1. Introduction
Human activity is causing an increase in the content of heavy metals in the environment (soil, water and air), posing a threat to living organisms [
1,
2,
3,
4,
5,
6,
7]. These elements are neither removed from the environment nor degraded, unlike other pollutants, which can be degraded by either chemical or biological means [
8]. Excessive content of heavy metals in the soil, especially in bioavailable forms, increases their uptake and causes them to accumulate in plants. This negatively affects germination, root growth, the development of above-ground organs, biomass production, and transpiration. Heavy metals also disturb control mechanisms at the gene level, inhibit the activity of enzymatic proteins, impair the functioning of metabolic pathways, and contribute to apoptosis [
9]. Zinc is a distinctive heavy metal that is essential for plants and animals in low concentrations, but becomes toxic for them above a critical concentration [
10]. Its content in the soil depends mainly on its quantity in the bedrock and on anthropogenic factors [
11]. Zinc is present in the soil solution, adsorbed on various minerals, e.g., clay minerals, oxides or carbonates, or bound to organic matter [
12,
13]. Soils with insufficient levels of zinc are also common throughout the world [
14,
15]. Zinc is an essential micronutrient for plants, in which it takes part in chlorophyll biosynthesis and gene expression and is a cofactor of many enzymes [
16,
17,
18,
19,
20]. It is a component of carbonic anhydrase and a stimulator of aldolase, enzymes which take part in carbon metabolism [
21]. It also influences the capacity for water uptake and transport, reduces the unfavourable effects of short periods of high temperatures and salt stress [
22], and increases the resistance of plants to fungal diseases [
23,
24]. Stress associated with zinc deficiency in plants, caused by its low bioavailability, reduces productivity and the nutritional quality of food [
25]. In the case of high content in the soil, however, zinc can be toxic for plants, and plants that have taken up excessive zinc exhibit symptoms similar to those occurring in the case of other toxic heavy metals, such as Cd or Pb [
26]. In most cases, excessive Zn generates reactive oxygen species and displaces other metals from active sites in proteins. Its toxicity is manifested in part by impaired seed germination, limited growth, and chlorotic and necrotic changes on the leaves [
27]. The mobility of zinc and thus its toxicity for plants can be reduced by increasing the content of organic matter in the soil. Its sources are organic fertilizers and organic waste materials used as fertilizers [
28]. They then form mineral–organic complexes (chelates), thereby reducing the bioavailability of zinc for plants [
3].
The aim of the study was to determine the effect of increasing levels of zinc application in combination with additional various organic fertilizers on the yield of cocksfoot and the content and uptake of the metal, as well as to determine the bioaccumulation factor and tolerance indices.
The research hypothesis was that the application of all organic substances to the soil would increase the tolerance of cocksfoot to increasing levels of zinc, reducing the accumulation of this heavy metal in the harvested biomass.
3. Results
Application of increasing amounts of zinc (0, 200, 400 and 600 mg Zn·kg
−1 of soil) and various organic fertilizers (cattle manure, chicken manure and spent mushroom substrate) significantly affected the yield of cocksfoot, the tolerance indices, zinc content and uptake by cocksfoot, and the bioaccumulation factor of the metal (
Table 2,
Table 3,
Table 4,
Table 5,
Table 6,
Table 7 and
Table 8).
Significantly the highest yield of cocksfoot was harvested following application of 200 mg Zn·kg−1 of soil. It was greater than the yield of plants that were not fertilized with zinc and those fertilized with 400 and 600 mg Zn·kg−1 of soil, by 6.2%, 12.0% and 19.2%, respectively.
Application of 600 mg Zn·kg−1 significantly reduced yield by 10.9% in comparison with the control treatment and by 16.1% and 6.0% compared to the yield following application of 200 and 400 mg Zn·kg−1 of soil. Yield was not significantly affected by application of 400 mg Zn·kg−1 of soil, but it was somewhat higher than in the control treatment.
All of the organic materials increased the yield of cocksfoot. Significantly the highest yields were obtained following application of chicken manure and cattle manure. They were 52.6% and 50.0% higher, respectively, than the yield obtained from the control treatment and 13.6% and 11.7% greater than the yield following application of spent mushroom substrate.
The cocksfoot yield decreased in successive years of the study. In the second and third year it was 62.1% and 46.0% of the yield in the first year.
Application of organic fertilizers, irrespective of their origin, did not reduce the negative impact of application of 600 mg Zn·kg−1 of soil on the yield of the grass. The negative effect of this amount of zinc on the yield of the plant was also shown to be significant only in the first year after its application.
The effect of various amounts of zinc and organic materials in the soil on the yield of cocksfoot was confirmed by the tolerance indices—the effect of increasing application of zinc in combination with various organic fertilizers (Zn/Org) and the effect of various organic fertilizers in combination with increasing application of zinc (Org/Zn) (
Table 3 and
Table 4). Values of these indices smaller or greater than 1 indicate that the effect of the factor on plants is negative or positive, respectively, while values close to 1 indicate a lack of effect.
Application of 400 and 600 mg Zn·kg−1 of soil significantly reduced the Zn/Org tolerance index in the first and third year of cocksfoot cultivation. This index was increased by spent mushroom substrate. The Zn/Org tolerance index was not found to be significantly influenced by the application of cattle manure or chicken manure, nor did it differ in different years of the study.
The Org/Zn tolerance index was higher following application of 400 mg Zn·kg−1 of soil than in the control treatment and after application of 200 and 600 mg Zn·kg−1 of soil. It was highest following the application of cattle manure and chicken manure, which confirms the positive effect of these fertilizers on cocksfoot, expressed as its yield. The Org/Zn tolerance index was the same in the second and third years of the study, but significantly lower than in the first year.
Application of increasing amounts of zinc increased its content in the cocksfoot biomass (
Table 5). Significantly the highest content of the metal was found in the plants harvested following application of 600 mg Zn·kg
−1 of soil. It was 232.0%, 53.9% and 27.4% higher, respectively, than the content in plants from the control treatment and those fertilized with 200 and 400 mg Zn·kg
−1 of soil. Application of all organic materials decreased the content of zinc in the test plant. Significantly the lowest content of zinc was noted following application of cattle manure and spent mushroom substrate. In successive years of the study, the zinc content in the biomass of cocksfoot decreased. In the second and third year it was 14.6% and 52.5% lower, respectively, than in the first year. All application rates of zinc increased its uptake by cocksfoot, calculated as the average from the three years of the study 3 (
Table 6), as well as the total uptake in the three-year cycle (
Table 7). On average in the three-year cycle, following application of 200, 400 and 600 mg Zn·kg
−1 of soil, the plants accumulated 145.3%, 163.2% and 208.3% more of this metal than plants from the control treatment. Total zinc uptake during the three years of the study was highest following application of 600 mg Zn·kg
−1 of soil. Following application of cattle manure, chicken manure and spent mushroom substrate, zinc uptake by cocksfoot was higher than in the control treatment. The most zinc was accumulated by plants fertilized with chicken manure. Zinc uptake decreased in successive years of the study, and in the second and third year of the study it was only 54.8% and 23.5% as high as in the first year.
In addition, the effect of increasing application of zinc and application of organic fertilizers on zinc accumulation in the biomass of the grass was shown to vary in successive years of the study. In the third year of the study, zinc uptake by plants was similar following application of 400 and 600 mg Zn·kg−1 of soil. In this year of the study, the uptake of zinc after the application of 600 mg of Zn was higher than in the control object and after the application of 200 mg of Zn. In the third year of the study, application of various organic fertilizers had no effect on the accumulation of zinc in cocksfoot.
Application of increasing amounts of zinc decreased its bioaccumulation factor in the test plant (
Table 8). It was significantly the lowest for the plants grown following application of 600 mg Zn·kg
−1 of soil. The bioaccumulation factor of zinc in cocksfoot was not affected by chicken manure or spent mushroom substrate, but was reduced by the application of cattle manure.
The bioaccumulation factor of zinc in cocksfoot was similar in the first and second years of the study and did not exceed 1. In the third year, it was significantly lower.
Correlation analysis revealed significant relationships between the zinc application rate and its content in and uptake by cocksfoot. A significant correlation between zinc content in the plant and its accumulation was noted as well (
Table 9).
4. Discussion
The yield and chemical composition of plants depend not only on the soil content of macroelements but on that of microelements and trace elements as well [
32,
33,
34]. Microelements perform very important physiological functions in plants, taking part in metabolism of proteins, carbohydrates, and sugars. They are also activators of enzymatic reactions. Important microelements for plant nutrition include zinc [
35,
36]. Deficiencies of this element are a major problem around the world, but in soil contaminated by mining and metallurgical activity, soil fertilized with wastewater sludge, and urban and suburban soils anthropogenically enriched with zinc, it can have toxic effects on plants [
37,
38,
39]. In the present study, cocksfoot responded with a significant increase in yield to the application of 200 mg Zn·kg
−1 of soil and with a small decrease following the application of 400 mg Zn·kg
−1 of soil. A significant decrease in yield followed the application of 600 mg Zn·kg
−1 of soil. The Zn/Org tolerance index was also reduced following the application of 400 and 600 mg Zn·kg
−1 of soil. A stimulating effect of soil application of zinc in the form of ZnSO
4 × 7H
2O at rates of 15, 30 and 45 kg of fertilizer per ha
−1 on maize yield was reported by Liu et al. [
40]. However, these levels of application were much lower per 1 kg of soil than in the present study. Ryegrass yield reduction following zinc application was demonstrated by Zalewska [
41]. In a pot experiment, the author tested zinc application rates from 25 to 400 mg Zn·kg
−1 of soil and found that even the smallest dose decreased the yield of the grass when it was grown on sand, but in the case of cultivation on sandy clay only the highest application rate caused decreased yield. Baran [
42] demonstrated that zinc had a negative effect on maize. In a pot experiment, the author applied zinc in the form of an aqueous solution of ZnSO
4 × 7H
2O in the amount of 0, 50, 250 and 750 mg Zn·kg
−1 of soil and observed a decrease in maize yield following application of just 50 mg Zn·kg
−1 of soil. Chaney [
43] notes that zinc toxicity thresholds marked in leaves depend on the species and even the variety of the plant. No information describing the toxicity level of zinc in soil for grasses has been found in the scientific literature. In an experiment by Long et al. [
44] the threshold of toxicity of this metal was 413 mg Zn·kg
−1 of soil for Chinese cabbage (
Brassica chinensis L.), 224 mg Zn·kg
−1 of soil for pok choi (
Brassica chinensis L.), and 272 mg Zn·kg
−1 of soil for celery (
Apium graveolens L.).
The bioavailability of zinc for plants depends in part on its total content in the soil, the amount and type of organic matter. High content of organic matter can limit zinc bioavailability due to adsorption by organic ligands [
11]. At the same time, application of organic fertilizers increases soil fertility, which increases crop yield [
45]. In the present study, all of the organic materials increased the yield of cocksfoot. The best effect on yield and the highest Org/Zn tolerance index were obtained following application of chicken manure and cattle manure, which may be linked to their chemical composition and the C:N ratio. Varied effects of organic fertilizers depending on their chemical composition were obtained in fertilization of grassland by Štýbnarová et al. [
46] and by Tong et al. [
47].
Application of spent mushroom substrate increased the Zn/Org tolerance index, which may indicate a minor protective effect counteracting the negative effect of high levels of zinc on cocksfoot.
The content of zinc in grasses is an important indicator of the fodder value of hay. Both a deficiency and a surplus of zinc in feed adversely affect the health of animals, especially ruminants, which are among the most sensitive to this metal [
48,
49]. Zinc content above 100 mg Zn·kg
−1 DW in feed can be harmful to animals due to various interactions.
As the amount of zinc applied to the soil was increased, its amount in the biomass of cocksfoot increased as well. In the first year of the study, the average content of the metal in the grass following its application, irrespective of the amount applied, was greater than 100 mg Zn·kg
−1 DW. In the second year of the study, this was the case only following application of 400 and 600 mg Zn·kg
−1 of soil. In the cocksfoot harvested in the third year of the study, the content of zinc did not exceed 100 mg Zn·kg
−1 DW. An increase in zinc content in plants grown on soil contaminated with this metal was obtained by Mishra et al. [
50]. Chaney [
43] reports that symptoms of zinc toxicity most often appear at concentrations of 300 mg Zn·kg
−1 DW in the aerial parts of plants, although some plants show symptoms of toxicity at concentrations of more than 100 Zn·kg
−1 DW. According to Marschner [
23], the zinc content in leaves exceeding the value of 300–600 mg Zn·kg
−1 DM is a toxic amount for plants. Research by Broadley et al. [
37] shows that toxicity symptoms often make themselves evident at leaf Zn concentrations higher than 300 mg·kg
−1 DM. The physiology of Zn phytotoxicity in leaves is complicated, resulting from Zn interference in chlorophyll biosynthesis, and other biochemical reactions.
While none of the organic fertilizers used in the experiment mitigated the negative effect of 600 mg Zn·kg
−1 of soil on the yield of cocksfoot, they did reduce its content of zinc. Its lowest content was noted following the application of cattle manure and spent mushroom substrate. This indicates potential binding and immobilization of zinc by organic matter applied to the soil together with the metal. Binding of zinc by organic matter has been reported by Fan et al. [
51], who showed that soil organic matter is the main factor determining sequestration of heavy metals in the soil.
The efficiency of accumulation of heavy metals in plants can be assessed by means of the bioaccumulation factor [
52]. The bioaccumulation factor (BAF) can be used to describe active transport of metals from the environment to plants and animals via metabolism [
53]. According to Netty al. [
54], a bioaccumulation factor of 1–10 indicates a hyperaccumulator plant, a value of 0.1–1 indicates a moderate accumulator plant, a value of 0.01–0.1 indicates a low accumulator plant, and a value of <0.01 indicates a non-accumulator plant. In the present study, the bioaccumulation factor of zinc in cocksfoot ranged from 0.092 to 0.808, which indicates moderate accumulation. In an experiment by Łukowski et al. [
30], the bioaccumulation factor of zinc in fodder grasses ranged from 0.07 to 1.55. Baran and Wieczorek [
55] obtained values for the factor ranging from 0.26 to 1.01 in monocotyledonous plants (on average 0.63) and from 0.41 to 0.89 in dicotyledonous plants (on average 0.83). In a study by Aladesanmi et al. [
56], the bioaccumulation factor of zinc in maize grown on soil contaminated with this metal ranged from 0.011 to 0.99. Klatka et al. [
29] found that the value of this index also depends on the species of plant. As the level of zinc application increased, its bioaccumulation factor in cocksfoot significantly decreased. This may indicate that plants have mechanisms counteracting excessive uptake of zinc. According to Emamverdian et al. [
57], a key element of these mechanisms is chelation of zinc through the formation of a metal complex of phytochelatins or metallothionein at the intra- and intercellular levels. This is followed by the removal of zinc ions from susceptible sites or sequestration of the ligand-zinc complex in the vacuoles.
Cattle manure showed a protective effect against accumulation of zinc, decreasing its bioaccumulation in the grass. This confirms the hypothesis that the organic matter contained in it binds the metal [
51].