Acute toxicity and sublethal effects of the mixture glyphosate ( Roundup ® Active ) and Cosmo-Flux ® 411 F to anuran embryos and tadpoles of four Colombian species

Glyphosate is the most widely used herbicide in the world with application in agriculture, forestry, industrial weed control, garden and aquatic environments. However, its use is highly controversial for the possible impact on not-target organisms, such as amphibians, which are vanishing at an alarming and rapid rate. Due to the high solubility in water and ionic nature, the glyphosate requires of surfactants to increase activity. In addition, for the control of coca (Erythroxylum coca) and agricultural weeds in Colombia, formulated glyphosate is mixed and sprayed with the adjuvant Cosmo-Flux®411F to increase the penetration and activity of the herbicide. This study evaluates the acute toxic and sublethal effects (embryonic development, tadpole body size, tadpole swimming performance) of the mixture of the formulated glyphosate Roundup® Active and Cosmo-Flux®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 pustulosus 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 more tolerant to the mixture than tadpoles, which could be likely attributed to the exclusion of chemicals by the embryonic membranes and the lack of organs, such as gills, which are sensitive to surfactants. Sublethal effects were observed for the tadpole body size, but not for the embryonic development and tadpole swimming performance. In microcosms, no toxicity (LC50 could not be estimated), or sublethal responses were observed at concentrations up to fourfold (14.76kg glyphosate a.e./ha) the highest field application rate of 3.69kg glyphosate a.e./ha. Thus, toxicity was less in the microcosms than in laboratory tests, which may be attributed to the presence of sediments and organic matter which rapidly adsorb glyphosate and surfactants such as POEA. It is concluded that the mixture of glyphosate (Roundup® 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. Rev. Biol. Trop. 63 (1): 223-233. Epub 2015 March 01.

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 coca, poppy crops (Solomon et al., 2007) and for the control of agricultural weeds (Solomon, Anadón, Cerdeira, Marshall, & Sanin, 2005).Nevertheless, during the aerial or hand spraying, this mix can affect aquatic animals such as amphibians, many of which are threatened with extinction (Mattoon, 2000;Collins & Storfer, 2003;Storfer, 2003).The life cycle and physiological characteristics of amphibians make them particularly susceptible to all environmental stressors (Cowman & Mazanti, 2000).Glyphosate formulations have been considered to induce genotoxic, morphological, biochemical, and physiological effects to anurans (Clements, Ralph, & Petras, 1997;Lajmanovich, Lorenzatti, Maitre, Enrique, & Peltzer, 2003;Cauble & Wagner, 2005;Bernal, Solomon, & Carrasquilla, 2009a;Mann, Hyne, Choung, & Wilson, 2009;Lenkowski, Sanchez-Bravo, & Mclaughlin, 2010;Williams & Semlitsch, 2010;Lajmanovich, Attademo, Peltzer, Junges, & Cabagna, 2011;Relyea, 2012), although low toxicity has been reported under realistic field conditions (Howe et al., 2004;Wojtaszek, Staznik, Chartrand, Stephenson, & Thompson, 2004;Bernal, Solomon, & Carrasquilla, 2009b).Other studies have concluded that the glyphosate and its formulated products represent moderate (Giesy, Dobson, & Solomon, 2000;Sparling, Linder, Bishop, & Krest, 2010) or negligible risk to aquatic organisms (World Health Organization International Program on Chemical Safety, 1994; Giesy et al., 2000;Solomon & Thompson, 2003).These differences could be attributed not only to experimental conditions, but also to types of commercial products tested.Overall, there is a strong debate about the impact of glyphosate on amphibians.Glyphosate is the active ingredient of several herbicide formulations, which contains surfactants such as the ethoxylated tallowamine (POEA) that vary in concentration and may have a greater toxicity than glyphosate itself (Bradberry, Proudfoot, & vale, 2004).The glyphosate acts as an inhibitor of enzymes involved in the synthesis of aromatic amino acids in growing plants, and must be absorbed through foliage; however, due to its high solubility in water and ionic nature, it does not penetrate easily through the hydrophobic cuticle of leaves.Therefore, surfactants are used in the formulated commercial products.In addition, for the control of coca in Colombia, formulated glyphosate is mixed and sprayed with 2.3% v/v of the adjuvant Cosmo-Flux ® 411F, which reduce the surface tension of leaves to increase the penetration and activity of the herbicide in coca plants (Solomon et al., 2007).The rate of application of formulated glyphosate is 3.69kg a.e./ha, which is greater than that recommended in agriculture (1.77kg a.e./ha) (Ministry of Environment, Housing and Territorial Development of Colombia, 2007).The toxic effect of Cosmo-Flux ® 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 lethal consequence at the concentrations sprayed in the field.Another study conducted by Rondón, Ramírez and Eslava (2007) in the fish Piaractus brachypomus, concluded that the Cosmo-Flux ® 411F produced anatomopathological effects and mortality, but at much larger concentrations (LC 50 =4 418mg/L) than those applied in the field.
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 microcosm conditions.Despite these data, there is no still information about the toxicity of the combined glyphosate and Cosmo-Flux ® 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 coca) 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 corn fields, which may be used as habitat by anurans.

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 adjuvant Cosmo-Flux ® 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 used in the field (Solomon et al., 2007;Bernal et al., 2009a).All solutions were prepared with the same dechlorinated tap water used to raise the embryos and tadpoles.

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 total of 50 embryos and 20 tadpoles per concentration.Biomass loading (defined as the total wet weight of tadpoles per liter of test water) was maintained below 0.6g/L as recommended in ASTM guidelines (1998).Test chambers were 2L glass bowls, containing 1L of test solution, which were indiscriminately positioned in an air-conditioned area of the laboratory, designed to maintain the environmental temperature (24±2ºC) throughout the experiments.After preliminary results of toxicity, the final nominal concentrations were: 0 (negative control: dechlorinated tap water), 325, 750, 1 500, 3 000 and 6 000μg glyphosate a.e./L.Test solutions were renewed daily by transferring the organisms to freshly prepared solutions during the 96h test experiments.
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 renewals (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 each of two microcosms for a total of 100 embryos and 50 tadpoles per concentration.Biomass loading was maintained below 0.6g/L as recommended in ASTM guidelines (1998).Microcosms were constructed from polyethylene plastic containers (70cm in diameter, 13cm depth; area: 0.1520m 2 ), which were placed randomly in an indoor but ventilated area of the laboratory at the University of Tolima, with an environmental temperature of 24±2°C and a photoperiod of approximately 12:12h light/dark cycle.At the bottom of the microcosms, 450g of soil, 645g of sand and 10L of dechlorinated tap water to a depth of 12cm above the sediment were added.Then, a fine screen nylon cloth (0.5mm mesh) was placed in the microcosms and pressed into the sediment with four or five small stones (approximately 250g), to facilitate the subsequent collection of embryos and tadpoles, and two leaves (leaf litter) and one macrophyte (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 were measured with a positive displacement pipette and then mixed with 150mL water.This amount was sprayed over the pools, at a height of approximately 20cm, with a small handheld pump-up garden sprayer of 3L capacity.Spray drift was minimized by spraying just in the center of the pool and with no wind.The physicochemical parameters of the water in the microcosms (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 (LC 50 ) and the associated 95% confidence intervals, using the TSK Trimmed Spearman-Karber method (version 1.5).Sublethal effects at 96h were assessed by measuring the tadpole body size, the stage of embryonic development, and the tadpole swimming performance, as this is expected to relate the ability to evade predators (Fitzpatrick, Reisen, & McCaslin, 2003;Walker et al., 2005).Twenty surviving animals, and ten animals for the laboratory experiments with tadpoles, were tested from the control and each of the tree experimental concentrations lower than the LC 50 .Photographs of individual

DISCUSSION
Amphibians are decreasing at an alarming rate around the world.Several factors have been suggested as possible cause of this declining, among them the herbicides.There is an extensive literature on the toxic effects of the glyphosate on amphibians (Plötner & Matschke, 2012); however, results are contradictory and consequently controversial points of view may be found about its use.Therefore, more information on the impact of ecologically relevant concentrations on survival and sublethal endpoints is essential to determine  properly the sensitivity of amphibians (Egea, Relyea, Tejedo, & Torralva, 2012).
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 of 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.LC 50 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 organic matter which rapidly adsorb the glyphosate (Relyea, 2004;Tsui & Chu, 2004;Wojtaszek et al., 2004) and surfactants such as POEA (Wang et al., 2005).In addition, residues of glyphosate decline rapidly within 24h after application (Trumbo, 2005).
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 pipiens 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 cause lysis of gill epithelial cells in the rainbow trout (Partearroyo, Pilling, & Jones, 1991), and affect the ability of the gills to maintain osmotic balance.
In the laboratory experiments, tadpole body size was reduced by the mix at concentrations lower than LC 50 .Impacts of pesticides on tadpole growth have been reported to be much more prevalent than tadpole survival in some amphibian species (Relyea, 2004) and it may affect populations in the long-term.For instance, slower growth can result in the death of populations if tadpoles are not able to metamorphose before their habitat dries (Wilbur & Collins, 1973).In addition, a smaller body size at metamorphosis has additional longterm fitness effects, such as reduced survival, smaller size at maturity, and lower egg production by females (Semlitsch, Scott, & Pechmann, 1988).Overall, a delay of embryonic development after exposure to the mixture of Roundup ® Active and Cosmo-Flux ® 411F was not observed.Chronic exposures to glyphosate formulations have increased the development time of Bufo americanus, Pseudacris triseriata (Williams & Semlitsch, 2010), and 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 concentrations tested (lower than LC 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 LC 50 values for tadpoles from Colombian frog species showed that sensitivity to glyphosate formulations is similar to that observed in other tropical and temperate species (Maltby, Blake, Brock, & van den Brink, 2005;Brain & Solomon, 2009;Relyea & Jones, 2009;Moore et al., 2012).This suggests that Roundup formulations have a relatively narrow toxicity range.In addition, that Cosmo-Flux ® 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 et al., 2000), which represents the level of chronic exposure expected to result in no deleterious effects to amphibians, the toxicity of the mix Roundup ® Active and Cosmo-Flux ® 411F to Colombian anurans would be low, as the 95% of Colombian embryos and tadpoles would have LC 50 greater than these values.On the other hand, neither toxic nor sublethal effects were observed in the microcosms at concentrations up to fourfolds (14.76kg glyphosate a.e./ha) the highest field application rate of 3.69kg glyphosate a.e./ha.We concluded, therefore, that the mix Roundup ® Active and Cosmo-Flux ® 411F, used as recommended in the field, will have negligible effects to anuran embryos and tadpoles of the study species.

ACKNOWLEDGMENT
Funding was provided by Fondo de Investigaciones de la Universidad del Tolima (Project number 490110) and a grant provided by COLCIENCIAS to Marcela Henao as Young Researcher.Collection permit was approved by Corporación Autónoma Regional del Tolima, CORTOLIMA (resolution number 2886 from July 21, 2011).Experiments were authorized by the Bioethics Committee of the Tolima University (October 18, 2011).The authors wish to thank Teófila María Triana and Jorge Luis Turriago for their valuable help in this research, and two anonymous reviewers for their constructive comments to improve the manuscript.

Fig. 1 .
Fig. 1.Toxicity values (LC 50 ) for embryos and tadpoles exposed to the mix Roundup ® Active and Cosmo-Flux ® 411F under laboratory conditions.The error bars correspond to the 95% confidence intervals.

Fig. 2 .
Fig. 2. Species sensitivity distribution of LC 50 values for Colombian embryos and tadpoles exposed to the mix Roundup ® Active and Cosmo-Flux ® 411F and for other glyphosate formulations as indicated in Brain and Solomon (2009), Bernal et al. (2009a), Relyea and Jones (2009) and Moore et al. (2012).

Fig. 3 .
Fig. 3. Mean of morphometric measurements of surviving larvae (raised from embryo experiments) (A) and tadpoles (B) exposed to the mix Roundup ® Active and Cosmo-Flux ® 411F under laboratory conditions.

TABLE 1
Mean and standard deviation of physicochemical parameters of the water used in the laboratory and microcosm experiments

TABLE 2
Percent mortality at glyphosate nominal concentrations in the mix Roundup ® Active and Cosmo-Flux ® 411F