RD 14156

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 21 March 2016 and 22 March 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Sponsor Lot/Batch no.: 53995610P10
- Expiration date of the lot/batch: 24 February 2020
- Purity test date: 26 January 2016

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:

no

Remarks:

Not a guideline requirement

Vehicle:

yes

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 Water Plc, 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.
- Pretreatment:
- Initial biomass concentration: 3.0 g/L

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

20-21”C

pH:

7.8

Dissolved oxygen:

6.4 i.e. >60% dissolved oxygen saturation level of 8.9 mgO2/L

Details on test conditions:

TEST SYSTEM
- Test vessel: conical flask
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: 500 mL
- Aeration: 0.5-1.0 L/min
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
- No. of vessels per vehicle control (replicates):
- No. of vessels per abiotic control (replicates): 3
- Sludge concentration (weight of dry solids per volume): 3.0 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 laboratory 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: Yes

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

2.9 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

not specified

Basis for effect:

inhibition of total respiration

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

8.5 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

not specified

Basis for effect:

inhibition of total respiration

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: 3.2, 10, 32 mg/L
- Other:

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)	O2 consumption rate (mgO2/L/h)	% Inhibition
Control R1	5.5	6	1.8	37.00	-
Control R2	5.3	6	1.8	35.00	-
Control R3	5.6	6	2.0	36.00	-
Control R4	5.1	5	2.0	37.20	-
Test 10	4.9	5	1.9	36.00	7
Test 100	5.3	5	2.2	37.20	[2]
Test 1000 R1	1.9	5	1.7	38.4	[6]
Test 1000 R2	5.8	6	2.2	36.00	1
Test 1000 R3	4.9	5	1.8	37.2	[2]
Ref 3.2	6.2	8	2.3	29.28	19
Ref 10	7.4	10	5.1	13.80	62
Ref 32	8.0	10	7.0	6.00	83

[ ] 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.

Executive summary:

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)".

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 measured temperatures of between 20 and 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 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 8.5 mg/L, 95% confidence limits 6.7 to 11 mg/L.

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