[No public or meaningful name is available]

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:

April 2013 - July 2013

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:

See below.

Principles of method if other than guideline:

The dissolved oxygen content was not above 60% to 70% of the dissolved oxygen saturation level of 8.9 mgO2/L in several of the vessels.
This deviation was considered to have had no adverse effect on the study as all oxygen consumption values were measured over the linear portion of the traces.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- 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 overnight prior to use in the test. On the day of collection the activated sewage sludge (10 liters) was fed synthetic sewage sludge (500 mL). The pH of the sample on the day of the test was 7.8 measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter. Determination of the suspended solids level of the activated sewage sludge was carried out by filtering a sample (100 mL) of the activated sewage sludge by suction through a pre-weighed GF/A filter paper using a Buchner funnel which was then rinsed 3 times with 10 mL of deionized reverse osmosis water and filtration continued for 3 minutes. The filter paper was then dried in an oven at approximately 105 ºC for at least one hour and allowed to cool before weighing. This process was repeated until a constant weight was attained. The suspended solids concentration was equal to 2.9 g/L prior to use.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

20 ± 2 ºC

pH:

between 7.0 and 8.0

Dissolved oxygen:

4.6 to 6.9 mg O2/L

Nominal and measured concentrations:

Nominal test concentrations of 100 and 1000 mg/L

Details on test conditions:

TEST SYSTEM- Test vessel: closed- Aeration: The mixture was aerated with clean, oil-free compressed air via narrow bore glass tubes at a rate of approximately 0.5 - 1 liter per minute.
OTHER TEST CONDITIONS- Adjustment of pH: The pH of this stock solution was measured to be pH 5.2 and was adjusted to pH 7.2 using 1.0 M NaOH.
- Photoperiod: All test vessels were shielded from the light during mixing.
TEST CONCENTRATIONS- Range finding study- Test concentrations: 100 and 1000 mg/L- Results used to determine the conditions for the definitive study: No statistically significant toxic effects were shown at 100 and 1000 mg/L. Therefore, it was considered justifiable not to perform a definitive test.

Reference substance (positive control):

yes

Remarks:

A reference item, 3,5-dichlorophenol, was tested at concentrations of 3.2, 10 and 32 mg/L in order to confirm the suitability of the inoculum.

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

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:

respiration rate

Details on results:

The dissolved oxygen concentrations after 30 minutes contact time in several of the vessels was below 60% of the dissolved oxygen saturation level of 8.9 mg O2/L. This deviation to the study plan was considered to have had no adverse effect on the study given that all oxygen consumption values were measured/calculated over the linear portion of the traces.
No statistically significant toxic effects were shown at 100 and 1000 mg/L. Therefore, it was considered justifiable not to perform a definitive test.
It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/L.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels: The reference item gave a 3-Hour EC50 value of 11 mg/L.

Reported statistics and error estimates:

One way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955) was carried out on the oxygen consumption data for the range­finding test after 3 hours for the control and all test concentrations to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS, 1999 - 2001).

No statistically significant toxic effects were shown at 100 and 1000 mg/L. Therefore, it was considered justifiable not to perform a definitive test.

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 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 according to the OECD Guidelines No. 209 and GLP.

Activated sewage sludge was exposed to an aqueous dispersion of the test item at concentrations of 100 and 1000 mg/L (3 replicates of the 1000 mg/L test concentration) for a period of 3 hours at a temperature of 20 ± 2 °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 11 mg/L.

Description of key information

Toxicity to microorganisms

In an OECD 209 study conducted according to GLP, the 3 hour toxicity of the test material to activated sewage sludge micro-organisms is EC50 greater than 1,000 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 1000 mg/L. Therefore, it is possible to conclude that no effects were observed on activated sludge microorganisms.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

1 000 mg/L