methyl 2-hydroxybut-3-enoate

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:

2021-08-2021 to 2021-08-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD guidance document No. 23 (ENV/JM/MONO(2000)6/REV1;)

Version / remarks:

8 February 2019

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 23 March 2006 (Annex 5 corrected 28 July 2011)

Deviations:

yes

Remarks:

in accordance with OEDC Guidance No. 23

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

01 March 2016

Deviations:

yes

Remarks:

in accordance with OEDC Guidance No. 23

Principles of method if other than guideline:

In accordance with OECD TG 201 vessels should be capped with air-permeable stoppers. However, due to the volatility of the test item flasks were sealed. This modification is in compliance with the OECD Guidance document No. 23.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Sampling method: Three replicate samples were taken from the test concentrations and six from the control group at each analytical occasion during the test.
- Sample storage conditions before analysis: no

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

ACCLIMATION
- Acclimation period: not necessary, cultivation at conditions of test
- Culturing media and conditions: same as test
- 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:

post exposure observations not performed

Test temperature:

22.8 - 23.8 °C

pH:

7.63 – 10.45 (pH shift due to closed test system)

Dissolved oxygen:

not specified

Salinity:

not applicable

Conductivity:

not specified

Nominal and measured concentrations:

Nominal: 0.70, 1.9, 5.1, 13.7, 37.0, 100 mg/L; Measured: nd*, nd*, nd*, 1.21, 3.31 and 18.51 mg/L, * = not determined

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmyer flask
- Type: closed, sealed by parafilm and aluminium foil
- Material, size, fill volume: glass, headspace minimized, 250 mL nominal volume, fill volume 200 mL
- Aeration: no aeration but cultures mixed with orbital shaker
- Initial cells density: approx. 10^4 cells/mL in each test flask
- Control end cells density: 85.0 x10^4/mL
- No. of vessels per concentration: 3
- No. of vessels per control: 6

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: continuously illuminated
- Light intensity and quality: 7143 lux

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: 24, 48 and 72 hours, manual cell counting using a microscopic method with a counting chamber

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.5
- Range finding study: performed
- Test concentrations: 0.01, 0.1, 1, 10, 100 mg/L (nominal)
- Results used to determine the conditions for the definitive study: yes

CULTURING APPARATUS
- Details on culturing apparatus used: climate chamber

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.21 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.21 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.36 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

7.31 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.:

2.55 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

2.8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

1.69 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

3.31 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

3.31 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Details on results:

- Exponential growth in the control: yes
- Observation of abnormalities: yes
- Unusual cell shape: pinches on cells and swollen cells at the highest test concentration after 24 h and 48 h of exposure, after 72 h additionally thin cells
- 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: the substance evaporates from the test system; evaporation was minimized by using a closed test system
- Effect concentrations exceeding solubility of substance in test medium: no

Results with reference substance (positive control):

72h ErC50: 0.62 mg/L (95 % confidence limits: 0.59 – 0.65 mg/L);
72h EyC50: 0.33 mg/L (95 % confidence limits: 0.32 – 0.34 mg/L)

Reported statistics and error estimates:

ECx values were determined by Probit-analysis using linear max. likelihood regression. For the determination of the NOEC/LOEC values, Dunnett´s multiple t-test (α = 0.05, 2-sided) was performed with growth rates and Welsh-t-test after Bonferroni-Holm (α = 0.05, 2-sided) was performed with yield. Pretesting on normal distribution was performed by Shapiro Wilk’s test and on variance homogeneity by Levene’s test.

Table 1: Calculation of exposure concentrations

Nominal concentration [mg/L]	Mean measured concentrations [mg/L]				Geometric mean [mg/L]
	0h	24h	48h	72h
Control	not detected	not detected	not detected	not detected	-
0.31	0.26	0.19	not detected	not detected	not determined
0.77	0.72	0.45	not detected	not detected	not determined
1.9	1.82	1.19	< LOQ	not detected	not determined
4.8	4.57	3.15	0.49	0.3 (LOQ/2)	1.21
12	11.4	8.2	4.26	0.3 (LOQ/2)	3.31
30	28.3	22.6	18	10.2	18.51

 

 

Table 2: Growth Rates and Yield calculated for the entire test duration (day 0-3)

Concentration			Growth rates	%inhibition	Yield	%inhibition
			(day-1)
Nominal	Exposure		day 0-3		day 0-3
control	-		1.45		76
			1.43		73
			1.49		87
			1.50		88
			1.50		90
			1.50		90
		Mean	1.48		84
		SD	0.0		7.5
		CV	2.1		8.9
0.31	nd		1.55	-4.8	104	-23.8
			1.47	0.5	82	2.4
			1.49	-0.9	87	-3.6
		Mean	1.51	-1.7	91	-8.3
		SD	0.0		11.5
		CV	2.7		12.7
0.77	nd		1.51	-2.1	92	-9.5
			1.48	0.2	83	1.2
			1.47	0.5	82	2.4
		Mean	1.49	-0.5	85.7	-2
		SD	0		5.5
		CV	1.4		6.4
1.9	nd		1.48	-0.1	84	0
			1.54	-4.2	101	-20.2
			1.47	0.5	82	2.4
		Mean	1.5	-1.3	89	-6
		SD	0		10.4	7.1
		CV	2.5		11.7	-4.8
4.8	1,21		1.46	1.6	78	11.9
			1.5	-1.1	88	4.8
			1.44	2.7	74
		Mean	1.46	1.1	80	4.8
		SD	0		7.2
		CV	2		9
12	3.31		1.13	23.4	29	65.5
			1.09	26.6	25	70.2
			1.03	30.4	21	75
		Mean	1.08	26.8	25	70.2
		SD	0.1		4
		CV	4.8		16
30	18.51		0.37	75.3	2	97.6
			0.23	84.4	1	98.8
			0.37	75.3	2	97.6
		Mean	0.32	78.3	1.7	98
		SD	0.1		0.6
		CV	24.3		34.6

 

VALIDITY OF TEST

The cell concentration in the control cultures should have increased exponentially by a factor of at least 16 within three days (72 hours), corresponding to a specific growth rate of 0.92 day^-1.

The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3) in the control cultures must not exceed 35 %.

The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7 %.

Validity criteria fulfilled:

yes

Conclusions:

In an Algae Growth Inhibition Test according to OECD 201 and EU Method C.3, the 72-hour EC10 and EC 50 towards Raphidocelis subcapitata based on growth rate was determined to be 1.70 mg/L and 7.31 mg/L, respectively. The 72-hour EC10 and EC 50 based on yield was determined to be 1.36 mg/L and 2.55 mg/L, respectively. The 72-hour NOEC both based on the growth rate and yield was 1.21 mg/L. All validity criteria of the test guideline were fulfilled. The study is thus regarded valid.

Executive summary:

The growth inhibiting effect of the test item towards the green algae Raphidocelis subcapitata was assessed in a study according to OECD TG 201, EU Method C.3 and GLP principles. Exponentially growing cells (approx.. 10⁴ cells per mL at start of the test) were exposed for 72 hours to nominal concentrations of 0.31, 0.77, 1.9, 4.8, 12.0 and 30 mg/L. Untreated control ran in parallel. The suitability of the test system was confirmed in a positive control using Potassium dichromate as reference compound. For determination of the test item concentrations, samples were taken from each test concentration and from the control at each day of the test. Concentrations were determined by HPLC method with UV detection. The corresponding calculated exposure concentrations (based on analytical measurements) were the followings: not determined, not determined, not determined, 1.21, 3.31 and 18.51 mg/L.  Biological results and endpoints are hence based on the measured concentrations (i.e. calculated exposure concentrations). The algal growth in relation to a control culture was determined over a fixed test period of 72 hours and, thus, over several algal generations. The test design included three replicates at each test concentration and six replicates for the control, respectively. Sealed 250 mL glass Erlenmeyer flasks filled up with 200 mL test liquid were used as test vessels. The algae cell concentration was determined by manual cell counting by microscope in each testing flask during the 72-hour test, at 24-hour intervals. To determine NOEC/LOEC values, Dunnett´s multiple t-test (for growth rate) and Welsh-t-test after Bonferroni-Holm (for yield), to determine the ECx values Probit analysis (for growth rate and yield) was used. As a result, the 72-hour EC10 and EC 50 based on growth rate was determined to be 1.70 mg/L and 7.31 mg/L, respectively. The 72-hour EC10 and EC 50 based on yield was determined to be 1.36 mg/L and 2.55 mg/L, respectively. The 72-hour NOEC both based on the growth rate and yield was 1.21 mg/L. All validity criteria of the test guideline were fulfilled. The study is thus regarded valid.

Description of key information

In an Algae Growth Inhibition Test according to OECD 201 and EU Method C.3, the 72-hour EC10 and EC 50 towards Raphidocelis subcapitata based on growth rate was determined to be 1.70 mg/L and 7.31 mg/L, respectively. The 72-hour EC10 and EC 50 based on yield was determined to be 1.36 mg/L and 2.55 mg/L, respectively. The 72-hour NOEC both based on the growth rate and yield was 1.21 mg/L. All validity criteria of the test guideline were fulfilled. The study is thus regarded valid.

Key value for chemical safety assessment

EC50 for freshwater algae:

7.31 mg/L

EC10 or NOEC for freshwater algae:

1.7 mg/L

Additional information

The growth inhibiting effect of the test item towards the green algae Raphidocelis subcapitata was assessed in a study according to OECD TG 201, EU Merthod C.3 and GLP principles. Exponentially growing cells (approx.. 10⁴ cells per mL at start of the test) were exposed for 72 hours to nominal concentrations of 0.31, 0.77, 1.9, 4.8, 12.0 and 30 mg/L. Untreated control ran in parallel. The suitability of the test system was confirmed in a positive control using Potassium dichromate as reference compound. For determination of the test item concentrations, samples were taken from each test concentration and from the control at each day of the test. Concentrations were determined by HPLC method with UV detection. The corresponding calculated exposure concentrations (based on analytical measurements) were the followings: not determined, not determined, not determined, 1.21, 3.31 and 18.51 mg/L.  Biological results and endpoints are hence based on the measured concentrations (i.e. calculated exposure concentrations). The algal growth in relation to a control culture was determined over a fixed test period of 72 hours and, thus, over several algal generations. The test design included three replicates at each test concentration and six replicates for the control, respectively. Sealed 250 mL glass Erlenmeyer flasks filled up with 200 mL test liquid were used as test vessels. The algae cell concentration was determined by manual cell counting by microscope in each testing flask during the 72-hour test, at 24-hour intervals. To determine NOEC/LOEC values, Dunnett´s multiple t-test (for growth rate) and Welsh-t-test after Bonferroni-Holm (for yield), to determine the ECx values Probit analysis (for growth rate and yield) was used. As a result, the 72-hour EC10 and EC 50 based on growth rate was determined to be 1.70 mg/L and 7.31 mg/L, respectively. The 72-hour EC10 and EC 50 based on yield was determined to be 1.36 mg/L and 2.55 mg/L, respectively. The 72-hour NOEC both based on the growth rate and yield was 1.21 mg/L. All validity criteria of the test guideline were fulfilled. The study is thus regarded valid.