Glycerol trimyristate

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 Aug - 31 Aug 2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Qualifier:

according to guideline

Guideline:

other: OECD Guidance Document No. 23 on Aquatic Toxicity of Difficult Substances and Mixtures, OECD 2000

GLP compliance:

yes (incl. QA statement)

Remarks:

LUBW Landesanstalt für Umwelt, Messungen und Naturschutz Baden-Württemberg, Karlsruhe, Germany

Analytical monitoring:

yes

Details on sampling:

- Analysis was performed in additional test vessels without Daphnia to avoid disturbance of the measurement by the organisms itself or excrements. At the start (0 h) and at the end (48 h) of the experimental phase samples (4 mL) were taken and filtered for DOC analysis with a 0.45 µm CA membrane filter (Whatman FP 30/0.45 (im). First the filter was washed with 100 mL bi-distilled water and with about 15 mL sample. For TOC the samples were measured without filtration.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Due to the sparingly soluble properties of the test item, the study was performed with WAFs ("water accommodated fraction") prepared with Daphnia medium similar to M4 described in OECD 202. The solid test item was weighted on a piece of polyethylene foil (rinsed with ethanol and H2O bidest. before). The solid fat was melted in the drying oven at about 50 °C. In addition the Daphia medium was heated to about 40 °C in the drying oven. The the melted test item was transferred together with the polyethylene foil into a defined volume of the heated Daphnia medium into a 1000 mL beaker. The suspension was stirred for 48 hours at 21 °C in the dark. For stirring a magnetic stirrer with a 2 cm stir bar was used. After stirring was stopped, the suspension was allowed to float and sediment for a period of 1 hour. After this sedimentation period the WAF with the highest loading rate was inhomogeneous and showed cloudy cords. The two next lower loading rates were turbid. And also the secong lowest loading rate was still a little bit turbid. The lowest loading rate was clear. At the bottom of the beaker and at the surface of the WAF weny orange coloured particles could be observed. They were not transferred into the tets vessels. The test solution was taken from the middle of the suspension in the beaker using a glass tube and transferred into the test vessels.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea
- Source: German Federal Environmental Agency, Marienfelde, Department IV 2.4; current breeding is held at Hydrotox GmbH since February 2007
- Age at study initiation: 25 min - 21 h 50 min
- Method of breeding: Twice a week the animals are placed into fresh Elendt-Medium M4 (according to OECD 202). They are kept at approximately 10 animals / 200 mL. Daily, they are fed with a suspension of the algae Desmodesmus subspicatus in order to keep the C-content in the incubation beakers at 0.1 mg C per Daphnia and day. The C-content oft the algal suspension is measured photometrically taking a correlation between measured TOC and photometrical chlorophyll determination as basis. The Daphnia are held at 20 ± 2 °C with a light / dark cycle of 16 / 8 h.
- Feeding during test: none

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test temperature:

20.6 - 21. 6 °C

pH:

7.69 - 8.49

Dissolved oxygen:

7.8 - 8.2 mg O2/L

Nominal and measured concentrations:

Nominal: control, 6.25, 12.5, 25, 50, and 100 mg/L (WAF, nominal loading rate)
Nominal loading rates of 6.25, 25 and 100 mg/L were measured at the beginning and at the end of the test. Mean measured concentrations were 1.7 mg/L (6.25 mg/L nominal), 4.3 mg/L (25 mg/L nominal) and 14.8 mg/L (100 mg/L nominal).

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Material, size, headspace, fill volume: 50 mL glass beakers filled with 20 mL test solution
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: no; similar to M4-medium accordig to OECD 202

OTHER TEST CONDITIONS
- Photoperiod: 16 h light, 8 h dark

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): After 24h and 48h, the swimming capability of Daphnia magna was determined.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

36.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

mobility

Remarks on result:

other: 95% CL: 32.2 - 40.8

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

5.6 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

4.3 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- 95 and 100% immobilization was observed at the highest loading rates (50 and 100 mg/L, nominal) whereas no immobilization was reported in any other treatment group.
- A correlation between measured loading rates and nominal loading rates for loading rates of 6.25, 25 and 100 mg/L was made. The parameters of the curve allowed the measured EC50 concentration.

Results with reference substance (positive control):

EC50 (<24h) = 2.88 mg/L (95% CL: 2.55 - 3.97 mg/L).
EC50 (<48h) = 1.58 mg/L (95% CL: 1.42 - 1.73 mg/L).

Reported statistics and error estimates:

The calculation of EC50 including probability values was performed with the statistical software ToxRat (ToxRat Solutions GmbH, Alsdorf) as far as it was mathematically possible.

Results of the accompanying analysis:

The analytical results show maximal 27.8% of the TOC concentration expected. That means that the test item is not completely soluble or dispersible. During the 48 hours test course almost no decrease of TOC/DOC concentrations was measured. Only marginal differences between 0 h and 48 h measurements occurred. In addition, in most cases only slightly higher TOC concentrations were measured compared to the DOC concentrations. The test item was stable during the test course of 48 hours, the mean measured loading rates were maximal 14.8 mg/L test item. TOC content of the test item was determined with 69.1%. From this the recovery rates and the measured test item concentration in the test solution were calculated. Measured TOC was in the range of about 14.0% - 27.8% of the TOC expected.

Table: Results of the Daphnia test

	A	B	C	D	E	Control
Loading Rate [mg/L]	100	50	25	12.5	6.25	0
Immobile Daphnia 24h	65%	40%	0%	0%	0%	0%
Immobile Daphnia 48h	100%	95%	0%	0%	0%	0%

Table: Results of the Daphnia test in detail (0 h - 48 h)

Concentration test item [mg/L]	Volume test solution [mL]	[h]	Number of immobile Daphnia / number Daphnia introduced				Immobile Daphnia total	Introduced Daphnia total	Daphnia immobile [%]
			1	2	3	4
100	20	24	5/5	2/5	4/5	2/5	13	20	65
		48	5/5	5/5	5/5	5/5	20	20	100
50	20	24	2/5	3/5	2/5	1/5	8	20	40
		48	5/5	5/5	5/5	4/5	19	20	95
25	20	24	0/5	0/5	0/5	0/5	1	20	0
		48	0/5	0/5	0/5	0/5	1	20	0
12.5	20	24	0/5	0/5	0/5	0/5	0	20	0
		48	0/5	0/5	0/5	0/5	0	20	0
6.25	20	24	0/5	0/5	0/5	0/5	0	20	0
		48	0/5	0/5	0/5	0/5	0	20	0
Control		24	0/5	0/5	0/5	0/5	0	20	0
		48	0/5	0/5	0/5	0/5	0	20	0

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Please refer to teh analogue justification provided in IUCLID section 13

Reason / purpose for cross-reference:

read-across source

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

36.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

mobility

Remarks on result:

other: 95% CL: 32.2 - 40.8

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

5.6 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

4.3 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

mobility

Reference 3

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

other: supporting information

Adequacy of study:

other information

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable, well documented publication/study report which meets basic scientific principles

Principles of method if other than guideline:

Phase diagrams were prepared using a monoglyceride (glycerol monocaprylocaprate: Capmul MCM® EP), a diglyceride (glycerol dicaprylate) and two triglycerides (glycerol tricaprylate: Captex 8000®; caprylic/capric triglycerides: Captex 355 EP/NF®) in combination with a common surfactant (PEG-35 castor oil: Cremophor EL®) and water.

GLP compliance:

no

Test organisms (species):

other: not relevant

Test type:

other: Phase behaviour of lipid/surfactant/water phases

Water media type:

not specified

Remarks on exposure duration:

Not relevant

Details on results:

Refer to "Any other information on results including tables"

Phase boundaries were first identified by visual observation upon dilution with water. The particle size was then determined in the region of higher water content (>70% w/w).

Monoglyceride/Surfactant/Water Phase Diagram

Glycerol monocaprylocaprate (Capmul MCM EP; ABITEC) and PEG-35 castor oil (Cremophor EL; BASF) were used as lipid and surfactant, respectively. Since Capmul MCM used in this phase diagram contains a mixure of monoglyceride (60 %) and diglyceride (35 %), it is indeed a pseudoternary phase diagram.

A clear liquid solution was observed at all compositions of lipid / surfactant containing up to 15 % water. This clear region represents water-in-oil (w/o) microemulsion at low water content and oil-in-water (o/w) microemulsion at high water content. Upon further dilution with water, this clear region turned to a milky-white emulsion when the lipid comprised 60 % or more of the lipid / surfactant blend. In contrast, the solutions remained clear throughout aqueous dilution up to 90 % when the initial lipid content was 20 % or less. At the intermediate lipid content of 30 and 50 %, the solutions remained clear up to 65 % dilution with water; after which, a milky white emulsion resulted. Furthermore, when the initial lipid content was in the middle of this range (40 %), the clear solution transformed into a gel at water concentrations between 45 and 60 %.

The particle size analysis performed shows that glycerol monocaprylocaprate formed microemulsion only at high surfactant concentrations (> 80 %, giving particle sizes of 13 to 30 nm).

Diglyceride/Surfactant/Water Phase Diagram

Medium chain diglyceride (glyceryl dicaprylate), PEG 35 castor oil (Cremophor EL) and water was used.

Clear regions representing water-in-oil (w/o) microemulsion were observed with all lipid / surfactant blends containing up to 20 % water except when the initial lipid content was greater than 80 %. When lipid / surfactant mixtures with an initial diglyceride content of 70 % or less were further diluted with water (25 to 60 %), they transitioned to a gel phase. Upon still further dilution, this gel phase transitioned into a microemulsion or emulsion (o/w).

The particle size data for mixtures diluted with water ranging from 70 to 99 % shows that microemulsions (< 100 nm) formed when the initial lipid content was 50 % or less, and emulsions formed at higher lipid / surfactant ratios (≥ 70 % lipid). At 90 % initial lipid concentration, the particle size was in the micron range (2 to 3 μm) upon dilution with water (≥ 99 %).

Triglyceride/Surfactant/Water Phase Diagrams

The triglycerides used in these part were glycerol tricaprylate (Captex 8000) and caprylic/capric triglycerides (Captex 355), the major difference between the two being Captex 8000 was prepared from caprylic acid (99 %) while Captex 355 was prepared from caprylic / capric acid (55:45).

Phase diagrams of the two triglycerides were qualitatively similar, although the clear region representing o/w microemulsion (starting at 60 % water) appeared to be slightly larger for Captex 8000 than Captex 355. It should, however be noted, that Captex 355 is slightly more hydrophobic than Captex 8000 because of the higher capric acid (C10) content but this difference may not be significant. Furthermore, the gel phase regions of the triglycerides were larger than that of the diglyceride.

The particle sizes within the emulsion regions were less than 0.5 μm (≥ 80 % water). The particle sizes when using Captex 355 at the lipid / surfactant ratio of 9:1 were the exception, ranging from 1.7 to 3.6 μm. Additionally the particle size decreased with increasing water content (≥ 70%). Microemulsions (< 200 nm) formed upon dilution with water (99 % w/w) when the initial lipid content was less than 50 %.

Conclusions:

The monoglyceride gave microemulsion (clear or translucent liquid) and emulsion phases, whereas di- and triglycerides exhibited an additional gel phase.
Among individual mono-, di- and triglycerides, the oil-in-water microemulsion region was the largest for the diglyceride.
Medium chain triglycerides may form microemulsions at very dilute concentrations (1 to 100 dilution).

Description of key information

No toxicity observed up to the limit of water solubility

Additional information

Endpoint summary for Glycerol trimyristate (CAS 555-45-3); Short-term toxicity to aquatic invertebrates

Since no studies investigating the short-term toxicity of Glycerol trimyristate (CAS 555-45-3) to aquatic invertebrates are available, in accordance with Regulation (EC) No 1907/2006 Annex XI, 1.5, a read-across to the structurally related source substance Glycerides C12-18 mono- and di- (CAS 91052-49-2) was conducted. The source substance is representative to evaluate the short-term toxicity of the target substance to aquatic invertebrates.

The target substance Glycerol trimyristate (CAS 555-45-3) is characterized by the alcohol component glycerol which is triply esterified by C14 fatty acid (myristic acid).

The source substance Glycerides C12-18 mono- and di- (CAS 91052-49-2) is characterized as a UVCB substance containing mainly C12 fatty acids and, to a lesser extent, C14-C18 fatty acids. The alcohol component is glycerol which is esterified mainly once and twice.

This read-across is justified in detail in the analogue justification in IUCLID section 13. In this case of read-across, the best suited (highest degree of structural similarity, nearest physico-chemical properties) read-across substance was used for the assessment.

The study with the source substance Glycerides C12-18 mono- and di- (CAS 91052-49-2) was performed according to OECD 202 and OECD Guidance Document No. 23 on Aquatic Toxicity of Difficult Substances and Mixtures (2000). Due to the low water solubility of the test material, WAFs ("water accommodated fraction") prepared with Daphnia medium similar to M4 described in OECD 202 were used to prepare the five test material loading rates. The test was performed under GLP conditions. Daphnia magna were exposed to five different loading rates (6.25, 12.5, 25, 50 and 100 mg test material/L) over a period of 48h in a static freshwater system.

After 48h, the mobility of Daphnia magna was assessed.An EC50 (48h) of 5.6 mg/L was determined. No immobility was observed in the three lowest loading rates or the control group. 95% and 100% immobilization were reported at the highest loading rates of 50 and 100 mg/L, respectively.

Nevertheless, the observed effects are above the water solubility (WS 3.3 mg/L) of the substance and might be caused by direct physical interference of test substance particles with test organisms (i.e. physical entrapment), rather than intrinsic toxicity. Scientific evidence showed that aquatic toxicity testing of this type of Glycerides is technically very difficult. In an article by Prajapati et al. (2012) (see Supporting information IUCLID section 6.1.3), the phase behaviour of lipid/surfactant/water phases was investigated, where medium-chain (C8-10) mono-, di- and triglycerides represent the lipid. Phase boundaries between lipids (monoglycerides, diglycerides, triglycerides), surfactant (PEG-35 castor oil) and water were established by visual inspection after an equilibration period, and the results expressed in phase diagrams. Viscosity and particle size distribution were measured. The mixtures with monoglyceride displayed two predominant phases: microemulsion and emulsion phases, whereas di- and triglycerides showed additionally a gel phase. Mixtures of monoglycerides and diglycerides, and of monoglycerides and triglycerides seemed to promote an increase of the microemulsion phase (in the 4 phases equilibrium). Particle size in these mixtures was found to be much smaller than in the monoglyceride sample alone. Microemulsions are solutions with an average particle size < 0.2 µm. This particle size would not be intercepted by a standard filter used in an aquatic toxicity test (generally, pore size of 0.45 µm). Due to their small size, based on visual inspection, clear or translucent solutions might be observed even when these microemulsions are present. Glycerides, C12-18 mono- and di- contains 40-70% C12 fatty acids and formation of microemulsions in test solutions is therefore possible for this substance.

Based on the available information, no toxicity of Glycerides, C12-18 mono- and di- (CAS No. 91052-49-2) to aquatic invertebrates up to the limit of the water solubility is expected.

Based on the results for the structurally related read-across substance (in accordance with Regulation (EC) No 1907/2006 Annex XI, 1.5) which is characterized by a similar ecotoxicological profile and comparable structure, it can be concluded that a similar toxicity range to aquatic invertebrates can be expected for the target substance Glycerol trimyristate (CAS 555-45-3).