Lyase, pectate

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 based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

August 1992 to November 1993

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 pectate lyase.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

Method: The test substance was dissolved in sterile nutrient medium to give an initial stock solution of 200 mg/L. This stock solution was further diluted with sterile nutrient medium to produce a series of solutions exactly twice the concentration of the intended exposure levels. 150 mL of algal preculture was mixed with 150 mL of each of these solutions to give final test series.
- Controls: medium.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: green alga
- Strain: CCAP 276/20
- Source (laboratory, culture collection): Culture Centre of Algae & Protozoa c/o Freshwater Biological Association, Cumbria, UK
- Method of cultivation:
Pre-culture: Sterile nutrient medium was inoculated from a master culture and incubated under continuous illumination (ca. 7000 lux) and stirring (orbital shaker) at 24 ± 1°C to give an algal suspension in log phase growth.
Culture conditions: Conical flasks each containing 100 mL test or control culture were loosely stoppered and placed at random in a Gallenkamp Illuminated Orbital Incubator. The cultures were incubated, without media renewal for 72 hours under continuous illuminatioon of approximately 7000 lux. The temperature was maintained at 24 ± 1°C and gaseous exchange and suspension of the algal cells was ensured by the action of the orbital shaker oscillating at 100 cycles per minute.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

23.9 - 24.2°C

pH:

7.5 - 10.2

Nominal and measured concentrations:

Nominal: 0, 6.25, 12.5, 25, 50 and 100 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Conical flasks (250 mL)
- Type (delete if not applicable): loosely stoppered
- Material, size, fill volume: glas, 250 mL, 100 mL
- Initial cells density: 8.2*10^4 cells/mL
- Control end cells density: 2.4*10^6 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes


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

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuous illumination
- Light intensity and quality: 7000 lux provided by 7 x 30 W "warm white" 1 metre fluorescent tubes


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Samples were taken at 0, 24, 48 and 72 hours and the absorbance measured at 665 nm using 4 cm cell path cuvettes in a Cecil 373 Series 2 Spectrophotometer. The cell densities of the control cultures at initiation and at termination were determined by direct counting with the aid of a haemocytometer (Improved Neubauer).


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Test concentrations: 6.25, 12.5, 25, 50 and 100 mg/L

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

24 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% CL 22-26 mg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

2.5 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

active enzyme protein

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

49 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL 42-57 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

5.2 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

active enzyme protein

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

12.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.3 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

active enzyme protein

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2.6 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

active enzyme protein

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): There were no abnormalities in the control cultures or any of the test cultures apart from the two highest exposure levels (50 and 100 mg/L), where cells were observed to be smaller than average and clumped.
- Any stimulation of growth found in any treatment: no

Conclusions:

Alpha-amylase is inhibitory to the growth of Desmodesmus subspicatus at concentrations in excess of 12.5 mg/L corresponding to 1.3 mg active enzyme protein (aep)/mL. The EbC50 (72h) value is 24 mg/L and the ErC50 (24-72h) is 49 mg/L corresponding to 2.5 and 5.2 mg aep/L, respectively.

Executive summary:

A study was conducted to assess the inhibitory effect of alpha-amylase on the growth of the unicellular green alga Desmodesmus subspicatus, Strain No. CCAP 276/20.

The study was conducted in accordance with OECD Guideline for Testing of Chemicals No. 201 and EEC Methods for the Determination of Ecotoxicity, EEC Directive 67/458 Annex VIII, Part C (87/302/EEC).

Algal cultures exposed to five test concentrations of alpha-amylase plus one untreated control were incubated on an orbital shaker under continuous illumination at 24 ± 1°C for 72 hours. Growth was monitored daily by measuring the absorbance of each culture at 665 nm.

The following values were derived from the data:

E_bC₅₀(72h):       24 mg/l corresponding to 2.5 mg active enzyme protein (aep)/L

E_rC₅₀(24-72h):      49 mg/l corresponding to 5.2 mg aep/L

NOEC 72h for AUC (Area under the curve): 12.5 mg/L corresponding to 1.3 mg aep/L

NOEC for the 24 -72 hour growth rate: 25 mg/L corresponding to 2.6 mg aep/L

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

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

Adequacy of study:

supporting study

Study period:

28 March 2011 - 12 March 2012

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 pectate lyase.

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 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):

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 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

Key result

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

Reference 3

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

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

Adequacy of study:

key study

Study period:

20 December 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 pectate lyase.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Certificate included in the study report.

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0, 220 mg TOS (Total Organic Solids)/L
- Sampling method: Triplicate samples, of 5 ml, were taken from control and the test stock solution (including the “no
algae” level) at 0 hours and from culture vessels at 72 hours (replicates pooled) for analysis. The samples were not filtered to remove algal cells before analysis. Additional samples were taken from flasks containing the “no algae” cultures at 72 hours (replicates pooled).
- 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 algal medium to give the nominal test concentration of 220 mg TOS/L .
- Controls: medium.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Culture Collection of Algae & Protozoa, SAMS Research Services Ltd, Argyll, Scotland.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

24°C

pH:

7.2 - 7.9

Nominal and measured concentrations:

nominal: 220 mg TOS/L
measured: 230 mg TOS/L

Details on test conditions:

TEST SYSTEM
- Test vessel: conical flask
- Type (delete if not applicable): loosely stoppered
- Material, size, headspace, fill volume: glass, 250 mL, fill volume:100 mL
- Initial cells density: control: 12087 cells/mL; 220 mg TOS/L: 15073 cells/mL
- Control end cells density: 1348333 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reverse osmosis purified/deionised water

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuous illumination
- Light intensity and quality: 7350-7540 Lux provided by fluorescent tubes


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] samples were taken at 24, 48 and 72 hours and the cell densities determined by direct counting using a haemacytometer (Improved Neubauer). Cell densities at 0 hours were to low to be counted using a haematocytometer and therefore estimation was conducted using a Coulter®Multisizer II particle counter.


TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 1.0, 10, 100 and 220 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.:

230 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: Total Organic Solids (TOS)

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

108.4 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Remarks:

active enzyme protein (aep)

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 230 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: TOS

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 108.4 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Remarks:

active enzyme protein (aep)

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 230 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: TOS

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 108.4 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Remarks:

active enzyme protein (aep)

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: no

Validity criteria fulfilled:

yes

Remarks:

The test was considered valid because cell concentrations in control cultures increased by a factor of 108 within 72 hours (mean cell density of controls at 0 and 72 hours: 1.2 x 10^4 and 1.3 x 10^6 cells/mL, respectively).

Conclusions:

The test substance did not inhibit the growth of Pseudokirchneriella subcapitata, Strain No. CCAP 278/4 at a concentration of 230 mg TOS/L under the conditions of this test.
The EbC50 (72 hours) was >230 mg TOS/L (108.4 mg aep/l) and the ErC50 (0 - 72 hours) was >230 mg TOS/L (108.4 mg aep/L).
The no-observed-effect concentration NOEC was ≥230 mg TOS/L (108.4 mg aep/L) (TOS = Total Organic Solids; aep = active enzyme protein).

Executive summary:

A study was conducted to assess the inhibitory effect of glucoamylase on the growth of the unicellular green alga Pseudokirchneriella subcapitata, Strain No. CCAP 278/4. Six algal cultures, with an initial cell count of approximately 1 x 10⁴ cells/mL, were exposed to the test substance at a nominal test concentration of 220 mg TOS/l. Mean measured concentration was 230 mg TOS/L. These cultures, together with an untreated control group of six replicates, were incubated in a Gallenkamp illuminated orbital incubator under continuous illumination at temperatures in the range of 24 ± 1°C for 72 hours. Cell numbers were counted daily to monitor growth. Test concentrations are based on Total Organic Solids (TOS). Verification of test concentrations was performed based on enzyme activity. The amount of measured enzyme (activity/g) was then converted to mg TOS/l in order to calculate the achieved measured concentrations. Measured concentrations ranged from 119 - 120% of nominal at 0 hours and from 91 - 93% at 72 hours. The high light intensity is thought to have been attributable to the 23% reduction in enzyme activity during the test. The median effective concentrations for inhibition of growth based on biomass and average specific growth rates (EbC50 and ErC50, respectively) were:

EbC50 (AUC 72 hours): >230 mg TOS/L or > 108.4 mg aep/L (TOS = Total Organic Solids; aep = active enzyme protein)

ErC50 (Growth rate 0 - 72 hours) >230 mg TOS/L or >108.4 mg aep/L

No-observed-effect concentration (NOEC): ≥230 mg TOS/L or 108.4 mg aep/L

Reference 4

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

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

Adequacy of study:

supporting study

Study period:

June 1992 to January 1994

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 pectate lyase.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes

Remarks:

Including certificate

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

Method: The test substance was dissolved in sterile nutrient medium. 50 mL of algal preculture was added to sterile nutrient medium containing appropriate amounts of test substance.
- Controls: medium.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: green alga
- Strain: CCAP 276/20
- Source (laboratory, culture collection): Culture Centre of Algae & Protozoa c/o Freshwater Biological Association, Cumbria, UK
- Method of cultivation:
Pre-culture: Sterile nutrient medium was inoculated from a master culture and incubated under continuous illumination (ca. 7000 lux) and stirring (orbital shaker) at 24 ± 1°C to give an algal suspension in log phase growth. The suspension was diluted to an absorbance of 0.042 prior to use.
Culture conditions: Conical flasks (250 mL) each containing 100 mL test or control culture were loosely stoppered to reduce evaporation. The cultures were incubated, without media renewal for 72 hours under continuous illuminatioon of approximately 7000 lux. The temperature was maintained at 24 ± 1°C. The gaseous exchange and suspension of the algal cells was ensured by the action of the orbital shaker.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

24 +/- 1°C

pH:

7.1 - 10.7

Nominal and measured concentrations:

Nominal: 0, 62.5, 125, 250, 500 and 1000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Conical flasks (250 mL)
- Type (delete if not applicable): loosely stoppered
- Material, size, fill volume: glass, 250 mL, 100 mL
- Initial cells density: 1.1*10^5 cells/mL
- Control end cells density: 2.0*10^6 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes


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

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuous illumination
- Light intensity and quality: 7000 lux


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Samples were taken at 0, 24, 48 and 72 hours and the absorbance measured at 665 nm. The cell densities of the control cultures at initiation and at termination were determined by direct counting with the aid of a haemocytometer.


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Test concentrations: 62.5, 125, 250, 500 and 1000 mg/L

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

320 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% CL 290-360 mg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

88.3 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

active enzyme protein

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

640 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL 560-730mg/L

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

176.6 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

active enzyme protein

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

125 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

34.5 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

active enzyme protein

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

500 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

138 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

active enzyme protein

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): There were no abnormalities in the control cultures or any of the test cultures apart that the initial cell count at 0 hours is higher than recomended under OECD guidelines. This was justified. Concentrations 500 and 1000 mg/mL at 24, 48 and 72 hours caused clumping of cells and cells size was reduced by concentration 1000 mg/L.
- Any stimulation of growth found in any treatment: yes, at 62.5 and 125 mg/L

Conclusions:

Peroxidase SP 491 inhibited growth of Desmodesmus subspicatus at concentrations above 125 mg/L corresponding to 34.5 mg active enzyme protein (aep)/mL. The EbC50 (72h) value is 320 mg/L and the ErC50 (24-72h) is 640 mg/L corresponding to 88.3 and 176.6 mg aep/L, respectively.

Executive summary:

This study was conducted to assess the inhibitory effect of alpha-amylase on the growth of the unicellular green alga Desmodesmus subspicatus, Strain No. CCAP 276/20.

The study was conducted in accordance with OECD Guideline for Testing of Chemicals No. 201 and EEC Methods for the Determination of Ecotoxicity, EEC Directive 67/458 Annex VIII, Part C (87/302/EEC).

Algal cultures exposed to five test concentrations of Peroxidase SP491 plus one untreated control were incubated on an orbital shaker under continuous illumination at 24 ± 1°C for 72 hours. Growth was monitored daily by measuring the absorbance of each culture at 665 nm.

The following values were derived from the data:

E_bC₅₀(72h): 320 mg/L corresponding to 88.3 mg active enzyme protein (aep)/L

E_rC₅₀(24-72h): 640 mg/l corresponding to 176.6 mg aep/L

NOEC 72h for AUC (Area under the curve): 125 mg/L corresponding to 34.5 mg aep/L

NOEC for the 24 -72 hour growth rate: 500 mg/L corresponding to 138 mg aep/L

Reference 5

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

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

Adequacy of study:

supporting study

Study period:

03 January 2006 - 09 February 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 pectate lyase.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

certificate included in report

Analytical monitoring:

yes

Details on sampling:

- Nominal concentrations:
Preliminary range finding: 0, 1, 10 and 100 mg TOS/L
Definitive test: 0, 4.6, 10, 22, 22 (no algae), 46 and 100 mg TOS/L
- Sampling method: At the start of the definitive test and after 72 hours, three samples (5 mL) were taken from control and test stock solutions for analysis. The samples were not filtered to remove algal cells before analysis. Additional samples were taken from flasks containing the "no algae" cultures at 0 and 72 hours (replicates pooled). 15 mL sample was also removed for analysis from the sub-sample of test material before and after centrifugation. Because by-product from the enzymatic action of Asparaginase is ammonia, ammonia level within the test were determined.
- 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 centrifuged at 4000 n/min for 15 min ( 1190 mg) was added directly to algal culture medium (1 L) to provide the test medium at a nominal concentration of 100 mg TOS/L. The algal preculture was mixed with control and the test solution at ratio of 2.2 mL per 500 mL to give strating cell density 10^4 cells/mL. Flask containing test substance at 22 mg TOS/mL without cells was prepared to test whether substance as lost due to adsorption or absoprtion at the algal cells.
- 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: Flasks containing nutrient medium were inoculated from a master culture and were maintained under continuous illumination (8670-9180 lux) in an orbital incubator at 24+/-C, to give an algal suspension in log phase groth, characterised by a cell density of 2.3x10^6 cell/mL.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

23-24°C

pH:

9.3-9.4

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.

- Nominal concentrations:
Preliminary range finding: 0, 1, 10 and 100 mg TOS/L
Definitive test: 0, 4.6, 10, 22, 22 (no algae), 46 and 100 mg TOS/L
- Measured concentrations:
Definitive test: 0, 2.5, 7.1, 18.2, 37.4 and 85.1 mg TOS/L

Details on test conditions:

TEST SYSTEM
- Test vessel: conical flask
- Type (delete if not applicable): Flasks were loosely stoppered.
- Material, size, fill volume: glass, 250 mL, 100 mL
- Initial cells density: 1 x 10^4 cells/mL
- Control end cells density: 1212500 cells/mL
- No. of vessels per concentration (replicates): 3
- 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)


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reverse osmosis purified/deionized water.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: continuous illumination
- Light intensity and quality: nominally 7740 to 8170 lux provided by 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 densities at 0 hours were too low to be counted using haemacytometer and therefore estimation was conducted using a Coulter Multisizer II particle counter.


TEST CONCENTRATIONS
- Range finding study: 1, 10, 100 mg TOS/L
- Test concentrations (nominal):4.6, 10, 22, 46 and 100 mg TOS/L
- Results used to determine the conditions for the definitive study: yes



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.:

7.1 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

other: TOS (Total Organic Solids)

Basis for effect:

other: growth rate, biomass and yield

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: TOS

Basis for effect:

other: growth rate, biomass and yield

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 85.1 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

other: TOS

Basis for effect:

other:

Remarks on result:

other: AUC

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 19.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: TOS

Basis for effect:

other:

Remarks on result:

other: AUC

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 85.1 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

other: TOS

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 19.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: TOS

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

26.8 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Remarks:

Active enzyme protein (AEP)

Basis for effect:

other: growth rate, biomass and yield

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.9 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Remarks:

Active enzyme protein (AEP)

Basis for effect:

other: growth rate, biomass and yield

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 26.8 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Remarks:

Active enzyme protein (AEP)

Basis for effect:

other: AUC

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Remarks:

Active enzyme protein (AEP)

Basis for effect:

other: AUC

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 26.8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Remarks:

Active enzyme protein (AEP)

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Remarks:

Active enzyme protein (AEP)

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: no

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the test, Asparaginase was not acutely toxic to Pseudokirchneriella subcapitata at an inital measured concentration of 7.1 mg TOS/L, equivalent to 3 mg TOS/L geometric mean measured concentration, 2.23 mg AEP/g and 0.9 mgAEP/g respectively.
Consequently, the 72-hour EbC50, ErC50 and EyC50 values for Asparaginase were estimated to be >85.1 mg TOS/L based on initial measured concentration , equivalent to >19.2 mg TOS/L based on the highest geometric mean measured concentration tested, 26.8 mg AEP/g and 6.0 mg AEP/g respectively. The “no observed effect concentration” was 7.1 mg TOS/L based on initial measured concentration, equivalent to 3 mg TOS/L based on geometric mean measured concentration, 2.23 mg AEP/g and 0.9 mg AEP/g respectively.

Executive summary:

The effect of Asparaginase 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 to Directive 92/69/EEC (O.J. No. L383A, 29.12.92) Part C, Method 3 “Algal Inhibition Test”, OECD Guideline for Testing of Chemicals No. 201 “Alga, Growth Inhibition Test”, and in compliance with GLP.

Three algal cultures (six controls), with an initial nominal cell density of 1 x 10^4 cells/mL, were exposed to Asparaginase at a nominal concentration up to 100 mg TOS (Total Organic Solids)/L. The test medium was prepared in OECD medium by a 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 23 to 24 °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.

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:

	Initial measured Asparaginase concentration mg TOS/L (mg AEP/g)	Geometric mean measured concentration mg TOS/L(mg AEP/g)
Area under the growth curve EbC50 (72 h)	>85.1 (26.8)	>19.2 (6.0)
Average specific growth rate ErC50 (0 - 72 h)	>85.1 (26.8)	>19.2 (6.0)
“No observed effect concentration”	7.1 (2.23)	3 (0.9)

Description of key information

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

Alpha amylase: Alpha-amylase was tested with Desmodesmus subspicatus. The ErC50 (24-72h) is 49 mg/L corresponding to 5.2 mg aep/L, respectively.

Cellulase: Under the conditions of the test, it 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 72h ErC50 values must be >100 mg TOS/L (> 52.1 mg aep/L).

Glucoamylase: Under the conditions of the test, it was not found to be acutely toxic to Pseudokirchneriella subcapitata at a nominal concentration of 230 mg TOS/L corresponding to 108.4 mg aep/L. Therefore, the 72h ErC50 must be >230 mg TOS/L (108.4 mg aep/L).

Peroxidase: Peroxidase SP 491 inhibited growth of Desmodesmus subspicatus at concentrations above 125 mg/L corresponding to 34.5 mg active enzyme protein (aep)/mL. The 72h ErC50 (24-72h) is 640 mg/L corresponding to 176.6 mg aep/L, respectively.

Asparaginase: Under the conditions of the test, it was not acutely toxic to Pseudokirchneriella subcapitata at an initial measured concentration of 7.1 mg TOS/L, equivalent to 3 mg TOS/L geometric mean measured concentration, 2.23 mg aep/g and 0.9 mg aep/g respectively. Consequently, the 72h ErC50 and EyC50 values for Asparaginase were estimated to be >85.1 mg TOS/L based on initial measured concentration, equivalent to >19.2 mg TOS/L based on the highest geometric mean measured concentration tested, 26.8 mg sep/g and 6.0 mg aep/g, respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:

5.2 mg/L