To further enhance the scientific rigor of environmental safety evaluation for pesticides, the national standard GB/T 31270.15-2025 "Test guidelines on environmental safety assessment of chemical pesticides — Part 15: Earthworm acute toxicity test" was officially released and came into force on May 1, 2026, replacing the previous 2014 version.
This revision comprehensively optimizes testing conditions, reagents and materials, data processing, and quality control. The new version removes the rigid requirement for test humidity, enhancing test flexibility. It also adds a normative references section and optimizes the list of reagents and equipment.
REACH24H Reminder
Companies conducting or planning earthworm acute toxicity tests for China pesticide registration must now comply with GB/T 31270.15-2025, which came into force on May 1, 2026. Data generated under the old 2014 methodology may no longer be accepted for new regulatory submissions.
1. Scope, Normative References, and Definitions
The 2025 revision introduces foundational structural changes to the standard, including expanded scope applicability, newly added normative references, and streamlined terminology definitions. These changes establish a more robust and transparent framework for the entire testing procedure.
1.1 Scope
Old Version (GB/T 31270.15-2014): Not applicable to volatile and poorly soluble chemical pesticides.
New Version (GB/T 31270.15-2025): Not applicable to volatile pesticides. The "poorly soluble" restriction has been removed, broadening the scope of testable substances.
1.2 Normative References
Old Version (GB/T 31270.15-2014): None.
New Version (GB/T 31270.15-2025): Added a dedicated Chapter 2 for normative references, citing key supporting standards including GB/T 6682 (water for analytical laboratory use), NY/T 52 (soil determination), NY/T 1121.2 (soil pH determination), and NY/T 4195.8 (cultivation of biological test organisms).
1.3 Terms and Definitions
Old Version (GB/T 31270.15-2014): Included definitions for clitellum, adult earthworm, median lethal concentration, test substance, and chemical pesticide.
New Version (GB/T 31270.15-2025): Removed the definitions for clitellum, adult earthworm, test substance, and chemical pesticide. Retains only the core term: median lethal concentration (LC50).
2. Test Conditions and Materials
The 2025 version significantly expands the specification of materials and equipment needed for testing, providing laboratories with clearer operational guidance while removing unnecessary constraints.
2.1 Test Conditions
Old Version (GB/T 31270.15-2014): Temperature: 20°C ± 2°C; relative humidity: 70% to 90%; light intensity: 400 lx to 800 lx.
New Version (GB/T 31270.15-2025): Removed the requirement for test humidity, enhancing operational flexibility. Temperature remains 20°C ± 2°C and light intensity remains 400 lx to 800 lx.
2.2 Reagents
Old Version (GB/T 31270.15-2014): None.
New Version (GB/T 31270.15-2025): Added a detailed reagent list including acetone, n-hexane, quartz sand, kaolin clay, calcium carbonate, chloroacetamide, and others, providing clear guidance on required chemicals and materials.
2.3 Test Organism
Old Version (GB/T 31270.15-2014): Recommended selecting adult Eisenia fetida for the test, with a body weight between 0.30 g and 0.60 g.
New Version (GB/T 31270.15-2025): Specifies the use of Eisenia fetida. Healthy adult earthworms over 2 months old with clitellum development (sexually mature) and a body weight between 300 mg and 600 mg should be used. The cultivation of Eisenia fetida shall comply with NY/T 4195.8, providing standardized rearing conditions.
2.4 Apparatus and Equipment
Old Version (GB/T 31270.15-2014): Only listed major apparatus: incubator, glass containers, and volumetric flasks.
New Version (GB/T 31270.15-2025): Provides a comprehensive equipment list with precision specifications: crystallizing dishes or beakers, environmental chambers or constant temperature rooms, mixing or blending equipment, electronic balances (readability: 0.01 g, 0.001 g, 0.0001 g), electronic scales (readability: 0.1 g), thermo-hygrometer or temperature/humidity recorders, pH meter, oven, and luxmeter.
2.5 Test Substance
Old Version (GB/T 31270.15-2014): The test substance should be pesticide formulations, technical materials, or pure substances. For substances poorly soluble in water, a small amount of organic solvent with low toxicity to earthworms could be used as an auxiliary solvent, with the amount generally not exceeding 0.1 mL(g)/L.
New Version (GB/T 31270.15-2025): The test substance shall be technical materials (technical concentrates), formulations, or pure substances. Prior to the test, information regarding the solubility of the test substance in water and organic solvents, stability in water and soil, photolysis stability, saturated vapor pressure, purity, n-octanol/water partition coefficient, and ready biodegradability test results should be obtained. This reflects a much stronger emphasis on pre-test characterization.
3. Test Procedures
The test procedures have undergone the most substantial revisions in the 2025 standard. Key changes span the range-finding test, limit test, and nearly every aspect of the definitive test — from concentration setting and replicates to soil preparation, organism handling, and observation protocols.
3.1 Range-finding Test
Old Version (GB/T 31270.15-2014): Under the same conditions as the definitive test, set several concentration groups with wide spacing to determine the minimum concentration causing 100% mortality and the maximum concentration causing 0% mortality of earthworms. The concentrations for the definitive test shall be set within this range.
New Version (GB/T 31270.15-2025): Set the treatment groups using a geometric series. Conduct the range-finding test under the same conditions as the definitive test to determine the concentration range for the definitive test. Replicates are not required for the range-finding test, simplifying the preliminary testing phase.
3.2 Limit Test
Old Version (GB/T 31270.15-2014): Set the upper limit concentration at 100 mg a.i./kg dry soil. If no earthworm mortality is observed, no further testing is required.
New Version (GB/T 31270.15-2025): Set the upper limit concentration at 1,000 mg test substance/kg dry soil. If the mortality rate of earthworms is not greater than 10%, no definitive test is required. The higher threshold reduces unnecessary testing for low-toxicity substances.
3.3 Definitive Test — Concentration Setting and Replicates
Old Version (GB/T 31270.15-2014): Set 5 to 7 concentration groups within the range determined by the range-finding test, plus one untreated control group. If an organic solvent is used, a solvent control group shall also be added. 3 replicates for each concentration group.
New Version (GB/T 31270.15-2025): Based on range-finding results or known toxicity data, set 5 to 7 treatment concentrations using a geometric series, with an untreated control group and, if applicable, a solvent control group. Each group shall have 4 replicates (up from 3), with 10 earthworms placed in each replicate.
3.4 Definitive Test — Test Organism Preparation
Old Version (GB/T 31270.15-2014): None (no specific preparation procedure specified).
New Version (GB/T 31270.15-2025): Prior to the test, the test earthworms shall be kept in artificial soil for 24 hours under the same conditions as the definitive test. Wash the earthworms clean and blot them dry with filter paper before weighing them. This acclimation step improves test consistency.
3.5 Definitive Test — Application Method of Test Substance
Old Version (GB/T 31270.15-2014): Place 500 g of soil in a glass container (soil depth ≥ 8 cm). Add the test substance solution and mix thoroughly. If an organic solvent is used, it must be fully evaporated. Adjust soil moisture to 30%–35% of dry soil weight. For poorly water-soluble substances, a small amount of organic solvent (≤ 0.1 mL(g)/L) could be used.
New Version (GB/T 31270.15-2025): Provides three distinct application methods based on solubility:
(1) For water-soluble test substances: prepare the test solution with water and mix thoroughly with artificial soil.
(2) For organic-solvent-soluble substances: dissolve in n-hexane or acetone, add dropwise to 10 g of quartz sand. After complete solvent evaporation, mix with pre-moistened artificial soil, and add deionized water to reach 750 g wet weight.
(3) For substances insoluble in both water and organic solvents: mix with quartz sand to a total of 10 g, then mix with pre-moistened artificial soil, and add deionized water to 750 g wet weight.
3.6 Definitive Test — Weight of Artificial Soil
Old Version (GB/T 31270.15-2014): Place 500 g of soil in a glass container (soil depth ≥ 8 cm).
New Version (GB/T 31270.15-2025): Add 750 g wet weight of treated artificial soil into a glass container, with soil depth not less than 8 cm. The specified "wet weight" replaces the previous dry weight-based measurement.
3.7 Definitive Test — Earthworm Body Weight
Old Version (GB/T 31270.15-2014): Weigh once at the start of the test — body weight between 0.30 g and 0.60 g.
New Version (GB/T 31270.15-2025): Weigh twice: once at the start of the test (body weight between 0.30 g and 0.60 g), and once at the end of the test for the surviving earthworms.
3.8 Definitive Test — Soil Moisture Content
Old Version (GB/T 31270.15-2014): Add deionized water to adjust soil moisture to 30%–35% of dry soil weight.
New Version (GB/T 31270.15-2025): If necessary, add water to adjust soil moisture to approximately 35% of dry soil weight. Soil moisture content shall be determined in accordance with NY/T 52, establishing a standardized measurement method.
3.9 Definitive Test — Incubation and Observation
Old Version (GB/T 31270.15-2014): The test lasts two weeks. On day 7 and day 14, pour out the soil, observe and record poisoning symptoms and dead earthworms (touch the posterior end with a needle; no response = dead), remove dead earthworms immediately. Calculate LC50 and 95% confidence limits based on mortality rates on day 7 and day 14.
New Version (GB/T 31270.15-2025): Test period remains 14 days. Key refinements include:
- Continuous light is provided to ensure earthworms remain in the artificial soil.
- Periodic water replenishment by weighing to maintain soil moisture.
- Observation method: Gently pour soil onto a glass dish or plate; pick out earthworms for examination.
- Death criterion: Earthworms are considered dead when they show no response of the anterior end to mechanical stimulus (changed from posterior end).
- After the 7-day examination, surviving earthworms and artificial soil are returned to the container to continue.
- At the end of the test, weigh surviving earthworms and determine the moisture content of the artificial soil.
4. Statistical Methods and Quality Control
The 2025 version modernizes the statistical analysis framework and tightens quality control requirements, ensuring more reliable and reproducible test results.
4.1 Statistical Methods
Old Version (GB/T 31270.15-2014): LC50 and 95% confidence limits for each observation time may be calculated using the Spearman-Kärber method, linear interpolation method, or probit analysis graph method. Relevant toxicity data calculation software may also be used.
New Version (GB/T 31270.15-2025): Select statistical methods such as the probit method or logistic regression method to calculate the LC50 and its 95% confidence interval (except for the limit test). Appropriate statistical software may be used. When mortality rates are 0% and 100% under two consecutive concentrations with a geometric ratio ≤ 2.0, their geometric mean shall be used as the LC50. This provides clearer statistical guidance and eliminates ambiguity around edge cases.
4.2 Quality Control
Old Version (GB/T 31270.15-2014): Mortality in the untreated control group shall not exceed 10%.
New Version (GB/T 31270.15-2025): Mortality in the untreated control group and the solvent control group shall not exceed 10%. The explicit inclusion of the solvent control group in the QC criteria ensures any solvent-related effects are properly monitored.
5. Reporting and Appendices
The 2025 standard significantly upgrades reporting requirements and makes notable changes to the appendices, including the removal of the toxicity classification framework.
5.1 Test Report
Old Version (GB/T 31270.15-2014): Basic reporting requirements.
New Version (GB/T 31270.15-2025): Report contents are more detailed and standardized, covering test conditions, raw data, statistical methods, and additional quality parameters. This aligns with international GLP practices and improves data transparency.
5.2 Preparation of Artificial Soil
Old Version (GB/T 31270.15-2014): Appendix A provided the components and proportions of artificial soil.
New Version (GB/T 31270.15-2025): Added the preparation method for artificial soil, in addition to the components and proportions, providing a complete step-by-step protocol for consistent soil preparation across laboratories.
5.3 Toxicity Classification
Old Version (GB/T 31270.15-2014): Appendix B specified four toxicity levels based on LC50 (14d) / (mg a.i./kg dry soil):
Extremely toxic: LC50 ≤ 0.1
Highly toxic: 0.1 < LC50 ≤ 1.0
Moderately toxic: 1.0 < LC50 ≤ 10
Slightly toxic: LC50 > 10
New Version (GB/T 31270.15-2025): Removed the appendix on toxicity classification.
6. Summary and Recommendations
The transition from GB/T 31270.15-2014 to GB/T 31270.15-2025 represents a comprehensive upgrade of the earthworm acute toxicity testing framework. Key improvements include expanded normative references, more detailed reagent and equipment specifications, refined test procedures with multiple application methods, enhanced statistical rigor, and more robust quality control criteria.
Companies conducting or commissioning earthworm acute toxicity tests for China pesticide registration should prioritize the following actions:
Update testing protocols — revise SOPs to reflect the new application methods, soil weight (750 g wet weight), replicate requirements (4 per group), and observation procedures.
Upgrade equipment — ensure balances meet the specified readability requirements (0.01 g, 0.001 g, 0.0001 g) and acquire additional equipment such as a luxmeter and pH meter if not already available.
Adopt new statistical methods — implement probit or logistic regression analysis in place of the older Spearman-Kärber approach, and prepare for the geometric mean calculation for specific mortality patterns.
Enhance reporting documentation — align test reports with the new, more comprehensive reporting requirements, including raw data, test conditions, and statistical methods.
Need help transitioning to the GB/T 31270.15-2025 standard?
With the May 1, 2026 effective date now passed, laboratories and pesticide registrants that have not yet completed their methodology transition should do so as soon as possible. REACH24H's agrochemical compliance team can assist with test protocol review, laboratory qualification assessment, and regulatory submission support to ensure compliance with the new standard.
Submit Your Compliance Inquiry View China Pesticide Service
浙公网安备 33011002014301号