Effects of the emulsifiable herbicide Dicamba on amphibian tadpoles: an underestimated toxicity risk?

The effects of exposure to the herbicide Dicamba (DIC) on tadpoles of two amphibian species, Scinax nasicus and Elachistocleis bicolor, were assessed. Mortality and biochemical sublethal effects were evaluated using acetylcholinesterase (AChE), glutathione S-transferase (GST), glutathione reductase (GR), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) activities and thyroid hormone (T4) levels. The LC50 value at 48h was 0.859 mg L-1 for S. nasicus and 0.221 mg L-1 for E. bicolor tadpoles. After exposure to sublethal DIC concentrations for 48 h, GST activity increased in S. nasicus but significantly decreased in E. bicolor with respect to controls. GR activity decreased only in S. nasicus at all the tested DIC concentrations. AChE activity was significantly inhibited in both S. nasicus and E. bicolor tadpoles at 48 h. DIC also caused significant changes in transamination, as evidenced by an increase in AST and ALT activities in both amphibian species. T4 levels were higher in DIC-treated tadpoles of both species than in controls. The DIC-induced biochemical alterations in glutathione system enzymes and transaminases indicate lesions in liver tissues and cellular function. Moreover, the observed AChE inhibition could lead to the accumulation of acetylcholine, excessively stimulating postsynaptic receptors, and the increase in T4 levels in both species may indicate an overactive thyroid. The commercial DIC formulation showed a high biotoxicity in the two amphibian native species after short-term exposure, controversially differing from the toxicity level indicated in the official fact sheet data. This fact highlights the need for an urgent re-categorization and reevaluation of DIC toxicity in native species.

Toxicity to Rhinella arenarum tadpoles (Anura, Bufonidae) of herbicide mixtures commonly used to treat fallow containing resistant weeds: glyphosate-dicamba and glyphosate-flurochloridone.

Glyphosate (GLY)-dicamba (DIC) and GLY-flurochloridone (FLC) are herbicide mixtures which are widely used for treating fallow containing glyphosate resistant weeds. The aim of this study was to evaluate the acute toxic effects and the prevailing interactions on stage 36 tadpoles of the anuran species Rhinella arenarum when exposed to equitoxic and non-equitoxic combinations of these herbicide combinations. Experiments were realized using the following combinations of commercial formulations: 48% GLY-based Credit® + 57.71% DIC-based Banvel® and 48% GLY-based Credit® + 25% FLC-based Twin Pack Gold®. GLY-DIC and GLY-FLC equitoxic mixtures were assayed mixing each constituent with an equivalent individual toxicity able to induce the same lethality effect. After 96 h of exposure, GLY-DIC and GLY-FLC equitoxic mixtures presented toxic unit 50 values (TU50 96h) of 1.74 (confidence interval: 1.58-1.92) and 1.54 (confidence interval: 1.46-1.62) respectively, indicating the presence of a weak antagonistic interaction as TU values were greater than 1. For their part, most non-equitoxic combinations of GLY-DIC and GLY-FLC tested did not significantly differ from additivity, the only exception being when DIC and FLC were fixed at 0.33 TUs, where a weak antagonism was observed. Overall, results indicate that the toxicity of both GLY-DIC and GLY-FLC mixtures to R. arenarum tadpoles vary from additive to slightly antagonistic, depending on the proportion of constituting herbicide formulations present in the mixture.

Genotoxicity by long-term exposure to the auxinic herbicides 2,4-dichlorophenoxyacetic acid and dicamba on Cnesterodon decemmaculatus (Pisces: Poeciliidae).

Long-term genotoxic effects of two auxinic herbicide formulations, namely, the 58.4% 2,4-dichlorophenoxyacetic acid (2,4-D)-based DMA® and the 57.7% dicamba (DIC)-based Banvel® were evaluated on Cnesterodon decemmaculatus. Primary DNA lesions were analyzed by the single-cell gel electrophoresis methodology. Two sublethal concentrations were tested for each herbicide corresponding to 2.5% and 5% of the LC5096h values. Accordingly, fish were exposed to 25.2 and 50.4 mg/L or 41 and 82 mg/L for 2,4-D and DIC, respectively. Fish were continuously exposed for 28 days with replacement of test solutions every 3 days. Genotoxicity was evaluated in ten individuals from each experimental point at the beginning of the exposure period (0 day) and at 7, 14, 21 and 28 days thereafter. Results demonstrated for first time that 2,4-D-based formulation DMA® induced primary DNA strand breaks after 7-28 days exposure on C. decemmaculatus regardless its concentration. On the other hand, DIC-based formulation Banvel® exerted its genotoxic effect after exposure during 7-14 days and 7 days of 2.5 and 5% LC5096h, respectively. The present study represents the first evidence of primary DNA lesions induced by two widely employed auxinic herbicides on C. decemmaculatus, namely 2,4-D and DIC, following long-term exposure.

Morpho and Cytological Differentiation of Calli of Eucalyptus grandis x Eucalyptus urophylla During Somatic Embryogenesis

ABSTRACT The aim of this study was to induce and analyze embryogenic calli from two types of explants (leaves and meristems) of the hybrid Eucalyptus grandis x Eucalyptus urophylla. Leaves and meristems of plants kept in a nursery were disinfected and inoculated in Petri dishes containing MS culture medium supplemented with different concentrations of the growth regulator dicamba (1.13, 4.52, and 9.04 µM) and without it. At 60 days of culturing, the calli were analyzed by scanning electron microscopy and at 90 days were evaluated by light microscopy in regard to the embryogenic characteristics of the cells. Different type of calli were induced in leaf explants, designated as Type I with light yellow coloring, Type II with dark yellow coloring, and Type III of brown coloring; however, only Type I had embryogenic characteristics. In the meristematic explants, only one type of callus was induced, and it had embryogenic characteristics. At 90 days of culturing, the formation of somatic embryos in the different embryogenic stages was observed and the formation of procambium, protoderm, and ground meristem tissues. At 150 days of culturing, the concentration of 1.13 µM of dicamba was prominent in the formation of somatic embryos in the different embryogenic stages.

Transcriptomic analysis of basidiocarp development in Ustilago maydis (DC) Cda.

Previously, we demonstrated that when Ustilago maydis (DC) Cda., a phytopathogenic basidiomycete and the causal agent of corn smut, is grown in the vicinity of maize embryogenic calli in a medium supplemented with the herbicide Dicamba, it developed gastroid-like basidiocarps. To elucidate the molecular mechanisms involved in the basidiocarp development by the fungus, we proceeded to analyze the transcriptome of the process, identifying a total of 2002 and 1064 differentially expressed genes at two developmental stages, young and mature basidiocarps, respectively. Function of these genes was analyzed with the use of different databases. MIPS analysis revealed that in the stage of young basidiocarp, among the ca. two thousand differentially expressed genes, there were some previously described for basidiocarp development in other fungal species. Additional elements that operated at this stage included, among others, genes encoding the transcription factors FOXO3, MIG3, PRO1, TEC1, copper and MFS transporters, and cytochromes P450. During mature basidiocarp development, important up-regulated genes included those encoding hydrophobins, laccases, and ferric reductase (FRE/NOX). The demonstration that a mapkk mutant was unable to form basidiocarps, indicated the importance of the MAPK signaling pathway in this developmental process.

Genotoxic effect of a binary mixture of dicamba- and glyphosate-based commercial herbicide formulations on Rhinella arenarum (Hensel, 1867) (Anura, Bufonidae) late-stage larvae.

The acute toxicity of two herbicide formulations, namely, the 57.71 % dicamba (DIC)-based Banvel(®) and the 48 % glyphosate (GLY)-based Credit(®), alone as well as the binary mixture of these herbicides was evaluated on late-stage Rhinella arenarum larvae (stage 36) exposed under laboratory conditions. Mortality was used as an endpoint for determining acute lethal effects, whereas the single-cell gel electrophoresis (SCGE) assay was employed as genotoxic endpoint to study sublethal effects. Lethality studies revealed LC5096 h values of 358.44 and 78.18 mg L(-1) DIC and GLY for Banvel(®) and Credit(®), respectively. SCGE assay revealed, after exposure for 96 h to either 5 and 10 % of the Banvel(®) LC5096 h concentration or 5 and 10 % of the Credit(®) LC5096 h concentration, an equal significant increase of the genetic damage index (GDI) regardless of the concentration of the herbicide assayed. The binary mixtures of 5 % Banvel(®) plus 5 % Credit(®) LC5096 h concentrations and 10 % Banvel(®) plus 10 % Credit(®) LC5096 h concentrations induced equivalent significant increases in the GDI in regard to GDI values from late-stage larvae exposed only to Banvel(®) or Credit(®). This study represents the first experimental evidence of acute lethal and sublethal effects exerted by DIC on the species, as well as the induction of primary DNA breaks by this herbicide in amphibians. Finally, a synergistic effect of the mixture of GLY and DIC on the induction of primary DNA breaks on circulating blood cells of R. arenarum late-stage larvae could be demonstrated.

Evaluation of the genotoxicity of a herbicide formulation containing 3,6-dichloro-2-metoxybenzoic acid (dicamba) in circulating blood cells of the tropical fish Cnesterodon decemmaculatus.

Acute toxicity and genotoxicity of the dicamba-based commercial herbicide formulation Banvel(®) were evaluated on Cnesterodon decemmaculatus (Pisces, Poeciliidae) exposed under laboratory conditions. A lethal effect was used as the end point for mortality, whereas frequency of micronuclei (MNs) and DNA single-strand breaks evaluated by the single cell gel electrophoresis assay were employed as end points for genotoxicity. Mortality studies revealed an LC50 96 h value of 1639 mg/L (range, 1471-1808) of dicamba. Furthermore, behavioral changes, e.g., gathering at the bottom of the aquarium, slowness in motion, abnormal swimming, and slow reaction, were observed. Whereas increased frequency of MNs was observed when 1229 mg/L dicamba was assayed for 48 h, no induction of MNs was observed in fish exposed to the herbicide for 96 h, regardless of the concentration of dicamba. Furthermore, other nuclear abnormalities, i.e., binucleated cells and lobed and notched nuclei, were induced in fish exposed for 48 h but not 96 h. Increase in the genetic damage index was observed in those treatments (lasting for both 48 and 96 h) within the 410-1229 mg/L dicamba concentration-range. This study represents the first evidence of acute lethal and sublethal effects exerted by dicamba on a piscine species native to Argentina. The results could indicate that dicamba-based formulation Banvel(®) is the less toxic emerging pollutant reported so far for C. decemmaculatus. Finally, our findings highlight the properties of this herbicide that jeopardize nontarget living species exposed to this agrochemical.

On-line monitoring of the photocatalytic degradation of 2,4-D and dicamba using a solid-phase extraction-multisyringe flow injection system.

A fully automated on-line system for monitoring the photocatalytic degradation of herbicides was developed using multisyringe flow injection analysis (MSFIA) coupled to a solid phase extraction (SPE) unit with UV detection. The calibration curves were linear in the concentration range of 100-1000 µg L(-1) for 3,6-dichloro-2-methoxybenzoic acid (dicamba) and 500-3000 µg L(-1) for 2,4-dichlorophenoxyacetic acid (2,4-D), while the detection limits were 30 and 135 µg L(-1) for dicamba and 2,4-D, respectively. The monitoring of the photocatalytic degradation (TiO2 anatase/UV 254 nm) of these two herbicides was performed by MSFIA-SPE system using a small sample volume (2 mL) in a fully automated approach. The degradation was assessed in ultrapure and drinking water with initial concentrations of 1000 and 2000 µg L(-1) for dicamba and 2,4-D, respectively. Degradation percentages of approximately 85% were obtained for both herbicides in ultrapure water after 45 min of photocatalytic treatment. A similar degradation efficiency in drinking water was observed for 2,4-D, whereas dicamba exhibited a lower degradation percentage (75%), which could be attributed to the presence of inorganic species in this kind of water.

Type II callus production and plant regeneration in tropical maize genotypes.

A total of 113 maize inbreds adapted to tropical conditions were evaluated for their tissue culture response. Additionally, four media combinations of 15 or 30 µM dicamba with or without 88 µM AgNO3 were used to study the effect of dicamba and AgNO3 on type II callus production and plant regeneration from 42 of the inbred lines. Inbreds 48, 389 and 1345 of the populations BR 105, BR 112, and Catete, respectively, showed a high capacity for type II callus production and plant regeneration. The production of type II calli increased significantly when the concentration of dicamba was changed from 15 to 30 µM and when AgNO3 was added to the medium. A synergistic effect between 88 µM AgNO3 and 30 µM dicamba (CM-30Ag medium) was observed, leading to additional production of type II callus. Medium CM-30Ag allowed the best tissue culture performance and plant regeneration capacity.