Mixture Toxicity Studies Research Articles

Mixture toxicity study of two metal oxide nanoparticles and chlorpyrifos on Eisenia andrei earthworms.

Co-exposure soil studies of pollutants are necessary for an appropriate ecological risk assessment. Here, we examined the effects of two-component mixtures of metal oxide nanoparticles (ZnO NPs or goethite NPs) with the insecticide chlorpyrifos (CPF) under laboratory conditions in short-term artificial soil assays using Eisenia andrei earthworms. We characterized NPs and their mixtures by scanning electron microscopy, atomic force microscopy,dynamic light scattering and zeta potential, and evaluated effects on metal accumulation, oxidative stress enzymes, and neurotoxicity related biomarkers in single and combined toxicity assays. Exposure to ZnO NPs increased Zn levels compared to control in single and combined exposure (ZnO NPs + CPF) at 72h and 7days, respectively. In contrast, there was no indication of Fe increase in organisms exposed to goethite NPs. One of the most notable effects on oxidative stress biomarkers was produced by single exposure to goethite NPs, showing that the worms were more sensitive to goethite NPs than to ZnO NPs. Acetylcholinesterase and carboxylesterase activities indicated that ZnO NPs alone were not neurotoxic to earthworms, but similar degrees of inhibition were observed after single CPF and ZnO NPs + CPF exposure. Differences between single and combined exposure were found for catalase and superoxide dismutase (goethite NPs) and for glutathione S-transferase (ZnO NPs) activities, mostly at 72h. These findings suggest a necessity to evaluate mixtures of NPs with co-existing contaminants in soil, and that the nature of metal oxide NPs and exposure time are relevant factors to be considered when assessing combined toxicity, as it may have an impact on ecotoxicological risk assessment.

An Environmentally Relevant Mixture of Perfluorooctanesulfonic Acid and Perfluorohexanesulfonic Acid Does Not Conform to Additivity in Northern Leopard Frogs Exposed Through Metamorphosis.

Per- and polyfluoroalkyl substances (PFAS) are chemicals associated with adverse health effects. At aqueous film-forming foam sites, they occur as mixtures, with perfluorooctanesulfonic acid (PFOS) and perfluorohexanesulfonic acid (PFHxS) commonly co-occurring in the highest concentrations. Although PFOS and PFHxS toxicities have been studied, few studies have tested their potential interaction. Using Rana pipiens, the present study compared toxicities of a 1:1 PFOS:PFHxS mixture to PFOS and PFHxS individually with the prediction that responses would be additive. Gosner stage 25 (GS 25) tadpoles were exposed through metamorphosis (GS 46) to 0.5 and 1 ppb PFOS or PFHxS alone or to a mixture of 0.5 ppb PFOS and 0.5 ppb PFHxS. Tadpoles were weighed and measured (snout-vent length [SVL]) at day 31, metamorphic climax (GS 42), and GS 46. These values were used to calculate the scaled mass index (SMI), a measure of body condition. Body burdens were quantified on day 31 and at GS 46. The PFOS and PFHxS body burdens were elevated relative to controls at GS 46. No effects were observed on survival, SVL, or mass. Single PFAS effects included a 17% reduction in SMI at day 31 (0.5 ppb PFHxS) and a 1.1-day longer metamorphic period (1 ppb PFHxS) relative to controls. Mixture results deviated from additivity-SMIs were higher than expected on day 31 and lower than expected at GS 42. In addition, time to GS 42 in the PFAS mixture exceeded expected additivity by 12 days. Results from a chronic exposure to a 1:1 PFOS:PFHxS mixture resulted in changes in body condition and length of metamorphosis that deviated from additivity. More PFAS mixture toxicity studies conducted at relevant ratios and concentrations are needed. Environ Toxicol Chem 2022;41:3007-3016. © 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Toxic Effect of Antibiotics on Freshwater Algal Systems and the Mechanisms of Toxicity: A Review

Antibiotics are used to treat bacterial infections in humans and animals and also act as a growth promoter for poultry. Due to incomplete metabolism, these antibiotics are excreted in the environment in their parental forms and accumulates in the aquatic ecosystem. Besides the evolution of antibiotic-resistant bacteria, these drugs can damage non-target organisms. Green algae are highly sensitive to different antibiotics. Damage in the algal population will cause imbalances in the ecosystems. Till now, the mechanisms of antibiotic toxicity towards algae have not been completely elucidated. It was observed that antibiotics mainly affected the photosynthetic machinery and decreased the carbon fixation process, finally resulting in algal growth inhibition. This present review deals with antibiotics classification, various routes of antibiotics exposure to the freshwater environment, sensitivity towards the different classes of antibiotics, possible Mode-of-Action (MOA) on algal systems, and gaps that need to be filled. Significant gaps include the unavailability of proper eco-toxicological data for antibiotics. Moreover, they exist in nature as complex mixtures, and their behavior in the ecosystem may vastly differ from the parent molecules. To improve our understanding of antibiotic responses mechanism in real-life scenarios, mixture toxicity studies may be the first step.

Per- and polyfluoroalkyl mixtures toxicity assessment “Proof-of-Concept” illustration for the hazard index approach

ABSTRACT The 2018 ATSDR mixture framework recommends three approaches including the hazard index (HI) for environmental mixture toxicity assessment. Per- and polyfluoroalkyls (PFAS) are found in our environment and general populations. Recent experimental mixture toxicity studies of perfluorooctane sulfonic acid (PFOS) and perfluorooctanoic acid (PFOA) and an assessment of 17 PFAS indicate the use of additivity for their joint toxicity assessment. The aim of this investigation was to detail the stepwise procedures and examine the extent and use of the HI approach for PFAS mixture assessment. Using estimated general public lifetime exposures (high, medium, and low), binary mixtures of PFOS and PFOA yielded, respectively, hazard indices (HIs) of 30.67, 8.33, and 3.63 for developmental toxicity; 10.67, 5.04, and 2.34 for immunological toxicity; 3.57, 1.68, and 0.78 for endocrine toxicity; 4.51, 1.73, and 0.79 for hepatic toxicity; and 15.08, 2.29, and 0.88 for reproductive toxicity. A heterogeneous mixture of PFOA, PFAS, dioxin (CDD), and polybrominated compounds (PBDE) for high exposure scenario yielded HIs of 30.99 for developmental, 10.77 for immunological, 3.64 for endocrine, 4.61 for hepatic, and 17.36 for reproductive effects. The HI values are used as a screening tool; the potential concern for exposures rises as HI values increase. For HI values >1, a follow-up including further analysis of specific exposures, use of internal dosimetry, and uncertainty factors is conducted before recommending appropriate actions. The HI approach appears suitable to address present-day PFAS public health concerns for initial assessment of multiple health effects, until further insights are gained into their mechanistic toxicology. The findings and conclusions in this article are those of the author(s) and do not necessarily represent the official position of the Centers for Disease Control and Prevention/the Agency for Toxic Substances and Disease Registry.

Application of a Framework for Grouping and Mixtures Toxicity Assessment of PFAS: A Closer Examination of Dose-Additivity Approaches.

Environmental occurrence and biomonitoring data for per- and polyfluoroalkyl substances (PFAS) demonstrate that humans are exposed to mixtures of PFAS. This article presents a new and systematic analysis of available PFAS toxicity study data using a tiered mixtures risk assessment framework consistent with United States and international mixtures guidance. The lines of evidence presented herein include a critique of whole mixture toxicity studies and analysis of dose-response models based on data from subchronic oral toxicity studies in rats. Based on available data to-date, concentration addition and relative potency factor methods are found to be inappropriate due to differences among sensitive effects and target organ potencies and noncongruent dose-response curves for the same effect endpoints from studies using the same species and protocols. Perfluorooctanoic acid and perfluorooctane sulfonic acid lack a single mode of action or molecular initiating event and our evaluation herein shows they also have noncongruent dose-response curves. Dose-response curves for long-chain perfluoroalkyl sulfonic acids (PFSAs) also significantly differ in shapes of the curves from short-chain PFSAs and perfluoroalkyl carboxylic acids evaluated, and additional differences are apparent when curves are evaluated based on internal or administered dose. Following well-established guidance, the hazard index method applied to perfluoroalkyl carboxylic acids and PFSAs grouped separately is the most appropriate approach for conducting a screening level risk assessment for nonpolymeric PFAS mixtures, given the current state-of-the science. A clear presentation of assumptions, uncertainties, and data gaps is needed before dose-additivity methods, including hazard index , are used to support risk management decisions. Adverse outcome pathway(s) and mode(s) of action information for perfluorooctanoic acid and perfluorooctane sulfonic acid and for other nonpolymer PFAS are key data gaps precluding more robust mixtures methods. These findings can guide the prioritization of future studies on single chemical and whole mixture toxicity studies.

Toxic Effects of the Mixture of Phthalates and Bisphenol A-Subacute Oral Toxicity Study in Wistar Rats.

Phthalates and bisphenol A, classified as endocrine disruptors, have weak estrogenic, anti-androgenic properties, and affect thyroid hormone regulation. The aim of this study on male rats was to compare the subacute toxic effects of low doses of single compounds (bis (2 –ethylhexyl) phthalate (DEHP), dibutyl phthalate (DBP), and bisphenol A (BPA)) with the effects of their mixture through different biochemical, hormonal, and hematological parameters. Rats were divided into five experimental groups: Control (corn oil), DEHP (50 mg/kg b.w./day), DBP (50 mg/kg b.w./day), BPA (25 mg/kg b.w./day), and MIX (50 mg/kg b.w./day DEHP + 50 mg/kg b.w/day DBP + 25 mg/kg b.w./day BPA). Animals were sacrificed after 28 days of oral treatment and blood was collected for further analysis. The results demonstrated that the mixture produced significant changes in lipid profile, liver-related biochemical parameters, and glucose level. Furthermore, the opposite effects of single substances on the thyroxine level have been shown in comparison with the mixture, as well as a more pronounced effect of the mixture on testosterone level. This study contributes to the body of knowledge on the toxicology of mixtures and gives one more evidence of the paramount importance of mixture toxicity studies, especially in assessing the endocrine disruptive effects of chemicals.

Critical assessment and integration of separate lines of evidence for risk assessment of chemical mixtures

Humans are exposed to multiple chemicals on a daily basis instead of to just a single chemical, yet the majority of existing toxicity data comes from single-chemical exposure. Multiple factors must be considered such as the route, concentration, duration, and the timing of exposure when determining toxicity to the organism. The need for adequate model systems (in vivo, in vitro, in silico and mathematical) is paramount for better understanding of chemical mixture toxicity. Currently, shortcomings plague each model system as investigators struggle to find the appropriate balance of rigor, reproducibility and appropriateness in mixture toxicity studies. Significant questions exist when comparing single-to mixture-chemical toxicity concerning additivity, synergism, potentiation, or antagonism. Dose/concentration relevance is a major consideration and should be subthreshold for better accuracy in toxicity assessment. Previous work was limited by the technology and methodology of the time, but recent advances have resulted in significant progress in the study of mixture toxicology. Novel technologies have added insight to data obtained from in vivo studies for predictive toxicity testing. These include new in vitro models: omics-related tools, organs-on-a-chip and 3D cell culture, and in silico methods. Taken together, all these modern methodologies improve the understanding of the multiple toxicity pathways associated with adverse outcomes (e.g., adverse outcome pathways), thus allowing investigators to better predict risks linked to exposure to chemical mixtures. As technology and knowledge advance, our ability to harness and integrate separate streams of evidence regarding outcomes associated with chemical mixture exposure improves. As many national and international organizations are currently stressing, studies on chemical mixture toxicity are of primary importance.

Effects of individual and combined toxicity of bisphenol A, dibutyl phthalate and cadmium on oxidative stress and genotoxicity in HepG 2 cells

Bisphenol A, dibutyl phthalate and cadmium can be found in environment simultaneously. Several studies suggested that they had genotoxic effect. In this study, mono-exposure and co-exposure treatments, designed by 3 × 3 full factorial, were established to determine the individual toxicity and binary mixtures’ combined effects on the oxidative stress and genotoxicity in HepG 2 cells. The highest oxidative damage was observed in the Cd treatments groups. Compared with control groups, the maximum level of reactive oxygen species and malondialdehyde were ∼1.4 fold and ∼2.22 fold respectively. And a minimum level of superoxide dismutase activity was found with the decrease of 43%. The mechanism that excessive oxidative stress led to the DNA damage was inferred. However, cells treated with BPA showed the worst DNA damage rather than Cd, which may because Cd mainly damages DNA repairing mechanism. For the joint effect, different interactions can be found in different biological endpoints for different combinations since different mechanisms have been clarified in mixture toxicity studies. It is sure that the co-exposure groups enhanced cytotoxicity, oxidative stress and genotoxicity compared to the mono-exposures. Synergistic and additive interactions were considered, which means greater threat to organisms when exposed to multiple estrogenic endocrine disruptors.

Interactive effects of PAHs and heavy metal mixtures on oxidative stress in Chlorella sp. MM3 as determined by artificial neural network and genetic algorithm

Mixture toxicity studies are very complex due to the complexity exhibited by the chemicals involved, and the net interaction effects are highly dependent on mixture combinations, exposure dose and the test organism. For assessing the toxicity of mixtures, factorial analysis has been widely used, while the usage of models developed by artificial neural network (ANN) analysis and genetic algorithm (GA) is very limited. We combined for the first time the factorial design experiment with ANN and GA to develop a model for predicting the interactive toxicological effects using a soil microalga, Chlorella sp. MM3. The chemicals included in the mixtures were two polyaromatic hydrocarbons (PAHs), phenanthrene and benzo[a]pyrene, and two heavy metals (HMs), cadmium and lead. Three biochemicals implicated in oxidative stress, viz., malondialdehyde (a measure for lipid peroxidation, LPO), catalase activity and proline accumulation were used as the toxicity criteria. Validation of the predicted results related to the biochemicals with the experimental data clearly indicated that the model developed with the combination of ANN and GA is greatly effective in predicting the toxicity of PAHs and HMs mixtures toward microalga with <10% relative error. Both catalase and LPO were found to be the promising biomarkers for predicting microalgal toxicity of PAHs and HMs mixtures. In addition, a significant positive correlation was evident between the removal of PAHs/uptake of HMs and LPO.

Environmental levels of Zn do not protect embryos from Cu toxicity in three species of amphibians

Contaminants often occur as mixtures in the environment, but investigations into toxicity usually employ a single chemical. Metal contaminant mixtures from anthropogenic activities such as mining and coal combustion energy are widespread, yet relatively little research has been performed on effects of these mixtures on amphibians. Considering that amphibians tend to be highly sensitive to copper (Cu) and that metal contaminants often occur as mixtures in the environment, it is important to understand the interactive effects that may result from multiple metals. Interactive effects of Cu and zinc (Zn) on amphibians have been reported as antagonistic and, conversely, synergistic. The goal of our study was to investigate the role of Zn in Cu toxicity to amphibians throughout the embryonic developmental period. We also considered maternal effects and population differences by collecting multiple egg masses from contaminated and reference areas for use in four experiments across three species. We performed acute toxicity experiments with Cu concentrations that cause toxicity (10–200 μg/L) in the absence of other contaminants combined with sublethal concentrations of Zn (100 and 1000 μg/L). Our results suggest very few effects of Zn on Cu toxicity at these concentrations of Zn. As has been previously reported, we found that maternal effects and population history had significant influence on Cu toxicity. The explanation for a lack of interaction between Cu and Zn in this experiment is unknown but may be due to the use of sublethal Zn concentrations when previous experiments have used Zn concentrations associated with acute toxicity. Understanding the inconsistency of amphibian Cu/Zn mixture toxicity studies is an important research direction in order to create generalities that can be used to understand risk of contaminant mixtures in the environment.

Evaluating the concentration addition approach for describing expected toxicity of a ternary metal mixture (Ni, Cu, Cd) using metal speciation and response surface regression

Environmental contextEnvironmental quality guidelines are often based on an ‘additive’ approach using single metal toxicity values. We evaluated the ‘additive’ approach by testing it on three priority pollutant metals (Ni, Cu, Cd), and found that the toxicity of the metal mixture was less than additive when dose was expressed as total metal concentration, but it was additive when dose was expressed as bioavailable metal. We suggest that for metal mixtures, a more realistic indicator of risk is provided by calculations based on the bioavailable form of metals. AbstractAquatic environments containing elevated metal concentrations, such as natural waters receiving mining effluents, are often a mixture of metals because mineral deposits are commonly an association of multiple metals. Water quality guidelines for protection of aquatic life are not designed for multiple toxicants but are overwhelmingly based on dose–response studies of a single toxicant and assuming additivity. Resolving the uncertainty in risk assessment for metal mixtures in waters surrounding Canada’s many current and legacy extractive mining sites is a high priority for both government and base metal mining companies. Our study evaluated the ‘concentration addition’ approach to predicting the chronic toxicity of a ternary metal mixture (Ni, Cu, Cd) to Lemna minor (a free-floating macrophyte used in biomonitoring of mining effluents) using either total metal concentration as dose or free-ion activity. The aim was to fill several data gaps in mixture toxicity studies, such as: inclusion of water chemistry to calculate metal speciation, test species other than the commonly studied rainbow trout and cladocerans, and test mixture effects on chronic toxicity. Results indicate that toxicity of Ni, Cu plus Cd to L. minor was less than additive (overestimated toxicity) when expressed as total metal concentration but was additive when expressed as free ion (the bioavailable form). We suggest that applying single-element quality guidelines ‘additively’ for plants is likely to overestimate risk to the ecosystem from metal mixtures, and that the use of a concentration addition approach based on the bioavailable form of metals provides a more realistic indicator of risk.

Quantifying synergy: a systematic review of mixture toxicity studies within environmental toxicology.

Cocktail effects and synergistic interactions of chemicals in mixtures are an area of great concern to both the public and regulatory authorities. The main concern is whether some chemicals can enhance the effect of other chemicals, so that they jointly exert a larger effect than predicted. This phenomenon is called synergy. Here we present a review of the scientific literature on three main groups of environmentally relevant chemical toxicants: pesticides, metal ions and antifouling compounds. The aim of the review is to determine 1) the frequency of synergy, 2) the extent of synergy, 3) whether any particular groups or classes of chemicals tend to induce synergy, and 4) which physiological mechanisms might be responsible for this synergy. Synergy is here defined as mixtures with minimum two-fold difference between observed and predicted effect concentrations using Concentration Addition (CA) as a reference model and including both lethal and sub-lethal endpoints. The results showed that synergy occurred in 7%, 3% and 26% of the 194, 21 and 136 binary pesticide, metal and antifoulants mixtures included in the data compilation on frequency. The difference between observed and predicted effect concentrations was rarely more than 10-fold. For pesticides, synergistic mixtures included cholinesterase inhibitors or azole fungicides in 95% of 69 described cases. Both groups of pesticides are known to interfere with metabolic degradation of other xenobiotics. For the four synergistic metal and 47 synergistic antifoulant mixtures the pattern in terms of chemical groups inducing synergy was less clear. Hypotheses in terms of mechanisms governing these interactions are discussed. It was concluded that true synergistic interactions between chemicals are rare and often occur at high concentrations. Addressing the cumulative rather than synergistic effect of co-occurring chemicals, using standard models as CA, is therefore regarded as the most important step in the risk assessment of chemical cocktails.

Realistic environmental mixtures of micropollutants in surface, drinking, and recycled water: Herbicides dominate the mixture toxicity toward algae

Mixture toxicity studies with herbicides have focused on a few priority components that are most likely to cause environmental impacts, and experimental mixtures were often designed as equipotent mixtures; however, real-world mixtures are made up of chemicals with different modes of toxic action at arbitrary concentration ratios. The toxicological significance of environmentally realistic mixtures has only been scarcely studied. Few studies have simultaneously compared the mixture effect of water samples with designed reference mixtures comprised of the ratios of analytically detected concentrations in toxicity tests. In the present study, the authors address the effect of herbicides and other chemicals on inhibition of photosynthesis and algal growth rate. The authors tested water samples including secondary treated wastewater effluent, recycled water, drinking water, and storm water in the combined algae assay. The detected chemicals were mixed in the concentration ratios detected, and the biological effects of the water samples were compared with the designed mixtures of individual detected chemicals to quantify the fraction of effect caused by unknown chemicals. The results showed that herbicides dominated the algal toxicity in these environmentally realistic mixtures, and the contribution by the non-herbicides was negligible. A 2-stage model, which used concentration addition within the groups of herbicides and non-herbicides followed by the model of independent action to predict the mixture effect of the two groups, could predict the experimental mixture toxicity effectively, but the concentration addition model for herbicides was robust and sufficient for complex mixtures. Therefore, the authors used the bioanalytical equivalency concept to derive effect-based trigger values for algal toxicity for monitoring water quality in recycled and surface water. All water samples tested would be compliant with the proposed trigger values associated with the appropriate guidelines.

Sediment quality guidelines: challenges and opportunities for improving sediment management

During the International Conference on Deriving Environmental Quality Standards for the Protection of Aquatic Ecosystems held in Hong Kong in December 2011, an expert group, comprising scientists, government officials, and consultants from four continents, was formed to discuss the important scientific and regulatory challenges with developing sediment quality guidelines (SQGs). We identified the problems associated with SQG development and made a series of recommendations to ensure that the methods being applied were scientifically defensible and internationally applicable. This document summarizes the key findings from the expert group. To enable evaluation of current SQG derivation and application systems, a feedback mechanism is required to communicate confounding factors and effects in differing environments, while field validation is necessary to gauge the effectiveness of SQG values in sediment quality assessments. International collaboration is instrumental to knowledge exchange and method advancement, as well as promotion of 'best practices'. Since the paucity of sediment toxicity data poses the largest obstacle to improving current SQGs and deriving new SQGs, a standardized international database should be established as an information resource for sediment toxicity testing and monitoring data. We also identify several areas of scientific research that are needed to improve sediment quality assessment, including determining the importance of dietary exposure in sediment toxicity, mixture toxicity studies, toxicity screening of emerging chemicals, how climate change influence sediments and its biota, and possible use of new toxicity study approaches such as high throughput omic-based toxicity screenings.

An Empirical Approach to Sufficient Similarity: Combining Exposure Data and Mixtures Toxicology Data

When assessing risks posed by environmental chemical mixtures, whole mixture approaches are preferred to component approaches. When toxicological data on whole mixtures as they occur in the environment are not available, Environmental Protection Agency guidance states that toxicity data from a mixture considered "sufficiently similar" to the environmental mixture can serve as a surrogate. We propose a novel method to examine whether mixtures are sufficiently similar, when exposure data and mixture toxicity study data from at least one representative mixture are available. We define sufficient similarity using equivalence testing methodology comparing the distance between benchmark dose estimates for mixtures in both data-rich and data-poor cases. We construct a "similar mixtures risk indicator"(SMRI) (analogous to the hazard index) on sufficiently similar mixtures linking exposure data with mixtures toxicology data. The methods are illustrated using pyrethroid mixtures occurrence data collected in child care centers (CCC) and dose-response data examining acute neurobehavioral effects of pyrethroid mixtures in rats. Our method shows that the mixtures from 90% of the CCCs were sufficiently similar to the dose-response study mixture. Using exposure estimates for a hypothetical child, the 95th percentile of the (weighted) SMRI for these sufficiently similar mixtures was 0.20 (i.e., where SMRI <1, less concern; >1, more concern).

Mixtures of endocrine disrupting contaminants modelled on human high end exposures: an exploratory study in rats

By diminishing the action of androgens during gestation, certain chemicals can induce irreversible demasculinization and malformations of sex organs in the male rat after gestational exposure. Studies with mixtures of such anti-androgens have shown that substantial combined effects occur even though each individual chemical is present at low, ineffective doses, but the effects of mixtures modelled based on human intakes have not previously been investigated. To address this issue for the first time, we selected 13 chemicals for a developmental mixture toxicity study in rats where data about in vivo endocrine disrupting effects and information about human exposures was available, including phthalates, pesticides, UV-filters, bisphenol A, parabens and the drug paracetamol. The mixture ratio was chosen to reflect high end human intakes. To make decisions about the dose levels for studies in the rat, we employed the point of departure index (PODI) approach, which sums up ratios between estimated exposure levels and no-observed-adverse-effect-level (NOAEL) values of individual substances. For high end human exposures to the 13 selected chemicals, we calculated a PODI of 0.016. As only a PODI exceeding 1 is expected to lead to effects in the rat, a total dose more than 62 times higher than human exposures should lead to responses. Considering the high uncertainty of this estimate, experience on lowest-observed-adverse-effect-level (LOAEL)/NOAEL ratios and statistical power of rat studies, we expected that combined doses 150 times higher than high end human intake estimates should give no, or only borderline effects, whereas doses 450 times higher should produce significant responses. Experiments indeed showed clear developmental toxicity of the 450-fold dose in terms of increased nipple retention (NR) and reduced ventral prostate weight. The 150-fold dose group exhibited significantly increased NR. These observations suggest that highly exposed population groups, especially women of reproductive age, may not be protected sufficiently against the combined effects of chemicals that affect the hormonal milieu required for normal male sexual differentiation.

Chronic mixture toxicity study of Clophen A60 and diethyl phthalate in male rats

Chemical mixtures are an important area of research as individuals are exposed to low doses of persistent chemical agents known as environmental pollutants throughout their life time. Polychlorinated biphenyls (PCBs) and diethyl phthalate (DEP) are ubiquitous environmental pollutants that could be present in the same environmental compartment; hence organisms may get simultaneously exposed to both. Therefore, a study was undertaken to see whether PCB and DEP together show interactive chronic mixture toxicity in male Wistar rats. Healthy male Wistar rats weighing 70–100 g were randomly assigned to four groups of six each. Control rats were fed on normal diet and water ad libitum. Oil control rats were maintained on a normal diet mixed with corn oil. Rats were given Clophen A60 (PCB) and DEP dissolved individually in corn oil mixed with the diet at 50 mg kg−1 of the diet/day, as well as a mixture in corn oil mixed with the diet both at 50 mg kg−1 of the diet/day. After 150 days of treatment animals were sacrificed and enzymes and other biochemical parameters in the serum and liver were assessed. Liver weight to body weight ratio showed a significant increase in Clophen A60 and in Clophen A60 + DEP treated rats. In the DEP, Clophen A60 and Clophen A60 + DEP treated groups there was significant increase in liver and serum alanine aminotransferase (ALT) and acid phosphatase (ACP) activity. Aspartate aminotransferase (AST) was significantly increased in the liver and serum of DEP treated rats only. Cholesterol levels were significantly increased only in the serum and the liver of DEP treated rats. Triglyceride levels were significantly increased in the serum of treated rats and only in the liver of Clophen A60 and Clophen A60 + DEP treated rats. Liver glycogen levels were significantly increased in DEP and Clophen A60 + DEP treated rats. In all treated animals, there was a significant decrease in liver glutathione reductase (GR). Histology of liver showed severe vacuolations, fatty degeneration and loss of hepatic architecture in Clophen A60 and Clophen A60 + DEP treated rats, whereas in DEP treated rats only loss of hepatic architecture and granular deposits in the hepatocytes was predominant with mild vacuolations of centrilobular and periportal area. It is evident from this study of mixture toxicity of Clophen A60 and DEP that there is no significantly enhanced toxicity due to the interaction of these two compounds. On the other hand, to some extent there is alleviation in toxicity as evidenced by enzyme ACP and AST levels in the liver. The hepatocellular damage and biliary congestion caused by these two compounds, which can be confirmed by significantly increased liver weights and elevated serum and liver enzyme levels as well as histology, was almost the same between individual and mixture treated group.

An isobole-based statistical model and test for synergism/antagonism in binary mixture toxicity experiments

Synergism and antagonism are often defined in relation to the model of Concentration Addition (CA). Hence, it is vital for the conclusion of mixture toxicity studies to be able to test whether an observed deviation from CA reflects a true deviation or whether it is simply due to random variation. In this paper we consider a non-linear regression model for the classical ray designs for binary mixture experiments. The model combines dose–response curves for each mixture in the experiment with an isobole model, describing possible deviations from CA. The method allows us to test whether the chosen isobole model is reasonable for the data and to test the hypothesis of CA. Furthermore, it provides us with a measure of the degree of synergism/antagonism. The method is flexible since both the dose–response relationships and the isobole model can be chosen arbitrarily. We demonstrate the use of the method on datasets where combinations of pesticides are tested on a floating plant, Lemna minor, and an algae, Pseudokirchneriella subcapitata. Furthermore, we conduct a simulation study in order to explore the power with which a specific deviation from CA can be distinguished in different test-systems.

Mixture toxicity of three toxicants with similar and dissimilar modes of action to Daphnia magna

Mixture toxicity of similar- and dissimilar-acting toxicants can be predicted by the models concentration addition (CA) and independent action (IA) using single substance toxicity data. Knowledge of the toxicants mode of action is thus required in order to use the models. In order to test the predictive capability of the models, we conducted Daphnia magna 48 h immobilization experiments with three toxicants with known modes of action (dimethoate, pirimicarb and linear alkyl benzene sulfonate) singly, and in binary and ternary mixtures. Our results indicate that CA and IA predict binary mixtures of similar- and dissimilar-acting toxicants equally well. CA and IA also equally predicted the ternary mixture consisting of both similar- and dissimilar-acting chemicals. The paper discusses the concept of mode of action and the implications the definition of mode of action has on the choice of reference model for mixture toxicity studies.

Is mixture toxicity measured on a biomarker indicative of what happens on a population level? A study with Lemna minor

For plants, pigment content has shown to be a remarkably consistent biomarker across chemicals with different modes of action. In this study, we evaluated the use of pigment content as endpoint in binary mixture toxicity studies compared to three growth endpoints on the floating plant Lemna minor. Six binary combinations of six herbicides with different mode of action were used. Data were tested against both the concentration addition (CA) and independent action (IA) reference models. For CA, two statistical approaches were used. The study showed that for some herbicide combinations the mixture toxicity measured on pigment content did not reflect the results measured on plant population growth, emphasizing the importance of measuring growth in parallel with biomarkers. CA explained the data just as well as IA, and the two different statistical models used to test the data in relation to CA showed very similar results.