Reaction products of diphenylamine with nonene, branched

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

from 5/12/2002 to 20/03/2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: EEC Commission Directive 87/302/EEC

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

impurity 1

Reference substance name:

Ar-nonyldiphenylamine

EC Number:

248-295-7

EC Name:

Ar-nonyldiphenylamine

Cas Number:

27177-41-9

Molecular formula:

C21H29N

IUPAC Name:

4-nonyl-N-phenylaniline

Test material form:

liquid: viscous

Remarks:

pale brown viscous liquid

Details on test material:

Reaction products of Benzenamine, N-phenyl, and Nonene, branched EC 253-249-4; CAS no.: 36878-20-3, followed by purification (distillation) with a 96% mono-alkyl content.

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature, dark
- Solubility in water: 0.0113 mg/l
- Reactivity of the test material with the incubation material used (e.g. plastic ware): Data suggested that the test material would adsorb to glassware (glass bottles were pre-conditioned with test concentration)

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): two different methods were used. First: test material was dispersed directly in water; Second: test material was dissolved in dimethylformamide first
- Final concentration of a dissolved solid, stock liquid or gel: 2.825 mg/25 ml (second)

Sampling and analysis

Analytical monitoring:

no

Details on sampling:

- Sampling method: after 30 min an aliquot was removed from the colonial flasks of each treatment and poured into a measuring vessel (250 ml , darkened glass BOD bottle). The respiration rate was measured while the content of the vessel was stirred constantly. It was returned to the test vessel after measurement

Test solutions

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method:
a) test material was dispersed directly in water and subjected to ultrasonication (30 min), sythetic sewage, activated sewage sludge and water were added, final concnetration = 1000 mg/l ;
b) a preliminary solution in dimethylformamide was prepared (2.825 mg/ 25 ml), 50 µl of the stock solution was dispersed with synthetic sewage, activated sewage sludge and water, final conmcentration = 0.0113 mg/l
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): dimethylformamide
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): solvent control = 100 µl/l
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): Oil globules of test material dispersed throughout with an oil slick on the surface were observed for the test concentration of 1000 mg/l
- Other relevant information: activated sewage sludge was centrifuged (3500 rpm, 10 min) and supernatant discarded prior to addition of activated sewage sludge "pellets" to the test vessels

Test organisms

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Laboratory culture: on collection day
- Name and location of sewage treatment plant where inoculum was collected: aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK
- Method of cultivation: maintenance under continuous aeration at 21 °C
- Preparation of inoculum for exposure: a) effect of dispersed material: pH= 7.5; determination of suspended solid level = sample was filtered (100 ml) through a pre - weighted GF/A filter paper and dried
b) effect at water solubility level: pH = 7.7; same procedure as in a)
- Initial biomass concentration: 4.0 g/l

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test conditions

Hardness:

100 mg/l [CaCO3 ] (hardness of water used for preparation of the test medium)

Test temperature:

room temperature = 21 °C

pH:

see table 1

Dissolved oxygen:

see table 1

Nominal and measured concentrations:

Test material nominal: 1000 mg/l (dispersed concentration), 0,0113 mg/l
Reference substance nominal: 3.2, 10, 32 mg/l

Details on test conditions:

TEST SYSTEM
- Test vessel: 500 ml colonial flask
- Type (delete if not applicable): not specified
- Material, size, headspace, fill volume: fill volume = 500 ml
- Aeration: every 15 min with compressed air (approx. 0.5 - 1 l/min)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 2
- No. of vessels per vehicle control (replicates): 1
- No. of vessels per reference substance (replicates): 1per concentration (3 in total)
- Sludge concentration (weight of dry solids per volume): 800 mg/ 500 ml
- Nutrients provided for bacteria: Synthetic sewage = 16 g Peptone, 11 g Meat extract, 3 g Urea, 0.7 g NaCl, 0.4 g CaCl2.2H2O, 0.2 g MgSO4.7H2O, 2.8 g K2HPO4 dissolved in 1 l water
- Nitrification inhibitor used (delete if not applicable): none

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: laboratory tap water was dechlorinated, filtered through an activated carbon filter and partly softened, the water was warmed to the required temperature prior use


OTHER TEST CONDITIONS
- Light intensity: normal laboratory lightning

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : pH value and oxygen consumption rate was measured at the end of the exposure, changes on the test preparations were observed throughout the test period

TEST CONCENTRATIONS
- Justification for using fewer concentrations than requested by guideline: results of the range finding study and preliminary dosing trials

RANGE FINDING STUDY
- Test concentrations: 100, 1000 mg/l and control
- Results used to determine the conditions for the definitive study: No significant effect on respiration was observed at either test concentration employed. Results of the preliminary dosing trials considered 0.0113 mg/l to be the highest employed concentration, which is consistent with the reported water solubility level of the test material.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorphenol

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

> 0.011 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Basis for effect:

inhibition of total respiration

Remarks on result:

other: concentration was equal to the reported water solubility level of the test material

Effect concentrations 2

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 0.011 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Basis for effect:

inhibition of total respiration

Remarks on result:

other: concentration was equal to the reported water solubility level of the test material

Effect concentrations 3

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Basis for effect:

inhibition of total respiration

Remarks on result:

other: concentration exceeded the water solubility of the test material

Effect concentrations 4

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Basis for effect:

inhibition of total respiration

Remarks on result:

other: concentration exceeded the water solubility of the test material

Details on results:

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: oil slick on the surface for 1000 mg/l treatment
- Effect concentrations exceeding solubility of substance in test medium: yes (1000 mg/l)
- Blank controls oxygen uptake rate:
a) (1000 mg/l) 0.5 - 0.53 mg O2/l/min
b) (0.0113 mg/l) 0.64 - 0.65 mg O2/l/min
- Coefficient of variation of oxygen uptake rate in control replicates:
a) (1000 mg/l) after 30 min = +- 1%, after 3 h = +- 3 %
b) (0.0113 mg/l) after 30 min = +- 1%, after 3 h = +- 1 %

Results with reference substance (positive control):

- Results with reference substance valid?: yes
- Relevant effect levels:
EC50 after 3 h = 12 mg/l (reported for dispersed test material 1000 mg/l)
EC50 after 3 h = 13 mg/l (reported for water solubility level 0.0113 mg/l)

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The results show that at a concentration in excess of the water solubility level of the test material, the test material has no effect on the respiration rate of the activated sewage sludge. Furthermore, at a concentration equivalent to the reported water solubility level of the test material, the test material has no effect on the respiration rate of the activated sewage sludge.

Executive summary:

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". A range-finding test conducted at test concentrations in excess of the water solubility of the test material (100 and 1000 mg/l) indicated that dispersed test material exhibited no adverse effect on the respiration rate of activated sewage sludge.

Therefore in order to satisfy the requirements of all Competent Authorities worldwide, the definitive test was conducted using two methods of addition of the test material to the test system:

a) activated sewage sludge was exposed to a dispersed test material concentration of 1000 mg/l, which was dispersed directly in water

b) acitive sewage sludge was was exposed to a nominal test concentration of 0.0113 mg/l, which is consitent with the repoted water solubility level of the test material. The concentration was prepared using a preliminary solution in auxiliary solvent to spike the test medium.

In each method the activated sewage sludge was exposed to the test material for a period of 3 hours at 21ºC with the addition of a synthetic sewage as a respiratory substrate. The rate of respiration was determined after 30 min and 3 h contact time and compared to data for the control and reference material, 3,5-dichlorophenol. In the test vessels of both methods no significant inhibition of respiration of the activated sewage sludge was observed.

3-Hour EC50 value was determined to be greater than 1000 mg/l. Correspondingly the No Observed Effect Concentration (NOEC) after 3 hours exposure was determined to be 1000 mg/l. These results show that at a concentration in excess of the water solubility level of the test material, the test material has no effect on the respiration rate of the activated sewage sludge.

In the test vessels prepared at a concentration equivalent to the reported water solubility level of the test material, no significant inhibition of respiration of the activated sewage sludge was observed. The 3-h EC50 value was determined to be > 0.0113 mg/l, the NOEC was determined to be 0.0113 mg/l. These results show that at a concentration equivalent to the reported water solubility level of the test material, the test material has no effect on the respiration rate of the activated sewage sludge.