Test substance application

Accurate and even application of test substance is absolutely critical to study success. If the application is highly variable or deviates significantly from the target application rate, the study results may be technically unusable and/or unacceptable to regulatory authorities. Accurate agrochemical application begins with careful calibration of the spray equipment. Hence Study Directors should be familiar with sprayer calibration techniques, even if they will not be personally making the applications. 

Spray nozzle type plays an important role in the success of agrochemical application. For broadcast applications to soil, flat fan nozzles should be used. Newer spray tips such as the DG TeeJet, XR TeeJet, Turbo TeeJet and similar nozzles supplied by Lechler and Hardy have provided acceptable results in a number of studies. For a given nozzle type, the lower the application pressure, the larger is the spray droplet size and the less potential for spray drift. Similarly, the closer the boom is positioned to the soil surface, the less is the potential for spray drift. Most applications are made with spray tips having 80° or 110° spray angles and boom heights of about 50 cm above the soil surface. 

A combination of techniques is typically used to verify the accuracy and precision of agrochemical applications to soil. For example, the catch-back method or passtime method is typically used in conjunction with analytical results from application verification monitors to confirm proper application. The catch-back method involves measuring the spray solution volume before and after application to double check that the desired volume of test solution was actually applied to the test plots. Experienced applicators are often able to apply within 2% of the targeted spray volume. 

Application verification (AV) monitors are devices that are placed within test plots to measure actual spray deposition that occurred during application. The main function of AV monitors is to show whether or not the intended amount of test material was actually deposited on the soil surface. Application monitors consisting of soil-filled containers, paper disks, polyurethane foam plugs, and glass Petri dishes have all been used successfully for this purpose. Prior to using a monitor in the field, it is important to determine that the test substance can indeed be successfully extracted from the monitor and that the compound will be stable on the monitor under field conditions 

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For studies involving test substance application to soil, there may be a requirement for more soil information than for studies where applications are made to foliage of established crops. The study protocol should describe any specific requirements relative to soil type selection and how to confirm the soil characteristics for the study. Most studies simply require that the soil be identified by its name (e.g., Keystone silt loam) and composition (e.g., percent sand, silt, and clay). This information can typically be acquired from farm records, a soil survey of the local area, or a typical soil analysis by a local soil analysis laboratory. In some instances, a GLP compliant soil analysis must be completed. The study protocol must clearly define what is needed and how it is to be obtained. Unless specified in the protocol, non-GLP sources are adequate to identify the soil and its characteristics. The source of the soil information should be identified in the field trial record. 

The four main phases involved in a field soil dissipation study are (I) planning and design phase, (II) field-conduct phase, (III) sample processing/analysis phase, and (IV) data handling/reporting phase. Each phase is vitally linked to the next and each is critical to study success. Results from an otherwise perfectly executed study may be made useless by uneven test substance application or improper sampling, sample handling, and/or analytical techniques. Each of these phases is discussed below. 

Each of the five main steps in field conduct (site selection, test plot layout, test substance application, sample collection, and sample storage/handling) is addressed below. 

Plot maintenance B Expertise must be available to maintain the test site and, if cropped, to take care of the crop For bare-soil studies, the soil surface must be carefully prepared prior to test substance application and kept weed-free without disturbing the test areas. If the test is cropped, the crop should be treated according to Good Agricultural Practice. In case of a soil accumulation study, the field may be cultivated and cropped each season for up to 6 years... 

Assuming proper soil surface preparation (i.e., smooth with no soil clods or crop debris) and test substance application, the diameter of the soil probe does not generally impact observed pesticide residue concentrations in soil or associated variability. Nevertheless, a minimum diameter of 5 cm for the upper soil probe is recommended to improve sampling under less than ideal conditions. Increasingly, researchers are using probes having diameters >5 cm with good results under a variety of field conditions. 

Protocols to determine exposure scenarios should require that application be made using normal practices. Test substance application must be thoroughly documented by researchers. Documentation should include weights and volumes of materials added to... 

Although laboratory data regarding homogeneity, stability, effects of adjuvants, etc. may be available from the sponsor, this information is frequently not made available at the site of test substance application. A GLP deviation listed in the compliance statement may be the only realistic alternative without resorting to heroic effort however, it must be kept in mind... 

Any portion of the study that is a "field study" may also be audited. A field study auditor would inspect many of the same items already mentioned, but at the field site location. These include the training and experience of the field personnel, the calibration and maintenance of equipment, the field management and operations, the test substance application, and the sampling. Special problems are sometimes encountered in the field because the site is physically displaced from the main site of the study. Despite the physical displacement, the equipment must still be calibrated and maintained, the protocols and SOPs must be followed, there must be proper record-keeping, and there must be regular inspections by the QAU. 

Procedures for chemical analysis - including procedures for analysis of test substances, application mixtures and samples... 

In more recent times chemically defined basal media have been elaborated, on which the growth of various lactic acid bacteria is luxuriant and acid production is near-optimal. The proportions of the nutrients in the basal media have been determined which induce maximum sensitivity of the organisms for the test substance and minimize the stimulatory or inhibitory action of other nutrilites introduced with the test sample. Assay conditions have been provided which permit the attainment of satisfactory precision and accuracy in the determination of amino acids. Experimental techniques have been provided which facilitate the microbiological determination of amino acids. On the whole, microbiological procedures now available for the determination of all the amino acids except hydroxy-proline are convenient, reasonably accurate, and applicable to the assay of purified proteins, food, blood, urine, plant products, and other types of biological materials. On the other hand, it is improbable that any microbiological procedure approaches perfection and it is to be expected that old methods will be improved and new ones proposed by the many investigators interested in this problem. 

Aliquots of water used for dilution are taken from plastic bucket and divided into other plastic buckets. A portion of the water is poured into the plastic bags containing the test materials. After thorough agitation, the concentrated test substance solution is poured into the plastic bucket and the plastic bag is rinsed twice with the rest of the water. After thorough agitation, the diluted test solution in the bucket is poured into the application equipment. 

Application of the test substance to the test system is without doubt the most critical step of the residue field trial. Under-application may be corrected, if possible and if approved by the Study Director, by making a follow-up application if the error becomes known shortly after the application has been made. Over-application errors can usually only be corrected by starting the trial again. The Study Director must be contacted as soon as an error of this nature is detected. Immediate communication allows for the most feasible options to be considered in resolving the error. If application errors are not detected at the time of the application, the samples from such a trial can easily become the source of undesirable variability when the final analysis results are known. Because the application is critical, the PI must calculate and verify the data that will constitute the application information for the trial. If the test substance weight, the spray volume, the delivery rate, the size of the plot, and the travel speed for the application are carefully determined and then validated prior to the application, problems will seldom arise. With the advent of new tools such as computers and hand-held calculators, the errors traditionally associated with applications to small plot trials should be minimized in the future. The following paragraphs outline some of the important considerations for each of the phases of the application. 

At times, unexpected events delay application of the test substance after the spray solution has been prepared. Most test substance spray solutions are stable for a reasonable period of time. However, the protocol, SOPs, specific test substance guidance... 

In a field residue study, commodities are grown on control plots located near the plots used to produce commodities treated with test substance. Care is taken to ensure that the only difference between control and treated commodities is that the former does not receive application of the pesticide and the latter does. Crop variety and growing conditions (including geographical location, soil, time of year, weather, etc.) are essentially identical for the control and the treated commodities. 

All application verification and soil samples must be individually labeled with unique sample identification (ID) and other identifying information such as study ID, test substance name, sample depth, replicate, subplot and date of collection, as appropriate. Proper study documentation requires that sample lists and labels be created prior to work commencing in the field. Water- and tear-resistant labels should be used since standard paper labels may become water-soaked and easily torn during sample handling. Sample lists should have the same information on them as the labels and are a convenient place to record plot randomization, initials of the individual who collected the sample, and date of collection. As such, the sample list is important in establishing chain of custody from the point of sample collection until its arrival at the laboratory. 

Protocols must specify calibration of application equipment before and after application to determine the rate of product delivery when application equipment was traveling at a constant ground speed. Liquid or emulsion samples should be collected from spray nozzles and granule collection should occur as the test substance exits the application equipment. Once the correct ground speed has been determined for a given application system, that speed is maintained throughout the application process. 

Protocols should require documentation of actual application practices and times. Samples of applied pesticides should be collected to document application rates to study sites. The test substance must be applied with typical equipment used for the crop, and the application must be made in accordance with the labeled use. Another variable that impacts such studies is the fact that most landowners have their own application equipment, which increases the variance in actual application rates among fields and may cause differential intra-field heterogeneity in application rates. 

If the label allows for multiple applications of a test substance, then a minimum of two applications at the shortest spray interval are required with the decline measured from the time of the second application. The application rate should be the highest legal label rate for each test site in the lowest gallons of spray solution per acre to provide the highest potential test substance residue levels. 

Application of the test substance to the target crop prior to harvest represents a worst-case scenario for potential exposure to workers for the crop category. 

A protocol should be designed to conform as closely as possible to all ERA requirements. The test substance is a typical end-use product and application and agronomic practices accurately reflect the label and normal crop culture in the areas where the study will be conducted. Dislodging leaf material with a surfactant in aqueous... 

The recommended sampling interval for most pesticides is 35 days after the final application or decline of test substance through two half-life degradations. For most pesticides, significant degradation takes place in the first week and the 35-day sampling... 

Tracer materials are defined as any product included in the test substance that can be recovered analytically for determining the drift from the application. This may be the active ingredient in an actual tank mix, or it may be a material added to the tank mix for subsequent detection. The selection of an appropriate tracer for assessing deposition rates in the field is critical to the success of a field study. Tracer materials such as low-level active ingredient products, colored dyes, fluorescent dyes, metallic salts, rare earth elements and radioactive isotopes have been used with varying degrees of success in the field. An appropriate tracer should have the following characteristics ... 

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