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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Mar 14, 2000 - Mar 17, 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Composition 0
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes
Test material information:
Composition 1
Analytical monitoring:
yes
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
Origin: Institute of Plant Physiology, University of Goettingen
- Algal inoculum 10000 cells/ml initial cell density
- Illumination: 120 µE/m2s
- Cell density measured with a microcelcounter after 24, 48 and 72 hours
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
pH:
8.0 - 10.3
Nominal and measured concentrations:
Nominal concentration: 1.25, 2.5, 5, 10 and 20 mg/l
Details on test conditions:
- Erlenmeyer flasks (300 ml) with stoppers
- 100 ml of test medium
- Light chamber with shaker

Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
16.2 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI: 15.504 - 16.537 mg/l
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
4.6 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
Since the original study on short-term toxicity to algae from 2000 only gives a range (> 9.8 < 18.2 mg/L) but not a distinct value for the ErC50 this value was determined by Probit analysis as ErC50 = 16.2 mg/L and quoted in the OECD SIDS report, 2003 (agreed by the ICCA initiative). The calculated ErC50 value is used as result of the underlying study on short-term toxicity to algae.

Results analysis by start of test and after 72 hours

 Nominal concentration [mg/l]  0 hours [mg/l]  72 hours [mg/l]
 control  < 2  3
 5 *w.algae  5  3
 5  4
 10  8
 20  15 13 
 40  29  26
 40 *w.algae  27  27

 *w.algae = without algal inoculum

All values refer to TOC determinations. According to the relevant product infomations 1 mg/l TOC equals to 1.3 mg/l of the test substance (empirical formula: C10H20O; molecular weight: 156,3g/mol).

Analysis of the test concentrations 5 mg/l, 10 mg/l, 20 mg/l, 40 mg/l plus a control; additional analysis of the highest and the lowest analytically determined test concentration without algal inoculum,at test start and after 72 hours of exposure. The test concentrations 1.25 mg/l and 2.5 mg/l were not analytically determined as the measured concentrations to be expected were below the detection limit of the TOC determination method (2 mg/l).

Before measuring TOC, the samples after 72 hours of exposure were filtered.

Validity criteria fulfilled:
yes
Conclusions:
During 72 hours exposure an ErC50 of 16.2 mg/L and a NOEC of 4.6 mg/l was determined.
Executive summary:

With a growth inhibition study according to EU Method C.3 on algae (Desmodesmus subspicatus former name: Scenedesmus subspicatus) which is equivalent to OECD Guideline for Testing of Chemicals No. 201 a 72-hour EC50 of 16.2 mg/l (based on the measured arithmetic mean) was determined for DL-Menthol (CAS no. 1490-04-6). The EC50 value is quoted in the OECD SIDS report, 2003 (agreed by the ICCA initiative).The NOEC resulting from the Dunnett test was 4.6 mg/l.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
Dec 10, 2002 - Dec 13, 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
yes (incl. certificate)
Remarks:
(2002-12-19)
Test material information:
Composition 1
Analytical monitoring:
yes
Details on sampling:
Schedule of sampling:
Stock solution at 0 hours
Control at 0 and 72 hours
Test concentration at 0 and 72 hours
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
Before the beginning of the study, the test substance was pulverized. A stock solution was prepared to give the desired series of test concentrations. To achieve this 124.9 mg of the test substance were added to 1 litre of dilution water and treated for 1 h in an ultrasonic bath and afterwards stirred for 24 h on a magnetic stirrer.
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: Desmodesmus subspicatus
- Strain: Non-axenic strain
- Source (laboratory, culture collection): "The collection of Algal Cultures" of the Institute of Plant Physiology at the University of Göttingen, Germany.
- Method of cultivation: Exponentially-growing stock cultures are maintained in the test facility under constant temperature conditions (23 +/- 2°C) at a light intensity in the range 60-120 μE/m2/s (measured in the range 400 to 700 nm using a spherical quantum flux meter). The nutrient medium (according to BRINGMANN & KÜHN (1977)) is renewed once a week. Cell density measurements are made using a microcell counter.

ACCLIMATION
- Acclimation period: Pre-cultures are set up three days before the start of a test.
- Culturing media and conditions (same as test or not): Same as test culture method (see above)
Test type:
static
Water media type:
not specified
Limit test:
no
Total exposure duration:
72 h
Test temperature:
21°C to 25°C +/- 2°C
pH:
At 0 hour: pH=8.2 to 8.5
At 72 hours: pH=8.6 to 10.4
Nominal and measured concentrations:
Nominal concentrations: 5.0, 10, 20, and 40 mg/l administered from stock solutions.
Measured concentrations (at 0 hour - at 72 hours): 4.6-4.5, 10-9.3, 19.9-17.6 and 41-36.7 mg/l
Details on test conditions:
TEST SYSTEM
- Test vessel: 300 ml Erlenmeyer flasks with stoppers
- Initial cells density: 10^4 cells/ml
- Control end cells density: 318889 cell/ml
- No. of vessels per concentration (replicates): 3 replicates per concentration
- No. of vessels per control (replicates): 6 replicates per concentration

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Photoperiod: continuous uniform illumination
- Light intensity and quality: light between 400-700 nm, light intensity in the range 60-120 μE/m2/s (equivalent to 4000 to 8000 lx)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: microcell counter or alternatively by means of a microscopic counting chamber

TEST CONCENTRATIONS
- Spacing factor for test concentrations: x2
- Test concentrations: see above
Reference substance (positive control):
no
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
20 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: reduction of growth
Remarks on result:
other: 95% confidence limits : 16-24 mg/l
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
21.4 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence limits : 21.4-21.5 mg/l
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
9.65 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: reduction of growth
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
9.65 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
18.75 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: reduction of growth
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
18.75 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Validity criteria fulfilled:
yes
Conclusions:
In the test using the freshwater algae Desmodesmus subspicatus an ErC50 (based on growth rate) of 21.4 mg/l was determined, whereas the No effect concentration (NOEC) was determined to be 9.65 mg/l. The substance needs to be considered harmful to algae.

Description of key information

Desmodesmus subspicatus ErC50 (72 h) = 16.2 mg/L (measured, based on growth); read-across
Desmodesmus subspicatus NOErC (72 h) = 4.6 mg/L (measured, based on growth); read-across

Key value for chemical safety assessment

EC50/LC50 for freshwater algae:
16.2 mg/L
EC10, LC10 or NOEC for freshwater algae:
4.6 mg/L

Additional information

Since no data for the target substance are available, the aquatic toxicity is assessed on the basis of data from two structurally similar menthol substances (stereo isomers).
Both studies with the source substances examined the impact on growth of the unicellular green algae Desmodesmus subspicatus according to standard guidelines and GLP. In the key study with D/L-menthol (CAS 1490-04-6) a growth inhibition of 50% (ErC50, 72 h) was observed in the range of > 9.8 < 18.2 mg/L test item concentration (measured arithmetic mean). By Probit analysis a defined value for the ErC50 (72 h) of 16.2 mg/L (measured arithmetic mean) was determined. The corresponding NOErC (72 h, based on growth rate) is reported with 4.6 mg/L (measured arithmetic mean).
This result is confirmed by the supporting study with L-Menthol (CAS 2216-51-5), where a slightly higher ErC50 (72 h) of 21.4 mg/L (measured arithmetic mean) was determined. The corresponding NOErC (72 h, based on growth rate) is reported to be 9.65 mg/L (measured arithmetic mean).
Based on the results from two structurally related source substances (in accordance to Regulation (EC) No 1907/2006 Annex XI, 1.5) it can be concluded that testing the target substance would result in comparable results.