[No public or meaningful name is available]

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

effects on growth of green algae

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January 11, 2021 to March 23, 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

2006

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Appearance/Physical state: Solid (deliquescent)
Batch: 209-653 (dried material from [Tradename] batch number 0001569403)
Purity: 94.5%
Expiry date: 03 December 2021
Storage conditions: Room temperature under nitrogen in the dark
Formulated concentrations were adjusted to allow for the stated impurity content (5.5%) of the test item.

Analytical monitoring:

yes

Details on sampling:

Samples were taken from the control and each test group from the bulk test preparation at 0 hours and from the pooled replicates at 72 hours for quantitative analysis. An additional sample of each test concentration was incubated alongside the test from which an aliquot was removed for analysis at 24 and 48 hours.

Vehicle:

no

Details on test solutions:

A nominal amount of test item (100 mg) was dissolved in culture medium and the volume adjusted to 1 L to give a 100 mg/L stock solution from which a series of dilutions was made to give further stock solutions of 32, 10, 3.2, 1.0 and 0.32 mg/L.

Test organisms (species):

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

Details on test organisms:

The test was carried out using Raphidocelis subcapitata strain CCAP 278/4. Liquid cultures of Raphidocelis subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland.

Approximately 3 to 4 days before the start of the test, inoculum cultures of algae was set up at an initial cell density of approximately 103 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 105 to 106 cells/mL.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

MgCl2.6H2O 12.16 mg/L; CaCl2.2H2O 4.41 mg/L; MgSO4.7H2O 14.6 mg/L

Test temperature:

24 ±1 °C

pH:

7.5 ± 0.1

Nominal and measured concentrations:

Nominal: 0.32, 1.0, 3.2, 10, 32 and 100 mg/L; Measured: 104-106% of nominal at 0 h, which declined at 24, 48 and 72 h. Measured (geometric mean) test concentration: 0.13, 0.42, 1.2, 3.3, 11 and 57 mg/L

Details on test conditions:

SYSTEM
- Glass conical flasks;
- three replicate flasks per concentration;
- Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell
density of 9.39 x 105 cells per mL;
- Inoculation of 450 mL of test medium with 2.4 mL of this algal suspension gave an initial nominal cell density of 5.00 x 103 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 ±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.


GROWTH MEDIUM
The culture medium was prepared using reverse osmosis purified deionized water* and the pH
adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl.
NaNO3 25.5 mg/L
MgCl2.6H2O 12.16 mg/L
CaCl2.2H2O 4.41 mg/L
MgSO4.7H2O 14.6 mg/L
K2HPO4 1.044 mg/L
NaHCO3 15.0 mg/L
H3BO3 0.186 mg/L
MnCl2.4H2O 0.415 mg/L
ZnCl2 0.00327 mg/L
FeCl3.6H2O 0.160 mg/L
CoCl2.6H2O 0.00143 mg/L
Na2MoO4.2H2O 0.00726 mg/L
CuCl2.2H2O 0.000012 mg/L
Na2EDTA.2H2O 0.30 mg/L

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Samples were taken at 24, 48 and 72 hours and the cell densities determined using a Coulter®
Multisizer Particle Counter. Three determinations were made for each sample. The nominal
inoculated cell concentration (5.00 x 103 cells/mL) was taken as the starting cell density.
To determine the potential effect of the test item on the appearance of algal cells, a sample
was removed from each test and control culture (replicates pooled) at the end of the test. The
shape and size of the algal cells was inspected microscopically and any abnormalities
recorded.


TEST CONCENTRATIONS
0.32, 1.0, 3.2, 10, 32 and 100 mg/L
range finding test: 0.10, 1.0, 10 and 100 mg/L for a period of 72 hours

Reference substance (positive control):

yes

Remarks:

A positive control (Covance study number 8461000) used potassium dichromate as the reference item at concentrations of 0.125, 0.25, 0.50, 1.0 and 2.0 mg/L. The positive control was conducted between 21 December 2020 and 24 December 2020.

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

42 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.9

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Remarks on result:

other: 95% confidence limit 1.6 - 2.1 mg/L

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

8.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence limit 7.2 - 11 mg/L

Analysis of the test preparations at 0 hours showed measured test concentrations range from 104% to 106% of nominal. A decline in measured test concentrations was observed after each 24-Hour period in the range of 34% to 76% of nominal at 24 hours, 20% to 53% of nominal at 48 hours and 10% to 24% of nominal at 72 hours. Given this decline in measured test concentrations it was considered justifiable to base the results on the geometric mean measured test concentrations in order to give a "worst case" analysis of the data. The geometric mean measured test concentrations were determined to be 0.13, 0.42, 1.2, 3.3, 11 and 57 mg/L.

Validity criteria fulfilled:

yes

Conclusions:

For the test substance, under the test conditions, EC50 values of 8.7 (7.2 - 11) mg/L and 1.9 (1.6 - 2.1) mg/L were observed for growth rate and yield, respectively.

For the test substance, under the test conditions, NOEC values of 1.2 mg/L and 0.42 mg/L were observed for growth rate and yield, respectively.

Executive summary:

A study was performed to assess the effect of the test item on the growth of the green alga Raphidocelis subcapitata (formerly known as 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" and Method C.3 of Commission Regulation (EC) No 761/2009.

 

Following a preliminary range-finding test and initial experiment, Raphidocelis subcapitata was exposed to aqueous solutions of the test item at concentrations of 0.32, 1.0, 3.2, 10, 32 and 100 mg/L (three replicate flasks per concentration) 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.

 

Analysis of the test preparations at 0 hours showed measured test concentrations range from 104% to 106% of nominal. A decline in measured test concentrations was observed after each 24-Hour period in the range of 34% to 76% of nominal at 24 hours, 20% to 53% of nominal at 48 hours and 10% to 24% of nominal at 72 hours. Given this decline in measured test concentrations it was considered justifiable to base the results on the geometric mean measured test concentrations in order to give a "worst case" analysis of the data. The geometric mean measured test concentrations were determined to be 0.13, 0.42, 1.2, 3.3, 11 and 57 mg/L.

 

For the test substance, under the test conditions, EC50 values of 8.7 (7.2 - 11) mg/L and 1.9 (1.6 - 2.1) mg/L were observed for growth rate and yield, respectively.

For the test substance, under the test conditions, NOEC values of 1.2 mg/L and 0.42 mg/L were observed for growth rate and yield, respectively.

Description of key information

A study was performed to assess the effect of the test item on the growth of the green alga Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata). The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2006) No 201.

Raphidocelis subcapitata was exposed to aqueous solutions of the test item at concentrations of 0.32, 1.0, 3.2, 10, 32 and 100 mg/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ±1 °C.
For the test substance, under the test conditions, EC50 values of 8.7 (7.2 - 11) mg/L and 1.9 (1.6 - 2.1) mg/L were observed for growth rate and yield, respectively.

For the test substance, under the test conditions, NOEC values of 1.2 mg/L and 0.42 mg/L were observed for growth rate and yield, respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:

8.7 mg/L

EC10 or NOEC for freshwater algae:

1.2 mg/L