Abstract:

Fathead minnow (Pimephales promelas) embryos and juveniles were exposed in vivo to the water accommodated fraction (WAF) of crude oil in the presence and absence of nanoparticle-based dispersants. An in vivo EROD assay was used to measure cyp1A activity of fish and it was expected that there would be gradations of response depending on the presence of the nanoparticle dispersants.

Suggested Citation:

Kurita, Hajime and Nancy Denslow. 2020. EROD assay using live fathead minnows (Pimephales promelas) exposed to nanoparticle dispersants in the absence and presence of the water accommodated fraction of crude oil. Distributed by: GRIIDC, Harte Research Institute, Texas A&M University–Corpus Christi. doi:10.7266/WXY0SF0D

Purpose:

To measure in vivo the increase in activity of cyp1A when fish are exposed to the water accommodated fraction (WAF) of crude oil and to see if the NPs decreased this activity.

Data Parameters and Units:

Dataset consists of 4 Excel files which are described with an extensive readme.

Methods:

Water accommodated fraction of the oil (WAF) preparation 1 mL of crude oil was weathered during 24-hour with reconstituted freshwater (Hanks 20% strength: 27 mM NaCl, 1 mM KCl, 0.05 mM Na2HPO4, 0.09 mM KH2PO4, 0.25 mM CaCl2, 0.2 mM MgSO4, 41 mM NaHCO3). The aqueous phase (the water accommodated fraction of the oil -WAF-) was separated using separation funnel. 1. WAF_Dose_response Four concentrations of WAF were prepared diluting the original WAF solution. The concentrations were established arbitrarily as 1%, 10%, 50% and 100% of WAF. The dilutions were made by mixing the appropriate volume of WAF into 20% Hanks solution. The 100% WAF was the original 24-hour weathered oil described above. 1-month old juvenile fathead minnows were exposed in 25ml glass beaker with the different WAF concentrations. In total 10 individuals were exposed per beaker during 24-hour. 2. SiO2HPG_mixing_Time Three different concentrations of NPs (at 2, 10, and 50 mg/L) were mixed with 100% WAF with different mixing times. One experiment consisted of 24-hour mixing of the different NPs concentrations with WAF. Another experiment included the same NP concentrations indicated above, but mixed with WAF for a few minutes before exposing to fish. 3. SEW-16_EROD & SEW-17_EROD NPs were re-suspended in 20% Hanks alone, and in 100% WAF at a concentration of 20 mg/L. Embryonic stage FHM (dechorionated) were used. Fish were exposed during 24-hour and without the prior mixing time. Embryos were dechorionated by using pronase and exposed to the different NPs at the concentration sindicated above. The experimental design is shown in Table 2 Exposure time was 24-hour and plate reading was carried after 2 hours of incubation with CYP1A substrate 7-ethoxyresorufin (7-ER).

Instruments:

Biotek Synergy H1 Hybrid multimode microplate reader -- software version 2.05.5

Error Analysis:

Standard error of the mean and Anova

Individual Files: