Mixture toxicity of zinc,cadmium, copper,and lead to the potworm Enchytraeus albidus

Central composite designs were used to develop surface response relationships for predicting the chronic toxicity of mixtures of zinc, cadmium, copper, and lead to the potworm Enchytraeus albidus. Surface response relationships were obtained for all binary mixtures of zinc, cadmium, copper, and lead as well as for mixtures with all four metals. For the binary mixture of zinc and cadmium, this approach and the toxit unit approach gave similar results. However, with the toxit unit approach no such surface response models can be developed. The experimental results indicated that effect predictions based on the concentration addition model were always higher than those obtained with the independent action model. The observed effect was lower than the effect predicted by the concentration addition model for all metal mixtures. Therefore it can be concluded that the concentration addition model represents a reasonable worst-case scenario for the risk assessment of metal mixtures in terrestrial ecosystems.     

Joint actions of carboxylic acid binary mixtures on Xenopus embryo development: Comparison of joint actions for malformation types

The joint action for malformations induced by 18 binary mixtures of four carboxylic acids was determined, using Xenopus embryos. The acids were selected to evaluate whether joint actions for mixtures changed when the data were analyzed by specific malformations or malformation syndromes as opposed to joint actions determined when any type of malformation was included in the analysis. Each of six binary combinations of the acids were tested three or four times in 96-h staticrenewal tests, starting with late-blastula stage embryos. Toxic unit analysis indicated that all but five of 56 possible analyses showed concentration addition joint actions. Concentration addition was observed for all instances in which microcephaly was included as a malformation (with or without other malformations being included) and for all possible mixtures where only eye defects were evaluated. In five instances, evaluating only abnormal gut coiling, the joint action was response addition, including all three mixtures for the valproic acid and pentanoic acid combination. The other 29 analyses which included abnormal gut coiling showed concentration addition. The results indicate that, for chemicals that are structurally similar and induce the same types of defects in frog embryos, there is no significant change in the joint action when the data is analyzed by specific malformations or syndromes rather than by including all malformations together. Abnormal gut coiling, a malformation induced by most toxic chemicals, appears to be less reliable for joint action determination, especially if it is the primary manifestation of abnormal development.     

A model of additive effects of mixtures of narcotic chemicals

Biological effects data with single chemicals are far more abundant than with mixtures. Yet, environmental exposures to chemical mixtures, for example near hazardous waste sites or non point sources, are very common and using test data from single chemicals to approximate effects of mixtures can be useful in environmental risk assessment. To facilitate the linkage, the Weibull function was used as a common model to link responses of single chemicals with the response of their mixtures. The present paper addresses the response of fish to mixtures of narcotic chemicals and a second paper addresses the developmental malformation of frog embryos when exposed to defined mixtures of teratogenic chemicals.Biological effects data with singly tested chemicals cannot be used directly to predict effects of mixtures. However, narcotic chemicals are known to produce an additive concentration effects in fish and the Weibull function with an additive concentration variable was used to model the effects of mixtures of these chemicals. The model produced good agreement with data over a wide range of chemicals and mixture ratios and provides a useful initial assessment of environmental effects of mixtures of narcotic chemicals.     

Combined toxicity of the mixtures of phenol and aniline derivatives to Vibrio qinghaiensis sp.-Q67

To test whether the dose addition and independent action models can predict the combined toxicity of the mixtures of phenol and aniline derivatives, six phenolic and two aniline derivatives were selected as the test components. The inhibition toxicity of the derivatives and their mixtures to Vibrio qinghaiensis sp.-Q67 indicated that all dose–response relationships could be effectively described by the Weibull function with correlation coefficients greater than 0.99. The combined toxicity of two equivalent-effect concentration ratio mixtures and eight uniform design concentration ratio mixtures could be predicted successfully by the dose addition model within 95% confidence intervals. However, it was also well predicted by the independent action model, especially at lower concentrations.     

The impact of Furadan 3G (carbofuran) applications on aquatic macroinvertebrates in irrigated rice in Senegal

Chemical compounds known to be teratogenic to frog embryos were tested singly and in binary mixtures and Weibull functions were used to model their concentration-response relationships. A separate Weibull function with an additive concentration variable modeled the mixtures using only single chemical test data. Seven chemicals in five binary mixtures at 3/1, 1/1, and 1/3 mixture proportions each were tested. The ratios of measured and calculated model parameters and median concentrations were estimated for comparing the results. The ratios ranged from 0.72 and 1.44 with an average at 0.99, an agreement that was qualitatively similar to modeling mixtures of narcotic chemicals which are known to produce additive concentration effects. The model reliability in determining mixture response classes and prediction of effects based on single chemical data was quantified. The model is useful for environmental risk assessment of chemical mixtures in hazardous waste sites and for the design of mixture experiments.     

Response predictions for organisms water-exposed to metal mixtures: a meta-analysis

To develop a multimetal toxicity model requires insight into the relationships between the composition of metal mixtures and their toxicological effects on organisms. As a first step in developing such a model, quantitative data from binary and higher mixture studies of Cu, Cd, and Zn were compiled and used to assess trends in toxicological effects on various organisms. The findings of this meta-analysis show relatively little occurrence of additive effects compared with antagonistic and synergistic effects. This observation held true irrespective of test species, environmental compartment, or metal concentrations in the mixture. However, the type of effect was significantly correlated with the metal combination tested and the selected toxicological endpoint. It was also found that different methods were used for assessing deviation from additivity in the various individual studies. For robust comparison, standardization on this point is required. Toxicological responses of organisms to metal mixtures were shown to be hard to predict and were often slightly less than or slightly more than additive. The interactions observed could not be explained by metal-metal interactions alone. We therefore conclude that with current scientific knowledge it is not yet possible to predict responses to metal mixtures in individual cases; at best this is possible only in terms of general patterns. Nevertheless, in the context of environmental risk policy, the assumption of additivity produces a conservative prediction of toxicity, because toxicity of a metal mixture will be either predicted correctly or overpredicted by default in approximately 75% of all cases. The use of models based on noninteraction is satisfactory from this regulatory perspective.     

Empirical modeling of an in vitro activity of polychlorinated biphenyl congeners and mixtures.

The goal of this research is to predict an in vitro activity of polychlorinated biphenyl (PCB) congeners and their mixtures and to describe the relationship between this activity and chemical structure. The test system used multiple PCB concentrations on each cell culture plate in a repeated measures design, which improved precision for comparing between concentration levels. A weighted regression that accounted for this experimental design feature was used in fitting a nonlinear dose-response exponential model to the PCB concentration-activity data from an in vitro test system in which 3H-phorbol ester binding was measured in cerebellar granule cells exposed to different PCB congeners to test for their effects on protein kinase C translocation. The model allowed for the minimum level to be less than control, a common slope, and the estimation of the log of the concentration that produces an activity 50% above the control activity (E50) for 36 congeners and 3 commercial mixtures. Next, a weighted logistic regression using a second order response model in the variables Clortho, Clpara, and Clmeta was used to relate the estimated log E50s to indicators of chemical structure. This model was preferred over models that might seem more mechanistically based because in internal validation, it attained a smaller PRESS statistic (the sum of squares between all observed and predicted observations) than other models. Evidently, this second order model makes more efficient use of parameters than other models considered. Plots of the predictions of the logistic second order response model versus log Kow confirm the usual pattern that congeners with intermediate levels of log Kow are the more active. The data of three commercial mixtures were included in this regression by assuming a common combination index (ratio of observed E50 to predicted E50, assuming dose addition). The logistic model suggests that congeners with one, two, or three chlorine substitutions at the ortho position are more active than other congeners. Also, congeners with log Kow between 5.2 and 6.6 are generally more active. The estimated combination index indicated that the joint action of PCB congeners in the three commercial mixtures was less than dose additive. The error sum of squares was significantly large, which may indicate a lack of fit of the logistic model. Empirical Bayes estimates (EBE) are weighted averages of model predictions and observations of E50s and can be better estimates than the fitted model when there is a lack of fit. The PRESS statistic for the EBE indicated larger prediction error than for the logistic model, but the EBE provided better estimates of commercial mixture E50s based on dose addition. This may indicate that the logistic model is not incorporating all the information in the single congener data needed to predict mixtures.     

Carcinogenic risk assessment of complex mixtures

Although procedures for assessing the carcinogenic risks associated with exposure to individual chemicals are relatively well developed, risk assessment methods for mixtures of chemicals are still in the developmental stage. In this paper, we examine the difficulties in assessing the risks of exposure to complex mixtures, with special reference to the potential for synergistic effects among the components of the mixture. Statistical models for describing the joint action of multiple exposures are reviewed, and their implications for low-dose risk assessment are examined. The potential use of pharmacokinetic models to describe the metabolism of mixtures is also considered. Application of these results in regulating mixtures of carcinogenic substances is illustrated using examples involving multiple contaminants in drinking water and polycyclic aromatic hydrocarbons produced from combustion sources.     

Use of simultaneous curve fitting and a four-parameter logistic model to evaluate the nutritional quality of protein sources at growth rates of rats from maintenance to maximum gain

A four-parameter logistic model was used to describe the dose-response relationship of rats fed diets containing 12 levels of casein, peanut meal or wheat gluten. The model was capable of accurately describing the entire response curve of rats fed diets containing each of the three protein sources. Incorporation of a technique known as parameter sharing into the curve-fitting process facilitated convergence of the parameter estimates for b (the response of rats fed a protein-free diet) and Rmax (maximum response) for all curves when compared with the values observed experimentally. Parameter sharing also provided a method by which the curves could be differentiated on a statistical basis. These data indicate that the relative value of a protein source is dependent on the concentration of the protein in the diet. The application of nonlinear models combined with parameter sharing provides a technique by which protein values can be evaluated at levels of animal response from maintenance to maximum growth.     

Single Versus Combined Lethal Effects of Three Agricultural Insecticides on Larvae of the Freshwater Insect Chironomus dilutus

Pesticides are currently regulated individually but are present in aquatic systems as mixtures of toxicants. In this study, the lethal effects of three agricultural insecticides-chlorpyrifos, imidacloprid and dimethoate-on Chironomus dilutus larvae were examined. Ninety-six-hour static bioassays were performed using single, binary, and ternary toxicant mixtures. The effects of the binary mixtures were investigated using the MIXTOX model to determine if the mixtures behaved additively, synergistically, or antagonistically. Dimethoate was much less toxic than chlorpyrifos or imidacloprid. Chlorpyrifos and dimethoate were not additive in mixture despite their common mode of action (MOA) and resulted in opposing effects (synergism and antagonism, respectively) when combined with imidacloprid. Synergism was observed in the ternary mixture. These results suggest that current mixture models based on MOA alone may not always be adequate in describing mixture effects, as in the current situation of mixtures with relatively few components. Other factors, such as water solubility, secondary MOA, and mechanisms of toxicity, should also be considered in future investigations of multichemical-mixture effects.     

Inter-species extrapolation of skin heating resulting from millimeter wave irradiation: modeling and experimental results

This study reports measurements of the skin surface temperature elevations during localized irradiation (94 GHz) of three species: rat (irradiated on lower abdomen), rhesus monkey (posterior forelimb), and human (posterior forearm). Two exposure conditions were examined: prolonged, low power density microwaves (LPM) and short-term, high power density microwaves (HPM). Temperature histories were compared with calculations from a bio-heat transfer model. The mean peak surface temperature increase was approximately 7.0 degrees C for the short-term HPM exposures for all three species/locations, and 8.5 degrees C (monkey, human) to 10.5 degrees C (rat) for the longer-duration LPM exposures. The HPM temperature histories are in close agreement with a one-dimensional conduction heat transfer model with negligible blood flow. The LPM temperature histories were compared with calculations from the bio-heat model, evaluated for various (constant) blood flow rates. Results suggest a variable blood flow model, reflecting a dynamic thermoregulatory response, may be more suited to describing skin surface temperature response under long-duration MMW irradiation.     

Reconstructing source polybrominated diphenyl ether congener patterns from semipermeable membrane devices in the Fraser River,British Columbia,Canada: comparison to commercial mixtures

Semipermeable membrane devices (SPMDs) were placed in the Fraser River near Vancouver, British Columbia, Canada, between August 6 and September 30, 1996. This location is near a large urban and industrial region (population 2,000,000) and is expected to be representative of other large, modern cities. After exposure to the ambient water column, SPMD samples were analyzed for a suite of 36 polybrominated diphenyl ether (PBDE) congeners plus all homologue groups from mono- through hexa-brominated. Observed congener patterns differed significantly from that of the commercial penta- and octa-BDE mixtures. A reconstruction approach was developed based on an aquatic transport model and utilizing published octanol-water partition coefficients, calculated SPMD uptake rates, and predicted water concentrations by using the EcoFate multimedia mass balance aquatic simulation model for the 13 major PBDE congeners. In combination, composite technical mixtures were created by combining commercial penta-BDE mixtures (Bromkal 70-SDE and Great Lakes Chemicals DE-71) with commercial octa-BDE mixtures (Bromkal 79-8DE and Great Lakes Chemicals DE-79) in their relative 2000 North American production volumes. The reconstructed SPMD patterns more closely approximated the composite technical mixtures and suggest that PBDEs in such an industrial region arise primarily from penta- and octa-BDE source mixtures.     

Mixture toxicity and gene inductions: can we predict the outcome?

As a consequence of the nature of most real-life exposure scenarios, the last decade of ecotoxicological research has seen increasing interest in the assessment of mixture ecotoxicology. Often, mixtures are considered to follow one of two models, concentration addition (CA) or response addition (RA), both of which have been described in the literature. Nevertheless, mixtures that deviate from either or both models exist; they typically exhibit phenomena like synergism, ratio or concentration dependency, or inhibition. Moreover, both CA and RA have been challenged and evaluated mainly for acute responses at relatively high levels of biological organization (e.g., whole-organism mortality), and applicability to genetic responses has not received much attention. Genetic responses are considered to be the primary reaction in case of toxicant exposure and carry valuable mechanistic information. Effects at the gene-expression level are at the heart of the mode of action by toxicants and mixtures. The ability to predict mixture responses at this primary response level is an important asset in predicting and understanding mixture effects at different levels of biological organization. The present study evaluated the applicability of mixture models to stress gene inductions in Escherichia coli employing model toxicants with known modes of action in binary combinations. The results showed that even if the maximum of the dose-response curve is not known, making a classical ECx (concentration causing x% effect) approach impossible, mixture models can predict responses to the binary mixtures based on the single-toxicant response curves. In most cases, the mode of action of the toxicants does not determine the optimal choice of model (i.e., CA, RA, or a deviation thereof).     

Measurement and modeling of the toxicity of binary mixtures in the nematode caenorhabditis elegans--a test of independent action

Ecological risk assessments must increasingly consider the effects of chemical mixtures on the environment as anthropogenic pollution continues to grow in complexity. Yet testing every possible mixture combination is impractical and unfeasible; thus, there is an urgent need for models that can accurately predict mixture toxicity from single-compound data. Currently, two models are frequently used to predict mixture toxicity from single-compound data: Concentration addition and independent action (IA). The accuracy of the predictions generated by these models is currently debated and needs to be resolved before their use in risk assessments can be fully justified. The present study addresses this issue by determining whether the IA model adequately described the toxicity of binary mixtures of five pesticides and other environmental contaminants (cadmium, chlorpyrifos, diuron, nickel, and prochloraz) each with dissimilar modes of action on the reproduction of the nematode Caenorhabditis elegans. In three out of 10 cases, the IA model failed to describe mixture toxicity adequately with significant or antagonism being observed. In a further three cases, there was an indication of synergy, antagonism, and effect-level-dependent deviations, respectively, but these were not statistically significant. The extent of the significant deviations that were found varied, but all were such that the predicted percentage effect seen on reproductive output would have been wrong by 18 to 35% (i.e., the effect concentration expected to cause a 50% effect led to an 85% effect). The presence of such a high number and variety of deviations has important implications for the use of existing mixture toxicity models for risk assessments, especially where all or part of the deviation is synergistic.     

Assessment of the Probabilistic Ecological Risk Assessment-Toxic Equivalent Combination Approach for Evaluating Pesticide Mixture Toxicity to Zooplankton in Outdoor Microcosms

The probabilistic ecological risk assessment-toxic equivalent (PERA-TE) combination approach was recently introduced in response to the increased demand for risk assessment approaches that can accommodate mixtures. The effectiveness and validity of the PERA-TE approach was assessed using two types of pesticide mixtures tested in outdoor microcosms. The first type of mixture consisted of pesticides with similar modes of action (the organophosphorus insecticides chlorpyrifos and diazinon) and the second of pesticides with different modes of action (chlorpyrifos, endosulfan, and trifluralin). To assess the toxicity of, and potential interaction within, each type of mixture, theoretically equitoxic TE mixtures were prepared in different proportional ratios. The TE mixtures were based on the 10th centile of acute toxicity effects distributions (data obtained from the literature) and a factor of the sum of the 90th centile field concentrations extrapolated from exposure distributions based on North American surface water monitoring data. Changes in zooplankton population abundances were used as the effect measure. The binary organophosphorus mixtures were equitoxic and conformed to the concentration addition model. The observed response trends of zooplankton exposed to the mixture of chemicals with different modes of action were a result of the susceptibility of individual taxa to the dominating pesticide in each mixture. Overall, the PERA-TE approach was not effective in predicting the toxicity and interaction of all mixture types and should be limited to assessing mixtures of chemicals with similar modes of action.     

Taking account of between-patient variability when modeling decline in Alzheimer's disease.

The pattern of deterioration in patients with Alzheimer's disease is highly variable within a given population. With recent speculation that the apolipoprotein E allele may influence rate of decline and claims that certain drugs may slow the course of the disease, there is a compelling need for sound statistical methodology to address these questions. Current statistical methods for describing decline do not adequately take into account between-patient variability and possible floor and/or ceiling effects in the scale measuring decline, and they fail to allow for uncertainty in disease onset. In this paper, the authors analyze longitudinal Mini-Mental State Examination scores from two groups of Alzheimer's disease subjects from Palo Alto, California, and Minneapolis, Minnesota, in 1981-1993 and 1986-1988, respectively. A Bayesian hierarchical model is introduced as an elegant means of simultaneously overcoming all of the difficulties referred to above.     

Application of benzo(a)pyrene and coal tar tumor dose-response data to a modified benchmark dose method of guideline development

Assessment of cancer risk from exposure to polycyclic aromatic hydrocarbons (PAHs) has been traditionally conducted by applying the conservative linearized multistage (LMS) model to animal tumor data for benzo(a)pyrene (BaP), considered the most potent carcinogen in PAH mixtures. Because it has been argued that LMS use of 95% lower confidence limits on dose is unnecessarily conservative, that assumptions of low-dose linearity to zero in the dose response imply clear mechanistic understanding, and that "acceptable" cancer risk rests on a policy decision, an alternative cancer risk assessment approach has been developed. Based in part on the emerging benchmark dose (BMD) method, the modified BMD method we used involves applying a suite of conventional mathematical models to tumor dose-response data. This permits derivation of the average dose corresponding to 5% extra tumor incidence (BMD0.05) to which a number of modifying factors are applied to achieve a guideline dose, that is, a daily dose considered safe for human lifetime exposure. Application of the modified BMD method to recent forestomach tumor data from BaP ingestion studies in mice suggests a guideline dose of 0.08 microg/kg/day. Based on this and an understanding of dietary BaP, and considering that BaP is a common contaminant in soil and therefore poses human health risk via soil ingestion, we propose a BaP soil guideline value of 5 ppm (milligrams per kilogram). Mouse tumor data from ingestion of coal tar mixtures containing PAHs and BaP show that lung and not forestomach tumors are most prevalent and that BaP content cannot explain the lung tumors. This calls into question the common use of toxicity equivalence factors based on BaP for assessing risk from complex PAH mixtures. Emerging data point to another PAH compound--H-benzo(c)fluorene--as the possible lung tumorigen.     

A review of independent action compared to concentration addition as reference models for mixtures of compounds with different molecular target sites

From a theoretical point of view, it has often been argued that the model of independent action (IA) is the most correct reference model to use for predicting the joint effect of mixtures of chemicals with different molecular target sites. The theory of IA, however, relies on a number of assumptions that are rarely fulfilled in practice. It has even been argued that, theoretically, the concentration addition (CA) model could be just as correct. In the present study, we tested the accuracy of both IA and CA in describing binary dose-response surfaces of chemicals with different molecular targets using statistical software. We compared the two models to determine which best describes data for 158 data sets. The data sets represented 98 different mixtures of, primarily, pesticides and pharmaceuticals tested on one or several of seven test systems containing one of the following: Vibrio fischeri, activated sludge microorganisms, Daphnia magna, Pseudokirchneriella subcapitata, Lemna minor, Tripleurospermum inodorum, or Stellaria media. The analyses showed that approximately 20% of the mixtures were adequately predicted only by IA, 10% were adequately predicted only by CA, and both models could predict the outcome of another 20% of the experiment. Half of the experiments could not be correctly described with either of the two models. When quantifying the maximal difference between modeled synergy or antagonism and the reference model predictions at a 50% effect concentration, neither of the models proved significantly better than the other. Thus, neither model can be selected over the other on the basis of accuracy alone.