[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 15, 2019 to April 18, 2019

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))

Version / remarks:

2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Details on sampling:

Measurement for each group, i.e. test substance, negative and positive controls
- After 10 min (except for 10 and 100 mg/L, which was 9 min)

Vehicle:

no

Details on test solutions:

TEST SUBSTANCE PREPARATION:
The test substance was dispersed directly in water. In order to aid weighing, the test substance was ground using a pestle and mortar prior to weighing. Nominal amounts of test substance (5, 50 and 500 mg (500 mg in triplicate)) were each separately dispersed in approximately 200 mL of deionized reverse osmosis water and sealed with Nescofilm prior to being subjected to ultrasonication for approximately 15 minutes followed by magnetic stirring for 24 h, at room temperature, in order to maximize the dissolved test substance concentration. All test vessels were shielded from the light during mixing. Synthetic sewage (16 mL), activated sewage sludge (250 mL) and water were added to a final volume of 500 mL to give the required concentrations of 10, 100 and 1000 mg/L (three replicates at 1000 mg/L).

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

A mixed population of activated sewage sludge micro-organisms was obtained to measure heterotrophic respiration from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK which treats predominantly sewage. 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, 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 (rinsed 3 times with 20 mL deionized reverse osmosis water prior to drying in an oven) using a Buchner funnel which was then rinsed three 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 1 h and allowed to cool before weighing. This process was repeated until a constant weight was attained. The suspended solids concentration was equal to 2.5 g/L and was adjusted to 3.0 g/L prior to the activated sewage sludge (8.25 L) being fed an aliquot of synthetic sewage (412.5 mL).
The pH of the sample on the day of the test was 7.5 measured using a Hach HQ40d Flexi handheld meter. The suspended solids level of the activated sewage sludge was checked prior to use to ensure the suspended solids concentration was equal to 3.0 g/L.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Remarks on exposure duration:

Continuous aeration

Test temperature:

20-21°C

pH:

6.8 to 7.2 (0 hours, test substance) and 7 to 7.3 (3 hours, test substance)

Dissolved oxygen:

Total respiration and heterotropic respiration: 8.9 mg O2/L

Nominal and measured concentrations:

0 (negative control), 10, 100 and 1000 (R1), 1000 (R2), 1000 (R3) mg/L

Details on test conditions:

At time "0" 16 mL of synthetic sewage was diluted to 250 mL with water and 250 mL of inoculum added in a 500 mL conical flask (first control). The mixture was aerated with clean, oil-free compressed air at a rate of 0.5 to 1.0 L/minute. Thereafter, at 15-minute intervals the procedure was repeated for the second control followed by the reference substance vessels with appropriate amounts of the reference substance. Finally, two further control vessels were prepared. The test was conducted under normal laboratory lighting in a temperature-controlled room at measured temperatures of approximately 21 °C.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Inhibition of respiration rate for heterotropic and total respiration

Key result

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

> 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of nitrification rate

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Inhibition of respiration rate for total, heterotrophic and nitrification respiration

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

ca. 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Inhibition of respiration rate for total, heterotrophic and nitrification respiration

Details on results:

Oxygen consumtion rate after 3 h contact time (Total respiration):
Control vessels: 36, 40, 40.5 and 42 mg O2/L/h.
Test substance vessels at concentrations of 10, 100 and 1000 (R1), 1000 (R2), 1000 (R3) mg/L: 42, 40, 36, 36 and 36 mg O2/L/hour, respectively

Oxygen consumtion rate after 3 h contact time (Heterotrophic respiration):
Control vessels: 22.2, 21, 21 and 22.2 mg O2/L/h.
Test substance vessels at concentrations of 10, 100 and 1000 (R1), 1000 (R2), 1000 (R3) mg/L: 24.67, 24, 21, 20.4 and 21 mg O2/L/hour, respectively

Oxygen consumtion rate after 3 h contact time (Nitrification respiration):
Control vessels: 19.8, 19.5, 19 and 13.8 mg O2/L/h.
Test substance vessels at concentrations of 10, 100 and 1000 (R1), 1000 (R2), 1000 (R3) mg/L: 17.3, 16, 15, 15.6 and 15 mg O2/L/hour, respectively

Results with reference substance (positive control):

Positive control vessels at concentrations of 3.2, 10 and 32 mg/L: 28.5, 16.80 and 4.80 mg O2/L/hour, respectively (total respiration), 21, 16.2 and 4.2 respectively (heterotropic respiration) and 7.5, 0.6 and 0.6 respectively (nitrification respiration)
EC50 value (3h) was found to be 7.4 (total respiration), 15 (heterotropic respiration) and 1.3 (nitrification respiration) mg/L

Validity criteria fulfilled:

yes

Conclusions:

Under the study conditions, the EC50 of the test substance for 3 h (in activated sludge) was determined to be >1000 mg/L (nominal) and NOEC was found to be 1000 mg/L.

Executive summary:

A study was conducted to determine the toxicity of the test substance 'mono- and di- C16-18 PSE and C16-18 AE10 PSE', to microorganisms according to OECD Guideline 209 (activated sludge respiration inhibition test), in compliance with GLP. Approximately 3 g/L of activated sewage sludge (domestic) were exposed to an aqueous dispersion of the test substance at concentrations of 10, 100 and 1000 mg/L (three replicates of the 1000 mg/L test concentration) with and without the presence of allylthiourea (ATU) for a period of 3 h. The measured temperatures ranged between 20 °C and 21 °C. The rates of respiration (total respiration, heterotrophic respiration and nitrification respiration) were determined after 3 h contact time and compared to data for the control and a reference substance, 3,5-dichlorophenol. The test substance had no effects on the total, heterotrophic, or nitrification respiration of the activated sewage sludge micro-organisms at any of the tested concentrations. Any additional testing above 1000 mg/L was considered to be unnecessary and unrealistic. The test was considered to be valid as all the validity criteria fulfilled, i.e., (a) the EC50 values of reference substance for total respiration, heterotrophic respiration and nitrification were determined to be 7.4, 15.1 mg/L and 7.4 mg/L respectively (b) the specific respiration rate of the blank controls was determined to be 26.42 mg O2/g sludge dw/h; (c) the coefficient of variation of oxygen uptake in the control group for total and heterotrophic respirations were determined to be 6.5 and 3.2% respectively by the end of the test. Under the conditions of the study, the 3 h EC50 and NOEC values for effects on total, heterotrophic and nitrification respiration of activated sewage sludge micro-organisms was >1000 and 1000 mg/L (nominal) respectively. (Envigo, 2019).

Description of key information

Based on the results of the study, the 3 h EC50 and NOEC values for effects on total, heterotrophic and nitrification respiration of activated sewage sludge micro-organisms was >1000 and 1000 mg/L (nominal) respectively.

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L

EC10 or NOEC for microorganisms:

1 000 mg/L

Additional information

A study was conducted to determine the toxicity of the test substance 'mono- and di- C16-18 PSE and C16-18 AE10 PSE', to microorganisms according to OECD Guideline 209 (activated sludge respiration inhibition test), in compliance with GLP. Approximately 3 g/L of activated sewage sludge (domestic) were exposed to an aqueous dispersion of the test substance at concentrations of 10, 100 and 1000 mg/L (three replicates of the 1000 mg/L test concentration) with and without the presence of allylthiourea (ATU) for a period of 3 h. The measured temperatures ranged between 20 °C and 21 °C. The rates of respiration (total respiration, heterotrophic respiration and nitrification respiration) were determined after 3 h contact time and compared to data for the control and a reference substance, 3,5-dichlorophenol. The test substance had no effects on the total, heterotrophic, or nitrification respiration of the activated sewage sludge micro-organisms at any of the tested concentrations. Any additional testing above 1000 mg/L was considered to be unnecessary and unrealistic. The test was considered to be valid as all the validity criteria fulfilled, i.e., (a) the EC50 values of reference substance for total respiration, heterotrophic respiration and nitrification were determined to be 7.4, 15.1 mg/L and 7.4 mg/L respectively (b) the specific respiration rate of the blank controls was determined to be 26.42 mg O2/g sludge dw/h; (c) the coefficient of variation of oxygen uptake in the control group for total and heterotrophic respirations were determined to be 6.5 and 3.2% respectively by the end of the test. Under the conditions of the study, the 3 h EC50 and NOEC values for effects on total, heterotrophic and nitrification respiration of activated sewage sludge micro-organisms was >1000 and 1000 mg/L (nominal) respectively. (Envigo, 2019).