Registration Dossier

Administrative data

Endpoint:
toxicity to soil microorganisms
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 January to 25 April 2007
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:
2007
Report Date:
2007

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 216 (Soil Microorganisms: Nitrogen Transformation Test)
Deviations:
yes
Principles of method if other than guideline:
In addition to the ECx values required by the guideline, the NOEC was determined. Due to the high test item concentrations tested that require a large volume of the solvent acetone, the amount of (spiked) sand added to the soil was increased from 1 to 4%. These deviations were considered not to affect the reliability of the study result.
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Batch number: 6541
Purity: 99.6%

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations:
10, 100 and 1000 mg/kg

- Sampling method:
not reported.

- Sample storage conditions before analysis:
Not reported.

Test substrate

Vehicle:
yes
Details on preparation and application of test substrate:
AMENDMENT OF SOIL
- Type of organic substrate:
Standard Soil Lufa 2.3

APPLICATION OF TEST SUBSTANCE TO SOIL
- Method:
The test item was insoluble in water. Therefore, stock solutions and dosage solutions were prepared by dissolving the test item in acetone. These solutions were used to spike quartz sand which was mixed into the soil after evaporation of the solvent. Two stock solutions (SL1 and SL2) were prepared by dissolving 2000 mg of the test item in 50 mL acetone for Solution SL1, and 633 mg of the test item in 20 mL acetone for SL2. Stock solutions SL3, SL4, and SL5 were prepared by filling up 20 mL volumetric flasks with acetone after having added either 5.0 mL of SL1 or 2.0 ml of SL2, or 2.0 mL of SL3, respectively.

To spike quartz sand with the test item, a volume of 25 mL (SL1) or 10 mL (SL2, SL3, SL4, and SL5) was used to soak 40 g of quartz sand, respectively (Table 3, see remarks section). All soaked sand samples were left under a fume hood until complete evaporation of the solvent. To achieve test soil specimens with nominal test item concentrations of 1000 mg/kg, 100 mg/kg, and 10 mg/kg dry weight, respectively, portions of 639.7 g of the bulk soil (594 g dry weight) were mixed with 24 g of the respective spiked sand. Thereafter, 62.1 mL of demineralised water were added to each batch of treated soil to achieve a water content of 50% of the maximum water holding capacity. A bulk soil sample was prepared by mixing 4.0 kg of soil (3.71 kg dry weight) with 18.6 g of finely ground lucerne meal (i.e. 5.0 g/kg soil dry weight).

To achieve test soil specimens with nominal test item concentrations of 316.2 mg/kg, and 31.6 mg/kg dry weight, respectively, portions of 465.2 g of the bulk soil (water content 7.69% of dry weight) were mixed with 18 g of the respective spiked sand. Thereafter, 45.3 mL of demineralised water were added to each batch of treated soil to achieve a water content of 50% of the maximum water holding capacity. No analytical samples were taken from these two test specimens. Deviating from the study plan, three of the nominal test item concentrations given in the Study Plan (10 mg/kg, 100 mg/kg, and 1000 mg/kg) were not met. Due to a minor calculation error, the actually applied concentrations were 9.71 mg/kg, 97.1 mg/kg, and 970.8 mg/kg, respectively. Since the resulting reduction of the concentrations is less than 3% compared to the target concentrations, all calculations are based on the nominal concentrations. This deviation is considered negligible for the outcome of the study.

After mixing the soil thoroughly with an electric stirrer, three portions of 176 g soil (150 g dry weight) were filled into glass vessels, closed with a lid (not air tight), and placed in a test chamber at 20 ± 2°C in the dark. The remaining soil was filled into three glass bottles for later chemical verification of the test item concentration in soil (performed separately from this study).

Test organisms

Test organisms (inoculum):
soil

Study design

Total exposure duration:
28 d

Test conditions

Test temperature:
20 ± 2ºC
Moisture:
50% of maximum water holding capacity.
Details on test conditions:
TEST SYSTEM
- Test container (type, material, size):
Glass jars with lids (volume 0.37 L, height 69 mm, diameter 100 mm) were used as test vessels in the N-transformation test. The lid allowed for some gas exchange to avoid anaerobic conditions in the vessel.

- Amount of soil:
594g

- No. of replicates per concentration:
3

- No. of replicates per control:
3

- No. of replicates per vehicle control:
3

SOIL INCUBATION
- Method: series of individual subsamples.
The soil was incubated in glass jars for a test period of 28 days at 20 ± 2°C in the dark.

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Geographical reference of sampling site (latitude, longitude):
Germany, Rheinland-Pfalz, Offenbach, Im Bildgarten No. 586

- History of site:
- Vegetation cover:
not reported, however the site was recorded as fallow soil for 2002, used for the cultivation of pumpkins from 2003 to 2005 and was uncultivated in 2006.

- Treatments with pesticides or fertilizers:
No pesticides were used on the site for at least 4 years prior to sampling.

- Accidental contamination:
not reported.

- Depth of sampling:
Depth: ca. 20 cm

- Soil texture
not reported

- Soil taxonomic classification:
Standard Soil Lufa 2.3 (sandy loam)

- Soil classification system:
not reported.

- pH (in water):
6.2 ± 0.3

- Maximum water holding capacity (in % dry weigth):
34.9 ± 3.0

- Initial nitrate concentration for nitrogen transformation test (mg nitrate/kg dry weight):
Mean concentration of nitrate in control samples on Day 0: 4.57 mg NO3/kg soil dry weight

- Cation exchange capacity (mmol/kg):
not reported.

- Pretreatment of soil:
Air drying (only until sieveable) on 11 – 15 December 2006; Final sieving to 2 mm on 18 December 2006; Adjustment to ca. 23 % of the maximum water holding capacity on December 19, 2006.

- Storage (condition, duration):
stored at 20 ± 2°C in the dark until January 16, 2006

- Initial microbial biomass as % of total organic C:
1.46

DETAILS OF PREINCUBATION OF SOIL (if any):
not reported.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Nitrate formation, measured at beginning and termination of test.

VEHICLE CONTROL PERFORMED:
yes

RANGE-FINDING STUDY
no
Nominal and measured concentrations:
Nominal: 10, 100, and 1000 mg/kg
measured: 10.4, 83.1, 750 mg/kg
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Duration:
28 d
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
nitrate formation rate
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
nitrate formation rate
Details on results:
The test was performed from January 16, 2007 (Day 0) to February 13, 2007 (Day 28). Nitrate was extracted from control, solvent control, and test item treated soil specimens on January 17, 2007 (Day 0), and on February 13, 2007 (Day 28).

Day 0: Mean concentrations of nitrate on day 0 of the test were below or slightly above the detection limit of 1.0 mg NO3/L which is equivalent to 5.0 mg kg-1 soil dry mass; therefore, it was decided not to measure all samples (Table 4).

Day 28: The mean nitrate concentrations on day 28 were in the range of 54.1 mg kg-1 to 62.3 mg kg-1 soil dry mass (Table 4, Figure 1) in the control and treated soil samples.

The variation of nitrate concentrations within the three control specimens was less than ± 15 % of the mean (Table 5). Therefore, the validity criteria defined in the guideline have been fulfilled.
Results with reference substance (positive control):
not applicable
Reported statistics and error estimates:
Normal distribution and homogeneity of variance was assessed using the R/S test and the Bartlett's test, respectively. ANOVA and Dunnett multiple t-tests were applied to detect differences between the pooled control and the test item concentrations, and to determine the NOEC. The Probit Analysis using linear maximum likelihood regression was used to determine EC50, EC20 and EC10 values. All tests were performed with the ToxRat® satatistical software Version 2.09.

Any other information on results incl. tables

Table 4. Mean concentration and standard deviation (SD) of nitrate in soil treated wit the test item and in non treated soil (control and solvent control) on day 0 and day 28 of the test.

Treatment [mg a.i. kg-1 soil] Day 0 [mg NO3. kg-1 soil dry weight]  SD Day 28 [mg NO3. kg-1 soil dry weight] SD
Control 0.0 4.57 0.15 56.18 5.57
Solvent Control 0.0 4.61 0.36 62.28 3.72
10 4.26 0.2 58.94 6.40
31.6 n.d. n.a. 54.22 3.13
100 4.23 0.22 54.06 1.29
316.2 n.d. n.a. 60.11 3.52
1000 5.57 0.52 59.64 5.38

Table 5:Mean, minimum and maximum nitrate concentration, and maximum deviation from the mean in the non treated soil (control) at the start (day 0) and at the end (day 28) of the test.

Time Concentration [mgkg-1soil dry weight] Max. deviation from mean (%)
Mean Min. Max.
Day 0 4.57 4.48 4.75 3.94
Day 28 56.18 50.70 61.83 10.07

Validity criteria.

The validity criterion concerning the variation between replicate control samples in the Nitrogen Transformation Test (concentration of nitrate) was fulfilled: the maximum deviation was 10.07%, and was therefore within the acceptable range of deviation of <15% (Table 2).

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
See remarks on results.
Conclusions:
The addition of HBCD ranging from 10.0 to 1000 mg/kg. d.w. soil (nominal concentrations) did not lead to any significant effect (ANOVA at p≤0.05) on the formation of nitrate in the treated soil samples as compared to the control soils. Correspondingly, it can be concluded that the EC50 of HBCD is >1000 mg/kg dry soil, whereas the NOEC is considered to be 1000 mg/kg dry soil.
Executive summary:

Report:

Förster, B. (2007) Hexabromocyclododecane (HBCD): Effects on Soil Microorganisms. ECT Oekotoxikologie GmbH, D-65439 Flörsheim, Germany, unpublished report number AU1BB, 20 pages plus an Appendix of 37 pages (Study Plan 11 pages; Analytical Letter Report 26 pages).

Amendments:

None.

Guideline:

OECD Guideline for the Testing of Chemicals No. 216 “Soil Microorganisms: Nitrogen Transformation Test”.

Deviations from the Guidelines:

The amount of quartz sand added to the soil was 4% instead of 1%.

Test Item:

Hexabromocyclododecane (HBCD); CAS RN: 3194-55-6; Batch No.: 6541; Content (a.i.): 99.6%.

Methods:

Respective amounts of the test item were diluted in acetone and mixed into quartz sand. After evaporation of the acetone the sand was mixed into sieved field soil (Lufa standard soil 2.3) that was amended with ground lucerne meal (5.0 g kg-1soil) and adjusted to a watercontent of 50% of the maximum water-holding capacity. The nominal test item concentrations were 10 mg kg-1, 31.6 mg kg-1, 100 mg kg-1, 316.2 mg kg-1, and 1000 mg kg-1soil dry weight. There were three replicates per test item concentration, and three per control and solvent control, each. The soil was incubated in glass jars for a test period of 28 days at 20 ± 2 °C in the dark. Soil nitrate concentrations were measured on day 0 (i.e. within 24-h after test start), and on day 28 of the test (28 days after test item application).

Findings:

The mean nitrate concentrations on day 28 were in the range of 54.1 mg kg-1 to 62.3 mg kg-1 soil dry mass in the control and treated soil samples.

The variation of the nitrate concentrations within the three control specimens was less than ± 15% of the mean. Therefore, the validity criteria defined in the guideline have been fulfilled.

The addition of HBCD ranging from 10 to 1000 mg/kg. d.w. soil (nominal concentrations) did notlead to any significant effect (ANOVA at p0.05) on the formation of nitrate in the treated soil samples as compared to the control soils.

Conclusions: It can be concluded that the EC50 of HBCD is >1000 mg/kg dry soil, whereas the NOEC is considered to be 1000 mg/kg dry soil.