1-Tetradecanaminium, N,N,N-trimethyl-, methyl carbonate (1:1)

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

other information

Study period:

2015-09-15 to 2015-09-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study under GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

154858-16-9

Cas Number:

154858-16-9

IUPAC Name:

154858-16-9

Constituent 2

Reference substance name:

Tetradecytrimethylammonium oxalate

IUPAC Name:

Tetradecytrimethylammonium oxalate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Each test item concentration and the control were analytically verified via LC-MS/MS from freshly prepared media after 0 hours and old test media after 72 hours. Separate replicates for test item analysis at the beginning of exposure were prepared without algae. For the test item analysis after 72 hours, samples were taken from the test replicates. Adsorption to the glass was tested after 72 hours.

Test solutions

Vehicle:

no

Details on test solutions:

Stock solution
The stock solution (10 mg/L test item) was prepared freshly with dilution water.

Test concentrations
5 concentrations were tested in a geometric series with a dilution factor of 3.16: 1.50 - 4.74 - 15.0 - 47.4 - 150 µg/L.
The concentrations were based on the results of a preliminary range finding test (non-GLP).

Control
Six replicates (without test item) were exposed to the same conditions as the test concentrations.

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

Hardness:

Dilution water: 0.24 mmol Ca+mg/L.

Test temperature:

min.: 21.5 max.: 22.0, mean value: 21.8

pH:

Geometric mean measured test item concentration pH-value
[µg/L] Start; 0 hours End; 72 hours
139 7.80 8.09
39.8 7.80 8.11
11.3 7.81 8.33
2.82 7.92 8.47
0.851 7.95 8.53
Control 8.00 8.58

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater conditions

Nominal and measured concentrations:

nominal concentrations: 1.50 - 4.74 - 15.0 - 47.4 - 150 µg/L
geometric mean measured concentrations: 0.851 – 2.82 – 11.3 – 39.8 – 139 µg/L

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 4847 cells/mL
- Control end cells density: Mean 1781103 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: Dilution water, composition acc. to the guideline
- Hardness: 0.24 mmol Ca+Mg/L
- pH-value: 8.1 +/- 0.2
- Culture medium: Nutrient Medium Z acc. to LÜTTGE et al. (1994)


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: 72.7



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
- Range finding study:

Biological Data

The results of the preliminary range finding test are presented below:

Results of the Range Finding Test (0-72 h)

Nominal test item
concentration
[µg/L] Growth Rate Inhibition [%] Yield Inhibition [%]
100 66 98
10.0 6 28
1.00 0 2



Measured concentrations of the Test Item in the Range Finding Test

The measured concentrations of the test item in the preliminary test are presented below:

Measured Exposure Concentrations of the Test Item during the Range Finding Test

Nominal 0 hours 72 hours
test item
concentration
[µg/L]
Meas. conc. [µg/L] % Meas. conc. [µg/L] %
100 94.6 95 71.1 71
10.0 9.99 100 5.20 52
1.00 0.815 82* 0.479 48*

Meas. Conc. = measured concentration, dilution factor taken into account
SysQL = 0.5 µg/L)
% = percentage of the nominal concentration of the test item
* = measured concentration < SysQL

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2.82 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

11.3 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

6.42 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 4.81 - 8.45

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

11.9 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 9.78 - 14.3

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

32.5 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 29.4 - 36.2

Effect concentrations 6

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.851 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 7

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

2.82 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 8

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.64 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 0.851 - 3.17

Effect concentrations 9

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

3 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 1.88 - 4.68

Effect concentrations 10

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

7.34 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 5.27 - 9.57

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
EC50 Values of the Reference Item
based on nominal concentrations mg/L, (0-72 hours)
Current Study Valid Range (average ± 3 x SD)
Growth Rate Inhibition
ErC50 0.546 0.671 ± 0.413
95 % confidence interval 0.496 - 0.606
Yield Inhibition
EyC50 0.281 0.317 ± 0.139
95 % confidence interval 0.220 - 0.347

Reported statistics and error estimates:

EC-values and statistical analyses
EC10-, EC20- and EC50-values with confidence intervals of growth rate and yield inhibition after 72 hours were calculated by non-linear sigmoidal dose-response regression.

NOEC, LOEC and statistical analyses
The values for NOEC/LOEC were determined by calculation of statistical significance of inhibition of growth rate and yield. As standard methods, 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. 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.

Software
The data for the tables in this report were computer-generated and have been rounded for presentation from the full derived data. Consequently, if calculated manually based on the given data minor deviations may occur from these figures.
Calculations were carried out using software
• Excel, MICROSOFT CORPORATION
• SigmaPlot, SPSS INC.
• GraphPad Prism, GRAPHPAD SOFTWARE, INC.

Any other information on results incl. tables

Cell Densities

Geometric mean measured test item concentration	Replicate	Cell density [cells/mL]
[µg/L]	No.	0 hours	24 hours	48 hours	72 hours
139	1	4847	8508	3899	2600
	2	4847	8435	4515	2187
	3	4847	8103	3221	6689
	Mean	4847	8349	3878	3825
39.8	1	4847	22737	26888	50888
	2	4847	24377	37861	59970
	3	4847	20760	39670	67556
	Mean	4847	22625	34806	59471
11.3	1	4847	32050	122098	404137
	2	4847	36822	183730	745491
	3	4847	35398	163824	661211
	Mean	4847	34757	156551	603613
2.82	1	4847	42037	276823	1120517
	2	4847	39506	262969	1473127
	3	4847	37775	266134	1720050
	Mean	4847	39773	268642	1437898
0.851	1	4847	53659	300404	1594400
	2	4847	38867	294920	1722455
	3	4847	41363	288570	1761901
	Mean	4847	44630	294631	1692919
Control	1	4847	51432	368521	1922860
	2	4847	48242	325900	1732990
	3	4847	41161	281537	1567797
	4	4847	48627	367039	1751318
	5	4847	51518	272695	1664537
	6	4847	53250	374476	2047116
	Mean	4847	49038	331695	1781103

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 [µg/L]	Replicate	Growth rate		Growth rate inhibition	Yield		Inhibition of yield
	No.	[d^-1]		[%]	[cells/mL]		[%]
139	1		-0.208	100		-2247	100
	2		-0.265	100		-2660	100
	3		0.107	95		1842	100
	Mean	(+)	-0.122	100	(+)	-1022	100
39.8	1		0.784	60		46041	97
	2		0.838	57		55123	97
	3		0.878	55		62709	96
	Mean	(+)	0.833	58	(+)	54624	97
11.3	1		1.47	25		399290	78
	2		1.68	15		740644	58
	3		1.64	17		656364	63
	Mean	(+)	1.60	19	(+)	598766	66
2.82	1		1.81	8		1115670	37
	2		1.91	3		1468280	17
	3		1.96	1		1715203	3
	Mean	(-)	1.89	4	(+)	1433051	19
0.851	1		1.93	2		1589553	11
	2		1.96	1		1717608	3
	3		1.97	0		1757054	1
	Mean	(-)	1.95	1	(-)	1688072	5
Control	1		1.99			1918013
	2		1.96			1728143
	3		1.93			1562950
	4		1.96			1746471
	5		1.95			1659690
	6		2.02			2042269
	Mean		1.97			1776256

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	2.36	1.97	1.65	1.99	0.356	17.8	16.7
	2	2.30	1.91	1.67	1.96	0.316	16.1
	3	2.14	1.92	1.72	1.93	0.211	11.0
	4	2.31	2.02	1.56	1.96	0.375	19.1
	5	2.36	1.67	1.81	1.95	0.368	18.9
	6	2.40	1.95	1.70	2.02	0.354	17.5
				Mean	1.97
				SD ±	0.03
				CV [%]	1.64

CV= Coefficient of variation

SD= Standard deviation

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study, Tetradecyltrimethylammonium oxalat was found to inhibit the growth of the freshwater green alga Desmodesmus subspicatus after 72 hours with the following effect values: The NOEC-values of the test item after 72 hours for inhibition of growth rate and yield were 2.82 and 0.851 µg/L, respectively. Consequently, the LOEC-values for inhibition of growth rate and yield were 11.3 and 2.82 µg/L, respectively. The EC50-values with 95 % confidence intervals of the test item for inhibition for growth rate (ErC50) and yield (EyC50) after 72 hours were determined to be 32.5 (29.4 – 36.2) mg/L and 7.34 (5.27 – 9.57) µg/L.
All effect levels are given based on the geometric mean measured concentrations of Tetradecyltrimethylammonium oxalat.

Executive summary:

The toxicity of Tetradecyltrimethylammonium oxalat (batch number:HS25022015) to the unicellular freshwater green alga Desmodesmus subspicatus was determined according to the principles of OECD 201 (2011) and Council Regulation (EC) 761/2009 Method C.3 at Dr.U.Noack-Laboratorien in 31157 Sarstedt, Germany from 2015-09-15 to 2015-09-21, with a definitive exposure phase from 2015-09-15 to 2015-09-18. 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 4847 cells/mL. Five test item treatments were prepared with the nominal concentrations 1.50 - 4.74 - 15.0 - 47.4 - 150 µg/L, corresponding to the geometric mean measured concentrations of 0.851 – 2.82 – 11.3 – 39.8 – 139 µg/L. Three replicates were tested for each test item concentration and six for the control. The environmental conditions were determined to be within the acceptable limits.

The test item concentration in the test and control solutions were analysed by LC-MS/MS at the start and the end of exposure. The measured concentrations were in the range of 95 – 103 % of the nominal concentrations at the test start and in the range of < LOQ to 85 % at the test end.
All effect values of Tetradecyltrimethylammonium oxalatare given based ongeometric mean measured test item concentrations.

NOEC, LOEC, EC-values and Confidence Intervals of Tetradecyltrimethylammonium oxalat(0-72 hours)

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

 

	Growth Rate Inhibition Geometric mean measured test item concentration [µg/L]
NOEC	2.82
LOEC	11.3
ErC10	6.42 (4.81 – 8.45)
ErC20	11.9 (9.78 – 14.3)
ErC50	32.5 (29.4 – 36.2)
	Inhibition of Yield Geometric mean measured test item concentration [µg/L]
NOEC	0.851
LOEC	2.82
EyC10	1.64 (0.851 – 3.17)
EyC20	3.00 (1.88 – 4.68)
EyC50	7.34 (5.27 – 9.57)