1,2,3-propanetriyl tris[(R)-12-(acetoxy)oleate]

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

Study period:

3 February 2018 - 16 February 2018

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:

23 November 2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

Council Regulation 440/2008, 30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Test item information
Appearance: Clear light amber liquid
Purity/Composition: >90%
Test item storage: At room temperature
For Certificate of Analysis see Appendix 4.

Additional information
Test Facility test item number: 209077/A
Purity/Composition correction factor: No correction factor required
Chemical name (IUPAC), synonym or trade name: Glyceryl Triacetyl Ricinoleate
CAS number: 101-34-8
Molecular formula: C63H110O12
Molecular weight: 1059
Highly reactive to water: Not indicated
Highly reactive to oxygen: Not indicated
Solubility in water: Insoluble
Stability in water: Stable

Analytical monitoring:

yes

Details on sampling:

Samples for possible analysis were taken from all test concentrations and the control according to the schedule below. In addition, the glass wool containing the undissolved residue was kept for possible analysis. The method of analysis is described in the appended Analytical Report (Appendix 2).
- Frequency: at t=0 h and t=48 h
- Volume: 1.0 mL from the approximate centre of the test vessels
- Storage: Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.
At the end of the exposure period, the replicates were pooled at each concentration before sampling.
Additionally, reserve samples of 1.0 mL were taken for possible analysis. If not used, these samples were stored in a freezer (≤-15°C) for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.

Vehicle:

no

Details on test solutions:

The batch of Dermol GTR tested was a clear light amber liquid with a purity >90% which was not completely soluble in test medium at the loading rates initially prepared. No correction was made for the purity/composition of the test item.
Preparation of test solutions started with loading rates individually prepared at 1.0, 10 and 100 mg/L. A two-day period of magnetic stirring was applied to ensure maximum dissolution of the test item in medium. The obtained mixtures were allowed to settle for a period of one hour. Thereafter, the aqueous Water Accommodated Fractions (WAFs) were collected by means of siphoning through glass wool and used as test concentrations. All test solutions were clear and colorless at the end of the preparation procedure.
Any residual volumes were discarded.

Preliminary Data
The water solubility of Dermol GTR at 20°C was determined to be <1.5 µg/L, using the slow-stirring flask method (Test Facility Study No. 20136813).

Test organisms (species):

Daphnia magna

Details on test organisms:

Test System
- Species: Daphnia magna (Crustacea, Cladocera) (Straus, 1820), at least third generation, obtained by a cyclical parthenogenesis under specified breeding conditions.
- Source: In-house laboratory culture with a known history.
- Reason for selection: This system has been selected as an internationally accepted invertebrate species.
- Validity of batch: Daphnids originated from a healthy stock, 2nd to 5th brood, showing no signs of stress such as mortality >20%, presence of males, ephippia or discoloured animals and there was no delay in the production of the first brood.
- Characteristics: For the test selection of young daphnids with an age of < 24 hours, from parental daphnids of more than two weeks old.

Breeding
- Start of each batch: With newborn daphnids, i.e. less than 3 days old, by placing about 250 of them into 5 litres of medium in an all-glass culture vessel.
- Maximum age of the cultures: 4 weeks
- Renewal of the cultures: After 7 days of cultivation half of the medium twice a week.
Temperature of medium: 18-22°C
- Feeding: Daily, a suspension of fresh water algae.
- Medium: M7, as prescribed by Dr. Elendt-Schneider (Elendt, B.-P., 1990: Selenium deficiency in Crustacea. An ultrastructural approach to antennal damage in Daphnia magna Straus. Protoplasma 154, 25-33).

Composition of medium M7:
Adjusted ISO medium: the following chemicals (analytical grade) are dissolved in tap water purified by Reverse Osmosis (RO-water, GEON Waterbehandeling, Berkel-Enschot, The Netherlands):
Macro salts: CaCl2.2H2O 211.5 mg/L
MgSO4.7H2O 88.8 mg/L
NaHCO3 46.7 mg/L
KCl 4.2 mg/L

Medium M7: trace elements, macronutrients and vitamins are added to freshly prepared ISO medium to reach the following concentrations:
Trace elements:
B 0.125 mg/L
Fe 0.05 mg/L
Mn 0.025 mg/L
Li, Rb and Sr 0.0125 mg/L
Mo 0.0063 mg/L
Br 0.0025 mg/L
Cu 0.0016 mg/L
Zn 0.0063 mg/L
Co and I 0.0025 mg/L
Se 0.0010 mg/L
V 0.0003 mg/L
Na2EDTA.2H2O 2.5 mg/L

Macro nutrients:
Na2SiO3.9H2O 10.0 mg/L
NaNO3 0.27 mg/L
KH2PO4 0.14 mg/L
K2HPO4 0.18 mg/L

Vitamins: Thiamine 75.0 µg/L
B12 1.0 µg/L
Biotin 0.75 µg/L

The hardness: 180 mg/L expressed as CaCO3 and the pH: 7.7 ± 0.3.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Post exposure observation period:

Not applicable

Hardness:

CaCO3: 180 mg/l

Test temperature:

19- 20 °C

pH:

7.9 - 81

Dissolved oxygen:

8.7 - 10

Salinity:

Not applicable

Conductivity:

Not reported

Nominal and measured concentrations:

Nominal: 1.0, 10.0, & 100.0 mg/l loading rate

Details on test conditions:

Test Concentrations
- Dermol GTR: WAFs individually prepared at loading rates of 1.0, 10 and 100 mg/L.
- Control: Test medium without test item or other additives.

Test Procedure and Conditions
- Test duration: 48 hours
- Test type: Static
- Test vessels: 60 mL, all-glass
- Medium: Adjusted ISO medium
- Number of daphnids: Control and limit concentration: 20 per test group
- WAFs prepared at 1.0 and 10 mg/L: 10 per test group
- Loading: 5 per vessel containing 50 mL of test solution
- Light: 16 hours photoperiod daily
- Feeding: No feeding
- Aeration: No aeration of the test solutions.
- Introduction of daphnids: 12 minutes after preparation of the test solutions.

Sampling for Analysis of Test Concentrations
Samples for possible analysis were taken from all test concentrations and the control according to the schedule below. In addition, the glass wool containing the undissolved residue was kept for possible analysis.
- Frequency: at t=0 h and t=48 h
- Volume: 1.0 mL from the approximate centre of the test vessels
- Storage: Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.
At the end of the exposure period, the replicates were pooled at each concentration before sampling.
Additionally, reserve samples of 1.0 mL were taken for possible analysis. If not used, these samples were stored in a freezer (≤-15°C) for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.

Measurements and Recordings
- Immobility (including mortality): At 24 hours and at 48 hours.
- pH and dissolved oxygen: At the beginning and at the end of the test, for the control and WAF prepared at a loading rate of 100 mg/L.
- Temperature of medium: Continuously in a temperature control vessel, beginning at the start of the test.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 0.11 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: Exceeds water solubility

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

> 0.11 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: Exceeds water solubility

Details on results:

Measured Concentrations
Samples taken from the WAF prepared at a loading rate of 100 mg/L were analysed. The measured concentration at the start of the test was 1.47 mg/L, which decreased to 0.53% of initial at the end of the test. Based on these results, effect parameters were based on the average exposure concentration calculated to be 0.11 mg/L at the limit concentration (see Table 1).
The measured concentrations during the entire test period exceeded the water solubility, determined to be < 1.5 µg/L (Test Facility Study No. 20136813). Hence, it is likely that the over-saturation explained the high decrease of the test item concentration during the test period. It can be stated that testing was performed at the maximum soluble concentration of test item in test medium with an average exposure concentration of 0.11 mg/L.

Immobility
Table 2 shows the responses recorded during the test. No immobility was observed in the control and at any test concentration throughout the test.
The responses recorded in this test allowed for reliable determination of an EC50.

Determination of Effect Concentrations
Table 3 shows the effect parameters based on the average exposure concentration.

Experimental Conditions
The results of measurement of pH and oxygen concentrations (mg/L) are presented in Table 4. These test conditions remained within the limits prescribed by the study plan (pH: 6 9, not varying by more than 1.5 units; oxygen: >3 mg/L at the end of the test).
The temperature continuously measured in a temperature control vessel varied between 19 and 20°C during the test, and complied with the requirements as laid down in the study plan (18-22°C, constant within 2°C).

Table 1: Average Exposure Concentration Versus Loading Rate

Dermol GTR WAF-specific loading rate (mg/L)	Measured concentrations (mg/L)		Average exposure conc. (mg/L)
	t=0h	t=48h
100	1.47	0.00783	0.11

Table 2: Number of Introduced Daphnids and Incidence of Immobility

Time (h)	Replicate	Dermol GTR; WAF-specific loading rate (mg/L)
		Control	1	10	100 [0.11]
0	A	5	5	5	5
	B	5	5	5	5
	C	5			5
	D	5			5
	Total introduced	20	10	10	20
24	A	0	0	0	0
	B	0	0	0	0
	C	0			0(1)
	D	0			0(1)
	Total immobilised	0	0	0	0
	Effect %	0	0	0	0
48	A	0	0	0	0
	B	0	0	0	0(1)
	C	0			0
	D	0			0
	Total immobilised	0	0	0	0
	Effect %	0	0	0	0

( ) – between brackets: number of daphnids observed trapped at the surface of the test solutions. These organisms were reimmersed into the respective solutions before recording of mobility.

[ ] – average exposure concentration (mg/L).

Table 3: Effect Parameters

Parameter	Dermol GTR Average exposure conc. (mg/L)
24h, 48h-EC50	>0.11

Table 4: pH and Oxygen Concentrations (mg/L) During the Test

Dermol GTR WAF-specific loading rate (mg/L)	Start (t=0 h)		End (t=48 h)
	pH	O2	pH	O2
Control	7.9	10	8.1	8.9
100 (0.11)	8	9.1	8.1	8.7

( ) – average exposure concentration (mg/L).

Validity criteria fulfilled:

yes

Conclusions:

In conclusion, the 48h-EC50 for Daphnia magna exposed to Dermol GTR exceeded the solubility limit in test medium with an average exposure concentration of 0.11 mg/L.

Executive summary:

The objective of the study was to evaluate Dermol GTR for its ability to generate acute toxic effects on the mobility ofDaphnia magnaduring an exposure period of 48 hours and, if possible, to determine the EC₅₀ at 24 and 48 hours of exposure.

The study procedures described in this report were based on the OECD guideline No. 202, 2004.In addition, procedures were based on the test methods described in the OECD series on testing and assessment number 23, 2000.

The batch of Dermol GTR tested was a clear light amber liquid with a purity >90% which was not completely soluble in test medium at the loading rates initially prepared.Water Accommodated Fractions (WAFs) were individually prepared at loading rates of 1.0, 10 and 100 mg/L and used as test concentrations.

A combined limit/range-finding test was performed. Twenty daphnids per group (5 per replicate, quadruplicate) were exposed to an untreated control and to a WAF prepared at a loading rate of 100 mg/L, in a limit test. In addition ten daphnids per group (5 per replicate, duplicate) were exposed to WAFs prepared at loading rates of 1.0 and 10 mg/L in the combined range-finding test. The total exposure period was 48 hours and samples for analytical confirmation of exposure concentrations were taken at the start and at the end of the test.

Samples taken from the WAF prepared at a loading rate of 100 mg/L were analysed. The measured concentration at the start of the test was 1.47 mg/L, which decreased to 0.53% of initial at the end of the test.Based on these results, effect parameters were based on the average exposure concentration calculated to be 0.11 mg/L at the limit concentration. It should be noted that the water solubility of the test item was determined to be <1.5µg/L(Test Facility Study No. 20136813), which likely explained the high decrease of the concentration during the test period. 

No immobility was observed in the control and at any test concentration throughout the test.

The study met the acceptability criteria prescribed by the study plan and was considered valid.

In conclusion, the 48h-EC₅₀ for Daphnia magna exposed to Dermol GTR exceeded the solubility limit in test medium with an average exposure concentration of 0.11 mg/L.

Description of key information

Study conducted to recognised testing guidelines with GLP certification.

Additional information

EC50 exceeds water solubility of the registered substance.