Esterification products of fatty acids, C18 (unsaturated) alkyl and adipic acid with pentaerythritol

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 May 2022 - 21 Feb 2023

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Remarks:

The water solubility of the test item is below the LOQ of the analytical method. The analytical method could not be improved.

Details on sampling:

Samples for possible analysis were taken from both the limit concentration and the control.
- Concentrations: 0, 2 mg/L (nominal water accommodated fraction (WAF) loading rate)
- Sampling method: 2.0 mL from the approximate centre of the test solutions at 0 and 72 h. At the end of the exposure period, the replicates with algae were pooled before sampling.
- Sample storage: Not applicable, samples were transferred to the analytical laboratory at the Test Facility and analysed on the day of sampling.

Compliance with the quality criteria regarding maintenance of actual concentrations was checked by preparing a test vessel at a WAF at a loading rate of 2.0 mg/L but without algae (abiotic control) and samples for analysis were taken at the start of exposure and at the end of the test period.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
Due to the very low solubility of the test material in test medium, a WAF was prepared. Loading rates were determined based on solubility pre-tests.

- Method: WAF
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): Tyndall effect negative

SOLUBILITY PRE-TESTS

A first pre-test was prepared with a loading rate of 100 mg/L, the solution was stirred slowly overnight, then it was stabilized for two hours. After siphoning through glass wool, a sample was taken and analysed to be around 0.11 µg/L, below the LOQ of the analytical method of 10 µg/L.

A second pre-test at a lower loading rate was prepared, to observe if a stable emulsion with measurable concentrations above the water solubility of the test material could be achieved. The test solution with a loading rate of 1.0 mg/L was stirred fast for a period of two hours, after which a sample was taken. Some suspended droplets were visible in the test solution. The test solution was agitated in an incubator for a three-day period, after which another sample was taken. After the samples were taken it was observed that the test material had separated out of the aqueous phase and was still present in undissolved form. The samples taken directly after stirring, at the start of the pre-test had measurable concentrations of test material, however after three days the concentrations were < LOQ. It is probable that the measured test material at the start of the pre-test was undissolved test material present as droplets in the aqueous phase.

A longer mixing period was not considered as no information was or could be obtained on stability of the test material in test medium.

PREPARATION OF WAF FOR THE DEFINITIVE TEST

Due to the very low solubility of the test material in test medium, a WAF was prepared at a loading rate of 2.0 mg/L, which is above the solubility limit of the test material. A loading rate of 2.0 mg/L represents a low amount of test material that can still be weighed and still allows for visual confirmation of the presence of undissolved test material.
- Loading rate: 0 (n=6), 2 mg/L (above water solubility) (n=6 with algae + 1 without algae)
- Description: The test item was weighted out on a watch glass after which the watch glass was added to the test medium.
- Stirring: A two-hour period of fast magnetic stirring was applied to ensure creating a homogeneous emulsion.
- Sampling method: The obtained mixture was checked for Tyndall effect. Thereafter, the aqueous WAF was immediately poured in the test vessels and used as the limit concentration.
- Test medium: M2

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain: NIVA CHL 1
- Source: In-house laboratory culture
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: Three 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.

ACCLIMATION
- Acclimation period/Culturing media and conditions: The pre-culture was maintained under the same conditions as used in the test.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

22 - 23 °C

pH:

8.0 - 8.1

Nominal and measured concentrations:

Nominal: 0 mg/L and 2 mg/L (WAF loading rate)
Measured (at 0 h): 0 mg/L and 45 µg/L
Measured (at 72 h): 0 mg/L and Geometric mean measured: 15 µg/L (applying half of the LOQ for the end result)

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL, all-glass
- Type: with aluminium caps, perforated for ventilation
- Fill volume: 50 mL
- Aeration: no
- Initial cells density: 1 x 10^4 cells/mL
- Control end cells density: 521.84 x 10^4 cells/mL (mean of all replicates)
- No. of vessels per concentration (replicates): 6
- No. of vessels per abiotic control, test item without algae (replicates): 1
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: M2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The culture medium was prepared using reverse osmosis purified deionized water.
- Culture medium different from test medium: no
- Intervals of water quality measurement: pH of the control and each test concentration (0 h and 72 h); temperature continuously in a temperature control vessel

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: continously
- Light intensity and quality: 82 to 91 µE.m-2.s-1, provided by TLD-lamps

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: 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) at 24, 48 and 72 h. Test medium was used as blank.


TEST CONCENTRATIONS
- Spacing factor for test concentrations: Limit test
- Justification for using less concentrations than requested by guideline: It was chosen to perform a limit test, as no toxicity was expected due to the low solubility of the test material in water.
- Range finding study : no, due to low water solubility of the test item

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

>= 2 mg/L

Nominal / measured:

nominal

Conc. based on:

other: WAF loading rate

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

> 2 mg/L

Nominal / measured:

nominal

Conc. based on:

other: WAF loading rate

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL20

Effect conc.:

> 2 mg/L

Nominal / measured:

nominal

Conc. based on:

other: WAF loading rate

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 2 mg/L

Nominal / measured:

nominal

Conc. based on:

other: WAF loading rate

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Effect concentrations exceeding solubility of substance in test medium: It should be noted that the actual solubility of the test material in medium proved to be well below the detection limit of the analytical method. The analytical method could not be further improved to enable measuring at lower levels. It was consequently decided to test a concentration above the solubility limit, but in the range of the analytical method possibilities.
- Observation of abnormalities (for algal test): 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.

Results with reference substance (positive control):

- Results with reference substance valid?: yes
- EC50: 0.95 mg/L
- Other: performed March 2022

Reported statistics and error estimates:

For determination of the NOELR 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 limit concentration compared with those obtained in the negative control revealed significant inhibition of growth rate or inhibition of yield (two-sample Mann-Whitney U-test, α=0.05, one-sided, smaller). This statistical comparison was preceded by a check on normal distribution of the data (Shapiro-Wilk’s Test) and a test for homogeneity of the variances (Levene’s test).
The ELx-values could not be determined because the observed effects were below 10%.
ToxRat Professional v 3.3.0 (ToxRat Solutions® GmbH, Germany) was used to perform the analysis.

Table 1          
Growth Rate and Percentage Inhibition for the Total Test Period

Loading rate (mg/L)	Mean(day^-1)	Std. Dev.	n	%Inhibition
Control	2.085	0.0198	6
2.0	2.062	0.0235	6	1.1

Table 2          
Growth Rate and Percentage Inhibition at Different Time Intervals

Loading rate (mg/L)	n	0 – 24 h		24 – 48 h		48 – 72h
		Mean(day^-1)	%Inhibition^#	Mean(day^-1)	%Inhibition	Mean(day^-1)	%Inhibition
Control	6	2.761		2.065		1.430
2.0	6	2.900	-5.0	1.868	9.6	1.419	0.78

^#Negative values indicate stimulation rather than inhibition.

Table 3: Validity criteria for OECD 201 (2006)

Criterion from the guideline	Outcome	Validity criterion fulfilled
The biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72-hour test period.	Biomass in the control increased by a factor of 522	yes
The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures must not exceed 35%	Mean coefficient of variation for section-by-section specific growth rates was 32%	yes
The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% in tests withPseudokirchneriella subcapitataandDesmodesmus subspicatus. For other less frequently tested species, the value should not exceed 10%.	The coefficient of variation of average specific growth rates was 0.95%	yes

Validity criteria fulfilled:

yes

Remarks:

For further details please refer to table 3 in “Any other information on results incl. tables”.

Description of key information

No effects up to and above the limit of water solubility (OECD 201)

Additional information

One experimental study is available investigating the toxicity of Esterification products of fatty acids, C18 (unsaturated) alkyl and adipic acid with pentaerythritol to freshwater algae. The study was performed according to OECD 201. Since the test substance is poorly soluble in water, a Water Accommodated Fraction (WAF) of 2.0 mg/L (nominal) was prepared. The nominal loading rate was analytically verified by UPLC/MS-MS analysis and resulted in a concentration of 0.045 mg/L at study initiation and depleted below the limit of quantification at the end of the study after 72 h.
After incubation, inhibition of growth of 1.1% was recorded at the limit WAF loading rate of 2.0 mg/L (nominal). Statistical analysis resulted in no significant difference between the control and the limit WAF loading rate of 2.0 mg/L (nominal). Thus, the NOELR (72 h) was set to 2.0 mg/L based on growth rate, which is above the water sobubility of the test substance.

In conclusion the test substance will not exhibit toxic effects to aquatic algae up to the limit of water solubility.