Pesticide toxicology currently focuses mainly on two areas: long-term effects of given compounds and cocktail effects of chemicals, including combined effects of pesticide active ingredients with their adjuvants, as seen in the case of glyphosate-based herbicides. In this study surface water pollution in an agricultural region of Hungary by glyphosate was determined by ELISA method, and cytotoxic effects on HEK293 and NE-4C cells by glyphosate, its formulated herbicide (ROUNDUP® ) and adjuvant (polyethoxylated tallowamine, POEA) were compared. ROUNDUP and POEA were found to be equitoxic at short exposures (LC50: 10-15 ng/ml in 6 hrs), while glyphosate occurred to be of 500-750-fold less toxicity

Darvas, Béla

Gyurcsó, Gergő

Klátyik, Szandra

Mörtl, Mária

Ottucsák, Marianna

Székács, András

Ottucsák Marianna

Klátyik Szandra

Gyurcsó Gergő

Mörtl Mária

Darvas Béla

Székács András

University of Szeged

21st International Symposium on Analytical and Environmental Problems  64  Environmental Monitoring of Glyphosate and Assessment of its Combined Cytotoxicity with Adjuvants  Marianna Ottucsák, Szandra Klátyik, Gergő Gyurcsó, Mária Mörtl, Béla Darvas, András Székács*  Agro-Environmental Research Institute, National Agricultural Research and Innovation Centre, Herman O. u. 15, H-1022 Budapest, Hungary *e-mail: a.szekacs@cfri.hu  Abstract Pesticide toxicology currently focuses mainly on two areas: long-term effects of given compounds and cocktail effects of chemicals, including combined effects of pesticide active ingredients with their adjuvants, as seen in the case of glyphosate-based herbicides. In this study surface water pollution in an agricultural region of Hungary by glyphosate was determined by ELISA method, and cytotoxic effects on HEK293 and NE-4C cells by glyphosate, its formulated herbicide (ROUNDUP®) and adjuvant (polyethoxylated tallowamine, POEA) were compared. ROUNDUP and POEA were found to be equitoxic at short exposures (LC50: 10-15 ng/ml in 6 hrs), while glyphosate occurred to be of 500-750-fold less toxicity.  Introduction  The role of pesticides in current industrial agriculture is to suppress damages in crop production by agricultural pests. To achieve such chemical protection, pesticides are used in formulations as mixtures of active ingredient(s) responsible for the main effect of the pesticide preparation with adjuvants added to improve physico-chemical properties, adsorption/penetration capability and other characte istics of the active ingredient(s) [1]. With the worldwide expansion of monoculture-based agriculture, overall pesticide consumption continuously grows not only affecting targeted crop yields, but also causing increasing chemical pressure on the environment. In addition, pesticide residues are often the source of chemical exposure as they enter the food chain uponagricultural practices. Due to this environmental load and subsequent exposure to numerous non-target organisms, surveys on pesticide residues and their side-effects are expanding [2], and consequently pesticides are subject to strict registration processes specified in corresponding international recommendations [3, 4] and legal regulations [5, 6], and pesticide residues are strictly regulated through their maximal residue levels (MRLs) in food and feed set upon evidence-based scientific risk assessment [7, 8]. General pesticide toxicology research in the 70’s was mostly focused on the acute effects. During the lat 80’s (with the development and extended use of the metabolic bacterial reverse mutation assay developed by Bruce Ames [9] and other microbial mutagenicity tests), attention turned towards mutagenicity, carcinogenicity, teratogenicity and epidemiological examination of pesticides, and toxicity requirements strictened towards candidate pesticide substances. Expanding knowledge justifies why active ingredients in plant protection products have to undergo regular re-assessment. During the past two decades, increasing interest has been expressed in fields, where sufficient knowledge was still lacking: the study of immunomodulant and endocrine disruptive effects [10]. Current toxicology focuses on two main areas: (i) long-term effects of chemicals at doses near the no observed effect level (NOEL) in exposures extending over long periods, even life-times, and (ii ) effects of numerous compounds exerting toxicity in parallel, so called cocktail effect. The latter type includes the case of combined effects of active ingredients with their adjuvants. These adjuvants are considered „inert” in erms of the main effects of the pesticide 21st International Symposium on Analytical and Environmental Problems  65  active ingredient, but they may cause substantial side-effects or increase side-effects of the active ingredients. Glyphosate Glyphosate is presently the largest selling herbicide active ingredient in the world, and its market continues to grow in line with restrictions/ban on other herbicides and the increase in the cultivation of glyphosate-tolerant (GT) transgenic crops [11]. By blocking the biosynthesis of essential aromatic amino acids thoug  inhibiting the shikimic acid metabolic pathway and also inhibiting photosynthesis, glyphosate shows general phytotoxicity, allowing its broad pre-emergent herbicide applications. As a result of the long-term, intensive use, our surveys indicate glyphosate as a common contaminant  rivers and other surface waters [12-13]. Formulated preparations of glyphosate have been indicated to exert harmful biological effects, for example endocrine disruption [14-15] or teratogenicity [16], even under its no-effect level (NOEL) upon extended chronic exposures. The current evaluation by the UN International Agency for Research on Cancer (IARC) classified glyphosate as probably carcinogenic to humans (Group 2A), based on “limited evidence” in human experiments and ”sufficient evidence” in animal-experiments [17]. In light of these adverse side effects, the recent re-approval of glyphosate has been claimed unacceptable [18]. As glyphosate has been reported as common surface water pollutant, and as substantial differences have been evidenced in the toxicity of glyphosate and its formulated herbicide products, attributed to side-effects of the adjuvants applied in formulation, our study aimed to evaluate the environmental occurrence of glyphosate in surface waters, and to comparatively assess cytotoxicity of glyphosate, its formulated herbicide preparation ROUNDUP® and common adjuvant (used also in ROUNDUP) polyethoxylated tallowamine (POEA).  Materials and Methods Glyphosate and ROUNDUP were obtained commercially, POEA was provided by Lamberti SpA (Albizzate, Italy), chemicals were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Glyphosate was determined in environmental samples by a commercial enzyme-linked immunosorbent assay (ELISA), cell viability was tested using the thiazolyl blue tetrazolium bromide (MTT) reduction assay detecting mitochondrial respiration intensity [19] on two cell types: a human embryonic kidney cell line expressing adenovirus-specific tumor antigen (HEK293) [20] and a mouse neuroectodermal stem cell line (NE-4C) [21]. HEK293 cells were purchased from Sigma-Aldrich Co., NE-4C cells were kindly provided by the Institute of Experimental Medicine of the Hungarian Academy of Sciences.  Immunochemical analysis of glyphosate Immunochemical analysis of glyphosate was performed using the ELISA kit by Abraxis LLC (Warminster, PA, USA), validated to determination of glyphosate in water (ground/surface/well water). Without sample extraction, water samples were derivatized (acetylated) prior to immunoanalysis, and then were pip tted onto the manufacturer-supplied 96-well microplates. Samples were incubated (30 min) on the microplate with glyphosate-specific antibodies, then IgG-specific second antibod es conjugated to a reporter enzyme were added, and upon further incubation (60 min) and washing, substrate (H2O2) and a chromophore were added, and optical density of the solutions was detected in a MULTISKAN ASCENT microplate reader (Labsystems, Finland). Glyphosate concentration was determined using analytical standard and sigmoid (logistic regression) calibration.  Cell culture work Cell lines were stored at -196oC in liquid nitrogen, and subsequently thawed in water bath, washed and cultured in buffer medium containing 10% fetal bovine serum. Cells were grown 21st International Symposium on Analytical and Environmental Problems  66  at 37oC (under an atmosphere of 5% CO2, 95% air) to 80% confluence, and prior to cytotoxicity measurement were passed through at leas one passage. Upon being washed with serum-free medium buffer, cells were exposed to various chemicals for up to 24 hrs [22]. The MTT cytotoxicity tests were carried out in 96-well microplates. Cells were incubated with glyphosate, ROUNDUP and POEA at various concentrations for 2, 6 and 24 hrs in buffer medium with or without serum added [23] and after washing, MTT (0.1 mg/ml in buffer medium) was added, and color development was detected at 570 nm.  Results and Discussion Determination of glyphosate Using the Abraxis ELISA method, glyphosate was detected above the practical limit of detection (LOD) (0.12 ng/ml) in half of the surface water samples collected in the autumn period from a maize growing agricultural region of Hungary. Glyphosate concentrations showed a somewhat bimodal pattern: concentrations were either below (or in some cases slightly above) the LOD, or were found alarmingly high (0.542±0.003 to 0.984±0.003 ng/ml). In contrast, surface water samples collected in the spring period (before intensive pre-emergent herbicide applications) were found predominantly not to contain glyphosate above the LOD, possibly due to a dilution effect in standing water bodies or in rivers. It has to be noted that the ELISA method is specific only to glyphosate, and it doesn’t detect its primary metabolite, aminomethylphosphonic acid (AMPA). According to manufacturer’s specifications, the cross-reactivity of the detection method for AMPA is slightly above 0.0001%. This means, analysis covers only the presenc /absence of the parent compound (glyphosate) and not its official residue level, glyphosate and its metabolite(s).  Cytotoxicity measurements ROUNDUP strongly suppressed cell viability, detectable even after 2 hrs of exposure, significantly dropped by 6 hrs, but with no further decay until 24 hrs. POEA caused similarly decreased cell viability above 5 ng/ml concentration, and the effect continuously increased from 2 to 24 hrs of exposure. LC50 values for ROUNDUP and POEA on NE-4C cells upon 6 hrs of exposition were found to be 15 and 10 ng/ml, respectively, but POEA caused more rapid cytotoxicity. In contrast, cytotoxicity of glyphosate (LC50: 7.5% µg/ml in 6 hrs) was 500-750-fold lower. ROUNDUP is well known to be cytotoxic by inhibiting mitochondrial respiration (LC50: 57.5 µg/ml) [22] and induced cell necrosis by a 15-fold increase in adenylate kinase release. The apoptotic effect of ROUNDUP was seen by an increase in caspase 3/7 activity by 6.29-8.24 times compared to the control level.  Conclusions Glyphosate-based herbicides present dual hazards in terms of environmental contamination and cytotoxicity. In this study, glyphosate was found in half of the surface water samples from a maize growing region of Hungary, with contamination as high as 0.984 ng/ml in a bimodal pattern. POEA, the main adjuvant of glyphosate, was found cytotoxic above 1 ng/ml concentration on human cell lines after 2 to 24 hrs of exposure. The results evidenced the hazard of glyphosate and even more of its adjuvant POEA on human cell viability, underlying the necessity of toxicological risk assessment of pesticide formulating adjuvants for their combined effects with pesticide residues. Thus, risk a sessment has to be extended to toxicological consequences of such parallel exposure.  Acknowledgments The authors thank Prof. Emilia Madarász (Institute of Experimental Medicine of the Hungarian Academy of Sciences) for the provision of NE-4C cells and facilities for 21st International Symposium on Analytical and Environmental Problems  67  cytotoxicity tests on this cell line. The work was supported by project K109865 of the Hungarian Scientific Research Fund (OTKA).  References [1] A.K. Hassall, The Biochemistry and Uses of Pesticides: Structure, Metabolism, Mode of Action and Uses in Crop Protection, VCH Publ., Lond, 1990, pp. 25-56. [2] United Nations Environment Programme, Clearing the Waters: A Focus on Water Quality Solutions, UNEP Division of Environmental Policy Implementation, Nairobi, Kenya, 2010, pp. 13-16. [3] Organisation for Economic Co-operation and Development (OECD), Water Quality and Agriculture Meeting the Policy Challenge, OECD Publ., Paris, France, 2012, p. 11. [4] Food and Agriculture Organization (FAO), International Code of Conduct on the Distribution and use of Pesticides, 2013, FAO, Rome, Italy, 2013, pp. 16-17. [5] U.S. Environmental Protection Agency (US EPA), Pesticide Registration Improvement Extension Act (PRIA 3), US EPA, Washington, DC, USA, 2012. [6] European Commission (EU), Regulation 284/2013 ( March 2013) Setting out the Data Requirements for Plant Protection Products, in Accordance with Regulation 1107/2009 Concerning the Placing of Plant Protection Products on the Market, 2013. [7] World Health Organization (WHO), International Programme on Chemical Safety, Principles for Modelling Dose-Response for the Risk Assessment of Chemicals, Annex I: Terminology, Environmental Health Criteria 239, World Health Organization, Geneva, Switzerland, 2009. [8] European Food Safety Authority (EFSA), Scientific Opinion on Risk Assessment Terminology, EFSA Scientific Committee, Parma, Italy, 2012. [9] B.N. Ames, F.D. Lee, W.E. Durston, Proc. Nat. Acad. Sci. U.S.A., 70 (3) (1973) 782-786. [10] T. Colborn, D. Dumanoski, J.P. Myers Our Stolen Future, Dutton, New York, NY, USA, 1996. [11] A. Székács, B. Darvas, Herbicides – Properties, Synthesis and Control of Weeds, InTech, Rijeka, Croatia (2012) 247-284. [12] M. Mörtl, Gy. Németh, J. Juracsek, B. Darvas, L. Kamp, F. Rubio, A. Székács, Microchem. J. 2013, 107: 143-151. [13] A. Székács, M. Mörtl, B. Darvas, J. Chem. (2015) Article ID 717948. [14] C. Gasnier, C. Dumont, N. Benachour, E. Clair, M.C. Chagnon, G.-E. Séralini, Toxicology 262 (3) (2009) 184-191. [15] M. Antoniou, M.E.E.-D.M. Habib, C.V. Howard, R.C. Jennings, C. Leifert, R.O. Nodari, C. Robinson, J. Fagan, Roundup and Birth Defects, Earth Open Source, Lancashire, UK, 2011. [16] A. Paganelli, V. Gnazzo, H. Acosta, S.L. López, A.E. 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Environmental monitoring of glyphosate and assessment of its combined cytotoxicity with adjuvants

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Environmental monitoring of glyphosate and assessment of its combined cytotoxicity with adjuvants

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