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 microorganisms

Link to relevant study record(s)

Reference

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 April - 28 April, 2009

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study, well documented report. Restriction due to no chemical analysis of test material.

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.6800 (Modified Activated Sludge, Respiration Inhibition Test for Sparingly Soluble Chemicals)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Issued by: the Department of Health of the Goverment of the United Kingdom Inspection date: 19 August 2008

Analytical monitoring:

no

Details on sampling:

- Concentrations: Nominal test concentrations were prepared at 10, 32, 100, 320 and 1000 mg/L
- Sampling method: The test material was not monitored in the test solutions
- Sample storage conditions before analysis: N.A.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)

-Test Material preparation :
The test material was dispersed directly in water. Amounts of test material (5, 16, 50, 160, and 500 mg) were each separately dispersed in approximately 250 mL of water and subjected to ultrasonication for approximately 15 minutes followed by prolonged mixing for 24 hours in order to maximize the dissolved test material concentration. All test vessels were shielded from the light during mixing. Synthetic sewage (16 ml), activated sewage sludge (200 ml) and water were added to a final volume of 500 ml to give the required concentrations of 10, 32, 100, 320, and 1000 mg/L. The control group was maintained under identical conditions but not exposed to the test material. Analysis of the concentration, homogeneity and stability of the test material in the test preparations was not appropriate to the Test Guideline.

-Preparation of Inoculum:
The activated sewage sludge samples was maintained on continuous aeration in the laboratory at a temperature of approximately 21C and was used on the day of collection. The pH of the sample was 7.6. Determination of the suspended solids level of the activated sewage sludge was carried out by filtering a sample (100 ml) of the activated sewage sludge by suction through a pre-weighed GF/A filter paper using a Buchner funnel that was then rinsed 3 times with 10mL of deionized reverse osmosis water and filtration continued for 3 minutes. The filter paper was then dried in an oven at approximately 105C for at least 1 hour and allowed to cool before weighing. This process was repeated until a constant weight was attained. The suspended solids concentration was equal to 3.7 g/L prior to use.


-Reference Material preparation:
For the purpose of the test 3,5-dichlorophenol was used as a reference material. Two stock solutions of 50 and 160 mg/L were prepared by dissolving the reference material directly in water with the aid of ultrasonication for approximately 15 minutes. Aliquots (10 and 100 mL) of the 160 mg/L stock solution were removed and dispersed with activated sewage sludge, synthetic sewage and water to give the final concentrations of 3.2 and 32 mg/L. Similarly a 100 mL aliquot of the 50 mg/L stock solution was used to prepare the 10 mg/L concentration. The volumetric flasks containing the reference material were inverted several times to ensure homogeneity of the solutions.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

A mixed population of activated sewage sludge micro-organisms was obtained from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK which treats predominantly domestic sewage. The sludge was maintained under continuous aeration at 21 deg. C until use. The pH of the sludge mixture was 7.6. The suspended solids concentration was equal to 3.3 g/L prior to use.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Hardness:

140 mg/l as CaCO3

Test temperature:

21ºC

pH:

7.3 - 7.8

Dissolved oxygen:

Between approximately 7.9 mg O2/L and 2.7 mg O2/L

Salinity:

Not applicable/freshwater

Nominal and measured concentrations:

Definitive Test: 0, 10, 32, 100, 320, and 1,000 mg/L nominal

Details on test conditions:

TEST SYSTEM
-Test Vessels: At time zero testing was carried out in 500 mL conical flasks, through which mixtures were aerated with oil-free compressed air at approx. 0.5 – 1 L/min. As each veseel reached 3 hours contact time an aliquot was removed from the conical flask and poured into the measuring vessel (250 ml darkened glass BOD bottle).

-Number of Vessels and Concentrations (Definitive Study):

Controls – two replicates containing inoculum
Reference – one each at 3.2, 10 and 32 mg/L 3,5-dichlorophenol

Test Material – one each at 10, 32, 100, 320, and 1000 mg/L nominal
Solvent control –
Biomass Loading Rate:


TEST MEDIUM / WATER PARAMETERS
-Test Water: Test water used for the definitive tests was laboratory tap water dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex water softener) giving water with a total hardness of approximately 140 mg/l as CaCO3. After dechlorination and softening the water was passed through a series of computer controlled plate heat exchangers to achieve the required temperature


OTHER TEST CONDITIONS
- Adjustment of pH: No.
- Photoperiod: No data.
- Light intensity: normal laboratory lighting


EXPOSURE
During testing, each mixture was aerated with clean, oil-free compressed air via narrow bore glass tubes at a rate of approx. 0.5 – 1 liter/min.
After the 3 hour contact time, an aliquot was removed from the conical test flask and transferred to a 250 mL darkened glass BOD bottle for measurement of dissolved oxygen.


EQUIPMENT:
-Measuring Vessel: 250 mL darkened glass BOD bottles

-Oxygen Meter: Yellow Springs DO meter with BOD probe


EFFECT PARAMETERS MEASURED (with observation intervals if applicable):

mg/L Oxygen was measured at start, and after 3 hours exposure. Observations on the test preparations throughout the test period were made at intervals of 0 hours, 30 minutes contact time, and 3 hours contact time.


TEST CONCENTRATIONS
Test concentrations: 0, 10, 32, 100, 320, 1000 mg/L nominal

Reference substance (positive control):

yes

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

250 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

94 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Duration:

3 h

Dose descriptor:

other: EC20

Effect conc.:

120 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Duration:

3 h

Dose descriptor:

other: EC80

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Details on results:

Observations on the test preparations of were made throughout the test period and large and fine particles of test material were visible in all test concenrations at time 0 hours. After 30 minutes and 3 hours contact time, no undissolved test material was visible in the 10 mg/L nominal test concentration.

Results with reference substance (positive control):

- Results with reference substance valid? Yes.

- Relevant effect levels: For the reference material, 3,5-dichlorophenol, the 3-Hour EC50 value was determined to be 7.1 mg/L with 95% confidence limits from 4.6 to 11 mg/L. The 3 hour NOEC for the reference material was determined to be 1.4 mg/L.

Table 1. Oxygen Consumption Rates and percentage Inhibition Values after 3 Hours Contact Time

Nominal Concentration (mg/L)	Initial O2(mg O2/l)	Measurement Period (minutes)	Final O2(mg O2/l)	O2 Consumption Rates (mg O2/l/min)	% Inhibition
Control            R1                     R2	6.6	10	3.0	0.36	--
	6.1	8	2.7	0.43	--
Test Material    10 32 100 320 1000	6.3	9	2.9	0.38	4
	6.5	10	3.0	0.35	11
	7.1	10	3.8	0.33	16
	7.6	10	5.9	0.17	57
	7.9	10	6.5	0.14	65
3,5-dichlorophenol 3.2 10 32
	7.1	10	4.4	0.27	32
	7.8	10	6.2	0.16	59
	8.3	10	7.6	0.07	82

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test material on the respiration of activated sewage sludge gave a 3-Hour EC50 of 250 mg/l. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 94 mg/L. It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/L. The reference material gave a 3-Hour EC50 value of 7.1 mg/l, 95% confidence limits 4.6 - 11 mg/L.

Executive summary:

GUIDELINE
A study was performed to assess the effect of the test material on the respiration of activated sewage sludge. The method followed that described in the OECD Guidelines for Testing of Chemicals (1984) No 209 "Activated Sludge, Respiration Inhibition Test", Method C.11 of Commission Regulation (EC) No. 440/2008 and US EPA Draft Ecological Effects Test Guidelines OPPTS 850.6800.

METHODS

Activated sewage sludge was exposed to an aqueous dispersion of the test material at concentrations of 10, 32, 100, 320 and 1000 mg/l for a period of 3 hours at a temperature of approximately 21C with the addition of a synthetic sewage as a respiratory substrate. The rate of respiration was determined after 3 hours contact time and compared to data for the control and a reference material, 3,5-dichlorophenol.

RESULTS

The effect of the test material on the respiration of activated sewage sludge gave a 3-Hour EC50 of 250 mg/l. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 94 mg/L. It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/L. The reference material gave a 3-Hour EC50 value of 7.1 mg/L (95% confidence limits 4.6 - 11 mg/L).

Description of key information

The effect of the test material on the respiration of activated sewage sludge gave a 3-Hour EC50 of 250 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 94 mg/L. It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/L. The reference material gave a 3-Hour EC50 value of 7.1 mg/l, 95% confidence limits 4.6 - 11 mg/L (OECD 209, EU Method C.11 and OPPTS 850.6800).

Key value for chemical safety assessment

EC50 for microorganisms:

250 mg/L

EC10 or NOEC for microorganisms:

94 mg/L

Additional information

GUIDELINE
A study was performed to assess the effect of the test material on the respiration of activated sewage sludge. The method followed that described in the OECD Guidelines for Testing of Chemicals (1984) No 209 "Activated Sludge, Respiration Inhibition Test", Method C.11 of Commission Regulation (EC) No. 440/2008 and US EPA Draft Ecological Effects Test Guidelines OPPTS 850.6800.

METHODS

Activated sewage sludge was exposed to an aqueous dispersion of the test material at concentrations of 10, 32, 100, 320 and 1000 mg/L for a period of 3 hours at a temperature of approximately 21 °C with the addition of a synthetic sewage as a respiratory substrate. The rate of respiration was determined after 3 hours contact time and compared to data for the control and a reference material, 3,5-dichlorophenol.

RESULTS

The effect of the test material on the respiration of activated sewage sludge gave a 3-Hour EC50 of 250 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 94 mg/L. It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/L. The reference material gave a 3-Hour EC50 value of 7.1 mg/L (95% confidence limits 4.6 - 11 mg/L).