Embriotoxicity and teratogenesis by glyphosate
Laboratorio de Neurociencia Molecular y Núcleo de Biotecnología y Marcadores Moleculares, Instituto Clemente Estable, Av. Italia 3318, Montevideo, Uruguay. E-mail: firstname.lastname@example.org
A growing body of evidence suggests that herbicides formulated in base of N-(phosphonomethyl)glycine (glyphosate), the most fequently used in the world, present considerable toxicity for algae, molluscs, fishes and amphybia (Quassinti et al., 2008). Its non-controlled use represents a real menace for biodiversity, natural fishery resources, and pisciculture.
It is of great interest to point out that non-permitted concentrations of this and other herbicides and/or their metabolites and/or coadjuvants have been found in waters and foods for human use, and these findings are a cause of concern as potential dangers for human and animal health, which must be strictly monitored. The most abundant glyphosate metabolite, the AMPA, is more toxic and more resistant to degradation than it. The aim of this work is to investigate and, when possible, quantify the effects of glyphosate on zebrafish (Danio rerio) development using internationally accepted tests (Hill et al, 2005; Baunbeck & Lammar, 2006).
Materials and Methods
Animals used. Zebrafish embryo produced in our facility according to internationally accepted procedures were used. We have 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) to quantify glyphosate effects on zebrafish development.
ZETA test. Specimens of one post-fecundation hour of life (hpf) were incubated in ELISA plates at 27.5 ± 1ºC, at a one-per-well ratio. Each well contained 1.0 ml of filtered, controlled aquarium water containing a pre-established final concentraciones (ccf) of glyphosate. After fixed, pre-stablished time intervals (12, 24, 48, 72 and 96 hpf), the presence of universally acepted final points of zebrafish development was computed (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.
Lethality. Lethality plots showed that from ccf of 25 microg/ml and higher, embryo lethality was significantly dependent on both the herbicide concentration and the time of exposure. At 24 hpf, lethalities of 35.4% were verified for ccf of 50 microg/ml, whereas it was 74% at ccf of 75 microg/ml, and 82% at ccf of 150 microg/ml, wheras in control embryos remained between 0-5%.
Ecclosion events. Although most morphologic-structural final points 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 microg/ml, corion effraction with embryo ecclosion appeared earlier, i.e., the ratio of eclosioned/total embryos at 72 hpf was significanly higher than in controls. Therefore, arrive in direct contact with the external aquatic medium, forms which are more immature than in control samples.
Cardiac and pericardic alterations. Embryionic cardiac frequency, as measured at 48 hpf, decreased significatintly, from ccf 25 microg/ml on, with regard to control specimens without glyphosate. At ccf of 50 microg/ml, cardiac and pericardic edema, and delays in the separation of the viteline sac, appear.
Physiopatologic and biochemical changes. At ccf of 75 microg/ml or higher, all the embryos showed at least one physiologic or structural developmental alteration, as, for instance, cardio-pericardic edema, circulatory delay, cholinesterase inhibition, or decreases in trunk flection movements at stadii in which they should be present.
Digestivo-viteline malformations. Morphologic alterations of the viteline sac, or its absence of separation, are frequently seen. Some of the morphologic alteratins are so important, that were incompatible with life.
Musculo-eskeletic malformations. Deformities of varied importance were frequent in both axial and apendicular skeleton. Fishes showed incurvations, “gibas”, and angles of the vertebral axis and varied deformities and abnormal divisions of tail and appendages.
Discussion and Conclusions
In summary, we have verified a group of changes in parameters of type functional, biochemical and morphologic. There is a correlation between high concentrations and more severe alterations encountered, with the exeption of the cases where the toxicity is so high that the embryos are unable to survive more than one minutes or hours.
Early ecclosion might also be viewed as an additional cause of lethality, since the corion barrier, which is normally protecting immature embryos fom the external medium, is opened earlier in herbicide-treated specimens (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).
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 1-3 orders of magnitude inferior to those determined in water sheds close to culturs in Argentina and Uruguay. Thus, in very exact measurements carried out in Pergamino, Argentina, in water bodies close to cultures treated with glyphosate, the levels found in the water varied along the year and along the day only between 0.1 and 0.7 mg/ml, i.e., they never reached levels under 0.1 mg/ml.
We are indebted to PEDECIBA (Biología) for its continued grant support to DRI and for fellowship support to AS.
Bortagaray, V., Barrios, L., Cruces Aramburu, R., Del Puerto, G., Ojeda, P., Saravia, A. Rodríguez-Ithurralde, D. (2009). Los ensayos de embriotoxicidad en pez cebra detectan una variedad de efectos teratogénicos del glifosato.
6th SBBM Jorn. Biotechnology Section, (Uruguay)
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