Formaldehyde, telomer with 1,3-benzenedimethanamine, 1,3-benzenediol and ethenylbenzene

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 2012-29 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)

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:

The batch of HK 128 tested was a red-violet waxy solid with a purity of 94-95%. Before weighing the test substance was treated with a mortar. 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. 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:

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:

yes

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.

Test temperature:

Between 18.6 and 20.5°C

pH:

Controls and reference substance series, before addition of sludge: 7.4 - 7.5.
Controls and reference substance series, after 3 hour exposure period: 7.8 - 8.4

Test substance series, before addition of sludge: 7.4 - 8.2.
Test substance series, after 3 hour exposure period: 7.7 - 8.4.

Dissolved oxygen:

The oxygen concentration at the start of measurement was at least 6.8 mg O2/l

(Mean) respiration rates:
Control: 46 mg O2/l/h
10 mg/l: 54 mg O2/l/h
100 mg/l: 50 mg O2/l/h
1000 mg/l: 41 mg O2/l/h

Nominal and measured concentrations:

Nominal concentrations, three loading rates: 10, 100 and 1000 mg/l

Details on test conditions:

TEST SYSTEM
- Type: open
- Material, size, headspace, fill volume: All glass
- Aeration:During exposure with clean, oil-free air
- No. of vessels per concentration (replicates): 10 mg/l (1), 100 mg/l (1), 1000 mg/l (3)
- No. of vessels per control (replicates): 2
- Biomass loading rate: 1.5 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 water).

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.

TEST CONCENTRATIONS
- Test concentrations in a combined limit/range-fdining test: 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

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

Details on results:

In the combined limit/range-finding test no statistically significant inhibition of the respiration rate of the sludge was recorded at a loading rate of 1000 mg HK 128 per litre (Two Sample t-Test : α=0.05).

There was no significant oxygen uptake from abiotic processes and the results at 1000 mg/l with a nitrification inhibitor showed that the heterotrophic inhibition of the respiration rate was comparable to the total inhibition.

Results with reference substance (positive control):

The EC50 of 3,5-dichlorophenol was in the accepted range of 2 to 25 mg/l for total respiration (5.3 mg/l).

Reported statistics and error estimates:

ECx
For the reference substance the percentage inhibition was plotted against the logarithm of the concentrations and the EC50 was determined using linear regression analysis.

For HK 128 no EC50 could be calculated because the test substance proved to be non-toxic (EC50 > a loading rate of 1000 mg/l).

NOEC estimation
The NOEC was based on statistical analysis of the data. Data obtained for the test concentrations were compared with those obtained for the control using TOXSTAT Release 3.5, 1996, D.D. Gulley, A.M. Boelter, H.L. Bergman.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this present test HK 128 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.

Description of key information

EC50 (3hrs) > 1000 mg/L

NOEC (3hrs) = 1000 mg/L

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L

EC10 or NOEC for microorganisms:

1 000 mg/L