Propanenitrile, 3-[[3-(isodecyloxy)propyl]amino]-

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:

2013-04-02 to 2013-05-03

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:

3-({3-[(2-methylnonyl)oxy]propyl}amino)propanenitrile

EC Number:

919-218-0

Cas Number:

72162-47-1

Molecular formula:

C16H32N2O

IUPAC Name:

3-({3-[(2-methylnonyl)oxy]propyl}amino)propanenitrile

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 3.16 - 10.0 - 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 the end of the test samples were taken directly from the test replicates per concentration level and control. The additionally prepared replicate per concentration level after 72 hours was incubated under test conditions. 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.
- Sample storage conditions before analysis:All samples (original and prepared) were stored at room temperature.

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. The stock solution was slightly turbid after the treatment.
- Eluate: Water of the river Innerste was used. The river water was replenished with compounds to achieve 50% of the OECD medium to enable a sufficient growth of the algae (total application volume 6.6 mL/L).

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

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.

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: 3.16 - 10.0 - 31.6 - 100 - 316 - 1000 µg/L (factor 10)
- 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): 4 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.: 22.0 max.: 23.5, mean value: 22.8 °C

pH:

Nominal concentration pH-value
Test item
[µg/L] Start; 0 hours End; 72 hours
1000 8.11 8.41
316 8.10 8.41
100 8.10 8.41
31.6 8.10 8.43
10.0 8.09 8.50
3.16 8.08 8.51
Control 8.07 8.51

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 882455 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: 65.0



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 76 99
0.100 52 94
0.0100 2 12

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

25.4 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: (22.5 - 28.7)

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 0.590 0.675 ± 0.440
95 %
confidence
interval 0.564 - 0.623
Yield Inhibition
EyC50 0.299 0.313 ± 0.139
95 %
confidence
interval 0.278 - 0.324
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 regression. Calculation of the confidence intervals EC10-, EC20- and EC50-values were carried out using standard procedures.
NOEC/LOEC was determined by calculation of statistical 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.

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		10688	19839	17503
		2		4332		10313	16512	15418
		3		4332		10436	17893	16034
		Mean		4332		10479	18081	16318
316		1		4332		11087	33196	22777
		2		4332		11996	25472	21774
		3		4332		11823	25504	21623
		Mean		4332		11635	28057	22058
100		1		4332		15484	29337	52761
		2		4332		12430	34583	68100
		3		4332		13842	34134	52220
		Mean		4332		13919	32685	57694
31.6		1		4332		19516	84686	361292
		2		4332		16097	86844	363258
		3		4332		24551	88259	490945
		Mean		4332		20055	86596	405165
10.0		1		4332		20478	174036	858218
		2		4332		23378	141655	968261
		3		4332		19261	168916	984207
		Mean		4332		21039	161536	936895
3.16		1		4332		23079	162965	885328
		2		4332		18997	153228	938321
		3		4332		21481	175614	980904
		Mean		4332		21186	163936	934851
Control		1		4332		18443	202391	939327
		2		4332		17946	171287	831643
		3		4332		17572	162122	881428
		4		4332		18339	148542	892656
		5		4332		18506	155417	775913
		6		4332		23871	188603	973765
		Mean		4332		19113	171394	882455

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		0.465	74		13171	99
	2		0.423	76		11086	99
	3		0.436	75		11702	99
	Mean	(+)	0.442	75	(+)	11986	99
316	1		0.553	69		18445	98
	2		0.538	70		17442	98
	3		0.536	70		17291	98
	Mean	(+)	0.542	69	(+)	17726	98
100	1		0.833	53		48429	94
	2		0.918	48		63768	93
	3		0.830	53		47888	95
	Mean	(+)	0.860	51	(+)	53362	94
31.6	1		1.47	17		356960	59
	2		1.48	17		358926	59
	3		1.58	11		486613	45
	Mean	(+)	1.51	15	(+)	400833	54
10.0	1		1.76	0		853886	3
	2		1.80	-2		963929	-10
	3		1.81	-2		979875	-12
	Mean	(-)	1.79	-1	(-)	932563	-6
3.16	1		1.77	0		880996	0
	2		1.79	-1		933989	-6
	3		1.81	-2		976572	-11
	Mean	(-)	1.79	-1	(-)	930519	-6
Control	1		1.79			934995
	2		1.75			827311
	3		1.77			877096
	4		1.78			888324
	5		1.73			771581
	6		1.81			969433
	Mean		1.77			878123

negative inhibitions = increase of growth

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	1.45	2.40	1.53	1.79	0.524	29.2	21.6
	2	1.42	2.26	1.58	1.75	0.443	25.3
	3	1.40	2.22	1.69	1.77	0.416	23.5
	4	1.44	2.09	1.79	1.78	0.325	18.3
	5	1.45	2.13	1.61	1.73	0.354	20.5
	6	1.71	2.07	1.64	1.81	0.229	12.7
				Mean	1.77
				SD ±	0.03
				CV [%]	1.55

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study 3-[[3-(Isodecyloxy)propyl]amino]-propanenitrile 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 both inhibition of growth rate and yield after 72 hours were for 10.0 µg/L. The LOEC-values for both inhibition of growth rate and yield after 72 hours were 31.6 µg/L. The EC50-value for inhibition of growth rate (ErC50) after 72 hours was 96.4 (88.9 – 105) µg/L. The EC50-value for inhibition of yield (EyC50) with 95 % confidence intervals after 72 hours was 30.2 (24.6 – 32.4) µg/L.
All effect levels are given based on nominal test item concentrations of 3-[[3-(Isodecyloxy)propyl]amino]-propanenitrile.

Executive summary:

The toxicity of 3-[[3-(Isodecyloxy)propyl]amino]-propanenitrile(batch number: F-212289)to the unicellular freshwater green alga Desmodesmus subspicatus was determined according to the principles of OECD 201 (2006) at Dr.U.Noack-Laboratorien in 31157 Sarstedt, Germany from 2013-04-02 to 2013-05-03, with the definitive exposure phase from 2013-04-30 to 2013-05-03. 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, 6 concentration levels were tested in a geometrical series with a dilution factor of √10 (nominal): 3.16 - 10.0 - 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.

3-[[3-(Isodecyloxy)propyl]amino]-propanenitrile has a 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 3-[[3-(Isodecyloxy)propyl]amino]-propanenitrileand the control were analytically verified by LC-MS/MS analysis at the start of the exposure (0 hours) and the end of the exposure (72 hours).
The measured concentrations of 3-[[3-(Isodecyloxy)propyl]amino]-propanenitrileat the start of the exposure were in the range of 84 % to 101 % of the nominal values. At the end of the exposure, they were in the range of LOQ_M to 72 % in samples with algae and 54 % to 77 % without algae.

The adsorption samples were analytically verified by LC-MS/MS at the end of the exposure. Adsorption to the glass walls was < 1 % for all measured samples.

NOEC, LOEC, EC-values and Confidence Intervals of 3-[[3-(Isodecyloxy)propyl]amino]-propanenitrile(0-72 hours)

                   based on nominal test item concentration [µg/L]

	Growth Rate Inhibition
NOEC	10.0
LOEC	31.6
ErC10	25.4 (22.5 – 28.7)
ErC20	38.7 (35.3 – 42.2)
ErC50	96.4 (88.9 - 105)
	Yield Inhibition
NOEC	10.0
LOEC	31.6
EyC10	18.5 (13.0 – 24.4)
EyC20	21.7 (15.4 – 26.2)
EyC50	30.2 (24.6 – 32.4)