BS EN 46-1:2016

BSI Standards Publication

Wood preservatives —
Determination of the
preventive action against
recently hatched larvae of
Hylotrupes bajulus (Linnaeus)
Part 1: Application by surface treatment
(laboratory method)
BS EN 46-1:2016 BRITISH STANDARD

National foreword
This British Standard is the UK implementation of EN 46-1:2016. It
supersedes BS EN 46-1:2009 which is withdrawn.
The UK participation in its preparation was entrusted to Technical
Committee B/515, Wood preservation.
A list of organizations represented on this committee can be
obtained on request to its secretary.
This publication does not purport to include all the necessary
provisions of a contract. Users are responsible for its correct
application.
© The British Standards Institution 2016. Published by BSI Standards
Limited 2016
ISBN 978 0 580 90448 6
ICS 71.100.50
Compliance with a British Standard cannot confer immunity from
legal obligations.
This British Standard was published under the authority of the
Standards Policy and Strategy Committee on 30 June 2016.
Amendments issued since publication
Date Text affected
 BS EN 46-1:2016

EUROPEAN STANDARD EN 46-1

NORME EUROPÉENNE
EUROPÄISCHE NORM June 2016

ICS 71.100.50 Supersedes EN 46-1:2009

English Version

Wood preservatives - Determination of the preventive

action against recently hatched larvae of Hylotrupes
bajulus (Linnaeus) - Part 1: Application by surface
treatment (laboratory method)
Produits de préservation du bois - Détermination de Holzschutzmittel - Bestimmung der vorbeugenden
l'action préventive contre les larves récemment écloses Wirkung gegenüber frisch geschlüpften Larven von
d'Hylotrupes bajulus (Linnaeus) - Partie 1: Application Hylotrupes bajulus (Linnaeus) - Teil 1: Anwendung
par traitement de surface (Méthode de laboratoire) durch Oberflächenverfahren
(Laboratoriumsverfahren)

This European Standard was approved by CEN on 5 January 2017.

CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this
European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references
concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN
member.

This European Standard exists in three official versions (English, French, German). A version in any other language made by
translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management
Centre has the same status as the official versions.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,
Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and
United Kingdom.

EUROPEAN COMMITTEE FOR STANDARDIZATION

COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG

CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels

© 2016 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 46-1:2016 E
worldwide for CEN national Members.
BS EN 46-1:2016
EN 46-1:2016 (E)

Contents Page

European foreword....................................................................................................................................................... 4
Introduction .................................................................................................................................................................... 5
1 Scope .................................................................................................................................................................... 6
2 Normative references .................................................................................................................................... 6
3 Terms and definitions ................................................................................................................................... 6
4 Principle ............................................................................................................................................................. 7
5 Test materials ................................................................................................................................................... 7
5.1 Biological material.......................................................................................................................................... 7
5.2 Products and reagents................................................................................................................................... 7
5.3 Apparatus........................................................................................................................................................... 7
6 Sampling ............................................................................................................................................................. 8
7 Test specimens ................................................................................................................................................. 8
7.1 Species of wood ................................................................................................................................................ 8
7.2 Wood quality ..................................................................................................................................................... 9
7.3 Provision of test specimens ......................................................................................................................... 9
7.4 Dimensions of test specimens .................................................................................................................... 9
7.5 Number of test specimens ............................................................................................................................ 9
8 Procedure........................................................................................................................................................ 10
8.1 Preparation of the test specimens ......................................................................................................... 10
8.1.1 Conditioning of the test specimens prior to sealing ........................................................................ 10
8.1.2 Sealing of the transverse faces ................................................................................................................ 10
8.1.3 Treatment of the test specimens ............................................................................................................ 10
8.1.4 Drying and conditioning of the test specimens after treatment ................................................. 12
8.2 Exposure of the test specimens to the insects ................................................................................... 12
8.3 Conditions and duration of the test ....................................................................................................... 13
8.4 Examination of the test specimens ........................................................................................................ 13
8.4.1 Examination ................................................................................................................................................... 13
8.4.2 Validity of the test ........................................................................................................................................ 14
9 Expression of results................................................................................................................................... 14
9.1 Evaluation of attack ..................................................................................................................................... 14
9.2 Toxic values .................................................................................................................................................... 14
10 Test report ...................................................................................................................................................... 15
Annex A (informative) Example of a test report ............................................................................................ 17
Annex B (informative) Technique for culturing Hylotrupes bajulus (Linnaeus)................................ 19
B.1 General ............................................................................................................................................................. 19
B.2 Obtaining parent beetles ........................................................................................................................... 19
B.3 Mating ............................................................................................................................................................... 19
B.4 Egg-Iaying........................................................................................................................................................ 19

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B.5 Hatching of eggs ............................................................................................................................................ 20

B.6 Larval development ..................................................................................................................................... 20
B.7 Enemies and parasites ................................................................................................................................ 21
Annex C (informative) Differentiation of heartwood and sapwood in Pinus species ....................... 22
C.1 Principle ........................................................................................................................................................... 22
C.2 Reagents ........................................................................................................................................................... 22
C.3 Apparatus ........................................................................................................................................................ 22
C.4 Procedure ........................................................................................................................................................ 22
Annex D (informative) Environmental, health and safety precautions within
chemical/biological laboratory ............................................................................................................... 23
Annex E (normative) Slow acting and deferred effect formulations – Extending the test
duration ........................................................................................................................................................... 24
Bibliography ................................................................................................................................................................. 26

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EN 46-1:2016 (E)

European foreword

This document (EN 46-1:2016) has been prepared by Technical Committee CEN/TC 38 “Durability of
wood and wood-based products”, the secretariat of which is held by AFNOR.

This European Standard shall be given the status of a national standard, either by publication of an
identical text or by endorsement, at the latest by December 2016, and conflicting national standards
shall be withdrawn at the latest by December 2016.

Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN [and/or CENELEC] shall not be held responsible for identifying any or all such patent
rights.

This document supersedes EN 46-1:2009.

Significant technical differences between this document and EN 46-1:2009 are as follows:

a) introduction of new harmonized specifications for wood quality;

b) option to omit control test specimens treated with the solvent or diluents only when the solvent or
diluents is water of drinking quality;

c) determination of validity of the test by using an X-ray apparatus added in 8.4.2.

The standard EN 46 is composed of two parts:

— EN 46-1, Wood preservatives ― Determination of the preventive action against recently hatched
larvae of Hylotrupes bajulus (Linnaeus) ― Part 1: Application by surface treatment (laboratory
method)

— EN 46-2, Wood preservatives ― Determination of the preventive action against recently hatched
larvae of Hylotrupes bajulus (Linnaeus) ― Part 2: Ovicidal effect (laboratory method)

EN 46 consists of two parts to enable preventive action of wood preservatives, against recently hatched
larvae of Hylotrupes bajulus, which are intended to be applied by surface treatment; Part 1 is required to
determine the larvicidial effect of preservatives and Part 2 is required to determine the ovicidal action
of the preservatives after egg-laying of young females.

According to the CEN/CENELEC Internal Regulations, the national standards organizations of the
following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria,
Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia,
France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta,
Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland,
Turkey and the United Kingdom.

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Introduction

This European Standard describes a laboratory method of testing which gives a basis for the
assessment of the preventive action of a wood preservative, when applied as a surface treatment,
against recently hatched larvae of Hylotrupes bajulus, whereas the method for determining the toxic
values against Hylotrupes bajulus (EN 47) provides a means of checking whether a preservative
prevents attack by these larvae and prevents their survival within totally impregnated wood.
This method makes it possible to determine whether recently hatched larvae are capable of boring
through the treated surface of a susceptible wood species and of surviving in the untreated part of the
test specimen. For this purpose, the procedure seeks to reproduce normal egg-laying conditions
existing in cracks in wood, which provide the principal egg-laying sites. It takes account of the fact that,
if larvae pass through the treated surface, they will then tunnel in the direction of the least protected
regions of the wood.
This laboratory method provides one criterion by which the value of a preservative can be assessed. In
making this assessment, the methods by which the preservative may be applied should be taken into
account. This test is of particular interest when applied to test specimens which have been subjected to
an ageing procedure. It is further recommended that results from this test should be supplemented by
those from other appropriate tests and, above all, by practical experience.
When products which are very active at low concentrations are used it is very important to take
suitable precautions to isolate and separate, as far as possible, operations involving chemical products,
other products, treated wood, laboratory apparatus and clothing. Suitable precautions should include
the use of separate rooms, areas within rooms, extraction facilities, conditioning chambers and special
training for personnel (see also Annex D for environmental, health and safety precautions).

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1 Scope
This European Standard specifies a method for the determination of the preventive action of a wood
preservative against recently hatched larvae of Hylotrupes bajulus (Linnaeus) when the preservative is
applied as a surface treatment to wood.
This method is applicable to:
— water-insoluble chemicals which are being studied as active insecticides;

— organic formulations, as supplied or as prepared in the laboratory by dilution of concentrates;

— organic water-dispersible formulations as supplied or as prepared in the laboratory by dilution of

concentrates; and

— water-soluble materials, for example salts.

The method is applicable whether or not the test specimens have been subjected to appropriate ageing
procedures.

2 Normative references
The following documents, in whole or in part, are normatively referenced in this document and are
indispensable for its application. For dated references, only the edition cited applies. For undated
references, the latest edition of the referenced document (including any amendments) applies.
EN 73, Wood preservatives — Accelerated ageing of treated wood prior to biological testing —
Evaporative ageing procedure

EN 84, Wood preservatives — Accelerated ageing of treated wood prior to biological testing — Leaching
procedure

EN ISO 3696, Water for analytical laboratory use — Specification and test methods (ISO 3696)

3 Terms and definitions

For the purposes of this document, the following terms and definitions apply.
3.1
representative sample
sample having its physical and/or chemical characteristics identical to the volumetric average
characteristics of the total volume being sampled

[SOURCE: EN 1001-2:2005, 4.71]

3.2
supplier
sponsor of the test (person or company providing the sample of wood preservative to be tested)

Note 1 to entry: Adapted from EN 1001–2:2005, 4.83.

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4 Principle
Depending on the test being carried out either:
— on a set of test specimens of a susceptible wood species that is surface treated with a solution of the
preservative; or

— if toxic values are to be determined, on several sets of test specimens of a susceptible wood species
that are surface treated with a series of solutions in which the concentration of preservative is
ranged in a given progression.

The treated test specimens are exposed to recently hatched larvae of Hylotrupes bajulus. The resulting
attack is observed and compared with those in untreated control test specimens. If the preservative has
been prepared in the laboratory by dilution of a concentrate or by dissolution of a solid, the resulting
attack is also compared to that in solvent or diluent treated control test specimens.

5 Test materials

5.1 Biological material

5.1.1 Hylotrupes bajulus (Linnaeus) larvae, within three days of hatching.

5.1.2 Source of larvae. Obtain the larvae from cultures reared, e.g. as described in Annex B.

5.1.3 Provision of larvae. Collect larvae from eggs laid by different females.

5.1.4 Choice of larvae. Use a mixed batch of these larvae for the test. Use ten larvae per treated test
specimen or control test specimen.

5.2 Products and reagents

5.2.1 Paraffin wax, for fixing the glass plate and for sealing the end faces of test specimens to be
treated with solutions in all cases in which water is the continuous phase.

NOTE Paraffin wax with a setting point of 52 °C to 54 °C has been found to be suitable.

5.2.2 Gelatin, for sealing the end faces of test specimens to be treated with solutions in which an
organic solvent is the continuous phase.

5.2.3 Water, complying with grade 3 of EN ISO 3696.

5.2.4 Solvent or diluent, a volatile liquid that will dissolve or dilute the preservative but does not
leave a residue in the wood at the end of the post-treatment conditioning period that has a toxic effect
on the insects.

CAUTION — Do not use benzene or other solvents which pose a health risk.
5.3 Apparatus

5.3.1 Culturing chamber, with air circulation, and controlled at (28 ± 2) °C and at a relative humidity
of (70 ± 5) %.

5.3.2 Conditioning chamber, well ventilated and controlled at (20 ± 2) °C and at a relative humidity
of (65 ± 5) %.

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The conditioning of test specimens may be carried out in the laboratory work area (see 5.3.3) provided
that this has the conditions specified for the conditioning chamber (see 5.3.2).
5.3.3 Laboratory work area, well ventilated, where treatment of the test specimens is carried out.

CAUTION — It is essential to follow safety procedures for handling flammable and toxic
materials. Avoid excessive exposure of operators to solvents or their vapours.
5.3.4 Testing chamber, ventilated and air conditioned, controlled at (22 ± 2) °C and at a relative
humidity of (70 ± 5) %.

5.3.5 Treatment vessels of a material that does not react with the preservative under test, for
example of glass for organic products and of polyethylene for salts containing fluorine.

5.3.6 Weights, to provide ballast for the test specimens.

The weights shall not react with any materials with which they come into contact during the test.
5.3.7 Safety equipment and protective clothing, appropriate for the test product and the test
solvent, to ensure the safety of the operator.

5.3.8 Glass plates, (48 ± 1) mm long and (25 ± 1) mm wide, intended to provide a lateral slit on the
test specimens.

5.3.9 Ordinary laboratory equipment, including a balance capable of weighing to an accuracy of

0,01 g.

5.3.10 Protective gloves

5.3.11 X-ray apparatus, (optional) with tungsten target and beryllium window, with voltage and
current continuously variable in the ranges:

— voltage: 10 kV to 50 kV;

— current: 0 mA to 15 mA.

6 Sampling
The sample of preservative shall be representative of the product to be tested. Samples shall be stored
and handled in accordance with any written recommendations from the supplier.
For the sampling of preservatives from bulk supplies, the procedure given in EN 212 should be used.

7 Test specimens

7.1 Species of wood

The reference species is Scots pine (Pinus sylvestris Linnaeus) 1).

Additional tests may be carried out using other species but, if so, this should be stated in the test report.

1) In southern European countries the pine species most frequently infested by Hylotrupes bajulus may be used as an
alternative, provided that the suitability of the species for use in the tests specified in this document has been demonstrated in
all aspects (development of larvae, resistance to impregnation, etc.).

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7.2 Wood quality

The wood shall be free from visible cracks, stain, decay, insect damage and other defects. The wood
shall not have been water-stored, floated, chemically treated or steamed. The wood shall originate from
trees preferably felled in winter. The trees shall be cut immediately after felling and the timber rapidly
air-dried or kiln dried at temperatures below 60 °C. The wood shall not have been stored for more than
five years.
The wood shall be exclusively sapwood containing little resin and having between 2,5 annual rings per
10 mm and eight annual rings per 10 mm. The proportion of latewood in the annual rings shall not
exceed 30 % of the whole.
It is recommended to use test specimens of similar growth rate within a single test.
7.3 Provision of test specimens 2)

Prepare planed strips having a cross-section of (25 ± 0,5) mm × (15 ± 0,5) mm removing a minimum of
2 mm from any faces exposed during drying. The longitudinal faces shall be parallel to the direction of
the grain. The annual rings shall have a contact angle of 45° ± 15° to the broad faces. Make transverse
cuts, neatly to give sharp edges and a fine-sawn finish to the end-grain surfaces, to give test specimens
(50 ± 0,5) mm long.
The test specimens shall originate from a minimum of three trees or shall be taken at random from a
stock originally of more than 500 test specimens.
7.4 Dimensions of test specimens

The dimensions of each test specimen after reaching equilibrium in the conditioning chamber (5.3.2)
shall be (50 ± 0,5) mm × (25 ± 0,5) mm × (15 ± 0,5) mm.
Mark each test specimen so that it can be identified throughout the test.
7.5 Number of test specimens

a) Six treated test specimens (no more than two originating from the same tree unless taken at
random from a stock of more than 500) for each preservative, each concentration and each
duration of treatment;

b) Three untreated control test specimens (each originating from a different tree unless taken at
random from a stock of more than 500) for a complete test of any given preservative;

c) Three control test specimens treated with the solvent or diluent (5.2.3 or 5.2.4) (each originating
from a different tree unless taken at random from a stock of more than 500) if a solvent or diluent
(including water) is used.

Control test specimens under c) may be omitted if the solvent or diluents is water of drinking quality.
When dipping is to be used (8.1.3.3) it is advisable to treat more than the specified number of test
specimens so that, after weighing, any test specimens with abnormally high or low retentions can be
rejected from the batch.
NOTE To gain further information on a formulation the manufacturer may find it useful to test a version of
the preservative where the active ingredient(s) has been removed.

2) For special tests, test specimens may be obtained according to a given series. As a result, it may be preferable to take test
specimens from pretreated strips. Where pretreated strips are used details should be included in the test report.

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8 Procedure

8.1 Preparation of the test specimens

8.1.1 Conditioning of the test specimens prior to sealing

Allow the test specimens to condition in the conditioning chamber (5.3.2) for a minimum of two weeks.
8.1.2 Sealing of the transverse faces

8.1.2.1 General

When treatment is to be by brushing or by pipette then only the transverse faces of test specimens shall
be sealed. When treatment is to be by dipping then all faces, except one 25 mm x 50 mm face, shall be
sealed. The material used for sealing shall be resistant to the penetration of wood preservatives under
test. The sealings specified in 8.1.2.2 and 8.1.2.3 have been proven as suitable.
8.1.2.2 For tests with solutions in which water is the continuous phase, apply three coats of the
paraffin wax (5.2.1) at about 90°C so that the first coat adheres closely to the wood and the successive
coatings bond to one another. Condition the sealed test specimens in the conditioning chamber (5.3.2)
for at least one day.

8.1.2.3 For tests with preservative solutions in which the continuous phase is an organic
solvent that dissolves paraffin wax, use the gelatine (5.2.2): apply the first coat as an aqueous solution
of 200 g/l at 40°C, then after a minimum of 8 h of drying, apply two further coats of an aqueous solution
of 300 g/l at 50°C. Condition the sealed test specimens in the conditioning chamber (5.3.2) for at least
one day.

8.1.3 Treatment of the test specimens

8.1.3.1 Preparation of the treatment solutions

8.1.3.1.1 Solid preservatives

— Water-soluble preservatives:

Dissolve the preservative in the water (5.2.3) to the required concentration, or in a series of
concentrations if toxic values are to be determined.
— Non-water-soluble preservatives:

Dissolve the preservative in an appropriate solvent (5.2.4) to the required concentration, or in a series
of concentrations if toxic values are to be determined.
All treatment solutions shall be freshly prepared.
8.1.3.1.2 Liquid preservatives

If appropriate, use the preservative without further preparation other than any necessary stirring. If it
is a concentrate or if toxic values are to be determined, dilute the preservative with the diluent to the
required working concentration, using the procedure specified by the manufacturer.
All treatment solutions shall be freshly prepared.

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8.1.3.1.3 Toxic values

If toxic values are to be determined, prepare a series of at least five concentrations by mass, distributed
evenly about the expected toxic values.
A solvent or diluent control, i.e. treatment at concentration = 0, shall also be used. If the approximate
toxic values are unknown, the concentrations shall form a widely spaced geometric progression for a
first test and a more closely spaced geometric or arithmetic progression for subsequent tests.
All treatment solutions shall be freshly prepared.
8.1.3.2 Treatment by brushing or by pipette

In the laboratory work area (5.3.3) apply the preservative either by brushing or by pipette to the
50 mm x 25 mm lateral face that would have been furthest from the centre of the tree.
Depending on the type of treatment, the volume (application by pipette) or mass (application by
brushing) of the treatment solution shall be determined to obtain the surface application specified by
the manufacturer. Place the specimens such that the 50 mm x 25 mm lateral face that is to be treated is
uppermost and apply the appropriate fluid uniformly to that face.
When the preservative is applied by brush, place the test specimens on a balance while being brushed
to determine the amount of preservative applied to the nearest 0,01 g.
When the preservative is applied by pipette, move the pipette across the fibre direction and the amount
of preservative applied shall be determined to the nearest 0,01 ml.
Several applications can be necessary to apply the required amount. In this case the coats should be
applied sufficiently quickly to avoid any solidification of certain substances which can impede the
penetration of further coats.
Care should be taken to avoid fluid running off the lateral face being treated.
8.1.3.3 Treatment by dipping

When the treatment of specimens is by dipping all faces, except one 25 mm x 50 mm face, shall be
sealed (see 8.1.2).
Weigh to the nearest 0,01 g each sealed test specimen, to obtain its initial mass.
Treat each test specimen in the treatment vessel (5.3.5) as follows:
Immerse completely in the solution. The dipping times to be used shall be one of the following, agreed
beforehand according to the purpose of the test:
— Either one 10 s period and/or two periods of 10 s at an interval of 24 h.

If the rate of solidification of some constituents of a preservative formulation would have the effect of
retarding its penetration during the second dipping, this interval has to be reduced. The interval
employed shall be mentioned in the test report.
— Or a period sufficient for a determined quantity to be retained by the test specimen 3).

Using forceps, remove each test specimen from the preservative fluid and sponge off fluid from all the
sealed faces of the specimen. Keeping the face that has not been sealed upper most, immediately weigh
to the nearest 0,01 g.

3) The dipping time depends upon the type of preservative and may extend to several hours for water-soluble preservatives.
The progress of absorption is monitored by successive weighings of the treated test specimens. For this long period dipping
the treated test specimens are immersed together and kept submerged by the weights (5.3.6).

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In the case of water-soluble chemicals, for example salts, and water-insoluble chemicals which are being
studied as active insecticides, calculate the mass of chemical retained for each test specimen from the
mass of solution absorbed and its concentration.
In the case of organic formulations or organic water-dispersible formulations, the retention is
expressed for each test specimen in terms of the corresponding mass of the formulation ready for use,
but if a concentrate is supplied the retention is expressed in terms of the solution prepared ready for
use as specified by the manufacturer.
Calculate the mass of preservative retained per unit area of unsealed wood surface.
8.1.4 Drying and conditioning of the test specimens after treatment

If the end-sealing has been damaged before or after treatment, reject the test specimens concerned
from the tests.
After treatment, condition the test specimens for four weeks in the environment specified for the
conditioning chamber (5.3.2). Arrange the test specimens on their lateral narrow faces, resting on glass
rods, not touching one another. Invert the test specimens twice a week.
NOTE The drying and conditioning of the test specimens depend on the nature of the product under test and
on the solvent or diluent used. For slow drying products it may be necessary to extend the conditioning process.

If, in the case of slow drying products, the conditioning period is extended, the extended conditioning
period shall be stated in the test report.
If the test specimens are to be subject to an ageing procedure (according to EN 73 or EN 84), this shall
be carried out after this drying procedure.
8.2 Exposure of the test specimens to the insects

After the conditioning period has ended place one of the glass plates (5.3.8) against the treated face of
each test specimen.
At one of the 50 mm edges, insert a spacer 0,35 mm thick between the glass plate and the test specimen
so as to leave a gap 0,35 mm wide (see Figure 1).
Fix the plate by dipping the transverse faces and the narrow longitudinal face opposite the wedge in the
paraffin wax (5.2.1) kept close to melting point so as to seal the openings at the edge of these faces.
After cooling remove the wedge. Next place the ten larvae in the gap thus provided in the middle part of
the test specimen.

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Dimensions in millimetres

Key
1 gap
2 small glass wedge plate or rod
3 glass plate (5.3.8)
4 sealed opening
5 transverse face sealed with paraffin wax or gelatine

Figure 1 — Test specimen fitted with its glass plate

8.3 Conditions and duration of the test

Place all the test specimens in the testing chamber (5.3.4), keeping the treated test specimens separate
from the untreated control test specimens and the solvent or diluent-treated control test specimens,
and also, if toxic values are being sought, keeping the different concentrations separate.
The total duration of the test, during which examinations and observations are carried out as described
in 8.4.1 is 12 weeks.
NOTE When slow acting or deferred effect insecticides, or products containing them, are to be evaluated the
total duration of the test according to EN 46–1 can be extended to 24 weeks (see Annex E).

8.4 Examination of the test specimens

8.4.1 Examination

Four weeks after the start of the test, carefully remove the glass plate and ascertain the tunnelling and
mortality rate of the larvae. Those larvae which have tunnelled leave a small quantity of wood dust at
the tunnel entrance. Any dead newly hatched larvae are completely dried up at the end of four weeks
and are dark in colour.
At least 70 % of the larvae in contact with the control test specimens shall have tunnelled, otherwise,
stop the test and begin again.
If, after four weeks:

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a) all the larvae are already dead on the surface of the treated test specimens and if the control test
specimens have been sufficiently attacked (see above), consider the test completed and determine
the number of live larvae in the control test specimens by cutting up the wood;

b) larvae have tunnelled in the treated test specimens and the control test specimens have also been
sufficiently attacked (see above), continue the test for a further eight weeks and then carry out a
final examination of the test specimens by cutting them up at the end of the total of 12 weeks.

For tests with several concentrations:

c) the test at any concentration is considered complete when, at the end of four weeks, all the larvae
are dead on the surface of the set of treated test specimens for that concentration;

d) continue the test for a further eight weeks on those concentrations for which the treated test
specimens contain larvae that have tunnelled as well as on the control test specimens. Carry out a
final examination by cutting up the wood at the end of the total of 12 weeks.

Final examination of the presence and size of larvae in the test specimens may be carried out using the
X-ray apparatus (5.3.11), if available. It shall be ensured that differentiation between live and dead
larvae (9.1) is possible when using the X-ray apparatus.
8.4.2 Validity of the test

The test shall be considered valid if at least 70 % of the larvae exposed to all of the untreated control
test specimens survive and, if applicable, at least 70 % of those exposed to all of the control test
specimens treated with the solvent or the diluent alone, survive. Otherwise, repeat the test.
Determination of validity may be carried out using the X-ray apparatus (5.3.11), if available. It shall be
ensured that differentiation between live and dead larvae is possible when using the X-ray apparatus.

9 Expression of results

9.1 Evaluation of attack

The extent of attack shall be evaluated in terms of:

— number of dead larvae not having tunnelled;

— number of dead larvae having tunnelled ;

— number of live larvae and the state of these larvae;

— number of larvae not retrieved.

9.2 Toxic values

If a range of concentrations of product are tested, the results shall be expressed as toxic values.
The toxic values of a preservative product are expressed as the following two loadings:
— mean mass of preservative retained per unit area in the set of test specimens treated with the
lowest concentration of the product in the series in which all larvae are dead in all of the test
specimens at the end of the test;

14
 BS EN 46-1:2016
EN 46-1:2016 (E)

— mean mass of preservative per unit area in the set of test specimens treated with the next lowest
concentration of the product in the series in which live larvae are found in any of the test specimens
at the end of the test.

Express the toxic values as g of preservative per m2 of treated wood surface (see 8.1.3.2 and 8.1.3.3),
and also state the corresponding concentrations of the preservative in the solvent or the diluent.

10 Test report
The test report shall include at least the following information (see also Annex A for an example):
a) number and date of this European Standard;

b) name of the supplier of the preservative under test;

c) specific and unique name or code of the preservative tested, with an indication of whether or not
the composition has been declared;

d) name and concentration of active ingredient;

e) if relevant, the solvent or diluent used;

f) species of wood used;

g) method of application and, if applicable, the concentration of preservatives tested, expressed as

mass fraction;

h) date of the application of the preservative;

i) for each test specimen treated

1) if dipping

i) the mass, in g, of solution absorbed;

ii) the corresponding mass of test product per unit surface area, in g/m2;

2) if treating by pipette

i) the volume of solution applied to the treated surface of the test specimen (or, where
necessary), the volume of solvent or diluent in ml;

ii) the corresponding amount of the product under test in g/m2 or ml/m2;

3) if brushing

i) the mass in g of solution applied to the treated surface of the specimen (or, where
necessary, the volume of solvent or diluents in ml);

ii) the corresponding amount of the product under test in g/m2 or ml/m2;

j) method of drying the test specimens;

15
BS EN 46-1:2016
EN 46-1:2016 (E)

k) any ageing procedures carried out, specifying the type, conditions and duration, with possible
reference to a standard;

l) date of insertion of the larvae into the test specimens;

m) date(s) of examination of the test specimens;

n) results obtained at each examination and each method of treatment for both treated test specimens
and control test specimens:

1) number of dead larvae not having tunnelled;

2) number of dead larvae having tunnelled;

3) number of live larvae and their condition;

4) number of larvae not retrieved;

o) if determined, the toxic values in g of preservative per m2 of treated wood surface, together with
the concentrations, as mass fraction, of treating solution to which these values correspond;

p) name of the organization responsible for the test report and the date of completion of the testing;

q) name and signature of the officer(s) in charge of testing;

r) following note:

“The interpretation and the practical conclusions that can be drawn from this test report demand a
specialized knowledge of the subject of wood preservation and, for this reason, this test report
cannot of itself constitute an approval certificate.”

s) any variation from the described test method and any factors that may have influenced the results.

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EN 46-1:2016 (E)

Annex A
(informative)

Example of a test report

Number and date of this document EN 46–1:2016

Name of supplier Company M
Name and type of product X product in organic solvent, ready for use,
formulation declared
Name and concentration of active ingredient Y mass fraction 1 %
Solvent or diluent employed Nil
Wood species Scots pine (Pinus sylvestris Linnaeus)
Concentration of preservative tested Product used undiluted
Date of dipping 2015–05–05
Dipping conditions Single dipping of 10 s
Amount of solution absorbed and preservative retained See Table A.1.
Method of drying As specified in EN 46–1
Ageing procedure previously carried out Evaporation test (with details)
Date of exposure 2015–09–01
Date of examination 2015–09–29
Results See Table A.1.
This report has been prepared by Laboratory L
Location and date X 2015–10–04
Name and signature of the officer(s) in charge Mrs Y

NOTE The interpretation and the practical conclusions that can be drawn from this test report demand a
specialized knowledge of the subject of wood preservation and, for this reason, this test report cannot of itself
constitute an approval certificate.

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EN 46-1:2016 (E)

Table A.1 — Results

Absorption Larvae recovered
Concentrations Mass or Mean Dead Live after
tested volume of retention of tunnelling
Type of test Not having Having Larvae not
specimens Mass fraction solution the recovered
absorbed preservative tunnelled tunnelled
% g/m2 or
g
ml/m2

Treated
1 100 0,30 75 10 0 0 0
2 0,30 75 10 0 0 0
3 0,35 87,5 10 0 0 0
4 0,30 75 10 0 0 0
5 0,28 70 10 0 0 0
6 0,32 80 9 0 0 1
Untreated
control
1 _ _ _ 0 0 10 0
2 _ _ _ 0 0 10 0
3 _ _ _ 0 0 10 0
Solvent _ _ _ _ _ _ _
treated
control

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 BS EN 46-1:2016
EN 46-1:2016 (E)

Annex B
(informative)

Technique for culturing Hylotrupes bajulus (Linnaeus)

B.1 General
Before undertaking culturing of Hylotrupes bajulus, a basic knowledge of the biology of this insect
should be acquired from literature and from official organizations conducting research into wood
preservation.

B.2 Obtaining parent beetles

A culture can be begun by taking larvae from naturally-infested wood and inserting them head first into
suitably sized drilled holes in pine sapwood blocks and allowing them to pupate in order to obtain
reproductive adults.
It is necessary to ensure that the insects have not been in contact with toxic products or with treated
wood at any stage in their development.
Adult beetles of the brown variety should be removed.

B.3 Mating
Place together a male and female adult on a face, cover them with a Petri dish lid and leave them in
daylight, as Hylotrupes bajulus is a diurnal insect.
Quite soon, the male will approach the female and mating will take place with the male uppermost.
Then separate the two insects as they will bite and damage one another if left confined together.
A male can fertilize two or three females a day.

B.4 Egg-Iaying
B.4.1 Isolate the females after fertilization and place them for egg-laying, using either of the
techniques given in B.4.2 and B.4.3.

B.4.2 Place the females in glass bottles containing small blocks of pine sapwood, resting on a filter
paper disc. The egg-Iaying will take place between the block and the filter paper on which they can be
seen easily.

B.4.3 Place the females on the surface of pine sapwood blocks introduced in an appropriate vessel and
which have been previously split into several pieces and then reassembled with adhesive tape placed at
one end, as shown in Figure B.1, so that open cracks remain which diminish in width from the free end
to the end bound with the tape. By removal of the tape, the different pieces of the block can be
separated easily and any eggs laid in the cracks can be seen. Place the females singly on each composite
block and cover with a glass lid.

It should be noted that mating repeated every day or every two days stimulates egg-Iaying.

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BS EN 46-1:2016
EN 46-1:2016 (E)

Once a day check if eggs have been laid. If so, remove the filter paper or the block which carries them
and give the female fresh blocks for further egg-laying, allowing continued mating in order to activate
egg-Iaying.
A favourable temperature for egg-Iaying is about 25 °C.

Key
1 adhesive tape

Figure B.1 — Block with adhesive tape

B.5 Hatching of eggs

Place the surfaces (discs of filter paper or split blocks) on which eggs have been laid in such a manner
that, on hatching, the young larvae fall into a glass vessel from which they cannot escape.
The optimum conditions for hatching are as follows:
— temperature: about 28 °C;

— relative humidity: about 80 %.

B.6 Larval development

Place the newly hatched larvae in culturing blocks, as described below. They should not have been
deprived of food for more than three days before being inserted in the block. Handle the larvae carefully
using a soft brush or vacuum tweezers.
As the larvae of Hylotrupes bajulus may eat one another, they should be kept apart during culturing by
having one larva per block. Insert each larva into a hole, pierced with a bradawl at right angles to the
grain of the wood, to a depth of 4 mm to 6 mm.
Culturing blocks shall be of pine sapwood. Prior to insertion of the larvae, impregnate the blocks under
vacuum with an aqueous solution of 10 g/l peptone and 5 g/l yeast (yeast can be replaced with 0,01 g/l
lactoflavine), then dry them. In this way, the duration of larval development can be shortened to about
one tenth of its natural length. The size of the blocks is not critical but they may conveniently be of
dimensions
50 mm × 25 mm × 15 mm, the length being parallel to the grain of the wood.
The growth of Hylotrupes bajulus is most rapid between 29 °C and 30 °C with an optimum relative
humidity of 97 % to 98 %, but very high humidities favour the development of moulds. It is therefore
preferable to use a relative humidity of (70 ± 5) %.

20
 BS EN 46-1:2016
EN 46-1:2016 (E)

When conditions of microclimate and nutrition are perfect, the male adult insects can appear at about
six months. However, as soon as the larvae reach a size such that the volume of the blocks is insufficient
to permit normal growth, it is preferable to transfer them into blocks of larger dimensions that are not
impregnated with peptone and yeast, in which larval growth can be continued until pupation.
The larvae of Hylotrupes bajulus exhibit a long diapause but pupate more rapidly when they are exposed
to low temperature. It is therefore advantageous to place the large blocks outdoors in winter at a
temperature of 5 °C to 10 °C. In this way, a mass emergence of insects can be obtained, with a high
proportion emerging within a short space of time. This is particularly suitable for establishing new
cultures.

B.7 Enemies and parasites

Take care to avoid infestation by hymenopterous parasites and coleopterous predators by closing the
rearing vessels with fine mesh grilles.
The insects likely to cause the greatest damage to Hylotrupes bajulus cultures are:
— Rhoptrocentrus piceus Marshall (Braconidae);

— Scleroderma domesticum Latreille (Bethylidae).

Experience has shown that special precautions against mites are unnecessary.
Insects collected from the field should undergo a severe quarantine before being introduced into the
culturing chamber.

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EN 46-1:2016 (E)

Annex C
(informative)

Differentiation of heartwood and sapwood in Pinus species

C.1 Principle
The phenolic compounds in the heartwood of Pinus species are detected by the formation of a coloured
complex with o-dianisidine.

C.2 Reagents
o-dianisidine diazonium chloride solution, 20 g/l.
Dissolve 2 g of o-dianisidine diazonium chloride in 100 ml of water. Prepare freshly as required.
NOTE o-dianisidine diazonium chloride stabilized with zinc chloride may be purchased as ‘Fast blue B salt’.

C.3 Apparatus
C.3.1 Spray equipment, capable of producing a fine even spray.

Both gas-powered aerosol units and small hand-powered units have been found to be suitable.
C.3.2 Soft paint brush, 5 mm to 50 mm wide, depending on the size of the timber section to be tested.

C.4 Procedure
Prepare the wood sample to give a clean surface and remove any loose sawdust. Brush or spray the
solution (C.2) onto the timber section.
CAUTION — It is advisable to wear protective goggles and to work under a fume hood when
carrying out spray tests.
The heartwood of most Pinus species is coloured magenta and the sapwood is coloured yellow.

22
 BS EN 46-1:2016
EN 46-1:2016 (E)

Annex D
(informative)

Environmental, health and safety precautions within chemical/biological

laboratory

When preparing this document, consideration was given to the minimization of environmental impacts
caused by the use of the methods of testing.
It is the users’ responsibility to use safe and proper techniques in handling materials in the methods of
testing specified in this document.
The following list is not exhaustive but users of this document may use it as a guide to the use of safe
and proper techniques. They should:
— investigate if European Directives, transposed European legislation and national laws, regulations
and administrative provisions apply;

— consult manufacturers/suppliers for specific details such as material safety data sheets and other
recommendations;

— use safety equipment and wear protective clothing, usually goggles and coats, appropriate for the
test product and the test chemicals, in all laboratory areas, to ensure the safety of the operator;

— be careful about flammable materials and substances that are toxic and/or human carcinogens and
generally take care during transportation, decanting, diluting and dealing with spillages;

— use a fume cupboard during preparation of organic solvent solutions;

— store, handle and dispose of chemicals in a safe and environmentally satisfactory manner: including
chemicals for laboratory test, test specimens, unused solvents and reagents that have to be
disposed of.

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BS EN 46-1:2016
EN 46-1:2016 (E)

Annex E
(normative)

Slow acting and deferred effect formulations – Extending the test duration

When slow acting or deferred effect insecticides, or products containing them, are to be evaluated the
total duration of the test can be prolonged. The duration of the test, as given in 8.3 and 8.4.1, can be
extended to 24 weeks but the modifications contained in this annex shall be adhered to. Fewer larvae
are exposed per test specimen to ensure that there is sufficient timber to support the larvae over the 24
week period. To allow the same number of larvae to be exposed during the test the number of test
specimens is doubled.
When slow acting or deferred effect formulations are tested the following alterations to the EN 46-1
method shall be made:
7.5 Number of test specimens
When the exposure period is to be 24 weeks use:
a) 12 treated test specimens (no more than four originating from each tree unless taken at random
from a stock of more than 500) for each preservative, each concentration and each duration of
treatment;

b) six untreated control test specimens (no more than two originating from each tree unless taken at
random from a stock of more than 500) for a complete test of any given preservative;

c) six control test specimens treated with the solvent or the diluent (5.2.3 or 5.2.4) (no more than two
originating from each tree unless taken at random from a stock of more than 500) if a solvent or
diluent (including water) is used.

When dipping is to be used (8.1.3.3) it is advisable to treat more than the specified number of test
specimens so that, after weighing, any test specimens with abnormally high or low retentions can be
rejected from the batch.
8.2 Exposure of the test specimens to the insects
When the exposure period is to be 24 weeks:
Place one of the glass plates (5.3.8) against the treated wide face of each test specimen. A glass plate is
also placed against one of the wide faces of control specimens.
At one of the 50 mm edges, insert a spacer 0,35 mm thick between the glass plate and the test specimen
so as to leave a gap 0,35 mm wide (see Figure 1).
Fix the plate by dipping the transverse faces and the narrow longitudinal face opposite the wedge in the
paraffin wax (5.2.1) kept close to melting point so as to seal the openings at the edge of these faces.
After cooling remove the wedge. Next place five larvae in the gap thus provided in the middle part of the
test specimen
8.3 Conditions and duration of the test
When slow acting or deferred effect insecticides, or products containing them, are to be evaluated and
the exposure period is to be 24 weeks:
Place all the treated test specimens in the testing chamber (5.3.4), keeping the treated test specimens
separate from the untreated control test specimens and the solvent or diluent-treated control test
specimens, and also, if toxic values are being sought, keeping the different concentrations separate.

24
 BS EN 46-1:2016
EN 46-1:2016 (E)

The total duration of the test, during which examinations and observations are carried out as described
in 8.4.1, is 24 weeks.
8.4.1 Examination
Four weeks after the start of the test, carefully remove the glass plate and ascertain the tunnelling and
mortality of the larvae. Those larvae which have tunnelled leave a small quantity of wood dust at the
tunnel entrance. Any dead, newly hatched larvae are completely dried up at the end of the four weeks
and are dark in colour.
At least 70 % of the larvae in contact with the control test specimens shall have tunnelled, otherwise
stop the test and begin again.
If, after four weeks:
a) all the larvae are already dead on the surface of the treated test specimens and if the control test
specimens have been sufficiently attacked (see above), consider the test completed and determine
the number of live larvae in the control test specimens by cutting up the wood.

b) larvae have tunnelled in the treated test specimens and the control test specimens have also been
sufficiently attacked (see above), continue the test for a further 20 weeks and then carry out the
final examination of the test specimens by cutting them up at the end of the total of 24 weeks.

For tests with several concentrations:

c) the test at any concentration is considered complete when, at the end of four weeks, all the larvae
are dead on the surface of the set of treated specimens for the concentration;

d) continue the test for a further 20 weeks on those concentrations for which the treated test
specimens contain larvae that have tunnelled as well as the control test specimens. Carry out a final
examination by cutting up the wood at the end of the total of 24 weeks.

10 Test report
The information contained in the test report shall include the following statement when an exposure
period of 24 weeks is used:
“This EN 46-1 testing has been conducted such that the normal exposure period has been extended to
24 weeks. This exposure period is appropriate for testing formulations, or insecticides, which are slow
acting or have a deferred effect.”

25
BS EN 46-1:2016
EN 46-1:2016 (E)

Bibliography

[1] EN 47, Wood preservatives — Determination of the toxic values against larvae of Hylotrupes
bajulus (Linnaeus) — (Laboratory method)

[2] EN 212, Wood preservatives — General guidance on sampling and preparation for analysis of
wood preservatives and treated timber

[3] EN 1001-1, Durability of wood and wood-based products — Terminology — Part 1: List of
equivalent terms

[4] EN 1001-2:2005, Durability of wood and wood based products — Terminology — Part 2:
Vocabulary

[5] EN ISO 835, Laboratory glassware — Graduated pipettes (ISO 835)

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