RD 14153

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 22 March 2016 and 23 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 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