Toxicity, Biodegradability and Detection Methods of Glyphosate; the Most Used Herbicide: A Systematic Review

SYSTEMATIC REVIEW Introduction: Glyphosate is known as the most used world's herbicides and contradictions exist over its classification as a probably carcinogenic for the human. This study aimed to review the newest evidences in toxicity, biodegradability and detection methods of glyphosate. Materials and Methods: To conduct this systematic review, databases such as Scopus, Web of Science, PubMed, and Google Scholar were searched to extract studies on the non-target toxicity, biodegradability and detection methods of glyphosate from 2000 to 2018. The applied key words included glyphosate, herbicide, biodegradation, and bio decomposition. The number of articles retrieved and reviewed was 84 and 23, respectively. Results: Glyphosate could cause endocrine disrupting effects, dermal irritation, embryo toxicity, electrolyte abnormalities, apoptosis, cardiovascular collapse, teratogenicity, and mutagenic effects. High-performance liquid chromatography, UV-visible spectroscopy, gas chromatography/ mass spectrometry, and ion-exchange liquid chromatography were techniques used for detecting glyphosate in soil and water. The biodegradation of glyphosate was performed by various bacteria and fungi microorganisms. Conclusions: Given the high consumption and low rates of biodegradation of glyphosate, more attention should be paid to its toxicity potential in the human's environment and health. Article History: Received: 28 January 2019 Accepted: 20 April 2019


Introduction
Annually, two million tons of pesticides are consumed only in 10 top pesticide consuming countries in the world 1 . Worldwide, 40 percent of pesticides are used as herbicides. Glyphosate is known as the most concerned herbicide in some countries such as Iran. Glyphosate has the chemical name of Glyphosate; N-(phosphonomethyl) glycine; 1071-83-6; Glyphosphate; Pondmaster; Roundup enolpyruvylshikimate-3-phosphate (EPSP) synthase 4,5 , antifungal agents, 6 and as an uncoupling agent . In terms of health aspects, the U.S. Environmental Protection Agency (USEPA) classified glyphosate originally as a carcinogen (Class C, possible human carcinogen) because of the increased incidence of renal tumours in the studied mice. Furthermore, the International Agency for Research on Cancer (IARC) announced that glyphosate was a plausible agent for human cancer. From the perspective of environmental issues, continued extensive use of glyphosate components in agricultural activities is a cause of fundamental risks such as bioaccumulation, biomagnification, and toxicity for the environment 7,8 . These risks potentially effect soil biological cycles 9 , non-target organisms , subsequent pollution of groundwater 10,11 , runoff, 12 and the residues on agricultural crops .
Recently, researchers showed an increased interest in related issues such as toxicity, degradation, biodegradation, fate, pathways, and health risks of glyphosate herbicides. However, toxicity, biodegradability, and detection method of glyphosate has been discussed rarely.
The aim of this paper was to review the newest evidences in toxicity, biodegradability, and detection methods of glyphosate.

Materials and Methods
This review was conducted using key terms of toxicity, biodegradation, and detection methods of glyphosate. In this regard, databases including Scopus, Web of Science, PubMed, and Google Scholar along with related published books in this filed were investigated. Considering the study aims, more detailed references with technical explanation and clarification on glyphosate toxicity, biodegradability, and detection method in soil and water were included; whereas, the unrelated articles and references were excluded. Some of the excluded articles were on other aspects of glyphosate, such as its removal or were published before year 2000. In addition, the articles published in languages other than Persian or English were excluded. Finally, in the biodegradation section, 14 articles were included for further investigation. Figure 1 shows the clinical features of glyphosate mentioned in the literature. As Figure  1 represents, human health risks caused by glyphosate exposure ranged from skin irritation to death. On the other hand, in the absence of human data, studies on experimental animals can provide the most reliable tools for detecting the important toxic properties of chemical compounds and for estimating the risks for human and environmental health. In addition, the experimental animals that received glyphosate through a variety of ways were at the risks of glyphosate toxicity 13 . In the literature, many animal toxicity studies were conducted to survey the toxicity of glyphosate. The results of animal toxicity studies are summarized in Table 1. Previous glyphosate biodegradation studies identified different species of microbial. Table 2 represents the microbial species and their isolation environments. These microbial species are isolated based on the morphology differences from a various glyphosate-contaminated environment. Moreover, Table 3 illustrates some of the main characteristics of glyphosate biodegradation. So far, different methods have been applied to measure glyphosate in different environments. Table 4 shows the detection methods and techniques of glyphosate in several environments.

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 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 associated with 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 cells 65 . 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 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 hypothalamipituitary-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 CO 2 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 glyphosatecontaining herbicide. McVey et al.'s study showed that eggs from Caenorhabditis elegans exposed to glyphosate resulted in larva with abnormal neuronal cell bodies 77 . 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 0 C, 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  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 15Nglyphosate, 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 chromatographytandem 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 26,86 . Further research is required to develop ultrasensitive, realtime, and robust detection methods to detect and measure glyphosate and its production.

Conclusion
Environmental pollution, bioaccumulation, biomagnification, 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 0 C. 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.