Terrestrial ecotoxicology aims to assess the effects of chemical substances on soil organisms, which are essential to the proper functioning of ecosystems, such as plants, microorganisms or invertebrates.
In this context, the OECD 208 test is a reference method for measuring the impact of a substance on the germination and growth of higher plants. This test is part of a battery of standardized tests used to characterize the ecotoxicological profile of a product, particularly within the framework of European regulatory requirements such as REACH, the authorization of plant protection products, or the evaluation of biodegradable materials.
To better understand the role and structure of these methods, consult our article dedicated to OECD guidelines in toxicology and ecotoxicology .
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An essential method for assessing the phytotoxicity and environmental safety of products
OECD Test 208: An Essential Standardized Method
OECD Guideline 208, first adopted in 2006 and revised in 2021, is specifically designed to test the impact of chemical substances on seedling emergence and early growth. It applies to a wide range of products: pure substances, formulations, mixtures, effluents, composts, etc. The test can be carried out in the laboratory, under controlled conditions, with standardized plant species to produce reproducible, scientifically robust results that can be used in regulatory dossiers.
Its value is twofold. On the one hand, it allows the identification of the harmful effects of a substance on plants representative of agricultural or natural ecosystems. On the other hand, it provides quantitative indicators (such as EC50 or CSEO) essential for calculating environmental risk, comparing the relative toxicity of several products, or justifying regulatory thresholds within the framework of hazard assessment.
A method used in a variety of industrial contexts
Beyond its traditional regulatory use, the OECD 208 test is increasingly being used by manufacturers for eco-design purposes or to demonstrate the environmental safety of their products. It can therefore be used:
- To demonstrate the non-toxicity of a new biodegradable material under composting conditions (e.g., plastic films, packaging, single-use items).
- To assess the potential impact of veterinary drug residues present in manure spread on the ground.
- To analyze the effects of complex mixtures of substances or formulated products, for example in biocides or multi-component plant protection products.
OECD 208 trial: a reference test for seedling emergence and growth
Objective and regulatory scope
OECD Trial 208 aims to measure the potential effects of a chemical on two critical stages of plant development: seedling emergence from seed and growth during the first few weeks of life. These parameters are essential because any disruption at this stage can compromise plant viability and affect the balance of terrestrial ecosystems.
This test is commonly required by health and environmental authorities as part of the environmental risk assessment of substances subject to registration. It is included in regulatory dossiers in several contexts:
- Authorisation files for plant protection products (Regulation (EC) No 1107/2009).
- Evaluation of biocidal substances (Regulation (EU) No 528/2012).
- REACH dossiers for substances produced or imported in quantities exceeding 10 tonnes per year.
- Environmental dossiers for veterinary medicinal products (Directive 2001/82/EC and Regulation (EU) 2019/6).
- Standards associated with compostable plastic materials, in relation to ASTM D6400 tests.
The OECD 208 trial is therefore a pivotal method, allowing the risk to be documented for non-target plants under reproducible experimental conditions.
Products tested and contexts of use
The test can be applied to a wide variety of substances and formulations, whether pure or in mixtures. Among the main products tested are:
- The active substances or co-formulants of plant protection products.
- Biocides for domestic, industrial or agricultural use.
- The compounds present in veterinary medicines, particularly when excreted by animals and spread via manure.
- Polymer or plastic materials designed to be biodegradable, particularly in composting environments.
- Industrial chemicals that may reach the soil (treatment agents, solvents, organic derivatives…).
In some cases, the test can be adapted to evaluate complex matrices such as composts, leachates or sludges, by incorporating these materials into the culture substrate.
Selection of plant species
To ensure the ecological relevance of the test, OECD guideline 208 recommends using a panel of plant species representative of taxonomic diversity. The choice of plants depends on the properties of the substance being tested, the intended exposure environment, and regulatory objectives. Generally, between six and ten species are selected, including both monocots and dicots.
Among the most commonly used species are:
- Allium cepa (onion)
- Zea mays (maize)
- Brassica napus (rapeseed)
- Cucumis sativus (cucumber)
- Glycine max (soybean)
- Helianthus annuus (sunflower)
- Solanum lycopersicon (tomato)
- Stellaria media (chickweed)
These species are chosen for their availability, their known sensitivity to various chemical agents, their homogeneous growth under controlled conditions, and their value as indicator plants of agricultural or semi-natural ecosystems.
Simulated exposure pathways
The plants can be exposed to the tested substance in different ways, depending on the type of product and the intended use scenario:
- For plant protection products, the substance is usually sprayed on the soil surface after sowing, which simulates an open field application.
- For veterinary medicines or general chemicals, it is incorporated into the soil before sowing, reproducing a scenario of exposure by leaching or via the spreading of contaminated manure or sludge.
- In the case of compostable products, the test may incorporate a compost containing the tested material, mixed with the soil in defined proportions (25 to 50%) in order to simulate post-composting exposure.
In all cases, the guideline requires that the applied concentration be analytically verified to ensure traceability and reproducibility of the tests. Preliminary range-finding tests can be performed to determine relevant concentrations before the final test.
Experimental design and test procedure
Growing conditions and controlled environment
The OECD 208 trial is conducted in a controlled environment to ensure the reproducibility and comparability of the results. The plants are grown in growth chambers, phytotrons, or greenhouses equipped with devices for regulating temperature, humidity, light, and irrigation.
The standard conditions recommended by the guideline are as follows:
- Temperature: 22 °C ± 10 °C (day/night variation allowed).
- Relative humidity: 70% ± 25%.
- Photoperiod: 16 hours of light per day.
- Luminous intensity: 350 ± 50 µE/m²/s (useful spectrum 400–700 nm).
The plants are grown in non-porous or glazed plastic pots, placed on trays or shelves to ensure even drainage. The positioning of the pots is regularly adjusted during the trial to minimize the effect of micro-variations in light or temperature within the growth chamber.
Watering is adapted to the species being tested and the nature of the substrate. It can be done by capillary action (soaking from the bottom) or by controlled spraying. In all cases, the control and treatment groups must be subjected to the same humidity and light conditions.
Nature of the growing medium
The choice of substrate is fundamental to ensuring uniform seedling growth and good exposure to the substance being tested. Two main options are possible:
- Natural soil : sandy silt, silty sand or sandy clay silt, with an organic carbon content ≤ 1.5%. This type of soil is generally sieved to 2 mm to remove coarse particles and pasteurized to limit the presence of pathogens.
- Artificial substrate : quartz sand, glass beads or mineral wool, used in some cases to reduce variability related to the chemical and biological properties of natural soil.
The electrical conductivity, pH, and particle size of the substrate are measured and recorded for each batch used. Nutrients may be added if necessary to prevent deficiency stress.
Organization of experimental units
Each tested condition (control and treatment groups) is replicated in several pots (replicates) to ensure the statistical robustness of the results. The protocol includes:
- A number of replicates ≥ 4 per condition.
- A total number of seeds ≥ 20 per condition.
- A sowing density adapted to the size of the seeds: 3 to 10 seeds per 100 cm².
For example, a pot with a diameter of 15 cm can hold:
- 1 to 2 corn or tomato seeds.
- 3 rapeseed or pea seeds.
- 5 to 10 onion, wheat or lettuce seeds.
It is essential to sow the same number of seeds in each pot and to ensure a homogeneous distribution, without overlapping, to limit the effects of competition.
Application of the tested substance
The application method depends on the chemical nature of the substance:
- Water-soluble substances : dissolved in water, then added to the soil by homogenous mixing or by watering.
- Poorly soluble substances : dissolved in a volatile solvent (acetone, ethanol), mixed with sand, then incorporated into the soil after evaporation of the solvent.
- Insoluble solid substances : directly mixed into the dry soil using a suitable mixing device.
For plant protection products, the substance is generally applied by spraying onto the soil surface after sowing. A second control group, containing the solvent, is sometimes required to isolate the effects related to the vehicle used (water, solvent, adjuvant, etc.).
The exact quantity of substance applied is verified analytically (by weighing or chemical assay), and the method of application (volume, equipment, concentration) is carefully recorded in the test report.
The testing period officially begins when 50% of the seedlings in the control group have emerged , marking “day 0” of the trial. From this point, the study lasts 14 to 21 days, depending on the growth of the species being tested.
Measured parameters and validity criteria
Ecotoxicological indicators monitored
Throughout the trial, several parameters are evaluated both quantitatively and qualitatively to determine the effect of the tested substance on the seedlings. These parameters are classified into two main categories: objective measurements (biometrics) and visual observations (phytotoxicity).
The most commonly measured indicators are as follows:
- Seedling emergence rate : number of seeds that produced a visible seedling, expressed as a percentage of the total number of seeds sown.
- Biomass of shoots : measured in fresh weight (wet weight) or in dry weight (dry weight) after drying at 60 °C until constant mass.
- Shoot height : measured from the collar to the tip of the stem, using a meter stick or calipers.
- Mortality rate : proportion of seedlings that died during the test period.
- Visible phytotoxicity : observation of symptoms such as chlorosis (yellowing), necrosis (tissue death), wilting, leaf or stem deformations, and abnormal growth.
These observations are made at regular intervals, generally every two to three days, and systematically at the end of the trial. They must be recorded in a structured manner, with supporting photos if possible, and a consistent rating according to a validated evaluation grid.
Some studies may also include monitoring the vegetative development stage according to the BBCH code, allowing each seedling to be precisely located in its growth cycle.
Test validity criteria
OECD Guideline 208 sets out several minimum conditions that must be met for a test to be considered valid. These criteria ensure that the experimental conditions are controlled and that the observed effects are indeed due to the substance being tested.
The main validity criteria are as follows:
- Emergence of controls ≥ 70% : in the untreated group, at least 70% of the seeds must have produced a seedling.
- Survival of control seedlings ≥ 90% : among the seedlings that emerged in the control group, at least 90% must survive until the end of the trial.
- Absence of phytotoxicity in the controls : the controls must not show any visible symptoms of stress or toxicity (except for normal variations in growth).
- Homogeneous growing conditions : temperature, light, watering and substrate must be identical between groups.
If these criteria are not met, the trial may be invalidated, or the data interpreted with caution. In some cases, a repetition of the trial may be required to ensure the robustness of the conclusions.
Additional recommendations for reliable measurement
To ensure accurate measurement and minimize bias, Good Laboratory Practices (GLP) recommend:
- To use seeds from the same batch, certified for their germination rate.
- To standardize the methods of harvesting, drying and weighing the shoots.
- To avoid variations in seeding density and sowing depth.
- To regularly record environmental conditions in the grow rooms.
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Analysis of results and interpretation of data
Statistical methods used
The analysis of data from the OECD 208 test relies primarily on the construction of dose-response curves . These curves allow for the evaluation of the effect of a substance on a measured parameter (such as biomass or emergence) as a function of concentration or application rate. From this relationship, several key values can be calculated.
The main statistical values extracted are:
- CEx (Effective Concentration) : concentration of the substance causing an effect x % relative to the control (e.g. CE25 or CE50).
- TEx (Effective Rate) : application rate producing the same effect, used when exposure is expressed in mg/kg of dry soil or in kg/ha.
- CSEO (Concentration No Observed Effect) : highest concentration tested that did not produce a statistically significant effect compared to the control.
- CMEO (Minimum Concentration with Observed Effect) : lowest concentration producing a significant effect.
Several statistical models can be used to adjust dose-response curves:
- Log-logistics model,
- Weibull model
- Logit or probit regression,
- Spearman-Karber method (and its abbreviated variants),
- Non-linear regression (Bruce-Versteeg type) for continuous effects.
The choice of model depends on the type of variable measured (on/off, continuous), the data distribution, and the number of concentrations tested. In all cases, the goodness of fit is assessed by the coefficient of determination (R²), which should ideally be greater than or equal to 0.7 for the most sensitive species.
Presentation and structuring of results
The test results must be presented clearly and in detail in a final report, which will serve as a basis for regulatory assessors. This report typically includes:
- A table of raw results : number of emerged seedlings, biomass, height, visual observations, for each replicate and each concentration.
- A summary table : averages per treatment, standard deviations, percentages of inhibition compared to the control.
- Graphical representations : dose-response curves, comparative histograms, photos of observed symptoms.
- Details of the statistical methodology , including the hypotheses used, the tests applied, the parameters estimated and their confidence intervals.
This rigorous presentation is essential to ensure data transparency and interpretation. In particular, it allows the competent authorities to verify the test's compliance with OECD guidelines and the principles of Good Laboratory Practice (GLP).
Use of data in risk assessment
The results obtained from OECD trial 208 are used in environmental risk assessment models for non-target plants. They allow, in particular:
- To estimate the margin of safety (ratio between the no-effect concentration and the predicted environmental concentration – PEC).
- To compare several substances with each other in terms of relative toxicity .
- To determine regulatory thresholds for placing chemical or plant protection products on the market.
- risk management measures , such as untreated areas or usage restrictions.
By combining robust experimental data with rigorous statistical analysis, the OECD 208 trial becomes a fundamental decision-making tool in the protection of terrestrial ecosystems. It helps ensure that products placed on the market do not pose an unacceptable risk to non-target plants in exposed areas.
Sectoral applications of the OECD 208 test
Plant protection products: a key regulatory requirement
In the agricultural sector, the OECD 208 test is systematically required in the registration dossiers for plant protection products, under Regulation (EC) No 1107/2009. It makes it possible to demonstrate that the active substances or commercial formulations do not present an unacceptable risk to non-target flora, in particular plants located on the edge of cultivated plots.
The results of this test are essential for:
- Identify the harmful effects on representative species of the environment (flowering plants, grasses, vegetables).
- Define untreated zones (ZNT) to protect adjacent habitats.
- Implement risk management measures in marketing authorizations.
YesWeLab assists manufacturers and distributors in carrying out OECD 208 tests under GLP (Good Laboratory Practice) conditions, with selection of suitable plant species and drafting of usable regulatory reports.
Veterinary medicines and organic fertilizers
When a veterinary medicinal product is administered to livestock, residues can be found in their excrement and subsequently in manure or slurry spreading. These chemical residues can come into contact with cultivated or wild plants. The OECD 208 test measures these indirect effects on plant growth.
The protocol can be adapted to simulate realistic conditions by applying the tested substance via treated manure (pre-incubated under anaerobic conditions) incorporated into the soil. This type of test is particularly relevant for:
- Evaluate the environmental impact of veterinary antibiotics, antiparasitics, or hormones.
- Validate the environmental compatibility of fertilizers of animal origin or digestates from methanization.
- Identify the threshold concentrations that should not be exceeded in organic inputs.
YesWeLab offers methodological adaptations to integrate these parameters, in accordance with the recommendations of the European Medicines Agency (EMA) and the VICH guidelines.
Evaluation of bioplastics and compostable materials
With the increasing use of biodegradable and compostable plastics, new ecotoxicity requirements have emerged. The OECD 208 test is integrated into standardized assessment protocols such as ASTM D6400 and EN 13432. It allows for the evaluation of the effects of plastic degradation products or additives on seed germination and growth.
In this context, the test is carried out by incorporating compost containing the tested material into standard substrates, then observing the germination of two plant species. The results confirm that:
- The plastic or the additive does not release phytotoxic substances.
- The final compost is compatible with agronomic use.
- Compostability certifications can be obtained (OK Compost, TÜV, etc.).
YesWeLab works with manufacturers of bio-based and compostable materials to validate the environmental compatibility of their products within the framework of industrial or regulatory certifications.
Biocidal products and chemical substances subject to REACH
The REACH regulation mandates the assessment of the environmental impact of chemicals, including their effect on terrestrial vegetation. The OECD 208 test may be required when registering a new molecule or for the assessment of a substance of very high concern (SVHC). In the case of biocides (EU Regulation 528/2012), it helps demonstrate safety for non-target plants in the event of drift or soil contamination.
This test is also used in impact studies for:
- Industrial areas that may discharge effluents onto the ground.
- Polluted sites to be diagnosed as part of a rehabilitation study.
- Substances used in the formulation of cleaning products, paints, inks, solvents, etc.
Thanks to its network of partner laboratories, YesWeLab offers a complete service for these uses, from the selection of experimental conditions to the statistical interpretation of results, including, if necessary, regulatory support for the drafting of REACH or biocides dossiers.
Conducting an OECD 208 test with YesWeLab: expertise, compliance, and tailored support
Multi-sector and regulatory expertise
YesWeLab supports manufacturers in a variety of sectors: agrochemicals, animal health, bioplastics, environment, regulated chemicals, etc. Each project is managed by a technical team capable of:
- Select the optimal experimental conditions according to the product being tested (active substance, formulation, complex matrix, compost, etc.).
- Identify the specific requirements of applicable regulations (REACH, phytosanitary regulations, biocides, VICH, compostability standards).
- Adapt the protocol in case of particular matrices (treated manure, poorly soluble products, volatile substances, multiple formulations).
The tests are entrusted to partner laboratories accredited according to the ISO 17025 standard and recognized according to good laboratory practices (GLP), guaranteeing the quality, traceability and validity of the results.
Simplified management thanks to the digital platform
One of YesWeLab's major strengths lies in its digital platform, designed to streamline communication and centralize test management. By entrusting an OECD 208 test to YesWeLab, manufacturers benefit from:
- A clear interface for searching, ordering and tracking analytical services.
- A dashboard that groups together ongoing tests, results received, and associated documents (protocols, reports, certificates).
- Automatic alerts for critical stages: quote validation, sample shipment, results availability.
This digital approach significantly reduces execution times and prevents communication errors or information loss. It also allows regulatory, quality, and R&D teams to monitor projects in real time.
Personalized support at every stage
YesWeLab does more than simply act as a logistics intermediary. Each client is supported by a dedicated technical contact person who is involved throughout the entire process:
- Upstream advice to verify feasibility, define the concentrations to be tested and the relevant plant species.
- Monitoring the progress of the trial, in conjunction with the selected laboratory, to ensure proper execution of the protocol.
- Assistance in interpreting statistical results and in drafting regulatory documents, where applicable.
This support is particularly useful for companies that do not have an internal ecotoxicology department, or that wish to outsource all or part of their environmental studies.
