We thank Khalied Ahmed, Laney McKnight and Drew Pinnix for field and laboratory assistance, as well as the Lake Wheeler Turfgrass Field Laboratory support staff, including Dustin Corbett and Marty Parish, for maintaining research areas. This work was partially supported by the North Carolina State University Center for Turfgrass Environmental Research and Education.

1. Field Plot Identification and Establishment
<ol>
	<li>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.</li>
	<li>Conduct preliminary residue analyses of foliage and soil matrices to ensure pesticide(s) of interest are not detectable.</li>
	<li>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.</li>
</ol>
2. Trial Initiation
<ol>
	<li>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.</li>
	<li>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.
	<ol>
		<li>Select the current minimum labeled spray application carrier volume to maximize foliar-spray retention.</li>
	</ol>
	</li>
	<li>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 (&#60;11 km/h) and forecasted precipitation
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>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.</li>
</ol>
3. Quality Control Samples
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>Produce spray application controls as described by Welsh et al.12 to ensure the appropriate amount of pesticide is applied to plots.</li>
	<li>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.</li>
</ol>
4. Sample Collection Considerations
<ol>
	<li>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.</li>
	<li>Following the day of application, collect samples at, or just after sunrise, when maximum turfgrass canopy moisture is typically present14.
	<ol>
		<li>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) &#8212; 0 h after spraying; 2) 0 DAT &#8212; 1 h after spraying; 3) 1 DAT; 4) 2 DAT; 5) 3 DAT; 6) 6 DAT; 7) 12 DAT; 8) 24 DAT14.</li>
	</ol>
	</li>
</ol>
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.
<img alt="Figure 1" src="/files/ftp_upload/55182/55182fig1.jpg" /> 
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. <a href="https://cloudfront-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55182/55182fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol>
	<li>Hand wipe 
	NOTE: Requires one person.
	<ol>
		<li>Calibrate hand wiping pressure to 2 kPa.
		<ol>
			<li>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).</li>
			<li>Quantify area of front- and backhand used for turfgrass sampling. Primarily use fingers to contact unique turfgrass blades while limiting them from laying over.
			<ol>
				<li>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.</li>
				<li>With a cotton glove on, press hand on the nonporous surface (Figure 1).</li>
				<li>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.</li>
			</ol>
			</li>
			<li>Combine wiping force mass with wiping area to calculate hand pressure.</li>
		</ol>
		</li>
		<li>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.</li>
		<li>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).</li>
		<li>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).
		<ol>
			<li>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.</li>
		</ol>
		</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 2" src="/files/ftp_upload/55182/55182fig2.jpg" /> 
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 &#60;415 cm2 area; however, this should be confirmed in pesticide- and site-specific conditions prior to experiment initiation. <a href="https://cloudfront-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55182/55182fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="2">
	<li>Modified California roller 
	NOTE: Requires three people. Apparatus building specifications found at Fuller et al.9
	<ol>
		<li>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 (&#62;200 thread count; 70 cm width by 99 cm length) is secured to.</li>
		<li>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.</li>
		<li>Have Person A and B secure plastic (6 mm) around the roller to prevent contamination across samples.</li>
		<li>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 &#177; 0.2 km/h.</li>
		<li>Have Person C remove the frame from the ground and take to a nearby sampling table.</li>
		<li>While Person C is holding the frame, have Person A visually inspect the cloth and remove grass/soil debris with tweezers.</li>
		<li>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.
		<ol>
			<li>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.</li>
		</ol>
		</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 3" src="/files/ftp_upload/55182/55182fig3.jpg" /> 
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. <a href="https://cloudfront-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55182/55182fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="3">
	<li>Soccer ball roll 
	NOTE: Requires one person.
	<ol>
		<li>Use a soccer ball roller frame constructed of polyvinyl chloride (PVC) pipe (schedule 40; 5 cm inner diam).
		<ol>
			<li>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.</li>
			<li>Lay the roller on a flat surface (aids with alignment) and secure both sides of the tee coupler to slip 90&#176; elbow fittings (Figure 3D) with 7 cm pipe length (Figure 3C).</li>
			<li>Insert 24 cm pipe lengths (Figure 3E) to the open end of the elbow fittings</li>
			<li>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.
			<ol>
				<li>Set distance between lag bolts to approximately 18.4 cm (can vary across brands/quality of soccer balls used).</li>
				<li>File/grind sharp point off lag bolt ends with a hand file/grinder so that soccer balls are not punctured when rolling (Figure 3G).</li>
			</ol>
			</li>
		</ol>
		</li>
		<li>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.</li>
		<li>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.
		<ol>
			<li>Spin ball by hand to ensure the ball roll is symmetrical.</li>
			<li>Press lightly on top of the mounted soccer ball to ensure it is adequately secured.</li>
		</ol>
		</li>
		<li>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).
		<ol>
			<li>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.</li>
		</ol>
		</li>
		<li>Roll the ball at a constant speed (1.6 &#177; 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.</li>
		<li>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.
		<ol>
			<li>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.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
6. Turfgrass Vegetation Collection
<ol>
	<li>Remove the plunger inside of the cup cutter to keep sampling equipment from contacting treated foliage (Figure 4A).</li>
</ol>
<img alt="Figure 4" src="/files/ftp_upload/55182/55182fig4.jpg" /> 
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. <a href="https://cloudfront-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55182/55182fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="2">
	<li>Harvest a turfgrass core of representative quality.
	<ol>
		<li>Use the cup cutter to outline the core and cut to 5-7.5 cm soil depth to aid with removal.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Milesi, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+and+modeling+the+biogeochemical+cycling+of+turf+grasses+in+the+United+States.">Mapping and modeling the biogeochemical cycling of turf grasses in the United States.</a> Environ. Manag. 36, 426-438 (2005).</li><li>Turgeon, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Turfgrass management. , (2008).</li><li>Cisar, J. L., Synder, R. H., Sartain, J. B., Borgert, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dislodgeable+residues+of+chloropyrifos+and+isazofos+and+implications+for+golfer+exposure.">Dislodgeable residues of chloropyrifos and isazofos and implications for golfer exposure.</a> Int. Turf. Soc. 9, 12-18 (2001).</li><li>Morgan, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+and+children's+exposure+to+2,4-D+from+multiple+sources+and+pathways.">Adult and children's exposure to 2,4-D from multiple sources and pathways.</a> J. Expos. Sci. Environ. Epidemiol. 18 (5), 486-494 (2008).</li><li>. Overview of Risk assessment in the Pesticide Program [Internet] Available from: <a target="_blank" href="https://www.regulations.gov/document?D=EPA-HQ-OPPT-2009-0157-0003">https://www.regulations.gov/document?D=EPA-HQ-OPPT-2009-0157-0003</a> (2016)</li><li>. OCSPP Harmonized Test Guidelines - Master List [Internet] Available from: <a target="_blank" href="https://www.epa.gov/sites/production/files/2015-11/documents/ocspp-testguidelines_masterlist-2015-11-19.pdf">https://www.epa.gov/sites/production/files/2015-11/documents/ocspp-testguidelines_masterlist-2015-11-19.pdf</a> (2015)</li><li>. Occupational and Residential Exposure Test Guidelines - OPPTS 875.1100 Dermal Exposure - Outdoor Available from: <a target="_blank" href="https://www.epa.gov/pesticide-science-and-assessing-pesticide-risks/overview-risk-assessment-pesticide-program">https://www.epa.gov/pesticide-science-and-assessing-pesticide-risks/overview-risk-assessment-pesticide-program</a> (1996)</li><li>Fuller, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modified+California+roller+for+measuring+transferable+residues+on+treated+turfgrass.">Modified California roller for measuring transferable residues on treated turfgrass.</a> Bull. Environ. Contam. Toxicol. 67 (6), 787-794 (2001).</li><li>Klonne, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+study+of+five+transferable+turf+residue+methods.">Comparative study of five transferable turf residue methods.</a> Bull. Environ. Contam. Toxicol. 67 (6), 771-779 (2001).</li><li>Rosenheck, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+a+standardized+sampling+technique+for+pesticide+transferable+turf+residues.">Determination of a standardized sampling technique for pesticide transferable turf residues.</a> Bull. Environ. Contam. Toxicol. 67 (6), 780-786 (2001).</li><li>Kim, D. S., Marshall, E. J. P., Brain, P., Caseley, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+crop+canopy+structure+on+herbicide+deposition+and+performance.">Effects of crop canopy structure on herbicide deposition and performance.</a> Weed Res. 51 (3), 310-320 (2011).</li><li>Jeffries, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+influencing+dislodgeable+2,4-D+plant+residues+from+hybrid+bermudagrass+(Cynodon+dactylon+L.+x+C.+transvaalensis.)+athletic+fields.">Factors influencing dislodgeable 2,4-D plant residues from hybrid bermudagrass (Cynodon dactylon L. x C. transvaalensis.) athletic fields.</a> PLoS One. 11 (2), 1-16 (2016).</li><li>Sears, M. K., Bowhey, C., Braun, H., Stephenson, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dislodgeable+residues+and+persistence+of+diazinon,+chloropyrifos+and+isofenphos+following+their+application+to+turfgrass.">Dislodgeable residues and persistence of diazinon, chloropyrifos and isofenphos following their application to turfgrass.</a> Pestic. Sci. 20 (3), 223-232 (1987).</li><li>Campillo, C., Prieto, M. H., Daza, C., Mo&#324;ino, M. J., Garc&#237;a, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+digital+images+to+characterize+canopy+coverage+and+light+interception+in+a+processing+tomato+crop.">Using digital images to characterize canopy coverage and light interception in a processing tomato crop.</a> HortSci. 43 (6), 1780-1786 (2008).</li></ol>

The authors have no disclosures to make.

Regulating agencies have not identified a specific method to quantify transferable pesticide residues from turfgrass. This research supports utilizing different methods based on site- and exposure process-specific criteria, as they all have utility for human risk assessments. However, they all have limitations that researchers should be cognizant of prior to their use. Lastly, turfgrass species is not a site selection parameter currently stated in transferable pesticide residue protocols, and this research builds on previous efforts suggesting its inclusion should be stated.
The described soccer ball roll method is relatively robust across samplers and only requires one individual to complete. It also mimics a very common occurrence in the most popular international sport, and has many implications for nondietary pesticide exposure from hand-to-mouth contact. Additionally, this concept could be applied with minimal modification for pesticide transfer onto objects associated with other sports. However, it is a comparably less aggressive method for pesticide transfer and consequently, should not be solely utilized in risk assessments.
Of the three methods presented, the modified California roller has been employed most extensively in turfgrass transferable residue research. It is the most robust approach across samplers, and transferred a similar amount of 2,4-D as other methods at the lowest mown turfgrass system evaluated, creeping bentgrass. This suggests that this method should be employed for transferable pesticide residue research on golf course putting greens, and potentially other closely mown systems such as golf course fairways/tees and athletic fields. The limitations of this method include potentially laying grass blades down as mowing height increases and the requirement of three individuals to complete. Additionally, the roller and frame preparation required between samples can be time consuming, and previous research has shown pesticide transfer fluctuates over relatively short timescales within a day as canopy moisture dissipates14. This may limit the amount of samples that can be collected at a given timing (i.e. reduced treatments), or add a confounding factor to data should samples be collected over an extensive amount of time. Researchers should be aware of this as they plan experiments utilizing the modified California roller.
Hand wiping over treated plant vegetation with cotton-based gloves is a method commonly used to measure transferable pesticide residues for workers in orchards and tobacco due to the high frequency of hand-to-vegetation contact associated with agricultural production. While this method has been utilized less in turfgrass systems, it provides a superior approach to quantify transferable residues in turfgrass systems at mowing heights commonly associated with commercial/residential lawns and parks. Additionally, hand-to-treated turfgrass contact is common for both nonoccupational and occupational risk assessments, as turfgrass systems are utilized for a variety of societal purposes. Of the three evaluated methods, hand wiping is the least reproducible method across samplers, which may require additional measures (replications, training, etc.) to produce conclusive results.
Although the evaluated methods vary widely in their execution, the critical steps within each protocol conceptually overlap. Sampling at a constant speed and pressure is paramount to produce reproducible data, as these influence sorbent material-pesticide transfer. Maintaining a constant speed is required of samplers across all three methods, while pressure is a point of concern for hand wiping only. Samplers should not put additional pressure on the modified California or soccer ball rollers, while hand wiping is an approach that takes substantial preliminary efforts to maintain consistency within, and across samplers. This is the greatest limitation to transferable residue research relying solely on hand wiping, and future research should identify a less subjective approach that provides its unique attribute of canopy penetration while minimally laying grass down.
The purpose of collecting turfgrass vegetation is to provide a reference point in addition to the amount of pesticide initially applied by accounting for dissipation between application and subsequent sample collection timings. Furthermore, quantifying pesticide residue in vegetation enhances explanation when nondetection occurs in transfer samples. Basically, it allows the researcher to determine if transfer did not occur because pesticide residue was sorbed in/on vegetation, making it nontransferable, or if residue was no longer detectable in/on vegetation.

Turfgrasses are grown on over 16.3 million ha in the contiguous United States (US) &#8212; exceeding the combined area of irrigated grain corn [(Zea mays L.) 2.5 million ha], soybeans [(Glycine max L.) 2.1 million ha] and cotton [(Gossypium hirsutum L.) 0.9 million ha] &#8212; and are utilized by the public with land uses including athletic fields, commercial/residential lawns, golf courses and parks1,2. They provide many positive societal attributes including dust control, heat dissipation, recreational surfaces and soil stabilization. However, pest encroachment may occur which requires the use of pesticide(s) to maintain the turf to an acceptable level3.
Plant canopies in established turfgrass systems intercept sprayed pesticides. Human-pesticide exposure is possible via various routes, including transferring from treated vegetation directly onto humans, as well as through indirect routes such as transfer onto maintenance equipment, pets and recreational items4,5. To address such concerns, human pesticide exposure risk assessments must be conducted prior to pesticide registration and re-registration in the U.S. to estimate the nature and probability of adverse health effects following exposure to contaminated environmental media6. Within the occupational and residential exposure test guidelines currently employed by the USEPA, foliar transferable residue dissipation tests (OPPTS 875.2100) are conducted to quantify pesticide residues on treated surfaces that can be transferred through various processes onto human skin/clothing or inhaled7,8.
Previous research efforts have compared several foliar transferable pesticide residue methods including a California roller (moving a roller of known mass over cotton cloth), drag sled (pulling a solid object of known mass with a piece of cloth attached to it), polyurethane foam roller (moving a roller of known mass covered with polyurethane foam) and shoe shuffling (attaching cotton cheesecloth to shoes), which are all conducted in a known area of pesticide-treated turfgrass9,10,11,12. Of the aforementioned methods, California roller-based approaches provide the most repeatable approach to quantify foliar transferable pesticide residues; however, comparably more aggressive approaches such as shoe shuffling can transfer more pesticide residue, which also has utility in risk assessments. Hand wiping methods provide a comparatively enhanced ability to contact unique treated turfgrass vegetation surfaces. This method provides more applicable data for nondietary pesticide ingestion, as hand-to-mouth contact is a common process associated with this exposure route.
Turfgrass canopy dynamics vary widely between both species and use sites. Species commonly vary in growth habit and season, as well as blade texture and density, which affect pesticide spray interception and physiological processes3,13. Management inputs can vary widely between use sites, and within a use site based on site-specific expectations. For example, bermudagrass (Cynodon spp.) is utilized in adapted climates as a golf course putting green surface which are typically irrigated and mown &#62;5 times per week (clippings collected) at 0.3 to 0.4 cm, as well as a nonirrigated utility ground cover which may be mown &#60;1 times per week (clippings returned) at 1.5 to 6.3 cm. Previous research has shown transferable foliar pesticide residues can vary between species within a use site, and is affected by irrigation and mowing practices14,15. Ultimately, variability between turfgrass systems inhibits the implementation of a universal method to quantify transferable foliar pesticide residues. Therefore, method selection to optimize human risk assessments should encompass pesticide-, process-, site- and species-specific criteria. The objective of this study was to characterize various methods used to quantify transferable foliar pesticide residues, and highlight conditions that should be considered when selecting a method for a given experiment.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>General</td>
			<td>---</td>
			<td>---</td>
		</tr>
		<tr>
			<td>Nitrile gloves</td>
			<td>Any</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Coolers</td>
			<td>Any</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Turf paint</td>
			<td>Any</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Field plot measuring equipment</td>
			<td>Any</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Protective foot apparel</td>
			<td>Any</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Whatman 3 MM Chr Chromatography Paper</td>
			<td>Fisher Scientific</td>
			<td>05-716-3E</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
		</tr>
		<tr>
			<td>Ball roll</td>
			<td>---</td>
			<td>---</td>
		</tr>
		<tr>
			<td>PVC pipe (5 cm inner diameter)</td>
			<td>Home Depot</td>
			<td>531137</td>
		</tr>
		<tr>
			<td>Hacksaw for PVC cutting</td>
			<td>Any</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>90 degree elbow (5 cm inner diameter)</td>
			<td>Home Depot</td>
			<td>RCE-2000-S</td>
		</tr>
		<tr>
			<td>Tee coupler (5 cm inner diameter)</td>
			<td>Home Depot</td>
			<td>PVC024001600HD</td>
		</tr>
		<tr>
			<td>PVC adhesive</td>
			<td>Any</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Lag bolt (0.6 cm diameter by 7 cm length)</td>
			<td>Home Depot</td>
			<td>801366</td>
		</tr>
		<tr>
			<td>Size 4 soccer ball</td>
			<td>Any</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Pressure gauge</td>
			<td>Any</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Hand air pump</td>
			<td>Any</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Fabric scissors</td>
			<td>Any</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Scott Rags-In-A-Box</td>
			<td>Uline</td>
			<td>S-12809</td>
		</tr>
		<tr>
			<td>Adhesive tape</td>
			<td>Any</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>8 oz sealable glass jar</td>
			<td>Any</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
		</tr>
		<tr>
			<td>Hand wipe</td>
			<td>---</td>
			<td>---</td>
		</tr>
		<tr>
			<td>100% cotton heavyweight inspection gloves</td>
			<td>Uline</td>
			<td>S-19284</td>
		</tr>
		<tr>
			<td>Digital camera</td>
			<td>Any</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>ImageJ software</td>
			<td>National Institutes of Health</td>
			<td>https://imagej.nih.gov/ij/</td>
		</tr>
		<tr>
			<td>Digital scale that measures up to 400 g</td>
			<td>Any</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Stopwatch</td>
			<td>Any</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Plastic bucket (23 cm diameter)</td>
			<td>Home Depot</td>
			<td>209313</td>
		</tr>
		<tr>
			<td>16 oz sealable glass jar</td>
			<td>Any</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
		</tr>
		<tr>
			<td>Modifed California roller</td>
			<td>---</td>
			<td>---</td>
		</tr>
		<tr>
			<td>Metal conduit (1.25 cm diameter by 1.8 m length)</td>
			<td>Home Depot</td>
			<td>101543</td>
		</tr>
		<tr>
			<td>PVC pipe (10 cm inner diameter)</td>
			<td>Home Depot</td>
			<td>531103</td>
		</tr>
		<tr>
			<td>Sand + reebar to bring roller to 14.5 kg</td>
			<td>Any</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>PVC plug</td>
			<td>Home Depot</td>
			<td>33403D</td>
		</tr>
		<tr>
			<td>Polyurethane foam sheet (1.25 cm thick) to cover PVC pipe</td>
			<td>Any</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>6 mm painter&#39;s plastic</td>
			<td>Any</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Plexiglass sheet (107 cm length by 76 cm width by 0.6 cm thick)</td>
			<td>Any</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Toggle clamps</td>
			<td>Any</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Metal nails (10 to 15 cm length) to secure frame to ground</td>
			<td>Any</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>100% cotton sheets (&#62; 200 threadcount)</td>
			<td>Any</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td>Any</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>32 oz sealable glass jar</td>
			<td>Any</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
		</tr>
		<tr>
			<td>Turfgrass vegetation core collection</td>
			<td>---</td>
			<td>---</td>
		</tr>
		<tr>
			<td>Lever action golf course cup cutter</td>
			<td>Par Aide Product Company</td>
			<td>1001-1</td>
		</tr>
		<tr>
			<td>Knife</td>
			<td>Any</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Fiskars Herb Scissors</td>
			<td>Home Depot</td>
			<td>96086966J</td>
		</tr>
		<tr>
			<td>Turf plug plastic container</td>
			<td>Any</td>
			<td>NA</td>
		</tr>
	</tbody>
</table>

protocols for quantifying transferable pesticide residues in turfgrass systems

Plant canopies in established turfgrass systems can intercept an appreciable amount of sprayed pesticides, which can be transferred through various routes onto humans. For this reason, transferable pesticide residue experiments are required for registration and re-registration by the United States Environmental Protection Agency (USEPA). Although such experiments are required, limited specificity is required pertaining to experimental approach. Experimental approaches used to assess pesticide transfer to humans including hand wiping with cotton gloves, modified California roller (moving a roller of known mass over cotton cloth) and soccer ball roll (ball wrapped with sorbent strip) over three treated turfgrass species (creeping bentgrass, hybrid bermudagrass and tall fescue maintained at 0.4, 5 and 9 cm, respectively) are presented. The modified California roller is the most extensively utilized approach to date, and is best suited for use at low mowing heights due to its reproducibility and large sampling area. The soccer ball roll is a less aggressive transfer approach; however, it mimics a very common occurrence in the most popular international sport, and has many implications for nondietary pesticide exposure from hand-to-mouth contact. Additionally, this approach may be adjusted for other athletic activities with limited modification. Hand wiping is the best approach to transfer pesticides at higher mowing heights, as roller-based approaches can lay blades over; however, it is more subjective due to more variable sampling pressure. Utility of these methods across turfgrass species is presented, and additional considerations to conduct transferable pesticide residue research in turfgrass systems are discussed.

Building on previous research efforts comparing transferable pesticide residue methods within a single turfgrass system, and turfgrass systems within a single transferable pesticide residue method, a field study (initiated May 24, 2016 in Raleigh, North Carolina, USA) was conducted to compare methods across turfgrass systems. In short, 2,4-D, a broadleaf herbicide used commonly in turfgrass systems, transfer from three turfgrass species (creeping bentgrass, Agrostis stolonifera L.; hybrid bermudagrass, Cynodon dactylon L. x C. transvaalensis Burtt-Davy; tall fescue, Lolium arundinaceum [Schreb.] S.J. Darbyshire) via three methods (hand wipe, modified California roller or soccer ball roll) was quantified immediately after application and a 1 h drying period, as well as 1 and 3 DAT. Creeping bentgrass mowing height simulated a golf course putting green at 0.4 cm, while hybrid bermudagrass and tall fescue were maintained at 5 and 9 cm, respectively, which is representative of commercial/residential lawns and parks. Research areas were not mown and covered during rainfall following broadcast 2,4-D spray application (1 kg acid equivalent ha-1).
Compared to previous reports evaluating transferable 2,4-D from treated turfgrass, data from the presented research suggest conditions were favorable for transfer across all methods. Averaged over methods, 2,4-D transfer immediately after application ranked creeping bentgrass (21% of applied) &#62; hybrid bermudagrass (16.4%) = tall fescue (15.1%), which aligns with canopy density trends across systems (Table 1; Figure 5). Averaged over turfgrasses, 2,4-D transfer immediately after application ranked hand wipe (21.2% of applied) &#62; modified California roller (16.8%) = soccer ball roll (14.4%), which agrees with previous efforts showing hand/shoe sampling can transfer more pesticide residue compared to other transfer techniques including drag- and roller-based methods10,11. A 1 h drying period resulted in a 2- to 4-fold decrease in transferable 2,4-D residues across turfgrasses and with modified California roller and hand wiping methods, while transfer decreased 36-fold from soccer ball roll. This decline agrees with Jeffries et al.14, who reported 2,4-D transfer via soccer ball roll decreased from 11.2% of the applied immediately after application on hybrid bermudagrass to 0.3% after a 1 h drying period. These data emphasize the effect sample collection time and method applied have in quantifying transferable residues from turfgrass. A soccer ball roll is a relatively specific process in turfgrass systems, and while it provides pertinent information for human exposure on an athletic field, it may not be as appropriate to solely utilize for general human exposure risk assessments as other methods.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td fo:text-align="center"></td>
			<td fo:text-align="center" style="text-align: center;">Creeping bentgrass</td>
			<td fo:text-align="center" style="text-align: center;">Hybrid bermudagrass</td>
			<td fo:text-align="center" style="text-align: center;">Tall fescue</td>
			<td fo:text-align="center" style="text-align: center;">Ball roll</td>
			<td fo:text-align="center" style="text-align: center;">Mod. Cal. roller</td>
			<td fo:text-align="center" style="text-align: center;">Hand wipe</td>
		</tr>
		<tr>
			<td fo:text-align="center"></td>
			<td colspan="6" fo:text-align="center" style="text-align: center;">_________________________ % dislodged of applied 2,4-D _________________________</td>
		</tr>
		<tr>
			<td fo:text-align="center">0 DAT - 0 h</td>
			<td fo:text-align="center" style="text-align: center;">21</td>
			<td fo:text-align="center" style="text-align: center;">16.4</td>
			<td fo:text-align="center" style="text-align: center;">15.1</td>
			<td fo:text-align="center" style="text-align: center;">14.4</td>
			<td fo:text-align="center" style="text-align: center;">16.8</td>
			<td fo:text-align="center" style="text-align: center;">21.2</td>
		</tr>
		<tr>
			<td fo:text-align="center">LSD0.05</td>
			<td colspan="3" fo:text-align="center" style="text-align: center;">_______________ 2.8 _______________</td>
			<td colspan="3" fo:text-align="center" style="text-align: center;">_______________ 2.8 _______________</td>
		</tr>
		<tr>
			<td fo:text-align="center">0 DAT - 1 h</td>
			<td fo:text-align="center" style="text-align: center;">5</td>
			<td fo:text-align="center" style="text-align: center;">6.8</td>
			<td fo:text-align="center" style="text-align: center;">4.9</td>
			<td fo:text-align="center" style="text-align: center;">0.4</td>
			<td fo:text-align="center" style="text-align: center;">7.7</td>
			<td fo:text-align="center" style="text-align: center;">8.5</td>
		</tr>
		<tr>
			<td fo:text-align="center">LSD0.05</td>
			<td colspan="3" fo:text-align="center" style="text-align: center;">_______________ 1.0 _______________</td>
			<td colspan="3" fo:text-align="center" style="text-align: center;">_______________ 1.0 _______________</td>
		</tr>
	</tbody>
</table>
Table 1: Transferable 2,4-D from field plots the day of application. Main effect of turfgrass species and sampling method on transferable 2,4-D data reported as percent of the initial application rate. Sample collections occurred immediately following application and after a 1 h drying time.
<img alt="Figure 5" src="/files/ftp_upload/55182/55182fig5.jpg" /> 
Figure 5: Turfgrass systems evaluated. Turfgrass canopy density and height can vary by system. Within the presented research, density (highest to lowest) ranked creeping bentgrass (A) &#62; hybrid bermudagrass (B) &#62; tall fescue (C); while height (highest to lowest) ranked tall fescue &#62; hybrid bermudagrass &#62; creeping bentgrass. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55182/55182fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Data from 1 and 3 DAT suggests sample collection methods do not transfer 2,4-D from treated vegetation similarly across turfgrass species, which was hypothesized due to varying canopy dynamics. Within a sample collection method at 1 DAT, 2,4-D transfer from hybrid bermudagrass (17.3 to 31.2% of applied) was greater than creeping bentgrass (10.6 to 16.2%) and tall fescue (8.1 to 20.9%), which is likely due in part to varying canopy dynamics and herbicide-physiological effects across species (Table 2). Elucidating this occurrence is beyond the scope of this experiment; however, it is highlighted to demonstrate the importance of turfgrass species selection for transferable pesticide residue research. Transferable 2,4-D did not vary between methods on creeping bentgrass at 1 or 3 DAT, which was the finest textured, lowest mown turfgrass evaluated. This allowed for relatively consistent sorbent material-treated vegetation contact across the three evaluated methods. 2,4-D transfer varied across methods in hybrid bermudagrass and tall fescue, with hand wiping resulting in the greatest transfer. Hybrid bermudagrass and tall fescue are coarser textured than creeping bentgrass, and were mown at higher heights (5 and 9 cm, respectively), which accentuates an inherent limitation of rolling-based methods of laying vegetation over (Figure 6). When this occurs, sorbent material-treated vegetation contact can be reduced and consequently, underestimate transferable residues.
<table fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td fo:text-align="center" style="text-align: center;"></td>
			<td colspan="4" fo:text-align="center" style="text-align: center;">____________________ 1 DAT - 7:00 EST ____________________</td>
		</tr>
		<tr>
			<td fo:text-align="center" style="text-align: center;">Turfgrass</td>
			<td fo:text-align="center" style="text-align: center;">Ball roll</td>
			<td fo:text-align="center" style="text-align: center;">Mod. Cal. roller</td>
			<td fo:text-align="center" style="text-align: center;">Hand wipe</td>
			<td fo:text-align="center" style="text-align: center;">LSD0.05</td>
		</tr>
		<tr>
			<td fo:text-align="center" style="text-align: center;"></td>
			<td colspan="4" fo:text-align="center" style="text-align: center;">____________ % dislodged of applied 2,4-D ____________</td>
		</tr>
		<tr>
			<td fo:text-align="center" style="text-align: center;">Creeping bentgrass</td>
			<td fo:text-align="center" style="text-align: center;">10.6</td>
			<td fo:text-align="center" style="text-align: center;">13.6</td>
			<td fo:text-align="center" style="text-align: center;">16.2</td>
			<td fo:text-align="center" style="text-align: center;">NS</td>
		</tr>
		<tr>
			<td fo:text-align="center" style="text-align: center;">Hybrid bermudagrass</td>
			<td fo:text-align="center" style="text-align: center;">17.3</td>
			<td fo:text-align="center" style="text-align: center;">20.9</td>
			<td fo:text-align="center" style="text-align: center;">31.2</td>
			<td fo:text-align="center" style="text-align: center;">2.2</td>
		</tr>
		<tr>
			<td fo:text-align="center" style="text-align: center;">Tall fescue</td>
			<td fo:text-align="center" style="text-align: center;">8.1</td>
			<td fo:text-align="center" style="text-align: center;">9.1</td>
			<td fo:text-align="center" style="text-align: center;">20.9</td>
			<td fo:text-align="center" style="text-align: center;">2.7</td>
		</tr>
		<tr>
			<td fo:text-align="center" style="text-align: center;">LSD0.05</td>
			<td fo:text-align="center" style="text-align: center;">3.2</td>
			<td fo:text-align="center" style="text-align: center;">3.2</td>
			<td fo:text-align="center" style="text-align: center;">5.2</td>
			<td fo:text-align="center" style="text-align: center;"></td>
		</tr>
		<tr>
			<td fo:text-align="center" style="text-align: center;"></td>
			<td colspan="4" fo:text-align="center" style="text-align: center;">____________________ 3 DAT - 7:00 EST ____________________</td>
		</tr>
		<tr>
			<td fo:text-align="center" style="text-align: center;">Turfgrass</td>
			<td fo:text-align="center" style="text-align: center;">Ball roll</td>
			<td fo:text-align="center" style="text-align: center;">Mod. Cal. roller</td>
			<td fo:text-align="center" style="text-align: center;">Hand wipe</td>
			<td fo:text-align="center" style="text-align: center;">LSD0.05</td>
		</tr>
		<tr>
			<td fo:text-align="center" style="text-align: center;"></td>
			<td colspan="4" fo:text-align="center" style="text-align: center;">____________ % dislodged of applied 2,4-D ____________</td>
		</tr>
		<tr>
			<td fo:text-align="center" style="text-align: center;">Creeping bentgrass</td>
			<td fo:text-align="center" style="text-align: center;">1.9</td>
			<td fo:text-align="center" style="text-align: center;">2.8</td>
			<td fo:text-align="center" style="text-align: center;">3.1</td>
			<td fo:text-align="center" style="text-align: center;">NS</td>
		</tr>
		<tr>
			<td fo:text-align="center" style="text-align: center;">Hybrid bermudagrass</td>
			<td fo:text-align="center" style="text-align: center;">1.9</td>
			<td fo:text-align="center" style="text-align: center;">4.8</td>
			<td fo:text-align="center" style="text-align: center;">7.6</td>
			<td fo:text-align="center" style="text-align: center;">2.1</td>
		</tr>
		<tr>
			<td fo:text-align="center" style="text-align: center;">Tall fescue</td>
			<td fo:text-align="center" style="text-align: center;">1.8</td>
			<td fo:text-align="center" style="text-align: center;">2</td>
			<td fo:text-align="center" style="text-align: center;">6.8</td>
			<td fo:text-align="center" style="text-align: center;">2.6</td>
		</tr>
		<tr>
			<td fo:text-align="center" style="text-align: center;">LSD0.05</td>
			<td fo:text-align="center" style="text-align: center;">NS</td>
			<td fo:text-align="center" style="text-align: center;">0.9</td>
			<td fo:text-align="center" style="text-align: center;">3.4</td>
			<td fo:text-align="center" style="text-align: center;"></td>
		</tr>
	</tbody>
</table>
Table 2: Transferable 2,4-D from field plots 1 and 3 days after application. Turfgrass species-by-sampling method interaction on transferable 2,4-D data reported as percent of the initial application rate. Sample collections occurred at 7:00:00 eastern standard time.
<img alt="Figure 6" src="/files/ftp_upload/55182/55182fig6.jpg" /> 
Figure 6: Tall fescue canopy following modified California roller sampling. An inherent limitation to this transferable pesticide residue sampling method is increasing potential for grass blades to lie down as canopy height increases. When this occurs, abaxial surfaces are not contacted by the cotton sorbent sheet, thus, potentially underestimating transferable residues. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55182/55182fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Protocols for Quantifying Transferable Pesticide Residues in Turfgrass Systems at JoVE.com

Protocols for Quantifying Transferable Pesticide Residues in Turfgrass Systems

Transferable pesticide residue experiments in turfgrass systems are integral components of human risk exposure assessments. Experimental approaches to measure transferable residues should be adjusted to the human interaction of interest and turfgrass system dynamics. Three transferable pesticide residue protocols are presented and the suitability across three turfgrass systems is discussed.

Plant canopies in established turfgrass systems can intercept an appreciable amount of sprayed pesticides, which can be transferred through various routes ...

protocols-for-quantifying-transferable-pesticide-residues-turfgrass

Nanyang Technological University

Research

JoVE Journal

Environment

7.2K Views.  North Carolina State University. Transferable pesticide residue experiments in turfgrass systems are integral components of human risk exposure assessments. Experimental approaches to measure transferable residues should be adjusted to the human interaction of interest and turfgrass system dynamics. Three transferable pesticide residue protocols are presented and the suitability across three turfgrass systems is discussed.

Video: Protocols for Quantifying Transferable Pesticide Residues in Turfgrass Systems

A Whole Cell Bioreporter Approach to Assess Transport and Bioavailability of Organic Contaminants in Water Unsaturated Systems

Bioavailability of contaminants is a prerequisite for their effective biodegradation in soil. The average bulk concentration of a contaminant, however, is not an appropriate measure for its availability; bioavailability rather depends on the dynamic interplay of potential mass transfer (flux) of a compound to a microbial cell and the capacity of the latter to degrade the compound. In water-unsaturated parts of the soil, mycelia have been shown to overcome bioavailability limitations by actively transporting and mobilizing organic compounds over the range of centimeters. Whereas the extent of mycelia-based transport can be quantified easily by chemical means, verification of the contaminant-bioavailability to bacterial cells requires a biological method. Addressing this constraint, we chose the PAH fluorene (FLU) as a model compound and developed a water unsaturated model microcosm linking a spatially separated FLU point source and the FLU degrading bioreporter bacterium Burkholderia sartisoli RP037-mChe by a mycelial network of Pythium ultimum. Since the bioreporter expresses eGFP in response of the PAH flux to the cell, bacterial FLU exposure and degradation could be monitored directly in the microcosms via confocal laser scanning microscopy (CLSM). CLSM and image analyses revealed a significant increase of the eGFP expression in the presence of P. ultimum compared to controls without mycelia or FLU thus indicating FLU bioavailability to bacteria after mycelia-mediated transport. CLSM results were supported by chemical analyses in identical microcosms. The developed microcosm proved suitable to investigate contaminant bioavailability and to concomitantly visualize the involved bacteria-mycelial interactions.

A whole cell bioreporter assay with Burkholderia sartisoli RP037-mChe was developed to detect fractions of an organic contaminant (i.e., fluorene) available for bacterial degradation after active transport by mycelia bridging air-filled pores in a water unsaturated model system.

Bioavailability of contaminants is a prerequisite for their effective biodegradation in soil. The average bulk concentration of a contaminant, however, is ...

A New Application of the Electrical Penetration Graph (EPG) for Acquiring and Measuring Electrical Signals in Phloem Sieve Elements

Electrophysiological properties of cells are often studied in vitro, after dissociating them from their native environments. However, the study of electrical transmission between distant cells in an organism requires in vivo, artifact-free recordings of cells embedded within their native environment. The transmission of electrical signals from wounded to unwounded areas in a plant has since long piqued the interest of botanists. The phloem, the living part of the plant vasculature that is spread throughout the plant, has been postulated as a major tissue in electrical transmission in plants. The lack of suitable electrophysiological methods poses many challenges for the study of the electrical properties of the phloem cells in vivo. Here we present a novel approach for intracellular electrophysiology of sieve elements (SEs) that uses living aphids, or other phloem-feeding hemipteran insects, integrated in the electrical penetration graph (EPG) circuit. The versatility, robustness, and accuracy of this method made it possible to record and study in detail the wound-induced electrical signals in SEs of central veins of the model plant Arabidopsis thaliana1. Here we show that EPG-electrodes can be easily implemented for intracellular electrophysiological recordings of SEs in marginal veins, as well as to study the capacity of SEs to respond with electrical signals to several external stimuli. The EPG approach applied to intracellular electrophysiology of SEs can be implemented to a wide variety of plant species, in a large number of plant/insect combinations, and for many research aims.

A New Application of the Electrical Penetration Graph EPG for Acquiring and Measuring Electrical Signals in Phloem Sieve Elements

Electrical Penetration Graph (EPG) is a well-established technique for studying the feeding behavior of stylet-bearing insects. Here we show a new application of EPG as a non-invasive tool for the acquisition of intracellular electrophysiology recordings of sieve elements (SEs), the cells that form the phloem vasculature in plants.

Electrophysiological properties of cells are often studied in vitro, after dissociating them from their native environments. However, the study of ...

Protocols for Robust Herbicide Resistance Testing in Different Weed Species

Robust protocols to test putative herbicide resistant weed populations at whole plant level are essential to confirm the resistance status. The presented protocols, based on whole-plant bioassays performed in a greenhouse, can be readily adapted to a wide range of weed species and herbicides through appropriate variants. Seed samples from plants that survived a field herbicide treatment are collected and stored dry at low temperature until used. Germination methods differ according to weed species and seed dormancy type. Seedlings at similar growth stage are transplanted and maintained in the greenhouse under appropriate conditions until plants have reached the right growth stage for herbicide treatment. Accuracy is required to prepare the herbicide solution to avoid unverifiable mistakes. Other critical steps such as the application volume and spray speed are also evaluated. The advantages of this protocol, compared to others based on whole plant bioassays using one herbicide dose, are related to the higher reliability and the possibility of inferring the resistance level. Quicker and less expensive in vivo or in vitro diagnostic screening tests have been proposed (Petri dish bioassays, spectrophotometric tests), but they provide only qualitative information and their widespread use is hindered by the laborious set-up that some species may require. For routine resistance testing, the proposed whole plant bioassay can be applied at only one herbicide dose, so reducing the costs.

A robust and flexible approach to confirm herbicide resistance in weed populations is presented. This protocol allows the herbicide resistance levels to be inferred and applied to a wide range of weed species and herbicides with minor adaptations.

Robust protocols to test putative herbicide resistant weed populations at whole plant level are essential to confirm the resistance status. The presented ...

LC-MS Analysis of Human Platelets as a Platform for Studying Mitochondrial Metabolism

Perturbed mitochondrial metabolism has received renewed interest as playing a causative role in a range of diseases. Probing alterations to metabolic pathways requires a model in which external factors can be well controlled, allowing for reproducible and meaningful results. Many studies employ transformed cellular models for these purposes; however, metabolic reprogramming that occurs in many cancer cell lines may introduce confounding variables. For this reason primary cells are desirable, though attaining adequate biomass for metabolic studies can be challenging. Here we show that human platelets can be utilized as a platform to carry out metabolic studies in combination with liquid chromatography-tandem mass spectrometry analysis. This approach is amenable to relative quantification and isotopic labeling to probe the activity of specific metabolic pathways. Availability of platelets from individual donors or from blood banks makes this model system applicable to clinical studies and feasible to scale up. Here we utilize isolated platelets to confirm previously identified compensatory metabolic shifts in response to the complex I inhibitor rotenone. More specifically, a decrease in glycolysis is accompanied by an increase in fatty acid oxidation to maintain acetyl-CoA levels. Our results show that platelets can be used as an easily accessible and medically relevant model to probe the effects of xenobiotics on cellular metabolism.

Here we show isolated human platelets can be used as an accessible ex vivo model to study metabolic adaptations in response to the complex I inhibitor rotenone. This approach employs isotopic tracing and relative quantification by liquid chromatography-mass spectrometry and can be applied to a variety of study designs.

Perturbed mitochondrial metabolism has received renewed interest as playing a causative role in a range of diseases. Probing alterations to metabolic ...

A Protocol for Collecting and Constructing Soil Core Lysimeters

Leaching of nutrients from land applied fertilizers and manure used in agriculture can lead to accelerated eutrophication of surface water. Because the landscape has complex and varied soil morphology, an accompanying disparity in flow paths for leachate through the soil macropore and matrix structure is present. The rate of flow through these paths is further affected by antecedent soil moisture. Lysimeters are used to quantify flow rate, volume of water and concentration of nutrients leaching downward through soils. While many lysimeter designs exist, accurately determining the volume of water and mass balance of nutrients is best accomplished with bounded lysimeters that leave the natural soil structure intact. 
Here we present a detailed method for the extraction and construction of soil core lysimeters equipped with soil moisture sensors at 5 cm and 25 cm depths. Lysimeters from four different Coastal Plain soils (Bojac, Evesboro, Quindocqua and Sassafras) were collected on the Delmarva Peninsula and moved to an indoor climate controlled facility. Soils were irrigated once weekly with the equivalent of 2 cm of rainfall to draw down soil nitrate-N concentrations. At the end of the draw down period, poultry litter was applied (162 kg TN ha-1) and leaching was resumed for an additional five weeks. Total recovery of applied irrigation water varied from 71% to 85%. Nitrate-N concentration varied over the course of the study from an average of 27.1 mg L-1 before litter application to 40.3 mg L-1 following litter application. While greatest flux of nutrients was measured in soils dominated by coarse sand (Sassafras) the greatest immediate flux occurred from the finest textured soil with pronounced macropore development (Quindocqua).

A detailed method for extraction and assembly of intact soil core lysimeters and their use for study of leachate and associated loss of nutrients from surface applied poultry litter is demonstrated.

Leaching of nutrients from land applied fertilizers and manure used in agriculture can lead to accelerated eutrophication of surface water. Because the ...

Implementation of Portable Emissions Measurement Systems (PEMS) for the Real-driving Emissions (RDE) Regulation in Europe

Vehicles are tested in controlled and relatively narrow laboratory conditions to determine their official emission values and reference fuel consumption. However, on the road, ambient and driving conditions can vary over a wide range, sometimes causing emissions to be higher than those measured in the laboratory. For this reason, the European Commission has developed a complementary Real-Driving Emissions (RDE) test procedure using the Portable Emissions Measurement Systems (PEMS) to verify gaseous pollutant and particle number emissions during a wide range of normal operating conditions on the road. This paper presents the newly-adopted RDE test procedure, differentiating six steps: 1) vehicle selection, 2) vehicle preparation, 3) trip design, 4) trip execution, 5) trip verification, and 6) calculation of emissions. Of these steps, vehicle preparation and trip execution are described in greater detail. Examples of trip verification and the calculations of emissions are given.

Implementation of Portable Emissions Measurement Systems PEMS for the Real-driving Emissions RDE Regulation in Europe

The European Commission has developed a Real-Driving Emissions (RDE) test procedure to verify pollutant emissions during real-world vehicle operation using the Portable Emissions Measurement Systems (PEMS). This paper presents the experimental procedures required by the newly-adopted RDE test.

Vehicles are tested in controlled and relatively narrow laboratory conditions to determine their official emission values and reference fuel consumption. ...

Vegetated Treatment Systems for Removing Contaminants Associated with Surface Water Toxicity in Agriculture and Urban Runoff

Urban stormwater and agriculture irrigation runoff contain a complex mixture of contaminants that are often toxic to adjacent receiving waters. Runoff may be treated with simple systems designed to promote sorption of contaminants to vegetation and soils and promote infiltration. Two example systems are described: a bioswale treatment system for urban stormwater treatment, and a vegetated drainage ditch for treating agriculture irrigation runoff. Both have similar attributes that reduce contaminant loading in runoff: vegetation that results in sorption of the contaminants to the soil and plant surfaces, and water infiltration. These systems may also include the integration of granulated activated carbon as a polishing step to remove residual contaminants. Implementation of these systems in agriculture and urban watersheds requires system monitoring to verify treatment efficacy. This includes chemical monitoring for specific contaminants responsible for toxicity. The current paper emphasizes monitoring of current use pesticides since these are responsible for surface water toxicity to aquatic invertebrates.

This article summarizes the design attributes and the effectiveness of treatment systems that treat urban stormwater and agriculture irrigation runoff to remove pesticides and other contaminants associated with aquatic toxicity.

Urban stormwater and agriculture irrigation runoff contain a complex mixture of contaminants that are often toxic to adjacent receiving waters. Runoff may ...

RGB and Spectral Root Imaging for Plant Phenotyping and Physiological Research: Experimental Setup and Imaging Protocols

Better understanding of plant root dynamics is essential to improve resource use efficiency of agricultural systems and increase the resistance of crop cultivars against environmental stresses. An experimental protocol is presented for RGB and hyperspectral imaging of root systems. The approach uses rhizoboxes where plants grow in natural soil over a longer time to observe fully developed root systems. Experimental settings are exemplified for assessing rhizobox plants under water stress and studying the role of roots. An RGB imaging setup is described for cheap and quick quantification of root development over time. Hyperspectral imaging improves root segmentation from the soil background compared to RGB color based thresholding. The particular strength of hyperspectral imaging is the acquisition of chemometric information on the root-soil system for functional understanding. This is demonstrated with high resolution water content mapping. Spectral imaging however is more complex in image acquisition, processing and analysis compared to the RGB approach. A combination of both methods can optimize a comprehensive assessment of the root system. Application examples integrating root and aboveground traits are given for the context of plant phenotyping and plant physiological research. Further improvement of root imaging can be obtained by optimizing RGB image quality with better illumination using different light sources and by extension of image analysis methods to infer on root zone properties from spectral data.

An experimental protocol is presented for assessment of soil grown plant root systems with RGB and hyperspectral imaging. Combination of RGB image time series with chemometric information from hyperspectral scans optimizes insights into plant root dynamics.

Better understanding of plant root dynamics is essential to improve resource use efficiency of agricultural systems and increase the resistance of crop ...

Development of New Methods for Quantifying Fish Density Using Underwater Stereo-video Tools

The use of video camera systems in ecological studies of fish continues to gain traction as a viable, non-extractive method of measuring fish lengths and estimating fish abundance. We developed and implemented a rotating stereo-video camera tool that covers a full 360 degrees of sampling, which maximizes sampling effort compared to stationary camera tools. A variety of studies have detailed the ability of static, stereo-camera systems to obtain highly accurate and precise measurements of fish; the focus here was on the development of methodological approaches to quantify fish density using rotating camera systems. The first approach was to develop a modification of the metric MaxN, which typically is a conservative count of the minimum number of fish observed on a given camera survey. We redefine MaxN to be the maximum number of fish observed in any given rotation of the camera system. When precautions are taken to avoid double counting, this method for MaxN may more accurately reflect true abundance than that obtained from a fixed camera. Secondly, because stereo-video allows fish to be mapped in three-dimensional space, precise estimates of the distance-from-camera can be obtained for each fish. By using the 95% percentile of the observed distance from camera to establish species-specific areas surveyed, we account for differences in detectability among species while avoiding diluting density estimates by using the maximum distance a species was observed. Accounting for this range of detectability is critical to accurately estimate fish abundances. This methodology will facilitate the integration of rotating stereo-video tools in both applied science and management contexts.

We describe a new method for counting fishes, and estimating relative abundance (MaxN) and fish density using rotating stereo-video camera systems. We also demonstrate how to use distance from camera (Z distance) to estimate species-specific detectability.

The use of video camera systems in ecological studies of fish continues to gain traction as a viable, non-extractive method of measuring fish lengths and ...

Ecosystem Fabrication (EcoFAB) Protocols for The Construction of Laboratory Ecosystems Designed to Study Plant-microbe Interactions

Beneficial plant-microbe interactions offer a sustainable biological solution with the potential to boost low-input food and bioenergy production. A better mechanistic understanding of these complex plant-microbe interactions will be crucial to improving plant production as well as performing basic ecological studies investigating plant-soil-microbe interactions. Here, a detailed description for ecosystem fabrication is presented, using widely available 3D printing technologies, to create controlled laboratory habitats (EcoFABs) for mechanistic studies of plant-microbe interactions within specific environmental conditions. Two sizes of EcoFABs are described that are suited for the investigation of microbial interactions with various plant species, including Arabidopsis thaliana, Brachypodium distachyon, and Panicum virgatum. These flow-through devices allow for controlled manipulation and sampling of root microbiomes, root chemistry as well as imaging of root morphology and microbial localization. This protocol includes the details for maintaining sterile conditions inside EcoFABs and mounting independent LED light systems onto EcoFABs. Detailed methods for addition of different forms of media, including soils, sand, and liquid growth media coupled to the characterization of these systems using imaging and metabolomics are described. Together, these systems enable dynamic and detailed investigation of plant and plant-microbial consortia including the manipulation of microbiome composition (including mutants), the monitoring of plant growth, root morphology, exudate composition, and microbial localization under controlled environmental conditions. We anticipate that these detailed protocols will serve as an important starting point for other researchers, ideally helping create standardized experimental systems for investigating plant-microbe interactions.

Ecosystem Fabrication EcoFAB Protocols for The Construction of Laboratory Ecosystems Designed to Study Plant-microbe Interactions

This article describes detailed protocols for ecosystem fabrication of devices (EcoFABs) that enable the studies of plants and plant-microbe interactions in highly controlled laboratory conditions.

Beneficial plant-microbe interactions offer a sustainable biological solution with the potential to boost low-input food and bioenergy production. A ...