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Toxicity to soil macroorganisms except arthropods

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Endpoint:
toxicity to soil macroorganisms except arthropods: long-term
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Remarks:
Similar to ISO 11268-1:1998 guideline study but with insufficient information available on background levels of tungsten, other metals, and environmental toxicants to assess toxicity of tungsten alone to evaluate toxicity results.
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
ISO 11268-1 (Effects of Pollutants on Earthworms. 1. Determination of Acute Toxicity Using Artificial Soil Substrate)
Version / remarks:
modified
GLP compliance:
not specified
Analytical monitoring:
no
Vehicle:
no
Details on preparation and application of test substrate:
- Method of mixing into soil (if used): mixed manually with tungsten powder
- Controls: Non-treated highway soil
Test organisms (species):
Eisenia fetida
Animal group:
annelids
Details on test organisms:
TEST ORGANISM
- Common name: Earthworm
- Source: Worm Farm Inc. Monroe Twp, NJ
- Age at test initiation (mean and range, SD): Not specified
- Weight at test initiation (mean and range, SD): Not specified

ACCLIMATION
- Acclimation period: Not specified
Study type:
extended laboratory study
Substrate type:
other: soil collected adjacent to highway exit
Limit test:
no
Total exposure duration:
80 d
Post exposure observation period:
2 days
Test temperature:
Room temperature
pH:
Varies with tungsten concentration, 4.80-7.25.
Moisture:
Not specified; DI water added daily.
Details on test conditions:
TEST SYSTEM
- Test container (material, size): glass jar, covered with filter paper
- Amount of soil or substrate: 750 g
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 3 or 4
- No. of replicates per control: 3 or 4
- One set of replicates ammended with 10,000 mg/kg CaCO3 in addition to the toxicant to buffer pH change.

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Geographic location: Highway soil (Exit 40, Hwy 78, The Plainfields, NJ)
- Pesticide use history at the collection site: Not specified
- Sampling depth (cm): 1-5 cm
- Organic carbon (%): 2 %
- Pretreatment of soil: Homogenized, plant roots and large particles removed, sieved through a 1 mm sieve.
- Storage (condition, duration): 4degree C until use- more than 1 year.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Mortality and tissue concentration of tungsten.

VEHICLE CONTROL PERFORMED: no


Nominal and measured concentrations:
Nominal-Control, 10, 100 and 10,000 mg/kg tungsten.
Reference substance (positive control):
no
Duration:
80 d
Dose descriptor:
NOEC
Effect conc.:
100 mg/kg soil dw
Nominal / measured:
nominal
Basis for effect:
mortality
Duration:
80 d
Dose descriptor:
LOEC
Effect conc.:
10 000 mg/kg soil dw
Nominal / measured:
nominal
Basis for effect:
mortality

Initial Test

- All Controls and worms in the 10 and 100 mg/kg level survived for 80 days.

- All worms in the 10,000 mg/kg soil died after 80 days.

- All worms in all concentrations survived after 80 days in the soils amended with 10,000 mg/kg CaCO3.

-The pH of the soil decreased over time from its initial value of 5.6 in all soils without a CaCO3 supplement. The largest drop observed for the highest W concentration where a final soil pH of 4.80 was measured. However, for the replicates containing the same W concentration and supplemented with CaCO3, the pH after 80 days was 7.25.

- Worms took up and accumulated tungsten in their tissue.

- Dry weight tissue concentrations were 1.52, 3.24, and 193.2 mg/kg tungsten for the 10, 100, and 10,000 mg/kg concentrations, respectively.

Second Test

- Soils were amended with tungsten as above, and aged 1 year before the introduction of the worms.

-The results of the second test are consistent with the results of the first test where all worms survived 14 days of exposure to soils amended with 10 -1000 mg/kg tungsten and all of them died off at the 10000 mg/kg level.

- Dry weight tissue concentrations were 3.45 and 25.9 mg/kg tungsten for the 10 and 100 mg/kg concentrations, respectively.

Conclusions:
Results suggest that metallic tungsten in soils has some adverse environmental effects at or above the threshold level of 1% mass basis ( i.e. 10000 mg/kg). Metallic tungsten levels of 1% (mass basis) resulted in the death of red worms.
Endpoint:
toxicity to soil macroorganisms except arthropods: long-term
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
2008-12-30 to 2009-07-23
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 222. A deviation in the protocol included the prepared diet was provided first to the test organisms on the day of test initiation and not one day later as specified by the protocol. There was no effect on the integrity of the study. 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 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:
REPORTING FORMAT FOR THE CATEGORY APPROACH
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 metal
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 222 (Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei))
Deviations:
yes
Remarks:
the prepared diet was provided first to the test organisms on the day of test initiation and not one day later as specified by the protocol but this did not affect the integrity of the study.
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:
no data
Vehicle:
no
Details on preparation and application of test substrate:
A 2.69 mg/mL primary standard was prepared on March 4, 2009, by transferring 5.3755 g of sodium tungstate (5.73 g corrected for purity) to a 2-L volumetric flask and bringing the flask to volume with deionized water. The 2.69 mg/mL primary standard was serially diluted with 2,000 mL volumes of deionized water to prepare working standards at concentrations of 0.169, 0.338, 0.675, and 1.35 mg/mL. A 931 mL aliquot of each working standard solution and the primary standard were added to 2,533.8 grams of air dry soil (equivalent to 2.5 kg of oven dry soil). The soil from the control and each treatment batch was mixed for approximately two to three minutes in a Hamilton Beach commercial mixer. This produced dosed soils with nominal concentrations of 0 (control), 62.9, 126, 251, 503, and 1,000 mg sodium tungstate/kg dry soil and a hydration level of approximately 60% of the soil water holding capacity (i.e., 38.6% moisture). Approximately 831 g of hydrated soil (approximately 600 g dry weight equivalent) was added to each replicate test vessel.
Test organisms (species):
Eisenia fetida
Animal group:
annelids
Details on test organisms:
TEST ORGANISM
- Common name: Earthworms (clitellate adults)
- Source: Vicker’s Farms, Orlando, Florida
- Age at test initiation (mean and range, SD): The adult animals selected for acclimation and testing were approximately 3 months of age with a developed clitellum.
- Weight at test initiation (mean and range, SD): 337 to 595 mg wet weight; initial mean weight of the treatment worms used in this exposure ranged from 411 to 450 mg/worm, based upon the replicate weights of ten worms.
- Weight of control worms at test initiation (mean and range, SD): initial mean weight of the control earthworms used for the definitive exposure was 462 mg/worm based upon the mean of the replicate weights of ten worms in each control replicate.
- The worms were maintained in commercial worm bedding prior to testing.
- An invertebrate diet similar to the diet utilized in the test was provided during the culture period.


ACCLIMATION
- Acclimation period: The adult animals selected for acclimation and testing were approximately 3 months of age with a developed clitellum. Four days prior to test initiation, adult earthworms were removed from the culture bedding and placed in artificial soil to acclimate the worms to the test conditions.
- Acclimation conditions (same as test or not): Same as test
- Health during acclimation (any mortality observed): No mortality observed
Study type:
extended laboratory study
Substrate type:
artificial soil
Limit test:
no
Total exposure duration:
56 d
Post exposure observation period:
no data
Test temperature:
19 to 21 degree C
pH:
Soil pH (day 0): 6.09 to 6.45
Soil pH (day 56): 5.59 to 5.73
Moisture:
Soil Moisture (day 0): 37.04 to 38.92%
Soil Moisture (day 56): 44.60 to 48.22%
Soil Moisture at 60% WHC: 38.6%
Details on test conditions:
TEST SYSTEM
- Test container (material, size): The test chambers were glass containers that have an approximate volume of 1.8 L.
- Amount of soil or substrate: A total of 500 or 600 g (dry weight) of prepared soil was added to each test chamber.
- No. of organisms per container (treatment): 10; Each replicate test chamber contained ten worms for a total of 40 worms per test substance treatment.
- No. of replicates per treatment group: four
- No. of replicates per control: Eight replicate test chambers were prepared for the control treatment; Each replicate test chamber contained ten worms for a total of 80 control worms.

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Composition (if artificial substrate): The artificial soil used for both definitive exposures was prepared to approximate a sandy loam soil by mixing the following ratio of constituents: 70% silica sand, 20% kaolin clay, and 10% sphagnum peat based on dry weight equivalents. Calcium carbonate, CaCO3, was added to the soil to adjust the pH to fall within the 6.5 ± 0.5 range specified by the protocol. The peat moss was sieved to a finely ground consistency and did not contain any visible plant remains.
- Organic carbon (%): low total content
- Maximum water holding capacity (in % dry weigth): The water holding capacity (WHC) was determined to be 64.4 mL per 100 grams dry soil for the soil used in the definitive exposure.
- Storage (condition, duration): After preparation, the soil was stored at room temperature.

OTHER TEST CONDITIONS
- Photoperiod: 16-hr light to 8-hr dark
- Light intensity: 560.8 to 708.1 lux

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: A 28-day range-finding test was conducted using nominal concentrations of 0 (control), 1.0, 10, 100, and 1,000 mg/kg dry soil. Two replicates were used for the control and test substance treatments with 10 worms added per replicate.
- Results used to determine the conditions for the definitive study: After 28 days there was no mortality or sublethal effects observed in the control or test substance treatments. At the termination of the range-finding test there was an average of 34, 33, 31, 30, and 28 cocoons per replicate for the control, 1.0, 10, 100, and 1,000 mg/kg dry soil treatments, respectively. Based on these results, a definitive test was performed at target nominal
concentrations of 0 (control), 62.5, 125, 250, 500, and 1,000 mg/kg dry soil.
Nominal and measured concentrations:
Calculated Nominal Soil Concentrations: 0 (Control), 62.9, 126, 251, 503, and 1,000 mg/kg dry soil
Reference substance (positive control):
yes
Remarks:
Tungsten ICP Standard
Duration:
56 d
Dose descriptor:
NOEC
Effect conc.:
1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
reproduction
Remarks on result:
other: The highest concentration tested
Duration:
56 d
Dose descriptor:
LOEC
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
reproduction
Remarks on result:
other: The highest concentration tested
Duration:
56 d
Dose descriptor:
EC10
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
reproduction
Remarks on result:
other: The highest concentration tested
Duration:
56 d
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
reproduction
Remarks on result:
other: The highest concentration tested
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: survival
Remarks on result:
other: The highest concentration tested
Duration:
28 d
Dose descriptor:
LOEC
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: suvival
Remarks on result:
other: The highest concentration tested
Duration:
28 d
Dose descriptor:
EC10
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: survival
Remarks on result:
other: The highest concentration tested
Duration:
28 d
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: survival
Remarks on result:
other: The highest concentration tested
Details on results:
- Mortality at end of exposure period: After 28 days of exposure to sodium tungstate, percent mortality of the adult worms was 0% in the control and all treatment.
- Other biological observations: Observations of general health and behavior of the worms were also noted (i.e., normal, lethargic, softness, absence of burrowing, and discoloration). All of the live earthworms were normal in appearance and behavior with the exception of five lethargic worms in the 1,000 mg/Kg dry soil treatment. No abnormal burrowing or avoidance behaviors were recorded during testing.
- The weights of surviving earthworms were measured on a replicate basis. The control worms gained an average of 2.164 g per replicate or 47% in replicate mass during the 28-day exposure . The treatment worms also gained weight during the study. The mean replicate weight gain of the surviving treatment animals ranged from 2.004 to 2.671 grams. The mean percent weight gain for the treatment animals ranged from 47 to 65% in replicate animal mass over the initial 28 days of exposure.
- At test termination, the number of juvenile worms will be determined by rinsing the test soil through a series of sieves (e.g., 710, 500, and 425 um mesh), collecting and enumerating the animals retained within the sieves.
- The average reproduction for the control animals was 547 juveniles per replicate. The percent coefficient of variation (%CV) for the control data was 14%. The average reproduction values were 447, 447, 483, 459, and 518 juveniles for nominal concentrations of 62.9, 126, 251, 503, and 1,000 mg sodium tungstate per kilogram dry soil treatments, respectively. There was no statistically significant reduction (p < 0.05) in the reproductive output of the adult worms exposed to the test substance. The EC10 and EC50 for reproduction was > 1,000 mg sodium tungstate/kg dry soil, the highest concentration tested. The NOEC and LOEC for reproduction was 1,000 and > 1,000 mg sodium tungstate/kg dry soil, respectively.
- The survival of the control adult animals was 100%, which met the acceptability of the test protocol and guidance document. Each control replicate containing 10 adult worms after the first 28 days of exposure produced more than the minimum acceptability criterion of 30 juvenile worms and the %CV value for the control reproduction was less than the maximum acceptability of 30% as specified by the protocol and the test guideline.
Results with reference substance (positive control):
- The analytical reference substance, Tungsten ICP Standard (1,000 ppm W) was received from Ricca Chemical Company on September 11, 2008, and was stored at room temperature. The sample was assigned ABC reference no. PS-22260. A certificate of analysis reported the concentration of solution to be 1,000 ppm W (Appendix A). This material was used to prepare analytical reference standard solutions. All solutions were corrected for the purity of the reference substance.

The reference substance (i.e., toxic standard) was carbendazim (received from Chem Service (West Chester, Pennsylvania)). The certificate of analysis showed the purity to be 99.4%
Reported statistics and error estimates:
All statistical analyses were performed using SAS software. Inferences of statistical significance were based upon p <=0.05. Since there was no mortality in the control or treatments the noobserved-effect concentration (NOEC) for the survival data was equal to the highest level tested. The reproduction NOEC value was estimated using a one-way analysis of variance (ANOVA) procedure and a one-tailed Dunnett's test. Prior to the Dunnett's test, a Shapiro-Wilk’s and the Levene’s tests were performed to determine data normality and homogeneity of the treatment variances. The reproductive data met the criterion for normality and homogeneity of variance, therefore a parametric analysis was performed on the reproductive data.

The soil moisture and pH were measured in samples collected from the parent control and treatment soils on day 0. At test termination (study day 56), the moisture content and soil pH were determined from samples of test medium collected from a single replicate test chamber (i.e., replicate A) for all treatment levels. The soil samples were collected prior to the enumeration of the juvenile worms. The pH was measured using a WTW Model pH 330i pH meter. The percent moisture was calculated as the difference between the wet and dry soil samples and presented as percent of the soil dry weight using a Mettler Toledo HR73 Halogen Moisture Analyzer. A continuous temperature record was measured with an electronic data logger in a chamber containing moistened control soil and placed in the water bath. Light intensity was measured at test initiation with a LI-COR Model LI-189 light meter equipped with a photometric sensor.

Validity criteria fulfilled:
yes
Conclusions:
The survival of the control adult animals was 100%, which met the acceptability of the test protocol and guidance document. Each control replicate containing 10 adult worms after the first 28 days of exposure produced more than the minimum acceptability criterion of 30 juvenile worms and the %CV value for the control reproduction was less than the maximum acceptability of 30% as specified by the protocol and the test guideline.

Under the conditions of this study and based upon nominal concentrations, the 56-day reproduction NOEC value was 1,000 mg sodium tungstate/kg dry soil and the estimated EC10 and EC50 value for average reproduction was > 1,000 mg sodium tungstate/kg, the highest concentration tested.
Executive summary:

No soil microorganisms toxicity data of sufficient quality are available for tungsten metal (target substance). However, soil microorganisms toxicity 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 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

The 56-day NOEC for earthworms (Eisenia fetida) found in a test conducted according to OECD 222, under GLP standards, and with analytical verification of the test concentrations was >=1000 mg sodium tungstate/kg soil dw (586 mg W/kg). Other studies with Eisenia fetida were not considered for the risk characterisation, as they were found to be less reliable.

Key value for chemical safety assessment

Additional information

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 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.