Sedolisin

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

09 August 2007 - 01 February 2008

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Justification for type of information:

Enzymes are of the same sub-sub class and similar toxicological properties are expected.

Qualifier:

according to guideline

Guideline:

ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)

Deviations:

yes

Remarks:

In order to stabilize the enzyme activity of the test product, the pH of the test solutions had to be maintained below 7.0 during the test period. This required a modification from the guideline. A different test medium was used.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

2006

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Details on sampling:

Concentrations: 0.11, 0.33, 1.1, 2.2, 5.6, 11.1, 33.3 mg enzyme concentrate dry matter/L.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Stock solutions of 500 mg/L of the test product were prepared by weighing out 500 mg of the test product and adding it to 1.0 litre of the test medium. The stock solution was used
for preparing a dilution series of the test product in the test medium. A freshly prepared
stock solution was used for each new dilution series prepared.
- Controls: test medium

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Unicellular green algae
- Strain: Pseudokirclmerie/la subcapitata (clone: NIV A, CHL I).
- Source (laboratory, culture collection): The algae was received from NIVA, Norway and cultured at DHI under the test conditions prior to the start of the test.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

23.0 ± 0.1 °C

pH:

6.0 - 6.5

Nominal and measured concentrations:

Nominal concentrations: 0.11, 0.33, 1.1, 2.2, 5.6, 11.1, 33.3 mg enzyme concentrate dry matter/L.
Measured concentrations: 0.0021, 0.042, 0.49, 1.4, 5.6, 13.4, 42.6 mg enzyme concentrate dry matter/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass flask with 100 mL test solution
- Initial cells density: approximately 9000 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: no
- Detailed composition if non-standard medium was used:
Ca(NO3)2, 4H2O: 50 mg/L
KNO3: 100 mg/L
NaNO3: 50 mg/L
Na2SO4: 40 mg/L
MgCl2, 6H2O: 50 mg/L
β-Na2 glycerophosphate: 100 mg/L
Na2EDTA: 5 mg/L
FeCl3, 6H2O: 0.5 mg/L
MnCl2, 4H2O: 5 mg/L
ZnCl2: 0.5 mg/L
CoCl2, 6H2O: 5 mg/L
Na2MoO4, 2H2O: 0.8 mg/L
H3BO3: 20 mg/L
Bicine: 500 mg/L

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Photoperiod: 72 h
- Light intensity and quality: 60-120 μmol * m-2 * sec-1

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: in-vivo fluorescence (by use of a Turner TD-700 Laboratory Fluorometer)

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (K2Cl2O7, Merck 4864, Batch No. K30614564 227). Concentrations: 0 (control), 0.20, 0.50, 1.0, 1.4, 2.0, 2.8, and 4.0 mg/L.

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

9.8 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

other: mg enzyme concentrate dry matter/L

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence intervals: 9.0-10.6 mg enzyme concentrate dry matter/L

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.41 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

other: mg enzyme concentrate dry matter/L

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence intervals: 0-34-0.48 mg enzyme concentrate dry matter/L

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.042 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

other: mg enzyme concentrate dry matter/L

Basis for effect:

growth rate

Details on results:

The test results show that "Savinase, batch PBI 48037" caused a significant inhibition of
the growth of Pseudokirchneriella subcapitata at concentrations of 0.49-42.6 mg enzyme concentrate dry matter/L (nominal: 1.11-
33.3 mg enzyme concentrate dry matter/L).

Results with reference substance (positive control):

NOEC: 0.20 mg/L
EC10: 0.60 [0.51 - 0.69] mg/L
EC50: 3.26 [3.04 - 3.54] mg/L

Reported statistics and error estimates:

95% confidence intervals (EC10 and EC50)

Validity criteria fulfilled:

yes

Remarks:

The sensitivity of the algae in the modified test medium was verified by testing the inhibitory effect of the reference substance, which indicated that the algae were less sensitive to potassium dichromate when grown in MA medium than in the ISO medium.

Conclusions:

Savinase, batch PBI 48037 was tested at the following nominal concentrations: 0 (control), 0.11, 0.33, 1.11, 2.22, 5.55, 11.1, 33.3 mg enzyme concentrate dry matter/L.
The analyses of Savinase performed on the test solutions at the start of the test, and
after 24, 48 and 72 hours, respectively, showed recoveries from 0.8% to 165%.
At the concentrations 0.0021 mg enzyme concentrate dry matter/L (nominal: 0.11 mg enzyme concentrate dry matter/L) and 0.042 mg enzyme concentrate dry matter/L (nominal: 0.33
mg enzyme concentrate dry matter/L), only a slight effect was observed on the growth rate of Pseudokirchneriella subcapitata. Significant inhibition of the growth was observed at concentrations from 0.49 - 42.6 mg enzyme concentrate dry matter/L (nominal: 1.11 - 3.33 mg enzyme concentrate dry matter/L).
NOEC for the growth rate was 0.042 mg enzyme concentrate dry matter/L (corresponding to 0.022 mg active enzyme protein/L). The EC10 was 0.41 mg enzyme concentrate dry matter/L [95%-CI: 0.34-0.48] (corresponding to 0.21 mg active enzyme protein/L) and the EC50 was 9.8 mg enzyme concentrate dry matter/L [95%-CI: 9.0-10.6] (corresponding to 5.02 mg active enzyme protein/L).

Executive summary:

The objective of this study was to investigate the effect of savinase, batch PBI 48037 on the inhibition of the growth of the freshwater micro algae Pseudokirchneriella subcapitata according to the OECD Guideline for the Testing of Chemicals No. 201 "Freshwater Alga and Cyanobacteria. Growth lnhibition Test" /1/ and further specified in the ISO International Standard 8692 "Water Quality - Fresh water algal growth inhibition test with unicellular green algae" /2/. The test was carried out in compliance with the OECD Principles of Good Laboratory Practice (GLP) /3/.

The test was carried out in 250-mL glass flasks with 100 mL of test solution. The
growth of the algae during 72 hours was followed by measuring the fluorescence at the beginning of the test and every 24 hours. In order to verify the actual test concentrations, samples of the test solutions were collected for chemical analysis. The actual test concentrations were verified by specific Savinase ELISA analysis.

The analyses of Savinase performed on the test solutions at the start of the test, and after 24, 48 and 72 hours, respectively, showed recoveries from 0.8% to 165%. At the concentrations 0.0021 mg enzyme concentrate dry matter/L (nominal: 0.11 mg enzyme concentrate dry matter/L) and 0.042 mg enzyme concentrate dry matter/L (nominal: 0.33 mg enzyme concentrate dry matter/L), only a slight effect was observed on the growth rate of Pseudokirchneriella subcapitata. Significant inhibition of the growth was observed at concentrations from 0.49 - 42.6 mg enzyme concentrate dry matter/L (nominal: 1.11 - 3.33 mg enzyme concentrate dry matter/L).
NOEC for the growth rate was 0.042 mg enzyme concentrate dry matter/L (corresponding to 0.022 mg active enzyme protein/L). The EC10 was 0.41 mg enzyme concentrate dry matter/L [95%-CI: 0.34-0.48] (corresponding to 0.21 mg active enzyme protein/L) and the EC50 was 9.8 mg enzyme concentrate dry matter/L [95%-CI: 9.0-10.6] (corresponding to 5.02 mg active enzyme protein/L).

 

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

28-03-2017 to 05-01-2018

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Remarks:

Oxygen concentration was not measured.

Qualifier:

according to guideline

Guideline:

ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)

Deviations:

no

Principles of method if other than guideline:

Only nominal concentrations.

GLP compliance:

no

Remarks:

See *justification for type of information'

Analytical monitoring:

no

Details on sampling:

- Concentrations: 11.9, 35.8, 59.6, 83.5, 119.2 mg enzyme concentrate dry matter/L

Vehicle:

no

Details on test solutions:

According to the MicroBiotests Inc SOP.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Unicellular green algae
- Strain: Pseudokirchneriella subcapitata
- Source (laboratory, culture collection): MicroBiotests Inc. The algal growth inhibition bioassay has been developed by the research teams at the Laboratory for Biological Research in Aquatic Pollution (LABRAP) at the University of Ghent in Belgium.
- Method of cultivation: According to the MicroBiotests Inc. SOP.
Algae are supplied as de-immobilized algal beads.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

23°C +/- 2 °C

pH:

8.1 +/- 0.2

Dissolved oxygen:

Media was saturated with air before use

Nominal and measured concentrations:

Nominal concentrations: 11.9, 35.8, 59.6, 83.5, 119.2 mg enzyme concentrate dry matter/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 25 mL calibrated flask
- Material, size, headspace, fill volume: 25 mL
- Initial cells density: 1*10^4 algae/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes.
- Detailed composition if non-standard medium was used: As described in OECD Guideline 201and the ISO/DIS Guideline 8692.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Photoperiod: 72 h
- Light intensity and quality: 10000 lux for sideway illumination or min. 3000-4000 lux for bottom illumination

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: spectrophotometer

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 119.2 mg/L

Nominal / measured:

nominal

Conc. based on:

other: mg enzyme concentrate dry matter/L

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

> 11.9 mg/L

Nominal / measured:

nominal

Conc. based on:

other: mg enzyme concentrate dry matter/L

Basis for effect:

growth rate

Details on results:

No significant growth inhibition and no abnormalities were observed.

Validity criteria fulfilled:

yes

Remarks:

Oxygen concentrations not measured.

Conclusions:

Sedolisin, batch PPF38268 was tested in concentrations 11.9, 35.8, 59.6, 83.5, 199.2 mg enzyme concentrate dry matter/L. It was found to inhibit growth rate at all concentrations tested after 72h exposure, whereas no significant inhibition was observed after 24 and 48h exposure. Inhibition of the growth rate at 72h was found to be approximately 3% at 11.9 mg enzyme concentrate dry matter/L, 34 % at 35.8 mg enzyme concentrate dry matter/L, 30% at 59.6 mg enzyme concentrate dry matter/L, and 12 % at 119.2 mg enzyme concentrate dry matter/L indicating only slightly toxicity towards algal growth. There was no dose-response relationship and because of the low observed toxicity towards algal growth no ErC50 value could be estimated and must be > 119.2 mg enzyme concentrate dry matter/L (equivalent to > 24.7 mg active enzyme protein/L). The NOEC value was determined to be < 11.9 mg enzyme concentrate dry matter/L (equivalent to < 2.5 mg active enzyme protein/L).

Executive summary:

The purpose of this study was to screen for toxicity of Sedolisin, batch PPF38268 towards algae. A 72h algal growth inhibition test was performed in long cell test vials with Pseudokirchneriella subcapitata. Algae are supplied as de-immobilized algal beads. The Algal tox kit test follows the prescriptions of the OECD "Algal growth inhibition test" (Guideline 201) and the ISO "Water Quality Freshwater Algal Growth Inhibition Tests with Unicellular Green Algae" (ISO Standard 8692). Endpoint of the study is percent inhibition (I%) of algal growth rate compared to the control.

IC50: Calculated/estimated 50% growth inhibition compared to control algae.

Sedolisin, batch PPF38268 was found to inhibit growth rate at all concentrations tested after 72h exposure, whereas no significant inhibition was observed after 24 and 48h exposure. Inhibition of the growth rate at 72h was found to be approximately 3% at 11.9 mg enzyme concentrate dry matter/L, 34 % at 35.8 mg enzyme concentrate dry matter/L, 30% at 59.6 mg anzyme concentrate dry matter/L, and 12 % at 119.2 mg enzyme concentrate dry matter/L indicating only slightly toxicity towards algal growth. There was no dose-response relationship and because of the low observed toxicity towards algal growth no ErC50 value could be estimated and must be > 119.2 mg enzyme concentrate dry matter/L (equivalent to > 24.7 mg active enzyme protein/L). The NOEC value was determined to be < 11.9 mg enzyme concentrate dry matter/L (equivalent to < 2.5 mg active enzyme protein/L).

Description of key information

Under the conditions of the test, Sedolisin, batch PPF38268 was found not to be toxic to Daphnia magna at a nominal concentration up to 119.2 mg enzyme concentrate dry matter/L (24.7 mg aep/L) after 48h exposure. Thus, no 48h EC50 could be calculated and must be > 119.2 mg enzyme concentrate dry matter/L (equivalent to > 24.7 mg active enzyme protein/L).

Subtilisin was used as read-across: The EC50 was 9.8 mg enzyme concentrate dry matter/L (corresponding to 5.02 mg active enzyme protein/L). NOEC for the growth rate was 0.042 mg enzyme concentrate dry matter/L.