Lipase, triacylglycerol

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:

experimental study

Adequacy of study:

key study

Study period:

17 August 1992-30 November 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test substance was dissolved in sterile nutrient medium to provide an initial stock solution of 304.6 mg enzyme concentrate dry matter/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 pre-culture was mixed with 150 mL of each of these solutions to give the 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 and Protozoa c/o Freshwater Biological Association, Cumbria, UK
- Method of cultivation: Sterile nutrient medium was inoculated from a master culture and incubated under continuous illumination (appr. 7000 lux) and stirring (orbital shaker) at 24 +/- 1°C to give an algal suspension in log phase growth characterised by an absorbanse of 0.380 (@665nm).
The suspension was diluted using sterile nutrient medium to an absorbance of 0.036 prior to use.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

23-25°C

pH:

7.5 - 10.3

Nominal and measured concentrations:

nominal: 9.5, 19, 38.1, 76.2 and 152.3 mg enzyme concentrate dry matter/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: 7.9 x 10^4 cells/mL
- Control end cells density: 2.3 x 10^6 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: Sterile nutrient medium as recommended in OECD Guideline No. 201

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

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: continuous illumination
- Light intensity and quality: approximately 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 cell densities measured at 665 nm using 4 cm path cuvettes in a Cecil 373 Series 2 Spectrophotometer. The cell densities of control cultures at initiation and at termination were determinated by direct counting in a haemacytometer.

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

38.1 mg/L

Nominal / measured:

nominal

Conc. based on:

other: enzyme concentrate dry matter

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

94.2 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): At the highesr exposure level of 152.3 mg enzyme concentrate dry matter/L cells were observed to be clumped. No abnormalities were obsereved at all other exposure concetrations inclduing controls.

Reported statistics and error estimates:

In order to estimate the concentration at which 50% inhibition occurs, a logistic model (Volund, A. 1978. Biometrics 34) was fitted to the percentage inhibition values based on the 'area under the curve' (AUC) at 72 hours and on the growth rate between 24 and 72 hours.
The 50% points were estimated by interpolation of the fitted curves and 95% confidence limits were produced by reference to Student's t distribution, using a pooled estimate of the error variance about the model (Draper and Smith, 1966. Applied Regression Ananlyisi (first ed.) John Wiley and Sons, Inc. New York).
A 'no observed effect level' was obtained using Williams test (Williams, D.A., 1971. Biometrics 27. Williams, D.A.1972. Biometrics 28). Bartletts test (Bartlett, M.S. 1973. J.Royal Stat.Soc.Suppl.4) with a 1% significance level was used for homogeneity of variance.

Validity criteria fulfilled:

yes

Conclusions:

Lipase, batch PPW3983 is inhibitory to the growth of Desmodesmus subspicatus. NOEC was 38.1 mg enzyme concentrate dry matter/L (equivalent to 6.2 mg active enzyme protein/L). The 72h ErC50 is 94.2 mg enzyme concentrate dry matter/L (equivalent to 15.5 mg active enzyme protein/L) and the EbC50 is 92.3 mg enzyme concentrate dry matter/L.

Executive summary:

The effect of Lipase, batch PPW3983 on the growth of the unicellular green alga Desmodesmus subspicatus was assessed in accordance with EEC Methods for the Determination of Ecotoxicity, EEC Directive 67/548 Annex VIII Part C (87/302/EEC) and OECD test guideline 201, and in compliance with GLP.

Algal cultures exposed to five test concentrations of Lipase 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:

 

	Nominal Lipase concentration (mg enzyme protein dry matter/L)
Area under the growth curve EbC50 (72 h)	92.3
Average specific growth rate ErC50 (0 - 72 h)	94.2
“No observed effect concentration”	38.1

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

5 September 2003 - 4 February 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

1984

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

certificate included in report

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 11.9, 26.2, 54.8, 119.2, 262.3 mg enzyme concentrate dry matter/L
- Sampling method: Triplicate samples of 5 mL were taken from control and test stock solutions at 72 hours (replicates pooled) for analysis. The samples were not filtered before analysis. Additional samples were taken from the flasks containing the 'no algae' cultures at 72 hours (replicates).
- 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 was dispersed in diluent water to give an initial concentration of 262.3 mg enzyme concentrate dry matter/L. Serial dilutions of this test solution were prepared with nutrient medium to give the remaining nominal test concentrations of 11.9, 26.2, 54.8, 119.2 mg enzyme concentrate dry matter/L. Algal pre-culture was mixed with the control and test solutions at a ratio of 5.13 mL per 1000 mL to give a cell density of approximately 10^4 cells/mL. Additional flasks containing the test substance at a nominal concentration of 54.8 mg enzyme concentrate dry matter/L but without presence of algal cells were also prepared in order to obtain information on the extent to which the test substance was lost by either adsorption onto or absorbtion by algal cells (referred to as 'no algae').
- Controls: Medium

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green alga Selenastrum capricornutum
- 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:

no

Total exposure duration:

72 h

Test temperature:

23 ± 1 °C

pH:

7.6 - 8.6

Nominal and measured concentrations:

nominal: 11.9, 26.2, 54.8, 119.2, 262.3 mg enzyme concentrate dry matter/L
Geometric mean measured concentrations: 4.5, 17.9, 53.7, 134.7, 289.7 mg enzyme concentrate dry matter/L
The measured analytical results of the dose solutions were not as expected. The high concentration samples were diluted between 20000 to 40000 times to be measurable in the low ELISA range and this could contribute to minor discrepancies from expected results.

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: 1102500 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 and OECD Procedure 201)

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

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: continuous illumination
- Light intensity and quality: 7400 - 7880 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Samples were taken at 24, 48 and 72 hours and the cell densitiesdetermined by direct counting using a haemocytometer. The presence of any abnormal cell numbers in each sample was also noted during screening of each test level.

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

262.3 mg/L

Nominal / measured:

nominal

Conc. based on:

other: enyzme concentrate dry matter

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 262.3 mg/L

Nominal / measured:

nominal

Conc. based on:

other: enyzme 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: no

Reported statistics and error estimates:

EC50s and EC10s could not be estimated because there was no evidence of inhibition at any concentration.
For AUC and growth rate, the F1 test for monotonicity (Healey 1999) was first applied. If this was not significant at the 5% level, then each treated group was compared with the control using a twosided Williams test for monotonic trend (Williams 1971,1972). If the monotonicity test was significant at the 5% level, indicating a non-monotone dose-response, then a two-sided Dunnett's test (Dunnett 1955, 1964) was applied instead.
The data were received analysed using SAS 8.2 (SAS Institute 1999).

RESULTS
As all concentrations had less than 10% inhibition, EC10 and EC50 estimates are >262.3 mg enzyme concentrate dry matter/L.
For growth rate, there was evidence of a non-monotonic dose-response relationship, so Dunnett’s test was used instead of Williams’ test. None of the concentrations were significantly different from control for either area under curve or growth rate.

The measured analytical results of the dose solutions were not as expected. The high concentration samples were diluted between 20000 to 40000 times to be measurable in the low ELISA range and this could contribute to minor discrepancies from expected results. The results are based on the nominal concentrations - which are the lower values - as a worse case.

Validity criteria fulfilled:

yes

Conclusions:

Lipase, batch PPW 21180 did not inhibit the growth of Selenastrum capricornutum, at any concentration under the conditions of this test. The ErC50 (0 – 72 hours) was > 262.3 mg enzyme concentrate dry matter/L (equivalent to 37.4 mg active enzyme protein/L).
The EbC50 (72 hours) was > 262.3 mg enzyme concentrate dry matter/L. The No-observed-effect concentration NOEC was 262.3 mg enzyme concentrate dry matter/L (equivalent to 37.4 mg active enzyme protein/L). The results are based on the nominal concentrations - which are the lower values - as a worse case.

Executive summary:

A study was conducted to assess the inhibitory effect of Lipase, batch PPW 21180 on the growth of the unicellular green alga Selenastrum capricornutum, Strain No. CCAP 278/4.

Triplicate algal cultures, with an initial cell count of approximately 1 x 10^4 cells/mL, were exposed to Lipase, batch PPW 21180 at nominal test concentrations of 11.9, 26.2, 54.8, 119.2, 262.3 mg enzyme concentrate dry matter/L. These cultures, together with one untreated control group of six replicates, were incubated in a Gallenkamp illuminated orbital incubator under continuous illumination at temperatures in the range of 23 ± 1°C for 72 hours. Cell numbers were counted daily to monitor growth.

Verification of test concentrations was performed based on enzyme activity. The amount of measured enzyme (ng/g) was then converted to mg enzyme concentrate dry matter/L in order to calculate the achieved measured concentrations. These results are based on geometric mean measured concentrations, which ranged from 43 – 164% of nominal at 0 hours and from 34 – 78% at 72 hours. The measured analytical results of the dose solutions were not as expected. The high concentration samples were diluted between 20000 to 40000 times to be measurable in the low ELISA range and this could contribute to minor discrepancies from expected results. The results are based on the nominal concentrations - which are the lower values - as a worse case.

The effective nominal concentrations for inhibition of growth based on biomass and average specific growth rates (EbC50 and ErC50, respectively) were:

	Lipase, PPW 21180 (mg enzyme concentrate dry matter/L)
Area under the growth curve EbC50 (72 h)	> 262.3
Average specific growth rate ErC50 (0 - 72 h)	> 262.3
“No observed effect concentration”	≥ 262.3

Description of key information

The lowest ErC50 for lipase was determined to be 94.2 mg enzyme concentrate dry matter/L (equivalent to 15.5 mg active enzyme protein/L).

NOEC was 38.1 mg enzyme concentrate dry matter/L (equivalent to 6.2 mg active enzyme protein/L).

Key value for chemical safety assessment

EC50 for freshwater algae:

15.5 mg/L

EC10 or NOEC for freshwater algae:

6.2 mg/L