Reaction mass of hexadecyl dihydrogen phosphate and cetyl alcohol (mono- C16 PSE and C16-OH)

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From February 29, 2000 to April 4, 2000

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

yes

Remarks:

acetone

Details on test solutions:

Nominal exposure concentraations were of: 1, 10, 100 mg/L.
The 100 mg/L nominal stock solution was prepared by dosing 0.1 mL of acetone onto 0.1 g of the test substance and adding 1 L of dilution water. To further aid dissolution the stock solution was stirred for approximately 3 h and sonicated for approximately 30 minutes. The 1 and 10 mg/L test media were prepared by dilution of the 100 mg/L test medium. Two control treatments were prepared, one containing acetone at the same concentration as the test media (0.1 mL/L) and one containing water only.

Test organisms (species):

Daphnia magna

Test type:

static

Water media type:

not specified

Limit test:

no

Total exposure duration:

48 h

Hardness:

CaCO3 216-221 mg/L

Test temperature:

19.3-21.0 °C

pH:

7.6-8.2

Dissolved oxygen:

86-93 %

Nominal and measured concentrations:

Nominal exposure concentraations: 1, 10, 100 mg/L.

Details on test conditions:

Five Daphnia magna less than 24 h old, were introduced into duplicate glass crystallising dishes, each containing approximately 100 mL of test medium. D. magna were observed after 24 and 48 h. D. magna were considered to be immobile if they did not swim during a 15 s period of observation.

Reference substance (positive control):

no

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

At the start of the exposure period the control, solvent control and 1 mg/L test treatments were clear and colourless and remained so for the duration of the study. At the start of the exposure period the 10 and 100 mg/L treatments were pale white and white hazy dispersions respectively with undissolved test substance present. At the end of the exposure period both 10 and 100 mg/L test treatments were colourless wth undissolved test substance present on the base of the test vessels.

Validity criteria fulfilled:

yes

Conclusions:

Under the study conditions, the 48 h EC50 for the test substance was determined to be greater than 100 mg/L (nominal).

Executive summary:

A study was conducted to determine acute toxicity potential of the test substance, 'mono- C16 PSE and C16-OH' (purity not specified), towards Daphnia magna, according to OECD Guideline 202, in compliance with GLP. The study was performed using 3 nominal concentrations of the test substance: 1, 10, 100 mg/L. The 100 mg/L nominal stock solution was prepared by dosing 0.1 mL of acetone onto 0.1 g of the test substance and adding 1 liter of dilution water. Subsequently the stock solution was stirred for approximately 3 h and sonicated for approximately 30 minutes. The 1 and 10 mg/L test media were prepared by dilution of the 100 mg/L test medium. Water was used as negative control and 0.1 mg/L of acetone in water as solvent control. Five Daphnia magna less than 24 h old, were introduced into duplicate glass crystallising dishes, each containing approximately 100 mL of test medium. Daphnia were exposed to the test substance for 48 h in a static test system. After 24 and 48 h, the immobilised Daphnia were counted. In the beginning and end of the experiment undissolved test substance was present in the medium at 10, 100 mg/L test concentrations. The test treatments showed no toxicity. Under the study conditions, the 48 h EC50 for the test substance was determined to be >100 mg/L (nominal) (Covance, 2000).

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

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

Adequacy of study:

key study

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. The study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.

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 test substance, mono- C16 PSE + C16 -OH for Daphnia magna is considered to be >0.34 mg/L (measured).

Executive summary:

A study was conducted to determine the acute toxicity of read across substance, mono- and di- C16 PSE, K+ and H3PO4 (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 >0.34 mg/L(measured) (Scymaris, 2018). Based on the results of the read across study, similar EC50 and NOEC values can be expected for the test substance, 'mono- C16 PSE and C16-OH'.

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:

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. The study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: EU guideline 92/69/EWG

Deviations:

not specified

GLP compliance:

yes

Analytical monitoring:

yes

Remarks:

Fluid chromatography of dichloromethane extract

Vehicle:

no

Details on test solutions:

Concentrations: 10, 30, 100, 1000, 3000 mg/L
Renewal of test solution: None
Exposure vessel type: 100 mL beakers covered with clockglasses

Test organisms (species):

Daphnia magna

Details on test organisms:

Test organism
Strain: Daphnia magna
Supplier: BioInternational B.V., NJ Horn, NL
Age: 6-24 h old
Feeding: Green algae during 6 hours prior to testing
Pretreatment: M4 medium / 20oC / 16h light - 8h dark
Feeding during test: No feeding according to EU guideline
Control group: 1 group (2 replicates)
Number of replicates: 2
Invertebrate per replicate: 10

Test type:

static

Water media type:

other: Water accommodated fractions

Limit test:

no

Total exposure duration:

48 h

Test temperature:

20.8-20.9 deg C

pH:

mean pH: 7.9

Dissolved oxygen:

99.2%

Nominal and measured concentrations:

0, 10, 30, 100, 1000, 3000 mg/L nominal Water accommodated fractions (WAF)
Measured concentrations of C12, C14, C16 and C18 were between 0.003 and 0.025 mg/L at 48 h for 10 and 100 mg/L dose groups.

Details on test conditions:

Intensity of irradiation: approx. 900 lux
Photoperiod: 16 h light/ 8 h darkness

Reference substance (positive control):

no

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

ca. 40 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Loading rates

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

EL100

Effect conc.:

> 3 000 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Loadin rates

Basis for effect:

mobility

Details on results:

Nominal test concentrations were 10, 30, 100, 1000, and 3000 mg/L. No solvent was used. Instead, the water accommodated fraction (WAF) was used in the test chambers. 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, is 3 mg/L, therefore the EC50 was greater than the solubility limit.

Validity criteria fulfilled:

yes

Conclusions:

Based on the results of the read across study, the 48 h EL50 of the test substance is considered to be 40 mg/L WAF (nominal).

Executive summary:

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 EL50 value can be considered for the test substance, mono - C16 PSE and C16-OH'.

Description of key information

Based on the available weight of evidence from studies on the main constituents, no acute toxicity to Daphnia is expected up to the highest soluble concentrations of the test substance, ‘mono- C16 PSE and C16-OH’. As a conservative approach, the lowest 48 h EC50 value of >0.34 mg/L (measured), based on the read across study, has been considered further for hazard/risk assessment.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.34 mg/L

Additional information

In absence of specific study with the test substance, the acute toxicity to Daphnia endpoint has been assessed based on studies for substances representative of the two main constituents, which can be categorised as phosphate esters (PSE) and alcohol. The results are presented below:

Constituent 1: PSE - read across studies:

Study 1:

A study was conducted to determine acute toxicity potential of the test substance, 'mono- C16 PSE and C16-OH' (purity not specified), towards Daphnia magna, according to OECD Guideline 202, in compliance with GLP. The study was performed using 3 nominal concentrations of the test substance: 1, 10, 100 mg/L. The 100 mg/L nominal stock solution was prepared by dosing 0.1 mL of acetone onto 0.1 g of the test substance and adding 1 liter of dilution water. Subsequently the stock solution was stirred for approximately 3 h and sonicated for approximately 30 minutes. The 1 and 10 mg/L test media were prepared by dilution of the 100 mg/L test medium. Water was used as negative control and 0.1 mg/L of acetone in water as solvent control. Five Daphnia magna less than 24 h old, were introduced into duplicate glass crystallising dishes, each containing approximately 100 mL of test medium. Daphnia were exposed to the test substance for 48 h in a static test system. After 24 and 48 h, the immobilised Daphnia were counted. In the beginning and end of the experiment undissolved test substance was present in the medium at 10, 100 mg/L test concentrations. The test treatments showed no toxicity. Under the study conditions, the 48 h EC50 for the test substance was determined to be >100 mg/L (nominal) (Covance, 2000).

Study 2:

A study was conducted to determine the acute toxicity of read across substance, mono- and di- C16 PSE, K+ and H3PO4 (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 test substance in the test vessels were measured at start (0 h) and end of the test (48 h), using the HPLC method. Except for the measured values of the top 2 test concentrations at 0 h (i.e., 0.43 and 0.68 mg/L), the remaining measurements at 0 and 48 h were found to be below the limit of quantification (LOQ). Therefore, the test concentrations were presented as mean measured concentrations i.e., 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 >0.34 mg/L(measured) (Scymaris, 2018).

Constituent 2: Alcohol:

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 available weight of evidence from studies on the main constituents, no acute toxicity to Daphnia is expected up to the highest soluble concentrations of the test substance, 'mono- C16 PSE and C16-OH'. Further, as a conservative approach, the lowest 48 h EC50 value of >0.34 mg/L (measured), based on study with the read across substance, ‘mono- and di- C16 PSE, K+ and H3PO4’, has been considered further for hazard/risk assessment.