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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
2002-09-27 to: 2002-09-27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
REPORTING FORMAT FOR THE CATEGORY APPROACH
1. HYPOTHESIS FOR THE CATEGORY APPROACH: The hypothesis is that properties are likely to be similar or follow a similar pattern because of the presence of a common metal ion, in this case tungstate.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES):
Source: Tungsten trioxide
Target: Tungstic acid
3. CATEGORY APPROACH JUSTIFICATION: See Annex 3 in CSR
4. DATA MATRIX: See Annex 3 in CSR
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
yes
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
PHYSICO-CHEMICAL PROPERTIES
- Water solubility: < 10 mg/L W03 at 20 °C
Analytical monitoring:
no
Details on sampling:
no data
Vehicle:
no
Details on test solutions:
no data
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Source: Non-axenic strain of the test species obtained from 'The Collection of Algal Cultures' of the Institute of Plant Physiology at the University of Gottingen (Germany).

- Exponetially- growing stock cultures are maintained in the test facility under constant temperature conditions (23 +/- 2 degrees C) at a light intesity in the range 400 to 700 nm using a spherical quantum flux meter. The nutrient medium (according to Bringmann and Kuhn, 1977) is renewed once a week. Cell density measurements are made using a microcell counter.

- Pre-cultures are set-up for three days before the start of the test. They are grown under identical exposure conditions as the stock cultures, exceptfrom the use of different nutrient medium.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Post exposure observation period:
no data
Hardness:
no data
Test temperature:
21 - 25 °C (can be maintained +/- 2 °C)
pH:
0 hours = 7.6
72 hours = 10.5
Dissolved oxygen:
no data
Salinity:
n/a
Nominal and measured concentrations:
Nominal: 1 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 300 mL Erlenmeyer flasks with stoppers
- Experimental design: 1 test concentration plus 1 control
- Initial cells density: Initial cell density in the test cultures approximately 10,000 cells per mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

OTHER TEST CONDITIONS
Light intensity: At the average of the test solutions, a light intensity in the range of 4000 to 8000 lx.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Solid NaHCO3 is added to the nutrient media to make up final concentation of 50 mg/L in the solutions of the pre-cultures and the test cultures.
Reference substance (positive control):
no
Key result
Duration:
72 h
Dose descriptor:
EC0
Effect conc.:
>= 1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: biomass and growth rate
Details on results:
Growth
Area under growth curve: 408333
Percentage increase of cell growth: -7.9%

Growth rate
Specific growth rate: 1.19
Percentage increase of cell growth rate: -3.7%

See attached results for final cell densities.
Results with reference substance (positive control):
n/a
Reported statistics and error estimates:
NOEC and LOEC were determined according to DUNNETT (1955, 1964)

All results are expressed in terms of nominal concentrations. Considering the low aqueous solubility of the test substance (< 1 mg/L WO3 under exposure conditions) no specific analysis was established.

Validity criteria fulfilled:
yes
Conclusions:
No toxic effects against algae at 1 mg/L. EC0: >= 1 mg/L
Executive summary:

No aquatic algae invertebrate toxicity data of sufficient quality are available for tungstic acid (target substance). However, aquatic algae invertebrate toxicity data are available for tungsten trioxide (source substance), which were used for read-across. Tungstic acid (WO3•H2O) is the hydrated form of tungsten trioxide (WO3). Based on Annex V, hydrates and water free forms (anhydrous) of compounds can be regarded as the same substance for registration purposes. Therefore, tungstic acid will be considered equivalent to WO3. For more details on the rationale, refer to the attached description of the read-across approach.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
2008-10-06 to: 2009-03-19
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Well documented, scientifically sound study that was conducted in accordance to GLP and OECD guideline 201 with no deviations to the protocol. The reliability of this study for the substance tested is a K1, but in application of read-across to a different substance ECHA’s guidance specifies that the score can be a maximum of K2. Due to lower transformation/dissolution results for tungsten metal (the target substance) than sodium tungstate (the source substance), the resulting toxicity potential would also be expected to be lower, so read-across is appropriate. In addition, read-across is justified because the classification and labelling is less severe for the target substance and the PBT/vPvB profile is the same. Finally, the dose descriptor for the target substance is expected to be sufficiently lower than the source chemical, and read-across to the source chemical is adequately protective. For more details refer to the attached description of the read-across approach.
Justification for type of information:
REPORTING FORMAT FOR THE CATEGORY APPROACH
1. HYPOTHESIS FOR THE CATEGORY APPROACH: The hypothesis is that properties are likely to be similar or follow a similar pattern because of the presence of a common metal ion, in this case tungstate.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES):
Source: Sodium Tungstate
Target: Tungstic acid
3. CATEGORY APPROACH JUSTIFICATION: See Annex 3 in CSR
4. DATA MATRIX: See Annex 3 in CSR
Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
no data
Analytical monitoring:
yes
Details on sampling:
- Concentrations: A volume of 10 mL was collected from each control and test substance treatment at 0 and 72 hours and transferred to a culture tube. The samples at 0 hour were collected from the parent solutions and the samples at 72 hours were collected after combining replicate solutions by treatment. An abiotic replicate of the lowest test substance treatment was also sampled at 72 hours.
- Sampling method: 10 mL taken from the parent solutions at time 0, and from replicate composites at 72 hours. Samples were centrifuged for 15minutes and a 0.05 mL volume of the supernatant was taken, diluted as necessary with 2 % HNO3, capped and shaken to mix, and analyzed by ICP-MS.
- Sample storage conditions before analysis: Room temperature
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 0.0200 g sodium tungstate was dissolved in 1,000 mL of freshwater algal nutrient medium. This primary standard was used as the highest test substance treatment and the five lower test substance treatments, each at a volume of 500 mL, were prepared individually using appropriate volumes of the primary standard and test medium.
- Controls: Algal nutrient medium only.

- The medium was prepared by the addition of appropriate reagent grade salts to autoclaved ABC reagent water. ABC reagent water is produced by passing reverse-osmosis water through a series of deionization tanks, a laboratory water purification system consisting of carbon, de-mineralization, and organic adsorption cartridges, and then through a 0.2-um filter. After preparation, the medium was pH-adjusted to 7.5 ± 0.1 using 0.1 N NaOH and filtered through a 0.45-um Millipore® filters.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella supcapitata (formerly known as Selenastrum capricornutum)
- Source (laboratory, culture collection): Department of Botany, Culture Collection of Algae, University of Texas at Austin
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: Cloned from existing culture derived from parent stock (identified as Selenastrum capricornutum)

ACCLIMATION
- Acclimation period: None necessary; culture media and conditions the same as test conditions.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
None
Hardness:
n/a
Test temperature:
22.9-24.0 °C
pH:
7.5-7.9 SU
Dissolved oxygen:
n/a
Salinity:
n/a
Nominal and measured concentrations:
Nominal- Control, 0.39, 0.85, 1.9, 4.1, 9.1, and 20 mg/L
Mean calculated concentrations-
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 250-mL Erlenmeyer flasks with foam stoppers and labeled with study number, treatment, replicate, and grid position. Each test substance treatment was replicated three times.
- Initial cells density: 5x10E3 cells/mL
- Control end cells density: 113x10E4; 224 times initial density
- No. of vessels per concentration (replicates): 3; 4 (which included the additional replication of the lowest test substance treatment.
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Autoclaved ABC reagent water; Deionized reverse osmosis water passed through carbon, de-mineralization, and organic absorbtion cartridges and then a 0.2 um filter.
- Culture medium different from test medium: No

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: Adjusted to 7.5 ± 0.1 with 0.1 N NaOH and filtered through 0.45 um filter
- Photoperiod: Continuous light
- Light intensity and quality: 7,923-8,290 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Growth rate and Yield, 24 hours
- Determination of cell concentrations: Direct microscopic counting with a hemacytometer

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Approx 2.2
- Justification for using less concentrations than requested by guideline:
- Range finding study
- Test concentrations: Range-finding test #1: Nominal concentrations of 0 (control), 0.010, 0.10, 1.0, 10, and 100 mg/L
Range-finding test #2: Nominal concentrations of 0 (control), 0.10, 1.0, 5.0, 10, and 50 mg/L
- Results used to determine the conditions for the definitive study: Yes
Reference substance (positive control):
no
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 17.7 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Remarks:
sodium tungstate
Basis for effect:
growth rate
Remarks on result:
other: 95 % CL could not be calculated
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
13.8 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Remarks:
sodium tungstate
Basis for effect:
growth rate
Remarks on result:
other: 95 % CL 13.0-14.6 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
5.76 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Remarks:
sodium tungstate
Basis for effect:
growth rate
Remarks on result:
other: 95 % CL 4.94-6.58 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.812 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Remarks:
concentration sodium tungstate
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
7.35 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Remarks:
sodium tungstate
Basis for effect:
biomass
Remarks on result:
other: 95 % CL 6.58-8.12 mg/L
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
2.02 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Remarks:
sodium tungstate
Basis for effect:
biomass
Remarks on result:
other: 95 % CL 1.54-2.50 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.95 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Remarks:
sodium tungstate
Basis for effect:
biomass
Remarks on result:
other: 95 % CL 0.626-1.27 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.812 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Remarks:
sodium tungstate
Basis for effect:
biomass
Details on results:
The number of algal cells in the control at test termination was greater than 16 times the number initially inoculated to verify logarithmic phase growth. The coefficient of variation for daily growth rates in the control replicates during the course of the test did not exceed 35%. The coefficient of variation of average specific growth rates during the whole test period in control replicate did not exceed 7%. Since the study was not conducted as a limit test, there were at least one test concentration exhibiting less than 50% decrease in growth and once concentration exhibiting greater than 50% decrease in growth relative to the control. The pH in the control did not increase more than 1.5 units during the study.
Reported statistics and error estimates:
All statistical analyses were performed with SAS software. NOEC values were calculated using ANOVA and Dunnett's tests. Shapiro-Wilk's and Levene'stests were conducted to test for normality and homogeneity of variance. EC estimates were calculated using a logistic model. Parametric analyses were performed on the 72-hour growth rate and yield data. Non-parametric analyses were performed on the 24- and 48-hour growth rate and yield data.
Conclusions:
The 72 hour EC50 for growth rate for P.subcapitata was >17.7 mg sodium tungstate/L, while the EC10 for growth rate was 5.76 mg sodium tungstate/L, with 95 % confidence limits of 4.94 and 6.58 mg/L. The 72 hour EC50 for yield was 7.35 mg/L, with 95 % confidence limits of 6.58 and 8.12 mg/L.
Executive summary:

No aquatic algae invertebrate toxicity data of sufficient quality are available for tungstic acid (target substance). However, aquatic algae invertebrate toxicity data are available for sodium tungstate (source substance), which will be used for read-across. Due to lower water solubility and lower toxicity for the target substance compared to the source substance, the resulting read-across from the source substance to the target substance is appropriate as a conservative estimate of potential toxicity for this endpoint. In addition, read across is appropriate because the classification and labelling is more protective for the source substance than the target substance, the PBT/vPvB profile is the same, and the dose descriptors are, or are expected to be, lower for the source substance. For more details, refer to the attached description of the read-across approach.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
2009-07-24 to 2010-02-11
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
GLP and Guideline study without deviations. Well documented. The reliability of this study for the substance tested is a K1, but in application of read-across to a different substance ECHA’s guidance specifies that the score can be a maximum of K2. Due to lower transformation/dissolution results for tungsten metal (the target substance) than sodium tungstate (the source substance), the resulting toxicity potential would also be expected to be lower, so read-across is appropriate. In addition, read-across is justified because the classification and labelling is less severe for the target substance and the PBT/vPvB profile is the same. Finally, the dose descriptor for the target substance is expected to be sufficiently lower than the source chemical, and read-across to the source chemical is adequately protective. For more details refer to the attached description of the read-across approach.
Justification for type of information:
REPORTING FORMAT FOR THE CATEGORY APPROACH
1. HYPOTHESIS FOR THE CATEGORY APPROACH: The hypothesis is that properties are likely to be similar or follow a similar pattern because of the presence of a common metal ion, in this case tungstate.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES):
Source: Sodium Tungstate
Target: Tungstic acid
3. CATEGORY APPROACH JUSTIFICATION: See Annex 3 in CSR
4. DATA MATRIX: See Annex 3 in CSR
Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Principles of method if other than guideline:
N/A
GLP compliance:
yes
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
N/A
Analytical monitoring:
yes
Details on sampling:
- Concentrations: All concentrations
- Sampling method: 10 ml was collected from each test solution and control at 0 and 72 hours
- Sample storage conditions before analysis: no data
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A primary stock solution was prepared in reagent water at a concentration of 2.45 mg sodium tunstate./ml. The solution was stored at room temperature when not in use.
- Controls: algal medium only
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): none
- Evidence of undissolved material (eg precipitate, surface film, etc): All sodium tungstate test solutions appeared clear and colorless, with no visible precipitates, surface films, or undissolved test substance at initiation and throughout the test.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Source (laboratory, culture collection): Original stock culture obtained from the Department of Botany, Culture Collection of Algae, University of Texas at Austin, Jan 13, 2009.
- Age of inoculum (at test initiation): 4 days
- Method of cultivation: The prepared cultures were maintained in a temperature-controlled environmental chamber under continuous light. Periodically, new cultures were cloned from an existing culture derived from the parent stock. All cultures were maintained under the same conditions as those used for testing.


ACCLIMATION
- Acclimation period: N/A
- Culturing media and conditions (same as test or not): same as test
- Any deformed or abnormal cells observed: no data
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
none
Hardness:
no data
Test temperature:
22.8-23.8 °C
pH:
7.4-8.4
Dissolved oxygen:
no data
Nominal and measured concentrations:
Nominal-Control, 13, 25, 50, 100, and 200 mg sodium tungstate/L
Initial measured- Control, 10.8, 21.4, 43.5, 87.9, and 173 mg sodium tungstate/L
Mean measured- Control, 10.9, 21.9, 41.6, 84.5 and 168 mg sodium tungstate/L
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 250 ml Erlenmeyer flasks with foam stoppers
- Aeration: no
- Initial cells density: 5000 cells/ml
- Control end cells density: 750000 cells/ml
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): N/A


GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition if non-standard medium was used: standard


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ABC well water passed through RO and then deionization tanks, a laboratory water purification system consisting of carbon, demineralization and organic absorption cartridges, and then passed through a 0.2 um filter. The water was autoclaved before use in preparation of algal medium. Characteristics below are for the ABC well water.
- Total organic carbon: no data
- Particulate matter: no data
- Metals: Ca- 83.1, Mg-33.1, K-7.93, Na-32.2 mg/L
- Pesticides: BDL
- Chlorine: no data
- Alkalinity: no data
- Ca/mg ratio: 1:2.5
- Conductivity: no data
- Culture medium different from test medium: no
- Intervals of water quality measurement: yearly, primary measurements taken at test initiation and test termination


OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: yes, to 7.5 with 0.1N HCl
- Photoperiod: continuous light
- Light intensity and quality: cool white fluorescent, 7936-8479 lux
- Salinity (for marine algae): N/A


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : growth rate and cell density, 24 h
- Determination of cell concentrations: direct microscopic counting with a hemacytometer
- Chlorophyll measurement: no
- Other: none


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Justification for using less concentrations than requested by guideline: N/A
- Range finding study: Not performed
Reference substance (positive control):
no
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
>= 54.1 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Remarks:
calculated sodium tungstate concentrations
Basis for effect:
growth rate
Remarks on result:
other: 95% CL 41.1 to 67.1
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
52.9 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
other: calculated sodium tungstate concentrations
Basis for effect:
growth rate
Remarks on result:
other: 95% CL 40.5-65.3 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
< 10.9 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
other: calculated sodium tungstate concentrations
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
< 10.9 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
other: calculated sodium tungstate concentrations
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
< 10.9 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
other: calculated sodium tungstate concentrations
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: none
- Any observations (eg precipitation) that might cause a difference between measured and nominal values: none
- Effect concentrations exceeding solubility of substance in test medium: none
Results with reference substance (positive control):
N/A
Reported statistics and error estimates:
NOEC values were estimated using one-way ANOVA procedure and two-tailed Dunnett's test. Shiro-Wilke's and Levene's tests were also used as appropriate. EC values were calculated using a logistic model. All statistical analysis was conducted with SAS software.
Validity criteria fulfilled:
yes
Conclusions:
The 72 hour ErC50 of the test substance to P.subcapitata was found to be 52.9 mg sodium tungstate/L, with 95% confidence limits of 40.5-65.3 mg sodium tungstate/L. The 72 hour NOErC was <10.9 mg sodium tungstate/L.
Executive summary:

No aquatic algae invertebrate toxicity data of sufficient quality are available for tungstic acid (target substance). However, aquatic algae invertebrate toxicity data are available for sodium tungstate (source substance), which will be used for read-across. Due to lower water solubility and lower toxicity for the target substance compared to the source substance, the resulting read-across from the source substance to the target substance is appropriate as a conservative estimate of potential toxicity for this endpoint. In addition, read across is appropriate because the classification and labelling is more protective for the source substance than the target substance, the PBT/vPvB profile is the same, and the dose descriptors are, or are expected to be, lower for the source substance. For more details, refer to the attached description of the read-across approach.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Study period:
3 June 2003 - 6 June 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Well documented, scientifically sound study that was conducted in accordance with GLP and OECD 201 & EC guidelines with no deviation to the protocols.
Justification for type of information:
1. HYPOTHESIS FOR THE CATEGORY APPROACH: The hypothesis is that properties are likely to be similar or follow a similar pattern because of the presence of a common metal ion, in this case tungstate.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES):
Source: Tungsten oxide
Target: Tungstic acid
3. CATEGORY APPROACH JUSTIFICATION: See Annex 3 in CSR
4. DATA MATRIX: See Annex 3 in CSR
Reason / purpose for cross-reference:
reference to other study
Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
-Water solubility: 118 mg/L (solubility test (NON-GLP) at DR.U.NOACK-LABORATORIUM showed no complete solubility at 100 mg/L under test conditions)
- log Pow: Not specified
- Density: Not specified
- Stability in water: Not specified
- pH-value: Approximately 6 at a concentration of 100 g/L in aqueous suspension
Analytical monitoring:
not specified
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- A saturated solution was tested, prepared with 100 mg/L and dilution water. The test solution (1 L) was shaken for 24 hours at 20 rpm (rotating shaker 3040, GFL) in a 1 L brown glass bottle. Undissolved particles of the test item were removed by filtration.
- The definitive test was performed with 1 dilution level and 3 replicates.
- Controls: A control with dilution water (without test item, six replicates) was tested under the same test conditions as the test group.
- Self Fluorescence: No self fluorescence has been found in the saturated solution in the definitive test.
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
Origin:
Sammlung von Algenkulturen (SAG),
Pflanzenphysiologisches lnstitut der Universität Göttingen,
Nikolausberger Weg 18, D-37073 Göttingen

Date of receipt:
1997-04-07

Method of cultivation:
Fresh stocks were prepared every month on Z-Agar. Light intensity amounted to 35-70 uE/m2/s for 24 h per day.

Culture medium:
Nutrient medium Z according to LÜTTGE et al (1994).
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Hardness:
Nominal: 0.48 mmol Ca+Mg/L (medium)
Test temperature:
23.5-24.2 (mean 23.9) °C
pH:
Test material: 8.04-8.62
Control: 8.15 - 8.66
Dissolved oxygen:
no data
Salinity:
no data
Nominal and measured concentrations:
0 (control) and 3.5 mg/L
Reference substance (nom.): 0.1, 0.2, 0.4, 0.8, 1.6, 3.2 mg/L
Details on test conditions:
- Test volume: 100 mL
- Test container: Erlenmeyer flasks, volume: 250 mL
- Test containers were placed on a rotary shaker and oscillated at approximately 70 rpm.
- Control end cells density: Cell density was measured via Chlorophyll-a-fluorescence (excitation at 435 nm, emission at 685 nm). Each replicate was measured 6-fold. The cell density was measured at the beginning of the test and every 24 hours. Filtrated culture medium was used as ground signal. The pH-value at the beginning of the test was measured out of one additional replicate of the limit concentration and control. At the end it was measured from a pool of all replicates. The room temperature was measured continuously by a hygrothermographe. Light intensities were measured prior to test start. Microscopic evaluation of the cells was determined at the start and end of the incubation. Alga cells were checked for any unusual cell shapes, color differences, differences in chloroplast morphology, flocculations, adherence of algae to test containers or aggregation of alga cells.

GROWTH MEDIUM
- A four day old preculture was used as inoculum. Incubation was performed in 500 mL Erlenmeyer flasks with test medium. For the start of the test the preculture was diluted with test medium to receive an initial cell concentration of approximately 1 xE4 cells/mL in the replicates. All algae were from the same source and have not been used in a previous study.

OTHER TEST CONDITIONS
- Light intensity and quality: Nominally 60-120 uE/m2 s
- Light regime: 24 h/d light
Reference substance (positive control):
yes
Remarks:
Potassium dichromate p.a. (Merck)
Key result
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
> 3.5 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
dissolved
Remarks:
mg tungsten/L
Basis for effect:
biomass
Remarks:
Inhibition of biomass growth
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 3.5 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
dissolved
Remarks:
mg tungsten/L
Basis for effect:
biomass
Remarks:
Inhibition of biomass growth
Key result
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
> 3.5 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
dissolved
Remarks:
mg tungsten/L
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 3.5 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
dissolved
Remarks:
mg tungsten/L
Basis for effect:
growth rate
Details on results:
-Microscopic evaluation of the cells at the start and end of the incubation period revealed no morphological abnormalities.
-Water quality parameters of pH-value, measured at 0 and 72 h, and room temperature, measured continuously, met the guideline requirements.
Results with reference substance (positive control):
- Results with reference substance valid? Yes

Reported statistics and error estimates:
The NOEC and LOEC were determined by calculation of statistical significance of biomass integrals and growth rates. One Way Analysis of Variance (ANOVA) (growth rates) and DUNNETT'S test (biomass integrals) were carried out for the determination of statistically significant differences compared to control replicates. When running a One Way Analysis of Variance, a Normality Test and an Equal Variance Test were done first. P-values for both Normality and Equal Variance Tests were 0.05. The a-value for ANOVA was a=0.05.
Validity criteria fulfilled:
yes
Conclusions:
In this study Tungsten oxide blue was found not to inhibit the growth of the freshwater green alga Desmodesmus subspicatus at the saturated solution level of 3.5 mg Tungsten/L (mean measured concentration).
Executive summary:

No aquatic algae toxicity data of sufficient quality are available for tungstic acid (target substance). However, aquatic algae toxicity data are available for tungsten oxide (source substance), which will be used for read-across. Due to lower water solubility and similar toxicity for the target substance compared to the source substance, the resulting read-across from the source substance to the target substance is appropriate as a conservative estimate of potential toxicity for this endpoint. In addition, read-across is appropriate because the classification and labelling is equally protective for the source and target substances, the PBT/vPvB profile is the same, and the dose descriptors are, or are expected to be, lower for the source substance. For more details, refer to the attached description of the read-across approach.

Description of key information

No data on effects on algae / cyanobacteria are availablefor tungstic acid; therefore, data for tungsten trioxide (WO3), tungsten oxide (also known as tungstem blue oxide or TBO) and sodium tungstate were evaluated for read-across. In a 72-h static test with D. subspicatus (green algae) and WO3, no effects were observed at the only concentration tested and, therefore, the 72-h EC0 was found to be ≥1 mg WO3/L for both growth rate and biomass (based on nominal concentrations). Due to the fact that the no mortality occurred in this test and, therefore, a definitive effect concentration does not exist for this assay, data from tungsten oxide were also evaluated for read-across.In a 72-h static assay with tungsten oxide and D. subspicatus, the 72-h NOEC was ≥3.5 mg W/L for both growth rate and biomass inhibition based on mean measured concentrations. A definitive effect concentration was not being obtained; therefore, data from sodium tungstate were evaluated for read-across. Two growth inhibition assays with sodium tungstate and P. subcapitata determined an ErC50 of 52.9 mg sodium tungstate (31.0 mg W/L), an ErC10 of 5.76 sodium tungstate (3.38 mg W/L) and a NOEC (based on growth rate) of 0.812 sodium tungstate (0.476 mg tungsten/L) based on measured concentrations. The ErC10 from this study was considered to be the most appropriate effect concentration and, therefore, will be used for classification and risk characterisation.

Key value for chemical safety assessment

EC50 for freshwater algae:
31 mg/L
EC10 or NOEC for freshwater algae:
3.38 mg/L
EC10 or NOEC for marine water algae:
0.476 mg/L

Additional information

No short-term (ie EC50) algal toxicity data are available for tungstic acid (target substance). However, short-term algal toxicity data are available for tungsten oxide (also known as tungsten blue oxide or TBO), tungsten trioxide (WO3 ) and sodium tungstate, which were evaluated for read-across. Tungstic acid (WO3•H2O) is the hydrated form of WO3. Based on Annex V, hydrates and water free forms (anhydrous) of compounds can be regarded as the same substance for registration purposes. Therefore, tungstic acid will be considered equivalent to WO3. For more details on the read-across rationale to WO3, tungsten oxide, and sodium tungstate, refer to the read-across category approach document (Annex 3).

No long-term (ie NOEC/EC10) algal toxicity data are available for tungstic acid (target substance). However, long-term algal toxicity data are available for tungsten trioxide, tungsten oxide, and sodium tungstate, which were evaluated for read-across. Tungstic acid (WO3•H2O) is the hydrated form of tungsten trioxide (WO3). Based on Annex V, hydrates and water free (anhydrous) forms of compounds can be regarded as the same substance for registration purposes. Therefore, tungstic acid will be considered equivalent to WO3. For more details on the read-across rationale to WO3, tungsten oxide and sodium tungstate, refer to the read-across category approach document (Annex 3).

Data for WO3 were evaluated for this read-across for this endpoint. In a 72-h static test with D. subspicatus (green algae), no effects were observed at the only concentration tested and, therefore, the 72-h EC0 was found to be ≥1 mg WO3/L for both growth rate and biomass (based on nominal concentrations). Due to the fact that no effects occurred in this test data for tungsten oxide were also evaluated for read-across.

Exposure to tungsten oxide in a static 72 -h assay with D. subspicatus caused no observable effects on growth rate and biomass at the only concentration tested and, therefore, the 72-h EC0 and NOEC were both ≥3.5 mg W/L (based on mean measured concentrations). Due to the fact that no effects occurred in this test, data for sodium tungstate were evaluated for read-across.

Two toxicity tests were performed with sodium tungstate using two different concentration ranges in order to bracket the desired test endpoints (EC values and NOEC values) for yield (biomass) and growth rate. In the first study, exposure to sodium tungstate at a concentration range of 10.9 to 168 mg/L (6.4 to 98.5 mg W/L) (mean measured values) resulted in statistically significant reductions in both yield and growth rate at all test concentrations, with inhibition of growth rate ranging from 23% at the lowest test concentration to 65% at the highest test concentration. The NOEC could not be determined, and the EC50 based upon growth rate was estimated to be 52.9 mg sodium tungstate/L (31 mg/L W) (mean measured concentration). In the second study, which tested concentrations of sodium tungstate between 0.35 and 17.7 mg sodium tungstate/L (0.21 and 10.4 mg W/L) (mean measured values), the inhibition of growth rate ranged from 0% at 0.81 mg sodium tungstate/L ( 0.47 mg W/L) to 24% at the highest concentration. The EC50 for growth rate was thus greater than 17.7 mg sodium tungstate/L (10.4 mg W/L).

The NOEC determination was problematic. The highest test concentration in which growth rate was not statistically significantly less than that in the control (ie the NOEC) was 0.81 mg sodium tungstate/L (0.47 mg W/L). This NOEC, however, must be considered as an artifact of the unusually low coefficient of variation (CV) that occurred in this test (0, 1 or 2% CV at all test concentrations). The next highest test concentration, 1.79 mg sodium tungstate/L (1.05 mg W/L), exhibited only a 4% inhibition of growth rate, and only 8% inhibition occurred at 3.83 mg sodium tungstate/L (2.24 mg W/L). These small effects are almost certainly not biologically significant, despite being statistically significant, due to the unusual precision of the test. As stated in Chapter R.10 of ECHA (2008c), when the statistical “power is high, it may occur that biologically unimportant differences are statistically significant“. Further, ECHA (2008c) notes that there has been a recommendation within OECD to phase out the use of the NOEC, which also has the disadvantage of being highly dependent upon the selection of test concentrations and does not allow for the estimate of a confidence interval. An alternative, and according to ECHA (2008c), the preferred method of expressing chronic toxicity, is the use of the EC10. The advantage of the use of a regression-based endpoint such as the EC10 is that information from the entire concentration-effect relationship is used and confidence intervals can be calculated. The ErC10 (ie the concentration bringing about a 10% inhibition of growth rate), as determined from the results of the second test with sodium tungstate, was 5.76 mg sodium tungstate/L (3.38 mg W/L) with 95% confidence limits of 4.94 (2.89 mg W/L) and 6.58 (3.86 mg W/L) mg /L. This value is considered to be more appropriate for estimating chronic toxicity than the NOEC for this endpoint.