Undergraduate Research and Creativity

URECA

2007-2008

Using in-ovo EROD Activity in the Embryos of Japanese Medaka
(Oryzias latipes) as a Sensitive Measure of Organic Contaminants in Sediment

John Gondek, Bingqi Cheng and Anne McElroy
Aquatic Toxicology Lab
School of Marine and Atmospheric Sciences

The study of contamination in marine ecosystems is important in understanding anthropogenic effects on the environment. Many contaminated soils contain agonists such as PCB’s and PAH’s. These toxicants are metabolized by the induction of CYP450 via the aryl hydrocarbon (Ah) receptor. Activity of this metabolic pathway can be measured through the fluorescent product, resorufin. This is produced by the enzyme ethoxyresorufin-O-deethylase (EROD) when combined with an Ah agonist and the substrate ethoxyresorufin (ER). Nacci et al. utilized the in-ovo EROD method to measure contamination of water using killifish (Fundulus heteroclitus). Our lab has been adapting this method using other fish species and by testing the exposure effects of contaminated sediment. In the embryonic fish used, the contamination levels were measured by the accumulation of the florescent byproduct in the gallbladder. The brighter the fluorescence, the more the metabolic pathway had been activated, indicating higher amount of toxicants.

Japanese Medaka were used since they are easily reared and produce eggs daily. For the first trial, fresh eggs were collected and exposed to sediment from Riker’s Island (known high contamination level) and sediment from a reference site, Flax Pond (control). These were then left on screens on the sediments for 4 days, rotating the screens daily. After the 4 days, the eggs were placed in individual vials with a set concentration of 7ER. Embryos were incubated for 24hrs in the dark. After incubation, the eggs were rinsed in ERM then imaged using a fluorescent microscope. The embryos were then allowed to incubate and imaged for 48hrs and 72hrs. This first trial compared the signal response measured and the time the eggs were incubated.

The second trial repeated the above steps but using sediments from 7 different sites (Port Jefferson, Jamaica Bay, Oyster Bay, Raritan Bay, Hudson River, Little Neck and Flax Pond). The first trial indicated 48hrs incubation and imaging provided the best fluorescence without much background response. The eggs from the above sites were then imaged using the technique above. The images were then semi-quantitatively measured by using computer software to measure pixel density of the fluorescent gall bladders in the image.

The results show that compared to the control, all other sediment collection sites showed a higher level of CYP450 induction. This indicates a higher level of contamination in the soils at these sites. The results from these tests indicate that animals living in polluted habitats show metabolic reactions to their environment and these responses can be used to measure the contamination levels of these sites.