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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:
4 February 1992 to 24 January 1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1994
Report date:
1994

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
OECD Guideline No. 201 and EEC Directive 67 /549 Annex VIII, Part C (87 /302/EEC).
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Reference substance name:
Active enzyme protein of peroxidase (EC no. 232-668-6, CAS no. 9001-05-2, EC name Peroxidase, Enzyme class no. 1.11.1.7)
Molecular formula:
Not applicable, see remarks.
IUPAC Name:
Active enzyme protein of peroxidase (EC no. 232-668-6, CAS no. 9001-05-2, EC name Peroxidase, Enzyme class no. 1.11.1.7)
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: crystalline
Details on test material:
- Name of test material (as cited in study report): SP 491 Peroxidase
- Substance type: UVCB
- Physical state: Dark brown crystaline solid
- Lot/batch No.: PPX 3806
- Expiration date of the lot/batch: 04 March 1994
- Storage condition of test material: In the refrigerator at 4 degrees of Celcius

Sampling and analysis

Analytical monitoring:
no

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Direct dispersion in water.
- Controls: Nutrient medium.

Test organisms

Test organisms (species):
Scenedesmus sp.
Details on test organisms:
TEST ORGANISM
Name: Scenedesmus subspicatus
Strain No. CCAP 276/20.
Source: Culture Centre of Algae & Protozoa c/o Freshwater Biological Association, Cumbria, U.K.
Pre-culture: Sterile nutrient medium (Appendix 1) was inoculated from a master culture and incubated under continuous illumination (≈7000 lux) and stirring (orbital shaker) at 24°C to give an algal suspension in log phase growth characterised by an absorbance of 0.95 (@ 665 nm). The suspension was diluted to an absorbance of 0.042 prior to use.

Study design

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

Test conditions

Hardness:
CaCl2.2H2O = 8 mg/L
MgCl2.6HO = 12 mg/L
Test temperature:
24±1⁰C
pH:
7.1-10.7
Nominal and measured concentrations:
62.5, 125, 250, 500 and 1000 mg/L (nominal).
Details on test conditions:
TEST SYSTEM
- Test vessels: 250 mL conical flasks containing 100 mL test solution and loosely stoppered to reduce evaporation.
- Type: Loosely stoppered to reduce evaporation.
- Aeration: None. Gaseous exchange and suspension of algal cells maintained by orbital shaker.
- Initial cells density: 1.1*10^5 cells/mL
- Control end cells density: 2*10^6 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
- Method of initiation: 50 mL algal pre-culture were added to 50 mL of sterile nutrient medium containing appropriate amounts of the test substance.

GROWTH MEDIUM
- Standard medium used: Yes.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: Continuous (approximately 7000 lux).

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: Direct counting with the aid of a haemacytometer.

TEST CONCENTRATIONS
- Concentrations: 62.5, 125, 250, 500 and 1000 mg/L (nominal).

Medium renewal: None.
Duration of exposure: 72 hours.
Measurement of growth: Samples were taken from the control cultures at 0, 24, 48 and 72 hours and the absorbance measured at 665 nm. The cell densities of the control and test cultures were determined at 0, 24, 48 and 72 hours by direct counting with the aid of a haemacytometer.
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
other: EbC50
Effect conc.:
320 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
other: EbC50
Effect conc.:
88.3 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
other: ErC50
Effect conc.:
640 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
other: ErC50
Effect conc.:
176.6 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
125 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: area under the curve
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
35.3 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: area under the curve
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
500 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
138 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Details on results:
Mean cell density of control @ 0 h: 1.1 x 10^5 cells/mL
Mean cell density of control @ 72 h: 2.0 x 10^6 cells/mL
The initial cell count for the cultures at 0 hours is higher than that recommended under OECD guidelines. However, we consider that the higher initial cell density is justified in order to improve the reliability of haemocytometer and spectrophotometer measurements, as a slight error in cell densities at 0 hours can lead to a large error in the "growth factor" estimate and subsequently to misleading conclusions on growth. It is our opinion that satisfactory growth was achieved in this study as indicated by an increase in mean control cell density from 1.1 x 10^5 cells/mL cells/ml to 2.0 x 10^5 cells/mL in 72 hours.

Observations: All test and control cultures were inspected at 72 hours. At the two highest test concentrations of 500 mg/L and 1000 mg/L, the cells were observed to be clumped at 24, 48 and 72 hours. At 72 hours in the highest test concentration (1000 mg/L) cells were also observed to be small. A stimulatory effect on algal growth rate was observed in the two lowest exposure levels (62.5 mg/L and 125 mg/L). Also at 72 hours those cells in the 62.5 mg/L exposure level were observed to be considerably larger than those in the control.
Reported statistics and error estimates:
In order to estimate the concentration at which 50% inhibition of growth occurs, a logistic model 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. In order to assess the adequacy of these models, the variance due to 'lack of fit' was compared with the within-group variance. F-tests were applied, with 1 and 10 degrees of freedom. 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. A 'no observed effect level' was obtained using Williams' test to compare the percentage inhibition in each treated group with the baseline (control) values. Bartlett's test with a 1% significance level was used to test for homogeneity of variance and this was not significant.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
In the current testing conditions, exposure of Scenedesmus suhspicatus to peroxidase for 72 hours in concentrations up to 1000 mg/L (276 mg/L active enzyme protein) produced the following EC50 and NOEC values:
EbC50 (72 h): 320 mg/L (95 % confidence limits 290 - 360 mg/L)
ErC50 (24 - 72 h): 640 mg/L (95 % confidence limits 560 - 730 mg/L)
"No observed effect level" for 72 hour AUC: 125 mg/L
"No observed effect level" for the 24 - 72 hour growth rate: 500 mg/L
Executive summary:

Scenedesmus suhspicatus was exposed to peroxidase for a period of 72 hours. The study was conducted according to OECD Guideline No. 201 and EEC Directive 67 /549 Annex VIII, Part C (87 /302/EEC). Five algeal cultures with an initial cell density of 1.1 x 10^5 cells/mL were exposed to peroxidase in concentrations 62.5, 125, 250, 500, 1000 mg/L and 17.3, 34.5, 69, 138, 276 mg/L active enzyme protein.There were 5 concentrations plus 1 control in each triplicate. The cultures were incubated for 72 hours under continuous illumination.

The cell densities of the control and test cultures were determined at 0, 24, 48 and 72 hours by direct counting with the aid of a haemacytometer.

The growth inhibition test showed the following values:

EbC50 (72 h): 320 mg/L (95 % confidence limits 290 - 360 mg/L); 88.3 mg/L active enzyme protein

ErC50 (24 - 72 h): 640 mg/L (95 % confidence limits 560 - 730 mg/L); 176.6 mg/L active enzyme protein

"No observed effect level" for 72 hour AUC: 125 mg/L; 35.5 mg/L active enzyme protein

"No observed effect level" for the 24-72 hour growth rate: 500 mg/L; 138 mg/L active enzyme protein