Dehydratase, carbonate

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:

20 July 2020 to 26 May 2021

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 that belong to the same enzyme sub-class, similar results are expected for carbonic anhydrase.

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004

Deviations:

no

GLP compliance:

yes

Remarks:

No claim of GLP compliance is made for the analysis of samples.

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 110.0, 220.2, 440.3, 880.7 and 1761.4 mg enzyme concentrate dry matter/L.
- Sample storage conditions before analysis: During the duration of the test, the samples were treated the same as the test concentrations. Thereafter, the samples were stored frozen until analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Dilution in water
- Controls: Elendt M4 medium
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): No

Test organisms (species):

Daphnia magna

Details on test organisms:

Common name: Daphnia magna
Strain: Straus
Source: In-house laboratory cultures. A clone of this species was originally supplied by the University of Sheffield, UK.
Age at study initiation: Gravid adults were isolated 24 hours prior to initiation of the test. Young daphnids produced overnight were used for testing.
- Feeding during test: No

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

250 mg/L CaCO3

Test temperature:

21-22°C

pH:

7.9-8.2 in control
7.4-8.1 in test concentrations

Dissolved oxygen:

8.7-9.0 mg O2/L in controls
7.4-8.6 mg O2/L

Nominal and measured concentrations:

110.0, 220.2, 440.3, 880.7 and 1761.4 mg enzyme concentrate dry matter/L (nominal concentrations).
Analysis of the test preparations at 0, 24 and 48 hours were performed by the Sponsor. No enzyme activity was detected in the control samples. Low recovery was observed for the lower concentrations in medium (6.25 and 12.5 mg aep/L) with both methods. This however was not the case for the samples prepared in reverse osmosis (RO) water, where recovery was > 85% for all concentrations of freshly prepared samples. Since similar results are expected for freshly prepared samples (0 hours and 24 hours fresh) in medium and RO water, an influence of medium on the enzyme assays cannot be ruled out for the lower concentrations. The two higher concentrations were generally good recovery (> 90%).

Details on test conditions:

TEST SYSTEM
- Test vessels: 150 mL glass vessels each containing approximately 100 mL test solution.
- Experimental design: 20 animals per test concentration. 5 test concentrations plus 1 control. Duplicate samples were prepared for enzyme activity analysis and stored in the test concentrations.
- Type: covered to reduce evaporation
- 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. Total hardness 250 mg/L as CaCO3.
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
Method of initiation: Daphnia were placed in the vessels containing the test preparations.
Loading: 20 mL test solution per organism.
Photoperiod: 16 h light: 8 h dark with with 20 minute dawn and dusk transition periods.
Medium renewal: None.
Duration of exposure: 48 hours.
Criterion of effect: Daphnia were considered to be immobilized if they were unable to swim for approximately 15 seconds after gentle agitation.

EFFECT PARAMETERS MEASURED: The numbers of mobile, immobile and floating daphnia were counted approximately 24 and 48h after the start of the study.
TEST CONCENTRATIONS
110.0, 220.2, 440.3, 880.7 and 1761.4 mg enzyme concentrate dry matter/L.

Reference substance (positive control):

no

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 1 761.4 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

other: enzyme concentrate dry matter

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

1 761.4 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

other: enzyme concentrate dry matter

Basis for effect:

mobility

Details on results:

Details on results
- Mortality of control: 0
- Other adverse effects control: No
- Abnormal responses: No
- Effect concentrations exceeding solubility of substance in test medium: No

Validity criteria fulfilled:

yes

Conclusions:

Exposure of Daphnia magna to Xanthan lyase, batch PPE55581 gave EC50 value of greater than 1761.4 mg enzyme concentrate dry matter/L (equivalent to 100 mg active enzyme protein/L). The No Observed Effect Concentration (NOEC) was the highest concentration tested, 1761.4 mg enzyme concentrate dry matter/L.

Executive summary:

A study was performed to assess the acute toxicity of Dispersin, batch PPW69725 to Daphnia magna. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphnia sp., Acute Immobilisation Test" referenced as Method C.2 of Commission Regulation (EC) No. 440/2008.

20 daphnids (4 replicates of 5 animals) were exposed to aqueous dispersions of the test item at nominal concentrations of 110.0, 220.2, 440.3, 880.7 and 1761.4 mg enzyme concentrate dry matter/L for 48 hours at a temperature of 21 °C to 22 °C under static test conditions. The number of immobilized Daphnia was recorded after 24 and 48 hours.

Analysis of the test preparations at 0 and 48 hours were performed by the Sponsor. The two higher concentrations had good recovery (> 90%).

Exposure of Daphnia magna to Xanthan lyase, batch PPE55581 gave EC50 value of greater than 1761.4 mg enzyme concentrate dry matter/L (equivalent to 100 mg active enzyme protein/L). The No Observed Effect Concentration (NOEC) was the highest concentration tested, 1761.4 mg enzyme concentrate dry matter/L.

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

15 September 2022 - 09 December 2022

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004

GLP compliance:

no

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION:
- Method: The test enzyme was be provided (deep frozen) in a container. The container was defrosted at room temperature on the day of the experiment start. The container was gently inverted a few times, followed by gentle magnetic stirring to avoid damage to the enzyme and to ensure homogeneity prior to preparation of the daily aliquots. The test item was dispersed in the test medium, from which serial dilutions were prepared for at least five concentrations, arranged in a geometric series with a factor preferably not exceeding 2.2.
- Controls: ISO Standard 6341 medium
- Test concentration separation factor: 2

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia Magna
- Age at study initiation: < 24 h
- Source: From test kit - provided as dormant eggs
- Feeding during test: No

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test temperature:

21 °C

pH:

7.8-8.8

Dissolved oxygen:

5.9-7.5 mg/L

Nominal and measured concentrations:

Nominal: 42.3, 84.6, 169.2, 338.3, 676.6 mg enzyme concentrate dry matter/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Multivell plates with 4 wells
- Material, size, headspace, fill volume: 10 mL fill volume
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16-hour light/8-hour dark
- Light intensity: 700-3000 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : organisms not able to swim within 15 seconds after gentle agitation with metal needle (even if they move the antennae) were considered immobile.

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

> 676.6 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.:

> 676.6 mg/L

Nominal / measured:

nominal

Conc. based on:

other: enzyme concentrate dry matter

Basis for effect:

mobility

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of the test item to the freshwater invertebrate Daphnia magna has been investigated based on nominal concentrations revealed no effect on immobilization. The 48-hour EC50 value was greater than the highest concentration tested 676.6 mg enzyme concentrate dry matter/L (equivalent to 100 mg active enzyme protein/L). The NOEC is 676.6 mg enzyme concentrate dry matter/L (equivalent to 100 mg active enzyme protein/L).

Executive summary:

A study was performed to assess the acute immobilization of Carbonic anhydrase, batch PPE93985 to Daphnia magna. 20 daphnids (4 replicates of 5 animals) were exposed to aqueous dispersions of the test item at nominal concentrations of 42.3, 84.6, 169.2, 338.3, 676.6 mg enzyme concentrate dry matter/L for 48 hours at a temperature of 21 °C under static test conditions in a 16-hour light and 8-hour dark cycle. The number of immobilized Daphnia was recorded after 24 and 48 hours. No verification of the test concentrations was performed.

Exposure of Daphnia magna to Carbonic anhydrase, batch PPE93985 based on nominal concentrations gave EC50 values of greater than 676.6 mg enzyme concentrate dry matter/L (equivalent to 100 mg active enzyme protein/L). The NOEC is 676.6 mg enzyme concentrate dry matter/L (equivalent to 100 mg active enzyme protein/L).

Description of key information

A GLP study was selected as read-across from Xanthan lyase. A non-GLP study is available on Carbonic anhydrase as a supporting study.

Exposure of Daphnia magna to Xanthan lyase, batch PPE55581 gave EC50 value of greater than 1761.4 mg enzyme concentrate dry matter/L (equivalent to 100 mg aep/L). The No Observed Effect Concentration (NOEC) was the highest concentration tested, 1761.4 mg enzyme concentrate dry matter/L (equivalent to 100 mg aep/L).

Exposure of Daphnia magna to Carbonic anhydraase, batch PPE93985 has been investigated based on nominal concentrations revealed no effect on immobilization. The 48-hour EC50 value was greater than the highest concentration tested 676.6 mg enzyme concentrate dry matter/L (equivalent to 100 mg active enzyme protein/L). The NOEC is 676.6 mg enzyme concentrate dry matter/L (equivalent to 100 mg active enzyme protein/L).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC50

Remarks:

enzyme concentrate dry matter

Effect concentration:

> 676.6 mg/L