Registration Dossier

Administrative data

Endpoint:
activated sludge respiration inhibition testing
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
13 - 14 June 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Rationale for read-across: in the environment, lime substances rapidly dissociate or react with water. These reactions, together with the equivalent amount of hydroxyl ions set free when considering 100mg of the lime compound (hypothetic example), are illustrated below: Ca(OH)2 <-> Ca2+ + 2OH- 100 mg Ca(OH)2 or 1.35 mmol sets free 2.70 mmol OH Ca(OH)2 + 2Ca2SiO4 +9CaCO3 + 13H2O <-> 14Ca2+ + 2SiO2 + 9CO2 + 28OH- 100 mg “Reaction mass of limestone and dicalcium silicate” or 0.08 mmol sets free 2.24 mmol OH- has to be noted that CO32- is not expected to directly release two hydroxyl ions under most environmental conditions (depends on CO2 concentrations and pH) and this is therefore a worst case assumption. From these reactions it is clear that the effect of "Reaction mass of limestone and dicalcium silicate" will be caused either by calcium or hydroxyl ions. Since calcium is abundantly present in the environment and since the effect concentrations are within the same order of magnitude of its natural concentration, it can be assumed that the adverse effects are mainly caused by the pH increase caused by the hydroxyl ions. Furthermore, the above mentioned calculations show that the base equivalents are within a factor 2 for lime (chemical), hydraulic and calcium hydroxide. As such, it can be reasonably expected that the effect on pH of "Reaction mass of limestone and dicalcium silicate" is comparable to calcium hydroxide for a same application on a weight basis. Consequently, read-across from calcium hydroxide to "Reaction mass of limestone and dicalcium silicate" is justified.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
IUPAC name: Calcium dihydroxide
PRECAL 50S
Active ingredient: Calcium dihydroxide
Batch no.: 7025
Content: 98.2% (w/w)
Pysical appearance: white powder
Water solubility: 1.26 g/l (20°C)
Date of receipt / quantity received: January 31, 2007 / approx. 2 kg
Storage requirements: store under dry conditions, tightly closed
Stability under correct storage conditions: January 30, 2009

Sampling and analysis

Analytical monitoring:
not required
Details on sampling:
After the test, the contents of the test vessels were poured into flat bottom flasks. Then oxygen measurements were conducted.

Test solutions

Vehicle:
no
Details on test solutions:
The dilution medium (deionised water) will be adapted to test temperature. For each test concentration, the dilution medium will be combined with the synthetic sewage feed (16 mL) in a way that a total volume of 300 ml will be obtained. The test item is weighed into a vessel and transferred to the test medium. The volume of the test solution will be large enough to prepare each test concentration level.
Then 200 ml of the microbial inoculum were added. The total mixture for each concentration and the two controls was 500 mL.

Test organisms

Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
Activated sludge from a sewage treatment plant will be used as the microbial inoculum for the test. The activated sludge is obtained preferentially from a sewage work treating predominantly domestic
sewage.
On the day the activated sludge is obtained from the sewage treatment plant it was washed with reconstituted water. After centrifugation of the sludge the supernatant was decanted. This procedure was
repeated three times.
The final concentration of active sludge in the test medium was 1.6 g/l.
If the activated sludge was not used the day of the collection, 50 ml synthetic sewage feed was added to each litre of the activated sludge and it was aerated with clean, oil-free air and kept at a temperature
of 20 ± 2°C. At the end of every storage day the sludge will be fed with 50 ml/l of synthetic sewage feed (maximum storage = 4 days).
The used microbial inoculum had a mixed liquor suspended solids level of 3.6 g/l. The final level in the test solustions was 1.44 g/l.
RECONSTITUTED WATER
According to OECD Guideline No. 203, prepared according the ECT-Standard Operation Procedure (SOP) A 2.1.
Used to keep the microbial inoculum before the period of the test.
Concentration of salts: 294.0 mg/l CaCl2.2H2O, 123.0 mg/l MgSO4.7H2O, 64.8 mg/l NaHCO3 and 5.75 mg/l KCl.
Water was prepared not longer than 4 weeks before it was used. During storage water was aerated.
SYNTHETIC SEWAGE FEED
Contains peptone, meat extract, urea, NaCl, CaCl2.2H2O, MgSO4.7H2O and K2HPO4.
Prepared not longer than 1 week before it was used.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h

Test conditions

Hardness:
235.8 mg/l CaCO3
Test temperature:
19.1-19.7 °C
pH:
7.4-12.6 (before contact time)
7.8-10.6 (after contact time)
Dissolved oxygen:
8.7 mg/l (at the beginning of the test)
Nominal and measured concentrations:
Nominal: 0, 62.5, 125, 250, 500 and 1000 mg/l
Details on test conditions:
Before use the pH of the activated sludge will be checked and adjusted if necessary to a pH 6.0-8.0 using sodium hydrogen carbonate (NaHCO3) solution.
Every 15 minutes one test vessel containing the test item will be prepared (starting with control 1 and ending with control 2):
- 16 ml of synthetic sewage feed was added to each test vessel.
- deionised water and/or the prepared stock solution was added to the test vessels to a total volume of 300 ml.
- the amount of test item necessary to result in the desired test concentration was added to the test medium and stirred.
- the pH was measured in the test medium.
- 200 ml microbial inoculum was added to each test vessel.
Test vessels: 1000 ml glass beakers.
Number of replicates per test item concentration: 1
Number of replicates in the control: 2
After 3 hours of incubation, the pH was measured. Afterwards the content of the vessels was poured into the measuring apparatus and the respiration rate was determined.
Reference substance (positive control):
yes
Remarks:
(3,5-dichlorophenol)

Results and discussion

Effect concentrationsopen allclose all
Duration:
3 h
Dose descriptor:
other: EC20
Effect conc.:
229.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: Lower 95%: 192.4 mg/l. Upper 95%: 252.6 mg/l.
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
300.4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: Lower 95%: 273.4 mg/l. Upper 95%: 348.9 mg/l.
Duration:
3 h
Dose descriptor:
other: EC80
Effect conc.:
393.9 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: Lower 95%: 341.6 mg/l. Upper 95%: 548.1 mg/l.
Details on results:
The biological findings were closely related to the initial pH of the test solutions, which increased from 7.4 in the controls to pH 9.2, 10.0, 11.6, 12.2, 12.6 at 62.5, 125, 250, 500 and 1000 mg/L, respectively.
Within the contact time of 3 hours, the initial pH was considerably lowered which is likely attributable to the reaction of the test item with CO2 in the medium to CaCO3. Therefore the initial pH is considered to be the main reason for the effects of the test item on the test organisms.
Results with reference substance (positive control):
The 3h- EC50 was in the accepted range of 5-30 mg/L : namely 7.8 mg/L.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
(The two control respiration rates within 15 per cent of each other: 0,4%. The EC50 (3 hours) of 3,5-dichlorophenol was in the accepted range of 5 to 30 mg/l: 7,8 mg/l.)
Conclusions:
The biological findings (inhibition of respiration) were closely related to the initial pH of the test solutions.
Within the contact time of 3 hours, the initial pH was considerably lowered which is likely attributable to the reaction of the test item with CO2 in the medium to Calcium carbonate. Therefore the initial pH is considered to be the main reason for the effects of the test item in the test organisms.