Analysis of Diphacinone Residue in Fish by LC/MS/MS Jingyu Chen, Binh Loo Hawaii Department of Agriculture Chemical Analysis Laboratory

2009 FERN National Training and Conference Dallas, Texas June 16-19, 2009

Introduction • Lehua Island,

•

•

Ni’ihau Island (a privately owned, shown in the background) is about half mile from Ni’ihau Most of the residents of Ni’ihau are native Hawaiian. Fish is a staple of the Hawaiian diet.

CB032574| Value RF| © Michael T. Sedam/Corbis Lehua Island off Niihau

An investigation of possible contamination

• January 17, 2009, the owner of Ni’ihau reported “tons of fish” washing onto their beaches.

• January 21, 2009, the owner reported a juvenile whale had beached on Ni’ihau.

Dead Fish Photos (1) (from owner of Niihau)

Dead Fish Photos (2)

Could Diphacinone be the culprit? Reasons for: • Rats prey on endangered birds and snails in •

Hawaii. Wildlife protection agencies used the rodenticide Diphacinone to eradicate rats on Lehua Island. – 3,900 pounds of diphacinone bait was aerial applied to Lehua Island on January 6, 2009 and again on January 13, 2009

• The dominant fish found dead were two species of trigger fish (humuhumu ‘eleele and the humuhumu hi’ukole)

– These fish stay close to the surface and would be attracted to bait

Reasons against Diphacinone: • Diphacinone doesn’t survive long in the marine • •

environment. It decomposes quickly in water and sunlight. [1] The rat bait used are low concentration: 0.005% Diphacinone. There were no heavy rains that would have caused rat bait to wash into the ocean during the period between the application of the rat bait and when the dead fish were found. [9]

Timeline for the Investigation • February 4, 2009, state water pollution and aquatic • • •

resources personnel collected the fish specimens from Ni’ihau. Department of Health screened the dead fish for organochlorines and organophophorus pesticides and nothing was detected. Residents of Ni’ihau expressed concern about food safety. Late March 2009, Hawaii Department of Agriculture was requested to test the fish specimens for diphacinone.

Role of the Chemical Analysis Laboratory • To ease the anxiety of Ni’ihau resident, DOA develop • •

plan to determine if the fish are contaminated with diphacinone CAL would develop a LC/MS/MS method to analyze the fish for diphacinone residue. CAL would analyze the following fish samples. – Trigger fish caught off shore of Honolulu to serve as a control. – The dead fish specimens collected from Ni’ihau shores. – Different species of fish caught off different area of Ni’ihau Island.

Properties of Diphacinone • Solubility: Insoluble in water

(ca. 0.3 mg/kg). In chloroform 204, toluene 73, xylene 50, acetone 29, ethanol 2.1, heptane 1.8 (all in g/kg).[1]

• Toxicity to fish: LC50 (96 hours) for rainbow trout 2.8, bluegill sunfish 7.6, channel catfish 2.1 mg/l. [1]

Review of the Analytical Methods of Diphacinone Residue • GC-ECD, GC-MS, Thin-layer

chromatography, Immunoassay[2-5]

Most commonly used: • HPLC-UV (PDA) [6,7] • LC-MS/MS[8]

Sample Preparation

Accelerated Solvent Extraction Condition • Oven temperature: • Pressure: • Static time: • Flush volume: • Nitrogen purge: • Static Cycles:

60 oC 1500 psi 5 min 60% of the cell volume 60 sec at 1500 psi 1

Accelerated Solvent Extraction Condition – Solvent Selection • Solvent selection: – Dichloromethane – Dichloromethane : Acetonitrile (50%:50%) – Acetonitrile – Acetonitrile : H2O (75%:25%) – Acetonitrile : H2O (60%:40%) – Acetonitrile : H2O (50%:50%)

• More acetonitrile, higher recovery

Accelerated Solvent Extraction Condition - Solvent Selection • Fish liver samples: Acetonitrile : H2O •

(50%:50%) Fish tissue samples: Acetonitrile : H2O (75%:25%), add 0.5ml DI water to 1ml extract before injection

LC/ESI-MS • Varian 1200 LC/MS/MS with ProStar Solvent • •

Delivery Modules Varian PorStar Auto sampler with 20ul sample loop Zorbax XDB C8, 2.1 X 150 mm, 5um column

HPLC Condition – Mobile phase • Pump A: 10mM ammonium acetate • Pump B: Methanol Time

%A

%B

0 min 5 min 6 min 9 min 10 min 15 min

45 40 45 40 45 45

55 60 95 95 55 55

Flow

0.2 0.2 0.2 0.2 0.2 0.2

ml ml ml ml ml ml

MS/MS Transition Q1FM

Q3FM

Capillary

Coll. Energy

Dwell Time

(-) 339.2

(-) 115.8

(-) 40V

42.0V

0.1ms

(-) 339.2

(-) 144.8

(-) 40V

22.0V

0.1ms

(-) 339.2

(-) 166.8

(-) 40V

24.0V

0.1ms

(-) 339.2

(-) 171.7

(-) 40V

22.0V

0.1ms

Mean Recovery and Relative Standard Deviations at 15 ppb level

Mean Recovery (%)

RSD (n=3)

Trigger Fish Liver

69.0

14.6

Trigger Fish Tissue

60.4

10.4

Limit of Detection (LOD) Instrumental LOD (ng/ml)

Method LOD (ng/g)

Fish Liver

1.0

3.0

Fish Tissue

1.0

4.5

Fish Tissue Extract

Standard at 5 ng/ml Prepared by Tissue Extract

Fish Liver Extract

Standard at 5 ng/ml Prepared by Liver Extract

Results • Four composite samples of the dead fish

specimens were analyzed. • A total of 42 fish tissue and liver samples of 8 species of fish from 9 area caught near Ni’ihau Island were analyzed. • No diphacinone residue was detected in these samples.

Conclusions • The analytical method developed to detect the diphacinone residue in fish liver and tissue samples is relative simple and efficient.

• The analyses of all the samples were completed in timely manner.

References 1. Chemistry, R.S.o., The Agrochemicals Handbook. 3rd ed. 1991: 2. 3. 4.

Unwin Brothers Limited, Old working, Surrey. Bullard, R.W., G. Holguin, and J.E. Peterson, Determination of

chlorophacinone and diphenadione residues in biological materials.

Journal of Agricultural and Food Chemistry, 1975. 23(1): p. 72-74. Duffield, P., et al., Warfarin and warfarin-alcohol levels in anticoagulated patients. Aust. N. Z. J. Med., 1979. 9: p. 534. Berny, J.P., T. Buronfosse, and G. Lorgue, Anticoagulant poisoning

in animals: A simple new high-performance thin-layer chromatographic (HPTLC) method for the simultaneous determination of eight anticoagulant rodenticides in liver samples.

J. Anal. Toxicol., 1995. 19: p. 576.

5. Mount, M.E., M.J. Kurth, and D.Y. Jackson, Production of antibodies and development of an immunoassay for the anticoagulant, diphacinone. J. Immunoassay, 1988. 9: p. 69. 6. Robert W. Gale, M.T., Carl E. Orazio, Determination of Diphacinone in Sea Water, Vertebrates, Invertebrates, and Bait Pellet Formulations Following Aerial Broadcast on Mokapu Island, Molokai, Hawaii. 2008, U.S. Department of the Interior; U.S.

7. 8.

9.

Geological Survey. p. 24. Thomas M. Primus, D.J.K., and John J. Johnston, Determination of Diphacinone Residues in Hawaiian Invertebrates. Journal of Chromatographic Science, 2006. 44: p. 1-5. Marek, L.J. and W.C. Koskinen, Multiresidue Analysis of Seven

Anticoagulant Rodenticides by High-Performance Liquid Chromatography/Electrospray/Mass Spectrometry. Journal of

Agricultural and Food Chemistry, 2007. 55(3): p. 571-576. TenBruggencate, J. (2009) Does Ni'ihau fish kill cause still lurk? https://www.blogcatalog.com/blog/raisingislands/ff455da4a48a1ed bdfab078a106987e2