Reaction mass of N1,N1'-(methanediyldibenzene-4,1 -diyl)diaryldiamine and 2-{4-[(4-aminophenyl)amino]benzyl}-N4-(4-{4-[(4-aminophenyl)amino]benzyl}phenyl)aryldiamine

Toxicity to soil macroorganisms except arthropods

Link to relevant study record(s)

Reference

Endpoint:

toxicity to soil macroorganisms except arthropods: short-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 January 2015 to 04 August 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: HJ/T 153-2004. The guidelines for the testing of chemicals [S]. Beijing: State Environmental Protection Administration, 2004.

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: CRC-MEP. The Guidelines for the Testing of Chemicals-Effects on Biotic Systems. [M], The second edition. Beijing: China Environment Press. 2013: 58-62.

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: GB/T 21809-2008, Chemicals-Test method of earthworm acute toxicity test [S]. Beijing: Standardization Administration of China, 2008.

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 207 (Earthworm, Acute Toxicity Tests)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.8 (Toxicity for Earthworms: Artificial Soil Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Method 9045D, Soil and waste pH [S].US Environmental Protection Agency, 2004.

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

no

Details on preparation and application of test substrate:

TEST SOIL
- Artificial soil test substrate contained the following components:
a) 10 % sphagnum peat (Pindstrup Mosebrug A/S; pH 6.0, no visible plant remains, finely ground, dried).
b) 20 % kaolin clay (CAS: 1332-58-7; Lot number: 20140708; Sinopharm Chemical Reagent Co. Ltd.; kaolinite content preferably above 30 per cent).
c) 70 % quartz sand (CAS: 14808-60-7; Lot number: 20140514; Sinopharm Chemical Reagent Co. Ltd.; fine sand was dominant with 80 per cent of the particles between 50 and 200 microns).
- The dry constituents were blended in the correct proportions and mixed thoroughly with a large-scale laboratory mixer.

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
- Eisenia foetida was adopted as the test species.
- The worms were obtained from Nanjing Earthworm Nursery, Pudong community, Changlu Street, Dachang District, Nanjing (Batch No: E20150204E).

ACCLIMATION OF TEST WORMS
- The earthworms were released from quarantine for 14 days and then the batch was turned into pre-culture (Batch No.: E20150204E).
- Before the test, the worms were acclimated in test soil for 34 days.
- Average weight range of worms used in the study was 307 to 417 mg.
- The worms were then pre-cultured in test soils for another 24 hours.
- The worms were washed quickly with distilled water and surplus water absorbed on filter paper before use.

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

14 d

Post exposure observation period:

Not applicable

Test temperature:

20.6 to 22.3 °C

pH:

5.61 to 6.47

Moisture:

26 to 35 %

Details on test conditions:

EQUIPMENT
- Electronic balance (MSI 05DU, METTLER TOLEDO, Switzerland)
- Glass spoutless beakers (1 L, 012 cm, Nanjing Jinzheng Teching Instrument Factory)
- Plastic films with small ventilation holes (25 cm x 38 cm, Suzhou Surong plastic product company)
- Climatic chambers (controllable to ± 2 °C with a light intensity of 400 lux to 800 lux, MMM, CLC222)
- Stainless plates (40 cm><25 cm, Nanjing Jinzheng Teching Instrument Factory)
- Filter paper: (09 cm, approximately 0.2 mm thick, medium grade, Hangzhou Whatman-Xinhua Filter Paper Co., Ltd.)
- Deionised water (resistivity 18.2 MΩ.cm)
- Water quality parameter analyzer (HQ30d, HACH Corporation, parameters including pH)
- Oven (DHG-9146A, Shanghai Jinghong laboratory devices CO., LTD.)

REFERENCE SUBSTANCE
- Reference substance: 2-Chloracetamide (CAS: 79-07-2; purity: ≥ 98.5%; Lot number: 20130905; Sinopharm Chemical Reagent Co. Ltd).
- The test with the reference substance is performed three times a year as a means of ensuring that the laboratory test conditions are adequate and have not changed significantly.
- The results of the most recent test are given in Table 3 and Figure 1 (attached).

PREPARATION OF TEST SUBSTATE
- The test substance was ground into powder and directly added into the soil.
- A mixture of the test substance and quartz sand (10 g total) was prepared and then mixed with 550 g dry weight of soil for each test container.
- The medium was thoroughly mixed and deionised water was added to give an overall moisture content of about 35 % of the dry weight.
- Moisture content was determined by weighing, drying a small sample at 105°C and re-weighing.
- Details of test substrate preparation at chosen concentrations are given in the table below.

RANGE-FINDING TEST
- A pre-test was performed with test substance concentrations of 100, 500 and 1000 mg/kg artificial soil (dry weight) to determine the range of concentrations to be tested in the study.
- Simultaneously one blank control was established under the same conditions.
- One test batch for each concentration and control was used (each with 10 individuals).
- The test worms (10 worms each) were firstly placed on the surface of test substrate (dry weight: 560 g each; Moisture content: 35% dry soil weight).
- After having been sealed with plastic films, containers were put into the chambers.
- The test duration was 14 days, and the test temperature was maintained at 20.1 to 22.5 °C with humidity of 80 to 85% and continuous light (intensity of 400 to 800 lux).

DEFINITIVE TEST
- Based on the results of the range-finding test, a definitive test was undertaken with test concentrations of 640, 800, 1000, 1250, 1500 mg/kg (dry weight of soil) plus blank control.
- Four replicates were used for each concentration and control (each with 10 individuals).
- The test worms (10 worms each) were firstly placed on the surface of test substrate (dry weight: 560 g each; Moisture content: 35% dry soil weight).
- After having been sealed with plastic films, containers were put into the chambers.
- The test duration was 14 days, and the test temperature was 20.6 to 22.3°C with humidity of 80 to 85% and continuous light (intensity of 400 to 800 lux).

OBSERVATIONS
- On the 7th day of exposure, the mortality was assessed by emptying test medium onto a stainless plate, sorting worms from the medium and testing their reaction to a mechanical stimulus at the front end.
- Worms were classified as dead when they do not respond to a gentle mechanical stimulus to the front end.
- Due to rapid decomposition under test conditions, missing earthworms were also considered dead.
- At the completion of this assessment, soils were replaced in the test container and the surviving earthworms were replaced on the same test substrate surface until the 14 day assessment.
- In addition, the number and type of behavioural and other pathological abnormalities (e g. lethargic or rigid worms) were determined during each mortality assessment on 7 d and 14 d.
- On day 0 and 14, the weight of the worms was measured.
- Worms were washed prior to weighing (with deionised water) and the excess water removed by placing the worms briefly on filter paper. The surviving worms were weighed using the same methods on day 14.
- Soil pH and the moisture were also measured on day 0 and 14.
- The pH value was determined according to “Soil and waste pH (Method 9045D)” (US EPA).
- The moisture content of the soil was determined by weighing and then drying a small sample at 105 °C and re-weighing.

VALIDITY OF TEST
- Control group: A blank control group, comprising the same number of test worms as each treatment, was also treated with the same methods while without using the test substance.
- Reference control: The 7-day and 14-day LC50 values of a reference substance (2-chloracetamide) were determined 3 times a year to assure that the laboratory test conditions were adequate and had not changed significantly. The results of this study are shown in Table 3 and Figure 1 (attached). The LC50 (7d) was 33.6 mg/kg and the LC50 (14d) was 25.1 mg/kg in the latest reference substance test.
- Validity of test result: Mortality in the untreated control group at the end of the study must not exceed 10 %. Otherwise the test should be considered invalid.

Nominal and measured concentrations:

- Nominal target concentrations of 0, 640, 800, 1000, 1250 and 1500 mg/kg dw soil

Reference substance (positive control):

yes

Remarks:

2-chloroacetamide

Duration:

7 d

Dose descriptor:

LC0

Effect conc.:

640 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

14 d

Dose descriptor:

LC0

Effect conc.:

640 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

7 d

Dose descriptor:

LC50

Effect conc.:

1 241 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: 95 % confidence limits 1145 to 1345 mg/kg dw soil

Key result

Duration:

14 d

Dose descriptor:

LC50

Effect conc.:

884 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Basis for effect:

mortality

Remarks on result:

other: 95 % confidence interval 847 to 922 mg/kg dw soil

Details on results:

TEST CONDITIONS
- During the test, artificial soils were used with the moisture content controlled in range of 26% to 35% with pH from 5.61 to 6.47 (see Table 1, attached).
- The test conditions were maintained as follows: temperature of 20.6 to 22.3 °C, humidity of 80% to 85% and continuous light with intensity of 400 to 800 lux.
- No unusual circumstances that might have affected the integrity and quality of the study were observed.
- Mortality was not observed in the control at the end of the study.
- The test system was considered to be valid.

EARTHWORM MORTALITY
- The average weights of the worms at start and end of exposure are shown in Table 2 (attached).
- Number of dead earthworms after 7 and 14 days is shown in Table 4 and Table 5 (attached).
- No dead worms were observed in the blank control.
- No dead worms were observed in treatments of 640 mg/kg dw soil, while all worms were dead in the test treatments of 1500 mg/kg dw soil after 14 d.
- The results showed that the 7 d-LC50 was 1241 mg/kg dry weight soil, and the 95% confidence interval was 1145 to 1345 mg/kg dw soil.
- The 14 d-LC50 was 884 mg/kg dw soil, and the 95% confidence interval was 847 to 922 mg/kg dw soil.

BEHAVIOURAL EFFECTS
- At the beginning of test substance administration, test worms soon disappeared into the test soil medium.
- During the test, all survival earthworms remained in the soil.

Results with reference substance (positive control):

- The LC50 (7d) was 33.6 mg/kg and the LC50 (14d) was 25.1 mg/kg in the latest reference substance test (see Table 3 and Figure 1, attached).

Reported statistics and error estimates:

DATA PROCESSING
- The LC50 and 95 % confidence intervals were calculated by the Trimmed Spearman-Karber Method (Version 1.5, USEPA).

Validity criteria fulfilled:

yes

Conclusions:

The highest concentration of test substance causing no mortality at 7 days was 640 mg/kg dw soil (7d-LC0) and the LC50 (7d) value was reported as 1241 mg/kg dw soil. The highest concentration of substance causing no mortality at 14 days was 640 mg/kg dw soil (14d-LC0) and the LC50 (14d) value was reported as 884 mg/kg dw soil.

Executive summary:

GUIDELINE

The test was conducted to determine the acute toxicity of the test substance to earthworm according to “The guidelines for the testing of chemicals” (HJ/T 153-2004) and “The Guidelines for the Testing of Chemicals-Effects on Biotic Systems” (the 2nd edition, Beijing: China Environment Press, 2013) with reference to Procedure 207 of the “Guidelines for Testing of Chemicals” of the OECD: “Earthworm, Acute Toxicity Tests” (1984) and EU Method C.8 (Toxicity for Earthworms: Artificial Soil Test).

 

METHODS

The study was designed to determine the acute toxicity of test substances to earthworm at target concentrations of 0, 640, 800, 1000, 1250 and 1500 mg/kg dw soil. In this test, earthworms were exposed in samples of the artificial soil to which a range of concentrations of the test substance had been applied. Mortality was assessed 7 days and 14 days after application. The study comprised a range-finding test followed by a definitive test to determine the median lethal concentration (LC50) of the test substance.The moisture content of artificial soils was controlled in the range 26 to 35 % and pH 5.61 to 6.47 were used during the test. Temperature was maintained at 20.6 to 22.3 °C, humidity was maintained at 80 to 85 % and continuous light with intensity of 400 to 800 lux was supplied.

 

RESULTS

The highest concentration of test substance causing no mortality at 7 days was 640 mg/kg dw soil (7d-LC0) and the LC50 (7d) value was reported as 1241 mg/kg dw soil. The highest concentration of substance causing no mortality at 14 days was 640 mg/kg dw soil (14d-LC0) and the LC50 (14d) value was reported as 884 mg/kg dw soil.

Description of key information

The highest concentration of test substance causing no mortality at 7 days was 640 mg/kg dw soil (7d-LC0) and the LC50 (7d) value was reported as 1241 mg/kg dw soil. The highest concentration of substance causing no mortality at 14 days was 640 mg/kg dw soil (14d-LC0) and the LC50 (14d) value was reported as 884 mg/kg dw soil (HJ/T 153 -2004, OECD 207 and EU Method C.8).

Key value for chemical safety assessment

Short-term EC50 or LC50 for soil macroorganisms:

884 mg/kg soil dw

Additional information

GUIDELINE

The test was conducted to determine the acute toxicity of the test substance to earthworm according to “The guidelines for the testing of chemicals” (HJ/T 153-2004) and “The Guidelines for the Testing of Chemicals-Effects on Biotic Systems” (the 2nd edition, Beijing: China Environment Press, 2013) with reference to Procedure 207 of the “Guidelines for Testing of Chemicals” of the OECD: “Earthworm, Acute Toxicity Tests” (1984) and EU Method C.8 (Toxicity for Earthworms: Artificial Soil Test).

 

METHODS

The study was designed to determine the acute toxicity of test substances to earthworm at target concentrations of 0, 640, 800, 1000, 1250 and 1500 mg/kg dw soil. In this test, earthworms were exposed in samples of the artificial soil to which a range of concentrations of the test substance had been applied. Mortality was assessed 7 days and 14 days after application. The study comprised a range-finding test followed by a definitive test to determine the median lethal concentration (LC50) of the test substance.The moisture content of artificial soils was controlled in the range 26 to 35 % and pH 5.61 to 6.47 were used during the test. Temperature was maintained at 20.6 to 22.3 °C, humidity was maintained at 80 to 85 % and continuous light with intensity of 400 to 800 lux was supplied.

 

RESULTS

The highest concentration of test substance causing no mortality at 7 days was 640 mg/kg dw soil (7d-LC0) and the LC50 (7d) value was reported as 1241 mg/kg dw soil. The highest concentration of substance causing no mortality at 14 days was 640 mg/kg dw soil (14d-LC0) and the LC50 (14d) value was reported as 884 mg/kg dw soil.