Biological evaluation of microbial toxin degradation by microinjected zebrafish (Danio rerio) embryos

Abstract

The use of microinjection of newly fertilized zebrafish eggs as an appropriate tool for qualifying the biodetoxification properties of toxin-degrading microbes was investigated. Ochratoxin A (OTA), bacterial degradation products of OTA and bacterial metabolites of the Cupriavidus basilensis ŐR16 strain were microinjected. Results showed that variations in the injected droplet size, and thus treatment concentrations, stayed within ±20%, moreover embryo mortality did not exceed 10% in controls, that is in accordance with the recommendations of the OECD 236 guideline. The highest lethality was caused by OTA with a significantly higher toxicity than that of bacterial metabolites or OTA degradation products. However, toxicity of the latter two did not differ statistically from each other showing that the observed mortality was due to the intrinsic toxicity of bacterial metabolites (and not OTA degradation products), thus, the strain effectively degrades OTA to nontoxic products. Sublethal symptoms also confirmed this finding. Results confirmed that microinjection of zebrafish embryos could be a reliable tool for testing the toxin-degrading properties of microbes. The method also allows comparisons among microbial strains able to degrade the same toxin, helping the selection of effective and environmentally safe microbial strains for the biodetoxification of mycotoxins in large scale.

Qualifying the T-2 Toxin-Degrading Properties of Seven Microbes with Zebrafish Embryo Microinjection Method

“…The first step was the determination of the T-2 toxicity baseline, which had to comply with the criteria described below. The maximum injectable volume for zebrafish embryos is 4.17 nL corresponding to a sphere diameter of 200 µm [46,47]. Concentration and injected volumes of mycotoxin should be selected, so that mortality values are interpretable above and below the baseline in every injected volume.…”

“…The second step was the examination of the toxicity of bacterial metabolites, which were tested with the same four injected volumes as the ones used for the analysis of mycotoxin baseline. Bacteria are able to produce toxic metabolites under normal living conditions [46]. The effects of these bacterial metabolites need to be examined.…”

“…Microinjection of zebrafish embryos (microinjector, capillary puller and parameters of capillary) was conducted as described by Csenki et al [46]. Briefly, one-cell embryos were injected with different volumes: sphere diameter of 52 µm corresponded to an injection volume of 0.074 nL, 150 µm to 1.77 nL, 180 µm to 3.05 nL and 200 µm to 4.17 nL.…”

“…This method uses microinjection to introduce Ochratoxin-A,ŐR16 primary and degradation metabolites at different volumes into newly fertilized zebrafish eggs and examine the lethal and sublethal effects on embryos at 72-and 120-h postinjection. Based on the results of this article, this method could be used for the classification of more bacterial strains by their degradation and detoxification capacity [46].…”

Mycotoxin Biodegradation Ability of the Cupriavidus Genus

“…Microinjection was conducted as described by Csenki et al . [ 35 ]. The changes were in the volume of injection, namely the sphere diameter of 50 µm corresponded to an injection volume of 0.074 nL, 75 µm to 0.22 nL, and 200 µm to 4.17 nL.…”