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Toxicity to soil macroorganisms except arthropods

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Several studies are available and indicate that nano- and microsized TiO2 (including anatase, rutile and mixed forms) in natural soil and artificial substrate do not affect survival, reproduction and growth of Eisenia fetida and Eisenia andrei as well as survival and reproduction of Enchytraeus crypticus up to at least 1000 mg/kg.

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Regarding toxicity to the reproduction of Eisenia fetida and Eisenia andrei, Schlich et al. (2012) and Wyrwoll et al. (2014) did not observe effects of nanosized and microsized TiO2 up to 1000 mg/kg dw soil:


In the study by Wyrwoll et al. (2014), dispersions of the microsized TiO2 material Tiona AT1 (200 nm, anatase), and of the nano-sized TiO2 materials Hombikat UV 100 (7-10 nm, anatase) and PC 105 (15-25 nm, anatase) in a natural test soil did not affect the survival of Eisenia fetida in the acute limit toxicity test (OECD 207) and did not affect growth and reproduction in the earthworm reproduction limit test (OECD 222) resulting in 28-d NOEC values of ≥ 1000 mg/kg soil dw (nominal) for survival and reproduction. Schlich et al. (2012) tested three different nano-sized TiO2 materials, i.e. NM 105 (21 nm, rutile/anatase), NM 101 (8 nm, anatase) and NM 103 (20 nm, rutile, coated), in a standard toxicity test according to OECD 222 (2004). The TiO2 materials did not reduce the number of offspring, and adults of Eisenia andrei when applied to the test system via powder or dispersion on feed or soil. Depending on the test setup, the 56-d and 28-d NOEC values ranged from ≥ 20 (dispersion) to ≥ 1000 mg/kg soil dw (powder; nominal). On the contrary, significant stimulation of reproduction was observed for some TiO2 materials (P25, NM 101) with increasing levels. However, the effect may be due to an improvement of the test soil.

Additionally, information is available on the toxicity of nanosized TiO2 to Enchytraeid worms from a study by Hund-Rinke et al. (2016): In a standard toxicity test according to OECD 220 (2004), the two different nano-sized TiO2 materials NM-104 (26 nm, rutile, Al-coated) and NM 105 (P25; 21 nm, anatase/rutile) did not affect the survival and reproduction of Enchytraeus crypticus (28 d EC10 and EC50 > 1000 mg TiO2 NP/kg soil dw (nominal)).

Several supporting studies on the toxicity of nanosized and microsized TiO2 to earthworm exist:

Micro-TiO2 material Titanium oxide-325 mesh (< 40 µm) did not affect survival and reproduction of Eisenia andrei and survival, reproduction and juvenile growth of Eisenia fetida in a sandy-loam soil resulting in 28-d, 56-d and 126 d NOEC values of ≥ 200 mg/kg dw (nominal) in a study by McShane et al. (2012). Higher concentrations were applied to an artificial substrate and the natural soil by dry-mixing resulting in 28-d and 56-d NOEC values of ≥ 10,000 mg/kg soil dw (nominal) for all endpoints. The toxicity of nanosized TiO2 material to the survival and reproduction of Eisenia andrei and Eisenia fetida was studied in artificial substrate and in a natural soil, the latter only with E. Andrei, according to OECD 207 and OECD 222, resulting in 28-d and 56-d NOEC values of ≥ 10,000 mg/kg soil dw (nominal) for adult survival and reproduction. However, tests were performed in the dark to avoid potential phototoxicity and thus potentially reducing the exposure of the worms. Under standard-light-darkness cycles, earthworms tend to remain in the soil under light conditions. Hence, results of these experiments may not be conservative and are considered supporting data.

Further, McShane et al. (2012) observed in an earthworm avoidance test with Eisenia fetida that the avoidance behaviour was not affected by microsized TiO2 at 10,000 mg/kg soil. Results for the three nano-sized TiO2 materials vary but indicate that earthworms do not avoid TiO2-NP concentrations up to 100 mg/kg soil but consistently avoid higher levels of 10,000 mg/kg soil.

Whitfield Aslund et al. (2012) used metabolomics to examine the response of Eisenia fetida earthworms raised from juveniles for 20−23 weeks in soil spiked with 20 or 200 mg/kg nano- and microsized TiO2. Data seem to indicate that earthworms exhibited changes in their metabolic profile relative to the control after exposure to 200 mg/kg of nano- and microsized TiO2. However, the correlation between the observed metabolomic response and ecologically relevant endpoints is missing to date.

Lapied, E. et al. (2011) exposed Lumbricus terrestris for 2-8 weeks to a TiO2 nanocomposite (TiO2 core coated with superposed layers of Al(OH)3 and PDMS) mixed into food or soil at concentrations up to 100 mg/kg. Apoptosis was measured by immunohistochemistry. Mortality and apoptotic frequency were not affected in soil-exposed worms. Based on Ti localization by XRF microscopy, nanoparticles appear to be rather inert and did not cross the intestinal epithelium/ chloragogenous matrix barrier to enter the coelomic liquid, or the cuticular barrier to reach the muscular layers.

The fact that direct exposure to TiO2 NPs did not impair the immuno-effectiveness of earthworm Eisenia veneta (Rosa) coelomocyte cells at concentrations well above those predicted for sewage sludge in the UK (25 mg/L) appears to be further evidence of the low potential of TiO2 nanoparticles for toxicity to earthworms (Johnson et al. 2011).

In sum and applying a weight of evidence approach, nano- and microsized TiO2 (rutile, anatase and mixed forms) appear to have a low potential for short- and long-term toxicity to soil annelids based on data available for different earthworm species and one enchytraeid species up to at least 1000 mg/kg dw soil.