Chymotrypsin

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:

13-05-2004 to 05-05-2005

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:

1984

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

no

Details on sampling:

Test concentrations were verified by chemical analysis conducted by Novozymes A/S. Triplicate 5 mL samples were taken from the control and each test concentration at 0 hours (fresh media), and 72 hours (expired media) and stored frozen before dispatching for analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A 885 mg sample of the test substance was dispersed directly into diluent water to give a concentration of 100 mg TOS/L.
- Controls: Nutrient medium

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Selenastrum capricornutum, (also known as Pseudokirchneriella subcapitata).
- Strain: Strain No. CCAP 278/4.
- Source (laboratory, culture collection): Culture Collection of Algae & Protozoa, CEH, Windermere, Cumbria, UK.

ACCLIMATION
- Acclimation period: Flasks containing sterile nutrient medium were inoculated from a master culture and were maintained under continuous illumination (7600 - 8080 lux) in an orbital incubator at 22 to 25°C, to give an algal suspension in log phase growth, characterised by a cell density of 3.0 x 10^6 cells/mL.
- Any deformed or abnormal cells observed: No

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Hardness:

Medium as recommended in Official Journal No. L383A Part C.3.

Test temperature:

22-25⁰C

pH:

6.9-7.4

Dissolved oxygen:

Not stated

Nominal and measured concentrations:

Nominal concentrations: 0.31, 0.63, 1.25, 2.5 and 5.0 mg TOS/L
Mean measured concentrations: 0.045, 0.11, 0.23, 0.40 and 1.3 mg TOS/L

Details on test conditions:

TEST SYSTEM
- Test vessels: 250 mL conical flasks containing 100 mL test solution and loosely stoppered.
- Type: Loosely stoppered.
- Aeration: None. Gaseous exchange and suspension of the algal cells were ensured by the continuous oscillation of the orbital shaker platform, at 130 revolutions per minute.
- Initial cells density: 1.1 * 10^4 cells/mL
- Control end cells density: 1.1 * 10^6 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- Method of initiation: Not stated

GROWTH MEDIUM
- Standard medium used: Medium as recommended in Official Journal No. L383A Part C.3.
- Detailed composition if non-standard medium was used: Medium as recommended in Official Journal No. L383A Part C.3.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: Continuous (7600 - 8080 lux)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Coulter® Multisizer II particle counter.

TEST CONCENTRATIONS
- Concentrations: 0.045, 0.11, 0.23, 0.40 and 1.3 mg TOS/L

Medium renewal: None.
Duration of exposure: 72 hours.
Measurement of growth: Samples were taken at 0, 24, 48 and 72 hours and the cell densities measured by direct counting using a Coulter® Multisizer II particle counter.

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

other: ErC50

Effect conc.:

2.4 other: mg TOS/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.45 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

active enzyme protein

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.64 mg/L

Nominal / measured:

nominal

Conc. based on:

other: TOS

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

other: EbC50

Effect conc.:

0.12 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

active enzyme protein

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.23 other: mg TOS/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

43.1 µg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

active enzyme protein

Basis for effect:

growth rate

Details on results:

Mean cell density of control @ 0 h: 1.1 x 10^4 cells/mL
Mean cell density of control @ 72 h: 1.1 x 10^6 cells/mL

- Exponential growth in the control: Yes
- Observation of abnormalities: No
- Unusual cell shape: No
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: Not stated
- Aggregation of algal cells: All test and control cultures were inspected at 72 hours.
- Any stimulation of growth found in any treatment: No

All results are expressed in terms of mean measured concentration. Measured concentrations ranged from 16 - 41% of nominal at 0 hours and from below the limit of analytical detection (0.041 mg TOS/L) to 13% of nominal at 72 hours. Test solutions were unable to be prepared by direct addition of the test material to the dilution material due to the low test concentrations. It was therefore necessary to dilute a concentrated stock solution. The low recoveries at 0 hours (fresh) were thought to be a result of using the dilution method.

De-activation of the enzyme was observed during the 72 hour exposure period. The three highest test levels lost between 18 and 30% of the enzyme activity. In the lowest test concentrations (0.31 and 0.63 mg) the enzyme activity was below the limit of analytical detection (0.041 mg TOS/L) after 72 hours. The denaturing of the enzyme was thought to be due to the relatively high temperature and light intensity conditions required for the study. It was unlikely to be a result of biodegradation due to the sterile nature of the study. Comparison of the measured concentrations of the test substance in flasks with and without algae indicated that the presence of algal cells had affected the stability of the exposure solutions. The initial nominal test concentrations not being achieved, and the reduction in enzyme activity over the course of the study, was not thought to have affected the outcome of the study as the results are calculated based on mean measured concentration.

Validity criteria fulfilled:

yes

Conclusions:

Protease 10R was tested for effects on the growth of the unicellular green alga Selenastrum capricornutum at concentrations 0.045, 0.11, 0.23, 0.40 and 1.3 mg TOS/L. The test revealed ErC50 (Growth rate 0-72 hours) 2.4 mg TOS/L equivalent to 0.45 mg aep/L (95% confidence limits 1.6 – 3.7 mg TOS/L).

Executive summary:

A study was conducted to assess the inhibitory effect of Protease 10R on the growth of the unicellular green alga Selenastrum capricornutum, Strain No. CCAP 278/4. Triplicate algal cultures, with an initial cell count of approximately 1 x 10^4 cells/mL, were exposed to Protease 10R at mean measured test concentrations of 0.045, 0.11, 0.23, 0.40 and 1.3 mg TOS/L. These cultures, together with one untreated control group of six replicates, were incubated in a Gallenkamp illuminated orbital incubator under continuous illumination at temperatures in the range of 23 ± 2°C for 72 hours. Cell numbers were counted daily to monitor growth.

Nominal test concentrations are based on Total Organic Solid (TOS), of which the test material contained 11.3%. Test concentrations were verified by determination of the enzyme activity. The amount of measured enzyme (ng/g) was converted to mg TOS/L in order to calculate the achieved measured concentrations. All results are expressed in terms of mean measured concentration. Measured concentrations ranged from 16 - 41% of nominal at 0 hours and from below the limit of analytical detection (0.041 mg TOS/L) to 13% of nominal at 72 hours. Test solutions were unable to be prepared by direct addition of the test material to the dilution material due to the low test concentrations. It was therefore necessary to dilute a concentrated stock solution. The low recoveries at 0 hours (fresh) were thought to be a result of the dilution method.

De-activation of the enzyme was observed during the 72 hour exposure period. The three highest test levels lost between 18 and 30% of the enzyme activity. In the lowest test concentrations (0.31 and 0.63 mg TOS/L) the enzyme activity was below the limit of analytical detection (0.041 mg TOS/L) after 72 hours. The denaturing of the enzyme was thought to be due to the relatively high temperature and light intensity conditions required for the study. It was unlikely to be a result of biodegradation due to the sterile nature of the study and the presence of algae did not appear to effect the stability of the exposure solutions. Measured concentrations were significantly lower than nominal values at both the start and end of the exposure period. However, this difference was not considered to have affected the outcome of the study as the results were calculated based on mean measured concentration.

The median effective concentrations for inhibition of growth based on biomass and average specific growth rates (EbC50 and ErC50, respectively) were:

Time (hours)                                          EC50 (mg TOS/L)                     95% confidence limits (mg TOS/L)

EbC50 (AUC 72 hours)                                   0.64                                                0.46 – 0.92

ErC50 (Growth rate 0-72 hours)                      2.4                                                  1.6 – 3.7

No-observed-effect concentration (NOEC): 0.23 mg TOS/L

Description of key information

Protease 10R was tested for effects on the growth of the unicellular green alga Selenastrum capricornutum at concentrations 0.045, 0.11, 0.23, 0.40 and 1.3 mg TOS/L. The test revealed ErC50 (Growth rate 0-72 hours) 2.4 mg TOS/L equivalent to 0.45 mg aep/L (95% confidence limits 1.6 – 3.7 mg TOS/L).

Key value for chemical safety assessment

EC50 for freshwater algae:

0.45 mg/L