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Toxicity to microorganisms

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The acute toxicity to aquatic microorganisms was tested using titanium dioxide and titanium tetrachloride. Hence, for titanium carbide this endpoint is derived by read-across from titanium dioxide and titanium tetrachloride.
The Tetrahymena growth inhibition test (Sauvant et al. 1995a, 1995b) shows the lowest effect value of EC50 (6 & 9 h) = 20 mg Ti/L.

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Additional information

Sauvant et al (1995a, 1995b) examined the toxicity of TiCl4 to Tetrahymena pyriformis by incubating the test organisms in the exponential growth phase for up to 9 hours with the test substance. Growth inhibition of Tetrahymena pyriformis was determined by measuring the cellular density. Growth of Tetrahymena pyriformis is inhibited by 50 % at 35, 20, and 20 mg Ti/L after 3, 6, and 9 h, respectively.


Adams et al. (2006) studied the toxic effects of TiO2 (particle size 330 nm) to Gram-positive bacteria Bacillus subtilis and Gram-negative bacteria E. coli with and without illumination (natural sunlight). The 14-h NOEC for TiO2 (330 nm, illumination) in Bacillus subtilis was determined to be 500 ppm. The next higher concentration of 1000 ppm caused 75 % inhibition. The 14-h NOEC for TiO2 (330 nm, illumination) in E. coli was determined to be 100 ppm. 44 ± 7.0 % and 46 ± 11.3 % inhibition could be observed at 1000 ppm and 2000 ppm, respectively. Illumination significantly (p < 0.05) enhanced toxicity of TiO2 to Bacillus subtilis and E. coli, with growth inhibition being 2.5-fold and 1.8-fold greater in the presence of light, respectively. Thus, the authors conclude that the antibacterial effect of TiO2 is related to the photocatalytic production of reactive oxygen species in addition to a supplementary mechanism as TiO2 toxicity also occurs under dark conditions.


Inhibition of TiO2 nanoparticles to Vibrio fischeri was investigated by measurement of luminescence (> Flash Assay) and growth (Heinlaan et al., 2008). Inhibition of the bacterial luminescence [%] was calculated after 30 min. NOEC (defined as < 20 % inhibition), EC20 and EC50 values were calculated. Subsequently, 72-h growth tests were conducted. Under the conditions of this study TiO2 is not inhibitory to growth and luminescence of V. fischeri up to concentrations of 20,000 mg/L.


Effects of TiO2 nanoparticles on Vibrio fischeri were also evaluated by Velzeboer et al. (2008) who exposed the test organisms at nominal concentrations of 0 (control) and 100 mg/L for up to 30 minutes. Fluorescence as compared to the control was determined after 0, 5, 15, and 30 minutes. Also in this test fluorescence was not affected.


According to ECHA Guidance R.7b "[...] single species tests with e.g. Vibrio fischeri (used in the MICROTOX® test), Pseudomonas fluorescens or Escherichia coli should be considered of low relevance for STPs. The tests with P. fluorescens and E. coli (Bringmann and Kühn 1960) cannot be used for determination of the PNECstp as they use glucose as a substrate (nor is E. coli a bacterium that will tend to multiply in an activated sludge environment). Likewise, Vibrio fisheri requires a high salinity environment. The information from such single-species screening tests may eventually be considered together with other existing data in a weight-of-evidence approach."

Available results were obtained in tests conducted above the solubility limit of the TiO2. In general, according to ECHA Guidance R.7b "Microbial toxicity testing above the solubility limit of a chemical is to be avoided, similar to toxicity test with higher organisms. It is also unrealistic because insoluble chemicals will be removed in the primary settling tank or fat trap of full scale installations, and thus will not reach the activated sludge. However, data from existing tests where the experimentally derived NOEC is higher than the aqueous solubility can still be used as valid information to derive a PNECstp. This can be justified because it is a conservative estimate unlikely to occur in practice, and because undissolved test substance is found to be less confounding in microbial tests than in tests with higher organisms." Therefore, the available data are used for substance assessment.

Furthermore, ECHA Guidance R.7b states that "Ciliate-based test data can be used for deriving a PNECstp in case these are the sole data available or in multiple-data situations where the ciliates have the lowest NOEC." Thus, as the Tetrahymena growth inhibition test (Sauvant et al. 1995a, 1995b) shows the lowest effect value, the EC50 (6 & 9 h) of 20 mg Ti/L is used in a WoE approach and is used as basis for substance assessment. 


Due to lower transformation/dissolution results for titanium carbide (the target substance) than titanium dioxide and titanium trichloride (the source substances), the resulting toxicity potential would also be expected to be lower. Therefore, the dose descriptors are expected to be sufficiently high for the target substance, and read-across to the source chemical is adequately protective. In fact, (eco-)toxicologically relevant release of Ti ions from titanium carbide is not expected as the concentration of soluble Ti ions was below the method detection limit (< 0.4 µg/L) in the T/D test. Thus, TiC is considered to be practically insoluble. Release of Ti ions to any ecotoxicologically relevant extent (and potential subsequent formation of soluble and/or insoluble Ti compounds) is not expected. Therefore, any toxic effects to aquatic microorganisms are not expected to arise from TiC and a PNECstp is not derived from the available data on TiCl4.