Chemical Mixtures: Greater-than-Additive Effects?

Referencing: Pesticide Mixtures, Endocrine Disruption, and Amphibian Declines: Are We Underestimating the Impact?

Various combinations of chemicals are being detected in the environment with increasing frequency. This has raised awareness that we are not exposed to individual chemicals in isolation and heightens concern that the toxicity of individual chemicals may not represent toxicity when the chemicals are present in combination. Of greatest concern is that chemicals in combination may elicit synergistic toxicity that goes undetected in evaluations of individual chemical toxicity.

In a recent article, Hayes et al. (2006) assessed the effects of nine pesticides individually (at 0.10 ppb) and in combination (each at 0.10 ppb) on time to foreleg emergence and time to complete tail resorption in Rana pipiens. Both end points are measures of larval development in frogs. The authors reported that the pesticide mixture had a much greater effect on these developmental parameters than did the individual chemicals; they concluded that estimating ecologic risk of pesticides on amphibians using studies that examine single pesticides may lead to gross underestimates of the role of pesticides in amphibian declines. Clearly, Hayes et al. implied that the combined effect of the nine pesticides is greater than the sum of the individual chemicals. But is this speculation of synergy warranted from these data?

To invoke synergy, one must—at a minimum—exclude the possibility of concentration or response additivity. Concentration additivity may appear as synergy when individual constituents, sharing the same mechanism of toxicity, in a mixture are all present below the threshold concentrations required for toxicity. However, in combination, the joint concentration of the constituents exceeds that threshold concentration, resulting in significant adversity. These experiments were not designed to assess concentration additivity, so no judgment can be made either in favor of or against the possibility that the toxicity of the mixture represented concentration additivity. However, individual responses to the nine pesticides were shown in graph form (Figure 1; Hayes et al. 2006), which allows for an assessment of response additivity for the mixture. Eight of nine pesticides prolonged the time to foreleg emergence, and nine of nine chemicals prolonged the time to tail resorption. However, these effects were not statistically significant, with the exception of the effects elicited by propiconzole.

We subjected these data to analyses for response additivity under the assumption that the observed effects were real but were not statistically significant due to the low power of the experimental design. A description of the response additivity model is available on our website [Computational Approach to the Toxicity Assessment of Mixtures (CATAM) 2006a] along with a mixtures toxicity calculator used in these analyses (CATAM 2006b). The response addition model predicted that the mixture of pesticides would prolong the time to foreleg emergence from 44 days to 60 days and the time to tail resorption from 56 days to 67 days. Hayes et al. (2006) determined that the pesticide mixture extended these developmental time points to 59 ± 2 days and 70 ± 2 days, respectively (mean ± SE; these values are our best estimates from Hayes et al.'s Figure 2). Thus, response addition alone explains the toxicity associated with a pesticide mixture. There is no need to invoke greater-than-additive effects and no need to raise concern that mixtures of these pesticides cause unexpected toxicity.

Toxicity of mixtures is a perplexing problem that warrants significant investigation. However, when assessing the toxicity of chemical mixtures, it is prudent to test the null hypothesis of no interactions. Only upon rejection of this hypothesis should the possibility of synergistic interactions be considered. In response to the question posed by Hayes et al. (2006) in the title of their article—"Pesticide Mixtures . . . Are We Underestimating the Impact?"—the evidence presented suggests that the answer is "no."

The authors declare they have no competing financial interests.

Gerald A. LeBlanc
Guirong Wang
Department of Environmental and Molecular Toxicology
North Carolina State University
Raleigh, North Carolina
E-mail: ga_leblanc@ncsu.edu

References

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Chemical Mixtures: Hayes Responds

LeBlanc and Wang point out that we did not demonstrate synergy; they stated that "to invoke synergy, one must—at a minimum—exclude the possibility of concentration or response additivity."In fact, we did not "invoke synergy": in our article, not once did we use the word "synergy." LeBlanc and Wang themselves "invoke synergy" simply to show that it cannot be invoked from the current study; their problem is with what they inferred from our article, not with any claims that we made.

LeBlanc and Wang also point out that our " . . . experiments were not designed to assess concentration additivity, so no judgment can be made either in favor of or against the possibility that the toxicity of the mixture represented concentration additivity." We agree. This was not the aim of the study. In fact, LeBlanc and Wang's thesis here is merely a restatement of our own conclusions:

The present effects of mixtures cannot be assigned to the categories of concentration additive or response additive . . . (Hayes et al. 2006; p. 47)

and

The examinations needed to characterize pesticide interactions as concentration additive or response additive . . . are difficult to design and carry out and present new challenges to regulators. Such studies are necessary, however . . . . (Hayes et al. 2006; p. 48)

Finally, LeBlanc and Wang examined our data for response additivity using a simple model and testing select parameters that fit their model while ignoring others. In our study we examined effects of nine pesticides alone (0.1 ppb) or in three different mixtures at 0.1 ppb and 10 ppb on leopard frog (Rana pipiens) larvae. Each treatment (30 larvae/tank) was replicated three times (1,350 larvae total). We assessed effects on 10 parameters: time to foreleg emergence (FLE) and time to complete tail resorption (TR), snout-vent length (SVL) and body weight (BW) at metamorphosis, mortality, gonadal development, thymus histology, disease rates, and the interaction between time to TR and SVL and BW at metamorphosis. Yet, according to LeBlanc and Wang, we simply

assessed the effects of nine pesticides individually (at 0.10 ppb) and in combination (each at 0.10 ppb) on time to foreleg emergence and time to complete tail resorption.

LeBlanc and Wang used their simple model to show that the effects of one of the pesticide mixtures on developmental time are predictable from the nonsignificant effects of the individual pesticides. Although they predicted the effects of a single pesticide mixture on a single variable, can their model predict the effects of even the single pesticides (propiconazole, -cyhalothrin, and atrazine) on the interaction between development and growth (Figure 5; Hayes et al. 2006), when none of these compounds significantly affected development alone and only atrazine affected size alone? Can their model predict the effects of atrazine plus S-metolachlor on the relationship between development and size or explain why the "inert" ingredients in the commercial mixture (Bicep II magnum; Syngenta Crop Protection U.S., Research Triangle Park, NC) appear to reduce this effect? Most certainly, the 70% meningitis infection rate in the surviving animals exposed to the nine-compound mixture cannot be predicted from exposure to the single pesticides, where disease rates were zero. The effect on development was the only parameter that fit LeBlanc and Wang's model and thus explains their reason for focusing on this single measure and ignoring the other nine parameters we measured.

In conclusion, the questions raised in our article (Hayes et al. 2006) can be answered only with empirical evidence obtained from appropriately designed and carefully conducted laboratory experiments, not by simplified models that ignore interactions between independent variables and relationships between dependent variables. Finally, and most important, our data clearly show that examining individual pesticides one at a time does not reveal the magnitude of effects of low-dose chronic exposure to pesticide mixtures and thus does not allow us to accurately assess their impacts on amphibians. Practically all of the chemicals we examined had no significant effects alone, but this was certainly not the case when they were combined. Whether or not these interactions are response additive, concentration additive, or synergistic is irrelevant to the real question: Are we underestimating the impact? If we continue to base assessments on examinations of single compounds, the answer is "yes."

The author declares he has no competing financial interests.

Tyrone B. Hayes
Department of Integrative Biology
University of California
Berkeley, California
E-mail: tyrone@berkeley.edu

References