N-(dodecylphenyl)naphthalen-1-amine

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:

25 August 2000 to 5 September 2000.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

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

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

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

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

No further details specified in the study report.

Analytical monitoring:

no

Details on sampling:

Not specified

Vehicle:

no

Details on test solutions:

For the purpose of the definitive study, the test material was dispersed directly in water.
An amount of test material (500 mg) was dispersed in approximately 250 ml of water and subjected to ultrasonication (approximately 30 minutes). Synthetic sewage (16 ml), activated sewage sludge (200 ml) and water were added to a final volume of 500 ml to give the required concentration of 1000 mg/l.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

A mixed population of activated sewage sludge micro-organisms was obtained on 5 September 2000 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Belper. Derbyshire, UK which treats predominantly domestic sewage.

Preparation of inoculum
The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of 21 °C and was used on the day of collection. The pH of the sample was 7.6 and the suspended solids equal to 3.9 g/l prior to use.

Test type:

not specified

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Post exposure observation period:

No post exposure observation period specified.

Hardness:

The test water used for both the range-finding and definitive studies 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 100 mg/l as CaCO3.

Test temperature:

The study was conducted under normal laboratory lighting in a temperature controlled room at 21 °C.

pH:

7.9 - 8.2

Dissolved oxygen:

Not required - respiration study.

Salinity:

Not specified

Conductivity:

Not specified

Nominal and measured concentrations:

Nominal concentration of 1000 mg/l

Details on test conditions:

Preparation of test system
At time "0" 16 ml of synthetic sewage was diluted to 300 ml with water and 200 ml of inoculum added in a 500 ml conical flask (first control). The mixture was aerated with compressed air via narrow bore glass tubes at a rate of approximately 0.5 - 1 litre per minute. Thereafter, at 15 minute intervals the procedure was repeated with appropriate amounts of the reference material being added. Finally a second control was prepared.
As each vessel reached 30 minutes contact time an aliquot was removed from the conical flask and poured into the measuring vessel (250 ml darkened glass Biological Oxygen Demand (BOD) bottle) and the rate of respiration measured using a Yellow Springs dissolved oxygen meter fitted with a BOD probe. The contents of the measuring vessel were stirred constantly by magnetic stirrer. The rate of respiration for each flask was measured over the linear portion of the oxygen consumption trace for an approximate 10 minute period (between approximately 7.7 mg O2/l and 1.6 mg O2/l. After measurement the contents of the BOD bottle were returned to the test vessel.
This procedure was repeated after 3 hours contact time. The study was conducted under normal laboratory lighting in a temperature controlled room at 21 °C.

Range-finding Studies
The test concentration to be used in the definitive study was determined by preliminary range-finding studies.
In the initial range-finding study activated sewage sludge micro-organisms were exposed to nominal test concentrations of 100 and 1000 mg/l. The test material was dispersed directly in water.
Using this approach the effect of an excess amount of undissolved test material can be assessed in order to determine whether the test material has any effect on the activated sewage sludge microorganisms exoenzymes, or whether the uptake of undissolved test material by processes such as phagocytosis has any adverse effect on the activated sewage sludge. Additionally, the effect of the test material as a whole on a waste water treatment facility and any possible contact toxicity can be determined.
Amounts of test material (50 and 500 mg) were separately dispersed in approximately 250 ml of water and subjected to ultrasonication (approximately 30 minutes). 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 100 and 1000 mg/l.
Data supplied by the Sponsor gave a water solubility level of 1.96 mg/l. Therefore in the second range-finding study activated sewage sludge micro-organisms were exposed to a test concentration of 1.96 mg/l. The test material was prepared using a preliminary solution in dimethylformamide. Using this approach the effect of the test material at the highest attainable test concentration that was a solution could be determined.
An amount of test material (196 mg) was dissolved in dimethylformamide and the volume adjusted to 10 ml to give a 196 mg/10 ml solvent stock solution. An aliquot (50 μl) of this solvent stock solution was dispersed with synthetic sewage (16 ml), activated sewage sludge (200 ml) and water, to a final volume of 500 ml, to give the required concentration of 1.96 mg/l. The volumetric flask containing the solvent stock solution was inverted several times to ensure homogeneity of the solution.
The control and solvent control groups were maintained under identical conditions but not exposed to the test material. The solvent control group was exposed to 100 μl/l of dimethylformamide.
A solvent control vessel was prepared containing 16 ml synthetic sewage, 300 ml water, 50 μl dimethylformamide and 200 ml of inoculum in a 500 ml conical flask and oxygen consumption rates were measured after 3 hours contact time only.
The control group was used for calculation of inhibition of respiration rate for the reference material prepared by dissolving the reference material directly in water. The solvent control was used for calculation of inhibition of respiration rate for the test material vessel prepared using a preliminary solution in auxiliary solvent.
A reference material, 3,s-dichlorophenol, was included in the range-finding study at concentrations of 3.2 and 32 mg/l in order to confirm the suitability of the inoculum.

Definitive Study
Based on the results of the range-finding study a "limit test" was conducted for the definitive study at a test concentration of 1000 mg/l (three replicate vessels) to confirm that at this concentration no effect on respiration of the activated sewage sludge was observed.
Observations were made on the test preparations throughout the study period, and the pH of the control, reference material and test material preparations was measured at the end of the exposure period prior to measurement of the oxygen consumption rate.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

Range-finding Studies
No significant effect on respiration was observed at any of the test concentrations employed in either of the range finding studies. A study to determine the General Physico-Chemical properties of the test material (Safepharm Laboratories Limited Project Number 445/283) gave a water solubility level of 1.96 mg/l. It was therefore considered justifiable to conduct the definitive study at a nominal concentration in excess of this value as the second range finding study confirmed that there was no effect on the respiration rate at the maximum attainable test concentration that was a solution. Based on this information, a single test concentration, in triplicate, of 1000 mg/l was selected for the definitive study.

Definitive Study
The following results were derived:
APAN EC50 (30 minutes): >1000 mg/l
Variation in respiration rates of controls 1 and 2 after 30 minutes: ± 4%.

APAN EC50 (3 hours): >1000 mg/l
Variation in respiration rates of controls 1 and 2 after 3 hours: ± 4%.

The No Observed Effect Concentration (NOEC) after 3 hours was 1000 mg/l.

The validation criteria for the control respiration rates and reference material EC50 values have been satisfied.
It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/l.
Observations made throughout the study period, see Table 6, showed that at the test concentration of 1000 mg/l the test material was visible as globules of test material on the surface of the test preparations. The test material was also visible adhering to the side of the test vessels. These observations were considered to be due to the insoluble nature of the test material in the test medium.

Results with reference substance (positive control):

3,5-dichlorophenol EC50 (30 minutes): 17 mg/l
3,5-dichlorophenol EC50 (3 hours): 10 mg/l

Reported statistics and error estimates:

Not specified

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test material on the respiration of activated sewage sludge micro-organisms gave a 3-Hour EC50 of greater than 1000 mg/l. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 1000 mg/l.

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", EEC Commission Directive 87/302/EEC and US EPA Draft Ecological Effects Test Guidelines OPPTS 850.6800.

 

Following preliminary range-finding studies, activated sewage sludge was exposed to an aqueous dispersion of the test material at a concentration of 1000 mg/l (three replicate flasks) for a period of 3 hours at 21 °C with the addition of a synthetic sewage as a respiratory substrate.

 

The rate of respiration was determined after 30 minutes and 3 hours contact time and compared to data for the control and a reference material, 3,5-dichlorophenol.

 

The effect of the test material on the respiration of activated sewage sludge gave a 3-Hour EC50 of greater than 1000 mg/l. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 1000 mg/l.

Description of key information

GLP accredited laboratory study performed in accordance with OECD Guideline No 209, EU Method C.11 and US EPA procedure OPPTS 850.6800.

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L

EC10 or NOEC for microorganisms:

1 000 mg/L

Additional information

A study was performed to assess the effect of the test material on the respiration of activated sewage sludge.

Following preliminary range-finding studies, activated sewage sludge was exposed to an aqueous dispersion of the test material at a concentration of 1000 mg/l (three replicate flasks) for a period of 3 hours at 21 °C with the addition of a synthetic sewage as a respiratory substrate.

 

The effect of the test material on the respiration of activated sewage sludge gave a 3-Hour EC50 of greater than 1000 mg/l. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 1000 mg/l.