Calcium dihydroxide

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 - 14 June 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: According to OECD 209. Well documented GLP study. Validity criteria fulfilled.

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

GLP compliance:

yes (incl. QA statement)

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.

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 (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.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

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)

Salinity:

not applicable

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

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.

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.

Description of key information

Klimisch 1 study; activated sludge respiration inhibition test according to OECD 209; nominal EC50(3h) = 300.4 mg Ca(OH)2/L (Egeler et al., 2007)
Klimisch 1 study; activated sludge respiration inhibition test according to OECD 209; nominal EC50(3h) >1000 mg CaCO3/L (Youngs, 2010)

Key value for chemical safety assessment

EC50 for microorganisms:

300.4 mg/L

Additional information

The toxicity to aquatic microorganisms of calcium dihydroxide was assessed in a study performed to OECD TG 209 under GLP (Egeler et al. 2007). Activated sewage sludge was exposed to calcium dihydroxide at nominal test concentrations of 0, 62.5, 125, 250, 500 and 1000 mg/L for 3 hours. 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. The 3-h EC50 was 300.4 mg/L.

The toxicity to aquatic micro-organisms of calcium carbonate (nano) was assessed in a study performed according to OECD TG 209 under GLP (Youngs, 2010). Activated sewage sludge was exposed to calcium carbonate at nominal test concentrations of 0, 10, 32, 100, 320 and 1000 mg/L for 3 hours. No toxic effects were seen at any concentration of calcium carbonate (nano) tested. Hence the 3-h EC50 was >1000 mg/L and the NOEC was 1000 mg/L. Calcium carbonate (nano) is therefore not toxic to aquatic microorganisms at concentrations up to 1000 mg/L. Evidence of undissolved test material was observed in some of the test vessels suggesting that the concentrations tested exceeded the maximum solubility of calcium carbonate in water.

Based on the results of the studies performed on calcium dihydroxide and calcium carbonate, it may be concluded that the acute toxicity to aquatic microorganisms of grades of calcium dihydroxide containing up to 35% calcium carbonate will be driven by the calcium dihydroxide content and that the results available for the calcium dihydroxide itself represent the worse-case for all grades.