Performance of a proposed determinative method for p-TSA in rainbow trout fillet tissue and bridging the proposed method with a method for total chloramine-T residues in rainbow trout fillet tissue.

Chloramine-T is an effective drug for controlling fish mortality caused by bacterial gill disease. As part of the data required for approval of chloramine-T use in aquaculture, depletion of the chloramine-T marker residue (para-toluenesulfonamide; p-TSA) from edible fillet tissue of fish must be characterized. Declaration of p-TSA as the marker residue for chloramine-T in rainbow trout was based on total residue depletion studies using a method that used time consuming and cumbersome techniques. A simple and robust method recently developed is being proposed as a determinative method for p-TSA in fish fillet tissue. The proposed determinative method was evaluated by comparing accuracy and precision data with U.S. Food and Drug Administration criteria and by bridging the method to the former method for chloramine-T residues. The method accuracy and precision fulfilled the criteria for determinative methods; accuracy was 92.6, 93.4, and 94.6% with samples fortified at 0.5X, 1X, and 2X the expected 1000 ng/g tolerance limit for p-TSA, respectively. Method precision with tissue containing incurred p-TSA at a nominal concentration of 1000 ng/g ranged from 0.80 to 8.4%. The proposed determinative method was successfully bridged with the former method. The concentrations of p-TSA developed with the proposed method were not statistically different at p < 0.05 from p-TSA concentrations developed with the former method.

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.

Liquid chromatographic determination of para-toluenesulfonamide in edible fillet tissues from three species of fish.

Chloramine-T (N-sodium-N-chloro-p-toluene-sulfonamide) is a candidate therapeutic drug for treating bacterial gill disease, a predominant disease of a variety of fish species. Research has been initiated to obtain the U.S. Food and Drug Administration's (FDA) approval for the use of chloramine-T on a variety of fish species. An attribute of a therapeutic aquaculture drug that must be characterized before the FDA approves its use is depletion of the drug's marker residue (the drug's parent compound or metabolite of highest concentration in an edible tissue). para-Toluenesulfonamide (p-TSA) is the primary degradation product and marker residue for chloramine-T in rainbow trout. To conduct residue depletion studies for chloramine-T in fish, a robust analytical method sensitive and specific for p-TSA residues in edible fillet tissue from a variety of fish was required. Homogenized fillet tissues from rainbow trout (Oncorhynchus mykiss), walleye (Stizostedion vitreum), and channel catfish (Ictalurus punctatus) were fortified at nominal p-TSA concentrations of 17, 67, 200, 333, and 1000 ng/g. Samples were analyzed by isocratic reversed-phase liquid chromatography (LC) with absorbance detection at 226 nm. Mean recoveries of p-TSA ranged from 77 to 93.17%; relative standard deviations ranged from 1.5 to 14%; method quantitation limits ranged from 13 to 18 ng/g; and method detection limits ranged from 3.8 to 5.2 ng/g. The LC parameters produced p-TSA peaks without coelution of endogenous compounds and excluded chromatographic interference from at least 20 chemicals and drugs of potential use in aquaculture.

A bridging study for oxytetracycline in the edible fillet of rainbow trout: analysis by a liquid chromatographic method and the official microbial inhibition assay.

Oxytetracycline (OTC) is a drug approved by the U.S. Food and Drug Administration (FDA) to control certain diseases in salmonids and catfish. OTC is also a likely control agent for diseases of other fish species and for other diseases of salmonids and catfish not currently on the label. One requirement for FDA to extend and expand the approval of this antibacterial agent to other fish species is residue depletion studies. The current regulatory method for OTC in fish tissue, based on microbial inhibition, lacks sensitivity and specificity. To conduct residue depletion studies for OTC in fish with a liquid chromatographic method, a bridging study was required to determine its relationship with the official microbial inhibition assay. Triplicate samples of rainbow trout fillet tissue fortified with OTC at 0.3, 0.6, 1.2, 2.4, 4.8, and 9.6 ppm and fillet tissue with incurred OTC at approximately 0.75, 1.5, and 3.75 ppm were analyzed by high-performance liquid chromatography (HPLC) and the microbial inhibition assay. The results indicated that the 2 methods are essentially identical in the tested range, with mean coefficients of variation of 1.05% for the HPLC method and 3.94% for the microbial inhibition assay.

Crop grouping: a proposal for public aquaculture.

Production in US aquaculture is limited by the few FDA-approved drugs available for use. The problem is compounded by the high costs and long time frames associated with extending the approved label of an existing drug to treat additional fish species. An FDA approved crop grouping plan could significantly reduce the costs associated with extending or expanding the label of a currently approved drug to other species. Before FDA could sanction such a plan, they require scientific data with which to make an informed decision. Under a crop grouping plan, a surrogate fish species would represent a single group of fish for the purposes of gaining drug approvals. The concept, if practical, would conserve substantial public resources expended to gain drug approvals and yet give regulators assurance that the extended label use provides necessary regulatory safeguards to protect human food safety and the environment. A crop grouping plan should include development of a data base that is sufficiently sensitive to discriminate differences of one group from another and yet would be able to identify potential similarities between like-species for grouping. The proposed crop grouping action plan should include data sets for fish grouped by temperature preference, activity level, and phylogenetic classification. Initially, two representative species would be identified for testing as surrogates for candidate groups; rainbow trout representing a coldwater, active, and conservative phylogenetic group, and channel catfish representing a warmwater, relatively sedentary and more phylogenetically advanced group. Additional species representing more primitive (sturgeon) and more advanced (striped bass and walleye) groups would be added. A waterborne (benzocaine) and an orally administered drug (sarafloxacin) would initially be tested among the major species groups. The integrity of the group will be tested by comparing the variability of response between major species against variability within cohort species identified for inclusion within each specific group.

Liquid chromatographic determination of oxytetracycline in edible fish fillets from six species of fish.

The approved use of oxytetracycline (OTC) in U.S. aquaculture is limited to specific diseases in salmonids and channel catfish. OTC may also be effective in controlling diseases in other fish species important to public aquaculture, but before approved use of OTC can be augmented, an analytical method for determining OTC in fillet tissue from multiple species of fish will be required to support residue depletion studies. The objective of this study was to develop and validate a liquid chromatographic (LC) method that is accurate, precise, and sensitive for OTC in edible fillets from multiple species of fish. Homogenized fillet tissues from walleye, Atlantic salmon, striped bass, white sturgeon, rainbow trout, and channel catfish were fortified with OTC at nominal concentrations of 10, 20, 100, 1000, and 5000 ng/g. In tissues fortified with OTC at 100, 1000, and 5000 ng/g, mean recoveries ranged from 83 to 90%, and relative standard deviations (RSDs) ranged from 0.9 to 5.8%. In all other tissues, mean recoveries ranged from 59 to 98%, and RSDs ranged from 3.3 to 20%. Method quantitation limits ranged from 6 to 22 ng/g for the 6 species. The LC parameters produced easily integratable OTC peaks without coelution of endogenous compounds. The method is accurate, precise, and sensitive for OTC in fillet tissue from 6 species of fish from 5 phylogenetically diverse groups.

Effect of temperature on the pharmacokinetics of benzocaine in rainbow trout (Oncorhynchus mykiss) after bath exposures.

The pharmacokinetics of benzocaine during bath exposures at 1 mg/L were determined in rainbow trout acclimated at 6 degrees C, 12 degrees C or 18 degrees C for at least 1 month. Individual fish were exposed to benzocaine in a recirculating system for 4 h and pharmacokinetic parameters were estimated in a unique manner from the concentration of benzocaine in the bath water vs. time curve. Elimination from plasma was also determined after the 4 h exposure. The uptake clearance and metabolic clearance increased with increased acclimatization temperatures (uptake clearance 581 +/- 179 mL/min/kg at 6 degrees C and 1154 +/- 447 mL/min/kg at 18 degrees C; metabolic clearance 15.2 +/- 4.1 mL/min/kg at 6 degrees C and 22.3 +/- 4.2 mL/min/kg at 18 degrees C). The apparent volume of distribution had a trend for increasing with temperature that was not significant at the 5% level (2369 +/- 678 mL/kg at 6 degrees C to 3260 +/- 1182 mL/kg at 18 degrees C). The elimination half-life of benzocaine in plasma was variable and did not differ significantly with temperature (60.8 +/- 30.3 min at 6 degrees C to 35.9 +/- 13.0 min at 12 degrees C). Elimination of benzocaine from rainbow trout is relatively rapid and even more rapid at higher acclimatization temperatures based on calculated metabolic clearances and measured plasma concentrations, but was not evident by measurement of terminal plasma half-lifes.

Determination of benzocaine in rainbow trout plasma.

A liquid chromatographic method is described for analysis of benzocaine (BZ), a proposed fish anesthetic, in rainbow trout plasma. Mean recoveries of BZ from plasma samples fortified at 44-10 100 ng/mL were 96-100%. The method detection limit is 10 ng/mL, and the limit of quantitation is 37 ng/mL. Acetylation of BZ occurs in whole blood after storage at room temperature (i.e., 21 degrees C) for 10 min. However, no acetylation of BZ was detected in plasma samples held at room temperature for 4 h. Mean method precision for plasma samples with incurred BZ residue is similar to that for fortified samples in the same concentration range (relative standard deviations of 0.9 and 1.2%, respectively).

Metabolism and elimination of benzocaine by rainbow trout, Oncorhynchus mykiss.

1. Branchial and urinary elimination of benzocaine residues was evaluated in adult rainbow trout, Oncorhynchus mykiss, given a single dorsal aortic dose of 14C-benzocaine hydrochloride. 2. Branchial elimination of benzocaine residues was rapid and accounted for 59.2% of the dose during the first 3 h after dosing. Renal elimination of radioactivity was considerably slower; the kidney excreted 2.7% dose within 3 h and 9.0% within 24 h. Gallbladder bile contained 2.0% dose 24 h after injection. 3. Of the radioactivity in radiochromatograms from water taken 3 min after injection, 87.3% was benzocaine and 12.7% was N-acetylated benzocaine. After 60 min, 32.7% was benzocaine and 67.3% was N-acetylated benzocaine. 4. Of the radioactivity in radiochromatograms from urine taken 1 h after dosing, 7.6% was para-aminobenzoic acid, 59.7% was N-acetylated para-aminobenzoic acid, 19.5% was benzocaine, and 8.0% was N-acetylated benzocaine. The proportion of the radioactivity in urine changed with time so that by 20 h, 1.0% was para-aminobenzoic acid and 96.6% was N-acetylated para-aminobenzoic acid. 5. Benzocaine and a more hydrophobic metabolite, N-acetylated benzocaine, were eliminated primarily through the gills; renal and biliary pathways were less significant elimination routes for benzocaine residues.

Plasma catecholamine concentrations in rainbow trout (Salmo gairdneri) at rest and after anesthesia and surgery.

The effects of surgery and anesthesia on concentrations of plasma epinephrine (E), norepinephrine (NE), and dopamine (DA) were investigated in rainbow trout fitted with dorsal aorta cannulae. Baseline catecholamines (CA) concentrations, established in resting rainbow trout, were 1.55 +/- 0.90 pmol/ml (mean +/- SD) for E, 2.07 +/- 1.26 for NE, and 1.33 +/- 0.87 for DA. These values were based on the pooled analyses of five individual fish taken over seven different sampling periods. The E:NE ratio in resting fish was always less than 1.0. In a second experiment, fish were subjected to dorsal aorta cannulation and sequential blood samples were taken immediately after surgery, and 6, 24, and 48 hr later. Plasma E concentrations were 36 times greater than baseline values in the first sample; NE was 15 times greater and DA was 41 times greater. After surgery, plasma concentrations of all CAs fell rapidly but values were still higher than baseline 6 hr after surgery, then were near baseline at 24 and 48 hr after surgery. The E:NE ratio was about 3.0 immediately after surgery, dropped to 1.8 at 6 hr, and was about 1.0 at 24 and 48 hr. In a third experiment, plasma CAs were determined in a group of five animals anesthetized with tricaine methanesulfonate (100 mg/ml) to advanced anesthesia, and then allowed to recover in flowing well water over a 12-hr observation period. Plasma E and NE concentrations in the fish during early anesthesia (1.14 +/- 0.14 min) were not significantly different from preanesthesia values.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of whole body and tissue blood volumes in rainbow trout (Salmo gairdneri) with(125)I bovine serum albumin and (51)Cr-erythrocyte tracers.

Total, packed cell and, plasma volume estimates were made for the whole body and selected tissues of rainbow trout by the simultaneous injection of radiolabelled trout erythrocyte ((51)Cr-RBC) and radioiodinated bovine serum albumin ((125)I-BSA) tracers. Blood volumes were estimated with both markers separately by the tracer-hematocrit method and as the combination of the(51)Cr-RBC packed cell and(125)I-BSA plasma volumes. Mean whole body blood volume was significantly less when calculated from the(51)Cr-RBC tracer data (3.52±0.78 ml/100 g; ±SD) than when calculated with the(125)I-BSA tracer (5.06±0.86 ml/100 g) or as the sum of the two volumes combined (4.49±0.60 ml/100 g). The whole body hematocrit (28±5%), estimated as the quotient of the(51)Cr-RBC volume divided by the sum of the(125)I-BSA and the(51)Cr-RBC volumes, also was significantly less than the dorsal aortic microhematocrit (36±4%). Estimates of total blood volumes in most tissues were significantly smaller when calculated from the(51)Cr-RBC data than when calculated by the other two methods. Tissue blood volumes were greatest in highly vascularized and well perfused tissues and least in poorly vascularized tissues. The relative degree of vascularization among tissues generally remained the same regardless of whether the red cell or the plasma tracer was used to calculated blood volume. It is not clear whether the expanded plasma volume is the result of the distribution of erythrocyte-poor blood into the secondary circulation or the result of extravascular exchange of plasma proteins.

Estimates of plasma, packed cell and total blood volume in tissues of the rainbow trout (Salmo gairdneri).

1. Total blood volume and relative blood volumes in selected tissues were determined in non-anesthetized, confined rainbow trout by using 51Cr-labelled trout erythrocytes as a vascular space marker. 2. Mean total blood volume was estimated to be 4.09 +/- 0.55 ml/100 g, or about 75% of that estimated with the commonly used plasma space marker Evans blue dye. 3. Relative tissue blood volumes were greatest in highly perfused tissues such as kidney, gills, brain and liver and least in mosaic muscle. 4. Estimates of tissue vascular spaces, made using radiolabelled erythrocytes, were only 25-50% of those based on plasma space markers. 5. The consistently smaller vascular volumes obtained with labelled erythrocytes could be explained by assuming that commonly used plasma space markers diffuse from the vascular compartment.