Kale is a nutrient-dense leafy vegetable associated with wide-ranging health benefits. It is tolerant of drought and temperature fluctuations, and could thus serve an increasingly important role in providing a safe and nutritious food supply during the climate crisis, while kale's ease of cultivation and ability to be grown in a wide range of soils make it a good fit for urban agriculture. In this pilot study we explored potential differences between kale grown at urban versus rural farms. We planted kale seedlings (Darkibor variety) at three urban and four rural farms in and around Baltimore City, Maryland, instructed farmers to cultivate them using their usual growing practices, harvested the kale from fields and points of distribution, and analyzed it for concentrations of carotenoids, vitamins C and K1, ten nutritional elements, and eight non-essential metals. Although sample sizes for some analyses were in some cases too small to produce statistically significant results, we identified potentially meaningful differences in concentrations of several components between urban and rural kale samples. Compared to urban samples, mean concentrations of carotenoids and vitamins were 22–38% higher in rural field samples. By contrast, mean concentrations for eight nutritional elements were higher in urban field samples by as much as 413% for iron. Compared to rural field samples, mean concentrations of nine non-essential metals were higher in urban samples, although lead and cadmium concentrations for all samples were below public health guidelines. Some urban-rural differences were more pronounced than those identified in prior research. For six elements, variance within urban and rural farms was greater than variance between urban and rural farms, suggesting urbanicity may not be the primary driver of some observed differences. For some nutrients, mean concentrations were higher than upper ranges reported in prior estimates, suggesting kale may have the potential to be more nutrient-dense than previously estimated. The nutritive and metals composition of this important crop, and the factors that influence it, merit continued investigation given its growing popularity.
Kale (Brassica oleracea var. acephala), widely considered a "superfood," is a nutrient-dense leafy vegetable associated with antioxidant, anticancer, cardiovascular, and gastrointestinal benefits [[
The popularity of kale has surged in recent decades. Bon Appétit magazine named 2012 the year of kale; a year later, "National Kale Day" was established in the US [[
The nutritive value and widespread popularity of kale are grounds for investigating the many factors that affect kale's beneficial properties. For example, studies have demonstrated that the nutrient and/or phytochemical composition of kale can vary based on cultivar [[
Environmental factors can also impact concentrations of non-essential metals and other harmful contaminants in kale. Urban areas, for example, often have high concentrations of industrial activity, waste incineration, building demolition, lead-based paint, vehicular emissions, tire wear, and other sources of harmful metals [[
Generally, the health benefits of consuming vegetables likely outweigh any risks associated with exposure to non-essential metals and other harmful contaminants; regardless, research and monitoring are important to ensure the safety of food supplies. An assessment of produce grown in Baltimore City, Maryland found some significant differences in concentrations of essential and non-essential (arsenic (As), barium (Ba), cadmium (Cd), chromium (Cr), lead (Pb)) metals in kale samples from urban-grown sources compared to peri-urban, grocery conventional, and grocery organic sources; however, differences were too small in magnitude to have any practical significance for health, and Cd and Pb concentrations in all samples were well below public health guidelines for exposure (no guidelines are available for As, Ba, and Cr) [[
Despite its popularity, raw kale is not included in the FDA's Total Diet Study [[
Building upon the existing body of evidence, we conducted a pilot study to assess levels of select health-relevant nutrients, nutritional elements, and non-essential metals in kale grown at two urban and four rural farms; and to gather data about site history, farming practices, environmental conditions, and other factors that might influence the properties of kale grown at those sites. The aim of the study was to explore the degree to which concentrations of these analytes might differ between urban and rural farms, and possible reasons for those differences. The analytes of interest were carotenoids, vitamin K
We identified and recruited seven farms for participation in this study. Three urban farms were identified through previous inclusion in the Safe Urban Harvests Study [[
For the purposes of this survey study, one variety of kale, Darkibor, was selected. Organic (F1) Darkibor kale seeds were acquired from a commercial seed vendor (Johnny's Selected Seeds, Maine, US). The seeds were sown in seedling starting trays filled with Fafard growing mix (Sun Gro Horticulture, MA, US) on 25 July 2019 in a growth chamber (temperature 25°C, relative humidity 60%, in darkness) at the US Department of Agriculture (USDA) Agricultural Research Service's Beltsville Agricultural Research Center laboratory in Beltsville, MD. Germination of the seeds was first noted two days after sowing. Plants were then grown under 14h/10h light/dark with an intensity of 160 μmol per m
Between 27–29 August 2019, we transplanted 32 seedlings at each farm using stainless steel trowels at a spacing of 12 inches (30 cm) in an area determined by the farmer, in configurations most suitable for the location, e.g., 1 row of 32 seedlings, 2x16, or 4x8. All plants were grown in open air, i.e., not in high tunnels/hoop houses. Farmers were otherwise instructed to grow and tend to the kale plants per usual practices for their farm (e.g., some farms applied pesticides, some installed and used row cover). Farmers were compensated $1,000 USD for the use of space, labor, and time in growing the kale.
At the time of transplanting, members of the study team verbally administered a baseline survey (provided in Supporting Information) to a representative at each farm. The survey included questions about farm history; prior soil testing for fertility or contaminants; and growing practices, e.g., irrigation, pest management, use of soil amendments, and USDA Organic certification.
After harvesting, a follow-up survey was emailed to the representative at each farm. The survey asked representatives to verify which practices—specifically irrigation, pest management, and use of soil amendments—had been used to grow the study kale and whether those differed from their typical kale growing practices reported in the baseline survey.
After at least one farmer notified the study team that their kale was ready to harvest (approximately six to seven weeks after transplanting), four kale samples were collected directly from each farm ("field samples") between 11–18 October 2019. The 12 largest and healthiest plants at each farm, selected based on a visual assessment of size and the fewest spots and yellow leaves, were harvested, and sets of three plants were randomly selected and combined into four composite samples. Two study team members took a photo of the plants prior to harvesting, removed each plant from the ground (including roots) using a shovel, and recorded the time of harvest. Each sample was placed in a five-gallon (approximately 19-liter) bucket and promptly transported via air-conditioned vehicle to a laboratory at the Johns Hopkins Bloomberg School of Public Health (BSPH), where it was immediately processed.
During the same week, two samples of kale from each farm were collected from the point of distribution ("market samples"), i.e., farmers market, mobile market, or donation site. Farmers obtained six bunches of market-quality kale, which were then randomly assigned to two composite samples of three bunches each. The kale in the market samples was harvested, processed, and transported to the point of distribution, following the usual practices for that farm (e.g., any washing, sanitizing, use of refrigerated storage and/or delivery). The study team picked up the harvested samples at the point of distribution and transported them to the BSPH laboratory, where they were immediately processed. When study team members picked up market samples, they verbally administered a market survey (provided in Supporting Information) to a representative of each farm. The survey included questions about the date and time of harvest, processing (e.g., washing), and use of refrigeration during transport.
Upon arrival at the BSPH laboratory, each sample was inspected by the study team. Leaves deemed edible by the study team (e.g., not yellow or covered in pests) were separated from the central stem and roots. Each sample (i.e., leaves without the central stem and roots) was then weighed, rinsed in deionized water, patted dry with WypAll lint-free surface wipes, laid out on a flat white surface, photographed, and weighed again.
The center rib of each leaf was removed with a stainless-steel knife on a plastic, consumer-grade kitchen cutting board. Following previously described procedures [[
Moisture was analyzed in all samples to assess differences on a fresh and dry mass basis. Ten elements essential for human health were selected for analysis: calcium, copper, iron, magnesium, manganese, molybdenum, phosphorous, potassium, sodium, and zinc. Vitamin C, vitamin K
Samples were packed in dry ice after removal from the -80°C freezer at the BSPH laboratory, shipped via overnight express to designated laboratories, verified to have arrived frozen upon receipt at each laboratory, and held at -60°C storage until analyzed. Vitamin C and moisture were analyzed at Virginia Tech (Phillips laboratory), vitamin K
Moisture was measured by vacuum drying 2 g subsamples to a constant weight at 635 mm Hg and 65–70°C, as adapted from Association of Official Analytical Chemists International [[
For each nutrient, 10–15% of the samples were assayed in duplicate with the laboratory blinded to the sample duplicates. Additionally, well-characterized in-house control materials, developed for the USDA National Nutrient Database/Food Data Central [[
At the time of seedling transplanting and weekly thereafter until harvesting, two members of the study team visited each farm to collect data on environmental conditions, totaling six visits over five weeks. During each visit, a DustTrak portable aerosol and dust monitor (TSI, Shoreview, MN) was used to measure mean concentrations of particulate matter (PM
On the first visit after transplanting, three Bluetooth-enabled HOBO data loggers (ONSET, Bourne, MA) were installed within each kale plot. Two were installed above ground to track light intensity and ambient air temperature. One was buried at root depth to track soil temperature. Loggers recorded light and temperature data at five- to 15-minute intervals from installation until they were stopped and retrieved at the time of harvesting.
Data management, analysis, and visualization were performed using Python version 3.6. For some visualizations Microsoft Excel was additionally used.
For some non-essential metals (As, Ni, Pb, U, V), results for some samples were below the detection limit (
For samples analyzed in duplicate for a given component, the mean was used as the sample result.
Analyte concentrations were reported by laboratories on a per fresh weight basis, e.g., mg vitamin C per 100 g fresh kale. Results herein are also reported on a per fresh weight basis, since this is the form in which kale is typically consumed, i.e., not dehydrated. In the Supporting Information and Supporting Data Tables we additionally present results after converting to dry weight, to control for moisture content. Fresh weight concentrations were converted to dry weight using the following formula adapted from the US Environmental Protection Agency (EPA) [[
Graph
Where C is the concentration of analyte a in sample i and W is the wet fraction for sample i.
For statistical analyses, sample descriptive variables included an identifier for each farm, farm type (urban, rural), and harvest location (field, market). Two-sided Mann-Whitney U tests were used to compare analyte concentrations between groups, e.g., samples from urban vs. rural farms, samples collected from farms vs. at market, or samples from farms using a particular growing practice vs. those that did not. One-way analysis of variance (ANOVA) was also used to compare differences between urban and rural samples; although this test is more appropriate for larger sample sizes and normally distributed data, it is useful for comparing variation between urban and rural samples to variation within urban and rural samples. Potential correlations between continuous variables, such as between different analyte concentrations, were assessed using Pearson's correlation tests.
Data from individual kale samples were clustered, i.e., we would expect analyte concentrations among kale samples from the same farm to be correlated, thus kale samples were not independent. To account for clustering, for the purpose of statistical testing with kale data we used the mean value for each cluster (i.e., farm), as previously recommended [[
Analyses of light and temperature were based on hourly means and included only those data that were common to every group (e.g., when checking for differences across farms, if a HOBO data logger at one farm was started 30 hours before those at the other farms, those first 30 hours were excluded from analyses). Estimates of peak hourly light and temperature for each day excluded days that did not have a full 24 hours of data, i.e., days when data loggers were either installed or removed. Wilcoxon signed-rank tests were used to compare two groups with repeated measures over time, i.e., PM concentrations, weekly collected rainfall, and mean hourly light and temperature between urban and rural farms.
With the exception of ANOVA, the aforementioned tests are all non-parametric and were used on the rationale that sample sizes were small. Light and temperature data had larger sample sizes, e.g., there were 907 mean hourly light intensity observations per farm; however, the results of Shapiro-Wilks tests indicated that the distributions of light and temperature data were non-normal and thus better suited for non-parametric testing.
The Johns Hopkins Bloomberg School of Public Health Institutional Review Board (IRB) reviewed and determined that this study did not require oversight as human subjects research.
Thirty-two kale samples were represented in the results (Table 1). These comprised 23 field samples and nine market samples, including four field samples and two market samples from each rural farm. After harvesting four field samples from urban farm U1, there was only enough kale remaining for one market sample. Urban farm U2 had a harlequin bug (Murgantia histrionica) infestation and only had enough kale for three field samples and no market samples. A third urban farm encountered significant pest issues that inhibited kale production; no kale was harvested from this farm and their survey results were excluded from the study. Sample weight, measured after removing the central stem and roots, washing, and drying, ranged from 319–1840 grams (mean: 923 g). S1 Fig in S1 File shows photos of cultivated kale at each farm.
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Table 1 Number of kale samples by farm. A third urban farm encountered significant pest issues that inhibited kale production; no kale was harvested from this farm and their survey results were excluded from the study. Farms were anonymized by assigning a unique identifier beginning with "U" for urban farms and "R" for rural farms.
Farm ID Number of field samples Number of market samples U1 4 1 U2 3 0 R1 4 2 R2 4 2 R3 4 2 R4 4 2
Four of the six eligible farms were established between 2009–2018, while two rural farms (R3, R4) were established in the 1980s. The urban farms were previously vacant lots with some history of residential use. The rural farms all had some history of use as a farm or garden prior to current management and their participation in this study.
Both urban farms had previously tested their soil for metals as part of their participation in the Safe Urban Harvests Study [[
All farms had at least two years of experience growing kale, and all reported growing at least two different varieties of kale since the establishment of their respective farms. Only one rural farm (R3) had prior experience growing the Darkibor variety of kale. All rural farms reported using cover crops; the urban farms did not. Other crops previously grown on study plots included turnips, beets, carrots, pattypan squash, tomatoes, garlic, strawberries, eggplant, and other brassicas. All farms reported rotating crop locations each year. Only one farm (R3) was USDA-certified Organic.
Participating farms did not consistently reply to survey questions about the scale of their operations, but one rural farm (R2) reported growing 600 square feet (0.01 acres) of kale. Another rural farm (R1) reported harvesting 150 kale plants in the previous year, less than the 600 plants harvested by one of the urban farms (U2).
Prior to planting, all farms reported applying soil amendments at least once per season, including compost, feather meal, worm castings, kelp, fish emulsion, and/or minerals. During kale cultivation, both urban farms (U1, U2) and one rural farm (R3) applied compost, two rural farms applied feather meal (R2, R3), and the research farm (R4) did not apply soil amendments.
During kale growth, one urban (U2) and three rural farms (R2, R3, R4) reported using pesticides. The other urban farm (U1) relied exclusively on guardian plants (e.g., marigolds), row cover, and other non-chemical deterrents. The other rural farm (R1) used pesticides but only prior to planting. The five farms using pesticides either before or after planting used kaolin clay, neem oil, pyrethrin, spinosad, and/or Bacillus thuringiensis, all of which are generally allowed for use under Organic Materials Review Institute standards [[
During kale cultivation, all urban farms used municipal water to irrigate. Three rural farms (R1, R2, R3) used well water and the research farm (R4) used filtered pond water. None of the urban farms and all of the rural farms used drip irrigation. Three rural farms irrigated once per week; the other farms irrigated as frequently as once per day, with variations based on weather patterns or stage of plant growth.
All farms except for the research farm (R4) identified as commercial, i.e., they grew and sold produce for profit, at least in part.
The duration of time between harvesting kale and the point at which flash-frozen homogenized samples were put into the freezer ranged from 1.8 to 6.8 hours for field samples and 2.9 to 26.4 hours for market samples. Three of the five farms with market samples (U1, R1, R3) brought kale to the respective distribution points the day after it was harvested. Of these, two rural farms noted that they kept the kale in refrigerated storage. The urban farm did not provide any information about their storage method (the survey requested information about refrigerated transport but not storage; when designing the survey we had assumed kale would be transported directly to market following harvest), but the kale was noticeably wilted upon collection by the research team. Only one farm reported using refrigerated transport.
Results for the quality control materials are summarized in S1 Table in S1 Data. All values were within the expected range for in-house control materials and the certified range for reference materials, with acceptable HorRat (≤3.0) [[
Among the carotenoids analyzed, lutein, zeaxanthin, and total beta-carotene are reported in this study. Beta-cryptoxanthin, lycopene, and alpha-carotene were detected at trace levels (less than ~0.1 mg/100g fresh weight), but the data had insufficient precision for quantitative results. Alpha-cryptoxanthin and gamma-carotene were also monitored but not detected (<0.01 mg/100g fresh weight) in any samples (lycopene was also <0.01 mg/100g in most samples). For vitamin K
Unless specified otherwise, all analyte concentrations and statistical tests reported in the manuscript and tables refer to fresh weight concentrations. Dry weight concentrations are provided in Supplementary Figures and Tables. Mean analyte concentrations for field samples, by farm urbanicity, are reported in Table 2. Results for individual farms are reported in Figs 1–3. Additional descriptive statistics and analyte concentrations for individual samples are provided in S2-S4 Tables in S1 Data.
Graph: Each dot represents one kale sample. Site identifiers (x-axis) with "U" represent urban farms, identifiers with "R" represent rural farms. P values are from Mann-Whitney U tests comparing field samples from urban and rural farms, using the mean value from each farm (N = 6).
Graph: Each dot represents one kale sample. Site identifiers (x-axis) with "U" represent urban farms, identifiers with "R" represent rural farms. P values are from Mann-Whitney U tests comparing field samples from urban and rural farms, using the mean value from each farm (N = 6). See S2 Fig in S1 File for dry weight concentrations.
Graph: Each dot represents one kale sample. Site identifiers (x-axis) with "U" represent urban farms, identifiers with "R" represent rural farms. P values are from Mann-Whitney U tests comparing field samples from urban and rural farms, using the mean value from each farm (N = 6). See S3 Fig in S1 File for dry weight concentrations. Chromium was not speciated between hexavalent chromium (a carcinogen) and trivalent chromium (an essential nutrient).
Graph
Table 2 Mean fresh weight analyte concentrations in field samples by urban/urban farms, with comparisons to prior estimates. Mean fresh weight analyte concentrations by urban/rural farm, with standard error (SE). Since there was only one market sample from urban farms, market samples are not included in means. Estimates from the current study are compared against prior estimates from the Safe Urban Harvests (SUH) study (
Group Analyte Unit Current study urban, mean ± SE Current study rural, mean ± SE SUH urban, mean SUH non-urban, mean USDA, mean (range) Moisture g/100g fresh wt 88.2 ± 0.7 85.2 ± 0.4 Carotenoids & vitamins Beta-carotene mg/100g fresh wt 3.20 ± 0.26 5.28 ± 0.21 2.87 (2.16–3.83) Lutein + zea. mg/100g fresh wt 8.09 ± 0.18 10.65 ± 0.32 6.26 (4.46–8.56) Phylloquinone mcg/100g fresh wt 228 ± 5 350 ± 7 390 (369–422) Vitamin C mg/100g fresh wt 113 ± 3 147 ± 4 93 (84–104) Nutritional elements Calcium mg/100g fresh wt 397 ± 54 374 ± 11 254 (203–281) Copper mg/100g fresh wt 0.07 ± 0.00 0.05 ± 0.00 0.06 0.27 0.05 (0.03–0.08) Iron mg/100g fresh wt 7.58 ± 2.02 1.61 ± 0.06 1.60 (0.77–3.61) Magnesium mg/100g fresh wt 50.3 ± 6.0 45.3 ± 1.2 32.7 (28.4–45.8) Manganese mg/100g fresh wt 0.57 ± 0.07 0.88 ± 0.06 0.38 0.62 0.92 (0.51–1.46) Molybdenum mg/100g fresh wt 0.03 ± 0.00 0.02 ± 0.00 Phosphorous mg/100g fresh wt 53.1 ± 2.1 52.8 ± 3.0 55 (47–62) Potassium mg/100g fresh wt 312 ± 15 441 ± 8 348 (301–389) Sodium mg/100g fresh wt 18.1 ± 3.3 8.6 ± 0.7 53 (16–107) Zinc mg/100g fresh wt 0.61 ± 0.02 0.39 ± 0.01 0.43 0.39 0.39 (0.20–0.57) Non-essential metals Arsenic ppm fresh wt 0.02 ± 0.00 0.01 ± 0.00 0.02 0.01 Barium ppm fresh wt 3.82 ± 0.67 4.49 ± 0.33 3.37 2.08 Cadmium ppm fresh wt 0.03 ± 0.01 0.02 ± 0.00 0.05 0.06 Chromium (total) ppm fresh wt 0.17 ± 0.04 0.04 ± 0.00 0.05 0.05 Lead ppm fresh wt 0.13 ± 0.02 0.01 ± 0.00 0.04 0.02 Nickel ppm fresh wt 0.09 ± 0.02 0.08 ± 0.01 0.09 0.15 Uranium ppb fresh wt 7.61 ± 1.52 5.00 ± 0.00 Vanadium ppm fresh wt 0.12 ± 0.04 0.02 ± 0.00
Since there was only one market sample from urban farms (Table 1), market samples were excluded from statistical comparisons between urban and rural kale, i.e., comparisons were only made using field samples. Since we had to use mean values for Mann-Whitney U tests, sample sizes (N = 6 mean values for each analyte, one per farm) were too small to produce statistically significant results from urban-rural comparisons, P values are provided in Figs 1–3 and S2, S3 Figs in S1 File and may be indicative of potentially meaningful differences that could be explored in larger studies.
Sample weights (measured after removing the central stem and roots, washing, and drying) were generally lower in urban field samples, while the mass ratios of leaves to stems were generally higher in urban samples. Mean weights of rural field samples were nearly twice those of urban field samples; by contrast, the mean ratio of leaf mass to stem mass was 76% higher in urban samples.
Moisture content was similar between urban and rural samples (means: 88 and 85%, respectively). Controlling for moisture content (i.e., comparing dry weight concentrations) did not meaningfully affect the overall conclusions from any statistical analyses, including differences in analyte concentrations between urban and rural samples, differences by growing practices, and correlation tests. Time between harvesting and storing processed samples in the freezer was not correlated with moisture content (P>0.05).
Sample sizes (N = 6 mean values for each analyte, one per farm) precluded the possibility of statistically significant differences in analyte concentrations between urban and rural field samples. However, P values were as low as 0.06 for arsenic and 0.11 for all carotenoids and vitamins, copper, iron, potassium, zinc, lead, and vanadium (Figs 2 and 3). Mean concentrations of carotenoids and vitamins were generally higher in rural samples. Compared to urban field samples, mean fresh weight concentrations of carotenoids and vitamins were 22–38% higher in rural field samples. By contrast, with the exception of manganese and potassium, mean concentrations of nutritional elements were higher in urban field samples by as much as 413% for iron.
Compared to rural field samples, with the exception of barium, mean concentrations of non-essential metals were higher in urban field samples by as much as 13 times for lead and six times for vanadium (Table 2, Fig 3). That said, lead and cadmium concentrations for all samples, including urban (mean Pb: 0.13 ppm, mead Cd: 0.03 ppm) and rural (mean Pb: 0.01 ppm, mean Cd: 0.02 ppm) field samples, were below the maximum levels (Pb: 0.3 ppm, Cd: 0.2 ppm; Fig 3) specified by the Food and Agriculture Organization of the United Nations (FAO) and the World Health Organization (WHO) [[
One-way ANOVA was used to compare variance between urban and rural farms to variance within urban and rural farms (S5 Table in S1 Data). For six elements (Ba, Ca, Cd, Mg, Ni, P), variance was greater within urban and rural farms; this can also be observed in Figs 2 and 3. These results suggest that differences in production conditions among farms may in some cases have a greater influence on analyte concentrations than farm location being rural or urban. For all 17 other analytes, however, variance was greater between urban and rural farms.
The only market sample provided by urban farms was collected at the market 19 hours after harvest, and had much higher concentrations of six elements (Ca, Cu, Mg, K, Na, Ba), and lower moisture content, compared to field samples from the same farm (Figs 1–3). Given this sample was an outlier in many regards, and because there was only one market sample from urban farms (Table 1), urban farms were excluded from statistical comparisons between field and market samples.
There were no significant differences (P>0.05) in sample weight, leaf:stem ratio, or moisture content between the four mean field and four mean market samples (N = 8), by farm, among rural farms. There were also no significant differences in analyte concentrations between mean field and mean market samples (N = 8) among rural farms, with the exception of dry weight (and not fresh weight) concentrations of calcium (P<0.05). Compared to field samples, mean fresh weight concentrations of nutritional elements and non-essential metals by farm were higher in market samples by as much as 80% for lead and chromium. Mean carotenoid and vitamins concentrations were similar between field and market samples (6% lower to 2% higher in market samples). Differences in moisture content were less than 1%.
The leaf:stem mass ratio was significantly positively correlated with fresh weight concentrations of some carotenoids and vitamin K
Graph: Levels of statistical significance for Pearson's correlations between sample properties (mass, moisture, and time to freezing; table rows) and fresh weight concentrations of nutrients and metals (table columns) in field samples, using the mean values from each farm (N = 6). See S4 Fig in S1 File for dry weight correlations.
There appeared to be some patterns in how certain groups of analytes correlated with others (Fig 5). Fresh weight concentrations of one carotenoid or vitamin, for example, consistently tracked with the others (P<0.05, top left corner of Fig 5); for example, samples high in vitamin K
Graph: Levels of statistical significance for Pearson's correlations between mean fresh weight concentrations of nutrients and metals in field samples, using the mean values from each farm (N = 6). See S5 Fig in S1 File for dry weight correlations.
Analyte concentrations in some cases differed significantly based on growing practices used (Fig 6). Carotenoid and vitamin concentrations, for example, were significantly different between field samples from farms that used drip irrigation vs. samples from farms that did not (P<0.05), with mean concentrations higher among the former. Only rural farms reported using drip irrigation, however, thus any associations between growing practices and analyte concentrations may be confounded by urbanicity. Farms that tested their soil for metals prior to planting generally had lower concentrations of carotenoids and vitamin K
Graph: Levels of statistical significance for Mann-Whitney U tests (N = 6) comparing mean mass, moisture, and fresh weight concentrations of nutrients and metals in field samples (table columns) from farms that used a growing practice vs. those that did not (table rows). Parenthesized values indicate the number of farms, out of six, following each growing practice. See S6 Fig in S1 File for dry weight differences.
Five-minute mean particulate matter concentrations (PM
Collected rainfall ranged from 0–2.2 cm per week (mean: 0.5 cm; median: 0 cm; S8 Fig in S1 File). There were no significant differences among farms or between urban and rural farms.
Peak hourly light intensity for each day ranged from 35–7739 lumens/ft
Peak hourly ambient and in-ground temperatures for each day ranged from 60–142 degrees F (16–61 degrees C) and 59–114 degrees F (15–46 degrees C), respectively, and generally declined with the change in season (S8 Fig in S1 File). Differences between urban and rural farms were highly significant (P<0.001) but negligible in magnitude (difference in overall means: <1 degree F).
Since there was only one market sample from urban farms, all comparisons to prior estimates (Table 2) are based on field samples only. The Safe Urban Harvests (SUH) study analyzed concentrations of nine metals in kale (among other produce items) from urban farms and community gardens in Baltimore City (N = 25) [[
A more direct comparison between kale from the two urban farms in this study and kale from the same two urban farms in SUH indicates metals concentrations were, with some exceptions, higher in the current study by as much as 96% for lead (farm U1); chromium was also 82% higher in this study for one of the farms (U2). These differences may be due to a wide range of factors, including changes in soil metals concentrations (and accordingly, plant uptake) between the two study periods, either due to environmental factors and/or changes in soil management practices; spatial differences in soil metals concentrations; and/or differences in plant uptake between different kale cultivars.
The SUH study also analyzed metals concentrations in conventional and organic kale samples from grocery stores, and samples from farmers market vendors from outside Baltimore City (N = 32). For five of the nine metals included in SUH, mean metals concentrations from rural farms in this study were between 20% and 81% lower compared to non-urban samples from SUH. Some differences are to be expected, in part because grocery store samples included in SUH likely represent industrial-scale farms that operate under qualitatively different conditions than those in the current study. Participating farms did not consistently reply to survey questions about the scale of their operations, but one rural farm (R1) reported harvesting 150 kale plants in a prior year compared to 600 plants from an urban farm (U2), while another rural farm (R2) reported growing 600 square feet (0.01 acres) of kale. For context, the average 2021 farm size in Maryland was 161 acres [[
Field samples from the current study were also compared to estimates of 11 nutritional components for raw kale provided in the USDA FoodData Central Foundation Foods database [[
Although small sample size (N = 6 mean values for each analyte, one per farm) precluded the possibility of statistically significant differences between urban and rural field samples, concentrations of carotenoids and vitamins were generally higher in rural samples, while concentrations of nutritional elements and non-essential metals were generally higher in urban samples, particularly for lead and vanadium. Given element concentrations in kale from urban farms in this study were generally higher than those from prior studies, and concentrations in kale from rural farms in this study were generally lower than those from prior studies, the urban-rural differences observed in this study are to be expected. For some elements, however, ANOVA results suggest differences in production conditions within a farm or among farms may have a greater influence on some analytes than farm location being rural or urban.
Neither environmental conditions nor surveys of growing practices revealed any obvious explanation as to why these differences occurred. Urban farms' use of municipal water is unlikely to explain differences in kale metal concentrations, since the overwhelming majority of irrigation water samples from the SUH study were well below public health guidelines for nine metals [[
Soil conditions could also explain urban-rural differences in kale. Urban soils may be subject to numerous factors that can decrease fertility, including compaction by buildings and heavy machinery which, in turn, can decrease porosity, suppress the activity of beneficial microbes, and inhibit the accumulation of soil organic carbon [[
To mitigate soil quality and contamination concerns, farmers may grow crops in raised beds filled with imported growing media. Both urban farms in the current study reported using some compost, but it was unclear whether they used imported growing media exclusively, or if it was added to native soil. The overwhelming majority (95%, among the 100 farms that responded to questions about soil) of Baltimore's farms and gardens participating in SUH reported growing crops in at least some soil, compost, or mulch brought in from off-site, and two-thirds (69%) used raised beds [[
The health implications of urban-rural differences in non-essential metals are difficult to gauge without a risk assessment that would additionally consider how much kale people typically consume, among other factors. The SUH assessment of nine metals in soil, produce, and irrigation water in 104 urban farms and gardens in Baltimore City did not identify cause for concern [[
Since there was only one market sample from urban farms, statistical comparisons between field and market samples were performed on rural samples only to mitigate confounding. Since field samples were transported directly to the lab for processing and freezing, kale samples harvested from the market might be expected to have lower vitamin and carotenoid concentrations due to the oxidation and/or degradation of those nutrients. There were not, however, any significant differences in levels of potentially labile nutrients (i.e., vitamins and carotenoids) between field and market samples, nor was time from harvesting to freezing correlated with concentrations of any vitamins or carotenoids.
It is also notable that the one urban market sample had much higher concentrations of six elements and lower moisture content compared to field samples from the same farm, which would be consistent with the samples losing moisture over the 19-hour period between harvest and collection at the market, concentrating the elements.
We also observed some statistically significant relationships among analyte concentrations and other properties of the kale samples. The ratio of leaf mass to stem mass, for example, was negatively correlated with some carotenoids and vitamin K
There also appeared to be patterns in how certain groups of analytes correlated with others in field samples. Fresh weight concentrations of one carotenoid or vitamin, for example, consistently tracked with the others; e.g., samples high in vitamin K
The sample size for this study was small (Table 1). Larger sample sizes would be needed to control for multiple potential confounding factors at once, including urbanicity, time between harvesting and freezing samples, and growing practices. We also had only one market sample from one urban farm (which was an outlier in moisture/element concentrations) versus two market samples from each rural farm, which could skew results, thus market samples were excluded from comparisons to prior studies and from most statistical analyses. Given the popularity of farmers markets, future research could explore the potential for nutrient loss between harvest and point of sale, with an eye toward helping producers make decisions about how best to preserve nutrient density.
Some open-ended questions for growers elicited responses that were not comparable across farms because of differences in how they may have been interpreted. In response to a question about pest management, for example, some growers mentioned row covers—which are used for pest management, but also to protect against the elements and retain moisture. Differences in how growers classify the primary use of row covers could have influenced their responses, thus we did not have reliable data on the use of these and certain other practices. Wherever possible, in lieu of open-ended questions, an expanded checklist of growing and supply chain practices (e.g., use of row covers, mulching film, and refrigerated storage for market samples) for use during both questionnaires and farm visits could aid in gathering more viable data, although this would have to be balanced against potentially longer times for survey administration.
Challenges for urban farms observed during this study, including the loss of kale plants and the exclusion of one urban farm due to pest infestations, reflect the realities of growing operations that are often under resourced, understaffed, and/or run by volunteers. More extreme temperatures due to the climate crisis, combined with heat island effects in urban areas, are likely to make urban agriculture an even more challenging endeavor. Future studies on urban agriculture should plan and account for these realities, while exploring policy interventions to help address them.
Kale is a hardy, resilient, and nutrient-dense crop that could serve an increasingly important role in providing an affordable, safe, and nutritious food supply, particularly within the growing urban and community farming movement, as well as in light of the climate crisis and associated effects on extreme weather patterns and food insecurity. With both urban and rural farms filling different roles and priorities in a changing food system, including climate adaptation and resilience, the safety and nutrient density of produce grown in these different environments is of critical importance. Although small sample sizes precluded statistical significance for some analyses, we observed non-significant but potentially meaningful differences between kale grown in urban and rural settings. Absent any compelling evidence to the contrary, however, the health benefits of a varied diet high in fruits and vegetables—regardless of urban or rural origins—likely outweigh any potential risks associated with exposure to non-essential metals, provided growing sites follow recommended practices for soil safety. Our findings also suggest kale in some cases may have the potential to be even more nutrient-dense than previously estimated, although those comparisons are based on a small number of fresh field samples (in this study) with retail samples (USDA) using potentially different varieties of kale. Further research with larger sample sizes could shed more light on the nutritive and metals composition of this important crop and the factors that influence it, particularly given its growing popularity.
S1 Data
S1-S5 Tables.
(XLSX)
S1 File
S1-S8 Figs and baseline farmer questionnaire.
(PDF)
S2 File
(DOCX)
Sauli Elingarami Academic Editor
27 Oct 2023
PONE-D-23-29655Nutrients and Non-Essential Metals in Darkibor Kale Grown at Urban and Rural Farms: A Pilot StudyPLOS ONE
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This work was supported by cooperative agreement 58-8040-8-018 between the US Department of Agriculture Agricultural Research Service and Virginia Tech, and by cooperative agreement 58-8040-8-021 between the U.S. Department of Agriculture Agricultural Research Service and Johns Hopkins University. Sara Lupolt was supported by a dissertation grant from the Johns Hopkins 21st Century Cities Initiative; a pilot award from the Johns Hopkins Education and Research Center for Occupational Safety and Health, supported by the National Institute for Occupational Safety and Health and the US Department of Agriculture Northeast Sustainable Agriculture Research and Education Program (GNE 19-209); and a Johns Hopkins Center for a Livable Future-Lerner Fellowship. Because this was a cooperative agreement, three USDA staff members (Naomi Fukagawa, Carrie Green, and Tiabao Yang) were involved in helping design, conduct, and co-author the study. Other funders had no role in preparing, reviewing, or editing the manuscript. Support for statistical consulting was made possible by The Johns Hopkins Institute for Clinical and Translational Research, funded in part by [Grant Number UL1 TR003098] the National Center for Advancing Translational Sciences, a component of the National Institutes of Health (NIH) and the NIH Roadmap for Medical Research.
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***
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Reviewer #1: General comment:
The pilot study discussed in this paper examines the nutritional elements, non-essential metals, and health-relevant nutrients in Darkibor kale grown in urban and rural settings. Despite its small sample size, the research reveals significant differences in kale composition between these two environments, which has important implications for the future of kale farming and nutrition.
Specific comments:
Constructive feedback:
The paper could be strengthened by providing a more comprehensive explanation of the methodology employed in the study. Additionally, including data on the actual concentrations of the identified elements and nutrients would help readers better grasp the significance of the findings.
While the study rightly mentions that concentrations of metals remained below public health guidelines, it would be beneficial to explicitly state the actual concentrations and compare them to those guidelines for clarity.
Summary:
The significance of kale as a climate-resilient crop is well articulated, and the study motivates further investigation in this area, which is crucial given the increasing popularity of this nutrient-dense vegetable. However, to improve its impact, the paper should provide more detailed information on the methodology and specific concentration levels of elements and nutrients.
Reviewer #2: The manuscript entitled "Nutrients and Non-Essential Metals in Darkibor Kale Grown at Urban and Rural Farms: A Pilot Study" has been reviewed. The manuscript is well written; however, before the final acceptance authors should execute the following minor corrections:
Reviewer #3: This study explored potential differences in nutritional elements, non-essential metals, and certain health-relevant nutrients in Darkibor kale grown at urban versus rural farms. The objectives are clear. The selection of farms for planting and survey are good representatives. The sample harvesting and processing, the analyses of different parameters are well-described. The results collected are meaningful. Although there are constraints such as small sample sizes, which precluded statistical significance for some analyses, this study identified non-significant but potentially meaningful differences in in concentrations of several components between urban and rural kale samples. The paper is well-written. It can be accepted for publication in "PLOS ONE": after making the following revision:
Reviewer #4: PONE-D-23-29655-Nutrients and Non-Essential Metals in Darkibor Kale Grown at Urban and Rural Farms: A Pilot Study
The words "Pilot study" may not be relevant in the publications.
Abstract
Lines 32-34 "In this pilot study we explored potential differences in nutritional elements, non-essential metals, and select health-relevant nutrients in Darkibor kale grown at urban versus rural farms"
What is the meaning of "pilot study" in this context?
What do authors mean by "Darkibor kale"- I guess Darkibor is a local name.
Lines 35-37 "Although small sample sizes precluded statistical significance for some analyses, we identified non-significant but potentially meaningful differences in concentrations of several components between urban and rural kale samples"
Clarify the meaning of "small sample sizes precluded statistical significance for some analyses'
The statements like "we identified non-significant but potentially meaningful differences..." This is quite confusing. Please present direct and explicit information, and given that this is an abstract, then should use catchy statements.
Lines 38-41 "Some urban-rural differences were more pronounced than those identified in prior research, although for some elements, variance within urban and rural farms was greater than variance between urban and rural farms"
Please rephrase the statement. See the bolded section.
Line 45: Introduction
This section was fairly well written though there were no compelling reasons to warrant research in Urban and Rural set up. For instance, are there more fumes as a result of vehicles and hence more heavy metal in urban areas? Let the background show previous work be reviewed showing the critical dissimilarities and find the gap that this work intended to fill. Please also clearly show the objective(s) of study and hypothesis/hypotheses (these should come out at the end of the section).
Line 102: Materials and Methods
This section is quite mixed up and quite confusing. This was supposed to be experimental research. Whereas in rural areas there were four (
The experimental design is not shown but even more fundamental is that the replication does not comes out. For the rural areas, we assume that the four farms represent 4 replicates; which make lots of sense. However, the same cannot be said about the urban area. The two farms cannot be considered as a sufficient replication as this is not analysable.
The authors did not take initial soil samples before the start of the experiments. In that case it is not possible to speculate the possible causes in differences in kales grown in the urban areas compared to rural areas.
Line 103 Farm selection
It is not clear why this is being reported in the manuscript. I fully acknowledge the effort made in the identification of the farms, but the information is not necessary for the purposes of this manuscript. The authors can explain how the population in the rural and urban affect the mineral elements in the soil and hence uptake by the plant varieties.
Line 114 Kale seedling and planting
This subtitle is confusing – unless there were two sets where there was direct planting and transplanting of the seedlings. Can the authors clarify this?
Line 117 There is need to clarify the statement "The seeds were sown in seedling starting trays filled with Fafard growing mix" – see the bolded words
Lines 125-126 "in configurations most suitable for the location, e.g., 1x24, 2x12, 3x8 or 4x6" – the authors need to clarify the spacing as shown in bold above. What these spacing/numbers stand for?
Line 130 Surveys on farm history and growing practices
This section may not be relevant unless during the process (survey), soil samples were picked and analysis made to check the chemical composition (including essential elements, beneficial elements, non-essential or toxic heavy metals and pesticides). Otherwise the way the section is presented, I do not find its relevance, but the authors can explain.
Line 161 "Kale sample processing and storage"
The authors need to justify the relevance of this section. Is it important that the vegetables be transported in refrigerated form yet tissue analysis (usual procedures) require heating at very high temperatures?
Line 180: Nutrient and non-essential metals analyses
In this section, there is need to clearly break down the methods- they cannot be lumped together- e.g. the methods for extraction of heavy metals is quite different compared for instance with the other essential elements. Method for P is totally different from say Ca and so they should be done as subsections. Then the vitamins and carotenoids are also determined using different protocols.
Still under this section, please explain the need for the vegetables being carried in ice box or freezer.
But looking at section starting from line 197- "analytical methods", then you will notice that there is no need for section on "Nutrient and non-essential metals analyses" above since section starting from line 197 is more comprehensive. Maybe have section as "Determination of minerals and vitamins in vegetables" to replace "analytical methods" and do away with "Nutrient and non-essential metals analyses"
However section in lines 209-213 need more elaboration – the analysis part of the metals is clear but the extraction methods not mentioned. A brief mention of the methods with citation of authors that the methods are adapted from can be quite useful.
Line 241: Data reporting and analyses
The contents should have been better be placed under "Results" section starting from section 286.
Why report in ppb instead of ppm? No wonder the metals were below the detectable limit leading to erroneous conclusion that the vegetables are safe (irrespective of whether cultivated in the urban or rural area (see abstract; line 41).
Line 255- there is a formula for conversion from fresh weight to dry weight- why not just dry the vegetables at a prescribed temperature till the weight is stable?
Line 286 Results
Line 287 Kale yields
In this section, reference is made of field samples and market samples – from materials and method section, such categorization was not evident. Can this be elaborated?
Table 1; These are raw data that have not been analysed and hence there is no point of presenting in this section.
Line 301: Farm history and growing practices
The section does fit under results – moreover, this would only be important if that history led to differences in factors like pH, organic carbon and nutrients as these would affect the availability of these elements to the crop. Can the authors explain the relevance of this section as far as the presentation of the results are concerned
Line 338; Kale sample processing and storage
This section does not belong to the "results" section. Is there any reason as to why this section is presented under "results" section?
Line 348: Analytical quality control
The authors present quite interesting results on different types of carotenoids. Unfortunately such differentiations/distinctions were not presented under "materials and methods' section.
Table 2: the heavy and essential elements are reported in terms of mg/100mg fresh weights. It is not common to report metal concentrations in fresh weights, instead dry weight is used. Any reasons for this exception?
And for the case when the concentration is in ppm fresh weight – why not mg/kg since we are dealing with solid particles and not liquid?
Note that here the reporting is in ppm yet in the materials and method the authors always made reference of ppb.
There is a column for "current mean- Urban" and "current mean, rural" – were these results analysed? Please use t-test to compare the two. But since the mean for urban had can only two replications, is it possible to have calculation using two replications?
In the figures, the symbols U1, U2, R1, R2, R3 and R4 are used yet there was no mention of these under "materials and method" section. It would have been polite for the authors to have figure captions where all these could be explained. Otherwise the way they are, it is not possible to interpret.
It is quite interesting that almost all the results show p-values more than 0.05 hence not significant. Is there possible reason? Could it be that calculation was not well done?
Discussion
Since the methodology needs serious reworking (a lot need to be cut out – especially the survey part and only remain with experimental components). And once the result section is more comprehensive and clear (cut out on survey and reduce the subsections under results; same with discussion). Once there is that kindly harmony, it is too premature to take a look at the discussion.
Reviewer #5: The material and method section is too narrative, and contains a lot of redundant information. It should be more concise and clear. E.g. Line 135 Redundant information. Line 162 the same.
Why authors choose different digestion methods for essential vs trace elements analysis?
Lines 516-519 already mentioned, not part of the discussion
Reviewer #6: In the manuscript entitled "Nutrients and Non-Essential Metals in Darkibor Kale Grown at Urban and Rural Farms: A Pilot Study", the authors studied a pilot plant to explore potential differences in nutritional elements, non-essential elements, and some health-relevant nutrients in kale grown at urban versus rural farms. The manuscript is very relevant and interesting. However, the authors need to clarify some parts before publication on Plos one.
The following are my comments and suggestions:
General comments:
The authors need to revise all the units and the effective numbers in the whole manuscript.
Abstract:
The abstract needs to contain all parts of the manuscript. So the authors need to improve this topic, including parts of the methodology. The authors need to insert some results (numbers) , for example, when the authors said "Concentrations of elements were generally higher in kale from urban farms", how much higher? A conclusion sentence about their work is necessary too.
Introduction:
The introduction is interesting and contextualizes the environmental problem for the reader. However, the authors should insert more recent references in the Introduction and it´s essential to describe the potentially toxic metal levels allowed in the national and international legislation. Also, when the authors said on page 4 lines 83-84"concentrations of essential and non essential (arsenic (As), barium (Ba), cadmium (Cd), chromium (Cr), lead (Pb)) metals in.." trivalente chromium is essential for the human being. The authors need to research more specific references about it. Please, pay attential to this part including about chemistry speciation.
Page 5: "Building upon the existing body of evidence.." The authors need to write more about the aim of the work. They determined a lot of importance parameters obtaining different kind of results, but what is the main question to be answered?
Materials and methods:
Page 06: The authors need to standardize the date. Sometimes the authors write day, month and year and other times no.
Page 10: The authors need to explain more about the digestion process, including more information as the weight of the samples, temperature, final volume, acid concentration, etc.
Page 10: The authors need to write more about organic and inorganic determination as lines of emissions, analytical concentration curve, detection and quantification limits. The authors need to mention the replicates here too.
Page 12, line 255: The authors need to include the reference for this equation.
Results and discussion
In general, the authors need to discuss more the results comparing with the literature. The authors need to explore further the parts containing discussion and results, for example, in the topic "Correlations among analytes", the authors said "We also observed some statistically significant relationships among analyte concentrations and other properties of the kale samples.", the authors need to discuss and to say more specifically about all the parameters studied. In the sentence, "Similarly, concentrations of elements and metals were in some cases correlated with one another. ", the authors need to specify the elements here. What´s the diference between metals and elements?
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11 Dec 2023
Please refer to the attached PDF for responses to reviewer comments.
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Sauli Elingarami Academic Editor
20 Dec 2023
Nutrients and Non-Essential Metals in Darkibor Kale Grown at Urban and Rural Farms: A Pilot Study
PONE-D-23-29655R1
Dear Dr. Keeve. E. Nachman,
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Reviewer #3: All comments have been addressed
Reviewer #7: All comments have been addressed
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3. Has the statistical analysis been performed appropriately and rigorously?
Reviewer #3: Yes
Reviewer #7: Yes
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The
Reviewer #3: Yes
Reviewer #7: Yes
***
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Reviewer #3: The authors have addressed all of my concerns with the original manuscript. The revised manuscript can be now accepted for publication.
Reviewer #7: Authors have addressed all the comments and suggestions of all reviewers and have done good efforts for improving the manuscript. Therefore, I consider that it is now I consider that their R1 version of the manuscript may be accepted for publication.
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Reviewer #3: No
Reviewer #7: No
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Sauli Elingarami Academic Editor
2 Apr 2024
PONE-D-23-29655R1
PLOS ONE
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The authors thank the Maryland farmers who participated in the study; Johns Hopkins Center for a Livable Future's research assistants Trent Dilka, Andrea Chiger, and Ruth Young for assistance with fieldwork and other support; Nancy Pennington, who did protocol and instrument training and assisted with distributions of samples and controls to labs; Ryan McGinty, who also assisted with distribution of samples and controls, and ran the moisture and vitamin C analyses; and Kit Carson at the Johns Hopkins Institute for Clinical and Translational Research, who provided guidance on statistical methods.
By Brent F. Kim; Sara N. Lupolt; Raychel E. Santo; Grace Bachman; Xudong Zhu; Tianbao Yang; Naomi K. Fukagawa; Matthew L. Richardson; Carrie Green; Katherine M. Phillips and Keeve E. Nachman
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