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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The range-finding study was conducted between 11 September 1998 and 14 September 1998 and the definitive study between 2 November 1998 and 6 November 1998.
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP study conducted in compliance with agreed test protocols following OECD Method 201. Acceptable restriction was that analytical measurements on the test material were not conducted.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report Date:
1999

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
Principles of method if other than guideline:
not applicable.
GLP compliance:
yes (incl. certificate)
Remarks:
1998-07-21

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Substance type: amber viscous liquid
- Physical state: liquid
- Analytical purity: no data
- Impurities (identity and concentrations): no data
- Composition of test material, percentage of components: no data
- Isomers composition: no data
- Purity test date: no data
- Lot/batch No.: no data
- Expiration date of the lot/batch: no data
- Stability under test conditions: no data
- Storage condition of test material: at room temperature in the dark
- Other: none
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
none

Sampling and analysis

Analytical monitoring:
no
Details on sampling:
- Concentrations: Based on the results of the range-finding study the following test concentrations were assigned to the definitive study 6.25, 12.5, 25, 50 and 100 mg/l loading rate WSF.
- Sampling method: 300 mL of each test sample was extracted with 3 (3 x 50 mL) portions of dichloromethane and the extracts filtered through anhydrous sodium sulphate. The combined extracts were evaporated to dryness and the residue redissolved in methanol (2 mL)
Stoarage conditions: ambient temperature in light and dark conditions for 72 hours.

Test solutions

Vehicle:
no
Details on test solutions:
For the purpose of the definitive study the test material was prepared in the dark as a Water Soluble Fraction (WSF).
Amounts of test material (62.5 and 125 mg) were added to the vortex created in 10 litres of culture medium to give loading rates of 6.25 and 12.5 mg/l. Similarly, amounts of test material (50, 100 and 200 mg) were added to the vortex created in 2 litres of culture medium to give loading rates of 25, 50 and 100 mg/l. Each loading rate was stirred for a period of 23 hours with a vortex depth of approximately 25% of the water column depth.
After 23 hours stirring, the mixtures were allowed to stand for 1 hour prior to the removal of the aqueous phase by siphon and subsequent filtration through 0.45 pm membrane filters to give the 6.25, 12.5, 25, 50 and 100 mg/l loading rate WSF.
An aliquot (1 litre) of each loading rate WSF was inoculated with algal suspension (5 ml) to give the test concentrations of 6.25, 12.5, 25, 50 and 100 mg/l loading rate WSF.

Test organisms

Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: green algae
- Strain: CCAP 276/20
- Source (laboratory, culture collection): CCAP, Institute of Freshwater Ecology, Ferry house, Ambleside, Cumbria.
- Age of inoculum (at test initiation): no data
- Method of cultivation: Cultures were maintained in the laboratory by the periodic replenishment of culture medium. The culture was maintained in the laboratory at a temperature of 21+/- 1°C under continuous illumination (intensity approximately 7000 lux) and constant aeration.

- Culturing media and conditions:
The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.

Culture medium:
NaNO3 25.5 mg/l
MgCl2.6H2O 12.164 mg/l
CaCl2.2H2O 4.41 mg/l
MgSO4.7H2O 14.7 mg/l
K2HPO4 1.044 mg/l
NaHCO3 15.0 mg/l
H3BO3 0.1855 mg/l
MnCl2.4H2O 0.415 mg/l
ZnCl2 0.00327 mg/l
FeCl3.6H2O 0.159 mg/l
CoCl2.6H2O 0.00143 mg/l
Na2MoO4.2H2O 0.00726 mg/l
CuCl2.2H2O 0.000012 mg/l
Na2EDTA.2H2O 0.30 mg/l
Na2SeO3.5H2O 0.000010 mg/l

The culture medium was prepared using reverse osmosis purified deionised water (Elga Optima 15 +) and the pH adjusted to 7.5 ± 0.1 with 0.1 N NaOH or HCl. The prepared media was sterilised by 0.22 µm membrane filtration and stored in darkness.

Pre-culture conditions gave an algal suspension in log phase growth characterised by a cell density of 3.75 x lo6 cells per ml. This suspension was diluted to a cell density of 2.05 x lo4 cells per ml prior to use. Addition of an aliquot (5 ml) of algal suspension to 1 litre of test medium gave a nominal cell density of 1 x IO4 cells per ml at initiation of the study. The flasks were covered with aluminium foil and incubated (Gallenkamp INR- 401-010W) at 24 f 1 'C under continuous illumination (intensity approximately 7000 lux) and constantly shaken at 100 rpm for 72 hours.
Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter@M ultisizer II Particle Counter.

- Any deformed or abnormal cells observed:
All test and control cultures were inspected microscopically at 72 hours. There were no abnormalities detected in any of the control or test cultures.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
none.

Test conditions

Hardness:
no data
Test temperature:
24 +/- 1°C
pH:
7.0-8.1
Dissolved oxygen:
no data
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal concentratins: 0, 6.25, 12.5, 25, 50, and 100 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: glass conical flasks
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 250 mL glass conical flasks containing 100 mL of culture medium
- Aeration: yes
- Initial cells density (test): 1.00 x 10^4 cells/mL
- Initial cells density (control): 1.21 x 10^4 cells/mL
- Control end cells density: 2.50 x 10^5 cells/mL
- No. of organisms per vessel: ca. 100 x 10^4 cells
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Standard medium used: no
- Detailed composition if non-standard medium was used:
NaNO3: 25.5 mg/L
MgCl2.6H2O: 12.164 mg/L
CaCl2.2H2O: 4.41 mg/L
MgSO4.7H2O: 14.7 mg/L
K2HPO4: 1.044 mg/L
NaHCO3: 15.0 mg/L
H3BO3: 0.1855 mg/L
MnCl2.4H2O: 0.415 mg/L
ZnCl2: 0.00327 mg/L
FeCl3.6H2O: 0.159 mg/L
CoCl2.6H2O: 0.00143 mg/L
Na2MoO4.2H2O: 0.00726 mg/L
CuCl2.2H2O: 0.000012 mg/L
Na2EDTA.2H2O: 0.30 mg/L
Na2SeO3.5H2O: 0.000010 mg/L

OTHER TEST CONDITIONS
- Sterile test conditions: yes/no
- Adjustment of pH: no
- Photoperiod: continuous
- Light intensity and quality: 7000 lux
- Salinity (for marine algae): not applicable

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: electronic particle counter
- Chlorophyll measurement: no data
- Other: none.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Justification for using less concentrations than requested by guideline:
- Range finding study
- Test concentrations:0, 1.0, 10.0, 100.0 mg/L
- Results used to determine the conditions for the definitive study: The results showed no effect on growth at the test concentrations of 1.0 and
10 mg/l loading rate WSF. However, growth was observed to be reduced at 100 mg/l loading rate WSF.
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
14 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 13-15
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
16 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 15-17
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
6.25 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Details on results:
none
Results with reference substance (positive control):
not applicable.
Reported statistics and error estimates:
none.

Any other information on results incl. tables

Table 1 and 2 are presented in attached document.

Table 1: Cell densities and pH values un the definitive study.

Table 2: Inhibition of growth rate and biomass.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Exposure of Scenedesmus subspicatus to the test material gave an EbLR50 (72 h) value of 14 mg/L loading rate WSF: 95% confidence limits 13 - 15 mg/L loading rate WSF and an ErLR50 (0-72 h) value of 16 mg/L loading rate WSF: 95% confidence limits 15 - 17 mg/L loading rate WSF. The No Observed Effect Loading Rate was found to be 6.25 mg/L loading rate WSF.
Executive summary:

Methods: A study was performed to assess the effect of the test material on the growth of the green alga Scenedesmus subspicatus. The method followed that described in the OECD Guidelines for Testing of Chemicals (1984) No 201, "Alga, Growth Inhibition Test" referenced as Method C.3 of Commission Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC).

Procedure: Following a preliminary range-finding study, Scenedesmus subspicatus was exposed to Water Soluble Fraction (WSF) of the test material at loading rates of 6.25, 12.5, 25, 50 and 100 mg/L for 72 hours, under constant illumination and shaking at a temperature of 24 +/-1°C. Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group using a Coulter@ Multisizer I I Particle Counter.

Results: Exposure of Scenedesmus subspicatus to the test material gave an EbLR50 (72 h) value of 14 mg/L loading rate WSF: 95% confidence limits 13 - 15 mg/L loading rate WSF and an ErLR50 (0-72 h) value of 16 mg/L loading rate WSF: 95% confidence limits 15 - 17 mg/L loading rate WSF. The No Observed Effect Loading Rate was found to be 6.25 mg/L loading rate WSF.

The concentration of the components of the test material being taken into the water by the preparation of a WSF were expected to be low given the low water solubility of the test material. Analysis at 0 and 72 hours showed the measured test concentrations to range from 1 % to 34% of nominal. The variation in measured concentration followed a concentration dependent effect with higher percentage of nominal concentrations being determined from the lower test concentrations. Due to the limited water solubility of the test material in the test media only a small amount of test material can enter the aqueous phase before the test medium becomes saturated. Whilst the amount which can enter the aqueous phase is constant regardless of the amount of test material present it becomes of proportionately greater significance as the nominal loading rate, ie the amount of test material present, decreases.

Therefore the measured test concentration, expressed as a percentage of nominal loading rate increased as the loading rate declined.