Chemosphere 100 (2014) 111–115

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Comparative and combined acute toxicity of butachlor, imidacloprid and chlorpyrifos on earthworm, Eisenia fetida Chen Chen a,1, Yanhua Wang b,1, Xueping Zhao b, Qiang Wang b,⇑, Yongzhong Qian a,⇑ a Key Laboratory of Agro-Product Quality and Safety of Ministry of Agriculture, Institute of Quality Standards and Testing Technology for Agro-Products, Chinese Academy of Agricultural Sciences, Beijing 100081, China b State Key Laboratory Breeding Base for Zhejiang Sustainable Pest and Disease Control/Key Laboratory for Pesticide Residue Detection of Ministry of Agriculture, Institute of Quality and Standard for Agro-Products, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China

h i g h l i g h t s  An earthworm assay was used to assess the combined toxicity of three pesticides.  Ecotoxicity essays were conducted in artificial soil and contact filter paper test.  9 out of 12 binary combinations were found to have deviation factor of less than 2.  The CA model does not markedly underestimate the toxicity of mixtures.

a r t i c l e

i n f o

Article history: Received 11 June 2013 Received in revised form 26 November 2013 Accepted 4 December 2013 Available online 27 December 2013 Keywords: Butachlor Imidacloprid Chlorpyrifos Acute mixture toxicity Eisenia fetida

a b s t r a c t Various pesticides have become widespread contaminants of soils due to their large applications in agriculture and homes. An earthworm assay was used to assess the acute toxicity of butachlor, imidacloprid and chlorpyrifos with different modes of action. Ecotoxicities of these pesticides were compared for earthworm Eisenia fetida separately and in combination in artificial soil and contact filter paper tests. Imidacloprid was the most toxic for E. fetida with LC50 (lethal concentration 50) values three orders magnitude lower than that of butachlor and chlorpyrifos in both tests. The toxicity of the mixtures was compared to that predicted by the concentration addition (CA) model. According to the CA model, the observed toxicities of all binary mixtures were less than additive. However, for all the mixtures in 14 d artificial soil test, and mixtures of butachlor plus chlorpyrifos and imidacloprid plus chlorpyrifos in 48 h contact filter paper test, the difference in toxicity was less than 30%, hence it was concluded that the mixtures conformed to CA. The combined effects of the pesticides in contact filter paper tests were not consistent with the results in artificial soil toxicity tests, which may be associated with the interaction of soil salts with the pesticides. The CA model provides estimates of mixture toxicity that did not markedly underestimate the measured toxicity, and therefore the CA model is the most suitable to use in ecological risk assessments of the pesticides. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Contaminants in natural systems, especially in agricultural lands, rarely occur as individual chemicals, but rather in mixtures (Altenburger et al., 2000; Backhaus et al., 2000; Muschal and Warne, 2003). Risk assessment from single-chemical exposures may not be useful for predicting toxicity to soil-dwelling organisms exposed to a variety of pollutants simultaneously or sequentially in the field (Jonker et al., 2004). As the awareness ⇑ Corresponding authors. Tel.: +86 10 82106550; fax: +86 10 82106551. E-mail addresses: [email protected] (Q. Wang), [email protected] (Y. Qian). 1 These authors contributed equally to this work. 0045-6535/$ - see front matter Ó 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.chemosphere.2013.12.023

and interest of scientists and regulatory policies in the toxicology and potential risks of combined exposures is growing, it is now widely recognized that the adverse effects caused by exposure to complex mixtures must be an integral part of environmental and human health risk assessment (Groten, 2000). Toxicity experiments using mixtures of contaminants may better reflect the real-world exposures of an ecosystem relative to experiments in which toxicants are tested individually (Spurgeon et al., 1994; Walter et al., 2002; Seghers et al., 2005). It is impossible to test every chemical combination, therefore it is desirable to be able to predict effects of mixtures from the knowledge on effects of single chemicals. For this purpose, a range of mathematical models have been developed (Jonker et al., 2005). Concentration addition (CA), also called dose addition, was

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founded on the assumption that mixture components each possess a similar pharmacological mode of action, and thus is most applicable for toxic substances that have the same molecular target site (Bliss, 1939). This model is based on a dilution principle, and has proven effective in several settings (Altenburger et al., 2000; Backhaus et al., 2000; Barata et al., 2006; Liu et al., 2009; Phyu et al., 2011). Butachlor is a chloroacetanilide herbicide for the control of annual grasses and broadleaf weeds acting by inhibiting elongase responsible for the elongation of very long-chain fatty acids and geranylgeranyl pyrophosphate cyclisation enzymes (Böger et al., 2000). It is applied frequently and extensively in China (Xu et al., 2007). Imidacloprid is the insecticide with the world’s fastest growing sales (Tomizawa and Casida, 2005) and is considered as a possible replacement for organophosphorus pesticides in many countries (Jemec et al., 2007). It is a relatively new systemic insecticide acting as an agonist at nicotinic acetylcholine receptors (nAChRs). Chlorpyrifos is a typical organophosphorus insecticide, known as acetylcholinesterase (AChE) inhibitors. Toxic effects are caused by the chemical disrupting normal nervous system function due to an excessive accumulation of acetylcholine in the synapse (Taylor and Brown, 1999). These pesticides are used widely on agricultural crops and are commonly recorded as contaminants in surface waters and agricultural areas. Although the effects of these pesticides individually on non-target organisms are quite well documented recently (Wang et al., 2012; He et al., 2013), their effects as mixtures have not been examined, despite their likelihood of co-occurrence in the environment (Bowmer et al., 1998). In this study we examined the toxicity of these pesticides as all possible binary mixtures to the earthworm, Eisenia fetida; determined the type of joint action that occurred in these mixtures and whether the use of the CA model of mixture toxicity effectively estimated the risk posed by the mixtures. Acute exposures were used with survival as the determinDaphnia magna ed end point.

refrigerator at 4 °C. The stock solutions were stored for up to 1 month. All working stock solutions were made immediately prior to use.

2.3. Toxicity test methods 2.3.1. Contact filter paper test Contact filter paper test was performed according to the OECD guideline (OECD, 1984). A piece of filter paper was placed in a 9 cm Petridish and treated with the test substance dissolved in 2 mL of acetone. After the solvent was evaporated, the piece of filter paper was remoistened with 2 mL distilled water, and one earthworm was placed on it. The dish was incubated in the light at 20 °C for 48 h and mortality was recorded. An earthworm was considered dead if it failed to respond to a gentle mechanical touch on the front end. Earthworms were held on wet filter paper for 24 h at 20 ± 1 °C in the dark to have the gut contents purged before the dose response test. A preliminary test was conducted to determine a concentration range for each chemical in which a 0–100% mortality of the earthworms was obtained. At least five concentrations and a control were included for each chemical. Ten replicates were used for each concentration. Acetone was used as the control. Treated earthworms were maintained at 20 ± 1 °C under 80–85% relative humidity in the dark.

2.2. Test chemicals

2.3.2. Artificial soil test Artificial soil consisted of 10% ground sphagnum peat (30% kaolinite) and 70% fines and was used for artificial soil tests (OECD, 1984). A small amount of calcium carbonate was added to adjust the pH to 6.0 ± 0.5. In toxicity tests, the water content was adjusted to 35%. For each tested concentration, the desired amount of pesticide was dissolved in 10 mL acetone and mixed into a small quantity of fine quartz sand. The sand was mixed for at least 1 h to evaporate the acetone and then mixed thoroughly with the premoistened artificial soil in a household mixer. The final moisture contents of artificial soil were adjusted to the descried level by the addition of distilled water. A total of 0.65 kg soil (equivalent to 0.5 kg dry artificial soil) was placed in a 500 mL glass jar (surface area 63.6 cm2) and 10 adult earthworms were added to each jar. Controls were prepared similarly but only with 10 mL acetone and no insecticide. The jars were loosely covered with polypropylene lids, allowing exchange of air, and stored at 20 ± 1 °C with 80–85% relative humidity under 400–800 lx of constant light. Survival was assessed at 7 and 14 d intervals after treatment. In a pilot trial, a range of concentrations of 0, 0.1, 1.0, 10, 100 and 1000 mg kg1 dry soil, were used to determine a concentration range that resulted in 0–100% mortality. Six test concentrations and a control were used to obtain the LC50 value. Three jars, each containing 10 adult earthworms, were used for each concentration. The earthworms were preconditioned for 24 h under the same conditions described above in the untreated soil before the dose– response test.

Butachlor (CAS-No. 23184-66-9; 90% TC) was supplied by Hangzhou Qingfeng Chemical Industrial Group (Hangzhou, Zhejiang, China). Chlorpyrifos (CAS-No. 2921-88-2; 96% TC) was purchased from Jiangsu Yangnong Agrochemical Group (Yangzhou, Jiangsu, China). Imidacloprid (CAS-No. 138261-41-3; 105827-789; 95.3% TC) was supplied by Nanjing Red Sun Chemical Co. Ltd. (Nanjing, Jiangsu, China). Stock and working stock solutions of each chemical were prepared in analytical-grade acetone (99% purity) and stored in a

2.3.3. Mixture toxicity On the basis of the measured LC50 values, a mixture ratio where 50% of the effect came from each individual pesticide was chosen. The 50:50% effect mixture ratio was chosen as this is the mixture ratio where the largest deviation from concentration addition can be observed. The 50:50% effects mixture ratio was tested at seven to eight doses with a separation factor of 2. The single pesticides were tested simultaneously.

2. Materials and methods 2.1. Test organisms The E. fetida is an invertebrate currently used for ecotoxicological assessment of substances in soil, which is the Organization for Economic Co-operation and Development (OECD) and International Standardization Organization (ISO) recommended earthworms test species (OECD, 1984, 2004; ISO, 1993). Adult earthworms (weighing between 350 and 500 mg) with well-developed clitella were purchased from the Animal Sciences College, Zhejiang University, China, and cultured in the laboratory in artificial soil according to OECD guidelines (OECD, 1984, 2004). Soils were mixed with decayed leaves and decomposed pig manure, and kept at room temperature (20 ± 1 °C). Soil water content was measured every week and moisture was adjusted to 35% of the maximum water-holding capacity by adding distilled water.

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2.4. Data analysis The concentration of each mixture that caused 50% lethality (LC50) was determined by fitting non-linear regression curves to the toxicity data (Phyu et al., 2011) with Prism 5.0 software. The fit of the optimal model was confirmed by a goodness of fit test. Two-parameter regression models were fitted, assuming a lower limit of 0. Significant level of mean separation (P 6 0.05) was based on non-overlap between the 95% confidence intervals of two LC50 values. The mortality of the earthworms as a function of concentration was described with a dose–response model with an upper limit of 1:

1 y¼  b 1 þ xe The parameter e is the concentration causing 50% death of the earthworms (LC50), and the parameter b denotes the relative slope around e (Nørgaard and Cedergreen, 2010). 2.5. Calculation of the toxicity according to the concentration addition (CA) model To address the toxicity effects of the mixtures or combination of stressors, the observed combined toxic effect was compared with an expected combined effect calculated from the single compound exposure, using the reference conceptual model of the CA described by Belden et al. (2007). This conceptual CA model is defined as a summation of the relative toxicities of the individual components in mixture (Groten, 2000), and is mathematically expressed as:

ECx;mix ¼

n X pi ECx i i¼1

!1

where ECx,mix is the effect concentration of the mixture eliciting x% effect, ECx,i denotes the concentration of the ith component when exists individually and elicits the same effect (x%) as the mixture, pi is the relative mass proportions of the ith component in the mixture. In the present study, for survival data, simply exchange ECx with LC50 (lethal concentration 50). 2.6. Determining the type of mixture joint action To address the toxicity effects of the mixtures of pesticides, the observed combined toxic effect was compared with an expected combined effect calculated from the single pesticide exposures, using the reference CA model. Significant departures from additive toxicity were used to define antagonistic and synergistic interactions between pesticides in mixtures (Hertzberg and MacDonell, 2002). Confidence intervals were determined using a linear interpolation method that did not assume any particular dose–effect model (USEPA, 2002). By substituting upper and lower 95% confidence intervals for the mixture and components into the CA equation, the confidence intervals for the ECx,mix can be determined. If the observed mixture toxicity value is inside of the 95% confidence intervals of the expected value of the concentration addition model then the mixture was considered to conform to the CA model. However, if an observed mixture toxicity value was outside of the 95% confidence intervals of the expected value then the mixture potentially may not conform to the model. This approach to determining conformity to the model can lead to very small biologically insignificant (albeit statistically significant) deviations from the model being classed as antagonistic or synergistic. Therefore, we also required that the expected and observed values differed in toxicity by at least

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30%. To enable a quantitative estimation of the difference in predicted and measured toxicity, the model deviation ratio (MDR) method (Belden et al., 2007) was used. For the CA model, the MDR was derived by dividing the predicted toxicity value (LC50) by the observed toxicity value. MDR values greater than 1.3 mean that the toxicity of the mixture conforms with synergism while values less than 0.7 are taken to conform to antagonism (Phyu et al., 2011). 3. Results 3.1. Single-pesticide exposures The LC50 values and associated parameters for the acute toxicity with artificial soil test (7 d and 14 d) and contact filter paper test (24 h and 48 h) from single exposures are presented in Tables 1 and 2. In artificial soil tests, among the tested pesticides, imidacloprid showed the highest toxicity, which was about two orders of magnitude more toxic than butachlor and chlorpyrifos with LC50 values of 3.15 (7 d) and 2.82 (14 d) mg kg1, followed by chlorpyrifos (with LC50 values of 421.3 and 384.9 mg kg1, respectively, in two time intervals), while butachlor exhibited the lowest toxicity, with LC50 values of 1709.7 and 1197.8 mg kg1, respectively, in 7 d and 14 d toxicity test. In contact filter paper test, imidacloprid also showed the highest acute toxicity. 3.2. Mixture exposures 3.2.1. Artificial soil test In 7 d test, the LC50 value of butachlor in mixtures ranged between 1378.1 to 1728.7 mg kg1 soil. Chlorpyrifos was more toxic than butachlor across all tests, with LC50 values ranging from 322.9 to 444.0 mg kg1 soil. Imidacloprid was the most toxic with LC50 values several orders of magnitude lower, with LC50 values ranging from 2.34 to 4.03 mg kg1 soil (Table 3). Based on 14 d test, lower LC50 values were observed in all the tests, with LC50 values of butachlor between 745.2 and 855.6 mg L1, that of chlorpyrifos ranged from 236.9 to 240.1 mg L1, and that of imidacloprid ranged from 1.72 to 1.99 mg L1, respectively (Table 3). 3.2.2. Contact filter paper test In 24 h test, imidacloprid still exhibited the most severe toxicity with LC50 values (ranging from 5.46 to 12.58 mg L1) several orders of magnitude lower than the other two pesticides. Similar LC50 values were observed for butachlor and chlorpyrifos, ranging from 2736.5 to 5556.2 mg L1 and 3693.8 to 4194.6 mg L1, respectively (Table 3). Based on 48 h test, imidacloprid was also the most toxic chemical, with LC50 values of 0.78 and 1.44 mg L1. While LC50 values for butachlor and chlorpyrifos were from 371.6 to 637.2 mg L1 and 525.1 to 569.4 mg L1, respectively (Table 3). 3.2.3. Concentration addition model For the mixtures in 7 d artificial soil tests, the predicted toxicity was significantly less than observed and had MDR values less than 0.7, suggesting an antagonistic joint action type (Table 4). Although the observed toxicity for all the binary pesticide combinations was less than additive in 14 d tests, the difference in toxicity was less than 30% (MDR = 0.71, 0.80, and 0.82, respectively), hence it was concluded that these mixtures conformed to CA. For the mixtures in contact filter paper tests, the expected toxicities of all the mixtures in 24 h test, and butachlor plus imidacloprid in 48 h test was significantly less than observed and had MDR values less than 0.7 and so was considered as antagonistic (Table 4).

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Table 1 Summary of parameters for the acute toxicity with artificial soil test of three pesticides to E. fetida. Pesticides

Butachlor Imidacloprid Chlorpyrifos

7d

14 d 2

Slope (SE)

Df (x )

LC50 (95% CI), mg kg

Slope (SE)

Df (x2)

LC50 (95% CI), mg kg1

5.04 (0.89) 12.04 (1.85) 12.09 (1.87)

4 (0.44) 4 (2.10) 4 (0.16)

1709.7 (1282.4–3016.4) 3.15 (2.86–3.71) 421.3 (380.7–501.9)

4.88(0.69) 9.99(1.37) 10.95(1.59)

4 (4.39) 4 (1.24) 4 (0.93)

1197.8 (993.8–1614.3) 2.82 (2.61–3.17) 384.9 (353.5–440.3)

1

Table 2 Summary of parameters for the acute toxicity with contact filter paper test of three pesticides to E. fetida . Pesticides

Butachlor Imidacloprid Chlorpyrifos

24 h

48 h

Slope (SE)

Df (x2)

LC50 (95% CI), mg L1

Slope (SE)

Df (x2)

LC50 (95% CI), mg L1

3.78 (0.74) 3.39 (0.88) 5.87 (1.89)

4 (1.78) 4 (0.13) 4 (0.032)

1368.4 (1017.2–2368.6) 5.84 (3.39–29.97) 5654.4 (2940.8–9584.9)

3.41 (0.61) 3.63 (0.66) 3.24 (0.57)

4 (0.29) 4 (0.62) 4 (1.44)

662.9 (503.2–873.4) 1.50 (1.16–1.99) 1015.9 (756.1–1334.4)

Table 3 The concentrations of butachlor, imidacloprid, and chlorpyrifos within binary mixtures producing 50% mortality (LC50) to E. fetida and the 95% confidence interval (95% CI) in 50:50% effect experiments. Compound

Butachlor Imidacloprid Butachlor Chlorpyrifos Imidacloprid Chlorpyrifos

Artificial soil test (95%CI), mg kg1

Contact filter paper test (95% CI), mg L1

7d

14 d

24 h

48 h

1728.7 (1260.4–3426.2) 4.03 (2.94–7.98) 1378.1 (1062.2–2175.8) 444.0 (342.2–701.1) 2.34 (1.98–3.08) 322.9 (273.5–425.2)

855.6 (718.1–1070.6) 1.99 (1.67–2.49) 745.2 (639.5–890.8) 240.1 (206.1–287.0) 1.72 (1.50–2.03) 236.9 (206.7–279.5)

5556.2 (2836.2–27843.7) 12.58 (6.42–63.04) 2736.5 (1551.4–10572.1) 4194.6 (2378.1–16205.4) 5.46 (3.23–17.12) 3693.8 (2186.7–11590.4)

637.2 (445.8–1599.1) 1.44 (1.01–3.62) 371.6 (285.2–557.1) 569.6 (437.2–653.9) 0.78 (0.59–1.12) 525.1 (405.1–761.6)

Table 4 Summaries of mixture toxicity assessments with artificial soil and contact filter paper tests using the concentration addition model for binary pesticide mixtures. Pesticide mixtures

Expected LC50

Observed LC50

7 d artificial soil test Butachlor + Imidacloprid Butachlor + Chlorpyrifos Imidacloprid + Chlorpyrifos

756.5 979.7 215.5

1732.7 1822.1 325.2

14 d artificial soil test Butachlor + Imidacloprid Butachlor + Chlorpyrifos Imidacloprid + Chlorpyrifos

603.9 790.8 194.7

857.6 985.3 238.6

MDR

Type of combined action

Expected LC50

MDR

Type of combined action

0.44 0.54 0.66

Antagonistic Antagonistic Antagonistic

24 h contact filter paper test 896.1 5568.8 2528.1 6931.1 2329.2 3699.3

0.16 0.39 0.63

Antagonistic Antagonistic Antagonistic

0.71 0.80 0.82

Concentration addition Concentration addition Concentration addition

48 h contact filter paper test 332.2 638.6 839.4 941.2 508.7 525.9

0.52 0.89 0.97

Antagonistic Concentration addition Concentration addition

Although the observed toxicity of the butachlor plus chlorpyrifos, and imidacloprid plus chlorpyrifos was less than additive, however, the difference in toxicity was less than 30% (MDR = 0.89 and 0.97) in 48 h test, hence it was concluded that these pairings conformed to CA. 4. Discussion The results from the present study showed that five of the pesticide mixtures conformed to the CA model. The relatively low percentages of mixtures that conform to the CA model could be interpreted as demonstrating the inadequacy of this model for ecological risk assessment of pesticide mixtures. While the CA model predicted or over-predicted the toxicity of all three tested mixtures (Norwood et al., 2003; Phyu et al., 2011). Therefore, risk assessments based on the CA model would provide an environmentally conservative (protective) assessment of the toxicity of the mixtures. Belden et al. (2007) found that eighty-eight percent of all experiments that evaluated the CA model had observed effective concentrations within a factor of 2 of predicted values. It is consistent with the results in the current study, in which 9 out of

Observed LC50

12 binary combinations were found to have MDR values between 0.5 and 1. Although the components of the tested pesticides mixtures may have different modes of action, CA model exhibited well performance to predict the mixture toxicity with the assumption that chemicals have the same mode of action (Backhaus et al., 2004; Belden et al., 2007; Liu et al., 2009). The results of mixture effect in contact filter paper test were consistent with the results in artificial soil toxicity tests by the concentration addition approach (Table 4). This antagonistic effect in soil toxicity tests may be associated with the interaction of soil salts with pesticides, but further mechanistic study is needed. As total chemical measures are inadequate for expressing the chemical exposure of earthworms in soil due to various abiotic and biotic modifying factors, it is necessary to include chemical bioavailability in the expression of exposures. Therefore, bioavailability needs to be considered for understanding exposures in soil systems and that biological and chemical measures of bioavailability must be correlated (Lanno et al., 2004). Since the compounds are present as a mixture in the environments, combined effects of pesticides mixtures should be taken into account to ecological risk assessment in soil environment.

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The artificial soil toxicity test need to be performed to predict the effect of soil contaminants in a real world situation since the filter contact test may have a poor relation of the soil tests (Heimbach, 1984). However, the filter paper test was included in the present study to understand the interaction of pesticides by eliminating the other effects associated with soil properties. Our work indicated that combined toxicity as well as comparative toxicity is influenced by bioassay procedures such as filter paper test and artificial soil toxicity test, and that soil toxicity test should be conducted to predict the mixture toxicity. 5. Conclusions The toxicity and type of joint action of binary mixtures of three pesticides (butachlor, imidacloprid and chlorpyrifos) to the earthworm E. fetida were determined using the concentration addition model in both contact filter paper and artificial soil test. The mixtures conformed to antagonism or additivity based on the CA model and 9 out of 12 binary combinations were found to have MDR values between 0.5 and 1. Besides, the CA model does not markedly underestimate the toxicity of any tested mixture. Therefore, the CA model accurately predicted the toxic effects of mixture to E. fetida. Acknowledgments The research was supported by Zhejiang Provincial Natural Science Foundation (No. LY13C03006), Opening Project Fund of State Key Laboratory Breeding Base for Zhejiang Sustainable Pest and Disease Control (No. 2010DS700124-KF1306) and the Innovation Project of Zhejiang Academy of Agricultural Sciences. References Altenburger, R., Backhaus, T., Boedeker, W., Faust, M., Scholze, M., Grimme, L.H., 2000. Predict ability of the toxicity of multiple chemical mixtures to Vibrio fischeri: mixtures composed of similarly acting chemicals. Environ. Toxicol. Chem. 19, 2341–2347. Backhaus, T., Altenburger, R., Boedeker, W., Faust, M., Scholze, M., Grimme, L.H., 2000. Predictability of the toxicity of a multiple mixture of dissimilarly acting chemicals to Vibrio fischeri. Environ. Toxicol. Chem. 19, 2348–2356. Backhaus, T., Arrhenius, A., Blanck, H., 2004. Toxicity of a mixture of dissimilarly acting substances to natural algal communities: predictive power and limitations of independent action and concentration addition. Environ. Sci. Technol. 38, 6363–6370. Barata, C., Baird, D.J., Nogueira, A.J.A, Soares, A., Riva, M.C., 2006. Toxicity of binary mixtures of metals and pyrethroid insecticides to Daphnia magna Straus. Implications formulti-substance risks assessment. Aquat. Toxicol. 78, 1–14. Belden, J., Gilliom, R., Lydy, M., 2007. How well can we predict the toxicity of pesticide mixtures to aquatic life? Integrat. Environ. Assess. Manage. 3, 364– 372. Bliss, C.I., 1939. The toxicity of poisons applied jointly. Ann. Appl. Biol. 26, 585–615. Böger, P., Matthes, B., Schmalfuß, J., 2000. Review: towards the primary target of chloroacetamides-new findings pave the way. Pest Manage. Sci. 56, 497–508. Bowmer, K.H., Korth, W., Scott, A., McCorkelle, G., Thomas, M., 1998. Pesticide monitoring in the irrigation areas of south-western NSW, 1990–1995. CSIRO Land and Water, Australia, p. 156. Groten, J.P., 2000. Mixtures and interactions. Food Chem. Toxicol. 38, S65–S71.

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