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Toxicity to soil microorganisms

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Description of key information

The NOEC for toxicity to soil microorganisms is 2930 mg Sb/kg dw (Smolders et al., 2007).

Key value for chemical safety assessment

Additional information

There are three studies available on antimony toxicity to microflora which included analytical confirmation of the exposure concentrations. However, as stated above, only the study by Smolders et al. (2007), which resulted in a bounded NOEC of 2930 mg Sb/kg dw, will be used when deriving PNECsoil. In addition, Guillamot et al (2014) evaluated the effect of antimony trioxide and potassium antimony tartrate trihydrate on the microbial communities of a contaminated and non-contaminated soil (andpoint: SIR), but the authors observed that SIR was more significantly affected by Sb input in a non-contaminated soil than in a long- term contaminated soil, and rthis after an exposure period of 3 days. After a 3-month incubation, positive effects of Sb2O3 occurred on SIR; no explanation was provided for this observation. No EC10 or EC50 were reported, and therefore the study is not included in the dossier.

Study 1

Smolders et al. (2007) studied the toxicity of Sb to native microorganisms in 31 week aged Sb2O3 amended soil (measured concentrations = 3, 90, 322, 999, 2930 and 10119 mg Sb/kg dw) using a nitrification assay based on ISO 14238-1 (1997). The test measures the Substrate Induced Nitrification rate (SIN), which is the nitrification rate induced after substrate (NH4+) supply measured during 28 days of incubation. Soils were preincubated for three days at a moisture content of 23%, taking into account the volume of (NH4)2SO4 (80 mg N/mL) that was added later. Afterwards (NH4)2SO4 was added to soil to a final concentration of 100 mg NH4-N/kg fresh soil by thoroughly mixing. Soils were incubated in an incubation room at 20°C. After 0, 7 (additional sampling compared to the ISO protocol) and 28 days, the NO3 - and NH4 + concentrations were measured colorimetrically in a shaken and centrifuged soil extract (soil in 12.5 mL KCl, 2 h end-over-end shaking, 15 min centrifugation at 3500g) and the SIN was calculated as the daily production of NO3-/kg soil, based on the measurements between day 0 and day 28. The nitrate production rate was also calculated in the initial period (0-7 days) during which there was unlimited substrate present; this endpoint is termed the Potential Nitrification Rate (PNR, also referred to as the nitrification potential in the literature). This means that two endpoints were defined: the SIN and the PNR. The ISO guideline only recommends the latter, but the PNR endpoint has been reported as being more sensitive than the SIN endpoint since the former is measured during the initial period after NH4+ addition, i.e. when the substrate is still abundantly present (Smolders et al.,2001). The test was performed in duplicate per soil. No validity criteria are given in the guideline ISO 14238-1 from 1997, but the nitrification rate in the control soil was considered to be within expected limits and the results are therefore considered reliable.

The SIN endpoint resulted in an unbounded NOEC of >10119 mg Sb/kg dw, while the PNR endpoint resulted in a bounded NOEC of 2930 mg Sb/kg dw and a LOEC of 10119 mg Sb/kg dw. The pore water concentrations corresponding to the NOEC and LOEC for the PNR were 18.7 and 54.4 mg Sb/L, respectively. It was not possible to derive an EC10 or EC50 for the PNR endpoint.

As already mentioned, when presenting the results for plants by the same authors (i.e. Smolders et al., 2007), the calculation of the PNECsoil needs to correct for the fact that the results were obtained from soils that were not fully equilibrated.

Study 2

Oorts et al. (2005) studied the toxicity of antimony to native microorganisms in freshly spiked soil (Sb2O3 or SbCl3; control (measured concentration of 0.6 mg Sb/kg dw, plus nominal added concentrations of 20, 50, 100, 200, 500, 1000 and 2000 mg Sb/kg dw.), and in five-year aged Sb2O3 amended soil (measured concentrations 0.4, 5.8, 28.4, 71.7 and 116.3 mg Sb/kg dw). Seven days after metal spiking, triplicate subsamples of each Sb concentration of each soil were amended with 100 mg NH4-N/kg fresh soil. The increase in soil NO3-N in a period of 28 days after substrate addition was measured by analysing the soil nitrate colorimetrically in a centrifuged soil extract (KCl, subsample, L/S = 2.5, 2 h end-cover-end shaking, n=3). Two different endpoints were measured: the substrate induced nitrification (SIN) and the potential nitrification rate (PNR). SIN was calculated as the percentage of added NH4-substrate transformed into NO3-N during 28 days (OECD 216). PNR (mg NO3-N/kg fresh soil/day) was calculated as the slope of the regression of soil nitrate concentration against time. The PNR endpoint has been reported as being more sensitive than the SIN endpoint since the former is measured during the initial period after NH4+ addition, i.e. when the substrate is still abundantly present (Smolders et al.,2001). For the SbCl3amended samples, PNR was calculated for a seven day incubation period, and for a ten day period for all the other soils. The incubation time selected was identical for each sample within a dose-response study (i.e. for each Sb dose).

There was no toxicity observed in either of the Sb2O3 spiked soils, i.e. the freshly spiked soil and the five-year aged soils, for either of the two endpoints PNR and SIN. However, the reason for the lack of response in the aged Sb2O3 soil might be that the microflora had adapted to the different Sb concentrations during the five years of aging, and that therefore no response is to be expected.This is in contrast to the freshly spiked SbCl3 soils where the NOECPNR was 73 mg Sb/kg dw (nominal added concentration of 100 mg Sb/kg dw) and the NOECSIN was 384 mg Sb/kg dw (nominal added concentration of 500 mg Sb/kg dw). The potential effect of the counter ion, i.e. Cl-, was tested separately. Increased concentrations of chloride decreased the PNR but not the SIN (a slight stimulation was observed with increasing concentrations of chloride). The measured PNR and SIN in the separate chloride toxicity experiment were about 70-80 % of the PNR and SIN in the control of the freshly spiked SbCl3 soils. In addition, the dose-response pattern with decreasing PNR with increasing concentrations of chloride (measured as electrical conductivity) differed. It is therefore not clear how much of the decrease in PNR resulted from increased concentrations of chloride. According to the authors (Oorts et al., 2005), about 50% of the decrease was explained by increased concentrations of chloride. The rest of the decrease in PNR and all of the decrease of SIN was proposed by Oorts et al (2005) to be due to reduced pH, since the nitrification process has been reported to be sensitive to changes in pH.

The response observed for SIN is easier to interpret since there was no observed effect of the counter ion. The observed response may therefore be assumed to result either from increased concentrations of Sb or decreased pH. The NOEC for SIN in the freshly spiked SbCl3 soil based on the measured Sb soil solution concentration was 13.1 mg Sb/L. This can be compared with the Sb soil solution concentration measured in the highest freshly Sb2O3 spiked soil, which was 13.9 mg Sb/L. Despite probable differences in toxic pressure in these two exposure regimes, the two results both appear to indicate that the toxicity to microorganisms measured via SIN is low. The reason for a large response in the freshly spiked SbCl3 soils is unclear as there was a relatively small increase in the measured Sb soil solution concentration from 13.1 mg Sb/L (pH = 6.68) with 96% substrate used, to 14.4 mg Sb/L (pH = 6.58) with 73% substrate used, to 15.85 mg Sb/L (pH = 5.94) with 15% substrate used. The decrease from 96% substrate used at pH = 6.58 to 73% substrate used at pH = 6.58 appears especially large if the reduction of pH is a major factor. This is since the decrease in pH from the control with 100% of the substrate used at pH = 7.04 to pH = 6.68 only reduced the substrate used to 96%.

There was no response of SIN in the aged Sb2O3 spiked soils up to soil solution concentrations of 4.2 mg Sb/L, which may be due to adapted microflora, but is also in agreement with the NOECs in the freshly spiked soils, since no toxicity was observed at that Sb concentration level.

Study 3

In this report by LISEC (2002), the effect of Sb2O3 on the nitrogen transformation activity of soil organisms was studied. The soil used was a sandy loam. The test substance was applied using quartz sand as a carrier. The test was performed with three replicates (with an additional fourth replicate for Sb measurement), and five concentrations (range: 2.6-823 mg Sb/mg dw) and a control (concentration below detection limit of 1.8 mg Sb/kg dw). The test was terminated after 28 days when the quantities of nitrate were determined. There were no significant difference in nitrogen transformation activity between the highest concentration used (823 mg Sb/kg dw) and the control, resulting in a LOEC > 823 mg Sb/kg dw. The value of this unbounded NOEC is unclear due tothe uncertainty associated with a lack of response using freshly spiked Sb2O3 soils. It is not possible to know how the Sb soil solution concentration changed (most probably increased) during the exposure period. It can therefore not be excluded that the absence of observed effects, resulting in an unbounded NOEC, may be the consequence of too low an Sb soil solution concentration for too long during the exposure period. To conclude, the results from this study are not considered valid.