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Description of methods used

1080 in water, soil, and biological materials

The method is used for the determination of trace amounts of sodium monofluoroacetate in water, soil, and biological samples. Aqueous extracts are obtained from tissue and plant samples by dispersing in an alcohol/water mixture, centrifuging, filtering, and passing through an ion exchange column to extract the 1080. Milk samples or milk made from milk powder are mixed with acetone and passed through an ion exchange column as for tissue samples.

Blood serum or plasma, and invertebrate samples, after precipitation of the protein, and urine samples, are added directly to 2% sodium chloride solution ready for derivatisation.

The 1080 in the aqueous extract or water sample is acidified with hydrochloric acid and converted to the dichloroaniline derivative with N,N'-dicyclohexylcarbodiimide and 2,4-dichloroaniline using ethyl acetate as the extraction solvent. The derivative is cleaned on a silica solid phase extraction cartridge to remove excess derivatising agent, eluted with toluene, and quantified by gas chromatography on a BP-5 capillary column with electron capture detection.

This test is IANZ accredited.

The limit of detection (LD) for different samples is given below:

1080 in powders, stock solutions and bait materials

A sample of bait is ground and a subsample taken and acidified. The monofluoroacetic acid is extracted with ethyl acetate, diluted in acetone, and derivatised with pentafluorobenzyl bromide at 55°C. The resulting solution is analysed on a gas chromatograph equipped with an electron capture detector.

Powder and solution samples are analysed by this method from dilute solutions (1000 μg/mL) using the same preparation as for 1080 standards.

1080 washdown sump-water samples are tested in the same way as for 1080 standard solutions.

This test is IANZ accredited.

The limit of detection for cereal and carrot bait is 0.0002% (0.002 mg/g).
The limit of detection for waste sump water is 5 μg/mL.

Compound 1080 grain bait assay. Denver Wildlife Research Center Method No. 8B May 1989.

Alpha-chloralose active ingredient in wheat bait

Wheat baits comprising wheat grains coated with alpha-chloralose are used as a bird repellent/toxicant. Samples of bait are extracted with methanol and the filtered extract is assayed by HPLC, using a C8 column and UV detector set at 203 nm.

Anthraquinone in carrot bait and formulations

A sample of carrot bait is homogenised with water thickened with Carbapol 941, and extracted in a heated ultrasonic bath, followed by shaking on a shaking machine with methanol, and then analysed by HPLC.

A sample of Avex suspension is diluted with methanol and water, ready for analysis by HPLC.

The analysis is carried out on a liquid chromatograph equipped with a C18 column and a UV/VIS detector.

Brodifacoum in stock solutions and bait materials

This method is for the assay of brodifacoum content of concentrates, cereal bait, paste bait and wax block bait.

A sample of concentrate (2.5% w/w) is weighed into a volumetric flask and diluted to 100 mL with methanol/water/acetic acid. This solution is then further diluted in the same solvent to achieve a suitable concentration for HPLC analysis.

A sample of cereal bait is ground and a subsample is weighed into a centrifuge tube. The mixture is extracted with methanol and a 2 M sodium hydroxide solution using an ultrasonic bath followed by shaking. After neutralising with acetic acid a small aliquot of the combined extracts is filtered and diluted in mobile phase for HPLC analysis.

A sample of wax block bait is ground and a subsample is weighed into a centrifuge tube. Anhydrous sodium sulphate is added and the mixture extracted with methanol using an ultrasonic bath followed by shaking. A small aliquot of the combined extracts is filtered and diluted in mobile phase for HPLC analysis.

A sample of paste or gel bait is mixed with anhydrous sodium sulphate and the mixture homogenised and extracted with methanol. A small aliquot of the combined extracts is filtered and diluted in mobile phase for HPLC analysis.

The analysis is carried out on a liquid chromatograph equipped with a C18 column, a fluorescence detector and a post-column reagent pump. Difenacoum is used as an internal standard for improved quantitation. A post-column pH switching technique, using 10% ammonia as the post-column reagent, is used to exploit the natural fluorescence of the rodenticide.

This test is IANZ accredited.

Brodifacoum in animal tissues

A sample of animal tissue (normally liver) is chopped and a subsample is mixed with anhydrous sodium sulphate in a beaker followed by extraction with chloroform/acetone/ammonia. The extracts are evaporated and taken up in chloroform/hexane for SPE clean-up on an aminopropyl column. The analyte is eluted from the column using 0.05 M TBAP in methanol, which is evaporated and the sample taken up in a mobile phase of methanol/water/acetic acid, for HPLC analysis.

A sample of invertebrate tissue is chopped and a subsample is mixed with anhydrous sodium sulphate in a beaker followed by chloroform/acetone/ammonia. The contents of the tube are shaken and centrifuged. The supernatant is decanted and the extraction process repeated twice more. The combined extracts are evaporated and taken up in chloroform/hexane for SPE clean-up on an aminopropyl column. The analyte is eluted from the column using 0.05 M TBAP in methanol, which is evaporated off and the sample taken up in mobile phase for HPLC analysis.

The analysis is carried out on a liquid chromatograph equipped with a C18 column and fluorescence detection. A post-column pH switching technique is used to exploit the natural fluorescence of this compound and difenacoum is used as an internal standard for improved quantitation.

This test is IANZ accredited.

The method detection limit (MDL) in liver tissue is

Brodifacoum in serum and plasma

The serum or plasma sample is extracted with acetonitrile which also removes the protein. Diethyl ether is added to remove water and the sample evaporated on a vacuum evaporator. The residue taken up in methanol is analysed by HPLC with fluorescence detection.

The least detectable level is taken as 0.02 μg/mL.

Brodifacoum in water

A water sample (500 mL of stream water and 50 mL of waste sump water) is passed through a C18 solid-phase extraction cartridge. The analyte is eluted with acetone, evaporated, taken up in methanol, and injected into an HPLC using post-column pH switching and fluorescence detection.

This test is IANZ accredited.

The method detection limit (MDL) is given below:

Bromadiolone in bait materials

This method is for the assay of bromadiolone in concentrates, cereal bait, paste bait and wax block bait.

A sample of concentrate is weighed into a volumetric flask and diluted with methanol/water/acetic acid. This solution is then further diluted in the same solvent to achieve a suitable concentration for HPLC analysis.

A sample of wax block bait is ground in a mortar and pestle and a subsample is weighed into a centrifuge tube. Anhydrous sodium sulphate and an internal standard are added and the mixture homogenised and extracted with methanol using an ultrasonic bath followed by shaking. A small aliquot of the combined extracts is filtered and diluted in mobile phase for HPLC analysis.

The analysis is carried out on a liquid chromatograph equipped with a C18 column, using fluorescence detection. Difenacoum is used as an internal standard for improved quantitation. A post-column pH switching technique, using 10% ammonia as the post-column reagent, is used to exploit the natural fluorescence of the rodenticide.

Rodenticides (see below) in animal tissues

The method determines the concentration of warfarin, coumatetralyl, bromadiolone, flocoumafen and brodifacoum in animal tissues using HPLC with fluorescence detection. A post-column pH switching technique is used to exploit the natural fluorescence of these compounds. Difenacoum is used as an internal standard.

A sample of animal tissue (normally liver) is chopped and a 1-g subsample is mixed with anhydrous sodium sulphate in a beaker and extracted with chloroform/acetone/ammonia. The extracts are evaporated and taken up in chloroform/hexane for SPE clean-up on an aminopropyl column. The analytes are eluted from the column using 10 mL of 0.05 M TBAP in methanol. The solvent is evaporated and the sample taken up in a mobile phase of methanol/water/acetic acid, for HPLC analysis.

This test is IANZ accredited.

The method detection limits (MDL) in liver tissue are:

Cholecalciferol in concentrates and bait material

This method is designed for the assay of cholecalciferol resin (71%), oil (37.5%) and baits (0.8%).

The sample of cholecalciferol resin is shattered with a mortar and pestle, weighed into a volumetric flask, dissolved in toluene and made up to volume. The solution is diluted with hexane/isopropyl alcohol, filtered and analysed by HPLC.

The sample of cholecalciferol oil concentrate is warmed, mixed and weighed into a volumetric flask, toluene added, ultrasonicated, and made up to volume. The solution is diluted with hexane/isopropyl alcohol, filtered and analysed by HPLC.

The sample of cereal bait is ground in a mortar and pestle and extracted with isooctane using sonication. The extract is diluted with hexane/isopropyl alcohol, filtered and analysed by HPLC.

The sample of aqueous paste or gel bait is weighed and treated with 1 N sodium hydroxide solution, diluted and extracted with toluene on a Varian ChemElut liquid/liquid extraction cartridge. The extract is filtered and diluted with hexane/isopropyl alcohol for analysis by HPLC.

The sample of ground wax-block bait or oily paste bait is weighed into a volumetric flask, toluene added, ultrasonicated, and made up to volume. The solution is diluted with hexane/isopropyl alcohol, filtered and analysed by HPLC.

The HPLC analysis is carried out on an HPLC equipped with a silica column and a UV detector set at 265 nm. The solvent is 3% isopropyl alcohol/hexane run isocratically at 1 mL/min.

Coumatetralyl in bait materials

The cereal bait is milled and duplicate samples extracted with methanol, centrifuged, made up to volume and analysed by HPLC.

The sample of paste bait is ground in a mortar and pestle with a silicaceous powdering agent and extracted with methanol. The extract is filtered, diluted as necessary and analysed on the HPLC as above.

The analysis is carried out on a liquid chromatograph equipped with a C18 column and a UV/VIS detector.

Diphacinone in bait materials

Samples of homogenised cereal bait or whole paste bait are extracted in a heated ultrasonic bath followed by shaking using a solvent mixture of acetonitrile/methanol/phosphoric acid. The extract is neutralised with triethanolamine buffer, filtered and injected into an HPLC using paired ion chromatography on a C8 column and a fixed wavelength UV detector at 284 nm.

Technical diphacinone is dissolved in methanol and diluted to approximately 2 μg/mL in mobile phase and analysed by HPLC.

Diphacinone in animal tissues

The method determines the levels of diphacinone in animal and invertebrate tissues using paired ion chromatography (PIC) and a UV detector at 284 nm.

A sample of animal tissue (normally liver) is weighed into a glass Oak Ridge tube. Chlorophacinone, to act as an internal standard, and anhydrous sodium sulphate are added and extracted with chloroform/acetone/formic acid. The extracts are evaporated and taken up in hexane/chloroform for clean-up on a carbograph SPE column followed by an aminopropyl column. The eluent is evaporated to dryness and taken up in mobile phase for quantification by HPLC analysis, using a C8 column and UV detector set at 284 nm.

A sample of insect (normally whole chopped insect/s) is weighed into a glass Oak Ridge tube. Chlorophacinone, to act as an internal standard, and anhydrous sodium sulphate are added and extracted with chloroform/acetone/formic acid. The combined extracts are evaporated and taken up in hexane/chloroform for clean-up on an aminopropyl SPE column. The analyte is eluted from the SPE column with TBAP in methanol, which is then evaporated to dryness and taken up in mobile phase for quantification by HPLC analysis, using a C8 column and UV detector set at 284 nm.

This test is IANZ accredited.

The method detection limit (MDL) is shown below:

PAPP in baits

For tablets, the test is carried out on individual tablets. The tablet is crushed and extracted by shaking with methanol, diluted and filtered for HPLC analysis.

A sample of fish paste bait 0.2% w/w is mixed and a subsample extracted by shaking with methanol, then filtered for HPLC analysis.

The analysis is carried out on a liquid chromatograph equipped with a C8 column and fluorescence detection.

PAPP in water

A 200-mL water sample is passed through a C18 solid-phase extraction cartridge. The analyte is eluted with methanol, evaporated, taken up in mobile phase and a subsample filtered and diluted for HPLC analysis using a C8 column and a UV/Vis detector set to 300 nm. The mobile phase is methanol/water buffered with ammonium acetate, acetic acid and triethanolamine.

The method detection limit (MDL) is shown below:

Pindone in bait materials and formulations

Duplicate samples of homogenised bait are extracted on a shaking machine with a solvent mixture of acetonitrile/methanol/phosphoric acid, then neutralised with triethanolamine buffer, and filtered.

Technical Pival or Pivalyn (the sodium salt) is dissolved in methanol or water as appropriate, and diluted in mobile phase. Samples are analysed by HPLC using paired ion chromatography on a C8 column, with a UV detector at 284 nm.

This test is IANZ accredited.

Pindone in animal tissues

The method determines the levels of pindone in animal tissues using paired ion chromatography (PIC) and a UV detector at 280 nm.

A sample of tissue (normally liver) is chopped and a 2-g subsample is ground in anhydrous sodium sulphate, transferred to a centrifuge tube and extracted with chloroform/acetone/formic acid. The combined extracts are evaporated and taken up in hexane/chloroform/acetone for application to a gel permeation column for clean-up. Diphacinone is added, to act as an internal standard, to the eluent from the column, which is evaporated and taken up in mobile phase for HPLC analysis, using a C8 column and UV detector set at 280 nm.

The method detection limit (MDL) is shown below:

Pindone in soil

A sample of sieved soil is extracted on a shaking machine with a solvent mixture of acetonitrile/methanol/phosphoric acid, neutralised with triethanolamine buffer, filtered and injected into an HPLC using paired-ion chromatography on an ODS column and a UV detector at 280 nm.

The limit of detection in soil is 0.5 μg/g.

Pindone in water

A water sample (100 mL of stream water or 10 mL waste sump water) is passed through a C18 solid-phase extraction cartridge. The analyte is eluted with 0.005 M tetrabutyl ammonium phosphate in methanol, evaporated, taken up in mobile phase and injected into an HPLC using a C8 column and UV detector set at 284 nm. Diphacinone is added to the sample to act as internal standard.

The method detection limits (MDL) are shown below:

Rotenone in water

The rotenone content is measured on 500-mL water samples stabilised with phosphoric acid received in amber glass bottles. The sample is passed through a C18 solid-phase extraction cartridge. The analyte is eluted with 5 mL methanol, filtered and tested by HPLC using a 5-µm C18 column, UV detector set at 295 nm and methanol/water (90/10) at 0.8 mL/min.

The method detection limit (MDL) is:

* May depend on the presence of interfering peaks.

Warfarin in bait materials

The cereal bait is milled and samples extracted with a solvent mixture of methanol/water/acetic acid, centrifuged, made up to volume and injected into an HPLC with post-column pH change and fluorescence detection.

The sample of paste bait is ground in a mortar and pestle with a silicaceous powdering agent and extracted with a solvent mixture of methanol/water/acetic acid. The extract is filtered and analysed on the HPLC as above.

Warfarin in serum/plasma

The method determines the levels of warfarin in serum and plasma using fluorescence detection. A post-column pH switching technique using a mixture of ammonia, methanol and water exploits the natural fluorescence of this compound. Coumatetralyl is used as an internal standard.

The serum or plasma sample is extracted in diethyl ether in the presence of hydrochloric acid. The extract is then dried down on a heating block fitted with an air blowdown system. The residue taken up in mobile phase is analysed by HPLC with fluorescence detection.

The method detection limit (MDL) is:

Zinc phosphide in bait materials

This method has been developed to assay the zinc phosphide content of cereal and paste bait.

An amount of cereal bait material is ground with a mortar and pestle. A small aliquot of the homogeneous mix is weighed into a glass centrifuge tube and toluene and 5% sulphuric acid is added. The tube is shaken and centrifuged. A GC vial is filled with the toluene mix for analysis using an HP5 column and nitrogen phosphorus detector.

The paste bait is thoroughly mixed and a small aliquot is weighed into a centrifuge tube and analysed in a similar way to the cereal bait.

An amount of granulated micro-encapsulated zinc phosphide (MEZP) is weighed directly into the 35-mL centrifuge tube. The tube is capped and shaken on a horizontal shaker. A GC vial is filled with the toluene mix for analysis.

The method detection limit (MDL) is:

Zinc phosphide in animal tissues

The zinc phosphide content of stomach samples is measured by this method. An amount of stomach contents is homogenised using the Ultra-Turrax and a small aliquot of the homogeneous mix is weighed into a centrifuge tube followed by toluene and 5% sulphuric acid. The tube is capped and shaken on a horizontal shaker. The tube is then centrifuged and a GC vial filled with the toluene solution for analysis using an HP5 column and nitrogen phosphorus detector.

The method detection limit (MDL) is:

Zinc phosphide in soil

A small homogeneous aliquot of soil is weighed into a centrifuge tube to which toluene and 5% sulphuric acid is added. The tube is capped and shaken on a horizontal shaker. The tube is then centrifuged and a GC vial filled with the toluene solution for analysis using an HP5 column and nitrogen phosphorus detector.

The method detection (MDL) level is:

Cinnamon content in bait materials

This method has been developed for the assay of the flavour content in concentrates, and cereal bait and carrot bait.

The sample of cereal bait is soaked in methanol, shaken, then allowed to stand overnight. The bait and solvent are then homogenised with an Ultra-Turrax homogeniser and shaken again. An aliquot of the extract is removed, filtered and diluted for analysis on a GLC with a flame ionisation detector.

The sample of cinnamon concentrate is diluted with methanol and analysed on the GLC as above.

Moisture content of bait materials

This method has been developed to measure the moisture content and solids content of cereal baits, paste, and gels.

The sample is weighed into a dish and placed in an oven at 103°C for a period of 20 hours. After heating it is allowed to cool in a dessicator and then reweighed. The sample is again heated for 5 hours, cooled and reweighed as before. The moisture content is calculated using the appropriate spreadsheet.

This test is IANZ accredited.