Municipal Sewage Sludge Effects on Maize Yield, Nitrogen Use Efficiency, and Soil Properties

Biosolids are frequently used as organic soil additives. This 2-year field study investigated the effects of sewage sludge (SWS) application on maize ( Zea mays L.) production, nitrogen accumulation, redistribution, and nitrogen use efficiency, as well as on grain element concentrations and soil properties. Treatments included three SWS rates (20, 40, and 80 Mg ha −1 year ‒ 1 ), mineral fertilizer (300 kg N ha −1 year ‒ 1 and 26 kg P ha −1 year ‒ 1 ), and an unamended control. Plant samples were collected at silking and maturity in both years, while soil samples were taken following maize harvest in the second year. Maize grain yield increased by 143 kg ha −1 for every Mg increase in SWS rate. Sewage sludge application resulted in maize grain yield comparable to or even greater than that of mineral fertilizer. Nitrogen uptake and translocation to grains increased quadrati-cally with increasing SWS rates. Nitrogen use efficiency increased with moderate SWS application (up to 40 Mg ha −1 ) but decreased with heavy application (80 Mg ha −1 ) compared to fertilizer, raising the risk of nitrogen losses. Compared to fertilizer, SWS resulted in similar or lower P and Cu concentration in the grain, as well as similar or higher K, but had no effect on N, Zn, Fe, and Mn concentration. Soil pH, NO 3 -N, and exchangeable K were similar in SWS and fertilizer, but soil Olsen P improved with SWS. There were no differences among treatments in DTPA-extractable Zn, Fe, and Cu, while Mn raised with SWS. Sewage sludge could be used to increase plant N reservoir at silking, N translocation to grains, and post-silking N uptake, allowing plants to meet their N requirements throughout the growing season. Even at 20 Mg ha −1 , sewage sludge can successfully replace mineral fertilizer in maize crop.


Introduction
Management of soil fertilization is a challenge for optimizing crop output, improving product quality, and ensuring sustainability of cropping systems. In the case of maize (Zea mays L.), a nutrient-demanding grain crop, efficient management of nutrients, particularly nitrogen (N), is considered a key strategy implemented in intensive production systems. Adequate N availability can support leaf growth, leaf area duration, and photosynthetic rate per unit area (Marschner 1995) resulting in enhanced biomass accumulation, grain yield, and grain protein content (Ray et al. 2020;Chen et al. 2015).
Municipal sewage sludge, which contains essential elements (N and P) or micronutrients, can be utilized for managing soil fertility of agricultural lands instead of mineral fertilizers. The fertility contribution of sewage sludge is determined by many factors, including sludge composition, method of sludge treatment prior to application, and the dynamics of sludge-N release (Rigby et al. 2016). Apart from adding nutrients to soil, sewage sludge promotes organic matter content, ameliorates physical and chemical properties (Goss et al. 2013), and is a cost-effective and environmentally friendly disposal method of this type of waste (Evans et al. 2004). However, using sewage sludge in agriculture may pose a risk of contamination of foods and the environment due to harmful contents, including heavy metals and pathogens (Usman et al. 2012).
Several studies have shown that sewage sludge improves the productivity of various field crops in semi-arid Mediterranean conditions, including bread wheat (Triticum aestivum L.) (Koutroubas et al. 2014), barley (Hordeum vulgare L.) (Antolín et al. 2005), cotton (Gossypium hirsutum L.) (Samaras et al. 2008), and sunflower (Helianthus annuus L.) (Koutroubas et al. 2020b). In maize, sewage sludge or sludge compost application has been shown to improve certain physiological crop characteristics, such as photosynthetic activity and stomatal conductivity (Moreira et al. 2020), resulting in enhanced grain yield and N uptake (Bozkurt et al. 2006;Motta and Maggiore 2013). Černý et al. (2012) found that applying sewage sludge at rates of 9.82 and 19.64 Mg ha -1 year -1 increased maize silage yield by 19 to 25% in comparison to control. Regarding the application amount, low sewage sludge rates (up to 9 Mg ha -1 ) are generally easier to manage and appear to be more economically and environmentally acceptable (Koutroubas et al. 2020b). However, due to the limited availability of sludge-N, whose the crop uptake percentage in the year of application ranges from 15 to 40% (Kelling et al. 1977;Binder et al. 2002), application of sewage sludge at low rates may not be enough to meet maize N needs, necessitating the use of supplementary inorganic fertilization (Motta and Maggiore 2013). In irrigated maize, sewage sludge application at 62 Mg ha −1 was found to be necessary for maximizing grain yield (Binder et al. 2002). Another factor affecting the efficiency of soil amendments is its spatial distribution (placement) into the soil. For example, the plant response to localizing a P source can depend on the type of P source (sewage sludge, sewage sludge ash, and triple super phosphate) (Lemming et al. 2016). Experimental data showed that localized application of sewage sludge close to the seed of maize increased root density in and around the fertilizer patch but did not enhance sludge-P uptake compared to the mixed source (Lemming et al. 2016).
Grain N in maize, like in other cereal crops, derives from remobilization of N absorbed in vegetative growth prior to silking and from post-silking N absorption. Differences in the relative contribution of each source to grain N have been reported as a function of the amount and form of N inputs as well as genotype. In this respect, presilking N remobilization, which typically accounts for 45 to 65% of grain N (Gallais et al. 2007;Ciampitti and Vyn 2012;Ning et al. 2017), is crucial for maize productivity when the availability of season or post-silking N is inadequate (Friedrich and Schrader 1979;Ta and Weiland 1992). In particular, maize N metabolism is adapted to post-silking N stress in order to maintain the ear or grain N at the expense of the vegetative organs N (Nasielski et al. 2019). With respect to genotype, modern stay-green maize varieties typically exhibit higher post-silking N uptake than varieties with early-senescing leaves (He et al. 2005;Pommel et al. 2006). Regardless of the N origin in the grain, the primary objective of maize growers is to synchronize the crop demands with soil N availability. This goal can be more readily achieved by implementing certain management strategies, such as optimizing N fertilizer application schedule and selecting the appropriate fertilizer type. In this context, split N application has been reported to be a sufficient and sustainable N management strategy for maize production, although its exact influence relies on N application rates. For example, Mueller et al. (2017) reported that late-split N applications with moderate or high N rates promoted N recovery, but not yield of maize plant. Furthermore, using a slow-release N fertilizer alone or in combination with a fulvic acid biostimulant has been suggested as a potential fertilization strategy for increasing maize growth and production .
A well-established index used to benchmark practices of N management in a specific cropping system is the nitrogen use efficiency (NUE), which is determined as the grain yield generated per N unit applied (Moll et al. 1982). In maize, Motta and Maggiore (2013) reported that NUE was increased with increasing N application up to 185.5 kg ha −1 , regardless of the fertilizer type (sewage sludge or urea) used. Binder et al. (2002), on the other hand, found that the efficiency of biosolids-N use decreased as the rate of biosolids increased, showing no differences with fertilizer-N use efficiency. A similar trend has been observed in other crops like sunflower, but at common agronomically doses (< 26 Mg sewage sludge ha −1 ), sludge-N use efficiency was greater than that of fertilizer-N (Koutroubas et al. 2020a).
Most of the research regarding the use of sewage sludge in agriculture has been focused mainly on the effects on grain yield, N uptake and soil properties. The findings of these studies, however, must be confirmed in various climatic locations or farming systems (Binder et al. 2002). In addition, few field studies dealt with the influence of sludge application on the overall N economy of maize, particularly between silking and maturity, as well as on the NUE in terms of total biomass. We hypothesized that, as part of a nutrient recovery and recycling strategy, sewage sludge would effectively substitute traditional mineral fertilization of maize grown in areas of the Mediterranean basin, where soils are typically deficient in organic matter. The aim of the present work was to compare, under field conditions, the effect of increasing rates of sewage sludge versus commonly applied mineral fertilization strategies on maize production, N accumulation, redistribution, and nitrogen use efficiency, as well as on grain element concentrations and soil properties.

Location Description and Municipal Sewage Sludge Properties
The research was carried out over two growing seasons in 2012 and 2013 on farmers' fields in Lepti-Orestiada, located in the region of Eastern Macedonia and Thrace, Greece. The previous crop was durum wheat (Triticum durum Desf). The soil was clay loam (Typic Xerofluvent) with pH 7.03, electrical conductivity 1.02 mS cm −1 , organic matter 1.6%, P (Olsen) 23 mg kg −1 , K (CH 3 COONH 4 ) 376 mg kg −1 , Zn (DTPA) 1.9 mg kg −1 , Fe (DTPA) 30.3 mg kg −1 , Mn (DTPA) 26.9 mg kg −1 , Cu (DTPA) 1.5 mg kg −1 , bulk density 1.6 g cm −3 , and cation exchange capacity 21.6 cmol c kg −1 . The region has a Mediterranean climate. Mean temperature of the entire growing season was 22.1 °C in 2012 and 22.8 °C in 2013, slightly higher than that of the long-term average (20.5 °C) ( Table 1). Total seasonal rainfall was higher in 2012 (142 mm) than in 2013 (118 mm), while measurements for both years were lower compared with longterm average rainfall (210 mm). The municipal sewage sludge (SWS) was acquired from the facility of wastewater treatment of Orestiada. Soil properties (0-25 cm) and sewage sludge properties were determined at the beginning of the experiments according to Rowell (1994) as reported by Koutroubas et al. (2020b). The main properties of the municipal SWS are given in Table 2.

Treatments, Experimental Design, and Crop Management
The hybrid DKC 60-40 (FAO maturity group 600) (Dekalb Bayer), a widely cultivated maize hybrid in the region, was used. There were five treatments, composed of an unamended control (C, neither SWS nor mineral fertilizer were added), mineral fertilizer (MF, 300 kg N ha -1 year -1 and 26 kg P ha -1 year -1 ), and three rates of municipal sewage sludge (20 (SWS1), 40 (SWS2), and 80 (SWS3) Mg DW ha -1 year -1 , corresponding to 150, 300, and 600 kg of N ha -1 year -1 , respectively). The treatments were performed on the same plots in both years. The sewage sludge was manually applied to the surface and incorporated into the soil two weeks before planting. One-third of the mineral N fertilizer (as urea) and the entire amount of P (as super phosphate) were broadcast applied and incorporated into the soil before sowing, while the remaining two-thirds of N fertilizer (as ammonium nitrate) was top-dressed at the eight to nine leaf stage of maize and incorporated into the soil by irrigation. Treatments were set using the randomized complete block (RCB) design and four replications. Sowing took place on 24 April 2012 and 12 May 2013 using 8.3 seeds per m 2 , following the regional recommendations. Each plot was 75 m 2 (10 m long by 7.5 m wide) and consisted of 10 rows. Supplemental irrigations were applied as required using a sprinkler system. The total amount of available water (rainfall plus irrigation) was about 420 mm in each growing season in accordance with the local recommendations for maize crop (Koutroubas et al. 2000).

Plant Sampling and Measurements
The primary phenological growth stages of maize were noted according to Ritchie and Hanway (1982). Plant samples were taken at silking and physiological maturity, each consisting of five consecutive plants that were ripped at the soil surface from a row within each plot. Initially, sampled plants were used to count the total number of leaves and main stem nodes per plant, as well as to determine plant height and stalk diameter. Plant height (m) was measured from the soil surface to the collar of the uppermost leaf. Stalk diameter (mm) was always determined between the first and second node above the soil surface. The plant samples were then split up at silking into leaves, stems, husks, tassels and ear-shoots, and into leaves, stems, husks, tassels, cobs and kernels at maturity. All samples were weighed after drying in an oven set to 70 °C. A portion of each sample was ground and used for chemical analyses. At maturity, the two central rows by each plot were harvested to determine grain yield. Crop growth rate to silking (CGR) was computed by dividing the increase in dry biomass by the number of days between emergence and silking. The concentration of N (g kg −1 ) in plants was measured using the Kjeldahl method (Bremner 1965). Grain phosphorus concentration was measured spectrophotometrically (882 nm) and grain potassium concentration was measured using a flame photometer (776 nm). After maize harvest in the second growing season, electrical conductivity (EC), pH, NO 3 -N, available P (extraction by 0.5 mol L −1 NaHCO 3 ), and exchangeable K (extraction by 1 mol L −1 CH 3 CHOONH 4 ) were determined in composite soil samples from each plot, consisting of three surface sub-samples (0-20 cm), according to Rowel (1994). Concentrations of Cu, Zn, Mn and Fe were determined in both soil samples and grains by atomic absorption spectrophotometry.

Calculation of Parameters Related with N Partitioning, Translocation, and Use
The following equations were used to evaluate the N partitioning, translocation, and NUE (Moll et al. 1982;Chen et al. 2015;Koutroubas et al. 2020a): Nitrogen translocation = NT, kg ha −1 = N content of vegetative parts at silking kg ha −1 − N content of vegetative parts at maturity kg ha −1 Nitrogen translocation ef f iciency(NTE, %) = NT kg ha −1 N of vegetative parts at silking kg ha −1 × 100 Nitrogen use ef f iciency NUE, kg kg −1 = Grain dry weight Gdw, kg ha −1 Sludge − or fertlizer − N applied to the soil kg ha −1 Nitrogen uptake ef f iciency NUpE, kg kg −1 = Total aboveground N at maturity NCM, kg ha −1 Sludge − or fertlizer − N applied to the soil kg ha −1 Nitrogen utilization ef f iciency NUtE, kg kg −1 = Gdw kg ha −1 NCM kg ha −1 Biomass production ef f iciency BPE, kg kg −1 = Total above ground dry matter at maturity kg ha −1 Sludge − or fertilizer − N appied to the soil kg ha −1 Nitrogen harvest index(NHI) = Grain N at maturity kg ha −1 NCM kg ha −1

Data Analysis
An over years analysis of variance was conducted following Steel and Torrie (1980), using the MSTAT-C statistical package (version 1.41). Before the combined analysis of data, the homogeneity of variances was examined by the Bartlett's test. To compare treatment means, the least significant difference was used. Regression equations and correlation coefficients were determined using standard statistical analysis.

Morphological Traits, Dry Matter, and Grain Yield
Seed emerging and crop establishment were uniform across treatments. Averaged across treatments, the time to emergence was 16 days in 2012 and 5 days in 2013. The time to silking was, on average, 73 days in 2012 and 66 days in 2013. Plant height, stalk diameter, and the number of ears per plant were consistent across years but there were significant differences among treatments (Table 3). Mean plant height and stalk diameter values increased significantly following the application of all soil amendments compared to the control. The same trend was observed for the number of ears per plant, but there were no differences with the unamended control when the lowest SWS rate was applied. When compared to MF, the application of SWS resulted in similar stalk diameter and number of ears per plant, as well as in similar (SWS1) or even higher (SWS2 and SWS3) plant height. In fact, plant height responded linearly to SWS application rates, while the response of stalk diameter was quadratic (Table 4). Plant height ranged from 1.98 to 2.55 m and was significantly correlated with stalk diameter (r = 0.931, P < 0.01). Both the total number of nodes per plant and the total number of leaves per plant were consistent across treatments, ranging from 13.0 to 13.8 and from 11.8 to 12.6, respectively (data not shown). Crop growth rate down to the silking stage was significantly higher in 2013 than in 2012 (183 vs 166 kg dry matter per ha per day). The application of soil amendments, either in organic or in inorganic form, increased the CGR up to the silking compared to the unamended control (Fig. 1a). As far as SWS is concerned, the CGR responded linearly to SWS application rate (Table 4) and was comparable to (SWS1 and SWS2) or even exceeded (SWS3) that noted with MF application (Fig. 1a). Plant height (r = 0.918, P < 0.01) and stalk diameter (r = 0.895, P < 0.01) were both significantly correlated with dry matter at silking.
The application of soil amendments resulted in more final dry matter (Table 5) and greater grain yield compared with the unamended control (Fig. 1b). Dry matter at maturity responded linearly to SWS application (Table 4) and was comparable to (SWS1) or even greater (SWS2 and SWS3) than those achieved with the MF (Table 5). Similarly, grain yield was enhanced linearly by the addition of SWS (Fig. 2), with increases ranging from 104% (for the SWS1) to 226% (for the SWS3) when compared with the unamended control. The corresponding increase of grain yield obtained with the MF was 95% (Fig. 1b). With regard to the year of the experimentation, final dry matter was, on average, substantially greater in 2013 than in 2012 (26.5 vs 24.6 Mg ha −1 ), whereas grain yield showed no changes between the 2 years. A significant correlation of grain yield with dry matter at silking was noted (r = 0.928, P < 0.01).

Nitrogen Uptake, Translocation, and Use
Nitrogen uptake at silking was substantially influenced by year and treatment (Table 5). On average, N content at silking was substantially greater in 2013 (204 kg ha −1 ) than in 2012 (167 kg ha −1 ). When compared to control, the addition of soil amendments, either organic or inorganic, significantly increased the cross-year mean N uptake at silking. In Table 3 Plant height, stalk diameter, and total number of ears of maize as affected by the application of soil amendments. Control, unamended control; SWS, sewage sludge applied at 20 Mg ha −1 (SWS1), 40 Mg ha −1 (SWS2), and 80 Mg ha −1 (SWS3); MF, mineral fertilizer applied at 300 kg N ha −1 and 26 kg P ha −1 over 2 years (2012 and 2013) SOV, source of variation; df, degrees of freedom; in each column, different letters within years or treatments indicate statistically significant differences at P < 0.05 the case of SWS, N uptake at silking followed a quadratic response to SWS application rate (Table 4) and was comparable to (SWS1 and SWS2) or higher (SWS3) than that noted with the MF (Table 5). At maturity, the cross-year mean total N uptake in soil amendment treatments (SWS or MF) was greater than that in the unamended control. In particular, total N uptake at maturity followed a quadratic response to SWS application rate (Table 4) and was always higher than that achieved with the MF (Table 5). Nitrogen harvest index was similar across years (Table 5). On average, the lowest rate of SWS reduced NHI compared with the unamended control, while SWS application had no effect on NHI compared with the MF. Nitrogen translocation was significantly greater in 2013 (129 kg ha −1 ) than in 2012 (103 kg ha −1 ), while no yearly differences were observed for the NTE (Table 6). Nitrogen translocation increased significantly with the application of soil amendments compared with the unamended control. In particular, N translocation had a quadratic response to SWS application rates (Fig. 3b), increasing up to the SWS2 treatment and reaching values similar to MF. Averaged across years, NTE ranged from 49% (SWS1) to 69% (MF) ( Table 6). When SWS was applied, NTE was similar to (SWS2 and SWS3) or lower (SWS1) relative to the unamended control as well as similar to (SWS2) or lower (SWS1 and SWS3) relative to the MF. There was a linear relationship between NT and N content at silking (Fig. 3a). Nitrogen use efficiency parameters (such as NUE, NUpE, NUtE and BPE) remained unchanged across years, but were significantly affected by treatments (Table 6). Both NUE and NUpE degreased in a quadratic pattern with increasing rates of applied-SWS (Table 4). Compared with the MF, the values of NUE and NUpE increased with SWS application at the SWS1 and SWS2 treatments, but not at the SWS3 treatment. The NUtE was consistent across soil amendments, apart from the lowest rate of SWS application, where it showed the smallest value. The BPE ranged between 117 and 151 kg kg −1 (Table 6) and was correlated with NUtE (r = 0.736, P < 0.05). Nitrogen use efficiency was correlated with NUpE (r = 0.979, P < 0.01) but not with NUtE (r = − 0.670, P > 0.05). Figure 4 shows the concentrations of the examined macroand micronutrients in grains at maturity the second year. Grain N concentration was not affected by treatments and ranged from 9.88 to 12.45 g kg −1 . In contrast, grain P and   Fig. 1 Maize crop attributes as affected by the application of soil amendments: a crop growth rate, b grain yield (means over 2 years with standard errors). C, unamended control; SWS, sewage sludge applied at 20 Mg ha −1 (SWS1), 40 Mg ha −1 (SWS2), and 80 Mg ha −1 (SWS3); MF, mineral fertilizer applied at 300 kg N ha −1 and 26 kg P ha −1 . The same letters denote non-significant differences at P < 0.05. In crop growth rate, the F value for each source of variation was 6.578 (P = 0.043) for year, 19.45 (P < 0.001) for treatments, and 0.183 (P = 0.945) for the year × treatment interaction. In grain yield, the F value for each source of variation was 1.593 (P = 0.254) for year, 26.98 (P < 0.001) for treatments, and 0.53 (P = 0.945) for the year × treatment interaction K concentrations significantly increased with the addition of SWS or MF relative to the unamended control. Grain K concentration rose with rising rates of SWS application and was comparable to (SWS1) or greater (SWS2 and SWS3) than that noted with the addition of MF. Regarding the grain P concentration, there was no consistent response to SWS application rate, and the values were comparable to that of MF only when SWS was applied at a moderate rate (SWS2 treatment).

Grain Nutrient Concentration and Soil Properties
Treatments had no effect on grain Zn and Fe concentrations, which ranged from 17.13 to 26.08 mg kg −1 and from 25.75 to 35.96 mg kg −1 , respectively. Soil amendments reduced grain Mn concentration compared with the unamended control, but no differences were observed between the SWS and MF treatments. Regarding Cu, grain concentration increased in the SWS2 and SWS3 treatments compared with the unamended control, but the values did not exceed that of the MF.
Soil properties at the end of the experimentation are given in Table 7. Soil pH dropped in the SWS1 and SWS2 treatments compared with the unamended control, with the reduction being comparable to that noted when the MF was applied. Compared with the unamended control, no changes in soil pH were observed with the SWS3 treatment. The application of SWS resulted in an increase of soil electrical conductivity compared with the unamended control or MF, which was significant only with the SWS1 and SWS2 treatments. The concentration of soil NO 3 -N ranged from 38 to 60 mg kg −1 , with marginal differences among treatments. Soil Olsen-P concentration increased significantly with the application of SWS compared with the unamended control or MF, with no differences among SWS application rates. Soil exchangeable K was not affected by the addition of soil amendments. Regarding the concentrations of DTPA extractable micronutrients, no differences were found among treatments for Zn, Fe, and Cu. In the case of Mn, DTPA extractable concentration increased with SWS application, significantly at the SWS1 treatment, but never exceeded the value obtained with the MF.

Discussion
Previous studies have demonstrated that SWS has an inhibitory effect on seed germination, which is primarily determined by the availability of heavy metals and their phytotoxicity (Wu et al. 2018;Walter et al. 2006), to which maize is particularly sensitive throughout the germination process (Deng et al. 2016). In the present study, the incorporation of SWS into the soil, even at the heavy dose of 80 Mg dry weight ha −1 , had no influence on seedling emergence and stand establishment of maize. The neutral effect of SWS application on crop establishment could be attributed to the low concentrations of heavy metals, which were substantially below the permitted limits for agricultural use (Council Directive 86/278/EEC 1986), as SWS was derived from a small town's municipal waste-water treatment plant that processed mostly domestic sewage.
The application of SWS increased height and stalk diameter of maize plants, traits that typically are closely associated with the agronomic performance of maize. Specifically, plant height was estimated to increase by 6 cm for each 10 Mg ha −1 increase in SWS application rate. Because the number of main stem nodes remained unchanged across treatments, the increased height of plants grown in soils amended with SWS was due to the longer internode length compared to the unamended control. In agreement with earlier studies (Mousavi and Nagy 2021;Abbasi et al. 2012), both height and stalk diameter were correlated with dry matter accumulation, indicating that tall plants with thick stems had a considerable capacity for storing assimilates, with confirmed positive effects on maize productivity (Ray et al. 2020). This finding is also supported by the close relationship between grain yield and early dry matter accumulation found in the present study, indicating the importance of added-SWS in improving maize productivity by enhancing source capacity. It is noteworthy that a considerable portion (5-34%) of assimilates required for grain filling of maize is supplied by remobilization of reserves stored in the vegetative organs up to the silking stage (He et al. 2005). However, despite the large height of plants grown in SWS-amended soil, no plant lodging was observed, most likely due to the thicker stalks of the plants, a trait that is adversely associated with plant lodging or breakage (Galindo et al. 2019). The variation in dry matter accumulation across treatments reflected mainly changes related to crop growth rate, given that the phenological development of maize showed no differences. In fact, early crop growth rate was proportional to SWS application rate, implying the importance of increasing nutrient availability on biomass accumulation (Koutroubas et al. 2014). Indeed, sufficient nutrient supply, particularly N, stimulates maize growth by increasing leaf area and photosynthetic surface, thus contributing to dry matter accumulation under both rainfed and irrigated conditions (Srivastava et al. 2018;Ray et al. 2020).
It is well known that the application of SWS increases grain yield of several crops (Binder et al. 2002;Eid et al. 2017;Koutroubas et al. 2020b). In the present study, SWS increased grain yield of maize by 104-226% compared with the unamended control, depending on the SWS application rate. Actually, grain yield of maize was increased by 143 kg ha −1 for every Mg ha −1 increase in SWS rate. Furthermore, grain yield obtained with SWS application was comparable to (SWS1) or even exceeded (SWS2 and SWS3) that noted with the MF treatment, showing an increase by 5-67%. The positive influence of SWS on grain yield has been linked not only to the supply of nutrients to plants, particularly N, but also to the improvement of soil properties (Goss et al. 2013). In terms of crop nutrition, although Table 6 Nitrogen translocation (NT), nitrogen translocation efficiency (NTE), biomass production efficiency (BPE), nitrogen utilization efficiency (NUtE), nitrogen use efficiency (NUE), and nitrogen uptake efficiency (NUpE) of maize as affected by the application of soil amendments. Control, unamended control; SWS, sewage sludge applied at 20 Mg ha −1 (SWS1), 40 Mg ha −1 (SWS2), and 80 Mg ha −1 (SWS3); MF, mineral fertilizer applied at 300 kg N ha −1 and 26 kg P ha −1 over 2 years (2012 and 2013) SOV, source of variation; df, degrees of freedom; in each column, different letters within years or treatments denote significant differences at P < 0.05 N fertilization was found to be efficient in enhancing grain yield of maize (Srivastava et al. 2018;Qian et al. 2016), excessive N fertilizer application, beyond optimal N rate, has been reported to inhibit or not consistently increase grain yield (Jin et al. 2012;Yu et al. 2021;Qiu et al. 2015). In the current study, the positive grain yield response to the application of SWS suggests that N availability did not surpass the optimal threshold, even when SWS was applied at the heavy rate, a fact that might be attributed to the gradual release of sludge-N during maize growth. However, balanced crop nutrition, a prerequisite for optimal plant growth, is questionable in the case of SWS, because the overall amount of SWS applied to agricultural soils is frequently based on crop N requirements, which can lead to over-application of P and K (Antoniadis et al. 2015). Apart from the positive response of grain yield, SWS application has been reported to improve N uptake by plants (Motta and Maggiore 2013;Bozkurt et al. 2006). In the present study, N uptake was closely related with maize dry matter, probably because biomass growth stimulated mineral nutrient uptake and assimilation (Abbasi et al. 2012). Concerning the soil amendments, the application of either SWS or MF to the soil resulted in a substantial increase of N uptake compared with the unamended control, indicating that N availability was a limiting factor for maize growth. It is well documented that early N uptake by maize in excess of vegetative growth requirements can support grain growth via N translocation (Nasielski et al. 2019). This was the case in the current study, where the enhanced early N uptake by maize plants grown in amended soil promoted N translocation to grains, contributing to higher grain-N content and grain yield than the unamended control, regardless of the type of amendment (SWS or MF).
In addition to ΝΤ from vegetative parts, post-silking N uptake seemed to be a considerable source for grain-N at maturity in plants grown in SWS-amended soil. This might be explained by the prolonged N availability late in the growing season, which was caused by the gradual release of sludge-N. Furthermore, given that N uptake is dependent on water availability (Gonzalez-Dugo et al. 2010), it is reasonable to hypothesize that the application of SWS facilitated postsilking N uptake by alleviating water deficits, as sludge has been reported to improve water-holding capacity of the soil (Koutroubas et al. 2020b). Overall, grain N content and grain yield were most related to NT rather than to the post-silking N uptake. These findings emphasize the importance of NT from vegetative tissues to grains in optimizing maize yield, as well as the suitability of SWS as an effective agronomic means of increasing plant N reservoir at silking and, secondarily, enhancing post-silking N uptake, thus allowing plants to meet their N requirements throughout the growing season.
Although the type of soil amendment had no apparent effect on the proportion of aboveground N removed by the grains (i.e., the NHI remained nearly constant across treatments), the net amount of N remaining in the field after harvest was frequently greater with the application of SWS. This finding indicates potentially larger N losses to the environment when compared with the application of the MF. In line with previous studies in several crops (Binder Table 7 Soil pH, electrical conductivity (EC), nitrate nitrogen (NO 3 -N), Olsen P, exchangeable K, and diethylenetriamine pentaacetic acid (DTPA)-extractable concentration of micronutrients after maize harvest as affected by the application of soil amendments.  Koutroubas et al. 2014), the NUE of maize declined in a curvilinear pattern with increasing rates of SWS, most likely due to the increased N supply that is typically inversely associated with NUE (Srivastava et al. 2018). However, when compared with the MF, the application of SWS at moderate rates (i.e., 20 and 40 Mg ha −1 ) improved NUE, probably owing to better synchronization of N availability and plant N demands due to the progressive supply of sludge-N throughout the growing season. Despite this, heavy SWS application (i.e., 80 Mg ha −1 ) had a significant negative impact on NUE, primarily due to the reduction of NUpE, which appeared to be the main component of the overall NUE. These findings suggest that excessive SWS application may enlarge the risk of N losses to the environment. In terms of NUtE, when the SWS rate of 40 Mg ha −1 was used to apply N at agronomic rates, maize produced 67 kg of grain yield per unit of N acquired, exhibiting an internal efficiency of N slightly higher than that of MF (60 kg of grain yield per unit of N acquired). Furthermore, because NUtE was closely related to BPE, there was a similar pattern in N utilization efficiency for grain yield and dry matter production. The application of soil amendments had an inconsistent influence on grain concentrations in macronutrients. Specifically, grain N concentration, upon which grain quality in terms of protein content is dependent, remained unchanged throughout treatments. Given the enhanced grain yield in soil amendment treatments, it appears that, despite increased N uptake following SWS or MF application, grain N concentration was maintained at levels comparable with that of the unamended control because of the dilution effect. In contrast to N, an increase in grain K concentration was evident after heavy SWS application compared with the MF, whereas grain P concentration in SWS treatments was similar to or even lower than that of MF, most likely due to microbial immobilization of P in soil (Lemming et al. 2016). Furthermore, because soil pH affects P availability, more research is needed to understand the potential impact of pH changes caused by the addition of SWS on P dynamics and uptake, as well as grain P concentration.
The accumulation of heavy metals resulting by land application of sewage sludge may raise concerns about their potential detrimental effects on plants and the environment. In this regard, it has been found that the sewage sludge application at heavy rates reduced grain yield of several crops, such as wheat (Tejada and González 2007) and barley (Fernández et al. 2009). Similarly, Koutroubas et al. (2020b) employing SWS from the same source as the present study observed that SWS rate beyond 18 Mg ha −1 decreased the achene yield of rainfed sunflower. The lack of SWS phytotoxicity on irrigated maize in the present study could be attributed to low concentrations of Cu, Zn, Fe, and Mn in maize grains, which were associated with high dry matter accumulation induced by adequate water supply and the resulting dilution effect. Besides, even heavy metals considered essential for plant growth appear to have a toxic effect on plants at high concentrations, but more research is needed to elucidate potential mechanisms of phytotoxicity for each plant species.
Soil amendments modified most soil properties at the end of the second year. In particular, SWS decreased soil pH compared with the unamended control, in accordance with previous studies (Eid et al. 2017;Kołodziej et al. 2015), with the reduction being more evident at moderate rates of SWS application and, in all cases, comparable to that noted when the MF was applied. The decrease in soil pH following the application of SWS compared to the unamended control was most likely caused by the production of acids during the microbial degradation of SWS (Brofas et al. 2000). However, soil pH in SWS treatments was consistently above the range that increases soil availability of heavy metals, as well as micronutrients necessary for plant development (Singh and Agrawal 2009). Soil concentrations of NO 3 -N and exchangeable K in SWS-added treatments were comparable to those obtained with the addition of MF. These findings suggested that the potential environmental risk posed by applying SWS, in terms of NO 3 -N leaching and excessive K build-up, was not greater than that posed by the MF. In comparison to moderate application rates, the heavy SWS rate resulted in a reduction of soil NO 3 -N concentration, which seems to be associated with the optimization of maize yield. Binder et al. (2002) reported little addition of NO 3 -N with biosolids rates lower or close to the recommended rate for maximum yield. Concerning Olsen P concentration in the soil, there was a considerable increase upon the application of SWS compared with the MF, in accordance with previous studies (Shaheen and Tsadilas 2013), suggesting an increased risk for dissolved phosphates in runoff.

Conclusions
The application of sewage sludge stimulated maize crop growth in early stages and increased plant nitrogen reservoir at silking, contributing to enhanced nitrogen translocation to grains during the filling period. Furthermore, sewage sludge improved post silking nitrogen uptake, allowing plants to meet their nitrogen requirements throughout the growing season, and resulting in grain yield that was similar to or even exceeded that obtained with mineral fertilizer. Findings support the hypothesis of the present study that domestic sewage sludge could substitute the mineral fertilizer in maize crop as part of a nutrient recovery and recycling strategy, without adverse effects on the soil. In practice, a rate of 20 Mg ha −1 year −1 may be managed more effectively by producers, in terms of transportation and field application, and is more acceptable in terms of environmental pollution risk compared to higher rates. Nevertheless, further research should be conducted to quantify the potential level of soil pollution caused by long-term land application of sewage sludge. Author Contribution Conceptualization: SDK; methodology: SDK; validation: SDK, VA, CAD, and SF; formal analysis: SDK, VA, and SF; resources: SDK; data curation: SDK, VA, and SF; writing-original draft preparation: SDK; writing-review and editing: SDK and CAD; supervision: SDK; funding acquisition: SDK. All authors have read and agreed to the published version of the manuscript.
Funding Open access funding provided by HEAL-Link Greece. This research was funded by the Region of Eastern Macedonia & Thrace, grant number 81069.

Data Availability
The data presented in this study are available on request from the corresponding author.

Conflict of Interest
The authors declare no competing interests.
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