Mycophenolic acid

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 05-December 06, 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well documented and reported study, conducted according to internationally accepted technical guidelines and in compliance with GLP in recognized contract research organization.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)

Version / remarks:

, 1989

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

, 1992

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

, 1984

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

37415-62-6

Cas Number:

37415-62-6

IUPAC Name:

37415-62-6

Constituent 2

Reference substance name:

6-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-2-benzofuran-5-yl)-4-methylhex-4-enoic acid, sodium salt

IUPAC Name:

6-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-2-benzofuran-5-yl)-4-methylhex-4-enoic acid, sodium salt

Details on test material:

- Name of test material (as cited in study report): ERL080/DS
- Test substance code (testing laboratory): 3105806
- Physical state: White powder
- Expiration date of the lot/batch: 28 February 2000
- Stability under storage conditions: Stable
- Storage condition of test material: In refrigerator protected from light
- Stability in vehicle: Water: at least 96 h

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Singular samples for analysis were taken from all test concentrations and the control. The samples were taken from the approximate centre of the test solutions.
Frequency: At the start and the end of the test. Extra samples were taken after the 24-hour dark period.
Volume: 10 ml
Storage: The samples taken at the start of the test and after the 24- hour period in the dark were stored in a deep freeze and analysed on the last day of exposure together with the samples taken at the end of the test.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- (Test) Water: ISO-medium (formulated according to ISO/IS 8692)
- Method: ERL080/DS was soluble in test medium at the concentrations tested. Stock solutions were prepared in test medium and used to prepare lower concentrations. During preparation the solutions were protected from light.
- Controls: Test medium without test substance or other additives (blank).

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: CCAP 278/4
- Source (culture collection): not indicated
- Age of inoculum (at test initiation): 4 days
- Method of cultivation: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light (4000-9000 lux) in a climate room at a temperature of 23 ± 2°C.

ACCLIMATION
- Acclimation period: 4 days
- Culturing media and conditions: same as test
- Any deformed or abnormal cells observed: not reported

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Remarks on exposure duration:

A 24-hour period in the dark to optimise exposure to the original substance, followed by 72 hours exposure under continuous illumination.

Post exposure observation period:

no data

Test conditions

Hardness:

not indicated; the composition of the test water was according to the guideline

Test temperature:

22-23 °C

pH:

test start: 8.5
test end: 8.4-8.8

Dissolved oxygen:

not applicable

Nominal and measured concentrations:

nominal concentration: 0.0046, 0.010, 0.022, 0.046, 0.10 and 0.22 mg/l
measured at test start (0h): 127-137% of the expected nominal concentrations
measured after 24 h: 97-123% of nominal (75-96% of initially measured)
measured at test end: 35-74% of nominal
geometric mean: 66-87% of nominal
for details see Table 1

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: 100 mL, all glass
- Aeration: continuous shaking
- Initial cells density: 1 x 10E4 cells/mL
- Control end cells density: 123.4 x 10E4 cells/mL
- No. of replicates:
3 replicates of each test concentration,
6 replicates of the controls,
1 replicate without algae at the highest test concentration,
1 replicate of each test concentration with algae for sampling at t=24h.


GROWTH MEDIUM
- Standard medium used: yes


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ISO-medium (formulated according to ISO/IS 8692)
- Culture medium different from test medium: no
- Intervals of water quality measurement: pH was measured at the beginning and at the end of the test; temperature was measured daily


OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: Continuous illumination
- Light intensity and quality: 3500 - 4000 lux; TLD-Iamps of the type 'GroLux' of 30 Watt (Sylvania)


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : cell density at 0, 24, 48, 72 and 96 hours exposure
- Determination of cell concentrations: counting chamber (0 h), spectrophotometer (24-96 h)


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.2
- Range finding study
- Test concentrations: 0.1 to 100 mg/I (first test) and 0.001 to 1.0 mg/I (second test), both with a spacing factor of 10
- Results used to determine the conditions for the definitive study: Complete inhibition of algal growth was recorded at 1.0 mg/l, while far more than 50 % inhibition was recorded at 0.1 mg/l. No significant reduction of the extinctions was observed at 0.01 mg/1. Based on these results the EC50 for algal cell growth was expected to be between 0.01 and 0.1 mg/I.

Reference substance (positive control):

yes

Remarks:

: potassium dichromate, 0, 0.18, 0.32, 0.56, 1.0, 1.8, 3.2 mg/L

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

0.068 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL 0.027-0.173 mg/L; EC value calculated between 24 and 96 h

Effect concentrations 2

Duration:

96 h

Dose descriptor:

EC10

Effect conc.:

0.012 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL 0.005-0.030 mg/L; EC value calculated between 24 and 96 h

Effect concentrations 3

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

0.01 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 0.009 mg/L based on geometric mean

Effect concentrations 4

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

0.017 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% CL 0.013-0.022 mg/L; EC value calculated between 24 and 96 h

Effect concentrations 5

Duration:

96 h

Dose descriptor:

EC10

Effect conc.:

0.008 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% CL 0.006-0.010 mg/L; EC value calculated between 24 and 96 h

Effect concentrations 6

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

0.005 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: < LOD based on geometric mean

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): none reported
- Any stimulation of growth found in any treatment: no

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EbC50 (0-72 h): 0.75 mg/I with a 95 % confidence interval ranging from 0.51 to 1.1 mg/I (ISO-Standard range: 0.20 to 0.75 mg/I).
- ErC50 (0-72 h): 1.5 mg/I with a 95 % confidence interval ranging from 1.0 to 2.1 mg/I (ISO-Standard range: 0.60 to 1.03 mg/I. Although the value for the ErC50 slightly exceeded this range, the lower limit of the 95 % confidence interval was within this standard range.).

Reported statistics and error estimates:

The percentages of growth inhibition and/or the percentages of growth rate reduction were set out against the logarithms of the corresponding concentrations of the test substance. If a test concentration related effect was present, calculation of the EC-values was based on the respective curves corresponding to the various observation times using linear regression analysis.
The NOEC was based on statistical analysis of the data. Data obtained for the test concentrations were compared with those obtained in the negative control using the ANOVA-Tukey test in combination with the Williams' test (TOXSTAT Release 3.5, 1996, D.D. Gulley, A.M. Boelter, H.L. Bergman).
Regression line for EC-values for growth inhibition: Y=117.61X-93.63 (multiple R=0.9796, n=9)
Regression line for EC-values for growth rate reduction: Y=57.39X+7.28 (multiple R=0.9366, n=15)

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

: Criteria of ISO 8692 (1989), EU Method C.3 (1992) and OECD Guideline 201 (1984): Increase of the cell concentrations in the control cultures by a factor of at least 16 within three days.

Executive summary:

In a 96-h toxicity study, green algae (Scenedesmus subspicatus) were exposed to ERL080/DS in accordance with OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test) of 1984 and the corresponding EC and ISO Guidelines (from 1992 and 1989, respectively). The study was performed in compliance with GLP. Reliability grade 1 was assigned. The test period included a 24-hour period in the dark to optimise exposure to the original substance followed by 72 hours exposure under continuous illumination. Nominal concentrations of 0 (control), 0.0046, 0.010, 0.022, 0.046, 0.10 and 0.22 mg/l were tested. The toxicity endpoints were calculated based on geometric mean concentrations of < 0.014 (LOD), 0.009, 0.017, 0.03, 0.084, 0.149, 0.186 mg/l.

Inhibtion (growth rates):
ErC50 (24 - 96 h):	0.068 mg/l
95 % confidence limit:	0.027-0.173
NOErC ( 0-72 h): (5 % level)	0.009 mg/l (nominal 0.01 mg/l)
Inhibition (AUC):
EbC50 (24 - 96 h):	0.017 mg/l
95 % confidence limit:	0.013-0.022
NOEbC (0-72 h): (5 % level)	<LOD mg/l (nominal 0.0046 mg/l)