2,2-Bis(2-aminoethoxy)propane

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:

2020-05-25 to 2020-05-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Annex 5 corrected 28 July 2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

01 March 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Sampling method: For determination of the test item concentrations in the test solutions, all three test vessel were analysed at the start and at the end of the study. In case of control samples, all six test vessel were analysed at the start and at the end of the test.
- Sample storage conditions before analysis: no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method:
The test solutions used in the test was prepared by mechanical dispersion without using of any solubilising agent.
For preparation of test solutions a stock solution of 100 mg/L (nominal concentration) was first prepared by dissolving an amount of 0.0817 g test item in 817 mL OECD medium. The test solutions of subsequent concentrations were prepared by appropriate dilution of this stock solutions
- Controls: negative (blank) control, positive control

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: 61.81 SAG (identical strains: CCAP 278/4; UTEX 1648; ATCC 22662)
- Source: The algae were supplied by the SAG: Collection of Algal Cultures, Inst. Plant Physiology, University of Göttingen, Untere Karspüle 2, D-37073 Göttingen, Germany.
- Age of inoculum: 3 days (exponentially growth)
- Method of cultivation: The stock cultures are small algal cultures that are planted on agar. These are transferred to fresh medium at least once every two months under standardised conditions according to the test guidelines. The pre-culture was prepared with OECD Medium, incubated under the conditions of the test and used when still exponentially growing, normally after an incubation period of 2-4 days. (The pre-culture was incubated for three days at this test.)

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

None

Hardness:

not specified

Test temperature:

22.5 – 23.3°C

pH:

7.59 – 9.60

Dissolved oxygen:

not specified

Salinity:

not relevant

Conductivity:

not specified

Nominal and measured concentrations:

nominal: 0 (blank control), 6.25, 12.5, 25, 50 and 100 mg/L
measured (at the start of the test): 6.167, 12.482, 25.222, 50.244 and 100.883 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Type: closed with permeable stoppers
- Material, size, fill volume: glass, ~250 mL nominal volume,100 mL fill volume
- Aeration: agitated by using an orbital shaker
- Initial cells density: 10^4 cells/mL in each test flask
- Control end cells density: 10^4 cells/mL
- No. of vessels per concentration: 3
- No. of vessels per control: 6

GROWTH MEDIUM
- Standard medium used: yes (OECD Medium)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reconstituted algal growth medium (OECD medium, according to OECD 201) prepared with deionised water

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continous illumination
- Light intensity and quality: 6988 lux, wavelength range: 400 – 700 nm

DETERMINATION OF CELL CONCENTRATION
- Determination of cell concentrations: counting chamber

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study: performed
- Test concentrations: 0 (control), 0.01, 0.1, 1, 10 and 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.:

> 100 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.:

21.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

12.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

38.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

7.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

12.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

39.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

13.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

- Exponential growth in the control: yes
- Observation of abnormalities: no
- Unusual cell shape: No morphological abnormalities were observed in the control and in the concentrations of 6.25, 12.5 and 25 mg/L during the test.
Swollen cells were observed together with the normal cells in the concentrations of 50 and 100 mg/L.
- Colour differences: no
- Flocculation: no
- Adherence to test vessels: no
- Aggregation of algal cells: no
- Other: none
- Any stimulation of growth found in any treatment: no
- Any observations 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):

72h ErC50: 0.52 mg/L (95 % confidence limits: 0.46 – 0.58 mg/L)
72h EyC50: 0.31 mg/L (95 % confidence limits: 0.28 – 0.35 mg/L)

Reported statistics and error estimates:

For the determination of the LOEC and NOEC, Jonckheere-Terpstra Test (for growth rate) and Dunnett-t test (for yield) was used by SPSS software. For the determination of the ECx values Probit analysis was used by SPSS software.

Table 1: Algal cell number (× 10⁴cell/mL)during the test period of 72 hours and Yield

Concentration [mg/L]		Number of Cells (× 10^4/mL)				Yield
		0 h	24 h	48 h	72 h
Control		1	3	10	49	48
		1	2	8	47	46
		1	3	8	36	35
		1	2	9	52	51
		1	2	6	44	43
		1	3	7	33	32
	Mean	1.00	2.50	8.00	43.50	42.5
	SD	0.00	0.55	1.41	7.50	7.50
6.25		1	4	8	43	42
		1	3	6	39	38
		1	3	7	36	35
	Mean	1.00	3.33	7.00	39.33	38.3
	SD	0.00	0.58	1.00	3.51	3.51
12.5		1	2	6	36	35
		1	3	6	29	28
		1	2	4	37	36
	Mean	1.00	2.33	5.33	34.00	33.0
	SD	0.00	0.58	1.15	4.36	4.36
25		1	2	6	32	31
		1	3	5	31	30
		1	3	5	28	27
	Mean	1.00	2.67	5.33	30.33	29.3
	SD	0.00	0.58	0.58	2.08	2.08
50		1	3	5	30	29
		1	2	5	18	17
		1	3	3	20	19
	Mean	1.00	2.67	4.33	22.67	21.7
	SD	0.00	0.58	1.15	6.43	6.43
100		1	2	3	11	10
		1	2	2	6	5
		1	3	3	4	3
	Mean	1.00	2.33	2.67	7.00	6.0
	SD	0.00	0.58	0.58	3.61	3.61

Table 2: The specific Growth Ratesduring the test period of 72 hours

Concentration [mg/L]		Growth Rates (µ)
		0 – 24 h	0 – 48 h	0 – 72 h
Control		0.0458	0.0480	0.0541
		0.0289	0.0433	0.0535
		0.0458	0.0433	0.0498
		0.0289	0.0458	0.0549
		0.0289	0.0373	0.0526
		0.0458	0.0405	0.0486
	Mean	0.0373	0.0430	0.0522
	SD	0.0093	0.0038	0.0025
6.25		0.0578	0.0433	0.0522
		0.0458	0.0373	0.0509
		0.0458	0.0405	0.0498
	Mean	0.0498	0.0404	0.0510
	SD	0.0069	0.0030	0.0012
12.5		0.0289	0.0373	0.0498
		0.0458	0.0373	0.0468
		0.0289	0.0289	0.0502
	Mean	0.0345	0.0345	0.0489
	SD	0.0098	0.0049	0.0019
25		0.0289	0.0373	0.0481
		0.0458	0.0335	0.0477
		0.0458	0.0335	0.0463
	Mean	0.0401	0.0348	0.0474
	SD	0.0098	0.0022	0.0010
50		0.0458	0.0335	0.0472
		0.0289	0.0335	0.0401
		0.0458	0.0229	0.0416
	Mean	0.0401	0.0300	0.0430
	SD	0.0098	0.0061	0.0037
100		0.0289	0.0229	0.0333
		0.0289	0.0144	0.0249
		0.0458	0.0229	0.0193
	Mean	0.0345	0.0201	0.0258
	SD	0.0098	0.0049	0.0071

Table 3: Growth Rates (µ) and percentage inhibition of µ during the test period

Concentration [mg/L]	Growth Rates (µ)and % Inhibition ofµ
	0–24 h		0–48 h		0–72 h
	µ	%	µ	%	µ	%
Control	0.0373	–	0.0430	–	0.0522	–
6.25	0.0498	-33.3	0.0404	6.1	0.0510	2.4
12.5	0.0345	7.5	0.0345	19.8	0.0489	6.4
25	0.0401	-7.5	0.0348	19.2	0.0474	9.3
50	0.0401	-7.5	0.0300	30.3	0.0430	17.7
100	0.0345	7.5	0.0201	53.4	0.0258	50.6

 

Table 4: Morphological changes of algal cells

Concentration[mg/L]	Morphological changes
	24 h	48 h	72 h
Control	- normal cells	- normal cells	- normal cells
6.25	- normal cells	- normal cells	- normal cells
12.5	- normal cells	- normal cells	- normal cells
25	- normal cells	- normal cells	- normal cells
50	- normal cells	- normal cells - some swollen cells	- normal cells - some swollen cells
100	- normal cells - swollen cells	- normal cells - swollen cells	- normal cells - swollen cells

 Table 5: Concentration of Test Item measured in the Test Solutions

Nominal concentration mg/L	Mean of the measured concentrations
	Start (May 25, 2020)		End (May 28, 2020)
	Concentration (mg/L)	% of the nominal	Concentration (mg/L)	% of the nominal
Control	not detected	-	not detected	-
6.25	6.167	99	5.065	81
12.5	12.482	100	10.686	85
25	25.222	101	22.318	89
50	50.244	100	51.060	102
100	100.883	101	102.957	103

Validity criteria fulfilled:

yes

Conclusions:

In an algae growth inhibition test according to OECD TG 201 with Raphidocelis subcapitata the 72-hour EC50 and EC10 for the growth rate was determined to be > 100 mg/L and 21.8 mg/L (nominal), respectively. The 72-hour EC50 and EC10 based on biomass were found to be 38.3 mg/L and 7.5 mg/L (nominal), respectively. The 72-hour NOEC was 12.5 mg/L (nominal) both for the growth rate and biomass.

Executive summary:

To determine the effect of the test item on the growth of a unicellular green algal species Raphidocelis subcapitata a growth inhibition test according to OECD TG 201 and EU Method C.3. was carried out in compliance with GLP principles. Exponentially growing cultures of Raphidocelis subcapitata were exposed for 72 hours to nominal test item concentration of 6.25, 12.5, 25, 50 and 100 mg/L over several generations under defined conditions. An untreated control group (OECD Test Medium without test item) was tested parallel with the treated cultures. The test design included three replicates at each test concentration and six replicates for the untreated control. The alga cell concentration was approximately 10⁴ cells/mL at the start of the test in all of the test cultures. The algal cell concentration was determined in 24-hour intervals by manual cell counting using a microscope. The nominal test item concentrations were analytically verified by using a HPLC-UV method. The measured concentrations remained within ± 20 % of the nominal over the test period of 72 hours. All validity criteria of the guidelines were met. The 72-hour EC50 and EC10 for the growth rate was determined to be > 100 mg/L and 21.8 mg/L in nominal, respectively. The 72-hour EC50 and EC10 based on biomass were found to be 38.3 and 7.5 mg/L in nominal, respectively. The 72-hour NOEC was 12.5 mg/L in nominal both for the growth rate and biomass.

Description of key information

In an algae growth inhibition test according to OECD TG 201 with Raphidocelis subcapitata the 72-hour EC50 and EC10 for the growth rate was determined to be > 100 mg/L and 21.8 mg/L in nominal, respectively. The 72-hour EC50 and EC10 based on biomass were found to be 38.3 and 7.5 mg/L in nominal, respectively. The 72-hour NOEC was 12.5 mg/L in nominal for both the growth rate and biomass.

Additional information

To determine the effect of the test item on the growth of a unicellular green algal species Raphidocelis subcapitata a growth inhibition test according to OECD TG 201 and EU Method C.3. was carried out in compliance with GLP principles. Exponentially growing cultures of Raphidocelis subcapitata were exposed for 72 hours to nominal test item concentration of 6.25, 12.5, 25, 50 and 100 mg/L over several generations under defined conditions. An untreated control group (OECD Test Medium without test item) was tested parallel with the treated cultures. The test design included three replicates at each test concentration and six replicates for the untreated control. The alga cell concentration was approximately 10⁴ cells/mL at the start of the test in all of the test cultures. The algal cell concentration was determined in 24-hour intervals by manual cell counting using a microscope. The nominal test item concentrations were analytically verified by using a HPLC-UV method. The measured concentrations remained within ± 20 % of the nominal over the test period of 72 hours. All validity criteria of the guidelines were met. The 72-hour EC50 and EC10 for the growth rate was determined to be > 100 mg/L and 21.8 mg/L, respectively. The 72-hour EC50 and EC10 based on biomass were found to be 38.3 and 7.5 mg/L, respectively. The 72-hour NOEC was 12.5 mg/L both for the growth rate and biomass.