Biological indicators of soil quality

Living soils Biological fertility of soils in large-scale crops

Here are several tests that provide an indicative result of the biological quality of a soil.

Role of organisms

Soil is a complex ecosystem in which mineral and organic matrices interact, liquid and gaseous fluids, macroscopic and microscopic organisms coexist. It is a living environment where multiple organisms live in community: bacteria, fungi, algae, animals or plants. They can be classified according to several criteria such as their size, their diet, their habitat or their ecological function.

All these elements can be measured by field tests or in the laboratory. Standardized or not, these tests inform us about the biological aspect (microbial respiration, faunal abundance...), the physical aspect of the soil (texture, structural state, infiltration, resistance, water presence...) and the chemical aspect (acidity or alkalinity, carbon content, nitrogen content...).

The fauna contributes to the formation and evolution of the soil. These organisms are the main agents of the nutrient cycle: they regulate the dynamics of the organic matter in the soil and the absorption of nutrients by vegetation, participate in carbon sequestration and greenhouse gas emissions, modify the physical structure of the soil and water regime, and improve plant health. All these functions guarantee good soil fertility and therefore good soil quality.

Classification of organisms

Four subgroups can be distinguished, differentiated according to their size:

The megafauna (organisms larger than 10 cm) such as mammals, reptiles…

The macrofauna (organisms from 4 to 80 mm) such as earthworms, insects, myriapods…
The mesofauna (organisms from 0.2 to 4 mm) such as mitess, springtails…
The microfauna (organisms smaller than 0.2 mm) such as protozoa, nematodes…

Indicators

The quality of this fauna results from genetic diversity, organism diversity and ecological diversity. This quality is evaluated using faunistic indicators to determine the diversity of fauna and using microbiological indicators to determine the biological activity, abundance and diversity of microorganisms.

Faunistic indicators make sense in the context of environmental risk assessment in contaminated/polluted sites, to report on soil fertility and the impact of different agricultural practices on soil quality.

Microbiological indicators make sense in the context of a yield decline, to understand the impact of inputs or activators on soil quality, to assess the quality of green spaces and to determine the impact of changing agricultural practices.

The results of these tests will be:

Abundance: the number of individuals per m² (overall or by categories).
Biomass: the mass of individuals per m² (g/m²).
Richness: the number of species.

Study of macrofauna

Study of earthworms (macrofauna)

Earthworms, earthworms, are living beings very important for soil life. Indeed, they provide many services thanks to their biological activity. They participate in the formation of topsoil: repeated mixing of the soil, incorporation of organic matter, effect of burrowing, casting (bioturbation containing many soil elements), overall erosion-sedimentation cycle with hydric and aerial transfers of fine soil particles brought to the surface.

The Bouché method

The sampling area is 1 m².

Gently clean the surface (cut vegetation and remove litter or organic clumps).

Apply 3 waterings of formalin solution or a chemical extractant (e.g., for 1m², use 10 liters of mustard solution) at 15-minute intervals.
Collect earthworms on the soil surface then perform a superficial scraping up to 1 cm depth to recover uncollected individuals. Perform a physical sampling on the area: extract a soil block (25x25x20 cm) and sort manually.
Place the found individuals in 4% formalin (for preservation) and proceed to species identification.

Equipment: A spade, basins, formalin.

: Fast.

: Fauna destruction.

Study of macro-invertebrates (average size over 10 mm)

The TSBF and IndVal method

Delimit a soil area of 25 cm side and 30 cm depth.
Divide the sample into 3 layers by depth: 0-10 cm, 10-20 cm and 20-30 cm.
It is possible to use a 0.2% formalin solution or a mustard-based solution to extract macro-invertebrates from the soil (up to 15 cm depth).
Manually sort the fauna then proceed to species identification.
Biological Indicator of Soil Quality: IBQS= ∑ln(Di+1)xSi IBQSЄ[0;20]. Di: average density of species i at a site (obtained with TSBF method). Si: indicator value of the taxon (with IndVal method).

Equipment: A spade, basins, formalin.

: Reliable.

:

Long.
Fauna destruction.
The "repellent" aspect of formalin reduces sampling quality (escape of part of the fauna).
Difficulty injecting the repellent in a precise volume (use a frame to limit the area).

Study of aerial insect populations with subterranean larvae

The emergence cage

Place the emergence cage on the soil. Wait a few days then collect the insects present in the trap. Proceed to identification.

Equipment: A trap or an emergence cage.

: Simple and easily transferable from one place to another.

: The study time is long (the transition from larva to adult stage takes several days).

Study of the entire macro and mesofauna

The Berlese-Tullgren extractor

This extraction is often done after sieving the soil sample. For mesofauna, extraction is performed on several small homogeneous samples.
Place a known volume of soil in a funnel whose outlet hole is closed by a mesh.
Place a container with a preservative liquid under the funnel outlet.
Place the assembly under an incandescent lamp (heat source).
Identify the species found.

Equipment: A funnel, 70% alcohol (a preservative liquid), a heat source.

: Easy, fast and inexpensive.

: Fauna destruction by the preservative liquid.

Study of enchytraeids (mesofauna)

The water funnel

Using a corer, take soil samples at different depths (in 5 cm increments).
Extract enchytraeids using a water funnel: place the sample in a 2 mm mesh nylon net on the funnel, itself placed on a test tube filled with water, cover the surface with a glass or plastic disc, avoiding trapping air bubbles, place the device under a heating lamp.
Enchytraeids flee (light and heat) the funnel seeking a more oxygenated environment at the bottom of the test tube. They are deposited in a petri dish and counted using a binocular magnifier and a graph paper.

Equipment: A corer, a funnel, a nylon net, a test tube, a glass disc, a heating lamp, a petri dish and a binocular magnifier.

: Effective.

Study of microfauna

Microorganisms play a key role in the recycling of organic matter in the soil and in the alteration of minerals into nutrients made available to plants. These microorganisms also help to stabilize soil aggregates and participate in water regulation circuits through the excretion of exo-polysaccharides, which contribute to the water retention of the soil. Microorganisms include: bacteria, archaea, yeasts, microalgae and filamentous fungi.

Microbiological indicators of soil have two main objectives: to determine the biological activity of a soil through the decomposition of organic matter and to determine the abundance and diversity of soil microorganisms.

Study of micro-arthropods (from 100 µm to a few mm)

The MacFadyen extractor

Push the corer as deeply as possible into the soil (if the soil is hard to penetrate, start by cutting the soil with a spade to make two parallel openings).
Open the corer then cut the core progressively with a serrated knife. Fix a cap on each side of the sample indicating the top and bottom of the core.
Extract the mesofauna using the MacFadyen extractor. Transfer micro-arthropods into preservation alcohol.
Sort and identify species under a binocular magnifier or microscope.

Equipment: A corer, a serrated knife, sampling boxes, a MacFadyen extractor, a binocular magnifier or microscope.

: Fauna destruction.

Study of nematodes

For all these methods, the first step is to collect soil samples from the 0-20 cm layer with a corer and ends with counting. Ultimately, we obtain abundance: number of individuals/m² (overall or by ecological categories), biomass: g/m² and richness: number of species.

Elutriation

Equipment: A corer, an Oostenbrink elutriator, sieves, a binocular magnifier or microscope.
Extract nematodes by elutriation: when a soil sample is placed in water, heavy soil particles settle faster than nematodes. During elutriation, a constant upward water flow prevents nematode sedimentation. To collect nematodes, pass the supernatant through a sieve and count.

Cobb or bucket method

Equipment : A corer, 2 buckets, 1 mm mesh sieves, a finer mesh sieve, a beaker or reading cup.
Mix the sample with water by vigorous stirring in bucket A then let settle.
When most of the mineral phase has settled at the bottom of bucket A, pour the supernatant water from bucket A into bucket B through a 1 mm sieve. Slowly pour the water from the second bucket onto a fine mesh sieve to retain nematodes.
Collect nematodes in a beaker or reading cup.

Seinhorst two-flask method

Equipment : A corer, beakers, sieves, Erlenmeyer flasks, funnels, a binocular magnifier or microscope.
Mix the sample with water in a beaker then pass the muddy suspension through a 1 to 2 mm mesh sieve to remove large debris.
Transfer it to an Erlenmeyer A equipped with a funnel. Fill with water then invert it over an Erlenmeyer B filled with water so that the funnel of A is submerged 1 to 2 cm in the water in B. Let stand about 10 minutes (soil falls from A to B).
Invert Erlenmeyer A over a beaker C full of water for 10 to 20 min. Invert B over a beaker D full of water. Then invert B over C. Contents of A and B are passed through a 40 to 50 μm sieve and that of C through a 90 to 100 μm sieve.
Collect nematodes.
50% of small nematodes are in Erlenmeyer A and the rest in B and C (which contains 75% of the larger ones). Beaker D contains almost no nematodes and its content is discarded.

Baermann funnel method and modified Baermann method

Equipment : A corer, funnels, a sieve, a flexible tube, a paper/fabric filter, a beaker, a binocular magnifier or microscope.
A sieve is held in the upper part of a funnel. The funnel tube end is equipped with a clamped flexible tube. A paper or fabric filter is placed in the sieve, the soil or plant material sample to be extracted is placed on the fabric. Water is poured into the funnel until the sample is covered.
After 12 hours to 3 days, mobile nematodes have crossed the fabric and are at the lower end of the funnel. They are collected in a beaker by loosening the clamp and taking a few ml.

Method
Elutriation		Sampling: beginner. Analysis: Expert.
Cobb / bucket	Simple.	Not precise: Sedimentation judged by eye. Extraction efficiency highly variable. Beginner.
Seinhorst	Allows recovery of small to medium-sized nematodes. The device separates the extract into several fractions: easier sieving.	Fragile. Sampling: beginner. Analysis: Expert.
Baermann	Allows recovery of mobile nematodes (if oxygenation is poor, their mobility is reduced: modified Baermann method (using a dish instead of a funnel)).	Inefficient. Expert.

Study of soil biological activity

To determine the biological activity of a soil, we rely on the decomposition of organic matter.

By observing the degradation of matter, we calculate:

A decomposition rate.
A carbon dioxide rate released by the mass loss of the partially or totally decomposed material (curves).

These tests are carried out both in the field and in the laboratory by observing the degradation of elements. A precise decomposition rate curve generally requires several data points over time, which implies the need to be close to the study site.

The cotton underwear method

Equipment: A white, organic cotton underwear.
Bury a white cotton underwear in the soil to be studied.
Wait about 2 months. Wash the underwear (to remove soil) and observe cotton decomposition.
Color and odors can be studied. They indicate differences in microbiological activity. Odor is often of the type "compost", "humus", "fungi" in the most active soils. A musty odor points more to a soil with problems such as root asphyxiation for example.

The tea bag method

Equipment: A tea bag (green tea, Rooibos) and a scale. It is possible to use different types of tea with slow or fast decomposition.
Bury a tea bag in the soil to be studied after weighing it.
Wait 3 months. Weigh the tea bag again and observe the decomposition of tea leaves.

LEVA-bag

Equipment : A nylon bag, straw or a LevaKit.
Bury a nylon bag (1 mm mesh) filled with straw in the soil to be studied after weighing it.
Wait 4 months. Weigh the nylon bag again and observe straw decomposition.

The Bait Lamina test

Equipment: perforated plastic strips, baits, light and a computer (matrix program).
Take 16 cm long PVC plastic strips perforated with 16 holes. Fill the holes with organic baits (cellulose + wheat bran + activated charcoal). Insert the strips vertically into the soil and leave them about 14 days.
Remove the strips from the soil, wash them (to remove soil residues) and place the strips near a light source to count the baits that have been eaten.
Scores are assigned related to bait consumption (2 - bait completely consumed; 1 - bait partially consumed; 0 - bait not consumed). They are reported on a 16 x 16 matrix. A percentage of feeding activity per soil layer and per replicate can thus be calculated. Then total feeding activity can be deduced.
The test duration depends on season, temperature, humidity and soil.


Method
Cotton underwear	Simple. Easy to use. Standardized method: allows comparison of different data. Analysis of odor and color.	Not an established scientific protocol.
Tea bag	Simple. Low cost. Practical. Standardized method: allows comparison of different data.	Not precise.
LEVA-bag	Simple. Easy to use. Standardized method: allows comparison of different data.	Not precise. Sensitive to pedoclimatic conditions.
Bait Lamina test	Any soil type. Low cost. Fast.	Impossible on floodable or very shallow soils. Difficult in extreme climatic or geographic conditions.

Study of active soil biomass

To estimate the active biomass of soil microorganisms, their abundance and diversity, one can study gas exchanges during respiration and the overall microbial diversity of an environment by DNA sequencing of all genomes present in that environment.

These measurements are done in the laboratory and require prior preparations.

Other indicators inform us about organism abundance and diversity such as the method by phospholipid fatty acid analysis (PLFA) and ether-phospholipid lipid analysis (PLEL) or the method by phospholipid fatty acid analysis (PLFA) using the simple PLFA extraction method (these will not be presented).

Study of gas exchanges

The respiratory activity of microorganisms depends on several factors, such as the moisture content of the environment and the availability of nutrients present in the soil.

The MicroRespTM and SituResp Technique

This technique measures: the basal respiration of the soil (active microorganisms in the soil catabolize the organic matter-nutrients present and accessible in this soil) and the microbial biomass (all active microorganisms catabolize the solubilized glucose distributed in excess and their CO₂ release is proportional to their biomass).

Equipment: Sampling equipment, sieves, deep well plates, seals, microplates, substrate, incubator, spectrophotometer.
The "catabolic fingerprint" of the soil (additions of various solubilized carbon substrates, different according to their composition and the origin they represent).

It is necessary to prepare the detection microplates, substrates, and deep well microplates:
- The detection microplates: these microplates must be prepared one week before use and must be used within three weeks thereafter. The plates must be stored in the dark in a sealed container containing soda lime (to remove CO₂) with a humid atmosphere to prevent desiccation of the gel in the plates.
- The deep well microplates: Weigh the empty deep well microplate. Each soil sample is then volumetrically and homogeneously distributed into the wells. Place the deep well microplate on a support. Position the dispensing device with its bottom plate on the deep well microplate. Deposit the soil aggregates on the system (after previously removing any remaining roots or plant debris). Gently "level off" the holes of the dispenser (with a gloved hand). Once the soil sample is distributed, slide the bottom plate to allow the soil to fall into each deep well of the microplate. Weigh the microplate again after each soil sample distribution to precisely know the average soil weight per well. It is possible to measure the respiration of several soil samples on the same microplate by filling only part of the microplate. Place parafilm on part of the deep well microplate and proceed as indicated above. The microplate is ready to receive the substrates.

Collect a soil sample. Mix the soil and sieve with a 2 mm mesh sieve.
Distribute the sample into deep well plates, put different carbon substrates in the wells (glucose, sucrose, trehalose, cellulose, starch, malate, oxalic acid, malic acid...). The substrates are prepared at a concentration of 120g/L. Substrates dissolved in distilled water must be stored at 4°C for a maximum of 15 days. Substrates, before contact with the soil, must be brought to the bioassay temperature. In each well, 25 µl of substrate will be added. Place a sealing gasket on the deep well plate, thus isolating each well. Cover the plate/gasket with a detection microplate containing an agar gel and a color indicator.
Incubate the microplates in the incubator at 29°C for 6 hours.
Measure the color change of the gel by spectrophotometer at a wavelength of 570 nm. The color change of the agar gel is proportional to the amount of CO2 released. The CO2 release from the so-called "control" wells (no substrate added) corresponds to the basal respiration of the soil.

The Fumigation-Extraction Method

Equipment: A vacuum incubator, a vacuum pump, a separatory funnel (for washing chloroform), small glass containers, a shaker table, a centrifuge, centrifuge tubes, a dissolved organic carbon (DOC) analyzer.
Take a soil sample with a known volume. Divide it into two batches: a control batch (non-fumigated) and a fumigated batch. The fumigated batch is treated with chloroform vapors for 16 hours (in a vacuum desiccator).
The organic carbon extraction is performed by shaking the sample in K2SO4 (0.05 N) for 45 min at 20°C. The extraction is followed by centrifugation at 6000g for 5 min to separate the soil pellet from the supernatant (in which the DOC will be quantified).
The dosage is measured by persulfate oxidation under UV radiation (Dohrman DC 80 device) of the soluble organic carbon.
The difference in soluble organic carbon between the two types of samples (fumigated and non-fumigated controls) gives the amount of "extractable" carbon of microbial origin. This amount can be converted into biomass using a proportionality coefficient (0.38) according to Chaussod and Houot (1993).


Method
MicroResp TM	Sensitivity and selectivity of substrates allow to differentiate activities and discriminate low levels. Simple. Low cost.	Lack of measurements (with standardized or normalized protocols in different contexts) which limits interpretation. Environmental variables have a significant influence on parameters.
Fumigation-extraction	Accurate. Can be linked to other measurements. Applicable to freshly collected non-dried soils.	Reference data remain to be shared and consolidated among different laboratories implementing this measurement. Chloroform vapors (fumigation) destroy the cells of living microorganisms.

Genome Studies

These methods proceed by direct extraction of DNA from the soil. Genome study allows to:

Make an inventory of species in difficult environments.
Inventory of species difficult to identify.
Allows detection and monitoring of invasive species.
Evaluate the ecological quality of environments.
Monitor the modification of distribution areas.

Warning, the presence of DNA does not always mean the presence of organisms.

The Metagenomic Method

Field sampling. DNA extraction. DNA sequencing. Sequence analysis.
Identification of functional aspects of the genome (without necessarily determining species).

The Metabarcoding Method

Field sampling. DNA extraction. DNA amplification (mitochondrial). Sequencing of amplified DNA. Sequence analysis.
Identification of the species present in the sample.

A Still Partial View

To determine the overall quality of a soil, the analysis of a single indicator is not relevant. Indeed, soil is a very complex environment and in constant interaction with the surrounding ecosystem. It is therefore perfectly logical to reason on a larger scale by considering several indicators at once, including indicators on soil biology, on physical properties, and on chemical properties.

Soil analysis can also be performed by experts. For example, there is a methodology developed by researchers from the IRD and Cirad to assess soil health by studying their biological activity: Biofunctool®. This method is based on a multi-criteria evaluation of the three essential functions for soil life and the organisms it contains: the carbon dynamics, the nutrient cycle, and the maintenance of soil structure.

This analysis can be performed either by Cirad and IRD experts (within the framework of services or research and development projects), or by agronomic experts who have received training (2023).

The indicators presented here are specific to soil horizons. There are other less soil-specific indicators, such as snails and so-called indicator plants.

Sources

INRAE, SOLAE, Mégane PEREZ (2021), Contribution to the appropriation and generalization of the use of soil quality evaluation indicators in Provence –Alpes – Côte d’Azur, rd-agri, https://opera-connaissances.chambres-agriculture.fr/doc_num.php?explnum_id=163817