Glycerides, C16-18 and C18-unsatd. mono-, di- and tri-, citrates, potassium salts

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-06-11 to 2012-06-12

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

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Directly weighing of the stock solution in an Erlenmeyer flask, addition of 200 mL demineralised water, overnight stirring
- Eluate: Synthetic waste water acc. to OECD 209
- Differential loading: Limit concentration of 1000 mg/L
- Controls: Triplicates at the beginning of the test + triplicates at the end of the test

Test organisms (species):

activated sludge

Details on inoculum:

- Preparation of inoculum for exposure: Non adapted activated sludge from the sewage plant at Hildesheim (Municipal sewage treatment plant of 31137 Hildesheim, Germany)
- Pretreatment: Washed twice with autoclaved tap water and an adjustment to a dry weight concentration of 3.0 g/L +/- 10 % was not necessary. The sludge was used within 24 h after sampling
- Dry sludge concentration: 2.72 g/L, corresponding to 1.36 g/L in the test vessel

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Hardness:

Not determined

Test temperature:

20.9 °C

pH:

pH-value of the activated sludge 7.72
pH-value of the synthetic waste water 7.44

Salinity:

Not determined

Details on test conditions:

TEST SYSTEM
- Test vessel: 500 mL Erlenmeyer flask, DIN 12380
- Aeration: Permanent
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
- Composition of test medium:
250 mL inoculum
16 mL synthetic waste water
234 mL demineralised water


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
After 3 h incubation, the oxygen concentration of each control, reference item and test item replicate was determined by means of oximeter immediately after transferring the solution to the measuring cell and the oxygen depletion was recorded for at least 3 minutes on a flatbed recorder.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: None, limit test concentration
- Range finding study
Inhibition of Respiration (Preliminary Test)
Nominal Test Item
Concentration
[mg/L] Pretreatment pH(test item in water) pH(test item in water) Inhibition

[%]
10 Stirring overnight - - -4
100 - - -10
1000 - - -25
1000 6.82 7.24 -17

Reference substance (positive control):

yes

Remarks:

Copper (II) sulphate pentahydrate p.a.

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

Remarks:

respiration rate

Details on results:

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels: EC50: 99.1 mg/L

Reported statistics and error estimates:

The NOEC was determined by calculation of statistical significance of the inhibition of respiration in comparison to the control. One Way Analysis of Variance (ANOVA) and DUNNETT’s test was used for NOEC calculations. When running a One Way Analysis of Variance a Normality test and an Equal Variance test were done first. P-values for both Normality and Equal Variance test are 0.05. The a-value (acceptable probability of incorrectly concluding that there is a difference) is a=0.05.

From the oxygen depletion the oxygen uptake rates were calculated in mg O₂/L×h, additionally the specific respiration rates based on the dry sludge concentration were calculated in
mg O₂/g×h.

The inhibition of the oxygen uptake rates of the test and reference item concentrations in % was calculated in comparison with the oxygen uptake rates of the control.

Oxygen Uptake Rates, Specific Respiration Rates and Inhibition of the Respiration
of the Control and the Limit Test Item Concentration

Test Item Concentration [mg/L]	Repl.	Oxygen Uptake Rate R [mg O2/L×h]	Specific Respiration Rate Rs [mg O2/g×h]	Inhibition [%]	Mean Inhibition [%]
Control	1	18.4	13.5	–	–
	2	18.8	13.8
	3	18.8	13.8
	4	20.0	14.7
	5	18.8	13.8
	6	17.6	12.9
1000	1	21.6		-16	-21
	2	23.2		-24
	3	22.8		-22

Oxygen Uptake Rates, Specific Respiration Rates and Inhibition of the Respiration
of the Control and the Reference Item Concentrations

Reference Item Concentration [mg/L]	Repl.	Oxygen Uptake Rate R [mg O2/L×h]	Specific Respiration Rate Rs [mg O2/g×h]	Inhibition [%]	Mean Inhibition [%]
Control	1	20.0	14.7	–	–
	2	18.8	13.8
	3	17.6	12.9
	4	18.4	13.5
	5	17.2	12.6
	6	16.0	11.8
58	1	12.8		29	24
	2	14.0		22
	3	14.0		22
100	1	9.2		49	50
	2	8.4		53
	3	9.2		49
180	1	3.6		80	79
	2	4.0		78
	3	4.0		78

Validity criteria fulfilled:

yes

Conclusions:

The NOEC of the test item (Glycerides, C16-18 mono-, di-, and tri-, hydrogenated, citrates, potassium salts) is 1000 mg/L.
The test item is not toxic at the limit concentration of 1000 mg/L to activated sludge of a municipal sewage treatment plant.

Executive summary:

A Respiration Inhibition Test with activated sludge according to OECD Guideline No. 209 was carried out with the test item (Glycerides, C16-18 mono-, di-, and tri-, hydrogenated, citrates, potassium salts) (batch number: 105618) from 2012-06-11 to 2012-06-12, with the definitive experimental phase on 2012-06-12 at the Dr. U. Noack-Laboratorien, 31157 Sarstedt, Germany. Test system was activated sludge of the municipal treatment plant of 31137 Hildesheim, Germany. The test was carried out under static conditions with the limit test item concentration of 1000 mg/L. The respiration rates of the control, reference and test item replicates were measured after a contact time of three hours, and the inhibitory effects of the test and reference item were determined in comparison to the control respiration rates. The mean inhibition of respiration for the test item replicates ranged from -16 % to -22 %.

In order to check the activity of the test system and the test conditions a reference test was carried out with copper (II) sulphate pentahydrate as reference item and the reference toxicity was determined. The EC₅₀-value for the reference item was 99.1 mg/L.

Table 1: NOEC-Value of Glyceryl StearateCitrate (Glycerides, C16-18 mono-, di-, and tri-, hydrogenated, citrates, potassium salts)

		Test Item Concentration [mg/L]
NOEC		1000

                                              

The NOEC of the test item Glyceryl Stearate Citrate (Glycerides, C16-18 mono-, di-, and tri-, hydrogenated, citrates, potassium salts) is 1000 mg/L. The test item is not toxic at the limit concentration of 1000 mg/L to activated sludge of a municipal sewage treatment plant.

Reference 2

Endpoint:

activated sludge respiration inhibition testing

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Read-across to K1 study therefore K2 is the highest Klimisch score that can be assigned

Justification for type of information:

Please see the read-across report attached to section 13 of this dossier.

Reason / purpose for cross-reference:

read-across source

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Directly weighing of the stock solution in an Erlenmeyer flask, addition of 200 mL demineralised water, overnight stirring
- Eluate: Synthetic waste water acc. to OECD 209
- Differential loading: Limit concentration of 1000 mg/L
- Controls: Triplicates at the beginning of the test + triplicates at the end of the test

Test organisms (species):

activated sludge

Details on inoculum:

- Preparation of inoculum for exposure: Non adapted activated sludge from the sewage plant at Hildesheim (Municipal sewage treatment plant of 31137 Hildesheim, Germany)
- Pretreatment: Washed twice with autoclaved tap water and an adjustment to a dry weight concentration of 3.0 g/L +/- 10 % was not necessary. The sludge was used within 24 h after sampling
- Dry sludge concentration: 2.72 g/L, corresponding to 1.36 g/L in the test vessel

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Hardness:

Not determined

Test temperature:

20.9 °C

pH:

pH-value of the activated sludge 7.72
pH-value of the synthetic waste water 7.44

Salinity:

Not determined

Details on test conditions:

TEST SYSTEM
- Test vessel: 500 mL Erlenmeyer flask, DIN 12380
- Aeration: Permanent
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
- Composition of test medium:
250 mL inoculum
16 mL synthetic waste water
234 mL demineralised water


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
After 3 h incubation, the oxygen concentration of each control, reference item and test item replicate was determined by means of oximeter immediately after transferring the solution to the measuring cell and the oxygen depletion was recorded for at least 3 minutes on a flatbed recorder.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: None, limit test concentration
- Range finding study
Inhibition of Respiration (Preliminary Test)
Nominal Test Item
Concentration
[mg/L] Pretreatment pH(test item in water) pH(test item in water) Inhibition

[%]
10 Stirring overnight - - -4
100 - - -10
1000 - - -25
1000 6.82 7.24 -17

Reference substance (positive control):

yes

Remarks:

Copper (II) sulphate pentahydrate p.a.

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

Remarks:

respiration rate

Details on results:

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels: EC50: 99.1 mg/L

Reported statistics and error estimates:

The NOEC was determined by calculation of statistical significance of the inhibition of respiration in comparison to the control. One Way Analysis of Variance (ANOVA) and DUNNETT’s test was used for NOEC calculations. When running a One Way Analysis of Variance a Normality test and an Equal Variance test were done first. P-values for both Normality and Equal Variance test are 0.05. The a-value (acceptable probability of incorrectly concluding that there is a difference) is a=0.05.

From the oxygen depletion the oxygen uptake rates were calculated in mg O₂/L×h, additionally the specific respiration rates based on the dry sludge concentration were calculated in
mg O₂/g×h.

The inhibition of the oxygen uptake rates of the test and reference item concentrations in % was calculated in comparison with the oxygen uptake rates of the control.

Oxygen Uptake Rates, Specific Respiration Rates and Inhibition of the Respiration
of the Control and the Limit Test Item Concentration

Test Item Concentration [mg/L]	Repl.	Oxygen Uptake Rate R [mg O2/L×h]	Specific Respiration Rate Rs [mg O2/g×h]	Inhibition [%]	Mean Inhibition [%]
Control	1	18.4	13.5	–	–
	2	18.8	13.8
	3	18.8	13.8
	4	20.0	14.7
	5	18.8	13.8
	6	17.6	12.9
1000	1	21.6		-16	-21
	2	23.2		-24
	3	22.8		-22

Oxygen Uptake Rates, Specific Respiration Rates and Inhibition of the Respiration
of the Control and the Reference Item Concentrations

Reference Item Concentration [mg/L]	Repl.	Oxygen Uptake Rate R [mg O2/L×h]	Specific Respiration Rate Rs [mg O2/g×h]	Inhibition [%]	Mean Inhibition [%]
Control	1	20.0	14.7	–	–
	2	18.8	13.8
	3	17.6	12.9
	4	18.4	13.5
	5	17.2	12.6
	6	16.0	11.8
58	1	12.8		29	24
	2	14.0		22
	3	14.0		22
100	1	9.2		49	50
	2	8.4		53
	3	9.2		49
180	1	3.6		80	79
	2	4.0		78
	3	4.0		78

Validity criteria fulfilled:

yes

Conclusions:

The NOEC of the test item (Glycerides, C16-18 mono-, di-, and tri-, hydrogenated, citrates, potassium salts) is 1000 mg/L.
The test item is not toxic at the limit concentration of 1000 mg/L to activated sludge of a municipal sewage treatment plant.

Executive summary:

A Respiration Inhibition Test with activated sludge according to OECD Guideline No. 209 was carried out with the test item (Glycerides, C16-18 mono-, di-, and tri-, hydrogenated, citrates, potassium salts) (batch number: 105618) from 2012-06-11 to 2012-06-12, with the definitive experimental phase on 2012-06-12 at the Dr. U. Noack-Laboratorien, 31157 Sarstedt, Germany. Test system was activated sludge of the municipal treatment plant of 31137 Hildesheim, Germany. The test was carried out under static conditions with the limit test item concentration of 1000 mg/L. The respiration rates of the control, reference and test item replicates were measured after a contact time of three hours, and the inhibitory effects of the test and reference item were determined in comparison to the control respiration rates. The mean inhibition of respiration for the test item replicates ranged from -16 % to -22 %.

In order to check the activity of the test system and the test conditions a reference test was carried out with copper (II) sulphate pentahydrate as reference item and the reference toxicity was determined. The EC₅₀-value for the reference item was 99.1 mg/L.

Table 1: NOEC-Value of Glyceryl StearateCitrate (Glycerides, C16-18 mono-, di-, and tri-, hydrogenated, citrates, potassium salts)

		Test Item Concentration [mg/L]
NOEC		1000

                                              

The NOEC of the test item Glyceryl Stearate Citrate (Glycerides, C16-18 mono-, di-, and tri-, hydrogenated, citrates, potassium salts) is 1000 mg/L. The test item is not toxic at the limit concentration of 1000 mg/L to activated sludge of a municipal sewage treatment plant.

Description of key information

The NOEC of the test item (Glycerides, C16-18 mono-, di-, and tri-, hydrogenated, citrates, potassium salts) is 1000 mg/l.
The test item is not toxic at the limit concentration of 1000 mg/l to activated sludge of a municipal sewage treatment plant.

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L

EC10 or NOEC for microorganisms:

1 000 mg/L

Additional information

A Respiration Inhibition Test with activated sludge according to OECD Guideline No. 209 was carried out with the test item (Glycerides, C16-18 mono-, di-, and tri-, hydrogenated, citrates, potassium salts) (batch number: 105618) from 2012-06-11 to 2012-06-12, with the definitive experimental phase on 2012-06-12 at the Dr. U. Noack-Laboratorien, 31157 Sarstedt, Germany. Test system was activated sludge of the municipal treatment plant of 31137 Hildesheim, Germany. The test was carried out under static conditions with the limit test item concentration of 1000 mg/l. The respiration rates of the control, reference and test item replicates were measured after a contact time of three hours, and the inhibitory effects of the test and reference item were determined in comparison to the control respiration rates. The mean inhibition of respiration for the test item replicates ranged from -16 % to -22 %.

In order to check the activity of the test system and the test conditions a reference test was carried out with copper (II) sulphate pentahydrate as reference item and the reference toxicity was determined. The EC₅₀-value for the reference item was 99.1 mg/L.

The NOEC of the test item (Glycerides, C16-18 mono-, di-, and tri-, hydrogenated, citrates, potassium salts) is 1000 mg/L.

The test item is not toxic at the limit concentration of 1000 mg/L to activated sludge of a municipal sewage treatment plant.