monoesters of C16 and C18 (branched and linear) fatty acids with decan-1-ol

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:

18 - 22 Dec 2017

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:

yes

Remarks:

The following GLP deviation is not considered to affect the integrity of the study or the validity of the conclusions drawn: The range-finding test completed prior to study initiation (Appendix 5) was undertaken outside of this claim for GLP compliance.

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United Kingdom (15 Nov 2016)

Analytical monitoring:

yes

Remarks:

gas chromatography / mass spectrometry

Details on sampling:

- Concentrations: Control, solvent control, 0.625, 1.25, 2.5, 5.0, and 10 mg/L (nominal)
- Sampling method: At the start of the test, 100 mL samples were taken from excess solutions. At the end of the test, one test vessel from each concentration was taken for whole sample analysis (all three test vessels taken for nominal 1.25 mg/L test concentration). The method of whole sample analysis was used due to the low solubility of the compound.

Vehicle:

yes

Remarks:

Tetrahydrofuran (THF) (100 µL/L per test vessel)

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A primary stock concentrate with a nominal concentration of 100 g/L was prepared by dissolving 1.00011 g test item in 10 mL tetrahydrofuran (solvent). Test solutions were then prepared by the direct addition of appropriate amounts of this concentrate to dilution water while stirring.
- Differential loading: Yes
- Controls: A solvent control was prepared in the sam way using solvent only. The (negative) control consisted of culture medium only.
- Chemical name of vehicle: The solvent tetrahydrofuran (THF) was used to assist in dosing due to the apparent low solubility of the test item in test media.
- Concentration of vehicle in test medium: 100 µL/L
- Evidence of undissolved material: The primary stock was a clear and colourless solution.The control, solvent control and the 0.625 and 1.25 mg/L test solutions were clear and colourless. The 2.5, 5.0 and 10 mg/L test solutions were colourless but with visible particles in a homogenous dispersion.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Unicellular green alga
- Strain: CCAP 278/4
- Source: In-house axenic laboratory cultures
- Age of inoculum: 3 d old culture of alga in the exponential growth phase
- Method of cultivation: Axenic cultures in AAP-medium

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

21.3 - 21.9 °C

pH:

7.10 - 7.27 ( 0 h)
7.30 7.73 (72 h)

Nominal and measured concentrations:

Control, solvent control, 0.625, 1.25, 2.50, 5.0 and 10.0 mg/L (nominal)
< LOQ, < LOQ, 0.28, 0.42, 0.72, 1.6 and 2.1 mg/L (arithmetic mean measured concentration)

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass conical flasks of 250 mL nominal capacity, filled with 100 mL test solution and closed with foam bungs.
- Initial cell density: 0.539E04 cells/mL (0.514 mL inoculum per test vessel)
- Control end cell density: 38.4E04 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): 6

GROWTH MEDIUM
- Standard medium used: Yes, AAP-medium

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: AAP medium
- Culture medium different from test medium: Culture medium same as test medium.
- Intervals of water quality measurement: The pH of the control and test solution was measured at the start of the test using the excess remaining after filling the test vessels. At the end of the test the pH was measured in one replicate vessel (with algae) from each test concentration. The temperature of the incubator was measured daily. Light intensity was measured once during the study, in each of the four representative positions.

OTHER TEST CONDITIONS
- Photoperiod: Continuous
- Light intensity and quality: "Cool-white" of approximately 6000 lux
- Other: Orbital shaking at 160 rpm. The position of each test vessel (replicate) in the incubator was randomised daily.

EFFECT PARAMETERS MEASURED:
Algal cell densities were measured as a surrogate for biomass.
- Determination of cell concentrations: Electronic particle counter (Coulter counter) (after 24, 48, and 72 h).
- Visual determination of cell aspect: Microscope (after 72 h)

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.0
- Range finding study: Yes, non-GLP
- Test concentrations: Control, solvent control, 0.001, 0.01, 0.1, 1.0, and 10
- Results used to determine the conditions for the definitive study: Yes,

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 2.1 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 2.1 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 2.1 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control: Yes
- Observation of abnormalities: Algae from the solvent control and the treatments appeared normal compared to the control algal cells
- Any stimulation of growth found in any treatment: No
- Any observations that might cause a difference between measured and nominal values: The test solutions at 2.5, 5.0 and 10 mg/L (nominal) were homogenous dispersions with visible particles.
- Effect concentrations exceeding solubility of substance in test medium: Yes

Reported statistics and error estimates:

Cell count data was statistically analysed with CETIS. Where appropriate, significant differences (p > 0.05) between the controls and test concentratinos were tested and ECx values with associated 95% confidence limits were determined.
Growth rates were examined by one-way analysis of variance and an Equal Variance Two-Sample t test to identify significant differences (p < 0.05) between the control and solvent control. There was no significant effect (p = 0.2334) between the control and solvent control. Therefore, the solvent control was compared to the treatments by one-way analysis of variance and Bonferroni adjusted t-test to identify signifcant differences (p < 0.05). ECx was calculated using the linear interpolation (ICPIN) method.

VALIDITY CRITERIA

The study fulfilled the validity criteria defined by OECD guideline 201 and is thus considered valid and reliable (Table 1).

 

Table 1: Validity criteria for OECD 201.

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.	Cell density increased by 71.2 (control) and 67.0 (solvent control) over the 72 h .	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%.	The mean coefficient of variation was 15% in the control and 17% in the solvent control.	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 with Pseudokirchneriella subcapitata.	The coefficient of variation of average specific growth rate as 2.2% in the control and 1.8% in the solvent control.	Yes

 

ANALYTICAL RESULTS

The measured test item concentrations in the test vessels are summarized in Table 2. The measured concentrations at the start of the test (0 h) were 83 – 160% of nominal and 0 – 13% at the end of the test (72 h).

Thus, effect values were based on mean measured test item concentrations. The arithmetic mean was calculated for the 0.625 mg/L (nominal) test concentration. Geometric means were calculated for the 1.25, 2.5, 5.0 and 10.0 mg/L (nominal) test concentrations.

 

Table 2. Analytical results.

Nominal test item concentration [mg/L]	measured test item concentration				Mean* measured concentration	Mean measured concentration
	0 h		72 h
	[mg/L]	% of nominal	[mg/L]	% of nominal	[mg/L]	[%]
Control	< LOQ	-	< LOQ	-	0	-
Solvent control	< LOQ	-	< LOQ	-	0	-
0.625	0.57	91	< LOQ	-	0.28	45
1.25	1.0^a	83	0.17^b	13	0.42	33
2.50	2.1	85	0.25	10	0.72	29
5.0	8.0^c	160	0.32	6	1.6	32
10.0	9.8	98	0.45	5	2.1	21

^*Arithmetic mean calculated for nominal 0.625 mg/L test concentration. Geometric means calculated for nominal 1.25, 2.5, 5.0 and 10.0 mg/L test concentrations.

^aMean of triplicate analyses: 0.99, 1.2 and 0.96 mg/L

^bMean of triplicate analyses: 0.21, 0.08 and 0.20 mg/L

^cMean of repeat analyses (1 sample): 7.9, 8.2 and 8.0 mg/L. The analysis for the nominal 5.0 mg/L 0 h sample was repeated multiple times due to the high concentration observed. Excess test solution for this concentration was resampled 24 h after the start of the test and analysed to confirm the accuracy of the 0 h results but was not used in the calculation of the measured concentration (resample concentration: 7.6 mg/L; 152% of nominal)

 

BIOLOGICAL RESULTS

The mean growth rates are summarized in Table 3. There was no significant difference (p < 0.05) between the solvent control and the culture medium control or any of the test concentrations. Based on these growth rates, a NOEC (72 h) of ≥ 2.1 mg/L, an ErC10 (72 h) of > 2.1 mg/L and an ErC50 (72 h) of > 2.1 mg/L were obtained (Table 4).

 

Table 3. Mean growth rates over the test period.

Nominal test item concentration	Mean measured test item concentration	Mean growth rate/d	Mean growth rate/d 95% confidence limits	Percentage of solvent control
[mg/L]	[mg/L]	0 – 72 h		[%]
Culture medium control	0	1.42	1.39-1.45	101
Solvent control	0	1.40	1.38-1.43	-
0.625	0.28	1.44	1.38-1.50	103
1.25	0.42	1.41	1.32-1.50	101
2.5	0.72	1.39	1.33-1.46	99
5.0	1.6	1.43	1.33-1.54	102
10	2.1	1.43	1.38-1.49	102

 

Table 4. Effect values.

	Test item concentration [mg/L]	95% confidence limits [mg/L]
NOEC	2.1	-
LOEC	> 2.1	-
ErC10	> 2.1	N/A
ErC20	> 2.1	N/A
ErC50	> 2.1	N/A

Validity criteria fulfilled:

yes

Remarks:

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

Conclusions:

The objective of this study was to determine the inhibition of the growth rate of the aquatic alga P. subcapitata by the present test item. The obtained effect values are a NOErC (72 h) of 2.1 mg/L, an ErC10 (72 h) of > 2.1 mg/L and an ErC50 (72 h) of > 2.1 (arithmetic mean measured, OECD 201).

Executive summary:

A 10 mg/L test concentration was selected as the highest concentration based on the dispersibility of the test substance observed in a non-GLP range-finding study. This concentration was dosed to the media via a solvent carrier (tetrahydrofuran (THF)) to assist in dosing the test compound. The concentration of solvent used in all exposure solutions with the exception of the control was 100 µL/L.

Control, solvent control and nominal concentrations of 0.625, 1.25, 2.5, 5.0 and 10 mg/L.

Control, solvent control and mean measured concentrations of 0.28, 0.42, 0.72, 1.6 and 2.1 mg/L.

Based on growth rate at the end of the test, the results obtained were:

Monoesters of C16 and C18 (branched and linear) fatty acids with decan-1-ol( mg/L)       

NOEC:    2.1

LOEC:   >2.1

ErC50:  >2.1

ErC20:  >2.1

ErC10:  >2.1

Based on yield at the end of the test, the results obtained were:

Monoesters of C16 and C18 (branched and linear) fatty acids with decan-1-ol (mg/L)       

NOEC:    2.1

LOEC:   >2.1

EyC50:  >2.1

EyC20:  >2.1

EyC10:  >2.1

Description of key information

No effects up to the limit of water solubility (measured, OECD 201, P. subcapitata)

Additional information

One study is available, in which the toxicity of Monoesters of C16 and C18 (branched and linear) fatty acids with decan-1-ol to aquatic algae was assessed according to OECD guideline 201 and GLP.

In a static test, the model organism P. subcapitata was exposed to nominal test item concentrations of 0.625, 1.25, 2.5, 5.0 and 10 mg/L including a control for 72 h. 10 mg/L was the highest concentration based on the dispersibility of the test item. This concentration was dosed to the media via the solvent carrier tetrahydrofuran (THF) to assist in dosing the compound. The concentration of solvent in all exposure solutions and the solvent control was 100 µL/L, except in the negative control. Test item concentrations were analytically verified by GC-MS at the start and end of the test.

The measured concentrations were 83 – 160% of nominal at the start of the study and 0 – 13% at the end of the study. Thus, results were based on the mean measured concentrations of 0.28, 0.42, 0.72, 1.6 and 2.1 mg/L. After 72 h of exposure, there was no significant difference between the control and solvent control (p = 0.2334) and no effects were observed on algal growth rates, resulting in a NOErC (72 h) of ≥ 2.1 mg/L for growth inhibition and no other effect values.