Formularity: Software for Automated Formula Assignment of Natural and Other Organic Matter from Ultrahigh-Resolution Mass Spectra.

Ultrahigh resolution mass spectrometry, such as Fourier transform ion cyclotron resonance mass spectrometry (FT ICR MS), can resolve thousands of molecular ions in complex organic matrices. A Compound Identification Algorithm (CIA) was previously developed for automated elemental formula assignment for natural organic matter (NOM). In this work, we describe software Formularity with a user-friendly interface for CIA function and newly developed search function Isotopic Pattern Algorithm (IPA). While CIA assigns elemental formulas for compounds containing C, H, O, N, S, and P, IPA is capable of assigning formulas for compounds containing other elements. We used halogenated organic compounds (HOC), a chemical class that is ubiquitous in nature as well as anthropogenic systems, as an example to demonstrate the capability of Formularity with IPA. A HOC standard mix was used to evaluate the identification confidence of IPA. Tap water and HOC spike in Suwannee River NOM were used to assess HOC identification in complex environmental samples. Strategies for reconciliation of CIA and IPA assignments were discussed. Software and sample databases with documentation are freely available.

Novel Interactions between Gut Microbiome and Host Drug-Processing Genes Modify the Hepatic Metabolism of the Environmental Chemicals Polybrominated Diphenyl Ethers.

The gut microbiome is a novel frontier in xenobiotic metabolism. Polybrominated diphenyl ethers (PBDEs), especially BDE-47 (2, 2', 4, 4'-tetrabromodiphenyl ether) and BDE-99 (2, 2', 4, 4',5-pentabromodiphenyl ether), are among the most abundant and persistent environmental contaminants that produce a variety of toxicities. Little is known about how the gut microbiome affects the hepatic metabolism of PBDEs and the PBDE-mediated regulation of drug-processing genes (DPGs) in vivo. The goal of this study was to determine the role of gut microbiome in modulating the hepatic biotransformation of PBDEs. Nine-week-old male C57BL/6J conventional (CV) or germ-free (GF) mice were treated with vehicle, BDE-47 or BDE-99 (100 µmol/kg) for 4 days. Following BDE-47 treatment, GF mice had higher levels of 5-OH-BDE-47 but lower levels of four other metabolites in liver than CV mice; whereas following BDE-99 treatment GF mice had lower levels of four minor metabolites in liver than CV mice. RNA sequencing demonstrated that the hepatic expression of DPGs was regulated by both PBDEs and enterotypes. Under basal conditions, the lack of gut microbiome upregulated the Cyp2c subfamily but downregulated the Cyp3a subfamily. Following PBDE exposure, certain DPGs were differentially regulated by PBDEs in a gut microbiome-dependent manner. Interestingly, the lack of gut microbiome augmented PBDE-mediated upregulation of many DPGs, such as Cyp1a2 and Cyp3a11 in mouse liver, which was further confirmed by targeted metabolomics. The lack of gut microbiome also augmented the Cyp3a enzyme activity in liver. In conclusion, our study has unveiled a novel interaction between gut microbiome and the hepatic biotransformation of PBDEs.

Discrimination in Degradability of Soil Pyrogenic Organic Matter Follows a Return-On-Energy-Investment Principle.

A fundamental understanding of biodegradability is central to elucidating the role(s) of pyrogenic organic matter (PyOM) in biogeochemical cycles. Since microbial community and ecosystem dynamics are driven by net energy flows, then a quantitative assessment of energy value versus energy requirement for oxidation of PyOM should yield important insights into their biodegradability. We used bomb calorimetry, stepwise isothermal thermogravimetric analysis (isoTGA), and 5-year in situ bidegradation data to develop energy-biodegradability relationships for a suite of plant- and manure-derived PyOM (n = 10). The net energy value (ΔE) for PyOM was between 4.0 and 175 kJ mol(-1); with manure-derived PyOM having the highest ΔE. Thermal-oxidation activation energy (Ea) requirements ranged from 51 to 125 kJ mol(-1), with wood-derived PyOM having the highest Ea requirements. We propose a return-on-investment (ROI) parameter (ΔE/Ea) for differentiating short-to-medium term biodegradability of PyOM and deciphering if biodegradation will most likely proceed via cometabolism (ROI < 1) or direct metabolism (ROI ≥ 1). The ROI-biodegradability relationship was sigmoidal with higher biodegradability associated with PyOM of higher ROI; indicating that microbes exhibit a higher preference for "high investment value" PyOM.

Generalized two-dimensional perturbation correlation infrared spectroscopy reveals mechanisms for the development of surface charge and recalcitrance in plant-derived biochars.

Fundamental knowledge of how biochars develop surface-charge and resistance to environmental degradation is crucial to their production for customized applications or understanding their functions in the environment. Two-dimensional perturbation-based correlation infrared spectroscopy (2D-PCIS) was used to study the biochar formation process in three taxonomically different plant biomass, under oxygen-limited conditions along a heat-treatment-temperature gradient (HTT; 200-650 °C). Results from 2D-PCIS pointed to the systematic, HTT-induced defragmenting of lignocellulose H-bonding network and demethylenation/demethylation, oxidation, or dehydroxylation/dehydrogenation of lignocellulose fragments as the primary reactions controlling biochar properties along the HTT gradient. The cleavage of OH(...)O-type H-bonds, oxidation of free primary hydroxyls to carboxyls (carboxylation; HTT ≤ 500 °C), and their subsequent dehydrogenation/dehydroxylation (HTT > 500 °C) controlled surface charge on the biochars; while the dehydrogenation of methylene groups, which yielded increasingly condensed structures (R-CH(2)-R →R═CH-R →R═C═R), controlled biochar recalcitrance. Variations in biochar properties across plant biomass type were attributable to taxa-specific transformations. For example, apparent inefficiencies in the cleavage of wood-specific H-bonds, and their subsequent oxidation to carboxyls, lead to lower surface charge in wood biochars (compared to grass biochars). Both nontaxa and taxa-specific transformations highlighted by 2D-PCIS could have significant implications for biochar functioning in fire-impacted or biochar-amended systems.

An index-based approach to assessing recalcitrance and soil carbon sequestration potential of engineered black carbons (biochars).

The ability of engineered black carbons (or biochars) to resist abiotic and, or biotic degradation (herein referred to as recalcitrance) is crucial to their successful deployment as a soil carbon sequestration strategy. A new recalcitrance index, the R(50), for assessing biochar quality for carbon sequestration is proposed. The R(50) is based on the relative thermal stability of a given biochar to that of graphite and was developed and evaluated with a variety of biochars (n = 59), and soot-like black carbons. Comparison of R(50), with biochar physicochemical properties and biochar-C mineralization revealed the existence of a quantifiable relationship between R(50) and biochar recalcitrance. As presented here, the R(50) is immediately applicable to pre-land application screening of biochars into Class A (R(50) ≥ 0.70), Class B (0.50 ≤ R(50) < 0.70) or Class C (R(50) < 0.50) recalcitrance/carbon sequestration classes. Class A and Class C biochars would have carbon sequestration potential comparable to soot/graphite and uncharred plant biomass, respectively, whereas Class B biochars would have intermediate carbon sequestration potential. We believe that the coupling of the R(50), to an index-based degradation, and an economic model could provide a suitable framework in which to comprehensively assess soil carbon sequestration in biochars.

Pyrogenic inputs of anthropogenic Pb and Hg to sediments of the Hood Canal, Washington, in the 20th century: source evidence from stable Pb isotopes and PAH signatures.

Combustion-derived PAHs and stable Pb isotopic signatures ((206)Pb/(207)Pb) in sedimentary records assisted in reconstructing the sources of atmospheric inputs of anthropogenic Pb and Hg to the Hood Canal, Washington. The sediment-focusing corrected peak fluxes of total Pb and Hg (1960-70s) demonstrate that the watershed of Hood Canal has received greater atmospheric inputs of these metals than its mostly rural land use would predict. The tight relationships between the Pb, Hg, and organic markers in the cores indicate that these metals are derived from industrial combustion emissions. Multiple lines of evidence point to the Asarco smelter, located in the Main Basin of Puget Sound, as the major emission source of these metals to the watershed of the Hood Canal. The evidence includes (1) similar PAH isomer ratios in sediment cores from the two basins, (2) the correlations between Pb, Hg, and Cu in sediments and previously studied environmental samples including particulate matter emitted from the Asarco smelter's main stack at the peak of production, and (3) Pb isotope ratios. The natural rate of recovery in Hood Canal since the 1970s, back to preindustrial metal concentrations, was linear and contrasts with recovery rates reported for the Main Basin which slowed post late 1980s.

Metal interactions at the biochar-water interface: energetics and structure-sorption relationships elucidated by flow adsorption microcalorimetry.

Plant-derived biochars exhibit large physicochemical heterogeneity due to variations in biomass chemistry and combustion conditions. However, the influence of biochar heterogeneity on biochar-metal interaction mechanisms has not been systematically described. We used flow adsorption microcalorimetry to study structure-sorption relationships between twelve plant-derived biochars and two metals (K(+) and Cd(2+)) of different Lewis acidity. Irrespective of the biochar structure, sorption of K(+) (a hard Lewis acid) occurred predominantly on deprotonated functional groups via ion exchange with molar heats of adsorption (ΔH(ads)) of -4 kJ mol(-1) to -8 kJ mol(-1). By comparison, although ion exchange could not be completely ruled out, our data pointed to Cd(2+) (a soft Lewis acid) sorption occurring predominantly via two distinct cation-π bonding mechanisms, each with ΔH(ads) of +17 kJ mol(-1). The first, evident in low charge-low carbonized biochars, suggested Cd(2+)-π bonding to soft ligands such as -C ═ O; while the second, evident in low charge-highly carbonized biochars, pointed to Cd(2+)-π bonding with electron-rich domains on aromatic structures. Quantitative contributions of these mechanisms to Cd(2+) sorption can exceed 3 times that expected for ion exchange and therefore could have significant implications for the biogeochemical cycling of metals in fire-impacted or biochar-amended systems.

Spatial and temporal baseline of perfluorooctanesulfonic acid retained in sediment core samples from Puget Sound, Washington, USA.

Per- and poly-fluorinated alkyl substances (PFAS) are a group of highly persistent synthetic chemicals utilized in many industrial and consumer products, and - significantly toward introduction to the marine environment - in fire-fighting foams. Recently, PFAS have been linked to adverse health effects, prompting the need to better understand transport, lability, and fate. Perfluorooctanesulfonic acid (PFOS), a manufactured PFAS and biodegradation product, partitions to marine sediments and thus can be used as a primary indicator toward regulatory efforts. The current study offers a spatial and temporal analysis of Puget Sound from cores collected adjacent Tacoma and Seattle, WA, as well as cores from central Hood Canal and Carr Inlet. Temporal fluxes reflected releases into the environment corresponding with initial production and subsequent increases in use. Biologically active layers ranged from 434 pg/g (Carr Inlet) to 824 pg/g (Hood Canal) PFOS, producing benthic community risk quotients between 0.11 and 0.17.

Occupational and dietary differences in hydroxylated and methoxylated PBDEs and metals in plasma from Puget Sound, Washington, USA region volunteers.

Electronic waste (E-waste) recycling is a rapidly growing occupation in the USA with the potential for elevated exposure to flame retardants and metals associated with electronic devices. We previously measured polybrominated diphenyl ethers (PBDEs) in plasma from E-waste workers and found them similar to non-E-waste workers. This study focused on structurally related PBDE derivatives, the hydroxylated (OH-PBDEs) and methoxylated (MeO-PBDEs) forms along with metals known to occur in E-waste. Humans can metabolize PBDEs and some MeO-PBDEs into OH-PBDEs, which is a concern due to greater health risks associated with OH-PBDEs. We measured 32 different OH-PBDEs and MeO-PBDEs in plasma samples provided by 113 volunteers living in the greater Puget Sound region of Washington State, USA. We measured 14 metals in a subset of 10 E-waste and 10 non-E-waste volunteers. Volunteers were selected based on occupational and dietary habits: work outdoors and consume above average amounts of seafood (outdoor), electronic waste recycling (E-waste) or non-specific indoor occupations (indoor). A two-week food consumption diary was obtained from each volunteer prior to blood sampling. OH-PBDEs were detected in all volunteers varying between 0.27 and 102 ng/g/g-lipid. The MeO-PBDEs were detected in most, but not all volunteers varying between n.d. - 60.4 ng/g/g-lipid. E-waste recyclers had OH-PBDE and MeO-PBDE plasma levels that were similar to the indoor group. The outdoor group had significantly higher levels of MeO-PBDEs, but not OH-PBDEs. Comparison of plasma concentrations of BDE-47 with its known hydroxylated metabolites suggested OH-PBDE levels were likely determined by biotransformation and at least two subpopulations identified differing in their apparent rates of OH-PBDE formation. The metals analysis indicated no significant differences between E-waste workers and non-E-waste workers. Our results indicate E-waste workers do not have elevated plasma levels of these contaminants relative to non-E-waste workers.

Metabolic Profiling Reveals Biochemical Pathways Responsible for Eelgrass Response to Elevated CO2 and Temperature.

As CO2 levels in Earth's atmosphere and oceans steadily rise, varying organismal responses may produce ecological losers and winners. Increased ocean CO2 can enhance seagrass productivity and thermal tolerance, providing some compensation for climate warming. However, the metabolic shifts driving the positive response to elevated CO2 by these important ecosystem engineers remain unknown. We analyzed whole-plant performance and metabolic profiles of two geographically distinct eelgrass (Zostera marina L.) populations in response to CO2 enrichment. In addition to enhancing overall plant size, growth and survival, CO2 enrichment increased the abundance of Calvin Cycle and nitrogen assimilation metabolites while suppressing the abundance of stress-related metabolites. Overall metabolome differences between populations suggest that some eelgrass phenotypes may be better suited than others to cope with an increasingly hot and sour sea. Our results suggest that seagrass populations will respond variably, but overall positively, to increasing CO2 concentrations, generating negative feedbacks to climate change.

A highly efficient uranium grabber derived from acrylic fiber for extracting uranium from seawater.

Acrylic fiber can be chemically converted to an amidoxime and carboxylate containing chelating adsorbent by a two-step synthesis method for extraction of uranium from seawater. A portion of the nitrile groups in the fiber is first converted to amidoxime using hydroxylamine followed by conversion of another portion of the nitrile groups to carboxylate with NaOH. At an optimized ratio of amidoxime/carboxylate (about 1 : 1), the chelating fiber in real seawater shows a higher uranium adsorption capacity and shorter saturation time compared with similar high-surface-area chelating fibers developed recently using a radiation-induced grafting method. The saturation capacity of uranium is estimated to be 7.73 grams per kilogram of the adsorbent at 20 °C and the half-saturation time is about 15.7 days. The fiber shows a vanadium/uranium ratio of about 1 in real seawater tests. The low vanadium adsorption capacity of the fiber is attributed to the branched-chain amidoxime groups formed by the specified amidoximation process. This simple and low-cost synthesis method can be scaled up to mass produce the chelating fiber for recovering metals from various aquatic environments including production of uranium from seawater.

Polybrominated diphenyl ethers (PBDEs) in plasma from E-waste recyclers, outdoor and indoor workers in the Puget Sound, WA region.

Polybrominated diphenyl ethers (PBDEs) were widely used as flame retardants in consumer products including electronic devices. Important routes of human exposure are contaminated food and contact with dust. In this study, we measured twelve PBDEs in household/workplace dust and blood plasma samples provided by 113 volunteers living in the Puget Sound region, WA and working at electronic waste (E-waste) recycling sites (n = 29) or non-specific indoor (n = 57) or outdoor occupations (n = 27). The volunteers in the outdoor group were also selected because of a history of high seafood consumption habits. Results indicated the sum PBDE levels varied between <2.5 and up to 310 ng g-1 lipid. E-waste recyclers were predominantly men, generally consumed low amounts of seafood, and had PBDE blood levels (geometric mean, GM = 26.56 ng g-1 lipid) that were similar to indoor workers (GM = 27.17 ng g-1 lipid). The sum PBDE levels were highest in the outdoor group (GM = 50.63 ng g-1 lipid). Dust samples from E-waste sites were highly enriched with BDE-209 and BDE-153 relative to non-E-waste businesses and homes. The concentrations of these BDE congeners in dust at E-waste sites were ∼32-39 times higher than in dust from other sites. However, the detection rate of BDE-209 in plasma was low across all groups (13%) and no statistical comparisons were made. Our results suggest that E-waste recyclers in this study population did not have elevated PBDE levels in comparison to volunteers working in other types of occupations.

Polybrominated diphenyl ethers and their hydroxylated and methoxylated derivatives in seafood obtained from Puget Sound, WA.

Synthetic polybrominated diphenyl ethers (PBDEs) are ubiquitous environmental contaminants and known to occur in most food items. Consumer fish products have been identified as having some of the highest PBDE levels found in USA food sources. Natural formation of hydroxylated (OH-) and methoxylated (MeO-) PBDEs are also known to occur in simple marine organisms, which may be bioaccumulated by seafood. In this study, we report findings of an initial survey of PBDE, OH-PBDE and MeO-PBDE content in common seafood items available to residents living in the Puget Sound region of Washington State. Seafood samples were either purchased from local grocery stores or caught off the coast of SE Alaska and in Puget Sound. The edible portions of the seafood were analyzed, which for finfish was white muscle (skinless fillets) and for shellfish, either the entire soft tissue (bivalves) or processed meat (calamari, shrimp and scallops). Results indicated that finfish typically had higher levels of PBDEs compared to shellfish with BDE-47 and BDE-99 as the most common congeners detected. Among shellfish, bivalves (clams and mussels) were notable for having much higher levels of OH- and MeO-PBDEs compared to other types of seafood with 6'-OH-BDE-47 and 2'-MeO-BDE-68 being the more common OH- and MeO- congeners, respectively. Based on our results and recent updates to daily fish consumption rates, estimated intake rates for Washington State residents will be between 34 and 644ngPBDEs/day, depending on species consumed. For the OH- and MeO- forms, daily exposure is much more variable but typically would range between 15 and 90ng/day for most seafood types. If shellfish are primarily consumed, OH-PBDE intake could be as high as 350ng/day. These daily intake rates for PBDEs are higher than most dietary intake rates calculated for populations in other world regions.

Stage change in binding of pyrene to selected humic substances under different ionic strengths.

Influence of ionic strength (I) on the pyrene binding constant (Koc) in six different humic substance (HS) solutions, three soil humic acids, one freshwater humic acid, one freshwater fulvic acid, and one freshwater natural organic matter was investigated by fluorescence quenching technique. Specific two-stage changes of Koc with increasing ionic strength were only observed in three soil humic acid solutions with varying to different extents. Two primary governing factors for the differences of these HS systems are binding potential and the abundance of acidic functional groups (carboxyl and phenolic) of HS. Experimental results demonstrated that within the ionic strength range studied in the present research, ionic strength had a major effect on molecular size of HS and brought about change of Koc. The salting-out effect did not. The change in molecular size was caused by the associations between cations and acidic groups of HS. Using pyrene as a probe, we also investigated the interaction of cation and dissolved HS and found relatively slow kinetics (at least 5 d), leading us to believe that the special cation-HS kinetics may influence the results of geochemical studies involving the measurement of ionic strength effect.

Effects of ionic strength on the binding of phenanthrene and pyrene to humic substances: three-stage variation model.

This study compared the effects of ionic strength on the binding constants (K(doc)) of selected polycyclic aromatic hydrocarbons (PAHs) (phenanthrene and pyrene) and a terrestrial humic acid (Leonardite Humic Acid) in different electrolyte solutions (KCl, KBr, MgCl(2) and MgSO(4)). Distinct trends were found in K(doc) variation depending upon the range of ionic strength resulting from added electrolytes. These trends demonstrated similar shapes for all the systems studied, while degree of variation increased with hydrophobicity of the PAHs. Furthermore, different types of electrolytes had different effects on the interactions between humic acid (HA) and the PAHs. These differences were primarily caused by types of cation, not anion. To describe the complicated effects of ionic strength on K(doc), we developed a three-stage variation model that encompasses increasing and decreasing trends and plateaus in K(doc) associated with ionic strength, as well as the mechanisms behind these trends, including the variation of HA structure configuration, HA aggregation and the salting-out effect. This model illustrated the importance of sufficient experimental data when interpreting the influence of ionic strength on the trends in K(doc) variation.

A centennial record of anthropogenic impacts and extreme weather events in southwestern Taiwan: evidence from sedimentary molecular markers in coastal margin.

A 100-year history of human and natural disturbances in southwestern Taiwan was reconstructed using a suite of molecular markers in four dated sediment cores from the upper slope region off the Gaoping River mouth. Trends in polycyclic aromatic hydrocarbons (PAHs) tracked Taiwan's industrialization/urbanization starting in the 1970s, and the enactment of environmental regulatory policies thereafter. The predominant pyrogenic sources include vehicular, smelter, and coal combustion but spatial differences are observed among sub-regions of the shelf. Profiles of lignin oxidation products (LOPs) point to a significant increase in terrestrial organic matter inputs driven by land development after the 1970s. Low lignin diagenetic signature ratios [(Ad/Al)v] in all sediments suggest quick transport of fresh plant material from land to sea via mountainous rivers. Shifts in PAHs, LOPs, and radionuclides in recent sediments reveal the deposition of turbidites resulting from typhoon-induced floods. Multiproxy analysis illustrates the interplay between anthropogenic activities and natural processes.

Acetone-butanol fermentation of marine macroalgae.

The objective of this study was to subject mannitol, either as a sole carbon source or in combination with glucose, and aqueous extracts of the kelp Saccharina spp., containing mannitol and laminarin, to acetone-butanol fermentation by Clostridium acetobutylicum (ATCC 824). Both mannitol and glucose were readily fermented. Mixed substrate fermentations with glucose and mannitol resulted in diauxic growth of C. acetobutylicum with glucose depletion preceding mannitol utilization. Fermentation of kelp extract exhibited triauxic growth, with an order of utilization of free glucose, mannitol, and bound glucose, presumably laminarin. The lag in laminarin utilization reflected the need for enzymatic hydrolysis of this polysaccharide into fermentable sugars. The butanol and total solvent yields were 0.12 g/g and 0.16 g/g, respectively, indicating that significant improvements are still needed to make industrial-scale acetone-butanol fermentations of seaweed economically feasible.

Influence of combustion conditions on yields of solvent-extractable anhydrosugars and lignin phenols in chars: implications for characterizations of biomass combustion residues.

Anhydrosugars, such as levoglucosan and its isomers (mannosan, galactosan), as well as the solvent-extractable lignin phenols (methoxylated phenols) are thermal degradation products of cellulose/hemicellulose and lignin, respectively. These two groups of biomarkers are often used as unique tracers of combusted biomass inputs in diverse environmental media. However, detailed characterization of the relative proportion and signatures of these compounds in highly heterogeneous plant-derived chars are still scarce. Here we conducted a systematic study to investigate the yields of solvent-extractable anhydrosugars and lignin phenols in 25 lab-made chars produced from different plant materials under different combustion conditions. Solvent-extractable anhydrosugars and lignin phenols were only observed in chars formed below 350°C and yields were variable across different combustion temperatures. The yields of mannosan (M) and galactosan (G) decreased more rapidly than those of levoglucosan (L) under increasing combustion severity (temperature and duration), resulting in variable L/M and L/(M+G) ratios, two diagnostic ratios often used for identification of combustion sources (e.g. hardwoods vs. softwoods vs. grasses). Our observations thus may provide an explanation for the wide ranges of values reported in the literature for these two ratios. On the other hand, the results of this study suggest that the ratios of the major solvent-extractable lignin phenols (vanillyls (V), syringyls (S), cinnamyls (C)) provide additional source reconstruction potential despite observed variations with combustion temperature. We thus propose using a property-property plot (L/M vs. S/V) as an improved means for source characterization of biomass combustion residues. The L/M-S/V plot has shown to be effective in environmental samples (soil organic matter, atmospheric aerosols) receiving substantial inputs of biomass combustion residues.

Combustion-derived substances in deep basins of Puget Sound: historical inputs from fossil fuel and biomass combustion.

Reconstructions of 250 years historical inputs of two distinct types of black carbon (soot/graphitic black carbon (GBC) and char-BC) were conducted on sediment cores from two basins of the Puget Sound, WA. Signatures of polycyclic aromatic hydrocarbons (PAHs) were also used to support the historical reconstructions of BC to this system. Down-core maxima in GBC and combustion-derived PAHs occurred in the 1940s in the cores from the Puget Sound Main Basin, whereas in Hood Canal such peak was observed in the 1970s, showing basin-specific differences in inputs of combustion byproducts. This system showed relatively higher inputs from softwood combustion than the northeastern U.S. The historical variations in char-BC concentrations were consistent with shifts in climate indices, suggesting an influence of climate oscillations on wildfire events. Environmental loading of combustion byproducts thus appears as a complex function of urbanization, fuel usage, combustion technology, environmental policies, and climate conditions.