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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP guideline study. Reliability assigned by a secondary source (OECD SIDS Initial Assessment Report). Only secondary source reviewed, as primary study was not available.
Qualifier:
according to
Guideline:
other: Directive 92/69/EEC, C.3, comparable to OECD TG 201
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:
no
Vehicle:
not specified
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
Test Organism:
- Common name: Scenedesmus subspicatus (new name: Desmodesmus subspicatus)
- Strain: CHODAT
-Stock culture: non-axenic strain obtained from the Institute of Plant Physiology at the University of Göttingen (Germany); exponentially-growing stock cultures maintained under constant temperature conditions (23 +/- 2 °C), light intensity (120 µE x m-2 x s-1); nutrient medium renewed once a week; cell density measured using a microcell counter (Sysmex F-300).
-Preculture: set up three days before test started; grown under identical exposure conditions except from the use of a 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:
N/A
Test temperature:
23 +/- 2 degree C
pH:
8.0 (at test start) - 10.4 (after 72 hours)
Dissolved oxygen:
no data
Salinity:
no data
Nominal and measured concentrations:
-Test concentration: 1 mg/L nominal
Details on test conditions:
TEST SYSTEM:
-Test vessel: 300 mL Erlenmeyer flasks with stoppers.
-Culturing apparatus: continuous uniform illumination, shaking facility.
-Light intensity: 120 uE x m-2 x s-1.
-Dilution water: deionized water "Millipore".
-Test cultures: final cell density 10E+4 cells/mL
-To prepare the stock solution an amount of the test substance was weighed into water, treated for 1 hour in a ultrasonic bath, afterwards stirred for 24 hours on a magnetic stirrer and finally filtered.
-Replicates: 3 (concentration), 6 (control)
-Test parameter: growth rate/ biomass
-Cell density measurement: via microcell counter (Sysmex F-300) Digitana.
Reference substance (positive control):
no
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 1 mg/L
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC0
Effect conc.:
>= 1 mg/L
Basis for effect:
growth rate
Details on results:
At a concentration of 1 mg/L no inhibition of algae growth was observed. An EC50 of >1 mg/L was derived from the test results.
The cell growth increased more than 16-fold after 72 h
Reported statistics and error estimates:
no data
Conclusions:
An EC50 of >1 mg/L was derived from the test results.
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 carbide (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 the 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:
1. HYPOTHESIS FOR THE ANALOGUE 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: Tungsten Carbide
3. CATEGORY APPROACH JUSTIFICATION: See Annex 1 in CSR
4. DATA MATRIX: See Annex 1 in CSR
Reason / purpose:
read-across: supporting information
Qualifier:
according to
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 degrees 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 and cyanobacteria toxicity to aquatic invertebrates data of sufficient quality are available for tungsten carbide (target substance). However, aquatic algae and cyanobacterias data are available for sodium tungstate (source substance), which will be used for reading 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 read-across category approach included in the Category section of this IUCLID submission and/or as an Annex in the CSR.

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:
other: see 'Remark'
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 carbide (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 the 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:
1. HYPOTHESIS FOR THE ANALOGUE 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: Tungsten Carbide
3. CATEGORY APPROACH JUSTIFICATION: See Annex 1 in CSR
4. DATA MATRIX: See Annex 1 in CSR
Reason / purpose:
read-across: supporting information
Qualifier:
according to
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 (e.g. 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 deg 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- 83-88% of nominal
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:
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:
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:
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 (e.g. 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 and cyanobacteria toxicity to aquatic invertebrates data of sufficient quality are available for tungsten carbide (target substance). However, aquatic algae and cyanobacterias data are available for sodium tungstate (source substance), which will be used for reading 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 read-across category approach included in the Category section of this IUCLID submission and/or as an Annex in the CSR.

Description of key information

In a 72-hr algal growth inbibition test with tungsten carbide, no toxic effects at the highest concentration tested (1 mg/L). Therefore, the ErC50 could not be ascertained and read-across to sodium tungstate was used for this endpoint.  Two growth inhibition tests using sodium tungstate, determined an ErC50 of 52.9 mg sodium tungstate (31.0 mg tungsten/L), an ErC10 of 5.76 sodium tungstate (3.38 mg tungsten/L), and a NOEC of 0.812 sodium tungstate (0.476 mg tungsten/L). The ErC10 from this study was considered to be more appropriate and will be carried forward to the classification and risk characterization.

Key value for chemical safety assessment

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

Additional information

Due to lower transformation/dissolution results for tungsten carbide (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 the 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.

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 the various growth parameters (yield and growth rate).

In the first study with sodium tungstate, conducted at a concentration range of 10.9 to 168 mg/L (mean measured values), statistically significant reductions in both yield and growth rate were observed 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 (mean measured concentration). In the second study, conducted at a concentration range of 0.35 to 17.7 mg/L (mean measured values), inhibition of growth rate ranged from 0% at the second lowest concentration (0.81 mg/L) to 24% at the highest concentration. The EC50 for growth rate was thus greater than 17.7 mg/L. The highest test concentration in which growth rate was not statistically significantly less than that in the control, i.e., the NOEC, was 0.812 mg/L. This NOEC, however, must be considered as an artifact of the unusually low coefficient of variation that occurred in this test (0, 1 or 2% CV at all test concentrations). The next highest test concentration to the NOEC, 1.79 mg/L, exhibited only a 4% inhibition of growth rate, and only 8% inhibition occurred at 3.83 mg/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 the ECHA guidance R10 (ECHA, 2008), when the statistical “power is high, it may occur that biologically unimportant differences are statistically significant“. 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, 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 EC10, as determined from the results of the second test with sodium tungstate, was 5.76 mg sodium tungstate/L with 95% confidence limits of 4.94 and 6.58 mg/L. This value is considered to be more appropriate for estimating a chronic toxicity than the NOEC for this endpoint.