Assessing single and joint effects of chemicals on the survival and reproduction of Folsomia candida (Collembola) in soil

Chemicals are often found in the environment as complex mixtures. There has been a large effort in the last decade to assess the combined effect of chemicals, using the conceptual models of Concentration Addition and Independent Action, but also including synergistic, antagonistic, dose-level and dose-ratio dependent deviations from these models. In the present study, single and mixture toxicity of atrazine, dimethoate, lindane, zinc and cadmium were studied in Folsomia candida, assessing survival and reproduction. Different response patterns were observed for the different endpoints and synergistic patterns were observed when pesticides were present. Compared with the previously tested Enchytraeus albidus and Porcellionides pruinosus, the mixture toxicity pattern for F. candida was species specific. The present study highlights the importance of studying toxicity of chemicals mixtures due to the observed potentiation of effects and confirms that for an adequate ecologically relevant risk assessment different organisms and endpoints should be included.     

Nature and prevalence of non-additive toxic effects in industrially relevant mixtures of organic chemicals

The concentration addition (CA) and the independent action (IA) models are widely used for predicting mixture toxicity based on its composition and individual component dose–response profiles. However, the prediction based on these models may be inaccurate due to interaction among mixture components. In this work, the nature and prevalence of non-additive effects were explored for binary, ternary and quaternary mixtures composed of hydrophobic organic compounds (HOCs). The toxicity of each individual component and mixture was determined using the Vibrio fischeri bioluminescence inhibition assay. For each combination of chemicals specified by the 2ⁿ factorial design, the percent deviation of the predicted toxic effect from the measured value was used to characterize mixtures as synergistic (positive deviation) and antagonistic (negative deviation). An arbitrary classification scheme was proposed based on the magnitude of deviation (d) as: additive (10%, class-I) and moderately (10 < d  30%, class-II), highly (30 < d  50%, class-III) and very highly (>50%, class-IV) antagonistic/synergistic. Naphthalene, n-butanol, o-xylene, catechol and p-cresol led to synergism in mixtures while 1, 2, 4-trimethylbenzene and 1, 3-dimethylnaphthalene contributed to antagonism. Most of the mixtures depicted additive or antagonistic effect. Synergism was prominent in some of the mixtures, such as, pulp and paper, textile dyes, and a mixture composed of polynuclear aromatic hydrocarbons. The organic chemical industry mixture depicted the highest abundance of antagonism and least synergism. Mixture toxicity was found to depend on partition coefficient, molecular connectivity index and relative concentration of the components.     

Evaluation on the toxicity of ionic liquid mixture with antagonism and synergism to Vibrio qinghaiensis sp.-Q67

Ionic liquids (ILs) are a fascinating group of new chemicals with the potential to replace the classical volatile organic solvents, stimulating many applications in chemical industry. In case ILs are released to the environment, possible combined toxicity should be taken into account and it is, however, often neglected up to now. In this paper, therefore, the concentration-response curves (CRCs) of four groups of IL mixtures with various mixture ratios to Vibrio qinghaiensis sp.-Q67 were determined using the microplate toxicity analysis and were compared to the CRCs predicted by an additive reference model, the concentration addition (CA) or independent action (IA), to identify the toxicity interaction. It is showed that most of the IL mixture rays displayed the classical addition while the remaining rays exhibited antagonism or synergism. Moreover, it is found that the pEC₅₀ values of the mixture rays exhibiting antagonism or synergism are well correlated with the mixture ratio of a certain IL therein.     

Assessing joint toxicity of chemicals in Enchytraeus albidus (Enchytraeidae) and Porcellionides pruinosus (Isopoda) using avoidance behaviour as an endpoint

Contamination problems are often characterized by complex mixtures of chemicals. There are two conceptual models usually used to evaluate patterns of mixture toxicity: Concentration Addition (CA) and Independent Action (IA). Deviations from these models as synergism, antagonism and dose dependency also occur. In the present study, single and mixture toxicity of atrazine, dimethoate, lindane, zinc and cadmium were tested in Porcellionides pruinosus and Enchytraeus albidus, using avoidance as test parameter. For both species patterns of antagonism were found when exposed to dimethoate and atrazine, synergism for lindane and dimethoate exposures (with the exception of lower doses in the isopod case study) and concentration addition for cadmium and zinc occurred, while the exposure to cadmium and dimethoate showed dissimilar patterns.This study highlights the importance of dose dependencies when testing chemical mixtures and that avoidance tests can also be used to asses the effects of mixture toxicity.     

The interaction effects of binary mixtures of benzene and toluene on the developing heart of medaka (Oryzias latipes)

The United States Environmental Protection Agency (USEPA) has pursued the estimation of risk of adverse health effects from exposure to chemical mixtures since the early 1980s. Methods used to calculate risk estimates of mixtures were often based on single chemical information that required assumptions of dose-addition or response-addition and did not consider possible changes in response due to interaction effects among chemicals. Full factorial designs for laboratory studies can produce interactions information, but these are expensive to perform and may not provide the information needed to evaluate specific environmentally relevant mixtures. In this research, groups of Japanese medaka (Oryzias latipes) embryos were exposed to binary mixtures of benzene and toluene as well as to each of these chemicals alone. Endpoint specific dose-response models were built for the hydrocarbon mixture under an assumption of dose-additivity, using the single chemical dose-response information on benzene and toluene. The endpoints included heart rate, heart rate progression, and lethality. Results included a synergistic response for heart rate at 72 h of development, and either additivity or antagonism for all other endpoints at 96 h of development. This work uses an established statistical method to evaluate the toxicity of an environmentally relevant mixture to ascertain whether interaction effects are occurring, thus providing additional information on toxicity.     

等浓度配比法研究苯酚、硝基苯和问硝基苯胺对发光菌的联合毒性作用

分别测定了苯酚、硝基苯和间硝基苯胺对发光菌的单一毒性，以及等浓度配比和等毒性配比的二元及三元混合体系的联合毒性，采用相加指数法对其联合效应进行了评价。结果表明，等浓度比和等毒性比混合体系的联合作用结果一致：苯酚＋间硝基苯胺二元体系为协同作用，其他各体系为相加作用。为简化联合毒性实验方法，建议在研究相关系列化合物的联合毒性作用机制中，可采用等浓度配比方法。     

等浓度配比法研究苯酚、硝基苯和间硝基苯胺对发光菌的联合毒性作用

分别测定了苯酚、硝基苯和间硝基苯胺对发光菌的单一毒性,以及等浓度配比和等毒性配比的二元及三元混合体系的联合毒性,采用相加指数法对其联合效应进行了评价。结果表明,等浓度比和等毒性比混合体系的联合作用结果一致:苯酚+间硝基苯胺二元体系为协同作用,其他各体系为相加作用。为简化联合毒性实验方法,建议在研究相关系列化合物的联合毒性作用机制中,可采用等浓度配比方法。     

A novel method dependent only on the mixture information (MIM) for evaluating the toxicity of mixture

Compound contamination and toxicity interaction necessitate the development of models that have an insight into the combined toxicity of chemicals. In this paper, a novel and simple model dependent only on the mixture information (MIM), was developed. Firstly, the concentration-response data of seven groups of binary and multi-component (pseudo-binary) mixtures with different mixture ratios to Vibrio qinghaiensis sp.-Q67 were determined using the microplate toxicity analysis. Then, a desirable non-linear function was selected to fit the data. It was found that there are good linear correlations between the location parameter (α) and mixture ratio (p) of a component and between the steepness (β) and p. Based on the correlations, a mixture toxicity model independent of pure component toxicity profiles was built. The model can be used to accurately estimate the toxicities of the seven groups of mixtures, which greatly simplified the predictive procedure of the combined toxicity.     

Quantification of joint effect for hydrogen bond and development of QSARs for predicting mixture toxicity

A QSAR model is successfully proposed to predict the toxicity effect on Photobacterium phosphoreum by nonpolar-narcotic-chemical mixtures and/or polar-narcotic-chemical mixtures. For nonpolar-narcotic-chemical mixtures and polar-narcotic-chemical mixtures, their corresponding hydrophobicity-based QSAR models are derived from regression analysis. Comparison of these two QSAR models make us believe that it is the joint effect of hydrogen bond in polar-narcotic-chemical mixture that leads to the difference between these two models. Such joint effect of hydrogen bond can be quantified as AMH and BMH by using the different partition coefficients of mixtures in various organic phase/water systems. And the regression analysis results convinced us that the introduction of AMH does improve the quality of the QSAR model with r2=0.948, S.E.=0.166 and F=745.201 at P=0.000 for total 84 mixtures.     

Joint action and lethal levels of toluene, ethylbenzene, and xylene on midge (Chironomus plumosus) larvae

Aquatic ecosystems are vulnerable to the exposure with petrochemicals such as toluene, ethylbenzene, and xylene (o-, m-, and p-xylene) (TEX) and their adverse effects. Considering the widespread use, occurrence, and high toxicity of TEX, the aim of this work was to investigate the differential toxicity of TEX against midge (Chironomus plumosus) larvae and reveal the joint action of binary and ternary mixtures of TEX using the predictive concentration addition model. More importantly, this research can afford the basic toxicity data and scientific reference for the establishment of water quality criteria or benchmark, water pollution control, and aquatic risk assessment. Single and joint toxic effects of TEX on C. plumosus larvae were investigated using a semi-static bioassay, and the type of joint effects of TEX was ascertained. In the single toxicant experiments, the toxicity of the three pollutants could be sequenced as ethylbenzene > xylene > toluene. Specifically, LC_50s of T, E, and X after a 48-h exposure were 64.9, 37.8, and 42.0 mg/L, respectively. In the binary mixture experiments, the interaction between toluene and ethylbenzene, ethylbenzene and xylene, and toluene and xylene was largely in conformity with partial additive or additive effect as determined by isobologram representation and toxic unit models. In the ternary mixture experiments, the interaction was basically dependent on the use of additive index and mixture toxicity index methods. However, the antagonistic and synergistic actions were not significant. Thus, the tertiary mixture interaction could be regarded as additive action. The concentration addition model could successfully predict the joint action of TEX mixtures on C. plumosus larvae. Particularly, the additive action of TEX on C. plumosus larvae can be further recommended to evaluate water quality criteria of TEX.     

Prediction of mixture toxicity with its total hydrophobicity

Based on the C18 Empore disk/water partition coefficient of a mixture, quantitative structure-activity relationships (QSARs) are presented, which are used to predict the toxicity of mixed halogenated benzenes to P. phosphoreum. The predicted toxicity of 10 other related mixtures based on the QSAR model, agree well with the observed data with r2 = 0.973, SE = 0.113 and F = 287.785 at a level of significance P < 0.0001. The joint effect of these chemicals is simple similar action and the toxicity of the mixtures can be predicted from total hydrophobicity and is independent of hydrophobicity of the components or the ratio of the individual chemicals.     

Individual and mixture toxicity of three pesticides; atrazine,chlorpyrifos, and chlorothalonil to the marine phytoplankton species Dunaliella tertiolecta

This study analyzed the toxicity of three pesticides (the herbicide atrazine, the insecticide chlorpyrifos and the fungicide chlorothalonil) individually, and in two mixtures (atrazine and chlorpyrifos; atrazine and chlorothalonil) to the marine phytoplankton species Dunaliella tertiolecta (Chlorophyta). A standard 96 h static algal bioassay was used to determine pesticide effects on the population growth rate of D. tertiolecta. Mixture toxicity was assessed using the additive index approach. Atrazine and chlorothalonil concentrations > or = 25 microg/L and 33.3 microg/L, respectively, caused significant decreases in D. tertiolecta population growth rate. At much higher concentrations (> or = 400 microg/L) chlorpyrifos also elicited a significant effect on D. tertiolecta population growth rate, but toxicity would not be expected at typical environmental concentrations. The population growth rate EC50 values determined for D. tertiolecta were 64 microg/L for chlorothalonil, 69 microg/L for atrazine, and 769 microg/L for chlorpyrifos. Atrazine and chlorpyrifos in mixture displayed additive toxicity, whereas atrazine and chlorothalonil in mixture had a synergistic effect. The toxicity of atrazine and chlorothalonil combined was approximately 2 times greater than that of the individual chemicals. Therefore, decreases in phytoplankton populations resulting from pesticide exposure could occur at lower than expected concentrations in aquatic systems where atrazine and chlorothalonil are present in mixture. Detrimental effects on phytoplankton population growth rate could impact nutrient cycling rates and food availability to higher trophic levels. Characterizing the toxicity of chemical mixtures likely to be encountered in the environment may benefit the pesticide registration and regulation process.     

Unexpected toxic interactions in the freshwater amphipod Gammarus pulex (L.) exposed to binary copper and nickel mixtures

To document the toxicity of copper and nickel in binary mixtures, freshwater amphipods Gammarus pulex were exposed to the metals given independently or as mixtures. Toxicity to Cu alone was relatively high: 96-h LC₁₀ and LC₅₀ were found at 91 and 196 μg L^?1, respectively. Toxicity to Ni alone was very low, with 96-h LC₁₀ and LC₅₀ of 44,900 and 79,200 μg L^?1, respectively. Mixture toxicities were calculated from single toxicity data using conventional models. Modeled toxicity was then compared with the measured toxicity of the binary mixture. Two kinds of mixtures were tested. Type I mixtures were designed as combinations of Cu and Ni given at the same effect concentrations, when taken independently, to identify possible interactions between copper and nickel. In type II mixtures, Cu concentrations varied from 0 to 600 μg L^?1 while the nickel concentration was kept constant at 500 μg L^?1 to mimic conditions of industrial wastewater discharges. Ni and Cu showed synergic effects in type I mixtures while type II mixtures revealed antagonistic effects. Low doses of Ni reduced Cu toxicity towards G. pulex. These results show that even for simple binary mixtures of contaminants with known chemistry and toxicity, unexpected interactions between the contaminants may occur. This reduces the reliability of conventional additivity models.     

化学物质对发光菌的联合毒性评价方法

毒性单位法(TU)的理论基础来源于剂量加和模型(DA),目前仅在二元联合毒性评价中广泛应用。为了确定TU模型适合评价的混合物类型,实验选取5种剂量效应曲线类型不同的物质,采用微板光度计测试了一元、二元混合物对发光菌青海弧菌-Q67(Vibrio-qinghaiensis sp.-Q67)的急性毒性。根据物质的剂量效应曲线形状将物质分为A、B、C 3类,利用毒性单位法(TU)和联合作用定义法分别对AA类、AB类、AC类、BC类混合物进行分析。结果表明,TU法仅适合于由剂量效应曲线接近直线的物质组成的混合物进行联合毒性的评价。以效应为基准、TU模型为框架建立了TU’模型,该模型可以满足对任何类型已知成分的混合物或者未知成分的实际水样之间的多元联合作用的评价。     

Toxic effect of metal cation binary mixtures to the seaweed Gracilaria domingensis (Gracilariales,Rhodophyta)

The macroalga Gracilaria domingensis is an important resource for the food, pharmaceutical, cosmetic, and biotechnology industries. G. domingensis is at a part of the food web foundation, providing nutrients and microelements to upper levels. As seaweed storage metals in the vacuoles, they are considered the main vectors to magnify these toxic elements. This work describes the evaluation of the toxicity of binary mixtures of available metal cations based on the growth rates of G. domingensis over a 48-h exposure. The interactive effects of each binary mixture were determined using a toxic unit (TU) concept that was the sum of the relative contribution of each toxicant and calculated using the ratio between the toxicant concentration and its endpoint. Mixtures of Cd(II)/Cu(II) and Zn(II)/Ca(II) demonstrated to be additive; Cu(II)/Zn(II), Cu(II)/Mg(II), Cu(II)/Ca(II), Zn(II)/Mg(II), and Ca(II)/Mg(II) mixtures were synergistic, and all interactions studied with Cd(II) were antagonistic. Hypotheses that explain the toxicity of binary mixtures at the molecular level are also suggested. These results represent the first effort to characterize the combined effect of available metal cations, based on the TU concept on seaweed in a total controlled medium. The results presented here are invaluable to the understanding of seaweed metal cation toxicity in the marine environment, the mechanism of toxicity action and how the tolerance of the organism.     

Novel approach to predicting hormetic effects of antibiotic mixtures on Vibrio fischeri

The determination of the hormetic effects of a mixture is quite difficult because of the moderate simulation and the complexity of measurement in low doses. In the present study, two typical models for mixture toxicity prediction, concentration additive (CA) and independent action (IA), were used to predict the hormetic effects of mixtures. The predictive power of those models was validated by the hormetic effects (24-h exposure) of antibiotic’s binary mixtures to Vibrio fischeri. The results showed that CA and IA were unable to predict the hormetic dose-response of mixture, especially those of the interactive mixtures. As an alternative, a novel model, which was named as “six-point” and developed based on the quantitative features in the determined dose-response curve and on the Quantitative Structure Activity Relationships (QSARs) approach, was proposed for predicting the hormetic effects of mixtures in low dose. The results indicated that the “six-point” model can accurately predict the mixture hormetic effects in low dose, not only for non-interactive mixtures but also for interactive mixtures. Therefore, the “six-point” model is a powerful tool to predict the mixture hormetic effects at low dose, and may offer an important approach in the environment risk assessment of mixtures.     

A comparison of mixture toxicity assessment: examining the chronic toxicity of atrazine, permethrin and chlorothalonil in mixtures to Ceriodaphnia cf. dubia

Pesticides predominantly occur in aquatic ecosystems as mixtures of varying complexity, yet relatively few studies have examined the toxicity of pesticide mixtures. Atrazine, chlorothalonil and permethrin are widely used pesticides that have different modes of action. This study examined the chronic toxicities (7-d reproductive impairment) of these pesticides in binary and ternary mixtures to the freshwater cladoceran Ceriodaphnia cf. dubia. The toxicity of the mixtures was compared to that predicted by the independent action (IA) model for mixtures, as this is the most appropriate model for chemicals with different modes of action. Following this they were compared to the toxicity predicted by the concentration addition (CA) model for mixtures. According to the IA model, the toxicity of the chlorothalonil plus atrazine mixture conformed to antagonism, while that of chlorothalonil and permethrin conformed to synergism. The toxicity of the atrazine and permethrin mixture as well as the ternary mixture conformed to IA implying there was either no interaction between the components of these mixtures and/or in the case of the ternary mixture the interactions cancelled each other out to result in IA. The synergistic and antagonistic mixtures deviated from IA by factors greater than 3 and less than 2.5, respectively. When the toxicity of the mixtures was compared to the predictions of the CA model, the binary mixture of chlorothalonil plus atrazine, permethrin plus atrazine and the ternary mixture all conformed to antagonism, while the binary mixture of chlorothalonil plus permethrin conformed to CA. Using the CA model provided estimates of mixture toxicity that did not markedly underestimate the measured toxicity, unlike the IA model, and therefore the CA model is the most suitable to use in ecological risk assessments of these pesticides.     

The joint effects of sulfonamides and their potentiator on Photobacterium phosphoreum: differences between the acute and chronic mixture toxicity mechanisms

Organisms are typically exposed to mixtures of chemicals over long periods of time; thus, chronic mixture toxicity analysis is the best way to perform risk assessment in regards to organisms. However, most studies focus on the acute mixture toxicity. To investigate the difference between chronic mixture toxicity and acute mixture toxicity, Photobacterium phosphoreum were exposed to chronic (24 h exposure) and acute (15 min exposure) toxicity of single sulfonamide (SA) and their potentiator (trimethoprim, TMP), both individually and mixtures (SA with TMP). A comparison of chronic vs. acute mixture toxicity revealed the presence of an interesting phenomenon, that is, that the joint effects vary with the duration of exposure; the acute mixture toxicity was antagonistic, whereas the chronic mixture toxicity was synergistic. Based on the approach of Quantitative Structure Activity Relationships (QSARs) and molecular docking, this phenomenon was proved to be caused by the presence of two points of dissimilarity between the acute and chronic mixture toxicity mechanism: (1) the receptor protein of SAs in acute toxicity was Luc, while in chronic toxicity it was Dhps, and (2) there is a difference between actual concentration of binding-Luc in acute toxicity and individual binding-Dhps in chronic toxicity. This deep insight into the difference between chronic and acute mixture toxicity will benefit environmental science, medical science, and other disciplines. The existence of these differences poses a challenge for the assessment of routine combinations in medicine, risk assessment, and mixture pollutant control, in which, previously, only a synergistic effect has been observed between SA and their potentiator.     

Assessment of combined antiandrogenic effects of binary parabens mixtures in a yeast-based reporter assay

To date, toxicological studies of endocrine disrupting chemicals (EDCs) have typically focused on single chemical exposures and associated effects. However, exposure to EDCs mixtures in the environment is common. Antiandrogens represent a group of EDCs, which draw increasing attention due to their resultant demasculinization and sexual disruption of aquatic organisms. Although there are a number of in vivo and in vitro studies investigating the combined effects of antiandrogen mixtures, these studies are mainly on selected model compounds such as flutamide, procymidone, and vinclozolin. The aim of the present study is to investigate the combined antiandrogenic effects of parabens, which are widely used antiandrogens in industrial and domestic commodities. A yeast-based human androgen receptor (hAR) assay (YAS) was applied to assess the antiandrogenic activities of n-propylparaben (nPrP), iso-propylparaben (iPrP), methylparaben (MeP), and 4-n-pentylphenol (PeP), as well as the binary mixtures of nPrP with each of the other three antiandrogens. All of the four compounds could exhibit antiandrogenic activity via the hAR. A linear interaction model was applied to quantitatively analyze the interaction between nPrP and each of the other three antiandrogens. The isoboles method was modified to show the variation of combined effects as the concentrations of mixed antiandrogens were changed. Graphs were constructed to show isoeffective curves of three binary mixtures based on the fitted linear interaction model and to evaluate the interaction of the mixed antiandrogens (synergism or antagonism). The combined effect of equimolar combinations of the three mixtures was also considered with the nonlinear isoboles method. The main effect parameters and interaction effect parameters in the linear interaction models of the three mixtures were different from zero. The results showed that any two antiandrogens in their binary mixtures tended to exert equal antiandrogenic activity in the linear concentration ranges. The antiandrogenicity of the binary mixture and the concentration of nPrP were fitted to a sigmoidal model if the concentrations of the other antiandrogens (iPrP, MeP, and PeP) in the mixture were lower than the AR saturation concentrations. Some concave isoboles above the additivity line appeared in all the three mixtures. There were some synergistic effects of the binary mixture of nPrP and MeP at low concentrations in the linear concentration ranges. Interesting, when the antiandrogens concentrations approached the saturation, the interaction between chemicals were antagonistic for all the three mixtures tested. When the toxicity of the three mixtures was assessed using nonlinear isoboles, only antagonism was observed for equimolar combinations of nPrP and iPrP as the concentrations were increased from the no-observed-effect-concentration (NOEC) to effective concentration of 80 %. In addition, the interactions were changed from synergistic to antagonistic as effective concentrations were increased in the equimolar combinations of nPrP and MeP, as well as nPrP and PeP. The combined effects of three binary antiandrogens mixtures in the linear ranges were successfully evaluated by curve fitting and isoboles. The combined effects of specific binary mixtures varied depending on the concentrations of the chemicals in the mixtures. At low concentrations in the linear concentration ranges, there was synergistic interaction existing in the binary mixture of nPrP and MeP. The interaction tended to be antagonistic as the antiandrogens approached saturation concentrations in mixtures of nPrP with each of the other three antiandrogens. The synergistic interaction was also found in the equimolar combinations of nPrP and MeP, as well as nPrP and PeP, at low concentrations with another method of nonlinear isoboles. The mixture activities of binary antiandrogens had a tendency towards antagonism at high concentrations and synergism at low concentrations.     

Interaction of nano-TiO2 with lysozyme: insights into the enzyme toxicity of nanosized particles

Background, aim, and scope  

Nanomaterials have been used increasingly in industrial production and daily life, but their human exposure may cause health risks. The interactions of nanomaterial with functional biomolecules are often applied as a precondition for its cytotoxicity and organ toxicity where various proteins have been investigated in the past years. In the present study, nano-TiO₂ was selected as the representative of nanomaterials and lysozyme as a representative for enzymes. By investigating their interaction by various instrumentations, the objective is to identify the action sites and types, estimate the effect on the enzyme structure and activity, and reveal the toxicity mechanism of nanomaterial.