only the 10 mg/kg treatment group maintained a level equal to control, and the SOD activity of the remaining three concentrations treatment
groups was inhibited. On day 21, similar with the artificial soil, the SOD activities were re-activated in all treatment groups. On day 28, no treatment groups differed significantly from the control except the 10 mg/kg treatment group.
Y. Shao et al. / Science of the Total Environment 686 (2019) 946–958
A Olive Tail Moment (OTM)
Fluvo-aquic soil
Artificial soil
1.0
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d
c 0.5
d
c b
b
b
c
10
20
b
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Concentration (mg/kg) Fig. 7. The OTM (Olive Tail Moment) value of earthworms exposed to [Bmim]BF4 after 7 (A), 14 (B), 21 (C), 28 (D) days in artificial soil and fluvo-aquic soil at diverse concentrations. The colors (black, red, blue, pink and green) mean the concentration of [Bmim]BF4 (0, 5, 10, 20 and 40 mg/kg). The line and little box within the box plot indicate the median and mean values. The boundaries of the box indicate the 25/75th percentiles. The whiskers indicate minimum and maximum. Least significant difference test was adopted with a significance of p b 0.05 between control and exposure treatments shown by a, b, c etc. Each group contained at least 10 values. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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When Xiong et al. (2013) studied the toxic impacts of copper sulfate on Eisenia fetida, and Duan et al. (2015) studied the toxic impacts of benzo[a]pyrene on Eisenia fetida, they all observed that the SOD activities in poisoned earthworms were first activated and then inhibited. The SOD activities were activated first, because excessive ROS induced the synthesis of anti-oxidase or activated SOD as a mechanism to resist oxidative stress. Then, the SOD activity decreased because the removal of ROS consumed some SOD. However, as the exposure time prolonged, increasing amounts of ROS forced the earthworms to activate more SOD to counter the impact, and SOD activity thus increased again. Ultimately, the SOD activity showed inhibition at higher concentrations, possibly because earthworms had higher ROS levels at higher concentrations (and more SOD was thus consumed) or because the ROS accumulated beyond the catalytic ability of SOD and inhibited the SOD activity. For these two soils, the SOD activity in earthworms in fluvo-aquic soil had a more obvious response. CAT activity in earthworms following exposure to [Bmim]BF4 over a 28-day period was presented in Fig. 3. After 7 d exposure to [Bmim]BF4 in artificial soil, the CAT activity in all earthworms was activated, and significant difference was observed only in the 20 and 40 mg/kg treatment groups. However, on day 14 and day 21, the activation effect disappeared, and the levels in all the treatment groups returned to control level. On day 28, all treatment groups showed a significant effect in artificial soil and an inapparent inhibitory effect in fluvo-aquic soil. POD activity following exposure to [Bmim]BF4 over a 28-day period was presented in Fig. 4. After 7 d exposure to [Bmim]BF4 in artificial soil, the POD activity in earthworms of all treatment groups was significantly higher than control, and the highest concentration treatment group had the biggest growth. On day 14, the POD activities were no longer activated. On day 21, the POD activities were decreased, while a significant activation was observed in the lowest concentration treatment group. On day 28, all the treatment groups were slightly inhibited. Meanwhile, after earthworms contacted [Bmim]BF4 in the fluvo-aquic soil, the POD activity was unchanged on day 7. On day 14, the POD activities were little activated. On day 21, the POD activity of all the treated groups was significantly inhibited, which lasted until day 28. CAT and POD, two enzymes with the same function, collectively remove excess H2O2 in earthworms. In previous studies, Du et al. (2014) researched the toxic effect of ILs [C8mim]Br in Danio rerio; under the induction of ILs, the CAT activity in zebrafish was first activated and then gradually inhibited to reduce the damage. Duan et al. (2015) researched the toxic impacts of benzo[a]pyrene on Eisenia fetida and confirmed that
the POD activities in earthworms were first activated and then gradually inhibited as the earthworms were poisoned. Similarly, in this study, H2O2 accumulated under the action of SOD, and both CAT and POD were thus activated to eliminate the effects of H2O2. After that, due to the different catalytic effects of CAT and POD, CAT disproportionated H2O2 directly, while POD catalyzed the oxidation of H2O2 with another substrate (Vidossich et al., 2012). Thus, CAT dominated the reaction and still maintained the activated state, but POD was gradually returned to the control's level on day 14. Beginning on day 21, with the prolonged exposure time, oxidative stress increased, and more O−2 and H2O2 accumulated in the earthworms. The adaptability and health of the earthworms were thus reduced, leading to toxic reactions that decreased the activities of CAT and POD; these changes were more obvious in the higher concentration groups. The inhibitory effect of enzymes in fluvo-aquic soil was more obvious. 3.4. Detoxification enzyme activities GST is an important metabolic detoxification enzyme which catalyzes the activity of reduced glutathione (GSH) to react with contaminants and increases the water solubility of toxicants which facilitates their elimination/excretion. GST also catalyzes the binding of GSH to electrophilic intermediate metabolites and reduces the possibility of such compounds to react with intracellular biological macromolecules, thus maintaining genomic integrity of cells (LaCourse et al., 2009). GST activity following exposure to [Bmim]BF4 over a 28-day period in earthworms was presented in Fig. 5. After 7 d exposure to [Bmim]BF4 in artificial soil, GST activities in earthworms were activated in all treatment groups, and the higher concentration treated groups showed the greater differences. On day 14, the activation was diminished, and the levels restored to the level of control. On days 21 and 28, the GST activity began to be inhibited, and all treatment groups showed significant differences on day 21. Meanwhile, when earthworms contacted [Bmim]BF4 in the fluvo-aquic soil, on day 7, GST activity was also activated and showed a concentrationeffect relationship. On day 14, the GST activity began to be inhibited in the 20 and 40 mg/kg treatment groups. After days 21 and 28, the inhibition was strengthened, all treatment groups were inhibited, and more obvious inhibitory effects were observed with increasing concentrations. In this study, the GST activities in earthworms increased first and then decreased. Lin et al. (2012) studied the physiological and molecular responses of Eisenia fetida to chlortetracycline, and Shao et al. (2012) studied the oxidative stress induced by endosulfan in zebrafish. Though
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the organisms were exposed to different contaminants and concentrations, they both observed that the GST activity in organisms increased first and then decreased. The GST activity increased first, possibly as an adaptive response of earthworms, and the enhanced GST detoxification effect helped to reduce the damage caused by ILs. After that, the detoxification process produced large amounts of intermediate metabolites, perhaps altering the composition of the GST subunit and reducing the GST activity. At the same time, GST was consumed as the substrate (Shao et al., 2018b,). Shao et al. (2019) studied oxidative stress and genotoxic effects in earthworms induced by five imidazolium bromide ILs with different alkyl chains. They find that, GST activity was stimulated at lower concentrations but inhibited at higher concentrations particularly after 28 days exposure to [C10mim]Br. Therefore, the GST activity was significantly inhibited in the second half of the experiment. In these two soils, the inhibitory effect of GST in the fluvo-aquic soil occurred earlier. This means that earthworms in fluvo-aquic soil were more seriously damaged. 3.5. Oxidative damage Exposure to [Bmim]BF4may result in the generation of ROS, enzyme stress and oxidative damage (such as LPO and DNA damage) (Shi and Zhou, 2010). [Bmim]BF4 exposure may lead to the accumulation of free radicals, which may oxidize unsaturated fatty acids, damaging the structure and function of biological membranes. As LPO may result in the formation of MDA, the intensity of LPO damage and the extent of oxidative damage can be inferred from changes in MDA content (Gill and Tuteja, 2010). MDA content in earthworms was shown in Fig. 6. After 7 d exposure to [Bmim]BF4 in the artificial soil, there was no significant change in the MDA content in any of the treatment groups. On day 14, the MDA content was significantly increased. On days 21 and 28, the MDA content in all treated groups was significantly increased, and the degree of the effect gradually increased. Which may be due to the combined effects of ROS and GST. As IL toxicity increased, ROS content increased and antioxidant enzymes were inhibited, causing significant increase in MDA content. Guo et al. (2015) researched the toxic effect of [C8mim]Cl on Eisenia fetida, Shao et al. (2018a) researched the toxicity of ILs ([C8mim]Br and [C8mim]BF4) on Eisenia fetida. The concentration they set was same as this study, and their experiments showed that ILs can induce serious LPO in earthworms, same as this study. The MDA content increased as the exposure time and dose increased. It can be speculated that the degree of LPO intensified. The LPO in fluvo-aquic soil was more obvious. [Bmim]BF4 may also cause DNA damage whereby ROS reacts with DNA molecules directly, resulting in DNA spiral structure damage. In addition, ROS reacts with LPO products and intermediate alkyl radicals, indirectly exacerbating further damage to DNA (Possalmai et al., 2007). OTM value in earthworms was shown in Fig. 7. After earthworms contacted [Bmim]BF4 in the artificial and the fluvo-aquic soil, the OTM value of nearly all these treatment groups was higher than control, and this value increased gradually as the dose and exposure time increasing. Dong et al. (2013) studied the DNA damage by [C10mim]Br in zebrafish and showed that ILs can continuously induce DNA damage. Shao et al. (2018a) also proved that [Omim]BF4 and [Omim]Br can cause DNA damage in Eisenia fetida and observed nearly a linear increase in OTM value with increasing concentration. In the present study, the DNA in earthworms was damaged, and the OTM value increased as the increasing dose and exposure times. Zhang et al. (2018) and Ma et al. (2019) verified the same results when studied the DNA damage by fluoxastrobin and pyraclostrobin respectively and they even find that the comet assay was the most sensitive biomarkers. Besides, DNA damage in the fluvo-aquic soil was more obvious than that in the artificial soil.
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3.6. Toxicity between the two soils As an integrated index, IBR stands for toxicity level of [Bmim]BF4. The IBR index histogram and a star plot under 40 mg/kg concentration on day 28 were shown in Fig. 8. The star plot including all biomarkers, the blue circle can be seen as the base line. Indices inside base line mean inhibited, while indices outside the base line mean activated. On day 28, under 40 mg/kg concentration, the IBR index in fluvoaquic (21.61) soil was higher than that in artificial soil (19.90), which indicates that [Bmim]BF4 can cause severer toxicity in fluvo-aquic soil. The responses of biomarkers of the star plot indicate that the toxicity effects were the increase of ROS and MDA contents, the inhibition of CAT, POD and GST activity, and the damage of DNA. Among all selected indicators, the bigger contribution indicators to IL toxicity were ROS and OTM. Combining the acute and subchronic experimental results, we can predict that [Bmim]BF4 showed greater toxicity in fluvo-aquic soil. Artificial soil experiments cannot fully represent the toxicity of ILs. This may be due to the fact that natural soils can introduce all sorts of variables to the tests, such as the soil organic matter, pH, cation exchange capacity (CEC) and ratio of organic carbon to total nitrogen (C:N), resulting in toxic differences, the most possibility is that increasing organic matter leads to decreasing toxicity (Amorim et al., 2005; Stepnowski et al., 2007). Schnug et al. (2014) also highlighted that the toxic effects of pesticide in the field are greater than laboratory. Specific impacts, such as whether all ILs exhibit these effects, still require further study. In the future, related experiments should be designed and ecotoxicity tests should be performed more in real environmental condition (other natural soils) to help understand the real danger of IL under natural conditions. At the same time, experiments should be designed to investigate what is the possible factor for the difference. 4. Conclusions The present study measured biochemical toxicity and genotoxicity in earthworms (Eisenia fetida) after exposure to [Bmim]BF4 in artificial soil and fluvo-aquic soil, and the primary conclusions were as follows: (1) [Bmim]BF4 can increase the ROS contents and cause the increase of MDA contents. (2) [Bmim]BF4 can stimulate the SOD activity and inhibit the activities of CAT, POD and GST. (3) [Bmim]BF4 above 5 mg/kg can cause DNA damage in earthworms. (4) IBR index indicated that the toxicity of [Bmim]BF4 in fluvo-aquic soil was higher than that in artificial soil when exposure to 40 mg/kg [Bmim]BF4 on day 28. (5) Combining all results, [Bmim]BF4 showed more toxicity in fluvoaquic soil to earthworms (Eisenia fetida) than that in artificial soil. (6) The authors declare no competing financial interest. Acknowledgments Funding: The present study was supported by “the National Key R&D Program of China [grant numbers: 2016YFD0800202], the National Natural Science Foundation of China under grants: 41771282 and 41701279, the Natural Science Foundation of Shandong Province, China under grants: ZR2017MD005 and ZR2017BB075, and the Special Funds of Taishan Scholar of Shandong Province, China”. All applicable international, national, and/or institutional guidelines for the care and use of earthworms were followed. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.scitotenv.2019.06.010.
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