Registration Dossier

Administrative data

Endpoint:
activated sludge respiration inhibition testing
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
16 August 2007 and 29 August 2007
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 the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

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:
EPA OPPTS 850.6800 (Modified Activated Sludge, Respiration Inhibition Test for Sparingly Soluble Chemicals)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Sponsor's identification: AS305BD
Description: dark brown, viscous liquid
Batch number: TS07002
Storage conditions: room temperature in the dark
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Not applicable

Sampling and analysis

Analytical monitoring:
no
Details on sampling:
not applicable.

Test solutions

Vehicle:
no
Details on test solutions:
For the purposes of the range-finding and definitive tests, the test material was dispersed directly into the test water.

RANGE-FINDING TEST
Amounts of test material (50 and 500 mg) were each separately dispersed, using a plastic disposable syringe, in approximately 250 mL of water and subjected to ultrasonication for approximately 15 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.

DEFINITIVE TEST
An amount of test material (500mg) was dispersed, using a plastic disposable syringe in approximately 250 mL of water and subjected to ultrasonication for 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. This method of preparation was performed for the three separate replicate test vessels.

Test organisms

Test organisms (species):
activated sludge
Details on inoculum:
A mixed population of activated sewage sludge micro-organisms was obtained on 16 August 2007 for the range-finding test and on 29 August 2007 for the definitive test 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
The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21°C and was used on the day of collection. The pH of the sample was 8.0 measured using a WTW pH/Oxi 3401 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 105 ºC 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
Post exposure observation period:
Not applicable.

Test conditions

Hardness:
140 mg/L as CaCO3
Test temperature:
21 ± 1 ºC
pH:
7.3-8.6
Dissolved oxygen:
See 'Remarks and results including tables and figures' below
Salinity:
Not applicable
Nominal and measured concentrations:
Based on the results of the reange-finding test, a 'limit test' was conducted at a concentration of 1000 mg/L to confirm whether at this concentration no effect on respiration of the activated sewage sludge was observed.
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): not reported
- Material, size, headspace, fill volume: glass, 500ml conical flask, 500ml fill volume
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: dechlorinated laboratory tap water

OTHER TEST CONDITIONS
- Adjustment of pH:
- Photoperiod: not reported
- Light intensity: normal laboratory lighting

SYNTHETIC SEWAGE
A synthetic sewage of the following composition was added to each test vessel to act as a respiratory substrate:
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 litre of water with the aid of ultrasonication

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
Oxygen consumption rates: Measured at 3 hours in range-finding study and every 30 minutes during the definitive study.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: limit test
- Range finding study
- Test concentrations: 100, 1000 mg/l
- Results used to determine the conditions for the definitive study: yes
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
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: 95% CL 6.0-9.9 mg/L
Duration:
3 h
Dose descriptor:
other: EC20
Effect conc.:
> 1 000 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
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:
See 'Effect concentrations' above and 'Remarks on results including tables and figures' below
Results with reference substance (positive control):
The reference material gave a 3-Hour EC50 value of 7.7 mg/L, 95% confidence limits 6.0 - 9.9 mg/L.
Reported statistics and error estimates:
In order to calculate the inhibitory effect of the reference materials, the respiration rate was expressed as a percentage of the two control respiration rates:

% inhibition = [1- 2RS/RC1 + RC2] x 100

where:
RS = oxygen consumption rate for test or reference sample
RC1 + RC2 = oxygen consumption rates for controls 1 and 2

The percentage inhibition values were plotted against concentration for the reference material only, a line fitted using the XIfit software package (IDBS) and the EC20, EC50 and EC80 values determined from the equation for the fitted line. 95% confidence limits were calculated for the the EC50 values using the method of Litchfield and Wilcoxon (1949). The NOEC was determined by inspection of the inhibition of respiarion rate data.

Any other information on results incl. tables

Table 1. Oxygen consumption rates and percentage inhibition values in the definitive test after 3 hours contact time.

Nominal concentration (mg/L) Intial O2 reading (mg O2/L) Measurement period (minutes) Final O2 reading (mg O2/L) O2consumption rates (mg O2/L/min) % inhibition
Control R1 6.3 9 2.5 0.42 -
R2 6.3 4 4.2 0.53 -
Test material 1000 R1 3.7 5 1.5 0.44 7
1000 R2 4.0 4 2.3 0.43 9
1000 R3 3.2 3 1.9 0.43 9
3,5-dichlorphenol 3.2 5.4 9 2.2 0.36 24
10 7.4 10 5.6 0.18 62
32 8.1 10 7.4 0.07 85

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 resulted in a 3h EC50 of >1000 mg/L and a 3h NOEC of 1000 mg/L.
Executive summary:

A study was performed to assess the effect of the test material on the respiration of activated sewage sludge in line with OCED Guideline 209. Following a range-finding study, activated sewage sludge was exposed to the test material at a concentratio of 1000 mg/l in three replicate flasks for a period of 3 hours.

The effect of the test material on the respiration of activated sewage sludge gave a 3h EC50 of >1000 mg/L and a NOEC of 1000 mg/L. It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/L.