Toxicity ranking and toxic mode of action evaluation of commonly used agricultural adjuvants on the basis of bacterial gene expression profiles

Abstract

The omnipresent group of pesticide adjuvants are often referred to as "inert" ingredients, a rather misleading term since consumers associate this term with "safe". The upcoming new EU regulation concerning the introduction of plant protection products on the market (EC1107/2009) includes for the first time the demand for information on the possible negative effects of not only the active ingredients but also the used adjuvants. This new regulation requires basic toxicological information that allows decisions on the use/ban or preference of use of available adjuvants. In this study we obtained toxicological relevant information through a multiple endpoint reporter assay for a broad selection of commonly used adjuvants including several solvents (e.g. isophorone) and non-ionic surfactants (e.g. ethoxylated alcohols). The used assay allows the toxicity screening in a mechanistic way, with direct measurement of specific toxicological responses (e.g. oxidative stress, DNA damage, membrane damage and general cell lesions). The results show that the selected solvents are less toxic than the surfactants, suggesting that solvents may have a preference of use, but further research on more compounds is needed to confirm this observation. The gene expression profiles of the selected surfactants reveal that a phenol (ethoxylated tristyrylphenol) and an organosilicone surfactant (ethoxylated trisiloxane) show little or no inductions at EC(20) concentrations, making them preferred surfactants for use in different applications. The organosilicone surfactant shows little or no toxicity and good adjuvant properties. However, this study also illustrates possible genotoxicity (induction of the bacterial SOS response) for several surfactants (POEA, AE, tri-EO, EO FA and EO NP) and one solvent (gamma-butyrolactone). Although the number of compounds that were evaluated is rather limited (13), the results show that the used reporter assay is a promising tool to rank commonly used agricultural adjuvants based on toxicity and toxic mode of action data.

Figure 1. Overview of major types of adjuvants.

Dotted squares represent selected groups, below one or more evaluated representatives. APEOs: alkyl phenol ethoxylates, ANEOs: alkyl amine ethoxylates, AEO: alcohol ethoxylates, FEO: fatty acid ethoxylates and EO: ethoxylated.

Figure 2. Description of differences in dose-respons curves, IC50 concentrations are equal but LOEC values differ due to differences in slope.

Figure 3. Bacterial dose response profile after exposure to an adjuvant (ethoxylated nonylphenol) and a reference compound (paraquat).

Figure 3a) ethoxylated nonylphenol, and 3b) paraquat. The y-axis denotes the induction at any given dose, the x-axis shows the different stress genes and the z-axis shows the applied concentrations in a ½ serial dilution. All data are means of three replicates (n = 3), c) Detailed results for significantly induced genes after exposure to ethoxylated nonylphenol meeting the criteria as mentioned in Material and Methods, bars indicate standard error. *Significantly different from solvent control (one-way ANOVA, Dunett's test, p<0.05).

Figure 4. Principal component analysis of FIS (fold induction score) dataset.

The first two components (PC1 and PC2) are shown. Individual points represent the gene expression pattern. This plot shows the possible presence of outliers, groups, similarities and other patterns in the data. Observations situated outside the ellipse are outliers. Blue dots: solvents, red dots: surfactants, green dots: reference compounds.