Fatty acids, C18-unsatd., trimers, reaction products with triethylenetetramine

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 (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:

At the start of the test, two samples (50 mL) were taken from freshly prepared uninoculated control and test media. After 72 hours, two samples (50 mL) of control and test media were taken from uninoculated replicate flasks. Samples were stored in a freezer prior to analysis.

Vehicle:

no

Details on test solutions:

REPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: water accommodated fractions (WAF)
15.7 mg/5L was dispersed in OECD medium in a glass vessel. The contents of the vessel was stirred for approximately 24 hours and then left to stand for approximately 24 hours. An aliquot (1200 mL) was then removed mid vessel to provide a Water Accomodated Fractions (WAF) at nominal loading rate of 3.13 mg/L. The WAF was then diluted to provide the test media at the lower levels.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland
- Age of inoculum (at test initiation): Three days. Algal cells used for inoculation were in the log phase of growth.
- Method of cultivation: Liquid slope cultures were stored in an illuminated refrigerator. Sterile algal nutrient medium was inoculated with cells aseptically removed from the slope culture; these primary liquid cultures were incubated for approximately three days in an orbital incubator under continuous illumination at nominal temperatures in the range 21 to 25°C. Subsequently, appropriate volumes of these primary cultures were aseptically transferred to fresh sterile algal nutrient medium to prepare secondary liquid cultures; these cultures were incubated, as stated above, for a further three days to provide an inoculum in the log phase of growth.

ACCLIMATION
- Acclimation period: 3 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:

25 mg/L as CaCO3

Test temperature:

22-23 °C

pH:

test start (0 h): 7.6-7.7
test end (72 h): 8.0-8.6

Nominal and measured concentrations:

nominal loading rates: 0.00931, 0.0298, 0.0954, 0.305, 0.977 and 3.13 mg/L as WAF
measured levels: The measured levels of TOC were below the limit of quantification of the analysis method after background correction for the control medium

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL conical flasks (autoclaved) containing 100 mL of inoculated test medium and loosly plugged with foam bungs
- Aeration/gaseous exchange: orbital incubator, oscillating at nominal 130 cycles per minute
- Initial cells density: 1x10⁴ cells/mL
- Control end cells density: 116x10⁴ cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: OECD medium according to guidelines, using filtered, dechlorinated tap water, softened and treated by reverse osmosis before microfiltration and purification (resistivity 18 Megohm/cm)
- Total organic carbon: 1.3 mg C/L
- Ca/Mg ratio: 1:1
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: continuous illumination
- Light intensity and quality: nominally 6292 to 6652 lux provided by 6 x 30 W “cool white” 1 metre fluorescent tubes. Light intensity (four corner positions and in a central position of the random block design) within the test area were determined each day. To minimise the impact of differences in light intensity across the test area on algal growth, control and test flasks were re-positioned in the test area each day during the test.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : cell densities measured at 24, 48 and 72 hours
- Determination of cell concentrations: electronic particle counter

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study
- Test concentrations: nominal loading rates of 1.0, 10 and 100 mg/L (range finder) and 0.0954, 0.305, 0.977, 3.13 and 10 mg/L (first study)
- Results used to determine the conditions for the definitive study:
range finder: inhibition of algal growth after 72 hours was 74 and 95% at 1.0 and 10 mg/L.
first study: growth was inhibited by 28% at 0.0945 mg/L, so a NOELR was not identified. Complete inhibition (100%) was observed at nominal loading rates of 0.977 mg/L and higher.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

0.45 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence limits: 0.3-0.5 mg/L; results are expressed in terms of loading rates

Key result

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

0.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence limits: 0.2-0.3 mg/L; results are expressed in terms of loading rates

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

0.03 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: results are expressed in terms of loading rates

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: stimulation of growth was observed for 24 to 48 hours and 48 to 72 hours daily growth rates (see attached backgrtound material (Table).

At the start of the test, the test medium was a colourless dispersion.

For details see "Attached background material" (Tables Cell densities and inhibition of growth (GAH0080).pdf; Effect concentrations (GAH0080).pdf).

Results with reference substance (positive control):

- 72-hour EbC50: 0.541 mg/L (typical range in this laboratory: 0.3 to 1 mg/L)

Reported statistics and error estimates:

Statistical analysis was perfomed using SAS 9.1 (SAS Institute 2002), using nominal loading rates. 95% confidence intervals were calculated using the likelihood ratio method. Williams’ test was used to compare each treated group with control unless there was evidence of a non-monotonic dose-response relationship, in which case Dunnett’s test was used.

Control cultures:

-cell concentration increased by a factor of 116;

-the mean coefficient of variation for daily growth rates ranged between 2.3 and 4.3%;

-the coefficient of variation for the average specific growth rates was 1.6% during the 72 hour exposure period

Validity criteria fulfilled:

yes

Remarks:

The criteria of OECD Guideline 201 and EU Method C.3 for biomass and growth rates were fulfilled.

Description of key information

effect values for average specific growth rates of Pseudokirchneriella subcapitata, 0-72 h, based on loading rates (OECD 201, EU C.3):

EL50: 0.45 mg/L, EL10: 0.3 mg/L, NOELR:

Additional information

EL50 for freshwater algae: 0.45 mg/L

EL10 for freshwater algae: 0.3 mg/L