[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:

4 May 2017 - 5 May 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

This study was performed in according with OECD Guideline N. 209 and in accordance with GLP principles

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

no

Remarks:

stock solution

Details on test solutions:

The batch of the test substance tested was a colourless to light yellow high viscous liquid with a purity of 97%. No correction was made for the purity/composition of the test item.
A stock solution of 10 g/L was prepared by adding 2.5 g test item to 250 mL of Milli-RO water (tap water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA). Magnetic stirring for 32 minutes was applied to accelerate dissolution and to ensure homogeneity. Volumes of the milky white stock solution (pH 5.0) corresponding to the test concentration were then added to the test media. Subsequently, synthetic medium, sludge and Milli-RO water were added resulting in the required loading rates. Optimal contact between the test item and test organisms was ensured by applying continuous aeration and stirring.
Any residual volumes were discarded.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

SOURCE:
Municipal sewage treatment plant: 'Waterschap Aa en Maas', 's- Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.

PREPARATION OF THE SLUDGE:
The sludge was coarsely sieved (1 mm) and allowed to settle. The supernatant was removed and ISO-medium was added. A small amount of the sludge was weighed and dried overnight at ca. 105°C to determine the amount of suspended solids (3.0 g/L of sludge, as used for the test). The pH was 7.9 on the day of testing. The batch of sludge was used one day after collection; therefore 50 mL of synthetic medium (=sewage feed) was added per litre of activated sludge at the end of the collection day. The sludge was kept aerated at test temperature until use

MEDIUM:
Adjusted ISO-medium, formulated using RO-water (tap water purified by reverse osmosis; GEON Waterbehandeling, Berkel-Enschot, The Netherlands) with the following composition:
CaCl2.2H2O 211.5 mg/L
MgSO4.7H2O 88.8 mg/L
NaHCO3 46.7 mg/L
KCl 4.2 mg/L

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Remarks on exposure duration:

During the exposure duration, aeration and stirring took place

Test temperature:

19 - 21°C

pH:

Before addition of sludge: 7.0 - 7.4.
After the 3 hour exposure period: 7.1 - 8.5

Nominal and measured concentrations:

Nominal - 0 (control), 10, 100 and 1000 mg/L

Details on test conditions:

VESSEL:
All glass open bottles/vessels.

WATER:
Milli-RO / Milli-RO water, Tap water purified by reverse osmosis (Millipore Corp., Bedford, Mass., USA).

SYNTHETIC MEDIUM:
16 g peptone
11 g meat extract
3 g urea
0.7 g NaCl
0.4 g CaCl2.2H2O
0.2 g MgSO4.7H2O
2.8 g K2HPO4
Dissolved in Milli-RO water, made up to 1 litre and filtered. The pH was within 7.5 ± 0.5.

AIR SUPPLY:
Clean, oil-free air.

AERATION:
The aeration was adjusted in such a way that the dissolved oxygen concentration at the start was above 60-70% saturation (60% of air saturation is > 5 mg/L at 20°C) and to maintain the sludge flocs in suspension.

PERFORMANCE OF THE TEST
The synthetic medium (= sewage feed, 16 mL) and an appropriate amount of the test item stock were mixed and made up to 250 mL with Milli-RO water. The pH was determined. Thereafter, 250 mL activated sludge was added. This was the start of the test.
After the 3-hour contact time the oxygen consumption was recorded for a period of approximately 10 minutes. During measurement, the sample was not aerated but continuously stirred on a magnetic stirrer.
The pH was determined in the remaining part of the reaction mixture. This procedure was repeated for all test/reference item concentrations and controls.
The medium temperature was recorded continuously in temperature control vessels. The temperature control vessels were identically prepared compared to the control vessels. A temperature control vessel with a REES sensor was placed in each fume cupboard of the climate room.

OXYGEN RECORDING
Determination of oxygen was performed with multiple oxygen sensors connected to a BlueBox (GO-Systemelektronik GmbH, Germany), a multichannel measuring and controlling system.

pH
was mesaured at the start and at the end of the test

TESTED CONCENTRATIONS:
10, 100, 1000 mg/L

REPLICATE:
the highest loading rate was tested in triplicate; the lowest loading rate consisted in one replicate. Blank control 6 replicates

MEDIUM for the sludge:
Adjusted ISO-medium, formulated using RO-water (tap water purified by reverse osmosis; GEON Waterbehandeling, Berkel-Enschot, The Netherlands) with the following composition:
CaCl2.2H2O 211.5 mg/L
MgSO4.7H2O 88.8 mg/L
NaHCO3 46.7 mg/L
KCl 4.2 mg/L


ACCEPTABILITY OF THE TEST
1.The mean control oxygen uptake rate exceeded 20 mg oxygen per one gram of activated sludge (dry weight of suspended solids) in an hour (39 mg oxygen per one gram of activated sludge).
The coefficient of variation of oxygen uptake in control replicates did not exceed 30% at the end of the definitive test (11%).
2.The EC50 of 3,5-dichlorophenol was in the accepted range of 2 to 25 mg/L for total respiration (4.7 mg/L, see also Appendix 3).
Since all criteria for acceptability of the test were met, this study was considered to be valid.
All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol (CAS n 591-35-5)

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:

inhibition of total respiration

Remarks:

respiration rate

Results with reference substance (positive control):

Results with reference substance are valid

All deviations that occurred during the study have been authorized/acknowledged by the Study Director, assessed for impact, and documented in the study records. Only minor SOP deviations that did not impact the quality or integrity of the study occurred during the course of the study.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this present study, the test substance was slightly toxic to waste water bacteria (activated sludge) at 1000 mg/L.
The EC50 was above 1000 mg/L.

Executive summary:

The toxicity of the test substance to microorganism was determined in a 3 h activated sludge respiration inhibition study carried out according to OECD Guideline N. 209 and GLP principles. Microorganisms in activated sludge were exposed to the test substance at nominal concentrations of 0 (control and solvent control), 10, 100, 1000 mg/L under static conditions in a combined limit/range-finding test.

The test revealed an inhibition of the respiration rate of 0%, 5% and avarage of 33% at the concentrations of 10, 100 and 1000 mg/L respectively. Therefore, the EC50 was above the highest concentration tested (1000 mg/L).

A statistically significant inhibition of the respiration rate of the sludge was recordedat a concentration of 1000 mg the test substance per litre. Therefore, a NOEC could not be determined. There was no oxygen uptake from abiotic processes.

All criteria for acceptability of the test were met and this study was considered to be valid. The present toxicity study is classified as reliable without restrictions according to the OECD test guideline.

Description of key information

A toxicity study to microorganism was performed according to OECD guideline No. 209 in an activated sludge respiration inhibition study and according to GLP principles. The 3h EC50 was determined to be > 1000 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L

Additional information

The toxicity of the test substance to microorganism was determined in a 3 h activated sludge respiration inhibition study carried out according to OECD Guideline N. 209 and GLP principles. Microorganisms in activated sludge were exposed to the test substance at nominal concentrations of 0 (control and solvent control), 10, 100, 1000 mg/L under static conditions in a combined limit/range-finding test.

0%, 5% and average of 33% of the respiration rate inibition was observed at the concentrations of 10, 100 and 1000 mg/L respectively. Therefore, the EC50 was above the highest concentration tested (1000 mg/L).

A statistically significant inhibition of the respiration rate of the sludge was recordedat a concentration of 1000 mg the test substance per litre.

All criteria for acceptability of the test were met and this study was considered to be valid. The present toxicity study is classified as reliable without restrictions according to the OECD test guideline