Registration Dossier

Administrative data

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26/01/2007-30/01/2007.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well documented report of a guideline study conducted according to GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material: ATBS
- Physical state: white powder
- Analytical purity: 99.4 %
- Lot/batch No.: 061202
- Expiration date of the lot/batch: 30/06/2007
- Stability under test conditions: stable
- Storage condition of test material: room temperature
- Date received: 18/01/2007

Sampling and analysis

Analytical monitoring:
no

Test solutions

Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: dilution
- Eluate: no
- Controls: yes
- Chemical name of vehicle : dechlorinated tap water
- Evidence of undissolved material (e.g. precipitate, surface film, etc): no

Test organisms

Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Laboratory culture: A sample of activated sludge was obtained the day before the start of the test from Worlingworth Sewage Treatment Works, which treats predominantly domestic waste.
- Method of cultivation: In the laboratory, the sample was maintained under aerobic conditions until required.
- Preparation of inoculum for exposure: On the day of collection, aliquots (25 ml) of the activated sludge were filtered through dried and preweighed Whatman GF IC filter papers which were then dried again at approximately 105°C for at least one hour, allowed to cool in a desiccator and reweighed. The mixed liquor suspended solids (MLSS) content of the activated sludge was then calculated
- Pretreatment: Synthetic sewage (50 mI/l) was added to the sample of activated sludge and the mixture aerated overnight. On the day of the test, the MLSS content of the sludge was determined and adjusted to 4 g/l by the addition of dechlorinated tap water. The pH and temperature of the sludge was also measured
- Initial biomass concentration: 4 g activated sludge/l

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h

Test conditions

Hardness:
200-250 mg/l as CaCO3
Test temperature:
Initial: 19.9-20.0°C
Final: 19.7-19.8°C
pH:
Initial:
-3,5-DCP stock solution (preparation): 7.9
-Activated sludge: 7.2
-ATBS stock solution: before adjustement: 2.2
-ATBS stock solution: after adjustement: 7.5
Dissolved oxygen:
Initial DO concentration in the culture (mgO2/l): 7.2-8.0
Initial measured DO concentration (mgO2/l): 6.5
Salinity:
Not specified
Nominal and measured concentrations:
Nominal concentrations: 10, 100 and 1000 (triplicate) mg/l
Details on test conditions:
TEST SYSTEM
- Test vessel: not specified
- Type: closed
- Material, size, headspace, fill volume: -BOD bottle : 270 ml
- Yellow Springs Instruments (YSI) dissolved oxygen meter
- Aeration: ca. one litre/minute (supply of oil-free compressed air)
- Type of flow-through: none (static)
- No. of vessels per concentration (replicates): 2 replicates for the 1000mg/l test material concentration
- No. of vessels per control: 2
- Biomass loading rate: 4 g/l
TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: tap water
- Chlorine: dechlorinated
- Hardness: 200 - 250 mg/l as CaCO3
OTHER TEST CONDITIONS
- Adjustment of pH: yes
EFFECT PARAMETERS MEASURED (with observation intervals if applicable): dissolved oxygen
TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
The prepared mixtures were covered loosely with aluminium foil and aerated for three hours in a thermostatically-controlled water bath, using a glass aerator connected to a laboratory supply of oil-free compressed air (ca. one litre/minute).
Following the exposure period, a well-mixed sample of each mixture was transferred to a biochemical oxygen demand (BOD) bottle (nominal capacity, 270 ml). The rate of oxygen consumption was measured, over a period of approximately ten minutes or until the dissolved oxygen concentration fell below 2 mg02/l, using a Yellow Springs Instruments (YSI) dissolved oxygen meter, with temperature probe and self-stirring bottle probe, connected to a chart recorder. The pH and temperature of the samples were measured at the start and end of the test.
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol (3,5-DCP)

Results and discussion

Effect concentrations
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
Remarks:
respiration rate
Details on results:
- Any observations that might cause a difference between measured and nominal values: None
- Effect concentrations exceeding solubility of substance in test medium: None
-other: ATBS was considered to have had no biologically significant inhibitory effect on the respiration rate of activated sludge at any of the concentrations employed in the test. Respiration rates were decreased by, at most, 9% in one mixture containing the test substance at 1000 mg/I. The EC20, EC50 and EC80 of the test substance could not, therefore, be calculated but these must be greater than 1000 mg/l, the highest level tested.
Results with reference substance (positive control):
- Results with reference substance valid. Yes. The three-hour EC50 for 3,5-DCP (14.5 mg/l) fulfilled the validity criterion relating to sensitivity to inhibition (acceptable EC50 range 5 to 30 mg/l), and that relating to the respiration rates in the control (variation not greater than 15%) was also satisfied
- Relevant effect levels: Sludge respiration rates were progressively reduced in the presence of increasing concentrations of 3,5-DCP. The 3-hour 50% effect concentration (EC50) for 3,5-DCP was calculated to be 14.5 mg/l (95% confidence limits 10.9-19.8 mg/l).
-other: The specific respiration rate of the control culture established at the end of the test (23.1 mg02/g/h) was 111 % of the rate of that established at the start (20.8 mg02/g/h).

Any other information on results incl. tables

Table 1: Dissolved oxygen concentrations and measurement times

Test mixture

Initial DO concentration in the culture (mg02/l)

Initial measured DO concentration (mg02/l)

Final measured DO concentration (mg02/l)

Measurement time (minutes)

 

 

 

 

 

 

Control (1)

7.4

6.5

2.5

7.2

ATBS (mg/l)

 

 

 

 

10

7.2

6.5

2.5

7.1

100

7.8

6.5

2.5

7.0

1000

7.4

6.5

2.5

6.8

1000

8.0

6.5

2.5

7.5

1000

7.5

6.5

2.5

7.0

3,5-DCP

 

 

 

 

{mgill

 

 

 

 

3

7.0

6.5

2.5

8.4

10

7.8

6.5

4.6

5.6

32

8.4

7.9

6.3

9.1

Control (2)

7.7

6.5

2.5

6.5

DO: Dissolved Oxygen

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
ATBS was considered to have had no biologically significant inhibitory effect on the respiration rate of activated sludge at any of the concentrations employed in the test. Therefore, the No Observed Effect Concentration was concluded to be 1000 mg/l, the highest level tested.