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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 August 1992-30 November 1993
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1993
Report date:
1993

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Reference substance name:
Active enzyme protein of Lipase, triacylglycerol (EC no. 232-619-9, CAS no. 9001-62-1, EC name: Lipase, triacylglycerol, Enzyme Class No.: 3.1.1.3)
Molecular formula:
Not applicable, see remarks
IUPAC Name:
Active enzyme protein of Lipase, triacylglycerol (EC no. 232-619-9, CAS no. 9001-62-1, EC name: Lipase, triacylglycerol, Enzyme Class No.: 3.1.1.3)
Constituent 2
Reference substance name:
Protein as a constituent of enzyme deriving from the fermentation or extraction process
Molecular formula:
Not available
IUPAC Name:
Protein as a constituent of enzyme deriving from the fermentation or extraction process
Constituent 3
Reference substance name:
Inorganic salts as a constituent of enzyme deriving from the fermentation or extraction process
Molecular formula:
Not available. See remarks.
IUPAC Name:
Inorganic salts as a constituent of enzyme deriving from the fermentation or extraction process
Constituent 4
Reference substance name:
Carbohydrates constituent of enzyme deriving from the fermentation or extraction process
Molecular formula:
Not available. See remarks.
IUPAC Name:
Carbohydrates constituent of enzyme deriving from the fermentation or extraction process
Constituent 5
Reference substance name:
Lipids as a constituent of enzyme deriving from the fermentation or extraction process
Molecular formula:
Not available. See remarks.
IUPAC Name:
Lipids as a constituent of enzyme deriving from the fermentation or extraction process
Test material form:
solid: particulate/powder
Details on test material:
- Lot/batch No.: PPW 3983
- Expiration date of the lot/batch: 2 September 1994
- Stability under test conditions: The test enzyme and dilutions in water (10% and 100%) are stable for at least 24 hours at room temperature
- Storage condition of test material: In the dark at 4⁰C

Sampling and analysis

Analytical monitoring:
no

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test substance was dissolved in sterile nutrient medium to provide an initial stock solution of 320 mg/L. This stock solution was further diluted with sterile nutrient medium to produce a series of solutions exactly twice the concentration of the intended exposure levels. 150 mL of algal pre-culture was mixed with 150 mL of each of these solutions to give the final test series.
- Controls: Medium

Test organisms

Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: green alga
- Strain: CCAP 276/20
- Source (laboratory, culture collection): Culture Centre of Algae and Protozoa c/o Freshwater Biological Association, Cumbria, UK
- Method of cultivation: Sterile nutrient medium was inoculated from a master culture and incubated under continuous illumination (appr. 7000 lux) and stirring (orbital shaker) at 24 +/- 1°C to give an algal suspension in log phase growth characterised by an absorbanse of 0.380 (@665nm).
The suspension was diluted using sterile nutrient medium to an absorbance of 0.036 prior to use.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h

Test conditions

Test temperature:
23-25°C
pH:
7.5 - 10.3
Nominal and measured concentrations:
nominal: 0, 10, 20, 40, 80 and 160 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: conical flask
- Type (delete if not applicable): Flasks were loosely stoppered.
- Material, size, fill volume: glass, 250 mL, 100 mL
- Initial cells density: 7.9 x 10^4 cells/mL
- Control end cells density: 2.3 x 10^6 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6


GROWTH MEDIUM
- Standard medium used: Sterile nutrient medium as recommended in OECD Guideline No. 201


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reverse-osmosis purified water

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: continuous illumination
- Light intensity and quality: approximately 7000 lux provided by 7 x 30 W "warm white" 1 metre fluorescent tubes


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Samples were taken at 0, 24, 48 and 72 hours and the cell densities measured at 665 nm using 4 cm path cuvettes in a Cecil 373 Series 2 Spectrophotometer. The cell densities of control cultures at initiation and at termination were determinated by direct counting in a haemacytometer.


Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 40 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: growth rate and biomass
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 7.4 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Remarks:
aep (active enzyme protein)
Basis for effect:
other: growth rate and biomass
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
97 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
17.6 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Remarks:
aep
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
99 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
18 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Remarks:
aep
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): At the highesr exposure level of 160 mg/L cells were observed to be clumped. No abnormalities were obsereved at all other exposure concetrations inclduing controls.

Reported statistics and error estimates:
In order to estimate the concentration at which 50% inhibition occurs, a logistic model (Volund, A. 1978. Biometrics 34) was fitted to the percentage inhibition values based on the 'area under the curve' (AUC) at 72 hours and on the growth rate between 24 and 72 hours.
The 50% points were estimated by interpolation of the fitted curves and 95% confidence limits were produced by reference to Student's t distribution, using a pooled estimate of the error variance about the model (Draper and Smith, 1966. Applied Regression Ananlyisi (first ed.) John Wiley and Sons, Inc. New York).
A 'no observed effect level' was obtained using Williams test (Williams, D.A., 1971. Biometrics 27. Williams, D.A.1972. Biometrics 28). Bartletts test (Bartlett, M.S. 1973. J.Royal Stat.Soc.Suppl.4) with a 1% significance level was used for homogeneity of variance.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Lipolase is inhibitory to the growth of Scenedesmus subspicatus (new name: Desmodesmus subspicatus) at concentration in excess of 40 mg/L or 7.4 mg active enzyme protein (aep)/L. The 72h EbC50 is 97 mg/L or 17.6 mg aep/L and the ErC50 is 99 mg /L or 18 mg aep/L.
Executive summary:

The effect of Lipolase on the growth of the unicellular green alga Scenedesmus subspicatus (new name 

Desmodesmus subspicatus) was assessed in accordance with EEC Methods for the Determination of Ecotoxicity, EEC Directive 67/548 Annex VIII Part C (87/302/EEC) and OECD test guideline 201, and in compliance with GLP.

Algal cultures exposed to five test concentrations of Lipolase plus one untreated control were incubated on an orbital shaker under continuous illumination at 24 ± 1°C for 72 hours. Growth was monitored daily by measuring the absorbance of each culture at 665 nm.

The following values were derived from the data:

 

 

Nominal Lipolase concentration

(mg/L)

Nominal Lipolase concentration

(mg aep/L)

Area under the growth curve

EbC50 (72 h)

97  17.6

Average specific growth rate

ErC50 (0 - 72 h)

99   

18

“No observed effect concentration”

 40  7.4