Concentration addition and response addition to analyze mixture toxicity: Is it worth testing?

Having gained ample knowledge on the toxicity of single chemicals and now recognizing that chemicals usually occur in mixtures in the environment, ecotoxicological studies are increasingly addressing chemical mixtures. To understand mixture toxicity, concentration addition (also called simple similar joint action) and response addition (also called independent action) approaches are commonly applied. Briefly, concentration addition assumes that chemicals of concern have the same mode of action, whereas response addition assumes they have different modes of action and act independently [1–3]. Although concentration addition and response addition have been tested extensively in ecotoxicological studies, we contend that they are just 2 of many ways to test mixture toxicity, and their use is not mandatory. This is particularly true when the toxic mechanisms of contaminants in mixtures are not well understood for the given species, exposure duration, exposure route (i.e., dietary and waterborne), exposure concentration level, measure of exposure used (i.e., tissue or exposure concentrations), and developmental stage of the animal/plant being tested. Indeed, toxic mechanisms are usually not well identified (with the exception of some organic compounds such as pesticides and estrogenic agents) and from a practical standpoint, numerous potential pathways are involved in toxic responses that change based on exposure route, concentration, duration, and so on. This makes it difficult to a priori determine which approach should be applied to the toxicity data set in hand. Therefore, both concentration addition and response addition approaches have been often applied to test the chemical interaction in the same data set. Such studies typically have concluded that the effects of chemical mixtures are better predicted by concentration addition than response addition or that the concentration addition approach provides a more conservative estimate of mixture toxicity than response addition [4]. The accumulation of these results allows us to suggest that applying concentration addition is the first practical choice in ecological risk assessment regardless of modes of action. However, what do they really mean for the data set tested given that it is uncertain that the assumptions regarding the mode of action are satisfied and that similarity in the modes of action of chemicals (i.e., meeting the assumption of the concentration addition model) does not preclude non-additive toxicity [3]? Furthermore, some have criticized that successfully applying the concentration addition approach may be a result of random chance, as potential antagonistic or potentiating interactions can manifest an outcome that mimics concentration addition predictions [5]. Thus, the evidence supporting the concentration addition and response addition approaches should be interpreted with caution. Another issuewe have observed is that by using concentration addition or response addition approaches, variations in the observed or predicted responses are frequently overlooked to test the additivity of the chemicalmixture [2]. Evenworse are cases in which the test of additivity is conducted by subjective inspection lacking any objective measure. This implies that caution is needed to interpret the line of evidence that has been accumulated supporting the concentration addition and response addition approaches. In addition, if the concentration addition approach is used to evaluate concentration–response relationships for mixtures by summing toxic units, the implicit assumption is that concentration–response curves for individual chemicals are parallel (seeKortenkampet al. [4] formore detailed information). Unfortunately, this assumption is also often overlooked.Again, it is unclear what the results of applying concentration addition mean in such cases. The use of concentration addition or response addition models dominate the literature in terms of assessing additivity; however, they are just 2 of several available methods for testing mixture toxicity [4,6–9]. For example, the chemical interaction can be tested in a classical statistical manner by using a generalized linear model or generalized linear mixed model (e.g., Iwasaki and Brinkman [6]) and amore comprehensive tool developed by Jonker et al. [7], both of which model the entire concentration–response relationships. The use of toxicokinetic and toxicodynamic modelling approaches have also been presented [8,9], avoiding the assumptions of concentration addition and response addition models and instead opting to model physiological processes of contaminant metabolism, detoxification, and distribution [10]. Applying multiple mechanistically based models has also been presented to model toxicity of metal mixtures [11]. Just as we choose from a list of statistical approaches to analyze non-mixture data, the choice of which approaches to take for testing mixture toxicity depends on the researcher’s needs. For example, what type of experimental design was applied? What would the researcher like to infer from the data? Is mixture additivity to be assessed, and does the researcher want to test additivity by performing the point-by-point comparisons or by evaluating the entire concentration–response relationships? It should be emphasized that we agree concentration addition and response addition approaches have a long history of use and accumulating further evidence would be of value. Nonetheless, we do not recommend that concentration addition and response addition approaches be simply applied to the data in hand without carefully recognizing and considering the underlying assumptions of the employed Published online in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/etc.3263 Environmental Toxicology and Chemistry, Vol. 35, No. 3, pp. 526–527, 2016 # 2016 SETAC Printed in the USA