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Toxicity to soil macroorganisms except arthropods

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Reference
Endpoint:
toxicity to soil macroorganisms except arthropods: short-term
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19/02/2018 - 25/04/2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 207 (Earthworm, Acute Toxicity Tests)
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Batch number : TROD7BB06
Analytical monitoring:
no
Vehicle:
no
Details on preparation and application of test substrate:
For the definitive test, 2989.4 mg of the test substance were dissolved in 40 mL of deionized water and the total volume was completed with deionized water to 1000 mL, resulting in a concentration of 2989.4 mg/L.
An aliquot of 188 mL of the test solution was added to 562 g of dry artificial soil in order to keep the moisture nearly 35%.
Test organisms (species):
Eisenia fetida
Animal group:
annelids
Details on test organisms:
The test system used in this study was adult earthworms from the species E. foetida, with age between two months to one year, cultivated at BIOAGRI's laboratory, with characteristic clitellum (a swollen ring-shaped glandular region).
Twenty-four hours before starting the test, the worms were removed from the cultivation containers and placed in moisturized artificial soil in a 5 L container, under the same environmental conditions of the culture. For both the treatments with the test substance and the controls average live weight range for individual earthworms was 480 to 530 mg at the beginning of the definitive test and 440 to 480 mg at the end of this test.

The initial culture of the test system was obtained from Santana Ranch, located in Anhumas, Campinas/SP — Brazil.

Twenty-four hours before starting the test, the worms were removed from the cultivation containers and placed in moisturized artificial soil in a 5 L container,
under the same environmental conditions of the culture.

For both the treatments with the test substance and the controls average live weight range for individual earthworms was 480 to 530 mg at the beginning of the
definitive test and 440 to 480 mg at the end of this test. For the test with the reference substance, the average live weight range for individual earthworms was
460 to 540 mg at the beginning of the test and 400 to 510 mg at the end of the test.
Study type:
laboratory study
Substrate type:
artificial soil
Limit test:
yes
Total exposure duration:
14 d
Remarks:
First observation at 7 days
Test temperature:
Between 19.2 to 21.7°C.
pH:
6.1
Moisture:
35%
Details on test conditions:
2 L polyethylene disposable test containers were used for the test.
The test was carried out using the static system and incubated inside an illuminated cabinet with a mean continuous light of 607 lux, within the intensity range of 400 to 800 lux and temperature of 20 ± 2°C, according to the Guideline OECD, 207 (1984). During the test period, the temperature was recorded daily, and was kept between 19.2 to 21.7°C.

The test substrate was artificial soil with the following composition:
• 10% ground sphagnum peat (pH between 5.5 to 6.0);
• 20% kaolin-clay (kaolinite content over 30 per cent);
• 70% industrial sand (fine sand was dominant with more than 50% of the particles between 50 and 200 pirn).
The dry constituents were blended in the correct proportions and mixed thoroughly. Moisture content was determined by drying at 105 ± 10°C and successive weighing a small sample.

Artificial soil was used as the test substrate to avoid any stress to the organisms. The artificial soil has the texture similar to the natural soil where the earthworms were cultivated. The pH of the artificial soil in the beginning of the definitive test was 6.1, and accordance with the range report by the OECD, 207 (1984) pH 6.0 ± 0.5.

For each treatment, four replicates were prepared by adding ten earthworms to each test container. The test containers were covered with perforated plastic lids. After 7 and 14 days the mortality was assessed by emptying the test medium onto a plate and sorting worms from the medium and testing their reaction to a mechanical stimulus placed at the front end of their body.
Nominal and measured concentrations:
nominal: 0 and 1000 mg/kg
Reference substance (positive control):
yes
Remarks:
chloroacetamide
Key result
Duration:
14 d
Dose descriptor:
LC50
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
During the evaluation, the earthworms were normal in appearance.

The maximum tested concentration of 1000 mg/kg of artificial soil of test substance did not cause any mortality to the earthworms during 14 days exposure period. Therefore, the 14-days L050 was determined to be considered higher than 1000 mg/kg of artificial soil.
The control treatment did not cause any mortality to the earthworms during 14 days exposure period. This result shows that the test was considered valid, in accordance with the validity criteria reported by the OECD, 207 (1984), which should not exceed 10 percent at the end of the test.
Results with reference substance (positive control):
The 14-days LC50 for reference substance chloroacetamide was 48.30 mg.kg-1, with 95% confidence limits of 40.56 to 57.51 mg.kg-1. This response to chloroacetamide confirms the sensitivity of the organisms used in this bioassay (ABNT, 2014), which is between 20 and 80 mg.kg-1.
Validity criteria fulfilled:
yes
Conclusions:
The 14-days LC50, of the acute toxicity test, for earthworm Eisenia foetida exposed to test substance MEAHA-Fe was considered higher than 1000 mg/kg of artificial soil.
Executive summary:

The objective of this study was to determine the acute toxicity of MEAHA-Fe to the earthworm Eisenia foetida (Savigny 1826). A range-finding test was carried out to determine the concentrations of the test substance that caused 100% and 0% mortality. According to the results obtained in the range-finding test, a definitive test was carried out with the following treatments: 0 (water control) and 1000 mg/kg of artificial soil. Forty earthworms per treatment were used in the definitive test, distributed into four replicates of 10 earthworms each. The test was carried out in polyethylene containers placed inside a room with continuous light for fourteen days. Mortality was determined at 7 and 14 days of incubation. Temperature and humidity were both measured throughout the test and were within acceptable limits. The control treatment did not cause any mortality to the earthworms during 14 days exposure period.

The highest tested concentration (1000 mg/kg of artificial soil) of test substance did not cause mortality to the test organisms. Therefore, the 14-days LC50 value, of the acute toxicity test, for earthworm E. foetida exposed to tested substance was considered greater than 1000 mg/kg of artificial soil.

Description of key information

In a standard OECD 207 test, the 14-days LC50, of the acute toxicity test, for earthworm Eisenia foetida exposed to test substance MEAHA-Fe was considered higher than 1000 mg/kg of artificial soil.

Key value for chemical safety assessment

Short-term EC50 or LC50 for soil macroorganisms:
1 000 mg/kg soil dw

Additional information