Protocols for Quantifying Transferable Pesticide Residues in Turfgrass Systems

1. Field Plot Identification and Establishment

1. Identify research areas representative of the turfgrass system of choice. Specifically consider appropriate turfgrass species and soil type, as well as management practices including fertility, irrigation and mowing height.
2. Conduct preliminary residue analyses of foliage and soil matrices to ensure pesticide(s) of interest are not detectable.
3. Lay out plots to appropriate dimensions for site and sample collection methods, and incorporate 0.5-1.0 m alleys on plot borders to allow for personnel foot traffic.
   NOTE: Plot dimensions can vary based on site-specific conditions, but should be a minimum of 3.3 m². This will allow for a 3.7 m x 0.5 m ball travel pathway, as well as an equivalent adjacent area for foot-traffic and vegetation sampling.

2. Trial Initiation

1. Twenty-four h prior to initiation mow turfgrass to the species-system specific height and collect clippings. Remove all remaining debris with a blower and irrigate to soil saturation.
   NOTE: Irrigating at this time should allow for optimal moisture (i.e. field capacity) at pesticide application.
2. Select pesticide application rate at the currently registered maximum labeled allowances for the site of interest. The application rate is product-specific and determined by the USEPA, and is stated by law on all pesticide container labels.
   1. Select the current minimum labeled spray application carrier volume to maximize foliar-spray retention.
3. At trial initiation, apply pesticide spray treatments to plots when climatic conditions favor retention on treated vegetation.
   NOTE: The proper personal protective equipment must be worn by applicators and nonapplicators near the test area during the treatment period. Always keep nonapplicator personnel upwind of spray application. Climatic conditions favoring retention on treated vegetation include canopy moisture (dry at application), wind speed (<11 km/h) and forecasted precipitation
   1. Cover plots during irrigation/precipitation through at least four sampling events.
      NOTE: Plots are covered to prevent pesticide wash from treated vegetation into turfgrass thatch and/or the soil surface, making it less dislodgeable and underestimating exposure risk. Covers can vary by material and design, but should be constructed of a nonporous material to keep plots dry and designed such that treated vegetation is not contacted (ex. do not lay tarps directly onto plots), which may compromise subsequent pesticide transfer sampling.
4. Collect hourly climatic data including air temperature, dew point, relative humidity, time of sunrise, wind direction and wind speed throughout the trial period to aid with data interpretation.

3. Quality Control Samples

1. Collect one sample (50 to 100 mL) from each pesticide container used in the research to ensure pesticide-product loading rates are correct. Record product name and lot number of each container.
2. Collect a sample of each pesticide spray tank mix applied by collecting output from each spray nozzle to make one composite sample (50 to 100 mL) per tank to ensure correct spray solution mixing procedures.
3. Produce spray application controls as described by Welsh et al.¹² to ensure the appropriate amount of pesticide is applied to plots.
4. Produce field fortification and blank samples as described by Welsh et al.¹² at each sampling event to quantify dissipation during the sample collection and storage processes, and to identify potential sources of contamination.
   NOTE: Fortification sample concentrations and solvents can vary based on compound- and analytical-specific considerations.

4. Sample Collection Considerations

1. Collect samples immediately after an application, and again once the pesticide has dried on the leaf surfaces. Depending on environmental conditions, this is typically 1 to 3 h after application.
2. Following the day of application, collect samples at, or just after sunrise, when maximum turfgrass canopy moisture is typically present¹⁴.
   1. Determine sample collection timings after the day of application based on specific pesticide physicochemical properties, with sampling intervals decreasing as pesticide persistence decreases. Schedule sample collection timings such that at least two consecutive nondetections occur by experiment completion.
      NOTE: Example from previous research quantifying transferable 2,4-dimethylamine salt (2,4-D; field half-life = 6.2 d): 1) 0 d after treatment (DAT) — 0 h after spraying; 2) 0 DAT — 1 h after spraying; 3) 1 DAT; 4) 2 DAT; 5) 3 DAT; 6) 6 DAT; 7) 12 DAT; 8) 24 DAT¹⁴.

5. Transferable Residue Sample Collection

NOTE: Nitrile gloves are worn throughout all sample collection procedures, and should be replaced as often as needed to prevent contamination.

Figure 1: Hand wiping pressure quantification. Approach used to quantify pressure from fronthand (A) and backhand (B) wiping motions. To do so, spray a green food coloring solution (water + 10% v/v food coloring + 1% v/v nonionic surfactant) over a nonporous surface (ex. glass or metal tray) and press hand as intended for pesticide dislodge sampling. Quantify the contact surface area by digital image analysis as described by Campillo et al.¹⁷ to determine proportion of green pixels per image of known area. Collect a turfgrass core from the intended research area when soil is at field capacity and secure it to a digital weight scale. Quantify the mass of downward force when hand wiping. Hand wiping pressure should not exceed 2 kPa. Please click here to view a larger version of this figure.

1. Hand wipe
   NOTE: Requires one person.
   1. Calibrate hand wiping pressure to 2 kPa.
      1. Calculate wiping force by securing a turfgrass core collected at soil field capacity to a digital scale and pressing to provide 150 to 200 g (tare prior to pressing).
      2. Quantify area of front- and backhand used for turfgrass sampling. Primarily use fingers to contact unique turfgrass blades while limiting them from laying over.
         1. Prepare a water-based solution of 10% v/v green food coloring + 1% v/v nonionic surfactant, and spray on a nonporous surface to create a thin film.
            NOTE: The nonionic surfactant is used to increase solution coverage over the nonporous surface by reducing surface tension.
         2. With a cotton glove on, press hand on the nonporous surface (Figure 1).
         3. Remove the glove and quantify area of hand in contact with the nonporous surface by taking a digital photo over a known area of each glove, and use ImageJ¹⁶ to select for green pixels similarly to Campillo et al.¹⁷. This will provide a percent of the total image in green pixels, which is used to calculate area.
      3. Combine wiping force mass with wiping area to calculate hand pressure.
   2. Calibrate hand wiping speed to 1.2 km/h by marking a 0.3 m distance on the ground. Adjust the hand speed such that wiping this distance takes 1 s.
   3. Mark sampling area (420 cm²) by placing a circular bucket (23 cm diameter) upside down where the open end is facing downward. Apply light pressure to the center of the bucket and with opposite hand paint around the outer edge of the bucket (using turf marking paint).
   4. With a cotton glove over a nitrile glove on the dominant hand, wipe over the sampling area for 30 s. Alternate front- and backhand surfaces, while adjusting directions perpendicularly (Figure 2).
      1. Repeat step 5.1.4 with a new cotton glove.
         NOTE: Variation in gloves required per sample can vary based on the pesticide of interest, turfgrass system and sampling area. This should be determined prior to experiment initiation via preliminary efforts collecting four to six gloves per sampling area immediately after application, as well as the following morning when canopy moisture is present to identify the number of gloves to transfer maximum pesticide residue.
   5. Following sample collection, store gloves in a glass jar temporarily on ice or dry ice in the field, and transfer to a freezer within 3 h following sample collection completion.

Figure 2: Hand wiping directions. Progression is intended to contact maximum unique treated vegetation surface area to glove. Two gloves are required to avoid overloading cotton when sampling <415 cm² area; however, this should be confirmed in pesticide- and site-specific conditions prior to experiment initiation. Please click here to view a larger version of this figure.

2. Modified California roller
   NOTE: Requires three people. Apparatus building specifications found at Fuller et al.⁹
   1. Use a roller (roller: 14.5 kg, 10 cm diameter by 61 cm length covered with 1.2 cm polyurethane foam; handle: 1.2 m length) and a frame (62 cm width by 91 cm length) that a cotton sheet (>200 thread count; 70 cm width by 99 cm length) is secured to.
   2. Have Persons B and C secure cotton sheet to frame with clamps and secure to sampling area with metal nails.
      NOTE: Cut cotton sheets prior to a sample collection event to reduce time for a given collection.
   3. Have Person A and B secure plastic (6 mm) around the roller to prevent contamination across samples.
   4. Without adding down pressure, have Person A roll the roller over the cotton cloth five times (one down and back roll equals one time). Roll speed should target 1.6 ± 0.2 km/h.
   5. Have Person C remove the frame from the ground and take to a nearby sampling table.
   6. While Person C is holding the frame, have Person A visually inspect the cloth and remove grass/soil debris with tweezers.
   7. Have Person A remove the cotton cloth, and with Person B fold it such that the side in contact with turfgrass is folded together and store in glass sample jar.
      1. Store the glass jar temporarily in a cooler on ice or dry ice in the field, and transfer to a freezer within 3 h following sample collection completion.
   8. Following sample collection, discard the plastic surrounding the roller, clean the frame, sampling table and tweezers with 1:1 ammonia:water, rinse with water and dry prior to repeating the process.

Figure 3: Soccer ball roller. Excluding A to B, all junctions are glued with PVC adhesive. Part C length can vary by supplier due to varying dimensions of parts B and D. Please click here to view a larger version of this figure.

3. Soccer ball roll
   NOTE: Requires one person.
   1. Use a soccer ball roller frame constructed of polyvinyl chloride (PVC) pipe (schedule 40; 5 cm inner diam).
      1. Secure the handle (122 cm length; Figure 3A) to a slip tee coupler (Figure 3B). This is the only junction not secured with PVC glue to make for easier storage.
      2. Lay the roller on a flat surface (aids with alignment) and secure both sides of the tee coupler to slip 90° elbow fittings (Figure 3D) with 7 cm pipe length (Figure 3C).
      3. Insert 24 cm pipe lengths (Figure 3E) to the open end of the elbow fittings
      4. Drill lag bolts (0.6 cm diam by 7.5 cm length; Figure 3F) through the end of the pipe perpendicular to the handle and parallel with the flat surface.
         1. Set distance between lag bolts to approximately 18.4 cm (can vary across brands/quality of soccer balls used).
         2. File/grind sharp point off lag bolt ends with a hand file/grinder so that soccer balls are not punctured when rolling (Figure 3G).
   2. Release soccer ball air pressure to 24 kPa. Although this will underinflate the ball when loose from the PVC roller, it will increase pressure to approximately 42 kPa when the ball volume is reduced after securing to the roller.
   3. Mount the soccer ball onto the roller by touching the ball endpoints (maximum diameter) to lag bolts.
      NOTE: To reduce overall sampling time and potential contamination, have one soccer ball per sample at a given collection timing.
      1. Spin ball by hand to ensure the ball roll is symmetrical.
      2. Press lightly on top of the mounted soccer ball to ensure it is adequately secured.
   4. Double-wrap a sorbent strip (5 cm width by 132 cm length) around the circumference of the ball that aligns with the PVC roller handle, which creates constant turfgrass-sorbent strip contact when rolling (Figure 3H).
      1. Secure the sorbent strip to the ball with adhesive tape strips (2.5 cm by 2.5 cm).
         NOTE: Cut sorbent strips prior to a sample collection event to reduce time for a given collection.
   5. Roll the ball at a constant speed (1.6 ± 0.2 km/h) over a 3.7 m distance of unique treated turfgrass. Depending on plot dimensions, this distance may require side-by-side rolls to complete. When doing so, take precaution to not contact vegetation that has yet to be sampled.
      NOTE: The 3.7 m ball roll distance may need to be adjusted based on the pesticide of interest and turfgrass system. This should be determined prior to experiment initiation via preliminary efforts collecting samples of varying distances immediately after application, as well as the following morning when canopy moisture is present to identify the optimal distance to transfer maximum pesticide residue.
   6. Following the ball roll, remove the sorbent strip and fold it such that the side in contact with turfgrass is folded together and store in a glass jar.
      1. Store the glass jar temporarily in a cooler on ice or dry ice in the field, and transfer to a freezer within 3 h following sample collection completion.

6. Turfgrass Vegetation Collection

1. Remove the plunger inside of the cup cutter to keep sampling equipment from contacting treated foliage (Figure 4A).

Figure 4: Turfgrass core collection. Golf course cup cutter is a robust, relatively cheap apparatus for turfgrass/soil collection. Remove the inside plunger used to eject cores prior to use for transferable pesticide residue sample collections so that vegetation is not inadvertently contacted, which may reduce pesticide residue concentrations. Please click here to view a larger version of this figure.

2. Harvest a turfgrass core of representative quality.
   1. Use the cup cutter to outline the core and cut to 5-7.5 cm soil depth to aid with removal.
   2. Cut with a knife (blade length: 10 to 15 cm) at a diagonal angle outside the edge of the core to remove the core from the ground. Pay special attention to prevent hand/knife contact with treated aboveground vegetation.
   3. Level the bottom soil surface with scissors so that it will sit flat in a sample container. Soil depth should allow enough space such that turfgrass vegetation does not contact the sample container lid.
      NOTE: When weather permits, the golf course cup cutter step (6.2.1 in protocol) is typically completed in the afternoon when foliage is dry before sample collection the following morning.