3-(isodecyloxy)propiononitrile

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-03-18 to 2013-03-21

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

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-(isodecyloxy)propiononitrile

EC Number:

264-840-1

EC Name:

3-(isodecyloxy)propiononitrile

Cas Number:

64354-92-3

Molecular formula:

C13H25NO

IUPAC Name:

3-(decylalkyl-(branched)oxy)-propiononitrile

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0.625 - 1.25 - 2.50 - 5.00 - 10.0 mg/L (factor 2), corresponding to 0.441 - 0.958 - 2.10 - 4.14 - 8.63 mg/L
(geometric mean measured)

- Sampling method: The concentrations of the test item were analytically determined from all test concentrations and the control at the start of the exposure (0 h) and at the end of the exposure (72 h). Samples at the start of the exposure were taken from an additional replicate without algae. At the end of the exposure samples were taken directly from the pooled test replicates.

- Sample storage conditions before analysis:
All original and diluted samples were stored at room temperature until sample preparation and until analysis, if necessary.

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 dilution water. 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: Dilution water
- Differential loading:
0.625 - 1.25 - 2.50 - 5.00 - 10.0 mg/L (factor 2, nominal)
0.441 - 0.958 - 2.10 - 4.14 - 8.63 mg/L (geometric mean measured)

- 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

Hardness:

0.24 mmol Ca+Mg/l

Test temperature:

min.: 22.0 max.: 23.0, mean value: 22.5 °C

pH:

Environmental Conditions

Geometric mean measured
test item concentration pH-value
[mg/L] Start; 0 hours End; 72 hours
8.63 8.00 7.56
4.14 8.02 7.58
2.10 8.05 7.65
0.958 8.05 7.76
0.441 8.05 7.93
Control 8.11 8.16

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 802428 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: 71



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 -3 -14
0.100 1 5
0.0100 0 1

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.829 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: (0.761 - 0.895)

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

Current 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:

NOEC/LOEC were 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 a-value (acceptable probability of incorrectly concluding that there is a difference) is a=0.05. Normality test failed for calculation of yield values. No transformation was found to pass Normality test.
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 of growth rate was estimated directly from the concentration levels. Calculation of the confidence intervals EC10-, EC20- and EC50-values of yield was carried out using standard procedures.

Any other information on results incl. tables

Cell Densities

	Geometric mean measured test item concentration		Replicate		Cell density [cells/mL]
[mg/L]		No.		0 hours		24 hours	48 hours	72 hours
8.63		1		4332		3231
		2		4332		3706
		3		4332		4127
		Mean		4332		3688	n.a.	n.a.
4.14		1		4332		8231	2985
		2		4332		9914	4014
		3		4332		9442	3765
		Mean		4332		9196	3588	n.a.
2.10		1		4332		12458	7866	5190
		2		4332		11694	13015	8414
		3		4332		11672	11181	10971
		Mean		4332		11941	10687	8192
0.958		1		4332		17088	48877	236105
		2		4332		15405	54821	343312
		3		4332		21799	62162	316529
		Mean		4332		18097	55287	298649
0.441		1		4332		22881	161588	799545
		2		4332		22327	131698	874899
		3		4332		25963	175942	1072405
		Mean		4332		23724	156409	915616
Control		1		4332		28872	164060	910186
		2		4332		19600	140938	728185
		3		4332		22585	137447	692363
		4		4332		26714	167255	847192
		5		4332		27321	174621	897574
		6		4332		22088	155700	739066
		Mean		4332		24530	156670	802428

Evaluation after 72 hours

                   Statistically significant differences of growth rates and yield compared to

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

Geometric mean measured test item concentration	Replicate	Growth rate		Growth rate inhibition	Yield		Inhibition of yield
[mg/L]	No.	[d^-1]		[%]	[cells/mL]		[%]
8.63	1		n.a.	100		n.a.	100
	2		n.a.	100		n.a.	100
	3		n.a.	100		n.a.	100
	Mean	(*)	n.a.	100	(*)	n.a.	100
4.14	1		n.a.	100		n.a.	100
	2		n.a.	100		n.a.	100
	3		n.a.	100		n.a.	100
	Mean	(*)	n.a.	100	(*)	n.a.	100
2.10	1		0.060	97		858	100
	2		0.221	87		4082	99
	3		0.310	82		6639	99
	Mean	(*)	0.197	89	(+)	3860	100
0.958	1		1.33	23		231773	71
	2		1.46	16		338980	58
	3		1.43	18		312197	61
	Mean	(+)	1.41	19	(+)	294317	63
0.441	1		1.74	0		795213	0
	2		1.77	-2		870567	-9
	3		1.84	-6		1068073	-34
	Mean	(-)	1.78	-2	(-)	911284	-14
Control	1		1.78			905854
	2		1.71			723853
	3		1.69			688031
	4		1.76			842860
	5		1.78			893242
	6		1.71			734734
	Mean		1.74			798096

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 h)	SD ±	CV [%]	Mean CV [%]
		section-by-section
		0 - 24 hours	24 - 48 hours	48 - 72 hours
Control	1	1.90	1.74	1.71	1.78	0.100	5.59	8.57
	2	1.51	1.97	1.64	1.71	0.239	14.0
	3	1.65	1.81	1.62	1.69	0.101	5.95
	4	1.82	1.83	1.62	1.76	0.118	6.72
	5	1.84	1.85	1.64	1.78	0.122	6.87
	6	1.63	1.95	1.56	1.71	0.211	12.3
				Mean	1.74
				SD ±	0.04
				CV [%]	2.25

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study 3-(Isodecyloxy)propionitrile was found to inhibit the growth of the freshwater green alga Desmodesmus subspicatus after 72 hours. The NOEC-value of the test item for inhibition of growth rate and inhibition of yield after 72 hours was for both 0.441 mg/L. The EC50-values of the test item for inhibition of growth rate (ErC50) and yield (EyC50) with 95 % Confidence Intervals after 72 hours were 1.31 (1.24 – 1.40) mg/L and 0.884 (0.516 – 0.951) mg/L, respectively.
All effect levels are given based on geometric mean measured concentrations of 3-(Isodecyloxy)propionitrile.

Executive summary:

The toxicity of 3-(Isodecyloxy)propionitrile(batch number: F-212252) 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 2013-03-18 to
2013-03-21. The aim of the study was to assess the effects on growth rate and yield 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 nominal dilution factor of 2 : 0.625 - 1.25 - 2.50 - 5.00 - 10.0 mg/L, corresponding to geometric mean measured test item concentrations of 0.441 - 0.958 - 2.10 - 4.14 - 8.63 mg/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.

The concentrations of the test item 3-(Isodecyloxy)propionitrile were analytically verified by LC-MS/MS at the start and the end of the exposure of all test concentrations and the control.
The measured concentrations of the test item 3-(Isodecyloxy)propionitrileat the start of the exposure were in the range of 85 to 97 % and after 72 hours in the range of 59 to 79 % of the nominal values. Therefore, all effect values given are based on the geometric mean measured test item concentrations of 3-(Isodecyloxy)propionitrile.

NOEC, LOEC, EC-values and 95 % Confidence Intervals of 3-(Isodecyloxy)propionitrile (0-72 hours)

                   based on geometric mean measured test item concentrations [mg/L]

	Growth Rate Inhibition Geometric mean measured test item concentration [mg/L]
NOEC	0.441
LOEC	0.958
ErC10	0.829 (0.761 – 0.895)
ErC20	0.970 (0.912 – 1.03)
ErC50	1.31 (1.24 - 1.40)
	Inhibition of Yield Geometric mean measured test item concentration [mg/L]
NOEC	0.441
LOEC	0.958
EyC10	0.687 (0.470 – 0.911)
EyC20	0.739 (0.483 – 0.920)
EyC50	0.884 (0.516 – 0.951)