Low concentration of HgCl2 drives rat hepatocytes to autophagy/apoptosis/necroptosis in a time-dependent manner

Mercury (Hg) is known to produce hepatotoxicity driving cells towards apoptosis. It was recently reported that low concentrations of Hg (5 μM) initiate autophagy in vitro within 30 min of incubation modulated by several autophagy-related gene proteins, and co-regulators through ubiquitination. The present study aimed to elucidate in vitro mode of cytotoxic responses including programed cell death in 5-μM Hg-treated rat hepatocytes. Autophagy proceeded from 30 min to 4 hr mediated by crosstalk between specific regulating factors of cell-death-signaling mechanisms. It was noted that after 4-hr incubation with 5 μM HgCl₂, cells were driven towards apoptosis followed by necroptosis within 6.5 hr. Receptor-interacting serine-threonine protein 3 (RIP3) and caspase-8 played a significant role in interlinking function. The positive role of caspase-8 with RIP3 significantly triggered caspase-3 via extrinsic apoptotic pathway. A shift from apoptosis to necroptosis occurred after 6 hr via tumor necrosis factor α-RIP3-caspase-8 pathway. No alteration in caspase-3 expression and presence of high-mobility group box 1 protein in nucleus indicated absence of apoptosis and necrosis in rat hepatocytes between 6.5 and 8 hr. Data indicated that cellular homeostasis is regulated by modulating different proteins and driving hepatocytes through autophagy to apoptosis to necroptosis in a time-dependent manner.     

Mercuric chloride effects on adult rat oval cells-induced apoptosis

In the present investigation, the toxicity of mercuric chloride (HgCl₂) was evaluated in adult oval cells isolated from rat utilizing the 2-acetylaminofluorene/partial hepatectomy technique. Isolated oval cells were incubated with 5 μM of HgCl₂ for 8 hr to elucidate in vitro cytotoxic responses. Recently, autophagic cell death was found in rat hepatocytes in vitro within 30 min of incubation with 5 μM of mercury (Hg) which triggered apoptosis and necroptosis in a time-dependent manner. Nuclear degradation occurred within 30 min of incubation and progressed with time until 8 hr. Apoptosis evidenced by activation of caspase-dependent pathway between 30 min to 8 hr of incubation was mediated via interchange of death domain signaling pathways. Receptor-interacting protein played a positive role to modulate the death domain receptors in the scenario of apoptotic death of oval cells until 6 hr. Autophagic marker proteins ATG12 and LC3B exerted a significant role in triggering apoptosis in 5 μM Hg-treated oval cells. No apparent expression of apoptosis-inducing-factor (AIF) and HMGB1 indicated absence of caspase-independent apoptosis and necrosis in the rat oval cells between 30 min and 8 hr. Thus a low concentration of Hg modulates programmed cell death in adult rat oval cells by altering expression of proteins involved in the molecular mechanisms of cellular functions.     

Protective role of Curcuma longa extract and curcumin on mercuric chloride-induced nephrotoxicity in rats: evidence by histological architecture

Mercury (Hg) is a potent nephrotoxin. The aim of this study was to investigate the protective role of Curcuma longa extract and curcumin against HgCl₂-induced nephrotoxicity. Male Sprague Dawley rats were administered HgCl₂ (12 μmol kg^?1, ip; once only) followed by treatment of Curcuma longa extract (200 mg kg^?1, po) and curcumin (80 mg kg^?1, po) for three days after 24 h of HgCl₂ administration. The present results showed that mercuric chloride administration caused an impairment of renal function system which was evident from significant increase in urea, creatinine, uric acid, and blood urea nitrogen concentration in serum. In addition, the swelling in glomerulus and degenerated renal tubules with obstructed lumen was also observed by acute mercuric chloride administration. Treatment with Curcuma longa extract and curcumin was effective in restoring all variables of kidney functions near to control group, which was consistent with kidney histoarchitecture. In conclusion, these results suggest that Curcuma longa extract and curcumin protect against HgCl₂-induced nephrotoxicity. This study could be important for the further understanding of mercury toxicity in renal tissues and in the development of better treatments for people and/or animals exposed to the metal.     

Comparison of cellular and molecular cytotoxic mechanisms of Cochlodinium polykrikoides in isolated trout and rat hepatocytes

Harmful algal blooms produced by the marine ichthyotoxic dinoflagellate Cochlodinium polykrikoides are responsible for mass mortalities of wild and farmed fish globally. This study compared the cytotoxic mechanisms of C. polykrikoides total extract on both trout and rat liver hepatocytes. Trout hepatocytes were more sensitive than rat hepatocytes against C. polykrikoides extract. The effective concentration 50 after 3 hour incubation (EC50_3hr) concentrations found for C. polykrikoides extract in trout and rat hepatocytes (i.e., 50% membrane lysis in 3 hr) were Eq. 1 cell/ml and Eq. 240 cell/ml, respectively. C. polykrikoides extract exposure in both isolated trout and rat hepatocytes resulted in membrane lysis, reactive oxygen species formation, glutathione depletion, collapse of mitochondrial membrane potential, ATP depletion, increase in adenosine diphosphate (ADP)/adenosine triphosphate (ATP) ratio, cytochrome c release into the hepatocyte cytosol, and activation of caspases cascade. Trout hepatocyte toxicity was also associated with lysosomal membrane injury. Mitochondrial permeability transition in both trout and rat hepatocytes produced cytochrome c release from the mitochondrial intramembrane space into the cytosol. Thus, the cytochrome c release triggered activation of caspase-3 and apoptosis. Finally, data demonstrated that C. polykrikoides extract may induce more apoptotic phenotype in rat than trout hepatocytes, which in the latter favored predominantly necrotic mode of cell death.     

Involvement of mitochondrial-mediated caspase-3 activation and lysosomal labilization in acrylamide-induced liver toxicity

Acrylamide (ACR) is a chemical frequently used in both industrial and synthetic processes and may be produced during food processing. ACR at very high concentrations is postulated to exert its toxicity through the stimulation of an oxidative stress. ACR in excessive doses induces the central nervous system, reproduction, and genetic toxicity. However, ACR effects on the liver, a major organ of drug metabolism, have not been adequately explored. In addition, the role of mitochondria in an ACR-mediated hepatotoxicity is still unclear. The aim of this study was to investigate the cytotoxic mechanisms attributed to ACR using isolated rat hepatocytes. Hepatocytes were isolated by the collagenase perfusion method and incubated with an EC50_2hr concentration of ACR for 3 hr. The EC50_{2 hr} of ACR on isolated rat hepatocytes was determined to be 1 mM. Based on our results, hepatocytes cytotoxicity of ACR (1 mM) was mediated by a reactive oxygen species formation and lipid peroxidation. Incubation of hepatocytes with ACR produced rapid hepatocyte glutathione depletion which is another marker of the cellular oxidative stress. ACR cytotoxicity was also associated with mitochondrial injury as evidenced by the decline of mitochondrial membrane potential and lysosomal membrane leakiness. Our results also showed that ACR induced caspase-3 activation, the final mediator of apoptosis signaling. These findings contribute to a better understanding underlying mechanisms involved in ACR hepatotoxicity originating from the oxidative stress and ending in mitochondrial/lysosomal damage and cell death signaling.     

Hypoxia-driven selective degradation of cellular proteins in jumbo squids during diel migration to the oxygen minimum zones

The jumbo squid, Dosidicus gigas, is an oceanic top predator in the eastern tropical Pacific that undergoes diel vertical migrations into mesopelagic oxygen minimum zones (OMZs). Besides glycogen breakdown, the pathways of the squid’s metabolic (suppression) strategy are poorly understood. Here, juvenile D. gigas were exposed to oxygen levels found in the OMZ off Gulf of California (1 % O₂, 1 kPa at 10 °C) with the aim to identify, via proteomic tools, eventual anaerobic protein degradation as potential energy source at such depths. Under hypoxia, total protein concentration decreased nonsignificantly from 79.2 ± 12.4 mg g^?1 wet mass to 74.7 ± 11.7 mg g^?1 wet mass (p > 0.05). Yet, there was a significant decrease in heat-shock protein (Hsp) 90 and α-actinin contents (p < 0.05). The lower α-actinin concentration at late hypoxia was probably related to decreased protection of the Hsp90 chaperon machinery resulting in increased ubiquitination (p < 0.05) and subsequent degradation. Thus, the present findings indicate that D. gigas might degrade, at least under progressing hypoxia, specific mantle proteins anaerobically to increase/maintain anaerobic ATP production and extend hypoxia exposure time. Moreover, the ubiquitin–proteasome system seems to play an important role in hypoxia tolerance, but further investigations are necessary to discover its full potential and pathways.     

Studies on mercuric sulfide,a component of cinnabar,a Chinese herbal medicine,on celluar functions in mouse lung and fibroblasts

Although the cytotoxic effects of mercuric chloride (HgCl₂) and methylmercury chloride (MeHg) have been extensively studied, the insoluble mercuric sulfide (HgS) has been the subject of fewer studies. Since the traditional Chinese mineral drug, cinnabar (containing >95% HgS) continues to be used as an ingredient for infant sedation, the pharmacological and toxicological effects of HgS need to be clarified. In previous experiments, HgS and cinnabar were shown to be absorbed from the gastrointestinal tract (GIT) and distributed in various tissues including the lungs. Thus, a preliminarily examination of whether HgS might exert any oxidative stress on a mouse lung was undertaken. HgS reduced GSH content and increased lipid peroxidation in the lung. Further studies on the cytotoxic effects and the possible mechanisms of action of HgS were compared with HgCl₂ and MeHg in cultured lung fibroblast V₇₉ cells. The results showed that HgS produced cytotoxicity at a concentration (400–1200 µM)in a dependent manner with IC₅₀ of 795.6 µM, as compared to HgCl₂ and MeHg, 8.1 µM and 5.9 µM, respectively. In addition, the HgS induced the phenomena of DNA fragmentation, increasing reactive oxygen species (ROS) and decreasing mitochondrial membrane potential, accompanied by decreased levels of intracellular ATP and GSH and higher lipid peroxidation levels, similar to HgCl₂ and MeHg, but with different toxicokinetic properties. These findings provide evidence for understanding the mechanisms underlying the toxic effects of HgS.     

Geological factors controlling occurrence and distribution of arsenic in groundwaters from the southern margin of the Duero Basin,Spain

Groundwater from springs and boreholes on the southern edge of the Cenozoic Duero Basin (DB) of Spain has concentrations of arsenic (As) which are commonly above the EC drinking-water limit of 10 μg/L and reach observed values up to 241 μg/L. Groundwater compositions within the sedimentary aquifer vary from Ca–HCO₃ type, variably affected by evaporation and agricultural pollution at shallow levels, to Na–HCO₃ compositions in deeper boreholes of the basin. Groundwater conditions are mainly oxidising, but reducing groundwaters exist in sub-basins within the aquifer, localised flow paths likely being influenced by basement structure. Arsenic concentrations are spatially variable, reaching up to 38 μg/L in springs of the Spanish Central System (SCS) basement aquifer and up to 62 μg/L in springs from the DB. Highest As concentrations are associated with the Na–HCO₃ compositions in deep boreholes (200–450 m depth) within the DB. These have high pH values (up to 9.6) which can give rise to associated elevated concentrations of V and U (up to 64 and 30 μg/L, respectively). In the deep borehole waters of the DB, oxidising flows derived from the mineralised igneous–metamorphic basement and discharging via major faults, and are considered the origin of the higher concentrations. Compositions are consistent with desorption of As and other anionic species from metal oxyhydroxides in an oxic environment. Under locally reducing conditions prevalent in some low-flow parts of the DB, an absence of detectable dissolved As is coincident with low or undetectable SO₄ concentrations, and consistent with loss via formation of authigenic sulphide minerals. Mitigation measures are needed urgently in this semi-arid region where provision of alternative sources of safe drinking water is logistically difficult and expensive.     

多效唑对水生生物的急性毒性评价研究

本文以斜生栅藻、大型水蚤和草鱼为试验生物,研究植物生长调节剂多效唑对水生生物的急性毒性效应。多效唑对斜生栅藻EC_(50)-96h为20.62mg/l;对大型水蚤LC_(50)-48h为21.86mg/h;对草鱼苗静态生物检测结果为LC_(50)—120h 16.33mg/l,流水生物检测结果为LC_(50)-96h 14.36mg/l,LC_(50)-120h 9.13mg/l。经多效唑处理后,藻的细胞形态也发生明显改变。根据农药的实验室毒性评价划分标准,多效唑对三种生物的毒性均属低毒;田间安全性预评价的结果也表明其毒性为低毒,故认为多效唑是一种对水生生物危害较小的农药。但其慢性毒性及长期积累效应,尚有待进一步研究。     

Chronic exposure effects of copper on growth,metamorphosis and thyroid gland,liver health in Chinese toad,Bufo gargarizans tadpoles

The impact of copper exposure on Chinese toad (Bufo gargarizans) tadpoles was investigated in this study. First, the 96h LC50 value of copper was 8.697 μM, by means of a 4 d acute toxicity test. Second, we studied the chronic effects of copper on B. gargarizans tadpoles at control, 0.025, 0.1 and 1.0 μM concentration. Survival, body length, body weight, developmental stage, incidence of metamorphic climax, and size at metamorphic climax were determined. In tadpoles developed to metamorphic climax (stage G42), liver and thyroid gland were assessed histologically. Copper at 0.1 and 1.0 μM could inhibit tadpole growth and prolong tadpole metamorphic progress relative to controls. Tadpole size (total length and weight) at stage G42 is also affected in the 0.1 and 1.0 μM treatments. In addition, histological examinations have revealed that 1.0 μM copper could cause significant pathological changes and hepatocytes degeneration in liver. Furthermore, histomorphological measurements indicated that copper at 0.1 and 1.0 μM reduced thyroid gland size, diameter and number of follicle. In conclusion, our study suggests that Cu could damage the liver and thyroid gland, so growth and metamorphosis of B. gargarizans tadpoles were inhibited resulted of disrupting liver metabolism and THs homeostasis.     

In vitro genotoxicity of extracellular extract from Nostoc piscinale

The extracellular extract obtained from 3 weeks incubation of the soil isolate cyanobacterium strain Nostoc piscinale GT-319 in BG-11 broth medium showed cytotoxic activity against Chinese Hamster Ovary (CHO) cells. Based on comet assay, a concentration of 333 µg mL^?1 (IC₅₀) produced DNA breakages in CHO cells. The concentration of 481 mg kg^?1 (LD₅₀) produced acute toxicity in mice at 48 h.     

Microstructure of the paper nautilus (Argonauta nodosa) shell and the novel application of electron backscatter diffraction (EBSD) to address effects of ocean acidification

Electron backscatter diffraction (EBSD) is a powerful microscopic technique to characterise the crystallography of biomineralisation. Here, we use high-resolution EBSD to characterise one of the least studied shells in the ocean, the female argonaut brood chamber, and to examine the changes in shell microstructure in response to incubation in decreased pH conditions. The thin (225 μm) shell of Argonauta nodosa is magnesium calcite with an average magnesium content of ca. 5.1 Wt % MgCO₃. EBSD and scanning electron microscopy (SEM) revealed that calcification of the shell is bidirectional with formation of irregular crystalline grains. Following a 2 week incubation in a range of pH treatments (pH, 8.1–7.2), shell fragment weight decreased by dissolution in pH ≤ 7.8. EBSD and SEM revealed altered shell crystallography and microstructure at pH ≤ 7.4 due to preferential etching down crystallite grain boundaries and a change in crystalline orientation on both the inner and outer shell surfaces. Our study highlights the value of EBSD for the detailed examination of biogenic carbonates and its potential use in the field of ocean acidification research.     

High seasonal variation of atmospheric C and particle concentrations in Delhi,India

The highly populated Indian regions are currently in a phase of rapid economic growth resulting in high emissions of carbonaceous aerosols. This leads to poor air quality and impact on climate. The chemical composition of carbonaceous aerosols has rarely been studied in industrial areas of India. Here, we investigated carbonaceous aerosols in particulate matter (PM) monthly in the industrial area of Delhi in 2011. The concentrations of organic C and elemental C in PM₁₀ fraction were analyzed. Results show a clear seasonal variability of organic and elemental C. PM₁₀ ranged 95.9–453.5 μg m^?3, organic C ranged 28.8–159.4 μg m^?3, and elemental C ranged 7.5–44.0 μg m^?3; those values were higher than reported values. Organic and elemental C were correlated with each other in pre-monsoon and winter seasons, implying the existence of similar emission sources such as coal combustion, biomass burning and vehicular exhaust. The annual average contribution of total carbonaceous aerosols in PM₁₀ was estimated as 62 %.     

Hematoxicity and hepatoxicity attributed to zinc and cadmium in the Norwegian rat

This study was carried out to determine changes in the blood and liver of rats given oral doses of cadmium (Cd) and zinc (Zn). Thirty 3 month old rats were kept six to a compartment in a well-demarcated five compartment cage labeled A, B, C, D, and E. After 2 weeks of acclimatization, and with ad libitum administration of water and feed, group A and B rats were, respectively, exposed to 100 mg CdSO₄ and 2200 mg ZnSO₄, respectively, mixed with their feed. Group C rats were fed with a combination of 100 mg CdSO₄ and 2200 mg ZnSO₄ mixed with their feed, while group D rats were exposed to 100 mg CdSO₄ mixed with their feed for 48 h, and after they were given 2200 mg ZnSO₄ for 96 h. Group E rats served as control and were fed a normal diet. Analysis of blood samples after 5 days showed that in rats of groups A, B, C, and D, WBC count increased from 6800 to 12,400 mm^?3 and platelets from 26,000 to 88,000 mm^?3. Reduction in blood counts were found for RBC from 13.8 to 4.7 × 10⁶ mm^?3, hemoglobin from 15.6 to 11.4 dL^?1, and PCV from 47 to 34%. ESR was the same 1.0 mm h^?1 in all groups. Severe damage to the liver was also shown by severe degeneration of hepatocytes and increase in Kuppfer cells. The above observations were mildest in the liver of group C rats. A reduction of body weight was observed in all treatment groups. Cd and Zn were found to be hematoxic and hepatotoxic in the Norwegian rat with Cd exhibiting greater toxicity than Zn. Toxicity of Cd was suppressed by Zn only when both metals were admistered at the same time, but a delay in intake of Zn (after 48 h) had little effect on Cd toxicity.     

Histopathological alterations of Nile tilapia, Oreochromis niloticus in acute and subchronic alachlor exposure

Histopathological alterations in Nile tilapia, Oreochromis niloticus, aged 3 months and subjected to acute and subchronic alachlor exposure were studied by light microscopy LC50 values of alachlor for 24 hr 48 hr, 72 hr and 96 hr were 963.6, 563, 448, and 381.9 microg l(-1), respectively and the maximum acceptable toxicant concentration (MATC) was 350 microg l(-1). Fish were exposed to 381.9 (acute) and 35 microg l(-1) (sub-chronic) of alachlor for 24, 48, 72 and 96 hr and 90 days, respectively Gill lamellae and kidney tubules were the primary target organs for the acute toxic effect of alachlor while in the subchronic exposure, the toxic effect on the gills was less marked than that of the kidneys and liver Gill alterations included edema of the epithelial cell system, aneurisms with some ruptures, hypertrophy and hyperplasia of epithelial cells. The liver showed hydropic swelling of hepatocytes and vacuolation. Lipid vacuoles were observed in hepatocytes in the second and third month of subchronic exposure. The kidney showed hydropic swelling of tubular cells, lipid vacuole accumulation in many tubules, and nuclear pyknosis. The findings of this study could be used as a guideline forbiomonitoring programs on populations of Nile tilapia cultured near alachlor contaminated areas.     

Copper sulphate use in South African traditional medicine

Copper (Cu) is an essential element to humans; however, exposure to elevated concentrations through occupational hazard and/or environmental means may be detrimental. This paper provides results of a cross-sectional study aimed to determine the prevalence of copper sulphate (CuSO₄) use in South African traditional medicine by traditional health practitioners (THPs) and details the use thereof. A total of 201 THPs were enrolled from two main municipal areas of KwaZulu-Natal (South Africa). Information on demographic characteristics of THPs, reasons for using or not using CuSO₄ as well as administration methods and age groups of recipients were collected. Of the 201 THPs interviewed, 145 (72 %) use CuSO₄ for healing purposes. The use of CuSO₄ was strongly associated with gender (p = 0.009) where the proportion of CuSO₄ users was higher for female than male THPs. CuSO₄ was reportedly administered to individuals of all ages, including infants and children. The main routes of administration were enema (n = 110; 76 %), oral (n = 40; 28 %) and use in bath (n = 40; 28 %). The reasons cited for use are diverse and included skin rashes (n = 43; 30 %), aches, pains and swelling (n = 38; 28 %) as well as sexually transmitted diseases (n = 28; 19 %). This study identified a high prevalence of THPs using CuSO₄ for healing purposes. These findings support the need to regulate South African traditional medicine to safeguard the user.     

Fluoride induces DNA damage and cytotoxicity in human hepatocellular carcinoma cells

Humans are primarily exposed to fluoride (Fl), a widespread environmental pollutant, via contaminated drinking water and foodstuffs. The aim of this study was to examine whether sodium fluoride (NaF) exerted cytotoxic effects in human hepatocarcinoma (HepG2) cells. HepG2 cells were incubated with different concentrations of NaF and reactive oxygen species (ROS) levels, cell cycle, apoptosis, and DNA damage determined. Concentration-dependent studies showed that exposure to HepG2 cells with different concentrations of NaF for 24 hr significantly decreased cell viability and intracellular antioxidant capacity. Furthermore, NaF exposure increased lipid peroxidation levels and accumulation of intracellular ROS; and lowered antioxidant glutathione concentrations. In addition to oxidative impairments, NaF treatment enhanced HepG2 cell death via apoptotic pathway as evidenced by DNA fragmentation and cell cycle arrest. Sodium fluoride treatment unregulated p53 level, and Bax and Bcl2 expression. Diminished cell viability and changes in cell cycle accompanied a rise in p53 expression.     

The influence of mercury on the burrowing behaviour ofCorophium volutator

The burrowing behaviour ofCorophium volutator (Pallas) has been recorded in sediments treated with seawater solutions of 0.001, 0.1, 10, and 1000 ppm HgCl₂. Heterotrophic sediment bacteria have also been counted. Non-choice and choice experiments have been conducted. In non-choice experiments no individuals burrowed in the sediment treated with 1000 ppm HgCl₂ solutions. The same numbers of individuals burrowed in the sediments treated with 0.001, 0.1, and 10 ppm HgCl₂ solutions as in the control. Bacterial numbers were lower in the sediments treated with 10 and 1000 ppm HgCl₂ solutions. Hence, a mercury concentration that killed bacteria (10 ppm) did not inhibit burrowing. In choice experiments, individuals avoided all the mercury-treated sediments, particularly that treated with 1000 ppm HgCl₂ solution. Bacterial numbers were only lower than the control in the sediments treated with 10 and 1000 ppm HgCl₂ solutions. Hence, individuals avoided treated sediments (0.001, 0.1 ppm HgCl₂) in which no bacterial mortality had occurred.     

Treating n-butane by activated carbon and metal oxides

In order to reduce environmental pollution and regulatory issues, the adsorption of n-butane from vehicle exhaust is one of the necessary requirements. When active carbon was mixed with different proportions of metal oxides, the adsorption capacity of n-butane was markedly improved. Infrared spectroscopy data provided information on functional groups derived from activated carbon composite metal oxide indicating that one of the best adsorption capacities for n-butane was obtained by mixing 3 g TiO₂, 2.7 g CuO, and 2.1 g NaBO₃·4H₂O with 30 g activated carbon. However, more effective results were obtained by mixing 6 g TiO₂, 2.1 g CuO, and 0.9 g NaBO₃·4H₂O with 30 g activated carbon. Data suggest that these chemical complexes may be effective in lowering n-butane pollution.     

Impact of natural and calcined starfish (<Emphasis Type="Italic">Asterina pectinifera</Emphasis>) on the stabilization of Pb,Zn and As in contaminated agricultural soil

Metal stabilization using soil amendments is an extensively applied, economically viable and environmentally friendly remediation technique. The stabilization of Pb, Zn and As in contaminated soils was evaluated using natural starfish (NSF) and calcined starfish (CSF) wastes at different application rates (0, 2.5, 5.0 and 10.0 wt%). An incubation study was conducted over 14 months, and the efficiency of stabilization for Pb, Zn and As in soil was evaluated by the toxicity characteristic leaching procedure (TCLP) test. The TCLP-extractable Pb was reduced by 76.3–100 and 91.2–100 % in soil treated with NSF and CSF, respectively. The TCLP-extractable Zn was also reduced by 89.8–100 and 93.2–100 % in soil treated with NSF and CSF, respectively. These reductions could be associated with the increased metal adsorption and the formation of insoluble metal precipitates due to increased soil pH following application of the amendments. However, the TCLP-extractable As was increased in the soil treated with NSF, possibly due to the competitive adsorption of phosphorous. In contrast, the TCLP-extractable As in the 10 % CSF treatment was not detectable because insoluble Ca–As compounds might be formed at high pH values. Thermodynamic modeling by visual MINTEQ predicted the formation of ettringite (Ca₆Al₂(SO₄)₃(OH)₁₂·26H₂O) and portlandite (Ca(OH)₂) in the 10 % CSF-treated soil, while SEM–EDS analysis confirmed the needle-like structure of ettringite in which Pb was incorporated and stabilized in the 10 % CSF treatment.