1,2,3-propanetriyl tris[12-(acetoxy)octadecanoate]

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:

4 February 2018 - 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

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

23 March 2006: corrected 28 July 2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

1,2,3-propanetriyl tris[12-(acetoxy)octadecanoate]

EC Number:

205-363-0

EC Name:

1,2,3-propanetriyl tris[12-(acetoxy)octadecanoate]

Cas Number:

139-43-5

Molecular formula:

C63H116O12

IUPAC Name:

1,3-bis({[12-(acetyloxy)octadecanoyl]oxy})propan-2-yl 12-(acetyloxy)octadecanoate

Test material form:

liquid

Specific details on test material used for the study:

- Appearance: Viscous amber liquid (determined by Charles River Den Bosch)
- Purity/Composition: >85%
- Test item storage: At room temperature

Additional information
- Test Facility test item number: 209080/A
- Purity/Composition correction factor: No correction factor required
- Chemical name (IUPAC), synonym or trade name: Glyceryl Triacetyl Hydroxystearate
- CAS number: 139-43-5
- Molecular formula: C63H116O12
- Molecular weight: 1065.6
- Highly reactive to water: Not indicated
- Highly reactive to oxygen: Not indicated
- Solubility in water: Insoluble
- Stability in water: Stable

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Samples for possible analysis were taken from all test concentrations and the control according to the schedule below. In addition, the glass wool containing the undissolved residue was kept for possible analysis.
- Frequency: at t=0 h, t=24 h and t=72 h
- Volume: 1.0 mL from the approximate centre of the test vessels
- Storage: Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.
At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.
Compliance with the quality criteria regarding maintenance of actual concentrations was checked by running a test vessel at the limit concentration (i.e. WAF prepared at a loading rate of 100 mg/L) but without algae and samples for analysis were taken at the start, after 24 hours of exposure and at the end of the test period.
Additionally, reserve samples of 1.0 mL were taken from all test solutions for possible analysis. If not already used, these samples were stored in a freezer (≤ -15°C) for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.

Test solutions

Vehicle:

no

Details on test solutions:

The batch of Hetester HCA tested was a viscous amber liquid with a purity >85% which was not completely soluble in test medium at the loading rates initially prepared. No correction was made for the purity/composition of the test item.
Preparation of test solutions for the second and final combined limit/range-finding test was based on what was observed during preparation of test solutions for the first non-valid combined limit/range-finding test, i.e. test solutions were still hazy after a settlement period of approximately two hours and undissolved material was present after siphoning off the Water Accommodated Fractions (WAFs).
Consequently, preparation of test solutions for the second combined limit/range-finding test started with loading rates individually prepared at 1.0, 10 and 100 mg/L. A two-day period of magnetic stirring was applied to ensure maximum dissolution of the test item in test medium. The obtained mixtures were allowed to settle for an overnight period. Thereafter, the aqueous Water Accommodated Fractions (WAFs) were collected by means of siphoning through glass wool and used as test concentrations. All test solutions were clear and colorless at the end of the preparation procedure.
After preparation, volumes of 50 mL were added to each replicate of the respective test concentration. Subsequently, 1 mL of an algal suspension was added to each replicate providing a cell density of 104 cells/mL.
Any residual volumes were discarded.

Test organisms

Test organisms (species):

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

Details on test organisms:

Test System
- Species: Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata), strain: NIVA CHL 1
- Source: In-house laboratory culture.
- Reason for selection: This system is a unicellular algal species sensitive to toxic items in the aquatic ecosystem and has been selected as an internationally accepted species.

Fresh Water Algae Culture
- Stock culture: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C.
- Light intensity: 60 to 120 µE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.
- Stock culture medium: M1; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tap-water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA) with the following composition:
NaNO3 500 mg/L
K2HPO4.3H2O 52 mg/L
MgSO4.7H2O 75 mg/L
Na2CO3.10H2O 54 mg/L
C6H8O7.H2O 6 mg/L
NH4NO3 330 mg/L
CaCl2.2H2O 35 mg/L
C6H5FeO7.xH2O 6 mg/L
H3BO3 2.9 mg/L
MnCl2.4H2O 1.81 mg/L
ZnCl2 0.11 mg/L
CuSO4.5H2O 0.08 mg/L
(NH4)6Mo7O24.4H2O 0.018 mg/L

- Pre-culture: 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 104 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
- Pre-culture medium: M2; according to the OECD 201 Guideline, formulated using Milli-RO water and with the following composition:
NH4Cl 15 mg/L
MgCl2.6H2O 12 mg/L
CaCl2.2H2O 18 mg/L
MgSO4.7H2O 15 mg/L
KH2PO4 1.6 mg/L
FeCl3.6H2O 64 µg/L
Na2EDTA.2H2O 100 µg/L
H3BO3 185 µg/L
MnCl2.4H2O 415 µg/L
ZnCl2 3 µg/L
CoCl2.6H2O 1.5 µg/L
CuCl2.2H2O 0.01 µg/L
Na2MoO4.2H2O 7 µg/L
NaHCO3 50 mg/L
Hardness (Ca+Mg) 0.24 mmol/L (24 mg CaCO3/L)
pH 8.1 ± 0.2

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test conditions

Hardness:

CaCO3: 24 mg/l

Test temperature:

21 - 23 °C

pH:

8.0 - 8.4

Dissolved oxygen:

Not reported

Salinity:

Not applicable

Conductivity:

Not reported

Nominal and measured concentrations:

1.0, 10.0, 100.0 mg/l loading rate

Details on test conditions:

Test Procedure and Conditions
- Test duration: 72 hours
- Test type: Static
- Test vessels: 100 mL, all-glass, containing 50 mL of test solution
- Medium: M2
- Cell density: An initial cell density of 1 x 104 cells/mL.
- Illumination: Continuously using TLD-lamps with a light intensity within the range of 86 to 87 µE.m-2.s-1.
- Incubation: Capped vessels were distributed at random in the incubator and daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.

Measurements and Recordings
- pH: At the beginning and at the end of the test.
- Temperature of medium: Continuously in a temperature control vessel.
- Appearance of the cells : At the end of the final test microscopic observations were performed on the limit concentration to observe for any abnormal appearance of the algae.

Recording of Cell Densities
At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with immersion probe (path length = 10 mm). Algal medium was used as blank and the extra replicates, without algae, as background for the treated solutions.
Cell densities were recorded at 24-hour intervals in the control and the limit concentration. Intermediate concentrations were measured only at the end of the exposure period.

INTERPRETATION
Acceptability of the Test
1. In the control, cell density increased by an average factor of at least 16 within the exposure period (i.e. 190).
2. The mean coefficient of variation for section-by-section specific growth rates in the control cultures did not exceed 35% (i.e. 19%).
3. The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures did not exceed 7% (i.e. 2.1%).
All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.

Data Handling
Calibration Curve
Quantification of cell densities was based on a calibration curve. Cell density was plotted versus extinction using spectrophotometric measurements of a minimum of six dilutions of an algal suspension with different cell densities. The calibration curve was composed using linear regression. The software automatically calculates the cell densities based on this curve for the spectrophotometric measurements at the various points in time during the test period.

Comparison of Average Growth Rates
The average specific growth rate for a specific period is calculated as the logarithmic increase in the biomass from the equation for each single vessel of controls and treatments:

µi-j = ((lnXj - lnXi) / (tj - ti)) * (day-1)

Where: µi-j = the average specific growth rate from time i to j
Xi = the biomass at time i
Xj = the biomass at time j
The average growth rate at each test item concentration is then compared with the control value and the percentage inhibition in growth rate is calculated:

%Ir = ((µC - µT) / µC) x 100

Where: %Ir = percent inhibition in average specific growth rate
µC = mean value for average specific growth rate in the control group
µT = average specific growth rate for the treatment replicate

Yield
The percent inhibition in yield is calculated for each treatment replicate as follows:

XIy = ((YC - YT) / YC) x 100

Where: %Iy = percent inhibition of yield
YC = mean value for yield in the control group
YT = value for yield for the treatment replicate

Determination of the Exposure Concentrations
In case concentrations measured were below the limit of quantification, the concentrations were determined by extrapolation of the calibration curve applied in the analysis of the samples provided that the response was within the calibration curve produced during validation of the analytical method.

ANALYSIS
Determination of the NOEC and Calculation of the EC50
For determination of the NOEC and the EC50 the approaches recommended in the OECD guideline 201 were used. An effect was considered to be significant if statistical analysis of the data obtained for the test concentration compared with those obtained in the control revealed significant inhibition of growth rate or inhibition of yield (Two-Sample t-Test Procedure, α=0.05, one-sided, smaller).
No EC50-values could be calculated because the measured effects were below 50%.
The calculations were performed with ToxRat Professional v. 3.2.1. (ToxRat Solutions® GmbH, Germany).

COMPUTERIZED SYSTEMS
Critical computerized systems used in the study are listed below or presented in the appropriate Phase Report. All computerized systems used in the conduct of this study have been validated; when a particular system has not satisfied all requirements, appropriate administrative and procedural controls were implemented to assure the quality and integrity of data.

Text Table 1: Critical Computerized Systems
System name Version No. Description of Data Collected and/or Analyzed
REES Centron SQL 2.0 Temperature, relative humidity and/or atmospheric pressure monitoring
Shimadzu Spectrophotometer UV-1800 including 1cm UVProbe 2.52 System control, data acquisition and processing

RETENTION OF RECORDS AND SAMPLES
All study-specific raw data, documentation, study plan and final reports from this study were archived at the Test Facility by no later than the date of final report issue unless otherwise specified in the study plan. At least five years after issue of the final report, the Sponsor will be contacted.

POSITIVE REFERENCE TEST
The objective of the study was to evaluate Potassium dichromate for its ability to generate toxic effects in Pseudokirchneriella subcapitata (strain: NIVA CHL-1) during an exposure period of 72 hours and, if possible, to determine the EC50 for inhibition of both growth rate and yield (Charles River Den Bosch Study Number 20143131).
Start of first exposure: 02 Jan 2018
Completion last exposure: 05 Jan 2018
The study procedures described in this report were based on the OECD guideline No. 201, Adopted March 23, 2006; Annex 5 corrected 28 July 2011 and ISO Standard 8692, Second edition, 01 October 2004.
This reference test was carried out to check the sensitivity of the test system used by Charles River Den Bosch to Potassium dichromate (Merck, Art. 1.04864, Batch K44879664).
Algae were exposed for a period of 72 hours to K2Cr2O7 (Potassium dichromate) concentrations of 0.18, 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L and to a control. The initial cell density was 1.0 x 104 cells/mL.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.039 µg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.039 µg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

Combined Limit/Range-Finding Test
A first combined limit/range-finding test was performed, which was not valid since the algae did not growth sufficiently in the control. Consequently, a second combined limit/range-finding test was performed, of which results are presented below.

Measured Test Item Concentrations
The results of analysis of the samples taken during the second and definitive combined limit/range-finding test are described in Table 2 of the appended Analytical Report.
Samples taken from the WAF prepared at loading rate of 100 mg/L were analysed. The measured concentration at the start of the test was estimated to be 0.039 µg/L by extrapolation of the calibration curve. No detectable test item concentrations were found after 24 and 72 hours of exposure. Nevertheless, it can be stated that testing was ultimately performed at the maximum soluble concentration of test item in test medium, since the water solubility was determined to be <1.5 µg/L. Based on these results, effects parameters were based on the initially estimated exposure concentration of 0.039 µg/L.

Mean Cell Densities
Figure 1 shows growth curves in the control and at the limit concentration of Hetester HCA. The individual and group mean cell densities measured at 24h intervals are given in Table 6.

Inhibition of Growth Rate and Inhibition of Yield
Table 1 shows group mean growth rates and the percentages of growth rate inhibition (total test period) whereas Table 2 shows the values at different time intervals. The group mean yields and the percentages of yield inhibition are summarized in Table 3 (see Appendix 1 for the individual values).
Growth rate and yield at the limit concentration were statistically significantly inhibited by 2.5 and 13%, respectively, at the end of the test. For growth rate, however, effects were considered as biologically not relevant (i.e. <10%) and therefore, the NOEC based on biological relevance was set at the TWA concentration of 0.039 µg/L.
Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to the limit concentration when compared to the control.

Determination of Effect Concentrations
Table 4 shows the effect parameters expressed as the estimated exposure concentration (µg/L) based on analytical measurements at the start of the test.

Experimental Conditions
Table 5 shows the pH recorded at the beginning and the end of the second combined limit/range-finding test. The pH was within the limits prescribed by the study plan (6.0-9.0, preferably not varying by more than 1.5 unit). During the exposure period the temperature measured in the incubator was generally maintained between 21 and 23°C. Temperature remained within the limits prescribed by the study plan (21-24°C, constant within 2°C).

Results with reference substance (positive control):

Overview of % inhibition of growth rate in the reference test:
Nominal conc.
K2Cr2O7 (mg/L) Mean Std. Dev. n %Inhibition
Control 1.605 0.0224 3
0.18 1.551 0.0144 3 3.4
0.32 1.402 0.0196 3 13
0.56 1.112 0.0424 3 31
1.0 0.653 0.0104 3 59
1.8 0.378 0.0728 3 76
3.2 0.050 0.0871 3 97

Overview of % inhibition of yield in the reference test:
Nominal conc.
K2Cr2O7 (mg/L) Mean Std. Dev. n %Inhibition
Control 122.4 8.41 3
0.18 103.9 4.53 3 15
0.32 66.1 3.96 3 46
0.56 27.3 3.58 3 78
1.0 6.1 0.22 3 95
1.8 2.2 0.73 3 98
3.2 0.2 0.33 3 100

Potassium dichromate inhibited growth rate of this fresh water algae species at nominal concentrations of 0.18 mg/L and higher.
The EC50 for growth rate inhibition (72h-ERC50) was 0.86 mg/L with a 95% confidence interval ranging from 0.84 to 0.88 mg/L. The historical ranges for growth rate inhibition lie between 0.82 and 2.3 mg/L. The observed 72h-ERC50 for the algal culture tested corresponds with this range.
The EC50 for yield inhibition (72h-EYC50) was 0.34 mg/L with a 95% confidence interval ranging from 0.34 to 0.35 mg/L. The historical ranges for yield inhibition lie between 0.20 and 1.1 mg/L. The observed 72h-EYC50 for the algal culture tested corresponds with this range.

Any other information on results incl. tables

Table 1: Growth Rate And Percentage Inhibition For The Total Test Period

Hetester HCA Loading rate (mg/l)	Mean	Std. Dev.	n	%Inhibition
Control	1.747	0.0362	6
1	1.708	0.0254	3	2.2
10	1.755	0.0182	3	-0.43
100 (0.039)	1.703	0.0171	6	2.5^#

^#– effect statistically significant but biologically not relevant (<10%), ( ) – Estimated exposure concentration at the start of the test (µg/L).

Table 2: Growth Rate And Percentage Inhibition At Different Time Intervals

Hetester HCA Loading rate (mg/L)	n	0 – 24 h		24 – 48 h		48 – 72h
		Mean	%Inhibition	Mean	%Inhibition	Mean	%Inhibition
Control	6	2.111		1.625		1.506
100 (0.039)	6	1.977	6.34	1.609	0.976	1.524	-1.197

( ) – Estimated exposure concentration at the start of the test (µg/L).

Table 3: Yield And Percentage Inhibition For The Total Test Period

Hetester HCA Loading rate (mg/L)	Mean	Std. Dev.	n	%Inhibition
Control	188.9	20.06	6
1	167.3	13.02	3	11
10	192.5	10.54	3	-1.9
100 (0.039)	164.8	8.48	6	13*

– effect was statistically significant, ( ) – Estimated exposure concentration at the start of the test (µg/L).

Table 4: Effect Parameters

Parameter (µg/L)	NOEC*	NOEC**	EC10	EC50
Growth rate	<0.039	0.039	>0.039	>0.039
Yield	<0.039	<0.039	<0.039	>0.039

* - based on statistical significance, ** - based on biological relevance

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In conclusion, under the conditions of the present study with Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata), no biologically relevant inhibition of growth rate was recorded at any of the concentrations of Hetester HCA tested.
The EC50 for growth rate inhibition (72h-ERC50) and yield inhibition (72h-EYC50) exceeded the maximum soluble concentration of test item in test medium, i.e. exceeded an initially estimated concentration of 0.039 µg/L.
The 72h-NOEC for growth rate inhibition was below 0.039 µg/L, based on statistical significance.
The 72h-NOEC for growth rate inhibition was 0.039 µg/L, based on biological relevance.
The 72h-NOEC for yield inhibition was below 0.039 µg/L, based on both statistical significance and biological relevance.

Executive summary:

The objectiveofthe study was to evaluate Hetester HCA for its ability to generate toxic effects inRaphidocelis subcapitata(formerly known asPseudokirchneriella subcapitata)during an exposure period of 72 hours and, if possible, to determine the NOEC, EC₁₀ and EC₅₀ for both inhibition of growth rate and inhibition of yield.

The study procedures described in this report were based on the OECD guideline No. 201, 2006; Annex 5 corrected 28 July 2011. In addition, procedures were based on the test methods described in the OECD series on testing and assessment number 23, 2000.

The batch of Hetester HCA tested was a viscous amber liquid with a purity >85% which was not completely soluble in test medium at the loading rates initially prepared.Water Accommodated Fractions (WAFs) were individually prepared at loading rates ranging from 1.0 to 100 mg/L and used as test concentrations.

A second combined limit/range-finding test was performed based on the results of a preceding combined limit/range-finding test. Six exponentially growing algal cultures per group were exposed to an untreated control and to a WAF prepared at a loading rate of 100 mg/L in the limit test. In addition, three replicates per group were exposed to WAFs prepared at 1.0 and 10 mg Hetester HCA per litre in a combined range-finding test. The initial algal cell density was 10⁴cells/mL.The total exposure period was 72 hours and samples for analytical confirmation of actual exposure concentrations were taken at the start, after 24 and 72 hours of exposure.

Samples taken from the WAF prepared at loading rate of 100 mg/L were analysed. The measured concentration at the start of the test was estimated to be 0.039 µg/L. No detectable test item concentrations were found after 24 and 72 hours of exposure. Nevertheless, it can be stated that testing was ultimately performed at the maximum soluble concentration of test item in test medium, since the water solubility was determined to be <1.5 µg/L.Based on these results, effects parameters were based onthe initially estimated exposure concentration of 0.039 µg/L.

Growth rate and yield at the limit concentration were statistically significantly inhibited by 2.5 and 13%, respectively, at the end of the test. For growth rate, however, effects were considered as biologically not relevant (i.e. <10%) and therefore, the NOEC based on biological relevance was set at the TWA concentration of 0.039 µg/L. 

The study met the acceptability criteria prescribed by the study plan and was considered valid.

The effect parameters (based on the TWA concentration) obtained in this study are summarized in the table below.

Parameter (µg/L)	NOEC*	NOEC**	EC10	EC50
Growth rate	<0.039	0.039	>0.039	>0.039
Yield	<0.039	<0.039	<0.039	>0.039

* - based on statistical significance, ** - based on biological relevance