Developmental toxicity of the fungicide ziram in zebrafish (Danio rerio).

Ziram is a broad spectrum pesticide that belongs to the class of dimethyl-dithiocarbamate (DTC) fungicides. The objectives of this study were to assess the effects of ziram in developing zebrafish. Ziram was highly toxic to zebrafish embryos, with a 96-h LC50 value of 1082.54 nM (∼0.33 mg/L). Zebrafish embryos at 6 h post-fertilization (hpf) were exposed to solvent control (0.1% DMSO), or one dose of 1, 10, 100, and 1000 nM ziram for 96 h. Ziram induced lethality in a dose-dependent manner, decreased hatching rate and heartbeat, and caused wavy deformities at 72 and 96 hpf at 100 and 1000 nM. Basal oxygen consumption rates of zebrafish at 24 hpf were decreased with 1000 nM, suggesting that ziram affects oxidative phosphorylation. We also measured the expression of transcripts associated with the oxidative stress response (sod1 and sod2) and dopamine receptor signaling at ∼96 h of exposure. There was no difference in the expression of genes related to oxidative stress, nor those related to the dopamine system. Locomotor activity was also assessed in larval zebrafish (7 dpf), and ziram increased total activity, the velocity in light zone, and total distance moved at 10 nM, while it decreased the mean time spent in the dark zone at 1 and 10 nM. Behavioral responses were dependent upon the time point and clutch examined. These data demonstrate that ziram negatively impacts embryonic development (i.e. mortality, hatching, heartbeat and notochord development) of zebrafish, decreases basal respiration of embryos, and alters behavioral responses in larvae.

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

Developmental neurotoxicity of maneb: Notochord defects, mitochondrial dysfunction and hypoactivity in zebrafish (Danio rerio) embryos and larvae.

Broad applications and exposure to the fungicide maneb can lead to toxicity in non-target organisms. Maneb is also associated with neurogenerative diseases such as Parkinson's disease (PD). The objectives of this study were to determine the acute toxicity of maneb to zebrafish by measuring mitochondrial bioenergetics, locomotor activity, and the expression of genes related to the oxidative damage response, as well as those related to dopamine signaling due to its association with PD. Zebrafish embryos at 6 h post-fertilization (hpf) were exposed to either solvent control (0.1% DMSO, v/v), or one dose of 0.1, 0.5, 1.0 and 10.0 µM maneb for 96 h. Maneb was moderately toxic to zebrafish embryos, and had a 96-h LC50 value of 4.29 μM (~ 1.14 mg/L). Maneb induced a dose-dependent increase in mortality, decreased hatching rate, and increased notochord deformity rate at both 1.0 and 10.0 µM after 72 and 96 h. Total body length was also significantly reduced with 1.0 µM maneb. A 50-60% decrease in mean basal oxygen consumption rate was also observed in embryos following a 24 hpf exposure to 10.0 µM maneb but oligomycin-induced ATP production and FCCP-induced maximum respiration remained unaffected. No change was detected in the expression levels of genes associated with oxidative stress (sod1 and sod2), nor those related to dopamine synthesis (th1), dopamine transporter (dat), dopamine receptors (drd1, drd2a, drd3, and drd4b). Thus, modifying the expression of these transcripts may not be a mechanism for maneb-induced developmental toxicity in zebrafish. To assess the potential for neurotoxicity, a dark photokinesis assay was conducted in larvae following 7 d exposure to 0.1, 0.5 and 1.0 μM maneb. Larvae exposed to 0.5 and 1.0 μM maneb showed signs related to hypoactivity, and this reduced activity is hypothesized to be associated with notochord defects as this deformity was prevalent at higher concentrations of maneb. Overall, these data demonstrate that maneb negatively affects embryonic development (i.e. notochord development), affects basal oxygen consumption rates of embryos, and induces hypoactivity in larval fish. This study improves understanding regarding the developmental neurotoxicity of the fungicide maneb to zebrafish.

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

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 ... -  《-》

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 ... -  《-》