Glycerol trimyristate

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 Oct - 19 Oct 2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition 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:

- Sampling method: Analysis was performed in additional test vessels without algae to avoid an influence of the algae on the measured concentrations. At the start and at the end of the experimental phase samples (4 mL) were taken and filtered with a 0.45 µm cellulose acetate (CA) membrane filter (Whatman FP 30/0.45 µm). First the filter was washed with 100 mL bi-distilled water and with 15 mL sample. Additional samples were measured at the end of the study (72h) from the original test vessels with algae to get an impression of the influence of the organisms on the exposure concentration. Algae were separated by filtration before analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Due to the low water solubility of the test item, the study was performed with WAFs ("water accommodated fraction") prepared with algal growth medium according to OECD 201. The test item is a solid fat which was weighted on a piece of polyethylene foil (rinsed with ethanol and H2O bidest. before). The solid fat was melted in a drying oven at about 50 °C. In addition the algal medium was heated to about 50 °C on a heated magnetic stirrer. Then the melted test item was transferred together with the polyethylene foil into a defined volume of the heated algal medium into a 2.000 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 turbid. The two next lower loading rates were turbid. And also the second lowest loading rate was still a little bit turbid. The lowest loading rate was clear. No particles could be observed in suspension which could affect the algae physically. Therefore the WAFs were not filtered to obtain the water soluble fractions (WSF).
The test solutions were taken from the middle of the suspension in the beakers using a glass tube and transferred into the test vessels.
The loading rates (LR) for the WAFs were chosen 1.11-fold higher than the nominal loading rates intended in the test solution so as to take into account the dilution by the algal inoculum suspension (10%). 45 mL test solution was transferred into each test vessel (100 mL wide necked Erlenmeyer flask) and 5 mL of the algae inoculum suspension (0.8 x 10E5 algae/mL) were added to obtain an algae starting concentration of 0.8 x 10^4 algae/mL. For the controls 5 mL algae inoculum suspension was added to 45 mL algal medium. This medium was stirred in the same way as the WAF.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Green alga
- Strain: SAG No. 86.81
- Source (laboratory, culture collection): German Environmental Agency (UBA), Berlin-Marienfelde, cultured in the laboratory of Hydrotox GmbH since June 2012.
- Method of cultivation: Twice a week the stock suspension is diluted 2:50 into fresh Holm-Hansen medium under axenic conditions to keep it in exponential growth.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

23.1 - 23.2 °C

pH:

7.06 - 7.76

Nominal and measured concentrations:

Nominal: control, 4.1, 9.7, 20.9, 45.7, and 99.4 mg/L (WAF, loading rates)
Measured loading rates ranged from 4.66 to 11.8 mg/L at a nominal loading rate of 99.4 mg/L and from < LOD to 1.52 mg/L for a nominal loading rate of 20.9 mg/L. Concentrations at the lowest nominal loading rate of 4.1 mg/L could not be detected (< LOD).

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed (sealed with a sterile cellulose stopper)
- Material, size, headspace, fill volume: 100 mL wide necked glass Erlenmeyer flask
- Initial cells density: 0.8 x 10E05 cells/mL
- Control end cells density: 7.2 x 10E05 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes, according to OECD guideline 201


OTHER TEST CONDITIONS
- Photoperiod: continuous
- Light intensity and quality: The flasks are illuminated laterally by eight fluorescent tubes (58 Watt each) which are separated from the incubation chamber by heat absorbing glass. Light intensity is continuously variable and was set on 45% of the maximum light intensity resulting in a mean light intensity of 120 µE/m2 s ± 5.7% (PAR)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Chlorophyll measurement: The algal growth was determined by measuring the chlorophyll fluorescence as a measure for algal biomass. At the start of the test and after 24, 48 and 72 hours, 200 µL samples were taken out of each test vessel and chlorophyll fluorescence was measured in a 96-well micro-plate for each sample using a fluorescence micro-plate reader (excitation wave length: 465 nm, emission wave length: 670 nm). The conversion factor between the chlorophyll fluorescence and the algal count, as required in the OECD guideline, was determined by correlating both parameters. Several different algal cell concentrations were measured using the same algal pre-culture as in the test. The algal count was determined for a cell size between 3 and 9 µm using a Coulter Counter 72.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

4.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

13 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

9.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

4.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

7.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

other: yield

Remarks on result:

other: 95% CL: 6.2 - 8.3

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

9.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

other: yield

Details on results:

- Growth inhibition was reported in all treatment groups except for the lowest nominal loading rate of 4.1 mg/L.

Results with reference substance (positive control):

EC50 (growth rate) = 0.94 mg/L
EC50 (yield) = 0.49 mg/L

Table: Mean growth rates and mean yields and its inhibitions compared to the control

	A	B	C	D	E	Control
Loading Rate mg/L	99.4	45.7	20.9	9.7	4.1	-
Number of replicates	3	3	3	3	3	6
Mean growth rate	0*	0*	0.395	0.965	1.506	1.505
Mean yield**	0*	0*	0.18	1.354	7.16	7.138
Mean inhibition of growth rate	100%	100%	73.7%	36.0%	0%	-
Mean inhibition of	100%	100%	97.5%	81.0%	-0.3%**	-

* no increase of algal chlorophyll fluorescence measured, microscopically no algal growth observed

** negative inhibition means growth propagation

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

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 Analogue Justification provided in Section 13.

Reason / purpose for cross-reference:

read-across source

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

4.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

13 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

9.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

4.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

7.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

other: yield

Remarks on result:

other: 95% CL: 6.2 - 8.3

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

9.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

other: yield

Reference 3

Endpoint:

toxicity to aquatic algae and cyanobacteria

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 type:

other: Phase behaviour of lipids/surfactants/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).

Additional information

Endpoint summary for Glycerol trimyristate (CAS 555-45-3); Toxicity to aquatic algae and cyanobacteria

Since no studies investigating the toxicity of Glycerol trimyristate (CAS 555-45-3) to aquatic algae and cyanobacteria 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 toxicity of the target substance to aquatic algae and cyanobacteria.

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 201 and OECD Guidance Document No. 23 on Aquatic Toxicity of Difficult Substances and Mixtures (2000). The test was performed under GLP conditions.

Green algal culture units of Desmodesmus subspicatus were exposed to five different loading rates (nominal: 4.1, 9.7, 20.9, 45.7, and 99.4 mg/L) over a period of 72 hrs in a static freshwater system.

Algal growth was determined using chlorophyll fluorescence measurement as a parameter for algal biomass. The EL50 (72h) was determined to be 13.0 mg/L for growth rate and 7.6 mg/L for yield. Growth inhibition was reported in all treatment groups except the lowest nominal loading rate of 4.1 mg/L. The NOEC was determined to be 4.1 mg/L for both growth rate and yield.

Nevertheless, the observed effects might be caused by direct physical interference of test substance particles with algae cells, rather than intrinsic toxicity. For this test, Water Accommodated Fractions (WAFs) were prepared by adding the test material into a defined volume of test medium, stirring for a period of 48 hours, followed by a sedimentation period of 1 hour. After the sedimentation period, the WAF with the highest loading rate (99.4 mg/L) was non-homogeneous and turbid. The next two lower loading rates (45.7 and 20.9 mg/L) were turbid, and also the second lowest loading rate (9.7 mg/L) was slightly turbid. The lowest loading rate (4.1 mg/L), the only one at which no effects were observed, was reported to be clear. The WAFs were not filtered for the final test. According to the authors of the report, at the highest loading rates (45.7 mg/L and 99.4 mg/L), algae cells were smaller and had a different shape (crumpled) compared to those in the control. Furthermore, at these two loading rates, a dense emulsion of oily drops was observed. This was confirmed by microscopic observation. At the middle loading rate of 9.7 mg/L, algae cells were larger than those in the control and small drops were reported. At a loading rate of 20.9 mg/L, the number of algae cells was significantly lower and the solution was turbid. In the lowest loading rate (4.1 mg/L) no difference compared to the control was observed. Based on the above information, mechanical disturbance of cells and cell growth cannot be excluded due to emulsified test material, which is very likely to have caused the observed effects.

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 6.1.5), 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 above information, the observed effects are expected to be caused by mechanical disturbance of the algae cells rather than due to intrinsic toxicity of the substance. No toxicity up to the highest attainable solubility (before microemulsion formation) of Glycerides, C12-18 mono- and di- is thus 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 algae and cyanobacteria can be expected for the target substance Glycerol trimyristate (CAS 555-45-3).