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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:
The study was conducted between 22 March 2016 and 23 March 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Sponsor Lot/Batch number: 5399661C10
- Expiration date of the lot/batch: 12 January 2010
- Purity test date:

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity:
- Specific activity:
- Locations of the label:
- Expiration date of radiochemical substance:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark
- Stability under test conditions:
- Solubility and stability of the test substance in the solvent/vehicle:
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
- Preliminary purification step (if any):
- Final dilution of a dissolved solid, stock liquid or gel:
- Final preparation of a solid:

FORM AS APPLIED IN THE TEST (if different from that of starting material)

OTHER SPECIFICS:
Analytical monitoring:
not required
Vehicle:
no
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Laboratory culture:
- Name and location of sewage treatment plant where inoculum was collected: Severn Trent Plc sewage treatment plant, Loughborough, Leicestershire, UK.
- Method of cultivation:
- Preparation of inoculum for exposure: The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21 ºC and was used on the day of collection. The pH of the sample was 7.9 measured using a Hach HQ40d Flexi handheld meter.
- Pretreatment:
- Initial biomass concentration: 3.0 g/L
Test type:
static
Water media type:
other: The test water used for the test was deionized reverse osmosis water containing less than 1 mg/L Dissolved Organic Carbon (DOC).
Limit test:
no
Total exposure duration:
3 h
Test temperature:
19 ”C
pH:
7.2-7.7 (after adjustment)
Dissolved oxygen:
6.3-7.5 in test vessels
Nominal and measured concentrations:
10 - 1000 mg/L nominal
Details on test conditions:
TEST SYSTEM
- Test vessel: conical flask
- Type (delete if not applicable): opend
- Material, size, headspace, fill volume:500 mL
- Aeration: 0.5-1.0L/min
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates):
- No. of vessels per abiotic control (replicates): 1
- Sludge concentration (weight of dry solids per volume): 3 g/L
- Weight of dry solids per volume of reaction mixture per unit of time:
- Nutrients provided for bacteria:
- Nitrification inhibitor used (delete if not applicable): none / N-allylthiourea
- Biomass loading rate:
TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reverse osmosis water
- Particulate matter:
OTHER TEST CONDITIONS
- Adjustment of pH:
- Photoperiod:
- Light intensity: normal laboratoru lighting
- Details on termination of incubation:
EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
TEST CONCENTRATIONS
- Spacing factor for test concentrations:
- Justification for using fewer concentrations than requested by guideline:
- Range finding study
- Test concentrations: 10, 100 and 1000 mg/L
- Results used to determine the conditions for the definitive study:
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Key result
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 on result:
not determinable
Details on results:

In the range finding test the dissolved oxygen concentrations after 30 minutes contact time in all vessels were above 70% 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.

Oxygen consumption rates and % inhibition values after 3h contact time in range finding test

 Nominal Concn. (mg/L)  Initial O2(mgO2/L)  Measurement period (mins)  Final O2(mgO2/L)  O2consumption rate (mgO2/L/h)  % Inhibition
 Control R1  6.1  7  2.1  31.29  -
 Control R2  5.6  7  1.9  31.71  -
 Control R3  5.5  6  2.2  33.00  -
 Control R4  5.3  6  2.0  33.00  -
 Test 10  4.7  5  1.9  33.60  [2]
 Test 100  5.4  6  2.0  31.00  [3]
 Test 1000 R1  5.5  6  2.2  33.00  0
 Test 1000 R2  6.0  7  2.3  31.71  4
 Test 1000 R3  5.3  6  1.9  34.00  [3]
 3,5 -dichlorophenol 3.2  6.3  10  2.2  24.60  25
 3,5 -dichlorophenol 10  7.8  10  5.6  13.20  60
 3,5 -dichlorophenol 32  8.2  10  7.4  4.80  85

[ ] Increase in respiration rate as compared to controls

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.
The test item was therefore considered not inhibitory in this assay.
Executive summary:

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 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. The test item was therefore considered not inhibitory in this assay.

Description of key information

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. The test item was therefore considered not inhibitory in this assay.

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L
EC10 or NOEC for microorganisms:
1 000 mg/L

Additional information