TESTING FOR PERSISTENT HERBICIDES IN FEEDSTOCKS AND COMPOST

PREVIOUS articles in this series (June 2013 and August 2013) have discussed the 2012 recurrence of persistent herbicide contamination of compost in Vermont and steps composters can take to manage the risks of dealing with contaminated feedstocks and compost. Persistent herbicides (PHs) are a class of herbicides that include pyridine carboxylic acid herbicides (also called plant-growth regulators or auxin-mimics) and they can be bioactive at levels as low as 1 part per billion (ppb), or roughly one-half teaspoon in an Olympic-sized swimming pool. These herbicides are not degraded fast enough in the composting process to reach nonbioactive levels in the normal time frame for composting. Problems with contaminated composts adversely affecting horticultural crops have been reported by several facilities after first surfacing in 1999.

Testing, or characterizing, feedstocks for composting is a standard operating procedure at many facilities. Feedstocks are normally tested for parameters important to composting recipe development, such as carbon and nitrogen concentrations and moisture content. Testing is also often done for parameters that can affect final product quality, such as heavy metals and soluble salts. These tests are normally conducted with standardized chemical analysis procedures. Numerous private laboratories around the country can do the analyses, as well as many of the State Agriculture laboratories.

However, testing for PHs is very difficult. One reason is that feedstocks and composts are considered complex matrices for laboratory analysis (a simple matrix would be a sugar solution in water). The term matrix refers to the components of a sample other than the chemical being sought (the analyte); the matrix can influence both how the sample is analyzed and the results obtained. Analyzing for PHs requires that the PH be extracted from the sample using solvents. Extraction solvents can be water, water/methanol mixtures, or one of several proprietary formulations. "If composts are produced with proper attention to good process management, the herbicide residue can be extracted with water with a revised extraction procedure we are developing," explains Mike Hastings, a Principal Research Scientist with Dow AgroSciences, which manufactures PHs including picloram, clopyralid and aminopyralid. "The difficulty is that when you extract compost with water, you get compost tea, and both the color and other compounds in the extracted solution cause complications with the analysis." In the case of feedstocks like grass clippings and vegetative material, the herbicide can bind inside the plant tissues, requiring more complex extraction techniques. This new analytical procedure being developed by Dow AgroSciences is in final development and will be published for use by State agricultural and other laboratories by the middle of 2014.

Hastings notes that because these herbicide compounds have small molecular weights and are highly polar (meaning they display a high affinity for the electrostatic attraction between two oppositely charged ions), they can be difficult to separate out following extraction. In the case of herbicide analysis, separating the herbicide from an extracted sample means passing the sample through solid-phase extraction cartridges to eliminate the complicating impurities. Once separated, the sample is then analyzed using liquid chromatography and mass spectrometry procedures. The equipment needed to analyze for these herbicides at detection levels corresponding to the levels of contaminant concern to composters is very expensive and not all commercial or state agriculture laboratories have that equipment. "A high-end laboratory with this type of equipment and well-trained chemists could analyze for these compounds, but it will likely cost $150-$200 per sample," he adds.

Another consideration is that State agricultural laboratories are tasked with a different mission. "Over 90 percent of the workload in state pesticide labs is to support the enforcement programs work related to drift issues or other pesticide misuse investigation cases," explains Cary Giguere, the Agrichemical Management Section Chief for the Vermont Department of Agriculture. "Most State agriculture labs really were not designed to do work-for-hire for composters or others. We were able to help investigate and analyze the trace-back sources of the herbicides that affected Green Mountain Compost, but that was part of our enforcement investigation." Chittenden Solid Waste District's Green Mountain Compost facility in Williston, Vermont had problems with PH contamination in 2012. (See Part I, "Unraveling The Maze of Persistent Herbicides In Compost," June 2013.)

The advantage of spending the money to have a sample of a feedstock tested is that the composter can manage the PH residue through recipe algebra (see "Composters Defend Against Persistent Herbicides, Part II," August 2013). The obvious disadvantage is deciding on whether to disclose a level of contamination in the final product going to market, particularly if it is below a No Observed Adverse Effect Level (NOAEL). The NOAEL is the highest tested concentration of a substance (like a drug or a chemical) at which no adverse effect is observed where higher concentrations would show an adverse effect. For example, assume the concentration of the herbicide was low enough so as not to damage the plants in a bioassay test conducted at the composting facility. Should the numerical value of that concentration be reported? One of the challenges in releasing information about the constituents in composts is that many people do not understand the meaning of the chemical data in laboratory reports (this has been demonstrated many times by potential customers of biosolids composts who have reviewed and questioned heavy metal concentrations in the final product quality analysis).

ADDITIONAL TEST METHODS

The other methods of testing for PHs are biological; one is called immunoassay, and the other is a bioassay test. An immunoassay method measures the concentration of a chemical (known as an antigen) in a solution through the use of antibodies, where the antibody recognizes and binds to that specific chemical (antigen) and to link the antibody to some sort of detectable label, like a color change or fluorescence under black light. The process is analogous to how the human body produces antibodies to counter invading bacteria or viruses. Antibodies and their respective antigens fit together like a key and lock (Figure 1).

Antibodies specific to detection of PH antigens must be derived from hybrid cell lines cultivated from laboratory animals inoculated with proteins carrying the PH compound of interest. Once the correct antibodies are isolated, identified and verified, they can be mass-produced using standard cell culture techniques. "Immunoassay techniques offered a lot of promise in the 1990s but they are only a screening tool," notes Hastings. "They rely on colorimetric determinations so are subject to the same color interference problems we find in chromatographic methods. You still have to confirm a positive result with a more rigorous analysis. That is why they were never well-accepted as a regulatory tool for enforcement procedures."

There has been some discussion in both industry and regulatory groups about the potential to develop rapid-response testing using an immunoassay procedure known as enzymelinked immunosorbent assay (ELISA). This would be analogous to the Solvita(TM) stability tests practiced by many composters. An ELISA test works on the principle of detection of an antigen in a liquid sample by a detection antibody linked to an enzyme (a catalyst) and its substrate (the chemical upon which the enzyme acts); the reaction between the antibody, the enzyme and the substrate produces a visible signal, such as a color change. ELISA testing is used in medical evaluations (for HIV, West Nile virus, tuberculosis antibodies etc.) and in detecting potential food allergens (milk, peanuts, walnuts, almonds, eggs). ELISA can be done in a laboratory or in the field. Field testing is done with a procedure known as Lateral Flow Immunochromatographic Assays, which is the same technology used in home pregnancy test kits, malaria tests, avian flu tests and other applications. To date, no commercial ELISA test kits are on the market for pyridine carboxylic acid herbicides but some research into developing such a kit has been funded by the U.S. Department of Agriculture.

The other biological testing method is bioassay. Bioassay testing is a procedure where the concentration or potency of a substance is measured by its effect on living cells or tissues. Many of the laboratories that composters use for product quality analysis can do germination testing for product maturity, which is a form of bioassay. Bioassay testing is being used by Green Mountain Compost in Vermont. Composters should realize that doing in-house bioassays requires 90 to 100 growing pots (to measure effects of different ratios of composts and soils and to have duplicate and triplicate pots to repeat and verify results) and two to three hours per week to properly analyze for impacts. Bioassays, when set up and run properly, are the best method for evaluating the potential impacts from herbicides. But there are a number of potential interferences from salinity, boron content, pH and maturity that can create false positives and false negatives.

Woods End Laboratories in Mt. Vernon, Maine does bioassay testing for herbicide impacts with its Plant-Injury Risk Management (PIRM) tool (Figure 2). "With this technique we've been able to accomplish virtual quantitation via bioassays, meaning you get a result that actually relates to plant damage; at the same time you can extrapolate to probable concentration for differing molecules," explains Will Brinton, Director of Woods End Farm and Laboratories. Brinton's PIRM tool links plantinjury ranks with compost application rates for insensitive, moderately sensitive, and sensitive crops. The output is the NOAEL for each category of crops, however, as Brinton notes, "in some cases the injury cannot be avoided, so no application rate is considered appropriate."

FINAL ASSESSMENT

So, where do we go from here? The reality for the composting industry is that these PHs are not going away, and chemical/biological testing for them is going to remain expensive. Therefore, composters should develop management protocols for dealing with them. Options include refusing to accept potentially problematic feedstocks, developing in-house bioassay tools (or using a commercial lab to do the bioassay), and seeking expansion of the prohibition on PH usage in a state, which can be done by working with the herbicide producer and the appropriate Association of American Pesticide Control Organization (AAPCO) official in each state (www.aapco.org/).

Other initiatives to consider include development of a "chain-ofcustody" program for problematic feedstocks, similar to the ones in place for handling hazardous chemicals. The composting facility, which is the last stop in the chain, would have details on where the feedstocks were sourced. "One challenge to developing a chain-of'Custody program is deciding who would run the program," notes Giguere. "Maybe that is something a nonprofit organization could do, similar to how the organic agriculture certification program is run?" Another idea being discussed is a chain-of-custody program offered as product stewardship with the producers of PHs, but that will be complicated by the fact that some of these herbicides have gone offpatent protection and there are now numerous producers.

Work is underway to develop faster analytical procedures, to understand the extent of PH degradation during active composting and to continue the ongoing dialogues between industry officials, state agriculture departments and composters. This type of collaboration will be important to help composters manage their feedstocks and their facilities in the future.

New testing methods may be available by mid2014, including molecularly imprinted polymers that simplify extraction and analysis procedures, and a "dipstick" to test for the presence, although not necessarily the concentration, of certain persistent herbicides.

Part III

Craig Coker

GREEN MOUNTAIN COMPOST LOOKS BACK, AND AHEAD

BioCycle sat down with Tom Moreau, General Manager of the Chittenden Solid Waste District in Williston, Vermont, to discuss the problems of persistent herbicides. The District's composting facility and staff have been at the center of this most recent incident involving the herbicides, starting in June 2012.

BioCycle: What analytical techniques seem to hold the most promise for detecting persistent herbicides in compost feedstock streams?

Moreau: Dow AgroSciences is in the midst of validating a new method of analyzing for clopyralid, picloram and aminopyralid in composts and manures. The new method is based upon use of molecularly imprinted polymers and significantly simplifies what has been a difficult extraction and analysis procedure requiring highly trained chemists and very sophisticated instrumentation. Dow hopes to have the new method validated and published by May 2014. This will allow for composters to ascertain the presence and concentration of all three of these compounds in incoming manures as well as in their composts down to about 1 part per billion on a dry weight basis.

Also, a company in Pennsylvania called Abraxis (www.abraxiskits.com/ index.php) is working on an ELISA test that will be able to detect the presence of Dow's clopyralid, picloram and aminopyralid in manures and composts. They have indicated to me that their "dipstick" kits will be available in the summer of 2014. This should allow composters to test for the presence, although not necessarily the concentration, of certain persistent herbicides in both manures and composts without going to an outside laboratory. And, DuPont has already worked with three commercial laboratories in the U.S. to accurately test for aminocyclopyrachlor, although that compound may have some delay in gaining approval for use on pasture land.

BioCycle: These PHs are coming up for reregistration with the U.S. EPA in 2014. It is a reasonable assumption that products like aminopyralid will not be taken off the market because of their impact on compost products. So what are reasonable requests that composters can make of the herbicide manufacturers and the EPA?

Moreau: After speaking with the USEPA, representatives from Dow AgroSciences and representatives from the Association of American Pesticide Control Officials (AAPCO), I believe that products like aminopyralid will not be taken off the market due to their relative safety compared to other herbicides and to their effectiveness in controlling invasive species and other broadleaf weeds. I understand that these compounds are used and desired by a significant portion of our agricultural industry. Therefore, composters will need to learn to coexist with these compounds. I believe that effective testing methodology should be required from the manufacturers as well as an upgraded label such as the one that we saw for Milestone in New England in 2013. There may be some additional product stewardship requirements such as establishing a private database and assistance program for composters that have been impacted. There are some very good points in both the AAPCO compost issue paper and the USCC Position Statement that, if implemented during the reregistration process, would significantly help protect composters.

BioCycle: What lessons were learned from Imprelis, and the fact that the damage it caused to trees (due to runoff from application on turf) led that product to be suspended by Dupont? For example, was the direct cause/effect much easier to prove analytically and in practice? Was it because it is a landscaping product versus an agricultural herbicide?

Moreau: Imprelis, or aminocyclopyrachlor, was on the market as a turfgrass or landscaping application for a short time, from November 2011 through August 2012, and not in all the states. Given that Scotts was licensed to use the compound in turfgrass applications, it had the potential for significant input as a compost feedstock in grass clippings. My subsequent conversations with representatives of DuPont indicate that they are now very sensitive to how such a product could be a problem to composters and will likely take that into account as they pursue registration for turf or pastureland usage.

BioCycle: What advice would you give to compost operators based on Green Mountain Compost's experience with aminopyralid. What are their best lines of defense and what are their best options for analyzing for presence of these herbicides?

Moreau: First, understand the nature of your feedstocks. If you accept manure, verify the sources of feed for the animals. Be especially careful of horse manure because many stables prefer a "grass only" or "weed free" hay. Second, if you suspect an issue with a particular manure, certain agronomy labs have good experience running bioassays on manures and can tell you the relative strength of the herbicides and how much of that material could be used in your feedstock recipe. Third, check the website of your State's Agriculture Department. They often publish how much of a persistent herbicide was used in your county. Fourth, routinely run bioassays on your composts both before and after the curing phase. We run our bioassays using at least two sensitive broadleaf species like beans, peas, tomatoes or clover and run the tests for at least four weeks in duration.

Composters should line up a laboratory that can test for the presence and concentration of persistent herbicides in both manures and composts. The lab could be your State Agricultural lab or a private commercial lab. For Dow AgroSciences' products, this should be much easier by mid-2014. Check on the Abraxiskits.com website to see if the dipstick tests for persistent herbicides are available after June 2014. Should your compost test positive for persistent herbicides with either a bioassay test or an actual chemical test, then cross check with the other type of test. For example, if your bioassay test using beans showed some distinct curling after three weeks, you should then send out some of the same compost or manure for a chemical test.

Another point is that we are seeing that low levels of persistent herbicides may be helped by dilution or by adding a source of activated carbon. For example, we have chemically tested some composts with clopyralid concentrations of up to 3 parts per billion dry weight that did not show impact in subsequent bioassay tests. Also, we have had noticeable impact with bioassays of compost with 1.5 ppb of aminopyralid and were able to alleviate the symptoms with addition of a high carbon wood ash.

"These compounds are used and desired by a significant portion of our agricultural industry. Therefore composters will need to learn to coexist with these compounds .... Effective testing methodology should be required from the manufacturers." - Tom Moreau, CSWD

Equipment needed to analyze for these herbicides at detection levels corresponding to the levels of contaminant concern to composters is very expensive and not always available at labs.

There has been discussion in both industry and regulatory groups about the potential to develop rapidresponse testing using an immunoassay procedure known as ELISA.

Craig Coker is a Contributing Editor to BioCycle and a Principal in the firm Coker Composting & Consulting (www.cokercompost.com), near Roanoke, VA. He can be reached at [email protected].