Reaction Mass of zirconium difluoride oxide and fluorozirconic acid

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:

2018-06-14 to 2018-07-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction Mass of zirconium difluoride oxide and fluorozirconic acid

EC Number:

947-897-3

Molecular formula:

(ZrOF2 )x(F6Zr.2H)y

IUPAC Name:

Reaction Mass of zirconium difluoride oxide and fluorozirconic acid

Test material form:

other: aqueous solution

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Identification: Reaction Mass of zirconium difluoride oxide and fluorozirconic acid
- Source and lot/batch No.of test material: 18-KTS-003
- Expiration date of the lot/batch: no data
- Weight % water: 73.8%
- Weight % Zr: 11.5%
- Weight % solids: 26.2%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, protected from light

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Sampling method: Samples of the test solutions were collected at approximately 0 and 96 hours to measure concentrations of both zirconium and fluoride using an ISE probe for fluoride and ICP-MS for zirconium. Samples at exposure initiation were collected from the individual batches of test solution prepared for each treatment and the control group prior to distribution into the test chambers. At exposure termination, samples were collected from the pooled biological replicates from each treatment and the control group. Additionally, at 0 and 96 hours of exposure, samples were collected from the pH adjusted replicates included in the 20 and 50 mg/L treatment groups. At each sampling interval, samples were collected in duplicate with one set of samples processed immediately for analysis, while the additional set of samples were stored under refrigerated conditions for potential future analysis.
At test initiation, samples were inadvertently not collected for fluoride analysis from the 20 and 50 mg/L test solutions prior to adjusting the pH of the test solutions and there was also insufficient test solution to collect samples for fluoride analysis from the 20 mg/L pH adjusted test solution. At test termination, only one sample was collected from the 20 mg/L pH adjusted replicate for fluoride analysis. These deviations from the protocol were not considered to be detrimental to the study since the duplicate samples collected for zirconium analysis were ultimately used for fluoride analysis.
- Sample storage conditions before analysis: stored in a refigerator until analysis.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A primary stock solution was prepared by dissolving 0.1002 g of Reaction Mass of zirconium difluoride oxide and fluorozirconic acid in 2000 mL of freshwater AAP medium to achieve a nominal concentration of 50 mg/L. The 50 mg/L primary stock was inverted at least twenty times to mix and then stirred with a Teflon-coated stir bar and magnetic stir plate while all subsequent dilutions were made. The 50 mg/L primary stock appeared clear and colorless at the time of preparation. Aliquots of the 50 mg/L primary stock were diluted with freshwater AAP medium to prepare five additional test solutions at nominal concentrations of 0.51, 1.3, 3.2, 8.0, and 20 mg/L. The 0.51, 1.3, 3.2, 8.0, and 20 mg/L test solutions were mixed by inversion and appeared clear, colorless, and otherwise unremarkable at the time of preparation. The negative control solution consisted of freshwater AAP medium without test substance added.
- Controls: yes
- Evidence of undissolved material (e.g. precipitate, surface film, etc): There was no evidence of surface slicks or particulates at test initiation or test termination.

Test organisms

Test organisms (species):

Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Raphidocelis subcapitata
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): Original algal cultures (UTEXID#1648) were obtained from the University of Texas at Austin. Algal cells used in this test were obtained from EAG Laboratories-Easton cultures that had been actively growing in culture medium for at least two weeks prior to test initiation.
- Age of inoculum (at test initiation): Algal cells were taken from a culture that had been transferred to fresh medium four days prior to test initiation
- Method of cultivation: cultivated under test conditions

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Test conditions

Hardness:

not reported

Test temperature:

23.55 to 24.31°C (mean 24.01°C)

pH:

Nominal test concentrations (mg/L) pH at t=0 pH at t=96h
negative control 7.3 9.3
0.51 7.3 9.4
1.3 7.2 9.1
3.2 7.1 8.5
8.0 6.9 7.7
20 6.4 6.9
50 4.6 4.5
20 - pH adjusted 7.2 7.2
50 - pH adjusted 7.1 7.2

Dissolved oxygen:

not reported

Salinity:

not relevant

Nominal and measured concentrations:

Final test:
nominal test concentrations:
0, 0.51, 1.3, 3.2, 8.0, 20, 50, 20 (pH adjusted), 50 (pH adjusted)

Measured concentrations of test item in Test Solution Samples at t= 0 h: Measured concentrations of test item in Test Solution Samples at t= 96h: Measured % of nominal: 82.4, 84.6, 81.3, 75.0, 55.0, 64.0, 75.0, 60.0

Measured Concentrations of fluoride in Test Solution Samples at t= 0 h: 0.045, 0.17, 0.38, 0.90, 2.01, 4.74, 21.2, 4.98, 11.02 mg/L
Measured Concentrations of fluoride in Test Solution Samples at t= 96 h: 0.043, 0.17, 0.41, 0.89, 2.0, 4.92, 21.0, 5.02, 11.42 mg/L
Measured concentrations of fluoride in test samples were about 10% of nominal concentrations but remained stable during the test period.

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Type (delete if not applicable): capped vessels, all-glass
- Material, size, headspace, fill volume: 250mL filled with 100 mL test solution plugged with sterile foam stoppers
- Initial cells density: 1.36 x 10^6 cells/mL
- Control end cells density: (mean)
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Freshwater AAP Algal Medium: Stock nutrient solutions were prepared by adding reagent-grade chemicals to purified EAG Laboratories-Easton well water. The test medium then was prepared by adding appropriate volumes of the stock nutrient solutions to purified well water (NANOpure® water). The pH of the medium was adjusted to 7.5 ± 0.1 with 0.1 NaOH and 10% HCl at the time of preparation. The medium was sterilized by filtration (0.22 µm) prior to use.
MgCl2•6H2O: 12.164 mg/L
CaCl2•2H2O: 4.410 mg/L
H3BO3: 0.1855 mg/L
MnCl2•4H2O: 0.4154 mg/L
ZnCl2: 3.27 µg/L
FeCl3•6H2O: 0.1598 mg/L
CoCl2•6H2O: 1.428 µg/L
Na2MoO4•2H2O: 7.26 µg/L
CuCl2•2H2O: 0.012 µg/L
Na2EDTA•2H2O: 0.300 mg/L
NaNO3: 25.50 mg/L
MgSO4•7H2O: 14.70 mg/L
K2HPO4: 1.044 mg/L
NaHCO3: 15.00 mg/L

- Culture medium different from test medium: no, algal cells were cultured and tested in freshwater AAP medium.
- Intervals of water quality measurement: temperature is continuously, pH at the beginning in all concentrations and the end of the test in the control and the limit concentration.

OTHER TEST CONDITIONS
- Adjustment of pH: yes
- Photoperiod: continuous illumination
- Light intensity and quality: The light intensity ranged from 3,910 to 4,410 lux, which was within the desired range of 4,300 lux ± 10%.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter]: Cell counts were performed using an electronic particle counter (Coulter Electronics, Inc.). Prior to conducting cell counts, the linearity of the instrument response was determined at settings previously established for Raphidocelis subcapitata. A primary counting standard containing R. subcapitata cells was prepared, the density was verified using a hemacytometer and a microscope, per laboratory standard operating procedures, and the standard was subsequently diluted to provide a series of seven counting standards for the determination of instrument linearity.
- effect calculated parameters: EbCx, ErCx and EyCx estimates and their corresponding 95% confidence intervals were determined using non-linear regression with treatment response (area under the growth curve, growth rate, and yield) and nominal test concentrations.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study
- Test concentrations range finding: 0.24, 0.81, 2.7, 9.0, 30 mg/L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

20 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% C.L. 18-22 mg/L

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.2 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
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Flocculation: After 96 hours of exposure, there were no observations of flocculation or aggregation of cells in any of the experimental groups.
- Adherence to test vessels: Adherence of cells to the test chambers was not observed in any of the experimental groups.
- Other:
*Cells present in the 0.51, 1.3, 3.2, 8.0, 20, and 50 mg/L treatment groups appeared normal relative to cells in the negative control. Cells present in the 20 mg/L pH adjusted replicate appeared normal, while cells present in the 50 mg/L pH adjusted replicate appeared enlarged relative to the cells presented in the negative control.
*Additional replicates in which the pH was adjusted to a neutral level were included in the exposure concentrations where the pH was <6.5. The responses of the pH adjusted replicates demonstrate that the toxicity observed from the test substance exposure is related to introduction to the test substance and not solely physical toxicity from the reduced pH.

- 72-h EC50 (biomass)*= 11 mg/L (95% C.L. 10-12 mg/L)
- 72-h EC10 (growth rate)* = 7.1 mg/L (95% C.L. 5.5-9.3 mg/L)
- 72-h EC50 (yield)*= 9.8 mg/L (95% C.L. 8.4-12.0 mg/L)
- 72-h EC10 (yield)* = 5.7 mg/L (95% C.L. 4.3-7.6 mg/L)
- 72-h NOEC (yield)* = 3.2 mg/L

- 96-h EC50 (biomass)*= 9.3 mg/L (95% C.L. 8.5-10.0 mg/L)
- 96-h EC10 (growth rate)* = 6.4 mg/L (95% C.L. 5.6-7.3 mg/L)
- 96-h EC50 (yield)*= 8.3 mg/L (95% C.L. 7.4-9.2 mg/L)
- 96-h EC10 (yield)* = 4.7 mg/L (95% C.L. 3.8-5.7 mg/L)
- 96-h NOEC (yield)* = 0.51 mg/L

* Results were based on nominal, total formulation concentrations since the test substance is considered to be a formulated product and the day 0 recoveries for zirconium ranged from 91.6 to 101% of nominal.

- After 72 hours, mean area under the growth curve and mean yield were significantly reduced (Dunnett’s Test; p ≤ 0.05) in the 8.0, 20, and 50 mg/L treatment groups when compared to the negative control.
- After 72 hour, mean growth rate was significantly reduced (Jonckheere-Terpstra Step-Down Trend Test; p ≤ 0.05) in the 8.0, 20, and 50 mg/L treatment group when compared to the negative control. Consequently, the 72-hour NOEC was determined to be 3.2 mg/L and was based on statistically significant reductions in all three endpoints.

Results with reference substance (positive control):

not examined

Reported statistics and error estimates:

The 72 and 96-hour area under the growth curve, growth rate and yield data were evaluated for normality and homogeneity of variance (α = 0.01) using Shapiro-Wilk’s and Levene’s tests, respectively. The responses of the treatment groups were compared to the negative control responses using Dunnett’s test (α = 0.05). In instances where the data violated assumptions of normality and/or homogeneity of variance the data was logarithmically transformed and reanalyzed. A non-parametric analysis, Johnckheere-Terpstra Step Down Trend Test (α = 0.05) was conducted with data that still violated assumptions of normality and/or homogeneity of variance after transformation.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

A 72-h growth inhibition test with the unicellular green alga Pseudokirchneriella subcapitata was performed wit the Reaction Mass of Zirconium Difluoride Oxide and Fluorozirconic Acid according to the OECD guideline 201 (GLP conditions). Based on nominal, total formulation concentrations, the 72-hour EC50 based on growth rate was determined to be 20 mg/L. The 72-hr NOEC based on growth rate was determined to be 3.2 mg/L. The results of the test can be considered reliable without restrictions.