Rui Qua,
Shu-Shen Liu*ab,
Fu Chenc and
Kai Lia
aKey Laboratory of Yangtze River Water Environment, Ministry of Education, College of Environmental Science and Engineering, Tongji University, Shanghai 200092, China. E-mail: ssliuhl@263.net; Tel: +86-021-65982767
bState Key Laboratory of Pollution Control and Resource Reuse, College of Environmental Science and Engineering, Tongji University, Shanghai 200092, China
cCollege of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang 310058, China
First published on 15th February 2016
Ionic liquids (ILs) and pesticides may coexist in ecosystems, because more and more people try to extract pesticides from various samples using ILs. Many studies have indicated that the toxicities of ILs and pesticides are time-dependent. However, people know little about the time-dependent toxicity of the mixtures of ILs and pesticides. Hence, the toxicities of two pesticides, two ILs and 20 binary pesticide–IL mixture rays at seven exposure times, 0.25, 2, 4, 6, 8, 10 and 12 h, to Vibrio qinghaiensis sp.-Q67 were determined by time-dependent microplate toxicity analysis. The effect residual ratio (ERR) was used to quantitatively evaluate the toxicological interaction between pesticide and IL. Ten mixture rays exhibited synergism or antagonism at different exposure times. Toxicological interaction is mixture ratio-dependent and time-dependent. These findings suggest that mixtures of IL and pesticide pose a threat to the aquatic environment. People should pay more attention to the environmental effect of mixtures of IL and pesticide.
To effectively analyze the effects of pesticides on organisms, it is necessary to extract and concentrate pesticides from various environmental samples through various extractants such as ionic liquids (ILs).8–10 For example, Parrilla Vazquez et al.11 used a mixture of CH3OH and 1-octyl-3-methyl-imidazolium hexafluorophosphate to extract benzoylurea insecticides from wastewater. Zhang et al.12 used 1-butyl-3-methylimidazolium tetrafluoroborate ([bmim]BF4) to extract triazine herbicides in vegetable samples. Although ILs were considered green solvents due to their negligible vapor pressure and reduced inflammability,13 the toxicities of some ILs with long side-chain on aquatic organism cannot be ignored due to high toxicity to aquatic organism. In recent years, there were many reports on the toxicities of ILs.14–16 Not only that, we also found that some ILs have different concentration-response profiles (CRPs) at different times.17,18 For example, the CRPs of 1-ethyl-3-methylimidazolium chloride ([emim]Cl) and 1-butyl-3-methylimidazolium chloride ([bmim]Cl) on Vibrio qinghaiensis sp.-Q67 (V. qinghaiensis) are from monotonically increasing sigmoid-type curves to non-monotonic biphasic curves (see Fig. 1) when the exposure time is from 15 min to 12 h.17
Hormesis (Fig. 1) having low-concentration stimulation and high-concentration inhibition19 is a very common phenomenon20,21 and contaminants always co-occur in ecosystems.22 Hence, it is worth understanding how compounds inducing hormesis interact with other compounds. Despite the increasing number of mixture toxicity researches about ionic liquids23,24 and many reports on the combined toxicities of pesticides,25,26 few people investigate the mixture toxicity of IL and another chemical. Zhang et al. found that the mixture of aldicarb and 1-benzyl-3-methylimidazolium tetrafluoroborate on the inhibition of bioluminescence of V. qinghaiensis exhibits antagonism, but they didn't study time-dependent combined toxicity of ionic liquid and pesticide.27
The aim of this paper is to examine what toxicological interaction will happen in the mixture of a IL and pesticide. Two pesticides, metalaxyl (MET) and simetryn (SIM),1,28–31 selected are widespread in water environment and two ILs, 1-ethyl-3-methylimidazolium chloride [emim]Cl and 1-ethyl-3-methylimida-zolium bromide ([emim]Br), not only are high water-soluble but also can induce hormesis.18 Considering that freshwater is closely related to people's daily lives and the pollutants in freshwater will directly (drinking and bathing) or indirectly (if water is used to irrigation, it is likely to contaminate soil and crops) affect human health, V. qinghaiensis, a freshwater bioluminescent bacterium, was selected as test organism. To investigate the effect of exposure time on combined toxicity, we used the time-dependent microplate toxicity analysis (t-MTA)17 to determine the toxicity and employed the effect residual ratio (ERR)32 method to evaluate the toxicological interaction at different effect levels.
No. | Chemicals | Abbr. | CAS RN | M.W.a | Purity (%) | Stock (mol L−1) |
---|---|---|---|---|---|---|
a M.W.: molecular weight. | ||||||
1 | 1-Ethyl-3-methylimidazolium bromide | [emim]Br | 65039-08-9 | 191.0 | 98.0% | 2.24 × 10−1 |
2 | 1-Ethyl-3-methylimidazolium chloride | [emim]Cl | 65039-09-0 | 146.6 | 97.0% | 2.82 × 10−1 |
3 | Metalaxyl | MET | 57837-19-1 | 279.3 | 98.7% | 7.16 × 10−3 |
4 | Simetryn | SIM | 1014-70-6 | 213.3 | 97.5% | 1.77 × 10−3 |
Four binary mixture systems, [emim]Br–MET, [emim]Cl–MET, [emim]Br–SIIM and [emim]Cl–SIIM, were constructed by one IL and one pesticide combination. For each mixture system, five rays (noted as R1, R2, R3, R4 and R5) were designed by the direct equipartition ray design (EquRay) procedure.33 The mixture ratios (pi,j),34 the ratio of the concentration of the jth component in the ith ray to the total concentration of the ray, of various components in 20 mixture rays and the concentrations of stocks were listed in Table 2.
No. | Mixture ray | pi,j (i = 1,2,…,20; j = 1,2,3,4) (%) | Concentration of stock (mol L−1) | |||
---|---|---|---|---|---|---|
[emim]Br | [emim]Cl | MET | SIM | |||
1 | [emim]Br–MET-R1 | 98.00 | 2.00 | 4.93 × 10−2 | ||
2 | [emim]Br–MET-R2 | 95.15 | 4.85 | 4.21 × 10−2 | ||
3 | [emim]Br–MET-R3 | 90.74 | 9.26 | 3.43 × 10−2 | ||
4 | [emim]Br–MET-R4 | 83.06 | 16.94 | 2.60 × 10−2 | ||
5 | [emim]Br–MET-R5 | 66.22 | 33.78 | 1.69 × 10−2 | ||
6 | [emim]Br–SIM-R1 | 99.21 | 0.79 | 4.51 × 10−2 | ||
7 | [emim]Br–SIM-R2 | 98.04 | 1.96 | 3.50 × 10−2 | ||
8 | [emim]Br–SIM-R3 | 96.16 | 3.84 | 2.57 × 10−2 | ||
9 | [emim]Br–SIM-R4 | 92.60 | 7.40 | 1.72 × 10−2 | ||
10 | [emim]Br–SIM-R5 | 83.35 | 16.65 | 9.19 × 10−3 | ||
11 | [emim]Cl–MET-R1 | 98.03 | 1.97 | 6.01 × 10−2 | ||
12 | [emim]Cl–MET-R2 | 95.22 | 4.78 | 4.95 × 10−2 | ||
13 | [emim]Cl–MET-R3 | 90.87 | 9.13 | 3.90 × 10−2 | ||
14 | [emim]Cl–MET-R4 | 83.27 | 16.73 | 2.84 × 10−2 | ||
15 | [emim]Cl–MET-R5 | 66.57 | 33.43 | 1.78 × 10−2 | ||
16 | [emim]Cl–SIM-R1 | 99.22 | 0.78 | 9.92 × 10−1 | ||
17 | [emim]Cl–SIM-R2 | 98.07 | 1.93 | 9.81 × 10−1 | ||
18 | [emim]Cl–SIM-R3 | 96.21 | 3.79 | 9.62 × 10−1 | ||
19 | [emim]Cl–SIM-R4 | 92.70 | 7.30 | 9.27 × 10−1 | ||
20 | [emim]Cl–SIM-R5 | 83.56 | 16.44 | 8.36 × 10−1 |
![]() | (1) |
For the non-monotonic biphasic (like J-shaped) CRC, concentration–toxicity data were fitted to the five parameters logistic equation (eqn (2)).37 The physical meaning of five parameters in the logistic equation was illustrated in Fig. 1.
![]() | (2) |
![]() | (3) |
![]() | ||
Fig. 2 Plots of EC10 (![]() ![]() ![]() ![]() ![]() ![]() |
The CRCs of two pesticides are typical monotonic S-shaped at any exposure time and can be described by Weibull function. Plots of the pEC10, pEC50 and pEC70 of two pesticides vs. time were shown in Fig. 3A and B. For MET, the pEC10, pEC50 and pEC70 increase at beginning and are basically unchanged after 2 h. For SIM, pEC10, the pEC50 and pEC70 don't change over exposure times. The fitted CRC model, statistics, EC10, EC50 and EC70 at seven exposure times were listed in Table S2.† The pEC50 of SIM changes over the time, which is consistent with results reported by Wang et al.39 In the present study, both ionic liquids induce hormesis at some exposure times, but the maximum stimulatory effects are different from the literatures. Zhang found that the maximum stimulation of [emim]Cl and [emim]Br at 12 h are 60.7%17 and 104%.18 Wang and we purchased freeze-dried V. qinghaiensis from the same producers, but Zhang purchased it from the other, this is probably the main reason for this results.
In [emim]Br–MET systems, EC10 of R1, R2 and R3 increase from 0.25 to 12 h. EC10 of R4 and R5 are unchanged during the exposure times. EC50 and EC70 of R1 and R2 increase from 0.25 to 2 h, but those of R3, R4 and R5 decrease. EC50 and EC70 of R1, R2, R3 and R4 increase after 4 h, while R5 is unchanged after 2 h.
In [emim]Cl–MET systems, the tendency of EC50 and EC70 between 0.25 and 2 h are similar with [emim]Br–MET. After 2 h, EC50 and EC70 of five rays are stable. EC10 of R1, R2 and R3 increase from 0.25 to 12 h. EC10 of R4 and R5 are unchanged during the exposure times.
In [emim]Br–SIM systems, EC50 and EC70 of R1, R2, R3 and R4 decrease after 4 h, while R5 are relatively constant after 4 h. The EC10 of R1 and R2 increase from 0.25 to 12 h, but the EC10 of R3, R4 and R5 are constant. The ZEP and ECmin of R1 and R2 are constant. The Emin of R1 and R2 both increase over time.
In [emim]Cl–SIM systems, the EC10 of R1, R2 and R3 increase from 0.25 to 12 h. EC10 of R4 and R5 are relatively constant. The EC50 and EC70 of five mixtures rays remain relatively unchange except at 2 h.
Ray | Effect (%) | [emim]Br–MET | [emim]Cl–MET | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
0.25 h | 2 h | 4 h | 6 h | 8 h | 10 h | 12 h | 0.25 h | 2 h | 4 h | 6 h | 8 h | 10 h | 12 h | ||
a —: there is no significant deviation between CA prediction and observation. | |||||||||||||||
R1 | 10 | — | — | — | — | — | — | — | — | — | — | — | — | — | — |
20 | — | — | — | — | — | — | — | — | — | — | — | — | — | — | |
30 | — | 8.28 | — | — | — | — | — | — | 9.86 | — | — | — | — | — | |
40 | — | 29.79 | — | — | — | — | — | — | 24.13 | — | — | — | 8.16 | — | |
50 | — | 37.30 | 8.29 | — | — | — | — | — | 27.24 | — | — | 2.44 | 14.65 | — | |
60 | — | 37.15 | 17.57 | — | — | — | — | — | 24.00 | 3.04 | 2.05 | 7.84 | 19.82 | 6.03 | |
70 | — | 33.23 | 26.24 | 6.40 | — | — | — | — | 18.87 | 6.38 | 2.92 | 11.15 | 18.52 | 8.03 | |
80 | — | 24.89 | 28.65 | 12.13 | — | — | — | — | 11.20 | 5.96 | 2.57 | 9.20 | 16.49 | 11.82 | |
R2 | 10 | — | — | — | — | — | — | −89.77 | — | — | — | — | — | — | — |
20 | — | — | — | — | — | — | −59.09 | — | — | — | — | — | — | — | |
30 | — | 10.71 | — | — | — | — | — | — | 14.58 | — | — | — | — | — | |
40 | — | 22.92 | — | — | — | — | — | — | 25.62 | — | — | — | |||
50 | — | 26.35 | 5.94 | — | — | — | — | — | 28.74 | 2.90 | — | 1.94 | — | ||
60 | — | 23.90 | 10.54 | — | — | — | — | — | 26.43 | 6.92 | — | 4.84 | — | ||
70 | — | 18.13 | 21.03 | 0.82 | — | — | — | — | 21.83 | 8.92 | 2.90 | — | 6.25 | 2.89 | |
80 | — | 8.62 | 22.14 | 5.24 | — | — | — | — | 14.28 | 7.82 | 3.31 | — | 5.30 | 0.03 | |
R3 | 10 | — | — | — | — | — | — | −91.99 | — | — | — | — | — | — | — |
20 | — | — | — | — | — | −69.87 | −67.39 | — | — | — | — | — | — | — | |
30 | — | — | — | — | −53.30 | −52.08 | — | — | — | — | — | — | |||
40 | — | 16.57 | — | — | — | — | −40.29 | — | 7.81 | — | — | — | — | — | |
50 | — | 26.58 | — | — | — | — | −31.06 | — | 11.98 | — | — | — | — | — | |
60 | — | 30.56 | 3.22 | — | — | — | — | — | 13.36 | — | — | — | — | — | |
70 | — | 31.04 | 8.84 | — | — | — | — | — | 12.83 | 3.09 | — | — | — | — | |
80 | — | 26.60 | 12.45 | — | — | — | — | — | 11.06 | 5.06 | — | — | — | — | |
R4 | 10 | — | — | — | — | −99.93 | −95.08 | −94.08 | — | — | — | — | — | −68.94 | −68.94 |
20 | — | — | — | — | −73.49 | −70.17 | −68.60 | — | — | — | — | −57.76 | −47.92 | −47.92 | |
30 | — | — | — | −44.28 | −58.67 | −56.83 | −55.19 | — | — | — | — | −44.99 | −37.08 | −37.08 | |
40 | — | — | — | −35.00 | −47.47 | −46.71 | −45.72 | — | — | — | — | −35.84 | −29.74 | −29.74 | |
50 | — | 4.18 | — | — | −38.71 | −38.73 | −37.75 | — | — | — | — | −28.55 | −23.77 | −23.77 | |
60 | — | 9.79 | — | — | −31.29 | −31.73 | −31.11 | — | — | — | — | −21.91 | −18.63 | −18.63 | |
70 | — | 13.25 | — | — | — | −25.47 | −25.29 | — | — | — | — | — | −14.24 | −14.24 | |
80 | — | 14.72 | — | — | — | — | — | — | 2.41 | — | — | — | — | — | |
R5 | 10 | — | — | — | — | — | — | — | −62.59 | — | — | — | — | — | — |
20 | — | — | — | — | — | — | — | −47.09 | — | — | — | — | — | — | |
30 | — | — | — | — | −44.24 | — | — | −39.36 | — | — | — | — | — | — | |
40 | — | — | — | −29.33 | −37.84 | −35.48 | −33.67 | −33.52 | — | — | −33.52 | −33.28 | −26.21 | −26.21 | |
50 | — | — | — | −25.97 | −33.04 | −31.95 | −30.18 | −29.13 | — | — | −29.42 | −29.79 | −24.51 | −24.51 | |
60 | — | — | — | −23.04 | −29.02 | −28.63 | −26.91 | −23.56 | — | — | −25.93 | −26.62 | −22.71 | −22.71 | |
70 | — | — | — | −19.94 | −24.90 | −25.11 | −23.75 | −19.65 | — | — | −22.41 | −23.09 | −20.29 | −20.29 | |
80 | — | — | — | −16.38 | −20.33 | −20.94 | −19.93 | −14.89 | — | — | −18.49 | −19.02 | −17.09 | −17.09 |
For the [emim]Br–MET systems, the ERR values of five mixture rays at the effect of 50% are positive (synergism) in the beginning and then decrease gradually and change sign as negative values (antagonism). For example, the ERR of R1 at the effect of 50% decreases gradually from 37.30 to 8.29 during 2 to 4 h and changes into zero (6–12 h). The ERR of R2 at the effect of 50% decreases from 26.35 to 5.94 (2–4 h) and changes into zero (6–12 h). The ERR of R5 at the effect of 50% is zero from 0.25 to 4 h and changes into negative (6–12 h).
In [emim]Cl–MET systems, the ERR of R1 is positive (2–12 h) except at 0.25 h. The ERR of R5 is negative at 0.25 h, and changes into zero (2–4 h), and negative (6–12 h). The ERRs of R1 and R2 are positive at most of the exposure times. The ERR of R3 is positive (2–4 h) and there is no deviation at other exposure times. The ERR of R4 is positive at 2 h and turns to negative (8–12 h).
Complex toxicological interaction was identified for [emim]Br–MET and [emim]Cl–MET at different exposure times. Toxicological interaction in the [emim]Br–MET and [emim]Cl–MET mixture system changed with the mixture ratios. For example, according to EC50, the R3 and R4 of [emim]Br–MET showed antagonism but R1 and R2 showed additive action at 12 h. A number of studies have proved that40,41 mixture ratio is an important factor to determine interaction.
Dilipkumar and Chuah42 indicated that the ratio of allelopathic crop water extracts in combination with herbicide is an important factor in influencing the potency of phytotoxic activity. Some people43 found that antagonism was larger for mixtures with higher proportions of mecoprop when they studied combined toxicity of mecoprop and terbuthylazine to Lemna minor. It should be pointed out that CA just predict the effective concentrations at the effects of greater than zero in this paper, because CA predictions are restricted to that the compounds also share an identical range of effects (the same minimum and maximum effect).44 We need further research to understand how to predict the hormesis effect of the mixture.
Toxicological interaction in the [emim]Br–MET and [emim]Cl–MET mixture system also changed over the exposure times. The results indicated that just examining concentration effect relationship of compounds is not enough, we need to understand the time-dependent toxicity if want to optimize risk assessments.45 Inhibition of bioluminescence in the V. qinghaiensis of the two ionic liquids, two pesticides and their mixtures changed over times. Organisms grow during the test could affect dilution and surface area to volume ratio, so that alter the toxicokinetics.46 Literatures47 indicated that even the organisms exposure to a binary mixture with stable concentration, the internal concentration usually change with time.
Footnote |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra27096k |
This journal is © The Royal Society of Chemistry 2016 |