Reaction mass of 1,2,2,6,6-pentamethylpiperidin-4-yl hexadecanoate and 1,2,2,6,6-pentamethylpiperidin-4-yl octadecanoate

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:

15 August 2012 to 19 August 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study conducted according to OECD and EC test methods

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 1,2,2,6,6-pentamethylpiperidin-4-yl hexadecanoate and 1,2,2,6,6-pentamethylpiperidin-4-yl octadecanoate

EC Number:

700-792-4

Molecular formula:

C26H51NO2, C28H55NO2

IUPAC Name:

Reaction mass of 1,2,2,6,6-pentamethylpiperidin-4-yl hexadecanoate and 1,2,2,6,6-pentamethylpiperidin-4-yl octadecanoate

Test material form:

other: SOLID

Details on test material:

- Name of test material (as cited in study report): T-1640L

- Physical state: SOLID
- Analytical purity: 98.74%
- Purity test date: 20 May 2011
- Lot/batch No.: 10121
- Expiration date of the lot/batch: 18 October 2013
- Storage condition of test material: Refrigerated in the dark under dry conditions
- Other:

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
None

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: WAFs prepared at nominal loading rates of 0.100, 0.300, 1.00, 3.13 and 10.0 mg/L
- Sampling method: Two samples taken from freshly prepared control and test media. After 72 hours, the contents of the replicate flasks for each group were pooled and further samples taken for analysis. Additional samples were also taken from the flasks containing T-1640L at nominal loading rates of 0.100 and 10.0 mg/L WAF, but with no algal cells, in order to obtain information on the extent of adsorption/absorption of the test substance by the algal cells.
- Sample storage conditions before analysis: All samples, not analysed, were stored in a freezer in case further attention was required.

Test solutions

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Two aliquots of the test substance (50 and 15.6 mg) were each weighed on to a cover slide. Each was then added to a respective volumetric flask (5 L) which was then filled with OECD dilution medium. The flasks were stirred for approximately 24 hours in the dark, and then left to stand for approximately 24 hours in the dark. An aliquot (2000 mL) was then removed mid-vessel of each flask to provide Water Accommodated Fractions (WAF) which were used as the test media with nominal loading rates of 10 and 3.13 mg/L, respectively.
- Eluate:
- Differential loading:
- Controls:
- Chemical name of vehicle (organic solvent, emulsifier or dispersant):
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)):
- Evidence of undissolved material (e.g. precipitate, surface film, etc):

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Algae
- Strain:CCAP 278/4
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd., Dunstaffnage Marine Laboratory, Dunbeg, Oban, Argyll, Scotland
- Method of cultivation: Sterile algal nutrient medium (Appendix 2) was inoculated with cells aseptically removed from the slope culture; these primary liquid cultures (100 mL) were incubated for approximately three days in an orbital incubator under continuous illumination at nominal temperatures in the range 21 to 24C to provide an inoculum in the log phase of growth, characterised by a cell density of 1.82 x 106 cells/mL.

ACCLIMATION
- Acclimation period:
- Culturing media and conditions (same as test or not): same as test
- Any deformed or abnormal cells observed: none

Study design

Test type:

static

Water media type:

freshwater

Total exposure duration:

72 h

Post exposure observation period:

none

Test conditions

Hardness:

N/A

Test temperature:

23 +/- 2C NOMINAL (19.7 - 23.6C actual)

pH:

7.49 - 8.97

Dissolved oxygen:

N/A

Salinity:

N/A

Details on test conditions:

TEST SYSTEM
- Test vessel: 250mL conical flask
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 100mL innoculated test medium or test medium alone
- Aeration: No
- Type of flow-through (e.g. peristaltic or proportional diluter): N/a
- Renewal rate of test solution (frequency/flow rate): not renewed
- Initial cells density: ca. 1 x 104 cells/mL
- Control end cells density: 1.44 x 106 cells/mL
- No. of organisms per vessel: N/a
- 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
- Detailed composition if non-standard medium was used:

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: filtered, dechlorinated tap water which had been softened and treated by reverse osmosis, before microfiltration and purification (resistivity of 18 Megohm/cm).
- Alkalinity: pH8


OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: No
- Photoperiod: continuous illumination.
- Light intensity and quality: nominally 4440 to 8880 lux
- Salinity (for marine algae): No

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: cell densities were measured using a Coulter Z Series Particle Count and Size Analyser

TEST CONCENTRATIONS
- Spacing factor for test concentrations: >3
- Justification for using less concentrations than requested by guideline:
- Range finding study
- Test concentrations: 0.01, 0.1, 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: effects on algal growth

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

9.23 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

3.51 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

1

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

1 h

Dose descriptor:

NOELR

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.06 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 6

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.02 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 7

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.007 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 8

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.007 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

Mean measured values are extrapolated values from mean measured concentration value from the highest test level

Any other information on results incl. tables

Concentrations of T-1640L were measured for samples from the test media at a nominal loading rate of 10.0 mg/L.  At the start of the test, the measured level was 1% of nominal, which decreased after 72 hours to 0.35% of nominal (or 25% of its initial starting value). The mean measured concentration was 0.0696 mg/L. This value was used to calculate extrapolated values for the lower test concentrations, giving values of 0.000696, 0.00209, 0.00696 and 0.0218 mg/L. 

After 72 hours, analysis of the sample of media containing T-1640L at nominal a loading rate 10.0 mg/L, which had been incubated without algal cells, gave a similar result to test media incubated in the presence of algal cells. This indicated that the presence of algal cells did not significantly affect the stability of the test substance. 

The mean coefficient of variation (CoV) for section by section daily growth rates in control cultures was 3.7 during the definitive test and the CoV for the average specific growth rates of control culture was 0.984 during the 72 hour exposure period. The factor increase for control growth at 72 hours was 183.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The effect of T-1640L on the growth of the unicellular green alga Pseudokirchneriella subcapitata was assessed over a 72 hour period.
Based on nominal loading rates, the 72-hour EL50 for growth rate and yield was 9.23 and 3.51 mg/L mg/L, respectively. The ‘no-observed effect loading rate’ for growth rate and for yield was 1.00 mg/L .
Based on measured/calculated values, the 72-hour EC50 for growth rate and yield was 0.06 mg/L and 0.02 mg/L, respectively. The ‘no-observed effect concentration’ for growth rate and for yield was 0.00696 mg/L.