Calcium glycerophosphate

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:

September 2020 - october 2020

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 (Carbon and Ammonium Oxidation))

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (manufacturer): Givaudan Lavirotte
- Lot/batch number of test material: Batch No 2000001347
- Purity: 92.1%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature. Keep only in the original container.
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions and during storage: stable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing : no
- Preliminary purification step : no
- Final concentration of liquid solution: test solutions were prepared to obtain the following nominal test concentrations: 100 and 1000 mg test item.L-1

Analytical monitoring:

no

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

The activated sludge concentration in all test, reference and blank mixtures was approx. 1.5 g.L-1 of suspended solids.

ORIGIN
Activated sludge was collected (24/09/2020) from a wastewater treatment plant (Aureilhan, France) receiving predominantly domestic sewage.

PRE-TREATMENT
Upon arrival in the test facility, the sludge was sieved (using a 1 mm2 mesh sieve), washed 3 times with chlorine-free tap water and re-suspended in tap water. The activated sludge was aerated and fed with the synthetic sewage feed (30 mL per L of sludge suspension) until the next day (final test). The dry weight of the sludge suspension was determined, then the sludge was diluted in chlorine-free tap water to obtain the required sludge solids concentration of approximately 3.0 g.L-1.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Test temperature:

Temperatures recorded continuously were situated between 19.4 and 20.2°C throughout the test
(average value: 19.7°C), and complied with the requirements as laid down in the study plan (20 ± 2 °C).

pH:

The pH of the reaction mixtures at the end of the test period ranged from 7.485 to 7.762

Nominal and measured concentrations:

Nominal concentrations are 100 and 1000 mg test item.L-1

Details on test conditions:

TEST SYSTEM
- Test vessel:
All-glass bottles of approximately 1 L capacity closed with PTFE screw caps, containing 500 mL of reaction mixture. Each test vessel was uniquely identified with study code, replicate number, start time of the test and type of series.
At the end of incubation, samples were withdrawn to measure the rate of decrease of dissolved O2 in completely filled all-glass BOD (Biological Oxygen Demand) bottles of approximately 300 mL capacity.

- Aeration:
All suspensions were aerated and stirred during the exposure period.

- Environment:
Controlled environment room (20°C ± 2°C)

- No. of vessels per concentration (replicates):
5 replicates bottles per test concentration containing synthetic sewage feed, test water, test item and inoculum (test mixtures)

- No. of vessels per control (replicates):
6 replicates bottles containing synthetic sewage feed, test water and inoculum (blank controls).
1 replicate per reference substance concentration containing synthetic sewage feed, test water, reference substance and inoculum (reference mixtures)

- Sludge concentration (weight of dry solids per volume):
The activated sludge concentration in all test, reference and blank mixtures was approx. 1.5 g.L-1 of suspended solids.

- Nutrients provided for bacteria:
A specific medium was prepared (composition detailed in the report attached).


WATER PARAMETERS
Deionised water, containing less than 1 mg.L-1 DOC (Dissolved Organic Carbon); determined value: 0.636 mg.L-1 DOC; DOC analysis was performed in accordance with ISO 17025.

ADMINISTRATION OF THE TEST AND REFERENCE SUBSTANCES
The test vessels were 1 L cylindrical glass bottles closed with PTFE screw caps. A magnetic stirring bar was placed in each test vessel. Blank controls were prepared with 16 mL synthetic sewage feed and 234 mL test water. Test mixtures were prepared by addition of the required amounts of test item in pre-filled test vessels (with synthetic sewage feed and test water). The 3,5-DCP stock solution was diluted with test water as necessary to obtain the required test concentrations into reference mixtures after filling with synthetic sewage and water. Then, 250 mL of sludge suspension were sequentially added in all test vessels. After addition of the inoculum, the test vessels were immediately closed and the mixtures were stirred under aeration for 3 hours.

Reference substance (positive control):

yes

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

>= 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Details on results:

Based on these results, the highest nominal test concentration without observed effects (= NOEC) was 1000 mg.L-1; the 3-hour EC50 was estimated to be > 1000 mg.L-1.

Validity criteria fulfilled:

yes

Conclusions:

The effect of test item CALCIUM GLYCEROPHOSPHATE on microorganisms from activated sludge was investigated in a respiration inhibition test. Under the experimental conditions, and based upon nominal concentrations, the 3-hour EC50 and the highest nominal concentration without observed effects values were determined to be greater than and equal to 1000 mg test item.L-1 respectively. Thus, the NOEC value was 1000 mg test item.L-1.

Executive summary:

Introduction. A study was performed to assess the effects of test item CALCIUM GLYCEROPHOSPHATE on microorganisms from activated sludge by performing a respiration inhibition test. The method followed was designed to be compliant with OECD Guideline for Testing of Chemicals No. 209, "Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation)" (1), referenced as Method C.11 of Commission Regulation No. 440/2008 (2).

 

Methods. A definitive test was performed following the results of a range-finding test. Microorganisms from activated sludge were exposed to the test item at nominal concentrations of 100 and 1000 mg test item.L-1 (five replicates for each concentration) and to six blank controls in order to determine a concentration with no significant effect and to demonstrate that EC50 was higher than 1000 mg.L-1. Furthermore, an exposure of the test organism to chemical 3,5-dichlorophenol (3,5-DCP) was performed in order to check the sensitivity of the batch of activated sludge used on the day of exposure. All the mixtures contained an activated sludge concentration of about 1.5 g.L-1 of suspended solids.

After 3 hours of exposure under aeration and stirring, samples of each mixture were poured into BOD bottles to continuously measure the oxygen concentration using an oxygen electrode and determine the rate of decrease of dissolved O2. The measurement of total oxygen uptake inhibition was determined only (carbon and ammonium oxidation not separated).

 

Results. The validity of the test was demonstrated by an oxygen uptake rate in the blank controls higher than 20 mg O2.g-1.h-1, a coefficient of variation of oxygen uptake rate in control replicates below 30% and an EC50 of 11.5 mg.L-1 for 3,5-DCP (within the prescribed range of 2 to 25 mg.L-1 for total respiration). At the end of the test, the 3-hour EC50 was estimated to be ≥ 1000 mg.L-1.

 

Conclusion. The effect of test item CALCIUM GLYCEROPHOSPHATE on microorganisms from activated sludge was investigated in a respiration inhibition test. Under the experimental conditions, and based upon nominal concentrations, the 3-hour EC50 and the highest nominal concentration without observed effects values were determined to be greater than and equal to 1000 mg test item.L-1 respectively. Thus, the NOEC value was 1000 mg test item.L-1.

Description of key information

The effect of CALCIUM GLYCEROPHOSPHATE on microorganisms from activated sludge was investigated in a respiration inhibition test. Under the experimental conditions, and based upon nominal concentrations, the 3-hour EC50 and the highest nominal concentration without observed effects values were determined to be greater than and equal to 1000 mg test item.L-1 respectively. Thus, the NOEC value was 1000 mg test item.L-1.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

1 000 mg/L