3-cyclohexylaminopropylamine

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:

2019-05-06 to 2019-07-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Five concentrations were tested in a geometrical series with factor of √10 in the concentration range of 0.316 - 1.00 - 3.16 - 10.0 - 31.6 - 100 mg/L.
- Sampling method: At the start, samples were taken from one additional replicate of the test item concentration and the control and at the end samples were taken from pooled replicates.
- Sample storage conditions before analysis: All samples were stored at room temperature until start of analysis, if necessary. Prepared samples were stored at room temperature until analysis.

Vehicle:

no

Details on test solutions:

Stock solution: 100 mg test item/L, freshly prepared with dilution water.
Dispersion treatment: The stock solution was agitated until the solution was visually clear. The stock solution was checked via laser beam for undissolved test
item. The tyndal effect was negative.
Test concentrations: The stock solution and further five concentration levels were tested in a geometrical series with a dilution factor of √10: 0.316 – 1.00 – 3.16 – 10.3 – 31.6 - 100.0 mg/L.
The concentration levels are based on the results of a preliminary range finding test (non-GLP).
Control: Six replicates (without test item) were tested under the same test conditions as the test item replicates.

Test organisms (species):

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

Details on test organisms:

Test organism Pseudokirchneriella subcapitata HINDÁK, CCAP 278/4

Synonyms Selenastrum capricornutum; Ankistrodesmus subcapitata; Raphidocelis subcapitata; Ankistrodesmus bibraianus (Experimental Phycology and Culture Collection of Algae at the University of Goettingen 2014)

Reason for the selection Pseudokirchneriella subcapitata is a suitable green alga species
of the test organism according to the guideline.

Origin Culture Collection of Algae and Protozoa (CCAP)
SAMS Research Services Ltd
Dunstaffnage Marine Laboratory
Dunbeg, OBAN; Argyll PA37 1QA; Scotland, UK

Cultivation at test facility Fresh stocks are prepared every month on Z-Agar. Light intensity amounted to 2567 - 5130 lux per day.

Culture medium Nutrient Medium Z according to LÜTTGE et al. (1994)
Preculture A four days old preculture, prepared in dilution water, was used as inoculum
ACCLIMATION
- Acclimation period: No
- Culturing media and conditions (same as test or not): Nutrient medium Z for culture and test medium according to OECDTG201
- Any deformed or abnormal cells observed: No

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

32 mg CaCO3/L

Test temperature:

21 - 24 °C controlled at ± 2°C

pH:

Control:
Start (0h): 7.97
End (72h): 9.21

Conductivity:

139 µS/cm

Nominal and measured concentrations:

Nominal concentration measured concentration

0 h:
Control < LOQ < LOQ (0.03 mg/L)
100 mg/L 99.3 mg/L
0.316 mg/L 0.311mg/L
72 h:
Control < LOQ < LOQ
100 mg/L 106 mg/L
0.316 mg/L 0.305 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Sterile glass Erlenmeyer flasks, volume: 250 mL
- Type (delete if not applicable): sealed with cotton wool plugs
- Material, size, headspace, fill volume: 100 mL
- Aeration: no
- Initial cells density: 5416 cells/mL
- Control end cells density: 2005038 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): 3
GROWTH MEDIUM
- Standard medium used: Nutrient medium Z according to LÜTTGE et al. (1994)
TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to OECDTG201
- Total organic carbon: < 2 mgC/L
- Alkalinity: pH = 7.97 (t= 0h)
- Conductivity: 139µS/cm
- Culture medium different from test medium: yes
- Intervals of water quality measurement: at start(0h) and at the end (72h) of study
OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: 24 hours/day light
- Light intensity and quality: Approximately 4440 to 8880 lux, corresponding to 60 to 120 µE*m-2*s-1
EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter]
- Chlorophyll measurement: The cell density was measured daily via Chlorophyll a- fluorescence, excitation at 436 nm, emission at 685 nm.
TEST CONCENTRATIONS
- Spacing factor for test concentrations: √10
- Justification for using less concentrations than requested by guideline:
- Range finding study: yes
- Test concentrations: 1; 10; 100 mg/L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

8.72 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

3.62 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

5.08 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

3.63 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

1.91 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.21 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): No morphological abnormalities observed.
- Unusual cell shape: No
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Other:
- Any stimulation of growth found in any treatment: 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
- ErC50: 0.701 mg/L (CI; 0.660 – 0.750)
- Other: EyC50 = 0.353 mg/l (CI; 0.312 – 0.394)

Evaluation after 72 hours of growth rates and yield inhibition    

Statistically significant differences of growth rate and yield inhibition 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.	100	(+)	n.a.	100
31.6	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
10.0	1		0.872	56		68576	97
	2		0.665	66		34388	98
	3		0.917	53		79475	96
	Mean	(+)	0.818	58	(+)	60813	97
3.16	1		1.84	7		1354312	32
	2		1.77	10		1082385	46
	3		1.80	8		1209975	39
	Mean	(+)	1.81	8	(+)	1215557	39
1.00	1		1.94	2		1815506	9
	2		1.91	3		1642692	18
	3		1.87	5		1479955	26
	Mean	(-)	1.91	3	(+)	1646051	18
0.316	1		1.97	0		1964759	2
	2		2.00	-2		2197157	-10
	3		1.98	-1		2063774	-3
	Mean	(-)	1.98	-1	(-)	2075230	-4
Control	1		1.98			2074386
	2		1.99			2116640
	3		1.99			2125598
	4		1.99			2134311
	5		1.92			1704759
	6		1.94			1842037
	Mean		1.97			1999622

n.a. = not applicable

Validity criteria fulfilled:

yes

Conclusions:

In this study, the EC50-values of the test item on the freshwater green alga Pseudokirchneriella subcapitata based on nominal test item concentrations after 72 hours were 8.72 (8.11 – 9.28) mg/L and 3.63 (3.06 – 4.42) mg/L, for inhibition of growth rate and yield (EyC50), respectively. The NOEC -values for growth rate and yield inhibition were 1.00 and 0.316 mg/L respectively.

Executive summary:

The toxicity of the test item 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. The aim of the study was the determination of 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 5416 cells/mL. Six concentrations were tested in a geometrical series with a factor of √10 in the concentration range of0.316 - 1.00 - 3.16 - 10.0 - 31.6 - 100 mg/L. A stock solution with a nominal concentration level of 100 mg test item/L was freshly prepared and agitated until the solution was visually clear.The stock solution was checked via laser beam (Tyndall effect) for undissolved test item. The Tyndall effect was negative. The solution was then used for the preparation of the dilutions.

Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditions were within the acceptable limits.

The measured concentrations in the test solutions at the start of the exposure were in the range of 95 to 103% of the nominal concentrations. At the end of exposure after 72 hours, the measured concentrations in the test item solutions were in the range of 96 to 106% of the nominal test item concentrations. As the concentrations remain in the range of ± 20% of the nominal concentrations, all effect values given are based on the nominal test item concentrations.

The EC₅₀-values of the test item on the freshwater green alga Pseudokirchneriella subcapitataafter 72 hours were 8.72 (8.11 – 9.28) mg/L and 3.63 (3.06 – 4.42) mg/L, for inhibition of growth rate and yield (EyC₅₀), respectively. The NOEC -values for growth rate and yield inhibition were 1.00 and 0.316 mg/L respectively.

Description of key information

The toxicity of the test item 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. The aim of the study was the determination of 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 5416 cells/mL. Six concentrations were tested in a geometrical series with a factor of √10 in the concentration range of0.316 - 1.00 - 3.16 - 10.0 - 31.6 - 100 mg/L. A stock solution with a nominal concentration level of 100 mg test item/L was freshly prepared and agitated until the solution was visually clear.The stock solution was checked via laser beam (Tyndall effect) for undissolved test item. The Tyndall effect was negative. The solution was then used for the preparation of the dilutions.

Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditions were within the acceptable limits.

The measured concentrations in the test solutions at the start of the exposure were in the range of 95 to 103% of the nominal concentrations. At the end of exposure after 72 hours, the measured concentrations in the test item solutions were in the range of 96 to 106% of the nominal test item concentrations. As the concentrations remain in the range of ± 20% of the nominal concentrations, all effect values given are based on the nominal test item concentrations.

The EC₅₀-values of the test item on the freshwater green alga Pseudokirchneriella subcapitataafter 72 hours were 8.72 (8.11 – 9.28) mg/L and 3.63 (3.06 – 4.42) mg/L, for inhibition of growth rate and yield (EyC₅₀), respectively. The NOEC -values for growth rate and yield inhibition were 1.00 and 0.316 mg/L respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:

8.72 mg/L

EC10 or NOEC for freshwater algae:

3.62 mg/L