Method of Determining Biocides Within Wood

Background to the Invention

The invention relates to a method for determining biocides within wood.

'Presently, chemical preservatives are added to wood, wood-based products and engineered

wood products to protect them. The preservatives can include insecticides, fungicides,

mouldicides and bacteriocides.

For example, the preservatives can be insecticides to protect the products from attack by

insect species. In order to ensure that such wood-based materials are treated with sufficient

insecticide to inhibit attack, it must be possible to quantify the amount of insecticide that is

infused into the material.

As is known in the art, the families of insecticide presently used are neonicotinoids,

synthetic pyrethroids, organophosphates and phenyl pyrazόles. As the potency of the

compounds within these families has increased, the quantity of the compounds required for

protection of timber related materials has decreased remarkably. The resultant low dosages

have made it difficult to detect the active ingredients in the timber related materials, let

alone quantify them.

Conventional methods of analysis for determination of the active content of wood-based

materials rely on chromatography, specifically gas chromatography (GC) and high

performance liquid chromatography (HPLC). Although such chromatographic techniques

yield excellent results for the analysis of the above mentioned insecticides in water or sap

extractions from vegetable based materials, they give reduced recoveries when applied to

the analysis of preservatives such as insecticides in wood and wood-based materials. This is

especially true in the presence of plastic or resin based adhesives or coatings in or on the

wood and wood-based materials.

Immunoassays such as enzyme linked immunosorbent assays are presently used for the

analysis of pesticides on wood, vegetables and surface treated seeds. The use of

immunoassay techniques using conventional immunoassay methodology in an effort to

analyse the amount of insecticide infused into wood-based materials has revealed that these

techniques are inadequate for infused insecticides due to the low recoveries.

The term "preservative" is understood to include not only the original active ingredient

contained within the timber treatment formulation but also biologically active derivatives of

the active ingredient that may arise during the timber treatment process or during the

lifetime of the treated product.

Summary of the Invention

It is therefore an object of the present invention to provide a method for determining

biocides within wood wherein a high recovery is achieved.

Broadly in one aspect the invention consists of a method for quantitation of chemical

preservatives used to treat wood, wood-based products and engineered wood products

characterised in the use of an enzyme linked immunosorbent assay.

The present invention is based on the surprising discovery that when aspects of the

immunoassay technology are combined with aspects of chromatography analyses when

applied to the analysis of preservatives in wood and wood-based materials, a very high

recovery is obtained.

In a preferred form of the invention high performance liquid chromatography extraction

methodology is applied to an enzyme linked immunosorbent assay ("ELISA") technique.

Surprisingly, the invention in this form yields high rates of extraction of imidacloprid relative

to conventional methodology for both high performance liquid chromatography and

immunoassay techniques.

Description of the Preferred Embodiment of the Invention

According to a preferred form of the invention the method includes the steps of reducing the

particle size, through mechanical means of the wood, wood-based material or engineered

wood product to be extracted, using organic liquids that can penetrate the wood matrix as

extraction solvents, filtration of extracts and preparation of standards using appropriate

untreated wood extracts.

Preferably the particle size of the wood-based material being analysed is reduced to between

1000 and 10 micrometres diameter or less. The reduction in particle size can be carried out

before extraction solvent using a Wiley mill or after extraction solvent is added using an

industrial type high shear blender like a Silverson_.

The extraction solvent is generally an organic solvent such as, but not restricted to,

methanol, dichloromethane, acetone, or toluene. Non water miscible solvents are then

stripped from the extract by evaporation under reduced pressure and taken into either water

or methanol prior to analysis with ELISA techniques. Water miscible solvents can be used

providing they are diluted into water by a ratio of 400:1 or greater.

The filtration of extracts can involve filtration with .02 micrometre pore filters.

The preparation of standards of known concentration using extracts from appropriate

untreated wood-based materials can be in accordance with techniques known within the art.

According to a further embodiment of the invention accelerated solvent extraction, and

soxhlet extraction can also be used.

More particularly a preferred embodiment of the invention includes the following steps.

a) Primary mechanical fragmentation of the treated wood, wood-based product or

engineered wood product.

b) Suspension of the wood particles in an appropriate extraction solvent.

c) Secondary fragmentation of the suspended wood particles in order to reduce particle

size.

d) Incubation of the suspended wood particles.

e) Separation of the extraction solvent from suspended wood solids.

f) Dilution of the extraction solvent into a suitable aqueous sample buffer.

g) Determination of the concentration of extracted and diluted preservative using an

enzyme-linked immunosorbent assay directed at the preservative of interest.

Primary mechanical fragmentation of the wood product (step a above) may include but is not

restricted to milling, grinding, shaving, shredding, or sanding or a combination of the above,

and may be achieved using equipment such as a Willey mill, or a Silverson as disclosed

above.

The extraction solvent used in step b above in the preferred form of the inventions is able to

both penetrate the wood matrix and solubilise the preservative of interest. Accordingly the

solvent may include various organic solvents, water, or aqueous mixtures containing

solubility aids such as salts, detergents, or a combination thereof.

Secondary mechanical fragmentation of the suspended wood particles (step c above) may

include but is not restricted to shearing in an in-line or batch type high shear mixer,

treatment with ultrasound, rapid pressurisation and depressurisation as in a French press or

a Manton Gaulin mixer, other methods of homogenisation known to those skilled in the art,

or a combination of treatments.

Incubation of the suspended wood particles (step d above) is in the preferred form of the

invention sufficient to allow equilibration of the preservative of interest between the solid

and liquid phases of the extraction mixture. It is to be noted that during the incubation the

mixture may be stirred, heated, treated with ultrasound, subjected to one or more freeze-

thaw cycles, evacuated, pressurised, or a combination of these treatments, in order to

facilitate diffusion of the preservative.

Separation of the extraction solvent and soluble compounds from the wood solids (step e

above) may be achieved by centrifugation, screening, sieving, decantation, or filtration.

Preferably the extraction solvent is separated by centrifugation and filtration through a 0.2

micron filter.

In order to be quantitated by ELISA technique the extracted preservative is preferably

presented in a predominantly aqueous phase. The extraction solvent containing the

preservative of interest may be diluted directly into a suitable aqueous sample buffer in the

case of a water miscible solvent (step f above), or in the case of a non-water miscible

solvent, the solvent may be removed by evaporation and the preservative re-dissolved in the

sample buffer.

Suitable aqueous sample buffers include water or, preferably, a buffered saline solution

containing additional compounds as "blocking agents" intended to reduce non-specific

binding reactions in the ELISA. Examples include phosphate-buffered saline or tris-buffered

saline, containing in addition one or more detergents, proteins, or synthetic polymers, in

concentrations and combinations known to those skilled in the art. It should be noted that

these blocking agents may be included in the aqueous sample buffer or may be added at a

later stage during execution of the ELISA.

Examples

The following example relates to the analyses of commercially manufactured and treated

sheets of laminated veneer lumber made of pinus radiata veneers and cured with phenol-

formaldehyde resin for the amount of imidacloprid present per one hundred grams of oven

dried wood sample.

Example 1.

Thus the present invention provides a method of determining biocides in wood, wood-based

products and wood engineered products where the method links extraction techniques most

commonly employed in GC and HPLC type techniques for analysing compounds in wood,

then using ELISA techniques to analyse these extractants. Thus there is a solvent (non-water

miscible) extraction technology followed by the water miscible analysis technology. In the

past there has been no attempt to employ ELISA form of analysis to wood extractions due to

the nature of the solvents used in extraction. The present invention, therefore, employs the

more powerful extraction techniques of solvents, but then by evaporating the solvent

moving to a water system where the extractant is analysed via a water miscible system.

According to the present invention therefore a recovery higher than normal methods of

analysis is achieved when immunoassay technology is combined with aspects of

chromatographic analyses. Thus, analysis of insecticides in wood and wood-based materials

when low dosages of insecticide have been applied can nevertheless yield surprisingly high

rates of extraction. Therefore, the present invention provides a method for determining

biocides within wood which represents a useful and surprising advance in the art.