Oxalonitrile

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

according to guideline

Guideline:

OECD Guideline 216 (Soil Microorganisms: Nitrogen Transformation Test)

Qualifier:

according to guideline

Guideline:

OECD Guideline 217 (Soil Microorganisms: Carbon Transformation Test)

GLP compliance:

yes

Duration:

28 d

Dose descriptor:

other: NH4-N and NO3-N

Effect conc.:

667 mg/kg soil ww

Conc. based on:

test mat.

Basis for effect:

nitrate formation rate

Conclusions:

The substance will be dispatched in the air and will get quickly diluted due to its high volatility. The substance will stay in the air based on its physical and chemical properties and will not transfer to other environmental compartments such as soil and water as confirmed by models in the environmental fate part of dossier.

Executive summary:

Soil Microorganism-nitrogen Transformation for Cyanogen (C₂N₂) Study using OECD 216 test method:

This study was performed to determine the effects of ethanedinitrile on nitrogen transformation by soil microorganisms to comply with the requirements for an ecotoxicology study on plant protection products ‘Effects on soil non-target microorganisms’. The study was conducted according to the OECD-Guideline for the testing of chemicals, Soil Micro-organisms: Nitrogen transformation test, Guideline 216, the OECD guideline for the testing of chemicals.Cyanogen was applied at the maximum application rate (MAR) of 667 mg kg^-1 soil and at 5 times the MAR (3,333 mg kg^-1 soil.) The test soil was a sandy loam obtained from a site near the USDA facility of Salinas, CA. Soil jars were induced with cyanogen in the lab according to the study protocol. All jars were capped with butyl rubber septa. All C₂N₂treated jars were ventilated after 24 hours of application. Day 0 samples from jars were extracted immediately after ventilation of fumigants. All the remaining jars were incubated at room temperature ± 20^oC and extracted following 7, 14 and 28 days of incubation. Ammonium nitrogen (NH₄-N) and nitrate nitrogen (NO₃-N) were measured on 0, 7, 14 and 28 days of extracted samples.

Analytical method performance was monitored by obtaining by duplicating the sample and analysis of reference standard. 

The method detection limit for NH₄-N and NO₃-N was set at 0.05 mg L^-1. Laboratory quality control reference samples (15.0 mg ml^-1of NH₄-N and 15.0 mg ml^-1of NO₃) were analysed concurrently along with samples and blanks. The recoveries for most compounds were within the acceptable range of 80 to 120%. The methods were therefore found acceptable for the use in this study.

The OECD 216 Guideline states in the “INITIAL CONSIDERATIONS” section that “If agrochemicals (e.g. crop protection products, fertilisers, forestry chemicals) are tested, both nitrogen transformation and carbon transformation tests are conducted.” Therefore, the results of the study should be interpreted along with the carbon transformation study.

The results from this study do not reflect total microbial activities. High concentrations of ammonium produced from the degradation of cyanogen can suppress nitrification of ammonium to nitrate. The Carbon transformation study accurately reflects total microbial activities in soil.

Total N transformation is higher in the ethanedinitrile treated soil compared with control soil. Similarly, the total N transformation in alfalfa amended ethanedinitrile soil was higher than alfalfa only treated soil. This shows that Total N transformation occurs at high rate in ethanedinitrile treated soil compared with all other non-ethanedinitrile treated soils.

The total N transformation comprised of both NH₄-N and NO₃-N concentrations. The NH4-N concentrations inethanedinitrile treated soils were higher than control and alfalfa treatment. The differences were greater than 25%. For NO₃-N, there were no difference between control and ethanedinitrile treated soil. But the NO₃-N concentrations in alfalfa-amended soils were higher on days 14 and 28 than all other treatments (control,ethanedinitrile, alfalfa +ethanedinitrile). NO₃-N concentrations were the lowest in alfalfa amended with high dose rate ofethanedinitrile, this is due to excess amount ofethanedinitrile (5 times above the recommended dose rate) used in this study that would have formed excess NH4-N toxic to nitrifying bacteria but it will not happen in commercial situation where the soil is treated with the recommended dose rate.

The NH₄-N concentration obtained for the control soil and alfalfa-amended soil was negligible but high for all ethanedinitrile-treated soils. NH₄-N concentration increased sharply with incubation period for all treated soils.

NO₃-N concentrations in alfalfa-amended soils were higher on days 14 and 28 than all other treatments. NO₃-N values were lowest for alfalfa-amended soil treated with high levels of ethanedinitrile. 

NH₄-N concentrations in ethanedinitrile-treated soils increased with increased incubation period. Differences between the controls and all ethanedinitrile-treated soils were greater than 25%. These results indicate that the addition of ethanedinitrile to soil reacts with water and forms additional NH₄N.

For NO₃-N, however, there were no differences between the control soils and the ethanedinitrile-treated soil until day 14. Maximum recovery of nitrogen transformation occurred in the alfalfa treated soil without ethanedinitrile. The alfalfa-amended ethanedinitrile-treated soil showed much less recovery of NO₃-N. However,NO₃-N concentrations in alfalfa-amended soils were higher on days 14 and 28 than all other treatments (control, ethanedinitrile, and alfalfa + plus ethanedinitrile). NO₃-N concentrations were the lowest in alfalfa amended with high rate of ethanedinitrile due to excess amount of ammonium produced from the degradation of ethanedinitrile (5 times above the recommended dose rate) used in this study.

Addendum

The Addendum lists the NH4-N and NO3-N concentrations in soil after 90 days. Nitrogen transformation results are shown for NH4-N and NO3-N, respectively, for 90 days compared to 28 days of incubation. These results show that the amount of NO3-N mineralized in the alfalfa-treated soils was identical to the amount of NO3-N mineralized in the alfalfa plus ethanedinitrile-treated soils. Although most of the NH4-N in the ethanedinitrile treatments was mineralized within 90 days, a small amount of nitrate was extracted possibly due to immobilization of the mineralized NO3-N by soil microorganisms. This study shows that ethanedinitrile has no long-term effect on microbial activities involved in N transformations in soil.

Soil Microorganism-Carbon Transformation for Cyanogen (C2N2) Study using OECD 217 test method (08_EDN_AJWA_Carbon_2015):

This study was performed to determine the effects of ethanedinitrile on carbon transformation by soil microorganisms to comply with the requirements for an ecotoxicology study on plant protection products ‘Effects on soil non-target microorganisms’. The study was conducted according to the OECD-Guideline for the testing of chemicals, Soil Micro-organisms: Nitrogen transformation test, Guideline 216, the OECD guideline for the testing of chemicals, soil microorganisms, Carbon Transformation test, Guidelines 217.

Ethanedinitrile was applied at the maximum application rate (MAR) of 667 mg kg^-1soil and at 5 times the MAR (3,333 mg kg^-1soil.) The test soil was a sandy loam obtained from a site near the USDA facility of Salinas, CA.

Soil jars were induced with ethanedinitrile in the lab according to the study protocol. The soils were incubated in the dark at ± 20^oC. All jars were capped with butyl rubber septa. All jars were analyzed for the amount of

C₂N₂ one hour after application and before ventilation for confirmation. CO₂was measured at different time intervals until the 28^th day.

Prior to sample analysis, the analytical method was validated at the Ajwa Analytical Laboratories in Salinas, CA using pure CO₂and C₂N₂ gases from Stainles Steel cylinders or Tedlar sampling bags. The recoveries for all concurrent fortified samples were in the acceptable range of 80 to 120%. The methods were therefore found acceptable for use in this study.

Analytical method performance was monitored by obtaining a six point calibration curve for CO₂ at the beginning and the end of each batch of samples analyzed. A calibration check on standard quality control samples was run after approximately 9 or 18 samples had been analyzed. A sample concentration that exceeded the calibration range was appropriately diluted and analyzed again such that it fell within the linear response range of the standards injected.

The Reporting Limit (RL) was set at 0.03 µg mL^-1for CO_2. Laboratory fortified quality control samples (1.0 mL CO₂per 120 mL bottle ≈ 4.0887µg mL^-1) of CO₂were analyzed concurrently along with samples and blanks in the micro GC. The recoveries for CO₂were within the acceptable range of 80 to 120%. 

The results from this study demonstrated that the treatment of soil with glucose addition releases more CO₂ at the beginning and glucose with ethanedinitrile treatment released more CO₂ at a later period. It was

Initially, the soil respiration rate was highest for the glucose amended soil. Carbon transformation in ethanedinitrile-treated soils was low until day 13 when it increased substantially, peaked and then began declining. Cumulative CO₂-C differences between the control and all treated soils were under 25% during the 28-day period.

There were no differences in cumulative CO₂-C between the control soil and the ethanedinitrile-treated soil during the28 day period, but the glucose-amended ethanedinitrile soil showed lower initial concentration of CO₂-C, increasing sharply after two weeks of ethanedinitrile applications.

Ethanedinitrile is very stable in dry air, but degrades quickly in soil and neutral pH water or high humidity. Fast degradation of ethanedinitrile under field condition is expected and results from this study represent realistic field application of ethanedinitrile.

Description of key information

The substance will be dispatched in the air and will get quickly diluted due to its high volatility. The substance will stay in the air based on its physical and chemical properties and will not transfer to other environmental compartments such as soil and water as confirmed by models in the environmental fate part of dossier.