4,4,7,7-tetraethoxy-3,8-dioxa-4,7-disiladecane

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:

2016-04-08 to 2016-08-09

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Test was conducted using concentrations above the limit of solubility of the parent substance (predicted solubility is 20 mg/l – test concentrations range from 6.25-100 mg/l). Test material stirred for 24 hours prior to test start. Significant hydrolysis will have occured prior to test start. Analysis is by TOC. therefore it is not clear to which substance organisms were exposed. [Hydrolysis half-life: 36 h at pH 7, 0.8 h at pH 4, 0.7 h at pH 5 and 0.5 h at pH 9 and 20-25°C (QSAR)]

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

All concentration levels and the control were analytically verified via analysis of total organic carbon (TOC, according to DIN EN 1484) at the start of the exposure (fresh media, 0 hours) and at the end of the exposure (old media, 72 hours). Since adsorptive properties of the test item cannot be foreclosed, analysis of abiotic replicates is recommended to account for possibly binding to the algal biomass. Separate replicates for the test item analysis after 0 and 72 hours were prepared without algae and incubated under test conditions.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Five stock solutions in demineralized water (pH 7 ± 0.2) were prepared in a geometric series with a factor of 2: 6.25 - 12.5 - 25.0 - 50.0 - 100 mg/L. For preparation the density of the test item was taken into account. For each stock solution the test item was pipetted slowly and steadily under the surface of the water to avoid concentration peaks leading to polymerization or other adverse effects. The pH was measured and adjusted to pH 7 ± 0.2 by addition of 1M HCl. After 2 and 4 hours of stirring undissolved test item were observed. Therefore, the dispersions was stirred for 24 hours with approximately 700 rpm at room temperature. After stirring for 24 hours at room temperature the components of the dilution water were added to the stock solutions (13 mL/L). The pH was adjusted accordingly.

- Controls: Six replicates (without test item) were tested under the same test conditions as the test vessels.

- Evidence of undissolved material (e.g. precipitate, surface film, etc.): The test solutions were clear and the test item was dissolved throughout the test period.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Freshwater green alga
- Strain: HINDÁK, SAG 61.81
- Source (laboratory, culture collection): Sammlung von Algenkulturen (SAG), Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, D-37073 Göttingen.
- Age of inoculum (at test initiation): A four day old preculture, prepared in dilution water, was used as inoculum.

ACCLIMATION
- Acclimation period:
- Culturing media and conditions (same as test or not): No differences stated

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

Nominal range: 21 - 24 °C, controlled at ± 2 °C
Room temperature [°C]: min.: 22.5 max.: 23.5 mean value: 22.0

pH:

Start; 0 hours: 7.84 - 8.20
End; 72 hours: 7.82 - 8.23

Nominal and measured concentrations:

Nominal concentration (mg/L): 6.25, 12.5, 25.0, 50.0, 100

Details on test conditions:

TEST SYSTEM
- Test vessel: Sterile Erlenmeyer flasks, sealed with cotton wool plugs.
- Material, size, headspace, fill volume: 250 mL. Test volume 100 mL
- Initial cells density: 6305 cells/mL
- No. of vessels per concentration (replicates): Three
- No. of vessels per control (replicates): Six

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: According to the guidelines (OECD TG 201 medium)
- Total organic carbon: Not reported
- Particulate matter: Not reported
- Metals: Not reported
- Pesticides: Not reported
- Chlorine: Not reported
- Alkalinity: Not reported
- Ca/mg ratio: Not reported
- Conductivity: Not reported
- Culture medium different from test medium: No differences stated
- Intervals of water quality measurement: The pH-value at the start of the exposure was measured in one additional replicate of each test item concentration and the control. After 24 and 48 hours an aliquot of each test item concentration and the control was taken out of the test replicates for pH measurement (approximately 1-2 mL per replicate). The pH measurement at the end of the exposure was conducted in a pooled sample of each test item concentration and the control.

OTHER TEST CONDITIONS
- Sterile test conditions: No
- Adjustment of pH: Yes
- Agitation: Test containers were placed on a rotary shaker and oscillated at approximately 70 rpm.
- Photoperiod: 24 hours/day light
- Light intensity and quality: 60 - 120 µE · m-2 · s-1. Mean value: 71.9; range of the measured values: 64.0 to 77 µE · m-2 · s-1.


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Chlorophyll measurement: Chlorophyll-a-fluorescence. Cell density was measured daily via Chlorophyll-a-fluorescence, 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 of 100 mg/L.
- Other: Microscopic evaluation of the cells at the start and end of the exposure was carried out. The cells were checked for unusual cell shapes, colour differences, differences in chloroplast morphology, flocculation, adherence of algae to test containers and agglutination of algae cells.


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study
- Test concentrations: 10, 50, 100 mg/l, plus 50 mg/l with 100 µL/L THF as solvent
- Results used to determine the conditions for the definitive study: 52% inhibition at 100 mg/l. Preliminary testing with and without solvent (THF) with the test item showed that no differences in solubility occurred between the two different approaches. Biological effects were similar between test item with and without solvent. Since the maximum concentration of 100 mg/L is not achievable with the use of solvent due to dilution of the test item, the approach without solvent is used in the test.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate is tested twice a year as reference item

Key result

Duration:

72 h

Dose descriptor:

EC50

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:

EC10

Effect conc.:

92.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: (95% CL 27.1 – >100)

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

81.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Remarks on result:

other: (95% CL 76.9 – 86.0)

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

50.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Remarks on result:

other: (95%CL < 6.25 – 54.1)

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

57 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Remarks on result:

other: (95% CL 53.4 – 60.3)

Details on results:

All effect values are given based on nominal test item concentrations.
Microscopic evaluation of the cells at start and end of the incubation period revealed no morphological abnormalities.

Results with reference substance (positive control):

The toxicity of potassium dichromate (SIGMA, 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 2016-04-26 to 2016-04-29 (with headspace). The reference item toxicity is in the valid range following test facility SOPS.
ErC50 (Growth rate inhibition) 0.460 mg/l (nominal) (95%CI 0.443 – 0.481); Valid range (average ± 3 x SD) 0.780 ± 0.473
EyC50 (Yield inhibition) 0.317 mg/l (nominal) (95%CI 0.208 – 0.347); Valid range (average ± 3 x SD) 0.411 ± 0.276

Table1. Measurement of Total Organic Carbon

Nominal Concentration (mg/L)	TOC in mg C/L at Test start (without algae), 0 hours	TOC in mg C/L Test end (without algae), 72 hours	TOC in mg C/L Expected value*	Ratio test start to expected value [%]
100	42.3	32.5	47.5	89
50.0	22.5	12.7	23.8	93
25.0	11.7	4.58	11.8	99
12.5	6.57	4.04	5.94	110
6.25	3.71	2.43	2.97	125
Control	< 2.00	< 2.00	< 2.00	-

Limit of quantification = 2.00 mg C/L

* = Calculated with factor 0.475 carbon content

 

 

Table2. Test results

 

Concentration (mg /L)	Mean cell density after 72 hours (cells/mL)	Mean yield after 72 hours (cells/mL)	% inhibition of yield relative to control	Mean growth rate 0-72 hours	% inhibition of growth rate relative to control
100	690457	(+) 684152	68	(+) 1.56	19
50.0	1946933	(-) 1940628	9	(-) 1.91	2
25.0	1759770	(+) * 1753465	18	(+) * 1.88	3
12.5	1900269	(-) 1893964	11	(-) 1.90	2
6.25	2123327	(-) 2117022	1	(-) 1.94	0
Control	2134193	2127888		1.94

 

Statistically significant differences of growth rates and yield compared to control values are marked (+), not significant differences are marked (-)

Negative inhibition = growth stimulation

* = biologically not significant

^#= outlier

 

 

Validity criteria fulfilled:

yes

Conclusions:

72 hour ErC50 and ErC10 values for freshwater green alga Pseudokirchneriella subcapitata of > 100 mg/L (nominal) and 92.9 mg/l (95% confidence limits: 27.1 – >100) (nominal) were determined in a reliable study conducted according to an appropriate OECD test guideline, with acceptable restrictions. The restrictions were that the test was conducted using concentrations above the limit of solubility of the parent substance (predicted solubility is 20 mg/l – test concentrations range from 6.25-100 mg/l). Test material stirred for 24 hours prior to test start. Significant hydrolysis will have occured prior to test start. Analysis is by TOC. therefore it is not clear to which substance organisms were exposed. [Hydrolysis half-life: 36 h at pH 7, 0.8 h at pH 4, 0.7 h at pH 5 and 0.5 h at pH 9 and 20-25°C (QSAR)]

Description of key information

Short-term toxicity to algae: 72 hr ErC50 >100 mg/L (highest concentration tested) and 72 hr ErC10 92.9 mg/L (nominal) (OECD 201). The parent substance hydrolyses at a moderate rate in water but only non-specific analytical evidence is available. The observations in this study are attributed to the exposure of test organisms to the hydrolysis products in the test system. The ErC50 is equivalent to >53 mg/L and the ErC10 is equivalent to 49 mg/L when expressed in terms of the silanol hydrolysis product.

Key value for chemical safety assessment

EC50 for freshwater algae:

53 mg/L

EC10 or NOEC for freshwater algae:

49 mg/L

Additional information

A 72-h ErC50 value of >100 mg/L (highest concentration tested) and ErC10 of 92.9 mg/L (nominal) have been determined for the effects of the registration substance on growth rate of Pseudokirchneriella subcapitata. In view of the test media preparation method (24-h stirring of test media, at pH 7) and exposure regime (static at pH 7.8 - 8.2) it is likely that the test organisms were exposed predominantly to the hydrolysis products of the tested substance during the exposure period, although the test organisms would have been exposed to significant levels of parent substance during the early phase of the exposure period. The cell density measurements across the time points of the study suggest that the inhibition observed at the highest test concentration is most significant between the 48h and 72h time points. The nominal test concentrations are supported by DOC analysis which showed that the total dissolved organics in the test medium (parent substance plus hydrolysis products) were substantially reduced by the end of the exposure period. The results may be expressed in terms of concentration of the hydrolysis product, 1,1,1,4,4,4-hexahydroxy-1,4-silabutane, by applying a molecular weight correction: (MW of silanol=186.27 / MW of parent=354.59) * 100 = >53 mg/L.

In terms of parent substance, the geometric mean concentration over time, for the highest concentration tested, can be estimated as 32 mg/L. The EC50 in terms of the parent substance can therefore be estimated to be >32 mg/L. It is not appropriate to use this value directly in the chemical safety assessment but it is useful for comparison of sensitivity with the available short-term aquatic results.

Above a concentration of approximately 1000 mg/L, oligomeric, cyclic or cross-linked condensation products could potentially form over time in the aqueous test media. No evidence of undissolved material is indicated in the study report for this test and in view that the maximum concentration was 100 mg/L of test substance (equivalent to 53 mg/L as silanol hydrolysis product) condensation phenomena are not expected to be a major issue, although it was noted that DOC recoveries appeared to be variable which could suggest the test solutions did not remain homogenous throughout the exposure phase. The effects observed in the test are attributed to hydrolysis products in the test system. Although the predicted limit of solubility has apparently been exceeded, undissolved material was not observed and significant physical fouling is not anticipated in this concentration range.