Fatty acids, tall-oil, reaction products with 2-[(2-aminoethyl)amino]ethanol

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

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)

Version / remarks:

Mar 2006 (corrected 2011)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

Dec 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: loading rates 4.6, 10, 22, 46, 100 and 220 µg/L and solvent control
- Sampling method: samples were taken from each loading rate and the solvent control at test start, 24, 48 and 72 hours. At test start, samples were taken from the freshly prepared test media without algae. For the samplings at 24 and 48 hours, a set of additional flasks containing the corresponding test medium with algae was incubated under conditions identical to the test. At the end of the test, the test media of the treatment replicates were pooled.
- Sample storage conditions before analysis: deep-frozen at about -20 ± 5 °C. The storage stability was proven in pre-experiments.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: water accommodated fractions (WAFs)
- Test media:
Stock solution and application solution for the highest loading rate: 440 mg/L in acetone, intensely stirred for 2 minutes.
Application solutions for the lower loading rates: prepared from the stock solution by a series of dilutions with acetone.
Preparation of test media: 0.5 mL of each application solution were spiked into the empty stirring vessels. The acetone was evaporated to dryness under nitrogen stream. Thereafter, 1000 mL test water (AAP medium) were added into the vessels in order to dissolve the test item, using ultrasonic treatment for 15 minutes and intense stirring for 24 hours at room temperature in the dark. These solutions were tested as WAFs.
- Controls: test water (AAP) without addition of test item, nor solvent
- Solvent controls: 0.5 mL acetone without test item were spiked into the empty stirring vessel. The further procedure was the same as used for preparation of the test media.
Test and control media were prepared just before the start of the test.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): All test media were clear solutions

The method of preparation of the test media was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: 61.81 SAG
- Source (laboratory, culture collection): SAG, Institute for Plant Physiology, University of Göttingen, 37073 Göttingen / Germany).
- Age of inoculum (at test initiation): An inoculum culture was set up three days before the start of the exposure. The algae were cultivated under the test conditions and kept in the exponential growth phase until inoculation of the test solutions.
- Method of cultivation: standardized conditions according to the test guidelines

ACCLIMATION
- Acclimation period: three days (pre-culturing)
- Culturing media and conditions (same as test or not): same

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

0.15 mM (15 mg CaCO3/L)

Test temperature:

23 °C

pH:

Control and solvent control
test start (0 h): 7.3 for both
test end (72 h): 7.9 and 7.7

treatments
test start (0 h): 7.3 – 7.4
test end (72 h): 7.4 – 8.0

Nominal and measured concentrations:

Nominal concentrations (loading rates): (4.6, 10)*, 22, 46, 100, and 220 µg/l
Measured concentrations (time weighted geometric means**): 3.2, 3.8, 6.4, and 32 µg/L

*: The samples of the loading rates of 4.6 and 10 µg/L were not analyzed since they were below the NOEC determined in this test and therefore not relevant for the interpretation of the biological results.
**: As a decrease of test item concentrations during the test period was observed, the time weighted geometric means were calculated from the measured concentrations from all sampling dates.

For details see attached document "Compilation of Analytical Results - AS-400112.pdf"

Details on test conditions:

TEST SYSTEM
- Test vessel: 75 mL Erlenmeyer flasks containing 30 mL test medium, covered with a glass lid
- Aeration: continuous shaking on an orbital shaker
- Initial cells density: 5000 cells/mL (1.34 x 10E4 relative fluorescence units, RFU)
- Control end cells density: 213 x 10E4 RFU
- Solvent control end cells density: 198 x 10E4 RFU
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
- No. of vessels per solvent control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes (AAP Medium)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: sterile purified water
- Culture medium different from test medium: no
- Intervals of water quality measurement: The light intensity was measured at the start of the test. The pH was measured and recorded in each treatment at the start and end of the test. The temperature in the incubator was monitored and recorded continuously. The appearance of the test media was also visually controlled and recorded daily.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: continuously illumination by LED light installed above the test flasks.
- Light intensity and quality: The light intensity at the level of the test solutions was approximately 75 µE s-1 m-2 (range: 73 to 76 µE s-1 m-2, measured at nine places in the experimental area). The light intensity over the incubation area was within a ±15 %-deviation from the average light intensity as recommended by the guideline.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : daily determination of the algal biomass
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter]
fluorescence measurement (SpectraMax I3x, Molecular Devices Ltd, Wokingham Berkshire/UK) at an excitation of 440 nm and emission of 680 nm. The measurements were performed at least in duplicate.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.2
- Range finding study
- Test concentrations: nominal loading rates of 0.010, 0.10, and 10 mg/L (first range finder, test media filtrated) and 7.0, 30 and 70 µg/L (second range finder, test media unfiltrated)
- Results used to determine the conditions for the definitive study:
Inhibition of average growth (I):
First range-finder: 100 % I at nominal 1.0 and 10 mg/L; < 10% I at nominal 0.1 and 0.01 mg/L
Second range-finder: 10 % I at nominal 0.07 mg/L; < 10% I at 7.0 and 30 µg/L

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

100 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

nominal loading rate

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

95% confidence interval: 69 - 170 µg/L

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

14 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

nominal loading rate

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

95% confidence interval: 10 - 20 µg/L

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

46 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

nominal loading rate

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no microscopic abnormalities
- Any stimulation of growth found in any treatment: no
- Effect concentrations exceeding solubility of substance in test medium: no

All test media were clear solutions throughout the test period.

For details see attached document "Compilation of Biological Results for Algal growth - AS-400112.pdf"

Results with reference substance (positive control):

- Results with reference substance valid? yes
- 72-hour ErC50: 1.3 mg/L (within the range 0.9 - 1.5 mg/L as recommended by the guidelines)

Reported statistics and error estimates:

Statistical analysis was performed using ToxRat Professional® (ToxRat® Solutions GmbH, Version 3.2.1).
The mean yield and growth rate of the solvent control were not statistically significantly different from the control after 72 hours (according to Student t-test, α = 0.05, two-sided). Therefore all treatments were compared to the pooled control. All treatments were included in the statistical calculations.
The 72-hour EC10, EC20 and EC50 values for the inhibition of average growth rate and yield and their 95% confidence intervals were calculated by Probit Analysis using linear weighted regression.
For the determination of the LOEC and NOEC, the average growth rate and yield at the test concentrations were compared to the pooled control values by the Welch's t-test or the Williams' t-test, where appropriate.

Validity criteria fulfilled:

yes

Remarks:

Increase of biomass >16 after 72 hours; mean coefficient of variation for section-by-section specific growth rate <35%; coefficient of variation for average specific growth rate <7%.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 µg/L

EC10 or NOEC for freshwater algae:

46 µg/L