TiO2 Photocatalysis Damages Lipids and Proteins in Escherichia coli

This study investigates the mechanisms of UV-A (315 to 400 nm) photocatalysis with titanium dioxide (TiO₂) applied to the degradation of Escherichia coli and their effects on two key cellular components: lipids and proteins. The impact of TiO₂ photocatalysis on E. coli survival was monitored by counting on agar plate and by assessing lipid peroxidation and performing proteomic analysis. We observed through malondialdehyde quantification that lipid peroxidation occurred during the photocatalytic process, and the addition of superoxide dismutase, which acts as a scavenger of the superoxide anion radical (O₂·⁻), inhibited this effect by half, showing us that O₂·⁻ radicals participate in the photocatalytic antimicrobial effect. Qualitative analysis using two-dimensional electrophoresis allowed selection of proteins for which spot modifications were observed during the applied treatments. Two-dimensional electrophoresis highlighted that among the selected protein spots, 7 and 19 spots had already disappeared in the dark in the presence of 0.1 g/liter and 0.4 g/liter TiO₂, respectively, which is accounted for by the cytotoxic effect of TiO₂. Exposure to 30 min of UV-A radiation in the presence of 0.1 g/liter and 0.4 g/liter TiO₂ increased the numbers of missing spots to 14 and 22, respectively. The proteins affected by photocatalytic oxidation were strongly heterogeneous in terms of location and functional category. We identified several porins, proteins implicated in stress response, in transport, and in bacterial metabolism. This study reveals the simultaneous effects of O₂·⁻ on lipid peroxidation and on the proteome during photocatalytic treatment and therefore contributes to a better understanding of molecular mechanisms in antibacterial photocatalytic treatment.     

Photopatterning Proteins and Cells in Aqueous Environment Using TiO2 Photocatalysis

Organic contaminants adsorbed on the surface of titanium dioxide (TiO₂) can be decomposed by photocatalysis under ultraviolet (UV) light. Here we describe a novel protocol employing the TiO₂ photocatalysis to locally alter cell affinity of the substrate surface. For this experiment, a thin TiO₂ film was sputter-coated on a glass coverslip, and the TiO₂ surface was subsequently modified with an organosilane monolayer derived from octadecyltrichlorosilane (OTS), which inhibits cell adhesion. The sample was immersed in a cell culture medium, and focused UV light was irradiated to an octagonal region. When a neuronal cell line PC12 cells were plated on the sample, cells adhered only on the UV-irradiated area. We further show that this surface modification can also be performed in situ, i.e., even when cells are growing on the substrate. Proper modification of the surface required an extracellular matrix protein collagen to be present in the medium at the time of UV irradiation. The technique presented here can potentially be employed in patterning multiple cell types for constructing coculture systems or to arbitrarily manipulate cells under culture.     

Photocatalytic Antibacterial Effects Are Maintained on Resin-Based TiO2 Nanocomposites after Cessation of UV Irradiation

Photocatalysis induced by TiO₂ and UV light constitutes a decontamination and antibacterial strategy utilized in many applications including self-cleaning environmental surfaces, water and air treatment. The present work reveals that antibacterial effects induced by photocatalysis can be maintained even after the cessation of UV irradiation. We show that resin-based composites containing 20% TiO₂ nanoparticles continue to provide a pronounced antibacterial effect against the pathogens Escherichia coli, Staphylococcus epidermidis, Streptococcus pyogenes, Streptococcus mutans and Enterococcus faecalis for up to two hours post UV. For biomaterials or implant coatings, where direct UV illumination is not feasible, a prolonged antibacterial effect after the cessation of the illumination would offer new unexplored treatment possibilities.     

Role of Visible Light-Activated Photocatalyst on the Reduction of Anthrax Spore-Induced Mortality in Mice

Background

Photocatalysis of titanium dioxide (TiO₂) substrates is primarily induced by ultraviolet light irradiation. Anion-doped TiO₂ substrates were shown to exhibit photocatalytic activities under visible-light illumination, relative environmentally-friendly materials. Their anti-spore activity against Bacillus anthracis, however, remains to be investigated. We evaluated these visible-light activated photocatalysts on the reduction of anthrax spore-induced pathogenesis.

Methodology/Principal Findings

Standard plating method was used to determine the inactivation of anthrax spore by visible light-induced photocatalysis. Mouse models were further employed to investigate the suppressive effects of the photocatalysis on anthrax toxin- and spore-mediated mortality. We found that anti-spore activities of visible light illuminated nitrogen- or carbon-doped titania thin films significantly reduced viability of anthrax spores. Even though the spore-killing efficiency is only approximately 25%, our data indicate that spores from photocatalyzed groups but not untreated groups have a less survival rate after macrophage clearance. In addition, the photocatalysis could directly inactivate lethal toxin, the major virulence factor of B. anthracis. In agreement with these results, we found that the photocatalyzed spores have tenfold less potency to induce mortality in mice. These data suggest that the photocatalysis might injury the spores through inactivating spore components.

Conclusion/Significance

Photocatalysis induced injuries of the spores might be more important than direct killing of spores to reduce pathogenicity in the host.     

Bactericidal Activity of Photocatalytic TiO2 Reaction: toward an Understanding of Its Killing Mechanism

When titanium dioxide (TiO₂) is irradiated with near-UV light, this semiconductor exhibits strong bactericidal activity. In this paper, we present the first evidence that the lipid peroxidation reaction is the underlying mechanism of death of Escherichia coli K-12 cells that are irradiated in the presence of the TiO₂ photocatalyst. Using production of malondialdehyde (MDA) as an index to assess cell membrane damage by lipid peroxidation, we observed that there was an exponential increase in the production of MDA, whose concentration reached 1.1 to 2.4 nmol · mg (dry weight) of cells⁻¹ after 30 min of illumination, and that the kinetics of this process paralleled cell death. Under these conditions, concomitant losses of 77 to 93% of the cell respiratory activity were also detected, as measured by both oxygen uptake and reduction of 2,3,5-triphenyltetrazolium chloride from succinate as the electron donor. The occurrence of lipid peroxidation and the simultaneous losses of both membrane-dependent respiratory activity and cell viability depended strictly on the presence of both light and TiO₂. We concluded that TiO₂ photocatalysis promoted peroxidation of the polyunsaturated phospholipid component of the lipid membrane initially and induced major disorder in the E. coli cell membrane. Subsequently, essential functions that rely on intact cell membrane architecture, such as respiratory activity, were lost, and cell death was inevitable.     

Differential Growth of and Nanoscale TiO2 Accumulation in Tetrahymena thermophila by Direct Feeding versus Trophic Transfer from Pseudomonas aeruginosa

Nanoscale titanium dioxide (TiO₂) is increasingly used in consumer goods and is entering waste streams, thereby exposing and potentially affecting environmental microbes. Protozoans could either take up TiO₂ directly from water and sediments or acquire TiO₂ during bactivory (ingestion of bacteria) of TiO₂-encrusted bacteria. Here, the route of exposure of the ciliated protozoan Tetrahymena thermophila to TiO₂ was varied and the growth of, and uptake and accumulation of TiO₂ by, T. thermophila were measured. While TiO₂ did not affect T. thermophila swimming or cellular morphology, direct TiO₂ exposure in rich growth medium resulted in a lower population yield. When TiO₂ exposure was by bactivory of Pseudomonas aeruginosa, the T. thermophila population yield and growth rate were lower than those that occurred during the bactivory of non-TiO₂-encrusted bacteria. Regardless of the feeding mode, T. thermophila cells internalized TiO₂ into their food vacuoles. Biomagnification of TiO₂ was not observed; this was attributed to the observation that TiO₂ appeared to be unable to cross the food vacuole membrane and enter the cytoplasm. Nevertheless, our findings imply that TiO₂ could be transferred into higher trophic levels within food webs and that the food web could be affected by the decreased growth rate and yield of organisms near the base of the web.     

A comparison of two methods for determining titanium dioxide marker content in broiler digestibility studies

The use of inert markers in broiler diets eliminates the need to quantitatively evaluate feed intake and excreta output to determine diet digestibility, and enables nutrient uptake at specific points along the gastrointestinal tract to be examined. Titanium dioxide (TiO₂) is commonly used for this purpose and measured using a UV-spectrophotometric assay. Two experiments were conducted to observe whether an inductively coupled plasma optical emission spectrophotometer (ICP-OES) assay is able to replace the UV-spectroscopy assay for rapid analysis of TiO₂ in broiler feed and ileal digesta samples. In the first experiment, TiO₂ was added at 5 g/kg to 19 broiler diets. Ross 308 male broilers (n=452) fed these diets were involved in a series of digestion studies to determine ileal digesta recovery of TiO₂. In the second experiment, defined amounts of TiO₂ were added to ileal digesta samples from Ross 308 male broilers (n=176) and TiO₂ recoveries were determined. The feed and ileal samples from both experiments were analysed by both UV-spectroscopy and ICP-OES, and relatedness of the findings from the two assays was determined. Overall relatedness of the two assays was strong for determination of TiO₂ concentration in both the broiler diets and ileal digesta samples (r=0.908 and r=0.884, respectively). Overall recovery of supplemented TiO₂ was 97.62% by the UV-spectroscopy assay and 98.77% by the ICP-OES assay. The ICP-OES assay in this study was as accurate as spectrophotometric determination for the quantification of TiO₂ content. The ICP-OES method can also be used to analyse several elements within one assay, with a single preparation step, and thus the measurement of TiO₂ may be incorporated into the analysis of other minerals. Time and resources dedicated to determining diet digestibility in broilers could be minimised by using the ICP-OES assay to replace the UV-spectroscopy assay when measuring TiO₂ concentration.     

Flexible polymer TiO2 modified film photocatalysts active in the photodegradation of azo-dyes in solution

This review summarizes the recent developments of thin film polymer coated photocatalysis with TiO₂ mediating the discoloration/degradation of the azo-dye Orange II under light irradiation. The stable anchoring of TiO₂ on non-heat resistant but chemically inert flexible polymer films is described. The nature of the polymer films used, the pretreatment of the film for the TiO₂ loading and the testing of the photocatalytic activity are addressed for different inert polymer films not having the conventional functional surface groups to bind TiO₂. The discoloration of Orange II in the presence of LDPE/TiO₂ is completed in about 10 h. This is a significantly longer times than the one observed for the same process when Tedlar/TiO₂ and Parylene/TiO₂ were used in the dye discoloration process. This points out to specific effects particular to each the polymer support used to graft the photoactive TiO₂ particles.     

Electrospun PVDF Nanofibers Decorated with Graphene and Titania for Improved Visible Light Photocatalytic Methanation of CO2

The photoreduction of carbon dioxide (CO₂) by a highly efficient Graphene@PVDF(polyvinylidene fluoride)@TiO₂(titania) electrospinning film photocatalysis system was explored, achieving an innovative combination of electrospinning and hydrothermal treatment using nanoscale pristine graphene sheets. TiO₂ was embedded in PVDF nanowire due to the inducement of element F, while the TiO₂ nanoparticles on PVDF facilitated transporting the photo-generated electron-hole pairs to the Ti-F groups, which performed as electron-trapping sites due to the strong electronegativity of fluorine. The possible mechanism of CO₂ reduction is depicted involving two distinct functional regions – the production regions (TiO₂) and the consumption regions (graphene sheets) of the photo-generated electrons. In this photocatalysis system, the photo-generated electrons are quickly captured and transferred in a timely manner, efficiently suppressing the recombination of photo-generated carriers. The electron reservoirs in the graphene sheets can then accelerate the photoreduction reaction and promote the conversion of CO₂ to CH₄ (methane), leading to a highly efficient photoreduction of CO₂ under visible light illumination, which is a promising material in new energy development, mitigating climate change.

     

Modulation of physiological responses with TiO<Subscript>2</Subscript> nano-particle in <Emphasis Type="Italic">Azolla pinnata</Emphasis> R.Br. under 2,4-D toxicity

The present work is emphasised with the herbicidal tolerance of Azolla pinnata R.Br. and its modulation with TiO₂ nano-particle. Both carbohydrate and nitrogen metabolism were effected with 2,4-D as herbicide and in few cases TiO₂-NP had recovered few detrimental effects. From the nutrient status in Azolla it recorded the recovery of nitrogen as well as potassium by TiO₂-NP but not in case of phosphorus. However, a conversion of nitrate to ammonium was more induced by TiO₂-NP under herbicidal toxicity. Similar results were obtained for inter-conversion of amino acid–nitrate pool, but no changes with glutamine synthase activity with TiO₂-NP. Initially, the effects of 2,4-D was monitored with changes of chlorophyll content but had not been recovered with nanoparticle. Photosynthetic reserves expressed as both total and reducing sugar were insensitive to TiO₂-NP interference but activity of soluble and wall bound invertase was in reverse trend as compared to control. The 2,4-D mediated changes of redox and its oxidative stress was ameliorated in plants with over expressed ADH activity. As a whole the Azolla bio system with TiO₂ supplementation may be useful in sustenance against 2,4-D toxicity through recovery of nitrogen metabolism. Thus, Azolla-TiO₂-NP bio system would be realised to monitor the herbicidal toxicity in soil and its possible bioremediation.     

Visible-Light-Induced Bactericidal Activity of a Nitrogen-Doped Titanium Photocatalyst against Human Pathogens

The antibacterial activity of photocatalytic titanium dioxide (TiO₂) substrates is induced primarily by UV light irradiation. Recently, nitrogen- and carbon-doped TiO₂ substrates were shown to exhibit photocatalytic activities under visible-light illumination. Their antibacterial activity, however, remains to be quantified. In this study, we demonstrated that nitrogen-doped TiO₂ substrates have superior visible-light-induced bactericidal activity against Escherichia coli compared to pure TiO₂ and carbon-doped TiO₂ substrates. We also found that protein- and light-absorbing contaminants partially reduce the bactericidal activity of nitrogen-doped TiO₂ substrates due to their light-shielding effects. In the pathogen-killing experiment, a significantly higher proportion of all tested pathogens, including Shigella flexneri, Listeria monocytogenes, Vibrio parahaemolyticus, Staphylococcus aureus, Streptococcus pyogenes, and Acinetobacter baumannii, were killed by visible-light-illuminated nitrogen-doped TiO₂ substrates than by pure TiO₂ substrates. These findings suggest that nitrogen-doped TiO₂ has potential application in the development of alternative disinfectants for environmental and medical usages.     

Enhanced phenol bioavailability by means of photocatalysis

Phenol was investigated for the ability of TiO₂ photocatalysis to increase its bioavailability as an electron donor for denitrification. The rate of nitrate removal by denitrification was increased by up to 2.6-fold by exposing phenol to photocatalysis for 30 min, although the rate decreased with increasing photocatalysis. The increased denitrification rate appeared to be associated with the photocatalytic production of carboxylic acids, but the slow down correlated to the production of catechol and hydroquinone.     

Photocatalytic antimicrobial activity of thin surface films of TiO(2), CuO and TiO (2)/CuO dual layers on Escherichia coli and bacteriophage T4

TiO₂-coated surfaces are increasingly studied for their ability to inactivate microorganisms. The activity of glass coated with thin films of TiO₂, CuO and hybrid CuO/TiO₂ prepared by atmospheric Chemical Vapour Deposition (Ap-CVD) and TiO₂ prepared by a sol–gel process was investigated using the inactivation of bacteriophage T4 as a model for inactivation of viruses. The chemical oxidising activity was also determined by measuring stearic acid oxidation. The results showed that the rate of inactivation of bacteriophage T4 increased with increasing chemical oxidising activity with the maximum rate obtained on highly active sol–gel preparations. However, these were delicate and easily damaged unlike the Ap-CVD coatings. Inactivation rates were highest on CuO and CuO/TiO₂ which had the lowest chemical oxidising activities. The inactivation of T4 was higher than that of Escherichia coli on low activity surfaces. The combination of photocatalysis and toxicity of copper acted synergistically to inactivate bacteriophage T4 and retained some self-cleaning activity. The presence of phosphate ions slowed inactivation but NaCl had no effect. The results show that TiO₂/CuO coated surfaces are highly antiviral and may have applications in the food and healthcare industries.     

Microplate-based assays for the evaluation of antibacterial effects of photocatalytic coatings

Three microplate-based viability assays for assessing the antibacterial effects of photocatalytic coatings were compared to the conventional colony count method. In the experimental design, cultured Escherichia coli were exposed to photocatalysis on various TiO₂ films in the presence of either UVA or visible light. The photocatalytic effects on the bacterial physiology were determined by real-time measurements of metabolic activity (XTT assay), biomass formation in the liquid medium (growth assay), and by assessing membrane integrity (with propidium iodide and SYTO 9 fluorescent nucleic acid binding dyes—BacLight assay). All three methods proved to be more sensitive and reproducible than colony count for the evaluation of the bactericidal effect of photocatalysis, XTT, and growth assay succeeded in detecting differences in both UVA and visible light-activated photocatalytic coatings. BacLight could efficiently detect the visible light-dependent photocatalytic effect on bacteria and identify membrane damage, but resulted inadequate for evaluating the UVA-dependent antibacterial effects. The described microplate-based evaluation methods proved being more effective and rapid than the colony count assay for assessing the antibacterial effect of various photocatalytic coatings.     

Sustainable approaches towards green synthesis of TiO2 nanomaterials and their applications in photocatalysis-mediated sensing to monitor environmental pollution

Nanomaterials are gaining enormous interests due to their novel applications that have been explored nearly in every field of our contemporary society. In this scenario, preparations of nanomaterials following green routes have attracted widespread attention in terms of sustainable, reliable, and environmentally friendly practices to produce diverse nanostructures. In this review, we summarize the fundamental processes and mechanisms of green synthesis approaches of TiO₂ nanoparticles (NPs). We explore the role of plants and microbes as natural bioresources to prepare TiO₂ NPs. Particularly, focus has been made to explore the potential of TiO₂-based nanomaterials to design a variety of sensing platforms by exploiting the photocatalysis efficiency under the influence of a light source. These types of sensing are of massive importance for monitoring environmental pollution and therefore for inventing advanced strategies to remediate hazardous pollutants and offer a clean environment.     

Degradation of reactive dyes in a photocatalytic circulating-bed biofilm reactor

Decolorization and mineralization of reactive dyes by intimately coupled TiO₂‐photocatalysis and biodegradation (ICPB) on a novel TiO₂‐coated biofilm carrier were investigated in a photocatalytic circulating‐bed biofilm reactor (PCBBR). Two typical reactive dyes—Reactive Black 5 (RB5) and Reactive Yellow 86 (RY86)—showed similar first‐order kinetics when being photocatalytically decolorized at low pH (～4–5) in batch experiments. Photocatalytic decolorization was inhibited at neutral pH in the presence of phosphate or carbonate buffer, presumably due to electrostatic repulsion from negatively charged surface sites on TiO₂, radical scavenging by phosphate or carbonate, or both. Therefore, continuous PCBBR experiments were carried out at a low pH (～4.5) to maintain high photocatalytic efficiency. In the PCBBR, photocatalysis alone with TiO₂‐coated carriers could remove target compound RB5 and COD by 97% and 47%, respectively. Addition of biofilm inside macroporous carriers maintained a similar RB5 removal efficiency, but COD removal increased to 65%, which is evidence of ICPB despite the low pH. ICPB was further proven by finding microorganisms inside carriers at the end of the PCBBR experiments. A proposed ICPB pathway for RB5 suggests that a major intermediate, a naphthol derivative, was responsible for most of the residual COD, while most of the nitrogen in the azo‐bonds (? N?N? ) was oxidized to N₂. Biotechnol. Bioeng. 2012; 109:884–893. © 2011 Wiley Periodicals, Inc.     

Bactericidal Performance of Visible-Light Responsive Titania Photocatalyst with Silver Nanostructures

Background

Titania dioxide (TiO₂) photocatalyst is primarily induced by ultraviolet light irradiation. Visible-light responsive anion-doped TiO₂ photocatalysts contain higher quantum efficiency under sunlight and can be used safely in indoor settings without exposing to biohazardous ultraviolet light. The antibacterial efficiency, however, remains to be further improved.

Methodology/Principal Findings

Using thermal reduction method, here we synthesized silver-nanostructures coated TiO₂ thin films that contain a high visible-light responsive antibacterial property. Among our tested titania substrates including TiO₂, carbon-doped TiO₂ [TiO₂ (C)] and nitrogen-doped TiO₂ [TiO₂ (N)], TiO₂ (N) showed the best performance after silver coating. The synergistic antibacterial effect results approximately 5 log reductions of surviving bacteria of Escherichia coli, Streptococcus pyogenes, Staphylococcus aureus and Acinetobacter baumannii. Scanning electron microscope analysis indicated that crystalline silver formed unique wire-like nanostructures on TiO₂ (N) substrates, while formed relatively straight and thicker rod-shaped precipitates on the other two titania materials.

Conclusion/Significance

Our results suggested that proper forms of silver on various titania materials could further influence the bactericidal property.     

Shape-Related Toxicity of Titanium Dioxide Nanofibres

Titanium dioxide (TiO₂) nanofibres are a novel fibrous nanomaterial with increasing applications in a variety of fields. While the biological effects of TiO₂ nanoparticles have been extensively studied, the toxicological characterization of TiO₂ nanofibres is far from being complete. In this study, we evaluated the toxicity of commercially available anatase TiO₂ nanofibres using TiO₂ nanoparticles (NP) and crocidolite asbestos as non-fibrous or fibrous benchmark materials. The evaluated endpoints were cell viability, haemolysis, macrophage activation, trans-epithelial electrical resistance (an indicator of the epithelial barrier competence), ROS production and oxidative stress as well as the morphology of exposed cells. The results showed that TiO₂ nanofibres caused a cell-specific, dose-dependent decrease of cell viability, with larger effects on alveolar epithelial cells than on macrophages. The observed effects were comparable to those of crocidolite, while TiO₂ NP did not decrease cell viability. TiO₂ nanofibres were also found endowed with a marked haemolytic activity, at levels significantly higher than those observed with TiO₂ nanoparticles or crocidolite. Moreover, TiO₂ nanofibres and crocidolite, but not TiO₂ nanoparticles, caused a significant decrease of the trans-epithelial electrical resistance of airway cell monolayers. SEM images demonstrated that the interaction with nanofibres and crocidolite caused cell shape perturbation with the longest fibres incompletely or not phagocytosed. The expression of several pro-inflammatory markers, such as NO production and the induction of Nos2 and Ptgs2, was significantly increased by TiO₂ nanofibres, as well as by TiO₂ nanoparticles and crocidolite. This study indicates that TiO₂ nanofibres had significant toxic effects and, for most endpoints with the exception of pro-inflammatory changes, are more bio-active than TiO₂ nanoparticles, showing the relevance of shape in determining the toxicity of nanomaterials. Given that several toxic effects of TiO₂ nanofibres appear comparable to those observed with crocidolite, the possibility that they exert length dependent toxicity in vivo seems worthy of further investigation.     

Influence of Several Sources and Amounts of Iron on DNA, Lipid and Protein Oxidative Damage During Anaemia Recovery

Nanoparticles (NPs) in agricultural systems can potentially be used as appropriate candidate for change in growth, development, productivity, and quality of plants. In the present study, we assessed the effect of TiO₂ NP concentrations (0, 2, 5, and 10 ppm) on changes of membrane damage indexes like electrolyte leakage index (ELI) and malondialdehyde (MDA) during cold stress (CS) 4 °C in sensitive (ILC 533) and tolerant (Sel 11439) chickpea (Cicer arietinum L.) genotypes. Aggregation of NPs within the vacuole and chloroplast indicated absorbed NPs in seedlings. Bioaccumulation of NPs showed that, under thermal treatments, the sensitive genotype had more permeability to NPs compared to the tolerant one, and TiO₂ content was higher during CS compared to optimum temperature. Physiological indexes were positively affected by NP treatments during thermal treatments. TiO₂ NP treatments (especially 5 ppm) caused a decrease in ELI during thermal treatments, whereas ELI content under CS treatment increased at 0 ppm TiO₂ in both genotypes. Under thermal treatments, although the genotype 11439 showed lower accumulation of MDA than ILC 533 genotype, a significant decrease was observed in MDA content at 5 ppm TiO₂. Results showed that TiO₂ treatments not only did not induce oxidative damage in sensitive and tolerant chickpea genotypes but also alleviated membrane damage indexes under CS treatment. It was suggested for the first time that TiO₂ NPs improved redox status of the genotypes under thermal treatments. New findings possibly would reveal the use of NPs generally or TiO₂ NPs especially for increase of cold tolerance in crops.     

Evaluation of titanium dioxide and chromic oxide as digestibility markers in ponies fed alfalfa hay in relation to marker dosing frequency

In equines, Cr₂O₃ is widely accepted as an indigestible marker, but there are health concerns regarding the carcinogenic properties of Cr₂O₃. Recently, TiO₂ has been suggested to be an alternative digestibility marker in equines. However, a comparison between Cr₂O₃ and TiO₂ has not been made in equines. Six Welsh pony geldings (initial BW: 254±3 kg; 7 years of age) fed chopped alfalfa hay were used to evaluate the use of TiO₂ (Ti) and Cr₂O₃ (Cr) as markers for calculating apparent digestibility and to investigate the effect of frequency of marker administration on the measurement of digestibility values. Diets contained 4.65 kg dry matter (DM) chopped alfalfa hay supplemented with minerals, vitamins, TiO₂ (3.3 g Ti/day) and Cr₂O₃ (3.2 g Cr/day). Ponies were dosed with either 3.3 g Ti and 3.2 g Cr once daily (DF1) or with 1.65 g Ti and 1.60 g Cr twice daily (DF2). After adaptation to the diets and procedures for 14 days, voluntary voided faeces were collected quantitatively over 7 days and analysed for moisture, ash, Ti and Cr. Apparent total tract DM digestibility (DMD) and organic matter digestibility (OMD) were calculated using the total faecal collection (TFC) and marker method (Ti and Cr). The overall mean cumulative faecal recovery of Cr and Ti (as % of intake) were 102.0% and 96.6%, respectively. Mean daily faecal recoveries of Cr as well as of Ti were not different (P=0.323; P=0.808, respectively) between treatments. Overall daily faecal recovery of Cr differed (P=0.019) from 100% when the marker was dosed once daily, whereas overall daily faecal recovery was similar to 100% for both administration frequencies when Ti was used as a marker. For both markers, the coefficient of variation of the mean faecal marker recovery between horses was lower when the markers were administrated twice per day. Across treatments, cumulative DMD and OMD estimated with Ti were similar (P=0.345; P=0.418, respectively) compared with those values determined by TFC method. When Cr was used, the calculated cumulative DMD tended (P=0.097) to be greater compared with those estimated with TFC, and cumulative OMD values were overestimated (P=0.013). Orally supplemented Ti recovery in the faeces of ponies fed chopped alfalfa hay with Ti administered once or twice daily was close to 100%, making it the preferred marker for digestibility trials in equines.