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

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Qualifier:

according to guideline

Guideline:

EU Method C.20 (Daphnia magna Reproduction Test)

Qualifier:

equivalent or similar to guideline

Guideline:

other: ISO 10706

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: all test concentrations
- Sampling method: The concentration of the test substance was determined in one representative test interval per week in each freshly prepared test solution (test vessels without daphnids) and in the 48 hour or 72 hour old test solutions before renewal (combined sample from test vessels with
daphnids).
- Sample storage conditions before analysis: the samples were analyzed immediately

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The necessary volume of the test substance was calculated from the density. Prior to use the test substance container was shaken to ensure homogeneity. The stock solution (20 mg/L) was prepared by pipetting 40 mg (42.1 µL based on the density) or 100 mg (105.3 µL) test substance into 2 L or 5 L of test medium and stirring for approximately 10 min. The stock solution was checked for the presence of any undissolved substance before diluting this stock solution to reach the lower test concentrations. By dilution of this stock solution with M4 medium the test concentrations were prepared.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): No. The stock solution appeared colorless-clear.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: water flea
- Source: Institut National de Recherche Chimique Appliquée (France, 1978), cultured continuously in test facility since 1978
- Age of parental stock (mean and range, SD): maximum 4 weeks
- Feeding during test: yes
- Food type: live green algae Desmodesmus subspicatus
- Amount: Day of test: 0-3 (0.10 mg TOC), 4-5 (0.12 mg TOC), 6-7 (0.14 mg TOC), 8-21 (0.20 mg TOC)
- Frequency: daily

ACCLIMATION
- Acclimation conditions (same as test or not): same as test

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

2.20 - 3.20 mmol/L

Test temperature:

20°C (±1°C)

pH:

7.5 - 8.5

Dissolved oxygen:

≥ 3mg/L

Nominal and measured concentrations:

Nominal: 0 (control), 2.7, 3.7, 5.2, 7.2, 10 mg/L
Measured (mean measured concentrations): 0 (control), 2.20 (81.5%), 3.09 (83.5%), 4.77 (91.7%), 6.79 (94.3%), 9.82 mg/L (98.2%)
(see detailed results in "any other information on results incl. tables")

Details on test conditions:

TEST SYSTEM
- Test vessel: glass beakers (100 mL nominal volume)
- Type: open (covered with petri plates)
- Aeration: none
- Material, size, headspace, fill volume: 50ml headspace, fill volume 50 mL
- Renewal rate of test solution (frequency/flow rate): semi static / 3 times per week
- No. of organisms per vessel: 1 parent
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10
- Biomass loading rate: 1 animal / 50 mL

OTHER TEST CONDITIONS
- Photoperiod: 16 light : 8 dark
- Light intensity: 620 -670 lux at a wave length of 400 - 750 nm
- Adjustement of pH: all stock solutions were adjusted with 1 molar HCl to a pH of approx. 8

TEST MEDIUM / WATER PARAMETERS
- pH: 7.5 - 8.5
- Total organic carbon: < 2mg/L
- Ca/mg ratio: about 4:1
- Conductivity: 550 - 650 µS/cm

RANGE-FINDING STUDY
- Results used to determine the conditions for the definitive study: In a preliminary Daphnia reproduction test a NOEC = 4.13 mg/L and LOEC = 9.09 mg/L was found (experimental conduct in accordance with GLP but without a GLP status). These results guided the concentration selection for the definitive test.

Reference substance (positive control):

yes

Remarks:

sodium chloride

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

(analytically verified)

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

7.2 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

(analytically verified)

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

(analytically verified)

Basis for effect:

mortality

Remarks:

(parental mortality)

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

7.2 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

(analytically verified)

Basis for effect:

mortality

Remarks:

(parental mortality)

Details on results:

- Mortality of parent animals: control: 0%
- Mean living young per surviving adult after 21 d: control: 167
- Time to first brood release or time to hatch: control: 8.2 d
- Effect concentrations exceeding solubility of substance in test medium: no
- The analytically measured concentrations of the test substance in the test water were within the range of ± 20% of the nominal concentration in all tested concentrations with the only exception that in aged test solutions concentrations decreased slightly with the largest decrease
in the lowest treatment group, which was 79% of the mean initial value. However, mean measured concentrations for the whole exposure period were within ±20% of the nominal concentration. Since both mean initial and overall mean concentrations were acceptable, the nominal concentrations can be used to evaluate the results of the study.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: EC50 (48h): 5.08 g/L (test date: 05 Mar 2013)
- Other: - remarks: normal response to the reference substance (NaCl)

EFFECT VALUES

Effect concentration [mg/L]
Endpoint	nominal		measured (arithm. mean)
	NOEC	LOEC	NOEC	LOEC
Reproduction	7.20	10.00	6.79	9.82
Mortality	7.20	10.00	6.79	9.82

 The data were not sufficient to calculate any ECx values.

ANALYTICAL CONCENTRATION CONTROL

Nominal concentration [mg/L]	Analytically measured concentration
	Mean initial^d		Mean 48/72h old		arithm. mean
	[mg/L]	[%]^a	[mg/L]	[%]^b	[mg/L]^c	[%]^a
0 (control)	<LOQ^e	–	<LOQ	–	<LOQ	–
2.7	2.46	91	1.95	79	2.20	82
3.7	3.33	90	2.86	86	3.09	84
5.2	4.93	95	4.60	93	4.77	92
7.2	6.93	96	6.65	96	6.79	94
10	9.93	99	9.70	98	9.82	98

^a: % of nominal concentration

^b: % of mean initial

^c: based on mean-initial value and mean-old value

^d: initial measured is the mean measured concentration at the start of each renewal period

^e: Limit of quantification (0.3 mg/L)

BIOLOGICAL RESULTS

Reproduction and Mortality Summary after 21 days

Nominal concentration [mg/L]	Reproduktion				Mortality
	per surviving parent		per introduced parent		Parent animals
	Mean living young	% effect^b	Mean living young	% effect
0.0	167 (5.9%)^a	-	167 (5.9%)	-	0
2.7	184	-	184	-	0
3.7	192	-	192	-	0
5.2	169	-	169	-	1
7.2	168	-	168	-	0
10.0	149*	11	124**	26	2*

* : p≤ 0.05; ** : p≤ 0.01

^a: Coefficient of variation for control fecundity based on surviving parent.

^b: Effect relative to control. (Only calculated for statistically significant effects.)

Other biological observations among surviving parent animals in each concentration after a 21 day exposure

Test conc. [mg/L, nominal]	Mean Growth Length[mm]	Immobile young [%]	Mean days to first brood	Aborted eggs [%]
0 (control)	4.51	0.2	8.2	1.0
2.7	4.59	0.3	8.0	0.7
3.7	4.30*	0.1	8.2	0.6
5.2	4.31	0.2	8.4	1.0
7.2	4.20**	0.7	8.1	0.9
10	3.23**	0.8	8.4	1.8

*: p ≤ 0.05; **: p ≤ 0.01 

No additional adverse effects or abnormal behavior were observed in any of the test groups.

OTHER OBSERVATIONS pH-values (always measured in replicate 1)

Nominal concentration [mg/L]	Day of the test
	0^a	3^b	10^a	12^b	17^a	19^b
0 (control)	8.0	8.1	7.9	8.0	7.9	7.9
2.7	8.0	8.3	8.0	8.0	8.0	7.9
3.7	8.1	8.2	7.9	8.1	8.0	8.0
5.2	8.0	8.3	8.1	8.1	8.0	8.0
7.2	8.1	8.3	8.1	8.2	8.0	8.1
10	8.1	8.2	8.0	8.2	8.1	8.2

^a: in the freshly prepared test solution

^b: before renewal in the old test solutions

Oxygen content [mg/L] (always measured in replicate 1)

Nominal concentration [mg/L]	Day of the test
	0^a	3^b	10^a	12^b	17^a	19^b
0 (control)	9.0	9.1	8.9	9.2	8.7	8.4
2.7	9.1	9.3	9.0	9.1	8.8	8.4
3.7	9.1	9.3	8.9	9.2	8.8	9.0
5.2	9.0	9.2	9.0	9.2	8.8	8.9
7.2	9.1	9.3	9.0	9.1	8.8	8.9
10	9.1	9.3	9.0	9.3	8.7	9.0

^a: in the freshly prepared test solution

^b: before renewal in the old test solutions

Total hardness [mmol/L] (always measured in replicate 1)

Nominal concentration [mg/L]	Day of the test
	0^a	3^b	10^a	12^b	17^a	19^b
0 (control)	2.41	2.45	2.43	2.46	2.45	2.46
2.7	2.43	2.45	2.43	2.46	2.45	2.48
3.7	2.41	2.43	2.41	2.45	2.45	2.46
5.2	2.43	2.45	2.41	2.46	2.45	2.48
7.2	2.41	2.43	2.43	2.46	2.45	2.48
10	2.41	2.43	2.41	2.46	2.45	2.50

^a: in the freshly prepared test solution

^b: before renewal in the old test solutions

Instantaneous temperature [°C]

Temperature was measured continuously during the exposure period in a

separate water filled vessel proximal to the test vessels. The temperature ranged from 19.7-20.4°C.

Validity criteria fulfilled:

yes

Description of key information

Chronic effects are not expected.

Additional information

To determine chronic effects of N4-Amine N,N’-Bis-(3-Aminopropyl)-ethylenediamine (CAS 10563 -26 -5) on aquatic invertebrates, a GLP-study according to OECD 211 was performed with Daphnia magna (21 d-reproduction test). The test concentrations were analytically verified. The mean measured concentrations for the whole exposure period were within ±20% of the nominal concentration. The NOEC was determined to be 7.2 mg/L (reproduction; nominal) (BASF SE, 2014; report no. 51E0402/04E011). All validity criteria were fulfilled in this study. Based on the NOEC of 7.2 mg/L chronic effects on aquatic invertebrates are not expected.