The purpose of this study was to determine the extent to which post harvest mushroom nitrate levels are influenced by the concentration of nitrate in water used to irrigate mushroom crops and if the use of irrigation water that is above the EPA limit for drinking water increases food safety risks to consumers. The Mushroom Good Agricultural Practices (MGAP) program has been successful in bringing the industry up to, and even exceeding, current produce farm food safety standards. Collaborative efforts between Penn State Extension and the American Mushroom Institute (AMI) have been valuable for educating regulators and auditors on how mushrooms are grown and what efforts the industry has taken to prevent microbial, chemical and physical contamination. Occasional disputes between auditors and growers over how to interpret MGAP standards may occur and resolution usually can be achieved by considering current knowledge of produce food safety risks and applying appropriate preventative controls. When information is lacking, issues may often be resolved by conducting appropriate scientific research.
A recent example of such a dispute is a restriction imposed by some auditors on nitrate levels in water used for pre-harvest mushroom irrigation. The auditor's perspective is that water used for irrigation should be potable, meaning that it meets U.S. and state government standards for drinking water which includes limits on nitrates. Mushroom growers, however, see this as an unreasonable requirement because no such limit is imposed on irrigation water used for other produce crops. Furthermore, reverse osmosis equipment necessary to lower nitrate levels can cost thousands of dollars for each well site and thus represents an unreasonable and selective financial burden on mushroom growers. The current MGAP standard for irrigation water (Standard 2.1) states "Water quality shall be known to be adequate for irrigation method and/or chemical application and potable drinking water shall be available for employees" (AMI 2010). Guidelines under the standard state that "water used for irrigation should meet U.S. EPA microbial standards for drinking water." The intent of the standard was to place limits only on microorganisms that can cause food borne disease and not on any particular chemical contaminant such as nitrates; this approach is consistent with farm food standards established for other fruit and vegetable crops.
Nitrates (NO3-) are nitrogen containing chemical compounds that are a natural part of the soil environment. Because they are very soluble in water, nitrates readily percolate into the ground water where they ultimately can enter well water supplies. Nitrates by themselves are not toxic; in fact some studies have shown that they might have beneficial health effects on the cardiovascular system of adults (McKnight et al. 1999, Lundberg et al. 2006). But in the soil, bacteria convert some of the nitrates into nitrites (NO2-) that can pose health risks to certain individuals. Foods that contain high levels of nitrates are of concern because enzymes in human saliva also convert nitrates to nitrites. The very young are especially sensitive to nitrites. At toxic levels, infants may acquire a potentially fatal blood condition known as methaemoglobinaemia, also known as "Blue Baby Syndrome." Based on toxicological studies that take into account estimates of daily water intake by individuals (WHO 2007), the Environmental Protection Agency (EPA) has established an allowable Maximum Contamination Level (MCL) of 10 mg NO3-N /L (ppm) in public drinking water supplies (EPA 1991, 2012).
It should be noted that in the United States, nitrate concentrations in government drinking water standards and surveys are expressed on an elemental nitrogen basis and are referenced using the term "NO3-N." On the other hand, plant tissue nitrate levels are generally based on the total weight of nitrogen and oxygen in the molecule and are written as "NO3" (1 mg NO3-N = 4.4 mg NO3). This convention is used to describe nitrate concentrations in water and mushrooms in this study.
Nitrate levels in most ground water sources are usually well below the MCL. In a survey of 607 wells located in Chester County, PA, nitrate levels ranged from undetectable to 45 mg NO3-N/L, although 75 percent of samples contained less than 6.5 mg NO3-N/L (Ludlow and Loper 2004). Levels tend to be higher in agricultural areas where the application of nitrogen fertilizers or animal manures to soils occurs or where there are high concentrations of farm animals. Lower levels are found in suburban areas where the main source of nitrates is leaking septic tanks, runoff from lawns and golf courses and waste disposal sites. At any location, higher levels can be expected where wells are poorly constructed or maintained such that they are open to surface water runoff (Robillard et al. 2001). Other surveys in Pennsylvania and throughout the United States have similarly shown that most well water samples are well below the EPA MCL with maximum levels nationwide reaching no higher than 60 mg NO3-N/L (Sharpe et al. 1985, Swistock et al. 1993, Swistock et al. 2009, Burow et al. 2010).
It has been estimated that about one third of the total amount of nitrates ingested by individuals is from drinking water with the remainder coming from cured meats and some vegetables (Chilvers et al., 1984). Nitrate levels are generally higher in leafy greens, celery, cabbage and radishes compared to other crops and there is some concern that infants who consume these vegetables in high amounts may be at risk (Santamaria 2006). Because nitrates are an essential plant nutrient, tissue levels are strongly influenced by the amount of nitrogen fertilizer applied during cultivation. Mushrooms are considered a very low source of nitrates in the human diet. Studies have shown that levels in leafy greens may be as much as 50 times higher than in mushrooms (Siciliano et al. 1975, Santamaria 2006, Shao-ting et al. 2007). This is not unexpected given that mushrooms do not use nitrates as a nutritional source of nitrogen (Iiyama et al. 1996). There are no U.S. regulatory limits on the amount of nitrates allowed in vegetables. However, the U.S. EPA has established a reference dose of 1.6 mg NO3-N per kg of body weight per day, which is equivalent to 7.0 mg NO3 per kg of body weight per day. (EPA 1991)
The purpose of this study was to determine the extent to which post harvest mushroom nitrate levels are influenced by the concentration of nitrate in water used to irrigate mushroom crops and if the use of irrigation water that is above the EPA limit for drinking water increases food safety risks to consumers.
Materials and Methods
Mushroom growing procedures
Composting and casing
Phase I compost was prepared at the Penn State Mushroom Test Demonstration Facility (MTDF) using a formula consisting of wheat straw-bedded horse manure, switch grass straw, gypsum and dried, pelletized poultry manure. Water was added to the ingredients as the components were added into a Jaylor® feed mixer. The materials were then placed in a forced aerated bunker for three days during which time compost temperatures reached 80°C (176°F). On day 3, the compost was removed from the bunker, returned to the Jaylor® mixer and distiller's grain and additional water was added. The compost was then placed back into the aerated bunker for an additional three days. On day 6, the compost was returned to the mixer and more water was added until the compost was approximately 75 percent moisture. At the end of the Phase I composting period, the compost was filled into 1.24 m2 (13.3 ft2) wooden trays, which were placed into a Phase II room at the Penn State Mushroom Research Center (MRC). During the seven day Phase II process, the compost was pasteurized to a temperature of at least 60°C (140°F) for two hours, followed by a 4-day conditioning period to remove ammonia. After Phase II was complete, a commercial supplement was mixed into the compost at the manufacturer's suggested rate and spawned with a commercial U1-type hybrid. Thirty six plastic tubs were filled with 50 pounds each of spawned and supplemented compost. The tubs were placed in a growing room at the MRC with humidity kept near 98 percent and compost temperatures maintained at approximately 25°C (77°F) during the 16-day spawn run. Following spawn run, a layer of sphagnum peat moss (ca. 80 percent moisture), pH adjusted with pulverized limestone, was added as a casing layer to the top of each tub. Compost temperatures were lowered to approximately 22°C (72°F) during casehold and humidity levels were maintained near 98 percent until day 11 after casing, at which time the humidity was lowered by removal of the humidifier.
Six nitrate levels were established for irrigation treatments. One treatment consisted of duplicate applications to three adjacent replicate tubs (6 treatments X 3 tubs per treatment X 2 duplicate treatments = 36 tubs) that were randomly assigned locations throughout the growing room. Water used to prepare nitrate solutions for irrigation was obtained in 5-gal jugs from a commercial supplier of spring water (Roaring Springs Bottling, Roaring Springs, PA). Sodium nitrate solutions (50-ml volumes) were prepared at concentrations calculated to achieve 15, 25, 40, and 65 ppm NO3-N after adding to individual jugs of water. The spring water was sampled from triplicate jugs and found to contain 4.9 + 0.1 NO3-N. Therefore, jugs of water to which no sodium nitrate was added were used as a treatment to represent a water source containing nitrate levels compliant with EPA regulations. Distilled water was used as a control treatment to represent water that contained no nitrates.
A roseface applicator was used to water the crop approximately every other day during casehold. For each irrigation treatment, water and the appropriate concentrated nitrate solution or distilled water were added to the applicator reservoir and thoroughly mixed. Actual nitrate levels of irrigation solutions were determined by sampling from the reservoir at two separate watering times (Table 1). The timing and amount of water added during each application was determined based on the experience of the researchers. Water treatments were also added to the crop between flushes, a standard cropping procedure for mushroom production.
Sample collection and analysis
Mushrooms were harvested, counted, and weighed daily during each of the three breaks. For each treatment, a 500-g sub-sample was taken randomly from the harvested mushrooms and each mushroom was wiped free of casing soil with a paper towel. The mushrooms were cut manually into 4-5 mm slices, accurately weighed in pre-weighed aluminum pans and dried under vacuum at 60oC (140°F) for 18 hours. The dried mushrooms were allowed to cool to room temperature, weighed again, and then ground in a small coffee grinder (Cuisinart, Model DCG-12BC) for 1 minute. Water and mushroom nitrate analysis was conducted at the Penn State Agricultural Analytical Services Laboratory using standard automated colorimetric (EPA 1993) and ion-selective electrode (Miller 1998) methods for water and mushroom samples, respectively. Total solids levels were used to convert mushroom dry weight levels to a fresh weight basis.
Target and actual NO3-N concentrations in water used for each irrigation treatment are shown in Table 1. No nitrates were detected in the distilled water. Actual levels were slightly lower than target levels for each of the sodium nitrate solutions, most likely due to preparation and sampling errors. However, the actual values are within ranges reported in several surveys of well water and therefore were used to plot the experimental data.
The effect of nitrate concentration (mg NO3-N/L) in irrigation water on post-harvest mushroom nitrate levels (mg NO3/kg) is shown in Figure 1. When all data from each of the three breaks were analyzed together, irrigation water nitrate concentration showed a significant (p<0.05) positive effect on mushroom nitrate levels. Mushroom nitrate levels, averaged over the three breaks between 0.0 to 61.4 mg NO3-N/L irrigation water, increased from 18.7 + 3.4 to 33.3 + 13.9 mg NO3/kg. However, the significance of the irrigation nitrate effect varied for each of the three breaks. At first break, mushroom nitrate levels were not significantly (p>0.05) affected by irrigation nitrate levels between 0 and 61.5 mg/L. For second and third break mushrooms, tissue nitrate levels were not significantly different between 0 and 36.6 mg NO3-N/L, but significantly increased when the irrigation nitrate concentration was raised to 61.5 NO3-N/L. From the appearance of the graph, it seems that mushroom nitrate levels generally increased between break one and three. However, a break effect was only significant (p<0.05) at irrigation water nitrate levels greater than 12.1 mg NO3-N/L. Mushroom solids and harvest yields were not affected significantly (p>0.05) by any of the irrigation treatments (data not shown).
Discussion and Recommendations
The EPA reference dose of 7.0 mg NO3 per kg of body weight per day can be used as a basis for estimating nitrate risk attributed to consumption of mushrooms. For example, it can be calculated that a 70 kg (154 lb) adult should consume no more than 490 mg of nitrates per day. The lowest and highest individual mushroom sample nitrate levels in this study were 14.9 + 3.9 (0.0 mg NO3-N/L water, second break) and 48.0 + 11.7 mg NO3/kg (61.5 mg NO3-N/L water, third break), respectively. From these values, it can be calculated that the amount of nitrates ingested from a single serving (1 cup = 0.070 kg, USDA 2011) of the mushrooms grown during this study would be 1.0 and 3.4 mg NO3, respectively. These values are only a small fraction of the EPA reference value which therefore strongly suggests that mushrooms grown under any of the irrigation conditions in this study do not represent a hazard to consumers. This conclusion is further supported by the fact that per capita consumption of mushrooms is lower than that for lettuce which contains higher amounts of nitrates. Mushroom growers should therefore be permitted to use well water sources for irrigation that exceed the EPA MCL of 10 NO3-N/L and there is no need to purchase and install expensive reverse osmosis equipment to lower nitrate levels. Growers should, however, continue to test mushroom irrigation water regularly to assure that it meets current EPA microbial standards for potable water.
- AMI. 2010. Mushroom Good Agricultural Practices (MGAP). Industry-Wide Food Safety Standards for Fresh Mushroom Growing, Harvesting, and Shipping. American Mushroom Institute and Penn State University. Revised May 2010. AMI web site. Accessed October 3, 2012.
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Table 1. Target and actual nitrate concentrations of mushroom irrigation solutions.
mg/L + SD
|0||0.00 + 0.00|
|<10||4.85 + 0.07|
|15||12.05 + 1.77|
|25||23.00 + 1.56|
|40||36.55 + 4.03|
|65||61.45 + 3.89|
Figure 1. Effect of mushroom irrigation water nitrate concentration (mg/L NO3-N) on post-harvest mushroom tissue nitrate levels (mg/L NO3). Data points represent the average + standard deviation of duplicate irrigation treatments.
Luke F. LaBorde Ph.D.1 and John A Pecchia Ph.D.2
1 Associate Professor, Department of Food Science, Penn State University
2 Assistant Professor, Department of Plant Pathology and Environmental Microbiology, Penn State University
In: Mushroom News. 60(11):4-11. November 2012.