Natural resources and bioeconomy studies 40/2025 Natural Resources Institute Finland 2023 Natural resources and bioeconomy studies 40/2025 BENCHMARKS soil sampling protocol Guidelines for agricultural and forest systems Sarah Symanczik, Martina Lori, Aurélie Bacq-Labreuil, Nicolas Beriot, Alexander Berlin, Else K. Bünemann, Pénélope Cheval, Rachel Creamer, Luís Cunha, Felix David, Paolo Di Lonardo, Sophia Götzinger, Jakub Hofman, Raisa Mäkipää, María Martínez-Mena, Julia Möller, Filipa Reis, Taru Sandén, Tiina Törmänen and Titia Mulder Natural Resources Institute Finland 2025 Natural resources and bioeconomy studies 40/2025 BENCHMARKS soil sampling protocol Guidelines for agricultural and forest systems Sarah Symanczik, Martina Lori, Aurélie Bacq-Labreuil, Nicolas Beriot, Alexander Berlin, Else K. Bünemann, Pénélope Cheval, Rachel Creamer, Luís Cunha, Felix David, Paolo Di Lonardo, Sophia Götzinger, Jakub Hofman, Raisa Mäkipää, María Martínez-Mena, Julia Möller, Filipa Reis, Taru Sandén Tiina Törmänen and Titia Mulder Natural resources and bioeconomy studies 40/2025 The work presented in this report is part of and supported by the European Union’s Horizon Europe research and innovation program under the Grant Agreement 101091010 for the Soil Mission project BENCHMARKS. Referencing instructions: Symanczik, S., Lori, M., Bacq-Labreuil, A., Beriot, N., Berlin, A., Bünemann, E.K., Cheval, P., Creamer, R., Cunha, L., David, F., Di Lonardo, P., Götzinger, S., Hofman, J., Mäkipää, R., Mar- tínez-Mena, M., Möller, J., Reis, F., Sandén, T., Törmänen, T. & Mulder, T. 2025. BENCHMARKS soil sampling protocol : Guidelines for agricultural and forest systems. Natural Resources and Bioeconomy Studies 40/2025. Natural Resources Institute Finland. Helsinki. 32 p. Sarah Symanczik ORCID ID, https://orcid.org/0000-0002-2773-3875 ISBN 978-952-419-063-3 (Online) ISSN 2342-7639 (Online) URN urn.fi/URN:ISBN:978-952-419-063-3 Copyright: Natural Resources Institute Finland (Luke) Authors: Sarah Symanczik, Martina Lori, Aurélie Bacq-Labreuil, Nicolas Beriot, Alexander Ber- lin, Else K. Bünemann, Pénélope Cheval, Rachel Creamer, Luís Cunha, Felix David, Paolo Di Lonardo, Sophia Götzinger, Jakub Hofman, Raisa Mäkipää, María Martínez-Mena, Julia Möller, Filipa Reis, Taru Sandén, Tiina Törmänen and Titia Mulder Publisher: Natural Resources Institute Finland (Luke), Helsinki 2025 Year of publication: 2025 Cover picture: BENCHMARKS CSS leaders Natural resources and bioeconomy studies 40/2025 3 Abstract Sarah Symanczik1, Martina Lori1, Aurélie Bacq-Labreuil2, Nicolas Beriot3, Alexander Berlin4, Else K. Bünemann1, Pénélope Cheval5, Rachel Creamer6, Luís Cunha7, Felix David6, Paolo Di Lonardo6, Sophia Götzinger8, Jakub Hofman9, Raisa Mäkipää10, María Martínez-Mena11, Julia Möller3, Filipa Reis7, Taru Sandén8, Tiina Törmänen10, Titia Mulder6 1 Department of Soil Sciences, Research Institute of Organic Agriculture (FiBL), Frick, Switzer- land 2 Genesis, Paris, France 3 Soil Physics and Land Management Group, Wageningen University, Wageningen, the Neth- erlands 4 Climate Farmers Academy gUG, Berlin, Germany 5 Université de Lorraine, INRAE, LSE, Nancy, France 6 Soil Biology Group, Wageningen University, Wageningen, the Netherlands 7 Centre for Functional Ecology, Associate Laboratory TERRA, Department of Life Sciences, University of Coimbra, Coimbra, Portugal 8 Department for Soil Health and Plant Nutrition, Austrian Agency for Health and Food Safety (AGES), Vienna, Austria 9 RECETOX, Faculty of Science, Masaryk University, Brno, Czech Republic 10 Natural Resources Institute Finland (LUKE), Helsinki, Finland 11 Soil and Water Conservation Research Group. CEBAS-CSIC, Campus Universitario de Espi- nardo, Murcia, Spain. This document presents a comprehensive sampling protocol to address spatial heterogeneity and variability of soil health indicators (SHI) across a site. Geo-referenced sampling points are identified using K-means sampling and integrating satellite and terrain data and existing data from the site to determine the sampling clusters. In addition, a random sample is obtained within the site. The sample numbers are tailored to the sampling area. To evaluate soil health, a diverse set of biological, chemical, and physical SHIs is proposed. To optimize soil samples for subsequent analyses, SHI-compliant sampling protocols are imple- mented, aligning with the BENCHMARKS sampling scheme. For baseline site characterisation, soil samples will be collected using BENCHMARKS proto- cols for bulk soil, bulk density, earthworms, and mesofauna. Depending on site-specific chal- lenges, additional samples may be collected using protocols tailored to plastic sampling or hydraulic property sampling. The document also provides detailed guidelines on sample processing, shipping, and storage tailored to each soil sampling protocol. Lastly, it outlines the SHIs recommended for assess- ment both in the laboratory and directly in the field. Keywords: BENCHMARKS soil sampling protocols, soil sample processing, sample storage, sample shipment, soil health indicators, laboratory analysis, variance sampling, sampling de- sign Natural resources and bioeconomy studies 40/2025 4 Contents 1. BENCHMARKS soil sampling design..................................................................... 5 2. BENCHMARKS sampling protocols ....................................................................... 6 2.1. Define the sampling area ..................................................................................................................................... 7 2.2. BENCHMARKS earthworm sampling protocol ............................................................................................. 7 2.3. BENCHMARKS mesofauna sampling protocol ............................................................................................. 8 2.4. BENCHMARKS bulk soil sampling protocol .................................................................................................. 8 2.5. BENCHMARKS bulk density sampling protocol ........................................................................................... 9 2.6. BENCHMARKS soil hydraulic property sampling protocol ...................................................................... 9 2.7. BENCHMARKS plastic sampling protocol ................................................................................................... 10 3. Sample processing, storage and shipping ......................................................... 11 4. BENCHMARKS soil health indicator catalogue ................................................. 15 5. Field-based assessments ...................................................................................... 18 6. Identification and registration of samples ........................................................ 19 7. Acknowledgements .............................................................................................. 20 References .................................................................................................................... 21 Appendix 1 ................................................................................................................... 26 Natural resources and bioeconomy studies 40/2025 5 1. BENCHMARKS soil sampling design The proposed sampling design is applied in agricultural and forest sites for a basic characteri- sation and to address spatial heterogeneity and variability of SHI across a site. The sampling objective for the variance sites is capturing the spatial variability within a site and allow com- parison of various sites which have different management practices. The sampling design was optimized in such a way that the land surface variability was optimally captured, given the constraint of the sample size. For this, K-means sampling was used (Brus et al., 2019), utilizing existing data from Sentinel 2 satellite data (Copernicus Sentinel 2) and Digital Elevation Model (DEM) data (Copernicus DEM). From the Sentinel satellite data, the NDVI (Normalized Difference Vegetation Index, 10 m resolution) and Principal Components of the VNIR-SWIR (Visible and Near-Infrared - Shortwave Infrared) images (20 m resolution) were derived, using the images from the year before sampling. The NDVI images came from the top of the grow- ing season, whereas the VNIR-SWIR images were taken from the month that showed the largest spectral variability in the Principal Components. From the DEM (30 m resolution), the elevation and the DEM-derivates slope and aspect were used. The DEM data was only used for sampling optimization if they portrayed sufficient spatial variability, e.g. this excluded sites on reclaimed land. For model validation and assessment of short-scale variability, an addi- tional random sample was allocated in the site. The sample numbers are tailored to the sampling area. In BENCHMARKS, a minimum sample size of 30 was adopted to ensure sufficient samples to be collected per site, allowing statisti- cal comparison of various sites. In general, we recommend to aim for a sampling density of 1 sample per ha for the K-means sampling (KS) and supplement this with a random (RS) half the size of the KS sample. For sites smaller than 3 ha, the total sample size remains 30 sam- ples but only random sampling is required, as in these small sites the k-means clustering us- ing the data described above does not result in a useful stratification of the field. Finally, we recommend for fields between 3 and 10 ha or prior information from land managers to con- sider using a k-means sampling with a sample density of 2 samples per ha, as this would im- prove the capturing of spatial heterogeneity. Natural resources and bioeconomy studies 40/2025 6 2. BENCHMARKS sampling protocols To assess soil health, a range of biological, chemical and physical SHIs are proposed. To en- sure that soil samples are optimized for subsequent analysis, we use SHIs-compliant sam- pling protocols from the BENCHMARKS sampling scheme (Figure 1). At each sampling site, samples are collected following the BENCHMARKS bulk soil, bulk density, earthworm and mesofauna sampling protocols. Where specific soil functions or site challenges dictate, addi- tional samples are taken following the plastic or hydraulic properties sampling protocols. In the BENCHMARKS variance sampling campaign, the earthworm sampling protocol was omit- ted due to the high number of sampled locations, which were not feasible to handle. Sampling is recommended before any major management operations (e.g., fertilisation or till- age), generally at the start of the growing season when baseline SHI values are most repre- sentative. However, timing may be adjusted based on climatic zone. Weather and soil-mois- ture conditions should be monitored to ensure the soil is neither excessively wet (to prevent compaction) nor overly dry (to prevent sampling bias and disturbance). To minimize disturb- ance and avoid cross-contamination between different sample types, the following overall sampling order should be followed, and the designated sampling locations for each type must be respected (no trampling). 1. earthworm sampling, 2. mesofauna sampling, 3. plastic sampling and then the remaining samplings. A list of soil sampling materials and equipment is provided in Appendix 1.1. For subsequent years, the sampling design will remain consistent, but sampling points will be slightly adjusted to avoid exact overlap with prior locations. The sampling timeline should also be maintained, allowing a flexibility of ±1 week to accommodate local weather condi- tions. Figure 1. BENCHMARKS sampling scheme. Overview of the sampling protocols suggested for soil health indicator (SHI) assessments. The upper box indicates the name of the sampling protocol and the box below specifies the type of soil sample to be collected, required sample condition, and the sample volume. * Sampling protocols chosen for selected sites to address specific challenges or study specific soil functions. Natural resources and bioeconomy studies 40/2025 7 2.1. Define the sampling area Find the geo-referenced point using a GPS or GIS-enabled device and mark it with a large stick. To delineate the sampling area, attach one end of a tape measure to the geo-refer- enced point and use it to draw a circle with a radius of 1 m around the center point. With a compass, define the three cardinal directions (north, east, south) with individual sticks to fa- cilitate the specific sampling protocols: the mesofauna sampling on the north side of the cir- cle, the hydraulic sampling on the south side and the earthworm and bulk density sampling on the east side (Figure 2). Figure 2. BENCHMARKS sampling approach. The composite bulk soil sample consisting of 15 subsamples is randomly sampled within a circle of 1 m radius. The mesofauna sample is taken on the north side of the circle, the hydraulic sample on the south side and the earthworm and bulk density sample on the east side. The composite plastic sample consisting of 15 subsam- ples randomly sampled within the 2 m circle avoiding plastic sampling materials and only at a depth of 0-10 cm. 2.2. BENCHMARKS earthworm sampling protocol On the east side of the sampling area, excavate a soil monolith of 20 cm x 20 cm x 20 cm with a spade and transfer it onto a large black plastic bag or tray. Earthworms present in the soil monoliths are hand-sorted and transferred to a 50 ml reaction tube (or similar air-tight con- tainer) filled with >90% ethanol for fixation. Before returning the soil into the earthworm pit, make a basic assessment of the soil texture following the FAO guidelines (FAO 2006) and take the bulk density samples (see section BENCHMARKS bulk density sampling protocol). Natural resources and bioeconomy studies 40/2025 8 2.3. BENCHMARKS mesofauna sampling protocol With the BENCHMARKS mesofauna sampling protocol, collect an undisturbed soil core at the north side of the sampling area using a PVC cylinder (5 cm diameter x 5 cm height, ~100 cm3 volume) from a depth of 5 cm. Remove the vegetation (upper 1-2 cm) or organic (O) layer (if present in forest systems). Extract the soil core by gently driving the PVC tube into the soil using a wooden block and a mallet. This avoids the compaction of soil. Remove the PVC cyl- inder from the soil with the help of a spade/spatula placed underneath the cylinder. Remove the excess soil around the PVC cylinder with a knife. Wrap the cylinder with plastic film and seal it with paper tape to preserve the soil structure inside the cylinders (make a few tiny holes with a needle on the top cover of each cylinder). Transfer samples into a labeled plastic bag and double pack each sample (put the labeled bag inside another plastic bag to prevent losing the label in case it detaches from the bag). Transport samples in a cooling box. To pre- vent compaction, either use separate cooling boxes for different samples or place buffer ma- terial between layers of samples. In forest systems, also collect a mesofauna sample from the O layer, if present. Use a split corer (5 cm diameter) to collect one core from the entire O layer. Take a photo of the core profile to complement the site characterisation using a ruler as a scale. Report the thickness of the O layer in the field observation protocol. Discard the mineral soil layer and transfer the O layer into a labelled plastic bag and double pack each sample (put the labeled bag inside another plastic bag to prevent losing the label in case it detaches from the bag). 2.4. BENCHMARKS bulk soil sampling protocol With the BENCHMARKS bulk soil sampling protocol, take a composite bulk soil sample of ap- proximately 2 kg at each sampling site. The composite sample consists of at least 15 subsam- ples taken with a soil corer (3‒5 cm diameter) randomly within the sampling area from a depth of 0‒20 cm in agricultural systems or 0‒20 cm and the O layer in forest systems. Before taking a soil core, remove vegetation, litter, stones, etc. from the soil surface. Collect the soil cores, separate the cores with a knife according to the required sampling depths and place each layer in the appropriate labelled plastic bag, which are placed in clearly labelled buckets. Repeat this procedure until all subsamples have been collected. Remove bigger stones (> 6 cm). Double pack each sample for transportation (put the labeled bag inside an- other plastic bag to prevent losing the label in case it detaches from the bag). Transport sam- ples in a cooling box cooled with freezer packs but avoid samples lying directly on the ice packs (add a layer of isolation material). In forest systems, also collect bulk samples from the O layer. Extract a soil core from the en- tire O layer using a soil corer (3‒5 cm diameter). Measure the depth of the organic layer with a ruler (record it in the field observation protocol as well as the diameter of the soil corer used). Take a picture. Transfer the entire volume of the O layer into a labelled plastic bag. Re- peat this procedure until all subsamples have been collected. Pack and transport samples as described for bulk soil samples. To avoid cross-contamination of samples, always wear laboratory gloves when touching the soil and equipment and try to touch the soil as little as possible. Sampling equipment needs Natural resources and bioeconomy studies 40/2025 9 to be cleaned with water and dried with tissue paper after each composite sample. In addi- tion, the first soil core taken at a new sampling site will be discarded. 2.5. BENCHMARKS bulk density sampling protocol In the earthworm pit, use a vertical measuring rod to identify the midpoint of the 0–20 cm depth increment perpendicular to the soil profile. Coat the outside of a metal cylinder (5 cm diameter x 5 cm height, 100 cm3 volume) with a very thin layer of Vaseline or grease (only if necessary for easier soil penetration). Insert the cylinder horizontally into the soil at the targeted depth using a wooden block and mallet (Figure 3). Once the cylinder is fully inserted, remove the surrounding soil gently and carefully extract the cylinder using a spade or shovel placed underneath if needed. Trimm any excess soil extending beyond each end of the cylinder with a straight-edged knife. Transfer the cyl- inders into a labeled plastic bag and double pack each sample (put the labeled bag inside an- other plastic bag to prevent losing the label in case it detaches from the bag). Repeat the procedure for all sampling depths, positioning the cylinder at the midpoint of each layer. Figure 3. BENCHMARKS bulk density sampling protocol. The bulk density cylinder is inserted horizontally at the midpoint of each sampling depth in the expanded earthworm pit. 2.6. BENCHMARKS soil hydraulic property sampling protocol With the BENCHMARKS hydraulic properties sampling protocol, collect undisturbed soil sam- ples at the south side of the sampling area using a metal cylinder of varying size depending on the subsequent analysis: • Cylinders of 7 cm diameter × 7 cm height, 270 cm³ volume (or 5 cm diameter × 5 cm height, 100 cm³ volume for HYPROP2) to determine water retention curves (equilib- rium method) • Cylinders of 8 cm diameter × 15 cm height, 670 cm³ volume (or 7.2 cm diameter × 6.2 cm height, 250 cm³ volume) unsaturated hydraulic conductivity and water reten- tion curves simultaneously (evaporation/wind’s method). Natural resources and bioeconomy studies 40/2025 10 Remove the vegetation or O layer (if present in forest systems) and level the soil using a spat- ula, scraping off 1–2 cm of the uppermost layer. This ensures that the inserted cylinders sam- ple the 0–10 cm soil layer effectively. Coat the outside of the metal cylinder with a thin layer of Vaseline or grease to reduce friction during insertion. Position the cylinder on the soil sur- face, optionally stacking a second cylinder or a 1–2 cm extension of equal diameter and thickness on top. Gently insert the cylinder into the soil using a wooden block and a mallet. Ideally, a specific sample ring insertion tool is available fitting the selected ring size. It is criti- cal that the insertion proceeds vertically and slowly to minimize soil compaction. Once the cylinder has been inserted 3‒4 cm, check that the inner soil surface aligns with the outer soil surface. If not, compaction has likely occurred, and the procedure must be repeated at a new location. Then, remove surrounding soil using a spatula to release lateral pressure on the cyl- inder. Continue to gently insert the remaining part of the cylinder into the soil. Extract the cylinder carefully using a spade or large spatula placed underneath. Trim excess soil from both ends of the cylinder using a sharp spatula or straight-edged knife. Wrap the sample tightly in plastic film, sealing both ends with paper tape to preserve structure and prevent soil loss. If available, use metal or plastics lids to close both ends before wrapping. If an empty space (1–2 cm) remains at the top, fill it with soft material (e.g. leaves, paper, etc.) to stabilize the soil inside. Transfer the cylinders into a labeled plastic bag and double pack each sample (put the labeled bag inside another plastic bag to prevent losing the label in case it detaches from the bag) and pad with cushioning material to protect the cylinders from vibration. 2.7. BENCHMARKS plastic sampling protocol The BENCHMARKS plastic sampling protocol follows the same procedure than the BENCH- MARKS bulk soil sampling protocol with some adaptations: i) sampling depth is restricted to 0‒10 cm and ii) do not use plastic tools and do not wear synthetic clothing, instead use metal, glass or wooden tools and wear cotton or other natural fibres or wear a cotton lab coat over your clothes to avoid contamination. For sample transport and storage, use e.g. al- uminium containers. Sampling equipment needs to be cleaned with water and dried with tis- sue paper after each composite sample. Natural resources and bioeconomy studies 40/2025 11 3. Sample processing, storage and shipping Processing of samples from the BENCHMARKS bulk soil sampling should be done latest the day after sampling (better the same day). Gently break big soil aggregates and mix the soil to take a homogeneous subsample of 150 g for aggregate stability analyses and of 500 g for chemical analyses (store at 4 °C until shipping by regular post), a subsample of 350 g from the most upper soil layer for nematode analyses (store at 4 °C, keep bags open until express shipping max. one week after sampling, see below) and a backup sample of 200 g (store lo- cally at 4 °C). Sieve a subsample of 500 g at 2 mm, or at 5 mm for clay-rich and peat soils, and take a subsample of 50 g for nitrogen mineralization analyses, 200 g for pollutant (per- sistent organic pollutants, pesticides and metals) analyses and 200 g for microbiological anal- yses. Ship sieved fresh samples immediately by express in a styropor/thermo box filled with cooling packs by express courier (e.g. DHL or FedEx and provide the tracking number to the recipient and inform beforehand to arrange shipping to preserve the characteristics of the samples. Take a subsample 10‒20 g for molecular analyses in a 15 ml or larger reaction tube. Do not compact the soil in the tube. Either fix the label additionally with transparent tape or write the sample ID by hand onto the tube, since labels like to detach when frozen (store at - 20 °C, express shipping dry ice). If available, freeze-dry the samples and ship by regular post. And take a backup sample of 20-30 g and store it locally at -20 °C. Air dry the remaining soil at max. 30 °C for 48 h or longer if needed. Sieve at 2 mm and take a subsample of 50 g for active carbon analysis and keep the remaining soil as backup (store locally at room tempera- ture). From forest sites, process samples from the O layer in the same way, but omit sieving and subsampling for specific analyses such as aggregates, microbial biomass, nitrogen mineraliza- tion, and active carbon, depending on the requirements. Figure 4 gives an overview of bulk soil sample processing steps and shipping conditions. Be- fore and after sieving, store samples in a cooling box. Ship samples in plastic bags (i.e. zip lock bags except those of the BENCHMARKS plastic sampling and those for molecular anal- yses) and double pack each sample (put the labeled bag inside another plastic bag to prevent losing the label in case it detaches from the bag). Clean all equipment (sieves, bowls, etc.) carefully with water to avoid contamination. Natural resources and bioeconomy studies 40/2025 12 Figure 4. Overview of bulk soil processing and shipping. Bulk soil processing steps required sample volumes and storage conditions and shipping conditions are shown. The dashed ar- row indicates that only a subsample of the fresh bulk soil sample is sieved at 2 mm (a or at 5 mm in case of clay-rich/peat soils) before drying. Snowflakes indicate that samples need to be stored at 4 °C until shipping, -20°C indicates storage at -20°C, runner indicates express shipping with cooling packs, runner with flash indicates express hipping on dry ice, house in- dicates that backup samples are stored locally at the partner institution in case they are needed later. N, nitrogen. Store soil cores from BENCHMARKS bulk density sampling in the cold room for max. one week until further processing. Weigh the cylinder to determine soil fresh weight (mf), place the cylinder containing the soil sample in an oven at 105 °C for 48 h until constant mass is reached. Transfer cylinders from oven into desiccator and allows to cool for 4 h. Weigh the cylinders after removal from the desiccator (mt). Remove the soil, clean cylinder and weigh empty cylinder (ms). Calculate bulk density as follows: ρ (g cm-3) = (mt-ms)/V. Store soil cores from BENCHMARKS hydraulic properties sampling in the cold room and ship them max. one week after sampling. Pack and ship the cores to protect them from vibration, shock, extreme heat and freezing. To do so, wrap the cores in cushioning material with a min- imum thickness of 2.5 cm around the sample and 5 cm on the bottom. If necessary, protect against heat or cold by shipping it in styropor/thermo box. To facilitate handling, it is recom- mended that packages are not made too large or heavy. Natural resources and bioeconomy studies 40/2025 13 Store cylinders from BENCHMARKS mesofauna sampling in a cold room (4-10 °C) and keep plastic bags open for aeration. Ship cylinders max. five days after sampling by express with cooling packs. Do not place cylinders directly onto the cooling packs (add a layer of isolation material) and avoid compacting the cylinders by directly staking them on top of each other, add a layer of buffer material (polystyrene or buffer foil with air cushion) on the first layer of cylinders and then add a second layer of cylinders. Store earthworms from BENCHMARKS earthworm sampling at room temperature in a fume hood or an open and well aerated place. Change ethanol after 24 h to avoid ethanol dilution due to the water content in the earthworms. Store samples from BENCHMARKS plastic sampling at 4 °C and process them at the latest 48 h after sampling. Freeze a subsample of 200 g at -20 °C in an aluminum coated paper bag or in a paper bag placed inside a plastic bag as backup. Dry the remaining samples, including the plastic-free control at 40 °C in an oven until constant weight. Avoid contamination by i) cleaning the oven before use, ii) avoid contact with plastic material, iii) try to wear no/few synthetic cloths during sample handling and iv) avoid possible sources of dust in the room with the oven and only keep the soil exposed to the environment during drying. Directly after drying, store samples in aluminum coated paper bags to avoid further contamination at room temperature until further processing. Sieve samples at 2 mm metal sieve into a metal con- tainer. Transfer 2 x 200 g soil into a labeled paper bag and double pack each sample for transportation. An overview of shipping details including sample properties and volume and shipping condi- tions (regular, express, express dry ice) is shown in Table 1. For each package, fill out an anal- ysis order (template provided in Appendix 1.3) and proforma invoice (template provided in Appendix 1.4) and send it together with the samples (the analysis order inside the package and the proforma invoice attached outside of the package). Before shipping the samples, in- form the recipient and pass over the tracking number of the parcel as soon as the parcel has been posted. Natural resources and bioeconomy studies 40/2025 14 Table 1. Overview of shipping requirements, sample properties and volumes per analysis type. Analysis Sample property and volume Shipping Chemical properties Moist, 500 g Regular Active carbon Air-dried, 50 g Regular Pollutants Moist, 200 g Expressa Nitrogen mineralisation Moist, 50 g Expressa Nematodes extraction and morphotyping Moist, 350 g Expressa Biological analyses Moist, 200 g Expressa DNA metabarcoding Moist-frozen, 20-30 g Express dry iceb Mesofauna extraction and morphotyping Moist, cores from mesofauna sampling Expressa Aggregates Moist, 150 g Regular-cautiousc Earthworm morphotyping Earthworms in >90% Ethanol Regular Soil hydraulic properties Moist, undisturbed cores of hydraulic property sampling Regular-cautiousc Plastic analyses Air-dried, 400 g from plastic sampling Regular; in paper bag aExpress: Ship samples in styropor box with ice packs by express courier (e.g. Fedex, DHL) and provide tracking number to recipient. bExpress on dry ice: Ship samples in styropor box with dry ice by express courier (e.g. Fedex, DHL) and provide tracking number to recipient. Only ship samples Mondays or Tuesdays. cRegular-cautious: Pack and ship soil cores to protect them from vibration, shock, extreme heat and freezing. Natural resources and bioeconomy studies 40/2025 15 4. BENCHMARKS soil health indicator catalogue For basic characterisation, and depending on the specific challenges of a site, a set of chemi- cal, physical, and biological SHIs is selected for analysis (Table 2, Table 3, Table 4). Each SHI is analysed in the same laboratory using standardized methods to ensure data comparability. Table 2. Methods used for the analysis of chemical soil health indicators in BENCHMARKS soil samples. Soil health indicator Methods Reference Cation exchange capacity Extraction in 0,1 mol/l BaCl2 followed by ICP- AES ÖNORM L 1086-1 Electrical conductivity Metal electrode in a 1:5 (W/V) suspension of soil in H2O extract ISO 11265:1994 pH Glass electrode in a 1:5 (W/V) suspension of soil in 0.01 M CaCl2 extract ISO 10390 Total nitrogen Elemental analysis using a CNS at 1250 °C ÖNORM EN 16168 Plant available phosphorus Sodium hydrogen carbonate extraction fol- lowed by spectral photometry ISO 11263 Plant available potassium Calcium-acetate-lactate extraction followed by flame photometry using a Segmented flow Analyser SAN ÖNORM L1087; Schüller, 1969 Copper, Iron, Mangan- ese, Molybdenum, Nickel, Zinc Aqua Regia extraction followed by ICP-OES NEN 6961: 2014; NEN 6966: 2005 Soil organic carbon Dry combustion at 900-1500 °C ÖNORM EN 15936 Active carbon Pyrolysis Rock-Eval coupled with the PAR- TYSOC model Cécillon et al., 2018; Cécillon et al., 2021; POM:MAOM Rapid particle size fractionation to determine labile vs. stable cycling soil organic carbon Baldock et al., 2013; Lavallee et al., 2020; Poeplau et al., 2018; Sander- man et al., 2013 Metals Aqua Regia extraction followed by ICP-MS Rotter et al., 2017 Pesticides QuEChERS method followed by LC-MS and GC-MS Geissen et al., 2021; Svobodová et al., 2018; Lehotay et al., 2005 Persistent organic pollutants Determination of persistent organic pollu- tants by GC-MS Tombesi et al., 2017; Llanos et al., 2022 Plastics Extraction of microplastics from soils with subsequent µ-FTIR analysis Foetisch et al., 2024 POM:MAOM, ratio of particulate organic matter and mineral associate organic matter. Natural resources and bioeconomy studies 40/2025 16 Table 3. Methods used for the analysis of physical soil health indicators in BENCHMARKS soil samples. Soil health indicator Methods Reference Soil texture Calculated from the mass and the volume of sole cores taken with rings of known volume ISO 11277:2020 Aggregate fractions Wet sieving method Elliot et al., 1986; Six et al., 1998 Bulk density Cylinder (gravimetric) method ISO 11272:2017 Soil water retention, un- saturated soil hydraulic conductivity Wind’s evaporation method, HYPROP2 Arya, 2002; Basile et al., 2006; Van Genuchten, 1980. Bin Shokrana and Ghane, 2020 Saturated soil hydraulic conductivity Constant head and falling head method (lab method), adapted single ring infiltrometer method (field method) Reynolds et al. 2002 Natural resources and bioeconomy studies 40/2025 17 Table 4 Methods used for the analysis of biological soil health indicators in BENCHMARKS soil samples. Soil health indicator Methods References Potentially mineralizable nitrogen Anaerobic incubation ÖNORM L1204 Microbial biomass carbon and nitrogen Chloroform-Fumigation Extraction Vance et al., 1987 Earthworms Hand sorting and morphological identification Sims and Gerard, 1985; Marcel-B. Bouché, 1972 Microarthropods MacFadyen extraction and morphological identification Macfadyen, 1962; Parisi et al., 2005; Vandewalle et al., 2010 Microarthropods DNA metabarcoding of microarthropods Shokralla et al., 2015; Elbrecht et al., 2019 Nematodes Extraction, counting and morphological identi- fication Bongers, 1994; Oostenbrink, 1960 Nematodes DNA metabarcoding of nematodes Stoeck et al., 2010 ; Shokralla et al., 2015 Microorganisms (bacteria, fungi) DNA extraction, 16S and ITS fragment PCR amplification and sequencing using Illumina or PacBio platforms Lori et al., 2023; Labouryie et al., 2023 Bacterial abundance qPCR of 16S marker gene Caporaso et al., 2012; Han et al., 2023 Fungal abundance qPCR of 18S marker gene Vainio and Hantula, 2000; Han et al., 2023 Nitrifying archaea qPCR of ammonia monooxygenasea (moA) functional genes Leininger et al., 2006; Schauss et al., 2009; Han et al., 2023 Nitrifying bacteria qPCR of ammonia monooxygenasea (moA) functional genes Rothauwe et al., 1997; Han et al., 2023 Nitrous oxide reducing bacteria qPCR of nitrous oxide reductase (nosZ, nosZII) functional gene Henry et al., 2006; Han et al., 2023 Proteolytic bacteria qPCR of alkaline metallopeptidase (apr) and neutral metallopeptidase (npr) functional genes Bach et al., 2001; Han et al., 2023 Urea-hydrolizing bacteria qPCR of urease (ureC) functional gene Gresham et al., 2007; Han et al., 2023 Natural resources and bioeconomy studies 40/2025 18 5. Field-based assessments Document each sampling site by photography from each cardinal direction. In addition, rec- ord information about climate and weather conditions, site description including information on landscape, land use, ground cover and human influence and describe soil surface charac- teristics such as coarse surface fragments, signs of soil erosion or soil surface sealing, etc. us- ing the BENCHMARKS field observation protocol (Appendix 1.2). Also report deviations from the original protocol. If the soil is characterised by a high quantity of stones, take a sample of 20 cm x 20 cm x 20 cm with a spade to quantify the amount of stones (in kg). If possible, take additional on-site measurements, depending on the research focus and avail- ability of tools (Table 5). Table 5. Overview of field-based assessments. Soil health indicator Methods Reference Soil profile Guidelines for soil description FAO (2006) Guidelines for soil description Soil erosion Visual observation FAO (2006) Guidelines for soil description Surface sealing Visual observation FAO (2006) Guidelines for soil description Natural resources and bioeconomy studies 40/2025 19 6. Identification and registration of samples Sampling points are identified by unique sample identifiers (IDs) previously assigned based on the geo-reference points. The sample IDs are composed of a site ID, treatment ID and profile ID. To distinguish individual samples collected at the same sampling point (profile ID), sample IDs are further amended by layer ID and a sample type ID (BULKS for bulk soil sam- ple; BDENS for bulk density sample, HYDRA for hydraulic property sample, PLASTIC for plastic sample, MFAU for mesofauna sample, EWORM for earthworm sample). To distinguish sam- ples collected in different years, the sampling date is added at the last position of the label ID. These sample IDs are used in each sampling campaign to record agro-environmental data related to each point on the data management platform. At each sampling point, surveyors document agro-environmental observations by filling in the BENCHMARKS field observation protocol and by taking photographs. All the data is then stored on the data management platform. Natural resources and bioeconomy studies 40/2025 20 7. Acknowledgements This research was funded by the European Union's Horizon Europe research and Innovation program, under Grant Agreement: 101091010, Project BENCHMARKS. This work has received funding from the Swiss State Secretariat for Education, Research and Innovation (SERI) under contract no. 22.00619. Natural resources and bioeconomy studies 40/2025 21 References Arya, L.M. 2002. Wind and hot-air methods. Methods of Soil Analysis 4: 916‒920. 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Methods in Ecology and Evolution 3(4): 613‒623. Natural resources and bioeconomy studies 40/2025 26 Appendix 1 1.1. Sampling and sample processing materials Common material and equipment for sampling Sampling • GPS (will be brought by sampling core team) • Camera (mobile phone also fine) • Field observation protocol (one for each sampling point, or only one + notebook to write down the in-field assessments) • Paper tape • Tape measure • Wooden scale of 2 m • Compass • Knife • Trowel/ hand shovel • Marker sticks: large (n=no of sampling points), small (n=no of sampling points x3) • Rope (min. 2.5 m) • Scissors Sample packaging and transport • Labelled plastic bags • Permanent markers • Cooling boxes (enough to fit all samples) • Freezer packs • Hand scale Cleaning • Lab gloves • Water • Tissue paper • Brush BENCHMARKS bulk soil sampling • Soil corer (min. 2-5 cm diameter) • Wooden device to get the soil core out of the corer • Big rubber/plastic hammer (when soil is dry) • Buckets (min. 1 per sampling depth + some spare) • Metal spoons Natural resources and bioeconomy studies 40/2025 27 BENCHMARKS bulk density sampling • Large spatula • Trowel • Rubber/plastic hammer • Wooden blocks (min. 4 cm thick) • Steel cylinder (5 cm diameter * 5 cm height) • Vaseline • Plastic film • Knife BENCHMARKS soil hydraulic properties sampling • Same as for bulk density sampling • Steel cylinder (7 cm diameter * 7 cm height) BENCHMARKS earthworm sampling • Spate • Large and stable plastic bags or large trays for hand-sorting of the earthworms • Tweezers • Labelled sample containers (e.g. Falcon tubes, or 100 ml beaker with air-tight lid) filled with 90% EtOH BENCHMARKS mesofauna sampling • PVC cylinders (5 cm x 5 cm) for undisturbed soil cores • Split corer for litter • Rubber/plastic hammer • Wooden block (min. 4 cm thick) • Knife • Plastic film • Needle BENCHMARKS plastic sampling • Soil corer (2-5 cm diameter) • Wooden or metal device to get the soil core out of the corer • Aluminium bowl/metal bucket • Hand shovel • Aluminium containers (n=no of sampling points) (example here) • If soil is dry: wooden blocks (min. 4 cm thick) + steel hammer Common material and equipment for sample processing • Sieves 2 mm OR 5 mm (clay or peat soils) • Bowls • Balance • Spoons • Aluminum trays or similar for air-drying the soil Natural resources and bioeconomy studies 40/2025 28 • Water • Tissue paper • Lab gloves (S/M/L) • Labelled bags • Additional bags • Cooling boxes • Cooling packs • Thermoboxes • Bubble wrap • Cooling packs Natural resources and bioeconomy studies 40/2025 29 1.2. BENCHMARKS field observation protocol Benchmarks field observation protocol Sample ID: ……………………… Date: ……………………………. Sampler: ………………………..... Sample site location Site ID: ………………. City: …………… Country: …………… Date: ……………… Coordinates (in degree, minutes, seconds) Longitude: ………. °/ ………….'/………….'' Latitude: ………. °/ ………….'/………….'' Altitude (m): ………………………………… Climate and weather conditions (see Table 2 in Guidelines for soil description, fao.org) Monthly mean temperature: ............ Monthly mean precipitation: ............. Present weather conditions: ................ Former weather conditions: ................ Site description (see Table 4, 8-11 in Guidelines for soil description, fao.org) Landscape/Topography: ………………........... Land use: …………………….................. Ground cover/crops: ………………….................... Human influence: ……………………….......... Soil description (see Table 14-20 in Guidelines for soil description, fao.org) Coarse surface fragments: …….. Surface cover: ............... Size classes: .............. Soil erosion: Category: …….. Area (%): ............ Degree: .............. Surface sealing: Thickness (mm): ………. Consistency: .............. Sample description Texture: □ Sandy □ Sandy-loam □ Loamy □ Clayey-loam □ Clayey □ Clay □ Peat Sample humidity: □ Dry □ Moist □ Wet Coarse fraction (%, in case of a high quantity of stones): ……………. Remarks/deviation from sampling protocol: …………………………………………………………………………………………………………………………………………………… ….……………………………………………………………………………………………………………………………………………… ………. Natural resources and bioeconomy studies 40/2025 30 Photographs Sample ID Sampling site photograph North facing photograph East facing photograph South facing photograph West facing photograph Additional photograph Additional photograph Natural resources and bioeconomy studies 40/2025 31 1.3. Template – Analysis order with shipping information Analysis order Recipient information: Institution: … Address: … Name: … Phone: … Email: … List of soil analysis: • … • … • … Required sample volume: … Soil conditions: ... Shipping conditions: … List of soil samples Site ID Land use type Sample ID Institution Sampler Sampling date Weather condition Sample weight (g) Natural resources and bioeconomy studies 40/2025 32 1.4. Template – Proforma invoice Sender Institution: Name: Address: Phone: Email: Recipient Institution: Name: Address: Phone: Email: Date: Proforma invoice Commission: Name of sender / Institution Content: Soil samples / For soil analyses / Scientific research Weight samples: ……. kg Weight packaging: ……. kg Value: Euro 1.00 No commercial value, for laboratory analysis only Country of origin: …. ------------------------------------------------------------- Add name here and signature above Natural resources and bioeconomy studies 40/2025 33 You can find us online luke.fi Natural Resources Institute Finland (Luke), Latokartanonkaari 9, 00790 Helsinki, Finland