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Assessing the biological reactivity of organic compounds on volcanic ash: Implications for human health hazard

Exposure to volcanic ash is a long-standing health concern for people living near active volcanoes and in distal urban areas. During transport and deposition, ash is subjected to various physicochemical processes that may change its surface composition and, consequently, bioreactivity. One such process is the interaction with anthropogenic pollutants; however, the potential for adsorbed, deleterious organic compounds to directly impact human health is unknown. We use an in vitro bioanalytical approach to screen for the presence of organic compounds of toxicological concern on ash surfaces and assess their biological potency. These compounds include polycyclic aromatic hydrocarbons (PAHs), polychlorinated dibenzo- p -dioxins and dibenzofurans (PCDD/Fs) and dioxin-like polychlorinated biphenyls (dlPCBs). Analysis of ash collected in or near urbanised areas at five active volcanoes across the world (Etna, Italy; Fuego, Guatemala; Kelud, Indonesia; Sakurajima, Japan; Tungurahua, Ecuador) using the bioassay inferred the presence of such compounds on all samples. A relatively low response to PCDD/Fs and the absence of a dlPCBs response in the bioassay suggest that the measured activity is dominated by PAHs and PAH-like compounds. This study is the first to demonstrate a biological potency of organic pollutants associated with volcanic ash particles. According to our estimations, they are present in quantities below recommended exposure limits and likely pose a low direct concern for human health.

Bulletin of Volcanology

Serendipitous insight into pre-hatch microstructure formation in larval lake whitefish (Coregonus clupeaformis) otoliths

Otoliths are useful tools to estimate daily age, growth, and ontogenetic shifts in the early life stages of fish, providing information to support management efforts. Little effort, however, has gone into the examination of pre-hatch increment development. To assess variation in pre-hatch otolith size and increment formation, lake whitefish ( Coregonus clupeaformis ) sagittal otoliths were examined at the time of hatch from the control group of a previous toxicology study. The otolith surface area, nucleus surface area, and increment counts were measured, displaying variability in pre-hatch size and increment formation. While the nucleus surface area did not vary based on incubation time, the number of pre-hatch increments and total surface area increased for delayed-hatched individuals despite being reared under near-identical conditions. Increment deposition, however, did not appear to be based on an endogenous daily circadian rhythm. These results refine our understanding of otolith microstructure during embryonic development and confound the interpretation of otoliths from wild-caught larvae.

Environmental Biology of Fishes

Mercury concentrations in Seaside Sparrows and Marsh Rice Rats differ across the Mississippi River Estuary

Mercury (Hg) concentrations and their associated toxicological effects in terrestrial ecosystems of the Gulf of Mexico are largely unknown. Compounding this uncertainty, a large input of organic matter from the 2010 Deepwater Horizon oil spill may have altered Hg cycling and bioaccumulation dynamics. To test this idea, we quantified blood concentrations of total mercury (THg) in Seaside Sparrows ( Ammospiza maritima ) and Marsh Rice Rats ( Oryzomys palustris ) in marshes west and east of the Mississippi River in 2015 and 2016. We also tested for a difference in THg concentrations between oiled and non-oiled sites. To address the potential confounding effect of diet variation on Hg transfer, we used stable nitrogen (δ 15 N) and carbon (δ 13 C) isotope values as proxies of trophic position and the source of primary production, respectively. Our results revealed that five to six years after the spill, THg concentrations were not higher in sites oiled by the spill compared to non-oiled sites. In both species, THg was higher at sites east of the Mississippi River compared to control and oiled sites, located west. In Seaside Sparrows but not in Marsh Rice Rats, THg increased with δ 15 N values, suggesting Hg trophic biomagnification. Overall, even in sites with the most elevated THg, concentrations were generally low. In Seaside Sparrows, THg concentrations were also lower than previously reported in this and other closely related passerines, with only 7% of tested birds exceeding the lowest observed effect concentration associated with toxic effects across bird species (0.2 µg/g ww ) . The factors associated with geographic heterogeneity in Hg exposure remain uncertain. Clarification could inform risk assessment and future restoration and management actions in a region facing vast anthropogenic changes.

Ecotoxicology

Environmental survey in the Tuul and Orkhon River basins of north-central Mongolia, 2010: Metals and other elements in streambed sediment and floodplain soil

Streambed sediment and subsurface floodplain soil were sampled for elemental analyses from 15 locations in river basins of north-central Mongolia during August 2010. Our primary objective was to conduct a reconnaissance-level assessment of potential inputs of toxicologically important metals and metalloids to Lake Baikal, Russia, that might originate from mining and urban activities within tributaries of the Selenga River in Mongolia. Samples were collected in triplicate from all sites, then dried, and sieved to <2 mm for analysis by portable X-ray florescence spectroscopy and by inductively coupled plasma mass spectrometry after digestion with concentrated nitric and hydrochloric acids. Arsenic, copper, and mercury were greatly elevated in sediment and floodplain soil collected from tributary streams located near two major mining operations. Lead and zinc were moderately elevated in streambed sediment and in floodplain soil obtained from a small tributary in the capital city of Ulaanbaatar, but those concentrations were considerably less than probable effects benchmarks. Historical and possibly present mining activities have led to considerable metal contamination in certain tributaries of the Orkhon River in north-central Mongolia; however, metals originating from those sources did not appear to be accumulating in sediments at our downstream-most sampling sites located near the border between Mongolia and Russia.

Environmental Monitoring and Assessment

Pre-mining trace element and radiation exposure to biota from a breccia pipe uranium mine in the Grand Canyon (Arizona, USA) watershed

The risks to wildlife and humans from uranium (U) mining in the Grand Canyon watershed are largely unknown. In addition to U, other co-occurring ore constituents contribute to risks to biological receptors depending on their toxicological profiles. This study characterizes the pre-mining concentrations of total arsenic (As), cadmium (Cd), copper (Cu), lead (Pb), mercury (Hg), nickel (Ni), selenium (Se), thallium (Tl), U, and zinc (Zn); radiation levels; and histopathology in biota (vegetation, invertebrates, amphibians, birds, and mammals) at the Canyon Mine. Gross alpha levels were below the reporting limit (4 pCi/g) in all samples, and gross beta levels were indicative of background in vegetation (<10–17 pCi/g) and rodents (<10–43.5 pCi/g). Concentrations of U, Tl, Pb, Ni, Cu, and As in vegetation downwind from the mine were likely the result of aeolian transport. Chemical concentrations in rodents and terrestrial invertebrates indicate that surface disturbance during mine construction has not resulted in statistically significant spatial differences in fauna concentrations adjacent to the mine. Chemical concentrations in egg contents and nestlings of non-aquatic birds were less than method quantification limits or did not exceed toxicity thresholds. Bioaccumulation of As, Pb, Se, Tl, and U was evident in Western spadefoot ( Spea multiplicata ) tadpoles from the mine containment pond; concentrations of As (28.9–31.4 μg/g) and Se (5.81–7.20 μg/g) exceeded toxicity values and were significantly greater than in tadpoles from a nearby water source. Continued evaluation of As and Se in biota inhabiting and forging in the mine containment pond is warranted as mining progresses.

Arizona

Assessing the impact of charr research past, present, and future

The 9th International Charr Symposium convened on 18–21 June 2018, in Duluth, Minnesota, USA to gather scientists with an interest in charr biology and management from the entire geographical range of the genus Salvelinus. The symposium was attended by 169 individuals from six countries, and included 99 oral and 32 poster presentations, 28 of which were published in the ensuing proceedings. Topic areas of presentations and publications included: (1) cultural anthropology; (2) genetics, evolution, taxonomy and systematics; (3) behavior, movement and habitat; (4) reproductive ecology, developmental ontogeny and physiology; (5) population ecology, dynamics and life history; (6) trophic ecology, parasites, predators, toxicology and pollution; and (7) management. Research reported in these proceedings has built upon the history of earlier symposiums and will be continued with the 10th symposium scheduled to convene in Nikko City, Tochigi Prefecture, Japan, in 2021.

Hydrobiologia

Determination of mono- and non-o,o′-chlorine substituted polychlorinated biphenyls in Aroclors and environmental samples

High resolution capillary gas chromatography (GC) is the best known technique for the separation of complex mixtures; however, no single GC column has yet separated all 209 congeners of polychlorinated biphenyls (PCBs). A number of coeluting PCB congener pairs exist, and even under favorable separation conditions such as with multidimensional GC, assignment of peak identities to known PCB structures is tedious, subject to errors from other contaminants, and requires enrichment to achieve the necessary detection limits for the most toxic PCB congeners. Routine analysis of PCBs is also complicated by coelution with other halogenated hydrocarbons such as naphthalenes, terphenyls, dibenzofurans, and pesticides. Therefore, techniques for class separations of PCBs prior to gas chromatography must be developed. These techniques should separate PCB congeners along lines that have environmental or toxicological significance. The unique ability of activated carbon to separate halogenated aromatics on the basis of molecular planarity and degree of halogenation has been demonstrated. We present a method that uses dispersed carbon on glass fibers and commercially available instrumentation to fractionate and determine mono- and non-o,o′-chlorine substituted PCB congeners in Aroclors and environmental samples.

Chemosphere

Seasonal variation in diagnostic enzymes and biochemical constituents of captive northern bobwhites and passerines

1. A variety of biochemical measurements were taken periodically in captive northern bobwhite ( Colinus virginianus L.), European starlings ( Sturnus vulgaris L.), red-winged blackbirds ( Agelaius phoeniceus L.) and common grackles ( Quiscalus quiscula L.) to determine whether baseline values remain sufficiently stable throughout the year for general clinical use in the absence of concurrent control specimens. 2. Variables included whole blood hemotacrit and hemoglobin, plasma lactate dehydrogenase, α-hydroxybutyrate dehydrogenase, aspartate aminotransferase, alinine aminotransferase, creatine kinase, butyrylcholinesterase, alkaline phosphatase, glucose, albumin, total protein, creatinine, urea nitrogen, uric acid, cholesterol, and triglycerides, and brain acetylcholinesterase. Butyrl- and acetylcholinesterase were included because of their specific uses in toxicology. 3. Significant seasonal differences were detected for each of the variables except brain acetylcholinesterase in at least one of the species. Significant species differences were detected during at least one season for all of the variables measured. 4. All species were maintained outdoors, but only northern bobwhites came into reproductive condition and showed sex-differences in the clinical variables during their normal breeding season. 5. It was concluded that reference values for the 18 clinical variables measured could be calculated from our data for adult specimens of the species studied, and that results for one species cannot be extrapolated with certainty to any other species. 6. Estimated normal bounds for each of the 18 variables measured by commonly used clinical procedures are presented for reproductively quiescent northern bobwhites, European starlings, red-winged blackbirds, and common grackles.

Comparative Biochemistry and Physiology, Part B: C

The environmental and medical geochemistry of potentially hazardous materials produced by disasters

Many natural or human-caused disasters release potentially hazardous materials (HM) that may pose threats to the environment and health of exposed humans, wildlife, and livestock. This chapter summarizes the environmentally and toxicologically significant physical, mineralogical, and geochemical characteristics of materials produced by a wide variety of recent disasters, such as volcanic eruptions, hurricanes and extreme storms, spills of mining/mineral-processing wastes or coal extraction by-products, and the 2001 attacks on and collapse of the World Trade Center towers. In describing these characteristics, this chapter also illustrates the important roles that geochemists and other earth scientists can play in environmental disaster response and preparedness. In addition to characterizing in detail the physical, chemical, and microbial makeup of HM generated by the disasters, these roles also include (1) identifying and discriminating potential multiple sources of the materials; (2) monitoring, mapping, and modeling dispersal and evolution of the materials in the environment; (3) understanding how the materials are modified by environmental processes; (4) identifying key characteristics and processes that influence the materials' toxicity to exposed humans and ecosystems; (5) estimating shifts away from predisaster environmental baseline conditions; and (6) using geochemical insights learned from past disasters to help estimate, prepare for, and increase societal resilience to the environmental and related health impacts of future disasters.

Book chapter

Depletion of isoeugenol residues from the fillet tissue of AQUI-S™ exposed rainbow trout ( Oncorhynchus mykiss )

There is a critical need in U.S. public aquaculture and fishery management for an approved sedative that allows for the immediate release of fish after sedation. AQUI-S TM is a fish anesthetic/sedative approved for use in several countries and until recently was being developed in the U.S. as a sedative for immediate release of fish after sedation. The U.S. National Toxicology Program reported that isoeugenol (the active ingredient in AQUI-S TM ) exposed male mice showed clear evidence of carcinogenicity, therefore efforts within the U.S. Department of Interior to develop AQUI-S TM as a sedative that allows for immediate release ceased. Despite the ruling, AQUI-S TM still has the potential to be approved as an anesthetic with a short withdrawal time. Among the data required to gain approval for use in the U.S. are data describing the composition and depletion of all AQUI-S TM residues from fish fillet tissue. A total residue depletion study for AQUI-S TM was conducted by exposing market-sized rainbow trout, Oncorhynchus mykiss (mean weight, 502.7 &plusmn; 54 g; s.d.) to 8.9 mg/L of 14 C-[URL]-isoeugenol for 60 min in 17 &deg;C water. The 14 C-[URL]-isoeugenol was mixed with a surfactant resulting in a mixture that mimicked AQUI-S TM . Groups of fish ( n = 6) were sampled immediately after the exposure (0-h sample group) and at 0.5, 1, 2, and 4 h thereafter. Total isoeugenol-equivalent residue concentrations in the fillet tissue were determined by oxidizing triplicate subsamples of homogenized skin-on fillet tissue from each fish to 14 CO 2 and enumerating the radioactivity by static liquid scintillation counting. Isoeugenol concentrations in fillet tissue were determined by extracting homogenized fillet tissue with solvents and determining the isoeugenol concentrations in the extracts with high performance liquid chromatography techniques. The mean total isoeugenol-equivalent residue concentrations in the 0, 0.5, 1, 2, and 4-h sample groups were 55.4, 32.0, 19.8, 11.3, and 4.9 &micro;g/g, respectively. The primary chemical residue in fillet tissue from all exposed fish was isoeugenol. The mean isoeugenol concentrations in the 0, 0.5, 1, 2, and 4-h sample groups were 48.9, 26.5, 15.3, 7.2, and 2.2 &micro;g/g, respectively. The percents of the total radioactivity classified as isoeugenol in the 0, 0.5, 1, 2, and 4-h tissue extracts were 95, 73, 73, 64, and 48%, respectively.

Aquaculture

Development of aquaculture protocols and gonadal differentiation of green sunfish (Lepomis cyanellus)

We provide detailed rearing methods and describe green sunfish ( Lepomis cyanellus ) gonadal development and histological differentiation for both sexes. Developing in-depth aquaculture protocols and describing the gonadal differentiation of green sunfish could facilitate strategies to control nuisance populations, enhance stocking programs, and provide information for this species' use in bioassay trials or toxicology studies. Our methods resulted in consistent year-round production of green sunfish and allowed us to identify the timing of their gonadal differentiation through histological assessment. Our spawning methods provided year-round volitional spawns from green sunfish broodstock. Our rearing methods involved weaning larval green sunfish off live nauplii and onto only artificial diets by 37 days post-hatch (dph). Most of the offspring generation reached sexual maturity by 213 dph. Green sunfish are gonochoristic, with testes and ovaries differentiating directly from undifferentiated gonads. Ovaries begin to differentiate by 39 dph and testes begin to differentiate by 69 dph. This information can provide biologists consistent means to produce this Centrachid and understand their gonadal development.

Aquaculture

Mercury speciation in the Mt. Amiata mining district (Italy): interplay between urban activities and mercury contamination

A fundamental step to evaluate the biogeochemical and eco-toxicological significance of Hg dispersion in the environment is to determine speciation of Hg in solid matrices. In this study, several analytical techniques such as scanning electron microscopy-energy dispersive X-ray spectroscopy (SEM-EDS), sequential chemical extractions (SCEs), and X-ray absorption spectroscopy (XANES) were used to identify Hg compounds and Hg speciation in samples collected from the Mt. Amiata Hg mining district, southern Tuscany, Italy. Different geological materials, such as mine waste calcine (retorted ore), soil, stream sediment, and stream water suspended particulate matter were analyzed. Results show that the samples were generally composed of highly insoluble Hg compounds such as sulphides (HgS, cinnabar and metacinnabar), and more soluble Hg halides such as those associated with the mosesite group. Other moderately soluble Hg compounds, HgCl 2 , HgO and Hg 0 , were also identified in stream sediments draining the mining area. The presence of these minerals suggests active and continuous runoff of soluble Hg compounds from calcines, where such Hg compounds form during retorting, or later in secondary processes. Specifically, we suggest that, due to the proximity of Hg mines to the urban center of Abbadia San Salvatore, the influence of other anthropogenic activities was a key factor for Hg speciation, resulting in the formation of unusual Hg-minerals such as mosesite.

Tuscany

Gene transcription patterns in response to low level petroleum contaminants in Mytilus trossulus from field sites and harbors in southcentral Alaska

The 1989 Exxon Valdez oil spill damaged a wide range of natural resources, including intertidal communities, and post-spill studies demonstrated acute and chronic exposure and injury to an array of species. Standard toxicological methods to evaluate petroleum contaminants have assessed tissue burdens, with fewer assays providing indicators of health or physiology, particularly when contaminant levels are low and chronic. Marine mussels are a ubiquitous and crucial component of the nearshore environment, and new genomic technologies exist to quantify molecular responses of individual mussels to stimuli, including exposure to polycyclic aromatic hydrocarbons (PAHs). We used gene-based assays of exposure and physiological function to assess chronic oil contamination using the Pacific blue mussel, Mytilus trossulus . We developed a diagnostic gene transcription panel to investigate exposure to PAHs and other contaminants and its effects on mussel physiology and health. During 2012–2015, we analyzed mussels from five field sites in western Prince William Sound, Alaska, with varying oil histories from the 1989 Exxon Valdez oil spill, and from three boat harbors in the area. Gene transcription patterns of mussels from harbors were consistent with elevated exposure to PAHs or other contaminants, whereas transcription patterns of mussels sampled from shorelines in areas affected by the oil spill indicated no PAH exposure.

Alaska

CYP1A protein expression and catalytic activity in double-crested cormorants experimentally exposed to Deepwater Horizon Mississippi Canyon 252 oil

Double-crested cormorants ( Phalacrocorax auritus , DCCO) were orally exposed to Deepwater Horizon Mississippi Canyon 252 (DWH) oil to investigate oil-induced toxicological impacts. Livers were collected for multiple analyses including cytochrome P4501A (CYP1A) enzymatic activity and protein expression. CYP1A enzymatic activity was measured by alkoxyresorufin O-dealkylase (AROD) assays. Activities specific to the O-dealkylation of four resorufin ethers are reported: benzyloxyresorufin O-debenzylase (BROD), ethoxyresorufin O-deethylase (EROD), methoxyresorufin O-demethylase (MROD), and pentoxyresorufin O-depentylase (PROD). CYP1A protein expression was measured by western blot analysis with a CYP1A1 mouse monoclonal antibody. In study 1, hepatic BROD, EROD, and PROD activities were significantly induced in DCCO orally exposed to 20 ml/kg body weight (bw) oil as a single dose or daily for 5 days. Western blot analysis revealed hepatic CYP1A protein induction in both treatment groups. In study 2 (5 ml/kg bw oil or 10 ml/kg bw oil, 21 day exposure), all four hepatic ARODs were significantly induced. Western blots showed an increase in hepatic CYP1A expression in both treatment groups with a significant induction in birds exposed to 10 ml/kg oil. Significant correlations were detected among all 4 AROD activities in both studies and between CYP1A protein expression and both MROD and PROD activities in study 2. EROD activity was highest for both treatment groups in both studies while BROD activity had the greatest fold-induction. While PROD activity values were consistently low, the fold-induction was high, usually 2nd highest to BROD activity. The observed induced AROD profiles detected in the present studies suggest both CYP1A4/1A5 DCCO isoforms are being induced after MC252 oil ingestion. A review of the literature on avian CYP1A AROD activity levels and protein expression after exposure to CYP1A inducers highlights the need for species-specific studies to accurately evaluate avian exposure to oil.

Ecotoxicology and Environmental Safety

Exploring relationships among stream health, human well-being, and demographics in Virginia, USA

Quantification of empirical relationships between ecosystem health and human well-being is uncommon at broad spatial scales. We used public data for Virginia (USA) counties to examine pairwise correlations among two indicators of stream health, thirteen indicators of human well-being, and four demographic metrics. Our indicators of stream health included the Virginia Stream Condition Index (VSCI) and the percentage of stream kilometers with a fish consumption advisory (%FCA); these measures are inversely related. VSCI and %FCA were correlated with some indicators of human health, safety and security, and living standards, as well as with some demographic metrics. VSCI was most strongly correlated (positively) with the percentage of a county’s population self-identifying as White; %FCA was most strongly correlated (positively) with overall mortality rate (number of deaths per 100,000 people). This exploratory study highlights the need for future multidisciplinary, multiscale studies to characterize toxicological, epidemiological, socioeconomic, and political linkages – including causal mechanisms – between ecosystem health and human well-being.

Virginia

Perfluorodecanesulfonate (PFDS) induces innate immune toxicity through the NF-κB pathway in early life stage zebrafish

Perfluorodecanesulfonate (PFDS), a long-chain polyfluoroalkyl substance (PFAS), is widely detected in aquatic environments and increasingly recognized for its environmental persistence and bioaccumulative potential; however, its immunotoxicity remains poorly understood in aquatic biota. In this study, early life stage zebrafish ( Danio rerio ) were exposed to environmentally relevant concentrations of PFDS and PFOS for 120 h to better characterize the adverse effects of PFDS on aquatic organisms. Additionally, the toxicological differences between PFDS and PFOS at the same exposure concentrations were compared, as PFDS is a known substitute for PFOS. PFDS bioaccumulated in zebrafish larvae at environmentally relevant concentrations, which disrupted immune function by altering the number of macrophages and neutrophils, inducing oxidative stress, and dysregulating immune markers such as interleukins and immunoglobulins. Mechanistically, PFDS activated the nuclear factor kappa B (NF-κB) signaling pathway, driving pro-inflammatory cytokine expression and immune dysfunction. Furthermore, the use of a NF-κB morpholino knockdown confirmed the role of the NF-κB pathway in mediating PFDS-induced immunotoxicity. These findings provide the first comprehensive evidence of PFDS-induced immunotoxicity being mediated through NF-κB activation, offering novel insights into the ecological risks of long-chain perfluorosulfonic acids. Notably, PFDS exhibited a stronger immunotoxic response relative to PFOS, indicating that its adverse effects may be more severe. Overall, these findings provide valuable insights for the ecological risk assessment of PFDS and the toxic potential that unregulated PFAS can have to aquatic systems.

Environment International

Per- and polyfluoroalkyl substances and pesticides in black bears (Ursus americanus) and grizzly bears (Ursus arctos horribilis) from Montana's Cabinet-Yaak ecosystem: A baseline assessment of emerging and legacy contaminants

Per- and polyfluoroalkyl substances (PFAS) and pesticides are globally distributed contaminants that persist in terrestrial food webs, yet baseline data for large omnivores in the continental U.S. remain limited. This study quantified PFAS and pesticides in whole blood from free-ranging black bears ( Ursus americanus , n = 15) and grizzly bears ( Ursus arctos horribilis , n = 9) in the Cabinet-Yaak Ecosystem of northwestern Montana. Using high-resolution mass spectrometry-based methods (GC-MS/MS and LC-MS/MS), 52 PFAS and 186 pesticides were analyzed. PFAS were detected in all bears, with total PFAS concentrations ranging from 79.5 to 317 pg/mL. PFAS profiles were dominated by long-chain perfluoroalkyl carboxylic acids, particularly PFOA, PFDA, and PFUnDA, with minimal contribution from short-chain compounds. Species was the strongest ecological predictor of PFAS concentrations, although variability across age, sex, and capture location were observed. Only two pesticides, piperonyl butoxide and permethrin, were detected at quantifiable concentrations (2070–12,600 pg/mL), and no correlations were observed between pesticide and PFAS concentrations, indicating independent exposure pathways. The predominance of long-chain PFAS suggests diffuse environmental sources and bioaccumulation within terrestrial food webs. Although measured concentrations were generally low relative to other wildlife toxicological studies, the persistence and protein-binding properties of long-chain PFAS warrant continued monitoring, particularly for long-lived omnivores. These results establish baseline contaminant concentrations for two apex omnivores in a remote ecosystem and highlight the utility of large mammals as integrative sentinels of environmental contaminant exposure for regional monitoring and wildlife conservation. Continued monitoring in the Cabinet-Yaak and across western North America will be critical for assessing temporal trends, identifying new contaminant sources, and evaluating ecological health in bear populations.

Montana

The effect of aluminium and sodium impurities on the in vitro toxicity and pro-inflammatory potential of cristobalite

Background Exposure to crystalline silica (SiO 2 ), in the form of quartz, tridymite or cristobalite, can cause respiratory diseases, such as silicosis. However, the observed toxicity and pathogenicity of crystalline silica is highly variable. This has been attributed to a number of inherent and external factors, including the presence of impurities. In cristobalite-rich dusts, substitutions of aluminium (Al) for silicon (Si) in the cristobalite structure, and impurities occluding the silica surface, have been hypothesised to decrease its toxicity. This hypothesis is tested here through the characterisation and in vitro toxicological study of synthesised cristobalite with incremental amounts of Al and sodium (Na) dopants. Methods Samples of synthetic cristobalite with incremental amounts of Al and Na impurities, and tridymite, were produced through heating of a silica sol-gel. Samples were characterised for mineralogy, cristobalite purity and abundance, particle size, surface area and surface charge. In vitro assays assessed the ability of the samples to induce cytotoxicity and TNF-α production in J774 macrophages, and haemolysis of red blood cells. Results Al-only doped or Al+Na co-doped cristobalite contained between 1 and 4 oxide wt% Al and Na within its structure. Co-doped samples also contained Al- and Na-rich phases, such as albite. Doping reduced cytotoxicity to J774 macrophages and haemolytic capacity compared to non-doped samples. Al-only doping was more effective at decreasing cristobalite reactivity than Al+Na co-doping. The reduction in the reactivity of cristobalite is attributed to both structural impurities and a lower abundance of crystalline silica in doped samples. Neither non-doped nor doped crystalline silica induced production of the pro-inflammatory cytokine TNF-α in J774 macrophages. Conclusions Impurities can reduce the toxic potential of cristobalite and may help explain the low reactivity of some cristobalite-rich dusts. Whilst further work is required to determine if these effects translate to altered pathogenesis, the results have potential implications for the regulation of crystalline silica exposures.

Environmental Research