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Administrative data

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 November 2008 and 27 November 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Valid/accepted methods. GLPStudy conducted to GLP in compliance with agreed protocols with no deviations from standard test guidelines and/or minor methodological deficiences which do not affect the quality of the 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
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 850.6800 (Modified Activated Sludge, Respiration Inhibition Test for Sparingly Soluble Chemicals)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Details on test material:
dark red powder
Batch Number MB-1-A
Storage: Room temperature in dark

Sampling and analysis

Analytical monitoring:
no

Test solutions

Vehicle:
no
Details on test solutions:
Range Finding Test
The test concentration to be used in the definitive test was determined by a preliminary range-finding test.

In the range-finding test activated sewage sludge micro-organisms were exposed to a series of test concentrations of 1.0, 10, 100 and 1000 mg/I. The test material was dissolved directly in water.

An amount of test material (2000 mg) was dissolved in water with the aid of ultrasonication for approximately 10 minutes and the volume adjusted to 1 litre to give a 2000 mg/I stock solution from which serial dilutions were made to give 200, 20 and 2.0 mg/I stock solutions. Aliquots (250 ml) of the 2.0, 20, 200 and 2000 mg/I stock solutions were each separately dispersed with synthetic sewage (16 ml), activated sewage sludge (200 ml) and water, to a final volume of 500 ml, to give the required concentrations of 1.0, 10, 100 and 1000 mg/I 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.

A reference material, 3,5-dichlorophenol (Sigma-Aldrich Batch No. 15402DH-367), was included in the range-finding test at concentrations of 3.2 and 32 mg/I in order to confirm the suitability of the inoculum.

An amount of reference material (160 mg) was dissolved in water with the aid of ultrasonication for approximately 20 minutes and the volume adjusted to 1 litre to give a 160 mg/I stock solution. Aliquots (10 and 100 ml) of the 160 mg/I stock solution were removed and dispersed with activated sewage sludge and water to give the final concentrations of 3.2 and 32 mg/I. The volumentric flask containing the reference material was inverted several time to ensure homogeneity of the solution.

Definitive Test
Based on the results of the range-finding test a "limit test" was conducted at a concentration of 1000 mg/I (three replicate vessels) to confirm that at this concentration no effect on respiration of the activated sewage sludge was observed.

For the purpose of the definitive test, the test material was dissolved directly in water. An amount of test material (2000 mg) was dissolved in water with the aid of ultrasonication for approximately 10 minutes and the volume adjusted to 1 litre to give a 2000 mg/I stock solution. Aliquots (3 x 250 ml) of this stock solution were separately 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 1000 mg/I in triplicate. The volumetric flask containing the stock solution was inverted several times to ensure homogeneity of the stock solution.

The control group was maintained under identical conditions but not exposed to the test material.

Analysis of the concentration, homogeneity and stability of the test material in the test preparations was not appropriate to the Test Guideline.

Test organisms

Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
Test species: mixed population of activated sewage sludge micro-organisms was obtained on 21 November 2008 for the range-finding test and on 27 November 2008 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.

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 7.8 measured using a VVTW 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°C 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.

Synthetic sludge added to test vessel to act as a respiratory substrate:
16g Peptone
11g Meat extract
3g Urea
0.7g NaCl
0.4g CaCl2*2H2O
0.2g MgSO4*7H2O
2.8g K2HPO4
Dissolved in 1 litre of water


Study design

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

Test conditions

Hardness:
140 mg/L CaCO3
Test temperature:
21 ± 1 °C
pH:
pH7.8 measured using a WTW pH/Oxi 3401 pH and dissolved oxygen meter
Dissolved oxygen:
In some instances, the initial and final dissolved oxygen concentrations were below those recommended in the test guidelines (6.5 mg 02/I and 2.5 mg 02/I respectively). This was considered to have had no adverse effect on the results of the study given that in all cases the oxygen consumption rate was determined over the linear portion of the oxygen consumption trace
Nominal and measured concentrations:
Measurements based on nominal concentrations only. In the range-finding test activated sewage sludge micro-organisms were exposed to a series of test concentrations of 1.0, 10, 100 and 1000 mg/I. The test material was dissolved directly in water.Based on the results of the range-finding test a "limit test" was conducted at a concentration of 1000 mg/I (three replicate vessels) to confirm that at this concentration no effect on respiration of the activated sewage sludge was observed.
Details on test conditions:
Laboratory tap water dechlorinated by passage through activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex water softener).
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol

Results and discussion

Effect concentrationsopen allclose all
Duration:
3 h
Dose descriptor:
other: EC20
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
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:
other: EC80
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):
3 hour EC20 2.7 mg/L
3 hour EC50 8.0 mg/L; 95% Confidence Limits 6.3-10 mg/L
3 hour EC80 24 mg/L

Variation in respiration rates of controls 1 and 2 after 3 hours contact time was ± 11%.

The validation criteria for the control respiration rates and reference material EC50 values were therefore satisfied.

Any other information on results incl. tables

  Nominal Concentration (mg/l) Initial 02 Reading (mg 02/1) Measurement Period (minutes)
Final 02 Reading (mg O2/l)
O2 Consumption Rates (mg O2/l/min)		 % inhibition
Control 0 - R1 5.8 7 2.2 0.51 ‑
0- R2 3.6 3 1.7 0.63 ‑
Test Material 1000 - R1 4.8 4 2.5 0.58 [2]
1000 - R2 5.9 6 2.3 0.6 [5]
1000 - R3 5.2 5 2.2 0.6 [5]
3,5-dichlorophenol 3.2 6.7 10 2.3 0.44 23
10 7.9 10 5.6 0.23 60
32 8.6 10 7.8 0.08 86

Values in parenthese indicate increase compared to controls

  EC, (3 Hours) (mg/I) 95% Confidence Limits (mg/I)
EC20 >1000 ‑
EC50 >1000 ‑
EC80 >1000 ‑
NOEC 1000 ‑

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
The validation criteria for the control respiration rates and reference material EC50 values were satisfied.
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:

In an Assessment of the inhibitory effect on the respiration of activated sewage sludge study (Harlan project number:

0959/0243) the test material had an EC50 of greater than 1000 mg/l. The No Observed Effect Concentration (NOEC)

after 3 hours exposure was 1000 mg/l.

The method followed the recommendations of the OECD Guidelines for Testing of Chemicals (1984) No 209

"Activated Sludge, Respiration Inhibition Test", Method C.11 of EEC Commission Directive 88/302/EEC and US EPA Draft Ecological Effects Test Guidelines OPPTS 850.6800.

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