Comparative toxicity assessment of glyphosate and two commercial formulations in the planarian Dugesia japonica

Abstract

Introduction: Glyphosate is a widely used, non-selective herbicide. Glyphosate and glyphosate-based herbicides (GBHs) are considered safe for non-target organisms and environmentally benign at currently allowed environmental exposure levels. However, their increased use in recent years has triggered questions about possible adverse outcomes due to low dose chronic exposure in animals and humans. While the toxicity of GBHs has primarily been attributed to glyphosate, other largely unstudied components of GBHs may be inherently toxic or could act synergistically with glyphosate. Thus, comparative studies of glyphosate and GBHs are needed to parse out their respective toxicity. Methods: We performed such a comparative screen using pure glyphosate and two popular GBHs at the same glyphosate acid equivalent concentrations in the freshwater planarian Dugesia japonica. This planarian has been shown to be a useful model for both ecotoxicology and neurotoxicity/developmental neurotoxicity studies. Effects on morphology and various behavioral readouts were obtained using an automated screening platform, with assessments on day 7 and day 12 of exposure. Adult and regenerating planarians were screened to allow for detection of developmentally selective effects. Results: Both GBHs were more toxic than pure glyphosate. While pure glyphosate induced lethality at 1 mM and no other effects, both GBHs induced lethality at 316 μM and sublethal behavioral effects starting at 31.6 μM in adult planarians. These data suggest that glyphosate alone is not responsible for the observed toxicity of the GBHs. Because these two GBHs also include other active ingredients, namely diquat dibromide and pelargonic acid, respectively, we tested whether these compounds were responsible for the observed effects. Screening of the equivalent concentrations of pure diquat dibromide and pure pelargonic acid revealed that the toxicity of either GBH could not be explained by the active ingredients alone. Discussion: Because all compounds induced toxicity at concentrations above allowed exposure levels, our data indicates that glyphosate/GBH exposure is not an ecotoxicological concern for D. japonica planarians. Developmentally selective effects were not observed for all compounds. Together, these data demonstrate the usefulness of high throughput screening in D. japonica planarians for assessing various types of toxicity, especially for comparative studies of several chemicals across different developmental stages.

Keywords: GBH; behavioral screening; ecotoxicology; flatworm; glyphosate; herbicide; neurotoxicity; new approach method.

FIGURE 1

Comparison of lethality in different exposure conditions. (A, B) Percent survival at day 7 for (A) adult and (B) regenerating planarians. (C, D) Percent survival at day 12 in (C) adult planarians and (D) regenerating planarians. Concentrations refer to glyphosate acid equivalent concentrations. Bars indicate the average of all replicates (colored dots; 2 for 96-well plates and 3 for bulk).

FIGURE 2

Comparison of toxicity of glyphosate, RR and RC exposure in adult and regenerating planarians. Heatmap comparing the lowest observed effect levels (LOELs) for glyphosate (GLY), RR, and RC exposure in adult (A) and regenerating (R) planarians in all tested endpoints. Percent time resting was calculated in both the dark (D) and blue (B) light periods of the phototaxis assay. NS: Noxious stimuli. Only concentration-dependent hits are shown. For simplicity, only the results from stickiness (Z) are shown as no significant hits were found in stickiness (A). The negative control, 100 μM L-ascorbic acid, showed no significant effects in any of the endpoints. All compiled data can be found in Supplementary File S1.

FIGURE 3

Sublethal effects of RC exposure. (A) Example images of a normal control planarian and the abnormal body shapes observed with 316 µM RC exposure in regenerating planarians. Abnormal body shapes (from left to right) are corkscrew/twisted, C-shape, contracted. Scale bar: 2 mm. (B) Concentration-response curves for adult (top) and regenerating (bottom) planarians exposed to RC. Symbols represent the mean of the speed in the dark during the phototaxis assay of n = 24 planarians. Error bars are the standard error. Speed was evaluated on both day 7 (d7, black solid line) and day 12 (d12, gray dashed line). 316 μM RC was lethal to adult planarians and thus no data are shown for adult planarians at this concentration. *p < 0.05 using a Welch’s ANOVA test followed by a pairwise Tamhane-Dunnett test comparing to the respective in-plate control population.

FIGURE 4

Effects of glyphosate and GBHs on biomarkers. (A) Percent normalized AChE activity in adult planarians after 12-day exposure to 100 µM glyphosate (GLY), RC, or RR. Exposure to 0.178 µM diazinon (DZN) was compared to its vehicle control 0.5% DMSO as a positive control for AChE inhibition. (B) Percent normalized GST activity in adult planarians exposed to 100 µM GLY, RC, or RR for 12 days. **p < 0.01 using a one-way ANOVA followed by a post hoc Dunnett test compared to the respective control population. Data shown as the mean of 4 biological replicates. Error bars represent the standard deviation.