Teratological Effect of Indoxacarb on Chick Embryos (Gallus gallus domesticus)

Poultry provide nutritious food containing protein of good quality. About 50 billion birds are raised annually as a source of meat and eggs worldwide. Poultry is an important source of income and employment for farmers. It also provides raw materials to some industries. Different synthetic chemicals are indiscriminately used by farmers in routine agricultural practices including insecticides. When such hazardous chemicals come in contact with farm animals though air current or water may affect their health thus reducing growth ratio and causing loss to farmers.
        
Indoxacarb is a new category of chemical with a different type of chemical activity. (McKinley et al., 2002). It belongs to the oxadiazine compound group. Indoxacarb interferes with many ion channels and inhibits the movement of sodium ions into nerve tissues. Indoxacarb is advertised by industry as a “reduced-risk” pesticide and new substitute of organophosphates. It is a general scale foliar insecticide used to control insect pests in their larval stages. These agricultural pests include the beet root army worm, cotton boll worm, the cabbage looper and leaf-hoppers. Indoxacarb is a non-systemic pesticide and is advertised as a reduced risk pesticide by the U.S. EPA (U.S. EPA, 2000). Indoxacarb is a highly effective new DuPont insecticide with broad-spectrum control of larval pests (Dinter and Wiles, 1999). Indoxacarb controls most Lepidoptera, as well as certain members of other insect orders, including Coleoptera, Hemiptera, Diptera, Orthoptera and Hymenoptera (BASF, 2014). Indoxacarb is especially active on foliar feeding lepidopteran larvae (Wing et al., 2000). When lepidopteran larvae ingest sprayed foliage or sprayed directly, they stop feeding and suffer through mild convulsions or a passive paralysis with no recovery (Wing et al., 1998). Indoxacarb is highly efficacious against tarnished plant bug and may be a new tool in an integrated pest management program for this pest in cotton (Teague et al., 2000). In cockroaches, indoxacarb test at the LD₃₀, affected the vitellogenesis by a reduction of ovarian contents of proteins, carbohydrates and lipids (Djemaous et al., 2015). In cat fleas, egg production is significantly reduced; egg viability and flea emergence is also significantly affected due to application of Indoxacarb. There was significant decrease in overall protein content in mantle, foot, gills, digestive glands and whole body tissue of Parreysia cylindrica due to acute and chronic exposure to pesticide Indoxacarb (Patil, 2011). They are also harmful to estuarine/marine fish and invertebrates with EC50 (50 percent effective concentrations) reaching from 0.0542 to > 0.37 mg/L. Prolonged toxicities range from 0.0036 to 0.25 mg/L for freshwater fish and invertebrates and from 0.017 to 0.042 mg/L for estuarine fish and invertebrates (U.S. EPA, 2000). The fish were exposed to sub-lethal concentration for 8 days and the significant changes in the biochemical constituents of the vital organs viz. gill, liver, brain, muscle and kidney were reported. Several behavioral changes during the period of exposure were also observed and noted in animals (Veeraiah et al., 2013).

Seventy five fertilized eggs were collected from a local farm and the eggs were divided into four groups (A, B, C, D) comprising of 15 eggs per group. Group A was control group without treatment whereas group B, C and D were treated with different doses (0.5, 1.0 and 1.5µL/1000µL/egg) of Indoxacarb respectively. Indoxacarb was available in white, slightly viscous liquid.
        
Doses were administered at day-1 of incubation. For administration of pesticide, all the eggs in each group were selected randomly without considering the size and color. Eggs were wiped with a sterile cotton pad moistened with 70% alcohol and marked according to their groups. A small hole was made in the shell of each egg by modified “window technique” (Jelink, 1977), with the help of sterile needle. Shell membrane was not ruptured. All bits of egg shell were removed with an aspirator. The chemical was injected with needle (1 inch long no. 27) inserted straight into the yolk sac of the eggs of respective group. Immediately after the injection, the hole in the shell was wrapped with adhesive tape. In order to avoid contamination, the process of injections was performed in a room with disinfected atmosphere using formaldehyde vapors. A medium sized incubator model D-113600B11 (Huanghua Faithful Instrument Co. Ltd) having a capacity of 100 eggs was used. The shelves of the incubator were covered by a layer of cotton onto which eggs were placed, to avoid the direct contact of eggs with shelf surface. The treated eggs were kept in an incubator with large ends up. The eggs were rotated normally after every 8 hours of incubation and were kept at an optimum temperature of 38 ±0.5°C and relative humidity of 60%. Ventilator was made open. Eggs were candled every four days and dead embryos were culled out.
        
Experiments were terminated and recoveries were made at the 12^th day of incubation.
        
The mortality rate of embryos was recorded. Weight of the embryos was measured by digital balance (SF-400). Measurements of crown-rump length, head diameter (anterior-posterior), eyes diameter, neck length, beak length etc. were done by digital Vernier calipers while measurements of fore limbs and hind limbs were done with the help of scale and compass. Qualitative anomalies viz. Microcephaly, Hydrocephaly, Hematomas formation, Micropthalmia, short upper beak, agnatha, micromelia, amelia, omphalocele and Ectopia cardis were also recorded. The organs were studied with the help of magnifying lens and naked eye depending upon the size of the embryo. The observational data was tabulated for comparison of development with control group. Embryos from day 12 of incubation were macro-photographed by using Digital Sony Camera (DSC-W530). The obtained data of different morphometric parameters were presented as Mean ± S.E (Standard Error). One way ANOVA was applied to test statistical significance by using Statistical Package for Social Sciences (Version 21). Differences were considered significant when p≤0.05.

Mortality rate
 
After recovery on 12^th day, mortality rate was recorded. The embryos that received 0.5, 1.0 and 1.5µl/1000µl of Indoxacarb showed 40, 50 and 70% mortality respectively (Table 1). The highest mortality rate (70%) of chick embryos was found with 1.5µl/1000µl dose. Gamil et al., (2011) reported that percentage pupation and adult emergence were significantly less than their equivalent control in Egyptian cotton leaf worm and mortality was higher among embryos treated with higher doses. In eastern subterranean termites, abnormal behavior and mortality was observed (Quarcoo et al., 2010).
 

 
Morphometric measurements
 
The mean and standard error of measurements of different parameters viz. body weight, crown-rump length and eyes diameter, anterior-posterior head diameter beak length, neck length, humerus, radius and ulna, metacarpus, femur, fibula and metatarsus length were noted for viable 12-days-old treated and chick embryos of control group (Table 2).
 
  
 
Wet body weight was significantly decreased as dose of pesticide increases. The wet body weight of all treated embryos B, C and D were lower (3.05±0.93 g, 3.10±1.2g, 0.90±0.00 g respectively) than negative control (4.21±0.63 g).
        
Crown rump length and anterior posterior head diameter of treated embryos was also significantly different (p≤0.05) from control group. At higher doses (1.5µl) of Indoxacarb crown rump length showed greater reduction. Eye diameter of control and treated (0.5µl, 1.0µl) were similar. However, group D showed significant reduction in eye diameter.
        
Gamil et al., (2011) observed that the toxic signs were dose dependent, as they were quite rapid with the higher concentrations and slower with the lower concentrations. He also showed that younger instars of Spodopter alittoralis were more susceptible than older ones. Beak length also showed significant difference among experimental groups. In embryos receiving dose of 0.5µg/1000µl, the length of beak was reduced in comparison to control. Beak length is lowered in other two groups when compared to control but the mean beak length of 1.0µg and 1.5µg were similar. In some embryos of group D beak was absent that indicates obstruction of beak formation at higher dose. While in other it was significantly lowered. Decrease in mean neck length was not directly proportional to increase in concentration of dose. The mean value of neck length was decreased in treated groups but this reduction was not proportional to increase in dose. In 0.5µg and 1.5µg treated group the mean of neck length were similar while in 1.0µg treated groups the value was higher as compared to other groups. The average length of forelimb (humerus, radius and metacarpus) of chick embryos revealed significant difference (p<0.05) among all treated groups of Indoxacarb. At higher concentration (1.5µg/1000µl) humerus, radius and ulna, metacarpus showed greater reduction. In some embryos complete absence of forelimb was recorded while in some it was not developed properly. The mean hind limb length of chicks treated with Indoxacarb also declined significantly.
 
Qualitative anomalies
 
Embryos recovered from control group showed all normal body parts having normal eyes, beak, legs and external auditory apparatus (ear). The external morphology of normal chick embryos, on 12^th embryonic day, discussed here are somewhat analogous to some scientists (Mobarak, 2009; Bellairs and Osmond, 1998). The frequency percentage of different anomalies observed in Indoxacarb treated embryos at different doses were presented in Table 3. In this trial, Fig A shows microcephaly, micropthalmia, agnathia and amelia in embryos whereas Fig B shows hematomas formation, swelling around eyes, meromelia and short beak. The embryos treated with 0.5µg/1000ml also had abnormal body coloration. Fig C shows wry neck, microcephaly, hematoma formation and short beak whereas Fig D shows edematous swelling, short beak, swelling around eyes and hematoma on chick embryo. Generalized hematomas (extravasations of blood) were noticed in embryos treated 1.5 µg/1000ml.
 

 

 

 

 

 
Indoxacarb decreased the hemoglobin content, leukocyte and erythrocyte counts in mice. These effects could be due to adverse effects of insecticide on bone marrow or direct destruction of blood cells. Similar decrease in hemoglobin content and erythrocyte counts was also observed in sub chronic toxicity of rats and dogs (Malek, 1997).
        
In the present study, embryos treated with 1.5µg/100ml showed microcephaly, hematoma formation, short beak, meromelia, agnathia, micropthalmia and abnormal body colouration. Indoxacarb treated chick embryos showed considerably high ratios of external deformities and all deformed embryos revealed one type or 2-4 types of malformations.

Indoxacarb treated embryos that examined at 12^th day of development, significantly indicated high percentages (compared to controls) of external malformations and each malformed embryos exhibited one type or 2-4 types of malformations. Therefore, it is evident from the results obtained that Indoxacarb (anthranilic diamide) insecticide is potentially dangerous to the development of birds and may be hazardous to other animals including human beings. Therefore more research is needed to evaluate the impacts of Indoxacarb on other animals including humans.

The authors are thankful to the Dean of Scientific Research, King Saud University, Riyadh, Saudi Arabia, for funding the project through the research group Project no RGP-262.