Subtilisin

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:

key study

Study period:

29 August - 20 October 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

1984

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Except for the analysis of the test material

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0.16, 0.35, 0.78, 1.71, 3.75 and 8.26 mg enzyme concentrate dry matter/L
- Sampling method: Triplicate samples (10 mL) 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 (0.78 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 2 L) 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 0.78 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: 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: 0.16, 0.35, 0.78, 1.71, 3.75 and 8.26 mg enzyme concentrate dry matter/L. Measured:
0.03, 0.07, 0.23, 1.09, 2.06, 7.19 mg enzyme concentrate dry matter/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: glass jar (120 mL capacity)
- 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: 1.07, 10.7 and 107 mg enzyme concentrate dry matter/L
- Results used to determine the conditions for the definitive study: yes.

Reference substance (positive control):

no

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.327 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: enzyme concentrate dry matter

Basis for effect:

mobility

Remarks on result:

other: 95% confidence limits: 0.25 and 0.458 mg enzyme concentrate dry matter/L

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.071 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: enzyme concentrate dry matter

Basis for effect:

mobility

Details on results:

- Immobilisation of control: 0
- Other biological observations: None

The geometric mean of the two highest concentrations was calculated by measurements of 0 hours fresh and 24 hours old. The 24 hours fresh and 48 hours old not sampled because all of the Daphnia were immobilised at 24 hours.

Reported statistics and error estimates:

95% Confidence intervals on EC50.

Validity criteria fulfilled:

yes

Conclusions:

The 48-hour EC50 of Subtilisin, batch PXI48489 for the immobilisation of Daphnia magna was 0.327 mg enzyme concentrate dry matter/L (95% confidence limits, 0.25 and 0.458 mg enzyme concentrate dry matter/L), corresponding to 0.172 mg active enzyme protein/L.
The 'no-observed effect concentration' NOEC was 0.071 mg enzyme concentrate dry matter/L. Media was renewed after 24 hours.

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 compliance with GLP, and in accordance with EC Methods for Determination of Ecotoxicity, Annex to Directive 92/69/EEC (O.J. No. L383A, 1992) Part C, Method 2 “Acute Toxicity For Daphnia” and the OECD Guideline for Testing of Chemicals No. 202, Part I “Daphnia Acute Immobilisation Test” (1984).

Daphnia magna 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 concentrations were verified using ELISA. At 1.71 to 8.26 mg enzyme concentrate dry matter/L, the measured concentrations in samples of freshly prepared media ranged between 76 and 92% of their nominal values. At the lower test levels (0.16 to 0.78 mg enzyme concentrate dry matter/L), the measured levels in freshly prepared media were lower, ranging between 14 and 52% of nominal. At 0.16 to 3.75 mg enzyme concentrate dry matter/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 0.35 mg enzyme concentrate dry matter/L where the measured level was maintained (91% of initial). At 8.26 mg enzyme concentrate dry matter/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. Measured concentrations: 0.03, 0.07, 0.23, 1.09, 2.06, 7.19 mg enzyme concentrate dry matter/L. 

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:

Time (hours)	Subtilisin concentrations (mg enzyme concentrate dry matter/L)		Subtilisin concentration (mg active enzyme protein/L)
	Nominal	Mean measured	Mean measured
24-hr EC50 (95% C.l.)	1.38 (1.26 & 1.71)	0.721 (0.637 & 1.09)
48-hr EC50 (95% C.l.)	0.927 (0.785 & 1.12)	0.327 (0.25 & 0.458)	0.1718
100% immobility	1.71	1.09
NOEC	0.35	0.071

 

The measurements of water quality (temperature, pH, dissolved oxygen, total hardness and alkalinity), remained within acceptable limits during the study.

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

3 December - 5 December 1992

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The test concentrations not verified.

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

1984

Deviations:

no

Remarks:

Study based on nominal concentrations only

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test substance was dissolved in test water to give an initial stock solution of 100 mg batch/L. Serial dilutions of this stock solution were prepared to give the desired series of exposure levels.
- 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:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

150 - 200 mg/L CaCO3

Test temperature:

nominal: 20 ± 2°C
measured: 21°C

pH:

measured: 7.8 - 8.0

Dissolved oxygen:

7.6 - 8.5 mg O2/L

Nominal and measured concentrations:

Nominal: 0.18, 0.31, 0.55, 0.98, 1.8, 3.1, 5.5, 9.8, 17.6 mg enzyme concentrate dry matter/L

Details on test conditions:

TEST SYSTEM
- Test vessel: glass jar
- Type (delete if not applicable): loosely covered with aluminium foil
- Material, size, headspace, fill volume: glass, fill volume 200mL
- Renewal rate of test solution (frequency/flow rate): renewal of test media after 24 hours (test period 48 hours)
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
Biomass loading rate: 20 mL test solution per organism.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Laboratory tap water filtered, dechlorinated and softened by passage through an Elga® water purification system. The water passes through a high pressure sand filter to remove colloids and particulate matter and a high grade activated carbon filter to remove chlorine and any organic contaminants. A proportion of the supply then passes through a water softener before final reverse osmosis treatment to produce a highly purified water supply. The two grades of dechlorinated water are then remixed in the ratio of approximately 1:1 to give a supply with the desired water hardness. Batches of softened water were "aged" with aeration for at least 12 hours prior to use by recirculating through a gravel filter bed.
- Alkalinity: HCO3 210 mg/L

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours light: 8 hours dark

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: 1.8

Reference substance (positive control):

no

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.9 mg/L

Nominal / measured:

nominal

Conc. based on:

other: enzyme concentrate dry matter

Basis for effect:

mobility

Remarks on result:

other: 95% confidence limits: 0.77 and 1.07 mg enzyme concentrate dry matter/L

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.55 mg/L

Nominal / measured:

nominal

Conc. based on:

other: enzyme concentrate dry matter

Basis for effect:

mobility

Details on results:

- Mortality of control: No control animals were immobilised.

Reported statistics and error estimates:

95% confidence intervals on EC50.

Validity criteria fulfilled:

yes

Conclusions:

The nominal 48-hour EC50 of the Subtilisin, batch PPA3980 for the immobilisation of Daphnia magna was 0.90 mg enzyme concentrate dry matter/L (95% confidence limits: 0.77 - 1.07 mg enzyme concentrate dry matter/L), equivalent to 0.092 mg/L. NOEC at 48 h was 0.55 mg enzyme concentrate dry matter/L.

Executive summary:

The acute toxicity of subtilisin to Daphnia magna was assessed under static exposure conditions, with renewal of the media after 24 hours. The study was conducted in compliance with GLP and in accordance with OECD Guideline for Testing of Chemicals No. 202, Pan 1 and EEC Methods for the Determination of Ecotoxicity, EEC Directive 67 /548 Annex V Part C.2 (84/449/EEC).

Groups of twenty, 1st instar Daphnia (less than 24 hours old) were exposed for 48 hours to 9 concentrations of subtilisin dissolved in water. The nominal concentrations were 0.18, 0.31, 0.55, 0.98, 1.8, 3.1, 5.5, 9.8 and 17.6 mg enzyme concentrate dry matter/L. The incidence of immobilisation was recorded for each test and control group at 24 hours and 48 hours and the following values determined:

48-hour EC50 (95% C.l.): 0.90 mg enzyme concentrate dry matter/L (0.77 & 1.07) corresponding to 0.092 mg active enzyme protein/L.

NOEC at 24 h: 3.1 mg enzyme concentrate dry matter/L.
NOEC at 48 h: 0.55 mg enzyme concentrate dry matter/L.

All results are expressed in terms of nominal concentration. All validity criteria were met

Reference 3

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

25 January - 27 January 1993

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The test concentrations were not verified.

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test substance was dissolved in test water to give an initial stock solution of 100 mg batch/L. Serial dilutions of this stock solution were prepared to give the desired series of exposure levels.
- 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:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

171 mg CaCO3/L

Test temperature:

nominal: 20 ± 2°C
measured: 21°C

pH:

measured: 8.1 - 8.3

Dissolved oxygen:

measured: 8.2 - 8.6 mg O2/L

Nominal and measured concentrations:

Nominal: 0.09, 0.17, 0.30, 0.53, 0.94, 1.70, 3.02, 5.28, 9.43 mg enzyme concentrate dry matter/L

Details on test conditions:

TEST SYSTEM
- Test vessel: glass jar
- Type (delete if not applicable): loosely covered with aluminium foil
- Material, size, headspace, fill volume: glass, fill volume 200 mL
- Renewal rate of test solution (frequency/flow rate): no renewal of test solution.
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
Biomass loading rate: 20 mL medium per organism.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Laboratory tap water filtered, dechlorinated and softened by passage through an Elga® water purification system. The water passes through a high pressure sand filter to remove colloids and particulate matter and a high grade activated carbon filter to remove chlorine and any organic contaminants. A proportion of the supply then passes through a water softener before final reverse osmosis treatment to produce a highly purified water supply. The two grades of dechlorinated water are then remixed in the ratio of approximately 1:1 to give a supply with the desired water hardness. Batches of softened water were "aged" with aeration for at least 12 hours prior to use by recirculating through a gravel filter bed.
- Alkalinity: HCO3 210mg/L

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours light: 8 hours dark
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: 1.8

Reference substance (positive control):

no

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

1.7 mg/L

Nominal / measured:

nominal

Conc. based on:

other: enzyme concentrate dry matter

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

2.4 mg/L

Nominal / measured:

nominal

Conc. based on:

other: enzyme concentrate dry matter

Basis for effect:

mobility

Remarks on result:

other: 95% confidence limits: 1.9 - 2.9 mg enzyme concentrate dry matter/L

Details on results:

- Mortality of control: No control animals were immobilised
Individual pH, temperature and dissolved oxygen values remained within acceptable limits throughout the duration of the study.

Reported statistics and error estimates:

95% confidence limits on EC50.

Validity criteria fulfilled:

yes

Conclusions:

The nominal 48-hour EC50 of Subtilisin, batch PPA3352 for the immobilisation of Daphnia magna was nominal 2.4 mg enzyme concentrate dry matter/L (equivalent to to 0.062 mg active enzyme protein/L). NOEC at 24 and 48 hours was 1.7 mg enzyme concentrate dry matter/L.

Executive summary:

The acute toxicity of subtilisin to Daphnia magna was assessed under static exposure conditions. The study was conducted in compliance with GLP and in accordance with OECD Guideline for Testing of Chemicals No. 202, Part 1 and EEC Methods for the Determination of Ecotoxicity, EEC Directive 67 /548 Annex V Part
C.2 (84/449/EEC).

Groups of twenty, 1st instar Daphnia (less than 24 hours old) were exposed for 48 hours to 9 concentrations of subtilisin dissolved in water. The nominal concentrations were: 0.09, 0.17, 0.30, 0.53, 0.94, 1.70, 3.02, 5.28 and 9.43 mg enzyme concentrate dry matter/L. The incidence of immobilisation was recorded for each test and control group at 24 hours and 48 hours and the following nominal values determined:

48-hour EC50 (95% c.l.): 2.4 mg enzyme concentrate dry matter/L (1.9 & 2.9), equivalent to 0.062 mg active enzyme protein/L. 24-hour EC50 (95% C.I.): 4.7 mg enzyme concentrate dry matter/L (3.9 & 5.8).

NOEC at 24 and 48h: 1.7 mg enzyme concentrate dry matter/L.

All concentrations are given in terms of nominal concentration. Individual pH, temperature and dissolved oxygen values remained within acceptable limits throughout the duration of the study.

Reference 4

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

29 January - 31 January 1993

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The test concentrations were not verified.

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Remarks:

The dissolved oxygen dropped to below 3 mg O2/L at 48 hours at the two highest concentrations. The test was still considered to be valid.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test substance was dissolved in test water to give an initial stock solution of 1000 mg batch/L. Serial dilutions of this stock solution were prepared to give the desired series of exposure levels.
- 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:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

171 mg CaCO3/L

Test temperature:

nominal: 20 ± 2°C
measured: 21°C

pH:

measured: 7.4 - 8.2

Dissolved oxygen:

measured: 2.1 - 8.7 mg O2/L

Nominal and measured concentrations:

Nominal: 5.3, 9.4, 17, 30, 53, 94, 170, 302 and 528 mg enzyme concentrate dry matter/L

Details on test conditions:

TEST SYSTEM
- Test vessel: glass jar
- Type (delete if not applicable): loosely covered with aluminium foil
- Material, size, headspace, fill volume: glass, fill volume 200mL
- Renewal rate of test solution (frequency/flow rate): no renewal of test media.
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
Biomass loading rate: 20 mL medium per organism.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Laboratory tap water filtered, dechlorinated and softened by passage through an Elga® water purification system. The water passes through a high pressure sand filter to remove colloids and particulate matter and a high grade activated carbon filter to remove chlorine and any organic contaminants. A proportion of the supply then passes through a water softener before final reverse osmosis treatment to produce a highly purified water supply. The two grades of dechlorinated water are then remixed in the ratio of approximately 1:1 to give a supply with the desired water hardness. Batches of softened water were "aged" with aeration for at least 12 hours prior to use by recirculating through a gravel filter bed.
- Alkalinity: HCO3 210mg/L

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours light: 8 hours dark
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: 1.8

Reference substance (positive control):

no

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

60.4 mg/L

Nominal / measured:

nominal

Conc. based on:

other: enzyme concentrate dry matter

Basis for effect:

mobility

Remarks on result:

other: 95% confidence limits: 53.8 - 68.8 mg/L

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

30.2 mg/L

Nominal / measured:

nominal

Conc. based on:

other: enzyme concentrate dry matter

Basis for effect:

mobility

Details on results:

- Mortality of control: No control animals were immobilised.
Individual pH, temperature and dissolved oxygen values remained within acceptable limits throughout the duration of the study, except for the two highest concentrations (302 and 528 mg enzyme concentrate dry matter/L) where the oxygen concentration fell < 2.8 mg O2/L. The test was still considered to be valid, since 100% immobilization was seen at concentrations 94 and 170 mg enzyme concentrate dry matter/L, where the O2 concentration was > 3 mg/L.

Reported statistics and error estimates:

95% confidence limits on EC50 values.

Validity criteria fulfilled:

not specified

Remarks:

The dissolved oxygen dropped to below 3 mg O2/L at 48 hours at the two highest concentrations. The test was still considered to be valid.

Conclusions:

The nominal 48-hour EC50 of the Subtilisin, batch PPA4046 for the immobilisation of Daphnia magna was 60.4 mg enzyme concentrate dry matter/L (95% confidence limits: 53.8 - 68.8 mg enzyme concentrate dry matter/L) corresponding to 12.4 mg active enzyme protein/L. NOEC at 24 and 48h was 30.2 mg enzyme concentrate dry matter/L.

Executive summary:

The acute toxicity of Subtilisin, batch PPA4046 to Daphnia magna was assessed under static exposure conditions. The study was conducted in compliance with GLP and in accordance with OECD Guideline for Testing of Chemicals No. 202, Part 1 and EEC Methods for the Determination of Ecotoxicity, EEC Directive 67 /548 Annex V Part C.2 (84/449/EEC).

Groups of twenty, 1st instar Daphnia (less than 24 hours old) were exposed for 48 hours to 9 concentrations of subtilisin dissolved in water. The nominal concentrations were: 5.3, 9.4, 17, 30, 53, 94, 170, 302, 528 mg enzyme concentrate dry matter/L. The incidence of immobilisation was recorded for each test and control group at 24 hours and 48 hours and the following values determined:

48-hour EC50 (95% C.l.): 60.4 mg enzyme concentrate dry matter/L (53.8 & 68.8) corresponding to 12.4 mg active enzyme protein/L.

24-hour EC50 (95% C.I.): 78.3 mg enzyme concentrate dry matter/L (64.1 & 94.3). 

NOEC at 24 and 48h: 30.2 mg enzyme concentrate dry matter/L.

All concentrations are given in terms of nominal concentration.
Individual pH, temperature and dissolved oxygen values remained within acceptable limits throughout the duration of the study, except for the two highest concentrations (302 and 528 mg enzyme concentrate dry matter/L) where the oxygen concentration fell < 2.8 mg O2/L. The test was still considered to be valid, since 100% immobilization was seen at concentrations 94 and 170 mg enzyme concentrate dry matter/L, where the O2 concentration was > 3 mg/L. Therefore, the study is deemed valid.

Description of key information

The geometrical mean of the EC50 values was 2.56 mg enzyme concentrate dry matter/L and the geometrical mean of the four values in active enzyme protein was 0.331 mg aep/L. 

• The 48-hour EC50 of Subtilisin, batch PXI48489 for the immobilisation of Daphnia magna was 0.327 mg enzyme concentrate dry matter/L (corresponding to 0.172 mg active enzyme protein/L).
  The 'no-observed effect concentration' NOEC was 0.071 mg enzyme concentrate dry matter/L. Media was renewed after 24 hours.


• (Reliability: 2) The nominal 48-hour EC50 of the Subtilisin, batch PPA3980 for the immobilisation of Daphnia magna was 0.90 mg enzyme concentrate dry matter/L, equivalent to 0.092 mg active enzyme protein/L. NOEC at 48 h was 0.55 mg enzyme concentrate dry matter/L.


• (Reliability: 2) The nominal 48-hour EC50 of Subtilisin, batch PPA3352 for the immobilisation of Daphnia magna was nominal 2.4 mg enzyme concentrate dry matter/L (equivalent to to 0.062 mg active enzyme protein/L). NOEC at 24 and 48 hours was 1.7 mg enzyme concentrate dry matter/L.


• (Reliability: 2) The nominal 48-hour EC50 of the Subtilisin, batch PPA4046 for the immobilisation of Daphnia magna was 60.4 mg enzyme concentrate dry matter/L, corresponding to 12.4 mg active enzyme protein/L. NOEC at 24 and 48h: 30.2 mg enzyme concentrate dry matter/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC50

Remarks:

enzyme concentrate dry matter

Effect concentration:

2.56 mg/L

Additional information

Four acute Daphnia magna immobilisation assays were performed to determine the effect of subtilisin on aquatic invertebrates. All studies were conducted in accordance with OECD and EC test guidelines, and in compliance with GLP. However, only the study conducted with Subtilisin, batch PXI48489 was rated as Klimisch 1 reliability, and the other studies were rated as Klimisch 2 reliability, since test concentrations were not verified.

The results from all studies showed that the EC50 values ranged from 0.9 to 60.4 mg enzyme concentrate dry matter/L and ranged from 61.5 µg aep/L to 12400 µg aep/L.