Lyase, pectin

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:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

1992-08-17 to 1993-06-11

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 pectin lyase.

Qualifier:

according to guideline

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 10000 mg/L. Serial dilutions of this stock solution were prepared with test water to give the desired series of exposure levels.
- Controls: test water

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia
- Strain: Daphnia magna Straus
- Source: Institute National de Recherche Chimique Appliqué (IRChA), France
- 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

Hardness:

171 mg CaCO3/L

Test temperature:

21 °C

pH:

7.7 - 8.1

Dissolved oxygen:

4.8 - 8.6 mg O2/L

Nominal and measured concentrations:

nominal. 320, 560, 1000, 1800, 3200 and 5600 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: glass jar
- Type (delete if not applicable): loosely covered with aluminium foil
- Material, fill volume: glass, 200 mL
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2

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 move 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. Chlorine levels remained below detection limit at all times.
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light: 8 hours dark.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8

Reference substance (positive control):

no

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

4 900 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CL: 3900 - 6200 mg/L

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

519 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.:

3 200 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

24 h

Dose descriptor:

NOEC

Effect conc.:

138 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

active enzyme protein

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

2 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CL: 1700-2300 mg/L

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

212 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

active enzyme protein

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

106 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

active enzyme protein

Basis for effect:

mobility

Details on results:

- Mortality of control: 0

Reported statistics and error estimates:

EC50 values and 95% confidence limits were calculated according to the method of Thompson and Weil (Thompson, W.R. & Weil, C.S., 1952, Biometrics 8; 51-54).
The NOEC (no-observed-effect concentration) is the highest concentration at and below which the incidence of immobilisation is equal or less than 10%.

Conclusions:

The 48 h EC50 value for alpha-amylase with Daphnia magna is 2000 mg test material/L corresponding to 212 mg active enzyme protein/L.

Executive summary:

A study was performed to assess the acute toxicity of alpha-amylase to Daphnia magna.

The study was conducted in accordance with OECD Guideline for Testing of Chemicals No.202, and in compliance with GLP.

Groups of 20, 1st instar Daphnia (less than 24 hours old) were exposed for 48 hours to six concentrations of the test substance dissolved in water. The incidence of immobilisation was recorded for each test and control group at 24 and 48 hours and the following values determined:

EC₅₀ (24h): 4900 mg/L corresponding to 519 mg active enzyme protein/L

EC₅₀(48h): 2000 mg/L corresponding to 212 mg active enzyme protein/L

NOEC (48h): 1000 mg/L corresponding to 106 mg active enzyme protein/L

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

supporting study

Study period:

28 March 2011 to 12 March 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study was conducted in accordance with EC Methods for Determination of Ecotoxicity, Annex to Commission Regulation (EC) No 440/2008 Part C, Method 2 "Daphnia sp. Acute Immobilistaion Test" and the OECD Guideline for Testing of Chemicals No.202, and in compliance with GLP.

Justification for type of information:

Due to the similarity between the two enzymes, similar results are expected for pectin lyase.

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Certificate included in report

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0 and 100 mg Total organic solids (TOS) per litre
- Sampling method: At the start from the test, three samples (15 mL) were taken from the freshly prepared control and test media. After 48 hours, the contents of the test vessels from each group were pooled and further three samples (15 mL) were taken for analysis.
- Sample storage conditions before analysis: Frozen

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test substance (878 mg) was dispersed in dilution medium (1L) in a volumetric flask. The contents of the flask were shaken before poured in to the test vessels.
- Controls: 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: Stock cultures are maintained in glass vessels containing approximately 0.5 to 0.8 litres of Elendt M4 culture medium in a temperature controlled laboratory at nominally 20 ± 2°C. A photperiod of 16 hours light: 8 hours dark, with periods of subdued light at the beginning and end of each light phase. Culture medium was renewed three times each week. Cultures were fed daily with a suspension of the unicellular green algae, Pseudokirchneriella subcapitata, to provide nominally 0.1 to 0.2 mg carbon per daphnid, per day. Culture conditions ensure that the stock animals reproduce by parthenogenesis. The day before the start of the test, all juvenile Daphnia were removed from the laboratory cultures. The following morning, juveniles produced by the egg-bearing adult Daphnia were removed from the culture vessels and held in a separate holding vessel. These animals were used in the test.
- Feeding during test: no

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Hardness:

270 mg/L as CaCO3

Test temperature:

20.4-20.8°C

pH:

7.61-8.05

Dissolved oxygen:

40-99% ASV (air saturation value)

Nominal and measured concentrations:

nominal: 0 and 100 mg TOS/L
At the start of the test, enzyme recovery was 89% of the nominal value. After 48 hours the recovery decreased to 41% of nominal value.

Details on test conditions:

TEST SYSTEM
- Test vessel: glass dishes
- Type (delete if not applicable): loosely covered
- Material, size, fill volume: glass, 120 mL, 100 mL
- Aeration: no
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: deionised, reverse osmosis water
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours light: 8 hours dark with periods of subdued light at the beginning and at the end of each light phase

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Daphnia were considered immobile if they were unable to swim within approximmately 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
- 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:

48 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: TOS (total organic solids)

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

>= 52.1 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

active enzyme protein (aep)

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: TOS

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 52.1 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

aep

Basis for effect:

mobility

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the test, Cellulase was not found to be acutely toxic to Daphnia magana at a nominal concentration of 100 mg TOS/L equivalent to 52.1 mg aep/L.

Executive summary:

The acute toxicity of Cellulase to Daphnia magna was assessed under static exposure conditions.

The study was conducted in accordance with EC Methods for Determination of Ecotoxicity, Annex to Commission Regulation (EC) No 440/2008 Part C, Method 2 “Daphnia sp. Acute Immobilisation Test” and the OECD Guideline for Testing of Chemicals No. 202, “Daphnia Acute Immobilisation Test” (2004).

A group of twenty Daphnia, less than 24 hours old, was exposed for 48 hours to Cellulase at a nominal concentration of 100 mg TOS (Total Organic Solids)/L equivalent to 52.1 mg aep (active enzyme protein)/L. The test medium was prepared in Elendt M4 medium by the direct addition of the test substance to the dilution medium.

At the request of the Sponsor, the test concentration was verified by analysis of the enzyme concentration, which was performed at the Sponsor’s laboratory.

Observations of the Daphnia in each control and test vessel were made after 24 and 48 hours.

No immobilisation or adverse effects on the Daphnia were noted at a nominal concentration of 100 mg TOS/L.

Based on these findings the following values have been estimated:

48-hour EC50 value :                                   >100 mg TOS/L or > 52.1 mg aep/L

“No observed effect concentration” :            ≥ 100 mg TOS/L or 52.1 mg aep/L

Reference 3

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

supporting study

Study period:

30 November 2005 - 30 May 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 pectin lyase.

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Certificate included in the study report.

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 220 mg TOS/L
- Sampling method: Triplicate samples (5mL) were taken from the control and test concentration at 0 and 24 hours (fresh media) and at 24 and 48 hours (expired media) for analysis.
- Sample storage conditions before analysis: frozen at - 20°C in the dark.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A sample of the test material weighing 1584 mg was dispersed directly in 1 litre of diluent water to give the nominal test concentration of 220 mg TOS/L.
- Controls: medium

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain: Straus
- Source: Daphnia magna used in this study were cultured in-house and were obtained from a strain originating from the Institute National de Recherche Chimique Appliqué (IRChA), France.
- Age at study initiation (mean and range, SD): less than 24 hours old.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Test temperature:

21-22°C

pH:

7.5-7.7

Dissolved oxygen:

7.6-8.2

Nominal and measured concentrations:

nominal: 220 mg TOS/L
measured: 255 mg TOS/L

Details on test conditions:

TEST SYSTEM
- Test vessel: glass jars
- Type (delete if not applicable): loosely covered
- Material, size, headspace, fill volume: glass, 250 ml, fill volume: 100 mL
- Aeration: no
- Renewal rate of test solution (frequency/flow rate): medium renewal after 24 hours
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates):4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elendt M4 medium

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

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Observations of the condition of the test and control Daphnia were made following 24 and 48 hours exposure. Daphnia were considered to be immobile if they were unable to swim within approximately 15 seconds following gentle agitation of the test vessel.

TEST CONCENTRATIONS
Range finding study
- Test concentrations: 1, 10, 100 and 220 mg TOS/L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

no

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 255 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

other: Total Organic Solids (TOS)

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 120.2 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Remarks:

active enzyme protein (aep)

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

255 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

other: TOS

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

120.2 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Remarks:

active enzyme protein (aep)

Basis for effect:

mobility

Details on results:

- Mortality of control: 0

Conclusions:

No immobilisation was observed following the exposure of Daphnia magna to the test substance at a concentration of 255 mg TOS/L. Due to the low toxicity of the test material, the 48-hour EC50 (immobilisation) value of the substance was unable to be calculated. Therefore the following values were assumed:
The 48-hour EC50 value of the test substance to Daphnia magna was > 255 mg TOS/L corresponding to > 120.2 mg aep/L (TOS = Total Organic Solids; aep = active enzyme protein).
The no-observed effect concentration (NOEC) was 255 mg TOS/L corresponding to 120.2 mg aep/L.

Executive summary:

The acute toxicity of the test substance to Daphnia magna was assessed under semi-static exposure conditions.The study was conducted according to OECD and EC guidelines, and in compliance with GLP.

A group of twenty, first instar Daphnia, less than 24 hours old, were exposed to the test substance dispersed in Elendt M4 medium for 48 hours at a nominal concentration of 220 mg Total Organic Solids (TOS)/L. Mean measured concentration was 255 mg TOS/L.

Verification of test concentrations was performed based on enzyme activity. The amount of measured enzyme (AGU/g) was then converted to mg TOS/L in order to calculate the achieved measured concentrations. Measured concentrations ranged from 118 - 119% of nominal at 0 hours (fresh), from 113 - 118 at 24 hours (expired), was 116% at 24 hours (fresh) and from 112 - 114% at 48 hours (expired). These results confirmed that the nominal exposure concentration was achieved and maintained satisfactorily for the 24-hour duration between each medium renewal.

The numbers of immobilised daphnids were recorded for each test and control group after 24 and 48 hours. All results are expressed in terms of mean measured concentration.

No immobilisation in either the control or the test group was noted throughout the exposure period, and therefore the following values were estimated:

48-hour EC50: >255 mg TOS/L corresponding to > 120 mg aep/L (TOS = Total Organic Solids; aep = active enzyme protein)

Lowest test concentration resulting in 100% immobilisation >255 mg TOS/L

No-observed effect concentration (NOEC): 255 mg TOS/L corresponding to 120 mg aep/L

Control immobilisation: 0%

Reference 4

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

supporting study

Study period:

1992-02-04 to 1992-12-02

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 pectin lyase.

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes

Remarks:

including certificate

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.
- Controls: test water

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia
- Strain: Daphnia magna Straus
- Source: Institute National de Recherche Chimique Appliqué (IRChA), France
- 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

Hardness:

150-200 mg CaCO3/L

Test temperature:

21 °C

pH:

7.6 - 8.0

Dissolved oxygen:

2.7 - 8.6 mg O2/L

Nominal and measured concentrations:

nominal. 5.6, 10, 18, 32, 56, 100, 180, 320, 560 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: glass jar
- Material, fill volume: glass, 200 mL
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Laboratory tap water filtered, dechlorinated by passage through activated carbon, softened by reverse osmosis and aged by recirculation through a gravel filter.
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light: 8 hours dark

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8

Reference substance (positive control):

no

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 560 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

24 h

Dose descriptor:

NOEC

Effect conc.:

> 560 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

95 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CL: 74-120 mg/L

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 154.5 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.:

> 154.5 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

active enzyme protein

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

26.2 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

active enzyme protein

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

8.8 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

active enzyme protein

Basis for effect:

mobility

Details on results:

- Mortality of control: 0

Reported statistics and error estimates:

EC50 values and 95% confidence limits were calculated according to the method of Thompson and Weil (Thompson, W.R. & Weil, C.S., 1952, Biometrics 8; 51-54).
The NOEC (no-observed-effect concentration) is the highest concentration at and below which the incidence of immobilisation is equal or less than 10%.

Conclusions:

The 48 h EC50 value for Peroxidase SP 491 with Daphnia magna is 95 mg test material/L, 26.2 mg AEP/g respectively.

Executive summary:

A study was performed to assess the acute toxicity of Peroxidase SP 491 to Daphnia magna. The study was conducted in accordance with OECD Guideline for Testing of Chemicals No. 202, and in compliance with GLP.

Groups of 2x10, 1st instar Daphnia (less than 24 hours old) were exposed for 48 hours to 10 concentrations of the test substance dissolved in water. The incidence of immobilisation was recorded for each test and control group at 24 and 48 hours and the following values determined:

EC₅₀ (24h): > 560 mg/L, corresponding to 154.6 mg AEP/g

EC₅₀(48h): 95 mg/L, corresponding to 26.2 mg AEP/g

NOEC (48h): 32 mg/L, corresponding to 8.8 mg AEP/g

Reference 5

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

supporting study

Study period:

03 January 2006 - 09 Fabruary 2007

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 pectin lyase.

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Certificate included in the study report.

Analytical monitoring:

yes

Details on sampling:

- Concentrations (based on dose range finding study): 4.6, 10, 22, 46 and 100 mg TOS/L
- Sampling method: Triplicate samples (5mL) were taken from the control and each test concentration at 0 and 24 hours (fresh media) and at 24 and 48 hours (expired media) for analysis.
- Sample storage conditions before analysis: frozen at - 20°C in the dark for 19-21 days befire dispatching to the Sponsor for analysis

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Sample of the test material was centrifuged at 4000 n/min-1 for 15 min to remove precipitat. The main activity was similar in the centrifuged and uncentrifuged sample. The centrifuged sample weighing 2380 mg was dispersed directly in 1 L of diluent water to give nominal concnentration 100 mgTOS/L. Serial dilution of this stock was performed to complete the test series.
- Controls: medium

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain: Straus
- Source: Daphnia magna used in this study were cultured in-house and were obtained from a strain originating from the Institute National de Recherche Chimique Appliqué (IRChA), France.
- Age at study initiation (mean and range, SD): less than 24 hours old.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test temperature:

20°C

pH:

7.0-7.7

Dissolved oxygen:

6.6-8.1

Nominal and measured concentrations:

nominal: 0, 4.6, 10, 22, 46 and 100 mg TOS/L
fresh measured: ND, 1.3, 3.5, 10.2, 24.5 and 81 mg TOS/L
expired measured: ND, 1.3, 3.5, 10.2, 24.5 and 81 mg TOS/L

Details on test conditions:

TEST SYSTEM
- Test vessel: glass jars
- Type (delete if not applicable): loosely covered
- Material, size, headspace, fill volume: glass, 250 ml, 20 mL test solution per organism, 100 mL
- Aeration: no
- Renewal rate of test solution (frequency/flow rate): medium renewal after 24 hours
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates):4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elendt M4 medium prepared using deionized water

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

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Observations of the condition of the test and control Daphnia were made following 24 and 48 hours exposure. Daphnia were considered to be immobile if they were unable to swim within approximately 15 seconds following gentle agitation of the test vessel.

TEST CONCENTRATIONS
Range finding study
- Test concentrations: 1, 10, and 100 TOS/L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

no

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 81 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

other: Total Organic Solids (TOS)

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 55 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: Total Organic Solids (TOS)

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

> 81 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

other: Total Organic Solids (TOS)

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

> 55 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.:

81 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

other: Total Organic Solids (TOS)

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

55 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: Total Organic Solids (TOS)

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 25.5 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Remarks:

active enzyme protein (AEP)

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 17.3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Remarks:

active enzyme protein (AEP)

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

> 25.5 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Remarks:

active enzyme protein (AEP)

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

> 17.3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Remarks:

active enzyme protein (AEP)

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

25.5 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Remarks:

active enzyme protein (AEP)

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

17.3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Remarks:

active enzyme protein (AEP)

Basis for effect:

mobility

Details on results:

- Mortality of control: 0

Conclusions:

No immobilisation was observed following the exposure of Daphnia magna to Asparaginase, PPV 24743 at a concentration of 81 mg TOS/L (equivalent to a geometric mean measured concentration 55 mg TOS/L), 25.5 mg AEP/g and 17.3 mg AEP/g respectively. Due to the low toxicity of the test material, the 48-hour EC50 (immobilisation) value of the substance was unable to be calculated. Therefore the following values were assumed:
The 48-hour EC50 value of Asparaginase, PPV 24743 with Daphnia magna was determined to be >81 mg TOS/L equivalent to >55 mg TOS/L, 25.5 mg AEP/g equivalent to a geometric mean measured concentration 17.3 mg AEP/g respectively.
The no-observed effect concentration (NOEC) was 81 mg TOS/L equivalent to 55 mg TOS/L, 25.5 mg AEP/g equivalent to a geometric mean measured concentration 17.3 mg AEP/g respectively.

Executive summary:

The acute toxicity of the test substance to Daphnia magna was assessed under semi-static exposure conditions.The study was conducted according to OECD and EC guidelines, and in compliance with GLP.

A group first instar Daphnia, less than 24 hours old, were exposed to the test substance dispersed in Elendt M4 medium for 48 hours at a nominal concentrations of 0, 4.6, 10, 22, 46 and 100 mg Total Organic Solids (TOS)/L. Mean fresh measured was ND, 1.3, 3.5, 10.2, 24.5 and 81 mg TOS/L and the mean expired measured was ND, 1.3, 3.5, 10.2, 24.5 and 81 mg TOS/L.

Verification of test concentrations was performed based on enzyme activity. The amount of measured enzyme (AGU/g) was then converted to mg TOS/L in order to calculate the achieved measured concentrations. Measured concentrations ranged from 29 - 100% of nominal at 0 hours (fresh), from 21 - 51% at 24 hours (expired), from 27 - 62% at 24 hours (fresh), and from 17 - 32% at 48 hours (expired). The variability of the freash measured concnetration indicates that dispersion of the test material in volumes of 0.5 til 1 L is difficult to achieve.

The numbers of immobilised daphnids were recorded for each test and control group after 24 and 48 hours. All results are expressed in terms of mean measured concentrations.

No immobilisation in either the control or the test group was noted throughout the exposure period, and therefore the following values were estimated:

48-hour EC50: > 81 mg TOS/L (fresh mean) equivalent to > 55 mg TOS/L (geometric mean), corresponding to > 25.5 mg aep/L equivalent to 17.3 mg aep/L

Lowest test concentration resulting in 100% immobilisation > 81 mg TOS/L equivalent to > 55 mg TOS/L(geometric mean), corresponding to > 25.5 mg aep/L equivalent to 17.3 mg aep/L

No-observed effect concentration (NOEC): 81 mg TOS/L equivalent to > 55 mg TOS/L(geometric mean), corresponding to > 25.5 mg aep/L equivalent to 17.3 mg aep/L

Control immobilisation: 0%

Reference 6

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

supporting study

Study period:

1994-03-31 to 1994-10-17

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 pectin lyase.

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes

Remarks:

including certificate

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.
- Controls: test water

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia
- Strain: Daphnia magna Straus
- Source: Institute National de Recherche Chimique Appliqué (IRChA), France
- 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:

19-21 °C

pH:

7.0 - 8.0

Dissolved oxygen:

5.2 - 9.0 mg O2/L

Nominal and measured concentrations:

nominal. 0 and 100 mg a.i/L

Details on test conditions:

TEST SYSTEM
- Test vessel: glass jar
- Material, fill volume: glass, 200 mL
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: M7 medium was prepared using analytical grade reagens and reverse osmosi purified water.
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light: 8 hours dark

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 20 mL test solution/organism

Reference substance (positive control):

no

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 0.062 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

Active enzyme protein (AEP)

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 0.062 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

Active enzyme protein (AEP)

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

>= 0.062 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

Active enzyme protein (AEP)

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

> 0.062 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

Active enzyme protein (AEP)

Basis for effect:

mobility

Details on results:

- Mortality of control: 0

Reported statistics and error estimates:

EC50 values and 95% confidence limits were calculated according to the method of Thompson and Weil (Thompson, W.R. & Weil, C.S., 1952, Biometrics 8; 51-54).

Conclusions:

The 48 h EC50 (immobilization) value for SP 472 Terminox with Daphnia magna is > 100 mg a.i/L, corresponding to > 0.062 mg AEP/L.

Executive summary:

The acute toxicity of SP 472 Terminox to Daphnia magna Straus was assessed under static exposure conditions, without renewal of the media until the end of study (48 hours). The study was conducted in accordance with OECD Guideline for Testing of Chemicals No.202, and in compliance with GLP.

Groups of 2x10, 1st instar Daphnia (less than 24 hours old) were exposed for 48 hours to 1 concentration of the test substance dissolved in M7 medium. The incidence of immobilisation was recorded for each test and control group at 24 and 48 hours and the following values determined:

EC₅₀ (24h): >100 mg a.i/L, corresponding to >0.062 mg AEP/L

EC₅₀(48h): >100 mg a.i/L, corresponding to >0.062 mg AEP/L

NOEC (48h): >or= 100 mg a.i/L, corresponding to >or=0.062 mg AEP/L

No immobilization was observed either in control or exposed animals.

Description of key information

The toxicity of pectin lyase to crustaceans has not been tested, however, a read-across was performed with other enzymes, namely alpha amylase, cellulase, glycoamulase, peroxidase, asparaginase, and catalase.

Due to the similarity between the enzymes, similar results are expected for pectin lyase.

Alpha amylase: The 48-h EC50 value for alpha-amylase with Daphnia magna was 2000 mg test material/L corresponding to 212 mg aep/L.

Cellulase: Under the conditions of the test, it was not found to be acutely toxic to Daphnia magna at a nominal concentration of 100 mg TOS/L equivalent to 52.1 mg aep/L.

Glucoamylase: No immobilisation was observed to Daphnia magna and therefore, the 48-hour EC50 value of the is >255 mg TOS/L corresponding to > 120.2 mg aep/L. The NOEC was 255 mg TOS/L corresponding to 120.2 mg aep/L.

Glucoamylase: The 48-h EC50 value for Daphnia magna is 95 mg test material/L corresponding to 26.2 mg aep/g.

Aspariginase: Due to the low toxicity of the test material, the 48-hour EC50 value should be >55 mg TOS/L, equivalent to 25.5 mg aep/g equivalent to a geometric mean measured concentration 17.3 mg aep/g. The NOEC is≥17.3 mg AEP/g.

Catalase: No immobilisation was observed to Daphnia magna and therefore, the 48 h EC50 value is >100 mg a.i/L, corresponding to >0.062 mg aep/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

26.2 mg/L