Reaction mass of (9-acetoxy-3,8,10-triethyl-7,8,10-trimethyl-1,5-dioxa-9-azaspiro[5.5]undec-3-yl)methyl palmitate and (9-acetoxy-3,8,10-triethyl-7,8,10-trimethyl-1,5-dioxa-9-azaspiro[5.5]undec-3-yl)methyl stearate

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:

2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, 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:

The degradation products of the test item were analytically determined. The test item could not be analyzed due to the low solubility and the fast
degradation of the test substance. Thus, the resulting degradation products will be more relevant for any toxic effects on the test organisms under
natural conditions.

Just before the start of the test:
- duplicate samples from each test medium (without algae)
- duplicate samples from the control (without algae)

After 72 hours:
- duplicate samples from each test medium (without algae)
(stability samples) - duplicate samples from the control (without algae)

For the 72-hour stability samples additional flasks with adequate volumes of the freshly prepared test media of all test concentrations and the
control were incubated under the same conditions as in the actual test but without algae. Sampling from the test beakers with algae was not
possible because the test media volumes were too small for the analytical requirements.

The concentration of the degradation products was analyzed in all test medium samples from the first sampling time (0 hours). At the end of the
test (72 hours) only the samples of the undiluted filtrate were analyzed. The 72-hour samples of the dilutions 1:2, 1:4, 1:8 and 1:16 were not
analyzed, since these concentrations were below the 72-hour NOEC determined in this test. From the control samples only one of the duplicate
samples was analyzed from each of both sampling times. The samples were analyzed immediately after sampling without prior storage.

Vehicle:

no

Details on test solutions:

According to the results of a pre-experiment (without GLP) the test item was not soluble at a concentration of 100 mg/L in test water and no
homogeneous dispersion could be prepared.

A filtrate of a supersaturated dispersion of the test item and dilutions of the filtrate were prepared. The undiluted filtrate and the dilutions 1:2, 1:4, 1:8 and 1:16 were tested. Additionally, a control was tested in parallel (test water without test item).

A supersaturated dispersion with a nominal concentration of 100 mg/L (= loading rate) was prepared by weighing 449 mg of the test item into 4.5 liters test water. No auxiliary solvent or emulsifier was used. The test item was mixed into the test water as homogeneously as possible by ultrasonic
treatment for 15 minutes and by intense stirring for 96 hours at room temperature in the dark to dissolve a maximum concentration of the test item
in the dispersion. The long stirring period of 96 hours was chosen because the concentration of dissolved degradation product continuously
increased over a stirring time of about 96 hours (results of a pre-test; without GLP).

The supersaturated dispersion of the test item was.filtered through a membrane filter (Schleicher & Schuell, Type NC45, pore size 0.45 microm) just
before the preparation of the test media. The undiluted filtrate of the supersaturated dispersion with the maximum concentration of dissolved
degradation product was used as the highest concentrated test medium. Additionally, adequate volumes of the filtrate were diluted with test water for the preparation of the test media with lower test item concentrations. No additional dilution step was inserted.

The test media were prepared just before addition of algae (= start of the test).

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: gree algae
- Strain: No. 85.81 SAG
- Source (laboratory, culture collection): Collection of Algal cultures (SAG, Insitute for Plant Physiology, University of Goettingen, D-37073 Goettingen, Germany)

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

0.24 mmol/L (= 24 mg/L) as CaCO3

Test temperature:

22 to 23 deg. C

Nominal and measured concentrations:

100 mg/L (loading rate)

Details on test conditions:

The algae were cultivated and tested in synthetic test water, prepared according to the test guidelines. Analytical grade salts were dissolved in sterile purified water to obtain the following final nominal concentrations:

Macro-nutrients:
NaHCO2 50.0 mg/L
CaCl2x2H20 18.0 mg/L
NH4CI 15.0 mg/L
MgSO4x7H2O 15.0 mg/L
MgCl2x6H2O 12.0 mg/L
KH2PO4 1.6 mg/L

Trace elements:
Na2EDTAx2H2O 100.0 microg/L
FeCU X 6 H2O 80.0 microg/L
MnCl2x4H20 415.0 microg/L
H3BO3 185.0 microg/L
Na2Mo04 X 2 H2O 7.0 microg/L
ZnCl2 3.0 microg/L
COCI2X6H2O 1.5 microg/L
CUCI2 X 2 H2O 0.01 microg/L

Calculated water hardness of the test water: 0.24 mmol/L (= 24 mg/L) as CaCO3. The test was started (0 hours) by inoculation of 10,000 algal cells per mL of test medium. These cells were taken from an exponentially growing pre-culture, which was set up three days prior to the test under the same conditions as in the test.

The test design included three replicates per test concentration and six replicates of the control. Volumes of 15 mL algal suspension for each
replicate were continuously stirred by magnetic stirrers in 50 mL Erlenmeyer flasks.

The flasks were covered with glass dishes. They were incubated in a temperature controlled water bath at a temperature between 22 and 23 °C, and continuously illuminated at a measured light intensity of about 8800 Lux (mean value), range: 7910 to 9500 Lux (minimum and maximum value of measurements at nine places distributed over the experimental area at the surface of the test media). This illumination was achieved by fluorescent tubes
(Philips TLD 36W-1/840), installed above the test flasks. The test vessels were labeled with the study number and all necessary additional information to assure unmistakable identification.

The test duration was 72 hours.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 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:

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

In conclusion, the test substance and its degradation products had no toxic effect on the algae up to their solubility limit in test water under the present test conditions.

Description of key information

With high probability acutely not harmful to aquatic organisms. No toxic effects were observed within the range of solubility.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

According to regulation (EC) No 1907/2006, Annex XI, paragraph 1.5., substances whose physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity may be considered as a group, or "category" of substances. Environmental effects or environmental fate may be predicted from data for reference substance(s) within the group by interpolation to other substances in the group (read-across approach). The substance consists of two main components, a component with a C18 chain (60%) and C16 chain (30%) component. The structure of both substances is the same except the chain length.

The substance with C18 is already notified under EC 445-990-9. It is expected that the structural difference between the octadecanoate and the hexadecanoate does not affect the physico-chemical properties, the toxicological and ecotoxicologocal profile as well as the environmental fate. Furthermore, it can be assumed that the degradation products of EC 445-990-9 are very similar to the degradation products of the mixture containing the octadecanoate and the hexadecanoate. Therefore read-across to EC 445-990-9 is scientifically justified for physico-chemical properties, the toxicological and ecotoxicologocal profile as well as environmental fate.