toxicokinetics of imidacloprid in rainbow trout of imidacloprid in rainbow trout john a. frew1, ......
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1. Compliance Services International, Lakewood, WA2. College of Pharmacy, University of Minnesota, Duluth, MN3. Mid-Continent Ecology Division, U.S. Environmental Protection
Agency, Duluth, MN4. School of Aquatic and Fishery Sciences, University of Washington,
Seattle, WA
Toxicokinetics of imidacloprid in rainbow trout
John A. Frew1, Jacob T. Brown2, Patrick N. Fitzsimmons3, Christian E. Grue4, Alex D. Hoffman3, John N. Nichols3
Goal
Characterize the disposition of imidacloprid (IMI) in fish
A D M E(Absorption, Distribution, Metabolism, Excretion)
Elimination
Disposition
(systemic exposure)
Objectives
• Conduct depuration studies with rainbow trout at “low”, “medium” and “high” asymptomatic dosing levels
• Quantitate residues in plasma, expired (branchial) water and urine; selected tissues
• Evaluate hepatic biotransformation of IMI
• Utilize measured residues in plasma to model primary kinetic parameters
• Utilize measured residues in expired water and urine to calculate branchial and renal clearances
• Conduct mass-balance analysis
• Contribution of mechanisms to overall clearance
Chambered fishUS EPA Mid-Continent Ecology Division
McKim & Goeden (1982)
• Cannula (dosing accuracy, multiple sampling intervals)• Expired (branchial) water• Urine
Depuration studies
Three dosing groups• “Low” = 47.6 μg/kg bw (n = 5)• “Medium= 117.5 μg/kg bw (n = 8)• “High” = 232.7 μg/kg bw (n = 5)
Low and High groups (48 h)• Plasma and expired water @ 1, 2, 4, 8, 16, 24, 36, 48 h
post-injection• Brain, kidney, liver, muscle and bile @ test termination
Medium group (36 h)• Plasma, expired water (n =8) and urine (n = 4) @ 0.5, 1,
2, 4, 8, 12, 16, 20,24, 36 h post-injection• Brain, kidney, liver, muscle and bile @ test termination
Samples flash-frozen in liquid N2 and stored @ -80 °C
Evaluation of hepatic biotransformation
S9 liver fractions
Incubate samples for 20, 40, 60, 80,100,120
minutes
Centrifugation @ 9,000g
Add 0.1 or 1.0 µM IMI
Mass Spec residue
quantitationSample storage @ -80°C
Substrate Depletion Assay
(Loss of chemical over time)
Analytical methods
• Sample preparation• Extraction: ACN/sonication• Additional clean-up: RP-SPE• Incorporation of D-4 IMI IS
• Residue quantitation• LC-MS/MS detection through adaptation of established
protocol (Schoning & Schmuck 2003)• LOQ: 1.0 µg/kg• LOD: 0.3 µg/kg
• Methods Validation• Intra- Reproducibility: 1.2–1.8%• Inter- Reproducibility: 0.2–3.9%
Plasma concentrations• Mean concentrations, medium dose (n = 8)• Time intervals 0.5 – 36 h post-injection
1
10
100
1000
0 5 10 15 20 25 30 35 40
Conc
entr
atio
n (u
g/L)
Time (h)
Plasma concentration-time profile
Terminal (elimination) phase
distribution phase (ends 4–6 h)
Toxicokinetic parameters
1Volume of distribution2Whole-body clearance
Dosinggroup
VD1
(L/kg)CLWB
2
(L/h/kg)t½
3
(h)
Low 1.72(0.07)
0.0217(0.0073)
66.98 (20.76)
Medium 2.23(0.30)
0.0270(0.0127)
68.35 (26.82)
High 1.81(0.25)
0.0195(0.0041)
68.1 (16.95)
(non-compartmental kinetic analysis)
3Whole-body half life
Branchial and renal clearance
Dosing group CLb1 (L/h)
Medium (36 h) 0.014 (0.0052)High (48 h) 0.0115 (0.0029)
CLr2 (L/h)
Medium (36 h) 0.0304 (0.0222)1Branchial clearance2Renal clearance
(expired water and urine residues)
Tissue Low Medium HighBrain 1.4 (0.1) 1.8 (0.4) 1.3 (0.1)Kidney 3.3 (0.3) 9.8 (4.6) 3.4 (0.3)Liver 3.0 (0.4) 3.9 (1.5) 3.0 (0.3)Muscle 1.7 (0.3) 2.2 (0.7) 1.6 (0.1)Bile 5.1 (1.0) 7.9 (3.1) 5.6 (0.3)Urine NA 5.0 (1.9) NA
Tissue:plasmaconcentration ratios
Concentration (μg/kg)
• Relative affinity consistent with modeled VD
Medium dose 0-36 h excretion(% dose)
36 h mass balance(% dose)
Branchial only(n = 8) 17.2 (3.9) 79.1 (20.4)
Renal only(n = 4) 31.2 (14.9) 83.8 (20.3)
Branchial and renal(n = 4) 48.0 (14.9) 100.6 (21.3)
Mass balance analysis
(account for amount of IMI in body at test termination)
Metabolism negligible in fish
Contributions to overall clearance
• S9 fractions• No measurable substrate depletion• Hepatic metabolism does not contribute to overall
clearance• Significant clearance route in mammals
• Renal clearance• ~ 66.6% of whole-body clearance
• Branchial clearance• ~ 33.3% of whole-body clearance
Practical applications
Environmental monitoring
Practical applications
Fish aquaculture• Paracitacide administered in food• Therapeutic dose• Residues in tissues/human consumption• Elimination: IMI in the environment
Conclusions
• Disposition• Rapidly distributed• Two/three-fold greater VD than predicted• Hepatic metabolism negligible• Mass balance: ~100% parent compound excreted• Clearance
• Renal: ~2/3rd; Branchial: ~1/3rd
• Practical applications• Environmental monitoring• Aquaculture usage: dosing, human health and
environmental concerns
Acknowledgements
• WACFWRU/USGS, US EPA (funding sources)• Dr. John Nichols (US EPA, Mid-continent Ecology
Division)• Dr. Christian Grue (University of Washington)• Dr. Martin Sadilek (UW Dept. of Chemistry Mass
Spec. Facility)• Patrick Fitzsimmons and Alex Hoffman (US EPA,
Mid-continent Ecology Division)• Jacob Brown (University of Minnesota/Duluth)