Sodium 1H-benzotriazolide

Toxicity to soil microorganisms

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to soil microorganisms

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 05 - July 03, 2020 (biological testing)
September 03, 2020 (study completion)

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:

OECD 216: 21.01.2000
EU C.21: 2004/73/EC

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Product name: 1,2,3 Benzotriazole Fine Granular
- Source (i.e. manufacturer or supplier) and lot/batch number of test material:
Connect Chemicals, Ratingen, Germany, Batch 190720
- Purity, including information on contaminants, isomers, etc.: 99.9 %

RADIOLABELLING INFORMATION (if applicable)
no

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Store cool (SDS)
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: stable
- Stability in the medium, i.e. sensitivity of the test material to hydrolysis and/or photolysis: assumed stable
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: assumed stable, Water solubility (25 °C): 20 g/L
- Reactivity of the test material with the incubation material used (e.g. plastic ware): assumed no reactivity

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
please refer to following sections

FORM AS APPLIED IN THE TEST (if different from that of starting material)
same as starting material

INFORMATION ON NANOMATERIALS
no nanomaterial

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable)
no biocide/pesticide

OTHER SPECIFICS
n/a

Analytical monitoring:

not specified

Details on sampling:

- Concentrations: 1.8, 4.5, 11.2, 28.0, 70.0
- Sampling method: samples were shook (30g dw) with 0.1M KCl solution at ratio of 5mL KCl solution/g dw for 60 min at 150rpm. Afterwards, the mixtures were filtered and liquid phases were analysed for nitrate photometrically (Spectroquant NOVA 400).
- Sample storage conditions before analysis: when the analysis could not be applied immediately, the soil samples were stored at <-18°C for 5d at a maximum.

Vehicle:

no

Details on preparation and application of test substrate:

APPLICATION OF TEST SUBSTANCE TO SOIL
- Method: the soil was sieved to a particle size of 2mm after 8d of storage (at 4-8°C until start of conditioning), soil conditioning at room temperature in the dark, soil moisture was 40-60% of maximum Water Holding Capacity (WHCmax). Sieved soil was amended with powdered plant material (lupine-green meal) at a plant-soil ratio of 5g plant/kg soil dw.

Test organisms (inoculum):

soil

Total exposure duration:

28 d

Test temperature:

20 ± 2 °C

Moisture:

start: 293 g H2O/kg soil dw)
end:

Organic carbon content (% dry weight):

0.93

Nitrogen content (% dry weight):

0.09

Details on test conditions:

TEST SYSTEM
- Testing facility: IME, Schmallenberg, Germany
- Test container (type, material, size): 1L wide neck glass bottles
- Amount of soil: 1600g dw (control and test replicates), 500g dw (nitrogen controls)
- No. of replicates per concentration: 3
- No. of replicates per control: 4
- No. of replicates per test item nitrogen control: 1 (for each test concentration and control)

SOIL INCUBATION
- Method: series of individual subsamples

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Geographical reference of sampling site (latitude, longitude): taken from reference soils at Fraunhofer Institute for Molecular Biology and Applied Ecology (IME) and is representative for an agricultural field soil
- Treatments with pesticides or fertilizers: > 1 year prior to sampling, no plant protection products and >6 and >3 months prior, no organic and mineral fertilizer, respectively, have been used.
- Accidental contamination: none
- Depth of sampling: n.a.
- Soil texture
- % sand: 73
- % silt: 20
- % clay: 7
- Soil classification: loamy sand soil
- pH (in water): 6.4
- Initial nitrate concentration for nitrogen transformation test (mg nitrate/kg dry weight): 6.6
- Maximum water holding capacity (in g H2O/kg soil dry weigth): 293
- Cation exchange capacity (mmol/kg): 16.2
- Pretreatment of soil: not specified
- Storage (condition, duration): at 4-8°C until start of conditioning, total indoor storage: 5 weeks
- Initial microbial biomass as % of total organic C: 1.0

SOIL PREPARATION
- Introduction of test item: quantity of test item required to obtain desired concentrations was applied as an aqueous stock solution of 500mg/250mL and mixed thoroughly with test substrate. 50mL of a solution with the requisite concentration was applied on the respective amount of test substrate (1600g dw and 500g dw, adjusted on 28% WHCmax, for assays with plant material and nitrogen controls, respectively). Subsequently, the requisite volume of water was applied to adjust the soil on 45% WHCmax.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- nitrate on days 0 and 28
- nitrogen controls at both sampling days, fourfold

RANGE-FINDING STUDY
- Test concentrations: not specified
- Results used to determine the conditions for the definitive study: no inhibition up to and including 2mg/kg soil dw

Nominal and measured concentrations:

nominal: 1.8, 4.5, 11.2, 28.0, 70.0 mg test item/kg soil dw

Reference substance (positive control):

no

Key result

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

8.1 mg/kg soil ww

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

nitrate formation rate

Duration:

28 d

Dose descriptor:

EC25

Effect conc.:

11 mg/kg soil ww

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

nitrate formation rate

Key result

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

15.4 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

nitrate formation rate

Details on results:

- Nitrate: Subtraction of "test item nitrogen controls" since only mineralised plant material should be included. Comparison of nitrate found in treated samples after 28 days with that found in controls. From that, the percent inhibition values for each test concentration were calculated. These percentages were plotted against concentration and statistical procedures are used to calculate ECx values (with confidence limits P=0.95 if possible) using standard procedures. Software used was ToxRat Professional by ToxRat Solutions GmbH

- Temperature: continuously measured with data logger, 20.0 - 20.5°C

	Control	1.8 mg TI per kg DM	4.5 mg TI per kg DM	11.2 mg TI per kg DM	28.0 mg TI per kg DM	70.0 mg TI per kg DM
Test start Nitrate [mg/kg]	19.9	19.9	20.7	18.5	16.2	19.2
Day 28 Nitrate [mg/kg] Deviation [%]	160.5	158.7 -1.1	124.7 -22.3	140.2 -12.6	-3.0 -101.8	1.5 -99.1

Validity criteria fulfilled:

yes

Remarks:

With a maximum of 11%, the variation between replicate control samples including powdered plant material was less than 15% at test start and end, therefor the study is valid.

Conclusions:

There was a concentration relating inhibition of nitrate formation after 28days of incubation due to the test item. The EC50 for the nitrogen transformation was assumed to be 15.4 mg Benzotriazole/kg dry soil (95% CL: 11.9 - 20.9 mg TI/kg dw).

Executive summary:

The effect of Benzotriazole on nitrogen transformation by soil microorganisms according to the OECD guideline 216, respectively. For this the nitrate content in the soil after an incubation period was measured.

Benzotriazole was incubated in loamy sand soil over a period of 28 days in concentrations of 1.8, 4.5, 11.2, 28.0 and 70.0 mg test item/kg dry mass soil, respectively. The concentrations applied were based on a non-GLP range finding test. A single application of test item was made and the effect on nitrate building rate was determined 28 days after start of incubation. Four replicates were prepared for a control and three for each concentration.

There was a concentration relating inhibition of nitrate formation after 28 days of incubation. The EC₅₀ for the nitrogen transformation was found to be 15.4 mg Benzotriazole per kg dry soil (95 % CL: 11.9 – 20.9 mg TI/kg DM).

Reference 2

Endpoint:

toxicity to soil microorganisms

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

See read-across justification in Section 13.2 titled "140704_NaBZT_rationale_RA 8.4.16".

Reason / purpose for cross-reference:

read-across source

Key result

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

9.6 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

nitrate formation rate

Duration:

28 d

Dose descriptor:

EC25

Effect conc.:

13 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

nitrate formation rate

Key result

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

18.3 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

nitrate formation rate

Details on results:

Indicated results are relevant for the target substance. These were derived by correcting the source substance results for molecular weight difference between source and target substances.

- Nitrate: Subtraction of "test item nitrogen controls" since only mineralised plant material should be included. Comparison of nitrate found in treated samples after 28 days with that found in controls. From that, the percent inhibition values for each test concentration were calculated. These percentages were plotted against concentration and statistical procedures are used to calculate ECx values (with confidence limits P=0.95 if possible) using standard procedures. Software used was ToxRat Professional by ToxRat Solutions GmbH

- Temperature: continuously measured with data logger, 20.0 - 20.5°C

	Control	1.8 mg TI per kg DM	4.5 mg TI per kg DM	11.2 mg TI per kg DM	28.0 mg TI per kg DM	70.0 mg TI per kg DM
Test start Nitrate [mg/kg]	19.9	19.9	20.7	18.5	16.2	19.2
Day 28 Nitrate [mg/kg] Deviation [%]	160.5	158.7 -1.1	124.7 -22.3	140.2 -12.6	-3.0 -101.8	1.5 -99.1

Validity criteria fulfilled:

yes

Remarks:

With a maximum of 11%, the variation between replicate control samples including powdered plant material was less than 15% at test start and end, therefor the study is valid.

Conclusions:

There was a concentration relating inhibition of nitrate formation after 28 days of incubation due to the test item. The EC50 for the nitrogen transformation was assumed to be 15.4 mg Benzotriazole/kg dry soil (95% CL: 11.9 - 20.9 mg TI/kg dw). The EC10 was 8.1 mg/kg soil dw.

As read-across has been shown to be applicable (IUCLID Section 13.2), but there are differences in molecular weight between the source and target substance the endpoint has been corrected for this difference, the target substance EC50 is 18.25 mg/kg soil dw. The EC10 was 9.6 mg/kg soil dw.

Executive summary:

A one-to-one read-across approach with correction for molecular weight has been used to derive endpoints for the target substance. The read-across was from benzotriazole (EC No.: 202-394-1) to sodium 1H-benzotriazolide (EC No. 239-269-6).  In line with ECHA guidance the reported endpoint values are those relevant to the target substance and have been corrected for molecular weight (ECHA How to prepare registration and PPORD dossiers, v10.0, 2021).  The only differences between the substances is the addition of a sodium salt in the target substance. However, at relevant environmental pH ca. 6-9, protonation will occur yielding the neutral form of the target substance (See Section 13.2 for more information). 

The effect of Benzotriazole on nitrogen transformation by soil microorganisms according to the OECD guideline 216 was studied. For this the nitrate content in the soil after an incubation period was measured.

Benzotriazole was incubated in loamy sand soil over a period of 28 days in concentrations of 1.8, 4.5, 11.2, 28.0 and 70.0 mg test item/kg dry mass soil, respectively. The concentrations applied were based on a non-GLP range finding test. A single application of test item was made and the effect on nitrate building rate was determined 28 days after start of incubation. Four replicates were prepared for a control and three for each concentration.

There was a concentration relating inhibition of nitrate formation after 28 days of incubation. The EC₅₀ for the nitrogen transformation was found to be 15.4 mg Benzotriazole per kg dry soil (95 % CL: 11.9 – 20.9 mg/kg soil dw). The EC₁₀ was 8.1 mg/kg soil dw.

As read-across has been shown to be applicable (IUCLID Section 13.2), but there are differences in molecular weight between the source and target substance the endpoint has been corrected for this difference, the target substance EC₅₀ is 18.25 mg/kg soil dw. The EC₁₀ was 9.6 mg/kg soil dw.

Description of key information

Soil microorganism nitrogen inhibition EC₁₀ = 9.60 mg/kg soil dw, OECD 216. Simon, 2020.

Soil microorganism nitrogen inhibition EC₅₀ = 18.3 mg/kg soil dw, OECD 216. Simon, 2020.

Key value for chemical safety assessment

Short-term EC50 for soil microorganisms:

18.3 mg/kg soil dw

Long-term EC10 or NOEC for soil microorganisms:

9.6 mg/kg soil dw

Additional information

A one-to-one read-across approach with correction for molecular weight has been used to derive endpoints for the target substance. The read-across was from benzotriazole (EC No.: 202-394-1) to sodium 1H-benzotriazolide (EC No. 239-269-6).  In line with ECHA guidance the reported endpoint values are those relevant to the target substance and have been corrected for molecular weight (ECHA How to prepare registration and PPORD dossiers, v10.0, 2021).  The only differences between the substances is the addition of a sodium salt in the target substance. However, at relevant environmental pH ca. 6-9, protonation will occur yielding the neutral form of the target substance (See Section 13.2 for more information). 

The effect of Benzotriazole on nitrogen transformation by soil microorganisms according to the OECD guideline 216 was studied. For this the nitrate content in the soil after an incubation period was measured.

Benzotriazole was incubated in loamy sand soil over a period of 28 days in concentrations of 1.8, 4.5, 11.2, 28.0 and 70.0 mg test item/kg dry mass soil, respectively. The concentrations applied were based on a non-GLP range finding test. A single application of test item was made and the effect on nitrate building rate was determined 28 days after start of incubation. Four replicates were prepared for a control and three for each concentration.

There was a concentration relating inhibition of nitrate formation after 28 days of incubation. The EC₅₀ for the nitrogen transformation was found to be 15.4 mg Benzotriazole per kg dry soil (95 % CL: 11.9 – 20.9 mg/kg soil dw). The EC₁₀ was 8.1 mg/kg soil dw.

As read-across has been shown to be applicable (IUCLID Section 13.2), but there are differences in molecular weight between the source and target substance the endpoint has been corrected for this difference, the target substance EC₅₀ is 18.25 mg/kg soil dw. The EC₁₀ was 9.6 mg/kg soil dw.