[No public or meaningful name is available]

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to fish

Type of information:

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

Adequacy of study:

weight of evidence

Study period:

From Janaury 14, 2013 to February 27, 2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

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 203 (Fish, Acute Toxicity Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Institut für Biologische Analytik und Consulting, IBACON GmbH, Arheilger Weg 17, 64380 Rossdorf, Germany

Analytical monitoring:

yes

Details on sampling:

Duplicate samples from the freshly prepared test medium of the only test concentration and the control were taken at the start of the test. For the determination of the stability of the test substance under the test conditions and of the maintenance of the test substance concentration during the test period, duplicate samples from the test medium of the only test concentration and the control were collected at the end of the test (after 96 h) from the approximate centre of the aquaria.

Vehicle:

no

Details on test solutions:

Preparation and application of test solution
- Method:
The test substance was not well soluble in test water. Therefore, a stock suspension of 100 mg test substance/L was prepared by suspending 1.0002 g of test substance in 10.0002 L test water for preparing the test concentration. The stock suspension was stirred for 24 h to dissolve as much test substance as possible. Then, the undissolved test substance was separated by filtration (0.45 µm cellulose acetate nitrate filter). The test media were prepared just before introduction of the test fish (= start of the test).

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

Test organism
- Common name: Rainbow trout
- Source: The test fish were obtained from Forellenzuchtbetrieb Störk, 88348 Bad Saulgau, Germany
- Age at study initiation: Juveniles
- Length at study initiation: 4.83 cm ± 0.30 cm
- Weight at study initiation: 1.12 ± 0.23 g
- Feeding during test: no

Acclimation
- Acclimation period: All fish were obtained and held in the laboratory for at least 12 d before the start of the test.
- Acclimation conditions: same as test conditions
- Feeding frequency: three times per week or daily until 24 h before the test was started
- Health during acclimation: During the last 7 d prior to the start of the test no fish died in the test fish batch. Therefore the mortalities in the fish batch were below 5 % and the fish batch was accepted.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Hardness:

2.5 mmol/L (= 250.0 mg/L) as CaCO3

Test temperature:

15°C

pH:

7.8 to 8.2

Dissolved oxygen:

93 to 98 % of the air saturation value

Nominal and measured concentrations:

A filtrate of a supersaturated stock suspension of nominal 100 mg/L. No measured concentrations are available (below LOD).

Details on test conditions:

Test system
- Test vessel: 12 L glass aquaria with 10 L test medium
- Aeration: The test media were slightly aerated during the test.
- No. of organisms per vessel: 7
- No. of vessels per concentration: 1
- No. of vessels per control: 1

Test medium / water parameters
- Chlorine: 2.0 mmol/L (= 294.0 mg/L)
- Alkalinity: 0.8 mmol/L
- Ca/mg ratio: 4: 1 (based on molarity)
- Conductivity: < 10 µScm-1
- Culture medium different from test medium: no

Other test conditions
- Adjustment of pH:
- Photoperiod: 16 h light: 8 h dark; 30 min dawn/dusk period was provided
- Light intensity: 780 to 810 lux

Test concentrations
- Test concentrations:
The test substance was not well soluble in test water. Therefore, a stock suspension of 100 mg test substance/L was prepared by suspending 1.0002 g of test substance in 10.0002 L test water for preparing the test concentration. The stock suspension was stirred for 24 h to dissolve as much test substance as possible. Then, the undissolved test substance was separated by filtration (0.45 µm cellulose acetate nitrate filter). The test media were prepared just before introduction of the test fish (= start of the test).

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: up to the water solubility of the test substance no mortality of the test animals occurred.

Key result

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

ca. 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: up to the water solubility of the test substance no mortality of the test animals occurred.

Key result

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

ca. 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Sublethal observations / clinical signs:

Results and discussion

Validity criteria of the study

Control: In the control no fish died until the end of the test.

Dissolved Oxygen Concentration: The dissolved oxygen concentration in the test media did not fall below 93 % of air saturation value during the test.

 

Biological results

Sublethal Effects: In the control and the only test concentration of nominal 100 mg test substance/L, all fish survived until the end of the experiment and showed no sublethal effects during the exposure time.

96 h LC50: > 100 mg test substance/L (nominal)

95 % Confidence Interval: Not determinable

96 h LC10: > 100 mg test substance/L (nominal)

95 % Confidence Interval: Not determinable

96 h LC0: 100 mg test substance/L (nominal)

96 h LC100: > 100 mg test substance/L (nominal)

96 h NOEC: 100 mg test substance/L (nominal)

96 h LOEC: > 100 mg test substance/L (nominal)

Table: Mortality and sublethal effects

Exposure time (h)	Effects	Nominal concentration (mg/L)
		Control	100
0 h	#Mortality	0	0
	#Sublethal effects	0	0
2 h	#Mortality	0	0
	#Sublethal effects	0	0
24 h	#Mortality	0	0
	#Sublethal effects	0	0
48 h	#Mortality	0	0
	#Sublethal effects	0	0
72 h	#Mortality	0	0
	#Sublethal effects	0	0
96 h	#Mortality	0	0
	#Sublethal effects	0	0

Analytical Results

Determination of the test substance: Based on the results of LC-MS/MS measurements the concentration of the test substance was determined using a calibration curve.

Calibration range: 0.005 to 0.15 mg test substance/L

Linearity of response: Correlation of peak area of different standard solutions with their corresponding concentrations, using a linear regression

Regression coefficient (r2): 0.9994

Calibration curve: y = 12112 * x + 10276 (see also Figure 1)

Limit of detection: 1.6 μg test substance/L

Limit of quantification: 18.83 μg test substance/L

The Limit of quantification was calculated according to DIN 32645 from the calibration curve. A preparation of fortified samples was not possible, since the test substance is not soluble in the test medium of the aquatic test.

Mean Recovery in the Fortified

Samples: 60 % (n = 10, RSD 16 %)

The measurements showed unsatisfying recoveries. This might be caused by the bad solubility of the test substance in test medium of the aquatic test.

As the solubility of the test substance in the test medium is low, the concentrations of dissolved test substance were below the limit of detection. However, all reported results refer to nominal concentrations.

Validity criteria fulfilled:

yes

Conclusions:

Based on the results of the read across study, the 96 h LC50 and NOEC for the PSE constituent of the test substance, can be considered to be >100 mg/L (nominal) and 100 mg/L (nominal), respectively.

Executive summary:

A study was conducted to determine the acute toxic potential of read across substance, mono- and di- C16 PSE, K+ (purity: ca. 85%) to rainbow trout (Oncorhynchus mykiss), according to OECD Guideline 203 and EU Method C.1, in compliance with GLP. Seven juvenile fish were exposed to the test substance at nominal loading rate of 100 mg/L for 96 h under static conditions. The test fish were observed at test start and after approximately 2, 24, 48, 72 and 96 h test duration for sublethal effects and mortality. As the test substance is poorly water soluble, the water-accommodated fraction (WAF) was tested. The quantification of the test substance was performed in duplicates using liquid chromatography (LC-MS/MS-method). The concentrations of dissolved test substance were found to be below the Limit of Detection (1.6 μg test substance/L). Therefore, all results were presented in terms of nominal loading rates. In the control and the only test concentration of nominal 100 mg test substance/L, all fish survived until the end of the experiment and showed no sublethal effects during the exposure time. The pH and the oxygen values were in normal ranges. Under the study conditions, the 96 h LC50 and NOEC for the read across substance were determined to be >100 mg/L (nominal) and 100 mg/L (nominal), respectively (Kuhl and Wydra, 2013). Based on the results of the read across study, similar 96 h LC50 and NOEC values can be expected for the PSE constituent of the test substance.

Reference 2

Endpoint:

short-term toxicity to fish

Type of information:

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

Adequacy of study:

weight of evidence

Study period:

From April 20, 1998 to April 24, 1998

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

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 203 (Fish, Acute Toxicity Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Two stock solutions were prepared in water at nominal 1 and 100 mg/L. applying magnetic stirring for ca. 23 h to achieve maximum soluble fraction of the test substance in the test medium. Subsequently, these stock solutions were transferred to a separating funnel and left tot stabilize for ca. 4 h. The water phase in the middle of the separating funnel was used for testing. Both solutions were clear and colourless.
- Eluate: no
- Differential loading: yes
- Controls: yes, water control

Test organisms (species):

Cyprinus carpio

Details on test organisms:

TEST ORGANISM
- Common name: carp
- Source: Obtained from Bio International, Roermond, The Netherlands (F1 from a single parent-pair nred om UV-treated water)
- Length at study initiation: 2.09 +/- 0.10 cm
- Weight at study initiation: 0.22 +/- 0.04 g
- Feeding during test: none

ACCLIMATION
- Acclimation period: At least 12 days after delivery.
- Acclimation conditions (same as test or not): same as test
- Type and amount of food: Commercial fish feed "Trouvit"
- Feeding frequency: daily
- Health during acclimation (any mortality observed): Mortality seven days prior to the start of the test was less than 5%.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

250 mg CaCO3/L

Test temperature:

20.1 - 20.6 °C

pH:

7.8 - 8.1

Dissolved oxygen:

6.9 - 9.3 mg O2/L

Nominal and measured concentrations:

Nominal: 0, 10 and 100 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: all-glass vessels
- Material, size, headspace, fill volume: all glass, 4 L, headspace: 1.5 L, fill volume: 2.5 L
- Aeration: no
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: 0.77 g/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ISO-medium, formulated using Milli-RO water (tap water purified by reverse osmosis) with the following composition: Ca2+ 80 mg/L, Mg2+ 12 mg/L, Na+ 15 mg/L, K+ 3 mg/L, Cl- 145 mg/L, SO4 2- 49 mg/L, HCO3 47 mg/L
- Culture medium different from test medium: no
- Intervals of water quality measurement: Oxygen, pH, nitrate and nitrite and ammonia concentration were measured once a week.

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

EFFECT PARAMETERS MEASURED: Mortality was observed after 2, 24, 48, 72 and 96 h.

Reference substance (positive control):

yes

Remarks:

Pentachlorophenol

Key result

Duration:

96 h

Dose descriptor:

LL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Water Accommodated Fraction (WAF)

Basis for effect:

mortality (fish)

Details on results:

- Mortality of control: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: A thin test substance film was observed on the surface after 24 h for the 10 mg/L test concentration after 24 h. A test substance film was observed on the surface for the 100 mg/L test concentration after 24 h.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Mortality: yes
- LC50: Between 0.10 - 0.15 mg/L

Validity criteria fulfilled:

not specified

Conclusions:

Based on the results of the read across study, similar LC50 value of >100 mg/L (nominal) can be expected for isoalkylester constituent.of the test substance.

Executive summary:

A study was conducted to determine the acute toxic potential of read across substance, ‘octyldodecyl isooctadecanoate’ (purity: assumed to be 100%) to Cyprinus carpio, according to OECD Guideline 203 and EU Method C.1, in compliance with GLP. Three replicates of seven fish were exposed to the read across substance at nominal loading rate concentrations of 0, 10 and 100 mg/L for 96 h under static conditions. The test fish were observed at test start and after approximately 2, 24, 48, 72 and 96 h test duration for mortality. As the read across substance is poorly water soluble, the water-accommodated fraction (WAF) was tested. No chemical analysis was performed, as it was not possible to detect the read across substance concentrations in the test medium using the available analytical techniques.No mortality was observed in the control and the test groups during the exposure period. The pH and the oxygen values were in normal ranges. Under the study conditions, the 96 h LL50 was determined to be >100 mg/L (nominal) (Notox, 1998). Based on the results of the read across study, similar LC50 and NOEC values can be expected for the isoalkylester constituent of the test substance.

Reference 3

Endpoint:

short-term toxicity to fish

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 14 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 203 (Fish, Acute Toxicity Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Remarks:

GC/MS with a Limit of quantification (LOQ) of 0.1 µg/L

Details on sampling:

- Concentrations: control, 100 mg/L
- Sampling method: Samplings out of the test aquaria were done by the staff of the analytical laboratory using a 'research 5000' pipette.
The aqueous samples of 5.0 mL were pipetted directly into the extraction (centrifuge) tubes; the pipette tips were rinsed multiple and the surface was wiped with a lint free paper towel prior taking the analytical sub-sample.
- Sample storage conditions before analysis: The samples were processed and measured directly after sampling without any time delay.

Vehicle:

no

Details on test solutions:

Preparation and application of test solution
- Method: Due to the very low water solubility of the test substance, a Water Accommodated Fraction (WAF) of the test substance was applied. The test substance pellets were crushed by mortar and pestle and the WAF prepared with the test substance powder. Therefore, 1050 mg test substance powder was weighed in a glass bottle. The bottle was filled with 10.5 L water. The solution was stirred slowly to avoid bubble and foam formation using a star shape magnetic stirring bar for about 48 h at room temperature (about 20 °C). After stirring the test solution was filtered by using a 0.2 µm filter (Sartolab 150v, Vacuum capsule sterile, Charge 21/000143, Sartorius). 10 L of the filtrate was used for the study resulting in a nominal concentration of 100 mg/L.
- Eluate: no
- Differential loading: yes
- Controls: yes, test medium control

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

Test organism
- Common name: zebra fish
- Source: Test facility bred.
- Length at study initiation: 2.2 - 2.6 cm (SD: 0.1 cm)
- Weight at study initiation: 0.11 g (mean; SD: 0.02 g)
- Method of breeding: The fish were held in water of the same quality as used in the test (purified drinking water) until the start of exposure. Fish were fed ad libitum throughout the holding period with live brine shrimp (Artemia spp.) nauplii and ground flake food TetraMin (Tetra Werke, Melle, Germany) once daily. No feeding of the fish occurred 24 h before test start.
- Feeding during test: no

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Hardness:

1 mmol/L

Test temperature:

23.1 - 23.2°C

pH:

7.9 - 8.6

Dissolved oxygen:

74 - 100%

Nominal and measured concentrations:

Nominal: control, 100 mg/L
Measured: < LOQ, 0.189 µg/L (0 h), < LOQ (96 h)

Details on test conditions:

Test system
- Test vessel Material, size, headspace, fill volume: full glass aquaria, 15 L, headspace: 5 L, fill volume: 10 L
- Aeration: slightly aerated via glass capillary
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: 0.07 g/L

Test medium / Water parameters
- Source/preparation of dilution water: Purified drinking water was used according to the OECD-Guideline. The purification includes filtration with activated charcoal, passage through a lime-stone column and aeration until oxygen saturation. To avoid copper contamination, plastic water pipes are used for the test facilities.
- Metals: < detection limit
- Chlorine: < 0.02 mg/L
- Alkalinity: 2 mmol/L
- Conductivity: 244.5 µS/cm
- Culture medium different from test medium: no

Other test conditions
- Adjustment of pH: no
- Photoperiod: 12 h light / 12 h dark

Effect parameters measured
Mortality and clinical signs (sub-lethal and abnormal behavior) were recorded at 3 h, 24 h, 48 h, 72 h and 96 h after fish addition.

Test concentrations
- Range finding study
- Test concentrations: 1, 10, 100 mg/L
- Results used to determine the conditions for the definitive study: No mortality, clinical signs or any distress were observed throughout the range-finding test.

Validity of the test
- Mortality in the control group must not exceed 10 % at the end of the test
- The dissolved oxygen concentration must have been at least 60 % of the air saturation value throughout the test in control and test vessels.

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 0.189 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

other: Water Accommodated Fraction

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

>= 0.189 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

other: Water Accommodated Fraction

Basis for effect:

mortality (fish)

Details on results:

- Behavioural abnormalities: No clinical signs nor mortality could be were observed throughout the study.
- Mortality of control: 0%

Validity
All validity criteria according the OECD guideline were met:
- Mortality did not exceed 10 % in the controls.
- The dissolved oxygen concentration was ≥ 60 % in test vessels throughout the test

Reported statistics and error estimates:

In the acute toxicity limit test no statistical analysis was performed to determine LC10 and LC50 values. The LC50 is reported as being > concentration tested. The evaluation of the effects is based on nominal loading and measured concentration for the test solution preparation.

Sublethal observations / clinical signs:

Results

The oxygen saturation in all test vessels was between 74% and 100%. The temperature was in the range of 23.1 until 23.2°C. The pH in the test vessels was between 7.9 and 8.6, thus was within the frame stated in the respective guideline. The pH value in the test vessel was not  affected by the test substance and remained constant until the end of the study.

Fish size at test start

Prior to test start, a subsample of the fish batch used in the test, was weighed out and lengths were measured. The fish lengths were in the range of 2.2 – 2.6 cm. The mean weight resulted in a loading of 0.07 g/L test medium, and therefore was in line with the density given by the respective guidelines.

Fish lengths [cm] and weights [g] at test start, n=7

	length [cm]	weight [g]
total min	2.2	0.08
total max	2.6	0.14
total mean	2.3	0.11
total SD	0.1	0.02

SD: Standard deviation

Effects of fish

Neither clinical signs nor mortality could be observed throughout the study.

Clinical signs during the test period of 96 h (number of fish per treatment n=7)

Nominal concentration  [mg/L]	Test duration
	3 h	24 h	48 h	72 h	96 h
Control	nd.	nd.	nd.	nd.	nd.
100	nd.	nd.	nd.	nd.	nd.

nd.: no symptoms detected

Cumulative mortality during the test period of 96 h (number of fish per treatment = 7)

Nominal	Test duration
concentration [mg/L]	3 h	24 h	48 h	72 h	96 h
Control	0	0	0	0	0
100	0	0	0	0	0

Effect concentrations

The effects of the test substance on fish were assessed in the acute limit study with zebrafish (Danio rerio), which were exposed to the Water Accommodated Fraction (WAF) of the test substance at the single treatment of nominal 100 mg test substance/L. The chemical analysis of the dissolved components of tetradecyl myristate in the WAF in the fresh and aged test media revealed measured concentrations of ≤0.01 % of the nominal loading of 100 mg/L and <LOQ at the start and the end of the test respectively. The LOQ was 0.1 µg/L. The LC50 of the test substance was determined to be >0.189 µg/L (LL50 >100 mg/L (nominal)). The NOEC based on the visual observation is reported as >=0.189 µg/L (NOEL >=100 mg/L).

Analytical concentrations in the stock solution at the beginning of the study and at the end of exposure.

Nominal conc. 100 mg/L	Samp- ling time	Conc. factor, FC	Quan. data, CGC/MS [ng/mL]	Analyzed a.s. conc., CW [µg/L]	Percent of nominal
Stock, 100 mg/L *)	t0 fresh test media	25.0	4.08	0.163	1.6E-4
Control		25.0	n.d. **)	< LOQ	-
Test media, 100 mg/L		25.0	4.73	0.189	1.9E-4
Control, sample 1	96 h, aged test media	25.0	n.d.	< LOQ	-
Control, sample 2		25.0	n.d.	< LOQ	-
Control, sample 3		25.0	n.d.	< LOQ	-
Test media, sample 1		25.0	n.d.	< LOQ	-
Test media, sample 2		25.0	n.d.	< LOQ	-
Test media, sample 3		25.0	n.d.	< LOQ	-

*) Analytical sub-sample was taken before the stock WAF was distributed into the test aquaria

**) n.d. = not detected

Conclusion

In the 96 h acute toxicity limit test, conducted according to the OECD guideline 203, zebrafish (Danio rerio) were exposed to the test substance at nominal concentration of 100 mg/L under static conditions for the period of 96 hours. Neither clinical signs nor mortality could be observed throughout the study. Based on the results of this study, the LC50 of the test substance was determined to be > 0.189 µg/L (LL50 > 100 mg/L (nominal)). The NOEC based on the visual observation is reported as >=0.189 µg/L (NOEL >=100 mg/L). This toxicity study is classified as acceptable and satisfies the guideline requirement for “Fish, Acute Toxicity Test”.

Validity criteria fulfilled:

yes

Conclusions:

Based on the results of the read across study, the LC50 and NOEC of the ester constituent of the test substance, were determined to be >100 mg/L (nominal or >0.189 µg/L measured) and >=100 mg/L (nominal or 0.189 µg/L measured), respectively.

Executive summary:

A study was conducted to determine the acute toxicity of the read across substance, tetradecyl myristate to zebrafish, according to the OECD guideline 203, in compliance with GLP. Due to the very low water solubility of the read across substance, a Water Accommodated Fraction (WAF) of the read across substance was applied. The read across substance was tested in a limit test at the nominal loading concentration of 100 mg a.s./L under static conditions for 96 h. Seven fish were used at the test concentration and in the control (dilution water). 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. The dissolved components of the read across substance in the WAF were analysed in the freshly prepared test solutions at test start and in the aged test media after 96 h at end of the test. The read across substance measured in the test media was <0.01 % of the nominal loading of 100 mg/L at the start and <LOQ at the end of the test. The effect evaluation was based on the concentration of 0.189 µg/L. There were no toxic effects of the read across substance on zebrafish at the loading of 100 mg read across substance/L (nominal) and 0.189 µg/L (measured). No clinical signs or mortality were observed throughout the study. No toxicity effect value LC50 for acute toxicity was calculated in this limit study with zebrafish exposed for a test period of 96 h under static conditions. Mortality did not exceed 10 % in the controls and the dissolved oxygen concentration was ≥ 60 % in test vessels throughout the test. Therefore, all validity criteria of the guideline were fulfilled (IME, 2012). Based on the results of the read across study, the LC50 and NOEC of theester constituent of the test substance, were determined to be >100 mg/L (nominal or >0.189 µg/L measured) and >=100 mg/L (nominal or 0.189 µg/L measured), respectively.

Reference 4

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1996

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

not specified

GLP compliance:

yes

Analytical monitoring:

not specified

Vehicle:

yes

Remarks:

Tetrahydrofuran

Details on test solutions:

Vehicle, solvent: Tetrahydrofuran
Concentration of vehicle, solvent: 100 uL/L

Dilution water
Source: Dechlorinated laboratory tap water
Aeration: Test vessels aerated via narrow bore glass tubes

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

Strain: Oncorhynchus mykiss
Supplier: Donnington Fish Farm, Upper Swell, Gloucestershire, UK
Weight: 1.20 g
Feeding: Commercial trout pellets
Pretreatment: Acclimatised to test conditions for 1 week prior to test
Feeding during test: None
Control group: 1 control and 1 solvent control group
Photoperiod: 16 h light and 8 h darkness
Renewal of test solution: Daily
Exposure vessel type: 20 L glass vessels
Number of replicates: 2
Fish per replicate: 10

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Post exposure observation period:

Mortalities and adverse reactions to exposure were recorded at 3, 6, 24, 48, 72 and 96 h

Hardness:

136 mg/L CaCO3

Test temperature:

13-14 deg C

pH:

7.4 - 7.9

Dissolved oxygen:

9.2 - 10.1 mg O2/L

Salinity:

80 mg/L

Conductivity:

405 uS/cm

Nominal and measured concentrations:

0.4 mg/L (nominal, limit of solubility)

Details on test conditions:

Number of replicates: 2
Fish per replicate: 10

Reference substance (positive control):

not specified

Key result

Duration:

96 d

Dose descriptor:

LC50

Effect conc.:

> 0.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mortality (fish)

Remarks on result:

other: i.e., equivalent to >0.38 mg a.i./L

Key result

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

ca. 0.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mortality (fish)

Remarks on result:

other: i.e., equivalent to 0.38 mg a.i./L

Validity criteria fulfilled:

not specified

Conclusions:

Under the study conditions, the 96 h LC50 and NOEC for the test substance were determined to be >0.4 mg/L (i.e., >0.38 mg a.i./L) and 0.4 mg/L (i.e., 0.38 mg a.i./L) (nominal), respectively.

Executive summary:

A study was conducted to determine the acute toxic potential of test substance, hexadecan-1-ol (purity: >95%) to Salmo gairdneri (Oncorhynchus mykiss), according to OECD Guideline 203, in compliance with GLP. Twenty Salmo gairdneri were exposed to the test substance at nominal concentration of 0.4 mg/L for 96 h under semi-static conditions. The solubility of the test substance was about 0.01 mg/L. Analytical monitoring data was not specified in the study. The test fish were observed for mortality and other adverse reactions after 3, 6, 24, 48, 72 and 96 h test duration. In the control and the only test concentration of nominal 0.4 mg/L, all fish survived until the end of the experiment and showed no sub lethal effects during the exposure time. The pH and the oxygen values were in normal ranges. Under the study conditions, the 96 h LC50 and NOEC values of the test substance were determined to be >0.4 mg/L (i.e., >0.38 mg a.i./L) and 0.4 mg/L (i.e., 0.38 mg a.i./L) (nominal), respectively (OECD SIDS, 2006).

Reference 5

Endpoint:

short-term toxicity to fish

Type of information:

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

Adequacy of study:

weight of evidence

Study period:

no information available

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

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:

other: US EPA 1975

Deviations:

not specified

GLP compliance:

not specified

Analytical monitoring:

yes

Details on sampling:

Concentrations of chemicals in water were measured in each tank throughout the test in all exposure treatments, although analysis details (methods and results) were not reported.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: the test substance was tested at a level below the limit of solubility, no details on the method of dispersion were provided in the text.

Test organisms (species):

Pimephales promelas

Details on test organisms:

Test organism
- Common name: fathead minnow
- Source: Environmental Research Laboratory-Duluth culture
- Age at study initiation (mean): 30d
- Weight at study initiation (mean): 0.12 g
- Feeding during test: none

Acclimation
- Acclimation period: not reported

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

56.3 mg/L as CaCO3

Test temperature:

25 ± 1°C

pH:

7.5

Dissolved oxygen:

>60%

Nominal and measured concentrations:

Five concentrations were tested in order to obtain 0 to 100% mortality, the values are not reported

Details on test conditions:

TEST SYSTEM
- Type of flow-through: proportional diluter
- No. of organisms per vessel: <30 fish per concentration
- No. of vessels per control: 2
- No. of vessels per concentration: 2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Lake Superior
- Alkalinity: 42.2 mg/L
- Intervals of water quality measurement: daily

EFFECT PARAMETERS MEASURED:
- Mortality was recorded at 1, 3, 6, 12, 24, 48, 72 and 96 h

Reference substance (positive control):

not specified

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

ca. 1.01 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

not specified

Basis for effect:

mortality (fish)

Details on results:

The mortality values in the control are not reported; however fish mortalities were monitored daily.

Reported statistics and error estimates:

The LC50 was calculated using the trimmed Spearman-Karber method (Hamilton et al. 1977) on a PDP-11/70 computer.

Validity criteria fulfilled:

not specified

Conclusions:

Based on the results of the read across study, the 96 h LC50 for the alcohol constituent is considered to be 1.01 mg/L (measured).

Executive summary:

A study was conducted to determine the acute toxicity to Pimephales promelas of the read across substance dodecanol, according to US EPA 1975 method. In a 96 h flow-through test, five concentrations were exposed to the test organism in replicates in order to obtain 0 to 100% mortality, however the values were not reported by the study author. The read across substance was tested at a level below the limit of solubility, no details on the method of dispersion were provided. Concentrations of chemicals in water were measured in each vessel in all exposure treatments throughout the test, although analysis results were not provided. The LC50 was calculated using the trimmed Spearman-Karber method on a PDP-11/70 computer. The mortality values in the control are not reported; however fish mortalities were monitored daily. Mortality was recorded at 1, 3, 6, 12, 24, 48, 72 and 96 h after exposure. Under the study conditions, the 96 h LC50 for the read across substance was determined to be 1.01 mg/L (measured) (Veith, 1983). Based on the results of the read across study, the 96 h LC50 for the test substance, hexadecanol is considered to be 1.01 mg/L (measured). Based on the results of the read across study, similar 96 h LC50 and NOEC values can be expected for the constituent alcohol.

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 100 mg/L (nominal) 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 96 h LC50 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 fish

Fresh water fish

Dose descriptor:

LC50

Effect concentration:

ca. 100 mg/L

Additional information

In the absence of short term fish toxicity study 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 toxic potential of read across substance, mono- and di- C16 PSE, K+ (purity: ca. 85%) to rainbow trout (Oncorhynchus mykiss), according to OECD Guideline 203 and EU Method C.1, in compliance with GLP. Seven juvenile fish were exposed to the test substance at nominal loading rate of 100 mg/L for 96 h under static conditions. The test fish were observed at test start and after approximately 2, 24, 48, 72 and 96 h test duration for sublethal effects and mortality. As the test substance is poorly water soluble, the water-accommodated fraction (WAF) was tested. The quantification of the test substance was performed in duplicates using liquid chromatography (LC-MS/MS-method). The concentrations of dissolved test substance were found to be below the Limit of Detection (1.6 μg test substance/L). Therefore, all results were presented in terms of nominal loading rates. In the control and the only test concentration of nominal 100 mg test substance/L, all fish survived until the end of the experiment and showed no sublethal effects during the exposure time. The pH and the oxygen values were in normal ranges. Under the study conditions, the 96 h LC50 and NOEC for the read across substance were determined to be >100 mg/L (nominal) and 100 mg/L (nominal), respectively (Kuhl and Wydra, 2013). Based on the results of the read across study, similar absence of acute toxicity to fish 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 to zebrafish, according to the OECD guideline 203, in compliance with GLP. Due to the very low water solubility of the read across substance, a Water Accommodated Fraction (WAF) of the read across substance was applied. The read across substance was tested in a limit test at the nominal loading concentration of 100 mg a.s./L under static conditions for 96 h. Seven fish were used at the test concentration and in the control (dilution water). 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. The dissolved components of the read across substance in the WAF were analysed in the freshly prepared test solutions at test start and in the aged test media after 96 h at end of the test. The read across substance measured in the test media was <0.01 % of the nominal loading of 100 mg/L at the start and <LOQ at the end of the test. The effect evaluation was based on the concentration of 0.189 µg/L. There were no toxic effects of the read across substance on zebrafish at the loading of 100 mg read across substance/L (nominal) and 0.189 µg/L (measured). No clinical signs or mortality were observed throughout the study. No toxicity effect value LC50 for acute toxicity was calculated in this limit study with zebrafish exposed for a test period of 96 h under static conditions. Mortality did not exceed 10 % in the controls and the dissolved oxygen concentration was ≥ 60 % in test vessels throughout the test. Therefore, all validity criteria of the guideline were fulfilled (IME, 2012). Based on the results of the read across study, the LC50 and NOEC of the ester constituent of the test substance, were determined to be >100 mg/L (nominal or >0.189 µg/L measured) and >=100 mg/L (nominal or 0.189 µg/L measured), respectively. Based on the results of the read across study, similar absence of acute toxicity to fish 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 toxic potential of read across substance, ‘octyldodecyl isooctadecanoate’ (purity: assumed to be 100%) to Cyprinus carpio, according to OECD Guideline 203 and EU Method C.1, in compliance with GLP. Three replicates of seven fish were exposed to the read across substance at nominal loading rate concentrations of 0, 10 and 100 mg/L for 96 h under static conditions. The test fish were observed at test start and after approximately 2, 24, 48, 72 and 96 h test duration for mortality. As the read across substance is poorly water soluble, the water-accommodated fraction (WAF) was tested. No chemical analysis was performed, as it was not possible to detect the read across substance concentrations in the test medium using the available analytical techniques.No mortality was observed in the control and the test groups during the exposure period. The pH and the oxygen values were in normal ranges. Under the study conditions, the 96 h LL50 was determined to be >100 mg/L (nominal) (Notox, 1998). Based on the results of the read across study, similar absence of acute toxicity to fish can be expected for the alkyl ester constituent of the test substance.

Constituent: Alcohol - read across study

A study was conducted to determine the acute toxicity to Pimephales promelas of the read across substance dodecanol, according to US EPA 1975 method. In a 96 h flow-through test, five concentrations were exposed to the test organism in replicates in order to obtain 0 to 100% mortality, however the values were not reported by the study author. The read across substance was tested at a level below the limit of solubility, no details on the method of dispersion were provided. Concentrations of chemicals in water were measured in each vessel in all exposure treatments throughout the test, although analysis results were not provided. The LC50 was calculated using the trimmed Spearman-Karber method on a PDP-11/70 computer. The mortality values in the control are not reported; however fish mortalities were monitored daily. Mortality was recorded at 1, 3, 6, 12, 24, 48, 72 and 96 h after exposure. Under the study conditions, the 96 h LC50 for the read across substance was determined to be 1.01 mg/L (measured) (Veith, 1983). Based on the results of the read across study, the 96 h LC50 for the test substance, hexadecanol is considered to be 1.01 mg/L (measured). Based on the results of the read across study, similar absence of acute toxicity to fish can be expected for the alcohol constituent of the test substance.

Supporting study on hexadecan-1-ol from OECD SIDS dossier:

A study was conducted to determine the acute toxic potential of test substance, hexadecan-1-ol (purity: >95%) to Salmo gairdneri (Oncorhynchus mykiss), according to OECD Guideline 203, in compliance with GLP. Twenty Salmo gairdneri were exposed to the test substance at nominal concentration of 0.4 mg/L for 96 h undersemi-static conditions.The solubility of the test substance was about 0.01 mg/L. Analytical monitoring data was not specified in the study. The test fish were observed for mortality and other adverse reactions after 3, 6, 24, 48, 72 and 96 h test duration. In the control and the only test concentration of nominal 0.4 mg/L, all fish survived until the end of the experiment and showed no sublethal effects during the exposure time. The pH and the oxygen values were in normal ranges. Under the study conditions, the 96 h LC50 and NOEC values of the test substance were determined to be >0.4 mg/L(i.e., >0.38 mg a.i./L) and 0.4 mg/L(i.e., 0.38 mg a.i./L) (nominal), respectively (OECD SIDS, 2006). Based on the results of the read across study, similar absence of acute toxicity to fish 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 LC50 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 100 mg/L (nominal) 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 96 h LC50 value of >100 mg/L (nominal) determined in a study with tetradecyl myristate has been considered further for hazard/risk assessment.