Mineral nitrogen fertilization effects on lettuce crop yield and nitrogen leaching

Scientia Horticulturae 255 (2019) 153–160

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Mineral nitrogen fertilization effects on lettuce crop yield and nitrogen leaching

T

⁎

Thiago de Barros Sylvestrea, Lucas Boscov Braosb, , Felipe Batistella Filhoc, Mara Cristina Pessôa da Cruzb, Manoel Evaristo Ferreirab a

Mosaic Brasil, Av. Roque Petroni Jr 999, 04707-910, São Paulo, Brazil Departamento de Solos e Adubos, Faculdade de Ciências Agrárias e Veterinárias, UNESP – Univ Estadual Paulista. Via de acesso Prof. Paulo Donato Castellane s/n, 14884-900, Jaboticabal, Brazil c Federal Institute of Education, Science, and Technology of São Paulo, Campus Matão. R. Estéfano D'avassi 625 N, 15991-502, Matão, Brazil b

A R T I C LE I N FO

A B S T R A C T

Keywords: Lactuca sativa Topdressing Nitrate Fertilization efficiency

The low efficiency of N fertilization in soils used to grow vegetables and the consequent need to add high levels of the nutrient are the major cause of nitrate contamination of groundwater and freshwater, which calls for practices to increase N fertilization efficiency in these crops. Two experiments were carried out in Jaboticabal, Brazil, to evaluate the effects of topdressing N fertilization on crisphead lettuce (Lactuca sativa) yield, nitrate accumulation in plant shoots, and soil nitrogen leaching in two growing seasons, summer and winter. Both experiments were designed as randomized blocks with seven treatments and five replications (35 plots). The treatments were seven different rates of topdressing N. The rates of N application were 0, 30, 60, 90, 120, 150, and 180 kg N ha -1 in the form of urea, applied 8 (20%), 16 (40%), and 24 (40%) days after transplanting. Lettuce was harvested 34 and 42 d after transplanting in the summer and winter, respectively. Plant nitrate concentrations were determined in dry matter. After the harvest, soil samples were collected at the 0-20, 20-40, and 40-60 cm layers to quantify nitrogen in the forms of ammonium and nitrate. The data were submitted to analysis of variance and nonlinear regression analysis. Better use of topdressing nitrogen fertilization for lettuce growth was observed at rates lower than 60 kg N ha -1. Rates of N up to 180 kg N ha -1 did not elevate lettuce nitrate concentrations over the levels allowed for consumption but increased the amount of nitrogen lost through leaching. Decreasing N rates resulted in a similar yield and lower nitrogen leaching, improving both the environmental and economic aspects.

1. Introduction Lettuce crops respond to nitrogen (N) fertilization with higher yields and quality improvement, which makes the application of high rates of N fertilizers a common practice in this crop. However, this procedure may result in nitrate (NO3-) accumulation in leaves and N losses due to leaching (Khoshgoftarmanesh et al., 2011). Nitrogen leaching from agricultural soils is one of the main causes of NO3- contamination in rivers and aquifers in São Paulo state (Gomes and Barizon, 2014). The lettuce yield response to N fertilization depends on the cultivar, soil, and climate conditions. The maximum yield for looseleaf lettuce was observed at the N dose of 165 kg N ha -1 (Thompson and Doerge, 1996), and the maximum yield for crisphead lettuce was observed at the N dose of 149 kg N ha -1 combined with 60 kg N ha -1 of N applied in the planting (Resende et al., 2005).

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The total N exported by lettuce crops is low, whereas the recommended N application rates are high, reaching up to 150 kg N ha -1 of N in São Paulo state (Trani et al., 1997). Aquino et al. (2007) observed that, for the N dose of 150 kg N ha -1, the total N exported to plant shoots was 57.9 kg ha -1, and the dry matter yield was 1,640 kg ha -1 . The difference between the applied and absorbed N suggests that 92.1 kg ha -1 of the nutrient stayed in the soil, liable to be lost in the drainage water. In addition to the economic loss due to the low recovery of N by plants, N leaching in agricultural land is a major source of NO3- contamination in freshwater (Di and Cameron, 2007). The superficial rooting system of lettuce (Jackson et al., 1994) combined with high levels of N fertilizer and irrigation, which are common in lettuce crop fields, are factors that intensify N leaching in lettuce crops. In the green belts (intensive vegetable-production areas) of the state of São Paulo,

Corresponding author. E-mail address: [email protected] (L.B. Braos).

https://doi.org/10.1016/j.scienta.2019.05.032 Received 18 September 2018; Received in revised form 30 April 2019; Accepted 13 May 2019 Available online 20 May 2019 0304-4238/ © 2019 Elsevier B.V. All rights reserved.

Scientia Horticulturae 255 (2019) 153–160

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months during which the crops were cultivated. Irrigation of the soil was performed using sprinklers whenever necessary. The experiments were placed side by side and designed as randomized blocks with five replications. The treatments were seven rates of N applied in the form of topdressing fertilization (0, 30, 60, 90, 120, 150, and 180 kg N ha -1). The N application was split into three parts that occurred on the 8th, 16th, and 24th day after planting and corresponded to 20%, 40%, and 40% of the total N dose, respectively. The N fertilizer used in the experiments was urea (46% N). Both experiments were carried out according to the same procedures. Before planting, the soil of all plots received the same doses of cattle manure and mineral fertilizers. The manure was applied at a rate of 10 t ha -1 and had the following characteristics: total N = 8.7 g kg-1; organic carbon (C) =228 g kg-1; C:N ratio = 14; and insoluble inorganic matter =551 g kg-1. The N rate applied via manure was 87 kg N ha -1. The mineral fertilizers used were urea, potassium chloride, and borax, applied at the rates of 40 kg ha -1 of N, 83 kg ha -1 of K, and 1 kg ha -1 of B. The fertilizers added to all plots were spread over the total area and incorporated after ploughing and harrowing. Five beds were prepared, with 0.5 m of spacing between each other and dimensions of 1.20 × 17.5 × 0.2 m3 (width × length × height). Each bed corresponded to an experiment block. Crisphead lettuce (cv. Vanda) seedlings were germinated in a greenhouse, in trays filled with substrate (Plantmax Hortaliças™) with the following characteristics: pH in water of 5.8; density of 450 kg m-3; electrical conductivity of 1.3 dS cm-1; and water holding capacity of 15%. The transplanting of seedlings to experiments 1 and 2 occurred on February 28 And June 6, 2009, respectively. The plot was 2.5-m-long and 1.2-m-wide (total area of 3.0 m2), with four crop rows arranged with a spacing of 0.3 m between rows and 0.25 m between plants within each row. The number of plants per m2 was 13.3, resulting in approximately 133,000 plants per ha (40 plants per plot). Only the two central rows were considered for the yield calculation and plant analysis. Experiment 1 was harvested 34 days after planting, on April 3rd, whereas experiment 2 was harvested 42 days after planting, on July 18th. The harvest was carried out before sunrise, between 6 and 7 am. Lettuce plants were harvested by cutting the entire plant close to the ground and weighed to determine the fresh matter yield (FMY). Three random plants from each plot were sampled to determine the total N uptake by plant shoots (Nup) and plant NO3- concentrations (plant NO3-). The plants were kept in a refrigerator for less than 24 hours and then washed and oven dried at 65 °C until their weight stabilized. The dry matter was weighed to quantify the moisture content, ground with a mortar and pestle, and sieved in a 0.355-mm mesh sieve. The dry matter N concentration was determined using the Kjeldahl digestion method followed by steam distillation and titration, according to Carmo et al. (2000). The plant NO3- concentration was obtained using water extraction followed by distillation, according to Mantovani et al. (2005). The Nup levels were calculated based on the N content in lettuce dry matter and yield, whereas the plant NO3- concentration was converted into mg of NO3- per kg of fresh matter using the moisture content. Immediately after the harvest, soil samples were collected from each

Brazil, problems involving NO3- leaching are recurrent. In these areas, vegetable crop fields are located near rivers and on soils with a shallow water table, conditions that increase NO3- contamination (Oliveira et al., 2001). One way to reduce the N loss by leaching is to decrease the amounts of N fertilizer used on lettuce crops (Lorensini et al., 2012). It has been reported that the use of lower amounts of N fertilizers reduced NO3leaching in the growth of vegetable crops and led to a small decrease in the crop yield (Aragão et al., 2012; Kurtz et al., 2012). Thus, water quality is maintained because of the reduced NO3- leaching, and the quality and quantity of vegetable crops are maintained at satisfactory levels. The high rates of N applied to vegetable crops are not only an environmental issue but also a human health issue. The intake of NO3is harmful to human health, and vegetable consumption is one of the main sources of this anion in human diet (Santamaria, 2006). Liu et al. (2014) observed that the N fertilizer application rate, type, and distribution influence the NO3- content of vegetables. Other factors such as the luminosity, soil moisture, and molybdenum availability can also impact the plant NO3- content (Kovács et al., 2015). The maximum NO3- concentrations allowed for human consumption in fresh/ groundwater (Gomes and Barizon, 2014) and open-field cropped lettuce fresh matter (Santamaria, 2006) are 10 mg L-1 and 2.5 g kg-1, respectively. Taking into consideration that lettuce plants absorb a small fraction of the total N applied and have a superficial root system, and that the recommended rates of N application in the form of organic and mineral fertilizers are usually high (Aguiar et al., 2014), the hypothesis of the present study is that applying N rates lower than those currently recommended will decrease the N leaching and NO3- content in lettuce fresh matter and will not affect crop yield significantly. The objective of the present investigation was to evaluate the effects of N fertilization on the yield, NO3- content, and N leaching in crisphead lettuce crops in two growing seasons.

2. Materials and methods Two field experiments were carried out in different growing seasons: experiment 1 was carried out from February to April 2009 (summer season), and experiment 2 was carried out from June to July 2009 (winter season). The experiments were conducted in the municipality of Jaboticabal, São Paulo state, Brazil, where the predominant soil types are oxisols and ultisols, the landscape is plain, and agricultural lands are mainly cropped under sugarcane and peanut. The coordinates of the locations where the experiments were carried out are 21°15′12″ South and 48°18′58″ West. Prior to the installation of the experiment, soil samples were collected for chemical and texture characterization (Camargo et al., 2009; Raij et al., 2001), the results of which are presented in Table 1. The soil was classified as a Hapludox (Soil Survey Staff, 2014), which is usually a deep soil (< 200 cm) with high contents of Fe and Al oxides in the clay fraction. The total rainfall and average temperature were 170 mm and 23 °C and 78 mm and 19 °C in the summer and winter seasons, respectively. Figs. 1 and 2 show the daily rainfall distribution, average temperature, sunshine duration, and global solar radiation in the

Table 1 Chemical attributes and texture of the soil in the area where the lettuce experiments were installed.. Expa

Resin Pb

OM

pH CaCl2

K+

Ca2+

Mg2+

1 2

mg dm-3 167 200

g dm-3 24 22

6.0 6.4

———————— mmolc dm-3 ——————— 2.6 52 19 1.6 51 12

a

H + Al

CEC

BS

Sand

Silt

Clay

15 16

89 81

% 83 80

340 -

20 -

640 -

Exp 1: experiment 1, carried out from February to April 2009 (summer season); Exp 2: experiment 2, carried out from June to July 2009 (winter season). Resin P: available phosphorus extracted with anion-exchange resin; OM: soil organic matter; pH: soil pH measured by suspending soil in a 0.01 M CaCl2 solution (1:2.5 soil solution ratio); K+, Ca2+, and Mg2+: exchangeable K, Ca, and Mg, respectively; H + Al: total acidity; CEC: cation-exchange capacity; BS: base saturation. b

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Fig. 1. Daily rainfall distribution (mm, scale on the left side) and average temperature (scale on the right side) during the summer (A) and winter (B) growing seasons in the year in which the experiments were carried out (2009).

Fig. 2. Daily sunshine duration (scale on the left side) and global solar radiation (scale on the right side) during the summer (A) and winter (B) growing seasons in the year in which the experiments were carried out (2009). 155

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T.d.B. Sylvestre, et al. n

Table 2 Mean values (n = 5) of lettuce yield, nitrogen uptake, and nitrate concentration in the fresh matter of lettuce harvested in summer and winter growing seasons. N rates

a

kg N ha-1 0 30 60 90 120 150 180 ANOVAc Treatments Blocks CV

2 ⎡ ∑ (Pi − Oi) ⎤ dw = 1 − ⎢ n i = 1 ' , 0 ≤ dw ≤ 1 ' 2⎥ ⎣ ∑i = 1 (|Pi | + |Oi |) ⎦

FMYb

Nup

Plant NO3-

FMY

Nup

Plant NO3-

t ha-1 12.8 c 15.9 b 20.3 A 19.9 a 18.8 Ab 19.2 A 16.9 b

kg ha-1 19.1 c 26.3 b 29.5 b 31.0 ab 33.2 A 32.5 a 32.2 A

mg kg-1 30 e 110 d 260 c 400 b 470 a 480 a 510 a

t ha-1 36.1 c 40.0 b 41.5 ab 42.4 ab 44.6 a 42.2 Ab 40.0 b

kg ha-1 71.3 c 78.2 bc 91.8 A 90.9 a 91.4 a 83.9 b 95.0 a

mg kg-1 40d 80d 180c 290 b 270 b 330 a 340 a

where n = number of observations; Pi = value estimated by the model; Oi = observed value; Pi’ = difference between the estimated value and the average of the observed values; and Oi’ = difference between the observed value and the average of the observed values. According to the definition, dw is always a positive value. When dw equals 0, there is total disagreement between the estimated and observed data, and when dw equals 1, there is a perfect agreement between the estimated and observed data. The results of Nup obtained from control and fertilized plots allowed the calculation of the apparent N recovery (ANR) (Eq. 4).

5.37** 1.7 ns 14.5 %

6.5** 5.6** 14.9 %

15.6** 0.6 ns 33.5 %

3.3* 1.6 ns 8.0 %

4.5** 3.0* 10.8 %

12.3** 5.5** 35.1 %

NFT − NCT ⎞ ANR (%) = ⎜⎛ ⎟ x 100 ⎝ applied N ⎠

Summer

Winter

N rates: rates of mineral nitrogen fertilizer applied in the form of urea; summer and winter: lettuce cropped in the summer and winter seasons, respectively. b FMY: lettuce fresh matter yield; Nup: nitrogen uptake by lettuce plants; Plant NO3-: nitrate concentration in lettuce fresh matter. All data shown are the average values from the five replications. c ANOVA: analysis of variance; **, *, and ns: significant at a probability of 1% and 5% and not significant, respectively; CV: coefficient of variation. Means followed by different letters do not differ statistically based on Tukey’s test (p < 0.05).

plot at the 0-20, 20-40, and 40-60 cm layers. Samples from each plot and depth were obtained by combining ten subsamples. The soil samples were stored in a freezer at −15 °C until the determination of soil ammonium (soil NH4+) and nitrate (soil NO3-) concentrations according to the method suggested by Bremner and Keeney (1965). The N leached was considered as the mineral N (NH4+ + NO3-) located at the 20-60 cm soil layer. The amount of N leached from the topdressing fertilization (NF loss) was estimated by calculating the difference between the mineral N content in the fertilized soil and in control treatments (0 kg N ha -1). The normality of the data was evaluated using the KolmogorovSmirnov test, and subsequently, data were submitted to analysis of variance (ANOVA) using the AgroEstat software (Barbosa and Maldonado Júnior, 2015). Linear and nonlinear regression analyses were performed using the Microsoft Excel 2016® software and the GRG2 method described by Lasdon et al. (1978).The responses of the variables FMY, Nup and plant NO3- as functions of the N topdressing fertilization rate were estimated and the equation parameters calculated. The Gaussian nonlinear model (Eq. 1) was fitted to the FMY results. −

3. Results The FMY, Nup, and plant NO3- values were significantly increased by the increase in N fertilizer rates (Table 2). Although Nup and plant NO3- continued to increase as a function of N rates, the FMY was higher for intermediate N rates. The Gaussian nonlinear model (peak model) was fitted to the FMY data and provided a good agreement between the estimated and observed results (dw values, Table 3), for both summer and winter growing seasons. Data fitting allowed estimation of the parameters described in Eq. (1), which were the estimated maximum yields (the sum of Y0 and A), 20.3 A nd 43.5 t ha -1, at the N rates of 97 A nd 109 kg N ha -1 (X0) for the summer and winter seasons, respectively. Using this equation, it was also possible to estimate the N rates that resulted in 90% of the maximum yield, which were 49 kg N ha -1 for the summer and 28 kg N ha -1 for the winter season. The fitting of the exponential model to Nup and plant NO3- data revealed good agreement and significance (Table 3). Data fitting provided estimates of all the parameters described in Eq. 2. The regressions fitted from each season’s dataset were compared by ANOVA and were considered different (p < 0.05). The estimated maximum Nup values (sum of Y0 and α) for the summer and winter seasons were 32.9 and 91.4 kg N ha -1, respectively. While the Nup regressions resulted in Y0 values higher than α ones, plant NO3- regressions resulted in higher α (Table 3). This pattern shows that the cropping season has a greater effect on Nup values than fertilization itself. Soil NH4+ levels were affected by the N fertilizer rate and sampling depth (Table 4). In both seasons, the increase in the N rate resulted in a linear increase in soil NH4+, although in the winter season, the values were slightly higher (Tables 3 and 4). In the summer season, the highest concentration of soil NH4+ was observed at the 20-40 cm layer, whereas in the winter season, the highest concentration was observed at the 0-20 cm layer. The N rates increased the soil NO3- concentration linearly (Tables 3 and 5). In the summer season, the highest contents of soil NO3- were observed at the 40-60 cm soil layer, whereas in the

(X − X0)2 2w 2

(1) -1

where Y = estimated fresh matter yield (kg ha ); Y0 = minimum yield (kg ha -1); A = model amplitude; X = N fertilizer dose (kg N ha -1); X0 = N fertilizer dose that resulted in the maximum yield (kg ha -1); and w = “bell” width. The maximum yield is the sum of Y0 and A. The exponential model with growth restraint (Eq. 2) was fitted to the data of the accumulation of total N and NO3- in lettuce shoots (Nup and plant NO3-).

Y = Y0 + α (1 − e−kX )

(4)

where NFT (kg ha -1) = Nup in the fertilized treatments (topdressing N fertilization); NCT (kg ha -1) = Nup in the control treatment (application of manure and urea before planting with no further N applications); and applied N = topdressing N rate applied after planting (30, 60, 90, 120, 150, or 180 kg N ha -1). In the statistical analysis of plant-related results (yield, plant N, and plant NO3-), the adopted experimental design was full randomized blocks, with seven treatments and five replications. For soil-related results (soil NH4+ and NO3-), the chosen design was split-plot with random blocks, with the N rates as plots (main treatments) and the soil sampling depth as subplots (secondary treatments). The means were compared with Tukey’s test (p < 0.05) and regression analysis. The cropping season effect was evaluated by comparing the fitted equations for summer and winter data using ANOVA.

a

Y = Y0 + A e

(3)

(2) -1

where Y = estimated Nup (kg ha ) or plant NO3- concentration (mg kg-1); Y0 = minimum value (without fertilization); α = maximum increase caused by fertilization (kg ha -1; mg kg-1); k = increase rate ((kg ha -1)-1; (mg kg-1)-1); and X = N fertilizer dose (kg N ha -1). The models were chosen based on their accuracy and all the nonlinear fittings. The fitting accuracy was measured using the Willmott index of agreement (dw), shown in Equation 3: 156

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Table 3 Models, parameters, accuracy, and significance of the regression analysis of yield, nitrogen uptake, nitrate content, soil nitrate concentration, and leached nitrogen as a function of nitrogen fertilizer rates in lettuce experiments cropped in the summer and winter seasons. Model / Variablea Gaussian FMY

Season

————————————— Equation parametersb —————————————

dw

F

R2

Summer Winter

Y0 (t ha-1) 6.9 28.3

A (t ha-1) 13.3 15.2

X0 (kg ha-1) 97 109

0.98 0.98

58.7** 72**

0.97 0.98

α (kg ha-1) 13.8 20.9 α (mg kg-1) 661 423.2

k 0.024 0.027 k 0.0088 0.0082

0.99 0.93

482** 16.6**

0.99 0.77

Summer Winter

Y0 (kg ha-1) 19.1 70.5 Y0 (mg kg-1) 4.2 23.0

0.99 0.99

180** 99.4**

0.97 0.95

Summer Winter

a (slope) 0.024 0.023

b (mg kg-1) 3.8 6.5

0.97 0.96

37.7** 29.1**

0.88 0.85

0.106 0.081 0.038 0.031 a (slope) 0.76 0.21

5.4 6.9 10.1 10.4 b (kg ha-1) 47.2 67.3

0.99 0.99 0.93 0.89

281** 128** 15.3* 10.1*

0.98 0.96 0.75 0.67

0.99 0.93

220** 16.5**

0.98 0.77

Exponential Nup Summer Winter Plant NO3-

Linear Soil NH4+

w (kg ha-1) 93 200

Soil NO3Summer Winter 0-20 cmc Winter 20-40 cm Winter 40-60 cm N leached Summer Winter

a Gaussian: Gaussian peak model used to estimate the lettuce yield response to the N dose; Exponential: exponential model used to estimate the lettuce N uptake and nitrate concentration response to the N dose; Linear: linear model used to estimate the soil ammonium and nitrate concentrations as well as the N leached. b Equation parameters: Gaussian, Y0 = minimum yield, A = maximum yield increase caused by N fertilization (amplitude), X0 = N rate that resulted in the maximum yield; Exponential, Y0 = minimum value (Nup or plant NO3-), α = maximum increase caused by fertilization, k = increase rate; dw: Wilmott index of agreement; F: significance value obtained using the F-test; * and **: significant at a probability of 5% and 1%, respectively; R2: coefficient of determination. c Statistical unfolding of the interaction between nitrogen rates and sampling depths (0-20, 20-40, and 40-60 cm).

4. Discussion

winter season, there was no effect of sampling depth on soil NO3- levels (Table 5). The ANR reached low values, especially in the summer season (Table 6), and in general, decreased as a function of N fertilizer rates. Consequently, the amounts of N leached and NF loss were high, accounting for 196 and 116 kg N ha -1 in the summer and winter seasons, respectively (Table 6). The greater leaching potential of the summer growing season was expressed as a higher slope in the linear fitting equation compared with the slope calculated for the winter season (Table 3).

The growth in the FMY and Nup values caused by the addition of N was expected because fertilization increases soil mineral N levels (Tables 4 and 5), leading to greater N absorption and better plant growth. The increase in plant NO3- observed after the N fertilizer use (Table 2) was also observed by Liu et al. (2014) and is a consequence of the increase in soil NO3- levels provoked by fertilization (Table 5). Although there was a significant increase in plant NO3- concentrations because of the application of N fertilizer, all values found were lower

Table 4 Mean values (n = 5) of soil ammonium concentrations after the harvest of crisphead lettuce cropped in two seasons as a function of the N fertilization rate and sampling depth. N ratesa

Summer 0-20 cm

-1

kg N ha 0 30 60 90 120 150 180 Mean ANOVAb N rates (N) Depth (D) NxD CV (plot) CV (split-plot)

Winter 20-40 cm

40-60 cm

————————————————————————————NH4+ 2.73 3.12 3.97 4.09 5.17 5.10 6.49 4.4 C

4.44 6.89 6.60 7.45 7.85 9.47 11.02 7.7 A

4.75 7.64 4.65 4.90 6.82 5.04 9.36 5.7 B

0-20 cm

20-40 cm

40-60 cm

(mg kg ) ———————————————————————————— 7.01 6.67 6.20 7.16 6.77 7.52 8.85 7.99 8.64 7.69 7.55 9.85 10.21 5.80 8.12 11.69 9.00 9.80 12.62 9.55 11.52 9.3 A 7.6 B 8.8 A -1

13.6** 10.46** 0.35 ns 14.6% 24.5%

10.1** 4.73* 1.1 ns 12.5% 13.1%

a

N rates: mineral nitrogen fertilizer rates applied in the form of urea; summer-autumn and winter cropping seasons; 0-20, 20-40, and 40-60 cm soil sampling depth. b N rates, Depth and N x D: significance values of N rates (main treatment), Depth (secondary treatment), and interaction between N rates and depth, respectively; CV: coefficient of variation; *, **, and ns: significant at a probability of 5% and 1% and not significant, respectively. Means followed by different letters do not differ statistically based on Tukey test (p < 0.05). 157

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Table 5 Mean values (n = 5) of soil nitrate concentrations after the harvest of crisphead lettuce as a function of the N fertilization rate and sampling depth, cropped in two seasons. N ratesa

Summer 0-20 cm

Winter 20-40 cm

40-60 cm

0-20 cm

20-40 cm

40-60 cm

————————————————————————————NO3- mg kg-1 ———————————————————————————— 3.3 5.52 9.75 7.97 9.99 10.39 4.9 7.47 12.89 9.11 11.22 11.70 6.8 11.46 15.40 10.80 13.63 13.21 10.2 16.28 19.41 13.38 13.84 12.26 12.6 19.21 20.68 17.15 13.01 14.23 16.1 22.57 21.55 20.79 13.96 12.69 24.8 21.84 31.15 20.79 18.88 18.17 11.2 A 14.9 B 18.7 C 14.3 13.5 13.2

kg N ha-1 0 30 60 90 120 150 180 Mean ANOVAb N rates (N) Depth (D) NxD CV (plot) CV (split-plot)

44.0** 32.38** 1.25 ns 15.1% 15.0%

32.5** 0.82 ns 3.73* 8.2% 9.3%

*, **, and ns: significant at a probability of 5% and 1% and not significant, respectively. Means followed by different letters do not differ statistically based on Tukey test (p < 0.05). a N rates: mineral nitrogen fertilizer rates applied in the form of urea; summer-autumn and winter cropping seasons; 0-20, 20-40, and 40-60 cm soil sampling depth. b N rates, Depth and N x D: significance values of N rates (main treatment), Depth (secondary treatment), and interaction between N rates and depth, respectively; CV: coefficient of variation.

20 cm soil layer (Tables 4 and 5). The low values of maximum increase caused by fertilization (α) can be explained by the addition of high N rates at the beginning of the experiment (87 A nd 40 kg N ha -1 via manure and urea, respectively), which supplied most of the crop requirement. The smaller effect of fertilization on the Nup (α) value in the summer season may have been caused by the loss of the applied N resulting from excessive rainfall (Fig. 1A). Although the increase in plant NO3- levels caused by fertilization (α) was higher, the maximum value of the plant NO3- concentration estimated by the regression (Y0 + α) was lower than the EC limits in both seasons. This result reveals that fertilization had a limited effect on plant NO3- concentrations, which also depend on the low N assimilation (conversion of mineral N into organic N) by plants (Kovács et al., 2015). The sunshine duration and the global solar radiation (Fig. 2) were suitable for NO3- assimilation, since nitrate accumulation is favored by low temperature and luminosity (Maynard et al., 1976). It is well documented that low temperatures and deficiencies of solar radiation and molybdenum are the major factors involving NO3- accumulation in vegetables (Kovács et al., 2015; Maynard et al., 1976). Ammonium is less likely to leach through the soil profile (Korsaeth, 2008), so the higher levels of soil NH4+ at deeper soil layers observed during the summer season (Table 4) may be a consequence of urea lixiviation before its conversion to ammonium (Zhao et al., 2009). The first and second topdressing urea applications (8 And 16 days after transplanting) were followed by intense rainfall, which favoured leaching of urea (Fig. 1A). Another reason for the lower NH4+ concentration in the superficial layer is the more favourable conditions for the nitrification process in this layer (aeration and temperature) (Sahrawat, 2008). The high levels of soil NH4+ in the superficial layer observed for the winter season, a result opposite to what was found for the summer season, occurred as a consequence of low rainfall precipitation, an unfavourable condition for leaching (Fig. 1B). Increases in soil NH4+ in the deep layers should be avoided because NH4+ is consumed in the nitrification process, leading to the acidification of these layers (Zhao et al., 2014). The low slope and intercept obtained in linear regression analysis of the soil NH4+ concentration as a function of N rates in the summer dataset (Table 3) suggest that increasing N fertilizer rates causes limited increases in the soil NH4+, probably due to the increase in the

Table 6 Apparent nitrogen fertilizer recovery by lettuce plants, total nitrogen leached, and nitrogen fertilizer loss in soil after the application of different amounts of nitrogen in the form of urea in two different growing seasons. N ratesa

kg N ha-1 0 30 60 90 120 150 180

Summer

Winter

ANRb

N leached

NF loss

ANR

N leached

NF loss

% 24 17 13 11 9 7

kg ha-1 49 73 90 117 134 150 196

kg ha-1 25 41 68 86 101 147

% 23 34 22 17 8 13

kg ha-1 66 74 86 87 82 91 116

kg ha-1 8 20 20 16 24 50

a N rates: mineral nitrogen fertilizer rates applied in the form of urea; summer and winter cropping seasons. b ANR: apparent nitrogen recovery; N leached: total nitrogen leached from the soil, calculated by considering all mineral nitrogen below the 0-20 cm soil layer, obtained as the difference between control (no nitrogen fertilization) and fertilized treatments.

than the limits established by the European Commission(Santamaria, 2006), as mentioned in the fourth paragraph of the introduction section. The higher values of FMY, Nup, and plant NO3- values (Table 2) observed in the winter season occurred because the winter climate is more suitable for lettuce cropping in the region, which results in better plant growth, higher Nup levels, and greater N assimilation. The high temperatures in summer (Fig. 1) were above the ideal range for lettuce, which is 18-25 °C during the day (Ryder, 1999). The manure dose used in the experiment (10 t ha -1) was only 25% of the minimum recommended dose and should lead to a greater crop response to the mineral topdressing N fertilization. However, the N fertilizer dose required to reach 90% of the maximum yield, which is considered a rational dose (Kirchmann and Bergström, 2001), was much lower than the recommended values, calculated as 49 and 28 kg N ha -1 N for the summer and winter growing seasons, respectively. The minimum Nup values (Y0) were considerably higher in the winter season, which may have been caused by the greater growth potential of this season and higher mineral N concentration in the 0158

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the mineral and organic fertilizer applied before seedling transplanting.

nitrification rate with increased temperature (Sahrawat, 2008). In contrast, the higher value of the intercept in the winter dataset indicates that this season had lower nitrification and leaching rates, resulting in higher levels of soil NH4+ in the 0-20 cm layer (Table 4), which also favoured N availability and uptake by lettuce plants (Table 2). The higher slope obtained for the summer dataset agrees with the higher nitrification rate and the N loss potential of this season. The relationship between the N rate and sampling depth observed in the soil NO3- levels of the winter season (Table 3) indicates that the applied N had a lower leaching potential during this season. Thus, the topdressing fertilization caused a greater increase in soil NO3- levels in the superficial layer compared with sub-superficial layers. The higher levels of mineral N (NH4+ and NO3-) in the superficial layer also contributed to the higher values of Nup during this season (Table 2). The absence of a soil depth effect in the winter season was caused by the low rainfall intensity (Fig. 1B), which does not imply that there was no NO3leaching, given that the soil NO3- levels increased at all soil depths. This result indicates that rainfall was not enough to drag all soil NO3- across the deepest layers, resulting in similar levels in all layers during this season. The decrease in ANR values as a function of N fertilizer rate (Table 6) occurred as a result of the limited potential of N uptake by the crop, defined as Y0 + α (Nup exponential equation, Table 3), leading to a non-use of the excess N. The winter season showed higher ANR values in comparison with the summer season because of the higher uptake of N by lettuce plants (Table 2). The lower ANR values of the summer season were also the reason for the greater N leaching in this period. The low Nup, combined with the higher levels of mineral N below the 0-20 cm soil layer, resulted in NF loss values, which accounted for more than half of the total N applied (Table 6). The slope of 0.76 obtained for the linear equation of N leaching for the summer season suggests that, for each kilogram of N applied as topdressing fertilizer, 0.76 kgs is lost (Table 3), a result that is consistent with the low Nup value and high leaching potential of this season (Chen et al., 2018). The same pattern was observed in the winter season (Table 3), but the lower slope was related to the high Nup value and less intense rainfall (Fig. 1B). The intercept values of both equations were high, showing that N losses were considerable even without topdressing N fertilization (Table 3). This pattern is caused by the application of fertilizer before the transplanting, a procedure that involves the use of mineral N and manure, resulting in higher concentrations of mineral N in the soil. Because lettuce seedlings have a superficial root system (Jackson et al., 1994), most of the applied N was inaccessible to the plants and susceptible to leaching. The lower intercept value calculated in the N leached equation for the summer season may have been caused by excess rainfall during this period (Fig. 1A), leading to N leaching below the 60 cm depth, which was the maximum depth sampled.

Acknowledgements The authors would like to express their gratitude to the São Paulo Research Foundation (FAPESP) for providing the first author with a master’s scholarship (grant #2008/52415-1). The authors thank the staff of the Agroclimatological Station, Exact Sciences Department, Faculdade de Ciências Agrárias e Veterinárias de Jaboticabal, for providing all the meteorological data used in this research. References Aguiar, A.T., de E, Gonçalves, C., Paterniani, M.E.A.G.Z., Tucci, M.L.S., Castro, C.E.Fde, 2014. Instruções agrícolas para as principais culturas econômicas, Boletim 200, 7th ed. Instituto Agronômico de Campinas. Instituto Agronômico de Campinas, Campinas. Aquino, L.A., Puiatti, M., Abaurre, M.E.O., Cecon, P.R., Pereira, P.R.G., Pereira, F.H.F., Castro, M.R.S., 2007. Yield, accumulation of nitrate, content and export of nutrients of lettuce cultivated under shade. Hortic. Bras. 25, 381–386. 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5. Conclusion The NO3- concentration in the lettuce fresh matter after the addition of topdressing N fertilizer was lower than the upper limit recommended for human consumption, even at the highest N application rate. However, the application of N fertilizer resulted in an increase in NO3leaching, which can lead to environmental and economic losses. The application of topdressing N rates lower than the recommended one (120 kg N ha -1) decreased leaching and increased the fertilizer recuperation by the lettuce crop. The predicted N rates required to reach 90% of the maximum yield were also lower than those currently recommended, supporting the that lowering the N rates does not reduce the lettuce yield while decreases the N leaching. The dose of 60 kg N ha -1 , which is half of the officially recommend, also provided good efficiency and lettuce yield. These findings also indicate that the guidelines should be reviewed to take into consideration the growing season and 159

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