This environmental toxicology report has been made by creating a collection of a range of sources on the biological effects of glyphosate. The executive summary brings into focus a general overview of the peer reviewed research which is found on the effects of glyphosate on a range of species and subjects.

The document is constructed of verbatim excerpts from the original texts along with hyperlinks to the text online where possible. For the purposes of edification I have drawn key themes out as headlines of the paper or study. Where relevant I have included source notations to indicate where further research can be found.

Introduction

This is a prospective document rather than a comprehensive study and the aim is to create a navigable sense of the range of published research on the effects of glyphosate. The aim of this document is also to gather together key technical information sufficient to inform and assist field testing. Please get in touch with any questions.

The work was brought together by request as glyphosate (also known as the herbicide RoundUp) is being used by councils across the United Kingdom on public spaces to kill vegetation.  These include places such as streets, public parks, primary and secondary schools, hospitals, colleges and universities but they also include agricultural spaces where the food supply is produced so it is a feature of a non-organic diet.

Since the International Agency for Research on Cancer (IARC) released its finding in 2015 concluding that there is sufficient evidence of glyphosate’s carcinogenicity there has been an explosion of investigations on what other health measures it has an impact on.  Glyphosate along with insecticides malathion and diazinon were categorised in the 2A section.  This category is used “when there is limited evidence of carcinogenicity in humans and sufficient evidence of carcinogenicity in experimental animals. Limited evidence means that a positive association has been observed between exposure to the agent and cancer but that other explanations for the observations (called chance, bias, or confounding) could not be ruled out”.

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The major concerns about this practice are for pets, children and infants, the elderly and health impaired however with the emergent research over several decades of application of this organophosphate herbicide, the science is showing that it has impacts on all life forms from soil bacterial populations to aquatic species to mammals.  Since the landmark legal ruling on Dwayne Johnson’s case in 2018 (the first successful law case against the chemical giants Monsanto on this matter) more and more law suits have been lodged further stimulating an abundance of scientific studies.

Environmental and medical toxicology is now a mature and advanced area of science which analyses these concerns and debates. This research digest document was assembled for community groups to open informed dialogues with their local authorities and managerial structures of the public spaces they (and their children and pets) occupy highlighting the issues with using glyphosate as an organophosphate herbicide.  Once managers  are made aware of the body of science it lays the foundation for future legal charges of negligence.

As a public health issue communities need to develop a better understanding of the medicine and science available on the chemicals to which they are being exposed.  It is not as complicated as people are led to believe and a measured effort of learning the meaning of the words which are used in reports and developing understandings of the connections between chemicals and specific health conditions enables individuals to make rational decisions and reasoned objections.

When Austria banned glyphosate in 2020 the manufacturers of Roundup changed the formulation to use agricultural vinegar – acetic acid – a toxicologically safe alternative to the organophosphate.  Agricultural vinegar is also cheaper so offers a cost saving to organisations and councils which switch to use it instead of glyphosate.  Wild Justice, the legal campaigners, have been running a series of investigations on glyphosate showing that it was detected in 84% of the sample of people’s urine.

Wild Justice point out in another article How to discover your own glyphosate levels: this website will send you a glyphosate testing kit that you can use in your own home and send off a urine sample to a laboratory in Germany which will send you your results.  The cost of the testing and postage is about £40 (depending on the £/euro exchange rate) so it is a significant investment.

Considering that it is marketed as a herbicide the manufacturers do not inform the public how it damages other forms of life such as bees and pollinating insects, pets and humans. The well known entomologist, Prof Dave Goulson, campaigned for a ban on the use of glyphosate in gardens saying: “Ban the use of pesticides in urban areas & end their sale for use in gardens. There is simply no need to spray poisons in our streets, parks & gardens for cosmetic purposes, where they harm bees & other wildlife & pose a risk to human health. Safe alternatives are available, where necessary.”

Alex Dunedin

Click here to download written report

Click here to download mindmap of report

Executive Summary

Glyphosate is an organophosphate compound which affects a range of species. There is a broad evidence base which has accumulated from over four decades of research detailing the biological effects of this widely used agrochemical. It is highly water soluble and has come to be almost ubiquitous in the landscape.

• • GBHs residues can be found in food
  • GBHs residues can be found in drinking-water
  • GBHs residues can be found in crops
  • GBHs residues can be found in animal feed
  • GBHs residues can be found in groundwater
  • GBHs residues can be found in rain
  • GBHs residues can be found in air

There is evidence of it acting in a toxic way to microbes, fungi, plants, fish, mammals and humans. Evidence demonstrates inhibition of acetylcholinesterase and changes to the normal metabolism of the neurotransmitter acetylcholine. There is also a significant evidence base showing that glyphosate partly acts through production of oxidative stress in the form of hydoxyl production and malondialdehyde.

Evidence is emerging that glyphosate impacts many health outcomes, including developmental and reproductive toxicity, endocrine disruption, host immunity, obesity and diabetes, gastrointestinal disorders, cardiovascular disorders and central nervous system dysfunction such as learning and memory impairment, anxiety, depression and autism. These chronic health outcomes may occur even at doses lower than established risk safety guidelines, in particular during critical development windows as denoted in the DOHaD paradigm.

Human epidemiological studies suggests a compelling link between exposures to (glyphosate-based herbicides) GBHs and increased risk for non-Hodgkin lymphoma (NHL)

Digest of Glyphosate Toxicology

The numbers in this digest refer to the papers listed in the following section where you can read verbatim excerpts from the original reference along with its citation information.  This is done so the reader can quickly assess the original context and locate the publication for their own analysis.  Below the digest and ‘Paper Index Point-by-Point’ you will find excerpts from each publication and paper dealt with sequentially; each paper has been given a sequential number which corresponds to the paragraph of information in this digest section.  You will find highlights drawn from the excerpt in the text box in each case.

1. N-(phosphonomethyl)glycine is a broad spectrum, post-emergence systemic herbicide used extensively over the past 45 years. Over time several plant species have developed resistance to its exposure.

2. Glyphosate is a herbicide which acts to cause shikimate to accumulation plants. It acts not just on plants but also in a range of species of life including fungus. It acts on microbial life such as malaria and toxoplasmosis changing the microbial community composition benefiting Proteobacteria and repressing species such as P. fluorescens. In susceptible plants it acts in a dose dependent manner to affect the biochemical formation of flavonoids, lignins, alkaloids and CO2 fixation.

3. Glyphosate is watersoluble. Rain within 6 hours of application causes transfer. It acts to control vegetation for 2 to 4 weeks with a half life of 60 days or less. It reacts with iron and galvanised steel

4. Glyphosate is reported to not be metabolized appreciably in mammals and is excreted largely unchanged in the urine. 60% of farmers had detectable levels found in urine on day of application. Urinary concentrations ranged from <1 to 233 ppb. Maximum systemic dose was estimated to be 0.004 mg kg−1.

5. Further studies showed that glyphosate is moderately absorbed through the gastrointestinal tract, undergoes minimal biotransformation and excreted via the kidneys. Symptoms from excessive exposure include gastro-intestinal irritation and damage, as well as dysfunction in several organ systems (e.g., lung, liver, kidney, CNS, and cardiovascular system).

6. Glyphosate has been demonstrated to affect aquatic life. Studies on the goldfish (Carassius auratus) show that exposure affect levels of the enzyme acetylcholinesterase and levels of the hydroxyl free radical. Its effects on acetylcholinesterase in the goldfish illustrate the classical effects of an organophosphate compound.

Glyphosate exposure also decreased the enzyme superoxide dismutase (SOD), which represents an integral part of biological antioxidant defence system. Malondialdehyde occurs naturally and is a marker for oxidative stress. Malondialdehyde significantly increased when exposed to glyphosate illustrating that its action is partly through mechanisms of oxidative stress.

7. China, with a population of 1.3 billion and an ever – expanding agricultural product demand, is now probably the second largest producer of pesticides in the world, exporting almost $1.2 billion in agrochemical products in 2004. Of the top ten global active ingredients, it is noteworthy that seven are herbicides, dominated by glyphosate, far and away the best selling agrochemical.

In wheat glyphosate inhibits an enzyme 25-hydroxycholesterol 7-alpha-hydroxylase (CYP71 C6v1). This is a part of the cytochrome P450 enzyme system which is involved in a wide range of detoxification processes. Many herbicides are metabolised by the cytochrome P450.

8. Glyphosate’s inhibition of cytochrome P450 (CYP) enzymes is an overlooked component of its toxicity to mammals. Glyphosate enhances the damaging effects of other food borne chemical residues and environmental toxins.

Negative impact on the body manifests slowly over time as inflammation damages cellular systems throughout the body. Exposure to glyphosate contributes to diseases of oxidative stress by virtue of its effects on oxidative radicals. Oxidative metabolism has consequences on various diseases including gastrointestinal disorders, obesity, diabetes, heart disease, depression, autism, infertility, cancer and Alzheimer’s disease.

In humans, only small amounts (~2%) of ingested glyphosate are metabolized to aminomethylphosphonic acid (AMPA), and the rest enters the blood stream until it is eventually eliminated through the urine. A now common practice of crop desiccation through herbicide administration shortly before the harvest assures an increased glyphosate presence in food sources.

The industry asserts that glyphosate is nearly nontoxic to mammals, and therefore it is not a problem if glyphosate is ingested in food sources. Acutely, it is claimed to be less toxic than aspirin. While short-term studies in rodents have shown no apparent toxicity, studies involving life-long exposure in rodents have demonstrated liver and kidney dysfunction and a greatly increased risk of cancer, with shortened lifespan.

9. A study had determined the effect of glyphosate-based herbicide (GBH) on acetylcholinesterase (AChE) enzyme activity, oxidative stress, and antioxidant status in Gammarus pulex. Gammarus pulex is a species of amphipod crustacean found in fresh water across much of Europe.

Glyphosate exposure produced oxidative stress in gammarus pulex. The marker for oxidative stress, Malondialdehyde level increased significantly (p < 0.05). The reduced glutathione (GSH) level, the acetylcholinesterase (AChE), the catalase (CAT), and the glutathione peroxidase (GPx) activities decreased compared with the control group (p < 0.05). G. pulex exposure to GBH for 24 h showed a temporary reduction in the superoxide dismutase (SOD). This is all consistent with glyphosates involvement with oxidative stress.

10. Glyphosate is a weak inhibitor of acetylcholinesterase in rats. This biochemical interaction alters the chief neurotransmitter system of acetyl choline. This is consistent with the neurotoxic effects of the organophosphate family of agrochemicals.

11. Teleostean fish Leporinus obtusidens (piava) were exposed to different concentrations of Roundup, a glyphosate. Results indicated that acetylcholinesterase (AChE) activity significantly decreased in the brain of fish exposed to all glyphosate concentrations tested, but in the muscle this parameter was not altered.

Fish exposed to all glyphosate concentrations showed a significant increase in hepatic glycogen and glucose, but a significant reduction in muscle glycogen and glucose. Levels of ammonia in both tissues increase in fish at all glyphosate concentrations. Exposure to this herbicide produced a decrease in all hematological parameters tested.

12. The toxic effect of sublethal concentrations of glyphosate was evaluated on acetylcholinesterase (AChE) activity in another fish species, Cnesterodon decemmaculatus. Inhibition ranged from 23 to 36%. The analytical determination of glyphosate in assay media by ion chromatography, was used to verify its stability. Fish survival was 100%, even at the highest concentration tested. Significant inhibitory effect on AChE activity was recorded even for the lowest herbicide concentration tested.

13. Numerous clinical and epidemiological data have reported the deleterious effects of glyphosate on learning and memory. In mice subchronic and chronic exposure to glyphosate based herbicides decreased discrimination index and the step-through-latency indicating recognition and retention memory impairments. Prominent decreases in Acetylcholinesterase, Superoxide Dismutase (SOD) and Peroxidase (PO) specific activities within the brain were found.

14. Evidence is emerging that they impact many health outcomes, including developmental and reproductive toxicity [46,47,48], endocrine disruption [49,50], host immunity [51,52,53], obesity and diabetes [7,54], gastrointestinal disorders [55], cardiovascular disorders [56,57] and central nervous system dysfunction such as learning and memory impairment [58], anxiety, depression [59] and autism [8]. These chronic health outcomes may occur even at doses lower than established risk safety guidelines, in particular during critical development windows as denoted in the DOHaD paradigm [60].

15. Deaths following ingestion of ‘Roundup’ alone were due to a syndrome that involved hypotension, unresponsive to intravenous fluids or vasopressor drugs, and sometimes pulmonary oedema, in the presence of normal central venous pressure. Accidental exposure was asymptomatic after dermal contact with spray (six cases). Mild oral discomfort occurred after accidental ingestion (13 cases).

16. Intentional ingestion (80 cases) resulted in erosion of the gastrointestinal tract (66%), seen as sore throat (43%), dysphagia (31%), and gastrointestinal haemorrhage (8%). Other organs were affected less often (non-specific leucocytosis 65%, lung 23%, liver 19%, cardiovascular 18%, kidney 14%, and CNS 12%).

17. Chronic as well as acute exposure to glyphosate based herbicides (GlySH) can lead to non-alcoholic fatty liver disease (NAFLD) and fulminant liver failure. It has been reported that chronic exposure to Glyphosate of more than 5 years’ duration due to consumption of food grains sprayed with this herbicide or inhalation of particles results in development of Fatty Liver, i.e., non-alcoholic fatty liver disease.

18. Human epidemiological studies suggests a compelling link between exposures to glyphosate-based herbicides (GBHs) and increased risk for non-Hodgkin lymphoma (NHL). Use of GBHs has increased dramatically worldwide in recent decades. In the United States alone, usage increased nearly sixteen-fold between 1992 and 2009

The practice of applying GBHs to crops shortly before harvest, so-called “green burndown,” began in the early 2000s to speed up their desiccation; as a consequence, crops are likely to have higher GBH residues. By the mid-2000s, green burndown became widespread, and regulatory agencies responded by increasing the permissible residue levels for GBHs.

Glyphosate and its metabolites persist in food [5–7], water [8], and dust [9], potentially indicating that everyone may be exposed ubiquitously. Non-occupational exposures occur primarily through consumption of contaminated food, but may also occur through contact with contaminated soil [9], dust [9] and by drinking or bathing in contaminated water [8]

In plants, glyphosate may be absorbed and transported to parts used for food; thus, it has been detected in fish [5], berries [6], vegetables, baby formula [7], and grains [10], and its use as a crop desiccant significantly increases residues. GBH residues in food persist long after initial treatment and are not lost during baking.

Average urinary glyphosate levels among occupationally exposed subjects range from 0.26-73.5 μg/L, whereas levels in environmentally exposed subjects have been reported between 0.13-7.6 μg/L [11]. Two studies of secular trends have reported increasing proportions of individuals with glyphosate in their urine over time [12, 13].

Given that more than six billion kilograms of GBHs have been applied in the world in the last decade [2], glyphosate may be considered ubiquitous in the environment [14]. Some epidemiological studies have reported an increased risk of NHL in GBH-exposed individuals [15–17]. We evaluated the all the published human studies on the carcinogenicity of GBHs and present the first meta-analysis to include the most recently updated Agricultural Health Study (AHS) cohort [24].

Paper Index via Point-by-Point Topic

Each of these papers and textbooks has been used to construct the above overview of the effects glyphosate is having in living species. To get to the original source of information you can click on the hyperlinks below to find the verbatim quote plus a link to the original text where possible.

Paper 1: Weed resistance to glyphosate

Glyphosate, N-(phosphonomethyl)glycine, is a broad-spectrum, postemergence systemic herbicide that has been used extensively over the past 35 years. However, the intense and prolonged use of the glyphosate herbicide has resulted in documented resistance to glyphosate in several weed populations [Nandula, V.K., Reddy, K.N., Duke, S.O., and Poston, D.H. (2005) Outlooks Pest Manag., 16(4), 183–187].

Page 96, Lamberth, C., & Dinges, J. (2012). Bioactive heterocyclic compound classes: Agrochemicals.

Paper 2: Glyphosate causes deficiencies of lignins, alkaloids and flavonoids

Glyphosate (N-[phosphonomethyl]glycine), mainly sold under the trade name Roundup, is a systemic broad-spectrum herbicide that inhibits 5-enolpyruvyl shikimate 3-phosphate synthase (EPSPS), a key enzyme in the biosynthesis of aromatic acids and secondary metabolites. Blockage of this pathway results in massive accumulation of shikimate in affected plant tissues leading to a deficiency of significant end-products such as lignins, alkaloids, and flavonoids and a decrease in CO2 fixation and biomass production in a dose dependant manner (Olesen & Cedergreen, 2010).

The formulation and adjuvants used to enhance the efficiency of the active compound can dramatically affect germination, growth, and propagation of fungal plant pathogens (Smith & Hallett, 2006; Weaver et al., 2006; Weaver, et al., 2009; Wyss et al., 2004). Interestingly, glyphosate has also been reported to control mammalian pathogenic fungi (Nosanschuk et al., 2001) and was active against apicomplexan parasites that cause diseases such as malaria and toxoplasmosis (Roberts et al., 2002).

There are several reports indicating that glyphosate inhibits fungal species involved in soilborne diseases. Sclerotium rolfsii, for instance, is a common soilborne plant pathogen known to persist on crop residues. Banana growers noted that rotting residues inadvertently sprayed with glyphosate had little mycelial growth and fewer sclerotia than those not sprayed with the herbicide. Growth of Sclerotium rolfsii was retarded on culture plates amended with benomyl or glyphosate, each at the commercial rate of application.

Both amended media reduced the radial growth of S. rolfsii compared to the control; however, glyphosate-amended medium had the greater inhibitory effect (Westerhuis et al., 2007). Radial growth of other pathogens such as Pythium ultimum and Fusarium solani f.sp. pisi was also retarded with increasing concentrations of the herbicide (Kawate et al., 1992), which also referred to conidial germination and sporulation in F. solani f.sp. glycines (Sanogo et al., 2000).

In contrast to the results described above, Harikrishnan and Yang (2001) found no negative effect of glyphosate on vegetative growth of several Rhizoctonia solani isolates and anastomosis groups. However, the herbicide influenced the production of fruiting bodies of this pathogen. The number of sclerotia produced was higher but these sclerotia remained smaller in the presence of the herbicide compared to the untreated control.

Even though inhibitory effects of glyphosate on several plant diseases have been reported, some pathogens were unaffected and/or glyphosate increased disease severity of host plants. In some cases, glyphosate affected growth and reproduction of a given pathogen in vitro but showed an adverse effect in the field. Glyphosate inhibited, for instance, the development of Nectria galligena mycelium in vitro but increased the number of lesions when apple shoots were inoculated with a mycelium derived from a medium containing glyphosate (Burgiel & Grabowski, 1996).

Thus, even though glyphosate exhibit a negative effect towards distinct pathogens in some test systems, this herbicide may show other effects in vivo. In greenhouse studies using glyphosate-resistant sugar beet, increased disease severity was observed following glyphosate application and inoculation with Rhizoctonia solani and Fusarium oxysporum (Larson et al., 2006). This increase in disease was not fungal mediated, since there was no direct effect of glyphosate on both fungal species as tested in in vitro studies. Thus, the herbicide seems to reduce the plant’s ability to protect itself against pathogens.

Glyphosate was also shown to be phytotoxic to sugarcane and herbicide treatment resulted in increased disease severity caused by Pythium arrenomanes (Dissanayake et al. 1998). Furthermore, glyphosate application caused injury and death of Lolium multiflorum as a result of increased Pythium root rot (Kawate and Appleby, 1987). Even sublethal doses of glyphosate inhibited the expression of resistance in soybean to Phytophthora megasperma f. sp. glycinea (Keen et al., 1982), in bean to Colletotrichum lindemuthianum (Johal & Rahe, 1990), and in tomato to Fusarium spp. (Brammal & Higgins, 1988). Furthermore, glyphosate applied to the soil increases the disease symptoms caused by Cylindrocarpon sp. in grapevine (Whitelaw-Weckert, 2010).

Despite the fact that glyphosate may have a direct effect on a crop plant and the respective pathogens, repeated glyphosate use has also an impact on the microbial community composition. Repeated applications favour species belonging to the group of Proteobacteria in glyphosate-treated soils than occurring in untreated control soils (Lancaster et al., 2010) glyphosate mineralisation was reduced when glyphosate was applied several times. Gimsing et al. (2004) found that glyphosate mineralisation rates are positively correlated with Pseudomonas spp. population size. However, results of Lancaster et al. (2010) indicate that a repeated application of glyphosate is associated with an increase of those soil microorganisms capable of metabolising the herbicide. Altered microbial community may repress Pseudomonas species such as the beneficial species P. fluorescens and may modulate plant-pathogen interactions as well.

2.1.2 Leaf pathogens There are several cases of inhibitory effects of glyphosate on certain leaf diseases in various crops. Transgenically modified wheat with tolerance to glyphosate showed very low infection rates regarding leaf rust caused by Puccinia triticina and stem rust caused by P. graminis f.sp. tritici when treated with field doses one day prior to inoculation with the pathogen (Anderson & Kolmer, 2005). The leaf rust control by glyphosate decreased with reduced application rates and longer periods of time between herbicide application and rust inoculation indicating a direct toxic effect. However, control of leaf rust in wheat conditioned by glyphosate is effective for at least 21 days (Anderson & Kolmer, 2005), but how glyphosate inhibits rust infection was not investigated.

The herbicide may act as a systemic fungitoxic compound itself or may induce a systemic resistance, since also nontreated leaves were protected after herbicide application. In wheat straw, Sharma et al. (1989) reported an inhibition of Pyrenophora tritici-repentis pseudothecia production by glyphosate. Glyphosate has been shown to reduce sporulation, growth, and disease development caused by other cereal fungal pathogen such as Septoria nodorum on wheat (Harris & Grossbard, 1979), Rhizoctonia root rot (Wong et al., 1993), and take-all of wheat caused by Gaeumannomyces graminis, as well as Rhynchosporium secalis and Drechslera teres on barley (Toubia-Rahme et al., 1995; Turkington et al., 2001). Feng et al. (2005) showed by using glyphosate-resistant wheat and soybeans that rust infections and symptoms caused by Puccinia striiformis f.sp. tritici, Puccinia triticina, and Phakopsora pachyrhizi, respectively, can be suppressed when plants had been sprayed with formulated glyphosate.

The authors proposed that when rust spores became exposed to the herbicide, glyphosate was able to inhibit fungal EPSPS, thus, through the same mechanism described for its herbicidal activity. Their studies with glyphosate-resistant wheat revealed that rust control activity of glyphosate is not mediated through the induction of SAR (systemic acquired resistance) genes, but that glyphosate provided both preventative and curative activities in greenhouse experiments and in the field. However, rust control seemed to depend on the systemic glyphosate concentration in the host plant during germination of rust spores and the first infection events. Thus, rust spores just entering the plant in order to receive nutrients have to be exposed to a lethal concentration of glyphosate.

Furthermore, field data obtained from glyphosate-resistant soybeans suggest that rust control by glyphosate is influenced by environmental conditions, and rust races may differ in glyphosate sensitivity (Feng et al., 2008). Also species-specific differences in glyphosate sensitivity seem to exist, so that rust control in soybean requires higher doses than rust control in wheat (Feng et al., 2008). There are also intra-specific variations in R. solani as shown by Verma and McKenzie (1985). Since glyphosate is originally used as an herbicide to prevent growth of unwanted weeds, the use of fungi and bacteria as biological control agents was tested as an alternative to chemical herbicides or, much more interesting, in combination with herbicides.

In many cases, weed control and disease incidence were enhanced when the biocontrol agent was applied after glyphosate treatment (Boyette et al., 2006; Boyette et al., 2008a; Boyette et al. 2008b). The authors demonstrated that an application of glyphosate prior to Myrothecium verrucaria provided better weed control in kudzu (Pueraria lobata), redvine (Brunnichia ovata), and trumpetcreeper (Campis radicans). This was also the case for green foxtail, which was sufficiently controlled when treated with glyphosate prior to Pyricularia setariae inoculation (Peng & Byer, 2005). These results suggest that timing of glyphosate application in relation to combined treatment with a bioherbicide is important. Wyss et al. (2004) reported that certain pesticides and their adjuvants affected spore germination and growth of Phomopsis amaranthicola, an effective bioherbicides against Amaranthus species.

Several herbicides such as glyphosate had also negative effects on spore germination of P. setariae (Peng & Byer, 2005). Thus, one strategy to overcome direct toxic effects of herbicides is a sequential rather than simultaneous application of the synthetic herbicide and the bioherbicides. Applying glyphosate prior to pathogen application would allow the absorption, translocation, and the full action of the herbicide (with minimised degradation) and reduces its possible toxicity to the biocontrol agent.

Furthermore, glyphosate interactions with bioherbicides were found to be synergistic. Sharon et al. (1992) showed that glyphosate suppressed the plant’s defence by lowering phytoalexin production and biosynthesis of other phenolics. Even a sublethal dose of glyphosate suppressed the shikimate pathway in sicklepod (Cassia abtusifolia) infected with Alternaria cassiae, thus reducing the resistance of this weed (Sharon et al., 1992). Numerous examples in the literature have correlated production or transformation of preformed phenolic compounds and plant defence. In most cases, an activation of the enzyme phenylalanine ammonia-lyase (PAL) plays a pivotal role, and compounds that inhibit PAL activity have caused increased susceptibility to disease (Hoagland, 2000). This seems also to refer to crop plants such as soybean. Glyphosate was able to block resistance to Phytophthora megasperma, even in an incompatible interaction by lowering the glyceollin production, an important phytoalexin and part of the resistance machinery in soybean (Keen et al., 1982).

Page 86. Andreas Kortekamp (Ed.) – Herbicides and Environment (2011) InTech

This result has been attributed to the products serving as a source of nutrients. It is worth stressing, however, that some studies have shown that glyphosate is toxic to some organisms, such as certain strains of nitrogenfixing bacteria (NFB).

Page 108. Andreas Kortekamp (Ed.) – Herbicides and Environment (2011) InTech

Herbicides are used in agricultural areas to induce the senescence of plants. The duration of the senescence phase deriving from herbicide application is very short, and the intervention may occur at any developmental stage. Therefore, it is possible that the behavioral standards of N are different from those observed in natural senescence conditions. Besides inducing senescence, glyphosate and glufosinate ammonium herbicides also affect the plant’s N metabolism. The herbicidal action of glyphosate is attributed to its inhibition of the EPSPs (enolpyruvylshikimate phosphate synthase) enzyme that is responsible for one of the synthesis phases of the tryptophan amino acids phenylalanine and tyrosine. However, as a secondary effect, there is an inhibition in the synthesis of phenolic compounds deriving from those amino acids, which increases the activity of the PAL (phenylalamine ammonia lyase) enzyme due to a response effect (Duke & Hoagland, 1985). The PAL acts in the lyase of phenylalanine and tyrosine amino acids, resulting in the formation of phenolic acids and NH4+ (Hoagland et al., 1979).

chemoorganotrophic microorganisms may be favored by the application of some herbicides, and such is the case for glyphosate and glufosinate ammonium. Laboratory studies have demonstrated the increase of N2O emission in soils treated with glyphosate (Bollag & Henninger, 1976; Yeomans & Bremner, 1985; Carlisle & Trevors, 1986), which might be attributable to the herbicide serving as a nutrient source to organisms and offering a competitive advantage to denitrificants through the killing of chemolithotrophic microorganisms.

Page 116 Andreas Kortekamp (Ed.) – Herbicides and Environment (2011) InTech

Paper 3: Glyphosate is highly water soluble

GLYPHOSATE

Glyphosate is the primary tactical herbicide that is used when feasible. Characteristics (see also Table 8-1):

· Relatively nonselective, translocated herbicide with little or no pre-emergence impact (crops can be planted/seeded directly onto treated areas following application)

· Applied as spray; visible effects usually occur within 7 to 10 days; however, effects are delayed by cool or cloudy weather. Available as a 41% solution that is further diluted with water before use.

· Applied through boom equipment in area spray missions on a spray-to-wet method.

· Controls vegetation for 2 to 4 weeks.

· Rainfall within 6 hours of application may wash it away.

· Mechanical agitators may cause excessive foaming.

· Corrosive to iron and galvanized steel and should not be stored for long periods in unlined containers. Flush sprayer parts with water after use.

· Leaching is very low and has strong soil absorption, but is subject to microbiological decomposition.

· Half life normally is 60 days or less.

Page 60, US Army Chemical School. (1996). Flame, riot control agents and herbicide operations. Washington, D.C: Govern.

Paper 4: Glyphosate is not metabolized by animals

There have been six published glyphosate biomonitoring studies (Abdelghani 1995 ; Acquavella et al. 2004 ; Centre de Toxicologie du Quebec 1988 ; Cowell and Steinmetz 1990a, 1990b ; Jauhiainen et al. 1991 ). The authors of each study quantified glyphosate in urine. Urine is an ideal medium for quantifying systemic dose because glyphosate is not metabolized by mammals and is excreted essentially unchanged in urine with a short half – life (Williams G. M. , R. Kroes , and I. C. Munro . 2000 . Safety evaluation and risk assessment of the herbicide Roundup ® and its active ingredient, glyphosate, for humans . Regulatory Toxicology and Pharmacology 31 : 117 – 165).

The most extensive biomonitoring study is the Farm Family Exposure Study (FFES), conducted by investigators at the University of Minnesota with guidance offered by an advisory committee of recognized international experts in exposure assessment (Acquavella et al. 2004 ). The study monitored farm families. Urine samples were collected the day before glyphosate was to be applied, the day of application, and for 3 days after application. The detection method was capable of detecting 1 part per billion (ppb) glyphosate.

In the FFES, 48 farmers applied a Roundup branded herbicide and provided 24 – h urine samples the day before, the day of, and for 3 days after the application. Approximately 50% of the applications were on more than 40 ha and application rates were at least 1 kg ha−1. Overall, 40% of the farmers did not have detectable glyphosate in their urine on the day of application. Some farmers did have detectable glyphosate in their urine samples, and the urinary concentrations ranged from <1 to 233 ppb. The maximum systemic dose was estimated to be 0.004 mg kg−1.

Page 16, Glyphosate Resistance in Crops and Weeds: History, Development, and Management (2010)

Paper 5: Glyphosate acts as a weak uncoupler of oxidative phosphorylation

Glyphosate (Round-Up) [N-(phosphonomethyl) glycine] (see Figure 15.6) is a widely used herbicide that interferes with amino acid metabolism in plants. In animals it is thought to act as a weak uncoupler of oxidative phosphorylation. Glyphosate is moderately absorbed through the gastrointestinal tract, undergoes minimal biotransformation, and is excreted via the kidneys. There have been several reports in the literature of intoxications, typically resulting from accidental or suicidal ingestion, following overexposure to the glyphosate-containing product Round-Up. Various signs and symptoms include gastro-intestinal irritation and damage, as well as dysfunction in several organ systems (e.g., lung, liver, kidney, CNS, and cardiovascular system).

Page 358. Williams, P. L., & John Wiley & Sons, Inc. (2000). Principles of Toxicology (Second Edition): Environmental and Industrial Applications. Wiley-Interscience.

Paper 6: Glyphosate affects acetylcholinesterase and produces oxidative stress

Roundup® is a glyphosate-based herbicide containing a mixture of surfactants. This paper evaluates the toxic effects of Roundup® and its main constituents on the goldfish, Carassius auratus, after 7 days exposure. Fish were exposed to 0.16, 0.032 and 0.0064 mg/L of Roundup® [containing 41% isopropylamine salt of glyphosate (G.I.S) and 18% polyoxyethylene amine (POEA)], G.I.S, and POEA. Their livers were taken for determining reactive oxygen species (ROS), superoxide dismutase (SOD) activity, malondialdehye (MDA) content and acetylcholinesterase (AChE) activity.

Hydroxyl radical (·OH) could be induced by exposing Roundup® at a rate of 43%–111%, G.I.S at 90%–124% and POEA at142%–157%. A decreased SOD activity was observed in fish exposed to G.I.S and POEA. The contents of MDA significantly increased when exposed to Roundup® at all concentrations, 0.16 mg/L G.I.S and 0.032 mg/L POEA. The exposure led to an inhibition of AChE in livers overall during the experimental periods. POEA was more toxic than Roundup® or G.I.S during this experiment. AChE and ·OH are supposed to be sensitive biomarkers of the exposure of Roundup® and its main constituents to C. auratus.

Fan, J. Y., Geng, J. J., Ren, H. Q., Wang, X. R., & Han, C. (2013). Herbicide Roundup® and its main constituents cause oxidative stress and inhibit acetylcholinesterase in liver ofCarassius auratus. Journal of Environmental Science and Health, Part B, 48(10), 844–850. doi:10.1080/03601234.2013.795841

Paper 7: Glyphosate inhibits cytochrome 71 C6v1 in wheat

China, with a population of 1.3 billion and an ever – expanding agricultural product demand, is now probably the second largest producer of pesticides in the world, exporting almost $1.2 billion in agrochemical products in 2004. Of the top ten global active ingredients, it is noteworthy that seven are herbicides, dominated by glyphosate, far and away the best selling agrochemical, while the most recently introduced product was fenoxaprop in 1984

Page 30. Andrew H. Cobb, John P. H. Reade – Herbicides and Plant Physiology (2010) Wiley-Blackwell

In recent years the rate of adoption of transgenic crops has been particularly rapid, with the effect, for example, of displacing the soybean herbicide market to a reliance on one chemical alone, glyphosate. Indeed, it is estimated that about half of the 72 million acres of soybeans sown in the USA in 1999 were tolerant to glyphosate. Sales of glyphosate reportedly rose by 25% in 1998 and other companies have responded by cutting costs of their soybean herbicides to compete. In addition, as the patent for glyphosate, owned by Monsanto in the USA, expired in 2000, many other companies have developed their own formulations to compete with existing products. Further developments in the global glyphosate market are likely. Furthermore, in 1999 transgenic versions of all major crops for tolerance to herbicides were sown in 70 million acres (28.5 million hectares) in the USA. In 2006, 102 million hectares of land was used to grow GM crops in the world, over 50% in the USA. According to Evans ( 2010 ), at the end of February 2010, 14 million farmers in 25 countries planted 134 million ha of GM crops in 2009, of which 13 million farmers were in developing countries. Thus, the major agrochemical companies have recently expanded their life science businesses or acquired new seed companies to exploit these major technological developments, for example with herbicides.

Transgenic versions of monocotyledon crops, especially wheat and rice, are reportedly in development, although the technological challenges are greater. The next generation of transgenic crops will feature aspects of crop quality as well as crop protection, with a projected market value of US$5 billion in 2020 (Thayer, 1999). Many improved agronomic traits have been developed in the laboratory and only time will tell which ones are adopted commercially. The list is almost endless (Evans, 2010 ). Protection of crops from insects and diseases, in addition to tolerance to herbicides, is well under way, and major advances have been reported in our understanding of drought, cold and salt tolerance at the molecular level. Enhanced antioxidant, fibre and lignin production, altered amino acid and lipid content and elevated starch synthesis have all been achieved in various crops in recent years. Such traits, coupled with, for example, herbicide tolerance, are seen as ways of increasing agricultural productivity in an environmentally benign fashion.

Metabolic Attack

This phase of metabolism aims to introduce or reveal chemically active groups, such as – OH or – COOH, which can undergo further reactions. The most common way in which plants attack herbicides is by hydroxylation of aromatic rings or of alkyl groups by a family of enzymes known as the cytochrome P450 mono – or mixed function oxidases (P450s).

The P450s are a very large family of enzymes now thought to be the largest family of enzymatic proteins in higher plants. They all have a haem porphyrin ring containing iron at a catalytic centre. These enzymes are responsible for the oxygenation of hydrophobic molecules, including herbicides, to produce a more reactive and hydrophilic product. The reaction utilises electrons from NADPH to activate oxygen by an associated enzyme, cytochrome P450 reductase. One atom from molecular oxygen is incorporated into the substrate (R), while the other is reduced to form water:

The enzymes are located on the cytoplasmic side of the endoplasmic reticulum and are anchored by their N – terminus (Figure 4.2 ). They are found in all plant cells but in very low abundance. This, coupled with their lability in vitro, has meant that they are difficult to study biochemically. All P450s have a highly conserved region of 10 amino acids surrounding the haem group and it is this region that is responsible for the binding of O2, its activation and the transfer of protons to form water.

The rest of the P450 amino acid sequences are highly variable and this probably explains the wide variety of reactions and substrate specificity shown by this enzyme superfamily. Their name is derived from the maximum absorbance at 450 nm when the reduced enzyme is bound to the inhibitor carbon monoxide, and the ‘ P ’ signifies protein. This inhibition by carbon monoxide is typically overcome by light. These features are all used in setting criteria for the involvement of P450 enzymes in herbicide metabolism. These criteria are:

• requirement of O2,
• requirement for NADPH,
• association of enzyme activity with the microsomal fraction produced by centrifugation at 100,000 × g, enriched in the endoplasmic reticulum,
• inhibition by CO, which is reversible by light,
• inhibition by anti – reductase antibodies, and
• inhibition of in vitro activity by known P450 inhibitors, including aminobenzotriazole, paclobutrazole, piperonyl butoxide and tetcyclasis.

The P450 proteins are between 45 and 62 kDa in size. While their amino acid sequences may vary considerably, their three – dimensional structure is highly conserved, especially in the haem – binding region. The haem binds to the protein at a cysteine residue and the flanking sequence (Figure 4.3 ) is a characteristic of all P450s. The conserved oxygen – binding sequence is about 150 residues upstream from the haem and consists of Ala or Gly – Gly – X – Asp or Glu – Thr – Thr or Ser.

In both haem – and oxygen – binding sequences, X denotes any other amino acid. When these conserved sequences were used to study P450s in plants, a surprisingly large number and diversity of P450s was found. Indeed, more than 500 plant P450 genes are now known in over 50 families, indicating that the P450s are the largest group of plant proteins. The precise roles of the proteins encoded by these genes is, however, largely unknown.

A nomenclature has been designed for P450 genes based on the identity of the amino acid sequences of the proteins they encode (Figure 4.4). The genes have been numbered in chronological order depending on their date of submission to the P450 nomenclature committee ( http://drnelson.utmem.edu/CytochromeP450.html ).

Typical families are numbered from CYP71 to CYP99. For example, CYP71 C6v1 in wheat is inhibited by glyphosate and CYP76B1 catalyses the dealkylation of the phenylurea herbicides (Wen – Sheng et al., 2005 ). The discovery of new P450 genes continues in plants. In contrast, only about 50 P450 genes in 17 families have been described in humans. So why are there so many P450s in plants? The answer seems to be that they play a very wide role in plant secondary metabolism.

They have been shown to be involved in the biosynthesis and metabolism of a wide variety of compounds, including terpenes, flavonoids, sterols, hormones, lignins, suberin, alkaloids and phytoalexins. They are also induced by pathogen attack, xenobiotics and by light – induced stress, unfavourable osomotic conditions, wounding and infection.

It is currently believed that herbicide molecules also fit the active sites of these P450s involved in biosynthesis, suggesting a broad diversity of substrate selectivity. Regarding their roles in herbicide metabolism, much remains to be done to establish substrate specificity and both the molecular and the metabolic regulation of these important enzymes. Such understanding will be invaluable in predicting and elucidating herbicide selectivity, as well as in the discovery and design of new selective herbicides.

The main reactions catalysed by P450s are shown in Figure 4.5 .

In herbicide metabolism these are hydroxylation and dealkylation, which progresses via a hydroxylation step. Examples of herbicides metabolised by P450s in plant systems include sulfonylureas (including primisulfuron, nicosulfuron, prosulfuron, triasulfuron and chlorimuron), substituted ureas (chlorotoluron, linuron), chloroacetanilides (metolachlor, acetochlor), triazolopyrimidines (flumetsulam), aryloxyphenoxypropionates (diclofop), benzothiadiazoles (bentazon) and imidazolinones (imazethapyr).

Selectivity to herbicides can be due to ability of the crop to metabolise herbicides via P450s, an ability that may not be possessed by susceptible weeds. In some cases, however, this metabolism is not enough to prevent crop damage, either because of low rates of P450 metabolism or phytotoxicity of products produced by these reactions.

Crop damage may only be prevented if reactions from Phase II (conjugation) are successful in carrying out further detoxification. Some Phase I reactions may be catalysed by peroxidases (E.C. 1.11.1.7) which are commonly found in leaves at high concentrations, being able to catalyse oxidations using hydrogen peroxide.

They are currently thought to be involved in proline hydroxylation, indole acetic acid (IAA) oxidation and lignification, and have been implicated in the metabolism of aniline compounds produced in the degradation of phenylcarbamate, phenylurea and acylaniline herbicides

Page 74. Andrew H. Cobb, John P. H. Reade – Herbicides and Plant Physiology (2010) Wiley-Blackwell

Paper 9: Glyphosate inhibits cytochrome P450 enzymes in mammals

Glyphosate, the active ingredient in Roundup®, is the most popular herbicide used worldwide. The industry asserts it is minimally toxic to humans, but here we argue otherwise. Residues are found in the main foods of the Western diet, comprised primarily of sugar, corn, soy and wheat. Glyphosate’s inhibition of cytochrome P450 (CYP) enzymes is an overlooked component of its toxicity to mammals. CYP enzymes play crucial roles in biology, one of which is to detoxify xenobiotics.

Thus, glyphosate enhances the damaging effects of other food borne chemical residues and environmental toxins. Negative impact on the body is insidious and manifests slowly over time as inflammation damages cellular systems throughout the body. Here, we show how interference with CYP enzymes acts synergistically with disruption of the biosynthesis of aromatic amino acids by gut bacteria, as well as impairment in serum sulfate transport.

Consequences are most of the diseases and conditions associated with a Western diet, which include gastrointestinal disorders, obesity, diabetes, heart disease, depression, autism, infertility, cancer and Alzheimer’s disease. We explain the documented effects of glyphosate and its ability to induce disease, and we show that glyphosate is the “textbook example” of exogenous semiotic entropy: the disruption of homeostasis by environmental toxins

Samsel A, Seneff S. Glyphosate’s Suppression of Cytochrome P450 Enzymes and Amino Acid Biosynthesis by the Gut Microbiome: Pathways to Modern Diseases. Entropy. 2013; 15(4):1416-1463. https://doi.org/10.3390/e15041416

Paper 10: Glyphosate produces oxidative stress and affects acetyl choline function

This study had determined the effect of glyphosate-based herbicide (GBH) on acetylcholinesterase (AChE) enzyme activity, oxidative stress, and antioxidant status in Gammarus pulex. Firstly, the 96-h LC50 value of glyphosate on G. pulex was determined and calculated as 403 μg/L. Subsequently, the organisms were exposed to sub-lethal concentrations (10, 20, and 40 μg/L) of the determined GHB for 24 and 96 h.

The samples were taken from control and GBH-treated groups at 24 and 96 h of study and analysed to determine the malondialdehyde (MDA) and reduced glutathione (GSH) levels, the AChE, superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) enzyme activities. In the G. pulex exposed to GBH for 24 and 96 h, the MDA level increased significantly (p < 0.05).

The GSH level, the AChE, the CAT, and the GPx activities decreased compared with the control group (p < 0.05). G. pulex exposure to GBH for 24 h showed a temporary reduction in the SOD. GBH exposure led to oxidative stress in the G. pulex as well as affected the cholinergic system of the organism. These results indicated that the parameters measured may be important indicators of herbicide contamination in G. pulex.

Pala, A. (2019). The effect of a glyphosate-based herbicide on acetylcholinesterase (AChE) activity, oxidative stress, and antioxidant status in freshwater amphipod: Gammarus pulex (Crustacean). Environmental Science and Pollution Research. doi:10.1007/s11356-019-06804-5

Paper 11: Glyphosate inhibits acetylcholinesterase

The current work evaluated the inhibitory potency of the herbicide glyphosate (GLP) on acetylcholinesterase (AChE) activity in male and female rat tissues. The AChE activity in brain was higher (p<0.05) than those observed in kidney (females: 2.2-fold; males: 1.9-fold), liver (females: 6-fold; males: 6.9-fold) and plasma (females: 14.7-fold; males: 25.3-fold). Enzyme activities were higher in presence of 10mM GLP compared to those measured at an equimolar concentration of the potent AChE inhibitor dichlorvos (DDVP). Moreover, IC50s for GLP resulted between 6×10(4)- and 6.8×10(5)-fold higher than those observed for DDVP. In conclusion, GLP is a weak inhibitor of AChE in rats.

Larsen KE, Lifschitz AL, Lanusse CE, Virkel GL. The herbicide glyphosate is a weak inhibitor of acetylcholinesterase in rats. Environ Toxicol Pharmacol. 2016 Jul;45:41-4. doi: 10.1016/j.etap.2016.05.012. Epub 2016 May 18. PMID: 27258137.

Paper 12: Glyphosate decreases acetylcholinesterase (L. obtusidens)

In this study, teleostean fish Leporinus obtusidens (piava) were exposed to different concentrations of Roundup, a glyphosate (acid equivalent) herbicide: 0 (control), 3, 6, 10, and 20 mg/L for 96 h (short-term). Acetylcholinesterase (AChE) activity was verified in brain and muscle tissues. Metabolic parameters in the liver and muscle (lactate, glycogen, glucose, protein, and ammonia), as well as some hematological parameters, were determined. Unexposed fish exhibited significantly higher brain AChE activity when compared to that of the muscle () (13.8±0.76 and 6.1±1.31 μmol/min/g protein, respectively).

Results indicated that AChE activity significantly decreased in the brain of fish exposed to all glyphosate concentrations tested, but in the muscle this parameter was not altered. In addition, fish exposed to all glyphosate concentrations showed a significant increase in hepatic glycogen and glucose, but a significant reduction in muscle glycogen and glucose. Lactate and protein of fish exposed to all glyphosate concentrations presented a significant decrease in the liver, but did not change significantly in the muscle.

Levels of ammonia in both tissues increase in fish at all glyphosate concentrations. Exposure to this herbicide produced a decrease in all hematological parameters tested. These results indicate that AChE activity as well as metabolic and hematological parameters may be good early indicators of herbicide contamination in L. obtusidens.

Glusczak, L., dos Santos Miron, D., Crestani, M., Braga da Fonseca, M., Araújo Pedron, F. de, Duarte, M. F., & Vieira, V. L. P. (2006). Effect of glyphosate herbicide on acetylcholinesterase activity and metabolic and hematological parameters in piava (Leporinus obtusidens). Ecotoxicology and Environmental Safety, 65(2), 237–241. doi:10.1016/j.ecoenv.2005.07.017

Paper 13: Glyphosate inhibits acetylcholinesterase at low concentrations (C. decemmaculatus)

The toxic effect of sublethal concentrations (1, 17.5 and 35 mg L(-1)) of pure glyphosate was evaluated on acetylcholinesterase (AChE) activity in the fish species, Cnesterodon decemmaculatus. Acute bioassays (96 h) under laboratory conditions were conducted and homogenates for each specimen corresponding to the anterior, middle and posterior body sections were performed. Fish survival was 100%, even at the highest concentration tested (35 mg L(-1)), in accordance with the low lethal toxicity reported for glyphosate.

However, a significant inhibitory effect on AChE activity was recorded even for the lowest herbicide concentration tested (1 mg L(-1)), in the homogenates corresponding to the anterior body section. The inhibition ranged from 23 to 36%. The analytical determination of glyphosate in assay media by ion chromatography, was used to verify its stability. These results indicate that AChE-a neurotoxicity biomarker-in C. decemmaculatus may be affected by exposure to environmentally relevant concentrations of glyphosate.

Menéndez-Helman RJ, Ferreyroa GV, dos Santos Afonso M, Salibián A. Glyphosate as an acetylcholinesterase inhibitor in Cnesterodon decemmaculatus. Bull Environ Contam Toxicol. 2012 Jan;88(1):6-9. doi: 10.1007/s00128-011-0423-8. PMID: 22002176.

Paper 14: Glyphosate exposure affects memory and behaviour (mice)

Numerous clinical and epidemiological data have reported the deleterious effects of glyphosate on learning and memory. The ability of this herbicide to cross the blood-brain barrier may have adverse effects on the structure and various functions of the nervous system. This study was conducted to highlight the effects of Glyphosate-based herbicide (GBH) on these two functions in mice treated daily with 250 or 500 mg/kg following acute (unique administration), subchronic (6 weeks) and chronic (12 weeks) treatments.

The integrity of learning and memory was assessed by using a specific behavioral test battery: Novel object recognition, Y-maze and passive avoidance tasks. The acetylcholinesterase (AChE) and anti-oxidant enzyme activities, especially superoxide dismutase (SOD) and peroxidase (PO) were evaluated. Our results indicated that unlike acute treatment, both subchronic and chronic exposure to GBH decreased discrimination index and the step-through-latency indicating recognition and retention memory impairments, respectively. In contrast, only chronic exposure affected working memory manifested by decreased spontaneous alternation. Furthermore, our results showed also a prominent decrease in AChE, SOD and PO specific activities within the brain of treated mice following repeated exposures.

bali, Y. A., Kaikai, N., Ba-M’hamed, S., & Bennis, M. (2019). Learning and memory impairments associated to acetylcholinesterase inhibition and oxidative stress following glyphosate based-herbicide exposure in mice. Toxicology. doi:10.1016/j.tox.2019.01.010

Paper 15: Glyphosate implicated in multiple negative health outcomes (humans)

Glyphosate-based herbicides (GBHs) can disrupt the host microbiota and influence human health. In this study, we explored the potential effects of GBHs on urinary metabolites and their interactions with gut microbiome using a rodent model. Glyphosate and Roundup (equal molar for glyphosate) were administered at the USA glyphosate ADI guideline (1.75 mg/kg bw/day) to the dams and their pups. The urine metabolites were profiled using non-targeted liquid chromatography—high resolution mass spectrometry (LC-HRMS).

Our results found that overall urine metabolite profiles significantly differed between dams and pups and between female and male pups. Specifically, we identified a significant increase of homocysteine, a known risk factor of cardiovascular disease in both Roundup and glyphosate exposed pups, but in males only. Correlation network analysis between gut microbiome and urine metabolome pointed to Prevotella to be negatively correlated with the level of homocysteine.

Our study provides initial evidence that exposures to commonly used GBH, at a currently acceptable human exposure dose, is capable of modifying urine metabolites in both rat adults and pups. The link between Prevotella-homocysteine suggests the potential role of GBHs in modifying the susceptibility of homocysteine, which is a metabolite that has been dysregulated in related diseases like cardiovascular disease or inflammation, through commensal microbiome.

GBHs are the most applied herbicides worldwide and humans are commonly exposed to these environmental chemicals at various doses. Environmental GBHs are ubiquitous and GBHs residues can be found in food [40], drinking-water [41], crops [4]2, animal feed [43], groundwater [3], rain [44] and even in air [45]. Although the effects of GBHs on human health are under intense public debate, evidence is emerging that they impact many health outcomes, including developmental and reproductive toxicity [46,47,48], endocrine disruption [49,50], host immunity [51,52,53], obesity and diabetes [7,54], gastrointestinal disorders [55], cardiovascular disorders [56,57] and central nervous system dysfunction such as learning and memory impairment [58], anxiety, depression [59] and autism [8]. These chronic health outcomes may occur even at doses lower than established risk safety guidelines, in particular during critical development windows as denoted in the DOHaD paradigm [60]. Environmental exposures may lead to changes in metabolism [20,61].

Hu, J., Lesseur, C., Miao, Y. et al. Low-dose exposure of glyphosate-based herbicides disrupt the urine metabolome and its interaction with gut microbiota. Sci Rep 11, 3265 (2021). https://doi.org/10.1038/s41598-021-82552-2

Paper 16: Deaths due to Roundup involve unresponsive hypotension and pulmonary oedema

Between 1 January 1980, and 30 September 1989, 93 cases of exposure to herbicides containing glyphosphate and surfactant (‘Roundup’) were treated at Changhua Christian Hospital. The average amount of the 41% solution of glyphosate herbicide ingested by non-survivors was 184 +/- 70 ml (range 85-200 ml), but much larger amounts (500 ml) were reported to have been ingested by some patients and only resulted in mild to moderate symptomatology.

Accidental exposure was asymptomatic after dermal contact with spray (six cases), while mild oral discomfort occurred after accidental ingestion (13 cases). Intentional ingestion (80 cases) resulted in erosion of the gastrointestinal tract (66%), seen as sore throat (43%), dysphagia (31%), and gastrointestinal haemorrhage (8%).

Other organs were affected less often (non-specific leucocytosis 65%, lung 23%, liver 19%, cardiovascular 18%, kidney 14%, and CNS 12%). There were seven deaths, all of which occurred within hours of ingestion, two before the patient arrived at the hospital.

Deaths following ingestion of ‘Roundup’ alone were due to a syndrome that involved hypotension, unresponsive to intravenous fluids or vasopressor drugs, and sometimes pulmonary oedema, in the presence of normal central venous pressure.

Talbot AR, Shiaw MH, Huang JS, Yang SF, Goo TS, Wang SH, Chen CL, Sanford TR. Acute poisoning with a glyphosate-surfactant herbicide (‘Roundup’): a review of 93 cases. Hum Exp Toxicol. 1991 Jan;10(1):1-8. doi: 10.1177/096032719101000101. PMID: 1673618.

Paper 17: Glyphosate can cause non-alcoholic fatty liver disease and liver failure

Background: Glyphosate containing herbicides are widely used the world over. They are marketed as nontoxic to humans, but numerous studies have showed that these glyphosate-based herbicides (GlySH) can cause multiorgan damage.1 Recent reports of animal studies on rats have raised a doubt of liver damage after long term exposure to GlySH.

Case Presentation: a young male had chronic exposure to Glyphosate for 5 years in the form of spraying GlySH in farm and eating cereals sprayed with GlySH. He developed fulminant liver failure after accidental consumption of glyphosate containing herbicide. His liver function deteriorated in spite of supportive treatment. He developed hepatorenal syndrome later and died.

Discussion: Studies done on rats have showed that chronic consumption of extremely low levels of a GlySH formulation (Roundup), at admissible glyphosate-equivalent concentrations, is associated with marked alterations of the liver proteome and metabolome.2 It has been reported that chronic exposure to Glyphosate of more than 5 years’ duration due to consumption of food grains sprayed with this herbicide or inhalation of particles results in development of Fatty Liver, i.e., non-alcoholic fatty liver disease.

Any acute insult can result in decompensation and development of fulminant liver failure. Although this herbicide is relatively safe, other complications like Acute renal failure, Acute pulmonary edema with respiratory distress and shock can also occur. Conclusion: Chronic as well as acute exposure to GlySH can lead to NAFLD and fulminant liver failure.

As there is no antidote to glyphosate, clinicians must depend only on intensive supportive management which might not always be fruitful as in our case. It is important to be aware of systemic complications of this commonly used herbicide so that appropriate preventive measures can be taken.

Khot, R., Bhise, A., Joshi, R., & Ambade, N. (2018). Glyphosate Poisoning with Acute Fulminant Hepatic Failure. Asia Pacific Journal of Medical Toxicology, 7, 86-88.

Paper 18: Epidemiological studies link glyphosate with non-Hodgkin lymphoma

Glyphosate is the most widely used broad-spectrum systemic herbicide in the world. Recent evaluations of the carcinogenic potential of glyphosate-based herbicides (GBHs) by various regional, national, and international agencies have engendered controversy. We investigated whether there was an association between high cumulative exposures to GBHs and increased risk of non-Hodgkin lymphoma (NHL) in humans.

We conducted a new meta-analysis that includes the most recent update of the Agricultural Health Study (AHS) cohort published in 2018 along with five case-control studies. Using the highest exposure groups when available in each study, we report the overall meta-relative risk (meta-RR) of NHL in GBH-exposed individuals was increased by 41% (meta-RR = 1.41, 95% confidence interval, CI: 1.13-1.75).

For comparison, we also performed a secondary meta-analysis using high-exposure groups with the earlier AHS (2005), and we calculated a meta-RR for NHL of 1.45 (95% CI: 1.11-1.91), which was higher than the meta-RRs reported previously. Multiple sensitivity tests conducted to assess the validity of our findings did not reveal meaningful differences from our primary estimated meta-RR.

To contextualize our findings of an increased NHL risk in individuals with high GBH exposure, we reviewed publicly available animal and mechanistic studies related to lymphoma. We documented further support from studies of malignant lymphoma incidence in mice treated with pure glyphosate, as well as potential links between glyphosate / GBH exposure and immunosuppression, endocrine disruption, and genetic alterations that are commonly associated with NHL or lymphomagenesis.

Overall, in accordance with findings from experimental animal and mechanistic studies, our current meta-analysis of human epidemiological studies suggests a compelling link between exposures to GBHs and increased risk for NHL.

Glyphosate is a highly effective broad spectrum herbicide that is typically applied in mixtures known as glyphosate-based herbicides (GBHs) and commonly sold under the trade names of Roundup® and Ranger Pro®. Use of GBHs has increased dramatically worldwide in recent decades. In the United States alone, usage increased nearly sixteen-fold between 1992 and 2009 [1].

Most of this increase occurred after the introduction of genetically modified glyphosate-resistant “Roundup-ready” crops in 1996 [1]. In addition, there have been significant changes in usage. In particular, the practice of applying GBHs to crops shortly before harvest, so-called “green burndown,” began in the early 2000s to speed up their desiccation; as a consequence, crops are likely to have higher GBH residues [2]. By the mid-2000s, green burndown became widespread, and regulatory agencies responded by increasing the permissible residue levels for GBHs [3, 4].

1.2. Ubiquitous Exposure in Humans

Glyphosate and its metabolites persist in food [5–7], water [8], and dust [9], potentially indicating that everyone may be exposed ubiquitously. Non-occupational exposures occur primarily through consumption of contaminated food, but may also occur through contact with contaminated soil [9], dust [9] and by drinking or bathing in contaminated water [8]. In plants, glyphosate may be absorbed and transported to parts used for food; thus, it has been detected in fish [5], berries [6], vegetables, baby formula [7], and grains [10], and its use as a crop desiccant significantly increases residues. GBH residues in food persist long after initial treatment and are not lost during baking.

Limited data exist on internal glyphosate levels among GBH-exposed individuals [11]. Average urinary glyphosate levels among occupationally exposed subjects range from 0.26-73.5 μg/L, whereas levels in environmentally exposed subjects have been reported between 0.13-7.6 μg/L [11]. Two studies of secular trends have reported increasing proportions of individuals with glyphosate in their urine over time [12, 13]. Given that more than six billion kilograms of GBHs have been applied in the world in the last decade [2], glyphosate may be considered ubiquitous in the environment [14].

1.3. Controversy Surrounding the Carcinogenic Potential of GBHs

Exposure to GBHs is reportedly associated with several types of cancer, among which the most-well studied in humans is non-Hodgkin lymphoma (NHL). Some epidemiological studies have reported an increased risk of NHL in GBH-exposed individuals [15–17]; however, other studies have not confirmed this association [18, 19]. GBHs have recently undergone a number of regional, national, and international evaluations for carcinogenicity in humans [20–23], resulting in considerable controversy regarding glyphosate and GBHs’ overall carcinogenic potential.

Hence, addressing the question of whether or not GBHs are associated with NHL has become even more critical. Here, we evaluated the all the published human studies on the carcinogenicity of GBHs and present the first meta-analysis to include the most recently updated Agricultural Health Study (AHS) cohort [24]. We also discuss the lymphoma-related results from studies of glyphosate-exposed animals as well as mechanistic considerations to provide supporting evidence for our analysis of the studies of human exposures to GBHs.

Zhang L, Rana I, Shaffer RM, Taioli E, Sheppard L. Exposure to glyphosate-based herbicides and risk for non-Hodgkin lymphoma: A meta-analysis and supporting evidence. Mutat Res. 2019 Jul-Sep;781:186-206. doi: 10.1016/j.mrrev.2019.02.001. Epub 2019 Feb 10. PMID: 31342895; PMCID: PMC6706269.