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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:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
2008-10-06 to: 2009-03-13
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 211 with no deviation 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 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 211 (Daphnia magna Reproduction 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: 10 mL from each control and treatment from freshly prepared solution on day 0, 9, 14 and 18; and from composites of spent solution on day 2, 11, 16, and 21, and transferred to a culture tube.
- Sampling method: 0.05 mL aliquot of each sample was taken, diluted if necessary with 2% HNO3, capped and shaken to mix.
- 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: 0.2002 g sodium tungstate dihydrate was dissolved in 2 L aged-blended freshwater. Test concentrations were diluted from this solution.
- Controls: Aged-blended freshwater only
- Evidence of undissolved material (e.g. precipitate, surface film, etc): None
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Water flea
- Source: In-house daphnid culture
- Age of parental stock (mean and range, SD): < 24 hours old
- Feeding during test
- Food type: Nanochloropis micro algae and/or the algal species Pseudokirchneriella subcapitata supplemented by an artificial diet that was prepared according to ABC standard operating procedure.
-Amount: 0.58 mL of a 3.0 x 10(7) cells/L, and 0.02 mL of 5mg/mL artificial diet
- Frequency: At least once a day

ACCLIMATION
- Acclimation period: None, because culturing conditions are the same as test conditions.
- Health during acclimation (any mortality observed): No adult mortality observed in the 48 prior to test initiation

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
- Individual adults were isolated in a container with fresh food suspension.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Post exposure observation period:
None
Hardness:
130-160 mg/L CaCO3
Test temperature:
19.2-19.9 degrees C
pH:
8.1-8.5 SU
Dissolved oxygen:
7.2-8.6 mg/L
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal-0, 6.5, 13, 25, 50, and 100 mg/L
Time-weighted Mean Calculated-
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 150 mL glass jars with plastic lids, 80 mL of control and treatment solutions. The test chambers were covered with plastic lids. The test chambers were maintained at 20 ± 1 degree C in a temperature-controlled water bath.
- Aeration: None
- Renewal rate of test solution (frequency): Every 2-3 days
- 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: ABC well water mixed with well water demineralized by reverse osmosis, then aged in a tank containing aquaticorganisms. Water was then passed through a sediment filter, a UV sterilizer, and aerated prior to use.
- Metals: B-0.415 mg/L, Ca-83.1 mg/L, Mg- 33.1 mg/L, K-7.93 mg/L, Na-32.2 mg/L; all others below DL.
- Pesticides: Below DL
- Alkalinity: 144-170 mg/L CaCO3
- Conductivity: 340-410 uS
- Culture medium different from test medium: No
- Intervals of water quality measurement: Daily

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 hr light, 8 hr dark, with 30 min simulated dawn and dusk periods
- Light intensity: 443 lux as measured on day 0 with a LI COR Model LI 189 light meter equipped with a photometric sensor.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Mortality, daily

VEHICLE CONTROL PERFORMED: no

RANGE-FINDING STUDY
- Range-finding study was not performed because previous data indicated low toxicity to aquatic organisms.
Reference substance (positive control):
no
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
immobilisation
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
immobilisation
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
immobilisation
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
92.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks:
Live young per surviving adult
Remarks on result:
other: 95% CL 86.8-97.4 mg/L
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
50 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks:
Live young per surviving adult
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks:
Live young per surviving adult
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth
Remarks:
Mean length per surviving adult
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
50 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth
Remarks:
Mean length per surviving adult
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth
Remarks:
Mean length per surviving adult
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
> 85.1 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Remarks:
mean calculated sodium tungstate concentrations
Basis for effect:
immobilisation
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
> 85.1 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Remarks:
mean calculated sodium tungstate concentrations
Basis for effect:
immobilisation
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
>= 85.1 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Remarks:
mean calculated sodium tungstate concentrations
Basis for effect:
immobilisation
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
78.7 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Remarks:
mean calculated sodium tungstate concentrations
Basis for effect:
reproduction
Remarks:
Live young per surviving adult
Remarks on result:
other: 95% CL= 74.5 to 83.0 mg/L
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
85.1 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Remarks:
mean calculated sodium tungstate concentrations
Basis for effect:
reproduction
Remarks:
Live young per surviving adult
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
44.2 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Remarks:
mean calculated sodium tungstate concentrations
Basis for effect:
reproduction
Remarks:
Live young per surviving adult
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
85.1 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Remarks:
mean calculated sodium tungstate concentrations
Basis for effect:
growth
Remarks:
mean length per surviving adult
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
44.2 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Remarks:
mean calculated sodium tungstate concentrations
Basis for effect:
growth
Remarks:
mean length per surviving adult
Details on results:
Biological Results:
- Mortality of parent animals: 10, 10, 0, 0, 0, and 20% in the 0 (control), 6.5, 13, 25, 50, and 100 mg/L treatments, respectively.
- Mean no. of offspring produced per female: 98, 115, 107, 109, 93, and 40 in the 0 (control), 6.5, 13, 25, 50, and 100 mg/L treatments, respectively.
- Mean lengths of daphnids: 4,2, 4.3, 4.3, 4.3, 4.2, and 3.7 mm in the control, 6.5, 13, 25, 50, and 100 mg/L treatments, respectively

- The mortality of the parent animal in the control did not exceed 20% at the end of the test.  The mean number of live young produced per parent animal surviving at the end of the test was ≥ 60.  The coefficient of variation around the number of living young produced per surviving parent animal in the control was 28%.  This study is classified as acceptable and satisfies the guideline requirement for a reproduction test with Daphnia magna.
Reported statistics and error estimates:
All statistical analyses were performed using SAS software. Inferences of statistical significance were based upon a p = 0.05 unless otherwise noted. The no-observed-effect concentration (NOEC) and lowest-observed-effect concentration (LOEC) for mortality/immobility data were determined by using a one-tailed Dunnett’s test and a Fisher’s exact test with the alternate hypothesis being that the mean for the parameter was reduced in comparison to the control mean. A Hochberg adjustment was used to control the experiment-wise error rate for the Fisher’s test at the same level (p = 0.05). Reproduction and length data were analyzed with a one-way analysis of variance (ANOVA) procedure and a one-tailed Dunnett’s test with the alternate hypothesis being that the mean for the parameter was reduced in comparison to the control mean. Prior to the Dunnett’s test, a Shapiro Wilk’s test and a Levene’s test were conducted to test for normality and homogeneity of variance, respectively, over treatments at each time point. The results from the Shapiro-Wilk’s and Levene’s tests indicated the assumptions of normality and homogeneity of variance were not met for the survival, reproduction, and length data. Therefore, these parameters were analyzed with a non parametric ANOVA and Dunnett’s test on the ranks of the values.Estimates of the EC50 concentration values and their 95% confidence limits for reproduction and length data were calculated by a four-parameter logistic (sigmoid-shaped) model with two parameters fixed (100 and 0% inhibition) that were fit to the data with percent inhibition as the dependent variable and log concentration as the independent variable.

Reported statistics and error estimates (Cont'd):

Estimates of the EC50 concentration values and their 95% confidence limits for immobilization data were calculated using the probit method and Trimmed Spearman-Karber method. When the p value for Goodness of Fit was >0.05 and there was no other evidence of questionable convergence, the probit method was selected for reporting.  When this criterion was not achieved, the Trimmed Spearman-Karber method was selected for reporting.

Conclusions:
The 21 day EC50 for survival of D. magna exposed to sodium tungstate dihydrate (reported as time-weighted mean calculated concentrations) was >85.1 mg/L, with a NOEC of >= 85.1 mg/L, and a LOEC of >85.1 mg/L. The 21 day EC50 based on number of young per surviving adult was 78.7 mg/L (95% CL 74.5-83.0 mg/L) , with a NOEC of 44.2 mg/L, and a LOEC of mg/L. The 21 day EC50 based on the mean length per surviving adult was >100 mg/L, with a NOEC of 50 mg/L, and a LOEC of 100 mg/L.
Executive summary:

No long-term toxicity to aquatic invertebrates data of sufficient quality are available for tungsten carbide (target substance). However, long-term to aquatic invertebrates 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

Tungsten carbide was not tested for chronic toxicity to invertebrates, and read-across to sodium tungstate was used for this endpoint. In a reproduction test using Daphnia magna and testing sodium tungstate, the 21-day NOEC for reproduction and growth was found to be 44.2 mg/L sodium tungstate/L (approximately 26 mg W/L).

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater invertebrates:
26 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 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.