Effects of Nitrogen and Zinc Fertilization on Alleviating Cadmium Accumulation in Durum Wheat Grains

ABSTRACT Cadmium (Cd) accumulation in durum wheat (Triticum durum L.) is of particular concern compared to the other commonly cultivated cereals. This study was conducted to determine the effects of zinc (Zn) and nitrogen (N) applications on grain Cd concentration of durum wheat grown in a Cd contaminated soil. The experimental design consisted of randomized plots with four replications. The treatments included low and adequate (0.05 and 5 mg kg−1) Zn doses, and inadequate, optimal and high (200, 400 and 600 mg kg−1) N doses. Cadmium application rates were 0 and 5 mg kg−1. The findings demonstrated that Cd application caused a significant increase in grain Cd concentrations, and a decrease in straw and grain yields under low and adequate doses of Zn and all N doses. However, increasing N applications with adequate soil Zn caused an increase in the straw and grain yields and a significant decrease in grain Cd concentration. The grain Cd concentration in low Zn and optimum N treatment under Cd application was 6206 µg kg−1, while the grain Cd concentration decreased by 26.3% (4574 µg kg−1) in the Zn and optimum N treatment. In addition, the grain Zn concentration of durum wheat under low soil Zn conditions was decreased in Cd application, whereas the grain Zn concentration increased in Cd application under adequate soil Zn concentration. The results revealed that combined application of N and Zn in Zn deficient soils is highly effective at decreasing Cd accumulation in wheat grains compared to individual applications.


Introduction
Accumulation of heavy metals in soils poses a severe threat to ecosystem services, and the impact of heavy metals may become more threatening depending on their toxicity and accumulation level (Liyu Yang, Wu, and Yang 2022). Agricultural production in heavy metal contaminated soils may cause the transfer of heavy metals into the food chain and serious health problems. Heavy metals are not required to sustain the lives of higher plants; therefore, accumulation of heavy metals has a significant adverse impact on the growth and development of most crops (Hembrom et al. 2020).
Cadmium (Cd) is an unessential and non-metabolic heavy metal that accumulates in soils due to anthropogenic activities such as mining activities, disposal of urban wastes, and the use of phosphate fertilizers in agricultural lands (Haider et al. 2021). The uptake and accumulation of Cd in the edible parts of crops is raising serious concerns for human health (Kaya et al., 2019). Like many other heavy metals, Cd is a non-degradable contaminant and does not easily leach from soil. High levels of Cd in soil inhibit plant growth, leading to a severe decrease in seed germination and the formation of plant necrosis, chlorosis, and stunted plants (Ismael et al. 2019). Therefore, the yield of various important crops throughout the world is hampered due to the high levels of Cd in agricultural soils (Sharma and Nagpal 2020). In addition, accumulation of Cd in edible crop parts has significant implications for human health (Danping Yang et al. 2016). The main problems encountered in plants exposed to Cd toxicity are decreased carbon fixation, chlorophyll content, and photosynthetic activities (Gallego et al. 2012). Cadmium toxicity impairs antioxidant enzyme activity, pigment and protein synthesis during photosynthetic processes, and stomatal conductance (El Rasafi et al. 2022). High levels of Cd in plants also encourage the production of reactive oxygen species, which damage plant membranes and destroy cell macromolecules and organelles (Abbas et al. 2018).
Cereals are the major food sources for humans worldwide. Wheat is the most commonly consumed cereal by more than half of the world's population, and the average annual global wheat production between 2016 and 2018 was around 779 million tons (Anonymous 2023). Cadmium uptake of wheat is higher compared to other cereal crops, and the Cd easily transfers to various above ground plant parts (Jafarnejadi et al. 2011). The accumulation of Cd in wheat grains reduces the nutritional value of cereal foods and poses a serious health concern to people whose diet primarily consists of foods prepared from wheat products (Yu et al. 2019). Durum wheat is one of the most widely used grains in the food industry due its high protein, vitamin, and fiber content and low glycemic index (Hammami and Sissons 2020). Therefore, the accumulation of Cd in cereals, is a growing concern, particularly in durum wheat. Although the cause is still not clear, durum wheat cultivars accumulate much more Cd in their grains compared to bread wheat cultivars (Erdem, Tosun, and Ozturk 2012).
Nitrogen (N) is an essential nutrient used in the synthesis of chlorophyll, proteins and a variety of secondary metabolites (Xiao et al. 2019). A strong correlation has been reported between the Cd concentration of grain and N application rates (Li et al. 2013). In contrast to the positive stimulating impact of N on grain Cd concentration, Landberg and Greger (2003) reported a negative relationship between N application and grain Cd content of wheat grown in both soil and nutrient solution. The decrease in grain Cd concentration has been attributed to a dilution effect caused by an improvement in wheat biomass production with the increased N application. Similar to the role of N in plants, zinc (Zn) is essential for respiration, the photosynthesis, activation of antioxidant enzymes, and the absorption of several other nutrients (Javed et al. 2016). Furthermore, Zn in the root zone may compete with Cd in transport mechanisms during absorption and translocation in plants due to the similar chemical characteristics of both nutrients (Gao and Grant 2012;Zhao et al. 2005). Since Zn and Cd have similar transport mechanisms, Javed et al. (2016) showed that Cd uptake of wheat roots and grain accumulation significantly decreased with Zn application to soil, while grain Zn concentration increased. Therefore, Zn application is considered to alleviate Cd toxicity by restricting Cd uptake of wheat plants (Hart et al. 2002).
The public awareness of contaminants is increasing, and concerns are raised to reduce heavy metal pollution and prevent the accumulation into the edible and non-edible parts of crops (Oladoye, Olowe, and Asemoloye 2022). Several extracts and other chemicals have been used to reduce the negative effects of heavy metals in soils, while the significance of mineral nutrition application in alleviating the adverse impacts of heavy metals is still unclear (Eijsackers et al. 2019). The following hypotheses tested in the study: (1) increasing N application in Zn sufficient soils increases grain and straw yields, (2) increasing N applications in Zn sufficient soils reduces grain Cd accumulation, and (3) Cd application reduces grein Zn and N concentration under Zn deficiency conditions.

Material and method
The study was conducted in a greenhouse equipped with an evaporative cooling system (average night and day temperatures of 22°C and 28°C) and natural sunlight conditions. The greenhouse was located between 40°19'39.5"N and 36°28'42.3" E latitudes. Wheat (Triticum durum, cv. Ege-88) seeds were sown in plastic pots filled with 3.2 kg of Zn-deficient soil. Before potting, the seeds were sterilized by soaking in a 20% sodium hypochlorite solution for 30 min and were rinsed three times in distilled water. The experimental soil had a clay-loam particle size distribution and a moderately alkaline reaction (pH 8.06). The soil was poor in organic matter content (1.08%) and moderately calcareous (12% CaCO 3 ). Diethylenetriamine Penta Acetic Acid (DTPA) extractable Zn and Cd concentrations of experimental soil were 0.17 and 0.05 mg kg −1 soil, respectively. The particle size distribution of experimental soil was determined by hydrometer method in a sedimentation cylinder, using sodium hexametaphosphate as dispersing agent (Bouyoucos 1962). The soil reaction (pH) was measured in a saturated paste (Rhoades 1983). Organic matter was determined by dichromate oxidation using the modified Walkley Black method (Nelson and Sommers 1996). CaCO 3 content was measured by Scheibler Calcimeter (Staff 1993). DTPA extractable concentrations of Zn and Cd were determined according to the method described by Lindsay and Norvell (1978).
The experimental design was completely randomized plots with four replications. Durum wheat was grown in low and sufficient Zn, insufficient, optimal, and high N conditions, and 0 and 5 mg Cd kg −1 applications. Plant nutrients required by wheat plants were homogeneously incorporated into the soil before sowing. Each pot contained 100 mg P kg −1 and 125 mg K kg −1 as KH 2 PO 4 , 2.5 mg Fe kg −1 as Fe-EDTA, 200 mg N kg −1 (for low N -N200), 400 mg N kg −1 (for optimum N -N400) and 600 mg N kg −1 (for high N -N600) as Ca(NO 3 ) 2 .4 H 2 O, 0.05 mg Zn −1 kg (for low Zn) and 5 mg Zn kg −1 (for adequate Zn) as ZnSO 4 .7 H 2 O, and 0 mg Cd kg −1 (Cd0) and 5 mg Cd kg −1 (Cd5) as 3(CdSO 4 ).8 H 2 O. Ten durum wheat seeds (Ege-88 genotype) were sown in each pot, and the number of plants was reduced to 4 per pot a few days following the emergence of seedlings.
Deionized water was used for irrigation. The spikes and shoots of wheat plants were harvested separately when plants completely senesced, and grains reached full maturity. The shoots were dried at 60°C in an oven to determine dry matter yield of straw (shoot). The grains in spikes were separated from the husks using a thresher and the grains were weighed to determine the grain yield for each treatment. Wheat grains were ground and digested using 2 ml of 35% H 2 O 2 and 5 ml of 65% HNO 3 in a closed vessel microwave system (Mars 6, CEM, Matthews, NC). An inductively coupled plasma optical emission spectrometry (ICP-OES) (ICAP7000 Plus Duo, Thermo) was used to determine the concentrations of Cd, Zn, Fe, Mn, and Cu in digested solutions (Jones et al., 1991). Nitrogen content of wheat grains was determined using a Thermo Scientific FlashSmart C/N Analyzer. Certified standard reference materials obtained from the National Institute of Standards and Technology (NIST, SRM Number 1573a; Gaithersburg, MD) were used to control the measurements of the C/N Analyzer.
The effects of treatments and interactions on yield (straw, grain) and grain mineral concentrations were assessed using variance analysis (ANOVA). The least significant difference (LSD) test (p < .01) was used to compare the means of yield and grain mineral contents for various treatments when the ANOVA indicated a significant difference between the treatments.

Results and discussion
Nitrogen and Zn applications, regardless of Cd applications, caused a statistically significant increase in the straw and grain yields of durum wheat (p < .01) (Figure 1a,b). Straw yield in Cd0 × low Zn x N200 interaction was 4.34 g plant −1 , while increased to 4.44 g plant −1 at the same Zn and Cd dose in N400 application and 4.80 g plant −1 in the N600 application ( Figure 1a). Similar to the N application, straw and grain yields significantly increased in the Zn application. The grain yield, which was 0.62 g plant −1 in low Zn x Cd0 × N200 interaction, increased by 82% to 1.13 g plant −1 at the optimum Zn application of the same Cd and N dose (Figure 1a). Many studies have documented an increase in crop yield with N application. Gray et al. (2002)  Previous studies have often reported a negative relationship between increasing doses of Cd to soil and plant yield (Erdem, Tosun, and Ozturk 2012;Zhou et al. 2020). Application of 5 mg Cd kg −1 to soil caused a statistically significant decrease in straw and grain yield of durum wheat variety (p < .01), especially under low Zn conditions (Figure 1a,b). The straw yield was 4.34 g plant −1 in the Cd0 × low Zn x N200 interaction, while straw yield decreased to 2.28 g plant −1 , causing a 47.5% decrease in the Cd5 × low Zn x N200 interaction. A similar trend was also recorded in the grain yield. The Cd application significantly decreased the yield under low Zn concentration (p < .01), while the yield increased with increasing N doses under adequate Zn treatment. Grain yields in Cd5 × low Zn x N200, N400 and N600 interactions were 0.56, 0.67 and 0.75 g plant −1 , respectively. A statistically significant increase of 60.7%, 56.7% and 82.7% was observed in grain yield under adequate Zn application with increasing doses of N, respectively (Figure 1b). The results showed that Cd caused a significant decrease in plant yield, especially at low Zn concentrations. The grain yield, which was 1.67 g plant −1 in an adequate Zn x Cd0 × N600 interaction, decreased by 55% to 0.75 g plant −1 at the low Zn x Cd5 × N600 dose (Figure 1b). However, the yield decrease could be prevented with Zn and N applications.
Cadmium is mobile in soil and plants, and can easily be absorbed and translocated to aerial parts and grains; therefore, higher accumulation of Cd in grains was reported in Zn deficient soils (Ismail Cakmak 2009). The findings demonstrated a substantial increase in grain Cd concentrations following the treatment of 5 mg Cd kg −1 (p < .01). The grain Cd content ranged from 278 g kg −1 in the Cd0 × low Zn x N200 interaction to 6441 g kg −1 in the Cd5 × low Zn x N200 interaction ( Figure 2). However, the grain Cd content of durum wheat under low-Zn concentrations was greater than an adequate Zn concentration. The grain Cd concentration in the Cd5 × low Zn x N600 interaction was 3352 g kg −1 Figure 1. The effect of N and Zn applications on (a) straw yield and (b) grain yield of durum wheat grown in Cd0 and Cd5 treatments. The differences between the means shown with different letters are significant at p < 0.01. Cd and Zn applications were statistically significant at p < 0.01. and 5303 g kg −1 in the Cd 5 × adequate Zn x N600 interaction. The findings revealed that Cd accumulation in the grains of durum wheat is higher in Zn deficiency conditions.
The grain Cd concentrations of durum wheat significantly decreased with the increase in N application dose (p < .01) (Figure 2). Grain Cd concentration in the Cd5 × low Zn x N200 interaction was 6441 µg kg −1 , and decreased to 6206 (3.65% reduction) and 5303 µg kg −1 (17.7% reduction) in the N400 and N600 doses, respectively. A similar decrease was noted with increasing N doses in adequate Zn concentrations of Cd5 treatment (Figure 2). The results revealed that the increase in straw and grain yields with the increase in N application doses caused a dilution of the accumulated Cd concentrations in wheat grains. The decrease in Cd concentration due to the dilution effect following adequate N fertilizer application has been reported in wheat (Landberg and Greger 2003), rice (Lin et al. 2011), and potato (Larsson Jönsson and Asp 2013).
The highest decrease in grain Cd concentrations with N applications was recorded in adequate Zn applications. Grain Cd concentrations in Cd5 × low Zn x N200, N400 and N600 interactions were 6441, 6206 and 5303 µg kg −1 , respectively, while grain Cd concentrations in adequate Zn application decreased to 5428 µg kg −1 (15.7% reduction), 4574 µg kg −1 (26.3% reduction), and 3352 µg kg −1 (36.8% reduction), respectively ( Figure 2). Numerous additional investigations have noted that application of Zn reduced the Cd concentration in wheat (Erdem, Tosun, and Ozturk 2012;Javed et al. 2016;Zhou et al. 2020). Javed et al. (2016) reported that Zn treatment in Cd-contaminated soil decreased Cd levels in roots, straw and grains by 74%. Similarly, Liu, Tjoa, and Römheld (2007) observed a substantial decrease in Cd concentration with Zn treatment only in Cd-stressed durum wheat plants. The findings can be linked to the prevention of Cd translocation from roots to shoots by Zn (Zhao et al. 2005), which is a chemically identical metal to Cd and competes with Cd for absorption by roots and translocation within plants (Gao and Grant 2012). The findings demonstrated that N treatments alone are insufficient to lower the Cd content in grains and that Zn should also be applied, particularly in Zn-deficient soils.
The grain Zn concentration of durum wheat grown under low Zn levels dramatically decreased in a 5 mg Cd kg −1 application, in line with the results of previous studies. The grain Zn concentration in the Cd0 × low Zn x N600 interaction was 23 mg kg −1 ; and it was 10.7 mg kg −1 in the Cd5 × low Zn x N600 interaction (Figure 3). The increasing Cd application doses in Zn-deficient soils tended to lower plant Zn concentrations. The Zn concentration of plants cultivated under adequate Zn concentrations was not affected by Cd application (Ismail Cakmak 2009). The findings confirmed that Cd dramatically decreased grain Zn content, particularly in Zn-deficient soils.
In addition to the decrease in grain Zn concentrations with Cd application, increased N application doses caused a significant increase in grain Zn concentrations in both low and adequate soil Zn concentrations (p < .01). The grain Zn concentration increased to 7.97, 16.1 and 23.0 mg kg −1 , respectively, with increasing N doses under low soil Zn concentration in Cd0 treatment (Figure 3). Previous research demonstrated that increasing N application can enhance grain Zn and Fe concentrations, and co-application of Zn and N had a stimulating impact on grain Zn concentration of durum wheat (Cakmak et al. 2010). In contrast, the increase in grain Zn concentrations with increasing N applications under low Zn x Cd5 interaction was smaller than that recorded under Cd0 × low Zn applications. The result can be attributed to the antagonistic effect of Cd on Zn absorption in low Zn concentrations. Cadmium in plants acts as an antimetabolite of Zn (El Rasafi et al. 2022). Therefore, a significant decrease in Zn concentrations of plant tissues, especially in roots, with the application of Cd has been reported by XE Yang et al. (2005) and Balen et al. (2011).
The grain N concentrations of durum wheat, regardless of Cd and Zn applications, increased significantly with N200, N400 and N600 applications (p < .01). The highest increase was obtained in Cd0 × adequate Zn interaction, while the least increase was recorded in Cd5 × low Zn treatment (Figure 4). The grain N concentration of durum wheat grown in both low and adequate Zn conditions under Cd treatment decreased significantly (Figure 4). Essential nutrients may not be absorbed under stressed conditions due to competition between Cd and Zn (mg kg -1 ) N200 N400 N600  essential nutrients for adsorption on root surfaces (Ertani et al. 2017). The grain N concentration in the Cd0 × low Zn x N600 interaction was 3.17%, while it decreased to 2.37% in the Cd5 × low Zn x N600 interaction. Similar results were observed under adequate Zn concentrations. Chaffei et al. (2004) reported a significant decrease in N concentration of tomato plants with increased Cd concentration (50 μM). Many studies indicated an adverse impact of Cd on the uptake as well as assimilation of nutrients in plants (Ismael et al. 2019;Mourato et al. 2019), and therefore, reported nutrient deficiency in plants grown in Cd accumulated soils (Khan et al. 2015).
The amount of N in grains dramatically decreased after Cd treatment, however, Zn application alleviated the Cd toxicity and resulted in an increase in grain N concentrations. The grain N content ranged from 2.14% in the Cd5 × low Zn x N400 interaction to 2.86% in the Cd5 × adequate Zn x N400 interaction (Figure 4). Zinc improves plant growth by improving photosynthetic efficiency, strengthening the membrane integrity of root cells, and increasing tissue antioxidant capacity (Hassan et al. 2005). A similar transport mechanism controls the absorption of Cd and Zn in the plasma membrane of root cells of bread and durum wheat seedlings (Hart et al. 2002). Therefore, positive effect of Zn on alleviating the Cd toxicity can be attributed to the direct suppression of Cd absorption.
The contents of Fe, Mn, and Cu in the grains of durum wheat increased considerably with increasing N application doses under Cd0-Cd5 × low-adequate Zn interactions (p < .01) ( Table 1). The most important three micronutrients that plants require for various biochemical activities are Mn, Fe, and Cu (Shukla et al. 2018). Cadmium has a significant interaction with the micronutrients. Therefore, excessive Cd content may prevent absorption and translocation of minerals in plants, which may lead to nutritional deficiencies (Zeng et al. 2020). In contrast, grain Mn and Cu concentrations considerably decreased when the soil was exposed to 5 mg Cd kg −1 . The grain Cu concentrations in Zn-deficient soil decreased considerably from 3.47 mg kg −1 in the Cd0 × low Zn x N200 interaction to 2.20 mg kg −1 in the Cd5 × low Zn x N200 interaction ( Table 1). The competition between Cd and Cu for the translocation has not been sufficiently explained in previous studies. Mwamba et al. (2016) noted that the ability of Brassica napus to absorb Cd was constrained by the presence of Cu. In contrast, Ma et al. (2015) reported higher Cd uptake by rice plants grown in cell culture solutions containing Cd and Cu compared to the solution containing Cd alone. Insufficient knowledge of the processes and interactions between Cd and Cu is the cause of the inconsistent results. In addition, little research has been conducted to investigate the role of Fe on the Cd adsorption of plants (Ma et al. 2015). The findings demonstrated that Cd decreased the Cu and Mn contents in durum wheat grains, whereas Zn and N treatments could prevent this decline. The differences between the means shown with different letters are significant at p <.01 Cd and Zn applications were statistically significant at p < .01.

Conclusions
In this study, straw and grain yields of durum wheat significantly decreased with Cd application to soil, while grain Cd concentration significantly increased, especially at low Zn concentration. On the other hand, increasing N application doses with adequate soil Zn concentrations caused an increase in straw and grain yields of durum wheat and a significant decrease in grain Cd concentrations. The highest decrease in grain Cd concentrations at adequate soil Zn concentrations was recorded at optimum and high N application doses. In addition, the toxic Cd level at low soil Zn concentration caused a decrease in grain Zn concentrations of durum wheat. However, the Zn concentration of durum wheat grain was not affected by Cd application in soils with adequate Zn concentrations, whereas the grain Zn concentration increased. A similar trend was also recorded for grain N concentration. The results revealed that N applications alone are not adequate to reduce the grain Cd concentration of durum wheat, and therefore, Zn fertilization should also be applied, especially in Zn deficient soils. The present study provides a useful method to reduce Cd accumulation in grain as well as increase the grain yield of durum wheat in Cdcontaminated soils.

Disclosure statement
No potential conflict of interest was reported by the authors.