Registration Dossier

Administrative data

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-07-27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
3,9-dimethyl-2,4,8,10-tetraoxa-3,9-diphosphaspiro[5.5]undecane 3,9-dioxide
EC Number:
221-088-9
EC Name:
3,9-dimethyl-2,4,8,10-tetraoxa-3,9-diphosphaspiro[5.5]undecane 3,9-dioxide
Cas Number:
3001-98-7
Molecular formula:
C7H14O6P2
IUPAC Name:
3,9-dimethyl-2,4,8,10-tetraoxa-3lambda5,9lambda5-diphosphaspiro[5.5]undecane-3,9-dione
Test material form:
solid: particulate/powder
Details on test material:
AFLAMMIT® PCO 962

Sampling and analysis

Analytical monitoring:
not required

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 1000 mg/L was weighed out directly in Erlenmeyer flasks.
- Controls: Test medium without test or reference item
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no

Test organisms

Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Name and location of sewage treatment plant where inoculum was collected: Municipal sewage treatment plant of D-31137 Hildesheim, Germany (back end of aeration tank)
- Method of cultivation: none
- Preparation of inoculum for exposure: none
- Pretreatment: The sludge was washed twice with chlorine free tap water and adjusted to a dry sludge concentration of 3.0 g/L ± 10 %. The sludge was used within 24 h after sampling.
- Initial biomass concentration: not determined

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Post exposure observation period:
no

Test conditions

Hardness:
not determined
Test temperature:
20.7°C
pH:
pH-value of the activated sludge: 7.23
pH-value of the synthetic waste water: 7.27
Dissolved oxygen:
not determined
Salinity:
not determined
Conductivity:
not determined
Nominal and measured concentrations:
1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 500 mL Erlenmeyer flasks, DIN 12380
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: 500 mL
- Aeration: Permanent, with a compressor which puts the air through a glass tube in the vessels, to keep the dissolved oxygen concentration above 60 - 70 % saturation and to maintain the sludge flakes in suspension.
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
- Sludge concentration (weight of dry solids per volume): dry sludge concentration of 2.78 g/L (corresponding to 1.39 g/L suspended solids in the test vessels)
- Nutrients provided for bacteria: Synthetic waste water according to OECD-Guideline 209
- Nitrification inhibitor used (delete if not applicable): none

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
- Particulate matter:

OTHER TEST CONDITIONS
- Adjustment of pH: The pH value of the activated sludge was determined prior to test start. The pH value of the synthetic waste water was determined prior to use and adjustment to 7.5 ± 0.5 was not necessary.
- Photoperiod: light
- Light intensity: not determined

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : After 3 h incubation, the oxygen concentration of each control, reference item and test item replicate was determined

TEST CONCENTRATIONS
- Spacing factor for test concentrations: limit test
- Range finding study : yes
- Test concentrations: 10, 100, 1000 mg/L
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
yes
Remarks:
Copper (II) sulphate pentahydrate

Results and discussion

Effect concentrations
Key result
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
Details on results:
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no
- Adsorption (e.g. of test material to the walls of the test container): no
- Blank controls oxygen uptake rate: specific respiration rate: 21.8 mg O2/gxh, oxygen uptake rate: 30.0-30.8 mg O2/gxh
- Coefficient of variation of oxygen uptake rate in control replicates: 1.1%
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: EC50 = 102 mg/L, 95 % Confidence Interval = 99.3 – 104 mg/L
- Other: This is in the recommended range of validity of 53 - 155 mg/L.
Reported statistics and error estimates:
The NOEC was determined by calculation of statistical significance of the inhibition of respiration in comparison to the control. One Way Analysis of Variance (ANOVA) was used for NOEC calculation. When running a One Way Analysis of Variance a Normality test and an Equal Variance test were done first. P-value for both tests was 0.05. The -value (acceptable probability of incorrectly concluding that there is a difference) is =0.05.

The ECx-value of the reference item was calculated by linear regression using software GraphPad Prism. Calculation of the confidence intervals for the EC50-value was carried out using standard procedures. The coefficient of variation of the oxygen uptake rates of the control replicates was calculated with standard methods using Excel.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The 3h-NOEC of the test item is ≥ 1000 mg/L.
The test substance caused no inhibiting effects on activated sludge of a municipal sewage treatment plant at the limit test item concentration of 1000 mg/L. No nitrification inhibition was observed at 1000 mg/L in the preliminary test and this endpoint was therefore not evaluated in the definitive test.