Trihexadecyl citrate

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 October 2017 to 12 February 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

23 November 2004

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

tris(2-octyldodecyl) citrate

EC Number:

603-121-2

Cas Number:

126121-35-5

Molecular formula:

C66H128O7

IUPAC Name:

tris(2-octyldodecyl) citrate

Test material form:

liquid

Details on test material:

Tris(octyldodecyl) citrate 99.87 %
Bis(octyldodecyl) citrate 0.13 %

Specific details on test material used for the study:

Test Substance Name: Citmol 320
Chemical Name: 2,3-propanetricarboxylic acid, 2-hydroxy-, 1,2,3-tris(2-octyldecyl)-ester
CAS Number: 126121-35-5
Batch Number: P7329
Purity: 99.39%
Receipt Date: 01 March 2017
Expiry Date: 31 August 2018
Storage on Receipt: Room Temperature (15 – 30°C)

Test substance details were supplied by the Sponsor. A Certificate of Analysis is presented in Appendix 4.

Based on the low solubility of the compound, which was stated as “insoluble in water” on the MSDS (material safety data sheet) a WAF preparation was considered most suitable (OECD 23).

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

The dilution water used in the study was Elendt M4 medium. Full preparation details are presented in Appendix 1. It was stated on page 11 of the protocol that the combined vitamin stock would be stored frozen, however, the facility now stores aliquots refrigerated. This was not considered to impact on study integrity as the health of the daphnia was assessed to be ok at study initiation, all control daphnia validity criteria was met and internal documents were followed.

Chemical Analysis
The Analytical Procedure, SMV 3201999-01V, used to confirm the concentration of Citmol 320 in samples of test media during this test, is summarised in Appendix 2. The analytical procedure was later updated to version 02 to amend a gradient program typo, to update stability information, to update the reagent supplier and to correct the conditions of the LC-TOF/MS system.
Concentrations of Citmol 320 were determined by diluting Elendt M4 medium samples with tetrahydrofuran (THF), then diluting further with diluent (4:1 v/v Elendt M4 medium: THF) as required to bring the response within the calibration range. Samples were analysed by injection onto a liquid chromatography time-of-flight mass spectrometry (LC-TOF/MS) system.
At the start of the test (0 hours), ca 20 mL samples of freshly prepared test media were taken from the control and each test concentration for chemical analysis.
At 24 hours, ca 8 mL samples were taken for chemical analysis from both freshly prepared media and the pooled old test media from the control and each test concentration.
At 48 hours, ca 8 mL samples were also taken for chemical analysis from the pooled old test media from the control and each test concentration.
In each case duplicate samples were taken, one for chemical analysis and one as a ‘back-up’ should further analysis be required.

Test solutions

Vehicle:

no

Details on test solutions:

Based on the low solubility of the compound, which was stated as “insoluble in water” on the MSDS (material safety data sheet) a WAF preparation was considered most suitable (OECD 23).

Range-finding Test
The range-finding test for which only the key findings have been reported was conducted at nominal test substance concentrations of 1.0, 10 and 100 mg/L LR WAF under static test conditions. A control group was also included. Duplicate test vessels were prepared for the control and each test concentration. Five juvenile daphnia (<24 hours old) were added to each test vessel.
No chemical analysis was performed for the range finding test.
Media Preparation Work
After the range-finder test was conducted without chemical analysis a media trial was conducted to assess test substance solubility and stability in the test system. Media were prepared in duplicate, using a WAF preparation method. New media samples were taken at 0 hours, media were then stored in test conditions for ca 48 hours. Old media samples were then taken to assess stability.

Based on the results of the range-finding test, the definitive test was conducted with a control group and the following test concentrations 2.84, 6.25, 12.5, 25, 50 and 100 mg/L LR WAF. The test was conducted with a semi static test system design. Media was prepared at 0 and 24 hours.
The test vessels were 100 mL glass beakers (filled to approximately 50mL). Four replicate test vessels were prepared for the control and each test concentration.
At the start of the test, amounts of test substance (2.83, 6.26, 12.50, 25.02, 50.03 and 100.04 mg) were separately added to 1000 mL of Elendt M4 medium. The preparations were stirred for ca 24 hours (with a vortex no deeper than ca 1 cm), and then allowed to settle for ca 1 hour. The final media were then syphoned from the mid-section (aqueous phase) of the vessel. A control treatment was prepared by adding Elendt M4 medium only to the control vessels. At 24 hours media were prepared using the same method, amounts of test substance (2.85, 6.25, 12.50, 24.99, 50.00 and 100.00 mg) were weighed for use at 24 hours. Daphnia were then transferred to the new media prepared at 24 hours.
Five juvenile Daphnia magna, less than 24 hours old, were added to each test and control vessel, using a wide bore glass pipette to avoid damaging the animals during transfer. The Daphnia magna were not fed during the test.
After 24 and 48 hours, the Daphnia magna in each test vessel were observed for evidence of immobility.
The observations differentiated between mobile and immobile daphnids. An individual was considered immobile if, when the contents of the test vessel were briefly agitated, it did not swim during a 15-second period of observation. In addition, Daphnia magna submerged in the body of the test media and those that were held at the surface of the test media were also recorded.

Water Quality and Environmental Conditions
The test was conducted with a 16-hour light: 8-hour dark photoperiod.
The pH, dissolved oxygen concentration (% air saturation value (ASV) and mg/L) and temperatures were determined in freshly prepared test media at the start of the test and at media renewal at 24 hours and in the old media at 24 and 48 hours. Continuous temperatures were measured using a digital (min/max) thermometer in an additional vessel maintained in the test area.
The freshly prepared media water quality measurements were performed on media sampled directly from preparation flasks. The old media water quality measurements were conducted using pooled replicate samples of old test media.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

Juvenile Daphnia magna (Straus), less than 24 hours old, were taken from healthy parental laboratory cultures, held and maintained at the test facility. The culture was originally obtained from Microbiotest, Belgium.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Post exposure observation period:

Not applicable

Test conditions

Test temperature:

18.8 to 21.9 °C

pH:

7.23 to 7.98

Dissolved oxygen:

8.31 - 9.30 mg/l

Salinity:

Not applicable

Conductivity:

not reported

Nominal and measured concentrations:

Chemical analysis showed the majority of concentrations of Citmol 320 to be below the LOQ, the few results that were above the LOQ and could be reported showed a decline in concentration over each renewal period. As results were below the LOQ and the media were prepared using a WAF preparation method results were based on nominal loading rates.

Details on test conditions:

The range-finding test for which only the key findings have been reported was conducted at nominal test substance concentrations of 1.0, 10 and 100 mg/L LR WAF under static test conditions. A control group was also included. Duplicate test vessels were prepared for the control and each test concentration. Five juvenile daphnia (<24 hours old) were added to each test vessel.
No chemical analysis was performed for the range finding test.

The results of the range-finding test are summarised below:
Nominal
concentration
(mg/L LR WAF) Cumulative immobilised Daphnia magna*
24 hours 48 hours % Immobility
R1 R2 R1 R2
Control 0 0 0 0 0
1.0 0 0 0 0 0
10 0 0 1 0 10
100 0 1 2 3 50
* Initial population of Daphnia magna: five per replicate
R Replicate
mg/L LR WAF loading rate water accommodated fraction

Chemical analysis was not conducted during the range-finding test.
The results of the range-finding test suggested that the 48-hour EC50 value would be between 10 and 100 mg/L LR WAF.

Reference substance (positive control):

yes

Remarks:

[State positive control substance]

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

24

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Remarks on result:

other: Derived empirically

Effect concentrations 2

Key result

Duration:

24

Dose descriptor:

other: NOEL

Effect conc.:

100 mg/L

Remarks on result:

other: Derived empirically

Effect concentrations 3

Key result

Duration:

24

Dose descriptor:

other: LOEL

Effect conc.:

> 100 mg/L

Remarks on result:

other: Derived empirically

Effect concentrations 4

Key result

Duration:

48

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Remarks on result:

other: Derived empirically

Effect concentrations 5

Key result

Duration:

48

Dose descriptor:

other: NOEL

Effect conc.:

100 mg/L

Remarks on result:

other: Derived empirically

Effect concentrations 6

Key result

Duration:

48

Dose descriptor:

other: LOEL

Effect conc.:

> 100 mg/L

Remarks on result:

other: Derived empirically

Details on results:

Definitive Test
Chemical Analysis
The results of the chemical analysis are presented in Table 1.
Example chromatograms of test samples, standard solution and a typical calibration line are presented in Appendix 2.
The limit of quantification (LOQ) for Citmol 320 in Elendt M4 medium using this method was 0.05 mg/L.
Analysis of the test media samples was conducted on fresh media at 0 and 24 hours, analysis of the corresponding old media was conducted at 24 and 48 hours.
Chemical analysis showed the majority of concentrations of Citmol 320 to be below the LOQ, the few results that were above the LOQ and could be reported showed a decline in concentration over each renewal period. As results were below the LOQ and the media were prepared using a WAF preparation method results were based on nominal loading rates. Smithers study number 3201997 found the water solubility to be (to be added once data is available).
Analysis of the test samples at 0, 24 and 48 hours are summarised in the table on the following page.



Nominal Conc. (mg/L LR WAF) Measured Concentration (mg/L)
0 hours (new) 24 hours (old) 24 hours (new) 48 hours (old)
Control - - - -
2.84 6.25 0.221 0.129 12.5 25 50 100 - Not Detected
LOQ Limit of quantification (0.05 mg/L)
mg/L LR WAF loading rate water accommodated fraction

Water Quality
Water quality determinations for pH, temperature and dissolved oxygen (% ASV and mg/L) during the definitive test are presented in Table 2.
The test vessel temperatures deviated from the specified maximum variation of 1°C. The deviations ranged from 0.4 – 0.8°C, this was not considered to impact study integrity as no daphnia immobility was observed during the test. The temperatures remained within the specified temperature range of 18 – 22°C.
The control group 0 hour oxygen levels were not measured at the time in error. This was not considered to impact study integrity as the same media was used to make up the control and all test concentrations which were all within acceptable limits. The 24 hour old control group result indicated good oxygen levels, the daphnia were observed as mobile in the control at 24 hours, also indicating good oxygen levels.

Test Media Descriptions
The test preparations were observed to be colourless solutions throughout the duration of the test.

Toxicity to Daphnia magna
The numbers of immobilised Daphnia magna at 24 and 48 hours, following exposure are presented in Table 3 and Table 4. The percentage immobility data is summarised on the next page.

Nominal concentration
(mg/L LR WAF) Cumulative immobilised Daphnia magna
(initial population: 5 Daphnia per replicate)
24 hours 48 hours
R1 R2 R3 R4 %
Immobility R1 R2 R3 R4 %
Immobility
Control 0 0 0 0 0 1 0 0 0 5
2.84 0 0 0 0 0 0 0 3* 1 20
6.25 0 0 0 0 0 0 0 0 0 0
12.5 0 0 0 0 0 0 0 0 0 0
25 0 0 0 0 0 0 1 0 1* 10
50 0 0 0 0 0 0 0 0 0 0
100 0 0 0 0 0 0 0 0 0 0
R Replicate
* Daphnia observed stuck to side of beaker, out of the test media, not considered to be caused by test substance toxicity.
mg/L LR WAF loading rate water accommodated fraction

No significant toxicity (>10%) was observed during the test. It is therefore considered that the results showed the test substance was not toxic to the test organisms at 100 mg/L LR WAF or at the limit of solubility in the media. Smithers study number 3201997 found the water solubility to be (to be added once data is available).

Validity Criteria
The validity criteria for control immobility (≤10%) and dissolved oxygen (≥3 mg/L) were both satisfied. The test is therefore considered valid.

Reported statistics and error estimates:

The validity criteria for control immobility (≤10%) and dissolved oxygen (≥3 mg/L) were both satisfied. The test is therefore considered valid.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The 48-hour acute toxicity of Citmol 320 to the freshwater planktonic crustacean, Daphnia magna, was determined in accordance with the requirements of OECD Chemicals Testing Guideline No. 202 Daphnia sp. Acute Immobilisation Test (adopted April 13 2004).
The validity criteria for control immobility (≤10%) and dissolved oxygen (≥3 mg/L) were both satisfied. The test is therefore considered valid.
Based on nominal loading rate concentrations, the 48-hour EL50 value was determined to be >100 mg/L LR WAF.
The corresponding No Observed Effect Loading rate (NOEL) was considered to be 100 mg/L LR WAF.
No toxicity was observed at the solubility level of the compound in test media.

Executive summary:

The objective of the study was to determine the 48-hour acute toxicity of Citmol 320 against the mobility of the freshwater planktonic crustacean, Daphnia magna.

The study was conducted in accordance with the requirements of OECD Chemicals Testing Guideline No. 202 Daphnia sp. Acute Immobilisation Test (adopted April 13 2004).

The test substance Citmol 320 is a multi-constituent substance with low water solubility and as such falls into the category of a “difficult substance” as defined by the OECD Guidance Document (OECD Series on Testing and Assessment, No. 23 (2000); Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures). Based on the recommendations of this guideline, the test substance was prepared as Water Accommodated Fractions (WAFs).

Based on the results of a range-finding test, for which the key results only have been reported, a definitive test was conducted at the following loading rate concentrations 2.84, 6.25, 2.5, 25, 50 and 100 mg/L LR WAF (loading rate water accommodated fraction). A control group was also included. The test was conducted under semi‑static test conditions with media preparation at 0 and 24 hours.

At the start of the test, five juvenile Daphnia magna were added to each test vessel. TheDaphnia magnain each test vessel were observed at 24 and 48 hours. The Daphnia magna were not fed during the test.

Analytical chemistry was performed during the definitive phase, however, due to the solubility of the compound being below the LOQ (limit of quantification) of the analytical method, which was 0.05 mg/L, the majority of the values could not be reported as they were not reliable. As media were prepared using a WAF preparation, the reported results were based on nominal loading rate concentrations (mg/L LR WAF).

Chemical analysis of the control and each test substance concentration was conducted on fresh media at 0 and 24 hours, analysis was conducted on the corresponding old media at 24 and 48 hours. Analysis of the test samples at 0, 24 and 48 hours are summarised in the table on the following page.

 

 

Nominal Conc. (mg/L LR WAF)	Measured Concentration (mg/L)
	0 hours (new)	24 hours (old)	24 hours (new)	48 hours (old)
Control	-	-	-	-
2.84	<LOQ	<LOQ	0.146	0.0730
6.25	0.221	0.129	<LOQ	<LOQ
12.5	<LOQ	<LOQ	<LOQ	<LOQ
25	<LOQ	<LOQ	<LOQ	<LOQ
50	<LOQ	<LOQ	0.258	0.163
100	<LOQ	<LOQ	0.049	<LOQ

-          Not Detected

LOQ   Limit of quantification (0.05 mg/L)

mg/L LR WAF          loading rate water accommodated fraction

The variability observed in the analytical results were considered to be due to the difficulties associated with analysis of multi-component compounds with low solubility. This was not considered to impact on the study integrity as there was no immobility observed during the test. 

The[1]EL₅₀values, the corresponding[2]NOEL and³LOEL values are presented in the following table.

Nominal Loading Rate (mg/L) Statistical Test   24-hour 48-hour   EL50 >100 mg/L >100 mg/L Derived Empirically NOEL 100 mg/L 100 mg/L LOEL >100 mg/L > 100 mg/L

The results indicated that there was no toxicity at the limit of solubility of the test substance.

The validity criteria for control immobility (≤10%) and dissolved oxygen (≥3 mg/L) were both satisfied. The test is therefore considered valid.

[1]Concentration resulting in 50% immobility of exposed Daphnia magna

[2]No observed effect level

³Lowest observed effect level