(4-amino-1-hydroxy-1-phosphonobutyl)phosphonic acid;hydrate

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:

2003-02-12 to 2003-06-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

sodium;(4-amino-1-hydroxy-1-phosphonobutyl)-hydroxyphosphinate

Cas Number:

121268-17-5

Molecular formula:

C4H12NaNO7P2 · 3 H2O

IUPAC Name:

sodium;(4-amino-1-hydroxy-1-phosphonobutyl)-hydroxyphosphinate

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: sponsor and Lot #005Y030

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test materials: At room temperature in the dark
- Solubility and stability of the test substance in the solvent/vehicle: 40 g active ingredient / L at 25 degrees Centrigrade

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: A stock solution of 10 mg active ingredient/L freshwater algal medium was prepared.
- Final dilution of a dissolved solid, stock liquid or gel: This stock solution was diluted down to concentrations of 0.041, 0.10, 0.26, 0.64, 1.6, 4.0, and 10 mg active ingredient / L.

FORM AS APPLIED IN THE TEST (if different from that of starting material)
0, 0.041, 0.10, 0.26, 0.64, 1.6, 4.0, and 10 mg active ingredient / L

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0.041, 0.10, 0.26, 0.64, 1.6, 4.0, and 10 mg active ingredient / L
- Sampling method: lgal growth was measured daily by direct cell counts using a hemacytometer counting chamber with a compound microscope. Morphological observations were also conducted on each test treatment using a compound microscope to detect abnormal cell morphology and coloration as compared to the controls.
- Sample storage conditions before analysis: Samples were analyzed immediately.

Test solutions

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Sterilized deionized water was enhanced with reagent-grade nutrients
- Controls: A negative vehicle control was used.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): water
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): This vehicle was the only solvent used in the test medium.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): None

Test organisms

Test organisms (species):

other: Selenastrum capricornutum

Details on test organisms:

TEST ORGANISM
- Common name: Green alga
- Strain: selenastrum capricornutum
- Source (laboratory, culture collection): The culture originated from an inoculum received from the Carolina Biological Supply Co., and has been maintained at Toxikon since January 4, 2002.
- Age of inoculum (at test initiation): 4 days old
- Method of cultivation: Cultured in freshwater algal medium (ASTM, 1990) under continuous illumination yielding approximately 120 micromols/m^2/s

ACCLIMATION
- Acclimation period: 4 days
- Culturing media and conditions (same as test or not): Same as test
- Any deformed or abnormal cells observed: None

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Remarks on exposure duration:

None

Post exposure observation period:

There was not a post exposure observation period.

Test conditions

Hardness:

Measurement not taken.

Test temperature:

21.3 to 21.9 degrees Centrigrade

pH:

7.1 to 7.6

Dissolved oxygen:

Measurement not taken.

Salinity:

Measurement not taken.

Conductivity:

Measurement not taken.

Nominal and measured concentrations:

Nominal Concetrations:
Preliminary Test:
Main Test: 0.041, 0.10, 0.26, 0.64, 1.6, 4.0, and 10 mg active ingredient / L

Measured Concentrations:
Not conducted as it is not required under guideline.

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Type (delete if not applicable): gas exchange caps
- Material, size, headspace, fill volume: Glass, 250 mL, 150 mL, 100 mL
- Aeration: Yes
- Initial cells density: 10,000 cells/mL
- Control end cells density:
- No. of organisms per vessel: 1,000,000 cells/vessel
- No. of vessels per concentration (replicates): 3
- No. of vessels per vehicle control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Sterilized deionized water enhanced with reagent-grade nutrients as described in ASTM (1990) and filtered through a 0.45 um membrane filter prior to use.
- Total organic carbon: <1.0 mg/L
- Particulate matter:
- Metals:
- Pesticides: <1.040 ug/L
- Chlorine: <1.0 mg/L
- Alkalinity:
- Ca/mg ratio: <1.0 mg/L
- Conductivity:
- Culture medium different from test medium: No
- Intervals of water quality measurement: Last collected February 12, 2003

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: To 7.5
- Photoperiod: Continuous
- Light intensity and quality: 94 to 116 umol/m^2/s

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: counting chamber
- Chlorophyll measurement: Not taken

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Geometric
- Range finding study
- Test concentrations: 0.010, 0.10, 1.0, 10 and 100 mg active ingredient/L
- Results used to determine the conditions for the definitive study:
After 72 hours of exposure, inhibition of algal growth in the range-finding test was -8.24% at 0.010 mg active ingredient/L, 16.9% at 0.10 mgactive ingredient/L, 29.2% at 1.0 mg active ingredient/L, 80.5% at 10' mg active ingredient/L, and 98.8% at 100 mg active ingredient/L

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

24 h

Dose descriptor:

EC50

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Remarks on result:

not determinable

Effect concentrations 2

Key result

Duration:

48 h

Dose descriptor:

EC50

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Remarks on result:

not determinable

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

ca. 5.5 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Effect concentrations 4

Key result

Duration:

24 h

Dose descriptor:

EC50

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

not determinable

Effect concentrations 5

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 10 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Effect concentrations 6

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 10 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Effect concentrations 7

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

ca. 4 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Details on results:

After 72 hours of exposure to ALENDRONATE SODIUM, the percentage inhibition of cell growth (based upon area under growth curve) compared to the control ranged from -3 percent (stimulation) at 0.10 mg active ingredient/L to 89 percent (inhibition) at 10 mg active ingredient/L. The growth curves of the controls exhibited a pattern of exponential growth during the 72-hour period. Cells at all testing concentrations appeared normal when compared to control cells for the duration of the study.

Results with reference substance (positive control):

No positive control was used during this test.

Reported statistics and error estimates:

EC50 values were calculated based on both biomass growth (comparison of areas under the growth curves) and on growth rates. EC50 values and 95 percent confidence limits were estimated by an EPA computer program (U.S. EPA, 1994) for calculating EC50 values by probit analysis.

In addition to the EC50 value, a no-observed-effect concentration (NOEC) was calculated using the statistical program ToxCalc (Version 5.01), with differences between cell density means determined by Dunnett's procedure. Statistical differences were determined at a probability level of 0.05.

For the 72 hour EC50 based on biomass inhibition, the 95% confidence interval was 3.9 to 8.6 mg active ingredient/L.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

This study validly assesed the test substance's potential toxicity to aquatic algae and cyanobacteria according to the specified guideline.