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Toxicity to microorganisms

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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014-10-08 to 2014-10-09
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
not required
Details on sampling:
Not applicable
Details on test solutions:
Range-finder
Nominal amounts of test item (5, 50 and 500 mg (in triplicate)) were each separately dispersed in approxmately 200 mL of deionised reverse osmosis water and subjected to ultrasonication for approximately 15 minutes followed by magnetic stirring for 24 hours, at temperatures between 20°C and 22°C, in orderto maximise the dissolved test item concentration. All test vessels were shielded from light during mixing. Synthetic sewage (16 mL), activated sewage sludge (250 mL) and water were added to a final volume of 500 mL to give the required concentrations of 10, 100 and 1000 mg/L (3 replicates).
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Test System: A mixed population of activated sewage sludge micro-organisms was obtained from the aeration stage of the Severn Trent Water Pic sewage treatment plant at Loughborough, Leicestershire, UK which treats predominantly domestic sewage.
- 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 CaCl2.2H20
0.2 g MgS04.7H20
2.8 g K2HPO4
dissolved in 1 liter of water with the aid of ultrasonication.
The pH of the synthetic sewage stock was 7.1. The pH value was measured using a Hach 160d handheld meter.
- 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 on the day of the test was 7.6 . Determination of the suspended solids level of the activated sewage sludge on the day of the test 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 deionized 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 one hour and allowed to cool before weighing. This process was repeated until a constant weight was attained. The suspended solids concentration was equal to 3.0 g/L prior to use.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Post exposure observation period:
None
Hardness:
Not stated
Test temperature:
20 - 22°C
pH:
6.0 - 7.2
Dissolved oxygen:
5.91 - 8.23 mg O2/L
Salinity:
Not applicable
Nominal and measured concentrations:
Range finder: 10, 100 and 100 mg/L
Details on test conditions:
- Preparation of Test System:
At time "0" 16 mL of synthetic sewage was diluted to 250 mL with water and 250 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 liter per minute. Thereafter, at 15 minute intervals the procedure was repeated for the second control followed by the reference item vessels with appropriate amounts of the reference item being added. The test item vessels were prepared as described above with two additional control vessels prior to test item preparation. Finally two further control vessels were prepared.
The test was conducted under normal laboratory lighting in a temperature controlled room at approximately 20 to 22°C.
Control groups maintained under identical conditions but not exposed to test item.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol (3.2, 10 and 32 mg/L)
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
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:
The dissolved oxygen concentrations after 30 minutes contact time in all vessels were above 60% of the dissolved oxygen saturation level of 8.9 mg O2/L
No statistically significant toxic effects were shown at all of the test concentrations employed. It was therefore considered justifiable not to perform a definitive test.
It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/L.
The coefficient of variation of oxygen uptake in the control vessels was 4.3% and the specific respiration rate of the controls was 23.15 mg oxygen per gram dry weight of sludge per hour. The validation criteria have therefore been satisfied.
The validation criterion for the reference item EC50 value was also satisfied (see Section 3.8). The EC50 value for the reference item after 3 hours contact time (7.2 mg/L) was within the range of 2 to 25 mg/L given in the validation criteria.
In some instances, the initial and final dissolved oxygen concentrations were outside those recommended in the test guidelines (7 mg O2/L and 2 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.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: EC50 (3h) = 7.2 mg/L
Validity criteria fulfilled:
yes
Conclusions:
The effect of the test item on the respiration of activated sewage sludge micro-organisms gave a 3-Hour EC50 value 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 item on the respiration of activated sewage sludge. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2010) No. 209 "Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation)".

Methods

Activated sewage sludge was exposed to an aqueous dispersion of the test item at concentrations of 10, 100 and 1000 mg/L (3 replicates of the 1000 mg/L test concentration) for a period of 3 hours at a temperature of approximately 20 °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 item, 3,5-dichlorophenol.

Results

The effect of the test item on the respiration of activated sewage sludge gave a 3-Hour EC50 value 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 item gave a 3-Hour EC50 value of 7.2 mg/L, 95% confidence limits 5.5 - 9.4 mg/L.

Description of key information

The effect of the test item on the respiration of activated sewage sludge gave a 3-Hour EC50 value 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 item gave a 3-Hour EC50 value of 7.2 mg/L, 95% confidence limits 5.5 - 9.4 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
1 000 mg/L

Additional information

A key study was performed to assess the effect of the test item on the respiration of activated sewage sludge. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2010) No. 209 "Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation)".

Activated sewage sludge was exposed to an aqueous dispersion of the test item at concentrations of 10, 100 and 1000 mg/L (3 replicates of the 1000 mg/L test concentration) for a period of 3 hours at a temperature of approximately 20 °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 item, 3,5-dichlorophenol.

The effect of the test item on the respiration of activated sewage sludge gave a 3-Hour EC50 value 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 item gave a 3-Hour EC50 value of 7.2 mg/L, 95% confidence limits 5.5 - 9.4 mg/L.