Tris[2-[[2,4,8,10-tetra-tert-butyldibenzo[d,f][1,3,2]dioxaphosphepin-6-yl]oxy]ethyl]amine

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Tris[2-[[2,4,8,10-tetra-tert-butyldibenzo[d,f][1,3,2]dioxaphosphepin-6-yl]oxy]ethyl]amine

EC Number:

279-459-6

EC Name:

Tris[2-[[2,4,8,10-tetra-tert-butyldibenzo[d,f][1,3,2]dioxaphosphepin-6-yl]oxy]ethyl]amine

Cas Number:

80410-33-9

Molecular formula:

C90H132NO9P3

IUPAC Name:

2-[(2,4,8,10-tetra-tert-butyldibenzo[d,f][1,3,2]dioxaphosphepin-6-yl)oxy]-N,N-bis{2-[(2,4,8,10-tetra-tert-butyldibenzo[d,f][1,3,2]dioxaphosphepin-6-yl)oxy]ethyl}ethanamine

Details on test material:

- Storage conditions: Room temperature
- Physical state/ color: Solid /white

Sampling and analysis

Analytical monitoring:

no

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Each test solution was prepared separately by directly adding test substance to test medium and stirring for about 2 days to ensure that the saturation concentration was reached. Loading concentrations: 0, 1.0, 3.2, 10, 32, 100, 320 mg/L

- Evidence of undissolved material (e.g. precipitate, surface film, etc.): At the end of the stirring period undissolved test substance was visible on the water surface, attached to the flask and dispersed in the aqueous phase in all test substance preparations. To avoid any physical effects of the undissolved fraction of the test substance on the test organisms and insure the testing of only the aqueous dissolved fraction of test substance, undissolved material was removed prior to exposure as recommended in OECD 23 to obtain water soluble fractions (WSF). Undissolved test substance was removed by filtration with a membrane filter (pore width 0.2 μm). The first 50-100 mL of filtered solution was discarded (used to condition the filter). The filtrates appeared colorless and clear. The test solution was filtered to remove undissolved substance. In a preliminary investigation the undissolved substance floated on the solution surface and could not be separated with centrifugation.

Test organisms

Test organisms (species):

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

Details on test organisms:

-Test species and strain: Pseudokirchneriella subcapitata KORSHIKOV (SAG 61.81) obtained from the SAG (Collection of algal cultures in Göttingen, Germany)
- Age (on study day 0): A stock algal culture is maintained continuously at the test facility. Before the exposure an inoculum culture is prepared from the stock culture and incubated for 4 days at 21 – 24 °C (max. temperature difference 2 °C). After this time, the inoculum culture is in exponential growth phase and can be used to initiate the test (study day 0).
- Supplier: Collection of algal cultures in University of Göttingen/Germany
- Inoculum (at test initiation): An inoculum culture in exponential growth phase was prepared with an aliquot of the stock algal culture added to sterile test media to provide an initial cell density of 0.5 x 104 cells/mL. The increase in biomass is verified to ensure that growth is within the normal range and algal cells are examined microscopically for normal morphology prior to use for test inoculation.
Inoculum culture cell density (4 days growth): 318 x 104 cells/mL Inoculum culture morphology: normal and healthy

INSERTION OF ORGANISMS:
The cell density of the inoculum culture in exponential growth phase was determined and then adjusted to 50 x 104 cells/mL To obtain the correct initial cell density in the test
replicates of 0.5 x 104 cells/mL and the correct nominal concentrations of the test solutions, inoculum culture was added to each test solution at a ratio of 1:100. The dilution by the addition of 1% inoculum was considered to be negligible for the evaluation.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

23.8-23.9°C

pH:

7.9 - 9.4

Nominal and measured concentrations:

0 (control), 1.0, 3.2, 10, 32, 100 and 320 mg/L as loading concentration based on test substance mass

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks (nominal volume 250 mL) plugged with gas permeable silicone sponge caps, 100 ml fill volume
- Initial cells density: 0.5 x 104 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
1 additional replicate per test group for initial concentration control analysis only. 1 additional uninoculated (without algae) replicate per test
group for background fluorescence correction and to determine the influence of the test design on test substance stability.


GROWTH MEDIUM
- Standard medium used: yes
The test medium (OECD medium) was prepared according to OECD Guideline for Testing of Chemicals, No. 201 Algal growth inhibition test, March 2006, Annex 3. The pH value of the test medium was 7.9 and was adjusted to 8.1 with 1 M NaOH. The test medium was sterilized after preparation.

OTHER TEST CONDITIONS
- Test chamber: controilled climate cabinet
- Adjustment of pH: yes for medium, no for test solution
- Photoperiod: 16:8
- Light intensity and quality: Average 7057 lux (within ±15% variability) at a wave length of 400 - 700 nm, vessels rearranged daily
- Shaking rate: continuously (85 rpm)
- Test parameter: Algal growth measured as in vivo chlorophyll-a fluorescence (pulsed excitation with light flashes having a wavelength of 430 nm)


TEST CONCENTRATIONS
The exposure was started after separation of the undissolved material by adding inoculum culture at a ratio of 1:100. The test solution after addition of algal inoculum is considered
equivalent to the nominal concentration since no more than 1% dilution occurs. The inoculated water soluble fractions of the test substance were distributed to the replicates
of each loading concentration, respectively. The pH of the test solutions was not adjusted after preparation or during the test.
- Range finding study
A slight effect was observed in a preliminary range-finding test with a loading concentration of 100 mg/L. The test concentrations were selected on the basis of a range finding test. The results of the 72 hour range finding test were: EfluorescenceC50 > 100 mg/L ; ErC50 > 100 mg/L
A 10% effect was observed at 100 mg/L therefore a full concentration range was performed including concentrations >100 mg/L in order to estimate the EC50. The effects in the range finding test might have been caused by an impurity. Therefore, the test solution was prepared following general guidance provided in OECD 23 in order to achieve saturated solutions of the test substance at different loading concentrations.
All of the test solutions were visibly clear and colorless after preparation. There was no visible undissolved test substance and no other remarkable observations through the end of exposure (72 h).


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
- Total organic carbon: 0.2-6.7mg/L, treatment groups comparable to control (no relevant quantities of soluble organic components from the test substance were present in the test solutions)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: The fluorescence of aliquots from each test vessel was measured using a Tecan Infinite 200Pro fluorometer in a 96-
well flat bottom black plate with the following parameters: fluorescence top reading; excitation / emission wavelength = 430 / 670 nm; excitation / emission bandwidth = 20 / 25 nm; flashes = 5; integration time = 20 μs; shaking duration = 15s; shaking amplitude = 6 mm. After the end of the exposure the control replicates were mixed and serially diluted by factor 2. The fluorescence of aliquots from the undiluted mixture and the dilutions were measured and in parallel cell density was determined by a direct microscopic count (two counts in a Neubauer haemocytometer). These data were used to derive a linear correlation between fluorescence and cell density. 0, 24, 48, 72h after exposure
- Algal morphology: The inoculum culture was observed microscopically at the start of the test to verify normal and healthy cells. At test termination, a pooled sample from each test concentration was examined and any abnormal appearance of the algae noted.
- Temperature: continuous measurement
- Light: Light homogeneity was evaluated by measuring light intensity at 5 locations within the incubation area at the start and end of the test. The light intensity did not vary by more than ± 15% over the incubation area.
- TOC: start and the end of exposure

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 320 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: based on loading concentration used for preparation of the water soluble fraction

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

> 320 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: based on loading concentration used for preparation of the water soluble fraction

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

>= 320 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: based on loading concentration used for preparation of the water soluble fraction

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 320 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: based on loading concentration used for preparation of the water soluble fraction

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

> 320 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: based on loading concentration used for preparation of the water soluble fraction

Effect concentrations 6

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

>= 320 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: based on loading concentration used for preparation of the water soluble fraction

Details on results:

The test substance had no inhibitory effect on algal yield or growth rate after 72-h exposure when tested at the limit of solubility under test conditions.
Algal Morphology: no remarkable observations

All tested loading concentrations were more than factor 10 above the limit of solubility of the test substance in water.
Yield and growth rate of algae in the lowest and the highest loading concentration were not statistically significantly different from the control group. Thus, the observed deviations shown in the section below were not an intrinsic effect of the dissolved fraction of the test substance. All deviations from the control were slight and in a range, which is within normal variability. Any variability attributable to inoculation, like slightly different volumes of inoculum or cell counts at start of exposure, was lower within the replicates of a test group than between replicates of different test groups. In conclusion, the observed statistically significant deviations might have been artifacts of the testing procedure.

This test was fully compliant with all the following validity criteria required by the corresponding test guidelines and is considered valid.
• The validity criterion for cell multiplication factor in the untreated control is > 16 in 72 hours. The cell multiplication factor in the untreated control (all replicates mixed together) was 368-fold after 72 hours
• The validity criterion for the mean coefficient of variation for section-by-section growth rates for each test day in the control cultures is ≤35%. The mean coefficient of variation for
section-by-section growth rates for each test day in the control cultures was 11.1%.
• The validity criterion for the coefficient of variation of average specific growth rates during the whole test period in replicate control cultures is ≤7%. The coefficient of variation of
average specific growth rates during the whole test period in replicate control cultures was 1.9%.

Results with reference substance (positive control):

The ErC50 (72 h) of the control substance potassium dichromate was 1.01 mg/L (Date of the last control experiment: 23 Oct 2017, project number: 60E0063/04E013)
The results from the reference substance test are compared to potassium dichromate EC50 values published in ISO test guideline 8692, which represent the typical response range for the algal species tested. According to the test ISO guideline 8692 the EC50 values of the reference substance potassium
dichromate should be in the range: ErC50 = 0.92 – 1.46 mg/L after 72 hours for Pseudokirchneriella subcapitata.
These results indicate that the algae are responding normally to toxicant stress.

Any other information on results incl. tables

Test Group  (loading concentration) [mg/L]	Mean Cell Density (cells/mL)x 10^4 at 72 hours	Mean yield at 72 hours	Mean specific growth rate at 72 hours
0 (control)	115	114	1.97
1.0	109	109	1.97
3.2	82.9	82.6*	1.89
10	85.6	85.2*	1.85*
32	87.5	87.2*	1.88
100	84.7	84.4*	1.89
320	112	112	1.99

Numbers with * are statistically significantly different than Control *p≤0.05

Even though these values are identified as statistically significant, they are not considered toxicologically significant and were not considered for evaluation of the point estimates. The weight of evidence supports this conclusion since no effect on growth or yield is observed in the highest test group (320 mg/L loading) and since the inhibition did generally not follow a concentration effect relationship.

The OECD test guideline 201 states that the use of average specific growth rate for estimating toxicity is scientifically preferred. Therefore, the observed reductions in algal yield in this study are not cause for concern and the results should be interpreted based on algal growth.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes