Atrazine is one of the most commonly used herbicides in the United States and is frequently found in freshwater ecosystems due to agricultural runoff. Its persistence in aquatic systems raises concerns about its impact on aquatic life, particularly during sensitive stages of development. This study examines the effects of short-term atrazine exposure on zebrafish (Danio rerio) embryos at specific developmental stages. Embryos were exposed to atrazine concentrations of 0.3 ppb, 3 ppb, and 30 ppb for 2 hours at four critical time points after fertilization (1–2 hpf, 6 hpf, 10 hpf, and 24 hpf). The primary outcomes measured were survival rates and hatching success. The results indicate that the effects of atrazine are stage-specific, with increased mortality and reduced hatching rates observed at the vulnerable developmental periods 6 hpf and 10 hpf. These findings support the hypothesis that even brief exposures to environmentally relevant concentrations of atrazine can have a negative impact on zebrafish embryonic development.

• Atrazine is a widely used herbicide that often contaminates aquatic environments.

• Zebrafish (Danio rerio) are a well-established model for studying vertebrate development and toxicology.

• Previous studies have shown that chronic atrazine exposure affects zebrafish behavior, reproduction, and morphology.

• Less is known about the effects of short-term, stage-specific exposure on early development, which this study aims to explore.

• Short-term, low-dose exposures especially during early development may lead to lasting physiological or behavioral effects even in the absence of visible malformations.

• Atrazine concentrations: 0.3 ppb, 3 ppb, and 30 ppb

• Exposure windows: 1–2 hpf, 6 hpf, 10 hpf, 24 hpf

• Exposure duration: 2 hours at each time point

• Post-treatment procedure: After exposure, embryos were thoroughly rinsed with water and returned to clean water to continue development.

• Zebrafish embryos were obtained from wild-type breeding pairs.

• Embryos were collected at the 1-cell stage and sorted under a microscope to separate fertilized from unfertilized eggs. 

• Embryos were housed in 6-well plates with 15 embryos per well in 5 ml of water. 

• Embryos were exposed for 2 hours at one of four developmental time points and then rinsed and placed back in clean water.

• Embryos were monitored for survival at 24 and 48 hpf, and hatching was assessed at 72 hpf.

• Data was recorded, and survival and hatching rates were calculated and analyzed to assess exposure effects.

Figure 1: Survival Rate (%) at 24 hpf

• Survival was highest in the control group (~99%).

• Treatments starting at 6 hpf - 0.3 ppb & 3 ppb showed a drop in survival (~86%).

• Noticeable dip in survival in treatments starting at 6 hpf and 10 hpf, especially at lower concentrations.

Figure 2: Survival Rate (%) at 48 hpf

• Most groups had survival rates above 90%.

• Treatments starting at 6 hpf – 3 ppb and 10 hpf – 3 ppb showed lower survival (~80–75%).

• Dip in survival seen in treatments starting at 6 hpf and 10 hpf, especially at 3 ppb.

Figure 3: Hatching Rate (%) at 72 hpf

• Control group showed the highest hatching rate.

• Lower hatching rates observed in treatments starting at 6 hpf – 3 ppb and 30 ppb.

• Lowest hatching seen in treatments starting at 6 hpf – 30 ppb.

• Lower survival observed in treatments starting at 6 hpf – 3 & 30 ppb (24 hpf) and 10 hpf – 3 ppb (48 hpf).

• The largest dips in survival and hatching occurred in treatments starting at 6 hpf and 10 hpf

• These stages coincide with key developmental events such as germ layer formation, axis specification, and early organ patterning, which could make embryos more susceptible to disruption.

• Both timing and dose influenced developmental outcomes.

• Conduct gene expression analysis on genes related to oxidative stress, endocrine disruption, and early morphogenesis.

• Measure developmental malformations such as body length and head size.

• Conduct behavioral assays at later developmental stages to assess potential neurological or motor effects of early atrazine exposure.

Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., & Schilling, T. F. (1995). Stages of embryonic development of the zebrafish. Developmental Dynamics, 203(3), 253–310. https://pubmed.ncbi.nlm.nih.gov/8589427/

Wirbisky, S. E., Weber, G. J., Sepúlveda, M. S., Lin, T.-L., Jannasch, A. S., & Freeman, J. L. (2016). An embryonic atrazine exposure results in reproductive dysfunction in adult zebrafish and morphological alterations in their offspring. Scientific Reports, 6(1), 21337. https://doi.org/10.1038/srep21337

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