[No public or meaningful name is available]

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

Study period:

April 2013 - July 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:

no

Details on test solutions:

- Range-finding Test:
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.
The range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to 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.
Amounts of test item (20 and 200 mg) were each separately added to the surface of 2 liters of culture medium to give the 10 and 100 mg/L loading rates respectively. 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 cm 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.

- Definitive Test :
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.
An amount of test item (250 mg) was added to the surface of 2.5 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 cm 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 W AF. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present. An aliquot (1 liter) of the WAF was inoculated with algal suspension (10.6 mL) to give the required test concentration of 100 mg/L loading rate WAF.
The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 72 hours.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella 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

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

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
- ErC50 (0 - 72h) = 1,2 mg/L (95% confidence limits 1 - 1,3 mg/L)
- Other: the results from the positive control with potassium dichromate were within the normal ranges for this reference item.

Reported statistics and error estimates:

A Student's t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) was carried out on the growth rate and yield data after 72 hours for the control and the 100 mg/L loading rate to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS, 1999 - 2001).

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:

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 No. 201 and GLP. Due to the low aqueous solubility and complex composition 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.

Exposure of Pseudokirchneriella subcapitata to the test item gave EL50 so values of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading Rate was 100 mg/L loading rate WAF.

Analysis of the 100 mg/L loading rate WAF test preparation at 0 hours showed a measured test concentration of 0.0038 mg/L was obtained. A significant decline in measured test concentration was observed at 72 hours to less than the limit of quantitation (LOQ) of the analytical method employed which was determined to be 0.00090 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.

Description of key information

In an OECD 201 study conducted according to GLP, the acute toxicity of Amides, C16-18 (even numbered), N-C16-18 (even numbered) alkyl, to aquatic algae (Pseudokirchneriella subcapitata) for a period of 72 hours 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.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

100 mg/L