Predicting Hormetic Effects of Ionic Liquid Mixtures ... - ACS Publications

Dec 31, 2010 - The concept of hormesis has generated considerable interest within the environmental and toxicological communities over the past decade...
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Predicting Hormetic Effects of Ionic Liquid Mixtures on Luciferase Activity Using the Concentration Addition Model Hui-Lin Ge,† Shu-Shen Liu,*,†,‡ Xiang-Wei Zhu,† Hai-Ling Liu,‡ and Li-Juan Wang† †

Key Laboratory of Yangtze River Water Environment, Ministry of Education, College of Environmental Science and Engineering, Tongji University, Shanghai 200092, China ‡ State Key Laboratory of Pollution Control and Resource Reuse, College of Environmental Science and Engineering, Tongji University, Shanghai 200092, China ABSTRACT: The concept of hormesis has generated considerable interest within the environmental and toxicological communities over the past decades. However, toxicological evaluation and prediction of hormesis in mixtures are challenging and only just unfolding. The hormetic effects of ten ionic liquids (ILs), singly and in mixtures in the ratios of their individual EC50, EC10, EC0, and ECm (maximal stimulatory effect concentration), on luciferase luminescence were determined by using microplate toxicity analysis. There was good agreement between the effects observed and predicted by concentration addition (CA) for all four mixtures. This evidence supports the use of CA model as a default approach for assessing the combined effect of chemicals at the molecular level. Focusing on the selected points of the concentration-response curves (CRCs) of mixtures, the mixtures of IL chemicals mixed at concentrations that individually showed stimulatory effects could produce inhibitory or no effects, and the mixture of IL chemicals mixed at concentrations that individually showed no effects could produce significant inhibitory effect. The three interesting phenomena in mixture hormesis may have important implications for current risk assessment practices.

’ INTRODUCTION Hormesis is a dose-response phenomenon characterized by low-dose stimulation and high-dose inhibition,1 which typically has a biphasic characteristic in the dose-response relationship as a J-shaped curve.2 In recent decades, a growing body of evidence has accumulated on hormetic effects of several chemicals for a number of biological end points of cellular systems and organisms.3 However, most of these reports have focused on individual chemicals, and mixtures represent the prevailing form of environmentally occurring contaminants such as in wastewaters, combustion exhausts, and industrial sludge.4 Only a few studies have been devoted to hormetic effects of mixtures of municipal solid waste landfill leachates,5 polychlorinated biphenyls (PCBs),6 four metals (As, Cd, Cr, and Pb),7 and persistent organic pollutants (POPs).8 Important questions are (i) Can the size and concentration range of the hormetic stimulation be predicted in mixtures of compounds?; (ii) Do we need to use biphasic concentrationresponse models for adverse effect risk assessment purposes?;9 and (iii) can the effect of a mixture of compounds with stimulatory and inhibitory effects still be predicted? Although these issues have been addressed in binary mixture inducing hormesis,10 it is necessary to further explore and validate this for the mixtures with more components. Toxicological evaluation and prediction of hormesis in mixtures are challenging and only just now unfolding.11 The evaluation and prediction of mixture effects are commonly based on two reference models for no interaction: concentration addition (CA) and independent action (IA). CA model assumes that the compounds interact with the same molecular target by means of the same mechanism.12 Alternatively, IA model assumes that the compounds act independently and have different modes of action.13 Commonly, CA and IA are used to r 2010 American Chemical Society

evaluate and predict the combined effects of compounds with monotonically S-shaped concentration-response curves (CRCs). Only in a few examples were CA and IA applied to mixtures of compounds showing J-shaped CRCs.9,11 Because the IA model will lose its probabilistic meaning when negative values (often referred to as a stimulatory response) are included,14 we only used the CA model to evaluate and predict the hormetic effects of mixtures in the present study. Ionic liquids (ILs) are a type of organic salts with low melting point and considered as green replacements for industrial volatile organic compounds (VOCs). The perceived environmentally friendly nature of ILs due to their negligible vapor pressure is now under scrutiny because they will probably enter the nonatmospheric environment such as water and soil15 and further cause toxicity to various organisms such as enzymes, bacteria, algae, mammalian cells, plants, invertebrates, and vertebrates.16 However, most of these studies focused on the toxicity of single ILs. Only a few studies were devoted to the assessment of biological effects of IL mixtures.17-19 Combined exposure to chemicals such as ILs in the environment is undoubtedly the rule. Before the likely industrial release of ILs into the environment, it is therefore necessary to determine how to predict their mixture effects. It was observed that more and more ILs present hormetic effects at the cellular level,20-24 and this observation was the basis of this. The aim of this paper was to examine if the hormetic effects of ILs occur at molecular level and if the hormetic mixture effects can be predicted by the CA model. This was achieved by Received: June 3, 2010 Accepted: December 14, 2010 Revised: December 11, 2010 Published: December 31, 2010 1623

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For J-shaped concentration-response model, a and b are the first median and slope in low concentration region, p and q are the second median and slope in high concentration region, m is the bottom, R2 is coefficient of determination, and RMSE is root-mean-square error. f is geometric dilution factor. All the units of C0, Cmax, Cmin, EC50, EC10, EC0, and ECm are mol/L. C0 is stock concentration, Cmax is maximum concentration tested, Cmin is minimum concentration tested, ECm is maximal stimulatory effect concentration, Em is maximal stimulatory effect, and EC50, EC10, and EC0 are the concentrations corresponding to 50, 10, and 0% effect.

-0.3266 0.9961 0.0136 0.57 2.67  10-1 1.07  10-1 2.14  10-4 1.07  10-1 6.10  10-2 4.73  10-2 1.47  10-2 -15.4 Mixm ECm ratio mixture 2.869  10

a

-0.2241 0.9989 0.0080 0.57 2.55  10-1 1.02  10-1 2.04  10-4 9.20  10-2 5.50  10-2 4.24  10-2 1.34  10-2 -12.9 16.34

11.85 109.3

8.836  10

-2

8.211  10 143.5 4.011  10 EC0 ratio mixture Mix0

-3

-0.3742 0.9982 0.0105 0.57 2.56  10-1 1.02  10-1 2.05  10-4 9.34  10-2 5.42  10-2 4.30  10-2 1.49  10-2 -16.3

-2

Mix10 EC10 ratio mixture 2.946  10

98.69 7.514  10

-3

13.29

-0.3336 0.9985 0.0093 0.57 2.70  10-1 1.08  10-1 2.16  10-4 9.66  10-2 5.30  10-2 4.01  10-2 1.46  10-2 -12.6 12.37

-2

96.10 7.863  10

-3

Mix50 EC50 ratio mixture 2.610  10

-0.5990 0.9975 0.0290 0.60 4.43  10-1 1.77  10-1 7.08  10-4 9.08  10-2 6.81  10-2 6.25  10-2 1.53  10-2 -48.3 19.89 4.164  10

-2

7.365  10 [HMIM]Cl IL10

148.5

-0.1484 0.9949 0.0290 0.70 1.76  10-2 7.05  10-3 1.41  10-4 5.09  10-3 4.02  10-3 3.60  10-3 1.30  10-3 -14.1

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-3

-2 -3

6.379  10-4 3071 [OMIM][BF4] IL9

4.910  10-3 632.5

26.65 -0.2748 0.9941 0.0202 0.66 1.67  10-1 6.66  10-2 6.66  10-4 6.40  10-2 4.26  10-2 3.58  10-2 7.56  10-3 -21.1 6.775 -0.5744 0.9996 0.0098 0.57 1.09 4.37  10-1 8.75  10-4 1.50  10-1 8.20  10-2 6.47  10-2 2.52  10-2 -15.1 2.466  10 3.267  10-4 [HMIM][BF4] [EPY]Cl

351.5 5.684  10 40.17 1.005  10-1

-0.2434 0.9948 0.0161 0.70 1.96  10-1 7.85  10-2 1.57  10-3 8.16  10-2 4.75  10-2 3.63  10-2 7.81  10-3 -14.8 17.35

IL7 IL8

-2

7.166  10 2.329  10

-3

[HPY]Cl IL6

307.9

-0.3456 0.9947 0.0351 0.57 8.56  10-2 3.42  10-2 6.85  10-5 1.92  10-2 1.51  10-2 1.39  10-2 3.45  10-3 -32.2 1.742  10-2 129.8

-2 -3

1.072  10-3 [OMIM]Cl IL5

880.2

-9.3

-0.3989 0.9994 0.0057 0.70 3.09  10-1 1.24  10-1 2.47  10-3 1.23  10-1 7.54  10-2 6.20  10-2 2.56  10-2 -16.3 10.66 6.505  10 [BMIM]Cl IL4

49.85 9.948  10

-2 -3

9.976 -0.4830 0.9968 0.0212 0.70 3.92  10-1 1.57  10-1 3.13  10-3 9.79  10-2 4.95  10-2 3.64  10-2 1.64  10-2 64.16 6.843  10-2 1.062  10-3 [BMIM]Br IL3

-15.2 1.11  10 3.87  10 5.03  10 8.90  10 1.78  10 8.92  10 -0.3230 0.9964 0.0132 0.57 2.23  10 14.04

8.863 -0.3304 0.9910 0.0168 0.57 3.18  10-1 1.27  10-1 2.54  10-4 1.50  10-1 8.86  10-2 7.05  10-2 1.72  10-2 -18.6 1.248  10-1

7.364  10

101.5

123.9

2.678  10-3

[EMIM][CF3SO3] 8.267  10 IL1

[EMIM]Cl

-2 -4

IL2

Em(%)

-2

ECm

-2

EC0

-2

EC10

-2

EC50

-4

Cmin

-2

Cmax

-1

C0 f RMSE R2 m q p b a IL or mixture no.

Table 1. Concentration-Response Models of Individual Ionic Liquids and Their Mixtures on the Inhibitory Effects of Luciferase Luminescence and Some Statisticsa

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investigating the effects of ten ILs and their mixtures on luciferase luminescence, comparing the effects of individual ILs to the effects of their mixtures, and comparing the mixture effects observed to those predicted by the CA model.

’ EXPERIMENTAL SECTION Reagents. Most of the ILs presenting the hormesis are imidazolium and pyridinium ILs which are water-stable and widely used in industry. For CA, the identical mechanisms of action are uniformly regarded as prerequisites. Consequently, eight imidazolium ILs were selected as the mixture components based on their chemical similarities. To maximize the number of mixture components, two pyridinium ILs were also included in the list of test chemicals. The ten ILs included five from Acros (Geel, Belgium), 1-ethyl3-methylimidazolium chloride ([EMIM]Cl, 98.2% purity), 1-butyl-3-methyl-imidazolium bromide ([BMIM]Br, 97.6% purity), 1-butyl-3-methylimidazolium chloride ([BMIM]Cl, 99% purity), 1-hexylpyridinium chloride ([HPY]Cl, 98.3% purity), and 1-ethylpyridinium chloride ([EPY]Cl, 98% purity); four from Merck (Darmstadt, Germany), 1-ethyl-3-methylimidazolium trifluoromethanesulfonate ([EMIM][CF3SO3], 99.1% purity), 1-octyl-3methylimidazolium chloride ([OMIM]Cl, g 98% purity), 1-hexyl3-methyl-imidazolium tetrafluoroborate ([HMIM][BF4], 99% purity), and 1-hexyl-3-methyl-imidazolium chloride ([HMIM]Cl, 99% purity); and one from Strem (Newburyport, MA), 1-methyl3-octylimidazolium tetrafluoroborate ([OMIM][BF4], 98% purity). The IL stock solutions were prepared by dissolving a proper amount (always lower than their water solubility) in Milli-Q water and stored in the dark at 4 °C. The stock solutions of IL mixtures were prepared by directly mixing the stock solutions of single ILs according to the concentration ratios assigned. The stock concentrations (C0) of ten ILs and their mixtures are listed in Table 1. Luciferase Toxicity Test. The toxicities of single substances and mixtures were expressed as percentage inhibition of the cellfree luciferase luminescence system. Luciferase luminescence is a process in which the luciferase catalyzes the oxidation of the substrate D-luciferin and the energy transfer from ATP to D-luciferin to yield light.25 Because the luciferase luminescence can be affected by chemicals, it was applied as a test system to characterize the toxicities of single compounds26 and mixtures.27 The chemicals used in the luciferase luminescence system include adenosine-50 -triphosphate (ATP-Na2, Sigma-Aldrich, St. Louis, MO, g 98.0% purity), the QuantiLum recombinant luciferase (cloned from North American firefly Photinus pyralis, catalog no. E1701, Promega, Madison, WI, > 95% purity), endotoxin-free D-luciferin (catalog no. E6551, Promega, Madison, WI, g 98.5% purity), and the glycylglycine buffer28 (pH 7.8, consisting of 50 mmol/L glycylglycine, 1 mmol/L MgSO4, 0.5 mmol/L EDTA, and 10 mmol/L DTT). Luciferase, luciferin, and ATP were separately stored in the glycylglycine buffer. It was found that the luciferase luminescence monotonously increased with the luciferase concentration (10-13 to 10-7 mol/L) or luciferin concentration (10-11 to 10-4 mol/L). However, ATP exerted a biphasic response on the luciferase luminescence, and the maximum relative light units (RLU) occurred at an ATP concentration of 1.1  10-4 mol/L. The final optimal conditions used in our luciferase toxicity test were as follows: the luciferase concentration of 6.6  10-8 mol/L, luciferin concentration of 1624

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Table 2. Concentration Ratios of Various ILs in Four Mixtures no. IL1

Mix50 1.02  10

Mix10 -1

Mix0 -2

9.62  10

Mixm -2

8.48  10-2

9.12  10

IL2 IL3

-1

1.72  10 1.12  10-1

-1

1.69  10 9.46  10-2

-1

1.66  10 8.58  10-2

1.31  10-1 1.25  10-1

IL4

1.41  10-1

1.44  10-1

1.46  10-1

1.96  10-1

2.21  10

-2

-2

-2

2.64  10-2

9.37  10

-2

-2

5.97  10-2

7.35  10

-2

-2

5.77  10-2

1.72  10

-1

-1

1.92  10-1

5.85  10

-3

-3

9.93  10-3

1.04  10

-1

-1

1.17  10-1

IL5 IL6 IL7 IL8 IL9 IL10

2.89  10

-2

9.08  10

-2

8.14  10

-1

1.57  10

-3

7.68  10

-1

1.30  10

3.28  10 8.55  10 8.44  10 1.52  10 8.48  10 1.47  10

1.3  10-5 mol/L, ATP concentration of 1.1  10-4 mol/L, pH 7.8, and 15 min exposure at 25 °C. Microplate Toxicity Analysis. According to the microplate toxicity analysis (MTA) developed in our previous study,29 the RLUs of the luciferase exposed to single ILs and their mixtures were determined on SpectraMax M5 reader (Molecular Devices Inc., USA) with a 96-well white flat-bottom microplate (Corning, USA). Twelve concentration series of IL chemicals and their mixtures in triplicate and twelve controls were arranged in a microplate. First, 100 μL of Milli-Q water was added to twelve wells of the first row in the microplate as controls, 100 μL solutions of IL chemicals and their mixtures of twelve gradient concentrations according to geometric dilution factors f (Table 1) were added to twelve column wells from the second to the fourth row. Then, 50 μL of ATP of 5.5  10-4 mol/L, 50 μL of luciferin of 6.5  10-5 mol/L (1 mg/50 mL), and 50 μL of luciferase of 3.3  10-7 mol/L (1 mg/50 mL) were added into each test well to reach the final test volume of 250 μL. Each microplate test was repeated at least twice. The RLUs of various treatments (with IL or mixture) and controls (without any IL) were determined after the exposure of 15 min at 25 °C. The toxicity of a treatment is expressed as an inhibition (E of x%), which is calculated as follows: E ¼ x% ¼ ð1 - L=L0 Þ  100%

ð1Þ

where L0 is an average of RLUs of the controls (12 replicates), L is an average of RLUs of the treatments (3 replicates). Statistical Analysis. The J-shaped CRC is described by eq 2 derived from a seven parameters logistic equation30 where the first top asymptote is fixed at zero and the second top asymptote is fixed at 100%. E ¼ m-

m 1-m þ bðC aÞ 1 þ 10 1 þ 10qðp - CÞ

ð2Þ

where a and b are the first median and slope in low-concentration region, p and q the second median and slope in high-concentration region, m is the bottom, and C is the concentration of test chemicals. The regression analysis was performed using nonlinear least-squares fit. The higher the coefficient of determination (R2) and the lower the root-mean-square error (RMSE), the better the fit. As a quantitative measure of the uncertainty, the observation-based 95% confidence interval was also determined.31 Experimental Design and Toxicity Prediction of Mixtures. Three equivalent effect concentration ratio (EECR) mixtures

Figure 1. J-shaped concentration-response curves for the inhibitory effects of ten ionic liquids on luciferase luminescence after 15 min of exposure. 0: Control; O: experimental data; -: biphasic model fit.

(named as Mix50, Mix10, and Mix0) and one ECm ratio mixture (Mixm) were designed. In the mixtures, the concentration ratios (Table 2) of various ILs are the ratios of their individual EC50, EC10, EC0 (zero or no effect concentration), and ECm (maximal stimulatory effect concentration) to the total concentration of the mixtures, respectively. It should be indicated that the Mixm is not an EECR mixture. The Mix0 and Mixm were especially designed for the nonmonotonic J-shaped CRCs. The aim of designing the Mix50 and Mix10 was to compare the mixture toxicity of ILs with J-shaped CRCs to those with classical S-shaped CRCs. Also, three EECR mixtures could be used to validate the hypothesis that a n-component mixture whose concentration equals the sum of ECx,i/n of the individual components will exactly produce an effect of x%, so meeting the assumption of CA.32 The CA model was employed to predict the mixture toxicity of ILs with J-shaped CRCs and the model can be formulated as eq 3:33 ECx, mix ¼ 1625

n X pi EC x, i i¼1

!-1 ð3Þ

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Figure 2. J-shaped concentration-response curves for the inhibitory effects of mixtures of ten ionic liquids on luciferase luminescence after 15 min of exposure. The arrows A-F in Figure 2 correspond to the panels A-F in Figure 3, respectively. 0: Control; O: experimental data; ---: biphasic model fit; -: concentration addition.

where n is the number of mixture components, ECx,i is the concentration of the ith component eliciting x% effect, ECx,mix is the concentration of the mixture eliciting x% effect, and pi is the concentration ratio of the ith component in the mixture.

’ RESULTS Hormetic Effects of Individual Ionic Liquids. All ten ILs presented J-shaped CRCs for their effects on luciferase luminescence (Figure 1). The fitted J-shaped CRC models and resulting parameters are listed in Table 1. [HMIM]Cl (IL10) presented the highest stimulatory effect (Em = -48.3%) while [BMIM]Br (IL3) elicited the lowest one (-9.3%). The J-shaped CRCs contain two key characteristics of the hormetic effects of the test chemicals. (i) There are two zeroeffect concentrations (EC0), one lower and one higher than the ECm because of the intersection between the J-shaped CRC and concentration axis. If not specified, EC0 referred specifically to the zero-effect concentration higher than ECm. (ii) There are two concentrations associated with the same stimulatory effect (x%) in the two opposite phases of the J-shaped CRC. We used ECxl and ECxr to denote the two concentrations lower and higher than ECm. Hormetic Effects of Ionic Liquid Mixtures. All four mixtures of ten ILs (Mix50, Mix10, Mix0, and Mixm) presented J-shaped CRCs (Figure 2), which were actually the reflection of the J-shaped CRCs of individual ILs. The fitted CRC models and resulting parameters of the four mixtures are given in Table 1. The concentration ratios of the ILs in the four mixtures are listed in Table 2. The four different mixtures have similar compositions because of the basically parallel CRCs of ten IL components. Because the stimulatory section below the effect of -9.3% (the lowest stimulatory effect of individual ILs) could not be predicted by the CA model, the CRCs predicted by CA (Figure 2)

were divided into two segments (left and right ones) where the concentrations in the right one are higher than EC-9.3r and those in the left one are lower than EC-9.3l. For the right segment, the CA effectively predicted the effects of three mixtures (Mix50, Mix10, and Mix0) but slightly overestimated those of another mixture (Mixm). For the left segment, the CA slightly underestimated the effects of all four mixtures (Mix50, Mix10, Mix0, and Mixm). Focusing on the selected points (marked by arrows A-F in Figure 2 which correspond to the panels A-F in Figure 3, respectively) of the CRCs of the four mixtures, three interesting phenomena were observed. First, three mixtures (Figure 3A, B and E) produce significant inhibitory effects where the concentrations of individual ILs are EC50,i/10, EC10,i/10, and ECm,i, respectively, and each IL only induces a stimulatory effect singly. Second, the mixture (Figure 3C) causes no effect where the concentrations of individual ILs are EC0,i/10 and each IL only induces a stimulatory effect singly. Third, the mixture (Figure 3D) causes a significant inhibitory effect where the concentrations of individual ILs are EC0,i and each IL induces no effect singly. The only exception (Figure 3F) different from the three phenomena above is the mixture producing the maximal stimulatory effect of ECm ratio mixture where the concentrations of various ILs are ECm,i/10 and each IL induces a stimulatory effect singly.

’ DISCUSSION Hormesis at the Molecular Level. In the present study, ten ILs and their mixtures presented hormetic effects on luciferase luminescence. To explore the possible mechanisms behind the hormetic effects, the molecular structures of ten ILs with J-shaped CRCs were compared with those of fifteen other ILs with S-shaped CRCs (data not shown). The results showed that most of the ILs with J-shaped CRCs belong to the imidazole ILs 1626

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Figure 3. Comparison of the effects on luciferase luminescence of mixtures with those of the ten individual ionic liquid (IL) where the concentrations of ILs correspond to 1/10 of individual EC50 (A), 1/10 of individual EC10 (B), 1/10 of individual EC0 (C), individual EC0 (D), individual ECm (E), and 1/10 of individual ECm (F), respectively. Obs is mixture effect observed. CA is mixture effect predicted by concentration addition. ?? is Mixture effect being not predicted by CA model. The error bar is 95% confidence interval of observed effects. * is Confidence interval not available.

with a chloride or bromide ion. In addition, the luciferase active sites contain a histidine residue having an imidazole group.34 Therefore, we presume that the ILs at low concentrations function as luciferase activators, and those at high concentrations (after the saturation of the luciferase active sites by ILs) function as luciferase inhibitors through preventing the substrate (e.g., luciferin) from entering the active sites, which is the possible mechanism of action for the hormetic effect of ILs. Hormesis in Mixtures. Because the CRCs of the ten ILs are basically parallel, these ILs are expected to exert the same mechanism of action and elicit similar toxic responses on luciferase molecules through competing for the luciferase active sites. The exposures of luciferase molecules to IL mixtures at sufficiently high concentrations change the low-concentration beneficial effects of the compounds into zero or harmful effects of mixtures. The good agreement between the effects observed and predicted by CA implies a good predictive power of the CA model for the hormesis in IL mixtures. The same mechanism of action of these

ILs on luciferase is probably the reason the CA model is well suited to predicting the hormetic effects. Unfortunately, the mixture effects (x) less than the lowest stimulatory effect (-9.3%) could not be predicted by CA due to the absence of some ECx,i (e.g., Figure 3F), which is still a problem needing to be addressed in the future.35 Nevertheless, it may be presumed that the Em of the mixture of hormetic compounds with the same mechanism of action will locate within the range of the Ems of the compounds (i.e., min Em,i e Em,mix e max Em,i) and the present study provides an example for this presumption. Implications. Regarding the three interesting phenomena shown in Results, the first phenomenon has been foreseen by Thayer et al.:36 the individual stimulatory responses are not always beneficial, and some may be harmful. For example, although TCDD can induce a low-dose beneficial effect, the mixture of numerous dioxin-like compounds with beneficial effects can still induce toxicity. Brian et al. also observed similar results.37 The second phenomenon could be helpful in controlling the discharge 1627

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Environmental Science & Technology of toxic wastewaters. If all chemicals in wastewaters present the hormetic effects and no toxic interaction, adjusting their concentrations to EC0,i/n (n denotes the number of mixture components) will theoretically achieve the discharge of nontoxic wastewaters. For the third phenomenon, it is well-known that it is impossible to determine EC0 from the typically S-shaped CRC, instead NOEC (no observed effect concentration) can be regarded as an approximation of EC0. Some evidence indicated that a large effect of mixtures could be induced from small exposures (at or below NOEC) of many compounds.38,39 However, NOEC is not the concentration really producing zero effect. To fill this gap we presented here an assessment of the combined effects of ten ILs at EC0 corresponding to a true zero effect concentration. Our results clearly showed that the EC0 mixture produced 100% effect (Figure 3D). This finding demonstrated that EC0 values are not regarded as the safe levels in risk assessment of environmental mixture. From the standpoint of risk assessment, J-shaped concentration-response relationship may be very important in characterizing the potential health risks of environmental pollutants and identifying the beneficial and harmful aspects of a hormetic effect in the related mixtures. For example, the mixtures consisting of the individual chemicals with beneficial effects could produce distinct combined effects including harm (Figure 3A, B and E), nothing (Figure 3C), and benefit (Figure 3F). Moreover, chemical mixture exposure is ubiquitous in the environment. Thus, whether an effect is adverse or beneficial should be identified in the context of mixtures.

’ AUTHOR INFORMATION Corresponding Author

*Tel: 86-21-65982767; e-mail: [email protected].

’ ACKNOWLEDGMENT We are thankful to the National High Technology Research and Development Program of China (2007AA06Z417), the National Natural Science Foundation of China (20777056, 20977065), and the Foundation of the State Key Laboratory of Pollution Control and Resource Reuse (PCRRK09002) for their financial support. We also thank four anonymous reviewers for helping to improve the manuscript. ’ REFERENCES (1) Calabrese, E. J.; Baldwin, L. A. Defining hormesis. Hum. Exp. Toxicol. 2002, 21, 91–97. (2) Qin, L. T.; Liu, S. S.; Liu, H. L.; Zhang, Y. H. Support vector regression and least squares support vector regression for hormetic dose-response curves fitting. Chemosphere 2010, 78, 327–334. (3) Calabrese, E. J. Hormesis: why it is important to toxicology and toxicologists. Environ. Toxicol. Chem./SETAC 2008, 27, 1451–1474. (4) Pagano, G.; Castello, G.; Gallo, M.; Borriello, I.; Guida, M. Complex mixture-associated hormesis and toxicity: The case of leather tanning industry. Dose-Response 2008, 6, 383–396. (5) Koshy, L.; Jones, T.; BeruBe, K. Bioreactivity of municipal solid waste landfill leachates - Hormesis and DNA damage. Water Res. 2008, 42, 2177–2183. (6) Love, O. P.; Shutt, L. J.; Silfies, J. S.; Bortolotti, G. R.; Smits, J. E. G.; Bird, D. M. Effects of dietary PCB exposure on adrenocortical function in captive American kestrels (Falco sparverius). Ecotoxicology 2003, 12, 199–208. (7) Gennings, C.; Carter, W. H.; Campain, J. A.; Bae, D. S.; Yang, R. S. H. Statistical analysis of interactive cytotoxicity in human epidermal

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