Comparison of Nitrate Content in LeafyVegetables from Organic and Conventional

Farms in California

June, 1999Revised Version

Dr. Joji MuramotoCenter for Agroecology and Sustainable Food Systems

University of California, Santa CruzSanta 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

1

EXECUTIVE SUMMARY

IntroductionThe 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 maximumlevels (limits) for nitrate content in lettuce and spinach. Though studies on this issue wereconducted 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 vegetablesis 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 oncertified organic farms in California.

Results

1) Market Sampling SurveyOrganic and conventionally produced Iceberg lettuce, Romaine lettuce, and spinach were

sampled in winter and summer from supermarkets (conventional), farmers market (organic), andnatural-food markets (organic) in Santa Cruz County, California.

Seasonal differences in nitrate content were significant only for Iceberg lettuce (higher inwinter). Conventionally produced spinach had higher nitrate levels than organic spinach, butconventional and organic lettuce nitrate levels were similar. Nitrate content (NO3 mg/kg freshweight) 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 inspinach 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 oursample of conventional and organic summer spinach exceeded EU limits, respectively, while nowinter spinach exceeded EU limits regardless of practice.

2) Farmer's Field SurveyFive 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 conventionaland organic fields (where we conducted paired field experiments), one conventional farm, andthree organic farms.

In paired fields, Romaine lettuce nitrate content was similar in conventional and organicfields despite differences in nitrate and potentially mineralizable nitrogen content in soils afterharvest.

Organic spinach grown using guano tended to have higher nitrate contents than thosegrown using compost only. However, spinach grown using guano on fields with sandy soilcontained the lowest nitrate contents. Nitrate levels in field-sampled lettuce and spinach weresimilar to those observed in our market samples.

3) Field ExperimentsA field study of fertilizer management factors affecting spinach nitrate content was

conducted at the Farm at the Center for Agroecology and Sustainable Food Systems, Universityof California, Santa Cruz. The study compared different rates of compost, compost with Chileannitrate, and commercial organic fertilizer applications. We used regression analysis to estimatethe maximum safe yield of each fertilizer practice, defined as "the maximum yield that contained

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nitrate lower than the EU limit (2500 mg/kg FW)".

There was no significant difference in average yield or nitrate content of spinach incompost or compost with Chilean nitrate plots, but nitrate content was more variable in thecompost 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 + standarderror was used to represent nitrate content. Nitrate content in spinach was positively correlatedwith yield, though commercial organic fertilizer produced lower yields at the same nitrogen ratesthan other treatments because of slower nitrification in the soil.

In conducting these experiments, we also observed that spinach harvested in theafternoon had significantly lower nitrate levels than morning harvests, despite slightly cloudyweather on the harvest day.

Conclusions1. 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 pre-

plant 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 ofseason and farming practice.

3

1. INTRODUCTIONNitrogen 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 (Maynardet 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 averageUS diet, vegetables contribute 87% of the total daily intake of nitrate (National Research Council1981). Nitrate is also formed by endogenous synthesis in the human intestine (Tannenbaum etal., 1978).

The potential health hazards of nitrate and nitrite are well studied. Nitrate can generallybe considered to be of relatively lower toxicity than nitrite. However, about 5% of dietary nitrate isconverted to nitrite in humans by bacterial and mammalian metabolic pathways (Walters andSmith, 1981). Potentially carcinogenic N-nitroso compounds can then be formed from nitrite andN-nitrosatable compounds endogenously. Nitrate intake may cause methemoglobinemia (alsoknown 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 Gangolliet al., (1994) for a recent review. Recently, a positive role of nitrate in the human body's defenseagainst 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, EuropeanCommission Regulation (EC) No. 194/97 set harmonized maximum levels (limits) for nitrate inlettuce and spinach. The limits vary according to season, with higher nitrate levels permitted inwinter-grown vegetables. For lettuce, different limits were set for glasshouse-grown and outdoorcrops (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)

Spinach (fresh) 1 November to 31 March1 April to 31 October

30002500

Spinach (canned and frozen) 2000

Lettuce* 1 October to 31 March1 April to 30 September

45003500

Lettuce with the exception ofoutdoor lettuce 1 May to 31 August 2500

* No separate limits have been established for different type of lettuces such as leaf type andhead type.

This regulation was amended in April 1999 based on results of monitoring carried out bythe Member States of the EU since the regulation was adopted (The Commission of theEuropean Communities, 1999). The amendment concluded to maintain the current maximumlimits for nitrates in spinach and lettuce. However, these levels will be reviewed in a three-yearperiod before 31 December 2001 for the first time, based on monitoring and the application ofcodes 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 ppmnitrate (dry weight) for spinach and asparagus, respectively. Between the late 1960s and early1980s, abundant studies on the nitrate issue at large were conducted in the US, including reportsby the National Research Council (National Research Council, 1972, 1978, 1981). However, nonitrate standards for vegetables have been introduced in the US.

Some Australian researchers (Lyons et al., 1994) and Asian countries such as Korea andJapan (Sohn and Yoneyama 1996) have also pointed out the importance of this problem.

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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 numerousenvironmental and practical factors. See Appendix 3.1 for detail of factors affecting nitrateaccumulation in spinach and Appendix 3.2 for those references. Of the factors studied, ingeneral, nitrogen fertilization and light intensity have been identified as the major factors whichinfluence nitrate levels in vegetables (Cantliffe, 1973b). In particular, light intensity and nitratecontent in soils before or at harvest are known to be critical factors in determining nitrate levels inspinach (Schuphan et al., 1967; Maynard et al., 1976).

In European countries, many studies have demonstrated that organically or biologicallygrown 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 organicfertilizers, however, may cause high nitrate levels in vegetables, depending on the types andamount 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 areknown 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 vegetablesregardless of the kind of fertilizers. There is also a higher risk of contaminating ground water withnitrates when overfertilization takes place. Therefore, fertilizing practices that produce vegetableswith 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 cropsin California (Hartz et al., 1994). Recently, Smith reported that nitrate content in fresh sap of theroot tissue of organic onions was significantly lower than that of conventional onions, althoughthere were no significant difference between their yields (Smith, 1996). However, to evaluate foodsafety, nitrate content in whole edible parts of vegetables must be examined. In 1978, Lorenzreported 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 movementin 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 grownleafy vegetables in California.

Therefore, the objectives of this project were to 1) demonstrate ranges and variability ofthe 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 vegetablesbased 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 oncertified 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 andin 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 ofretailers. Part 2 (Chapter 3), the farmer's field survey, was conducted to examine therelationships of farming practice and nitrate content in lettuce and spinach. Finally, a fieldexperiment using spinach grown with different types and levels of organic fertilizers wasconducted (Chapter 4). Based on the results obtained, methods to reduce nitrate concentration inspinach for organic growers were discussed at the end (Chapter 5).

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2. MARKET SAMPLING SURVEY

2.1 GoalsThe 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) assessnitrate 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 Methods2.2.1 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 comparethe 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 (Table2.1). Sampling was replicated twice within each season at intervals of two weeks. Iceberg lettuceand Romaine lettuce were selected as the most commonly purchased head lettuce and leaflettuce, respectively. When organic Iceberg lettuce was not available, organic green leaf lettucewas sampled.

Table 2.1 Sampling method for market survey

Factor Level

Type of vegetable (3*) Iceberg lettuce, Romaine lettuce, and spinach** Season (2x2) Winter and summer. [2 replications per season]

Practice (2) Conventional and organic Brand (3) 3 different brands (growers) per practice Sample (5) 5 sub-samples (bunches or plants) per brand

Total number 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 EuropeanCommission 1997). In this study, to evaluate variability of nitrate content within sub-samples, fiveplants or bundles of sub-samples were analyzed separately and averaged to produce one sampledata point. Total number of sub-samples and samples was 360 and 72, respectively (Table 2.2).

Table 2.2 Numbers of samples and sub-samplesVegetable 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. Organicvegetables were usually purchased at the farmers market, based on the assumption that themarket is the most common place to buy organic vegetables. The farmers market also offered the

6

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 atnatural-food markets in Santa Cruz County. No organic vegetables were purchased atsupermarkets in this project. See Appendix 4 for the list of sampled markets and Appendix 5 forthe 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 samplingday 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, assumingdifferent brand samples were grown by different growers. When three different brands were notavailable on a sampling day, as happened only with summer organic Iceberg lettuce, twosamples of the same brand were purchased at different markets. Five plants or bundles (sub-samples) of target vegetables were purchased at each market. Based on interviews, theinformation about the price, the grower or the distributor, the date of unloading of samples, andstorage method after unloading was recorded for each sample. Origins of samples were tracedand confirmed by calling the grower or the distributor of each sample.

2.2.2 Pretreatment and Nitrate AnalysisAll 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 werewashed 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 freshweight 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 anotherhalf was used for moisture measurement. Moisture content was determined by the differencebetween weights before and after heating at 60-70 °C for 48 hrs. For spinach, leaf-blades andpetioles 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 asneeded prior to homogenization. Fifty to 100 grams of sub-sample were weighed and placed intoa mixer. Deionized water, nine times the sub-sample's weight, was added and the water and sub-sample were homogenized for several minutes. A 30 gram sample of homogenate was placed ina 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 additionof decoloring agent. The sample was centrifuged at 3,500 rpm for 3 min. The supernatant wasthen filtered with filter paper Whatmann #1 and nitrate concentration in the filtrate was determinedcolorimetrically by a flow injection analysis system (Lachat Instruments, 1992). Nitrate contentwas 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 fromnitrate content in leaf-blades and petioles and the weight of each part.

2.2.3 Statistical AnalysisSample 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 (SPSS1996).

2.2.4 Comparison with Other DataTo 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 thepresent data.

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2.3 Results2.3.1 Origin of Samples

Sample origins showed a clear seasonal trend (Figure 2.1). In winter, regardless of thekind of vegetable, 67 - 84% of the conventional samples and 42 - 50% of the organic sampleswere 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

1). Winter

2). Summer

Figure 2.1 Origin of samplesPlots 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 foreach pie chart, respectively.

$

$

$$$$$

$

Yuma, AZ

Org. Lettuce

DV42%

CC8%

N/A17%

SC33%

Central Coast (CC)

South Coast (SC)Desert Valley (DV)

Mexicali, Mexico

Conv. Spinach

DV33%

SC33%

AZ+Mex34%

Org. Spinach

DV50%

SC33%

N/A17%

Conv. Lettuce

DV25%

SC8%

AZ+Mex59%

N/A8%

$

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

South Coast (SC)

Conv. SpinachSC

17%

CC50%

N/A33%

Org. Spinach

CC100%

Conv. Lettuce

CC75%

N/A25%

Org. Lettuce

CC100%

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(Oxnard). No local lettuce or spinach was available during the winter sampling period, except forone organic Romaine purchased at the farmers market. In summer, all of the organic and most ofthe conventional samples were grown on California’s Central Coast (Davenport, Gilroy,Greenfield, Hollister, Salinas, Santa Cruz, Soledad, and Watsonville) although there was oneconventional spinach sample from the South Coast of California (Oxnard).

2.3.2 Storage Days and Conditions at MarketsExcept for some natural food stores that received produce directly from growers, little

reliable information was obtained regarding the period between harvest and unloading ofvegetables 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 2.2days. minimum 0 days, maximum 5 days) reflecting frequent deliveries and turnover atsupermarkets that are more. Refrigeration temperatures varied between 1.7 and 10 °C, anddisplay rack temperatures ranged from 0.6 to 11 °C.

2.3.3 Plant Size and Moisture ContentAt 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, Iceberglettuce, 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 andpractices 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 CruzCounty, 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) ofspinach.

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 insummer 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 seasonsand practices

Vegetable Season PracticeWinter Summer Mann-

Whitney testaConv. Org. Mann-

Whitney testIceberg 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 VariationAverage 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,

9

spinach (n=24) contained an average of 2170 mg/kg FW of nitrate (Table 2.5). Nitrate content inspinach 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 oflettuces and spinach were 30% and 42% greater than those in samples, respectively. Coefficientof 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 inSanta 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 ContentThe 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 theirminimum and maximum values, and CVs. It should be noted that even in the same season andthe same practice, there was a wide range of nitrate content in spinach samples. In particular, theranges 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 highas five times that of the minimum (600 mg/kg FW) (Table 2.7).

Only iceberg lettuce showed a significant seasonal difference in nitrate content; onaverage, winter samples contained 52% higher nitrate than summer. The effect of managementpractice on nitrate content was significant only in spinach. That is, conventionally grown spinachcontained significantly higher levels of nitrate than organically grown samples (Table 2.8). SeeAppendix 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 9.7Green-leaf total 2 786 780 790 0.90 1.19 1.11 1.28 9.7Iceberg 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 25Winter total 10 973 760 1300 15 2.19 1.59 2.95 19Summer 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 31Summer total 12 641 480 1100 26 1.93 1.27 2.96 29Conventional total 12 839 520 1100 24 2.06 1.27 2.96 23Organic 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 29Winter total 12 1100 820 1500 20 1.96 1.32 2.85 21Summer 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 62Summer total 12 1050 580 1700 34 2.08 0.66 3.92 43Conventional total 12 1090 770 1700 23 2.10 1.70 3.22 21Organic 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 34Grand 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 43Winter total 12 2010 890 2900 30 2.54 0.92 3.68 33Summer 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 71Summer total 12 2330 600 3400 42 3.82 0.65 5.42 46Conventional total 12 2540 1500 3400 23 3.78 2.09 5.42 30Organic total 12 1810 600 3000 47 2.58 0.65 5.25 63Grand 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 differentseasons and practices

Vegetable Season PracticeWinter Summer Mann-

Whitney testaConv. Org. Mann-

Whitney testIceberg 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 RegulationNitrate content in lettuces and spinach purchased at various markets in Santa Cruz

County, California was contrasted with the maximum levels set by European CommissionRegulation (EC) No. 194/97 (Table 1.1). Regardless of season and practice, none of the lettucesamples exceeded the maximum levels. On the other hand, 29% of all spinach samplescontained nitrate levels exceeding the maximum EC standards. Summer-grown spinach inparticular tended to exceed the maximum levels, with 83% of conventional and 33% of organicspinach sampled in summer containing levels of nitrate above the maximum (Table 2.9). Nowinter-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 CommissionRegulation

SeasonMaximum levels

in ECR* (EC) No.194/97NO3 mg/kg FW

Practice nExceeding themaximum level

% (n)Conventional 6 0% (0)Organic 6 0% (0)Winter** 3000Winter total 12 0% (0)Conventional 6 83% (5)Organic 6 33% (2)Summer** 2500Summer 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 USNot 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 nitratecontent in head lettuce and spinach from the present study (market survey) was 1 to 2 % greaterthan 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 leavesof head lettuce that are usually removed in the field and contain higher nitrate than the innerportion (Lorenz 1978).

Much more data are available on nitrate content in lettuce and spinach purchasedelsewhere in the US. Richardson reported nitrate and nitrite content in various foods almost ahundred years ago (Richardson 1907). After that, most data were reported from the late 1960s tothe late 1970s, when substantial studies were conducted on this issue in the US (Table 2.11). Itshould be noted, however, that sampling numbers in these studies were very small (1 to 20). Inaddition, no sub-samples were taken, with each plant (lettuce) or bundle (spinach) treated as asample.

With regard to lettuce, the maximum nitrate content reported was 3500 mg/kg FW inRichardson's survey (Richardson 1907), which was as great as twice that of the maximum nitratecontent in lettuce found in the present study. However, since the moisture content in Richardson'slettuce was as low as 85% on average, the maximum nitrate content in lettuce on a dry weightbasis was 4.5%, which was lower than the maximum value found in the present study. For nitratecontent in spinach, on the other hand, the maximum value was 4700 mg-NO3/kg on a freshweight 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, takenfrom conventional and organic produce in winter and summer in California, appear to rangewidely enough to cover most data reported in the US.

12

Table 2.10 Ranges of nitrate content in lettuce and spinach sampled in California.Vegetable Year

reportedAnalysis by Sampling location

(Source)Samplingseason

N ratelbs/acre

Plant part NO3 % DW

Lettuce 1962 Zink andYamaguchi

Salinas Valley, CA(farmer's fields)

Apr. MayJune.

Aug. Sep.Oct.

121 - 300 shoots ofhead lettucea

0.88 - 2.9

1974 Lorenz Davis, CA(field experiments)

May, Oct.Nov. Dec.

0 - 500 heads 0.12 - 2.0

1999 Muramoto Santa Cruz, CA(markets)

Jan. Feb. Aug. Sep.

notavailable

heads 1.3 - 3.0

1999 Muramoto Soledad, CA(farmer's field)b

Sep. 137 heads 2.4

Spinach 1966 Zink Salinas Valley, CA(farmer's fields)

Apr. 180 - 220 top 0.93 - 3.2

1974 Lorenz Davis, CA(field experiments)

May, Oct.Nov. Dec.

0 - 500 leaf-bladespetioles

0.13 - 2.2 0.24 -10

1999 Muramoto Santa Cruz, CA(markets)

Jan. Feb. Aug. Sep.

notavailable

leaf-bladespetioles

top

0.49 - 3.21.2 -12

0.64 - 5.4

1999 Muramoto Central Coast of CA(farmer's field)c

Sep. Oct. 26 - 120 leaf-bladespetioles

0.23 - 1.9 1.5 - 7.6

1999 Muramoto Santa Cruz, CA(field experiments)d

Oct. 0 - 348 leaf-bladespetioles

0.9 - 1.9 2.9 - 4.2

For the sake of easier comparison, values were converted and rounded from original data asneeded.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.

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

Yearreported

Analysis by Samplinglocation

Samplingmonth

Lettucea Spinach

n NO3 mg/kg FW NO3 % DW n NO3 mg/kg FW NO3 % DW1907 Richardson Chicago, IL Sep. 5 400 - 3500 0.45 - 4.5 5 310 - 3800 0.30 - 3.91949 Wilson Ithaca, NY May 2 400 - 1800 4 300 - 24001967 Jackson et al. Washington D.C. (not available) 5 490 - 890 2 240 - 240

1967 Brown and Smith Columbia, MO May, Jun. Jul.Sep.

notavailable

0.09 - 4.7 notavailable

0.31 - 2.9

1971 Lee et al. (not available) (not available) 1 280 1 2100

1972 Maynard and Barker Amherst, MA (not available) 1 750 1 2300

1975 Siciliano et al. Philadelphia, PA Dec. Jan. 3 1100 - 1400 7 2200b

19761978

Minotti Ithaca, NY Apr. - Nov. 15 440 - 850c

740 - 2700d20 1300 - 4700

1999 Muramoto Santa Cruz, CA Jan. Feb.Aug. Sep.

48(240)e

490 - 1700 (330 - 1900) f

0.66 - 3.9 (0.37 - 4.9)f

24(120)e

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.

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2.4 Discussion2.4.1 Nitrate Content in Lettuce

Although nitrate concentration were relatively low, winter Iceberg lettuce containedsignificantly higher nitrate than summer (Table 2.8), and moisture content in summer lettuce wasgreater than that found in winter samples (Table 2.4). Therefore, if we can assume that moisturecontent of the samples reflected their original moisture regime at harvest, low nitrate in summersamples 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 soiltemperatures at selected locations where samples grew in winter and summer during theirpossible growth periods. El Centro, one of the origins of the winter lettuce, experienced coolertemperatures and weaker but constant solar radiation (142 W/m2 on average) than did summergrowing locations such as south Salinas (see Appendix 6 for climate of other origins).

There was no significant difference in nitrate content in lettuces between farmingpractices. Stopes et al (1988) compared nitrate content in organic and conventional vegetablesincluding lettuce sampled during winter in Great Britain. They found that the peak nitrate contentmight be lower in organically produced vegetables, although there is considerable variation. Zinkand Yamaguchi’s (1962) studies at Salinas Valley, California, reported that no relationship wasfound between the nitrate content of the aboveground portion of the head lettuce and the amountof nitrogen applied. They suggested that nitrate content of the plant was largely dependent uponthe 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-samplesranged from 330 to 1900 mg/kg FW. Previous data on nitrate content in lettuce purchased in theUS 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 mg-

NO3/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 thosefrom the 1907 study (the era before synthetic fertilizers) on a dry weight basis (Table 2.11). It isimpossible, however, to conclude above trend since sampling number in the previous studies wasvery 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 greaternitrate than organic spinach (Table 2.8) suggests that nitrogen fertilizer applications may be themain cause of the difference. Further survey at conventional spinach fields is needed to confirmactual causes. Positive effects of organic fertilizers on nitrate accumulation in spinach have beenreported by some studies (Barker 1975; Stopes et al., 1989). As slow release nitrogen fertilizers,some organic fertilizers may reduce nitrate accumulation in plants. However, easilydecomposable organic fertilizers such as blood meal and guano might increase nitrateaccumulation in the same way as conventional chemical fertilizers (Termine et al., 1987; seeChapter 3), especially with excessive application rates. Some relationships between nitratecontent in organic spinach and farming practices are examined in the following chapters. Cantliffeand Phatak (1974b) reported that application of herbicides such as cycloate, alachlor, and lenacilstimulates nitrate accumulation in spinach. Whether such herbicides are currently used byspinach growers in California should be explored.

2.4.3 Comparison with MLECRNone 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 lettucetended to be greater than those in the US lettuce. It was especially true for greenhouse-grownlettuce during winter. This contrast is explained by differences between the US and Europe insolar radiation, the degree of dependence on greenhouse-grown lettuce, and the variety oflettuce. See Appendix 7 for details of comparison across the US, Europe, and several othercountries’ 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

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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).

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.

16

MLECR (MAFF UK 1999). In particular, conventional spinach exceeded MLECR morethan organic spinach, due perhaps to some of the reasons discussed above.

As climatic conditions, production methods, and eating habits vary widely in differentparts of the European Union, this EC Regulation allows for an optional derogation from themaximum levels, for a transitional period, for nitrate in lettuce and spinach grown and sold inindividual Member States. This is provided that growers follow Codes of Good AgriculturalPractice and that the nitrate concentrations in lettuce and spinach do not pose a risk to publichealth. As of August 1998, the UK, Ireland, Finland and Belgium are applying this optionalderogation. It should be noted that lettuce and spinach imported from other Member States andthird countries must comply with the maximum levels set by this EC Regulation, as must produceexported to other Member States (MAFF UK 1998a).

Therefore, this could not only be a safety issue, but also a trade issue. A small amount ofspinach 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 Unionfrom Monterey County, where most conventional summer spinach sampled in this survey wasgrown.

2.4.4 Sampling MethodsOne 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 invegetables 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 compositesingle samples. Maynard et al. (1976) noticed in their review that average values based on asufficient number of samples will largely circumvent the problem of variability. In this survey, inorder to demonstrate difference in ranges when we took different number of sub-samples, eachsub-sample was analyzed separately. As mentioned before, ranges of nitrate content in sub-samples 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 inone-carton (24 plants) of vegetables based on CV within sub-samples (see Appendix 8 fordetail).

Sampling frequency is another issue that must be taken into account when evaluatingthis 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. InCalifornia, however, as seen last year (during El Niño), it is possible to have rainy weather affectnitrate 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 toits rather constant Mediterranean climate, nitrate content trend can differ from year to yeardepending on the weather (MAFF UK 1996). In addition, this survey did not sample organicvegetables sold at traditional supermarkets, a trend that is on the rise. Therefore, future studiesshould test the reproducibility of trends discussed above, using more frequent samplingthroughout 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 ofcontinuous heavy rain due to El Niño in 1998. See Appendix 6 for weather conditions ofsample's origins.

17

3. FARMER’S FIELD SURVEY

3.1 GoalThe 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 typesand amounts, on nitrate content in lettuce and spinach.

3.2 MethodsFive 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 farmingplan database of the California Certified Organic Farmers and information obtained from farmersat farmers market were used to select farms to be surveyed. Diversity of fertilizing practicesamong farms and nitrate content of their produce sampled in the market survey were also takeninto account in the selection. All the farms are located in Monterey County and Santa CruzCounty, California.

Farm surveys were conducted in September and October 1998. Fields that had lettuce orspinach 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 andcomposited as one sample. At the other fields, 5 to 20 lettuce plants or 10 to 60 spinach plantsfrom 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 ateach point where plant samples were removed. Soil profile characteristics were observed andrecorded 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 sampledand soils taken from the same layer of the same field were composited, except for the pairedfields, where soils sampled from each location were treated separately. Soil samples were alsoput into a cooler box right after sampling. Plant samples were treated and analyzed as describedin Chapter 2. Irrigation water was also sampled where it was available.

Soil samples were mixed, homogenized, and separated into halves. Half of each samplewas air-dried and passed through a 2 mm sieve and used to determine pH (1:1) and electricalconductivity (EC 1:1) (Smith and Doran, 1996). The other half was passed through a 2 mm sievewithout drying, and used to determine nitrate and ammonium (2M KCl extraction followed bydetermination using flow injection method (Lachat Instruments 1993a, b)), potentiallymineralizable nitrogen (PMN) (aerobic incubation method (Drinkwater et al., 1996)), and moisturecontent. PMN was measured in duplicate for surface layers. All the soil data are expressed on adry weight basis. Nitrate content in irrigation water was measured using the FIA method (LachatInstruments 1992). Results from paired fields were analyzed using a one-way ANOVA for thedifference between conventional and organic fields.

3.3 Results3.3.1 Comparison between Conventional and Organic Romaine Lettuce Fields

A pair of conventional and organic fields that have been managed by the same growerwas 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).

18

Table 3.1 Summary of conventional and organic fields surveyedConventional Organic

Crop Romaine lettuce Romaine lettuceVariety Gladiator GladiatorFarming history of the field Conventional many years Organic over 10 yearsField acreage (Romaine field) 30 acre (30 acre) 50 acre (0.7 acre)Seeding date Aug. 6, 1998 Aug. 12, 1998Sampling date Sep. 30, 1998 Sep. 30, 1998Growth period (by sampling) 55 days 49 daysPlanting density (#/acre) 22,000 30,000Fertility practices Preplant*:(application date) Sidedress

14 -7-14; 408 lbs/acre (7/27)*20-0-0; 40 gallons/acre (9/3)*

NoneMeat 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/acreBoth 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 andwater 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 **Irrigation waterf 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: Electrical conductivity. d: Potentially mineralizable nitrogen. e: Penetration resistance.f: Ground water. g: Not Detected.

Total nitrogen application rate at each field was similar (84 lbs-N/acre at conventional. 89lbs-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 withcommercial organic fertilizers.

Soil types of these fields differed (Table 3.2), with Elder sandy loam at the conventionalfield and Pinto loam at the organic field. At a depth from 0 to 12 inches, the conventional fieldshowed 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 theorganic fields’ topsoil reached 13 mg-N/kg, which was 6 times as high as that of the conventionalfield.

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

19

above, there was no significant difference between the nitrate content in Romaine lettuce (Table3.2). The nitrate contents were lower than the maximum limits of MLECR. However, these levelswere 70 to 90% higher than the average nitrate content in market-sampled summer Romainelettuce. 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 LettuceOne 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 theirpractices and nitrate content in lettuce, soil, and irrigation water (Table 3.3). C1 is located on theedge 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 apreplant and sidedress of 6-20-20. The C1 field had clay soil with pH 6.8 and slightly highelectrical conductivity of 1.2 dS/m for the topsoil (0 to 6 inches deep). Nitrate content was 33 mg-N/kg in topsoil and 9.9 mg-N/kg in subsoil (6 to 12 inches deep). PMN content in topsoil was 9mg-N/kg. Irrigation water (ground water) contained 11 mg-N/L (= 49 mg-NO3 /L) of nitrate, whichexceeds 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 ofmarket-sampled Iceberg lettuce (794 mg/kg FW). According to the grower, to suppress mildewdisease, nitrate concentration in plant tissue was tested at this field by the company and nitrogenfertilization was restricted based on the results.

Table 3.3 Farming practices and nitrate content in lettuce, soil, and irrigation water at farmer'sfields

Farms C1 [Conventional] O2 [Organic]Location Soledad, CA Watsonville, CAField acreage (of lettuce) 20 acre (20 acre) 9 acre (0.05 acre>)Farming history Conventional for 15 years Organic for 15 yearsCrop Iceberg lettuce Romaine lettuceVariety Vienas Darkland CosGrowth period from seeding 72 days about 80 daysPlanting density #/acre 23,000 30,000NO3 mg/kg FW 830 2,200Fresh weight g/plant 657 294Fertility practices Preplant Sidedress

6-20-20; 350 lbs/acre6-20-20; 350 lbs/acre X 315-8-4; 350 lbs/acre X 1

Compost; 5 to 7 t/acre[turkey & cow manure, grapepomace, 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 NO3-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 PR kg/cm2 1.2 0.1(6 - 12 inches) pH 1:1 6.6 6.6 EC 1:1 dS/m 0.79 0.40 NO3-N mg/kg DW 9.89 28.7 NH4-N mg/kg DW 1.71 2.16 PR kg/cm2 1.4 3.6Irrigation water NO3-N mg/kg (source)

11.0(ground water)

1.38(ground water)

*: Raw materials of compost. **: Estimated from compost analysis data.See footnotes of Table 3.2 for others.

20

Table 3.4 Comparison of market and field sample grown by the same growers.- Romaine lettuce -

Grower O2 [Organic]Sample Market-1 Market-2 FieldSampling date Aug. 26 Sep. 9 Sep. 23NO3 mg/kg FW 1200 1600 2200Fresh weight g/plant 349 444 294Moisture % 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 textureof the Romaine field’s soil was sandy. Topsoil pH was 6.8. Nitrate content in soil was the highestamong surveyed fields, reaching 48 mg-N/kg in topsoil and 29 mg-N/kg in subsoil. PMN contentin 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 concentrationamong all the Romaine lettuce sampled (including market samples), while nitrate content inmarket-sampled summer Romaine lettuce from the same field was 1200 and 1600 mg/kg (Table3.4). Fresh weight of field-sampled O2 Romaine lettuce was lower than that of market samples.

3.3.3 Comparison of Organic Spinach FieldsIn 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 considerabledifference in nitrate content across samples (Table 2.7). Therefore, to demonstrate the effects ofdifferences in fertility practices between high-nitrate organic spinach and low-nitrate organicspinach, four organic farms growing spinach were surveyed for their practices, nitrate content inspinach, soils, and irrigation water.

All four farms are located in Santa Cruz County and Monterey County; Table 3.5 showsthe outline of the farms and the survey results. The surveyed organic farms consisted of threedifferent types of fertility practices; compost only (O1 and O2), compost + commercial organicnitrogen fertilizers (O3), and commercial organic nitrogen fertilizers only (O5). Farms O3 and O5were selected because of the low-nitrate content and high-nitrate content of their market-sampledsummer spinach, respectively. The number of years these fields had been managed with organicpractices ranged from 4 to 13. Although the growers grew different varieties, all of the spinachwas the smooth leaf type. The following trends were observed with regard to the effect of fertilitymanagement 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 onlycompost was used.

This trend coincided with the high nitrate content in market-sampled summer spinachfrom O5 (Table 3.6). A grower interview confirmed that both the market- and the field-sampledspinach from O5 were grown at the same field, but on different dates. The fresh weight of spinachgrown 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 fieldwas as low as one third of the others (26 lbs-N/ acre. Table 3.5). According to the informationfrom the farm, the grower was cutting back nitrogen application rates to eliminate heavyaccumulation of nitrate in the soil, which occurred early in the season of the sampling year. Nodata 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 wascompletely opposite the trend found in the market survey, which displayed the lowest nitratecontent in summer organic spinach (Table 3.6). In addition, fresh weight of field-sampled spinachwas also much higher than that of the market samples. In fact, fresh weight of this spinach (33.5grams per plant) was the highest among all summer organic spinach sampled at markets andfields. According to the grower, the field where market sampled spinach was grown was differentfrom the field surveyed. Although they use almost the same fertility practices at both fields, the

21

Table 3.5 Farming practices and nitrate content in spinach, soil, and irrigation water at farmer's fields surveyedFarms O1 [Organic] O1 [Organic] O2 [Organic] O3 [Organic] O5 [Organic]Location King City, CA King City, CA Watsonville, CA Santa Cruz, CA Watsonville, CAField acreage (of spinach) 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)Organically managed 6 years 6 years 13 years 9 years 4 yearsCrop Spinach

(first harvest)aSpinach

(second harvest)aSpinach

(first harvest)aSpinach Spinach

Variety Kerdion RZ Kerdion RZ Space Bossanova Nordic IVGrowth period 46 days 46 days 70 days 44 days about 30 daysSpace between plants 1 to 3 inches 1 to 3 inches 2 - 4 inches about 1 inch about 1 inchNO3 (whole) mg/kg FW 1300 220 470 2200 2700NO3 (leaf-blades) mg/kg FW 920 110 240 1500 1800NO3 (petioles) mg/kg FW 2300 670 1400 4000 5000Fresh weight g/plant 15.6 11.0 18.0 33.5 5.7LPRb FW/FW 3.1 4.1 3.9 2.6 2.6Fertility preplantpractices sidedress[A] or top dress [B]

Compost; 5 t/acre[chicken manure,grape pomace,green waste] c

Compost; 5 t/acre[chicken manure,grape pomace,green waste] c

Compost; 5 to 7t/acre[turkey & cow manure,grape pomace, woodshavings]d

Compostc ; 5 t/acre

Bird guano;300 lbs[B]Foliar feeding [B]

Meat&bone meal;150 lbs/acreSea-bird guano;150 lbs/acre [A]

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

Soil type Sandy loamf Sandy loamf Baywood loamy sand Watsonville loam Pinto loam(0 - 6 pH 1:1 7.1 7.4 6.8 7.0 6.9inches) 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 7.1inches) 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(source) mg/kg

0.40(city water)

1.38 (ground water)

0.36 (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.

22

field not surveyed is located in the valley, which has cooler temperatures and more sandy soilthan the surveyed field (soil type of un-surveyed field is Elder sandy loam). They applied 5tons/acre of compost at pre-planting and 300 lbs/acre of guano as top-dressing, as well as doinga small amount of foliar feeding. Total N application rate was 120 lbs-N/acre. (Table 3.5). Soilanalysis 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 organicfields surveyed. PMN content in this field’s topsoil was as low as 58% of the average of the otherorganic 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 FieldSampling date Aug. 19 Sep. 2 Sep. 23 Aug. 19 Sep. 2 Oct. 7NO3 mg/kg FW 600 620 2200 2500 2900 2700Fresh weight g/plant 17.4 18.5 33.5 6.8 10.4 5.7LPR * FW/FW 3.0 2.7 2.6 1.7 1.6 2.6Moisture % 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 soilwith cooler climate) than the field from which sample was taken in field survey. In O5, both marketand 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 spinachcontained considerably lower nitrates (220 mg/kg) than the first one (1200 mg/kg). It was alsofound that nitrate level in the topsoil under the second harvest spinach was extremely low (0.23NO3-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. Althoughthis sample was first harvest, unfortunately it was collected at the very end of the harvest andmight not be representative of this field. Nitrate content in the topsoil and the sub soil was 13.4and 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 whichappeared to be an acceptable range for spinach growth. Ammonium content was less than 5 mg-N/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 Discussion3.4.1 Lettuce Fields

Although soil type was different between fields, nitrate content in conventional andorganic Romaine lettuce at paired fields was not significantly different. It agreed with the trendand levels of nitrate content in lettuces found in the market survey. The grower sidedressedreadily available organic fertilizers (meat + bone meal and guano) rather than basal application ofcompost for organic Romaine. These fertilizers might cause the comparable nitrate content seenin the organic and conventional plants.

High nitrate content (over 2000 mg/kg) in Romaine lettuce was observed at the O2organic field, although this figure was still lower than the EU safe limit (Table 1.1). Although it wasmarketable size, the lower fresh weight of field samples compared to those of market samplessuggests that field samples were harvested at an earlier age (Table 3.4). Accumulation of nitrateand PMN in soils, which is considered a result of long-term compost applications, may explain a

23

part of this nitrate accumulation in lettuce. High summer temperature stimulates mineralizationand nitrification in soils, and may affect nitrate content in plants. Since the sample was taken froma limited space, this result does not represent the whole O2 field. However, this case shows thepotential 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 beencontinuously applied for more than 10 years.

3.4.2 Spinach FieldsThe 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. Thatis, although spinach grown using guano tended to contain higher nitrate than spinach grownusing compost, this was not always the case. Spinach grown on sandy soils using guano showedthe lowest nitrate concentration among all organic spinach. Although further research is neededto determine the actual factor involved, it is obvious that differences in soil texture may affectnitrate movement in soils, and hence, nitrate content in plants. In other words, growing cropssuch as spinach may require higher levels of readily available nitrogen on fields with sandy thanon loam or clay fields. However, growers should apply readily available fertilizers carefully inorder to avoid nitrate accumulation in soils and plants. Soil nitrate testing may provide usefulinformation to determine whether crops need additional nitrogen.

Leaf blade harvest, which was practiced at O1 and O2, is an effective way to eliminateexcessive nitrate in spinach if such a practice fits with the farm’s marketing strategy. For example,if this method is applied on O3 and O5, which had 2200 and 2700 mg/kg FW of nitrate in wholespinach 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 isrelated to low nitrate content in the soil (Table 3.5). If soil contains a high concentration of nitrateat second harvest due to sidedressing with N fertilizer, mineralization and nitrification from soilnitrogen, or other factors, nitrate content in the second harvest spinach will also be high.

24

4. FIELD EXPERIMENT

4.1 GoalThe 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 ratesof organic fertilizers to produce spinach with acceptable nitrate contents compatible with optimumyields.

4.2 MethodsA 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 Elkhornsandy loam and has been in certified organic production since 1974. The experimental field hadbeen 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)

Layer(depth cm)

pH(H2O)1:1

ElectricalconductivitydS/cm 1:1

NO3-Nmg/kgDW

NH4-Nmg/kgDW

1 ( 0 - 15) 6.3 0.43 59.9 8.652 (15 - 30) 6.4 0.22 19.5 7.303 (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 organicfertilizers was established (Table 4.2). Three types of fertilizers; compost (made at the CASFSFarm 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; 7-7-7 dry analysis, made from guano, bone meal, chicken manure. OF) were tested. As applicationrates, four levels of none (control), standard (87lbs total N/ha, Std), twice the standard (174lbstotal N/ha. 2 x Std), and four-times the standard (348lbs total N/ha. 4 x Std) were established. Atthe Compost + CN plot, Chilean nitrate was sidedressed at a rate of 10% of the total nitrogeninput. At the OF plot, application rates of total N/acre on each level were about half that of theother plots, taking into account the high mineralization rate of organic fertilizers. Total number oftreatments 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 theplots.

Prior to the experiment, weeds were plowed in May 1998. A day after light sprinklerirrigation, compost and organic fertilizers were applied and incorporated by rotor tiller on August25. On August 31, 11 lbs per acre of spinach (Spinacia oleracea), cultivar Daehnfeldt (semi-Savoy type) was seeded by machine. Plant density was adjusted at intervals of 6 inches aftergermination. 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 to15 cm depth) composited from four points per plot (two soils per bed x 2) were taken every week.The samples were refrigerated at 4 °C until analysis. Nitrate and ammonium in soils wereanalyzed 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 weekduring 2 weeks after seeding to harvest. On harvest day, to examine diurnal change of nitratecontent in spinach, 10 plants per plot (5 plants per bed x 2) were sampled both in the morning

25

Table 4.2 Treatments of the field experiment.

# Fertilizer Rate tons/acre[tons/ha]

N % T-N lbs./acre [kg/ha]

1 Control 0 0 02 Compost 5.0 [12] 0.79 87 [98]3 Compost +

CN*4.5 [11]0.054[0.061]

0.7916

78+8.7=87[88+10=98]

4 Organic fertilizer 0.36 [0.89] 5.4 43 [48]5 Compost 10 [25] 0.79 174 [196]6 Compost +

CN*9 [22] +0.11 [0.12]

0.7916

157+17=174[176+20=196]

7 Organic fertilizer 0.72[1.8] 5.4 86 [96]8 Compost 20 [50] 0.79 348 [392]9 Compost +

CN*18 [45] +0.22 [0.24]

0.7916

314+35=348[353+39=392]

10 Organic fertilizer 1.5 [3.6] 5.4 171 [193]* : 10% of nitrogen budget

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 meterzone of each plot were harvested and weighed to measure yield (kg/m2). All the soil and plantsamplings were conducted in the middle 4 meter zone of each plot to avoid areas that might becontaminated by the adjacent plot.

Data were analyzed by ANOVA method (α = 0.05). We used regression analysis toestimate the maximum safe yield of each fertilizer practice, defined as "the maximum yield thatcontained 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 nitratecontent, taking into account its variability.

The relationship between nitrate in soils and spinach was examined using the plantavailable-N method, where plant available-N is defined as N-dressing plus the amount ofavailable N (NO3-N) in the soil at sowing in 0 to 60 cm deep soils (kg-N/ha, Breimer 1982).

10 4

8 3

5 2

6 1

7 9

2 3

1 9

8 7

4 5

6 10

26

4.3 Results4.3.1 Plant Development The average germination rate of spinach was below 50%. Consequently, plant density wasadjusted at intervals of 6 inches, which was far wider than spinach fields surveyed in the previouschapter. Maximum leaf length at the Compost plot increased almost linearly after 2 weeks toharvest. In the Compost + CN plot, changes in maximum leaf length shifted slightly upward afterChilean Nitrate application at 4 weeks after seeding. Development of maximum leaf length at theOF plot was slower than Control plot by 3 weeks after seeding, and even after that, were lowerthan the Compost plot. Especially, 4 x Std. of OF plot had the smallest maximum leaf lengthamong all the fertilized plots through the whole growth period (Figure 4.2). See Figure 4.3 forclimate during the growth period.

Figure 4.2 Changes in maximum leaf length of spinach

Figure 4.3 Climatic condition during field experiment at Santa Cruz, California.On left figure, precipitation (bar), maximum air temperature (bold line), and minimum airtemperature (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)

0

5

10

15

20

0 1 2 3 4 5 6 7

Weeks after seeding

Max

imum

leaf

leng

th (

cm)

Control

Compost Std.

Compost+C.N.Std.Org.Fer. Std.

Compost 2xStd.

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

Compost 4xStd.

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

Chilean NitrateSidedressing

0

40

80

120

8/31/98 9/20/98 10/10/98

Pre

cipi

tatio

n m

m

0

10

20

30

40

Air em

perature C

0

100

200

300

400

8/31/98 9/20/98 10/10/98

Sol

ar R

adia

tion

W/m2

0

10

20

30

40

Soil tem

perature C

27

4.3.2 YieldSpinach 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/acreof compost application increased spinach yield 106% (0.53 kg/m2) and 161% (0.67 kg/m2) ofControl plot (0.26 kg/m2), respectively. However, compost application at 4 x Std rate didn’tincrease the yield from 2 x Std rate. There was no significant difference in yield between anyapplication rates, except control.

Spinach yield in the Compost + CN plot was slightly higher than that of the Compost plotat each rate, although it was not significant. Yields of the Organic Fertilizer plot and Control plotwere significantly lower than those of the Compost plot and Compost + CN plot. Quadratic curveswere selected as regression curves of Compost and Compost + CN plots due to the highersignificance (Figure 4.4).

4.3.3 Fresh Weight and Leaf Blade/Petiole Ratio of SpinachLeaf 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 fertilizedplots also tended to be greater than that of market spinach (Table 4.3).

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

y = 4.43E-09x3 - 9.74E-06x2 + 0.00392x + 0.2550

y = 1.93E-08x3 - 1.76E-05x2 + 0.00500x + 0.2550

0.0

0.2

0.4

0.6

0.8

1.0

0 100 200 300 400

Total N application rate (N lbs/acre)

Yie

ld (k

g/m

2 )

Compost

Com.+ CN

Org. Fert.

Std.

2 x Std.

4 x Std.

Cont.

28

Table 4.3 Fresh weight and leaf blade /petiole ratio of spinach

Treatment Fresh Weightg/plant

LPR*FW/FW

Control 15 5.2Compost 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 ContentNitrate 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).

Figure 4.5 Changes in nitrate content in spinach associated with kind of fertilizers and theirapplication rate as nitrogen. On X axis, from left to right, each nitrogen application ratecorresponds 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 (α = 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 plotwas the lowest among fertilized plots, ranging from 1400 to 1800 mg/kg FW. However, there wasno significant difference in nitrate content of spinach across any type of fertilizers and applicationrates, 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 afresh weight basis and a dry weight basis, respectively. See Figure 4.7 for diurnal changes in

y = -3.16E-06x3 - 0.0124x2 + 6.27x + 1500

y = 1.27E-05x3 - 0.0231x2 + 8.24x + 1500

1000

1500

2000

2500

3000

0 100 200 300 400

Total N application rate (N lbs/acre)

NO

3 (m

g/kg

FW

)

Compost

Com.+ CN

Org. Fert.

29

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

Figure 4.6 Correlation of nitrate content in spinach and its yield.

Table 4.4 Effect of sampling time on nitrate content in spinach

Sampling time Moisture%

Leaf-blade NO3 (mg/kg) FW DW

Petiole NO3 (mg/kg) FW DW

Whole NO3 (mg/kg) FW DW

Morning 89.6 1670 16300 3750 36200 2010 19600Afternoon

(Relative change)88.9

(-0.8%) 1390 12600 (-17%) (-23%)

3840 35800 (+3.1%) (-0.4%)

1820 16500(-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.

Figure 4.7 Changes in solar radiation and air temperature on the harvest day at UC Santa Cruz.Left figure shows diurnal change in solar radiation (W/m2),. Right figure shows changes in air-temperature (degree C). (UCSC Applied Sciences Met Station 1999)

R2 = 0.87***

0

0.2

0.4

0.6

0.8

0 1000 2000 3000

Nitrate content (NO3 mg/kg FW)

Yie

ld (k

g/m

2 )

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

30

0

100

200

300

400

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

N-m

g/kg

0

100

200

300

400

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

N-m

g/kg

0

100

200

300

400

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

N-m

g/kg

0

100

200

300

400

-1 0 1 2 3 4 5 6

WEEKS AFTER PLANTING

N-m

g/kg

0

100

200

300

400

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

N-m

g/kg

0

100

200

300

400

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

N-m

g/kg

0

100

200

300

400

-1 0 1 2 3 4 5 6

WEEKS AFTER PLANTING

N-m

g/kg

0

100

200

300

400

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

N-m

g/kg

0

100

200

300

400

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

N-m

g/kg

0

100

200

300

400

-1 0 1 2 3 4 5 6

WEEKS AFTER PLANTING

N-m

g/kgNH4-N NO3-N

Control

Compost-Std.

Compost-2 x Std.

Compost-4 x Std.

Compost+C.N.-Std.

Compost+C.N.-2 x Std.

Compost+C.N.-4 x Std.

Org. Fert.-Std.

Org. Fert.-2 x Std. Org. Fert.-

4 x Std.

31

4.3.5 Maximum Safe YieldThe 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 particularexperiment, 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 4.4and 4.5).

4.3.6 Nitrate and Ammonium Content in SoilsIn 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 1weeks after seeding. By compost application, nitrate content in the soils increased proportionally to itsapplication rates. That is, at every 5 tons/acre of compost applied, nitrate in the soil increased by about15 mg-N/, which was approximately equivalent to the amount contained in the compost when it wasapplied. At 5 weeks after seeding, since soil samples were taken from the spots where Chilean nitratewas sidedressed, a considerable increase in nitrate was observed, associated with the Chilean nitrateapplication rates. Even after harvest (6 weeks after seeding), soil nitrate content reached 170 mg-N/kgand 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 growthperiod.

In contrast, in the soils in the OF plot, a significant ammonium concentration was detected. Onseeding day, ammonium in the soils accumulated to levels as high as 150 and 300 mg-N/kg at 2 x Stdand 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 NBreimer (1982) demonstrated that nitrate content in spinach was correlated positively with "plant-

available nitrogen" by field experiments and on-farm experiments conducted in the Netherlands. Hereported the critical level of plant-available N at which 10% yield reduction of maximum yield keepingnitrate content in spinach below 2500 mg/kg FW was 225 kg-N/ha.

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

0

1000

2000

3000

0 100 200 300

Plant Available N kg/ha

NO

3 in

spi

nach

mg/

kg F

W

Control

Compost

Compost+CN

Org. Fert.

32

Plant available-N was calculated for the field experiment and was compared with nitrate contentin 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 inspinach 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 lowerthan 2500 mg/kg FW. However, since nitrate content in the maximum yield plot was variable, the criticallevel of plant-available N at which nitrate content can reach 2500 mg/kg FW was considered to bebetween 100 and 150 kg-N/ha in this experiment.

4.4 DiscussionA 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 inspinach was greater in the Compost + CN plot than in the Compost plot. Therefore, the maximum safeyield in the Compost + CN plot was lower than that in the Compost plot when we use "mean + standarderror" as nitrate content. California Certified Organic Farmers (CCOF) restricts application of Chileannitrate to 20% of the total nitrogen budget due to concern over sodium accumulation in soils. SinceChilean nitrate is more soluble than guano, it should be applied carefully to prevent not only sodium buildup in soils but also nitrate accumulation in crops and soils.

The "plant available-N" method warrants further investigation in California. However, it doesn'ttake into account nitrogen mineralization from soil and organic fertilizers. Although it was assumed thatthe growth period of spinach was short enough to neglect those effects, Breimer found that data fromsome farmers’ fields didn't fit well into the correlation because of the history of heavy application ofmanure. In this experiment, the amount of nitrate increased in soils of Compost applied plots mostlycoincided with the amount of nitrate derived from compost when it was applied. From the changes in soilnitrate levels, mineralization of nitrogen in compost during the growth period was considered to benegligible. Thus, we could find a good correlation between plant available-N and nitrate content inspinach, 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 atplanting 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 atthe highest rate (4 x Standard) of Compost applied plots. Future studies, including evaluation of changesin soil physical characteristics at a high rate of compost application, may be needed to demonstrate thereason.

A significant "morning-evening" harvest effect on nitrate content in spinach was found in thisexperiment regardless of slightly cloudy weather on harvest day. This effect is well known on days withfine weather when spinach contains high level of nitrate (Breimer 1982). California receives abundantsolar radiation throughout year. Therefore, afternoon to evening harvest is recommended as a way toreduce 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 contentin spinach. Lorenz (1978) suggested that wider plant density leads to spinach with larger leaf-blades andshorter petioles, and hence lowers nitrate content in a whole plant. If the germination rate had beenhigher and the spinach had been grown at the standard interval of 1 to 2 inches in this experiment, thenitrate content might have been much higher.

In this experiment, a fallowed field was used because no option was available at that time. Due toslow nitrification in the soil, consequently, spinach growth at the OF plot was severely suppressed. Afurther experiment at a cultivated field, with regular nitrification activity in the soil, is desirable. What theresults suggested is a significant effect of nitrification on nitrate content in spinach, in addition to type andapplication rate of organic fertilizers. Although type and rate of nitrogen fertilizers influence the nitratecontent 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 localgrowing conditions.

33

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 contentcultivar, appropriate nitrogen fertilization, application of ammoniacal fertilizer associated with applicationof nitrification inhibitor, split nitrogen application rather than single basal application, utilization of slow-release fertilizer, plant tissue testing, afternoon harvest, petiole removal, and less dense planting. Organicgrowers can use some of those methods (Table 5.1).

Pre-plant soil nitrate testing may provide useful information for determining appropriateapplication rate of organic fertilizers. In that case, mineralization rate of those organic fertilizers also mustbe taken into account (Chaney et al., 1992). Some on-farm soil nitrate testing methods such as the nitratestrip test and electrical conductivity method are available (Sarrantonio et al., 1996). Preliminary results ofthis study suggest that such tests are particularly useful for fields which have been organically managedfor more than 10 years, for growers who wants to use commercial organic certified fertilizers such asguano and Chilean nitrate, and for the fields where residual nitrate from previous crops is considered tobe significant.

In addition, as seen in Chapter 3, soils in organic fields tended to contain greater potentiallymineralizable 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 mineralN, especially during times of reduced crop uptake, could indicate susceptibility to N losses throughleaching. Organically managed soils are sometimes characterized by higher levels of PMN, in conjunctionwith 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 washigh in both nitrate and PMN, suggesting variability of N dynamics across actual organic fields dependingon their fertility management. Although the laboratory incubation method used in this study cannotsimulate field conditions (Rice and Havlin, 1994), PMN in soils, in conjunction with mineral N, appears tobe important for evaluating nitrogen dynamics in organic fields' soils. In situ nitrogen mineralization fromorganic fertilizer and soil may be estimated to some extend using simulation models. For example, adynamic simulation model has been applied successfully to compare productivity and N leaching acrossbiodynamic 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 andSpiers, 1984). On tissue testing in organic vegetable systems, however, Smith (1996) found that nitratecontent in organic onion tissue may not be as good an indicator of crop nitrogen as it is in conventionalsystems. Maynard and Hochmuth (1997) noticed that "organic managed crops may show lower petiole-nitrate (NO3-N) concentrations. Total macro-nutrient concentration of whole leaves is the preferredmethod of evaluating nutrient sufficiency under organic fertility management". Therefore, future studyshould evaluate whether we can apply conventional critical nitrate levels for spinach to organically grownspinach.

Cultivar selection for reducing nitrate content in spinach based on leaf-types appears notconsistent (Maynard et. al., 1976).

Other methods such as compost based fertility management, afternoon to evening harvest, leaf-blades harvest, and less dense planting can also be employed to lower nitrate accumulation, althoughsome of these methods may result in lower yield.

34

Table 5.1 Methods to reduce nitrate concentration in spinach in organic systems

Method Merit Condition and/or demerit

Organic fertilizer application basedon pre-plant soil nitrate testing

• May increase N efficiency• On-farm methods such as EC meter

method, and nitrate strip test areavailable

• Further studies are needed for establishingthreshold values of nitrate and PMN level in soils

• Other local factors such as soil texture, nitrificationactivity of soil, irrigation practice, light environment,and temperature may also affect

Compost based fertilitymanagement

• Safer than readily available nitrogenfertilizers even at high application rate

• Need large quantities• Pre-plant soil nitrate test is recommended for fields

where have been managed organically for years(>10 years?)

Afternoon to evening harvest • 10% reduction (FW) in this fieldexperiment

• 28% reduction (DW) by harvest after 12hr of light instead of 0 hr (Cantliffe1972b)*

• Effective when NO3 in plant is high on fine weather

Petioles removal (leaf-bladeharvest)

• On average, 31% reduction (FW) in thismarket-sampling survey

• 28% (cv. America), and 45% (cv. Hybrid424) reduction (FW) (Olday et al., 1976)*

• Lower yield

* Adopted from Maynard 1978

35

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.

36

ACKNOWLEDGEMENTS

I thank K. Adachi, D. Bowen, L. Jackson, R. Smith and M. Werner for initiating discussion on thistopic and E. Ellis, S. Gliessman, and J. Halliday, for valuable advice and support while the research wasbeing 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 deepappreciation to the growers in farmer's field survey and produce managers of sample markets in marketsampling 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 forAgroecology and Sustainable Food Systems, University of California Santa Cruz provided excellent fieldwork 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 ofthis manuscript. I thankfully acknowledge funding from Organic Farming Research Foundation (OFRF).

37

APPENDIX 1 Toxicity and Regulations of Nitrate in Vegetables

A. Toxicity of nitrateNitrite 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 resultin coma and ultimately death depending on the extent of the hypoxia, which is the inability of red bloodcells to carry oxygen to tissues (Caudill et al., 1990). Particularly, methemoglobinemia can betroublesome in infants under three months of age, although it is inconsequential in adults. Sinios andWodsak (1965) (NRC 1972) reported 15 cases of methemoglobinemia (1 of them fatal) caused by nitrateand nitrite in spinach in European countries during 1959 to 1965. However, no other cases ofmethemoglobinemia due to nitrate and nitrite in vegetables has been reported. In contrast, numerouscases of methemoglobinemia caused by drinking water have been reported. According to Heathwaite etal. (1993), the World Health Organization (WHO) reported 2000 cases of infant methemoglobinemia and160 fatalities caused by drinking water containing more than 25 mgL-1of NO3-N from 1945 to 1985. Nitratepoisoning of groundwater clearly contributes to national infant mortality statistics in the US (Johnson andKross, 1990; Kross et al., 1995).

Many N-nitrosamine compounds have been shown carcinogenic in a range of laboratory animalspecies. Endogenous N-nitrosamine formation has been demonstrated in human volunteers on a diet richin 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, noconclusive epidemiological evidence has been reported on a causal association between nitrate exposureand human cancer risk (Gangolli et al., 1994). One explanation for this contradiction is the fact that mostof the vegetables that are major sources of nitrate also contain vitamin C, which is a strong inhibitor ofendogenous N-nitrosamine formation (Mirvish, 1972). Therefore, consumption of nitrate from sources lowin vitamin C (e. g., water) might be a higher risk than that posed by vitamin-C rich vegetables (Hotchkisset al., 1992).

B. Two ADIs for nitrateThe 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 fora 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 alsoestablished 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 benefitsof vegetables and the lack of data on the possible effects of vegetable matrices on the bioavailability ofnitrate. They considered "it inappropriate to compare exposure to nitrate from vegetables directly with theADI 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 (JECFA1995). Spinach should not be introduced to such infants (Phillips 1968).

Following recommendations and minority point of view of European Environmental ResearchOrganization (EERO) Training and Assessment in 1994 (Gangolli et al., 1994) shows the discrepancy ofopinions among scientists with regard to nitrate toxicity in humans, nitrate intake evaluations, and relatedregulations;Recommendations;1) There is little justification for recommending a drastic reduction in the present levels of nitrate found invegetables.2) The reported mild hypertrophy of the adrenal ozna glomerulosa produced by nitrite treatment in the ratneeds to further investigation with a view to elucidating the relevance of this finding in terms of humanhealth risk.3) Further human studies should be undertaken to delineate the genetic characteristics andenvironmental factors regulating enzyme systems mediating the endogenous syntheses and metabolic

38

disposition of nitrate, nitrite and N-nitroso compounds. This information would be particularly useful foridentifying individuals and population groups likely to be at 'high risk' from an increased body burden andtoxicity of nitrate, nitrite, and N-nitroso compounds.4) Preweaned infants on formulation diets and therefore deriving nitrate mainly from drinking waterconstitute a special 'at risk' group. Every effort should be made to ensure that nitrate levels in drinkingwater supplies do not exceed the EC prescribed limit.Minority point of view;Although current epidemiological data provide conflicting evidence regarding the potential long-termhealth risks of nitrate levels encountered in the diet, it is widely accepted that the reduction of dietarynitrate is a desirable preventive measure. The latter assumption is corroborated by the vast majority ofexperimental data which show that dietary nitrate is a major determinant for in-vivo formation of N-nitrosocompounds. 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 insuch 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 hasbeen investigated (Dykhuizen et al., 1996; Duncan et al., 1997).

C. Risk assessment of nitrate exposure from leafy vegetable consumptionAlthough 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 2500mg/kg FW already exceeds the ADI for NO3 by 13%. Moreover, calculated for NO2 (with 5% nitrateconversion) 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 drinkingwater 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 contentin lettuce and spinach throughout the year were estimated for each farming practice using probabilityfitting 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 ofexceeding 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 asfollows: 93 g /day and 123 g/day for conventional and organic spinach, 202 g/day and 205 g/day forconventional and organic Romaine lettuce, and 262 g/day and 298 g/day for conventional and organicIceberg lettuce, respectively.This showed the average amount (g/day) that could reach ADI for nitrate by eating only lettuce or spinachas salad. This example suggests that "organic only" consumer can eat 30g/day more of spinach orIceberg 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 lettuceeveryday. NRC (1981) estimated that nitrate intake from lettuce and spinach accounts 39% of total nitrateintake. For real assessment, therefore, nitrate content in all other sources must be taken into account, aswell as their average daily consumption amount.

Appendix 1.1 Probability distribution of nitrate content in conventional and organicvegetables purchased at Santa Cruz, California, 1998.

IB; Iceberg lettuce, RM; Romain lettuce, SP; spinach, CONV; conventional, ORG; organic.

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

footnote of Appendix 1.1 for legend.

39

0.00

0.25

0.50

0 2500 5000

NO3 mg/kg fresh weight

Pro

babi

lity IB_CONV

IB_ORG

RM_CONV

RM_ORG

SP_CONV

SP_ORG

0%

25%

50%

75%

100%

0 100 200 300 400 500

Consumption amount (grams/day)

Pro

babi

lity

of e

xcee

ding

AD

I for

N

O3

(%) IB_CONV

IB_ORGRM_CONV

RM_ORGSP_CONV

SP_ORG

40

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

Vegetable Germany(Guide)

Netherlands(Maximum)

Switzerland(Guide)

Austria(Maximum)

Russia(Maximum)

EC(Maximum)

Lettuce 3000 3000(S) 3500 3000(S) 2000(O) 3500(4-10)4500(W) 4000(W) 3000(G) 4500(11-3)

2500(O,5-8)Spinach 2000 3500(S) 3500 2000(<7) 2000(O) 2500(4-10)

4500(W) 3000(>7) 3000(G) 3000(11-3)2500(1995) 2000(P)

Red beet 3000 4000(4-6) 3000 3500(S) 14000(O)3500(7-3) 4500(W)

Radish 3000 3500(S)4500(W)

Endive 3000(S) 2500(S)Cabbage 875 1500 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 bythe end of June. >7:harvest from July. 1995: from 1995. 4-10:1 April to 31 October. 11-3:1 November to 31March. 5-8:1 May to 31 August.Data from Scharpf 1991(cited from Sohn and Yoneyama 1996) and MAFF UK (1999).

41

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

Factor Nitrate content in spinachLow - High Mechanism and/or condition

Environmental Light High - Low Nitrate reductase in plant requires light energy to be active

Temperature Low - High Stimulation of N mineralization and nitrification in soil and respiration ofplant at high temperature

Water stress Low - High Indirect effect on nitrate reductase activity by water stress

Season Spring to summer - Fall to winter Low light intensity and short duration of daytime in fall and winter

Soil characteristics Low PMN* - High PMN

Slow nitrification - Fast nitrificationLow NO3 - High NO3

Higher PMN in soil provides more NH4, hence NO3 to plantFaster nitrification in soil provides NO3 to plant soonerHigher NO3 in soil provides more NO3 to plant

Location Low latitude - High latitude Outdoor - Greenhouse

Less solar radiation at higher latitude in fall and winterLow light intensity and warm temperature in greenhouse

Practical N application rate Low - High High N application causes high NO3 in plants

N form Slow release - Soluble Slow release N fertilizers provide NO3 to plants gradually

Nitrification inhibitor High - Low Limited supply of NO3 to plant by inhibiting nitrification in soil

N application timing

Basal dressing - Side dressing Side dressing - Basal dressing

N sidedressing prior to harvest may increase NO3 in plants when it isexcessive. Reverse also may happen if basal application is excessive.

K application Low - High Stimulation of plant growth by K application

Cl application High - Low Antagonistic effect of Cl- on NO3 - suppresses NO3

-absorption by plant

Variety Smooth type - Savoy type Not always but some smooth types have higher nitrate reductase activitythan Savoy types

Herbicides Low - High Cycloate, alachlor, and lenacil decrease some phase of nitrate reduction

Plant age Pre mature - Fully matureFully mature - Pre mature

NO3 in plant increases with age when NO3 in soil is sufficient to excessNO3 in plant decreases with age when NO3 in soil is optimum or deficient

Time of harvest Evening - Morning Nitrate is reduced during daytime. It is effective especially when plantcontains high NO3 under fine and cool weather

Planting density Thin - Dense Leaf blade / petiole ratio will decrease when plant is seeded densely

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.

42

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 suchas cultivars (Cantliffe, 1973a; Barker et al., 1974; Maynard and Barker, 1974; Olday et al., 1976), nitrogenapplication (Brown and Smith, 1966, 1967; Barker and Maynard, 1971; Lorenz, 1978; Greenwood andHunt, 1986), potassium application (Regan et al., 1968), nitrification inhibitor (Mills et el., 1976; Bakr andGawish, 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 havebeen identified as the major factors which influence nitrate levels in vegetables (Cantliffe, 1973b). Inparticular, light intensity and nitrate content in soils before or at harvest are known to be critical factors indetermining nitrate levels in spinach (Schuphan et al., 1967).

In hydroponic systems, cultivation method for growing low-nitrate lettuce and spinach using pre-harvest transfer to N-free (Mozafar, 1996) or N-reduced solution (Andersen and Nielsen, 1992) has beenput into practice. One night period of supplement lighting prior to harvest to reduce shoot nitrate contentswas demonstrated to be effective in hydroponic greenhouse-grown spinach in the winter months inGerman (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.

43

APPENDIX 4 List of Sampling Markets in Santa Cruz County*

Certified Farmers MarketMarket Address Opening1. Santa Cruz City Parking Lot Wed. 2:30pm-6:30pm

Natural Foods RetailersRetailer Address TEL1. Aptos Natural Foods 7506 Soquel Dr. AP 685-33342. New Leaf (Capitola) 1210 41st. Av. CP 479-79873. New Leaf (Mission) 2351 Mission St. SC 426-13064. New Leaf (Downtown) 1134 Pacific Av. SC 425-17935. Stapleton's of Santa Cruz 415 River SC 425-58886. Food Bin 1130 Mission St. SC 423-55267. Staff of Life 1305 Water St. SC 423-8632

General Grocery MarketsRetailer Address TEL1. Deluxe Food of Aptos 783 Rio Del Mar Bl. AP 688-74422. Albertson’s Food Centers 1710 41st Av. CP 476-17173. Lucky Food Center 1475 41st Av. CP 462-69174. Safeway 16 Rancho Del Mar 688-2775

Shopping Ctr. AP5. Safeway 2650 41st Av. SQ 475-18976. Nob Hill Foods 222 Mount Hermon Rd. SV 438-16107. Zanotto's family Markets 14 Victor Sq. SV 438-43248. Safeway 253 Mount Hermon Rd. SV 438-74319. Safeway 6255 Graham Hill Rd. FT 335-353210. Shoppers Corner 622 Soquel Av. SC 423-139811. Zanotto's family Markets 700 Front SC 423-499412. Lucky Food Center 911 Soquel Av. SC 426-685213. Safeway 2111 Mission St. SC 429-981114. Safeway 117 Morrissey Bl. SC 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.

APPENDIX 5.1 Data and Information about Lettuce Sub-samples

Vegetable Season Practice Date # Grower Producing area Market Days after Refrigerator Rack Price Wrappingreceived degree C degree C $/plant

Green-leaf Winter Organic 21-Jan-98 1 Op4 Holtville, CA Or4 1.0 5.0 10.5 $1.69 None2 Op7 n.a. Or6 0.0 $1.80 None

Organic total 0.5 $1.75Winter total 0.5 $1.75

Green-leaf total 0.5 $1.75Iceberg Winter Conventional 21-Jan-98 1 Cp4 Yuma, AZ Cr10 0.0 6.1 7.2 - 10 $0.99 None

2 Cp3 Yuma, AZ Cr2 0.0 4.4 0.6 $1.29 Film3 Cp6 Yuma, AZ Cr13 1.0 1.7 - 3.3 1.7 - 3.3 $1.39 Film

2-Feb-98 4 Cp4 Yuma, AZ Cr18 0.0 9.4 11.1 $0.79 None5 Cp9 n.a. Cr11 0.0 3.3 7.2 $0.49 Net6 Cp6 Yuma, AZ Cr13 0.0 1.7 - 3.3 1.7 - 3.3 $0.79 Film

Conventional total 0.2 $0.96Organic 21-Jan-98 1 Op4 Holtville, CA Or4 5.0 5.0 10.5 $2.69 Film

2-Feb-98 2 Op9 El Centro, CA Or2 2.0 3.3 - 5.6 3.3 - 5.6 $1.49 Film3 Op8 El Centro, CA Or5 2.0 7.8 3.3 - 4.4 $1.49 Film4 Op4 Holtville, CA Or4 3.0 5.0 10.5 $1.39 Film

Organic total 3.0 $1.77Winter total 1.3 $1.28Summer Conventional 24-Aug-98 1 Cp19 n.a. Cr11 3.0 3.3 7.2 $0.49 Net

2 Cp11 Salinas, CA Cr3 0.0 4.4 $0.89 Film3 Cp4 Salinas, CA Cr18 0.0 9.4 11.1 $0.79 None

8-Sep-98 4 Cp19 n.a. Cr13 1.0 1.7 - 3.3 1.7 - 3.3 $0.79 Film5 Cp20 Watsonville, CA Cr10 1.0 6.1 7.2 - 10 $0.69 None6 Cp4 Salinas, CA Cr2 1.0 4.4 0.6 $0.99 Film

Conventional total 1.0 $0.77Organic 24-Aug-98 1 Op13 Watsonville, CA Or5 3.0 7.8 3.3 - 4.4 $1.29 None

5 Op16 Santa Cruz, CA Or8 4.0 $0.65 Film6 Op13 Watsonville, CA Or2 2.0 3.3 - 5.6 3.3 - 5.6 $1.49 None

8-Sep-98 2 Op15 Hollister, CA Or5 4.0 7.8 3.3 - 4.4 $1.29 Film3 Op16 Santa Cruz, CA Or8 5.0 $1.29 Film4 Op16 Santa Cruz, CA Or1 0.0 $0.99 Film

Organic total 3.0 $1.17Summer total 2.0 $0.97

Conventional total 0.6 $0.87Organic total 3.0 $1.41Iceberg total 1.7 $1.11Romain Winter Conventional 21-Jan-98 1 Cp5 Thermal, CA Cr10 0.0 6.1 7.2 - 10 $0.99 None

2 Cp4 Yuma, AZ Cr2 0.0 4.4 0.6 $1.39 None3 Cp1 Oxnard, CA Cr13 1.0 1.7 - 3.3 1.7 - 3.3 $0.79 None

4-Feb-98 4 Cp11 Yuma, AZ Cr3 1.0 4.4 $0.79 None5 Cp10 El Centro (or Oxnard) Cr13 1.0 1.7 - 3.3 1.7 - 3.3 $0.69 None6 Cp8 Thermal, CA Cr11 1.0 3.3 7.2 $0.99 None

Conventional total 0.7 $0.94Organic 21-Jan-98 1 Op5a Oxnard, CA Or4 5.0 5.0 10.5 $1.69 None

2 Op6 Gilroy, CA Fm1 $1.25 None3 Op5b Oxnard, CA Or6 0.0 $1.66 None

4-Feb-98 4 Op5b Oxnard, CA Or2 1.0 3.3 - 5.6 3.3 - 5.6 $1.49 None5 Op5a Oxnard, CA Or6 0.0 $1.09 None6 Op7 n.a. Or5 1.0 7.8 3.3 - 4.4 $1.49 None

Organic total 1.4 $1.45Winter total 1.0 $1.19Summer Conventional 26-Aug-98 1 Cp18 Soledad, CA Cr5 0.0 4.4 10.6 $0.69 None

2 Cp4 Salinas, CA Cr2 2.0 4.4 0.6 $0.59 None3 Cp12 Salinas or Pajaro Valley, CACr10 1.0 6.1 7.2 - 10 $0.39 None

9-Sep-98 4 Cp21 n.a. Cr11 1.0 3.3 7.2 $1.29 None5 Cp11 Salinas, CA Cr3 0.0 4.4 $0.69 None6 Cp23 n.a. Cr18 1.0 9.4 11.1 $0.89 None

Conventional total 0.8 $0.76Organic 26-Aug-98 1 Op2 Greenfields, CA Fm1 $1.00 None

2 Op11 Santa Cruz, CA Fm1 $0.75 None3 Op12 Watsonville, CA Fm1 $0.85 None

9-Sep-98 4 Op17 Davenport, CA Fm1 $0.55 None5 Op12 Watsonville, CA Fm1 $0.85 None6 Op2 Greenfields, CA Fm1 $0.80 None

Organic total $0.80Summer total 0.8 $0.78

Conventional total 0.8 $0.85Organic total 1.4 $1.12Romain total 0.9 $0.99Grand conventional total 0.7 $0.86Grand organic total 2.6 $1.24Grand winter total 1.1 $1.28Grand summer total 1.6 $0.90Grand total 1.3 $1.11

44

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

Vegetable Season Practice Date # Grower Market n NO3 mg/kg FW Moisture %Av. Min. Max. C.V.* Av. Min. Max. C.V.*

Green-leaf Winter Organic 21-Jan-98 1 Op4 Or4 5 792 670 960 14.8 93.7 93.0 95.0 0.792 Op7 Or6 5 780 670 880 9.72 93.0 92.6 93.4 0.33

Organic total 10 786 670 960 11.9 93.4 92.6 95.0 0.71Winter total 10 786 670 960 11.9 93.4 92.6 95.0 0.71

Green-leaf total 10 786 670 960 11.9 93.4 92.6 95.0 0.71Iceberg Winter Conventional 21-Jan-98 1 Cp4 Cr10 5 962 850 1100 9.73 95.3 94.9 95.6 0.32

2 Cp3 Cr2 5 1100 1000 1200 6.43 95.8 95.1 96.6 0.663 Cp6 Cr13 5 1010 930 1200 10.9 95.6 95.4 96.0 0.25

2-Feb-98 4 Cp4 Cr18 5 870 570 1000 20.3 94.7 93.4 95.4 0.825 Cp9 Cr11 5 908 820 1000 8.72 95.6 95.2 95.9 0.316 Cp6 Cr13 5 976 910 1100 8.09 95.1 94.4 96.3 0.76

Conventional total 30 970 570 1200 12.7 95.3 93.4 96.6 0.65Organic 21-Jan-98 1 Op4 Or4 5 900 730 1000 12.0 96.1 95.2 96.7 0.60

2-Feb-98 2 Op9 Or2 5 942 830 1000 8.27 95.3 94.9 95.7 0.393 Op8 Or5 5 1300 1200 1400 5.44 95.5 94.4 96.3 0.764 Op4 Or4 5 764 620 890 13.1 95.1 93.9 95.6 0.76

Organic total 20 977 620 1400 22.5 95.5 93.9 96.7 0.72Winter total 50 973 570 1400 17.1 95.4 93.4 96.7 0.68Summer Conventional 24-Aug-98 1 Cp19 Cr11 5 774 730 840 5.30 95.7 95.2 96.3 0.47

2 Cp11 Cr3 5 1090 940 1200 8.58 96.2 95.8 96.6 0.403 Cp4 Cr18 5 516 410 690 24.4 95.9 95.6 96.5 0.36

8-Sep-98 4 Cp19 Cr13 5 680 630 740 6.15 96.7 95.6 97.5 0.775 Cp20 Cr10 5 564 470 670 15.7 96.1 95.4 96.8 0.546 Cp4 Cr2 5 622 480 820 21.8 97.1 96.3 97.4 0.45

Conventional total 30 707 410 1200 29.8 96.3 95.2 97.5 0.67Organic 24-Aug-98 1 Op13 Or5 5 484 330 580 21.3 96.2 95.7 96.8 0.46

5 Op16 Or8 5 664 610 730 7.94 96.3 95.7 97.3 0.696 Op13 Or2 5 614 580 680 6.47 96.3 95.6 96.8 0.49

8-Sep-98 2 Op15 Or5 5 484 360 560 15.8 97.2 96.5 97.6 0.403 Op16 Or8 5 574 500 760 18.4 97.8 97.5 98.1 0.254 Op16 Or1 5 632 490 750 17.6 96.7 96.0 97.3 0.65

Organic total 30 575 330 760 18.4 96.8 95.6 98.1 0.76Summer total 60 641 330 1200 27.8 96.5 95.2 98.1 0.75

Conventional total 60 839 410 1200 25.8 95.8 93.4 97.5 0.83Organic total 50 736 330 1400 34.6 96.3 93.9 98.1 0.97Iceberg total 110 792 330 1400 30.2 96.0 93.4 98.1 0.92Romain Winter Conventional 21-Jan-98 1 Cp5 Cr10 5 1160 1100 1300 7.71 93.7 93.4 93.9 0.23

2 Cp4 Cr2 5 1060 1000 1100 5.17 95.1 94.6 96.2 0.693 Cp1 Cr13 5 982 940 1000 2.73 94.6 94.3 94.9 0.25

4-Feb-98 4 Cp11 Cr3 5 1200 1100 1300 5.89 94.5 94.1 94.9 0.405 Cp10 Cr13 5 888 840 950 5.71 94.7 93.9 95.3 0.586 Cp8 Cr11 5 914 880 980 4.35 95.0 94.5 95.9 0.60

Conventional total 30 1030 840 1300 12.6 94.6 93.4 96.2 0.66Organic 21-Jan-98 1 Op5a Or4 5 1460 1400 1600 6.13 94.8 94.5 95.1 0.26

2 Op6 Fm1 5 1280 1200 1400 6.54 94.7 93.8 95.3 0.603 Op5b Or6 5 1340 1200 1500 8.51 93.4 92.8 94.0 0.49

4-Feb-98 4 Op5b Or2 5 1270 950 1600 20.0 93.3 92.3 93.9 0.685 Op5a Or6 5 862 730 1000 15.4 93.9 93.3 94.2 0.386 Op7 Or5 5 826 720 930 11.6 93.7 93.2 94.1 0.40

Organic total 30 1170 720 1600 23.6 94.0 92.3 95.3 0.77Winter total 60 1100 720 1600 20.4 94.3 92.3 96.2 0.79Summer Conventional 26-Aug-98 1 Cp18 Cr5 5 1040 990 1100 5.65 94.9 94.1 95.8 0.79

2 Cp4 Cr2 5 774 540 1100 26.7 95.3 95.0 96.0 0.433 Cp12 Cr10 5 878 780 1000 9.20 94.9 93.6 95.5 0.76

9-Sep-98 4 Cp21 Cr11 5 1320 1200 1400 8.30 95.9 95.4 96.5 0.475 Cp11 Cr3 5 1110 870 1300 14.3 95.2 94.6 95.6 0.436 Cp23 Cr18 5 1720 1500 1900 8.62 93.2 92.7 93.7 0.47

Conventional total 30 1140 540 1900 29.9 94.9 92.7 96.5 1.1Organic 26-Aug-98 1 Op2 Fm1 5 800 460 960 24.5 91.4 87.8 95.1 3.7

2 Op11 Fm1 5 582 470 770 20.6 90.7 86.8 93.7 2.93 Op12 Fm1 5 1210 450 1700 40.9 94.9 94.6 95.2 0.23

9-Sep-98 4 Op17 Fm1 5 708 480 1000 31.5 94.8 94.4 95.1 0.305 Op12 Fm1 5 1580 1300 1700 10.4 95.9 95.4 97.3 0.826 Op2 Fm1 5 846 720 960 12.0 95.7 95.3 96.2 0.34

Organic total 30 954 450 1700 43.5 93.9 86.8 97.3 2.8Summer total 60 1050 450 1900 37.0 94.4 86.8 97.3 2.2

Conventional total 60 1090 540 1900 24.0 94.7 92.7 96.5 0.89Organic total 60 1060 450 1700 34.5 93.9 86.8 97.3 2.1Romain total 120 1080 450 1900 29.5 94.3 86.8 97.3 1.6Grand conventional total 120 963 410 1900 28.0 95.3 92.7 97.5 1.0Grand organic total 120 904 330 1700 38.3 94.9 86.8 98.1 2.0Grand winter total 120 1020 570 1600 21.0 95.5 86.8 98.1 2.0Grand summer total 120 844 330 1900 43.0 94.7 92.3 96.7 1.0Grand total 240 933 330 1900 33.3 95.1 86.8 98.1 1.6*: C.V.= standard diviation / average x 100

45

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

Vegetable Season Practice Date # Grower Market n Fresh weight g/plant Non edible part % FWAv. Min. Max. C.V.* Av. Min. Max. C.V.*

Green-leaf Winter Organic 21-Jan-98 1 Op4 Or4 5 301 258 363 15.3 4.7 3.9 5.5 142 Op7 Or6 5 196 156 226 14.4 3.5 2.8 4.0 16

Organic total 10 249 156 363 26.4 4.1 2.8 5.5 21Winter total 10 249 156 363 26.4 4.1 2.8 5.5 21

Green-leaf total 10 249 156 363 26.4 4.1 2.8 5.5 21Iceberg Winter Conventional 21-Jan-98 1 Cp4 Cr10 5 484 406 575 15.8 1.9 1.7 2.3 11

2 Cp3 Cr2 5 635 571 759 11.7 2.1 1.7 2.5 163 Cp6 Cr13 5 589 537 661 9.25 2.0 1.5 2.4 19

2-Feb-98 4 Cp4 Cr18 5 528 352 669 26.2 1.9 1.4 2.6 245 Cp9 Cr11 5 590 475 742 19.1 2.0 1.4 2.6 226 Cp6 Cr13 5 612 483 853 25.5 2.9 2.5 3.2 9.0

Conventional total 30 573 352 853 19.5 2.1 1.4 3.2 23Organic 21-Jan-98 1 Op4 Or4 5 627 571 709 9.74 2.5 1.8 3.2 24

2-Feb-98 2 Op9 Or2 5 491 404 587 14.9 2.0 1.2 2.5 283 Op8 Or5 5 640 505 741 13.6 1.9 1.6 2.2 134 Op4 Or4 5 605 439 727 18.3 2.1 1.9 2.6 13

Organic total 20 591 404 741 16.7 2.1 1.2 3.2 23Winter total 50 580 352 853 18.2 2.1 1.2 3.2 23Summer Conventional 24-Aug-98 1 Cp19 Cr11 5 705 585 821 14.9 4.0 2.3 5.3 33

2 Cp11 Cr3 5 778 674 831 7.78 4.0 3.7 4.6 8.33 Cp4 Cr18 5 570 384 744 25.3 5.8 4.8 7.4 17

8-Sep-98 4 Cp19 Cr13 5 826 630 918 14.2 4.9 3.4 6.1 205 Cp20 Cr10 5 491 361 758 33.3 4.0 3.2 5.4 236 Cp4 Cr2 5 842 698 1110 19.2 4.4 3.5 6.1 23

Conventional total 30 702 361 1110 25.4 4.5 2.3 7.4 25Organic 24-Aug-98 1 Op13 Or5 5 570 473 668 16.0 4.4 2.8 6.7 33

5 Op16 Or8 5 635 552 724 10.3 4.5 3.1 4.9 176 Op13 Or2 5 397 253 617 34.5 4.3 2.4 5.9 32

8-Sep-98 2 Op15 Or5 5 804 565 926 19.2 4.4 3.3 5.2 173 Op16 Or8 5 681 582 842 15.4 5.4 4.3 6.6 184 Op16 Or1 5 665 556 779 15.9 5.3 4.6 5.7 8.0

Organic total 30 625 253 926 26.1 4.7 2.4 6.7 22Summer total 60 664 253 1110 26.2 4.6 2.3 7.4 23

Conventional total 60 638 352 1110 25.3 3.3 1.4 7.4 45Organic total 50 611 253 926 23.0 3.7 1.2 6.7 41Iceberg total 110 626 253 1114 24.3 3.5 1.2 7.4 43Romain Winter Conventional 21-Jan-98 1 Cp5 Cr10 5 427 365 494 14.1 3.8 3.3 4.4 15

2 Cp4 Cr2 5 482 327 695 34.0 3.7 2.8 4.2 163 Cp1 Cr13 5 340 244 462 25.6 3.9 3.0 5.3 22

4-Feb-98 4 Cp11 Cr3 5 514 340 606 20.3 5.2 2.9 6.9 305 Cp10 Cr13 5 409 262 518 26.3 5.3 3.6 7.4 276 Cp8 Cr11 5 578 468 688 17.6 6.1 5.7 6.8 7.6

Conventional total 30 458 244 695 27.5 4.7 2.8 7.4 28Organic 21-Jan-98 1 Op5a Or4 5 415 344 544 18.3 5.0 3.1 6.1 24

2 Op6 Fm1 5 576 402 654 18.0 7.0 5.9 7.7 133 Op5b Or6 5 385 276 456 18.2 4.5 3.8 5.7 16

4-Feb-98 4 Op5b Or2 5 270 217 323 15.4 4.2 3.9 4.8 8.05 Op5a Or6 5 321 285 359 10.2 4.2 3.3 4.7 146 Op7 Or5 5 282 220 419 28.1 5.3 2.9 6.9 28

Organic total 30 375 217 654 33.1 5.0 2.9 7.7 26Winter total 60 417 217 695 31.4 4.9 2.8 7.7 27Summer Conventional 26-Aug-98 1 Cp18 Cr5 5 624 527 710 12.2 9.4 9.0 10 3.8

2 Cp4 Cr2 5 452 394 523 10.9 9.5 6.9 12 223 Cp12 Cr10 5 451 373 508 13.1 9.2 8.0 10 8.6

9-Sep-98 4 Cp21 Cr11 5 542 474 592 8.32 9.4 8.7 10 7.65 Cp11 Cr3 5 555 321 660 24.3 11 8.2 13 166 Cp23 Cr18 5 344 300 383 11.5 8.9 8.0 9.8 8.6

Conventional total 30 495 300 710 23.1 9.5 6.9 13 13Organic 26-Aug-98 1 Op2 Fm1 5 603 558 649 5.61 11 11 12 4.3

2 Op11 Fm1 5 431 378 485 10.9 12 11 13 5.73 Op12 Fm1 5 349 295 409 12.8 11 10 13 9.4

9-Sep-98 4 Op17 Fm1 5 304 287 339 6.72 15 14 17 7.65 Op12 Fm1 5 444 337 483 13.7 9.2 7.9 9.8 8.36 Op2 Fm1 5 732 554 921 18.9 11 10 12 4.3

Organic total 30 477 287 921 34.1 12 7.9 17 17Summer total 60 486 287 921 28.7 11 6.9 17 18

Conventional total 60 476 244 710 25.3 7.1 2.8 13 39Organic total 60 426 217 921 35.8 8.3 2.9 1.7 44Romain total 120 451 217 921 30.8 7.7 2.8 17 43Grand conventional total 120 557 244 1110 29.3 5.2 1.4 13 56Grand organic total 120 489 156 926 37.3 6.0 1.2 17 60Grand winter total 120 471 156 853 33.0 3.7 1.2 7.7 45Grand summer total 120 575 253 1110 31.4 7.6 2.3 17 44Grand total 240 523 156 1110 33.6 5.6 1.2 17 59*: C.V.= standard diviation / average x 100

46

APPENDIX 5.2 Data and Information about Spinach Sub-samples

Season Practice Date # Grower Producing area Market Days after Refrigerator Rack Pricereceived degree C degree C $/bunch

Winter Conventional 27-Jan-98 1 Cp4 Yuma, AZ Cr10 1 7.2 - 10 6.1 $0.792 Cp7 Coachelle, CA Cr2 1 4.4 0.6 $1.293 Cp8 Thermal, CA Cr11 1 3.3 7.2 $0.99

9-Feb-98 4 Cp1 Oxnard, CA Cr5 1 4.4 10.6 $0.595 Cp13 Mexicali, Mexico Cr13 0 1.7 - 3.3 1.7 - 3.3 $0.59

14-Feb-98 6 Cp14 Oxnard, CA Cr13 0 1.7 - 3.3 1.7 - 3.3 $0.59Conventional total 0.7 $0.81Organic 27-Jan-98 1 Op5 Oxnard, CA Or5 0 7.8 3.3 - 4.4 $1.49

2 Op7 n.a. Or7 3 5.6 5.6 $1.993 Op3 Coachelle, CA Or4 4 5.0 10.5 $1.99

9-Feb-98 4 Op4 Holtville, CA Or2 0 3.3 - 5.6 3.3 - 5.6 $1.595 Op5a Oxnard, CA Or5 2 7.8 3.3 - 4.4 $1.196 Op3 Coachelle, CA Or7 2 5.6 5.6 $1.09

Organic total 1.8 $1.56Winter total 1.3 $1.18Summer Conventional 19-Aug-98 1 Cp15 Salinas, CA Cr10 3 7.2 - 10 6.1 $0.99

2 Cp7 Salinas, CA Cr2 4 4.4 0.6 $0.953 Cp16 n.a. Cr3 2 4.4 $0.89

2-Sep-98 4 Cp7 Salinas, CA Cr11 2 3.3 7.2 $0.995 Cp1 Oxnard, CA Cr5 0 4.4 10.6 $0.796 Cp16 n.a. Cr2 1 4.4 0.6 $0.99

Conventional total 2.0 $0.93Organic 19-Aug-98 1 Op11 Santa Cruz, CA Fm1 $0.75

2 Op6 Gilroy, CA Fm1 $1.003 Op10 Watsonville, CA Or7 3 5.6 5.6 $1.29

2-Sep-98 4 Op11 Santa Cruz, CA Fm1 $0.505 Op6 Gilroy, CA Fm1 $1.006 Op10 Watsonville, CA Or7 3 5.6 5.6 $1.19

Organic total 3.0 $0.96Summer total 2.5 $0.94Conventional total 1.3 $0.87Organic total 2.1 $1.26Grand total 1.7 $1.06

47

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

Season Practice Date # Grower Market n NO3 mg/kg whole plant FW Moisture % whole plantAv. Min. Max. C.V.* Av. Min. Max. C.V.*

Winter Conventional 27-Jan-98 1 Cp4 Cr10 5 2840 2400 3400 14.2 92.2 91.8 92.5 0.302 Cp7 Cr2 5 2220 2100 2300 3.77 92.5 92.1 92.7 0.243 Cp8 Cr11 5 1460 1200 1700 12.4 93.0 92.7 93.3 0.25

9-Feb-98 4 Cp1 Cr5 5 2200 2100 2400 5.57 93.1 92.7 93.6 0.365 Cp13 Cr13 5 2880 2400 3200 10.2 91.2 90.7 91.7 0.49

14-Feb-98 6 Cp14 Cr13 5 1780 1500 2600 26.8 92.2 91.5 92.7 0.51Conventional total 30 2230 1200 3400 26.4 92.4 90.7 93.6 0.77Organic 27-Jan-98 1 Op5 Or5 5 1740 900 2400 31.6 91.1 90.3 91.6 0.57

2 Op7 Or7 5 1540 1300 1700 11.8 91.4 91.3 91.6 0.153 Op3 Or4 5 890 310 1200 41.2 90.1 88.9 91.2 1.01

9-Feb-98 4 Op4 Or2 5 2640 2200 2900 10.9 92.2 90.1 94.2 1.655 Op5a Or5 5 1680 680 2500 50.0 90.9 90.2 92.4 1.076 Op3 Or7 5 2300 2000 2800 13.7 92.2 91.6 93.1 0.73

Organic total 30 1800 310 2900 39.7 91.3 88.9 94.2 1.21Winter total 60 2010 310 3400 34.0 91.8 88.9 94.2 1.16Summer Conventional 19-Aug-98 1 Cp15 Cr10 5 3180 2400 4100 20.8 94.1 93.9 94.4 0.20

2 Cp7 Cr2 5 2700 2400 3000 11.1 94.7 94.6 95.0 0.193 Cp16 Cr3 5 2960 2400 3700 16.0 92.8 92.5 93.3 0.35

2-Sep-98 4 Cp7 Cr11 5 3400 3000 3700 7.50 93.7 93.0 94.4 0.545 Cp1 Cr5 5 2040 990 3200 49.4 93.6 93.4 94.0 0.316 Cp16 Cr2 5 2840 2000 4000 25.7 93.6 93.1 94.6 0.64

Conventional total 30 2850 990 4100 25.2 93.8 92.5 95.0 0.73Organic 19-Aug-98 1 Op11 Fm1 5 592 200 1300 81.8 91.2 89.9 92.2 1.02

2 Op6 Fm1 5 1360 820 2000 31.7 93.1 92.8 93.6 0.363 Op10 Or7 5 2460 1400 3500 30.4 94.4 94.0 94.8 0.30

2-Sep-98 4 Op11 Fm1 5 620 130 900 59.8 91.8 91.5 92.2 0.315 Op6 Fm1 5 2860 2700 3100 6.35 94.4 93.9 94.6 0.326 Op10 Or7 5 3000 2800 3300 7.07 94.3 94.2 94.3 0.05

Organic total 30 1820 130 3500 60.5 93.2 89.9 94.8 1.49Summer total 60 2330 130 4100 45.3 93.5 89.9 95.0 1.20Conventional total 60 2540 990 4100 28.5 93.1 90.7 95.0 1.05Organic total 60 1810 130 3500 50.8 92.3 88.9 94.8 1.69Grand total 120 2170 130 4100 41.5 92.7 88.9 95.0 1.47

Season Practice Date # Grower Market n Fresh weight g/plant Leafblade/petiole ratio FW/FWAv. Min. Max. C.V.* Av. Min. Max. C.V.*

Winter Conventional 27-Jan-98 1 Cp4 Cr10 5 12.8 10.8 15.2 13.5 1.2 1.2 1.2 2.82 Cp7 Cr2 5 12.2 11.3 13.0 6.14 1.7 1.7 1.8 1.53 Cp8 Cr11 5 11.1 7.0 14.6 26.1 1.4 1.2 1.6 11

9-Feb-98 4 Cp1 Cr5 5 22.9 15.4 32.6 27.7 1.5 1.4 1.7 7.15 Cp13 Cr13 5 29.5 20.4 48.1 36.9 1.3 1.2 1.5 8.1

14-Feb-98 6 Cp14 Cr13 5 32.1 13.8 42.8 40.7 1.9 1.4 2.3 19Conventional total 30 20.1 7.0 48.1 55.0 1.5 1.2 2.3 20Organic 27-Jan-98 1 Op5 Or5 5 9.18 6.7 11.7 21.0 2.4 2.1 2.5 8.8

2 Op7 Or7 5 25.2 12.8 35.4 35.9 1.9 1.7 2.2 113 Op3 Or4 5 9.86 7.8 11.8 15.4 1.7 1.6 1.9 8.8

9-Feb-98 4 Op4 Or2 5 24.6 14.3 33.3 28.6 1.4 1.3 1.5 6.35 Op5a Or5 5 14.8 11.8 17.4 14.4 1.9 1.6 2.3 146 Op3 Or7 5 14.9 12.3 17.8 13.6 1.4 1.3 1.8 14

Organic total 30 16.4 6.7 35.4 48.1 1.8 1.3 2.5 21Winter total 60 18.3 6.7 48.1 53.2 1.6 1.2 2.5 23Summer Conventional 19-Aug-98 1 Cp15 Cr10 5 13.2 10.0 16.0 18.2 1.7 1.4 2.1 17

2 Cp7 Cr2 5 12.3 10.1 14.3 12.7 1.6 1.4 1.7 7.43 Cp16 Cr3 5 20.3 13.1 32.7 36.4 2.8 2.2 3.3 14

2-Sep-98 4 Cp7 Cr11 5 11.5 10.0 12.8 9.28 1.6 1.4 1.9 115 Cp1 Cr5 5 32.2 22.3 39.5 19.8 1.8 1.7 1.9 4.36 Cp16 Cr2 5 9.18 7.0 13.0 27.2 1.4 1.1 1.6 17

Conventional total 30 16.4 7.0 39.5 54.0 1.8 1.1 3.3 29Organic 19-Aug-98 1 Op11 Fm1 5 17.4 10.9 27.9 36.3 3.0 2.7 3.2 6.4

2 Op6 Fm1 5 15.0 12.2 19.2 17.5 1.6 1.4 1.8 123 Op10 Or7 5 6.83 4.9 7.8 16.2 1.7 1.5 2.0 9.9

2-Sep-98 4 Op11 Fm1 5 18.5 14.2 23.9 19.4 2.7 2.4 3.2 125 Op6 Fm1 5 10.3 8.6 12.1 15.0 1.8 1.6 1.9 7.36 Op10 Or7 5 10.4 8.3 12.2 13.5 1.6 1.3 1.9 14

Organic total 30 13.1 4.9 27.9 39.9 2.1 1.3 3.2 29Summer total 60 14.8 4.9 39.5 50.3 1.9 1.1 3.3 30Conventional total 60 18.3 7.0 48.1 55.4 1.7 1.1 3.3 27Organic total 60 14.8 4.9 35.4 46.4 1.9 1.3 3.2 27Grand total 120 16.5 4.9 48.1 53.2 1.8 1.1 3.3 28*: C.V. = Standard diviation / Average x 100

48

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

Season Practice Date # Grower Market n NO3 mg/kg leaf blade FW Moisture % leaf bladeAv. Min. Max. C.V.* Av. Min. Max. C.V.*

Winter Conventional 27-Jan-98 1 Cp4 Cr10 5 1900 1600 2300 15.3 91.1 90.6 91.4 0.3832 Cp7 Cr2 5 1460 1300 1600 7.8 91.6 91.2 91.9 0.3033 Cp8 Cr11 5 994 830 1100 11.5 92.1 91.8 92.5 0.362

9-Feb-98 4 Cp1 Cr5 5 1200 1100 1400 11.8 92.5 92.1 92.9 0.3935 Cp13 Cr13 5 1900 1600 2200 13.4 90.1 89.5 90.7 0.591

14-Feb-98 6 Cp14 Cr13 5 964 770 1200 18.5 91.5 90.7 92.1 0.617Conventional total 30 1400 770 2300 30.8 91.5 89.5 92.9 0.947Organic 27-Jan-98 1 Op5 Or5 5 1210 740 1600 25.6 90.7 90.0 91.3 0.512

2 Op7 Or7 5 1070 830 1200 14.6 90.7 90.3 91.0 0.2923 Op3 Or4 5 754 260 1000 39.7 89.7 88.7 90.5 0.867

9-Feb-98 4 Op4 Or2 5 1700 1400 1900 11.8 90.7 87.2 93.8 2.735 Op5a Or5 5 1070 460 1600 45.3 90.2 88.8 92.8 1.776 Op3 Or7 5 1360 1200 1600 11.2 90.9 89.4 92.1 1.27

Organic total 30 1190 260 1900 33.3 90.5 87.2 93.8 1.43Winter total 60 1300 260 2300 32.7 91.0 87.2 93.8 1.32Summer Conventional 19-Aug-98 1 Cp15 Cr10 5 2140 1700 3000 23.5 93.2 92.8 93.5 0.382

2 Cp7 Cr2 5 1500 1300 1700 13.3 93.9 93.7 94.3 0.2603 Cp16 Cr3 5 2400 1900 2900 18.2 92.1 91.8 92.5 0.282

2-Sep-98 4 Cp7 Cr11 5 2120 1700 2300 11.7 92.7 91.7 93.6 0.7385 Cp1 Cr5 5 1250 560 2100 53.1 92.9 92.6 93.2 0.2616 Cp16 Cr2 5 1580 1200 2200 23.8 92.5 91.5 93.8 0.936

Conventional total 30 1830 560 3000 31.4 92.9 91.5 94.3 0.787Organic 19-Aug-98 1 Op11 Fm1 5 462 140 1100 91.9 90.8 89.4 91.8 0.973

2 Op6 Fm1 5 744 420 1200 39.0 92.3 91.9 92.9 0.4273 Op10 Or7 5 1950 930 2800 34.2 93.6 93.1 93.8 0.335

2-Sep-98 4 Op11 Fm1 5 419 93 640 57.8 91.3 90.9 91.6 0.2965 Op6 Fm1 5 2100 1900 2400 10.1 93.5 92.8 93.8 0.3926 Op10 Or7 5 1940 1600 2200 11.9 93.2 92.9 93.4 0.251

Organic total 30 1270 93 2800 65.0 92.5 89.4 93.8 1.28Summer total 60 1550 93 3000 49.0 92.7 89.4 94.3 1.08Conventional total 60 1620 560 3000 33.9 92.2 89.5 94.3 1.16Organic total 60 1230 93 2800 52.2 91.5 87.2 93.8 1.73Grand total 120 1420 93 3000 43.9 91.8 87.2 94.3 1.51

Season Practice Date # Grower Market n NO3 mg/kg petiole FW Moisture % petioleAv. Min. Max. C.V.* Av. Min. Max. C.V.*

Winter Conventional 27-Jan-98 1 Cp4 Cr10 5 4020 3400 4700 13.1 93.5 93.3 93.9 0.2332 Cp7 Cr2 5 3460 3300 3600 3.30 94.0 93.8 94.2 0.1873 Cp8 Cr11 5 2080 1800 2300 9.25 94.3 94.0 94.5 0.188

9-Feb-98 4 Cp1 Cr5 5 3760 3600 4000 4.83 94.1 93.7 94.5 0.3145 Cp13 Cr13 5 4160 3500 4500 9.56 92.7 92.1 93.3 0.507

14-Feb-98 6 Cp14 Cr13 5 3200 2500 4400 22.9 93.5 93.1 94.0 0.455Conventional total 30 3450 1800 4700 23.2 93.7 92.1 94.5 0.653Organic 27-Jan-98 1 Op5 Or5 5 2880 1300 3800 33.1 92.0 91.1 92.9 0.944

2 Op7 Or7 5 2360 2100 2700 11.0 92.8 92.5 93.0 0.2123 Op3 Or4 5 1130 400 1700 43.2 90.8 89.2 92.6 1.35

9-Feb-98 4 Op4 Or2 5 3860 3300 4200 8.90 94.2 94.0 94.7 0.3165 Op5a Or5 5 2840 1100 4200 49.3 91.9 91.0 92.5 0.6586 Op3 Or7 5 3640 3100 4300 12.8 94.2 94.1 94.4 0.147

Organic total 30 2790 400 4300 41.1 92.7 89.2 94.7 1.51Winter total 60 3120 400 4700 33.2 93.2 89.2 94.7 1.27Summer Conventional 19-Aug-98 1 Cp15 Cr10 5 4920 3800 5700 17.0 95.5 95.2 96.0 0.342

2 Cp7 Cr2 5 4580 4000 5200 10.5 96.1 95.9 96.2 0.1753 Cp16 Cr3 5 4580 3200 6500 26.6 94.7 94.5 95.3 0.371

2-Sep-98 4 Cp7 Cr11 5 5500 5000 6200 8.72 95.2 94.9 95.6 0.2655 Cp1 Cr5 5 3380 1700 5100 47.1 94.9 94.5 95.5 0.4656 Cp16 Cr2 5 4560 3400 6500 25.7 95.0 94.6 95.8 0.490

Conventional total 30 4590 1700 6500 25.0 95.3 94.5 96.2 0.571Organic 19-Aug-98 1 Op11 Fm1 5 988 230 2000 72.1 92.4 91.2 93.6 1.27

2 Op6 Fm1 5 2260 1500 3300 29.5 94.4 94.0 94.8 0.3223 Op10 Or7 5 3340 2200 4600 26.6 95.8 95.6 96.2 0.250

2-Sep-98 4 Op11 Fm1 5 1170 220 1700 62.4 92.9 92.4 93.8 0.5425 Op6 Fm1 5 4220 4000 4500 5.14 96.0 95.6 96.1 0.1926 Op10 Or7 5 4700 4400 5000 6.02 95.9 95.8 96.0 0.109

Organic total 30 2780 220 5000 56.0 94.6 91.2 96.2 1.65Summer total 60 3680 220 6500 44.4 94.9 91.2 96.2 1.27Conventional total 60 4020 1700 6500 28.3 94.5 92.1 96.2 1.04Organic total 60 2780 220 5000 48.7 93.6 89.2 96.2 1.87Grand total 120 3400 220 6500 40.9 94.0 89.2 96.2 1.57*: C.V. = standard diviation / average x 100

49

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

Season Practice Date # Grower Market Non edible part % FWn. Av. Min. Max. C.V.*

Winter Conventional 27-Jan-98 1 Cp4 Cr10 5 4.0 2.8 5.1 292 Cp7 Cr2 5 1.2 0.9 1.5 253 Cp8 Cr11 5 5.6 3.4 7.5 28

9-Feb-98 4 Cp1 Cr5 5 6.2 3.6 8.5 325 Cp13 Cr13 5 1.2 0.6 1.8 37

14-Feb-98 6 Cp14 Cr13 5 2.5 1.1 4.7 54Conventional total 30 3.4 0.6 8.5 67Organic 27-Jan-98 1 Op5 Or5 5 0.83 0.7 1.1 19

2 Op7 Or7 5 3.0 0.4 5.8 703 Op3 Or4 5 2.6 1.1 3.7 42

9-Feb-98 4 Op4 Or2 5 1.5 0.3 2.9 645 Op5a Or5 5 2.0 0.9 3.7 556 Op3 Or7 5 1.0 0.7 1.4 28

Organic total 30 1.8 0.3 5.8 73Winter total 60 2.6 0.3 8.5 77Summer Conventional 19-Aug-98 1 Cp15 Cr10 5 3.9 1.1 6.5 54

2 Cp7 Cr2 5 3.1 2.9 3.4 7.33 Cp16 Cr3 5 1.2 0.4 2.7 76

2-Sep-98 4 Cp7 Cr11 5 6.5 4.6 9.5 285 Cp1 Cr5 5 1.7 0.6 2.6 516 Cp16 Cr2 5 9.3 1.7 18 73

Conventional total 30 4.3 0.4 18 93Organic 19-Aug-98 1 Op11 Fm1 5 0.90 0.7 1.2 24

2 Op6 Fm1 5 0.00 0.0 0.0 0.003 Op10 Or7 5 1.4 0.8 2.4 41

2-Sep-98 4 Op11 Fm1 5 3.3 1.4 6.1 595 Op6 Fm1 5 0.0 0.0 0.0 0.006 Op10 Or7 5 7.3 2.3 16 71

Organic total 30 2.2 0.0 16 150Summer total 60 3.2 0.0 18 120Conventional total 60 3.9 0.4 18 84Organic total 60 2.0 0.0 16 130Grand total 120 2.9 0.0 18 100

50

APPENDIX 6.1 Climate at Market Sample's Origins (Winter1)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).51

0

40

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120

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/97

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/98

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98

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cipi

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40Holtville (Brawley)

Temperature C

0

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Yuma, AZ

Temperature C

0

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El Centro

Temperature C

0

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/97

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Thermal

Temperature C

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/97

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/98

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/98

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98

Pre

cipi

tatio

n m

m

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40Oxnard (Hueneme)

Temperature C

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

52

0

40

80

120

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

Pre

cipi

tatio

n m

m

0

10

20

30

40

Oxnard (Hueneme)

Temperature C

0

40

80

120

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

Pre

cipi

tatio

n m

m

0

10

20

30

40Freedom

Temperature C

0

40

80

120

6/1/

98

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/98

6/21

/98

7/1/

98

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/98

7/21

/98

7/31

/98

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/98

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/98

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/98

9/9/

98

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cipi

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n m

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10

20

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40

Santa Cruz

Temperature C

0

40

80

120

6/1/

98

6/11

/98

6/21

/98

7/1/

98

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/98

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/98

7/31

/98

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/98

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/98

8/30

/98

9/9/

98

Pre

cipi

tatio

n m

m

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10

20

30

40North Salinas

Temperature C

0

40

80

120

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

Pre

cipi

tatio

n m

m

0

10

20

30

40Watsonville

Temperature C

APPENDIX 6.2 Climate at Market Sample's Origins (Summer1. Continued). Precipitation (bar) and maximum (bold line), minimum (thin line) temperatureat areas where market samples grew during their estimated growth period.

(UC IPM California Weather Database 1999).

53

0

40

80

120

6/1/

98

6/11

/98

6/21

/98

7/1/

98

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/98

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/98

7/31

/98

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/98

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/98

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/98

9/9/

98

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cipi

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n m

m

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20

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40South Salinas

Temperature C

0

40

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120

6/1/

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/98

6/21

/98

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/98

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/98

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/98

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/98

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/98

8/30

/98

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98

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cipi

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n m

m

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20

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40

Hollister

Temperature C

0

40

80

120

6/1/

98

6/11

/98

6/21

/98

7/1/

98

7/11

/98

7/21

/98

7/31

/98

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/98

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/98

8/30

/98

9/9/

98

Pre

cipi

tatio

n m

m

0

10

20

30

40

Gilroy

Temperature C

0

40

80

120

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/

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Pre

cipi

tatio

n m

m

0

10

20

30

40

Soledad

Temperature C

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

0

100

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400

11/1

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ar R

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tion

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40El Centro

Temperature C

0

100

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400

11/1

/97

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/98

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98

Sol

ar R

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W/m

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40Holtville (Brawley)

Temperature C

0

100

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400

11/1

/97

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/98

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/98

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98

Sol

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tion

W/m

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40Thermal

Temperature C

0

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400

11/1

/97

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/97

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/98

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1/30

/98

2/9/

98

Sol

ar R

adia

tion

W/m

2

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10

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40Oxnard (Hueneme)

Temperature C

Appendix 6.4Climate at Market Sample's Origin (Summer2)Solar radiation (bar) and maximum (bold line),minimum (thin line) soil temperature at areas where summer samples grewduring their estimated growth period.

(UC IPM California Weather Database 1999).

55

0

100

200

300

4006/

1/98

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/98

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/98

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98

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/98

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Sol

ar R

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tion

W/m

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40

Oxnard (Hueneme)

Temperature C

0

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400

6/1/

98

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/98

6/21

/98

7/1/

98

7/11

/98

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/98

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/98

8/10

/98

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/98

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/98

9/9/

98

Sol

ar R

adia

tion

W/m

2

0

10

20

30

40

Santa Cruz

Temperature C

0

100

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400

6/1/

98

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/98

6/21

/98

7/1/

98

7/11

/98

7/21

/98

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/98

8/10

/98

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/98

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/98

9/9/

98

Sol

ar R

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tion

W/m

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0

10

20

30

40North Salinas

Temperature C

0

100

200

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400

6/1/

98

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/98

6/21

/98

7/1/

98

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/98

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/98

7/31

/98

8/10

/98

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/98

8/30

/98

9/9/

98

Sol

ar R

adia

tion

W/m

2

0

10

20

30

40Hollister

Temperature C

0

100

200

300

400

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

Sol

ar R

adia

tion

W/m

2

0

10

20

30

40

South Salinas

TemperatureC

56

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

A. LettuceSubstantial 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 (Appendix7.1). In particular, the average nitrate content exceeding 1500 mg/kg FW was found in lettuce grown ingreenhouses in fall to winter (Finland (Ahonen et al., 1987) and UK in Appendix 7.1). Moreover, themaximum nitrate content in lettuce grown in greenhouses in the Netherlands and UK during winterreached more than 5000 mg/kg FW .

A number of studies have shown that light influences nitrate accumulation in plants considerablyby 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 hightemperatures that stimulate mineralization and nitrification of nitrogen in soils in greenhouses duringwinter (Roorda van Eysinga, 1984; MAFF UK, 1998a). In the US, Minotti (Maynard et al. 1976) alsoreported 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 onesixth that of Southern California (Appendix 7.2). Interestingly, even under such low solar radiation, nitratecontent in winter lettuce grown in the UK did not differ significantly from that of summer lettuce when theywere grown outdoors (Appendix 7.1).

Variety appears to be another factor. For example, in UK, Iceberg lettuce is grown outdoors aswell as the US, and Round lettuce (also known as flat lettuce), which are the most commonly grown andconsumed lettuce in the UK, are grown throughout the year, predominantly in glass-houses (MAFF UK1996, 1997a). They reported that Iceberg lettuce tended to have lower nitrate concentrations than othervarieties and this may be explained in part by the practice of removing the outer leaves of these lettucesat harvest. As mentioned before, wrapper leaves of head type lettuce contains higher nitrate than innerpart (Lorenz 1978). Thus, this contrast may be explained by differences between the US and Europe insolar 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 oflettuce 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 casein the US (Maynard et al. 1976, Lorenz 1978). MLECR also established a lower maximum nitrate limit forspinach 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 (Appendix7.1).

B. SpinachIn 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, andleaf-blades, where nitrate reduction and assimilation take place, might be one of the reasons for spinach’srelatively high nitrate concentration. Certain vegetables, because of a very efficient uptake system, aninefficient reductive system, or an unfavorable combination of both, tend to accumulate more nitrate thanothers (Maynard et al., 1976).

Nitrate content in spinach grown in European countries was similar to that seen in the US, exceptfor the maximum value reported in the Netherlands (5400 mg/kg FW) . This trend differs from that oflettuce as discussed above. Cantliffe (1974a) showed that nitrate content in spinach was lower thanlettuce when those were grown in greenhouse by applying the same amount of nitrogen.

57

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

Lettucea (NO3 mg/kg FW) Spinach (NO3 mg/kg FW)Country Yearreported

Analysis by Origin ofsamples

Samplingseason n Average Range n Average Range

CA, USA Aug-Sep 24 [5]b 840 490 - 1700 (330 - 1900)c 12 [5] 2300 600 - 3400

(130 - 4100)USA 1999 Muramoto

AZ & CA,USAd Jan-Feb 24 [5] 1000 760 - 1500

(570 - 1600) 12 [5] 2000 890 - 2900(310 - 3400)

Germany 1984 Kampee Germany (not available) 162g 1600 230 - 3300 85g 840 20 - 2700

Summer 41 [1] 890 85 - 2500Finland 1987 Ahonen

et al. FinlandFallf 54 [1] 1900 280 - 3500

Netherlands 1988 CCRXe Netherlands (not available) 1682g,h 420 - 5500 866g,h 800 - 5400

Apr-Sep 70 [10] 2400 270 - 4200UKGreenhouse Oct-Mar 112 [10] 3100 600 - 5300UK 1999 Ysart

et al.UK Outdoor May-Octi 131 [10] 1100 50 - 3200 34 [10] 1900 50 - 4700

Australia 1994 Lyons et al. Queensland Aug-June, Dec 18 [1] 290 - 1500For 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.

58

South Coast of California Southern England

Appendix 7.2 Comparison of climate between California and EnglandMonthly 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)

0

100

200

300

400

JAN

MA

R

MA

Y

JUL

SE

P

NO

VSol

ar r

adia

tion

W/m

2

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Air tem

perature C0

100

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JAN

MA

R

MA

Y

JUL

SE

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NO

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adia

tion

W/m

2

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Air tem

perature C

59

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). Usingthose average CVs, number of sub-samples to obtain average nitrate content in plants in a carton (24plants 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 ofsamples calculated for Iceberg lettuce, Romaine lettuce and spinach were 6, 6, and 12 within ± 10%, and3, 3, and 5, within ± 25%, respectively. When we used five sub-samples, the average sampling error wasestimated as ± 13 to 14% for lettuces and ± 27 % for spinach at a confidence level of 0.95. To obtainaverage nitrate content in plants in a carton within 20% of error, we need to take at least five lettuceplants 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 sub-samples 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 3Spinach (whole plant) 24 5.0 83 12 5Spinach (leaf-blades) 26 8.0 93 - -

Spinach (petioles) 23 3.5 71 - - * CV % = Standard deviation / Average x 100. ** Normal distribution assumed.

60

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