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Earthworm reproduction test:

The chronic effects of TBBS on the earthworm Eisenia fetida were evaluated under laboratory test conditions using an artificial soil substrate containing 5% w/w peat content. In the present study no adverse effects on either mortality, biomass development and morphological changes were observed at concentrations up to and including 750 mg test item/kg soil dry weight. In terms of effects on earthworm reproduction the NOEC was determined as 133.4 mg test item/kg soil (dw) and LOEC as 237.2 mg test item/kg soil (dw). Since inhibition of reproduction was <50%, no EC50 value was calculated; the EC50 was regarded to be >750 mg test item/kg soil dw.

Terrestrial Plant Seedling Emergence and Seedling Growth Test:

The test item had no adverse effect on either seedling emergence or seedling survival. It induced however adverse effects on shoot fresh weight of higher terrestrial plants at concentrations of ≥4.0 mg/kg soil dry weight (= LOEC). The lowest NOEC of 3.16 mg/kg soil dry weight was estimated by a conservative evaluation of the diverging results of two test runs with Pisum sativum.

The lowest EC10 and EC50 were calculated to be 1.7 and 41.8 mg/kg soil dry weight, respectively, and were seen with Allium cepa. While neither seedling emergence nor seedling survival in test item treated differed significantly from emergence or survival in the control, soil shoot fresh weight was clearly affected. At test item concentrations of ≥4.0 mg/kg soil dry weight shoot fresh weight was significantly reduced by 17.8% in the test species Pisum sativum (pea) which with regard to the LOEC turned out to be the most sensitive among the six tested species in test run DT1. Allium cepa (onion) and the monocotyledonous Avena sativa (oats) revealed reduced shoot weight at concentrations of 12.6 mg/kg soil dry weight and above. The lowest EC10 and EC50 of 1.7 mg/kg soil dw and 41.8 mg/kg soil dw, respectively, was found with A. cepa.

Surprisingly, in the repeated test (DT2) with A. sativa and P. sativum adverse effects on shoot fresh weight occurred only in P. sativum and at the highest tested concentration (316 mg/kg soil dry weight) although seeds, soil and overall growth conditions were the same in both test runs, DT1 and DT2. Due to the diverging results in the DT1 and DT2 test with Pisum sativum there remains some uncertainty regarding the test item concentration that causes an adverse effect on shoot fresh weight. A conservative evaluation would be to consider both, the LOEC from DT1 (4.0 mg/kg soil dw) and the results from DT2. Consequently the overall NOEC would be 3.16 mg/kg soil dw. Visual damages of P sativum seedlings were seen at ≥400 mg/kg soil in DT1 and at ≥100 mg/kg soil in DT2.

Effects on Soil Microorganisms - N-transformation test (OECD 216):

The study was conducted in order to determine possible effects of TBBS on soil microorganisms through measuring microbial nitrate formation in treated versus untreated soils after 28 days of incubation following OECD guideline No. 216. Nitrate concentration in soil increased with increasing Vulkacit NZ/TBBS concentration up to and including 205 mg/kg soil dry weight probably due to the introduction of the test item containing nitrogen. Nitrate concentration in soil was statistically significantly reduced at test item concentrations of 328 mg/kg soil dry weight and above. The EC10, EC20 and EC50 were 248, 286 and 377 mg/kg soil dry weight, respectively. The 28d-NOEC was 205 mg/kg soil dry weight. Therefore, TBBS is considered to have no long term adverse effect on nitrogen transformation in soil at concentrations up to and including 205 mg/kg soil dry weight.

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