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Ecotoxicological information

Toxicity to soil microorganisms

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toxicity to soil microorganisms
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 216 (Soil Microorganisms: Nitrogen Transformation Test)
In order to avoid false positive results by reaction of the test substance with nitrite, ammonia reduction was used too as endpoint for the determination of inhibiting effects beside the formation of nitrate.
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
Details on preparation and application of test substrate:
- Type of organic substrate: Loamy sand according to DIN (supplied bei LuFa Speyer)

- Method: The test substance was grounded in a mortar. Than the required amount of the test substance for each test concentration were balanced with 5 g quartz sand as carrier (equal to 10 g/kg dry weight) and mixed well. The test substance was grounded in a mortar. Than the required amount of the test substance for each test concentration were balanced with 5 g quartz sand as carrier (equal to 10 g/kg dry weight) and mixed well. 14 portions of the prepared soil were blended with a mixer with quartz sand containing the test substance in the scheduled test concentrations and powdered ammonium sulfate (grounded in a mortar). 2 portions of the soil were mixed with quartz sand without the test substance and ammonium sulfate for blank control assays. The substrate proposed in OECD Guideline 216 (powdered plant matter) was not used because, as suggested in the guideline, there is not need to determine potential effect of the test substance (not being an agricultural chemical) on carbon transformation in soil. The potential variability in the carbon transformation process, which acts on the powdered plant matter to release ammonium, could therefore be avoided by direct application of a known quantity of a readily-available ammonium source. Therefore, in the modified test design, the plant meal amendment was omitted in all treatments, and not just the treatment associated with highest concentration of the test substance.

Test organisms (inoculum):
Total exposure duration:
28 d
Test temperature:
20.7-21.6 °C (2 times the scheduled temperature range was failed temporary with 22.9 °C and 23.3 °C. These filatures had no influence to the test.)
40 ± 5% of the WHCmax
Details on test conditions:
- Testing facility: BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany
- Amount of soil: approx. 579 g
- No. of replicates per concentration: 2
- No. of replicates per control: 2

- Method: bulk

- History of site: The soil was not treated with crop protection products for more than 4 years. The sampling field was not cultivated for more than 4 years and no fertilizer application was performed during the last 4 years before sampling the soil.
- Vegetation cover: no data
- Treatments with pesticides or fertilizers: no
- Accidental contamination: no
- C/N ratio: The C:N ratio of the organic matter naturally present in the test soil was 9:1.1
- Depth of sampling: approx. 20 cm
- Soil classification system: Loamy sand according to DIN, sandy loam according to USDA
- pH (in water): 7.3 ± 0.1 (0.01M CaCl2)
- Initial nitrate concentration for nitrogen transformation test (mg nitrate/kg dry weight): The initial nitrate concentration for the nitrogen transformation test was determined using triplicate sample analyses from duplicate control test vessels at Day 0. The measured nitrate concentrations ranged from 217.89 to 230.19 mg/kg d.w. (5.6 % difference between lowest and highest values)
- Cation exchange capacity: 16.6 ± 2.8 meq/100 g
- Pretreatment of soil: 48 h before start of the test 9576.11 g of the soil were mixed with 846 mL deionized water.
- Storage (condition, duration): The soil was stored from 12 Oct 2012 to 15 Oct 2012 in a cooled storage room in a closed plastic sack at a temperature of 6.7 °C to 7.8 °C in the dark until usage.
- Initial microbial biomass as % of total organic C: 484.4 ± 11.8 mg carbon/kg Dw/h
Nominal and measured concentrations:
10, 20, 50, 125, 250, 500 and 1000 mg of the test substance per kg dry weight of soil (nominal)
Reference substance (positive control):
28 d
Dose descriptor:
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
other: ammonium reduction
28 d
Dose descriptor:
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
other: ammonium reduction
Details on results:
- Effect concentrations exceeding solubility of substance in test medium: no
- no effects on nitrate formation were detected but nor EC-values were derived as only ammonia reduction results were used for statistical ECx avaluation.

- The basis for the reported effect endpoint was chosen as ammonium disappearance, and not nitrate formation, because the test substance has been shown to react directly with nitrite (a transient intermediate in the transformation of ammonium to nitrate). The biological transformation of ammonium to nitrate proceeds through nitrite as an intermediate. The study did not include analyses of, or information relating directly to, formation of nitrite. However, if inhibition of nitrite formation were to have occurred, this would be reflected in the results obtained for nitrate formation as a function of applied test substance and time. 

- The standard test design of OECD Guideline 216 involves the catabolism of soil organic matter and a plant matter amendment, which is presumed to result in release of ammonium, which is subsequently oxidized to nitrate. It is well-known that primary aromatic amines, such as the test substance, can react directly with nitrite to form a stable diazonium compound. It is also well-known that the biological transformation of ammonium to nitrate (i.e., nitrification in soil) occurs via a transient nitrite intermediate. Where the presence of the reactive aromatic amine test substance is concurrent with presence of active biological nitrification, a reduced yield of nitrite (and thus nitrate) could be observed which is not directly caused by inhibition or toxicity to the nitrifying microorganisms. In other words, even where the test substance may not have a direct or indirect effect on conversion of ammonium to nitrate (nitrification), such an effect could be inferred if reduced yield of nitrate were observed as a result of unquantified reaction of the transient nitrite intermediate with the test substance.

Validity criteria fulfilled:
Nitrification in soil is not inhibited by the test substance neither be measuring the ammonia decrease nor by the formation of nitrate as endpoints.

Description of key information

Nitrifying bacteria are not inhibited by the test substance.

Key value for chemical safety assessment

Short-term EC50 for soil microorganisms:
1 000 mg/kg soil dw
Long-term EC10 or NOEC for soil microorganisms:
1 000 mg/kg soil dw

Additional information

Inhibition of nitrogen cycle in soils was investigates in a GLP test according to OECD guideline 216 (BASF, 2013). After 20 days the EC10 and EC50 was estimated to be >1000 mg/kg soil (d.w.), related to the decrease of ammonia. No adverse effect was observed up to the highest tested concentration. Regarding the nitrate formation, no effects were detected as well.