N-[3-(isodecyloxy)propyl]propane-1,3-diamine

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

other information

Study period:

2013-03-25 to 2013-03-28

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

N-[3-(isodecyloxy)propyl]propane-1,3-diamine

EC Number:

276-432-0

EC Name:

N-[3-(isodecyloxy)propyl]propane-1,3-diamine

Cas Number:

72162-46-0

Molecular formula:

C16H36N2O

IUPAC Name:

(3-aminopropyl)({3-[(2-methylnonyl)oxy]propyl})amine

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 10 - 31.6 - 100 - 316 - 1000 µg/L (factor √10)
- Sampling method: All test item concentrations and the control were analytically verified via LC-MS/MS at the beginning and end of the test. Samples at test start were taken from an additional replicate without algae. At end of the test samples were taken directly from the pooled test replicates. The sorption of the test item on glass was quantified. Separate replicates of the three highest concentration levels were prepared without algae. After sampling for the test medium analysis (aqueous phase), the glassware of the three highest concentration levels was emptied and rinsed. The concentration of the test item in this solution was measured and the adsorbed test item amount was calculated from this concentration. Before this procedure the vessels were carefully rinsed with demineralized water to remove all test item dissolved in remaining test media. Additionally sorption to the walls of the glass container was determined from one test replicate (prepared with algae) of a concentration level close to the EC50-value. The samples being measured are listed below.

Schedule of Specific Test Item Analysis

Sampling time[h] 0 h (fresh media) 72 h (old media)
Appropriate dosage Aqueous phase Aqueous phase (with algae): All test media and control
levels and control (without algae):
All test media and control Glass surface (with algae):
one test replicate close to the EC50-value

aqueous phase and glass surface (without algae):
three concentration levels

Stock solution Aqueous phase ---------

- Sample storage conditions before analysis:All original samples were stored at room temperature. Prepared samples were stored at room temperature until analysis.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution of 10 mg/L was freshly prepared with test medium. The stock solution was treated with ultrasound for 5 min at room temperature, mixed with an ultraturrax (19600 rpm for 1 min) and stirred with a magnetic stirrer with 1100 rpm for 2 hours at room temperature.

- Eluate: Water of the river Innerste was used. The water parameters and the geographical site are given below. The river water was supplemented with 50 % of the OECD medium to enable a sufficient growth of the algae (total application volume 6.5 mL/L).
The natural river water was stored at 15 ± 2 °C and at - 18  2 °C for at least 24 hours. Freezing was found to be suitable to minimize the content of vital natural algae cells of the water as well as to reduce microbial (bacterial) activity.


River Innerste
Location Amtshof K514, km 26,
31157 Ruthe-Sarstedt, Germany
Latitude:52.25°, Longitude: 9.83°
Sampling Date 2012-09-17
Weather on Day of Sampling Sunny, slightly clouded; approx. 18 °C
Colour Yellowish to brownish, clear
pH 7.56
Conductivity [µS/cm] 898
Dissolved Oxygen [mg O2/L] 8.83
NPOC [mg C/L] 3.13
Ammonium-N [mg N/L] 0.05
Nitrate-N [mg N/L] 1.70
Total Nitrogen [mg N/L] 2.70
o-Phosphate-p [mg P/L] < 0.20
Total Phosphate [mg P/L] < 0.20
Suspended Matter [mg/L] 16.4
Total Hardness [mg CO3/L] 283
Storage conditions 15 ± 2°C

Composition of Dilution Water

Component Concentration [mg/L]
NH4Cl 15
MgCl2  6 H2O 12
CaCl2  2 H2O 18
MgSO4  7 H2O 15
KH2PO4 1.6
FeCl3  6 H2O 0.064
Na2EDTA  2 H2O 0.1
H3BO3 0.185
MnCl2  4 H2O 0.415
ZnCl2 3 x 10-3
Na2MoO4  2 H2O 7 x 10-3
CoCl2  6 H2O 1.5 x 10-3
CuCl2  2 H2O 1 x 10-5
NaHCO3 50
pH 8.1  0.2

- Differential loading: 10.0 - 31.6 - 100 - 316 - 1000 µg/L
- Controls: Six replicates (without test item) were tested under the same test conditions as the test replicates.

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Desmodesmus subspicatus Chodat
- Strain: SAG 86.81
- Source (laboratory, culture collection): SAG Pflanzenphysiologisches Institut der Universitaet Goettingen, Nikolausberger Weg 18, D-37073 Goettingen, Germany
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: Fresh stocks were prepared every month on Z-Agar. Light intensity amounted to 35 - 70 µE x m-2 x s-1 for 24 h per day.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

No

Test conditions

Test temperature:

min.: 21.0 max.: 22.0, mean value: 21.5 °C

pH:

Nominal test item pH-value
concentration
[µg/L] Start; 0 hours End; 72 hours
1000 8.09 8.25
316 8.12 8.23
100 8.10 8.25
31.6 8.13 8.22
10.0 8.13 8.20
Control 8.14 8.22

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater conditions

Nominal and measured concentrations:

See window "Any other information on materials and methods incl. tables"

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type (delete if not applicable): open, cotton wool plugs
- Material, size, headspace, fill volume: sterile 250 mL Erlenmeyer flasks, test volume 100 mL
- Aeration: Test containers were placed on a rotary shaker and oscillated at appr. 70 rpm
- Type of flow-through (e.g. peristaltic or proportional diluter): None
- Renewal rate of test solution (frequency/flow rate): One application at test start
- Initial cells density: Nominal: approximately 2 - 5 x 103 cells/mL, Actual: mean 4332 cells/mL
- Control end cells density: Mean 1113080 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6



GROWTH MEDIUM
- Standard medium used: yes



TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
Composition of Dilution Water
according to the guideline
Component Concentration [mg/L]
NH4Cl 15
MgCl2  6 H2O 12
CaCl2  2 H2O 18
MgSO4  7 H2O 15
KH2PO4 1.6
FeCl3  6 H2O 0.064
Na2EDTA  2 H2O 0.1
H3BO3 0.185
MnCl2  4 H2O 0.415
ZnCl2 3 x 10-3
Na2MoO4  2 H2O 7 x 10-3
CoCl2  6 H2O 1.5 x 10-3
CuCl2  2 H2O 1 x 10-5
NaHCO3 50
pH 8.1  0.2




OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 24 h
- Light intensity and quality: 60 - 120 µE x m-2 x s-1, mean value: 66.4



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] fluorimeter, Cell density and self fluorescence was measured daily via Chlorophyll-a- fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as background signal.



TEST CONCENTRATIONS

Results of the Range Finding Test (0-72 hours)
Nominal Test Item Concentration
[mg/L] Growth Rate Inhibition [%] Yield Inhibition [%]
100 100 100
10.0 100 100
1.00 100 100
0.100 42 90
0.0100 -1 -8

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

58.5 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: (43.0 - 75.6)

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test):
- Unusual cell shape: No
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Other: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

- Results with reference substance valid? Yes

Actual Study Valid Range (average ± 3 x SD)
Growth Rate Inhibition
ErC50 0.631 0.682 ± 0.452
95 %
confidence
interval 0.583 - 0.687
Yield Inhibition
EyC50 0.256 0.314 ± 0.144
95 %
confidence
interval 0.247 - 0.265
SD = Standard deviation

Reported statistics and error estimates:

EC10-, EC20- and EC50-values of the growth rate and yield inhibition after 72 hours were calculated by sigmoidal dose-response, respectively. Calculation of the confidence intervals EC10-, EC20- and EC50-values were carried out using standard procedures.

NOEC/LOEC was determined by calculation of statistical analyses significance of growth rate and yield. As standard One Way Analysis of Variance (ANOVA) and DUNNETT’s test were used for NOEC/LOEC calculations. When running a One Way Analysis of Variance a Normality test and an Equal Variance test were done first. The SHAPIRO-WILK-Test was used to test for normally distributed populations. The Levene median test was used for equal variance. P-values for both Normality and Equal Variance tests are 0.05. The -value (acceptable probability of incorrectly concluding that there is a difference) is =0.05. For growth rate the Equal Variance Test failed. No transformation was found to pass this test.

Any other information on results incl. tables

Cell Densities

	Nominal test item concentration		Replicate		Cell density [cells/mL]
[µg/L]		No.		0 hours		24 hours	48 hours	72 hours
1000		1		4332
		2		4332
		3		4332
		Mean		4332		n.a.	n.a.	n.a.
316		1		4332		2731
		2		4332		3256
		3		4332		4429
		Mean		4332		3472	n.a.	n.a.
100		1		4332		18091	60033	111664
		2		4332		17654	59639	125886
		3		4332		19440	63470	178848
		Mean		4332		18395	61047	138799
31.6		1		4332		21101	183049	793066
		2		4332		22340	160248	629306
		3		4332		24239	183754	758125
		Mean		4332		22560	175684	726832
10		1		4332		20641	172344	1067279
		2		4332		21359	202504	1127097
		3		4332		21865	196560	1094074
		Mean		4332		21288	190469	1096150
Control		1		4332		21198	182030	1166063
		2		4332		23101	186257	1079827
		3		4332		20286	169079	1034193
		4		4332		22994	182055	1102188
		5		4332		20689	186389	1162666
		6		4332		23507	191088	1133544
		Mean		4332		21963	182816	1113080

Evaluation after 72 hours

                   Statistically significant differences of growth rates and yield compared to

                   control values are marked (+), not significant differences are marked (-).

Nominal test item concentration	Replicate	Growth rate		Growth rate inhibition	Yield		Inhibition of yield
[µg/L]	No.	[d^-1]		[%]	[cells/mL]		[%]
1000	1		n.a.	n.a.		n.a.	n.a.
	2		n.a.	n.a.		n.a.	n.a.
	3		n.a.	n.a.		n.a.	n.a.
	Mean	n.t.	n.a.	n.a.	n.t.	n.a.	n.a.
316	1		n.a.	n.a.		n.a.	n.a.
	2		n.a.	n.a.		n.a.	n.a.
	3		n.a.	n.a.		n.a.	n.a.
	Mean	n.t.	n.a.	n.a.	n.t.	n.a.	n.a.
100	1		1.08	41.4		107332	90.3
	2		1.12	39.3		121554	89.0
	3		1.24	32.9		174516	84.3
	Mean	(+)	1.15	37.9	(+)	134467	87.9
31.6	1		1.74	6.09		788734	28.9
	2		1.66	10.3		624974	43.6
	3		1.72	6.91		753793	32.0
	Mean	(+)	1.71	7.75	(+)	722500	34.8
10	1		1.84	0.74		1062947	4.13
	2		1.85	-0.24		1122765	-1.26
	3		1.84	0.29		1089742	1.71
	Mean	(-)	1.84	0.26	(-)	1091818	1.53
Control	1		1.87			1161731
	2		1.84			1075495
	3		1.83			1029861
	4		1.85			1097856
	5		1.86			1158334
	6		1.86			1129212
	Mean		1.85			1108748

negative inhibitions = increase of growth

n.a. = no applicable

n.t. = not tested, due to 100 % mortality

Section-by-Section and Average Specific Growth Rates of the Control Group (0 – 72 hours)

	Replicate No.	Specific growth rate [d^-1]			Mean (0 - 72 hours)	SD ±	CV [%]	Mean CV [%]
		section-by-section
		0 - 24 hours	24 - 48 hours	48 - 72 hours
Control	1	1.59	2.15	1.86	1.87	0.281	15.1	13.7
	2	1.67	2.09	1.76	1.84	0.219	11.9
	3	1.54	2.12	1.81	1.83	0.289	15.8
	4	1.67	2.07	1.80	1.85	0.204	11.0
	5	1.56	2.20	1.83	1.86	0.319	17.1
	6	1.69	2.10	1.78	1.86	0.212	11.4
				Mean	1.85
				SD ±	0.02
				CV [%]	0.84

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study N-[3-(Isodecyloxy)propyl]propane-1,3-diamine was found to inhibit the growth of the freshwater green alga Desmodesmus subspicatus after 72 hours with the following effect values: The NOEC-values for inhibition of growth rate and yield after 72 hours were for both 10 µg/L. The LOEC-values for inhibition of growth rate and yield after 72 hours were both 31.6 µg/L. The EC50-value for inhibition of growth rate (ErC50) after 72 hours was 114 (107 – 137) µg/L. The EC50-value for inhibition of yield (EyC50) with 95 % confidence intervals after 72 hours was 40.9 (37.0 45.6) µg/L.
All effect levels are given based on nominal test item concentrations of N-[3-(Isodecyloxy)propyl]propane-1,3-diamine.

Executive summary:

The toxicity of N-[3-(Isodecyloxy)propyl]propane-1,3-diamine(batch number: F-212290)to the unicellular freshwater green alga Desmodesmus subspicatus was determined according to the principles of OECD 201 (2006) at Dr.U.Noack-Laboratorienin 31157 Sarstedt, Germany from 2013-03-25 to 2013-03-28. The aim of the study was to assess the effects on growth rate and yield in natural river water over a period of 72 hours.

The study was conducted under static conditions with an initial cell density of 4332 cells/mL. Based on a preliminary test, 5 concentration levels were tested in a geometrical series with a dilution factor of √10 (nominal): 10 - 31.6 - 100 - 316 - 1000 µg/L. Three replicates were tested for each test item concentration and six replicates for the control. Environmental conditions were determined to be within the acceptable limits.

N-[3-(Isodecyloxy)propyl]propane-1,3-diamine has low water solubility and sorbs to organic and inorganic materials by different mechanisms. The sorption processes are mostly non-linear, means are concentration dependent. Due to these properties the test item was difficult to test in artificial water (e.g. sorption to the test organism and walls of the test vessel). Natural river water contained particulate as well as dissolved organic carbon to which the test item could sorb partially preventing that the test item settled onto surfaces. The sorbed fraction of the test item was difficult to extract from the test system which normally led to low analytical recoveries. Nevertheless the test item was present in the test system and therefore available for exposure (dissolved in water and sorbed). Sorption of the test item to the glass ware of the test system was monitored as appropriate. Due to the properties of the test item nominal concentrations were used instead of measured ones.

All test concentrations of N-[3-(Isodecyloxy)propyl]propane-1,3-diamineand the control were analytically verified by LC-MS/MS analysis at the start of the exposure (0 h) and the end of the exposure (72 h).
The measured concentrations of N-[3-(Isodecyloxy)propyl]propane-1,3-diamineat the start of the exposure were in the range of 70 % to 108 % of the nominal values. At the end of the exposure, they were in the range of LOQ_M to 88 % in the samples with algae and 69 to 91 % in the samples without algae. Adsorption analytically verified at the end of the exposure was negligible. All measured concentration were below 1 % of the nominal test item concentration.

NOEC, LOEC, EC-values and Confidence Intervals of N-[3-(Isodecyloxy)propyl]propane-1,3-diamine (0-72 hours)                  

based on nominal test item concentrations [µg/L]

                               

	Growth Rate Inhibition
NOEC	10
LOEC	31.6
ErC10	58.5 (43.0 – 75.6)
ErC20	77.1 (62.6 – 86.7)
ErC50	114 (107 - 137)
	Yield Inhibition
NOEC	10
LOEC	31.6
EyC10	16.5 (12.9 – 19.8)
EyC20	22.5 (19.4 – 25.7)
EyC50	40.9 (37.0 – 45.6)