Evaluation of a Method for Quantifying Eugenol Concentrations in the Fillet Tissue from Freshwater Fish Species.

AQUI-S 20E(®) (active ingredient, eugenol; AQUI-S New Zealand Ltd, Lower Hutt, New Zealand) is being pursued for approval as an immediate-release sedative in the United States. A validated method to quantify the primary residue (the marker residue) in fillet tissue from AQUI-S 20E-exposed fish was needed. A method was evaluated for determining concentrations of the AQUI-S 20E marker residue, eugenol, in freshwater fish fillet tissue. Method accuracies from fillet tissue fortified at nominal concentrations of 0.15, 1, and 60 µg/g from six fish species ranged from 88-102%. Within-day and between-day method precisions (% CV) from the fortified tissue were ≤8.4% CV. There were no coextracted compounds from the control fillet tissue of seven fish species that interfered with eugenol analyses. Six compounds used as aquaculture drugs did not interfere with eugenol analyses. The lower limit of quantitation (LLOQ) was 0.012 µg/g. The method was robust, i.e., in most cases, minor changes to the method did not impact method performance. Eugenol was stable in acetonitrile-water (3 + 7, v/v) for at least 14 days, in fillet tissue extracts for 4 days, and in fillet tissue stored at ~ -80°C for at least 84 days.

Chronic toxicity of diphenhydramine hydrochloride to a freshwater mussel, Lampsilis siliquoidea, in a flow-through, continuous exposure test system.

Freshwater mussel populations are declining in North America. Potential anthropogenic stressors may be contributing to the declines and may include the continual presence of pharmaceutical compounds in waterways. Diphenhydramine hydrochloride (DH) is an over-the-counter antihistamine marketed under several name brand products including the common U.S. trademarked product, Benadryl™. The toxicity of DH to freshwater mussels was assessed by initiating an unprecedented 28 day, continuous exposure trial with 1 day old mussels. Results indicated that the survival and growth of Lampsilis siliquoidea was not impacted by DH concentrations ≤121 µg/L after 28 days of continuous exposure. With the successful completion of this study, the techniques are now verified to evaluate the toxicity of waterborne compounds initiating 28-day chronic exposures with 1 day old mussels.

Chronic toxicity of erythromycin thiocyanate to Daphnia magna in a flow-through, continuous exposure test system.

Approval of a new animal drug application for AQUAMYCIN 100(®) (erythromycin thiocyanate; ET) to treat freshwater salmonid species with bacterial kidney disease is being pursued in the US. As part of the approval process, ET's impact on an aquatic environment had to be described in an environmental assessment. The environmental assessment was lacking data to characterize the effect ET would have on a chronically exposed aquatic invertebrate organism. A major step to fulfilling the environmental assessment was completed after conducting a comprehensive study continuously exposing Daphnia magna to ET for 21 days. Results indicated that the no observable effect concentration for ET was 179 µg/L.

Chronic toxicity of diphenhydramine hydrochloride and erythromycin thiocyanate to daphnia, Daphnia magna, in a continuous exposure test system.

Diphenhydramine hydrochloride (DH; Benadryl™, an over-the-counter antihistamine) and erythromycin thiocyanate (ET; a commonly used macrolide antibiotic) are pharmaceutical compounds whose chronic toxicity to Daphnia magna had not been characterized. Continuous exposure to DH concentrations about 5 times greater than the maximum reported environmental concentration of 0.023 µg/L for 21 days or to ET concentrations about 40 times the maximum reported environmental concentration of 6 µg/L for 21 days did not significantly impact D. magna survival and production. In this study the no observable effect concentration for DH was 0.12 µg/L and for ET was 248 µg/L.

Evaluation of a method for determining concentrations of isoeugenol, an AQUI-S residue, in fillet tissue from freshwater fish species.

AQUI-S is a fish anesthetic/sedative that is approved for use in a number of countries throughout the world and has the potential for use in the United States. The active ingredient in AQUI-S is isoeugenol. A method for determining isoeugenol concentrations in edible fillet tissue is needed for regulatory purposes, including surveillance and potential use in studies fulfilling human food safety data requirements if U.S. Food and Drug Administration approval is pursued. A method was developed and evaluated for determining isoeugenol concentrations in fillet tissue using relatively common procedures and equipment. The method produced accurate and precise results with fillet tissue from 10 freshwater fish species. The percentage of isoeugenol recovered from samples fortified with isoeugenol at nominal concentrations of 1, 50, and 100 microg/g for all species was always >80 and <97%. Within-day precision for samples fortified at those same concentrations was < or =10%, and day-to-day precision was < or =4.0%. Method precision with fillet tissue containing biologically incurred isoeugenol was < or =8.1%. There were no or minimal chromatographic interferences in control fillet tissue extracts from 9 of the 10 species. The method detection limits for all but one species ranged from 0.004 to 0.014 microg/g, and the quantitation limits ranged from 0.012 to 0.048 microg/g.

Chronic toxicity of hydrogen peroxide to Daphnia magna in a continuous exposure, flow-through test system.

A flow-through, continuous exposure test system was developed to expose Daphnia magna to an unstable compound. 35% Perox-Aid is a specially formulated hydrogen peroxide (a highly oxidative chemical) product approved for use in U.S. aquaculture and therefore has the potential to be released from aquaculture facilities and pose a risk to aquatic invertebrates. The study objective was to assess the effects of 35% Perox-Aid on an aquatic invertebrate by evaluating the survival, growth, production, and gender ratio of progeny from a representative aquatic invertebrate continuously exposed to 35% Perox-Aid. The study design consisted of 6 treatment groups (10 test chambers each) with target hydrogen peroxide concentrations of 0.0, 0.32, 0.63, 1.25, 2.5, and 5.0 mg L(-1). The study was initiated with <24-h-old Daphnia (1 daphnid per chamber) that were exposed to hydrogen peroxide for 21 days. Hydrogen peroxide concentrations < or =1.25 mg L(-1) had no significant effect on Daphnia time to death compared to controls and no significant effect on the time to first brood production and the number of broods produced. Concentrations < or =0.63 mg L(-1) had no significant effect on the total number of young produced. Concentrations > or =0.32 mg L(-1) had a negative effect on Daphnia growth. Hydrogen peroxide had no significant effect on the gender ratio of young produced. All second generation Daphnia were female. A continuous discharge of hydrogen peroxide into aquatic ecosystems is not likely to affect cladocerans if the concentration is maintained at < or =0.63 mg L(-1) for less than 21 days.

Development and validation of a gas chromatography/mass spectrometry procedure for confirmation of para-toluenesulfonamide in edible fish fillet tissue.

Chloramine-T is a disinfectant being developed as a treatment for bacterial gill disease in cultured fish. As part of the drug approval process, a method is required for the confirmation of chloramine-T residues in edible fish tissue. The marker residue that will be used to determine the depletion of chloramine-T residues from the edible tissue of treated fish is para-toluenesulfonamide (p-TSA), a metabolite of chloramine-T. The development and validation of a procedure for the confirmation of p-TSA is described. Homogenized fish tissue is dried by mixing with anhydrous sodium sulfate, and the mixture is extracted with methylene chloride. The extract is passed through a silica gel solid-phase extraction column, from which p-TSA is subsequently eluted with acetonitrile. The acetonitrile extract is evaporated, and the oily residue is dissolved in hexane. The hexane solution is shaken with fresh acetonitrile. The acetonitrile solution is evaporated and the residue is redissolved in dilute potassium hydroxide solution. The aqueous solution is extracted with methylene chloride to further remove more of the fat co-extractive. The aqueous solution is reacted with pentafluorobenzyl bromide in presence of tetrabutylammonium hydrogensulfate. The resulting di-(pentafluorobenzyl) derivative of p-TSA is analyzed by gas chromatography/mass spectrometry. This method permits the confirmation of p-TSA in edible fish tissue at 20 ppb.