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Short-term toxicity to aquatic invertebrates

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Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
29 August - 20 October 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
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 0.726 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 100 mL
- 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
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
Reported statistics and error estimates:
Statistical analysis was performed using SAFEStat LD50 application, SAS 8.2 (SAS Institute, 1999)
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). This corresponds to 170 µg aep/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. This corresponds to 36.7 µg aep/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

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
29 - 31 January 1993
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
GLP compliance:
yes
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/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:
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: 5.6, 10, 18, 32, 56, 100, 320 and 560 mg/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 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
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
83 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% confidence limits: 68 - 100 mg/L
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
64 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% confidence limits: 57 - 73 mg/L
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
12.4 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Remarks:
active enzyme protein
Basis for effect:
mobility
Duration:
24 h
Dose descriptor:
NOEC
Effect conc.:
32 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
32 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
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 64 mg/L correponding to 12.4 mg active enzyme protein/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 accordance with EC and OECD Guidelines, and in compliance with GLP. Groups of twenty, 1st instar Daphnia (less than 24 hours old) were exposed for 48 hours to nine concentrations of subtilisin dissolved in water. The incidence of immobilisation was recorded for each test and control group at 24 hours and 48 hours and the following effect values were determined:

48-hour EC50 (95% CI): 64 mg/L (57 & 73) or 12.4 mg active enzyme protein/L

NOEC at 24 and 48 h: 32 mg/L

All concentrations are given in terms of nominal concentration.

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
3 - 5 Dec 1992
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
GLP compliance:
yes
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/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:
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, 0.32, 0.56, 1.0, 1.8, 3.2, 5.6, 10 and 18 mg/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
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
> 18 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
0.92 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% confidence limits: 0.79 and 1.1 mg/L
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
90 µg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Remarks:
active enzyme protein
Basis for effect:
mobility
Duration:
24 h
Dose descriptor:
NOEC
Effect conc.:
3.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
0.56 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
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 0.92 mg/L, corresponding to 90 µg aep/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 accordance with EC and OECD Guidelines, and in compliance with GLP. Groups of twenty, 1st instar Daphnia (less than 24 hours old) were exposed for 48 hours to nine concentrations of subtilisin dissolved in water. 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.): 0.92 mg /L (0.79 & 1.1) corresponding to 90 µg aep/L

NOEC at 24 h: 3.2 mg/L

NOEC at 48 h: 0.56 mg/L

All results are expressed in terms of nominal concentration

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:
guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
GLP compliance:
yes
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/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: 0.1, 0.18, 0.32, 0.56, 1.0, 1.8, 3.2, 5.6 and 10 mg/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): 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 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 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
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% confidence limits: 4.1 - 6.2 mg/L
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
2.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% confidence limits: 2.0 - 3.1 mg/L
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
620 µg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Remarks:
active enzyme protein
Basis for effect:
mobility
Duration:
24 h
Dose descriptor:
NOEC
Effect conc.:
1.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
1.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
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 2.5 mg/L correponding to 620 µg active enzyme protein/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.

Groups of twenty, 1st instar Daphnia (less than 24 hours old) were exposed for 48 hours to nine concentrations of subtilisin dissolved in water. 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% CI): 2.5 mg/L (2.0 & 3.1) or 620 µg active enzyme protein/L

NOEC at 24 and 48 h: 1.8 mg/L

All concentrations are given in terms of nominal concentration.

Description of key information

The EC50 (48 h) of 90 µg aep/L (nominal) for acute toxicity to aquatic invertebrates was determined in the source study by Douglas (1993b).

CLP Conclusion: Aquatic Acute 1, H400, M-factor: 10

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
90 µg/L

Additional information

Thermomycolin is sharing the same structural features and the same catalytic reactions as subtilisin as explained in the Analogue Approach Justification document provided in Section 13. Thus, based on the read-across from the structural analogue substance Subtilisin (CAS 9014-01-0) the target substance Thermomycolin (CAS 52233-31-5) is also considered to possess the same aquatic toxicity potential as the source substance.

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 most recent study was rated as Klimisch 1 study because test concentrations were not verified in the three other studies of older date and thus these studies were rated as Klimisch 2. The results from all studies showed that the EC50 values ranged from 90 µg aep/L to 12400 µg aep/L with a geometric mean of 586 µg aep/L. Although not determined in the Klimisch 1 study, the most sensitive effect value of 90 µg aep/L is considered for assessment and classification purposes, thus representing a worst case approach.

CLP Conclusion: Aquatic Acute 1, H400, M-factor: 10