4.1. Biomass Yield and S Uptake
Figure 1a shows relative yields increased in all fertilized treatments. Except for Cont, all fertilized treatments received uniformly 120 kg N ha
−1 year
−1 and produced comparable yields, while only two treatments received S fertilizer. Based on the fact, that all N fertilized treatments were statistically the same, we can deduce that S was not a limiting nutrient in our trials.
Relative sulfur uptake (
Figure 1b) showed more differences, as treatments amended with S fertilizer have the highest relative S uptake. Cont treatment provided uptake of 3.38 kg S ha
−1 year
−1. Meanwhile application of 142 kg S ha
−1 year
−1 in the form of AS resulted in uptake of 9.84 kg S ha
−1 year
−1, while application of UAN + MgS (84 kg S ha
−1 year
−1) increased S uptake to 8.31 kg S ha
−1 year
−1 (increase by 291% and 246% over Cont, respectively). A similar increase of S uptake was present in the study of Sakal et al. [
38], where a dose of 40 kg S ha
−1 increased S uptake of maize by up to 9.44 kg S ha
−1 over unfertilized treatment, which provided uptake of 2.58 kg S ha
−1 in trials with maize and wheat crop rotation. Overall, the increase in S uptake was also noted on treatments UAN and UAN + PK, providing increase of 192% and 180%, respectively. Weil and Mughogho [
30] presented similar results, as they also observed increase in S uptake on treatments fertilized by 80 kg of N ha
−1. Generally, the application of N increases yields and, thus, produces higher uptake of other nutrients [
39], including S [
40].
Table 4 describes these results in more detail. Interestingly, uptake on each treatment shows descending tendencies in periods 2002–2007, 2008–2013, and 2014–2019. During these periods, yields on individual treatments also show a significant decrease (not present in this study); therefore, reduced uptake can be attributed to reduced yields. The reduction of yields over time can be partially attributed to N dose. Our trials were previously investigated by Černý et al. [
41], where they focused on N uptake. They found that uptake of N can reach up to 185 kg N ha
−1, while yearly inputs are 120 kg N ha
−1, so the nitrogen dose might not be high enough to properly supply the crops. Other possible explanations can include a change of the crop hybrid (viz. materials and methods) and precipitation, which can cause mobilization of N in soil and possibly N leaching [
42,
43], as well as optimal growth of the crop itself and better nutrient availability [
44].
Given the results of correlation analysis (
Table 5), it seems that the S
ads fraction correlates best with total S content in plant biomass (r = 0.747;
p < 0.01), followed closely by S
av (r = 0.725;
p < 0.01) and S
W (r = 0.674;
p < 0.01). S
av is calculated as the sum of S
W and S
ads, thereby explaining the very similar coefficients of these fractions. This is generally in agreement with other works, stating that S
W, S
ads, and their sum Sav are very important fractions in plant nutrition [
17,
35] (as discussed later). S
M3 correlates only moderately with the content of S in biomass. Since Mehlich 3 includes HNO
3, it is possible that it extracts some part of the S
t content that is usually not available to plants. Relationship between the mentioned fractions and S biomass content may not represent the reality well enough in this study due to the small sample set size. Recently, Sedlář et al. [
45] correlated content of S in maize biomass with content of S in S
W and S
M3 fractions at Pearson´s correlation coefficient values of r = 0.961 and 0.804, respectively, at
p < 0.001 in a pot experiment. In field trials, correlation coefficients were measured at r = 0.174 (insignificant) and 0.629 (
p < 0.05) for S
W and S
M3, respectively. In their experiments, S biomass content was determined early during vegetation (BBCH 16–18), while in this study, maize was sampled quite a bit later (BBCH 75). Differences in sampling period can explain some of the differences, as content in S is quite variable in soil and plants across the vegetation season [
46]. In addition, Sedlář et al. [
45] collected maize from farmers scattered across the Czech Republic using both a crop rotation system and monoculture, bringing even more variability into the evaluation. On the other hand, this study focuses solely on maize from long-term monoculture on one station only.
4.2. Topsoil and Subsoil Sulfur Content
Results of two-way ANOVA (
Table 6) revealed significant interaction of year and treatment on content of S in all soil S fractions, with the exception of S
t. This was caused by a high content of S on AS treatment in the soil in the year 2019 (
Figure 2 and
Figure 3), which resulted in significant interaction of (AS treatment × year 2019 pair). Possible causes of such increase in content of S on this treatment are discussed later. These results would suggest that once inputs of S reach such levels, as with the inputs of S on AS treatment in our study (
Table 3), the S starts to cumulate in S
w, S
ads, S
av, and S
M3 fractions over time. UAN + PMgS treatment received S fertilizer as well. Interactions of (UAN + PMgS × any year pairs) were rarely significant and, in almost all cases, not different from pairs with (treatments without S fertilizer and any year). This suggests that the fertilizer rate on UAN + PMgS is not high enough to cause cumulation of S in topsoil.
Soil samples were collected after crop harvest. During crop growth, levels of S (especially S
av) would be high enough for sufficient crop growth in spring. Grobler et al. [
47] mention that maize is capable of producing sufficient yields with 10 mg S
av kg
−1 of soil. S
av in our trials (
Figure 3a) never reached lower values than 10 mg S
av kg
−1 even after harvest (on average around 44 kg S
av ha
−1), when S pools were already drained. Considering the results of S
M3 extraction (
Figure 4 in topsoil and
Figure 5d in subsoil), S
M3 extracted a very similar amount of sulfur to S
av. Kulhánek et al. [
13] investigated the relationship between S
av and S
M3 and found a very close Pearson correlation (r = 0.882;
p < 0.001). Using the Pearson correlation analyses, we also found a very close relationship of these two fractions in this study (r = 0.958 in 1997 and 0.990 in 2019, both at
p < 0.001). This high correlation can be caused by the fact that each treatment received the same amount of fertilizer each year for the entire duration of the trials, as well as all treatments of this study being located on the same soil type, while Kulhánek et al. [
13] investigated a wider variety of soil types with much more varied fertilizer inputs. A similar relationship was also reported in other studies [
48,
49]. As mentioned in the Results, no differences between S
av and S
M3 in topsoil and subsoil (Tukey;
p < 0.05) were found. This makes the Mehlich 3 method suitable for evaluating available S content in soil.
Kulhánek et al. [
50]—in Czech—developed a certified methodology for determination of plant available S using Mehlich 3 extractant. They proposed criteria (5 categories in total) for evaluation of plant available S using S
M3 content in soil. The results of our study place S
M3 content in topsoil in the “Satisfactory” category in 1997. Having said this, UAN and UAN + PK treatments have 20.7 and 20.4 mg S kg
−1, which technically puts them on the border of the “Low” and “Satisfactory” categories (ranges for “Low” and “Satisfactory” categories are 11–20 and 21–30 mg S kg
−1). On the other hand, in the year 2019 the S
M3 content is ranked in the “Low” category in all treatments, since the content of S
M3 has decreased in general with the exception of the AS treatment. In subsoil, there is no temporal comparison, since we only have results from 2019 (
Figure 5d). Using the categories proposed by Kulhánek et al. [
50], Cont and UAN are in the “Low” category, UAN + PK is ranked in the “Satisfactory” category, and UAN + PMgS and AS are ranked in the “High” content category.
Overall, S pools of different S fractions on all treatments in topsoil seem to be decreasing in time with the exception of the AS treatment (
Figure 2 and
Figure 3), which shows an increase in S
w, S
ads, and S
av fractions, while maintaining even levels of S
t.
It is obvious that (aside from AS) all treatments suffered a decrease of S pools over time (
Table 7). In 2019, the S
w remaining pool in the Cont, UAN, and UAN + PK treatments ranged from 23 to 33% of S
w determined in the samples of the year 1997. S
ads content on these treatments ranged 58–73% as compared to 1997 and in 2019. S
av content ranged from 31–43% of S
av in 1997. Lastly, S
t content for Cont, UAN, and UAN + PK treatments in our trials in 2019 reached 73–79% of those in 1997. This conforms to Balík et al. [
9], who investigated sulfur status in soils on 10 different sites in the Czech Republic. They found that S
w, S
ads, S
av, and S
t content decreased in time to 32, 61, 50, and 92%, respectively (comparing the year 1981 with 2007). Changes in mineral S fractions were evaluated by Kulhánek et al. [
19] in a long-term field experiment (comparing the year 1996 with 2014) with a potato, wheat, and barley crop rotation and several fertilizers. They describe a decreasing tendency of S
av content in the entire trial; however, S
w content increased following the application of sewage sludge after potatoes. In other crops, S
w always showed a decrease.
Indeed, the application of organic matter into soil can increase its water holding capacity [
51] and, therefore, temporarily increase the S
w fraction. In our experiment, no organic fertilizers were applied to the soil during the entire experiment. Changes of C
SOM in our monoculture trial were investigated by Balík et al. [
52]. Initial C
SOM levels decreased from 1.26% in 1993 to 0.98% in Cont and 0.93% in AS in topsoil in 2019. Subsoil C
SOM was reported at 0.73% in Cont and 0.65% in AS in 2019. This is caused by cultivation of maize for silage, since very little post-harvest residues are present (just stubble and roots), and C
SOM presents net loss. Considering the current content of C
SOM in Cont topsoil (0.98%) and subsoil (0.73%) [
52], it is possible to calculate their ratio at 1.34 as well as a ratio of current S
t levels in topsoil (157 mg S kg
−1–
Figure 3b) and subsoil (115 mg S kg
−1–
Figure 6), where we get the ratio 1.36. It seems that S
t content is bound in the same way in organic matter in topsoil and subsoil. After all, organic bound S can make up to 90% of S
t [
12]. Loss of the S
av fraction from 1997 to 2019 in topsoil (
Figure 3a) amounted roughly to 16.2–24.0 mg S kg
−1). Loss of S
t (
Figure 3b) ranged from 24 to 59 mg S kg
−1 in all treatments except AS. This suggests that loss of S from the organic fraction was present and partially contributed to loss in S
t. In arable systems where soil organic matter is not accumulating, there is little opportunity for inorganic SO
42− ions immobilization into organic matter [
53]. The S
t loss can probably be attributed to leaching of the SO
42− anion, which is discussed later.
Even the Fallow treatment shows interesting results. Content in all S fractions is highest in 1997, but lower in 2008. This treatment has no crop production, so the decrease in the period 1997–2008 can be attributed to leaching of S from atmospheric depositions. Unlike the other non-AS treatments, the decrease of S was not present from 2008 to 2019. The most likely cause is the decrease of atmospheric S depositions in recent years [
10]. These results are similar to those of Gourav et al. [
33], where a higher content of S
w and S
t was present on the Fallow treatment than on Cont after 48 years of experiment with maize monoculture. In our study, the Cont treatment of the S
av fraction amounted to 16.2, 8.5, and 6.9% of S
t in 1997, 2008, and 2019, respectively. S
av for the Fallow treatment made up 16.5, 10.1, and 9.4% in 1997, 2008, and 2019, respectively. UAN, UAN + PK, and UAN + PMgS behaved very similarly to Cont. It is clear, that treatments with maize production and S leaching had a higher influence over the decrease of S
av pools than that of Fallow, which is only influenced by leaching.
The AS treatment seems to show opposing results. The increase of S
ads in topsoil in this treatment (
Figure 2b) can be explained, e.g., by a decrease of pH that causes increased sorption of SO
42− [
54]. AS fertilization indeed decreases the pH of soil [
55]. This, however, does not explain the increase of S
w and S
t fractions. A possible explanation may be the combination of site and rainfall variability as well as annual application of 142 kg S per hectare (which can be considered as a very high dose), which could cause the accumulation of sulfur in this treatment over time. Influence of time was deemed significant earlier (
Table 6) and seems to support this thought.
The S content in all fractions (
Figure 5) in subsoil was increased in 2019 on UAN + PMgS and AS treatments over those without S fertilization. Knights et al. [
53] and Zayed et al. [
56] report that surplus S can be accumulated in subsoil, which could explain that the highest increase overall was present on the AS treatment that received the highest amounts of S inputs, followed by UAN + PMgS, which received the second highest inputs of S. This can also explain the increased content of S
t in 1997 on the AS treatment (
Figure 6). Even though we evaluate data from 1997, the experiment and fertilizer applications started in 1993, so AS was applied for several years before the first data are presented in this study. Leaching of S from previous years could have increased S content in the subsoil in 1997 on this treatment in subsoil.
S
w content in subsoil on AS and UAN + PMgS treatments had increased by 42.7 and 16.5 mg S kg
−1 over Cont (
Figure 5a). Since soil samples were taken after harvest, it is possible that soil was sampled during a downward movement of S, which was in surplus in topsoil. This could also explain the increase in S
ads (
Figure 5b). The S
av (
Figure 5c) content on Cont, UAN, and UAN + PK is on par; however, S
t content (
Figure 6) is somewhat lower for UAN and UAN + PK in comparison with Cont. It is possible that a higher uptake of S by plants in the UAN and UAN + PK treatments could cause mineralization and upward movement of S from the subsoil [
51]. Accumulation of total S over time is visible in the subsoil on Fallow in comparison with other non-S-fertilized treatments (
Figure 6). Since there is no crop production on Fallow, we assume that maize roots have influenced the other treatments. Indeed, maize roots can grow as deep as the subsoil layer (30–60 cm) [
57,
58] and could, therefore, cause a decrease in subsoil S content.
4.3. Sulfur Status and Movement in Topsoil and Subsoil
Values of S
t in the topsoil and subsoil (
Table 8) are calculated in kg S ha
−1. Therefore, temporal changes represent identical tendencies for the soil S content in
Figure 3b and
Figure 6.
From
Table 8, it is clear that all treatments except AS show a decrease in S
t content in the topsoil. Negative S balance can be attributed to crop growth and leaching [
24]. Cont is influenced by leaching and maize growth, while Fallow is only influenced by leaching; therefore, it is understandable that Fallow showed the lowest loss of S. A slightly positive balance is noticeable in AS. A possible explanation may be the combination of site and rainfall variability as well as annual application, as was mentioned above in the discussion of topsoil and subsoil sulfur content. In the subsoil Cont, Fallow, and UAN + PK treatments, we measured the increase in S content. It is possible that leached sulfates from the topsoil were absorbed or accumulated in organic matter.
Table 9 describes the movement of S in the topsoil and subsoil. The calculation presented in this table is based on a comparison of the original S status in soil and total S inputs during trials with the status of S at the end of the experiment with total plant uptake during the experiment. Using this method, we calculated that on all treatments in topsoil, an extensive S loss is present. In subsoil, the trend is the same, with the exception of the Fallow treatment that seems to have stable values. We consider the loss from subsoil in
Table 9 equal to the total S lost from the topsoil and subsoil profile (0–60 cm depth). The Fallow treatment registered the smallest loss of 11 kg S ha
−1, while Cont, UAN, and UAN + PK lost 308, 367, and 199 kg S ha
−1 during the entire trial, respectively. UAN + MgS and AS provided losses of 2193 and 3271 kg S ha
−1 during the entire trial or 95.3 and 142 kg S ha
−1 year
−1, respectively. This loss can certainly be attributed to leaching of SO
42− anions as described in Riley et al. [
24], where a three-year pot experiment with AS application and undisturbed soil sample was established with a focus on S leaching. Here, the control treatment received a total input of 22 kg S ha
–1 in terms of depositions, while the AS treatment received an additional 50 kg S ha
−1 in the form of AS fertilizer (in total 72 kg S ha
−1 was added). It was found that 78% of S applied on AS was leached in the first year and it increased to 96% after three years. When loss by leaching and plant uptake were added together (55 and 107 kg S ha
−1 in Cont and AS, respectively), the total S outputs in AS and even Cont treatments exceeded the total inputs at the end of the trial by 35 and 33 kg S ha
−1, respectively. The authors explain this by net S mineralization from organic bound S in soil. Using these data, we calculated a ratio (in%) of outputs (uptake and leaching) over inputs (deposition and fertilizer) and obtained 253% for Cont and 149% for AS. The same calculation was performed for Cont, AS, and UAN + PMgS treatments with the result of 221%, 102%, and 114%, respectively. Furthermore, S uptake (
Table 4) did not reach the levels of fertilizer inputs (
Table 3) and mobile sulfates were generally susceptible to percolation in AS and UAN + PMgS treatments. Leaching of sulfates was obviously present. Riley et al. [
24] measured the presence of organic bound S in leachates. It makes sense in the context of our study, since Balík et al. [
52] evaluated changes in organic bound S on the same trials as this study and found a decrease of organic matter content that reduces space for net SO
42− accumulation. This lack of S retention was also demonstrated in Rothamsted long-term experiments [
51], where applications of 52–220 kg S ha
−1 year
−1 over 150 years had not increased organic, nor inorganic, pools of S. In addition, we demonstrated (
Figure 3b) that S
t content in our trials is decreasing and organic bound S is, of course, part of this fraction [
17].
Bergholm and Majdi [
25] also report S leaching during a six-year experiment. However, their results differ from those of ours. Out of a total S input of 821 kg S ha
−1 in the form of depositions and AS fertilizer, up to 339 kg S ha
−1 was leached (41.2%) in comparison with Cont, where inputs were 133 kg S ha
−1 in the form of atmospheric S depositions, and leaching amounted to 79 kg S ha
−1 (59%). Accumulation in AS and Cont reached 317.1 and 8.1 kg S ha
−1, respectively. Calculation of outputs (leaching and accumulation) over inputs ratio was performed resulting in 60% and 65% for AS and Cont treatments, respectively. These results differ from those in our study or in Riley et al. [
24]. This difference is due to the fact, that the Bergholm and Majdi [
25] study was conducted on forest soil. Authors measured accumulation of S in the forest floor (that is very rich in organic matter) and also in mineral soil. On the other hand, Riley et al. [
24] as well as our study conducted an experiment on arable soil. They found leaching of organic bound S and a recent study by Balík et al. [
52] found a decrease of soil organic matter on our trials. There are other factors that can influence the movement of S in soil, like an increase in precipitation being capable of increasing movement of nutrients in soil [
26,
27]. Soil texture can have an influence on water retention and, therefore, leaching as well. The soil texture in our trials was Loam with 5.4% clay content. In Riley et al. [
24], the soil texture was Sandy loam of 0–40 cm depth with clay content around 7.5% and Loamy sand of 40–60 cm with clay content around 5.5%. In Bergholm and Majdi [
25], the soil texture was also Loamy sand with 4% clay. In the end, it is possible to state that the majority of the disparity can be attributed to vastly different conditions between forest and arable soil.
Comparing the results of our maize monoculture long-term field experiment with a pot experiment [
24] and forest trial [
25] is indeed difficult due to the very different conditions that apply to each of these respective environments. Even though these trials are so different, what they share in common is the fact that a significant part of sulfur fertilizers is leached and unavailable to plants and may even be contaminating ground waters [
59].
In general, to reduce the leaching of sulfates, it is recommended to use fertilizers with lower solubility, like elemental S, which is less susceptible to leaching [
24,
60], or performing the application in several smaller doses [
24]. However, Santoso et al. [
60] mention that fast growing crops like maize show significantly lower S uptake if fertilized by slowly soluble fertilizers, especially if applied in split doses.