Betamethasone

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:

2003-2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study following Testing guideline.

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Reference 2

Reference Type:

secondary source

Title:

The Selenastrum capricornutum Printz Algal Assay Bottle Test

Author:

Miller, W.E.; Greene, J.C.; Shiroyama, T.

Year:

1978

Bibliographic source:

U.S. Environmental Protection Agency Publication EPA-600/9-78-018, Corvallis, OR.

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Betamethasone

EC Number:

206-825-4

EC Name:

Betamethasone

Cas Number:

378-44-9

Molecular formula:

C22H29FO5

IUPAC Name:

9-fluoro-11,17,21-trihydroxy-16-methylpregna-1,4-diene-3,20-dione

Details on test material:

- Name of test material (as cited in study report): Betamethasone
- Substance type: Organic
- Physical state: Off-white crystalline powder
- Lot/batch No.: H032754
- Stability under test conditions: The concentration of Betamethasone was measured at 0 and 48 hours by HPLC method
- Storage condition of test material: The sample was stored at ambient temperature, in the container in which it was received, until required for testing
- Other: The substance was received and assigned the Brixham test substance number 03-0210

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
PHYSICO-CHEMICAL PROPERTIES
- Melting point:
- Boiling point:
- Vapour pressure:
- Water solubility (under test conditions):20 mg/l was selected as highest test concentration
- Henry's law constant:
- log Pow:
- pKa:
- Stability in water:
- Stability in light:
- pH dependance on stability:

OTHER PROPERTIES (if relevant for this endpoint)
- Results of test for ready biodegradability:
- Other:

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: nominal concentration 1.3, 2.5, 5.0, 10, and 20 mg/l
- Sampling method:
- Sample storage conditions before analysis: The test solution was prepared in the sterile culture medium at 24°C, centrifuged, and directly used for the experiment

Test solutions

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A 2000 ml volume of the highest nominal test concentration (20 mg/l) was prepared by direc addition of betamethasone to sterile culture medium. this stock solution was given 5 minutes ultrasonic treatment and stirred overnight using PTFE coated magnetic stirring bar.
- Eluate: Prepared culture medium for Alga Selenastrum capricornutum (ATCC 22662)
- Differential loading:
- Controls:
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Culture medium based on that described by Miller et al. (Reference)
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): Stock solution 20 mg/l; Final test concentrations: 1.3, 2.5, 5.0, 10, and 20 mg/l
- Evidence of undissolved material (e.g. precipitate, surface film, etc):

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Selenastrum capricornutum
- Strain: ATCC 22662
- Source (laboratory, culture collection): laboratory cultures mantained under axenic conditions
- Age of inoculum (at test initiation): 4 day old culture in the exponential growth phase
- Method of cultivation: growth in the medium

ACCLIMATION
- Acclimation period:
- Culturing media and conditions (same as test or not):
- Any deformed or abnormal cells observed:

Study design

Test type:

other: shaken

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

Assessment was made at 24, 48, and 72 h after the commencement of the test

Test conditions

Test temperature:

The hourly temperature measurements remained within the range 24 ± 2°C for all the 72 hours

pH:

At the start of the test the pH ranged from 7.30 to 7.39, and at the end of the test was in the range from 10.18 to 10.64.

Nominal and measured concentrations:

Nominal concentrations of 1.3, 2.5, 5.0, 10, and 20 mg/l were analyses by HPLC at the start of the test and at 72 hour in the culture medium pre- and post-centrifugation.

Details on test conditions:

TEST SYSTEM
- Test vessel: Borosilicate glass conical flasks of 250 ml nominal capacity closed with polyuretanefoam bungs.
- Type: closed
- Material, size, headspace, fill volume: Each flask contained 100 ml of the test solution.
- Aeration:
- Type of flow-through (e.g. peristaltic or proportional diluter):
- Renewal rate of test solution (frequency/flow rate):
- Initial cells density: Measured by Coulter counter model Z1, as 10600 cells/ml
- Control end cells density:
- No. of organisms per vessel: 10600 cells/ml
- No. of vessels per concentration (replicates): 3 vessels for each concentration
- No. of vessels per control (replicates): 6 vessels
- No. of vessels per vehicle control (replicates):

GROWTH MEDIUM
- Standard medium used: yes, as reported in the EPA solution description
- Detailed composition if non-standard medium was used:

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: prepared from sterile starting materials
- Total organic carbon:
- Particulate matter:
- Metals:
- Pesticides:
- Chlorine:
- Alkalinity:
- Ca/mg ratio:
- Conductivity:
- Culture medium different from test medium:
- Intervals of water quality measurement:

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH:
- Photoperiod: Cultures were incubated under continuous "cool-white" illumination
- Light intensity and quality: 6880 lux (by cosine receptor), also measured as 90.06 μ Einsteins∙m-2∙s-1
- Salinity (for marine algae):

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: electronic particle counter
- Chlorophyll measurement:
- Other:

TEST CONCENTRATIONS
- Spacing factor for test concentrations:
- Justification for using less concentrations than requested by guideline:
- Range finding study
- Test concentrations: 1.3, 2.5, 5.0, 10, and 20 mg/l
- Results used to determine the conditions for the definitive study:

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

other: ErC50

Effect conc.:

> 20 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

based on logarithmic growth rate over the test period

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

20 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

based on logarithmic growth rate over the test period

Remarks on result:

other: P=0.05

Effect concentrations 3

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 20 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

based on logarithmic growth rate over the test period

Remarks on result:

other: P=0.05

Effect concentrations 4

Duration:

72 h

Dose descriptor:

other: EbC50

Effect conc.:

> 20 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

based on areas under the growth curve

Effect concentrations 5

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

20 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

based on areas under the growth curve

Remarks on result:

other: P=0.05

Effect concentrations 6

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 20 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

based on areas under the growth curve

Remarks on result:

other: P=0.05

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no abnormality observed
- Unusual cell shape: no abnormality observed
- Colour differences: no abnormality observed
- Flocculation: no abnormality observed
- Adherence to test vessels: no abnormality observed
- Aggregation of algal cells: no abnormality observed
- Other:
- Any stimulation of growth found in any treatment:
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values:
- Effect concentrations exceeding solubility of substance in test medium:

Any other information on results incl. tables

ALGAL CELL DENSITIES

Nominal concentration of betamethasone    (mg/l)	Replicate	Algal cell density      (x 10^4 cells/ml)
		24 hours	48 hours	72 hours
Culture medium control	A B C D E F	7.23 7.16 6.74 7.54 6.77 6.03	58.5 61.8 60.1 62.9 57.0 49.1	309 295 306 320 234 228
	Mean	6.91	58.2	282
1.3	A B C	6.80 6.61 7.34	56.5 59.4 63.4	246 296 274
	Mean	6.92	59.8	272
2.5	A B C	7.82 7.72 7.54	63.6 63.2 62.2	339 312 245
	Mean	7.69	63.0	299
5.0	A B C	7.10 7.83 7.27	61.3 71.6 62.8	296 338 272
	Mean	7.40	65.2	302
10	A B C	7.30 7.25 7.71	57.3 60.3 62.8	314 309 318
	Mean	7.42	60.1	314
20	A B C	6.77 7.07 6.92	57.6 62.4 58.7	279 270 296
	Mean	6.92	59.6	282

MEAN AREA UNDER THE GROWTH CURVE

Nominal concentration of betamethasone(mg/l)	Mean area under growth curve (0 -72 hours) (P=0.05)	Percentage of culture medium control
Culture medium control	203	-
1.3	200	98.3
2.5	217	107
5.0	221	109
10	222	109
20	205	101

MEAN GROWTH RATES OVER THE TEST PERIOD

Nominal concentration of betamethasone(mg/l)	Mean growth rate (0 -72 hours) (P=0.05)	Percentage of culture medium control
Culture medium control	1.86	-
1.3	1.85	99.4
2.5	1.88	101
5.0	1.88	101
10	1.90	102
20	1.86	100

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

As less than 50% inhibition was observed over the range of concentrations tested, the 72 hour EbC50 is considered to be greater than the highest concentration tested (20 mg/l).
As less than 50% inhibition was observed over the range of concentrations tested, the 72 hour ErC50 is considered to be greater than the highest concentration tested (20 mg/l).