Co-Formulants in Glyphosate-Based Herbicides Disrupt Aromatase Activity in Human Cells below Toxic Levels

Abstract

Pesticide formulations contain declared active ingredients and co-formulants presented as inert and confidential compounds. We tested the endocrine disruption of co-formulants in six glyphosate-based herbicides (GBH), the most used pesticides worldwide. All co-formulants and formulations were comparably cytotoxic well below the agricultural dilution of 1% (18-2000 times for co-formulants, 8-141 times for formulations), and not the declared active ingredient glyphosate (G) alone. The endocrine-disrupting effects of all these compounds were measured on aromatase activity, a key enzyme in the balance of sex hormones, below the toxicity threshold. Aromatase activity was decreased both by the co-formulants alone (polyethoxylated tallow amine-POEA and alkyl polyglucoside-APG) and by the formulations, from concentrations 800 times lower than the agricultural dilutions; while G exerted an effect only at 1/3 of the agricultural dilution. It was demonstrated for the first time that endocrine disruption by GBH could not only be due to the declared active ingredient but also to co-formulants. These results could explain numerous in vivo results with GBHs not seen with G alone; moreover, they challenge the relevance of the acceptable daily intake (ADI) value for GBHs exposures, currently calculated from toxicity tests of the declared active ingredient alone.

Keywords: JEG3 cells; aromatase; co-formulant; endocrine disruption; glyphosate-based herbicide; pesticide.

Figure 1

Dose-dependent cytotoxic effects of various glyphosate-based herbicide formulations or co-formulants or the declared active ingredient (dAI) glyphosate alone (G) in the JEG3 human cell line.

Figure 2

Inhibition of cell respiration by co-formulants and formulations at similar levels. * p < 0.05, ** p < 0.01, *** p < 0.001. (A) 100 ppm of Glyfos is compared to the content in POEA (10 ppm) and G (48.6 ppm); (B) 100 ppm of Glyfos is compared to the content in POEA (10 ppm) and G (48.6 ppm); (C) 2000 ppm of Medallon is compared to the content in APG (800 ppm) and G (975 ppm); (D) 100 ppm of the formulations are compared to the content in G (48.6 ppm). Two co-formulants were also tested around their LC₅₀ (QAC, 58 ppm; POE-APE, 222 ppm, see Table 1).

Figure 3

Membrane integrity disruption by formulations and co-formulants. * p < 0.05, ** p < 0.01, *** p < 0.001. (A) 100 ppm of Glyfos is compared to the content in POEA (10 ppm) and G (48.6 ppm); (B) 100 ppm of Glyfos is compared to the content in POEA (10 ppm) and G (48.6 ppm); (C) 2000 ppm of Medallon is compared to the content in APG (800 ppm) and G (975 ppm); (D) 100 ppm of the formulations are compared to the content in G (48.6 ppm); Two co-formulants were also tested around their LC₅₀ (see Table 1): (E) QAC, 58 ppm; (F) POE-APE, 222 ppm.

Figure 4

Aromatase inhibition by formulations and their co-formulants alone at similar levels. * p < 0.05, ** p < 0.01, *** p < 0.001. (A) 25 ppm of Glyfos is compared to the content in POEA (2.5 ppm) and glyphosate (16 ppm); (B) 300 ppm of Medallon is compared to the content in APG (120 ppm) and glyphosate (146 ppm). These concentrations are 1.2–2 times below the NOEC and 1.4–3.4 below the toxicity threshold (LOEC).

Figure 5

Inhibition of aromatase by co-formulants and formulations. * p < 0.05, ** p < 0.01, *** p < 0.001. (A) 25 ppm of the co-formulant QAC is compared to the negative control (C) and to the positive control formestane (F); (B) 100 ppm of the co-formulant POE-APE is compared to the negative control (C) and to the positive control formestane (F); (C) 50 ppm of formulations of glyphosate-based herbicides R WeatherMAX—R WMAX—Glyfos, R Classic, Kapazin and Total is compared to the negative control (C) and to the positive control formestane (F). Glyphosate alone (isopropyl ammonium salt form) was also tested at its dose present in the formulations (24.3 ppm).