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.

Molecular responses differ between sensitive silver carp and tolerant bighead carp and bigmouth buffalo exposed to rotenone.

Some species of fish are more tolerant of rotenone, a commonly used non-specific piscicide, than others. This species-specific tolerance to rotenone has been thought to be associated with the uptake and the efficiency at which the chemical is detoxified. However, rotenone stimulates oxidative stress and superoxides, which are also toxic. Understanding the modes in which fish physiologically respond to rotenone is important in developing improved protocols for its application in controlling aquatic nuisance species. Using a molecular approach, we investigated the physiological and molecular mechanisms of rotenone resistance. Species-specific responses were observed when rotenone-sensitive silver, Hypophthalmichthys molitrix, and both rotenone-resistant bighead carp, Hypophthalmichthys nobilis, and bigmouth buffalo, Ictiobus cyprinellus, were exposed to rotenone. Rotenone levels in plasma were highest 90 min after exposure in both silver carp and bigmouth buffalo, but bigmouth buffalo tolerated over twice the burden (ng mL(-1) g(-1)) than silver carp. Expression of genes related with detoxification (cyp1a and gst) increased in silver carp, but either decreased or remained the same in bighead carp. Genes linked with oxidative stress in the cytosol (gpx, cat and sod1) and hsp70 increased only in silver carp after a 6-h exposure. Expression of genes associated with oxidative stress in the mitochondria (sod2 and ucp2) differed between silver carp and bighead carp. Expression of sod2 changed minimally in bighead carp, but expression of ucp2 linearly increased to nearly 85-fold of the level prior to exposure. Expression of sod2 and ucp2 did not change until 6 h in silver carp. Use of sod1 and sod2 to combat oxidative stress results in hydrogen peroxide production, while use of ucp2 produces nitric oxide, a chemical known to inhibit apoptosis. We conclude that the mechanism at which a fish handles oxidative stress plays an important role in the tolerance to rotenone.

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.

Residues of the lampricides 3-trifluoromethyl-4-nitrophenol and niclosamide in muscle tissue of rainbow trout.

Rainbow trout (Oncorhyncus mykiss) were exposed to the (14)C-labeled lampricide 3-trifluoromethyl-4-nitrophenol (TFM) (2.1 mg/L) or niclosamide (0.055 mg/L) in an aerated static water bath for 24 h. Fish were sacrificed immediately after exposure. Subsamples of skin-on muscle tissue were analyzed for residues of the lampricides. The primary residues in muscle tissue from fish exposed to TFM were parent TFM (1.08 +/- 0.82 nmol/g) and TFM-glucuronide (0.44 +/- 0.24 nmol/g). Muscle tissue from fish exposed to niclosamide contained niclosamide (1.42 +/- 0.51 nmol/g), niclosamide-glucuronide (0.0644 +/- 0.0276 nmol/g), and a metabolite not previously reported, niclosamide sulfate ester (1.12 +/- 0.33 nmol/g).

Rapid loss of lampricide from catfish and rainbow trout following routine treatment.

Rainbow trout (Oncorhynchus mykiss) and channel catfish (Ictalurus punctatus) were exposed to 3-trifluoromethyl-4-nitrophenol (TFM) and Bayluscide (niclosamide) during a sea lamprey control treatment of the Ford River, located in the upper peninsula of Michigan. Caged fish were exposed to a nominal concentration of 0.02 mg/L of niclosamide for a period of approximately 12 h. Samples of fillet tissue were collected from each fish species before treatment and at 6, 12, 18, 24, 48, 96, and 192 h following the arrival of the block of chemical at the exposure site. The fish were dissected, homogenized, extracted, and analyzed by high-performance liquid chromatography. The major residues found in the fillet tissues were TFM and niclosamide. Niclosamide concentrations were highest 12 h after arrival of the chemical block for rainbow trout (0.0395 +/- 0.0251 microg/g) and 18 h after arrival of the chemical block for channel catfish (0.0465 +/- 0.0212 microg/g). Residues decreased rapidly after the block of lampricide had passed and were below the detection limits in fillets of rainbow trout within 24 h and channel catfish within 96 h after the arrival of the lampricide.