N-{3-[C12-14-(even numbered)-alkyloxy]propyl}propane-1,3-diamine

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:

from 2021-08-09 to 2021-08-10 at the test facility

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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 5.62.
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.
The control was tested with and without ATU. Three replicates each
were tested, three at the beginning of the measurements and three
at the end.
The reference item Copper (II)-sulfate pentahydrate was tested with
three concentrations within the concentration range 58 - 180 mg/L
using 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
using 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: Municipal sewage treatment plant of 31137 Hildesheim, Germany
Non-adapted activated sludge from the sewage plant at Hildesheim is well suited as it receives predominantly municipal sewage and hardly any industrial chemical waste.
- Receipt: 2021-08-09
- Pretreatment: The sludge was washed twice and adjusted with chlorine free tap water if necessary. The sludge was used within 24 h after sampling.
- Dry sludge concentration: 3.03 g/L, corresponding to 1.52 g/L in the test vessels for replicates without ATU and 3.00 g/L, corresponding to 1.50 g/L in the test vessels for replicates with ATU.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Hardness:

Not determined.

Test temperature:

20.9 °C without ATU, 20.9 °C with ATU

pH:

pH-value of the activated sludge for test without ATU 7.34
pH-value of the activated sludge for test with ATU 7.35
pH-value of the synthetic waste water 7.20

Salinity:

Not determined.

Details on test conditions:

TEST SYSTEM
- Test vessel: 5
- 1000 mL laboratory bottles with screw cap, ISO 4796
- Aeration: 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.
- 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 and 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 1 - 5.62 - 31.6 - 178 - 1000 mg/L
The test item was weighed out on bent object slides.

Test item concentration [mg/L] 1 5.62 31.6 178 1000
Test item [mg] 0.5 2.8 15.8 89 500
Synthetic waste water [mL] 16
Ultrapure water [mL] Filled up to 250 mL
Inoculum [mL] 250


Test item concentration [mg/L] 1 5.62 31.6 178 1000
Test item [mg] 0.5 2.8 15.8 89 500
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
 

The reference item concentration (total respiration) was diluted out of a stock solution.
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
Demineralized water [mL] Filled up to 250 mL
Inoculum [mL] 250

*) Concentration stock solution: 300 g/L


The reference item concentration (nitrification) was diluted out of a stock solution.

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
Demineralized water [mL] Filled up to 250 mL
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
Demineralized water [mL] Filled up to 250 mL
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) :
The oxygen concentration in the measurement chamber was measured by the oximeter by optical read-out of the sensor spots at least every 5 seconds. The data was recorded using the software PreSens Measurement Studio 2. For each replicate, the oxygen uptake rate was calculated for a period of at least 3 minutes. A linear section was chosen for this calculation.

TEST CONCENTRATIONS

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

In the preliminary range finding test, additional replicates were prepared to determine the inhibition of nitrification. One replicate each of 10 – 100 - 1000 mg/L was measured with the addition of Allylthiourea (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 - 16 2 26
100 - 75 70 78
1000 8.20 99 98 100

Since there is inhibition of nitrification with certain difference from heterotrophic respiration, the test was need to be performed with the determination of both endpoints.

Reference substance (positive control):

yes

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

5.62 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

5.62 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of nitrification rate

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

31.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of heterotrophic respiration

Key result

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

21.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of nitrification rate

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

33.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

116 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of heterotrophic respiration

Duration:

3 h

Dose descriptor:

other: EC20

Effect conc.:

45.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Duration:

3 h

Dose descriptor:

other: EC20

Effect conc.:

127 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of heterotrophic respiration

Duration:

3 h

Dose descriptor:

other: EC20

Effect conc.:

31.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of nitrification rate

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

48.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of nitrification rate

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

151 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of heterotrophic respiration

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

77 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Results with reference substance (positive control):

- Results with reference substance valid? Yes

Reported statistics and error estimates:

The EC-values of the test item and the reference item were calculated by sigmoidal dose-response regression and the EC50-value of the reference item by linear regression using software GraphPadPrism. Calculation of the confidence intervals for the EC50-value was 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.

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 for Total Respiration and Nitrification and Multiple Sequentially-rejective Median (2x2-Table) Test After Bonferroni-Holm for Heterotrophic Respiration). Prior to running the multiple test, a normality test and a variance homogeneity test were performed.
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

Oxygen Uptake Rates, Specific Respiration Rates and Inhibition of the Respiration

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 to the oxygen uptake rates of the control. The results are listed in the tables below.

 

Oxygen Uptake Rates and Specific Respiration Rates of the Total Respiration of the Control

	Repl.	Oxygen	Oxygen Uptake Rate RT	Specific Respiration Rate Rs
		Concentration*
		[mg O2/L]	[mg O2/(L×h)]	[mg O2/g×h]
Control	1	5.84	33.5	22
	2	5.85	33.6	22.1
	3	6.41	33.4	22
	4	6.88	31.7	20.9
	5	6.79	32.5	21.4
	6	6.72	30.3	19.9
	Mean value		32.5	21.4
	Standard deviation		1.19
	CV [%]		3.66

*) 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	Repl.	Oxygen	Oxygen Uptake Rate RT	Inhibition	Mean Inhibition
		Concentration*
[mg/L]		[mg O2/L]	[mg O2/(L×h)]	[%]	[%]
1	1	6.43	34	-5	-3
	2	6.03	32.2	1
	3	6.15	33.2	-2
	4	6.79	33	-2
	5	6.61	34.5	-6
5.62	1	5.86	25.1	23	3
	2	5.36	32.2	1
	3	6.09	33.4	-3
	4	6.51	34	-5
	5	6.32	33.1	-2
31.6	1	5.77	30.2	7	8
	2	5.19	29.1	10
	3	5.78	29.1	10
	4	5.88	30.3	7
	5	5.39	30	8
178	1	8.24	3.8	88	89
	2	8.11	3.9	88
	3	8.2	4.1	87
	4	8.24	3.7	89
	5	8.29	2.4	93
1000	1	8.77	0.7	98	98
	2	8.72	0.9	97
	3	8.62	0.2	99
	4	8.7	0.9	97
	5	8.67	0.8	98

*) determined at start of the measurement

Repl. = Replicate

 

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

	Repl.	Oxygen	Oxygen Uptake Rate RH
		Concentration*
		[mg O2/L]	[mg O2/(L×h)]
Control	1	7.56	14.9
	2	7.32	14.4
	3	7.48	14.9
	4	7.86	14.5
	5	7.44	14.6
	6	7.38	13.7
	Mean value		14.5
	Standard deviation		0.4
	CV [%]		2.76

*) determined at start of the measurement

CV = Coefficient of variation

 

Oxygen Uptake Rates and Inhibition of the Heterotrophic Respiration

Test Item	Repl.	Oxygen	Oxygen Uptake Rate RH	Inhibition	Mean Inhibition
Concentration		Concentration*	[mg O2/L×h]		[%]
[mg/L]		[mg O2/L]		[%]
1	1	7.25	15.3	-6	-7
	2	7.06	15.1	-4
	3	7.39	15.3	-6
	4	7.38	16.4	-13
	5	7.28	15.7	-8
5.62	1	7.14	16.1	-11	-5
	2	6.92	15.4	-6
	3	7.2	15.2	-5
	4	7.25	14.5	0
	5	6.96	15	-3
31.6	1	7.75	21.1	-46	-7
	2	7.05	14.5	0
	3	7.12	14.1	3
	4	7.28	13.7	6
	5	7.35	14	3
178	1	8.01	8.3	43	74
	2	8.22	2.8	81
	3	8.26	2.3	84
	4	8.24	2.4	83
	5	8.25	2.7	81
1000	1	8.74	0.9	94	93
	2	8.64	0.8	94
	3	8.64	1.2	92
	4	8.63	1	93
	5	8.71	0.9	94

*) determined at start of the measurement

 

Calculated Oxygen Uptake Rates of the Nitrification of the Control

	Repl.	Oxygen Uptake Rate RN
		[mg O2/(L×h)]
Control	1	19
	2	19.1
	3	18.9
	4	17.2
	5	18
	6	15.8
Mean value		18
Standard deviation		1.3
CV [%]		7.2

CV = Coefficient of variation

 

Oxygen Uptake Rates and Inhibition of the Nitrification

Test Item	Rep	Oxygen Uptake Rate RN	Inhibition	Mean Inhibition
Concentration		[mg O2/L×h]		[%]
[mg/L]			[%]
1	1	18.4	-2	1
	2	16.6	8
	3	17.6	2
	4	17.4	3
	5	18.9	-5
5.62	1	9.9	45	9
	2	17	6
	3	18.2	-1
	4	18.8	-4
	5	17.9	1
31.6	1	14.7	18	21
	2	13.6	24
	3	13.6	24
	4	14.8	18
	5	14.5	19
178	1	0.1	99	100
	2	0.2	99
	3	0.4	98
	4	0	100
	5	-1.3	100
1000	1	-0.3	100	100
	2	-0.1	100
	3	-0.8	100
	4	-0.1	100
	5	-0.2	100

Validity criteria fulfilled:

yes

Conclusions:

The NOEC of C12 – C14 alkyletherdiamine (CAS No.: 68187-46-2) for Total Respiration is 5.62 mg/L.
The EC10 for Total Respiration is 33.8 mg/L and the EC50 is 77.0 mg/L.

The NOEC for heterotrophic respiration is 31.6 mg/L.
The EC10 for heterotrophic respiration is 116 mg/L and the EC50 is 151 mg/L.

The NOEC for nitrification is 5.62 mg/L.
The EC10 for nitrification is 21.8 mg/L and the EC50 is 48.8 mg/L.

Executive summary:

A respiration inhibition test with activated sludge according to OECD Guideline No. 209 was carried out withthe test item C12 – C14 alkyletherdiamine (CAS No.: 68187-46-2) (batch number: S20210420) from 2021-08-09 to 2021-08-10 at the test facility. The test system was activated sludge of the municipal treatment plant of 31137 Hildesheim, Germany. Based on the results of the preliminary test, the study was carried out under static conditions with the test item concentrations 1 - 5.62 - 31.6 - 178 - 1000 mg/L with and without N-Allylthiourea. 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 -3 % to 98 %. The mean inhibition of the heterotrophic respiration for the test item replicates ranged from -7 % to 93 %. The mean inhibition of nitrification for the test item replicates ranged from 1 % to 100 %.

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 105 mg/L, which is in agreement with the criteria. The EC50-value for the reference item N-Methylaniline was 1.73 mg/L.

 

 Endpoints of C12 – C14 alkyletherdiamine (CAS No.: 68187-46-2) for Inhibition of Respiration in [mg Test item/L]

	Total Respiration		Heterotrophic Respiration		Nitrification
	EC-values	CI	EC-values	CI	EC-values	CI
		P = 95 [%]		P = 95 [%]		P = 95 [%]
	[mg/L]	[mg/L]	[mg/L]	[mg/L]	[mg/L]	[mg/L]
EC10	33.8	27.3 – 44.7	116	33.2 – 490	21.8	< 1 – 30.3
EC20	45.7	37.7 – 57.8	127	35.4 – 500	31.2	19.8 – 86.2
EC50	77	63.3 – 93.7	151	39.3 – 544	48.8	36.8 – 106

 

Description of key information

The results from one test performed according to the OECD guideline 209 is available. The lowest 3h- EC50 could be determined from the results to be 48.8 mg/L for nitrification and the 3h-EC10 value was 21.8 mg/L.  

Key value for chemical safety assessment

EC50 for microorganisms:

48.8 mg/L

EC10 or NOEC for microorganisms:

21.8 mg/L