BIOservicES Soil sampling procedures and methods

Continuous research is imperious in our mission to understand the interconnection between soil organisms and the delivery of multiple soil ecosystem functions and services at different scales. An important part of research is soil sampling, and gathering consistent samplings in all of our European experimental sites is crucial to understanding how different regions, climates, types of soils, and soil pressures affect soil health.

We can identify regional trends and variations through standardised sampling and analysis methods, providing valuable insights into the factors that support or challenge soil health. This research contributes to developing sustainable land management practices that protect soil health and enhance ecosystem resilience across the continent.

For this reason, WP 2 – Soil biodiversity and ecosystem functions and services across biogeographic areas and land uses, has defined harmonised soil sampling procedures for each indicator, such as microbiota, nematodes, mesofauna, macrofauna, soil structure, or physicochemical properties. Beyond the soil sampling procedure, soil storage and shipment, different analysis types, were defined to ensure a cohesive approach for all of the experimental sites.

As a result, in the BIOservicES project, an extensive sampling was agreed between April and May 2024, which will be repeated in April-May 2026 in specific land uses and regions to assess the temporary dynamics. Before the sampling, we should follow a few steps:

1. Recording Vegetation

First, we record the main vegetation species within the sampling area, which covers a circular plot with a diameter of 4 meters. If the area is cropland, we note the crop(s) present during sampling. Additionally, we document vegetation richness by counting the different plant species within the sampling area. Estimating the percentage of vegetation cover in the plot helps us understand how much of the area is covered by plants.

2. Monitoring Weather Conditions

On the sampling day, we record the prevailing weather conditions, such as whether it is sunny, cloudy, rainy, windy, or foggy. These observations are essential as they can influence soil properties and the organisms present in the soil.

3. Capturing Images and Videos

To provide a comprehensive overview of the study site, our team captures photos and videos following the guidelines created by the Communication Team. These visuals help convey each sampling spot’s context, specific details, and different sampling types.

4. Adhering to Best Practices in Sampling

During the soil sampling process, we follow several best practices to avoid contamination and maintain data integrity:

• Avoid areas near edges, such as roads or pathways, which can introduce bias into the sample.
• Disinfecting sampling equipment with 70% ethanol between samples ensures cleanliness and prevents cross-contamination.
• Disposable gloves are used to handle samples, further reducing the risk of contamination.
• Proper labelling using official codes on all bags, tubes, and containers ensures that data can be accurately tracked and analysed.

Additionally, we record the geographical coordinates (decimal) of the central point of the sampling scheme in each area to provide precise location data. For some methods, like LUCAS sampling with an auger, we may sample additional points within the circle to gather sufficient samples.

In the field, to better understand the soil ecosystem and its inhabitants, we use specific techniques for collecting and analysing different types of fauna:

Earthworms and Macrofauna

We excavate a square pit with an area of 25×25 cm and a depth of 20 cm. The soil is separated into two layers: 0-10 cm (for collecting earthworms and macrofauna) and 10-20 cm (for earthworms only). The soil samples are placed on different plastic sheets for sorting.

The earthworms and macrofauna are carefully hand-sorted from the soil, roots, and litter using plastic tweezers or forceps. Earthworms are then rinsed, dried, and preserved in a formalin and ethanol solution. Other macrofauna are preserved in labelled test tubes with ethanol.

To expel deep-burrowing earthworms, we introduce 2.5 L of AITC solution into the pit. The expelled earthworms are collected, treated, and preserved as described above.

Mesofauna

We use a soil core sampler to collect soil cores of 4 cm diameter and 0-20 cm depth. These cores are separated into two depth levels (0-10 and 10-20 cm) and placed in beakers or properly labelled containers.

Samples are transported to the lab in cool boxes and extracted quickly. They are kept at 4°C at the lab and processed within four weeks.

Our soil sampling guidelines include techniques for collecting soil samples for physicochemical properties, microbiota, and nematodes:

Soil Structure, Bulk Density, and SOC

Samples are collected at 0-30 cm depths using a plastic liner auger. Plastic liners are labelled and sealed to prevent movement during storage and transport. We have increased soil depth up to 30 cm to analyse soil organic carbon to be aligned with IPCC and LUCAS recommendations for C farming monitoring.

Physicochemical Properties, Microbiota, and Nematodes

We collect around 3 kg of composite sample from five LUCAS points at 0-20 cm depth using an auger and homogenise the samples using a 5-mm sieve.

To ensure the integrity and reliability of soil sampling data, specific protocols must be followed for the preservation and storage of samples. Thus, our storage and shipment guidelines include:

1. Earthworms and Macrofauna

• Earthworms: After collecting the earthworms, they are treated with a fixing solution of a 1:1 ratio of 4% formalin and 96% ethanol. After ensuring the earthworms remain still for at least 2 minutes, they are transferred to a storage solution of 4% formalin.

• Macrofauna (Isopods, Myriapods, Insects, and Spiders): These are preserved and stored in 70% ethanol to maintain their condition and facilitate further analysis.

2. Mesofauna Sampling and Preservation

• Soil Samples: Unextracted soil samples should be shipped immediately in cool packs to retain their quality. Once extracted and preserved, mesofauna samples are shipped in boxes at ambient temperature.

• Extraction: The extraction process is conducted via the Kempson/MacFayden extractor, and the mesofauna samples are preserved in plastic vials with screw caps containing 96% ethanol.

• Storage: Mesofauna samples must be kept at 4°C in the lab for a maximum of four weeks. Promptly extracting the animals will ensure sample quality.

3. Physicochemical Properties and Microbiota

• Paper Bags for Physicochemical Properties: Around 2 kg of soil samples are air-dried and stored in paper bags to avoid plastic contamination, since microplastics will be analysed in the samples.

• Rigid Containers for Aggregates: Around 300 g of aggregates are air-dried, returned to rigid containers, and then kept at room temperature.

• Eppendorf Tubes for Microbiota: Microbiota samples are stored in tubes with DNA/RNA Shield and can be kept at room temperature for up to 30 days. After 30 days, they should be stored at -20ºC.

• Falcon Tubes or Ziploc Bags for Mineral N and Microarthropods: Around 40 g of soil samples are stored at -20ºC (or -80ºC if kept for over a week).

• Plastic Bags for Nematodes: Nematode samples should be kept at 4°C and not frozen. They must be processed within two weeks.

• Falcon Tubes for Viable Bacteria: Around 10 g of soil samples are stored at -20ºC and should be shipped within one week.

These protocols guarantee that the samples are preserved appropriately for subsequent analysis, enabling precise and reliable data collection for earthworms, macrofauna, mesofauna, and other soil components.