Registration Dossier

Administrative data

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April 2017
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))
Version / remarks:
Adopted 22 July 2010
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
N-[4-[(4,6-dimethylpyrimidin-2-yl)sulfamoyl]phenyl]-2-methyl-prop-2-enamide
EC Number:
611-915-5
Cas Number:
59941-98-9
Molecular formula:
C16 H18 N4 O3 S
IUPAC Name:
N-[4-[(4,6-dimethylpyrimidin-2-yl)sulfamoyl]phenyl]-2-methyl-prop-2-enamide
Test material form:
solid: particulate/powder
Remarks:
white crystalline

Sampling and analysis

Analytical monitoring:
no

Test solutions

Vehicle:
no
Details on test solutions:
The test item was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 10 g/L. Therefore, the required amount of V123109 was added directly to the dark brown test-bottles containing 200 mL Milli-RO water (tap water purified by reverseosmosis; Millipore Corp., Bedford, Mass., USA). The test item – Milli-RO water mixtures were magnetically stirred for a period of 25 minutes. 16 mL synthetic medium to obtain 50 mL and 250 mL sludge was added to this solution, resulting in the required loading rates. Optimal contact between the test item and test organisms was ensured by applying continuous aeration and stirring.

Test organisms

Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Laboratory culture: /
- Name and location of sewage treatment plant where inoculum was collected: Municipal sewage treatment plant: 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands
- Method of cultivation: /
- Preparation of inoculum for exposure: The sludge was coarsely sieved (1 mm) and allowed to settle. The supernatant was removed and ISO-medium was added. 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 7.7 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.
- Pretreatment: /
- Initial biomass concentration: /

Study design

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 approximately 10 minutes. During measurement, the sample was not aerated but continuously stirred on a magnetic stirrer.

Test conditions

Test temperature:
The temperature continuously measured in the temperature control vessels ranged between 20 and 22°C during the test
pH:
The pH in all test vessels, before addition of sludge was between 7.5 and 7.7. After the 3 hour exposure period the pH was between 7.2 and 8.2.
Nominal and measured concentrations:
loading rates of 10, 100 and 1000 mg/L were tested
Details on test conditions:
TEST SYSTEM
- Test vessel: All glass open bottles/vessels.
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: /
- Aeration: The aeration was adjusted in such a way that the dissolved oxygen concentration at the start was above 60-70% saturation (60% of air saturation is > 5 mg/L at 20°C) and to maintain the sludge flocs in suspension.
- No. of vessels per concentration (replicates): The highest loading rate was tested in triplicate, lower loading rates consisted of one replicate.
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): /
- No. of vessels per abiotic control (replicates): 1
- Sludge concentration (weight of dry solids per volume): suspended solids (3.0 g/L of sludge)
- Weight of dry solids per volume of reaction mixture per unit of time: /
- Nutrients provided for bacteria: /
- Biomass loading rate: /
TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: /
- Particulate matter: /
OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: /
- Light intensity: /
- Details on termination of incubation: /
EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : oxygen consumption
TEST CONCENTRATIONS
- Spacing factor for test concentrations: /
- Justification for using fewer concentrations than requested by guideline: /
- Range finding study: Combined Limit/Range-Finding Test
- Test concentrations: 10, 100 and 1000 mg/L
- Results used to determine the conditions for the definitive study: /
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol

Results and discussion

Effect concentrationsopen allclose all
Duration:
3 h
Dose descriptor:
other: NOELR
Effect conc.:
1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Duration:
3 h
Dose descriptor:
EC50
Remarks:
for 3,5-dichlorophenol
Effect conc.:
3.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Details on results:
The combined limit/range-finding test showed 11%, 8% and an average of 15% inhibition of the respiration rate at 10, 100 and 1000 mg/L, respectively. For V123109 no ELR50-value could be calculated because the test item proved to be non-toxic (ELR50 > 1000 mg/L)
No statistically significant inhibition of the respiration rate of the sludge was recorded at a concentration of 1000 mg V123109 per litre. Therefore, the NOELR was considered to be 1000 mg/L (see also Appendix 5).
There was no oxygen uptake from abiotic processes.

Furthermore, the mean control oxygen uptake rate was 29 mg O2/g.h, with a coefficient of variation of 13%, which was within the acceptable range.
Results with reference substance (positive control):
Functioning of the test system was checked by testing 3,5-dichlorophenol which showed an acceptable EC50 (3.3 mg/L).

Any other information on results incl. tables

Experimental Conditions

The pH values in the individual vessels are presented inTable 3ofAppendix 2.The pH in all test vessels, before addition of sludge was between 7.5 and 7.7. After the 3 hour exposure period the pH was between 7.2 and 8.2.

The temperature continuously measured in the temperature control vessels ranged between 20 and 22°C during the test, and complied with the requirements as laid down in the study plan (20 ± 2°C.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of this present test V123109 was not toxic to waste water bacteria (activated sludge) at a loading rate of 1000 mg/L (NOELR). The ELR50 was above 1000 mg/L.
Executive summary:

The objective of the study was to evaluate V123109 for its ability to adversely affect aerobic microbial treatment plants and, if possible, to determine the ELR50and/or the no-observed effect loading rate (NOELR).

The influence of V123109 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 on the OECD guideline No. 209, 2010.

The batch of V123109 tested was a white crystalline powder with a purity of >95%. No correction was made for the purity/composition of the test item.

The test item was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 10 g/L. Therefore, weight amounts were added to the test-bottles containing Milli-RO water. The test itemMilli-RO water mixtures were magnetically stirred for a period of 25 minutes and subsequently, synthetic medium, sludge and Milli-RO water were added to obtain the required loading rates. Optimal contact between the test item and test medium was ensured by applying continuous aeration and stirring during the 3-hour exposure period. Thereafter, oxygen consumption was recorded for approximately 10 minutes.

In a combined limit/range-finding test loading rates of 10, 100 and 1000 mg/L were tested. The highest loading rate was tested in triplicate, lower loading rates consisted of one replicate. Furthermore, at 1000 mg/L an abiotic control (1 replicate) was tested. Responses were compared to the controls.

No statistically significant inhibition of the respiration rate of the sludge was recorded at a loading rate of 1000 mg V123109 per litre in the combined limit/range-finding test and subsequent testing was therefore not required.

The batch of activated sludge was tested for sensitivity with the reference item 3,5-dichlorophenol, and showed normal sensitivity. The study met the acceptability criteria prescribed by the study plan and was considered valid.

In conclusion, V123109 was not toxic to waste water (activated sludge) bacteria at a loading rate of 1000 mg/L (NOELR). The ELR50was above 1000 mg/L.