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The effects of 1-hexyl-3-methylimidazolium bromide on embryonic development and reproduction in Daphnia magna
Ecotoxicology and Environmental Safety 190 (2020) 110137
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The effects of 1-hexyl-3-methylimidazolium bromide on embryonic development and reproduction in Daphnia magna
T
Miao Yua, Chuanhu Liub, Honghao Zhaoc, Yanjing Yangc, Jinhui Sunc,∗ a
College of Fisheries, Henan Normal University, Xinxiang, Henan, 453007, China Teacher Development Center, Xinxiang University, Xinxiang, Henan, 453003, China c College of Fisheries, Tianjin Agricultural University, Tianjin, 300384, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Ionic liquids Daphnia magna Embryonic development Reproduction Acute and chronic toxicity
Ionic liquids (ILs) are acknowledged as green chemicals and favorable substitutes for volatile organic solvents, which are currently used. However, previous studies have shown that these compounds had toxicological impacts on aquatic organisms. To investigate the effects of 1-hexyl-3- methylimidazolium bromide ionic liquid ([C6mim]Br) on embryonic development and reproduction in water flea (Daphnia magna), a series of exposure experiments were conducted, including acute toxicity, maternal exposure, and chronic exposure tests. In acute toxicity experiment, D. magna neonates exhibited developmental abnormalities in the shell spine and the second antennae in a concentration-dependent manner after exposure to [C6mim]Br. The results in maternal exposure test also revealed a certain embryo-toxicity in response to [C6mim]Br in D. magna. However, the toxicity was lower than that conveyed by direct acute exposure, this indicated that the IL could act directly on organism. During the 21 days chronic exposure, the 1.6 mg/L exposure caused marked drop in the survival, molts and the number of the first brood of D. magna. Meanwhile, the total number of offspring was significantly declined in 1.6 mg/L concentration treatment groups, whereas increased in 0.2 mg/L groups. Generally, abnormalities in the offspring were significantly increased across all of the treatment groups in contrast to the control. No effect on sex differentiation was found during the experiments. These findings suggested that [C6mim]Br could affect embryonic development and reproduction in D. magna, and provided references for further study on the mechanisms underlying toxicological effects of ILs and the assessment of their potential environmental risks.
1. Introduction Ionic liquids (ILs) are salts that contain organic cations and various anions with melting points below 100 °C, which present favorable properties such as negligible vapor pressure, high thermal and chemical stability, non-flammability, and excellent solubility (Fuller et al., 1997; Ranke et al., 2004; Shimada et al., 2017; Moyer et al., 2018). The ILs have been considered as alternatives to traditional volatile organic solvents (VOCs), being studied by industrial scientists as potential media for storing and transporting highly toxic or flammable gases (Freemantle, 2005). They have shown promise for new industrial applications, acting as solvent and reaction media, thermal fluids, lubricants, plasticizers, dispensants, surfactants, antimicrobial, anticorrosion and electropolishing agents in plating industry and pharmaceutical industries, have the potential to be useful for inorganic and material synthesis, catalysed reactions, bioactive compounds, energy generation and storage, separations and extractions for materials
∗
as different as those from the nuclear industry (Albinet et al., 2010; Frade and Afonso, 2010; Ventura et al., 2017; Welton, 2018). Among various ILs, imidazolium-based ILs have been extensively applied and their aqueous solutions have been also studied by various experimental techniques such as density, conductivity and thermodynamic measurements (Frade and Afonso, 2010; Kusano et al., 2013; Zhang et al., 2017). Although ILs that consist entirely of ions are considered as green solvents (Modelli et al., 2008; Das and Roy, 2014), many investigations have reported that these compounds conveyed toxicity to aquatic organisms such as bacteria (Ventura et al., 2011; Wu et al., 2019), algae (Ma et al., 2010), water fleas Daphnia magna (Bernot et al., 2005a; Costa et al., 2015), freshwater snails (Bernot et al., 2005b) and fish (Li et al., 2011; Dong et al., 2013). Among these organisms, D. magna is a freshwater planktonic filter feeder that plays a critical role in the trophic transfer of energy (from plants to animals) in ecosystems (Mellors, 1975; Dodson and Hanazato, 1995). Besides, it is a widely
Corresponding author. College of Fisheries, Tianjin Agricultural University, No. 22, St. Jinjing, Xiqing District, Tianjin, 300384, China. E-mail address: [email protected] (J. Sun).
https://doi.org/10.1016/j.ecoenv.2019.110137 Received 3 October 2019; Received in revised form 5 December 2019; Accepted 25 December 2019 0147-6513/ © 2019 Published by Elsevier Inc.
Ecotoxicology and Environmental Safety 190 (2020) 110137
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the total numbers of deaths, abnormities, as well as the deformity phenotypes and sex ratios in each treatment were recorded, respectively. A neonate is presumed dead if the movement distance within 15 s is shorter than its physical length (Sheng et al., 2004).
accepted model organism for toxicity testing due to its rapid reproduction rate and sensitivity to changing conditions (Ventura et al., 2013). Being one of the most commonly employed aquatic invertebrates in toxicity studies, D. magna has already been applied to evaluate the toxicity of several types of ILs (Bernot et al., 2005a; Wells and Coombe, 2006; Zhang et al., 2017). Some reports have proved that increase in the alkyl chain length leads to a greater negative impact on D. magna (Zhang et al., 2017). For the same alkyl chain length, the aromatic cations (imidazolium and pyridinium) normally are more toxic than non-aromatic ILs, such as pyrrolidinium, piperidinium, phosphonium and ammonium (Ventura et al., 2013; Wang et al., 2015). Additionally, the imidazolium-based ILs were found to significantly influence the antioxidant system including SOD, CAT, GPX, GST, GSH and MDA levels in the organism, exert a toxic effect on the development of D. magna (Yu et al., 2009). Moreover, the longer the IL exposure lasted, the more difficult it was for D. magna to recover (Luo et al., 2008). However, the effects of additional types of ILs on the embryonic development, reproduction, and maternal inheritance in this organism remain insufficient, and little attention has been given to whether the influences of ILs on D. magna are mediated by maternal exposure. For this work, the acute and chronic toxicities of 1-hexyl-3-methylimidazolium bromide on D. magna were investigated. Meanwhile, the trans-generational effects of IL on this organism were evaluated. The results of this study will contribute to future research on aquatic toxicology, provide better understanding the effects of various ILs on aquatic organisms, and its potential hazards to aquatic systems.
2.4. Maternal exposure test The details and methods of maternal exposure experiment were processed in accordance to the previous literature (LeBlanc et al., 2000). Three replicates were set up for each treatment group (i.e. 0, 0.2, 0.4, 0.8, and 1.6 mg/L [C6mim]Br), the treated concentrations were identical to the acute exposure test. For each replicate, ten neonates (ages 6–24 h) were placed in a 50 mL glass beaker with 20 mL exposure solution, and fed daily. The exposure solution was changed every 3 days. Following 7–10 days exposure, 30 embryos at stage 1 were collected from each treatment, contingent on their availability, then were transferred to a medium with no test compound later. Following 72 h, the total numbers of deaths, abnormities, as well as deformity phenotypes and the sex ratios in each treatment were recorded, respectively (LeBlanc et al., 2000). 2.5. Chronic exposure test Neonates (aged 6–24 h) were chronically exposed to five concentrations of [C6mim]Br, which were same as the design in acute and maternal exposure tests. Ten replicates were assigned to each concentration. For each replicate, one neonate (aged 6–24 h) was individually incubated in a 50 mL glass beaker containing 20 mL of test medium. All of the treatments and control were terminated after 21 days. During the 21-day exposure period, the tested animals were fed and transferred to fresh exposure medium daily. The observations for each replicate were recorded every day, e.g. the survival rate, molting frequency, number of first-brood, total number and the abnormality rate of offspring.
2. Materials and methods 2.1. Chemicals The 1-hexyl-3-methylimidazolium bromide ([C6mim]Br) ionic liquid was synthesized as described by Bonhộte et al. (1998) and prepared at the School of Chemistry and Environmental Science at Henan Normal University, in Xinxiang, Henan, China. The purity of the IL was determined to be > 99%. Additional reagents were obtained from various commercial sources and were of analytical grade.
2.6. Statistics All data was performed using Excel 2010 and SPSS 19.0 for statistical analysis. The results were expressed as means ± standard deviation (SD). A one-way analysis of variance and Dunnett's test were applied, to compare whether there are differences between the treatments and the control. Statistical significance was set at p < 0.05.
2.2. Test animals D. magna samples were collected from the Wei River in Xinxiang, China. A single clone of D. magna Straus was selected and maintained in parthenogenetic cultures in our laboratory. The culture for D. magna was created based on previous studies by Luo et al. (2008) and Yu et al. (2009). D. magna were grown at 22 ± 1 °C with a 16:8 h light: dark photoperiod in an illumination incubator and were fed daily with the living unicellular green algae Chlorella vulgaris at a concentration of 5 × 105 cells/mL medium. The collection of the eggs and the identification of embryonic development stages were referenced to the methods of Kast-Hutcheson et al. (2001).
3. Results 3.1. Lethal effects of acute and maternal exposure of [C6mim]Br on D. magna The lethality rates of [C6mim]Br in various concentrations to D. magna embryos, caused by both acute and maternal exposures are presented in Fig. 1. Generally, the results illustrated that higher concentrations of the IL led to the death of the organisms. For this test, the significant lethal effect was only observed in the 1.6 mg/L concentration groups for both acute and maternal exposure tests, compared to the control and the other treatment groups (p < 0.05). On the other hand, by the pairwise comparison between acute and maternal 1.6 mg/L [C6mim]Br exposures, the lethal rate in acute exposure was higher than that in maternal exposure.
2.3. Acute exposure test Dechlorinated tap water was used as the culture medium, with total hardness 7.2 ± 0.2 mmol/L, alkalinity 12.4 ± 0.3 mmol/L, and pH 8.1 ± 0.2. Neonates (aged 6–24 h) born from parthenogenetic females were used for the three parts of exposure experiment, where five different concentration treatments of [C6mim]Br (0, 0.2, 0.4, 0.8, and 1.6 mg/L) were established according to a previous study (Yu et al., 2009). For each concentration, three replicates with ten neonates per replicate were employed. During the 72 h exposure period, all treatments were conducted in an illuminated incubator at a constant temperature (22 ± 1 °C) and normal photoperiod (16 h light: 8 h dark), with no food provided and no medium changed. The embryos were subjected to microscopic examination every 24 h to determine the developmental stages and any abnormalities. After 72 h static exposure,
3.2. Malformation effect of acute and maternal exposure of [C6mim]Br on D. magna The results of the malformation rate of D. magna embryos in response to acute and maternal exposures of different levels of [C6mim]Br are shown in Fig. 2. The malformation rates of D. magna embryos dramatically elevated with increasing of [C6mim]Br concentration 2
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characteristic of most deformities is the curved shell spine, except for one of the neonates, where curved shell spines together with underdeveloped second antennal setae was observed in 0.4 mg/L exposure group. An embryonic development arrest existed under the 1.6 mg/L treated concentration (Fig. 3; Table 1). 3.3. Chronic effects of [C6mim] Br on D. magna survival, molting rate, and offspring The chronic toxic effects of [C6mim]Br on D. magna are presented in Table 2. The results revealed that the survival rate of D. magna decreased when the organism was chronically exposed to higher concentrations of [C6mim]Br (0.8 and 1.6 mg/L). This meant that the IL was increasingly toxic at higher concentrations, which could lead to the death of the organisms. The molting rate also significantly reduced under higher [C6mim]Br concentrations, especially under the 1.6 mg/L treatment compared to the control (p < 0.05). The numbers of firstbrood offspring declined significantly, in contrast to the control, following treatment with the 1.6 mg/L concentration of IL (p < 0.05). However, the total number of offspring significantly increased under the 0.2 mg/L concentration, while it substantially decreased under the 1.6 mg/L treatment (p < 0.05). Compared with the control, the malformation rates of the offspring had significant increases in all [C6mim] Br exposure groups. It suggested that this kind of IL might cause abnormal offspring to some extent, where the abnormal phenotypes mainly manifested in curved shell spines and underdeveloped second antennal setae. Nevertheless, no effect on sexual differentiation was observed throughout the experiments. All of the offspring were female.
Fig. 1. The lethality rate of acute and maternal exposure of [C6mim]Br to D. magna. Note: Acute direct exposure of daphnid embryos to concentrations of [C6mim]Br following their removal from the brood chamber were conducted, as well as maternal exposure of the female parent to concentrations of [C6mim] Br, followed by incubation of embryos in an IL-free medium ex vivo. Exposures were concluded when at least 90% of the control embryos reached stage 6. Data is shown as means ± SD from three separate experiments. * indicates a significant difference (p < 0.05).
4. Discussion ILs have garnered a great deal of interest as alternatives to organic solvents for many chemical processes. However, previous studies have confirmed that these compounds convey toxicity to aquatic organisms (Pretti et al., 2006; Cho et al., 2008a, 2008b; Pham et al., 2010; Nu'aim and Bustam, 2018). For this study, the results revealed that certain concentrations of [C6mim]Br were toxic to D. magna, causing abnormalities and death of embryos, which is in accordance with previous conclusion (Couling et al., 2006; Samori et al., 2007; Yu et al., 2009; Stolte et al., 2012; Wang et al., 2015). Abnormal phenotypes such as curved shell spines and undeveloped second antennae were also observed in our work, which might occur during late-stage toxicity (stages 4–6) as described by Kast-Hutcheson et al. (2001). Meanwhile, other reasonable explanations for the deformities or abnormalities detected in our study are that physiological mechanisms related to stress and fitness of developmental stability of organisms, e.g. the fluctuating asymmetry. The early-stage embryo toxicity characterized by arrested egg development was also found under the 1.6 mg/L concentration in maternal exposure. Both early and late-stage embryo toxicities verified the risks of this IL to aquatic organisms, and by extrapolation, to ecosystems. Both of the lethal and malformation effects of [C6mim]Br on D. magna embryos in acute and maternal tests were intensified under higher [C6mim]Br concentrations, and their toxic effects under acute exposure were much higher than those for maternal exposure at the same IL concentration. This testified that the toxicity of acute exposure was higher than maternal exposure, and the IL could directly influence the embryonic development of daphnids. Meanwhile, the female parent could protect their offspring from the toxicity of [C6mim]Br at a certain extent when the embryos were in the brood chamber. It has been proposed that reproductive parameters were more sensitive than survival during chronic exposure to toxicants (Villarroel et al., 2003). Our results demonstrated that the survival rate of this organism decreased under the highest concentration (1.6 mg/L) group during the chronic exposure, and the molting rate also declined significantly for this group, in contrast to the control. Since the molting
Fig. 2. Malformation rates of D. magna acute and maternal exposed to different concentrations of [C6mim]Br. Note: Direct acute exposure of daphnid embryos to concentrations of [C6mim]Br following their removal from the brood chamber were conducted, as well as maternal exposure of the female parent to concentrations of [C6mim]Br followed by incubation of embryos in an IL-free medium ex vivo. Exposures were concluded when at least 90% of the control embryos reached stage 6. Data is shown as means ± SD. from three separate experiments. * indicates a significant difference (p < 0.05).
under both exposure tests. In addition, the malformation rates under acute exposure were all significantly higher than those analyzed in maternal exposure test under the same IL concentration (p < 0.05). Further, significant increases in the malformation rates in comparison with the control were observed from the lowest concentration (0.2 mg/ L) to the highest (1.6 mg/L) for direct acute exposure. For maternal exposure, the significant increases in malformation rates were only obtained in 0.8 and 1.6 mg/L [C6mim]Br concentration groups, in contrast to the control (p < 0.05). Our results also discovered that both acute and maternal exposures could result in variable abnormal phenotypes (Fig. 3) and their higher proportions (Table 1). For the acute exposure experiment, the abnormal embryos were primarily characterized by curved shell spines and underdeveloped second antennal setae. For the 0 and 0.2 mg/L IL concentration groups, all of the abnormal embryos exhibited curved shell spines. For the 0.4–1.6 mg/L groups, 81%–89% of the abnormal embryos showed curved shell spines, with the remains exhibiting curved shell spines together with underdeveloped second antennal setae. However, during the maternal exposure experiment, the specific 3
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Fig. 3. Abnormal D. magna neonates following exposure to [C6mim]Br. Note: A: control. The neonate has normal shell spine and the second antennal setae. B: curved shell spine. C: curved shell spine together with losing second antennal setae (arrow indicates abnormality). Scale bars = 1 mm. D: developmental arrest (arrow indicates abnormality).
from parthenogenesis to gamogenesis in response to the environmental challenges, such as the lack of resources or crowding (Kim et al., 2011). However, [C6mim]Br had no effects on sex differentiation of D. magna in present study. No male D. magna neonates were observed in all concentration groups after treatment, proving that the tested IL concentrations did not meet the conditions for the production of male individuals. Another reasonable explanation for the finding is that this kind of IL was not associated with the genes and hormones that determined sex. The influence of [C6mim]Br and other ILs on the sexdifferentiation of D. magna requires further investigation.
rate is related to D. magna growth, the reduced molting rate likely inhibited the growth, subsequent development and reproduction of the organism. Our experiments also verified that both the number of first brood and total number of offspring declined significantly under exposure to the highest concentration of IL. These findings were coincided with previous studies, where the population of the first brood and total number of offspring in the organism decreased significantly with increasing imidazolium-based IL concentrations (Bernot et al., 2005a). Our results were also consistent with the research of Luo et al. (2008), who had confirmed that [C8mim]Br could slow the growth and decrease the reproductive ability of D. magna under chronic IL exposure. As relates to the rationality behind the toxic effects of ILs, some researchers speculated that the ILs could alter the physical properties of lipid bilayers, enhancing the permeability of membrane for external ions, since they are water-soluble (Pretti et al., 2006). Thus, we assumed that [C6mim]Br might have the same toxic effects and produce similar physiological influences, could easily enter the body and cells of D. magna, conveying damage to the growth and development of the organism. Further, the same class of ILs, namely, [C4mim]Br, [C6mim] Br and [C10mim]Br, might disrupt cellular metabolism by altering CYP1A1, CYP1A2, CYP3A4 and GST enzyme activities in HepG2 cells (liver hepatocellular cells) (Zhang et al., 2015), in a similar metabolic pattern triggered by [C8mim]Br. However, paradoxically, the total number of offspring in 0.2 mg/L concentration group was significantly higher than that in the control, which is probably due to the hormesis, and the toxic exposed organisms might have constituted an adaptive biological protection against the lower concentration of toxicity. In this experiment, the abnormal phenotypes primarily exhibited curved shell spines and underdeveloped second antennal setae, which suggested that the [C6mim]Br may disturb the formation of normal organs by acting on the genes related to the structural development and functional regulation. Interestingly, the probability of abnormal second antennal setae increased with the increasing concentration of IL during acute exposure. This demonstrated that the toxicity of IL ([C6mim]Br) might initially act on the shell spine, then on the second antennal setae. Additionally, the malformation rate of the offspring under chronic exposure was obviously higher than that for maternal exposure, which illustrated that the female parent could accumulate the toxicity of [C6mim]Br and pass it on to their next generation. As we all known, daphnids could adjust their reproductive strategies
5. Conclusions This study examined the toxic effects of the acute, maternal, and chronic exposures of five different concentrations of [C6mim]Br to the freshwater crustacean D. magna. Our results revealed that this IL could cause abnormal development and a decline in reproductive capacity in D. magna. Moreover, the maternal exposure would give rise to the accumulation of toxicity, which is subsequently passed on to the nextgeneration. Further investigations into the mechanisms of the biochemical toxic and genotoxic effects of [C6mim]Br on D. magna are urgently needed.
Declaration of interest The authors declare that there are no competing financial interests.
Author contributions Jinhui Sun contributed to the study design. Miao Yu had roles in culturing fish, collecting samples, data collection and analysis. The manuscript was written through contributions of MiaoYu and Honghao Zhao. Other authors did valuable assistance in data analysis. All authors have given approval to the final version of the manuscript, decided to submit the work for publication. The authors thank Ruiling Li and Ningning Wang for their valuable assistance in fish culture and tissues sampling.
Table 1 The total numbers of abnormal D. magna neonates under acute and maternal [C6mim]Br exposures. Groups
Acute exposure
Concentrations (mg/L)
curved shell spine 1 7 13 20 24
0 0.2 0.4 0.8 1.6
Maternal exposure curved shell spine and underdeveloped second antennal setae 0 0 3 3 3
curved shell spine 1 1 2 5 6
Note: the total number of abnormal D. magna with corresponding type of deformities was counted. 4
curved shell spine and underdeveloped second antennal setae 0 0 1 0 0
development arrest 0 0 0 0 1
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Table 2 Chronic effects of [C6mim]Br on D. magna survival, molting rate, and offspring. IL concentration (mg/L)
0
0.2
Survival rate (%) Molting frequency The number of first-brood offspring The total number of offspring Malformation rate of offspring
100 10.25 ± 0.50 a 13.00 ± 2.16 a 67.00 ± 1.00 b 4.40 ± 0.94 a
100 10.00 12.50 72.50 17.48
± ± ± ±
0.71 1.29 1.91 2.66
a a a b
0.4
0.8
1.6
100 9.80 ± 0.45 a 12.00 ± 1.83 ab 66.00 ± 2.45 b 17.23 ± 3.57 b
80 9.60 ± 0.55 ab 13.50 ± 1.29 a 64.00 ± 3.61 bc 20.01 ± 9.26 b
60 8.67 ± 0.58 b 9.33 ± 1.15 b 61.00 ± 2.65 c 44.98 ± 8.28 c
Note: For the chronic exposure test (n = 10), one D. magna was selected as female parent for each replicate. The data were represented by the mean ± S.D. The different lowercase letters indicated the significant differences between concentration groups (p < 0.05).
Acknowledgements
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This project was supported by the National Science Foundation of Henan Province (102300410104), the Open Fund of Tianjin Key Lab of Aquatic Ecology and Aquaculture (TJAE201806), and the Starting Fund for Doctoral Research of Henan Normal University (5101229170135), China. References Albinet, A., Papaiconomou, N., Estager, J., Suptil, J., Draye, M., Besombes, J.L., 2010. A new ozone denuder for aerosol sampling based on an ionic liquid coating. Anal. Bioanal. Chem. 396 (2), 857–864. https://doi.org/10.1007/s00216-009-3243-5. Bernot, R.J., Brueseke, M.A., Evans-White, M.A., Lamberti, G.A., 2005a. Acute and chronic toxicity of imidazolium-based ionic liquids on D. magna. Environ. Toxicol. Chem. 24 (1), 87–92. https://doi.org/10.1897/03-635.1. Bernot, R.J., Kennedy, E.E., Lamberti, G.A., 2005b. Effects of ionic liquids on the survival, movement, and feeding behavior of the freshwater snail, Physa acuta. Environ. Toxicol. Chem. 24 (7), 1759–1765. https://doi.org/10.1897/04-614r.1. Bonhôte, P., Dias, A.P., Armand, M., Papageorgiou, N., Kalyanasundaram, K., Grätzel, M., 1998. Hydrophobic, highly conductive ambient-temperature molten salts. Inorg. Chem. 37 (1). https://doi.org/10.1021/ic971286k. 166-166. Cho, C.W., Jeon, Y.C., Pham, T.P., Vijayaraghavan, K., Yun, Y.S., 2008a. The ecotoxicity of ionic liquids and traditional organic solvents on micro alga Selenastrum capricornutum. Ecotoxicol. Environ. Saf. 71 (1). https://doi.org/10.1016/j.ecoenv.2007. 07.001. 0-71. Cho, C.W., Pham, T.P.T., Jeon, Y.C., Yun, Y.S., 2008b. Influence of anions on the toxic effects of ionic liquid to a phytoplankton Selenastrum capricornutum. Green Chem. 10 (1), 67–72. https://doi.org/10.1039/B705520J. Costa, S.P.F., Pinto, P.C.A.G., Saraiva, M.L.M.F.S., Rocha, F.R.P., Santos, J.R.P., Monteiro, R.T.R., 2015. The aquatic impact of ionic liquids on freshwater organisms. Chemosphere 139, 288–294. https://doi.org/10.1016/j.chemosphere.2015.05.100. Couling, D.J., Bernot, R.J., Docherty, K.M., Dixon, J.K., Maginn, E.J., 2006. Assessingthe factors responsible for ionic liquid toxicity to aquatic organism via quantitative structure-property relationship modeling. Green Chem. 8 (1). https://doi.org/10. 1039/b511333d. 82-0. Das, R.N., Roy, K., 2014. Predictive modeling studies for the ecotoxicity of ionicliquids towards the green algae Scenedesmus vacuolatus. Chemosphere 104, 170–176. https:// doi.org/10.1016/j.chemosphere.2013.11.002. Dodson, S.I., Hanazato, T., 1995. Commentary on effects of anthropogenic and natural organic chemicals on development, swimming behavior, and reproduction of Daphnia, a key member of aquatic ecosystems. Environ. Health Perspect. 103 (Suppl. 4), 7–11. https://doi.org/10.1289/ehp.95103s47. Dong, M., Zhu, L.S., Zhu, S.Y., Wang, J.H., Wang, J., Xie, H., Du, Z.K., 2013. Toxic effects of 1-decyl-3-methylimidazolium bromide ionic liquid on the antioxidant enzyme system and DNA in zebrafish (Danio rerio) livers. Chemosphere 91 (8), 1107–1112. https://doi.org/10.1016/j.chemosphere.2013.01.013. Frade, R.F., Afonso, C.A., 2010. Impact of ionic liquids in environment and humans: an overview. Hum. Exp. Toxicol. 29 (12), 1038–1054. https://doi.org/10.1177/ 0960327110371259. Freemantle, M., 2005. Ionic liquids make splash in industry. Chem. Eng. News 83 (31), 33–38. https://doi.org/10.1021/cen-v083n031.p033. Fuller, J., Carlin, R.T., Osteryoung, R.A., 1997. The room temperature ionic liquids 1ethyl-3-methylimidazolium tetrafluoroborate: electrochemical couples and physical properties. J. Electrochem. Soc. 144 (11), 3881–3886. https://doi.org/10.1149/1. 1838106. Kast-Hutcheson, K., Rider, C.V., LeBlanc, G.A., 2001. The fungicide propiconazole interferes with embryonic development of the crustacean Daphnia magna. Environ. Toxicol. Chem. 20 (3), 502–509. https://doi.org/10.1002/etc.5620200308. Kim, J.K., Kim, Y.H., Lee, S.W., Kwak, K.H., Chung, W.J., Choi, K.H., 2011. Determination of mRNA expression of DMRT93B, vitellogenin, and cuticle 12 in Daphnia magna and their biomarker potential for endocrine disruption. Ecotoxicology 20 (8), 1741–1748. https://doi.org/10.1007/s10646-011-0707-0. Kusano, T., Fujii, K., Tabata, M., Shibayama, M., 2013. Small-angle neutron scattering study on aggregation of 1-alkyl-3-methylimidazolium based ionic liquids in aqueous solution. J. Solut. Chem. 42 (10), 1888–1901. https://doi.org/10.1007/s10953-0130080-0.
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The effects of 1-hexyl-3-methylimidazolium bromide on embryonic development and reproduction in Daphnia magna
T
Miao Yua, Chuanhu Liub, Honghao Zhaoc, Yanjing Yangc, Jinhui Sunc,∗ a
College of Fisheries, Henan Normal University, Xinxiang, Henan, 453007, China Teacher Development Center, Xinxiang University, Xinxiang, Henan, 453003, China c College of Fisheries, Tianjin Agricultural University, Tianjin, 300384, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Ionic liquids Daphnia magna Embryonic development Reproduction Acute and chronic toxicity
Ionic liquids (ILs) are acknowledged as green chemicals and favorable substitutes for volatile organic solvents, which are currently used. However, previous studies have shown that these compounds had toxicological impacts on aquatic organisms. To investigate the effects of 1-hexyl-3- methylimidazolium bromide ionic liquid ([C6mim]Br) on embryonic development and reproduction in water flea (Daphnia magna), a series of exposure experiments were conducted, including acute toxicity, maternal exposure, and chronic exposure tests. In acute toxicity experiment, D. magna neonates exhibited developmental abnormalities in the shell spine and the second antennae in a concentration-dependent manner after exposure to [C6mim]Br. The results in maternal exposure test also revealed a certain embryo-toxicity in response to [C6mim]Br in D. magna. However, the toxicity was lower than that conveyed by direct acute exposure, this indicated that the IL could act directly on organism. During the 21 days chronic exposure, the 1.6 mg/L exposure caused marked drop in the survival, molts and the number of the first brood of D. magna. Meanwhile, the total number of offspring was significantly declined in 1.6 mg/L concentration treatment groups, whereas increased in 0.2 mg/L groups. Generally, abnormalities in the offspring were significantly increased across all of the treatment groups in contrast to the control. No effect on sex differentiation was found during the experiments. These findings suggested that [C6mim]Br could affect embryonic development and reproduction in D. magna, and provided references for further study on the mechanisms underlying toxicological effects of ILs and the assessment of their potential environmental risks.
1. Introduction Ionic liquids (ILs) are salts that contain organic cations and various anions with melting points below 100 °C, which present favorable properties such as negligible vapor pressure, high thermal and chemical stability, non-flammability, and excellent solubility (Fuller et al., 1997; Ranke et al., 2004; Shimada et al., 2017; Moyer et al., 2018). The ILs have been considered as alternatives to traditional volatile organic solvents (VOCs), being studied by industrial scientists as potential media for storing and transporting highly toxic or flammable gases (Freemantle, 2005). They have shown promise for new industrial applications, acting as solvent and reaction media, thermal fluids, lubricants, plasticizers, dispensants, surfactants, antimicrobial, anticorrosion and electropolishing agents in plating industry and pharmaceutical industries, have the potential to be useful for inorganic and material synthesis, catalysed reactions, bioactive compounds, energy generation and storage, separations and extractions for materials
∗
as different as those from the nuclear industry (Albinet et al., 2010; Frade and Afonso, 2010; Ventura et al., 2017; Welton, 2018). Among various ILs, imidazolium-based ILs have been extensively applied and their aqueous solutions have been also studied by various experimental techniques such as density, conductivity and thermodynamic measurements (Frade and Afonso, 2010; Kusano et al., 2013; Zhang et al., 2017). Although ILs that consist entirely of ions are considered as green solvents (Modelli et al., 2008; Das and Roy, 2014), many investigations have reported that these compounds conveyed toxicity to aquatic organisms such as bacteria (Ventura et al., 2011; Wu et al., 2019), algae (Ma et al., 2010), water fleas Daphnia magna (Bernot et al., 2005a; Costa et al., 2015), freshwater snails (Bernot et al., 2005b) and fish (Li et al., 2011; Dong et al., 2013). Among these organisms, D. magna is a freshwater planktonic filter feeder that plays a critical role in the trophic transfer of energy (from plants to animals) in ecosystems (Mellors, 1975; Dodson and Hanazato, 1995). Besides, it is a widely
Corresponding author. College of Fisheries, Tianjin Agricultural University, No. 22, St. Jinjing, Xiqing District, Tianjin, 300384, China. E-mail address: [email protected] (J. Sun).
https://doi.org/10.1016/j.ecoenv.2019.110137 Received 3 October 2019; Received in revised form 5 December 2019; Accepted 25 December 2019 0147-6513/ © 2019 Published by Elsevier Inc.
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the total numbers of deaths, abnormities, as well as the deformity phenotypes and sex ratios in each treatment were recorded, respectively. A neonate is presumed dead if the movement distance within 15 s is shorter than its physical length (Sheng et al., 2004).
accepted model organism for toxicity testing due to its rapid reproduction rate and sensitivity to changing conditions (Ventura et al., 2013). Being one of the most commonly employed aquatic invertebrates in toxicity studies, D. magna has already been applied to evaluate the toxicity of several types of ILs (Bernot et al., 2005a; Wells and Coombe, 2006; Zhang et al., 2017). Some reports have proved that increase in the alkyl chain length leads to a greater negative impact on D. magna (Zhang et al., 2017). For the same alkyl chain length, the aromatic cations (imidazolium and pyridinium) normally are more toxic than non-aromatic ILs, such as pyrrolidinium, piperidinium, phosphonium and ammonium (Ventura et al., 2013; Wang et al., 2015). Additionally, the imidazolium-based ILs were found to significantly influence the antioxidant system including SOD, CAT, GPX, GST, GSH and MDA levels in the organism, exert a toxic effect on the development of D. magna (Yu et al., 2009). Moreover, the longer the IL exposure lasted, the more difficult it was for D. magna to recover (Luo et al., 2008). However, the effects of additional types of ILs on the embryonic development, reproduction, and maternal inheritance in this organism remain insufficient, and little attention has been given to whether the influences of ILs on D. magna are mediated by maternal exposure. For this work, the acute and chronic toxicities of 1-hexyl-3-methylimidazolium bromide on D. magna were investigated. Meanwhile, the trans-generational effects of IL on this organism were evaluated. The results of this study will contribute to future research on aquatic toxicology, provide better understanding the effects of various ILs on aquatic organisms, and its potential hazards to aquatic systems.
2.4. Maternal exposure test The details and methods of maternal exposure experiment were processed in accordance to the previous literature (LeBlanc et al., 2000). Three replicates were set up for each treatment group (i.e. 0, 0.2, 0.4, 0.8, and 1.6 mg/L [C6mim]Br), the treated concentrations were identical to the acute exposure test. For each replicate, ten neonates (ages 6–24 h) were placed in a 50 mL glass beaker with 20 mL exposure solution, and fed daily. The exposure solution was changed every 3 days. Following 7–10 days exposure, 30 embryos at stage 1 were collected from each treatment, contingent on their availability, then were transferred to a medium with no test compound later. Following 72 h, the total numbers of deaths, abnormities, as well as deformity phenotypes and the sex ratios in each treatment were recorded, respectively (LeBlanc et al., 2000). 2.5. Chronic exposure test Neonates (aged 6–24 h) were chronically exposed to five concentrations of [C6mim]Br, which were same as the design in acute and maternal exposure tests. Ten replicates were assigned to each concentration. For each replicate, one neonate (aged 6–24 h) was individually incubated in a 50 mL glass beaker containing 20 mL of test medium. All of the treatments and control were terminated after 21 days. During the 21-day exposure period, the tested animals were fed and transferred to fresh exposure medium daily. The observations for each replicate were recorded every day, e.g. the survival rate, molting frequency, number of first-brood, total number and the abnormality rate of offspring.
2. Materials and methods 2.1. Chemicals The 1-hexyl-3-methylimidazolium bromide ([C6mim]Br) ionic liquid was synthesized as described by Bonhộte et al. (1998) and prepared at the School of Chemistry and Environmental Science at Henan Normal University, in Xinxiang, Henan, China. The purity of the IL was determined to be > 99%. Additional reagents were obtained from various commercial sources and were of analytical grade.
2.6. Statistics All data was performed using Excel 2010 and SPSS 19.0 for statistical analysis. The results were expressed as means ± standard deviation (SD). A one-way analysis of variance and Dunnett's test were applied, to compare whether there are differences between the treatments and the control. Statistical significance was set at p < 0.05.
2.2. Test animals D. magna samples were collected from the Wei River in Xinxiang, China. A single clone of D. magna Straus was selected and maintained in parthenogenetic cultures in our laboratory. The culture for D. magna was created based on previous studies by Luo et al. (2008) and Yu et al. (2009). D. magna were grown at 22 ± 1 °C with a 16:8 h light: dark photoperiod in an illumination incubator and were fed daily with the living unicellular green algae Chlorella vulgaris at a concentration of 5 × 105 cells/mL medium. The collection of the eggs and the identification of embryonic development stages were referenced to the methods of Kast-Hutcheson et al. (2001).
3. Results 3.1. Lethal effects of acute and maternal exposure of [C6mim]Br on D. magna The lethality rates of [C6mim]Br in various concentrations to D. magna embryos, caused by both acute and maternal exposures are presented in Fig. 1. Generally, the results illustrated that higher concentrations of the IL led to the death of the organisms. For this test, the significant lethal effect was only observed in the 1.6 mg/L concentration groups for both acute and maternal exposure tests, compared to the control and the other treatment groups (p < 0.05). On the other hand, by the pairwise comparison between acute and maternal 1.6 mg/L [C6mim]Br exposures, the lethal rate in acute exposure was higher than that in maternal exposure.
2.3. Acute exposure test Dechlorinated tap water was used as the culture medium, with total hardness 7.2 ± 0.2 mmol/L, alkalinity 12.4 ± 0.3 mmol/L, and pH 8.1 ± 0.2. Neonates (aged 6–24 h) born from parthenogenetic females were used for the three parts of exposure experiment, where five different concentration treatments of [C6mim]Br (0, 0.2, 0.4, 0.8, and 1.6 mg/L) were established according to a previous study (Yu et al., 2009). For each concentration, three replicates with ten neonates per replicate were employed. During the 72 h exposure period, all treatments were conducted in an illuminated incubator at a constant temperature (22 ± 1 °C) and normal photoperiod (16 h light: 8 h dark), with no food provided and no medium changed. The embryos were subjected to microscopic examination every 24 h to determine the developmental stages and any abnormalities. After 72 h static exposure,
3.2. Malformation effect of acute and maternal exposure of [C6mim]Br on D. magna The results of the malformation rate of D. magna embryos in response to acute and maternal exposures of different levels of [C6mim]Br are shown in Fig. 2. The malformation rates of D. magna embryos dramatically elevated with increasing of [C6mim]Br concentration 2
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characteristic of most deformities is the curved shell spine, except for one of the neonates, where curved shell spines together with underdeveloped second antennal setae was observed in 0.4 mg/L exposure group. An embryonic development arrest existed under the 1.6 mg/L treated concentration (Fig. 3; Table 1). 3.3. Chronic effects of [C6mim] Br on D. magna survival, molting rate, and offspring The chronic toxic effects of [C6mim]Br on D. magna are presented in Table 2. The results revealed that the survival rate of D. magna decreased when the organism was chronically exposed to higher concentrations of [C6mim]Br (0.8 and 1.6 mg/L). This meant that the IL was increasingly toxic at higher concentrations, which could lead to the death of the organisms. The molting rate also significantly reduced under higher [C6mim]Br concentrations, especially under the 1.6 mg/L treatment compared to the control (p < 0.05). The numbers of firstbrood offspring declined significantly, in contrast to the control, following treatment with the 1.6 mg/L concentration of IL (p < 0.05). However, the total number of offspring significantly increased under the 0.2 mg/L concentration, while it substantially decreased under the 1.6 mg/L treatment (p < 0.05). Compared with the control, the malformation rates of the offspring had significant increases in all [C6mim] Br exposure groups. It suggested that this kind of IL might cause abnormal offspring to some extent, where the abnormal phenotypes mainly manifested in curved shell spines and underdeveloped second antennal setae. Nevertheless, no effect on sexual differentiation was observed throughout the experiments. All of the offspring were female.
Fig. 1. The lethality rate of acute and maternal exposure of [C6mim]Br to D. magna. Note: Acute direct exposure of daphnid embryos to concentrations of [C6mim]Br following their removal from the brood chamber were conducted, as well as maternal exposure of the female parent to concentrations of [C6mim] Br, followed by incubation of embryos in an IL-free medium ex vivo. Exposures were concluded when at least 90% of the control embryos reached stage 6. Data is shown as means ± SD from three separate experiments. * indicates a significant difference (p < 0.05).
4. Discussion ILs have garnered a great deal of interest as alternatives to organic solvents for many chemical processes. However, previous studies have confirmed that these compounds convey toxicity to aquatic organisms (Pretti et al., 2006; Cho et al., 2008a, 2008b; Pham et al., 2010; Nu'aim and Bustam, 2018). For this study, the results revealed that certain concentrations of [C6mim]Br were toxic to D. magna, causing abnormalities and death of embryos, which is in accordance with previous conclusion (Couling et al., 2006; Samori et al., 2007; Yu et al., 2009; Stolte et al., 2012; Wang et al., 2015). Abnormal phenotypes such as curved shell spines and undeveloped second antennae were also observed in our work, which might occur during late-stage toxicity (stages 4–6) as described by Kast-Hutcheson et al. (2001). Meanwhile, other reasonable explanations for the deformities or abnormalities detected in our study are that physiological mechanisms related to stress and fitness of developmental stability of organisms, e.g. the fluctuating asymmetry. The early-stage embryo toxicity characterized by arrested egg development was also found under the 1.6 mg/L concentration in maternal exposure. Both early and late-stage embryo toxicities verified the risks of this IL to aquatic organisms, and by extrapolation, to ecosystems. Both of the lethal and malformation effects of [C6mim]Br on D. magna embryos in acute and maternal tests were intensified under higher [C6mim]Br concentrations, and their toxic effects under acute exposure were much higher than those for maternal exposure at the same IL concentration. This testified that the toxicity of acute exposure was higher than maternal exposure, and the IL could directly influence the embryonic development of daphnids. Meanwhile, the female parent could protect their offspring from the toxicity of [C6mim]Br at a certain extent when the embryos were in the brood chamber. It has been proposed that reproductive parameters were more sensitive than survival during chronic exposure to toxicants (Villarroel et al., 2003). Our results demonstrated that the survival rate of this organism decreased under the highest concentration (1.6 mg/L) group during the chronic exposure, and the molting rate also declined significantly for this group, in contrast to the control. Since the molting
Fig. 2. Malformation rates of D. magna acute and maternal exposed to different concentrations of [C6mim]Br. Note: Direct acute exposure of daphnid embryos to concentrations of [C6mim]Br following their removal from the brood chamber were conducted, as well as maternal exposure of the female parent to concentrations of [C6mim]Br followed by incubation of embryos in an IL-free medium ex vivo. Exposures were concluded when at least 90% of the control embryos reached stage 6. Data is shown as means ± SD. from three separate experiments. * indicates a significant difference (p < 0.05).
under both exposure tests. In addition, the malformation rates under acute exposure were all significantly higher than those analyzed in maternal exposure test under the same IL concentration (p < 0.05). Further, significant increases in the malformation rates in comparison with the control were observed from the lowest concentration (0.2 mg/ L) to the highest (1.6 mg/L) for direct acute exposure. For maternal exposure, the significant increases in malformation rates were only obtained in 0.8 and 1.6 mg/L [C6mim]Br concentration groups, in contrast to the control (p < 0.05). Our results also discovered that both acute and maternal exposures could result in variable abnormal phenotypes (Fig. 3) and their higher proportions (Table 1). For the acute exposure experiment, the abnormal embryos were primarily characterized by curved shell spines and underdeveloped second antennal setae. For the 0 and 0.2 mg/L IL concentration groups, all of the abnormal embryos exhibited curved shell spines. For the 0.4–1.6 mg/L groups, 81%–89% of the abnormal embryos showed curved shell spines, with the remains exhibiting curved shell spines together with underdeveloped second antennal setae. However, during the maternal exposure experiment, the specific 3
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Fig. 3. Abnormal D. magna neonates following exposure to [C6mim]Br. Note: A: control. The neonate has normal shell spine and the second antennal setae. B: curved shell spine. C: curved shell spine together with losing second antennal setae (arrow indicates abnormality). Scale bars = 1 mm. D: developmental arrest (arrow indicates abnormality).
from parthenogenesis to gamogenesis in response to the environmental challenges, such as the lack of resources or crowding (Kim et al., 2011). However, [C6mim]Br had no effects on sex differentiation of D. magna in present study. No male D. magna neonates were observed in all concentration groups after treatment, proving that the tested IL concentrations did not meet the conditions for the production of male individuals. Another reasonable explanation for the finding is that this kind of IL was not associated with the genes and hormones that determined sex. The influence of [C6mim]Br and other ILs on the sexdifferentiation of D. magna requires further investigation.
rate is related to D. magna growth, the reduced molting rate likely inhibited the growth, subsequent development and reproduction of the organism. Our experiments also verified that both the number of first brood and total number of offspring declined significantly under exposure to the highest concentration of IL. These findings were coincided with previous studies, where the population of the first brood and total number of offspring in the organism decreased significantly with increasing imidazolium-based IL concentrations (Bernot et al., 2005a). Our results were also consistent with the research of Luo et al. (2008), who had confirmed that [C8mim]Br could slow the growth and decrease the reproductive ability of D. magna under chronic IL exposure. As relates to the rationality behind the toxic effects of ILs, some researchers speculated that the ILs could alter the physical properties of lipid bilayers, enhancing the permeability of membrane for external ions, since they are water-soluble (Pretti et al., 2006). Thus, we assumed that [C6mim]Br might have the same toxic effects and produce similar physiological influences, could easily enter the body and cells of D. magna, conveying damage to the growth and development of the organism. Further, the same class of ILs, namely, [C4mim]Br, [C6mim] Br and [C10mim]Br, might disrupt cellular metabolism by altering CYP1A1, CYP1A2, CYP3A4 and GST enzyme activities in HepG2 cells (liver hepatocellular cells) (Zhang et al., 2015), in a similar metabolic pattern triggered by [C8mim]Br. However, paradoxically, the total number of offspring in 0.2 mg/L concentration group was significantly higher than that in the control, which is probably due to the hormesis, and the toxic exposed organisms might have constituted an adaptive biological protection against the lower concentration of toxicity. In this experiment, the abnormal phenotypes primarily exhibited curved shell spines and underdeveloped second antennal setae, which suggested that the [C6mim]Br may disturb the formation of normal organs by acting on the genes related to the structural development and functional regulation. Interestingly, the probability of abnormal second antennal setae increased with the increasing concentration of IL during acute exposure. This demonstrated that the toxicity of IL ([C6mim]Br) might initially act on the shell spine, then on the second antennal setae. Additionally, the malformation rate of the offspring under chronic exposure was obviously higher than that for maternal exposure, which illustrated that the female parent could accumulate the toxicity of [C6mim]Br and pass it on to their next generation. As we all known, daphnids could adjust their reproductive strategies
5. Conclusions This study examined the toxic effects of the acute, maternal, and chronic exposures of five different concentrations of [C6mim]Br to the freshwater crustacean D. magna. Our results revealed that this IL could cause abnormal development and a decline in reproductive capacity in D. magna. Moreover, the maternal exposure would give rise to the accumulation of toxicity, which is subsequently passed on to the nextgeneration. Further investigations into the mechanisms of the biochemical toxic and genotoxic effects of [C6mim]Br on D. magna are urgently needed.
Declaration of interest The authors declare that there are no competing financial interests.
Author contributions Jinhui Sun contributed to the study design. Miao Yu had roles in culturing fish, collecting samples, data collection and analysis. The manuscript was written through contributions of MiaoYu and Honghao Zhao. Other authors did valuable assistance in data analysis. All authors have given approval to the final version of the manuscript, decided to submit the work for publication. The authors thank Ruiling Li and Ningning Wang for their valuable assistance in fish culture and tissues sampling.
Table 1 The total numbers of abnormal D. magna neonates under acute and maternal [C6mim]Br exposures. Groups
Acute exposure
Concentrations (mg/L)
curved shell spine 1 7 13 20 24
0 0.2 0.4 0.8 1.6
Maternal exposure curved shell spine and underdeveloped second antennal setae 0 0 3 3 3
curved shell spine 1 1 2 5 6
Note: the total number of abnormal D. magna with corresponding type of deformities was counted. 4
curved shell spine and underdeveloped second antennal setae 0 0 1 0 0
development arrest 0 0 0 0 1
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Table 2 Chronic effects of [C6mim]Br on D. magna survival, molting rate, and offspring. IL concentration (mg/L)
0
0.2
Survival rate (%) Molting frequency The number of first-brood offspring The total number of offspring Malformation rate of offspring
100 10.25 ± 0.50 a 13.00 ± 2.16 a 67.00 ± 1.00 b 4.40 ± 0.94 a
100 10.00 12.50 72.50 17.48
± ± ± ±
0.71 1.29 1.91 2.66
a a a b
0.4
0.8
1.6
100 9.80 ± 0.45 a 12.00 ± 1.83 ab 66.00 ± 2.45 b 17.23 ± 3.57 b
80 9.60 ± 0.55 ab 13.50 ± 1.29 a 64.00 ± 3.61 bc 20.01 ± 9.26 b
60 8.67 ± 0.58 b 9.33 ± 1.15 b 61.00 ± 2.65 c 44.98 ± 8.28 c
Note: For the chronic exposure test (n = 10), one D. magna was selected as female parent for each replicate. The data were represented by the mean ± S.D. The different lowercase letters indicated the significant differences between concentration groups (p < 0.05).
Acknowledgements
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