1-Decanaminium, N-decyl-N,N-dimethyl-, hexanedioate (2:1)

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:

20 March 2017 - 04 May 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

28 July 2011

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Reference substance 001

Cas Number:

210420-85-2

Constituent 2

Reference substance name:

N-methyldidecylamine

EC Number:

230-990-1

EC Name:

N-methyldidecylamine

Cas Number:

7396-58-9

Molecular formula:

C21H45N

IUPAC Name:

N-decyl-N-methyldecan-1-amine

Constituent 3

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

Dihydrogen oxide

impurity 1

Reference substance name:

Unknown impurities

Molecular formula:

Not available

IUPAC Name:

Unknown impurities

Test material form:

solid

Details on test material:

- Appearance: Slightly yellow gel
- Storage conditions: At room temperature; container flushed with nitrogen

Specific details on test material used for the study:

Correction factor for purity: 1.081
Solubility in water: > 1000 g/L

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Single samples for possible analysis were taken from all test concentrations and the control according to the schedule below.
Frequency at t=0 h, at t=24 h and t=72 h
Volume 2.0 mL
Storage Samples were stored in a freezer (=-15°C) until analysis at the analytical laboratory of the Test Facility.
At the end of the exposure period, the replicates were pooled at each concentration before sampling.
Compliance with the Quality criteria regarding maintenance of actual concentrations was checked by running a test vessel at an intermediate item concentration but without algae and samples for analysis were taken at the start, after 24 hours of exposure and at the end of the test period.
No samples for analysis were taken from the control group in the range-finding test.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Correction for purity: A correction factor of 1.081 was applied (reported concentrations represent corrected values).
- Method: Direct application to medium.
Preparation of test solutions started with 100 mg/L (highest test concentration in combined limit/range-finding) or 10 mg/L (range-finding and final test) applying a one to two-hour period of low-speed magnetic stirring to accelerate dissolution of the test item in medium and to prevent possible micelle formation. Lower test concentrations were prepared by subsequent dilutions in test medium. All test solutions were clear and colorless at the end of the preparation procedure.
Test solutions were prepared twice for each experiment. The first volume was used to pre-incubate the test vessels for one day as to minimize any possible adsorption of the test item to the glass surfaces.
50 mL of test solution and 1 mL of algal suspension were added to each replicate.
- Controls: Test medium without test item or other additives.

Test organisms

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: NIVA CHL 1
- Source: In-house laboratory culture.
- Method of cultivation: Algae stock cultures were started by inoculating growth medium (M1) with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C. 4 days before the start of the test, cells from the algal stock culture were inoculated in culture medium (M2) at a cell density of 1 x 10^4 cells/mL. The pre-culture was maintained under the same conditions as used in the test.


ACCLIMATION
- Acclimation period: no

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

24 mg CaCO3/L

Test temperature:

22.8-23.7°C

pH:

7.9-8.2 at t=0 h
7.9-8.0 at t=72 h

Nominal and measured concentrations:

Nominal concentrations: 0.32, 1.0, 3.2, 10, 32 and 100 µg/L
Geometric mean measured concentrations: 0.45, 0.78, 1.7, 5.1, 5.8, 18 and 83 µg/L, respectively corresponding to nominal concentrations above.
The test concentrations were based on measurement of two components (1-Decanaminium,N-decyl-N,N-dimethyl and Hexanedioate, which, according to spectral data, respresented 70.5% and 15.7% of the test item, resp.). Since the results based on Hexanedioate were nearly all below the limit of detection, the average measured concentration was calculated using only the measured concentrations of 1-Decanaminium,N-decyl-N,N-dimethyl at t=0 and t=72 h.

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL, all-glass
- Fill volume: 50 mL, pre-incubated with test material for 24 hours
- Aeration: yes
- Initial cells density: 1.0 x 10^4
- Control end cells density: 119.7 x 10^4
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- Additional replicates: 1 or 2 replicates of each test concentration without algae and 1 extra replicate of each test group with algae for sampling after 24 hours.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Milli-RO water (tapwater purified by reverse osmosis)
- Culture medium different from test medium: no, M2, according to OECD 201
- Intervals of water quality measurement: pH: at the beginning and at the end of the test; temperature of medium: continuously in a temperature control vessel; appearance of the cells: at the end of the final test

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Light intensity and quality: Continuously using TLD-lamps with a light intensity within the range of 82 to 87 µE/m^2/s.

EFFECT PARAMETERS MEASURED: Growth rate and yield at t=72 h.
- Determination of cell concentrations: At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with immersion probe (pathlength =20 mm).

COMBINED LIMIT/RANGE-FINDING TEST
- Test concentrations: 0.10, 1.0, 10 and 100 mg/L.
- Results: since growth was absent in all tested concentrations, a range-finding test was conducted to determine the doses for the final test.

RANGE FINDING TEST
- Test concentrations: 0.0010, 0.01, 0.1 and 1.0 mg/L.
- Results used to determine the conditions for the definitive study: yes, based on the results the EC50 was expected to be between 0.00010 mg/L and 0.010 mg/L.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (Feb 2017)

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

2.8 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI: 2.6-3.1

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.5 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI: 1.3-1.6

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.78 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control: yes
- Observation of abnormalities (for algal test): no
- Analysis based on 1-Decanaminium,N-decyl-N,N-dimethyl showed that concentrations were at or close to nominal at the start of the test. After 24 hours concentrations, especially at the lower end, decreased to or below the LOD. However, analyses at the end of the test provided better interpretable concentrations that were at or above those measured after 24 hours and none were below the LOD (36-99% of initial). It was therefore decided to omit the 24-hour results which consequently provided an average test range that showed a much more logical tendency.
- Analysis based on Hexanedioate showed that nearly all concentrations were below the LOD as was expected based on the LOD and the previous experiments. Estimated values, which were close to nominal, could only be determined for the highest concentration at the start and after 24 hours (111% and 103% of initial). These results could therefore not be used to reliably calculate average exposure concentrations for the test item.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- 72h-ErC50: 1.2 mg/L (95% CI: 1.1 - 1.2 mg/L)
- Other: results were within the historical range.

Reported statistics and error estimates:

For determination of the NOEC and the EC50 the approaches recommended in the OECD guideline 201 were used. An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the negative control revealed significant inhibition of growth rate or inhibition of yield (Multiple Sequentially-rejective Welsh-t-test After Bonferroni-Holm, a=0.05, one-sided, smaller).
Additionally, the EC10 and EC20 were determined to meet the recommendations as put down in "A Review of Statistical Data Analysis and Experimental Design in OECD Aquatic Toxicology Test Guidelines" by S. Pack, August 1993.
Calculation of ECx values was based on probit analysis using linear maximum likelihood regression with the percentages of growth rate inhibition and the percentages of yield inhibition versus the logarithms of the corresponding average measured concentrations of the test item.
The calculations were performed with ToxRat Professional v. 3.2.1. (ToxRat Solutions® GmbH, Germany).

Any other information on results incl. tables

Table 1 Measured Concentrations Versus Nominal Concentrations

1-Decanaminium, N-decyl-N,N-dimethyl-, hexanedioate (2:1) concentration (µg/L)	Measured concentration (µg/L)
	t=0h	t=72 h	Average (µg/L)
0.32	0.45	0.45		0.45
1.0	0.97	0.63		0.78
3.2	2.55	1.18		1.7
10	8.40	3.11		5.1
10 (wa)	8.56	3.95		5.8
32	29.2	10.7		18
100	96.5	70.7		83

wa = without algae

Table 2 Growth Rate And Percentage Inhibition For The Total Test Period

Average meas. test item concentration^#(µg/L)	Mean	Std. Dev	n	% Inhibition
Control	1.586	0.0827	6
0.45	1.593	0.0544	3	-0.4
0.78	1.549	0.0454	3	2.3
1.7	1.334	0.0272	3	15.9*
5.1	0.206	0.1991	3	87.0*
18	0.000	0.0000	3	100.0*
83	0.000	0.0000	3	100.0*

* - effect was statistically significant
^#Test item = 1-Decanaminium, N-decyl-N,N-dimethyl-, hexanedioate (2:1)

Table 3 Growth Rate And Percentage Inhibition At Different Time Intervals

Average meas. test item^#concentration (µg/L)	n	0 – 24 h		24 – 48 h		48 – 72h
		Mean	%Inhibition	Mean	%Inhibition	Mean	%Inhibition
Control	6	1.670	0.0	1.273	0.0	1.815	0.0
0.45	3	1.600	4.2	1.468	-15.3	1.712	5.7
0.78	3	1.550	7.2	1.260	1.0	1.836	-1.2
1.7	3	0.159	90.5	1.763	-38.4	2.081	-14.7
5.1	3	0.000	100.0	0.000	100.0	0.617	66.0
18	3	0.000	100.0	0.073	94.3	-0.073	104.0
83	3	0.000	100.0	0.000	100.0	0.000	100.0

^#Test item = 1-Decanaminium, N-decyl-N,N-dimethyl-, hexanedioate (2:1)

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

In the controls: cell density increased > 16 fold (i.e. 120), the CV of variation for section-by-section specific growth rates was < 35% (i.e. 19.2%) and the CV of average specific growth rates during the whole test period was < 7% (i.e. 5.2%).

Conclusions:

The 72h-ErC50 of 1-Decanaminium, N-decyl-N,N-dimethyl-, hexanedioate (2:1) towards algae, was 2.8 µg/L based on geometric mean measured concentrations. The NOEC for growth rate was 0.78 µg/L.