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Sub-lethal effects of six neonicotinoids on avoidance behavior and reproduction of earthworms (Eisenia fetida)
Ecotoxicology and Environmental Safety 162 (2018) 423–429
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Sub-lethal effects of six neonicotinoids on avoidance behavior and reproduction of earthworms (Eisenia fetida)
T
Jing Gea,b, Yuanzhuo Xiaob, Yangyang Chaib, Haijuan Yanb, Ruohan Wub, Xing Xinb, ⁎ Donglan Wangb, Xiangyang Yua,b, a Key Laboratory of Food Quality and Safety of Jiangsu Province-State Key Laboratory Breeding Base/Key Laboratory of Control Technology and Standard for Agroproduct Safety and Quality, Ministry of Agriculture, P.R. China, Nanjing 210014, China b Institute of Food Quality and Safety, Jiangsu Academy of Agricultural Sciences, 50 Zhongling Street, Nanjing 210014, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Neonicotinoids Eisenia fetida Avoidance behavior Chronic toxicity Reproduction
Avoidance behavior of earthworms (Eisenia fetida) against six neonicotinoids (NEOs) (acetamiprid, dinotefuram, clothianidin, thiacloprid, nitenpyram, imidacloprid) was studied following the protocol of ISO. The results showed obvious avoidance behavior of E. fetida against the tested insecticides, and the medium effective concentration for avoidance behavior (EC50) of the six pesticides was 0.14, 0.55, 0.91, 7.87, 1.32 and 0.77 mg/kg, respectively. Compared to the acute toxicity, avoidance behavior was more sensitive as an indicator of soil contamination with NEOs. Chronic toxicity of above six NEOs to E. fetida was also evaluated; cocoon production, hatchability, cocoon weight and adult weight were all affected in the test. Cocoon production and hatchability were more sensitive than cocoon weight and adult weight. The reproduction of earthworms were significantly reduced with a 56 d half-maximal effective hatchability concentration (EC50) of 0.37, 0.74, 1.30, 3.57, 1.20 and 0.70 mg/kg (acetamiprid, dinotefuram, clothianidin, thiacloprid, nitenpyram, imidacloprid), respectively. Most of the tested NEOs were highly toxic to E. fetida. Avoidance behavior and reproduction damage of E. fetida was observed at very low concentrations. The existing levels of pollution with NEOs in soil frequently exceed the lowest observed adverse effect concentrations, which are likely to have negative biological and ecological impacts on earthworms.
1. Introduction Neonicotinoids (NEOs) has been widely used all over the world since they were registered in the early 1990s, for instance, imidacloprid-containing products alone which has been dominating the insecticide market are registered for use on more than 140 crops in 120 countries (Jeschke and Nauen, 2008). Together with other members in neonicotinoid family, such as clothianidin, acetamiprid, dinotefuran, nitenpyram, thiamethoxam, thiacloprid, they represent the best-selling class of insecticide on the global market (Jeschke et al., 2011). The acute toxicity of NEOs to mammals is low, however, NEOs are highly toxic to many invertebrates, including non-target aquatic species (Liess and Beketov, 2011; Pestana et al., 2009; Roessink et al., 2013) and pollinators such as bees (Pisa et al., 2015; Tsvetkov et al., 2017). NEOs are reported to have long half-lives in soil typically from a few days to in excess of 1000 days (range 28–1250 for imidacloprid; 7–353 days for thiamethoxam; 148–6931 days for clothianidin; 3–74 days for thiacloprid and 31–450 days for acetamiprid) (Goulson, 2013), therefore the
⁎
accumulation could easily happen when used repeatedly (Bonmatin et al., 2015). Hence, concerns have been raised regarding the environmental fate and effects of NEOs, such as soil persistence, effects on pollinators and other non-target invertebrates etc. (Goulson, 2013) The toxicity of NEOs to both target and non-target organisms, such as mammals, birds, fish, insects, annelids etc. have been investigated in many studies (De Cant and Barrett, 2010; Luo et al., 1999; MotaSanchez et al., 2006；Renaud et al., 2018). Some of them are proven highly toxic to soil invertebrates (de Lima e Silva et al., 2017) and even show constancy in toxicity for survival and reproduction for three generations (van Gestel et al., 2017). Earthworms play an important role in agricultural soils to maintain and improve soil structure and fertility (Lee, 1985). Earthworms can be exposed to NEOs by direct contact when applied, contaminated soil, seed etc. Globally, about 60% of NEOs is used as a seed dressing in farming (Jeschke et al., 2011). NEOs can easily bind to soil particles at its second phase of its loss from agricultural soil (Goulson, 2013), which can pose a risk to earthworm survival and behavior changes, and
Corresponding author at: Institute of Food Quality and Safety, Jiangsu Academy of Agricultural Sciences, 50 Zhongling Street, Nanjing 210014, China. E-mail address: [email protected] (X. Yu).
https://doi.org/10.1016/j.ecoenv.2018.06.064 Received 10 January 2018; Received in revised form 20 June 2018; Accepted 20 June 2018 0147-6513/ © 2018 Published by Elsevier Inc.
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Table 1 Test concentrations of neonicotinoids in the avoidance test. Pesticides
7 d-LC50 (mg/kg）
Nominal/Measured concentrations (mg/kg.soil dw) 0.5%
Imidacloprid Clothianidin Nitenpyram Acetamiprid Dinotefuran Thiacloprid
4.0 6.0 8.0 1.2 3.0 120
0.02/0.002 0.03/0.02 0.04/0.017 0.006/0.003 0.015/0.003 0.60/0.57
1% 0.04/0.007 0.06/0.03 0.08/0.044 0.012/0.007 0.03/0.01 1.2/1.11
5%
10%
0.2/0.16 0.3/0.24 0.4/0.26 0.06/0.036 0.15/0.096 6.0/6.31
0.4/0.34 0.6/0.5 0.8/0.36 0.12/0.087 0.3/0.21 12/12.8
20% 0.8/0.8 1.2/1.1 1.6/1.01 0.240.18 0.6/0.48 24/20.9
The values of measured concentrations are means of three replicates.
further cause the disruption of soil fertility maintenance processes. The accumulation and persistence of NEOs in soil can extend the exposure period for earthworms (Broznic et al., 2012; Gupta and Gajbhiye, 2007). A range of different endpoints, including mortality, reproduction, growth, molecular response, behavior, have been investigated. Avoidance behavior has been recognized as a more sensitive endpoint than mortality, growth etc. (Hund-Rinke et al., 2003) and may be as sensitive as reproduction (Van Gestel, 1992). Avoidance behavior of earthworms against heavy metals, nanoparticles has been extensively studied (Brami et al., 2017; Syed et al., 2017), however, there are only a handful of studies about avoidance behavior of earthworm against NEOs (Alves et al., 2013; Dittbrenner et al., 2012, 2011; Capowiez and Berard, 2006), and most of them only studied imidacloprid. The purpose of the present study was to comprehensively investigate the effects of 6 widely used NEOs on avoidance behavior of E. fetida at sub lethal concentrations; meanwhile, the effect of long term exposure to low concentrations of 6 NEOs on E. fetida reproduction was also investigated.
Table 2 Test concentrations of neonicotinoids in chronic toxicity test. Pesticides
Imidacloprid Dinotefuran Clothianidin Nitenpyram Acetamiprid Thiacloprid
14d-LC50 (mg/kg)
3.6 3.0 5.5 8.0 1.2 12.0*
Nominal/Measured concentrations (mg/kg.soil dw) 5%
10%
20%
50%
0.18/0.15 0.15/0.096 0.275/0.24 0.40/0.26 0.06/0.036 0.60/0.57
0.36/0.25 0.30/0.21 0.55/0.52 0.80/0.36 0.12/0.087 1.20/1.11
0.72/0.70 0.60/0.48 1.10/0.98 1.60/1.01 0.24/0.18 2.40/2.29
1.80/1.56 1.50/1.10 2.75/2.66 4.00/3.09 0.60/0.49 6.00/6.31
“*” data from the preliminary test; the values of measured concentrations are means of three replicates.
E. fetida earthworms were originally purchased from Jurong Earthworm Farm (Nanjing, China). The earthworms were allowed to acclimatize for 7 days to the artificial climate chamber (20 ± 1 °C with a dark: light ratio of 10:14 h with illumination of 400–800 lx; humidity: 80–85%). Adult earthworms with visible clitellum and an individual wet weight of 300–500 mg were randomly selected for the tests. Artificial soil used for the soil tests consisted of 10% ground sphagnum peat (< 0.5 mm), 20% kaolinite clay (> 50% kaolinite), 70% fine sand (OECD, 1984, 2004) and the water content was adjusted to 30% (OECD, 2004). A small amount of calcium carbonate was added to adjust the pH to 6.0 ± 0.5. The Predicted Environmental Concentration (PEC) of the studied neonicotinoids in soil was calculated. Single application with 75 (imidacloprid), 375 (clothianidin), 75 (nitenpyram), 90 (acetamiprid), 180 (dinotefuran), 500 (thiacloprid) g active ingredient (a.i.) per hectare, homogeneous distribution in the top 5 cm of soil, no crop interception and a soil density of 1.5 kg l−1.
soil according to ISO guidelines (ISO, 2008). Tests were conducted at 5 concentrations based on 7-d LC50, which was 0.5%, 1%, 5%, 10% and 20% of 7-d LC50 (Table 1). A stock solution was used to make dilution series and each replicate of the soil was fortified with test chemicals individually. At the beginning of the avoidance and reproduction experiment, the concentration of NEOs in fortified soil was measured, and the results were also listed in Table 1 and 2, respectively. All the treatments were performed with 4 replicates. A two section unit was used for the test, one half of the unit was filled with 300 g contaminated soil, and the other filled with 300 g control soil. Then the separator in the middle of the unit was removed and 10 earthworms were placed on the separating line. The units were then closed with transparent perforated lids. The tests were conducted in dark in incubators at 20 ± 1 °C for 48 h. The water content of the soil was maintained at 30%. The earthworms were not fed during the test. At the end of 48 h test period, the control and the contaminated soil sections were carefully separated and the number of earthworms was determined in each section of the units. Individuals found between the sections (on the separating line) were counted as 0.5 for each side. Dead earthworms were classified as escaped animals. A control with clean soil (without pesticides) on both sides of the units was also carried out in the test. Dual-control experiment was carried out before the formal test at two concentrations of 0.5% and 20% of 7-d LC50, control with clean soil was also carried out at the same time. The distribution of earthworms in clean soil and contaminated soil was studied.
2.2. Test chemicals
2.4. Reproduction test
Imidacloprid (95.3%), clothianidin (98.0%), acetamiprid (96.8%), dinotefuran (98.0%), nitenpyram (97.5%), thiamethoxam (97.9%), thiacloprid (97.2%) were purchased from Shandong Luba Chemical Co., Ltd. (Shandong, China)
Based on the 14d-LC50 of acute toxicity tests, four different concentrations (5%, 10%, 20% and 50% of 14d-LC50) were set to study the effect of NEOs on reproduction of E. fetida (Table 2). The procedure of dosing soil was the same as described in avoidance test. Each treatment was performed in 4 replicates. Ten adult earthworms (300–500 mg with a clitellum) were selected for reproduction test according to OECD 222 guideline (OECD, 2004). A total of 500 g soil treated with tested chemicals was placed in a 1 L glass jar and 10 adult earthworms were added to each jar. Controls were prepared similarly but without tested
2. Materials and methods 2.1. Test organisms and soil
2.3. Avoidance test Acute toxicity tests were conducted ahead to obtain 7-d LC50 and 14-d LC50 values. The avoidance tests were performed in the artificial 424
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avoidance response of E. fetida to imidacloprid, clothianidin, nitenpyram and dinotefuran was all below 60% at the tested concentrations, while over 60% at (and above) 0.24 and 12.0 mg/kg for acetamiprid and thiacloprid, respectively.
chemicals. The jars was covered with gauze, and stored at 20 ± 1 °C with a relative humidity of 80% under 400–800 lx of constant light. Sterile cow manure at 0.50 g earthworm−1 wk−1 was supplied as food. At day 28, ten adult earthworms were removed from each container; while cocoons in each container were counted, measured and preserved to incubate in the container for another 4 weeks without feeding. At the end of the experiment period, the numbers of juveniles hatched from the cocoons and unhatched cocoons were determined. The adult earthworms were weighed before and after the test. Adult earthworm weight, cocoon number, cocoon weight, hatchability and hatchlings per cocoon were investigated.
3.2. Effect of NEO on reproduction of E. fetida Reproduction parameters of E. fetida exposed to six tested NEOs are listed in Table 4. All the six tested pesticides had significant effect on one or more reproduction parameters of E. fetida. Mean cocoon numbers and hatchability were affected more than the other two parameters. Significant differences were observed of imidacloprid on mean cocoon weight and hatchability at 10% of 14d-LC50 and above. There were significant differences of dinotefuran, nitenpyram, thiacloprid on mean cocoon numbers at 10% of 14d-LC50 and above. All tested pesticides had a highly significant effect on cocoon production at the highest concentration. The EC50 values of hatchability of E. fetida were much lower than 14d-LC50 values of the acute toxicity (Table 5). As shown in Table 5, the avoidance behavior as an endpoint is as sensitive as reproduction. Compared with other NEO, dinotefuran and clothianidin could significantly reduce the body weight of E. fetida at 20% of 14d-LC50 and above. The inhibitory rate of clothianidin on body weight was highest (48%) at 1.1 mg/kg. NOEC and LOEC of different reproduction parameters of NEOs to E. fetida are shown in Table 6. Cocoon production of E. fetida was the most sensitive reproduction indicators for dinotefuran, nitenpyram, acetamiprid and thiacloprid, while juvenile number was the most sensitive indicators for imidacloprid and clothianidin. Other than the indicators mentioned above, NEOs also affected the appearance of the cocoons (Fig. 2). The cocoons were observed to be smaller and thinner with increased concentrations of the tested NEOs.
2.5. Detection of NEO in soil samples The extraction method of NEOs from artificial soil samples was adjusted from Abdel-Ghany's method (2016). The concentration of NEOs in soil samples was detected with an HPLC-MS/MS. The detailed information of instrument parameters can refer to Support information. 2.6. Data assessment Avoidance test: In the avoidance test, for each replicate the net response (NR) (expressed as percentage) was calculated as NR = [(C - T) / 10] × 100, where C = sum of earthworms observed in the control soil; T = sum of earthworms observed in treated soil; 10 = total number of earthworms per replicate. A positive net response indicates avoidance and a negative net response indicates an attraction to the chemical tested in a given concentration. According to ISO guidelines (ISO, 2008), describing the testing of chemicals, attraction reactions (the worms prefer the soil treated with the test chemical) have to be considered as 0% of avoidance. Prior to analysis, data were tested for homogeneity of variance (Levene's test) and for normal distribution (Kolmogorove Smirnov test). The highest no observed effect concentration (NOEC) and lowest observed effect concentration (LOEC) value was determined using ANOVA and Dunnett's test. The median effective concentration of avoidance response (AC50) values and its 95%-confidence limits were determined with Excel 2013 and DPS 7.05 using Trimmed Spearmane Karber method (Hamilton et al., 1977). These calculations were based on the mean net response of the four replicates. Chi-square tests were applied to determine whether the avoidance response of the earthworms towards a test soil was significant or not. The null hypothesis was no significant different between control and test soils, and the alternate hypothesis was that significant different existed in the two soils. When a significant difference of p < 0.05 was obtained, a soil was considered to be avoided. Reproduction toxicity test: The median effective concentration (EC50) values and its 95%-confidence limits were determined with Excel 2013 and DPS 7.05; significance analysis between control and experiment group were performed in SPSS with Dunnett's test (p < 0.05，0.01 or 0.001), and NOEC, LOEC were also calculated in SPSS.
4. Discussion Behavior and reproduction of earthworms can be affected by pesticides at sub-lethal concentrations with long term exposure; however, the actual risk of harmful effects on earthworms still depend on the exposure concentration, exposure duration and route of exposure under field conditions (Pisa et al., 2015). Avoidance response was recently considered as a more sensitive endpoint than many other standard endpoints such as mortality, growth and even reproduction (Syed et al., 2017). In many instances, exposure to NEOs contaminated soils below lowest-observed-effect concentrations (LOECs) for mortality and reproduction can still provoke an avoidance response from earthworms (Alves et al., 2013; Dittbrenner et al., 2012). In this work, the behavioral AC50 was about 5.1–15.2 times lower than the 7 d-LC50 of acute toxicity in the artificial soil test, indicating that avoidance behavior was much more sensitive than survival effects as an endpoint. It was consistent with those previous studies mentioned above. Among the behavioral effects (burrowing, avoidance behavior, cast production and weight change) investigated previously, avoidance behavior ranked the second most sensitive endpoint after burrowing (Pisa et al., 2015). According to the NR equation, the avoidance response of 60% corresponds to 80% of the E. fetida in the control soil, which suggests the contaminated soil is inhospitable for E. fetida to live. Special attention needs to be paid to acetamiprid, because the avoidance response was greater than 60% when the tested concentration was at 10% of 7d-LC50 (measured concentrations: 0.087 mg/kg.soil dw), which is accessible under practical conditions. The results showed the potential to use avoidance behavior as an endpoint for toxicity tests, since it could be linked to effects at the ecosystem level (Capowiez and Berard, 2006). However, the influence (avoidance or attraction) of various odorous compounds (Zirbes et al., 2010) to E. fetida should be taken into account under practical conditions. Only a few studies tested sub-lethal effects of NEOs on earthworm reproduction and the results showed obvious reductions in fecundity at
3. Results 3.1. Effect of neonictinoids on avoidance behavior of E. fetida In the dual control test, 100% survival of E. fetida was recovered. No significant difference (p > 0.05) in the distribution of earthworms was observed between both sides of the chamber with either clean or treated soils. The effects of 6 neonicotinoids on avoidance behavior of E. fetida are shown in Fig. 1 and Table 3. Significant avoidance behavior of E. fetida was observed towards all the tested NEO at even low concentrations (4–20% of 7 d-LC50). The AC50 of acetamiprid, dinotefuran, imidacloprid, clothianidin, nitenpyram and thiacloprid was 0.77, 0.91, 1.32, 0.14, 0.55, 7.87 mg/kg, respectively. The order was in accordance with the acute toxicity test results. As can be seen in Fig. 1, the 425
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Fig. 1. Avoidance or attraction response of E. fetida to 6 neonicotinoids, A: Imidacloprid; B: Clothianidin; C: Nitenpyram; D: Acetamiprid; E: Thiacloprid; F: Dinotefuran. The values are means of four replicates. “*”: p < 0.05; “**”: p < 0.01 (Chi-square tests).
the reproduction test. Among the reproduction parameters measured in the present study (cocoon numbers per earthworm, cocoon weight, hatchability, adult earthworm weight), cocoon production and hatchability were more sensitive than the rest. Similar results have been reported suggesting a significant decrease in cocoon production caused by imidacloprid and thiacloprid at 1.91 and 0.51 mg/kg, respectively (Capowiez and Berard, 2006). Mean cocoon weight and adult weight were affected at comparatively higher tested concentrations. The results from the present study deviated from the previous study, in which mean cocoon weight was more sensitive than cocoon production and hatchability (Wang et al., 2015). The EC50 of the hatchability was in the order of acetamiprid > imidacloprid ≈ dinotefuran > nitenpyram ≈ clothianidin > thiacloprid, which was in accordance with the acute toxicity test. Even though thiacloprid was not a very toxic pesticide to earthworm, it could affect cocoon production at 10% of 14d-LC50 and above (> 1.1 mg/kg, measured concentration). Dinotefuran, the third generation NEO, was also investigated in the present study. The toxicity of dinotefuran ranked in the middle of all the tested NEOs, and it could affect cocoon production significantly at 0.3 mg/kg. Our results showed
Table 3 Effect of 6 neonicotinoids on the avoidance behavior of E. fetida (mg/kg). Pesticides
7 d-LC50a
AC50
NOEC
LOEC
PECsoil
Imidacloprid Clothianidin Nitenpyram Acetamiprid Dinotefuran Thiacloprid
4.0(3.5–4.4) 6.0(5.3–8.6) 8.0(7.3–9.1) 1.2(1.0–1.5) 3.0(2.6–3.6) 120(79.6–196)
0.77 0.91 1.32 0.14 0.55 7.87
0.4 0.6 0.04 0.012 > 0.6 6.0
0.8 1.2 0.08 0.06 > 0.6 12
0.1 0.5 0.1 0.12 0.24 0.67
a
7 d-LC50 of the artificial soil test.
low concentrations (Alves et al., 2013; Gomez-Eyles et al., 2009; Wang et al., 2015; Renaud et al., 2018; de Lima e Silva et al., 2017). Since imidacloprid has been in use for much longer time than other NEO members, its toxicity on earthworms has been extensively studied, however, information on the toxicity of other NEOs on soil fauna in scientific literature have been scant (Goulson, 2013; Renaud et al., 2018). In the present study, 6 commonly used NEOs were all studied in 426
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Table 4 Reproduction parameters measured for E. fetida exposed to difference concentrations of tested neonicotinoids. Concentrations (mg/kg) control 0 Imidacloprid 0.18 0.36 0.72 1.8 Dinotefuran 0.15 0.3 0.6 1.5 Clothianidin 0.275 0.55 1.1 2.75 Nitenpyram 0.4 0.8 1.6 4 Acetamiprid 0.06 0.12 0.24 0.6 Thiacloprid 0.6 1.2 2.4 6.0
Mean cocoons per E. fetida
Mean cocoon weight(mg)
Mean hatchlings per cocoon
Inhibitory rate of adult weight (%)
4.03 ± 0.32
10.93 ± 0.38
2.07 ± 0.08
11.22 ± 5.56
3.90 3.50 2.71 1.03
± ± ± ±
0.10 0.20 1.02* 0.55***
10.50 ± 0.63 9.77 ± 0.49* 9.42 ± 0.51** 8.75 ± 0.25**
1.92 1.77 1.30 1.04
± ± ± ±
0.14 0.16* 0.03*** 0.08***
8.95 ± 0.42 12.90 ± 0.65 20.80 ± 2.71* 28.61 ± 6.35**
3.17 2.55 1.71 1.29
± ± ± ±
0.63 0.22* 0.96** 0.17***
10.55 ± 0.5 9.42 ± 1.00 9.30 ± 0.86 8.76 ± 0.58*
1.95 1.90 0.97 0.17
± ± ± ±
0.15 0.95 0.42 0.16**
14.15 17.41 32.12 31.49
± ± ± ±
1.99 4.12 1.53*** 5.86**
3.54 3.06 2.65 1.94
± ± ± ±
0.19 1.23 0.31 0.54**
10.24 ± 0.87 9.87 ± 1.35 9.17 ± 1.04 8.38 ± 0.54*
2.00 1.46 1.24 0.98
± ± ± ±
0.51 0.22 0.21* 0.18**
12.89 26.47 48.04 30.73
± ± ± ±
4.29 6.69 21.89** 1.58**
3.33 2.45 1.46 0.85
± ± ± ±
0.65 0.76* 0.34** 0.83***
10.55 ± 0.11 10.48 ± 0.46 8.89 ± 1.02* 8.56 ± 0.92**
1.92 1.52 1.08 0.76
± ± ± ±
0.47 0.50 0.22* 0.25**
4.08 ± 14.65 5.32 ± 5.79 13.13 ± 3.06 25.99 ± 4.80
3.55 2.78 2.07 1.15
± ± ± ±
0.72 0.73 0.64** 0.28***
10.39 ± 0.97 9.66 ± 0.25 9.61 ± 1.38 9.07 ± 0.07*
2.02 1.77 1.42 0.89
± ± ± ±
0.14 0.36 0.15** 0.12***
12.24 10.89 14.48 24.41
± ± ± ±
2.43 5.44 5.10 1.65*
3.68 2.82 1.74 0.33
± ± ± ±
0.37 0.26** 0.71*** 0.11***
10.11 ± 0.96 9.69 ± 0.74 9.19 ± 0.73* 8.67 ± 0.76*
1.91 1.60 1.37 0.67
± ± ± ±
0.18 0.33 0.30* 0.29***
10.97 12.42 16.60 25.14
± ± ± ±
7.93 4.06 1.94 1.31*
* p＜0.05. ** p＜0.01. *** p＜0.001; n = 4. Table 5 EC50 (mg/kg) of different endpoints of E. fetida exposed to neonicotinoid insecticides.
Table 6 NOEC and LOEC of different toxicity indicator (cocoon number/cocoon weight/ juvenile number/adult weight) of neonicotinoids to earthworm.
Pesticides
Acute toxicity 14dLC50(95% CI)
Hatchability EC50(95% CI)
Avoidance AC50
Pesticides
14d-LC50 (mg/kg)
NOEC/ LOEC
Cocoon production
Cocoon weight
Juvenile number
Adult weight
Imidacloprid Dinotefuran Clothianidin Nitenpyram Acetamiprid Thiacloprid
3.59(3.07–4.20) 3.0(2.65–3.38) 5.51(4.55–6.66) 8.0(7.17–8.93) 1.12(0.95–1.31) 56.6(34.10–94.47)
0.70(0.5–0.75) 0.74(0.65–0.84) 1.30(1.12–1.53) 1.20(1.06–1.34) 0.37(0.26–0.53) 3.57(2.96–4.29)
0.77 0.91 1.32 0.14 0.55 7.87
Imidacloprid
3.6
Dinotefuran
3.0
Clothianidin
5.5
Nitenpyram
8.0
Acetamiprid
1.2
Thiacloprid
12
NOEC LOEC NOEC LOEC NOEC LOEC NOEC LOEC NOEC LOEC NOEC LOEC
0.36 0.72 0.15 0.3 1.1 2.75 0.4 0.8 0.12 0.24 0.6 1.2
0.18 0.36 0.6 1.5 1.1 2.75 0.8 1.6 0.24 0.6 0.6 1.2
0.18 0.36 0.6 1.5 0.55 1.1 0.8 1.6 0.12 0.24 1.2 2.4
0.36 0.72 0.3 0.6 0.55 1.1 >4 >4 0.24 0.6 2.4 6
that reproductive parameters and avoidance behavior were almost equally sensitive as endpoints for studying the adverse effect of NEOs. The results show that the toxicity of different NEOs to the earthworms can vary by up to a factor of 50, the reason of which is rarely studied. However, there is some research done with other animals, such as bees. The toxicity differences were even found to be about 20 times to earthworms (de Lima e Silva et al., 2017) and 1000 times to bees between imidacloprid and thiacloprid (Manjon et al., 2018). Previous work on honeybees has suggested that it is due to the different rates of these compounds (Brunet et al., 2005; Alptekin et al., 2016), and some of the related enzymes have also been identified recently (Manjon et al., 2018). There is also another assumption about the different toxicity of NEOs to insects. NEOs are nerve toxins which act on the nAChR. Different insect nAChR subunits have different sensitivity to different NEOs; therefore it is likely that different subunits may be activated with distinctive effects on their toxicity (Thany et al., 2006). To avoid or minimize the harm of NEOs to bees, seed coating NEOs formulation was recommended and widely used (Tomizawa and Casida, 2005).
Note: The unit for NOEC and LOEC is mg/kg.
However, this strategy increases the risks to soil fauna, such as earthworms. PEC values of the tested NEOs are shown in Table 3. PEC values of acetamiprid and nitenpyram are greater than their LOEC values of the avoidance test. Special attention needs to be paid to these two NEOs. It is worth noting that the PEC values are calculated with single application, thus the actual concentrations in soil could easily get higher than PCE values with multiple applications. Bonmatin et al. (2005) found that concentrations of imidacloprid in some soil samples exceeded 0.1 mg/kg in UK. Soil dwelling organisms are expected to be exposed to mixtures of NEOs in the range from 0.001 to > 0.1 mg/kg. When imidacloprid was applied at 20 and 80 g active ingredient ha−1, 427
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5. Conclusions In summary, comprehensive study of 6 NEOs on the earthworms’ avoidance behavior and reproduction was carried out in the present work. Avoidance behavior and reproduction harm on the earthworms were observed at very low concentrations. Avoidance behavior was as sensitive as reproduction as a toxicity indicator of NEOs pollution in soil. Both avoidance behavior and reproduction were more sensitive than other indicators such as mortality and growth. Since NEOs are extensively used all over the world and they are persistent in soil, it is easy to have extended exposure period under practical field conditions. Long term low-level concentrations of NEOs in soils may pose high risks to the earthworms by affecting their behavior and reducing their fecundity. Empirical field studies of earthworm responses to realistic field concentrations are still needed to evaluate the risk more accurately. Since the earthworms contribute the most biomass in soil and they are also at the bottom of the land food chain, special attention needs to be paid on the use of NEOs for the sustainable development of the ecological system. The mechanism of different toxicity of different NEOs to earthworms still needs to be further studied. Acknowledgements The work is financially supported by the National Natural Science Foundation of China [grant number: 31601660] and Jiangsu key R & D plan [grant number: B2016367]. Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at doi:10.1016/j.ecoenv.2018.06.064. References Abdel-Ghany, M.F., Hussein, L.A., El Azab, N.F., El-Khatib, A.H., Linscheid, M.W., 2016. Simultaneous determination of eight neonicotinoid insecticide residues and two primary metabolites in cucumbers and soil by liquid chromatography-tandem mass spectrometry coupled with QuEChERS. J. Chromatogr. B 1031, 15–28. Alptekin, S., Bass, C., Nicholls, C., Paine, M.J., Clark, S.J., Field, L., Moores, G.D., 2016. Induced thiacloprid insensitivity in honeybees (Apis mellifera L.) is associated with up-regulation of detoxification genes. Insect Mol. Biol. 25, 171–180. Alves, P.R.L., Cardoso, E.J.B.N., Martines, A.M., Sousa, J.P., Pasini, A., 2013. Earthworm ecotoxicological assessments of pesticides used to treat seeds under tropical conditions. Chemosphere 90, 2674–2682. Bonmatin, J.M., Moineau, I., Charvet, R., Collin, M.E., Fleche, C., Bengsch, E.R., 2005. Behavior of imidacloprid in fields. Toxicity for honey bees. Environmental chemistry: Green Chemistry and Pollutants in Ecosystems. Springer, New York. Bonmatin, J.M., Giorio, C., Girolami, V., Goulson, D., Kreutzweiser, D.P., Krupke, C., 2015. Environmental fate and exposure; neonicotinoids and fipronil. Environ. Sci. Pollut. Res. Int. 22, 35–67. Brami, C., Glover, A.R., Butt, K.R., Lowe, C.N., 2017. Effects of silver nanoparticles on survival, biomass change and avoidance behaviour of the endogeic earthworm Allolobophora chlorotica. Ecotoxicol. Environ. Safe. 141, 64–69. Broznic, D., Marinic, J., Tota, M., Juresic, G.C., Petkovic, O., Milin, C., 2012. Hysteretic behavior of imidacloprid sorption-desorption in soils of croatian coastal regions. Soil Sediment Contam. 21, 850–871. Brunet, J.L., Badiou, A., Belzunces, L.P., 2005. In vivo metabolic fate of [14C]-acetamiprid in six biological compartments of the honeybee, Apis mellifera L. Pest Manag. Sci. 61, 742–748. Capowiez, Y., Berard, 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. Safe. 64, 198–206. Chowdhury, S., Mukhopadhyay, S., Bhattacharyya, A., 2012. Degradation dynamics of the insecticide: clothianidin (Dantop 50 % WDG) in a tea field ecosystem. B. Environ. Contam. Tox. 89, 340–343. Dankyi, E., Gordon, C., Carboo, D., Fomsgaard, I.S., 2014. Quantification of neonicotinoid insecticide residues in soils from cocoa plantations using a QuEChERS extraction procedure and LC-MS/MS. Sci. Total Environ. 499, 276–283. De Cant, J., Barrett, M., 2010. Clothianidin Registration of Prosper t400 Seed Treatment on Mustard Seed (Oilseed and Condiment) and Poncho/votivo Seed Treatment on Cotton. United States Environmental Protection Agency. Dittbrenner, N., Schmitt, H., Capowiez, Y., Triebskorn, R., 2011. Sensitivity of Eisenia fetida in comparison to Aporrectodea caliginosa and Lumbricus terrestris after imidacloprid exposure. Bodymass change and histopathology. J. Soil Sediment 11, 1000–1010.
Fig. 2. Cocoons of E. fetida exposed to difference concentrations of neonicotinoid insecticides.
the residues in soil samples 7 days after application was 4.29 and 7.81 mg/kg, respectively (Sharma and Singh, 2014). The half-life of imidacloprid is longer than 10 days, which means that earthworms could suffer from long term exposure of imidacloprid at a concentration higher than LOEC. In another study, NEOs residues were studied in cucumber fields. The residues of acetamiprid, imidacloprid, nitenpyram, clothianidin and thiacloprid in soil 21 days after the application were 0.28, 0.85, 0.28, 0.27 and 0.38 mg/kg, respectively (AbdelGhany et al., 2016). Compared with the NOEC and LOEC values in the present study, the residue of imidacloprid was higher than LOEC of all the reproduction indicators, which could pose a potential risk to the reproduction of earthworms. In West African cocoa farms (Dankyi et al., 2014), imidacloprid was the most frequently detected NEOs in the soil, in which the concentrations ranged from 0.004 to 0.25 mg/kg. The highest concentration (0.25 mg/kg) of imidacloprid in soil is about the LOEC level (measured concentration: 0.25 mg/kg) in the present study, and reproduction harm was observed at this concentration. Soil samples from a tea plantation treated with clothianidin had average concentraions of up to 0.45 mg/kg (Chowdhury et al., 2012). Ramasubramanian (2013) reported clothianidin concentrations in soils of 0.51–0.88 mg/kg by 3 days after double applications. Potential risks can be expected with multiple applications. Not all of the other members of NEOs were detected as frequently as imidacloprid, but given their excessive use in recent years and similar persistence, long term sub-lethal exposure and combined toxicity to earthworms is expected under practical conditions.
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Dittbrenner, N., Capowiez, Y., Kohler, H.R., Triebskorn, R., 2012. Stress protein response (hsp70) and avoidance behaviour in Eisenia fetida, aporrectodea caliginosa and lumbricus terrestris when exposed to imidacloprid. J. Soil Sediment 12, 198–206. van Gestel, C., de Lima e Silva, C., Lam, T., Koekkoek, J., Lamoree, M., Verweij, R., 2017. Multigeneration toxicity of imidacloprid and thiacloprid to Folsomia candida. Ecotoxicology 26, 320–328. Gomez-Eyles, J.L., Svendsen, C., Lister, L., Martin, H., Hodson, M.E., Spurgeon, D.J., 2009. Measuring and modelling mixture toxicity of imidacloprid and thiacloprid on caenorhabditis elegans and Eisenia fetida. Ecotoxicol. Environ. Safe. 72, 71–79. Goulson, D., 2013. Review: an overview of the environmental risks posed by neonicotinoid insecticides. J. Appl. Ecol. 50, 977–987. Gupta, S., Gajbhiye, V.T., 2007. Persistence of acetamiprid in soil. Bull. Environ. Contam. Toxicol. 78, 349–352. Hamilton, M.A., Russo, R.C., Thurston, R.V., 1977. Trimmed spearman-karber method for estimating median lethal concentrations in toxicity bioassays. Environ. Sci. Technol. 11, 714–719. Hund-Rinke, K., Achazi, R., Rombke, J., Wamecke, D., 2003. Earthworm avoidance test: results of a laboratory comparison test. J. Soil Sediment 3, 7–12. ISO, 2008. Soil Quality: Avoidance Test for Testing the Quality of Soils and Effects of Chemicals on Behavior-Part 1: Test With Earthworms (Eisenia fetida and Eisenia andrei). International Organization for Standardization, Geneve, Switzerland. Jeschke, P., Nauen, R., 2008. Neonicotinoids-from zero to hero in insecticide chemistry. Pest Manag. Sci. 64, 1084–1098. Jeschke, P., Nauen, R., Schindler, M., Elbert, A., 2011. Overview of the status and global strategy for neonicotinoids. J. Agr. Food Chem. 59, 2897–2908. de Lima e Silva, C., Brennan, N., Brouwer, J., Commandeur, D., Verweij, R., van Gestel, C., 2017. Comparative toxicity of imidacloprid and thiacloprid to different species of soil invertebrates. Ecotoxicology 26, 555–564. Lee, K., 1985. Earthworms: their ecology and relationship with soils and land use. Sydney. Liess, M., Beketov, M., 2011. Traits and stress: keys to identify community effects of low levels of toxicants in test systems. Ecotoxicology 20, 1328–1340. Luo, Y., Zang, Y., Zhong, Y.A., Kong, Z.M., 1999. Toxicological study of two novel pesticides on earthworm eisenia foetida. Chemosphere 39, 2347–2356. Manjon, C., Troczka, B.J., Zaworra, M., Beadle, K., Randall, E., Hertlein, G., Saurabh Singh, K., Zimmer, C.T., Homem, R.A., Lueke, B., Reid, R., Kor, L., Kohler, M., Benting, J., Williamson, M.S., Emyr Davies, T.G., Field, L.M., Bass, C., Nauen, R., 2018. Unravelling the molecular determinants of bee sensitivity to neonicotinoid insecticides. Curr. Biol. 28, 1137–1143. Mota-Sanchez, D., Hollingworth, R.M., Grafius, E.J., Moyer, D.D., 2006. Resistance and cross-resistance to neonicotinoid insecticides and spinosad in the colorado potato beetle, Leptinotarsa decemlineata (say) (coleoptera: chrysomelidae). Pest Manag. Sci.
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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Sub-lethal effects of six neonicotinoids on avoidance behavior and reproduction of earthworms (Eisenia fetida)
T
Jing Gea,b, Yuanzhuo Xiaob, Yangyang Chaib, Haijuan Yanb, Ruohan Wub, Xing Xinb, ⁎ Donglan Wangb, Xiangyang Yua,b, a Key Laboratory of Food Quality and Safety of Jiangsu Province-State Key Laboratory Breeding Base/Key Laboratory of Control Technology and Standard for Agroproduct Safety and Quality, Ministry of Agriculture, P.R. China, Nanjing 210014, China b Institute of Food Quality and Safety, Jiangsu Academy of Agricultural Sciences, 50 Zhongling Street, Nanjing 210014, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Neonicotinoids Eisenia fetida Avoidance behavior Chronic toxicity Reproduction
Avoidance behavior of earthworms (Eisenia fetida) against six neonicotinoids (NEOs) (acetamiprid, dinotefuram, clothianidin, thiacloprid, nitenpyram, imidacloprid) was studied following the protocol of ISO. The results showed obvious avoidance behavior of E. fetida against the tested insecticides, and the medium effective concentration for avoidance behavior (EC50) of the six pesticides was 0.14, 0.55, 0.91, 7.87, 1.32 and 0.77 mg/kg, respectively. Compared to the acute toxicity, avoidance behavior was more sensitive as an indicator of soil contamination with NEOs. Chronic toxicity of above six NEOs to E. fetida was also evaluated; cocoon production, hatchability, cocoon weight and adult weight were all affected in the test. Cocoon production and hatchability were more sensitive than cocoon weight and adult weight. The reproduction of earthworms were significantly reduced with a 56 d half-maximal effective hatchability concentration (EC50) of 0.37, 0.74, 1.30, 3.57, 1.20 and 0.70 mg/kg (acetamiprid, dinotefuram, clothianidin, thiacloprid, nitenpyram, imidacloprid), respectively. Most of the tested NEOs were highly toxic to E. fetida. Avoidance behavior and reproduction damage of E. fetida was observed at very low concentrations. The existing levels of pollution with NEOs in soil frequently exceed the lowest observed adverse effect concentrations, which are likely to have negative biological and ecological impacts on earthworms.
1. Introduction Neonicotinoids (NEOs) has been widely used all over the world since they were registered in the early 1990s, for instance, imidacloprid-containing products alone which has been dominating the insecticide market are registered for use on more than 140 crops in 120 countries (Jeschke and Nauen, 2008). Together with other members in neonicotinoid family, such as clothianidin, acetamiprid, dinotefuran, nitenpyram, thiamethoxam, thiacloprid, they represent the best-selling class of insecticide on the global market (Jeschke et al., 2011). The acute toxicity of NEOs to mammals is low, however, NEOs are highly toxic to many invertebrates, including non-target aquatic species (Liess and Beketov, 2011; Pestana et al., 2009; Roessink et al., 2013) and pollinators such as bees (Pisa et al., 2015; Tsvetkov et al., 2017). NEOs are reported to have long half-lives in soil typically from a few days to in excess of 1000 days (range 28–1250 for imidacloprid; 7–353 days for thiamethoxam; 148–6931 days for clothianidin; 3–74 days for thiacloprid and 31–450 days for acetamiprid) (Goulson, 2013), therefore the
⁎
accumulation could easily happen when used repeatedly (Bonmatin et al., 2015). Hence, concerns have been raised regarding the environmental fate and effects of NEOs, such as soil persistence, effects on pollinators and other non-target invertebrates etc. (Goulson, 2013) The toxicity of NEOs to both target and non-target organisms, such as mammals, birds, fish, insects, annelids etc. have been investigated in many studies (De Cant and Barrett, 2010; Luo et al., 1999; MotaSanchez et al., 2006；Renaud et al., 2018). Some of them are proven highly toxic to soil invertebrates (de Lima e Silva et al., 2017) and even show constancy in toxicity for survival and reproduction for three generations (van Gestel et al., 2017). Earthworms play an important role in agricultural soils to maintain and improve soil structure and fertility (Lee, 1985). Earthworms can be exposed to NEOs by direct contact when applied, contaminated soil, seed etc. Globally, about 60% of NEOs is used as a seed dressing in farming (Jeschke et al., 2011). NEOs can easily bind to soil particles at its second phase of its loss from agricultural soil (Goulson, 2013), which can pose a risk to earthworm survival and behavior changes, and
Corresponding author at: Institute of Food Quality and Safety, Jiangsu Academy of Agricultural Sciences, 50 Zhongling Street, Nanjing 210014, China. E-mail address: [email protected] (X. Yu).
https://doi.org/10.1016/j.ecoenv.2018.06.064 Received 10 January 2018; Received in revised form 20 June 2018; Accepted 20 June 2018 0147-6513/ © 2018 Published by Elsevier Inc.
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Table 1 Test concentrations of neonicotinoids in the avoidance test. Pesticides
7 d-LC50 (mg/kg）
Nominal/Measured concentrations (mg/kg.soil dw) 0.5%
Imidacloprid Clothianidin Nitenpyram Acetamiprid Dinotefuran Thiacloprid
4.0 6.0 8.0 1.2 3.0 120
0.02/0.002 0.03/0.02 0.04/0.017 0.006/0.003 0.015/0.003 0.60/0.57
1% 0.04/0.007 0.06/0.03 0.08/0.044 0.012/0.007 0.03/0.01 1.2/1.11
5%
10%
0.2/0.16 0.3/0.24 0.4/0.26 0.06/0.036 0.15/0.096 6.0/6.31
0.4/0.34 0.6/0.5 0.8/0.36 0.12/0.087 0.3/0.21 12/12.8
20% 0.8/0.8 1.2/1.1 1.6/1.01 0.240.18 0.6/0.48 24/20.9
The values of measured concentrations are means of three replicates.
further cause the disruption of soil fertility maintenance processes. The accumulation and persistence of NEOs in soil can extend the exposure period for earthworms (Broznic et al., 2012; Gupta and Gajbhiye, 2007). A range of different endpoints, including mortality, reproduction, growth, molecular response, behavior, have been investigated. Avoidance behavior has been recognized as a more sensitive endpoint than mortality, growth etc. (Hund-Rinke et al., 2003) and may be as sensitive as reproduction (Van Gestel, 1992). Avoidance behavior of earthworms against heavy metals, nanoparticles has been extensively studied (Brami et al., 2017; Syed et al., 2017), however, there are only a handful of studies about avoidance behavior of earthworm against NEOs (Alves et al., 2013; Dittbrenner et al., 2012, 2011; Capowiez and Berard, 2006), and most of them only studied imidacloprid. The purpose of the present study was to comprehensively investigate the effects of 6 widely used NEOs on avoidance behavior of E. fetida at sub lethal concentrations; meanwhile, the effect of long term exposure to low concentrations of 6 NEOs on E. fetida reproduction was also investigated.
Table 2 Test concentrations of neonicotinoids in chronic toxicity test. Pesticides
Imidacloprid Dinotefuran Clothianidin Nitenpyram Acetamiprid Thiacloprid
14d-LC50 (mg/kg)
3.6 3.0 5.5 8.0 1.2 12.0*
Nominal/Measured concentrations (mg/kg.soil dw) 5%
10%
20%
50%
0.18/0.15 0.15/0.096 0.275/0.24 0.40/0.26 0.06/0.036 0.60/0.57
0.36/0.25 0.30/0.21 0.55/0.52 0.80/0.36 0.12/0.087 1.20/1.11
0.72/0.70 0.60/0.48 1.10/0.98 1.60/1.01 0.24/0.18 2.40/2.29
1.80/1.56 1.50/1.10 2.75/2.66 4.00/3.09 0.60/0.49 6.00/6.31
“*” data from the preliminary test; the values of measured concentrations are means of three replicates.
E. fetida earthworms were originally purchased from Jurong Earthworm Farm (Nanjing, China). The earthworms were allowed to acclimatize for 7 days to the artificial climate chamber (20 ± 1 °C with a dark: light ratio of 10:14 h with illumination of 400–800 lx; humidity: 80–85%). Adult earthworms with visible clitellum and an individual wet weight of 300–500 mg were randomly selected for the tests. Artificial soil used for the soil tests consisted of 10% ground sphagnum peat (< 0.5 mm), 20% kaolinite clay (> 50% kaolinite), 70% fine sand (OECD, 1984, 2004) and the water content was adjusted to 30% (OECD, 2004). A small amount of calcium carbonate was added to adjust the pH to 6.0 ± 0.5. The Predicted Environmental Concentration (PEC) of the studied neonicotinoids in soil was calculated. Single application with 75 (imidacloprid), 375 (clothianidin), 75 (nitenpyram), 90 (acetamiprid), 180 (dinotefuran), 500 (thiacloprid) g active ingredient (a.i.) per hectare, homogeneous distribution in the top 5 cm of soil, no crop interception and a soil density of 1.5 kg l−1.
soil according to ISO guidelines (ISO, 2008). Tests were conducted at 5 concentrations based on 7-d LC50, which was 0.5%, 1%, 5%, 10% and 20% of 7-d LC50 (Table 1). A stock solution was used to make dilution series and each replicate of the soil was fortified with test chemicals individually. At the beginning of the avoidance and reproduction experiment, the concentration of NEOs in fortified soil was measured, and the results were also listed in Table 1 and 2, respectively. All the treatments were performed with 4 replicates. A two section unit was used for the test, one half of the unit was filled with 300 g contaminated soil, and the other filled with 300 g control soil. Then the separator in the middle of the unit was removed and 10 earthworms were placed on the separating line. The units were then closed with transparent perforated lids. The tests were conducted in dark in incubators at 20 ± 1 °C for 48 h. The water content of the soil was maintained at 30%. The earthworms were not fed during the test. At the end of 48 h test period, the control and the contaminated soil sections were carefully separated and the number of earthworms was determined in each section of the units. Individuals found between the sections (on the separating line) were counted as 0.5 for each side. Dead earthworms were classified as escaped animals. A control with clean soil (without pesticides) on both sides of the units was also carried out in the test. Dual-control experiment was carried out before the formal test at two concentrations of 0.5% and 20% of 7-d LC50, control with clean soil was also carried out at the same time. The distribution of earthworms in clean soil and contaminated soil was studied.
2.2. Test chemicals
2.4. Reproduction test
Imidacloprid (95.3%), clothianidin (98.0%), acetamiprid (96.8%), dinotefuran (98.0%), nitenpyram (97.5%), thiamethoxam (97.9%), thiacloprid (97.2%) were purchased from Shandong Luba Chemical Co., Ltd. (Shandong, China)
Based on the 14d-LC50 of acute toxicity tests, four different concentrations (5%, 10%, 20% and 50% of 14d-LC50) were set to study the effect of NEOs on reproduction of E. fetida (Table 2). The procedure of dosing soil was the same as described in avoidance test. Each treatment was performed in 4 replicates. Ten adult earthworms (300–500 mg with a clitellum) were selected for reproduction test according to OECD 222 guideline (OECD, 2004). A total of 500 g soil treated with tested chemicals was placed in a 1 L glass jar and 10 adult earthworms were added to each jar. Controls were prepared similarly but without tested
2. Materials and methods 2.1. Test organisms and soil
2.3. Avoidance test Acute toxicity tests were conducted ahead to obtain 7-d LC50 and 14-d LC50 values. The avoidance tests were performed in the artificial 424
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avoidance response of E. fetida to imidacloprid, clothianidin, nitenpyram and dinotefuran was all below 60% at the tested concentrations, while over 60% at (and above) 0.24 and 12.0 mg/kg for acetamiprid and thiacloprid, respectively.
chemicals. The jars was covered with gauze, and stored at 20 ± 1 °C with a relative humidity of 80% under 400–800 lx of constant light. Sterile cow manure at 0.50 g earthworm−1 wk−1 was supplied as food. At day 28, ten adult earthworms were removed from each container; while cocoons in each container were counted, measured and preserved to incubate in the container for another 4 weeks without feeding. At the end of the experiment period, the numbers of juveniles hatched from the cocoons and unhatched cocoons were determined. The adult earthworms were weighed before and after the test. Adult earthworm weight, cocoon number, cocoon weight, hatchability and hatchlings per cocoon were investigated.
3.2. Effect of NEO on reproduction of E. fetida Reproduction parameters of E. fetida exposed to six tested NEOs are listed in Table 4. All the six tested pesticides had significant effect on one or more reproduction parameters of E. fetida. Mean cocoon numbers and hatchability were affected more than the other two parameters. Significant differences were observed of imidacloprid on mean cocoon weight and hatchability at 10% of 14d-LC50 and above. There were significant differences of dinotefuran, nitenpyram, thiacloprid on mean cocoon numbers at 10% of 14d-LC50 and above. All tested pesticides had a highly significant effect on cocoon production at the highest concentration. The EC50 values of hatchability of E. fetida were much lower than 14d-LC50 values of the acute toxicity (Table 5). As shown in Table 5, the avoidance behavior as an endpoint is as sensitive as reproduction. Compared with other NEO, dinotefuran and clothianidin could significantly reduce the body weight of E. fetida at 20% of 14d-LC50 and above. The inhibitory rate of clothianidin on body weight was highest (48%) at 1.1 mg/kg. NOEC and LOEC of different reproduction parameters of NEOs to E. fetida are shown in Table 6. Cocoon production of E. fetida was the most sensitive reproduction indicators for dinotefuran, nitenpyram, acetamiprid and thiacloprid, while juvenile number was the most sensitive indicators for imidacloprid and clothianidin. Other than the indicators mentioned above, NEOs also affected the appearance of the cocoons (Fig. 2). The cocoons were observed to be smaller and thinner with increased concentrations of the tested NEOs.
2.5. Detection of NEO in soil samples The extraction method of NEOs from artificial soil samples was adjusted from Abdel-Ghany's method (2016). The concentration of NEOs in soil samples was detected with an HPLC-MS/MS. The detailed information of instrument parameters can refer to Support information. 2.6. Data assessment Avoidance test: In the avoidance test, for each replicate the net response (NR) (expressed as percentage) was calculated as NR = [(C - T) / 10] × 100, where C = sum of earthworms observed in the control soil; T = sum of earthworms observed in treated soil; 10 = total number of earthworms per replicate. A positive net response indicates avoidance and a negative net response indicates an attraction to the chemical tested in a given concentration. According to ISO guidelines (ISO, 2008), describing the testing of chemicals, attraction reactions (the worms prefer the soil treated with the test chemical) have to be considered as 0% of avoidance. Prior to analysis, data were tested for homogeneity of variance (Levene's test) and for normal distribution (Kolmogorove Smirnov test). The highest no observed effect concentration (NOEC) and lowest observed effect concentration (LOEC) value was determined using ANOVA and Dunnett's test. The median effective concentration of avoidance response (AC50) values and its 95%-confidence limits were determined with Excel 2013 and DPS 7.05 using Trimmed Spearmane Karber method (Hamilton et al., 1977). These calculations were based on the mean net response of the four replicates. Chi-square tests were applied to determine whether the avoidance response of the earthworms towards a test soil was significant or not. The null hypothesis was no significant different between control and test soils, and the alternate hypothesis was that significant different existed in the two soils. When a significant difference of p < 0.05 was obtained, a soil was considered to be avoided. Reproduction toxicity test: The median effective concentration (EC50) values and its 95%-confidence limits were determined with Excel 2013 and DPS 7.05; significance analysis between control and experiment group were performed in SPSS with Dunnett's test (p < 0.05，0.01 or 0.001), and NOEC, LOEC were also calculated in SPSS.
4. Discussion Behavior and reproduction of earthworms can be affected by pesticides at sub-lethal concentrations with long term exposure; however, the actual risk of harmful effects on earthworms still depend on the exposure concentration, exposure duration and route of exposure under field conditions (Pisa et al., 2015). Avoidance response was recently considered as a more sensitive endpoint than many other standard endpoints such as mortality, growth and even reproduction (Syed et al., 2017). In many instances, exposure to NEOs contaminated soils below lowest-observed-effect concentrations (LOECs) for mortality and reproduction can still provoke an avoidance response from earthworms (Alves et al., 2013; Dittbrenner et al., 2012). In this work, the behavioral AC50 was about 5.1–15.2 times lower than the 7 d-LC50 of acute toxicity in the artificial soil test, indicating that avoidance behavior was much more sensitive than survival effects as an endpoint. It was consistent with those previous studies mentioned above. Among the behavioral effects (burrowing, avoidance behavior, cast production and weight change) investigated previously, avoidance behavior ranked the second most sensitive endpoint after burrowing (Pisa et al., 2015). According to the NR equation, the avoidance response of 60% corresponds to 80% of the E. fetida in the control soil, which suggests the contaminated soil is inhospitable for E. fetida to live. Special attention needs to be paid to acetamiprid, because the avoidance response was greater than 60% when the tested concentration was at 10% of 7d-LC50 (measured concentrations: 0.087 mg/kg.soil dw), which is accessible under practical conditions. The results showed the potential to use avoidance behavior as an endpoint for toxicity tests, since it could be linked to effects at the ecosystem level (Capowiez and Berard, 2006). However, the influence (avoidance or attraction) of various odorous compounds (Zirbes et al., 2010) to E. fetida should be taken into account under practical conditions. Only a few studies tested sub-lethal effects of NEOs on earthworm reproduction and the results showed obvious reductions in fecundity at
3. Results 3.1. Effect of neonictinoids on avoidance behavior of E. fetida In the dual control test, 100% survival of E. fetida was recovered. No significant difference (p > 0.05) in the distribution of earthworms was observed between both sides of the chamber with either clean or treated soils. The effects of 6 neonicotinoids on avoidance behavior of E. fetida are shown in Fig. 1 and Table 3. Significant avoidance behavior of E. fetida was observed towards all the tested NEO at even low concentrations (4–20% of 7 d-LC50). The AC50 of acetamiprid, dinotefuran, imidacloprid, clothianidin, nitenpyram and thiacloprid was 0.77, 0.91, 1.32, 0.14, 0.55, 7.87 mg/kg, respectively. The order was in accordance with the acute toxicity test results. As can be seen in Fig. 1, the 425
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Fig. 1. Avoidance or attraction response of E. fetida to 6 neonicotinoids, A: Imidacloprid; B: Clothianidin; C: Nitenpyram; D: Acetamiprid; E: Thiacloprid; F: Dinotefuran. The values are means of four replicates. “*”: p < 0.05; “**”: p < 0.01 (Chi-square tests).
the reproduction test. Among the reproduction parameters measured in the present study (cocoon numbers per earthworm, cocoon weight, hatchability, adult earthworm weight), cocoon production and hatchability were more sensitive than the rest. Similar results have been reported suggesting a significant decrease in cocoon production caused by imidacloprid and thiacloprid at 1.91 and 0.51 mg/kg, respectively (Capowiez and Berard, 2006). Mean cocoon weight and adult weight were affected at comparatively higher tested concentrations. The results from the present study deviated from the previous study, in which mean cocoon weight was more sensitive than cocoon production and hatchability (Wang et al., 2015). The EC50 of the hatchability was in the order of acetamiprid > imidacloprid ≈ dinotefuran > nitenpyram ≈ clothianidin > thiacloprid, which was in accordance with the acute toxicity test. Even though thiacloprid was not a very toxic pesticide to earthworm, it could affect cocoon production at 10% of 14d-LC50 and above (> 1.1 mg/kg, measured concentration). Dinotefuran, the third generation NEO, was also investigated in the present study. The toxicity of dinotefuran ranked in the middle of all the tested NEOs, and it could affect cocoon production significantly at 0.3 mg/kg. Our results showed
Table 3 Effect of 6 neonicotinoids on the avoidance behavior of E. fetida (mg/kg). Pesticides
7 d-LC50a
AC50
NOEC
LOEC
PECsoil
Imidacloprid Clothianidin Nitenpyram Acetamiprid Dinotefuran Thiacloprid
4.0(3.5–4.4) 6.0(5.3–8.6) 8.0(7.3–9.1) 1.2(1.0–1.5) 3.0(2.6–3.6) 120(79.6–196)
0.77 0.91 1.32 0.14 0.55 7.87
0.4 0.6 0.04 0.012 > 0.6 6.0
0.8 1.2 0.08 0.06 > 0.6 12
0.1 0.5 0.1 0.12 0.24 0.67
a
7 d-LC50 of the artificial soil test.
low concentrations (Alves et al., 2013; Gomez-Eyles et al., 2009; Wang et al., 2015; Renaud et al., 2018; de Lima e Silva et al., 2017). Since imidacloprid has been in use for much longer time than other NEO members, its toxicity on earthworms has been extensively studied, however, information on the toxicity of other NEOs on soil fauna in scientific literature have been scant (Goulson, 2013; Renaud et al., 2018). In the present study, 6 commonly used NEOs were all studied in 426
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Table 4 Reproduction parameters measured for E. fetida exposed to difference concentrations of tested neonicotinoids. Concentrations (mg/kg) control 0 Imidacloprid 0.18 0.36 0.72 1.8 Dinotefuran 0.15 0.3 0.6 1.5 Clothianidin 0.275 0.55 1.1 2.75 Nitenpyram 0.4 0.8 1.6 4 Acetamiprid 0.06 0.12 0.24 0.6 Thiacloprid 0.6 1.2 2.4 6.0
Mean cocoons per E. fetida
Mean cocoon weight(mg)
Mean hatchlings per cocoon
Inhibitory rate of adult weight (%)
4.03 ± 0.32
10.93 ± 0.38
2.07 ± 0.08
11.22 ± 5.56
3.90 3.50 2.71 1.03
± ± ± ±
0.10 0.20 1.02* 0.55***
10.50 ± 0.63 9.77 ± 0.49* 9.42 ± 0.51** 8.75 ± 0.25**
1.92 1.77 1.30 1.04
± ± ± ±
0.14 0.16* 0.03*** 0.08***
8.95 ± 0.42 12.90 ± 0.65 20.80 ± 2.71* 28.61 ± 6.35**
3.17 2.55 1.71 1.29
± ± ± ±
0.63 0.22* 0.96** 0.17***
10.55 ± 0.5 9.42 ± 1.00 9.30 ± 0.86 8.76 ± 0.58*
1.95 1.90 0.97 0.17
± ± ± ±
0.15 0.95 0.42 0.16**
14.15 17.41 32.12 31.49
± ± ± ±
1.99 4.12 1.53*** 5.86**
3.54 3.06 2.65 1.94
± ± ± ±
0.19 1.23 0.31 0.54**
10.24 ± 0.87 9.87 ± 1.35 9.17 ± 1.04 8.38 ± 0.54*
2.00 1.46 1.24 0.98
± ± ± ±
0.51 0.22 0.21* 0.18**
12.89 26.47 48.04 30.73
± ± ± ±
4.29 6.69 21.89** 1.58**
3.33 2.45 1.46 0.85
± ± ± ±
0.65 0.76* 0.34** 0.83***
10.55 ± 0.11 10.48 ± 0.46 8.89 ± 1.02* 8.56 ± 0.92**
1.92 1.52 1.08 0.76
± ± ± ±
0.47 0.50 0.22* 0.25**
4.08 ± 14.65 5.32 ± 5.79 13.13 ± 3.06 25.99 ± 4.80
3.55 2.78 2.07 1.15
± ± ± ±
0.72 0.73 0.64** 0.28***
10.39 ± 0.97 9.66 ± 0.25 9.61 ± 1.38 9.07 ± 0.07*
2.02 1.77 1.42 0.89
± ± ± ±
0.14 0.36 0.15** 0.12***
12.24 10.89 14.48 24.41
± ± ± ±
2.43 5.44 5.10 1.65*
3.68 2.82 1.74 0.33
± ± ± ±
0.37 0.26** 0.71*** 0.11***
10.11 ± 0.96 9.69 ± 0.74 9.19 ± 0.73* 8.67 ± 0.76*
1.91 1.60 1.37 0.67
± ± ± ±
0.18 0.33 0.30* 0.29***
10.97 12.42 16.60 25.14
± ± ± ±
7.93 4.06 1.94 1.31*
* p＜0.05. ** p＜0.01. *** p＜0.001; n = 4. Table 5 EC50 (mg/kg) of different endpoints of E. fetida exposed to neonicotinoid insecticides.
Table 6 NOEC and LOEC of different toxicity indicator (cocoon number/cocoon weight/ juvenile number/adult weight) of neonicotinoids to earthworm.
Pesticides
Acute toxicity 14dLC50(95% CI)
Hatchability EC50(95% CI)
Avoidance AC50
Pesticides
14d-LC50 (mg/kg)
NOEC/ LOEC
Cocoon production
Cocoon weight
Juvenile number
Adult weight
Imidacloprid Dinotefuran Clothianidin Nitenpyram Acetamiprid Thiacloprid
3.59(3.07–4.20) 3.0(2.65–3.38) 5.51(4.55–6.66) 8.0(7.17–8.93) 1.12(0.95–1.31) 56.6(34.10–94.47)
0.70(0.5–0.75) 0.74(0.65–0.84) 1.30(1.12–1.53) 1.20(1.06–1.34) 0.37(0.26–0.53) 3.57(2.96–4.29)
0.77 0.91 1.32 0.14 0.55 7.87
Imidacloprid
3.6
Dinotefuran
3.0
Clothianidin
5.5
Nitenpyram
8.0
Acetamiprid
1.2
Thiacloprid
12
NOEC LOEC NOEC LOEC NOEC LOEC NOEC LOEC NOEC LOEC NOEC LOEC
0.36 0.72 0.15 0.3 1.1 2.75 0.4 0.8 0.12 0.24 0.6 1.2
0.18 0.36 0.6 1.5 1.1 2.75 0.8 1.6 0.24 0.6 0.6 1.2
0.18 0.36 0.6 1.5 0.55 1.1 0.8 1.6 0.12 0.24 1.2 2.4
0.36 0.72 0.3 0.6 0.55 1.1 >4 >4 0.24 0.6 2.4 6
that reproductive parameters and avoidance behavior were almost equally sensitive as endpoints for studying the adverse effect of NEOs. The results show that the toxicity of different NEOs to the earthworms can vary by up to a factor of 50, the reason of which is rarely studied. However, there is some research done with other animals, such as bees. The toxicity differences were even found to be about 20 times to earthworms (de Lima e Silva et al., 2017) and 1000 times to bees between imidacloprid and thiacloprid (Manjon et al., 2018). Previous work on honeybees has suggested that it is due to the different rates of these compounds (Brunet et al., 2005; Alptekin et al., 2016), and some of the related enzymes have also been identified recently (Manjon et al., 2018). There is also another assumption about the different toxicity of NEOs to insects. NEOs are nerve toxins which act on the nAChR. Different insect nAChR subunits have different sensitivity to different NEOs; therefore it is likely that different subunits may be activated with distinctive effects on their toxicity (Thany et al., 2006). To avoid or minimize the harm of NEOs to bees, seed coating NEOs formulation was recommended and widely used (Tomizawa and Casida, 2005).
Note: The unit for NOEC and LOEC is mg/kg.
However, this strategy increases the risks to soil fauna, such as earthworms. PEC values of the tested NEOs are shown in Table 3. PEC values of acetamiprid and nitenpyram are greater than their LOEC values of the avoidance test. Special attention needs to be paid to these two NEOs. It is worth noting that the PEC values are calculated with single application, thus the actual concentrations in soil could easily get higher than PCE values with multiple applications. Bonmatin et al. (2005) found that concentrations of imidacloprid in some soil samples exceeded 0.1 mg/kg in UK. Soil dwelling organisms are expected to be exposed to mixtures of NEOs in the range from 0.001 to > 0.1 mg/kg. When imidacloprid was applied at 20 and 80 g active ingredient ha−1, 427
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5. Conclusions In summary, comprehensive study of 6 NEOs on the earthworms’ avoidance behavior and reproduction was carried out in the present work. Avoidance behavior and reproduction harm on the earthworms were observed at very low concentrations. Avoidance behavior was as sensitive as reproduction as a toxicity indicator of NEOs pollution in soil. Both avoidance behavior and reproduction were more sensitive than other indicators such as mortality and growth. Since NEOs are extensively used all over the world and they are persistent in soil, it is easy to have extended exposure period under practical field conditions. Long term low-level concentrations of NEOs in soils may pose high risks to the earthworms by affecting their behavior and reducing their fecundity. Empirical field studies of earthworm responses to realistic field concentrations are still needed to evaluate the risk more accurately. Since the earthworms contribute the most biomass in soil and they are also at the bottom of the land food chain, special attention needs to be paid on the use of NEOs for the sustainable development of the ecological system. The mechanism of different toxicity of different NEOs to earthworms still needs to be further studied. Acknowledgements The work is financially supported by the National Natural Science Foundation of China [grant number: 31601660] and Jiangsu key R & D plan [grant number: B2016367]. Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at doi:10.1016/j.ecoenv.2018.06.064. References Abdel-Ghany, M.F., Hussein, L.A., El Azab, N.F., El-Khatib, A.H., Linscheid, M.W., 2016. Simultaneous determination of eight neonicotinoid insecticide residues and two primary metabolites in cucumbers and soil by liquid chromatography-tandem mass spectrometry coupled with QuEChERS. J. Chromatogr. B 1031, 15–28. Alptekin, S., Bass, C., Nicholls, C., Paine, M.J., Clark, S.J., Field, L., Moores, G.D., 2016. Induced thiacloprid insensitivity in honeybees (Apis mellifera L.) is associated with up-regulation of detoxification genes. Insect Mol. Biol. 25, 171–180. Alves, P.R.L., Cardoso, E.J.B.N., Martines, A.M., Sousa, J.P., Pasini, A., 2013. Earthworm ecotoxicological assessments of pesticides used to treat seeds under tropical conditions. Chemosphere 90, 2674–2682. Bonmatin, J.M., Moineau, I., Charvet, R., Collin, M.E., Fleche, C., Bengsch, E.R., 2005. Behavior of imidacloprid in fields. Toxicity for honey bees. Environmental chemistry: Green Chemistry and Pollutants in Ecosystems. Springer, New York. Bonmatin, J.M., Giorio, C., Girolami, V., Goulson, D., Kreutzweiser, D.P., Krupke, C., 2015. Environmental fate and exposure; neonicotinoids and fipronil. Environ. Sci. Pollut. Res. Int. 22, 35–67. Brami, C., Glover, A.R., Butt, K.R., Lowe, C.N., 2017. Effects of silver nanoparticles on survival, biomass change and avoidance behaviour of the endogeic earthworm Allolobophora chlorotica. Ecotoxicol. Environ. Safe. 141, 64–69. Broznic, D., Marinic, J., Tota, M., Juresic, G.C., Petkovic, O., Milin, C., 2012. Hysteretic behavior of imidacloprid sorption-desorption in soils of croatian coastal regions. Soil Sediment Contam. 21, 850–871. Brunet, J.L., Badiou, A., Belzunces, L.P., 2005. In vivo metabolic fate of [14C]-acetamiprid in six biological compartments of the honeybee, Apis mellifera L. Pest Manag. Sci. 61, 742–748. Capowiez, Y., Berard, 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. Safe. 64, 198–206. Chowdhury, S., Mukhopadhyay, S., Bhattacharyya, A., 2012. Degradation dynamics of the insecticide: clothianidin (Dantop 50 % WDG) in a tea field ecosystem. B. Environ. Contam. Tox. 89, 340–343. Dankyi, E., Gordon, C., Carboo, D., Fomsgaard, I.S., 2014. Quantification of neonicotinoid insecticide residues in soils from cocoa plantations using a QuEChERS extraction procedure and LC-MS/MS. Sci. Total Environ. 499, 276–283. De Cant, J., Barrett, M., 2010. Clothianidin Registration of Prosper t400 Seed Treatment on Mustard Seed (Oilseed and Condiment) and Poncho/votivo Seed Treatment on Cotton. United States Environmental Protection Agency. Dittbrenner, N., Schmitt, H., Capowiez, Y., Triebskorn, R., 2011. Sensitivity of Eisenia fetida in comparison to Aporrectodea caliginosa and Lumbricus terrestris after imidacloprid exposure. Bodymass change and histopathology. J. Soil Sediment 11, 1000–1010.
Fig. 2. Cocoons of E. fetida exposed to difference concentrations of neonicotinoid insecticides.
the residues in soil samples 7 days after application was 4.29 and 7.81 mg/kg, respectively (Sharma and Singh, 2014). The half-life of imidacloprid is longer than 10 days, which means that earthworms could suffer from long term exposure of imidacloprid at a concentration higher than LOEC. In another study, NEOs residues were studied in cucumber fields. The residues of acetamiprid, imidacloprid, nitenpyram, clothianidin and thiacloprid in soil 21 days after the application were 0.28, 0.85, 0.28, 0.27 and 0.38 mg/kg, respectively (AbdelGhany et al., 2016). Compared with the NOEC and LOEC values in the present study, the residue of imidacloprid was higher than LOEC of all the reproduction indicators, which could pose a potential risk to the reproduction of earthworms. In West African cocoa farms (Dankyi et al., 2014), imidacloprid was the most frequently detected NEOs in the soil, in which the concentrations ranged from 0.004 to 0.25 mg/kg. The highest concentration (0.25 mg/kg) of imidacloprid in soil is about the LOEC level (measured concentration: 0.25 mg/kg) in the present study, and reproduction harm was observed at this concentration. Soil samples from a tea plantation treated with clothianidin had average concentraions of up to 0.45 mg/kg (Chowdhury et al., 2012). Ramasubramanian (2013) reported clothianidin concentrations in soils of 0.51–0.88 mg/kg by 3 days after double applications. Potential risks can be expected with multiple applications. Not all of the other members of NEOs were detected as frequently as imidacloprid, but given their excessive use in recent years and similar persistence, long term sub-lethal exposure and combined toxicity to earthworms is expected under practical conditions.
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