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Ecotoxicological information

Toxicity to soil macroorganisms except arthropods

Administrative data

Endpoint:
toxicity to soil macroorganisms except arthropods: long-term
Type of information:
experimental study
Adequacy of study:
key study
Study period:
not reported.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 207 (Earthworm, Acute Toxicity Tests)
Deviations:
yes
Qualifier:
according to
Guideline:
EPA OPPTS 850.6200 (Earthworm Subchronic Toxicity Test)
Deviations:
yes
Principles of method if other than guideline:
Two deviations occurred from SOP No. ARS-432-R1. The SOP required that each sample be analyzed in duplicate. Each sample was analyzed in triplicate, as specified in the analytical protocol. In addition, the SOP required that a representative sample be labeled as a "lab/reference standard" and be analyzed. Pass or failure of subsequent samples in the study was to be determined by comparison to this original sample. The representative sample was not designated nor analyzed and no comparisons to subsequent samples were made. The pass/fail criterion for the test article in this study was outlined in the analytical protocol and was not based on a "lab/reference standard". The pass/fail criterion in SOP No. ARS-432-R1 was not applicable to this study. These deviations did not affect the quality or integrity of the data generated.
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
The test material consists of 5.79% alpha isomer, 19.2% beta isomer and 74.89% gamma isomer.

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Samples of the test soils were collected on days 0 (test initiation), 28, and 56 (test termination) for analytical confirmation of the concentration of HBCD. The day 0 samples were collected from the parent soils. Homogeneity samples were collected from the top, middle, and bottom of the low and high treatment soils after mixing and hydration and just prior to test initiation. The day 28 and 56 samples were collected from composite samples of each treatment level soil. The soil samples were collected in glass vials, which were hand labeled by treatment with a permanent marker. A paper label, which detailed the sample type, date collected, collector, amount of sample collected, was adhered to the vial over the hand written label. This procedure prevented the destruction of the paper label during the sample collection procedure. During the labeling of the soil samples collected on day 56, the control and level 1 paper labels were accidentally switched. The potential for a switch in the labels for these two samples was noticed during the analytical verification of these samples, which resulted in HBCD concentrations in the control sample at a level anticipated for level 1 and no quantifiable HBCD concentrations in the level 1 sample. There was also no evidence of contamination in the stability spike samples, which were prepared from the same control sample after the analytical samples were collected. After review of the actual sample vials it was confirmed that the hand written labels did not agree to the paper labels for only the control and level 1 samples and that the hand written labels correctly identified these samples.

Storage stability spiked samples were prepared from control soil at nominal concentrations approximating the low and high treatment levels for each sampling time point. A primary spiking solution at a nominal concentration of 50.0 mg/mL was prepared in acetone, which was utilized in the preparation of the high concentration storage stability samples. A dilution of the primary spiking solution in acetone was used to prepare a 0.781 mg/mL solution, which was utilized in the preparation of the low concentrations storage stability samples. The spiking solutions were prepared on April 24, 2002, and were refrigerated when not in use. The nominal concentrations of the low concentration stability samples were 78.1, 78.1, and 78.0 mg/kg dry soil for the 0-, 28-, and 56-day fortifications. The nominal concentrations of the high concentration storage stability samples were 5,000, 4,990, and 4,980 mg/kg dry soil for the 0-, 28-, and 56-day fortifications. The storage stability samples were stored frozen along with the test samples until analysis.

After 28 days of exposure to HBCD, the surviving worms were composited by treatment and placed in glass dishes containing wet paper towels in order to allow the worms to adequately purge their gut contents. After 48 hours, the worms were rinsed in deionized water, gently blotted dry, transferred to labeled glass vials, and were stored frozen until analysis.

All of the test soil and worm tissue samples were packed with dry ice and shipped frozen to Wildlife International, Ltd., Easton, Maryland. The soil and worm samples collected from days 0 and 28 were frozen upon receipt. The soil samples collected on day 56 were received at ambient temperatures due to complete sublimation of the dry ice during shipment.

Test substrate

Vehicle:
no
Details on preparation and application of test substrate:
The soils were prepared by directly adding 0.1766, 0.3534, 0.7044, 1.4063, 2.8125, 5.6250, and 11.2500 g of pulverized HBCD to 2.305 kg air dried soil (2.25 kg of dry soil), which resulted in nominal soil concentrations of 78.5, 157, 313, 625, 1,250, 2,500, and 5,000 mg HBCD per kg dry soil.

Test organisms

Test organisms (species):
Eisenia fetida
Animal group:
annelids
Details on test organisms:
TEST ORGANISM
- Common name:
Earthworm

- Source:
Vicker's Farms, Orlando, Florida

- Age at test initiation (mean and range, SD):
7-8 months. The supplier confirmed that the animals were from a synchronous population where the animals were no greater than four weeks apart in age.

- Weight at test initiation (mean and range, SD):
The control earthworms used for this experiment had an initial mean weight of 433.2 mg/worm. The initial mean weight of the treatment worms ranged from 354.0 to 502.6 mg/worm.

ACCLIMATION
- Acclimation period:
1 week

- Acclimation conditions (same as test or not):
Same as test.

- Health during acclimation (any mortality observed):
No mortality was observed during the acclimation period.

Study design

Study type:
laboratory study
Substrate type:
artificial soil
Limit test:
no
Total exposure duration:
28 d
Post exposure observation period:
All surviving adult worms within a test replicate on day 28 are rinsed to remove soil, weighed as a group, and the weight loss/gain of the worms will be calculated. The surviving worms will be composited by treatment and placed in a glass container with wet filter paper and allowed to purge their gut contents for a period up to 48 hours then frozen for tissue analysis of the test substance, if applicable.

After the removal of the adults (test day 28), the test soil is placed back into the test chambers and incubated for another 28 days (test day 56). The number of juvenile worms will be determined at test termination by rinsing the test soil through a series of sieves (e.g., 710, 500, and 425 gm mesh), collecting, and enumerating the animals retained within the sieves.

Test conditions

Test temperature:
20 ± 2 deg C
pH:
6.55-6.67 (day 0); 5.50-5.68 (termination)
Moisture:
The water holding capacity (i.e., WHC) of the soil used in the definitive test was determined to be 56.8 mL per 100 grams dry soil. The percent moisture of the soil at 60% of its WHC was calculated as 22.2%.
Details on test conditions:
TEST SYSTEM
- Test container (material, size):
The test chambers will be approximately 1.8-L glass containers.

- Amount of soil or substrate:
A total of 500 or 600 g (dry weight) of prepared soil will be added to each test chamber.

- No. of organisms per container (treatment):
10

- No. of replicates per treatment group:
4
- No. of replicates per control:
8

- No. of replicates per vehicle control:
Not applicable

COMPOSITION AND PROPERTIES OF ARTIFICIAL SOIL:
The artificial soil used for the test 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. The peat moss was sieved to a finely ground consistency and did not contain any visible plant remains. 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. After preparation the soil was stored at room temperature. The water holding capacity (i.e., WIHC) of the soil used in the definitive test was determined to be 56.8 mL per 100 grams dry soil. The percent moisture of the soil at 60% of its WHC was calculated as 22.2%. Agvise Laboratories, Northwood, North Dakota characterized the artificial soil.

Artificial Soil Characterization:
Sandy Loam (International Textural Class; Hydrometer method)
Percent Sand: 80
Percent Silt: 8
Percent Clay: 12
% Organic Matter (Carbon): 7.4 (4.3)
Disturbed Bulk Density: 0.97 gm/cc
Cation Exchange Capacity: 8.4 (meq/100 g)

OTHER TEST CONDITIONS
- Photoperiod:
16 hours light/8 hours dark

- Light intensity:
400-800 LUX

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
reproduction and survival rates at 28-days (measured at 28-days).

TEST CONCENTRATIONS
- Spacing factor for test concentrations:
x2

- Justification for using less concentrations than requested by guideline:
Not applicable.

- Range finding study
- Test concentrations:
The nominal test concentrations for the range-finding test will be 50, 100, 500, 1000, and 5000 mg/kg dry soil.

- Results used to determine the conditions for the definitive study:
yes
Nominal and measured concentrations:
Range finding test:
Nominal: 50, 100, 500, 1000, and 5000 mg/kg dw soil.

Main test:
Nominal: 0 (control), 78.5, 157, 313, 625, 1,250, 2,500, and 5,000 mg/kg dw soil at test initiation.
Measured: <1.22 (control), 73.7, 165, 268, 592, 1,290, 2,460, and 4,680 mg/kg dw soil at test initiation.
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
4 190 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
28 d
Dose descriptor:
EC50
Effect conc.:
> 4 190 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
56 d
Dose descriptor:
NOEC
Effect conc.:
128 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
56 d
Dose descriptor:
EC10
Effect conc.:
21.6 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: 95% CL 0.000468 - 110 mg/kg soil dw
Duration:
56 d
Dose descriptor:
EC50
Effect conc.:
771 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: 95% CL 225 - 4900 mg/kg soil dw
Details on results:
- Mortality at end of exposure period:
0%

- Changes in body weigth of live adults (% of initial weight) at end of exposure period:
The control worms gained an average of 0.418 g per replicate or 10% in replicate mass during the 28 days of adult worm exposure. The mean replicate weight gain of the surviving treatment animals ranged from 0.018 to 0.808 grams. These gains represented average increases of 0.4 to 19% in replicate animal mass over the initial 28 days of exposure.

- No. of offspring produced:
The average reproduction in the control replicates was 72 juveniles per replicate (Table 6). The coefficient of variation for the control data was 16%. The average reproduction was 61, 60, 49, 31, 26, 26, and 30 juveniles per replicate for treatment levels 51.5, 128, 235, 543, 1,070, 2,020, and 3,990 mg HBCD/kg, respectively.

- No. of unhatched cocoons:
Not reported.

- Morphological abnormalities:
None reported.

- Behavioural abnormalities:
None reported.

- Other biological observations:
None reported.
Results with reference substance (positive control):
Not applicable.
Reported statistics and error estimates:
The statistical analyses were performed using SAS for Windows, Release 6.12 and ToxCalc version 5.0. The survival data was not analyzed statistically due to the low level of mortality (i.e., <= 5%) within any of the treatment levels. 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 satisfied the assumptions of normality and homogeneity of variance and the ANOVA was performed on the raw data.

The exposure to HBCD did not adversely affect (i.e., >10% effect) adult worm survival. Therefore, the 10 and 50 percent effect concentrations (i.e., EC10 and EC50) for this parameter were estimated to be greater than the highest 28-day mean measured concentration tested. The reproductive EC10 and EC50 concentrations and their corresponding 95% confidence limits were calculated using probit analysis.

Any other information on results incl. tables

Study validity

No control group exceeded 10% mortality during the initial 28 days of the test.

Each control replicate produced at least 30 juveniles.

The percent coefficient of variance (CV for the control reproduction was ≤30%.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
See remarks on results incl tables and figures section.
Conclusions:
Under the conditions of this study, the 28-day mortality NOEC value was 4,190 mg HBCD per kilogram of dry soil. The estimated EC10 and EC50 values for adult earthworm survival were >4,190 mg/kg dry soil. After 28 days of exposure, measured concentrations of HBCD in the worm tissue samples were <0.200 (control), 3.40, 7.32, 16.8, 15.3, 510, 71.2, and 150 µg HBCD per gram of tissue. The bioaccumulation factors for the treatment worms ranged from 2.6 x 10E+2 to 6.9 x 10E-2. Therefore, HBCD did not bioaccumulate (i.e., BAF values < 1.0) within the worm tissues during the 28-day exposure. The 56-day reproduction NOEC value was 128 mg HBCD per kilogram of dry soil (Table 7). The estimated EC10 and EC50 values for average reproduction were 21.6 (95% confidence limits of 0.000468 to 110 mg/kg) and 771 mg/kg dry soil (95% confidence limits of 225 to 4,900 mg/kg), respectively.
Executive summary:

The effect of Hexabromocyclododecane (HBCD) on the mortality and reproductive rates of Eisenia fetida (earthworms) over a 28 day exposure period was examined.

The study was conducted using a range of concentrations of HBCD (78.5, 157, 313, 625, 1,250, 2,500, and 5,000 mg HBCD per kg dry soil) in artifical soil. The adult earthworms were given a 28 day exposure period in the HBCD test soil and then removed, washed and assessed for mortality rates. The adult earthworms were then allowed to expunge their gut contents for 48 hours and then frozen for analysis.

The test soil was incubated for a further 28 days to allow un-hatched offspring to mature. After the 28 day incubation period the number of juvenile offspring was assessed and the mean reproductive rates of the adults calculated.

Under the conditions of this study, the 28-day mortality NOEC value was 4,190 mg HBCD per kilogram of dry soil. The estimated EC10 and EC50 values for adult earthworm survival were >4,190 mg/kg dry soil. After 28 days of exposure, measured concentrations of HBCD in the worm tissue samples were <0.200 (control), 3.40, 7.32, 16.8, 15.3, 510, 71.2, and 150µg HBCD per gram of tissue. The bioaccumulation factors for the treatment worms ranged from 2.6 x 10-2 to 6.9 x 10-2. Therefore, HBCD did not bioaccumulate (i.e., BAF values < 1.0) within the worm tissues during the 28-day exposure. The 56-day reproduction NOEC value was 128 mg HBCD per kilogram of dry soil. The estimated EC10 and EC50 values for average reproduction were 21.6 (95% confidence limits of 0.000468 to 110 mg/kg) and 771 mg/kg dry soil (95% confidence limits of 225 to 4,900 mg/kg), respectively.