Bull Environ Contam Toxicol (2014) 93:452–455 DOI 10.1007/s00128-014-1345-z
Toxicological Responses of the Earthworm Eisenia fetida to 18-Crown-6 Under Laboratory Conditions Yongtao Du • Pinhua Rao • Yinsheng Li • Jiangping Qiu • Weiguo Qiu • Hao Tang • Murray A. Potter
Received: 25 January 2014 / Accepted: 30 July 2014 / Published online: 7 August 2014 Ó Springer Science+Business Media New York 2014
Abstract The earthworm Eisenia fetida was exposed to artificial soil supplemented with 18-crown-6 (1,4,7,10,13,16hexaoxacyclooctadecane) to investigate its effects on earthworm mortality, growth, avoidance, burrowing behavior and respiration. The results revealed that 18-crown-6 had the potential to negatively affect the behavior of earthworms. The 7-d LC50 was 585 mg kg-1 soil. Avoidance behavior was the most sensitive endpoint, with a 48-h EC50 of 120 mg kg-1 soil. Growth, burrow length and respiration showed general decreases with increasing 18-crown-6 concentrations. Behavioral endpoints and respiration may be regarded as sensitive parameters in evaluating the toxicity of this chemical to earthworms. Keywords 18-crown-6 Burrowing behavior Respiration Avoidance Mortality 18-Crown-6 (1,4,7,10,13,16-hexaoxacyclooctadecane) is an artificial macrocyclic poly-ether. The synthesis of macrocyclic poly-ethers was first described by Pedersen (1967) who coined the name ‘‘crown ether’’ for these compounds. Structurally, crown ethers possess a hydrophobic ring
Y. Du P. Rao (&) School of Chemistry and Chemical Engineering, Shanghai University of Engineering Science, Longteng Rd. 333, Songjiang District, Shanghai 201620, China e-mail: [email protected] Y. Du Y. Li J. Qiu W. Qiu H. Tang School of Agriculture and Biology, Shanghai Jiaotong University, Shanghai 200240, China M. A. Potter Institute of Agriculture and Environment, Massey University, Private Bag 11-222, Palmerston North 4442, New Zealand
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surrounding a hydrophilic cavity that enables them to form a stable complex with metal ions and sulfonamides. Crown ethers have been used as ion-chelating resins, in ion-selective electrodes, in promoting phase transfer reactions in binary solvent systems, and in biological systems as pharmacological and antibacterial agents (Arenaz et al. 1989). In short, crown ethers have a variety of applications in diverse fields and thus might readily be released into the environment. Pedersen (1967) was the first to report toxicity data on crown ethers. Since then, some studies have reported physiological effects of crown ethers. Leong et al. (1974) described toxic effects of the inhalation of 12-crown-4 on rats; and several articles have reported the toxicity of crown ethers on mice (Hendrixson et al. 1978) and rabbits (Gad et al. 1978). Arenaz et al. (1992) found that crown ethers were not significantly genotoxic in mammalian cells despite their cytotoxicity. However, to our knowledge, no studies have examined their toxicological impacts on earthworms in soil. Earthworms are common soil organisms that can be found in most environments and play a crucial role in controlling substance cycling and energy transformation in terrestrial ecosystems (Hong and James 2009). They are thus considered by the Organization for Economic Cooperation and Development (OECD 1984) to be suitable bioindicators of soil pollution. Here, we exposed the earthworm Eisenia fetida to artificial soil contaminated with 18-crown-6. Mortality, growth, avoidance, burrowing behavior and respiration were investigated. The purpose was to obtain a more comprehensive understanding of the effects of 18-crown6 on earthworms and to provide more information about the potential ecological risks of 18-crown-6 on soil ecosystems.
Bull Environ Contam Toxicol (2014) 93:452–455
Materials and Methods We used OECD artificial soil (OECD 1984) comprised of 10 % sphagnum peat, 20 % kaolinite and 70 % quartz sand. A small amount of calcium carbonate was added to adjust the pH to 6.0 ± 0.5. The moisture content was adjusted to approximately 30 %. 18-Crown-6 (99 % purity) was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Adult earthworms that possessed a clitellum and had an individual wet weight of between 300 and 450 mg were selected for testing. An acute toxicity test was used in accordance with OECD guidelines (OECD 1984). A range of concentrations were used in pre-trials to determine the suitable concentrations that produced 0 %–100 % mortality. Based on those results, 0, 200, 300, 400, 500, 600, 700, 800, 900 and 1,000 mg 18-crown-6 kg-1 dry soil were selected for the acute toxicity tests to determine the 50 % mortality concentration (i.e., LC50). Each container held ten adult earthworms and 300 g dry soil. The containers were held at a constant temperature (20 ± 1°C). After 7 days, earthworms were sorted by hand and mortality rates were determined. Each concentration was replicated five times. The avoidance tests were conducted in plastic containers based on ISO (2005). One half of the container was filled with 300 g dry contaminated soil; the other was filled with the same amount of control soil. Five replicates were used for each treatment. Ten adult earthworms were placed carefully at the mid-line of each test container. The containers were then covered with transparent perforated lids and stored at 20 ± 1°C. After 48 h, the numbers of earthworms on each side were recorded. The burrowing activity of earthworms was conducted by means of two-dimensional (2D) terraria. These consisted of two glass sheets (30 cm 9 42 cm) separated by 3-mmthick pieces of glass sheets and filled by 2 mm sieved soil in which earthworm movement and behavior could be observed (Fig. 1a). At the beginning of the experiment, one
453
individual was weighed and introduced into each terrarium. All terraria were kept at 20 ± 1°C in a dark climate-controlled chamber for 7 days. The behavior (location of the earthworm) was recorded six times a day for 7 days. At the end of the experiment, earthworms were reweighed and burrow length and depth were recorded for each terrarium. Five replicates were analyzed at each concentration of 0, 50, 100, 150 and 200 mg 18-crown-6 kg-1 dry soil. Body mass change was expressed as a percentage of the initial mass. Earthworm respiration activity (20 ± 1°C) was recorded on the 1st, 3rd, 5th and 7th day of exposure to five different concentrations of 18-crown-6 (0, 50, 100, 150 and 200 mg kg-1 dry soil). Soil (300 g) and 10 earthworms were placed into 1,000 mL jars (Fig. 1b). Air entering these jars was first passed through KOH solution to remove CO2. The CO2 produced by earthworms was captured using 4 mL of 1 N KOH solution, which was titrated with 0.1 N HCl to quantify the amount of CO2. The change in the volume of CO2 produced by earthworms was calculated from the following equation: DCx = (Vx-V1)/V1, where V1 = the volume of CO2 on day 1 and Vx = the volume of CO2 on other days. Each concentration was replicated five times. It should be mentioned that earthworms ingest the soil to obtain food. Thus, the toxicity of 18-crown-6 to earthworms may result from its uptake by either the skin or alimentary canal. The added total 18-crown-6 concentration (i.e., nominal concentration of 18-crown-6) in soils was used in the figures and discussion. The median effective concentration (EC50) and median lethal concentration (LC50) were obtained by probit analysis (US EPA Probit Analysis, Version 1.5; Cincinnati, USA). SPSS 13 (SPSS Inc., Chicago, USA) was used for statistical analyses. One-way ANOVA followed by LSD multiple comparison was used to determine statistical differences between controls and treated groups. The results were expressed as mean ± standard deviation (SD), and the level of significance was p \ 0.05.
Fig. 1 Experimental devices for determining burrowing behavior (a) and respiration (b) in earthworms
123
454
Bull Environ Contam Toxicol (2014) 93:452–455
Results and Discussion The acute toxicity test has played an essential role in examining the toxic effects of chemicals on earthworms and in assessing their environment. As observed in Table 1, no mortality occurred within the 200 mg kg-1 dose group after the 7-d exposure, while 100 % mortality occurred in the 1,000 mg kg-1 dose group. 18-Crown-6 markedly decreased the survival of earthworms in a dose-dependent manner after 7 days. The 7-d LC50 for 18-crown-6 was calculated as 585 mg kg-1 dry soil. 18-Crown-6 exhibited neurologic toxicity in eukaryotes following intravenous and intraperitoneal administration in rats, mice and rabbits (Gad et al. 1978). Hendrixson et al. (1978) found that oral LC50 values in mice were 710 mg kg-1 for 18-crown-6, 1,020 for 15-crown-5 and 3,150 for 12-crown-4. It was suggested that selective metal ion interactions might be the source of the toxic effects of crown ethers (Hendrixon et al. 1978). It was also reported that despite the highly toxic nature of the crown ethers, these compounds were not genotoxic in prokaryotes as measured by reversion of the his locus in Salmonella typhimurium (Arenaz et al. 1989). Body mass change can serve as a indicator of the general health of earthworms and as an indicator of chemical stress. According to Table 2, 18-crown-6 had a negative impact on the growth of earthworms after 7 days. Higher 18-crown-6 concentrations resulted in smaller weight
gains. Significant differences in comparison to the control groups were found after 7 d exposure to 18-crown-6 concentrations of 150 and 200 mg kg-1 dry soil (p \ 0.05). Effects of 18-crown-6 on burrow length are shown in Fig. 2a. In non-contaminated control soil, burrow length
Table 1 Earthworm mortality (mean ± SD, n = 5) during 7 days of exposure to various concentrations of 18-crown-6 in artificial soils 18-crown-6 concentration (mg kg-1 dry soil)
Day 7 mortality (%)
0
0
200 300
0 2.8 ± 1.5
400
24.5 ± 6.4
500
36.2 ± 10.5
600
48.7 ± 12.4
700
62.2 ± 10.4
800
78.5 ± 7.6
900
95.8 ± 4.3
1,000
100 ± 0
Fig. 2 Toxicological responses of earthworms to 18-crown-6. a burrow length; b avoidance; c the change in the volume of CO2 produced by the earthworms. (statistical significance vs. control group: *p \ 0.05, **p \ 0.01, ***p \ 0.001)
Table 2 Body mass change and maximal burrow depths (mean ± SD, n = 5) of earthworms in 2D terraria in relation to the concentration of 18-crown-6 in soils 18-crown-6 concentration (mg kg-1 dry soil) 0 Body mass change (%) Maximal depth (cm)
50
96.3a ± 5.6 a
41.1 ± 1.5
100
94.6ab ± 10.2 a
38.3 ± 4.4
150
88.4ac ± 16.5 ab
34.8
± 10.6
Values indicated by different letters are significantly different from each other at the 5 % level
123
200
81.5bc ± 11.6 27.5
ab
± 14.5
78.2c ± 6.4 23.4bc ± 9.5
Bull Environ Contam Toxicol (2014) 93:452–455
increased continuously with time, reflecting general burrowing activity. However, in the presence of higher concentrations of 18-crown-6, the burrowing activity of earthworms was inhibited. Burrowing activity almost stopped completely toward the end of the trial period at the higher concentrations of 18-crown-6 (100, 150, 200 mg kg-1 dry soil), which was similar to results from previous studies using pesticides (Capowiez and Berar 2006). Significant differences of burrow length were observed after 7 d (168 h) exposure to 18-crown-6 concentrations of 150 and 200 mg kg-1 dry soil in comparison to the control group (p \ 0.05). Meanwhile, the average burrow depth made by earthworms was observed to decrease with increasing 18-crown-6 exposure concentrations (Table 2). Significant differences of burrow depth were observed at the concentration of 200 mg 18-crown6 kg-1 dry soil compared to the control group (p \ 0.05). Changes in burrow depth are likely to be ecologically important because of its influence on water and gas transfer in soil, which has the potential to affect the entire ecosystem (Bastardie et al. 2003). The present study provides further evidence that earthworm burrowing behavior is a sensitive and promising indicator of 18-crown-6 toxicity. Avoidance responses of earthworms to 18-crown-6 were studied and the net response (NR) showing avoidance of earthworms to 18-crown-6 in soils is presented in Fig. 2b. As observed, avoidance responses of earthworms increased with increasing 18-crown-6 concentrations, which indicated that 18-crown-6 had the potential to be harmful to earthworms and their environment. The 48-h EC50 was calculated as 120 mg 18-crown-6 kg-1 dry soil. The avoidance NR values showed dose-dependent increases at all of the 18-crown-6 concentrations that were tested. The avoidance endpoint was the most sensitive of all test endpoints. The change in the volume of CO2 produced by earthworms was used to assess respiration activity in this study. Preliminary tests showed that differences in 18-crown-6 concentrations had little detectable effect on respiration activity of earthworms during the first 24 h (day 1) of exposure. Therefore, respiration activity on the first 24 h (day 1) was used as a reference value. Experimental results clearly showed an inverse relationship between respiration activity and 18-crown-6 concentration on days 3, 5 and 7 (Fig. 2c), with high concentrations of 18-crown-6 having dramatic effects on respiration. Respiration was significantly
455
affected at 18-crown-6 concentration C100 mg kg-1, with changes increasing as exposure time increased. In conclusion, 18-crown-6 was shown to adversely affect the survival, growth, avoidance, burrowing behavior and respiration activity of the earthworm under laboratory conditions. The behavioral endpoints and respiration activity may be regarded as sensitive parameters for evaluating the toxicity of this chemical to earthworms. Acknowledgments This research was supported by the National Natural Science Foundation of China (Nos. 41071170, 31172360, 41111130195), Innovation Projects from Shanghai Municipal Education Committee, China (12YZ153) and the State Key Laboratory of Pollution Control and Resource Reuse Foundation (PCRRF12019). We also thank the Tripartite Funding from Education New Zealand and Ministry of Education of China.
References Arenaz P, Bitticks L, Pannell K, Garcia S (1989) Genotoxic potential of crown ethers in Salmonella typhimurium. Mutagenesis 4:437–438 Arenaz P, Bitticks L, Pannell KH, Garcia S (1992) Genotoxic potential of crown ethers in mammalian cells: induction of sisterchromatid exchange. Mutat Res 280:109–115 Bastardie F, Capowiez Y, de Dreuzy JR, Cluzeau D (2003) X-ray tomographic and hydraulic characterization of burrow by three earthworm species in repacked soil cores. Appl Soil Ecol 24:3–16 Capowiez Y, Berar A (2006) Assessment of the effects of imidacloprid on the behavior of two earthworm species (Aporrectodea nocturna and Allolobophora icterica) using 2D terraria. Ecotoxicol Environ Saf 64:198–206 Gad SC, Conroy WJ, Mckelvey JA, Turney KA (1978) Behavioral and neuropharmacological toxicology of the macrocyclic ether 18-crown-6. Drug Chem Toxicol 1:339–353 Hendrixson RR, Mack MP, Palmer RA, Ottolenghi A, Ghirardelli RG (1978) Oral toxicity of the cyclic polyethers 12-crown-4, 15-crown5 and 18-crown-6 in mice. Toxicol Appl Pharmacol 44:263–268 Hong Y, James SW (2009) Some new Korean megascolecoid earthworms (Oligochaeta). J Nat Hist 43:1229–1256 ISO (2005) Soil quality-avoidance test for testing the quality of soil sand toxicity of chemicals-test with earthworms. International Organization for Standardization, Geneva Leong BK, Ts’o TO, Chenoweth MB (1974) Testicular atrophy from inhalation of ethylene oxide cyclic tetramer. Toxicol Appl Pharmacol 27:342–354 OECD (1984) Organization of Economic and Cooperative Development. Guidelines for the testing of chemicals, No. 207. Earthworm acute toxicity tests Pedersen CJ (1967) Cyclic polyethers and their complexes with metal salts. J Am Chem Soc 89:7017–7036
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Toxicological Responses of the Earthworm Eisenia fetida to 18-Crown-6 Under Laboratory Conditions Yongtao Du • Pinhua Rao • Yinsheng Li • Jiangping Qiu • Weiguo Qiu • Hao Tang • Murray A. Potter
Received: 25 January 2014 / Accepted: 30 July 2014 / Published online: 7 August 2014 Ó Springer Science+Business Media New York 2014
Abstract The earthworm Eisenia fetida was exposed to artificial soil supplemented with 18-crown-6 (1,4,7,10,13,16hexaoxacyclooctadecane) to investigate its effects on earthworm mortality, growth, avoidance, burrowing behavior and respiration. The results revealed that 18-crown-6 had the potential to negatively affect the behavior of earthworms. The 7-d LC50 was 585 mg kg-1 soil. Avoidance behavior was the most sensitive endpoint, with a 48-h EC50 of 120 mg kg-1 soil. Growth, burrow length and respiration showed general decreases with increasing 18-crown-6 concentrations. Behavioral endpoints and respiration may be regarded as sensitive parameters in evaluating the toxicity of this chemical to earthworms. Keywords 18-crown-6 Burrowing behavior Respiration Avoidance Mortality 18-Crown-6 (1,4,7,10,13,16-hexaoxacyclooctadecane) is an artificial macrocyclic poly-ether. The synthesis of macrocyclic poly-ethers was first described by Pedersen (1967) who coined the name ‘‘crown ether’’ for these compounds. Structurally, crown ethers possess a hydrophobic ring
Y. Du P. Rao (&) School of Chemistry and Chemical Engineering, Shanghai University of Engineering Science, Longteng Rd. 333, Songjiang District, Shanghai 201620, China e-mail: [email protected] Y. Du Y. Li J. Qiu W. Qiu H. Tang School of Agriculture and Biology, Shanghai Jiaotong University, Shanghai 200240, China M. A. Potter Institute of Agriculture and Environment, Massey University, Private Bag 11-222, Palmerston North 4442, New Zealand
123
surrounding a hydrophilic cavity that enables them to form a stable complex with metal ions and sulfonamides. Crown ethers have been used as ion-chelating resins, in ion-selective electrodes, in promoting phase transfer reactions in binary solvent systems, and in biological systems as pharmacological and antibacterial agents (Arenaz et al. 1989). In short, crown ethers have a variety of applications in diverse fields and thus might readily be released into the environment. Pedersen (1967) was the first to report toxicity data on crown ethers. Since then, some studies have reported physiological effects of crown ethers. Leong et al. (1974) described toxic effects of the inhalation of 12-crown-4 on rats; and several articles have reported the toxicity of crown ethers on mice (Hendrixson et al. 1978) and rabbits (Gad et al. 1978). Arenaz et al. (1992) found that crown ethers were not significantly genotoxic in mammalian cells despite their cytotoxicity. However, to our knowledge, no studies have examined their toxicological impacts on earthworms in soil. Earthworms are common soil organisms that can be found in most environments and play a crucial role in controlling substance cycling and energy transformation in terrestrial ecosystems (Hong and James 2009). They are thus considered by the Organization for Economic Cooperation and Development (OECD 1984) to be suitable bioindicators of soil pollution. Here, we exposed the earthworm Eisenia fetida to artificial soil contaminated with 18-crown-6. Mortality, growth, avoidance, burrowing behavior and respiration were investigated. The purpose was to obtain a more comprehensive understanding of the effects of 18-crown6 on earthworms and to provide more information about the potential ecological risks of 18-crown-6 on soil ecosystems.
Bull Environ Contam Toxicol (2014) 93:452–455
Materials and Methods We used OECD artificial soil (OECD 1984) comprised of 10 % sphagnum peat, 20 % kaolinite and 70 % quartz sand. A small amount of calcium carbonate was added to adjust the pH to 6.0 ± 0.5. The moisture content was adjusted to approximately 30 %. 18-Crown-6 (99 % purity) was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Adult earthworms that possessed a clitellum and had an individual wet weight of between 300 and 450 mg were selected for testing. An acute toxicity test was used in accordance with OECD guidelines (OECD 1984). A range of concentrations were used in pre-trials to determine the suitable concentrations that produced 0 %–100 % mortality. Based on those results, 0, 200, 300, 400, 500, 600, 700, 800, 900 and 1,000 mg 18-crown-6 kg-1 dry soil were selected for the acute toxicity tests to determine the 50 % mortality concentration (i.e., LC50). Each container held ten adult earthworms and 300 g dry soil. The containers were held at a constant temperature (20 ± 1°C). After 7 days, earthworms were sorted by hand and mortality rates were determined. Each concentration was replicated five times. The avoidance tests were conducted in plastic containers based on ISO (2005). One half of the container was filled with 300 g dry contaminated soil; the other was filled with the same amount of control soil. Five replicates were used for each treatment. Ten adult earthworms were placed carefully at the mid-line of each test container. The containers were then covered with transparent perforated lids and stored at 20 ± 1°C. After 48 h, the numbers of earthworms on each side were recorded. The burrowing activity of earthworms was conducted by means of two-dimensional (2D) terraria. These consisted of two glass sheets (30 cm 9 42 cm) separated by 3-mmthick pieces of glass sheets and filled by 2 mm sieved soil in which earthworm movement and behavior could be observed (Fig. 1a). At the beginning of the experiment, one
453
individual was weighed and introduced into each terrarium. All terraria were kept at 20 ± 1°C in a dark climate-controlled chamber for 7 days. The behavior (location of the earthworm) was recorded six times a day for 7 days. At the end of the experiment, earthworms were reweighed and burrow length and depth were recorded for each terrarium. Five replicates were analyzed at each concentration of 0, 50, 100, 150 and 200 mg 18-crown-6 kg-1 dry soil. Body mass change was expressed as a percentage of the initial mass. Earthworm respiration activity (20 ± 1°C) was recorded on the 1st, 3rd, 5th and 7th day of exposure to five different concentrations of 18-crown-6 (0, 50, 100, 150 and 200 mg kg-1 dry soil). Soil (300 g) and 10 earthworms were placed into 1,000 mL jars (Fig. 1b). Air entering these jars was first passed through KOH solution to remove CO2. The CO2 produced by earthworms was captured using 4 mL of 1 N KOH solution, which was titrated with 0.1 N HCl to quantify the amount of CO2. The change in the volume of CO2 produced by earthworms was calculated from the following equation: DCx = (Vx-V1)/V1, where V1 = the volume of CO2 on day 1 and Vx = the volume of CO2 on other days. Each concentration was replicated five times. It should be mentioned that earthworms ingest the soil to obtain food. Thus, the toxicity of 18-crown-6 to earthworms may result from its uptake by either the skin or alimentary canal. The added total 18-crown-6 concentration (i.e., nominal concentration of 18-crown-6) in soils was used in the figures and discussion. The median effective concentration (EC50) and median lethal concentration (LC50) were obtained by probit analysis (US EPA Probit Analysis, Version 1.5; Cincinnati, USA). SPSS 13 (SPSS Inc., Chicago, USA) was used for statistical analyses. One-way ANOVA followed by LSD multiple comparison was used to determine statistical differences between controls and treated groups. The results were expressed as mean ± standard deviation (SD), and the level of significance was p \ 0.05.
Fig. 1 Experimental devices for determining burrowing behavior (a) and respiration (b) in earthworms
123
454
Bull Environ Contam Toxicol (2014) 93:452–455
Results and Discussion The acute toxicity test has played an essential role in examining the toxic effects of chemicals on earthworms and in assessing their environment. As observed in Table 1, no mortality occurred within the 200 mg kg-1 dose group after the 7-d exposure, while 100 % mortality occurred in the 1,000 mg kg-1 dose group. 18-Crown-6 markedly decreased the survival of earthworms in a dose-dependent manner after 7 days. The 7-d LC50 for 18-crown-6 was calculated as 585 mg kg-1 dry soil. 18-Crown-6 exhibited neurologic toxicity in eukaryotes following intravenous and intraperitoneal administration in rats, mice and rabbits (Gad et al. 1978). Hendrixson et al. (1978) found that oral LC50 values in mice were 710 mg kg-1 for 18-crown-6, 1,020 for 15-crown-5 and 3,150 for 12-crown-4. It was suggested that selective metal ion interactions might be the source of the toxic effects of crown ethers (Hendrixon et al. 1978). It was also reported that despite the highly toxic nature of the crown ethers, these compounds were not genotoxic in prokaryotes as measured by reversion of the his locus in Salmonella typhimurium (Arenaz et al. 1989). Body mass change can serve as a indicator of the general health of earthworms and as an indicator of chemical stress. According to Table 2, 18-crown-6 had a negative impact on the growth of earthworms after 7 days. Higher 18-crown-6 concentrations resulted in smaller weight
gains. Significant differences in comparison to the control groups were found after 7 d exposure to 18-crown-6 concentrations of 150 and 200 mg kg-1 dry soil (p \ 0.05). Effects of 18-crown-6 on burrow length are shown in Fig. 2a. In non-contaminated control soil, burrow length
Table 1 Earthworm mortality (mean ± SD, n = 5) during 7 days of exposure to various concentrations of 18-crown-6 in artificial soils 18-crown-6 concentration (mg kg-1 dry soil)
Day 7 mortality (%)
0
0
200 300
0 2.8 ± 1.5
400
24.5 ± 6.4
500
36.2 ± 10.5
600
48.7 ± 12.4
700
62.2 ± 10.4
800
78.5 ± 7.6
900
95.8 ± 4.3
1,000
100 ± 0
Fig. 2 Toxicological responses of earthworms to 18-crown-6. a burrow length; b avoidance; c the change in the volume of CO2 produced by the earthworms. (statistical significance vs. control group: *p \ 0.05, **p \ 0.01, ***p \ 0.001)
Table 2 Body mass change and maximal burrow depths (mean ± SD, n = 5) of earthworms in 2D terraria in relation to the concentration of 18-crown-6 in soils 18-crown-6 concentration (mg kg-1 dry soil) 0 Body mass change (%) Maximal depth (cm)
50
96.3a ± 5.6 a
41.1 ± 1.5
100
94.6ab ± 10.2 a
38.3 ± 4.4
150
88.4ac ± 16.5 ab
34.8
± 10.6
Values indicated by different letters are significantly different from each other at the 5 % level
123
200
81.5bc ± 11.6 27.5
ab
± 14.5
78.2c ± 6.4 23.4bc ± 9.5
Bull Environ Contam Toxicol (2014) 93:452–455
increased continuously with time, reflecting general burrowing activity. However, in the presence of higher concentrations of 18-crown-6, the burrowing activity of earthworms was inhibited. Burrowing activity almost stopped completely toward the end of the trial period at the higher concentrations of 18-crown-6 (100, 150, 200 mg kg-1 dry soil), which was similar to results from previous studies using pesticides (Capowiez and Berar 2006). Significant differences of burrow length were observed after 7 d (168 h) exposure to 18-crown-6 concentrations of 150 and 200 mg kg-1 dry soil in comparison to the control group (p \ 0.05). Meanwhile, the average burrow depth made by earthworms was observed to decrease with increasing 18-crown-6 exposure concentrations (Table 2). Significant differences of burrow depth were observed at the concentration of 200 mg 18-crown6 kg-1 dry soil compared to the control group (p \ 0.05). Changes in burrow depth are likely to be ecologically important because of its influence on water and gas transfer in soil, which has the potential to affect the entire ecosystem (Bastardie et al. 2003). The present study provides further evidence that earthworm burrowing behavior is a sensitive and promising indicator of 18-crown-6 toxicity. Avoidance responses of earthworms to 18-crown-6 were studied and the net response (NR) showing avoidance of earthworms to 18-crown-6 in soils is presented in Fig. 2b. As observed, avoidance responses of earthworms increased with increasing 18-crown-6 concentrations, which indicated that 18-crown-6 had the potential to be harmful to earthworms and their environment. The 48-h EC50 was calculated as 120 mg 18-crown-6 kg-1 dry soil. The avoidance NR values showed dose-dependent increases at all of the 18-crown-6 concentrations that were tested. The avoidance endpoint was the most sensitive of all test endpoints. The change in the volume of CO2 produced by earthworms was used to assess respiration activity in this study. Preliminary tests showed that differences in 18-crown-6 concentrations had little detectable effect on respiration activity of earthworms during the first 24 h (day 1) of exposure. Therefore, respiration activity on the first 24 h (day 1) was used as a reference value. Experimental results clearly showed an inverse relationship between respiration activity and 18-crown-6 concentration on days 3, 5 and 7 (Fig. 2c), with high concentrations of 18-crown-6 having dramatic effects on respiration. Respiration was significantly
455
affected at 18-crown-6 concentration C100 mg kg-1, with changes increasing as exposure time increased. In conclusion, 18-crown-6 was shown to adversely affect the survival, growth, avoidance, burrowing behavior and respiration activity of the earthworm under laboratory conditions. The behavioral endpoints and respiration activity may be regarded as sensitive parameters for evaluating the toxicity of this chemical to earthworms. Acknowledgments This research was supported by the National Natural Science Foundation of China (Nos. 41071170, 31172360, 41111130195), Innovation Projects from Shanghai Municipal Education Committee, China (12YZ153) and the State Key Laboratory of Pollution Control and Resource Reuse Foundation (PCRRF12019). We also thank the Tripartite Funding from Education New Zealand and Ministry of Education of China.
References Arenaz P, Bitticks L, Pannell K, Garcia S (1989) Genotoxic potential of crown ethers in Salmonella typhimurium. Mutagenesis 4:437–438 Arenaz P, Bitticks L, Pannell KH, Garcia S (1992) Genotoxic potential of crown ethers in mammalian cells: induction of sisterchromatid exchange. Mutat Res 280:109–115 Bastardie F, Capowiez Y, de Dreuzy JR, Cluzeau D (2003) X-ray tomographic and hydraulic characterization of burrow by three earthworm species in repacked soil cores. Appl Soil Ecol 24:3–16 Capowiez Y, Berar A (2006) Assessment of the effects of imidacloprid on the behavior of two earthworm species (Aporrectodea nocturna and Allolobophora icterica) using 2D terraria. Ecotoxicol Environ Saf 64:198–206 Gad SC, Conroy WJ, Mckelvey JA, Turney KA (1978) Behavioral and neuropharmacological toxicology of the macrocyclic ether 18-crown-6. Drug Chem Toxicol 1:339–353 Hendrixson RR, Mack MP, Palmer RA, Ottolenghi A, Ghirardelli RG (1978) Oral toxicity of the cyclic polyethers 12-crown-4, 15-crown5 and 18-crown-6 in mice. Toxicol Appl Pharmacol 44:263–268 Hong Y, James SW (2009) Some new Korean megascolecoid earthworms (Oligochaeta). J Nat Hist 43:1229–1256 ISO (2005) Soil quality-avoidance test for testing the quality of soil sand toxicity of chemicals-test with earthworms. International Organization for Standardization, Geneva Leong BK, Ts’o TO, Chenoweth MB (1974) Testicular atrophy from inhalation of ethylene oxide cyclic tetramer. Toxicol Appl Pharmacol 27:342–354 OECD (1984) Organization of Economic and Cooperative Development. Guidelines for the testing of chemicals, No. 207. Earthworm acute toxicity tests Pedersen CJ (1967) Cyclic polyethers and their complexes with metal salts. J Am Chem Soc 89:7017–7036
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