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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Identification: 2,2'-[propane-1,3-diylbis(oxy)]bis(3",5,5"-tri-tert-butyl-5'-methyl-1,1':3',1"-terphenyl-2’-ol)
Batch: 1502501005
CAS number: 1042662-40-7
Purity: 98.1%
Physical state/Appearance: Sponsor - solid powder, white, odorless
Envigo - white powder
Expiry Date: 01 February 2025
Storage conditions: room temperature in the dark
Analytical monitoring:
yes
Details on sampling:
Samples were taken at beginning and end of the test.
Vehicle:
no
Details on test solutions:
Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication and high shear mixing. Given this, and at the request of the Sponsor, a slow stir saturated solution method of preparation was selected for testing.
Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to solutions of the test item at nominal concentrations of 6.25, 12.5, 25, 50 and 100% v/v saturated solution (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ±1 °C. The test item solutions were prepared by stirring an excess (50 mg/L) of test item in culture medium with the aid of magnetic stirring at a rate such that a dimple was formed at the media surface for 24 hours. After the stirring period any undissolved test item was removed by filtration (0.2 µm Sartorius Sartopore filter, first approximate 1 liter discarded in order to pre-condition the filter) to produce a 100% v/v saturated solution of the test item. This saturated solution was then further diluted as necessary, to provide the remaining test groups.
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), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland. Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were kept under constant agitation by orbital shaker (approximately 150 rpm) and constant illumination at 24 ±1 °C.
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 10^3 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (approximately 150 rpm) and constant illumination at 24 ±1 °C until the algal cell density was approximately 10^4 to 10^5 cells/mL.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
24 ±1 °C.
Nominal and measured concentrations:
Nominal: 6.25, 12.5, 25, 50 and 100% v/v saturated solution.
Details on test conditions:
Exposure Conditions
As in the range-finding test 250 mL glass conical flasks were used. Six flasks each containing 100 mL of solution were used for the control group and 3 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.
Pre-culture conditions gave an algal suspension in log phase growth. Inoculation of 900 mL of test medium with algal suspension gave an initial nominal cell density of 5.00 x 10^3 cells per mL.
The flasks were plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ±1 °C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 to 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 other: % v/v saturated solution
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 100 other: % v/v saturated solution
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
Analysis of the test preparations at 0 and 72 hours showed measured test concentrations of less than the limit of quantification (LOQ) of the analytical method employed were obtained which was determined to be 0.010 mg/L. This does not infer that no test item was in solution, just that any dissolved test item present was at a concentration of less than the LOQ.
Given this it was considered appropriate to calculate results based on the nominal test concentrations only.
Results with reference substance (positive control):
A positive control (Envigo study number RD71YQ) used potassium dichromate as the reference item at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L.
Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.
Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results:

ErC50 (0 to 72 hour) : 1.6 mg/L; 95% confidence limits 1.4 to 1.8 mg/L
EyC50 (0 to 72 hour) : 0.77 mg/L; 95% confidence limits 0.68 to 0.87 mg/L

No Observed Effect Concentration based on growth rate: 0.25 mg/L
No Observed Effect Concentration based on yield: 0.25 mg/L
Lowest Observed Effect Concentration based on growth rate: 0.50 mg/L
Lowest Observed Effect Concentration based on yield: 0.50 mg/L

The results from the positive control with potassium dichromate were within the normal ranges for this reference item.
Validity criteria fulfilled:
yes
Conclusions:
This study showed that there were no toxic effects at saturation.
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 that described in 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) No 761/2009.

Methods

Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication and high shear mixing. Given this, and at the request of the Sponsor, a slow stir saturated solution method of preparation was selected for testing.

Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to solutions of the test item at nominal concentrations of6.25,12.5,25,50and100% v/v saturated solution (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ±1 °C. The test item solutions were prepared by stirring an excess (50 mg/L) of test item in culture medium with the aid of magnetic stirring at a rate such that a dimple was formed at the media surface for 24 hours. After the stirring period any undissolved test item was removed by filtration (0.2 µm Sartorius Sartopore filter, first approximate 1 liter discarded in order to pre-condition the filter) to produce a100% v/v saturated solution of the test item. This saturated solution was then further diluted as necessary, to provide the remaining test groups.

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

Analysis of the test preparations at 0 and 72 hours showed measured test concentrations of less than the limit of quantification (LOQ) of the analytical method employed were obtained which was determined to be 0.010 mg/L. This does not infer that no test item was in solution, just that any dissolved test item present was at a concentration of less than the LOQ.

Given this it was considered appropriate to calculate results based on the nominal test concentrations only.

Exposure of Pseudokirchneriella subcapitata to the test item gave EC50values based on the nominal test concentrations of greater than100% v/v saturated solution. The No Observed Effect Concentration (NOEC) was determined to be100% v/v saturated solution.

This study showed that there were no toxic effects at saturation.

Description of key information

A study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. The method followed that described in 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) No 761/2009.

Analysis of the test preparations at 0 and 72 hours showed measured test concentrations of less than the limit of quantification (LOQ) of the analytical method employed were obtained which was determined to be 0.010 mg/L. This does not infer that no test item was in solution, just that any dissolved test item present was at a concentration of less than the LOQ.

Given this it was considered appropriate to calculate results based on the nominal test concentrations only.

Exposure of Pseudokirchneriella subcapitata to the test item gave EC50values based on the nominal test concentrations of greater than100% v/v saturated solution. The NOEC was determined to be100% v/v saturated solution.

This study showed that there were no toxic effects at saturation.

Key value for chemical safety assessment

Additional information