3-methyl-2-butenyl salicylate

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

See read-across justification attached to section 13.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.791 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: values recalculated based on molecular weight of source and target substance

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.614 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: values recalculated based on molecular weight of source and target substance

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.173 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: growth rate and yield

Remarks on result:

other: values recalculated based on molecular weight of source and target substance

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.733 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: values recalculated based on molecular weight of source and target substance

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.417 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: values recalculated based on molecular weight of source and target substance

Validity criteria fulfilled:

yes

Conclusions:

In the study the test item was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values (based on geometric mean measured test item concentrations): The EC50 and EC10-values for inhibition of growth rate were 0.791 mg/L and 0.614 mg/L respectively. The NOEC-values for both inhibition of growth rate and yield after 72 hours were 0.173 mg/L, respectively. All values were recalculated based on molecular weight of source and target substance.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2017-06-12 to 2017-06-15

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:

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:

- Nominal concentrations: 0.100 - 0.316 - 1.00 - 3.16 - 10.0 mg/L, and a control
- Sampling method: All concentration levels and the controls were analytically verified via HPLC-DAD at the start (0 hours) and at the end of the exposure (72 hours) with algae.
- Sample storage conditions before analysis: All original samples were stored at at 6 ± 2 °C. Prepared samples were stored at room temperature in an autosampler until analysis.

Vehicle:

yes

Remarks:

demineralized water

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A stock solution with a concentration of 10 mg/L test item was prepared once with demineralized water (adjusted to pH 4 + 0.2) in a brown glass bottle and stirred for 72 hours at room temperature, 1100 rpm. The stock solution was checked after stirring via laser beam (Tyndall effect) for undissolved test item (formation of an emulsion). The Tyndall effect was negative.
- Test concentrations: 0.100 - 0.316 - 1.00 - 3.16 - 10.0 mg/L
- Control: Six replicates (without test item) were incubated under the same conditions as the test concentrations.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: KORSHIKOV, SAG 61.81
- Method of cultivation: Fresh stocks are prepared every month on Z-Agar. Light intensity amounted to 2567 - 5130 lux for 24 hours per day

ACCLIMATION
- Acclimation period: three days
- Culturing media and conditions (same as test or not): Nutrient medium Z, same as test

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

0.24 mmol Ca+Mg/L

Test temperature:

22.0 – 23.0 °C, mean value 22.5°C

pH:

7.99 to 8.22 at test start
8.21 to 9.45 at test end

Nominal and measured concentrations:

Nominal concentrations: 0.100 - 0.316 - 1.00 - 3.16 - 10.0 mg/L
Geometric mean measured concentrations: 0.0557 - 0.163 - 0.526 - 2.11 - 6.65 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Sterile headspace flasks
- Type: closed
- Material, size, headspace, fill volume: volume: 59 mL, with aluminium tops with PTFE seals, fill volume: 59 mL without headspace
- Initial cells density: 6839 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes, As recommended in OECD TG 201

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: 24 hours/day light
- Light intensity and quality: range of the measured values: 4955 to 6023 lux, mean value: 5634 Lux

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: Chlorophyll a-fluorescence
- Microscopic evaluation: at the start and the end of the incubation period;
- Physico-chemical Properties: The pH-values at the start of exposure were measured in one additional replicate of each test item concentration and the control. At the end of exposure, the pH-values were measured from pooled samples of each test item concentration and the control. The room temperature was measured continuously. Light intensity was measured prior to the start of exposure.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Range finding study: yes
- Test concentrations: 0.001, 0.01, 0.1, 1.00, 10.0 mg/L
- Results used to determine the conditions for the definitive study: All concentration levels were visually clear throughout the exposure period. Growth rate inhibition was 100% at 10.0 mg/L.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.745 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.:

0.578 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.163 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: growth rate and yield

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.69 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.393 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

- Microscopic evaluation: revealed no morphological abnormalities.
- Physicochemical data: The environmental conditions (pH-value, room temperature, light intensity) were determined to be within the acceptable limits. The test item solutions were visually clear throughout the exposure phase.
- Chemical analysis of test item concentrations: The measured concentrations of the test item were between 71% and 78% at the start of the exposure (0 hours) and between < LOQ and 63% at the end of the exposure (72 hours).

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50: most recent test: growth rate 0.559 mg/L (95% CI: 0.538 - 0.581), yield 0.320 mg/L (95% CI: 0.242 - 0.341)

Results non-GLP range finding test:

Nominal concentration (mg/l)	Growth rate inhibition (%)	Yield inhibition (%)
10	100	100
1	1	2
0.1	-1	-5
0.01	1	2
0.001	1	5

Mean cell densities (cells/mL):

Geometric mean measured concentration (mg/L)	0h	24h	48h	72h
6.65	6839	n.a.	n.a.	n.a.
2.11	6839	5544	4673	n.a.
0.526	6839	27193	186870	635090
0.163	6839	33228	244536	755280
0.0557	6839	32504	249674	774875
Control	6839	35466	278360	814181

Evaluation of effection growth rate and yield after 72h (mean):

Geometric mean measured concentration (mg/L)	Growth rate (d-1)	inhibition of growth rate (%)	Yield (cells/mL)	Inhibition of yield (%)
6.65	n.a.	100	n.a.	100
2.11	n.a.	100	n.a.	100
0.526	1.51	5	628251	22
0.163	1.56	2	748441	7
0.0557	1.58	1	768036	5
Control	1.59		807342

Measured concentrations and percentage of the nominal concentration of test item in fresh medium (0hours) and 72hours, aged with algae:

Sample	0 hours fresh medium		72 hours old medium
Nominal concentration of the test item [mg/L]	Meas. conc. [mg/L]		Meas. conc. [mg/L]	[%]	Geometric mean measured concentration [mg/L]^1^)
10.0	7.08	71	6.23	62	6.65
3.13	2.24	71	1.98	63	2.11
1.00	0.724	72	0.382	38	0.526
0.316	0.235	74	0.114	36	0.163
0.100	0.0775^2)	78	< LOQ		0.0557
Control	< LOQ		< LOQ

Meas. Conc. = Measured concentration of the test item (dilution factor taken into account), mean value oftwo injections

%= Percentage of the nominal concentration of the test item

LOQ= Limit of quantification (0.0800mg test item/L)

¹⁾= Measured concentrations < 70% LOQ were taken into account with half of the LOQ

²⁾= < LOQ, but > 70% LOQ

Validity criteria fulfilled:

yes

Conclusions:

In this study, test item was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values (based on geometric mean measured test item concentrations): EC50-values for inhibition of growth rate (ErC50) and yield (EyC50) being 0.745 mg/L and 0.690 mg/L, respectively, EC10-values ErC10 = 0.578 mg/L and EyC10 = 0.393 mg/L, and NOEC-values for both inhibition of growth rate and yield after 72 hours were 0.163 mg/L.

Executive summary:

The toxicity of test item to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 under static conditions. The main test was carried out with five concentrations in a geometrical series with a dilution factor of 10: 0.100 - 0.316 - 1.00 -3.16 - 10.0 mg/L. The test media were clear throughout the test period. The concentrations of the test item in all test concentrations and the control were analytically verified by HPLC-DAD at the start and the end of exposure.

The measured concentrations of test item in the fresh media (0 h) were between 71 and 78% of the nominal values. The measured concentrations in the old media (72 h) were in the range of <LOQ to 63% of the nominal values. Therefore, the evaluation based on geometric mean measured test item concentrations was considered the most fitting evaluation.

In this study, test item was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values (based on geometric mean measured test item concentrations): EC50-values for inhibition of growth rate (ErC50) and yield (EyC50) being 0.745 mg/L and 0.690 mg/L, respectively, EC10-values ECr10 = 0.578 mg/L and EyC10 = 0.393 mg/L, and NOEC-values for both inhibition of growth rate and yield after 72 hours were 0.163 mg/L.

Description of key information

In a static short term study the surrogate test item was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values (based on geometric mean measured test item concentrations): The EC50 and EC10-values for inhibition of growth rate were 0.791 mg/L and 0.614 mg/L respectively. The NOEC-values for both inhibition of growth rate and yield after 72 hours were 0.173 mg/L, respectively. All values were recalculated based on molecular weight of source and target substance

Key value for chemical safety assessment

EC50 for freshwater algae:

0.791 mg/L

EC10 or NOEC for freshwater algae:

0.614 mg/L

Additional information

The toxicity of the surrogate 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 NOEC, LOEC, EC10, EC20- and EC50-values of growth rate and yield over a period of 72 hours. The study was conducted under static conditions with an initial cell density of 6839 cells/mL. A stock solution of 10 mg test item/L was freshly prepared with demineralized water and was stirred for 72 hours at 1100 rpm and room temperature. The definitive test was carried out with five concentrations in a geometrical series with a dilution factor of V10: 0.100 - 0.316 - 1.00 -3.16 - 10.0 mg/L. Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditions were within the acceptable limits. The test media were clear throughout the test period. The concentrations of the test item in all test concentrations and the control were analytically verified by HPLC-DAD at the start and the end of exposure. The measured concentrations of the test item in the fresh media (0 h) were between 71 and 78% of the nominal values. The measured concentrations in the old media (72 h) were in the range of <LOQ to 63% of the nominal values. Therefore, the evaluation based on geometric mean measured test item concentrations was considered the most fitting evaluation. In this study the read across test item was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values (based on geometric mean measured test item concentrations): The EC50-values with 95 % confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 0.745 (0.542 - 1.51) mg/L and 0.690 (0.547 - 1.55) mg/L, respectively. The EC10-values with 95 % confidence intervals for inhibition of growth rate (ErC10) and yield (EyC10) after 72 hours were 0.578 (0.528 - 1.41) mg/L and 0.393 (< 0.0557 - 0.519) mg/L, respectively. The NOEC-values for both inhibition of growth rate and yield after 72 hours were 0.163 mg/L, respectively.

All values were recalculated based on molecular weight of source and target substance.