Registration Dossier

Administrative data

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 January 2011 - 14 April 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: According to OECD and in accordance with GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
ISO 8192 (Water quality - Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Description Orange powder
Batch MB-1
Test substance storage At room temperature protected from light
Stability under storage conditions Stable

Sampling and analysis

Analytical monitoring:
no

Test solutions

Vehicle:
no
Details on test solutions:
The batch of E-Y110 tested was an orange powder with a purity of 95.5% and indicated to be light sensitive. Preparation was as much as possible performed under low light conditions.

A stock solution of 0.5 g/l was prepared by adding 125.34 mg test substance to 250 ml of Milli-RO water (tap water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA). Thorough mixing (vortex) was used to accelerate dissolution and to ensure homogeneity. Volumes of the orange stock solution corresponding to the test concentration were then added to the test media.
A concentration of 100 mg/l was tested in duplicate.

Test organisms

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 (4.0 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 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.

Study design

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.

Test conditions

Test temperature:
The temperature of the test medium was between 19.4 and 19.9°C
pH:
Controls: 7.6-7.8
Test substance series: 7.7-7.8
Dissolved oxygen:
Oxygen consumption:
Controls series:54-56 mg O2/l/h
Test substance series: 49-51 mg O2/l/h
Nominal and measured concentrations:
A nominal concentration of 100 mg/L was tested in duplicate
Details on test conditions:
TEST SYSTEM
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: All glass, approximately 300 ml oxygen bottles (measuring) and
1 litre test bottles(exposure)
- Aeration:During exposure with clean, oil-free air
- No. of vessels per concentration (replicates):2
- No. of vessels per control (replicates):2/ series
- Biomass loading rate:4 g/l of sludge as used for the test
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 2.5 and 6.5 mg O2/l.
TEST CONCENTRATIONS
- Test concentrations: limit study at a loading rate of 100 mg/l
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol

Results and discussion

Effect concentrationsopen allclose all
Duration:
3 h
Dose descriptor:
IC50
Effect conc.:
> 100 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:
IC10
Effect conc.:
ca. 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: 7 and 11% inhibition at 100 mg/l
Details on results:
7 and 11% inhibition of respiration rate of the sludge was recorded at 100 mg E-Y110 per litre in the duplicate measurements. Therefore, no further testing was needed. Hence, the EC50 of E-Y110 exceeded 100 mg/l (based on nominal concentrations).
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 38 % inhibition at 5.0 mg/l to 83% at 30 mg/l. Relevant effects levels: The EC50 of 3,5-dichlorophenol was: 7.0 mg/l.
Reported statistics and error estimates:
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 E-Y110 no EC50 could be calculated because the test substance proved to be non-toxic (EC50 >100 mg/l).

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of this present test, E-Y110 was slightly inhibitory to waste water (activated sludge) bacteria at a nominal concentration of 100 mg/l (7 and 11% inhibition of respiration rate of the sludge). The 3-hour EC50 of E-Y110 exceeded a nominal concentration of 100 mg/l.
Executive summary:

The influence of E-Y110 on the respiration rate of activated sludge was investigated after a contact time of 3 hours. The study procedures described in this report were based onthe OECD guideline No. 209, 1984.In addition, the procedures were designed to meet the test methods of the CouncilRegulation (EC) No. 440/2008 of, Publication No. L142, Part C11 and ISO Standard 8192 (2007). A concentration of 100 mg/l was tested in duplicate. Two controls without test substance were tested in each test series. Optimal contact between the test substance and test medium was ensured applying continuous aeration and stirring during the 3-hour exposure period. Thereafter, oxygen consumption was measured and recorded for approximately 10 minutes. 7 and 11% inhibition of respiration rate of the sludge was recorded at 100 mg E-Y110 per litre in the duplicate measurements. Therefore, no further testing was needed. Hence, the EC50of E-Y110 exceeded 100 mg/l (based on nominal concentrations). The batch of activated sludge was checked for sensitivity by testing the reference substance 3,5-dichlorophenol. Since all criteria for acceptability of the test were met, this study was considered to be valid.