Levomenol

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:

2017-09-18 to 2017-10-06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Sampling method: Samples of each test medium concentration were taken directly after preparation and after 24h, 48h, 72h and stored in a freezer at =< -20 °C. Samples with algae were centrifuged (4000g for 20 minutes) and the supernatant was stored. One replicate was analyzed, the second replicate was used as backup in case repeated analysis was needed.
- Sample storage conditions before analysis: Samples without algae were directly stored at =< -20 °C in a freezer until analysis. Supernatant was stored =< -20 °C in a freezer until analysis.

Vehicle:

yes

Remarks:

reconstituted water (as cited in the study)

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test item was weighed in a calibrated flask and vehicle (reconstituted water, aerated for at least 24 hours prior preparation of the test item concentrations) was added. The preparation was stirred with a magnetic stirrer for 24 hours. Then the formulation was given into a separating funnel for 1 hour. The middle fraction in the separating funnel was used for the preparation of the different test item concentrations. The pH was not adjusted.
- Controls: Yes, blank control
- Chemical name of vehicle: Reconstituted water (for details please see "any other information on materials and methods incl. tables")

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: 61.81 SAG
- Source: "Pflanzenphysiologisches Institut der Universität Göttingen", Germany
- Age of inoculum (at test initiation): 3 days old pre-culture
- Method of cultivation: Permanent algae cultures; pre-culture and the algae cultures of the study were cultivated in an air-conditioned room with water temperature of 21.0 - 24.0 °C.

ACCLIMATION
- Culturing media and conditions: Same as test

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

None

Hardness:

Reconstituted water: 0.24 mmol/L (=24 mg/L) as CaCO3

Test temperature:

23.7 to 25.6 °C

pH:

7.82 to 9.50

Dissolved oxygen:

Not specified

Salinity:

Not applicable

Conductivity:

Not specified

Nominal and measured concentrations:

Nominal: 0.0 (control), 1.0, 3.2, 10.0, 31.6 and 100.0 mg/L
Measured concentrations (geometric mean, derived according to OECD Guidance Document No 23): not determined (control), 0.12, 0.42, 1.41, 4.90 and 14.45 mg/L

Details on test conditions:

TEST SYSTEM
- Vessel type: open
- Fill volume: 100 mL
- Initial cell density: 10 000 cells/mL
- No. of vessels per concentration (replicates): 3 (with algae), 1 (without algae, used for pH-value measurement and chemical analysis without algae), 2 (with algae, used for for analysis of the test item preparations with algae after 24 and 48 hours, respectively)
- No. of vessels per control (replicates): 6 (with algae), 1 (without algae, used for pH-value measurement and chemical analysis without algae), 2 (with algae, used for for analysis of the test item preparations with algae after 24 and 48 hours, respectively)
- No. of vessels per vehicle control (replicates):

GROWTH MEDIUM
- Standard medium used: Yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Milli-Q-water

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Light intensity and quality: 7392 Lux +- 4.7% and 7915 Lux +- 3.7% prior to and at the end of the exposure period, respectively

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: electronic particle counter
- Chlorophyll measurement: No


TEST CONCENTRATIONS
- Range finding study: Yes
- Test concentrations: 1, 10 and 100m mg/L
- Results used to determine the conditions for the definitive study: Yes

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (Experiment: 2017-09-12 to 2017-09-15)

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.76 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence limits [mg/L]: 0.52 - 1.01

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

1.33 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence limits [mg/L]: 1.00 - 1.64

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

3.8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence limits [mg/L]: 3.26 - 4.43

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.38 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% confidence limits [mg/L]: 0.31 -0.44

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

0.52 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% confidence limits [mg/L]: 0.45 - 0.59

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.96 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% confidence limits [mg/L]: 0.98 - 1.04

Details on results:

- Exponential growth in the control: Yes
- Observation of abnormalities: No
- Unusual cell shape: No
- Colour differences: No
- Any stimulation of growth found in any treatment: Yes, at the 1.0 mg/L level, a slight increase both in growth rate and yield compared to the negative control (0 mg/L) was noted

Results with reference substance (positive control):

72-h EC10 [mg/L]: 0.36 (growth rate) and 0.31 (yield)
72-h EC50 [mg/L]: 0.91 (growth rate) and 0.48 (yield)
72-h NOEC [mg/L]: 0.4 (growth rate and yield)

Table 1: Nominal and corresponding concentrations of analytical control samples without algae

Nominal test item concentration [mg/L]	Analytical results
	0h		24h		48h		72h
	Meas. conc. [mg/L]	[%]	Meas. conc [mg/L]	[%]	Meas. conc. [mg/L]	[%]	Meas. conc. [mg/L]	[%]
1.0	0.151	15.1	0.138	13.8	0.121	12.1	0.108	10.8
3.2	0.522	16.3	0.458	14.3	0.410	12.8	0.355	11.1
10.0	1.741	17.4	1.576	15.8	1.486	14.9	1.311	13.1
31.6	5.882	18.6	5.094	16.1	5.312	16.8	4.500	14.2
100.0	17.925	17.9	11.985	12.0	12.472	12.5	14.410	14.4

Table 2: Nominal and corresponding analytical concentrations of samples inoculated with algae are given in the following table.

Nominal test item concentration [mg/L]	Analytical results								Geometric mean [mg/L]^3)
	0h^1)		24h		48h		72h
	Meas. conc. [mg/L]	[%]	Meas. conc [mg/L]	[%]	Meas. conc. [mg/L]	[%]	Meas. conc. [mg/L]	[%]^2)
0	<LOQ	-	<LOQ	-	<LOQ	-	<LOQ	-	-
1.0	0.151	15.1	0.134	13.4	0.108	10.8	0.077	7.7	0.12
3.2	0.522	16.3	0.420	13.1	0.405	12.7	0.335	10.5	0.42
10.0	1.741	17.4	1.451	14.5	1.352	13.5	1.227	12.3	1.41
31.6	5.882	18.6	4.925	15.6	4.745	15.0	4.379	13.9	4.90
100.0	17.925	17.9	13.630	13.6	13.692	13.7	14.859	14.9	14.45

Validity criteria fulfilled:

yes

Conclusions:

The test item showed a 72-h EC50 of 3.80 mg/L for the growth rate and 0.96 mg/L for the yield of the unicellar green algae Pseudokirchneriella subcapitata. The 72-h EC10 was determined to be 0.76 mg/L and 0.38 mg/L for the growth rate and the yield, respectively.

Executive summary:

To assess the effect of the test item on the growth and the growth rate of the green algae species Pseudokirchneriella subcapitata, an Algae Growth Inhibition Test according to OECD Guideline 201 and EU Method C.3 was carried out in compliance with GLP principles. The study comprised of one control group with six replicates and five test item groups with three replicates each. The nominal test item concentration of 1.0, 3.2, 10.0, 31.6 and 100.0 mg/L were prepared in reconstituted water. The initial algae cell density in each replicate was 10 000 cells per mL. The growth of the algae was determined over an exposure peroid of 72 hours and compared with the growth of the control group. Samples of the control media were taken directly after preparation and then daily and stored at =< -20 °C until further analysis of the test item concentration via HPLC with UV detection. The recoveries of the test item concentrations in all test media with algae (0h, 24h, 48h, 72h) ranged from 7.7% to 15.6% of the nominal concentration. Furthermore, the recoveries of the test item concentrations in all test media without algae ranged from 10.8% to 18.6% of the nominal concentrations over the time period of 72 hours and, thus, the effect values were determined based on the geometric mean of the measured concentrations. Under the conditions of the study, the test item showed a 72-h EC50 of 3.80 mg/L for the growth rate and 0.96 mg/L for the yield of the unicellar green algae Pseudokirchneriella subcapitata. The 72-h EC10 was determined to be 0.76 mg/L and  0.38 mg/L for the growth rate and the yield, respectively. All validity criteria of the test guidelines were fulfilled.

Description of key information

The test item showed a 72-h EC50 of 3.80 mg/L for the growth rate and 0.96 mg/L for the yield of the unicellar green algae Pseudokirchneriella subcapitata. The 72-h EC10 was determined to be 0.76 mg/L and  0.38 mg/L for the growth rate and the yield, respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:

3.8 mg/L

EC10 or NOEC for freshwater algae:

0.76 mg/L

Additional information

To assess the effect of the test item on the growth and the growth rate of the green algae species Pseudokirchneriella subcapitata, an Algae Growth Inhibition Test according to OECD Guideline 201 and EU Method C.3 was carried out in compliance with GLP principles. The study comprised of one control group with six replicates and five test item groups with three replicates each. The nominal test item concentration of 1.0, 3.2, 10.0, 31.6 and 100.0 mg/L were prepared in reconstituted water. The initial algae cell density in each replicate was 10 000 cells per mL. The growth of the algae was determined over an exposure peroid of 72 hours and compared with the growth of the control group. Samples of the control media were taken directly after preparation and then daily and stored at =< -20 °C until further analysis of the test item concentration via HPLC with UV detection. The recoveries of the test item concentrations in all test media with algae (0h, 24h, 48h, 72h) ranged from 7.7% to 15.6% of the nominal concentration. Furthermore, the recoveries of the test item concentrations in all test media without algae ranged from 10.8% to 18.6% of the nominal concentrations over the time period of 72 hours and, thus, the effect values were determined based on the geometric mean of the measured concentrations. Under the conditions of the study, the test item showed a 72-h EC50 of 3.80 mg/L for the growth rate and 0.96 mg/L for the yield of the unicellar green algae Pseudokirchneriella subcapitata. The 72-h EC10 was determined to be 0.76 mg/L and  0.38 mg/L for the growth rate and the yield, respectively. All validity criteria of the test guidelines were fulfilled.