Embryotoxicity and teratogenesis in zebrafish embryos exposed in vitro to glyphosate-type herbicides


1Viviana Bortagaray, 1Rosa Cruces Aramburu, 1Luisa Barrios, 1Pilar Ojeda,1Gabriela del Puerto, 1,2Daniel Rodríguez-Ithurralde

1Laboratorio de Neurociencia Molecular y Núcleo de Biotecnología y Marcadores Moleculares, Instituto Clemente Estable, Av. Italia 3318, Montevideo, Uruguay. E-mail: drit@iibce.edu.uy

2Cell and Molecular Biology Subarea, Biology Area, Programa de Desarrollo de las Ciencias Básicas (PEDECIBA), Montevideo, Uruguay. E-mail: drithurralde@gmail.com

Introduction

A growing body of evidence suggests that herbicides formulated in base of  N-(phosphonomethyl)glycine (glyphosate), the most frequently used herbicide in the world, present considerable toxicity for algae, molluscs, fishes and amphybia (Quassinti et al., 2008).  Its non-controlled use represents, thererefore, a real menace for biodiversity, natural fishery resources,  and pisciculture.

Glyphosate causes zebrafish spinal kyphosis

Fig. 1. Spinal kyphosis in zebrafish exposed to glyphosate (ccf 25 mg/l) from 1.5 to 96 hpf.

It is of great interest to point out that non-allowed concentrations of this and other herbicides and/or their metabolites and/or coadjuvants have been found in waters and foods for human and/or animal use. In addition, glyphosate final concentrations (ccf) of 3-37 mg/l were found in water samples upon soil infiltration assays and ccf of 0.21-1.51 mg/l were encountered in water samples taken in the vicinity of  soils associated with direct sowing soybean cultivation (Peruzzo et al., 2008, 2009).  These findings, taken together, are a cause of concern as  they show a potential danger for human and animal health, which  must be strictly monitored.  In addition, the most abundant glyphosate metabolite, the AMPA,  is more toxic and more resistant to degradation than glyphosate.

The aim of this work was to investigate and, when possible, quantify using controled, globally accepted laboratory bioassays, the effects of  known  concentrations of glyphosate on zebrafish (Danio rerio) development (Hill et al., 2005; Baunbeck & Lammar, 2006).

Materials and Methods

Animals used. Zebrafish embryo of the dram strain, produced in our facility according to internationally accepted procedures were used.

ZETA test.  We applied our previously validated, multi-parametric, Zebrafish Embryo Toxicity Assay (ZETA), which allows measuring numerous morphologic, physiologic and biochemical parameters in hundreds of simultaneous  samples with sensitivity, high reproductibility and low cost (Ojeda et al., 2007; Rodriguez-Ihurralde., 2007, 2010). Specimens  of  1-1.5  post-fertilization hours of life (hpf) were incubated in ELISA plates  at 27.9 ± 1ºC,  at a one embryo-per-well ratio. Each plate well contained 1.0 ml of filtered, controlled aquarium water containing a pre-established final concentration (ccf) of glyphosate.  In most experiments, ccf  (in μg of glyphosate per ml) used were:  0.00, 6.2, 12.5, 25, 50,  75 and 150.  In other experiments ccf (μg/ml) used were: 0.00, 0.055, 0.11, 0.22, 0.45, 0.9, 1.8 and 3.6. After fixed, pre-stablished time intervals (12, 24, 48, 72 and 96 hpf), the presence of universally accepted final points of zebrafish development was computed  (Hill et al., 2005; Baunbeck & Lammar, 2006; Ojeda et al., 2007; Bortagaray et al., 2009).

Biochemical assays. All biochemical determinations were run in triplicate. Total cholinesterase, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) assays were performed as described in Karlsson et al. (1984), using acetylthiocholine as a substrate. This method is a modification of that of Augustinsson et al. (1978), which is in turn a chromophore modification of Ellman et al. (1961).

Results

Lethality. Lethality plots showed that from ccf of 25 μg/ml  and higher, embryo lethality was significantly dependent on both herbicide concentration and time of exposure. At 24 hpf, lethalities of 35.4% were verified for ccf of 50 μg/ml, whereas it was 74% at ccf  of 75 μg/ml, and 82% at ccf of 150 μg/ml, wheras it remained between 0-5% in control embryos.

Ecclosion events. Although most morphologico-structural endpoints exhibited delays in their time of appearance in embryos exposed to glyphosate, ecclosion events were, as a general rule, accelerated. At ccf between 12.5-50 μg/ml, corion effraction  with embryo ecclosion appeared earlier, i.e., the ratio of ecclosioned/total embryos at 72 hpf was significantly higher than in controls.  Therefore, embryos in a more immature form than those in control samples arrive in direct contact with the  external aquatic medium.

Cardiac and pericardic alterations. Embryonic cardiac frequency, as measured at 48 hpf, decreased significantly, from ccf 25 μg/ml on, with regard to control  specimens without glyphosate. At ccf of 50 μg/ml, cardiac and pericardic edema, and  delays in the separation of  the viteline sac were evident .

Physiopathologic and biochemical changes. At glyphosate ccf of 75 μg/ml or higher, all embryos presented at least one physiological or structural developmental alteration.  The most frequent change was cardio-pericardic edema and circulatory delay, although cholinesterase inhibition and decrease in trunk flection movements at stadii in which they should be present, were very frequently recorded.

Digestivo-viteline malformations. Morphologic alterations of the viteline sac, or its complete absence of separation,  were frequently seen. Some of the morphologic alterations were so important that were incompatible with life.

Musculo-skeletic malformations. Deformities of varied importance were frequent in both axial and apendicular skeleton.  Fishes showed incurvations, “gibas”, and angles of  the vertebral axis as well as varied  deformities and abnormal divisions of  tail and appendages.

Discussion and Conclusions

In summary, we have verified a group of  changes in functional, biochemical and morphologic parameters.  We found an apparent correlation between high herbicide concentrations and more severe alterations encountered, with the exception of  cases where the toxicity is so high that the embryos are unable to survive  more than minutes or hours.

Early  ecclosion  might also be viewed as a potential additional cause of lethality for wildlife species, since the corion barrier, which is  normally protecting immature embryos fom the external medium, is opened earlier in herbicide-treated  specimens  as seen in this and in a previous paper of our group (Ojeda et al., 2007).  This early release of immature forms to the water, when occurs in the usual environment of real water bodies, might cause massive deaths of embryos under the effect of other biological, physical or chemical agents, that can enter in direct contact with the embryo (Ojeda et al., 2007; Rodríguez-Ithurralde et al., 2007).  Inhibition of cholinestareses, although in the order of  20-30% of control specimens in our assay, might, in the “natural” environment be potentiated by other anticholinesterase agents as organophosphate insecticides, which are in some cases realtively resistant to water degradation, and therefore  AChE actions on neurodevelopment  (Olivera et al., 2003) might be contributing to some of the structural phenotypic alterations.

It is relevant to point out that we have demonstrated lethal and sub-lethal toxic effects, as well as teratogenic effects, at glyphosate ranks of concentration which have been previously reported as atoxic.  The concentrations used in our experiments are in the same order of magnitude as those  directly determined in water  bodies close to cultures in the Argentineian pampasic region (Peruzzo et al., 2008, 2009).  For instance, glyphosate concentrations found in water samples taken upon soil infiltration assays performed at Pergamino, Argentina, were between 2,9 and 37.1 mg/l.  These authors  also show that in water bodies close to cultures treated with glyphosate,  its levels varied along the year and along the day only between 0.1 and 0.7 mg/l,  i.e., they never reached levels under 0.1 mg/l (Peruzzo et al., 2008, 2009).  In this context,  and focussing on the consequences exposure of mammals to herbicides, it is of great interest to point out that cutaneous-muccosal absortion occurs in humans, as evidenced by local and systemic clinical signology demonstrated by Burger and Fernández (2004). These authors also reported neuromuscular clinical symphtoms in the same subjects, but were unable to find any physiopathological explanation for them.

Acknowledgements

We are indebted to PEDECIBA (Biología) for its continued grant support to DRI and for fellowship support to AS.


References

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Peruzzo, P., Marino, D., Cremonte, C., da Silva, M. Porta, A. & Ronco, A. (2008). Levels of glyphosate in surface waters and soils associated with direct sowing soybean cultivation un north pampasic region of Argentina. Environm. Pollut. 156, 61-66.

Peruzzo, P., Marino, D., Cremonte, C., da Silva, M. Porta, A. & Ronco, A. (2009).  Impacto de pesticidas en aguas superficiales y sedimentos asociados a cultivos por siembra directa. Conf. Internacional Usos Múltiples del Agua. Inst. Cinara.

Quassinti, L., Maccari, E., Murri, O. & Bramucci, M., 2008. Effects of paraquat and glyphosate on steroidogenesis in gonads of the frog Rana sculenta in vitro.

Rodríguez-Ithurralde, D. (2007). Biomarcadores en peces autóctonos y exóticos para el monitoreo eco-toxicológico de los recursos acuáticos. Actas Fisiol. (Montevideo), 11, 53-53.

Rodríguez-Ithurralde, D. (2010).  The use of selected fish species for water quality monitoring bioassays, with special reference to the zebrafish embryo toxicity assay (ZETA).  Urug. Cong. Zool., 1: 53.

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About The Editor
Science Editor, Scientific Researcher, Neurobiologist, Neurochemist. Specialized in Neurochemistry, Molecular Neurobiology, Developmental Neurobiology, Developmental Toxicology

One Response to Embryotoxicity and teratogenesis in zebrafish embryos exposed in vitro to glyphosate-type herbicides

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