Sodium 4(or 5)-methyl-1H-benzotriazolide

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:

03 February - 16 March

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC) No 440/2008 of May 30, 2008, C.21: Soil Microorganisms: Nitrogen transformation Test

Qualifier:

according to guideline

Guideline:

OECD Guideline 216 (Soil Microorganisms: Nitrogen Transformation Test)

Version / remarks:

Version 2000

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Remarks:

soil nitrate concentration.

Details on sampling:

Sampling: three Sample per treatment directly after application and at test end.
Storage: deep-frozen as particle-free liquid extracts until analysis.

Vehicle:

yes

Details on preparation and application of test substrate:

Soil: The soil characteristics and cultivation requirements of the natural soil used in the study are in accordance with the OECD guideline 216. More details on soil type characteristics and preparation under heading Details on Test condition.

APPLICATION OF TEST SUBSTANCE TO SOIL
Different Tolyltriazole test concentration were prepared by dispersing the test item in the organic solvent acetone, before applied on surface of adequate amounts (10 g of sand/kg dry soil) of quartz sand. After evaporating the solvent for 20 h the test item-quartz sand mixture was thoroughly mixed with the prepared soil (amended by lucerne meal) to the final test substrate.

Total exposure duration:

28 d

Test temperature:

Temperature 17.9-20.9 °C with a mean of 20.2 °C

Moisture:

Water content of soil at test initiation: 45 % of MWC and was controlled weekly by weighing. Losses of water were compensated by addition of purified water followed by mixing of the samples.

Organic carbon content (% dry weight):

0.65

Nitrogen content (% dry weight):

0.07

Details on test conditions:

TEST SYSTEM
- Testing facility: Innovative Environmental Services (IES) Ltd, Benkenstrasse 260, 4108 Witterswil, Switzerland
- Test vessels (type, material, size), glass incubation flasks (1 L) stoppered with cotton wool plugs.
- Amount of soil: 170.8 g wet soil with 45 % MWHC corresponding to 147.4 g dry soil
- Study treatments: five test item concentrations, one control, one solvent control and one reference item concentration
- No. of vessels per treatment: two


SOURCE AND PROPERTIES OF SUBSTRATE (if soil):
- Soil type: Standard soil type 2.3 - Sandy loam - full fill requirements of the test guidelines (No cultivation, fertilization or pesticide usage on soil sampling site for at least 4 Years)
- Supplier: LUFA Speyer, Germany
- Batch: F 2.3 0220; Sampling date 08 January 2020, Date of packaging 20 January 2020
- Sampling depth (cm): Approx.: 20 cm
- Preparation after sampling: Drying at room temperature and sieved to mesh size 2 mm
- Organic carbon (%): 0.65 ± 0.08
- Nitrogen (%): 0.07 ± 0.02
- pH: 6.1 ± 0.4
- Particle size of sand: 0.063 – 2.0 mm
- Maximum water holding capacity (in g/100 g dry soil): 35.2 ± 1.8
- CEC (in meq/100 g): 6.8 ± 1.4
- Initial nitrate concentration for nitrogen transformation test (mg nitrate/kg dry weight):
- Pretreatment of soil: soil was pre-moistened to approx. 40 % MWHC and acclimatised to test conditions (dark, approx. 20 °C) for two days until test start. After soil was amended with powdered lucerne meal (C:N ratio of approx. 15.8:1) in amounts of approx. 5 g/kg dry soil as a source of organic nitrogen before application of Tolyltriazol.
- Storage (condition, duration): Storage condition at test facility prior testing: in the dark under aerobic conditions for ten days at approx. 4 °C.
- Initial microbial biomass as % of total organic C: The microbial biomass was 2.5 % of the organic C content of the used test soil (Sampling at test initial and measured by the fumigation method)


DETAILS OF PREINCUBATION OF SOIL (if any):
- see above bulit point Pretreatment of soil

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Nitrate concentration and the rate of nitrate formation in the treated soil samples in relation to control

VEHICLE CONTROL PERFORMED: yes

Nominal and measured concentrations:

Nominal 10, 32, 100, 320 and 1000 mg/kg dry soil

Reference substance (positive control):

yes

Remarks:

Name: Nitrapyrin, 2-Chloro-6-(trichloromethyl)pyridine (Sigma-Aldrich, Germany). Batch No.: MKCH4320; Expiration Date: 11 November 2024. Purity: 99.9 %. Material form: White to off-white powder. Storage Conditions (Supplier): Keep container tightly closed

Duration:

28 d

Dose descriptor:

EC25

Effect conc.:

240 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Nitrate concentration

Key result

Duration:

28 d

Dose descriptor:

EC25

Effect conc.:

48.7 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

nitrate formation rate

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

1 178 mg/kg soil dw

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Nitrate concentration

Key result

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

117 mg/kg soil dw

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

nitrate formation rate

Details on results:

Control:
In the control and solvent control, the mean nitrate concentration, differed statistically significantly from each other, while the nitrate formation rate did not. Therefore, the controls were pooled for testing of differences in nitrate formation rate but not pooled for nitrate concentrations. In the latter, the solvent control was used for testing of differences.

ENDPOINT SPECIFIC RESULTS
Nitrate Concentration:
In the test Substrate treated with Tolyltriazole, mean nitrate concentrations decreased in concentration-effect relationship. Even though, differences in the nitrate concentrations in comparison to the solvent control after 28 days were only statistically significant at the highest test concentration 320 and 1000 mg Tolyltriazole/ kg dry soil. The EC50 and EC25 for nitrate concentration after 28 day were 1178 (95 % CI: 891-1896) and 240 (95 % CI: 209-301) mg Tolyltriazole/ kg dry soil, respectively.

Nitrate Formation Rate
In the test Substrate treated with Tolyltriazole, mean nitrate formation rate decreased in concentration-effect relationship. At the highest test concentrations (320 and 1000 mg Tolyltriazole/ kg dry soil), the nitrate Formation Rate after 28 days were statistically significant smaller than in the pooled control. The EC50 and EC25 for nitrate formation rate after 28 day were 117 (95 % CI: 74.5-202) and 48.7 (95 % CI: 28.6-76.9) mg Tolyltriazole/ kg dry soil, respectively.

Results with reference substance (positive control):

Nitrate Concentration:
The mean nitrate concentration at DAT28 in the reference item treatment was -89 % smaller
than in the solvent control and differed statistically significantly from the solvent control.

Nitrate Formation Rate:
The mean nitrate formation rate in the reference item treatment was -346 % lower than in the
pooled control and differed statistically significantly from the pooled control.

Reported statistics and error estimates:

Statistics: The two-sample Student t-test (α = 0.05) was used to test differences between control and solvent control (two-sided) and between the reference item and solvent control ( one-sided smaller).
The multiple tests (α = 0.05) was used to test differences between the test item treatments and control(s) (Williams t-test (one-sided smaller)).
Calculations of EC25 and EC50 were performed using Probit analysis and maximum likelihood regressions for nitrate concentration at Day 28 as well as linear weighted regression for soil nitrate formation rate.
The 95 % Confidence intervals were determined for each endpoint.

Validity criteria fulfilled:

yes

Conclusions:

This study fulfilled the validity criterion since the coefficient of variation of the controls regarding nitrate concentration was < 15 %. Based on experimental observations the effect concentrations for Nitrate formation rate EC50(28d) = 117 mg/ kg dw and for nitrate concentration EC50(28d) = 1178 mg/ kg dw, were calculated.

Executive summary:

In this GLP study, according to OECD guideline 216, soil microorganisms were exposed for 28 days to soil treated with the test item Tolyltriazole to assess potential long-term effects on the nitrogen transformation activity.

An EC₅₀ of 1178 [mg/ kg dry soil] was determined for nitrate concentration, while the EC₅₀ for nitrate formation rate was tenfold lower 117 [mg/ kg dry soil].

Tolyltriazole decrease the nitrate concentration and transformation rate significantly only at higher tested concentrations (320 and 1000 mg Tolyltriazole/ kg dry soil). These results indicate that Tolyltriazole may inhibit nitrogen transformation activity of soil microorganisms at concentrations ≥ 320 mg/kg dry soil.

Description of key information

Both key values were determined in an OECD 216 study based on the observed nitrate formation rate after 28 days.

Key value for chemical safety assessment

Short-term EC50 for soil microorganisms:

117 mg/kg soil dw

Long-term EC10 or NOEC for soil microorganisms:

48.7 mg/kg soil dw