Abstract:

Adult female fathead minnow (Pimephales promelas) were treated by gavage with water, or WAF (water accommodated fraction of oil) alone or in the presence of 50 mg/L hyperbranched polyglycidol (HPG)-nanoparticle dispersant. The treatment occurred four times over two weeks and consisted of 15 ul gavage of each of the treatments described above. There were 8 fish per treatment group. After the treatment, the fish were euthanized with MS222 and severance of the spinal cord and livers were removed and placed into RNAlater. RNA was extracted and analyzed for expression of Cyp1a (Cytochrome P450 1A). The results indicated that the WAF alone induced Cyp1A expression but that the treatment of WAF plus HPG-NP decreased Cyp1A below that shown for WAF alone.

Suggested Citation:

Kurita, Hajime and Nancy Denslow. 2020. Gavage of fathead minnows (Pimephales promelas) with nanoparticle-based dispersants and water accommodated fraction (WAF) of crude oil. Distributed by: GRIIDC, Harte Research Institute, Texas A&M University–Corpus Christi. doi:10.7266/7F0JADMH

Purpose:

Female adult fathead minnows (Pimephales promelas) were treated with nanoparticle-based dispersants alone and in the presence of the water accommodated fraction of crude oil to determine if the nanoparticles can diminish the bioavailability of crude oil PAHs.

Data Parameters and Units:

The headers are Cq Mean, ΔCT, ΔΔCT, and Fold change. Content= control; WAF; WAF+NP

Methods:

WAF was prepared by mixing 1 ml crude Macondo oil with 1 L water for one week with a stir bar in a hood. Then the WAF was separated from the crude oil with a separatory funnel. A portion of the WAF was then mixed with NP to generate a solution of WAF+NP at a concentration of 50 mg/L. 8 adult female fathead minnows were gavaged with water, WAF or WAF+50mg/L of HPG-NP. Gavage was carried out four times during a two-week period, with each gavage consisting of 15 ul of the solutions. Then, livers were removed and immediately preserved in RNAlater for RNA extraction. RNA was extracted from the livers using STAT-60 and checked by Nanodrop and by gel electrophoresis. Only RNA samples that passed quality control by gel electrophoresis were used for qPCR. This meant that we had at least livers from 4 fish for analysis without pooling. RNA were treated with DNase (Turbo DNA free kit from ThermoFisher) and then converted to cDNA using qScript (QuantaBio). cDNA were diluted 1 to 20 for Cyp1A and 1 to 100 for the rpl8 housekeeping gene. And then they were placed in the BioRAD q PCR instrument for amplification. Data analysis was performed using the delta delta Ct method to determine relative amplification values compared to control.

Instruments:

BioRAD qPCR CFX Connect Real Time PCR Detection System

Error Analysis:

Standard deviation and standard error of the means were calculated for each group. Data were analyzed using ANOVA for significance.

Individual Files: