Evaluation of a proposed determinative method to determine concentrations of the 
AQUI-S marker residue in fillet tissue from coolwater and warmwater fish species

The work described in this protocol will be conducted as a regulated study.  The data will 
be submitted to the Center for Veterinary Medicine (CVM) to support acceptance of the 
described method as a determinative method.  The work described in this protocol 
assumes that isoeugenol will be selected as the marker residue for AQUI-S.  A 
tolerance concentration for the AQUI-S marker residue has not been established, but is 
projected to be >1 ug/g and <100 ug/g.

	There is a critical need in U.S. public aquaculture and fisheries management for 
an anesthetic with zero withdrawal time.  Such an anesthetic 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 minimal stress and released immediately.  A zero withdrawal time 
anesthetic 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 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-S as a fish anesthetic with a zero withdrawal time is 
being pursued in the U.S.  As part of the approval process, a marker residue for AQUI-
S must be selected from AQUI-S 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 in a variety of fish species from the 
colwater, coolwater, and warmwater temperature groups 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-
S.
	Assuming isoeugenol is selected as the marker residue, a tolerance concentration 
for isoeugenol will eventually be declared by the Center for Veterinary Medicine (CVM) 
after all mammalian toxicology tests are completed and evaluated.  Teratogenicity and 
carcinogenicity tests with isoeugenol have been 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-S marker residue will not be 
established until 2006.  Despite the lack of a tolerance concentration for the AQUI-S 
marker residue, we will conduct work that will address data requirements for a proposed 
AQUI-S marker residue determinative method.
	A method was developed at the UMESC to determine isoeugenol concentrations 
in fillet tissue from rainbow trout, a coldwater fish species.  The method must be 
evaluated with fillet tissue from coolwater and warmwater fish species before it can be 
considered as the determinative method for the AQUI-S marker residue.  This study 
will evaluate the method with guidance described in CVM guideline 3, General 
principles for evaluating the safety of compounds used in food-producing animals, 
section IV, Guideline for approval of a method of analysis for residues.  The 
isoeugenol concentrations chosen for investigation include a relatively wide range of 
concentrations and were chosen by incorporating current knowledge of isoeugenol 
concentrations in rainbow trout fillet tissue after exposing fish to AQUI-S.

Objectives
	The following objectives were established to support a determinative method for 
isoeugenol in edible fillet tissue from coolwater and warmwater fish species:  (1) evaluate 
method interferences from naturally endogenous compounds extracted from control fillet 
tissue from coolwater and warmwater fish species; (2) evaluate method systematic error 
by assessing percent recovery with fillet tissue from coolwater and warmwater fish 
species fortified with isoeugenol at 1, 50, and 100 ug/g; (3) evaluate method repeatability 
by assessing within-day and day-to-day precision with fillet tissue from coolwater and 
warmwater fish species fortified with isoeugenol at 1, 50, and 100 ug/g; (4) evaluate 
method repeatability with biologically incurred isoeugenol residues in fillet tissue from 
coolwater and warmwater fish species; (5) evaluate method detection and quantitation 
limits with fillet tissue from coolwater and warmwater fish species; (6) evaluate the 
stability of isoeugenol in fillet tissue extracts fillet tissue from coolwater and warmwater 
fish species; and (7) evaluate the stability of isoeugenol in fillet tissue fillet tissue from 
coolwater and warmwater fish species containing biologically incurred isoeugenol 
residues.

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