Magnesium octadecylbenzenesulphonate

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:

16-may-2014 till 06-June-2014

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF THE 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 24 to 24¾ hours. Subsequently, 16 ml synthetic medium (= sewage feed), 250 ml sludge and Milli-RO water up to 500 ml were added resulting in the required loading rates. 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 (1 mm), 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 7.0 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.

INITIAL BIOMASS CONCENTRATION: 1.5 g dry weight / liter in the final test mixture

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Post exposure observation period:

Oxygen consumption was measured and recorded for 10 to 12 minutes after the 3 hours exposure period.

Test temperature:

- The temperature continuously measured in temperature control vessels varied between 16.8 and 19.8°C during the test

pH:

- The pH in all test vessels, before addition of sludge was between 7.3 and 7.6. After the 3 hour exposure period the pH was between 6.7 and 7.7.

Dissolved oxygen:

- The dissolved oxygen concentration at the start was above 60-70% saturation (>60% of air saturation is >5 mg/l at 20°C).

- The respiration rate was calculated from the linear part of the respiration curve (in mg O2/l/h)

- Mean respiration rates:
Blank control: 46 mg O2/l/h
Nitrification control: 27 mg O2/l/h
1000 mg/l: 49 mg O2/l/h
1000 mg/l + ATU (nitrification inhibitor): 24 mg O2/l/h

Nominal and measured concentrations:

Nominal concentrations:
Blank control, Nitrification control, 1000 mg/l and 1000 mg/l + ATU (nitrification inhibitior)

Details on test conditions:

TEST SYSTEM
- Test vessel material, size, fill volume: All glass, 1 Liter bottles, filled with 500 ml test solution.
- Type: open
- Aeration:During exposure with clean, oil-free air
- No. of vessels per concentration (replicates):3
- No. of vessels per control (replicates): 6 blank control vessels, 2 nitrification control vessels.

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

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

Remarks on result:

other: -

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

Remarks on result:

other: -

Details on results:

In the limit test no significant inhibition was observed at 1000 mg/l (STUDENT-t test for Homogeneous Variances, apha = 0.05, one sided smaller). Results at 1000 mg/l with a nitrification inhibitor showed that the heterotrophic respiration was 9% inhibited. Based on the results of the total and heterotrophic inhibition, nitrification was calculated not to be inhibited (-30%).

Results with reference substance (positive control):

- Results with reference substance valid? Yes

- The 3h EC50 for 3,5-dichlorophenol was 5.2 mg/l

Reported statistics and error estimates:

ECx:
For the reference substance calculation of ECx values was based on probit analysis using linear max. likelihood regression with the percentages of respiration inhibition versus the logarithms of the corresponding concentrations of the test substance.

For EXP1313100 no EC50-values could be calculated because the test substance proved to be non-toxic (EC50 > 1000 mg/l)

NOEC determination
An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the blank control revealed significant inhibition of the respiration rate (Student-t test for Homogeneous Variances, a=0.05, one-sided, smaller).

The calculations were performed with ToxRat Professional v. 2.10.05 (ToxRat Solutions® GmbH, Germany).

Acceptability of the test:

- The mean blank control oxygen uptake rate exceeded 20 mg oxygen per one gram of activated sludge (dry weight of suspended solids) in an hour.

- The coefficient of variation of oxygen uptake in blank control replicates did not exceed 30% at the end of the definitive test.

- The EC₅₀ of 3,5-dichlorophenol was in the accepted range of 2 to 25 mg/l for total respiration (5.2 mg/l limit test).

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this present test EXP1313100 was not toxic to waste water (activated sludge) bacteria at a loading rate of 1000 mg/l (NOEC).

The EC50 was above 1000 mg/l.

Description of key information

Under the conditions of this present test, no toxicity to waste water (activated sludge) bacteria was observed at a loading rate of 1000 mg/l (NOEC). The EC50 was above 1000 mg/l.