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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:
December 04, 2020 to March 23, 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021
Report date:
2021

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
other: OECD Series on Testing and Assessment No. 23: "Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures"
Version / remarks:
December 15, 2000
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
adopted March 2006, corrected July 28, 2011
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
4-(trifluoromethoxy)aniline
EC Number:
207-317-5
EC Name:
4-(trifluoromethoxy)aniline
Cas Number:
461-82-5
Molecular formula:
C7H6F3NO
IUPAC Name:
4-(trifluoromethoxy)aniline
Test material form:
liquid

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Sampling:
Duplicate samples from the freshly prepared test media (without algae) of all test concentrations, from the control were taken at the start of the test. For the determination of the stability of the test item under the test conditions and of the maintenance of the test item concentrations during the test period, duplicate samples from the test media of all test concentrations, from the control (containing algae) were taken at the end of the test (after the 72 hours test period) by pouring together the contents of the test beakers of each treatment.The samples were diluted by a factor of two with acetonitrile. Additional samples of the control and of the dilution solvent were aken at each sampling without any sample treatment.
Storage: All samples were stored in a freezer (≤ - 20 °C), protected from light until analysis was performed. Afterwards the samples were again stored deep frozen up to the date of the final report.

Test solutions

Vehicle:
no
Details on test solutions:
The test medium of the highest test concentration of nominal 100 mg test item/L was prepared by dissolving 54.2 mg test item into 542 mL test water by intense stirring for 15 minutes. Adequate volumes of this test medium were diluted with test water to prepare the test media of the other desired test concentrations.The test media were prepared just before introduction of the algae (= start of the test).

Test organisms

Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
Species: Pseudokirchneriella subcapitata (KORSHIKOV), Strain No. 61.81 SAG, formerly known as Selenastrum capricornutum, and recently renamed as Raphidocelis subcapitata (KORSHIKOV)
Origin: The algae were originally supplied by the „Sammlung von Algenkulturen, Albrecht-von-Haller-Institut für Pflanzenwissenschaften, Universität Göttingen", 37073 Göttingen, Germany.
Breeding Conditions: The algae were cultivated in the laboratories of ibacon under standardised conditions according to the test guidelines.

Study design

Test type:
static
Water media type:
other: re-constituted water; OECD Medium
Limit test:
no
Total exposure duration:
72 h

Test conditions

Hardness:
Calculated water hardness of the test water: 0.24 mmol/L (= 24 mg/L) as CaCO3.
Test temperature:
Water temperature was: 21.4 to 22.0 °C
pH:
The pH was measured in all test item concentrations and the control at the start and the end of the test.
The pH in the control was: 8.0 at test start and 9.0 at test end
The pH at different test item concentrations was:7.8 at test start and 7.7 to 8.8 at test end
Nominal and measured concentrations:
Test Concentrations: 100, 32, 10, 3.2 and 1.0 mg test item/L (spacing factor 3.16) and a control, corresponding to following geometric mean measured concentrations of the test item: 69.5, 20.5, 6.00, 1.96 and 0.675 mg test item/L, and a control.
Details on test conditions:
Test Units: Erlenmeyer flasks of 50 mL volume with approximately 50 mL of test mediumcovered with watch glasses

Introduction of Algae: The test was started (0 hours) by inoculation of a biomass of nominal 5000 algal cells per mL test medium. These cells were taken from an exponentially growing pre-culture, which was set up 3 days prior to the test start under the same conditions as in the test.
Replicates: The test was performed with three replicates per test concentration and six replicates in the control.
Test Procedure: The test units were continuously stirred by magnetic stirrers. The flasks were covered with watch glasses and incubated in a water bath. The flasks were placed in a random order and were repositioned each day to minimize differences in test conditions.
Blanks: Additionally, one replicate of each test concentration and of the control was prepared without algae to provide a "blank".
Exposure Time: 72 hours
Determination of the Cell Density: Cell density was determined by cell counting via microscope. Therefore, defined volumes of the algal suspensions from all replicates were sampled after 24, 48 and 72 hours of exposure, and were not replaced

GROWTH MEDIUM
Standard medium used: yes (OECD medium)
Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: The pH of the untreated test water was checked before test start and adjusted from 7.9 to 8.0 using 1 M NaOH.
- Photoperiod:
Continuous illumination
Light Intensity: The light intensity was measured once during the test at 6 positions distributed over the experimental area at the surface of the test media.
Mean light intensity: 4978 lux (range: 4440 to 5370 lux)

Pre-experiments were performed to determine a suitable concentration range and to establish suitable methods to prepare the test solutions.
Reference substance (positive control):
yes
Remarks:
For the evaluation of the quality of the algae and the experimental conditions, the reference item potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
1.96 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.675 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
1.38 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
1.84 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.:
3.17 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
1.96 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.675 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.72 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Key result
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
0.92 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
1.47 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Details on results:
Growth Inhibition: The 72-hour EyC50 was calculated to be 1.47 mg test item/L and the ErC50 3.17 mg test item/L. The 72-hour EyC10 was calculated to be 0.720 mg test item/L and the ErC10 1.38 mg test item/L. The 72-hour NOEyC was determined to be 0.675 mg test item/L and the associated 72-hour LOEyC was 1.96 mg test item/L. The 72-hour NOErC was determined to be 0.675 mg test item/L and the associated 72-hour LOErC was 1.96 mg test item/L.
Signs of Phytotoxicity evaluated by Microscopic Observations: The microscopic examination of the shape of the algal cells after 72 hours of test duration did not show any difference between the algae that had been growing up to a nominal test concentration of nominal 10 mg test item/L and the algal cells in the control. At the nominal concentrations of 32 and 100 mg test item/L cells showed a grainy structure.
Results with reference substance (positive control):
Results of the Most Recent Test with Pseudokirchneriella subcapitata performed with the Reference Item Potassium Dichromate

72 h EC50 (yield): 0.402 mg test item/L
72 h EC50 (growth rate): 0.878 mg test item/L
72 h EC50 (biomass): 0.449 mg test item/L

72 h NOEC (yield): 0.2 mg test item/L
72 h NOEC (growth rate): 0.2 mg test item/L
72 h NOEC (biomass): 0.2 mg test item/L

72 h LOEC (yield): 0.63 mg test item/L
72 h LOEC (growth rate): 0.63 mg test item/L
72 h LOEC (biomass): 0.63 mg test item/L
Reported statistics and error estimates:
Based on the calculated cell densities, the 72-hour ErC50 and the 72-hour EyC50 (see Definitions), the corresponding EC20 and EC10 values and where possible their 95 %-confidence limits were calculated by Probit analysis.For the determination of the 72-hour LOEC and the 72-hour NOEC, the calculated growth rates and yields at each test concentration weretested for significant differences compared to the control values by Williams t-test. The software used to perform the statistical analysis was ToxRat Professional, Version 3.3.0, ToxRat Solutions GmbH

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
Cell Density Increase in Control Cultures: 204.9-fold increase within 72 h Coefficient of Variation of Sectional (Daily) Growth Rates in Control Cultures: 9.8 % Coefficient of Variation of Average Growth between Control Replicates: 3.1 %
Conclusions:
The influence of the test item on the growth of the freshwater green algae Pseudokirchneriella subcapitatawas assessed in a static concentration-response test. The 72-hour EyC50 was calculated to be 1.47 mg test item/L and the 72-hour ErC50 value was calculated to be 3.17 mg test item/L. The 72-hour NOEyC was determined to be 0.675 mg test item/L and the associated 72-hour LOEyC was 1.96 mg test item/L. The 72-hour NOErC was determined to be 0.675 mg test item/L and the associated 72-hour LOErC was 1.96 mg test item/L. The initial concentrations and the maintenance of the exposure concentrations during the test were verified in the analytical part. All reported results refer to geometric mean concentrations, since the test item concentrations were not within ± 20 % of the nominal ormeasured initial concentrations during the test.
Executive summary:

Test Purpose: The purpose of this test was to determine the inhibitory effect of the test item on the growth of the freshwater green algae Pseudokirchneriella subcapitata. For this purpose, exponentially growing cultures of this unicellular green algal species were exposed to various concentrations of the test item under defined conditions. The inhibition of growth in relation to control cultures was determined over a test period of 72 hours, and thus over several algal generations.The test method of application and the test system are recommended by the test guidelines and Pseudokirchneriella subcapitatais one of the recommended test species. The purpose of the analytical part of this study was to verify the concentrations of the test item in the test medium.


Test Species: Pseudokirchneriella subcapitata, Strain No. 61.81 SAGformerly known as Selenastrum capricornutum, and recently renamed as Raphidocelis subcapitata (KORSHIKOV).


Test Design: This study encompassed 6 treatment groups (5 dose rates of the test item and a control) with three replicates per test concentration and six replicates for the control. At test start 50 mL of the test mediawere inoculated with nominal 5000 algal cells per mL test medium and defined volumes of the algal suspensions were sampled after 24, 48 and 72 hours for determination of cell densities by cell counting via microscope.


Endpoints:Yield and growth rate of the algae


Test Concentrations:100, 32, 10, 3.2 and 1.0 mg test item/L (spacing factor 3.16)and a control, corresponding to following geometric mean measured concentrations of the test item: 69.5, 20.5, 6.00, 1.96 and 0.675 mg test item/L, and a control.


Test Conditions: Water temperature: 21.4 to 22.0 °C; pH values in the control at test start: 8.0, pH values at test end: 9.0, pH values in the test item treatments at test start: 7.8, pH values in the test item treatments at test end: 7.7 to 8.8; continuous illumination; mean light intensity: 4978 lux (4440 to 5370 lux).


Results:


72 h EC50 (yield): 1.47 mg test item/L
72 h EC50 (growth rate): 3.17 mg test item/L


72 h NOEC (yield): 0.675 mg test item/L
72 h NOEC (growth rate): 0.675 mg test item/L


72 h LOEC (yield): 1.96 mg test item/L
72 h LOEC (growth rate): 1.96 mg test item/L



The quantification of the test item in the test samples was performed using liquid chromatography with UV detection.At the start of the test,recoveries of the nominal test concentration varied between 87 and 96 % (all test concentrations considered). After 72hours test duration, the recoveries of the nominal values varied between 40 and 53 % (all test concentrations considered). The test item concentrations were dosed correctly and werefound to be not stable during the test period.


Conclusion: The influence of the tst item on the growth of the freshwater green algae Pseudokirchneriella subcapitatawas assessed in a static concentration-response test. The 72-hour EyC50was calculated to be 1.47 mg test item/L and the 72-hour ErC50value was calculated to be 3.17 mg test item/L. The 72-hour NOEyC was determined to be 0.675 mg test item/L and the associated 72-hour LOEyC was 1.96 mg test item/L. The 72-hour NOErC was determined to be 0.675 mg test item/L and the associated 72-hour LOErC was 1.96 mg test item/L. The initial concentrations and the maintenance of the exposure concentrations during the test were verified in the analytical part. All reported results refer to geometric mean concentrations, since the test item concentrations were not within ± 20 % of the nominal or measured initial concentrations during the test