Fatty acids, coco, tetraesters with bis[2,2-bis(hydroxymethyl)butyl] adipate

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:

29 March 2012 to 29 April2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in accordance with recognised guideline

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

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:

1,6-bis({2,2-bis[(undecyloxy)methyl]butyl}) hexanedioate

EC Number:

700-772-5

Cas Number:

1190961-28-4

Molecular formula:

N/A - too complex

IUPAC Name:

1,6-bis({2,2-bis[(undecyloxy)methyl]butyl}) hexanedioate

Details on test material:

- Physical state: straw colored liquid
- Expiration date of the lot/batch: 17 September 2012
- Storage condition of test material: room temperature in the dark

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 100 mg/L
- Sampling method: samples were taken from the uninoculated control and 100mg/L loading rate WAF test group at 0 and 72 hours for TOC analysis. Duplicate samples were taken
- Sample storage conditions before analysis: samples were kept at -20 deg C.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)

- Method: the test the test item was prepared as a Water Accommodated Fraction (WAF). An amount of test item (200 mg) was added to the surface of 2 liters of culture medium to give the 100 mg/L loading rate. After the addition of the test item, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixture allowed to stand for 1 hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 em from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75-100 mL discarded) to give the 100 mg/L loading rate WAF. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present.

- Controls: concurrenr untreated

- Evidence of undissolved material (e.g. precipitate, surface film, etc): none

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Green alga
- Strain: Desmodesmus subspicatus, strain CCAP 278/4
- Source (laboratory, culture collection): Liquid cultures were obtained from the Culture Collection of Algae and Protozoa (CCAP), Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland.
- Method of cultivation: Cultures were maintained in the laboratory at a temperature of 24 ± 1 °C under constant aeration and constant illumination. Prior to the start of the test sufficient master culture was added to culture media contained in conical flasks to give an initial cell density of 10^3 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 -150 rpm) and constant illumination until the algal cell density was approximately 10^4 to 10^5 cells/mL.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test conditions

Hardness:

no data

Test temperature:

Temperature was maintained at 24 ± 1 °C throughout the test

pH:

The pH value of the control cultures was observed to increase from pH 8.0 at 0 hours to pH 8.2 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines.

Dissolved oxygen:

no data

Salinity:

no data

Nominal and measured concentrations:

Nominal loading rates were assigned to the definitive test at 100 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass conical flasks were used, each containing 100 mL of test preparation
- Type (delete if not applicable): closed
- Initial cells density: 5 x 10^3 cells per mL a
- Control end cells density:
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6


- Test vessel:
As in the range finding tests 250 ml glass conical flasks were used. Six flasks each containing 100 ml of test preparation were used for the control and three flasks each containing 100 ml were used for each treatment group. The control group was maintained under identical conditions but not exposed to the test item. Inoculation of test medium with the algal suspension gave an initial nominal cell density of 5E3 cells per ml and had not significant dilution effect on the final test concentration. The flasks were plugged with polyurethane foam bungs and incubated at 24 degrees C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380-730 nm) and constantly shaken at approximately 150 rpm for 72 hours. Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter Multisizer Particle Counter.



GROWTH MEDIUM
The culture medium used for the range finding and definitive tests was the same as that used to maintain the stock culture. Stock solutions of the culture medium was prepared using reverse osmosis purified deionized water and the pH adjusted to 7.5.

- Standard medium used: yes

Reference substance (positive control):

yes

Remarks:

A positive control used potassium dichromate as the reference material at 0.25, 0.5, 1.0, 2.0 and 4.0 mg/L. Exposure conditions +data evaluation for the positive control were similar to the definitive test.

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
Observation of abnormalities (for algal test):
All test and control cultures were inspected microscopically at 72 hours. There were no abnormalities detected in any of the control or test cultures at 72 hours.

Any other information on results incl. tables

Table. Inhibition of Growth Rate and Yield in the Definitive Test

NominalLoadingRate (mg!L)	GrowthRate (cells/mL/hour)		Yield (cells/mL)
	0-72h	%Inhibition	0-72h	%Inhibition*
Control                  R1 R2 R3 R4 R5 R6 Mean SD	0.075 0.071 0.067 0.070 0.077 0.074 0.072 0.004	-	1.13E+06 8.30£+05 5.99E+05 7.95E+05 1.29E+06 9.90E+05 9.39E+05 2.50E+05	-
100                        R1 R2 R3 R4 R5 R6 Mean SD	0.075 0.072 0.070 0.070 0.071 0.071 0.072 0.002	[4]: increased 0 3 3 1 1 1	1.07E+06 8.79E+05 7.82E+05 7.83E+05 8.35E+05 8.25E+05 8.62E+05 1.08E+05	8

[Increase in growth as compared to controls]

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Exposure of Pseudokirchneriella subcapitata to the test item gave ELso values of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading Rate was 100 mg/L loading rate WAF.

Executive summary:

Introduction

A study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata.  The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) 761/2009.

Methods

Due to the low aqueous solubility and complex nature of the test item for the purposes of the test the test item was prepared as a Water Accommodated Fraction (WAF).

Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to a Water Accommodated Fraction (WAF) of the test item, at a single nominal loading rate of 100 mg/L (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter Multisizer Particle Counter.

Results

Exposure of Pseudokirchneriella subcapitata to the test item gave ELso values of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading Rate was 100 mg/L loading rate WAF.

It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L.

Total Organic Carbon (TOC) analysis of the test preparations was performed at 0 and 72 hours. Given the background level of carbon in the control vessels and also the low level of carbon in the test vessels, it was considered that the results gave no evidence of the presence of test item in the WAF.

Therefore, given that the toxicity cannot be attributed to a single component or a mixture of components but to the test item as a whole the results were based on nominal loading rates only.