The occurrence of nitrate and nitrite in Mediterranean fresh salad vegetables and its modulation by preharvest practices and postharvest conditions

Accepted Manuscript The occurrence of nitrate and nitrite in Mediterranean fresh salad vegetables and its modulation by preharvest practices and postharvest conditions Marios C. Kyriacou, Georgios A. Soteriou, Giuseppe Colla, Youssef Rouphael PII: DOI: Reference:

S0308-8146(19)30272-9 https://doi.org/10.1016/j.foodchem.2019.02.001 FOCH 24278

To appear in:

Food Chemistry

Received Date: Revised Date: Accepted Date:

30 March 2018 28 January 2019 3 February 2019

Please cite this article as: Kyriacou, M.C., Soteriou, G.A., Colla, G., Rouphael, Y., The occurrence of nitrate and nitrite in Mediterranean fresh salad vegetables and its modulation by preharvest practices and postharvest conditions, Food Chemistry (2019), doi: https://doi.org/10.1016/j.foodchem.2019.02.001

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The occurrence of nitrate and nitrite in Mediterranean fresh salad

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vegetables and its modulation by preharvest practices and postharvest

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conditions

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Marios C. Kyriacoua,†,*, Georgios A. Soterioua,†, Giuseppe Collab, Youssef Rouphaelc

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aDepartment

of Vegetable Crops, Agricultural Research Institute, Nicosia, Cyprus

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bDepartment

of Agriculture and Forestry Sciences, University of Tuscia, 01100 Viterbo,

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Italy

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cDepartment

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Italy

of Agricultural Sciences, University of Naples Federico II, 80055 Portici,

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*Corresponding author. Tel.: +35722403221; Fax.: +35722316770

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E-mail address: [email protected] (Marios C. Kyriacou)

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†Authors

of equal contribution

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Running title: Nitrate/nitrite in Mediterranean vegetables

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Abstract

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Winter and summer nitrate/nitrite concentrations in 11 salad vegetables were surveyed

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using a validated HPLC-DAD method. Nitrate was highest in rocket, both in winter

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(x̅=3974 mg kg-1 fw) and summer (x̅=3819 mg kg-1 fw). High nitrate accumulators

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included spinach, purslane, chards, dill, coriander and parsley. Wide intra-species

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variability and levels in excess of permitted maxima highlighted the importance of

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monitoring vegetable production methods to protect consumer health. Occurrence of

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detectible nitrite (14-352 mg kg-1 fw) was most frequent in winter head cabbage. Three

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additional experiments examined the seasonal effects of nitrogen (N) fertilization rate,

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application method, formulation and postharvest storage on nitrate and nitrite levels in

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lettuce, rocket and spinach. Violation of current nitrate limits is likely when total N

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exceeds 200 kg ha-1, particularly in rocket and spinach. Postharvest nitrate reduction

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requires exogenous microbial nitrate reductase activity, which is unlikely to be achieved

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without visible loss of quality.

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Keywords: Cold storage, HPLC-DAD, lettuce, nitrogen fertilization, rocket, spinach.

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1. Introduction

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Natural compounds such as nitrate (NO3-) and nitrite (NO2-), which are involved in the

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nitrogen (N) cycle, are also present in a wide range of foodstuffs as well as drinking water

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and, to a lesser extent, food additives (EFSA, 2008). For nitrate, intake is predominant via

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vegetables, in particular leafy greens, which tend to accumulate higher concentrations in

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their leaves compared to bulbs, fruits, roots, seeds and tubers. (Colla, Kim, Kyriacou, &

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Rouphael, 2018). Among widely consumed leafy greens, lettuce, rocket and spinach are

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considered typical nitrate-accumulating species (Colla et al., 2018).

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According to the scientific risk assessment on nitrate in vegetables, requested by the

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European Commission and adopted by the European Food Safety Authority (EFSA, 2008),

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nitrate per se is relatively harmless, since the toxic threshold (>7-35 g) for nitrate is much

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higher (100-fold) than the acceptable daily intake of 3.7 mg nitrate kg-1 body weight

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adopted by the joint FAO/WHO Expert Committee on Food Additives (FAO/WHO, 2003a,

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2003b) and the European Union. Moreover, consumption of nitrate-containing plant foods

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has been linked to certain beneficial effects on human health, such as lowering of blood

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pressure and improved cardiovascular function (Hord, Tang, & Bryan, 2009). However,

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nitrate reaction products and metabolites, including nitrite, nitric oxide and N-nitroso

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compounds, have potentially adverse health implications associated most notably with

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gastric and bladder cancers as well as the methaemoglobinaemia syndrome (Colla et al.,

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2018; EFSA, 2008; Parks, Huett, Campbell, & Spohr, 2008). These concerns make nitrate

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of regulatory importance and have prompted the European Commission to set maxima for

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nitrate to regulate commercialization of nitrate-accumulating leafy greens (EC, 2006), i.e.

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summer lettuce (Lactuca sativa L.) grown in open air (3000 mg kg-1 fw); summer lettuce

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grown under cover (4000 mg kg-1 fw); winter lettuce grown in open air (4000 mg kg-1 fw);

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winter lettuce grown under cover (5000 mg kg-1 fw); ‘Iceberg’ type lettuce grown in open

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air (2000 mg kg-1 fw); ‘Iceberg’ type lettuce grown under cover (2500 mg kg-1 fw); fresh

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spinach (Spinacia oleracea, 3500 mg kg-1 fw); salad and wild rocket (Eruca sativa,

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Diplotaxis sp., Brassica tenuifolia, Sisymbrium tenuifolium, 6000-7000 mg kg-1 fw).

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The accumulation of nitrate in raw leafy vegetables depends upon several preharvest

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factors, such as the genetic material (species and genotype), cultural practices (i.e., amount,

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timing, concentration, method and form of N application), environmental conditions during

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the growth cycle (i.e., light intensity and quality, air temperature and carbon dioxide

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concentration), developmental stage at harvest and, also, diurnal variation (Colla et al.,

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2018; Colonna, Rouphael, Barbieri, & De Pascale, 2016; Fallovo, Rouphael, Rea,

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Battistelli, & Colla, 2009; Petropoulos, Constantopoulou, Karapanos, Akoumianakis, &

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Passam, 2011; Santamaria, 2006). However, the three main preharvest determinants of

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nitrate in leafy vegetables are plant genetics, light intensity and N supply (Amr & Hadidi,

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2001).

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Uptake of nitrate by the roots and its transfer through the transpiration stream to the

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leaves, where accumulation and assimilation take place, is terminated at harvest (Riens &

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Heldt, 1992). Putative postharvest reduction of nitrate must, therefore, rely on endogenous

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conversion of accumulated nitrate to nitrite, which may be influenced by postharvest

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handling and storage conditions. Critical postharvest factors for nitrate reduction include

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product sanitation treatments (Dangour, Dodhia, Hayter, Allen, Lock, & Uay, 2009; Ilić &

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Šunić, 2015) and storage temperature (Alexander et al., 2008; Ekart, Hmelak Gorenjal,

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Madorran, Lapajne, & Langerholc, 2013). However, the temperature range effective in

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eliciting postharvest changes in nitrate and nitrite levels seems associated with species

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perishability and storage duration (Chung et al., 2003; Ferrante, Incrocci, Maggini,

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Tognoni, & Serra, 2003; Ilić & Šunić, 2015; Kim & Ishii, 2007; Konstantopoulou et al.,

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2010; Koukounaras, Siomos, & Sfakiotakis, 2010). Moreover, reported changes in nitrate

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levels occurring during postharvest storage are often marked by inconsistency, as they

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depend on the overall quality of stored products and are favoured by adverse storage

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conditions (Chung, Chou, & Hwang, 2004; Lin & Yen, 1980).

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Mediterranean countries are characterized by climatic conditions ranging from

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temperate to sub-tropical, which are ideal for open-field leafy vegetable production

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throughout most of the year. In addition, this region is characterized by high irradiance

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intensity across growing seasons, which promotes high nitrate reductase activity and,

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presumably, decreased nitrate accumulation in leafy vegetables grown under both

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greenhouse and open-field conditions. However, currently, there is a scarcity of

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information about the range of nitrate and nitrite levels occurring in common salad crops

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produced in south Mediterranean countries and available to the retail market during winter

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and summer seasons. In addition, a composite examination of the relative effects of critical

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preharvest practices, such as N application rate, form and method of application, and

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postharvest storage conditions on nitrate and nitrite levels in leafy vegetables, is lacking.

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In order to examine seasonal variability in the nitrate and nitrite levels, a market survey

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was performed using a validated HPLC-DAD method on 11 salad vegetables

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representative of the south Mediterranean market during the winter and summer seasons.

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Furthermore, two open-field experiments were carried out to assess the effect of N

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application rate, method of application (basal or top-dressing) and N form on the nitrate

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and nitrite levels in three leafy vegetables (lettuce, rocket and spinach) currently regulated

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for maximum nitrate levels according to European Commission Regulation Nº 1881/2006

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and subsequent amendments. A fourth experiment examined the combined effects of N

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application rate and postharvest storage on the nitrate and nitrite concentrations of lettuce,

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spinach and rocket. The knowledge gained from this research will contribute toward

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defining the potential variability in nitrate and nitrite levels of salad vegetables produced in

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Mediterranean countries and the extent to which this variability is governed by agro-

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environmental and postharvest factors. Such knowledge might prove critical in developing

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appropriate agricultural and postharvest practices for controlling nitrate and nitrite

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accumulation in salad vegetables and promoting their safe consumption.

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2. Materials and methods

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2.1. Market survey of seasonal variation in nitrate and nitrite levels of eleven salad crops

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A market survey was performed during the winter and spring-summer periods, as

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defined by European Commission Regulation (EC) No 1881/2006, on 11 non-packaged,

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commonly-consumed fresh salad vegetables that were purchased repeatedly from eight

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different retail chain stores in Cyprus. The number of samples per species, retail outlet and

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purchase round varied according to availability. Sampled vegetables were: head cabbage

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(Brassica oleracea L. var. capitata L. cv. Bajonet), cellery leaves and stalks [Apium

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graveolens L. var. dulce (Mill.) DC. cv. Verde Da Taglio], coriander (Coriandrum sativum

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L., local landrace), dill (Anethum graveolens L. cv. Domino), chards (Beta vulgaris L.

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subsp. vulgaris, local landrace), Cos type lettuce (Lactuca sativa L. var. longifolia cv. Paris

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Island), purslane (Portulaca oleracea L., local landrace), parsley [Petroselinum crispum

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(Mill.) Fuss cv. Gigante D’Italia], rucola [Eruca vesicaria L. Cav. subsp. sativa (Mill.)

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Thell. cv. Green Salad] and spinach (Spinacia oleracea L. cv. Nagano). Single samples,

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consisting of single heads or bunches, were placed in open plastic bags and stored in cool

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boxes to avoid wilting during transfer to the Agricultural Research Institute postharvest

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laboratory in Nicosia, which was - in all cases - completed within an hour of purchase.

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Samples visually defective were discarded and impurities, such as soil particles, were

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removed. Samples were rinsed sequentially in tap water and distilled water, dried in a

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manually-operated spin drier, and treated for analyses, as described below.

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2.2. Sample preparation, nitrate and nitrite analysis

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The edible part of each head or bunch was retained by removing any damaged or

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conspicuously senescent leaves and trimming the basal 5 cm of the petioles from each

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bunch. The remaining fresh mass was blended into a thick homogenous slurry using a Vita

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Prep 3 blender (Vita-Mix Corp., Cleveland, USA) operated at low speed to prevent

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foaming. Homogenate samples not immediately extracted for analyses were placed in 50

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ml sterilized Falcon tubes, frozen in liquid nitrogen and stored at -80 °C.

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From each homogenate, three subsamples of 1.0 g were weighed on a Precisa XT120A

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analytical balance, then suspended in 40 ml ultrapure water in 50 ml falcon tubes and

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agitated gently (80 rpm) for 20 min at 80 °C in a shaking waterbath (OLS200, Grant

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Instruments, Shepreth, UK). The suspension was allowed to cool before being passed

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through Whatman-1 filter papers (GE Healthcare UK Limited, Little Chalfont, UK) and

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made up to a final volume of 100 ml with additional ultrapure water. Nitrate and nitrite

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analysis of fresh samples was performed by liquid chromatography following a

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modification of the method of Chou, Chung, and Hwang (2003).

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An Agilent HPLC system (Agilent Technologies, Santa Clara, USA) equipped with a

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1200 Series quaternary pump and a 1260 Series diode array detector (DAD) commanded

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by ChemStation software was employed. Separation was performed on a 4.6 × 250 mm 5-

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micron Agilent Zorbax Eclipse DDB-C18 column thermostated at 30 °C. A mobile phase

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of 0.01 M octyl-ammonium phosphate was used, which consisted of 0.01 M octyl-amine in

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20% methanol aqueous solution corrected to pH 6.5 with phosphoric acid and vacuum

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filtered through a Durapore® 0.45 μm membrane filter (Merck Millipore, Darmstadt,

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Germany). Samples were passed through Agilent PVDF 13 mm 0.22 μm syringe filters

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before being loaded. Injection volume was 10 μL and an isocratic flow rate was set at 1.0

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ml min-1 with a total run time of 14.0 min. Two injections per subsample were performed.

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Absorbance was recorded at 220 nm. Typical retention times for nitrite and nitrate were 7.9

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and 9.8 min, respectively (Fig. 1). Quantification was performed against external nitrate

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and nitrite standard calibrating curves of 1-100 mg L-1 with a coefficient of determination

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(R2) >0.9999. Recovery trials performed under the same operating conditions were, in all

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cases, in the order of 100%. For nitrate, the limit of detection (LOD) was 0.150 mg L-1 and

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the limit of quantification (LOQ) was 0.501 mg L-1. For nitrite, the LOD was 0.092 mg L-1

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and the LOQ was 0.031 mg L-1.

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2.3. N rate, mode of application, formulation and seasonal effects on nitrate and nitrite

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2.3.1. Base and top-dressing N rate effects on lettuce, rocket and spinach

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Experiments for lettuce (Lactuca sativa L. cv. Paris Island), rocket (Eruca sativa cv.

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Green Salad) and spinach (Spinacia oleracea L. cv. Nagano) were carried out in successive

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winter (December 2015 - February 2016) and spring-summer (April - June 2016) periods,

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conforming to the two periods prescribed by European Commission Regulation (EC) No

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1881/2006 for setting maximum nitrate levels in these crops.

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The experiments examined the effects of base-dressing rate (0, 100, 150, 200 kg N ha-1)

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and top-dressing rate (0, 50, 100, 150 kg N ha-1) on nitrate and nitrite concentrations of

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fresh edible tissues from field-grown lettuce, rocket and spinach. All base-dressing

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applications of N were in the form of ammonium nitrate (34.5-0-0; NH4NO3) and were

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combined with 120 kg P2O5 ha-1 and 50 kg K2O ha-1 in the form of triple superphosphate

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[0-0-48; (Ca(H2PO4)2)] and potassium sulphate (0-0-52; K2SO4).

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All base-dressings were incorporated into the soil mechanically prior to planting. Top-

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dressing rates were split in two applications, except for spring-summer rocket where a

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single application was dictated by the short crop cycle. Crop duration was approximately

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40, 45 and 84 days during the winter season and 41, 24 and 46 days during the spring-

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summer season for spinach, rocket, and lettuce, respectively. Top-dressing rates were

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applied in the form of ammonium nitrate solutions delivered through the irrigation system.

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Each plant was irrigated by a Netafim® labyrinth-type dripper (Hatzerim, Israel) supplying

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about 3.8 L h-1 at a pressure of 152 kPa.

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Each experimental plot accommodated 72 plants arranged in four rows spaced 0.30 m

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apart, with plants spaced 0.33 m within rows, resulting in a density of approximately

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101000 plants ha-1. The two outer rows and the first and last plant of each middle row were

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‘guards’ and were not sampled. There were four replicate plots per treatment arranged in a

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randomized design.

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Lettuce plants consisted of a single seedling delivering a single head, while rocket and

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spinach plants were planted as aggregates of seven seedlings delivering one bunch. All

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seedlings were produced by Solomou Nurseries (Nicosia, Cyprus) and were transplanted to

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the field at the second to third true leaf stage. Standard pest and disease control practices

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were applied. Harvesting was performed when the fresh weight for lettuce heads, spinach

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bunches and rocket bunches reached a minimum of 400, 150 and 170 g, respectively. In

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each experimental plot, eight plants were sampled, harvested between 07:00-09:00 hours.

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The experiments were performed at the Zygi Experimental Station (34° 44' 00" N; 33°

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20' 15" E) of the Agricultural Research Institute of Cyprus, where the prevailing climate is

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typical Mediterranean. Natural precipitation fell mostly between November and March.

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Mean day-time temperature were 11-17 ºC during winter and 19-25 ºC during spring. The

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mean daily solar radiation during the winter growing season was 11.3 Mj/m2 and the

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radiation range during harvest of the three cultivated species was 11.4-11.6 Mj/m2. The

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mean daily solar radiation during the spring growing season was 23.3 Mj/m2 and the

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radiation range during harvest of the three cultivated species was 25.0-25.3 Mj/m2.

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2.3.2. Base-dressing N formulation effect on winter rocket

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The experiment to examine the effect of nitrogenous fertilizer formulations, delivered

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as base-dressings, on the nitrate and nitrite concentrations of the edible tissue of rocket

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(Eruca sativa cv. Green Salad) was carried out during the winter season (January 15th –

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March 6th). Four inorganic N fertilizers were applied, as base-dressings, at the rate of 100

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kg N ha-1: ammonium nitrate (34.5-0-0), ammonium sulphate (21-0-0) and slow release

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urea ENtec (46-0-0). Moreover, a base-dressing treatment with organic fertilizer (10-3-3),

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in the form of granulated chicken manure, was examined, also at 100 kg N ha-1. All N

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base-dressing treatments were incorporated mechanically into the soil prior to planting

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with supplementary triple superphosphate (0-0-48) and potassium sulphate (0-0-52) at 120

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kg P2O5 ha-1 and 50 kg K2O ha-1. The experiment was set up in a randomized design with

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four replicate plots per treatment. Sampling was performed as in the previous experiments

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described above.

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2.4. Postharvest storage of fresh samples

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The effects of total N rate (0, 100, 200, 250 and 300 kg N ha-1 applied as combined

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base and top-dressings) and postharvest storage were examined on nitrate and nitrite

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concentrations of winter spinach, summer and winter rocket, and summer lettuce. Base-

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dressing N applications were applied in all treatments, except the control, at 100 kg N ha-1,

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delivered in the form of ammonium nitrate, accompanied by supplemental 120 kg P2O5 ha-1

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and 50 kg K2O ha-1, as described above. 200, 250 and 300 kg ha-1 total N rate treatments

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were attained with supplemental top-dressings of 100, 150 and 200 kg N ha-1 delivered in

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the form of ammonium nitrate through the irrigation system. Samples comprised

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aggregates of eight heads (lettuce) or bunches (spinach, rocket). One sample corresponding

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to each storage treatment was harvested from each of the four randomized (CRD) replicate

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plots for the five fertilizer treatments. Samples were placed in open, biaxially oriented

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polypropylene bags (BOPP) and transferred, in a refrigerated van set at 10 °C, to the

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facilities of the Agricultural Research Institute within 1 hour from harvest.

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Samples were subsequently packaged in 40-micron thickness BOPP bags sized 50 × 70

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cm perforated with 4.0 mm holes at 10000 holes m-2 density. Initially, the bags were placed

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open inside cold chambers to allow for temperature equilibration and avoid condensation.

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Cold chambers were kept dark and set at 5 ± 0.5 ºC for lettuce (0, 2, 3, 4 days), spinach (0,

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4, 8, 12 days) and rocket (0, 5, 10, 15 days), and at 22 ± 0.5 ºC for lettuce (0, 2, 3, 4 days)

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while ambient relative humidity was maintained at 90% by means of Carel, UE001PD000

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humidifying systems (Carel Industries S.p.A., Brugine PD, Italy). At the end of each

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storage interval, samples were weighed, inspected for overall appearance and,

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subsequently, prepared for analysis, as described above.

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2.5. Statistical analysis

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Market survey data were subjected to descriptive statistics; frequencies of samples with

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detectible nitrite and nitrate levels above legal limits are reported. Data obtained from

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factorial experiments were subjected to analysis of variance (ANOVA). Where a

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significant effect was noted, and in the absence of any interactions, mean comparisons

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were performed according to Tukey–Kramer HSD test. All statistical analyses were

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executed using SAS 9.1. statistical package (SAS Institute, Cary, NC, USA).

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3. Results

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3.1. Seasonal variation in nitrate and nitrite levels of common salad crops

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A market survey conducted on 11 salad vegetables procured from eight chain retail

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stores during the summer season revealed a mean nitrate content across products of 2009

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mg kg-1 fw, with the lowest mean content in celery stalks (334 mg kg-1 fw) and the highest

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in rocket (3819 mg kg-1 fw; Table 1). During the winter season, the same products

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purchased from the same retail outlets, demonstrated an overall mean nitrate content of

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1892 mg kg-1 fw, with the lowest mean content encountered in head cabbage (251 mg kg-1

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fw) and the highest in rocket (3974 mg kg-1 fw). Maximum nitrate levels encountered in

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lettuce, spinach and rocket - crops presently covered by European Commission (EC)

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Regulation No 1881/2006 - were 2151, 5767 and 6946 mg kg-1 fw, respectively, in the

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summer, and 2497, 2698 and 8279 mg kg-1 fw, respectively, in the winter. Mean nitrate

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levels for summer and winter samples of lettuce, spinach and rocket were below the

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maxima set by the EC Regulation No 1881/2006. However, individual samples in excess of

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permitted levels were identified in 1/39 samples of summer rocket and in 1/25 samples of

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spinach whereas, among winter samples, excess was identified in only 3/44 rocket samples.

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Concerning the occurrence of nitrite, quantifiable levels in the summer season were

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detected in 1/20 samples of head cabbage (49 mg kg-1 fw) and in 1/14 samples of dill (12

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mg kg-1 fw). In the winter season, quantifiable levels of nitrite were identified in 10/20

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samples of head cabbage (mean = 135 mg kg-1 fw), in 1/40 samples of coriander (58 mg

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kg-1 fw), in 3/24 samples of dill (mean = 35 mg kg-1 fw) and in 5/48 samples of spinach

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(mean = 52 mg kg-1 fw). None was in excess of permitted levels.

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3.2. Effects of base-dressing and top-dressing N rates on nitrate and nitrite contents

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In the summer season, spinach nitrate levels increased in response to base-dressing N

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rate above 100 kg ha-1 (Table 2), whereas top-dressing had no significant effect on nitrate

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levels. In the case of winter spinach, nitrate levels were affected both by base-dressing and

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top-dressing. Nitrate levels were increased by all base-dressing N applications by an

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average of 89.9% compared with the control. A trend for increased nitrate levels was

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observed when N rates increased from 100 to 200 kg ha-1, but mean differences were not

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statistically significant. Nitrate levels demonstrated a trend for increase with increasing

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top-dressing N rates, but nitrate levels significantly higher than control were attained only

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at 100 and 150 kg ha-1. All treatment nitrate means were below the 3500 mg kg-1 maximum

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set for summer and winter spinach alike, as stated by EC Regulation No 1881/2006. In

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summer spinach, no individual samples exceeded the nitrate maximum of 3500 mg kg-1.

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However, quantifiable nitrite levels of 42.8, 271.2 and 99.3 mg kg-1 were detected in three

295

samples corresponding to 200, 250 and 300 kg ha-1 total N rate, respectively, (Table 3).

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Quantifiable nitrite levels were not detected in any winter spinach samples, although five

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samples - corresponding to 250-350 kg ha-1 total N rate - exceeded nitrate maximum,

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ranging from 3434.8 to 3946.0 mg kg-1.

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Summer season lettuce nitrate levels incurred a significant increase with respect to

300

base-dressing N rates, whereas top-dressing had no effect (Table 2). In the winter crop,

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both base and top-dressing N applications affected nitrate levels of the harvested fresh

302

product. All base-dressing N rates resulted in higher nitrate levels than the control, with

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200 kg N ha-1 producing the highest (1553 mg kg-1) and accounting for a 67.3% increase

304

compared with the control. Top-dressing with 50 kg N ha-1 did not change nitrate levels in

305

the final product significantly compared to the control, however the 100 and 150 kg ha-1 N

306

rates yielded similar nitrate levels, which were on average 38.0% higher than the control.

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No samples were detected with quantifiable nitrite or nitrate levels exceeding the maxima

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set by EC Regulation No 1881/2006 for summer (3000 mg kg-1) and winter (4000 mg kg-1)

309

lettuce grown in open air (Table 2).

310

Nitrate levels in summer rocket were affected by both base-dressing and top-dressing N

311

(Table 2). Significant increases in nitrate levels were obtained at 150 and 200 kg N ha-1

312

base-dressing, which were 70.2% and 76.6% higher than the control. A trend for increased

313

nitrate levels was apparent with increasing top-dressing N rates, but a significant increase

314

(67.6%) in nitrate compared to the control was, in fact, only observed at 150 kg N ha-1. In

315

winter rocket, nitrate levels increased significantly with increasing base-dressing rate

316

between 0-150 kg N ha-1 with a mean increase of 37.8% (1830 mg kg-1) at 150-200 kg ha-1

317

compared to the control. Top-dressing of winter rocket raised nitrate levels (1041 mg kg-1)

318

significantly by 18.7% only at the maximal rate of 150 kg N ha-1. Quantifiable nitrite levels

319

were not detected in either summer or winter rocket. However, excess nitrate (6190.2 and

320

6418.9 mg kg-1) were found in two samples of summer rocket, corresponding to 250 and

321

350 kg ha-1 total N rate, and 10 samples of winter rocket ranging from 7073.4 to 8892.1 mg

322

kg-1 and corresponding to total N rates of 150 to 350 kg ha-1 (Table 3).

323 324

3.3. Effects of N fertilizer formulations on rocket nitrate and nitrite contents

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All the nitrogenous fertilizers tested as base-dressing applications at 100 kg N ha-1 on

326

field grown winter rocket increased mean nitrate levels significantly compared to the

327

control (Table 4). No differentiation was observed in nitrate levels between the inorganic

328

and organic fertilizer formulations applied. All samples were found below the 7000 mg

329

nitrate kg-1 maximum set by European Commission Regulation (EC) No 1881/2006 in

330

winter rocket and nitrite levels were not quantifiable in any sample.

331 332

3.4. Effects of N rate and postharvest storage on nitrate concentrations

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Total N rate, delivered as 100 kg ha-1 base-dressing and the rest as top-dressing, had a

334

significant effect on postharvest nitrate levels of winter spinach held at 5 °C, summer and

335

winter rocket held at 5 °C, and summer lettuce held at 5 and 22 °C (Table 5). Postharvest

336

nitrate levels in winter spinach increased linearly with increasing N rates, from 698 mg kg-1

337

in the control to 3087 mg kg-1 at the top N rate of 300 kg N ha-1. Linear responses in

338

postharvest nitrate levels to total N rate were also demonstrated in summer rocket, with

339

mean nitrate concentration ranging from 1885 to 3781 mg kg-1, and in winter rocket, with

340

mean nitrate concentration ranging from 3524 to 6982 mg kg-1. Linear responses of

341

postharvest nitrate levels to total N application rate were also observed in summer lettuce,

342

ranging from 412 to 1563 mg kg-1 in samples held postharvest at 5 °C, and from 339 to

343

1708 mg kg-1 in samples held postharvest at 22 °C. Violation of the nitrate maxima set by

344

European Commission Regulation (EC) No 1881/2006 occurred in 12 winter spinach

345

samples ranging from 3477 to 4403 mg kg-1, two summer rocket samples (6149 and 6190

346

mg kg-1) and 20 samples of winter rocket, ranging from 6969 to 9282 mg kg-1 (Table 3).

347

Postharvest storage of winter spinach for 0-12 d at 5 °C, summer and winter rocket for 0-

348

15 d at 5 °C and summer lettuce held for 0-4 d at both 5 and 22 °C had no significant effect

349

on the levels of nitrate in these products. Moreover, no quantifiable levels of nitrite were

350

detected in any of the products held under the conditions described.

351 352

4. Discussion

353

4.1. Seasonal variation in nitrate and nitrite levels of common salad crops

354

Leafy vegetables constitute unequivocally food sources high in nitrate. The

355

translocation of nitrate from the roots to the epigeal plant organs is mainly passive,

356

facilitated by the transpiration stream through the xylem, and terminates in the leaf laminae

357

(Colla et al., 2018). A survey conducted on winter and summer market vegetables in the

358

context of the present study focused on leafy vegetables, stalk vegetables and herbs, all of

359

which are common Mediterranean salad ingredients. The highest mean nitrate levels were

360

identified in rocket, both during winter (3974 mg kg-1 fw) and summer (3819 mg kg-1 fw).

361

Less conventional leafy vegetables, such as purslane and chards, and fresh herbs, including

362

dill, coriander (cilantro) and parsley, were also high nitrate accumulators in the winter, and

363

dill, chards, coriander, spinach and parsley in the summer. Although lettuce is considered a

364

nitrate accumulating species (Colla et al., 2018; EFSA, 2008), it was ranked second lowest

365

both in summer (843 mg kg-1 fw) and winter (1144 mg kg-1 fw) in this study.

366

Several previous studies have pointed to the importance of irradiance and

367

photosynthesis in furnishing electrons to support active uptake of nitrate and the activity of

368

nitrate reductase, and in furnishing carbon skeletons for assimilation of ammonium ions

369

resulting from nitrite reduction (Cavaiuolo & Ferrante, 2014). Such activity is likely to be

370

favoured during the spring-summer period, as opposed to the lower irradiance and

371

photoperiod of the winter period, which is conducive to nitrate accumulation (Santamaria,

372

2006). Studies performed under controlled environments have demonstrated that reduced

373

light intensity can adversely affect nitrate reductase activity and promote nitrate

374

accumulation in species such as lettuce and spinach (Fallovo et al., 2009; Proietti,

375

Moscatello, Leccese, Colla, & Battistelli, 2004).

376

In the present study, six out of 11 species surveyed (rocket, lettuce, purslane, celery

377

leaves, celery stalks and chards) demonstrated higher mean nitrate content in winter than

378

summer, in contrast to other five species (spinach, dill, parsley, cabbage and coriander).

379

Moreover, much higher variability was observed between intra-species samples than

380

between seasons overall, which highlights potentially significant differences in the nitrate

381

contents of products from different producers (Guadagnin, Rath, & Reyes, 2005). Our

382

findings also support the premise that effects of irradiance on nitrate levels are minimal,

383

compared to the effects of principle agronomic factors, such as N fertilization, provided

384

light intensity exceeds a species-specific critical level (Cantlife, 1972). In the case of Swiss

385

chards, this was defined as ≈200 μmol m-2 s-1 (Parks et al., 2008). Such levels of light

386

intensity can be met throughout the year in south Mediterranean countries like Cyprus, and

387

offers a plausible explanation for limited seasonal variation in nitrate levels. In contrast, the

388

high variability in nitrate levels encountered among intra-species samples highlight the

389

importance of agricultural factors, such as N supply, harvest stage and possibly time of

390

harvest within the day (Colla et al., 2018). Rocket and spinach samples in excess of

391

permitted rates (EC, 2006) suggest that such agricultural factors warrant closer

392

examination in the Mediterranean environment, despite high light intensity light prevailing

393

during the production period.

394

Concentration of nitrite in plant tissues is cytotoxic and the presence of nitrite in vivo is

395

usually limited through the coordinated reduction of endogenous nitrite with that of nitrate

396

(Colla et al., 2018; Riens & Heldt, 1992). Nitrite levels in horticultural commodities are

397

more erratic and have not been linked consistently to specific genotypic or agro-

398

environmental factors, but nitrite is most commonly detected in spinach. In a survey of

399

nitrate and nitrite concentrations in raw vegetables in the United States, Nunez de

400

Gonzalez et al. (2015) reported nitrite levels ranging from 0.1 to 1.2 mg kg-1 fw, with the

401

exception of spinach, which contained up to 8.0 mg kg-1 fw. In a market survey of

402

Australian leafy vegetables by Parks et al. (2008), nitrite concentrations were 0-37.5 mg

403

kg-1 fw while levels >1 mg were detected in only 6/165 samples of baby leaf spinach, pak

404

choi, tatsoi and rocket. A similar survey conducted by Iammarino, Taranto, and Cristino

405

(2014) on 39 fresh spinach and 75 lettuce samples from the Italian market identified

406

quantifiable nitrite in 7/39 spinach samples (9.5-197.5 mg kg-1 fw) and in 1/75 lettuce

407

samples (66.5 mg kg-1 fw). In the current study, nitrite was detected in one sample of dill

408

and one cabbage out of the 330 summer samples. In the winter season, nitrite was detected

409

in 19/368 samples, of which 10 accounted for cabbage alone (14-352 mg kg-1 fw), five for

410

spinach, three for dill, and one for coriander. While the occurrence of nitrite coincided, in

411

some cases, with high nitrate levels (e.g. dill, spinach), in others (e.g. cabbage) it did not;

412

there was no detectible nitrite in nitrate-accumulating species, such as rocket. The high

413

incidence of quantifiable nitrite in head cabbage samples, especially during the winter,

414

invites particular interest and might be explained on the basis of morphology. The multi-

415

layered structure of the cabbage head with opaque, chlorophyllous leaves on the outside,

416

restricts light transmission towards the inner leaves. Nitrite reductase activity, on the other

417

hand, is coupled to the function of Photosystem I and is, thus, highly dependent on light

418

(Riens & Heldt, 1992). Under restricted light irradiance, nitrite reductase activity is

419

suppressed, and nitrite accumulation might, therefore, take place.

420 421

4.2. Effects of base-dressing and top-dressing N rates on nitrate and nitrite contents

422

Nitrogen fertilizers, when applied, are undoubtedly the main source of nitrate uptake

423

and accumulation in edible plant parts. Nitrate uptake in excess of assimilation capacity

424

will inevitably lead to nitrate accumulation (Colla et al., 2018). However, accumulation of

425

nitrate in response to N fertilizers has been less well documented for field grown leafy

426

vegetables (Wang & Li, 2004) compared with vegetables cultivated in pots (Liu, Sung,

427

Chen, & Lai, 2014; Petropoulos, Olympios, & Passam, 2008) and crops produced under

428

controlled, soil-less environments (Konstantopoulou et al., 2010). Generally, it has been

429

shown that nitrate accumulates in the edible parts of leafy vegetables in response to

430

increasing N rates, but there has been little emphasis on the timing of N application (Colla

431

et al., 2018). This factor is particularly critical under field conditions with respect to crop

432

season and crop cycle duration, since control of nitrate uptake is less effective than in soil-

433

less environments. In soil-less agriculture, the use of nitrate-free solution for several days

434

before harvest has been successful in reducing nitrate levels in leafy vegetables

435

(Borgognone, Rouphael, Cardarelli, Lucini, & Colla, 2016).

436

In the current study, nitrate levels in spinach, lettuce and rocket were affected by base-

437

dressing N rate, in both summer and winter crops. Nitrate levels in lettuce and spinach

438

summer crops were not affected by top-dressing N rate but, in the case of summer rocket,

439

this effect was significant. Top-dressing N rate had a significant effect on nitrate levels in

440

all three winter crops. Thus, it is apparent that the longer cycle of the winter crops allows

441

more time for nitrate uptake and may, additionally, compensate for nitrate leaching from

442

the root zone. Brief summer crop cycles do not provide adequate response time to top-

443

dressing applications, except for nitrophilous crops like rocket. No samples of lettuce

444

grown in open air were found to contain excess nitrate, as set by Commission Regulation

445

(EC) No. 1882/2006 for summer (3000 mg kg-1 fw) and winter (4000 mg kg-1 fw; EC,

446

2006). Two samples of summer rocket treated with 250 and 350 kg N ha-1 and 10 samples

447

of winter rocket treated with 150-350 kg N ha-1 were found to contain excess nitrate (6000

448

and 7000 mg kg-1 fw, respectively). In the case of spinach, only five winter samples treated

449

with 250-350 kg N ha-1 exceeded the maximum limit set at 3500 mg kg-1 fw.

450

Our results indicate that violating the current legal limits for nitrate is likely, even under

451

Mediterranean climatic conditions, particularly for rocket and spinach, when total nitrogen

452

supply rates exceed 200 kg N ha-1. Longer crop cycles during autumn and winter combined

453

with nitrogenous top-dressing applications render these crops more prone to nitrate

454

accumulation. On the other hand, detectible levels of nitrite, ranging from 42.8 to 271.2 mg

455

kg-1 fw, were identified only in three samples of summer spinach. These levels are in

456

agreement to those reported for market vegetables previously (Iammarino et al., 2013;

457

Parks et al., 2008). However, the low incidence of detectible nitrite levels supports the

458

suggestion that, as a highly cytotoxic compound (Riens & Heldt, 1992), nitrite in leafy

459

vegetables should be minimised but not necessarily linked to high nitrate levels.

460 461

4.3. Effects of N fertilizer formulations on winter rocket nitrate and nitrite contents

462

Besides the N rate and time of application, nitrate uptake and accumulation might be

463

also influenced by the form of N in nitrogenous fertilizers, which may be nitrate,

464

ammonium, urea or other organic forms or combinations of these (Liu et al., 2014; Pavlou,

465

Ehaliotis, & Kavvadias, 2007). Santamaria and Elia (1997) reported that replacement of

466

nitrate with ammonium in soil-less agriculture of endive reduced nitrate accumulation. Use

467

of ammoniacal fertilizers or fertilizers containing nitrate and ammonium N was also found

468

to reduce nitrate levels in green onion grown under soil-less system (Inal & Tarakcioglu,

469

2001). Wang and Li (2004) reported that nitrate concentrations in field-grown Pekking

470

cabbage and spinach were higher when nitrate-based rather than ammoniacal nitrogenous

471

formulations were applied. However, reports on field-grown vegetables are scarce and

472

overall less consistent.

473

In their review on factors affecting nitrate accumulation in plants, Renseigné, Umar,

474

and Iqbal (2007) categorized nitrogenous fertilizers based on nitrate levels found in the

475

laminae of leafy vegetables in the following order: urea > ammonium carbonate >

476

ammonium nitrate > ammonium sulphate. In our study, all nitrogenous fertilizers tested for

477

base-dressing application on field-grown winter rocket increased mean nitrate levels

478

significantly compared to the control. No other significant differentiation was observed in

479

nitrate levels among the nitrogenous fertilizers tested, which included both inorganic

480

(ammonium nitrate, ammonium sulphate) and organic (urea, chicken manure) forms.

481

All treatments were applied at 100 kg N ha-1 and all samples analysed were found

482

below the 7000 mg nitrate kg-1 fw maximum set for winter rocket, (EC, 2006), and no

483

quantifiable nitrite levels were detected in any sample. In the case of organic fertilizers, the

484

release of nitrogen in forms accessible to plants is slower compared to inorganic fertilizers

485

(Herencia, García-Galavís, Dorado, & Maqueda, 2011), which is the usual reason

486

purported when nitrate levels are found to be lower in organically vs. conventionally grown

487

crops (Dangour et al., 2009; Nunez de Gonzalez et al., 2015). This rule is, however, liable

488

to frequent exceptions. For instance, higher nitrate levels were found in organic compared

489

to non-organic rocket and green salad (a mixture of endive and prickly lettuce) by De

490

Martin and Restani (2003). Comparing lettuce and arugula produced under organic,

491

conventional and hydroponic systems, Guadagnin et al. (2005) found the order of nitrate

492

contents was hydroponic > conventional > organic whereas, in the case of watercress, the

493

order was organic = hydroponic > conventional. Our findings suggest that when N

494

fertilization is applied as a base-dressing, in either inorganic or organic forms at moderate

495

rates (≈100 kg N ha-1), nitrate levels are not markedly differentiated and may be kept

496

within the permitted range.

497 498

4.4. Effects of N rate and postharvest storage on nitrate and nitrite concentrations

499

Nitrate levels in winter spinach increased from 698 to 3087 mg kg-1 in response to

500

increasing N rate from zero to 300 kg N ha-1. Similar responses in mean nitrate content

501

were also demonstrated by summer rocket (1885-3781 mg kg-1 fw), winter rocket (3524-

502

6982 mg kg-1 fw) and summer lettuce (412-1563 mg kg-1 fw and 339-1708 mg kg-1 fw).

503

These responses are overall in agreement with the premise that nitrate accumulation in

504

edible parts of leafy vegetables increases in response in response to increasing N rates

505

(Colla et al., 2018). Moreover, levels in excess of the nitrate maxima were identified in 12

506

winter spinach samples, two summer rocket samples, and 20 samples of winter rocket (EC,

507

2006), all having been subject to applications of 200-300 kg N ha-1. This further highlights

508

the prominent role of N fertilization in driving nitrate accumulation in salad crops and

509

reiterates that violating the legal limits for nitrate is possible even under Mediterranean

510

climatic conditions, especially for rocket and spinach, when the total N rate exceeds 200 kg

511

N ha-1.

512

The preharvest accumulation of nitrate in vegetables depends on uptake in excess of its

513

reduction and subsequent assimilation. Corresponding levels of nitrate and nitrite reductase

514

activities prohibit the accumulation of nitrite to phytotoxic levels during plant growth

515

(Riens & Heldt, 1992). However, the transport of nitrate, taken up by the roots through the

516

transpiration stream to the leaves where assimilation takes place, is interrupted after

517

harvest and as is the diurnal variation in leaf nitrate corresponding to fluctuations in the

518

transpiration stream. Putative postharvest reduction of nitrate may utilize concentrations

519

built up in the cytosol and vacuole at the time of harvest while the temperature dependence

520

of this process seems to be a function of species and storage duration (Alexander et al.,

521

2008; Ekart et al., 2013). Absence of nitrate reduction in rocket during dark storage at 4 or

522

15 °C was reported by Kim and Ishii (2007) and Ferrante et al. (2003). Also, Koukounaras

523

et al. (2010) identified no consistent effect of storage on nitrate content after 14 days at 0, 5

524

and 10 °C in the dark, while Chung et al. (2004) found no changes in nitrate and nitrite

525

contents of spinach, crown daisy, Chinese spinach and non-heading Chinese cabbage after

526

seven days at 5 °C. In the case of lettuce (cv. Parris Island), nitrate levels were unaltered

527

after 10-day storage at 5 and 10 °C (Konstantopoulou et al., 2010), and after 15-day

528

storage at 1 °C (Siomos et al., 2000). Our results indicate that 12-day storage of winter

529

spinach at 5 °C, 15-day storage of summer and winter rocket at 5 °C and 4-day storage of

530

summer lettuce at 5 and 22 °C had no effect on nitrate contents, moreover quantifiable

531

nitrite was not detected in any of our postharvest samples.

532

Cold storage seems to inhibit changes in nitrate and nitrite levels, whereas changes

533

observed during storage at ambient temperature are linked to overall quality of the

534

products. Decreased nitrate levels were observed in Chinese cabbage (Brassica chinensis

535

L.), field mustard (Brassica campestris L.) and broad-leaf mustard (Brassica juncea L. var.

536

rugose (Roxb.) Kitam.), but not water convolvulus (Ipomoea aquatic Forsk), kept in plastic

537

bags for three days at 26 °C, at which temperature samples senesced rapidly and were

538

rendered unmarketable in just two days (Lin & Yen, 1980); storage of the same species at

539

15 °C or lower had no effect on the activities of nitrate and nitrite reductases. Analogous

540

declines in nitrate concentration of Chinese spinach and non-heading Chinese cabbage after

541

three days at 22 °C, corresponding with increases in nitrite levels, was reported by Chung

542

et al. (2004), but no information was provided on the use of packaging or the quality of

543

these vegetables at the time nitrite increases were recorded. However, based on their

544

hypothesis that nitrate reductase activity was due to microbial proliferation, product quality

545

must also have been unacceptable. Hicks, Stall, and Hall (1975) attributed the reduction of

546

nitrate to nitrite in carrots containing high concentrations of nitrite to high counts of

547

facultative anaerobic bacteria. From this information, it might be concluded that

548

postharvest reduction of nitrate to nitrite is primarily temperature-dependent and relates to

549

the shelf-life potential of crops and their quality during storage. Foremost, the postharvest

550

accumulation of nitrite relates to the activity of nitrate reductase of exogenous microbial

551

origin and is unlikely to occur in the absence of visible quality deterioration.

552

Unsurprisingly, in the case of frozen leafy, stalk, root and tuberous vegetables, no

553

conversion of nitrate to nitrite was observed during 3 months of storage (Schuster & Lee,

554

1987).

555 556

5. Conclusion

557

A market survey conducted on 698 samples of 11 Mediterranean salad vegetables

558

during winter and summer revealed wider intra-species rather than seasonal variability, and

559

nitrate levels in excess of permitted values, as set by European Commission Regulation

560

1881/2006 (and subsequent amendments), for rocket and spinach. This study highlights the

561

importance of monitoring production methods, particularly with respect to rate and mode

562

of nitrogen application. The low incidence of detectible nitrite confirms that it is present at

563

very low levels in leafy vegetables and is not necessarily associated with high nitrate

564

levels. The high frequency of detectible nitrite in specific vegetables, notably head

565

cabbage, is likely due to their light-restricting morphology and deserves further

566

investigation. The nitrate levels of summer and winter open-field grown spinach, lettuce

567

and rocket are affected by N base-dressing. Longer crop cycles during autumn and winter

568

combined with nitrogenous top-dressing applications render these crops more prone to

569

nitrate accumulation. Violating the current legal limits for nitrate is possible even under

570

Mediterranean climatic conditions when total N rates exceed 200 kg ha-1. Postharvest

571

reduction of nitrate to nitrite relates to the activity of nitrate reductase of exogenous

572

microbial origin and is unlikely to occur without visible loss of quality.

573 574

References

575

Alexander, J., Benford, D., Cockburn, A., Cravedi, J. P., Dogliotti, E., Domenico, A. D., et

576

al. (2008). Nitrate in vegetables Scientific Opinion of the Panel on Contaminants in the

577

Food chain. EFSA Journal, 689, 1–79.

578

Amr, A., & Hadidi, N. (2001). Effect of cultivar and harvest data on nitrate (NO3) and

579

nitrite (NO2) content of selected vegetables gown under open field and greenhouse

580

conditions in Jordan. Journal of Food Composition and Analysis, 14, 59–67.

581

Borgognone, D., Rouphael, Y., Cardarelli, M., Lucini, L., & Colla, G. (2016). Changes in

582

biomass, mineral composition, and quality of cardoon in response to NO3-:Cl- ratio and

583

nitrate deprivation from the nutrient solution. Frontiers in Plant Science, 7, 978.

584

Cantliffe, D.J. (1972). Nitrate accumulation in spinach grown under different light

585

intensities. Journal of the American Society for Horticultural Sciences, 97, 152–154.

586

Cavaiuolo, M., & Ferrante, A. (2014). Nitrates and glucosinolates as strong determinants

587

of the nutritional quality in rocket leafy salads. Nutrients, 6, 1519–1538.

588

Chou, S. S., Chung, J. C., & Hwang, D. F. (2003). A high performance liquid

589

chromatography method for determining nitrate and nitrite levels in vegetables. Journal

590

of Food and Drug Analysis, 11, 233-238.

591

Chung, J. C., Chou, S. S., & Hwang, D. F. (2004). Changes in nitrate and nitrite content of

592

four vegetables during storage at refrigerated and ambient temperatures. Food Additives

593

& Contaminants, 21, 317–322.

594

Chung, S. Y., Kim, J. S., Kim, M., Hong, M. K., Lee, J. O., Kim, C. M., et al. (2003).

595

Survey of nitrate and nitrite contents of vegetables grown in Korea. Food Additives &

596

Contaminants, 20, 621–628.

597 598 599 600

Colla, G., Kim, H. J., Kyriacou, M. C., & Rouphael, Y. (2018). Nitrates in fruits and vegetables. Scientia Horticulturae, 237, 221-238. Colonna, E., Rouphael, Y., Barbieri, G., & De Pascale, S. (2016). Nutritional quality of ten leafy vegetables harvested at two light intensities. Food Chemistry, 199, 702–710.

601

Dangour, A. D., Dodhia, S. K., Hayter, A., Allen, E., Lock, K., & Uay, R. (2009).

602

Nutritional quality of organic foods: a systematic review. The American Journal of

603

Clinical Nutrition, 90, 680–685.

604

De Martin, S., & Restani, P. (2003). Determination of nitrates by a novel ion

605

chromatographic method: occurrence in leafy vegetables (organic and conventional)

606

and exposure assessment for Italian consumers. Food Additives & Contaminants, 20,

607

787–792.

608

EFSA (2008). Opinion of the scientific panel on contaminants in the food chain on a

609

request from the European commission to perform a scientific risk assessment on

610

nitrate in vegetables. The EFSA Journal, 689, 1–79

611

Ekart, K., Hmelak Gorenjal, A., Madorran, E., Lapajne, S., & Langerholc, T. (2013). Study

612

on the influence of food processing on nitrate levels in vegetables. EFSA Supporting

613

Publications 10, 12.

614

European Commission (EC). (2006). European Commission Regulation (EC) No

615

1882/2006 of 19 December 2006 laying down methods of sampling and analysis for the

616

official control of the levels of nitrates in certain foodstuffs. Official Journal of the

617

European Union, 364, 25–31.

618

Fallovo, C., Rouphael, Y., Rea, E., Battistelli, A., & Colla, G. (2009). Nutrient solution

619

concentration and growing season affect yield and quality of Lactuca sativa L. var.

620

acephala in floating raft culture. Journal of the Science Food and Agriculture, 89,

621

1682–1689.

622

FAO/WHO - Food and Agriculture Organisation of the United Nations/World Health

623

Organization. (2003a). Nitrate (and potential endogenous formation of N-nitroso

624

compounds). WHO Food Additive series 50, Geneva: World Health Organisation.

625

Available at URL: http://www.inchem.org/documents/jecfa/jecmono/v50je06.htm

626

FAO/WHO - Food and Agriculture Organisation of the United Nations/World Health

627

Organization. (2003b). Nitrite (and potential endogenous formation of N-nitroso

628

compounds). WHO Food Additive series 50, Geneva: World Health Organisation.

629

Available at URL: http://www.inchem.org/documents/jecfa/jecmono/v50je05.htm.

630

Ferrante, A., Incrocci, L., Maggini, R., Tognoni, F., & Serra, G. (2003). Preharvest and

631

postharvest strategies for reducing nitrate content in rocket (Eruca sativa). Acta

632

Horticulturae, 628, 153-159.

633

Guadagnin, S. G., Rath, S., & Reyes, F. G. R. (2005). Evaluation of the nitrate content in

634

leaf vegetables produced through different agricultural systems. Food Additives &

635

Contaminants, 12, 1203–1208.

636

Herencia, J. F., García-Galavís, P. A., Dorado, J. A. R., & Maqueda, C. (2011).

637

Comparison of nutritional quality of the crops grown in an organic and conventional

638

fertilized soil. Scientia Horticulturae, 129, 882–888.

639 640

Hicks, J. R., Stall, R. E., & Hall, C. B. (1975). The association of bacteria and nitrites in carrot juice. Journal of the American Society for Horticultural Sciences, 100, 402.

641

Hord, N. G., Tang, Y., & Bryan, N. S. (2009). Food sources of nitrates and nitrites: the

642

physiologic context for potential health benefits. American Journal of Clinical

643

Nutrition, 90, 1-10.

644

Iammarino M, Di Taranto A, & Cristino M. (2014). Monitoring of nitrites and nitrate

645

levels in leafy vegetables (spinach and lettuce): a contribution to risk assessment.

646

Journal of the Science Food and Agriculture, 15, 773–778.

647 648

Ilić, Z.S., & Sunić, L. (2015). Nitrate content of root vegetables during different storage conditions. Acta Horticulturae, 1079, 659–665.

649

Inal, A., & Tarakcioglu, C. (2001). Effects of nitrogen forms on growth, nitrate

650

accumulation, membrane permeability, and nitrogen use efficiency of hydroponically

651

grown bunch onion under boron deficiency and toxicity. Journal of Plant Nutrition, 24,

652

1521–1534.

653

Kim, S. J., & Ishii, G. (2007). Effect of storage temperature and duration on glucosinolate,

654

total vitamin C and nitrate contents in rocket salad (Eruca sativa Mill.). Journal of the

655

Science Food and Agriculture, 87, 966–973.

656

Konstantopoulou, E., Kapotis, E., Salachas, G., Petropoulos, S. A., Karapanos, I. -C., &

657

Passam, H. C. (2010). Nutritional quality of greenhouse lettuce at harvest and after

658

storage in relation to N application and cultivation season. Scientia Horticulturae, 125,

659

93.e1–93.e5.

660

Koukounaras, A., Siomos, A. S., & Sfakiotakis, E., 2010. Effects of 6‐BA Treatments on

661

yellowing and quality of stored rocket (Eruca sativa Mill.) leaves. Journal of Food

662

Quality, 33, 768–779.

663

Lin, J. K., & Yen, J. Y. (1980). Changes in the nitrate and nitrite contents of fresh

664

vegetables during cultivation and post-harvest storage. Food and Cosmetics

665

Toxicology, 18, 597–603.

666

Liu, C. W., Sung, Y., Chen, B. C., & Lai, H. Y. (2014). Effects of nitrogen fertilizers on

667

the growth and nitrate content of lettuce (Lactuca sativa L.). International Journal of

668

Environmental Research and Public Health, 11(4), 4427-4440.

669

Nunez de Gonzalez, M. T., Osburn,W. N., Hardin, M. D., Longnecker, M., Garg, H. K.,

670

Bryan, N. S., et al. (2015). A survey of nitrate and nitrite concentrations in

671

conventional and organic-labeled raw vegetables at retail. Journal of Food Science, 80,

672

C942–C949.

673

Parks, S. E., Huett, D. O., Campbell, L. C., & Spohr, L. J. (2008). Nitrate and nitrite in

674

Australian leafy vegetables. Australian Journal of Agricultural Reasearch, 59, 632–

675

638.

676

Pavlou, G. C., Ehaliotis, C. D., & Kavvadias, V. A. (2007). Effect of organic and inorganic

677

fertilizers applied during successive crop seasons on growth and nitrate accumulation

678

in lettuce. Scientia Horticulturae, 111, 319–325.

679

Petropoulos, S. A., Olympios, C. M., & Passam, H. C. (2008). The effect of nitrogen

680

fertilization on plant growth and the nitrate content of leaves and roots of parsley in the

681

Mediterranean region. Scientia Horticulturae, 118(3), 255-259.

682

Petropoulos, S. A., Constantopoulou, E., Karapanos, I., Akoumianakis, C. A., & Passam,

683

H. C. (2011). Diurnal variation in the nitrate content of parsley foliage. International

684

Journal of Plant Production, 5, 431-438.

685

Proietti, S., Moscatello, S., Leccese, A., Colla, G., & Battistelli, A. (2004). The effect of

686

growing spinach (Spinacia oleracea L.) at low light intensities on the amounts of

687

oxalate, ascorbate and nitrate in their leaves. Journal of Horticultural Science &

688

Biotechnology, 79, 606–609.

689

Renseigné, N., Umar, S., & Iqbal, M. (2007). Nitrate accumulation in plants, factors

690

affecting the process, and human health implications. A review. Agronomy for

691

Sustainable Development, - 27, 45-57.

692 693 694 695

Riens, B., & Heldt, H. W. (1992). Decrease of nitrate reductase activity in spinach leaves during a light-dark transition. Plant Physiology, 98, 573–577. Santamaria, P. (2006). Nitrate in vegetables: toxicity, content, intake and EC regulation. Journal of the Science Food and Agriculture, 86, 10 –17.

696

Santamaria, P., & Elia, A. (1997). Producing nitrate-free endive heads: Effect of nitrogen

697

form on growth, yield and ion composition of endive, Journal of the American Society

698

for Horticultural Sciences, 122, 140–145.

699

Schuster, B. E., & Lee, K. (1987). Nitrate and nitrite methods of analysis and levels in raw

700

carrots, processed carrots and in selected vegetables and grain products. Journal of

701

Food Science, 52, 1632–1636.

702 703 704 705

Siomos, A. S. (2000). Nitrate levels in lettuce at three times during a diurnal period. Journal of Vegetable Crop Production, 6, 37–42. Wang, Z., & Li, S. (2004). Effects of nitrogen and phosphorus fertilization on plant growth and nitrate accumulation in vegetables. Journal of Plant Nutrition, 27, 539-556.

707

Figure Captions

708

Fig. 1. HPLC-DAD chromatographic separation of nitrite (NO2-) and nitrate (NO3-) ions

709

performed on a reverse phase C18 column at an isocratic flow rate of 1.0 ml min-1 of 0.01

710

M octyl-ammonium phosphate (pH 6.5) and absorbance recorded at 220 nm.

711 712

713 714

Table 1

715

Nitrate and nitrite concentrations of eleven fresh vegetable products sampled from local

716

supermarkets during the winter and summer seasons.

Summer (April 1 - September 30)

Product nomenclature*

-1

-1

Nitrate (mg kg fw) Common name

Alternative common name

Binomial

N

Cabbage

white cabbage, red cabbage, Shetland cabbage celery, wild celery

Brassica oleracea L. var. capitata L.

20

-

Celery leaves

Apium graveolens L.

31

-

Celery stalks

celery, stalk celery

Apium graveolens L. var. dulce (Mill.) DC.

19

-

Coriander

cilantro, Chinese parsley

Coriandrum sativum L.

25

Dill

garden dill

Anethum graveolens L.

14

Chards

beet, beetroot, field beet, fodder beet, foliage beet, mangel, mangold, red beet, root beet, Sicilian broad-rib beet, spinach chard, sugarbeet, Swiss chard, yellow beet

Beta vulgaris L. subsp. vulgaris

Lettuce

Cos type lettuce

Purslane

common purslane, littile hogweed, portulaca-weed garden parsley arugula, edible rocket, garden rocket, Italian cress, rocket-salad, Roman rocket, rugula, salad rocket spinach

Parsley Rucola

717 718 719 720 721 722 723 724

Spinach

f >NO3 Mean ± SE

Nitrite (mg kg fw)

Nitrate

Min

Max

f NO2

Mean

Min

Max

N

1017 ± 82

286

1686

1/20

49

49

49

20

1433 ± 188

65

3362

-

-

-

-

36

-

334 ± 77

13

1155

-

-

-

-

15

-

-

2523 ± 190

671

3843

-

-

-

-

40

-

-

3015 ± 278

932

4432

1/14

12

12

12

24

-

50

-

2780 ± 135 1057

5568

-

-

-

-

45

-

Lactuca sativa L. var. longifolia

49

-

843 ± 91

60

2151

-

-

-

-

49

-

Portulaca oleracea L.

25

-

1533 ± 157

148

2783

-

-

-

-

11

-

Petroselinum crispum (Mill.) Fuss

33

-

2340 ± 163

934

4583

-

-

-

-

36

-

Eruca vesicaria (L.) Cav. subsp. sativa (Mill.) Thell.

39

1/39

3819 ± 222 1529

6946

-

-

-

-

44

3/44

Spinacia oleracea L.

25

1/25

2463 ± 185 1149

5767

-

-

-

-

48

-

*Alternative common names and binomials according to GRIN taxonomy of the U.S. National Plant Germplasm System (https://npgsweb.arsgrin.gov/gringlobal/taxon/taxonomysearch.aspx) f>NO3 = frequency of samples found in excess of legal nitrate limits according to Commission Regulation (EC) No. 1258/2011 fNO2 = frequency of samples with nitrite concentration above LOQ (0.03 mg kg-1 FW)

f >NO3 M -

Table 2 Effects of basal and top dressing N rates on fresh weight and nitrate content of field grown spinach, lettuce and rocket during the summer and winter seasons.

Source of variance

Spinach Nitrate (mg kg1 fw)

Lettuce Nitrate (mg kg1 fw)

Rocket Nitrate (mg kg1 fw)

kg N ha1

Summer

Basal 0 100 150 200

371 ± 82 b 617 ± 95 b a 680 ± 59 b 976 ± 113 a ***

838 118 1366 125 1706 128 2103 127 ***

±

1325 255 1408 156 1687 142 1589 114

±

c ± b ± a b ± a

2000 ± 273 2891 ± 430 3404 ± 370 3532 ± 361 *

b a b a a

Top dressing 0 50 100 150

547 ± 66 680 ± 98 792 ± 134 908 ± 144

2308 ± 374 2975 ± 331 2676 ± 334 3868 ± 416 *

± ± ±

b a b a b a

kg N ha1

Winter

Basal 0 100 150 200

Top

1279 178 2549 104 2614 191 2889 105 ***

± b

928 ± 96

c

a

1264 ± 66

a

1431 ± 63

b a b

a

1553 ± 66 ***

± ± ± a

4839 ± 350 5812 ± 179 6584 ± 155 6754 ± 204 ***

c b a a

dressing 0 50 100 150

1900 222 2115 211 2581 197 2759 186 ***

± c ± b c ± a b ± a

1056 107

± b

1206 ± 84

b

1467 ± 79

a

1447 ± 63 ***

a

5560 ± 453 5859 ± 266 5969 ± 172 6601 ± 170 **

b b a b a

* Significant effect at the 0.05 level, ** 0.01 level, *** 0.001 level. Data represent means ± standard error of four replicates (N=4). Means within each column followed by different letters denote significant differences (P < 0.05) according to Tukey-Kramer HSD test.

725

Table 3

726

Overall descriptive statistics (range and mean) for nitrate and nitrite concentrations in

727

experimental samples of lettuce, rocket and spinach, including occurrence of samples

728

found in excess of legal nitrate limits according to Commission Regulation (EC) No.

729

1258/2011 and samples with nitrite concentration above the limit of quantification (LOQ=

730

0.03 mg kg-1 FW). PH denotes postharvest samples.

Crop

Season

EC Reg. 1881 /2006 maximum nitrate levels (mg kg-1 fw)

Analysed samples

Nitrite samples > LOQ

Nitrite range

Nitrite mean±SE

Ni ra

n

n

(mg kg-1 fw)

(mg kg-1 fw)

(mg k

Lettuce

S

3000

61

0

-

0

78 -

Lettuce

W

4000

64

0

-

0

172 -

Rocket

S

6000

64

0

-

0

200 -

Rocket

W

7000

64

0

-

0

2133

Spinach

S

3500

60

3

43-271

138 ± 69

86 -

Spinach

W

3500

61

0

-

0

44 -

Rocket

S - PH

6000

64

0

-

0

216 -

Rocket

W - PH

7000

80

0

-

0

1551

Spinach

W - PH

3500

157

0

-

0

33 -

Lettuce

S - PH

3000

147

0

-

0

70 -

731 732

35

734

Table 4

735

Effect of base-dressing formulation on nitrate content of field grown winter rocket.

Base-dressing formulation

Rate (kg N

ha-1)

Nitrate (mg kg-1 fw) *

736 737 738 739 740 741

Ammonium nitrate (34.5-0-0)

100

5452 ± 277 a

Ammonium sulphate (21-0-0)

100

6067 ± 273 a

Urea Ntec (46-0-0) Granulated chicken manure (10-33) Control

100

5349 ± 30 a

100 0

5407 ± 410 a 3393 ± 96 b

* Significant effect at the 0.05 level Data represent means ± standard error of four replicates (N=4). Means within each column followed by different letters denote significant differences (P < 0.05) according to TukeyKramer HSD test.

36

743

Table 5

744

Effects of total N rate application (0-300 kg ha-1) and postharvest storage of winter spinach

745

(5 °C), summer and winter rocket (5 °C) and summer lettuce (5 and 22 °C) on nitrate

746

concentrations.

Spinach

Winter

Rocket

Summer

Source of variance

Nitrate

Source of variance

Nitrate

Nitrate

kg-1

kg-1

mg

kg-1

fw

N rate (kg ha1)

***

N rate (kg ha1)

0

696 ± 77 d

0

1509 ± 103 2616 ± 92 2800 ± 70 3054 ± 115

100 200 250 300 Storage (d)

c

100

b

200

ab

250

a

300

mg

4 8 12

fw

mg

*** 1885 ± 341 1634 ± 262 2952 ± 399 3934 ± 423 3781 ± 278

Lettuce Source of variance

fw

N rate (kg ha1)

*** b b ab a a

3524 ± 451 5390 ± 225 6386 ± 275 6734 ± 244 6982 ± 260

c

0

b

100

ab

200

a

250

a

300

Storage (d)

Storage (d)

5 °C 0

Winter

2075 ± 139 2196 ± 187 2358 ± 192 1935 ± 140

5 °C 2681 ± 349 2873 ± 372 2922 ± 388 2874 ± 393

0 5 10 15

5 °C 5441 ± 327 5821 ± 362 5959 ± 444 5993 ± 422

747

*** Significant effect at the 0.001 level. Data represent means ± standard error of four

748

replicates (N=4). Means within each column followed by different letters denote

749

significant differences (P < 0.05) according to Tukey-Kramer HSD test.

750 751 752 753

Highlights  Winter/summer market survey determined the NO3-/NO2- levels in 11 salad vegetables 37

0 2 3 4

754

 Excessive NO3- was identified in rocket and spinach and high NO2- in head cabbage

755

 Long winter crop cycles and N top-dressing encourage NO3- accumulation

756

 Violating current NO3- limits is likely when total N rates exceed 200 kg ha-1

757

 Postharvest NO3-reduction is exogenous and unlikely without visible quality loss

758

38