Forty years with glyphosate
Department of Ecotoxicology and Environmental Analysis, Plant Protection Institute, Hungarian Academy of Sciences Hungary
6. Adverse environmental effects of glyphosate
6.1 Glyphosate and Fusarium species
Sanogo and co-workers (2000) observed that crop loss in soy due to infestation by Fusarium solani f. sp. glycines increased after glyphosate applications.
Kremer and co-workers (2005) described a stimulating effect of the root exsudate of GR soy sampled after glyphosate application on the growth of Fusarium sp. strains. Treatments caused concentration dependent increase on the mycelium mass of the fungus. Nonetheless, Powel and Swanton (2008) could not confirm these observations in their field study.
Kremer and Means (2009) claim that certain fungi utilise glyphosate released from plant roots into the soil as a nutritive, which facilitates their growth. Soil manganese content also affects the above consequence of glyphosate through chelating with the compound and thus, modifying its effects. Considering the fact that numerous plant pathogenic Fusarium species produce mycotoxins, an increasing proportion of these species is far not favourable as a side-effect.
Johal and Huber (2009) lists numbersome plant pathogens (e.g., Corynespora cassicola or Sclerotinia sclerotiorum on soy) they claim to grow increasingly after glyphosate treatments, and the list contains several Fusarium species (F. graminearum, F. oxysporum, F. solani). They hypothesize that glyphosate causes disturbances in microelement metabolism in plants, and in parallel, deteriorate the defense system of the plants, thereby increasing the virulence of certain plant pathogens. Zobiole and co-workers (2011) confirmed the above effects by their observation that glyphosate treatments facilitate colonisation of Fusarium species on the soy roots, but reduces the fluorescent Pseudomonas fraction of the rhizosphere, the level of manganese reducing bacteria and of the indoleacetic acid producing rhizobacteria. As a combined result of these effects, root and overall plant biomasses were found to be reduced.
6.2 Toxicity of glyphosate to aquatic ecosystems and amphibians
Substances occurring in surface waters deserve special attention by ecotoxicologists, as they enter a matrix that is the habitat of numerous aqueous organisms and the basis of our drinking water reserves. Drinking water is an irreplaceable essential part of our diet, and is a possible vehicle for chronic exposure (the basis of chronic diseases) in daily contact/consumption.
Glyphosate has been known to cause toxicity to microalgae and other aquatic microorganisms (Goldsborough and Brown 1988; Austin et al., 1991; Anton et al., 1993; Sáenz et al., 1997; DeLorenzo et al., 2001; Ma 2002; Ma et al., 2002; Ma et al., 2003), in fact a green algal toxicity test has been proposed for screening herbicide activity (Ma & Wang, 2002). In contrast, cyanobacteria have been found to show resistance against glyphosate (López-Rodas et al., 2007; Forlani et al., 2008). Tsui and Chu (2003) tested the effect of glyphosate, its most common polyoxyethyleneamine (POEA) type formulating materials, polyethoxylated tallowamines, and the formulated glyphosate preparation (Roundup) on model species from aquatic ecosystems, bacteria (Vibrio fischeri), microalgae (Selenastrum capricornutum, Skeletonema costatum), protozoas (Tetrahymena pyriformis, Euplotes vannus) and crustaceans (Ceriodaphnia dubia, Acartia tonsa). The most surprising result of the study was that the assumedly inert detergent formulating agent, POEA was found to be the most toxic component. In light of this it is far not surprising that Cox and Surgan (2006) and Reuben (2010) propounded the question, why tests only on the active ingredients are necessary to be specified in the documentation required by the Environmental Protection Agency of the Unites States (US EPA), when several of the used formulating components are known to exert biological activity.
Although acute toxicity and genotoxicity of glyphosate have been evidenced to certain fish (Langiano & Martinez, 2008; Cavalcante et al., 2008), glyphosate shows favourable acute toxicity parameters on most vertebrates, and therefore, has been classified as III toxicity category by US EPA. The European discretion is stricter, listing the compound among substances causing irritation (Xi) and severe ocular damage (R41). It has to be noted, however, that that model species of neither amphibians, not reptilians are represented in the toxicological documentations required nowadays. It may not be surprising, therefore, that after atrazine (Hayes et al., 2002; 2010), glyphosate is the second herbicide active ingredient that is questioned due to its detrimental effects on the animal class, considered the most endangered on Earth, amphibians.
Mann and Bidwell (1999) studied the toxicity of glyphosate on tadpoles of four Australian frogs (Crinia insignifera, Heleioporus eyrei, Limnodynastes dorsalis and Litoria moorei). The toxicity of Roundup and its 48-hour LC50 values were found to be 3-12 mg glyphosate equivalent/l. Tolerance of the adult frogs was substantially greater. A glyphosate-based formulated herbicide preparation (VisionMAX) caused no significant effects on the juvenile adults of the green frogs (Lithobates clamitans) when applied at field application doses, only marginal differences in statistics of infection rates and liver somatic indices in relation to exposure estimates (Edge et al., 2011). Chen et al. (2004) observed that the toxicity of glyphosate on the frog species Rana pipiens was greatly affected by lacking food resources and the pH of the medium as stress factors. Relyea (2005a) reported tadpole (Bufo americanus, Hyla versicolor, Rana sylvatica, R. pipiens, R. clamitans and R. catesbeiana) mortality related to glyphosate applications. The effect, occurred at 2-16 mg glyphosate equivalent/l concentrations, was linked with the stress caused by the predator of the tadpoles, salamander Notophthalmus viridescens. Later Relyea and Jones (2009) included further frog species (Bufo boreas, Pseudacris crucifer, Rana cascadea, R. sylvatica) into the study, and found LC50 values to be 0.8-2 mg glyphosate equivalent/l. Testing four salamander species (Amblystoma gracile, A. laterale, A. maculatum and N. viridescens), the corresponding values ranged between 2.7 and 3.2 mg glyphosate equivalent/l. In this case, glyphosate was formulated with detergent POEA. Further studies also shed light on the fact that another stress factor, population density, playing an important part in the competition of the tadpoles increased the toxic effect of glyphosate (Jones et al., 2010). Lajmanovich and coworkers (2010) detected lowered enzymatic activities (e.g., acetylcholine esterase and glutathion-S-transferase) in a frog species, Rhinella arenarum upon glyphosate treatments.
Sparling and co-workers (2006) detected lowered fecundity of the eggs of the semiaquatic turtle, red-eared slider (Trachemys scripta elegans) if treated with glyphosate at high doses.
6.3 Teratogenic activity of glyphosate
The teratogenicity of the pesticide preparations containing glyphosate deserves special attention. The very first examples of observed teratogenicity of glyphosate preparations have also been linked to amphibians. Using the so-called FETAX assay, Perkins and coworkers (2000) observed a formulation dependent teratogenic effect of glyphosate on embryos of the frog species Xenopus laevis. The concentrations that triggered the effect were relatively high (the highest dose applied in the study was 2.88 mg glyphosate equivalent/l), but not irrealisticly high with respect to field doses of glyphosate, indicating, that high allowed agricultural doses cause glyphosate levels close to the safety margin. Lajmanovich and co-workers (2005) studied the effects of a glyphosate preparation (Glyfos) on the tadpoles of Scinax nasicus, and found that a 2-4-day exposure to 3 mg/l glyphosate caused malformation in more than half of the test animals. The treatment was carried out nearly at the LC50 level of glyphosate. Dallegrave and co-workers (2003) found fetotoxic effects on rats treated with glyphosate at very high, 1000 mg/l concentration on the 6th-15th day after fertilisation. Nearly half of the newborn rat progeny in the experiments were born with skeletal development disorders.
Testing the effects of glyphosate preparations on the embryos of the sea urchin, Sphaerechinus granularis, Marc and co-workers (2004a) observed a collapse of cell cycle control. Inhibition affects DNA synthesis in the G2/M phase of the first cell cycle (Marc et al., 2004b). The authors estimate that glyphosate production workers inhale 500-5000-fold level of the effective concentration in these experiments. A marked toxicity of the formulating agent POEA has also been observed on sea urchins (Marc et al., 2005). The very early DNA damage was claimed to be related to tumour formation by Bellé and co-workers (2007), and the authors consider the sea urchin biotest they developed as a possible experimental model for testing this effect. Jayawardena and co-workers (2010) described nearly 60% developmental disorders on the tadpoles of a Sri Lanka frog (Polpedates cruciger) upon treatment with 1 ppm glyphosate.
The teratogenicity of herbicides of glyphosate as active ingredient have been tested lately on amphibian (X. laevis) and bird (Gallus domesticus) embryos. Applied with direct injection at sublethal doses caused modification of the position and pattern of rhobomeres, the area of the neural crest decreased, the anterior-posterior axis shortened and the occurrence of cephalic markers was inhibited at the embryonic development stage of the nervous system. As a result, frog embryos became of characteristic phenotype: the trunk is shortened, head size is reduced, eyes were improperly or not developed (microphthalmia), and additional cranial deformities occurred in later development. Similar teratogenic effects were seen on embryos of Amniotes e.g., chicken. These developmental disorders may be related to damages of the retinoic acid signal pathway, resulting in the inhibition of the expression of certain essential genes (shh, slug, otx2). These genes play crucial roles in the neurulation process of embryogenesis (Paganelli et al., 2010). These findings were later debated by several comments. On behalf of the producers, Saltmiras and co-workers (2011) questioned certain conclusions in the work of Paganelli and co-workers (2010), claiming that the standardised pilot teratogenicity tests, carried out under good laboratory practice (GLP) by the manufacturers, have been evaluated by independent experts of several international organisations. They also considered the dosages used by Paganelli and co-workers exceedingly high, and the mode of application (microinjection) irrealistic in nature. Similar criticism has been voiced by Mulet (2011) and Palma (2011). In his answer, Carrasco (2011) emphasised their opinion that the company representatives ignore scientific facts supporting teratogenicity of atrazine, glyphosate and triadimefon through retinoic acid biosynthesis. He also emphasized that of 180 research reports of Monsanto, 150 are not public, or have never been presented to the scientific community. He also included that they obtained similar phenotypes in their studies with microinjection, than by incubation of the preparations. As a follow-up, Antoniou and co-workers (2011) compiled an extensive review of 359 studies and publications on the teratogenicity and birth defects caused by glyphosate, and heavily criticize the European Union for not banning glyphosate, but rather postponing its re-evaluation until 2015 European Commission, 2010).
6.4 Genotoxicity of glyphosate
Occupational exposure to pesticides, including glyphosate as active ingredient, may lead to pregnancy problems even through exposure of men (Savitz et al., 1997). Such phenomenon has been first described in epidemiology with Vietnam War veterans exposed to Agent Orange with phenoxyacetic acid type active ingredients contaminated with dibenzodioxins. Although glyphosate has been claimed not to be genotoxic and its formulation Roundup “causing only a week effect” (Rank et al., 1993; Bolognesi et al., 1997), Kale and co-workers (1995) observed mutagenic effects of Roundup in Drosophila melanogaster recessive lethal mutation tests. Lioi and co-workers (1998) described increasing sister chromatide exchange in human lymphocytes with increasing glyphosate doses. Walsh and co-workers (2000) detected in murine tumour cells the inhibitory activity of Roundup on the biosynthesis of a protein (StAR) participating in the synthesis of sex steroids. This reduced the operation of the cholesterol – pregnenolon – progesteron transformation pathway to a minimal level. As it often happens in exploring mutagenic effects of chemical substances, additional studies have not found glyphosate mutagenic, and therefore, it is not so listed in the GAP2000 program compiled from US EPA/IARC databases. However, Cox (2004) describes chronic toxicity profile of several substances applied in the formulation of glyphosate.
Studying the activity of dehydrogenase enzymes in the liver, heart and brain of pregnant rats, Daruich and co-workers (2001) concluded that glyphosate causes various disorders both in the parent female and in the progeny. According to results of the study by Benedettia and co-workers (2004), aminotransferase enzyme activity decreased in the liver of rats, impairing lymphocytes, and leading to liver tissue damages. In in vitro tests McComb and co-workers (2008) found that glyphosate acts in the mitochondria of the rat liver cells as an oxidative phosphorylation decoupling agent. Mariana and co-workers (2009) observed oxidative stress status decay in the blood, liver and testicles upon injection administration of glyphosate, possibly linked to reproductional toxicity.Prasad and co-workers (2009) detected cytotoxic effects, as well as chromosomal disorders and micronucleus formation in murine bone-marrow. Poletta and co-workers (2009) described genotoxic effects of Roundup on the erythrocytes in the blood of caimans, correlated with DNA damages.
According to the survey of De Roos and co-workers (2003), the risk of the incidence of non- Hodgkin lymphoma is increased among pesticide users. As the authors found it, this applies to herbicide preparations with glyphosate as active ingredient. Focusing the study solely on glyphosate preparations a year later in the corn belt of the United States, of the majority of malignant diseases, only the incidence of abnormal plasma cell proliferation (myeloma multiplex, plasmocytoma) showed a slight rise (De Roos et al., 2004). Myeloma represents approximately 10% of the malignant haematological disorders. Although the cause of the disease is not yet known, its risk factors include autoimmune diseases, certain viruses (HIV and Herpes), and the frequent use of certain solvents as occupational hazard. On the basis of murine skin carcinogenesis, George and co-workers (2010) reported that glyphosate may act as a skin tumour promoter due to the induction of several special proteins.
6.5 Hormone modulant effects of glyphosate and POEA
Studying chronic exposure of tadpoles of Rana pipiens, Howe and co-workers (2004) found that in addition to developmental disorders, gonads in 15-20% of the treated animals developed erroneously, and these animals showed intersexual characteristics. Arbuckle and co-workers (2001) registered increased risk of abortion in agricultural farms after glyphosate applications. In addition, excretion of glyphosate has been determined in the urine of agricultural workers and their family members (Acquavella et al., 2004). Richard and co-workers (2005) evidenced toxicity of glyphosate on the JEG3 cells in the placenta. Formulated Roundup exerted stronger effect than glyphosate itself. Glyphosate inhibited aromatase enzymes of key importance in estrogen biosynthesis. This effect has also been evidenced in in vitro tests by binding to the active site of the purified enzyme. The formulating agent in the preparation enhanced the inhibitory effect in the microsomal fraction. Benachour and co-workers (2007) tested the effect of glyphosate and Roundup Bioforce on various cell lines, and also determined the aromatase inhibiting effect of glyphosate and the synergistic effect of the formulating agent. They suppose that the hormone modulant effect of Roundup may affect human reproduction and fetal development. Testing these human cell lines, Benachour and Séralini (2009) found that glyphosate alone induces apoptosis, and POEA and AMPA applied in combination exert synergistic effects, similarly to the synergy seen for Roundup. The synergy was reported to be further acerbated with activated Cry1Ab toxin related to that produced by insect resistant GM plants, raising concern regarding stacked genetic event GM crops exerting both glyphosate tolerance and Cry1Ab based insect resistance (Mesnage et al., 2011). Moreover, the combined effect caused cell necrosis as well. Effect enhancement is likely to be explained by the detergent activity of POEA facilitating the penetration of glyphosate through cell membranes and subsequent accumulation in the cells. The aromatase inhibitory effect of the formulated preparation was four-fold, as compared to the neat active ingredient. The authors consider it proven, that POEA, previously believed to be inert, is far not inactive biologically. As the authorised MRL of glyphosate in forage is as high as 400 mg/kg, Gasnier and co-workers (2009) studied in various in vitro tests, what effects this may cause in a human hepatic cell line. All treatments indicated a concentration-dependent effect in the toxicity tests were found genotoxic in the comet assay for DNA damages, moreover, displayed antiestrogenic and antiandrogenic effects.
6.6 Glyphosate resistance of weeds
Frequent applications of glyphosate and the spread of GT crops outside of Europe escalate the occurrence of glyphosate in the environment, exerting severe selection pressure on the weed species. It has been well known that certain weeds have native resistance against glyphosate e.g., the common lambsquarters (Chenopodium album), the velvetleaf (Abutilon theophrasti) and the common cocklebur (Xanthium strumarium).
The first population of GT Lolium rigidum was described in 1996 by Pratley and co-workers in Australia. This was followed in 1997 by GT goosegrass (Eleusine indica) in Malaysia (Lee & Ngim, 2000), GT horseweed (Conyza canadensis) in the United States (VanGessel, 2001), GT Italian ryegrass (Lolium multiflorum) in Chile (Perez & Kogan, 2003). Further known GT weed species include Echinochloa colona (2007), Urochloa panicoides (2008) and Chloris truncata (2010) in Australia; Conyza bonariensis (2003) and ribwort plantain (Plantago lanceolata, 2003) in South Africa; ragweed (Ambrosia artemisifolia, 2004), Ambrosia trifida (2004), Amaranthus palmeri (2005), Amaranthus tuberculatus (2005), summer cypress (Bassia scoparia, 2007) and annual meadow grass (Poa annua, 2010) in the United States; Conyza sumatrensis (2009) in Spain; Johnsongrass (Sorghum halepense) (2005), Italian ryegrass (Lolium perene, 2008) in Argentina; Euphorbia heterophyla (2006) in Brazil; Parthenium hysterophorus (2004) in Colombia and Digitaria insularis (2006) in Paraguay (Heap, Epubl). GT Johnsongrass was reported in a continuous soybean field in Arkansas, United States (Riar et al., 2011). Price (2011) claims that agricultural conservation tillage is threatened in the United States by the rapid spread of GT Palmer amaranth (Amaranthus palmeri [S.] Wats.) due to wide range cultivation of transgenic, GT cultivars and corresponding broad use of glyphosate. GT amaranths were first identified in Georgia, and later reported in nine states, Alabama, Arkansas, Florida, Georgia, Louisiana, Mississippi, North Carolina, South Carolina, and Tennessee, and a closely related GT amaranth, common waterhemp (Amaranthus rudis Sauer) in four states, Illinois, Iowa, Minnesota, and Missouri. Moreover, GT Italian ryegrass populations collected in Oregon, United States appeared to show cross-resistance to another phosphonic acid type herbicide active ingredient, glufosinate (Avila-Garcia & Mallory-Smith, 2011).
Powles and co-workers (1998) described a L. rigidum population resisting 7-11-fold dosage of glyphosate in Australia. Shrestha and Hemree (2007) found GT subpopulations of 5-8 leaf stage Conyza canadensis surviving only 2-4-fold glyphosate doses. According to Powles (2008), it is not coincidental that in countries, where GT crops are on the rise (Argentina and Brazil), the occurrence of GT weeds is more frequent. Moreover, he considers this one of the main obstacles of the spread of GT crops in the agricultural practice. Glyphosate tolerance is an inherited property, therefore, accumulation of weeds in the treated areas is to be expected. Genomics studies of the GT populations revealed that mutation of the gene (epsps) encoding the target enzyme responsible for tolerance is not infrequent in nature. (The mutant alleles (mepsps, 2mepsps) responsible for tolerance has been found in maize as well, see Table 2.). Reduced or modified uptake or translocation of glyphosate has also been observed, and the metabolic fate of the compound may also become altered in the cell (Shaner, 2009), possibly resulting in GT populations. It is not difficult to predict, that prolonged cultivation of GT crops will necessitate supplemental herbicide administrations with active ingredients other than glyphosate.