Amines, N-(C16-18 and C18-unsatd. alkyl)-N,N',N'-trimethyltrimethylenedi-

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2017-04-25 to 2017-05-03, with definitive exposure phase from 2017-04-25 to 2017-04-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

The four highest concentration levels and the control were analytically verified via LC-MS/MS at the start (0 hours) and at the end of the exposure (72 hours) with algae. The lowest concentration level (0.500 µg/L) could not be analysed, because the Limit of Quantification (LOQ) is 2 µg/L. The implementation of a lower LOQ was technical not possible.
Adsorption to glass was analytically verified at the concentration levels of 5.00 and 15.8 µg/L with algae at the end of the exposure.
The vessels were rinsed carefully with deionized water before analysis to remove adhering algae.

Vehicle:

no

Details on test solutions:

Test item
N-C16-18-alkyl-(evennumbered, C18 unsaturated) trimethylpropane-1,3-diamine

Stock solution, media preparation
10.0 mg test item/L was freshly prepared with test medium. The dispersion was treated with ultrasound for 30 minutes at 40 °C.

Test concentrations (nominal)
Five concentration levels were tested in a geometrical series with a dilution factor of √10 (approximately 3.16): 0.500 - 1.58 - 5.00 - 15.8 - 50.0 µg/L. The concentration levels are based on the results of a preliminary range finding test (non-GLP, Erlenmeyer flasks, with headspace in natural river water).
 
Control
Six replicates (without test item) were exposed under the same conditions as the test concentrations.

Test container and pre-treatment
Sterile Erlenmeyer flasks (vol. 250 mL) with cotton wool plugs were used. A coating phase (saturation of the test container) was carried out. The test container were pre-treated with the appropriate test solutions for 24 hours under test conditions. Before the start of the exposure, the test containers were emptied and refilled with freshly prepared test solutions.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata HINDÁK
- Strain: SAG 61.81
- Source (laboratory, culture collection): SAG, Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, D-37073 Göttingen
- Age of inoculum (at test initiation): 4 days
- Method of cultivation: Fresh stocks were prepared every month on Z-Agar. Light intensity amounted to 2567 – 5130 lux corresponding to 35 - 70 µE ∙ m-2 ∙ s-1 for 24 h per day.

Test type:

static

Water media type:

freshwater

Remarks:

Natural river water

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

No

Hardness:

Not specified

Test temperature:

min.: 22.0 max.: 22.5 mean value: 22.3

pH:

Nominal test item concentration pH-value
[µg/L] Start; 0 hours End; 72 hours
50.0 8.24 8.22
15.8 8.13 8.20
5.00 8.13 8.71
1.58 8.13 8.68
0.500 8.13 8.69
Control 8.16 8.76

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater conditions

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 10^3 cells/mL, Actual: mean 6839 cells/mL
- Control end cells density: Mean 1243395 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:
Test medium
Water of the river Innerste was used and its properties in terms of organic matter content and suspended solids are considered typical for European rivers and are close to those in the EU risk assessment methods. The water parameters and the geographical site are given in the table below. The river water was supplemented with 50% of the standard OECD medium to enable a sufficient growth of the algae (see table below). The supplemental dilution water compounds were applied in a concentrated solution (6.5 mL/L river water) in order to avoid changes of the parameters of the river water.
 
Storage conditions
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.

Water Parameters of the River Water
River Innerste
Location Amtshof K514, km 26,
31157 Ruthe-Sarstedt, Germany
Latitude:52.25°, Longitude: 9.83°
Sampling Date 2016-08-09
Weather on Day of Sampling Cloudy; 18°C
Colour Yellowish to brownish, clear
pH 7.89
Conductivity [µS/cm] 1123
Dissolved Oxygen [mg O2/L] 6.97
DOC [mg C/L] 2.71
TOC [mg C/L] 3.33
Ammonium-N [mg NH4-N/L] < 0.04
Nitrate-N [mg NO3-N/L] 2.1
o-Phosphate-p [mg P/L] < 0.05
Total Phosphate [mg P/L] 0.05
Suspended Matter [mg/L] 12.8
Total Hardness [mg CO3/L] 272
Storage conditions 15 ± 2°C
NPOC = non purgeable organic carbon

Supplemental Dilution Water
Final concentrations
Component Concentration [mg/L]
NH4Cl 7.5
MgCl2 . 6 H2O 6.0
CaCl2 . 2 H2O 9.0
MgSO4 . 7 H2O 7.5
KH2PO4 0.8
FeCl3 . 6 H2O 0.032
Na2EDTA . 2 H2O 0.05
H3BO3 0.0925
MnCl2 . 4 H2O 0.208
ZnCl2 1.5 x 10-3
Na2MoO4 . 2 H2O 3.5 x 10-3
CoCl2 . 6 H2O 0.75 x 10-3
CuCl2 . 2 H2O 0.5 x 10-5
NaHCO3 25

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 24 h
- Light intensity and quality: Approximately 4440 to 8880 lux, corresponding to 60 to 120
µE ∙ m-2 ∙ s-1, mean value: 6163 lux,

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
-Chlorophyll a-fluorescence
The cell density was measured daily via Chlorophyll a-fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as a background signal. No self-fluorescence was found in the preliminary range finding test in the test concentration of 1 mg/L.

Reference substance (positive control):

yes

Remarks:

(Potassium dichromate)

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

5 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

15.8 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

13.2 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: CI: 5.16 - 40.9

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

14.2 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: CI: 5.49 - 41.4

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

16.1 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: CI: 5.97 - 43.0

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

5 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

15.8 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

12.1 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: CI: < 0.500 - 34.6

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

4.98 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: CI: 4.98 - 34.9

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

13.8 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: CI: 5.27 - 36.2

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: The test solutions were visually clear throughout the exposure period.
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

The toxicity of potassium dichromate (SIGMA, batch number MKBV0900V, purity 99.0 %, CAS RN 7778-50-9) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined over a period of 72 hours from 2017-04-18 to 2017-04-21. The reference item toxicity is in the valid range following test facility SOPS.

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 0.559 0.754 ± 0.518
95 % confidence interval 0.538 – 0.581
Yield inhibition
EyC50 0.320 0.398 ± 0.291
95 % confidence interval 0.242 – 0.341
SD = Standard deviation

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 sigmoidal dose-response regression.

NOEC- and LOEC-values
The NOEC / LOEC was determined by calculation of statistical significance of biomass integrals, growth rate and yield.
The following statistical tests were conducted:
Hypothesis tests:
Monotonicity was done by trend analysis by contrasts (significance level 0.05) for yield.
Shapiro-Wilk’s test on normal distribution was done with a significance level 0.01 for growth rate and yield.
Levene’s test on variance homogeneity was done with a significance level 0.01 for growth rate and yield.
William’s t-test on significant differences was done with a significance level 0.05 for yield.
Welsh-t-test after Bonferroni-Holm was done with a significance level 0.05 for growth rate.

Software
The data for the tables in this report were computer-generated and have been rounded for presentation from the fully 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.
- ToxRat, ToxRat Solutions GmbH
- GraphPad Prism, GRAPHPAD SOFTWARE, INC.

Cell Densities

Nominal test item concentration		Replicate		Cell density [cells/mL]
[µg/L]		No.		0 hours		24 hours		48 hours		72 hours
50.0		1		6839		<LOQcell density		<LOQcell density		<LOQcell density
		2		6839		<LOQcell density		<LOQcell density		<LOQcell density
		3		6839		<LOQcell density		<LOQcell density		<LOQcell density
		Mean		6839		n.a.		n.a.		n.a.
15.8		1		6839		11096		45632		90856
		2		6839		15380		21171		237703
		3		6839		10701		15649		87597
		Mean		6839		12392		27484		138719
5.00		1		6839		27672		255555		1237948
		2		6839		26920		187069		962845
		3		6839		27813		212496		1671844
		Mean		6839		27468		218373		1290879
1.58		1		6839		26838		212566		1102971
		2		6839		39400		311845		1277741
		3		6839		30028		367884		1569932
		Mean		6839		32089		297432		1316881
0.500		1		6839		24275		144782		1105283
		2		6839		29270		180819		1046587
		3		6839		29384		260131		1214832
		Mean		6839		27643		195244		1122234
Control		1		6839		27968		361722		1376991
		2		6839		43259		325761		1273645
		3		6839		35923		422588		1544212
		4		6839		25053		285049		1281721
		5		6839		29925		211899		1024116
		6		6839		25963		261103		959685
		Mean		6839		31349		311354		1243395

 

< LOQ_{cell density}= below the limit of quantification (2736 cells/mL)                    n.a. = data not determinable

 

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		Inhibition of growth rate	Yield		Inhibition of yield
[µg/L]	No.	[d^-1]		[%]	[cells/mL]		[%]
50.0	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
15.8	1		0.862	50		84017	93
	2		1.18	32		230864	81
	3		0.850	51		80758	93
	Mean	(+)	0.965	44	(+)	131880	89
5.00	1		1.73	0		1231109	0
	2		1.65	5		956006	23
	3		1.83	-6		1665005	-35
	Mean	(-)	1.74	0	(-)	1284040	-4
1.58	1		1.69	2		1096132	11
	2		1.74	-1		1270902	-3
	3		1.81	-5		1563093	-26
	Mean	(-)	1.75	-1	(-)	1310042	-6
0.500	1		1.70	2		1098444	11
	2		1.68	3		1039748	16
	3		1.73	0		1207993	2
	Mean	(-)	1.70	2	(-)	1115395	10
Control	1		1.77			1370152
	2		1.74			1266806
	3		1.81			1537373
	4		1.74			1274882
	5		1.67			1017277
	6		1.65			952846
	Mean		1.73			1236556

                                                                               n.a. = data not determinable

Section-by-Section and Average Specific Growth Ratesof 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	1.41	2.56	1.34	1.77	0.686	38.8	29.3
	2	1.85	2.02	1.36	1.74	0.339	19.5
	3	1.66	2.47	1.30	1.81	0.598	33.1
	4	1.30	2.43	1.50	1.74	0.604	34.6
	5	1.48	1.96	1.58	1.67	0.254	15.2
	6	1.33	2.31	1.30	1.65	0.572	34.7
				Mean	1.73
				SD ±	0.06
				CV [%]	3.47

 

SD = Standard deviation  CV = Coefficient of variation

Validity criteria fulfilled:

yes

Conclusions:

In this study, N-C16-18-alkyl-(evennumbered, C18 unsaturated) trimethylpropane-1,3-diamine was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values (based on nominal test item concentrations): The EC50-values with 95 % confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 16.1 (5.97 – 43.0) µg/L and 13.8 (5.27 – 36.2) µg/L, respectively. The NOEC-values for both inhibition of growth rate and yield after 72 hours were 5.00 µg/L, respectively.
All effect levels are based on nominal test item concentrations. There was no significant sorption to glass were in the test vessels. Therefore, the nominal test item concentrations were uses as basis for the evaluation.

Executive summary:

The toxicity of N-C16-18-alkyl-(evennumbered, C18 unsaturated) trimethylpropane-1,3-diamine (Batch no.:211401A Morris) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 266/2016/Method C.3 from 2017-04-25 to 2017-05-03, with definitive exposure phase from 2017-04-25 to 2017-04-28 at the test facility.The aim of the study was the determination of NOEC, LOEC, EC₁₀- , EC₂₀- and EC₅₀-values of growth rate and yield over a period of 72 hours.

 

The test item N-C16-18-alkyl-(evennumbered, C18 unsaturated) trimethylpropane-1,3-diamineis an amber liquid UVCB substance, which is dispersible in water. The study was conducted under static conditions with an initial cell density of 6839 cells/mL. A stock solution of 10.0 mg/L was freshly prepared with test medium (natural river water, which was supplemented with the mineral components of the dilution water ) and treated with ultrasound for 30 minutes at 40 °C. Five concentrations were tested in a geometrical series with a dilution factor of √10:0.500 - 1.58 - 5.00 - 15.8 - 50.0 µg/L. Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditionswere within the acceptable limits.

 

The concentrations of three components of thetest itemN-C16-18-alkyl-(evennumbered, C18 unsaturated) trimethylpropane-1,3-diaminewere analytically verified by LC-MS/MS at the start and the end of the exposure.Adsorption to the glass wall was investigated at the start and the end of the exposure for the concentrations 5.00 µg/L and 15.8 mg/L.

The measured concentrations of the three components of thetest itemN-C16-18-alkyl-(evennumbered, C18 unsaturated) trimethylpropane-1,3-diamineat the start of the exposure (0 hours) were in the range of 106 to 191% of the nominal values. The measured concentrationsthe three compounds of the test item at the end of the exposure (72 hours) were in the range of < LOQ to 10% of the nominal value. Adsorption to the glass wall was found to be negligible.

 

The aquatic ecotoxicity tests with N-C16-18-alkyl-(evennumbered, C18 unsaturated) trimethylpropane-1,3-diamine were performed in river water to allow a PEC_aquatic,bulk/PNEC_aquatic,bulk approach and is considered to be conservative but more environmentally realistic than the standard method. This approach is based on PEC estimations representing 'total aquatic concentrations'. To characterize the risk to the aquatic compartment the PEC_aquatic,bulk is compared with the PNEC_aquatic,bulk derived from river water ecotoxicity studies (ECETOC, 2003).

 

In order to class standard laboratory toxicity study valid, it is of particular importance that – besides information on test substance, test method / conditions and test organism used – suitable precautions are taken to prevent the loss of test substance by adsorption and that exposure concentrations are based upon measured levels.

For ecotoxicity tests performed using the bulk approach, however, adsorption to suspended matter and DOC is acceptable and only adsorption to glassware should be accounted for. For a valid bulk approach test the concentration-effect relationship should be based on the sum of adsorbed and dissolved substance in the volume of the medium tested. One of the advantages of the bulk approach tests with these difficult substances is that in the presence of suspended matter, humic acids and/or algae, the residual sorption to glassware will be negligible. The results of these bulk approach tests are therefore much easier to interpret and more realistic, and if compared to PEC_bulkclearly provide a more appropriate assessment of risks for the environment. An individual test design was applied. Natural river water was used as dilution medium.

 

All effect values given are based on the nominal test item concentrations ofN-C16-18-alkyl-(evennumbered, C18 unsaturated) trimethylpropane-1,3-diamine.

 

 NOEC, LOEC, EC-values and 95 % Confidence Intervals ofN-C16-18-alkyl-(evennumbered, C18 unsaturated) trimethylpropane-1,3-diamine (0 - 72 hours)

                  based on nominal test item concentrations [µg/L]

	Growth Rate Inhibition Nominal test item concentration [µg/L]
NOEC	5.00
LOEC	15.8
ErC10	13.2 (5.16 – 40.9)
ErC20	14.2 (5.49 – 41.4)
ErC50	16.1 (5.97 – 43.0)
	Yield Inhibition Nominal test item concentration [µg/L]
NOEC	5.00
LOEC	15.8
EyC10	12.1 (< 0.500 – 34.6)
EyC20	12.7 (4.98 – 34.9)
EyC50	13.8 (5.27 – 36.2)

Description of key information

One toxicity to quatic algae test is available for N-C16-18-alkyl-(evennumbered, C18 unsaturated) trimethylpropane-1,3-diamine. The ErC10 and ErC50 observed in this study are respectively 13.2 and 16.1 µg a.i./L based on measured initial concentrations.

Key value for chemical safety assessment

EC50 for freshwater algae:

16.1 µg/L

EC10 or NOEC for freshwater algae:

13.2 µg/L

Additional information

The toxicity of N-C16-18-alkyl-(evennumbered, C18 unsaturated) trimethylpropane-1,3-diamine freshwater green alga Pseudokirchneriella subcapitata was determined according to OECD 201 and EC No. 266/2016/Method C.3. The aim of the study was the determination of NOEC, LOEC, EC10- , EC20- and EC50-values of growth rate and yield over a period of 72 hours.

 

N-C16-18-alkyl-(evennumbered, C18 unsaturated) trimethylpropane-1,3-diamineis an amber liquid UVCB substance, which is dispersible in water. The study was conducted under static conditions with an initial cell density of 6839 cells/mL. A stock solution of 10.0 mg/L was freshly prepared with test medium (natural river water, which was supplemented with the mineral components of the dilution water ) and treated with ultrasound for 30 minutes at 40 °C. Five concentrations were tested in a geometrical series with a dilution factor of

√10: 0.500 - 1.58 - 5.00 - 15.8 - 50.0 µg/L. Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditions were within the acceptable limits.

 

The aquatic ecotoxicity tests with N-C16-18-alkyl-(evennumbered, C18 unsaturated) trimethylpropane-1,3-diamine were performed in river water to allow a PECaquatic,bulk/ PNECaquatic,bulk approach (ECETOC 2003) and is considered to be conservative but more environmentally realistic than the standard method.

The results of these bulk approach tests are much easier to interpret and more realistic, and when compared to PECbulk clearly provide a more appropriate assessment of risks for the environment.

Following the bulk approach and considering that biodegradation as possible reason for the observed decrease is very unlikely considering the short time frame of refreshment and the fact that the observed sorption to glassware is negligible shows that the test organisms were fully exposed to the test substance during the test. It is therefore considered justified to use the nominal test concentrations to derive the effect values.

 

All effect values given are based on the nominal test item concentrations of N-C16-18-alkyl-(evennumbered, C18 unsaturated) trimethylpropane-1,3-diamine.

Under the study conditions, the 72h ErC10 and ErC50 are 13.2 and 16.1 µg/L respectively.