glutamyl endopeptidase

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:

experimental study

Adequacy of study:

supporting study

Study period:

08-11-2013 - 04-02-2016

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)

Version / remarks:

ALGALTOXKIT F™ Freshwater Toxicity supplied by MicroBioTests Inc.

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

ALGALTOXKIT F™ Freshwater Toxicity supplied by MicroBioTests Inc.

Deviations:

no

Principles of method if other than guideline:

Only nominal concentrations.

GLP compliance:

no

Analytical monitoring:

no

Details on sampling:

- Concentrations: 0.01, 0.1, 1, 10, 100 mg TOS/L.
- Sampling method: According to the MicroBiotests Inc SOP.

Vehicle:

no

Details on test solutions:

According to the MicroBiotests Inc SOP.

Test organisms (species):

Pseudokirchneriella subcapitata (previous names: Raphidocelis 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:

According to the formula of the OECD Guideline 201 "Alga, Growth Inhibition Test" and the ISO/DIS Guideline 8692 "Water Quality - Freshwater Algal Growth Inhibition Tests with Unicellular Green Algae".

pH:

Within limits.

Nominal and measured concentrations:

Nominal concentrations

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: According to the formula of the OECD Guideline 201 "Alga, Growth Inhibition Test" and the ISO/DIS Guideline 8692 "Water Quality - Freshwater Algal Growth Inhibition Tests with Unicellular Green Algae".

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH:
- 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.:

> 14.3 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 other: mg TOS/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.14 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1 other: mg TOS/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

No significant growth inhibition and no abnormalities were observed.

Validity criteria fulfilled:

yes

Conclusions:

Glutamyl endopeptidase batch PPA 27269 was tested with Pseudokirchneriella subcapitata. It inhibited algal growth at 10 mg TOS/L, whereas no inhibitory effect was detected at lower concentrations.
The inhibitory effect at 10 mg TOS/L was below 50% and thus no ErC values could be determined (and must be above 100 mg TOS/L (14.3 mg aep/L). The NOEC was determined to be 1 mg TOS/L (0.14 mg aep/L).

Executive summary:

The purpose of this study was to screen for toxicity of Glutamyl endopeptidase batch PPA 27269 towards algae. A 72h algal growth inhibition test is performed in long cell test vials with Pseudokirchneriella subcapitata. Algae are supplied as de-immobilized algal beads. The Algal toxkit 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.

Validity: OECD 201 in turn indicates that the control growth rate must be at least 0.92 per day, i.e. a multiplication factor of 16.

Glutamyl endopeptidase inhibited algal growth at 10 mg TOS/L, whereas no inhibitory effect was detected at lower concentrations. The inhibitory effect at 10 mg TOS/L was below 50% and thus no ErC values could be determined (and must be above 100 mg TOS/L (14.3 mg aep/L). The NOEC was determined to be 1 mg TOS/L (0.14 mg aep/L).

Reference 2

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:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Due to the similarity between the two enzymes, similar results are expected for glutamyl endopeptidase.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

except for the analysis of the test material

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0.15, 0.33, 0.726, 1.60, 3.51 and 7.73 mg TOS/L
- Sampling method: Triplicate samples (10 mL) taken at the start of the definitive test from the freshly prepared control and test 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 (0.726 to 7.73 mg TOS/L) the test substance (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 0.726 mg TOS/L was diluted to provide test media at 0.15 and 0.33 mg TOS/L.
- Controls: one algal nutrient medium control group.

Test organisms (species):

Pseudokirchneriella subcapitata (previous names: Raphidocelis 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 (0.15 to 0.726 mg TOS/L) had decreased to between 5.01 and 5.11 and at 1.6 and 7.73 mg TOS/L pH values had increased to 7.33 to 7.50.

Nominal and measured concentrations:

Nominal: 0.15, 0.33, 0.726, 1.60, 3.51 and 7.73 mg TOS/L
Measured: 0.0728, 0.137, 0.249, 0.566, 2.62 and 6.87 mg TOS/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. 10000 cells/mL
- Control end cells density: 1260000 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
- Test concentrations: 1, 10 and 100 mg TOS/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.:

0.513 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

other: total organic solids

Basis for effect:

biomass

Remarks on result:

other: 95% confidence limits 0.441 - 0.606 mg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

290 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Remarks:

active enzyme protein

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.073 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

other: total organic solids

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

41 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Remarks:

active enzyme protein

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.48 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

other: total organic solids

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence limit 1.13 - 2.00 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

830 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Remarks:

active enzyme protein

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.566 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

other: total organic solids

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

317 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Remarks:

active enzyme protein

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): Yes
- Observation of abnormalities (for algal test): At 3.51 and 7.73 mg TOS/L the algal cells showed signs of cell lysis.
- Any stimulation of growth found in any treatment: No

Validity criteria fulfilled:

yes

Conclusions:

After 72 hours of exposure to the test substance, the EbC50 and ErC50, respectively were nominally 1.44 and 2.60 mg TOS/L. Based on the initial measured levels, the 72-hour EbC50 and ErC50 were 0.513 and 1.48 mg TOS/L corresponding to 290 and 830 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 0.33 and 1.6 mg TOS/L; equivalent to 0.0728 and 0.566 mg TOS/L as initial measured.

Executive summary:

The effect of the test substance 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 x 10⁴/mL, were exposed to the test substance,at nominal concentrations of 0.15, 0.33, 0.726, 1.60, 3.51 and 7.73 mg/L based on Total Organic Solid (TOS). 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 (0.0728, 0.137,0.249, 0.566, 2.62 and 6.87 mg TOS/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: 1.44 mg TOS/L (1.25,1.64)              

initial measured: 0.513 mg TOS/L (0.441; 0.606) or 290 µg aep/L

No-observed effect concentration:

nominal: 0.33 mg TOS/L

initial measured: 0.0728 mg TOS/L or 41 µg aep/L

Average specific growth rate

E_rC₅₀(0-72 h):

nominal: 2.60 mg TOS/L (2.27, 3.04)              

initial measured: 1.48 mg TOS/L (1.13; 2) or 830 µg aep/L

No-observed effect concentration:

nominal: 1.6 mg TOS/L

initial measured: 0.566 mg TOS/L or 317 µg aep/L

Description of key information

Glutamyl endopeptidase batch PPA 27269 was tested with Pseudokirchneriella subcapitata. It inhibited algal growth at 10 mg TOS/L, whereas no inhibitory effect was detected at lower concentrations.

The inhibitory effect at 10 mg TOS/L was below 50% and thus no ErC values could be determined (and must be above 100 mg TOS/L (14.3 mg aep/L). The NOEC was determined to be 1 mg TOS/L (0.14 mg aep/L).

Another similar enzyme - subtilisin – was tested with Pseudokirchneriella subcapitata. The 72-hour ErC50 was 1.48 mg TOS/L corresponding to 830 µg aep/L. The NOEC was 0.566 mg TOS/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

830 µg/L