Toxicity, Biodegradability and Detection Methods of Glyphosate; the Most Used Herbicide: A Systematic Review
 
Mohsen Ansari ^{1, 2}, Behnam Hatami ¹, Samaneh Sedighi Khavidak ^3*
 
1 Environmental Science and Technology Research Center, Department of Environmental Health Engineering, School of Public Health, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
² Student Research Committee, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
³ Department of Biology, Khorramabad Branch. Islamic Azad University, Khorramabad, Iran.
 

Citation: Ansari M, Hatami B, Sedighi Khavidak S. Toxicity, Biodegradability and Detection Methods of Glyphosate as the Most Used Herbicide: A Systematic Review. J Environ Health Sustain Dev. 2019; 4(2): 731-43.
 

Figure 1: The clinical features of glyphosate
 
Table 1: The newest toxicity profiles of glyphosate against different animals

 

 
Table 2: Microorganisms involved in the biodegradation of glyphosate Microbial  Species

Table 3: Biodegradation characteristics of glyphosate

Table 4: Analytical techniques applied for detection and quantitative estimation of glyphosate residues

NA: Not Available
 

Discussion
Toxicity of glyphosate against living organisms
human health effects
evidence for carcinogenicity
The evidences over carcinogenicity of glyphosate in humans were reported by several national and international agencies ⁴¹. The IARC’s study showed a limited evidence on carcinogenicity of glyphosate in humans ⁴². However, in some case-control studies, a positive evidence association was observed between occupational exposure to glyphosate and non-hodgkin lymphoma ^{43, 44}. Therefore, IARC interpreted all the evidences in order to justify the theory of glyphosate carcinogenicity ⁴⁵. The theory of glyphosate carcinogenicity was confirmed, although it has limited evidences in humans. In fact, IARC relied on the evidences of carcinogenicity in animals and strong mechanistic evidences of genotoxicity and oxidative stress as a reasonable reason to accept carcinogenicity in humans. In another study, the Environmental Protection Agency (EPA) conducted a systematic review study over the carcinogenicity of glyphosate based on the Agency’s own Cancer Guidelines and other related papers. It finally reported that glyphosate was not carcinogenic to humans ⁴⁶.
Human toxicity
The most glyphosate toxicity studies were conducted on patients who ingested the commercial product "Round-up" consisting of a mixture of glyphosate (as an isopropylamine salt) and a surfactant (polyoxyethyleneamine). The U.S. Environmental Protection Agency (USEPA) reported that the chronic Reference Dose (cRfD) of glyphosate was 1.75 mg of glyphosate in mg/kg/day ⁴⁷. However, several studies showed potential adverse health effects on humans. According to Figure 1, glyphosate is known as an endocrine disruptor ^{48, 49}. Considering this reason, the level of testosterone, 17β-estradiol, and total protein, as indexes of endocrine disreputability, significantly decreased (p ≤ 0.05) by exposure to glyphosate ⁵⁰. In addition, the concentrated solutions of glyphosate can also cause dermal irritation ⁵¹. Most human cases were intoxicated through ingestion, inhalation, and skin contact in the previous studies. Ingestion of glyphosate can result in acute kidney injury, electrolyte abnormalities, acidosis ⁵², and cardiovascular collapse ⁵³. Human lymphocytes, with or without metabolic activation, were proved by negative genotoxicity of glyphosate ⁵⁴. A number of modern diseases are associated with exposure to the glyphosate toxicity. Gluten sensitivity or intolerance, as a known Celiac disease, is a complex disorder affected by a variety of risk factors. Exposure to glyphosate can be considered as an environmental factor ⁵⁵. The mechanism of glyphosate toxicity appears intricate and complicated. In this regard, Zhan et al. reported that presence of surfactants in the glyphosate compound aggravated this complexity ⁵⁶. Weng et al. showed that the surfactant component of glyphosate was contributed by rhabdomyolysis (a serious syndrome can lead to serious complications such as renal (kidney failure) and compartment syndrome ⁵⁷. In addition, Sribanditmongkol et al. described that the toxic effects of the surfactant polyoxyethylene amine (POEA) and glyphosate were caused by their capability to erode tissues ⁵⁸. Although  glyphosate toxicity related to the central nervous system is still unknown, Malhotra et al. showed that its probability was a  reversible encephalopathy to the direct neuronal toxic effects of glyphosate ⁵⁹. The toxicokinetics properties of glyphosate are also complicated. Respiratory failure, metabolic acidosis, tachycardia, elevated creatinine level, and hyperkalemia are known as sings of the refractory cardiopulmonary failure ⁶⁰. Moon et al.’s pathological findings in glyphosate fatality indicated that the gastric mucosa of anterior fundus showed hemorrhage and the small intestines identified the bowel obstruction ⁶¹. The ingestion of glyphosate can result in acute kidney injury. Garlich et al. showed that hemodialysis should be considered because ingestion of glyphosate is associatedwith severe acidosis and acute kidney injury ⁶².The results of Mink et al.’s epidemiologic review showed no significant positive correlation and causal relation between exposure to glyphosate and diseases of non-cancer respiratory debases, diabetes, myocardial infarction, reproductive and developmental outcomes, rheumatoid arthritis, thyroid, and Parkinson's disease (PD) ⁶³. Chen, et al.’s surveillance study indicated that Paraquat (one of the most widely used herbicides) and glyphosate are known as mild caustic agents that can injure the oesophagus. Injuries of the oesophagus caused by glyphosate have only grades 1, 2a, and 2b. The glyphosate commercial formulation was more cytotoxic than the only active component; this condition implies that the additive plays the main role in the glyphosate toxicity when the additive was added to the glyphosate commercial formulation ⁶⁴. However, glyphosate induces some adverse formations in the structure of micronucleus and some risky modification in the chemical structure of DNA in a buccal epithelial cell line (TR146). In addition, glyphosate can alter some functional activity of human placental JEG3 cells⁶⁵. The glyphosate exposure and risk of lymph hematopoietic cancer (LHC) including non-Hodgkin lymphoma (NHL), Hodgkin lymphoma (HL), multiple myeloma (MM), and leukemia were assessed by Chang and Delzell ⁶⁶. They found that Meta-relative risks (meta-RRs) were statistically positive for the association between the contrast without facing and risk of NHL and MM. These associations were statistically null for HL, leukemia, and NHL subtypes except B-cell lymphoma. Sorahan conducted a re-analysis of US Agricultural Health Study (AHS) data to find the relation between multiple myeloma and glyphosate use. According to Sorahan’s study, positive association confirmed in some previous can be rejected due to their limited data. In this regard, their results showed no statistically significant trends for multiple myeloma risks in relation to application of the glyphosate ⁶⁷.
Animal toxicity studies
Rats and mice
Rats and mice are the most studied experimental animals in glyphosate toxicology. So far, many types of the immunological, biochemical, genetic, and histopathology examinations have been carried out on rats and mice. The results reported that exposure to glyphosate did not affect the uterine weight, but could modulate the expression of estrogen-sensitive genes ⁶⁸. The EPA screening assay results (in the Endocrine Disruptor Screening Program) for 52 pesticide chemicals showed no detectable evidence of disruption in the thyroid pathway. In contrary, de Souza et al.’s showed that by glyphosate exposure to thyroid-stimulating hormone (TSH), expression of genes associated with thyroid hormone was disrupted during the perinatal period in male rats. Oxidative stress is one of the most frequently used biochemical examinations in analysing the potential cytotoxicity of chemical compounds and refers to the difference between existence of free radicals and body ability in detoxifying the risky effects of free radicals ⁶⁹. In rats, the lipid peroxidation (LPO) level is considered as an oxidative stress response ⁷⁰. In rats, roundup is probably a better antioxidant disruptor than an active ingredient glyphosate. Hence, a typical response to stress and inflammation is increased by exposure to a sub-lethal concentration of glyphosate ⁷¹. Consequently, the antioxidant defence system is activated due to the increase in hydrogen peroxide generation. Therefore, the glyphosate-contained herbicides disrupts the normal biochemical function of liver and kidney ⁷². Enzyme assay measures either the consumption of substrate or by-product for the whole time of cell's life. Therefore, this assay can help to have a better understanding of the chemicals' toxic effects. The results of enzymatic activity in the pregnant rats and their foetuses who were exposed to glyphosate showed that maternal exposure to glyphosate during pregnancy caused some functional abnormalities. The abnormalities were observed in isocitrate dehydrogenase-NADP dependent glucose-6-phosphate dehydrogenase and malic dehydrogenase affected liver, heart, and brain of the pregnant rats and their foetuses ⁷³. In a study by Ait Bali et al., mice were subjected to behavioural and immunohistochemical tests to investigate their sub-chronic and chronic exposure to glyphosate. The results showed that unlike acute exposure, both sub-chronic and chronic exposure to glyphosate induced a decrease in body weight gain and locomotors activity, while they increased anxiety and depression-like behaviour levels. In addition, the immunohistochemically findings showed that the chronic treatment induced only a reduction of TH-immunoreactivity. However, both sub-chronic and chronic exposure reduced serotonin -immunoreactivity in the dorsal raphe nucleus, basolateral amygdala, and ventral medial prefrontal cortex ⁷⁴. Cattani et al. studied exposure to glyphosate herbicide and depressive-like behaviour by developed the glutamate excitotoxicity and oxidative stress tests in adult offspring. The results of Cattani et al.’s study showed that glyphosate exposure caused oxidative stress and affected cholinergic and glutamatergic neurotransmission in offspring hippocampus from immature and adult rats ¹⁴.
Rabbits
A few glyphosate toxicity studies were conducted on rabbits.  However, some results indicated that glyphosate toxicity had some effects on sperm quality. The adverse effects may be caused due to the cytotoxic effects of glyphosate on spermatogenesis directly and/or via hypothalami-pituitary-testis axis indirectly, which controls the reproductive efficiency ⁷⁵. Prenatal development of rabbits' cardiovascular status was affected when glyphosate posed a risk for cardiovascular malformations ⁷⁶.
Fishes
The glyphosate embryotoxicity was not well known as its oxidative stress effects. Therefore, Zebral et al. estimated the effect of exposure to glyphosate on the odontesthes humanises embryonic development. They found that exposure to concentration for 96 h (0.36-0.54 mg/L) reduced the eye diameter and the distance between eyes of odontesthes humanises. In addition, main result of Zebral et al.’s study indicated that exposure to glyphosate (0.54mg/L) caused high mortality rates of fish embryos ¹⁶. Sulukan et al. assessed body malformations during embryonic development on zebra fishes. Their results determined that glyphosate decreased CO₂ extraction and subsequently led to developing respiratory acidosis condition. In this condition, reactive oxygen species (ROS) level, as a carbonic anhydrase (CA) inhibitor increased. Finally, embryonic malformations were caused by ROS and inhibition of CA ¹⁸.
Other animals
McVey et al. conducted an exposure study to find the adult nervous system disorder in relation to Caenorhabditis elegans eggs exposed to glyphosate-containing herbicide. McVey et al.’s study showed that eggs from Caenorhabditis elegans exposed to glyphosate resulted in larva with abnormal neuronal cell bodies ⁷⁷.
Glyphosate biodegradation
Table 2 shows that bacterial species were used in most glyphosate degradation studies. These microbial species had the ability to grow on media containing glyphosate as carbon, nitrogen, or phosphorus sources. Furthermore, Table 3 indicates that the minimum value of pH for optimum glyphosate degrading is 5 and Penicillium oxalicum is responsible for it. Table 3 also indicates that the minimum and maximum required temperature for optimal glyphosate biodegradation were recorded at 19 and 60 ⁰C, respectively. In addition, the highest and lowest removal rates of glyphosate were 58.8 and 33.92 percent, respectively. The time required for maximum glyphosate removal was calculated between 0.62 and 32 days with a mean of 16.9 days. The strain could grow well in a wide range of pH (4 to 6.5) and the optimum growth was observed at pH range of 5-5.5. The results of kinetic investigation showed that the kinetic data of the glyphosate biodegradation process were characterized by the rate constants (k) of 0.0740, 0.0434, and 0.0946/day for strains GA07, GA09, and GC04, respectively ²¹. Nourouzi et al. observed that with increase in initial glyphosate concentration, the percentage of glyphosate degradation decreased from 100 to 98 percent and from 20 to 10 percent when the pH and initial inoculums size were constant, respectively. Nourouzi et al. suggested that the Haldane model was more suitable for prediction of the growth inhibition kinetic of glyphosate ²⁶. Bacteria and fungus microorganisms were studied in glyphosate biodegradation due to their ability to degrade glyphosate as carbon or phosphorus or nitrogen sources. Accessibility to carbon, phosphorous, and nitrogen (CNP) sources is a significant issue in determining the biodegradation capability of pesticides in the soils. Moreover, due to high requirement of nutrients, microorganisms have to adapt themselves to the alternative nutrient sources when certain nutrients are deficient in the medium ⁷⁸. In the biodegradation studies, glyphosate was added to the cultivation medium as carbon, phosphorous, or nitrogen sources by bacteria and fungi ⁷⁹. Arfarita et al. reported a continuous tense increase in the growth of utilizing bacterial species when glyphosate was used as a phosphorus source and glucose was applied as the carbon source ²⁴. Castro et al. reported the glyphosate biodegradation as a sole source of phosphorous by fungal strains. Castro et al.’s study elaborated that the filamentous fungi belonging to the Fusarium genre consumed glyphosate effectively as the source of phosphorous by increasing the biomass observed during the assay; even at a high concentration this compound supported the growth of Fusarium ³¹. Shushkova et al. added the glyphosate to MS1 medium, instead of NH₄Cl, as a source of nitrogen and phosphorus. Shushkova et al. provided a further support for the hypothesis that changing the type of phosphorus source in the inoculum medium affected both the growth of culture and the decrease of glyphosate concentration. Furthermore, the highest level of biomass production and the maximum amount of utilized glyphosate were observed when the glyphosate was used as a phosphorus source ²⁵. Moneke et al. explained the effect of adding glyphosate, as a carbon and phosphorous sources in sole or combined forms on the glyphosate biodegradability. Moneke et al.’s study showed that glyphosate degradation by Pseudomonas fluorescens was significantly more than Acetobacter sp., while glyphosate was used as carbon and phosphorus sources . The isolated Acetobacter sp., Azotobacter sp., and Alcaligenes sp. bacteria were grown on the salt medium containing glyphosate as a sole phosphorus source. However, Escherichia sp. did not have any noticeable growth on the medium ²⁹. Finally, biodegradation was categorized under the two terms of bio-mineralization and biotransformation. In the bio-mineralization process, the organic compounds are completely degraded and converted to an inorganic material such as water and carbon dioxide ⁸⁰. However, in biotransformation, a part of the organic compounds is degraded and the remaining is converted into other simple organic compounds ⁸¹. By reviewing related studies, it can be concluded that the glyphosate biodegradation is not classified in the bio-mineralization process.
Environmental detection of glyphosate
Depending on soil composition, glyphosate persists in soil for a long time (a few days to several months, or even one year) ⁸². However, the average of glyphosate’s half-life in water may differ from a few days to 91 days ⁸³. The mentioned half-life indicates that glyphosate has a good detection capability. Therefore, different methods were used to measure glyphosate in different environments. Table 4 represents analytical techniques applied for detection and quantitative estimation of glyphosate residues include derivatization with fluorenylmethyl chloroformate, stable isotope co-labeled 13C3 15N-glyphosate, direct detect, derivatization with fluorenylmethyl chloroformate (FMOC-Cl), vanadate-molybdate, derivatization reaction with acetic acid/TMOA, methamidophos assay, determination of turbidity, N -acylated derivatives.  Glyphosate detection in various media was performed through complex analytical procedures. As shown in Table 4, liquid chromatography-tandem mass spectrometry (LC-MS/MS), UPLC-MS i-Class system, high-performance liquid chromatography (HPLC), UV-visible spectroscopy (in 265 nm), UV-visible spectroscopy (in 880 nm), gas chromatography mass spectrometry, gas chromatography, UV-visible spectroscopy (in 660 nm), and Ion-exchange liquid chromatography are techniques coupled with the above methods. Table 4 highlights that the spectroscopy measures were the major instrumental methods for detecting residue glyphosate. Another detection method was the separation processes such as chromatography. Application of appropriate methods in pesticide detection can provide more precision for the analyser and saves time, cost, and energy to improve the performance of integrated pesticide management ⁸⁴. Sun et al. and Al-Rajab et al. used high-performance liquid chromatography to separate, identify, and quantify glyphosate and its product amino methyl phosphoric acid (AMPA) in a mixture form ^{35, 85}. Moreover, Zhao et al. detected the glyphosate in the culture liquid and soil by HPLC ²¹.  Nourouzi et al. and Zhao et al. used chromatography technique for detecting the glyphosate in soil environment ^{26, 86}. Further research is required to develop ultrasensitive, real-time, and robust detection methods to detect and measure glyphosate and its production.
Conclusion
Environmental pollution, bioaccumulation, bio-magnification, and hazard effects on living organisms are the inevitable results of glyphosate increased use. A crucial need exists to restrict the global use of herbicides and their impacts on the living organisms, water, and soil should be equally recognized. The expected glyphosate biodegradation depends on several key factors such as pH, temperature, and CNP values. The optimum condition of glyphosate biodegradation was obtained when pH was at 5 and temperature was from 19 to 60 ⁰C. Accessibility to carbon, phosphorous, and nitrogen (CNP) is considered as a life crucial factor for microorganisms' growing. Therefore, glyphosate surfactant herbicides, which contains CNP in its composition can be degraded easily by microorganisms. The field and concentration monitoring of glyphosate and its derivatives are facilitated mainly by high-performance liquid chromatography, UV-visible spectroscopy, gas chromatography/ mass spectrometry, and ion-exchange liquid chromatography techniques.
Acknowledgements
This work was not possible without the support of Environmental Science and Technology Research Center, Department of Environmental Health Engineering, Shahid Sadoughi University of Medical Sciences.
Conflict of Interest
Authors declare no conflict of interests.
Funding
Authors declare no financial and material support in this work.
 
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References