3,9-dimethyl-2,4,8,10-tetraoxa-3,9-diphosphaspiro[5.5]undecane 3,9-dioxide

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:

from 2016-09-19 to 2016-09-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)

Version / remarks:

now called EPA Guideline 712-C-006: OCSPP 850.4500 (2012)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

3,9-dimethyl-2,4,8,10-tetraoxa-3,9-diphosphaspiro[5.5]undecane 3,9-dioxide

EC Number:

221-088-9

EC Name:

3,9-dimethyl-2,4,8,10-tetraoxa-3,9-diphosphaspiro[5.5]undecane 3,9-dioxide

Cas Number:

3001-98-7

Molecular formula:

C7H14O6P2

IUPAC Name:

3,9-dimethyl-2,4,8,10-tetraoxa-3λ⁵,9λ⁵-diphosphaspiro[5.5]undecane-3,9-dione

Test material form:

solid: particulate/powder

Details on test material:

AFLAMMIT® PCO 962

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Sampling method: The concentration of the test item and the control were determined at the start of the exposure (0 h) and daily during the exposure via LC-MS/MS. At the end of the exposure, additional samples without algae and the adsorption of the test item to algae cells were determined. Test medium containing algae cells was filtrated using glass fiber filter and the filter containing the algae cells was extracted with acetonitrile to verify adsorption of the test item on algae cells. No significant sorption to algae cells could be determined.
- Sample storage conditions before analysis: All original samples were stored at room temperature until preparation. Prepared samples were stored in an autosampler at room temperature until analysis.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 100 mg test item/L were freshly prepared with dilution water. Agitation until the solution was visually clear
- Controls: Dilution water without test item incubated under the same conditions as the limit test item concentration
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green alga
- Strain: SAG 61.81
- Source (laboratory, culture collection): Sammlung von Algenkulturen, Pflanzenphysiologisches Institut der Universität Göttingen, 37073 Göttingen, Germany.
- Age of inoculum (at test initiation): A three day old preculture, prepared in dilution water, was used as inoculum.
- Method of cultivation: Fresh stocks are prepared every month on Z-Agar. Light intensity amounted to 35-70 μE ·m-2 ·s-1 for 24 hours per day.

ACCLIMATION
- Acclimation period: not necessary
- Culturing media and conditions (same as test or not): similar
- Any deformed or abnormal cells observed: no

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Test conditions

Hardness:

not determined

Test temperature:

24.6-25.4°C

pH:

7.42-8.90

Dissolved oxygen:

not determined

Salinity:

not relevant

Conductivity:

not determined

Nominal and measured concentrations:

100 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Sterile Erlenmeyer flasks, volume: 250 ml, sealed with cotton wool plugs. The flasks were positioned randomly and repositioned daily. Test containers were placed on a rotary shaker and oscillated at approximately 100 rpm.
- Material, size, headspace, fill volume: Test volume 100 ml
- Aeration: no
- Initial cells density: Nominal: approximately 10000 cells/ml, actual: 10026 cells/ml
- Control end cells density: 5803471 cells/ml
- No. of vessels per concentration (replicates): eight
- No. of vessels per control (replicates): eight

GROWTH MEDIUM
- Standard medium used: yes (Threefold concentrated AAP medium according to the guidelines)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: demineralised water.
- Culture medium different from test medium: no
- Intervals of water quality measurement: Temperature measured continuously, pH at start and end of test.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: prior to testing with addition of 1 M NaOH and HCI
- Photoperiod: 24 hours/day light
- Light intensity and quality: 3655 – 4945 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Chlorophyll measurement: The cell density was measured daily via Chlorophyll-afluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as background signal. No self-fluorescence was found in the preliminary range finding test at the concentration level of 100 mg/L.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

96 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Key result

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

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

Results with reference substance (positive control):

- Results with reference substance are valid. The reference item toxicity is in the valid range following test facility SOPS.
- EC50: 0.460 mg/L, 72 h; valid range: 0.780 ± 0.473 (growth rate)

Reported statistics and error estimates:

EC-values of the growth rate and yield inhibition were estimated empirically based on the results of the only treatment level (Limit test design).
NOEC/LOEC were determined by calculation of statistical significance of growth rate and yield. As standard a t-test was used for NOEC/LOEC calculations. When running a t-test a Normality test and an Equal Variance test were done first. The SHAPIRO-WILK-Test was used to test for normally distributed populations. The Levene median test was used for equal variance. P-values for both Normality and Equal Variance tests are 0.05. The -value (acceptable probability of incorrectly concluding that there is a difference) is =0.05.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study AFLAMMIT® PCO 962 was found not to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata.