Subtilisin

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

effects on growth of green algae

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

17 August 1992 - 10 November 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Remarks:

Test concentrations were not verified

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

Method of preparation: The test substance was dissolved in sterile nutrient medium to give an initial stock solution of 3019 mg enzyme concentrate dry matter/L. This stock solution was further diluted with sterile nutrient medium to produce series of solutions exactly twice the concentration of the intended exposure levels. 150 mL of algal preculture was mixed with 150 mL of each of these solutions to give the final test series.
Controls: medium

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): Culture Centre of Algae & Protozoa c/o Freshwater Biological Association, Cumbria, U.K.
Pre-culture: 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 characterized by an absorbance of 0.670 (at 665 nm).

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

24.0 - 24.6 °C

pH:

7.9 - 9.9

Nominal and measured concentrations:

Nominal: 94, 189, 377, 755 and 1509 mg enzyme concentrate dry matter/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL conical flasks
- Type (delete if not applicable): loosely stoppered
- Material, size, headspace, fill volume: glas, 250 mL (fill volume 100 mL)
- Initial cells density: 8.4 x 10^4 cells/mL
- Control end cells density:1.85 x 10^6 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reverse-osmosis purified water
- Culture medium different from test medium: No

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- 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) : Growth rate . Samples were taken at 0, 24, 48 and 72 hours and the cell densities of the cultures determined by direct counting with the aid of a haemacytometer (Improved Neubauer).

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Test concentrations: 94, 189, 377, 755 and 1509 mg enzyme concentrate dry matter/L

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 1 509 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:

NOEC

Effect conc.:

> 1 509 mg/L

Nominal / measured:

nominal

Conc. based on:

other: enzyme concentrate dry matter

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): Cell debris was present at 1509 mg enzyme concentrate dry matter/L.

Validity criteria fulfilled:

not specified

Conclusions:

Savinase in inhibitory to the growth of Scenedesmus subspicatus at concentrations in excess of 189 mg enzyme concentrate dry matter/L. The ErC50 (24-72 h) is > 1509 mg enzyme concentrate dry matter/L corresponding to 39.4 mg active enzyme protein/L. The NOEC is > 1509 mg enzyme concentrate dry matter.

Executive summary:

A study was conducted to assess the inhibitory effect of savinase on the growth of the unicellular green alga Scenedesmus subspicatus, Strain No. CCAP 276/20.

The study was conducted in accordance with OECD Guideline for Testing of Chemicals No. 201 and EEC Methods for the Determination of Ecotoxicity, EEC Directive 67/458 Annex VIII, Part C (87/302/EEC).

Algal cultures exposed to five test concentrations of savinase 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 direct counting with a haemacytometer.

The following values were derived from the data:

 

E_rC₅₀(24-72h):         > 1509 mg enzyme concentrate dry matter/L (equivalent to 39.4 mg active enzyme protein/L)

E_bC₅₀(72h):              755 mg enzyme concentrate dry matter/L

 

NOEC for the 24 -72 hour growth rate:               > 1509 mg enzyme concentrate dry matter/L

NOEC 72h for AUC (Area under the curve):         189 mg enzyme concentrate dry matter/L

Reference 2

Endpoint:

effects on growth of green algae

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

17 August 1992 - 7 December 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Remarks:

Concentrations were not verified.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

Method of preparation: The test substance was dissolved in sterile nutrient medium to give an initial stock solution of 195 mg enzyme concentrate dry matter/L. This stock solution was further diluted with sterile nutrient medium to produce series of solutions exactly twice the concentration of the intended exposure levels. 150 mL of algal preculture was mixed with 150 mL of each of these solutions to give the final test series.
Controls: medium

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): Culture Centre of Algae & Protozoa c/o Freshwater Biological Association, Cumbria, U.K.
Pre-culture: 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 characterized by an absorbance of 0.660 (at 665 nm).

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

24.1 - 24.4

pH:

7.6 - 10.6

Nominal and measured concentrations:

Nominal: 6.11, 12.2, 24, 49 and 97.7 mg enzyme concentrate dry matter/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL conical flasks
- Type (delete if not applicable): loosely stoppered
- Material, size, headspace, fill volume: glas, 250 mL (fill volume 100 mL)
- Initial cells density: 7.5 x 10^4 cells/mL
- Control end cells density: 2.5 x 10^6 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reverse-osmosis purified water
- Culture medium different from test medium: No

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- 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) : Growth rate . Samples were taken at 0, 24, 48 and 72 hours and the absorbance measured at 665 nm using a Cecil 373 Series 2 Spectrophotometer.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Test concentrations: 6.11, 12.2, 24, 49 and 97.7 mg enzyme concentrate dry matter/L

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

76 mg/L

Nominal / measured:

nominal

Conc. based on:

other: enzyme concentrate dry matter

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence limits: 61 - 96 mg enzyme concentrate dry matter/L

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

24 mg/L

Nominal / measured:

nominal

Conc. based on:

other: enzyme concentrate dry matter

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): No

Validity criteria fulfilled:

not specified

Conclusions:

Esperase in inhibitory to the growth of Scenedesmus subspicatus. The ErC50 (24-72 h) is 76 mg enzyme concentrate dry matter/L corresponding to 7.8 mg active enzyme protein/L. The NOEC is 24 mg enzyme concentrate dry matter.

Executive summary:

A study was conducted to assess the inhibitory effect of esperase on the growth of the unicellular green alga Scenedesmus subspicatus, Strain No. CCAP 276/20.

The study was conducted in accordance with OECD Guideline for Testing of Chemicals No. 201 and EEC Methods for the Determination of Ecotoxicity, EEC Directive 67/458 Annex VIII, Part C (87/302/EEC).

Algal cultures exposed to five test concentrations of esperase 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:

 

E_rC₅₀(24-72h):         76 mg enzyme concentrate dry matter/L (equivalent to 7.8 mg active enzyme protein/L)

E_bC₅₀(72h):              34 mg enzyme concentrate dry matter/L

 

NOEC for the 24 -72 hour growth rate:               24 mg enzyme concentrate dry matter/L

NOEC 72h for AUC (Area under the curve):         12 mg enzyme concentrate dry matter/L

Reference 3

Endpoint:

effects on growth of green algae

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

17 August 1992 - 7 December 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Remarks:

Test concentrations were not verified

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

Method of preparation: The test substance was dissolved in sterile nutrient medium to give an initial stock solution of 1886 mg enzyme concentrate dry matter/L. This stock solution was further diluted with sterile nutrient medium to produce series of solutions exactly twice the concentration of the intended exposure levels. 150 mL of algal preculture was mixed with 150 mL of each of these solutions to give the final test series.
Controls: medium

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): Culture Centre of Algae & Protozoa c/o Freshwater Biological Association, Cumbria, U.K.
Pre-culture: 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 characterized by an absorbance of 0.545 (at 665 nm).

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

24.1 - 24.6 °C

pH:

7.6 - 9.1

Nominal and measured concentrations:

Nominal: 58.9, 118, 236, 472 and 943 mg enzyme concentrate dry matter/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL conical flasks
- Type (delete if not applicable): loosely stoppered
- Material, size, headspace, fill volume: glas, 250 mL (fill volume 100 mL)
- Initial cells density: 8.7x10^4 cells/mL
- Control end cells density: 1.8x10^6 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reverse-osmosis purified water
- Culture medium different from test medium: No

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- 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) : Growth rate . Samples were taken at 0, 24, 48 and 72 hours and the absorbance measured at 665 nm using a Cecil 373 Series 2 Spectrophotometer.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Test concentrations: 58.9, 118, 236, 472 and 943 mg enzyme concentrate dry matter/L

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

339 mg/L

Nominal / measured:

nominal

Conc. based on:

other: enzyme concentrate dry matter

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence limits: 283 - 405 mg enzyme concentrate dry matter/L

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

118 mg/L

Nominal / measured:

nominal

Conc. based on:

other: enzyme concentrate dry matter

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): At 236 mg enzyme concentrate dry matter/L cell debris was present and the cells were observed to be clumped and at the two highest exposure levels, 472 and 943 mg enzyme concentrate dry matter/L, cell debris was present and cells were observed to be pale and clumped.

Validity criteria fulfilled:

not specified

Conclusions:

Alcalase in inhibitory to the growth of Scenedesmus subspicatus. The ErC50 (24-72 h) is 339 mg enzyme concentrate dry matter/L corresponding to 69.5 mg active enzyme protein/L. The NOEC is 118 mg enzyme concentrate dry matter.

Executive summary:

A study was conducted to assess the inhibitory effect of alcalase on the growth of the unicellular green alga Scenedesmus subspicatus, Strain No. CCAP 276/20.

The study was conducted in accordance with OECD Guideline for Testing of Chemicals No. 201 and EEC Methods for the Determination of Ecotoxicity, EEC Directive 67/458 Annex VIII, Part C (87/302/EEC).

Algal cultures exposed to five test concentrations of Alcalase 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:

 

E_rC₅₀(24-72h):         339 mg enzyme concentrate dry matter/L (equivalent to 69.5 mg active enzyme protein/L)

E_bC₅₀(72h):              245 mg enzyme concentrate dry matter/L

 

NOEC for the 24 -72 hour growth rate:               118 mg enzyme concentrate dry matter/L

NOEC 72h for AUC (Area under the curve):         12.1 mg enzyme concentrate dry matter/L

Reference 4

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 June 2007 - 31 January 2008

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

23 March, 2006

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. A different test medium than the one described in the guideline was therefore used.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0.11, 0.33, 1.1, 2.2, 5.6, 11.1 and 33.3 mg enzyme concentrate dry matter/L.
- Sampling method: Samples were taken from flasks identical to the test flasks at the start of the test, after 25.5 and 47 hours and at the termination of the test (71 hours). Subsamples of 8-10 mL were collected in 12 mL tubes and stored at -20 °C until the end of the test and hereafter sent frozen to the analytical laboratory. In addition freshly prepared test solutions were collected for chemical analysis at the initiation of the test and forwarded to the analytical laboratory immediately.
- Sample storage conditions before analysis: frozen

Vehicle:

no

Details on test solutions:

PREPARATION OF TEST SOLUTIONS:
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.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: clone: NIVA, CHL 1
- Source (laboratory, culture collection): received from NIVA, Norway and cultured at DHI under the test conditions.
- Age of inoculum (at test initiation): three days

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

23.0 ± 0.1

pH:

6.0 - 6.5

Nominal and measured concentrations:

Nominal: 0.11, 0.33, 1.1, 2.2, 5.6, 11.1 and 33.3 mg enzyme concentrate dry matter/L.
Measured: 0.0021, 0.042, 0.49, 1.44, 5.59, 13.4 and 42.6 mg enzyme concentrate dry matter/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass flasks
- Type (delete if not applicable): loosely plugged.
- Material, size, headspace, fill volume: 250 mL flask, 100 mL fill volume
- Initial cells density: approx. 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
beta-Na2glycerophosphate: 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: yes
- Adjustment of pH: yes (adjusted to pH 6.0)
- Photoperiod: continuous illumination
- Light intensity and quality: fluorescent light with an intensity of approx. 60-120 μmpl x m^(-2) x sec^(-1)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : cell density. Samples were taken at 24, 48 and 72 ± 2 hours.
- Determination of cell concentrations: Turner TD-700 laboratory flourometer.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: approx. 2-3

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

9.77 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

other: enzyme concentrate dry matter

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence limits: 8.95 - 10.6 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: enzyme concentrate dry matter

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.41 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

other: enzyme concentrate dry matter

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence limits: 0.34 - 0.48 mg enzyme concentrate dry matter/L

Details on results:

The results of the analysis of Savinase performed on the frozen test solutions collected at the start of the test, and after 24, 48 and 72 hours, respectively, showed recoveries from 0.8% to 165% of the nominal test concentration. A decreasing recovery was observed with decreasing enzyme concentrations, as well as a decrease over time.

Results with reference substance (positive control):

- Results with reference substance valid: yes
- EC50: 3.26 mg/L (95% CI: 3.04-3.54)

Reported statistics and error estimates:

The EC10 and EC50 were calculated by use of the computer program 'TOXEDO'. The NOEC was estimated by use of variance analysis according to Dunnett's procedure.

Validity criteria fulfilled:

yes

Conclusions:

The test results showed that Savninase, batch PBI 48037 caused a significant inhibition of the growth rate of Pseudokirchneriella subcapitata with an EC50 of 9.77 mg enzyme concentrate dry matter/L (corresponding to 5.02 mg active enzyme protein/L) and the NOEC was 0.042 mg enzyme concentrate dry matter/L, based on initial measured concentrations.

Executive summary:

The test product Savinase, batch PBI 48037 was tested for inhibitory effect on the growth of the micro algae Psudokirchneriella subcapitata according to the OECD Guideline for the Testing of Chemicals No. 201, which is further specified in the ISO International Standard 8692 "Water Quality - Fresh water algal growth inhibition test with unicellular green algae".

 

The test substance was tested at the following nominal concentrations: 0.11, 0.33, 1.1, 2.2, 5.6, 11,1 and 33.3 mg enzyme concentrate dry matter/L. 

 

The test was carried out in 250 mL glass flasks with 100 mL of test solution. The growth of the algae during the 72 hours was followed by measuring the flourescence 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 test results can be summarized as follows: 

• 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%. According to the analysis report, a decreasing recovery is observed with decreasing enzyme concentrations as well as a decrease over time. 


• At the measured concentrations 0.0021 and 0.042 mg enzyme concentrate dry matter/L, only a slight effect was observed on the growth rate. Significant inhibition of the growth rate was observed at concentrations from 0.49-42.6 mg enzyme concentrate dry matter/L.


• NOEC for the growth rate was 0.042 mg enzyme concentrate dry matter/L. The EC10 was 0.41 mg enzyme concentrate dry matter/L and the EC50 was 9.77 mg enzyme concentrate dry matter/L corresponding to 5.02 mg active enzyme protein/L. 

Reference 5

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 August - 13 October 2006

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The effect concentrations were based on initial analytical measurements only, as the test item concentration at the end of the test fell below the detection limit for some concentrations.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

1984

Deviations:

no

Remarks:

The measured concentrations were based on the initial fresh concentration preparations only.

GLP compliance:

yes (incl. QA statement)

Remarks:

except for the analysis of the test material

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0.16, 0.35, 0.78, 1.71, 3.75 and 8.26 mg enzyme concentrate dry matter/L
- Sampling method: Triplicate samples (10 mL) taken at the start of the definitive test and from the freshly-prepared control and test media and at 72 hours (expired media).
- Sample storage conditions before analysis: frozen at ca. -20°C

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test substance was supplied as a frozen liquid and an aliquot of the test substance was thawed. At the four highest concentrations (0.78 to 8.26 mg enzyme concentrate dry matter/L) the test substance batch (19.9, 21.9, 48.1 and 106 mg) was dispersed in dilution medium (1 or 2L) in a volumetric flask. The contents of the flasks were inverted 30 times to mix the contents. These preparations were than used directly in the test. At the lower levels, aliquots (206 and 454 mL) of the preparation made at 0.78 mg enzyme concentrate dry matter/L was diluted to provide test media at 0.16 and 0.35 mg enzyme concentrate dry matter/L.
- Controls: one algal nutrient medium control group.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd., Dunstaffnage Marine Laboratory, Dunbeg, Oban, Argyll, Scotland
- Age of inoculum (at test initiation): three days

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22.4 - 24.8°C

pH:

The pH of the culture medium was adjusted to pH 6 by addition of 0.1M hydrochloric acid. The pH of the control media was 6.02 at 0 hours and 6.65 at 72 hours. At 72 hours, the pH of the test media at the three lowest levels (0.16 to 0.78 mg enzyme concentrate dry matter/L) had decreased to between 5.01 and 5.11 and at 1.71 and 8.26 mg enzyme concentrate dry matter/L pH values had increased to 7.33 to 7.50.

Nominal and measured concentrations:

Nominal: 0.16, 0.35, 0.78, 1.71, 3.75 and 8.26 mg enzyme concentrate dry matter/L
Measured (initial): 0.0778, 0.146, 0.266, 0.605, 2.80 and 7.34 mg enzyme concentrate dry matter/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL conical flask
- Type (delete if not applicable): loosely plugged with a foam bung
- Material, size, headspace, fill volume: 250 mL conical flask, 100 mL fill volume
- Initial cells density: appr. 1.13 x 10^4 cells/mL
- Control end cells density: 1.26 x 10^6 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Sterile algal nutrient medium as recommended in Official Journal No. L383A Part C.3 and OECD Procedure 201. Prepared in-house using filtered, dechlorinated tap water which had been softened and treated by reverse osmosis, before microfiltration and purification (resistivity of 18 Megohm/cm).

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: yes, adjusted to pH 6 with 0.1M hydrochloric acid
- Photoperiod: continuous illumination
- Light intensity and quality: 6000-7000 lux provided by a 6 x 30 W 'cool white' 1 metre fluorescent tubes; mean measured values in the range 6080-6730 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Cell density. Samples were taken at 24, 48 and 72 hours.
- Determination of cell concentrations: Cell densities were measured using a haemocytometer (Improved Neubauer).

TEST CONCENTRATIONS
- Spacing factor for test concentrations: approximately 2.2
- Range finding study
- Test concentrations: 1.1, 11 and 107 mg enzyme concentrate dry matter/L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.548 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

other: enzyme concentrate dry matter

Basis for effect:

other: Area under the curve

Remarks on result:

other: 95% confidence limits 0.471 - 0.648 mg enzyme concentrate dry matter/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.078 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

other: enzyme concentrate dry matter

Basis for effect:

other:

Remarks:

Area under the curve

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.58 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

other: enzyme concentrate dry matter

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence limit 1.21 - 2.14 mg enzyme concentrate dry matter/L

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.605 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

other: enzyme concentrate dry matter

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): At 3.75 and 8.26 mg enzyme concentrate dry matter/L (nominal) the algal cells showed signs of cell lysis to those in the control group.
- Any stimulation of growth found in any treatment: no

Reported statistics and error estimates:

95% confidence limits on EC50.

Validity criteria fulfilled:

yes

Conclusions:

After 72 hours of exposure to Subtilisin, batch PXI48489, the EbC50 and ErC50, respectively were nominally 1.54 and 2.78 mg enzyme concentrate dry matter/L. Based on the initial measured levels, the 72-hour EbC50 and ErC50 were 0.548 and 1.58 mg enzyme concentrate dry matter/L corresponding to 288 and 831 µg aep/L, respectively.
The "no observed effect concentration" (NOEC) of the test substance for the area under the growth curve and growth rate respectively, were 0.35 and 1.7 mg enzyme concentrate dry matter/L; equivalent to 0.0778 and 0.605 mg enzyme concentrate dry matter/L as initial measured.

Executive summary:

The effect of Subtilisin, batch PXI48489 on the growth of the unicellular green alga Pseudokirchneriella subcapitata was assessed. The study was conducted in accordance with OECD and EC guidelines, and in compliance with GLP.

Triplicate algal cultures, with an initial cell density of 1.13 x 10⁴/mL, were exposed to the test substance,at nominal concentrations of 0.16, 0.35, 0.78, 1.71, 3.75 and 8.26 mg/L based on enzyme concentrate dry matter. The test media were prepared in OECD algal nutrient medium; because the activity of the active component of the enzyme was reduced under alkaline conditions, the pH of the OECD medium was adjusted to approximately 6 before use. The cultures were incubated in an orbital incubator under continuous illumination at temperatures ranging from 22.4 to 24.8°C for 72 hours.

The test concentrations were verified by the Sponsor based on the determination of the test substance by ELISA. Since the measured concentrations were lower than nominal values at the start of the exposure period, and little or no test substance was detected at the end, the test results have been calculated in terms of both the nominal and initial measured concentrations (0.0778, 0.146, 0.266, 0.605, 2.80 and 7.34 mg enzyme concentrate dry matter/L).

The test results are expressed in terms of the area under the growth curve and growth rate.

The following values were derived from the data:

Area under growth curve

E_bC₅₀(72 h):

Nominal (95% C.I.): 1.54 mg enzyme concentrate dry matter/L (1.34, 1.75). 

Initial measured (95% C.I.): 0.55 mg enzyme concentrate dry matter/L (0.47; 0.65) equivalent to 0.288 mg active enzyme protein/L.

NOEC:

Nominal: 0.35 mg enzyme concentrate dry matter/L

Initial measured: 0.078 mg enzyme concentrate dry matter/L.

 

Average specific growth rate

 

E_rC₅₀(0-72 h):

Nominal (95% C.I.): 2.78 mg enzyme concentrate dry matter/L (2.43, 3.25).  

Initial measured (95% C.I.): 1.58 mg enzyme concentrate dry matter/L (1.21; 2.14) or 0.831 mg aep/L.

NOEC:

Nominal: 1.7 mg enzyme concentrate dry matter/L

Initial measured: 0.605 mg enzyme concentrate dry matter/L, or 0.318 mg aep/L.

Description of key information

• (Reliability 2) Based on the initial measured concentrations, after 72 hours of exposure to Subtilisin, batch PXI48489, the ErC50 was 1.58 mg enzyme concentrate dry matter/L, equivalent to 831 µg active enzyme protein (aep)/L. The 72-h NOEC of the test substance (as initially measured) for the growth rate was 0.605 mg enzyme concentrate dry matter/L, equivalent to 0.317 mg active enzyme protein/L.


• (Reliability 2) Based on the initial measured concentrations, after 72 hours of exposure to Savinase, batch PBI48037, the ErC50 of 9.77 mg enzyme concentrate dry matter/L corresponding to 5.02 mg active enzyme protein/L and the NOEC was 0.042 mg enzyme concentrate dry matter/L.


• (supporting study) Based on the nominal concentrations, after 72 hours of exposure to Alcalase, batch PPA 4046, the ErC50 of 339 mg enzyme concentrate dry matter/L corresponding to 69.5 mg active enzyme protein/L and the NOEC was 118 mg enzyme concentrate dry matter/L.


• (supporting study) Based on the nominal concentrations, after 72 hours of exposure to Esperase, batch PPA 3980, the ErC50 of 76 mg enzyme concentrate dry matter/L corresponding to 7.8 mg active enzyme protein/L and the NOEC was 24 mg enzyme concentrate dry matter/L.


• (supporting study) Based on the nominal concentrations, after 72 hours of exposure to Savinase, batch PPA 3352, the ErC50 of > 1509 mg enzyme concentrate dry matter/L corresponding to > 39.4 mg active enzyme protein/L and the NOEC was > 1509 mg enzyme concentrate dry matter/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

1.58 mg/L

EC10 or NOEC for freshwater algae:

0.605 mg/L