Meinertz, J.R., G.R. Stehly, and S.L. Greseth. 2001. Development of a Regulatory 
Method for p-TSA in the Edible Fillet Tissue of Rainbow Trout; Evaluation of Readiness 
to Perform and Bridging with a Previously Reported Method to Determine Chloramine-T 
and p-TSA Concentrations in Rainbow Trout Tissue. Final report submitted to the 
UMESC archives January 17, 2001 and submitted to the Center for Veterinary Medicine, 
U.S. Food and Drug Administration, September 28, 2001. 715 pages.
Summary
Waterborne exposure to chloramine-T is an effective treatment for controlling bacterial 
gill disease in fish. Currently data are being generated in an effort to gain U.S. Food and 
Drug Administration (FDA) approval for use of chloramine-T in aquaculture. As part of 
the data required for an approval, depletion of the marker residue of chloramine-T (para-
toluenesulfonamide; p-TSA) from the edible fillet tissue of exposed fish must be 
characterized. Before a depletion study can be conducted, a regulatory method for 
determining p-TSA concentrations in fish fillet tissue must be validated according to 
FDA guidelines.
A method developed at the UMESC to determine p-TSA concentrations in edible fillet 
tissue of three freshwater fish species was submitted to the FDA for review. Although the 
FDA response was favorable, the FDA suggested appending the data supporting the 
method before the method is used in p-TSA residue depletion studies. The research 
requested by the FDA included the following tasks: 1) evaluate the method accuracy and 
precision with rainbow trout (Oncorhynchus mykiss) edible fillet tissue fortified with p-
TSA at 500 (0.5X the expected 1000 ng/g tolerance limit for p-TSA), 1000 (1X the 
expected tolerance limit), and 2000 ng/g (2X the expected tolerance limit); 2) evaluate 
the method precision with edible fillet tissue from at least five rainbow trout containing 
incurred p-TSA after exposing the fish to chloramine-T; and 3) bridge the proposed 
regulatory method with a method used by Dawson and Gingerich (1991) by mimicking 
the exposure of rainbow trout conducted for the original chloramine-T total residue 
depletion study (Dawson and Gingerich, 1991) and comparing the p-TSA concentrations 
in the fillet tissue of fish exposed in this study with data from the study by Dawson and 
Gingerich (1991).
The method accuracy was 92.6 % with samples fortified at 500 ng/g, 93.4 % with 
samples fortified at 1000 ng/g, and 94.6 % with samples fortified at 2000 ng/g. The 
method precision with tissue containing incurred p-TSA at a nominal concentration of 
1000 ng/g ranged from 0.80 to 8.4 %.
Concentrations of p-TSA in fillet tissue determined with the proposed regulatory method 
compared well with p-TSA concentrations determined with the method used by Dawson 
and Gingerich (1991). The mean p-TSA concentrations in fillet tissue from fish exposed 
to 20 mg/L of chloramine-T for 1 h were 140 ng/g at 1 h after the exposure, 110 ng/g at 3 
h, 100 ng/g at 6 h, and 82 ng/g at 12 h. The concentrations of p-TSA found in this study 
were not statistically different at p < 0.05 to the concentrations of p-TSA found in the 
fillet tissue of juvenile rainbow trout exposed by Dawson and Gingerich (1991).