Soil structure and soil organic matter in water-Stable aggregates under different application rates of biochar

ore, incorporating biochar into soils re- quires that other organic and mineral fertiliz- ers are artificially supplemented. As for agriculture sustainability, combin- ing biochar with a N fertilizer appears to be a promising practice offering a possibility of higher carbon sequestration rates. Since the interaction between biochar, mineral fertilizer and soil is a complex process, additional re- search is necessary. The objectives of this study were to (i) quantify the effects of biochar and biochar in combination with N fertilizer on the soil struc- ture parameters, the proportion of water-stable aggregates (WSA) and SOM in WSA, and (ii) evaluate the dynamic changes of proportion of WSA and SOM in aggregates in relation with doses of biochar and biochar with N fertilizer. 2. Material and Methods Description of study site The field experiments were conducted at the experimental site of the Slovak University of Agriculture Nitra, Dolna Malanta Nitra (48 o19′00″N; 18o09′00″E). The site has a tem- perate climate, with a mean annual air tem- perature of 9.8°C, and the mean annual pre- cipitation is 540 mm. The geological substra- tum consists of little bedrock materials such as biotite, quartz, diorite, triassic quartzites with phyllite horizonts, crinoid limestones and sandy limestone with high quantities of fine materials. The young Neogene deposits con- sist of various clays, loams and sand gravels on which loess was deposited during the Vietnam Journal of Earth Sciences, 40(2), 97-108 99 Pleistocene epoch. The soil at this site is clas- sified as Haplic Luvisol according to the Soil Taxonomy (WRB, 2014). The soil has 9.13 g kg -1 of soil organic carbon, pH is 5.71 and the texture is silt loam (sand: 15.2%, silt: 59.9% and clay: 24.9%). Experimental design and field management The soil had been cultivated for over 100 years classic conventional agriculture tech- niques before the experiment. The experiment was established in March 2014 and experi- mental field is shown in Figure 1. As is shown in Table 1 the experiment consisted of seven treatments. The study was set up in the field research station as a total of 21 plots each with an area of 24 m 2 (4 m × 6 m). Each set of seven plots was arranged in a row and treated as a replication, and the interval between neighboring replications was 0.5 m. To main- tain consistency, ploughing and mixing treat- ments were also performed in control plots where no biochar and N fertilizer were applied. A standard N fertilizer (Calc-Ammonium nitrate with dolomite, LAD 27) was used in this experiment. The doses of the level 1 were calculated on required aver- age crop production using balance method. The level 2 included additional 100% of N in the year 2014 and additional 50% of N in the year 2016. The biochar used in this study was acquired from Sonnenerde, Austria. The bio- char was produced from paper fiber sludge and grain husks (1:1 w/w). As declared by the manufacturer, the biochar was produced at a pyrolysis temperature of 550°C applied for 30 minutes in a Pyreg reactor. The pyrolysis product has particle sizes between 1 to 5 mm. On average, it contains 57 g kg -1 of Ca, 3.9 g kg -1 of Mg, 15 g kg -1 of K and 0.77 g kg -1 of Na. The total C content of the biochar sample is 53.1 %, while the total N content is 1.4 %, the C:N ratio is 37.9, the specific surface area (SSA) is 21.7 m 2 g -1 and the content of ash is 38.3 %. On average, the pH of the biochar is 8.8. The spring barley (Hordeum vulgare L.) and spring wheat (Triticum aestivum L.) were sown in 2014 and 2016, respectively. Figure 1. Field site location and an areal view of experimental plots Vladimir Simansky, et al./Vietnam Journal of Earth Sciences 40 (2018) 100 Table 1. The investigated treatments Treatment Description B0N0 no biochar, no N fertilization B10N0 biochar at rate of 10 t ha–1 B20N0 biochar at rate of 20 t ha–1 B10N1 biochar at rate of 10 t ha–1 with N: dose of N were, 40 and 100 kg N ha–1 in 2014 and 2016, respectively B20N1 biochar at rate of 20 t ha–1 with N: dose of N were, 40 and 100 kg N ha–1 in 2014 and 2016, respectively B10N2 biochar at rate of 10 t ha–1 with N: dose of N were, 80 and 150 kg N ha–1 in 2014 and 2016, respectively B20N2 biochar at rate of 20 t ha–1 with N: dose of N were, 80 and 150 kg N ha–1 in 2014 and 2016, respectively Sampling and measurements Soil samples were collected from the top- soil (0-20 cm) in all treatments. Sampling of soil was conducted monthly to cover the whole growing season of spring barley (sam- pling dates: 17 April, 15 May, 16 June, and 13 July in 2014) as well as in 2016 to cover the whole spring growing season of wheat (sam- pling dates: on 20 April, 17 May, 22 June, and 18 July). Thus, for the 2014 treatments, sam- pling was conducted at one, two, three and four months after biochar application, while for the 2016 treatments, sampling was con- ducted at 26, 27, 28 and 29 months after bio- char application. The soil samples were carefully taken us- ing a spade to avoid disruption of the soil ag- gregates. The samples were mixed to produce an average representative sample from each plot. Roots and large pieces of crop residues were removed. The collected soil samples were transported to the laboratory and large clods were gently broken up along natural fracture lines. The samples were air-dried at laboratory temperature 20 o C to obtain undis- turbed soil samples. We used the Baksheev method (Vadjunina and Korchagina, 1986) to determine the water-stable aggregates (WSA). The soil organic carbon (SOC) and the labile carbon (CL) were analyzed in all fraction sizes of the WSA (Loginow et al., 1987; Dziado- wiec and Gonet, 1999). The indexes of aggre- gate stability (Sw), mean weight diameters of aggregates for dry (MWDd) and wet sieving (MWDW), as well as vulnerability coefficient (Kv) were calculated according to following equations (1-4): claysilt sandWSA Sw    09.0 (1) where: Sw denotes aggregate stability and WSA is the content of water-stable aggregates (%).    n i iid wxMWD 1 (2) where: MWDd is the mean weight diameter of aggregates for dry sieving (mm), xi is the mean diameter of each size fraction (mm) and wi is the portion of the total sample weight within the corresponding size fraction, and n is the number of size fractions.    n i iW WSAxMWD 1 (3) where: MWDw is mean weight diameter of WSA (mm), xi is mean diameter of each size fraction (mm), and WSA is the portion of the total sample weight within the corresponding size fraction, and n is the number of size frac- tions. w d v MWD MWD K  (4) where: Kv is the vulnerability coefficient, MWDd is the mean weight diameter of aggre- Vietnam Journal of Earth Sciences, 40(2), 97-108 101 gates for dry sieving (mm), and MWDw is the mean weight diameter of WSA (mm). Statistics The data was analyzed by ANOVA tests using a software package Statgraphics Centu- rion XV.I (Statpoint Technologies, Inc., USA). Comparisons were made using the method of least significant differences (LSD) at the probability level P = 0.05. The Mann- Kendall test was used to evaluate the trends in the proportions of WSA and the contents of SOC and CL in the WSA. 3. Results and discussion Proportion of water-stable aggregates and soil structure parameters Parameters of soil structure such as MWDw, Kv, Sw, as well as WSAma and WSAmi as a result of biochar amendment are shown in Table 2. Our findings confirm the results of Atkinson et al. (2010) i.e. biochar exerted pos- itive effects on soil structure. However, the effects of biochar on soil structure largely de- pend on the properties of biochar that may vary considerably due to the variations in feedstock materials, pyrolysis conditions, etc. (Purakayastha et al. 2015; Heitkötter and Marschner 2015). In our case, the proportion of WSAma decreased in the following order: B20N2 > B10N0 > B20N1 > B20N0 > B10N1 > B0N0 > B10N2. The index of ag- gregate stability increased in the following or- der: B10N2 < B0N0 < B10N1 = B20N1 < B10N0 = B20N0 <B20N2. The one-way ANOVA test did not show any significant dif- ferences between the treatments in terms of Kv and MWDw (Table 2). Compared to the B0N0, only the B20N2 treatment significantly in- creased the proportion of WSAma and reduced the proportion of WSAmi. Furthermore, our results suggest that biochar did not enhance the formation of WSAmi, since the particle sizes of the biochar were within the range of 1 to 5 mm. These findings agree with those of Herath et al. (2013) who also observed that biochar applied after 295 days of incubation did not enhance the formation of micro- aggregates. Brodowski et al. (2006) stated that incorporation of biochar into soil contributes to the formation of micro-aggregates. Gener- ally, organic amendments added to soil are accompanied with an increase in microbially- produced polysaccharides (Angers et al., 1993), especially those from fungi (Tiessen and Stewart, 1988) which can increase the stability of aggregates and the content of WSAma (Herath et al., 2013; Soinne et al., 2014). In our study, a statistically significant effect on Sw was observed in the treatment with 20 t biochar ha -1 combined with 2 nd level of N fertilization. The reasons for a higher ag- gregate stability can be explained by the ap- plication of higher doses of biochar together with nitrogen. Fertilizer application generally improves soil aggregation (Munkholm et al., 2002). An improved nutrient management in- creases biomass and enhances the growth of roots and their activity (Abiven et al., 2015). The increased aggregate stability can be ex- plained by the enhanced root activity and the direct effect of biochar acting as a binding agent of soil particles (Brodowski et al., 2006). The higher root biomass through exu- dates and moving soil particles help aggregate formation (Bronick and Lal, 2005). The effects of various rates of biochar and biochar with various levels of N fertilizer on the individual size fractions of the WSAma are shown in Table 3. The B20N2 treatment showed a robust decrease (by 27%) in WSAma between 0.5 and 0.25 mm, but on the other hand, the content of WSAma with particle sizes between 3 and 2 mm increased by 56% com- pared to B0N0. Formation of soil aggregates is a function of biological activity and time, and it is unlikely to occur immediately upon biochar application (Herath et al., 2013). Vladimir Simansky, et al./Vietnam Journal of Earth Sciences 40 (2018) 102 Table 2. Parameters of soil structure (mean and standard deviation) Treatments %WSAma %WSAmi Sw Kv MWDd MWDW B0N0 72.0±6.78ab 28.6±6.78bc 0.82±0.08a 4.29±0.90ab 2.97±0.69a 0.72±0.21ab B10N0 75.6±9.70abc 24.4±9.70abc 0.88±0.11ab 3.33±0.62a 2.90±0.37a 0.90±0.24 b B20N0 75.4±10.5abc 24.6±10.5abc 0.88±0.12ab 3.99±1.92ab 2.85 ±0.13a 0.87±0.38ab B10N1 75.2±6.54abc 24.8±6.54abc 0.87±0.08ab 3.48±0.90a 3.07±0.43 a 0.94 ±0.28b B20N1 76.3±8.68bc 23.7±8.68ab 0.87±0.13ab 3.37±1.31a 2.73±0.43 a 0.90±0.31b B10N2 68.0±6.93a 32.0±6.93c 0.79±0.08a 4.75±1.68b 2.74 ±0.48a 0.62 ±0.17a B20N2 80.3±7.40c 19.7±7.40a 0.93±0.09b 3.05±0.69a 2.88±0.43 a 0.98±0.25b Different letters (a, b, c) between lines indicate that treatment means are significantly different at P<0.05 according to LSD test Table 3. Percentage contents of individual size fraction of water-stable macro-aggregates (mean and standard devia- tion) Treatments Individual size fractions of water-stable macro-aggregates in mm >5 5-3 3-2 2-1 1-0.5 0.5-0.25 B0N0 2.44±1.58ab 3.81±1.23ab 7.87±3.41ab 15.0±7.75ab 25.5±5.03a 17.5±4.60bc B10N0 3.62±1.22ab 5.91±2.10ab 11.0±4.23bc 17.4±7.35ab 22.5±3.52a 15.2±3.90ab B20N0 3.19±1.02ab 5.42±1.98ab 11.1±5.89bc 16.3±7.16ab 23.9±5.14a 15.5±4.87abc B10N1 4.70±1.30b 6.16±3.16ab 10.5±4.16abc 15.7±5.25ab 21.0±3.31a 17.2±5.65bc B20N1 4.10±1.13ab 4.50±1.35ab 10.6±4.35abc 19.3±7.65b 23.4±5.32a 14.3±3.12ab B10N2 2.06±0.93a 3.29±1.09a 6.34±3.29a 11.7±7.30a 24.9±4.95a 19.7±4.19c B20N2 3.421.23ab 6.66±2.39b 12.3±4.64c 21.8±7.13b 23.5±5.58a 12.7±2.80a Different letters (a, b, c) between lines indicate that treatment means are significantly different at P<0.05 according to LSD test The biochar in our experiments has rather coarse particle sizes with diameters ranging from 1 to 5 mm, which may pose limitations to the soil-microbe-biochar interactions. Fur- thermore, the conversion to WSAma with par- ticle sizes 0.5-0.25 mm might therefore be dif- ficult and can happen only after a certain amount of time. Applying biochar with no N fertilization at the rates of 10 and 20 t ha -1 did not affect the proportion of WSAma. A combi- nation of biochar applied at 10 t ha -1 with both levels of N fertilizer had no significant effect on the proportion of WSAma compared to the B0N0 treatment. The effect of N fertilizer on the WSAma was confirmed only in the case of the B10N2 treatment. The proportion of WSAma with particle sizes ranging from 3 to 2 mm decreased by 42%, and increased by 30% for the size fraction 0.5-0.25 mm compared to the B10N0 treatment. The Mann-Kendall test identified a significant trend in the WSA (Table 4). The proportion of WSAma with par- ticle diameters of 2 to 1 mm did not change during the growing season in 2014 and 2016. The content of WSAma with particle sizes between 1 and 0.5 mm decreased, whereas the content of WSAma with particle sizes above 5 mm increased during the investigated periods in all treatments except the B10N2 and the B20N2 treatments. The proportions of WSAma with particle sizes between 5 and 3 mm and between 3 and 2 mm increased in the B20N0, B10N1 and the B20N1 treatments over the growing seasons. Our findings show that sole biochar and biochar with the combination of N fertilizer do not explain the changes in the WSAma with particle sizes between 2 to 1 mm. The propor- tion of the WSAma with larger particle sizes increased over the investigated periods. On the contrary, the proportion of the WSA with small size fractions decreased during the growing periods. A stable trend was observed in the proportion of the WSA in both the B10N2 and B20N2 treatments. This means that biochar with a higher N fertilizer content may be responsible for the stabilized propor- tion of WSA (Table 4). Vietnam Journal of Earth Sciences, 40(2), 97-108 103 Table 4. Dynamics of individual size fraction of water-stable aggregates and soil organic carbon and labile carbon in water-stable aggregates during investigated period (Mann-Kendall test) Treatments Individual size fractions of water-stable aggregates in mm >5 5-3 3-2 2-1 1-0.5 0.5-0.25 <0.25 B0N0 increased increased increased stable/no trend decreased stable/no trend stable/no trend B10N0 increased stable/no trend decreased stable/no trend decreased stable/no trend stable/no trend B20N0 increased increased increased stable/no trend decreased stable/no trend decreased B10N1 increased increased increased stable/no trend decreased decreased stable/no trend B20N1 increased increased increased stable/no trend decreased stable/no trend stable/no trend B10N2 stable/no trend stable/no trend stable/no trend stable/no trend stable/no trend stable/no trend stable/no trend B20N2 stable/no trend stable/no trend stable/no trend stable/no trend stable/no trend stable/no trend stable/no trend Content of soil organic carbon in water-stable aggregates B0N0 stable/no trend decreased stable/no trend stable/no trend increased stable/no trend stable/no trend B10N0 decreased stable/no trend stable/no trend stable/no trend stable/no trend stable/no trend increased B20N0 stable/no trend stable/no trend stable/no trend stable/no trend stable/no trend stable/no trend stable/no trend B10N1 increased decreased stable/no trend stable/no trend stable/no trend increased increased B20N1 stable/no trend stable/no trend stable/no trend stable/no trend increased increased increased B10N2 stable/no trend stable/no trend stable/no trend stable/no trend stable/no trend stable/no trend stable/no trend B20N2 stable/no trend stable/no trend decreased stable/no trend stable/no trend stable/no trend stable/no trend Content of labile carbon in water-stable aggregates B0N0 increased increased increased increased increased increased increased B10N0 increased increased increased increased increased increased increased B20N0 increased increased increased increased increased increased increased B10N1 increased increased increased increased increased increased increased B20N1 increased increased increased increased increased increased increased B10N2 increased increased increased increased increased increased increased B20N2 increased increased increased increased increased increased increased Contents of soil organic matter in water- stable aggregates Organic amendments are known to in- crease the content of SOC (Agegnehu et al., 2016). Soil particles tend to form aggregates accompanying with occluded biochar (Brodowski et al., 2006). This could be the main reason of the elevated C content in the aggregates (Blanco-Canqui and Lal, 2004). Results of our study showed that different rates of biochar and biochar with different levels of N fertilization affected the distribu- tion of SOC and CL content in aggregates (Figure 2 and 3), ranging from 8.80 to 15.8 g Vladimir Simansky, et al./Vietnam Journal of Earth Sciences 40 (2018) 104 kg -1 and from 1.11 to 1.65 g kg -1 for biochar treatments, and from 9.70 to 15.6 g kg -1 and from 0.99 to 1.81 g kg -1 for biochar with N fertilization treatments. In all treatments, the content of SOC in WSAmi was lower than WSAma. The SOC in WSAmi were 10.5, 8.80, 10.6, 9.70, 11.1, 10.4 and 11.5 g kg -1 of SOC in the B0N0, B10N0, B20N0, B10N1, B20N1, B10N2 and B20N2 treatments, re- spectively. The largest size class of WSA (> 5 mm) contained the largest CL in all treatments, with 1.54, 1.54, 1.65, 1.57, 1.59, 1.66 and 1.81 g kg -1 of CL in the B0N0, B10N0, B20N0, B10N1, B20N1, B10N2 and B20N2 treatments, respectively, while the smallest size class of WSA (< 0.25 mm or WSAmi) contained the lowest CL pool in all treatments (Figure 3). Figure 2. Contents of soil organic carbon in individual size fractions of water-stable aggregates (mean and standard deviation); Different letters (a, b, c, d) between columns (the same color) indicate that treatment means are signifi- cantly different at P<0.05 according to LSD test Generally, the higher content of SOC is ac- companied with a higher occurrence of WSAma and WSAmi. The importance of SOC content in the formation of aggregates is well known (Kodesova et al., 2015). In the study of Liu and Zhou (2017), macro- and micro-aggregation was significantly improved by using organic amendments. The large aggregates contained the largest pool of C in manure treatments (Simansky, 2013). Tisdall and Oades (1980) and Six et al. (2004) found higher concentra- tions of organic C in macro-aggregates than in micro-aggregates. Decomposition of roots and hyphae occurs within macro-aggregates. Elliott (1986) suggested that macro-aggregates have elevated C concentrations because of the or- ganic matter binding micro-aggregates into macro-aggregates and the organic matter is “qualitatively more labile and less highly pro- cessed” than the organics stabilizing micro- aggregates. Based on Mann-Kendall test, the temporal behavior of SOC in WSA in relation to application of biochar or biochar with N fer- tilizer was different during the investigated pe- riod (Table 4). A considerable decrease in SOC with WSAma >5 mm and an increase in SOC with WSAmi when 10 t ha -1 of biochar was ap- plied. During the investigated period, the appli- cation of 20 t ha -1 of biochar as well as 10 and 20 t ha -1 of biochar combined with the second level of N fertilization had no effect on the re- distribution of SOC in WSA. The SOC in 0 5 10 15 20 25 B0N0 B10N0 B20N0 B10N1 B20N1 B10N2 B20N2c o n te n t o f s o il o rg a n ic c a rb o n (g k g -1 ) treatments >5 5-3 3-2 2-1 1-0.5 0.5-0.25 <0.25 a a a b a bc b b a a a a a a a b c b b cd b a a a a a a b b b b b c c c a b c b b b b a b b b b d d Vietnam Journal of Earth Sciences, 40(2), 97-108 105 WSAmi gradually increased after applying bio- char combined with the first level of N fertili- zation during the investigated period. CL in WSA significantly increased in all size frac- tions of WSA and in all treatments (Table 4) during the investigated period. The dynamic of CL changes significantly due to different soil management practices (Benbi et al., 2012). Therefore, the CL is used as a sensitive indica- tor of changes in SOM (Benbi et al., 2015) and aggregate stability (Simansky, 2013). As a re- sult, the decomposition of the organic matter increases CL, eventually enhancing aggregation (Bronick and Lal, 2005). Figure 3. Contents of labile carbon in individual size fractions of water-stable aggregates (mean and standard devia- tion); Different letters (a, b, c, d) between columns (the same color) indicate that treatment means are significantly different at P<0.05 according to LSD test 4. Conclusions Elevated doses of biochar with a higher level of N fertilizer application significantly increased the index of aggregate stability and the proportion of water-stable macro- aggregates, especially in the size fractions from 3 to 2 mm. On the other hand, less wa- ter-stable macro-aggregates within the frac- tion from 0.5 to 0.25 mm were observed. Ap- plication of N fertilizer at a higher level sig- nificantly decreased the proportions of water- stable macro-aggregates within the size frac- tions of 3-2 mm. On the contrary, increasing rates of N application increased the proportion of water-stable aggregates with sizes from 0.5 to 0.25 mm. During the investigated period, the proportion of larger macro-aggregates in- creased, while the proportion of smaller mac- ro-aggregates 1-0.5 mm decreased. Our findings show that the effect of SOM in the WSA can be significantly enhanced. Dosing biochar at higher rates resulted in a higher content of soil organic carbon and la- bile carbon in the WSA. It can be concluded that the higher content of SOM delivered through biochar led to more WSAma and WSAmi. The temporal dynamics of CL in WSA is more pronounced than in SOC. The content of CL measured within all size frac- tions of the WSA increased in all treatments. Generally, the higher content of SOC is accompanied with a higher occurrence of WSAma and WSAmi. The importance of SOC content in the formation of aggregates is well known (Kodesova et al., 2015). In the study of Liu and Zhou (2017), macro- and micro-aggregation was significantly improved by using organic amendments. The large aggregates contained the largest pool of C in manure treatments (Simansky, 2013). Tisdall and Oades (1980) and Six et al. (2004) found higher concentrations of organic C in macro-aggregates than in micro-aggregates. Decomposition of roots and hyphae occurs within macro-aggregates. Elliott (1986) suggested that macro-aggregates have elevated C concentrations because of the organic matter binding micro-aggregates into macro-aggregates and the organic matter is “qualitatively more labile and less highly processed” than the organics stabilizing micro-aggregates. Based on Mann-Kendall test, the temporal behavior of SOC in WSA in relation to application of biochar or biochar with N fertilizer was different during the investigated period (Table 4). A considerable decrease in SOC with WSAma >5 mm and an increase in SOC with WSAmi when 10 t ha -1 of biochar was applied. During the investigated period, the application of 20 t ha-1 of biochar as well as 10 and 20 t ha-1 of biochar combined with the second level of N fertilization had no effect on the re-distribution of SOC in WSA. The SOC in WSAmi gradually increased after applying biochar combined with the first level of N fertilization during the investigated period. CL in WSA significantly increased in all size fractions of WSA and in all treatments (Table 4) during the investigated period. The dynamic of CL changes significantly due to different soil management practices (Benbi et al., 2012). Therefore, the CL is used as a sensitive indicator of changes in SOM (Benbi et al. 2015) and aggregate stability (Simansky, 2013). As a result, the decomposition of the organic matter increases CL, eventually enhancing aggregation (Bronick and Lal, 2005). Generally, the higher content of SOC is accompanied with a higher occurrence of WSAma and WSAmi. The importance of SOC content in the formation of aggregates is well known (Kodesova et al., 2015). In the study of Liu and Zhou (2017), macro- and micro-aggregation was significantly improved by using organic amendments. The large aggregates contained the largest pool of C in manure treatments (Simansky, 2013). Tisdall and Oades (1980) and Six et al. (2004) found higher concentrations of organic C in macro-aggregates than in micro-aggregates. Decomposition of roots and hyphae occurs within macro-aggregates. Elliott (1986) suggested that macro-aggregates have elevated C concentrations because of the organic matter binding micro-aggregates into macro-aggregates and the organic matter is “qualitatively more labile and less highly processed” than the organics stabilizing micro-aggregates. Based on Mann-Kendall test, the temporal behavior of SOC in WSA in relation to application of biochar or biochar with N fertilizer was different during the investigated period (Table 4). A considerable decrease in SOC with WSAma >5 mm and an increase in SOC with WSAmi when 10 t ha -1 of biochar was applied. During the investigated period, the application of 20 t ha-1 of biochar as well as 10 and 20 t ha-1 of biochar combined with the second level of N fertilization had no effect on the re-distribution of SOC in WSA. The SOC in WSAmi gradually increased after applying biochar combined with the first level of N fertilization during the investigated period. CL in WSA significantly increased in all size fractions of WSA and in all treatments (Table 4) during the investigated period. The dynamic of CL changes significantly due to different soil management practices (Benbi et al., 2012). Therefore, the CL is used as a sensitive indicator of changes in SOM (Benbi et al. 2015) and aggregate stability (Simansky, 2013). As a result, the decomposition of the organic matter increases CL, eventually enhancing aggregation (Bronick and Lal, 2005). Generally, the higher content of SOC is accompanied with a higher occurrence of WSAma and WSAmi. The importance of SOC content in the formation of aggregates is well known (Kodesova et al., 2015). In the study of Liu and Zhou (2017), macro- and micro-aggregation was significantly improved by using organic amendments. The large aggregates contained the largest pool of C in manure treatments (Simansky, 2013). Tisdall and Oades (1980) and Six et al. (2004) found higher concentrations of organic C in macro-aggregates than in micro-aggregates. Decomposition of roots and hyphae occurs within macro-aggregates. Elliott (1986) suggested that macro-aggregates have elevated C concentrations because of the organic matter binding micro-aggregates into macro-aggregates and the organic matter is “qualitatively more labile and less highly processed” than the organics stabilizing micro-aggregates. Based on Mann-Kendall test, the temporal behavior of SOC in WSA in relation to application of biochar or biochar with N fertilizer was different during the investigated period (Table 4). A considerable decrease in SOC with WSAma >5 mm and an increase in SOC with WSAmi when 10 t ha -1 of biochar was applied. During the investigated period, the application of 20 t ha-1 of biochar as well as 10 and 20 t ha-1 of biochar combined with the second level of N fertilization had no effect on the re-distribution of SOC in WSA. The SOC in WSAmi gradually increased after applying biochar combined with the first level of N fertilization during the investigated period. CL in WSA significantly increased in all size fractions of WSA and in all treatments (Table 4) during the investigated period. The dynamic of CL changes significantly due to different soil management practices (Benbi et al., 2012). Therefore, the CL is used as a sensitive indicator of changes in SOM (Benbi et al. 2015) and aggregate stability (Simansky, 2013). As a result, the decomposition of the organic matter increases CL, eventually enhancing aggregation (Bronick and Lal, 2005). Generally, the higher content of SOC is accompanied with a higher occurrence of WSAma and WSAmi. The importance of SOC content in the formation of aggregates is well known (Kodesova et al., 2015). In the study of Liu and Zhou (2017), macro- and micro-aggregation was significantly improved by using organic amendments. The large aggregates contained the largest pool of C in manure treatments (Simansky, 2013). Tisdall and Oades (1980) and Six et al. (2004) found higher concentrations of organic C in macro-aggregates than in micro-aggregates. Decomposition of roots and hyphae occurs within macro-aggregates. Elliott (1986) suggested that macro-aggregates have elevated C concentrations because of the organic matter binding micro-aggregates into macro-aggregates and the organic matter is “qualitatively more labile and less highly processed” than the organics stabilizing micro-aggregates. Based on Mann-Kendall test, the temporal behavior of SOC in WSA in relation to application of biochar or biochar with N fertilizer was different during the investigated period (Table 4). A considerable decrease in SOC with WSAma >5 mm and an increase in SOC with WSAmi when 10 t ha -1 of biochar was applied. During the investigated period, the application of 20 t ha-1 of biochar as well as 10 and 20 t ha-1 of biochar combined with the second level of N fertilization had no effect on the re-distribution of SOC in WSA. The SOC in WSAmi gradually increased after applying biochar combined with the first level of N fertilization during the investigated period. CL in WSA significantly increased in all size fractions of WSA and in all treatments (Table 4) during the investigated period. The dynamic of CL changes significantly due to different soil management practices (Benbi et al., 2012). Therefore, the CL is used as a sensitive indicator of changes in SOM (Benbi et al. 2015) and aggregate stability (Simansky, 2013). As a result, the decomposition of the organic matter increases CL, eventually enhancing aggregation (Bronick and Lal, 2005). Generally, the higher content of SOC is accompanied with a higher occurrence of WSAma and WSAmi. The importance of SOC content in the formation of aggregates is well known (Kodesova et al., 2015). In the study of Liu and Zhou (2017), macro- and micro-aggregation was significantly improved by using organic amendments. The large aggregates contained the largest pool of C in manure treatments (Simansky, 2013). Tisdall and Oades (1980) and Six et al. (2004) found higher concentrations of organic C in macro-aggregates than in micro-aggregates. Decomposition of roots and hyphae occurs within macro-aggregates. Elliott (1986) suggested that macro-aggregates have elevated C concentrations because of the organic matter binding micro-aggregates into macro-aggregates and the organic matter is “qualitatively more labile and less highly processed” than the organics stabilizing micro-aggregates. Based on Mann-Kendall test, the temporal behavior of SOC in WSA in relation to application of biochar or biochar with N fertilizer was different during the investigated period (Table 4). A considerable decrease in SOC with WSAma >5 mm and an increase in SOC with WSAmi when 10 t ha -1 of biochar was applied. During the investigated period, the application of 20 t ha-1 of biochar as well as 10 and 20 t ha-1 of biochar combined with the second level of N fertilization had no effect on the re-distribution of SOC in WSA. The SOC in WSAmi gradually increased after applying biochar combined with the first level of N fertilization during the investigated period. CL in WSA significantly increased in all size fractions of WSA and in all treatments (Table 4) during the investigated period. The dynamic of CL changes significantly due to different soil management practices (Benbi et al., 2012). Therefore, the CL is used as a sensitive indicator of changes in SOM (Benbi et al. 2015) and aggregate stability (Simansky, 2013). As a result, the decomposition of the organic matter increases CL, eventually enhancing aggregation (Bronick and Lal, 2005). Generally, the higher content of SOC is accompanied with a higher occurrence of WSAma and WSAmi. The importance of SOC content in the formation of aggregates is well known (Kodesova et al., 2015). In the study of Liu and Zhou (2017), macro- and micro-aggregation was significantly improved by using organic amendments. The large aggregates contained the largest pool of C in manure treatments (Simansky, 2013). Tisdall and Oades (1980) and Six et al. (2004) found higher concentrations of organic C in macro-aggregates than in micro-aggregates. Decomposition of roots and hyphae occurs within macro-aggregates. Elliott (1986) suggested that macro-aggregates have elevated C concentrations because of the organic matter binding micro-aggregates into macro-aggregates and the organic matter is “qualitatively more labile and less highly processed” than the organics stabilizing micro-aggregates. Based on Mann-Kendall test, the temporal behavior of SOC in WSA in relation to application of biochar or biochar with N fertilizer was different during the investigated period (Table 4). A considerable decrease in SOC with WSAma >5 mm and an increase in SOC with WSAmi when 10 t ha -1 of biochar was applied. During the investigated period, the application of 20 t ha-1 of biochar as well as 10 and 20 t ha-1 of biochar combined with the second level of N fertilization had no effect on the re-distribution of SOC in WSA. The SOC in WSAmi gradually increased after applying biochar combined with the first level of N fertilization during the investigated period. CL in WSA significantly increased in all size fractions of WSA and in all treatments (Table 4) during the investigated period. The dynamic of CL changes significantly due to different soil management practices (Benbi et al., 2012). Therefore, the CL is used as a sensitive indicator of changes in SOM (Benbi et al. 2015) and aggregate stability (Simansky, 2013). As a result, the decomposition of the organic matter increases CL, eventually enhancing aggregation (Bronick and Lal, 2005). Generally, the higher content of SOC is accompanied with a higher occurrence of WSAma and WSAmi. The importance of SOC content in the formation of aggregates is well known (Kodesova et al., 2015). In the study of Liu and Zhou (2017), macro- and micro-aggregation was significantly improved by using organic amendments. The large aggregates contained the largest pool of C in manure treatments (Simansky, 2013). Tisdall and Oades (1980) and Six et al. (2004) found higher concentrations of organic C in macro-aggregates than in micro-aggregates. Decomposition of roots and hyphae occurs within macro-aggregates. Elliott (1986) suggested that macro-aggregates have elevated C concentrations because of the organic matter binding micro-aggregates into macro-aggregates and the organic matter is “qualitatively more labile and less highly processed” than the organics stabilizing micro-aggregates. Based on Mann-Kendall test, the temporal behavior of SOC in WSA in relation to application of biochar or biochar with N fertilizer was different during the investigated period (Table 4). A considerable decrease in SOC with WSAma >5 mm and an increase in SOC with WSAmi when 10 t ha -1 of biochar was applied. During the investigated period, the application of 20 t ha-1 of biochar as well as 10 and 20 t ha-1 of biochar combined with the second level of N fertilization had no effect on the re-distribution of SOC in WSA. The SOC in WSAmi gradually increased after applying biochar combined with the first level of N fertilization during the investigated period. CL in WSA significantly increased in all size fractions of WSA and in all treatments (Table 4) during the investigated period. The dynamic of CL changes significantly due to different soil management practices (Benbi et al., 2012). Therefore, the CL is used as a sensitive indicator of changes in SOM (Benbi et al. 2015) and aggregate stability (Simansky, 2013). As a result, the decomposition of the organic matter increases CL, eventually enhancing aggregation (Bronick and Lal, 2005). 0 0.5 1 1.5 2 2.5 B0N0 B10N0 B20N0 B10N1 B20N1 B10N2 B20N2 c o n te n t o f la b ile c a rb o n ( g k g -1 ) treatments >5 5-3 3-2 Generally, the higher content of SOC is accompanied with a higher occurrence of WSAma and WSAmi. The importance of SOC content in the formation of aggregates is well known (Kodesova et al., 2015). In the study of Liu and Zhou (2017), macro- and micro-aggregation was significantly improved by using organic amendments. The large aggregates contained the largest pool of C in manure treatments (Simansky, 2013). Tisdall and Oades (1980) and Six et al. (2004) found higher concentrations of organic C in macro-aggregates than in micro-aggregates. Decomposition of roots and hyphae occurs within macro-aggregates. Elliott (1986) suggested that macro-aggregates have elevated C concentrations because of the organic matter binding micro-aggregates into macro-aggregates and the organic matter is “qualitatively more labile and less highly processed” than the organics stabilizing micro-aggregates. Based on Mann-Kendall test, the temporal behavior of SOC in WSA in relation to application of biochar or biochar with N fertilizer was different during the investigated period (Table 4). A considerable decrease in SOC with WSAma >5 mm and an increase in SOC with WSAmi when 10 t ha-1 of biochar was applied. During the investigated period, the application of 20 t ha-1 of biochar as well as 10 and 20 t ha-1 of biochar combined with the second level of N fertilization had no effect on the re-distribution of SOC in WSA. The SOC in WSAmi gradually increased after applying biochar combined with the first level of N fertilization during the investigated period. CL in WSA significantly increased in all size fractions of WSA and in all treatments (Table 4) during the investigated period. The dynamic of CL changes significantly due to different soil management practices (Benbi et al., 2012). Therefore, the CL is used as a sensitive indicator of changes in SOM (Benbi et al. 2015) and aggregate stability (Simansky, 2013). As a result, the decomposition of the organic matter increases CL, eventually enhancing aggregation (Bronick and Lal, 2005). 2-1 1-0.5 0.5-0.25 <0.25 a a a ab a a a a a a a a a a a a a a a a a a a a a a a ab a a b a a a c a a a a a a a a a b b b b b Vladimir Simansky, et al./Vietnam Journal of Earth Sciences 40 (2018) 106 Water-stable aggregates are a significant pool of SOM. The rising content of CL during decomposition of biochar enhances the aggre- gation processes. Our findings confirmed the fact that biochar is responsible for carbon se- questration within the WSA. Acknowledgements This study was partially supported by the Slovak Research and Development Agency under the project No. APVV-15-0160, and the Scientific Grant Agency (VEGA) - project No. 1/0604/16 and 1/0136/17. References Abiven S., Hund A., Martinsen V., Cornelissen G., 2015. Biochar amendment increases maize root sur- face areas and branching: a shovelomics study in Zambia. Plant Soil, 342, 1-11. 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