Effects of Chlorpyrifos on Chicken Embryo Development

Allison Himiob, Srushti Chavan, Chris Wang

Introduction

•Organophosphate pesticides, such as chlorpyrifos (CHF), are known to bioaccumulate, disturb environmental factors, and show neurotoxicity effects, yet are highly used in agricultural and house products.1

•Among these pesticides, CHF is the most abundant active ingredient in most insecticides to agriculturally control a broad spectrum of insects.2

•The metabolism of CHF inhibits acetylcholinesterase (AChE) interactions with cholinergic nerve synapses in mammals, resulting in an overactivation of cholinergic receptors.3

•Studies have shown bone and limb malformations, reduced brain size, and heart arrhythmias in chicken embryos exposed to CHF.4

•EPA banned the use of CHF, but the Eighth Circuit Court objected to the lack of evidence and lifted the ban.5

•We hypothesize low levels of chlorpyrifos slow embryo heart rate, reduce embryo size, and affect blood content.

Methods

Eggs were injected with chlorpyrifos in the air sac and incubated for 7 days at 37ºC. After incubation, heart rate, RBC formation, embryo weight, body length, and head were observed and recorded.

Heart rate: Used the Egg Buddy and recorded 3 times in 10-second intervals then the average was taken

RBC Blood Smear: Blood was collected, smeared onto a slide, and stained.

Embryo body and head length: Photos of embryos were captured and uploaded to ImageJ to measure.

Results

A total of 60 eggs were used in this experiment to observe the effects of nontoxic amounts of chlorpyrifos on chick embryo development. We observed the most significant effect of chlorpyrfios on heart rate and with some signficance on body length. Other variables showed little to no indication of a statistical relationship between chlorpyrifos and chick embryo development. All figures are presented with outliers but p-value statistics are calculated after removing outliers.

Heartrate

Heart rate showed the most statistically significant results out of all the variables tested (p-value: > 0.01). We observed a negative trend between increasing concentrations of chlorpyrfios injected and heart rate measured with large statistical differences between the control to low concentration, low concentration to medium concentartion, and medium concentration to high concentration.

Body Length

Body length data suggested some statistical relationship between chlorpyrifos and the measured length of chick embryos (p-value = 0.15). We observed the lengths of the chick embryos to actually increase within the treatments groups compared to the control group, but no clear conclusions can be made at this time.

Head Length

Head size data suggested little to no relationship between chlorpyrifos and the measured length of chick embryos (p-value = 0.34). We also observed no trend in head size as concentrations of chlorpyrifos increased.

Rate of Development

The rates of development measured between treatment groups suggested little to no statistical relationship between chlorpyrifos and the measured length of chick embryos (p-value = 0.26). While we didn't observe any quantitative trends in our experiment, we did notice that as concentrations of chlorpyrifos increased, development became more abnormal, with more malformations and weaker, and more fragile embryonic bodies.

Viability Rates

The viability rates measured between treatment groups suggested little to no statistical relationship between chlorpyrifos and the measured length of chick embryos (p-value = 0.33). We also observed no clear trend between treatment groups suggesting some sort of relationship.

Red blood Cell (RBC) Counts

RBCs were unchanging between treatment groups and control. Figure A represents control group, Figure B represents low concentration group (0.01 µg), Figure C represents medium concentration group (0.05 µg), and Figure D represents high concentration group (0.1 µg). We observed no malformations or abnormal developments between all groups suggesting chlorpyrifos had no effect on the RBC count of the chick embryos. We did observe most of the red blood cells to be immature in every treatment group.

Discussion & Conclusion

The aim of this study was to examine the impact of different concentrations of Chlorpyrifos on chicken embryonic development. The results of this experiment show that chlorpyrifos affects the heart rate and body length of chicken embryos, but does not affect body weight, head length, or RBC production.

As we predicted, we found the heart rate of chicken embryos is negatively correlated with the concentration of chlorpyrifos. This indicates that chlorpyrifos has a significant negative effect on the cardiovascular system. In other studies, the combined use of chlorpyrifos and cypermethrin led to higher probability of congenital heart defects in chicken embryos(Verma et al., 2020). Therefore, we suspect that one of the target organs of Chlorpyrifos is the heart. However, there still need more research to confirm this point.

We observed that body length was affected by Chlorpyrifos, but no trend was observed. Further research is still needed to investigate the association between Chlorpyrifos and body length. We have no evidence to support the idea that Chlorpyrifos affects head length and weight.

We found chlorpyrifos had no effect on RBC formation, but we saw many immature RBCs in the blood smear. According to another study, the RBC counts of broiler chicken treated with different concentrations of chlorpyrifos decreased with increasing concentration (Ahmad et al., 2014). According to the study, we suspect that because the incubation time of the chicken embryos we used was insufficient, their hematopoietic function was still incomplete, and therefore Chlorpyrifos did not affect RBC formation. To prove this, in future study we will let them incubate for a longer time, so that they can fully develop their internal organs.

Chlorpyrifos can cause chronic environmental damage, and as a pesticide, it can remain on crops and be ingested by humans. Children and even infants are still developing, and their systems are not yet fully formed, which means they are more vulnerable to the harmful effects of Chlorpyrifos. Therefore, determining a safer dose of chlorpyrifos is of great significance for protecting the environment and the healthy development of infants and young children.

Reference

1. Nandhini, A. R., Harshiny, M., & Gummadi, S. N. (2021). Chlorpyrifos in environment and food: a critical review of detection methods and degradation pathways. Environ Sci Process Impacts, 23(9), 1255-1277. https://doi.org/10.1039/d1em00178g

2. Das, S., & Adhya, T. K. (2015). Degradation of chlorpyrifos in tropical rice soils. Journal of Environmental Management, 152, 36-42. https://doi.org/https://doi.org/10.1016/j.jenvman.2015.01.025

3. Lockridge, O. (2013). Chapter Five - Noncholinesterase Protein Targets of Organophosphorus Pesticides. In J. C. Fishbein & J. M. Heilman (Eds.), Advances in Molecular Toxicology (Vol. 7, pp. 179-205). Elsevier. https://doi.org/https://doi.org/10.1016/B978-0-444-62645-5.00005-5

4. Chandra Sekaran, S. P., Thotakura, B., Jyothi, A. K., Manickam, S., Chanemougavally, J., Prabhu, K., & Gopalan, D. H. (2023). Effect of chlorpyrifos and its metabolites on skeletal system development of chick embryo. Birth Defects Research, 115(11), 1063-1078. https://doi.org/https://doi.org/10.1002/bdr2.2170

5.https://www.epa.gov/ingredients-used-pesticideproducts/chlorpyrifos#:~:text=2021%20%E2%80%93%20Final%20Tolerance%

20Rule,to%20reflect%20the%20tolerance%20revocation.

6. Ahmad, M. Z., Ahrar, K., Tariq, J. M., & Hussain, I. (2014, December 31). Impact of chlorpyrifos on health biomarkers of Broiler Chicks. Pesticide Biochemistry and Physiology. https://www.sciencedirect.com/science/article/pii/S004835751400251X

7. Verma U;Khaire K;Desai I;Sharma S;Balakrishnan S; (n.d.). Early embryonic exposure to chlorpyrifos-cypermethrin combination induces pattern deficits in the heart of domestic hen. Environmental toxicology. https://pubmed.ncbi.nlm.nih.gov/33270332/

Authors

Srushti Chavan

770-378-5415

srushtichn@gmail.com

Allison Himiob

770-508-6396

allisonhimiob@gmail.com

Chris Wang

718-559-9259

xintun0510@gmail.com

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