Registration Dossier

Administrative data

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
8th December 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:
2010
Report Date:
2010

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:
Date of GLP inspection: 15/09/2009 Date of Signature on GLP certificate: 26/11/09

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Sponsor's identification :DVS005u (aka Weston 705)
Description : clear colourless viscous liquid
Batch number : MW9F23A901
Date received : 5 November 2009
Storage conditions :approximately 4ºC, in the dark, under nitrogen.

Sampling and analysis

Analytical monitoring:
no
Details on sampling:
- Concentrations: 10, 32, 100, 320 and 1000 mg/l

- Sampling method:
Observations such as oxygen consumption rates, percentage inhibition values, pH and appearance were made on the test preparations, the control and reference material at 0 hours, 30 minutes after contact time and 3 hours after contact time.

- Sample storage conditions before analysis: not specified in report

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)

- Method:
Test material preparation - For the purpose of this test, the test material was dispersed directly in water.

Amounts of test material (5, 16, 50, 160 and 500 mg) were each separately 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 concentrations of 10, 32, 100, 320 and 1000 mg/l.

- Differential loading:
Not applicable.

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

- Chemical name of vehicle (organic solvent, emulsifier or dispersant):
Not applicable.

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

- Evidence of undissolved material (e.g. precipitate, surface film, etc): Observations made at 0 hours prior to the addition of activated sewage sludge and synthetic sludge showed that the test concentration of 10 mg/l contained a lear water column with very small globules of test material on the surface, the test concentration of 32 mg/l contained a clear water colum with large and small globules of test material on the surface and the test concentration of 100 mg/l contained a clear water column with large globules of test material on the surface. the test concentration of 320 mg/l contained a clear water column with very large globules of test material on the surface and a thick oily layer of test material was visible on the surface in the 1000 mg/l test concentration.
Observations made after 30 minutes and 3 hours contact time for the test concentration of 10 mg/l showed the vessel contained a dark brown dispersion with few very small globules of test material on the surface. The test concentration of 32 mg/l contained a dark brown dispersion with small globules of test material on the surface, the test concentration of 100 mg/l contained a dark brown dispersion with an oily layer of test material on the surface. The test concentration of 320 mg/l contained a dark brown dispersion with an oily layer of test material on the surface and an oily layer of test material was visible on the surface and large globules were visible dispersed throughout in the 1000 mg/l test concentration.

Test organisms

Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Laboratory culture:
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 3.8 g/l prior to use.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Post exposure observation period:
Not applicable.

Test conditions

Hardness:
140 mg/l CaCO3
Test temperature:
21 ± 1°C.
pH:
7.5 for the test material at the start of the test.
7.4 to 7.7 after 3 hours exposure
Dissolved oxygen:
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.
Relatively low initial oxygen readings were observed in the test vessels (see Table 1) which could possibly be due to insoluble material coating the membrane of the oxygen probe. It was not possible to calculate oxygen consumption rates for the nominal test concentration of 1000 mg/l due to extremely low initial and final oxygen readings and therefore the EC50 value for the test material was taken as being greater than 320 mg/l.
Salinity:
Not applicable.
Nominal and measured concentrations:
10, 32, 100, 320 and 1000 mg/l
Details on test conditions:
TEST SYSTEM

Preparation of test system
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. The test material vessels were prepared as described in Section 3.4.1. Finally a second control was prepared.
As each vessel reached 3 hours contact time an aliquot was removed from the conical flask and poured into the measuring vessel (250 ml darkened glass Biological Oxygen Demand (BOD) bottle) and the rate of respiration measured using a Yellow Springs dissolved oxygen meter fitted with a BOD probe. The contents of the measuring vessel were stirred constantly by magnetic stirrer. The rate of respiration for each flask was measured over the linear portion of the oxygen consumption trace (where possible between approximately 6.5 mg O2/l and 2.5 mg O2/l). In the case of a rapid oxygen consumption, measurements may have been outside this range but the oxygen consumption was always within the linear portion of the respiration curve. In the case of low oxygen consumption, the rate was determined over an approximate 10 minute period.
The test was conducted under normal laboratory lighting in a temperature controlled room at 21±1°C.

TEST MEDIUM / WATER PARAMETERS
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.

OTHER TEST CONDITIONS
- Adjustment of pH: - not required

- Photoperiod: -3 hours

- Light intensity: Normal laboratory lighting.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
In order to calculate the inhibitory effect of the test and 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 Xlfit software package (IDBS) and the EC15, EC20, EC50 and EC80 values determined from the equation for the fitted line.
The EC15, EC20, EC50 and EC80 values for the test material were determined by inspection of the inhibition of respiration rate data.
95% confidence limits were calculated for the EC50 values for the reference material only using the method of Litchfield and Wilcoxon (Litchfield and Wilcoxon 1949).
The No Observed Effect Concentration (NOEC) was determined by inspection of the inhibition of respiration rate data.
The results of the study are considered valid if (i) the two control respiration rates are within 15% of each other and (ii) the EC50 (3-Hour contact time) for 3,5-dichlorophenol lies within the range 5 to 30 mg/l.


TEST CONCENTRATIONS
- Spacing factor for test concentrations: not stated in report

- Justification for using less concentrations than requested by guideline: - not applicable

- Range finding study -not conducted

- Test concentrations: 10, 32, 100, 320 and 1000 mg/l

- Results used to determine the conditions for the definitive study: - not applicable
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol

Results and discussion

Effect concentrationsopen allclose all
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 320 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.:
320 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
Range-finding Test - not applicable

Definitive Test
Oxygen consumption rates and percentage inhibition values for the control, test and reference materials are given in Table 1. The pH values of the test preparations at the start and end of the exposure period are given in Table 2, and observations made on the test preparations throughout the study are given in Table 3.

Percentage inhibition is plotted against concentration for the reference material, 3,5 dichlorophenol only (Figure 1).
The following results were derived:
DVS005u (aka Weston 705)
3,5-dichlorophenol
ECx (3 Hours)
(mg/l) 95% Confidence Limits (mg/l) ECx (3 Hours)
(mg/l) 95% Confidence Limits (mg/l)
EC20 >320 - 2.6 -
EC50 >320 - 7.9 6.1 - 10
EC80 >320 - 25 -
NOEC 320 - 2.1 -

Variation in respiration rates of controls 1 and 2 after 3 hours contact time was ± 4%.

The validation criteria for the control respiration rates and reference material EC50 values 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.
Relatively low initial oxygen readings were observed in the test vessels (see Table 1) which could possibly be due to insoluble material coating the membrane of the oxygen probe. It was not possible to calculate oxygen consumption rates for the nominal test concentration of 1000 mg/l due to extremely low initial and final oxygen readings and therefore the EC50 value for the test material was taken as being greater than 320 mg/l.
Observations made at 0 hours (see Table 3) prior to the addition of activated sewage sludge and synthetic sewage showed that the test concentration of 10 mg/l contained a clear water column with very small globules of test material on the surface, the test concentration of 32 mg/l contained a clear water column with large and small globules of test material on the surface and the test concentration of 100 mg/l contained a clear water column with large globules of test material on the surface. The test concentration of 320 mg/l contained a clear water column with very large globules of test material on the surface and a thick oily layer of test material was visible on the surface in the 1000 mg/l test concentration.
Observations made after 30 minutes and 3 hours contact time for the test concentration of 10 mg/l showed the vessel contained a dark brown dispersion with few very small globules of test material on the surface. The test concentration of 32 mg/l contained a dark brown dispersion with small globules of test material on the surface, the test concentration of 100 mg/l contained a dark brown dispersion with an oily layer of test material on the surface. The test concentration of 320 mg/l contained a dark brown dispersion with an oily layer of test material on the surface and an oily layer of test material was visible on the surface and large globules were visible dispersed throughout in the 1000 mg/l test concentration


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 7.9 mg/l, 95% confidence limits 6.1 - 10 mg/l.

- Other:
None.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The effect of the test material on the respiration of activated sewage sludge gave a 3 Hour EC50 of greater than 320 mg/l. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 320 mg/l.
The reference material gave a 3-Hour EC50 value of 7.9 mg/l, 95% confidence limits
6.1 - 10 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 Commission 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 dispersion 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 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 EC50 of greater than 320 mg/l. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 320 mg/l.

The reference material gave a 3-Hour EC50 value of 7.9 mg/l, 95% confidence limits
6.1 - 10 mg/l.