Elucidating the effects of pure glyphosate and a commercial formulation on early life stages of zebrafish using a complete biomarker approach: All-or-nothing!

The search for new methods in the toxicology field has increased the use of early life stages of zebrafish (Danio rerio) as a versatile organism model. Here, we use early stages of zebrafish to evaluate glyphosate as pure active ingredient and within a commercial formulation in terms of oxidative stress. Biomarkers involved in the oxidative status were evaluated along with other markers of neurotoxicity, genotoxicity, cytotoxicity, energy balance and motor performance, and the selected tools were evaluated by its sensitivity in determining early-warning events. Zebrafish embryos exposed to glyphosate active ingredient and glyphosate-based formulation were under oxidative stress, but only the commercial formulation delayed the embryogenesis, affected the cholinergic neurotransmission and induced DNA damage. Both altered the motor performance of larvae at very low concentrations, becoming larvae hypoactive. The energy balance was also impaired, as embryos under oxidative stress had lower lipids reserves. Although data suggest that glyphosate-based formulation has higher toxicity than the active ingredient itself, the most sensitive biomarkers detected early-warning effects at very low concentrations of the active ingredient. Biochemical biomarkers of defense system and oxidative damage were the most sensitive tools, detecting pro-oxidant responses at very low concentrations, along with markers of motor performance that showed high sensitivity and high throughput, suitable for detecting early effects linked to neurotoxicity. Alterations on morphology during embryogenesis showed the lowest sensitivity, thus morphological alterations appeared after several alterations at biochemical levels. Tools evaluating DNA damage and cell proliferation showed mid-sensitivity, but low throughput, thus they could be used as complementary markers.

Evaluation of extracts from Coccoloba mollis using the Salmonella/microsome system and in vivo tests.

The common everyday use of medicinal plants is an ancient, and still very widespread practice, whereby the need for studies on their possible toxicity and mutagenic properties. The species Coccoloba mollis has been much used in phytotherapy, mainly in cases involving loss of memory and stress. In order to investigate its genotoxic and mutagenic potential, ethanolic extracts from the leaves and roots underwent Salmonella/microsome assaying (TA98 and TA100 strains, with and without exogenous metabolism - S9), besides comet and micronucleus tests in vivo.There was no significant increase in the number of revertants/plate of Salmonella strains in any of the analyzed root-extract concentrations, although the extract itself was extremely toxic to the Salmonella TA98 strain in the tests carried out with S9 (doses varying from 0.005 to 0.5 µg/plate). On the other hand, the leaf-extract induced mutations in the TA98 strain in the absence of S9 in the highest concentration evaluated, although at very low mutagenic potency (0.004 rev/ µg). Furthermore, there was no statistically significant increase in the number of comets and micronuclei, in treatments involving Swiss mice. It was obvious that extracts of Coccoloba mollis, under the described experimental conditions, are not mutagenic.