Registration Dossier

Administrative data

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 16 May 2008 and 21 May 2008.
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:
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:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by SI 2004/0994)). Date of inspection: 21/08/2007. Date of signature: 01/08/2008

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Sponsor's identification :Hatcol 1760
Description : clear colourless slightly viscous liquid
Batch number : A25983
Date received : 4 February 2008
Storage conditions :room temperature in the dark

Sampling and analysis

Analytical monitoring:
no
Details on sampling:
Not applicable

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
Test material dispersed directly in water. An amount of test material (500 mg) was dispersed 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 concentration of 1000 mg/l.
This method of preparation was performed for the three separate replicate test vessels.
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.

- Eluate:

- Differential loading: not applicable

- Controls:
For the purpose of the test a reference material, 3,5-dichlorophenol 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 15 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.

- Chemical name of vehicle: not applicable

- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): not applicable

- Evidence of undissolved material: no

Test organisms

Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Laboratory culture: no

- Method of cultivation: not applicable

- 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 7.8 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.

- Pretreatment: not applicable

- Initial biomass concentration: The suspended solids concentration was equal to 3.7 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°C
pH:
7.8
Nominal and measured concentrations:
In the range-finding test 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.

Based on the results of the range-finding test a "limit test" was conducted at a 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.
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 500 ml conical flask
- Aeration: 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 applicable
- Renewal rate of test solution: not applicable
- No. of organisms per vessel: not applicable
- No. of vessels per concentration (replicates): four
- No. of vessels per control (replicates): duplicate
- No. of vessels per vehicle control (replicates): not applicable
- Biomass loading rate: not applicable


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
The test water used for both the range-finding and definitive tests 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 140 mg/l as CaCO3. 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: Average of 1.429 mg/l (Range: 1.090 to 1.900 mg/l)

- Particulate matter:
Turbidity: average of <0.120 NTU. (Range: <0.050 to 1.710 NTU)

- Metals: total metals not stated in report

- Pesticides: average of 0.019 µg/l (Range: 0.000 to 0.092 µg/l)

- Chlorine:
Free chlorine: average of 0.197 mg/l (Range: 0.010 to 0.460 mg/l)
Total chlorine: average of 0.274 mg/l (Range: 0.020 to 0.510 mg/l)

- Alkalinity:
- Ca/mg ratio: not stated in report
- Conductivity: average of 403.577 µS/cm at 20°C (Range: 289.000 to 504.000 µScm at 20°C)

- Culture medium different from test medium: not applicable

- Intervals of water quality measurement: 0 hours, 30 minutes and 3 hours


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


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 100 and 1000 mg/l
- Justification for using less concentrations than requested by guideline: not applicable
- Range finding study
- Test concentrations: 100 and 1000 mg/l
- Results used to determine the conditions for the definitive study: Based on the results of the range-finding test a "limit test" was conducted at a 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.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorphenol

Results and discussion

Effect concentrationsopen allclose all
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
meas. (initial)
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:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: none
- Effect concentrations exceeding solubility of substance in test medium: yes. Data supplied by the Sponsor indicated that the test material was a mixture of components and had a water solubility of less than 0.1 mg/l. It was therefore considered justifiable to conduct the definitive test at a single nominal test concentration of 1000 mg/l in excess of the water solubility of the test material in order to ensure that the toxicity of the test material as a whole was determined.
Results with reference substance (positive control):
- Results with reference substance valid?: Yes
- Relevant effect levels: The reference material gave a 3-Hour EC50 value of 8.5 mg/l, 95% confidence limits
6.8 - 11 mg/l.

Any other information on results incl. tables

For Hatcol 1760, it was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/l.

Variation in respiration rates of controls 1 and 2 after 30 minutes was ± 10%, and ± 3% after 3 hours contact time.

The validation criteria for the control respiration rates and reference material EC50values were therefore satisfied.

In some instances, the initial and final dissolved oxygen concentrations were below those recommended in the test guidelines (6.5 mg O2/l and 2.5 mg O2/l 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.

Observations made throughout the test period showed that at the test concentration of 1000 mg/l there was a dark brown dispersion with oily globules of test material on the surface at 0 hours, 30 minutes and 3 hours contact time. This was considered to be due to the insoluble nature of the test material in the test media. However the test material was dispersed with the aid of ultrasonication in the test diluent for approximately 15 minutes prior to the addition of the synthetic sewage, activated sewage sludge and water and each vessel was aerated with compressed air at a rate of approximately 0.5-1 litre per minute to ensure that there was maximum contact between the test material and activated sewage sludge in the test medium.

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 EEC Commission Directive 88/302/EEC and US EPA Draft Ecological Effects Test Guidelines OPPTS 850.6800.

Methods.Following a preliminary range-finding test, 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 a temperature of approximately 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.

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 8.5 mg/l, 95% confidence limits
6.8 - 11 mg/l.