Biochar counteracts nitrification inhibitor DMPP–mediated negative effect on spinach (Spinacia oleracea L.) growth

Ecotoxicology and Environmental Safety 191 (2020) 110243

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Biochar counteracts nitrification inhibitor DMPP–mediated negative effect on spinach (Spinacia oleracea L.) growth

T

Jamal Sheikhia, Hossein Mirsyed Hosseinia,∗∗, Hassan Etesamia,∗, Aziz Majidib a b

Department of Soil Science, University College of Agriculture and Natural Resources, University of Tehran, 31587-77871, Tehran, Iran Agricultural Research and Education and Natural Resource Center, West Azerbaijan, Iran

A R T I C LE I N FO

A B S T R A C T

Keywords: Biochar–amended soil 3,4–dimethylpyrazole phosphate Nitrate reductase Nitrite reductase Nitrogen use efficiency Wheat straw biochar

The use of nitrification inhibitors (NIs) such as 3,4–dimethylpyrazole phosphate (DMPP) has been suggested to diminish agricultural soil nitrate (NO3−) loss and increase nitrogen (N) use efficiency (NUE). However, the yield of ammonium (NH4+)–sensitive plants such as spinach (Spinacia oleracea L.) may be adversely affected by the application of NIs at high N levels and, on the other hand, the efficiency of the NIs may also be affected by soil amendments such as biochar. These two issues are still not adequately addressed. The aim of this study was to evaluate the effect of different N levels including DMPP or not in a calcareous soil with and without amendment of wheat straw biochar on spinach yield, NUE, nitrate concentration of spinach leaf, activity of enzymes nitrate reductase (NR) and nitrite reductase (NiR), and soil ammonium (NH4+) and NO3− concentration under greenhouse conditions. This experiment was carried out with different N rates factor at seven levels (un–fertilized, N0; fertilized with 50 mg N kg−1 soil, N50; fertilized with 75 mg N kg−1 soil, N75; fertilized with 100 mg N kg−1 soil, N100; fertilized with N50 + DMPP; fertilized with N75 + DMPP; and fertilized with N100 + DMPP) and biochar (BC) factor at two levels (0, 0%BC; and 2% (w/w), 2%BC) with six replications over a 56–day cultivation period of spinach. Results showed that the application of DMPP had no significant effect on the yield of spinach plant at low and medium levels of N (50 and 75 mg N kg−1 soil), but decreased the yield of this plant at the higher level of N (100 mg N kg−1 soil). However, application of BC decreased the negative effect of DMPP on spinach yield as the yield in spinach plants fertilized with N75 + DMPP and N100 + DMPP significantly increased. Both application of DMPP and addition of BC to soil decreased leaf NO3− concentration by 29.2% and 16.3% compared to control, respectively. Biochar compared to control decreased NR activity by 46.3%. With increasing N rate, NR and NiR activities increased, but DMPP decreased the activities of both enzymes. Biochar reduced the efficiency of DMPP as soil NH4+ concentration was higher in the treatments containing DMPP without BC at 56 days after planting. Biochar and DMPP could increase the quality of spinach plant through decreasing the leaf NO3− concentration. In general, wheat straw biochar counteracted DMPP–mediated negative effect on growth of spinach plant at high level of N by decreasing the efficiency of this inhibitor. These results provide the useful information for managing the application rate of N fertilizers including DMPP in biochar–amended soil.

1. Introduction Production of vegetables like leafy green vegetables plays an important role in making secure food availability and safety at the world. Spinach (Spinacia oleracea L.) is an important source of vitamins, carotenoids, organic acids, alkaline minerals, and antioxidants (Gong et al., 2019), and is also rich in essential nutrients for human health (Tang et al., 2019). During the last three decades, due to increasing the consumption of vegetable crops, the harvested areas and production of

∗

the vegetables have ceaselessly augmented at the rates of 7.0% and 9.5% per year, respectively. Nitrogen (N) is an essential element for the growth and yield of vegetable crops and is often a limiting factor for the crop production, as only a low fraction of the atmospheric nitrogen (N2) is available to plants through biological N2 fixation (White and Brown, 2010). The use of chemical nitrogenous fertilizers in vegetable fields has significantly increased the crop yield. However, nitrogen use efficiency (NUE) in the plant is low (~32% for spinach) (Canali et al., 2014) and contributes to environmental pollution and health risks (i.e.,

Corresponding author. Corresponding author. E-mail addresses: [email protected] (H. Mirsyed Hosseini), [email protected] (H. Etesami).

∗∗

https://doi.org/10.1016/j.ecoenv.2020.110243 Received 13 September 2019; Received in revised form 20 January 2020; Accepted 21 January 2020 0147-6513/ © 2020 Elsevier Inc. All rights reserved.

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accumulation of NH4+ in the soil, in calcareous soils amended with biochar, which results in increased sorption of NH4+ in soil, on nitrification and the yield of vegetable crops such as spinach plants. Recently, reduced nitrification inhibitory effect of DMPP and the subsequent mitigation of N2O emissions at both low and high soil water content conditions following the combined application of DMPP and biochar were reported by Fuertes-Mendizábal et al. (2019). Provided that biochar has been proven to reduce the efficiency of DMPP by adsorbing the DMPP molecules and increase higher sorption NH4+ compared to NO3− in soil, the aim of the research was to evaluate the effect of different N levels including DMPP or not in a calcareous soil with and without amendment of wheat straw biochar on yield of spinach plant, as an ammonium sensitive plant, and the efficiency of the nitrification inhibitor DMPP. Also in this study, the effect of the combined application of DMPP and this biochar on the activity of enzymes involved in N assimilation (nitrate reductase and nitrite reductase) in aerial parts of spinach plant was determined.

accumulation of excess nitrate in soil and groundwater) (Kumar et al., 2015). High N rates (global mean of 220 kg N ha−1 in each cultivation season), intensive production, and management practices such as repeated irrigation and tillage and manifold cultivation–harvest cycles during year are some of the main characteristics of vegetable fields (Rashti et al., 2015; Yang et al., 2019). All of these operations are a source of N loss in these fields. Nitrification, which increases the susceptibility of reactive–N loss from agroecosystems/vegetable fields, is an aerobic process that in the first step of nitrification NH4+ is oxidized to hydroxylamine (NH2OH) by the enzyme ammonia monooxygenase (AMO) and afterwards to nitrite NO2− by the hydroxylamine oxidoreductase (HAO). In the second step of nitrification, nitrite oxidizing bacteria oxidize NO2− to NO3− using enzyme nitrite oxidoreductase (NOR). Ammonia–monooxygenase synthesis is codified by the amoA gene found in ammonia–oxidizing archaea (AOA) and ammonia–oxidizing bacteria (AOB) (Ruser and Schulz, 2015). Nitrification inhibitors, which reduce the oxidation of NH4+ to NO3− by hampering AMO activity, the metabolic activity and growth of ammonia–oxidizing bacteria, are proposed as tools to reduce N loss, thereby increasing NUE by plants (Abalos et al., 2014). Among the nitrification inhibitors, 3,4–dimethylpyrazole phosphate (DMPP) is known as the most effective inhibitor due to having a high efficiency at low application rates and low mobility in soil, and longer duration of activity (Benckiser et al., 2013; Chaves et al., 2006; Zerulla et al., 2001). Although the main mechanisms yet remain obscure, it has been suggested that DMPP can act as a chelating compound that reduces the availability of cupper (Cu), the co–factor of AMO (Ruser and Schulz, 2015), and postpones the microbial oxidation of NH4+ to NO3− by inactivating the enzyme AMO (Gilsanz et al., 2016; Ruser and Schulz, 2015). It is known that nitrification inhibitors like DMPP can increase NUE and crop productivity by 12.9% and 7.5%, respectively. The efficiency of these inhibitors depends on the environmental and management factors such as irrigation systems, applied N fertilizer rates (Abalos et al., 2014) and soil properties like soil texture (Guardia et al., 2018), organic matter (Singh et al., 2008), and soil aeration (Balaine et al., 2015). In addition to the factors mentioned above, recently soil amendments such as biochar, a carbon–containing material obtained from the thermo–chemical conversion of biomass in an oxygen–restricted environment, have been known to affect the efficacy of DMPP. For example, in a study (FuertesMendizábal et al., 2019), the application of Pinus taeda biochar remarkably diminished the nitrification inhibitory impact of DMPP due to the adsorption of DMPP on biochar surfaces and, as a result a decrease in its availability to AOB and AOA (Keiblinger et al., 2018). However, there is still a considerable disagreement about the biochar effect on the efficiency of nitrification inhibitors. In previous studies, role of biochar in reducing NO3− loss and N2O emission (Fuertes-Mendizábal et al., 2019; Van Zwieten et al., 2015) and enhancing NUE (Cai and Chang, 2015) has also been reported. It is well known that biochars are potentially effective sorbents for NH4+ and NO3− in water treatment and soil applications (Fidel et al., 2018; Khalil et al., 2018). In addition, it has been found that the sorption of NH4+ by biochar is higher than that of NO3− in high pH (> 7.0) in soil (Fidel et al., 2018). Despite the fact that many various statements have been suggested (Fuertes-Mendizábal et al., 2019), the mechanisms by which biochar can affect soil N–cycling processes remain obscure. Depending on biochar feedstock, method of pyrolysis, soil type and properties (i.e., pH, organic matter, and soil texture), soil water content and agricultural system, the biochar effect on increasing or decreasing N loss varies (Borchard et al., 2014; Cayuela et al., 2015; Spokas and Reicosky, 2009). It has been demonstrated that nitrification inhibitors may have the negative effect on the growth and yield of plants (Casar et al., 2010) such as spinach. This can be due to the sensitivity of spinach plant to high concentrations of ammonium (Lasa et al., 2002; Xing et al., 2015). Little research has been undertaken to evaluate the effect of different N levels including nitrification inhibitors like DMPP, which results in the

2. Materials and methods 2.1. Soil sampling and analysis The soil used in this study was a sandy loam calcareous soil (sand, 59%; silt, 30%; and clay, 11%), which was collected from the surface horizon (0–20 cm) of a farm land (35°54′ N; 50°53′ E) in Karaj, Iran. Roots and stones were removed from this soil and the soil was sieved at 4 mm. The soil was then air–dried, homogenized and kept at 4 °C until used. This soil had a pH of 7.6; the electrical conductivity (EC) of 0.68 dS m−1; the organic carbon (O.C) of 0.58%; cation exchange capacity (CEC) of 10.80 cmolc kg−1; NH4+ content, 4.90 mg kg−1; NO3− content, 8.50 mg kg−1; total nitrogen (TN), 0.041%; available phosphorus (P), 10.68 mg kg−1; exchangeable potassium (K), 380 mg kg−1; available iron (Fe), 2.30 mg kg−1; available manganese (Mn), 6.80 mg kg−1; available copper (Cu), 1.20 mg kg−1; available zinc (Zn), 1.40 mg kg−1; calcium carbonate equivalent (CCE), 11.6%; population size of AOB, 4.37 log MPN g−1 soil; and population size of nitrite–oxidizing bacteria (NOB), 4.07 log MPN g−1 soil. In this experiment, soil texture was evaluated via the pipette method (Gee and Bauder, 1987). The EC was measured in a saturated solution extract (Rhoades, 1996) and pH in a 1:2.5 mixture of soil and deionized water using a glass electrode (EYELA, Japan) (Thomas, 1996). The organic C of this soil was measured by oxidizing the organic material with chromic acid and titrating the excess dichromate (Nelson and Sommers, 1996) and soil CEC was also determined using the ammonium acetate method (Sumner and Miller, 1996). Soil NH4+ content was quantified colorimetrically by the Berthelot reaction (Mulvaney, 1996) and the nitrate (NO3−) content estimated colorimetrically by vanadium (III) chloride reduction (Doane and Horwáth, 2003). Total N was analyzed by the Kjeldahl method (Bremner, 1996) and the available P was determined according to the published method (Watanabe and Olsen, 1965). Exchangeable K was determined using a flame photometer following soil extraction with 1 N ammonium acetate (Thomas, 1996), and micronutrients (Fe, Zn, Cu, and Mn) were extracted using a DTPA and their concentration were measured by using flame atomic absorption spectrophotometry (Lindsay and Norvell, 1978). Soil CCE was measured by neutralization with hydrochloric acid (Loeppert and Suarez, 1996) and population size of autotrophic nitrifying bacteria (AOB and NOB) were measured by most probable number (MPN) method (Schmidt and Belser, 1996). 2.2. Biochar preparation Wheat straw residues were collected from agricultural research fields of Tehran University, Iran, air–dried, and then were cut into small pieces (1 cm) and oven–dried at 65 °C. The sieved (a uniform 1 mm size fraction) and ground wheat straw was slow–pyrolyzed and converted to 2

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biochar at 450 °C at a rate of approximately 20 °C min−1 and residence time of 4 h under oxygen– restricted conditions using a muffle furnace according to Ok et al. (2015). The reactor was filled with the raw materials and tightly sealed to minimize oxygen content at reaction. Some basic physical and chemical properties of the biochar such as pH and EC using 1:20 biochar: deionized H2O, total C, N and H contents using dry combustion analysis using an elemental analyzer (PerkinElmer 2400 II, Massachusetts, USA), O.C calculated as total C – inorganic C, ash content, apparent density, surface acidic groups and CEC by the ammonium acetate were determined using standard methods provided by the International Biochar Institute (Definition, 2015). This biochar had a pH of 9.25; EC of 4.68 dS m−1; O.C, 67.80%; total C, 69.40%; total N, 0.34%; H, 3.10%; C/N, 199.4; H/O.C, 0.55; apparent density, 0.13 g cm−3; ash, 17.10%; CEC, 24.70 cmolc kg−1; and acidic groups, 1683 μmol H+ g−1.

Plant Breeding Research Institute (SPBRI) and disinfected. Ten germinated healthy seeds of spinach were planted in each pot and then thinned to five uniform seedlings in each pot 10 DAP. To remove the possible error of evaporation, light, temperature, transpiration and other factors as far as possible to warrant uniformity of the conditions, the place of the treatments and replications of each treatment were replaced in rotation. Based on a weighted method, the plants were irrigated daily with distilled water, providing 80% moisture content of field capacity. The duration of planting in the greenhouse was 56 days. 2.4. Measurements 2.4.1. Chlorophyll index, number of leaf, and plant yield Chlorophyll index, number of leaf, and plant yield (plant shoot fresh weight) were measured at the end of plant growth period (56 days after planting). Due to the relationship between the chlorophyll and N content in plant, the chlorophyll index was measured by Chlorophyll Meter (SPAD–502) prior to plant harvest. For this purpose, chlorophyll index was recorded randomly in four recently matured leaves in each pot, and then their means as a chlorophyll index in each pot were reported. Number of plant leaves in each pot was also counted and recorded. To measure spinach plant yield, the plant shoot was cut at the soil surface, and the fresh weight was recorded without delay.

2.3. Experimental design and set–up A completely randomized design with factorial arrangement (a 7 × 2 factorial design) in six replications was arranged to evaluate the effect of different N levels including DMPP or not in a potted calcareous soil with and without amendment of wheat straw biochar on the yield of spinach plant and DMPP efficiency. Of these six replicates, three replicates were used for plant and soil sampling at different time stages of plant growth and the other three replicates were used to obtain spinach chlorophyll index, number of leaf, and plant yield. In this study, the effects of the various treatments on parameters such as plant yield, chlorophyll index, leaf number, NUE, nitrogen physiologic efficiency (NPE), and nitrogen agronomic efficiency (NAE) 56 days after planting (DAP) and on parameters such as leaf nitrate concentration, leaf protein content and the activity of nitrate reductase (NR) and nitrite reductase (NiR) of spinach shoot 28, 42, and 56 DAP were investigated. Therefore, the analysis of the data obtained at different sampling dates and at the end of the plant growth period was carried out by a split–plot design in time with factorial arrangement and a completely randomized design with factorial arrangement, respectively. The research was carried out in a research greenhouse under 270–350 μmol m−2 s−1 of photosynthetic photon flux with a 12 h photoperiod and controlled temperature (21/18 °C day/night temperature) and relative humidity (70–75%) located at the Agricultural and Natural Resources Research Center, West Azerbaijan, Iran. The experiment treatments included (i) different N rates factor at seven levels (un–fertilized, N0; fertilized with 50 mg N kg−1 soil (equivalent to 150 kg N ha−1); N50; fertilized with 75 mg N kg−1 soil (equivalent to 200 kg N ha−1); N75; fertilized with 100 mg N kg−1 soil (equivalent to 250 kg N ha−1); N100; fertilized with N50 + DMPP; fertilized with N75 + DMPP; and fertilized with N100 + DMPP); and (ii) biochar (BC) factor at two levels (0, 0%BC; and 2% (w/w), 2%BC). In this assay, N without DMPP and N containing DMPP were used as sulfate ammonium (21% NH4+–N and 24% S) and NovaTec®Solub 21 (21% NH4+–N, 0.8% DMPP, and 24% S), respectively. The selected soil was filled into the polyethylene pots (25 cm × 25 cm × 30 cm). Biochar (on a dry weight basis) was mixed thoroughly into the soil (3 kg) and pots were filled with the same soil according to related treatments. Soil mixing was also performed in control treatment. Before planting, to stabilize microbial activity and chemical equilibria, the soil and the biochar soil mixture were moistened up to 60% moisture content of field capacity and pre–incubated for 7 days at room temperature (25 °C). Prior to planting and based on soil analysis, essential nutrients, except for N, including 20 mg P kg−1 (triple superphosphate), 10 mg Fe kg−1 (Fe–EDDHA), 10 mg Zn kg−1 (zinc sulfate), 5 mg Mn kg−1 (manganese sulfate), and 5 mg Cu kg−1 (copper sulfate) were added to all pots uniformly. Nitrogen fertilizer treatments were then added and uniformly mixed according to related treatments at planting time. In this experiment, spinach plant (Spinacia oleracea L. cv. Viroflay) was used as a test plant. The seeds of this variety were obtained from the Seed and

2.4.2. Leaf nitrate concentration Leaf nitrate concentration of spinach plant was measured 28, 42, and 56 days after planting using method of Cataldo et al. (1975) and the protocol provided by Zhao and Wang (2017). After preparation, the samples were measured at 410 nm with spectrophotometer (Shimadzu, UV/VS 3100, Japan). Leaf nitrate concentration was calculated from the standard curve using the regression equation and expressed as mg kg−1 fresh weight (FW) of plant. 2.4.3. Leaf protein content After 28, 42, and 56 DAP, the leaf protein content of spinach plant was estimated using the specific conversion factor of 4.39 determined for vegetables, including spinach plant by Fujihara et al. (2001). Leaf protein content was calculated from Eq. (1). Protein (g kg−1) = shoot N content (g kg−1) × 4.39

(1)

To measure the shoot N concentration after 28, 42, and 56 DAP, the plant samples were dried in an oven at 65 °C until constant weight and were then ground to fine powder using a mixer mill. Total N of these samples was determined by the Kjeldahl method (Bremner, 1996; Jones, 2001). Shoot N content was expressed as g kg−1 dry weight (DW) of plant. 2.4.4. Nitrogen use efficiency After 56 DAP, nitrogen use efficiency (NUE), N agronomic efficiency (NAE), N physiologic efficiency (NPE) were calculated according to Eq. (2), Eq. (3), and Eq. (4), respectively. NUE (%) = [(NFT – NCT)/Na] × 100

(2)

Where, NFT, NCT, and Na are the N uptake in N treatment, the N uptake in control treatment, and the N applied in soil, respectively. NAE (g g−1) = (YFT – YCT)/Na)

(3)

Where, YFT and YCT are the plant yield in N treatment and control treatment, respectively. NPE (g g−1) = (YFT – YCT) / (NFT – NCT)

(4)

The uptake of spinach shoot nitrogen was calculated according to Eq. (5). N uptake (mg pot-1) = Spinach shoot dry matter yield (mg 3

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pot−1) × shoot N concentration

(5)

Table 1 The effect of biochar (BC) and different nitrogen (N) rates (with and without DMPP; NI) on the yield, chlorophyll index and leaf numbers of spinach plant after 56 days after planting.

2.4.5. Activity of nitrate reductase and nitrite reductase After 28, 42, and 56 DAP, activity of two enzymes of nitrate reductase (NR) and nitrite reductase (NiR) was assayed according to the methods presented by Jaworski (1971) and Miflin (1967), respectively. Nitrate reductase activity and activity of NiR were expressed as μmol NO2− formed g−1 FW h−1 and μmol NO2− removed h−1 mg−1 protein, respectively.

Biochar (BC) levels (%) Nitrogen (N) rates

0 Plant yield (g pot

N0 N50 N75 N100 N50 + NI N75 + NI N100 + NI Mean

2.4.6. Soil ammonium and nitrate concentration Soil samples were collected from the soil of pots (from the bottom and the top of the soil of pots) 0, 14, 28, 42, and 56 DAP. About 10 g of each sample was extracted with 50 mL of 2 M KCl by shaking on a reciprocal shaker for 1 h and filtering through a Whatman No. 42 filter paper and kept in 4 °C. Another 10 g of each sample was oven–dried at 105 °C to calculate the soil moisture content. The NH4+ and NO3− concentration of the soil extracts were analyzed colorimetrically by the Berthelot reaction for NH4+ (Mulvaney, 1996) and vanadium (III) chloride reduction for NO3− (Doane and Horwáth, 2003). Briefly, for assaying NH4+ a 0.1 mL of sample or standard solution was added to a test tube, and then 1 mL of EDTA reagent was added to the test tube and swirled to mix its contents. The contents of the tube test were mixed after adding 4 mL of sodium salicylate–sodium nitroprusside reagent and 2 mL of buffered hypochlorite to this sample. After 1 h, its absorbance at 667 nm was measured by a spectrophotometer (Shimadzu, UV/VS 3100, Japan). The NH4+ concentration of samples was determined by utilizing the equation obtained via linear regression of the concentration of the standards on the corresponding absorbance measurements. For assaying NO3−, a 0.1 mL of sample extract or standard solution was added to the test tube, and then 5 mL of the reaction mixture containing vanadium (III) chloride, 1 M HCl, 0.2% N–1–naphthylethylenediamine dihydrochloride, and 2% sulfanilamide solution was added to it. For further color development, the samples were placed in a standard drying oven at 45 °C for 1 h. The samples were then read at a wavelength of 540 nm by a spectrophotometer (Shimadzu, UV/VS 3100, Japan). Nitrate concentration was calculated by linear regression equation of the concentration of the standards versus the corresponding measured absorbance. For measuring initial nitrite concentration of samples, all the above steps were performed without using vanadium (III) chloride, and were then subtracted from the NO3− concentration of samples. At the end of this experiment, total TN, O.C, EC, and pH of post–harvest soil were also measured according to the above methods.

10.7 34.2 44.5 54.8 35.6 41.5 46.4 38.2

± ± ± ± ± ± ± ±

−1

2

Mean

15.7 ± 0.85 g 40 ± 1.18 def 47.8 ± 0.66 c 58.3 ± 1.78 ab 40.9 ± 1.13 de 57.1 ± 2.92 ab 61.6 ± 0.82 a 45.9 ± 3.31 A

13.2 ± 1.21 37.1 ± 1.41 46.1 ± 0.93 56.6 ± 1.14 38.3 ± 1.46 49.3 ± 3.73 54 ± 3.6 A

22.7 37.8 44.9 47.4 34.6 43.1 45.7 39.5

± ± ± ± ± ± ± ±

0.78 0.55 0.74 1.54 0.39 0.24 1.08 1.98

23.3 ± 0.54 D 37 ± 0.81 C 44.2 ± 0.69 B 48.3 ± 0.92 A 36 ± 0.72 C 44.5 ± 0.75 B 48.5 ± 1.40 A

49.7 58.0 64.7 64.7 59.7 61.7 62.0 60.0

± ± ± ± ± ± ± ±

2.4 d 1.53 c 0.88 abc 0.33 abc 1.2 bc 1.2 abc 0.58 abc 1.15 A

)

0.4 g 0.32 f 1.11 cd 0.54 b 1.56 ef 0.62 de 2.43 cd 2.92 B

E D C A D B

Chlorophyll index N0 N50 N75 N100 N50 + NI N75 + NI N100 + NI Mean

23.9 36.2 43.5 49.3 37.4 45.9 51.4 41.1

± ± ± ± ± ± ± ±

0.73 1.53 1.15 1.01 0.69 0.89 0.77 1.23

e d c ab d bc a A

e d bc abc d c bc B

Leaf number (pot−1) N0 N50 N75 N100 N50 + NI N75 + NI N100 + NI Mean

45.3 59.7 63.7 65.0 61.7 65.7 66.7 61.1

± ± ± ± ± ± ± ±

0.33 0.67 0.88 1.53 2.03 1.76 1.76 1.59

d bc abc ab abc ab a A

47.5 58.8 64.2 64.8 60.7 63.7 64.3

± ± ± ± ± ± ±

1.45 0.83 0.60 0.70 1.14 1.31 1.33

C B A A AB A A

N0, un–fertilized; N50, fertilized with 50 mg N kg−1 soil; N75, fertilized with 75 mg N kg−1 soil; N100, fertilized with 100 mg N kg−1 soil; N50 + NI, fertilized with N50 + DMPP; N75 + NI, fertilized with N75 + DMPP; and N100 + NI, fertilized with N100 + DMPP. Values followed by the same small case letters or capital letters are not significantly different within rows or columns according to Duncan's multiple range test (DMRT) at P ≤ 0.05.

and treated with 100 mg N kg−1 including DMPP (N100 + NI), respectively (Table 1); although there was no significant difference between N100 + NI + 2%BC with treatments of N100 + 2%BC and N75 + NI + 2%BC. Application of BC (2%BC) increased the plant yield by 20% compared to without BC (0%BC). Also, application of different N levels gradually increased the plant yield compared to control (Table 1). At the treatments of 0%BC, the effect of ammonium sulfate containing DMPP was not significant on plant yield at levels of 50 and 75 mg N kg−1 compared to equal levels without DMPP. However, application of 100 mg N kg−1 containing DMPP decreased plant yield by 15% compared to equal levels without DMPP. At the treatments of 2% BC, the effect of ammonium sulfate containing DMPP was not significant on plant yield at level of 50 mg N kg−1 compared to equal levels without DMPP. But, application of 75 and 100 mg N kg−1 containing DMPP increased plant yield by 19.5 and 5.6%, respectively, compared to the equal levels without DMPP (Table 1), although there was no significant difference between 100 mg N kg−1 containing DMPP and 100 mg N kg−1 without DMPP. The highest chlorophyll index was obtained by addition of 100 mg N kg−1 containing DMPP (N100 + NI) at level of 0%BC (Table 1). Application of 2%BC reduced chlorophyll index by 3.9% compared to 0%BC, but had no significant effect on the number of spinach plant leaf. Addition of 50, 75, and 100 mg N kg−1 increased chlorophyll index by 58.8, 88.7, and 107% compared to control plants, respectively. Also, addition of 50, 75, and 100 mg N kg−1 increased the

2.5. Statistical analysis The data were analyzed with Statistical Analysis System (SAS Institute Inc., Cary, NC, version 9.4) for two–way analysis of variance (ANOVA), and the means ± the standard error (SE) were compared by a Duncan's multiple range test (P ≤ 0.05). The Pearson test (two–tailed) at P ≤ 0.05 was used for analyzing correlation among measured parameters. Any differences between the mean values were considered to be significant at P ≤ 0.05. 3. Results 3.1. Plant yield, chlorophyll index, and leaf number The interaction of biochar (BC) and various N rates (with and without DMPP) on the yield, chlorophyll index, and leaf number of spinach plant was significant (P < 0.05) after 56 DAP (Table S1). The lowest and highest yield of spinach plant were observed in the control plants (without application of BC and N) and the plants treated with BC 4

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Fig. 1. Effect of biochar (BC; 0%BC and 2%BC) and different nitrogen levels (with and without DMPP) on nitrate (NO3−) concentration of spinach leaf after 28, 42, and 56 days after planting. N0, un–fertilized; N50, fertilized with 50 mg N kg−1 soil; N75, fertilized with 75 mg N kg−1 soil; N100, fertilized with 100 mg N kg−1 soil; N50 + NI, fertilized with N50 + DMPP; N75 + NI, fertilized with N75 + DMPP; and N100 + NI, fertilized with N100 + DMPP. Vertical bars represent the standard error (SE) of the mean of each treatment (n = 3) and the same small case letters are not significantly different according to Duncan's multiple range test (DMRT) at P ≤ 0.05.

treatments compared to 0%BC treatments was higher significantly by 4.2% (Fig. 2). The effect of sampling dates on leaf protein content (Fig. 2) showed that with increasing the sampling dates, leaf protein content significantly decreased as leaf protein content was lower than 9.6 and 15.5% at 42 and 56 DAP compared to at 28 DAP, respectively.

number of leaves by 23.8, 35.2 and 36.4% compared to control, respectively. However, ammonium sulfate containing DMPP had no significant effect on chlorophyll index and the number of plant leaves compared to ammonium sulfate without DMPP (Table 1). 3.2. Leaf nitrate concentration

3.4. Nitrogen use efficiency The three–way interaction of various treatments (BC, various N rates, and sampling dates) on leaf nitrate (NO3−) concentration was significant (P < 0.01) (Table S2). The highest of nitrate concentration of spinach leaf was observed in the treatment of 0%BC with application of 100 mg N kg−1 without DMPP (N100) after 42 DAP (Fig. 1). Nitrate concentration of spinach leaf gradually increased by increasing N levels (Fig. 1). Leaf nitrate concentration was enhanced by 161.5, 200, and 293% in plants treated with 50, 75, and 100 mg N kg−1 without DMPP and by 126.6, 143, and 178% in plants treated with 50, 75, and 100 mg N kg−1 with DMPP, respectively, compared to control. In addition, leaf nitrate concentration was significantly reduced in plants treated with ammonium sulfate containing DMPP compared to plants treated with ammonium sulfate without DMPP as application of 50, 75, and 100 mg N kg−1 with DMPP decreased leaf nitrate concentration by 13.35, 19.1, and 29.2%, respectively, compared to the equal levels of N without DMPP (Fig. 1). Application of 2%BC decreased leaf nitrate concentration by 16.3% compared to 0%BC (Fig. 1). Also, leaf nitrate concentration at 42 DAP was 21.7% higher than that at 28 DAP. In addition, there was no significant difference in the leaf concentration of nitrate at 42 and 56 DAP (Fig. 1).

The interaction of BC and different N rates (with and without DMPP) on NUE, NPE, and NAE was not significant (P < 0.05) (Table S1). Nitrogen use efficiency in 2%BC was higher significantly by 10.2% compared to 0%BC (Fig. 3A), while NPE in 0%BC compared to 2%BC was higher significantly (Fig. 3B). Also, 2%BC had no significant effect on NAE (Fig. 3C). The highest NUE was observed in the treatment of 100 mg N kg−1 and the lowest NUE was observed in the treatment of 50 mg N kg−1 (Fig. 3D). Nitrogen physiologic efficiency gradually decreased by increasing N levels as the highest and lowest NPE were observed in the treatment of 50 mg N kg −1 and 100 mg N kg−1, respectively (Fig. 3E). Application of 50 mg N kg−1 containing DMPP significantly increased NAE compared to 50 mg N kg without DMPP. Application of 75 mg N kg−1 containing DMPP had no significant effect on NAE compared to 75 mg N kg−1 without DMPP, but application of 100 mg N kg−1 containing DMPP decreased NAE compared to 100 mg N kg−1 without DMPP (Fig. 3F). 3.5. Activity of nitrate reductase (NR) and nitrite reductase (NiR) The three–way interaction of various treatments (BC, various N rates, and sampling dates) on NR and NiR activity of spinach shoot was significant (P < 0.01) (Table S2). The highest of NR activity of spinach plant was obtained in the treatment of 0%BC with application of 100 mg N kg−1 without DMPP at 28 DAP (Fig. 4A). Also, the highest of NiR activity of spinach plant was observed in 0%BC with application of 100 and 75 mg N kg−1 without DMPP at 28 DAP (Fig. 4B). Application of 2%BC decreased NR activity compared to 0%BC by 46.3%, but had no significant effect on NiR activity (Fig. 4A and B). Enhanced N rates in treatments without DMPP increased NR and NiR activities (Fig. 4A and B). Application of ammonium sulfate containing DMPP compared to ammonium sulfate without DMPP decreased the activity of both enzymes as 50, 75, and 100 mg N kg−1 with DMPP compared to the equal

3.3. Leaf protein concentration The three–way interaction of various treatments (BC, various N rates, and sampling dates) on protein content of spinach leaf was significant (P < 0.01) (Table S2). The highest of protein content of spinach leaf was observed in treatment of 2%BC with application of 100 mg N kg−1 with and without DMPP at 28 DAP (Fig. 2). Increasing N levels gradually increased protein content of spinach leaf, as application of 50, 75, and 100 mg N kg−1 increased leaf protein content at treatments without DMPP by 31.1, 54.8, and 86.3% compared to without N (N0), respectively. However, DMPP had no significant effect on protein content of spinach leaf. Leaf protein content in 2%BC 5

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Fig. 2. Effect of biochar (BC; 0%BC and 2%BC) and different nitrogen levels (with and without DMPP) on protein content of spinach leaf after 28, 42, and 56 days after planting. N0, un–fertilized; N50, fertilized with 50 mg N kg−1 soil; N75, fertilized with 75 mg N kg−1 soil; N100, fertilized with 100 mg N kg−1 soil; N50 + NI, fertilized with N50 + DMPP; N75 + NI, fertilized with N75 + DMPP; and N100 + NI, fertilized with N100 + DMPP. Vertical bars represent the standard error (SE) of the mean of each treatment (n = 3) and the same small case letters are not significantly different according to Duncan's multiple range test (DMRT) at P ≤ 0.05.

4. Discussion

levels without DMPP decreased NR activity by 25.3, 44.9, and 50.4% and NiR activity by 24.8, 34.8, and 31.6%, respectively (Fig. 4A and B). According to Fig. 4, with increasing sampling dates in the plants treated with various N levels without DMPP, the activity of the enzymes gradually was also reduced.

The results of this assay showed that wheat straw BC application significantly increased the yield of spinach plant (and leaf protein), which are consistent with the results of study of Li et al. (2016), in which they showed that corn straw BC after 50 DAP increased yield of spinach plant by 63.7% compared to control in an alkaline soil. This increase of 63.7% over control in the soil was attributed to the presence of nutrients in biochar for spinach plant growth. Other studies have also reported increased yield in other plants as a result of biochar application. For example, Palansooriya et al. (2019) in a review study reported that the biochar application rates at the range of 0.1–67.5 t ha−1 resulted in augmented crop yields from 2 to 143% with an average of 38.1%. Mandal et al. (2016) in a study in Australia with biochar application on wheat growth showed that biochar increased nitrogen uptake from urea fertilizer by 33.16% and attributed this increase to the biochar ability in nitrogen availability. Biochar is reported to affect the N dynamics in soil by changing the rates of the transformation processes (Clough et al., 2013). The addition of biochar into the soil may moderate soil temperature, enhance soil moisture and aeration, and thus stimulate nitrifier activities (Ulyett et al., 2014). Biochar has been proven to have great potential for reducing gaseous ammonium N loss from soils due to the increased NH3 adsorption capacity of biochar. High sorption capacity of biochars is also due to their high surface area and surface functional groups (Mandal et al., 2016). Biochar can also increase N fertilizer retention in the soil so that plants can get more N from it (the ability of biochars to supply more N). It is known that NH4+ ions adsorbed onto the cation exchange sites of biochar will be available for plants through cation exchange reactions in the later stage of plant growth (Mandal et al., 2016; Taghizadeh-Toosi et al., 2012). Biochar used in this study decreased leaf NO3− concentration compared to control. Decreased chlorophyll index with application of biochar has also been reported (Akhtar et al., 2014; Asai et al., 2009; Ventura et al., 2013). For example, Alburquerque et al. (2013) showed that olive tree branches biochar decreased the chlorophyll index, while wheat straw biochar had no significant effect on this index. There are two reasons for reducing chlorophyll index as a result of biochar application. The first is the adsorption of inorganic N (NH4+ and NO3−) on biochar surfaces, which may leads to diminished N availability to plant and the second is the increase in soil C/N ratio with the addition

3.6. Soil ammonium and nitrate concentration The three–way interaction of various treatments (BC, various N rates, and sampling dates) on soil NH4+ and NO3− concentration was significant (P < 0.01) (Table S3). In the treatments without DMPP (Table S4, and Fig. 5A and B), soil NH4+ concentration at the 2%BC level at 14 and 28 DAP was higher significantly compared to that at 0% BC level. In the treatments containing DMPP, soil NH4+ concentration decreased gradually over time (Table S4, and Fig. 5A and B) and the highest soil NH4+ concentration at 56 DAP was obtained in the treatments of 75 and 100 mg N kg−1 at 0%BC level. The concentration of remaining NH4+ in the soil at the end of the experiment (56 DAP) at the 2%BC level was lower than the concentration of NH4+ at 0%BC level. However, soil NH4+ concentration in the treatments of 75 and 100 mg N kg−1 containing DMPP was higher significantly compared to equal treatments without DMPP (Table S4, and Fig. 5A and B). In the treatments without DMPP, the trend of soil NO3− concentration changes during the experiment (Table S5, and Fig. 5C and D) showed the lowest soil NO3− concentration at 0 DAP, and the highest soil NO3− concentration at 14 DAP at the level of 0%BC and at 28 DAP at the level of 2%BC, respectively. Also, 56 DAP, soil NO3− concentration at 2%BC level was lower than that at 0%BC level (Table S5, and Fig. 5C and D).

3.7. Total N, organic carbon, EC and pH of post–harvest soil Analyses of variance (ANOVA) showed that only the effect of BC treatment was significant on total N, O.C, EC, and pH of post–harvest soil (P < 0.01) (Table S6). The highest values of TN, O.C, EC, and pH in the soil were obtained at 2%BC level and their lowest values were observed at 0%BC level as application of 2%BC compared to 0%BC increased the soil TN by 0.225 g kg−1, O.C by 11.3 g kg−1, EC by 2.08 dS m−1, and pH by 0.57 units, respectively (Fig. 6A, B, C, and D). 6

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Fig. 3. Effect of biochar (BC; 0%BC and 2%BC) on nitrogen use efficiency (NUE), nitrogen physiologic efficiency (NPE), and nitrogen agronomic efficiency (NAE) of spinach plant (A, B, and C) and effect of different nitrogen levels (with and without DMPP) on NUE, and NPE, and NAE of spinach plant (D, E, and F) after 56 days after planting. N0, un–fertilized; N50, fertilized with 50 mg N kg−1 soil; N75, fertilized with 75 mg N kg−1 soil; N100, fertilized with 100 mg N kg−1 soil; N50 + NI, fertilized with N50 + DMPP; N75 + NI, fertilized with N75 + DMPP; and N100 + NI, fertilized with N100 + DMPP. Vertical bars represent the standard error (SE) of the mean of each treatment (n = 3) and the same small case letters are not significantly different according to Duncan's multiple range test (DMRT) at P ≤ 0.05.

DMPP. Xing et al. (2015) also reported decreased spinach yield with increasing ammonium/nitrate ratio in the plant. In our experiment, the decrease in spinach plant yield at 100 mg N kg−1 with DMPP compared to equal level without DMPP could be due to the high susceptibility of spinach plant to high ammonium concentrations. Lasa et al. (2002) and Lasa et al. (2001) introduced spinach plant as a highly susceptible plant to ammonium nutrition and attributed this decline in spinach plant yield to ammonium accumulation in the shoot of this plant. These authors showed that ammonium accumulation in spinach shoots is correlated with reduced plant growth. However, in the present study, application BC reduced negative effect of DMPP on spinach yield as application of BC increased spinach yield at levels of 75 and 100 mg N kg−1 containing DMPP compared to equal levels without BC. Oladele et al. (2019) also showed that rice husk biochar in combination with urea fertilizer increased rice yield and yield components, and they suggested that biochar amendment together with N had the potential to increase rice productivity and soil nutrient availability. Improved yield with application of BC at levels of 75 and 100 mg N kg−1 containing DMPP may be due to the effect of BC on adsorption or immobilization of inorganic N (Mandal et al., 2016; Taghizadeh-Toosi et al., 2012) or on adsorption DMPP (Fuertes-Mendizábal et al., 2019). This BC action

of biochar, which leads to soil N immobilization and consequently reduced N uptake by the plants (Akhtar et al., 2014; Lehmann et al., 2002). In this study, chlorophyll index significantly had positive correlation with total N (r = 0.88) and NO3− (r = 0.88) of plant, total N (r = 0.37) and NO3- (r = 0.74) of soil, and NH4+ of soil (r = 0.37) (Table S7). Liu et al. (2006) and Muchecheti et al. (2016) also showed that the relationship between shoot N content and chlorophyll index by SPAD readings was linear as they introduced the chlorophyll index as an indicator of N status of spinach plant. In this study, application of ammonium sulfate containing DMPP in biochar–free treatments at low N level (50 mg N kg−1) and at medium N level (75 mg N kg−1) had no significant effect on spinach yield but reduced the yield of spinach plant at higher N level (100 mg N kg−1). Since spinach plant is a plant sensitive to high concentrations of ammonium (Xing et al., 2015), DMPP decreased its yield by preventing or slowing the microbial conversion of NH4+ to NO3− and accumulation of ammonium in soil. The results of this experiment are in agreement with the results of previous studies (Canali et al., 2014; Casar et al., 2010). Casar et al. (2010) showed that the use of 100 kg N ha−1 (34 mg N kg−1) of ammonium source (100% N) containing DMPP had no significant effect on spinach yield compared to N level without

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Fig. 4. Effect of biochar (BC; 0%BC and 2%BC) and different nitrogen levels (with and without DMPP) on nitrate reductase (NR) activity (A) and nitrite reductase (NiR) activity (B) of spinach plant after 28, 42, and 56 days after planting. N0, un–fertilized; N50, fertilized with 50 mg N kg−1 soil; N75, fertilized with 75 mg N kg−1 soil; N100, fertilized with 100 mg N kg−1 soil; N50 + NI, fertilized with N50 + DMPP; N75 + NI, fertilized with N75 + DMPP; and N100 + NI, fertilized with N100 + DMPP. Vertical bars represent the standard error (SE) of the mean of each treatment (n = 3) and the same small case letters are not significantly different according to Duncan's multiple range test (DMRT) at P ≤ 0.05.

from 61.53 to 77.7% (Cao et al., 2019). Mandal et al. (2016) also showed that poultry litter biochar application could increase nitrogen retention in soil and NUE through decreasing N volatilization from soil and improving nitrogen uptake by wheat plant. The NUE at the level of 50 mg N kg−1 containing DMPP was significantly higher than that at the level of 50 mg N kg−1 without DMPP in this study. Also, the highest NAE was observed in 50 mg N kg−1 containing DMPP, which indicates a positive effect of DMPP on increasing N fertilizer utilization at the lowest N dose. The results of this experiment are consistent with the results of study of Alonso-Ayuso et al. (2016), in which they showed that NUE and NAE were significantly higher at N level (130 kg N ha−1) containing DMPP compared to equal level without DMPP. But there were no significant

may be the reason for the reduction of ammonium in the environment and thus its negative effect on spinach yield. In a study (Fidel et al., 2018), it was found that biochars in high pH (similar to the soil pH used in this study) tend to adsorb ammonium than nitrate. As mentioned above, NH4+/NO3− ions adsorbed onto the cation exchange sites of biochar may be available for plants through cation exchange reactions in the later stage of plant growth (Mandal et al., 2016; TaghizadehToosi et al., 2012). In this study, the application of 2%BC significantly increased NUE. Pereira et al. (2017) with biochar application of pine chips and walnut husk on lettuce (Lactuca sativa L.) yield showed that biochar at high levels of nitrogen application (225 kg ha−1) increased NUE. In another report, the biochar application of apple tree branches increased NUE

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Fig. 5. Changes of ammonium (NH4+) and nitrate (NO3−) of soil during the spinach plant growth as affected by biochar (0%BC: without biochar; and 2%BC: with biochar) and various N levels (with and without DMPP). N0, un–fertilized; N50, fertilized with 50 mg N kg−1 soil; N75, fertilized with 75 mg N kg−1 soil; N100, fertilized with 100 mg N kg−1 soil; N50 + NI, fertilized with N50 + DMPP; N75 + NI, fertilized with N75 + DMPP; and N100 + NI, fertilized with N100 + DMPP. The same small case letters are not significantly different according to Duncan's multiple range test (DMRT) at P ≤ 0.05.

Fig. 6. Effect of biochar (0%BC: without biochar and 2%BC: with biochar) on total nitrogen (TN), organic carbon (O.C), electrical conductivity (EC), and pH of post–harvest soil. Vertical bars represent the standard error (SE) of the mean of each treatment (n = 3) and the same small case letters are not significantly different according to Duncan's multiple range test (DMRT) at P ≤ 0.05. 9

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changes in NUE and NAE at higher N levels (170 kg N ha−1). Quemada et al. (2013) in a meta–analysis study in irrigated agricultural systems concluded that the use of nitrification inhibitors reduced nitrate leaching by 27% but did not increase the yield and NUE. In contrast, Abalos et al. (2014) indicated that the use of nitrification inhibitors increased yield and NUE. However, in some studies, it has been reported reduced NUE and NAE under the application of N containing DMPP for spinach (Canali et al., 2014). The activity of NR and NiR enzymes, the amount of plant (leaf) protein and NUE depend on the amount of soil nitrogen and its forms (ammonium and nitrate). For example, NR activity is known as an indicator of soil nitrate content (Högberg et al., 1986; Lata et al., 1999). In this study, application of BC decreased leaf nitrate concentration and activity of enzyme nitrate reductase (NR) in the spinach plant aerial part. Decreased leaf nitrate concentration and activity of NR enzyme by BC can be due to two causes, one of which is a BC effect on reducing soil nitrification rate and the other is a BC effect on the immobilization or adsorption of mineral nitrogen (nitrate and ammonium) (Mandal et al., 2016; Taghizadeh-Toosi et al., 2012). In this context, Li et al. (2016) showed that the concentration of nitrate in leaves of spinach plant increased with the biochar application. Maroušek et al. (2018) showed that the biochar application increased yield of red beet, but reduced the plant nitrate concentration by 35% compared to control at the same time. These researchers reported that this increase and this decrease were due to the biochar–mediated improvement of the physical and chemical properties of soil and the immobilization of soil mineral N, respectively. In the present study, the use of (NH4)2SO4 (ammonium sulfate) containing DMPP also decreased the concentration of spinach leaf nitrate and enzymes activity as compared to (NH4)2SO4 without DMPP, which shows the DMPP effect on nitrification process of soil. These results are consistent with the results of Casar et al. (2010), Canali et al. (2014), and Zerulla et al. (2001). These researchers reported that the use of ammonium containing DMPP reduced the nitrate concentration of spinach plant leaf. Karwat et al. (2019) showed that NR activity in leaves of plants fed by the ammonium containing DMPP was significantly lower than that of the nitrate–fed plants. Huérfano et al. (2016) also reported a decrease in NO3− concentration in lettuce plant leaf using nitrification inhibitor DMPP, without having a significant effect on yield. The decreasing effect of biochar and DMPP on the activity of these enzymes varied depending on the concentration of N used (a lower affect at higher N concentrations). DMPP decreased activity of the NR and NiR enzymes by preventing or slowing the microbial conversion of NH4+ to NO3− (a decrease in substrates for the enzymes). It is known that the nitrate uptake by plant induces the expression of NR enzyme gene while the ammonium or glutamine ions slow down or stop its activity (Ogawa et al., 1999). In this case, Ogawa et al. (1999) stated that the activity of the NR was limited after addition of glutamine to the spinach culture medium containing nitrate, but the activity of NiR was not affected. In another study, Shah et al. (2017) showed that the activity of NR and NiR enzymes increased with increasing NO3− application in barley genotypes at 22 DAP. In an experiment, Chen et al. (2004) also reported the increased NR activity with increasing cytosol nitrate at low levels of nitrogen (150 mg kg−1). In addition, the activity of NR enzyme did not change significantly at higher concentrations of cytosolic nitrogen, and in some cases, its activity decreased. In general, in the present experiment, no nitrate concentration above the permissible limit (2 g NO3− per kg fresh weight of spinach, recommended by National Standard Organization of Iran) was observed in any of these treatments. However, the use of biochar and DMPP increased the quality of spinach edible consumption by reducing nitrate concentration in the leaves of the plant. In relation to the effect of BC on efficiency of DMPP, it was found that the exchangeable NH4+ concentration of soil was higher in the treatments without BC at the end of this experiment (56 days after planting). With regard to the result, it can be concluded that the use of

BC has reduced the effectiveness of this nitrification inhibitor. Regarding the effect of wheat straw BC on efficiency of NI, it can be said that reduced efficiency of DMPP by BC may be due to the immobilization or sorption of inorganic N (Gao et al., 2018; Nguyen et al., 2017) and or the sorption of DMPP by BC (Keiblinger et al., 2018). Li et al. (2015) showed that the combined use of biochar and nitrification inhibitor nitrapyrin increased N2O emission rate compared to the use of nitrapyrin alone, indicating a biochar effect on decreasing nitrapyrin inhibitory efficacy. There has been limited research on the interaction effect of biochar and nitrification inhibitors, in particular DMPP. The results of our experiment are consistent with the results of Fuertes-Mendizábal et al. (2019), where the application of biochar of wood chips (pine) reduced the efficiency of DMPP and consequently diminished the amount of N2O emissions. Biochar application alters several biogeochemical parameters such as nitrogen speciation, nutrient availability, pH and microbial communities in soils and these changes in soil properties can affect the process of nitrification (Fuertes-Mendizábal et al., 2019). One of the reasons for the reduction of efficiency of DMPP in our study may be due to the sorption of this inhibitor by BC as Keiblinger et al. (2018) by investigation the application effect of several types of biochars (wood chips, wheat straw, and pruned branches of vineyard) in soil showed that the sorption of DMPP increased by application of these biochars and the highest sorption was found at low temperatures of pyrolysis (400 °C). These researchers finally concluded that the sorption of DMPP by soil–biochar mixtures might decrease its availability for microbes, which could reduce its inhibitory effect on the nitrification rate, although sorption of DMPP protects it from decomposition. According to this and the low temperature of pyrolysis (450 °C) in our experiment, the sorption of DMPP may have occurred. Biochar has also reduced the effectiveness of other inhibitors, such as nitrapyrin (Li et al., 2015) and dicyandiamide (DCD) (Shi et al., 2015), but in some cases increased the effectiveness of DCD (He et al., 2018), and or had no significant effect on DCD (Treweek et al., 2016). It is known that soil inorganic N (NH4+ and NO3−) can be adsorbed on the surfaces of biochar based on acid functional groups of biochar (i.e., carboxylic, hydroxyl, lactone, and lactol groups) (Nguyen et al., 2017) and or can be immobilized by biochar due to a wide range of C:N ratios of biochar (Gao et al., 2018). According to these findings, when inorganic N fertilizers containing nitrification inhibitors, such as ammonium sulfate containing DMPP, are applied together with biochar to soils, ammonium can be adsorbed and or immobilized by biochar. These biochar traits can also reduce the efficiency of nitrification inhibitors. The results of our experiment are no exception, because the used biochar in this study contains the acid functional groups (1683 μmol H+ g−1) and high C/N (199.4). In fact, sorption of DMPP and immobilization of inorganic N (such as NH4+) are not separated, and these two processes are interconnected, which in this way affects the efficiency of nitrification inhibitors such DMPP. Role of biochars in improving soil physical and chemical properties has been well reviewed in previous studies (Ding et al., 2017). The biochar used in this study, similar to most biochars, also resulted in an increase in TN, O.C, EC, and pH of the soil. Correlation between parameters (Table S7) showed that spinach yield had a significant positive correlation with chlorophyll (r = 0.86), leaf number (r = 0.79), leaf protein content (r = 0.89), shoot nitrogen (r = 0.89) and total soil nitrogen (r = 0.47), activity of enzymes (r = 0.46 for NR and 0.66 for NiR), and soil nitrate (r = 0.58). There was a significant positive correlation between plant leaf nitrate and activity of enzymes of NR and NiR (r = 0.64 for NR and r = 0.55 for NiR). In addition, there was a significant negative relationship between soil pH, EC, and O.C with NR enzyme activity. Given that in this experiment, biochar application increased pH and EC, and biochar also decreased NR enzyme activity, a negative correlation between pH and EC with NR enzyme activity is acceptable. The correlation between soil O.C with plant NR activity and soil nitrate was a significant negative 10

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relationship, which may also be due to adsorption or immobilization of inorganic nitrogen by biochar. Plant and soil nitrate, and NR activity had a significant negative correlation with soil pH. Since the reason for the increase in soil pH in the present experiment was due to addition of biochar to soil and it has also been found that biochar can either adsorb or immobilize soil mineral nitrogen, it is reasonable to observe a negative relationship between these parameters and soil pH. To sum up, DMPP and biochar through decreasing the NO3− concentration of the spinach plant leaf increased the quality of spinach plant and BC also reduced negative effect of DMPP on growth of spinach at high level of N, however, decreased the efficiency of DMPP. To our knowledge, this is the first study demonstrating a counteracting effect between DMPP and biochar and their effect on the yield of spinach plant.

Balaine, N., Clough, T.J., Kelliher, F.M., Van Koten, C., 2015. Soil aeration affects the degradation rate of the nitrification inhibitor dicyandiamide. Soil Res. 53, 137–143. Benckiser, G., Christ, E., Herbert, T., Weiske, A., Blome, J., Hardt, M., 2013. The nitrification inhibitor 3, 4-dimethylpyrazole-phosphat (DMPP)-quantification and effects on soil metabolism. Plant Soil 371, 257–266. Borchard, N., Spokas, K., Prost, K., Siemens, J., 2014. Greenhouse gas production in mixtures of soil with composted and noncomposted biochars is governed by charassociated organic compounds. J. Environ. Qual. 43, 971–979. Bremner, J., 1996. Nitrogen–total. In: Sparks, D.L. (Ed.), Methods of Soil Analyses, Part 3, Chemical Methods. Soil Science Society of America and American Society of Agronomy, Madison, WI, USA, pp. 1085–1122. Cai, Y., Chang, S.X., 2015. Biochar effects on soil fertility and nutrient cycling. Biochar: Prod. Char. Appl. 246–271. Canali, S., Diacono, M., Ciaccia, C., Masetti, O., Tittarelli, F., Montemurro, F., 2014. Alternative strategies for nitrogen fertilization of overwinter processing spinach (Spinacia oleracea L.) in Southern Italy. Eur. J. Agron. 54, 47–53. Cao, H., Ning, L., Xun, M., Feng, F., Li, P., Yue, S., Song, J., Zhang, W., Yang, H., 2019. Biochar can increase nitrogen use efficiency of Malus hupehensis by modulating nitrate reduction of soil and root. Appl. Soil Ecol. 135, 25–32. Casar, C., Muñoz-Guerra, L.M., Ordiales, E., López, J., 2010. Ammoniun fertilisers with nitrification inhibitors improve the nutritional quality of horticultural crops for industrial processing. Environmentally Friendly and Safe Technologies for Quality of Fruit and Vegetables. pp. 68–72. Cataldo, D., Maroon, M., Schrader, L., Youngs, V., 1975. Rapid colorimetric determination of nitrate in plant tissue by nitration of salicylic acid. Commun. Soil Sci. Plant Anal. 6, 71–80. Cayuela, M., Jeffery, S., Van Zwieten, L., 2015. The molar H: corg ratio of biochar is a key factor in mitigating N2O emissions from soil. Agric. Ecosyst. Environ. 202, 135–138. Chaves, B., Opoku, A., De Neve, S., Boeckx, P., Van Cleemput, O., Hofman, G., 2006. Influence of DCD and DMPP on soil N dynamics after incorporation of vegetable crop residues. Biol. Fertil. Soils 43, 62–68. Chen, B.-M., Wang, Z.-H., Li, S.-X., Wang, G.-X., Song, H.-X., Wang, X.-N., 2004. Effects of nitrate supply on plant growth, nitrate accumulation, metabolic nitrate concentration and nitrate reductase activity in three leafy vegetables. Plant Sci. 167, 635–643. Clough, T.J., Condron, L.M., Kammann, C., Müller, C., 2013. A review of biochar and soil nitrogen dynamics. Agronomy 3, 275–293. Definition, I.B.I.S.P., 2015. Product Testing Guidelines for Biochar that Is Used in Soil (Aka IBI Biochar Standards) Version 2.1. International Biochar Initiative (IB). Ding, Y., Liu, Y., Liu, S., Huang, X., Li, Z., Tan, X., Zeng, G., Zhou, L., 2017. Potential benefits of biochar in agricultural soils: a review. Pedosphere 27, 645–661. Doane, T.A., Horwáth, W.R., 2003. Spectrophotometric determination of nitrate with a single reagent. Anal. Lett. 36, 2713–2722. Fidel, R.B., Laird, D.A., Spokas, K.A., 2018. Sorption of ammonium and nitrate to biochars is electrostatic and pH-dependent. Sci. Rep. 8, 17627. Fuertes-Mendizábal, T., Huérfano, X., Vega-Mas, I., Torralbo, F., Menéndez, S., Ippolito, J.A., Kammann, C., Wrage-Mönnig, N., Cayuela, M.L., Borchard, N., 2019. Biochar reduces the efficiency of nitrification inhibitor 3, 4-dimethylpyrazole phosphate (DMPP) mitigating N 2 O emissions. Sci. Rep. 9, 2346. Fujihara, S., Kasuga, A., Aoyagi, Y., 2001. Nitrogen‐to‐protein conversion factors for common vegetables in Japan. J. Food Sci. 66, 412–415. Gao, S., DeLuca, T.H., Cleveland, C.C., 2018. Biochar additions alter phosphorus and nitrogen availability in agricultural ecosystems: a meta-analysis. Science of The Total Environment. Gee, G., Bauder, J., 1987. Particle size analysis. Methods of Soil Analysis. Part I. Physical and Mineralogical Methods. Soil Science Society of America, American Society of Agronomy, Madison, WI, pp. 377–411. Gilsanz, C., Báez, D., Misselbrook, T.H., Dhanoa, M.S., Cárdenas, L.M., 2016. Development of emission factors and efficiency of two nitrification inhibitors, DCD and DMPP. Agric. Ecosyst. Environ. 216, 1–8. Gong, Q., Wang, L., Dai, T., Zhou, J., Kang, Q., Chen, H., Li, K., Li, Z., 2019. Effects of copper on the growth, antioxidant enzymes and photosynthesis of spinach seedlings. Ecotoxicol. Environ. Saf. 171, 771–780. Guardia, G., Marsden, K.A., Vallejo, A., Jones, D.L., Chadwick, D.R., 2018. Determining the influence of environmental and edaphic factors on the fate of the nitrification inhibitors DCD and DMPP in soil. Sci. Total Environ. 624, 1202–1212. He, L., Bi, Y., Zhao, J., Pittelkow, C.M., Zhao, X., Wang, S., Xing, G., 2018. Population and community structure shifts of ammonia oxidizers after four-year successive biochar application to agricultural acidic and alkaline soils. Sci. Total Environ. 619, 1105–1115. Högberg, P., Granström, A., Johansson, T., Lundmark-Thelin, A., Näsholm, T., 1986. Plant nitrate reductase activity as an indicator of availability of nitrate in forest soils. Can. J. For. Res. 16, 1165–1169. Huérfano, X., Menéndez, S., Bolaños-Benavides, M.M., González-Moro, M.B., Estavillo, J.M., González-Murua, C., 2016. The nitrification inhibitor 3, 4-dimethylpyrazole phosphate decreases leaf nitrate content in lettuce while maintaining yield and N2O emissions in the Savanna of Bogotá. Plant Soil Environ. 62, 533–539. Jaworski, E.G., 1971. Nitrate reductase assay in intact plant tissues. Biochem. Biophys. Res. Commun. 43, 1274–1279. Jones Jr., J.B., 2001. Laboratory Guide for Conducting Soil Tests and Plant Analysis. CRC press. Karwat, H., Sparke, M.-A., Rasche, F., Arango, J., Nuñez, J., Rao, I., Moreta, D., Cadisch, G., 2019. Nitrate reductase activity in leaves as a plant physiological indicator of in vivo biological nitrification inhibition by Brachiaria humidicola. Plant Physiol. Biochem. 137, 113–120. Keiblinger, K.M., Zehetner, F., Mentler, A., Zechmeister-Boltenstern, S., 2018. Biochar application increases sorption of nitrification inhibitor 3, 4-dimethylpyrazole

5. Conclusions This study demonstrates that application of ammonium sulfate containing DMPP had no significant effect on the yield of spinach plant at low and medium levels of N (50 and 75 mg N kg−1 soil) but decreased the yield of this plant at the higher level of N (100 mg N kg−1 soil). However, application of wheat straw biochar reduced the negative effect of DMPP on spinach yield at high N level (100 mg N kg−1 soil + DMPP) and significantly increased spinach yield at 75 and 100 mg N kg−1 levels. According to the results of this study, the combined application of DMPP and biochar could significantly reduce the nitrification inhibitory effect of DMPP and the subsequent mitigation of negative effect of DMPP on growth of spinach plant, probably due to the adsorption of DMPP on biochar surfaces. The results of this study can be used to determine the proper level of N fertilizers (especially when using N fertilizers including nitrification inhibitors such as DMPP) for cultivation by those farmers that also use biochar as an amendment in their agricultural land. To clarify the kinetics of sorption of DMPP by biochar and DMPP efficiency in the soils with different characteristics in the presence of various biochars, further studies under field conditions/controlled conditions are recommended for the practical use of nitrification inhibitors such as DMPP by farmers. Author contribution All the authors have contributed substantially (the same) and approved the final submission. Acknowledgment We wish to thank University of Tehran for providing funding and the necessary facilities for this study. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ecoenv.2020.110243. References Abalos, D., Jeffery, S., Sanz-Cobena, A., Guardia, G., Vallejo, A., 2014. Meta-analysis of the effect of urease and nitrification inhibitors on crop productivity and nitrogen use efficiency. Agric. Ecosyst. Environ. 189, 136–144. Akhtar, S.S., Li, G., Andersen, M.N., Liu, F., 2014. Biochar enhances yield and quality of tomato under reduced irrigation. Agric. Water Manag. 138, 37–44. Alburquerque, J.A., Salazar, P., Barrón, V., Torrent, J., del Campillo, M.d.C., Gallardo, A., Villar, R., 2013. Enhanced wheat yield by biochar addition under different mineral fertilization levels. Agron. Sustain. Dev. 33, 475–484. Alonso-Ayuso, M., Gabriel, J., Quemada, M., 2016. Nitrogen use efficiency and residual effect of fertilizers with nitrification inhibitors. Eur. J. Agron. 80, 1–8. Asai, H., Samson, B.K., Stephan, H.M., Songyikhangsuthor, K., Homma, K., Kiyono, Y., Inoue, Y., Shiraiwa, T., Horie, T., 2009. Biochar amendment techniques for upland rice production in Northern Laos: 1. Soil physical properties, leaf SPAD and grain yield. Field Crop. Res. 111, 81–84.

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analysis of strategies to control nitrate leaching in irrigated agricultural systems and their effects on crop yield. Agric. Ecosyst. Environ. 174, 1–10. Rashti, M.R., Wang, W., Moody, P., Chen, C., Ghadiri, H., 2015. Fertiliser-induced nitrous oxide emissions from vegetable production in the world and the regulating factors: a review. Atmos. Environ. 112, 225–233. Rhoades, J.D., 1996. Salinity: electrical conductivity and total dissolved solids. Methods Soil Anal. part 3—Chem. Methods 417–435. Ruser, R., Schulz, R., 2015. The effect of nitrification inhibitors on the nitrous oxide (N2O) release from agricultural soils—a review. J. Plant Nutr. Soil Sci. 178, 171–188. Schmidt, E.L., Belser, L.W., 1996. Autotrophic nitrifying bacteria. In: Page, A.L. (Ed.), Methods of Soil Analysis, Part 2: Microbiological and Biochemical Properties. Soil Science Society of America and American Society of Agronomy, Madison, W.I., pp. 159–177. Shah, J.M., Bukhari, S.A.H., Zeng, J.-b., QuAN, X.-y., Ali, E., Muhammad, N., Zhang, G.p., 2017. Nitrogen (N) metabolism related enzyme activities, cell ultrastructure and nutrient contents as affected by N level and barley genotype. J. Integ. Agric. 16, 190–198. Shi, Y., Zhang, L., Zhao, M., 2015. Effect of biochar application on the efficacy of the nitrification inhibitor dicyandiamide in soils. BioResources 10, 1330–1345. Singh, J., Saggar, S., Giltrap, D., Bolan, N.S., 2008. Decomposition of dicyandiamide (DCD) in three contrasting soils and its effect on nitrous oxide emission, soil respiratory activity, and microbial biomass—an incubation study. Soil Res. 46, 517–525. Spokas, K.A., Reicosky, D.C., 2009. Impacts of sixteen different biochars on soil greenhouse gas production. Ann. Environ. Sci. 3, 179–193. Sumner, M.E., Miller, W.P., 1996. Cation exchange capacity and exchange coefficients. Methods Soil Anal. part 3—Chem. Methods 1201–1229. Taghizadeh-Toosi, A., Clough, T.J., Sherlock, R.R., Condron, L.M., 2012. Biochar adsorbed ammonia is bioavailable. Plant Soil 350, 57–69. Tang, L., Hamid, Y., Sahito, Z.A., Gurajala, H.K., He, Z., Feng, Y., Yang, X., 2019. Evaluation of variation in essential nutrients and hazardous materials in spinach (Spinacia oleracea L.) genotypes grown on contaminated soil for human consumption. J. Food Compos. Anal. 79, 95–106. Thomas, G.W., 1996. Soil pH and soil acidity. In: Sparks, D.L. (Ed.), Methods of Soil Analysis. Part 3. Chemical Methods. Soil Science Society of America, Madison, WI, pp. 475–490. Treweek, G., Di, H.J., Cameron, K.C., Podolyan, A., 2016. Effectiveness of the nitrification inhibitor dicyandiamide and biochar to reduce nitrous oxide emissions. N. Z. J. Agric. Res. 59, 165–173. Ulyett, J., Sakrabani, R., Kibblewhite, M., Hann, M., 2014. Impact of biochar addition on water retention, nitrification and carbon dioxide evolution from two sandy loam soils. Eur. J. Soil Sci. 65, 96–104. Van Zwieten, L., Kammann, C., Cayuela, M.L., Singh, B.P., Joseph, S., Kimber, S., Donne, S., Clough, T., Spokas, K., 2015. Biochar effects on nitrous oxide and methane emissions from soil. Biochar for Environmental Management: Science and Technology, second ed. Earthscan Books Ltd, London, New York, pp. 489–520. Ventura, M., Sorrenti, G., Panzacchi, P., George, E., Tonon, G., 2013. Biochar reduces short-term nitrate leaching from a horizon in an apple orchard. J. Environ. Qual. 42, 76–82. Watanabe, F., Olsen, S., 1965. Test of an ascorbic acid method for determining phosphorus in water and NaHCO3 extracts from soil 1. Soil Sci. Soc. Am. J. 29, 677–678. White, P.J., Brown, P.H., 2010. Plant nutrition for sustainable development and global health. Ann. Bot. 105, 1073–1080. Xing, S., Wang, J., Zhou, Y., Bloszies, S.A., Tu, C., Hu, S., 2015. Effects of NH4+-N/NO3--N ratios on photosynthetic characteristics, dry matter yield and nitrate concentration of spinach. Exp. Agric. 51 (1), 151–160. Yang, T., Li, F., Zhou, X., Xu, C., Feng, J., Fang, F., 2019. Impact of nitrogen fertilizer, greenhouse, and crop species on yield-scaled nitrous oxide emission from vegetable crops: a meta-analysis. Ecol. Indicat. 105, 717–726. Zerulla, W., Barth, T., Dressel, J., Erhardt, K., von Locquenghien, K.H., Pasda, G., Rädle, M., Wissemeier, A., 2001. 3, 4-Dimethylpyrazole phosphate (DMPP)–a new nitrification inhibitor for agriculture and horticulture. Biol. Fertil. Soils 34, 79–84. Zhao, L.F., Wang, Y., 2017. Nitrate assay for plant tissues. Bio Protoc 7, e2029.

phosphate in soil. Environ. Sci. Pollut. Control Ser. 25, 11173–11177. Khalil, A., Sergeevich, N., Borisova, V., 2018. Removal of ammonium from fish farms by biochar obtained from rice straw: isotherm and kinetic studies for ammonium adsorption. Adsorpt. Sci. Technol. 36, 1294–1309. Kumar, R., Parmar, B.S., Walia, S., Saha, S., 2015. Nitrification Inhibitors: Classes and its Use in Nitrification Management, Nutrient Use Efficiency: from Basics to Advances. Springer, pp. 103–122. Lasa, B., Frechilla, S., Aparicio-Tejo, P.M., Lamsfus, C., 2002. Alternative pathway respiration is associated with ammonium ion sensitivity in spinach and pea plants. Plant Growth Regul. 37, 49–55. Lasa, B., Frechilla, S., Lamsfus, C., Aparicio-Tejo, P., 2001. The sensitivity to ammonium nutrition is related to nitrogen accumulation. Sci. Hortic. 91, 143–152. Lata, J.-C., Durand, J., Lensi, R., Abbadie, L., 1999. Stable coexistence of contrasted nitrification statuses in a wet tropical savanna ecosystem. Funct. Ecol. 13, 762–768. Lehmann, J., da Silva Jr., J.P., Rondon, M., Cravo, M.d.S., Greenwood, J., Nehls, T., Steiner, C., Glaser, B., 2002. Slash-and-char-a feasible alternative for soil fertility management in the central Amazon. Proceedings of the 17th World Congress of Soil Science. pp. 1–12 Thailand. Li, B., Fan, C., Xiong, Z., Li, Q., Zhang, M., 2015. The combined effects of nitrification inhibitor and biochar incorporation on yield-scaled N 2 O emissions from an intensively managed vegetable field in southeastern China. Biogeosciences 12, 2003–2017. Li, Z.-F., Wang, Q., Zhang, W.-J., Du, Z.-L., He, X.-H., Zhang, Q.-Z., 2016. Contributions of nutrients in biochar to increase spinach production: a pot experiment. Commun. Soil Sci. Plant Anal. 47, 2003–2007. Lindsay, W.L., Norvell, W.A., 1978. Development of a DTPA soil test for zinc, iron, manganese, and copper 1. Soil Sci. Soc. Am. J. 42, 421–428. Liu, Y.-J., Tong, Y.-P., Zhu, Y.-G., Ding, H., Smith, F.A., 2006. Leaf chlorophyll readings as an indicator for spinach yield and nutritional quality with different nitrogen fertilizer applications. J. Plant Nutr. 29, 1207–1217. Loeppert, R., Suarez, L., 1996. Carbonate and gypsum. In: Sparks, D.L. (Ed.), Methods of Soil Analysis. Part 3. Chemical Methods. Soil Science Society of America, Madison, WI, pp. 437–474. Mandal, S., Thangarajan, R., Bolan, N.S., Sarkar, B., Khan, N., Ok, Y.S., Naidu, R., 2016. Biochar-induced concomitant decrease in ammonia volatilization and increase in nitrogen use efficiency by wheat. Chemosphere 142, 120–127. Maroušek, J., Kolář, L., Vochozka, M., Stehel, V., Maroušková, A., 2018. Biochar reduces nitrate level in red beet. Environ. Sci. Pollut. Control Ser. 25, 18200–18203. Miflin, B., 1967. Distribution of nitrate and nitrite reductase in barley. Nature 214, 1133. Muchecheti, F., Madakadze, C., Soundy, P., 2016. Leaf chlorophyll readings as an indicator of nitrogen status and yield of spinach (Spinacia oleracea L.) grown in soils amended with Luecaena leucocephala prunings. J. Plant Nutr. 39, 539–561. Mulvaney, R., 1996. Nitrogen—inorganic forms. Methods of Soil Analysis Part 3—Chemical Methods. pp. 1123–1184. Nelson, D.W., Sommers, L.E., 1996. Total carbon, organic carbon, and organic matter. Methods Soil Anal. part 3—Chem. Methods 961–1010. Nguyen, T.T.N., Xu, C.-Y., Tahmasbian, I., Che, R., Xu, Z., Zhou, X., Wallace, H.M., Bai, S.H., 2017. Effects of biochar on soil available inorganic nitrogen: a review and metaanalysis. Geoderma 288, 79–96. Ogawa, K., Shiraishi, N., Ida, S., Nakagawa, H., Komamine, A., 1999. Effects of glutamine on the induction of nitrate reductase and nitrite reductase in cultured spinach cells. J. Plant Physiol. 154, 46–50. Ok, Y.S., Uchimiya, S.M., Chang, S.X., Bolan, N., 2015. Biochar: Production, Characterization, and Applications. CRC press. Oladele, S., Adeyemo, A., Awodun, M., 2019. Influence of rice husk biochar and inorganic fertilizer on soil nutrients availability and rain-fed rice yield in two contrasting soils. Geoderma 336, 1–11. Palansooriya, K.N., Ok, Y.S., Awad, Y.M., Lee, S.S., Sung, J.-K., Koutsospyros, A., Moon, D.H., 2019. Impacts of biochar application on upland agriculture: a review. J. Environ. Manag. 234, 52–64. Pereira, E.I.P., Conz, R.F., Six, J., 2017. Nitrogen utilization and environmental losses in organic greenhouse lettuce amended with two distinct biochars. Sci. Total Environ. 598, 1169–1176. Quemada, M., Baranski, M., Nobel-de Lange, M., Vallejo, A., Cooper, J., 2013. Meta-

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