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Ecotoxicological information

Toxicity to microorganisms

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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From March 31, 2010 to April 16, 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to standard guidelines in compliance with GLP.
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:
Since PETIA was hardly soluble in water, test concentrations were prepared separately in Milli-RO water (tap water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA).

Two 1 L test bottles were filled with 250 ml of test substance mixtures with initial loading rates of 200 mg/L. These mixtures were stirred in closed brown glass bottles for approximately 26 h. Subsequently, 16 ml synthetic sewage feed, 200 ml sludge and Milli-RO water up to 500 ml were added resulting in a loading rate of 100 mg/L (see also section 6.4). This loading rate was tested in duplicate. 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 de Maaskant', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.
- Preparation of the sludge: The sludge was coarsely sieved, washed and diluted with ISO-medium. A small amount of sludge was weighed and dried overnight at ca. 105°C to determine the amount of suspended solids (3.8 and 3.6 g/L of sludge, as used for test 1 and 2, respectively). The pH was 8.2 and 7.9 on the day of testing, for experiments 1 and 2, respectively. The batch of sludge was used one day after collection; therefore 50 ml of synthetic 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.
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 approximately 10 minutes afther the 3 hours exposure period
Test temperature:
Experiment 1: between 19.0 and 20.1°C
Experiment 2: between 19.4 and 20.3°C
pH:
Experiment 1: 8.3-8.5
Experiment 2: 7.6-7.9
Dissolved oxygen:
Oxygen consumption:
Experiment 1:
Controls PETIA series: 56-62 mg O2/L/h
PETIA series: 46-60 mg O2/l/h

Experiment 2:
Controls PETIA series: 52-56 mg/ O2/l/h
PETIA series: 48-54 mg O2/l/h
Nominal and measured concentrations:
Nominal concentration: a loading rate of 100 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: All glass, approximately 300 ml oxygen bottles (measuring) and 1 L test bottles (exposure)
- Type (delete if not applicable): open
- Aeration: During exposure with clean, oil-free air.
- No. of organisms per vessel: 2
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2/ series
- Biomass loading rate:
Experiment 1: 1.52 g/L suspended solids in final test mixture
Experiment 2: 1.44 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) and subsequently passed over activated carbon and ion-exchange cartridges (Milli-Q) (Millipore Corp., Bedford, Mass., USA).

OTHER TEST CONDITIONS
- Adjustment of pH: no

EFFECT PARAMETERS MEASURED (with observation intervals if applicable)
The respiration rate from each vessel, in mg O2/L/h, was calculated from the linear part of the respiration curve, which was generally between 2.5 and 6.5 mg O2/L

TEST CONCENTRATIONS
- Test concentrations: Limit test at a loading rate of 100 mg/L. Since the difference between the duplicate values was significant the experiment was repeated.
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 100 other: mg/L (loading rate)
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:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks on result:
not determinable
Details on results:
In the first experiment, no inhibition of respiration rate of the sludge was recorded at 100 mg PETIA/L. In the duplicate measurement, inhibition of respiration rate of the sludge of 22% was recorded. Since the difference between the duplicate values was significant, the experiment was repeated.

In the second experiment, 11% inhibition of respiration rate of the sludge was recorded at a loading rate of 100 mg PETIA/L. In the duplicate measurement no inhibition of respiration rate of the sludge was recorded. Therefore, no further testing was needed. Hence, the EC50 of PETIA exceeded a loading rate of 100 mg/L.
Results with reference substance (positive control):
- Results with reference substance valid: yes
- The EC50 of 3,5-dichlorophenol was in the accepted range of 5-30 mg/L (7.4 and 6.0 mg/L, for experiments 1 and 2, respectively.
The inhibitory effect of 3,5 Dichlorophenol on aerobic waste water (activated sludge) bacteria increased with increasing concentration, ranging from a 37% and 44% inhibition at 5,0 mg/L to 83% and 84% at 30 mg/L.

None

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of this present test, the 3h EC50 of the substance exceeded a loading rate of 100 mg/L. The NOEC was 100 mg/L.
Executive summary:

A study was conducted to determine the toxicity of PETIA to activated sludge according to OECD Guideline 209. In the first experiment, no inhibition of respiration rate of the sludge was recorded at a nominal loading rate of 100 mg/L. In the duplicate measurement, an inhibition of respiration rate of the sludge of 22% was recorded. Since the difference between the duplicate values was significant, the experiment was repeated. Under the conditions of this study, the 3h EC50 of the substance exceeded a loading rate of 100 mg/L. The NOEC was 100 mg/L ( Desmares-Koopmans, 2010).

Description of key information

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
100 mg/L

Additional information

A study was conducted to determine the toxicity of PETIA to activated sludge according to OECD Guideline 209. In the first experiment, no inhibition of respiration rate of the sludge was recorded at a nominal loading rate of 100 mg/L. In the duplicate measurement, an inhibition of respiration rate of the sludge of 22% was recorded. Since the difference between the duplicate values was significant, the experiment was repeated. Under the conditions of this study, the 3h EC50 of the substance exceeded a loading rate of 100 mg/L. The NOEC was 100 mg/L ( Desmares-Koopmans, 2010).