2,2-bis[[(2-hexyl-1-oxodecyl)oxy]methyl]-1,3-propanediyl bis(2-hexyldecanoate)

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

Semi-static test design

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 04549/MA
- Expiration date of the lot/batch: Feb 2018 (Retest date)
- Purity test date: Dec 2015

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: None
- Preliminary purification step (if any): No
- Final dilution of a dissolved solid, stock liquid or gel: Not applicable
- Final preparation of a solid: Not applicable

Analytical monitoring:

yes

Details on sampling:

Samples of 5 mL were taken from each additional vessel for TOC-analysis (without test organisms in order to not disturb the TOC-analysis) of the treatment and NC at 0 h, at 24 h before and after renewal of test solutions and at 48 h, and were subsequently measured using a TOC-analyser. Of each treatment, 2 of the 4 samples from 0 h, 24 h old, 24 h new and 48 h were analysed directly after sampling and 2 samples were stored as retain samples in the freezer at ≤ -18 °C until finalization of the study.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: WAF
The test item solutions (one for the test start and one for the renewal after 24 h) were prepared by adding 100 mg test item to 1000 mL M4 medium and shaking for 24 h using an overhead
shaker at 20.8 - 21.4 °C in the dark. The test item solutions were filtered through a fibre glass filter with a retaining range till 0.6 μm. The filter was prepared by rinsing with purified water and preconditioning with ca. 250 mL test solution, to reduce adsorption of the test item. The negative control (NC; M4 medium) was treated in the same way as the test item solution.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Waterflea (Daphnia magna Straus)
- Justification for species other than prescribed by test guideline: Not applicable
- Source: In-house culture (received from Federal Environment Agency, department IV 2.4, Berlin)
- Feeding during test: No

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Test temperature:

20.6 - 21.4 °C

pH:

7.6 - 7.9

Dissolved oxygen:

7.4 - 7.6 mg/L

Nominal and measured concentrations:

100 mg/l Loading rate

Details on test conditions:

TEST SYSTEM
- Test vessel: 50 mL glass beakers
- Fill volume: 40 mL
- Aeration: No
- Type of flow-through (e.g. peristaltic or proportional diluter): Not applicable
- Renewal rate of test solution (frequency): After 24h exposure (semi-static)
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): Not applicable
- Additional vessels for analytical measurements: 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Standard M4 medium
- Culture medium different from test medium: No
- Intervals of water quality measurement: Temperature continously; pH/O2 at 0h and 48h

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 h light : 8 h dark

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Immobility at 0h, 24h, 48h

VEHICLE CONTROL PERFORMED: no

RANGE-FINDING STUDY
A preliminary test without GLP was performed before start of this GLP-study. Nominal loading rates of 100, 200 and 400 mg/L test item were tested and resulted in a NOELR ≥ 400 mg/L.

Reference substance (positive control):

no

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate

Basis for effect:

mobility

Remarks on result:

other: limit test

Duration:

48 h

Dose descriptor:

LOELR

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate

Basis for effect:

mobility

Remarks on result:

other: limit test

Duration:

48 h

Dose descriptor:

LOELR

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate

Basis for effect:

mobility

Remarks on result:

other: limit test

Details on results:

- Mortality of control: 2 daphnids in control immobile after 48 h test period
- Other biological observations: In the test item treatment, some daphnids were trapped at the surface but still vital and mobile until the end of the test (48 h). As a fraction of the daphnids at the surface could dive beneath
the surface during the evaluation of mobility, this effect was not quantified. The phenomenon of animals trapped at the surface was not observed in the control.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Quantitative measurement of actual test concentration not possible, as measured concentrations were below LOQ. These measurements indicate that only a very small fraction of the test item was dissolved in the test medium. This result is in conformity with the test item properties, which state insolubility of the test item in water.
- Effect concentrations exceeding solubility of substance in test medium: Yes, therefore results given as effect loading rate.

Results with reference substance (positive control):

No

Reported statistics and error estimates:

As the immobility in the test item treatment (100 mg/L loading rate) was 15 % and showed no statistically significant difference to the control, the ELR50 is > 100 mg/L (24 h and 48 h). The effect loading rates after 24 h and 48 h exposure were calculated with the statistical software ToxRat Professional 3.2.1.

Immobility after 24 h and 48 h exposure

Nominal test item loading rate [mg/L]	Mobile Daphnia (replicates 1 - 4)												Immobility [%]
	0 h				24 h				48 h				24 h	48 h
	1	2	3	4	1	2	3	4	1	2	3	4
0 (Control)	5	5	5	5	5	5	5	5	5	5	5	3	0	10
100	5	5	5	5	5	5	5	5	4	3	5	5	0	15

Validity criteria fulfilled:

yes

Remarks:

Immobilisation of the test organisms in the control at the end of the test was ≤ 10 % and dissolved oxygen concentration in the control and the test item treatment at the end of the test was ≥ 3 mg/L.

Conclusions:

A semi-static Daphnia magna acute immobilisation test was performed using a single loading rate of 100 mg/L.The results indicate a NOELR (48h) ≥ 100 mg/l and hence, an ELR50 > 100 mg/L.

Executive summary:

The acute aquatic toxicity of 2,2-bis[[(2-hexyl-1-oxodecyl)oxy]methyl}-1,3-propanediyl bis(2-hexyldecanoate) was assessed in an OECD 202 - Daphnia study.

The semi-static Daphnia magna acute immobilisation test was performed under GLP by using a single loading rate of 100 mg/L (renewal of test solutions after 24 h).

The test item solutions were prepared by adding the test item to M4 medium and shaking for 24 h and following results were obtained:

Effect Loading Rate	Nominal test item loading rate [mg/L]
	24 h	48 h
ELR50	>100	>100
NOELR	≥ 100	≥ 100
LOELR	> 100	> 100

As the immobility in the test item treatment (100 mg/L loading rate) was 15 % and showed no statistically significant difference to the control, the ELR50 is > 100 mg/L (24 h and 48 h).

The measured TOC-concentrations in the test item treatment (100 mg/L loading rate) as well as in the control were below the limit of quantification and in the same range. These measurements indicate that only a very small fraction of the test item was dissolved in the test medium. This result is in conformity with the test item properties, which state the insolubility of the test item in water.

Description of key information

No toxic effects to aquatic invertebrates up to the limit of water solubility (< 0.05 mg/L).

Additional information

In addition to the study with the registered substance itself, resulting in no effects up to the limit of water solubility, two additional studies investigating the short-term toxicity to aquatic invertebrates of fatty acids, C16-18 and C18-unsatd., tetraesters with pentaerythritol (CAS 68604-44-4) are available (Wierich, 1998; Junghans, 2010).

The short-term toxicity to aquatic invertebrates (Wierich, 1998) was investigated under static conditions and GLP according to EU Method C.2 using Daphnia magna as test organism . The WAF was prepared by adding the appropriate amount of test substance with subsequent stirring and filtering. A nominal test concentration of 100 mg/L was tested, corresponding to < 10 mg/L (detection limit). No immobilization was observed in the treatment and the control throughout the test period of 48 h. Hence, the 48 h-EL50 is determined to be > 100 mg/L based on the nominal test concentration. This result is confirmed by another supporting study, conducted according to OECD 202 (Junghans, 2010). Also in this limit test an EL50 > 100 mg/L based on nominal values could be determined.

Based on these results, it can be concluded that 2,2-bis[[(2-hexyl-1-oxodecyl)oxy]methyl]-1,3-propanediyl bis(2-hexyldecanoate) (CAS 60623 -04 -3) will not exhibit short-term effects to aquatic invertebrates up to the limit of water solubility.