Does Mosquito Control Spray Harm Embryonic Development?

1. Ethics statement

This experiment strictly followed the guidelines of Institutional Animal Care and Use

Committee and were conducted at the College of Veterinary Medicine, University of Georgia, Athens, GA, USA.

2. Egg collection

[Diagnostic & Research Center of University of Georgia, Athens, GA. Eggs of approximately similar size between 50 grams to 60 grams were selected and distributed into four groups (n = 20 per group): (i) Control group with no injection, (ii) 10μg BN group, (iii) 50μg BN group, and (iv) 150 μg BN group. All eggs are placed in the incubator (Brinsea Mini Advance) set at 37±0.5°C (avian body temperature) until they are ready to be administered BN.

3. Preparations of Bifenthrin solution  

A stock solution of Bifenthrin at a concentration of 1 mg/mL was prepared by dissolving 10 mg of BN in 1 mL of DMSO, then diluting in 9 mL of saline to avoid any undesired toxicity caused by DMSO. Our study dosages of BN were then drawn from the stock solution with adjusting volumes that match the target concentrations, then performed serial dilution.

4. Dose administration

All eggs at 48h of incubation (embryonic day 2) were weighted to divide into groups of eggs that weigh approximately equal in size, and then sterilized by 70% ethanol and Kim wipe to wipe off visible dirts, and contaminants under normal laboratory conditions with dry air to assist the evaporation of alcohol and prevent it from disturbing the egg shell. The prolate end of each egg is then pierced by egg pierced tool, and a 18-gauge needle is used to remove 1 mL of the albumen, then using 3M Tegaderm to cover the pierce to prevent dehydration or airborne contamination until ready to be administered BN. The target concentrations of Bifenthrin of our study are 10μg, 50μg, and 150μg, hence the volumes of injection are 10μL, 50μL, and 150μL, respectively as follow the solution dilution formula. Bifenthrin environmental concentrations are detected at about 0.11 mg/kg (Zhang et al., 2020), and therefore we allotted the concentration of 10μg to be relevant to the environmental concentration, and 150μg to be our highest concentration. Each dosage of BN is then administered to 20 eggs per group using sterilized small needle injection at the existing pierce on the prolate end. The site of injection was closed using a fresh 3M Tegaderm, then weighted to collect the weight after injection and placed in the incubator.

5. Embryo collection and observation

Embryos were collected one week after the injection (embryonic day 9) and weighted on the scale to obtain final weight and compared to the initial weight after injection. Using fine scissors and tapes, we cut a small window on the egg shell to expose the chicken embryo, then transferred the whole embryonic content into warm Syracuse glass dishes with CMRL media with the chick embryos facing upward to allow observation of their heart without disrupting their body system. The chick heart rate was measured using Olympus SZX9 microscope with camera recording the beating heart within a 60-second interval. Any observation of heart arrhythmias or deformities were also recorded. After this process, the chicken embryo would then be extracted from other embryonic content using an egg spoon, forceps, and micro dissecting scissors to isolate the body of the chick embryo from the chorioallantoic membrane. We, then, observed any morphological defects in the BN-treated groups and compared to the control group with no injection. To quantify the overall development of chick embryos, we measured the body length of each group using small-increment rulers. Then, finding the means of HR, and body length of four groups to perform a one-way ANOVA test to determine the difference in the control group and the other BN-treated groups.

We predicted that increasing concentrations of Bifenthrin on chicken embryos would affect the heart rate, body length, and the length of the top of the eye to the top of the head. The results from this study show that at high, medium, and low concentrations of Bifenthrin slowed down the heart rate significantly. Eggs injected with higher concentration of Bifenthrin had a much slower heart rate than the medium and low concentrations. We found no evidence to support that Bifenthrin impacted body length. However, the head development measured from top of eye to top of head, was influenced by the amount of Bifenthrin injected. These results show that Bifenthrin does have an effect on heart rate, and morphological development. 

Another study carried out on HoneyBees showed that Bifenthrin decreased the bees' fecundity, slowing down the rate at which they matured (Dai et al., 2010). In our research the eggs injected with high concentration of Bifenthrin (15μl) had a slower heart rate, larger head development. This suggests that Bifenthrin could over-stimulate the head to grow, while also slowing down the heart rate. Another study looking at the effects of Bifenthrin on aquatic species found that Bifenthrin exhibits high acute toxicity. Bifenthrin caused developmental toxicity along with neurobehavioral toxicity on non-target organisms (Yang, Wu, & Wang, 2018). Likewise a study conducted on two-spotted spider mites revealed that Bifentrhin decreased their reproductive rate, which suggests that Bifentrhin can also decrease overall development (Zhang et al., 2015). Following the injection of the 15μl Bifenthrin eggs on average they had larger head development when compared to the 10μl (medium) and 01μl (low) concentration of Bifenthrin. The 15μl eggs also had a slower heart rate when compared to the control and other treatment groups. These results indicate that Bifenthrin has a negative impact on embryonic development. 

One of the major limitations on this research was the sample size. Over the course of 6 weeks only 43 eggs were injected with different concentrations of Bifenthrin (14 eggs of low, 14 eggs for medium and 15 eggs for high)  and 9 were used as a control. The small sample size can lead to inaccurate conclusions. This study provides good ground work for future studies to improve upon. For future studies we would like to see this experiment carried out again with more trials and larger sample size. Along with that, observation of the chicken embryos at different stages of development. Injecting the embryos with different concentrations of BF to coincide with different levels found in the environment. 

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