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Comparison of Nitrate Content in Leafy Vegetables from Organic and Conventional Farms in California

June, 1999 Revised Version

Dr. Joji Muramoto Center for Agroecology and Sustainable Food Systems University of California, Santa Cruz Santa Cruz, CA 95064 831/459-2506

CONTENTS

EXECUTIVE SUMMARY.................................................................................................................1 1. INTRODUCTION.........................................................................................................................3 2. MARKET SAMPLING SURVEY..................................................................................................5 3. FARMER'S FIELD SURVEY......................................................................................................17 4. FIELD EXPERIMENT................................................................................................................24 5. GENERAL DISCUSSION..........................................................................................................33 6. CONCLUSION...........................................................................................................................35 ACKNOWLEDGEMENTS..............................................................................................................36 APPENDIX 1 Toxicity and Regulations of Nitrate in Vegetables..................................................37 APPENDIX 2 Guide and Maximum Tolerated Nitrate Concentrations of Vegetable....................40 APPENDIX 3.1 Summary of Environmental and Practical Factors Affecting Nitrate Content in Spinach and Their Mechanisms.....................................................41 APPENDIX 3.2 Factors Affecting Nitrate Content in a Plant........................................................42 APPENDIX 4 List of Sampling Markets in Santa Cruz County.....................................................43 APPENDIX 5.1 Data and Information about Lettuce Sub-samples..............................................44 APPENDIX 5.2 Data and Information about Spinach Sub-samples.............................................47 APPENDIX 6.1-4 Climate at Market Sample's Origins.................................................................51 APPENDIX 7 Regional Comparison of Nitrate Content in Lettuce and Spinach..........................56 APPENDIX 8 Sampling Number...................................................................................................59 REFERENCES..............................................................................................................................60

EXECUTIVE SUMMARY

Introduction The potential health hazards of human nitrate intake are well studied. Leafy vegetables are the main source of dietary nitrate, and in 1997, the European Union established the maximum levels (limits) for nitrate content in lettuce and spinach. Though studies on this issue were conducted in the USA from the late 1960's to early 1980's, regulations have yet to be introduced. Moreover, the relative nitrate content of organic versus conventionally produced leafy vegetables is unknown in California. Project Goals 1. To demonstrate ranges and variability of the nitrate content of lettuce and spinach sold as " organically grown" and " conventionally grown" in Santa Cruz County. 2. To assess the relative safety of California leafy vegetables based on European standard (EU) nitrate limits. 3. To examine relationships between farming practice and nitrate content in leafy vegetables on certified organic farms in California.

Results 1) Market Sampling Survey Organic and conventionally produced Iceberg lettuce, Romaine lettuce, and spinach were sampled in winter and summer from supermarkets (conventional), farmers market (organic), and natural-food markets (organic) in Santa Cruz County, California. Seasonal differences in nitrate content were significant only for Iceberg lettuce (higher in winter). Conventionally produced spinach had higher nitrate levels than organic spinach, but conventional and organic lettuce nitrate levels were similar. Nitrate content (NO3 mg/kg fresh weight) was highest in spinach (average 2170; range 130 to 4100), followed by Romaine lettuce (1080; 450 to 1900), and Iceberg lettuce (792; 330 to 1400). Nitrate content was more variable in spinach than lettuce and observed ranges were similar to those in previous US studies. Though nitrate levels in lettuce samples never exceeded EU limits, 83% and 33% of our sample of conventional and organic summer spinach exceeded EU limits, respectively, while no winter spinach exceeded EU limits regardless of practice. 2) Farmer's Field Survey Five farms in Santa Cruz and Monterey Counties were surveyed for nitrate content in leafy vegetables, as well as soil characteristics and fertility practices: one with both conventional and organic fields (where we conducted paired field experiments), one conventional farm, and three organic farms. In paired fields, Romaine lettuce nitrate content was similar in conventional and organic fields despite differences in nitrate and potentially mineralizable nitrogen content in soils after harvest. Organic spinach grown using guano tended to have higher nitrate contents than those grown using compost only. However, spinach grown using guano on fields with sandy soil contained the lowest nitrate contents. Nitrate levels in field-sampled lettuce and spinach were similar to those observed in our market samples. 3) Field Experiments A field study of fertilizer management factors affecting spinach nitrate content was conducted at the Farm at the Center for Agroecology and Sustainable Food Systems, University of California, Santa Cruz. The study compared different rates of compost, compost with Chilean nitrate, and commercial organic fertilizer applications. We used regression analysis to estimate the maximum safe yield of each fertilizer practice, defined as "the maximum yield that contained 1

nitrate lower than the EU limit (2500 mg/kg FW)". There was no significant difference in average yield or nitrate content of spinach in compost or compost with Chilean nitrate plots, but nitrate content was more variable in the compost with Chilean nitrate plots. Thus, the maximum safe yield of compost plots (0.70 kg/m2) was higher than compost with Chilean nitrate plots (0.58 kg/m2) when the average + standard error was used to represent nitrate content. Nitrate content in spinach was positively correlated with yield, though commercial organic fertilizer produced lower yields at the same nitrogen rates than other treatments because of slower nitrification in the soil. In conducting these experiments, we also observed that spinach harvested in the afternoon had significantly lower nitrate levels than morning harvests, despite slightly cloudy weather on the harvest day. Conclusions 1. Conventional spinach nitrate levels exceed the maximum levels specified by European Commission Regulation (Table 1.1) much more often than organic spinach. 2. Organic spinach grown using guano and Chilean nitrate tends toward higher nitrate levels than spinach grown using compost. 3. Spinach nitrate levels are affected by the rate and type of nitrogen fertilizers applied, and also by soil nitrification activity, soil texture, and harvest time. 4. Organic growers may reduce nitrate concentration in spinach using methods such as preplant soil nitrate testing, compost based fertility management, afternoon to evening harvest, and petiole removal. 5. California-sampled Iceberg and Romaine lettuce have safe nitrate levels regardless of season and farming practice.

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1. INTRODUCTION

Nitrogen is the main limiting factor for most field crops, and nitrate is the major form of nitrogen absorbed by crop plants. Farmers often use nitrogen fertilizers to increase crop yields. Consequently, many vegetables and forage crops accumulate high levels of nitrate. In particular, leafy vegetables such as spinach, lettuce, and celery contain nitrate at significant levels (Maynard et al., 1976). Leaf and stem tissues accumulate the most nitrate, followed by roots (Lorenz, 1978). Nitrite content in vegetables is usually very low compared to nitrate (Aworh et al., 1980; Hunt and Turner 1994). Vegetables are generally considered the largest source of dietary nitrate. In the average US diet, vegetables contribute 87% of the total daily intake of nitrate (National Research Council 1981). Nitrate is also formed by endogenous synthesis in the human intestine (Tannenbaum et al., 1978). The potential health hazards of nitrate and nitrite are well studied. Nitrate can generally be considered to be of relatively lower toxicity than nitrite. However, about 5% of dietary nitrate is converted to nitrite in humans by bacterial and mammalian metabolic pathways (Walters and Smith, 1981). Potentially carcinogenic N-nitroso compounds can then be formed from nitrite and N-nitrosatable compounds endogenously. Nitrate intake may cause methemoglobinemia (also known as "blue baby disease"), which can be troublesome in infants under three months of age, although it is inconsequential in adults. See Appendix 1 for detail of nitrate toxicity, and Gangolli et al., (1994) for a recent review. Recently, a positive role of nitrate in the human body's defense against pathogenic bacteria has been investigated (Duncan et al., 1997). To protect human health, most European countries regulate nitrate content in vegetables (Appendix 2). In 1997, to eliminate trade barriers across the European Union, European Commission Regulation (EC) No. 194/97 set harmonized maximum levels (limits) for nitrate in lettuce and spinach. The limits vary according to season, with higher nitrate levels permitted in winter-grown vegetables. For lettuce, different limits were set for glasshouse-grown and outdoor crops (The Commission of the European Communities, 1997. Table 1.1). Table 1.1 Summary of the maximum levels in European Commission Regulation (EC) No. 194/97 Product Harvest Period Max. nitrate levels (mg/kg Fresh product) 1 November to 31 March 3000 Spinach (fresh) 1 April to 31 October 2500 Spinach (canned and frozen) 2000 1 October to 31 March 4500 Lettuce* 1 April to 30 September 3500 Lettuce with the exception of 1 May to 31 August 2500 outdoor lettuce * No separate limits have been established for different type of lettuces such as leaf type and head type. This regulation was amended in April 1999 based on results of monitoring carried out by the Member States of the EU since the regulation was adopted (The Commission of the European Communities, 1999). The amendment concluded to maintain the current maximum limits for nitrates in spinach and lettuce. However, these levels will be reviewed in a three-year period before 31 December 2001 for the first time, based on monitoring and the application of codes of good (agricultural) practice to reduce nitrate levels in vegetables. In the US, the US Public Health Service (1962) suggested limits of 3600 and 50 ppm nitrate (dry weight) for spinach and asparagus, respectively. Between the late 1960s and early 1980s, abundant studies on the nitrate issue at large were conducted in the US, including reports by the National Research Council (National Research Council, 1972, 1978, 1981). However, no nitrate standards for vegetables have been introduced in the US. Some Australian researchers (Lyons et al., 1994) and Asian countries such as Korea and Japan (Sohn and Yoneyama 1996) have also pointed out the importance of this problem. 3

Nitrate content in a plant represents a dynamic balance between rates of absorption, assimilation and translocation (Maynard et al., 1976). Therefore, it is affected by numerous environmental and practical factors. See Appendix 3.1 for detail of factors affecting nitrate accumulation in spinach and Appendix 3.2 for those references. Of the factors studied, in general, nitrogen fertilization and light intensity have been identified as the major factors which influence nitrate levels in vegetables (Cantliffe, 1973b). In particular, light intensity and nitrate content in soils before or at harvest are known to be critical factors in determining nitrate levels in spinach (Schuphan et al., 1967; Maynard et al., 1976). In European countries, many studies have demonstrated that organically or biologically grown vegetables have lower nitrate contents than conventionally grown crops (Ahrens et.al., 1983, Vogtmann et.al., 1983; Stopes et.al., 1988, 1989; Leclerc et.al., 1991). Even organic fertilizers, however, may cause high nitrate levels in vegetables, depending on the types and amount of organic fertilizer applied (Maga et.al., 1976; Knorr and Vogtmann, 1983; Termine et.al., 1987). Mineralization rates of organic fertilizers vary widely by their C/N ratio and lignin contents (Chaney et. al., 1992), and easily decomposable organic fertilizers such as blood meals are known to affect plant growth in ways similar to mineral nitrogen fertilizers (Termine et.al., 1987). In general, overfertilization with nitrogen causes higher nitrate content in vegetables regardless of the kind of fertilizers. There is also a higher risk of contaminating ground water with nitrates when overfertilization takes place. Therefore, fertilizing practices that produce vegetables with low nitrate content compatible with optimum yields must be developed (Greenwood, 1990; Sohn and Yoneyama, 1996). Nitrate sap tests have been widely used to develop fertilizer recommendations for crops in California (Hartz et al., 1994). Recently, Smith reported that nitrate content in fresh sap of the root tissue of organic onions was significantly lower than that of conventional onions, although there were no significant difference between their yields (Smith, 1996). However, to evaluate food safety, nitrate content in whole edible parts of vegetables must be examined. In 1978, Lorenz reported nitrate content of many kinds of vegetables produced at experimental fields in Davis, California (Lorenz, 1978). His work coincided with the beginning of the organic farming movement in California. Since then, no studies have been conducted on nitrate levels in vegetable crops, especially on the differences between nitrate content of organically and conventionally grown leafy vegetables in California. Therefore, the objectives of this project were to 1) demonstrate ranges and variability of the nitrate content in lettuce and spinach sold as " organically grown" and " conventionally grown" in Santa Cruz County, California; 2) evaluate the relative safety of California leafy vegetables based on the maximum levels of European Commission Regulation (EC) No. 194/97; and 3) examine relationships between farming practices and nitrate content in leafy vegetables on certified organic farms in California. To accomplish these objectives, a three-part study was employed. Part 1 (Chapter 2) consisted of a market sampling survey to demonstrate the range of nitrate content in lettuce and in spinach sold as "organically grown" and "conventionally grown" in Santa Cruz County, California. As a case study, Santa Cruz County has the advantage of having many kinds of retailers. Part 2 (Chapter 3), the farmer's field survey, was conducted to examine the relationships of farming practice and nitrate content in lettuce and spinach. Finally, a field experiment using spinach grown with different types and levels of organic fertilizers was conducted (Chapter 4). Based on the results obtained, methods to reduce nitrate concentration in spinach for organic growers were discussed at the end (Chapter 5).

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2. MARKET SAMPLING SURVEY 2.1 Goals

The goals of the market sampling survey were to 1) measure the nitrate content of lettuce and spinach sold as " organically grown" and " conventionally grown" in Santa Cruz County, California; 2 ) compare the results with data from studies conducted in the US; and 3) assess nitrate levels in Californian lettuce and spinach by comparison with the maximum levels (limits) set by the European Commission Regulation (EC) No. 194/97.

2.2 Methods

2.2.1 Sampling The maximum nitrate levels specified by European Commission Regulation (EC) No 194/97 (MLECR) for lettuce and spinach take into account seasonal variations (Table 1.1). To compare the results of this study with MLECR, a sampling method which factors in type of vegetable (Iceberg lettuce, Romaine lettuce, and spinach), season (winter and summer), practice (conventional and organic), and brand (3 different brands per practice) was established (Table 2.1). Sampling was replicated twice within each season at intervals of two weeks. Iceberg lettuce and Romaine lettuce were selected as the most commonly purchased head lettuce and leaf lettuce, respectively. When organic Iceberg lettuce was not available, organic green leaf lettuce was sampled. Table 2.1 Sampling method for market survey Factor Type of vegetable (3*) Season (2x2) Practice (2) Brand (3) Sample (5) Total number Level Iceberg lettuce, Romaine lettuce, and spinach** Winter and summer. [2 replications per season] Conventional and organic 3 different brands (growers) per practice 5 sub-samples (bunches or plants) per brand 4x2x2x2x3x5 = 480 determinations** 3x2x2x2x3x5 = 360 sub-samples 3x2x2x2x3 = 72 samples

* Number of levels. ** For spinach, leaf-blades and petioles were analyzed separately. MLECR uses at least ten heads of lettuce or ten bundles of spinach as sub-samples, which are combined and homogenized to produce one representative test sample (The European Commission 1997). In this study, to evaluate variability of nitrate content within sub-samples, five plants or bundles of sub-samples were analyzed separately and averaged to produce one sample data point. Total number of sub-samples and samples was 360 and 72, respectively (Table 2.2). Table 2.2 Numbers of samples and sub-samples Vegetable Winter Summer Conv.* Org.** Conv. Org. Green-leaf lettuce 0 (0)** 2 (10) 0 (0) 0 (0) Iceberg lettuce 6 (30) 4 (20) 6 (30) 6 (30) Romaine lettuce 6 (30) 6 (30) 6 (30) 6 (30) Spinach 6 (30) 6 (30) 6 (30) 6 (30) * Conv. = conventional. Org. = organic. ** Number of sub-samples (= one plant (lettuce) or bundle (spinach)). Conventional vegetables were purchased at supermarkets in Santa Cruz County. Organic vegetables were usually purchased at the farmers market, based on the assumption that the market is the most common place to buy organic vegetables. The farmers market also offered the 5

chance to speak with growers directly to obtain information regarding their farming practices. When target organic vegetables were not available at the farmers market, they were purchased at natural-food markets in Santa Cruz County. No organic vegetables were purchased at supermarkets in this project. See Appendix 4 for the list of sampled markets and Appendix 5 for the price and other information of each sample. As a rule, both conventional and organic vegetables were purchased on the same day. Prior to sampling, the brands of target vegetables that each market received on each sampling day were surveyed by calling the produce manager of sample markets. Based on this information, three supermarkets and the necessary number of natural food markets were selected, assuming different brand samples were grown by different growers. When three different brands were not available on a sampling day, as happened only with summer organic Iceberg lettuce, two samples of the same brand were purchased at different markets. Five plants or bundles (subsamples) of target vegetables were purchased at each market. Based on interviews, the information about the price, the grower or the distributor, the date of unloading of samples, and storage method after unloading was recorded for each sample. Origins of samples were traced and confirmed by calling the grower or the distributor of each sample. 2.2.2 Pretreatment and Nitrate Analysis All sub-samples were put into cooler boxes immediately after purchase and brought to the Agroecology Laboratory, University of California, Santa Cruz. As needed, sub-samples were washed to remove soil and blotted on paper. Fresh weight per plant (lettuce) or bundle (spinach) was measured. For spinach, the number of plants per bundle was counted to calculate fresh weight per plant. Dead leaves and non-edible parts were removed and weighed. A half plant (lettuce) or bundle (spinach) of each sub-sample was taken for nitrate determination and another half was used for moisture measurement. Moisture content was determined by the difference between weights before and after heating at 60-70 °C for 48 hrs. For spinach, leaf-blades and petioles were detached and weighed separately to determine leaf-blade/petiole ratio (FW/FW), and nitrate and moisture content were determined for each part. For nitrate analysis, sub-samples were chopped and mixed with a food processor as needed prior to homogenization. Fifty to 100 grams of sub-sample were weighed and placed into a mixer. Deionized water, nine times the sub-sample's weight, was added and the water and subsample were homogenized for several minutes. A 30 gram sample of homogenate was placed in a centrifuge tube, and 0.5 ML of decoloring agents Carrez 2 and 1 (Adriaanse and Robbers 1969) were added successively, and the tube was capped, and shaken well by hand after each addition of decoloring agent. The sample was centrifuged at 3,500 rpm for 3 min. The supernatant was then filtered with filter paper Whatmann #1 and nitrate concentration in the filtrate was determined colorimetrically by a flow injection analysis system (Lachat Instruments, 1992). Nitrate content was expressed as milligrams nitrate per kilograms on a fresh weight basis (mg NO3 /kg FW) unless otherwise stated. Nitrate concentration in spinach as a whole plant was calculated from nitrate content in leaf-blades and petioles and the weight of each part. 2.2.3 Statistical Analysis Sample data were compared to MLECR. Coefficients of Variation (CV = standard deviation / average x 100) were calculated to indicate variation within sub-samples and factors. Differences between seasons (winter vs. summer), and between farming practices (organic vs. conventional) were analyzed for sample data using a non-parametric Mann-Whitney test (SPSS 1996). 2.2.4 Comparison with Other Data To evaluate the results in historical contexts, information on nitrate content in lettuce and spinach reported in California and elsewhere in the US was collected and compared with the present data.

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2.3 Results

2.3.1 Origin of Samples Sample origins showed a clear seasonal trend (Figure 2.1). In winter, regardless of the kind of vegetable, 67 - 84% of the conventional samples and 42 - 50% of the organic samples were grown in Southern California (Coachella, El Centro, Holtville, and Thermal), Yuma, Arizona, or Mexicali, Mexico. The rest of the winter samples were grown on the South Coast of California

Conv. Spinach

SC 33% AZ +Mex 34%

Conv. Lettuce

N/A SC 8% 8%

DV 25%

DV 33%

AZ +Mex 59%

Central Coast (CC) $

Org. Lettuce

N/A 17% CC 8% DV 42%

Org. Spinach

N/A 17% DV 50% SC 33%

South Coast (SC) $ $ $ Desert Valley (DV) $ $ SC $ 33% $ Mexicali, Mexico Yuma, AZ 1). Winter

Conv. Spinach

N/A 33% SC 17%

Conv. Lettuce

N/A 25%

CC 50%

Org. Spinach

$ $$ Central Coast (CC) $$ $ $ $

CC 100%

CC 75%

Org. Lettuce

South Coast (SC) $

CC 100%

2). Summer Figure 2.1 Origin of samples Plots show location of cities where samples grew. See Appendix 5 for name of cities. CC = Central Coast, SC = South Coast, DV + Desert Valley, AZ + Mex = Yuma Arizona + Mexicali Mexico, N/A = Samples whose origins were not available. Lettuce includes green-leaf, Iceberg, and Romaine. Lettuce; n = 12, spinach; n = 6 for each pie chart, respectively.

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(Oxnard). No local lettuce or spinach was available during the winter sampling period, except for one organic Romaine purchased at the farmers market. In summer, all of the organic and most of the conventional samples were grown on California' Central Coast (Davenport, Gilroy, s Greenfield, Hollister, Salinas, Santa Cruz, Soledad, and Watsonville) although there was one conventional spinach sample from the South Coast of California (Oxnard). 2.3.2 Storage Days and Conditions at Markets Except for some natural food stores that received produce directly from growers, little reliable information was obtained regarding the period between harvest and unloading of vegetables at markets. The period from unloading to sampling was shorter at supermarkets (average 0.9 days. minimum 0 days, maximum 4 days) than at natural food stores (average 2.2 days. minimum 0 days, maximum 5 days) reflecting frequent deliveries and turnover at supermarkets that are more. Refrigeration temperatures varied between 1.7 and 10 ° and C, display rack temperatures ranged from 0.6 to 11 ° C. 2.3.3 Plant Size and Moisture Content At purchase time, 64% of Iceberg lettuce samples were wrapped with plastic film. Other vegetables had no wrapping. Average fresh weight (grams/plant) of Green-leaf lettuce, Iceberg lettuce, Romaine lettuce, and spinach was 249g, 626g, 451g, and 16.5g, respectively (Table 2.3). With regard to fresh weight of samples, there was no significant difference across seasons and practices for any vegetable. Leaf-blade/petiole ratio of spinach (FW/FW) averaged 1.8. Table 2.3 Fresh weight and moisture content of lettuce and spinach purchased in Santa Cruz County, California, 1998. Vegetable n Fresh weight g/plant Moisture % Average CV % Average CV % Green-leaf lettuce* 2 (10)** 249 30 (26)** 93.4 0.6 (0.7)** Iceberg lettuce 22 (110) 626 18 (24) 96.0 0.8 (0.9) Romaine lettuce 24 (120) 451 27 (31) 94.3 1.4 (1.6) Spinach (whole plant) 24 (120) 16.5 46 (53) 92.7 1.4 (1.5) Spinach (leaf-blades) 24 (120) [1.8]*** [26(28)]*** 91.8 1.3 (1.5) Spinach (petioles) 24 (120) 94.0 1.5 (1.6) * Organic green-leaf lettuce was sampled twice in winter when organic Iceberg was not available. ** Number in ( ) indicates values for sub-samples. *** Leaf-blade/petiole ratio (FW/FW) of spinach. Moisture content (%) ranged from an average of 93% (spinach) to 96% (Iceberg lettuce). Although variability of moisture content was relatively small, Iceberg and spinach sampled in summer contained significantly more moisture than those sampled in winter (Table 2.4). Table 2.4 Comparison of moisture content (%) in lettuce and spinach across different seasons and practices Vegetable Season Practice Winter Summer MannConv. Org. MannWhitney testa Whitney test Iceberg 95.4 96.5 *** 95.8 96.3 n.s. Romaine 94.3 94.4 n.s. 94.7 93.9 n.s. Spinachb 91.8 93.5 ** 93.1 92.3 n.s. Values are average moisture content % w/w. a : ***;Significant at 0.1% level. **; Significant at 1% level. n.s.; Not significant. b : Whole plant of spinach. 2.3.4 Nitrate Content and Variation Average nitrate content in green-leaf lettuce (n=2), Iceberg lettuce (n=22), and Romaine lettuce (n=24) was only 786, 792, and 1080 (NO3 mg/kg FW), respectively. On the other hand, 8

spinach (n=24) contained an average of 2170 mg/kg FW of nitrate (Table 2.5). Nitrate content in spinach petioles reached 3400 mg/kg FW, more than twice the nitrate content of leaf-blades. Ranges (difference of maximum and minimum) of nitrate content in sub-samples of lettuces and spinach were 30% and 42% greater than those in samples, respectively. Coefficient of Variation (CV) of nitrate content was also higher in spinach (37%) than in lettuces (27-29 %) (Table 2.5). Table 2.5 Summary of nitrate content in lettuces and spinach (NO3 mg/kg FW) purchased in Santa Cruz County, California, 1998. Vegetable n Average Minimum Maximum CV % Green-leaf lettuce 2 (10)* 786 780 (670)* 792 (960)* 1.1 (12)* Iceberg lettuce 22 (110) 792 484 (330) 1300 (1400) 29 (30) Romaine lettuce 24 (120) 1080 582 (450) 1720 (1900) 27 (30) Spinach (whole plant) 24 (120) 2170 592 (130) 3400 (4100) 37 (41) Spinach (leaf-blades) 24 (120) 1420 419 (93) 2400 (3000) 39 (44) Spinach (petioles) 24 (120) 3400 988 (220) 5500 (6500) 36 (41) * Number in ( ) indicates the value for sub-samples. See Table 2.3 for other footnotes. 2.3.5 Effects of Seasons and Farming Practices on Nitrate Content The averages of nitrate content in samples for each practice and season are shown in Table 2.6 (lettuces) and 2.7 (spinach) on a fresh and a dry weight basis, along with their minimum and maximum values, and CVs. It should be noted that even in the same season and the same practice, there was a wide range of nitrate content in spinach samples. In particular, the ranges of nitrate content tended to be greater in organic spinach than in conventional samples. For instance, in summer organic spinach, maximum nitrate content (3000 mg/kg FW) was as high as five times that of the minimum (600 mg/kg FW) (Table 2.7). Only iceberg lettuce showed a significant seasonal difference in nitrate content; on average, winter samples contained 52% higher nitrate than summer. The effect of management practice on nitrate content was significant only in spinach. That is, conventionally grown spinach contained significantly higher levels of nitrate than organically grown samples (Table 2.8). See Appendix 5 for sub-sample's data.

Table 2.6 Nitrate content in lettuce samples purchased in Santa Cruz County, California, 1998. Vegetable Season Practice n NO3 mg/kg FW NO3 % DW Ave. Min. Max. CV % Ave. Min. Max. CV % Green-leaf Winter Organic 2 786 780 790 0.90 1.19 1.11 1.28 9.7 Green-leaf total 2 786 780 790 0.90 1.19 1.11 1.28 9.7 Iceberg Winter Conventional 6 970 870 1100 8.2 2.15 1.67 2.71 16 Organic 4 977 760 1300 23 2.25 1.59 2.95 25 Winter total 10 973 760 1300 15 2.19 1.59 2.95 19 Summer Conventional 6 707 520 1100 30 1.98 1.27 2.96 30 Organic 6 575 480 660 13 1.88 1.32 2.81 31 Summer total 12 641 480 1100 26 1.93 1.27 2.96 29 Conventional total 12 839 520 1100 24 2.06 1.27 2.96 23 Organic total 10 736 480 1300 35 2.03 1.32 2.95 28 Iceberg total 22 792 480 1300 29 2.05 1.27 2.96 25

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Table 2.6 Nitrate content in lettuce samples purchased in Santa Cruz County, California, 1998 (continued) Vegetable Season Practice n NO3 mg/kg FW NO3 % DW Ave. Min. Max. CV % Ave. Min. Max. CV % Romain Winter Conventional 6 1030 890 1200 13 1.94 1.70 2.24 11 Organic 6 1170 820 1500 23 1.99 1.32 2.85 29 Winter total 12 1100 820 1500 20 1.96 1.32 2.85 21 Summer Conventional 6 1140 770 1700 30 2.27 1.70 3.22 25 Organic 6 954 580 1600 39 1.89 0.66 3.92 62 Summer total 12 1050 580 1700 34 2.08 0.66 3.92 43 Conventional total 12 1090 770 1700 23 2.10 1.70 3.22 21 Organic total 12 1060 580 1600 31 1.94 0.66 3.92 46 Romain total 24 1080 580 1700 27 2.02 0.66 3.92 34 Grand total 48 936 480 1700 31 2.00 0.66 3.92 31 Table 2.7 Nitrate content in spinach samples purchased in Santa Cruz County, California, 1998. Season Practice n NO3 mg/kg whole plant FW NO3 % whole plant DW Ave. Min. Max. CV % Ave. Min. Max. CV % Winter Conventional 6 2230 1500 2900 25 2.92 2.09 3.68 21 Organic 6 1800 890 2600 34 2.16 0.92 3.47 43 Winter total 12 2010 890 2900 30 2.54 0.92 3.68 33 Summer Conventional 6 2850 2000 3400 17 4.65 3.23 5.42 19 Organic 6 1820 600 3000 61 3.01 0.65 5.25 71 Summer total 12 2330 600 3400 42 3.82 0.65 5.42 46 Conventional total 12 2540 1500 3400 23 3.78 2.09 5.42 30 Organic total 12 1810 600 3000 47 2.58 0.65 5.25 63 Grand total 24 2170 600 3400 37 3.18 0.65 5.42 47 Table 2.8 Comparison of nitrate content in lettuce and spinach (NO3 mg/kg FW) across different seasons and practices Vegetable Season Practice Conv. Org. MannWinter Summer MannWhitney test Whitney testa Iceberg 973 641 *** 839 736 n.s. Romaine 1100 1050 n.s. 1090 1060 n.s. Spinach 2010 2330 n.s. 2540 1810 * Values are average nitrate content NO3 mg/kg FW. a : ***; Significant at 0.1% level. *; Significant at 5% level. n.s.; Not significant. 2.3.6 Contrasting with Maximum Levels of European Commission Regulation Nitrate content in lettuces and spinach purchased at various markets in Santa Cruz County, California was contrasted with the maximum levels set by European Commission Regulation (EC) No. 194/97 (Table 1.1). Regardless of season and practice, none of the lettuce samples exceeded the maximum levels. On the other hand, 29% of all spinach samples contained nitrate levels exceeding the maximum EC standards. Summer-grown spinach in particular tended to exceed the maximum levels, with 83% of conventional and 33% of organic spinach sampled in summer containing levels of nitrate above the maximum (Table 2.9). No winter-grown spinach samples exceeded maximum levels of 3000 NO3 mg/kg FW.

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Table 2.9 Percentage of spinach exceeding maximum levels in European Commission Regulation Maximum levels Exceeding the Season in ECR* (EC) No.194/97 Practice n maximum level NO3 mg/kg FW % (n) Conventional 6 0% (0) Organic 6 0% (0) Winter** 3000 Winter total 12 0% (0) Conventional 6 83% (5) Organic 6 33% (2) Summer** 2500 Summer total 12 58% (7) Conventional total 12 42% (5) Organic total 12 17% (2) Grand total 24 29% (7) * ECR = European Commission Regulation. ** Winter; 1 Nov to 31 Mar. Summer; 1 April to 31 Oct.. 2.3.7 Comparison with Other Data Sampled in California and the US Not many data are found with regard to nitrate content in whole edible parts of lettuce and spinach grown in California. A few examples are listed in Table 2.10. Maximum nitrate content in head lettuce and spinach from the present study (market survey) was 1 to 2 % greater than that of Zink and Yamaguchi (1962), Zink (1966), and Lorenz (1974) on a dry weight basis. Note that nitrate content in lettuce reported by Zink and Yamaguchi (1962) included outer leaves of head lettuce that are usually removed in the field and contain higher nitrate than the inner portion (Lorenz 1978). Much more data are available on nitrate content in lettuce and spinach purchased elsewhere in the US. Richardson reported nitrate and nitrite content in various foods almost a hundred years ago (Richardson 1907). After that, most data were reported from the late 1960s to the late 1970s, when substantial studies were conducted on this issue in the US (Table 2.11). It should be noted, however, that sampling numbers in these studies were very small (1 to 20). In addition, no sub-samples were taken, with each plant (lettuce) or bundle (spinach) treated as a sample. With regard to lettuce, the maximum nitrate content reported was 3500 mg/kg FW in Richardson's survey (Richardson 1907), which was as great as twice that of the maximum nitrate content in lettuce found in the present study. However, since the moisture content in Richardson's lettuce was as low as 85% on average, the maximum nitrate content in lettuce on a dry weight basis was 4.5%, which was lower than the maximum value found in the present study. For nitrate content in spinach, on the other hand, the maximum value was 4700 mg-NO3/kg on a fresh weight basis reported in 1978, and was 6.6% on a dry weight basis of the present study. Thus, when we use the ranges of sub-samples, the nitrate contents found in the present study, taken from conventional and organic produce in winter and summer in California, appear to range widely enough to cover most data reported in the US.

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Vegetable

Table 2.10 Ranges of nitrate content in lettuce and spinach sampled in California. Year Analysis by Sampling location Sampling N rate Plant part NO3 % DW reported (Source) season lbs/acre shoots of Lettuce 1962 Zink and Salinas Valley, CA Apr. May 121 - 300 0.88 - 2.9 head lettucea Yamaguchi (farmer's fields) June. Aug. Sep. Oct. 1974 Lorenz Davis, CA May, Oct. 0 - 500 heads 0.12 - 2.0 (field experiments) Nov. Dec. 1999 1999 Muramoto Muramoto Zink Lorenz Muramoto Santa Cruz, CA (markets) Soledad, CA (farmer's field)b Salinas Valley, CA (farmer's fields) Jan. Feb. not Aug. Sep. available Sep. Apr. 137 180 - 220 0 - 500 heads heads top leaf-blades petioles leaf-blades petioles top leaf-blades petioles leaf-blades petioles 1.3 - 3.0 2.4 0.93 - 3.2 0.13 - 2.2 0.24 -10 0.49 - 3.2 1.2 -12 0.64 - 5.4 0.23 - 1.9 1.5 - 7.6 0.9 - 1.9 2.9 - 4.2

Spinach

1966 1974 1999

Davis, CA May, Oct. (field experiments) Nov. Dec. Santa Cruz, CA (markets)

Jan. Feb. not Aug. Sep. available 26 - 120 0 - 348

1999 1999

Muramoto Muramoto

Central Coast of CA Sep. Oct. (farmer's field)c Santa Cruz, CA (field experiments)d Oct.

For the sake of easier comparison, values were converted and rounded from original data as needed. a : Includes outer leaves. b : Conventional farm. See Chapter 3 for detail. c: Organic farms. See Chapter 3 for detail. d : Organic farming field experiment at UCSC. See Chapter 4 for detail.

12

Table 2.11 Ranges of nitrate content in lettuce and spinach purchased in the U.S.

Year reported 1907 1949 1967 1967 1971 1972 1975 1976 1978 1999 Analysis by Sampling location Sampling month n Richardson Wilson Jackson et al. Brown and Smith Lee et al. Maynard and Barker Siciliano et al. Minotti Muramoto Chicago, IL Sep. Ithaca, NY May Washington D.C. (not available) Columbia, MO (not available) Amherst, MA Philadelphia, PA Ithaca, NY Santa Cruz, CA 5 2 5 Lettuce

a

Spinach NO3 % DW 0.45 - 4.5 n 5 4 2 not available 1 1 7 20 0.66 - 3.9 (0.37 - 4.9) f 24 (120) e NO3 mg/kg FW 310 - 3800 300 - 2400 240 - 240 NO3 % DW 0.30 - 3.9

NO3 mg/kg FW 400 - 3500 400 - 1800 490 - 890

May, Jun. Jul. not Sep. available (not available) 1 (not available) Dec. Jan. Apr. - Nov. Jan. Feb. Aug. Sep. 1 3 15 48 (240) e

0.09 - 4.7 280 750 1100 - 1400 440 - 850 c 740 - 2700 d 490 - 1700 (330 - 1900) f

0.31 - 2.9 2100 2300 2200

b

1300 - 4700 600 - 3400 (130 - 4100) f 0.65 - 5.4 (0.16 - 6.6) f

For the sake of easier comparison, values were converted and rounded from original data as needed. a : Both leaf type and head type was included in lettuce. b: Average value. c: Inner leaves. d: Outer leaves. e : ( ) = Number of sub-samples. All other studies used one plant (lettuce) or bundle (spinach) as one sample. f : ( ) = Range of nitrate content in sub-samples.

13

2.4 Discussion

2.4.1 Nitrate Content in Lettuce Although nitrate concentration were relatively low, winter Iceberg lettuce contained significantly higher nitrate than summer (Table 2.8), and moisture content in summer lettuce was greater than that found in winter samples (Table 2.4). Therefore, if we can assume that moisture content of the samples reflected their original moisture regime at harvest, low nitrate in summer samples might be explained in part by dilution along with faster growth under the warmer climate. Figures 2.2 and 2.3 show the amount of precipitation, global solar radiation, and air and soil temperatures at selected locations where samples grew in winter and summer during their possible growth periods. El Centro, one of the origins of the winter lettuce, experienced cooler temperatures and weaker but constant solar radiation (142 W/m2 on average) than did summer growing locations such as south Salinas (see Appendix 6 for climate of other origins). There was no significant difference in nitrate content in lettuces between farming practices. Stopes et al (1988) compared nitrate content in organic and conventional vegetables including lettuce sampled during winter in Great Britain. They found that the peak nitrate content might be lower in organically produced vegetables, although there is considerable variation. Zink and Yamaguchi' (1962) studies at Salinas Valley, California, reported that no relationship was s found between the nitrate content of the aboveground portion of the head lettuce and the amount of nitrogen applied. They suggested that nitrate content of the plant was largely dependent upon the time of application of nitrogen fertilizer and on the growth rate of the plant. In the present study, nitrate concentration in Iceberg and Romaine lettuce sub-samples ranged from 330 to 1900 mg/kg FW. Previous data on nitrate content in lettuce purchased in the US was mostly agreed with this range. 2.4.2 Nitrate Content in Spinach Nitrate content in spinach sub-samples from this study ranged from 130 to 4100 mgNO3/kg FW. These values were similar to those reported from previous US studies. Furthermore, the values from this study are close to 1970s data on a fresh weight basis and greater than those from the 1907 study (the era before synthetic fertilizers) on a dry weight basis (Table 2.11). It is impossible, however, to conclude above trend since sampling number in the previous studies was very limited. In this survey, no significant seasonal difference was found in nitrate content in spinach (Table 2.8). On the other hand, the fact that conventional spinach contained significantly greater nitrate than organic spinach (Table 2.8) suggests that nitrogen fertilizer applications may be the main cause of the difference. Further survey at conventional spinach fields is needed to confirm actual causes. Positive effects of organic fertilizers on nitrate accumulation in spinach have been reported by some studies (Barker 1975; Stopes et al., 1989). As slow release nitrogen fertilizers, some organic fertilizers may reduce nitrate accumulation in plants. However, easily decomposable organic fertilizers such as blood meal and guano might increase nitrate accumulation in the same way as conventional chemical fertilizers (Termine et al., 1987; see Chapter 3), especially with excessive application rates. Some relationships between nitrate content in organic spinach and farming practices are examined in the following chapters. Cantliffe and Phatak (1974b) reported that application of herbicides such as cycloate, alachlor, and lenacil stimulates nitrate accumulation in spinach. Whether such herbicides are currently used by spinach growers in California should be explored. 2.4.3 Comparison with MLECR None of the lettuce samples in this study exceeded the MLECR. In fact, when we compare the US data with data from European countries, nitrate content in European lettuce tended to be greater than those in the US lettuce. It was especially true for greenhouse-grown lettuce during winter. This contrast is explained by differences between the US and Europe in solar radiation, the degree of dependence on greenhouse-grown lettuce, and the variety of lettuce. See Appendix 7 for details of comparison across the US, Europe, and several other countries'data. On the other hand, 29% of all spinach samples exceeded current MLECR (Table 2.9). This rate is similar to that seen in the UK in 1998, when 30% of spinach samples exceeded 14

120 Precipitation mm 80 40 0 11/1/97

120 Precipitation mm

El Centro

40

Precipitation mm

120

Oxnard (Hueneme)

40 Air temperature C 30 20

Air temperature C

30 20 10 0 2/9/98

40

80 40 0 6/1/98

120

10 0 9/9/98

40

11/26/97

12/21/97

1/15/98

6/26/98

7/21/98

8/15/98

Precipitation mm

Oxnard (Hueneme) Oxnard (Pueneme)

South Salinas South Salinas

Air temperature C

Air temperature C

30 80 20 40 10 0 11/1/97 0 2/9/98

30 80 20 40 10 0 6/1/98 0 9/9/98

11/26/97

12/21/97

1/15/98

6/26/98

7/21/98

8/15/98

Winter

Summer

Figure 2.2 Precipitation (bar) and maximum (bold line), minimum (thin line) air temperature at areas where market samples grew during their estimated growth period.

(UC IPM California Weather Database 1999).

Solar Radiation W/m

400 300 200 100 0 11/1/97

Solar Radiation W/m2

El Centro

40 Soil temperature C 30 20 10 0 2/9/98

400 300 200 100 0 6/1/98

Oxnard (Hueneme)

2

40 Soil temperature C 30 20 10 0 9/9/98

11/26/97

12/21/97

1/15/98

6/26/98

7/21/98

8/15/98

South Salinas

Solar Radiation W/m2

Solar Radiation W/m2

400 300 200 100 0 11/1/97

Oxnard (Hueneme)

40 Soil temperature C 30 20 10 0 2/9/98

400 300 200 100 0 6/1/98

40 Soil temperature C 30 20 10 0 9/9/98

11/26/97

12/21/97

1/15/98

6/26/98

7/21/98

8/15/98

Winter

Summer

Figure 2.3 Solar radiation (bar) and maximum (bold line), minimum (thin line) soil temperature (15 cm depth) at areas where market samples grew during their estimated growth period. (UC IPM California Weather Database 1999).

15

.

MLECR (MAFF UK 1999). In particular, conventional spinach exceeded MLECR more than organic spinach, due perhaps to some of the reasons discussed above. As climatic conditions, production methods, and eating habits vary widely in different parts of the European Union, this EC Regulation allows for an optional derogation from the maximum levels, for a transitional period, for nitrate in lettuce and spinach grown and sold in individual Member States. This is provided that growers follow Codes of Good Agricultural Practice and that the nitrate concentrations in lettuce and spinach do not pose a risk to public health. As of August 1998, the UK, Ireland, Finland and Belgium are applying this optional derogation. It should be noted that lettuce and spinach imported from other Member States and third countries must comply with the maximum levels set by this EC Regulation, as must produce exported to other Member States (MAFF UK 1998a). Therefore, this could not only be a safety issue, but also a trade issue. A small amount of spinach was exported from the US to the UK during December to April (MAFF UK 1996). Between 1994 to 1997, according to Monterey County Agricultural Committee (Bohn 1999, personal communication), there is no record of spinach being exported to the European Union from Monterey County, where most conventional summer spinach sampled in this survey was grown. 2.4.4 Sampling Methods One problem with comparing nitrate content data across studies is the difference in number of sub-samples per sample. For example, most market sampling studies for nitrate in vegetables conducted in the US took just one sample without sub-samples (See Table 2.11). Analyzing MLECR, on the other hand, requires at least ten sub-samples to make composite single samples. Maynard et al. (1976) noticed in their review that average values based on a sufficient number of samples will largely circumvent the problem of variability. In this survey, in order to demonstrate difference in ranges when we took different number of sub-samples, each sub-sample was analyzed separately. As mentioned before, ranges of nitrate content in subsamples of lettuces and spinach were 30% and 42% greater than those in samples, respectively. It is also possible to evaluate representativeness of sub-samples for average nitrate content in one-carton (24 plants) of vegetables based on CV within sub-samples (see Appendix 8 for detail). Sampling frequency is another issue that must be taken into account when evaluating this type of study. In the present study, two extremes of winter (Jan. and Feb.) and summer (Aug. and Sep.) were chosen as sampling seasons and sampling was done twice per season. In California, however, as seen last year (during El Niño), it is possible to have rainy weather affect nitrate accumulation in winter-grown plants, an effect that might not apply in spring and fall*. Although less difference might be expected in California than northern European countries due to its rather constant Mediterranean climate, nitrate content trend can differ from year to year depending on the weather (MAFF UK 1996). In addition, this survey did not sample organic vegetables sold at traditional supermarkets, a trend that is on the rise. Therefore, future studies should test the reproducibility of trends discussed above, using more frequent sampling throughout a year and using samples taken from a wider range of markets.

*: Note that winter sampling of this market survey was conducted just before beginning of continuous heavy rain due to El Niño in 1998. See Appendix 6 for weather conditions of sample's origins.

16

3. FARMER' FIELD SURVEY S 3.1 Goal

The goal of the farmer's field survey was to demonstrate the effects of environmental factors such as soil and water quality, as well the effects of farming practices and fertilizer types and amounts, on nitrate content in lettuce and spinach.

3.2 Methods

Five farms were chosen for the field survey; one farm that had both organic and conventional fields (paired fields), three organic farms, and one conventional farm. The farming plan database of the California Certified Organic Farmers and information obtained from farmers at farmers market were used to select farms to be surveyed. Diversity of fertilizing practices among farms and nitrate content of their produce sampled in the market survey were also taken into account in the selection. All the farms are located in Monterey County and Santa Cruz County, California. Farm surveys were conducted in September and October 1998. Fields that had lettuce or spinach ready to harvest within a few days to a week, at maximum, were selected for survey. Information on field, crop, and fertility management was determined from grower interviews. For the paired fields, plants and soils were sampled from four locations in each field, taking into account size of the fields. From each location, six plants were sampled and composited as one sample. At the other fields, 5 to 20 lettuce plants or 10 to 60 spinach plants from 2 to 6 locations were sampled, according to field size, which varied from 2 beds to 30 acres. Plant samples were immediately transferred to cooler boxes. After plant samplings, a shallow soil profile of 12 to 15 inches depth was collected at each point where plant samples were removed. Soil profile characteristics were observed and recorded and penetration resistance of each layer was measured using a pocket penetrometer (CL-700A, Soil test Inc. USA). Surface layer soil and sub-surface layer soil were then sampled and soils taken from the same layer of the same field were composited, except for the paired fields, where soils sampled from each location were treated separately. Soil samples were also put into a cooler box right after sampling. Plant samples were treated and analyzed as described in Chapter 2. Irrigation water was also sampled where it was available. Soil samples were mixed, homogenized, and separated into halves. Half of each sample was air-dried and passed through a 2 mm sieve and used to determine pH (1:1) and electrical conductivity (EC 1:1) (Smith and Doran, 1996). The other half was passed through a 2 mm sieve without drying, and used to determine nitrate and ammonium (2M KCl extraction followed by determination using flow injection method (Lachat Instruments 1993a, b)), potentially mineralizable nitrogen (PMN) (aerobic incubation method (Drinkwater et al., 1996)), and moisture content. PMN was measured in duplicate for surface layers. All the soil data are expressed on a dry weight basis. Nitrate content in irrigation water was measured using the FIA method (Lachat Instruments 1992). Results from paired fields were analyzed using a one-way ANOVA for the difference between conventional and organic fields.

3.3 Results

3.3.1 Comparison between Conventional and Organic Romaine Lettuce Fields A pair of conventional and organic fields that have been managed by the same grower was selected to compare the effect of farming practices on nitrate content in Romaine lettuce. Both fields are located in Pajaro Valley, Watsonville and the same variety of Romaine lettuce ("Gladiator") was grown in the same season (Table 3.1).

17

Table 3.1 Summary of conventional and organic fields surveyed Conventional Organic Crop Romaine lettuce Romaine lettuce Variety Gladiator Gladiator Farming history of the field Conventional many years Organic over 10 years Field acreage (Romaine field) 30 acre (30 acre) 50 acre (0.7 acre) Seeding date Aug. 6, 1998 Aug. 12, 1998 Sampling date Sep. 30, 1998 Sep. 30, 1998 Growth period (by sampling) 55 days 49 days Planting density (#/acre) 22,000 30,000 Fertility practices Preplant 14 -7-14; 408 lbs/acre (7/27)* None *:(application date) Sidedress 20-0-0; 40 gallons/acre (9/3)* Meat and bone meal; 510 lbs/acre (9/12)* Seabird guano; 150 lbs/acre (9/12)* Total nitrogen application rate 84 lbs N/acre 89 lbs N/acre Both fields located in Pajaro Valley, Watsonville, California and managed by the same growers. Table 3.2 Comparisons of nitrate content and some other characteristics of lettuce, soil and water between conventional and organic fields managed by the same grower. Sample Item Conventional Organic ANOVAa Romaine lettuce NO3 mg/kg FW 1880 1750 n.s. Fresh weight g/plant 330 310 n.s. Moisture % 93.9 94.7 * Soil Soil type Elder sandy loam Pinto loam (0 - 6 inches)b pH 1:1 7.1 6.7 *** ECc 1:1 dS/m 0.22 0.38 * NO3-N mg/kg DW 6.3 22 * NH4-N mg/kg DW 4.7 4.8 n.s. PMNd mg/kg DW 2.1 13 *** PRe kg/cm2 3.1 0.35 *** (6 - 12 inches)b pH 1:1 7.0 6.5 ** ECc 1:1 dS/m 0.28 0.33 n.s. NO3-N mg/kg DW 7.3 16 n.s. NH4-N mg/kg DW 4.5 3.0 n.s. PRd kg/cm2 3.2 1.0 ** f Irrigation water NO3-N mg/L n.d.g 0.15 n.a. All values are averages. Replication of each data was 4. a : One-way ANOVA Test. See footnote of Table 2.4 and 2.7 for symbols. b: Depth. c d e : Electrical conductivity. : Potentially mineralizable nitrogen. : Penetration resistance. f g : Ground water. : Not Detected. Total nitrogen application rate at each field was similar (84 lbs-N/acre at conventional. 89 lbs-N/acre at organic). In the conventional field, most nitrogen was applied at preplant with a 14 7-14 fertilizer. In the organic field, in contrast, nitrogen was supplied by sidedressing with commercial organic fertilizers. Soil types of these fields differed (Table 3.2), with Elder sandy loam at the conventional field and Pinto loam at the organic field. At a depth from 0 to 12 inches, the conventional field showed significantly higher soil pH and penetration resistance than the organic field. Surface soil (0 to 6 inches deep) of the organic fields contained significantly higher nitrate and PMN (Potentially Mineralizable Nitrogen) than conventional soil. In particular, the PMN content in the organic fields'topsoil reached 13 mg-N/kg, which was 6 times as high as that of the conventional field. Average nitrate content of conventional and organic Romaine was 1880 and 1750 mg/kg, respectively. Regardless of the different fertilizing practices and soil characteristics mentioned 18

above, there was no significant difference between the nitrate content in Romaine lettuce (Table 3.2). The nitrate contents were lower than the maximum limits of MLECR. However, these levels were 70 to 90% higher than the average nitrate content in market-sampled summer Romaine lettuce. Fresh weight of Romaine lettuce was also not significantly different between practices. Nitrate concentration in irrigation water of these fields was under 0.2 mg-N/L. 3.3.2 Nitrate in Conventional Iceberg Lettuce and Organic Romaine Lettuce One conventional Iceberg lettuce field (C1), conventionally managed for 15 years, and one organic Romaine lettuce field (O2), organically managed for 15 years, were surveyed for their practices and nitrate content in lettuce, soil, and irrigation water (Table 3.3). C1 is located on the edge of the Salinas Valley, in the city of Soledad; O2 is in Watsonville. The grower at C1 applied 137 lbs/acre of nitrogen for Iceberg lettuce using mainly a preplant and sidedress of 6-20-20. The C1 field had clay soil with pH 6.8 and slightly high electrical conductivity of 1.2 dS/m for the topsoil (0 to 6 inches deep). Nitrate content was 33 mgN/kg in topsoil and 9.9 mg-N/kg in subsoil (6 to 12 inches deep). PMN content in topsoil was 9 mg-N/kg. Irrigation water (ground water) contained 11 mg-N/L (= 49 mg-NO3 /L) of nitrate, which exceeds the California maximum contaminant level for nitrate in drinking water (45 mg-NO3 /L). Nitrate content in Iceberg lettuce of C1 was 830 mg/kg, a figure close to the average of market-sampled Iceberg lettuce (794 mg/kg FW). According to the grower, to suppress mildew disease, nitrate concentration in plant tissue was tested at this field by the company and nitrogen fertilization was restricted based on the results. Table 3.3 Farming practices and nitrate content in lettuce, soil, and irrigation water at farmer's fields Farms C1 [Conventional] O2 [Organic] Location Soledad, CA Watsonville, CA Field acreage (of lettuce) 20 acre (20 acre) 9 acre (0.05 acre>) Farming history Conventional for 15 years Organic for 15 years Crop Iceberg lettuce Romaine lettuce Variety Vienas Darkland Cos Growth period from seeding 72 days about 80 days Planting density #/acre 23,000 30,000 NO3 mg/kg FW 830 2,200 Fresh weight g/plant 657 294 Fertility practices Preplant Compost; 5 to 7 t/acre 6-20-20; 350 lbs/acre Sidedress 6-20-20; 350 lbs/acre X 3 [turkey & cow manure, grape 15-8-4; 350 lbs/acre X 1 pomace, wood shavings]* Total N application rate 137 lbs N/acre 90 to 120 lbs N/acre** Soil type Cropley silty clay Baywood loamy sand (0 - 6 inches) pH 1:1 6.8 6.8 EC 1:1 dS/m 1.17 0.68 NO 3-N mg/kg DW 32.7 48.4 NH4-N mg/kg DW 4.69 2.99 PMN mg/kg DW 9.01 35.0 2 PR kg/cm 1.2 0.1 (6 - 12 inches) pH 1:1 6.6 6.6 EC 1:1 dS/m 0.79 0.40 NO 3-N mg/kg DW 9.89 28.7 NH4-N mg/kg DW 1.71 2.16 2 PR kg/cm 1.4 3.6 Irrigation water NO 3-N mg/kg 11.0 1.38 (source) (ground water) (ground water) *: Raw materials of compost. **: Estimated from compost analysis data. See footnotes of Table 3.2 for others.

19

Table 3.4 Comparison of market and field sample grown by the same growers. - Romaine lettuce Grower O2 [Organic] Sample Market-1 Market-2 Field Sampling date Aug. 26 Sep. 9 Sep. 23 NO3 mg/kg FW 1200 1600 2200 Fresh weight g/plant 349 444 294 Moisture % 94.9 95.9 93.9 All values are average. At the O2 farms, the grower applied 5 to 7 tons/acre of farm-made compost. The texture of the Romaine field' soil was sandy. Topsoil pH was 6.8. Nitrate content in soil was the highest s among surveyed fields, reaching 48 mg-N/kg in topsoil and 29 mg-N/kg in subsoil. PMN content in topsoil was 35 mg-N/kg, which was also the highest of the farms surveyed. Romaine lettuce grown at O2 contained 2200 mg/kg of nitrate, the highest concentration among all the Romaine lettuce sampled (including market samples), while nitrate content in market-sampled summer Romaine lettuce from the same field was 1200 and 1600 mg/kg (Table 3.4). Fresh weight of field-sampled O2 Romaine lettuce was lower than that of market samples. 3.3.3 Comparison of Organic Spinach Fields In the market sampling survey, it was found that spinach contained higher nitrate levels than lettuces, and even in organic spinach sampled in the same season, there was considerable difference in nitrate content across samples (Table 2.7). Therefore, to demonstrate the effects of differences in fertility practices between high-nitrate organic spinach and low-nitrate organic spinach, four organic farms growing spinach were surveyed for their practices, nitrate content in spinach, soils, and irrigation water. All four farms are located in Santa Cruz County and Monterey County; Table 3.5 shows the outline of the farms and the survey results. The surveyed organic farms consisted of three different types of fertility practices; compost only (O1 and O2), compost + commercial organic nitrogen fertilizers (O3), and commercial organic nitrogen fertilizers only (O5). Farms O3 and O5 were selected because of the low-nitrate content and high-nitrate content of their market-sampled summer spinach, respectively. The number of years these fields had been managed with organic practices ranged from 4 to 13. Although the growers grew different varieties, all of the spinach was the smooth leaf type. The following trends were observed with regard to the effect of fertility management on nitrate content in spinach. 1. High nitrate content in spinach grown using guano (O3 and O5) Nitrate content in spinach of O3 (2200mg/kg) and O5 (2700mg/kg), both grown using guano, were considerably higher than those of O1 (1300mg/kg) and O2 (470mg/kg), where only compost was used. This trend coincided with the high nitrate content in market-sampled summer spinach from O5 (Table 3.6). A grower interview confirmed that both the market- and the field-sampled spinach from O5 were grown at the same field, but on different dates. The fresh weight of spinach grown at O5 was one of the lowest among all spinach sampled at both markets and fields, showing a tendency by the grower to harvest prematurely. Nitrogen application rate at the field was as low as one third of the others (26 lbs-N/ acre. Table 3.5). According to the information from the farm, the grower was cutting back nitrogen application rates to eliminate heavy accumulation of nitrate in the soil, which occurred early in the season of the sampling year. No data were available on the degree of nitrate accumulation in the soil at pre-plant. On the other hand, the high nitrate content in field-sampled spinach at O3 was completely opposite the trend found in the market survey, which displayed the lowest nitrate content in summer organic spinach (Table 3.6). In addition, fresh weight of field-sampled spinach was also much higher than that of the market samples. In fact, fresh weight of this spinach (33.5 grams per plant) was the highest among all summer organic spinach sampled at markets and fields. According to the grower, the field where market sampled spinach was grown was different from the field surveyed. Although they use almost the same fertility practices at both fields, the 20

Table 3.5 Farming practices and nitrate content in spinach, soil, and irrigation water at farmer's fields surveyed

O1 [Organic] O1 [Organic] O2 [Organic] O3 [Organic] O5 [Organic] King City, CA King City, CA Watsonville, CA Santa Cruz, CA Watsonville, CA 3 acre (0.1 acre) 3 acre (0.1 acre) 4.5 acre (0.05 acre>) 10 acre (0.2 acre) 5 acre ( 5 acre) 6 years 6 years 13 years 9 years 4 years Spinach Spinach Spinach Spinach Spinach a a a (first harvest) (second harvest) (first harvest) Variety Kerdion RZ Kerdion RZ Space Bossanova Nordic IV Growth period 46 days 46 days 70 days 44 days about 30 days Space between plants 1 to 3 inches 1 to 3 inches 2 - 4 inches about 1 inch about 1 inch NO3 (whole) mg/kg FW 1300 220 470 2200 2700 NO3 (leaf-blades) mg/kg FW 920 110 240 1500 1800 NO3 (petioles) mg/kg FW 2300 670 1400 4000 5000 Fresh weight g/plant 15.6 11.0 18.0 33.5 5.7 LPRb FW/FW 3.1 4.1 3.9 2.6 2.6 c Meat&bone meal; Compost; 5 t/acre Compost; 5 t/acre Compost; 5 to 7t/acre Compost ; 5 t/acre Fertility preplant 150 lbs/acre practices [turkey & cow manure, [chicken manure, [chicken manure, Sea-bird guano; Bird guano;300 lbs[B] sidedress[A] grape pomace, grape pomace, wood grape pomace, 150 lbs/acre [A] Foliar feeding [B] or top dress [B] green waste] c shavings] d green waste] c Total N application rate 110 lbs N/acre 110 lbs N/acre 90 to 120 lbs N/acre 120 lbs N/acre 26 lbs N/acree f f Soil type Sandy loam Sandy loam Baywood loamy sand Watsonville loam Pinto loam (0 - 6 pH 1:1 7.1 7.4 6.8 7.0 6.9 inches) EC 1:1 dS/m 0.48 0.32 0.33 0.34 0.47 NO3-N mg/kg DW 7.63 0.23 13.4 21.3 16.6 NH4-N mg/kg DW 3.16 2.72 5.14 4.83 3.25 PMN mg/kg DW 13.4 13.2 15.6 8.00 13.0 PR kg/cm2 1.0 -8 0.7 2.3 (6 - 12 pH 1:1 7.0 6.7 7.0 7.1 inches) EC 1:1 dS/m 0.53 0.35 0.43 0.58 NO3-N mg/kg DW 20.7 17.1 37.2 19.3 NH4-N mg/kg DW 5.27 2.23 2.51 1.75 PR kg/cm2 4.5 3.3 4.5 Irrigation water NO3-N 0.40 1.38 0.36 (source) mg/kg (city water) (ground water) (creek water) (ground water) a : These growers harvest only leaves of spinach. b: Leaf-blade/petiole ratio. c: Commercial compost. d: Farm made compost. e : Since the field soil had too much nitrogen earlier in the year, the grower was cutting back N fertilizers. f: Reclaimed land. g : Not sampled. See footnotes of Table 3.2 and 3.3 for others. Farms Location Field acreage (of spinach) Organically managed Crop

21

field not surveyed is located in the valley, which has cooler temperatures and more sandy soil than the surveyed field (soil type of un-surveyed field is Elder sandy loam). They applied 5 tons/acre of compost at pre-planting and 300 lbs/acre of guano as top-dressing, as well as doing a small amount of foliar feeding. Total N application rate was 120 lbs-N/acre. (Table 3.5). Soil analysis showed that this field had the highest nitrate accumulation both in topsoil (21.7 mg-N/kg) and subsoil (37.2 mg-N/kg) but the lowest PMN (8.0 mg-N/kg) in the topsoil among all organic fields surveyed. PMN content in this field' topsoil was as low as 58% of the average of the other s organic fields. Table 3.6 Comparison of market and field sample grown by the same growers. - Spinach Grower O3 [Organic] O5 [Organic] Sample Market-1 Market-2 Field Market-1 Market-2 Field Sampling date Aug. 19 Sep. 2 Sep. 23 Aug. 19 Sep. 2 Oct. 7 NO3 mg/kg FW 600 620 2200 2500 2900 2700 Fresh weight g/plant 17.4 18.5 33.5 6.8 10.4 5.7 LPR * FW/FW 3.0 2.7 2.6 1.7 1.6 2.6 Moisture % 91.2 91.8 89.6 94.4 94.3 91.4 All values are averages. In O3, market samples were grown at different field (more sandy soil with cooler climate) than the field from which sample was taken in field survey. In O5, both market and field spinach was grown at the same field. *: Leaf-blade/petiole ratio. 2. Low nitrate content in spinach grown using compost only (O1 and O2) At O1 and O2, spinach was grown using 5 to 7 tons/acre of compost only, and these growers harvested only spinach leaves. First and second harvests of spinach were sampled from different parts of a bed in the O1 field and their nitrate content was compared. Results show that the second harvest spinach contained considerably lower nitrates (220 mg/kg) than the first one (1200 mg/kg). It was also found that nitrate level in the topsoil under the second harvest spinach was extremely low (0.23 NO3-N mg/kg) compared to that of topsoil under the first harvest spinach (7.63 NO3-N mg/kg). Spinach sampled at O2 also showed a very low nitrate content of 470 mg/kg. Although this sample was first harvest, unfortunately it was collected at the very end of the harvest and might not be representative of this field. Nitrate content in the topsoil and the sub soil was 13.4 and 17.1 mg-N/kg, respectively. At all the spinach fields, soil pH at 0 to 12 inches deep ranged from 6.7 to 7.4 which appeared to be an acceptable range for spinach growth. Ammonium content was less than 5 mgN/kg at all the fields, indicating that the nitrification process had been taking place normally. Nitrate concentration in irrigation water was lower than 1.5 mg/L at O1, O2, and O3 (irrigation water at O5 was not available) (Table 3.5).

3.4 Discussion

3.4.1 Lettuce Fields Although soil type was different between fields, nitrate content in conventional and organic Romaine lettuce at paired fields was not significantly different. It agreed with the trend and levels of nitrate content in lettuces found in the market survey. The grower sidedressed readily available organic fertilizers (meat + bone meal and guano) rather than basal application of compost for organic Romaine. These fertilizers might cause the comparable nitrate content seen in the organic and conventional plants. High nitrate content (over 2000 mg/kg) in Romaine lettuce was observed at the O2 organic field, although this figure was still lower than the EU safe limit (Table 1.1). Although it was marketable size, the lower fresh weight of field samples compared to those of market samples suggests that field samples were harvested at an earlier age (Table 3.4). Accumulation of nitrate and PMN in soils, which is considered a result of long-term compost applications, may explain a 22

part of this nitrate accumulation in lettuce. High summer temperature stimulates mineralization and nitrification in soils, and may affect nitrate content in plants. Since the sample was taken from a limited space, this result does not represent the whole O2 field. However, this case shows the potential of nitrate accumulation in crops and soils at the fields based on compost application. That is, even compost-based applications may not always be safe when compost has been continuously applied for more than 10 years. 3.4.2 Spinach Fields The results of the organic spinach field survey show that not only the kind of organic fertilizers applied, but also soil characteristics, can significantly affect nitrate levels in plants. That is, although spinach grown using guano tended to contain higher nitrate than spinach grown using compost, this was not always the case. Spinach grown on sandy soils using guano showed the lowest nitrate concentration among all organic spinach. Although further research is needed to determine the actual factor involved, it is obvious that differences in soil texture may affect nitrate movement in soils, and hence, nitrate content in plants. In other words, growing crops such as spinach may require higher levels of readily available nitrogen on fields with sandy than on loam or clay fields. However, growers should apply readily available fertilizers carefully in order to avoid nitrate accumulation in soils and plants. Soil nitrate testing may provide useful information to determine whether crops need additional nitrogen. Leaf blade harvest, which was practiced at O1 and O2, is an effective way to eliminate excessive nitrate in spinach if such a practice fits with the farm' marketing strategy. For example, s if this method is applied on O3 and O5, which had 2200 and 2700 mg/kg FW of nitrate in whole spinach plants, nitrate concentration would drop to 1500 and 1800 mg/kg FW, respectively. The decrease in nitrate content in spinach observed in the second harvest at O1 is related to low nitrate content in the soil (Table 3.5). If soil contains a high concentration of nitrate at second harvest due to sidedressing with N fertilizer, mineralization and nitrification from soil nitrogen, or other factors, nitrate content in the second harvest spinach will also be high.

23

4. FIELD EXPERIMENT 4.1 Goal

The goals of the field experiment were to 1): demonstrate the effect of farming practices, especially organic fertilizing practices, on nitrate content in spinach. 2): estimate application rates of organic fertilizers to produce spinach with acceptable nitrate contents compatible with optimum yields.

4.2 Methods

A field experiment was conducted at the Farm of the Center for Agroecology and Sustainable Food Systems (CASFS), University of California, Santa Cruz. The soil is Elkhorn sandy loam and has been in certified organic production since 1974. The experimental field had been fallowed for 10 years. Chemical characteristics of the soil profile are shown in Table 4.1. Table 4.1 Chemical characteristics of the soil profile at the experimental field. (pre-fertilizing) NO3-N NH4-N Layer pH(H2O) Electrical (depth cm) conductivity mg/kg mg/kg 1:1 dS/cm 1:1 DW DW 1 ( 0 - 15) 6.3 0.43 59.9 8.65 2 (15 - 30) 6.4 0.22 19.5 7.30 3 (30 - 45) 6.5 0.13 2.69 3.40 Data are averages of two profiles. A randomized block designed experiment with two factors of kind and rate of organic fertilizers was established (Table 4.2). Three types of fertilizers; compost (made at the CASFS Farm from horse manure and chicken manure; pH 6.2, total nitrogen 0.7% FW, moisture 34%), compost with Chilean nitrate (Compost + CN), and commercial organic fertilizer (N-P2O5-K2O; 77-7 dry analysis, made from guano, bone meal, chicken manure. OF) were tested. As application rates, four levels of none (control), standard (87lbs total N/ha, Std), twice the standard (174lbs total N/ha. 2 x Std), and four-times the standard (348lbs total N/ha. 4 x Std) were established. At the Compost + CN plot, Chilean nitrate was sidedressed at a rate of 10% of the total nitrogen input. At the OF plot, application rates of total N/acre on each level were about half that of the other plots, taking into account the high mineralization rate of organic fertilizers. Total number of treatments was ten and each treatment had two replications (20 plots total). Plot size was 16.5 m2 (6m x 2.75m) with two beds having two rows per bed. Figure 4.1 shows the arrangement of the plots. Prior to the experiment, weeds were plowed in May 1998. A day after light sprinkler irrigation, compost and organic fertilizers were applied and incorporated by rotor tiller on August 25. On August 31, 11 lbs per acre of spinach (Spinacia oleracea), cultivar Daehnfeldt (semiSavoy type) was seeded by machine. Plant density was adjusted at intervals of 6 inches after germination. Chilean nitrate was sidedressed at the Compost + CN plot (treatments #3, 6, and 9) 4 weeks after seeding. Harvest was carried out 6 weeks after seeding on October 12, 1999. During the growth period, sprinkler irrigation was applied 9 times (0.27 inches each on average) and plots were weeded mechanically and manually as needed. To trace the changes of nitrate and ammonium content in soils, surface soil samples (0 to 15 cm depth) composited from four points per plot (two soils per bed x 2) were taken every week. The samples were refrigerated at 4 ° until analysis. Nitrate and ammonium in soils were C analyzed by the method described in Chapter 3. Moisture of the fresh soils was also determined. Maximum leaf length of 20 plants per plot (10 plants per bed x 2) was measured once a week during 2 weeks after seeding to harvest. On harvest day, to examine diurnal change of nitrate content in spinach, 10 plants per plot (5 plants per bed x 2) were sampled both in the morning

24

Table 4.2 Treatments of the field experiment. # Rate tons/acre [tons/ha] 1 Control 0 2 Compost 5.0 [12] 3 Compost + 4.5 [11] CN* 0.054[0.061] 4 Organic fertilizer 0.36 [0.89] 5 Compost 10 [25] 6 Compost + 9 [22] + CN* 0.11 [0.12] 7 Organic fertilizer 0.72[1.8] 8 Compost 20 [50] 9 Compost + 18 [45] + CN* 0.22 [0.24] 10 Organic fertilizer 1.5 [3.6] * : 10% of nitrogen budget Fertilizer N% T-N lbs./acre [kg/ha] 0 87 [98] 78+8.7=87 [88+10=98] 43 [48] 174 [196] 157+17=174 [176+20=196] 86 [96] 348 [392] 314+35=348 [353+39=392] 171 [193]

0 0.79 0.79 16 5.4 0.79 0.79 16 5.4 0.79 0.79 16 5.4

10 8 5 6 7

4 3 2 1 9

2 1 8 4 6

3 9 7 5 10

Figure 4.1 Arrangement of the plots. Number shows the treatment number in Table 4.2. (between 9 to 11am) and in the afternoon (between 2 to 3 pm). The plants in the middle 2 meter zone of each plot were harvested and weighed to measure yield (kg/m2). All the soil and plant samplings were conducted in the middle 4 meter zone of each plot to avoid areas that might be contaminated by the adjacent plot. Data were analyzed by ANOVA method ( = 0.05). We used regression analysis to estimate the maximum safe yield of each fertilizer practice, defined as "the maximum yield that contained nitrate lower than the maximum limit of European Committee Regulation (MLECR, 2500 NO3 mg/kg FW)". For this purpose, "mean (average) + standard error" was used for nitrate content, taking into account its variability. The relationship between nitrate in soils and spinach was examined using the plant available-N method, where plant available-N is defined as N-dressing plus the amount of available N (NO3-N) in the soil at sowing in 0 to 60 cm deep soils (kg-N/ha, Breimer 1982).

25

4.3 Results

4.3.1 Plant Development The average germination rate of spinach was below 50%. Consequently, plant density was adjusted at intervals of 6 inches, which was far wider than spinach fields surveyed in the previous chapter. Maximum leaf length at the Compost plot increased almost linearly after 2 weeks to harvest. In the Compost + CN plot, changes in maximum leaf length shifted slightly upward after Chilean Nitrate application at 4 weeks after seeding. Development of maximum leaf length at the OF plot was slower than Control plot by 3 weeks after seeding, and even after that, were lower than the Compost plot. Especially, 4 x Std. of OF plot had the smallest maximum leaf length among all the fertilized plots through the whole growth period (Figure 4.2). See Figure 4.3 for climate during the growth period.

20

Chilean Nitrate Sidedressing

Control Compost Std. Compost+C.N. Std. Org.Fer. Std. Compost 2xStd.

15 Maximum leaf length (cm)

10

Compost+C.N. 2xStd. Org.Fert. 2xStd. Compost 4xStd.

5

Compost+C.N. 4xStd. Org.Fert.4xStd.

0 0 1 2 3 4 5 6 7 Weeks after seeding

Figure 4.2 Changes in maximum leaf length of spinach

120 Precipitation mm

40

2 Solar Radiation W/m

400

Air emperature C

40 Soil temperature C 30 20 10 0 10/10/98

30 80 20 40 10 0 8/31/98 0 10/10/98

300 200 100 0 8/31/98

9/20/98

9/20/98

Figure 4.3 Climatic condition during field experiment at Santa Cruz, California. On left figure, precipitation (bar), maximum air temperature (bold line), and minimum air temperature (thin line). On right figure, global solar radiation (bar), maximum soil temperature (bold line), and minimum soil temperature (thin line). (UC IPM California Weather Database 1999)

26

4.3.2 Yield Spinach yield ranged from 0.26 kg/m2 to 0.70 kg/m2. In the Compost plot, spinach yield increased along with compost application rate up to 2 x Std. That is, 5 tons/acre and 10 tons/acre of compost application increased spinach yield 106% (0.53 kg/m2) and 161% (0.67 kg/m2) of Control plot (0.26 kg/m2), respectively. However, compost application at 4 x Std rate didn' t increase the yield from 2 x Std rate. There was no significant difference in yield between any application rates, except control. Spinach yield in the Compost + CN plot was slightly higher than that of the Compost plot at each rate, although it was not significant. Yields of the Organic Fertilizer plot and Control plot were significantly lower than those of the Compost plot and Compost + CN plot. Quadratic curves were selected as regression curves of Compost and Compost + CN plots due to the higher significance (Figure 4.4). 4.3.3 Fresh Weight and Leaf Blade/Petiole Ratio of Spinach Leaf blade/petiole ratio (LPR) was as high as 4.9 to 5.2, whereas the ratio from market sampled spinach averaged 1.8, with a maximum of 3.3. Fresh weight of the spinach in fertilized plots also tended to be greater than that of market spinach (Table 4.3).

1.0

4 x Std. 2 x Std.

y = 1.93E-08x - 1.76E-05x + 0.00500x + 0.2550

3 2

0.8

Std.

Yield (kg/m ) 0.6

2

y = 4.43E-09x - 9.74E-06x + 0.00392x + 0.2550 0.4 Compost 0.2 Com.+ CN

3

2

Cont.

0.0 0 100 200 300

Org. Fert.

400

Total N application rate (N lbs/acre)

Figure 4.4 Changes in yield of spinach associated with kind of fertilizers and their application rate as nitrogen. On X axis, from left to right, each nitrogen application rate corresponds to application rate of Control, Standard, 2 x Standard, and 4 x Standard, respectively. Error bars indicate average ± standard error. Quadratic regression curves were fitted for Compost plots and Compost + CN plots.

27

Table 4.3 Fresh weight and leaf blade /petiole ratio of spinach Fresh Weight LPR* g/plant FW/FW Control 15 5.2 Compost 40 (35 ­ 46)*** 4.9 (4.7 ­ 5.0) Compost+C.N.** 42 (38 ­ 45) 5.1 (5.0 ­ 5.2) Org. Fert. 21 (17 ­ 28) 5.0 (4.7 - 5.2) Market Samples 17 (4.9 ­ 48) 1.8 (1.1 ­ 3.3) *: LPR; Leaf-blade/Petiole Ratio. **: C.N.= Chilean Nitrate. ***: Average (Minimum ­ Maximum). 4.3.4 Nitrate Content Nitrate content in spinach ranged from 1500 mg/kg FW to 2300 mg/kg FW as an average of morning and afternoon sampling (Figure 4.5).

3000

Treatment

y = 1.27E-05x - 0.0231x + 8.24x + 1500 2500 NO3 (mg/kg FW)

3

2

2000 y = -3.16E-06x - 0.0124x + 6.27x + 1500

3 2

1500

Compost Com.+ CN Org. Fert.

1000 0 100 200 300 400 Total N application rate (N lbs/acre)

Figure 4.5 Changes in nitrate content in spinach associated with kind of fertilizers and their application rate as nitrogen. On X axis, from left to right, each nitrogen application rate corresponds to application rate of Control, Standard, 2 x Standard, and 4 x Standard, respectively (See Figure 4.3). Error bar shows average ± standard error. It showed strong positive correlation with the yield ( = 0.001. Figure 4.6). The highest nitrate concentration was detected in the Compost + CN plot at a rate of 2 x Std (9 tons/acre of compost + 0.11 tons /acre of Chilean nitrate). Nitrate in spinach in the OF plot was the lowest among fertilized plots, ranging from 1400 to 1800 mg/kg FW. However, there was no significant difference in nitrate content of spinach across any type of fertilizers and application rates, except Control plot. In spite of cloudy weather on the sampling day, compared to morning sampled spinach, nitrate content in afternoon sampled spinach (whole plant) was as low as 10% and 16% on a fresh weight basis and a dry weight basis, respectively. See Figure 4.7 for diurnal changes in

28

solar radiation and air temperature on the harvest day. In addition, most nitrate content reduction took place in leaf-blades, where nitrate was reduced, rather than in petioles (Table 4.4).

0.8 Yield (kg/m ) 0.6 0.4 0.2 0 0 1000 2000 3000 Nitrate content (NO3 mg/kg FW)

Table 4.4 Effect of sampling time on nitrate content in spinach Moisture Leaf-blade NO3 (mg/kg) Petiole NO3 (mg/kg) Whole NO3 (mg/kg) % FW DW FW DW FW DW Morning 89.6 1670 16300 3750 36200 2010 19600 Afternoon 88.9 1390 12600 3840 35800 1820 16500 (Relative change) (-0.8%) (-17%) (-23%) (+3.1%) (-0.4%) (-9.6%) (-16%) F testa *** ** ** n.s. n.s. n.s. ** All values are averages of all plots. Sampling date: 12 October 1998. Morning; 9 ­ 10:30am. Afternoon; 2:30 ­ 3:30pm. a Significant at ***; 0.1% level. **;1% level. n. s. ; not significant. Sampling time Figure 4.6 Correlation of nitrate content in spinach and its yield.

R2 = 0.87***

2

Figure 4.7 Changes in solar radiation and air temperature on the harvest day at UC Santa Cruz. 2 Left figure shows diurnal change in solar radiation (W/m ),. Right figure shows changes in airtemperature (degree C). (UCSC Applied Sciences Met Station 1999)

29

400 300 200 100 0

N-mg/kg

Control

-1

0

1

2

3

4

5

6

WEEKS AFTER PLANTING

400 300 N-mg/kg 200 100 0 -1 0 1 2 3 4 5 WEEKS AFTER PLANTING 6

400 N-mg/kg

N-mg/kg

400

CompostStd.

300 200 100 0 -1

Compost2 x Std.

300 200 100 0

Compost4 x Std.

0 1 2 3 4 5 WEEKS AFTER PLANTING

6

-1

0 1 2 3 4 5 WEEKS AFTER PLANTING

6

400

400

300 N-mg/kg 200 100 0 -1 0 1 2 3 4 5 WEEKS AFTER PLANTING 6

N-mg/kg

200 100 0 -1 0 1 2 3 4 5 WEEKS AFTER PLANTING 6

N-mg/kg

Compost+C.N.Std.

300

Compost+C.N.2 x Std.

400 300 200 100 0 -1

Compost+C.N.4 x Std.

0 1 2 3 4 5 WEEKS AFTER PLANTING

6

400 N-mg/kg 300 200 100 0 -1 0 1

N-mg/kg

Org. Fert.Std.

NH4-N NO3-N

400 300 200 100 0

Org. Fert.2 x Std.

N-mg/kg

400 300 200 100 0

Org. Fert.4 x Std.

2

3

4

5

6

-1

0

1

2

3

4

5

6

-1

0

1

2

3

4

5

6

WEEKS AFTER PLANTING

WEEKS AFTER PLANTING

WEEKS AFTER PLANTING

Figure 4.8 Changes in nitrate and ammonium ion content in topsoils at each plot during growth period of spinach.

30

4.3.5 Maximum Safe Yield The maximum safe yield was calculated using regression analysis for compost and compost + Chilean nitrate. Based on quadratic regression curves obtained, maximum safe yields, in this particular experiment, were estimated as 0.70 kg/m2 in the Compost plot, and 0.58 kg/m2 in the Compost + CN plot, respectively. Because of greater standard error in nitrate content in spinach in the Compost + CN plot, maximum safe yield at the Compost plot was 21% higher than that in the Compost + CN plot (Figure 4.4 and 4.5). 4.3.6 Nitrate and Ammonium Content in Soils In the Control plot, initial nitrate content in the soil was 80 mg-N/kg and it gradually decreased to 60 mg-N/kg by harvest (Figure 4.8). A small amount of ammonium was detected only during ­1 to 1 weeks after seeding. By compost application, nitrate content in the soils increased proportionally to its application rates. That is, at every 5 tons/acre of compost applied, nitrate in the soil increased by about 15 mg-N/, which was approximately equivalent to the amount contained in the compost when it was applied. At 5 weeks after seeding, since soil samples were taken from the spots where Chilean nitrate was sidedressed, a considerable increase in nitrate was observed, associated with the Chilean nitrate application rates. Even after harvest (6 weeks after seeding), soil nitrate content reached 170 mg-N/kg and 390 mg-N/kg at Compost + CN of 2 x Std rate plot and Compost + CN of 4 x Std rate plot, respectively. Very little ammonium was detected in the soils of any Compost plot throughout the growth period. In contrast, in the soils in the OF plot, a significant ammonium concentration was detected. On seeding day, ammonium in the soils accumulated to levels as high as 150 and 300 mg-N/kg at 2 x Std and 4 x Std rate of OF plot, respectively. Although nitrification took place gradually, it took 4 (Std rate) to 5 (2 x Std rate) weeks after seeding to complete. In the 4 x Std plot, 40 mg-N/kg of ammonium, accompanied by 230 mg-N/kg of nitrate, were found in the soil after harvest. 4.3.7 Plant Available N Breimer (1982) demonstrated that nitrate content in spinach was correlated positively with "plantavailable nitrogen" by field experiments and on-farm experiments conducted in the Netherlands. He reported the critical level of plant-available N at which 10% yield reduction of maximum yield keeping nitrate content in spinach below 2500 mg/kg FW was 225 kg-N/ha.

3000

NO 3 in spinach mg/kg FW

2000

1000

Control Compost Compost+CN Org. Fert.

0 0 100 Plant Available N kg/ha 200 300

Figure 4.9 Correlation between plant available-N and nitrate content in spinach (mg/kg FW). Bars indicate standard error of averages. See text for definition of plant available-N.

31

Plant available-N was calculated for the field experiment and was compared with nitrate content in spinach (Figure 4.9). For nitrate content in soils 15 to 60cm deep, the average of two profiles was used (Table 4.1). A significant positive correlation was found between plant-available N and nitrate content in spinach when the highest rates (4 x Standard) of Compost plot and Compost + CN plot were eliminated. This result suggests the existence of a limiting factor other than available N at these plots. In this case, average nitrate content in spinach in the maximum yield plot (Compost, Standard x 2) was slightly lower than 2500 mg/kg FW. However, since nitrate content in the maximum yield plot was variable, the critical level of plant-available N at which nitrate content can reach 2500 mg/kg FW was considered to be between 100 and 150 kg-N/ha in this experiment.

4.4 Discussion

A sidedress of Chilean nitrate, at a rate of 10% of the nitrogen budget, did not significantly increase nitrate content in spinach over the Compost plot. However, variability of nitrate content in spinach was greater in the Compost + CN plot than in the Compost plot. Therefore, the maximum safe yield in the Compost + CN plot was lower than that in the Compost plot when we use "mean + standard error" as nitrate content. California Certified Organic Farmers (CCOF) restricts application of Chilean nitrate to 20% of the total nitrogen budget due to concern over sodium accumulation in soils. Since Chilean nitrate is more soluble than guano, it should be applied carefully to prevent not only sodium build up in soils but also nitrate accumulation in crops and soils. The "plant available-N" method warrants further investigation in California. However, it doesn't take into account nitrogen mineralization from soil and organic fertilizers. Although it was assumed that the growth period of spinach was short enough to neglect those effects, Breimer found that data from some farmers'fields didn't fit well into the correlation because of the history of heavy application of manure. In this experiment, the amount of nitrate increased in soils of Compost applied plots mostly coincided with the amount of nitrate derived from compost when it was applied. From the changes in soil nitrate levels, mineralization of nitrogen in compost during the growth period was considered to be negligible. Thus, we could find a good correlation between plant available-N and nitrate content in spinach, except in plots with the highest application rate. Future studies on depth of soils to be sampled, and methods for evaluating mineralization of nitrogen from soils and organic fertilizers are necessary. Application of a nitrate strip test or electrical conductivity method for estimating nitrate content in soils at planting may provide a practical on-farm tool to manage nitrate concentration in spinach (See Chapter 5). Figure 4.9 suggests the existence of limiting factor(s) for spinach growth other than nitrogen at the highest rate (4 x Standard) of Compost applied plots. Future studies, including evaluation of changes in soil physical characteristics at a high rate of compost application, may be needed to demonstrate the reason. A significant "morning-evening" harvest effect on nitrate content in spinach was found in this experiment regardless of slightly cloudy weather on harvest day. This effect is well known on days with fine weather when spinach contains high level of nitrate (Breimer 1982). California receives abundant solar radiation throughout year. Therefore, afternoon to evening harvest is recommended as a way to reduce nitrate content in spinach, especially when a crop is thought to have a high nitrate concentration. Low germination affected planting density, leaf-blade/petiole ratio of spinach, and nitrate content in spinach. Lorenz (1978) suggested that wider plant density leads to spinach with larger leaf-blades and shorter petioles, and hence lowers nitrate content in a whole plant. If the germination rate had been higher and the spinach had been grown at the standard interval of 1 to 2 inches in this experiment, the nitrate content might have been much higher. In this experiment, a fallowed field was used because no option was available at that time. Due to slow nitrification in the soil, consequently, spinach growth at the OF plot was severely suppressed. A further experiment at a cultivated field, with regular nitrification activity in the soil, is desirable. What the results suggested is a significant effect of nitrification on nitrate content in spinach, in addition to type and application rate of organic fertilizers. Although type and rate of nitrogen fertilizers influence the nitrate content of spinach in organic fields, other environmental and practical factors, as listed in Appendix 3.1, should also be taken into account in determining the main factors influencing nitrate content under local growing conditions.

32

5. GENERAL DISCUSSION;

How Can Organic Growers Reduce Nitrate Accumulation in Spinach? Lorenz (1978) listed methods to reduce nitrate content in spinach; use of lower-nitrate content cultivar, appropriate nitrogen fertilization, application of ammoniacal fertilizer associated with application of nitrification inhibitor, split nitrogen application rather than single basal application, utilization of slowrelease fertilizer, plant tissue testing, afternoon harvest, petiole removal, and less dense planting. Organic growers can use some of those methods (Table 5.1). Pre-plant soil nitrate testing may provide useful information for determining appropriate application rate of organic fertilizers. In that case, mineralization rate of those organic fertilizers also must be taken into account (Chaney et al., 1992). Some on-farm soil nitrate testing methods such as the nitrate strip test and electrical conductivity method are available (Sarrantonio et al., 1996). Preliminary results of this study suggest that such tests are particularly useful for fields which have been organically managed for more than 10 years, for growers who wants to use commercial organic certified fertilizers such as guano and Chilean nitrate, and for the fields where residual nitrate from previous crops is considered to be significant. In addition, as seen in Chapter 3, soils in organic fields tended to contain greater potentially mineralizable nitrogen (PMN) than conventional fields. Similar trends have been found in other studies (Doran et al., 1987; Drinkwater et al., 1995). Large PMN in conjunction with high concentration of mineral N, especially during times of reduced crop uptake, could indicate susceptibility to N losses through leaching. Organically managed soils are sometimes characterized by higher levels of PMN, in conjunction with lower instantaneous mineral N pools compared with soils receiving conventional mineral fertilizers (Drinkwater et al., 1996). What we found in Chapter 3, however, was a case of an organic field that was high in both nitrate and PMN, suggesting variability of N dynamics across actual organic fields depending on their fertility management. Although the laboratory incubation method used in this study cannot simulate field conditions (Rice and Havlin, 1994), PMN in soils, in conjunction with mineral N, appears to be important for evaluating nitrogen dynamics in organic fields' soils. In situ nitrogen mineralization from organic fertilizer and soil may be estimated to some extend using simulation models. For example, a dynamic simulation model has been applied successfully to compare productivity and N leaching across biodynamic management, conventional management, and permanent grassland (Droogers and Bouma, 1997). A nitrate strip test for plant sap nitrate is also available (Scaife and Stevens, 1983; Prasad and Spiers, 1984). On tissue testing in organic vegetable systems, however, Smith (1996) found that nitrate content in organic onion tissue may not be as good an indicator of crop nitrogen as it is in conventional systems. Maynard and Hochmuth (1997) noticed that "organic managed crops may show lower petiolenitrate (NO3-N) concentrations. Total macro-nutrient concentration of whole leaves is the preferred method of evaluating nutrient sufficiency under organic fertility management". Therefore, future study should evaluate whether we can apply conventional critical nitrate levels for spinach to organically grown spinach. Cultivar selection for reducing nitrate content in spinach based on leaf-types appears not consistent (Maynard et. al., 1976). Other methods such as compost based fertility management, afternoon to evening harvest, leafblades harvest, and less dense planting can also be employed to lower nitrate accumulation, although some of these methods may result in lower yield.

33

Table 5.1 Methods to reduce nitrate concentration in spinach in organic systems Method Organic fertilizer application based on pre-plant soil nitrate testing Merit · May increase N efficiency · On-farm methods such as EC meter method, and nitrate strip test are available Condition and/or demerit · Further studies are needed for establishing threshold values of nitrate and PMN level in soils · Other local factors such as soil texture, nitrification activity of soil, irrigation practice, light environment, and temperature may also affect · Need large quantities · Pre-plant soil nitrate test is recommended for fields where have been managed organically for years (>10 years?) · Effective when NO3 in plant is high on fine weather

Compost based fertility management

· Safer than readily available nitrogen fertilizers even at high application rate

Afternoon to evening harvest

· 10% reduction (FW) in this field experiment · 28% reduction (DW) by harvest after 12 hr of light instead of 0 hr (Cantliffe 1972b)* · On average, 31% reduction (FW) in this market-sampling survey · 28% (cv. America), and 45% (cv. Hybrid 424) reduction (FW) (Olday et al., 1976)*

Petioles removal (leaf-blade harvest)

· Lower yield

* Adopted from Maynard 1978

34

6. CONCLUSIONS

1. Conventional spinach nitrate levels exceed the maximum levels specified by European Commission Regulation much more often than organic spinach. 2. Organic spinach grown using guano and Chilean nitrate tend toward higher nitrate levels than spinach grown using compost. 3. Spinach nitrate levels are affected by the rate and type of nitrogen fertilizers applied, and also by soil nitrification activity, soil texture, and harvest time. 4. Organic growers may reduce nitrate concentration in spinach using some or all of the methods listed in Table 5.1. 5. California-sampled Iceberg and Romaine lettuce have safe nitrate levels regardless of season and farming practice.

35

ACKNOWLEDGEMENTS

I thank K. Adachi, D. Bowen, L. Jackson, R. Smith and M. Werner for initiating discussion on this topic and E. Ellis, S. Gliessman, and J. Halliday, for valuable advice and support while the research was being conducted. G. Ysart provided me valuable information about European Commission Regulations. R. Franks, M. Los Huertos, and F. Rein supported laboratory analysis of this research. I extend my deep appreciation to the growers in farmer's field survey and produce managers of sample markets in market sampling survey. I thank E. Frayjo, B. McElroy, A. Mok, and D. O'Briene for assisting the farmers fields' survey. P. Goldman, J. Leap, D. Oretsky, N. Vail and 1998 apprentices of the farms at Center for Agroecology and Sustainable Food Systems, University of California Santa Cruz provided excellent field work for the field experiment. Seeds for the field experiment was supplied by Gowan Seed Company. Weather data were cited from following web-sites; · NASA Langley Research Center (Appendix 7.2): http://eosweb.larc.nasa.gov/DATDOCS/SSE_description.html · UC IPM California Weather Database (Figure 2.2-3, Appendix 6.1-4): http://www.ipm.ucdavis.edu/ · UCSC Applied Science Met Station (Figure 4.7): http://sapphire.cse.ucsc.edu/cgi-bin/reinaslite-site?asmet I thank M. Brown, E. Ellis, P. Fujiyoshi, S. Gliessman, and M. Los Huertos for reviewing earlier drafts of this manuscript. I thankfully acknowledge funding from Organic Farming Research Foundation (OFRF).

36

APPENDIX 1 Toxicity and Regulations of Nitrate in Vegetables A. Toxicity of nitrate

Nitrite exhibits three types of toxic effects: the formation of methemoglobin; carcinogenicity; and hypertrophy of the adrenal zona glomerulosa in rats (JECFA 1995). Methemoglobinemia (also known as "blue baby disease") is a syndrome of elevated methemoglobin level and high blood nitrate, and is frequently associated with acute diarrhea. It can result in coma and ultimately death depending on the extent of the hypoxia, which is the inability of red blood cells to carry oxygen to tissues (Caudill et al., 1990). Particularly, methemoglobinemia can be troublesome in infants under three months of age, although it is inconsequential in adults. Sinios and Wodsak (1965) (NRC 1972) reported 15 cases of methemoglobinemia (1 of them fatal) caused by nitrate and nitrite in spinach in European countries during 1959 to 1965. However, no other cases of methemoglobinemia due to nitrate and nitrite in vegetables has been reported. In contrast, numerous cases of methemoglobinemia caused by drinking water have been reported. According to Heathwaite et al. (1993), the World Health Organization (WHO) reported 2000 cases of infant methemoglobinemia and 160 fatalities caused by drinking water containing more than 25 mgL-1of NO3-N from 1945 to 1985. Nitrate poisoning of groundwater clearly contributes to national infant mortality statistics in the US (Johnson and Kross, 1990; Kross et al., 1995). Many N-nitrosamine compounds have been shown carcinogenic in a range of laboratory animal species. Endogenous N-nitrosamine formation has been demonstrated in human volunteers on a diet rich in fish containing high levels of amines and high-nitrate lettuce and spinach (Van Maanen et al., 1998). Consequently, it is most likely that humans are also susceptible to their carcinogenic action. However, no conclusive epidemiological evidence has been reported on a causal association between nitrate exposure and human cancer risk (Gangolli et al., 1994). One explanation for this contradiction is the fact that most of the vegetables that are major sources of nitrate also contain vitamin C, which is a strong inhibitor of endogenous N-nitrosamine formation (Mirvish, 1972). Therefore, consumption of nitrate from sources low in vitamin C (e. g., water) might be a higher risk than that posed by vitamin-C rich vegetables (Hotchkiss et al., 1992).

B. Two ADIs for nitrate

The European Commission's (EC) Scientific Committee for Food (SCF) in 1995 established Acceptable Daily Intake (ADI) for the nitrate ion of 3.65 mg/kg body weight (equivalent to 219 mg/day for a 60 kg person) taking into account potentially N-nitrosamine formation (European Commission, 1997). On the other hand, the Joint FAO/WHO Expert Committee on Food Additives (JECFA) has also established Acceptable Daily Intakes (ADI) of nitrate and nitrite as 0 - 3.7 NO3 mg/kg body weight and 0 0.06 NO2 mg/kg body weight (JECFA, 1995). Although this ADI is almost the same value with one of SCF, this is based only on toxicity studies and not on possible carcinogenic N-nitrosamine formation. Moreover, JECFA is taking a cautious position about nitrate intake from vegetables because of well-known benefits of vegetables and the lack of data on the possible effects of vegetable matrices on the bioavailability of nitrate. They considered "it inappropriate to compare exposure to nitrate from vegetables directly with the ADI and hence to derive limits for nitrate in vegetables directly from it". Note that JECFA stated ADI does not apply to infants younger than 3 months of age (JECFA 1995). Spinach should not be introduced to such infants (Phillips 1968). Following recommendations and minority point of view of European Environmental Research Organization (EERO) Training and Assessment in 1994 (Gangolli et al., 1994) shows the discrepancy of opinions among scientists with regard to nitrate toxicity in humans, nitrate intake evaluations, and related regulations; Recommendations; 1) There is little justification for recommending a drastic reduction in the present levels of nitrate found in vegetables. 2) The reported mild hypertrophy of the adrenal ozna glomerulosa produced by nitrite treatment in the rat needs to further investigation with a view to elucidating the relevance of this finding in terms of human health risk. 3) Further human studies should be undertaken to delineate the genetic characteristics and environmental factors regulating enzyme systems mediating the endogenous syntheses and metabolic

37

disposition of nitrate, nitrite and N-nitroso compounds. This information would be particularly useful for identifying individuals and population groups likely to be at 'high risk' from an increased body burden and toxicity of nitrate, nitrite, and N-nitroso compounds. 4) Preweaned infants on formulation diets and therefore deriving nitrate mainly from drinking water constitute a special 'at risk' group. Every effort should be made to ensure that nitrate levels in drinking water supplies do not exceed the EC prescribed limit. Minority point of view; Although current epidemiological data provide conflicting evidence regarding the potential long-term health risks of nitrate levels encountered in the diet, it is widely accepted that the reduction of dietary nitrate is a desirable preventive measure. The latter assumption is corroborated by the vast majority of experimental data which show that dietary nitrate is a major determinant for in-vivo formation of N-nitroso compounds. Human intake of nitrate originates primarily from certain vegetables high in nitrate. Unnecessarily high peak body burdens should be prevented by setting maximal allowable nitrate levels in such foods. These should not exceed levels that reflect good agricultural practices. Recently, a positive role of nitrate in the human body's defense against pathogenic bacteria has been investigated (Dykhuizen et al., 1996; Duncan et al., 1997).

C. Risk assessment of nitrate exposure from leafy vegetable consumption

Although JECFA takes a cautious position for such comparison, assuming 60 kg body weight, either ADI means that the ingestion of only 100g of fresh vegetables with a NO3 concentration of 2500 mg/kg FW already exceeds the ADI for NO3 by 13%. Moreover, calculated for NO2 (with 5% nitrate conversion) this would mean an exposure of 12.5 mg NO2 or an exceeding of the ADI for NO2 by 250%. In the Netherlands, many people (particularly children) exceed the ADI for nitrate (Feskens, 1996). Certain Asian populations, vegetarians and those exposed to high concentration of nitrate in their drinking water are also likely to have dietary intakes of nitrate above the ADI. As an example, based on the data of the present study, probability distributions of nitrate content in lettuce and spinach throughout the year were estimated for each farming practice using probability fitting software "BEST FIT" (Palisade 1997). Normal distributions, truncated at 0 (minimum) and 3000 (maximum for lettuce) or 5000 (maximum for spinach), were selected as probability distribution functions (Appendix 1.1). Then correlations between daily consumption amount (FW g /day) and probability of exceeding ADI (219 mg/day for a 60 kg person. SCF 1995) were calculated (Appendix 1.2). Consequently, the amount of spinach that exceeds ADI for nitrate at probability of 50% was estimated as follows: 93 g /day and 123 g/day for conventional and organic spinach, 202 g/day and 205 g/day for conventional and organic Romaine lettuce, and 262 g/day and 298 g/day for conventional and organic Iceberg lettuce, respectively. This showed the average amount (g/day) that could reach ADI for nitrate by eating only lettuce or spinach as salad. This example suggests that "organic only" consumer can eat 30g/day more of spinach or Iceberg lettuce than "conventional only" consumer, avoiding excess nitrate exposure over ADI. Apparently, however, it is unlikely that one would eat 100 grams of spinach or 200 to 300 grams of lettuce everyday. NRC (1981) estimated that nitrate intake from lettuce and spinach accounts 39% of total nitrate intake. For real assessment, therefore, nitrate content in all other sources must be taken into account, as well as their average daily consumption amount.

38

0.50

Probability

IB_CONV 0.25 IB_ORG RM_CONV RM_ORG SP_CONV SP_ORG 0.00 0 2500 NO3 mg/kg fresh weight 5000

Appendix 1.1 Probability distribution of nitrate content in conventional and organic vegetables purchased at Santa Cruz, California, 1998. IB; Iceberg lettuce, RM; Romain lettuce, SP; spinach, CONV; conventional, ORG; organic.

Probability of exceeding ADI for NO 3 (%)

100%

75% IB_CONV 50% IB_ORG RM_CONV 25% RM_ORG SP_CONV SP_ORG 0% 0 100 200 300 400 500 Consumption amount (grams/day)

Appendix 1.2 Probability of exceeding ADI for nitrate by consuming lettuce or spinach grown by different practices. As ADI, 199 mg NO 3 per person (60kg) was assumed. See footnote of Appendix 1.1 for legend.

39

APPENDIX 2 Guide and Maximum Tolerated Nitrate Concentrations of Vegetables (mg NO3 kg-1 in fresh weight)

Vegetable Lettuce Germany (Guide) 3000 Netherlands (Maximum) 3000(S) 4500(W) 3500(S) 4500(W) 2500(1995) 4000(4-6) 3500(7-3) Switzerland (Guide) 3500 Austria (Maximum) 3000(S) 4000(W) 2000(<7) 3000(>7) 3500(S) 4500(W) 3500(S) 4500(W) 2500(S) 1500 Russia (Maximum) 2000(O) 3000(G) 2000(O) 3000(G) 14000(O) EC (Maximum) 3500(4-10) 4500(11-3) 2500(O,5-8) 2500(4-10) 3000(11-3) 2000(P)

Spinach Red beet Radish Endive Cabbage

2000 3000 3000

3500 3000

3000(S) 875

900(S) 500(W) Carrot 1500 400(S) 250(W) S:summer. W:winter. O:outdoor. G:greenhouse . P:processed product (preserved/frozen). <7:harvest by the end of June. >7:harvest from July. 1995: from 1995. 4-10:1 April to 31 October. 11-3:1 November to 31 March. 5-8:1 May to 31 August. Data from Scharpf 1991(cited from Sohn and Yoneyama 1996) and MAFF UK (1999).

40

Appendix 3.1 Summary of Environmental and Practical Factors Affecting Nitrate Content in Spinach and Their Mechanisms

Factor Environmental Light Temperature Water stress Season Nitrate content in spinach Low High High Low Low Low High High Mechanism and/or condition Nitrate reductase in plant requires light energy to be active Stimulation of N mineralization and nitrification in soil and respiration of plant at high temperature Indirect effect on nitrate reductase activity by water stress Low light intensity and short duration of daytime in fall and winter Higher PMN in soil provides more NH4, hence NO3 to plant Faster nitrification in soil provides NO3 to plant sooner Higher NO3 in soil provides more NO3 to plant Less solar radiation at higher latitude in fall and winter Low light intensity and warm temperature in greenhouse High N application causes high NO3 in plants Slow release N fertilizers provide NO3 to plants gradually Limited supply of NO3 to plant by inhibiting nitrification in soil N sidedressing prior to harvest may increase NO3 in plants when it is excessive. Reverse also may happen if basal application is excessive. Stimulation of plant growth by K application Antagonistic effect of Cl- on NO3 - suppresses NO3-absorption by plant Not always but some smooth types have higher nitrate reductase activity than Savoy types Cycloate, alachlor, and lenacil decrease some phase of nitrate reduction NO3 in plant increases with age when NO3 in soil is sufficient to excess NO3 in plant decreases with age when NO3 in soil is optimum or deficient Nitrate is reduced during daytime. It is effective especially when plant contains high NO3 under fine and cool weather Leaf blade / petiole ratio will decrease when plant is seeded densely

Spring to summer - Fall to winter Low PMN* - High PMN Slow nitrification - Fast nitrification Low NO3 - High NO3 Low latitude - High latitude Outdoor - Greenhouse Low High Soluble Low

Soil characteristics

Location Practical N application rate N form Nitrification inhibitor N application timing K application Cl application Variety Herbicides Plant age Time of harvest

Slow release High -

Basal dressing - Side dressing Side dressing - Basal dressing Low High Smooth type Low High Low Savoy type High

Pre mature - Fully mature Fully mature - Pre mature Evening Morning

Planting density Thin - Dense Effect of each factor on nitrate content in spinach when all other factors keep constant. Modified from Breimer (1982) and Lorenz (1978). * PMN = Potentially Mineralizable Nitrogen.

41

APPENDIX 3.2 Factors Affecting Nitrate Content in a Plant

Viets and Hageman (1971) reviewed factors affecting the accumulation of nitrate in plants. A number of environmental factors, including drought (Younis et al., 1965; Huffaker et al., 1970), temperature (Cantliffe, 1972c), light (Schuphan et al., 1967; Cantliffe, 1972a,b; Scaife and Schloemer, 1994), and soil type (Raikova and Petkov 1996) influence nitrate content in plant. Farming practices such as cultivars (Cantliffe, 1973a; Barker et al., 1974; Maynard and Barker, 1974; Olday et al., 1976), nitrogen application (Brown and Smith, 1966, 1967; Barker and Maynard, 1971; Lorenz, 1978; Greenwood and Hunt, 1986), potassium application (Regan et al., 1968), nitrification inhibitor (Mills et el., 1976; Bakr and Gawish, 1997), slow-release fertilizer (Takebe et al., 1996) and herbicides (Cantliffe and Phatak, 1974b) affect nitrate accumulation in plant. Of the factors studied, nitrogen fertilization and light intensity have been identified as the major factors which influence nitrate levels in vegetables (Cantliffe, 1973b). In particular, light intensity and nitrate content in soils before or at harvest are known to be critical factors in determining nitrate levels in spinach (Schuphan et al., 1967). In hydroponic systems, cultivation method for growing low-nitrate lettuce and spinach using preharvest transfer to N-free (Mozafar, 1996) or N-reduced solution (Andersen and Nielsen, 1992) has been put into practice. One night period of supplement lighting prior to harvest to reduce shoot nitrate contents was demonstrated to be effective in hydroponic greenhouse-grown spinach in the winter months in German (Steingrover et al., 1986). Post-harvest storage length and condition (Phillips, 1968; Lee et al., 1971; Heisler et al., 1974; Aworh et al., 1980; Poulsen et al., 1995) and cooking (Phillips, 1968; MAFF UK, 1992: MAFF UK, 1998b) are also known to alter nitrate content in vegetables.

42

APPENDIX 4 List of Sampling Markets in Santa Cruz County* Certified Farmers Market

Market 1. Santa Cruz Address City Parking Lot Opening Wed. 2:30pm-6:30pm

Natural Foods Retailers

Retailer 1. Aptos Natural Foods 2. New Leaf (Capitola) 3. New Leaf (Mission) 4. New Leaf (Downtown) 5. Stapleton's of Santa Cruz 6. Food Bin 7. Staff of Life Address 7506 Soquel Dr. AP 1210 41st. Av. CP 2351 Mission St. SC 1134 Pacific Av. SC 415 River SC 1130 Mission St. SC 1305 Water St. SC TEL 685-3334 479-7987 426-1306 425-1793 425-5888 423-5526 423-8632

General Grocery Markets

Retailer 1. Deluxe Food of Aptos 2. Albertson' Food Centers s 3. Lucky Food Center 4. Safeway 5. Safeway 6. Nob Hill Foods 7. Zanotto's family Markets 8. Safeway 9. Safeway 10. Shoppers Corner 11. Zanotto's family Markets 12. Lucky Food Center 13. Safeway 14. Safeway Address 783 Rio Del Mar Bl. AP 1710 41st Av. CP 1475 41st Av. CP 16 Rancho Del Mar Shopping Ctr. AP 2650 41st Av. SQ 222 Mount Hermon Rd. SV 14 Victor Sq. SV 253 Mount Hermon Rd. SV 6255 Graham Hill Rd. FT 622 Soquel Av. SC 700 Front SC 911 Soquel Av. SC 2111 Mission St. SC 117 Morrissey Bl. SC TEL 688-7442 476-1717 462-6917 688-2775 475-1897 438-1610 438-4324 438-7431 335-3532 423-1398 423-4994 426-6852 429-9811 426-7489

* : Minimum requirement for market to be sampled; 1: Natural food store must have produce regularly. 2: General market must always have spinach and lettuce except running off.

43

APPENDIX 5.1 Data and Information about Lettuce Sub-samples

Vegetable Green-leaf Season Winter Practice Organic Date 21-Jan-98 # Grower 1 Op4 2 Op7 Market Days after Refrigerator Rack received degree C degree C Holtville, CA Or4 1.0 5.0 10.5 n.a. Or6 0.0 0.5 0.5 0.5 Yuma, AZ Cr10 0.0 6.1 7.2 - 10 Yuma, AZ Cr2 0.0 4.4 0.6 Yuma, AZ Cr13 1.0 1.7 - 3.3 1.7 - 3.3 Yuma, AZ Cr18 0.0 9.4 11.1 n.a. Cr11 0.0 3.3 7.2 Yuma, AZ Cr13 0.0 1.7 - 3.3 1.7 - 3.3 0.2 Holtville, CA Or4 5.0 5.0 10.5 El Centro, CA Or2 2.0 3.3 - 5.6 3.3 - 5.6 El Centro, CA Or5 2.0 7.8 3.3 - 4.4 Holtville, CA Or4 3.0 5.0 10.5 3.0 1.3 n.a. Cr11 3.0 3.3 7.2 Salinas, CA Cr3 0.0 4.4 Salinas, CA Cr18 0.0 9.4 11.1 n.a. Cr13 1.0 1.7 - 3.3 1.7 - 3.3 Watsonville, CA Cr10 1.0 6.1 7.2 - 10 Salinas, CA Cr2 1.0 4.4 0.6 1.0 Watsonville, CA Or5 3.0 7.8 3.3 - 4.4 Santa Cruz, CA Or8 4.0 Watsonville, CA Or2 2.0 3.3 - 5.6 3.3 - 5.6 Hollister, CA Or5 4.0 7.8 3.3 - 4.4 Santa Cruz, CA Or8 5.0 Santa Cruz, CA Or1 0.0 3.0 2.0 0.6 3.0 1.7 Thermal, CA Cr10 0.0 6.1 7.2 - 10 Yuma, AZ Cr2 0.0 4.4 0.6 Oxnard, CA Cr13 1.0 1.7 - 3.3 1.7 - 3.3 Yuma, AZ Cr3 1.0 4.4 El Centro (or Oxnard) Cr13 1.0 1.7 - 3.3 1.7 - 3.3 Thermal, CA Cr11 1.0 3.3 7.2 0.7 Oxnard, CA Or4 5.0 5.0 10.5 Gilroy, CA Fm1 Oxnard, CA Or6 0.0 Oxnard, CA Or2 1.0 3.3 - 5.6 3.3 - 5.6 Oxnard, CA Or6 0.0 n.a. Or5 1.0 7.8 3.3 - 4.4 1.4 1.0 Soledad, CA Cr5 0.0 4.4 10.6 Salinas, CA Cr2 2.0 4.4 0.6 Salinas or Pajaro Valley, CACr10 1.0 6.1 7.2 - 10 n.a. Cr11 1.0 3.3 7.2 Salinas, CA Cr3 0.0 4.4 n.a. Cr18 1.0 9.4 11.1 0.8 Greenfields, CA Fm1 Santa Cruz, CA Fm1 Watsonville, CA Fm1 Davenport, CA Fm1 Watsonville, CA Fm1 Greenfields, CA Fm1 0.8 0.8 1.4 0.9 0.7 2.6 1.1 1.6 1.3 Producing area Price $/plant $1.69 $1.80 $1.75 $1.75 $1.75 $0.99 $1.29 $1.39 $0.79 $0.49 $0.79 $0.96 $2.69 $1.49 $1.49 $1.39 $1.77 $1.28 $0.49 $0.89 $0.79 $0.79 $0.69 $0.99 $0.77 $1.29 $0.65 $1.49 $1.29 $1.29 $0.99 $1.17 $0.97 $0.87 $1.41 $1.11 $0.99 $1.39 $0.79 $0.79 $0.69 $0.99 $0.94 $1.69 $1.25 $1.66 $1.49 $1.09 $1.49 $1.45 $1.19 $0.69 $0.59 $0.39 $1.29 $0.69 $0.89 $0.76 $1.00 $0.75 $0.85 $0.55 $0.85 $0.80 $0.80 $0.78 $0.85 $1.12 $0.99 $0.86 $1.24 $1.28 $0.90 $1.11 Wrapping None None

Organic total Winter total Green-leaf total Iceberg Winter Conventional

21-Jan-98

2-Feb-98

1 2 3 4 5 6 1 2 3 4

Cp4 Cp3 Cp6 Cp4 Cp9 Cp6 Op4 Op9 Op8 Op4

None Film Film None Net Film Film Film Film Film

Conventional total Organic 21-Jan-98 2-Feb-98

Organic total Winter total Summer Conventional

24-Aug-98

8-Sep-98

1 2 3 4 5 6 1 5 6 2 3 4

Cp19 Cp11 Cp4 Cp19 Cp20 Cp4 Op13 Op16 Op13 Op15 Op16 Op16

Net Film None Film None Film None Film None Film Film Film

Conventional total Organic 24-Aug-98

8-Sep-98

Organic total Summer total Conventional total Organic total Iceberg total Romain Winter Conventional

21-Jan-98

4-Feb-98

1 2 3 4 5 6 1 2 3 4 5 6

Cp5 Cp4 Cp1 Cp11 Cp10 Cp8 Op5a Op6 Op5b Op5b Op5a Op7

None None None None None None None None None None None None

Conventional total Organic 21-Jan-98

4-Feb-98

Organic total Winter total Summer Conventional

26-Aug-98

9-Sep-98

1 2 3 4 5 6 1 2 3 4 5 6

Cp18 Cp4 Cp12 Cp21 Cp11 Cp23 Op2 Op11 Op12 Op17 Op12 Op2

None None None None None None None None None None None None

Conventional total Organic 26-Aug-98

9-Sep-98

Organic total Summer total Conventional total Organic total Romain total Grand conventional total Grand organic total Grand winter total Grand summer total Grand total

44

APPENDIX 5.1 Data and Information about Lettuce Sub-samples (continued)

Vegetable Season Practice Green-leaf Winter Organic Date # Grower Market Or4 Or6 n 5 5 10 10 10 5 5 5 5 5 5 30 5 5 5 5 20 50 5 5 5 5 5 5 30 5 5 5 5 5 5 30 60 60 50 110 5 5 5 5 5 5 30 5 5 5 5 5 5 30 60 5 5 5 5 5 5 30 5 5 5 5 5 5 30 60 60 60 120 120 120 120 120 240 Av. 792 780 786 786 786 962 1100 1010 870 908 976 970 900 942 1300 764 977 973 774 1090 516 680 564 622 707 484 664 614 484 574 632 575 641 839 736 792 1160 1060 982 1200 888 914 1030 1460 1280 1340 1270 862 826 1170 1100 1040 774 878 1320 1110 1720 1140 800 582 1210 708 1580 846 954 1050 1090 1060 1080 963 904 1020 844 933 NO3 mg/kg FW Min. Max. 670 960 670 880 670 960 670 960 670 960 850 1100 1000 1200 930 1200 570 1000 820 1000 910 1100 570 1200 730 1000 830 1000 1200 1400 620 890 620 1400 570 1400 730 840 940 1200 410 690 630 740 470 670 480 820 410 1200 330 580 610 730 580 680 360 560 500 760 490 750 330 760 330 1200 410 1200 330 1400 330 1400 1100 1300 1000 1100 940 1000 1100 1300 840 950 880 980 840 1300 1400 1600 1200 1400 1200 1500 950 1600 730 1000 720 930 720 1600 720 1600 990 1100 540 1100 780 1000 1200 1400 870 1300 1500 1900 540 1900 460 960 470 770 450 1700 480 1000 1300 1700 720 960 450 1700 450 1900 540 1900 450 1700 450 1900 410 1900 330 1700 570 1600 330 1900 330 1900 C.V.* 14.8 9.72 11.9 11.9 11.9 9.73 6.43 10.9 20.3 8.72 8.09 12.7 12.0 8.27 5.44 13.1 22.5 17.1 5.30 8.58 24.4 6.15 15.7 21.8 29.8 21.3 7.94 6.47 15.8 18.4 17.6 18.4 27.8 25.8 34.6 30.2 7.71 5.17 2.73 5.89 5.71 4.35 12.6 6.13 6.54 8.51 20.0 15.4 11.6 23.6 20.4 5.65 26.7 9.20 8.30 14.3 8.62 29.9 24.5 20.6 40.9 31.5 10.4 12.0 43.5 37.0 24.0 34.5 29.5 28.0 38.3 21.0 43.0 33.3 Av. 93.7 93.0 93.4 93.4 93.4 95.3 95.8 95.6 94.7 95.6 95.1 95.3 96.1 95.3 95.5 95.1 95.5 95.4 95.7 96.2 95.9 96.7 96.1 97.1 96.3 96.2 96.3 96.3 97.2 97.8 96.7 96.8 96.5 95.8 96.3 96.0 93.7 95.1 94.6 94.5 94.7 95.0 94.6 94.8 94.7 93.4 93.3 93.9 93.7 94.0 94.3 94.9 95.3 94.9 95.9 95.2 93.2 94.9 91.4 90.7 94.9 94.8 95.9 95.7 93.9 94.4 94.7 93.9 94.3 95.3 94.9 95.5 94.7 95.1 Moisture % Min. Max. 93.0 95.0 92.6 93.4 92.6 95.0 92.6 95.0 92.6 95.0 94.9 95.6 95.1 96.6 95.4 96.0 93.4 95.4 95.2 95.9 94.4 96.3 93.4 96.6 95.2 96.7 94.9 95.7 94.4 96.3 93.9 95.6 93.9 96.7 93.4 96.7 95.2 96.3 95.8 96.6 95.6 96.5 95.6 97.5 95.4 96.8 96.3 97.4 95.2 97.5 95.7 96.8 95.7 97.3 95.6 96.8 96.5 97.6 97.5 98.1 96.0 97.3 95.6 98.1 95.2 98.1 93.4 97.5 93.9 98.1 93.4 98.1 93.4 93.9 94.6 96.2 94.3 94.9 94.1 94.9 93.9 95.3 94.5 95.9 93.4 96.2 94.5 95.1 93.8 95.3 92.8 94.0 92.3 93.9 93.3 94.2 93.2 94.1 92.3 95.3 92.3 96.2 94.1 95.8 95.0 96.0 93.6 95.5 95.4 96.5 94.6 95.6 92.7 93.7 92.7 96.5 87.8 95.1 86.8 93.7 94.6 95.2 94.4 95.1 95.4 97.3 95.3 96.2 86.8 97.3 86.8 97.3 92.7 96.5 86.8 97.3 86.8 97.3 92.7 97.5 86.8 98.1 86.8 98.1 92.3 96.7 86.8 98.1 C.V.* 0.79 0.33 0.71 0.71 0.71 0.32 0.66 0.25 0.82 0.31 0.76 0.65 0.60 0.39 0.76 0.76 0.72 0.68 0.47 0.40 0.36 0.77 0.54 0.45 0.67 0.46 0.69 0.49 0.40 0.25 0.65 0.76 0.75 0.83 0.97 0.92 0.23 0.69 0.25 0.40 0.58 0.60 0.66 0.26 0.60 0.49 0.68 0.38 0.40 0.77 0.79 0.79 0.43 0.76 0.47 0.43 0.47 1.1 3.7 2.9 0.23 0.30 0.82 0.34 2.8 2.2 0.89 2.1 1.6 1.0 2.0 2.0 1.0 1.6

21-Jan-98 1 Op4 2 Op7

Organic total Winter total Green-leaf total Iceberg Winter Conventional

21-Jan-98 1 2 3 2-Feb-98 4 5 6 Conventional total Organic 21-Jan-98 1 2-Feb-98 2 3 4 Organic total Winter total Summer Conventional 24-Aug-98 1 2 3 8-Sep-98 4 5 6 Conventional total Organic 24-Aug-98 1 5 6 8-Sep-98 2 3 4 Organic total Summer total Conventional total Organic total Iceberg total Romain Winter Conventional 21-Jan-98 1 2 3 4-Feb-98 4 5 6 Conventional total Organic 21-Jan-98 1 2 3 4-Feb-98 4 5 6 Organic total Winter total Summer Conventional 26-Aug-98 1 2 3 9-Sep-98 4 5 6 Conventional total Organic 26-Aug-98 1 2 3 9-Sep-98 4 5 6 Organic total Summer total Conventional total Organic total Romain total Grand conventional total Grand organic total Grand winter total Grand summer total Grand total *: C.V.= standard diviation / average x 100

Cp4 Cp3 Cp6 Cp4 Cp9 Cp6 Op4 Op9 Op8 Op4

Cr10 Cr2 Cr13 Cr18 Cr11 Cr13 Or4 Or2 Or5 Or4

Cp19 Cp11 Cp4 Cp19 Cp20 Cp4 Op13 Op16 Op13 Op15 Op16 Op16

Cr11 Cr3 Cr18 Cr13 Cr10 Cr2 Or5 Or8 Or2 Or5 Or8 Or1

Cp5 Cp4 Cp1 Cp11 Cp10 Cp8 Op5a Op6 Op5b Op5b Op5a Op7

Cr10 Cr2 Cr13 Cr3 Cr13 Cr11 Or4 Fm1 Or6 Or2 Or6 Or5

Cp18 Cp4 Cp12 Cp21 Cp11 Cp23 Op2 Op11 Op12 Op17 Op12 Op2

Cr5 Cr2 Cr10 Cr11 Cr3 Cr18 Fm1 Fm1 Fm1 Fm1 Fm1 Fm1

45

APPENDIX 5.1 Data and Information about Lettuce Sub-samples (continued)

Vegetable Season Green-leaf Winter Practice Organic Date 21-Jan-98 # Grower 1 Op4 2 Op7 Market Or4 Or6 n 5 5 10 10 10 5 5 5 5 5 5 30 5 5 5 5 20 50 5 5 5 5 5 5 30 5 5 5 5 5 5 30 60 60 50 110 5 5 5 5 5 5 30 5 5 5 5 5 5 30 60 5 5 5 5 5 5 30 5 5 5 5 5 5 30 60 60 60 120 120 120 120 120 240 Fresh weight g/plant Av. Min. Max. C.V.* 301 258 363 15.3 196 156 226 14.4 249 156 363 26.4 249 156 363 26.4 249 156 363 26.4 484 406 575 15.8 635 571 759 11.7 589 537 661 9.25 528 352 669 26.2 590 475 742 19.1 612 483 853 25.5 573 352 853 19.5 627 571 709 9.74 491 404 587 14.9 640 505 741 13.6 605 439 727 18.3 591 404 741 16.7 580 352 853 18.2 705 585 821 14.9 778 674 831 7.78 570 384 744 25.3 826 630 918 14.2 491 361 758 33.3 842 698 1110 19.2 702 361 1110 25.4 570 473 668 16.0 635 552 724 10.3 397 253 617 34.5 804 565 926 19.2 681 582 842 15.4 665 556 779 15.9 625 253 926 26.1 664 253 1110 26.2 638 352 1110 25.3 611 253 926 23.0 626 253 1114 24.3 427 365 494 14.1 482 327 695 34.0 340 244 462 25.6 514 340 606 20.3 409 262 518 26.3 578 468 688 17.6 458 244 695 27.5 415 344 544 18.3 576 402 654 18.0 385 276 456 18.2 270 217 323 15.4 321 285 359 10.2 282 220 419 28.1 375 217 654 33.1 417 217 695 31.4 624 527 710 12.2 452 394 523 10.9 451 373 508 13.1 542 474 592 8.32 555 321 660 24.3 344 300 383 11.5 495 300 710 23.1 603 558 649 5.61 431 378 485 10.9 349 295 409 12.8 304 287 339 6.72 444 337 483 13.7 732 554 921 18.9 477 287 921 34.1 486 287 921 28.7 476 244 710 25.3 426 217 921 35.8 451 217 921 30.8 557 244 1110 29.3 489 156 926 37.3 471 156 853 33.0 575 253 1110 31.4 523 156 1110 33.6 Av. 4.7 3.5 4.1 4.1 4.1 1.9 2.1 2.0 1.9 2.0 2.9 2.1 2.5 2.0 1.9 2.1 2.1 2.1 4.0 4.0 5.8 4.9 4.0 4.4 4.5 4.4 4.5 4.3 4.4 5.4 5.3 4.7 4.6 3.3 3.7 3.5 3.8 3.7 3.9 5.2 5.3 6.1 4.7 5.0 7.0 4.5 4.2 4.2 5.3 5.0 4.9 9.4 9.5 9.2 9.4 11 8.9 9.5 11 12 11 15 9.2 11 12 11 7.1 8.3 7.7 5.2 6.0 3.7 7.6 5.6 Non edible part % FW Min. Max. C.V.* 3.9 5.5 14 2.8 4.0 16 2.8 5.5 21 2.8 5.5 21 2.8 5.5 21 1.7 2.3 11 1.7 2.5 16 1.5 2.4 19 1.4 2.6 24 1.4 2.6 22 2.5 3.2 9.0 1.4 3.2 23 1.8 3.2 24 1.2 2.5 28 1.6 2.2 13 1.9 2.6 13 1.2 3.2 23 1.2 3.2 23 2.3 5.3 33 3.7 4.6 8.3 4.8 7.4 17 3.4 6.1 20 3.2 5.4 23 3.5 6.1 23 2.3 7.4 25 2.8 6.7 33 3.1 4.9 17 2.4 5.9 32 3.3 5.2 17 4.3 6.6 18 4.6 5.7 8.0 2.4 6.7 22 2.3 7.4 23 1.4 7.4 45 1.2 6.7 41 1.2 7.4 43 3.3 4.4 15 2.8 4.2 16 3.0 5.3 22 2.9 6.9 30 3.6 7.4 27 5.7 6.8 7.6 2.8 7.4 28 3.1 6.1 24 5.9 7.7 13 3.8 5.7 16 3.9 4.8 8.0 3.3 4.7 14 2.9 6.9 28 2.9 7.7 26 2.8 7.7 27 9.0 10 3.8 6.9 12 22 8.0 10 8.6 8.7 10 7.6 8.2 13 16 8.0 9.8 8.6 6.9 13 13 11 12 4.3 11 13 5.7 10 13 9.4 14 17 7.6 7.9 9.8 8.3 10 12 4.3 7.9 17 17 6.9 17 18 2.8 13 39 2.9 1.7 44 2.8 17 43 1.4 13 56 1.2 17 60 1.2 7.7 45 2.3 17 44 1.2 17 59

Organic total Winter total Green-leaf total Iceberg Winter Conventional

21-Jan-98

2-Feb-98

1 2 3 4 5 6 1 2 3 4

Cp4 Cp3 Cp6 Cp4 Cp9 Cp6 Op4 Op9 Op8 Op4

Cr10 Cr2 Cr13 Cr18 Cr11 Cr13 Or4 Or2 Or5 Or4

Conventional total Organic 21-Jan-98 2-Feb-98

Organic total Winter total Summer Conventional

24-Aug-98

8-Sep-98

1 2 3 4 5 6 1 5 6 2 3 4

Cp19 Cp11 Cp4 Cp19 Cp20 Cp4 Op13 Op16 Op13 Op15 Op16 Op16

Cr11 Cr3 Cr18 Cr13 Cr10 Cr2 Or5 Or8 Or2 Or5 Or8 Or1

Conventional total Organic 24-Aug-98

8-Sep-98

Organic total Summer total Conventional total Organic total Iceberg total Romain Winter Conventional

21-Jan-98

4-Feb-98

1 2 3 4 5 6 1 2 3 4 5 6

Cp5 Cp4 Cp1 Cp11 Cp10 Cp8 Op5a Op6 Op5b Op5b Op5a Op7

Cr10 Cr2 Cr13 Cr3 Cr13 Cr11 Or4 Fm1 Or6 Or2 Or6 Or5

Conventional total Organic 21-Jan-98

4-Feb-98

Organic total Winter total Summer Conventional

26-Aug-98

9-Sep-98

1 2 3 4 5 6 1 2 3 4 5 6

Cp18 Cp4 Cp12 Cp21 Cp11 Cp23 Op2 Op11 Op12 Op17 Op12 Op2

Cr5 Cr2 Cr10 Cr11 Cr3 Cr18 Fm1 Fm1 Fm1 Fm1 Fm1 Fm1

Conventional total Organic 26-Aug-98

9-Sep-98

Organic total Summer total Conventional total Organic total Romain total Grand conventional total Grand organic total Grand winter total Grand summer total Grand total *: C.V.= standard diviation / average x 100

46

APPENDIX 5.2 Data and Information about Spinach Sub-samples

Season Winter Practice Conventional Date 27-Jan-98 # Grower 1 2 3 4 5 6 1 2 3 4 5 6 Cp4 Cp7 Cp8 Cp1 Cp13 Cp14 Op5 Op7 Op3 Op4 Op5a Op3 Producing area Yuma, AZ Coachelle, CA Thermal, CA Oxnard, CA Mexicali, Mexico Oxnard, CA Oxnard, CA n.a. Coachelle, CA Holtville, CA Oxnard, CA Coachelle, CA Market Cr10 Cr2 Cr11 Cr5 Cr13 Cr13 Or5 Or7 Or4 Or2 Or5 Or7 Days after received 1 1 1 1 0 0 0.7 0 3 4 0 2 2 1.8 1.3 3 4 2 2 0 1 2.0 Refrigerator degree C 7.2 - 10 4.4 3.3 4.4 1.7 - 3.3 1.7 - 3.3 7.8 5.6 5.0 3.3 - 5.6 7.8 5.6 Rack degree C 6.1 0.6 7.2 10.6 1.7 - 3.3 1.7 - 3.3 3.3 - 4.4 5.6 10.5 3.3 - 5.6 3.3 - 4.4 5.6 Price $/bunch $0.79 $1.29 $0.99 $0.59 $0.59 $0.59 $0.81 $1.49 $1.99 $1.99 $1.59 $1.19 $1.09 $1.56 $1.18 $0.99 $0.95 $0.89 $0.99 $0.79 $0.99 $0.93 $0.75 $1.00 $1.29 $0.50 $1.00 $1.19 $0.96 $0.94 $0.87 $1.26 $1.06

9-Feb-98 14-Feb-98 Conventional total Organic 27-Jan-98

9-Feb-98

Organic total Winter total Summer Conventional 19-Aug-98 1 2 3 4 5 6 1 2 3 4 5 6 Cp15 Cp7 Cp16 Cp7 Cp1 Cp16 Op11 Op6 Op10 Op11 Op6 Op10 Salinas, CA Salinas, CA n.a. Salinas, CA Oxnard, CA n.a. Santa Cruz, CA Gilroy, CA Watsonville, CA Santa Cruz, CA Gilroy, CA Watsonville, CA Cr10 Cr2 Cr3 Cr11 Cr5 Cr2 Fm1 Fm1 Or7 Fm1 Fm1 Or7

2-Sep-98

7.2 - 10 4.4 4.4 3.3 4.4 4.4

6.1 0.6 7.2 10.6 0.6

Conventional total Organic 19-Aug-98

3

5.6

5.6

2-Sep-98

Organic total Summer total Conventional total Organic total Grand total

3 3.0 2.5 1.3 2.1 1.7

5.6

5.6

47

APPENDIX 5.2 Data and Information about Spinach Sub-samples (continued)

Season Practice Winter Conventional Date 27-Jan-98 # 1 2 3 4 5 6 1 2 3 4 5 6 Grower Cp4 Cp7 Cp8 Cp1 Cp13 Cp14 Op5 Op7 Op3 Op4 Op5a Op3 Market Cr10 Cr2 Cr11 Cr5 Cr13 Cr13 Or5 Or7 Or4 Or2 Or5 Or7 n 5 5 5 5 5 5 30 5 5 5 5 5 5 30 60 5 5 5 5 5 5 30 5 5 5 5 5 5 30 60 60 60 120 NO3 mg/kg whole plant FW Av. Min. Max. C.V.* 2840 2400 3400 14.2 2220 2100 2300 3.77 1460 1200 1700 12.4 2200 2100 2400 5.57 2880 2400 3200 10.2 1780 1500 2600 26.8 2230 1200 3400 26.4 1740 900 2400 31.6 1540 1300 1700 11.8 890 310 1200 41.2 2640 2200 2900 10.9 1680 680 2500 50.0 2300 2000 2800 13.7 1800 310 2900 39.7 2010 310 3400 34.0 3180 2400 4100 20.8 2700 2400 3000 11.1 2960 2400 3700 16.0 3400 3000 3700 7.50 2040 990 3200 49.4 2840 2000 4000 25.7 2850 990 4100 25.2 592 200 1300 81.8 1360 820 2000 31.7 2460 1400 3500 30.4 620 130 900 59.8 2860 2700 3100 6.35 3000 2800 3300 7.07 1820 130 3500 60.5 2330 130 4100 45.3 2540 990 4100 28.5 1810 130 3500 50.8 2170 130 4100 41.5 Moisture % whole plant Av. Min. Max. C.V.* 92.2 91.8 92.5 0.30 92.5 92.1 92.7 0.24 93.0 92.7 93.3 0.25 93.1 92.7 93.6 0.36 91.2 90.7 91.7 0.49 92.2 91.5 92.7 0.51 92.4 90.7 93.6 0.77 91.1 90.3 91.6 0.57 91.4 91.3 91.6 0.15 90.1 88.9 91.2 1.01 92.2 90.1 94.2 1.65 90.9 90.2 92.4 1.07 92.2 91.6 93.1 0.73 91.3 88.9 94.2 1.21 91.8 88.9 94.2 1.16 94.1 93.9 94.4 0.20 94.7 94.6 95.0 0.19 92.8 92.5 93.3 0.35 93.7 93.0 94.4 0.54 93.6 93.4 94.0 0.31 93.6 93.1 94.6 0.64 93.8 92.5 95.0 0.73 91.2 89.9 92.2 1.02 93.1 92.8 93.6 0.36 94.4 94.0 94.8 0.30 91.8 91.5 92.2 0.31 94.4 93.9 94.6 0.32 94.3 94.2 94.3 0.05 93.2 89.9 94.8 1.49 93.5 89.9 95.0 1.20 93.1 90.7 95.0 1.05 92.3 88.9 94.8 1.69 92.7 88.9 95.0 1.47

9-Feb-98 14-Feb-98 Conventional total Organic 27-Jan-98

9-Feb-98

Organic total Winter total Summer Conventional

19-Aug-98

2-Sep-98

1 2 3 4 5 6 1 2 3 4 5 6

Cp15 Cp7 Cp16 Cp7 Cp1 Cp16 Op11 Op6 Op10 Op11 Op6 Op10

Cr10 Cr2 Cr3 Cr11 Cr5 Cr2 Fm1 Fm1 Or7 Fm1 Fm1 Or7

Conventional total Organic 19-Aug-98

2-Sep-98

Organic total Summer total Conventional total Organic total Grand total

Season Practice Winter Conventional

Date 27-Jan-98

# 1 2 3 4 5 6 1 2 3 4 5 6

Grower Cp4 Cp7 Cp8 Cp1 Cp13 Cp14 Op5 Op7 Op3 Op4 Op5a Op3

Market Cr10 Cr2 Cr11 Cr5 Cr13 Cr13 Or5 Or7 Or4 Or2 Or5 Or7

n 5 5 5 5 5 5 30 5 5 5 5 5 5 30 60 5 5 5 5 5 5 30 5 5 5 5 5 5 30 60 60 60 120

9-Feb-98 14-Feb-98 Conventional total Organic 27-Jan-98

9-Feb-98

Organic total Winter total Summer Conventional

19-Aug-98

2-Sep-98

1 2 3 4 5 6 1 2 3 4 5 6

Cp15 Cp7 Cp16 Cp7 Cp1 Cp16 Op11 Op6 Op10 Op11 Op6 Op10

Cr10 Cr2 Cr3 Cr11 Cr5 Cr2 Fm1 Fm1 Or7 Fm1 Fm1 Or7

Conventional total Organic 19-Aug-98

2-Sep-98

Organic total Summer total Conventional total Organic total Grand total *: C.V. = Standard diviation / Average x 100

Fresh weight g/plant Av. Min. Max. 12.8 10.8 15.2 12.2 11.3 13.0 11.1 7.0 14.6 22.9 15.4 32.6 29.5 20.4 48.1 32.1 13.8 42.8 20.1 7.0 48.1 9.18 6.7 11.7 25.2 12.8 35.4 9.86 7.8 11.8 24.6 14.3 33.3 14.8 11.8 17.4 14.9 12.3 17.8 16.4 6.7 35.4 18.3 6.7 48.1 13.2 10.0 16.0 12.3 10.1 14.3 20.3 13.1 32.7 11.5 10.0 12.8 32.2 22.3 39.5 9.18 7.0 13.0 16.4 7.0 39.5 17.4 10.9 27.9 15.0 12.2 19.2 6.83 4.9 7.8 18.5 14.2 23.9 10.3 8.6 12.1 10.4 8.3 12.2 13.1 4.9 27.9 14.8 4.9 39.5 18.3 7.0 48.1 14.8 4.9 35.4 16.5 4.9 48.1

C.V.* 13.5 6.14 26.1 27.7 36.9 40.7 55.0 21.0 35.9 15.4 28.6 14.4 13.6 48.1 53.2 18.2 12.7 36.4 9.28 19.8 27.2 54.0 36.3 17.5 16.2 19.4 15.0 13.5 39.9 50.3 55.4 46.4 53.2

Leafblade/petiole ratio FW/FW Av. Min. Max. C.V.* 1.2 1.2 1.2 2.8 1.7 1.7 1.8 1.5 1.4 1.2 1.6 11 1.5 1.4 1.7 7.1 1.3 1.2 1.5 8.1 1.9 1.4 2.3 19 1.5 1.2 2.3 20 2.4 2.1 2.5 8.8 1.9 1.7 2.2 11 1.7 1.6 1.9 8.8 1.4 1.3 1.5 6.3 1.9 1.6 2.3 14 1.4 1.3 1.8 14 1.8 1.3 2.5 21 1.6 1.2 2.5 23 1.7 1.4 2.1 17 1.6 1.4 1.7 7.4 2.8 2.2 3.3 14 1.6 1.4 1.9 11 1.8 1.7 1.9 4.3 1.4 1.1 1.6 17 1.8 1.1 3.3 29 3.0 2.7 3.2 6.4 1.6 1.4 1.8 12 1.7 1.5 2.0 9.9 2.7 2.4 3.2 12 1.8 1.6 1.9 7.3 1.6 1.3 1.9 14 2.1 1.3 3.2 29 1.9 1.1 3.3 30 1.7 1.1 3.3 27 1.9 1.3 3.2 27 1.8 1.1 3.3 28

48

APPENDIX 5.2 Data and Information about Spinach Sub-samples (continued)

Season Practice Winter Conventional Date 27-Jan-98 # 1 2 3 4 5 6 1 2 3 4 5 6 Grower Cp4 Cp7 Cp8 Cp1 Cp13 Cp14 Op5 Op7 Op3 Op4 Op5a Op3 Market Cr10 Cr2 Cr11 Cr5 Cr13 Cr13 Or5 Or7 Or4 Or2 Or5 Or7 n 5 5 5 5 5 5 30 5 5 5 5 5 5 30 60 5 5 5 5 5 5 30 5 5 5 5 5 5 30 60 60 60 120 NO3 mg/kg leaf blade FW Av. Min. Max. C.V.* 1900 1600 2300 15.3 1460 1300 1600 7.8 994 830 1100 11.5 1200 1100 1400 11.8 1900 1600 2200 13.4 964 770 1200 18.5 1400 770 2300 30.8 1210 740 1600 25.6 1070 830 1200 14.6 754 260 1000 39.7 1700 1400 1900 11.8 1070 460 1600 45.3 1360 1200 1600 11.2 1190 260 1900 33.3 1300 260 2300 32.7 2140 1700 3000 23.5 1500 1300 1700 13.3 2400 1900 2900 18.2 2120 1700 2300 11.7 1250 560 2100 53.1 1580 1200 2200 23.8 1830 560 3000 31.4 462 140 1100 91.9 744 420 1200 39.0 1950 930 2800 34.2 419 93 640 57.8 2100 1900 2400 10.1 1940 1600 2200 11.9 1270 93 2800 65.0 1550 93 3000 49.0 1620 560 3000 33.9 1230 93 2800 52.2 1420 93 3000 43.9 Av. 91.1 91.6 92.1 92.5 90.1 91.5 91.5 90.7 90.7 89.7 90.7 90.2 90.9 90.5 91.0 93.2 93.9 92.1 92.7 92.9 92.5 92.9 90.8 92.3 93.6 91.3 93.5 93.2 92.5 92.7 92.2 91.5 91.8 Moisture % leaf blade Min. Max. C.V.* 90.6 91.4 0.383 91.2 91.9 0.303 91.8 92.5 0.362 92.1 92.9 0.393 89.5 90.7 0.591 90.7 92.1 0.617 89.5 92.9 0.947 90.0 91.3 0.512 90.3 91.0 0.292 88.7 90.5 0.867 87.2 93.8 2.73 88.8 92.8 1.77 89.4 92.1 1.27 87.2 93.8 1.43 87.2 93.8 1.32 92.8 93.5 0.382 93.7 94.3 0.260 91.8 92.5 0.282 91.7 93.6 0.738 92.6 93.2 0.261 91.5 93.8 0.936 91.5 94.3 0.787 89.4 91.8 0.973 91.9 92.9 0.427 93.1 93.8 0.335 90.9 91.6 0.296 92.8 93.8 0.392 92.9 93.4 0.251 89.4 93.8 1.28 89.4 94.3 1.08 89.5 94.3 1.16 87.2 93.8 1.73 87.2 94.3 1.51

9-Feb-98 14-Feb-98 Conventional total Organic 27-Jan-98

9-Feb-98

Organic total Winter total Summer Conventional

19-Aug-98

2-Sep-98

1 2 3 4 5 6 1 2 3 4 5 6

Cp15 Cp7 Cp16 Cp7 Cp1 Cp16 Op11 Op6 Op10 Op11 Op6 Op10

Cr10 Cr2 Cr3 Cr11 Cr5 Cr2 Fm1 Fm1 Or7 Fm1 Fm1 Or7

Conventional total Organic 19-Aug-98

2-Sep-98

Organic total Summer total Conventional total Organic total Grand total

Season Practice Winter Conventional

Date 27-Jan-98

# 1 2 3 4 5 6 1 2 3 4 5 6

Grower Cp4 Cp7 Cp8 Cp1 Cp13 Cp14 Op5 Op7 Op3 Op4 Op5a Op3

Market Cr10 Cr2 Cr11 Cr5 Cr13 Cr13 Or5 Or7 Or4 Or2 Or5 Or7

n 5 5 5 5 5 5 30 5 5 5 5 5 5 30 60 5 5 5 5 5 5 30 5 5 5 5 5 5 30 60 60 60 120

9-Feb-98 14-Feb-98 Conventional total Organic 27-Jan-98

9-Feb-98

Organic total Winter total Summer Conventional

19-Aug-98

2-Sep-98

1 2 3 4 5 6 1 2 3 4 5 6

Cp15 Cp7 Cp16 Cp7 Cp1 Cp16 Op11 Op6 Op10 Op11 Op6 Op10

Cr10 Cr2 Cr3 Cr11 Cr5 Cr2 Fm1 Fm1 Or7 Fm1 Fm1 Or7

Conventional total Organic 19-Aug-98

2-Sep-98

Organic total Summer total Conventional total Organic total Grand total *: C.V. = standard diviation / average x 100

NO3 mg/kg petiole FW Av. Min. Max. C.V.* 4020 3400 4700 13.1 3460 3300 3600 3.30 2080 1800 2300 9.25 3760 3600 4000 4.83 4160 3500 4500 9.56 3200 2500 4400 22.9 3450 1800 4700 23.2 2880 1300 3800 33.1 2360 2100 2700 11.0 1130 400 1700 43.2 3860 3300 4200 8.90 2840 1100 4200 49.3 3640 3100 4300 12.8 2790 400 4300 41.1 3120 400 4700 33.2 4920 3800 5700 17.0 4580 4000 5200 10.5 4580 3200 6500 26.6 5500 5000 6200 8.72 3380 1700 5100 47.1 4560 3400 6500 25.7 4590 1700 6500 25.0 988 230 2000 72.1 2260 1500 3300 29.5 3340 2200 4600 26.6 1170 220 1700 62.4 4220 4000 4500 5.14 4700 4400 5000 6.02 2780 220 5000 56.0 3680 220 6500 44.4 4020 1700 6500 28.3 2780 220 5000 48.7 3400 220 6500 40.9

Av. 93.5 94.0 94.3 94.1 92.7 93.5 93.7 92.0 92.8 90.8 94.2 91.9 94.2 92.7 93.2 95.5 96.1 94.7 95.2 94.9 95.0 95.3 92.4 94.4 95.8 92.9 96.0 95.9 94.6 94.9 94.5 93.6 94.0

Moisture % petiole Min. Max. 93.3 93.9 93.8 94.2 94.0 94.5 93.7 94.5 92.1 93.3 93.1 94.0 92.1 94.5 91.1 92.9 92.5 93.0 89.2 92.6 94.0 94.7 91.0 92.5 94.1 94.4 89.2 94.7 89.2 94.7 95.2 96.0 95.9 96.2 94.5 95.3 94.9 95.6 94.5 95.5 94.6 95.8 94.5 96.2 91.2 93.6 94.0 94.8 95.6 96.2 92.4 93.8 95.6 96.1 95.8 96.0 91.2 96.2 91.2 96.2 92.1 96.2 89.2 96.2 89.2 96.2

C.V.* 0.233 0.187 0.188 0.314 0.507 0.455 0.653 0.944 0.212 1.35 0.316 0.658 0.147 1.51 1.27 0.342 0.175 0.371 0.265 0.465 0.490 0.571 1.27 0.322 0.250 0.542 0.192 0.109 1.65 1.27 1.04 1.87 1.57

49

APPENDIX 5.2 Data and Information about Spinach Sub-samples (continued)

Season Winter Practice Conventional Date 27-Jan-98 # 1 2 3 4 5 6 1 2 3 4 5 6 Grower Cp4 Cp7 Cp8 Cp1 Cp13 Cp14 Op5 Op7 Op3 Op4 Op5a Op3 Market Cr10 Cr2 Cr11 Cr5 Cr13 Cr13 Or5 Or7 Or4 Or2 Or5 Or7 n. 5 5 5 5 5 5 30 5 5 5 5 5 5 30 60 5 5 5 5 5 5 30 5 5 5 5 5 5 30 60 60 60 120 Non edible part % FW Av. Min. Max. 4.0 2.8 5.1 1.2 0.9 1.5 5.6 3.4 7.5 6.2 3.6 8.5 1.2 0.6 1.8 2.5 1.1 4.7 3.4 0.6 8.5 0.83 0.7 1.1 3.0 0.4 5.8 2.6 1.1 3.7 1.5 0.3 2.9 2.0 0.9 3.7 1.0 0.7 1.4 1.8 0.3 5.8 2.6 0.3 8.5 3.9 1.1 6.5 3.1 2.9 3.4 1.2 0.4 2.7 6.5 4.6 9.5 1.7 0.6 2.6 9.3 1.7 18 4.3 0.4 18 0.90 0.7 1.2 0.00 0.0 0.0 1.4 0.8 2.4 3.3 1.4 6.1 0.0 0.0 0.0 7.3 2.3 16 2.2 0.0 16 3.2 0.0 18 3.9 0.4 18 2.0 0.0 16 2.9 0.0 18 C.V.* 29 25 28 32 37 54 67 19 70 42 64 55 28 73 77 54 7.3 76 28 51 73 93 24 0.00 41 59 0.00 71 150 120 84 130 100

9-Feb-98 14-Feb-98 Conventional total Organic 27-Jan-98

9-Feb-98

Organic total Winter total Summer Conventional 19-Aug-98 1 2 3 4 5 6 1 2 3 4 5 6 Cp15 Cp7 Cp16 Cp7 Cp1 Cp16 Op11 Op6 Op10 Op11 Op6 Op10 Cr10 Cr2 Cr3 Cr11 Cr5 Cr2 Fm1 Fm1 Or7 Fm1 Fm1 Or7

2-Sep-98

Conventional total Organic 19-Aug-98

2-Sep-98

Organic total Summer total Conventional total Organic total Grand total

50

Precipitation mm Precipitation mm 120 40 80 0 120 40 80 0 120 11/1/97 11/11/97 11/21/97 12/1/97 12/11/97 12/21/97 12/31/97 1/10/98 1/20/98 1/30/98 2/9/98 Temperature C Temperature C 0 0 10 0 10 20 30 40 20 30 40 12/21/97 12/31/97 1/10/98 1/20/98 1/30/98 2/9/98 Temperature C 0 10 20 30 40 12/11/97 12/1/97 11/21/97 11/11/97 11/1/97 40 80 0 11/1/97 11/11/97 11/21/97 12/1/97 12/11/97 12/21/97 12/31/97 1/10/98 1/20/98 1/30/98 2/9/98

Precipitation mm

Yuma, AZ

El Centro

Holtville (Brawley)

Precipitation mm 120 40 80 40 0 11/1/97 11/1/97 11/11/97 11/21/97 12/1/97 12/11/97 12/21/97 12/31/97 1/10/98 1/20/98 1/30/98 1/30/98 2/9/98 2/9/98 0 Temperature C 10 20 30 40 0 10 11/11/97 11/21/97 12/1/97 12/11/97 12/21/97 12/31/97 1/10/98 1/20/98

Precipitation mm 120 80

Temperature C

51 (UC IPM California Weather Database 1999).

Thermal

Oxnard (Hueneme)

Precipitation (bar) and maximum (bold line), minimum (thin line) air temperature at areas where market samples grew during their estimated growth period.

20

30

40

APPENDIX 6.1 Climate at Market Sample's Origins (Winter1)

Precipitation mm 120 40 80 0 120 120 40 0 6/1/98 6/11/98 6/21/98 7/1/98 7/11/98 7/21/98 7/31/98 8/10/98 8/20/98 8/20/98 8/30/98 9/9/98 0 Temperature C 0 10 20 30 40 9/9/98 Temperature C 10 20 30 40 8/30/98 9/9/98 Temperature C 0 10 20 30 40 8/20/98 8/30/98 80 40 80 0 6/1/98 6/11/98 6/21/98 7/1/98 7/11/98 7/21/98 7/31/98 8/10/98 8/10/98 7/31/98 7/21/98 7/1/98 6/21/98 6/11/98 6/1/98

Precipitation mm

Precipitation mm

Freedom

Oxnard (Hueneme)

North Salinas

7/11/98 Precipitation mm 0 120 40 80 0 6/1/98 6/11/98 6/21/98 7/1/98 7/11/98 7/21/98 7/31/98 8/10/98 8/20/98 8/30/98 9/9/98 0 Temperature C 10 20 30 40 6/1/98 6/11/98 6/21/98 7/1/98 7/11/98 7/21/98 7/31/98 8/10/98 8/20/98 8/30/98 9/9/98 0

Precipitation mm 120 40 80

Temperature C

52 (UC IPM California Weather Database 1999).

Santa Cruz

Watsonville

Precipitation (bar) and maximum (bold line), minimum (thin line) air temperature at areas where market samples grew during their estimated growth period.

APPENDIX 6.2 Climate at Market Sample's Origins (Summer1)

10

20

30

40

Soledad

120 Precipitation mm 80 40 0 6/1/98 7/1/98 6/11/98 6/21/98 7/11/98 7/21/98 7/31/98 8/10/98 8/20/98 8/30/98 9/9/98

South Salinas

40 Precipitation mm Temperature C 30 20 10 0

120 80 40 0 6/1/98 7/1/98 6/11/98 6/21/98 7/11/98 7/21/98 7/31/98 8/10/98 8/20/98 8/30/98 9/9/98 40 Temperature C 30 20 40 0 6/1/98 7/1/98 6/11/98 6/21/98 7/11/98 7/21/98 7/31/98 8/10/98 8/20/98 8/30/98 9/9/98 10 0

40 Temperature C 30 20 10 0

Hollister

Precipitation mm 30 20 40 0 6/1/98 7/1/98 6/11/98 6/21/98 7/11/98 7/21/98 7/31/98 8/10/98 8/20/98 8/30/98 9/9/98 10 0 Precipitation mm 120 80 40 Temperature C 120 80

Gilroy

APPENDIX 6.2 Climate at Market Sample's Origins (Summer1. Continued).

Precipitation (bar) and maximum (bold line), minimum (thin line) temperature at areas where market samples grew during their estimated growth period.

(UC IPM California Weather Database 1999).

53

El Centro

400 Solar Radiation W/m2 300 200 100 0 11/1/97 12/1/97 1/10/98 1/20/98 11/11/97 11/21/97 12/11/97 12/21/97 12/31/97 1/30/98 2/9/98 40 Solar Radiation W/m2 Temperature C 30 20 10 0 400 300 200 100 0 11/1/97

Holtville (Brawley)

40 Temperature C 30 20 10 0 12/1/97 1/10/98 1/20/98 11/11/97 11/21/97 12/11/97 12/21/97 12/31/97 1/30/98 2/9/98 40 Temperature C 30 20 10 0 11/1/97 12/1/97 1/10/98 1/20/98 11/11/97 11/21/97 12/11/97 12/21/97 12/31/97 1/30/98 2/9/98

400 Solar Radiation W/m2 300 200 100 0 11/1/97 12/1/97

Thermal

Oxnard (Hueneme)

40 Temperature C 30 20 10 0 1/10/98 1/20/98 400 Solar Radiation W/m2 300 200 100 0

11/11/97

11/21/97

12/11/97

12/21/97

Appendix 6.3 Climate at Market Sample's Origin (Winter2)

Solar radiation (bar), and maximum (bold line), minimum (thin line) soil temperature (15 cm depth) at areas where winter market samples grew during their estimated growth period.

(UC IPM California Weather Database 1999). 54

12/31/97

1/30/98

2/9/98

Solar Radiation W/m 100 200 300 400 100 200 300 400 0 0 100 6/1/98 6/1/98 200 300 400 0 6/1/98 6/11/98 6/11/98 6/11/98 6/21/98 6/21/98 6/21/98 7/1/98 7/1/98 7/1/98 7/11/98 7/11/98 7/11/98 7/21/98 7/21/98 7/31/98 7/21/98

2

Solar Radiation W/m Solar Radiation W/m

2 2

North Salinas

Oxnard (Hueneme)

South Salinas

7/31/98 8/10/98

7/31/98

8/10/98 8/20/98

8/10/98

8/20/98 8/30/98

8/20/98

8/30/98

8/30/98 9/9/98 0 10 20 30 40 Temperature C Solar Radiation W/m 100 200 300 0

2

9/9/98 TemperatureC 0 10 20 30 40

9/9/98 Temperature C Solar Radiation W/m 100 200 300 400 0 400 6/1/98 6/1/98 6/11/98 6/11/98 6/21/98 6/21/98 7/1/98 7/1/98 7/11/98 7/11/98 7/21/98 7/21/98 7/31/98 7/31/98 8/10/98 8/10/98 8/20/98 8/20/98 8/30/98 8/30/98 9/9/98 9/9/98 Temperature C 0 10 20 30 40 Temperature C 0 10 20 30 40

2

0

10

20

30

40

55

Santa Cruz

Hollister

Solar radiation (bar) and maximum (bold line), minimum (thin line) soil temperature at areas where summer samples grew during their estimated growth period.

(UC IPM California Weather Database 1999).

Appendix 6.4 Climate at Market Sample's Origin (Summer2)

APPENDIX 7. Regional Comparison of Nitrate Content in Lettuce and Spinach A. Lettuce

Substantial data on nitrate content in vegetables have been reported in Europe. Some of these results showed lettuce containing considerably higher nitrate levels than were found in the US (Appendix 7.1). In particular, the average nitrate content exceeding 1500 mg/kg FW was found in lettuce grown in greenhouses in fall to winter (Finland (Ahonen et al., 1987) and UK in Appendix 7.1). Moreover, the maximum nitrate content in lettuce grown in greenhouses in the Netherlands and UK during winter reached more than 5000 mg/kg FW . A number of studies have shown that light influences nitrate accumulation in plants considerably by affecting nitrate reductase activity (Viets and Hageman 1971, Blom-Zandstra 1989. Appendix 3.1). This "greenhouse effect" is explained, therefore, by low light intensity, short daylight duration, and high temperatures that stimulate mineralization and nitrification of nitrogen in soils in greenhouses during winter (Roorda van Eysinga, 1984; MAFF UK, 1998a). In the US, Minotti (Maynard et al. 1976) also reported an extreme nitrate accumulation in lettuce grown in a greenhouse (maximum of 7.5% NO3 DW), although very little lettuce is grown in greenhouses in the US even in winter. Global solar radiation in southern England in January was as low as 23 W/m2 and was about one sixth that of Southern California (Appendix 7.2). Interestingly, even under such low solar radiation, nitrate content in winter lettuce grown in the UK did not differ significantly from that of summer lettuce when they were grown outdoors (Appendix 7.1). Variety appears to be another factor. For example, in UK, Iceberg lettuce is grown outdoors as well as the US, and Round lettuce (also known as flat lettuce), which are the most commonly grown and consumed lettuce in the UK, are grown throughout the year, predominantly in glass-houses (MAFF UK 1996, 1997a). They reported that Iceberg lettuce tended to have lower nitrate concentrations than other varieties and this may be explained in part by the practice of removing the outer leaves of these lettuces at harvest. As mentioned before, wrapper leaves of head type lettuce contains higher nitrate than inner part (Lorenz 1978). Thus, this contrast may be explained by differences between the US and Europe in solar radiation, the degree of dependence on greenhouse-grown lettuce, and the variety of lettuce. Such regional differences in nitrate accumulation in lettuce were reflected in the evaluation of lettuce as a nitrate accumulator. That is, in Europe, lettuce is grouped as a highest nitrate accumulator, as is spinach (Corre and Breimer 1979 cited by Blom-Zandstra 1989), but this is not necessarily the case in the US (Maynard et al. 1976, Lorenz 1978). MLECR also established a lower maximum nitrate limit for spinach than that for lettuce (Table 1.1). Lettuce grown under semitropical to tropical climate in Australia, on the other hand, showed a similar range of nitrate content as those of the present study (Appendix 7.1).

B. Spinach

In contrast to lettuce, spinach is regularly regarded as one of the highest nitrate accumulators. Its distinctive shoot structure, consisting of petioles, where nitrate passes through and accumulates, and leaf-blades, where nitrate reduction and assimilation take place, might be one of the reasons for spinach' s relatively high nitrate concentration. Certain vegetables, because of a very efficient uptake system, an inefficient reductive system, or an unfavorable combination of both, tend to accumulate more nitrate than others (Maynard et al., 1976). Nitrate content in spinach grown in European countries was similar to that seen in the US, except for the maximum value reported in the Netherlands (5400 mg/kg FW) . This trend differs from that of lettuce as discussed above. Cantliffe (1974a) showed that nitrate content in spinach was lower than lettuce when those were grown in greenhouse by applying the same amount of nitrogen.

56

Appendix 7.1 Geographical and seasonal comparison of nitrate content in lettuce and spinach

Country Year Analysis by reported Origin of samples CA, USA USA 1999 Muramoto AZ & CA, d USA Germany Finland Netherlands UK Greenhouse UK Outdoor Sampling season Aug-Sep Jan-Feb (not available) Summer Fall

f

Lettuce (NO3 mg/kg FW) n 24 [5]

b

a

Spinach (NO 3 mg/kg FW) n 12 [5] 12 [5] 85

g

Average 840 1000 1600 890 1900

Range 490 - 1700 c (330 - 1900) 760 - 1500 (570 - 1600) 230 - 3300 85 - 2500 280 - 3500 420 - 5500

Average 2300 2000 840

24 [5] 162

g

Range 600 - 3400 (130 - 4100) 890 - 2900 (310 - 3400) 20 - 2700

Germany Finland Netherlands

1984 1987 1988

Kampe

e

Ahonen et al. CCRX Ysart et al.

e

41 [1] 54 [1] 1682

g,h

(not available) Apr-Sep Oct-Mar May-Octi

866

g,h

800 - 5400

70 [10] 112 [10] 131 [10]

2400 3100 1100

270 - 4200 600 - 5300 50 - 3200 34 [10] 1900 50 - 4700

UK

1999

Australia 1994 Lyons et al. Queensland Aug-June, Dec 18 [1] 290 - 1500 For the sake of easier comparison, values were converted and rounded from original data as needed. Blue Italic letters and values indicate fall to winter samples and regular letters shows the other seasons or season not specified. Market and/or field samples were included. a : Lettuce includes both leaf type and head type. b : [ ] = Number of sub-samples per one sample. c: ( ) = Range of nitrate content in sub-samples. d : One spinach sample grew at Mexicali, Mexico. e: Cited by Schuddeboom (1993). f : Mostly grown in greenhouses. g: Number of sub-samples per one sample was not available. h : Data during1972 to 1987. i: For spinach, Jun. to Feb.

57

2

2

Air temperature C

Solar radiation W/m

Solar radiation W/m

400 300 200 100 0 JAN JUL NOV MAR MAY SEP

30 20 10 0

400 300 200 100 0 JAN JUL MAR MAY NOV SEP

30 20 10 0

Air temperature C

South Coast of California

Southern England

Appendix 7.2 Comparison of climate between California and England

Monthly average during March, 1985 through December, 1988 Original data; Solar radiation (bar) = Average total horizontal surface down. Air temperature (line) = Average near surface air temperature. (NASA Langley Research Center 1997)

58

APPENDIX 8 Sampling Number

Even within five sub-samples of the same brand purchased at the same time and market, average CV of nitrate content reached 12 to 13 % in lettuces, and 24% in spinach (Appendix 8.1). Using those average CVs, number of sub-samples to obtain average nitrate content in plants in a carton (24 plants or bundles) within ± 10 and 25% of sampling error was calculated assuming normal distribution (Cochran, 1977). It was also assumed that five sub-samples were taken from the same carton. Number of samples calculated for Iceberg lettuce, Romaine lettuce and spinach were 6, 6, and 12 within ± 10%, and 3, 3, and 5, within ± 25%, respectively. When we used five sub-samples, the average sampling error was estimated as ± 13 to 14% for lettuces and ± 27 % for spinach at a confidence level of 0.95. To obtain average nitrate content in plants in a carton within 20% of error, we need to take at least five lettuce plants and ten bundles of spinach from a carton containing 24 plants or bundles. Appendix 8.1 Coefficient of Variation (CV %) of nitrate content in lettuces and spinach within sub-samples (NO3 mg/kg FW. n = 5) purchased in Santa Cruz County, California, 1998, Number of subsamples for obtaining population mean of nitrate content in plants in a carton (24 plants) within ± 10% and ± 25% of sampling error at confidence level of 0.95. Vegetable Coefficient of Variation (%)* Number of sub-samples** Average Minimum Maximum ± 10% ± 25% Iceberg lettuce 12 4.7 25 6 3 Romaine lettuce 13 3.7 41 6 3 Spinach (whole plant) 24 5.0 83 12 5 Spinach (leaf-blades) 26 8.0 93 Spinach (petioles) 23 3.5 71 * CV % = Standard deviation / Average x 100. ** Normal distribution assumed.

59

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