Evaluation of an analytical method to determine concentrations of isoeugenol in the 
edible fillet tissue from cold, cool, and warm water fish species

There is a critical need for an anesthetic/sedative with zero withdrawal time in U.S. 
public aquaculture and fishery management.  Such an anesthetic/sedative would have 
applications for nearly all fish species.  For field studies, including population estimates 
and age and growth studies conducted by federal and state conservation agencies, fish 
could be handled with a minimum of stress and released immediately.  A zero withdrawal 
time anesthetic/sedative would increase the effectiveness of stocking programs by 
decreasing stress related mortalities that occur during and after transport of fish.  The 
availability of an anesthetic/sedative with no withdrawal time would also allow human 
consumption of spawned fish, such as Pacific salmon, immediately after exposure.  
Currently, Finquel (tricane methanesulphonate or MS-222) is the only fish anesthetic 
approved by the U.S. Food and Drug Administration (FDA).  Use of this anesthetic to 
collect field data, spawn fish, or sedate fish during transport is constrained by a 21-day 
withdrawal period.
	The approval of AQUI-STM as a fish anesthetic/sedative with a zero withdrawal 
time is being pursued in the U.S.  As part of the approval process, a marker residue for 
AQUI-STM must be selected from the residues extracted from edible fillet tissue from 
exposed fish.  After selection of the marker residue, a determinative method for the 
marker residue must be developed and validated according to FDA guidelines.  
Thereafter, AQUI-STM marker residue depletion studies can be conducted.
	Recently, a total residue depletion study was conducted at the Upper Midwest 
Environmental Sciences Center (UMESC).  Data from the study indicated that isoeugenol 
was the primary residue in extracts from rainbow trout (Oncorhynchus mykiss) skin-on 
fillet tissue after exposure.  Since isoeugenol was the primary residue in rainbow trout 
fillet tissue, we will assume isoeugenol will be selected as the marker residue for AQUI-
STM.
	Assuming isoeugenol is selected as the marker residue, a tolerance concentration 
for isoeugenol will be declared by the Center for Veterinary Medicine (CVM) after all 
mammalian toxicology tests are completed and evaluated.  Teratogenicity and 
carcinogenicity tests with isoeugenol were conducted by the National Toxicology 
Program (NTP).  Data from those tests are undergoing review by NTP.  Based on current 
information, a tolerance concentration for the AQUI-STM marker residue will not be 
established until 2006.
	A method was developed at the UMESC to determine isoeugenol concentrations 
in rainbow trout skin-on fillet tissue.  The intent of the work described in this protocol is 
to conduct a cursory evaluation of the performance of the same method with edible fillet 
tissue from additional cold water fish species as well as cool and warm water fish species.  
Evaluation will be limited to the method’s accuracy and precision with fortified tissue 
and the presence of compounds in the extracts that could interfere with the 
chromatography of isoeugenol.
	Because a tolerance concentration for isoeugenol has not been determined, data 
generated in this study will be considered supportive to data that will be generated during 
a definitive determinative method validation study.  The isoeugenol concentrations 
chosen for investigation for the current protocol include a relatively wide range of 
concentrations.  The concentrations were chosen by incorporating current knowledge of 
isoeugenol concentrations in rainbow trout fillet tissue after exposure to AQUI-STM.

Objectives
	The following objectives were established to support a liquid chromatography 
(LC) determinative method for isoeugenol in edible fish fillet tissue: (1) evaluate the 
method accuracy and precision with control edible fillet tissue from brook trout 
(Salvelinus fontinalis), lake trout (Salvelinus namaycush), walleye (Stizostedion vitreum), 
yellow perch (Perca flavescens), channel catfish (Ictalurus punctatus), and hybrid striped 
bass (Morone saxatilis x M. chrysops) fortified with isoeugenol at 1, 50, and 100 ug/g, 
(2) evaluate fillet tissue extracts by LC to determine the presence of compounds in the 
extracts that could interfere with the chromatography of isoeugenol at 261 nm, and (3) 
evaluate fillet tissue extracts by LC to determine the presence of compounds that could 
produce interfering ions generated with a mass spectrometer.