2,2,4(or 2,4,4)-trimethylhexane-1,6-diamine

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:

2015-07-20 to 2015-07-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study with GLP standard

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: A stock solution of 1000 mg/L of the test item was prepared in methanol and diluted to 10 mg/L with acetonitrile : HPLC water
(50 : 50) containing 1 % formic acid. This solution was further diluted with acetonitrile : HPLC water (50 : 50) containing 1 % formic acid to 6
concentrations.
- Sampling method: All concentration levels and the control were analytically verified via LC-MS/MS after 0 hours (start of exposure) and 72 hours
(end of exposure). Additionally samples of the concentration level 40.0 mg/L were taken and analysed after 24 hours.
Separate replicates for the test item analysis at the beginning of exposure were prepared without algae. For the test item analysis after 24 and
72 hours, samples were taken out of the test replicates.
- Sample storage conditions before analysis: All samples were stored at room temperature until sample preparation and until start of analysis, if
necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution of 100 mg/L was freshly prepared with dilution water.
- Media preparation: Direct addition of the test item and shaking until the test item was completely dissolved.
- Test concentrations (nominal): 5 concentrations were tested in a geometrical series with a dilution factor of
2.5: 2.56 - 6.40 - 16.0 - 40.0 - 100 mg/L. The concentrations were based on the results of a preliminary range finding test (non-GLP).
- Control: Six replicates (without test item) were exposed under the same conditions as the test concentrations

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata HINDAK
- Strain: SAG 61.81
- 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 from Z-Agar. Light intensity amounted 35 - 70 µE x m-2 x s-1 for 24 h per day.
- Culture medium: Nutrient medium Z according to LÜTTGE et al. (1994)

ACCLIMATION
- Culturing media and conditions (same as test or not): Not the same, standard growth medium
- Any deformed or abnormal cells observed: No

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

None

Hardness:

Not measured

Test temperature:

Room temperature were determined to be within the acceptable limits. See table "Environmental conditions" ( other informations on results)

pH:

pH-value were determined to be within the acceptable limits. See table "Environmental conditions" ( other informations on results)

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater

Nominal and measured concentrations:

Please refer to information on materials and methods

Details on test conditions:

TEST SYSTEM
- Test vessel: sterile 250 mL Erlenmeyer flasks, sealed with cotton wool plugs, test volume 100 mL
- Aeration: Test containers were placed on a rotary shaker and oscillated at appr. 70 rpm
- Renewal rate of test solution: One application at test start
- Initial cells density: Nominal: approximately 5 x 103 - 104 cells/mL
Current: 6153 cells/mL
- Control end cells density: Table Cell Densities, see "other informations on results"
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): None


GROWTH MEDIUM
- Standard medium used: Yes



TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: According to the guideline (OECD)

Composition of Dilution water (OECD)

Component Concentration [mg/L]
NH4Cl 15
MgCl2 x 6 H2O 12
CaCl2 x 2 H2O 18
MgSO4 x 7 H2O 15
KH2PO4 1.6
FeCl3 x 6 H2O 0.064
Na2EDTA x 2 H2O 0.1
H3BO3 0.185
MnCl2 x 4 H2O 0.415
ZnCl2 3 x 10-3
Na2MoO4 x 2 H2O 7 x 10-3
CoCl2 x 6 H2O 1.5 x 10-3
CuCl2 x 2 H2O 1 x 10-5
NaHCO3 50
pH-value 8.1 +/- 0.2
This medium had a nominal hardness of 0.24 mmol Ca+Mg/L.


OTHER TEST CONDITIONS
- Sterile test conditions: no
-pH-Control: The pH-value at the start of exposure was measured in one additional replicate of each test item concentration and the control. At the end of exposure, the pH-value was measured from pooled samples of each test item concentration and the control after measurement of the cell
density.
- Photoperiod: 24 h
- Light intensity and quality: 60 - 120 µE x m-2 x s-1, Light intensity was measured prior to the start of exposure.
- Room temperature: The room temperature was measured continuously.


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.
- Microscopic evaluation: Microscopic evaluation of the cells at the start and the end of exposure was carried out. The cells were checked for unusual cell shapes, colour differences, differences in chloroplast morphology, flocculation, adherence of algae to test containers and agglutination
of algae cells.



TEST CONCENTRATIONS
- Range finding study: preliminary range finding test (non-GLP)
- Results used to determine the conditions for the definitive study:
Nominal test item
concentration
[mg/L] Growth Rate Inhibition Yield Inhibition
[%] [%]

100 100 100
10.0 4 18
1.00 0 -1
0.100 -1 -6
pH-Control 25 77
(100 mg/L, adjusted
to pH 8.10)

- Test concentrations: 5 concentrations were tested in a geometrical series with a dilution factor of
2.5: 2.56 - 6.40 - 16.0 - 40.0 - 100 mg/L.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

16 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

40 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

43.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 40.1 – 81.3

Details on results:

The study meets the validity criteria of the guideline
- Exponential growth in the control: yes

Results with reference substance (positive control):

- Results with reference substance valid? Yes
Current Study Valid Range (average ± 3 x SD)
Growth Rate inhibition
ErC50 0.613 0.810 ± 0.404
95 % confidence interval 0.589 - 0.638
Yield inhibition
EyC50 0.281 0.421 ± 0.274
95 % confidence interval 0.234 - 0.314

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 -value (acceptable probability of incorrectly concluding that there is a difference) is =0.05.

NOEC, LOEC, EC-values and 95 % Confidence Intervals of test item (0 - 72 hours)

                             based on nominal test item concentrations [mg/L]         

	Growth Rate Inhibition Nominal test item concentration [mg/L]
NOEC	16.0
LOEC	40.0
ErC10	34.5 (19.3 – 39.9)
ErC20	37.6 (20.9 – 39.9)
ErC50	43.5 (40.1 – 81.3)
	Yield Inhibition Nominal test item concentration [mg/L]
NOEC	16.0
LOEC	40.0
EyC10	33.9 (16.2 – 77.3)
EyC20	35.0 (16.6 – 77.7)
EyC50	37.1 (17.2 – 79.6)

Cell Densities

Nominal test item concentration	Replicate	Cell density [cells/mL]
[mg/L]	No.	0 hours	24 hours	48 hours	72 hours
100	1	6153	<LOQ	<LOQ	<LOQ
	2	6153	<LOQ	<LOQ	<LOQ
	3	6153	<LOQ	<LOQ	<LOQ
	Mean	6153	n.a.	n.a.	n.a.
40.0	1	6153	4193	31856	363365
	2	6153	4529	30338	355635
	3	6153	3879	30655	313613
	Mean	6153	4200	30950	344204
16.0	1	6153	25301	246985	2108352
	2	6153	28475	306136	2506122
	3	6153	24546	277969	2251228
	Mean	6153	26107	277030	2288567
6.40	1	6153	23583	304255	2173769
	2	6153	23740	243034	2184996
	3	6153	24041	250253	2273431
	Mean	6153	23788	265847	2210732
2.56	1	6153	25549	256939	2158967
	2	6153	16985	164772	1790236
	3	6153	24270	239691	2490348
	Mean	6153	22268	220467	2146517
pH-Control                         (100 mg/L,adjusted to pH 8.10)	1	6153	7056	11701	22366
	2	6153	6222	8016	19312
	3	6153	6780	9214	16718
	Mean	6153	6686	9644	19465
Control	1	6153	25681	294188	2282651
	2	6153	23351	263970	2215164
	3	6153	22548	253038	2074483
	4	6153	22122	249588	2256246
	5	6153	25474	232152	2029199
	6	6153	23119	222800	2026314
	Mean	6153	23716	252623	2147343

< LOQ = below the limit of quantification (2736 cells/mL)                n.a. = not applicable

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
[mg/L]	No.	[d^-1]		[%]	[cells/mL]		[%]
100	1		n.a.	100		n.a.	100
	2		n.a.	100		n.a.	100
	3		n.a.	100		n.a.	100
	Mean	(n.a.)	n.a.	100	(n.a.)	n.a.	100
40.0	1		1.36	30		357212	83
	2		1.35	31		349482	84
	3		1.31	33		307460	86
	Mean	(+)	1.34	31	(+)	338051	84
16.0	1		1.95	0		2102199	2
	2		2.00	-3		2499969	-17
	3		1.97	-1		2245075	-5
	Mean	(-)	1.97	-1	(-)	2282414	-7
6.40	1		1.96	0		2167616	-1
	2		1.96	0		2178843	-2
	3		1.97	-1		2267278	-6
	Mean	(-)	1.96	-1	(-)	2204579	-3
2.56	1		1.95	0		2152814	-1
	2		1.89	3		1784083	17
	3		2.00	-3		2484195	-16
	Mean	(-)	1.95	0	(-)	2140364	0
pH-Control                         (100 mg/L,adjusted to pH 8.10)	1		0.430	78		16213	99
	2		0.381	80		13159	99
	3		0.333	83		10565	100
	Mean	(+)	0.382	80	(+)	13312	99
Control	1		1.97			2276498
	2		1.96			2209011
	3		1.94			2068330
	4		1.97			2250093
	5		1.93			2023046
	6		1.93			2020161
	Mean		1.95			2141190

n.a. = not applicable (cell density below the limit of quantification (2736 cells/mL))

 

Environmental Conditions

Nominal test item concentration		pH-value
[mg/L]		Start; 0 hours	End; 72 hours
100		9.97	8.31
40.0		8.85	8.28
16.0		8.34	8.30
6.40		8.20	8.29
2.56		7.98	8.30
pH-Control (100 mg/L, neutralized)		8.11	8.04
Control		7.96	8.50
Room temperature [°C]:	min.: 22.0	max.: 22.5		mean value: 22.3
Light intensity [µE x m^-2x s^-1]	n = 33	mean value: 71.0 range of the measured values: 61.6 to 77.9 equalling -13.2 to 9.77 %		CV [%]: 7.34

Validity criteria fulfilled:

yes

Conclusions:

In this study, test item 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 43.5 (40.1 – 81.3) mg/L and 37.1 (17.2 – 79.6) mg/L, respectively. The NOEC-values for both inhibition of growth
rate and yield after 72 hours were 16.0 mg/L. The highest test concentration (100 mg/L) tested at an initial pH value 8.1 caused a comparable
inhibition of growth as the replicates without adjustment of the pH value.
All effect level are based on nominal concentration which had been analytically vertified.

Executive summary:

The toxicity of test item to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 761/2009/Method C.3 at Dr. U. Noack-Laboratorien, Sarstedt (Germany).

The study was conducted under static conditions at five concentrations in a geometrical series with a dilution factor of 2.5 (nominal): 2.56 - 6.40 - 16.0 - 40.0 - 100 mg/L. Additionally the neutralized concentration level of 100 mg/L was tested. The measured concentrations in the old media (72 h) were in the range of 88 to 118 % of the nominal values of all concentrations so that all effect values given are based on the nominal test item concentrations of test item.

In this study, the test substance was found to inhibit the growth of the freshwater green alga 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 (E_rC₅₀) and yield (E_yC₅₀) after 72 hours were 43.5 (40.1 – 81.3) mg/L and 37.1 (17.2 – 79.6) mg/L, respectively. The NOEC-values for both inhibition of growth rate and yield after 72 hours were 16.0 mg/L. The highest test concentration (100 mg/L) tested at an initial pH value 8.1 caused a comparable inhibition of growth as the replicates without adjustment of the pH value.

Description of key information

The growth inhibition effects of the test item on Scendesmus subspicatus were investigated in a study conducted by Hüls AG (1996) according to national guidance documents (UBA 1984). Seven test concentrations + control were tested over a period of 72 hours and the cell growth (by means of turbidity) was determined at daily intervals. The EC50 (biomass, 72 hours) was determined as 29.5 mg/L and the EC10 (biomass, 72 hours) indicating that the test substance may be harmful to algae.
A second growth inhibition study on alga was conducted in 2015. The toxicity of test item to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 761/2009/Method C.3 at Dr. U. Noack-Laboratorien, Sarstedt (Germany). 
The study was conducted under static conditions at five concentrations in a geometrical series with a dilution factor of 2.5 (nominal): 2.56 - 6.40 - 16.0 - 40.0 - 100 mg/L. Additionally the neutralized concentration level of 100 mg/L was tested. The measured concentrations in the old media (72 h) were in the range of 88 to 118 % of the nominal values of all concentrations so that all effect values given are based on the nominal test item concentrations of test item. 
In this study, the test substance was found to inhibit the growth of the freshwater green alga 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 43.5 (40.1 – 81.3) mg/L and 37.1 (17.2 – 79.6) mg/L, respectively. The NOEC-values for both inhibition of growth rate and yield after 72 hours were 16.0 mg/L. The highest test concentration (100 mg/L) tested at an initial pH value 8.1 caused a comparable inhibition of growth as the replicates without adjustment of the pH value.

Key value for chemical safety assessment

EC50 for freshwater algae:

43.5 mg/L

EC10 or NOEC for freshwater algae:

16 mg/L

Additional information

The latest alga growth inhibition study that was conducted in 2015 on request by ECHA and is regarded as key study because the older study is characterised by methodical deficiencies.