N,N'-bis(3-aminopropyl)ethylenediamine

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:

15 Feb 2012 to 16 Feb 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

Application of test substance: Direct addition by initial weight into the test flasks. The test substance was added in the required amounts according to the test concentrations directly to the test vessels with about 234 mL demineralized water.

Reference substance: 500.0 mg of the reference substance was added to about 800 mL of demineralized water and stirred at room temperature until the reference substance was completely dissolved. The pH value of the stock solution was measured and adjusted to 7.2 with 1 M sodium hydroxide solution. Following this the stock solution was made up to 1L with demineralized water. The stock solution appeared yellowish and no undissolved reference substance was visible.

Aliquots of the reference substance stock solution were dosed to the test vessels and made up with demineralized water to a volume 234 mL. 16 mL synthetic medium were dosed to each test vessel with test substance and reference substance afterwards. To prepare the blank control assays 234 mL of demineralized water and 16 mL synthetic medium were mixed. The pH-values in all test vessels were checked and adjusted.

After addition of 250 mL of inoculum suspension the incubation was started by aeration of the test vessels with pressured air. The vessels for the blank control assays were prepared to the same procedure without addition of test- or reference substance.

Test organisms (species):

activated sludge, domestic

Details on inoculum:

Activated sludge from the municipal wastewater treatment plant of Mannheim, Germany was collected on 15 Feb 2012 from the aeration tank of the plant. The activated sludge suspension was sieved with a fine woven mesh (mesh size about 1 mm). This suspension was pre-aerated over night at room temperature. At the next day the sludge suspension was washed once with tap water and the suspension was adjusted to 3 g/L dry matter.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

180 min

Test temperature:

19.8 to 20.3 °C

pH:

blanc and reference assays: pH 7.1 to 7.2
test item assays: initial: pH 8.5 to 10.8; adjusted: pH 7.2; after incubation: pH 7.7 to 8.2

Dissolved oxygen:

The content of oxygen at the start of the measurements was > 7 mg/L.

Nominal and measured concentrations:

1000, 500, 250, 125, 62.5 mg/L as nominal concentration based on test substance without correction of purity or composition and blank controls.

Details on test conditions:

TEST SYSTEM
- Test vessel: 500 mL glass beakers (nominal volume 1 L)
- No. of vessels per blank control (replicates): 4
- No. of vessels per reference substance concentration (replicates): 2
- No. of vessels per test substance concentration (replicates): 2
- Oxygen concentration during aeration: > 2 mg/L
- Oxygen concentration immediately before measurement: > 7.0 mg/L
- Duration of the measurement of oxygenconsumption: 5 - 10 min
- Sludge concentration in the test vessel: 1.5 g/L
- Synthetic medium: 16 mL/test vessel of 100-fold concentrated OECD medium

After 3 hours incubation at 20 ± 2°C the mixtures in the test vessels were placed subsequently for oxygen measuring. The total oxygen consumption of the two blank controls (BC1+2) were measured at first. The total oxygen consumption of the reference substance assays (RS) were measured in the sequence RS1+2, RS3+4 and RS5+6. Then the total oxygen consumption of the test substance assays were measured in the sequence TS1+2, TS3+4, TS5+6, TS7+8 and TS9+10. The oxygen consumption of the blank control BC3+4 were measured at last. No abiotic control was tested. The oxygen uptake was measured for a period of 5 to 10 minutes or until the oxygen concentration fell below 2 mg/L.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol (1, 10, 100 mg/L)

Duration:

180 min

Dose descriptor:

EC10

Effect conc.:

34 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: 95% CL (lower/upper): 10.7/110

Duration:

180 min

Dose descriptor:

other: EC20

Effect conc.:

98 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: 95% CL (lower/upper): 47.1/201.7

Duration:

180 min

Dose descriptor:

EC50

Effect conc.:

720 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: 95% CL (lower/upper): 415.2/1245.9

Duration:

180 min

Dose descriptor:

other: EC80

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: no CL95%

Details on results:

- Effect concentrations exceeding solubility of substance in test medium: no

Results with reference substance (positive control):

The EC50 of 3,5-dichlorophenol was 6.5 mg/L. These results indicate that the microorganisms are responding normally to toxicant stress.

Reported statistics and error estimates:

The consumption rate (mg O2/L*6 minutes) of each test concentration and the blank controls were determined after 3 hours of exposure. The consumption rates (mg O2/L*h) were used to calculate the inhibition effects at particular test concentrations compared to the mean consumption rate of all controls.
The coefficient of variation of the four replicates of blank control was 17.8 % O2 consumption.

The consumption rates were used for the determination of the ECx by the probit method based on Finney [1] with the software TOXRAT Professional 2.10 [2]. The effect concentrations were given with an accuracy of one respectively two significant digits.

Results for controls and reference substance

Oxygen consumption

Treatment	BC1	BC2	BC3	BC4	RS1	RS2	RS3	RS4	RS5	RS6
Concentration of test substance	-	-	-	-	1		10		100
(mg/L)
O2 concentration at test start	7.8	7.9	6.5	7.4	6.8	7.4	7.4	7.6	8.3	8.0
(mg O2/L)
O2 concentration after 6 min	4.0	4.0	2.5	2.5	3.0	3.5	6.0	6.0	8.0	7.7
(mg O2/L)
O2 consumption rate	38	39	40	49	38	39	14	16	3	3
(mg O2/L*h)
Specific O2 consumption rate	25	26	27	33	25	26	9	11	2	2
(mg O2/g*h)
Inhibition of respiration	-	-	-	-	8	6	66	61	93	93
compared to control (%)

Results for test substance

Oxygen consumption

Treatment	TS1	TS2	TS3	TS4	TS5	TS6	TS7	TS8	TS9	TS10
Concentration of test substance	62.5		125		250		500		1000
(mg/L)
O2 concentration at test start	7.2	6.9	7.3	7.7	6.4	7.2	7.2	7.3	6.8	7.3
(mg O2/L)
O2 concentration after 6 min	3.5	2.5	4.5	4.5	4.0	4.5	5.0	5.0	5.0	5.0
(mg O2/L)
O2 consumption rate	37	44	28	32	24	27	22	23	18	23
(mg O2/L*h)
Specific O2 consumption rate	25	29	19	21	16	18	15	15	12	15
(mg O2/g*h)
Inhibition of respiration	11	-6	33	23	42	35	47	45	57	45
compared to control (%)

pH values

Treatment	TS1	TS2	TS3	TS4	TS5	TS6	TS7	TS8	TS9	TS10
Concentration of test substance (mg/L)	62.5		125		250		500		1000
pH values measured before correction	8.5	8.5	9.0	9.0	9.6	9.6	10.2	10.2	10.8	10.8
pH values corrected before addition of inoculum	7.3	7.2	7.2	7.2	7.3	7.3	7.2	7.2	7.2	7.2
pH values measured after incubation time	7.7	7.7	7.8	7.8	8.1	8.1	8.2	8.2	8.2	8.2

Validity criteria fulfilled:

yes

Description of key information

3-h EC10 =  34 mg/L (OECD 209; domestic activated sludge; BASF SE, 2012, report no. 08G0402/04G008)

Key value for chemical safety assessment

EC50 for microorganisms:

720 mg/L

EC10 or NOEC for microorganisms:

34 mg/L

Additional information

The microbial toxicity of N,N'-bis(3 -aminopropyl)ethylenediamine (CAS 10563 -26 -5) was studied in two tests: 1) GLP guideline study, respiration inhibition test with activated sludge, OECD 209; 2) non-GLP guideline study, Pseudomonas putida growth inhibition according to Bringmann-Kühn, German Industrial Standards DIN 38412, part 8.

The respiration inhibition test used activated sludge as inoculum taken from a wwtp treating predominantly domestic sewage. The 3 -h EC10 was determined to be 34 mg/L, the 3 -h EC50 was 720 mg/L (BASF SE, 2012; report no. 08G0402/04G008).

In the growth inhibition test, the test species was Pseudomonas putida. The test concentrations were not analytically verified. The 17 -h EC10 was determined to be 3.97 mg/L, the 17 -h EC50 was 8.8 mg/L (BASF AG, 1990; report no. 1/89/0502). This result will be used as supporting study.

The OECD 209 study was selected as key study since this result was determined under GLP according to an international guideline. The PNECs derived from the EC10 values of both studies are very similar (OECD 209: 3.4 mg/L; P.putida: 4.0 mg/L).