Dextransucrase

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

other information

Justification for type of information:

The toxicity to aquatic algae and cyanobacteria of the present target substance, dextransucrase IUBMB 2.4.1.5, has not been determined but another enzyme (Cellulase, IUBMB3.2.1.4) has been analyzed and used as a source substance for read-across.
The conclusion is that the target substance dextransucrase IUBMB 2.4.1.5 is considered not to be acutely toxic to Pseudokirchneriella subcapitata at a concentration of 100 mg TOS (total organic solids)/L or 52.1 mg aep (active enzyme protein)/L.
Justification for read-across: Please see attached justification.

Reason / purpose for cross-reference:

read-across source

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 110.1 mg/L

Nominal / measured:

nominal

Conc. based on:

other: enzyme concentrate dry matter

Basis for effect:

other: growth rate

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 March 2011 - 12 March 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Reliability 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

Deviations are considered not to have affected the validity and integrity of the study.

GLP compliance:

yes (incl. QA statement)

Remarks:

certificate included in report

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0 and 110.1 mg enzyme concentrate dry matter/L (limit test).
- Sampling method: At the start of the definitive test, three samples (15 mL) were taken from the freshly-prepared control and test media. After 72 hours, the contents of the replicate flasks for each group were pooled and further samples taken for analysis. Additional samples were also taken from a flask containing Cellulase at a nominal concentration of 110.1 mg enzyme concentrate dry matter/L but with no algal cells, in order to obtain information on the extent of adsorption/absorption of the test substance by the algal cells.
- 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 added directly to algal culture medium (1 L) to provide the test medium at a nominal concentration of 110.1 mg enzyme concentrate dry matter/L. An aliquot (8.6 mL) of the algal inoculum was added to a portion (800 mL) of the test medium to give an initial cell density of 1 x 104 cells/mL. An aliquot (100 mL) of the appropriate inoculated test medium was added to each of the test vessels.
- Controls: Medium

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: green alga Pseudokirchneriella subcapitata
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd., Dunstaffnage Marine Laboratory, Dunbeg, Oban, Argyll, Scotland
- Method of cultivation: The liquid slope cultures were stored in an illuminated refrigerator. Sterile algal nutrient medium was inoculated with cells aseptically removed from the slope culture; these primary liquid cultures (100 mL) were incubated for approximately three days in an orbital incubator under continuous illumination at nominal temperatures in the range 21 to 25°C. Subsequently, appropriate volumes of these primary cultures were aseptically transferred to fresh sterile algal nutrient medium to prepare secondary liquid cultures; these cultures were incubated, as stated above, for a further three days to provide an inoculum in the log phase of growth, characterised by a cell density of 9.3 x 10^5 cells/mL.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

21.8-24.1°C

pH:

7.54-7.96

Nominal and measured concentrations:

nominal: 0 and 110.1 mg enzyme concentrate dry matter/L
At the start of the test, enzyme recovery was 104% of the nominal value. After 72 hours, the recovery decreased to 90% of nominal.

Details on test conditions:

TEST SYSTEM
- Test vessel: conical flask
- Type (delete if not applicable): Flasks were loosely plugged with with foam bungs.
- Material, size, fill volume: glass, 250 mL, 100 mL
- Initial cells density: 1 x 10^4 cells/mL
- Control end cells density: 1372083 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6


GROWTH MEDIUM
- Standard medium used: yes (sterile algal nutrient medium as recommended in Official Journal No. L383A Part C.3 and OECD Procedure 201)


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Filtered, dechlorinated tap water which had been softened and treated by reverse osmosis, before microfiltration and purification (resistivity of 18 Megohm/cm).

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: continuous illumination
- Light intensity and quality: nominally 4440 to 8880 lux provided by 6 x 30 W "cool white" 1 metre fluorescent tubes


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Samples were taken at 24, 48 and 72 hours and the cell densities measured using a haemocytometer. The estimate of cell numbers in each sample was based on the mean of four consecutive counts. The presence of any abnormal cell numbers in each sample was also noted during screening of each test level.


TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 1.1, 11, 110.1 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:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 110.1 mg/L

Nominal / measured:

nominal

Conc. based on:

other: enzyme concentrate dry matter

Remarks:

aep (active enzyme protein)

Basis for effect:

other: growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 110.1 mg/L

Nominal / measured:

nominal

Conc. based on:

other: enzyme concentrate dry matter

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Any stimulation of growth found in any treatment: at 110.1 mg enzyme concentrate dry matter/L there was a significant stimulation at 24 to 48h, but no significant stimulation after 72h of exposure.

Reported statistics and error estimates:

The mean coefficient of variation (CoV) for daily growth rates in control cultures ranged between 5.87 and 8.18 during the definitive test and the CoV for the average specific growth rates of the control culture was 1.46 during the 72h period.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the test, Cellulase, batch PPC31776 was not found to be acutely toxic to Pseudokirchneriella subcapitata at a nominal concentration of 110.1 mg enzyme concentrate dry matter/L. Consequently, the 72-hour EbC50, ErC50 and EyC50 values could not be calculated and must be >110.1 mg enzyme concentrate dry matter/L (equivalent to 52.1 mg active enzyme protein)/L and the “no observed effect concentration” was ≥ 110.1 mg enzyme concentrate dry matter/L (equivalent to 52.1 mg active enzyme protein)/L.

Executive summary:

The effect of Cellulase, batch PPC31776 on the growth of the unicellular green alga Pseudokirchneriella subcapitata was assessed under non-axenic conditions.

The study was conducted in accordance with EC Methods for Determination of Ecotoxicity, Annex IV to Commission Regulation (EC) No 761/2009 (O.J. No. L220/36, 2009) Part C, Method 3 “Freshwater Algae and Cyanobacteria, Growth Inhibition Test” and Procedure 201 of the “Guidelines for Testing of Chemicals” of the Organisation for Economic Co-operation and Development: Freshwater Alga and Cyanobacteria, Growth Inhibition Test” (2006).

Six algal cultures, with an initial nominal cell density of 1 x 10^4 cells/mL, were exposed  to Cellulase, batch PPC31776 at a nominal concentration of 110.1 mg enzyme concentrate dry matter/L.

The test medium was prepared in OECD medium by the direct addition of the test substance to the dilution medium. The cultures were incubated in an orbital incubator under continuous illumination at temperatures ranging from 21.8 to 24.1 °C for 72 hours.

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.

Cell numbers were counted daily to monitor growth. The test results are expressed in terms of the area under the growth curve, growth rate and yield.

The following values were derived from the data:

	Nominal Cellulase concentration (mg enzyme concentrate dry matter/L)	Nominal Cellulase concentration (mg active enzyme protein/L)
Area under the growth curve EbC50 (72 h)	>110.1	>52.1
Average specific growth rate ErC50 (0 - 72 h)	>110.1	>52.1
Yield EyC50 (0 - 72 h)	>110.1	>52.1
“No observed effect concentration”	≥ 110.1	≥ 52.1

Description of key information

Under the conditions of the test, Cellulase, batch PPC31776 was not found to be acutely toxic to Pseudokirchneriella subcapitata at a nominal concentration of 110.1 mg enzyme concentrate dry matter/ L.

Consequently, the 72-hour EbC50, ErC50 and EyC50 values could not be calculated and must be >110.1 mg enzyme concentrate dry matter/L (equivalent to 52.1 mg active enzyme protein)/L and the “no observed effect concentration” was ≥ 110.1 mg enzyme concentrate dry matter/L (equivalent to 52.1 mg active enzyme protein)/L.

Because all enzymes are built up of amino acids, the physical and chemical characteristics are very similar for different enzymes, and hence read-across from other enzymes should be fully applicable. In general, enzymes exhibit the same ecotoxicological properties which are confirmed by ecotoxicity studies performed in the industry.

Therefore, Dextransucrase is considered not to be acutely toxic to Pseudokirchneriella subcapitata at

a nominal concentration of 110.1 mg enzyme concentrate dry matter/ L.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

110.1 mg/L

Additional information

The effect of Cellulase on the growth of the unicellular green alga Pseudokirchneriella subcapitatawas assessed under non-axenic conditions.

The study was conducted in accordance with EC Methods for Determination of Ecotoxicity, Annex IV to Commission Regulation (EC) No 761/2009 (O.J. No. L220/36, 2009) Part C, Method 3 “Freshwater Algae and Cyanobacteria, Growth Inhibition Test” and Procedure 201 of the “Guidelines for Testing of Chemicals” of the Organisation for Economic Co-operation and Development: Freshwater Alga and Cyanobacteria, Growth Inhibition Test” (2006).

Six algal cultures, with an initial nominal cell density of 1 x 10^4 cells/mL, were exposed 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 OECD medium by the direct addition of the test substance to the

dilution medium. The cultures were incubated in an orbital incubator under continuous illumination at temperatures ranging from 21.8 to 24.1°C for 72 hours.

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.

Cell numbers were counted daily to monitor growth. The test results are expressed in terms of the area under the growth curve, growth rate and yield.

The following values were derived from the data:

	Nominal Cellulase concentration (mg TOS/L)	Nominal Cellulase concentration (mg aep/L)
Area under the growth curve EbC50 (72 h)	>100	>52.1
Average specific growth rate ErC50 (0 - 72 h)	>100	>52.1
Yield EyC50 (0 - 72 h)	>100	>52.1
“No observed effect concentration”	≥ 100	≥ 52.1