Pullulanase

Short-term toxicity to aquatic invertebrates

Administrative data

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:

Non-proteolytic enzymes are in general of low to non toxicity towards aquatic organisms and since the enzyme belongs to the same sub-subclass as pullulanase i.e 3.2.1, it can be considered very similar to pullulanase . Thus read-across is fully applicable.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Active enzyme protein of alpha-amylase (EC no. 232-565-6, CAS no. 9000-90-2, EC name: Amylase, alpha-, Enzyme Class No.: 3.2.1.1)

Molecular formula:

Not available

IUPAC Name:

Active enzyme protein of alpha-amylase (EC no. 232-565-6, CAS no. 9000-90-2, EC name: Amylase, alpha-, Enzyme Class No.: 3.2.1.1)

Constituent 2

Reference substance name:

Protein as a constituent of enzyme deriving from the fermentation or extraction process

Molecular formula:

Not available. See remarks.

IUPAC Name:

Protein as a constituent of enzyme deriving from the fermentation or extraction process

Constituent 3

Reference substance name:

Inorganic salts as a constituent of enzyme deriving from the fermentation or extraction process

Molecular formula:

Not available. See remarks.

IUPAC Name:

Inorganic salts as a constituent of enzyme deriving from the fermentation or extraction process

Constituent 4

Reference substance name:

Carbohydrates constituent of enzyme deriving from the fermentation or extraction process

Molecular formula:

Not available. See remarks.

IUPAC Name:

Carbohydrates constituent of enzyme deriving from the fermentation or extraction process

Constituent 5

Reference substance name:

Lipids as a constituent of enzyme deriving from the fermentation or extraction process

Molecular formula:

Not available. See remarks.

IUPAC Name:

Lipids as a constituent of enzyme deriving from the fermentation or extraction process

Test material form:

solid: particulate/powder

Details on test material:

- Lot/batch No.: PPY 4048
- Expiration date of the lot/batch: 1994-09-07
- Stability under test conditions: The test material is stable for at least 24 hours at room temperature and at 4 degree Celcius.
- Storage condition of test material: in darkness at 4⁰C

Sampling and analysis

Analytical monitoring:

no

Test solutions

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

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

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test conditions

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


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

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

Effect concentrations 2

Key result

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

Effect concentrations 3

Key result

Duration:

24 h

Dose descriptor:

NOEC

Effect conc.:

3 200 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 4

Key result

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

Effect concentrations 5

Key result

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

Effect concentrations 6

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

Effect concentrations 7

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 8

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%.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

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