Toxicity Ranking and Toxic Mode of Action Evaluation of Commonly Used Agricultural Adjuvants on the Basis of Bacterial Gene Expression Profiles Ingrid Nobels 1 *, Pieter Spanoghe 2 , Geert Haesaert 3,4 , Johan Robbens 1 , Ronny Blust 1 1 Laboratory for Ecophysiology, Biochemistry and Toxicology, Department of Biology, University of Antwerp, Antwerp, Belgium, 2 Department of Crop Protection Chemistry, Ghent University, Ghent, Belgium, 3 Department of Biosciences and Landscape Architecture, University College Ghent, Ghent, Belgium, 4 Department of Plant Production, Ghent University, Ghent, Belgium 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. Citation: Nobels I, Spanoghe P, Haesaert G, Robbens J, Blust R (2011) Toxicity Ranking and Toxic Mode of Action Evaluation of Commonly Used Agricultural Adjuvants on the Basis of Bacterial Gene Expression Profiles. PLoS ONE 6(11): e24139. doi:10.1371/journal.pone.0024139 Editor: Baochuan Lin, Naval Research Laboratory, United States of America Received March 15, 2011; Accepted August 1, 2011; Published November 18, 2011 Copyright: ß 2011 Nobels et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was financially supported by the Federal Public Service of Health, Food chain safety and Environment, Belgium (acronym: ADDIT, projectnumber R-04/001). The funders had no role in study design, data collection and analysis, or preparation of the manuscript. An official approval of the manuscript from the Belgian Federal Public Service of Health, Food chain safety and Environment was needed to allow publication of the results. Competing Interests: The authors have declared that no competing interests exist. * E-mail: ingrid.nobels@ua.ac.be Introduction Adjuvants are compounds that modify the effects of other compounds without having any direct effects themselves. In most cases they are added to a pesticide formulation to increase the performance of the active ingredients or to make the formulation chemically more stable [1]. Depending on the usage, two different types of adjuvants are distinguished, spray adjuvants and formulation additives. Spray adjuvants also called tank mix adjuvants are added in the spray tank along with the pesticide(s) just before application on the field. The second type of adjuvants called formulation additives or inert ingredients are part of the pesticide formulation [1,2]. Besides solvents, surfactants and especially non-ionic surfactants make up the largest group of adjuvants, a simplified overview of the most important chemical classes is listed in Figure 1. This large and heterogeneous group of chemicals is used in pesticides, detergents, personal care and many other products. Due to their variety in applications, adjuvants are the chemicals that are produced and consumed in the largest volumes in the world and most of them end up in detectable levels dispersed in different environmental compartments (soil, water, sediment) and in our food chain [3,4]. Nevertheless, there is a lack in current (pesticide) legislation concerning the use and allowable residue levels of adjuvants. Current regulation concerning the placing of plant protection products on the market, Directive 91/414/EEC, does not specifically deal with adjuvants. The upcoming new regulation (EG) 1107/2009 replaces the Directives 79/117/EEG and 91/ 414/EEG and will apply from June 2011. The new regulation acknowledges the need for more (eco)toxicological information regarding all the components of plant protection products and claims a better protection of human, animal and environmental health by applying the precautionary principle. Adjuvants will make part of future pesticide risk evaluations and a list of forbidden adjuvants for use in crop protection will be constructed PLoS ONE | www.plosone.org 1 November 2011 | Volume 6 | Issue 11 | e24139