tris(2-octyldodecyl) citrate

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

8 November 2017 to 20 March 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 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

28 July 2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

1,2,3-tris(2-octyldodecyl) 2-hydroxypropane-1,2,3-tricarboxylate

EC Number:

603-121-2

Cas Number:

126121-35-5

Molecular formula:

C66H128O7

IUPAC Name:

1,2,3-tris(2-octyldodecyl) 2-hydroxypropane-1,2,3-tricarboxylate

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
Purity: 99.39%
Receipt Date: 01 March 2017
Expiry Date: 31 August 2018
Storage on Receipt: Room Temperature (15 – 30°C)

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Test Vessel Sampling
At approximately 24-hour intervals after the start of the incubation period, pre determined volumes of test media (1.0 mL at 24 hours and 0.5 mL at 48 and 72 hours) were removed from each incubated test vessel, and transferred to individually identified cell counting vials. The contents of each vial were diluted to a 10 mL final volume with an electrolyte solution. The cell density of the vial contents was then determined using a particle counter (Z2 Coulter Counter®).

Test solutions

Vehicle:

no

Details on test solutions:

Preliminary Solubility Trial
The test substance Citmol 320 was a UVCB (Substance of Unknown or Variable composition, Complex reaction products or Biological materials) 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 and the low solubility of the compound, stated as insoluble in water on the material safety data sheet (MSDS), the test substance was prepared as Water Accommodated Fractions (WAFs).

Test Procedures
Range-finding Test
The range-finding test was conducted with a control and nominal test substance concentrations of 1.0, 10 and 100 mg/L LR WAF.

Duplicate test vessels were prepared for the control and each test group. The test concentrations and control were inoculated with sufficient algal cells to achieve an initial algae cell concentration of 1 × 104 cells/mL. Vessels were prepared for the control, 1.0 mg/L LR WAF and 100 mg/L LR WAF concentrations which were not inoculated with algae and were used to determine background counts.

Media Preparation Work
After the range-finder test was conducted without chemistry analysis a media trial was conducted to assess test substance solubility/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 under test conditions for ca 72 hours. Old media samples were then taken to assess stability.
Definitive Test
Based on the results of the range-finding test and an initial definitive test which was repeated as a NOEC for yield could not be calculated and the chemistry batches for the analytical samples failed to meet acceptance criteria. Only the key results have been reported from the range-finder and initial definitive tests, the definitive test was conducted with a control and nominal test concentrations of 1.0, 3.2, 10, 32 and 100 mg/L LR WAF.
Six test vessels were prepared for the control (EC medium only) and three replicate test vessels were prepared for each test concentration, each with an initial cell concentration of 1 × 104 cells/mL. A blank vessel (not inoculated with algae) was prepared during the definitive test at control only.
At the start of the test, amounts of test substance (1.00, 3.21, 10.02, 32.00 and 100.04 mg) were separately added to 1000 mL of EC medium, test substance was weighed onto a glass slide which was then suspended at the top of the beaker, syphon tubes were added before the glass slides and test substance were added to avoid contamination of the tubes. The preparations were then stirred for ca 24 hours (with a vortex no deeper than 1 cm), media were 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 in the same manner with EC medium only.
The appearance, colour and behaviour of the test substance in the test media were recorded at the start and end of the test.
Algae cell counts were performed at 24, 48 and 72 hours during the test.
Test Vessel Preparation
The test vessels were sterile autoclaved glass 250 mL Erlenmeyer (conical) flasks, into which 100 mL of the appropriate control or test media was added. Sterile foam bungs were used to cover the top of the vessels.
Each test and control vessel was inoculated with sufficient Pseudokirchneriella subcapitata cells to achieve a starting algae cell concentration of 1 × 104 cells/mL. An additional inoculated vessel was prepared for the control and each test concentration for initial water quality analysis.

Test organisms

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

Test Organism
The test organism, Pseudokirchneriella subcapitata (Strain 278/4), was originally obtained from the Culture Collection of Algae and Protozoa (CCAP) and is a representative species of the freshwater aquatic phytoplankton. This species is recommended for testing in accordance with the OECD regulatory guidelines.
Prior to testing, duplicate starter cultures were prepared and incubated under test conditions (as detailed in Appendix 2) to obtain sufficient algal cells in exponential growth and to achieve a starting algae cell density of 1 × 104 cells/mL.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test conditions

Test temperature:

22.5 - 23.8 °C (21 - 24 °C target)

pH:

The pH of the test solutions ranged from 6.79 to 7.70 at test initiation in solutions with algae. The pH tended to increase relative to increases in algal densities, which is typical for tests conducted with P. subcapitata. The pH in the control increased by a maximum of 3.3 units, which exceeds the OECD 201 guideline requirement of an increase, not greater than 1.5 units. This change in pH was not considered to be detrimental to this study since increases in pH of greater than 1.5 units are commonly observed and this change had no impact on the growth of the control, which met the established validity criteria for the test.

Nominal and measured concentrations:

Chemical analysis showed concentrations of Citmol 320 to be below the LOQ and media were prepared using a WAF preparation method. Therefore, results were based on nominal loading rates
Nominal: 1.0, 3.2, 10, 32 and 100 mg/l [Loading rate]
Measured mean: < LOQ mg/l

Details on test conditions:

Range-finding Test
The range-finding test was conducted with a control and nominal test substance concentrations of 1.0, 10 and 100 mg/L LR WAF.
Duplicate test vessels were prepared for the control and each test group. The test concentrations and control were inoculated with sufficient algal cells to achieve an initial algae cell concentration of 1 × 104 cells/mL. Vessels were prepared for the control, 1.0 mg/L LR WAF and 100 mg/L LR WAF concentrations which were not inoculated with algae and were used to determine background counts.

Range-finder test
Key results from the range-finding test are summarised in Table 1.
At the start of the test, the control and test concentration were observed to be colourless solutions. At 72 hours, the control and test concentration were observed to be green, homogenous hazy dispersions of algal cells.
Chemical analysis was not conducted during the range-finding test.
The results of the range-finding test suggested that the 72-hour EL50 values for yield and specific growth rate were likely to be between 1.0 and 10 mg/L LR WAF and >100 mg/L LR WAF, respectively, based on nominal loading rate test substance concentration.

Media Preparation Work
After the range-finder test was conducted without chemistry analysis a media trial was conducted to assess test substance solubility/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 under test conditions for ca 72 hours. Old media samples were then taken to assess stability.
Definitive Test
Based on the results of the range-finding test and an initial definitive test which was repeated as a NOEC for yield could not be calculated and the chemistry batches for the analytical samples failed to meet acceptance criteria. Only the key results have been reported from the range-finder and initial definitive tests, the definitive test was conducted with a control and nominal test concentrations of 1.0, 3.2, 10, 32 and 100 mg/L LR WAF.
Six test vessels were prepared for the control (EC medium only) and three replicate test vessels were prepared for each test concentration, each with an initial cell concentration of 1 × 104 cells/mL. A blank vessel (not inoculated with algae) was prepared during the definitive test at control only.
At the start of the test, amounts of test substance (1.00, 3.21, 10.02, 32.00 and 100.04 mg) were separately added to 1000 mL of EC medium, test substance was weighed onto a glass slide which was then suspended at the top of the beaker, syphon tubes were added before the glass slides and test substance were added to avoid contamination of the tubes. The preparations were then stirred for ca 24 hours (with a vortex no deeper than 1 cm), media were 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 in the same manner with EC medium only.
The appearance, colour and behaviour of the test substance in the test media were recorded at the start and end of the test.
Algae cell counts were performed at 24, 48 and 72 hours during the test.
Test Vessel Preparation
The test vessels were sterile autoclaved glass 250 mL Erlenmeyer (conical) flasks, into which 100 mL of the appropriate control or test media was added. Sterile foam bungs were used to cover the top of the vessels.
Each test and control vessel was inoculated with sufficient Pseudokirchneriella subcapitata cells to achieve a starting algae cell concentration of 1 × 104 cells/mL. An additional inoculated vessel was prepared for the control and each test concentration for initial water quality analysis.

Environmental Conditions
All flasks were loosely-capped and incubated in a temperature and light controlled incubator. The vessels were placed on an orbital shaker (ca 100 rpm) under conditions of constant light (4440 to 8880 Lux), using florescent tube lights, emitting light across the visible portion of the spectrum (400 - 700 nm). The light intensity within the test area was monitored at the start and end of the test.
At the start of the test, the pH of freshly prepared test media was determined. The pH in each test vessel was also determined at the end of the test.
The incubator temperature was set within the range 21 to 24°C and maintained within ± 2°C for the duration of the test. The temperature was recorded continuously using a digital thermometer.


[copy and paste section on range-finding test, test system, test conditions, test media, controls, observations, effects measured]

Things to include:
- Details on the range finding test: [if applicable]
- Water source
- Water characteristics:
- Total organic carbon:
- Particulate matter:
- Metals:
- Pesticides:
- Chlorine:
- Acidity / Alkalinity:
- Culture medium for organisms different from test medium:
- Photo period:
- Light intensity: [Lux]

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Immobilisation

VEHICLE CONTROL PERFORMED: yes/no

Reference substance (positive control):

yes

Remarks:

[State positive control substance]

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EL50

Remarks:

EyL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

NOELR

Remarks:

NOEL

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

Specific growth

Effect concentrations 4

Key result

Duration:

72 h

Dose descriptor:

EL50

Remarks:

ErL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Chemical Analysis
The results of the chemical analysis are presented in Table 2.
Example chromatograms of test samples, standard solution and a typical calibration line are presented in Appendix 3.
The limit of quantification (LOQ) for Citmol 320 in EC medium using this method was 0.05 mg/L.
Analysis of the test media samples was conducted on fresh media at 0 hours, analysis of the corresponding old media was conducted at 72 hours.
Chemical analysis showed concentrations of Citmol 320 to be below the LOQ and media were prepared using a WAF preparation method. Therefore, 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 and 72 hours are summarised in the table below.

Nominal Concentration (mg/L LR WAF) Measured Concentration (mg/L)
0 hours (new) 72 hours (old)
Control - -
1.0 3.2 10 32 100
LOQ= 0.05 mg/L (LOQ = limit of quantification)
- None detected

The sensitivity of the method could not be increased to achieve a lower LOQ, meaning there was no scope of having reportable results for the analytical samples, the reasons for the sensitivity issues are discussed below.

Citmol 320 is a high molecular weight ester of citric acid. The molecular weight is too high to permit analysis by GC/MS (Gas chromatography–mass spectrometry) as it won’t elute off the column, therefore, LC/MS (Liquid chromatography–mass spectrometry) was used as the analytical technique. However, compounds such as Citmol 320 suffer significant issues regarding LC/MS analysis. The molecular structure is of a type that does not ionise efficiently. This has the effect of reducing the basic sensitivity of the analysis. Whilst efforts were made to improve the sensitivity (by changes to injection solvent, dilution regime and injection volume), the fundamental nature of the compound limited the improvements that could be achieved. Citmol 320 is highly insoluble, hence aqueous solutions contain very low concentrations. This, combined with the innately low sensitivity that molecule exhibits in LC/MS analysis, meant that it was not possible to measure the actual concentrations of the solutions in media generated during the study. Based on the analytical results it was considered that the solubility of the test substance was below the LOQ.

Test Media Descriptions
The freshly prepared test media at 0 hours appeared as colourless solutions.
The appearance of the test media following the 72-hour exposure period are summarised in the table below.
Chart 1. Test media descriptions at the end of the definitive test
Nominal Concentrations
(mg/L LR WAF) Colour of inoculated test medium State of test medium
Control green homogenous hazy dispersion of algal cells
1.0
3.2
10
32
100

Growth Test
Individual replicate data for cell density and mean treatment cell density are presented in Table 4. Results of the growth test, as specific growth rate, area under curve and yield are presented in Table 5 to Table 8.
Growth curves of Pseudokirchneriella subcapitata exposure to Citmol 320 are presented in Figure 1.

Parameter Toxicity Values (mg/L LR WAF)
72 hours Confidence limits Statistical Test
(LCL-UCL)
Growth Rate EyL10 >100 NC Linear Interpolation (ICPIN)
EyL20 >100 NC Linear Interpolation (ICPIN)
EyL50 >100 NC Linear Interpolation (ICPIN)
NOEL 100 NA Derived empirically
Yield ErL10 2.46 NC – 6.40 Linear Interpolation (ICPIN)
ErL20 6.28 1.95 – 12.2 Linear Interpolation (ICPIN)
ErL50 >100 NC Linear Interpolation (ICPIN)
NOEL 3.2 NA Derived empirically

NC = Not calculated
NA = Not applicable
LCL = Lower confidence limits
UCL = Upper confidence limits

The 72-hour yield (EyLx) and growth rate (ErLx) toxicity values, with corresponding NOEL values, are presented in the table below:
The results indicated that there was no inhibition at the limit of solubility of the test substance (estimated to be (to be added once data is available)) relative to the growth rate, it was considered that 40% inhibition was seen at the limit of solubility of the compound relative to yield.
Based on nominal loading rate concentrations, the 72-hour EyL50 and ErL50 values were calculated to be >100 mg/L LR WAF.
The corresponding NOEL values for yield and specific growth rate after 72 hours were 3.2 and 100 mg/L LR WAF, respectively.
All validity criteria were met therefore the test was considered valid.


[Copy and paste the results section of the report]

Things to include:
- Exponential growth in the control: yes/no
- Observation of abnormalities:
- Unusual cell shape:
- Colour differences:
- Flocculation:
- Adherence to test vessels:
- Aggregation of algal cells:
- Other:
- Any stimulation of growth found in any treatment:
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values:
- Effect concentrations exceeding solubility of substance in test medium:

Results with reference substance (positive control):

See attached data.

Reported statistics and error estimates:

Validity Criteria
The validity criteria specified in the OECD 201 Test Guideline (Reference 1) are;
i. To achieve ≥16 fold exponential increase in biomass in the control replicates within the 72-hour test period. In this test, the cell density increase (as a surrogate for biomass) was approximately 377 fold over the 72 hours.
ii. The mean coefficient of variation for the control replicate sectional (daily) specific growth rates must not exceed 35%. This was determined to be 30.75% in this test.
iii. The replicate coefficient of variation of average specific growth rates during the whole test period in the control replicate cultures must not exceed 7%. This was 1.32% in this test.
Statistics are attached.

Any other information on results incl. tables

Environmental Conditions

The results of the environmental conditions and pH determined during the test are presented inTable 3. 

Temperatures remained within 21 - 24 °C, and were maintained within ± 2ºC range established for the test, temperature range during the test was 22.5 – 23.8 °C.

The light intensity was measured at the start and end of the test; Lux at the start of the test ranged from 5220 to 6560 Lux and the Lux at the end of the test ranged from 5935 to 6430 Lux (three readings carried out at each time point). 

The pH of the test solutions ranged from 6.79 to 7.70 at test initiation in solutions with algae. The pH tended to increase relative to increases in algal densities, which is typical for tests conducted withP. subcapitata. The pH in the control increased by a maximum of 3.3 units, which exceeds the OECD 201 guideline requirement of an increase, not greater than 1.5 units. This change in pH was not considered to be detrimental to this study since increases in pH of greater than 1.5 units are commonly observed and this change had no impact on the growth of the control, which met the established validity criteria for the test.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The effects of Citmol 320 on the growth of the unicellular green alga, Pseudokirchneriella subcapitata, were determined during a 72-hour growth inhibition toxicity test conducted in accordance with OECD Chemicals Testing Guideline No. 201 Alga, Growth Inhibition Test (adopted 23 March 2006) (Annex 5 corrected 28 July 2011) (Reference 1).

As media were prepared as water accommodated fractions and results could not be determined by chemical analysis, results were based on nominal loading rate concentrations.

Based on nominal loading rate concentrations, the 72-hour EyC50 and ErC50 values were calculated to be >100 mg/L LR WAF, respectively.

The corresponding NOEL values for yield and specific growth rate after 72 hours were 3.2 and 100 mg/L LR WAF, respectively.

All validity criteria were met therefore the test was considered valid.

Smithers study number 3201997 found the water solubility to be (to be added once data is available, including discussion of impact on study and what it means for the results of the study).

Executive summary:

The objective of the study was to determine the effects of the test substance against algal growth by exposing the green alga,Pseudokirchneriella subcapitata,at the exponential growth phase to the test substance duringa 72-hour test period.  

The test was conducted in accordance with OECD Chemicals Testing Guideline No. 201. Alga, Growth Inhibition Test (adopted 23 March 2006, Annex 5 corrected 28 July 2011).

The test substance Citmol 320 was a UVCB (Substance of Unknown or Variable composition, Complex reaction products or Biological materials), a mixture comprised of diasterio-isomers (as it has three chiral centres), it also has 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, an initial definitive test was conducted atnominal test concentrations of 1.0, 3.2, 10, 32 and 100 mg/L LR WAF (loading rate water accommodated fraction). Six replicate vessels were prepared for the control and three replicate vessels were prepared for the test concentrations. However, the test was repeated as a NOEC for yield could not be calculated and the chemistry batches for the analytical samples failed to meet acceptance criteria.

Based on the results of a range-finding test and an initial definitive test, for which the key results only have been reported, the definitive test was conducted atnominal test concentrations of 1.0, 3.2, 10, 32 and 100 mg/L LR WAF (loading rate water accommodated fraction). Six replicate vessels were prepared for the control and three replicate vessels were prepared for the test concentrations.

Test vessels (250 mL conical flasks) were prepared containing 100 mL of the appropriate test or control medium. Each test vessel was inoculated with
1 × 10⁴ algae cells/mL and incubated at 21 – 24°C (under 4440 – 8880 Lux light intensity) for 72 hours with cell counts at 24-hour intervals.

Analytical chemistry was performed during the definitive test, 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 values could not be reported as they were not reliable. As media were prepared using a WAF preparation results were based on nominal loading rate concentrations (mg/L LR WAF).Smithers study number 3201997 found the water solubility to be(to be added once data is available).

 

Chemical analysis was conducted on fresh media at 0 hours and the corresponding old media at 72 hours. Analysis of the test samples at 0 and 72 hours are summarised in the table on the following page.

Nominal Concentration  (mg/L LR WAF)		Measured Concentration (mg/L)
		0 hours (new)	72 hours (old)
Control		-	-
1.0		<LOQ	<LOQ
3.2		<LOQ	<LOQ
10		<LOQ	<LOQ
32		<LOQ	<LOQ
100		<LOQ	<LOQ
LOQ =	0.05 mg/L (LOQ = limit of quantification)
-	None detected

The 72-hour yield (E_yL_x), area under the growth curve (E_bL_x) and growth rate (E_rL_x) toxicity values, with corresponding no observed effect loading rates (NOEL) are presented in the table below.  

Parameter			Toxicity Values (mg/L LR WAF)		Statistical Test
			72 hours	Confidence limits (LCL-UCL)
Growth Rate		EyL10	>100	NC	Linear Interpolation (ICPIN)
		EyL20	>100	NC
		EyL50	>100	NC
		NOEL	100	NA	Derived empirically
Yield		ErL10	2.46	NC – 6.40	Linear Interpolation (ICPIN)
		ErL20	6.28	1.95 – 12.2
		ErL50	>100	NC
		NOEL	3.2	NA	Derived empirically
NC	Not calculated
NA	Not applicable
LCL	Lower confidence limits
UCL	Upper confidence limits

The results indicated that there was no inhibition at the limit of solubility of the test substance (estimated to be(to be added once data is available)) relative to the growth rate, it was considered that 40% inhibition was seen at the limit of solubility of the compound relative to yield.

Based on nominal loading rate concentrations, the 72-hourE_yL₅₀and E_rL₅₀values were calculated to be >100 mg/L LR WAF.

The corresponding NOEL values for yield and specific growth rate after 72 hours were 3.2 and 100 mg/L LR WAF, respectively.

All validity criteria were met therefore the test was considered valid.