Fate of Pesticide Residues During Brewing

40 Fate of Pesticide Residues During Brewing Simón Navarro and Nuria Vela Department of Agricultural Chemistry, Geology and Pedology, School of Chemistry, University of Murcia, Campus Universitario de Espinardo, Murcia, Spain

Abstract This chapter emphasizes the influence of the different phases of beer-making and the removal of pesticide residues. The effect of their presence on beer quality and health impact is also assessed. With this aim, based on the current data an overview of the behavior and fate of agrochemical residues during the brewing stages (malting, mashing, boiling, fermentation, and stabilization of the beer) is presented. The methodology for analysis of pesticide residues and their metabolites in raw materials, wort, and beer and the main aspects of the food safety and policy in the European Union (EU) are summarized. Depending on the stage involved and the physical–chemical properties (mainly KOW [as log P] value, water solubility, vapor pressure and Henry’s law constant) of the residue in the raw materials, differences in the final fate of the residues are observed. As a general rule, the malsters should devote special attention to the residues of hydrophobic pesticides with KOW 2 because they can remain on the malt. On the contrary, brewers should control residues of hydrophilic pesticides with KOW  4 because they can be carried over into beer. Thus, the monitoring and surveillance of pesticide residues with KOW ranging from 2 to 4 (most of them) during the brewing process is essential to get a “healthy drink.” Additionally, the influence of pesticide residues on flavor, sugar content, acidity, color or total polyphenol, and flavonoid contents has been pointed out. Therefore, the knowledge of the behavior of pesticide residues must be intended to (i) provide information on the transfers of residues from barley to malt, wort, and beer to calculate concentration or reduction factors during each process; (ii) achieve a realistic estimate of the dietary intake of pesticide residues; (iii) propose maximum residue limits (MRLs) for residues in beer when necessary; and (iv) avoid alterations in the beer quality.

List of Abbreviations ASE CAC CCP CEC CI

Accelerated solvent extraction Codex Alimentarius Commission Control critical point Commission of the European Communities Chemical ionization

Beer in Health and Disease Prevention ISBN: 978-0-12-373891-2

CID DMG EBDC ECD EFSA ESD ETU EU FAO GAP GC GC/MS GLP HACCP HPLC IARC IPM LC LC/MS MAE MRL MRM MSD MSPD NPD OHA OHT OTA PBDC PLE PTU QA QC RAC SBI SFE SPE SPME UNIDO WHO WTO

Collision-induced dissociation Danish Malting Group Ethylene bisdithiocarbamate Electron capture detection European Food Safety Authority Element-selective detector Ethylenethiourea European Union Food and Agriculture Organization Good agricultural practices Gas chromatography Gas chromatography/mass spectrometry Good laboratory practice Hazard analysis critical control point High performance liquid chromatography International Agency for Research on Cancer Integrated pest management Liquid chromatography Liquid chromatography/mass spectrometry Microwave-assisted extraction Maximum residue limit Multiresidue method Mass spectrometric detection Matrix solid-phase extraction Nitrogen phosphorus detection Hydroxy atrazine Hydroxy terbuthylazine Ochratoxin A Propylene bisdithiocarbamate Pressurized liquid extraction Propylenethiourea Quality assurance Quality control Raw agricultural commodity Sterol biosynthesis-inhibiting Supercritical fluid extraction Solid-phase extraction Solid-phase microextraction United Nations Industrial Development Organization World Health Organization World Trade Organization Copyright © 2009 Elsevier Inc. All rights of reproduction in any form reserved

416 Beer Stability and Spoilage

Introduction For years there has been a great deal of publicity highlighting the health benefits of a moderate consumption of beer. The influence of beer on health is related to the absence of negative attributes and the presence of positive attributes. Beer, a wholesome beverage that has been a staple part of our diet for many thousands of years, contains a number of components such as antioxidants, which can be beneficial to health. Furthermore, the nutritive aspects of beer include its low sugar content and significant amount of vitamins and minerals (Sendra and Carbonell, 1998; Baxter and Hughes, 2001). One of the most important factors contributing to the public perception of beer as a “healthy drink” has been the accumulation of studies showing that moderate drinkers have lower death rates from all causes, especially from cardiovascular-related diseases, than either non-drinkers or heavy drinkers (Fagrell et al., 1999). The quality of raw materials (barley, hops, water, and yeasts) has a decisive influence on the quality of the final product (Kunze, 2004). Knowledge of the properties of the raw materials and their effects on the process and the product provides the basis for their handling and processing. Therefore, it is important to assess the pollution load of barley and how pesticide residue evolves during the malting process. The ingredients used for brewing must not be allowed to act as a transmitter of unacceptable pollutants that are a risk for the beer consumer and animals. Barley pests Because of their high content of starch and storage proteins, barley grains represent an attractive source of nutrients for insects and microbial pathogens. The vulnerability of the grain to insect and pathogen attack is expected to increase during germination, when amino acids, fermentable carbohydrates, nitrogenous bases, and other degradation products of reserve polymers accumulate in the starchy endosperm (Fincher and Stone, 1993). Good weed control is essential to the crop to make efficient use of moisture and to prevent weed seeds from contaminating the harvest. Furthermore a range of head, root, leaf, and stem diseases may affect barley, depending on climate, environment, and farm history. Some of the more common diseases include crown rot, rust, smut, root rot, net blotch, and nematode infection. Finally, armyworms, cutworms, or mites cause important damage to cereals in some areas (Theaker et al., 1989; Domínguez, 2004). Additionally, barley with a moisture content of about 14% may be attacked by fungi during storage especially by Aspergillus and Penicillium spp. These genera produce secondary metabolites such as ochratoxin A (OTA); it has been shown to be teratogenic and immunosuppressive. The International Agency for Research on Cancer (IARC) lists OTA as possibly carcinogenic to humans, group 2B (IARC, 1993). Some of these metabolites cause gushing, a very severe

quality defect, where the beer spontaneously gushes from a bottle on opening. It is therefore important that malting barley is stored under conditions that prevent fungal growth (DMG, 1999). Use of pesticides on barley For the reasons mentioned earlier, pesticides are widely used in different combinations at many stages of cultivation and also during postharvest storage. From the past decades there has been, in the developed countries, an increasing concern over possible dangers to human health and/or the environment resulting from the excessive or inappropriate use of chemical pesticides. At present, good agricultural practices (GAP) are formally recognized in the international regulatory framework for reducing risks associated with the use of pesticides, taking into account public and occupational health, environmental, and safety considerations. The International Code of Conduct on the Distribution and Use of Pesticides (the worldwide guidance document on pesticide management for all public and private entities engaged in, or associated with, the distribution and use of pesticides adopted for the first time in 1985 by the Twentyfifth Session of the FAO Conference) focuses on risk reduction, protection of human health and the environment, and support for sustainable agricultural development by using pesticides in an effective manner and applying integrated pest management (IPM) strategies (FAO, 2002). The environmental and health impact of pesticides is being reduced through the implementation of a number of concrete programs on pesticide management, such as residue analysis, product standards setting and methods to analyze them, prevention of accumulation of obsolete stocks of pesticides and means to dispose them, and exchange of information on national actions taken to control pesticides. Presence of pesticide residues in raw materials The problem is that traces of these pesticides may remain in the beer produced from the treated ingredients, although the residues may also come from the soil itself and the water used; a problem that affects the brewing industry in several countries. During the first step (malting), some residues of pesticides having log KOW 2 (as log P) would remain on malt as indicated by some authors (Miyake et al., 2002). After mashing and boiling, the pesticides on the malt can pass into the wort in different proportions, depending on the process used, although it should be noted that the removal of material in the form of trub and spent grains tends to reduce the level of pesticides, which are often relatively insoluble in water (Hack et al., 1997; Miyake et al., 1999; Navarro et al., 2005a, 2006). Finally, if pesticide residues, especially some fungicides, are present in the brewer wort, they may cause organoleptic alterations

Fate of Pesticide Residues During Brewing 417

to the finished beer and have toxic effects on the consumer ( Jones et al., 1988; Navarro et al., 2007a).

Analysis of Pesticide Residues in Raw Materials and Beer The widespread use of pesticides in cereals and hops had led to the presence of pesticide residues in beer. Public concern over pesticide residues in malt beverages and beers has been increasing such that it has become a significant food safety issue. Routine analyses of the composition of the raw materials used in the malting and brewing processes aim to assist quality control (QC). Validation protocols are essential for the analyst to provide the proper documentation of analytical results required to meet the QA/QC criteria of GLP/GMP and of various regulation agencies such as the Codex Alimentarius Commission (CAC) (Sherma, 1999). Additional analyses are performed to detect trace amounts of undesirable compounds such as pesticides to confirm that they are not present in the raw materials used in production. For this, multiresidue methods (MRMs) are needed to reliably and rapidly detect and quantitate as many pesticides as possible in the most cost-effective manner (Ghosh et al., 2005). Traditional batch liquid–liquid solvent extraction in a separatory funnel, Soxhlet extraction, or ultrasonic solvent extraction methods have been progressively substituted by more modern sample preparation methods in the past years (Sherma, 1999). These include general solid-phase extraction (SPE), polymeric reversed-phase (RP)SPE, disk format SPE, automated SPE, matrix solid-phase dispersion (MSPD), solid-phase microextraction (SPME), membrane filtration, solvent extraction flow injection, miniaturized liquid–liquid extraction, accelerated solvent extraction (ASE) or pressurized liquid extraction (PLE), microwaveassisted extraction (MAE), and water or supercritical fluid extraction (SFE). Methods and instruments for modern extraction methods have been reviewed ( Jordan, 1998). Residues in extracts are most commonly separated by gas chromatography (GC) or high performance liquid chromatography (HPLC) using ultraviolet (UV) absorbance, nitrogen phosphorus detection (NPD), or electron capture detection (ECD). However, although these elementselective detectors (ESDs) provide low ppb detection limits and are easy to operate, the data do not provide sufficient information to confirm a compound’s presence with confidence. Owing to the universal nature of mass spectrometric detection (MSD), a mass spectrometer provides additional information and increases confidence in the assignment of compound identity (Meng, 2001). GC/MS has features to enhance specificity, such as chemical ionization (CI), selective ion monitoring (SIM), or MS/MS. Even with SIM, where multiple ions are monitored (MIM), the matrix may contain similar ions at the same retention time, so more stringent selectivity must be invoked to remove the matrix ions from the mass spectrum, which eliminates false positives and raises

concentration values from matrix interferences. MS/MS does just that by ejecting all except the ion of interest out of the group. Then, a collision-induced dissociation (CID) energy is applied to fragment the ion into a very unique product ion spectrum (Butler and Conoley, 2005). However, although many established MRMs for analysis of food samples have used GC, some water-soluble pesticides that may be very important in beverages such as wine or beer are not suitable for analysis or their recoveries are very low. For these watersoluble pesticides, there are many analytical methods using liquid chromatography (LC). Recently, MS was found to be superior to other LC detectors, in particular, tandem mass spectrometry (MS/MS) was found to be far superior to any other method because of its high selectivity and sensitivity with the advantage of unequivocal analyte identification due to the ion selection of two mass analyzers. Also, as a single analytical procedure is desirable to screen beverage samples, the LC–MS/MS method allows the quantitation of many pesticides in beer without any sample preparation other than centrifugation. Many analytical methods using these techniques have been proposed in recent years for analysis of pesticide residues in cereals, malt, hops, and beer (Williams et al., 1994; Hack et al., 1996; Tadeo et al., 2000; Hengel and Shibamoto, 2002; Wong et al., 2004; Trösken et al., 2005; Omote et al., 2006; Vela et al., 2007).

Food Safety and Policy in the European Union The agro-food sector is of major importance for the European economy as a whole. The food and drink industry is a leading industrial sector in the European Union (EU), with an annual production worth almost €600 billion, or about 15% of total manufacturing output. An international comparison shows the EU as the world’s largest producer of food and drink products. Production of beer worldwide was forecast to increase to an annual growth rate of 2.3% through 2005 to a volume of about 153 billion liters. Concretely, about 34% of the world beer production in 2004 (530 million Hl) was manufactured in Europe – Germany, Great Britain, and Spain being the main producers of the EU (Barth-Haas Group, 2005). The contamination of food by chemical hazards is a worldwide public health concern and is a leading cause of trade problems internationally. Contamination may occur through environmental pollution, as in the case of toxic metals, polychlorinated biphenyls (PCBs), and dioxins, or through the intentional use of chemicals such as pesticides, animal drugs, and other agrochemicals. Food additives and contaminants resulting from food manufacturing and processing can also adversely affect health. The EU’s food policy is based on high food safety standards, which serve to protect and promote the health of the consumer. Regulation (EC) No. 396/2005 on maximum residue levels of pesticides in or on food and feed of plant and animal origin emphasizes the importance of carrying out further work

418 Beer Stability and Spoilage

to develop a methodology to take into account cumulative and synergistic effects of pesticides on human health. The EU food safety policy must be based on a comprehensive, integrated approach. This means throughout the food chain (“farm to table”), across all food sectors, between the Member States, at the EU external frontier and within the EU, in international and EU decision-making fore, and at all stages of the policy-making cycle. The pillars of food safety contained in the White Paper (scientific advice, data collection and analysis, regulatory and control aspects, as well as consumer information) must form a seamless whole to achieve this integrated approach (CEC, 2000). HACCP system in the food industry The hazard analysis critical control point (HACCP) system is a relatively new approach to the prevention and control of foodborne diseases. The HACCP system identifies specific hazards and preventative measures for their control to ensure the safety of food. HACCP is a tool to assess hazards and establish control systems that focus on preventative measures rather than relying mainly on end-products testing. Table 40.1 summarizes the seven HACCP principles. For many years public health and food control authorities worldwide, as well as international organizations such as Food and Agriculture Organization (FAO), World Health Organization (WHO), and United Nations Industrial Development Organization (UNIDO) have promoted the application of the HACCP system. FAO/WHO CAC adopted at its 20th Session (1993), the Guidelines for the Application of the HACCP System (CAC/GL 18-1993). This session emphasized that the work of Codex has increased in importance with the establishment of the World Trade Organization (WTO) and the WTO Agreement on the Application of Sanitary and Phytosanitary Measures coming into force. According to this Agreement; Codex standards;

Table 40.1 Principles of the hazard analysis critical control point (HACCP) system 1.

2. 3. 4.

5.

6. 7.

Conduct a hazard analysis. Steps in the process where significant hazards can occur including preventive measures. Identify the critical control points (CCPs) in the process. Establish critical points for preventive measures associated with each identified CCP. Establish CCP monitoring requirements and procedures for using the results of monitoring to adjust the process and maintain control. Establish corrective actions to be taken when monitoring indicates that there is a deviation from an established critical limit. Establish effective record-keeping procedures that document the HACCP system. Establish procedures for verification that the HACCP system is working correctly.

guidelines and recommendations relating to food additives, veterinary drug and pesticide residues, contaminants; methods of analysis and sampling; and codes and guidelines of hygienic practice have been recognized as the reference for international food safety requirements, and thus as a benchmark for national requirements. The Food Hygiene Directive (93/43/EEC) obliges food businesses in the EU to implement systems that are based on the principles of HACCP. Although non-EU suppliers of food products do not legally have to comply directly with the EU directive on food hygiene, they are affected by the Euhygiene rules. It was clearly confirmed that food businesses in Europe, implementing systems to ensure that hazards are identified and controls are in place, have become increasingly selective in dealing with their (foreign) suppliers and request a strict application of HACCP in the request countries of origin of imported products. In some cases, they have even set out additional hygienic requirements for their suppliers regarding specific product(s). Food businesses in Europe and other industrialized countries, applying systems to assure food safety, will not buy any raw material if they think that, even after sorting and processing, it could make food unfit for human consumption. Any raw material or processed food product that is only suspected or known to be infected or contaminated with parasites or foreign substances will not be accepted. Therefore, it is important to carry out a HACCP plan (a document describing the activities developed in accordance with the principles of HACCP to ensure control of hazards, which are significant for food safety in the product under consideration and its intended use) in order to reveal the weaknesses of the production line of beer and to suggest the critical limits in compliance with legislation and the corresponding preventive and corrective measures (Kourtis and Arvanitoyannis, 2001).

Effects of Brewing on the Pesticide Residues Depending on the type of process involved and the chemical nature of the residue in the raw agricultural commodity (RAC), differences in the nature of the residue in the processed commodities and the RAC may have to be determined. Once the nature of the residues formed during processing has been clarified and active ingredients and relevant metabolites to be analyzed have been identified, processing studies are conducted with RACs that normally undergo processing in the home or under commercial conditions. The process may be physical or may involve the use of heat or chemicals (Timme and Waltz-Tylla, 2003). These types of processing are intended to (i) provide information on the transfer of residues from RACs to the processed product, in order to calculate reduction/concentration factors; (ii) enable a more realistic estimate to be made of the

Fate of Pesticide Residues During Brewing 419

dietary intake of pesticide residues; and (iii) establish MRLs for residues in processed products when necessary. Figure 40.1 summarizes the principal steps of the brewing process. More detailed descriptions can be found in Eaton (2006). Dissipation of pesticide residues during storage of barley, malt, and spent grains When appropriate application methods of agrochemicals are not followed, pesticide residues on barley can be above the maximum residue limits (MRLs) established by the governments of each country, and the time for pesticide dissipation is also high. Desmarchelier et al. (1980) reported the losses of several pesticides (bioresmethrin, carbaryl, fenitrothion, d-fenothrin, methacrifos, and pirimiphos-methyl) from barley

Barley grains

Malting

Malted barley

Steeping

Steeped grains

Germination

Germinated grains

Kilning

Kilned grains

First wort Lautering

Mashing

Spent grains Sweet wort

Second wort

Boiling Brewer wort

Spent hops Primary fermentation

Young beer

Spent yeasts

Lagering Matured beer Clarification, filtration Stabilized beer Carbonation, packaging, storage Finished beer

Figure 40.1 Scheme illustrating the principal stages of the brewing process.

after storage and malting finding losses of 58–100%. Other authors show that after pesticide application using some insecticides (phentoate, fenitrothion, and ethiofencarb) and fungicides (triflumizole, mepromil, propiconazole, and triadimefon), common in barley cultivation, more than 80% of residues of phentoate and fenitrothion (organophosphorus insecticides) remained after 2 months of grain storage at room temperature, whereas the loss of other pesticides ranged from 28% to 85%, and metabolites of triadimefon (triadimenol) and triflumizole (TF-6-1) increased slightly (Miyake et al., 2002). Several models (Timme and Frehse, 1980; Timme et al., 1986; Morton et al., 2001) are used to describe the decay of pesticides in different matrices. Probably, the most commonly used model to describe loss of grain protectants is the following equation, where Rt is the residue at time t, R0 the residue at time zero, and K the rate constant (Desmarchelier, 1977, 1978). ln Rt  ln R0  Kt Navarro et al. (2007b) found a good linear correlation (r  0.95) between ln Rt and time when they studied the dissipation of several pesticides over 3 months of malt storage at 20  2°C. Additionally, a perfect correlation (r 0.99) between the analytical and theoretical concentration calculated (R0) at 0 days was observed, which indicates that the model is valid. According to the calculated values for the rate constant (K ), the following dissipation rate was observed: myclobutanil propiconazole fenitr othion trifluralin pendimethalin malathion nuarimol with half-lives ranging from 244 to 1,533 days. A moist by-product from the brewing industry, made up of spent grains, is widely fed to ruminant animals used as a buffer or as a forage or concentrate replacer. Therefore, although the nutritional potential of the spent grains for animals has been demonstrated, it is important to ascertain the pollution load of the same and how any pesticide residues evolve during storage. To know the dissipation rate of pesticide residues in the spent grains, Navarro et al. (2005a, 2006) studied their disappearance during storage (3 months). In all cases, there was a good linear correlation (r 0.96) between residue level and time. The necessary times to reach their respective MRLs in barley were 408, 515, 958, 711, 719, and 934 days for nuarimol, myclobutanil, propiconazole, fenitrothion, trifluralin, and pendimethalin, respectively, which indicates a high persistence level and minimum degradation for all compounds, especially for propiconazole. In the case of malathion, the initial residue was below its MRL. Decline of pesticide residues from barley to malt (malting) Common malting operations involve four basic stages: barley intake, drying and storage, steeping, germination

420 Beer Stability and Spoilage

Table 40.2 Carryover of some pesticide residues after each stage of malting Stage Pesticides

Log POW *

Steeping

Germination

Kilning

References

Ethiofencarb Mepronil Phentoate Triadimefon Triadimenol Triflumizole

2.04 3.66 3.69 3.11 3.08 4.36

3 24 27 24 36

1 6 4 5 13 11

5 30 18 30 47 9

Miyake et al. (2002)

Propiconazole

3.65

50

10

55

Miyake et al. (2002)

55

43

30

Navarro et al. (2007b)

Fenitrothion Malathion Myclobutanil Nuarimol Pendimethalin Trifluralin

3.43 2.75 2.94 3.18 5.18 3.07

52 45 59 64 85 80

31 20 42 57 67 65

13 14 36 51 49 50

Navarro et al. (2007b)

* Tomlin (2003).

140 120 % Carryover into steeped grains

and kilning (Bamforth and Barclay, 1993). The process commences with the steeping of barley in water to achieve a moisture level sufficient to activate metabolism in the embryonic and aleurone tissues, leading in turn to the development of hydrolytic enzymes. Germination is generally targeted to generate the maximum available extractable material by promoting endosperm modification through the development, distribution, and action of enzymes. Finally, after a period of germination sufficient to achieve even modification, the “green malt” is kilned to arrest germination and stabilize the malt by lowering moisture levels, typically to less than 5%. Table 40.2 shows some bibliographical data relative to pesticide decline during malting. Although in general terms steeping reduces residue levels significantly, the carryovers for dinitroaniline herbicides (pendimethalin and trifluralin) vary from 80% to 85% in steeped grains. Both are hydrophobic pesticides because of their high log POW ( 5) and low solubility (0.2–0.3 mg/l). In consequence, a low proportion of their residues (10–15%) are removed with the steeping water. Regarding the organophosphorus pesticides, 55% of malathion (log POW  2.7) is removed from the barley grains after steeping whereas 48% of fenitrothion residues (log POW  3.5) are removed in this stage (Navarro et al., 2007b). Other published data show carryover of 43% for fenitrothion after steeping process whereas other organophosphorus insecticide such as phentoate remains in lower proportion (27%) as indicated by Miyake et al. (2002). On the contrary, other insecticide of the same family as pirimiphos-methyl remains in high proportion (90%) after steeping (Collins and Armitage, 2006). Some fungicides such as nuarimol (pyrimidine), myclobutanil, and propiconazole (triazole) are removed from the barley grains (after steeping) in proportions ranging from 30% to

100 80 60 40 20 0 20 40 1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

log POW values

Figure 40.2 Correlation between carryover of some pesticides after steeping and their log POW values according to the data shown in Table 40.1 (solid line is 95% confidence interval and dash line 95% prediction interval).

41%, this is expected bearing in mind their respective coefficients between n-octanol and water (POW values). Miyake et al. (2002) found higher percentages of elimination for some azole fungicides, specifically 50%, 62%, and 76% for propiconazole, triflumizole, and triadimefon, respectively. These data are supported by the relationship between amounts removed after steeping and log POW of the pesticides used in this study, as can be seen in Figure 40.2. The carryover of hydrophilic pesticides (low log POW) such us malathion is lower, whereas carryovers of hydrophobic pesticides (pendimethalin and trifluralin) are higher. Miyake et al. (1999) suggest that brewers should pay attention to

Fate of Pesticide Residues During Brewing 421

Table 40.3

Carryover of some pesticide residues after mashing and boiling

Pesticide

Log POWa

Sweet wort

Spent grains

Brewer wort

Spent hops

References

Atrazine Terbutylazine

2.5 3.2

45 12

55 80

42 7

20 40

Hack et al. (1997)

Triadimenol

3.1

36

NDb

ND

ND

Miyake and Tajima (2003)

BDL 17 BDL 10 8 BDL 35 BDL BDL 97 1 1 BDL 84 2 8

3 3 45 10 BDL 70 30 50 60 3 BDL BDL 70 14 68 54

BDL 4 3 BDLc BDL 18 64 3 BDL 95 20 10 BDL 50 6 30

BDL 32 37 BDL BDL 60 1 7 10 2 BDL 3 50 3 12 15

Miyake et al. (1999)

Captafol Chlorpyrifos Deltamethrin Diclofuanid Dichlorvos Dicofol Fenobucarb Fenvalerate Flucythrinate Glyphosate Oxamyl Parathion-methyl Permethrin Pirimicarb Pirimiphos-methyl a-BHC

3.8 4.7 4.6 3.7 1.9 4.3 2.8 5.0 6.2 3.2 0.4 3.0 6.1 1.7 4.2 4.0

Malathion

2.7

20 7

35 40

15 4

5 ND

Miyake et al. (1999) Navarro et al. (2006)

Myclobutanil Nuarimol Propiconazole

2.9 3.2 3.6

9 6 4

38 26 42

8 6 4

ND ND ND

Navarro et al. (2005a)

Fenitrothion Pemdimethalin Trifluralin

3.4 5.2 5.1

4 1 1

30 21 17

3 1 1

ND ND ND

Navarro et al. (2006)

a

Tomlin (2003). Not determined. c Below detection limit. b

the residues of hydrophilic pesticides on malt with POW values 4 because they can be carried over into beer; the steeping stage of the malting process being of special interest. The same authors (Miyake et al., 2002) showed that pesticides with log POW 2 can remain on malt. Therefore, the control of pesticides with POW values ranging from 2 to 4 must be very important for maltster and brewers. Removal and transference of pesticide residues from malt to sweet wort (mashing) As a general rule, about 200 g of grains are used to produce 1 litre of wort at 12° Brix, although this amount varies according to whether a higher or lower alcoholic content is desired. Any residues present in the grain, even if completely transferred to the beer, should, therefore, undergo dilution by a factor of 5 although log POW values of pesticides should be kept in mind (Miyake et al., 1999). Taking into account the low solubility of most pesticide residues in water and their tendency to be easily adsorbed on the suspended matter, as in wine making, the presence of residues in beer should be very low (Farris et al., 1992).

The carryovers of residues for several pesticides after mashing are shown in Table 40.3. During mashing, the soluble substances (sugars, amino acids, and peptides) produced in malting and mashing are extracted into the liquid fraction (sweet wort), which is then separated from the residual solid particles (spent grains). According to Navarro et al. (2005a), at the end of the mashing phase, the remaining percentages are below 10% of the amount recorded in malt; propiconazole showing the greatest decrease (to 4%). On the contrary, the retained amounts on spent grain is relatively high (38%, 42%, and 26% for myclobutanil, propiconazole, and nuarimol, respectively, all the compounds having KOW 2) (Figure 40.3). Similar behavior was observed for atrazine and terbuthylazine during mashing, when 70% and 90%, respectively, were adsorbed on spent grain (Hack et al., 1997). In a general way, adsorption affinity depends on the polarity of the compounds: the more polar the pesticide, the lower the adsorbed amount (Hengel and Shibamoto, 2002). It is necessary to bear in mind that maceration of the malt and adjuncts produces a great quantity of suspended matter, which could adsorb the pesticide residues and, if the recorded levels allow it, the spent grains could be used as animal feeds, implying

422 Beer Stability and Spoilage

120 100

% Carryover into spent grains

80 60 40 20 0 20 40 60 80 4

2

0

2

4

6

8

log POW values

Figure 40.3 Correlation between carryover of some pesticides after mashing and their log POW values according to the data shown in Table 40.3 (solid line is 95% confidence interval and dash line 95% prediction interval).

a commercial use of this by-product. As shown in Table 40.3, the amounts remaining of dinitroaniline herbicides practically disappear after mashing (1% of the initial amount in sweet wort), whereas the remaining percentages of fenitrothion and malathion in the sweet wort are greater (4.2% and 6.6%, respectively). On the contrary, the retained amounts on spent grain were relatively high (22%, 17%, 30%, and 40% for pendimethalin, trifluralin, fenitrothion, and malathion, respectively). Other pesticides such as glyphosate (organophosphorus) and pirimicarb (carbamate) were found in sweet wort in proportions higher than 80%. On the contrary, no residues of pyrethroid compounds (fenvalerate, deltamethrin, permethrin, and flucythrinate) were obtained from sweet wort; they were recovered from the spent grains. In the case of oxamyl, dichlorvos, parathion-methyl, chlorpyrifos, dichlofuanid, and captafol, pronounced losses were observed, possibly due to thermal degradation, evaporation, or chemical reactions with some wort components during the mashing process (Miyake et al., 1999). Removal and transference of pesticide residues from sweet wort to brewer wort (boiling) Table 40.3 shows the carryovers of several pesticides in brewer wort and spent hops. As can be seen, a very small decrease (10%) has been observed in the residual content after the wort had been boiled for myclobutanil, nuarimol, and penconazole residues, which points to the stability of the three compounds at temperatures higher than 100°C (Navarro et al., 2005a). The carryovers of pendimethalin and trifluralin recorded from the brewer wort were lower than 30% of their content in sweet wort after wort boiling. With regard to the fall of organophosphorus compounds at this time, the levels of fenitrothion and malathion were 83% and 65%,

respectively, of the content in wort after mashing (Navarro et al., 2006). Other authors (Miyake et al., 1999) show that the percentages of residues for glyphosate, fenobucarb, and pirimicarb into the cold wort were prominently high showing a great stability at temperatures above 100°C, whereas dicofol and pyrethroid insecticides were mainly recorded in the spent hops, and dichlorvos, dichlofuanid, and captafol completely disappear, probably due to the higher temperature of the boiling process in this case. However, the sum of residues for oxamyl, parathion-methyl, and chlorpyrifos in cold wort and spent hops was slightly higher than that of the mashing process. Authors assumed this behavior on the basis that these pesticides can react with some components or proteins in the sweet wort but not in the cold wort. The fate of pesticide residues from hop to wort during boiling has also been studied. Some researchers have demonstrated that no pesticides added to the hops were detected in the young beer after wort boiling (Miyake et al., 1999). Other work carried out by Navarro et al. (2005b) shows that residues of malathion, methidathion, and fenamiphos were below their detection limits in the young beer after addition of spiked hop pellets (2 g/g) to the boiling wort, whereas 1 g/l was recorded for fenarimol. In most of the cases, the absence of pesticide residues is due to their losses during boiling and the high dilution of hops. Other studies carried out with field-treated hops with several pesticides show that residues of tebuconazole, Z- and E-dimethomorph were lower than 31% of the amount expected, bearing in mind these were only the diluted residues. Subsequent analysis showed that 84–109% of pesticide residues (chlofenapyr, quinoxyfen, pyridaben tebuconazole, fenarimol, and both Z- and E-dimethomorph) remain on the spent hops (Hengel and Shibamoto, 2002), which is explained by the highly lipophilic components of hops such as waxes and resins. In Europe, processing studies on hops are only required when residues are higher than 5 mg/kg of dried cones because of the high dilution factor – around 250 (Timme and Waltz-Tylla, 2003). Evolution of pesticide residues during fermentation With regard to the influence of the fermentative process on the elimination of pesticide residues (Table 40.4), a significant reduction has been observed for propiconazole residues (47% of the content recorded in brewer wort) but much less for other fungicides such as myclobutanil and nuarimol (around 20%) (Navarro et al., 2005a). However, no residues of dinitroaniline herbicides were found in young beer fermented with bottom-yeasts, although there was a significant reduction in the cases of organophosphorus insecticides fenitrothion and malathion (58% and 71% of the content recorded in brewer wort) (Navarro et al., 2006). Other fermentation studies showed that top-fermenting yeasts (Saccharomyces cerevisiae) had a much greater ability

Fate of Pesticide Residues During Brewing 423

Table 40.4 Carryover of some pesticide residues after fermentation Pesticide

Log POWa

Young beerb

Spent yeast c

References Hack et al. (1997)

Atrazine Terbutylazine

2.5 3.2

95 (76) 100 (50)

ND ND

Chlorfenapyr Quinoxyfen Tebuconazole Fenarimol Pyridaben Z-dimethomorph E-dimethomorph

4.8 4.7 3.7 3.7 6.4 2.7 2.6

BDL BDL 55 41 BDL 70 75

34 62 58 48 43 22 23

Hengel and Shibamoto (2002)

Triadimenol Captafol Chlorpyrifos Deltamethrin Diclofuanid Dichlorvos Dicofol Fenobucarb Fenvalerate Flucythrinate Glyphosate Oxamyl Parathion-methyl Permethrin Pirimicarb Pirimiphos-methyl -BHC

3.1 3.8 4.7 4.6 3.7 1.9 4.3 2.8 5.0 6.2 3.2 0.4 3.0 6.1 1.7 4.2 4.0

43

ND

Miyake and Tajima (2003)

BDLd 12 BDL 17 65 BDL 94 BDL BDL 110 30 60 BDL 50 40 110

BDL 16 15

Miyake et al. (1999)

BDL 10 BDL 11 2 BDL BDL 4 11 BDL 6 30

Malathion

2.7

58 20

2 ND

Miyake et al. (1999) Navarro et al. (2006)

Myclobutanil Nuarimol Propiconazole

2.9 3.2 3.6

78 82 52

ND ND ND

Navarro et al. (2005a)

Fenitrothion Pemdimethalin Trifluralin

3.4 5.2 5.1

35 BDL BDL

ND ND ND

Navarro et al. (2006)

a

Tomlin (2003). Values in parenthesis for atrazine and terbuthylazine are using top-yeasts. c Not determined. d Below detection limit. b

to convert triazine herbicide residues into their hydroxylated products than bottom-fermenting yeasts (Saccharomyces carlsbergensis) (Hack et al., 1997). For Miyake et al. (1999), no significant reduction was observed during the fermentation process for some group of pesticides. Other pesticides such as chlofenapyr, quinoxyfen, and pyridaben show a dramatic drop after addition to the pitching wort, being below detection limit after fermentation, whereas tebuconazole, fenarimol, and both Z- and E-dimethomorph had relatively high residue recoveries; their carryover, at the end of the fermentation, ranging from 41% to 75% (Hengel and Shibamoto, 2002). These results suggest that there is a relationship between KOW and the amount of pesticides found in the young beer and the trub as can be seen in Figure 40.4. The losses during fermentation may be attributed to biotic metabolism of the yeast and abiotic degradation from the anaerobic environment created by fermentation

(Cabras et al., 1995). Also, according to the Henry’s law constants (the tendency of a compound to volatilize from aqueous solution to air), those pesticides with high vapor pressure and low solubility in water may escape into the atmosphere (Mackay et al., 1979). This process is also favoredby the constant evolution of CO2 during the first few days of the fermentation. Decrease of pesticide residues during maturation phase (lagering), filtration, and beer storage No important decrease on the residual levels has been observed in any case after maturation and filtration. Nuarimol decreased its concentration (by 10%) with regard to the young beer. However, fenitrothion and malathion decrease their contents with regard to the young beer by

250

1,060

200

1,050

150

1,040 Specific gravity

% Carryover into young beer

424 Beer Stability and Spoilage

100

50

1,030

1,020 0 1,010 50 100 4

I

II

III

IV

V

1,000 2

0

2

4

6

8

0

2

4

log POW values

Figure 40.4 Correlation between carryover of some pesticides after fermentation and their log POW values according to the data shown in Table 40.4 (solid line is 95% confidence interval and dash line 95% prediction interval).

33% and 37%, respectively (Navarro et al., 2005a; Navarro et al., 2006). Hack et al. (1997) did not find loss of triazines after filtration. Finally, after the storage period (3 months), the concentrations of myclobutanil and fenitrothion fall sharply (50% and 75%, respectively), whereas the decrease observed for nuarimol and myclobutanil, being lower than 25% of the amount in the finished beer is less pronounced, and malathion residues are below detection limits.

Influence of Pesticide Residues on the Beer Quality During fermentation, yeast metabolizes sugars into energy, alcohol, carbon dioxide, secondary by-products, and more yeast. Those fermentation by-products have a considerable effect on the taste, aroma, and other characteristics of the beer. Beer flavor is a very complex subject. More than 800 compounds have been identified that contribute to the characteristic flavor of beer. Some pollutants, such as pesticides, can alter the normal fermentative process, being able to cause in certain cases sluggish and even stuck fermentation, although it will depend to a great extent on the initial concentrations in the malted barley, physical–chemical characteristics of each product, and beer-making procedure. In consequence, the organoleptic properties of the beer should be modified as in other fermented beverages such as wine (Cabras et al., 1987; Navarro, 2000). Flavor assessment is very important in quality control of beer. One of the most important tools is the sensory analysis by a panel of well-trained tasters. In some cases, the harsh astringent flavor observed in some beer samples was found to be due to products (metabolites) derived from pesticides present in the raw materials. Thus, residues of

6

8

10

12

Days Propiconazole

Nuarimol

Myclobutanil

Blank

Fenarimol

Figure 40.5 Evolution of specific gravity (n = 3) vs. time during fermentation phases (I: initial, II: low krausen, III: high krausen, IV: krausen collapsing, and V: collapsed foam) for blank and treated samples. Source: Navarro et al. (2007a).

up to 5 mg/kg of carbaryl were found on treated barley and up to 41 g/l of carbaryl-derived 1-naphtol were found in beer. Removal of up to 90% of the carbaryl and all of the 1-naphtol occurred during malting. Some tasters were able to consistently identify beer containing 20 g/l of 1-naphtol ( Jones et al., 1988). According to Navarro et al. (2007a), a marked influence of some pesticides in the fermentation rate has been observed (see Figure 40.5 where the evolution of specific gravity with time is shown for blank and treated samples). As can be seen, from the fourth day onward, the fermentation prematurely ceases (stuck fermentation; i.e., the premature termination of fermentation before all fermentable sugars have been metabolized) in the samples with propiconazole residues compared with the blank. No significant differences in the evolution of specific gravity has been found for samples fermented with myclobutanil residues whereas for samples treated with fenarimol and nuarimol residues the fermentative kinetic is quicker, from 2 to 6 days, probably due to the rapid assimilation of nitrogen by the yeasts. The four compounds are sterol biosynthesis-inhibiting (SBIs) fungicides. They inhibit the cytochrome P450 monooxygenase, which catalyzes the oxidative C14 demethylation of 24-methylenedyhydrolanosterol in the biosynthesis pathway, and are a widely applied class of antifungal agents because of their broad therapeutic window, wide spectrum of activity, and low toxicity (Koller, 1988). Some authors suggest that the complex nitrogen composition of the medium may create conditions resembling those responsible for inducing sluggish/ stuck fermentation (Batistote et al., 2006). The sugars in wort are not all fermented in the same proportion. Figure 40.6 shows the evolution of fermentable

Fate of Pesticide Residues During Brewing 425

Glucose

Fructose 2.5

14 Blank Propiconazole Myclobutanil Nuarimol Fenarimol

12

10

Blank Propiconazole Myclobutanil Nuarimol Fenarimol

2.0

1.5

(g/l)

(g/l)

8

6

1.0

4 0.5 2

0

0.0 0

2

4

6

8

10

12

14

16

0

2

4

6

Days

8

10

12

14

16

Days

Maltose

Maltotriose 20

70 Blank Propiconazole Myclobutanil Nuarimol Fenarimol

60

50

Blank Propiconazole Myclobutanil Nuarimol Fenarimol

15

(g/l)

(g/l)

40 10

30

20

5

10

0

0 0

2

4

6

8

10

12

14

16

Days

0

2

4

6

8

10

12

14

16

Days

Figure 40.6 Change in glucose, fructose, maltose, and maltotriose content (n = 3) vs. time during fermentation for blank and samples treated with fungicides (error bars are 95% confidence intervals). Source: Navarro et al. (2007a).

carbohydrates during fermentation according to Navarro et al. (2007a). Since yeast has to hydrolyze sugar polymers before it can use them, it always attacks hexoses first. For this reason, the yeasts assimilate a greater proportion of glucose during the first 96 h. No significant differences were observed between the blank sample and those treated with myclobutanil, nuarimol, and fenarimol residues whereas in the case of propiconazole, a delay in the glucose consumption was observed after 4 days. Sucrose was easily fermented by yeast in all cases because the enzyme that decomposes it, invertase, is located in the cell wall and sucrose is therefore treated as a start of fermentation sugar by the yeast. No significant differences were observed between the blank and the other samples although assimilation of this sugar was to some extent slower in the blank sample during the first 48 h. Fructose assimilation follows a different behavior among glucose and sucrose. Samples with nuarimol and fenarimol (pyrim-idine fungicides) residues reduced this sugar quicker

than those with propiconazole and myclobutanil (triazole fungicides) residues. The slowest assimilation corresponds to the blank sample. In all cases, the highest reduction occurs from 24 to 216 h. Maltose exhibits similar behavior although the biggest consumption takes place between 96 and 216 h, during the main fermentation. It is important to remark that after the fourth day the consumption of this sugar decreased drastically by the yeasts in the sample with propiconazole residues with logical bearing in mind that fermentation was stopped at this time. Finally, maltotriose is the last sugar assimilated by the yeasts. No significant differences were observed when comparing the behavior of the blank sample and those with residues of nuarimol and fenarimol. However, triazole fungicides, especially propiconazole, have a marked influence on the assimilation of this sugar by the yeasts. The effect of some pesticide residues on the pH and color of the beer has also been observed (Navarro et al., 2007a).

426 Beer Stability and Spoilage

Thus, the pH values at the end of the fermentation were 4.1 for the blank sample and 3.0, 3.7, 3.8, and 3.9 for those containing residues of propiconazole, myclobutanil, fenarimol, and nuarimol, respectively. In this case also, the presence of propiconazole alters the final quality of the beer. pH values below 4.0 cause an acidic beer taste and must be avoided, in particular, acidification by microbial infections during fermentation. Therefore, maturation should be completely excluded. Regarding color, at the beginning of the process 5.35 EBC units were recorded. Although a slight increase after 2 days of fermentation can be observed in all cases, color of the beer falls about 1–1.5 EBC units during fermentation. This is possibly due to the decoloration of some substances caused by the drop in pH, and absorption of highly colored compounds in the yeast cells or precipitation in the vessel bottom (Kunze, 2004). As a result of a decrease in pH during fermentation or adsorption on the yeast cells, a number of colloidally dissolved bitter substances and polyphenols can precipitate as surface active compounds on the CO2 bubbles in the foam head (Kunze, 2004). As a consequence of their low solubility at a pH below 5 and temperatures lower than 10°C, the -acids are not isomerized during the boiling of the wort precipitate. For this reason, in the study carried out by Navarro et al. (2007a), the values of bitterness are below detection limits in all cases. Regarding the total polyphenol and flavonoid contents found after fermentation, significant differences have been observed between the samples containing residues of triazole fungicides and the others, especially in the case of propiconazole due to the stuck fermentation after 4 days of beginning. In other cases such as carbaryl residues during brewing its metabolization to 1-naphtol confers, above 20 g/l, a characteristic harsh astringent flavor to the beer ( Jones et al., 1988). Bearing in mind the aforementioned laser, if the pitching wort contains SBIs, especially triazole compounds, it is important to use fining agents such as activated charcoal, bentonite, or polyvinylpolypyrrolidon (PVPP) to eliminate or at least reduce their concentration in the wort since they can alter the quality of the beer. Some results obtained by Pérez et al. (2006) confirm that the use of activated charcoal reduce considerably the level of these compounds in the wort. Specifically, more than 80% and 70% of myclobutanil and propiconazole residues, respectively, can be removed.

This is the case of triadimenol and TF-6-1, metabolites of triadimefon and triflumizole, respectively. Both have been found in beer (Miyake and Tajima, 2003). Similar behavior has been observed for triazine compounds such as atrazine and terbuthylazine. Hydroxy analogs (OHA and OHT) were mainly detected in top-fermented beers. Monitoring of these herbicides, mainly in the brewing water, is essential because like atrazine these polar degradation products are classified as possible human carcinogens (Hack et al., 1997). The ethylene bisdithiocarbamate (EBDC) or propylene bisdithiocarbamate (PBDC) fungicides are often used to illustrate the formation of toxicologically relevant metabolites during processing procedures. The conversion of EBDCs and PBDCs into ethylenethiourea (ETU) and propylenethiourea (PTU) is particularly favored by high pH and heat (Timme and Waltz-Tylla, 2003) although the formation of ETU by thermal degradation in aqueous medium can be greatly reduced by the addition of copper sulfate by the formation of a stable cupric EBDC complex (Lesage, 1980). A study carried out with hops treated with radiolabeled EBDCs showed that parent fungicides (maneb/propineb) are mainly changed to ETU/PTU. ETU and PTU are EBDC and PBDC degradation products with carcinogenic effects (Nitz et al., 1984). Therefore, studies to characterize the behavior of pesticide residues during brewing are necessary to perform a more realistic dietary risk assessment.

Summary Points ●

● ●

●

●

Toxicological Risk of Pesticide Residues on Beer In some cases, pesticide metabolites produced during the brewing phases have the same or more toxicity than their parent compounds and they can persist during fermentation. Agrochemical metabolites are generally water-soluble because most of them have hydroxyl or amine groups.

●

Introduction – Barley pests – Use of pesticides on barley – Presence of pesticide residues in raw materials Analysis of pesticide residues in raw materials and beer Food Safety and Policy in the EU – HACCP system in the food industry Effects of brewing on the pesticide residues – Dissipation of pesticide residues during storage of barley, malt, and spent grains – Decline of pesticide residues from barley to malt (malting) – Removal and transference of pesticide residues from malt to sweet wort (mashing) – Evolution of pesticide residues during fermentation – Decrease of pesticide residues during maturation phase (lagering), filtration, and beer storage Influence of Pesticide Residues on the beer quality Toxicological risk of pesticide residues on beer

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