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Ecotoxicological information

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study conducted uder GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Report date:
2011

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

1
Reference substance name:
Hydrogenated rosin alcohols
Cas Number:
2156595-41-2
IUPAC Name:
Hydrogenated rosin alcohols
Test material form:
liquid: viscous
Details on test material:
Sponsor's identification: Technical Hydroabietyl Alcohol
Description: Light amber coloured extremely viscous liquid
Lot number: HR-078
Purity: 100% (UVCB)
Expiry date: 21 December 2012
Storage conditions: Room temperature in the dark

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
The pH of each control and test flask was determined at initiation of the test and after 72 hours exposure. 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. Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.

Test solutions

Vehicle:
no
Details on test solutions:
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, 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. Using this approach, aqueous media are prepared by mixing the test item with water for a prolonged period. Pre-study work showed that a preparation period of 24 hours was sufficient to ensure equilibration between the test item and water phase.

To prepare the WAF solutions for the range finding study an amount of test item (20 mg) was weighed onto a glass slide and suspended within 2 litres of culture medium to give the 10 mg/L loading rate WAF. A further amount of test item (200 mg) was added to the surface of 2 litres of culture medium via a disposable syringe to give the 100 mg/L loading rate WAF. 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 mixtures 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 10 and 100 mg/L loading rate WAFs. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present. In the definitive limit test the 100 mg/L WAF was prepared in the same manner as above.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
The test was carried out using Pseudokirchneriella subcapitata strain CCAP 278/4. Liquid cultures of Pseudokirchneriella subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland. Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were maintained in the laboratory under constant aeration and illumination at 21 ± 1C. 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 10e3 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 ± 1C until the algal cell density was approximately 10e4 - 10e5 cells/mL.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h

Test conditions

Test temperature:
24 ± 1°C
pH:
7.6-8.0
Details on test conditions:
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). A range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to a series of nominal loading rates of 10 and 100 mg/L for a period of 72 hours. The test was conducted in 250 ml glass conical flasks each containing 100 ml of test preparation and plugged with polyurethane foam bungs to reduce evaporation. Two replicate flasks were prepared for each control and test concentration. An aliquot (500 ml) of each of the loading rate WAFs was separately inoculated with algal suspension (9.6 ml) to give the required test concentrations of 10 and 100 mg/L loading rate WAF. The control group was maintained under identical conditions but not exposed to the test item. The flasks were plugged with polyurethane foam bungs and incubated at 24 ± 1°C under continuous illumination provided by warm white lighting (380 - 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.

Based on the result of the range-finding test a "limit test" was conducted at a single loading rate of 100 mg/L to confirm that no effect on algal growth was observed. As in the range-finding test 250 ml glass conical flasks were used. Six flasks each containing 100 ml of test preparation were used for the control and 100 mg/L loading rate WAF treatment group. The control group was maintained under identical conditions but not exposed to the test item. Pre-culture conditions gave an algal suspension in log phase growth characterised by a cell density of 2.15 x 10e5 cells per mi. Inoculation of 1 litre of test medium with 23.3 ml of this algal suspension gave an initial nominal cell density of 5 x 10e3 cells per ml and had no significant dilution effect on the final test concentration.
The flasks were plugged with polyurethane foam bungs and incubated at 24 ± 1C 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.

Results and discussion

Effect concentrationsopen allclose all
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
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
The cell densities and percentage inhibition of growth values from the exposure of Pseudokirchneriella subcapitata to the test item during the range-finding test showed no effect on growth at 10 and 100 mg/L loading rate WAF. In the definitive limit tests the growth rate (r) and yield (y) of Pseudokirchneriella subcapitata were also not affected by the presence of the test item over the 72-Hour exposure period.

In the range-finding study chemical analysis of the 100 mg/L loading rate WAF test preparation at 0 and 72 hours showed measured test concentrations to range from 0.065 to 0.12 mg/L. In the definitive Limit test chemical analysis of the 100 mg/L loading rate WAF test preparation at 0 and 72 hours showed measured test concentrations to range from 0.056 to 0.15 mg/L. 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.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Executive summary:

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". 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 EL50 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. Chemical analysis of the 100 mg/L loading rate WAF test preparation at 0 and 72 hours showed measured test concentrations to range from 0.056 to 0.15 mg/1.