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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
Study period:
August-September 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Qualifier:
according to
Guideline:
other: EPA Ecological Effects Test Guidelines, OCSPP 850.5400
Version / remarks:
Algal Toxicity, Tiers I and II, EPA 712-C-96-164, April 1996.
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Batch 44034
Description: Cream to light grey powder
Expiry date: 30 November 2019
Storage conditions: Controlled room temperature (15-25 ºC, below 70 RH%).

Sampling and analysis

Analytical monitoring:
yes

Test solutions

Vehicle:
yes
Details on test solutions:
Formulation
A stock solution with a nominal concentration of 100 mg/L was prepared with direct addition of the test item, mixed into the test medium (OECD Medium) using ultrasonic bath for approximately one minute. As a Limit Test was carried out, further dilution of stock solution was not be performed.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Strain number: 61.81 SAG (identical strains: CCAP 278/4; UTEX 1648; ATCC 22662)
Source: The algae were supplied by the SAG: Collection of Algal Cultures, Inst. Plant Physiology, and University of Göttingen, GERMANY. Cultured under standardised conditions (see OECD 201) in the Ecotoxicological Laboratory of Citoxlab Hungary Ltd.
Justification of species: The species of Pseudokirchneriella subcapitata used, being a fast-growing species, is convenient for culturing and testing and is a recommended species by relevant guidelines.
Breeding conditions: Stock cultures are small algal colonies that are inoculated onto agar regularly. These are transferred to fresh agar medium at least once every two months and are maintained under standardised conditions according to the test guidelines.
The pre-culture is intended to give a quantity of algae suitable for the inoculation of test cultures. The pre-culture was prepared with the OECD algal growth medium, incubated under the same conditions as the test and used when still growing exponentially, after an incubation period of three days. When the algal cultures contain deformed or abnormal cells, they were discarded.

Study design

Water media type:
other: Reconstituted algal growth medium (OECD medium, according to OECD 201) was used as dilution water for both the range finding and definitive tests.
Limit test:
yes
Total exposure duration:
72 h

Test conditions

Test temperature:
Culture temperature was checked at the beginning of the experiment and each day thereafter in a flask filled with water, in the climatic chamber. In addition, water temperature was continuously measured (with a min/max thermometer) within the climate chamber. The temperature was in the range of 22.7 – 22.8 °C measured in the flask and between 22.2 and 23.2 °C measured within the climate chamber.
pH:
The pH was checked at the beginning and at the end of the test in each test vessels. The pH of the control medium was not increased by more than 1.5 units during the test. The range of the pH was 7.22 – 8.95 during the experiment.
Nominal and measured concentrations:
Nominal concentrations
[mg/L] 0.1 1 10 100
Details on test conditions:
Light Intensity

The algal culture flasks were continuously illuminated. The light intensity at the position occupied by algal culture flasks during the test was about 7594 lux (equivalent to ~103 E/m2/s), which was ensured with fluorescent lamps (with a spectral range of 400-700 nm). The differences in light intensity between the test vessels did not exceed  15 % and therefore provided equal conditions for each test vessel.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
72 h
Dose descriptor:
EC50
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:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Details on results:
There were no any observed morphological deviations during the experiment.
Results with reference substance (positive control):
For the evaluation of the quality of the algae and validation of the experimental conditions, Potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.
The date of the last study (Study Code: 18/178-022AL) with the reference item Potassium dichromate is (Batch Number: A0345704): 18 – 21 June 2018.
The 72h ErC 50: 0.99 mg/L, (95 % confidence limits: 0.91 – 1.07 mg/L)
The 72h EbC 50: 0.69 mg/L, (95 % confidence limits: 0.64 – 0.75 mg/L)
The 72h EyC 50: 0.60 mg/L, (95 % confidence limits: 0.55 – 0.65 mg/L)

These values are within the range of laboratory ring test data (see ISO Guideline No. 8692).
Reported statistics and error estimates:
VALIDITY
The cell density in the control cultures increased by the factor of 70.33 within three days.
The mean coefficient of variation for section-by-section specific growth rates (days 0-1; 1-2; 2-3) in the control cultures was 12.13 %.
The coefficient of variation of average specific growth rates during the whole test period (day 0-3) in the control cultures was 0.86 %.
All validity criteria were met, therefore the study can be considered as valid.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The effect of Sodium [(6-hydroxy-4-methyl-2-oxo-2H-1-benzopyran-7-yl)oxy]acetate was assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum), over an exposure period of 72 hours.
Under the conditions of this algal growth inhibition test the calculated endpoints for the effect of Sodium [(6-hydroxy-4-methyl-2-oxo-2H-1-benzopyran-7-yl)oxy]acetate were the following:
The 72h EbC50 value (biomass): > 100 mg/L (nominal)
The 72h ErC50 value (growth rate): > 100 mg/L (nominal)
The 72h EyC50 value (yield): > 100 mg/L (nominal)

The 72h No-Observed Effect Concentration (NOEC): 100 mg/L (nominal)
The 72h Lowest Observed Effect Concentration (LOEC): > 100 mg/L (nominal)
Executive summary:

The effect of Sodium [(6-hydroxy-4-methyl-2-oxo-2H-1-benzopyran-7-yl)oxy]acetate test item was assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata, over an exposure period of 72 hours.

Because a toxic response was not observed during the preliminary concentration range-finding test, a Limit Test was carried out using only one test concentration of 100 mg/L (nominal) and one control group.

The test concentration was analytically determined at the start and at the end of the experiment. Measured concentration was 97.6 mg/L at the start and 95.7 mg/L at the end of the experiment.

As the analytically measured concentration deviated not more than 20 per cent from the nominal, the biological results are based on the nominal test item concentration.

The test design included six replicates at test concentration and six replicates for the untreated control.

Statistical comparisons of biomass, average specific growth rates and yield in control and in treated groups were carried out using 2 Sample t-Test (α = 0.05) by TOXSTAT software.

The ErC50, EbC50 and EyC50 values of the test item were determined directly from the raw data.