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Toxicity to soil microorganisms

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Description of key information

OECD 216, Nitrogen transformation assay: EC10(28d): could not be determined, EC25(28d) > 1000 mg/kg soil d.w., EC50(28d) > 1000 mg/kg soil d.w.
OECD 217, Carbon transformation assay: EC10(28d): 29.94 mg/kg soil d.w. (95 % CL: 0.83 - 83.65 mg/kg soil d.w.), EC20(28d): 264 mg/kg soil d.w. (95 % CL: 102.98 - 616.35 mg/kg soil d.w.), EC50(28d) > 1000 mg/kg soil d.w.

Key value for chemical safety assessment

Long-term EC10 or NOEC for soil microorganisms:
29.94 mg/kg soil dw

Additional information

The toxicity of Fe(Na)HBED towards soil microorganisms was investigated in a nitrogen transformation assay, which was conducted in accordance to OECD Guideline 216 / EU Method C.21 (Swoboda, 2008) as well as in a carbon transformation assay, conducted according to OECD Guideline 217 / EU Method C.22 (Swoboda, 2008). No deviations from the corresponding guidelines are reported and the validity criteria were fulfilled. Both studies were conducted under certificated GLP compliance, thus both are regarded as "key information" with reliability Klimisch 2 (reliable without restriction).

In both experiments, freshly collected soil, manually cleared from large objects and sieved to particle size equal to 2 mm was used for testing. The sampling place has not been used for agricultural purposes during the past five years, no plant protection products, as well as organic and inorganic fertilisers have been used. Samples were taken down to 20 cm depth.

In the nitrogen transformation assay, amendment was done with lucerne as an organic substrate. Five concentrations were tested: 24, 64, 160, 400 and 1000 mg/kg of soil. The test soil as well as controls was incubated in three replicates. At the beginning and after 28 days of incubation, the quantities of nitrate were determined. The principle of the method was based on the spectrophotometric measurement of the nitrate ions concentration in soil extract of the 1 % potassium sulphate (VI) solution. Analysis was done by measurement of the intensity of yellow colour, generated in reaction with phenyldisulponic acid with spectrophotometer. The nitrate formation rate in each treatment was compared with that in control, and the per cent deviation from the control was calculated. The EC25(28d) as well as the EC50(28d) was determined to be greater than 1000 mg/kg. The EC10(28d) could not be evaluated based on the obtained results.

In the carbon transformation assay three replicates of each test concentration and control were incubated in darkness at a temperature range of 20 - 21 °C. The containers were covered with moistened polypropylene material and the surface of the soil was moistened with distilled water. After 28 days, the Substrate-Induced Respiration (SIR) was used to determine the intensity of soil respiration. During respiration in a closed system, carbon dioxide (which is generated while respiration) is bound to an absorber (45 % KOH), which results in a pressure drop. This pressure drop is proportional to the soil respiration. For this purpose, 100 g samples were thoroughly mixed with 200 mg glucose/kg and incubated for 12 h at a temperature around 20 °C. Measurement was started one hour after glucose addition and during a time duration of 12 h the total quantities of oxygen consumption and mean respiration rates were determined. Immediately after introduction of the test substance into soil the statistical significance of difference (P < 0.05) between control and soil treated with the test substance at concentrations of 64 - 1000 mg/kg was observed. In these concentrations decrease of inspiration activity was observed and the highest one occurred in the highest test concentration and was equal to 7 %. After 28 days, the statistical significance of difference (P < 0.05) between control and soil treated with the test material was observed in every concentration of tested material. Decrease of respiration activity as compared to control ranged from 9.8 for 24 and 64 mg/kg to 26.9 % for 1000 mg/kg. Analysis of variance (ANOVA) was used for evaluation and the statistical significance (P < 0.05) of differences was assessed by post hoc comparison of means using lowest significant differences (LSD-test) with help of the program STATISTICA v5.0. The EC-values were evaluated with the help of the computer program ToxRatPro Version 2.09. As results, the following values are reported, including 95 % CL in brackets: EC10(28d): 29.94 (0.83 - 83.65) mg/kg soil, EC20(28d): 264 (102.98 - 616.35) mg/kg soil and EC50(28d) > 1000 mg/kg soil.