Hydroxyacetone

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:

2018/01/30-2018/01/30

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

Deviations:

yes

Remarks:

Temperature in a range of 20.4 - 23.0 °C instead of 20.0 ± 2.0 °C, stated as uncritical.

Qualifier:

according to guideline

Guideline:

EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, 55116 Mainz (15.05.2018)

Specific details on test material used for the study:

The test item was stored in a closed vessel at room temperature (20 ± 5°C).

Analytical monitoring:

no

Details on sampling:

- Concentrations: 3200, 1000, 320, 100 and 32 mg/L
- Sampling method: In the control vessels, 16 mL nutrient solution was mixed with 234 mL water. The positive control and the treatments were prepared by putting the appropriate amount of positive control solution respectively test item into the test vessel, adding 16 mL nutrient solution and water to give 250 mL. Then, in 5 minutes intervals, 250 mL inoculum was added and closed with sealed lids. After 3 hours, the content of the first vessel was shaken vigorously for 30 seconds, the narrow neck bottle was filled to the rim with an aliquot of the content of the first vessel, a magnetic stirrer was added, and the oxygen electrode was tightly fitted into the vessel. Care was taken that no air could reach the contents of the vessel. The oxygen concentration of the vessel was measured every 5–10 seconds over a period of 3-5 minutes. The interface of the oxygen meter was connected to a personal computer, feeding the measured values with their corresponding times in a table. The following vessels were measured likewise in 5 minutes intervals.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test item was pipetted into the test vessels, using the density 1.07 g/cm3 given by the sponsor.
- Controls: Blank control, positive control

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Name and location of sewage treatment plant where inoculum was collected: The sludge was taken from the activation basin of the sewage treatment plant in D-67480 Edenkoben.
- Method of cultivation: Upon arrival in the test facility, the sludge was filtrated, washed with tap water 3 times and re-suspended in tap water. The activated sludge was aerated until usage in the test and fed daily with 50 mL synthetic sewage feed /L.
- Preparation of inoculum for exposure: On the day before the experiment, the inoculum was taken from its source, washed, aerated and the dry matter was determined. Volume was adapted to the desired content of dry matter. The nutrient solution was thawed and the sludge was fed with 50 mL nutrient solution / L sludge. On the day of the experiment, the dry matter was determined once more. The stock solution of the positive control was prepared.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Hardness:

0.71 mmol/L

Test temperature:

20.4 - 23.0 °C

pH:

7.7-8.0

Conductivity:

174 µS/cm (at 25 °C)

Nominal and measured concentrations:

nominal: 3200, 1000, 320, 100 and 32 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: SCHOTT flasks
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 2000 mL
- Aeration: no , in order to ensure sufficient oxygen supply, a large headspace was provided and the mixtures were stirred vigorously
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 1
- No. of vessels per blank control (replicates): 2 replicates before and 2 after measuring positive control and test item
- Sludge concentration (weight of dry solids per volume): 1.29 g/L
- Nutrients provided for bacteria: adding 16 mL nutrient solution and water to give 250 mL test solution. All chemicals used in the test were “analytical grade” or “for use in microbiology”.
Composition of nutrient solution (synthetic sewage):
Peptone 16.0 g
Meat Extract 11.0 g
Urea 3.0 g
NaCl 0.7 g
CaCl2*2H2O 0.4 g
MgSO4*7H2O 0.2 g
K2HPO4 2.8 g
Deionised water ad 1000 mL
The pH of the solution was 7.0 and therefore within the range of 7.5 ± 0.5.
The nutrient solution was frozen immediately after preparation.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: tap water from Wasserwerk Maikammer

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : NOEC, 3h-EC10, 3h-EC50
TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study : The concentrations were based on a non-GLP range finding test.
- Test concentrations: 3200, 1000, 320, 100 and 32 mg/L

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol (CAS-No. 591-35-5) was used as positive control. A stock solution in deionised water containing 500 mg/L was freshly prepared for the experiment.

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Key result

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

470 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

other:

Remarks:

confidence interval 280 - 730 mg/L

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 3 200 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

- Blank controls oxygen uptake rate: *35.76 mg oxygen per gram sludge in 1 hour (* Mean O2 consumption 46.13 mg/(L*h)
- Coefficient of variation of oxygen uptake rate in control replicates: 6.0 %

Results with reference substance (positive control):

- Results with reference substance valid? yes, all values lie within the recommended range of 2 – 25 mg/L.
- Relevant effect levels: 3h-EC50 = 11 mg/L

Reported statistics and error estimates:

For the treatments with the test item concentrations 320 and 100 mg/L, it was tested whether the differences between treatment and blank control were significant. For this determination, the values of the O2 consumption were used. As equality of variance is given, the t-test is used.
For the calculation of the EC10 and EC50, the percentage inhibition was plotted versus concentration in a Gauß-logarithmic diagram. EC10 and EC50 were determined from the x-values of the regression line at y = 10% and y = 50%.

All validity criteria were met. For the estimation of the EC50 of the positive control, a good statistical correspondence of the data with the dose-response-equation was found. The positive control gave an EC50 of 11 mg/L, which lies within the recommended range of 2 – 25 mg/L. The coefficient of variation of oxygen uptake rate in control replicates was below 30 % at the end of the test. The oxygen uptake rate of the blank controls was above 20 mg O₂ per gram activated sludge in 1 hour. No inconsistencies in the dose-response estimation were observed. Therefore, no further experiment was performed in order to discern between inhibition of nitrificators and inhibition of total population. No observations were made which might cause doubts concerning the validity of the study outcome. The result of the test can be considered valid.

Validity criteria fulfilled:

yes

Conclusions:

The following results for the test item Hydroxyacetone were determined:
3h NOEC = 100 mg/L
3h EC10 = 470 mg/L (confidence interval 280 - 730 mg/L)
3h EC50 > 3200 mg/L

Executive summary:

The inhibition of the respiration of activated sludge by hydroxyacetone was investigated according to OECD guideline 209 and U C.11, in compliance with GLP criteria. Hydroxyacetone was tested using 4 concentrations ranging from 3200 to 32 mg/L nominal concentration in an OECD 209 test. Duration of the test was 3 hours. Activated sludge was used as inoculum. It was taken from a domestic sewage treatment plant and washed before usage. The dry matter was determined as 2.58 g suspended solids/L, giving a concentration of 1.29 g suspended solids/L in the test. One valid experiment was performed. 3,5-Dichlorophenol was used as positive control. Four concentrations were tested; an EC50 of 11 mg/L (95% confidence interval: 7.2 – 16 mg/L) was determined, which lies within the recommended range of 2 – 25 mg/L stated in the OECD guideline. The following results for the test item hydroxyacetone were determined:

3h NOEC = 100 mg/L

3h EC10 = 470 mg/L (confidence interval 280 - 730 mg/L)

3h EC50 > 3200 mg/L

Description of key information

activated sludge, OECD 209, GLP, EC10 = 470 mg/L, EC50 > 3200 mg/L

Key value for chemical safety assessment

EC50 for microorganisms:

3 200 mg/L

EC10 or NOEC for microorganisms:

470 mg/L

Additional information

The inhibition of the respiration of activated sludge by hydroxyacetone was investigated according to OECD guideline 209 and U C.11, in compliance with GLP criteria. Hydroxyacetone was tested using 4 concentrations ranging from 3200 to 32 mg/L nominal concentration in an OECD 209 test. Duration of the test was 3 hours. Activated sludge was used as inoculum. It was taken from a domestic sewage treatment plant and washed before usage. The dry matter was determined as 2.58 g suspended solids/L, giving a concentration of 1.29 g suspended solids/L in the test. One valid experiment was performed. 3,5-Dichlorophenol was used as positive control. Four concentrations were tested; an EC50 of 11 mg/L (95% confidence interval: 7.2 – 16 mg/L) was determined, which lies within the recommended range of 2 – 25 mg/L stated in the OECD guideline. The following results for the test item hydroxyacetone were determined:

3h NOEC = 100 mg/L

3h EC10 = 470 mg/L (confidence interval 280 - 730 mg/L)

3h EC50 > 3200 mg/L