Tris(2-methylheptyl) 2-hydroxypropane-1,2,3-tricarboxylate

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:

12/9/2017 to 1/12/2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

OECD Chemicals Testing Guideline No.201 Alga, Growth Inhibition Test
(adopted 23 March 2006) (Annex 5 corrected 28 July 2011)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Tris(2-ethylhexyl) 2-hydroxypropane-1,2,3-tricarboxylate

EC Number:

230-457-3

EC Name:

Tris(2-ethylhexyl) 2-hydroxypropane-1,2,3-tricarboxylate

Cas Number:

7147-34-4

Molecular formula:

C30H56O7

Test material form:

liquid

Specific details on test material used for the study:

Test Substance

Chemical Name: Tris(2-methylheptyl) 2-hydroxypropane-1,2,3-tricarboxylate
Batch Number: P7803
Purity: 94%
Receipt Date: 12 June 2017
Expiry Date: 23 January 2019
Storage on Receipt: Room temperature (15 - 30°C)

Test substance details were supplied by the Sponsor. A Certificate of Analysis is
presented in Appendix 2.

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.

Test organisms

Test organisms (species):

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

Details on test organisms:

The axenic strain of Pseudokirchneriella subcapitata (CCAP 278/4) was obtained from a concentrated liquid slope culture from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd., Oban, UK.
On receipt from the CCAP, the slope culture was stored in the fridge at 2-8°C. A typical shelf life for each slope was 8 months, after which the slopes were discarded.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test conditions

Test temperature:

21.5 - 22.6 °C (see data for individual experiments)

pH:

7.73 to 7.75 at test initiation
pH in the control increased by a maximum of 0.68 pH units.

Dissolved oxygen:

NA

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.184 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

ca. 0.184 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Any other information on results incl. tables

RESULTS

 

Range-finder test

Key results from the range-finding test are summarised in table 1.

At the start of the test, the control and each test concentration were observed to be colourless solutions. At 72 hours, the control and each test concentrations were observed to be green, homogenous hazy dispersions of algal cells.

Chemical analysis was not conducted during the range-finder test.

The results of the range-finding test suggested that the 72-hour EC₅₀values for yield, specific growth rate and area under the curve (biomass) were likely to be greater than 100% saturated solution.

Media Trial

Media trial results are summarised below:

 

Nominal concentration (% saturated solution)	Measured Concentrations (mg/L)
	0 hours	48 hours
100	0.209	0.244

LOQ (limit of quantification) = 0.01 mg/L

* Batch failed due to analytical recovery sample results failing to meet acceptance criteria. Therefore, results for information purpose only.

 

Results showed the test substance was stable in test conditions over the 72-hour test period. Therefore, it was not considered necessary to include additional analytical vessels in the definitive test.

Definitive Test

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 4. The limit of quantification (LOQ) was 0.01 mg/L.

Media preparation work indicated that

Concentrations of Tricaprylyl citrate

was stable in the test system over a 72-hour period, however, the analytical batched failed to meet QC criteria and were therefore DNU and could not be reported. This was not considered to impact on study integrity as the compound was shown to be stable in the definitive test.

Analysis of the freshly prepared test solution at 0 hours showed a measured concentration of 0.184 mg/L.

Analysis of the 72-hour test solution showed a measured concentration of 0.181 mg/L.

Analysis of the 72-hour test solutions which were not inoculated with algae showed measured concentration of 0.183 mg/L.

Chemical analysis showed the test substance was stable over the 72-hour test period. But concentrations were outside of 80-120% of nominal concentrations. Therefore, results were based on initial measured concentrations (based on guidance from study guideline). Chemical analysis of the blank vessels at 72 hours indicated that the algae was not adhering to the test substance.

Environmental Conditions

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

Temperatures remained within 21 - 24ºC, and were maintained within ± 2ºC range established for the test, temperature range during the test was 21.5 – 22.6°C.

The light intensity was measured at the start and end of the test; 6490 Lux at the start and 7965 Lux at the end of the test (mean of two readings carried out at each time point).

The pH of the control and test solutions ranged from 7.73 to 7.75 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 0.68 pH units.

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 (% saturated solution)	Colour of inoculated test medium	State of test medium
Control	green	Homogenous hazy dispersion of algal cells
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 7.

Growth curves of Pseudokirchneriella subcapitata exposure to the test substance are presented in Figure 1.

The 72-hour yield (E_yC_x), area under the growth curve (E_bC_x) and growth rate (E_rC_x) toxicity values, with corresponding NOEC values, are presented in the table below.

 

Parameter		72 hours	Confidence limits (LCL - UCL)	Statistical Test
Yield	EyC10	0.136	0.0819 - NC	Linear Interpolation (ICPIN)
	EyC20	>0.184	NC - NC
	EyC50	>0.184	NC - NC
	NOEC	0.184	NC - NC	Equal variance t Two sample
Area Under the Curve (Biomass)	EbC10	0.109	0.0727 - NC	Linear Interpolation (ICPIN)
	EbC20	>0.184	NC - NC
	EbC50	>0.184	NC - NC
	NOEC	0.184	NC - NC	Equal variance t Two sample
Area Under the Curve (Biomass)	ErC10	>0.184	NC - NC	Linear Interpolation (ICPIN)
	ErC20	>0.184	NC - NC
	ErC50	>0.184	NC - NC
	NOEC	0.184	NC - NC	Equal variance t Two sample

NC Not calculated

LCL Lower confidence limit

UCL Upper confidence limit

 

Based on initial measured concentrations, the 72-hour E_yC₅₀, E_bC₅₀and E_rC₅₀values were calculated to be > 0.184 mg/L, respectively.

The corresponding NOEC values for yield, biomass and specific growth rate after 72 hours were 0.184 mg/L.

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

 

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 45 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 21.25% 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 1.88% in this test.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

CONCLUSION
The effects of Tricaprylyl citrate 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).
Chemical analysis showed Bernel Ester TCC was stable over the 72-hour test period. But concentrations were outside of 80-120% of nominal concentrations. Therefore, results were based on initial measured concentrations (based on guidance from study guideline).
Based on initial measured concentrations, the 72-hour EyC50, EbC50and ErC50values were calculated to be > 0.184 mg/L, respectively.
The corresponding NOEC values for yield, biomass and specific growth rate after 72 hours were 0.184 mg/L.
All validity criteria were met therefore the test was considered valid.

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 during a 72-hour test period.

The test was conducted in accordance with OECD Chemicals Testing Guideline No. 201(Reference 1).

The definitive limit test was conducted at a nominal test concentration of 100% saturated solution. Six replicate vessels were prepared for the control and 100% saturated solution test group.

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. Blank vessels were also prepared, these vessels were not inoculated with algae and were for establishing back ground counts.

Measured test concentrations were determined from samples of test solution from treatment and control groups at the beginning of the test (new media) and the test end (old media).

Analysis of the test samples at 0 and 72 hours are summarised in the table below.

 

Nominal concentration (% saturated solution)	Measured Concentrations (mg/L)
	0 hours (New)	72 hours (Old)	72 hours un-inoculated (Old)
Control	-	-	-
100	0.184	0.181	0.183

 

Chemical analysis showed

Concentrations of Tricaprylyl citrate

was stable over the 72-hour test period. But concentrations were outside of 80-120% of nominal concentrations. Therefore, results were based on initial measured concentrations (based on guidance from study guideline). Chemical analysis of the blank vessels at 72 hours indicated that the algae was not adhering to the test substance.

The 72-hour yield (EyCx), area under the growth curve (EbCx) and growth rate (ErCx) toxicity values, with corresponding no observed effect concentration (NOEC) values, are presented in the table on the next page.

  

Parameter		72 hours	Confidence limits (LCL - UCL)	Statistical Test
Yield	EyC10	0.136	0.0819 - NC	Linear Interpolation (ICPIN)
	EyC20	>0.184	NC - NC
	EyC50	>0.184	NC - NC
	NOEC	0.184	NC - NC	Equal variance t Two sample
Area Under the Curve (Biomass)	EbC10	0.109	0.0727 - NC	Linear Interpolation (ICPIN)
	EbC20	>0.184	NC - NC
	EbC50	>0.184	NC - NC
	NOEC	0.184	NC - NC	Equal variance t Two sample
Area Under the Curve (Biomass)	ErC10	>0.184	NC - NC	Linear Interpolation (ICPIN)
	ErC20	>0.184	NC - NC
	ErC50	>0.184	NC - NC
	NOEC	0.184	NC - NC	Equal variance t Two sample

NC Not calculated

LCL Lower confidence limit

UCL Upper confidence limit

 

Based on initial measured concentrations, the 72-hour E_yC₅₀, E_bC₅₀and E_rC₅₀values were calculated to be > 0.184 mg/L, respectively.

The corresponding NOEC values for yield, biomass and specific growth rate after 72 hours were 0.184 mg/L.

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