IPPC - International Plant Protection Convention



PMRG RESEARCH GUIDELINE:GUIDELINES FOR THE DEVELOPMENT OF FUMIGATION TREATMENTS (Format discussed at 2015 PTTEG meeting)Contents: TOC \o "1-3" \h \z \u INTRODUCTION PAGEREF _Toc486521236 \h 3Scope PAGEREF _Toc486521237 \h 3References PAGEREF _Toc486521238 \h 3Definitions PAGEREF _Toc486521239 \h 3Background PAGEREF _Toc486521240 \h 3Outline of Requirements PAGEREF _Toc486521241 \h 4Part I: GENERAL INFORMATION PAGEREF _Toc486521242 \h 41. Organization and researchers PAGEREF _Toc486521243 \h 42. Test insects PAGEREF _Toc486521244 \h 43. Test host PAGEREF _Toc486521245 \h 54. Fumigation enclosures PAGEREF _Toc486521246 \h 65. Analysis of fumigation parameters PAGEREF _Toc486521247 \h 65.1. Calibration of temperature analyzer PAGEREF _Toc486521248 \h 65.2. Measurement of temperature during treatment PAGEREF _Toc486521249 \h 75.3. Calibration of pressure analyzer PAGEREF _Toc486521248 \h 65.4. Measurement of pressure during treatment PAGEREF _Toc486521249 \h 75.5 Calibration of fumigant analyzer5.6 Measurement of fumigant concentrations during treatment5.7 Measurement of fumigation duration.Part II: GUIDELINES FOR TEST PROCEDURES PAGEREF _Toc486521250 \h 86. Host infestation and pest development PAGEREF _Toc486521251 \h 86.1. Purpose PAGEREF _Toc486521252 \h 86.2. Methods PAGEREF _Toc486521253 \h 86.3. Reporting PAGEREF _Toc486521254 \h 97. Most fumigant-tolerant life stage PAGEREF _Toc486521255 \h 107.1. Purpose PAGEREF _Toc486521256 \h 107.2. Methods PAGEREF _Toc486521257 \h 117.3. Reporting PAGEREF _Toc486521258 \h 127.4. Others PAGEREF _Toc486521259 \h 138. Exploratory testing PAGEREF _Toc486521260 \h 148.1. Purpose PAGEREF _Toc486521261 \h 148.2. Methods PAGEREF _Toc486521262 \h 148.3. Reporting PAGEREF _Toc486521263 \h 159. Cofirmatory testing PAGEREF _Toc486521264 \h 169.1. Purpose PAGEREF _Toc486521265 \h 169.2. Methods PAGEREF _Toc486521266 \h 179.3. Reporting PAGEREF _Toc486521267 \h 1810. Optional testing 10.1 Commercial-scale validation 10.2 Residues 10.3 Quality evaluationsINTRODUCTIONScopeThis research guideline provides technical procedures for the development of cold disinfestation treatments against quarantine fruit fly species in host commodities.ReferencesIPPC. 1997. International Plant Protection Convention. Rome, IPPC, FAO.ISPM 5. Glossary of phytosanitary terms. Rome, IPPC, FAO. ISPM 18. 2003. Guidelines for the use of irradiation as a phytosanitary measure. Rome, IPPC, FAO.ISPM 28. 2007. Phytosanitary treatments for regulated pests. Rome, IPPC, FAO.APPPC RSPM No.1. 2004. Guidelines for the development of heat disinfestation treatments of fruit fly host commodities. Bangkok, APPPC, FAOJapan MAFF. Guideline for experiment of development on quarantine cold treatment. 2014.Japan MAFF. Minimum requirement for the report of cold treatment. 2014.ECCT report 2013. Report: Expert Consultation on Cold Treatments December, 2013 in Buenos Aires, Argentina 2-6 December, 2013. IPPC, FAOASTM E1847-96(2013). Standard Practice for statistical Analysis of Toxicity Tests Conducted Under ASTM Guidelines, ASTM International, West Conshohocken, PA, 2013, ASTM E563-11(201?).USDA APHIS. 2014. Guidelines for Fumigation Research to Control Surface-Feeding Insects and Mites. V 1.0WHO. 2014. Calibration of temperature control and monitoring devices. Technical supplement to WHO Technical Report Series, No.961, 2011. Annex 9: Model guidance for the storage and transport of time and temperature-sensitive pharmaceutical products. Bond, E. J. 1984. Manual of fumigation for insect control: Chapter 2; Principles of fumigation. FAO Plant Production and Protection Paper No. 54. Food and Agriculture Organization on the United Nations. Rome. Finney, D.J., 1971. Probit Analysis, third ed. Cambridge University Press, Cambridge.Couey, M.C., Chew, V., 1986. Confidence limits and sample size in quarantine research. J. Econ. Entomol. 79(4), 887-890.DefinitionsDefinitions of phytosanitary terms used in the present standard can be found in ISPM 5 (Glossary of phytosanitary terms), ISPM 18 (Guidelines for the use of irradiation as a phytosanitary measure), ISPM 28 (Phytosanitary treatments for regulated pests) and APPPC RSPM No.1. (Guidelines for the development of heat disinfestation treatments of fruit fly host commodities). ASTMBackgroundPhytosanitary treatments are annexed to ISPM 28 after adoption by the CPM, however, the TPPT has faced challenges during the evaluation of treatment submissions because many of them do not address all requirements in Section 3 of ISPM 28. To help the TPPT assess these submissions on cold treatment, IPPC organized the Expert Consultation on Cold Treatments (ECCT) meeting (Dec 2-6, 2013) and urged the participants to discuss the best experimental design and methods to determine efficacy as a common approach around the world. In ECCT meeting, the group agreed to compile a research guideline for development of cold disinfestation treatments. This approach was expanded to include other types of phytosanitary treatments at the Phytosanitary Measures Research Group meeting (Aug 17-20, 2015). Herein, is a description of fumigation guidelines to aid the development of expertise and technical cooperation between the contracting parties.Outline of Requirements The development of a phytosanitary fumigation treatment involves a number of distinct steps. For the TPPT to evaluate a submission and the CPM to adopt draft annexes to ISPM 28 detailed information on experimental facilities and equipment, target pest, regulated article, and methodology should be available. The following steps are provided as a general guideline for the adoption of a fumigation phytosanitary treatment.Determination of the most fumigant-tolerant life stage(s) of the target species that is relavent to the marketing of the host.Indentification of the fumigation parametrics (dose, time, pressure, temperature) that provide a specified efficacy toward the most fumigant-tolerant life stage(s) of the target species that is relavent to the marketing of the host (exploratory fumigations). Demonstration of the fumigation treatment at a specified efficacy (confirmatory fumigations).Presention of an operationl fumigation scheduleCommercial-scale validation of the operational fumigation.Evaluation of marketing effects (fruit quality) and regulatory compliance (e.g., Maximum Residue Levels-MRL, approved technologies) of the fumigation treatment. –why is quality research required, this is market/consumer driven. Niether FAO or Researchers control marketing and consumer choice. General information (researcher, pest, regulated article-host, treatment facility, etc.) should be described as in Part I. In Part II, additional details are described (examples are provided).Part I: GENERAL INFORMATION1. Organization and researchersInformation on the laboratory, organization and researchers involved in producing the data should be provided in a document (refer to ISPM 28-3.1 Summary information).< Documentation >Location of the laboratorLaboratory affiliation(s)Name and contact person (4) Experiment year2. Test organism (pest)The pest species should be reliably identified, preferably by a suitable expert, and voucher specimens should be archived.Field-captured pests used in testing, which may or may not infest the host at the time of sourcing, should be established with an appropriate founder population and a description of potential species, parasites, diseases, and agrochemicals with potential to confound treatment efficacy.If a laboratory colony of the pest is established, it should be with an appropriate founder population, preferably collected from a large quantity of naturally-infested/infected host from different locations (field sites, regions). The founder population should be of a sufficient size, which may vary across pest species, and should be agreed upon by contracting parties. (e.g. 100 to 1 000 individuals).The laboratory colony should be regularly replenished with new field-captured specimens or by replacement with new founder populations to maintain genetic diversity, reflective of field populations.The health of the pests, either field-collected or from the colony, should be regularly checked by monitoring fecundity, developmental time and developmental success (Life History Table-LHT) for applicable life stages.< Documentation >(1) Scientific name of pest (insect, arachnid, microbe)(2) Origin of the colony - Location, date of collection of host (name and amount). (3) Rearing method - Temperature, humidity and lighting used to rear. - rearing media- Life History Table relevant to rearing & treatment conditions3. Hosts Each host, or subject article, should be identified and agreed upon by the contracting parties. If plant or animal, identification should be to species, and potentially below species level in certain cases. Hosts used in fumigation testing should be mostly free from non-target infestation/infection, pesticides, adjuvants, and any major non-pest disorder. Scientific justification must be provided, and agreed upon by the contracting parties, to allow for the “extrapolation/interpolation” of efficacy results to a host not included in the testing. < Documentation >(1) Scientific (Botanical) name and variety or cultivar of host- Physical Characteristics - relevant to fumigation (e.g., volume, surface area, maturity)- Morphological Characteristics, relevant to identification (e.g., color, seasonality, photographs) (2) Origin- Location and date host was sourced (relative to commercial harvest)- Maturity at sourcing (relative to commercial harvest) and infestation/infection (natural, simulated, or inoculated (vide infra)) supported with quantifiable metrics (as firmness, sugar, acidity, colour, starch index.etc)(3) Storage conditions from sourcing/harvest thorough treatment evaluation - Temperature, humidity and duration, etc.4. Fumigation enclosuresInformation on fumigation enclosure (chambers, tarpaulins, controlled-atmosphere (CA) rooms, pre-cooling rooms, etc.) used for the exploratory as well as confirmatory testing should be detailed.< Documentation >(1) Address, management, accreditations of facility where the enclosure are located(2) Manufacturer, specifications, and dimensions of the enclosure (and facility/room housing the facility if relevant) (3) Listing of enclosure certifications, when relevant, with date of most recent certification (eg pressure test, gas-tightness test)(4) List, in chronological order, key procedural aspects of operating each enclosure as well as application and ventilation processes. (3) Description of analytical instrumentation to measure temperature, pressure, and fumigant concentration (type, amount, resolution, intervals) as well as respective recording devices for each fumigation enclosure (and/or facility).(4) Details for loading target pest and host into fumigation enclosure as well as concomitant non-treated controls (vide infra), including a relation to commercial practice. See Bond (1984) for calculation of the load factor.(5) Rationale for relating data across: enclosure types (e.g., chamber versus tarpaulin), enclosure sizes (e.g., laboratory- versus commercial-scale chamber), load types, load amounts, or combinations thereof.5. Analysis of fumigation parameters5.1. Calibration of temperature analyzerTemperature sensors and recording devices should be calibrated by ice-water immersion method immediately prior to the start of confirmatory testing. Sensor (accuracy/precision within +/- 0.3°C) should be used (for more details refer e.g. ASTM E563-11).An example of calibration data is below: Example 1. Calibration of sensors (Ice water immersion) March 29, 2017 ──────────────────────────────────────────────　 Readings* Calibration Sensor no. ─────────────────────────── factor** Rep. 1 Rep. 2 Rep. 3 ────────────────────────────────────────────── 1 0.1 0.1 0.1 - 0.1 2 0 0 0 0 3 - 0.1 - 0.1 - 0.1 0.1 4　 - 0.1　 - 0.1 0 0.1 5 - 0.1 0 0 0----- ──　 ──　 ──　 ─ ── ────────────────────────────────────────────── *Readings: at least 3 times at 3-5 minutes intervals after the stability of readings.** Calibration factor = (True temperature: 0 °C) - (Reading)5.2. Measurement of temperature during treatmentRelevant temperatures associated with the host, target pest, or the enclosure should be measured at temporal intervals (e.g., every 15 minutes), using temperature calibrated sensors and recording devices, sufficient to formulate equilibrium and/or kinetic descriptors of temperature at pertinent locations over the course of treatment. More rigor in measurement is favorable during confirmatory studies, relative to exploratory studies. Less rigor in measurement and recording of temperature should be allowed during temperature-controlled storage events, agreed upon by the contracting parties to be independent of the said fumigation treatment.5.2.1. Host temperatureSensors to monitor internal temperature of a host are required if the pest develops internally, within the host, or externally, with proximity to the surface of the host. Non-infested/infected hosts should be distributed within the structure, in analogous fashion to the distribution of the infested/infected hosts. At least 10 sensors should be used for each confirmatory test. Sensor distribution should be relevant to the positioning of the host/pest in the enclosure, as well as in non-infested host undergoing treatment a and infested but non-treated hosts.5.2.2. Air temperatureAir temperature in enclosure headspace should be measured, with preference to air delivery and return positions when applicable. The temperature of the air surrounding the fumigation enclosure should also be measured when applicable.5.2.3. Recording of temperature dataNumerical data of the temperature (calibrated) should be tabulated with time. Authentic copies of (analogue and/or digital) data recordings should be available for review. The beginning and commencement of treatment point should be identified on temperature record/data. Temperature records for each replication of the confirmatory tests are critical to establishing a treatment schedule.< Documentation >Description of temperature measurement device as well as calbatraion procedure and methodology.- Procedures and pictures, for example ice-water immersion etc., and results of calibration.(2) Measurement of temperature during treatment and statistical description (number of sensors required depending on the type of trial, localization, frequency of recording, average temperature)- Temperature record for all tests (3) Rationale for temperature variation, when applicable.(4) Rationale for relating temperature data, and ultimately efficacy, across: enclosure types (e.g., chamber versus tarpaulin), enclosure sizes (e.g., laboratory- versus commercial-scale chamber), load types, load amounts, or combinations thereof.An example tabulation of temperature measurements is below: Example 2. Chamber air and fruit core temperatures during the treatment at 2.0±0.5°C* All temperature data are corrected by calibration factor.──────────────────────────────────────────────────────── Air temperature Fruit temperature ───────────　 ───────────────── Date Time Sensor no. Sensor no. Remarks 1 2 3 13 14 (Supply) (Return) (Top) (Mid) (Bottom) ────────────────────────────────────────────────────────29-3-2016 09:00AM 15.3 18.2 22.5 ── 22.3 22.5 ← loading infested/non- 29-3-2016 10:00AM 6.3 6.5 20.0 ── 20.8 20.3 infested fruits (=precooling 29-3-2016 11:00AM 5.6 5.9 19.5 ── 19.9 19.0 start) ──　 ──　 ────── ─── ─── ───　 30-3-2016 07:00PM 0.9 1.4 2.2 ── 2.4 2.2 30-3-2016 08:00PM 1.0 1.4 2.1 ── 2.2 2.1 30-3-2016 09:00PM 1.0 1.3 2.0 ── 2.1 2.0 ← Start of treatment at e.g. 2.0°C30-3-2016 10:00PM 1.1 1.5 2.0 ── 2.0 2.0 30-3-2016 11:00PM 1.2 1.4 1.9 ── 1.9 2.0 ─── ─── ───── ─── ─── ──── 17-4-2016 07:00PM 1.1 1.6 2.0 ── 2.0 1.9 17-4-2016 08:00PM 1.1 1.5 1.9 ── 2.0 1.8 17-4-2016 09:00PM 1.1 1.4 1.9 ── 2.0 1.8 ← End of e.g. 2.0°C for 18 daysand unloading infested fruits.─────────────────────────────────────────────────────────5.3. Calibration of pressure analyzerFor fumigation treatments requiring other than ambient atmospheric conditions (ca. 1 atm, 101325 Pa), such as a vacuum, pressure sensors and recording devices must be calibrated using procedures and instruments with ISO 3567 accreditation prior to the start of confirmatory testingCalibration data can be recorded as above for temperature (see Example 1).5.4. Measurement of pressure during treatmentPressure within the fumigation enclosure should be measured at temporal intervals (e.g. every 15 minutes), using calibrated devices, sufficient to formulate equilibrium and/or kinetic descriptors of pressure over the course of treatment. Over the course of fumigation treatment, the target value of pressure shall be within a certain range agreed between contracting parties. If no range has been specified, the realized pressure value shall be within ± 5 % (RSD) of the agreed upon initial pressure.5.4.1 Recording of pressure dataNumerical data of the pressure (calibrated) should be tabulated with time. Authentic copies of (analogue and/or digital) data recordings should be available for review. The beginning and commencement of treatment point should be identified on pressure record/data. Pressure records for each replication of the confirmatory tests are critical to establishing a treatment schedule.< Documentation >Description of pressure measurement device as well as calbatraion procedure and methodology.- Procedures and pictures and results of calibration.(2) Measurement of pressure during treatment and statistical description (frequency of recording, average)- pressure record for all tests (3) Rationale for pressure variation, when applicable.(4) Rationale for relating pressure data, and ultimately efficacy, across: enclosure types (e.g., chamber versus tarpaulin), enclosure sizes (e.g., laboratory- versus commercial-scale chamber), load types, load amounts, or combinations thereof.An example tabulation of pressure and fumigant concentration measurements is below: Example 3. On-site confirmatory test (Stockton, CA, January 22, 1997), codling moth diapausing larvae versus methyl bromide in inshell walnuts (vacuum quarantine treatment). Showing methyl bromide concentrations during fumigation, CT products, temperatures and pressures during the 4-h fumigation.5.5. Calibration of fumigant analyzerFumigant detectors, sensors and recording devices should be calibrated immediately prior to the start of a confirmatory trial. Calibration is encouraged before the start of exploratory testing. Efficacy data derived from the application of a nominal dosage, which is an approximation as opposed to the measured value, should be avoided. At minimum, a 5-point concentration-detector response should be used for calibration (to determine fumigant concentration) and the analysis of the response curve should result in a correlation coefficient, r2, ≥ 0.95. Calibration standards should made by diluting known volumes of fumigant, or alternatively authentic standards, into volumetric gas vessels. Replicate concentration-detector responses of the same calibration series should not yield a RSD’s > 5%. An example calibration between fumigant amount and detector response is below: Example 4. The response of the flame-ionization detector to serial dilutions of methyl bromide at various concentrations following gas-sampling injection onto a gas chromatograph with flame ionization detection, preceding trial X of trial type Y on dated z.5.6. Measurement of fumigant concentrations during treatmentFumigant concentration in the enclosure should be measured at temporal intervals (e.g., every 15 minutes), using detectors/sensors and recording devices, sufficient to formulate equilibrium and/or kinetic descriptors of concentration over the course of treatment. More rigor in measurement is favorable during confirmatory studies, relative to exploratory studies. 5.6.1. Recording concentration dataRecord of fumigant concentration should be tabulated temporally, over the duration of the fumigation treatment. Authentic copies of (analogue and/or digital) data recordings should be available upon request from the contracting parties. The beginning and commencement of treatment should be identified on the fumigant concentration record/data. Records of gas concentration for each replication of the commercial-scale confirmatory tests are critical to establishing a treatment schedule. For each temporal interval, at least 4 points of gas measurement should preferably be used for a confirmatory treatment trial. When not monitored continuously over the course of fumigation, duplicate measurements of each point and temporal interval should be attempted. Distribution of sampling locatuions should be relevant to the positioning of the host/target species in the enclosure.< Documentation >(1) Description of fumigant source, standards, measurement device, calibration procedure and methodology.- Procedures and pictures.(2) Measurement of fumigant during treatment and statistical description of measurement. Number of measurements required depending on the type of trial, localization, frequency of recording, average concentrations.- Temperature record for all tests (3) Rationale for concentration variation, when applicable.(4) Rationale for relating concentration data, and ultimately efficacy, across: enclosure types (e.g., chamber versus tarpaulin), enclosure sizes (e.g., laboratory- versus commercial-scale chamber), or combinations thereof.An example tabulation of fumigant concentration and pressure measurements was provided in Example 3. guidelines FOR TEST PROCEDURES6. Host infestation and pest development6.1. PurposeHosts should be infested/infected with a known age of specimens to maximize the relative amount of the most-fumigant tolerant life stage present during confirmatory testing. It is critical to quantify the how many specimens of the most-fumigant tolerant life stage are subjected to the treatment, as well as how many of the specimens of the most-fumigant tolerant life stage survive the treatment, particularly during studies where survivors are required, or at least more likely. For each host, a life history table (LHT) of the pest should be generated, or accessed from published literature, to demonstrate the chronology of development with and without the proposed treatment, as it relates to quantifying treatment efficacy. Note the importance of temperature on development in a given host. As such, the temperature regime (e.g., 27 1°C) LHT should be equivalent to the temperature regime used for pest development, before and after treatment, during confirmatory tests. 6.2. MethodsIn addition to using naturally-infested hosts, there are two main methods for obtaining infested fruit; artificial inoculation and simulated natural infestation. Every effort should be made to simulate natural conditions as far as possible. For natural infestation/infection, infested/infected hosts are collected from the field and used in testing, with a subset of the collected host retained as non-treated controls to quantitatively estimate the number of specimens receiving treatment that are of the most fumigant-tolerant life stage.For artificial inoculation, specimens of a finite timespan in development, typically inclusive of the most fumigant-tolerant life stage, are placed into/onto the host. In certain cases, the most fumigant-tolerant life stage can be placed into/onto the host.For simulated natural infestation/infection, a host is exposed to the reproductive life stage of the pest (e.g., 2 000 adults/host) for a defined period (e.g., 1-2 hours) to establish infestation/infection of a finite timespan in development, synchronized to maximize presence of the most fumigant-tolerant life stage in the host. With all infestation scenarios, host quality and pest density per host should be considered to favor pest development and survival. Ideally, the host(s) used in developmental and efficacy trails should represent the host(s) in the marketing channel. 6.3. Reporting< Documentation >Description of host, pest rearing, pest development (i.e., LHT) during infestation and incubation,.- Procedures and pictures (2)Provide detailed information of type of infestation, number of pest per host, number of infested and non-infested hosts per replicate trail, number of replicate trials, condition during infestation (duration, temperature, RH), storage conditions of infested hosts- post treatment (temperature, humidity and duration, etc.), characteristics used to identify the different ages and life stages of the pest, etc.). (3) Data on development rates within or across replicates should be tabulated.An example tabulation of pest development is below:Example 5. A life history table for Rhagoletis medax infesting blueberries at ( 27 1°C) showing heterogeneity in the chronology of development from an 24-h oviposition period through pupation. Since pre-treatment infestation and post-treatment incubation was under these conditions, one can potentially determine the quantity of the most-fumigant tolerant life stage subjected to a treatment, as well as the probable life stage of a specimen that survives the treatment.Example 6. On five separate occasions, the probability of each life stage being present in a 48- to 96-h old cohort of SWD infesting sweet cherry was evaluated just before a confirmatory fumigation by dissection ( ± s; egg, 0.011 ± 0.006; 1st, 0.058 ± 0.018; 2nd, 0.226 ± 0.036; 3rd, 0.651 ± 0.035, the most fumigant-tolerant life stage; pupa, 0.051 ± 0.015). 7. Most fumigant-tolerant life stage7.1. PurposeThe most fumigant-tolerant life stage of the pest should be established by comparing the relative susceptibility of the life stages applicable to marketing the host. Thereafter, the most fumigant-tolerant, marketing-relevant, life stage will be used: to identify efficacious treatment parameters during exploratory testing, during the confirmation of exploratory results, and during commercial-scale validation (when appropriate).7.2. Methods7.2.1 Developmental stage of the pest A life history table (LHT) of the pest should be generated, specific to the host for which the treatment is being conducted, to demonstrate the chronology of development. The LHT is developed so that predictive models exist to quantitatively estimate the life stage distribution of specimens entering treatment versus surviving the treatment. 7.2.2 Preparation of infested/infected hostsWhen more than a single life stage is applicable to a marketing channel, hosts should be infested/infected using the same method (artificial inoculation, natural infestation, etc.) to achieve a representative age group/life stage of the pest for testing. It is not uncommon for life stages of a pest to localize in different areas of the host, and this life stage-specific localization should be reflected. Effort should be made to concomitantly fumigate all age groupings/life stages, to minimize the variability in fumigant concentrations that can potentially occur during the comparative evaluation of susceptibility.7.2.3 Fumigation parametersFumigation parameters should be selected to be compliant with the regulatory requirements of the respective parties, whenever possible. The lowest temperature with commercial/operational potential should be selected to determine the most fumigant-tolerant age group/life stage. Testing should be replicated for each proposed pressure. The applied dose of fumigant, as well as the duration of the fumigation, should be selected to result in survivorship of specimens, or at least specimens of the most fumigant-tolerant life stage. The relationship between applied dose (C ) and duration (t), as related to toxicological efficacy, can vary for each life stage. Therefore, when establishing the most fumigant-tolerant life stage, it is critical to align the selection of the applied dose and duration with those ultimately proposed for commercial/operational utilization. Across trials, effort should be made to record fumigant concentrations as described above and have a consistent load factor across all trials (or justification for inconsistency).7.2.4 Mortality evaluation and efficacy determinationThe duration between treatment and the evaluation of treatment efficacy may vary for each life stage, as identified in the LHT, as well as by the mode and mechanism of fumigant action. With respect to diagnosing survivorship/mortality, a variety of approaches may need to be considered, depending on the host-pest complex, the most-fumigant tolerant life stage, the fumigant, marketing considerations (only one stage present), or regulatory considerations (when does an inspection occur, action triggered). It is recommended to refer to technical and regulatory precedence. Techniques/methodology should be agreed upon by the respective parties. Mathematical treatment of the number of treated pests, as well as the control mortality of pests – if applicable- must be provided. There are a variety of ways one can determine the most fumigant-tolerant life stage: directly through comparing the relative survival of life stages subject (concomitantly) to a fumigation, indirectly via survivorship-when development is predictable (see Example 7), or via comparing responses of life stages to treatment gradients (see Example 8 & 9). One of the most popular approaches, because it can be used to establish Most fumigant-tolerant life stage as well as the fumigation parameters required for it control, is the use of probit analysis- or by some other adequate statistical method of analysis- allowing for the calculation of the parameters (e.g., dose, duration, exposure) required for a particular level of control in a given population (e.g., 50, 95, 99, 99.9968%).7.3. ReportingTest results are recorded in a table such as follows. (Sample: Table 4). (1) Provide detailed information of methodology including figures and photographs (2) Provide detailed information of type of infestation, number of pest per host, number of infested and non-infested hosts per replicate trail, number of replicate trials, condition during infestation (duration, temperature, RH), storage conditions of infested hosts, post treatment (temperature, humidity and duration, etc.)(3)Mathematical treatment of the number of treated pests, as well as the control mortality of pests must be provided- when relevant.(4)The most-fumigant tolerant life stage(s) should be identified with statistical justification.Examples for determining the most fumigant-tolerant life stage is provided below:Example 7. A series of exploratory treatments were conducted to determine the relative susceptibility of 0- to 48- (Probability of each life stage (PLS) where LS = eggs, 1st instar, 2nd instar, 3rd instar, pupae; 0.57, 0.35, 0.08, 0.0; panel A), 24- to 72- (0.15, 0.39, 0.39, 0.07, 0.0; panel B), 96- to 144- (0.0, 0.0, 0.12, 0.84, 0.04; panel C), or 192- to 240-h old spotted wing drosophila (SWD) specimens (0.0, 0.0, 0.03, 0.30, 0.67; panel D), toward 1-h fumigation with a steady-state concentration of ~ 14 g m-3 (10,000 ppmv) ozone ([O3]ss) and ambient atmospheric CO2 levels ([CO2]ss = ca. 0.5 gm-3 = 400 ppmv (?LL-1)) at ca. 3°C (37.4°F) and 67.5 kPa. Third instar larvae, which was the most abundant life stage in 96- to 144-h old specimens, were identified as the most fumigation-tolerant life stage based on “indirect methods” described in Walse et al. (2012). Results clearly indicate that within the 96- to 144-h old grouping, adult emergence was synchronized at the temporal interval associated with 3rd instar larvae being present at the time of fumigation. For comparison, note the relatively lower, both total and proportional, emergence of the most prevalent life stage in each of the other age groupings (dashed box).Example 8. Probit analysis of the Ct exposure-mortality response for spotted wing drosophila (SWD) age groups infesting sweet cherries following exploratory fumigations with methyl bromide (MB) for 2 h at treatment temperature of 8.3 or 15.6 (± 0.5) ?C (). Example 9. Lethal exposure ratios (LERs) were calculated with 95% confidence intervals (CI) and used to identify difference in methyl bromide (MB)-tolerance across ages. LERs calculated for 48- to 96-h old SWD relative to 0- to 48-h and 0- to 24-h old specimens across exposures at pulp temperature of 8.3 ± 0.5 C paralleled a ratio of 1 and superseded 1, respectively. Results suggest that larger larvae (3rd >>>2nd instar), which were often observed to feed completely submerged within cherries containing 48- to 96-h old specimens, are more MB-tolerant than the younger larvae (1st >>>2nd instar) and eggs more frequently observed near the fruit periphery and chamber headspace. 7.4. OthersWhen a single treatment is proposed for multiple hosts or cultivars of the same host, after the most fumigant-tolerant stage has been determined, the scenario exhibiting the least mortality should be used in subsequent exploratory and confirmatory testing.Documentation (1)Provide detailed information of methodology including figures and photographsMathematical treatment of the number of treated pests, as well as the control mortality of pests must be provided- when relevant.The most-fumigant tolerant life stage(s) should be identified with statistical justification.8. Exploratory testing 8.1. PurposeA series of exploratory tests, typically at the laboratory-scale, should be conducted to determine the fumigation parameters required to control the most fumigant-tolerant life stage(s) of the pest in the host, during subsequent confirmatory trails, at level of efficacy that is agreed upon by the respective parties. These tests can be, but are not necessarily, coupled to the identification of the most fumigation-tolerant life stage, as discussed above.8.2. Methods8.2.1 Developmental stage of pest The most fumigant-tolerant life stage should be used.8.2.2 Infestation/infection of hostInfestation/infection should be that used to maximize the treatment of the most fumigant-tolerant life stage in the marketing channel.8.2.3 Fumigation parameters As was described above in Section 7, it is critical to align the selection of the applied dose and duration with those ultimately proposed for commercial/operational utilization. Include a minimum of five dose (or duration) scenarios, each of which should be replicated, that yield < 100% mortality, as well as scenarios expected to cause 100% mortality, along with corresponding non-treated control in each replicate. The analytical devices used to monitor the fumigation parameters should be clearly described. With respect to fumigant concertation measurement, refer to 5.6.1. A single point of measurement in the enclosure headspace, preferably analyzed in duplicate at each temporal interval, is typically adequate for exploratory treatment tests. 8.2.4 Mortality evaluation and efficacy determinationRefer to section 7, Examples 10 & 11.8.3. Reporting< Documentation >Provide detailed information of type of infestation, number of pest per host, number of infested and non-infested hosts per replicate trail, number of replicate trials, condition during infestation (duration, temperature, RH), storage conditions of infested hosts, post treatment (temperature, humidity and duration, etc.Mathematical treatment of the number of treated pests, as well as the control mortality of pests must be provided- when relevant. The most-fumigant tolerant life stage(s) should be targeted.Indentification of the fumigation parametrics (dose, time, pressure, temperature) that provide a specified efficacy toward the most fumigant-tolerant life stage(s) of the target species that is relavent to the marketing of the host (exploratory fumigations). See Example 8 for a representative data series that was used identify the fumigation parametrics (dose, time, pressure, temperature) resulting in pest control.9. Confirmatory testing 9.1. Purpose A single, or series of, commercial-scale tests are conducted to confirm the fumigation parameters proposed to result in operational control of most fumigant-tolerant life stage of the pest in the host at level of efficacy that is agreed upon by the respective parties. 9.2. Methods9.2.1 Developmental stage of pest The most fumigant-tolerant life stage(s) should be used.9.2.2 Infestation/infection of hostInfestation/infection should be that used to maximize the treatment of the most fumigant-tolerant life stage in the marketing channel.9.2.3 Fumigation parameters As was described above in section 7, parameters are selected for commercial/operational utilization. The analytical devices used to monitor the fumigation parameters should be clearly described. With respect to gas (fumigant) concertation measurement, refer to 5.6.1. Four points of measurement, one in the structure headspace and three within the load, should be analyzed in duplicate at each temporal interval.9.2.4 Mortality evaluation and efficacy determinationRefer to section 7, Examples 10 & 11.9.3. Reporting< Documentation >Provide detailed information of type of infestation, number of pest per host, number of infested and non-infested hosts per replicate trail, number of replicate trials, condition during infestation (duration, temperature, RH), storage conditions of infested hosts, post treatment (temperature, humidity and duration, etc.Mathematical treatment of the number of treated pests, as well as the control mortality of pests must be provided- when relevant. The most-fumigant tolerant life stage(s) should be targeted.Demonstration of the fumigation treatment at a specified efficacy (confirmatory fumigations).Presention of an operationl fumigation scheduleExample 10. Efficacy data related to 2-h methyl bromide fumigations at pulp temperatures (T) of 8.3 to 17.2 (± 0.5) ?C and exposures (Ct) ranging from 84.4 to 123.4 mgL-1h resulted in a grand sum of 2 survivors from 223,009 ± 4,580 treated ()(probit 9.62). Mean values of k SPT (m h-1) at each, or across, treatment temperature not connected by the same letter are significantly different (Tukey-Kramer HSD). Note that the estimated treated population was estimated based on the emergence of adults from infested, but non-treated control hosts.Example 11. Efficacy analysis and parametrics associated with confirmatory fumigations of adult bean thrips (BT), Caliothrips fasciatus, (Pergande) infesting navels of sweet oranges, Citrus Sinensis (L.) having pulp temperature (), T, and applied doses of ca. 0.5 gm-3 (300 ppmv) delivered by three formulations of cylinderized phosphine (1.6% (v/v) balanced in nitrogen- Scheme 1, VAPORPH3OS - Scheme 2, and ECOFUME - Scheme 3) with respective carbon dioxide levels, [CO2] (ppmv = ?LL-1), as well as headspace concentrations of phosphine, [PH3] (ppmv = ?LL-1). Note that the estimated treated population was estimated based on correction for control mortality.10. Optional testing 10.1 Commercial-scale validationContracting parties, typically at the request of the importer, may agree to a commercial-scale confirmatory test of the proposed phytosanitary treatment, often supervised. 10.2 ResiduesContracting parties, typically at the request of the importer, may agree to provide residue data associated with the proposed phytosanitary treatment. 10.3 Quality evaluationsContracting parties, typically at the request of the importer, may agree to provide quality data associated with the proposed phytosanitary treatment – to ensure marketability of the host. ................
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