2-Propanol, 1,3-bis[(3-methyl-2-buten-1-yl)oxy]-

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

effects on growth of green algae

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-09-28 to 2021-10-28

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:

adopted 2006-03-23, Annex 5 corrected: 2011-07-28

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: all test item concentrations and control (0 h and 72 h), retain samples were taken
- Sample storage conditions before analysis: Deep frozen

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: 60 minutes stirring of stock solution at 100 mg/L
- Controls: Test medium control
- Concentration of final test solutions: 100, 50.0, 25.0, 12.5, 6.25 mg/L and control
- Test concentration separation factor: 2.0
- Evidence of undissolved material: Solutions were clear and transparent

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata (Korshikov) Hindák (Sphaeropleales: Selenastraceae), formerly known as Selenastrum capricornutum, and renamed as Raphidocelis subcapitata (Korshikov) Nygaard, Komárek, J.Kristiansen & O.M.Skulberg
- Strain: SAG 61.81
- Source (laboratory, culture collection): MBM Sciencebridge GmbH, Hans-Adolf-Krebs-Weg 1, D-37077 Göttingen, Germany
- Age of inoculum (at test initiation): 3 to 4 days before start of the test, test medium was inoculated with the test organism and held under test conditions in order to produce a pre-culture in the state of exponential growth.
- Method of cultivation: The algae are grown semi-continuously in sterile cultures in the laboratory. Old medium is periodically replaced by fresh mineral solution in order to keep the algae in an exponential growth state. Stock cultures are ordered regularly from our commercial supplier.

ACCLIMATION
- Culturing media and conditions (same as test or not): same

Test type:

static

Water media type:

freshwater

Remarks:

AAP-Medium (according to Annex 3 of OECD 201)

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22.6 – 23.3 °C

pH:

Control: 7.42 – 8.63
Test solutions: 7.40 - 8.91

Nominal and measured concentrations:

Nominal: 100, 50.0, 25.0, 12.5, 6.25 mg/L and control
Measured (geometric mean): 90.5, 43.0, 20.9, 9.59, 4.04 mg/L and control

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks with aluminium caps
- Type (delete if not applicable): open
- Fill volume: ~ 50 mL
- Aeration: By continuous agitation: Test vessels were placed in an incubator on a pivoted bogie which turns around and induces shaking by regular sudden stops
- Initial cells density: 0.59 × 10^4 cells/mL
- Control end cells density: 74.42 × 10^4 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: AAP-Medium (according to Annex 3 of OECD 201)
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Sterile test conditions: yes/no
- Adjustment of pH: yes (The pH was adjusted to 7.5 ± 0.1 with NaOH or HCl.)
- Photoperiod: Continous illumination
- Light intensity and quality: 94.7 µEm-2s-1 (mean light intensity)

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: At test start (0 hours) the number of cells in each control replicate was determined in duplicate. At defined time periods (24, 48 and 72 hours), the number of cells in each replicate was determined in duplicate. The determination was performed by fluorescence measurement. The fluorescence measurements were performed with a fluorescence microplate reader (infinite 200Pro) with an emission wavelength of 670 nm and evaluated with Tecan i-control (Software for Tecan Readers Tecan i-control, 2.0.10.0).

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.0
- Test concentrations: 100, 50.0, 25.0, 12.5, 6.25 mg/L

CULTURING APPARATUS
-Details on culturing apparatus used: The incubation took place in a temperature controlled light incubator. The incubator is equipped with a pivoted bogie consisting of several platforms. The bogie turns around passing laterally placed fluorescent tubes, which enables uniform lighting of the test vessels.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate is tested as the toxic reference item in a separate study twice a year to confirm the sensitivity of the test organism against compounds with known effects under the test conditions.

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

9.59 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

Growth rate and yield

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

58.7 mg/L

95% CI:

51.4 - 68.1

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 90.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

66.7 mg/L

95% CI:

60.9 - 73.6

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

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

Remarks:

Growth rate and yield

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Unusual cell shape: The morphology of the algae cells was investigated microscopically at test end. The cells were considered normal for the control and up to and including a nominal test item concentration of 50.0 mg/L. At test item concentration 100 mg/L (nominal) deformed cells which were bloated or were narrowed at one end of the cell were observed.
- Any stimulation of growth found in any treatment: yes, at 6.25 and 12.5 mg/L
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: All solutions appeared to be clear and transparent.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- ErC50: 1.55 mg/L
- Other: The EC50 values calculated in this reference test were considered to be within an acceptable range therefore it can be considered that the test organism is sensitive.

Reported statistics and error estimates:

The statistical evaluation of NOEC/LOEC for the 72 hours period was performed for growth rate and yield using SAS® (2016).

A test for normality of the data was performed by calculating the Shapiro-Wilks statistic and the homogeneity of variance of the data was evaluated by the Levene’s test. The NOEC and LOEC were determined by using a multiple comparison method (Bonferroni-Holms corrected Welch test, left sided, for growth rate and yield).

The EC10, 20, 50-values for growth rate and yield after 72 h were determined with ToxRat Professional, Version 3.3.0. ToxRat Professional, Version 3.3.0 performs calculations with unrounded values.

The EC10, 20-values for growth rate and EC10, 20, 50-values for yield were determined by probit analysis using maximum linear likelihood regression.

Due to an inhibition of growth rate below 50 % at all test concentrations, a statistical determination of the EC50-values was not appropriate and the value was considered to be higher than the highest test item concentration.

Validity Criteria of the Study

Biomass: Cell numbers, measured in the controls between 0 and 72 hours, were found to increase by a factor of 126.14 (corresponding to a specific growth rate of 1.61263 d^-1), which exceeds the threshold of 16 (corresponding to a specific growth rate of 0.92 d^-1).

Coefficient of variation (section by section): The mean coefficient of variation for the section-by-section specific growth rates (hours 0 - 24, 24 - 48 and 48 - 72) in the control cultures was 7 % and did not exceed 35 %.

Coefficient of variation (average growth rate): The coefficient of variation of average growth rate in replicate control cultures was 0.7 % and did not exceed 7 % for the whole test period.

 

Table 1:       Average cell numbers for each sampling time and concentration

Conc.	Average cell numbers [10^4/mL]
[mg/L]	0 h	24 h	48 h	72 h
Control	0.59	2.64	14.52	74.42
6.25	0.59	2.89	17.59	91.19
12.5	0.59	2.70	18.73	83.20
25.0	0.59	2.41	15.00	68.91
50.0	0.59	2.22	11.83	46.17
100	0.59	1.82	7.94	23.91

 

Table 2:          Percentage inhibition of growth rate

Conc.	% Inhibition of growth rate
[mg/L]	0 h – 24 h	0 h – 48 h	0 h – 72 h
Control	0.0	0.0	0.0
6.25	-6.0	-5.9	-4.1
12.5	-1.6	-7.9	-2.3
25.0	5.9	-0.9	1.6*
50.0	11.4	6.5	10.1*
100	24.8	19.2	23.7*

* Statistically significant different to the control

 

Table 3:          Percentage inhibition of yield

Conc.	% Inhibition of yield
[mg/L]	0 h – 24 h	0 h – 48 h	0 h – 72 h
Control	0.0	0.0	0.0
6.25	-12.2	-22.0	-22.7
12.5	-2.9	-30.2	-11.9
25.0	11.2	-3.4	7.5*
50.0	20.5	19.3	38.3*
100	40.0	47.2	68.4*

* Statistically significant different to the control

 

Table 4:          pH measurements during the test

Conc.	t = 0 h	t = 72 h
[mg/L]
Control	7.42	8.63
6.25	7.42	8.76
12.5	7.42	8.91
25.0	7.42	8.80
50.0	7.40	8.72
100	7.41	8.36

 

Table 5:          Temperature during the test

Time	Temperature [°C]
[h]	min	max
0	22.9
24	22.6	23.2
48	22.6	23.3
72	22.6	23.3

 

Table 6:          Individual cell numbers

	Cell numbers [10^4/mL]
Conc.	0 h	24 h	48 h	72 h	Yield
[mg/L]					0 h – 72 h
	0.62	2.78	14.41	79.20	78.58
	0.59	2.49	15.50	73.24	72.65
Control	0.57	2.53	13.87	73.34	72.77
	0.63	2.71	14.03	75.14	74.51
	0.57	2.71	14.50	72.10	71.53
	0.56	2.59	14.78	73.50	72.94
Mean	0.59	2.64	14.52	74.42	73.83
	0.59	2.79	19.01	91.78	91.19
6.25	0.59	2.82	16.13	80.54	79.95
	0.59	3.05	17.63	101.24	100.65
Mean	0.59	2.89	17.59	91.19	90.60
	0.59	2.80	20.29	81.42	80.83
12.5	0.59	2.64	17.62	84.12	83.53
	0.59	2.66	18.28	84.06	83.47
Mean	0.59	2.70	18.73	83.20	82.61
	0.59	2.41	13.55	70.18	69.59
25.0	0.59	2.33	16.65	68.78	68.19
	0.59	2.50	14.81	67.78	67.19
Mean	0.59	2.41	15.00	68.91	68.32
	0.59	2.09	10.95	40.94	40.35
50.0	0.59	2.35	12.65	55.12	54.53
	0.59	2.23	11.88	42.44	41.85
Mean	0.59	2.22	11.83	46.17	45.58
	0.59	1.94	9.30	27.00	26.41
100	0.59	1.90	8.16	25.10	24.51
	0.59	1.63	6.35	19.62	19.03
Mean	0.59	1.82	7.94	23.91	23.32

 

Table 7:          Control, growth rates

	Growth rates, µ [d^-1]
	0 h – 24 h	0 h – 48 h	0 h – 72 h
	1.50049	1.57298	1.61667
	1.43992	1.63424	1.60712
Control	1.49034	1.59592	1.61908
	1.45898	1.55162	1.59380
	1.55907	1.61813	1.61339
	1.53148	1.63655	1.62570
Mean	1.49671	1.60157	1.61263
Std. Dev.	0.04427	0.03438	0.01108
CV [%]	3.0	2.1	0.7

Std. Dev.: Standard deviation

CV: Coefficient of variation

 

Table 8:          Control, daily growth rates

	Growth rates, µ [d^-1]
	0 h – 24 h	24 h – 48 h	48 h – 72 h
	1.50049	1.64547	1.70405
	1.43992	1.82856	1.55290
Control	1.49034	1.70151	1.66538
	1.45898	1.64425	1.67816
	1.55907	1.67720	1.60391
	1.53148	1.74162	1.60401
Mean	1.49671	1.70644	1.63474
Std. Dev.	0.04427	0.07016	0.05696
CV [%]	3.0	4.1	3.5

Std. Dev.: Standard deviation

CV: Coefficient of variation

 

Table 9:       Coefficient of variation (CV) of daily growth rates in the control

Mean daily growth rates µ [d^-1]	Std. Dev.  [d^-1]	CV [%]
1.61667	0.10479	6
1.60713	0.19991	12
1.61908	0.11294	7
1.59380	0.11798	7
1.61339	0.05963	4
1.62570	0.10674	7
	Mean CV	7

Std. Dev.: Standard deviation

CV: Coefficient of variation

 

Table 10:        Calibration data for cell numbers

Cell number [10^4/mL] ^1)	Fluorescence measurement ^2)				Mean	Std. Dev.	CV [%]
	Replicate 1	Replicate 2	Replicate 3	Replicate 4
73.00	10735.00	11052.00	10969.00	11095.00	10962.75	160.59	1.46
37.25	5652.00	5706.00	5651.00	5647.00	5664.00	28.08	0.50
17.25	3060.00	2923.00	2887.00	2899.00	2942.25	79.91	2.72
10.50	1437.00	1525.00	1398.00	1497.00	1464.25	57.43	3.92
4.88	753.00	779.00	783.00	765.00	770.00	13.71	1.78
2.31	384.00	425.00	365.00	393.00	391.75	25.05	6.39
1.19	117.00	191.00	188.00	224.00	180.00	45.06	25.03
0.56	114.00	85.00	103.00	98.00	100.00	12.03	12.03
0.19	58.00	58.00	59.00	64.00	59.75	2.87	4.80
0.06	18.00	26.00	22.00	18.00	21.00	3.83	18.24
0.00	-1.00	-1.00	0.00	2.00	0.00	1.41	- ^3)

¹⁾ The cell numbers were counted with a Neubauer chamber

²⁾ Measured with a TECAN Reader

³⁾ Not calculable due to a mean of zero

Std. Dev.: Standard deviation

CV: Coefficient of variation

 

Table 11:        Composition of test medium AAP-Medium

Substance	Conc. in test medium
	[mg/L]
NaHCO3	15.0
K2HPO4	1.044
MgSO4·7H2O	14.6
NaNO3 CaCl2·2H2O MgCl2·6H2O	25.5 4.41 12.16
H3BO3 MnCl2·4H2O ZnCl2 CoCl2·6H2O CuCl2·2H2O Na2MoO4·2H2O FeCl3 ·6H2O Na2EDTA·2H2O	0.186 0.415 0.00327 0.00143 0.000012 0.00726 0.160 0.300

 

Table 12:        Determined concentration of DPNG

Test item nominal [mg/L]	DPNG nominal [mg/L]	Sampling	DPNG found		Mean measured test item conc. [mg/L]
			[mg/L]	% of nominal
Control	0	0 h fresh	< LOD	-	-
		72 h aged	< LOD	-
6.25	6.18	0 h fresh	3.66	59	4.04
		72 h aged	4.41	71
12.5	12.4	0 h fresh	10.4	84	9.59
		72 h aged	8.66	70
25.0	24.7	0 h fresh	20.9	85	20.9
		72 h aged	20.3	82
50.0	49.4	0 h fresh	41.4	84	43.0
		72 h aged	43.7	88
100	98.8	0 h fresh	89.2	90	90.5
		72 h aged	90.0	91

- = not calculated; LOD = 0.188 mg/L DPNG, LOQ = 0.625 mg/L DPNG

Validity criteria fulfilled:

yes

Conclusions:

Significant inhibitory effects were determined for growth rate and yield at test item concentrations of 25.0 mg/L (nominal) and 20.9 mg/L (mean measured) and above. The overall LOEC was therefore determined to be 25.0 mg/L (nominal) and 20.9 mg/L (mean measured), the corresponding NOEC was set at 12.5 mg/L (nominal) and 9.59 mg/L (mean measured).
The EC10-value for growth rate (ErC10) was determined to be 52.7 mg/L (nominal) and 45.6 mg/L (mean measured). The EC10-value for yield (EyC10) was 24.9 mg/L (nominal) and 20.6 mg/L (mean measured).
The EC20-value for growth rate (ErC20) was determined to be 86.2 mg/L (nominal) and 77.2 mg/L (mean measured). The EC20-value for yield (EyC20) was 35.0 mg/L (nominal) and 29.5 mg/L (mean measured).
The EC50-value for growth rate (ErC50) was considered to be > 100 mg/L (nominal) and > 90.5 mg/L (mean measured). The EC50-value for yield (EyC50) was determined to be 66.7 mg/L (nominal) and 58.7 mg/L (mean measured).

Description of key information

The objective of this valid GLP study was to determine the effects of the test item on the growth of the single cell green alga Pseudokirchneriella subcapitata. The EC₅₀-value for growth rate (E_rC₅₀) was considered to be > 100 mg/L (nominal) and > 90.5 mg/L (mean measured). 

Key value for chemical safety assessment

EC50 for freshwater algae:

90.5 mg/L