Sub-lethal toxicity assessment of the phenylurea herbicide linuron in developing zebrafish (Danio rerio) embryo/larvae.

Due to run-off and rain events, agrochemicals can enter water catchments, exerting endocrine disruption effects and toxicity to aquatic organisms. Linuron is a phenylurea herbicide used to control a wide variety of vegetative weeds in agriculture in addition to residential applications. However, there are few studies that quantify its toxicity to early developmental stages of fish. The objectives of this study were to assess the acute toxicity of linuron to zebrafish embryos/larvae by measuring mortality, morphological deformities, oxidative respiration, gene expression, and locomotor activity via the Visual Motor Response test. Zebrafish embryos at ~6-h post-fertilization (hpf) were exposed to either embryo rearing medium (ERM), or one dose of 0.625, 1.25, 2.5, 5, and 10 μM linuron for up to 7 days post-fertilization (dpf) depending on the assay. Zebrafish larvae exposed to linuron displayed pericardial edema, yolk sac edema, and spinal curvature. Oxidative respiration assessments in embryos using the Agilent XFe24 Flux Analyzer revealed that linuron decreased mean basal respiration and oligomycin-induced ATP-linked respiration in 30 hpf embryos at 20 μM after a 24-hour exposure. In 7 dpf larvae, transcript abundance was determined for 6 transcripts that have a role in oxidative respiration (atp06, cox1, cox4-1, cox5a1, cytb, and nd1); the relative abundance of these transcripts was not altered with linuron treatment. A Visual Motor Response test was conducted on 7 dpf larvae to determine whether linuron (0.625 to 5 μM) impaired locomotor activity. Larval activity in the dark period decreased in a dose dependent manner and there were indications of hypoactivity as low as 1.25 μM. Transcript abundance was thus determined for tyrosine hydroxylase (th1) and glutamic acid decarboxylase 67 (gad1b), two rate limiting enzymes that control the production of dopamine and gamma-aminobutyric acid respectively. The mRNA levels of gad1b (p = 0.019) were reduced with increasing concentrations of linuron while th1 (p = 0.056) showed a similar decreasing trend, suggesting that neurotransmitter biosynthesis may be altered with exposure to linuron. This study improves knowledge related to the toxicity mechanisms for linuron and is the first to demonstrate that this anti-androgenic chemical impairs oxidative respiration and exerts neurotoxic effects associated with neurotransmitter biosynthesis during early development. These data are significant for environmental risk assessment of agrochemicals.

Maharaj S ，El Ahmadie N ，Rheingold S ，El Chehouri J ，Yang L ，Souders CL 2nd ，Martyniuk CJ ... -  《-》

Assessing sub-lethal effects of the dinitroaniline herbicide pendimethalin in zebrafish embryos/larvae (Danio rerio).

Pendimethalin is a dinitroaniline herbicide used to control broadleaf weeds by inhibiting the formation of microtubules during cell division. Its use on a variety of crops leads to its potential entry into aquatic environments, but little is known about its sub-lethal toxicity to early developmental stages of aquatic vertebrates. To address this knowledge gap, we assessed the toxicity of pendimethalin to zebrafish embryos and larvae by measuring mortality, developmental abnormalities, oxidative respiration, reactive oxygen species, gene expression, and locomotor activity following exposure to the herbicide throughout early development. Embryos at ~6 h post-fertilization (hpf) were exposed to either a solvent control (0.1% DMSO, v/v), embryo rearing medium (ERM), or one dose of either 1, 2.5, 5, or 25 μM pendimethalin for up to 7-days post fertilization depending on the bioassay. Exposure to 25 μM pendimethalin resulted in high prevalence of spinal curvature, tail malformations, pericardial edema, and yolk sac edema at 4 dpf, while exposure to 5 μM pendimethalin induced pericardial edema and lordosis in the fish exposed over 7 dpf. Exposure to pendimethalin up to 5 μM did not negatively impact oxidative respiration (e.g., basal respiration, oligomycin-induced ATP production) in embryos following a 24-h exposure. Pendimethalin did not induce reactive oxygen species at concentrations of 1-5 μM. Levels of transcripts associated with oxidative respiration and damage response were altered in 7d-larvae; cox1 mRNA was increased in larvae fish exposed to 1 μM while cox5a1 and sod2 mRNA were decreased with 2.5 μM exposure. The Visual Motor Response (VMR) test for light-dark response revealed that larval activity in the dark period was reduced for zebrafish exposed to >1 μM pendimethalin compared to ERM and DMSO solvent control groups. These data inform on the sub-lethal toxicity of pendimethalin to early stages of fish embryos and larvae.

Wang S ，Lopez S ，El Ahmadie N ，Wengrovitz AS ，Ganter J ，Zhao YH ，Souders CL 2nd ，Martyniuk CJ ... -  《-》

Investigation into the sub-lethal effects of the triazole fungicide triticonazole in zebrafish (Danio rerio) embryos/larvae.

Global use of azole fungicides is expected to increase over the next several years. Triticonazole is a triazole fungicide that is used for turf protection, residential, and other commercial applications. As such, it can enter local rural and urban water systems via run-off and rain events. Early life stages of aquatic organisms can be susceptible to pesticides that enter the water, but in the case of triticonazole, data on the potential for subacute toxicity are lacking. Here, we determined the effects of triticonazole on development, oxygen consumption rates, and locomotor activity in zebrafish to address this knowledge gap. Wild-type zebrafish (ABTu strain) embryos and larvae were exposed to triticonazole (1-100 μM) in early development for different lengths of time depending on the assay conducted. Triticonazole did not affect survival nor induce significant deformity (pericardial edema, skeletal defects) in zebrafish at doses up to 100 μM. Oxygen consumption rate was measured in embryos after 24 and 48 hour exposure to triticonazole beginning at ∼6 hpf using the XFe flux analyzer. Triticonazole did not affect basal respiration, oligomycin-induced ATP linked respiration, FCCP-induced maximum respiration, proton leak, spare capacity, nor non-mitochondrial respiration at doses up to 100 μM for 24 hours, even for exposure up to 250 μM for 48 hours. To determine whether the fungicide affected larval swimming activity, the visual motor response test was conducted following triticonazole exposure for 6 days. Larval zebrafish exposed to triticonazole showed hypoactivity in the dark following a 100 μM treatment, suggesting that the fungicide can affect the locomotor activity of zebrafish, albeit at relatively high levels. Given the fact that sublethal biological responses were absent at lower environmentally relevant concentrations, we conclude that triticonazole, relative to other triazole fungicides and types of pesticides, exhibits a relatively low risk of toxicity to the early life stages of fish.

Souders CL 2nd ，Perez-Rodriguez V ，El Ahmadie N ，Zhang X ，Tischuk C ，Martyniuk CJ ... -  《-》

Developmental toxicity of the triazole fungicide cyproconazole in embryo-larval stages of zebrafish (Danio rerio).

Cao F ，Souders CL 2nd ，Li P ，Pang S ，Qiu L ，Martyniuk CJ ... -  《-》

Sub-lethal effects of the triazole fungicide propiconazole on zebrafish (Danio rerio) development, oxidative respiration, and larval locomotor activity.

Propiconazole is a triazole fungicide used in agriculture. Via run-off, it can enter the aquatic environment, and can adversely affect organisms. However, data are scarce on how propiconazole may affect early developmental life stages of fish. The objectives of this study were to evaluate the potential sub-lethal effects of propiconazole during zebrafish development. Wildtype zebrafish (ABTu strain) embryos and larvae were exposed to propiconazole (0.1-100 μM) for up to 150 hours post fertilization (hpf) depending upon the endpoint measured. Propiconazole decreased survival and induced hypopigmentation in fish at 100 μM compared to the water and solvent controls. Pericardial edema was also noted in embryos and larvae (beginning at 2-3 dpf) exposed to 100 μM propiconazole. To visualize the effects of propiconazole on the circulatory system in more detail, we exposed transgenic zebrafish (globin-LCR:eGFP) to the fungicide. Hematopoietic changes were observed within 48 h of exposure to 100 μM, and localization of blood cells in the cardic region became diffuse, indicating pooling of blood in the pericardial region. We measured oxidative respiration in embryos as sufficient ATP is needed for development. Exposure to 100 μM propiconazole (~6-30 hpf) reduced basal respiration (~50%), oligomycin-induced ATP linked respiration (~70%), proton leak (~30%), and non-mitochondrial respiration (~50%), indicating compromised mitochondrial bioenergetics. A Visual Motor Response (VMR) test was used to measure dark photokinesis behavior in larval fish exposed to propiconazole for a 6-day period. Larval fish exposed to the highest concentration in the assay (10 μM) showed evidence of hypoactivity. This study demonstrates that propiconazole can induce hypopigmentation in zebrafish, disrupt mitochondrial bioenergetics, and can alter locomotor activity. However, these sub-lethal responses were observed at concentrations above what is typically detected in the environment.

Souders CL 2nd ，Xavier P ，Perez-Rodriguez V ，Ector N ，Zhang JL ，Martyniuk CJ ... -  《-》