Introduction
Diatomaceous earth (DE), a sedimentary deposit of silica-rich diatom frustules, is mined globally for a range of purposes, including its commercial value in the filler and filter aid industries [
1,
2]. Diatomite deposits are mainly composed of amorphous silica or opal diatom skeletons – the dominant diatom species of which can vary among deposits [
2] – and can be interspersed with contaminant minerals, such as clays or carbonates [
1]. Extracted material is processed by a series of grinding, calcination and classification techniques to give a wide range of end products. During this process, the diatomite deposit (unprocessed DE) is treated at ~1000 °C with (flux-calcined) or without (calcined) the presence of a fluxing agent, usually sodium carbonate [
1,
3]. During calcination, the amorphous silica is converted to crystalline silica, predominantly in the form of cristobalite [
2,
3]. The potential for crystalline silica to cause silicosis is well established [
4‐
6], and quartz and cristobalite are classified as Group 1 carcinogens [
7,
8]; therefore, exposure to processed DE has the potential to cause chronic respiratory disease.
A number of epidemiology studies assessing the respiratory DE hazard show increased mortality [
9,
10], pneumoconiosis [
11‐
14], increased risk of lung cancer [
9,
10,
15] and other lung diseases [
16] in DE workers, compared to unexposed populations. These findings are supported by radiographic evidence, which shows a strong relationship between exposure and the risk of opacities in chest X-rays indicative of silicosis [
17]. However, these studies only focused on DE deposits from California, USA, and little is known about how the physicochemical characteristics of DE vary globally. As such, these data may not inform the health hazard of DE worldwide; the few studies at other locations are much less detailed. The largest of these studies, in Iceland, showed a non-significant increase in lung cancer for DE workers [
18]. Beskow [
19] and Ebina et al. [
20] found signs of silicosis in DE workers in Sweden and Japan, but these were based on a small number of cases; whereas, Joma et al. [
21] found no signs of pneumoconiosis in DE-exposed workers in the Netherlands, but airflow in the lung was reduced after exposure.
Some of the above studies include evidence of silicosis-type pathology [
12,
19,
20,
22], or strong, positive correlations between crystalline silica content and level of observed disease [
9,
17,
23]. However, other clinical and epidemiological studies have demonstrated no link between crystalline silica exposure and the pathogenicity observed in DE workers [
14], and showed pathological changes in the lungs which differ from typical quartz-induced silicosis [
22]. Generally, crystalline silica content is shown to be very low in unprocessed samples (0–4 wt.%), high in calcined samples (>10 wt.%), and highest in flux-calcined samples (>20 wt.%) (e.g. [
9,
24]). It is often difficult to distinguish among these different types of DE exposures in epidemiological studies.
Toxicology studies allow differences between processed and unprocessed DE to be studied systematically. As with epidemiological studies, there is discrepancy among toxicological studies as to whether crystalline silica content is the determinant factor for disease. This knowledge is essential for the effective risk management of worker safety.
Many in vitro studies indicate that the cytotoxicity of DE does not correlate with crystalline silica content, as unprocessed and calcined DE exert a greater cytotoxic effect than cristobalite-rich flux-calcined DE in a number of in vitro assays on different cell lines [
24‐
27]. However, work by Elias and co-workers has shown that cristobalite-rich DE samples (both flux-calcined and those calcined at high temperature) have increased pro-carcinogenic potential in Syrian hamster embryos compared to unprocessed DE and DE calcined at lower temperature [
26,
28]. Exposure to both unprocessed and flux-calcined DE also resulted in an increase in abnormal nuclei formation (a marker of genotoxicity) in Chinese hamster ovary cells, with the damage more significant than quartz and cristobalite standards [
24].
In vivo studies of unprocessed, amorphous DE show it has the potential to be pathogenic, causing acute/sub-acute inflammation in rats 60 days after a single intratracheal injection of 10 mg DE, an effect that decreased with time [
29]. While inhalation of unprocessed DE (170 million particles per cubic foot (mppcf)) by guinea pigs for 39–44 h/week for 24 months resulted in fibrosis, calcined, cristobalite-rich DE was seen to cause more severe fibrosis more rapidly [
30]. However, in a separate study, exposure via inhalation to 5–50 mppcf flux-calcined DE, consisting of 61 % cristobalite, for 30 h/week up to 30 months caused no body weight loss or pulmonary fibrosis in rats, guinea pigs or dogs [
31].
Previous studies have focussed on few samples and single locations and, therefore, have not been able to investigate the source-dependent compositional and morphological variability of DE and the effect of this variability on toxicity endpoints. This study overcomes this limitation by determining the potential toxicity of DE sourced from seven quarries across the world, and investigating the physicochemical characteristics of the material to understand the properties that affect DE toxicity. Samples chosen cover a spectrum of deposit types, purities and processing techniques. Physicochemical analyses assessed sample composition, morphology, particle size and surface area. Haemolysis allowed for the assessment of particle-induced membrane damage, while cytotoxicity and cytokine release from macrophages were used to assess potential particle toxicity and the potential to induce inflammation.
Materials and methods
Nineteen DE samples were sourced from mines around the world to account for the global variability of DE deposits (Table
1). A range of DE products that encompass the spectrum of compositional characteristics and processing techniques available were selected using geochemical data obtained though the European Industrial Minerals Association (IMA-Europe). The sample set comprised unprocessed, calcined and flux-calcined samples, and included filler and filter aid grades of DE, which are determined by post-calcination size classification. Samples were chosen from a range of source deposits, including those with impurities (carbonates and clays). Unprocessed samples, and the equivalent sample post-processing, were sourced where possible in order to directly establish the effects of processing.
Table 1
Information for diatomaceous earth samples, including: particle size distribution, surface area and chemical composition
DE_05#a
| Spain | U | Filler | 11.3 | 38.8 | 7.5 | 0.7 | 90.47 | 0.05 | 0.91 | 0.38 | 0.34 | 6.96 | 0.65 | 0.14 |
DE_11b
| Spain | U | Filler | 12.4 | 47.3 | 6.3 | 0.4 | 87.26 | 0.05 | 1.40 | 0.42 | 0.49 | 8.96 | 1.04 | 0.17 |
DE_13c
| France | U | Filter aid | 7.8 | 28.0 | 22.9 | 0.2 | 89.02 | 0.64 | 4.42 | 3.22 | 0.36 | 0.66 | 0.38 | 0.35 |
DE_15d
| France | U | Filter aid | 7.9 | 28.5 | 23.8 | 0.3 | 87.40 | 0.70 | 4.73 | 3.99 | 0.49 | 0.74 | 0.19 | 0.34 |
DE_16d
| France | C | Filter aid | 6.7 | 25.0 | 3.8 | 0.1 | 88.03 | 0.66 | 4.50 | 3.86 | 0.39 | 0.66 | 0.17 | 0.33 |
DE_18 | China | C | Filter aid | 7.3 | 28.1 | 3.1 | 0.1 | 93.49 | 0.15 | 3.71 | 1.67 | 0.35 | 0.08 | −0.04 | 0.50 |
DE_20#
| Mexico | C | Filter aid | 6.8 | 25.3 | 4.0 | 0.1 | 92.00 | 0.24 | 5.07 | 1.95 | 0.34 | 0.30 | −0.13 | 0.20 |
DE_21 | USA-1e
| C | Filter aid | 6.2 | 21.7 | 5.7 | 0.2 | 87.83 | 0.32 | 6.57 | 1.81 | 1.02 | 0.54 | 0.51 | 1.02 |
DE_22 | USA-1 | C | Filler | 25.4 | 71.5 | 10.6 | 0.2 | 93.24 | 0.17 | 3.82 | 1.15 | 0.65 | 0.17 | 0.03 | 0.59 |
DE_23 | USA-1 | C | Filter aid | 21.8 | 65.5 | 6.8 | 0.2 | 93.88 | 0.15 | 3.03 | 1.05 | 0.69 | 0.30 | 0.21 | 0.47 |
DE_24#
| Mexico | C | Filter aid | 12.1 | 45.0 | 5.9 | 0.2 | 93.63 | 0.19 | 3.89 | 1.49 | 0.33 | 0.22 | 0.01 | 0.18 |
DE_06a
| Spain | FC | Filler | 8.5 | 34.4 | 1.1 | 0.1 | 91.27 | 0.03 | 0.67 | 0.35 | 0.27 | 6.25 | 1.00 | 0.09 |
DE_07 | USA-1 | FC | Filler | 10.6 | 31.6 | 1.3 | 0.0 | 91.67 | 0.15 | 3.00 | 1.07 | 0.53 | 0.29 | 2.69 | 0.48 |
DE_08#
| USA-2e
| FC | Filler | 9.2 | 28.7 | 1.3 | 0.1 | 93.07 | 0.06 | 1.12 | 1.99 | 0.51 | 0.30 | 2.89 | 0.03 |
DE_09 | Chile | FC | Filler | 9.8 | 33.4 | 1.5 | 0.1 | 93.04 | 0.10 | 1.82 | 0.95 | 0.23 | 1.29 | 2.15 | 0.34 |
DE_10#
| Mexico | FC | Filler | 8.7 | 35.3 | 1.7 | 0.1 | 93.64 | 0.10 | 2.05 | 0.80 | 0.14 | 0.32 | 2.59 | 0.12 |
DE_12b
| Spain | FC | Filler | 8.5 | 33.4 | 1.0 | 0.1 | 90.67 | 0.03 | 1.12 | 0.35 | 0.31 | 6.64 | 0.69 | 0.10 |
DE_14c
| France | FC | Filter aid | 6.4 | 17.0 | 1.1 | 0.1 | 87.36 | 0.58 | 4.10 | 2.61 | 0.40 | 0.62 | 3.17 | 0.39 |
DE_17 | China | FC | Filter aid | 4.6 | 11.4 | 1.0 | 0.0 | 91.68 | 0.13 | 3.12 | 1.44 | 0.27 | 0.11 | 2.22 | 0.51 |
Separation of the fine fraction
Fine fractions (close to PM10) were separated from 5 bulk samples for toxicological assessment alongside their bulk counterparts (Table
1). The samples, which comprise a range of crystalline silica contents, bulk impurities, and processing techniques, were separated by dry-resuspension as previously described by Moreno et al. [
32]. Briefly, the sample was suspended in a horizontal rotating drum with a baffle (1.5 rpm). Airflow of 6 l/min was passed through this system, which carried suspended particles through a gravitational settling chamber, where coarser particles were deposited, and the fine fraction continued through a Negretti elutriation filter system and was collected on a polycarbonate filter. The particle size distribution of the separated fine fractions was determined by SEM image analysis.
Physicochemical characterisation
Physiochemical analyses were carried out on bulk samples (because of the mass required by some techniques). The crystalline silica polymorph and relative crystalline silica contents were measured by X-ray diffraction position sensitive detection (XRD-PSD; Bruker D8 Advance, Durham University) from 5 to 90° 2θ. Samples were ground to a fine powder and compacted into a well using the knife-edge of a spatula to ensure random crystal orientation [
33]. The intensity of the primary peak (26.6 °2θ for quartz and 21.8 °2θ for cristobalite) was used as a proxy for the relative amounts of crystalline silica. To account for the different diffraction intensities of quartz and cristobalite, the peak heights of pure-phase quartz and cristobalite standards, run under the same conditions, were used to normalise the relative peak heights in the samples. This allowed the addition of peak intensities of quartz and cristobalite to give relative total crystalline silica contents amongst the samples. Bulk chemical compositions were measured by X-ray fluorescence (XRF; PANalytical Axios Advanced X-ray fluorescence spectrometer, University of Leicester). Particle sections were produced in polished resin blocks, coated with 25 nm carbon, and imaging and elemental analysis were performed by scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS) at 15 kV (Hitachi SU-70 FEG SEM, Durham University). The elemental composition of cristobalite in individual particles, and amorphous material in cristobalite-containing particles was measured in samples where these particles were clearly seen.
Particle size distributions were analysed by laser diffraction using a Coulter LS analyser (Durham University), with polarization intensity differentiation scattering (PIDS) to analyse particles in the range of 0.04–2000 μm diameter (all samples fell within this range). Data are presented as cumulative volume (c.v.) % and are an average of two 90 s measurements, analysed by Fraunhofer theory.
Qualitative analysis of dominant diatom morphology was performed on all samples by mounting particles on polycarbonate discs, which were adhered to aluminium stubs by carbon pads. These were coated with 25 nm gold/palladium and imaged at 8 kV by SEM. Quantitative assessment (1000–2000 particles per sample) of particle size and the abundance of fibre-like particles (defined by an aspect ratio >3 [
34]), was conducted on the five fine fractions used in toxicology experiments and their corresponding bulk samples (DE_05, DE_08, DE_10, DE_20 and DE_24).
Surface area was analysed by nitrogen adsorption measurements at 77 K using a TriStar 3000 instrument (Durham University). Samples were dried in nitrogen gas at 120 °C overnight. The Brunauer-Emmett-Teller (BET) theory was applied to measurements at relative pressures of 0.05–0.24, and the results are the mean of three repeated measurements.
In vitro toxicology
Haemolysis is a measure of the ability of particles to rupture cell membranes, and has been shown to be a good indicator of the pro-inflammatory potential of crystalline silica [
35]. Haemolysis was performed on all 19 bulk samples and 5 separated fine fractions by treating red blood cells with 63–1000 μg/ml DE powder for 1 h. A volume of 1 ml of sheep blood in Alsever’s solutions (Oxoid Ltd.) was centrifuged at 5000 rpm for 2 min and the supernatant removed. Isolated red blood cells were washed three times with saline and 100 μl of cells were added to 3.6 ml saline. Particle suspensions of 1 mg/ml particles in saline were sonicated for 20 min, serially diluted to final concentrations, and 150 μl of the particle suspensions added to 96 well plates in triplicate. Next, 75 μl of the prepared blood was added to each well, the plate covered and placed on an orbital shaker for 1 h. Post-exposure, the plate was centrifuged at 250 rcf for 5 min, 100 μl of the supernatant transferred to a new plate and absorbance measured at 540 nm (SpectraMax M5, Heriot-Watt University).
Cytotoxicity was measured using alamarBlue® (a measure of mitochondrial enzyme activity) and lactate dehydrogenase (LDH; a measure of membrane integrity) assays (Heriot-Watt University). These assays were performed on the five fine fractions, their corresponding bulk samples, two bulk samples chosen due to their haemolytic potential (DE_11 and DE_22), as well as a further unprocessed bulk sample (DE_15). The samples were suspended in RPMI medium containing 10 μl/ml L-glutamine, 10 μl/ml penicillin and streptomycin, and 10 % bovine foetal serum (complete medium), and sonicated for 20 min. J774 macrophages were seeded in a 96 well plate (5 × 104 cells per well) and exposed to 100 μl of DE suspension in concentrations of 8, 16, 31, 63, 125, 250, and 500 μg/ml for 24 h at 37 °C and 5 % CO2. Following exposure, the supernatant was removed and stored at −80 °C for LDH and cytokine analysis. A solution of 1 mg/ml alamarBlue® reagent (resazurin sodium salt; Sigma) in saline was diluted 1:10 in complete medium and 100 μl added to the cells. The plate was incubated for 4 h and fluorescence measured at excitation at 560 nm and emission at 590 nm. LDH release from macrophages was measured by adding 10 μl of cell supernatant to 50 μl 1 mg/ml NADH in 0.75 mM sodium pyruvate, incubating for 30 min at 37 °C in 5 % CO2, adding 50 μl of 2 mg/ml 2,4-dinitrophenylhydrazine in 1 M HCl and incubating for 20 min at room temperature in the dark, before adding 50 μl 4 M sodium hydroxide and measuring the absorbance at 550 nm.
Cytokine production was measured as a marker of inflammation using BD™ Cytometric Bead Array cytokine flex sets (bead based immunoassay; BD Biosciences, Heriot-Watt University). Flow cytometry was used to discriminate between different bead populations based on size and fluorescence, according to the manufacturer’s instructions. Keratinocyte chemoattractant (KC), interleukin 1β (IL-1β), tumour necrosis factor alpha (TNF-α) and interleukin 10 (IL-10) were measured for cells treated with 125 μg/ml or lower of the fine fractions of DE only. TNF-α and IL-1β are pro-inflammatory cytokines associated with silica induced toxicity [
36,
37], IL-10 is an anti-inflammatory cytokine providing information on the balance between pro- and anti-inflammatory signalling, and KC induces neutrophil and macrophage chemotaxis. For samples demonstrating cytotoxicity, the LC20 concentration, two times the LC20 and half the LC20 would generally be assessed to investigate cytokine production. However, in low toxicity samples, where an LC20 was not reached, only a concentration of 125 μg/ml was chosen to evaluate the inflammatory response.
Cells treated with the five fine fractions were imaged by light microscopy. J774 cells (2.5 × 105) were treated with 600 μl of 63 μg/ml DE in a 24 well plate. Cells were scraped from the plate and 50 μl of the cell suspension diluted in 300 μl saline and centrifuged onto a microscope slide at 1160 rcf for 5 min. The treated cells were dried and stained with Diff-Quik (Fisher Scientific). Briefly, slides were dipped in methanol, then Eosin G in phosphate buffer and Thiazine dye in phosphate buffer, rinsed with H2O and air dried.
In all assays, Triton-X was used as a positive control and untreated cells as a negative control. DQ12 was used as a positive crystalline silica standard and TiO2 as a negative particle standard. A calcite standard was also used in the alamarBlue® and haemolysis assays, due to a high calcite content in some samples. Results are presented as the relative percentages of the positive and negative controls.
Statistical analysis
Student’s t-test and ANOVA general linear model with a Tukey’s post-hoc test were performed to determine the significance of differences among samples in the in vitro assays (Minitab 15). Pearson’s correlation test was used to determine significant correlations amongst different physicochemical characteristics and toxicological results (* p <0.05, ** p <0.01, *** p <0.001).
Conclusions
This study, the first to systematically characterise the physicochemical properties and potential toxicity of a range of globally sourced DE samples, shows that the toxic potential of DE varies by processing technique and source. Flux-calcined samples were unreactive, whereas unprocessed and calcined DE had variable reactivity.
No correlation was observed between crystalline silica content and DE’s potential toxicity, despite previously being implicated in epidemiological studies of DE exposure. The dearth of crystalline silica at the particle surface, due to its crystallisation within an amorphous matrix, its presence in a heterogeneous dust, and impurities within the crystalline silica likely reduce the potential reactivity of these crystalline silica-bearing particles.
It is likely that a number of physicochemical properties play a role in DE toxicity. Calcium-rich phases may be important in the toxicity of some unprocessed samples, and iron or amorphous phases may be involved in calcined DE toxicity. Surface area, especially, was correlated to calcined and flux-calcined DE reactivity here, and the importance of surface reactivity and the unique particle morphologies of DE merits further investigation.
Although no single physicochemical property of DE considered here could be linked to its potential toxicity, a clear outcome of this study is that the crystalline silica content, alone, should not be used to determine the DE hazard, nor should it be assumed that it is the cause of disease observed in epidemiological or clinical studies without further investigation.
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Competing interests
The research has been part-funded by IMA Europe, an umbrella organisation representing a number of diatomite companies in all non-commercial issues related to the properties and safe use of minerals. Samples were provided by one diatomite company. Neither IMA Europe nor the company had input to the experimental design or research conducted but have seen a draft version of this manuscript.
Authors’ contributions
CN designed the study, performed physicochemical analyses at Durham University and in vitro toxicology assays at Heriot-Watt University, and drafted the manuscript. CJH conceived and designed the study, supervised CN, and participated in physicochemical characterisation. DED advised on study outcomes, and helped with physicochemical analyses. AK performed cytokine analysis and AK and DB participated in in vitro assays. VS and DB participated in the design of the study and VS supervised CN. All authors helped draft the manuscript, read and approved the final version.