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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:
10 July 2017 - 03 October 2018
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)
Analytical monitoring:
yes
Details on sampling:
Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to Water Accommodated Fractions (WAFs) containing the test material over a range of nominal loading rates of 6.25, 12.5, 25, 50 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.

The samples were stored frozen prior to analysis. Given that concentrations of close to the limit of quantification (LOQ) were observed in the preliminary work, when samples were analyzed promptly following collection, it was considered unnecessary to conduct any storage stability analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Nominal amounts of test item (12.5, 25, 50, 100 and 200 mg) were each separately added to the surface of 2 liters of culture medium to give the 6.25, 12.5, 25, 50 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 95 hours and the mixtures allowed to stand for 1 hour.
An aliquot (500 mL) of each of the loading rate WAFs was separately inoculated with algal suspension (5.2 mL) to give the required test concentrations of 6.25, 12.5, 25, 50 and 100 mg/L loading rate WAF.
- Differential loading: following loading rates were employed in the definitive test: 6.25, 12.5, 25, 50 and 100 mg/L
- Controls: The control group was maintained under identical conditions but not exposed to the test item
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): none
- Test concentration separation factor: 2
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): None. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present.
- Other relevant information: /
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
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.

The master cultures were maintained in the laboratory under constant aeration and illumination at 21 ± 2°C.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
24 ± 1 ºC
pH:
The pH value of the control cultures was observed to increase from pH 7.4 at 0 hours to pH 8.5 at 72 hours.
Nominal and measured concentrations:
Nominal loading rates of 6.25, 12.5, 25, 50 and 100 mg/L
Analysis of the 100 mg/L loading rate WAF test preparations at 0 and 72 hours showed that measured concentrations of less than the Limit of Quantification (LOQ) of the analytical method, determined to be 0.012 mg/L.
Details on test conditions:
As in the range-finding tests, 250 mL glass conical flasks were used. Six flasks each containing 100 mL of test preparation were used for the control and three flasks each containing 100 mL were used for each treatment group.

TEST SYSTEM
- Test vessel: 250 mL glass conical flasks
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: The flasks were then plugged with polyurethane foam bungs and incubated (INFORS Multitron¿ Version 2 incubator) at 24 ± 1 ºC
- Aeration: none
- Initial cells density: Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 4.85 x 105 cells per mL.
- Control end cells density:
Nominal cell density of control at 0 hours : 5.00 x 103 cells per mL
Mean cell density of control at 72 hours : 9.65 x 105 cells per mL
The mean coefficient of variation for section by section specific growth rate for the control cultures was 7% and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35%.


GROWTH MEDIUM
- Standard medium used: yes
The culture medium was prepared using reverse osmosis purified deionized water* and the pH adjusted to 7.5 with 0.1N NaOH or HCl.


TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: no
- Intervals of water quality measurement: no renewals

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: none
- Photoperiod: continuous illumination
- Light intensity and quality: approximately 7000 lux provided by warm white lighting (380 – 730 nm)


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Samples were taken at 24, 45 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter. The shape and size of the algal cells was inspected microscopically and any abnormalities recorded.
- Other: The appearance of the test media was recorded daily.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Based on the results of the range-finding test the following loading rates were employed in the definitive test: 6.25, 12.5, 25, 50 and 100 mg/L.
- Justification for using less concentrations than requested by guideline: NA
- Range finding study
- Test concentrations: The range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to nominal loading rates of 0.010, 0.10, 1.0, 10 and 100 mg/L for a period of 72 hours. Two replicate flasks were prepared for each control and test concentration.
- Results used to determine the conditions for the definitive study: The results showed no effect on growth at 0.010, 0.10, 1.0 and 10 mg/L loading rate WAF. However, growth was observed to be reduced at 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
Remarks:
yield and biomass
Key result
Duration:
72 h
Dose descriptor:
LOELR
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
yield and biomass
Details on results:
Chemical Analysis of Test Loading Rates
Chemical 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, which was determined to be 0.012 mg/L, were obtained. This does not indicate that no test item was in solution, just that any the measured dissolved test item was present at a concentration of less than the LOQ.
Any dissolved test item may have been comprised of one or several components of the test item. Given that toxicity cannot be attributed to a single component or mixture of components but to the test item as a whole, the results were based on nominal loading rates only.
Measured concentrations were observed in the range-finding test. Given that concentrations of less than the LOQ were observed in the definitive test and also in the Acute Toxicity to Daphnia Magna test performed on this test item (Envigo Study Number CH46FV), it was considered that the measured concentrations in the range finding test were due to undissolved particles of test item in the media. It was also considered that the presence of these undissolved particles contributed to the toxicity observed in the range-finding test. During the definitive test, microscopic observations did not show any undissolved particles of test item in the media.

Growth Data
That the growth rate (r) and yield (y) of Pseudokirchneriella subcapitata (CCAP 278/4) were not affected by the presence of the test item over the 72-Hour exposure period.

Inhibition of Growth Rates
ErL10 (0 - 72 h): >100 mg/L loading rate WAF
ErL20 (0 - 72 h): >100 mg/L loading rate WAF
ErL50 (0 - 72 h): >100 mg/L loading rate WAF
There were no statistically significant differences (P>=0.05), between the control and any of the loading rate WAF test groups and the "No Observed Effect Loading Rate" (NOEL) based on growth rate was therefore 100 mg/L loading rate WAF.

Inhibition of Yield
EyL10 (0 - 72 h): >100 mg/L loading rate WAF
EyL20 (0 - 72 h): >100 mg/L loading rate WAF
EyL50 (0 - 72 h): >100 mg/L loading rate WAF
There were no statistically significant differences (P>=0.05), between the control and any of the loading rate WAF test groups and the "No Observed Effect Loading Rate" (NOEL) based on yield was therefore 100 mg/L loading rate WAF.
Results with reference substance (positive control):
Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results:

ErC50 (0 – 72 h): 1.4 mg/L; 95% confidence limits 1.2 – 1.5 mg/L
EyC50 (0 – 72 h): 0.60 mg/L; 95% confidence limits 0.52 – 0.69 mg/L

No Observed Effect Concentration (NOEC) based on growth rate: 0.25 mg/L
No Observed Effect Concentration (NOEC) based on yield: 0.25 mg/L
Lowest Observed Effect Concentration (LOEC) based on growth rate: 0.50 mg/L
Lowest Observed Effect Concentration (LOEC) 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.
Reported statistics and error estimates:
There were no statistically significant differences (P>=0.05), between the control and any of the loading rate WAF test groups and the "No Observed Effect Loading Rate" (NOEL) based on growth rate was therefore 100 mg/L loading rate WAF.

All statistical analyses were performed using the SAS computer software package (SAS, 1999 - 2001).
Validity criteria fulfilled:
yes
Remarks:
- mean coefficient of variation for section by section specific growth rates : 7% < 35%. - coefficient of variation for average specific growth rate (0 – 72 h) : 1% <7%. - the control cultures increased by a factor of 193 after 72 hours > 16.
Conclusions:
The effect of the test material on the growth of Pseudokirchneriella subcapitata was investigated and the 72-hour EL50 values based on growth rate, yield and biomass were determined to be greater than 100 mg/L loading rate WAF. The No Observed Effect Loading Rate was 100 mg/L loading rate WAF.
Executive summary:

A GLP study was performed to assess the effect of the test item (EC =953-378-2), 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" and Method C.3 of Commission Regulation (EC) 761/2009.


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).



Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to Water Accommodated Fractions (WAFs) containing the test item over a range of nominal loading rates of 6.25, 12.5, 25, 50 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 100 mg/L loading rate WAF test preparations at 0 and 72 hours showed that measured concentrations of less than the Limit of Quantification (LOQ) of the analytical method, determined to be 0.012 mg/L, were obtained,. This does not indicate that no test item was in solution, just that the measured dissolved test item was present at a concentration of less than the LOQ.


Any dissolved test material may have been comprised of one or several components of the test item. 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.


Exposure of Pseudokirchneriella subcapitata to the test material resulted in EL50values 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

An experimental study was carried out to determine the aquatic toxicity of the registered substance on freshwater Pseudokirchneriella subcapitata according to the TG OECD 201 and GLP. The effect on the growth of Pseudokirchneriella subcapitata was investigated and the 72-hour EL50 values based on growth rate, yield and biomass were determined to be greater than 100 mg/L loading rate WAF.  The No Observed Effect Loading Rate was 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

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