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Long-term toxicity to aquatic invertebrates

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Reference
Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2008-10-06 to: 2009-03-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Well documented, scientifically sound study conducted in accordance to GLP and OECD guideline 211 with no deviation to the protocol.
Qualifier:
according to guideline
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:
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.

Description of key information

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.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
50 mg/L

Additional information