The Ramazzini Institute 13-week pilot study on glyphosate and Roundup administered at human-equivalent dose to Sprague Dawley rats: effects on the microbiome

Abstract

Background: Glyphosate-based herbicides (GBHs) are broad-spectrum herbicides that act on the shikimate pathway in bacteria, fungi, and plants. The possible effects of GBHs on human health are the subject of an intense public debate for both its potential carcinogenic and non-carcinogenic effects, including its effects on microbiome. The present pilot study examines whether exposure to GBHs at doses of glyphosate considered to be "safe" (the US Acceptable Daily Intake - ADI - of 1.75 mg/kg bw/day), starting from in utero, may modify the composition of gut microbiome in Sprague Dawley (SD) rats.

Methods: Glyphosate alone and Roundup, a commercial brand of GBHs, were administered in drinking water at doses comparable to the US glyphosate ADI (1.75 mg/kg bw/day) to F0 dams starting from the gestational day (GD) 6 up to postnatal day (PND) 125. Animal feces were collected at multiple time points from both F0 dams and F1 pups. The gut microbiota of 433 fecal samples were profiled at V3-V4 region of 16S ribosomal RNA gene and further taxonomically assigned and assessed for diversity analysis. We tested the effect of exposure on overall microbiome diversity using PERMANOVA and on individual taxa by LEfSe analysis.

Results: Microbiome profiling revealed that low-dose exposure to Roundup and glyphosate resulted in significant and distinctive changes in overall bacterial composition in F1 pups only. Specifically, at PND31, corresponding to pre-pubertal age in humans, relative abundance for Bacteriodetes (Prevotella) was increased while the Firmicutes (Lactobacillus) was reduced in both Roundup and glyphosate exposed F1 pups compared to controls.

Conclusions: This study provides initial evidence that exposures to commonly used GBHs, at doses considered safe, are capable of modifying the gut microbiota in early development, particularly before the onset of puberty. These findings warrant future studies on potential health effects of GBHs in early development such as childhood.

Keywords: Early developmental stage; Glyphosate; Gut microbiome; Roundup.

Fig. 1

Timeline of the experimental animal treatment and fecal sample collection

Fig. 2

The overall microbiome diversity. a, b, and c are non-metric dimensional scaling (NMDS) plots visualize the overall microbiome dissimilarities (beta-diversity) between individual rat across time. a All samples from SD dams (pink) and pups (green) of three treatment groups; b All samples from SD dam rats only. Colors indicate sample collection timepoint. BM: before mating; GD 5: gestation day 5; GD 13: gestation day 13; LD 7: lactation day 7; and LD 14: lactation day 14. c All samples from SD pup rats only. Colors indicate sample collection timepoint. PND 7 to PND 125: postnatal day 7 to postnatal day 125. d Box plots show the mean and variance of the within-community diversity (alpha-diversity) measured by Shannon index in three treatment groups across all time of collections

Fig. 3

The effect of glyphosate exposure on overall microbiome diversity. a NMDS plots visualize the overall microbiome dissimilarities (beta-diversity) between individual rat of three treatments at PND 31 and PND 57. b PERMANOVA test is performed to test the significance among all three treatments (displayed in NMDS plots) and between two treatments (values are listed in tables). The p-values in parenthesis were adjusted for genders. G: glyphosate; R: Roundup; C: control water

Fig. 4

Differential microbial features selected via LEfSe between treatment. a Clad plots visualize the significant differential taxa features from phylum (inner circle) to genus (outer circle) at PND 31 and PND 57. Color indicates the more abundant taxa under each condition. b The table lists the overall abundance of the significant differential taxa between treatment across time