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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April 2013
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
Principles of method if other than guideline:
In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test items, a modification of the standard method for the preparation of aqueous media was performed. An approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996, OECD 200 and Singer et al 2000), is to expose organisms to a Water Accommodated Fraction (WAF) of the test item in cases where the test item is a complex mixture and is poorly soluble in water and in the permitted auxiliary solvents and surfactants.
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 100 mg/L
- Sampling method: Samples were taken from the control and the 100 mg/L loading rate WAF test group (replicates R1 - R6 pooled) at 0 and 72 hours for quantitative analysis.
- Sample storage conditions before analysis: The 0-Hour test samples were stored at approximately -20°C prior to analysis, the 72-Hour test samples were analyzed on the date of receipt.
Vehicle:
not specified
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
Using a Water Accommodated Fraction (WAF) approach, aqueous media are prepared by mixing the test item with water for a prolonged period. Pre-study work showed that the maximum amount of dissolved test item was achieved after a preparation period of 24 hours. At the completion of mixing and following a 1-Hour settlement period, the test item phase is separated by siphon and the test organisms exposed to the aqueous phase or WAF (which may contain dissolved test item and/or leachates from the test item). Exposures are expressed in terms of the original concentration of test item in water at the start of the mixing period (loading rate) irrespective of the actual concentration of the test item in the WAF.

Pre-solubility work was carried out to determine whether stirring for a prolonged periods produced significantly higher measured test concentrations in the WAF.

A WAF of nominal loading rate of 100 mg/L was prepared, in duplicate, in deionized reverse osmosis water. One loading rate was stirred for a period of 23 hours and the other for a period of 95 hours. After a 1-Hour standing period the mixtures were then removed by siphon and the concentration of the test item in the 100 mg/L loading rate WAFs was verified by chemical analysis.

Amounts of test item (10, 40 and 200 mg) were each separately added to the surface of 10, 4 and 2 litres of reconstituted water to give the 1.0, 10 and 100 mg/L loading rates respectively. After the addition of the test item, the reconstituted water 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 mixtures allowed to stand for 1 hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present. The aqueous phase or WAF was removed by mid-depth siphoning (the first approximate 75-100 mL discarded) to give the 1.0, 10 and 100 mg/L loading rate WAFs.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM- Common name: Pseudokirchneriella subcapitata-
Strain: CCAP 278/4- Source (laboratory, culture collection): Liquid cultures of Pseudokirchneriella subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland.:-.
ACCLIMATION- Acclimation period: Prior to the start of the test sufficient master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of approximately 1E+03 cells/mL.
The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 – 150 rpm) and constant illumination at 24 ± 1°C until the algal cell density was approximately 1E+04 – 1E+05 cells/mL.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Test temperature:
24 ± 1 ºC
pH:
7.6-8.7
Nominal and measured concentrations:
Range-finding Test: Nominal 10 and 100 mg/L
Definitive Test: Nominal 100 mg/L
Details on test conditions:
OTHER TEST CONDITIONS- Adjustment of pH: the pH adjusted to 7.5 ± 0.1 with 0.1 N NaOH or HCl- EFFECT PARAMETERS MEASURED (with observation intervals if applicable):- Determination of cell concentrations: Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.
TEST CONCENTRATIONS- Range finding study: - Test concentrations: 10, 100 mg/L - Results used to determine the conditions for the definitive study: The results showed no effect on growth at 10 and 100 mg/L loading rate WAF.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
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.
- Effect concentrations exceeding solubility of substance in test medium: Observations on the test media were carried out during the mixing and testing of the WAFs.
At both the start and end of the mixing period, and following a 1-Hour standing period, the 100 mg/L loading rate WAF was observed to have formed a clear colourless media column with flakes of test item floating at the media surface. Microscopic examination of the WAF showed there to be no micro-dispersions of test item present.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
Validity criteria fulfilled:
yes
Conclusions:
The effect of the test material on the growth of Pseudokirchneriella subcapitata has been investigated and gave EL50 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:

The effect of the test material on the growth of Pseudokirchneriella subcapitata has been investigated and gave EL50 values of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading Rate was 100 mg/L loading rate WAF.

Description of key information

The acute toxicity of the test material to aquatic algae Effect Loading (EL)50 greater than 100 mg/L loading rate WAF, and the No Observed Effect Loading Rate is 100 mg/L loading rate WAF.

Key value for chemical safety assessment

EC50 for freshwater algae:
100 mg/L
EC10 or NOEC for freshwater algae:
100 mg/L

Additional information

In an OECD 201 study, conducted according to GLP , the acute toxicity of n-octadecylstearamide, UVCB to aquatic algae (Pseudokirchneriella subcapitata) is Effect Loading (EL)50 greater than 100 mg/L loading rate WAF, and the No Observed Effect Loading Rate is 100 mg/L loading rate WAF (Harlan Laboratories Ltd., 2013).