VARCLIREFOR Technical document

1 VARCLIREFOR FORWARDS project Grant Agreement G-04-2023-3 The Project under this Agreement is funded under Grant Agreement 101084481 — FORWARDS — HORIZON-CL6-2022-CLIMATE-01 Deliverable 1.2: VARCLIREFOR Final technical report René Verburg, Marijke van Kuijk, Robin Aanstoot (Utrecht University) 2 Preface This Deliverable 1.2 describes the methods applied in the VARCLIREFOR project, funded within the FORWARDS consortium. The VARCLIREFOR project was conducted between May 2024 (M1) and August 2025 (M16) in the Utrechtse Heuvelrug area, which is a ca. 20,000 ha large forest area found in the central part of the Netherlands. The forests on the Utrechtse Heuvelrug are dominated by P. sylvestris, but local codominance of other species occur like P. menziesii, B. pendula, Q. robur and F. sylvatica. Most forest is located on former sandy (glacial) moraines, and soils are highly affected by continuous nitrogen deposition from traffic, industry and agriculture, soil acidification and increasing drought periods due to climate change. In most areas, only deep ground water levels are found, indicating most of the forest is solely rain fed and phreatic water stored in the upper soil layers are the most important water sources for vegetation growth. The VARCLIREFOR project studied survival and growth of naturally regenerated and planted tree saplings in new field trials in one experimental site on the Utrechtse Heuvelrug area. Furthermore, patterns in soil factors in existing forest plots within the larger forest area where studied. New trials (new forest and sapling plots) where located in one of the Forest Management Units (FMU) of the Dutch State Forestry Service (FMU unit ‘AUS’), while the existing trials ca. 781 forest plots) where located in three separate FMUs (‘AUS’, ‘AML’ and ‘VUU’). Data collected during the execution of VARCLIREFOR are stored in datafiles and uploaded to the Forest Observatory of FORWARDS. The authors 3 Table of contents Preface ........................................................................................................................................ 2 1. Introduction ............................................................................................................................. 4 2.Plot setup and sapling planting in WP2........................................................................................ 6 2.1 Existing plots within the WP2 working area ........................................................................... 6 2.2 Additional semi-permanent plots ......................................................................................... 6 2.2.1 Additional plot level data .................................................................................................. 7 2.2.2 Tree level data of alive and standing dead trees ................................................................. 9 2.2.3 Lying dead wood ............................................................................................................ 10 2.2.4 Natural regeneration ...................................................................................................... 11 2.3 Sapling planting ................................................................................................................. 11 2.3.1 Sapling treatment ........................................................................................................... 13 2.3.2 Sapling measurements .................................................................................................... 14 2.4 Natural recruitment measurements ................................................................................... 15 3. Soil sampling and field measurements ..................................................................................... 16 3.1 Soil sampling in WP2 ......................................................................................................... 16 3.2 Soil sampling in WP3 ......................................................................................................... 16 3.3 Litter collection and processing in WP3............................................................................... 16 3.4 Light availability ................................................................................................................ 16 3.5 Decomposition measurements ........................................................................................... 17 3.5.1 Tea Bag Preparation, deployment, and processing ........................................................ 17 3.5.2 Decomposition time series .......................................................................................... 18 3.5.3 Tea Bag Index parameter calculations .......................................................................... 20 3.6 Soil fauna feeding activity .................................................................................................. 20 4. Soil and nutrient processing..................................................................................................... 23 4.1 Soil Analyses ..................................................................................................................... 23 4.1.1 Soil pH........................................................................................................................ 23 4.1.2 Anorganic nitrogen: NOx, NO3, NO2, NH4, PO4................................................................ 24 4.1.3 Cation concentrations ................................................................................................. 24 4.1.4 Soil organic carbon and C:N ratio ................................................................................. 24 4.1.5 Soil Organic Matter Content ........................................................................................ 25 5. WP 3 Forest plot data, processing and calculations ................................................................... 26 5.1. Plot layout existing field trials (WP3).................................................................................. 26 5.2 Tree growth measurements ............................................................................................... 26 5.3 Allometric relations ........................................................................................................... 27 4 1. Introduction The VARCLIREFOR project consists of two work packages, each with its unique aim and methodologies. This deliverable describes the applied methods (field and laboratory techniques) carried out in the work packages 2 and 3. All data were collected in the National Park Utrechtse Heuvelrug in three forest management units: Vuursche (VUU), Austerlitz (AUS) and AmerongenLeersum (AML). WP2 Work package 2 focused on newly established field trials in the forest management unit of Austerlitz, designed to evaluate tree species that may be better adapted to the increasingly dry conditions of the Utrechtse Heuvelrug. In these experimental plots, saplings of various species were planted in a controlled setup. Among these are so-called “rich litter species” — species known to produce nutrient-rich litter that may contribute to the development of a more favorable organic layer in the soil. This layer plays a key role in supporting soil biodiversity and improving nutrient cycling, potentially enhancing soil quality and ecosystem resilience under changing climatic conditions. The experimental setup consisted of the addition of a combination of nutrients and mycorrhiza in the planting holes of planted saplings to support the growing conditions. To better understand the growing conditions for the planted saplings, a range of soil and environmental parameters were measured within the plots. These include canopy cover, soil organic matter content, pH, soil organic carbon and CN ratio, NH4-N, NO3-N, NO2-N and NOx-N and several cations. Additionally, soil decomposition activity was assessed using the Tea Bag Index (TBI), which provides insight into microbial activity and organic matter breakdown in the topsoil. Alongside the planted saplings, the natural regeneration of tree species was monitored over a period of four months, with a focus on height growth and survival. The working area of WP2 also contained a range of existing plots that offer historical data on tree diameter at breast height (DBH) and historical growth. A subset of these plots also contain data on dead trees, lying dead wood and rejuvenation. WP3 Work Package 3 focuses on tree growth-, soiland environmental data from three forest management units on the Utrechtse Heuvelrug: Vuursche, Austerlitz, and Amerongen-Leersum. As in WP2, a comprehensive set of parameters was collected to characterize site conditions, in a random set of 150 plots. Sampling in these plots included litter biomass, canopy cover, soil organic matter content (SOM), pH, soil organic carbon and C/N ratio, concentrations of NH4-N, NO3-N, NO2-N and NOx-N and in a sub-set of these selected plots, various cation concentrations. In WP3, bait lamina strips were used in addition to the Tea Bag Index (TBI) method to measure decomposition rates, providing information on the feeding activity of soil invertebrates. These measurements provide a baseline for understanding how local abiotic factors influence forest dynamics and soil processes across different forest stands. The field measurements are complemented by long-term plot data on tree growth measurements derived from ca 800 plots in these three forest management units. These data included diameter at breast height (DBH) measurements recorded in three different years that covered a time span of roughly 30 years, and tree height measurements of a selection of trees in these plots. This dataset allows for the analysis of growth trends over time in relation to environmental variables. In addition, 5 historical meteorological data were integrated to help interpret how long-term climatic conditions may have influenced forest development and site productivity. 6 2.Plot setup and sapling planting in WP2 2.1 Existing plots within the WP2 working area The WP2 working area includes 11 plots that fall within the same grid of plots as the WP3 plots as described in section 5.1. Data in these plots is collected using the same methodology as in the plots of WP3 in three different periods (1994, 2006, 2019). In 2023, using the same methodology, 23 new plots were established and monitored for tree DBH of ca. 20 trees. The original plots have the format Location_LetterNumber (AUS_G18), the new 2023 plots have the format Location_number (AUS_1). Note that in these plots, data on the exact location of individual trees was not collected. In 2024, 38 additional semi-permanent plots were installed in the WP2 working area, using a slightly different monitoring methodology that allows for individual tree monitoring, as described in section 2.2. Table 2.1: Overview of plot types and used monitoring methodology in the WP2 working area. Plot type Methodology Individual tree location recorded Existing plots (1994, 2006, 2019) See section 5.1. No 2023 existing plots See section 5.1. No Additional semi-permanent plots See section 2.2. Yes 2.2 Additional semi-permanent plots In November and December 2024 (M7-M8 of the project), 38 new semi-permanent circular plots were installed in the forest management unit of Austerlitz. The plots are located in 11 ‘demonstration stands’, i.e. in 7 stands dominated by Pinus sylvestris (Scots pine) (28 plots total) and 4 stands dominated by Quercus rubra (American oak) (10 plots total) (see Figure 2.1). Measurements took place at plot level and at tree level, as described below. 7 Figure 2.1: Location of 38 semi-permanent circular plots in the FMU of Austerlitz, established in NovDec 2024. 2.2.1 Additional plot level data In Austerlitz, in each demonstration stand a 1 ha area was defined in which 2 or 4 circular plots were installed. Circular plots consisted of 15m radius circles and nested 8m radius circles (see Figure 2.2). 8 Figure 2.2: The semi-permanent circular plots consist of a circle with 15m radius in which trees ≥10cm DBH were measured and a smaller circle with 8m radius in which trees ≥5cm DBH were measured. Per plot the following was determined: • coordinates of the center • year of origin • dominant tree species • average height of 2-4 individuals of dominant species • development phase of the vegetation, i.e. successional stage • canopy cover Coordinates of the center of each plot were recorded with a GPS. The year of origin/establishment was estimated based on data of the state forest service. The dominant tree species was determined and defined as the tree species that is most abundant. Height of 2-4 individuals of the dominant species was determined by using laser equipment and averaged. The development phase of the forest consists of several classes of successional stages: Table 2.2: classification of successional stages Class Successional stage 1 open phase (no trees) 2 establishment phase (young trees present) 3 thicket phase 4 pole stage (closed forest) 5 tree/climax phase 6 deterioration phase 9 We determined canopy cover from the center point of three individual trees that were measured for extra traits in May 2025 (see 2.2.2). Canopy cover was measured with the CanopyApp available at the AppleStore, at a height of 1.8m. Additionally, in the 8m radius plots the following was measured: • tree cover • shrub cover The tree cover (above 6m) and shrub cover (0.5-6m) consist of the following percentage classes: Table 2.3: percentage classes of tree and shrub cover Code Cover in % 1 0-0.1 2 0.1-1 3 1-5 4 5-10 5 10-25 6 25-50 7 50-75 8 75-90 9 90-100 2.2.2 Tree level data of alive and standing dead trees Within the 15m radius circle plots for trees with a dbh ≥10cm and within the nested 8m radius circle plots for trees with a dbh ≥5cm, the following variables were measured: • species • DBH • angle and distance from center Each tree was identified to species level and DBH was measured with a caliper. The location of each tree was determined using the polar coordinate system. The distance from the centre of the plot to the tree (centre of the trunk base) was measured with a measuring taper. The deviation (angle) of each tree is determined clockwise (right) in degrees relative to north using a compass, where north is at 0°. Standing dead trees were also measured in the 15m radius circle plots. Standing dead wood was measured in the same way as the alive standing trees but are recorded on a separate tab in the datafile. In March 2025 (M13) we selected 3 random trees in/near each plot for additional measurements including: • crown dimensions 16 3. Soil sampling and field measurements 3.1 Soil sampling in WP2 In the WP2 experimental location and the new field trial section a selection of the 153 planted sapling groups was made so that 64 groups were selected for soil analyses. Five planting groups per species were randomly selected for sampling. In total, 65 soil samples were collected during the initial measurement period. Soil was extracted at four different points within the planting group using a soil auger, collecting the top 20 cm of soil. These four subsamples were combined to form one composite sample per group. To ensure our measurements were not influenced by the treatments given to the saplings, the samples were taken at least 0.5m away from the planted saplings. Upon arrival at the laboratory, all soil samples were stored at 4°C in a cold room to preserve their physiochemical properties for subsequent analyses. 3.2 Soil sampling in WP3 In WP3, a total of ca. 800 semi-permanent plots exist in the three forest management units, and a stratified random selection of 150 plots were made in such way that for each FMU ca 50 plots were sampled for soil properties. Environmental data was collected to assess abiotic factors influencing decomposition dynamics. Soil samples and light availability measurements were taken directly in the field, while additional data on forest structure and composition of the established plots were earlier provided by the State Forestry Service. In each of the 150 selected plots three soil samples were collected using a cylindrical auger. For each of the three soil sub-samples, the organic matter layer thickness was measured with a taper and recorded in the field before extracting the top 20 cm of soil. The sub-samples were then combined into a single composite sample per plot and put in labeled plastic bags for transport. Upon arrival at the laboratory, all soil samples were stored at 4°C in a cold room to preserve their physiochemical properties for subsequent analyses. 3.3 Litter collection and processing in WP3 In each of the 150 existing plots, two litter samples were taken, for which each sub-sample a 25x25 cm grid was placed on the forest floor and all litter material (leaves, twigs, seeds) were collected and put in a plastic bag. The bags were stored at 4°C in a cold room for further processing. Litter samples were separated in fractions of leaves, twigs and seeds. After separation, all sub samples were oven dried (Thermo Scientific Heratherm OMS180) at 45°C for 48 hours and weighed afterwards to determine the dry weight of each litter fraction. 3.4 Light availability For the 150 existing plots light availability was measured using a densiometer (Baudry et al., 2013). Three measurements were taken per plot to assess tree canopy density. The percentage of covered canopy was determined by recording the proportion of squares on the densiometer without 17 vegetation, which was then multiplied by a correction factor (1.04) to estimate total canopy cover. The three measurements were averaged to obtain one canopy cover parameter for the plot. 3.5 Decomposition measurements Due to problems in the supply of Beta-glucosidase enzymes for decomposition experiments, the TeaBag Index (TBI) methodology was applied to measure in-field decomposition rates. The TBI is a standardized method used to assess early-stage organic matter decomposition and stabilization in soils and sediments (Keuskamp et al., 2013). It involves burying bags containing green and rooibos tea—representing labile and recalcitrant plant materials, respectively—for a fixed period. Changes in mass over time are used to calculate decomposition rates and stabilization factors. The standard green tea as used in the research by Keuskamp et al. (2013) (Lipton Indonesian tea Sencha tradition, EAN8722700055525) is no longer available, and alternative green tea is only available in degradable bags. We therefore transferred the tea from degradable bags to self-made mesh bags with the same mesh size as the original tea bags as prescribed for the TBI (200 micron). We used an alternative green tea that is prescribed as a suitable alternative by the Tea Bag Index organization (Lipton Japanese Sencha: EAN 5063270101797). However, the authors of the earlier mentioned TBI research are still developing suitable parameters to derive k and S values from decomposition data of this tea. For the most accurate k and S calculations, these parameters can be recalculated using updated parameters in the future. However, due to a change in testing material (new type of green tea), a decomposition time series test had to be applied. Green and Rooibos mesh bags were buried in duplo in each of the 150 existing field trials (2 green , 2 rooibos). In the new field trials In Austerlitz where the saplings were planted, one rooibos and one green tea mesh bag was buried in each of the 64 plots where also soil measurements were performed. 3.5.1 Tea Bag Preparation, deployment, and processing Mesh bags were constructed using 200-micron nylon mesh, which was cut into uniform squares (approximately 5 cm x 5 cm), with three sides glued together using hot glue, leaving one side open for adding litter. Each mesh bag contained ca. 3 g of green tea (representing labile material) and one containing ca. 3 g of rooibos tea (representing recalcitrant material). Exact tea weights were recorded a-priori using a precision balance. Each bag was labeled with a unique identifier indicating location, plot, and tea type (“G” for green tea, and “R” for rooibos tea). The weight of each completed mesh bag was also measured and recorded. In the field, the teabags were buried per plot using standardized protocol. Teabags were buried approximately 1-1.5 m North from the plot’s central tree. Bags were buried at the soil surface at a uniform depth of approximately 20 cm, and 15 cm apart in a square. Each burial location was marked with a skewer and the central tree was marked with a ribbon to facilitate retrieval. Litterbags remained in situ for exactly 91 days. Exact burial and retrieval dates can be found in table 3.1. 18 Table 3.1: burial and retrieval dates of mesh tea bags IN: Mesh tea bags OUT: Mesh tea bags 25-4-2025 25-7-2025 28-4-2025 28-7-2025 29-4-2025 29-7-2025 30-4-2025 30-7-2025 2-5-2025 1-8-2025 7-5-2025 6-8-2025 8-5-2025 7-8-2025 9-5-2025 8-8-2025 12-5-2025 11-8-2025 13-5-2025 12-8-2025 After exactly 91 days, mesh bags were retrieved in the same order as their deployment to ensure temporal consistency. Using a shovel, teabags were carefully removed, and adhering soil particles were brushed off in the field. Retrieved bags were placed in sealed containers and transported to the lab to prevent contamination or further decomposition during transit. In the lab, all soil particles were removed from the outside of the bags. Mesh bags were then carefully opened and the contents were spread out over a large paper sheet, allowing for identification and removal of ingrown root material. were placed in separate paper cups for drying and weighing. The tea in paper cups was oven-dried at 70°C for 48 hours to standardize moisture content. After removal out of the oven, warm tea attracts moist from the air. The increase in weight due to moisture attraction within the first 10 minutes was considered negligible. Therefore, each paper cup was removed from the oven separately and weighed immediately after. The final dry weight of the material was then measured using a precision balance. Two compensations factors affecting decomposition needed to take place. First, the tea before deployment was not dried in the oven at 70°C for 48 hours in advance, as was done with the tea after deployment. We did follow this process for 5 tea samples to determine an average moisture content in the green tea. The average of this moisture content was used to compensate the weights before deployment with. 𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷 𝑡𝑡𝐷𝐷𝑡𝑡 𝐵𝐵𝐵𝐵𝐵𝐵𝐵𝐵𝐵𝐵𝐵𝐵 = 𝑀𝑀𝑀𝑀𝐷𝐷𝑀𝑀𝑡𝑡 𝑤𝑤𝐷𝐷𝐷𝐷𝑤𝑤ℎ𝑡𝑡 𝑡𝑡𝐷𝐷𝑡𝑡 𝐵𝐵𝐵𝐵𝐵𝐵𝐵𝐵𝐵𝐵𝐵𝐵 ∗𝑡𝑡𝑎𝑎𝐷𝐷𝐷𝐷𝑡𝑡𝑤𝑤𝐷𝐷 𝑚𝑚𝑀𝑀𝐷𝐷𝑀𝑀𝑡𝑡𝑚𝑚𝐷𝐷𝐷𝐷 % A second compensation was applied for possible influx of very small soil particles that may have passed through the 200 micron mesh openings. In 15 plots we therefore buried a bag and retrieved it directly after. These bags were brushed of carefully, contents were emptied in a paper cup, dried at 70°C for 48 hours and compensated for soil moisture. Any increase in weight that was recorded was deemed influx of soil particles in the mesh bags. These weight differences were averaged to obtain a compensation factor for soil influx. 𝑆𝑆𝑀𝑀𝐷𝐷𝑆𝑆 𝑐𝑐𝑀𝑀𝑚𝑚𝑐𝑐𝐷𝐷𝑐𝑐𝑀𝑀𝑡𝑡𝑡𝑡𝐷𝐷𝐷𝐷 𝑡𝑡𝐷𝐷𝑡𝑡 𝐴𝐴𝐵𝐵𝐴𝐴𝐵𝐵𝐵𝐵 = 𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷 𝑤𝑤𝐷𝐷𝐷𝐷𝑤𝑤ℎ𝑡𝑡 𝑡𝑡𝐷𝐷𝑡𝑡 𝐴𝐴𝐵𝐵𝐴𝐴𝐵𝐵𝐵𝐵 ∗𝑡𝑡𝑎𝑎𝐷𝐷𝐷𝐷𝑡𝑡𝑤𝑤𝐷𝐷 𝑀𝑀𝑀𝑀𝐷𝐷𝑆𝑆 𝐷𝐷𝑐𝑐𝑖𝑖𝑆𝑆𝑚𝑚𝑖𝑖 % 3.5.2 Decomposition time series After a certain amount of time all labile fractions in green tea have decomposed and the weight stabilizes, after which the parameters in section 3.5.3. can be calculated. In order to check whether the weight loss of green tea indeed stabilized over time, several time series were done. Teabags of 19 japanese sencha, indonesian sencha and rooibos were buried in 5 different plots. In each plot 5 bags were burried and retrieved after 7, 14, 29, 68 or 130 days. The weight loss of each bag was recorded. The time series setup was based on the research by Keuskamp et al. (2013) and measurement days were chosen accordingly; teabags were measured at 0, 4, 7, 14, 29, 68 and 130 days. For the green tea used in this experiment, Japanese sencha, a stabilization of decomposition took place between 67 and 130 days as shown in table 3.2. Figure 3. 1 shows the time series plot, showing the fraction of remaining tea over time. Individual points represent observed values from each experimental plot. The lines represent the monotonic means per tea type. The Japanese green sencha tea as used in this experiment meets the necessary assumption that green tea decomposition stabilizes after 90 days, with no further significant decomposition. With this assumption met, decomposition parameters can be calculated. Table 3.2: Tea bag index time series average results of remaining tea weight fractions over time for different tea types. Days Rooibos remaining fraction Japanese Green remaining fraction Indonesian Green remaining fraction 0 1.00 1.00 1.00 4 1.00 0.94 0.92 7 0.98 0.89 0.89 14 0.91 0.71 0.67 29 0.85 0.53 0.54 68 0.80 0.35 0.39 130 0.63 0.32 0.30 Figure 3.1: Tea bag index time series results for different tea types. Individual points represent the remaining fractions in each of the 5 time series plots. Lines represent the monotonic means through these points. 20 3.5.3 Tea Bag Index parameter calculations The TBI results in two parameters that together describe the decomposition in the soil: the stabilization factor S and the decomposition rate k. The stabilization factor is calculated as following: 𝑆𝑆= 1 −�𝑡𝑡𝑤𝑤 𝐻𝐻𝑤𝑤� Where: • S = Stabilisation factor • ag = fraction of decomposed green tea • Hg = hydrolysable fraction of green tea The decomposition rate k is calculated as following: 𝑘𝑘=𝐿𝐿𝑐𝑐�𝑡𝑡𝐷𝐷 𝑊𝑊𝑡𝑡−(1−𝑡𝑡𝐷𝐷)� 𝑡𝑡 In which = 𝑡𝑡𝐷𝐷 =𝐻𝐻𝐷𝐷∗(1 −𝑆𝑆) Where: • k = decomposition rate • ar = predicted labile fraction red tea • Wt = fraction remaining red tea • t = burial period • Hr = hydrolysable fraction of red tea • S = stabilization factor It is important to note that the hydrolysable fraction of both green and red tea for the tea types used are not yet up to date, and based on the original tea types as prescribed by Keuskamp et al. (2023). When updated parameters become available through their research, the k and S can be updated with the tea weight loss data that is provided. Also: in some plots k or S could not be calculated. According to the authors of the Tea Bag Index this is a result of a fast breakdown of labile material in the rooibos teabags, in which the tea moves from the first to the second phase of decomposition. The formula will then calculate a negative k or no k at all. 3.6 Soil fauna feeding activity The bait lamina test was used to complement decomposition experiments to the extent to assess soil faunal feeding activity in each plot. This method involves filling standardized strips with bait material to measure feeding activity via perforation consumption over time (Kratz, 1998). Two strips were deployed per plot. This action was performed two times, in 2024 and 2025, with some overlap in plots. 21 The bait material was prepared by mixing a standardized substrate consisting of 70% cellulose, 25% wheat bran, and 5% activated carbon. For this study, oats were used as the wheat bran source and were ground into fine particles using a series of sieves with decreasing mesh sizes (0.5 mm, 0.2 mm, and finally 0.12 mm). The activated carbon was sourced from Norit pills, which were crushed into a fine powder using a spoon to achieve the desired consistency. The prepared dry components were then thoroughly mixed to ensure uniform distribution. Distilled water was gradually added to the mixture to form a paste-like consistency suitable for filling the bait lamina strips. The bait material was then stored in an airtight container to maintain its moisture and consistency until it was applied to the strips. The bait lamina strips were constructed using standardized polyvinyl chloride (PVC) strips with 16 evenly spaced perforations (2 mm diameter) along their length. The empty strips were aligned on an aluminum sheet and taped at the upper portion of each strip (above the perforations) to keep them in place. The prepared bait mixture was applied directly onto the perforated area of the strips using a spatula. The bait was pressed firmly against the strips to ensure that each perforation was completely filled. This process was repeated for all strips. Excess bait on the surface of the strips was carefully wiped away to prevent obstruction or contamination during deployment. Once all perforations were filled, the strips were placed in an oven set at 40°C for 48 hours to dry the bait within the holes. After drying, any holes that had lost bait (as the material is fragile and can fall out during handling) were refilled following the same procedure. The strips were then dried again to secure the bait in place. After fully prepared, strips were stored in a labeled plastic container to protect them from moisture or damage until deployment. In the field, two bait lamina strips were deployed per plot, with strips placed on opposite sides of the plot’s central tree to account for spatial variability. Strips were inserted vertically into the soil using a small slit made with a trowel. Insertion depth was such that the topmost hole was just below ground level ensuring that the perforations were buried at uniform and exposed to the same environmental conditions. A marker was placed near each strip for identification during retrieval. Strips remained in the soil for two weeks, allowing adequate time for soil fauna to interact with the bait. Exact burial and retrieval dates for the 2025 dataset can be found in table 3.3. Table 3.3: Burial and retrieval dates of bait lamina strips in the 2025 measuring series. IN: Bait lamina 2025 OUT: bait lamina 2025 26-6-2025 10-7-2025 30-6-2025 14-7-2025 11-6-2025 25-6-2025 24-6-2025 8-7-2025 13-6-2025 27-6-2025 25-6-2025 9-7-2025 After two weeks, strips were retrieved in the same order as their deployment. Strips were carefully taken out of the soil to avoid disturbing the bait or altering the perforations. Retrieval was performed systematically to ensure consistency, with each strip being gently removed and immediately assessed for bait consumption. For each perforation (16 in total), data was recorded as follows: • “0” for an empty hole (bait fully consumed) • “0.5” for a half-filled hole (bait partially consumed) • “1” for a full hole (no bait consumed) 22 For each plot, the strip was divided in four depth sections: hole 1-4-, hole 5-8, hole 9-12, hole 13-16. For each groups the proportion of non-filled/filled was determined, which is a measure for the feeding activity. This was also done for the whole strip in total. The results for both strips were averaged to obtain an average total feeding activity per plot as well as average feedings activities per depth section per plot. 23 4. Soil and nutrient processing Soil and nutrient analysis was conducted in laboratories at the faculty of Geosciences and Natural Sciences, Utrecht University, using standardized protocols to ensure consistency and reliability. An overview of types of measurements, methods and used equipment can be found in table 4.1. Table 4.1: Overview of applied methods of soil, litter and decomposition analysis. Measurement Method Equipment Unit pH Measurement of soil in KCl extract Mettler Toledo SevenCompact Duo pH (KCl) C:N ratio Analysis of dried, ground and acidified soil samples Heto PowerDry LL3000; SCALA TFD5503;CN-analyzer (NA1500, Fisons Instruments); Herzog HP-Ma automatic pulverizer Soil C/N ratio, soil C (%) and soil N (%) Soil Organic Matter Calculated as weight loss after 4 hours heating at 550°C Heto PowerDry LL3000; SCALA TFD5503TGA701; Thermogravimetric Analyzer Soil organic Matter (%) NH 4 -N, NH 3 -N, NH2-N, NOx-N, PO4P Sample in KCl in discrete analyzer Thermo Fisher Scientific Gallery NOₓ-N (mg N/kg soil) PO₄-P (mg P/kg soil) Al, Ca, Fe, K, Mg, Na Heto PowerDry LL3000;SCALA TFD5503; Herzog HP-Ma automatic pulverizer, Perkin-Elmer Avio 500 ICP-OES Al, Ca, Fe, K, Mg, Na (mg/L) Litter Oven drying Thermo Scientific Heratherm OMS180, precision balance Biomass (g/m2 surface area) Teabags Oven drying Thermo Scientific Heratherm OMS180, precision balance g 4.1 Soil Analyses Soil samples were analyzed to determine their physical and chemical properties using the following methods: 4.1.1 Soil pH Soil pH was measured using a 1 M KCl solution to assess exchangeable acidity (Van Reeuwijk, 1992). 20 mL of 1M KCl was added to exactly 8 grams of soil in a 50 mL Greiner tube. The mixture was shaken for 2 hours using a reciprocating shaker. The suspension was centrifuged and the electrode was 24 immersed in the supernatant. The soil pH (pH-KCl) was measured using a Mettler Toledo SevenCompact Duo pH meter. pH readings were recorded once stabilized (±0.1 unit accuracy), with fluctuations not exceeding 0.1 unit per 30 seconds. 4.1.2 Anorganic nitrogen: NOx, NO3, NO2, NH4, PO4 Nitrogen and phosphate concentrations were measured using a 1 M KCl solution. 20 mL of 1M KCl was added to exactly 8 grams of soil in a 50 mL Greiner tube. The mixture was shaken for 2 hours using a reciprocating shaker. The supernatant resulting from the preparation of the pH measurement was filtered through a 0,45 µm glasfiber syringe filter. The concentrations of ammonium (NH₄⁺), total nitrogen oxides (NOₓ) and nitritie (NO₂⁻) were measured using a discrete analyzer (Thermo Fisher Scientific Gallery). Nitrate (NO₃⁻) concentration was derived from the NOₓ and NO₂⁻ measurements. The moisture percentage in the soil that was derived from the freeze-drying procedures at 4.1.4 was used to calculate NOx-N, NO3-N, NO2-N and NH4-N in g per kg of dry soil using using the following formula (similar for all measurements): 𝑁𝑁𝐻𝐻4−𝑁𝑁 𝑀𝑀𝐷𝐷 𝑁𝑁𝐵𝐵𝑖𝑖−𝑁𝑁 𝑀𝑀𝐷𝐷 𝑁𝑁𝐵𝐵3−𝑁𝑁 𝑀𝑀𝐷𝐷 𝑁𝑁𝐵𝐵2−𝑁𝑁 𝑀𝑀𝐷𝐷 𝑃𝑃𝐵𝐵4−𝑃𝑃 =𝑡𝑡∗𝑀𝑀 1000 ∗𝐿𝐿 𝑚𝑚∗(100 +𝑤𝑤) 100 Where: • NH₄-N, NO₃-N, NO₂-N, NOₓ-N, PO4-P,= nitrogen or phosphor concentrations in mg kg⁻¹ dry soil • a = concentration of NH₄⁺, NO₃⁻, or NO₂⁻ measured in the diluted sample (μmol/L) • M = atomic mass of N or P in g/mol • m = mass of wet soil used in the suspension (g) • L = volume of liquid used in suspension (ml) • w = percentage of water content based on freeze-dried soil 4.1.3 Cation concentrations Approximately 20 grams of soil was weighed using a precision balance into a pre-weighed 50 mL Greiner tube. The tubes were then placed in one of the two available freeze dryers (Heto PowerDry LL3000 and SCALA TFD5503) for 48 hours. The samples were subsequently ground in the Herzog HPMa automatic pulverizer. Exact weights of samples for analysis were recorded using a precision balance. The concentrations of Ca, Al, K, Na, Mg and P were measured using a ICP-OES analyzer (Perkin-Elmer Avio 500). 4.1.4 Soil organic carbon and C:N ratio Approximately 20 grams of soil was weighed using a precision balance into a pre-weighed 50 mL Greiner tube. The tubes were then placed in one of the two available freeze dryers (Heto PowerDry LL3000 and SCALA TFD5503) for 48 hours. The weight was then measured again to determine the soil moisture content. The samples were subsequently ground in the Herzog HP-Ma automatic pulverizer. A small portion of each sample was weighed using a precision balance and transferred to silver cups. In order to measure soil organic carbon content, potential CaCO3 was removed from the sample through acidification by placing the samples in a desiccator with a small beaker of 20ml 37% 25 fuming HCl underneath it for 6 hours. The percentage carbon (C) and nitrogen (N) was subsequently determined using a CN-analyzer (NA1500, Fisons Instruments), and the C:N ratio was calculated by dividing these two values. Note that in the results some values have an * or two times ** before the value. * indicates a measurement that was in between the Limit of Quantification (LOQ) and the Below Effective Concentration (BEC). In practice this means that these values have an error of >10% and should thus be interpretate as an estimate. ** indicates a value that is below the LOQ and thus cannot be considered accurate. 4.1.5 Soil Organic Matter Content Approximately 20 grams of soil was weighed using a precision balance into a pre-weighed 50 mL Greiner tube. The tubes were then placed in one of the two available freeze dryers (Heto PowerDry LL3000 and SCALA TFD5503) for 48 hours. The weight was then measured again to determine the soil moisture content. The samples were subsequently grinded in the Herzog HP-Ma automatic pulverizer. A portion of these soil samples were used to determine the percentage of organic matter, by measuring the weight loss after four hours of heating at 450 degrees Celsius (TGA701 Thermogravimetric Analyzer). The samples were then stepwise heated to 1000°C (550-800-1000°C), which resulted in additional organic matter mass loss at each temperature. Decrease in weight was recorded at each step as a percentage of the initial sample weight. Total soil organic matter percentage was calculated by summation of these weight losses.