Jeffery R. Meinertz

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.

Liquid chromatographic determination of benzocaine and N-acetylbenzocaine in the edible fillet tissue from rainbow trout

A method was developed for determining benzocaine and N-acetylbenzocaine concentrations in fillet tissue of rainbow trout. The method involves extracting the analytes with acetonitrile, removing lipids or hydrophobic compounds from the extract with hexane, and providing additional clean-up with solid-phase extraction techniques. Analyte concentrations are determined using reversed-phase high-performance liquid chromatographic techniques with an isocratic mobile phase and UV detection. The accuracy (range, 92 to 121%), precision (R.S.D., < 14%), and sensitivity (method quantitation limit, < 24 ng/g) for each analyte indicate the usefulness of this method for studies characterizing the depletion of benzocaine residues from fish exposed to benzocaine.

Effects of Immobilization by Electricity and MS-222 on Brown Trout Broodstock and Their Progeny

Abstract To determine the effects of electrically and chemically induced immobilization on postspawn broodstock and their progeny, age-2 and age-3 female broodstock and age-2 male broodstock of brown trout Salmo trutta were immobilized with electricity or tricaine methanesulfonate (MS-222), stripped of their eggs or milt, and weighed. Eggs taken from electrically immobilized females were fertilized with milt taken from age-2 males that were immobilized with electricity, and eggs taken from females immobilized with MS-222 were fertilized with milt taken from age-2 males that were immobilized with MS-222. After spawning, the mortality and weight of broodstock were compared twice over a 6-month period. Egg viability and growth of offspring fry from each treatment group were also compared. Electricity induced complete and consistent immobilization in brown trout broodstock. Electrically immobilized fish were more easily handled than fish immobilized with MS-222; however, electrically immobilized fish survival...

A simple analytical procedure to replace HPLC for monitoring treatment concentrations of chloramine-T on fish culture facilities

Abstract Concentrations of chloramine-T must be monitored during experimental treatments of fish when studying the effectiveness of the drug for controlling bacterial gill disease. A surrogate analytical method for analysis of chloramine-T to replace the existing high-performance liquid chromatography (HPLC) method is described. A surrogate method was needed because the existing HPLC method is expensive, requires a specialist to use, and is not generally available at fish hatcheries. Criteria for selection of a replacement method included ease of use, analysis time, cost, safety, sensitivity, accuracy, and precision. The most promising approach was to use the determination of chlorine concentrations as an indicator of chloramine-T. Of the currently available methods for analysis of chlorine, the DPD ( N , N -diethyl- p -phenylenediamine) colorimetric method best fit the established criteria. The surrogate method was evaluated under a variety of water quality conditions. Regression analysis of all DPD colorimetric analyses with the HPLC values produced a linear model ( Y =0.9602 X +0.1259) with an r 2 value of 0.9960. The average accuracy (percent recovery) of the DPD method relative to the HPLC method for the combined set of water quality data was 101.5%. The surrogate method was also evaluated with chloramine-T solutions that contained various concentrations of fish feed or selected densities of rainbow trout. When samples were analyzed within 2 h, the results of the surrogate method were consistent with those of the HPLC method. When samples with high concentrations of organic material were allowed to age more than 2 h before being analyzed, the DPD method seemed to be susceptible to interference, possibly from the development of other chloramine compounds. However, even after aging samples 6 h, the accuracy of the surrogate DPD method relative to the HPLC method was within the range of 80–120%. Based on the data comparing the two methods, the U.S. Food and Drug Administration has concluded that the DPD colorimetric method is appropriate to use to measure chloramine-T in water during pivotal efficacy trials designed to support the approval of chloramine-T for use in fish culture.

Effects of temperature on the elimination of benzocaine and acetylated benzocaine residues from the edible fillet of rainbow trout (Oncorhynchus mykiss)

The effect of temperature (7 degrees C and 16 degrees C) on the extent of accumulation and the elimination of benzocaine (BNZ) and its metabolite, acetylated benzocaine (AcBNZ), in the fillet tissue of rainbow trout was investigated. Residues were measured after bath exposure to an anesthetizing concentration of benzocaine (30 mg/l for 5 min) followed by a maintenance concentration (15 mg/l for 30 min). Immediately after exposure, the BNZ concentration in fillet tissue was approximately 27 micrograms/g at both temperatures; AcBNZ was 0.3 microgram/g at 7 degrees C and 0.6 microgram/g at 16 degrees C. The rates for elimination (alpha and beta) of BNZ and AcBNZ were not significantly different between the two temperatures. Terminal half-lives of elimination for BNZ were 1.62 h at 7 degrees C and 1.63 h at 16 degrees C; half-lives for AcBNZ were 2.36 h at 7 degrees C and 2.77 h at 16 degrees C.

Pharmacokinetics of benzocaine in rainbow trout (Oncorhynchus mykiss) after intraarterial dosing

Abstract The pharmacokinetics of benzocaine were analyzed in rainbow trout ( Oncorhynchus mykiss ) after intraarterial bolus administration of benzocaine at nominal concentrations of 6 and 9 mg/kg. Distributive phase pharmacokinetic parameters were difficult to estimate. Benzocaine concentrations at 2 min after dosing were highly variable and decreased rapidly within the first 10 min. Benzocaine concentrations were near or below the quantitation limit 90 min after dosing. A three-compartment pharmacokinetic model best described benzocaine concentrations in plasma. The apparent volume of distribution at steady state and total body clearance increased with respect to dose. Since the mean residence time was similar at both dosages, the differences in the apparent volume of distribution at steady state and total body clearance may have been the result of data variability or the degree of anaesthesia rather than dose dependency. The model parameters indicated that distribution of benzocaine outside of the plasma was limited. Despite the initial rapid distribution and elimination of benzocaine from plasma, terminal phase benzocaine elimination was relatively slow (terminal elimination phase half-lives of 89 and 109 min).

Oxytetracycline depletion from skin-on fillet tissue of coho salmon fed oxytetracycline medicated feed in freshwater at temperatures less than 9°C

Abstract Oxytetracycline (OTC) is a broad spectrum antibacterial agent approved in the USA for treating certain bacterial diseases in salmonids cultured in freshwater at temperatures greater than or equal to 9°C. This study was conducted to provide the information necessary to expand the OTC label to include treatment of diseased salmonids cultured in freshwater at temperatures below 9°C. The study was designed to treat juvenile coho salmon ( Oncorhynchus kisutch ) with OTC-medicated feed and determine the depletion of OTC from the skin-on fillet tissue. Oxytetracycline depletion was evaluated in juvenile coho salmon (weight range, 13–62 g) fed OTC-medicated feed at a rate of 88.2 mg OTC/kg body weight/day for 10 days. Pairs of skin-on fillets were taken from individual fish on days 4 and 10 during the treatment phase and on days 1, 4, 8, 14, and 19 during the depletion phase. Water temperatures during the study period ranged from 4.1°C to 8.5°C. The OTC concentrations in medicated feed and skin-on fillets were determined with high-performance liquid chromatography methods. The maximum mean OTC concentration in fillet tissue was 932 ng/g, 1 day after the last treatment and decreased to 32 ng/g 19 days after the last treatment. The log-linear loss of OTC from the fillet tissue was biphasic with a terminal phase half-life of 4.9 days.

Depletion of the chloramine-T marker residue, para-toluenesulfonamide, from skin-on fillet tissue of hybrid striped bass, rainbow trout, and yellow perch

Abstract Waterborne exposure to n-sodium-n-chloro-p-toluenesulfonamide (chloramine-T) is an effective treatment for controlling fish mortalities caused by bacterial gill disease (BGD). Currently, data are being generated to gain United States Food and Drug Administration (FDA) approval for the use of chloramine-T in aquaculture. As part of the data required for an approval, depletion of the chloramine-T marker residue (para-toluenesulfonamide [p-TSA]) from the edible fillet tissue of exposed fish must be determined. Hybrid striped bass (Morone saxatilis×Morone chrysops; mean weight 357 g), rainbow trout (Oncorhynchus mykiss; mean weight 457 g), and yellow perch (Perca flavescens; mean weight 144 g) were exposed to 20 mg/l of chloramine-T for 60 min on 4 consecutive days (the most aggressive treatment expected for approved use in the United States). Groups of fish (n=15 or 19) were sampled immediately after the last treatment and periodically through 48 or 168 h after the treatment phase. Duplicate subsamples of skin-on fillet tissue from each fish were analyzed for p-TSA. Mean p-TSA concentrations in fillet tissue from fish sampled immediately after the last treatment were 142 ng/g (hybrid striped bass), 97 ng/g (rainbow trout), and 150 ng/g (yellow perch). Mean p-TSA concentrations at terminal sample times were 94 (168 h; hybrid striped bass), 74 (48 h; rainbow trout), and 35 ng/g (168 h; yellow perch). The half-lives of p-TSA in fillet tissue from fish near or at market size were 11.4 (hybrid striped bass), 4.3 (rainbow trout), and 3.2 days (yellow perch).

Survival and Growth of Newly Transformed Lampsilis cardium and Lampsilis siliquoidea in a Flow-Through, Continuous Feeding Test System

Abstract: A test system was evaluated for assessing chronic toxicity of waterborne chemicals with early life stage mussels. To determine if the test system could result in ≥80% survival in a control (unexposed) group, fat mucket mussels (Lampsilis siliquoidea Barnes, 1823) and plain pocketbook mussels (L. cardium Rafinesque, 1820) 1 day post transformation were stocked into test chambers (250 mL beakers, water volume, 200 mL, 21 °C, 40 mussels of 1 species per chamber) within a test system constructed for conducting chronic, continuous exposure, flow-through toxicity tests. The test system contained 60 chambers containing silica sand, 30 chambers with L. siliquoidea, and 30 with L. cardium. Each chamber in the continuous feeding system received 1 of 6 food types prepared with concentrated algal products. After 28 days, mussels were harvested from chambers to assess survival and growth. For L. siliquoidea, mean survival ranged from 34 to 80% and mean shell length ranged from 464 to 643 µm. For L. cardium, mean survival ranged from 12 to 66% and mean shell length ranged from 437 to 612 µm. The maximum mean growth rate for L. siliquoidea was 12.7 µm/d and for L. cardium was 11.8 µm/d. When offered a continuous diet of Nannochloropsis, Tetraselmis, and Chlorella for 28 days in the test system, the survival of 1 day post transformation L. siliquoidea was 80%. The test system can be easily enhanced with a pumping system continuously delivering test chemical to the test systems flow stream allowing for chronic toxicity tests with 1 day post transformation mussels.

Florfenicol Residues in Rainbow Trout after Oral Dosing in Recirculating and Flow-through Culture Systems

Aquaflor is a feed premix for fish containing the broad spectrum antibacterial agent florfenicol (FFC) incorporated at a ratio of 50% (w/w). To enhance the effectiveness of FFC for salmonids infected with certain isolates of Flavobacterium psychrophilum causing cold water disease, the FFC dose must be increased from the standard 10 mg·kg⁻¹ body weight (BW)·d⁻¹ for 10 consecutive days. A residue depletion study was conducted to determine whether FFC residues remaining in the fillet tissue after treating fish at an increased dose would be safe for human consumption. Groups of Rainbow Trout Oncorhynchus mykiss (total n = 144; weight range, 126-617 g) were treated with FFC at 20 mg·kg⁻¹ BW·d⁻¹ for 10 d in a flow-through system (FTS) and a recirculating aquaculture system (RAS) each with a water temperature of ∼13°C. The two-tank RAS included a nontreated tank containing 77 fish. Fish were taken from each tank (treated tank, n = 16; nontreated tank, n = 8) at 6, 12, 24, 48, 72, 120, 240, 360, and 480 h posttreatment. Florfenicol amine (FFA) concentrations (the FFC marker residue) in skin-on fillets from treated fish were greatest at 12 h posttreatment (11.58 μg/g) in the RAS and were greatest at 6 h posttreatment (11.09 μg/g) in the FTS. The half-lives for FFA in skin-on fillets from the RAS and FTS were 20.3 and 19.7 h, respectively. Assimilation of FFC residues in the fillets of nontreated fish sharing the RAS with FFC-treated fish was minimal. Florfenicol water concentrations peaked in the RAS-treated tank and nontreated tanks at 10 h (453 μg/L) and 11 h (442 μg/L) posttreatment, respectively. Monitoring of nitrite concentrations throughout the study indicated the nitrogen oxidation efficiency of the RAS biofilter was minimally impacted by the FFC treatment.