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Long-term toxicity to aquatic invertebrates

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Reference
Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2021-02-12 - 2021-07-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Version / remarks:
OECD, 2012, Guideline for Testing of Chemicals, Daphnia magna Reproduction Test, Test Guideline 211, Adopted 2 October 2012
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Sampling method: On day 0, 7, 14, and 20 samples of the freshly prepared test solutions were taken from each treatment level and the control prior to division into replicate vessels; on day 1, 8, 15 and 21 samples of the aged test solutions (24 h) were taken composite of all available replicate vessels within each treatment level and the control.
One sample was taken from each exposure solution at each interval. Additional aliquots of the exposure solutions were also collected at each sampling interval and stored refrigerated
(one set) and frozen (one set) as archive samples. Samples were taken from the approximate midpoint from the surface, bottom, and sides of the vessels with a pipette.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
Primary Stock Concentration: 10 mg/L, prepared daily
Stock Volume: 3.0 L
Stock Vessel: Glass beaker
Amount of Test Substance Added (for example): 0.0300 g (tested as 100%)
Diluent: Fortified laboratory well water
The primary stock solution was mixed using a magnetic stir plate and Teflon-covered stir bar for approximately 5 minutes and also ultrasonificated for approximately 5 minutes. Exposure solutions were prepared from dilutions of the 10 mg/L primary stock solution. Exposure solutions were mixed for approximately 1 minute using a glass rod prior to division into replicate exposure vessels. Freshly prepared solutions were divided into 10 replicate vessels, each labeled with the study number, treatment level/control identification, and replicate designation. Exposure solutions were renewed daily. Freshly-prepared exposure solutions for each treatment level and the control were prepared following the procedures outlined above.
- Controls: Dilution water only maintained under the same conditions as the treatment level solutions.
- Test concentration separation factor: 2
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): The stock solution was observed to be slightly cloudy and colorless with no visible undissolved test substance following preparation.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna
- Age at study initiation (mean and range, SD): <24 hours old at exposure initiation
- Stage and instar at study initiation: neonates
- Method of breeding:
Culture water: The culture water was prepared by fortifying well water based on the formula for hard water (U.S. EPA, 1975) and filtering it through an Amberlite XAD-7 resin column to remove any potential organic contaminants.
Culture vessel: 1.0-L glass beaker containing approximately 0.80 L of water
Food type: Unicellular green algae, Raphidocelis subcapitata (6 × 107 cells/mL) and a suspension of YTC (yeast, trout chow, and wheat grass)
Feeding Frequency: Once daily during culture
Ephippia Observed: No
Immobilization 48 Hours Prior to Initiation: 0%
Representative samples of the food source were periodically analyzed for the presence of pesticides, PCBs, and toxic metals by Energy Laboratories, Inc., Billings, Montana (U.S. EPA, 1997). None of these compounds have been detected at concentrations considered toxic in any of the samples analyzed, in agreement with ASTM (2007) standard practice. Based on the analysis for pesticides, the food source was considered to be of acceptable quality since all analyte concentrations were below levels of concern.
- Source: The Daphnia magna used in this toxicity test were obtained from laboratory cultures maintained at the test facility.
- Age of parental stock (mean and range, SD): not specified
- Feeding during test
- Food type and amount:
Algal suspension (Raphidocelis subcapitata (6 × 107 cells/mL): 300 μL (test days 0 through 21)
Yeast, trout chow, and wheat grass (YTC) suspension: 200 μL
Equivalent to 0.2 mg carbon/daphnid/day
- Frequency: Once daily

ACCLIMATION
- Acclimation period:
- Acclimation conditions (same as test or not):
- Type and amount of food:
- Feeding frequency:
- Health during acclimation (any mortality observed):

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES, INCLUDING CULTURING CONDITIONS: Daphnids were obtained for testing by removing all immature daphnids from the culture vessels, thus isolating sexually mature daphnids approximately 24 hours prior to exposure initiation. Young produced by these daphnids within 24 hours of isolation were used in the exposure.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Test temperature:
19 to 22 °C in all freshly prepared and aged test solutions
pH:
freshly prepared test solutions: 7.3 to 8.3
aged test solutions (24h): 7.6 to 8.3
Dissolved oxygen:
freshly prepared test solutions: 83 to 110 % of Saturation
aged test solutions (24h): 70 to 95% of Saturation
Conductivity:
Control: 680 to 720 µS/cm
10 mg/L test solution: 700 to 770 µS/cm
Nominal and measured concentrations:
Nominal Concentrations: 0.63, 1.3, 2.5, 5.0, and 10 mg/L
Nominal Concentrations as Fluoride: 0.30, 0.59, 1.2, 2.4, and 4.7 mg F/L
Nominal Concentrations as Titanium: 0.14, 0.28, 0.56, 1.1, and 2.2 mg Ti/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 100-mL clear glass beakers
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: approximately 80 mL fill volume
- Aeration: None
- Renewal rate of test solution (frequency/flow rate): Exposure solutions were renewed daily.
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
Water Type: Fortified laboratory well water
pH: 7.5 to 8.0
Dissolved Oxygen: 8.5 to 10 mg/L
Conductivity: 700 to 790 μS/cm
Total Hardness as CaCO3: 170 to 180 mg/L
- Total organic carbon: 1.1 mg/L, Month measured: April 2021
- Alkalinity: 88 to 92 mg/L as CaCO3
- Culture medium different from test medium: Yes, the culture water was prepared by fortifying well water based on the formula for hard water (U.S. EPA, 1975) and filtering it through an Amberlite XAD-7 resin column to remove any potential organic contaminants.
- Intervals of water quality measurement: Representative samples of the dilution water source 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 (2007) standard practice. Daphnid cultures are maintained in water from the same source as the dilution water utilized in this study and have successfully survived and reproduced over several generations. The acceptable performance of the daphnid cultures, in combination with the previously mentioned analyses, confirmed the acceptability of this dilution water for use during the conduct of bioassays.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours light: 8 hours dark with 15- to 30-minute transition period; controlled with an automatic timer
- Light intensity: 15 to 19 μE m-2 s-1; measured with a Li-Cor BioSciences Model LI-250A photometer and quantum sensor
- Other: The study was conducted in an environmental chamber set to maintain temperature at 20 +/- 2 °C.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The number of immobilised adult daphnids and observations of abnormal behavior (e.g., presence of males, ephippia, or aborted broods) were recorded daily, if observed. Immobilisation is defined as the inability of daphnids to swim or if there is no observed movement of appendages or post-abdomen within 15 seconds of gentle agitation of the exposure vessel. The day of appearance of first brood in each exposure vessel was recorded. Live offspring were counted and removed daily beginning with the appearance of the first brood. The presence of aborted eggs and immobile offspring were recorded if observed and were removed. Observations of physical characteristics of exposure and control solutions (e.g., precipitate, cloudy solution) were recorded whenever the test organisms were observed.
At exposure termination (day 21), the total body length of each surviving adult daphnid was measured. Daphnids were measured (to the nearest 0.05 mm) from the apex of the head to the base of the carapace spine using an Olympus SZH-ILLD-200 dissecting scope and calibrated ocular.
Besides, the dissolved oxygen, pH, and temperature were measured in new solutions at initiation, in both the new and aged solutions at each renewal period, and in each aged solution at exposure termination.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study
A 21-day preliminary range-finding exposure was conducted at the test facility under static-renewal conditions (daphnids <24 hours old, five replicates, one daphnid per replicate exposure vessel). Stock and exposure solutions were prepared daily as described for the definitive exposure.
- Test concentrations: nominal 10, 2.0, 0.40 and 0.080 mg/L and a control
- Results used to determine the conditions for the definitive study: Based on the results of the chronic range-finder and in consultation with the Study Sponsor, nominal concentrations of 0.63, 1.3, 2.5, 5.0, and 10 mg/L, and a dilution water control, were selected for the definitive static-renewal exposure.
Reference substance (positive control):
yes
Remarks:
A reference test with the reference substance Potassium dichromate was performed outside of the scope of this study to evaluate the sensitivity of the Daphnia magna population at the test facility.
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
> 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth
Remarks:
Total body length of parental daphnids
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
>= 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth
Remarks:
Total body length of parental daphnids
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
> 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
>= 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
>= 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks:
Total living offspring per surviving female
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
> 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks:
Total living offspring per surviving female
Results with reference substance (positive control):
- Results with reference substance valid? This result of the reference test was within the expected range for daphnids and potassium dichromate.
- Relevant effect levels: EC50 (24h) = 3.2 mg/L, with a 95% confidence interval of 2.0 to 5.0 mg/L (historical mean = 2.3 mg/L with a range of 0.68 to 3.5 mg/L)
Reported statistics and error estimates:
Determination of NOEC and LOEC:
At the termination of the study, data obtained on organism survival, reproduction, and growth (total body length) were statistically analyzed to identify significant treatment-related effects. Analyses were performed using the individual response values.
All statistical analyses were conducted at the 95% level of certainty, except tests to assess normality and homogeneity of variance, where the 99% level of certainty was applied.
CETIS Version 1.9 (Ives, 2020) was used to perform the statistical computations.The highest test concentration that elicited no statistically significant difference between the exposed organisms and the appropriate control (No-Observed-Effect Concentration, NOEC) that was interpreted as dose related and biologically relevant. The lowest test concentration that elicited a statistically significant effect on organism performance interpreted as biologically relevant is defined as the Lowest-Observed-Effect Concentration (LOEC).

Determination of ECx
The nominal concentrations tested and the corresponding data for survival, reproduction, and growth (total body length) derived from the definitive exposure were used to estimate the 21-day median effect concentrations (EC50), the EC20, and EC10 values and their corresponding 95% confidence intervals, when possible. The EC50, EC20, and EC10 values are defined as the concentration of test substance in dilution water that caused a 50, 20, and 10% reduction, respectively, in survival, reproduction or growth (total body length) of the test organism population at the stated time interval. CETIS Version 1.9 (Ives, 2020) was used to calculate the ECx values and 95% confidence intervals to assess if any test concentrations caused a ≥10, 20, or 50% reduction of survival, reproduction, or growth in the test population. If possible, EC10, EC20, and EC50 values were calculated using a nonlinear regression model. The test concentrations bracketed the EC values so that (i) t
Validity criteria fulfilled:
yes
Conclusions:
The study was conducted under GLP according to OECD Guideline 211 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 long-term toxicity of the test substance towards daphnia.
The objective of this exposure was to evaluate the chronic effects of potassium hexafluorotitanate (IV) on the survival, reproduction, and growth (mean total body length) of the daphnid, Daphnia magna. In a semi-static test with daily water renewal neonates (< 24 h) of the freshwater invertebrate Daphnia magna were exposed to the test substance at concentrations of nominal 10, 5.0, 2.5, 1.3 and 0.63 mg/L corresponding to 4.7, 2.4, 1.2, 0.59 and 0.30 mg F-/L and 2.2, 1.1, 0.56, 0.28 and 0.14 mg Ti/L and a control for a period of 21 days 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 concentrations of 4.9, 2.3, 1.1, 0.59 and 0.29 mg F-/L and geometric mean concentrations of 1.7, 0.86, 0.44, 0.24 and 0.11 mg Ti/L were determined. Based on the obtained biological results, no effects were observed for the parameter survival, reproduction and growth (mean total body length). Accordingly, the 21-day No-Observed-Effect Concentration (NOEC) and Lowest-Observed-Effect Concentration (LOEC) were determined to be ≥ 10 and >10 mg/L, respectively, for all
endpoints. The highest concentration tested was the maximum concentration required by the guideline and, thus, no further testing is necessary.
Executive summary:

The study was performed under GLP according to OECD TG 211. The purpose of this study was to evaluate the chronic effects of the registered substance on the survival, reproduction, and growth (mean total body length) of the daphnid, Daphnia magna during an exposure period of 21 days. Therefore, female Daphnia (<24 hours old) taken from an in-house laboratory culture of the test facility, were exposed in a semi-static daily renewal test to aqueous test media containing the test item at concentrations of nominal 10, 5.0, 2.5, 1.3 and 0.63 mg/L corresponding to 4.7, 2.4, 1.2, 0.59 and 0.30 mg F-/L and 2.2, 1.1, 0.56, 0.28 and 0.14 mg Ti/L and a control under defined conditions. The mortality of adults and the number of offspring per introduced adult were observed daily in each of the 10 replicates of each test concentration and compared with corresponding parameters in the control. Exposure solutions were analysed for two components, fluoride and titanium using ICP-MS and ISE, respectively, and these 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 4.9, 2.3, 1.1, 0.59 and 0.29 mg F-/L and geometric mean concentrations of 1.7, 0.86, 0.44, 0.24 and 0.11 mg Ti/L were determined. The results of this study are based on nominal concentrations of potassium hexafluorotitanate (IV).


Based on the obtained results, the following endpoints were determined:


EC50 (21d) mortality > 10 mg//L


NOEC (21d) mortality ≥ 10 mg//L


LOEC (21d) mortality > 10 mg//L


 


EC50 (21d) reproduction > 10 mg//L


NOEC (21d) reproduction ≥ 10 mg//L


LOEC (21d) reproduction > 10 mg//L


 


EC50 (21d) growth > 10 mg//L


NOEC (21d) growth ≥ 10 mg//L


LOEC (21d) growth > 10 mg//L

Description of key information

Long-term toxicity to aquatic invertebrates: NOEC (21d) >/= 10 mg/L (nominal) for Daphnia magna based on mortality, reproduction and growth (semi-static daily renewal, OECD 211, GLP)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Dose descriptor:
NOEC
Effect concentration:
>= 10 mg/L

Additional information

Dipotassium hexafluorotitanate


A study was performed under GLP according to OECD TG 211 with the registered substance. The purpose of this study was to evaluate the chronic effects of the registered substance on the survival, reproduction, and growth (mean total body length) of the daphnid, Daphnia magna during an exposure period of 21 days. Therefore, female Daphnia (<24 hours old) taken from an in-house laboratory culture of the test facility, were exposed in a semi-static daily renewal test to aqueous test media containing the test item at concentrations of nominal 10, 5.0, 2.5, 1.3 and 0.63 mg/L corresponding to 4.7, 2.4, 1.2, 0.59 and 0.30 mg F-/L and 2.2, 1.1, 0.56, 0.28 and 0.14 mg Ti/L. 20.3, 6.44, 2.05, 0.65 and 0.206 µg/L and a control under defined conditions. The mortality of adults and the number of offspring per introduced adult were observed daily in each of the 10 replicates of each test concentration and compared with corresponding parameters in the control. Exposure solutions were analysed for two components, fluoride and titanium using ICP-MS and ISE, respectively, and these 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 4.9, 2.3, 1.1, 0.59 and 0.29 mg F-/L and geometric mean concentrations of 1.7, 0.86, 0.44, 0.24 and 0.11 mg Ti/L were determined. The results of this study are based on nominal concentrations of potassium hexafluorotitanate (IV).


Based on the obtained results, the following endpoints were determined:


EC50 (21d) mortality > 10 mg//L


NOEC (21d) mortality ≥ 10 mg//L


LOEC (21d) mortality > 10 mg//L


 


EC50 (21d) reproduction > 10 mg//L


NOEC (21d) reproduction ≥ 10 mg//L


LOEC (21d) reproduction > 10 mg//L


 


EC50 (21d) growth > 10 mg//L


NOEC (21d) growth ≥ 10 mg//L


LOEC (21d) growth > 10 mg//L