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 18 February 2008 and 25 February 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not effect 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 (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Sponsor's identification: C-Y9
Description: orange powder
Lot number: 071217
Purity: 95.8%
Date received: 24 January 2008
Storage conditions: room temperature, in the dark until 11 February 2008, thereafter at room temperature, in the dark over silica gel.

Sampling and analysis

Analytical monitoring:
no
Details on sampling:
- Concentrations:
Activated sewage sludge was exposed to an aqueous solution of the test material at a concentration of 1000 mg/l (three replicate flasks).

- Sampling method:
As each vessel reached 30 minutes 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.

This procedure was repeated after 3 hours contact time.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
Definitive Test - based on the results of the range-finding test a limit test was conducted at a concentration of 1000 mg/l.

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 15 minutes and the volume adjusted to 1 litre to give a 2000 mg/l stock solution. An aliquot (250 ml) of this stock solution was dispersed (in triplicate) 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/l. 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.

- 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/I 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.










Test organisms

Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
Test Species:
A mixed population of activated sewage sludge micro-organisms was obtained on 18 February 2008 for the range-finding test and on 25 February 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.

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 7.9 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 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 3.9 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:
Observations were made on the test preparations throughout the test period, and the pH of the control, reference material and test material preparations were measured using a WTW pH/Oxi 3401 pH and dissolved oxygen meter at 0 hours and prior to measurement of the oxygen consumption rate after 3 hours contact time.

Test conditions

Hardness:
The test water had a total hardness of approximately 140 mg/l CaCO3.
Test temperature:
The test was conducted under normal laboratory lighting in a temperature controlled room at 21±1°C.
pH:
Test material solutions ranged from 7.8 to 8.1.
Dissolved oxygen:
Final O2 Reading (mg O2/l) after 3 hours contact time ranged from 2.4 to 3.0 for test material solutions.
Salinity:
Not applicable.
Nominal and measured concentrations:
Range-finding test:
Nominal concentrations: 1.0, 10, 100 and 1000 mg/l.

Definitive test:
Nominal concentration: 1000 mg/l
Details on test conditions:
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. Finally a second control was prepared.

As each vessel reached 30 minutes 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 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. After measurement the contents of the BOD bottle were returned to the test vessel.

This procedure was repeated after 3 hours contact time.

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 l248D 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: 1.429 mg/l
- Pesticides: 0.019 µg/l
- Chlorine: 0.274 mg/l
- Conductivity: 403 µS/cm at 20°C

OTHER TEST CONDITIONS
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) :
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.


TEST CONCENTRATIONS
- Range finding study:
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/l. The test material was dissolved directly in water.

- 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/I (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-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:
growth inhibition
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Details on results:
Range-finding Test:
Oxygen consumption rates and percentage inhibition values for the control, test and reference materials in the range-finding test are given in Table 1 (see attached background material).

No significant effect on respiration was observed at any of the test concentrations employed.
Based on this information, a single test concentration, in triplicate, of 1000 mg/l was selected for the definitive test.

Definitive Test:
Oxygen consumption rates and percentage inhibition values for the control, test and reference materials in the definitive test are given in Tables 2 and 3 (see attached background material). The pH values of the test preparations at the start and end of the exposure period are given in Table 4 (see attached background material), and observations made on the test preparations throughout the study are given in Table 5 (see attached background material).

Percentage inhibition is plotted against concentration for the reference material, 3,5-dichlorophenol only (Figures 1 and 2 - see attached background material).

The following results were derived:
EC20 (30 minutes and 3 hours): >1000 mg/l
EC50 (30 minutes and 3 hours): >1000 mg/l
EC80 (30 minutes and 3 hours): >1000 mg/l
NOEC (30 minutes and 3 hours): 1000 mg/l

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

















Results with reference substance (positive control):
3,5-dichlorophenol:
EC20 (30 minutes): 4.3 mg/l
EC50 (30 minutes): 12 mg/l (95% confidence limits: 9.5-15 mg/l).
EC80 (30 minutes): 34 mg/l

EC20 (3 hours): 4.1 mg/l
EC50 (3 hours): 12 mg/l (95% confidence limits: 9.5-15 mg/l)
EC80 (3 hours): 27 mg/l

Variation in respiration rates of controls 1 and 2 after 30 minutes and 3 hours contact time was ± 0%.

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 O2/1 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 (see table 5 - attached background information) showed that all test material vessels contained dark yellow/orange/brown dispersions with no undissolved test material visible.










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 C11 of EEC Commission Directive88/3021EECand 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 solution 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 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.

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

The reference material gave a 3 -hour EC50 value of 12 mg/l.