1,3-dimethyl-1,3-diphenylurea

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

activated sludge respiration inhibition testing

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

7.11.-16.11.2011

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable guideline study, tested with the source substance Ethyl centralite. According to the ECHA guidance “Practical guide 6" the reliability was changed to RL2 to reflect the fact that this study was conducted on a read-across substance.

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
See attached document in IUCLID Section 13 - RA Justification document.pdf

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See attached document in IUCLID Section 13 - RA Justification document.pdf

3. ANALOGUE APPROACH JUSTIFICATION
See attached document in IUCLID Section 13 - RA Justification document.pdf

4. DATA MATRIX
See attached document in IUCLID Section 13 - RA Justification document.pdf, Table 1: Data matrix

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)

Deviations:

yes

Remarks:

See Overall remarks

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
The test substance is non-soluble in water in concentrations needed for the test. For this reason the direct dosing of test substance was used. The substance was weighed into reaction container where 100 mL of water were introduced.
In the preliminary test the concentrations were in the range of 46 – 1 000 mg.L-1 with a geometric progression factor of 2.2.
In the main test the concentrations were in the range of 100 – 1 000 mg.L-1 with a geometric progression factor of 1.8.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

TEST SYSTEM
The activated biological sludge containing mixed culture of microorganisms obtained from the second step of sewage treatment plant of Pardubice was used for the testing. The wastewater processed by the sewage treatment plant is predominantly municipal.

PREPARATION OF THE INOCULUM
The activated sludge was collected two days before the day of testing. After the sample collection the sludge was washed with potable water for 0.5 hour and subsequently decanted for 0.5 hour. This procedure was repeated three times in total.
Further the sludge was modified by addition of 50 mL of cultivation medium per 2 L of diluted sludge suspension at permanent aeration till the day of test.
The dry weight was determined from 10 mL of sludge suspension after 0.5-hour sedimentation.
Before the test the sludge was suspended in water up to concentration about 4 000 mg
of sludge dry weight per litre. The pH adjustment to 6.0 was carried out. In that way modified sludge suspension was aerated until the use.

JUSTIFICATION FOR THE SELECTION OF THE TEST SYSTEM
The activated sludge obtained is in conformity with the recommendations of the test guidelines.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

180 min

Test temperature:

20+/-2°C

pH:

approx. 6-8

Nominal and measured concentrations:

Main test: 100, 180, 320, 560 and 1 000 mg.L-1

Details on test conditions:

TEST SYSTEM
- Test vessel: aerated apparatus with glass ends
- Type (delete if not applicable): closed
- Aeration: with filtered pressured air, airflow rate 0.8 L/min
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 2
- Sludge dose on concentration of 4000 mg.L-1 : 380 mL.L-1

WATER PARAMETERS
- deionized water

OTHER TEST CONDITIONS
- Adjustment of pH: was not carried out
- Photoperiod: daily lighting

TEST PROCEDURE
100 mL of deionized water, the defined dose of the test or reference substance, 16 mL of the synthetic nutrient medium and 200 mL of the prepared sludge suspension were introduced into each test container. The volume was replenished up to 500 mL with deionized water.
The control samples were prepared only from the same volume of nutrient medium and sludge and replenished up to 500 mL with water.
The check of abiotic decomposition was performed at the highest concentration of the test substance and nutrient medium without sludge and with addition of 1 mL of mercury chloride solution.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): respiration rate of activated sludge

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8
- Range finding study
- Test concentrations: 46, 100, 220, 460 and 1 000 mg.L-1
- The preliminary test was performed to determine the test substance concentrations to be used in the main test.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Duration:

180 min

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: EC50 = 8.7 mg.L-1

Validity criteria fulfilled:

yes

Conclusions:

On the basis of the effective concentrations describing the respiration inhibition of activated sludge caused by the test substance, Centralit, the EC50 was found to be: EC50 > 1 000 mg.L-1
Under the current experimental conditions, all tested concentrations in the main test provided no significant inhibition of respiration rate (see table 6). From available data it was not possible to calculate the EC50 value.

Executive summary:

The influence of the test substance, Centralit, on the respiration rate of activated sludge was investigated after a contact time of 3 hours.

TEST PERFORMANCE

Test was performed according to: Method C.11 - Activated Sludge Respiration Inhibition Test, Council Regulation (EC) No. 440/2008, published in O.J.L 142, 2008.

The preliminary test was performed using 5 concentrations in range 46 – 1 000 mg.L-1 with a geometric factor of 2.2 to determine the concentrations to be used in the main test.

In the main test, 5 concentrations of the test substance in geometric progression with factor of 1.8 (100, 180, 320, 560, 1 000 mg.L-1) were used together with two control experiments (without the test substance), one at the beginning and one at the end of the test.

3,5-dichlorophenol was used as reference substance. Five concentrations in geometric progression with a factor of 1.4 (5.2, 7.2, 10, 14 and 19 mg.L-1) were selected.

The abiotic decomposition was not detected even at the highest concentration of the test substance.

The pH value during the main test was in the range of 7.1 to 7.9.

 

RESULTS OF THE RESPIRATION INHIBITION TEST

On the basis of the effective concentrations describing the respiration inhibition of activated sludge caused by the test substance, Centralit, the EC50 was found to be: EC50 > 1 000 mg.L-1

Under the current experimental conditions, all tested concentrations in the main test provided no significant inhibition of respiration rate. Therefore it was not possible to calculate the EC50 value.

Description of key information

Test was performed according to: Method C.11 - Activated Sludge Respiration Inhibition Test, Council Regulation (EC) No. 440/2008, published in O.J.L 142, 2008.

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L

Additional information

On the basis of the effective concentrations describing the respiration inhibition of activated sludge caused by the read -across substance Ethyl centralite, the EC50 was found to be: EC50 > 1 000 mg.L-1 (all tested concentrations in the main test provided no significant inhibition of respiration rate).