Application of a Solar UV/Chlorine Advanced Oxidation Process to Oil Sands Process-Affected Water Remediation

The solar UV/chlorine process has emerged as a novel advanced oxidation process for industrial and municipal wastewaters. Currently, its practical application to oil sands process-affected water (OSPW) remediation has been studied to treat fresh OSPW retained in large tailings ponds, which can cause significant adverse environmental impacts on ground and surface waters in Northern Alberta, Canada. Degradation of naphthenic acids (NAs) and fluorophore organic compounds in OSPW was investigated. In a laboratory-scale UV/chlorine treatment, the NAs degradation was clearly structure-dependent and hydroxyl radical-based. In terms of the NAs degradation rate, the raw OSPW (pH ∼ 8.3) rates were higher than those at an alkaline condition (pH = 10). Under actual sunlight, direct solar photolysis partially degraded fluorophore organic compounds, as indicated by the qualitative synchronous fluorescence spectra (SFS) of the OSPW, but did not impact NAs degradation. The solar/chlorine process effectively removed NAs (75–84% removal) and fluorophore organic compounds in OSPW in the presence of 200 or 300 mg L^–1 OCl^–. The acute toxicity of OSPW toward Vibrio fischeri was reduced after the solar/chlorine treatment. However, the OSPW toxicity toward goldfish primary kidney macrophages after solar/chlorine treatment showed no obvious toxicity reduction versus that of untreated OSPW, which warrants further study for process optimization

Comparison of the Acute Immunotoxicity of Nonfractionated and Fractionated Oil Sands Process-Affected Water Using Mammalian Macrophages

OSPW is a complex mixture of inorganic and organic substances and its principal toxic components have yet to be fully characterized. Previously, we showed in vitro that the oil sands process-affected water (OSPW) organic fraction (OF) caused a concentration-dependent immunotoxicity in mammals. In the present study we further explore the immunotoxicological properties of OSPW in mammals using a series of in vitro bioassays. Specifically, using the RAW 264.7 mouse macrophage cell line we show that whole OSPW containing naphthenic acid (NA) concentrations ranging from 12 to 18 mg/L, significantly inhibited cell proliferation, reduced cell viability, and was directly cytotoxic, whereas the exposure of cells to equivalent doses of the OSPW-OF had no measurable effects. Whole OSPW exposures also caused morphological changes in RAW 264.7 cells, and at sublethal doses (i.e., 10 mg/L) it induced the early expression of the stress genes <i>hmox1</i> and <i>gadd45</i>. In addition, at NA concentrations of 10 mg/L, whole OSPW but not the OSPW-OF had significant effects on pro-inflammatory cytokine mRNA levels and cytokine protein secretion activities. Finally, whole OSPW also impaired the ability of RAW 264.7 cells to perform phagocytosis. Overall, we demonstrate that exposure to whole OSPW (at NA doses ranging from 10 to 20 mg/L), but not the OSPW-OF caused both cytotoxic and immunomodulatory changes in mouse macrophages. This suggests that the complex mixture of inorganic and organic components found in whole OSPW are acutely toxic at much lower doses than we previously reported for the OSPW-OF (i.e., 50 mg/L) due to unknown additive and/or synergistic interactions that likely occur between the various components present in whole OSPW

Fish and Mammalian Phagocytes Differentially Regulate Pro-Inflammatory and Homeostatic Responses <em>In Vivo</em>

<div><p>Phagocytosis is a cellular mechanism that is important to the early induction of antimicrobial responses and the regulation of adaptive immunity. At an inflammatory site, phagocytes serve as central regulators for both pro-inflammatory and homeostatic anti-inflammatory processes. However, it remains unclear if this is a recent evolutionary development or whether the capacity to balance between these two seemingly contradictory processes is a feature already displayed in lower vertebrates. In this study, we used murine (C57BL/6) and teleost fish (<em>C. auratus</em>) <em>in vitro</em> and <em>in vivo</em> models to assess the evolutionary conservation of this dichotomy at a site of inflammation. At the level of the macrophage, we found that teleost fish already displayed divergent pro-inflammatory and homeostatic responses following internalization of zymosan or apoptotic bodies, respectively, and that these were consistent with those of mice. However, fish and mice displayed significant differences <em>in vivo</em> with regards to the level of responsiveness to zymosan and apoptotic bodies, the identity of infiltrating leukocytes, their rate of infiltration, and the kinetics and strength of resulting antimicrobial responses. Unlike macrophages, significant differences were identified between teleost and murine neutrophilic responses. We report for the first time that activated murine, but not teleost neutrophils, possess the capacity to internalize apoptotic bodies. This internalization translates into reduction of neutrophil ROS production. This may play an important part in the recently identified anti-inflammatory activity that mammalian neutrophils display during the resolution phase of inflammation. Our observations are consistent with continued honing of inflammatory control mechanisms from fish to mammals, and provide added insights into the evolutionary path that has resulted in the integrated, multilayered responses that are characteristic of higher vertebrates.</p> </div

Apoptotic bodies downregulate murine neutrophil ROS production in a contact dependent manner.

<p>Goldfish (left) and C57BL/6 mice (right) were injected intraperitoneally with zymosan (2.5 mg). Activated peritoneal cells from were harvested by peritoneal lavage and subpopulations were isolated by density centrifugation. (A) Separated neutrophil or mononuclear populations were incubated with labeled apoptotic bodies for 2 h and internalization was analyzed. n = 4; * p<0.05 and ** p<0.01; Mφ/M  =  macrophage/monocyte. (B) Isolated populations were cultured for 2 h in the presence of the indicated stimuli. Conditions denoted within brackets were contained within a 4 μm transwell. After 2 h, responder cells outside the transwells were harvested and respiratory burst was assayed using DHR.</p

Pro-inflammatory (zymosan) and homeostatic (apoptotic bodies) stimuli differentially impact leukocyte infiltration profiles in goldfish and mice.

<p>Goldfish and C57BL/6 mice were injected intraperitoneally with saline, apoptotic bodies (5×10⁶) or zymosan (2.5 mg). Apoptotic bodies were also pre-injected 4 h before zymosan injections. Goldfish leukocyte populations were defined by imaging flow cytometry (area, internal complexity, and morphology) and staining patterns with Sudan Black and an anti-CSF-1R antibody (Figure S3). Murine cells were defined based on surface markers for neutrophils (F4/80⁻/Gr1⁺/CD11b⁺), monocytes (F4/80^lo/Gr1^+/−/CD11b⁺), macrophages (F4/80^hi/Gr1^+/−/CD11b⁺) and lymphocytes (F4/80⁻/Gr1⁻; CD3, B220, NK1.1; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047070#pone.0047070.s004" target="_blank">Figure S4A</a>). n = 4; * p<0.05 and ** p<0.01 compared to control; + p<0.05 and ++ p<0.01 compared to zymosan. No- no internalized particle; AB- apoptotic body; Zy- zymosan.</p

Murine neutrophil respiratory burst antimicrobial responses are most greatly affected by the presence of apoptotic bodies.

<p>(A) C57BL/6 mice were injected intraperitoneally with saline, apoptotic bodies (5×10⁶) or zymosan (2.5 mg). Apoptotic bodies were also pre-injected 0, 2, or 4 h before zymosan injections. Cells from injected mice were harvested by peritoneal lavage and respiratory burst was assayed with DHR in peritoneal cell subpopulations based on forward scatter and side scatter profiles (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047070#pone.0047070.s004" target="_blank">Figure S4B</a>). n = 4; * p<0.05 and ** p<0.01 compared to control; + p<0.05 and ++ p<0.01 compared to zymosan. No- no internalized particle; A.B.- apoptotic body; Zy- zymosan. (B) Histograms show representative DHR responses for total leukocytes, neutrophils and monocytes/macrophages. We found that the high responders were predominantly neutrophils (>90%). Pre-incubation with apoptotic bodies resulted in a preferential switch in the neutrophil population from high responders to mid/low responders.</p

<i>In vivo</i> administration of zymosan induces a marked infiltration of leukocytes that is linked to high levels of respiratory burst.

<p>Goldfish (left) and C57BL/6 mice (right) were injected intraperitoneally with 2.5 mg of zymosan. Cells were harvested by peritoneal lavage at 0 h (saline alone), 8, 24 and 48 h and counted (top row). Injection of zymosan resulted in a marked increase in cell numbers isolated from the peritoneum that peaked at 48 h for mice and 24 h for goldfish. Respiratory burst in isolated cells at these time points was determined with DHR (bottom row). n = 4; * p<0.05; ** p<0.01.</p

Impact of Ozonation on Naphthenic Acids Speciation and Toxicity of Oil Sands Process-Affected Water to <i>Vibrio fischeri</i> and Mammalian Immune System

Oil sands process-affected water (OSPW) is the water contained in tailings impoundment structures in oil sands operations. There are concerns about the environmental impacts of the release of OSPW because of its toxicity. In this study, ozonation followed by biodegradation was used to remediate OSPW. The impacts of the ozone process evolution on the naphthenic acids (NAs) speciation and acute toxicity were evaluated. Ion-mobility spectrometry (IMS) was used to preliminarily separate isomeric and homologous species. The results showed limited effects of the ozone reactor size on the treatment performance in terms of contaminant removal. In terms of NAs speciation, high reactivity of NAs with higher number of carbons and rings was only observed in a region of high reactivity (i.e., utilized ozone dose lower than 50 mg/L). It was also found that nearly 0.5 mg/L total NAs was oxidized per mg/L of utilized ozone dose, at utilized ozone doses lower than 50 mg/L. IMS showed that ozonation was able to degrade NAs, oxidized NAs, and sulfur/nitrogenated NAs. Complete removal of toxicity toward <i>Vibrio fischeri</i> was achieved after ozonation followed by 28-day biodegradation period. In vitro and in vivo assays indicated that ozonation reduced the OSPW toxicity to mice

<i>In vivo</i> administration of apoptotic bodies leads to a more dramatic reduction of pro-inflammatory respiratory burst responses in teleost fish compared to mice.

<p>Goldfish and C57BL/6 mice were injected intraperitoneally with saline, apoptotic bodies (5×10⁶) or zymosan (2.5 mg) and incubated for 24 h. Apoptotic bodies were also pre-injected 0, 2, or 4 h before zymosan injections to assess the contributions of kinetics to these responses. Cells from injected animals were harvested by peritoneal lavage and respiratory burst was assayed with DHR. n = 4; * p<0.05 and ** p<0.01 compared to PBS (saline) control; + p<0.05 and ++ p<0.01 compared to zymosan. No- no internalized particle; A.B.− apoptotic body; Zy- zymosan.</p

Goldfish myeloid cell respiratory burst responses are most affected by the presence of apoptotic bodies.

<p>(A) Goldfish were injected intraperitoneally with saline, apoptotic bodies (5×10⁶) or zymosan (2.5 mg). Apoptotic bodies were also pre-injected 0, 2, or 4 h before zymosan injections. Cells from injected goldfish were harvested by peritoneal lavage and respiratory burst was assayed with DHR in peritoneal cell subpopulations based on forward scatter and side scatter profiles. n = 4; * p<0.05 and ** p<0.01 compared to control; + p<0.05 and ++ p<0.01 compared to zymosan. No- no internalized particle; A.B.- apoptotic body; Zy- zymosan. (B) Representative histograms show a single peak in DHR responses.</p