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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:
Range-finding study: 19th August 1999 Definitive study: 25th August 1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or methodological deficiencies, which do not affect the quality of relevant results.
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
Qualifier:
according to
Guideline:
EPA OPPTS 850.6800 (Modified Activated Sludge, Respiration Inhibition Test for Sparingly Soluble Chemicals)
Deviations:
no
Principles of method if other than guideline:
Not applicable.
GLP compliance:
yes (incl. certificate)
Remarks:
Date of inspection: 23rd March 1998 Date of signature: 21st July 1998
Specific details on test material used for the study:
No additional information
Analytical monitoring:
no
Details on sampling:
Not applicable, as analysis of the concentration, homogeneity and stability of the test material in the test preparations was not appropriate to the Test Guideline.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: An amount of test material (1000 mg) was dissolved in water, with the aid of ultrasonication and vigorous shaking for approximately 45 minutes and the volume was adjusted to 1 litre to give a 1000 mg/ stock solution. Aliquots of 50, 90, 160 and 280 ml were each separetel dispersed with activated sewage sludge (equivalent to 800 mg suspended solids), synthetic sewage (16 ml) and water to a final volume of 500 ml to give the test concentrations of 100, 180, 320 and 560 mg/l.
Similarly, a 450 ml aliquot of a second 1000 mg/l stock solution prepared as above was used to prepare the 900 mg/l test concentration.

- Differential loading: not applicable

- Controls: for the purpose of the study, a reference material, 3,5-dichlorophenol was used. Two stock solutions of 50 and 160 mg/l were prepared by direct solution in water with the aid of ultrasonic disruption. Aliquots (10 and 100 ml) of the 160 mg/l stock solution were removed and dispersed iwth activated sewage sludge (equivalent to 800 mg suspended solids), sewage sludge 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.

- Chemical name of vehicle (organic solvent, emulsifier or dispersant): not applicable, as dissolved directly in water

- 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): none
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 21°C and was used on the day of collection.
In order to minimise the dilution of the initial stock solutions by the addition of the relatively large volumes of activated sewage sludge required to give an initial loading rate of 1.60 g/l the activated sewage sludge was centrifuged (3500 rpm, 5 minutes) and the supernatent discarded prior to addition to the test vessels.
- Pretreatment: not applicable
- Initial biomass concentration: The suspended solids concentration of the sample was equal to 4.0 g/l prior to use.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Post exposure observation period:
Not applicable
Hardness:
Approximately 100 mg/l as CaCO3
Test temperature:
21°C
pH:
7.6
Dissolved oxygen:
100 mg/l: Initial reading 6.5 mg O2/l Final reading: 1.6 mg O2/l
180 mg/l: Initial reading 5.1 mg O2/l Final reading: 1.9 mg O2/l
320 mg/l: Initial reading 5.5 mg O2/l Final reading: 1.8 mg O2/l
560 mgl/: Initial reading 5.7 mg O2/l Final reading: 1.6 mg O2/l
900 mg/l: Initial reading 6.8 mg O2/l Final reading: 5.0 mg O2/l
Salinity:
Not applicable, as freshwater study
Nominal and measured concentrations:
Range-finding study:
Nominal concentrations of 1.0, 10, 100 and 1000 mg/l

Definitive study:
100, 180, 320, 560 and 900 mg/l
Details on test conditions:
TEST SYSTEM
- Test vessel: conical flasks
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 500 ml conical flask with 16 ml synthetic sewage diluted to 300 ml with water
- Aeration: the mixture was aerated with compressed are 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 (frequency/flow rate): not applicable
- No. of organisms per vessel: not applicable
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 1
- 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 anf definitive studies was laboratory tap water dechlorinated by passage through an activated carbon filter.

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

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Oxygen consumption rate

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 100, 180, 320, 560 and 900 mg/l
- Justification for using less concentrations than requested by guideline: not applicable
-
Range finding study
- Test concentrations: Nominal concentrations of 1.0, 10, 100 and 1000 mg/l
- Results used to determine the conditions for the definitive study: Significant inhibition of respiration was observed at 1000 mg/l, however, there were no effects on respiration at 1.0, 10 and 100 mg/l.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
840 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: respiration inhibition
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
320 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: respiration inhibition
Details on results:
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values:
Range-finding study: At concentrations in excess of 1000 mg/l the test material was observed tp form a cloudy dispersion in water and not a solution.

- Effect concentrations exceeding solubility of substance in test medium: During the preliminary solubility work conducted, the maximum limit of water solubility of the test material was determined to be 1000 mg/l under these test conditions by visual inspection.
Results with reference substance (positive control):
- Results with reference substance valid? Yes

- Relevant effect levels: 3hr EC50 = 11 mg/l
Reported statistics and error estimates:
No statistics used

No other information.

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 840 mg/l. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 320 mg/l.
Executive summary:

Methods.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", EEC Commission Directive 87/320/EEC and US EPA Draft Ecological Effects Test guidelines OPPTS 850.8600

Procedure: Following a preliminary range-finding study, activated sewage sludge was exposed to an aqueous solution of the test material at concentrations of 100, 180, 320, 560 and 900 mg/l for a period of 3 hours at 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.

Conclusions: The effect of the test material on the respiration of activated sewage sludge micro-organisms gave a 3-Hour EC50 of greater than 840 mg/l. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 320 mg/l.

Description of key information

OECD Guideline 209, EU Method C11, EPA OPPTS 850.6800, GLP, key study, validity 1:

3h-EC50 (activated sludge) > 840 mg/L.

3h-NOEC (activated sludge) = 320 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:
840 mg/L
EC10 or NOEC for microorganisms:
320 mg/L

Additional information

Based on the results of one key study (SPL, 1999), performed according to OECD Guideline 209, EU Method C11 and EPA OPPTS 850.6800 with GLP statement, the effect of the test material on the respiration of activated sewage sludge micro-organisms gave a 3 -hour EC50 of greater than 840 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 320 mg/L.