cyclic C12/14-Glucamide;cyclic N-methyl-N-dodecanoyl/tetradecanoyl-glucamine;anhydro-N-dodecanoyl/tetradecanoyl-N-methylglucamine;Amides, C12-C14, N-(1-deoxy-D-glucitol-1-yl)-N-methyl, dehydrated

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:

2021-02-01 to 2021-02-02

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

Specific details on test material used for the study:

Test item cyclic Glucamide C12-14
Batch number S148866
Purity (certified) 100 % (UVCB)
Active ingredient cyclic N-methyl-N-dodecanoyl/tetradecanoyl-glucamines incl. isomers
pH 9-10
Sum formula main compounds: C19H37NO5 (C12-acyl-derivative); C21H41NO5 (C14-acyl-derivative)
Water solubility soluble/miscible
Appearance slightly turbid brown solid mass / gel
Expiry date 2022-09-30
Recommended storage Room temperature, dry

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION

Based on the results of the preliminary test the definitive study was carried out with 5 concentrations within the range of 1 to 1000 mg/L (with and without ATU) in a geometrical series and with a dilution factor of approx. 5.6.

The test item concentrations were weighed out on bent object slides and were transferred in the test vessels. All concentrations were stirred overnight in 200 mL ultrapure water at 400 rpm.

Triplicates of the control (without ATU) without test item were included at the beginning and at the end of the replicates without ATU.

Triplicates of the control (with ATU) without test item were included at the beginning of the replicates with ATU and at the end of the test.

The reference item Copper (II)-sulfate pentahydrate was tested with three concentrations within the concentration range 58 - 180 mg/L and a dilution factor of approximately 1.8 (without ATU).

The reference item N-Methylaniline was tested with three concentrations within the concentration range 0.1 – 1.8 – 32 mg/L and a dilution factor of 18 (with and without ATU).

A fresh sample of activated sludge taken from the sewage plant Hildesheim was used for the test procedure. It was washed twice and adjusted with chlorine free tap water to a dry sludge concentration of 3.0 g/L ± 10 %.

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)
- Receipt: 2021-02-01
- Pretreatment:The sludge was washed twice with chlorine free tap water and adjusted to a dry sludge concentration of 3.0 g/L ± 10 %. The sludge was used within 24 h after sampling.
- Dry sludge concentration: 3.11 g/L (corresponding to 1.56 g/L in the test vessels) without ATU
3.10 g/L (corresponding to 1.55 g/L in the test vessels) with ATU

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Hardness:

Not determined

Test temperature:

21.4 °C without ATU
21.1 °C with ATU

pH:

pH-value of the activated sludge for test without ATU 7.51
pH-value of the activated sludge for test with ATU 7.53
pH-value of the synthetic waste water 7.19

Salinity:

Not determined

Details on test conditions:

TEST SYSTEM
- Test vessel: 5
- 1000 mL laboratory bottles with screw cap, ISO 4796
- Shaking of the laboratory bottles at 150 rpm to keep the dissolved oxygen concentration above 60 - 70 % saturation and to maintain the sludge flakes in suspension. The flasks were closed with screw caps.
- Pretreatment: Stirring overnight in 200 mL ultrapure water at 400 rpm

- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 6 (Triplicates at the beginning of the test + triplicates at the end of the test.)
- No. of vessels per reference item Copper-II-Sulfat (replicates): 9 (three concentrations within the range 58 - 180 mg/L and a dilution factor 1.8)
- No. of vessels per reference item N-Methylaniline (replicates): 9 (three concentrations within the range 0.1 - 32 mg/L and a dilution factor 18)
- Composition of test medium:

Test concentrations [mg/L] 1 - 5.6 - 32 - 180 - 1000 mg/L
The test item was weighed out on bent object slides.

Test item concentration [mg/L] 1 5.6 32 180 1000
Test item [mg] 0.5 2.8 16.0 90 500
Synthetic waste water [mL] 16
Ultrapure water [mL] Filled up to 250 mL
Inoculum [mL] 250


Preparation Table for the Test Item with ATU
Test item concentration [mg/L] 1 5.6 32 180 1000
Test item [mg] 0.5 2.8 16.0 90 500
Synthetic waste water [mL] 16
ATU solution [mL]* 2.5
Ultrapure water [mL] Filled up to 250 mL
Inoculum [mL] 250
*) Concentration of the ATU solution: 2.32 g/L


Dilution Table for the Reference Item (Total Respiration)
Reference item concentration [mg/L] 58 100 180
Stock solution [µL]* 96.7 166.7 300
Synthetic waste water [mL] 16
Demineralised water [mL] Filled up to 250
Inoculum [mL] 250
* Stock solution with 300 g/L

Dilution Table for the Reference Item (Nitrification) without ATU
Reference item concentration [mg/L] 0.1 1.8 32
Stock solution [mL]* 0.05 0.9 16
Synthetic waste water [mL] 16
Ultrapure water [mL] Filled up to 250
Inoculum [mL] 250
*) Concentration stock solution: 1 g/L

Dilution Table for the Reference Item (Nitrification) with ATU
Reference item concentration [mg/L] 0.1 1.8 32
Stock solution [mL]* 0.05 0.9 16
Synthetic waste water [mL] 16
ATU solution [mL]** 2.5
Ultrapure water [mL] Filled up to 250
Inoculum [mL] 250

*) Concentration stock solution: 1 g/L
**) Concentration of the ATU solution: 2.32 g/L


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 every 5 seconds after transferring the solution to the measuring chamber and the oxygen depletion was recorded for at least 5 minutes with the software PreSens Measurement Studio 2.

TEST CONCENTRATIONS

Inhibitions in the Preliminary Tests
A preliminary range finding test was carried out with the test item concentrations of 10 – 100 – 1000 mg test item/L with one replicate each. The pH was measured in the replicate with 1000 mg test item/L. The flasks were aerated for 3 hours.

In the preliminary test, additional replicates were prepared to determine the inhibition of nitrification. One replicate each of 10 – 100 – 1000 mg test item/L were measured with addition of Allylthiourea (ATU).

Inhibitions in the Preliminary Test (with / without ATU)
Nominal Test Item Concentration
[mg/L] pH
(test item in medium) Inhibition of total respiration (without ATU)
[%] Inhibition of heterotrophic respiration (with ATU)
[%] Inhibition of nitrification
[%]
10 - 32 -7 70
100 - 62 63 62
1000 7.46 77 76 78

Since a clear difference between the inhibition of nitrification and the inhibition of heterotrophic respiration was observed at a concentration of 10 mg/L, the test was need to be performed with the determination of both endpoints.

A fresh sample of activated sludge taken from the sewage plant Hildesheim was used for the test procedure. It was washed twice and adjusted with chlorine free tap water to a dry sludge concentration of 3.0 g/L ± 10 %.

The oxygen uptake rates of all samples were determined following an incubation period of 3 h.

Reference substance (positive control):

yes

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

5.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of heterotrophic respiration

Remarks:

respiration rate

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

5.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of nitrification rate

Remarks:

respiration rate

Key result

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

19.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Key result

Duration:

3 h

Dose descriptor:

other: EC20

Effect conc.:

27.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

57.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Key result

Duration:

3 h

Dose descriptor:

other: EC80

Effect conc.:

162 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration tate

Key result

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

123 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of heterotrophic respiration

Remarks:

respiration rate

Key result

Duration:

3 h

Dose descriptor:

other: EC20

Effect conc.:

131 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of heterotrophic respiration

Remarks:

respiration rate

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

153 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of heterotrophic respiration

Remarks:

respiration rate

Key result

Duration:

3 h

Dose descriptor:

other: EC80

Effect conc.:

188 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of heterotrophic respiration

Remarks:

respiration rate

Key result

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

7.83 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of nitrification rate

Remarks:

respiration rate

Key result

Duration:

3 h

Dose descriptor:

other: EC20

Effect conc.:

10.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of nitrification rate

Remarks:

respiration rate

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

21.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of nitrification rate

Remarks:

respiration rate

Key result

Duration:

3 h

Dose descriptor:

other: EC80

Effect conc.:

63.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of nitrification rate

Remarks:

respiration rate

Results with reference substance (positive control):

- Results with reference substance valid? Yes

Reported statistics and error estimates:

The NOEC for the oxygen uptake rate was determined with the software ToxRat Professional as specified below.
The NOEC was determined by calculation of statistical significance of the oxygen uptake rate in comparison to the control using statistical standard procedure as normality test (Shapiro-Wilk), variance homogeneity test (Levene’s test) and a multiple test (Williams Multiple Sequential t-test).
The P-value for all tests was 0.01. The a-value (acceptable probability of incorrectly concluding that there is a difference) is a=0.05

The EC-values of the test item and the reference item were calculated by sigmoidal dose-response regression and linear regression, respectively, using software GraphPad Prism. Calculations of the confidence intervals for the EC50-values were carried out using standard procedures. The coefficient of variation of the oxygen uptake rates of the control replicates was calculated with standard methods using Excel.

The calculation of the oxygen uptake rates (R) and inhibition of respiration rates (I) was performed according to the guideline.

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 and Specific Respiration Rates of the Total Respiration of the Control

	Repl.	Oxygen  Concentration*	Oxygen Uptake Rate RT	Specific Respiration Rate Rs
		[mg O2/L]	[mg O2/(L×h)]	[mg O2/g×h]
Control	1	5.3	30.4	19.5
	2	5.5	30.4	19.5
	3	5.6	29.4	18.9
	4	6.0	30.1	19.4
	5	5.7	29.1	18.7
	6	6.1	28.7	18.5
	Mean value		29.7	19.1
	Standard deviation		0.7
	CV [%]		2.3

*) determined at start of the measurement

Repl. = Replicate ; CV = Coefficient of variation

 

Oxygen Uptake Rates and Inhibition of the Total Respiration of the Test Item Concentrations

 

Test Item Concentration	Rep	Oxygen  Concentration*	Oxygen Uptake Rate RT	Inhibition	Mean Inhibition
[mg/L]		[mg O2/L]	[mg O2/(L×h)]	[%]	[%]
1	1	6.74	32.2	-8	-7
	2	5.93	31.1	-5
	3	6.44	30.7	-3
	4	6.69	31.1	-5
	5	7.29	33.9	-14
5.6	1	5.95	30.8	-4	-3
	2	5.56	29.4	1
	3	5.86	31.8	-7
	4	6.03	30.1	-1
	5	5.23	30.7	-3
32	1	5.38	25.1	15	26
	2	5.65	20.4	31
	3	6.15	21.8	27
	4	5.83	20.9	30
	5	5.64	22.1	26
180	1	7.95	3.7	88	82
	2	7.82	5.7	81
	3	7.63	5.6	81
	4	8.04	5.3	82
	5	7.54	6.9	77
1000	1	8.20	3.4	89	89
	2	8.13	3.3	89
	3	8.10	3.5	88
	4	8.18	3.0	90
	5	8.31	3.7	88

*) determined at start of the measurement

Repl. = Replicate

 

 Oxygen Uptake Rates of the Heterotrophic Respiration of the Control Replicates with ATU

	Rep	Oxygen  Concentration*	Oxygen Uptake Rate RH
		[mg O2/L]	[mg O2/(L×h)]
Control	1	6.86	15.4
	2	6.88	16.1
	3	6.85	15.1
	4	7.37	15.6
	5	7.03	15.6
	6	7.41	15.0
	Mean value		15.5
	Standard deviation		0.4
	CV [%]		2.3

*) determined at start of the measurement

CV = Coefficient of variation

 

Oxygen Uptake Rates and Inhibition of the Heterotrophic Respiration of the Test Item Concentrations

Test Item Concentration [mg/L]	Rep	Oxygen  Concentration* [mg O2/L]	Oxygen Uptake Rate RH [mg O2/L×h]	Inhibition [%]	Mean Inhibition [%]
1	1	6.70	15.9	-3	-7
	2	6.47	16.6	-7
	3	6.84	16.4	-6
	4	6.87	15.9	-3
	5	7.25	17.8	-15
5.6	1	6.11	17.4	-12	-8
	2	6.06	16.7	-8
	3	6.21	15.0	3
	4	6.31	17.4	-12
	5	6.44	17.5	-13
32	1	6.59	17.0	-10	-9
	2	6.41	17.1	-10
	3	6.58	16.8	-8
	4	6.70	16.8	-8
	5	6.71	17.2	-11
180	1	8.19	3.9	75	76
	2	8.08	4.1	74
	3	8.22	3.5	77
	4	8.22	3.4	78
	5	8.31	3.7	76
1000	1	8.49	1.9	88	90
	2	8.44	1.9	88
	3	8.35	1.9	88
	4	8.44	1.6	90
	5	8.53	0.5	97

*) determined at start of the measurement

 

Calculated Oxygen Uptake Rates of the Nitrification of the Control

	Rep	Oxygen Uptake Rate RN
		[mg O2/(L×h)]
Control	1	15.0
	2	14.3
	3	14.3
	4	14.5
	5	13.5
	6	13.7
Mean value		14.2
Standard deviation		0.5
CV [%]		3.5

CV = Coefficient of variation

 

 

Oxygen Uptake Rates and Inhibition of the Nitrification of the Test Item Concentrations

Test Item Concentration [mg/L]	Rep	Oxygen Uptake Rate RN [mg O2/L×h]	Inhibition [%]	Mean Inhibition [%]
1	1	15.7	-10	-8
	2	14.6	-3
	3	14.2	0
	4	14.6	-3
	5	17.4	-22
5.6	1	14.0	1	3
	2	12.6	11
	3	15.0	-6
	4	13.3	6
	5	13.9	2
32	1	8.1	43	64
	2	3.4	76
	3	4.8	66
	4	3.9	72
	5	5.1	64
180	1	0.0	100	88
	2	2.0	86
	3	1.9	87
	4	1.6	89
	5	3.2	78
1000	1	1.8	87	87
	2	1.7	88
	3	1.9	86
	4	1.4	90
	5	2.1	85

Oxygen Uptake Rates, Inhibition of the Total Respiration of the Reference Item Copper (II) sulphate pentahydrate Concentrations

Reference Item Concentration	Repl.	Oxygen  Concentration*	Oxygen Uptake Rate RT	Inhibition	Mean Inhibition
[mg/L]		[mg O2/L]	[mg O2/(L×h)]	[%]	[%]
58	1	6.57	19.9	33	36
	2	5.79	18.9	36
	3	6.22	17.7	40
100	1	6.20	15.3	48	51
	2	6.38	15.1	49
	3	6.51	13.1	56
180	1	7.50	5.4	82	80
	2	7.54	6.4	78
	3	7.55	5.7	81

*) determined at start of the measurement

Repl. = Replicate

 

 

 Oxygen Uptake Rates, Inhibition of the Nitrification of the Reference Item N-Methylaniline Concentrations

Reference Item Concentration	Repl.	Oxygen Uptake Rate RN	Inhibition	Mean Inhibition
[mg/L]		[mg O2/(L×h)]	[%]	[%]
0.1	1	11.3	20	25
	2	11.6	18
	3	8.9	37
1.8	1	6.3	55	49
	2	8.9	37
	3	6.5	54
32	1	-1.3	100	100
	2	-0.9	100
	3	-0.6	100

Repl. = Replicate

 

 

Validity criteria fulfilled:

yes

Conclusions:

The NOEC of cyclic Glucamide C12-14 for Total Respiration is 5.6 mg/L.
The EC10 for Total Respiration is 19.1 mg/L and the EC50 is 57.2 mg/L.
The NOEC for Heterotrophic respiration is 32 mg/L.
The EC10 for Heterotrophic respiration is 123 mg/L and the EC50 is 153 mg/L.
The NOEC for Nitrification is 5.6 mg/L.
The EC10 for nitrification is 7.83 mg/L and the EC50 is 21.9 mg/L.

Executive summary:

A respiration inhibition test with activated sludge according to OECD Guideline No. 209 was carried out with the test item cyclic Glucamide C12-14 (batch number: S148866) from 2021-02-01 to 2021-02-02 at the test facility. The test system was activated sludge of the municipal treatment plant of 31137 Hildesheim, Germany. The test was carried out under static conditions with the test item concentrations 1 - 5.6 - 32 - 180 - 1000 mg/L with and without ATU. 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 total respiration for the test item replicates ranged from -7 % to 89 %. The mean inhibition of the heterotrophic respiration for the test item replicates ranged from -9 % to 90 %. The mean inhibition of nitrification for the test item replicates ranged from -8 % to 88 %.

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 and N-Methylaniline (with and without ATU) as reference item and the reference toxicity was determined. The EC50-value for the reference item copper (II) sulphate pentahydrate was 87.2 mg/L. The EC50-value for the reference item N-Methylaniline was 0.98 mg/L.

 Endpoints of cyclic Glucamide C12-14 for Inhibition of Respiration

	Total Respiration		Heterotrophic Respiration		Nitrification
	EC-values [mg/L]	CI  P = 95 [%] [mg/L]	EC-values [mg/L]	CI  P = 95 [%] [mg/L]	EC-values [mg/L]	CI  P = 95 [%] [mg/L]
NOEC	5.6	–	32	–	5.6	–
EC10	19.1	16.1 – 22.2	123	38.3 – 180	7.83	5.55 – 11.3
EC20	27.1	24.0 – 30.1	131	40.3 – 180	10.7	7.80 – 14.4
EC50	57.2	50.6 – 66.6	153	44.1 – 180	21.9	17.2 – 26.9
EC80	162	122 – 210	188	46.4 – 589	63.7	44.1 – 110

 

Description of key information

The NOEC of cyclic Glucamide C12-14 for Total Respiration is 5.6 mg/L.
The EC10 for Total Respiration is 19.1 mg/L and the EC50 is 57.2 mg/L.
The NOEC for Heterotrophic respiration is 32 mg/L.
The EC10 for Heterotrophic respiration is 123 mg/L and the EC50 is 153 mg/L.
The NOEC for Nitrification is 5.6 mg/L.
The EC10 for nitrification is 7.83 mg/L and the EC50 is 21.9 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:

57.2 mg/L

EC10 or NOEC for microorganisms:

19.1 mg/L