Conditioning Film and Early Biofilm Succession on Plastic Surfaces.

In the context of environmental plastic pollution, it is still under debate if and how the "plastisphere", a plastic-specific microbial community, emerges. In this study, we tested the hypothesis that the first conditioning film of dissolved organic matter (DOM) sorbs selectively to polymer substrates and that microbial attachment is governed in a substrate-dependent manner. We investigated the adsorption of stream water-derived DOM to polyethylene terephthalate (PET), polystyrene (PS), and glass (as control) including UV-weathered surfaces by Fourier-transform ion cyclotron mass spectrometry. Generally, the saturated, high-molecular mass and thus more hydrophobic fraction of the original stream water DOM preferentially adsorbed to the substrates. The UV-weathered polymers adsorbed more polar, hydrophilic OM as compared to the dark controls. The amplicon sequencing data of the initial microbial colonization process revealed a tendency of substrate specificity for biofilm attachment after 24 h and a clear convergence of the communities after 72 h of incubation. Conclusively, the adsorbed OM layer developed depending on the materials' surface properties and increased the water contact angles, indicating higher surface hydrophobicity as compared to pristine surfaces. This study improves our understanding of molecular and biological interactions at the polymer/water interface that are relevant to understand the ecological impact of plastic pollution on a community level.

Weathering Plastics as a Planetary Boundary Threat: Exposure, Fate, and Hazards.

We described in 2017 how weathering plastic litter in the marine environment fulfils two of three criteria to impose a planetary boundary threat related to "chemical pollution and the release of novel entities": (1) planetary-scale exposure, which (2) is not readily reversible. Whether marine plastics meet the third criterion, (3) eliciting a disruptive impact on vital earth system processes, was uncertain. Since then, several important discoveries have been made to motivate a re-evaluation. A key issue is if weathering macroplastics, microplastics, nanoplastics, and their leachates have an inherently higher potential to elicit adverse effects than natural particles of the same size. We summarize novel findings related to weathering plastic in the context of the planetary boundary threat criteria that demonstrate (1) increasing exposure, (2) fate processes leading to poorly reversible pollution, and (3) (eco)toxicological hazards and their thresholds. We provide evidence that the third criterion could be fulfilled for weathering plastics in sensitive environments and therefore conclude that weathering plastics pose a planetary boundary threat. We suggest future research priorities to better understand (eco)toxicological hazards modulated by increasing exposure and continuous weathering processes, to better parametrize the planetary boundary threshold for plastic pollution.

Pan-European inter-laboratory studies on a panel of in vitro cytotoxicity and pro-inflammation assays for nanoparticles.

The rapid development of nanotechnologies and increased production and use of nanomaterials raise concerns about their potential toxic effects for human health and environment. To evaluate the biological effects of nanomaterials, a set of reliable and reproducible methods and development of standard operating procedures (SOPs) is required. In the framework of the European FP7 NanoValid project, three different cell viability assays (MTS, ATP content, and caspase-3/7 activity) with different readouts (absorbance, luminescence and fluorescence) and two immune assays (ELISA of pro-inflammatory cytokines IL1-ß and TNF-α) were evaluated by inter-laboratory comparison. The aim was to determine the suitability and reliability of these assays for nanosafety assessment. Studies on silver and copper oxide nanoparticles (NPs) were performed, and SOPs for particle handling, cell culture, and in vitro assays were established or adapted. These SOPs give precise descriptions of assay procedures, cell culture/seeding conditions, NPs/positive control preparation and dilutions, experimental well plate preparation, and evaluation of NPs interference. The following conclusions can be highlighted from the pan-European inter-laboratory studies: Testing of NPs interference with the toxicity assays should always be conducted. Interference tests should be designed as close as possible to the cell exposure conditions. ATP and MTS assays gave consistent toxicity results with low inter-laboratory variability using Ag and CuO NPs and different cell lines and therefore, could be recommended for further validation and standardization. High inter-laboratory variability was observed for Caspase 3/7 assay and ELISA for IL1-ß and TNF-α measurements.

Towards sensible toxicity testing for nanomaterials: proposal for the specification of test design.

During the last decade, nanomaterials (NM) were extensively tested for potential harmful effects towards humans and environmental organisms. However, a sound hazard assessment was so far hampered by uncertainties and a low comparability of test results. The reason for the low comparability is a high variation in the (1) type of NM tested with regard to raw material, size and shape and (2) procedures before and during the toxicity testing. This calls for tailored, nanomaterial-specific protocols. Here, a structured approach is proposed, intended to lead to test protocols not only tailored to specific types of nanomaterials, but also to respective test system for toxicity testing. There are existing standards on single procedures involving nanomaterials, however, not all relevant procedures are covered by standards. Hence, our approach offers a detailed way of weighting several plausible alternatives for e.g. sample preparation, in order to decide on the procedure most meaningful for a specific nanomaterial and toxicity test. A framework of several decision trees (DT) and flow charts to support testing of NM is proposed as a basis for further refinement and in-depth elaboration. DT and flow charts were drafted for (1) general procedure-physicochemical characterisation, (2) choice of test media, (3) decision on test scenario and application of NM to liquid media, (4) application of NM to the gas phase, (5) application of NM to soil and sediments, (6) dose metrics, (S1) definition of a nanomaterial, and (S2) dissolution. The applicability of the proposed approach was surveyed by using experimental data retrieved from studies on nanoscale CuO. This survey demonstrated the DT and flow charts to be a convenient tool to systematically decide upon test procedures and processes, and hence pose an important step towards harmonisation of NM testing.

Versailles project on advanced materials and standards (VAMAS) interlaboratory study on measuring the number concentration of colloidal gold nanoparticles.

We describe the outcome of a large international interlaboratory study of the measurement of particle number concentration of colloidal nanoparticles, project 10 of the technical working area 34, "Nanoparticle Populations" of the Versailles Project on Advanced Materials and Standards (VAMAS). A total of 50 laboratories delivered results for the number concentration of 30 nm gold colloidal nanoparticles measured using particle tracking analysis (PTA), single particle inductively coupled plasma mass spectrometry (spICP-MS), ultraviolet-visible (UV-Vis) light spectroscopy, centrifugal liquid sedimentation (CLS) and small angle X-ray scattering (SAXS). The study provides quantitative data to evaluate the repeatability of these methods and their reproducibility in the measurement of number concentration of model nanoparticle systems following a common measurement protocol. We find that the population-averaging methods of SAXS, CLS and UV-Vis have high measurement repeatability and reproducibility, with between-labs variability of 2.6%, 11% and 1.4% respectively. However, results may be significantly biased for reasons including inaccurate material properties whose values are used to compute the number concentration. Particle-counting method results are less reproducibile than population-averaging methods, with measured between-labs variability of 68% and 46% for PTA and spICP-MS respectively. This study provides the stakeholder community with important comparative data to underpin measurement reproducibility and method validation for number concentration of nanoparticles.

Laminar Pipe Flow with Mixed Convection under the Influence of Magnetic Field.

Magnetic influence on ferronanofluid flow is gaining increasing interest from not only the scientific community but also industry. The aim of this study is the examination of the potentials of magnetic forces to control heat transfer. Experiments are conducted to investigate the interaction between four different configurations of permanent magnets and laminar pipe flow with mixed convection. For that purpose a pipe flow test rig is operated with a water-magnetite ferronanofluid. The Reynolds number is varied over one order of magnitude (120-1200). To characterise this suspension, density, solid content, viscosity, thermal conductivity, and specific heat capacity are measured. It is found that, depending on the positioning of the magnet(s) and the Reynolds number, heat transfer is either increased or decreased. The experiments indicate that this is a local effect. After relaxation lengths ranging between 2 and 3.5 lengths of a magnet, all changes disappeared. The conclusion from these findings is that magnetic forces are rather a tool to control heat transfer locally than to enhance the overall heat transfer of heat exchangers or the like. Magnetically caused disturbances decay due to viscous dissipation and the flow approaches the basic state again.

Quality of nanoplastics and microplastics ecotoxicity studies: Refining quality criteria for nanomaterial studies.

It is becoming increasingly important to develop assessment criteria for the quality of nanoplastics studies. This study is an attempt to establish such criteria based on those developed for engineered nanomaterials, the GUIDEnano and DaNa criteria being two representatives. These criteria were applied to studies on polystyrene nanoparticles (PS NPs), which currently represent the majority of studies on nanoplastics. We compiled a list of existing nanomaterial-related criteria that are not fully relevant to PS NPs and propose additional nanoplastic-specific criteria targeting polymer chemical composition, source, production and field collection, impurities/chemical additives, density, hydrophobicity, colour, and chemical leaching. For each criterion, scientific justification is provided. We conclude that the existing study quality assessments originally developed for nano(eco)toxicity studies can, through refinements, be applied to those dealing with nanoplastics studies, with a further outlook on microplastics. The final quality criteria catalogue presented here is intended as a starting point for further elaborations considering different purposes of an assessment.

Physico-chemical characterization in the light of toxicological effects.

Toxicological investigations on nanoparticles require a comprehensive physico-chemical characterization to get useful information about the powder as well as the behavior of the suspended nanoparticles in water and physiological media. Therefore, we characterized the often used TiO(2) P25 and developed procedures to get stable, homogeneous, and well-defined nanoparticle suspensions. A titration of the zeta potential as a function of the pH yielded the conclusion that the TiO(2) suspension is stable at a pH of 4 or lower. In this region the zeta potential is higher than 30 mV, which guarantees a high stability of the suspended particles. Hence, a stable TiO(2) initial suspension was prepared in 0.1 mM HCl having a mean particle size of 170 +/- 5 nm, which was determined by dynamic light scattering. Furthermore, the initial suspension was added to different physiological media (0.9% NaCl solution, phosphate-buffered saline [PBS], Hanks balanced salt solution [HBSS], Dulbecco's modified Eagle's medium [DMEM]) for studying the agglomeration behavior. As a result, the agglomeration kinetics at the same TiO(2) concentration is independent of the used media. Investigations with PBS containing bovine serum albumin (BSA) and DMEM supplemented with 10% FBS revealed that these protein additions inhibit the agglomeration of the particles. Thus, the physiological media contains particles that are stabilized through the steric or electrosteric effect of BSA and of the proteins in FBS, respectively. Consequently, the particles keep their size from the initial suspension. Finally, our procedure demonstrated on TiO(2) P25 results in homogeneously suspended particles in physiological media. This definite status of the particles means an improvement for toxicological testing and understanding.

From the sea to the laboratory: Characterization of microplastic as prerequisite for the assessment of ecotoxicological impact.

The presence of microplastic (MP) in the aquatic environment is recognized as a global-scale pollution issue. Secondary MP particles result from an ongoing fragmentation process governed by various biotic and abiotic factors. For a reliable risk assessment of these MP particles, knowledge about interactions with biota is needed. However, extensive testing with standard organisms under reproducible laboratory conditions with well-characterized MP suspensions is not available yet. As MP in the environment represents a mixture of particles differing in properties (e.g., size, color, polymer type, surface characteristics), it is likely that only specific particle fractions pose a threat towards organisms. In order to assign hazardous effects to specific particle properties, these characteristics need to be analyzed. As shown by the testing of particles (e.g. nanoparticles), characteristics other than chemical properties are important for the emergence of toxicity in organisms, and parameters such as surface area or size distribution need consideration. Therefore, the use of "well-defined" particles for ecotoxicological testing (i.e., standard particles) facilitates the establishment of causal links between physical-chemical properties of MP particles and toxic effects in organisms. However, the benefits of well-defined particles under laboratory conditions are offset by the disadvantage of the unknown comparability with MP in the environment. Therefore, weathering effects caused by biological, chemical, physical or mechanical processes have to be considered. To date, the characterization of the progression of MP weathering based on powder and suspension characterization methods is in its infancy. The aim of this commentary is to illustrate the prerequisites for testing MP in the laboratory from 3 perspectives: (i) knowledge of particle properties; (ii) behavior of MP in test setups involving ecotoxicological test organisms; and (iii) accordingly, test conditions that may need adjustment. Only under those prerequisites will reliable hazard assessment of MP be feasible. Integr Environ Assess Manag 2017;13:500-504. © 2017 SETAC.

Dispersion of Nanomaterials in Aqueous Media: Towards Protocol Optimization.

The sonication process is commonly used for de-agglomerating and dispersing nanomaterials in aqueous based media, necessary to improve homogeneity and stability of the suspension. In this study, a systematic step-wise approach is carried out to identify optimal sonication conditions in order to achieve a stable dispersion. This approach has been adopted and shown to be suitable for several nanomaterials (cerium oxide, zinc oxide, and carbon nanotubes) dispersed in deionized (DI) water. However, with any change in either the nanomaterial type or dispersing medium, there needs to be optimization of the basic protocol by adjusting various factors such as sonication time, power, and sonicator type as well as temperature rise during the process. The approach records the dispersion process in detail. This is necessary to identify the time points as well as other above-mentioned conditions during the sonication process in which there may be undesirable changes, such as damage to the particle surface thus affecting surface properties. Our goal is to offer a harmonized approach that can control the quality of the final, produced dispersion. Such a guideline is instrumental in ensuring dispersion quality repeatability in the nanoscience community, particularly in the field of nanotoxicology.

An interlaboratory comparison of nanosilver characterisation and hazard identification: Harmonising techniques for high quality data.

Within the FP7 EU project NanoValid a consortium of six partners jointly investigated the hazard of silver nanoparticles (AgNPs) paying special attention to methodical aspects that are important for providing high-quality ecotoxicity data. Laboratories were supplied with the same original stock dispersion of AgNPs. All partners applied a harmonised procedure for storage and preparation of toxicity test suspensions. Altogether ten different toxicity assays with a range of environmentally relevant test species from different trophic levels were conducted in parallel to AgNP characterisation in the respective test media. The paper presents a comprehensive dataset of toxicity values and AgNP characteristics like hydrodynamic sizes of AgNP agglomerates and the share (%) of Ag(+)-species (the concentration of Ag(+)-species in relation to the total measured concentration of Ag). The studied AgNP preparation (20.4±6.8 nm primary size, mean total Ag concentration 41.14 mg/L, 46-68% of soluble Ag(+)-species in stock, 123.8±12.2 nm mean z-average value in dH2O) showed extreme toxicity to crustaceans Daphnia magna, algae Pseudokirchneriella subcapitata and zebrafish Danio rerio embryos (EC50<0.01 mg total Ag/L), was very toxic in the in vitro assay with rainbow trout Oncorhynchus mykiss gut cells (EC50: 0.01-1 mg total Ag/L); toxic to bacteria Vibrio fischeri, protozoa Tetrahymena thermophila (EC50: 1-10 mg total Ag/L) and harmful to marine crustaceans Artemia franciscana (EC50: 10-100 mg total Ag/L). Along with AgNPs, also the toxicity of AgNO3 was analyzed. The toxicity data revealed the same hazard ranking for AgNPs and AgNO3 (i.e. the EC50 values were in the same order of magnitude) proving the importance of soluble Ag(+)-species analysis for predicting the hazard of AgNPs. The study clearly points to the need for harmonised procedures for the characterisation of NMs. Harmonised procedures should consider: (i) measuring the AgNP properties like hydrodynamic size and metal ions species in each toxicity test medium at a range of concentrations, and (ii) including soluble metal salt control both in toxicity testing as well as in Ag(+)-species measurements. The present study is among the first nanomaterial interlaboratory comparison studies with the aim to improve the hazard identification testing protocols.

Investigations on mobility of carbon colloid supported nanoscale zero-valent iron (nZVI) in a column experiment and a laboratory 2D-aquifer test system.

Nanoscale zero-valent iron (nZVI) has recently gained great interest in the scientific community as in situ reagent for installation of permeable reactive barriers in aquifer systems, since nZVI is highly reactive with chlorinated compounds and may render them to harmless substances. However, nZVI has a high tendency to agglomerate and sediment; therefore it shows very limited transport ranges. One new approach to overcome the limited transport of nZVI in porous media is using a suited carrier colloid. In this study we tested mobility of a carbon colloid supported nZVI particle "Carbo-Iron Colloids" (CIC) with a mean size of 0.63 µm in a column experiment of 40 cm length and an experiment in a two-dimensional (2D) aquifer test system with dimensions of 110 × 40 × 5 cm. Results show a breakthrough maximum of 82 % of the input concentration in the column experiment and 58 % in the 2D-aquifer test system. Detected residuals in porous media suggest a strong particle deposition in the first centimeters and few depositions in the porous media in the further travel path. Overall, this suggests a high mobility in porous media which might be a significant enhancement compared to bare or polyanionic stabilized nZVI.

Transport of carbon colloid supported nanoscale zero-valent iron in saturated porous media.

Injection of nanoscale zero-valent iron (nZVI) has recently gained great interest as emerging technology for in-situ remediation of chlorinated organic compounds from groundwater systems. Zero-valent iron (ZVI) is able to reduce organic compounds and to render it to less harmful substances. The use of nanoscale particles instead of granular or microscale particles can increase dechlorination rates by orders of magnitude due to its high surface area. However, classical nZVI appears to be hampered in its environmental application by its limited mobility. One approach is colloid supported transport of nZVI, where the nZVI gets transported by a mobile colloid. In this study transport properties of activated carbon colloid supported nZVI (c-nZVI; d50=2.4µm) are investigated in column tests using columns of 40cm length, which were filled with porous media. A suspension was pumped through the column under different physicochemical conditions (addition of a polyanionic stabilizer and changes in pH and ionic strength). Highest observed breakthrough was 62% of the injected concentration in glass beads with addition of stabilizer. Addition of mono- and bivalent salt, e.g. more than 0.5mM/L CaCl2, can decrease mobility and changes in pH to values below six can inhibit mobility at all. Measurements of colloid sizes and zeta potentials show changes in the mean particle size by a factor of ten and an increase of zeta potential from -62mV to -80mV during the transport experiment. However, results suggest potential applicability of c-nZVI under field conditions.

Comparative evaluation of particle properties, formation of reactive oxygen species and genotoxic potential of tungsten carbide based nanoparticles in vitro.

Tungsten carbide (WC) and cobalt (Co) are constituents of hard metals and are used for the production of extremely hard tools. Previous studies have identified greater cytotoxic potential of WC-based nanoparticles if particles contained Co. The aim of this study was to investigate whether the formation of reactive oxygen species (ROS) and micronuclei would help explain the impact on cultured mammalian cells by three different tungsten-based nanoparticles (WC(S), WC(L), WC(L)-Co (S: small; L: large)). The selection of particles allowed us to study the influence of particle properties, e.g. surface area, and the presence of Co on the toxicological results. WC(S) and WC(L)/WC(L)-Co differed in their crystalline structure and surface area, whereas WC(S)/WC(L) and WC(L)-Co differed in their cobalt content. WC(L) and WC(L)-Co showed neither a genotoxic potential nor ROS induction. Contrary to that, WC(S) nanoparticles induced the formation of both ROS and micronuclei. CoCl(2) was tested in relevant concentrations and induced no ROS formation, but increased the rate of micronuclei at concentrations exceeding those present in WC(L)-Co. In conclusion, ROS and micronuclei formation could not be associated with the presence of Co in the WC-based particles. The contrasting responses elicited by WC(S) vs. WC(L) appear to be due to large differences in crystalline structure.

Evaluating the cytotoxicity of palladium/magnetite nano-catalysts intended for wastewater treatment.

Palladium/magnetite nanoparticulate catalysts were developed for efficient elimination of halogenated organic pollutants from contaminated wastewater. Particle recovery from treated water can be ensured via magnetic separation. However, in worst-case scenarios, this catalyst removal step might fail, leading to particle release into the environment. Therefore, a toxicological study was conducted to investigate the impact of both pure magnetite and palladium/magnetite nanoparticle exposure upon human skin (HaCaT) and human colon (CaCo-2) cell lines and a cell line from rainbow trout gills (RTgill-W1). To quantify cell viability after particle exposure, three endpoints were examined for all tested cell lines. Additionally, the formation of reactive oxygen species was studied for the human cells. The results showed only minor effects of the particles on the tested cell systems and support the assumption that palladium/magnetite nano-catalysts can be implemented for a new wastewater treatment technology in which advantageous catalyst properties outweigh the risks.

Physical-chemical characterization of tungsten carbide nanoparticles as a basis for toxicological investigations.

One task in risk assessment of engineered nanoparticles is toxicological studies. A suitable interpretation of these investigations demands a comprehensive physical-chemical characterization. Here, we present an approach to gain well-dispersed nanoparticles in physiological media. Therefore, a step-by-step procedure is demonstrated on two different tungsten carbide nanopowders which can be transferred to other powders. The procedure includes a comprehensive powder characterization, followed by a preparation of a non-physiologic, electrostatically stable nanoparticle suspension and finally closes with investigations of the particles' behavior in different physiological media. Our study showed that the particles agglomerate in protein-free media. In this context, dependencies of mass- and surface-based nanoparticle concentrations as well as of different physiological media were analyzed. In the presence of bovine serum albumin (BSA) or serum, the agglomeration process is decelerated or, at the appropriate protein amount, prevented.

Agglomeration of tungsten carbide nanoparticles in exposure medium does not prevent uptake and toxicity toward a rainbow trout gill cell line.

Due to their increased production and use, engineered nanoparticles are expected to be released into the aquatic environment where particles may agglomerate. The aim of this study was to explore the role of agglomeration of nanoparticles in the uptake and expression of toxicity in the rainbow trout (Oncorhynchus mykiss) gill cell line, RTgill-W1. This cell line was chosen as model because it is known to be amenable to culture in complete as well as greatly simplified exposure media. Nano-sized tungsten carbide (WC) with or without cobalt doping (WC-Co), two materials relevant in the heavy metal industry, were applied as model particles. These particles were suspended in culture media with decreasing complexity from L15 with 10% fetal bovine serum (FBS) to L15 to L15/ex, containing only salts, galactose and pyruvate of the complete medium L15. Whereas the serum supplement in L15 retained primary nanoparticle suspensions, agglomerates were formed quickly in L15 and L15/ex. Nevertheless, scanning electron microscopy (SEM) coupled with energy dispersive X-ray (EDX) elemental analysis revealed an uptake of both WC and WC-Co nanoparticles into RTgill-W1 cells irrespective of the state of agglomeration of nanoparticles. The localisation seemed to be restricted to the cytoplasm, as no particles were observed in the nucleus of cells. Moreover, reduction in cell viability between 10 and 50% compared to controls were observed upon particle exposure in all media although the pattern of impact varied depending on the medium and exposure time. Short-term exposure of cells led to significant cytotoxicity at the highest nominal particle concentrations, irrespective of the particle type or exposure medium. In contrast, long-term exposures led to preferential toxicity in the simplest medium, L15/ex, and an enhanced toxicity by the cobalt-containing WC nanoparticles in all exposure media. The composition of the exposure media also influenced the toxicity of the cobalt ions, which may dissolve from the WC-Co nanoparticles, with cells reacting much more sensitively toward cobalt ions in the absence of FBS. However, the toxicity observed by ionic cobalt alone did not explain the toxicity of the WC-Co nanoparticles, suggesting that the combination of metallic Co and WC is the cause of the increased particle toxicity of WC-Co. Taken together, our findings indicate that minimal exposure media can lead to rapid agglomeration of nanoparticles but that agglomeration does not prevent uptake into cells and the expression of toxicity.

Toxicity of tungsten carbide and cobalt-doped tungsten carbide nanoparticles in mammalian cells in vitro.

BACKGROUND: Tungsten carbide nanoparticles are being explored for their use in the manufacture of hard metals. To develop nanoparticles for broad applications, potential risks to human health and the environment should be evaluated and taken into consideration. OBJECTIVE: We aimed to assess the toxicity of well-characterized tungsten carbide (WC) and cobalt-doped tungsten carbide (WC-Co) nanoparticle suspensions in an array of mammalian cells. METHODS: We examined acute toxicity of WC and of WC-Co (10% weight content Co) nanoparticles in different human cell lines (lung, skin, and colon) as well as in rat neuronal and glial cells (i.e., primary neuronal and astroglial cultures and the oligodendrocyte precursor cell line OLN-93). Furthermore, using electron microscopy, we assessed whether nanoparticles can be taken up by living cells. We chose these in vitro systems in order to evaluate for potential toxicity of the nanoparticles in different mammalian organs (i.e., lung, skin, intestine, and brain). RESULTS: Chemical-physical characterization confirmed that WC as well as WC-Co nanoparticles with a mean particle size of 145 nm form stable suspensions in serum-containing cell culture media. WC nanoparticles were not acutely toxic to the studied cell lines. However, cytotoxicity became apparent when particles were doped with Co. The most sensitive were astrocytes and colon epithelial cells. Cytotoxicity of WC-Co nanoparticles was higher than expected based on the ionic Co content of the particles. Analysis by electron microscopy demonstrated presence of WC nanoparticles within mammalian cells. CONCLUSIONS: Our findings demonstrate that doping of WC nanoparticles with Co markedly increases their cytotoxic effect and that the presence of WC-Co in particulate form is essential to elicit this combinatorial effect.