Isohexadecyl 12-[(1-oxooctadecyl)oxy]octadecanoate

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 Feb - 10 Jun 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Version / remarks:

Oct 2012

Deviations:

no

Remarks:

only minor deviations without effects on the integrity of the study

GLP compliance:

yes (incl. QA statement)

Remarks:

Secrétariat général du GIPC-DGE-SI, Ivry-sur-Seine, CEDEX, France (24 Apr 2017)

Analytical monitoring:

yes

Remarks:

LC-MS

Details on sampling:

- Concentrations: Limit concentration (old and new medium)
- Sampling method: 40 mL was sampled from each preparation at test medium renewal

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test item was applied as water saturated solutions. A newly saturated solution was used at each renewal of the treatment solutions. The saturation was obtained by coating approximately 1 g of quartz sand with 20 µL test item, which was then poured into a 50 mL burette and filled with M4 mineral water and allowed to stand undisturbed for approximately 1 h. The flow through pf the column was set at approximately 1 mL/min and a total volume of approximately 500 mL was collected and distributed as 40 mL aliquot volumes. The same coated quartz sand portion was used for the entire test period and successive 500 mL water saturated volumes, but each volume of saturated solution was prepared with freshly prepared M4 mineral medium.
- Controls: The control units received 40 mL of M4 mineral medium. In this case the mineral medium was used as neat, without passing through untreated quartz sand.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Planktonic crustacean
- Source: Commercial breeder and regularly sub-cultured at the testing facility in M4 mineral medium
- Age of daphnids at test start: < 24 h (not first brood, derived from a healthy stock)
- Breeding conditions: Similar to test (20 ± 2.0 °C), 16 h light (15 - 20 µE*m^-2*s^-1), 8 h dark cycle. Adult females were transferred to fresh water on the day before test initiation.
- Feeding during test: Yes
- Food type: Algal cells (Chlorella sp.)
- Amount: 0.1 - 0.2 mg Carbon/Daphnia/day
- Frequency: Three times per week or according to media changes

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

21 d

Test temperature:

20.0 ± 2.0 °C

pH:

6.8 - 8.1 (control)
6.9 7.9 (treatment)

Dissolved oxygen:

6.5 - 9.4 mg/L (control)
6.3 - 8.9 mg/L (treatment)

Nominal and measured concentrations:

Control and 7.0 µg/L (nominal)
Control and 5.5 - 8.1 µg/L (measured values in old medium)
Control and 5.9 - 8.8 µg/L (measured in new medium)

Details on test conditions:

TEST SYSTEM
- Test vessel: 60 mL EPA glass tubes filled with 40 mL test medium
- Renewal rate of test solution: 2-3 d, i.e. on Day 0, 3, 5, 7, 10, 12, 14, 17, 19, and 21
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: M4 mineral medium was freshly prepared from pure water and mineral stock solutions
- Culture medium different from test medium: Culture medium same as test medium
- Intervals of water quality measurement: Day 0, 3, 10, 17 and 21

OTHER TEST CONDITIONS
- Photoperiod: 16 h light, 8 h dark
- Light intensity: 15 - 20 µE*m^-2*s^-1

EFFECT PARAMETERS MEASURED:
- Offspring: 1-2 d intervals
- Mortality
- Time to production of first brood
- Number of aborted broods
- Presence of males or ephippia

TEST CONCENTRATIONS
- Justification for using less concentrations than requested by guideline: A limit test was performed because the test item was poorly soluble in water.
- Range finding study: No

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

< 7 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

- Behavioural abnormalities: No
- Mortality of control: 10% after 21 d (within acceptable range)
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Other: The reproductive performance of the daphnids should be significantly reduced at 0.2 mg/L.

Reported statistics and error estimates:

The NOEC was derived from F-variance analysis at 5%-confidence level of mean produced offspring in each treated group as compared to the control group.

VALIDITY CRITERIA

The study fulfilled the validity criteria prescribed by the guideline and is thus considered valid and reliable (Table 1). Several deviations to the study plan occurred (see below), which, however, did not affect the integrity of the study.

 

Table 1: Validity criteria for OECD 211.

Criterion from the guideline	Outcome	Validity criterion fulfilled
The mortality of the parent animals in the control (femaleDaphnia) does not exceed 20% at the end of the test.	Mortality in the controls was 10%.	Yes
The mean number of living offspring produced per parent animal surviving at the end of the test is ≥ 60 in the control.	The mean number of live offspring produced in the controls was 62.8.	Yes
The dissolved oxygen concentration at the end of the test should be ≥ 3.0 mg/L in control and test vessels.	The dissolved oxygen concentration at the end of the test was ≥ 3.0 mg/L in control and test vessels.	Yes

 

STUDY PLAN DEVIATIONS

Several deviations from the study plan occurred, which, however, did not alter the quality and integrity of the study. The deviations included:

1.     The volume of mineral medium was reduced to 40 mL instead of 50 – 100 mL/unit because of the duration of the preparation of the water saturated stock solution. A total volume of 480 mL was the maximum volume which was possible to produce at 1 mL/min within less than 8 h prior to the treatment application and this allowed only for 40 mL/unit for a total of 11 units.

2.     The daphnids were fed living cells of Chlorella sp. instead of Scenedesmus subspicatus, according to the breeding practices of the testing facility.

3.     The test guidelines were revised on Oct 2012.

4.     The measured test item concentrations ranged from 78.5 – 126.5% of the nominal value and were thus slightly wider than the recommend range of 80 – 120%. This was considered an acceptable deviation because outer high values were observed in two cases only, on Day 12 and on Day 14 in the new media (121.0% and 126.5% of mean value, respectively) and the outer low value only occurred at the end of the test in the old medium (78.5% of mean value).

 

ANALYTICAL RESULTS

Single measured values ranged between 5.5 and 8.8 µg/L, which corresponds to 78.5 to 126.5% of the mean measured value (Table 2).

The stability of the test item throughout the test period was considered acceptable even though the amplitude was slightly higher than the recommended 80 – 120% interval. Outer values were only observed on Day 12 and 14 in new media and at the end of the test in old medium (Table 2).

 

Table 2. Measured test item concentrations

Days	Old medium		New medium
	measured concentration [µg/L]	% of mean value	measured concentration [µg/L]	% of mean value
0	-	-	7.8	112.1
3	7.6	109.9	6.6	95.0
5	5.9	85.4	6.9	98.7
7	5.5	79.8	6.1	87.4
10	6.7	96.0	6.2	89.0
12	7.9	114.1	8.4	121.0
14	8.1	116.1	8.8	126.5
17	7.2	104.2	7.9	113.6
19	6.1	88.3	5.9	84.7
21	5.5	78.5	-	-

 

BIOLOGICAL RESULTS

MORTALITY

At the end of the test, mortality was 10% in the water control (Table 3). No mortality was observed in the limit concentration. The NOEC based on mortality was > 7.0 µg/L

 

Table 3. Cumulative mortality [%]

	Days
	0	3	5	7	10	12	14	17	19	21
water control	0	0	0	0	0	0	0	10	10	10
reference treatment	0	0	0	0	0	0	0	0	0	10
test item limit concentration	0	0	0	0	0	0	0	0	0	0

 

OFFSPRING PRODUCTION

The mean offspring produced per parent animal was 62.8 in the control with a standard deviation of 9.4 (15.3% of the mean). In the test item treatment the mean offspring per parent animal was only 26.1 with a standard deviation of 13.4 (51.5% of the mean). At a 5% confidence level, F-variance analysis showed that the reproductive performance was significantly reduced (58.4% inhibition compared to the control) (Table 4).

 

TIME TO PRODUCTION OF FIRST BROOD

In the water control, test item treatment and reference treatment the first brood was observed on Day 8.

 

CONCLUSION

No mortality was observed in the test item treatments but mean offspring per parent animal was significantly reduced. The reproductive performance was reduced by 58.5% in the test item treatment. Thus, the NOEC (21 d) for reproduction was < 7 µg/L (nominal).

Validity criteria fulfilled:

yes

Remarks:

For further details please refer to “Any other information on results incl. tables”.

Description of key information

NOEC (21 d) < 7.0 µg/L (nominal, OECD 211, D. magna)

Additional information

One study is available, in which the long-term toxicity of Isohexadecyl 12-[(1-oxooctadecyl)oxy]octadecenoate (CAS 97338-28-8) was assessed according to OECD guideline 211 and GLP.

Based on the poor water solubility of the test item, a chronic limit test with a medium renewal rate of 2 to 3 d was performed, in which the model organism Daphnia magna was exposed to a limit concentration of 7.0 µg/L test item for 21 d. The test item was applied as water saturated solutions prepared in M4 mineral medium. A newly saturated solution was used at each renewal of the treatment solutions. Saturated solutions were obtained by collecting 500 mL volumes from a flow-through (1 mL/min) burette column filled with sand quartz, which had previously been coated with test item. The same coated quartz sand portion was used for the entire test period to produce successive 500 mL water saturated aliquots of saturated test item solutions. Test item concentrations were analytically verified by HPLC-MS at every renewal in old and fresh test medium. At the end of the test, the total number of living offspring produced per live parent animal was assessed.

The calculated mean measured concentration was 7.0 µg/L with a standard deviation of 1.0 µg/L. The measured test item concentrations in old and new medium ranged between 5.5 and 8.8 µg/L, corresponding to 78.5 to 126.5% of the mean measured value. Though the amplitude of the measured test item concentrations was slightly above the recommended range of 80 to 120% of nominal, the test item was considered to be stable throughout the duration of the test. Outliers were only observed on Day 12 and 14 in new media and at test end in old medium.

After 21 d, no mortality was observed in the limit concentration treatments but the reproductive performance (mean offspring per parent animal) was significantly reduced by 58.5%, resulting in a NOEC (21 d) for reproduction of < 7.0 µg/L (nominal).