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.

Comparative Assessment of the Toxicity of Brominated and Halogen-Free Flame Retardants to Zebrafish in Terms of Tail Coiling Activity, Biomarkers, and Locomotor Activity.

BDE-47, a flame retardant that is frequently detected in environmental compartments and human tissues, has been associated with various toxic effects. In turn, information about the effects of aluminum diethyl-phosphinate (ALPI), a halogen-free flame retardant from a newer generation, is limited. This study aims to assess and compare the toxicity of BDE-47 and ALPI to zebrafish by analyzing the tail coiling, locomotor, acetylcholinesterase activities, and oxidative stress biomarkers. At 3000 µg/L BDE-47, the coiling frequency increased at 26-27 h post-fertilization (hpf), but the burst activity (%) and mean burst duration (s) did not change significantly. Here, we considered that the increased coiling frequency is a slight neurotoxic effect because locomotor activity was impaired at 144 hpf and 300 µg/L BDE-47. Moreover, we hypothesized that oxidative stress could be involved in the BDE-47 toxicity mechanisms. In contrast, only at 30,000 µg/L did ALPI increase the catalase activity, while the motor behavior during different developmental stages remained unaffected. On the basis of these findings, BDE-47 is more toxic than ALPI.

Phenothiazinium dyes for photodynamic treatment present lower environmental risk compared to a formulation of trifloxystrobin and tebuconazole.

The widespread use of conventional chemical antifungal agents has led to worldwide concern regarding the selection of resistant isolates. In this scenario, antimicrobial photodynamic treatment (APDT) has emerged as a promising alternative to overcome this issue. The technique is based on the use of a photosensitizer (PS) and light in the presence of molecular oxygen. Under these conditions, the PS generates reactive oxygen species which damage the biomolecules of the target organism leading to cell death. The great potential of APDT against plant-pathogenic fungi has already been reported both in vitro and in planta, indicating this control measure has the potential to be widely used in crop plants. However, there is a lack of studies on environmental risk with ecotoxicological assessment of PSs used in APDT. Therefore, this study aimed to evaluate the environmental toxicity of four phenothiazinium PSs: i) methylene blue (MB), ii) new methylene blue N (NMBN), iii) toluidine blue O (TBO), and iv) dimethylmethylene blue (DMMB) and also of the commercial antifungal NATIVO®, a mixture of trifloxystrobin and tebuconazole. The experiments were performed with Daphnia similis neonates and zebrafish embryos. Our results showed that the PSs tested had different levels of toxicity, with MB being the less toxic and DMMB being the most. Nonetheless, the environmental toxicity of these PSs were lower when compared to that of NATIVO®. Furthermore, estimates of bioconcentration and of biotransformation half-life indicated that the PSs are environmentally safer than NATIVO®. Taken together, our results show that the toxicity associated with phenothiazinium PSs would not constitute an impediment to their use in APDT. Therefore, APDT is a promising approach to control plant-pathogenic fungi with reduced risk for selecting resistant isolates and lower environmental impacts when compared to commonly used antifungal agents.

Early Life Stage Assays in Zebrafish.

Fish embryo toxicity (FET) test using zebrafish (Danio rerio) has been established as an alternative assay to animal experimentation. The FET assay enables the assessment of multiple morphological endpoints during the development of zebrafish early life stages, showing high impact to the field of ecotoxicology on risk assessment of chemicals and pollutants. Moreover, it is also applied to screening drug-induced toxicity and human diseases, due to the high genetic and physiological orthology between zebrafish and humans. Here, we describe FET test, with all steps and several adaptations involved in the methodological procedures. To demonstrate the efficiency of this method, results using the reference substance 3,4-dichloroaniline (DCA) were included to demonstrate sublethal and teratogenic malformations on zebrafish embryos. Thus, there is a strong tendency for using FET tests as a replacement strategy of traditional tests in toxicology and ecotoxicology.

Acetylcholinesterase (AChE) Activity in Embryos of Zebrafish.

Acetylcholinesterase (AChE) is a useful biomarker for organophosphate and carbamate pesticides exposure. The inhibition of this enzyme has been associated with neurotoxicity and alterations at higher levels of biological organization, such as behavior and development impairments. In this chapter, we describe the methodologies for analyses of AChE activity in pools of 96 h of embryos of zebrafish (Danio rerio) using a spectrophotometric method adapted to 96-well microtiter plates.

A comparison of developmental toxicity of brominated and halogen-free flame retardant on zebrafish.

Brominated diphenyl ethers (BDEs) are halogenated flame retardants. Several concerns related to persistence and toxicity of BDEs have been resulted in a growing need of BDEs replacement. The use of halogen-free flame retardants (HFFR) has increased as a safer alternative, but little information is available on their toxic potential for environmental health and for developing organisms. Therefore, the aim of this study was to evaluate and compare the toxicity of three congeners of BDEs (BDE-47, BDE-99 and BDE-154) with an HFFR (aluminum diethylphosphinate, ALPI) on zebrafish (Danio rerio) by assessing endpoints of lethality, sub-lethality and teratogenicity at the earlier stages of development. The highest tested concentration of BDE-47 (12.1 mg/L) induced pericardium and yolk sac edemas that first appeared at 48 h post-fertilization (hpf) and then were mostly reabsorbed until 144 hpf. BDE-47 also showed a slight but non-significant tendency to affect swim bladder inflation. The rate of edemas increased in a concentration-dependent manner after exposure to BDE-99, but there were no significant differences. In addition, the congener BDE-99 also presented a slight and non-significant effect on swim bladder inflation, but only at the highest concentration tested. Regarding BDE-154 exposure, the rate of edemas and swim bladder inflation were not affected. Finally, in all ALPI exposure concentrations (0.003 up to 30 mg/L), no sub-lethal or teratogenic effects were observed on developing organisms until 96 hpf. Although further studies are needed, our results demonstrate that when comparing the developmental toxicity induced by flame retardants in zebrafish, the HFFR ALPI may be considered a more suitable alternative to BDE-47.

Ecotoxicological assessment of glyphosate-based herbicides: Effects on different organisms.

Glyphosate-based herbicides are the most commonly used worldwide because they are effective and relatively nontoxic to nontarget species. Unlimited and uncontrolled use of such pesticides can have serious consequences for human health and ecological balance. The present study evaluated the acute toxicity and genotoxicity of 2 glyphosate-based formulations, Roundup Original (Roundup) and Glyphosate AKB 480 (AKB), on different organisms: cucumber (Cucumis sativus), lettuce (Lactuca sativa), and tomato (Lycopersicon esculentum) seeds, and microcrustacean Artemia salina and zebrafish (Danio rerio) early life stages. For the germination endpoint, only L. esculentum presented significant sensitivity to AKB and L. sativa to Roundup, whereas both formulations significantly inhibited the root growth of all species tested. Both AKB and Roundup induced significant toxicity to A. salina; both are classified as category 3, which indicates a hazard for the aquatic environment, according to criteria of the Globally Harmonized Classification System. However, Roundup was more toxic than AKB, with 48-h median lethal concentration (LC50) values of 14.19 mg/L and 37.53 mg/L, respectively. For the embryo-larval toxicity test, Roundup proved more toxic than AKB for the mortality endpoint (96-h LC50 values of 10.17 mg/L and 27.13 mg/L, respectively), whereas for the hatching parameter, AKB was more toxic than Roundup. No significant genotoxicity to zebrafish larvae was found. We concluded that AKB and Roundup glyphosate-based formulations are phytotoxic and induce toxic effects in nontarget organisms such as A. salina and zebrafish early life stages. Environ Toxicol Chem 2017;36:1755-1763. © 2016 SETAC.

Ecotoxicity and environmental risk assessment of larvicides used in the control of Aedes aegypti to Daphnia magna (Crustacea, Cladocera).

Dengue transmitted by mosquitoes of the genus Aedes, species aegypti, is a major public health concern in Brazil. The chemical control of the mosquito larvae has been performed with the larvicide temephos since 1967. However, vector resistance was reported to temephos in several Brazilian states, and the Ministry of Health ordered the replacement of this larvicide by diflubenzuron (DFB), an inhibitor of chitin synthesis. Both insecticides are diluted in water with larvae and are able to reach aquatic environments in which they subsequently adversely damage nontarget organisms. The aims of this study were to (1) determine the acute toxicity (EC50) and environmental risk (RQ) of DFB and temephos to the microcrustacean Daphnia magna, and (2) evaluate the chronic toxicity (no-observed-effect concentration [NOEC] and lowest-observed-effect concentration [LOEC]) of these larvicides to D. magna. The experiments were performed according to a completely randomized design. The estimated 48-h EC50 of temephos was 0.15 µg/L (lower limit = 0.1 and upper limit = 0.2 µg/L) and the 48-h EC50 of DFB was 0.06 µg/L (lower limit = 0.03 and upper limit = 0.1 µg/L). RQ values were 4.166.7 to DFB and 6.666.6 to temephos. NOEC and LOEC values were respectively 2.5 and 5 ng/L for DFB, and respectively 6.2 and 12.5 ng/L for temephos. Thus, temephos and DFB are classified as highly toxic to Daphnia magna and pose a high environmental risk to this species. Mortality of D. magna was observed at concentrations lower than those used in the field to control A. aegypti larvae.