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Toxicity to aquatic algae and cyanobacteria

Administrative data

toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09/05/2000 - 30/08/2000
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study according to international guideline (ISO International Standard 8692) under GLP, validity criteria met.
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)
Principles of method if other than guideline:
not relevant
GLP compliance:

Test material

Constituent 1
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Details on test material:
- Name of test material (as cited in study report): Dihydrolinalool
- Physical state: Liquid
- Storage condition of test material: In refrigerator, protected from light
- Stability under storage conditions: Stable
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Not relevant

Sampling and analysis

Analytical monitoring:
Details on sampling:
- Concentrations: Nominal: 1.0, 2.2, 4.6, 10, 18, 32, 56 and 100 mg/l
- Sampling method: During the final test samples for analysis were taken from 1, 10, 100 mg/I and the blank-control. At the end of the exposure, the replicates incubated with algae were pooled at each concentration and the pooled solution was used for analysis. Samples of 10 ml were taken after 0, 24 and 96 hours of exposure.
- Sample storage conditions before analysis: Not applicable, samples were analysed on the day of sampling.

Test solutions

Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The standard test procedures required generation of test solutions which contained completely dissolved test substance concentrations or stable and homogeneous mixtures or dispersions. The testing of concentrations that would disturb the test system were prevented as much as
possible (e.g. film of the test substance on the water surface). DIHYDROLINALOOL in water was soluble at the concentrations tested. Stocks were
prepared in test medium at 100 mg/l. The lower concentrations were prepared by further dilution of aliquots taken from these stocks.
- Controls: Yes, test medium without test substance or other additives (blank control)

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
- Common name: Selenastrum capricornutum
- Strain: CCAP 278/4
- Age of inoculum (at test initiation): 3-4 days
- Method of cultivation: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light (4000-9000 lux) in a climate room at a temperature of 23 ± 2°C.

- Acclimation period: 3-4 days
- Culturing media and conditions (same as test or not): yes

Study design

Test type:
Water media type:
Limit test:
Total exposure duration:
96 h
Remarks on exposure duration:
initial period of 24 hours in the dark followed by 72 hours under continuous illumination.
Post exposure observation period:
Not relevant

Test conditions

Ca + Mg: 24 mg/l CaCO3
Test temperature:
23.7 °C at the start of the experiment, between 23.5 and 24 degrees Celsius at the end of the experiment.
in blank 8.4 at the start of the experiment, 10.1 at the end of the experiment.
in exposures 8.4 at the start of the experiment, 8.1-10.2 at the end of the experiment.
Dissolved oxygen:
No data
Not relevant
Nominal and measured concentrations:
Nominal: 1.0, 2.2, 4.6, 10, 18, 32, 56 and 100 mg/l
Measured (0 h, only for nominal 1, 10 and 100 mg/l with and without algae): 1.17, 10.6, 115 and 119 mg/l
Measured (24 h, only for nominal 1, 10 and 100 mg/l and without algae): 1.00, 9.15, 109 and 109 mg/l
Measured (96 h, only for nominal 1, 10 and 100 mg/l and without algae): 0.738, 8.18, 54 and 96 mg/l
Details on test conditions:
- Test vessel:
- Material, size, headspace, fill volume: 100 ml, all-glass
- Aeration: No data
- Initial cells density: 10,000 cells/ml
- Control end cells density: 1,897,000 cells/ml
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 6

- Standard medium used: Yes, M2-medium according to ISO standard 8692. MiliQ-water contained: NH4CI: 15 mg/I; MgCl2.6H2O: 12 mg/l; CaCI2.2H2O: 18 mg/l; MgSO4.7H20: 15 mg/I; KH2PO4: 1.6 mg/I; FeCI3.6H20: 80 ug/I; Na2EDTA.2H2O: 100 ug/I; H3BO3: 185 ug/l; MnCl2.4H20: 415 ug/l; ZnCl2: 3 ug/I; CoCl2.6H20:; CuCl2.2H20: 0.01 ug/l; Na2MoO4.2H2O: 7 ug/I and NaHCO3: 50 mg/I.

- Source/preparation of dilution water: Tap water purified by reverse osmosis and then passed over activated carbon and ion-exchange cartridges.
- Culture medium different from test medium: No
- Intervals of water quality measurement: Temperature: daily; pH: at the start and end of the experiment.

- Photoperiod: 24 hours of darkness followed by 72 hours of continuous illumination.
- Light intensity and quality: TLD-lamps pf the type "GroLux" of 30 Watt (Sylvania) yielding 9,400-10,500 lux with a light intensity within the range of
94 to 102 uE/m^2 S

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: At the beginning of the test cells were counted using a microscope using a counting chamber, further
measurements were done with a photospectrometer.
- Chlorophyll measurement: spectrophotic measurement at 720 nm using a Varian Cary 50 single beam spectrophotometer with immersion probe
(pathlength = 20 mm). Algal medium was used as blank.

- Spacing factor for test concentrations: 1.8
Reference substance (positive control):
Potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
96 h
Dose descriptor:
Effect conc.:
21 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
Remarks on result:
other: 95%CL: 8.2 - 53 mg/l
96 h
Dose descriptor:
Effect conc.:
78 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95%CL: 38 - 160 mg/l
96 h
Dose descriptor:
Effect conc.:
3.7 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
Remarks on result:
other: 95%CL: 1.4 - 9.9 mg/l
96 h
Dose descriptor:
Effect conc.:
24 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95%CL: 12 - 48 mg/l
Details on results:
- Exponential growth in the control (for algal test): yes
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- EC50: 0.49 mg/l
Reported statistics and error estimates:
For determination of the NOEC and the EC50 the approaches recommended in the OECD guideline (201, adopted 7 June 1984) were used. An effect
was considered to be significant if statistical analysis of the data revealed significant inhibition of growth or reduction of growth rate for the test
concentrations compared with those obtained in the negative control (ANOVA, Tukey test, Bonferroni t-Test, TOXSTAT Release 3.5, 1996, D.D. Gulley, A.M. Boelter, H.L. Bergman). Additionally, the EC10 was determined to meet the recommendations as put down in "A Review of Statistical Data Analysis and Experimental Design in OECD Aquatic Toxicology Test Guidelines" by S. Pack, August 1993.

For statistical analysis, see attachment.

Any other information on results incl. tables

Nominal concentration (mg/l) % inhibition area under growth curve (0-96h) % inhibition growth rate (0-96h)
1 12.3 2.5
1 5.6 1.3
1 11.2 3.8
2.2 3.6 0.3
2.2 18 4.5
2.2 -3.5 -3.3
4.6 13.6 1.5
4.6 29.9 8.1
4.6 -0.8 -2
10 29.9 6.3
10 29.1 6.6
10 32.8 6.4
18 40.2 8.8
18 40.3 9
18 29.5 4.8
32 54.1 14.2
32 61.7 19.2
32 52.3 13.3
56 78.7 33.3
56 59.7 16.8
56 80.7 35.1
100 96.1 68.5
100 93.9 69.1
100 94.9 63.8

Applicant's summary and conclusion

Validity criteria fulfilled:
Cell density increased by an average factor >16 in controls, concentrations of the test substance remained stable during the test period and pH increased more than 1.5 units only in the blank control and at the lowest concentration. Concentration in test
The 96h-EC50 (growth rate) of Dihydrolinalool to Selenastrum capricornutum is 78 mg/l.
Executive summary:

The toxicity of Dihydrolinalool towards Selenastrum capricornutum was investigated according to ISO International Standard 8692 under GLP. Validity criteria for the test were met. However, the pH increased more than 1.5 units during the experiment in the blank vessels and the vessels with the lowest concentration of test material. This increase >1.5 did not have an effect on the validity of the results. The 96h-EC50 (growth rate) was found to be 78 mg/l (38 - 160 mg/l).