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Toxicity to aquatic algae and cyanobacteria

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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, 4-phytase IUBMB 3.1.3.26, 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 4-phytase IUBMB 3.1.3.26 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:
read-across source
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 52.1 mg/L
Nominal / measured:
nominal
Conc. based on:
other: active ingredient/ active enzyme protein (aep)
Basis for effect:
other: growth rate, biomass and yield
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:
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. certificate)
Remarks:
certificate included in report
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0 and 100 mg TOS/L
- 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 containingCellulase at a nominal concentration of 100 mg TOS/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 100 mg TOS/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):
Pseudokirchneriella subcapitata (previous names: Raphidocelis 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 100 mg TOS/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, 10, 100 mg TOS/L
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
no
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
other: TOS (Total Organic Solids)
Basis for effect:
other: growth rate, biomass and yield
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 52.1 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Remarks:
aep (active enzyme protein)
Basis for effect:
other: growth rate, biomass and yield
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
other: TOS
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 52.1 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Remarks:
aep
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
other: TOS
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 52.1 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Remarks:
aep
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
other: TOS
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 52.1 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Remarks:
aep
Basis for effect:
other: yield
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 100 mg TOS/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 was not found to be acutely toxic to Pseudokirchneriella subcapitata at a nominal concentration of 100 mg TOS/L equivalent to 52.1 mg aep/L.
Consequently, the 72-hour EbC50, ErC50 and EyC50 values for Cellulase could not be calculated but must be >100 mg TOS/L equivalent to 52.1 mg aep/L and the “no observed effect concentration” was ≥ 100 mg TOS/L equivalent to 52.1 mg aep/L.
Executive summary:

The effect of Cellulase 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 at a nominal concentration of 100 mg TOS (Total Organic Solids)/L or 52.1 mg active enzyme protein (aep)/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

Description of key information

The toxicity to aquatic algae and cyanobacteria of the present target substance, 4 -phytase IUBMB 3.1.3.26, has not been determined but another enzyme (Cellulase, IUBMB3.2.1.4) has been analyzed and used as a source substance for read-across.

Under the conditions of the test, cellulase (structural analogue) was not found 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 and thus no EC50 values could be determined.

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, 4 -phytase 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.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:
52.1 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