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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:
April 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
Section 2
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
2-[2,4-bis(tert-pentyl)phenoxy]-N-(3,5-dichloro-4-ethyl-2-hydroxyphenyl)butyramide
EC Number:
300-634-0
EC Name:
2-[2,4-bis(tert-pentyl)phenoxy]-N-(3,5-dichloro-4-ethyl-2-hydroxyphenyl)butyramide
Cas Number:
93951-12-3
Molecular formula:
C28H39Cl2NO3
IUPAC Name:
2-[2,4-bis(tert-pentyl)phenoxy]-N-(3,5-dichloro-4-ethyl-2-hydroxyphenyl)butyramide
Test material form:
solid
Details on test material:
Batch 44029
colour : White to off-white
retest date : 01/06/2019
Specific details on test material used for the study:
Storage conditions: Controlled room temperature (15-25oC, ≤70% relative humidity), protected from light and humidity (store in a tightly closed container)

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Duplicate samples (from three replicates) were taken (2 x ~2 mL) in glass tubes at the beginning and at the end of the experiment from the control and at the applied test concentration level.
After sampling, samples were frozen and kept approximately at -20°C at the Test Facility. One set of the samples was sent to the Test Site for analysis and one set was retained as a back-up at the Test Facility, if required for any confirmatory analyses (discarded after satisfactory results were obtained on the first set of).

Test solutions

Vehicle:
yes
Remarks:
Reconstituted algal growth medium
Details on test solutions:
Formulation
Because the test item is poorly soluble in water, test solutions were prepared using a saturated solution method according to the Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, OECD No. 23.
Saturated test item solution (nominal loading rate of 100.0 mg/L) was prepared by dispersing/dissolving the amount of test item into the test medium (OECD Medium) two days before the start of the experiment. This solution was shaken for about 24 hours at approximately 30°C and then equilibrated for about 24 hours at approximately 20°C. The non-dissolved test material was removed by filtration through a fine (0.22 μm) filter to give the appropriate 100% saturated solution.
As a Limit Test was carried out, further dilution of stock solution was not performed.

Test organisms

Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella 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.
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

Test type:
not specified
Water media type:
other: OECD medium, according to OECD 201
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 22.4 – 22.8 °C measured in the flask and between 21.9 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 vessel. The pH of the control medium was not increased by more than 1.5 units during the test. The range of the pH was 7.63 – 8.90 during the experiment.
Nominal and measured concentrations:
Analysis of test medium was performed at the start and at the end of the experiment, but the test item concentration could not be measured. The measured concentration was below the Limit of Quantification (LOQ = 0.02 μg/L) at the start and below the Limit of Detection (LOD = 0.01 μg/L) at the end of the experiment. The biological results are based on the nominal test item concentration.
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 7934 lux (equivalent to ~107 μE/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
Remarks:
biomass
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
Remarks:
growth rate
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
Remarks:
yield
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Key result
Duration:
72 h
Dose descriptor:
NOEC
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:
LOEC
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:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Details on results:
As a Limit test was performed the ErC50, EbC50 and EyC50 values of the test item were not calculated and determined from the raw data.
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.
For the determination of the LOEC and NOEC, the calculated mean biomass, growth rates and yield at the test concentrations were tested on significant differences to the control values by 2 Sample t-Test (α = 0.05) by TOXSTAT software.
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: 19/009-022AL) with the reference item Potassium dichromate is (Batch Number: A0345704): 22 – 25 January 2019.
The 72h ErC 50: 0.87 mg/L, (95 % confidence limits: 0.80 – 0.95 mg/L)
The 72h EbC 50: 0.62 mg/L, (95 % confidence limits: 0.57 – 0.68 mg/L)
The 72h EyC 50: 0.52 mg/L, (95 % confidence limits: 0.48 – 0.57 mg/L)
These values are within the range of laboratory ring test data (see ISO Guideline No. 8692).
Reported statistics and error estimates:
There were no observed morphological deviations during the experiment.

Any other information on results incl. tables

The cell density in the control cultures increased by the factor of 67.33 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 7.87 %.

The coefficient of variation of average specific growth rates during the whole test period (day 0-3) in the control cultures was 0.58 %.

All validity criteria were met; therefore, the study can be considered as valid.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Based on the results of this study, the test item 80ACQ had no toxic effect at saturation; the EC50 results and the LOEC are higher than the solubility level of the test item in the test medium.
Executive summary:

The effect of 80ACQ 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 at the solubility level of the test item in the test medium (100 mg/L nominal loading rate, 100 % saturated solution) and one control group.

Analysis of test medium was performed at the start and at the end of the experiment, but the test item concentration could not be measured. The measured concentration was below the Limit of Quantification (LOQ = 0.02 μg/L) at the start and below the Limit of Detection (LOD = 0.01 μg/L) at the end of the experiment. The biological results are based on the nominal test item concentration.

The test design included six replicates at the 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.

Under the conditions of this algal growth inhibition test the calculated endpoints for the effect of 80ACQ were the following:

The 72h EbC50 value (biomass): > 100.0 mg/L nominal loading rate

The 72h ErC50 value (growth rate): > 100.0 mg/L nominal loading rate

The 72h EyC50 value (yield): > 100.0 mg/L nominal loading rate

The 72h No-Observed Effect Concentration (NOEC): 100.0 mg/L nominal loading rate

The 72h Lowest Observed Effect Concentration (LOEC): > 100.0 mg/L nominal loading rate