Fatty acids, C16-18, 2-butyloctyl esters

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:

2015-06-30 - 2015-09-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: well documented study performed according to OECD Guideline and GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Separate replicates for the test item analysis at the beginning of exposure were prepared without algae. For the test item analysis after 72 hours, samples were taken out of the test replicates. All original samples were stored at room temperature before preparation, if necessary. Prepared samples were stored in an autosampler at room temperature until analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
One water accommodated fraction (WAF) was prepared with a accommodated fraction nominal loading of the test item of 100 mg/L.
For the limit loading level, an appropriate amount of the test item was weighed out on a glass slide and inserted in a brown glass bottle with an appropriate amount of dilution water. This dispersion was shaken for at 24 hours with 20 rpm at room temperature. After a separation phase of 24 hour the WAF was taken from the homogeneous liquid phase from the middle of the glass flask. The WAF was checked via laser beam (Tyndall effect) for undissolved test item (formation of an emulsion).

- Method: A saturated solution was prepared with dilution water (loading: 100 mL test item/L). The test item was placed on the surface of the dilution water in a closed bottle and slowly stirred for 24 hours (the formation of an emulsion was avoided).
- Controls: dilution water
- Evidence of undissolved material (e.g. precipitate, surface film, etc): The test media were turbid throughout the test period.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Green alga
- Strain: Pseudokirchneriella subcapitata HINDAK, SAG 61.81
- Source (laboratory, culture collection): Sammlung von Algenkultutren (SAG), Pflanzenphysiologisches Institut der Universität Göttingen, Germany
- Age of inoculum (at test initiation): a three day old preculture was used
- Method of cultivation: fresh stocks were prepared every month on Z-Agar, light intensity amounted 35 - 70 µE/m²s for 24 h per day; Nutrient medium Z according to LÜTTGE et al. (1994)

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Hardness:

0.24 mmol Ca+Mg/L

Test temperature:

21 - 24 °C

pH:

8.1 +- 0.2

Dissolved oxygen:

no data

Salinity:

freshwater test according to guideline

Nominal and measured concentrations:

Nominal: 100 mg/l of saturated solution (WAF)
Measured: 11.3 mg/L (geometric mean)

Details on test conditions:

TEST SYSTEM
- Initial cells density: approximately 6153 cells/ml
- Control end cells density: cells/ml (mean)
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

TEST MEDIUM
- Dilution water: according to OECD test guideline 201

OTHER TEST CONDITIONS
- Photoperiod: 24 h/d light
- Light intensity and quality: 60 - 120 µE/m²s

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell desnity: chlorophyll a-fluorescence, excitation at 436 nm, emission at 685 nm, Dilution water was used as a background signal. No self-fluorescence was found in the preliminary range finding test at the water accommodated fractions of 1.00 to 100 mg/L.
- Measurement intervall: at 0, 24, 48 and 72 hours

Microscopic evaluation of the cells at the start and the end of exposure was carried out. The cells were checked for unusual cell shapes, colour differences, differences in chloroplast morphology, flocculation, adherence of algae to test containers and agglutination of algae cells.

TEST CONCENTRATIONS


- Range finding study
- Test concentrations: 100; 10; 1 mg/L
- Results used to determine the conditions for the definitive study:
100 mg/L: - 3 % growth rate inhibition, means growth stimulation
10 mg/L: 0 % growth rate inhibition
1 mg/L; - 6 % growth inhibtion, means growth stimulation

Reference substance (positive control):

yes

Remarks:

potassium dichromate (experimental date 2015-04-14 to 2015-04-17)

Key result

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

> 11.3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 11.3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

11.3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate

Basis for effect:

other: yield inhibition

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Adherence to test vessels: no
- Aggregation of algal cells: no
- Any stimulation of growth found in any treatment: no (slight increase of growth rate in lower concentrations, but within coefficient of variation of average growth in replicate control cultures)
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: turbid solution
- Effect concentrations exceeding solubility of substance in test medium: n.a.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- ErC50: 0.613 mg/l/72h
- EyC50: 0.281 mg/l/72h

Reported statistics and error estimates:

No statistics performed

Table 1: Cell densities
Nominal loading rate [mg/L]	Cell density x 10^4 [cells/mL]
	0 h	24 h	48 h	72 h
control	0.6153	2.2655	19.8001	200.6850
100	0.6153	2.3025	19.4055	180.6533
Table 2: Evaluation after 72 h
Nominal loading rate [mg/L]	Growth rate [d^-1]	Rate-related inhibition [%]	Yield x 10^4 [cells/mL]	Inhibition of yield [%]
control	1.93	-	200.0967	-
100	1.89	1.89	180.0380	10
* = biologically significant
negative inhibitions = increase of growth

all not biologically significant

Validity criteria fulfilled:

yes

Conclusions:

In this study Fatty acids, C16-18, 2-butyloctyl esters was found not to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours.

No effects were found up to the maximum water solubility level (estimated ≤ 5*10-7 mg/L), which is below the nominal loading level of the saturated solution with 100 mg/L. Therefore, no effects were found in the range of water solubility of the test item.

Executive summary:

The toxicity of Fatty acids, C16 -18, 2 -butyloctyl esters to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 761/2009 Method C.3. The aim of the study was the determination of NOEL, LOEL, EL10- , EL20- and EL50-values of growth rate and yield over a period of 72 hours in a limit test. The study was conducted under static conditions with an initial cell density of 6153 cells/mL. One Water Accommodated Fraction* (WAF) was prepared with a nominal loading of 100 mg/L. Six replicates were tested for the limit test item loading and six replicates for the control. The environmental conditions were within the acceptable limits. The test media were turbid throughout the test period. The concentrations of the test item Fatty acids, C16 -18, 2 -butyloctyl esters in the limit test item loading and the control were analytically verified by GC-MS at the start and the end of exposure. The measured concentration of Fatty acids, C16-18, 2-butyloctyl esters in the water accommodated fraction was 19.2 mg/L at the start of the exposure (0 h) and 6.67 mg/L at the end of the exposure (72 h). No effects were found up to the maximum water solubility level (estimated ≤ 5*10-7 mg/L), which is below the nominal loading level of 100 mg/L. Therefore, no effects were found in the range of water solubility of the test item. The NOEC-values for inhibition of both growth rate and yield after 72 hours were 11.3 mg/L, corresponding to a nominal test item concentration of 100 mg/L. The EC50-values for inhibition of both growth rate (ErC50) and yield (EyC50) after 72 hours were > 11.3 mg/L, corresponding to a nominal test item concentration of > 100 mg/L. It should be considered that the measured concentrations are higher than the calculated water solubility. Per definition of the WAF all terms related to concentration level will be given as loading level because partly dissolved compounds and mixtures cannot be related to concentrations.

At the water accommodated fraction of the test item Fatty acids, C16 -18, 2 -butyloctyl esters with a nominal loading rate of 100 mg/L, no inhibitory effects on the growth rate were observed for Pseudokirchneriella subcapitata. The ErL50 corresponds to a nominal loading rate of > 100 mg test item/L.

Description of key information

In an OECD 201 study Fatty acids, C16-18, 2-butyloctyl esters was found not to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

Additional information

The toxicity of Fatty acids, C16 -18, 2 -butyloctyl esters to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 761/2009 Method C.3. One Water Accommodated Fraction* (WAF) was prepared with a nominal loading of 100 mg/L. No effects were found up to the maximum water solubility level (estimated ≤ 5*10-7 mg/L), which is below the nominal loading level of 100 mg/L. Therefore, no effects were found in the range of water solubility of the test item. The NOEC-values for inhibition of both growth rate and yield after 72 hours were 11.3 mg/L, corresponding to a nominal test item concentration of 100 mg/L. The EC50-values for inhibition of both growth rate (ErC50) and yield (EyC50) after 72 hours were > 11.3 mg/L, corresponding to a nominal test item concentration of > 100 mg/L. It should be considered that the measured concentrations are higher than the calculated water solubility. Per definition of the WAF all terms related to concentration level will be given as loading level because partly dissolved compounds and mixtures cannot be related to concentrations.