Octadecyl docosanoate

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:

08 Jan – 02 Mar 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:

2006, corrected 2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesanstalt für Umwelt, Messungen und Naturschutz Baden-Württemberg, Karlsruhe, Germany

Test material

Test material information

Constituent 1

Reference substance name:

Octadecyl docosanoate

EC Number:

246-115-1

EC Name:

Octadecyl docosanoate

Cas Number:

24271-12-3

Molecular formula:

C40H80O2

IUPAC Name:

octadecyl docosanoate

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Sampling method: Analytical samples were taken from the highest test concentration and from the control at 0 hours (initial value) from fresh test solutions and after 24 hours and 72 hours from aged test solutions.
- Sample storage conditions before analysis: All samples were stored deep frozen until they were transferred to the analytical laboratory.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances):
- Method: The test item was applied as saturated solution. A stock solution was prepared by directly weighing 100 mg in 1000 mL test medium. This stock solution was stirred in the dark at room temperature for 24 h (based on OECD Series on Testing and Assessment No. 23). After the settling of undissolved test item and phase separation, the necessary volume for the test was withdrawn via a Teflon tube from the medium level of the stock solution. Algae were added to the stock solution resulting in target cell densities of 0.5 × 10E+04 cells per mL. The test item solutions with concentrations between 10 and 0.010 mg/L were made by diluting the appropriate solutions with AAPmedium containing algae cells with a target density of 0.5 × 10E+04 cells per mL. Approximately 50 mL of the prepared solutions were transferred to each test vessel.
- Controls: blank control without test material
- Eluate: no
- Differential loading: no
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): After stirring, white deposits were observed at the vessel wall and on the surface of the solution. Subsequently the undissolved test item was allowed to sediment and/or float for a period of 1.5 h until the phases had separated. The necessary volumes for the test were withdrawn from the clear phase of the stock solution.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: freshwater green algae
- Strain: SAG 61.81
- Source: MBM Sciencebridge GmbH, Hans-Adolf-Krebs-Weg 1, D-37077 Göttingen, Germany
- Age of inoculum (at test initiation): 3-4 days
- Method of cultivation: The algae are grown semi-continuously in sterile cultures in the laboratory. Old medium is periodically replaced by fresh mineral solution in order to keep the algae in an exponential growth state.
- Conditions of cultivation: Illumination: continuously (approx. 4440 - 8880 lux at cell culture level or 60 - 120 μEm-2s-1); Temperature: 21 - 24 °C; Culture flasks: 100 mL Erlenmeyer flasks; CO2 supply by shaking on a rotating shaker, approximately 105 rpm

ACCLIMATION
- Acclimation period: 3-4 days
- Culturing media and conditions: same as test

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test conditions

Test temperature:

22.6 – 23.1 °C

pH:

Control: 7.53 – 7.88
Treatment: 7.47 - 7.85

Nominal and measured concentrations:

nominal: 100, 10.0, 1.00, 0.0100 mg/L and control
measured: 0.532 mg/L for nominal 100 mg/L; < LOD for control; other test concentration levels were not analysed.

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks (100 mL) with aluminium caps were filled up with ~ 50 mL test solution
- Initial cells density: 0.5 × 10E+04 cells/mL (targeted)
- Control end cells density: 28.70 × 10E+04 cells/mL (mean)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: AAP-Medium (according to Annex 3 of OECD 201)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to guideline
- Intervals of water quality measurement: Measurements of pH-value were performed at test start and end, the temperature was measured continuously and recorded daily.

OTHER TEST CONDITIONS
- Adjustment of pH: pH was adjusted to 7.5 ± 0.1 with NaOH or HCl, if necessary
- Photoperiod: continuous light
- Light intensity and quality: 75.9 μEm-2s-1 (mean)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: fluorescence measurement (fluorescence microplate reader (infinite 200Pro) with an emission wavelength of 670 nm and evaluated with Tecan i-control (Software for Tecan Readers Tecan i-control, 1.11.1.0)). Additionally, the morphological appearance of the algae cells was observed microscopically at the end of the test.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10 (limit test design)
- Range finding study: The test is performed in a range-finding limit test design.

Reference substance (positive control):

yes

Remarks:

Potassium Dichromate; tested regularly

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.532 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI could not be determined

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI could not be determined

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 0.532 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI could not be determined

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI could not be determined

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.532 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% CI could not be determined

Effect concentrations 6

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% CI could not be determined

Effect concentrations 7

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 0.532 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% CI could not be determined

Effect concentrations 8

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% CI could not be determined

Effect concentrations 9

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 0.532 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: growth rate and yield

Effect concentrations 10

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: growth rate and yield

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test):The cells were considered normal for the control and up to and including a nominal concentration of 100 mg/L.
For further details on biological results, please refer to section "Any other information on results incl. tables".

Results with reference substance (positive control):

EC50 (growth rate) = 1.23 mg/L (95% CI: 1.11 - 1.36 mg/L)
EC50 (yield) = 0.685 mg/L (95% CI: 0.610 - 0.764 mg/L)

Reported statistics and error estimates:

The statistical evaluation for the 72 hours period was attempted for growth rate and yield using SAS® (2002–2010). A test for normality of the data was attempted by calculating the Shapiro-Wilk statistic and the homogeneity of variance of the data was evaluated by calculating the Levene Test. The NOELR and LOELR as well as NOEC and LOEC were determined by using a multiple comparison method (Dunnetts-t-test, left sided, for growth rate and for yield). The calculation of the EL10, 20, 50-values as well as EC10, 20, 50-values was not indicated since the inhibition was below 10 % at all test concentrations for both, yield and growth rate.

Any other information on results incl. tables

Table 1: Average cell numbers for each sampling time and concentration

Nominal concentration [mg/L]	Average cell numbers [10^4/mL]
	0 h	24 h	48 h	72 h
Control	0.53	1.49	5.22	28.70
0.0100	0.53	1.72	5.83	34.82
0.100	0.53	1.82	6.74	39.44
1.00	0.53	1.62	6.59	37.41
10.0	0.53	1.83	6.90	41.05
100	0.53	1.80	7.66	43.84

Table 2: Percentage inhibition of growth rate

Nominal concentration [mg/L]	% Inhibition of growth rate
	0 – 24 h	0 – 48 h	0 – 72 h
Control	0.0	0.0	0.0
0.0100	-14.1	-5.0	-4.9
0.100	-18.9	-11.1	-7.9
1.00	-9.0	-9.7	-6.6
10.0	-20.4	-11.9	-9.0
100	-18.3	-16.6	-10.4

Table 3: Percentage inhibition of yield

Nominal concentration [mg/L]	% Inhibition of yield
	0 – 24 h	0 – 48 h	0 – 72 h
Control	0.0	0.0	0.0
0.0100	-25.3	-13.2	-21.7
0.100	-35.8	-32.7	-38.1
1.00	-14.7	-29.5	-30.9
10.0	-36.8	-36.1	-43.8
100	-33.7	-52.4	-53.7

Table 4: Determined concentration of octadecyl docosanoate

Test item	Octadecyl docosanoate	Sampling	Octadecyl docosanoate		Geometric mean	Actual concentration
nominal [mg/L]		[h]	[mg/L]	% of nominal	[mg/L] ^1)	[mg test item/L]
Control	0	0 fresh	< LOD	-	-	-
		24 aged	< LOD	-
		72 aged	< LOD	-
100	92.0	0 fresh	1.28	1.39	0.489	0.532
		24 aged	0.734	0.798
		72 aged	0.164	0.178

- = not calculated; LOD = 0.0300 mg/L octadecyl docosanoate; LOQ = 0.100 mg/L octadecyl docosanoate

1) According to OECD Guidance document No. 23 (2000)

Table 5: Validity criteria

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.	factor of 54.15	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%	27%	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 and Desmodesmus subspicatus. For other less frequently tested species, the value should not exceed 10%.	0.7%; did not exceed 7% for the whole test period	yes

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

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