Reaction products of tetrabromophthalic anhydride with 2,2'-oxydiethanol and methyloxirane

Toxicity to soil microorganisms

Link to relevant study record(s)

Reference

Endpoint:

toxicity to soil microorganisms

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-10-16 - 2020-12-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 216 (Soil Microorganisms: Nitrogen Transformation Test)

Version / remarks:

21st January 2000

Deviations:

yes

Remarks:

The temperature was between 22 and 23°C for 16 hours and microbial biomass and nitrate content was not measured prior to pre-incubation. This does not impact the results of the study.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Remarks:

Analytical method validation, Sneckenberger et al. 2020

Details on sampling:

- Collection: Day 0 and 28
- Sampling method: A single sample of approximately 5.5 grams (5.0 grams dry soil) was removed from each test chamber at each sampling interval.

An analytical method was validated independently to determine the concentrations of the test material in acetone stock solutions as used in the present study. Acetone stock solutions samples were prepared by weighing the appropriate mass of test substance and bringing to a final volume with acetone.

Vehicle:

yes

Details on preparation and application of test substrate:

All test chambers were incubated under aerobic conditions in the dark in a temperature-controlled room set at approximately 20°C for five days prior to application of the test substance.

AMENDMENT OF SOIL
- Type of organic substrate: Alfalfa
ground and sieved to ≤710 μm, and dried in an oven. C/N ratio is 14.5 : 1, stored under ambient conditions.

APPLICATION OF TEST SUBSTANCE TO SOIL
- Method: Quartz sand was used as carrier. After dosing, soils were thoroughly homogenised using stainless steel spatulas.

VEHICLE:
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Acetone
- Concentration of vehicle in test medium (stock solution and final test solution): stock solution: 20.37 mg/mL.
The dose mixes for control were prepared by mixing 1 g of quartz sand with 5 mL of acetone in glass vials. The dose mixes for treatment were prepared by mixing 1 g of quartz sand with 5 mL of SAYTEX RB-79 stock solution (ID: 15864-102720-1).
- Evaporation of vehicle before use: yes. The solvent was removed under nitrogen flow in a fume hood at room temperature.

The final concentration of dose mixes for treatment (IDs: 15864-102720-mix Rep A, 15864-102720-mix Rep B and 15864-102720-mix Rep C) was 101.6 mg SAYTEX RB-79 per gram of quartz sand. This method was used due to the low water solubility and the physical-chemical properties (highly viscous) of the test item and considered suitable based on the results obtained in the non-GLP DRF study

Test organisms (inoculum):

soil

Total exposure duration:

28 d

Test temperature:

20°C

Moisture:

The measured moisture contents of the soil samples ranged from 10.0% to 11.7%, and were equivalent to 47.5% to 55.4% of the maximum water holding capacity (WHC) during the test.

Organic carbon content (% dry weight):

1

Details on test conditions:

TEST SYSTEM
- Testing facility: Eurofins EAG Agroscience, LLC
- Test container (type, material, size): 8-ounce French-square glass bottles with perforated aluminum foil lids.
- Amount of soil: ~ 108.0 g moist soil (equivalent to 100 g dry soil)
- No. of replicates per concentration: 3
- No. of replicates per control: 3

SOIL INCUBATION
- Method: bulk / series of individual subsamples
Following direct addition of the test substance, the test chambers with treated and untreated (control) soils were covered with perforated aluminum foil lids and incubated in the dark at a temperature of 20 ± 2°C for 28 days.

SOURCE AND PROPERTIES OF SUBSTRATE
- Geographical reference of sampling site: Greensboro, Maryland, outdoor soil plot
- History of site: The original collection area has not been actively farmed for at least 5 years.
- Vegetation cover: covered by grasses
- Treatments with pesticides or fertilisers: Approximately 15 meters from a farmed area. No known treatment with crop protection product or fertiliser has occurred in the past five years.
- Accidental contamination: Not likely, the soil has been maintained in this outdoor soil plot since 2012.
- Other: The soil for this study was collected from this plot on October 23, 2020.
- Soil texture
- % sand: 71.4
- % silt: 18.1
- % clay: 10.5
- Soil taxonomic classification: sandy loam
- Soil classification system: USDA classification
- pH (in water): 7.4
- Bulk density: 1.32 g/cc
- Initial nitrate concentration for nitrogen transformation test: 2.48 mg/kg
- Maximum water holding capacity (in % dry weigth): 21.1
- Cation exchange capacity (mmol/kg): 4.2 meq/100 g
- Pretreatment of soil: sieved through a 2-mm screen
- Initial microbial biomass as % of total organic C: 239.9 µg/g; The microbial biomass was greater than 1% of the organic carbon content.

DETAILS OF PREINCUBATION OF SOIL: Water was added to adjust the moisture content to approximately 50% of the maximum water holding capacity, and test chambers were acclimated to test conditions for five days. Test chambers were amended with dried, ground alfalfa at 5.0 g/kg of dry soil as a source of nitrogen. The concentration of test substance added was calculated assuming uniform incorporation to a depth of 5 cm and a nominal soil bulk density of 1.3 g/cm³.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Day 0 and 28, soil nitrate analysis

VEHICLE CONTROL PERFORMED: no

RANGE-FINDING STUDY
- Test concentrations: 0, 5.0, 10.0, 50.0, 100, 500 and 1000 mg/kg
- Results used to determine the conditions for the definitive study: An increase in nitrate formation was detected, but no inhibition of the nitrate formation > 25 % at any test concentrations by SAYTEX RB-79 within 0 – 1000 mg/kg.

Nominal and measured concentrations:

0 and 1000 mg/kg dry soil (nominal)

Reference substance (positive control):

no

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

Details on results:

Soil Moisture Measurements
The measured moisture contents of the soil for Day 0 and Day 28 samples ranged from 10.0% to 11.7%, and were equivalent to 47.5% to 55.4% of the maximum water holding capacity (WHC) during the test. The soil moisture contents were within the range specified in the protocol (40 to 60% WHC).

Soil pH Measurements
Before the study the batch of soil had a pH of 7.4 (Agvise). Throughout the test, the soil pH ranged from 5.54 to 5.81.

Soil Nitrate Analyses
The mean nitrate concentrations in the control soil were 162.8 and 359.7 mg NO3/kg on Day 0 and 28, respectively.
The mean nitrate concentrations in soils treated with SAYTEX RB-79 at 1000 mg/kg dry soil were 161.5 and 434.5 mg NO3/kg on Day 0 and 28, respectively.
The mean nitrate formation rate in control soils was 7.033 mg NO3/kg/day after 28 days. The mean nitrate formation rates in soils treated with SAYTEX RB-79 at 1000 mg/kg dry soil was 9.749 mg NO3/kg/day after 28 days.

Reported statistics and error estimates:

Statistical comparisons of measured nitrate concentrations were made at each sample interval in Microsoft Excel using Analysis of variance (ANOVA).
The coefficient of variation (CV) among control replicates at all sampling intervals was <15% and therefore, the test is considered valid.

Validity criteria fulfilled:

yes

Conclusions:

The study was conducted under GLP according to OECD Guideline 216 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation or deviations from the guidelines, the validity criteria were met. Hence, the results can be considered as reliable to assess the long-term toxicity of the test substance towards soil microorganisms.
After 28 days, the difference of nitrogen transformation rates between soils treated with SAYTEX RB-79 at 1000 mg/kg dry soil and controls was 38.61% indicating stimulation by the test substance. Therefore, the EC50 value for inhibition of nitrate formation by SAYTEX RB-79 was greater than 1000 mg/kg dry soil (the highest concentration tested) and SAYTEX RB-79 at the concentrations tested can be evaluated as having no long-term adverse effect on nitrogen transformation in soils.

Executive summary:

This study was conducted to assess the effects of SAYTEX RB-79 on the nitrogen transformation activity of soil microorganisms in aerobic surface soils and meets the conditions set in OECD Guideline 216 and conforms to GLP requirements. The study was conducted using a single soil type (sandy loam). Test systems were dosed with SAYTEX RB-79 at nominal concentrations of 0 and 1000 mg/kg dry soil based on the results obtained in a non-GLP dose range finding (DRF) study, and incubated at approximately 20ºC for 28 days. Soil samples were collected on Days 0 and 28, and extracted with 25 mL of water. Extracts were centrifuged and the supernatants were analysed to determine nitrate concentration using ion chromatography. The mean nitrate concentrations in the control soil were 162.8 and 359.7 mg NO₃/kg on Day 0 and 28, respectively. The mean nitrate concentrations in soils treated with SAYTEX RB-79 at 1000 mg/kg dry soil were 161.5 and 434.5 mg NO₃/kg on Day 0 and 28, respectively. Mean nitrate formation rates were calculated, and are presented in units of mg NO₃/kg dry soil per day. The difference in nitrate formation rates between treatments and untreated controls, was calculated for each treatment and resulted in a mean formation rate of 7.033 mg NO₃/kg dry soil/day for the control and 9.749 mg NO₃/kg dry soil/day for the 1000 mg/kg dry soil treatment,

 

The EC₅₀ value for inhibition of nitrate formation after 28 days was greater than 1000 mg/kg dry soil. SAYTEX RB-79 at the concentrations tested can be evaluated as having no long-term adverse effect on nitrogen transformation in soils. As noted in the non-GLP DRF study, an increase in nitrate formation was detected, but no inhibition of the nitrate formation by SAYTEX RB-79 was observed.

Description of key information

Nitrogen transformation test, OECD 216, GLP, aerobic surface soil, sandy loam, 0 and 1000 mg/kg dry soil, 28 days: EC50 > 1000 mg/kg dry soil

Key value for chemical safety assessment

Long-term EC10 or NOEC for soil microorganisms:

1 000 mg/kg soil dw

Additional information

This study was conducted to assess the effects of SAYTEX RB-79 on the nitrogen transformation activity of soil microorganisms in aerobic surface soils and meets the conditions set in OECD Guideline 216 and conforms to GLP requirements. The study was conducted using a single soil type (sandy loam). Test systems were dosed with SAYTEX RB-79 at nominal concentrations of 0, and 1000 mg/kg dry soil based on the results obtained in a non-GLP dose range finding (DRF) study, and incubated at approximately 20º C for 28 days. Soil samples were collected on Days 0 and 28, and extracted with 25 mL of water. Extracts were centrifuged and the supernatants were analysed to determine nitrate concentration using ion chromatography. The mean nitrate concentrations in the control soil were 162.8 and 359.7 mg NO₃/kg on Day 0 and 28, respectively.The mean nitrate concentrations in soils treated with SAYTEX RB-79 at 1000 mg/kg dry soil were 161.5 and 434.5 mg NO₃/kg on Day 0 and 28, respectively. Mean nitrate formation rates were calculated, and are presented in units of mg NO₃/kg dry soil per day. The difference in nitrate formation rates between treatments and untreated controls, was calculated for each treatment and resulted in a rate of 7.033 mg NO₃/kg dry soil/day for the control and 9.749 mg NO₃/kg dry soil/day for the 1000 mg/kg dry soil treatment. 

The EC₅₀ value for inhibition of nitrate formation after 28 days was greater than 1000 mg/kg dry soil. SAYTEX RB-79 at the concentrations tested can be evaluated as having no long-term adverse effect on nitrogen transformation in soils. As noted in the non-GLP DRF study, an increase in nitrate formation was detected, but no inhibition of the nitrate formation by SAYTEX RB-79 was observed.