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Diss Factsheets

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:
26 Apr - 29 Apr 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 guideline
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

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Sampling method: Duplicate samples from the freshly prepared test media (without algae) of all test concentrations and 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 and 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.
- Sample storage conditions before analysis: All samples remained undiluted until sample preparation. All samples were stored in a freezer (≤ - 20 °C), protected from light until analysis was performed.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION: The test medium of the highest test concentration of nominal 100 mg test item/L was prepared by dissolving 50.1 mg test item into 510 mL test water by intense stirring for 20 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).
Appearance of the Test Item in Test Medium: There were no remarkable observations.
Test concentration: 100, 32, 10, 3.2 and 1.0 mg test item/L and a control
In the control, test water was used without addition of the test item.

Test organisms

Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Name/Strain: Pseudokirchneriella subcapitata (KORSHIKOV), Strain No. 61.81 SAG, formerly known as Selenastrum capricornutum, and recently renamed as Raphidocelis subcapitata (KORSHIKOV)
- Source (laboratory, culture collection): The algae were originally supplied by the „Sammlung von Algenkulturen, Albrecht-von-Haller-Institut für Pflanzen-wissenschaften, Universität Göttingen", 37073 Göttingen, Germany.
- Age of inoculum (at test initiation): 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.
- Method of cultivation: The algae were cultivated in the laboratories of ibacon under standardised conditions according to the test guidelines.

ACCLIMATION
- Acclimation period: no

Study design

Test type:
static
Water media type:
freshwater
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: 21.9 to 23.4 °C
pH:
The pH was measured in all test item concentrations and the control at the start and the end of the test.
pH value in the control at test start: 8.0,
pH value in the control at test end: 10.4
pH values in the test item treatments at test start: 7.6 to 8.1,
pH values in the test item treatments at test end: 7.9 to 10.3
Nominal and measured concentrations:
Nominal concetration: 100, 32, 10, 3.2 and 1.0 mg test item/L (spacing factor 3.16), corresponding to following geometric mean measured concentrations of the test item:
100, 33.1, 11.6, 2.76 and 0.881 mg test item/L, and a control.
Details on test conditions:
Test Vessels: Erlenmeyer flasks of 50 mL volume with nominal 50 mL of test medium loosely covered 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 incubated in a water bath, 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" for the spectrophotometric measurements. The additional replicates were incubated under the same conditions as described above. The blank values were subtracted from the absorption measured in the samples containing algae in order to eliminate absorption caused by the test item.

OTHER TEST CONDITIONS
Light Regime: 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: 5614 lux (range: 5427 to 5890 lux)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Determination of the Cell Density: The cell density on each observation time was determined by spectrophotometric measurement. Therefore, defined volumes of the algal suspensions from all replicates and from the blanks were sampled after 24, 48 and 72 hours of exposure, and were not replaced. The algal cell densities were calculated by subtracting the absorption of the blanks, from each of the measured absorption of the test media (with algae).
Based on the counted cell densities and the absorption from an algal suspension and its dilutions, a linear regression was performed for the calculation of the cell densities of the replicates during the test.

No pre-experiments were performed.
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:
EC50
Effect conc.:
70.9 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.:
21.2 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
2.76 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
32.1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
11.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
18.4 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
yield
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
7.15 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
yield
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
4.37 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
yield
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
2.76 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
yield
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
11.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
yield
Details on results:
Growth Inhibition: The 72-hour EyC50 was calculated to be 18.4 mg test item/L and the ErC50 70.9 mg test item/L. The 72-hour EyC10 was calculated to be 4.37 mg test item/L and the ErC10 21.2 mg test item/L. The 72-hour NOEyC was determined to be 2.76 mg test item/L and the associated 72-hour LOEyC of 11.6 mg test item/L. The 72-hour NOErC was determined to be 2.76 mg test item/L and the associated 72-hour LOErC is 11.6 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 100 mg test item/L and the algal cells in the control. Thus, the shape of the algal cells was not obviously affected up to this test concentration, the highest concentration tested .
Results with reference substance (positive control):
Results of the Most Recent Test with Pseudokirchneriella subcapitata performed with the Reference Item Potassium Dichromate in February 2021

72-hour EC50 (Yield): 0.393 mg/L
72-hour NOEC (Yield): 0.2 mg/L
72-hour LOEC (Yield): 0.63 mg/L
72-hour EC50 (Growth rate): 0.835 mg/L
72-hour NOEC (Growth rate): 0.2 mg/L
72-hour LOEC (Growth rate): 0.63 mg/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 were tested 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.

Any other information on results incl. tables

Validity Criteria





























Criterionvalue requiredvalue obtainedfullfilled
Cell Density Increase in control Culturesat least by factor of 16 within 72 h370.1 fold within 72 hyes
Coefficient of Variation of Sectional (Daily) Growth Rates in Control Cultures< 35%18.7%yes
Coefficient of Variation of Average Growth between Control Replicates< 7%0.8 %yes

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
see any other information on results incl. tables
Conclusions:
The influence of DFPP malate on the growth of the freshwater green algae Pseudokirchneriella subcapitata was assessed in a static concentration-response test. The 72-hour EyC50 was calculated to be 18.4 mg test item/L, and the 72-hour ErC50 value was calculated to be 70.9 mg test item/L. The 72-hour NOEyC was determined to be 2.76 mg test item/L and the associated 72-hour LOEyC was 11.6 mg test item/L. The 72-hour NOErC was determined to be 2.76 mg test item/L and the associated 72-hour LOErC was 11.6 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 not all test item concentrations remained within ± 20 % of the nominal concentrations during the test.
Executive summary:

Test Species: Pseudokirchneriella subcapitata, Strain No. 61.81 SAG formerly 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 media were 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 spectrophotometric measurement.


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), corresponding to following geometric mean measured concentrations of the test item: 100, 33.1, 11.6, 2.76 and 0.881 mg test item/L, and a control.


Test Conditions: Water temperature: 21.9 to 23.4 °C; pH value in the control at test start: 8.0, pH value in the control at test end: 10.4; pH values in the test item treatments at test start: 7.6 to 8.1, pH values in the test item treatments at test end: 7.9 to 10.3; continuous illumination; mean light intensity: 5614 lux (5427 to 5890 lux).


Biological results









































Parameter



Yield



Growth Rate



 



mg test item/L



mg test item/L



72-hour EC50



18.4



70.9



72-hour EC20



7.15



32.1



72-hour EC10



4.37



21.2



72-hour NOEC



2.76



2.76



72-hour LOEC



11.6



11.6



 


Analytical results: The quantification of the test item DFPP malate in the test samples was performed using liquid chromatography with UV detection. At the start of the test 102 % of the nominal test concentrations were found (average of all test concentrations). After 72 hours test duration, 96 % of the nominal value was determined (average of all test concentrations). During the test the algae were exposed to a mean of 99 % of nominal.