Mannanase, endo-1,4-β-

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 August 2000 - 7 February 2001

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

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Test solutions were prepared by adding a volume (1.127 mL) of mannanase to a 1 litre volumetric flask and bringing to volume with algal medium. The contents were then shaken to ensure homogenous solution, producing a 105.8 mg TOS/L nominal stock solution. The required test concentrations were prepared by serial dilution of the stock solution with algal medium.
- Controls: Algal medium

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green alga
- Strain: Pseudokirchneriella subcapitata CCAP 278/4
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), Ambleside, Cumbria, UK
- Method of cultivation: Transfers of alga were made into fresh algal growth medium to provide suitable axenic subcultures, which were in exponential phase of growth for test inoculations.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

23-25°C

pH:

7.58 - 8.64

Nominal and measured concentrations:

nominal: 6.6, 13.2, 26.5, 52.9 and 105.8 mg enzyme concentrate dry matter/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flask with foil lid
- Material, size, fill volume: glass, 250 mL, 50 mL
- Initial cells density: 0.09 x 10^5 cells/mL
- Control end cells density: 15.76 x 10^5 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6


GROWTH MEDIUM
- Detailed composition if non-standard medium was used: 15 mg NH4CL/L, 12 mg MgCl2•6H2O/L, 18 mg CaCl2•2H2O/L, 15 mg MgSO4•7H2O/L, 1.6 mg KH2PO4/L, 0.08 mg FeCl3•6H2O/L, 0.1 mg Na2EDTA•2H2O/L, 0.185 mg H3BO3/L, 0.415 mg MnCl2•4H2O/L, 0.003 mg ZnCl2/L, 0.0015 mg CoCl2•6H2O/L, 1x10^-5 mg CuCl2•2H2O/L, 0.007 mg Na2MoO4•2H2O/L and 50 mg NaHCO3/L


OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: continuous uniform illumination
- Light intensity and quality: 9240 lux provided by a Solex SL 100 lux meter

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Samples were taken at 24, 48 and 72 hours and the cell concentrations determined using a Compound Light Microscope and Improved Neubauer Counting Chambers.


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Based on the outcome of a preliminary limit test (100 mg TOS/L) where there was a greater than 20% reduction in final cell numbers a definitive test was performed.

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 105.8 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:

NOEC

Effect conc.:

26.5 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
- Any stimulation of growth found in any treatment: no

Reported statistics and error estimates:

The area under the growth curve (AUC) and growth rate values from 0 to 72h were analysed separately for homogeneity of variance using Levene's test (Levene, 1960) at a 1% significance level. If there was no evidence of heterogeneity of variance, the AUC and growth rate values were then analysed using analysis of variance (ANOVA) techniques (Snedecor and Cochran, 1980). Following the ANOVA pairwise comparisons were performed between the control and each concentration of test material using Dunetts procedure (Dunnett, 1964) at 5% significance level.
If there was evidence of hetereogeneity of variance, the AUC and growth rate values were then transformed using either a logarithmic or a square root transformation. The transformed values were then re-analysed for separately for homogeneity using Levene's test at a 1% significance level. If there was no evidence of heterogeneity of variance, the transformed AUC and growth rate values were to be analysed in an identical way to the untransformed values.
If the variability between concentrations was still hetereogenous after both log and square root transformation a non-parametric analysis of variance based on the ranked data was to be performed using a one-tailed Dunnett's test (Conover and Iman, 1981) at a 5% significance level.
The % inhibition values were calculated using the mean value for control and the mean value for for each concentration. A probit transformation was applied and probit transformed data subjected to a regression procedure against logarithmically transformed concentrations of test material with a Newton-Raphson maximum likelihood iterative procedure (Finney, 1971) was used to obtain parameter estimates. From the fitted value EC50 value was estimated.
The goodness of the fit of the probit model was checked via Person chi-squared test.

Validity criteria fulfilled:

not specified

Conclusions:

Under the conditions of the test, the 72h EC50 value of Mannanase, batch PPE6432 to R. subcapitata growth rate could not be determined and must be >105.8 mg enzyme concentrate dry matter/L (equivalent to 55.5 mg aep/L), based on nominal concentrations.
The No Observed Effect Concentration (NOEC) for both growth rate and area under the growth curve was calculated as 26.5 mg enzyme concentrate dry matter/L.

Executive summary:

The effect of Mannanase, batch PPE6432 on the growth of R. subcapitata (formerly known as Pseudokirchneriella subcapitata and Selenastrum capricornatum), a freshwater green alga, was determined over a 72h period, in accordance with OECD (1984) Guideline 201 and EC (1992) Guideline C3 and in compliance with GLP.

The nominal concentrations of Mannanase tested were 6.6, 13.2, 26.5, 52.9 and  105.8 mg enzyme concentrate dry matter/L. The pH of test solutions and the temperature range of the environmental chamber were maintained within protocol specification throughout the study period. Illumination provided by artificial daylight fluorescent tubes giving a light intensity of 9240 lux.

The results are reported as daily cell concentrations (cells/mL), average specific growth rates/day and areas under growth curves (cells.h/mL). In addition, EC50 and the no observed effect concentration (NOEC) were calculated.

The following values were derived from the data:

	Nominal Mannanase concentration (mg enzyme concentrate dry matter/L)	Nominal Mannanse concentration (mg active enzyme protein/L)
Area under the growth curve EbC50 (0-72 h)	49.1 (46.6 - 55.4)	25.7 (22.9 - 29.1)
Average specific growth rate ErC50 (0 - 72 h)	>105.8	>55.5
“No observed effect concentration”	26.5	13.9

Description of key information

Under the conditions of the test, the 72h EC50 value of Mannanase, batch PPE6432 to R. subcapitata growth rate could not be determined and must be >105.8 mg enzyme concentrate dry matter/L (equivalent to 55.5 mg aep/L), based on nominal concentrations.

The No Observed Effect Concentration (NOEC) for both growth rate and area under the growth curve was calculated as 26.5 mg enzyme concentrate dry matter/L (equivalent to 13.9 active enzyme protein).

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

13.9 mg/L