SUBMISSION FORM OUTLINE of REQUIREMENTS National Plant

SUBMISSION FORM

OUTLINE OF REQUIREMENTS

National Plant Protection Organizations (NPPOs) and Regional Plant Protection Organizations (RPPOs) may submit data and other information for the evaluation of efficacy, feasibility and applicability of treatments. The information should include a detailed description of the treatment, including efficacy data, the name of a contact person, and the reason for the submission. Treatments that are eligible for evaluation include mechanical, chemical, irradiation, physical and controlled atmosphere treatments. The efficacy data should be clear and should preferably include data on the treatment under laboratory or controlled conditions as well as under operational conditions. Information on feasibility and applicability of the proposed treatment(s) should include items on cost, commercial relevance, level of expertise required to apply the treatment and versatility.

Submissions with complete information will be considered by the Technical Panel on Phytosanitary Treatments (TPPT), and if the treatment is deemed acceptable, it will be recommended to the CPM for adoption.

Process for Treatment Development and Adoption The development process is initiated by a call for topics for standards (including topics for treatments) according to the "IPPC standard setting procedure" and the "Procedure and criteria for identifying topics for inclusion in the IPPC standard setting work programme" (provided in the International Plant Protection Convention procedural manual).

In particular, the following points apply to treatments: - Once a topic for treatments (e.g. treatments for fruit flies or for pests on wood) has been added to the IPPC standard-setting work programme, the IPPC Secretariat, under direction of the Standards Committee (with recommendations from the TPPT), will call for the submissions and data on treatments on that topic. - NPPOs or RPPOs submit treatments (accompanied by relevant information as requested in section 3) to the Secretariat. - Only submissions of treatments that are deemed by the NPPO or RPPO to meet the requirements listed in this standard should be submitted, and it is recommended that these treatments have been approved for national use before their submission. Treatments include, but are not limited to, mechanical, chemical, irradiation, physical (heat, cold) and controlled atmosphere treatments. NPPOs and RPPOs should take into account other factors when considering phytosanitary treatments for submission, such as the effects on human health and safety, animal health and the impact on the environment (as described in the preamble and Article I.1 of the IPPC, 1997)1. Effects on the quality and intended use of the regulated article should also be considered. - Treatment submissions will be evaluated based on the requirements listed in section 3. If the volumes of submissions are high, the relevant TPPT criteria listed in the International Plant Protection Convention procedural manual will be applied to determine the priority for reviewing submissions. - Treatments that meet the requirements listed in section 3 will be recommended and the treatment submitted, along with a report and a summary of the information evaluated, to the Standards Committee and in turn to the IPPC standard setting process. - The CPM will adopt or reject a treatment. If adopted, the treatment is annexed to this standard.

Requirements for Phytosanitary Treatments For the purpose of this standard, phytosanitary treatments should fulfil the following requirements: - be effective in killing, inactivating or removing pests, or rendering pests infertile or for devitalization associated with a regulated article. The level of efficacy of the treatment should be stated (quantified or expressed statistically). Where experimental data is unavailable, other evidence that supports the efficacy (i.e. historical and/or practical information/experience) should be provided. - be well documented to show that the efficacy data has been generated using appropriate scientific procedures, including an appropriate experimental design. The data supporting the treatment should be verifiable, reproducible, and based on statistical methods and/or on established and accepted international practice; preferably the research should have been published in a peer-reviewed journal. - be feasible and applicable for use primarily in international trade or for other purposes (e.g. to protect endangered areas domestically or for research).

1 Contracting parties may have obligations related to treatments under other international agreements, e.g. The Montreal Protocol on Substances that Deplete the Ozone Layer (1999) and/or the Rotterdam Convention (1998). Proposed by: (Name of country or regional/international organization)

Contact: (Contact information for an individual able to clarify issues relating to this submission, including sources of efficacy data) Name: ...... Position and organization:...... Mailing address: ...... Phone:...... Fax:...... Email: ......

Treatment description Treatment name (provide enough detail to identify the treatment; for example, cold treatment of citrus for Mediterranean fruit fly): Microwave disinfesting treatment of wood and wooden packaging materials (WPM) Treatment type (for example, chemical, irradiation, heat, cold): Microwave irradiation Target commodity(ies)/regulated article(s): Logs/trunks and all WPM such as: pallets, dunnage, crating, packing blocks, drums, cases, load boats, pallet collars and skids in use in international deliveries. Target pest(s): Larvae of insects like Buprestydae, Cerambycidae, Curculionidae, Scolytidae. Larvae and adults of nematodes like Parasitaphelenchidae, Rhabditidae, Panagrolaimidae, Neotylenchidae, Cephalobidae, Aphelencoididae, Monhysteridae, Diplogasteridae, Mononchidae. Schedule (include brief description such as active ingredient, dose, time and temperature): The treatment consists in putting the logs or the WPM into a shielded reverberating chamber where it is irradiated by microwaves; the electromagnetic field produced into the treatment chamber is stochastically uniform, homogeneous and isotropic. The microwaves heating exploits the typical properties of some chemical substances, that is the capacity to absorb the energy carried by electromagnetic waves and transform it into thermal energy. Thermal and non-thermal microwave effects (also referred to as athermal effects) lead to the killing of the pests infesting wood. During the laboratory tests, the power and the exposure time of the trunks (diameter Ø≤19 cm; length ≤69 cm; weight ≤10,557 Kg) to the microwaves were settled every time on the basis of some elements such as presence/absence of bark, size and weight of samples. For the smaller trunks without bark a reverberating chamber with one magnetron was used (maximum power 6.0 Kw), while the other trunks were introduced into a 3-magnetron chamber (maximum power 18.0 Kw). LAB TESTS In order to reach the mortality of pests, electromagnetic waves at the frequency of 2.45 GHz were emitted for the exposure time ranging from 120’’to 360’’ and using the variable power range 3-9 kW.

OPERATIONAL CONDITIONS In the treatment tests on pallets (carried out in the 3-magnetron chamber) the exposure time changes with the increasing of the power and the decreasing of treated mass. As showed in the graph on the right, the exposure time decreases using a higher power but the function is not linear.

Microwave treatment Exposure time-Rf power of pallet at different power 550 80 500 70 450 400 60 Mean Temp. 350 50 1pallet 6 kW 300 2 pallet 40 Mean Temp.1 250 1 pallet 30 pallet 12 kW 200 20 Mean Temp. Time (sec) 150 10 100

Temperature (°C) 0 1pallet 18 kW 50 0 0 0 0 0 0 0 0 Mean Temp.2 0 3 6 90 50 70 00 90 12 1 18021024 2 3 33 36 3 42045048 510 pallet 6 kW 0 5 10 15 20

Time (s) Power(kW)

Efficacy data in support of the submission of a phytosanitary treatment The source of all efficacy data (published or unpublished) should be provided in the submission. Supporting data should be presented clearly and systematically.

The experience or expertise in the subject area of the laboratory, organization and/or scientist(s) involved in producing the data, and whether the research utilized a quality assurance or accreditation programme in the development and/or testing of the phytosanitary treatment, will be considered when evaluating the data submitted. Any claims on the efficacy must be substantiated by data. Efficacy data under laboratory/controlled conditions The life-cycle stage of the target pest for the treatment should be specified. Usually, the life stage(s) associated with the regulated article moving in trade is the stage for which a treatment is proposed and established. In some circumstances, e.g. where several life stages may occur on the regulated article, the most resistant life stage of the pest should be used for testing a treatment. However, practical considerations should be taken into account, as well as pest control strategies aimed at exploiting more vulnerable or otherwise specific stages of a pest. If efficacy data is submitted for a life stage that is not considered to be the most resistant (e.g. if the most resistant life stage is not associated with the regulated article), rationale for this should be provided. The efficacy data provided should specify the statistical level of confidence supporting efficacy claims made for treatment of the specified life stage.

Where possible, data should be presented on methods used to determine the effective dose/treatment to demonstrate the range of efficacy of the treatment (e.g. dose/efficacy curves). Treatments can normally be evaluated only for the conditions under which they were tested. However, additional information can be provided to support any extrapolation if the scope of a treatment is to be extended (e.g. extension of the range of temperatures, inclusion of other varieties or pest species). Where the information provided is adequate to demonstrate the effectiveness of the treatment, only a summary of relevant preliminary laboratory tests will be required. The materials and methods used in the experiments should be suitable for the use of the treatment at the stated efficacy.

The data provided should include detailed information on, but not limited to, the following elements: Pest information Identity of the pest to the appropriate level (e.g. genus, species, strain, biotype, physiological race), life stage, and if laboratory or field strain was used

Larvae of insects like Buprestydae, Cerambycidae, Curculionidae, Scolytidae. Larvae and adults of nematodes like Parasitaphelenchidae, Rhabditidae, Panagrolaimidae, Neotylenchidae, Cephalobidae, Aphelencoididae, Monhysteridae, Diplogasteridae, Mononchidae.

The Bursaphelenchus genus includes the four species of B. mucronatus, B. leoni Baujard, B. sexdentati Ruhm and B. teratospicularis Kakuliya et Devdariani that were extracted from the samples of conifers; while the B. eremus was found in the oak.

All the xylophagous insect species extracted from the wood were mature or near to maturity larvae, with the exception of the Scolytus intricatus present as larvae and adults. The following insects were found and isolated: Panagrolaimide Rhabditidae Arhopalus syriacus Acanthocinus griseus Orthotomicus erosus Monochamus galloprovincialis Pissodes castaneus Phaenops cyanea Cerambycidae Chrysobotris sp. Scolytus intricatus

Conditions under which the pests are cultured, reared or grown

The pests under experimentation derived from Northern and central Italy and infested the wood belonging to different plant species like Pinus pinaster, Quercus robur and Populus. In particular they were present in the subcortical zone and in the woody tissues of these plants. Sample materials were gathered and stored in a greenhouse at the Istituto Sperimentale per la Zoologia Agraria, Cascine del Riccio-Firenze (I).

Biological traits of the pest relevant to the treatment (e.g. viability, genetic variability, weight, developmental time, development stage, fecundity, freedom from disease or parasites)

Method of natural or artificial infestation

The trunks used during the lab tests were naturally infested.

Determination of most resistant species/life stage (in the regulated article where appropriate)

As regards the phytoparasitic nematodes (gen. Bursaphelenchus), tests were performed only evaluating the abundance of larvae and adults: in this group, differently from the root-knot nematodes, the egg is not a resistant life-cycle stage and therefore it is sufficient to take into consideration the number of dead larvae and adults in order to evaluate the effectiveness of the disinfesting treatment. Likewise for the more harmful xylophagous Coleoptera (group of a particular phytosanitary interest), the observations mainly focused on larvae: the larva represents the longest stage in the development and the more easily and frequently transported one by packaging materials while the egg of these insects is not a resistant stage.

Regulated article information Type of regulated article and intended use The MW treatment can be applied on all types of wood and wooden packaging material such as: pallets, dunnage, crating, packing blocks, drums, cases, load boats, pallet collars and skids in use in international deliveries.

Botanical name for plant or plant product • type/cultivar (where varietal differences impact on treatment efficacy, data should be provided). The requirement for varietal testing should be based on evidence to support the requirement. The experimentation was carried out on various types of trunks deriving from different types of plants. These were labelled using MF (1-15) and IM (1,2) for plants of Pinus pinaster, QMF (1,2) for Quercus robur and P(3-6) for Populus.

Tab. 1 – Photographs of the wood samples of conifer used for the treatment. Pinus pinaster Aiton (MF and IM). MF 9 MF 1 (with bark)

MF 10 MF 2 (with bark)

MF 11 MF 3 (with bark)

MF 12 MF 5 (with bark)

MF 13 MF 6 (with bark)

MF 7 MF 14 (with bark) (with bark)

MF 8 MF 15 (with bark) (with bark)

IM 1 IM 2 (with bark) (with bark)

Tab. 2 Photographs of the wood samples of broad-leaves used for the treatment. Quercus robur (QMF); Populus sp. (P).

QMF 1 P3 (with bark) (with bark)

QMF 2 P4 (with bark) (with bark)

P 1 P 5 (with bark) (with bark)

P 2 P 6 (with bark) (with bark)

• conditions of the plant or plant product, for example: ◦ whether it was free from non-target pest infestation, non-pest disorder or pesticide residue ◦ size, shape, weight, stage of maturity, quality etc. ◦ whether infested at a susceptible growth stage.

Before proceeding with the disinfesting treatment the wood samples were weighed by means of an electronic balance and data relative to the diameter (of the larger cut section) and length were recorded in the following tables.

Tab. 3 Pinus pinaster

No. SUB- TOTAL PRESENT PRESENT INSECTS SAMPLE ORIGIN SAMPLES WEIGHT NEMATODES (larvae (larvae) (Kg) and adults) Panagrolaimide Monochamus MF 1 Montefalcone (PISA) 4 10 Rhabditidae galloprovincialis

Rhabditidae Bursaphelenchus leoni MF 2 Montefalcone (PISA) 6 13 M. galloprovincialis B. mucronatus B. teratospicularis Rhabditidae MF 3 Montefalcone (PISA) 3 10 M. galloprovincialis

MF 5 Montefalcone (PISA) 4 7,5 M. galloprovincialis Rhabditidae Arhopalus syriacus Rhabditidae M.galloprovincialis MF 6 Montefalcone (PISA) 5 16,5 B. leoni Acanthocinus griseus B. mucronatus Orthotomicus erosus Pissodes castaneus Rhabditidae MF 7 Montefalcone (PISA) 6 27,5 O. erosus B. leoni (with bark) A. griseus B. sexdentati Cephalobidae Pissodes castaneus Diplogasteridae MF 8 Montefalcone (PISA) 2 7,0 Orthotomicus erosus Rhabditidae (with bark) Cerambycidae Aphelenchoididae B. leoni Cephalobidae MF 9 Monochamus Diplogasteridae Montefalcone (PISA) 2 16,0 (with bark) galloprovincialis Rhabditidae Aphelenchoididae Cephalobidae MF 10 Acanthocinus griseu Diplogasteridae (con Montefalcone (PISA) 2 10,5 Arhopalus syriacuss Rhabditidae corteccia) Aphelenchoididae Cephalobidae Diplogasteridae MF 11 Acanthocinus griseus Montefalcone (PISA) 2 9,2 Rhabditidae (with bark) Arhopalus syriacus Aphelenchoididae B. leoni Cephalobidae MF 12 Acanthocinus griseu Diplogasteridae Montefalcone (PISA) 2 6,2 (with bark) Phaenops cyanea Rhabditidae Aphelenchoididae Cephalobidae Diplogasteridae MF 13 Monochamus Rhabditidae Montefalcone (PISA) 2 6,0 (with bark) galloprovincialis Mononchidae Aphelenchoididae Neotylenchidae Cephalobidae Acanthocinus griseus MF 14 Rhabditidae Montefalcone (PISA) 2 4,1 Arhopalus syriacus (with bark) Aphelenchoididae Phaenops cyanea Neotylenchidae Cephalobidae MF 15 Montefalcone (PISA) 2 11,3 Cerambycidae Rhabditidae (with bark) Aphelenchoididae IM 1 Impruneta A. syriacus Panagrolaimidae 5 9,5 (with bark) (FIRENZE) A. griseus Rhabditidae Panagrolaimidae IM 2 Impruneta Rhabditidae 6 43,7 Chrysobotris sp. (with bark) (FIRENZE) B. leoni B. mucronatus

Tab. 4 Quercus robur (QMF); Populus sp. (P).

TOTAL PRESENT No. SUB- WEIGHT PRESENT INSECTS NEMATODES SAMPLE ORIGIN SAMPLES (Kg) (larvae*) (larvae and adults)

Cerambycidae Cephalobidae QMF 1 Scolytus Montefalcone (PISA) 3 8,3 Monhysteridae (with bark) intricatus(*larve, adulti Neotylenchidae e uova) QMF 2 Montefalcone (PISA) 3 10,5 Cerambycidae Cephalobidae (with bark) Scolytus Bursaphelenchus sp. intricatus(*larve, adulti e uova) Cascine del Riccio P 1 Cerambycidae (FIRENZE) 3 18,3 Cephalobidae (with bark) Buprestidae

Cascine del Riccio P 2 Buprestidae (FIRENZE) 3 55,5 Cephalobidae (with bark) Cerambycidae

Cascine del Riccio P3 Buprestidae (FIRENZE) 3 21,8 Cephalobidae (with bark) Saperda charcarias

Cascine del Riccio P4 Buprestidae (FIRENZE) 3 6,9 Cephalobidae (with bark) Cerambycidae

P 5 Cascine del Riccio Buprestidae 3 16,2 Cephalobidae (with bark) (FIRENZE) Cerambycidae Cascine del Riccio P 6 (FIRENZE) 1 1,6 Buprestidae Cephalobidae (with bark)

Experimental parameters Level of confidence of laboratory tests provided by the method of statistical analysis and the data supporting that calculation (e.g. number of subjects treated, number of replicate tests, controls)

Before proceeding with the lab test each sample was cut into smaller trunk portions in order to carry out replicate tests. In total 16 samples of Pinus pinaster (55 trunk portions), 2 samples of Quercus robur (6 trunk portions) and 6 samples of Populus sp. (16 trunk portions) were used during the experimentation. (see the tables above)

Experimental facilities and equipment

Facilities and equipment for pests isolation:

The wood for extraction was drawn from each of the samples of Pinus pinaster, Quercus cerris and Populus sp. by means of a drill bit of 10 mm Ø .

As regards the individuation of nematodes the Baermann funnel and the method through centrifuge were used

Baermann funnel - the extraction method based on the motility of living nematodes - The device consists of a funnel (10-13 cm Ø) with a piece of rubber tubing attached to the stem and closed by a Mohr’s clip. The funnel is placed on a support and filled with tap water. The sample (cut into small pieces of 10-15g) rolled in a paper-wool filter is then submerged in the water of the funnel and hold on a support of a rigid choline net. Nematodes come out from the wood, pass through the water and gather on the bottom of the stem. The extraction lasts 48 hours at a temperature of 22-24 °C. For this period of time the device is left undisturbed, but a small quantity of water (10ml) is tapped off after 24 hours and replaced with the same quantity. At the end of the extraction period the same amount of water is gathered and mixed with the previous amount. The volume of liquids which has been obtained can be opportunely reduced after a suitable period of decantation (at least 1 2 hours).

The centrifugal flotation allows to extract both dead and alive nematodes. Centrifuge - 10 gr of chopped wood are dispersed in 750 ml of water using a vibromixer (60’’) at 20.000 g. Then the content passes through a sieve with a 1 mm Ø aperture collocated on a beaker and is washed with such a quantity of water which allows to reach a total volume of 1000 ml. The content of the beaker is centrifuged. One half of the content is collected in a centrifuge tube (500 ml) containing also 3 ml of kaolin and then spun by a stirrer. This mechanic agitator is carefully washed after each process.

1st step

The mixture is centrifuged for 5’ at a centrifugal force of 1800 g. The supernatant is discarded at the end of the process.

2nd step

Wood is subject to an homogenization with a sucrose solution sp. gr. of 1,15 (401 gr/lt H2O) by means of a stirrer for 60’’. The suspension is centrifuged for 5’ at the same centrifugal force. The supernatant recovered is poured on a 15 µm-aperture sieve for the washing and the collecting of nematodes.

For the identification and the calculation of nematodes by the optical microscope, the suspensions obtained before the treatment were reduced to a volume of 1 ml. Parts of 1/5 or 1/10 were picked up through a micro-pipette and were distributed on a plate. A complete analysis of the liquid, obtained through the two methods of extraction, was carried out.

The dry weight of the samples after extraction has been calculated and considered as fiducial parameter of the other numerical data. The dry weight was determined by weighing the parts of the samples exposed to dry heat (105°C) for 24 hours.

Facilities and equipment for temperature measuring.

The surface temperature of samples was monitored by means of an infrared thermo-camera, Flir Thermacam SC2000, (calibrated in terms of thermal emissivity for wooden materials: 0,90), while the inner temperature of the samples’ volume by means of magnesium oxide (MgO) thermo-resistances inserted into the holes drilled on their surface and cut section. Two holes (3.5 mm Ø each) were performed by means of drill just at the middle of the logs’ length and at 5-6 cm in depth. For larger samples, (with diameter > 12 cm), three holes were drilled with the third one realized on the cut surface.

Before proceeding with the disinfesting treatment the wood samples were weighed by means of an electronic balance and the diameter (of the larger cut section) and length data were recorded to remark the weight differences before and after the treatment due to moisture losses.

Facilities and equipment for microwave treatment.

The disinfesting treatments were carried out using two prototypes of shielded reverberation chamber in Emitech’s laboratories; these were projected and realized by Emitech itself.

For the smaller trunks and the ones without bark the reverberation chamber with one magnetron shown in Fig. 1 was used (power max 6.0 Kw); while the 3-magnetron chamber (power max 18.0 Kw), shown in Fig. 2 was used for the other trunks.

Fig. 1 one-magnetron reverberation chamber Fig. 2 three-magnetron reverberation chamber

Experimental design (e.g. randomized complete block design)

Experimental conditions (e.g. temperature, relative humidity, diurnal cycle)

The room temperature, during the trials, was of 18 °C while the surface temperature of the samples, before the treatments, was of 14 °C.

Monitoring of critical parameters (e.g. exposure time, dose, temperature of regulated article and ambient air, relative humidity)

The critical parameters valid for the microwave treatment are: exposure time, internal and external temperature, power of the electromagnetic energy at the frequency of microwaves. Both the inner and the external temperatures of samples were monitored using a E660 IKD thermocouple for the inner temperature and an infrared thermo-camera, Flir Thermacam SC2000, (calibrated in terms of thermal emissivity for wooden materials: 0,90) for the surface temperature. In addition to the data recorded on the basis of temperature changes and humidity losses (deductible through weight variations), the visual analysis of the samples allowed to remark the leaking out of resin at the end of the treatment as it is shown in picture no 7.

Tests showed a high effectiveness of MW treatments in reverberation chambers to control nematodes and xylophagous insects. The effectiveness was evaluated in terms of pests mortality.

Methodology to measure the effectiveness of the treatment on Nematodes The wood for extraction was drawn from each of the samples of Pinus pinaster, Quercus cerris and Populus sp. by means of a drill bit of 10 mm Ø. In order to verify the number of nematodes present in the wood after the treatment the method through centrifuge was used, in addition to the the Baermann funnel.

The extracted nematodes have been categorized in terms of species (for the Bursaphelenchus genus) and in terms of family and sometimes of genus (for the other nematodes).

As regards the samples of P. pinaster, colonized by B. mucronatus, those without bark (MF2 and MF6) were exposed to treatments at 3 kW and at 4,5 kW for periods of time which were respectively 180” or 360” and 180” or 240”, while those with bark (IM2) were treated with 9 or 13,5 kW of power for 360”.

The extraction by centrifuge after the treatment, showed a constant and significant presence of dead B. mucronatus specimens in particular the number ranged from 0,6 to 9,2 nematodes/gr of dry wood in the samples with bark and increased from 38,5 to 366,6 in the samples without bark. The temperatures reached in the wood ranged from a minimum of 72°C to a maximum of 108°C in the sample MF2, from 52°C to 103°C in the sample MF6 and from 51°C to 110°C in the sample IM2.

The extraction through the Baermann funnel for the collection of alive specimens showed that 12/14 samples resulted exempt, while the presence of 0.1 B. mucronatus specimens (larvae) per gram of dry weight was found only in two samples: MF2 E ed MF6 D.

The data in the sample MF6 D is explained by the fact that the maximum temperatures recorded by the probes (P1 = 54°C, P2 = 47°C) were inferior with respect to the threshold considered as lethal temperature (65°C for 120” in water-bath); while in the sample MF2 E temperatures (P1 = 98°C, P2 = 107°C) were much higher; anyway the value can be considered insignificant if compared to the 214,3 dead specimens/gram isolated by the centrifuge method.

The action of treatments in MW reverberating chamber was completely successful for the other species of Bursaphelenchus. The post-treatment check, performed through the centrifugal method, showed the presence of a relevant number of dead specimens in trunks with and without bark as well, while no living form was found during the check carried out with the method of Baermann. As for the B. teratospicularis species, whose presence was very low and found only in the sample MF6, there was no trace in the treated wood.

As for the other forms which frequently occur in deteriorating wood and/or sawdust and excrements produced by xylophagous insects, the Cephalobidae, Diplogasteridae, Panagrolaimidae and Rhabditis often elude the action of microwaves.

Anyway treatments reaching temperatures which exceed 110°C are effective on these saprophytic forms or at least they reduce them significantly.

Methodology to measure the effectiveness of the treatment on Xylophagous insects After the treatment in the reverberation chamber and the sampling of wooden materials for nematological tests, the trunk sections were entirely examined and trunks with bark were debarked for a preliminary analysis of the specimens present in this part of the samples.

In order to check the MW effects on xylophagous insects present in wood, all samples were progressively sectioned and all specimens present inside them were collected. All dead and alive specimens were examined one by one for the species identification in the case of Monochamus galloprovincialis, Arhopalus syriacus, Acanthocinus griseus, Saperda carcharias, Phaenops cyanea, Pissodes castaneus, Orthotomicus erosus and Scolytus intricatus, while in the other cases Family and Genus were individuated.

High infestation levels by one or several species of xylophagous Coleoptera were present in the treated portions of trunk.

As regards the main species, Monochamus galloprovincialis, a maximum of 17 larvae was found in the wooden tissues in the galleries or in the pupal cells of the samples MF2A, MF2C e MF2E.

In the trunks with bark, the highest presence of insects was counted in the samples MF7B of Orthotomicus (94 larvae), MF7F Pissodes (49 larvae), IM1A Arhopalus (67 larvae) and QMF1C Scolytus intricatus (177 larvae and adults).

After the treatments in the reverberating chambers, a 100% of mortality was recorded for all the xylophagous Coleoptera in the samples treated for 360”, regardless of the power applied (3 – 4, 5 – 9 – 13,5 KW).

Considering each species, it is possible to remark that, of the 142 larvae of Monochamus in the samples MF1 (A,B,C,D), MF2 (A,B,C,D,E,F) MF3 (A, B,C), MF6 (A,B,C,E), MF9 (A) e MF13 (A, B) only 2 specimens were found alive in the sample MF3C treated with a power of 3 Kw, for 180”. The thermometrical analysis of this sample revealed the achievement of maximum values which were lower than the established temperatures (max. temperature of the detectors: P1 55°C; P2 = 38°C).

Likewise, for Acanthocinus griseus, the second species belonging to the Cerambycidae family, 2 alive specimens were found in the sample MF6D treated for 180”, at a power of 4.5KW and temperature values lower than the established ones were recorded (max. temperature of the detectors: P1 = 54°C; P2 = 47°C). Moreover, three alive larvae of A. griseus were found in the sample IM1E with bark (exposure time 180”, power 4.5 Kw, P1 = 80°C, P2 = 66°C).

One alive but not very mobile larva of Arhopalus was also found in the sample IM2A which was partly covered by bark and underwent two subsequent treatments (exposure time 180”, power 9 KW, P1 = 73°C, P2 = 53°C).

Only 3 larvae of 173 Coleoptera Curculioniodae, Pissodes castaneus, were found alive on a log treated for 180”, at 4.5Kw. In this case the thermal threshold was just exceeded, P1 = 79° e P2 = 80°.

As regards the other two species of Coleoptera tested on coniferous wood, the Buprestidae Phaenops cyanea and the Scolitidae Orthotomicus erosus (which has only a subcortical development), no alive specimens were found in the samples.

The tests performed on trunks of Quercus and Populus showed very high mortality levels in each treatment: the power ranged from 6 to 12 Kw and the exposure time from 60” to 540”.

In particular, as for Buprestydae and Cerambycidae, treatments employing 8 Kw for 360” were sufficient to guarantee a total disinfestation of the material with bark. The same exposure time coupled with a lower power was sufficient to achieve a 100% mortality in the case of Scolytid on Quercus.

Determination of efficacy over a range of critical parameters, where appropriate, such as exposure time, dose, temperature, relative humidity and water content, size and density.

The aforesaid tests showed that mortality was obtained for each species under treatment with some variation in the application of power and exposure times. (See the following graphs)

As regards Bursaphelenchus mucronatus, very rare alive specimens were found using a power of 3 and 4.5 Kw; while all nematodes were killed with a power of 9 Kw and exposure time of 360”.

A power of 6 Kw for 360” was sufficient to reach a total mortality of another Bursaphelenchus species, associated with the oak, that was recorded for the first time in Italy during the collection of materials for the treatments in the reverberation chamber.

All specimens of the Coleoptera Cerambycidae, in particular the noxious Monochamus galloprovincialis, were killed with only 3 Kw for 360”.

In the case of Coleoptera Buprestidae there was an even higher efficacy in treatments of coniferous wood: 100% mortality with a power of 3 Kw for 180’’.

A power of 4.5 Kw for 360” was enough to exert a total control of the material containing Weevil larvae.

For the last group, the conifer and broad-leaves bark beetles, included in the Scolytidae, a 100 % mortality of larvae was reached by applying a power of 6 and 9 Kw, respectively, for 240’’.

For the last group taken into consideration, included among the Scolytidae of the conifers and broad-leaves, a 240’’ exposure at 6 KW and 9 KW of power, respectively, permitted to reach a 100% mortality.

alive larvae no treatment 100%

75% Monochamus galloprovinci 50% alis 3KW Acanthocineu 25% s griseus 4,5 KW 0% Arhopalus 4,5 t° 180" 360" KW exposure time

Tab. 5 – Cerambycidae larvae mortality in logs of maritime pine treated in the microwave reverberation chamber.

alive larvae

100% 75% no treatment

50% treated 3 KW 25%

0% t° 180" exposure time

Tab. 6 - Buprestidae larvae mortality in logs of maritime pine treated in the microwave reverberation chamber.

alive larvae

100%

no treatment 75%

50% treated 4,5 kw

25%

0% t° 180" 360" exposure time

Tab. 7 – Pissodes castaneus DeGeer (Curculionidae) larvae mortality in logs of maritime pine treated in the microwave reverberation chamber.

alive larvae and adults no treatment

100%

75% Scolytus 50% intricatus (oak) 6 KW 25% Orthotomicus 0% erosus (pine) 9 t° 240" KW

exposure time

Tab. 8 – Scolytidae larvae and adults mortality in logs of English oak and maritime pine treated in the microwave reverberation chamber.

alive larvae and adults 100%

75%

50%

25%

0% t° 180" 360" 3KW 88 0,01 0,01

exposure time

Tab. 9 – Bursaphelenchus mucronatus (Parasitaphelenchidae): adults and larvae mortality in logs treated of maritime pine in the microwave reverberation chamber.

alive larvae and adults 20

0 t° 180" 360"

4,5 KW 15 0,1 0

exposure time

Tab. 10 – Bursaphelenchus mucronatus (Parasitaphelenchidae): adults and larvae mortality in logs treated of maritime pine in the microwave reverberation chamber.

alive larvae and adults

0 t° 180" 9 KW 10,6 0 exposure time

Tab. 11 – Bursaphelenchus mucronatus (Parasitaphelenchidae): adults and larvae mortality in logs treated of maritime pine in the microwave reverberation chamber.

alive larvae and adults 20

0 t° 180" 3KW 40,3 0 4,5KW 47,7 0 exposure time

Tab. 12 – Bursaphelenchus leoni (Parasitaphelenchidae): adults and larvae mortality in logs treated of maritime pine in the microwave reverberation chamber.

alive larvae and adults 20

0 t° 240" 9 KW 76,8 0 exposure time

Tab. 13 – Bursaphelenchus sexdentati (Parasitaphelenchidae): adults and larvae mortality in logs treated of maritime pine in the microwave reverberation chamber.

alive larvae and adults 10

0 t° 360" 6 KW 3,8 0 exposure time

Tab. 14 – Bursaphelenchus sp. (Parasitaphelenchidae): adults and larvae mortality in oak logs treated in the microwave reverberation chamber.

Tests carried out on samples with and without bark showed the same efficacy. A higher power (9-13.5 kW) for the same treatment times is sufficient in order to obtain an effective disinfesting treatment of the samples with bark .

Efficacy data using operational conditions Treatments may be submitted for evaluation without going through the processes outlined in section 3.2.1 when there is sufficient efficacy data available from the operational application of the treatment. When a treatment has been developed under laboratory conditions, it should be validated by testing under operational or simulated operational conditions. Results of these tests should confirm that the application of the treatment schedule achieves the stated efficacy under conditions in which the treatment will be used.

Where treatment specifications differ for trials under operational conditions, the test protocol modifications should be indicated. Supporting data may be presented from preliminary tests to refine the treatment schedule to establish the effective dose (e.g. temperature, chemical, irradiation) under operational conditions.

In some cases the method of achieving the effective dose will be different from the method established under laboratory conditions. Data that supports any extrapolation of laboratory results should be provided. The data provided should include detailed information on, but not limited to, the following elements: Pest information Identity of the pest to the appropriate level (e.g. genus, species, strain, biotype, physiological race), life stage, and if laboratory or field strain was used Larvae of insects like Buprestydae, Cerambycidae, Curculionidae, Scolytidae. Larvae and adults of nematodes like Parasitaphelenchidae, Rhabditidae, Panagrolaimidae, Neotylenchidae, Cephalobidae, Aphelencoididae, Monhysteridae, Diplogasteridae, Mononchidae.

Conditions under which the pests are cultured, reared or grown

Biological traits of the pest relevant to the treatment (e.g. viability, genetic variability, weight, developmental time, development stage, fecundity, freedom from disease or parasites)

Method of natural or artificial infestation

Natural infestation

Determination of most resistant species/life stage (in the regulated article where appropriate)

Regulated article information Type of regulated article and intended use

The MW treatment can be applied on all types of wood and wooden packaging material such as: pallets, dunnage, crating, packing blocks, drums, cases, load boats, pallet collars and skids in use in international deliveries.

Material used in the operational application of the treatment Nine standard pallets piled and placed inside the test reverberation chamber. Each pallet weighed 18 kg; its dimensions were 800 x 1200 mm. The total pile weight was 162 kg.

• conditions of the plant or plant product, for example: ◦ whether it was free from non-target pest infestation, non-pest disorder or pesticide residue. ◦ size, shape, weight, stage of maturity, quality etc. ◦ whether infested at a susceptible growth stage.

Each pallet weighed 18 kg; its dimensions were 800 x 1200 mm.

Experimental parameters Level of confidence of operational tests provided by the method of statistical analysis and the data supporting that calculation (e.g. number of subjects treated, number of replicate tests, controls)

Experimental facilities and equipment - Shielded reverberation chamber with microwave generators that can emit a maximum power of 18 kW; - infrared thermo-camera Flir Thermacam SC2000 interfaced with a computer elaborating the temperature trend (for the surface temperature of pallets); - 9-resistance Pt 100 Thermometer, made of magnesium oxide, with a diameter of 2,5 mm: to measure the inner temperature of pallets we drilled a core sampling (3 mm diameter and 4 cm depth) in the central part of the blocks of each pallet; - HT GSC 57 multimeter, equipped with amperometric clamps for the measurement of the electrical consumption; - sensors for the material thermo-hygrometric control before and after treatment; these sensors allow a better calibration of the thermal microwave process and the actual control of the irradiated material thermo-hygrometric conditions.

Experimental design (e.g. randomized complete block design)

Experimental conditions (e.g. temperature, relative humidity, diurnal cycle)

Methodology to measure the effectiveness of the treatment (e.g. whether mortality is the proper parameter, whether the end-point mortality was assessed at the correct time, the mortality or sterility of the treated and control groups) Mortality is the proper parameter to measure the effectiveness of the treatment. To be sure of reaching it, it is important to heat pests over their lethal temperature (56-60°C). For this reason the pallet surface temperature was monitored by means of an infrared thermo-camera, while the inner temperature was measured by means of a 9-resistance Pt 100 Thermometer.

Determination of efficacy over a range of critical parameters, where appropriate, such as exposure time, dose, temperature, relative humidity and water content, size and density. The following table shows that after only 6 minutes of microwaves irradiation at 6 kW the average temperature reached in the pallet is > 60° C. This value is sufficient to assure the mortality of pests whose lethal temperature ranges from 56°C to 60 °C.

Tab. 15 CORE TEMPERATURE (°C) SURFACE TEMPERATURE (°C) Power (kW) Time (sec) P1 P1

6 360 69°C 56°C

The unique characteristic of the heating through microwaves is that heat propagates from the core to the surface of the object. The inner temperature is, for this reason, always higher than the external one. Anyway after some minutes of irradiation the distribution of heat results almost uniform and the disinfestation can be considered effective in every part of the object.

Factors that affect the efficacy of the treatment (e.g. for post-harvest treatments: packaging, packing method, stacking, timing of treatments (pre/post packaging or processing, in transit, on arrival)). The circumstances of the treatment should be stated, for example the efficacy of a treatment may be affected by packaging, and data should be provided to support all the circumstances that are applicable. The stacking of pallets affects negatively the efficacy of the treatment because there is a lesser penetration depth of microwaves in the pallets which are situated at the middle of the pile. For this reason a most effective treatment can be carried out in dynamic systems where pallets are treated singularly and continuously while moving on a conveyor belt, for example. By wrapping the object under treatment with thermal-insulating materials, it is possible to reach the target temperature more rapidly and to maintain it for a longer time.

Monitoring of critical parameters (e.g. exposure time, dose, temperature of regulated article and ambient air, relative humidity). For example: • the number and placement of gas sampling lines (fumigation) • the number and placement of temperature/humidity sensors.

Treatment tests were carried out on nine standard pallets inside a laboratory shielded reverberation chamber with microwave generators that can emit a maximum power of 18 kW. Treatments time and energetic consumptions were evaluated and the following parameters were constantly monitored. • pallet surface temperature: monitored by means of an infrared thermo-camera, Flir Thermacam SC2000, interfaced with a computer elaborating the temperature trend as a graphic; • inner temperature of each pallet: a 9-resistance Pt 100 Thermometer, made of magnesium oxide, with a diameter of 2,5 mm, was used. In order to measure the inner temperature we drilled a core sampling (3 mm diameter and 4 cm depth) in the central part of the blocks of each pallet; • electrical consumption: it was measured by means of an HT GSC 57 multimeter, equipped with amperometric clamps.

Tests were subdivided in four steps, each one lasted 3 minutes and at the end of each step all aforesaid parameters were measured.

Fig. 4: Inner temperature measurement points Fig. 3: Instruments for thermal measurements

All results are indicated in the following tables and charts:

Tab. 16 : Trend of the pallet inner temperatures Time Temperatures P1 P2 P3 P4 P5 P6 P7 P8 P9 0 min 15°C 15°C 15°C 15°C 15°C 15°C 15°C 15°C 15°C 3 min 30°C 25°C 22°C 24°C 25°C 23°C 22°C 21°C 26°C 6 min 55°C 50°C 58°C 46°C 51°C 42°C 41°C 42°C 55°C 9 min 65°C 56°C 62°C 52°C 57°C 48°C 47°C 50°C 61°C 12 min 75°C 63°C 66°C 60°C 61°C 60°C 60°C 62°C 65°C

Fig. 5 Trend of the pallet inner temperatures

The thermographic images in figure 6 represent the surface temperature distribution at the end of the treatment steps. These images emphasize the uniform thermal distribution after 12' of treatment and the MW heating selectivity.

Fig. 6: Thermographic images in 4 treatment steps.

The second purpose of the tests was to estimate the actual energetic consumption associated with the achievement of the aforesaid temperatures.

Tab. 17 : Absorbed energy Treatment duration Absorbed energy 3 min. 3 kWh

6 min. 5,947 kWh

9 min. 8,957 kWh

12 min. 11,96 kWh

In addition, any special procedures that affect the success of the treatment (e.g. to maintain the quality of the regulated article) should be included. It is remarkable that the microwave treatment in shielded reverberation chambers is successfully applicable to pallets notwithstanding their metallic elements, especially nails present in the contact points between tables and blocks. Inside a microwaves reverberation chamber an object is irradiated from all directions, since the electromagnetic field is the sum of infinite plane waves having an omni-directional propagation; this condition derives from the generation of a statistically homogeneous and isotropic electromagnetic field, due to the use of electromagnetic waves agitators (“stirrers”). In the specific case of the electromagnetic field incident on wood, which is a dielectric dissipative material, the field propagates omni directionally in the treatment area creating electric currents in the wooden material that consequently heats up. The electromagnetic field incident on conductive materials (e.g. nails, screws or metallic junctions present inside the wooden material) provokes some superficial currents on the metal which have a high density and are concentrated in the spots where the conductor has a minor section, producing the so-called “point effect”. Moreover, there is density of dissipated power in the dielectric material (wood) near the metallic junction elements due to these currents; such electric dissipation produces, for the Joule effect, a localized overheating, higher than the overall mean temperature of the irradiated object. To confirm that, some thermographic images were taken showing the disinfestation of a pallet in a microwave reverberation chamber treated with 9kW of power for 360 seconds. In the figure below there is a pallet at the end of the treatment with a surface average temperature of 75° C and the indication of the temperatures in each spot (SP01 – SP02 – SP03 – SP04) near the nails in the junction points; this temperature is about 10-20 °C higher than the mean temperature reached into the pallet (LI01 – LI02).

Tab. 16 Thermographic image of a pallet at the end of treatment and indication of the temperatures reached in the metallic elements (e.g. nails) and near them.

Fig. 7: Detail of a pallet at the end of the treatment. The picture puts into evidence a great leak of resin. Although temperatures reach very high levels, they do not produce any breakings and burnings near the metallic elements (nails).

As regards the treatment effects on the commodity, the tests with microwaves carried out on standard pallets of 18 Kg, in laboratory conditions, do not show any alteration; the only change to be remarked is the leak of resin at the end of the treatment that crystallizes very quickly in a few minutes. In order to confirm that we have started a collaboration with CRIL (Centro Ricerche Imballaggi in legno e Logistica – Italian testing agency for wooden packaging materials) to evaluate the actual MW effects on the strength of nailed joints.

Feasibility and applicability Information should be provided, where appropriate, to evaluate if the phytosanitary treatment is feasible and applicable. This includes such items as: Procedure for carrying out the phytosanitary treatment (including ease of use, risks to operators, technical complexity, training required, equipment required, facilities needed). Treatment procedures should adequately describe the method for applying the treatment in a commercial setting.

The microwave treatment is highly versatile: in the static MW treatment chamber it is possible to disinfest pallets, dunnage, crating, packing blocks, cases, load boards, pallet collars but also raw wooden material before its processing. The user-friendliness is another factor contributing to the feasibility of the microwave treatment: the device is completely automated and can be managed through a dedicated software so that the operator has only to supervise the disinfesting process. The risks to the operators and to the environment are significantly inferior compared with the other treatments such as the MB fumigation (chemical risk) and the HT (risk deriving from the presence of fuel) since microwaves are completely confined in the shielded treatment chamber.

Tunnel for MW disinfestations

This configuration, particularly suitable for large industrial pallet production establishments, has been studied for in line treatments. The tunnel can be installed at the end of the pallet production chain and just before the automatic stacking system. The system is equipped with a device for the internal transfer of pallets, already used in the production of pallets. Thanks to this, the production line is not modified, but only extended in the final part.

The tunnel is a modular microwave shielded structure composed of electrically connected shielded elements which assure the high shielding efficacy of the system. Each unit is provided with one or more microwave power generators, a system of electromagnetic waves agitators (stirrers) and aspiration and humidity evacuation systems.

The dimensions in section have been studied in order to optimize the process of electromagnetic reverberation and to allow the treatment of standard pallets. The uniformity of the electromagnetic shield is always guaranteed and the irradiation process can be continuative, avoiding the down periods due to the loading / unloading of the treatment chamber. This system typology allows to adjust time of treatments to time of production (180-360 pallet/h).

If necessary, the tunnel can be multiplied with other parallel treatment lines in case of very fast production lines (more than 360 pallet/h).

The system can be provided with sensors for the material thermo-hygrometric control before and after treatment; these sensors allow a better calibration of the thermal microwave process and the actual control of the irradiated material thermo-hygrometric conditions.

Cost of typical treatment facility and operational running costs if appropriate

The cost of typical treatment facility is about € 400.000,00. This cost is referred to 6-units version of the disinfestation tunnel whose features are:

Tecnical characteristics - Mi.Sy.A. (6-units version) Unit Size LxWxH: 1350x6000x1100 [mm] Total Size LxWxH: 1350x36000x1100 [mm]

Productive capacity: 180 [pallets/h] Total weight: 3000 [kg] Electrical power of 95 [kW] installation: RF Power at 2.45 GHz: 84 [kW]

At the end of the process, the energy absorbed by the system for the treatment is 11,96kWh; assuming that the electric power cost is 11 Cent.€/kWh. (in Italy: cost of electrical energy for medium voltage supply <=500 kW - source ENEL 2006) the treatment for the 9 pallets costs €1,32, in particular the average cost for a single pallet is about € 0,15.

Commercial relevance, including affordability This technology could be considered almost affordable if we consider the remarkable saving of time and resources it allows. Due to the unique characteristics of microwave energy, the transition period between commencing treatment and achieving the target temperature across the profile of the wood can last a few minutes and permits therefore a relatively low consumption of energy.

Extent to which other NPPOs have approved the treatment as a phytosanitary measure

Availability of expertise needed to apply the phytosanitary treatment

The device is completely automated and can be managed through a dedicated software so that the operator has only to supervise the disinfesting process. Anyway a minimum knowledge in the fields of informatics and electronics is needed. Expertise is also needed for the device’s maintenance.

Versatility of the phytosanitary treatment (e.g. application to a wide range of countries, pests and commodities)

The MW treatment allows to disinfest a wide range of wooden materials. The treatment is effective not only against the pests listed in ISPM 15 but can be effective against other pests whose lethal temperature ranges from 55 to 60°C. The system can be applied in all the countries in which the use of microwaves at the frequency of 2.45 GHz is allowed: in other cases is necessary to substitute the microwave generator.

The degree to which the phytosanitary treatment complements other phytosanitary measures (e.g. potential for the treatment to be used as part of a systems approach for one pest or to complement treatments for other pests)

Consideration of potential indirect effectsErrore. Il segnalibro non è definito. (e.g. impacts on the environment, impacts on non- target organisms, human and animal health) The exposure to microwaves has also some secondary effects such as the wood desiccation and the elimination of the so-called “blue phenomenon” in the wood (study in progress). Its efficacy against mouldings and fungi can be related to direct and indirect effects of desiccation. Further interesting considerations that support the use of this technology are the possibility to handle the treated material immediately after the end of the treatment because there are no residues of electromagnetic energy: no electromagnetic pollution is produced in the working area, since the emitted electromagnetic energy is completely confined inside the microwave shielded chamber. Both the environment and operators are completely safeguarded.

Applicability of treatment with respect to specific regulated article/pest combinations

Technical viability Two prototypes of the device have been realized. - The static MW treatment chamber is an ISO STD 20’ container for the disinfestations of pallets, dunnage, crating, packing blocks, cases, load boards, pallet collars but also raw wooden material before its processing. - The tunnel is a modular microwave shielded structure particularly suitable for large industrial pallet production establishments. This configuration, has been studied for in line treatments. The tunnel can be installed at the end of the pallet production chain and just before the automatic stacking system. The standard tunnel is composed of 6 units and is 36m long but its size can be changed with respect to the needs. Each unit has been appropriately projected in order to optimize the reverberation process and to allow the treatment of standard pallets. The device is managed by means of a touch screen on a control panel so that the operator has only to supervise the disinfesting process; the device is completely automated by means of a programmed logic (PLC) and its remote operation is possible thanks to a dedicated software.

Phytotoxicity and other effects on the quality of regulated articles There are no residues of electromagnetic energy in the treated material immediately after the end of the treatment because the irradiated energy is absorbed by the dielectric material (wood) and converted into heat. The wood’s quality of packaging materials is preserved even at very high temperatures (85°C) and despite the presence of metallic elements (nails, screws etc.). In order to confirm this last point, we have started a collaboration with CRIL (Centro Ricerche Imballaggi in legno e Logistica – Italian testing agency for wooden packaging materials).

Consideration of the risk of the target organism having or developing resistance to the treatment It is improbable that pests develop any resistance to the treatment because its principle is based on the mortality provoked by direct and indirect effects that microwaves have on living cells.

Supporting Information and References

Copies of all relevant supporting information and references should be supplied with the submission, preferably in PDF format for ease of subsequent distribution.

Evaluation of Submitted Treatments Submissions will be considered by the TPPT only when the information outlined in section 3 is fully addressed. The information provided will be evaluated against the requirements in section 3.

Due respect for confidentiality will be exercised when the confidential nature of information is indicated. In such cases, the confidential information within the submission should be clearly identified. Where confidential information is essential for the adoption of the treatment, the submitter may be requested to release the information. If the release of the information is not granted, the adoption of the treatment may be affected.

Treatments will be adopted only for the regulated articles and target species for which they were tested and for the conditions under which they were tested, unless data is presented to support extrapolation (e.g. to apply the treatment to a range of pest species or regulated articles).

If the submission fails to meet the requirements outlined in section 3, the reason(s) will be communicated to the contact identified on the submission. There may be a recommendation to provide additional information or to initiate further work (e.g. research, field testing, analysis).

Send submissions to: E-mail: [email protected] Fax: (+39) 06 5705 6347 Mail: IPPC Secretariat AGPP) Food and Agriculture Organization of the UN Viale delle Terme di Caracalla 00100 Rome, Italy