Improvement in Day Zero Recoveries in Field Soil Dissipation Studies

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Improvement in Day Zero Recoveries in Field Soil Dissipation Studies Using Larger Diameter Soil Samples Ashok K. Sharma,* Harry J. Strek,† and Aldos C. Barefoot Stine Haskell Research Center, E. I. DuPont de Nemours and Co., Newark, Delaware 19711, United States ABSTRACT: Obtaining acceptable recovery of the applied test substance at zero time in field soil dissipation studies has been a subject of considerable interest among scientists conducting regulatory field studies. In particular, achieving recoveries of ≥90% in soil samples collected immediately after applications in most studies has been elusive. This study investigated a modified soil sampling method, which could be used not only on day zero but for the entire study duration, to see if the recoveries in soil samples, especially in the early stages, can be improved. The modified sampling system has demonstrated that recoveries averaging 90% are possible and can be routinely obtained on day zero. Description of this modified sampling procedure and statistical analysis of the data collected for day zero samples are discussed. KEYWORDS: day zero recoveries, field dissipation studies, large diameter soil cores

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INTRODUCTION Field dissipation studies for crop protection chemicals are an important and expensive portion of the environmental fate studies required for registration of new agrochemicals. Typically, a test substance is applied to a leveled bare soil plot of suitable size, and then soil samples are collected in 6 in. depth segments to a depth of 36 in.1,2 or more. Soil sampling is typically initiated immediately after application (zero time samples) and continued until substantial dissipation of the applied chemical and its metabolites has been demonstrated. To meet the study objectives, it is essential to achieve a nearly quantitative recovery of the applied chemical in zero time samples, commonly referred to as day zero samples. As noted in the EPA’s guideline1 “time zero concentration lays the foundation for all subsequent sampling and is used to build confidence that the pesticide was applied uniformly and accurately”. Using the commonly employed soil sampling procedures, in which soil cores of approximately 2 in. diameter are collected in 6 in. depth segments, the uppermost 6 in. deep sample should have 100% of the applied chemical at time zero. However, such day zero samples frequently fail to show acceptable recovery. Zero time recoveries are often below 75−80% and display high levels of variability. This shortcoming has been a subject of several symposia at agrochemical scientific conferences4 and the EPA’s rejection rate analysis,3 as well as several presentations at scientific conferences.5,6 Low recoveries at day zero have been considered a significant deficiency in the conduct of field dissipation studies, because they imply either gross errors in the chemicals’ application process, poor sampling technique, or deficient analytical methods. Although all of these factors can cause poor recoveries in theory, application methods and analytical methods were set aside as major causes because the test substance applications are typically conducted by skilled field and laboratory personnel and always initiated under favorable weather conditions. Analytical methods employed are usually robust and typically deliver 80−110% recoveries in concurrent © 2014 American Chemical Society

analysis with 10−15% standard deviation; otherwise, the methods would be unacceptable for regulatory authorities. On the basis of the analytical methods’ capabilities averaging 90% recovery, day zero soil sample recoveries of equal magnitude should be achievable. Before embarking on attempts to improve the recoveries, we analyzed the data available from a large number of our own field studies as well as information from recent symposia. Data compiled in appendices of ref 4 provided a good starting point to formulate a working hypothesis. The hypothesis was that better recoveries in soil cores could be achieved if the amount of treated soil, as a percentage of the total sample collected, could be increased substantially by not only increasing the sampled area but reducing the sample depth at the same time. Our statistical analysis of data in ref 4 indicated that day zero recoveries based on the secondary sample type (Figure 1), such

Figure 1. Recovery in day zero samples by sample type box plot of data from ref 4. Large circle = mean. Received: Revised: Accepted: Published: 4090

February April 13, April 15, April 15,

4, 2014 2014 2014 2014

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the degradation kinetics always employ the soil core data but also because it is the only device used over the entire study duration. If day zero recoveries in soil cores are unreliable, one cannot be certain at which later sampling intervals the recoveries begin to be reliable. The secondary devices can verify application rates; however, they can be used only at day zero but not for later sampling intervals. We postulated that if a chemical can be applied accurately as often supported by the recoveries in secondary monitoring devices, then for a vast majority of the agrochemicals, and for most of the formulations employed, recoveries equivalent to the average recovery delivered by the analytical method should be achievable in soil samples.

as Petri dishes and foam pads, were consistently good but soil samples collected to 6 in. depth were frequently quite deficient. A different perspective of the same data, illustrated in Figure 2,

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MATERIALS AND METHODS

We assumed that low recoveries in 6 in. deep soil cores having a 2 in. diameter were possibly due to two shortcomings of these soil samples. First, the portion of soil in a 6 in. deep sample carrying the applied test chemical on day zero was miniscule (approximately 1%) as compared with the total amount of soil sampled. Unless homogenization procedures capture this entire residue and mixe it uniformly in the entire sample, lower recoveries would be encountered. One possible solution was to collect the uppermost soil sample to depth of 2 in., instead of the traditional 6 in. depth. Second, a 2 in. surface area for sampling the uppermost soil sample appeared inadequate because the small surface area in a field study was vulnerable to spot-to-spot variability of the residue present on soil surface. Therefore, we decided to collect a larger surface area of at least 4 in. or possibly 6 in. diameter to a depth of only 2 in. for the uppermost sample. Although soil cores of larger than 2 in. diameter have been attempted by many working in this area, sampling has generally been to a typical 6 in. depth. Perhaps for that reason, we are not aware of any published studies that have demonstrated consistently high recoveries in large-diameter soil samples. In particular, increasing the collection of treated soil as a percentage of total sample by collecting a shallower depth was considered an essential element of sampling the uppermost soil core. To our knowledge a systematic study that utilized a combination of larger surface area and shallow depth for sampling the uppermost soil sample has not been reported in peer-reviewed literature. Initially, we decided to evaluate a comparison of a shallower uppermost depth of only 2 in. instead of 6 in. and a larger surface area

Figure 2. Recovery in day zero samples by sampled area [data from ref 4 and select DuPont studies].

focused on the surface area sampled without regard to the device used. It also suggested that samples with larger surface area had a higher probability of achieving good recovery. In our opinion, secondary devices frequently achieved good recoveries primarily because they have a larger surface area than soil cores. In our own experience also, secondary devices such as Petri dishes or papers, which had a larger surface area, and in which the applied test substance could be analyzed without the encumbrances associated with the soil homogenization and matrix extractions, were generally successful in obtaining good day zero recoveries. However, 2 in. diameter soil cores collected to 6 in. depth, a method used consistently at all sampling intervals, invariably showed day zero recoveries that seldom exceeded 75−80%. Our objective, therefore, was to determine if the soil sampling method or sampling device could be modified so that the day zero recoveries in soil cores collected could be improved substantially. This was important not only because

Figure 3. Large-diameter soil sampling devices. 4091

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⎛ ⎞ ⎜g ⎟ residue ⎜ ⎟ ⎜ ha ⎟ ⎝ ⎠

of 4−6 in. diameter in parallel with traditional 2 in. on day zero to determine if such sampling leads to better day zero recoveries. To test this hypothesis, we conducted multiple types of soil sampling within the same studies at four sites each in the United States and European Union. In conformance with regulatory guidelines, three replicate samples, each composed of five cores sampled from randomly selected areas, were collected at each location and for each sample type. Within each study, at least two types of soil samples were collected for the uppermost soil horizon. One of the sample types was a set of traditional 2 in. (5 cm) cores with standard 6 in. (15 cm) depth; a second set was of either medium core (4 in. diameter) or large core (6 in. diameter), both to a 2 in. depth. Collection of the 2 in. depth soil samples was performed using the device displayed in Figure 3. Such devices, which had ca. 4 in. internal diameter in the European studies and ca. 6 in. diameter in the U.S. studies, were collected by pressing the device into the soil until the upper lip was in line with the soil surface. Soil inside this sampler was collected using a spoonlike implement by scraping soil to a 2 in. depth. The depth sampled was easily noted when soil up to the bottom of this device had been removed. After uppermost depth had been collected, the device remained in place until all lower depth soil sampling had been completed. In each case, lower depths were sampled as usual, namely, 2 in. diameter samples for all deeper soil samples, although the depth of these lower segments was changed slightly from the regular 6 in. (illustrated in Figure 4).

( ) × wt of 5 cores (g) × area 1 ha (sq cm) surface area of 5 cores (sq cm) × conversion factor ( )

ppb in soil sample =

ng g

ng g

g found × 100 ( ha ) recovery (%) = ( hag ) applied

One calculation for illustrative purposes is shown below: ppb in soil = 209.10

soil weight of 5 cores (g) = 7146.9

inside diameter of 2 in. deep core = 15.3 cm (radius = 7.65cm) surface area of 5 cores = 5 × pi × (7.65 cm)2 = 919.269 sq cm area of 1 ha in sq cm = 108 sq cm Therefore g/ha in sample =

7146.9 g × 209.100 ppb × 108 sq cm/ha (109 ng/g × 919.269 sq cm) = 162.57 g/ha

amount applied = 200 g/ha recovery (%) = (162.57 × 100)/200 = 81.3% Soils weights and areas used were for five cores because five replicate cores were collected and combined to obtain one sample for analysis as is customary in regulatory studies. When multiple samples of each type were collected in a study, the replicates were not averaged for statistical analysis and the residues were not corrected for fresh fortification recoveries. The analytical method employed in the initial investigation provided 92% recovery with 10% standard deviation. Recovery numbers obtained were rounded to one decimal place for use in statistics. All statistical analysis was conducted using Minitab software, version 16.1.

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RESULTS AND DISCUSSION Results from the initial trials (four in the United States and four in the European Union), where a small diameter (2.25 in. surface diameter) and an additional either 3.8 or 5.75 in. diameter sample had been collected for the uppermost depth, are displayed in Figure 5. These samples were collected concurrently from the same test sites and for the same chemical applied. Samples collected were analyzed by using the same method for comparison of recoveries on day zero (note 3.75 and 5.75 in. diameter samples are sometimes referred to as 4 or 6 in. samples for simplicity). Data summarized in Figure 5 (24 samples of each type) illustrated that larger diameter soil cores collected to a shallower depth were indeed more accurate in demonstrating the day zero residues. Whereas small-diameter samples found an average 80% recovery for all samples collected, the larger cores averaged >90%. Statistically the difference in recoveries was significant when small-core data were compared with medium (3.75 in.) or larger cores (5.75 in.). In the studies conducted in the United States, we even analyzed a separate set of 10 large-diameter samples as single replicates and compared them with their respective 5-sample composites (obtained by combining 5 large cores from one

Figure 4. Traditional and modified soil sampling schemes (depth segments in inches). All soil samples were five replicate cores combined to generate one sample. The combined sample, typically weighing 2−3 kg for 2 in. cores, 4−5 kg for 4 in. cores, and 7−8 kg for 6 in. cores, was homogenized using a mechanical soil mixer with added dry ice to prevent chemical changes during homogenization. After homogenization, a subsample weighing approximately 500 g was saved for analysis and stored in a freezer until the start of analysis. Typically, 10 g aliquots of homogenized soil were extracted and analyzed using analytical methods specific for the test substance applied. For the purpose of this paper, only the uppermost depth samples are reported because the test substance resides only in the uppermost samples on day zero. Remaining lower depth samples were also analyzed and submitted to various regulatory authorities. Because the depth segments using the two devices were different, a comparison of the data by these two methods was possible by converting the residues obtained in parts per billion into mass per unit area (g/ha or lb/acre) and were then calculated as percent of applied amounts. Equations for such calculations are shown below: 4092

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field studies have shown consistently improved recoveries (Figure 7). Results from more than 40 field studies (N = 161 is

Figure 5. Day zero recoveries using small- and large-diameter devices.

subplot) to see if each individual sample also achieved the desired results of acceptable recovery. Even the single-replicate samples (Figure 6) showed an average recovery in the 90%

Figure 7. Day zero recoveries using larger diameter 2 in. deep cores.

the total number of day zero samples from all studies) conducted over 5 years and with four different test substances applied at different use rates are summarized in Figure 7. It should be noted that all four test substances were nonvolatile (chlorsulfuron, penthiopyrad, chlorantraniliprole, and cyantraniliprole) but had significantly different physicochemical properties. Even though water solubility, dissipation rates, or formulations used varied substantially among these compounds, we do not believe such properties have a significant impact on the capability of achieving good day zero recoveries. For statistical analysis, 80 and 110% were arbitrarily chosen as the lower and upper specification limits (LSL and USL, respectively) because most of the analytical method recoveries were in the same range, and average analytical method recovery of about 90% for all four compounds was set as the target. In addition, choice of lower and upper specification limits for recoveries in the 80−110% range was based on the assumption that such results are considered acceptable by most regulatory authorities. Selection of a somewhat less strict specification of 70% for the LSL and 120% for the USL would also be acceptable, and Figure 7 shows essentially all results obtained are within the less strict range. We still encounter 2 in. depth samples. In conclusion, instead of collecting 2 in. diameter soil cores to a 6 in. depth for the uppermost soil sample (a standard practice), a larger diameter device and collection of a shallower depth for the uppermost soil core can be used to achieve a significant improvement in day zero recoveries.

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AUTHOR INFORMATION

Present Address †

(H.J.S.) Bayer Crop Sciences, Germany.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge contributions made by Fred Rice of ABC Laboratories, Columbia, MO, USA; Jeff Old of Charles River Laboratories, Scotland, UK; and Mick Qualls of Qualls Research, Ephrata, WA, USA.

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REFERENCES

(1) U.S. EPA Office of Pesticide Programs. Fate, Transport and Transformation Test Guidelines; Washington, DC, USA, 2008; OPPTS 835.6100. (2) European Union. Council Directive 91/414/EEC, Rev 7.0, Annex 1, Fate and Behavior in the Environment, 2007; 7.1.1.2.2, Field Studies. (3) U.S. EPA. Pesticide Reregistration Rejection Rate Analysis, Environmental Fate; Washington, DC, USA, 1993; EPA 738-R-93-010. (4) Graham, D. G.; Clay, V.; Jackson, S. H.; Jones, R. Field soil dissipation studies: the measurement of zero-time residues. In Terrestrial Field Dissipation Studies; ACS Symposium Series 842; Arthur, E. L., Barefoot, A. C., Clay, V. E., Eds.; American Chemical Society: Washington, DC, USA, 2003. (5) Portions of this work presented at the following conferences: Sharma, A. K.; Strek, H. J.; Barefoot, A. C. ACS National Meeting, Washington, DC, 2005. Huber, A.; Strek, H. J.; Sharma, A. K. XIII Symposium. Pesticide Chemistry−Environmental Fate and Human Health, Univ. Sacre Cuore, Piacenza, Italy, 2007. (6) U.S. Environmental Protection Agency and Pest Management Regulatory Agency (PMRA), Canada. ACS National Meeting, Anaheim, CA, USA, 2004. (7) Device in Figure 8 was fabricated by Mick Qualls, Qualls Agricultural Laboratory, 3975 Dodson Road N., Ephrata, WA, USA. (e-mail: [email protected]).

Figure 8. Recoveries based on test substance and application rates.

recoveries for all test compounds, regardless of the use rate, chemical structure, or formulation used. Use of 6 in. diameter devices for sampling the uppermost 2 in. of soil as a standard practice is currently carried out using our most recent sampler7 displayed in the bottom part of Figure 3. This sampler is a two-piece device, in which the inner piece with four quadrants is placed inside the outer casing, shown on the left side of the figure. The assembled device is pressed into soil with a hydraulic press. The lip at the top of the outer casing stops the device at a 2 in. depth. Technicians can grab the handle and rotate the inner piece of this sampler by approximately 30°, which cleanly separates the uppermost 2 in. of soil from the column below. The inner piece can be lifted and held above a bag into which soil is dislodged by gently hitting the device with a rubber hammer. The inside device quadrants assist in not only separating the uppermost 2 in. of soil but also help keep the soil in place while the inside portion is lifted. If a portion of the soil falls within the outer casing, it is recovered using a spoonlike implement. An important point worth reiterating is that large-diameter samplers are effective in combination with a shallow (2 in.) depth only, and the 6 in. diameter is perhaps the upper limit beyond which any further increase in area sampled begins to create homogenization issues. Five replicate cores (6 in. diameter−2 in. depth] composited to obtain one sample generate soil samples that weigh 7−9 kg. The homogenization equipment in use at most facilities we work with can handle samples of 2−10 kg, but work best for 2−5 kg sample sizes. Larger diameter cores collected to 6 in. depths will lead to substantially large sample weights (around 20 kg), and homogenization issues will again negate the advantage offered 4094

dx.doi.org/10.1021/jf500607r | J. Agric. Food Chem. 2014, 62, 4090−4094