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Ecotoxicological information

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:
1999-03-23 - 1999-06-08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: OECD Guidelines for Testing Chemicals, 1993 Section 2 - 207
Deviations:
no
GLP compliance:
no
Specific details on test material used for the study:
4924 mg of the test substance was dissolved in 20 mL of dimethyl sulphoxide (DMSO) and completed with destilled and deionized water to a total volume of 1,000 mL, resulting on a concentration of 4924 mg/L, then it was prepared others stock solutions to range-finding. To assist the solubilization eight drops of Tween-80 and 15 minutes of magnetic stirred were used.
For the definitive test, specific amount of product were weight and solubilized, 255 mL of each stock solution were added and mixed with 495 g of artificial soil in proportions described in the raw data.
Concentrations are reported here as milligrams (mg) of test material per kilograms (kg) of dry weight of artificial soil.
Analytical monitoring:
not specified
Details on sampling:
not specified
Vehicle:
not specified
Details on preparation and application of test substrate:
The test substrate was artificial soil, that gave toxicity data more representative of natural exposure of earthworms.
The artificial soil test substrate was prepared, as follows:
10% sphagnum peat (pH 5.5 - 6), no visible plant remains, finely ground, dried to measured misture content.
20% Dusk red latosol (LR)
70% industrial sand ( fine sand should be dominant with more than 50 % of the particles between 50 and 200 microns).
The artificial soil test involved keeping earthworms in samples of a precisely defined artificial soil to which a range of concentrations of the test substance had been applied.
The dry constituents were blended in the correct proportions and mixed thoroughly. Moisture content was determined by drying a small sample at 105° C and re- weighing. Deionized water was addded to give an overall moisture content of about 35% of dry weight, and the medium was thoroughly mixed. The complete mixture remained moist but not so wet that water appeared when the artificial soil was compressed.
Test organisms (species):
Eisenia fetida
Animal group:
annelids
Details on test organisms:
Stock culture
The test organism used was earthworms E. foetida, at least two months old that exibit the characteristic clitellum (a swollen ring-shaped glandular region). The earthworms were hold in a 250-Liter tank with 120 Liters of cow manure. Wheat flake (0.5 kilograms) and fruits like avocado pear and papaya were added each twice a week. Temperature and humidity of the box were controlled daily.

Acclimation
Before the test, the worms were washed in tap water and blot dried with filter paper, the average fresh weight of the worms was obtained, and they were kept in dry weight of artificial soil for 24 hours at 20 °C.
Study type:
laboratory study
Substrate type:
artificial soil
Limit test:
yes
Total exposure duration:
14 d
Test temperature:
20 +/- 2 °
pH:
not specified
Moisture:
not specified
Details on test conditions:
Test System
The range-finding was carried out in a static system in illuminated cabinet with a light intensity of 400 to 800 lux and temperature variation of 2°C.

Testing Procedure
An preliminary range-finding test was carried out using a 2-L glass chamber. Quantity of test substance corresponding to 750 g wet weight of artificial soil were mixed for each test container. The test solution was prepared according TEST MATERIAL. Ten earthworms were transferred to each glass chamber. The concentrations used were: 23.89; 238.89; and 2388.89 mg/kg of dry weight of artificial soil, with two replicates.
The glass chambers were covered with a perforated plastic film to avoid the escape of earthworms, prevent the test medium from drying and kept under the test conditions for 14 days.
The experiment was carried out in a continuous light environment at 20 ± 2 °C. The 7 and 14-day mortalities were assessed by emptying test medium onto a plate, sorting worms from the medium and testing their reaction to a mechanical stimulus at the front end. After the 7-day assessment worms and medium were replaced in the test container until revaluation in 14 days of incubation. Any behavioural or pathological symptoms noted were reported. At the death rate of the earthworms were determined, considering dead the worm that showed no reaction to a needle sting.
Based on the results of the range-finding test, the final test using four replicates for each treatment was carried out with the following effective concentrations: 123.43; 255.88; 504.87; 1017.24 and 2049.67 mg/Kg dry weight of artificial soil.
Control and solvent control were carried out together in this test.
All standard weights, dilutions and the artificial soil quantity for each concentration were listed in the raw data.

Reference Test
The reference test with chloroacetamid (SIGMA) was conducted to verify the sensitivity of test organism. It was carried out as mentioned above in the final test. A chloroacetamid standard was included in this test at levels of 23.89, 47.78, 71.67, 95.56, 119.44, and 143.33 mg/Kg dry weight artificial soil.

Glassware preparation
All glassware used in testing was thoroughly washed with detergent and rinsed with tap water. This was followed by a rinse with acetone followed by rinsing with a 10% solution of nitric acid to remove any residue and washed again in tap water followed by rinsing with distilled and deionized water.
Reference substance (positive control):
yes
Key result
Duration:
14 d
Dose descriptor:
LC50
Effect conc.:
351.77 mg/kg soil dw
Nominal / measured:
not specified
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
7 d
Dose descriptor:
LC50
Effect conc.:
563.39 mg/kg soil dw
Nominal / measured:
not specified
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
14 d
Dose descriptor:
LC100
Effect conc.:
1 017.24 mg/kg soil dw
Nominal / measured:
not specified
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
123.43 mg/kg soil dw
Nominal / measured:
not specified
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
The concentration that causes the death of 50% of the earthworms in relation to the control, LC50 after 7 and 14 days of exposure were 563.39 and 351.77 mg/Kg dry weight of artificial soil.
The highest tested level that had no effect on the earthworms was 123.43 mg/Kg dry weight of artificial soil in 14 days of exposure and the lowest concentration that showed 100% mortality was 1017.24 mg/Kg dry weight of artificial soil.
For tested concentrations, behavioural or pathological symptoms in worms were not observed during test period.
Results with reference substance (positive control):
The chloroacetamid 14-day LC50 was 98.47 mg/Kg dry weight of artificial soil (95% CL = 88.94 - 109.02 mg/Kg dry weight of artificial soil).
Validity criteria fulfilled:
yes
Conclusions:
The results indicate that under the test conditions, that 7 and 14-day LC50 were 563.39 and 351.77 mg/Kg dry weight of artificial soil., respectivelly. The highest tested concentration that showed no effect was 123.43 mg/Kg and the lowest concentration that showed 100% mortality was 1017.24 mg/ Kg dry weight of artificial soil. As a quality check, the earthworms were challenged with a reference compound, chloroacetamid. The results indicate that the 14-day LC50 was 98.47 mg of chloroacetamid/Kg dry weight of artificial soil (95% CL = 88.94 - 109.02 mg/Kg dry weight of artificial soil).
Executive summary:

The objective of this study was to determine the toxicity on a selected specie of earthworm E. foetida. The study was conducted not according GLP but according to OECD Guidelines for Testing Chemicals, 1993 Section 2 - 207. Fourty earthworms per test effective concentration were used 123.43; 255.88; 504.87; 1017.24 and 2049.67 mg/Kg dry weight of artificial soil in chambers incubated in continuous light room during fourteen days. Control and solvent control were added in this study. Incubation temperature and humidity were measured throughout the test and were within acceptable limits.

The results indicate that under the test conditions, that 7 and 14-day LC50 were 563.39 and 351.77 mg/Kg dry weight of artificial soil., respectivelly. The highest tested concentration that showed no effect was 123.43 mg/Kg and the lowest concentration that showed 100% mortality was 1017.24 mg/ Kg dry weight of artificial soil. As a quality check, the earthworms were challenged with a reference compound, chloroacetamid. The results indicate that the 14-day LC50 was 98.47 mg of chloroacetamid/Kg dry weight of artificial soil (95% CL = 88.94 - 109.02 mg/Kg dry weight of artificial soil).

Description of key information

The study was conducted not according GLP but according to OECD Guidelines for Testing Chemicals, 1993 Section 2 - 207.

The results indicate that under the test conditions, that 7 and 14-day LC50 were 563.39 and 351.77 mg/Kg dry weight of artificial soil., respectivelly. The highest tested concentration that showed no effect was 123.43 mg/Kg and the lowest concentration that showed 100% mortality was 1017.24 mg/ Kg dry weight of artificial soil. As a quality check, the earthworms were challenged with a reference compound, chloroacetamid. The results indicate that the 14-day LC50 was 98.47 mg of chloroacetamid/Kg dry weight of artificial soil (95% CL = 88.94 - 109.02 mg/Kg dry weight of artificial soil).

Key value for chemical safety assessment

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

Additional information

The objective of this study was to determine the toxicity on a selected specie of earthworm E. foetida. The study was conducted not according GLP but according to OECD Guidelines for Testing Chemicals, 1993 Section 2 - 207. Forty earthworms per test effective concentration were used 123.43; 255.88; 504.87; 1017.24 and 2049.67 mg/Kg dry weight of artificial soil in chambers incubated in continuous light room during fourteen days. Control and solvent control were added in this study. Incubation temperature and humidity were measured throughout the test and were within acceptable limits.

The results indicate that under the test conditions, that 7 and 14-day LC50 were 563.39 and 351.77 mg/Kg dry weight of artificial soil, respectively. The highest tested concentration that showed no effect was 123.43 mg/Kg and the lowest concentration that showed 100% mortality was 1017.24 mg/ Kg dry weight of artificial soil. As a quality check, the earthworms were challenged with a reference compound, chloroacetamid. The results indicate that the 14-day LC50 was 98.47 mg of chloroacetamid/Kg dry weight of artificial soil (95% CL = 88.94 - 109.02 mg/Kg dry weight of artificial soil).