Registration Dossier

Administrative data

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 January 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Inspection date: 21 August 2007; Date of signature: 15 October 2007

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Sponsor's identification : H-CB sodium salt
Description : blue solid
Lot number : MB-2
Date received : 04 December 2008
Storage conditions : room temperature in the dark

Sampling and analysis

Analytical monitoring:
no
Details on sampling:
- Concentrations:
- Sampling method: Not applicable as no analysis conducted. Analysis of the concentration, homogeneity and stability of the test material in the test preparations was not appropriate to the Test Guideline.

Test solutions

Vehicle:
not specified
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: For the purpose of the definitive test, the test material was dissolved directly in water. The following test concentrations were assigned to the test: 10, 32, 100, 320, and 1000 mg/l. An amount of test material (2000 mg) was dissolved in water with the aid of ultrasonication for approximately 15 minutes and the volume adjusted to 1 litre to give a 2000 mg/l stock solution from which serial dilutions were made to give 200 and 20 mg/l stock solutions. An aliquot (250 ml) of the 20 mg/l 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 10 mg/l. Similarly, aliquots (80 and 250 ml) of the 200 mg/l stock solution and aliquots (80 and 250 ml) of the 2000 mg/l stock solution were used to prepare the test concentrations of 32, 100, 320 and 1000 mg/l respectively. The volumetric flasks containing the stock solutions were inverted several times to ensure homogeneity of the stock solutions.
The control group was maintained under identical conditions but not exposed to the test material.

- Controls: 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 clean, oil-free 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.

Reference material preparation
For the purpose of the test a reference material, 3,5-dichlorophenol (Sigma-Aldrich Batch No. 15402DH-367) was used. 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 20 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

Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Method of cultivation: A mixed population of activated sewage sludge micro-organisms was obtained on 21 January 2009 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK which treats predominantly domestic sewage.

- Preparation of inoculum for exposure: The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21ºC and was used on the day of collection. The pH of the sample was 7.5 measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter. 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 which was then rinsed 3 times with 10 ml of deionised reverse osmosis water and filtration continued for 3 minutes. The filter paper was then dried in an oven at approximately 105ºC 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 4.0 g/l prior to use.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h

Test conditions

Hardness:
140 mg/l as CaCO3
Test temperature:
21°C
pH:
7.6
Dissolved oxygen:
3.5 - 4.3 mg O2/l
Salinity:
Not applicable, test carried out dechlorinated laboratory tap water.
Nominal and measured concentrations:
Nominal:
10, 32, 100, 320 and 1000 mg/l

Measured:
>1000
Details on test conditions:
TEST SYSTEM
- Test vessel: 500 ml conical flask
- Aeration: yes, the mixture was aerated with clean, oil-free compressed air via narrow bore glass tubes at a rate of approximately 0.5 – 1 litre per minute.
- Type of flow-through (e.g. peristaltic or proportional diluter): Not stated.
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 2
- No. of vessels per vehicle control (replicates): 1

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The test water used for the definitive test was laboratory tap water dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex water softener). After dechlorination and softening the water was then passed through a series of computer controlled plate heat exchangers to achieve the required temperature.
- Total organic carbon: 0.810 - 1.380, mean: 1.041 mg/l
- Pesticides: 0.00 - 0.001, mean: 0.00 µg/l
- Chlorine: Free chlorine: 0.030 - 0.550, mean: 0.231; Total chlorine: 0.80 - 0.610, mean: 0.297 mg/l
- Conductivity: 421.385 µS/cm at 20°C

OTHER TEST CONDITIONS
- Photoperiod: Not applicable.

TEST CONCENTRATIONS
- Test concentrations: 10, 32, 100, 320 and 1000 mg/l
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol

Results and discussion

Effect concentrationsopen allclose all
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
1 000 mg/L
Nominal / measured:
nominal
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Results with reference substance (positive control):
- Results with reference substance valid? yes

Please see Table 1 below for further details.

Any other information on results incl. tables

Oxygen consumption rates and percentage inhibition values for the control, test and reference materials are given in Table 1 below. 

Table1              Oxygen Consumption Rates and Percentage Inhibition Values in the Definitive Test after 3 Hours Contact Time

Nominal

Concentration

(mg/l)

Initial O2

Reading

(mg O2/l)

Measurement Period

(minutes)

Final O2Reading

(mg O2/l)

O2Consumption Rates

(mg O2/l/min)

% Inhibition

Control

R1

4.3

3

2.4

0.63

-

 

R2

4.4

4

1.8

0.65

-

Test Material

10

3.7

2

2.4

0.65

[2]

 

32

3.5

2

2.3

0.60

6

 

100

3.8

3

1.9

0.63

2

 

320

3.8

3

1.9

0.63

2

 

1000

4.3

3

2.3

0.67

[5]

3,5-dichlorophenol

3.2

5.1

5

2.4

0.54

16

 

10

6.7

10

3.3

0.34

47

 

32

7.5

10

6.3

0.12

81

[Increase in respiration rate as compared to controls]

R1– R2= Replicates 1 to 2

Applicant's summary and conclusion

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:

Introduction. 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 Council 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 solution 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 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 EC50of greater than 1000 mg/l. The No Observed Effect Concentration (NOEC) after 3 hours exposure was1000mg/l.

It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/l.

The reference material gave a 3-Hour EC50value of 11 mg/l, 95% confidence limits 9.1 - 13 mg/l.