3-hydroxypropyl octanoate

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:

2020-02-07 to 2020-05-04

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, corrected 2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

Commission Regulation (EU) No 2016/266 of December 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 3.2, 10, 32 and 100 mg/L

- Sampling method: For measurement of the actual concentrations of the test item, duplicate samples were taken from the test media at all test concentrations and the control at the start (without algae) and at the end of the test (with algae). For sampling at the end of the test, the test medium of the treatment replicates was pooled.

- Sample storage conditions before analysis: All samples were frozen (at -20 ± 5 °C) immediately after sampling and stored until analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test medium of the highest nominal concentration of 100 mg/L was prepared by dissolving 99.98 mg of the test item completely in 1000 mL of test water using ultrasonic treatment for 15 minutes and intense stirring for 15 minutes at room temperature in the dark. The test media of the lower test item concentrations were prepared in a series of further dilution steps. The test media were prepared just before the start of the test.

- Controls: A control (test water without test item) was tested in parallel

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain: 61.81 SAG
- Source: Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, 37073 Göttingen /Germany)
- Age of inoculum: 3 days (exponentially growth)
- Method of cultivation: The algae were cultivated under the test conditions and were kept in the exponential growth phase until inoculation of the test solutions.

ACCLIMATION
- Culturing media and conditions: same as test

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

15 mg/L as CaCO3

Test temperature:

22 °C

pH:

7.5 to 8.2

Dissolved oxygen:

not specified

Salinity:

not relevant

Conductivity:

not specified

Nominal and measured concentrations:

Nominal: control, 1.0, 3.2, 10, 32, 100 mg/L;
Mean Measured (geometric mean): n.a., n.a., 0.021, 0.33, 1.1, 80 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Type: closed
- Material, size, fill volume: glass, 75 mL, 30 mL
- Initial cells density: 5000 cells/mL
- Control end cells density: 5000 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: reconstituted algal growth medium (OECD medium, according to OECD 201)

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuous
- Light intensity and quality: ~ 67 μE s-1 m-2

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: electronic particle counter

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.125
- Range finding study: performed
- Test concentrations: 0.1, 1.0, 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:

48 h

Dose descriptor:

EC50

Effect conc.:

3.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % Confidence Interval: 2.6 - 5.8

Key result

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

0.12 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % Confidence Interval: 0.077 - 0.17

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.33 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

1.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

1.9 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95 % Confidence Interval: 1.3 - 2.7

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

0.066 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95 % Confidence Interval: 0.043 – 0.096

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.33 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

1.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

- Exponential growth in the control: yes
- Observation of abnormalities: no
- Unusual cell shape: The shape and size of the algal cells were obviously not affected by the test item up to at least the mean measured test concentration of 1.1 mg/L (nominal 32 mg/L)
- Colour differences: no
- Flocculation: no
- Adherence to test vessels: no
- Aggregation of algal cells: no
- 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

Reported statistics and error estimates:

The 48-hour EC10, EC20 and EC50 values for the inhibition of average growth rate and yield and their 95% confidence intervals were calculated by Probit Analysis using linear weighted regression.
For the determination of the 48-hour LOEC and NOEC, the average growth rate and yield at the test concentrations were compared to the control values by Williams t-test and Welch t-test with Bonferroni-Holm-adjustment, where appropriate.
Statistical analysis was performed using ToxRat Professional®.

Table 1: Biomass of Algae

Nominal Test Item Concentration (mg/L)	Mean Measured Test Item Concentration (mg/L)	Repl. No.	Biomass of Algae*
			24 Hours	48 Hours	72 Hours**
Control	--	1	2.11	13.4	62.4
		2	1.89	13.6	57.9
		3	2.52	13.0	60.6
		4	2.13	14.4	63.2
		5	2.08	13.6	58.4
		6	2.13	13.5	60.3
		Mean SD	2.14	13.6	60.5
			0.204	0.447	2.09
1.0	n.a	1	2.15	16.4	64.5
		2	2.54	14.2	62.1
		3	2.10	16.0	63.9
		Mean SD	2.26	15.5	63.5
			0.238	1.15	1.28
3.2	0.021	1	1.46	13.0	59.7
		2	1.57	13.5	60.6
		3	2.19	13.7	61.7
		Mean SD	1.74	13.4	60.7
			0.397	0.358	1.00
10	0.33	1	1.77	12.4	55.1
		2	1.69	15.5	53.1
		3	2.04	14.1	54.5
		Mean SD	1.83	14.0	54.2
			0.185	1.56	1.02
32	1.1	1	1.73	6.04	20.3
		2	1.37	4.00	10.8
		3	1.55	6.49	19.2
		Mean SD	1.55	5.51	16.8
			0.181	1.33	5.18
100	80	1	0.468	0.306	0.249
		2	0.468	0.634	0.121
		3	0.525	0.490	0.176
		Mean SD	0.487	0.477	0.182
			0.0331	0.164	0.0643

n.a.: Not analyzed since the inhibition of growth rate and yield was below the 48 and 72-hour NOEC.

SD: Standard deviation

*: The biomass was determined by fluorescence measurement (mean of duplicate measurements per replicate) and is given as relative fluorescence units (x 104). At the start of the test, the initial cell density was 5000 algal cells/mL, corresponding to 0.696 x 104 relative fluorescence units.

**: The biological results after 72 hours were not used for the calculation of the ECx and NOEC/LOEC values due to the decrease of the substance after 48 hours.

Table 2: Average Growth Rates (μ)

Nominal Test Item Concentration (mg/L)	Mean Measured Concentration (mg/L)	Average Growth Rate μ [day-1] and Inhibition Ir [%]
		0-24 h		0-48 h		0-72 h**
		μ [day-1]	Ir [%]	μ [day-1]	Ir [%]	μ [day-1]	Ir [%]
Control	---	1.121	0.0	1.485	0.0	1.488	0.0
1.0	n.a	1.177	-4.9	1.552	-4.5	1.505	-1.1
3.2	0.021	0.900*	19.7	1.479	0.4	1.489	-0.1
10	0.33	0.965*	14.0	1.499	-1.0	1.452	2.4
32	1.1	0.798*	28.9	1.024*	31.1	1.048*	29.6
100	80	-0.358*	131	-0.211*	114	-0.461*	131

n.a.:     Not analyzed since the inhibition of growth rate and yield was below the 48 and 72-hour NOEC.

*:          Mean value statistically significantly lower than in the control (according to a Williams t-test one-sided smaller, α = 0.05).

**:        The biological results after 72 hours were not used for the calculation of the ECx and NOEC/LOEC values due to the decrease of the substance after 48 hours.

Note:    Percentage inhibition values in excess of 100 % are obtained when the biomass at the end of the interval is lower than at the start of the interval.

Table 3: Yield (Y)

Nominal Test Item Concentration (mg/L)	Mean Measured Concentration (mg/L)	Average Growth Rate μ [day-1] and Inhibition Ir [%]
		0-24 h		0-48 h		0-72 h**
		Y	Iy [%]	Y	Iy [%]	Y	Iy [%]
Control	---	1.4	0.0	12.9	0.0	59.8	0.0
1.0	n.a	1.6	-8.4	14.8	-15.3	62.8	-5.1
3.2	0.021	1.0*	27.9	12.7	1.4	60.0	-0.3
10	0.33	1.1*	21.5	13.3	-3.4	53.5^#	10.5
32	1.1	0.9*	40.8	4.8*	62.6	16.1^#	73.1
100	80	-0.2*	114.4	-0.2	101.7	-0.5^#	100.9

n.a.:     Not analyzed since the inhibition of growth rate and yield was below the 48 and 72-hour NOEC.

*:          Mean value statistically significantly lower than in the control (according to a Williams t-test one-sided smaller, α = 0.05).

^#:          Mean value statistically significantly lower than in the control (according to a Welch t-test one-sided smaller, α = 0.05).

**:        The biological results after 72 hours were not used for the calculation of the ECx and NOEC/LOEC values due to the decrease of the substance after 48 hours.

Note:    Percentage inhibition values in excess of 100 % are obtained when the biomass at the end of the interval is lower than at the start of the interval.

Table 4: Section-by-Section Growth Rates

Nominal Test Item Concentration (mg/L)	Mean Measured Concentration (mg/L)	Average Growth Rate μ [day-1] and Inhibition Ir [%]
		0-24 h		0-48 h		0-72 h**
		μ [day-1]	Ir [%]	μ [day-1]	Ir [%]	μ [day-1]	Ir [%]
Control	---	1.121	0.0	1.849	0.0	1.494	0.0
1.0	n.a	1.177	-4.9	1.928	-4.2	1.409	5.7
3.2	0.021	0.900	19.7	2.057	-11.3	1.511	-1.1
10	0.33	0.965	14.0	2.034	-10.0	1.357	9.2
32	1.1	0.798	28.9	1.250	32.4	1.097	26.6
100	80	-0.358	132	-0.063	103	-0.961	164

n.a.:     Not analyzed since the inhibition of growth rate and yield was below the 48 and 72-hour NOEC.

**:        The biological results after 72 hours were not used for the calculation of the ECx and NOEC/LOEC values due to the decrease of the substance after 48 hours.

Note:    Percentage inhibition values in excess of 100 % are obtained when the biomass at the end of the interval is lower than at the start of the interval.

 

Table 5: Impact of the test item on the growth of the freshwater green algal species Pseudokirchneriella subcapitata (results are based on mean measured concentrations of the test item)

Endpoint after 48 Hours	Growth Rate [mg/L]	Yield [mg/L]
EC10 95 % Confidence Interval	0.12 0.077 – 0.17	0.066 0.043 – 0.096
EC20 95 % Confidence Interval	0.38 027 – 0.53	0.21 0.15 – 0.29
EC50 95 % Confidence Interval	3.7 2.6 – 5.8	1.9 1.3 – 2.7
NOEC	0.33	0.33
LOEC	1.1	1.1

Analytical results

Table 6: Results for the Range-Finding Test

Sampling Day/Sample Age [day/hours]	Nominal Concentration of Test Item Cnom [mg/L]	Measured Concentration of Test Item X [µg/L]	Sample Preparation Factor F	Determined Concentration of Test Item c [mg/L]	% from Initially Measured [%]
0/0 (fresh)	Control	n.d.	126	< LOQ	n.a.
	1	7.74	126	0.977	98
	10	81.1	126	10.2	102
	100	38.7	2561	103	103
1/24 (aged)	1	5.78	126	0.729	73
2/48 (aged)	1	n.d.	2.1	< LOQ	n.a.
3/72 (aged)	Control	n.d.	2.1	< LOQ	n.a.
	1	n.a.	2.1	< LOQ	n.a.
	10^a	n.a.	51.3	< LOQ	n.a.
	100	65.2	504	32.8	33

LOQ: 0.002 mg test item /L (LOQ in range-finding test)

n.d. = not detected

n.a. = not applicable

The tabulated values of the samples represent rounded results obtained by calculation using the exact raw data.

 

Table 7: Results for Spiked Samples

Nominal Concentration of Test Item Cnom [mg/L]	Measured Concentration of Test Item X [µg]	Sample Preparation Factor F	Determined Concentration of Test Item C [mg/L]	Recovery Rate R [%]	Accuracy (Average Recovery) [%]	Precision (Relative Standard Deviation of Recovery) [%]
0	n.d.	2	n.a.	< LOQ	n.a.	-
0.00728	4.09	2	0.00817	101	113	6.1
0.00728	3.84	2	0.00769	104
0.00728	3.97	2	0.00794	109
0.00728	4.51	2	0.00901	124
0.00728	4.13	2	0.00827	114
121	183	669	122	101	105	2.4
121	189	669	127	104
121	191	669	127	105
121	193	669	129	107
121	194	669	130	107
Acceptance Target:					80 - 120	≤ 10

LOQ: 0.00365 mg test item /L

n.d. = not detected

n.a. = not applicable

The tabulated values of the samples represent rounded results obtained by calculation using the exact raw data.

 

Table 8: Results for Centrifuged Spiked Samples

Nominal Concentration of Test Item [day/hours]	Measured Concentration of Test Item in the Spiked Sample x [µg/L]	Sample Preparation Factor F	Determined Concentration of Test Item c [mg/L]	Recovery Rate R [%]
0.00728	3.71	2	0.00741	102
121	179	669	120	99

 

Table 9: Results for Test Samples

Sampling Day/Sample Age [day/hours]	Nominal Concentration of Test Item Cnom [mg/L]	Measured Concentration of Test Item X [µg/L]	Sample Preparation Factor F	Determined Concentration of Test Item c [mg/L]	% of Nominal Concentration [%]
0/0 (fresh)	Control	n.d.	2	< LOQ	n.a.
	3.2	93:5	31	2:86	89
	10	95:7	102	9:76	98
	32	158	302	31:8	100
	100	153	669	102	102
1/24 (aged)	Control	n.d.	2	< LOQ	n.a.
	3.2	n.d.	2	< LOQ	n.a.
	10	20.0	102	2.04	20
	32	103	202	20.9	65
	100	144	669	96.3	96
2/48 (fresh)	Control	n.d.	2	< LOQ	n.a.
	3.2	n.d.	2	< LOQ	n.a.
	10	n.d.	2	< LOQ	n.a.
	32	n.d.	2	< LOQ	n.a.
	100	77.1	669	51.6	52
2/72 (aged)	control	n.d.	2	< LOQ	n.a.
	3.2	n.d.	2	< LOQ	n.a.
	10	n.d.	2	< LOQ	n.a.
	10^a	n.d.	2	< LOQ	n.a.
	10^b	n.d.	2	< LOQ	n.a.
	32	n.d.	2	< LOQ	n.a.
	100	2.69	2	0.00538	0.00538

LOQ: 0.00365 mg test item/L

a: without algae

b: without algae, without light

n.d. = not detected

n.a. = not applicable

The tabulated values of the samples represent rounded results obtained by calculation using the exact raw data.

 

Validity criteria fulfilled:

yes

Conclusions:

The test item had acute toxic effects on Pseudokirchneriella subcapitata in a 72-hour static test performed according to OECD 201.
Due to the almost complete decrease of the test item after 72 hours and since the biomass in the control already increased by a factor of 19.5 over the first 48 hours it was decided, to calculate the mean measured concentrations as the geometric mean of the concentrations measured at the sampling dates after 0, 24 and 48 hours. It was also decided to reflect the biological 48-hour endpoints based on these mean measured concentrations.
Therefore, the 48-h EC50 for growth rate was calculated to be 3.7 mg/L with 95 % confidence limits of 9.0 and 12 mg/L. The 48-h EC10 for growth rate was determined to be 0.12 mg/L (95 %-CL: 0.077 - 0.17).

Executive summary:

The impact of the test item on the growth of the freshwater green algal species Pseudokirchneriella subcapitata was investigated in a 72 -hour static test according to the OECD Guideline 201 (2006, corrected 2011) and the Commission Regulation (EU) No. 2016/266, C3. The nominal test item concentrations tested were 1.0, 3.2, 10, 32 and 100 mg/L. Additionally, a control (test water without test item) was tested in parallel.

The analytical measurements from test media at the start of the test show the correct preparation of the test media. As the substance is highly instable, the test item concentration decreased distinctly during the test period of 72 hours. At the end of the test, all test concentrations were below the limit of quantification (LOQ = 0.00365 mg/L), with the exception of the highest test concentration of 100 mg/L, where 0.005 % of the nominal concentration could be measured.

Due to the almost complete decrease of the test item after 72 hours and since the biomass in the control already increased by a factor of 19.5 over the first 48 hours it was decided, to calculate the mean measured concentrations as the geometric mean of the concentrations measured at the sampling dates after 0, 24 and 48 hours. It was also decided to reflect the biological 48-hour endpoints based on these mean measured concentrations.

Photodegradation could be excluded, since a decrease of the test item was also determined in the additional samples incubated without light but it seems that the test item is not hydrolytically stable in test water.

The samples of the nominal test concentration of 1.0 mg/L were not analyzed since the concentrations were below the 48-hour NOEC determined in this test and, thus, were not relevant for the interpretation of the biological results.

Due to the decrease of the test item concentrations during the test period of 72 hours, the biological results were related to the mean measured concentrations.

The 48-hour NOEC for growth rate and yield was determined to be 0.33 mg/L, since at this concentration the growth rate and yield of the algae after 48 hours were not significantly lower than in the control. The LOEC for growth rate and yield was determined to be 1.1 mg/L. The 48-h EC50 for growth rate was calculated to be 3.7 mg/L with 95 % confidence limits of 9.0 and 12 mg/L. The 48-h EC10 for growth rate was determined to be 0.12 mg/L (95 %-CL: 0.077 - 0.17).

Description of key information

The test item had acute toxic effects on Pseudokirchneriella subcapitata  in a 72-hour static test performed according to OECD 201.

Due to the almost complete decrease of the test item after 72 hours and since the biomass in the control already increased by a factor of 19.5 over the first 48 hours it was decided, to calculate the mean measured concentrations as the geometric mean of the concentrations measured at the sampling dates after 0, 24 and 48 hours. It was also decided to reflect the biological 48-hour endpoints based on these mean measured concentrations.

Therefore, the 48-h EC50 for growth rate was calculated to be 3.7 mg/L with 95 % confidence limits of 9.0 and 12 mg/L. The 48-h EC10 for growth rate was determined to be 0.12 mg/L (95 %-CL: 0.077 - 0.17).

Key value for chemical safety assessment

EC50 for freshwater algae:

3.7 mg/L

EC10 or NOEC for freshwater algae:

0.12 mg/L

Additional information

The impact of the test item on the growth of the freshwater green algal species Pseudokirchneriella subcapitata was investigated in a 72 -hour static test according to the OECD Guideline 201 (2006, corrected 2011) and the Commission Regulation (EU) No. 2016/266, C3. The nominal test item concentrations tested were 1.0, 3.2, 10, 32 and 100 mg/L. Additionally, a control (test water without test item) was tested in parallel.

The analytical measurements from test media at the start of the test show the correct preparation of the test media. As the substance is highly instable, the test item concentration decreased distinctly during the test period of 72 hours.

At the end of the test, all test concentrations were below the limit of quantification (LOQ = 0.00365 mg/L), with the exception of the highest test concentration of 100 mg/L, where 0.005 % of the nominal concentration could be measured.

Due to the almost complete decrease of the test item after 72 hours and since the biomass in the control already increased by a factor of 19.5 over the first 48 hours it was decided, to calculate the mean measured concentrations as the geometric mean of the concentrations measured at the sampling dates after 0, 24 and 48 hours. It was also decided to reflect the biological 48-hour endpoints based on these mean measured concentrations.

Photodegradation could be excluded, since a decrease of the test item was also determined in the additional samples incubated without light but it seems that the test item is not hydrolytically stable in test water.

The samples of the nominal test concentration of 1.0 mg/L were not analyzed since the concentrations were below the 48-hour NOEC determined in this test and, thus, were not relevant for the interpretation of the biological results.

Due to the decrease of the test item concentrations during the test period of 72 hours, the biological results were related to the mean measured concentrations.

The 48-hour NOEC for growth rate and yield was determined to be 0.33 mg/L, since at this concentration the growth rate and yield of the algae after 48 hours were not significantly lower than in the control. The LOEC for growth rate and yield was determined to be 1.1 mg/L. The 48-h EC50 for growth rate was calculated to be 3.7 mg/L with 95 % confidence limits of 9.0 and 12 mg/L. The 48-h EC10 for growth rate was determined to be 0.12 mg/L (95 %-CL: 0.077 - 0.17).