Acute toxicity and sublethal effects of the mixture glyphosate (Roundup® Active) and Cosmo-Flux®411F to anuran embryos and tadpoles of four Colombian species
Toxicidad aguda y efectos subletales de la mezcla glifosato (Roundup® Activo) y Cosmo-Flux® 411Fen embriones y renacuajos de cuatro especies de anuros colombianos
Liliana Marcela Henao Muñoz1*, Claudia Marsela Montes Rojas1 & Manuel Hernando ]]>
1
Abstract
Glyphosate is the ]]>
Erythroxylum coca) and agricultural weeds in Colombia, formulated glyphosate is mixed and sprayed with the adjuvant ]]>
®411F to anuran embryos and tadpoles of four Colombian species under 96h laboratory standard tests and microcosms, which are more similar to field conditions as they include soil, sand and macrophytes. In the laboratory, embryos and tadpoles of Engystomops ]]>
were the most tolerant (LC50=3 904µg a.e./L; LC50=2 799µg a.e./L, respectively), while embryos and tadpoles of Hypsiboas crepitans (LC50=2 203µg a.e./L; LC50=1 424µg a.e./L, respectively) were the most sensitive. R. humboldti and R. marina presented an intermediate toxicity. Embryos were significantly ]]>
® Active) and Cosmo-Flux®411F, as used in the field, has a negligible toxic effect to embryos and tadpoles of the species tested in this study. ]]>
Key words: amphibians, ecotoxicology, herbicides, surfactants, survival.
Resumen
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El glifosato es el herbicida más usado en el mundo con aplicaciones para la agricultura, control de malezas forestales, industriales, en jardines y ambientes acuáticos. Sin embargo, su uso es altamente controversial por el posible impacto sobre organismos no blanco, como los anfibios, los cuales están desapareciendo a una tasa alarmantemente rápida. Debido a su alta solubilidad en agua y naturaleza iónica, el glifosato requiere de surfactantes para incrementar su actividad. Además, ]]>
Erythroxylum coca y de malezas en la agricultura en Colombia, el glifosato formulado es mezclado y rociado con el coadyuvante Cosmo-Flux®411F para incrementar la penetración y actividad del herbicida. Este estudio evalúa los efectos ]]>
® Activo con el Cosmo-Flux®411F en embriones y renacuajos de cuatro especies de anuros colombianos, bajo pruebas de 96h en condiciones estándar ]]>
Engystomops pustulosus fueron los más tolerantes (CL50=3 904µg a.e./L; CL50=2 799µg a.e./L, respectivamente), mientras que los embriones y renacuajos de Hypsiboas ]]>
fueron los más sensibles (CL50=2 203µg a.e./L; CL50=1 424µg a.e./L, respectivamente). R. humboldti y R. marina presentaron una toxicidad ]]>
® Activo) y Cosmo-Flux®411F, como se aplica en ]]>
Glyphosate is the most commonly used herbicide in the world with many commercial formulations for application in agriculture (Duke & Powles, 2008; Plötner & Matschke, 2012). However, the use of glyphosate is also highly controversial for its possible impact on not-target organisms. In Colombia, the mixture of formulated glyphosate and the adjuvant Cosmo-Flux®411F is used to eradicate ]]>
®411F, ]]>
®411F has been recently evaluated in Colombian anuran embryos by Henao, Arango and Bernal (2013), where they found that this adjuvant did not exert any ]]>
Piaractus brachypomus, concluded that the Cosmo-Flux®411F produced anatomopathological effects and mortality, but at much larger concentrations (LC50=4 418mg/L) than those applied in the field.
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Toxicity studies assessing the effect of glyphosate formulations on amphibians have been mainly performed on larval stages (see review from Plötner & Matschke, 2012), less on juveniles and adults (Mann & Bidwell, 1999; Relyea, 2005; Bernal et al., 2009b), and a few on embryonic stages. For example, Edginton, Sheridan, Stephenson, Thompson and Boermans (2004) evaluated the interactive effect of pH and the glyphosate (Vision®) in embryos and larvae of Rana clamitans, Rana pipiens, Bufo americanus and Xenopus laevis. They reported that larvae were more sensitive to glyphosate than embryos. Triana, Montes and Bernal (2013), on the other hand, evaluated the effect of glyphosate (Roundup® Active) to anuran embryos of four Colombian species, and showed that it was highly toxic in laboratory experiments but less in ]]>
®411F, as sprayed in field, to embryos from Colombian anurans. Bernal et al. (2009a,b) carried out a previous work on the effect of this mix to anuran larvae, juveniles and adults, but they used the formulated glyphosate GLY-41 which is applied to eradicate illicit plantations such as coca (Erythroxylum ]]>
) and opium poppy (Papaver somniferum).
This study was conducted to evaluate the acute toxicity and sublethal effects (embryonic development, tadpole body size, tadpole swimming performance) of the mixture of Roundup® Active and the adjuvant Cosmo-Flux®411F to embryos and tadpoles of four Colombian anuran species. Roundup® Active was selected for this study as this herbicide is widely used in agriculture in Colombia to remove weeds of rice, cotton, soybean and ]]>
Materials and Methods
Test organisms: Between two and four freshly egg masses for each study species were collected in the ]]>
Rhinella humboldti (Gallardo, 1965) in Payandé (04°17’51” N - 75°05’48” W), Engystomops pustulosus (Cope, 1864) in Mariquita (04°26’20” N - 75°13’56” W), and Hypsiboas ]]>
(Wied-Neuwied, 1824) and Rhinella marina (Linnaeus, 1758) in Potrerillo (04°15’00” N - 74°59’00” W). These species were selected as they occur in areas where the mixture of Roundup® Active and Cosmo-Flux®411F is ]]>
Test substances: The commercial formulation of glyphosate, Roundup® Active, which is commonly sprayed by Colombian farmers, was used. It contains 446g/L of potassium salt of N-(phosphonomethyl) glycine, equivalent to 363g/L of glyphosate acid at 20°C. Unfortunately, there is no information about the inactive ingredients in the technical fact sheet of the commercial product, so the presence and proportion of POEA are unknown. The ]]>
®411F contains a mixture of linear and aryl polyethoxylates (17% v/v) and isoparaffins (83% v/v) (Cosmoagro, 2013) and is sold in a concentration of 170g/L at 20°C. We purchased these products from agricultural retailers. Cosmo-Flux®411F was added to the stock test solution at 2.3% v/v to prepare a mixture as ]]>
Test procedure-laboratory experiments: A total of 25 embryos and 10 tadpoles per species, with its replicates, were placed separately in each test chambers for a ]]>
Fluorescent lights (Phillips TLT 20W/54RS) were used for illumination of test bowls. A photoperiod of 12:12h light/dark cycle was controlled by an automatic timer (General Electric PM621). Temperature, dissolved oxygen, conductivity and pH were measured in each test bowl at the beginning, at approximately 24h intervals, and the end of the experiments, including before and after ]]>
Table 1). Temperature was measured in test bowls using a liquid-in-glass thermometer. Dissolved oxygen was measured using a portable dissolved oxygen meter (Hanna HI 9146); conductivity and pH were measured using a conductimeter (Hanna HI 8033) and membrane pH meter (HI 8314).
Test procedure-microcosms: A total of 50 embryos and 25 tadpoles per species were placed separately in ]]>
Pistia stratiotes) were incorporated. All these materials were obtained from the botanical garden of the University of Tolima, a nonagricultural area. Embryos (Gosner stage ten) and tadpoles (Gosner stage 25) were placed in the water in the middle of the microcosms, which were immediately sprayed with the mix Roundup® Active and Cosmo-Flux®411F, to provide a range of concentration equivalent to 0 (negative control: dechlorinated tap water), 3.69, 7.38, 14.76 and 29.52kg glyphosate a.e./ha.
The amounts of the test substances ]]>
Table 1) were similar to those tested in laboratory, and measured at 24, 48, 72 and 96h after the application of the mix. At the 96h of exposure, the nylon mesh was carefully removed from the microcosms and the surviving animals were counted.
Measurements of toxicity and sublethal effects: Mortality was based on the accumulated number of dead and missing animals to 96h of exposure. Toxicity values were estimated through the mean lethal concentration (LC50) and the associated 95% confidence intervals, using the TSK Trimmed Spearman-Karber method (Version 1.5). Sublethal effects at 96h were ]]>
50. Photographs of individual tadpoles were taken to measure the body size characters: total length (TL), head width (HW), corporal length (CL) and tail length (tL), using the software ImageJ ]]>
Results
In laboratory experiments, embryos and tadpoles of E. ]]>
were the most tolerant to the mix (embryos: LC50=3 904µg a.e./L; tadpoles: LC50=2 799µg a.e./L), while embryos and tadpoles of H. crepitans (embryos: LC50=2 203µg a.e./L; tadpoles: LC50=1 424µg a.e./L) were the most ]]>
Table 2). R. marina (embryos: LC50=2 270µg a.e./L; tadpoles: LC50=2 170µg a.e./L), and R. humboldti (embryos: LC50=3 336µg a.e./L; tadpoles: LC50=2 121µg a.e./L) presented an intermediate toxicity. Embryos were significantly more tolerant to the mix than tadpoles in three of the four species, but in R. marina there was not a statistical significance at the 95% confidence interval (Fig. 1). The LC50 values for embryos and tadpoles were combined with other ]]>
Fig. 2). The fifth centile of the toxicity distribution was 883μg a.e./L, indicating that 95% of Colombian species are within the toxicity range reported to glyphosate formulations.
Morphometric measurements of larvae (obtained from embryos after 96h exposure) and tadpoles were statistically significant among laboratory treatments (embryos: ]]>
2=0.532, p=0.0001; tadpoles: Hotelling, T2=0.46, p<0.0001). For each species, there were significant differences between concentrations (0, 325, 750, 1 500µg a.e./L) for all four morphometric measures (TL, HW, CL, tL) (ANOVA, p<0.05) (Fig. 3), although a decreasing in tadpole body size at the highest sublethal ]]>
Fig. 3). A significant delay in embryonic development was only found between the greatest experimental concentration (organisms in stage 24) and control (organisms in stage 25) (ANOVA, F=4.75, p<0.0043) for R. humboldti. Other species and concentrations did not show any significant change in embryonic development. ]]>
In microcosms, mortality was less than 50% for embryos and tadpoles of all the studied species (Table 2), and consequently LC50 values could not be calculated by the TSK method. Additionally, no statistic differences were found among treatments for ]]>
T2=0.49, p=0.9514), tadpole body size (Hotelling, T2=0.03, p=0.8705), embryonic development (ANOVA, p>0.05), nor tadpole swimming performance (Laboratory: Hotelling, T2=1.4, p=0.1083; Microcosms: Hotelling, T2=2.5, p=0.0827).
Discussion
Amphibians are decreasing at an ]]>
]]>
In this study, embryos and tadpoles of E. pustulosus were the most resistant to the application of the mix Roundup® Active and Cosmo-Flux®411F, while embryos and tadpoles ]]>
H. crepitans showed the greatest sensitivity. These results are in concordance with Bernal et al. (2009a), who found that larvae (stage 25) of E. pustulosus were the most tolerant to the mix GLY-41 and Cosmo-Flux, whereas H. crepitans were the most sensitive. LC50 values could not be calculated for microcosms, this due to the low mortality to exposure concentrations which were up to fourfold the highest field application rate of 3.69kg glyphosate a.e./ha. Toxicity of frog embryos and tadpoles was less in the microcosms than in laboratory tests. The reason for this may be attributed to the presence of sediments and ]]>
Anuran embryos were less sensitive than tadpoles to the mix Roundup® Active and Cosmo-Flux®411F under the same standard laboratory conditions, with the exception of R. marina. This is consistent with Edginton et al. (2004), who found that embryos (Gosner stage eight) of Xenopus laevis., Bufo americanus, Rana clamitans and Rana ]]>
exposed to glyphosate formulated Vision® were less sensitive than larvae (Gosner stage 25). The greater sensitivity to pesticides of larval stage as compared with the embryonic stage of amphibians has been also reported by Hall and Swineford (1980), Berrill et al. (1993), Berrill, Bertram, McGillivary, Kolohon and Pauli (1994), Berrill, Coulson, McGillivary and Pauli (1998), Edginton et al. (2004). Surfactants have been demonstrated to ]]>
In the laboratory experiments, tadpole body size was reduced by the mix at concentrations lower than LC50. Impacts of pesticides on tadpole growth have been reported to be ]]>
® Active and Cosmo-Flux®411F was not observed. Chronic exposures to glyphosate formulations have increased the development time of Bufo americanus, Pseudacris triseriata ]]>
Rana pipiens (Howe et al., 2004), but also accelerated the metamorphosis for Rana cascadae (Cauble & Wagner, 2005). Differences in the biology of the species, the sublethal concentrations and the glyphosate formulations could explain these contradictory results. Tadpole swimming performance was not significant among any of the ]]>
50), either in laboratory or microcosm experiments. This result was unexpected as other studies have shown that exposure to pesticides caused a significant decrease in the swimming speed for tadpoles of Rana blairi (Bridges, 1997) and Rana berlandieri (Punzo, 2005). It is possible that this commercial formulation Roundup® Active is of low toxicity and does not affect the tadpole swimming performance.
The LC50 values for tadpoles from Colombian frog species showed that sensitivity to glyphosate formulations is similar to that observed in other tropical and ]]>
®411F does not significantly increase the toxicity of the glyphosate formulations. At the level of glyphosate considered to be protective of human health (700μg/L a.i., 572μg/L a.e.) (Williams & Semlitsch, 2010), or the amphibian chronic toxicity reference value (TRV) of 740μg/L a.e. (Giesy ]]>
® Active and Cosmo-Flux®411F to Colombian anurans would be low, as the 95% of Colombian embryos and tadpoles would have LC50 greater than these ]]>
® Active and Cosmo-Flux®411F, used as recommended in ]]>
Acknowledgment
Funding was provided by Fondo de Investigaciones de la Universidad del Tolima (Project number 490110) ]]>
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1. Grupo de Herpetología, Eco-Fisiología & Etología, Departamento de Biología, Universidad del Tolima, Calle 42 Barrio Santa Helena Ibagué, Colombia; lmhenaom@ut.edu.co, cmmontesr@ut.edu.co, mhbernal@ut.edu.co
Received 20-XII-2013. Corrected 25-VII-2014. Accepted 26-VIII-2014.
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