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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:

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

Adequacy of study:

weight of evidence

Study period:

From January 02, 2018 to January 04, 2018

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

KL2 due to RA

Justification for type of information:

Refer to section 13 of IUCLID for details on the read-across justification.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Remarks:

High-performance liquid chromatography (HPLC)

Details on sampling:

To demonstrate that nominal exposure concentrations were being achieved the concentrations of test substance in the test vessels were measured using the high-performance liquid chromatography method. At study start, samples were taken from excess test solutions and at study end from the test vessels of the dilution water control, solvent control and each test concentration.

Vehicle:

yes

Remarks:

Water

Details on test solutions:

The study was run with a dilution water control and nominal exposure concentrations of 0.625, 1.25, 2.5, 5 and 10 mg/L. A primary stock concentrate of Test substance, with a nominal concentration of 10 mg/L, was prepared by adding a nominal 0.020 g of test substance (actual weight 0.02015 g) and making up to 2000 mL volume with dilution water in a volumetric flask. The stock was sonicated for 45 minutes and the resultant stock observed to be a homogenous, colourless dispersion with fine particles visible. The stock was used directly as the 10 mg/L test solution and used to prepare the other test solutions by the direct addition of the appropriate amount to dilution water in a volumetric flask. The control consisted of dilution water only. All test solutions were clear and colourless. In all cases the final solutions contained nutrients.

Test organisms (species):

Daphnia magna

Details on test organisms:

The test organism was the freshwater crustacean, Daphnia magna, obtained from continuous laboratory cultures held at Scymaris. The stock cultures of D. magna were maintained in a reconstituted water medium, the same as the test dilution water, at a temperature of 20 ± 2°C. The cultures were maintained in 2 L glass vessels with a working volume of 1.6 L. A photoperiod of 16 h light:8 h dark, with 20-minute transition periods was provided. The D. magna cultures were fed on a mixed algae diet of Chlorella vulgaris, strain CCAP 211/12 and Pseudokirchneriella subcapitata, strain CCAP 278/4. The D. magna cultures were fed a daily ration depending on the age and density of the culture. Culture conditions were such that the D. magna reproduction was by diploid parthenogenesis. D. magna <24 h old, obtained from a single culture vessel, were used for testing. The parent animals were 28 ± 1 d old and had been maintained with a twice weekly renewal of reconstituted water medium since birth. The test organisms and the culture from which they were obtained showed no evidence of disease before the test period.

Test type:

static

Water media type:

other: Elendt's M4 D. magna medium

Limit test:

no

Total exposure duration:

48 h

Test temperature:

20±1°C

pH:

7.92 to 8.03

Dissolved oxygen:

8.97 to 9.44 mg/L

Nominal and measured concentrations:

Control and 0.625, 1.25, 2.5, 5 and 10 mg/L (nominal)
Control and 0, 0, 0, 0.21 and 0.34 mg/L (measured)
A 10 mg/L test concentration was selected as the highest concentration based on the dispersibility observed in a non-GLP range finding test

Details on test conditions:

Apparatus
Glass beakers of 250 mL nominal capacity were used as test vessels, with four replicates per test concentration. Each vessel contained 200 mL of test solution providing a depth of approximately 60 mm. The beakers were covered with loose fitting glass lids. The positions of the treatments were randomly allocated within the test area.

Reference substance (positive control):

no

Key result

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 0.34 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat. (total fraction)

Basis for effect:

mobility

Remarks on result:

other: Calculation method: Direct observation from data

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 0.34 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat. (total fraction)

Basis for effect:

mobility

Remarks on result:

other: Calculation method: Direct observation from data

Key result

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

ca. 0.34 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat. (total fraction)

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

> 0.34 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat. (total fraction)

Basis for effect:

mobility

Details on results:

Analytical data
The limit of quantification (LOQ) in this study was 0.4 mg/L for all test solutions. The instrument LOQ was 0.2 mg/L but during analysis, samples from the control and each test concentration were diluted x2, doubling the LOQ. Analytical calibrations were constructed using a minimum of 5 calibration levels, with a minimum R2 value of 0.995. The maximum percentage difference from nominal concentration for standards at the LOQ is less than 20% and less than 15% at levels greater than the LOQ. All analytical values are quoted to two significant figures and percentages to the nearest integer. On the basis of the analytical data the mean measured concentrations were used for the calculation and reporting of results.

Biological data
Based on immobility compared to the control (p <0.05) the 48 h No Observed Effect Concentration (NOEC) was determined to be 0.34mg/L and the Lowest Observed Effect Concentration (LOEC) was >0.34 mg/L. There was no immobility observed in the dilution water control. No other symptoms of toxicity were observed. It was observed that test substance was visible on the surface of the test vessels as a raft of fine particles at 24 h and 48 h in a dose dependant amount.

Table 1: Analytical results

Nominal concentration of test substance (mg/L)	Measured concentration of test substance (mg/L)				Mean measured concentration (mg/L)	Mean measured concentration (%)
	0 h		48 h
	(mg/L)	% of nominal	(mg/L)	% of nominal
Control	<LOQ	-	<LOQ	-	0	-
0.625	<LOQ	-	<LOQ	-	0	0
1.25	<LOQ^a	-	<LOQ^b	-	0	0
2.5	<LOQ	-	<LOQ	-	0	0
5	0.43	9	<LOQ	-	0.21	4
10	0.68	7	<LOQ	-	0.34	3

All analytical measurements quoted to 2 significant figures. Arithmetic means are used due to the zero values present.

^a Mean of triplicate analyses: <LOQ, <LOQ, <LOQ mg/L.

^b Mean of triplicate analyses: <LOQ, <LOQ, <LOQ mg/L.

The limit of quantification (LOQ) in this study was 0.4 mg/L for all test solutions. The instrument LOQ was 0.2 mg/L but during analysis, samples from the control and each test concentration were diluted ×2, doubling the LOQ. Values in Table 1 have been corrected for these dilutions.

Table 2: Daphnia magna response

Time (h)	Nominal concentration of test substance (mg/L)	Mean measured concentration of test substance (mg/L)	Number immobilised per replicate				Total number tested	Total number immobilised	Percentage immobilised
			A	B	C	D
24	Control	0	0	0	0	0	20	0	0
	0.625	0	0	0	0	0	20	0	0
	1.25	0	0	0	0	0	20	0	0
	2.5	0	0	0	0	0	20	0	0
	5	0.21	0	0	0	0	20	0	0
	10	0.34	0	0	0	0	20	0	0
48	Control	0	0	0	0	0	20	0	0
	0.625	0	0	0	0	0	20	0	0
	1.25	0	0	0	0	0	20	0	0
	2.5	0	0	0	0	0	20	0	0
	5	0.21	0	0	0	0	20	0	0
	10	0.34	0	0	0	0	20	0	0

Validity criteria

As no immobility or other signs of stress were observed in the control and dissolved oxygen concentration remained at ≥3 mg/L, this test has satisfied all OECD Guideline 202 validity criteria.

Validity criteria fulfilled:

yes

Conclusions:

Based on the results of read across study, the 48 h EC50 of the read across substance for Daphnia magna was determined to be >10 mg/L (nominal or >0.34 mg/Lmeasured)

Executive summary:

A study was conducted to determine the acute toxicity of read across substance, 'Reaction mass of mono- and di- hexadecyl phosphate esters, potassium salts and phosphoric acid' (100%), to Daphnia magna, according to the OECD Guideline 202, in compliance with GLP. Twenty test organisms were exposed to each nominal read across substance concentrations of 0, 0.625, 1.25, 2.5, 5 and 10 mg/L for 48 h under static conditions. The concentrations of read across substance in the test vessels were measured using the high-performance liquid chromatography method. The mean measured concentrations of read across substance were determined to be 0, 0, 0, 0, 0.21 and 0.34 mg/L. Immobilisation was recorded at 24 and 48 h and compared with control values. Based on immobility compared to the control (p <0.05), the 48 h NOEC was determined to be 0.34 mg/L (measured) and the LOEC was >0.34 mg/L (measured). No other symptoms of toxicity were observed. Based on the study results, the 48 h EC50 was calculated to be >0.34 mg/L (measured). As no immobility or other signs of stress were observed in the control and dissolved oxygen concentration remained at ≥3 mg/L, this test has satisfied all validity criteria. Under the study conditions, the 48 h EC50 of the read across substance for Daphnia magna was determined to be >10 mg/L (nominal or >0.34 mg/Lmeasured) (Scymaris, 2018). Based on the results of the read across study, similar EC50 and NOEC values can be expected for the phosphate ester constituents of the test substance.

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

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

Adequacy of study:

weight of evidence

Study period:

1998

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

KL2 due to RA

Justification for type of information:

Refer to section 13 of IUCLID for details on the read-across justification.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

yes

Remarks:

Only two replicates were used instead of four as recommended.

Qualifier:

according to guideline

Guideline:

ISO 6341 (Water quality - Determination of the Inhibition of the Mobility of Daphnia magna Straus (Cladocera, Crustacea))

Deviations:

yes

Remarks:

The pH of the dilution water was slightly higher (8.0 +/- 0.2).

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

no

Remarks:

Using the available analytical techniques, it was not possible to detect concentrations of the test substance in the test medium. Therefore, no chemical analysis was performed.

Vehicle:

no

Details on test solutions:

Preparation and application of test solutions
- Method: The test solutions were prepared separately and mixed with test medium for a defined period. Subsequently, the test solutions were allowed to stabilize in a separating funnel. The water phase in the middle of the separating funnel was used for testing and was identified as the Water Accommodated Fraction (WAF).
- Eluate: no
- Differential loading: yes
- Controls: yes, water control
- Evidence of undissolved material: All final test solutions were clear and colourless.

Test organisms (species):

Daphnia magna

Details on test organisms:

Test organism
- Common name: water flea
- Age at study initiation: < 24 h
- Method of breeding: Daphnia were held at a temperature of 18 – 22 °C. A new batch was started with newborn animals, i.e. less than 3 d old, by placing about 250 of them into 30 L of medium in an all-glass culture vessel. The maximum age of culture was 4 weeks. After 7 d of cultivation half of the medium was renewed. Daphnids were fed daily with a suspension of fresh water algae.
- Feeding during test: no

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Test temperature:

21.3 °C

pH:

7.8 - 7.9

Dissolved oxygen:

8.1 - 8.6 mg O2/L

Nominal and measured concentrations:

Nominal: 0, 86 mg/L (corresponding to 100 µL/L)

Details on test conditions:

Test system
- Test vessel: all-glass vessels
- Material, size, fill volume, headspace: all-glass, 100 mL, Fill volume: 80 mL, Headspace: 20 mL
- Aeration: no
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2

Test medium/Water parameter
- Source/preparation of dilution water: Medium was prepared according to ISO guideline in milli-RO water
- Culture medium different from test medium: no

Other test condition
- Photoperiod: 16 h light / 8 h dark

Effects parameters measured
Immobility was recorded after 24 and 48 h.

Test concentrations
- Range finding study
- Test concentrations: control, WAFs at 10 and 100 mg/L (range finding study 1); control, WAFs at 0.86 and 8.6 mg/L (range finding study 2)
- Results used to determine the conditions for the definitive study: At WAFs prepared at 10 and 100 mg/L daphnids trapped at the surface of test solutions. At WAFs prepared at 0.86 and 8.6 mg/L the stabilisation period was increased to 24 h to overcome daphnids becoming trapped at the surface. No effects were observed in the second range-finding study.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

> 86 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Water Accommodated Fraction (WAF)

Basis for effect:

mobility

Details on results:

- Mortality of control: no
- Effect concentrations exceeding solubility of substance in test medium: The test solutions at 86 mg/L (nominal) exceeded the maximum water solubility of the test substance.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50/LC50: 48 h EC50 was 0.5 mg/L (95% Confidence Limit: 0.4 - 0.6 mg/L)

Validity criteria fulfilled:

yes

Conclusions:

Based on the results of read across study, the 48 h EL50 value of the constituents 'linear or branched esters' for Daphnia magna is considered to be >86 mg/L (nominal) WAF.

Executive summary:

A study was conducted to determine the acute toxicity of read across substance, octyldodecyl isooctadecanoate (purity: assumed to be 100%), to Daphnia magna, according to OECD Guideline 202 and EU Method C.2, in compliance with GLP. Twenty test organisms each were exposed to nominal concentration of 86 mg/L (i.e., corresponding to 100 µL/L) for 48 h under static conditions. Potassium dichromate was used as positive control substance. Using the available analytical techniques, it was not possible to detect concentrations of the read across substance in the test medium. Therefore, no chemical analysis was performed. The test solutions at 86 mg/L (nominal) exceeded the maximum water solubility of the test substance, therefore concentration identified as Water Accommodated Fraction (WAF) was considered for reporting the result. Immobilisation was recorded at 24 and 48 h and compared with control values. Based on immobility compared to the control, the 48 h EL50 was determined to be >86 mg/L (nominal) WAF. No other symptoms of toxicity were observed. Positive control substance results were considered valid. As no immobility or other signs of stress were observed in the control and dissolved oxygen concentration remained at ≥3 mg/L, this test had satisfied all the validity criteria. Under the study conditions, the 48 h EL50 of the read across substance for Daphnia magna was determined to be >86 mg/L (nominal) WAF (Notox, 1998). Based on the results of read across study, similar EL50 value can be expected for the constituents 'linear or branched esters'.

Reference 3

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

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

Adequacy of study:

weight of evidence

Study period:

From 10 September, 2012 to 12 September, 2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

KL2 due to RA

Justification for type of information:

Refer to section 13 of IUCLID for details on the read-across justification.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

yes

Remarks:

The study author stated that changes had no effect on the outcome of the study

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

yes

Remarks:

The study author stated that changes had no effect on the outcome of the study

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Remarks:

GC/MS

Details on sampling:

- Concentrations: control, 100 mg/L. The concentrations of tetradecyl myristate in the test media were analysed at test start directly after distribution of the stock test substance WAF into the 60 mL glass beakers (fresh test media at test start). Furthermore, a second sample of the stock WAF was taken at this time to investigate a possible 'wall effect' of the used glass beakers. Another sample was taken at the end of the test after 48 h (aged test media at test end).
- Sampling method: As the volume of the analytical sub-sample required for sampling was large compared to the total test media volume, the test substance concentration was determined from samples taken from additionally prepared glass beakers. Five millilitres were pipetted directly into 20 mL extraction (centrifuge) tubes and 15 mL of purified water added. The pipette tips were rinsed multiple times and the surface wiped with a lint free paper towel prior taking the analytical sub-sample.
- Sample storage conditions before analysis: The samples were measured immediately after sampling without further storage.

Vehicle:

no

Details on test solutions:

Preparation and application of test solution
- Method: The test substance pellets were crushed by mortar and pestle and the Water Accommodated Fraction (WAF) prepared with the test item powder. A test preparation with a loading of 100 mg/L dilution water was prepared according to the OECD guideline No. 23, and stirred slowly at 300 rpm to avoid bubble and foam formation using a stirring bar on a magnetic stirrer for 48 h at room temperature (about 20 °C). Finally, the test preparation was filtered using a 0.2 µm membrane filter (Sartolab 150V, vacuum capsule sterile - Sartorius) to separate insoluble parts from the aqueous phase in accordance with the OECD guideline No. 29. The aqueous phase (WAF) was drawn off and used for testing. The control consisted of dilution water only and was not stirred for 48 h.
- Eluate: no
- Differential loading: yes
- Controls: yes, dilution water control

Test organisms (species):

Daphnia magna

Details on test organisms:

- Common name: water flea
- Source: Obtained from the German Federal Environment Agency, Institut für Wasser-, Boden- und Lufthygiene, Germany. Specimens used in the test were bred in the laboratory at Fraunhofer IME.
- Age at study initiation (mean and range, SD): < 24 h
- Method of breeding: Adult Daphnia, at least 3 weeks old, were separated from the stock population by sieving. Batches of 30 - 50 animals were held at room temperature in ca. 1.8 L of dilution water for one week. During this week, the daphnids were fed daily with an algal suspension (Desmodesmus subspicatus) and LiquizellR. Algae growing in the log-phase were centrifuged and the pellet was re-suspended in a few mL of medium. Thirty milliliters of this suspension was given to 1 L of medium. The water was changed once per week. Newborn Daphnia (between 4 - 23 h old) were removed by wide-bore pipette (to avoid damage) and isolated in fresh dilution water for at least 1 h prior to being added randomly to the test vessels containing the appropriate test or control media.
- Feeding during test: no

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Test temperature:

20.2 - 20.4°C

pH:

7.91 - 8.49

Dissolved oxygen:

6.3 - 8.2 mg O2/L

Nominal and measured concentrations:

Nominal: control, 100 mg/L
Measured: < LOQ, 0.128 µg/L (geometric mean)

Details on test conditions:

Test system
- Test vessel
- Type: covered with a glass pane
- Material, size, headspace, fill volume: glass, 60 mL, headspace: 10 mL, fill volume: 50 mL
- Aeration: no
- 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: Purified drinking water was used according to the OECD guideline. The purification included filtration with activated charcoal, passage through a lime-stone column and aeration. To avoid copper contamination, plastic water pipes were used for the test facilities. The water was aerated to the point of oxygen saturation.
- Chlorine: < 0.02 mg/L
- Alkalinity: 2 mmol/L
- Conductivity: 244.5 µS/cm
- Nitrate: 1.7 mg/L
- Nitrite: 0.005 mg/L
- Ammonium: < 0.01 mg/L
- Phosphate: 0.73 mg/L
- Culture medium different from test medium: no
- Intervals of water quality measurement: regularly

Other test conditions
- Adjustment of pH: no
- Photoperiod: 16 h light / 8 h dark
- Light intensity: 518 - 536 lux

Test procedure and incubation conditions
Four replicates of five daphnids per test vessel were used per test concentration and control. The test vessels were 60 mL glass beakers. All replicates per test concentration were held in a rack covered with a glass pane and the position of the racks randomised within the growth cabinet. Approximately 50 mL of test medium was added to each replicate test vessel followed by the Daphnia (less than 24 h old). The daphnids were exposed to the test substance under static conditions for a period of 48 hours. The test solutions were not exchanged. The daphnids were maintained under a light/dark cycle of 16/8 h. The light intensity, temperature, pH, and oxygen saturation were measured at test start and end. The incubation conditions for temperature must be between 18 – 22°C with less than 1°C variation, less than 1.5 units variation for pH, and more than 3 mg/L for oxygen saturation of the holding water, respectively. The test vessels were not aerated during the test.

Effect parameters measured
After 24 h and 48 h, the number of immobile animals in each beaker was counted. The animals were considered to be immobile if they were not able to swim within 15 sec of gentle agitation of the test vessels. Any abnormalities in appearance and behaviour were also recorded.

Test concentration
- Range finding study
- Test concentrations: 1, 10 and 100 mg/L (nominal WAF)
- Results used to determine the conditions for the definitive study: EL50 > 100 mg/L

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Water accommodated fraction

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

> 0.128 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: Water accommodated fraction

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Water accommodated fraction

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

>= 0.128 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: Water accommodated fraction

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

LOELR

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Water accommodated fraction

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

LOELR

Effect conc.:

> 0.128 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: Water accommodated fraction

Basis for effect:

mobility

Details on results:

- Other biological observations: No signs of disease or stress were observed. All surviving specimens gave the impression of healthy condition.
- Mortality of control: 0%

Validity of the test
The test is valid because:
- Mortality in the control did not exceed 10% (0%)
- Dissolved oxygen concentration at the end of the aging period (48 h) was 3 mg/L in the control and test vessels

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50/LC50: EC50 (24 h): 0.87 mg/L

Reported statistics and error estimates:

Numerical values in this report were frequently rounded to a smaller degree of precision (number of digits) than used in the actual calculation. Minor differences in results obtained from calculations with such rounded values in comparison to those obtained with higher precision values are possible. They are, however, well within the limits of the experimental accuracy and thus of no practical concern. For the immobilisation endpoint, the NOEC, LOEC, and ECx values were determined. If it is not possible to determine EC50 because no inhibition was observed, the EC50 will be reported as being more than the highest measured concentration tested. Calculations for the determination of ECx-values were performed by probit analysis using the number of immobile daphnids recorded at 24 and 48 h. Threshold concentrations (NOEC, LOEC) for mobility at 48 h were determined by pairwise comparisons using Fisher’s Exact Binominal Test with Bonferroni correction. The computer software ToxRat® was used for the statistical evaluations.

Results

 

Test substance concentrations

The chemical analysis of tetradecyl myristate in the fresh and aged test media revealed measured concentrations of ≤0.01% of the nominal loading of 100 mg/L. The LOQ was 0.01 µg/L. The effect evaluation was based on the geometric mean measured concentration of 0.128 µg/L and the nominal loading of 100 mg/L.

Measured concentrations of tetradecyl myristate [µg/L]

Nominal loading	Test duration				Geometric mean
	Mean measured 0 h		Mean measured 48 h
[mg/L]	[µg/L]	% nominal	[µg/L]	% nominal	[µg/L]	[%]
100	0.172	1.72 E-4	0.096	9.59 E -5	0.128	1.28 E-4
Control	<LOQ	-	<LOQ	-	-	-

 

Environmental conditions

With a temperature of 20.4°C at test start and 20.2°C at the end of the test, the optimal range was maintained. The oxygen saturation was between 6.3 - 8.2 mg/L and pH was within the range of 7.91 – 8.49 the in the control and test media. The light intensities ranged from 518 – 536 lux. All water quality criteria were met.

 

Physical/pathological symptoms and changes inbehaviour

Neither significant adult mortality nor signs of disease or stress were observed. All surviving specimens gave the impression of healthy condition.

Immobilisation of Daphnia magna

At 24 and 48 h, no immobilisation effects were observed in any treatment. As there was 0% immobilisation at the highest test concentration (100 mg/L nominal loading: 0.128 µg/L geometric mean measured concentration) no statistical analysis was completed. The NOEC of the test substance is >=0.128 µg/L and the EC₅₀ >0.128 µg/L. In terms of the nominal loading, the NOEL is >=100 mg/L and the EL₅₀ >100 mg/L.

Immobility raw data

Geometric mean conc. [mg/L]	Control	100
0 h	5	5
	5	5
	5	5
	5	5
Number of replicates:	4	4
Total Introduced:	20.00	20.00
24 h	0	0
	0	0
	0	0
	0	0
Number of replicates:	4	4
Total Immobile:	0.00	0.00
48 h	0	0
	0	0
	0	0
	0	0
Number of replicates:	4	4
Total Immobile:	0.00	0.00

NOEL, LOEL, and EL50 values for immobilisation Daphnia magna by test substance at 48 h

	Test substance
Geometric mean measured concentration [µg/L]		Nominal loading [mg/L]
NOEC / NOEL	>=0.128	>=100
LOEC / LOEL	>0.128	>100
EC50/ EL50	>0.128	>100

n.d.: not determined due to mathematical reasons or inappropriate data as immobilisation was less than 50%.

 

Validity criteria fulfilled:

yes

Conclusions:

Based on the results of the read across study, the 48-h EC50 and NOEC values of the read across substance were determined to be >100 mg/L (nominal or >0.128 µg/L measured) and 100 mg/L (nominal or 0.128 µg/L measured), respectively.

Executive summary:

A study was conducted to determine the acute toxicity of the read across substance, tetradecyl myristate to Daphnia magna, according to the OECD Guideline 202, in compliance with GLP. Daphnia magna were exposed under static conditions to the read across substance for 48 h. Four replicates of five individuals were tested for each treatment. Based on the results of a pre-test a limit test was conducted with a single test loading of 100 mg/L and a control of dilution water only. A water accommodated fraction (WAF) of the read across substance was prepared. The analytical determination of tetradecyl myristate was carried out by gas chromatography (GC) and electron impact ionization mass spectrometry detection (GC/EI-MS) using the mass spectrometer in the single ion monitoring (SIM) mode, which resulted in a measured geometric mean concentration of 0.128 µg/L. No immobilization was recorded after 48 h resulting in an EL50 of >0.128 µg/L (measured, geometric mean) and >100 mg/L (nominal), respectively. resulted in a measured geometric mean concentration of 0.128 µg/L. Under the conditions of the study, the 48-h EC50 and NOEC values of the read across substance were determined to be >100 mg/L (nominal or >0.128 µg/L measured) and 100 mg/L (nominal or 0.128 µg/L measured), respectively. Thus it can be concluded that no toxicological effects on aquatic invertebrates are expected up to the limit of water solubility for tetradecyl myristate ( Schlechtriem, 2012). Based on the results of read across study, similar effect levels can be expected for the constituents 'linear or branched esters'.

Description of key information

Based on the available weight of evidence information from studies for substances representative of the main constituents, a similar absence of toxicity up to the highest tested or highest soluble concentrations can be expected for the test substance. However, given the UVCB nature of the test substance with variable solubilities for individual constituents, the respective nominal values have been considered for hazard/risk assessment. The phosphate esters and alkyl ester constituents did not show toxicity at >10 mg/L or >100 mg/L (nominal) respectively and the alcohols did not show toxicity at their maximum soluble concentrations. Further, considering that the alkyl esters make up more than 80% of the composition, the 48 h EC50 value of >100 mg/L (nominal) determined in a study with tetradecyl myristate has been considered further for hazard/risk assessment.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC50

Effect concentration:

> 100 mg/L

Additional information

In the absence of short-term toxicity to aquatic invertebrates with the test substance, the endpoint has been assessed based on studies for read across substances representative of the main constituents, which can be categorised as phosphate esters (PSE i.e., mono- and di C16 PSE, K+: 10 -40%), alkyl esters (i.e., C16-18 linear and branched fatty acid esters: 39 -87%) andalcohols (i.e., C16 -18 linear or branched alcohol: 10 -30%). The results are presented below:

 

Constituent: PSE - read across study:

A study was conducted to determine the acute toxicity of read across substance, 'mono- and di- C16 PSE, K+ and H3PO4' (purity: 100%), to Daphnia magna, according to the OECD Guideline 202, in compliance with GLP. Twenty test organisms were exposed to each nominal read across substance concentrations of 0, 0.625, 1.25, 2.5, 5 and 10 mg/L for 48 h under static conditions. The concentrations of read across substance in the test vessels were measured using the high-performance liquid chromatography method. The mean measured concentrations of read across substance were determined to be 0, 0, 0, 0, 0.21 and 0.34 mg/L. Immobilisation was recorded at 24 and 48 h and compared with control values. Based on immobility compared to the control (p <0.05), the 48 h NOEC was determined to be 0.34 mg/L (measured) and the LOEC was >0.34 mg/L (measured). No other symptoms of toxicity were observed. Based on the study results, the 48 h EC50 was calculated to be >0.34 mg/L (measured). As no immobility or other signs of stress were observed in the control and dissolved oxygen concentration remained at ≥3 mg/L, this test has satisfied all validity criteria. Under the study conditions, the 48 h EC50 of the read across substance for Daphnia magna was determined to be >10 mg/L (nominal or >0.34 mg/Lmeasured) (Scymaris, 2018). Based on the results of the read across study, similar absence of acute toxicity to daphnia can be expected for the phosphate ester constituent of the test substance.

Constituent: Alkyl ester (linear) - read across study:

A study was conducted to determine the acute toxicity of the read across substance, 'tetradecyl myristate' (purity: 100%) to Daphnia magna, according to the OECD Guideline 202, in compliance with GLP. Daphnia magna were exposed under static conditions to the read across substance for 48 h. Four replicates of five individuals were tested for each treatment. Based on the results of a pre-test a limit test was conducted with a single test loading of 100 mg/L and a control of dilution water only. A water accommodated fraction (WAF) of the read across substance was prepared. The analytical determination of tetradecyl myristate was carried out by gas chromatography (GC) and electron impact ionization mass spectrometry detection (GC/EI-MS) using the mass spectrometer in the single ion monitoring (SIM) mode, which resulted in a measured geometric mean concentration of 0.128 µg/L. No immobilization was recorded after 48 h resulting in an EL50 of >0.128 µg/L (measured, geometric mean) and >100 mg/L (nominal), respectively. resulted in a measured geometric mean concentration of 0.128 µg/L. Under the conditions of the study, the 48-h EC50 and NOEC values of the read across substance were determined to be >100 mg/L (nominal or >0.128 µg/L measured) and 100 mg/L (nominal or 0.128 µg/L measured), respectively. Thus it can be concluded that no toxicological effects on aquatic invertebrates are expected up to the limit of water solubility for tetradecyl myristate (Schlechtriem, 2012).

Based on the results of the read across study, similar absence of acute toxicity to daphnia can be expected for the alkyl ester constituent of the test substance.

Constituent: Alkyl ester (branched) - read across study:

A study was conducted to determine the acute toxicity of read across substance, 'octyldodecyl isooctadecanoate (purity: assumed to be 100%)', to Daphnia magna, according to OECD Guideline 202 and EU Method C.2, in compliance with GLP. Twenty test organisms each were exposed to nominal concentration of 86 mg/L (i.e., corresponding to 100 µL/L) for 48 h under static conditions. Potassium dichromate was used as positive control substance. Using the available analytical techniques, it was not possible to detect concentrations of the read across substance in the test medium. Therefore, no chemical analysis was performed. The test solutions at 86 mg/L (nominal) exceeded the maximum water solubility of the test substance, therefore concentration identified as Water Accommodated Fraction (WAF) was considered for reporting the result. Immobilisation was recorded at 24 and 48 h and compared with control values. Based on immobility compared to the control, the 48 h EL50 was determined to be >86 mg/L (nominal) WAF. No other symptoms of toxicity were observed. Positive control substance results were considered valid. As no immobility or other signs of stress were observed in the control and dissolved oxygen concentration remained at ≥3 mg/L, this test had satisfied all the validity criteria. Under the study conditions, the 48 h EL50 of the read across substance for Daphnia magna was determined to be >86 mg/L (nominal) WAF (Notox, 1998). Based on the results of the read across study, similar absence of acute toxicity to daphnia can be expected for the alkyl ester constituent of the test substance.

 

Constituent: Alcohol - read across studies:

Study 1:A study was conducted to determine the long-term toxicity to aquatic invertebrates of the read across substance, Octan-1-ol (purity not specified), according to the method equivalent or similar to OECD Guideline 211. The read across substance at 0.4 to 50 mg/L nominal concentration was exposed to daphnia magna for 21 days under semi-static conditions in freshwater. Four replicates per read across substance and control group were used, where each vessel contained five test organisms. Samples were taken twice from selected concentration levels of the test series during the test period and analysed chemically. The first sampling was taken before the 7th day previous to any offspring appearance, the second sample was taken between Day 16 and Day 21. No further details on analytical methods were presented. Mortality, reproduction rate and appearance of offspring were evaluated daily. Student's t-test and U-test were used for determining the NOEC of reproduction rate and parent mortality. Primarily, the results were expressed with reference to the nominal concentration. If however, the chemical analysis showed a loss of tested substance greater than 20%, then the lowest analysed concentration (minimum value) obtained during the test was also reported for the NOEC. Based on mortality, reproduction rate and appearance of offspring, the 21-d NOEC was determined to be at 1.6 mg/L (nominal concentration), which was corresponding to 1 mg/L (minimum measured value). The most sensitive endpoint was the appearance of first offspring. Mortality of parent animals were noted to be 7.1%, while time to first brood release or time to hatch was 7-8 days for the appearance of first offspring and brood size was 88 offspring. Therefore, the study validity criteria were fulfilled. Under the study conditions, based on the effects of the read across substance on reproduction and survival of the freshwater test organism Daphnia magna, the 21-day NOEC value was determined to be at 1 mg/L (measured) (Kuhn, 1989). Based on the results of the read across study, similar absence of acute toxicity to daphnia can be expected for the alcohol constituent of the test substance.

Study 2: A study was conducted to determine the long term toxicity to aquatic invertebrates of the read across substances,'tetradecanol (Purity: 99.5%) and pentadecanol (Purity: 99.5%)', according to the OECD Guideline 211, in compliance with GLP. Ten Daphnia magna (24 h old) were exposed to four concentrations of each read across substances: 25, 69, 190, 500 µg C14 alcohol/L and 30, 65, 140 and 300 µg C15 alcohol/L and dilution water as control for a period of 21–23 days. The entire test was conducted under sterile conditions as much as possible (e.g., test solutions were added to sterilized test vessels to minimize presence of bacteria and algae that naturally degrade alcohol). Parental Daphnia magna were visually assessed for immobility and any other abnormalities in appearance and behaviour every day. At study termination, the length of the exposed adults was measured by digital photography and image analysis and compared with those of the control animals. The newborn Daphnia magna in each beaker were counted, inspected for abnormalities and removed at each daily renewal of the test solutions. The following endpoints observed in the reproduction test were evaluated quantitatively: mortality (immobility) of parental generation, age at first brood, total number of offspring per replicate, cumulative number of live offspring per surviving female at the time of recording, intrinsic rate of increase (r), and individual length of adults. All the test concentrations were sampled for chemical analysis three times a week at renewal of the test media (representative samples). Measurement was performed by GC–MS method and effect concentrations of the read across substance were based on mean measured concentrations. At the highest treatment level, 30% mortality was observed with C14 alcohol and no mortality occurred with C15 alcohol. Adult body length exhibited no significant dose related effects. The mean age at first brood was between for all test concentrations of C15 alcohol and between 8.1 and 8.4 days for C14 alcohol. No offspring mortality occurred. The cumulative number of offspring was significantly reduced by 14-15% at the third test concentrations (190 µg/L for C14 alcohol and 140 µg/L for C15 alcohol). At the highest test concentrations (500 µg/L for C14 alcohol and 300 µg/L for C15 alcohol), the decrease of reproduction per surviving female cumulated to 19-20%. In consequence, the cumulative number of offspring was considered to be the most sensitive endpoint. All validity criteria for the performance of the controls were fulfilled. However, for C15, the quality criterion for controls of 60 newborns per female was reached by Day 23 (although survival and other quality criteria were not impaired). The statistical power of the tests was generally high (B <0.2) due to the low variability of reproduction, the coefficients of variation of cumulative offspring number ranging from 6.5% to 14% in all treatments without mortality with C14 and C15. Under the study conditions, the 21-d EC10 values for C14 and C15 alcohol were determined to be 39 and >63 µg/L (measured) based on mortality and 6.3 and 12 µg/L (measured) based on effects on reproduction respectively (Schafers, 2009). Based on the results of the read across study, similar absence of acute toxicity to daphnia can be expected for the alcohol constituent of the test substance.

Supporting acute toxicity studies on alcohol from OECD SIDS dossier:

Study 1: A study was conducted to determine the acute toxicity of read across substance, 'Alcohols, C12-18' (purity not specified), to Daphnia magna, according to the EU Guideline 92/69/EWG, in compliance with GLP. Twenty test organisms were exposed to each nominal test substance concentrations of 0, 10, 30, 100, 1000, 3000 mg/L water accommodated fractions (WAF) for 48 h under static conditions. No solvent was used and instead, the water accommodated fraction (WAF) was used in the test chambers. The concentrations of test substance in the test vessels were measured using the fluid chromatography of dichloromethane extract. Measured concentrations in samples collected from the 10 and 100 mg/L chambers were less than 1% of nominal. The solubility of the lowest carbon chain length in this compound, C12, was 3 mg/L, therefore the EL50 was greater than the solubility limit. Immobilisation was recorded at 24 and 48 h and compared with control values. Based on the study results, the 48 h EL50 was calculated to be 40 mg/L WAF (nominal). As no immobility or other signs of stress were observed in the control and dissolved oxygen concentration remained at 99.3% mg/L, the test met all validity criteria. Under the study conditions, the 48 h EL50 was determined to be 40 mg/L (WAF; nominal) (OECD SIDS, 2006). Based on the results of the read across study, similar absence of acute toxicity to daphnia can be expected for the alcohol constituent of the test substance.

Study 2: A study was conducted to determine the acute toxicity of read across substance, 1-Decanol (purity not specified), to Daphnia magna, according to a method similar to OECD Guideline 202. Twenty daphnia magna were exposed to the range of test substance concentrations for 48 h, under static conditions. No analytical monitoring of the test substance was performed in the study. Immobilisation is recorded at 24 h and 48 h and compared with control values. The results were analysed in order to calculate the EC50 at 48 h. Under the study conditions, the 48 h EC50 and EC100 was determined to be 2.9 and 29 mg/L respectively (nominal) (OECD SIDS, 2006). Based on the results of the read across study, similar absence of acute toxicity to daphnia can be expected for the alcohol constituent of the test substance.

Further, data across several alcohols indicate that, alcohols with a carbon chain length greater than C14 are expected to be non-toxic at the solubility limit (Schäfers et al., 2009). Therefore, given the low solubilities of the constituents hexadecan-1-ol (0.0412 mg/L) and isostearyl alcohol (0.01745 mg/L) the L(E)C50 value can be considered to be >0.0412 mg/L. Further, the presence of branched alcohols in the test substance are not expected to show difference in toxicity compared to the linear alcohols.

Overall, based on the available weight of evidence information from studies for substances representative of the main constituents, a similar absence of toxicity up to the highest tested or highest soluble concentrations can be expected for the test substance, ‘Reaction products of hexadecyl dihydrogen phosphate, dihexadecyl hydrogen phosphate, hexadecan-1-ol, stearic acid, esters of C18 (branched and linear) fatty acids with C18 (branched and linear) alcohols, and potassium hydroxide’. However, given the UVCB nature of the test substance with variable solubilities for individual constituents, the respective nominal values have been considered for hazard/risk assessment. The phosphate esters and alkyl ester constituents did not show toxicity at >10 mg/L or >100 mg/L (nominal) respectively and the alcohols did not show toxicity at their maximum soluble concentrations. Further, considering that the alkyl esters make up more than 80% of the composition, the 48 h EC50 value of >100 mg/L (nominal) determined in a study with tetradecyl myristate has been considered further for hazard/risk assessment.