Comparative Lipid Peroxidation and Apoptosis in Embryo‐Larval Zebrafish Exposed to 3 Azole Fungicides, Tebuconazole, Propiconazole, and Myclobutanil, at Environmentally Relevant Concentrations

Abstract

Azole fungicides have entered the aquatic environment through agricultural and residential runoff. In the present study, we compared the off‐target toxicity of tebuconazole, propiconazole, and myclobutanil using embryo‐larval zebrafish as a model. The aim of the present study was to investigate the relative toxicity of tebuconazole, propiconazole, and myclobutanil using multiple‐level endpoints such as behavioral endpoints and enzymatic and molecular biomarkers associated with their mode of action. Zebrafish embryos were exposed to azoles at environmentally relevant and high concentrations, 0.3, 1.0, and 1000 µg/L, starting at 5 h postfertilization (hpf) up to 48 hpf, as well as 5 d postfertilization (dpf). Relative mRNA expressions of cytochrome P450 family 51 lanosterol‐14α‐demethylase, glutathione S‐transferase, caspase 9, phosphoprotein p53, and BCL2‐associated X protein were measured to assess toxicity attributable to fungicides at the mRNA level, whereas caspase 3/7 (apoptosis) and 3,4‐methylene​dioxy​amphetamine (lipid peroxidation) levels were measured at the enzymatic level. Furthermore, mitochondrial dysfunction was measure through the Mito Stress test using the Seahorse XFe24 at 48 hpf. In addition, light to dark movement behavior was monitored at 5 dpf using Danio Vision® to understand adverse effects at the organismal level. There was no significant difference in the light to dark behavior with exposure to azoles compared to controls. The molecular biomarkers indicated that propiconazole and myclobutanil induced lipid peroxidation, oxidative stress, and potentially apoptosis at environmentally relevant concentrations (0.3 and 1 µg/L). The results from the mitochondrial respiration assay indicated a slight decrease in spare respiratory capacity with an acute exposure (48 hpf) to all 3 azoles at 1000 µg/L. Based on the present results, propiconazole and myclobutanil are acutely toxic compared to tebuconazole in aquatic organisms at environmentally relevant concentrations. Environ Toxicol Chem 2019;38:1455–1466. © 2019 SETAC

Isoliquiritigenin induces oxidative stress and immune response in zebrafish embryos

“…The excessive production of ROS activates inflammation. 24 In the immune system, iNOS is an early response enzyme in the inflammatory process and is often used as an inflammatory marker. 25 Its main product, NO, is a mediator of inflammation and immune regulation.…”

Toxicity Testing of Effluent-Dominated Stream Using Predictive Molecular-Level Toxicity Signatures Based on High-Resolution Mass Spectrometry: A Case Study of the Lubbock Canyon Lake System

“…At the end of the 5 dpf exposure period, zebrafish larvae from individual replicates were euthanized by prolonged submersion in 200 mg/L of tricaine methanesulfonate at a pH of 7. Details of total RNA extraction and cDNA synthesis are discussed elsewhere . RT-PCR was performed to determine the mRNA expression of genes from the predicted pathways based on our proposed workflow (Table S9).…”

“…Details of total RNA extraction and cDNA synthesis are discussed elsewhere. 46 RT-PCR was performed to determine the mRNA expression of genes from the predicted pathways based on our proposed workflow (Table S9). Fold change in transcript expression was determined using 2 −ΔCt and presented as fold ±SE relative to control.…”

Developmental Neurotoxicity and Behavioral Screening in Larval Zebrafish with a Comparison to Other Published Results

“…Using the zebrafish model, we had two main goals for this study: to screen a library of chemicals for developmental neurotoxicity, and to compare our findings with previously published results for those chemicals. The specific chemical library was chosen because (1) some of the chemicals have been associated with developmental neurotoxicity in mammals [ 26 ]; (2) many of the chemicals were tested by other investigators within the U.S. Environmental Protection Agency (EPA) using in vitro assays for developmental neurotoxicity potential [ 27 , 28 , 29 ]; and/or (3) some of the chemicals have been tested by investigators external to EPA using zebrafish assays [ 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 , 57 , 58 , 59 , 60 , 61 , 62 , 63 ]. The first aspect of this study was to screen the library of 61 chemicals in zebrafish embryos/larvae to determine if the chemical (maximum nominal concentration = 120 µM) produced developmental toxicity (lethality, non-hatching or malformations) and/or neurotoxicity (changes in larval locomotor activity).…”