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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Administrative data

toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1996-11-19 to 1996-11-22
1 (reliable without restriction)

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
according to guideline
EU Method C.3 (Algal Inhibition test)
Version / remarks:
as described in Commission Directive 92/69/EEC
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
C29 H24 O7
Specific details on test material used for the study:
- Description : off white solid

Sampling and analysis

Analytical monitoring:
Details on sampling:
- Sampling method: Water samples were taken from the solvent control and the 0.10 mg/L test group (replicates R1 - R3 and R4 - R6 pooled) at 0 and 72 hours for quantitative analysis.
- Sample storage conditions before analysis: not specified

Test solutions

Details on test solutions:
- Method: An amount of test material (50 mg) was dispersed in solvent and the volume adjusted to 50 mL to give a 50 mg/50 mL solvent stock solution. An aliquot (200 µL) of this stock solution was then dispersed in 2 litres of algal suspension to give the 0.10 mg/L test concentration.
- Differential loading: no
- Controls: blank control (without test item), solvent control (with solvent but without test item)
- Chemical name of vehicle: dimethylformamide
- Concentration of vehicle in test medium: 100 µL/L
- Test concentration separation factor: not applicable (limit test)
- Evidence of undissolved material: no

Test organisms

Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
- Common name: Desmodesmus subspicatus
- Strain: CCAP 276/20
- Source: Culture Centre for Algae and Protozoa (CCAP), Institute of Freshwater Ecology, Ferry House, Ambleside, Cumbria
- Age of inoculum: not specified, preculture conditions gave an algal suspension in log phase growth characterised by an absorbance of 1 .247 (at 665 nm)
- Method of cultivation: cultures maintained in laboratory by replenishment of culture medium approximately once per week; culture maintained in laboratory at 21 + 1 °C under continuous illumination (intensity approximately 2000 lux) and constant aeration

- Acclimation period: not necessary, culturing conditions same as test
- Culturing media and conditions: same as test
- Any deformed or abnormal cells observed: not specified

Study design

Test type:
Water media type:
Limit test:
Total exposure duration:
72 h

Test conditions

not specified
Test temperature:
Test temperature was maintained at 24 +/- 1 °C
The pH values of the control and test cultures (see Table 2) were observed to increase from pH 8.0 at 0 hours to pH 10.0 - 10.3 at 72 hours. This effect is
considered to be due to the large number of cells in the log phase of growth respiring oxygen and producing carbonates and bicarbonates as part of
photosynthesis/respiration which in solution give rise to alkaline conditions.
Dissolved oxygen:
not specified
not applicable
not specified
Nominal and measured concentrations:
nominal: 0.10 mg/L; measured (at 72 hrs): 0.084 mg/L (replicates 1 - 3) and 0.094 mg/L (replicates 4 - 6), corresponding to 84 % and 94 % of nominal concentration, respectively
Details on test conditions:
- Test vessel: conical flasks
- Type: closed (with aluminium foil)
- Material, size, fill volume: glass, 250 mL nominal volume, 100 mL fill volume
- Aeration: shaken at 100 rpm for 72 hours
- Initial cells density: 10^4 cells/mL
- No. of vessels per concentration: 6
- No. of vessels per control: 3
- No. of vessels per vehicle control 3

- Detailed composition:
NH4CI: 15 mg/L
MgCl2.6H2O: 12 mg/L
CaCl2.2H2O: 18 mg/L
MgSO4.7H2O: 15 mg/L
KH2PO4: 1.6 mg/L
FeCl3.6H2O: 0.08 mg/L
Na2EDTA.2H2O: 0.1 mg/L
H3BO3: 0.185 mg/L
MnCl2.4H2O: 0.415 mg/L
ZnCl2: 3 x 10^-3 mg/L
CoCl2: 6H2O: 1.5 x 10^-3 mg/L
ZnCl2: 3 x 10^-3 mg/L
CoCl2.6H2O: 1.5 x 10^-3 mg/L
CuCl2.2H2O: 1 x 10^-5 mg/L
Na2MoO4.2H2O: 7 x 10^-3 mg/L
NaHCO3: 50 mg/L

The pH of this medium after equilibration with air is approximately 8.

- Sterile test conditions: not specified
- Adjustment of pH: no
- Photoperiod: continuously illuminated
- Light intensity: 7000 lux

- Determination of cell concentrations: Samples were taken at 0, 24, 48 and 72 hours and the absorbance measured at 665 nm using a Jenway 6100 Spectrophotometer. The cell densities of the control cultures at 0, 24, 48 and 72 hours, were determined by direct counting with the aid of a haemocytometer to confirm that the absorbance values were sufficiently well correlated with cell density values to be used to monitor the growth of the test cultures.

- Test concentration: 0.10 mg/L
- Justification for using less concentrations than requested by guideline: The highest attainable concentration using DMF as solvent was applied. This experimental design conforms to a "limit test" to confirm that at the highest attainable test concentration of 0.10 mg/L no effect on growth was observed.
- Range finding study: performed at 0.10 mg/L for a period of 72 hours. Results used to determine the conditions for the definitive study: yes

-Details on culturing apparatus used: Gallenkamp INR - 401-010W
Reference substance (positive control):

Results and discussion

Effect concentrationsopen allclose all
Key result
72 h
Dose descriptor:
Effect conc.:
> 0.1 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
Key result
72 h
Dose descriptor:
Effect conc.:
> 0.1 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
72 h
Dose descriptor:
Effect conc.:
>= 0.1 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
Key result
72 h
Dose descriptor:
Effect conc.:
>= 0.1 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
There were no abnormalities detected in any of the control or test cultures.
- Exponential growth in the control: yes
- Observation of abnormalities: no
- Unusual cell shape: no
- Colour differences: no
- Flocculation: not reported
- Adherence to test vessels: not reported
- Aggregation of algal cells: no
- Any observations that might cause a difference between measured and nominal values: not specified
- Effect concentrations exceeding solubility of substance in test medium: the effect concentration of 0.1 mg/L could only be reached by using DMF as solvent; this concentration exceeds the water solubility of the substance which is <0.0352 mg/L (see IUCLID section 4.8).
Results with reference substance (positive control):
not applicable (positive control not performed)
Reported statistics and error estimates:
A Students t-test was carried out on the area under the growth curve data at 72 hours for the solvent control and the 0.10 mg/L test concentration group to determine any statistically significant differences between the test and control groups.

Any other information on results incl. tables

Justification of applied test concentration

The test concentration of 0.10 mg/l was the highest attainable test concentration that could be prepared due to the limited solubility of the test material in water and auxiliary solvent and having due regard to the amount of auxiliary solvent permitted in the study under the OECD Cuideline. The limited water solubility is reported in IUCLID sectin 4.8 and is below 0.0352 mg/L.

The test concentration of 0.10 mg/l used during this study was prepared with the aid of an auxiliary solvent, dimethylformamide. The concentration of 0.10 mg/l was considered to be close to the true limit of solubility for the test material given that the freshly prepared test preparations for the range-finding study were observed to be clear colourless solutions.
During preliminary solubility work, at concentrations in excess of 0.10 mg/l a
marked precipitation of the test material was observed after addition of the solvent
stock solution to water. The use of other various recognised auxiliary solvents did
not allow the preparation of a higher test concentration.

Cell concentration of control cultures

The following data show that the cell concentration of the control cultures increased by a factor of 63 and the cell concentration of the solvent control cultures increased by a factor of 52 during the test in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours:

Mean cell density of control at 0 hours: 1.23 x 10^4 cells/ml
Mean cell density of control at 72 hours: 7.78 x 10^4 cells/ml
Mean cell density of solvent control at 0 hours: 1.55 x 10^4 cells/ml
Mean cell density of solvent control at 72 hours: 8.01 x 10^4 cells/ml

Table 1: Inhibition of growth

Nominal Concentration (mg/L)Area under curve at 72 h% inhibitionGrowth rate (0 - 24 h)% inhibition
Solvent Control15.5-0.067-

[increase in growth as compared to controls]


Table 2: Absorbance and pH values in the definitive study

Nominal Concentration (mg/L)pHAbsorbance valuespH
0 h0 h24 h48 h72 h72 h
Solvent ControlR18.00.0220.1120.1890.77510.1

R1 - R6 = Replicates 1 - 6


Table 3: Mean cell density and absorbance values of the control cultures in the definitive study

 0 h 24 h 48 h72 h
ControlMean cell density* (cells/mL)1.23 x 1041.05 x 1052.38 x 1057.78 x 105
Mean absorbance value0.0220.1060.2320.767
Solvent controlMean cell density* (cells/mL)1.55 x 1041.11 x 1052.44 x 1058.01 x 105
Mean absorbance value0.0220.1090.2160.752

*Mean cell density values represent the mean number of cells per ml calculated from the mean of the cell counts from 3 fields of view for each of the replicate flasks.


Table 4: Analytical results: All the test sample results have been corrected for the apparent levels determined in the solvent control samples.

SamplesNominal concentration (mg/L)Concentration found (mg/L)Expressed as a % of the nominal concentration
0 hoursSolvent Control< LOQ-
 0.10 R1 - R30.122122
 0.10 R4 - R60.08686
72 hoursSolvent Control< LOQ-
 0.10 R1 - R30.08484
 0.10 R4 - R60.09494

LOQ : Limit of quantitation
R1 - R6: Replicates 1 - 6


Applicant's summary and conclusion

Validity criteria fulfilled:
A GLP compliant study was performed to assess the effect of the test material on the growth of the green alga Scenedesmus subspicatus following OECD TG 201. Following a preliminary range-finding study, Scenedesmus subspicatus was exposed to an aqueous dispersion of the test material at a concentration of 0.10 mg/l (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1°C. Samples of the algal populations were removed daily and absorbance values determined for each control and treatment group.
Exposure of Scenedesmus subspicatus to the test material gave EC50 values of greater than 0.10 mg/l and correspondingly the No Observed Effect Concentration was greater than or equal to 0.10 mgl/l. The test concentration of 0.10 mg/l was the highest attainable test concentration that could be prepared due to the limited solubility of the test material in water and auxiliary solvent and having due regard to the amount of auxiliary solvent permitted in the test under the OECD Guidelines. Analysis of the test solutions at 0 and 72 hours showed the measured test concentrations to be in excess of the required 80 % of nominal test concentrations and so the results are based on nominal test concentrations alone.