Magnesium glycerophosphate

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

February 01, 2018 - March 02, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Version / remarks:

July 22, 2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Magnesium glycerophosphate

EC Number:

213-149-3

EC Name:

Magnesium glycerophosphate

Cas Number:

927-20-8

Molecular formula:

C3H9O6P.Mg

IUPAC Name:

magnesium glycerophosphate

Test material form:

solid: particulate/powder

Details on test material:

Batch: INVG003917

Specific details on test material used for the study:

Test Item
The test item and the information concerning the test item were provided by the Sponsor:
Name: Magnesium glycerophosphate
Batch No.: INVG003917
CAS No.: 927-20-8
Purity: 96.5% (w/w)
Certificate of Analysis Date: September 1, 2017
Aggregate State: Solid
Colour: White
Solubility: ca. 80g/L
Expiry Date: June 10, 2021
Storage Conditions at Test Facility: At 20  5°C, in the dark

Sampling and analysis

Analytical monitoring:

no

Details on sampling:

Measurement of Respiration Rate:

For the measurement of the respiration rate a well-mixed sample of test medium from each flask was poured into a Karlsruher flask after exactly 3 hours incubation time and was not further aerated during measurement. The oxygen concentration was then measured with an oxygen electrode and recorded for about ten minutes. During measurement, the samples were continuously stirred on a magnetic stirrer. The oxygen consumption (in mg O2 L-1 minute-1) was determined over a period up to 10 minutes in the range between approximately 8.2 – 1.5 mg O2/L.

Test solutions

Vehicle:

no

Details on test solutions:

Identification Synthetic Activated Test Stock Water Final
Sewage Sludge item* solution** conc.
(mL) (mL) (mg) (mL) (mL) (mg/L)
control 16 250 --- --- 234 ---
control 16 250 --- --- 234 ---
3,5-Dichlorophenol 16 250 --- 0.5 233.5 1
3,5-Dichlorophenol 16 250 --- 2.0 232 4
3,5-Dichlorophenol 16 250 --- 8.0 226 16
control 16 250 --- --- 234 ---
control 16 250 --- --- 234 ---
test item 16 250 500 --- 234 1000
test item 16 250 500 --- 234 1000
test item 16 250 500 --- 234 1000
control 16 250 --- --- 234 ---
control 16 250 --- --- 234 ---

Test organisms

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

Species / Origin:
Activated sludge from a domestic waste water treatment plant was supplied by the municipal sewage treatment plant Bensheim, Germany.

Conditioning:
The activated sludge used for this study was used as collected, but coarse particles were removed by settling for a short period (30 minutes) and then the upper layer decanted. During holding prior to use the sludge was fed daily with 50 mL synthetic sewage (see below) per litre and kept aerated at room temperature overnight.
An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to its dry weight determined. Based on the sludge dry matter, calculated amounts of wet sludge were suspended in pure water to yield a concentration equivalent to 3 g/L on dry weight basis. This level gave a concentration of 1.5 g/L suspended solids in the test medium. The pH of the activated sludge was 6.4 and therefore no adjustment was necessary.

Study design

Test type:

static

Water media type:

other: Deionised water

Limit test:

yes

Total exposure duration:

3 h

Post exposure observation period:

10 minutes

Test conditions

Test temperature:

20 ± 2°C

pH:

Test Treatment Concentration pH-Value
Flasks mg/L Start End

1 Control - 6.3 7.3
2 Control - 6.3 7.3
3 3.5-DCP 1 6.3 7.5
4 3.5-DCP 4 6.3 7.5
5 3.5-DCP 16 6.3 7.3
6 Control - 6.4 7.4
7 Control - 6.4 7.3
13 TS 1000 7.0 7.3
14 TS 1000 7.0 7.3
15 TS 1000 7.0 7.3
16 Control - 6.2 7.3
17 Control - 6.3 7.4

Dissolved oxygen:

O2 [mg/l] *
Start Ende
7.1 6.6
7.3 7.8
7.2 7.1
7.3 7.6
7.4 8.2
6.8 7.4
7.1 7.8
6.0 7.1
6.0 8.1
6.2 8.1
5.9 8.3
6.0 7.6
*dissolved oxygen at test start and test end

Salinity:

not determined

Conductivity:

not determined

Nominal and measured concentrations:

not determined

Details on test conditions:

Test Units

Type and Size: Glass flasks of approximately 1 litre volume and Karlsruher flasks of 250 mL volume
Identification: Each test unit was uniquely identified with the study number, treatment and replicate number.

Test Conditions

Surrounding Type: Controlled environment room
Temperature: 20 ± 2°C
Aeration: Compressed air (1.017 litre per minute)
Recording: Test conditions (temperature) was recorded continuously with suitable instruments, documented in the raw data and reported in the final report. (Short-term deviations (< 2 hours) from the recommended temperature range normally don’t result in major disturbances of the test performance and were not reported).

Reference substance (positive control):

yes

Remarks:

Name: 3,5-Dichlorophenol Lot No.: MKBZ0947V

Results and discussion

Effect concentrations

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

Toxicity of Test item

The mean respiration rates of the activated sludge treated with the test item at a concentration of 1000 mg/L differed by less than 10% from control.
Conclusion: At a nominal test concentration of 1000 mg test item/L less than 10% inhibition was observed after three hours of incubation. The mean respiration rate was 6.5% at three replicates of 1000 mg Magnesium glycerophosphate/L.
The respiration rate of the activated sludge to Magnesium glycerophosphate at the rate of 1000 mg/L did not show substantial biological effects.
Magnesium glycerophosphate at the rate of 1000 mg/L had no toxic effects on respiration activity of activated sludge.
Therefore it can be assumed that the NOEC is above 1000 mg/L.

Results with reference substance (positive control):

Toxicity of 3,5-Dichlorphenol

Inhibition of Respiration Rate:
The following nominal concentrations of the positive control 3,5-Dichlorophenol were tested on the same activated sludge and under identical conditions as the test item: 1, 4 and 16 mg/L. In comparison to the controls the respiration rate of the activated sludge was moderately inhibited by 2.2% at the lowest nominal concentration of 1 mg/L. At the nominal concentrations of 4 and 16 mg reference item/L, the respiration rate was inhibited by 28.4% and 80.6%, respectively.

The 3-hour EC50 of 3,5-Dichlorophenol was calculated to be 6.9 mg/L.
Parameter EC10 EC20 EC50
Value [mg/l] 2.0 3.1 6.9
lower 95%-cl 2.0 3.0 6.9
upper 95%-cl 2.1 3.1 7.0

Reported statistics and error estimates:

Statistical Analysis:

The software used to perform the statistical analysis was ToxRat Professional®, Version 3.2.1, ToxRat Solutions GmbH. The EC50 including the 95 %-confidence limits of the reference item was calculated by Probit Analysis. Shapiro Wilk’s Test, Levene’s Test and Williams Multiple Sequential t-Test were used for the determination of normal distribution, homogeneity and calculation of the NOEC.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

At a nominal test concentration of 1000 mg test item/L less than 10% inhibition was observed after three hours of incubation. The mean respiration rate was 6.5% at three replicates of 1000 mg Magnesium glycerophosphate/L.
The respiration rate of the activated sludge to Magnesium glycerophosphate at the rate of 1000 mg/L did not show substantial biological effects.
Magnesium glycerophosphate at the rate of 1000 mg/L had no toxic effects on respiration activity of activated sludge.
Therefore it can be assumed that the NOEC is above 1000 mg/L.

Executive summary:

Title:	Magnesium glycerophosphate: Toxicity to Activated Sludge in a Respiration Inhibition Test (Limit Study)
Guidelines/Recommendations:	-     Commission Regulation (EC) No 440/2008, Method C.11: "Activated Sludge Respiration Inhibition Test", Official Journal of the European Union No.L 142/559-563, dated -     OECD Guideline for Testing of Chemicals, No. 209: "Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation)", adopted July 22, 2010 -     Water quality - Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation (ISO 8192:2007); German version EN ISO 8192:2007
Purpose:	The influence of the test item Magnesium glycerophosphate on the activity of activated sludge was evaluated by measuring the respiration rate under defined conditions. The respiration rate (oxygen consumption) of an aerobic activated sludge fed with a standard amount of synthetic sewage was measured in the presence of one concentration of the test item after an incubation period of 3 hours. This limit test was performed in compliance with the test guidelines to demonstrate that the test item had no toxic effect on activated sludge up to at least this concentration.
Test Concentrations:	Nominal 1000 mg Magnesium glycerophosphate/L; 1, 4 and 16 mg 3,5-Dichlorophenol/L and six inoculum controls
Results:
Toxicity of Test Item:	The mean respiration rate of activated sludge treated with the test item at a concentration of 1000 mg/L differed from the mean respiration rate in the control by less than 10%.
Conclusion:	Therefore Magnesium glycerophosphate at the rate of 1000 mg/L had no toxic effects on respiration activity of activated sludge. It can be assumed that the NOEC is below 1000 mg/L.
Toxicity of 3,5-Dichlorophenol:	The positive control 3,5-Dichlorophenol was tested in the same way as the test item. The 3-hour EC50was found to be 6.9 mg/L thus being in the range of 2 - 25 mg/L recommended by the test guidelines. The result confirms the suitability of the activated sludge used.