Pesticides

"Some of the most important publications by scientific teams supported by CRIIGEN or by CRIIGEN members who have participated in them.

Traditionally, when a scientific study is cited, only the name of the first author is given. Exceptionally, all authors are cited to thank them for their efforts, cooperation and support of CRIIGEN.

(Authors who are members of CRIIGEN are underlined)

2023

A Roundup herbicide causes high mortality and impairs development of Chrysoperla carnea (Stephens) (Neuroptera: Chrysopidae). N Defarge, M Otto, A Hilbeck. Sci Total Environ. 2023. DOI : 10.1016/j.scitotenv.2022.161158

"A Roundup herbicide causes high mortality and impairs the development of Chrysoperla carnea larvae. Chrysoperla carnea larvae".

Some Roundups can be considered insecticides...

Ecotoxicological testing of herbicides focuses on non-target plants and higher animals, while direct effects on arthropods are only superficially tested on the basis of contact exposure. However, oral exposure, as we show in our case, can be highly relevant for systemic pesticides, such as GBH. More specifically, in cropping systems comprising GBH-tolerant genetically modified crops, these herbicides and their degradation products are present both inside and outside crop plants, and are therefore ingested by crop-associated arthropod fauna. We tested the effects of oral absorption of the Roundup WeatherMax formulation on Chrysoperla carnea larvae, a model organism in ecotoxicity testing programs. Long-term oral exposure of C. carnea larvae throughout their juvenile life stages was tested at concentrations ranging from 0.001 to 1% dilution, below the 1.67% recommended for field applications. Inhibition of metamorphosis was observable at 0.1%, but at a concentration of 0.5%, GBH significantly impaired cocoon formation and led to massive lethal malformations. At a GBH concentration of 1%, half the individuals remained permanent larvae and no adults hatched alive. The effects observed followed a clear dose-response relationship. The hazard caused by the direct insecticidal action of GHB after oral absorption is highly relevant to environmental safety and reveals a gap in regulatory risk assessments that should be urgently addressed, particularly in light of the current insect decline.

2022

Comparative Toxicogenomics of Glyphosate and Roundup Herbicides by Mammalian Stem Cell-Based Genotoxicity Assays and Molecular Profiling in Sprague-Dawley Rats. Robin MesnageMariam Ibragim, Daniele Mandrioli, Laura Falcioni, Eva Tibaldi, Fiorella Belpoggi, Inger Brandsma, Emma Bourne, Emanuel Savage, Charles A Mein, Michael N Antoniou. Toxicol Sci. 2022. Doi: 10.1093/toxsci/kfab143.

"Comparative toxicogenomics of glyphosate and Roundup herbicides by mammalian stem cell-based genotoxicity assays and molecular profiling in Sprague-Dawley rats".

Whether glyphosate-based herbicides (GBHs) are more potent than glyphosate alone in activating the cellular mechanisms underlying carcinogenesis remains controversial. Since GBHs are more cytotoxic than glyphosate, we thought they might also be more capable of activating carcinogenic pathways...

We tested this hypothesis by comparing the effects of glyphosate with those of Roundup GBH in vitro and in vivo. Firstly, glyphosate was compared with representative GBHs, namely MON 52276 (European Union), MON 76473 (UK) and MON 76207 (USA), using the mammalian stem cell-based ToxTracker system. Here, MON 52276 and MON 76473, but not glyphosate and MON 76207, activated oxidative stress and deployed protein responses. Secondly, liver molecular profiling was performed in female Sprague-Dawley rats exposed to glyphosate or MON 52276 (at 0.5, 50 and 175 mg/kg bw/day glyphosate) for 90 days. MON 52276, but not glyphosate, increased hepatic steatosis and necrosis. MON 52276 and glyphosate altered gene expression in the liver, reflecting TP53 activation by DNA damage and regulation of circadian rhythm. The genes most affected in the liver were also altered in the kidneys. Small RNA profiling in the liver showed a decrease in miR-22 and miR-17 following MON 52276 ingestion. Glyphosate decreased miR-30, while miR-10 levels increased. Liver DNA methylation profiling revealed 5,727 and 4,496 differentially methylated CpG sites between control animals and animals exposed to glyphosate and MON 52,276, respectively. The formation of apurinic/apyrimidine DNA damage in the liver increased with glyphosate exposure. Overall, our results show that Roundup formulations induce more biological changes related to carcinogenesis than glyphosate.

2022

Glyphosate and its formulations Roundup Bioflow and RangerPro alter bacterial and fungal community composition in the rat caecum microbiome. Robin MesnageSimona Panzacchi , Emma Bourne , Charles A Mein, Melissa J Perry, Jianzhong Hu, Jia Chen, Daniele Mandrioli, Fiorella Belpoggi, Michael N Antoniou. Front Microbiol. 2022. Doi: 10.3389/fmicb.2022.888853.

"Glyphosate and its formulations Roundup Bioflow and RangerPro alter bacterial and fungal community composition in the rat cecal microbiome".

The potential health consequences of glyphosate-induced alterations to the gut microbiome have become a topic of intense debate. In a multifaceted study of the toxicity, carcinogenicity and multigenerational effects of glyphosate and its commercial herbicide formulations, we assessed changes in bacterial and fungal populations in the cecal microbiota of rats exposed ...

... before birth to adulthood (13 weeks after weaning) at three doses of glyphosate. (0.5, 5, 50 mg/kg body weight/day), or to formulated herbicide products Roundup Bioflow and RangerPro at the same glyphosate-equivalent doses. The caecal bacterial microbiota was assessed by 16S rRNA sequencing, while the fungal population was determined by ITS2 amplicon sequencing. Results showed that both fungal and bacterial diversity were affected by Roundup formulations in a dose-dependent manner, while glyphosate alone significantly altered only bacterial diversity. At taxon level, a reduction in Bacteroidota abundance, marked by alterations in levels of Alloprevotella, Prevotella and Prevotellaceae UCG-003, was concomitant with an increase in levels of Firmicutes (e.g. Romboutsia, Dubosiella, Eubacterium brachy group or Christensenellaceae) and Actinobacteria (e.g. , Enterorhabdus, Adlercreutzia or Asaccharobacter). Treponema and Mycoplasma levels were also reduced by pesticide treatments. Analysis of fungal composition indicated that the abundance of the rat intestinal commensal Ascomycota Kazachstania was reduced, while the abundance of Gibberella, Penicillium, Claviceps, Cornuvesica, Candida, Trichoderma and Sarocladium was increased by exposure to Roundup formulations, but not to glyphosate. Altogether, our data suggest that glyphosate and its Roundup RangerPro and Bioflow induced profound changes in the composition of the cecal microbiome by affecting the fitness of key commensals, reducing competition and allowing opportunistic fungi to thrive in the gut, particularly in animals exposed to herbicide formulations. This further indicates that changes in the composition of the gut microbiome could influence the long-term toxicity, carcinogenicity and multigenerational effects of glyphosate-based herbicides.

2022

The surfactant co-formulant POEA in the glyphosate-based herbicide RangerPro but not glyphosate alone causes necrosis in Caco-2 and HepG2 human cell lines and ER stress in the ToxTracker assay. Robin MesnageScarlett Ferguson, Inger Brandsma, Nynke Moelijker, Gaonan Zhang, Francesca Mazzacuva, Anna Caldwell, John Halket, Michael N Antoniou. Food Chem Toxicol. 2022. Doi: 10.1016/j.fct.2022.113380.

"The surfactant co-formulant POEA in the glyphosate herbicide RangerPro, but not glyphosate alone, causes necrosis in human Caco-2 and HepG2 cell lines and ER stress in the ToxTracker assay."

The toxicity of co-formulants present in glyphosate-based herbicides (GBH) has been widely discussed, leading the European Union to ban polyoxyethylene tallow amine (POEA). We have identified the most commonly used POEA, known as POE-15 tallow amine (POE-15), in the widely used American GBH RangerPro. Cytotoxicity tests...

... using human intestinal epithelial Caco-2 and hepatocytic HepG2 cell lines showed that RangerPro and POE-15 are much more cytotoxic than glyphosate alone. RangerPro and POE-15, but not glyphosate, induced cell necrosis in both cell lines, and glyphosate and RangerPro, but not POE-15, induced oxidative stress in HepG2 cells. We then tested these pesticide ingredients in the ToxTracker assay, a system used to evaluate a compound's carcinogenic potential, to assess their ability to induce DNA damage, oxidative stress and an unfolded protein response (endoplasmic reticulum, ER stress). RangerPro and POE-15, but not glyphosate, induced ER stress. We conclude that toxicity resulting from exposure to RangerPro is therefore multifactorial, involving both POE-15-induced ER stress and glyphosate-induced oxidative stress. Our observations reinforce the need to test both co-formulants and active ingredients in commercial pesticides to inform the enactment of more appropriate regulations and thus better protection of the public and the environment.

2022

Glyphosate exposure in early pregnancy and reduced fetal growth: a prospective observational study of high-risk pregnancies. Roy R Gerona, Jill L Reiter, Igor Zakharevich, Cathy Proctor, Jun Ying, Robin Mesnage, Michael Antoniou, Paul D Winchester. Observational Study Environ Health. 2022. Doi: 10.1186/s12940-022-00906-3.

"Glyphosate exposure in early pregnancy and reduced fetal growth: a prospective observational study of high-risk pregnancies."

Prenatal exposure to glyphosate (GLY) is associated with adverse reproductive outcomes in animal studies. Little is known about the effects of GLY exposure during pregnancy in the human population. This study aims to establish baseline urine GLY levels in a cohort of high-risk, racially diverse pregnancies, and to assess the relationship between prenatal GLY exposure and fetal development and birth outcome...

...Random first-trimester urine samples were collected from high-risk pregnant women between 2013 and 2016 as part of the Indiana Pregnancy Environmental Exposures Study (PEES). Demographic and clinical data were extracted from maternal and infant medical records. Urine glyphosate levels were measured as an indicator of GLY exposure and quantified by liquid chromatography tandem mass spectrometry. Key outcome variables included gestation-adjusted birth weight percentile (BWT%ile) and neonatal intensive care unit (NICU) admission. Relationships between key outcome variables and GLY exposure were assessed using univariate and multivariate linear and logistic regression models.

Results: Urinary GLY levels above the detection limit (0.1 ng/mL) were found in 186 out of 187 pregnant women (99%). Further analyses were limited to 155 pregnant women who had delivered a single baby. The mean age of participants was 29 years, and the majority were non-Hispanic whites (70%) or non-Hispanic blacks (21%). The mean (± SD) urinary GLY level was 3.33 ± 1.67 ng/mL. Newborns' BWT %iles were negatively related to GLY (adjusted slope ± SE = -0.032 + 0.014, p = 0.023). Infants born to women living outside the greater central Indiana metropolitan area were more likely to have lower percent BWT associated with maternal first-trimester GLY levels (slope ± SE = -0.064 ± 0.024, p = 0.007). The adjusted odds ratio for NICU admission and maternal GLY levels was 1.16 (95% CI: 0.90, 1.67, p = 0.233).

Conclusion: GLY was found in 99% of pregnant women in this Midwestern cohort. Higher maternal GLY levels in the first trimester were associated with lower BWT percentages and higher risk of NICU admission. The findings warrant further research into the effects of GLY exposure on human pregnancies in larger population studies.

2022

Cytotoxicity Mechanisms of Eight Major Herbicide Active Ingredients in Comparison to Their Commercial Formulations. Scarlett Ferguson, Robin Mesnage, Michael N Antoniou. Toxics. 2022. Doi: 10.3390/toxics10110711.

"Cytotoxicity mechanisms of eight major herbicidal active ingredients compared to their commercial formulations".

Commercial pesticide formulations contain co-formulants, which are generally considered to have no toxic effects in mammals. This study aims to compare the toxicity of 8 major herbicidal active ingredients, namely glyphosate, dicamba, 2,4-D, fluroxypyr, quizalofop-p-ethyl, pendimethalin, propyzamide and metazachlor, with a typical commercial formulation of each active ingredient...

... Cytotoxicity and oxidative stress capacity were assessed in human hepatoma HepG2 cells. Using an MTT assay, formulations of glyphosate (Roundup Probio), fluroxypyr (Hurler), quizalofop-p-ethyl (Targa Super) and dicamba (Hunter) were more toxic than the active ingredient alone. Metazachlor and its Sultan formulation showed similar cytotoxicity profiles. Cytotoxicity profiles were comparable in immortalized human fibroblasts. Toxilight necrosis tests showed that the metazachlor formulation (Sultan50C) caused significant membrane disruption compared with the active ingredient. Reactive oxygen species generation was detected for glyphosate, fluroxypyr, pendimethalin, quizalofop-p-ethyl, the 2,4-D formulation (Anti-Bindweed) and dicamba and its Hunter formulation. Further testing of quizalofop-p-ethyl and its Targa Super formulation in the ToxTracker test system revealed that both products induced oxidative stress and an unfolded protein response. In conclusion, these results show that most of the herbicide formulations tested in this study are more toxic than their active ingredients in human tissue culture cell model systems. The results add to a growing body of evidence, implying that commercial herbicide formulations, and not just their active ingredients, should be evaluated as part of regulatory pesticide risk assessment.

2022

Impacts of dietary exposure to pesticides on faecal microbiome metabolism in adult twins. Robin MesnageRuth C E Bowyer, Souleiman El Balkhi, Franck Saint-Marcoux, Arnaud Gardere, Quinten Raymond Ducarmon, Anoecim Robecca Geelen, Romy Daniëlle Zwittink, Dimitris Tsoukalas, Evangelia Sarandi, Efstathia I Paramera, Timothy Spector, Claire J Steves, Michael N Antoniou. Environ Health. 2022. Doi : 10.1186/s12940-022-00860-0.

"Impacts of dietary pesticide exposure on fecal microbiome metabolism in adult twins".

Dietary habits have a profound influence on the metabolic activity of gut microorganisms and their influence on health. Concerns have been raised as to whether consumption of pesticide-contaminated food may contribute to the development of chronic disease by affecting the gut microbiome. We conducted the first pesticide biomonitoring survey of the UK population...

... then used the results to carry out the first pesticide association study on the composition and function of the gut microbiome from the TwinsUK register.

Dietary exposure to 186 common insecticide, herbicide or fungicide residues and the fecal microbiome of 65 twin couples in the UK was studied. We assessed whether dietary habits, geographical location or rural/urban environment are associated with pesticide residue excretion. The composition and metabolic activity of the fecal microbiota were assessed using metagenomics and metabolomics respectively. We performed a targeted urinary metabolomics analysis to assess whether urinary excretion of pesticides was also associated with physiological changes.

Residues of pyrethroid and/or organophosphate insecticides were found in all urine samples, while the herbicide glyphosate was found in 53% of individuals. Food frequency questionnaires showed that organophosphorus residues were higher with increased consumption of fruit and vegetables. A total of 34 associations between pesticide residue concentrations and fecal metabolite concentrations were detected. Glyphosate excretion was positively associated with increased overall bacterial species richness, as well as with fatty acid metabolites and phosphate levels. The insecticide metabolite Br2CA, reflecting deltamethrin exposure, was positively associated with the phytoestrogens enterodiol and enterolactone, and negatively associated with certain N-methyl amino acids. Urinary metabolomics performed on a subset of samples revealed no association with the excretion of pesticide residues.

Conclusion: Consumption of conventionally grown fruit and vegetables leads to higher pesticide intake with unknown long-term health consequences. Our results highlight the need for future dietary intervention studies to understand the effects of pesticide exposure on the gut microbiome and possible health consequences.

2022

Comparative analysis of detection techniques for glyphosate in urine and in water. Velot CPoitou F, Spiroux de Vendômois J. (2022) Environmental Sciences Europe. DOI 10.1186/s12302-022-00637-9

" Comparative analysis of glyphosate detection techniques in urine and water ".

Glyphosate is the declared active ingredient in the world's most widely used herbicides, and is therefore widely present in the environment. Urinary glyphosate levels represent a relevant biomarker for individual exposure to glyphosate-based herbicides. However, the measurement of GLY levels in urine is controversial, as different detection methods have led to contradictory results...

... notably in the case of enzyme-linked immunosorbent assay (ELISA) versus liquid chromatography coupled with tandem mass spectrometry (LC/MS-MS) for urine, and ELISA versus high-performance liquid chromatography coupled with fluorescence detection (HPLC/Fluo) for water.

We compared ELISA to LC/MS-MS or HPLC/Fluo by submitting identical urine and water samples to two laboratories (Biocheck, Germany and Labocéa, France respectively), spiked or unspiked with precise concentrations of glyphosate, but also with two chemically similar molecules: glycine and aminomethylphosphonic acid, respectively analogue and primary metabolite of GLY.

Both laboratories claimed a similar threshold of quantification (LOQ) for glyphosate: 0.08 and 0.05 ng/mL respectively. Each of the methods tested was found to be specific for glyphosate and therefore gave no results.

cross-detection with glycine and aminomethylphosphonic acid. However, these methods showed differences in both reproducibility and reliability depending on the matrix used (water or urine).

Although the ELISA method gave less precise results than the HPLC/Fluo technique when applied to water samples, the glyphosate concentrations measured in urine were much more reliable and reproducible with ELISA technology than those obtained with LC/MS-MS.

2022

Cytotoxicity Mechanisms of Eight Major Herbicide Active Ingredients in Comparison to Their Commercial Formulations. Ferguson S, Mesnage R, Antoniou MN. Toxics. 2022. doi: 10.3390/toxics10110711.PMID: 36422919.

" Cytotoxicity mechanisms of eight major herbicide active ingredients compared to their commercial formulations ".

Cytotoxicity and oxidative stress capacity were assessed in human hepatoma HepG2 cells. Using an MTT assay, formulations of glyphosate (Roundup Probio), fluroxypyr (Hurler), quizalofop-p-ethyl (Targa Super) and dicamba (Hunter) were more toxic than the active ingredient alone. Metazachlor and its Sultan formulation showed similar cytotoxicity profiles. Cytotoxicity profiles were comparable in immortalized human fibroblasts. Toxilight necrosis tests showed that the metazachlor formulation (Sultan50C) caused significant membrane disruption compared with the active ingredient. Reactive oxygen species generation was detected for glyphosate, fluroxypyr, pendimethalin, quizalofop-p-ethyl, the 2,4-D formulation (Anti-Bindweed) and dicamba and its Hunter formulation. Further testing of quizalofop-p-ethyl and its Targa Super formulation in the ToxTracker test system revealed that both products induced oxidative stress and an unfolded protein response. In conclusion, these results show that most of the herbicide formulations tested in this study are more toxic than their active ingredients in human tissue culture cell model systems. The results add to a growing body of evidence, implying that commercial herbicide formulations, and not just their active ingredients, should be evaluated as part of regulatory pesticide risk assessment.

2022

Comparative Toxicogenomics of Glyphosate and Roundup Herbicides by Mammalian Stem Cell-Based Genotoxicity Assays and Molecular Profiling in Sprague-Dawley Rats. Mesnage RIbragim M, Mandrioli D, Falcioni L, Tibaldi E, Belpoggi F, Brandsma I, Bourne E, Savage E, Mein CA, Antoniou MN. Toxicol Sci. 2022. doi: 10.1093/toxsci/kfab143.PMID: 34850229

" Comparative toxicogenomics of glyphosate and Roundup herbicides by mammalian stem cell-based genotoxicity assays and molecular profiling in Sprague-Dawley rats."

2022

Comparative Toxicogenomics of Glyphosate and Roundup Herbicides by Mammalian Stem Cell-Based Genotoxicity Assays and Molecular Profiling in Sprague-Dawley Rats. Robin Mesnage 1, Mariam Ibragim, Daniele Mandrioli, Laura Falcioni, Eva Tibaldi, Fiorella Belpoggi, Inger Brandsma, Emma Bourne, Emanuel Savage, Charles A Mein, Michael N Antoniou. Toxicol Sci. 2022. Doi: 10.1093/toxsci/kfab143.

"Comparative toxicogenomics of glyphosate and Roundup herbicides by mammalian stem cell-based genotoxicity assays and molecular profiling in Sprague-Dawley rats".

... We tested this hypothesis by comparing the effects of glyphosate with those of Roundup GBH in vitro and in vivo. Firstly, glyphosate was compared with representative GBHs, namely MON 52276 (European Union), MON 76473 (UK) and MON 76207 (USA), using the mammalian stem cell-based ToxTracker system. Here, MON 52276 and MON 76473, but not glyphosate and MON 76207, activated oxidative stress and deployed protein responses. Secondly, liver molecular profiling was performed in female Sprague-Dawley rats exposed to glyphosate or MON 52276 (at 0.5, 50 and 175 mg/kg bw/day glyphosate) for 90 days. MON 52276, but not glyphosate, increased hepatic steatosis and necrosis. MON 52276 and glyphosate altered gene expression in the liver, reflecting TP53 activation by DNA damage and regulation of circadian rhythm. The genes most affected in the liver were also altered in the kidneys. Small RNA profiling in the liver showed a decrease in miR-22 and miR-17 following MON 52276 ingestion. Glyphosate decreased miR-30, while miR-10 levels increased. Liver DNA methylation profiling revealed 5,727 and 4,496 differentially methylated CpG sites between control animals and animals exposed to glyphosate and MON 52,276, respectively. The formation of apurinic/apyrimidine DNA damage in the liver increased with glyphosate exposure. Overall, our results show that Roundup formulations induce more biological changes related to carcinogenesis than glyphosate alone.

2021

Use of Shotgun Metagenomics and Metabolomics to Evaluate the Impact of Glyphosate or Roundup MON 52276 on the Gut Microbiota and Serum Metabolome of Sprague-Dawley Rats. Robin MesnageMaxime Teixeira, Daniele Mandrioli, Laura Falcioni, Quinten Raymond Ducarmon, Romy Daniëlle Zwittink, Francesca Mazzacuva, Anna Caldwell, John Halket, Caroline Amiel, Jean-Michel Panoff, Fiorella Belpoggi, Michael Nicolas Antoniou. Environ Health Perspect. 2021. Doi : 10.1289/EHP6990.

"Using metagenomics and metabolomics to assess the impact of glyphosate or Roundup MON 52276 on the gut microbiota and serum metabolome of Sprague-Dawley rats".

There is intense debate as to whether glyphosate can inhibit the shikimate pathway of gastrointestinal microorganisms, with potential health implications. We tested glyphosate and or its herbicide formulation Roundup MON 52276 to see if it affected the rat gut microbiome...

We combined metagenomics of the caecal microbiome with metabolomics of serum and cecum to assess the effects of glyphosate [0.5, 50, 175 mg/kg body weight (bw) per day or MON 52276 at the same glyphosate-equivalent doses, in a 90-day toxicity test in rats.

Results: Treatment with glyphosate and MON 52276 resulted in caeca accumulation of shikimic acid and 3-dehydroshikimic acid, suggesting inhibition of the 5-enolpyruvylshikimate-3-phosphate synthase of the shikimate pathway in the gut microbiome.

This study highlights the power of multi-omics approaches to study the toxic effects of pesticides. Multi-omics studies revealed that glyphosate and MON 52276 inhibited the shikimate pathway in the rat gut microbiome. Our results could be used to develop biomarkers for epidemiological studies to assess the effects of glyphosate herbicides on humans. https://doi.org/10.1289/EHP6990.

2020

Transcriptome profiling of the fungus Aspergillus nidulans exposed to a commercial glyphosate-based herbicide under conditions of apparent herbicide tolerance. Robin MesnageNathalie Oestreicher, Florence Poirier, Valérie Nicolas, Céline Boursier, Christian Vélot. Environ Res. 2020. Doi : 10.1016/j.envres.2020.109116.

" Transcriptome profiling of the fungus Aspergillus nidulans exposed to a commercial glyphosate herbicide under conditions of apparent herbicide tolerance ".

Glyphosate-based herbicides, such as Roundup®, are the most widely used non-selective broad-spectrum herbicides. The release of these compounds in large quantities into the environment is likely to affect soil quality and health, not least because of their non-targeted effects on a wide range of organisms, including soil micro-organisms. It has already been demonstrated that...

the filamentous soil fungus Aspergillus nidulans, a well-characterized experimental model organism that can be used as a bio-indicator of agricultural soil health, is strongly affected by Roundup GT Plus (R450: 450 g/L glyphosate). at concentrations well below the recommended agricultural application rate, including at a dose that causes no macroscopic effects. In this study, we determined alterations in the transcriptome of A. nidulans when exposed to R450 at a dose corresponding to the no-observed-adverse-effect level (NOAEL) for macroscopic parameters. A total of 1,816 distinct genes had their expression altered. The biological functions most affected were protein synthesis, amino acid and secondary metabolism, stress response, and detoxification pathways via cytochrome P450, glutathione-S-transferases and ABC transporters. These results partly explain the molecular mechanisms underlying the alterations in growth parameters detected at higher concentrations for this ascomycete fungus. In conclusion, our results highlight molecular perturbations in a soil fungus under conditions of apparent herbicide tolerance, and thus confirm the need to question the principle of "substantial equivalence" when applied to herbicide-tolerant plants.

2019

Insight into the confusion over surfactant co-formulants in glyphosate-based herbicides. Robin MesnageCharles Benbrook, Michael N Antoniou. Review Food Chem Toxicol. 2019. Doi: 10.1016/j.fct.2019.03.053.

"Insights into the confusion surrounding surfactant co-formulants in glyphosate-based herbicides".

Glyphosate is the declared active ingredient in glyphosate-based herbicides (GBH). Other chemicals in GBHs are presumed inert by regulatory authorities and are largely ignored in pesticide safety assessments. We identified surfactants in a representative sample of GBH formulations and compared their acute toxic effects...

The first-generation polyethoxylated amine (POEA) surfactants (POE-tallowamine) contained in Roundup are significantly more toxic than glyphosate, and have raised concerns about human health risks, particularly for highly exposed applicators. From the mid-1990s onwards, first-generation POEAs were gradually replaced by other POEA surfactants, ethoxylated etheramines, which showed less non-targeted toxic effects. Persistent concerns about surfactant toxicity were at least partially alleviated in the European Union by the introduction of propoxylated quaternary ammonium surfactants. This class of POEA surfactants is around 100 times less toxic to aquatic ecosystems and human cells than previous GBH-POEA surfactants. As the composition of GBH is legally classified as confidential business information, confusion over the identity and concentrations of co-formulants is common, and descriptions of the substances tested in published studies are often erroneous or incomplete. In order to clear up this confusion, legislation requiring disclosure of the chemical composition of pesticide products could be enacted. Research is needed to understand the health implications of ingesting these substances.

2019

Insight into the confusion over surfactant co-formulants in glyphosate-based herbicides. Robin Mesnage, Charles Benbrook, Michael N Antoniou. Review Food Chem Toxicol. 2019 Jun. Doi: 10.1016/j.fct.2019.03.053.

"Insights into the confusion surrounding surfactant co-formulants in glyphosate-based products"

Glyphosate is the active ingredient in glyphosate-based herbicides (GBH). Other chemicals in GBHs are presumed inert by regulatory authorities and are largely ignored in pesticide safety assessments. We identified surfactants in a representative sample of GBH formulations and compared their acute toxic effects...

... The first-generation polyethoxylated amine (POEA) surfactants (POE-tallowamine) contained in Roundup are significantly more toxic than glyphosate, and have raised concerns about human health risks, particularly for highly exposed applicators. From the mid-1990s onwards, first-generation POEAs were gradually replaced by other POEA surfactants, ethoxylated etheramines, which showed less non-targeted toxic effects. Persistent concerns about surfactant toxicity were at least partially alleviated in the European Union by the introduction of propoxylated quaternary ammonium surfactants. This class of POEA surfactants is around 100 times less toxic to aquatic ecosystems and human cells than previous GBH-POEA surfactants. As the composition of GBH is legally classified as confidential business information, confusion over the identity and concentrations of co-formulants is common, and descriptions of the substances tested in published studies are often erroneous or incomplete. In order to clear up this confusion, legislation requiring disclosure of the chemical composition of pesticide products could be enacted. Research is needed to understand the health implications of ingesting these substances.

2018

Ignoring Adjuvant Toxicity Falsifies the Safety Profile of Commercial Pesticides. Robin Mesnage, Michael N Antoniou. Review Front Public Health. 2018. Doi: 10.3389/fpubh.2017.00361.

"Ignoring adjuvant toxicity falsifies the safety profile of commercial pesticides".

Commercial pesticide formulations do not always contain a single ingredient. Rather, they are cocktails of chemicals, composed of a designated pesticidal "active ingredient" and "other ingredients", the latter also collectively referred to as "adjuvants". These include surfactants, antifoaming agents, colorants and so on. Some adjuvants are added to influence the absorption and stability of the active ingredient, thus enhancing its pesticidal action...

... Currently, health risk assessment for pesticides in the European Union and the USA focuses almost exclusively on the declared active ingredient. However, adjuvants can also be toxic in their own right, with numerous adverse health effects reported in humans and the environment. Despite the known toxicity of adjuvants, they are regulated differently from active ingredients, their toxic effects being generally ignored. Adjuvants are not subject to an acceptable daily intake and are not included in the assessment of health risks associated with dietary exposure to pesticide residues. Here, we illustrate this gap in risk assessment with reference to glyphosate, the most widely used pesticide active ingredient. We also study the case of neonicotinoid insecticides, strongly suspected of being involved in bee and bumblebee colony collapse disorder. Study authors sometimes use the name of the active ingredient (e.g. glyphosate) when testing a commercial formulation containing several ingredients (active ingredient plus adjuvant). This leads to confusion in scientific literature and regulatory circles, and misrepresents the safety profile of commercial pesticides. Urgent action is needed to lift the veil on the presence of adjuvants in human food and body fluids, as well as in the environment (such as air, water and soil), and to characterize their toxicological properties. This must be accompanied by precautionary regulatory measures to protect the environment and the human population in general from certain toxic adjuvants currently lacking in risk assessments.

2017

Is it time to reassess current safety standards for glyphosate-based herbicides? Laura N Vandenberg, Bruce Blumberg, *Michael N Antoniou, Charles M Benbrook, Lynn Carroll, Theo Colborn, Lorne G Everett, Michael Hansen, Philip J Landrigan, Bruce P Lanphear, Robin Mesnage**, Frederick S Vom Saal, Wade V Welshons, John Peterson Myers*. Review J Epidemiol Community Health. 2017. Doi: 10.1136/jech-2016-208463.

"Is it time to reassess current safety standards for glyphosate-based herbicides?"

The use of glyphosate-based herbicides (GBH) increased approximately 100-fold between 1974 and 2014. Further increases are expected due to the widespread emergence of glyphosate-resistant weeds, increased application of GBHs and their use as pre-harvest desiccants. Current safety assessments are largely based on studies conducted over 30 years ago...

... We reviewed information on GBH use, exposures, mechanisms of action, toxicity and epidemiology. Human exposures to glyphosate are increasing, and a number of in vitro and in vivo studies question the basis for the current safety assessment of glyphosate and GBH. We conclude that current safety standards for GBHs are outdated and may not protect public health or the environment. To improve safety standards, the following are urgently needed: (1) human biomonitoring of glyphosate and its metabolites; (2) prioritization of glyphosate and GBHs for hazard assessments, including toxicological studies using state-of-the-art approaches; (3) epidemiological studies, particularly on occupationally exposed agricultural workers, pregnant women and their children; and (4) assessments of GBHs in commercially used formulations, recognizing that herbicide mixtures are likely to have effects not predicted by the study of glyphosate alone.

2017

Facts and Fallacies in the Debate on Glyphosate Toxicity. Robin Mesnage, Michael N Antoniou. Front Public Health. 2017. Doi: 10.3389/fpubh.2017.00316

"Facts and fallacies in the glyphosate toxicity debate"

The safety profile of the herbicide glyphosate and its commercial formulations is controversial. Criticism has been published by consultants and employees of companies marketing glyphosate-based herbicides in support of glyphosate's re-approval by regulatory agencies...

...These authors conclude that glyphosate is safe at levels below permitted regulatory limits. On the other hand, studies conducted by academic scientists independent of the industry report toxic effects below regulatory limits, as well as shortcomings in the current regulatory assessment of risks associated with glyphosate exposure. Two authors in particular (Samsel and Seneff) have published a series of commentaries proposing that long-term exposure to glyphosate is responsible for numerous chronic diseases (including cancers, diabetes, neuropathies, obesity, asthma, infections, osteoporosis, infertility and birth defects). ). The aim of this review is to examine the evidence for these alleged negative health effects, and the mechanisms that are thought to underlie them. We found that these authors inappropriately employ a deductive reasoning approach based on syllogism. We found that their conclusions are not supported by the available scientific evidence. Thus, the mechanisms and wide range of conditions proposed as resulting from glyphosate toxicity presented by Samsel and Seneff in their comments are at best unsubstantiated theories, speculation or simply incorrect. This misrepresentation of glyphosate toxicity misleads the public, the scientific community and regulators alike. Although there is evidence that glyphosate-based herbicides are toxic below regulatory safety limits, Samsel and Seneff's arguments largely serve to distract rather than give rational direction to much-needed future research into the toxicity of these pesticides, particularly at ingestion levels. which are typical of human populations.

2017

Proteomic analysis of the soil filamentous fungus Aspergillus nidulans exposed to a Roundup formulation at a dose causing no macroscopic effect: a functional study. Florence Poirier, Céline Boursier, Robin Mesnage, Nathalie Oestreicher , Valérie Nicolas, Christian Vélot. Environ Sci Pollut Res Int. 2017 doi: 10.1007/s11356-017-0217-6.

" Proteomic analysis of the filamentous soil fungus Aspergillus nidulans exposed to a Roundup formulation at a dose causing no macroscopic effects: a functional study."

Roundup® is a glyphosate-based herbicide (GBH) used worldwide in both agriculture and private gardens. As such, it is a major source of environmental contamination, particularly of water and soil, and can impact a number of non-target organisms essential to the balance of ecosystems...

The filamentous soil fungus Aspergillus nidulans has been shown to be strongly affected by a commercial formulation of Roundup® (R450), containing 450 g/L glyphosate (GLY), at doses well below the recommended agricultural application rate. In the present study, we used two-dimensional gel electrophoresis combined with mass spectrometry to analyze proteomic profile changes in A. nidulans exposed to R450 at a dose corresponding to the no-observed-adverse-effect level (NOAEL) for macroscopic parameters (31.5 mg/L GLY among adjuvants). Comparative analysis revealed a total of 82 differentially expressed proteins between control and R450-treated samples, 85% of which (70) were unambiguously identified. Their molecular functions were mainly attributed to cellular detoxification and stress response (16%), protein synthesis (14%), amino acid metabolism (13%), glycolysis/gluconeogenesis/glycerol metabolism/pentose phosphate pathway (13%) and Krebs cycle TCA/acetyl-CoA synthesis/ATP metabolism (10%). These results provide new insights into the toxicity induced by higher doses of this herbicide in the soil model organism A. nidulans. To our knowledge, this study represents the first evidence of modulation of protein expression, and hence of possible metabolic disruption, in response to herbicide treatment at a dose that causes no visible effects. These data are likely to challenge the concept of "substantial equivalence" when applied to herbicide-tolerant plants.

2017

Multiomics reveal non-alcoholic fatty liver disease in rats following chronic exposure to an ultra-low dose of Roundup herbicide. Robin Mesnage, George Renne, Gilles-Eric Séralini, Malcolm Ward, Michael N Antoniou. Sci Rep. 2017. Doi : 10.1038/srep39328.

" Multiomic studies reveal non-alcoholic hepatic steatosis in rats following chronic exposure to an ultra-low dose of Roundup herbicide ".

Impairment of liver function by low doses of environmentally friendly glyphosate-based herbicides (GBH) remains a controversial and unresolved issue. This study was carried out to find out...

Nous avons précédemment montré que des rats ayant reçu pendant 2 ans 0,1 ppb (dilution équivalente à 50 ng/L de glyphosate ; 4 ng/kg de poids corporel/jour d'apport quotidien) d'une formulation Roundup GBH présentaient des signes d'atteinte hépatique accrue, comme l'indiquent les analyses anatomorphologiques et sanguines. /Changements biochimiques urinaires et profilage du transcriptome. Nous présentons ici une étude multiomique combinant des analyses hépatiques du métabolome et du protéome pour mieux comprendre la pathologie induite par le Roundup. Les protéines significativement perturbées (214 sur 1906 détectées, q < 0,05) étaient impliquées dans le métabolisme des organo-azotés et dans la β-oxydation des acides gras. Les perturbations du protéome reflétaient la prolifération des peroxysomes, la stéatose et la nécrose. L’analyse du métabolome (55 métabolites altérés sur 673 détectés, p < 0,05) a confirmé les conditions lipotoxiques et le stress oxydatif en montrant une activation des systèmes piégeurs de radicaux libres glutathion et ascorbate. De plus, nous avons trouvé des altérations des métabolites associées à des caractéristiques d'hépatotoxicité telles que les dipeptides γ-glutamyl, les acylcarnitines et les dérivés de la proline. Dans l'ensemble, les perturbations du métabolome et du protéome ont montré un chevauchement substantiel avec les biomarqueurs de la stéatose hépatique non alcoolique et sa progression vers la stéatohépatose et confirment ainsi un dysfonctionnement hépatique résultant d'une exposition chronique à très faible dose de GBH.

2017

Toxicity of formulants and heavy metals in glyphosate-based herbicides and other pesticides. N Defarge, J Spiroux de Vendômois, G E Séralini. Toxicol Rep. 2017. Doi : 10.1016/j.toxrep.2017.12.

" Toxicity of formulants and heavy metals in glyphosate herbicides and other pesticides ".

The main pesticides used worldwide are glyphosate-based herbicides (GBH), and their toxicity is highly controversial. To understand their mode of action, the comparative herbicidal and toxicological effects of glyphosate (G) alone and 14 of its formulations were studied in this work, as a model for the pesticides...

GBHs are water mixtures, generally with 36% to 48% G claimed as the active ingredient. As with other pesticides, 10-20% of GBH is made up of chemicals. We had previously identified these by mass spectrometry and found that they were mainly families of petroleum-based oxidized molecules, such as POEA, and other contaminants. We exposed plants and human cells to the components of formulations, both mixed and separated, and measured toxicity and human cellular endocrine disruption below the experimentally measured threshold of direct toxicity. Contrary to popular opinion, G was only slightly toxic to plants at the dilutions recommended for agricultural use. In the short term, the strong herbicidal and toxic properties of its formulations were exerted by the POEA family of formulants alone. The toxic effects and endocrine-disrupting properties of the formulations were mostly due to the formulants and not to G. In this work, we also identified by mass spectrometry the heavy metals arsenic, chromium, cobalt, lead and nickel, known to be toxic. and endocrine disruptors, as contaminants in 22 pesticides, 11 of them G-based. This could also explain some of the pesticides' adverse effects. In chronic in vivo regulatory experiments used to establish acceptable daily doses of pesticides, G or other active ingredients reported in pesticides are assessed alone, without the formulants. In the light of these new data, this assessment method appears insufficient to guarantee safety. Taken together, these results shed new light on the toxicity of these major herbicides and of pesticides in general.

2017

Sex-dependent impact of Roundup on the rat gut microbiome. Veronica L Lozano, Nicolas Defarge, Louis-Marie Rocque, Robin Mesnage, Didier Hennequin, Renaud Cassier, Joël Spiroux de Vendômois, Jean-Michel Panoff , Gilles-Eric Séralini, Caroline Amiel. Toxicol Rep. 2017. Doi : 10.1016/j.toxrep.2017.12.005.

" Gender-dependent impact of Roundup on the rat gut microbiome ".

A growing body of research suggests that dysbiosis of the gut microbiota is induced by environmental pollutants, such as pesticides, which may play a role in the development of metabolic disorders...

We examined the long-term effects of 3 doses of the herbicide Roundup(R) (composed of glyphosate and formulants) on the gut microbiota of male and female Sprague-Dawley rats. A total of 141 bacterial families were identified using a 16S sequencing analysis approach. OPLS-DA analysis revealed an increase in the Bacteroidetes S24-7 family and a decrease in Lactobacillaceae in 8 of 9 females treated with 3 different doses of R (n = 3, for each dose). These effects were confirmed by sequence-based repetitive PCR fingerprinting showing a clustering of treated females. A culture-based method showed that R had a direct effect on rat gut microbiota. Culturable species showed different sensitivities to R, including the presence of a highly tolerant or resistant strain identified as Escherichia coli by 16S rRNA sequencing. The high tolerance of this E. coli strain is explained by the absence of the EPSPS gene (target enzyme encoding glyphosate), as shown by DNA amplification. Overall, these disturbances in the gut microbiome overlapped considerably with those associated with liver dysfunction in other studies. In conclusion, we have revealed that an environmental concentration of R (0.1 ppb) and two other concentrations (400 ppm and 5000 ppm) have a sex-dependent impact on the composition of the rat gut microbiome and therefore warrant further investigation.

2016

Co-Formulants in Glyphosate-Based Herbicides Disrupt Aromatase Activity in Human Cells below Toxic Levels. Nicolas Defarge, Eszter Takács , Verónica Laura Lozano , Robin Mesnage, Joël Spiroux de Vendômois, Gilles-Eric Séralini, András Székács. Int J Environ Res Public Health. 2016. Doi: 10.3390/ijerph13030264.

" Co-formulants in glyphosate-based herbicides disrupt aromatase activity in human cells below toxic levels ".

Pesticide formulations contain declared active ingredients and coformulants presented as inert, confidential compounds. We tested the endocrine disruption of coformulants in six glyphosate-based herbicides (GBH), the most widely used pesticides in the world.

All co-formulants and formulations were comparatively cytotoxic well below the 1% agricultural dilution (18 to 2,000-fold for co-formulants, 8 to 141-fold for formulations), and not the declared active ingredient, glyphosate (G), alone. The endocrine-disrupting effects of all these compounds were measured on the activity of aromatase, a key enzyme in the balance of sex hormones, below the toxicity threshold. Aromatase activity was reduced both by the coformulants alone (polyethoxylated tallow amine-POEA and alkyl polyglucoside-APG) and by the formulations, from concentrations 800 times lower than the agricultural dilutions; while G only exerted an effect at 1/3 of the agricultural dilution. It was shown for the first time that endocrine disruption caused by GBH could be due not only to the declared active ingredient, but also to co-formulants. These results could explain many in vivo results with GBH not observed with G alone; moreover, they call into question the relevance of the Acceptable Daily Intake (ADI) value for GBH exposures, currently calculated from toxicity tests on the declared active ingredient alone.

2016

Multiple effects of a commercial Roundup® formulation on the soil filamentous fungus Aspergillus nidulans at low doses: evidence of an unexpected impact on energetic metabolism. Valérie Nicolas, Nathalie Oestreicher, Christian Vélot. Environ Sci Pollut Res Int. 2016. Doi: 10.1007/s11356-016-6596-2.

" Multiple effects of a Roundup® commercial formulation on the filamentous soil fungus Aspergillus nidulans at low doses: evidence of an unexpected impact on energy metabolism ".

Soil microorganisms are highly exposed to glyphosate-based herbicides (GBH), particularly Roundup®, which is widely used worldwide. However, studies on the effects of GBH formulations on specific soil microbial species outside the rhizosphere are scarce. This study evaluated...

... the toxicity of a commercial formulation of Roundup® (R450), containing 450 g/L glyphosate (GLY), on the filamentous soil fungus Aspergillus nidulans, an experimental model micro-organism. The median lethal dose (LD50) on solid medium was between 90 and 112 mg/L of GLY (among the adjuvants, also included in the Roundup® formulation), corresponding to a dilution percentage around 100 times lower than that used in agriculture. The LOAEL and NOAEL (lowest no-observed-adverse-effect levels) associated with morphology and growth were 33.75 and 31.5 mg/L GLY respectively among the adjuvants. The R450 formulation proved far more active than technical GLY. At LD50 and lower concentrations, R450 altered growth, cell polarity, endocytosis and mitochondria (mean number, total volume and metabolism). In contrast to the depletion of mitochondrial activity reported in animal studies, R450 stimulated mitochondrial enzymatic activity, revealing a different mode of action from Roundup® on energy metabolism. These mitochondrial perturbations were also evident at a low dose corresponding to the NOAEL for macroscopic parameters, indicating that these mitochondrial biomarkers are more sensitive than those of growth and morphology. Overall, our data indicate that the toxic effects of GBH on filamentous soil fungi, and thus potential degradation of soil ecosystems, can occur at doses well below the recommended agricultural application rate.

2015

Transcriptome profile analysis reflects rat liver and kidney damage following chronic ultra-low dose Roundup exposure. Robin Mesnage, Matthew Arno, Manuela Costanzo, Manuela Malatesta, Gilles-Eric Séralini, Michael N Antoniou. Environ Health. 2015. doi: 10.1186/s12940-015-0056-1.

" Transcriptome profile analysis reflects rat liver and kidney damage following chronic exposure to Roundup at very low doses ".

A new study confirms that the concentration of Roundup present in tap water causes effects on the livers and kidneys of laboratory rats. Prof. Séralini's team published the first long-term toxicity study of the herbicide Roundup on laboratory rats in 2012, then republished it in 2014. Using organs from that same study, two researchers from King's College London, Dr. Robin Mesnage and Dr. Michael Antoniou, from CRIIGEN, have published an in-depth analysis of the effects of Roundup on genes regulating liver and kidney function...

Background: Glyphosate-based herbicides (GBH) are the main pesticides used worldwide. Converging evidence suggests that GBH, such as Roundup, presents a particular risk to liver and kidney health, although environmentally relevant low doses have not been examined. To address this issue, a 2-year study in rats administered 0.1 ppb Roundup (equivalent to 50 ng/L glyphosate) via drinking water (giving a daily intake of 4 ng/kg body weight/day glyphosate) was conducted. A markedly increased incidence of anatomorphological and biochemical changes in blood and urine was indicative of hepatic and renal structure and functional pathology. To confirm these results, we performed a microarray analysis of the liver and kidney transcriptome of the same animals.

Résultats : L'expression des groupes de transcription 4224 et 4447 (un groupe de sondes correspondant à un gène connu ou putatif) s'est avérée altérée respectivement dans le foie et le rein (p < 0,01, q < 0,08). Les modifications de l'expression des gènes variaient de -3,5 à 3,7 fois dans le foie et de -4,3 à 5,3 fois dans les reins. Parmi les 1319 groupes de transcription dont l'expression était altérée dans les deux tissus, un enrichissement ontologique en 3 catégories fonctionnelles parmi 868 gènes a été trouvé. Premièrement, les gènes impliqués dans l’épissage des ARNm et les petits ARN nucléolaires étaient pour la plupart régulés positivement, ce qui suggère une perturbation de l’activité normale du spliceosome. L'analyse au microscope électronique des hépatocytes a confirmé une perturbation structurelle nucléolaire. Deuxièmement, les gènes contrôlant la structure de la chromatine (en particulier les histones-lysine N-méthyltransférases) étaient pour la plupart régulés positivement. Troisièmement, les gènes liés au complexe I de la chaîne respiratoire et au cycle de l’acide tricarboxylique étaient pour la plupart régulés négativement. L'analyse des voies suggère une modulation des voies de signalisation mTOR et phosphatidylinositol. Les perturbations génétiques associées à l'administration chronique de Roundup à très faible dose reflètent une condition lipotoxique du foie et des reins et une croissance cellulaire accrue qui peuvent être liées à la régénération en réponse à des effets toxiques causant des dommages aux tissus. Les altérations observées dans l'expression des gènes étaient compatibles avec une fibrose, une nécrose, une phospholipidose, un dysfonctionnement de la membrane mitochondriale et une ischémie, qui sont en corrélation avec et confirment ainsi les observations de pathologie faites aux niveaux anatomique, histologique et biochimique.

Conclusion: Our results suggest that chronic exposure to a GBH in an established laboratory animal toxicity model system at an ultra-low environmental dose can result in liver and kidney damage with potentially significant implications for the health of animal and human populations.

2015

Laboratory Rodent Diets Contain Toxic Levels of Environmental Contaminants: Implications for Regulatory Tests. Mesnage R, Defarge N, Rocque LM, Spiroux de Vendômois J, Séralini GE. PLoS One. 2015. Doi : 10.1371/journal.pone.0128429.

" Diets of laboratory rodents contain toxic levels of environmental contaminants: implications for regulatory testing ".

Regulatory toxicological studies compare the impact of a product on a group of "test" animals against a group of "control" animals. Is the food of the "control" animals free of potentially toxic products? That's the question answered by this publication...

With the help of accredited laboratories, we have analyzed laboratory animal feed using standardized methods. This food, sourced from all five continents, is usually considered balanced and hygienic. The scope of the study is exceptional: 13 common samples of rat kibble were examined for traces of 262 pesticides, 4 heavy metals, 17 dioxins and furans, 18 PCBs and 22 GMOs. All the samples were contaminated with very high doses of some of these substances. These pollutants, which are typical of industrial food, can explain the level of tumors and diseases classified as natural in rats by the industry, allowing them to overlook the visible side effects of the industrial products they process. If this knowledge were to be taken into account by regulations, it could have far-reaching consequences for understanding the health effects of additives, contaminants and pesticides in food produced by intensive agriculture. Here again, CRIIGEN is open to comments from companies developing quality products, and from NGOs, to bring this issue to the attention of the authorities.

2014

Letter to the Editor regarding " Delaney et al., 2014 ": uncontrolled GMOs and their associated pesticides make the conclusions unreliable. Robin Mesnage, Nicolas Defarge, Joël Spiroux de Vendômois, Gilles-Eric Séralini. Food Chem Toxicol 2014. Doi : 10.1016/j.fct.2014.07.003

" Letter to the editor regarding "Delaney et al., 2014": uncontrolled GMOs and their associated pesticides make conclusions unreliable ".

We are concerned about the characterization of the diet tested in the study by Delaney et al. (2014), investigating the subchronic health effects of genetically modified Roundup-tolerant canola (DP-Ø73496-4) on rats. The conclusion can be used by regulatory authorities. Purina Certified Rodent LabDiet 5002 was not tested for the presence of other Roundup-tolerant GMOs and Roundup herbicide residues. According to our accredited PCR analyses, this control diet also contained 18% NK603 Roundup-tolerant maize and 14.9% MON810 (a GMO-producing modified Bt insecticide). We also found 110 ppb glyphosate and 200 ppb AMPA (the main metabolite of glyphosate). Although their toxicities are debated (Seralini et al., 2014), the uncontrolled presence of pesticide residues and other GMOs makes the study inconclusive. Any animal parameters measured after eating GM canola cannot be compared with controls eating a diet containing other GMOs with the same characteristic, and are not taken into account. By the standards of the editor of Food and Chemical Toxicology (Hayes, 2014), this study (Delaney et al., 2014) should be retracted.

Conclusiveness of toxicity data and double standards. Séralini, G.-E., Mesnage, R., Defarge, N., Spiroux, J. (2014) Food and Chem. Tox. 69:357-359.

" Conclusive toxicity data and double standards ".

We comment on the arguments of Mr Hayes, director of Food and Chemical Toxicology (Hayes2014) who took the decision to retract our study (Seralini et al., 2012). This publication highlights a double standard and inequity in choices to retract scientific publications.

2014

Letter to the Editor regarding " Delaney et al., 2014 ": uncontrolled GMOs and their associated pesticides make the conclusions unreliable. Robin Mesnage, Nicolas Defarge, Joël Spiroux de Vendômois, Gilles-Eric Séralini. Food Chem Toxicol 2014. Doi : 10.1016/j.fct.2014.07.003

" Letter to the editor regarding "Delaney et al., 2014": uncontrolled GMOs and their associated pesticides make conclusions unreliable ".

Conclusiveness of toxicity data and double standards. Séralini, G.-E., Mesnage, R., Defarge, N., Spiroux, J. (2014) Food and Chem. Tox. 69:357-359.

" Conclusive toxicity data and double standards ".

2014

Republished study: Long-term toxicity of a Roundup herbicide and a Roundup-tolerant genetically modified maize. Séralini, G.E., Clair, E., Mesnage, R., Gress, S., Defarge, N., Malatesta, M. Hennequin, D. Spiroux de Vendômois, J. (2014) Environ Sci Eur. 2014. doi: 10.1186/s12302-014-0014-5.

"Re-edited study: long-term toxicity of a Roundup herbicide and a Roundup-tolerant genetically modified maize".

An exceptional case!

2014

Conflicts of interests, confidentiality and censorship in health risk assessment: the example of an herbicide and a GMO. Gilles-Eric Séralini, Robin Mesnage, Nicolas Defarge, Joël Spiroux de Vendômois. Editorial Environ Sci Eur. 2014. doi: 10.1186/s12302-014-0013-6.

" Conflicts of interest, confidentiality and censorship in health risk assessment: the example of a herbicide and a GMO ".

We studied the long-term toxicity of Roundup-tolerant GM maize (NK603) and a whole formulation of Roundup pesticide at environmentally relevant levels from 0.1 ppb. This has caused turmoil in the scientific editorial world, highlighting conflicts of interest...

Our study was first published in Food and Chemical Toxicology (FCT) on September 19, 2012. The first wave of criticism arrived within a week, mainly from plant biologists with no toxicology experience. We responded to all these criticisms. The debate then encompassed scientific arguments, and a wave of ad hominem and potentially defamatory comments appeared in various journals by authors with serious but undisclosed conflicts of interest. At the same time, FCT recruited a former Monsanto employee as its new Associate Editor for Biotechnology after he had sent a letter to FCT complaining about our study. Not least because of this, FCT requested a post-hoc analysis of our raw data. On November 19, 2013, the editor requested the withdrawal of our study while acknowledging that the data were not incorrect and that there was no fault, fraud or intentional misinterpretation in our raw data set - an unusual, if not unprecedented, action in scientific publishing. The publisher argued that no conclusions could be drawn because we studied 10 rats per group for 2 years, because they were Sprague Dawley rats and because the data were inconclusive on cancer. This, however, was known at the time our study was submitted. However, our study was never considered a carcinogenicity study. We never used the word "cancer" in our paper. The present opinion is a summary of the debate that led to this retraction, as it is a historical example of conflicts of interest in scientific assessments of products marketed worldwide. We also show that the decision to retract cannot be rationalized on any discernible scientific or ethical basis. Censorship of health risk research undermines the value and credibility of science, so we are republishing our article.

2013

Cytotoxicity on human cells of Cry1Ab and Cry1Ac Bt insecticidal toxins alone or with a glyphosate-based herbicide. R Mesnage, E Clair, S GressC Then, A Székács, G-E Séralini. J Appl Toxicol. 2013 doi : 10.1002/jat.2712. Epub 2012 Feb 15.

" Cytotoxicity on human cells of Cry1Ab and Cry1Ac Bt insecticidal toxins alone or with a glyphosate-based herbicide ".

The study of the combined effects of pesticides represents a challenge for toxicology. In this study we investigate the cumulative effects of glyphosate-based herbicides and insecticides...

In the case of the new and growing generation of stacked-trait genetically modified (GM) plants, residues of glyphosate-based herbicides (such as Roundup) are present in Roundup-tolerant edible plants (particularly maize) and mixed with modified Bt insecticidal toxins that are produced by the GM plants themselves. The potential side effects of these combined pesticides on human cells are investigated in this work. Here, for the first time ever, we tested the Cry1Ab and Cry1Ac Bt toxins (10 ppb to 100 ppm) on the human embryonic kidney cell line 293, and their combined actions with Roundup, over 24 h, on three biomarkers of cell death: measurements of mitochondrial succinate dehydrogenase, adenylate kinase release by membrane alterations and caspase 3/7 inductions. Cry1Ab induced cell death at 100 ppm and above. For Cry1Ac, under such conditions, no effect was detected. Roundup tested alone from 1 to 20,000 ppm was necrotic and apoptotic from 50 ppm, well below agricultural dilutions (lethal concentration at 50% 57.5 ppm). The only significant combined effect measured was that Cry1Ab and Cry1Ac reduced Roundup-induced caspase 3/7 activations; this could delay the activation of apoptosis. The trend was the same for the other markers. In these results, we argue that engineered Bt toxins are not inert on non-target human cells, and that they may exhibit side effects in combination with other pesticide residues specific to genetically modified plants.

2013

Ethoxylated adjuvants of glyphosate-based herbicides are active principles of human cell toxicity. R Mesnage, B Bernay, G-E Séralini. Toxicology. 2013. Doi : 10.1016/j.tox.2012.09.006.

" Ethoxylated adjuvants in glyphosate-based herbicides are active ingredients in human cell toxicity ".

This study, along with others, contributed to the withdrawal by Anses of 132 glyphosate-based herbicides containing POE-Tallowamine.

Pesticides are always used in formulations as mixtures of an active ingredient with adjuvants. Glyphosate, the active ingredient in the world's leading pesticide, is a herbicide that is supposed to be specific to plant metabolism. Its adjuvants are generally considered to be inert diluents...

As side effects have been claimed for all these compounds, we studied the potential active ingredients for toxicity on human cells for 9 glyphosate-based formulations. To do this, we detailed their compositions and toxicities, and as controls we used a major adjuvant (the polyethoxylated suifamine POE-15), glyphosate alone, and a total formulation without glyphosate. This was carried out after 24 hours of exposure on hepatic (HepG2), embryonic (HEK293) and placental (JEG3) cell lines. We measured mitochondrial activity, membrane degradation and caspase 3/7 activity. Adjuvant compositions were analyzed by mass spectrometry. We demonstrate here that all formulations are more toxic than glyphosate, and have experimentally separated three groups of differentially toxic formulations according to their ethoxylated adjuvant concentrations. Of these, POE-15 is clearly the most toxic to human cells, although others cannot be ruled out. It begins to be active with dose-dependent adverse effects on cell respiration and membrane integrity between 1 and 3 ppm, at environmental/occupational doses. We further demonstrate that POE-15 induces necrosis from its first micellisation process, unlike glyphosate which is known to promote endocrine disrupting effects after its entry into cells. Overall, these results call into question the establishment of guideline values such as the acceptable daily intake of glyphosate, when these are mainly based on a long-term in vivo test of glyphosate alone. Given that pesticides are always used with adjuvants likely to modify their toxicity, the need to assess all their formulations as mixtures becomes obvious. This calls into question the notion of pesticide active ingredients for non-target species.

2013

Answers to critics: Why there is a long term toxicity due to a Roundup-tolerant genetically modified maize and to a Roundup herbicide. Gilles-Eric Séralini, Emilie Clair, Robin Mesnage, Steeve Gress, Nicolas Defarge, Manuela Malatesta, Didier Hennequin, Joël Spiroux de Vendômois (2013). Food Chem. Toxicol. 53,461-468.

" Answers to critics: Why there is long-term toxicity from genetically modified Roundup-tolerant corn and Roundup herbicide "

Our recent work (Séralini et al., 2012) remains the most detailed study to date of the lifelong consumption of a genetically modified agricultural organism (GMO) and its associated herbicide. Nevertheless, this study has generated a violent polemic against us. We have responded to all the attacks in this publication...

This is particularly true for NK603 maize, for which only one 90-day marketing test has previously been carried out with the same rat strain (Hammond et al., 2004). This is also the first detailed long-term research on mammals exposed to a highly diluted pesticide in its total formulation with adjuvants. This may explain why 75% of our initial criticisms within a week, among the publication's authors, came from plant biologists, some developing patents on GMOs, even though this was a toxicological article on mammals, and the Monsanto company, which owns both NK603 GM maize and Roundup (R) herbicide. Our study has limitations like any other, and here we carefully respond to all the criticisms from agencies, consultants and scientists that have been directed at the publisher or ourselves. At this level, a full debate would be biased if the mammalian toxicity tests of NK603 and R obtained by Monsanto were to remain confidential and therefore unavailable in electronic format for the entire scientific community to conduct an independent review of the raw data. In our article, the conclusions on the long-term toxicity of NK603 and Roundup were based on statistically highly discriminating results at biochemical level in the treated groups compared to controls, as these results corresponded well in blind analysis to the pathologies observed in the organs, i.e. were in turn linked to deaths by anatomopathologists. GM NK603 and GM R cannot be considered safe at this time.

2011

Defined plant extracts can protect human cells against combined xenobiotic effects. Céline Gasnier, Claire Laurant, Cécile Decroix-Laporte, Robin Mesnage, Emilie Clair, Carine Travert, Gilles-Eric Séralini. J Occup Med Toxicol. 2011 doi: 10.1186/1745-6673-6-3.

" Defined plant extracts can protect human cells from combined xenobiotic effects ".

Representative pollutants of common environmental contaminants induce intracellular toxicity in human cells, which is usually amplified in combination. We wanted to test common pathways of intoxication and detoxification in human embryonic and hepatic cell lines...

We used various pollutants such as Roundup residues, Bisphenol-A and Atrazine, as well as five specific herbal extracts called Circ1, Dig1, Dig2, Sp1 and Uro1 to understand whether or not specific molecular actions had taken place.

The kidneys and liver are major detoxification organs. We studied human embryonic kidney and liver cell lines E293 and HepG2. The intoxication was caused on the one hand by a formulation of one of the world's most widely used herbicides, Roundup 450 GT+ (glyphosate and specific adjuvants), and on the other by a mixture of Bisphenol-A and Atrazine, all present in surface waters. The preventive and curative effects of The preventive and curative effects of plant extracts were also measured on mitochondrial succinate dehydrogenase activity, on the entry of radiolabelled glyphosate (in Roundup) into cells, and on cytochromes P450 1A2 and 3A4 as well as glutathione-S-transferase.

Results: Clear pollutant toxicity was observed in both cell lines at very low sub-agricultural dilutions. Prevention of such phenomena occurred within 48 hours with the plant extracts tested, with success rates ranging from 25 to 34% for Roundup-intoxicated E293, and surprisingly up to 71% for HepG2. In contrast, after intoxication, no plant extract was able to restore E293 viability within 48 hours. However, two herbal combinations restored Bisphenol-A/Atrazine-intoxicated HepG2 up to 24-28%. Analysis of the underlying mechanisms revealed that the plant extracts were unable to prevent radiolabeled glyphosate from entering cells; however, Dig2 restored Roundup-disrupted CYP1A2 activity and had only a slight preventive effect on CYP3A4 and no effect on glutathione S-transferase.

In conclusion: environmental pollutants have intracellular effects that can be prevented, or partly cured, by specific herbal extracts in two human cell lines. This appears to be due at least in part to modulation of cytochromes P450.

2010

Dig1 protects against cell death provoked by glyphosate-based herbicides in human liver cell lines. Céline Gasnier, Nora Benachour, Emilie Clair, Carine Travert, Frédéric Langlois, Claire Laurant, Cécile Decroix-Laporte, Gilles-Eric Séralini. Jour. Occ. Med and Tox. 2010 doi : 10.1186/1745-6673-5-29.

" Dig1 protects against glyphosate herbicide-induced cell death in human liver cell lines ".

Pesticides used worldwide containing various adjuvants, such as Roundup formulations of glyphosate-based herbicides, can cause toxicity in vivo and in human cells. In order to understand their effects on liver cells, a major detoxification organ, we studied their mechanism of action and their possible protection by specific extracts of medicinal plants called Dig1...

The cytotoxicity pathways of four glyphosate herbicide formulations were investigated using human liver cell lines HepG2 and Hep3B, well-known models for studying the effects of xenobiotics. We monitored mitochondrial succinate dehydrogenase activity and caspases 3/7 for cell death and protection by Dig1, as well as cytochromes P450 1A1, 1A2, 3A4 and 2C9 and glutathione-S-transferase to address the mechanism of action.

All four Roundup formulations induce liver cell death, with adjuvants having more potent effects than glyphosate alone. Dig 1, which is non-cytotoxic and does not induce caspases on its own, is able to prevent Roundup-induced cell death in a time-dependent manner, with a significant efficacy of up to 89%, within 48 hrs. Furthermore, we demonstrated that it prevents caspase 3/7 activation and CYP3A4 enhancement, not GST reduction, but slightly inhibits CYP2C9 when added before Roundup.

In conclusion, Roundup is capable of inducing intracellular disruption in liver cell lines at various levels, but a mixture of Dig1 medicinal plant extracts can protect human cell lines to some extent against these pollutants. Together, the system provides a tool for studying intoxication and hepatic detoxification.

Two cases of birth defects overlapping Stratton-Parker syndrome after multiple pesticide exposure. Robin Mesnage, Emilie Clair, Joël Spiroux de Vendômois and Gilles-Eric Séralini 2010. Case Reports Occup Environ Med doi : 10.1136/oem.2009.052969.

" Two cases of congenital malformations overlapping Stratton-Parker syndrome after multiple pesticide exposure ".

In this study we suggest possible environmental origins for this syndrome. Indeed, the impact of synergistic pollution during embryonic development cannot be excluded...

Stratton-Parker syndrome is rare and includes growth retardation, imperforation and often genital anomalies, but no genetic origin is known. Two new cases have been reported in the same family, which sprays 1.3 tonnes of pesticides a year without protection. These pesticides include many endocrine disruptors with similar combined effects on the development of laboratory animals. We know that the emergence of a pathology is always potentially multi-factorial. We suggest possible environmental origins for this syndrome; a synergistic pollution effect during embryonic development cannot be ruled out. We need to further develop an active precautionary approach in cases of scientific uncertainty, and improve epidemiological and toxicological knowledge of commercial pesticide mixtures.

2009

Glyphosate-based herbicides are toxic and endocrine disruptors in human cell lines. Céline Gasnier, Coralie Dumont, Nora Benachour, Emilie Clair, Marie-Christine Chagnon, Gilles-Eric Séralini. Toxicology. 2009 doi: 10.1016/j.tox.2009.06.006.

" Glyphosate-based herbicides are toxic and endocrine disruptors of human cell lines ".

In this study we wanted to assess the toxicity or endocrine disruptor status of glyphosate-based herbicides...

Glyphosate-based herbicides are the most widely used in the world, marketed in a variety of formulations. Their residues are frequent pollutants in the environment. What's more, these herbicides are widespread on most transgenic consumer plants, modified to tolerate high levels of these compounds in their cells. Up to 400 ppm of their residues are accepted in some animal feeds. We exposed human HepG2 liver cells, a well-known model for studying xenobiotic toxicity, to four different formulations and to glyphosate, which is usually tested alone in chronic in vivo regulatory studies. We measured cytotoxicity with three assays (Alamar Blue, MTT, ToxiLight), as well as genotoxicity (comet assay), anti-estrogenic (on ERalpha, ERbeta) and anti-androgenic (on AR) effects using gene reporter assays. We also checked the conversion of androgens to estrogens by aromatase activity and mRNA. All parameters were perturbed at sub-agricultural doses with all formulations within 24 hours. These effects were more dependent on formulation than on glyphosate concentration. Initially, we observed endocrine disruption of human cells as early as 0.5 ppm on the androgen receptor in MDA-MB453-kb2 cells for the most active formulation (R400), then from 2 ppm transcriptional activities on both estrogen receptors were also inhibited on HepG2. Aromatase transcription and activity were disrupted from 10 ppm. Cytotoxic effects began at 10 ppm with the Alamar Blue assay (the most sensitive) and DNA damage at 5 ppm. A true cellular impact of glyphosate herbicide residues in food, feed or the environment must therefore be taken into account, and their classification as carcinogenic/mutagenic/reprotoxic discussed..

2009

Glyphosate-based herbicides are toxic and endocrine disruptors in human cell lines. Céline Gasnier, Coralie Dumont, Nora Benachour, Emilie Clair, Marie-Christine Chagnon, Gilles-Eric Séralini. Toxicology. 2009 doi: 10.1016/j.tox.2009.06.006.

" Glyphosate-based herbicides are toxic and endocrine disruptors of human cell lines ".

In this study we wanted to assess the toxicity or endocrine disruptor status of glyphosate-based herbicides...

2008

Factors to consider before production and commercialization of aquatic genetically modified organisms: the case of transgenic salmon. Olivier Le Curieux-Belfond, Louise Vandelac, Joseph Caron, Gilles-Éric Séralini (2008). Environmental Science & Policy doi.org/10.1016/j.envsci.2008.10.001

Numerous genetically modified plants have been developed, and four of them (soybean, maize, cotton and rapeseed), representing over 99% of commercial crops, are widely grown, mainly in the USA and America. However, all over the world, policy is still under development regarding the authorization of modified and/or cloned plants and animals for food or feed use, and their release into the environment. Commercial animal projects...

...the most advanced involve various species of fish, which are easier to transform genetically, notably because conception and development take place in water and with easy access to numerous eggs. An application for approval to market genetically modified (GM) salmon has been submitted to the US Food and Drug Administration (FDA). In the meantime, questions have been raised about the impacts of GM salmon, modified for productivity, on aquaculture, wildlife, ecosystems and human health. Here, we review these scientific studies and health, environmental, social and economic arguments. This article analyzes the current gaps in our knowledge of the impacts of transgenic fish, and proposes the legislative guidelines needed to protect the environment and human health, should the commercialization of animal genetically modified organisms (GMOs) be authorized.

2007

Cytotoxic effects and aromatase inhibition by xenobiotic endocrine disrupters alone and in combination. Nora Benachour, Safa Moslemi, Herbert Sipahutar, Gilles-Eric Seralini. Toxicology and Applied Pharmacology (2007). doi.org/10.1016/j.taap.2007.03.033

" Cytotoxic effects and aromatase inhibition by xenobiotic endocrine disruptors alone and in combination ".

Xenobiotics can cause long-term adverse effects in humans, particularly at the embryonic level, raising questions about their exposure levels, combined effects and crucial endpoints. We focused on possible interactions between xenobiotic endocrine disruptors, cell viability and androgen metabolism.

Thus, we tested aroclor 1254 (A1254), atrazine (AZ), o,p′-DDT, vinclozoline (VZ), p,p′-DDE, bisphenol A (BPA), chlordecone (CD), nonylphenol (NP), tributyline oxide (TBTO) and diethylstilbestrol (DES) for cell toxicity against human embryonic cells and activity against cellular aromatase, but also on placental microsomes and purified equine enzyme. Cell viability was affected within 24 h by all xenobiotics with a threshold at 50 µM (except TBTO and DES, threshold 10 µM), and aromatase was inhibited at non-toxic doses. In combination, a synergy was observed, reducing toxicity threshold values to 4-10 μM and aromatase activity by 50% in some cases. In placental microsomes, the most active xenobiotics rapidly inhibit microsomal aromatase in a manner independent of NADPH metabolism. Prolonged exposure to low doses in cells generally results in a 50-fold increase in aromatase inhibition. These xenobiotics may act by inhibition of the active site or by allosteric effects on the enzyme. Bioaccumulation is a feature of some xenobiotics, notably chlordecone, DDT and DDE, and low-level chronic exposures can also affect cellular signalling mechanisms. This new information on the mechanism of action of these xenobiotics will help improve molecular design to provide safer compounds for use in the (human) environment.

2005

Differential effects of glyphosate and roundup on human placental cells and aromatase. Sophie Richard, Safa Moslemi, Herbert Sipahutar, Nora Benachour, Gilles-Eric Seralini. Comparative Study Environ Health Perspect. 2005. Doi : 10.1289/ehp.7728.

" Differential effects of glyphosate and Roundup on human placental cells and aromatase."

We conclude that endocrine and toxic effects of Roundup, not just glyphosate, can be observed in mammals. We suggest that the presence of Roundup adjuvants enhances the bioavailability and/or bioaccumulation of glyphosate.

Roundup is a glyphosate-based herbicide used worldwide, including on most genetically modified plants designed to tolerate it. Its residues can thus enter the food chain, and glyphosate is found as a contaminant in rivers. Some farm workers using glyphosate have problems with pregnancy, but its mechanism of action in mammals has been called into question. We show here that glyphosate is toxic to human JEG3 placental cells within 18 hours at concentrations lower than those found in agriculture, and this effect increases with concentration and time or in the presence of Roundup adjuvants. Surprisingly, Roundup is always more toxic than its active ingredient. We tested the effects of glyphosate and Roundup at lower, non-toxic concentrations on aromatase, the enzyme responsible for estrogen synthesis. The glyphosate herbicide disrupts aromatase activity and mRNA levels and interacts with the active site of the purified enzyme, but the effects of glyphosate are facilitated by the Roundup formulation in microsomes or cell culture. We conclude that the endocrine and toxic effects of Roundup, not just glyphosate, can be observed in mammals. We suggest that the presence of Roundup adjuvants enhances the bioavailability and/or bioaccumulation of glyphosate.