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)