glutamyl endopeptidase

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

24-03-2011 to 13-04-2011

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

DAPHTOXKIT F™ magna supplied by MicroBioTests Inc.

Principles of method if other than guideline:

The concentrations are nominal concentrations.

GLP compliance:

no

Analytical monitoring:

no

Details on sampling:

- Concentrations: 4.6, 10, 21, 46, 100 mg TOS/L

Vehicle:

no

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia
- Strain: Daphnia magna Straus
- Source: MicroBioTests Inc. Daphnia magna have been developed by the research teams at the Laboratory for Biological Research in Aquatic Pollution (LABRAP) at the Ghent University in Belgium.
- Age at study initiation (mean and range, SD): less than 24 hours
- Feeding during test: no

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test temperature:

20-25°C

pH:

Within limits

Nominal and measured concentrations:

4.6, 10, 21, 46, 100 mg TOS/L

Details on test conditions:

TEST SYSTEM
The test was conducted according to DAPHTOXKIT F MAGNA Crustacean Toxicity Screening Test for Freshwater with a test system supplied by MicroBioTests Inc.
http://www.microbiotests.be/SOPs/Daphtoxkit%20magna%20F%20SOP%20-%20A5.pdf

- Test vessel: The bioassays are conducted in disposable multiwell test plates with 30 test wells.
- Type: closed
- Material, size, headspace, fill volume: 10 mL
- Aeration: The Standard Freshwater (ISO medium) must be aerated for at least 15 minutes prior to using it.
- No. of organisms per vessel: 5
- No. of vessels per concentration and control (replicates): Each plate is provided with 4 wells for the controls and 4 wells (A,B,C,D) for each toxicant concentration.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ISO medium, formula according to ISO 6341

Composition :
vial 1 : NaHCO3 (129.5 mg - dissolved in 2 L = 67.75 mg/L)
vial 2 : CaCl2.2H2O (588 mg - dissolved in 2 L = 294 mg/L)
vial 3 : MgSO4.7H2O (246.5 mg - dissolved in 2 L = 123.25 mg/L)
vial 4 : KCl (11.5 mg - dissolved in 2 L = 5.75 mg/L)

Reference substance (positive control):

no

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.7 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

5 other: mg TOS/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

> 4.6 other: mg TOS/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

> 0.66 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mobility

Details on results:

Details on results
- Mortality of control: 0
- Other adverse effects control: No
- Abnormal responses: No
- Effect concentrations exceeding solubility of substance in test medium: No
Daphnia were considered to be immobile if they were unable to swim within 15 seconds following gentle touch of the animal with a pin.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the test, testing of glutamyl endopeptidase, batch PPA27269 to Daphnia magna revealed an 48h EC50 value of 5 mg TOS/L (0.7 mg aep/L). The NOEC (48h) was determined to be > 4.6 mg/L (0.66 mg aep/L).

Executive summary:

The purpose of this study was to screen glutamyl endopeptidase, batch PPA27269 for the test material concentration estimated to immobilise 50 % of the Daphnia magna after 24 and 48 hours exposure. DAPHTOXKIT F™ magna was supplied by MicroBioTests Inc. The testing of the glutamyl endopeptidase, batch PPA27269 was performed as a screening with a broad range of concentrations 9.2, 20, 42, 92, 100 mg TOS/L. The acceptance criterion for a valid test is that the spontaneous immobility in the control is below or equal to 10%.

Under the conditions of the test, testing of glutamyl endopeptidase, batch PPA27269 to Daphnia magna revealed an 48h EC50 value of 5 mg TOS/L (0.7 mg aep/L). The NOEC (48h) was determined to be > 4.6 mg/L (0.66 mg aep/L).

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 August - 20 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 202 (Daphnia sp. Acute Immobilisation 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 were taken from control and test media at four different occasions (freshly prepared media at 0 and 24 hours, and expired media at 24 and 48 hours).
- Sample storage conditions before analysis: frozen (ca. -20°C)

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test media were freshly prepared on two occasions during the definitive test (0 and 24 hours). The test substance was supplied as a frozen liquid and an aliquot of the test substance was thawed on each of these occasions. 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 then 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: dilution medium

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain: Straus
- Source: Institute National de Recherche Chimique Appliqué (IRChA), France
- Age at study initiation (mean and range, SD): Less than 24 hours old.
- Method of breeding: Parthenogenesis ensured by stock culture conditions.
- Feeding during test: No feeding during test.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

226 mg/L CaCO3

Test temperature:

nominal: 20 ± 2°C
measured: 19.8 - 21.3°C

pH:

measured: 6.07-7.10

Dissolved oxygen:

measured: 97-102 % ASV (air saturation value)

Nominal and measured concentrations:

Nominal: 0.15, 0.33, 0.726, 1.60, 3.51, 7.73 mg TOS/L
Measured:
At 1.60 to 7.73 mg TOS/L the measured concentrations in samples of freshly prepared media ranged between 76 and 92% of their nominal values. At the lower test levels (0.15 to .726 mg TOS/L), the measured levels in freshly prepared media were ranging between 14 and 52% of the nominal.
At 0.15 to 3.51 mg TOS/L, the measured levels in samples of expired (24-hours old) media ranged between 27 and 91% of their starting value. At 7.73 mg TOS/L the measured level was 105% of the starting value.

Details on test conditions:

TEST SYSTEM
- Test vessel: glass jar
- Type (delete if not applicable): loosely covered
- Material, size, headspace, fill volume: glass, fill volume 100mL
- Renewal rate of test solution (frequency/flow rate): renewal of test media after 24 hours (test period 48 hours)
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
Biomass loading rate: 20 mL medium per organism.


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elendt M4 medium. The media was prepared in deionised, reverse osmosis water and pH was adjusted to approximately 6 before use.
- Alkalinity: 43 mg/L CaCO3

OTHER TEST CONDITIONS
- Adjustment of pH: yes, to pH 6
- Photoperiod: 16 hours light: 8 hours dark with periods of subdued lighting at the beginning and end of each light phase.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
Daphnia were considered to be immobile if they were unable to swim within approximately 15 seconds following gentle agitation of the test vessel.
The numbers of mobile, immobile and floating Daphnia were counted approximately 24 and 48 hours after the start of the study.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.2
- Range finding study
- 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:

24 h

Dose descriptor:

EC50

Effect conc.:

1.29 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Totaal Organic Solids (TOS)

Basis for effect:

mobility

Remarks on result:

other: 95% confidence limits: 1.18 and 1.6 mg TOS/L

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

0.675 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: Total Organic Solids (TOS)

Basis for effect:

mobility

Remarks on result:

other: 95% confidence limits: 0.596 and 1.02 mg TOS/L

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.868 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Total Organic Solids (TOS)

Basis for effect:

mobility

Remarks on result:

other: 95% confidence limits: 0.735 and 1.05 mg TOS/L

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.306 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: Total Organic Solids (TOS)

Basis for effect:

mobility

Remarks on result:

other: 95% confidence limits: 0.23 and 0.429 mg TOS/L

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

170 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Remarks:

active enzyme protein

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

1.6 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Total Organic Solids (TOS)

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

1.02 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: Total Organic Solids (TOS)

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.33 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Total Organic Solids (TOS)

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.066 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: Total Organic Solids (TOS)

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

36.7 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Remarks:

active enzyme protein

Basis for effect:

mobility

Details on results:

- Mortality of control: No control animals were immobilised

Validity criteria fulfilled:

not specified

Conclusions:

The 48-hour EC50 of the test substance for the immobilisation of Daphnia magna was nominal 0.868 mg TOS/L (95% confidence limits of 0.735 and 1.05 mg TOS/L). Based on the mean measured levels, the 48-hour EC50 was 0.306 mg TOS/L (95% confidence limits, 0.23 and 0.429 mg TOS/L).
The 'no-observed effect concentration' of the test substance with Daphnia magna was nominally 0.33 mg TOS/L. Based on the mean measured levels, the NOEC was 0.066 mg TOS/L.

Executive summary:

The acute toxicity of subtilisin to Daphnia magna was assessed under semi-static exposure conditions, with renewal of the media after 24 hours. The study was conducted in accordance with EC and OECD Guidelines, and in compliance with GLP.

Daphnia magna 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 concentrations were verified using ELISA. At 1.60 to 7.73 mg TOS/L, the measured concentrations in samples of freshly prepared media ranged between 76 and 92% of their nominal values. At the lower test levels (0.15 to 0.726 mg TOS/L), the measured levels in freshly prepared media were lower, ranging between 14 and 52% of nominal. At 0.15 to 3.51 mg TOS/L, the measured levels in samples of expired (24-hours-old) media had decreased (to between 27 and 77% of their starting values) except on one occasion at 0.33 mg TOS/L where the measured level was maintained (91% of initial). At 7.73 mg TOS/L, the measured level was maintained (105% of the starting value). The loss in enzyme activity was not considered to have affected the outcome of the study as the results have been calculated in terms of both the nominal and mean measured concentrations.

Observations of the Daphnia in each control and test vessel were made after approximately 24 and 48 hours. Based on these findings the following values have been estimated:

24-hour EC50 (95% c.l.): nominal 1.29 mg TOS/L (1.18 & 1.6)

measured (geom. mean) 0.675 mg TOS/L (0.596 & 1.02) or 375 µg aep/L

48-hour EC50 (95% c.l.): nominal 0.868 mg TOS/L (0.735 & 1.05)

measured (geom. mean) 0.306 mg TOS/L (0.23 & 0.429) or 170 µg aep/L

100% immobility: nominal 1.60 mg TOS/L

measured (geom. mean) 1.02 mg TOS/L or 567 µg aep/L

0% immobility / NOEC: nominal 0.33 mg TOS/L

measured (geom. mean) 0.066 mg TOS/L or 36.7 µg aep/L

Reference 3

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

other information

Justification for type of information:

Glutamyl endopeptidase is a proteolytic enzyme and belongs to the same enzyme sub-subclass (IUB 3.4.21) as subtilisin. Data from the supporting algal growth inhibition and daphnia immobilisation study with glutamyl endopeptidase indiccate that the ecotoxicity profile of glutamyl endopeptidase is similar to subtilisin and thus support read-across to subtilisin.

Reason / purpose for cross-reference:

read-across source

Conclusions:

Based on the supporting study and read-across to subtilisin glutamyl endopeptidase is toxic to aquatic invertebrates with an 48hEC50 value of 170 mg aep/L.

Description of key information

Under the conditions of the test, testing of glutamyl endopeptidase batch PPA27269 to Daphnia magna revealed an 48h EC50 value of 5 mg TOS/L (0.7 mg aep/L). The NOEC (48h) was determined to be > 4.6 mg/L (0.66 mg aep/L).

Another similar proteolytic enzyme – subtilisin belonging to the same enzyme sub-subclass – was tested using Daphnia magna. The EC value was 0.306 mg TOS/L (0.17 mg aep/L), and NOEC was 0.066 mg TOS/L (36.7 µg aep/L).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

170 µg/L