Effect of long-term fertilisation of Calcisols on organic carbon sequestration in marine climate in Estonia

ABSTRACT The aim of this work was to investigate the effect of long-term (45-year) fertilization of the crop rotation on soil organic carbon concentration (SOC%) changes in different periods of the experiment and its trend under conditions of more northerly marine climate changes. In this work, the data of the long-term NPK fertilization experiment of sandy loam Calcisols located in Northern Estonia (longitude 58.584816, latitude 24.422128) have been used. The crop rotation was divided into six blocks: potato-spring barley-spring barley under sowing with forage grasses – 1y and 2y forage grasses-winter rye. Different combinations of mineral (N 40—180; P 18—54; K 50—105 kg ha−1) and organic (manure (FYM) 30 and 60 Mg ha−1) fertilizers were used in the study. Two periods were observed: the last 20y vegetation period was 10% warmer and 15% less rainy than the earlier period. Under conditions of climate warming, the yield of barley and potatoes increased the most, especially with manure application. SOC% increased from 2.01 to 2.04 under the influence of mineral fertilizers, and from 2.08 to 2.22 with mineral + FYM. The soil is sustainable if manure is used at least 5–10 Mg ha−1 per y−1.


Introduction
Calcareous soils are mostly found in the semi-arid tropics, subtropics, Mediterranean, etc., the conditions of which are not comparable to the conditions of our experimental area, which is affected by the cyclonic activity developing in the North Atlantic Ocean (Kask 1996).Soil organic carbon sequestration may be affected differently by the natural conditions of a geographic location.The Mediterranean, characterised mainly by a special distribution of temperature and precipitation.Annual precipitation decreases from the western Atlantic areas to the eastern region with a dry Mediterranean climate, with values ranging from 170 to 2000 mm y −1 .Muñoz-Rojas et al. ( 2012) identified a decrease in organic carbon in Calsisols arable soils in the Mediterranean region, Andalusia, -24% in the 51year period.Keel et al. (2019) found an average soil C loss of 0.29 Mg C ha −1 per year in eleven long-term field trials on Swiss arable and permanent grasslands involving mineral or organic fertilisation and tillage.Riley et al. (2022) research showed that in SE Norway, the general SOC mass in the soil after 28 years of cropping with arable and mixed dairy rotations decreased from 98 to 89 Mg ha −1 , which means a loss of 0.3% yr −1 of the initial SOC.It was found that the SOC mass was unchanged when the input was 4 Mg C ha −1 yr −1 , but decreased when the inputs were smaller.
Changes in soil OC stock are long-term, so long-term experiments are critical for assessing them.Soil quality is defined through a number of soil chemical, physical and biological properties and their interactions (Adhikari and Hartemink 2016).Among these, soil organic carbon (SOC) is considered to be one of the most relevant and universal indicators for assessing soil quality (Bünemann et al. 2018) and soil degradation (Lorenz et al. 2019).Long-term studies show that fertilisation of plants with composts or manure increases soil carbon storage, while the use of synthetic fertilisers leads to a decrease or the same level of soil carbon (Nafziger and Dunker 2011).Organic nitrogen sources support carbon sequestration in the soil because they feed the microbes responsible for carbon storage.Synthetic nitrogen sources, on the other hand, promote the dominance of bacteria, which quickly convert ammonia into nitrate, which is easily lost from the soil (Carey et al. 2016).Organic matter promotes the growth and reproduction of soil microflora and -fauna, transforming the soil into a living system (Meena et al. 2017).Soil structure, aeration, nutrient storage, water storage, plant health and productivity, microbial biomass and activity, and carbon sequestration are all associated with SOC (Comerford et al. 2013;Murphy 2015;Adhikari and Hartemink 2016).
The aim of this work is to improve the knowledge of the effect of long-term (45-y) fertilisation on soil organic carbon (SOC) sequestration in calcisols in northern maritime climates and to prevent problems related to global climate warming.

Study site and soil
Crop rotation and fertilisation trials were established (1965) to determine the needs for Estonian soil fertilisation.The current study is based on data from A. Piho (1973) (IUSS 2015).Crop rotation consisted of six blocks as follows: potato (Solanum tuberosum), spring barley (Hordeum vulgare), spring barley under sown with red clover (Trifolium pratense) + timothy (Phleum), first-year (1y) forage grasses, second-year (2y) forage grasses, and winter rye.The size of each trial plot is 56 m 2 (7.5 × 7.5 m).
Traditional agrotechnical measures were used, including soil plowing to a depth of 0.22 m and chemical plant protection measures.Mineral fertilisers were applied prior to sowing during soil tillage.Manure was applied by autumn plowing for the following year's potato crop.Cereal straw was returned to the soil by plowing.The applications of fertilisers, crop sowing (end of April), and harvesting was performed at the optimal time (August).
The study used 11 fertiliser treatments: four NPK (kg ha −1 ) fertilisers and two levels of manure (FYM, Mg ha −1 ), in four replicates.Throughout the experimental period, the same fertilisation scheme was used according to the culture.Rotation takes 3 years.The fertilisation of crops in the crop rotation and the doses of fertilisers are given in Table 1.
The following are the crop rotation test treatments: Soil sampling and analysis Soil samples (1 sample consisted of 15 partial samples) were collected from the upper soil layer (depth 0-0.2 m) during the study period (45y) 6 times in three repetitions, i.e 18 per variant of analysis, and in 2021, they were also taken from a depth of 0.2-0.4 m.The soil samples were air-dried and sieved to a thickness of 2 mm.The following chemical properties of the soil samples were determined in an accredited laboratory at the Estonian Agricultural Research Centre: Soil organic carbon concentration (SOC%) as determined by the sulfochromic method; soil nitrogen (N) as determined by ISO 11261.The SOC content was between 1.9 and 2.1% at the beginning of the study.Since changes in SOC content are known to occur very slowly and with small fluctuations, to clarify the influence of weather conditions on SOC%, the vegetation period of the experiment was divided into two, both periods were characterised by the average results of three SOC% determinations.The amount of organic carbon (OC) dissolved (DOC) in water was measured to estimate the labile fraction of soil organic matter.10 g of air-dry soil was shaken for 1 h with 30 ml of distilled water, then centrifuged and filtered through a 0.45 μm filter.The OC content dissolved in water was determined using a varioMAX elemental analyzer from the resulting solution.The following formula (Kauer et al. 2015) was used to calculate the proportion of OC dissolved in soil water to total SOC content (DOCp): where DOC is water-dissolved soil organic carbon (mg g -1 ) and SOC is total soil organic carbon (mg g -1 ).

Sampling and analysis of crop yields
Trial plot yield and dry matter determination, 32.5 m 2 of the crop was harvested, of which 1 kg was taken for analysis.

The weather conditions
Estonia is characterised by a transitional climate from maritime to continental.The average annual air temperature is 6.7°C, and the average precipitation is 696 mm.The average annual water evaporation is less than the precipitation.Temperatures and precipitation were measured during the vegetation period (Figure 1) using an automatic weather station near the experimental area.

Statistical analysis
The likelihood of a difference between treatments was found at a level of 95% confidence in the LSD 0.05 (LSD; least significant difference) test MS Excel.

Results
Looking at the SOC sequestration results of the first and second half of the 45-year test period (Figure 2), we see that the SOC% increased the most in the last period when mineral fertiliser and manure were used together, i.e. by 0.11-0.15percentage points.The dynamics of changes in the experimental period shows that compared to the results of the NPK + FYM variant and the unfertilised variant, SOC% increased by 10-13% in the first experimental period and by 18-21% in the second period, and by 3-8% and 8-13%, respectively, in the case of mineral fertilisers without manure.
The SOC content is influenced by the plants growing on the soil and their yield, as well as by the organic matter they leave in the soil.Table 2 shows the yield of crops in the crop rotation during the experimental period.Compared to the first period of the 2nd test period, the vegetation weather in the second period was 10% warmer and 15% drier (Figure 1).Under these conditions, in the second experimental period, spring barley yield increased by 10-44% and potato yield by 27-36% depending on fertilisation.At the same time, the yield of 2y forage grass in the decreased by 7-54%.
At the end of the experimental period, the effect of fertilisation on C/N, subsoil SOC concentration and DOC content was also explained (Table 3).SOC% increased consistently in both soil and subsoil when manure was applied.The test soil's C/N ratio was primarily 11-12:1.The percentage of water-soluble SOC in the examined soil was low, remaining at 1% in the plow layer.

Discussion
SOC content is known to increase rather slowly, which was also confirmed by the results of our long-term experiment (Figure 2).The results also revealed that the increase in SOC% was more intensive in the second half of the experiment, including unfertilised and low fertiliser (N60P22K50) SOC% decreased by 1%.At the same time, the grain harvest also increased (Table 2).Only the main crop was removed from the field, straw, stalks and roots remained in the soil, enriching the soil with carbon.The yield depends on  fertilisation, but the yield is even more affected by weather conditions.While fertilisation improved the yield of different crops in the same experiment by 1.3-2.6 times, weather conditions caused yield fluctuations of up to 6.4 times (Loide 2019).Comparing long-term weather data revealed that the last, 20-year vegetation period is 10% warmer and 15% drier than the previous period (Table 1).The resistance of crops to changes in weather conditions is a variable factor that greatly affects yield.The amount of organic matter remaining in the soil, including SOC, depends on the yield.Soil quality is critical to ecosystem long-term functionality, productivity, and resilience to current climate change.Despite its importance, soil is being lost and degraded worldwide at an enormous rate (Grill et al. 2021).In our experiment (Figure 2), carbon accumulation was positive when using fertilisers, but using only mineral fertilisers, the result is poor despite growing clover in the crop rotation.The purpose of growing fodder grass in the crop rotation is to use the surplus of nutrients from the previous crops in addition to fodder production.When humidity decreases, it is more difficult for plants to absorb nutrients from the soil and form crops.However, legumes have been found to retain soil organic carbon twice as efficiently as nitrogen fertiliser (Veloso et al. 2018).Minimum, critical levels of SOC between 5 and 25 g SOC kg −1 for crop production have been reported (Hijbeek et al. 2017).A critical range of 29.7 38.0 27.9 *-000unfertilised; NPK (N-Nitrogen, P-Phosphorus, K-Potassium) is the average of NPK fertilisation variants of N60P22K50, N90P22K50 and N120P33K75 kg ha −1 , because changes in soil organic carbon concentration -SOC% were marginal there; manure: FYM 30 Mg ha −1 and FYM 60 Mg ha −1 .
Table 3.The effect of fertilisation (NPK kg ha −1 and manure (FYM) Mg ha −1 ) on the content of stable and labile forms of organic carbon in sandy loam Calcisols in the 0-0.20 and 0.20-0.40m soil layer in Kuusiku.SOC between 10 and 20 g SOC kg −1 was previously reported in the review by Loveland and Webb (2003).
In our experiment (Figure 2), the critical limit of SOC%, 2%, was exceeded when 30 Mg ha −1 farmyard manure was used in the crop rotation in addition to mineral fertilisers (1 time in 6 years), which indicates the healthy state of the soil according to the study by Grill et al. (2021).Riley et al. (2022) studies show a decline in SOC in many countries, despite expectations to the contrary -SOC is expected to decline sharply in Europe between 1990 and 2080 due to temperature increase.Thus, with the change of weather conditions, securing the reserves of organic matter in the soil becomes more and more relevant.
Since changes in the total supply of SOC are longterm, it has been found that it is more practical to determine changes in different fractions of soil organic matter (Guimaraes et al. 2013).The most common fractions of soil organic matter are labile, water-soluble and inactive.The labile fraction of soil organic matter consists of easily degradable compounds, mineralises quickly and is not stable in the soil.In the experimental area of our study (Table 3), where farmyard manure was used, DOC accounted for only 1% of SOC, which is important for sustainable agriculture.The low DOC content is the result of using composted manure.Composted manure contains less of the more labile fraction (Gómez-Brandón et al. 2008).Thus, SOC is estimated to be mostly stable and permanent in our experiment when composted manure is used as needed in addition to mineral fertilisers.Soil organic matter with a larger and more stable fraction is more durable and environmentally friendly because it binds more C in a more stable form and is not sensitive to the effects of external conditions, such as global warming.However, despite its importance, according to Grill et al. (2021), soil is disappearing and degrading worldwide at an enormous rate, which must be attributed to climate warming.Soil temperature (Effects … 2013) has a major influence on microbial activity and thus the rate of decomposition.Microbial activity in soil at or below 0°C is very low or absent.Above 0°C, microbial activity increases with increasing temperature and the rate increases significantly at around 10°C.The optimal rate of microbial activity is between 25 and 35°C, although different microbial species have different optimal temperatures.As a result of increased soil temperature, large decreases in SOC have been predicted in Europe between 1990 and 2080 (Smith et al. 2005;Jones et al. 2009;Wiesmeier et al. 2016).Soil loss, and especially SOC loss, is one of the key environmental issues of this century, which together with climate change poses serious risks to ecosystem sustainability and food security in many regions of the world (Cherlet et al. 2018).Wiesmeier et al. (2016) results showed that C inputs must increase by 29% to maintain available SOC stocks in agricultural soils.
The microbial population requires food with a C/N ratio of approximately 15:1 to meet energy (C) and protein (N) needs, respectively, during the decomposition of organic material.Materials with a C/N ratio of about 15:1 would have no immediate effect on the amount of N available to plants (Qian and Schoena 2002).The soil C/N ratio (Table 3) in our experiment ranged from 11 to 13:1 in both the top and bottom soil layers, indicating that there is more nitrogen in the soil than microbes need for life, this free nitrogen can be used by plants.
Projected climate change may affect soil moisture and temperature regimes.At the ecosystem level, soil affects vegetation through its effects on water availability, element cycling, and soil temperature regime (Cheddadi et al. 2001).In our work, where the vegetation period of the last 20 years was 10% warmer and 15% less rainy compared to the previous period, the yield of crops (spring barley, potatoes) increased, especially when manure was used, except for forage hay, which need more water to form crops.At the same time, the SOC% increased minimally only with mineral fertilisers: from 2.01 to 2.04%, but with mineral + FYM from 2.08 to 2.22% under conditions of climate warming.The share of DOC from SOC in the studied soil was less than 1%, which suggests that the majority of the share of SOC is more stable, permanent and sustainable in the soil perspective, especially if manure is used together with mineral fertilisers at least 5-10 Mg ha −1 y −1 , which ensures of an optimal 2% SOC level in Calcisols in a northern marine climate under conditions of global climate warming.

Figure 2 .
Figure 2. Effect of long-term fertilisation (NPK kg ha −1 and manure (FYM) Mg ha −1 ) on soil organic carbon concentration, SOC%, in the NPK fertilisation experiment of the crop rotation; LSD95%level of 95% confidence in the LSD0.05 (LSD; least significant difference) test MS Excel.

Table 2 .
Overview and 1996yield changes in the long-term fertilisation experiment of the crop rotation in the years1976-1995and 1996-2021 on Calcisol loamy soil in Kuusiku.