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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:
22.05.08 - 03.07.08
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
Analytical monitoring:
yes
Details on sampling:
After the 3-hour contact time, a well mixed sample of the contents was poured into a 300 ml oxygen bottle, and the flask was sealed with an oxygen electrode connected to a recorder, forcing the air out of the vessel. Oxygen consumption was measured and recorded for approximately 10 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.
This procedure was repeated for the duplicate concentration. Two controls without test substance were tested in each test series, one at the start and
one at the end.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
The batch of Anhydrothymidine tested was a white powder with a purity of 99.7%. Since Anhydrothymidine was well soluble in water a stock solution of 5 g/l was prepared by dissolution 1.2502 g Anhydrothymidine in 250 ml Milli-RO water (tap water purified by reverse osmosis). Volumes of the stock solution corresponding to the test concentration were then added to the test media. A concentration of 1000 mg/l was tested in duplicate.
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 inoculum for exposure: 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 (4.3 g/l of sludge, as used for the test). The pH was 6.2 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.
- Initial biomass concentration: 4.3 g/l
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Remarks on exposure duration:
aeration and stirring took place
Post exposure observation period:
After the exposure period, oxygen consumption was measured and recorded for approximately 10 minutes.
Measurement of pH.
Test temperature:
18.1 and 18.7°C
pH:
7.4 - 7.5
Dissolved oxygen:
8.5 - 8.9 mg O2/l
Details on test conditions:
TEST SYSTEM
- Test vessel: All glass, approximately 300 ml oxygen bottles and 1 litre test bottles.
- Aeration: yes
- No. of organisms per vessel: The synthetic sewage feed (16 ml), activated sludge (200 ml) and an adequate amount of the test substance stock were mixed and made up to 500 ml with Milli-RO water in a 1 litre bottle.
- No. of vessels per concentration (replicates): This procedure was repeated for the duplicate concentration. Two controls without test substance were tested in each test series, one at the start and one at the end.


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: tap-water purified by reverse osmosis


OTHER TEST CONDITIONS
- Adjustment of pH: 7.5 ± 0.5.



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
The respiration rate from each vessel, in mg O2/l/hr

TEST CONCENTRATIONS
0, 1000 mg/l
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol CAS 591-35-5
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
Details on results:
No (significant) inhibition of respiration rate of the sludge was recorded at 1000 mg Anhydrothymidine per litre. The duplicate measurement confirmed the result of the first measurement. Therefore, no further testing was needed.
Hence, the EC50 of Anhydrothymidine exceeded 1000 mg/l (based on nominal concentrations).
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels:
EC50 = 6.2 mg 3,5-dichlorophenol / L
Reported statistics and error estimates:
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.
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 Anhydrothymidine no EC50 could be calculated because the test substance proved to be non-toxic (EC50 > 1000 mg/l).
Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of this present study, the test substance was not toxic to waste water (activated sludge) bacteria at a nominal concentration of 1000 mg/l.

Description of key information

Anhydrothymidine was not toxic to waste water bacteria at a nominal concentration of 1000 mg/l.

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L

Additional information