In Vivo Toxicity Assessment of Hybrid Diatomite Nanovectors Using Hydra vulgaris as a Model System.

Drug nanocarriers based on nanostructured materials are very promising for precision and personalized medicine applications. Diatomite porous biosilica has been recently proposed as a novel and effective material in formulations of drug systems for oral and systemic delivery. In this paper, the cytotoxicity of hybrid diatomite silica functionalized nanovectors is assessed in vivo in a living model organism, the cnidarian freshwater polyp Hydra vulgaris. Hydra specimens are exposed to modified diatomite nanoparticles by prolonged incubation within their medium. Uptake and toxicological effects on Hydra are examined from viability and genetic points of view. High concentrations, up to 3.5 g L-1 for 72 h, of diatomite modified nanoparticles do not affect Hydra morphology nor do growth rate and the genetic analysis confirm the biosafety of this material, opening the way to new applications in nanomedicine.

Bioactivity of diatomaceous earth against the subterranean termite Reticulitermes chinensis Snyder (Isoptera: Rhinotermitidae).

The effects of diatomaceous earth (DE) on the penetrating behavior, tunneling behavior, mortality, and body surface characteristics of the subterranean termite Reticulitermes chinensis were investigated in this study. Our results show that the workers of R. chinensis were able to penetrate 1- and 2-mm layers of dry DE but not 3-mm layers. After treatment with dry DE for 6 h, the mortality of termites reached 100%, which was significantly higher than in the treatment with DE with a 10 and 25% moisture content and treatment with sand of three different moisture contents. The tunneling distances of workers in DE with 10, 25, and 50% moisture contents were all significantly shorter than those in sand with the same moisture contents (10, 25, and 50%), indicating that DE has a good suppressing effect on the tunneling behavior of workers. After treatment with dry DE for different times (1, 3, and 6 h), many DE particles adhered to the bodies of workers, whereas no particles adhered to the body of workers in the case of treatment with dry sand. The treatment with dry DE for 6 h resulted in the death of all workers, which presented conspicuous abdominal shrinkage, whereas workers treated with sand had no significant mortality and no obvious abdominal shrinkage. In summary, we suggest that dry DE has ideal insecticidal activity against the subterranean termite R. chinensis and can be further exploited for controlling termites inside houses.

Estimating Counterfactual Risk Under Hypothetical Interventions in the Presence of Competing Events: Crystalline Silica Exposure and Mortality From 2 Causes of Death.

Exposure to silica has been linked to excess risk of lung cancer and nonmalignant respiratory disease mortality. In this study we estimated risk for both these outcomes in relation to occupational silica exposure as well as the reduction in risk that would result from hypothetical interventions on exposure in a cohort of exposed workers. Analyses were carried out using data from an all-male study population consisting of 2,342 California diatomaceous earth workers regularly exposed to crystalline silica and followed between 1942 and 2011. We estimated subdistribution risk for each event under the natural course and interventions of interest using the parametric g-formula to adjust for healthy-worker survivor bias. The risk ratio for lung cancer mortality, comparing an intervention in which a theoretical maximum exposure limit was set at 0.05 mg/m3 (the current US regulatory limit) with the observed exposure concentrations, was 0.86 (95% confidence interval: 0.63, 1.22). The corresponding risk ratio for nonmalignant respiratory disease mortality was 0.69 (95% confidence interval: 0.52, 0.93). Our findings suggest that risks from both outcomes would have been considerably lower if historical silica exposures in this cohort had not exceeded current regulatory limits.

Effectiveness of an improved form of insecticide-based diatomaceous earth against four stored grain pests on different grain commodities.

The effectiveness of Grain-Guard, an improved form of diatomaceous earth (DE), with low risk to the environment, was evaluated against the storage pests Liposcelis paeta, Cryptolestes ferrugineus, Rhyzopertha dominica, and Tribolium castaneum on four grain commodities, wheat, rice, maize and sorghum. The overall mortality of stored grain pests increased with the rise of application rate and exposure intervals of diatomaceous earth and decreased over 120 days of post-treatment period. Our results revealed that mortality of adults 14 days post-disclosure was > 80% on wheat at the start of post-treatment than rice, maize, and sorghum, respectively. During the first 60 days of post-treatment, adult mortality increased, whereas later on, a steady decrease in adult mortality was observed. Considerable differences in dose rates were observed on mortality levels along with grain commodities. Following 14 days of exposure, all adults of four species were dead on wheat at 100 ppm and on rice at 150 ppm except maize and sorghum. The suppression of progeny was noticeably higher at the beginning of post-treatment duration while after 60-days of post-treatment, progeny numbers started to increase with the rise in post-treatment durations. This new improved DE formulation was found to be effective at dose rates that are extensively lower than required with previous DE formulations and will contribute to lower the risk of health and environment.

Longitudinal study of parasite-induced mortality of a long-lived host: the importance of exposure to non-parasitic stressors.

Hosts face mortality from parasitic and environmental stressors, but interactions of parasitism with other stressors are not well understood, particularly for long-lived hosts. We monitored survival of flour beetles (Tribolium confusum) in a longitudinal design incorporating cestode (Hymenolepis diminuta) infection, starvation and exposure to the pesticide diatomaceous earth (DE). We found that cestode cysticercoids exhibit increasing morphological damage and decreasing ability to excyst over time, but were never eliminated from the host. In the presence of even mild environmental stressors, host lifespan was reduced sufficiently that extensive degradation of cysticercoids was never realized. Median host lifespan was 200 days in the absence of stressors, and 3-197 days with parasitism, starvation and/or DE. Early survival of parasitized hosts was higher relative to controls in the presence of intermediate concentrations of DE, but reduced under all other conditions tested. Parasitism increased host mortality in the presence of other stressors at times when parasitism alone did not cause mortality, consistent with an interpretation of synergy. Environmental stressors modified the parasite numbers needed to reveal intensity-dependent host mortality, but only rarely masked intensity dependence. The longitudinal approach produced observations that would have been overlooked or misinterpreted if survival had only been monitored at a single time point.

Inorganically modified diatomite as a potential prolonged-release drug carrier.

Inorganic modification of diatomite was performed with the precipitation product of partially neutralized aluminum sulfate solution at three different mass ratios. The starting and the modified diatomites were characterized by SEM-EDS, FTIR, thermal analysis and zeta potential measurements and evaluated for drug loading capacity in adsorption batch experiments using diclofenac sodium (DS) as a model drug. In vitro drug release studies were performed in phosphate buffer pH6.8 from comprimates containing: the drug adsorbed onto the selected modified diatomite sample (DAMD), physical mixture of the drug with the selected modified diatomite sample (PMDMD) and physical mixture of the drug with the starting diatomite (PMDD). In vivo acute toxicity testing of the modified diatomite samples was performed on mice. High adsorbent loading of the selected modified diatomite sample (~250mg/g in 2h) enabled the preparation of comprimates containing adsorbed DS in the amount near to its therapeutic dose. Drug release studies demonstrated prolonged release of DS over a period of 8h from both DAMD comprimates (18% after 8h) and PMDMD comprimates (45% after 8h). The release kinetics for DAMD and PMDMD comprimates fitted well with Korsmeyer-Peppas and Bhaskar models, indicating that the release mechanism was a combination of non-Fickian diffusion and ion exchange process.

Formation of a vitreous phase at the surface of some commercial diatomaceous earth prevents the onset of oxidative stress effects.

To understand the effect of the commercial processing of diatomaceous earths (DEs) on their ultimate surface structure and potential toxicity, we investigated the influence of the industrial processing and the nature of the deposit. Two flux calcined specimens from different deposits, DE/1-FC and DE/2-FC, and the simply calcined sample DE/1-C, from the same deposit as DE/1-FC, were compared in both their bulk and their surface properties. X-ray diffraction (XRD) analysis in a heating chamber revealed the presence of cristobalite in all samples, more abundant on the flux calcined ones. The crystal lattice is probably imperfect, as the alpha-beta transition, visible by XRD in DE/1-FC and DE/2-FC, is not detected by differential scanning calorimetry. Progressive etching with HF solutions suggests that most of the crystalline phase is at the core and not at the outer region of the samples. The combined use of spectroscopic (UV-vis and IR) and calorimetric techniques (heat of adsorption of water as a measure of hydrophilicity) reveals that DE/1-FC and DE/2-FC particles have an external layer of glass, absent in DE/1-C, where iron impurities act as network-forming and sodium ions as modifier species, with few patches of a hydrophobic phase, the latter relatable to a heated pure silica phase. When tested on a macrophage cell line (MH-S) in comparison with appropriate positive and negative controls (an active and an inactive quartz dust, respectively), only DE/1-C exhibited a cell damage and activation similar to that of active quartz (measured by lactate dehydrogenase release, peroxidation of membrane lipids and synthesis of NO). It is likely that the presence of a vitreous phase mitigates or even eliminates the cellular responses of silica in DE.

Comparison of different silicas of natural origin as possible insecticides.

Many insect-pests have developed resistances to pesticides. Therefore, there is always a need for new plant protection substances. For example the physically active insecticide diatomaceous earth (DE) gained much attention as an alternative insecticide in stored products. DE is a naturally occurring silica, which acts by destroying the insect's cuticle by absorbing the protective wax layer. This results in body water loss and ultimately the insect's death by desiccation. The silica-based materials tested were the commercial DE product Fossil Shield 90.0s, Advasan, and a formulation newly developed by the Urban Horticultural Department at Humboldt University, called Al-06. The trials were performed in small covered plastic boxes. Test substances were either dusted onto the surface of the boxes (E. vigintioctopunctata, S. litura) or mixed into rice medium (S. oryzae). The mortality was observed after 1, 2, 4, 7, 14, and 28 days. Untreated insects served as control. The first test series showed that some AL-06-formulations and FS90.0s were very effective against adults of S. oryzae and S. litura and larvae of E. vigintioctopunctata. For adult Epilachna beetles, we could not detect any differences between the treatments. The highest mortality rate in S. oryzae trials occurred with FS90.0s (100%) after 21 days. The same efficiency was achieved after 2 days with some AL-06 formulations against S. litura and E. vigintioctopunctata. The results of this study indicate that silica dusts can effectively control insect pests from different orders. At higher dosages, all materials resulted in higher insect mortality rates. It was also found that some substances did not perform well under higher rel. humidity; therefore, the conclusion was drawn that hydrophilic substances saturate with water from the surrounding air and lose their insecticidal efficacy. Earlier studies have proven that particles with a larger surface area are more effective than particles with smaller surfaces. As a result, the most effective substances in the field trials were the ones containing the small particles, since there is a larger surface area available to interact with the insects' cuticles. Further studies will be conducted to analyse the relevance of water saturation of substances in order to examine their effectiveness under greenhouse conditions. Greenhouse experiments are generally considered to study practicability of silica dusts in horticulture. Perhaps the silica dusts will show phytotoxic side effects.

Acaricidal effect of a diatomaceous earth formulation against Tyrophagus putrescentiae (Astigmata: Acaridae) and its predator Cheyletus malaccensis (Prostigmata: Cheyletidae) in four grain commodities.

Laboratory bioassays were conducted to evaluate the effect of the diatomaceous earth (DE) formulation SilicoSec (Biofa GmbH, Münsingen, Germany), against two stored-product mite species, Tyrophagus putrescentiae (Shrank) and the predator Cheyletus malaccensis Oudemans. For this purpose, DE was applied in wheat, oat, rye, and maize, at the dose rates 0, 0.5, 1, and 2 g/kg grain. The mortality of the exposed mites was assessed after 24 h, 48 h, 7 d, and 14 d of exposure in the treated substrate. After this interval, the treated grains were checked for oviposition or progeny. The tests were conducted at 80% RH and at two temperatures, 20 and 25 degrees C. Generally, for both species, mortality was higher at 25 degrees C than at 20 degrees C. For T. putrescentiae, at both temperatures, the mortality in grains treated with the highest DE rate was 100% after only 24 h of exposure, with the exception of maize at 20 degrees C, where mortality was 91.7%. The mortality of C. malaccensis after 24 h of exposure to the treated grains, in the absence of prey, did not exceed 29% at any of the temperature- grain-dose combinations, whereas no mites were dead in rye and maize treated with 0.5 and 1 g of DE. Even after 14 d of exposure at the highest DE rate, mite mortality did not reach 100%. The presence of T. putrescentiae individuals as prey in the treated substrate enhanced C. malaccensis survival. Hence, after 14 d of exposure, the mortality of C. malaccensis, in wheat, oat, rye, and maize treated with the highest DE rate was 51.7, 59.7, 70, and 36.9, respectively. No progeny production was recorded in the treated substrate for T. putrescentiae; in contrast, oviposition and F1 progeny were recorded for C. malaccensis. Our results suggest that the use of C. malaccensis with low doses of DE may be an appealing integrated pest management (IPM) approach against T. putrescentiae, and probably against other stored-grain mite species.

Surface reactivity, cytotoxic, and morphological transforming effects of diatomaceous Earth products in Syrian hamster embryo cells.

In order to evaluate the effect of thermal treatments on the surface reactivity and carcinogenic potential of diatomaceous earth (DE) products, the physicochemical features of some specimens--derived by heating the same original material--were compared with their cytotoxic and transforming potency. The samples were an untreated DE (amorphous) progressively heated in the laboratory at 900 degrees C (DE 900) and 1200 degrees C (DE 1200) and a commercial product manufactured from the same DE (Chd) from which the finer fraction (< 10-microm diameter) was separated (Chd-F). Quartz (Min-U-Sil 5) and a vitreous silica (amorphous) smoothed up with hydrofluoric acid and were used as positive and negative controls, respectively. All samples were analyzed for their degree of crystallization, for their ability to release free radicals and reactive oxygen species, and for their cytotoxic and transforming potencies in Syrian hamster embryo (SHE) cells. X-ray diffractometry showed that DE 900, like DE, was still amorphous, whereas DE 1200 as well as the commercial product (Chd) were partially crystallized into cristobalite. The ability of the dust to release hydroxyl (*OH) radicals in the presence of hydrogen peroxide, as revealed by the spin-trapping technique, was as follows: Chd-F, DE 1200 > Chd > DE 900 > DE, suggesting that on heating, the surface acquires a higher potential for free radical release. Most of the silica samples generated COO* radicals from the formate ion, following homolytic rupture of the carbon-hydrogen bond, in the presence of ascorbic acid. A concentration-dependent decrease in cell proliferation and colony-forming efficiency was observed in SHE cultures treated with Chd-F, Chd, and DE. Heating abolished DE cytotoxicity but conferred a transforming ability to thermal treated particles. DE was the only sample that did not induce morphological transformation of cells. According to their transformation capacity, the samples were classified as follows: Chd-F > Chd, DE 1200 > DE 900 >> DE. Taken together, the reported results suggest that (1) the transforming potential of a biogenic amorphous silica is related to the thermal treatment that transforms the original structure in cristobalite and generates surface active sites; (2) the reactivity of samples in releasing *OH radicals correlates to their transforming ability; (3) the finer fraction of the commercial product is significantly more toxic and transforming than the coarse dust; and (4) opposite to silica dusts of mineral origin, which loose both cytotoxicity and transforming ability upon heating, heated diatomite acquires a cell-transforming potency. DE products should be thus considered a set apart of silica-based potentially toxic materials.

Electrostatic application of inert silica dust based insecticides onto plant surfaces.

One of the most effective naturally occurring insecticide powders is diatomaceous earth (DE), which contains above 96 % of silica (silicon dioxide SiO2). In recent days, the possibility to use new improved DE formulations for plant protection in horticulture has been the focus of research. For aphids and other under-leaf insects only insecticides deposited on leaf undersides are effective. We tested electrostatic application of different silica containing dusts onto the cruciferous crop pak-choi (Brassica chinensis). The materials tested were Fossil Shield 90.0s, Advasan, Biobeck PA910, and a formulation newly developed by the Urban Horticultural Section at Humboldt University called "Al-06". Silica materials were tested for their effect on plant photosynthesis and efficacy against the mustard beetle (Phaedon cochleariae F.). All materials have been effective in contact experiments against tested insects. However, significant differences were observed between materials after application onto plant leaves. Fossil Shield, Advasan, and Al-06 application resulted in a good coverage and in high protection against the mustard beetle. Biobeck PA910 was easily removed by wind from leaf surfaces and did not protect the plants well. However, photosynthesis has been reduced in treated plants and remained at a lower level even after dust removal. Experimental results are critically discussed in the view of future potential for crop protection programs.

Effect of diatomaceous earths Fossil Shield and Silico-Sec on the egg laying behaviour of Callosobruchus maculatus (F.) (Coleoptera: Bruchidae).

The pulse beetle, Callosobruchus maculatus (F.) is a destructive pest of pulses in both storage and field. It is well known that diatomaceous earth (DE) kill the insects by locally absorbing the epicuticular lipid layers leading to high rate of water loss through the cuticle. However, the effectiveness of DE depends on its ability to kill the adults before copulation and egg-laying. Newly emerged virgin males and females of Callosobruchus maculatus (F.) were exposed to the DEs, Fossil-Shield and Silico-Sec on 30 treated mungbeans (Vigna radita (L)). Fecundity, number of beans used for egg-laying and beans without eggs were evaluated after four days; the number of unhatched eggs was evaluated after ten days. It was determined, that the fecundity of female insects decreased sigmoidely with increasing rate of DE content. Percentages of unhatched eggs and seeds without eggs increased with increasing DE dosages. However, the maximum egg densities (eggs per used secd) occurred at 1200 mg DE/kg for Fossil-Shield and Silico-Sec. The reason for such DE-stimulated behaviour of egg laying expressed as a number of seeds with eggs of C. maculatus is not known, but it may be related to the stress caused by the inert dusts or to the reduction of both chemical and physical (tactile) stimuli. Treatment with DEs altered the surface texture of the beans and caused less cohesion between eggs and the seed surface. Only few larvae managed to penetrate into the grains, possibly due to increased grain roughness and repellent effect of DE. A relatively high number of eggs were laid on the surface of those beans where the amount of dust had been locally reduced by adults' movement and their pick up of DE. Therefore, several larvae tried to penetrate into these treated beans, causing a high larval density per partially cleaned bean. All these reasons lead to a progeny decline.

In vitro toxicity of respirable-size particles of diatomaceous earth and crystalline silica compared with asbestos and titanium dioxide.

The relationship between particle characteristics and in vitro toxicity was investigated using Chinese hamster ovary cells. Test dusts included respirable natural (Nat) and flux-calcined (FC) diatomaceous earth (DE), quartz, cristobalite, TiO2, and chrysotile and crocidolite asbestos. All dusts elicited a qualitatively similar, concentration-dependent response: particle uptake, induction of micro- and polynuclei, and reduction in cell proliferation. However, similar mass concentrations of the dusts yielded a 35-fold range of toxicity: chrysotile > crocidolite > Nat DE > FC DE > quartz > Cristobalite > TiO2. In vitro toxicity did not correlate with crystalline silica content, surface area, composition, volume, particles/cm2, or fibrous geometry. Toxicity was closely associated with the number of particles/cm2 culture surface that had at least one dimension > 7.5 mu. Thus particle size but not shape could be a determinant of in vitro toxicity. Particle size might also impact in vivo pathogenesis.

An ultrastructural study of acute and long-term lung response to commercial diatomaceous earth.

The acute pulmonary effects of intratracheally instilled particles of calcined diatomaceous earth were found to include a pronounced neutrophil invasion of the bronchioles by 4 h after exposure which remained well developed through 1 day post-exposure. The number of macrophages and neutrophils in the alveoli continued to increase through 1 day post-exposure and remained above control values through 7 days post-exposure. The number of macrophages, many of which contained diatomaceous earth, remained elevated for the duration of the experiment. Most phagocytosis of the particles was carried out by macrophages, with minor participation by neutrophils. Many of the reactive macrophages in the groups with post-exposure periods longer than 2 h showed various types of pathological alterations. A few particles were found in type I epithelial cells. Oedematous changes were observed in some type I epithelial cells and proliferation of type II epithelial cells was evident in some alveoli, particularly those near the respiratory bronchioles. Mild diffuse fibrosis was first observed at 6 months and was still present at 15 months but remained confined to the areas containing the diatomaceous earth.