30170234 pdf BRITISH STANDARD BS EN 1390 2006 Incorporating corrigendum no 1 Wood preservatives — Determination of the eradicant action against Hylotrupes bajulus (Linnaeus) larvae — Laboratory method[.]
BRITISH STANDARD BS EN 1390:2006 Incorporating corrigendum no Wood preservatives — Determination of the eradicant action against Hylotrupes bajulus (Linnaeus) larvae — Laboratory method The European Standard EN 1390:2006 has the status of a British Standard Confirmed April 2012 ICS 71.100.50 BS EN 1390:2006 National foreword This British Standard is the UK implementation of EN 1390:2006 It supersedes DD ENV 1390:1995 and BS 5219:1975 which are withdrawn The UK participation in its preparation was entrusted by Technical Committee B/515, Wood preservatives, to Subcommittee B/515/1, Preservative preconditioning and biological testing A list of organizations represented on this subcommittee 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 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 31 July 2006 Amendments issued since publication Amd No Date Comments 17378 31 October 2007 Addition to supersession details Corrigendum No © BSI 2007 ISBN 978 580 60059 EN 1390 EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM June 2006 ICS 71.100.50 Supersedes ENV 1390:1994 English Version Wood preservatives - Determination of the eradicant action against Hylotrupes bajulus (Linnaeus) larvae - Laboratory method Produits de préservation du bois - Détermination de l'action curative contre les larves d'Hylotrupes bajulus (Linnaeus) Méthode de laboratoire Holzschutzmittel - Bestimmung der bekämpfeden Wirkung gegenüber Larven von Hylotrupes bajulus (Linnaeus) Laboratoriumsverfharen This European Standard was approved by CEN on 24 May 2006 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 Central Secretariat 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 Central Secretariat has the same status as the official versions CEN members are the national standards bodies of Austria, Belgium, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom EUROPEAN COMMITTEE FOR STANDARDIZATION COMITÉ EUROPÉEN DE NORMALISATION EUROPÄISCHES KOMITEE FÜR NORMUNG Management Centre: rue de Stassart, 36 © 2006 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members B-1050 Brussels Ref No EN 1390:2006: E EN 1390:2006 (E) Contents Page Foreword Introduction Scope Normative references Terms and definitions Principle 5 Test materials .6 Sampling .7 Test specimens Procedure .9 Validity of the test 12 10 Expression of results 12 11 Test report 13 Annex A (informative) Example of a test report 14 Annex B (informative) Technique for culturing Hylotrupes bajulus (Linnaeus 16 Annex C (informative) Environmental, health and safety precautions within chemical/biological laboratory 19 Bibliography 20 EN 1390:2006 (E) Foreword This document (EN 1390:2006) has been prepared by Technical Committee CEN/TC 38 “Durability of wood and derived materials”, 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 2006, and conflicting national standards shall be withdrawn at the latest by December 2006 This document supersedes ENV 1390:1994 Significant technical differences between this standard and ENV 1390:1994 are as follows: a) introduction of new harmonised specifications for the test specimens used in the diverse biological tests; b) separation of the method according to the expected test periods for fast and slow acting preservatives and for deferred acting preservatives respectively; c) admission of the terms given in EN 1001-1and the definitions of EN 1001-2; d) introduction of an informative Annex to take account of consideration for minimisation of environmental and health hazards caused by the use of this biological test According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom EN 1390:2006 (E) Introduction This document describes a laboratory method of testing which gives a basis for the assessment of the eradicant action of fast and slow acting wood preservatives and of deferred acting wood preservatives against Hylotrupes bajulus It allows determination of the lethal effect of a surface application of a preservative product on a population of large larvae previously introduced into the test specimens The method simulates conditions in practice where a beam is treated, which is only slightly attacked and where cutting away has not exposed insect tunnels This represents a severe test of the product In some particular instances, for example where the preservative is to be used on timbers of large dimensions, laminated beams, blockboard, plywood and other panel products, other test methods can be used to obtain complementary information on the effectiveness of the eradicant action of a product Such methods lie outside the scope of this document This laboratory method provides one criterion by which the value of a product can be assessed In making this assessment the methods by which the preservative may be applied should be taken into account lt is further recommended that results from this test should be supplemented by those from other appropriate tests, and above all by comparison with practical experience When products that 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 C for environmental, health and safety precautions) EN 1390:2006 (E) Scope This document specifies a method for the determination of the eradicant action of a surface application of a fast and a slow acting wood preservative product or a deferred acting wood preservative product on timber infested with larvae of Hylotrupes bajulus (Linnaeus) This method is applicable to: organic formulations, as supplied or as prepared in the laboratory by dilution of concentrates, or organic water-dispersible formulations, as supplied or as prepared in the laboratory by dilution of concentrates, or water-soluble products, for example, salts NOTE An ageing procedure cannot be combined with this method Normative references The following referenced documents are indispensable for the application of this document For dated references, only the edition cited applies For undated references, the latest edition of the referenced document (including any amendments) applies EN ISO 3696,Water for analytical laboratory use - Specification and test methods (ISO 3696:1987) ISO 835-1:1981, Laboratory glassware - Graduated pipettes - Part 1: General requirements Terms and definitions For the purposes of this document, the following terms and definitions apply 3.1 representative sample sample with physical and/or chemical characteristics identical to the volumetric average characteristics of the total volume being sampled [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) [Adapted from EN 1001-2:2005, 4.83] Principle Insertion of larvae of Hylotrupes bajulus into test specimens After a period of time to allow the larvae to establish themselves in the test specimens, treatment of these test specimens by brushing or pipetting of the test preservative product After the time necessary for the preservative to act effectively, assessment of the mortality of the larvae compared with that of larvae in untreated control test specimens EN 1390:2006 (E) Test materials 5.1 Biological material 5.1.1 Hylotrupes bajulus (Linnaeus) larvae 5.1.2 Source of larvae The larvae shall preferably be obtained from cultures reared according to the method described in Annex B NOTE Larvae can also be taken from naturally infested wood, in which case they should be transferred into sapwood of pine and stored for at least four weeks under the rearing conditions specified in Annex B Do not use the larvae in the test if they have not fed normally during this storage period 5.1.3 Provision of larvae Carefully split or crumble infested blocks to extract larvae Keep the larvae separate from one another in glass receptacles for two or three days in the culturing chamber (5.3.1) to check they are healthy 5.1.4 Choice of larvae Use only healthy larvae in the test NOTE A healthy larva can be recognized by ivory-white colour, its firm consistency and rounded appearance, and by the absence of wounds or bites, which show up as dark marks Healthy larvae react to the touch by vigorous movement and attempts to bite Reject any larvae, which are shrunken or aged which have recently moulted, or which are in a pre-pupal stage Weigh each larva and place it in a glass receptacle marking the receptacle with the weight of the larva Make up two groups with the weight ranges: - 51 mg to 100 mg and -101 mg to 150 mg NOTE Larvae with a mass larger than 150 mg in mass are unsuitable as they can pupate during the course of the test 5.2 Products and reagents 5.2.1 Paraffin wax, for sealing the relevant surfaces of specimens to be treated with solutions in which water is the continuous phase NOTE Paraffin wax with a setting point of 52 °C to 54 °C has been found suitable 5.2.2 Gelatine, for sealing the relevant surfaces of specimens to be treated with solutions in which an organic solvent is the continuous phase 5.2.3 Water, complying with grade 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 EN 1390:2006 (E) 5.3 Apparatus 5.3.1 Culturing chamber, with air circulation, controlled at (28 ± 2) °C and at a relative humidity of (70 ± 5) % 5.3.2 Ventilated fume cupboard, in which the specimens are treated with an input air temperature of (20 ± 5) °C and a maximum air speed, measured at the input opening with the sash in the approximate operating position, of 0,5 m/s CAUTION - It is essential to follow safety procedures for handling flammable and toxic materials Avoid excessive exposure of operators to solvents or their vapours 5.3.3 Testing chamber, ventilated and controlled at (21 ± 2) °C and at a relative humidity of (75 ± 5) % 5.3.4 Drill and twist drills, with mm, mm and mm diameters 5.3.5 Pipettes as specified in ISO 835-1:1981, Class B - graduated pipette with no waiting time, with a capacity ml and an accuracy of ± 0,05 ml 5.3.6 Safety equipment and protective clothing, appropriate for the test product and the test, to ensure the safety of the operator 5.3.7 Ordinary laboratory equipment, including a balance capable of weighing to an accuracy of mg 5.3.8 Rectangular cover with sides, constructed either of glass, plastics, plywood and of a height not less than 200 mm and with an open face of sufficient size to cover all the treated specimens from a single test 5.3.9 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: mA to 15 mA 5.3.10 Protective gloves Sampling The sample of preservative shall represent the product to be tested Samples shall be stored and handled in accordance with any written recommendations from the supplier NOTE For the sampling of preservatives from bulk supplies, the procedure given in EN 212 should be used Test specimens 7.1 Species of wood The reference species is Scots pine (Pinus sylvestris Linnaeus)1) NOTE 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 species of pine 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 standard has been demonstrated in all aspects (development of larvae, resistance to impregnation, etc.) EN 1390:2006 (E) 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 wood shall not have been stored for more than five years NOTE Wood that has been kiln dried at temperatures below 60 °C may be used 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 NOTE It is recommended to use test specimens of a similar growth rate within a single test 7.3 Provision of test specimens Prepare planed strips having a cross-section of (100 ± 2) mm x (25 ± 2) mm removing a minimum of mm from any surface exposed during drying The longitudinal faces shall be parallel to the direction of the grain The annual rings shall be parallel to the broad faces (contact angle of less than 35 °), (see Figure 1) Make transverse cuts neatly to give sharp edges and a fine-sawn finish to the end-grain surfaces, to make test specimens (150 ± 2) 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 100 test specimens 7.4 Dimensions of test specimens The test specimen at (12 ± 2) % (m/m) moisture content shall be (150 ± 2) mm x (100 ± 2) mm x (25 ± 1) mm NOTE A moisture meter of the two-pronged electrical conductivity type is suitable for assessing moisture content Mark each test specimen so that it can be identified throughout the test 7.5 Number of test specimens 7.5.1 For fast and slow acting wood preservatives, test duration 12 weeks or 24 weeks Use: a) for each wood preservative, each concentration and each method of treatment -10 treated test specimens; b) for a single test of each wood preservative - two untreated control test specimens 7.5.2 For deferred acting wood preservatives, test duration 52 weeks Use: a) for each wood preservative, each concentration and each method of treatment- 20 treated test specimens; b) for a single test of each wood preservative -four untreated control test specimens EN 1390:2006 (E) Procedure 8.1 Preparation of the test specimens Using the drill (5.3.4), drill vertically three holes, 30 mm deep, into each cross section of each test specimen, positioning the holes as shown in Figure For each hole choose the twist drill diameter so as to provide a hole size which will accommodate the size of larva selected (8.2) Place the test specimens in the testing chamber (5.3.3) for one week Dimensions in millimetres Figure 1a: frontal perspective view Figure 1b: horizontal section Key plan of section in Fig 1b insertion hole for larva estimated test duration Figure 1: Example of a test specimen 8.2 Insertion of Iarvae into the test specimens For fast and slow acting wood preservatives, allocate three larvae from the 51 mg to 100 mg mass range and three larvae from the 101 mg to 150 mg mass range to each test specimen Carefully insert the larvae (5.1) head first into the appropriately sized holes For deferred acting wood preservatives, allocate one larva from the 51 mg to 100 mg mass range and two larvae from the 101 mg to 150 mg mass range to each test specimen (or vice versa) Carefully insert the larvae (5.1) head first into the appropriately sized holes Insert one larva from one mass range into the middle hole at one end of the test specimen and two larvae from the other mass range into outer holes of the other end of the test specimen Seal the insertion holes with plugs of cotton wool Incubate the test specimens for one week in the testing chamber (5.3.3), then remove the cotton wool plugs and determine whether each larva has bored, replacing larvae, which have not bored lf any larvae are replaced then incubate all test specimens for a further week in the testing chamber (5.3.3) 8.3 Sealing of the surfaces not to be treated Seal the 100 mm x 150 mm pith face and the two cross sections EN 1390:2006 (E) 1) For tests with preservative solutions in which water is the continuous phase, apply three coats of 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 2) 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 with an aqueous solution (200 g/l) at about 40 °C, then after a minimum of h of drying, apply two further coats of an aqueous solution (300 g/l) at about 50 °C 8.4 Incubation of the test specimens Incubate all the sealed test specimens in the testing chamber (5.3.3) for a maximum of two weeks from the date at which larvae were first inserted 8.5 Treatment of test specimens 8.5.1 8.5.1.1 Preparation of the treatment solution Solid wood preservatives Water-soluble wood preservatives: dissolve the wood preservative in the water (5.2.3) to the required working concentration Non-water-soluble wood preservatives: dissolve the wood preservative in an appropriate solvent (5.2.4) to the required working concentration 8.5.1.2 Liquid wood preservatives If appropriate, use the wood preservative without further preparation other than any necessary stirring If it is a concentrate, dilute the wood preservative with the diluent to the required working concentration, using the procedure specified by the manufacturer For the dilution of water-based wood preservatives use the water specified in 5.2.3 All treatment solutions shall be freshly prepared 8.5.2 Application of the treatment solution Remove the test specimens to be treated from the testing chamber (5.3.3) after completion of the incubation period (8.4) Determine the actual area of each unsealed surface to be treated taking into account any possible encroachment of the sealing compound NOTE The total area to be treated is theoretically 225 cm2 Determine the volumes or masses of the treatment solution (8.5.1) to be applied to each unsealed face to give the application rate specified by the supplier NOTE The quantity of the treatment solution to be applied should be realistic in view of the field of application and the manufacturer's instructions In the fume cupboard (5.3.2), using either the pipette (5.3.5) or a brush, apply the appropriate calculated volume or mass of the treatment solution (8.5.1) to each of the unsealed faces as uniformly as possible Apply the treatment solution to each unsealed face whilst keeping that face in a horizontal and upward facing position Allow any surface liquid to be absorbed into each face before treating the next face NOTE If the required quantity cannot be applied in one application, the treatment solution may be applied in successive applications at appropriately close intervals so as to avoid solidification of any substances hindering the penetration of the subsequent applications 10 EN 1390:2006 (E) lf a brush application is used, weigh the test specimens during application to determine the mass applied From the quantity of the treatment solution applied to each unsealed face of each treated test specimen, determine and record the average application rate in g/m2 (brush application), or ml/m2 (pipette application) of the treated test specimens Complete the treatment of all faces of each set of test specimens within h 8.6 Drying of the test specimens Immediately after treatment, place the treated test specimens with the larger treated face upwards on a wire rack raised above the base of the fume cupboard on (10 ± 2) mm thick spacers Contact between treated specimens is to be prevented Place the cover (5.3.8) over the test specimens and retain the test specimens in the fume cupboard (5.3.2) for h whilst maintaining air speed (see Figure 2) After drying store the test specimens in the testing chamber (5.3.3) with their larger treated faces upwards Dimensions in millimetres Key Grill Rectangular cover with sides Treated test specimen Figure 2: Arrangement of treated specimens (10 or 20 parallel specimens) for drying in the fume cupboard 8.7 Duration of test Keep the test specimens in the testing chamber either for 12 weeks for fast acting wood preservatives, for 24 weeks if the product being tested is slow-acting or for 52 weeks if it has a deferred action NOTE The test may be stopped earlier if information is available that all larvae in the preservative treated test specimens are dead 11 EN 1390:2006 (E) 8.8 Examination of the test specimens 8.8.1 Examination without radiography At the end of the test period (8.7) carefully cut up the test specimens Determine and record the following values for each test specimen: a) number of dead and moribund larvae; b) number of live larvae; c) number of pupating larvae; d) number of larvae not recovered; e) number of adult beetles present inside the test specimen NOTE 8.8.2 If it is uncertain whether particular larvae are moribund or live, the procedure in 8.8.3 may be adopted Examination with radiography (optional) Using the X-ray apparatus (5.3.9), radiograph the test specimens after weeks and 12 weeks in the case of fast acting products or 20 weeks and 24 weeks in the case of slow-acting products or 48 weeks and 52 weeks in the case of deferred acting products Estimate the number and condition of larvae in each test specimen at each examination At the final examination record: a) number of dead larvae; b) number of larvae which, though apparently alive, have not moved since the previous examination; c) number of actively boring larvae; d) number of larvae unaccounted for, if any; e) number of adult beetles present inside each test specimen 8.8.3 Verification of condition of recovered larvae lf it is uncertain whether recovered larvae are live or dead then place these larvae into untreated wood blocks and store them for two weeks in the testing chamber (5.3.3) lf they are not able to bore normally after this period then they shall be classified as dead Validity of the test The results shall be accepted as valid provided that at least live larvae of 12 are recovered in total from the two untreated control test specimens 10 Expression of results Express results as average percentage mortality in the treated test specimens and in the untreated control test specimens as recorded in 8.8 12 EN 1390:2006 (E) 11 Test report The test report shall include at least the following (see also Annex A for an example): a) number and date of this document (EN 1390:2006); b) name of the supplier of the wood preservative under test; c) type of wood preservative as fast or slow acting or as deferred acting wood preservative; d) specific and unique name or code of the wood preservative tested, with an indication of whether or not the formula has been declared; e) solvent or diluent used, if any; f) species of wood used; g) date of the first insertion of larvae into the test specimens; h) where relevant, the concentration(s) of the wood preservative expressed as a mass fraction; i) type of treatment (pipette or brush) and the number of applications; j) total quantity of wood preservative applied to the test specimens in g/m2 or ml/m2; k) date of the application of the wood preservative; l) date of examination of the test specimens, whether the examination has been carried out by cutting of the test specimens or by X-ray; m) results of the examination of each treated test specimen and control test specimen: - number of beetles recovered; - number of live larvae recovered; - number of dead or moribund larvae recovered; - number of larvae undergoing pupation; - number of larvae not recovered; n) name of the organisation responsible for the test report and the completion date of the test; o) name and signature of officer(s) in charge of testing; p) following note: "The interpretation and the practical conclusions that can be drawn from this test report demand a specialised knowledge of the subject of wood preservation and for this reason, this test report cannot of itself constitute an approval certificate" The test report shall also list any variation from the described test method, as well as any factors that may have influenced the results 13 EN 1390:2006 (E) Annex A (informative) Example of a test report Number and date of this document EN 1390:2006 Name of supplier company S Name and type of product X-preservative ,fast acting ,in the form of an organic solvent, ready for use, formula not declared Solvent or diluent used none Species of wood used Scots pine (Pinus sylvestris Linnaeus) Date of first insertion of larvae 2002-07-15 Concentration of the wood preservative tested wood preservative used undiluted Type of treatment and number of applications applied in two coats using a pipette Quantity of wood preservative applied to the test specimens 300 ml/m2 Date of application of the wood preservative 2002-07-29 Date of examination of the test specimens 2002-10-21 Results see Table A.1 This report has been prepared by the institute VWX Location and date Y 2002-10-28 Name and signature of officer(s) in charge Mrs Z The interpretation and the practical conclusions that can be drawn from this test report demand a NOTE specialised knowledge on the subject of wood preservation and for this reason, this test report cannot of itself constitute an approval certificate 14 300 Control (0) 0 0 0 0 0 0 A B C D E F G H I J 0 6 6 6 6 6 60 11 per test per test total total specimen specimen larvae Recovered insects adult beetles No Dosage ml/m² Test specimen Treatment 5 5 6 0 per test specimen 53 total Dead or moribund larvae Table A.1: Results 1 1 per test specimen 11 total Living larvae 83,3 83,3 100 83,3 83,3 100 66,7 100 83,3 100 0 per test specimen 15 88,3 average Mortality % EN 1390:2006 (E) EN 1390:2006 (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 in wood preservation B.2 Obtaining parent beetles In order to obtain reproductive adults, create a cultureby taking larvae from naturally infested wood and insert them head first into suitably sized drilled holes in pine sapwood blocks and allow them to pupate 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 surface, cover them with a Petri dish lid and, as Hylotrupes bajulus is a diurnal insect, leave them in daylight 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-Iaying 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 so that they may 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 removing 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 each day or every two days stimulates egg-Iaying Check once a day to see 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-Iaying, allowing continued mating, to activate egg-Iaying A favourable temperature for egg-Iaying is about 25 °C 16 EN 1390:2006 (E) Key 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, upon hatching, 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 85 % B.6 Larval development Place the newly-hatched larvae in culturing blocks, as described below They shall not have been deprived of food for more than days before being inserted in the blocks Handle the larvae carefully using a soft brush or vacuum tweezers As the larvae of Hylotrupes bajulus eat one another, they should be kept apart during culturing by having only 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 mm to 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 10 g/l yeast (the yeast may be replaced with 0,01 g/l lactoflavine), then dry them 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 can conveniently be of dimensions 50 mm x 25 mm x 15 mm, the length being parallel to the grain of the wood The growth of Hylotrupes bajulus is most rapid between 28 °C and 30 °C with an optimum relative humidity of 97 % to 98 %, however, as very high humidity favours the development of moulds it is preferable to use a relative humidity of (70 ± 5)% When conditions of microclimate and nutrition are perfect, the male adult insects can appear at about months As soon as the larvae reach a size such that the volume of the blocks is insufficient to permit normal 17 EN 1390:2006 (E) growth, it is preferable to transfer them into blocks of larger dimensions that are not impregnated with peptone and yeast, so that 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 temperatures It is therefore advantageous to place the large blocks outdoors in winter at a temperature between °C to 10 °C 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: Rhoptocentrus 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 18