Peroxidase

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

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

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

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Direct dispersion in water.
- Controls: Nutrient medium.

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.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Hardness:

CaCl2.2H2O = 8 mg/L
MgCl2.6HO = 12 mg/L

Test temperature:

24±1⁰C

pH:

7.1-10.7

Nominal and measured concentrations:

61, 122, 244, 488 and 976 mg enzyme concentrate dry matter/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: 61, 122, 244, 488 and 976 mg enzyme concentrate dry matter/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

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

488 mg/L

Nominal / measured:

nominal

Conc. based on:

other: enzyme concentrate dry matter

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

624.6 mg/L

Nominal / measured:

nominal

Conc. based on:

other: enzyme concentrate dry matter

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

122 mg/L

Nominal / measured:

nominal

Conc. based on:

other: enzyme concentrate dry matter

Basis for effect:

other: area under the curve

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 488 and 976 mg enzyme concentrate dry matter/L, the cells were observed to be clumped at 24, 48 and 72 hours. At 72 hours in the highest test concentration (976 mg enzyme concentrate dry matter/L) cells were also observed to be small. A stimulatory effect on algal growth rate was observed in the two lowest exposure levels (61 and 122 mg enzyme concentrate dry matter/L). Also at 72 hours those cells in the 61 mg enzyme concentrate dry matter/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.

Validity criteria fulfilled:

yes

Conclusions:

In the current testing conditions, exposure of Scenedesmus subspicatus to Peroxidase for 72 hours in concentrations up to 976 mg enzyme concentrate dry matter/L produced the following EC50 and NOEC values:
ErC50 (24 - 72 h): 624.6 mg enzyme concentrate dry matter/L (95 % confidence limits 546.6 - 712.5 mg enzyme concentrate dry matter/L); equivalent to 176.6 mg active enzyme protein/L.

EbC50 (72 h): 312.3 mg enzyme concentrate dry matter/L (95 % confidence limits 283 - 351.4 mg enyzme concentrate dry matter/L).

"No observed effect level" for 72 hour AUC: 122 mg enzyme concentrate dry matter/L.

"No observed effect level" for the 24-72 hour growth rate: 488 mg enzyme concentrate dry matter/L.

Executive summary:

Scenedesmus subspicatus 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 61, 122, 244, 488 and 976 mg enzyme concentrate dry matter/L.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:

ErC50 (24 - 72 h): 624.6 mg enzyme concentrate dry matter/L (95 % confidence limits 546.6 - 712.5 mg enzyme concentrate dry matter/L); equivalent to 176.6 mg active enzyme protein/L.

EbC50 (72 h): 312.3 mg enzyme concentrate dry matter/L (95 % confidence limits 283 - 351.4 mg enyzme concentrate dry matter/L).

"No observed effect level" for 72 hour AUC: 122 mg enzyme concentrate dry matter/L.

"No observed effect level" for the 24-72 hour growth rate: 488 mg enzyme concentrate dry matter/L.

Description of key information

In the current testing conditions, exposure of Scenedesmus subspicatus to Peroxidase for 72 hours in concentrations up to 976 mg enzyme concentrate dry matter/L produced the following EC50 and NOEC values:
ErC50 (24 - 72 h): 624.6 mg enzyme concentrate dry matter/L (95 % confidence limits 546.6 - 712.5 mg enzyme concentrate dry matter/L); equivalent to 176.6 mg active enzyme protein/L.

EbC50 (72 h): 312.3 mg enzyme concentrate dry matter/L (95 % confidence limits 283 - 351.4 mg enyzme concentrate dry matter/L).

"No observed effect level" for 72 hour AUC: 122 mg enzyme concentrate dry matter/L.

"No observed effect level" for the 24-72 hour growth rate: 488 mg enzyme concentrate dry matter/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

624.6 mg/L