Matt Hengel. Washington Hop Commission

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1 Evaluation of Input Residue and Biological Material Impacts from Commercially and Organically Grown Hops on Different Commercial Beer Production Methods Ann George Doug Walsh Matt Hengel Washington Hop Commission

2 Purpose of Study As the market for specialty beers has grown, questions have been raised regarding the impact of higher hopping rates and changes in the timing of hop additions during the brewing process on the quantity of pesticide (and other) contaminants in beer. This study sought to address those questions.

3 Project Launched Washington State Department of Agriculture Specialty Crop Block Grant Collaboration between Dr. Doug Walsh, IR-4 State Liaison Representative, Washington State University Dr. Matt Hengel, Western Region IR-4 Laboratory Coordination and oversight by U.S. Hop Industry Plant Protection Committee

4 Pesticide Selection Rationale Commonly used by growers Heavier-than-average usage rate Maximum residue levels on the higher end of the scale Range of polarity (solubility)

5 Hops In Our Study We used hops grown in conventional, organic, and untreated yards to brew beers, defined for this study as follows: Conventional - Hops produced at WSU IAREC that received a relatively heavy regime of commercially available pesticide applications. Organic - Hops purchased from a WSDA organically certified hop grower. Untreated - Hops from a rehabilitated hopyard that had received no pesticide applications for over 5 years (aka, the James butterfly garden).

6 With All 3 Hop Types We brewed beers using late additions of: fresh/green hops dried whole hops, and pelletized hops and compared the pesticide residues in these beers to beers brewed using additions of: dried hops during the traditional kettle boil stage, and beers brewed with no hops at all.

7 Pesticide residues were analyzed by Dr. Hengel from all 3 growing systems On freshly harvested wet hops On hops dried in our research kiln at WSU to 8% moisture.

8 In 2012 we established plots in a 2 acre cv. Cascade block These plots received 120# N (dry urea) and an additional 120# N by chemigation in 2012 and 2013

9 Most pesticides were applied with our airblast sprayer in 90 gallons per acre water carrier. Pesticides selected were among the suspect pesticides. These are pesticides that are polar in nature and tend to get applied at high concentrations of active ingredient per acre. All applications were made by our EPA certified Good Laboratory Practices applicators following GLP compliant protocols. The only deviation from GLP is that we did not pay $$ for a quality assurance evaluation

10 Detail of FUNGICIDE applications to conventional block of hops. All applications were by airblast sprayer at 90 gallons per acre.

11 Detail of INSECTICIDE/MITICIDE applications to conventional block of hops. All applications were by airblast sprayer at 90 gallons per acre.

12 Detail of HERBICIDE applications to conventional block of hops. All except carfentrazone ethyl were applied by airblast sprayer at 90 gallons per acre.

13 Detail of FUNGICIDE applications to ORGANIC block of hops.

14 Detail of INSECTICIDE/MITICIDE applications to ORGANIC block of hops.

15 Hops from all 3 growing systems were harvested, transported to our Wolf picker at WSU IAREC, and then stripped. An aliquot of hops from each plot was frozen for pesticide residue analysis at UC Davis by Dr. Hengel

16 All the rest of the hops were then dried to 8% moisture in our research floor dryer (aka research hop kiln) An aliquot of hops from each plot was frozen for residue analysis at UC Davis by Dr. Hengel. The rest of the hops were kept for brewing and pelletizing

17 An aliquot of dried hops was then taken from hops harvested from each plot and pelletized in our research hop grinder/pelletizer. We can load this pelletizer with a minimum of 150g of whole dried cones and get 100g of pellets

18 All the beers in this study were brewed in the WSU Research Brewery located in the Hop Research Facility at WSU IAREC

19 Brewing: Test beers were divided into four treatments with different hopping regimens. 1. (60 Minute Boil) Hops were added to the wort 15 minutes after the start of boil and boiled for 60 minutes. 2. (5 Minute Boil) Hops were added 70 minutes after the start of boil and boiled for 5 minutes. 3. (Flameout) Hops were added at flameout, immediately after the end of the boil. 4. (Dry/wet Hop) Hops were not added to the brew kettle, but were added to the secondary fermenting container immediately before racking.

20 Brewing: Each of these four treatments were brewed using: fresh (wet) conventionally treated hops, dried conventionally treated whole hop cones, dried organically treated whole hop cones, dried untreated whole hop cones, and hop pellets formed from conventionally treated hops. * beers were also brewed with no hops added as untreated control beers

21 Hopping rates by brewing method Brewing method Dry hops Pelletized hops Wet hops* 60 minute boil 70 g 70 g 350 g (1.0 lb/ barrel) (1.0 lb/ barrel) (5.0 lb/ barrel) 5 minute boil 36.6 g (0.5 lb/ barrel) 36.6 g (0.5 lb/ barrel) g (2.5 lb/ barrel) Flame out 73.2 g 73.2 g g (1.0 lb/ barrel) (1.0 lb/ barrel) (5.0 lb/ barrel) Dry or wet 7 days 73.2 g (1.0 lb/ barrel) 73.2 g (1.0 lb/ barrel) g (5.0 lb/ barrel) No hops 0 g 0 g 0 g */Fresh hops are typically five times heavier than dried hops

22 Analytical Method (Hops) Homogenize 0.5g Hops + 15 ml ACN Solid Phase Extraction Concentrate LC-MS/MS ESI * Hengel, J. Am. Soc. Brew. Chem. 69(3): , 2011

23 Analytical Method (Hops)

24 Analytical Method (Beer)

25 Analytical Method (Beer) Developed and validated method in conjunction with Dr. Steve Thun at Pacific Agricultural Laboratory (Portland, OR). Designed method to screen for ~ 30 pesticides currently used on hops. Limit of Quantitation = 0.5 ppb

26 Analytical Method (Beer) 20 ml Beer+ 5 ml ACN + Salt Shake LC-MS/MS ESI

27 Analytical Method (Beer)

28 Instrumentation (Hops and Beer) The utilization of liquid chromatography coupled to a tandem mass spectrometer (LC- MS/MS) gives us good pesticide specificity and is amendable to a wide range of pesticides.

29 Some key pesticide terms! EPA evaluates pesticides thoroughly to ensure that they will not harm human health or the environment. Pesticides that pass this evaluation are granted a "registration. Registration permits the sale and use of a pesticide according to requirements set by EPA to protect human health and the environment.

30 More key pesticide terms! Before registering a pesticide on a food crop, EPA sets a tolerance, or MRL (maximum residue limit). The tolerance is the amount of pesticide residue allowed to remain in or on a treated food commodity. Hops are legally a food commodity.

31 In setting a tolerance, EPA must make a safety finding that the pesticide can be used with reasonable certainty of no harm. To make this finding, EPA considers many factors including these: The toxicity of the pesticide and its breakdown products How much of the pesticide is applied and how often How much of the pesticide (i.e., the residue) remains in or on food by the time it is marketed and prepared What are the cumulative risks of a lifetime of consuming residue of the pesticide And safety provisions are made for the most vulnerable (e.g., children and the elderly).

32 Tolerance The tolerance is the residue level that triggers enforcement actions. That is, if residues are found above that level, the commodity could be subject to seizure. Tolerances are set based on worst-case criteria. These include: That the pesticide is applied at its maximum permitted use rate That the pesticide is applied the maximum number of times permitted on the label That the crop is harvested at the shortest pre-harvest interval permitted on the pesticide label following application. EPA ensures that the tolerance established for a pesticide s residue is safe!

33 Pesticide tolerances on hops All pesticide tolerances granted on hops are based on: Residues of parts per million On hop cones dried to 8 to 10% moisture Parts per Dry hops million Chemical Conventional Organic Nontreated US Tolerance Imidacloprid ND ND ND 6 Mefenoxam ND ND ND 4 Boscalid ND Dimethomorph ND ND ND 60 Bifenazate ND ND 15 Spirotetramat ND ND Trace 10 Carfentrazone-ethyl ND ND ND 0.1 Pyraclostrobin ND ND 23 Triflumizole ND ND 11 Quinoxyfen ND ND 3 Etoxazole ND Pesticide residues detected on hops dried to 8% moisture by Dr. Hengel. ND= no residues detected. Trace= some residue detected, but below Dr. Hengel s level of quantification.

34 Wet Hops Chemical Conventional Organic Nontreated Imidacloprid ND ND ND Mefenoxam ND ND ND Boscalid Trace ND Dimethomorph ND ND ND Bifenazate ND Trace * Spirotetramat ND ND ND Carfentrazone-ethyl ND ND ND Pyraclostrobin ND ND Triflumizole ND ND Quinoxyfen ND ND Etoxazole ND Trace * Pesticide residues detected on wet hops from this study by Dr. Hengel. ND= no residues detected. Trace= some residue detected but below Dr. Hengel s level of quantification. */ These detects are from spray drift from a nearby commercial wine grape vineyard and are a testament to Dr. Hengel s analytical abilities. I have the spray records from the vineyards. The viticulturist is someone I know well.

35 Pesticide residues of boscalid and bifenazate detected in beers at parts per billion. (All other pesticides were below Dr. Hengel s level of quantification) Hop Source Brewing Technique Boscalid Bifenazate Mean Square df Hops used ** 64.9** Error Untreated 60 Minute Boil ND ND Untreated 5 Minute Boil ND ND Untreated Flame out ND 0.06± Untreated Dry 7 days ND ND Organic 60 Minute Boil 0.21± ND Organic 5 Minute Boil 0.37±0.37 ND Organic Flame out 0.39±0.39 ND Organic Dry 7 days 0.74±0.74 ND Conventional dry 2 60 Minute Boil 6.83± ±0.97 a Conventional dry 5 Minute Boil 5.79± ±0.82 a Conventional dry Flame out 8.60± ±0.83 ab Conventional dry Dry 7 days 8.96± ±0.68 b Conventional pellet 3 60 Minute Boil 5.95± ±0.87 Conventional pellet 5 Minute Boil 4.86± ±0.86 Conventional pellet Flame out 8.28± ±1.00 Conventional pellet Dry 7 days 8.32± ±0.67 Conventional wet 4 60 Minute Boil 8.28±1.91 a 10.30±1.81 a Conventional wet 5 Minute Boil 8.67±2.31 a 11.07±2.28 a Conventional wet Flame out 11.11±2.19 ab 11.77±1.98 a Conventional wet Wet 7 days 17.27±3.84 b 22.48±4.24 b **/ p<0.01, 1/ All values are in parts per billion (ppb); 2/ 1-way ANOVA of brewing technique within conventional dry hops df= 3,12, F=2.92 and p>0.05 for boscalid and F=3.68 and p>0.05 for bifenazate; 3/ 1-way ANOVA of brewing technique within conventional pelletized hops df= 3,12, F=3.64 and p<0.05 for boscalid and F=1.96 and p<0.05 for bifenazate. Means of residues detected in beers not followed by a common letter are significantly different at p< / 1-way ANOVA of brewing technique within conventional wet hops df= 3,12, F=3.41 and p<0.05 for boscalid and F=4.35 and p<0.05 for bifenazate. Means of residues detected in beers not followed by a common letter are significantly different at p<0.05.

36 Keeping It In Perspective What does a Parts Per Million (ppm) look like?

37 Parts Per Million 1 ppm would be roughly equivalent to 1 drop in 15.5 gallons of beer.

38 What about Parts Per Billion? 1 ppb would be roughly equivalent to 1 drop in 15,500 gallons of beer.

39 Parts Per Billion 1 ppb would be roughly equivalent to 1 kernel of corn in a silo measuring 45 ft tall with a diameter of 16 ft.

40 Residue Impact on Beer and Us Highest residue found in beer was ppb (ng/ml, nanogram/milliliter) of bifenazate. Current Acceptable Daily Intake (ADI) for bifenazate is 10.0 * µg/kg bw (microgram/kilogram of body weight) * EFSA Journal 2012;10(10):2920 [45 pp.]

41 Residue Impact on Beer and Us Let s say we go out and enjoy a few pints, say six. Determine bifenazate dosing from beer. 6 pints = 6 x 16 oz x ml/oz = 2,839 ml 2839 ml x ng/ml = 63,814 ng 63,814 ng = 63.8 µg of bifenazate in 6 pints.

42 Residue Impact on Beer and Us ADI for average person. 62 kg * (~136 lbs) 10 µg/kg bw x 62 kg bw = 620 µg/day Compare dose from beer to ADI By our results 6 pints of beer results in an adi of 63.8 µg of bifenazate. This is 10% of the permitted 620 µg/day * Walpole et al. BMC Public Health 2012, 12:439

43 Conclusions In the final analysis for pesticide residues present in beer, only two pesticides, bifenazate and boscalid were detected above Dr. Hengel s level of quantification in parts per billion that could be analyzed statistically. Wet hopping with the conventional hops consistently made the greatest contribution of boscalid and bifenazate to the beers compared to conventional dry or conventional pellet. In all cases these were far below levels with any health or legal ramifications.

44 Acknowledgements Dan & Geoff Groenendale, and Ruth Hendersen, Washington State University Steve Thun and Rick Jordan, Pacific Agricultural Laboratory

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