5,7-Di-t-butyl-3-[3,5-dimethyl-4-[(1,3,7,9-tetra-t-butyl-5-methyl-5H-benzo[d][1,3,2]benzodioxaphosphocin-11-yl)oxy]phenyl]-3H-benzofuran-2-one

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:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

The limit concentration and the control were analytically verified via LC-MS/MS at the start of the exposure (0 hours). Care was taken to only analyze dissolved test item: The samples were filtered to remove all undissolved test item (membrane filter 0.45 μm, RC, MACHEREY-NAGEL). Since the measured concentration was below the lowest calibration level at the start of the exposure (0 hours), analytical monitoring at the end of the exposure was waived and the test item concentrations was considered below the lowest calibration level in the old media, too. No method validation was performed either.

Vehicle:

no

Details on test solutions:

Stock solution, media preparation
The limit concentration 100 mg/l (dispersion) was freshly prepared with dilution water directly in the test vessels. Since the test item is sparingly soluble with a water solubility limit very likely below the analytical detection limit, a worst case approach was applied to ensure that the solubility limit under test conditions was maintained during the exposure period. Test item in excess of the water solubility limit was present in the test vessels throughout the exposure period. To prepare the test item dispersions, 10 mg of the test item were weighed into each test vessel and 100 ml of the dilution water were added. These dispersions were stirred with a magnetic stirrer at approximately 750 rpm for 72 hours at room temperature. The test solutions were filtrated.

Test concentrations
One limit concentration of 100 mg/l was tested. The concentration level was based on the results of a preliminary range finding test (non-GLP), which demonstrated that the undissolved part of the test item had no adverse effects on the test organisms.

Test organisms (species):

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

Details on test organisms:

Test organism
Pseudokirchneriella subcapitata HINDAK, SAG 61.81

Synonyms
Selenastrum capricomutum; Ankistrodesmus subcapitata; Raphidocelis subcapitata; Ankistrodesmus bibraianus (Experimental Phycology and Culture Collection of Algae at the University of Goettingen 2014)

Reason for the selection of the test organism
Pseudokirchneriella subcapitata is a suitable green alga species according to the guideline.

Origin
Sammlung von Algenkulturen (SAG) Pflanzenphysiologisches lnstitut der Universität Göttingen
Nikolausberger Weg 18, D-37073 Göttingen

Cultivation at test facility
Fresh stocks are prepared every month on Z-Agar. Light intensity amounted to 2590 - 5180 lux (35 - 70 μE · m^-2 · s^-1) for 24 hours per day.

Culture medium
Nutrient medium Z according to L0TTGE et al. (1994)

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Hardness:

n/a

Test temperature:

22 - 23.5 deg. C

pH:

Control: 8.09 - 9.13
100 mg/L: 7094 - 9.22

Dissolved oxygen:

n/a

Salinity:

n/a

Conductivity:

n/a

Nominal and measured concentrations:

Nominal: 100 mg/L
Measured: < calibration (calibration range 1 - 25 µg/L)

Details on test conditions:

Stock solution, media preparation
The limit concentration 100 mg/l (dispersion) was freshly prepared with dilution water directly in the test vessels. Since the test item is sparingly soluble with a water solubility limit very likely below the analytical detection limit, a worst case approach was applied to ensure that the solubility limit under test conditions was maintained during the exposure period. Test item in excess of the water solubility limit was present in the test vessels throughout the exposure period. To prepare the test item dispersions, 10 mg of the test item were weighed into each test vessel and 100 ml of the dilution water were added. These dispersions were stirred with a magnetic stirrer at approximately 750 rpm for 72 hours at room temperature. The test solutions were filtrated.

Test concentrations
One limit concentration of 100 mg/l was tested. The concentration level was based on the results of a preliminary range finding test (non-GLP), which demonstrated that the undissolved part of the test item had no adverse effects on the test organisms.

Control
Six replicates (without test item) were tested under the same test conditions as the test item replicates.

Reference Item
Potassium dichromate is tested twice a year as a reference

Test method
Static procedure

Duration of the test
72 hours

Replicates
Six replicates for the limit concentration and the control

Test container
Sterile Erlenmeyer flasks, volume: 250 ml, sealed with cotton wool plugs

Test volume
100 ml
Preculture
A three day old preculture, prepared in dilution water, was used as inoculum.

Initial cell density
Nominal: approximately 5 x 103 – 104 cells/ml
Actual: 6839 cells/ml

Application
Application was carried out by adding appropriate volumes of the stock solution to the replicate test vessels. The inoculum was added separately to each replicate.

Incubation
The flasks were positioned randomly and repositioned daily.

Temperature
Nominal range: 21 - 24 °C, controlled at ± 2 °C

Agitation
Test containers were placed on a rotary shaker and oscillated at approximately 70 rpm.

Light intensity (target)
Approximately 4440 to 8880 lux, corresponding to 60 to 120 μE*m-2*s-1

Light regime
24 hours/day light

Light homogeneity (target)
Within ± 15% over incubation area

Reference substance (positive control):

yes

Remarks:

potassium dichromate (SIGMA ALDRICH, batch number MKBV0900V, purity 99.0 %, CAS RN 7778-50-9)

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL20

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

The toxicity of the test item to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 266/2016/Method C.3 from 2017-06-16 to 2017-06-22, with the definitive exposure phase from 2017-06-19 to 2017-06-22 at the test facility. The aim of the study was to assess the effects on growth rate and yield over a period of 72 hours. The study was conducted under static conditions with an initial cell density of 6839 cells/ml. The limit concentration 100 mg/L was freshly prepared with dilution water directly in the test vessels. Since the test item is sparingly soluble with a water solubility limit very likely below the analytical detection limit, a worst case approach was applied to ensure that the solubility limit under test conditions was maintained during the exposure period. Test item in excess of the water solubility limit was present in the test vessels throughout the exposure period. To prepare the test dispersions, 10 mg of the test item were weighed into each test vessel and 100 ml of the dilution water were added. These dispersions were stirred with a magnetic stirrer at approximately 750 rpm for 72 hours at room temperature. The test solutions were not filtrated. Six replicates were used for the limit concentration and the control. The environmental conditions were within the acceptable limits. The test media were clear and test item was observed on the surface of water throughout the test period. The limit concentration and the control were analytically verified by LC-MS/MS at the start of the exposure. To only analyse dissolved test item, the samples were filtrated to remove all undissolved test item (membrane filter 0.45 μm, RC, MACHEREY-NAGEL). In consequence of the low water solubility the measured concentration of the test item at the start of the exposure (0 hours) was below the lowest calibration level (LCL) of the nominal value. Therefore, the test item concentrations can be considered to be lower than the calibration range at the end of the exposure, too.

Results with reference substance (positive control):

The toxicity of potassium dichromate (SIGMA ALDRICH, batch number MKBV0900V, purity 99.0 %, CAS RN 7778-50-9) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined over a period of 72 hours from 2017-04-18 to 2017-04-21. The reference item toxicity is in the valid range following test facility SOPs.

Validity criteria fulfilled:

yes

Conclusions:

The validity criteria of the OECD TG 201 are fulfilled. The study is valid.

Description of key information

There is a high probability that the product is not acutely harmful to aquatic algae and cyanobacteria.

Additional information

The toxicity to aquatic algae and cyanobacteria was determined in a static toxicity study according to OECD TG 201 under consideration of GLP. The test was conducted was limit test with one concentration group of 100 mg/L. The concentration level was based on the results of a preliminary range finding test, which demonstrated that the undissolbed part of the test item had no adverse effects on the test organisms. After an exposure time of 72h the EC10 and EC50 were determined as >100 mg/L. No toxic effects could be recorded.