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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Link to relevant study record(s)

Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2021-02-11 - 2021-09-09
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:
OECD Guideline for Testing of Chemicals, Freshwater Alga and Cyanobacteria, Growth Inhibition Test, Test Guideline 201, 28 July 2011
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Remarks:
The test substance contains fluoride (concentration: 47.4%) and titanium (concentration: 22.4%). These two components were used for the analytical confirmation of the exposure concentrations.
Details on sampling:
- Concentrations: all test concentrations and control
- Sampling method:
Sampling Intervals and Solution Composition: 0-Hour samples were taken from exposure solutions in volumetric flasks prior to division into replicate vessels; after 24- and 72-Hour composite samples of all replicates within each treatment and control were taken, as well as an additional replicate for solution without algae. One sample was taken from each solution at each interval.
Additional samples of the test solutions were also collected at each sampling interval and stored refrigerated (one set) and frozen (one set) as archive samples
Sampling Location: Approximate midpoint from the surface, bottom, and sides of the vessel
Sampling Device: Pipette
Quality Control (QC) Samples: Six samples per sampling interval (three for titanium analysis and three for fluoride analysis), prepared in dilution water at nominal concentrations approximating
the test concentration range.
An additional four sets of QC samples (two sets for titanium analysis and two sets for fluoride analysis) were prepared for frozen (one set of each active) and refrigerated (one set of each active) archives and stored with exposure solutions.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
Primary Stock Solution Concentration: 32 mg/L
Stock Solution Vessel and Volume: 2-L volumetric Flask
Amount of Test Substance Added: 0.0646 g (0.640 g as active ingredient)
Diluent: AAP medium
The test substance was brought to full volume with AAP medium, and after shaking and inversion of the volumetric flask, the primary stock solution was observed to be clear and colorless with a small amount of visible undissolved test substance at the bottom of the vessel.
The primary stock solution was then sonicated for approximately 5 minutes and was observed to
be clear and colorless, with no visible undissolved test substance following preparation. The solution was mixed vigorously with a magnetic stir plate and Teflon-coated stir bar for
approximately 5 minutes, and the solution remained clear and colorless, with no visible
undissolved test substance following preparation.
The final stock solution was used to prepare the test media of the other desired test concentrations. The stock solution was spiked directly into full volume of dilution medium. After mixing with a magnetic stir plate and Teflon-coated stir bar for approximately 5 minutes, all exposure solutions were observed to be clear and colorless with no visible undissolved test substance.
- Controls: AAP medium only maintained under the same conditions as the treatment level solutions.
- Test concentration separation factor: 3.2
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): No, the final stock solution was clear and colorless, with no visible undissolved test substance following preparation.
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Raphidocelis subcapitata, formerly Pseudokirchneriella subcapitata
- Strain: 1648
- Source (laboratory, culture collection): UTEX The Culture Collection of Algae at the University of Texas, Austin, Texas
- Age of inoculum (at test initiation): Three days since previous transfer
- Method of cultivation: Algal stock cultures were maintained in stock culture at the test facility

ACCLIMATION
- Acclimation period: Three days
- Culturing media and conditions (same as test or not):
Growth medium: AAP medium, prepared with sterile, deionised source water
pH: 7.5 ± 0.1, initial pH adjusted with dilute hydrochloric acid or sodium hydroxide prior to use if necessary
Vessels: 250-mL glass Erlenmeyer flasks covered with stainless steel caps which permitted gas exchange (volume = 100 mL per vessel)
Photoperiod: None, continuous illumination (65 to 72 μE/m2/S)
Temperature: 23 to 24 °C, controlled using an environmental chamber
Aggitation: Continuous at a rate of 100 ± 10 rpm on an orbital shaker
- Any deformed or abnormal cells observed: not specified
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
23 to 24°C
pH:
0 Hour: 7.4 for control and concentrations of nominal 0.11, 0.34 and 1.0 mg/L, 6.7 at 3.3 mg/L, 5.7 at 10 mg/L and 4.0 at 32 mg/L;
72 Hour: 8.1 for control and the concentration of 0.11 mg/L, 8.0 at 0.34 mg/L, 7.5 at 1.0 mg/L, 7.3 at 3.3 mg/L, 6.2 at 10 mg/L and 3.7 at 32 mg/L
Conductivity:
0 Hour: 93 to 160 µS/cm
Nominal and measured concentrations:
Nominal Concentrations: 0.11, 0.34, 1.0, 3.3, 10, and 32 mg/L
Nominal Concentrations as Fluoride: 0.052, 0.16, 0.47, 1.6, 4.7 and 15 mg F/L
Nominal Concentrations as Titanium: 0.025, 0.076, 0.22, 0.74, 2.2, and 7.2 mg Ti/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 250-mL Erlenmeyer glass flasks, fitted with stainless steel caps which permitted gas exchange
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: 100 mL fill volume per replicate
- Initial cells density: 10,000 cells/mL
- Control end cells density: 989,000 cells/mL (mean)
- No. of vessels per concentration (replicates): Three replicates (A, B, and C) per treatment level
- No. of vessels per control (replicates): Six replicates (A, B, C, D, E, and F) for the control

GROWTH MEDIUM
- Standard medium used: Yes, AAP medium

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
Water Type (Cukture): Algal Assay Procedure (AAP) medium prepared with sterile, deionised source water
Water Type (Testing): AAP medium
Dilution Water Source (Culture and Testing): Town of Wareham water
pH: 7.5 ± 0.1, initial pH adjusted with dilute hydrochloric acid (HCl) or sodium hydroxide (NaOH) prior to use if necessary
- Total organic carbon: 0.78 mg/L (measured in May 2021)
- Culture medium different from test medium: no
- Intervals of water quality measurement: Representative samples of the dilution water source used in the preparation of the culture medium were analyzed periodically for the presence of pesticides, PCBs, and toxic metals by Eurofins Lancaster Laboratories Environmental, Lancaster, Pennsylvania (U.S. EPA, 1997). None of these compounds have been detected at concentrations that are considered toxic in any of the water samples analyzed in agreement with ASTM guidelines (ASTM, 2007).

OTHER TEST CONDITIONS
- Sterile test conditions: No
- Adjustment of pH: No
- Photoperiod: None (continuous illumination)
- Light intensity and quality: Approximately 4600 to 5800 lux, measured at exposure initiation using a Fisher Scientific Traceable dual-range light meter; Photosynthetically-Active Radiation: 64 to 76 μE/m2/S, measured at initiation and at each 24-hour interval during the exposure period using a Li-Cor BioSciences Model LI-189 photometer and radiation sensor LI-190R; Bulb Type: Premira VitaLux fluorescent bulbs
- Other:
Control of Bias: Random placement of exposure vessels and position reassignment daily, based on computer-generated random numbers.
Agitation: Continuous, 100 ± 10 rpm on an orbital shaker, rate monitored and recorded daily

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: At each subsequent 24-hour interval, cell counts were conducted on each replicate solution of the treatment levels and the controls using a hemacytometer (Neubauer Improved) and Leica DM 500 compound microscope. Generally, one sample was removed from each flask, and one count was determined for each sample. One or more hemacytometer fields, each 0.10 × 0.10 cm in surface area, 0.010-cm deep and containing 0.00010 mL of culture, were counted for each sample until at least 400 algal cells or four fields were counted. Cell density, expressed as cells/mL, was calculated for each test vessel by dividing the number of cells counted by the number of fields examined. Observations of the health of the algal cells were also made and recorded at each 24-hour interval.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study
Prior to initiating the definitive exposure, two preliminary range-finding exposures were conducted at the test facility. Two exposure vessels were established for each concentration.
- Test concentrations: Exposure solutions were prepared at nominal concentrations of 0.010, 0.10, 1.0, 10, and 100 mg/L for the first preliminary range-finding exposure. For the second preliminary range-finding exposure solutions were prepared at 1.0, 10, and 100 mg/L, and another 100 mg/L with pH adjustment.
- Results used to determine the conditions for the definitive study: The first preliminary range-finding exposure was conducted with solutions that were not pH adjusted, which in consultation with the Study Sponsor, elicited a second preliminary range-finder to be conducted with pH adjusted solutions in order to determine whether the toxicity observed in the first exposure were test substance related or pH related. The second preliminary range-finding exposure was then conducted and at termination, observations of precipitation/undissolved particulates were noted in all replicates that had been pH adjusted making cell counting difficult and highly variable.
Recommended per the guideline, solutions are not to be pH adjusted if an impact to the test material solubility is likely to occur (OECD, 2011). It was determined that the solubility of the test substance in the second preliminary range-finding exposure would greatly impact the analytical results for the definitive exposure and therefore determined that the definitive exposure would be conducted without pH adjusting the solutions.

Based on these results and in consultation with the Study Sponsor the nominal concentrations were selected for the definitive exposure.

CULTURING APPARATUS
-Details on culturing apparatus used: The test was conducted in an environmental chamber set to maintain temperature at 24 ± 2 °C.
Reference substance (positive control):
yes
Remarks:
A reference test was performed at the test facility from 23 to 27 November 2020 with Zinc chloride (ZnCl2) as reference substance outside of the scope of this study to evaluate the health and sensitivity of the test organism Raphidocelis subcapitata.
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
2.1 mg/L
95% CI:
>= 1.7 - <= 2.7
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
yield
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
1 mg/L
95% CI:
>= 0.51 - <= 1.4
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
yield
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.6 mg/L
95% CI:
>= 0.17 - <= 1
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
yield
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
yield
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.34 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
yield
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
11 mg/L
95% CI:
>= 8.8 - <= 13
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
2.4 mg/L
95% CI:
>= 1.8 - 3.8
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
1.4 mg/L
95% CI:
>= 1.1 - <= 1.8
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
3.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): Cells exposed to 0.11, 0.34, 1.0, and 3.3 mg/L nominal treatment levels tested and the control were observed to be normal. The 10 mg/L nominal treatment level cells were observed to be small compared to the control at the 72 hour interval and throughout the exposure period. The 32 mg/L nominal treatment level cells were observed to be grossly misshapen and fragmented at all intervals (24 hours, 48 hours, and 72 hours).
- Any stimulation of growth found in any treatment: A slight but not significant stimulation of growth was determined for the lower test concentrations of nominal 0.11 and 0.34 mg/L, which is considered to be a reason of biological variation and not treatment related.
- Effect concentrations exceeding solubility of substance in test medium: At each observation interval, control, and 0.11, 0.34, 1.0, and 3.3 mg/L nominal exposure solutions were observed to be the same as at exposure initiation (clear and colourless without precipitate). At 24 hours, the 32 mg/L nominal exposure solution was observed to be cloudy. At 48 and 72 hours, the 10 mg/L nominal exposure solution was observed to have precipitate and the 32 mg/L nominal exposure solution was observed to be cloudy with precipitate.
Results with reference substance (positive control):
The results of the reference test were within the expected range for Raphidocelis subcapitata; therefore, the culture is considered of appropriate health and sensitivity for use in toxicity testing.
EC50 (96h) = 0.098 mg Zn/L, with a 95% confidence interval of 0.089 to 0.11 mg Zn/L (historical mean = 0.096 mg Zn/L, March 2005 to present)
Reported statistics and error estimates:
Prior to NOEC and LOEC determinations, the data were first checked for normality using
Shapiro-Wilk’s Test (U.S. EPA, 2002) and for homogeneity of variance using Bartlett’s Equality
of Variance Test (U.S. EPA, 2002). If the data sets passed the tests for homogeneity and
normality, then a parametric statistical test, e.g., Dunnett’s Multiple Comparison Test
(U.S. EPA, 2002) was used to determine the NOEC and LOEC. If the data did not pass the tests
for homogeneity and normality, then the NOEC and LOEC were determined using an
appropriate non-parametric statistical test, e.g., Dunn’s Test with Bonferroni-Holm’s Adjustment
(U.S. EPA, 2002). All statistical determinations were made at the 95% level of certainty, except
in the case of Shapiro-Wilk’s and Bartlett’s Equality of Variance Tests, where the 99% level of
certainty was applied.
If possible, EC10, EC20, and EC50 values were calculated using a nonlinear regression model.
The test concentrations bracketed the EC values so that the EC value came from interpolation
rather than an extrapolation. The EC10, EC20, and EC50 values were estimated so that (i) the
95% confidence intervals reported for the EC value did not contain zero and was not overly
wide, (ii) the 95% confidence interval for the predicted mean at the EC10, EC20, or EC50 value did
not contain the control mean, and (iii) there was no significant lack-of-fit of regression model to
the data. If no concentration resulted in a 10, 20, or 50% reduction, the EC values were
empirically estimated to be greater than the highest concentration tested. CETIS Version 1.9
(Ives, 2020) was used to perform all statistical calculations.
Validity criteria fulfilled:
yes
Conclusions:
The study was conducted under GLP according to OECD Guideline 201 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation or deviations from the guidelines, the validity criteria were met. Hence, the results can be considered as reliable to assess the toxicity of the test substance towards algae.
Exponentially growing cells of the freshwater green algae Raphidocelis subcapitata were exposed to the test item added to test water at concentrations of nominal 32, 10, 3.3, 1.0, 0.34 and 0.11 mg/L corresponding to 15, 4.7, 1.6, 0.47, 0.16 and 0.052 mg F-/L and 7.2, 2.2, 0.74, 0.22, 0.076 and 0.025 mg Ti/L, respectively. A water control was running in parallel for a period of 72 hours under defined conditions. Exposure solutions were analysed for two components, fluoride and titanium, using ICP-MS and ISE, respectively. Based on the analytical recoveries in the freshly prepared and aged test solutions mean measured concentrations of 16, 4.7, 0.46, 0.16 and 0.055 mg F-/L and 6.2, 1.9, 0.65, 0.22, 0.062 and 0.020 mg Ti/L were determined. The results of this study are based on nominal concentrations of potassium hexafluorotitanate (IV).Based on the obtained biological results a significant inhibition of algal yield and growth rate was observed at the end of the 72 hour study period, relative to control cultures grown under identical conditions. Accordingly, the 72h EC50 was determined to be 2.1 mg/L for the parameter yield and 11 mg/L for the parameter growth rate. The 72h NOEC was determined to be 0.34 and 1.0 mg/L for yield and growth rate, respectively, with associated LOEC`s of 1.0 and 3.3 mg/L.
Executive summary:

The acute toxicity of the registered substance to aquatic algae was tested under GLP according to OECD guideline 201 in a static freshwater test with Raphidocelis subcapitata (former name: Pseudokirchneriella subcapitata) as test organism. Exponentially growing cells of the freshwater green algae were exposed to the test item added to test water at concentrations of nominal 32, 10, 3.3, 1.0, 0.34 and 0.11 mg/L corresponding to 15, 4.7, 1.6, 0.47, 0.16 and 0.052 mg F-/L and 7.2, 2.2, 0.74, 0.22, 0.076 and 0.025 mg Ti/L, respectively, for  period of 72 hours. A water control was running in parallel under test conditions. Exposure solutions were analysed for fluoride and titanium using ICP-MS and ISE, respectively, and the obtained results were used to appropriately evaluate the preparation of the exposure solutions. Based on the analytical recoveries in the freshly prepared and aged test solutions mean concentrations of 16, 4.7, 1.6, 0.46, 0.16 and 0.055 mg F-/L and 6.2, 1.9, 0.65, 0.22, 0.062 and 0.020 mg Ti/L were determined. The results of this study are presented based on nominal concentrations of potassium hexafluorotitanate (IV), and are reported as the 72-hour EC10, EC20, and EC50 values for average specific growth rate and yield, denoted as ErC10, ErC20, ErC50, and EyC10, EyC20, EyC50, respectively. The 72-hour No-Observed-Effect Concentration (NOEC) and Lowest-Observed-Effect Concentration (LOEC) values were also determined for the parameters average specific growth rate and yield. Based on the obtained biological results a significant inhibition of algal yield and growth rate was observed at the end of the 72 hour study period, relative to control cultures grown under identical conditions. Accordingly, the 72h EC50 was determined to be 2.1 mg/L for the parameter yield and 11 mg/L for the parameter growth rate. The 72h NOEC was determined to be 0.34 and 1.0 mg/L for yield and growth rate, respectively, with associated LOEC`s of 1.0 and 3.3 mg/L.

Description of key information

Key_Toxicity to aquatic algae and cyanobacteria: ErC50 (72h) = 11 mg/L and ErC10 (72h) = 1.4 mg/L (nominal) for Raphidocelis subcapitata based on the average growth rate (static, OECD 201, GLP, 2021)

Key value for chemical safety assessment

EC50 for freshwater algae:
11 mg/L
EC10 or NOEC for freshwater algae:
1.4 mg/L

Additional information

Dipotassium hexafluorotitanate


The acute toxicity of the registered substance to aquatic algae was tested under GLP according to OECD guideline 201 (2021) in a static freshwater test with Raphidocelis subcapitata (former name: Pseudokirchneriella subcapitata) as test organism. Exponentially growing cells of the freshwater green algae were exposed to the test item added to test water at concentrations of nominal 32, 10, 3.3, 1.0, 0.34 and 0.11 mg/L corresponding to 15, 4.7, 1.6, 0.47, 0.16 and 0.052 mg F-/L and 7.2, 2.2, 0.74, 0.22, 0.076 and 0.025 mg Ti/L, respectively, for  period of 72 hours. A water control was running in parallel under test conditions. Exposure solutions were analysed for fluoride and titanium using ICP-MS and ISE, respectively, and the obtained results were used to appropriately evaluate the preparation of the exposure solutions. Based on the analytical recoveries in the freshly prepared and aged test solutions mean concentrations of 16, 4.7, 1.6, 0.46, 0.16 and 0.055 mg F-/L and 6.2, 1.9, 0.65, 0.22, 0.062 and 0.020 mg Ti/L were determined. The results of this study are presented based on nominal concentrations of potassium hexafluorotitanate (IV), and are reported as the 72-hour EC10, EC20, and EC50 values for average specific growth rate and yield, denoted as ErC10, ErC20, ErC50, and EyC10, EyC20, EyC50, respectively. The 72-hour No-Observed-Effect Concentration (NOEC) and Lowest-Observed-Effect Concentration (LOEC) values were also determined for the parameters average specific growth rate and yield. Based on the obtained biological results a significant inhibition of algal yield and growth rate was observed at the end of the 72 hour study period, relative to control cultures grown under identical conditions. Accordingly, the 72h EC50 was determined to be 2.1 mg/L for the parameter yield and 11 mg/L for the parameter growth rate. The 72h EC20 and EC10 were determined to be 1.0 and 0.60 mg/L for the parameter yield and 2.4 and 1.4 mg/L for the parameter growth rate, respectively. The 72h NOEC was determined to be 0.34 and 1.0 mg/L for yield and growth rate, respectively, with associated LOEC`s of 1.0 and 3.3 mg/L.


 


Supporting information: The toxicity of dipotassium hexafluorotitanate to algae (Pseudokirchneriella subcapitata) was tested according to OECD 201 (2012); respective EC 10 and EC50 values were 1.31 mg/L and 10.81 mg/L.