a Department of Pharmacology, Instituto de Ciências Básicas da Sáude, Universidade Federal do Rio Grande do Sul, Porto Alegre/RS, Brazil. ^bDepartment of Pharmacology, Instituto de Ciências Biológicas, Centro Politécnico, Universidade Federal do Paraná, Curitiba/PR, Brazil. Address for correspondence:Eliane Dallegrave, Mestre em Ciências Veterinárias, Departamento de Farmacologia, Instituto de Ciências Básicas da Saúde,
Universidade Federal do Rio Grande do Sul Rua Sarmento Leite / 500, sala 202, 90046-900 - Porto Alegre/RS. Brasil. Email: elianed@vortex.ufrgs.br

Dallegrave E, Mantese FD, Dalsenter PR, Langeloh A: Oral acute toxicity of glyphosate in Wistar rats, Online Journal of Veterinary Research 1:29-34, 2002 The acute toxicity of glyphosate (N-phosphonomethylglycine with the surfactant polyoxyethyleneamine:RoundupÒ) was determined 2, 4, 6, 8, 10, 12 and 24 hours after the oral administration of 1, 2, 3 or 4g/kg to Wistar rats. Each animal was observed for immediate toxicity up to 12h and for death up to 24h. Signs of toxicity included salivation, diarrhea, gasping, blood-stained nose discharges and seizures. Post mortem examination showed lung hyperemia and liver congestion. The oral acute LD50was found to be 31g/kg (95%confidence limits = 1.81 – 2.95) in Wistar rats, a value 54% lower than that reported in the literature (> 5g/kg).

KEYWORDS: Glyphosate; N-phosphonomethylglycine; polyoxyethyleneamine; DL₅₀; rats; Acute toxicity; RoundupÒ .

The herbicide Roundup® is a water-based product which contains glyphosate (N-phosphonomethylglycine) as the active ingredient and polyoxyethyleneamine (POEA) as the surfactant agent (Menkes et al 1991). Glyphosate is marketed as a non-selective, broad spectrum, post-emergence herbicide, used to control weeds in emerged grasses, broad-leaf weeds, pastures and cultures as rice, corn and soy (Smith and Oehme 1992). Numerous acute toxicity studies have been performed to determine LD50 values of glyphosate and herbicide formulations containing this active ingredient (WHO 1994). The oral acute LD50 in rats of the glyphosate and/or Roundup® formulation used in United States has been reported to be greater than 5g/kg (WHO 1994). This value suggests that glyphosate and its formulations have minor toxicity by oral administration (WHO 1994). However, the formulation of Roundup®varies in different countries. The commercial product marketed in Brazil contains glyphosate 36% and polyoxyethyleneamine 18%. Teratogenicity studies [segment II (EPA 1996)] previously developed in our laboratory using this Roundup® formulation administered to pregnant Wistar rats, showed that 50% of the dams treated orally with 1g/kg/day died between treatment days 2 and 9(unpublished data). The aim of the present study was to determine the acute LD50 of glyphosate-Roundup® as marketed in Brazil administered orally to Wistar rats.

Adult male and female Wistar rats bred at our own Department were used, in accordance with the rules of the Ethics and Experimental Animals Committee. All animals were individually housed in polyethylene cages, with sawdust bedding. Animals were maintained in a colony room with temperature of 22 ± 2° C, controlled humidity and 12h-light/dark cycle. Adult rats (90 days old, 200-280g), were fasted overnight, and provided free access to water. Five groups of 14 animals (7 males and 7 females) were treated orally (1.1ml/100gbody weight) with water or 1, 2, 3 or 4g/kg of glyphosate. The various solutions of the Roundup® formulation (lot: BS 1096/98 – 36% glyphosate and 18% w/v polyoxyethyleneamine) were prepared by addition of appropriate volumes of distilled water.

Each animal was observed for one minute at 0, 2, 4, 6, 8, 10 and 12h after dosing, for the presence of respiratory, digestive and neurological alterations. Moreover number of deaths at 24h were noted. Animals that died within 12h of administration were immediately necropsied, and analyzed for macroscopical alterations in lungs, liver and kidneys.

Data are shown as cumulative frequency of death and/or toxicity signs. The LD50, 95% confidence limits and correlation index were calculated by the Litchfield and Wilcoxon method (computer software: LETAL.EXE, US. 1.3. FKS, 1991). Statistical analysis for toxic effects (salivation, diarrhea, gasping, blood-stained nose discharges and seizures) was done by chi-square test. Statistical significance was set at P < 0.05 (Zar 1999).

Salivation, diarrhea, blood-stained nose discharges and seizures were the signs of toxicity observed only in glyphosate treated groups (Table 1).

Table 1. Toxicity signs following oral administration of glyphosate-RoundupÒ or distillilled water to Wistar rats
 

Values indicate the number of animals with the toxic effect/total number of animals remaining alive. N= 14. * Significantly different from control group (P < 0.05) by chi-square test.

Table 1 shows that in controls, two rats exhibited gasping after oral administration and other controls did not exhibit any abnormality. In rats treated with 1g/kg glyphosate, gasping was observed at 2h in 21.4% followed by diarrhea in 64.3% of the animals. Diarrhea was most frequent in this group (P = 0.003). Blood-stained nose discharges occurred in 14.3% of the animals.

Immediately after 2g/kg dose administrations, salivation occurred in 21.4% and gasping in 35.7% increasing to 50% gasping at 4, 10 and 12h (P < 0.001). Diarrhea occurred in 30.8%, reaching 60.0% at 8h (P = 0.004). Blood-stained nasal discharges and seizures were observed in 7.7% reaching 20% by 4h.

Rats given 3g/kg, 35.7% exhibited salivation and gasping immediately after oral administration (P = 0.03). Diarrhea appeared in 23.1% 2h, reaching 33.3% by 8h (P = 0.03). Rats treated with 4g/kg glyphosate exhibited salivation (70%) and gasping (50%) immediately (P = 0.002 and P = 0.008, respectively and also exhibited blood-stained nose discharges (11.1%) and seizures (33.3%) (P = 0.03).

Death occurred in all glyphosate dose-groups 30min to 24h (Table 2).
 

Post mortem examination showed lung hyperemia and liver congestion in all animals treated with glyphosate. The oral acute LD50 calculated for glyphosate (Roundup®) was 2.31g/kg, (95% confidence limits: 1.81 – 2.95) and the correlation index was 0.948.

The results showed that the oral acute toxicity of glyphosate (Roundupâ formulation commercialized in Brazil) was 54% lower than those previously reported by others (WHO 1994; referring to unpublished technical reports from Bio/Dynamics Inc., 1988 and NOTOX 1987; LD50 oral acute being > 5g/kg). Toxic signs were salivation, diarrhea, gasping, blood-stained nose discharges and seizures. Salivation and gasping appeared initially followed by diarrhea, blood-stained nose discharges and seizures in some rats, with increasing frequency with higher doses of glyphosate.

According to Bababunmi et al (1978) and Olorunsogo and Bababunmi (1980), the administration of glyphosate by oral or intraperitoneal route caused severe stress, increased respiration, elevated rectal temperatures, and occasional asphyxia and seizures in mice and rats. Lung hyperemia was a common lesion observed in the glyphosate groups. The primary mechanism of the acute glyphosate toxicity was likely to be an uncoupling of oxidative phosphorylation in rat mitochondria. For the same researchers (Bababunmi et al 1978), the median lethal dose of glyphosate was 4.704g/kg in rats, 49% higher than that found in this study.

Acute toxicity studies vary considerably between laboratories, rats strain, age, gender, however, other factors are to be considered such as the commercial formulation with different concentrations of surfactant. Adam et al (1997) determined the oral and intratracheal toxicity of Roundupâ and its components to rats. The oral administration of polyoxiethyleneamine-POEA (1g/kg), mixture of glyphosate (2g/kg) + POEA (1g/kg), or Roundupâ (containing 2g/kg glyphosate and 1g/kg POEA) produced diarrhea and blood-stained nose discharges. In the group that received 2g/kg glyphosate, only transient diarrhea was observed. Both POEA and glyphosate caused lung hemorrhages and lung epithelial cell damage. Death was only seen in the POEA group after 24h. The authors suggested that the polyoxyethyleneamine and preparations that contained POEA were more toxic than glyphosate. The results agree with those reported by Adam et al (1997) and showed that the oral acute toxicity of glyphosate-Roundupâ formulation to Wistar rats was highest than the glyphosate alone. According to Sawada et al. (1988), the oral LD50 of polyoxyethyleneamine (POEA) falls within the range of 1 to 2g/kg.

The proportion of glyphosate to surfactant in Roundupâ formulation varies in different countries or regions. In Asia,  Roundupâ is available as 41% glyphosate and 15-18% POEA, and in North America the concentrated product contains 18% glyphosate and 7% POEA (Martinez et al 1990). In Brazil, Roundupâ formulation contains glyphosate 36% and POEA 18%. Tominack (2000) calls attention to the need of evaluating other ingredients formulated along with the active ingredient in pesticides. For example, surfactants are nearly universally present in herbicide formulations or added prior to its application. In the United States, the strategy of the Environmental Protection Agency has been to categorize currently used formulation ingredients into four lists based on their toxicology profiles. This suggests that other ingredients, like surfactants, needed a careful evaluation for its potential toxicity, either alone or as formulations. Accordingly, the present study showed that Roundupâ glyphosate formulation used in Brazil is more toxic than previously reported.

We are grateful to Ph.D. Maria Beatriz Cardoso Ferreira for technical assistance and Ph.D. Elaine Elisabetsky for English revision. This work was partly supported by grants from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Fundação de Amparo à Pesquisa do Rio Grande do Sul (FAPERGS) and Universidade Federal do Rio Grande do Sul (UFRGS) / Brasil.

Adam A, Marzuki A, Rahman HÁ and Aziz MA (1997), The oral and intratracheal toxicities of Roundup and its components to rats, Vet. Human Toxicol. 39 p 147-151
Bababunmi EA, Olorunsogo OO, Bassir O (1978), Toxicology of glyphosate in rats and mice, Toxicol. Appl. Pharmacol. 45 p 319-320
EPA-Environmental Protection Agency (1996), Guidelines for Reproductive Toxicity Risk Assessment – EPA/630/R-96/009, p 1-162
Litchfield JT, and Wilcoxon F (1987), Confidence limits of ED₅₀, In: Tallarida RJ and Murray RB (1987), Manual of pharmacologic calculations with computer programs, Springer-Verlag, New York, p 153-166
Martinez TT, Long WC, Hiller H (1990), Comparison of the toxicology of the herbicide Roundup by the oral and pulmonary routes of exposure, Proc. West Pharmacol. Soc. 33 p 193-197
Menkes DB, Temple WA and Edwards IR (1991), Intentional self-poisoning with glyphosate-containing herbicides, Hum. Exp. Toxicol. 10 p 103-107
Olorunsogo OO, Bababunmi E A (1980), Innibition of succinate-linking reduction of pyridine nucleotide in rat liver mitochondria "in vivo" by N-(phosphonomethyl)glycin, Toxicol. Lett. 7 p 149-152
Sawada Y, Nagai Y, Ueyama M et al (1988), Problable toxicity of the surface active agent in commercial herbicide containing glyphosate [letter], Lancet. p 289
Smith EA and Oehme FW (1992), The biological activity of glyphosate to plants and animals: a literature review, Vet. Human Toxicol 34 p 531-543
Tallarida RJ and Jacob LS (1987), Quantal dose-response: probits In: Tallarida RJ and Murray RB (1987), Manual of pharmacologic calculations with computer programs, Springer-Verlag, New York, p 31-35
Tominack RL (2000), Herbicide formulations, Journal of Toxicology–Clinical Toxicology 38 p 129-135
WHO-World Health Organization (1994), Glyphosate, Environmental Health Criteria 159 p 1-177
Zar JH (1999), Biostatistical analysis, Prentice Hall, New Jersey, p 486-515