RSS-4, DRY SUBSTANCE

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:

29 March 2012

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:

no

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

ISO 8192 (Water quality - Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

The test substance was not completely soluble in test medium at the loading rates initially prepared. Therefore, 1-Litre test bottles were filled with 200 mL of test substance mixtures in Milli-RO water (tap water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA) with initial loading rates of 2.5 times the final loading rate. These mixtures were stirred in closed dark brown bottles for approximately 24 hours. Subsequently, 16 mL synthetic medium, 250 mL sludge and Milli-RO water up to 500 mL were added resulting in the required loading rates (see also section 6.4). Optimal contact between the test substance and test organisms was ensured applying continuous aeration and stirring.

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 inoculum for exposure: The sludge was coarsely sieved, washed and diluted with ISO-medium. 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 7.6 on the day of testing. The batch of sludge was used one day after collection; therefore 50 mL of synthetic medium was added per litre of activated sludge at the end of the collection day. The sludge was kept aerated at test temperature until use.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Post exposure observation period:

After the 3-hour contact time, the oxygen consumption was recorded for a period of 11 minutes. During measurement, the sample was not aerated but continuously stirred on a magnetic stirrer. The pH and temperature were determined in the remaining part of the reaction mixture. This procedure was repeated for all test/reference substance concentrations/loading rates and controls.

Test temperature:

18.6-20.8 °C

pH:

Before addition of sludge: 7.2-7.5
After 3 h exposure: 7.2-8.4

Dissolved oxygen:

Oxygen consumption:
Controls test substance series: 44-47 mg O2/L/h
Test substance series: 42-49 mg O2/L/h
The oxygen concentration at the start of measurements was at least 6.9 mg O2/L

Nominal and measured concentrations:

Nominal: loading rate of 10, 100 and 1000 mg/L

Details on test conditions:

TEST SYSTEM
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: All glass
- Aeration: During exposure with clean, oil-free air
- No. of vessels per concentration (replicates): at a loading rate of 10 and 100 mg/L only one replicate was used. For the highest test concentration (1000 mg/L) triplicates were used.
- No. of vessels per control (replicates): duplicate
- Biomass loading rate: 3.0 g/L suspended solids in final test mixture

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap-water purified by reverse osmosis (Milli-RO)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The respiration rate from each vessel, in mg O2/L/hr, was calculated from the linear part of the respiration curve, which was generally between 2 and 7 mg O2/L.
Furthermore the respiration rate was also expressed as the amount of oxygen consumed per g dry weight of sludge per hour (Rs in mg O2/g.h).

TEST CONCENTRATIONS
- Test concentrations: Combined limit/range-finding test with loading rates of 10, 100 and 1000 mg/L

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

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

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 valid: Yes
The inhibitory effect of 3,5-dichlorophenol on aerobic waste water (activated sludge) bacteria increased with increased with increasing concentration , ranging from a 45 % inhibition at 5.0 mg/L to 91 % at 30 mg/L.
Relevant effects levels: The EC50 of 3,5-dichlorophenol was: 5.3 mg/L

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this present test Magnesium bis(2-ethylbutanoate) was not toxic to waste water (activated sludge) bacteria at or below a loading rate of 1000 mg/L (NOEC). The EC50 exceeded a loading rate of 1000 mg/L.

Executive summary:

In a 3 h activated sludge respiration inhibition study that was carried out in accordance with OECD guideline 209 and Good Laboratory Practice, activated sludge was exposed to Magnesium bis(2-ethylbutanoate) at a loading rate of 10, 100 and 1000 mg/L under static conditions. The resulting NOEC value was 1000 mg/L and the EC50 was >1000 mg/L.

Since the test was carried out according to OECD guideline and GLP and all criteria for acceptability and validity of the test were fulfilled. it is classified as reliable without restrictions.

Description of key information

In a 3 h activated sludge respiration inhibition study that was carried out in accordance with OECD guideline 209 and Good Laboratory Practice, activated sludge was exposed to 





  
  

Magnesium bis(2-ethylbutanoate) at a loading rate of 10, 100 and 1000 mg/L under static conditions.
The resulting NOEC value was 1000 mg/L and the EC50 was >1000 mg/L.
Since the test was carried out according to OECD guideline and GLP and all criteria for acceptability and validity of the test were fulfilled. it is classified as reliable without restrictions.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

1 000 mg/L