Zero-Carbon and Carbon-Neutral Fuels: A Review of Combustion Products and Cytotoxicity

Abstract

The use of zero-carbon and carbon-neutral fuels reduces emissions of conventional pollutants, but their emissions can be toxic and have various adverse effects on human health. This article reviews the possible combustion products of zero-carbon and carbon-neutral fuels, as well as their cytotoxic effects and potential health risks. At the same time, the review outlines biological models and toxicity detection methods commonly used in pollutant toxicity studies. Metals, nitrogen oxides (NO_X), and ammonia (NH₃) emitted from the combustion of metal fuels, hydrogen fuels, and ammonia fuels in zero-carbon fuels are harmful to human health. Exhaust emissions from carbon-neutral fuels, particularly biodiesel, and their blends with gasoline/diesel are cytotoxic, leading to severe cellular damage, such as oxidative damage, inflammatory responses, DNA damage, cell death, or apoptosis. Moreover, the normal function of the human body’s respiratory, cardiovascular, immune, digestive, urinary, and nervous systems may also be impacted by these fuel emissions according to cytotoxic research. Cytotoxicity of fuel combustion products is usually related to the fuel type, time, dose, and cell line used in the experiment. This review provides some ideas for the exhaust emission management of zero-carbon and carbon-neutral fuels and human health assessment. It also presents a theoretical and experimental basis for further research, including in vivo experiments.

1. Introduction

Since the beginning of the 21st century, energy demand and consumption around the world have increased with the rapid growth of the global population and the development of industry and transportation [1]. According to the Energy Institute (EI), fossil fuels (oil, coal, and gas) remained a major source of primary energy in 2022, accounting for about 82% of the world’s primary energy consumption [2]. Due to the non-renewable nature of fossil fuels and the growing depletion of fossil fuel reserves, there is a global energy crisis [3]. According to a 2021 report by the International Energy Agency (IEA), fossil fuel combustion produces CO, CO₂, NO_x, SO_x, HC, various trace metal elements, volatile organic compounds (VOCs), and particulate matter (PM) [4]. High greenhouse gas emissions have caused global warming, melting glaciers, and rising sea levels [5]. Additionally, these harmful pollutants also contribute to serious air pollution, which unavoidably puts human health at risk by increasing the incidence of diseases like asthma, pneumonia, lung cancer, stroke, and heart disease [6]. World Health Organization (WHO) data show that almost all of the global population (99%) breathe air that exceeds WHO guideline limits [7]. Air pollution has become a major problem that needs to be urgently addressed.

From the perspective of sustainable development and environmental protection, many countries have introduced strict policies and emission standards to reduce the consumption of fossil fuels and the emission of motor vehicle exhaust pollutants [8,9,10,11,12]. Researchers are actively looking for ways to reduce pollutant emissions from engines, including engine modifications, vehicle exhaust after-treatment, and improvements in combustion technologies [13]. In addition, countries are developing clean, renewable alternative energy sources, such as solar, wind, hydro, ocean, geothermal, and biomass, to reduce fossil fuel consumption [14]. However, they are confronted with challenges such as intermittent production or geographical dependence [15,16]. Therefore, they need to be processed and converted into secondary energy use, which means that their energy is stored in a secondary energy carrier.

Solar fuel, electric fuel, and biomass fuel are secondary energy carriers that have attracted much attention in recent years. Solar fuels are produced directly (photocatalytic) or indirectly (photoelectrochemical, solar thermochemical) by solar energy [17,18,19,20]. Electric fuels are gas/liquid fuels or synthetic fuels produced with renewable electricity as energy and carbon dioxide as the main carbon source [21,22]. Biomass fuel is a renewable fuel produced by photosynthetic organisms (microalgae, macroalgae, bacteria, plants, etc.) or fungi through biological processes [23,24,25,26]. Solar fuel uses renewable solar energy, and the electricity used for electric fuel production also comes from renewable energy [27]. Both, in turn, can re-reduce the CO₂ produced by combustion back into fuel [28]. In this sense, solar fuel and electric fuel belong to the category of zero-carbon fuel and carbon-neutral fuel. The CO₂ released into the atmosphere when biomass fuel is burned can be reabsorbed by photosynthetic organisms, which means that the biomass fuel consumes the CO₂ produced by combustion during its production [29]. In this way, biomass fuels do not have any net impact on the carbon concentration of the biosphere, achieving carbon neutrality in the whole life-cycle process. Therefore, biomass fuel can also be called a carbon-neutral fuel.

Recently, the goal of “carbon neutrality” has been proposed, which is the pursuit of “net-zero emissions” of carbon dioxide in energy and transportation systems. To achieve this, it is crucial to increase the use of zero-carbon fuels and carbon-neutral fuels [30]. Zero-carbon fuels are fuels that do not produce carbon emissions when burned. Carbon-neutral fuel refers to the fuel produced by using renewable energy to re-reduce the emitted CO₂ through electrocatalysis, photocatalysis, thermal catalysis, and other technologies [31]. Researchers are trying to explore and apply binary or multi-component blends of zero-carbon fuels/carbon-neutral fuels with gasoline/diesel [32,33,34,35]. Conventional pollutants emitted by engines are significantly reduced by using these alternative fuels [32,33,34,35].

When evaluating their toxicity to humans, zero-carbon fuels, carbon-neutral fuels, and their blends’ combustion chemistry need to be studied in depth. It is necessary to take into account the content of various pollutants created by combustion, new changes in the physical and chemical characteristics of pollutants, and the production of potentially harmful pollutants. Therefore, this paper first briefly reviews the categories of gas-phase and particulate-phase pollutants emitted by zero-carbon and carbon-neutral fuel combustion.

Currently, appropriate chemical analysis and bioassays have been performed to understand the toxicity of fuel exhaust [36,37,38]. However, in vivo toxicity studies in humans and animals with zero-carbon/carbon-neutral fuels are limited due to ethical and safety concerns. The exhaust toxicity of these new fuels is currently concentrated in in vitro studies, especially in vitro cytotoxicity studies. The damage and death caused by pollutants can eventually manifest as changes at the cellular level [39]. Pathological changes at the cellular level may alter or inhibit the functioning of various systems in the body, which may eventually lead to disease [40]. Therefore, this article focuses on the in vitro cytotoxic effects of carbon-neutral fuel exhaust pollutants and their effects on human health. These toxic effects involve cytotoxicity, oxidative stress or oxidative damage, inflammatory response, and genotoxicity (carcinogenicity and mutagenicity), ultimately affecting the normal functioning of the respiratory, circulatory, immune, and nervous systems. These cytotoxicity studies can predict acute and chronic toxicity in vivo, preliminarily assess the harm of fuel combustion products to human health, provide prerequisites for animal testing, and offer a theoretical basis for subsequent human-exposure experiments. Finally, toxicity detection methods and major biological exposure models are briefly introduced. In summary, understanding the strength of the toxic effects of zero-carbon and carbon-neutral fuels can help us better assess the impact of combustion products on human health, improve fuel composition, design effective engine and after-treatment systems, develop new emissions regulations, and implement effective prevention strategies to protect public health.

2. Zero-Carbon and Carbon-Neutral Fuel Combustion Products

2.1. Zero-Carbon Fuel Combustion Products

2.1.1. Metal Fuels

Metal fuels (e.g., aluminum, magnesium, iron, beryllium, and lithium) have been proposed as recyclable carriers for a large number of green renewable energy sources [27,41]. Ideally, the complete combustion products of metals are metal oxides without producing other waste products, including CO₂ [42]. As shown in Figure 1, a zero-carbon electrolysis process powered by primary renewable energy re-reduces metal oxides recovered or captured from combustion or reaction products to an unlimited amount of metal fuels [27]. In addition, metal combustion can lead to NOx emissions [43,44].

2.1.2. Hydrogen

Hydrogen (H₂) is an efficient, environmentally friendly, and sustainable energy source, often considered an ideal electric fuel or solar fuel [27]. Green hydrogen, produced using renewable energy, is expected to be at the heart of future zero-carbon energy carriers (Figure 1) [45,46,47,48]. Hydrogen does not contain carbon, and its products of complete combustion in the air are mainly water, producing no hydrocarbons and carbon oxides [49]. However, hydrogen combustion produces NO_x due to the high combustion temperature [50].

2.1.3. Ammonia

Ammonia (NH₃) is representative of zero-carbon fuels (Figure 1). Green ammonia is carbonless ammonia produced by electrolysis using renewable energy or biomass gasification [51,52,53]. NH₃ only produces N₂ and H₂O when completely burned in pure oxygen [54]. However, there are also significant NOx and unburned NH₃ emissions when NH₃ is burned in an engine [54,55,56]. Dual-fuel combustion of standard hydrocarbon fuels and NH₃ also generates carbon emissions and higher NH₃ and NOx emissions [57].

2.2. Carbon-Neutral Fuel Combustion Products

2.2.1. Alcohol Fuels

Alcohol fuels refer to liquid fuels containing alcohol, mainly methanol, ethanol, propanol, butanol, and so on. As shown in Figure 1, alcohol fuels have the potential to be used in a near-carbon-neutral manner when they are produced from captured carbon dioxide and renewable energy sources [58,59,60]. Not only can they be burned in engines as fuel separately, but they are also often used as additives to be blended with gasoline or diesel to make a blended fuel [61].

Table 1. Combustion products of alcohol fuels, ether fuels, and biodiesel.

Product Type	Alcohols	Ethers	Biodiesel
Conventional Products	CO, CO2, H2O, NOx, HC	CO, CO2, H2O, NOx, HC	CO, CO2, H2O, NOx, HC
Aldehydes	formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde, acrolein, etc.	formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde, acrolein, etc.	formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde, acrolein, etc.
Alcohols 1	methanol, ethanol, propanol, butanol, etc.	methanol, ethanol, propanol, butanol, etc.	methanol, ethanol, propanol, butanol, etc.
Acids	formic acid, acetic acid, etc.	formic acid, acetic acid, etc.	formic acid, acetic acid, etc.
Ketones	acetone	acetone	acetone
Olefins	ethylene, propylene, etc.	ethylene, propylene, etc.	ethylene, propylene, etc.
Ethers 2	alkyl ethers	methyl tert-butyl ether, dimethyl ether, etc.	alkyl ethers
Esters	methyl formate, ethyl formate, etc.	methyl formate, ethyl formate, etc.	methyl formate, ethyl formate, methyl 3-butenoate, methyl 4-pentenoate, methyl hexanoate, methyl enanthate, methyl heptanoate, methyl nonanoate, 2-methyl propyl 2-acrylate, butyl acetate, etc.
Peroxide	-	H2O2, HPMF, etc.	-
MAHs	-	-	benzene, toluene, xylene, methylbenzene, dimethylbenzene, mesitylene, ethylbenzene, styrene, propylbenzene, etc.
PAHs	-	-	naphthalene, acenaphthylene, acenaphthene, fluorene, phenanthrene, anthracene, fluoranthene, pyrene, benzo(a)anthracene chrysene, benzo(b)fluoranthene, benzo(a)pyrene, indeno(1,2,3-cd)pyrene, dibenzo(a, h)anthracene, benzo(g, hi)perylene, etc.
PM	SOF	SOF	soot, SOF, sulfate, metal particles, PAHs, etc.

¹ Alcohols in alcohol fuel combustion products refer to unburned alcohols. ² Ethers in ether fuel combustion products refer to unburned ethers.

lists the combustion products of alcohol fuels. The constituent elements of alcohol are C, H, and O, so the main combustion products in engines are CO, CO₂, and H₂O. Moreover, there are other conventional contaminants: NOx and HC, such as methane (CH₄), ethane (C₂H₄), and acetylene (C₂H₂) [62]. The unconventional pollutants produced by the combustion of alcohol fuels mainly include aldehydes, ketones, acids, unburned alcohols, enols, and olefins [63,64,65,66,67,68,69,70,71,72,73,74]. Alcohols do not contain unsaturated bonds such as C=C, so their combustion does not produce soot [75]. Furthermore, alcohols also do not contain sulfur and there are no sulfur oxides in the combustion products [75].

2.2.2. Ether Fuels

Ethers are expected to become renewable fuels and additives for future advanced internal combustion engines. Ethers can be produced from a variety of feedstocks, such as fossil fuels, residual oil, scrap, biomass, and renewable energy [76,77]. As shown in Figure 1, like alcohol fuels, ether fuels may also achieve the goal of carbon neutrality when they are produced from captured carbon dioxide and renewable energy sources. Methyl tert-butyl ether (MTBE) and dimethyl ether (DME) are currently the two most commonly used ether fuels [78]. Table 1 lists the combustion products of ethers. The main combustion products are CO₂ and H₂O. The incomplete combustion products also include CO, NOx, HC, PM, and incompletely burned ethers [79,80]. Ethers do not contain unsaturated bonds and sulfur, so they do not produce soot and sulfur oxides [79]. However, as an oxygenated fuel, ether also has some unconventional emissions: carbonyl products (aldehydes, ketones, acids, esters), peroxides such as hydrogen peroxide (H₂O₂) and hydroperoxymethyl formate (HPMF), olefins, alkynes, alcohols, enols, benzene, and other pollutants [81,82,83,84,85,86,87,88,89,90].

2.2.3. Biodiesel

Biodiesel is a typical carbon-neutral biomass fuel (Figure 1). Biodiesel is often referred to as a methyl or ethyl-ester-fuel-containing vegetable fat, animal fat, or other triacylglycerol-containing substances [91]. Biodiesel can be used directly or mixed with other fuels [92]. Biodiesel is environmentally friendly and produces fewer harmful pollutants when burned. Table 1 shows the types of pollutants produced by biodiesel combustion. Biodiesel is mainly composed of three elements, namely C, H, and O, so the main combustion products are CO₂ and H₂O. The composition of biodiesel is complicated due to the residue of alcohols, alkalis, acids, and other impurities in the preparation process of biodiesel. Therefore, biodiesel often produces CO, NO_x, HC (methane, ethane, ethylene, 1-butene, 1,3-butadiene, acetylene, propyne, etc.), PM, and other incomplete combustion products [93,94]. Biodiesel combustion also produces some unconventional pollutants: carbonyl substances (aldehydes, ketones, carboxylic acids, esters), monocyclic aromatic hydrocarbons (MAHs), polycyclic aromatic hydrocarbons (PAHs) [94,95,96,97,98,99]. Unconventional pollutants are more harmful to the human body although they account for a small proportion of all products. Biodiesel has low or no sulfur content and does not contain aromatic hydrocarbons, so its combustion produces low levels of sulfides and aromatic hydrocarbons [100].

2.2.4. Blended Fuels

Since pure fuel has its shortcomings in physical and chemical properties, the direct use of pure fuel may reduce the performance of the engine, and the effect often does not meet expectations. Therefore, depending on the advantages and disadvantages of each fuel characteristic, binary or multivariate blends are prepared to improve fuel characteristics and optimize engine performance at an acceptable cost. Currently, commonly mixed fuels are gasoline/diesel–biodiesel, gasoline/diesel–alcohol/ether, biodiesel–alcohol/ether, and gasoline/diesel–biodiesel–ester/alcohol.

Table 2. Trends in pollutant emissions from the combustion of common fuel blends.

Trends	Gasoline/Diesel– Alcohol/Ether		Gasoline/Diesel– Biodiesel		Biodiesel– Alcohol/Ether 1		Gasoline/Diesel –Biodiesel –Alcohol/Ether 2		Gasoline/Diesel –Biodiesel –Alcohol/Ether 3
CO	↓ ↑	[101,102,103,104,105,106,107,108] [109,110,111]	↓ ↑	[112,113,114,115,116,117,118,119,120] [121,122]	↓	[123,124]	↓	[125,126,127]	↓	[106,110,125,128,129,130]
CO2	↓ ↑	[101,102,110] [104,106,131,132]	↓ ↑	[133] [112,117]	-	-	↓ ↑	[125] [132]	↓	[110,128,130,134]
NOx	↓ ↑	[101,102,107,109,110,111,135,136,137,138,139] [106]	↓ ↑	[121] [112,117,118,119,120]	↓	[123,124,138,140]	↓	[127]	↓ ↑	[125,128,130,134,141] [121]
HC	↓ ↑	[101,102,104,106,108,109,110,131,132,136,137] [111]	↓ ↑	[112,113,114,115,117,118,119,120] [121,122]	↓	[123,124]	↓	[126,127,132]	↓	[106,110]
PM	↓	[107,108,110,111,142]	↓ ↑	[112,114,115,119,143,144,145] [121]	↓	[123,146,147]	-	-	↓	[110,128,129,134,144,145,148,149,150]

¹ Alcohol/ether is added to biodiesel. The arrow direction shows the emission trend in the blends relative to biodiesel. ² Biodiesel and alcohol/ether are added to gasoline/diesel. The arrow direction shows the emission trend in the blends relative to gasoline/diesel. ³ Alcohol/ether is added to the mixture of gasoline/diesel and biodiesel. The arrow direction shows the emission trend in the total blends relative to the mixture of gasoline/diesel–biodiesel.

shows the trends in regulated pollutant emissions after the use of some experimental fuel blends. The emission trends in CO, CO₂, NOx, HC, and PM measured by different experiments are inconsistent. This may be due to different types, volume ratios, and test conditions of alcohol/ether and biodiesel added to gasoline or diesel fuel.

3. Toxicity of Zero-Carbon Fuels

Currently, there are few in vitro cytotoxicity studies on exhaust from engines fueled with metal, hydrogen, and ammonia. The health risk potential of these fuels can be inferred from the toxicity of the single pollution produced by the fuels. The combustion of all three fuels produces NO_x. Common NO_x with greater health effects are nitric oxide (NO), nitrogen dioxide (NO₂), and nitrous oxide (N₂O) [151]. Exposure to NOx, even for short periods, can lead to a variety of respiratory diseases: inflammatory reactions in the lungs and bronchi, respiratory symptoms (cough, wheezing, dyspnea), asthma, and severe acute respiratory syndrome [152]. Furthermore, NO_x plays a role in the formation of ozone (O₃), which can cause severe respiratory diseases and reduce lung function in exposed populations [153]. Unburned NH₃ is emitted in large quantities during the use of ammonia fuel. Exposure to NH₃ for some time can have serious effects on human health, with signs of exposure manifesting as eye irritation, skin inflammation, severe cough, and burning of the nose, throat, and respiratory tract [154,155]. Severe exposure to high concentrations of NH₃ can lead to lung disease, permanent blindness, and even death [154,155].

4. Toxicity of Biodiesel and Biodiesel Blends

4.1. Types of Cytotoxic Effects

The toxic effects of emissions from biodiesel and its fuel blends involve cytotoxicity, oxidative stress, inflammatory response, and genotoxicity (carcinogenicity and mutagenicity) (Figure 2). In terms of cytotoxicity, cell morphological changes, the release of cytoplasmic components (e.g., LDH and AK), mitochondrial activity (e.g., MTT, MTS, XTT, and WST-1 assays), lysosomal integrity (neutral red uptake), adenosine triphosphate (ATP) levels, and the number of apoptosis and necrotic cells (trypan blue, propidium iodide and annexin staining) are often used as indicators of cytotoxicity or cell viability [156,157,158]. Biodiesel treatment increased the content of LDH and decreased cell viability [159]. A PM concentration-dependent increase in the number of necrotic cells was observed after exposure to PM of biodiesel blends [160,161].

Oxidative stress was assessed by measuring ROS production, depletion of free radical scavengers or antioxidant enzymes, and proteins that characterize cellular oxidative stress [162,163]. The inflammatory response was measured by measuring inflammatory markers, such as cyclooxygenase-2 (COX-2), interleukins (IL-1, IL-4, IL-6, IL-8, IL-10, IL-13), tumor necrosis factor (TNF-α, TNF-β), monocytes chemotactic protein-1 (MCP-1/CCL2), macrophage inflammatory protein-2 (MIP-2), interferon-γ (INF-γ), and exogenous metabolic enzyme (CYP1A1, CYP1B1) [163,164,165]. Cell culture studies have shown an increase in intracellular ROS production following exposure to PMs from the combustion of hydrogenated vegetable oil (HVO), rapeseed methyl ester (RME), fatty acid methyl ester (FAME), bean-based biodiesel, and other biodiesel fuels [161,163,166,167,168,169]. It has been suggested that engine type, rather than biodiesel type, is an important factor in ROS production [169]. Cells exposed to biodiesel increased the expression of SOD1 in the superoxide dismutase gene [170]. One study observed an immediate increase in transcripts coding for heme oxygenase-1 (HO-1/HMOX1) proteins associated with oxidative stress in cells after exposure to biodiesel [159]. Two other studies have also demonstrated that biodiesel fuel increased HMOX1 gene expression in cells [171,172]. The GSH/GSSG ratio fell after 48 h of cell culture in mediums containing biodiesel exhaust particles [173]. Exposure to different types of biodiesel exhaust induced the release of pro-inflammatory factors (e.g., IL-6, IL-8) and expression of the CYP1A1 gene [160,170,171,174].

In terms of genotoxicity, DNA damage is mainly detected by comet assay (CA)/single-cell gel electrophoresis (SCGE), micronucleus (MN) assay, agarose gel electrophoresis, cytoplasmic division blocking micronucleus (CBMN) assay, products formed after DNA damage (γ-H2AX, 8-OhdG), and Ames test [175]. After exposure to biodiesel, comet assays revealed increased levels of DNA strand break (SB) and formamidopyrimidine DNA glycosylase (FPG)-sensitive sites, causing cellular DNA damage [169]. Other findings support this conclusion [176,177].

4.2. Exposure of Specific Cell Lines in Different Biological Systems

4.2.1. Exposure of Respiratory System Cell Lines

The effects of biodiesel exhaust on the respiratory system are usually analyzed using various types of airway epithelial cells and alveolar cells [178]. The airway epithelium acts as the first line of defense against inhalation injury (viral, bacterial, air pollutants, and other environmental pollutants) [179]. After Mullins et al. exposed human epithelial cell line (NuLi-1 and 10KT) cultures to diluted exhaust from the combustion of 100% FAME and 20% FAME mixtures, significant apoptosis and loss of cell viability occurred, and IL-6 and IL-8 production increased [180]. Exposure to exhaust gases from 100% soy biodiesel or 20% soy biodiesel–diesel blends induced cell deaths and the release of immune mediators from human-respiratory-tract epithelial cells compared to air control and ultra-low-sulfur diesel (ULSD) engine exhaust [181]. Aqueous solutions of extractable organic matter (EOM) from PM of soy methyl ester (SME) or soy ethyl ester (SEE) can increase the release of pro-inflammatory cytokines IL-8 and IL-6 by respiratory epithelial cells [182]. Similarly, Traviss et al. found that B20 exhaust gases from waste yellow oils and used cooking oils increased IL-6 and IL-8 gene expression in human bronchial epithelial cells (BEAS-2B) [183]. Studies have indicated that after 6 h of exposure to soy biodiesel, BEAS-2B cells did not exhibit any change in the expression of cytokine genes (IL-6, IL-8, TNF, MCP-1, IL-1b) [181]. However, MCP-1 and IL-8 gene expression was upregulated in human alveolar basal epithelial cells (A549) [181].

Exposure to combustion particles from FAME biodiesel blends can cause DNA damage and chromosomal damage in A549 cells and BEAS-2B cells [177]. Cells exhibit increased levels of single-strand breaks (SSBs), increased MN frequency, or dysregulation of gene expression associated with DNA damage signaling pathways, but do not induce double-strand breaks and oxidative DNA damage (FPG) in cells [177]. This is supported by Cervena and Barraud, whose findings found that REM biodiesel exhaust induced DNA damage in BEAS-2B cells and A549 cells [184,185]. Furthermore, the DNA of epithelial cell lines (HepG2) was severely damaged by PM organics from diesel +10% biodiesel (D+BIO) and diesel +10% HVO (D+HVO), according to Valentina et al. [186]. BEAS-2B cells exhibit cytotoxicity and DTT activity (oxidative potential) and develop oxidative stress (HMOX-1 gene expression upregulation) after exposure to pure HVO and HVO–biodiesel blend PM emissions [187]. Another study also observed that BEAS-2B cells exhibited mild cytotoxicity associated with alterations in ATP/ADP ratios, increased expression of oxidative stress marker genes (NQO1 and HO-1), inflammatory marker genes (IL-8 and IL-6), and xenobiotic metabolic marker genes (CYP1A1 and CYP1B1) after 3 h of exposure to second-generation biofuel (B2G) extracted from lignocellulosic biomass [188]. Betha et al. also noted that biodiesel (B100) from waste cooking oil (WCO) caused higher cytotoxicity and oxidative stress in A549 cells than ULSD [173].

Steiner et al. performed experiments on a complex three-dimensional cell model of the human airway epithelium and reported severe oxidative stress and pro-inflammatory responses in cells after exposure to 100% RME engine exhaust [172]. Landwehr et al. exposed primary airway epithelial cells to diluted exhaust gases from engines fueled with biodiesel made from soybean oil, rapeseed oil, waste cooking oil, tallow oil, palm oil, and cottonseed oil. Shea butter biodiesel was the most toxic, with exposure leading to a significant decrease in cell viability and increased release of several immune mediators, including IL-6 and IL-8 [189]. In one study, PAHs in biodiesel exhaust were cytotoxic in human alveolar epithelial cells (HPAEpiC), manifested by increased oxidative stress (SOD, GSH, and ROS), decreased cell viability, increased extracellular LDH, and apoptosis promotion [190]. During the FTP-75 test cycle, RME’s PM extract was slightly more toxic to L929 cells (mouse lung fibroblasts) than biodiesel [191].

After exposing human bronchial epithelial cells cultured at the air–liquid interface (ALI) to the emissions of coconut oil and diesel fuel mixture (D90C10, D85C15, D80C20), compared with filtered air exposure, the metabolic viability of the cells was reduced, IL-8 and IL-6 secretion increased, and CYP1A1 gene expression significantly increased [170]. D80C20 decreased the percentage of viable cells, but there was no significant effect on the expression of the apoptotic and anti-apoptotic biomarkers genes (CASP3, BCL2) [170]. The 24 h exposure of primary human airway epithelial cells grown on the ALI to diluted exhaust gases from 100% canola biodiesel and 100% or 20% shea butter biodiesel blends increased protein release, indicating damage to epithelial cells [192]. Exposure to shea butter biodiesel exhaust induced increased permeability of ALI cultures [192].

This evidence suggests that biodiesel exhaust exposure leads to loss of airway epithelial cell viability, cell death, or apoptosis, ultimately affecting the normal functioning of the respiratory system. Biodiesel may increase the permeability of the epithelial barrier, leading to the loss of the defensive function of the epithelial barrier. The resulting damage can invade the underlying lung tissue, resulting in irreversible damage to the lung parenchymal tissue, such as a reduced gas-exchange area and impaired lung function, accelerating the development of respiratory diseases, such as asthma, pneumonia, emphysema, and chronic obstructive pulmonary disease [193]. In addition, biodiesel exhaust exposure also causes oxidative stress and inflammatory reactions in the respiratory tract and lung cells, which may cause bronchitis, pneumonia, and even systemic inflammation. Notably, biodiesel exhaust causes DNA damage and reduces the DNA repair capacity of respiratory cells, resulting in mutations. The mutations may lead to irregular cell proliferation and promote tumor development, increasing the risk of morbidity and death associated with lung cancer.

4.2.2. Exposure of Cardiovascular System Cell Lines

In the cardiovascular system, intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) are mechanistic intermediates of cancer and vascular disease. PM from the combustion of conventional diesel significantly raised the expression of ICAM-1 and VCAM-1 protein in human-umbilical-cord endothelial cells (HUVECs), but no such increase was seen in the PM from the combustion of 20% biodiesel [169]. Exposure to biodiesel exhaust PM triggers the loss of primary human-vascular-smooth-muscle cells (hVSMCs), which is strongly associated with the apoptosis of vascular wall cells [194]. It is speculated that biodiesel exhaust may cause damage to cardiovascular cells in the circulatory system, promote the occurrence of cardiovascular disease, and accelerate the development of atherosclerotic plaque. The underlying mechanism for this damage may be the ability of ultrafine particles emitted by biodiesel to bypass the respiratory barrier and the cerebral barrier and directly enter the bloodstream, directly causing inflammation of the brain and/or systemic inflammation of the respiratory and cardiovascular systems [195,196].

4.2.3. Exposure of Immune System Cell Lines

In terms of the immune system, an innate immune response barrier consists of neutrophils, monocytes/macrophages, and natural killer (NK) cells [197]. These cells are essential in maintaining immune homeostasis and tolerance [198]. They are the first line of defense against pathogens, contaminants, or inhaled particulate matter and are considered sentinel cells in immune response, responsible for initiating and resolving the immune response to the invading substance [199]. They mediate non-specific immune responses that are strong and quick enough to prevent infection without inducing damaging inflammation [200]. Therefore, due to the ubiquitous presence of macrophages throughout the human body, including the lungs, the toxic effects of fuel combustion emissions on macrophages or monocyte-derived macrophages (MDMs) are often considered.

Jalava et al. found that PM from RME and HVO caused dose-dependent fragmentation of chromosomal DNA strands in mouse macrophages (RAW264.7), with the highest concentrations (150 and 300 mg/mL) of PM causing cytotoxicity and apoptosis [161,166]. Another study also reported the toxicity of exposure to water-soluble components extracted from exhaust PMs of diesel and biodiesel blends (BD30) on RAW 264.7 cells [201]. ROS levels increased and cell viability was significantly reduced (15–70%) after exposure to 100 μL PM for 24 h compared to untreated cell controls [201]. Furthermore, exposure of rat alveolar macrophages (AMs) to soybean biodiesel blend (B20) exhaust particles induced a dose-dependent increase in inflammatory signaling [202]. B20 exposure resulted in elevated prostaglandin E₂ (PGE₂) release at lower particle concentrations compared to low-sulfur petroleum diesel (PDEP) [202]. Previous studies also reported a significant increase in TNF-α levels and dose-dependent increases in chemokine MIP-2 concentrations after exposure of the RAW264.7 cell line to RME and HVO biofuels compared with the control group [161]. ROS production increases when human monocytes (THP-1) are exposed to PM from 20% animal fat or RME blends, while CCL2 and IL-8 gene expressions remain unaffected [169]. Another study of THP-1 cells showed that exposure to PM from 20% waste oil biodiesel–80% petroleum diesel blends for 24 h can trigger an inflammatory response comparable to petroleum diesel particles [183]. Human U937-derived macrophages exposed to biodiesel emission samples produced with heavy-duty diesel vehicles elevated inflammatory markers (COX-2 and IL-8) and upregulated CYP1A1 gene expression [203]. Tooker et al. found that exposure to PAHs during the differentiation of monocytes (U937) into macrophages (MDMs) did not change the number of MDMs, but exposure to a unique mixture of PAHs resulted in altered immune markers [204]. The percentage of DNA migration and the degree of cell damage increased compared to controls after human lymphocytes were exposed to EOM from PM of biodiesel blends (72% soybean oil and 28% palm oil) [205]. Metals associated with the organic composition of biodiesel may play an important role in molecular mechanisms involved in cell proliferation and immune response [206].

It can be seen that biodiesel exposure can cause damage to immune cells, triggering oxidative stress, inflammatory responses, and DNA damage. These injuries lead to functional deficiencies, such as phagocytosis, which may provoke allergies, autoimmune diseases, and various bacterial infections.

4.2.4. Exposure of Skin and Other System Cell Lines

Biodiesel exhaust can enter the body through skin contact [207]. The biofuels produced by esterifying fried vegetable oils showed more cytotoxic activity against A431 skin cells when compared to those produced by esterifying waste animal fats [208]. Biodiesel may be associated with kidney and liver diseases. PM from 20% biodiesel–mineral diesel blends is mutagenic and cytotoxic to human embryonic kidney 293T cells (HEK 293T) [209]. Cavalcante et al. reported that the exposure of the ZFL zebrafish liver cell line to a soluble portion of biodiesel exhaust particles increased ROS production, glutathione S-transferase activity, and DNA damage, resulting in cytotoxic, biochemical, and genotoxic changes in ZFL cells [210].

4.3. Degree of Cytotoxic of Biodiesel

4.3.1. Lower Toxicity Than Diesel

Some evidence suggests that biodiesel engine exhaust has low or no cytotoxic effects. BEAS-2B cells did not show any toxic effects after exposure to extracts from biodiesel exhaust particles, and levels of IL-6 and IL-8 protein secretion did not change [206]. Similarly, HepG2 cell lines did not exhibit cytotoxic after exposure to soluble organics extracted from exhaust particles of three fuels (ULSD, ULSD+13% HVO, ULSD+20% HVO) [211]. Some studies have found that biodiesel is less toxic than diesel. Biodiesel engine exhaust PM is safer for human lung cells than its mineral diesel counterpart [172]. Under real conditions, cytotoxicity was 8.5 times higher after exposure of the BEAS-2B cell line to PM from petroleum diesel for 24 h than that of PM from a 20% waste oil biodiesel blend [212].

In terms of mutagenicity, exhaust from RME is less mutagenic than that from diesel [191]. This may be due to a lower concentration of highly polar mutagens (PAHs, aromatic amines, nitroaromatic hydrocarbons, and oxygenated PAHs) emitted by biodiesel–mineral diesel blends compared to the benchmark mineral diesel, resulting in a lower mutagenic potential for exhaust emissions [213]. In terms of oxidative stress and inflammatory response, PM from a 20% animal fat or RME blend was less or equal in cell ROS production, DNA damage, and CCL2 and IL-8 gene expression compared to PM from conventional diesel fuel (D100) combustion [169]. Novotna et al. observed high levels of DNA strand breaking and oxidative damage DNA after 4 h exposure of A549 cells to DCM organics extracted from petroleum diesel and pure biodiesel [214]. While these results indicate the potential health benefits of biodiesel, the toxic effects of each biodiesel exhaust gas may vary depending on the chemical composition and organ systems examined.

4.3.2. Higher Toxicity Than Diesel

A few studies have come to the opposite conclusion, finding that biodiesel is more toxic than diesel [215]. Miriam et al. reported that PM from B50 (50% RME+50% diesel) resulted in increased cytotoxicity and IL-6 release from BEAS-2B cells compared to PM from diesel [160]. PM organics from biodiesel blends (diesel+10% HVO) exhibit higher genotoxicity compared to conventional ULSD [186]. The particles emitted by biodiesel engines are more harmful than those emitted by diesel engines, possibly because they contain more intermediate combustion products, NPs, toxic trace metals, and harmful PAHs [209].

4.3.3. Causes of the Toxicity Contradiction

The reasons for the contradiction of biodiesel toxicity may be related to factors such as biodiesel feedstock type, biodiesel content, gas and particle-phase properties, exposure time, engine conditions, and other factors. Even though biodiesel is not a major component of fuel, the amount of biodiesel in the fuel blend can significantly affect the cytotoxicity of exhaust gases [192]. The particulate-phase extract of 10% palm oil biodiesel induced cell death at lower doses and was more cytotoxic than the particulate-phase extract of 20% palm oil biodiesel [216]. Vaughan et al. reported that a low proportion of coconut oil–diesel blends (10% and 15%) reduced inflammation and increased antioxidant expression compared with conventional diesel, while a high proportion of coconut oil blends (20%) reduced cell viability and increased inflammation [170]. There is a difference in the toxicity of biodiesel gas-phase emissions and particulate-phase emissions to cells. Palm oil biodiesel gas-phase extracts showed higher cytotoxicity and genotoxicity in A549 cells but lower in the particulate phase [216]. The toxicity of biodiesel semi-volatile particles is higher than that of solid particles [217].

The toxicity of biodiesel has a time effect, and different exposure times activate different regulatory pathways, resulting in different toxic effects [218]. Water-soluble fractions from biodiesel and its blends with diesel are cytotoxic to human cell lines. Leme et al. reported a dose-dependent response to toxic effects in human T-cell leukemia (Jurkat) and human liver cancer cells (HepG2) in water systems contaminated with pure biodiesel and its diesel blend (B5) [219]. The presence or absence of after-treatment systems (particulate traps and oxidation catalysts) is critical to the health effects of exhaust gas exposure. The results of cytotoxicity experiments found that the cytotoxicity, oxidative, and pro-inflammatory properties of biodiesel vehicle exhaust equipped with a diesel particulate filter (DPF) were reduced, which may be due to the reduction in PM mass and particle number due to the application of DPF [160]. Different driving conditions also have some influence on the toxicity of biodiesel. Particles collected under rural driving conditions had more cytotoxic and inflammatory responses but lower oxidative potential than those collected under urban driving conditions [160].

5. Toxicity of Alcohol and Alcohol Blends

The physicochemical properties and toxicity of PM from oxygenated fuel blend change because the thermochemistry properties of oxygenated fuels alter the combustion chemistry. The addition of oxygenated fuels (alcohols/ethers) to gasoline/diesel can reduce PM emissions [142]. Determining changes in PM properties caused by different types of oxygenated fuel mixtures can help assess PM toxicity.

5.1. Exposure of Respiratory System Cell Lines

Several experiments have investigated the toxic effects of gasoline–alcohol fuels on respiratory cell lines. One study used MN assay and CA of A549 cells to compare the genotoxicity of gasoline and methanol exhaust. The results showed that methanol vehicle exhaust did not cause DNA damage and chromosomal damage to A549 cells, while gasoline vehicle exhaust had strong cytotoxicity and genotoxicity [220]. Liang et al. also found that gasoline exhaust is more genotoxic than methanol exhaust, and methanol exhaust is more cytotoxic than gasoline exhaust within a certain dose range [221]. A 72% and 83% reduction in genotoxicity was seen from ethanol–gasoline blends (E10, E85) compared to gasoline [222]. Some dysregulated genes and processes were identified, such as oxidative stress, lipid and steroid metabolism, PPARα signaling, and immune response after 4 h exposure of BEAS-2B cells to subtoxic doses of PM extract from E15 (85% gasoline + 15% ethanol) [223]. On the other hand, after 24 h exposure, genes and pathways associated with the metabolism of PAHs and aromatic hydrocarbon receptors (AhR) were activated, and multiple genes and pathways associated with cancer promotion and progression were dysregulated [223].

Bisig et al. found that multicellular human lung models consisting of human bronchial cells (16HBE14o-), supplemented with human-monocyte-derived dendritic cells (MDDCs) and monocyte-derived macrophages (MDMs), did not cause observable cytotoxicity, morphological changes, oxidative stress, and DNA damage after 6 h exposure to aerosols excreted from ethanol–gasoline mixture (E10, E85) [224]. However, an increase in cell death, oxidative stress, and pro-inflammatory cytokines was observed in cells exposed to diesel exhaust [224]. Rossner et al. exposed the BEAS-2B cell line to complete emissions of PM produced by the gasoline–20% ethanol mixture (E20) and found that lipid oxidation was reduced, but concentrations of AA metabolites and chemokines with anti-inflammatory effects increased [225]. Sima et al. investigated the toxic effects of OME from PM produced by gasoline blended with different ethanol contents and found that 5-day exposure induced significant cytotoxicity in BEAS-2B cells and low cytotoxicity in 3D cell models of human airways (MucilAir™) [226]. Compared to the control group, ROS production decreased, CYP1A1 gene expression was upregulated, and histone phosphorylation was not significantly increased in two cell cultures, i.e., double-stranded DNA damage was not evident [226]. Arias et al. reported that soluble organic fractions (SOFs) extracted from PM produced by two fuels (ULSD, ULSD+13% butanol) had no cytotoxic activity against HepG2 cells and produced genotoxic reactions at the same level [211].

These studies have shown low levels of oxidative stress, cytotoxic effects, and genotoxic effects caused by alcohols and their fuel mixtures on respiratory system cell lines. However, these fuel emissions can cause an inflammatory response in cells. In summary, the addition of ethanol to gasoline has advantages in reducing the risk of respiratory diseases from engine exhaust.

5.2. Exposure of Immune System Cell Lines

One study reported that the exposure of RAW264.7 cells to PM from ethanol–gasoline blends (E10, E8) decreased cell viability and metabolic viability and raised the level of the inflammatory response (TNF-α and MIP-2) but did not increase oxidative stress levels compared to control cells [227]. Another study found that rat alveolar macrophages (NR8383) produced cytotoxic responses similar to environmental aerosols but secreted more TNF-α after being exposed to PM from three ethanol–gasoline blends (E10, E30, and E78) [228]. The E78 mixture showed minimal oxidation potential, while E30 showed the greatest oxidation potential [228]. Natural killer (NK) cells eliminate pathogen infection through secretory pathways and death-receptor-mediated pathways, as well as by secreting immunomodulatory cytokines, such as interferon (IFN)-γ and granzyme B [229]. In addition, NK cells, which can directly or indirectly attack and kill tumor cells, have been described as the main effector cells against cancer in innate immunity [230]. Co-culture models consisting of bronchial epithelial cells (ECs) and NK cells did not detect toxic effects after being exposed to an 85% ethanol–gasoline mixture (E85) compared to air controls for the short term [231]. This evidence suggests that alcohol fuel emissions have damaging effects on macrophages and may lead to systemic inflammation and immune system disease. However, alcohol fuel emissions are not significantly toxic to NK cells, indicating that this fuel may have an advantage in reducing cancer incidence.

5.3. Exposure of Other System Cell Lines

Agarwal et al. reported that the cytotoxicity, ROS production potential, and mutagenicity of PM from 10% ethanol–gasoline blends on human embryonic kidney (HEK) 293 cells were lower than those of gasoline [142]. This suggests that alcohol fuel may cause less damage to the kidney organs than gasoline.

6. Toxicity of Gasoline/Diesel—Biodiesel—Alcohol/Ether Blends

The characteristics of particulate matter can change when biodiesel and ethanol are added to diesel, and ethanol has a higher influence on these changes than biodiesel [232]. PM from DB10 (90% diesel + 10% biodiesel) vehicles reduced RAW 264.7 macrophage death (−60.8%) compared to PM from pure diesel (D100) vehicles, while PM from DE10 (90% diesel + 9% ethanol + 1% biodiesel) vehicles increased cell death (84.1%) [232]. This suggests that alcohol blends may be more immunotoxic than biodiesel blends. Ternary fuel blends can reduce the genotoxicity associated with PM. Yang et al. showed that PM10 produced by fuel blends composed of waste cooking oil/butanol/diesel (B10W20, B10W40) significantly reduced diesel-induced genotoxicity of A549 cells and diesel-induced mutagenicity of CHO-K1 cells [233]. Diluted engine exhaust from grapeseed, bran, or coconut biodiesel blended with 10% (v/v) diethylene glycol dimethyl ether (DGDME) resulted in reduced permeability, increased cell damage, and increased IL-6 and IL-8 release of primary human airway epithelial cells growing at the gas–liquid interface [234]. Collectively, these results suggest that these fuel blends are cytotoxic. Blends of biodiesel with oxygenated fuels may be a potential alternative fuel for diesel engines as they have less cytotoxic effects than diesel.

7. Biological Models and Methods for Toxicity Assessment

7.1. Biological Models

Air pollution caused by vehicle emissions has posed a huge threat to the ecological environment and biological health. Vehicle emissions affect a broad spectrum, from biogeochemical cycles to microbial communities, and from plant development to animal and human health. Under such circumstances, there is an increasing need to develop and validate microbial, plant, animal, and human-based toxicity testing models and methods. This multifaceted assessment can help better predict the actual impact of vehicle exhaust emissions on ecosystems and understand their effects from the cellular level to the organismal level [235].

As shown in Figure 3, bacteria, yeast, filamentous fungi, and protozoa are commonly used microbial models [236]. Model plants that have been used for air pollutant assessment include Arabidopsis, tobacco, wheat, rice, soybean, maize, and sorghum [237,238,239]. The plants evaluated for toxicity of aquatic organisms are mainly algae, such as chlorella and microalgae [240,241]. The selection of plant species is related to geographical factors, and researchers usually choose common plant species in the area to be studied. Model animals currently commonly used for toxicity studies are small rodents (e.g., mice and rats), Drosophila, and nematodes (e.g., C. elegans) [242,243,244,245]. The model animals commonly used in aquatic environment toxicity assessments mainly include zebrafish, Daphnia magna, and Artemia [246,247,248]. Animal model experiments have made many important contributions to our understanding of the relationship of entire organisms to pollutants, the human disease process caused by pollutants, and the prediction of the impact of pollutants on human health [249]. However, toxic effects in humans may differ significantly from those in animals due to distinct species characteristics [250]. Animal models ignore the detailed changes that occur at the level of individual cells.

In vitro, cell models have evolved from simple cell-free biochemical assays to monoculture systems under submerged conditions to the latest and advanced multicellular cultures grown at air–liquid interfaces. Monoculture cell lines commonly used in respiratory toxicity studies include human nasal epithelial cells (HNECs), human bronchial epithelial cell lines (HBECs) (e.g., BEAS-2B, HBEC3KT, 16-HBE, NL-20), human alveolar basal epithelial cells (e.g., A549, NCI-H441, HPAEpiC), human lung adenocarcinoma cell line (CALU-3), and mouse lung fibroblast cell line (L929) [191,251,252,253,254,255]. Cell models commonly used in other human toxicity studies are the human epidermal carcinoma cell line (A431), normal human keratinocytes (NHKs), the human hepatocellular carcinoma cell line (HepG2), mouse macrophages (RAW264.7), the human lymphoblastoid cell line (MCL-5), the human monocytic cell line (U937, THP-1) and monocyte-derived macrophages (MDM), vascular endothelial cells (VECs), human pulmonary artery endothelial cells (HPAECs), human embryonic kidney cells (HEK 293T), and the pheochromocytoma line (PC12) [208,211,254,256,257,258,259,260,261]. Although the monoculture system under immersion conditions is easy to operate and low in cost, it cannot reflect the actual in vivo cell–cell interaction, cannot truly simulate the exposure process, and is difficult to expose cells to gaseous or vaporized exhaust pollutants [262,263]. In recent years, the air–liquid interface (ALI) in vitro cell-exposure technology has been a way to expose cells to all or part of the components of engine exhaust particulate matter and gas-phase substances [264]. The system more realistically simulates in vivo inhalation exposure conditions [265]. Additionally, cell culture systems used to study air-pollution-mediated respiratory toxicity also include microfluidic organ chips, organoids, and human precision-cut tissue slices [266,267,268,269].

7.2. Toxicity Detection Methods

Toxicity testing on plants involves all aspects of plant growth, development, and reproduction, for example, plant growth indicators (seed germination rate, plant height, root length, plant phenotype, biomass, leaf quality), physiological and biochemical indicators (chlorophyll content, carotenoid content, proline content, sugar content, relative water content of leaves, photosynthesis parameters), oxidative stress indicators, and genotoxicity indicators (DNA damage) [238,270,271,272,273,274].

In vivo toxicity assessments in mammals and humans are often evaluated using animal experiments, epidemiological studies, and human clinical studies [275,276]. By exposing laboratory animal models to an artificially defined environment, the effects of pollutant exposure on animal behavior, the pathological changes in tissues and organs, the impact of pollutant exposure on the morbidity and mortality of certain human diseases, and the carcinogenicity of pollutants can be evaluated [277,278,279,280,281].

In vitro toxicity assessment in mammals and humans typically includes cytotoxicity, oxidative stress, inflammatory biomarkers, genotoxicity, neurotoxicity, and reproductive toxicity. Cytotoxicity can be mainly divided into the following aspects of detection [282,283,284,285,286,287,288,289]: (i) Cell morphological damage observations commonly use instruments, such as microscopes, including optical microscope (OM), electron microscope (EM), scanning tunneling microscope (STM), and atomic force microscope (AFM); (ii) Cell death identification commonly uses trypan blue staining; (iii) Apoptosis and cell cycle are often measured using fluorescent dye probes and flow cytometry, and the expression levels of proteins closely associated with apoptosis are detected using Western blot and RT-PCR; (iv) Detection of metabolic or proliferative activity (WST-1 assay, XTT assay, MTT assay, CCK-8 assay, Alamar Blue assay, AB detection) and detection of DNA synthesis ([³H]-TdR method, BrdU immunoassay, EdU labeling method) usually use cell tracing, which can be performed by various techniques such as in vivo imaging, microplate or flow cytometry; (v) The detection methods of cell membrane damage usually include the following: changes in cell membrane permeability based on lactate dehydrogenase (LDH) detection, comparison of membrane surface structure changes before and after contamination exposure by transmission electron microscope (TEM) and atomic force microscope, evaluation of cell membrane fluidity based on lipid diffusion coefficient and fluorescence recovery rate, and rapid detection of cell membrane potential changes based on fluorescent dyes and fluorescence microscopy; (vi) The effects of contaminants on cell morphology and cell and organelle movement were studied by (a) Coomassie blue staining, (b) immunofluorescence staining, and (c) RT-PCR and Western blot with microscopy and flow cytometry; (vii) Organelle damage mainly focuses on mitochondrial damage of mitochondrial morphology and mitochondrial membrane potential; (viii) Real-time cell analyzer (RTCA) is a new technology for detecting cytotoxicity. It is a transient cell-inductance continuous recording system based on impedance. The resistance is closely related to the cells’ adhesion, quantity, volume size, and morphological changes. The cytotoxic response is dynamically monitored in real time by detecting the resistance value.

Detection of oxidative damage typically includes several aspects [287,290,291,292,293,294,295,296]: (i) Intracellular radicals can be determined directly by electron spin resonance (ESR) and chemiluminescence; (ii) Lipid peroxidative damage is usually reflected by the content of malondialdehyde (MDA), a by-product determined by thiobarbituric acid reactive substances (TBARS); (iii) Protein oxidative damage is often determined by traditional detection techniques, such as colorimetry, spectrophotometry, or enzyme-linked immunosorbent assay (ELISA), to determine hydrazone generated by the reaction of 2,4-dinitrophenylhydrazine with protein carbonyls; (iv) DNA oxidative damage is most commonly detected by the biomarker 8-hydroxydeoxyguanosine (8-OHdG); (v) Changes in key protein expression in multiple signaling pathways involved in oxidative damage are detected by Western blotting. The method for measuring the inflammatory response is described in Section 4.1. At present, there are some mature and systematic experimental methods for in vitro genotoxicity studies [297,298,299,300]: the Ames test for bacterial mutagenicity, the comet assay/single-cell gel electrophoresis, DNA double-strand break analysis, micronucleus test, chromosomal aberration test, gene mutation test, giant DNA adduct formation or oxidative DNA damage detection, genome-wide gene expression analysis, the occurrence of apoptosis and subdiploid DNA content analyzed by annexin V/PI staining, and the detection of relevant gene expression changes by RT-PCR.

8. Conclusions and Prospect

The metal particles produced by the combustion of metal fuels and the NH₃ produced by the combustion of ammonia fuels are toxic and can negatively affect human health. Hydrogen fuel generally does not adversely affect the environment and human health. It is worth noting that the combustion of these three types of zero-carbon fuels leads to the production of NOx, which can cause respiratory diseases in humans. Future research on metal fuel will require a detailed assessment of toxic metal and NO_x emission levels, as well as consideration of advanced combustion strategies or after-treatment technologies to mitigate emissions. Further use of ammonia fuel also requires controlling NH₃ and NO_x emissions, developing new NH₃ combustion systems, and establishing more complete after-treatment systems.

Biodiesel has negative effects on various cell lines, including those in the immunological, circulatory, and respiratory systems, according to in vitro cytotoxicity studies. However, there are conflicting results in the literature regarding biodiesel-induced cytotoxicity, which may be related to differences in fuel type, fuel content, exposure time, engine configuration, and PM emissions sampling. These results cannot be directly compared due to different experimental conditions. It is necessary to establish more effective assessment methods for pollutant emissions assessment methods to compare the toxicity of different types of biodiesels. Most studies have shown that biodiesel and its fuel blends cause lower cytotoxicity than diesel, suggesting potential health benefits for biodiesel when used as an alternative to diesel. The use of alcohols and their fuel mixtures can reduce the cytotoxicity of diesel or gasoline engine exhaust gases and does not even cause adverse cellular responses. From a human health point of view, alcohol fuels can be considered carbon-neutral alternatives. When biodiesel and alcohol are added to gasoline or diesel as additives, the toxic effects vary due to the changing proportions. However, in comparison, the fuel blends with these additives are less toxic than pure gasoline or pure diesel. Unfortunately, the cytotoxicity of ethers and their fuel blends has been less studied. Further research is needed to determine whether their toxicity has the same effect as alcohol fuels.

It is recommended that more detailed studies be conducted in the future on toxic components in carbon-neutral fuel emissions to reduce toxic component emissions. In addition, the toxicity of respiratory system cell lines and immune system cell lines have been well studied. Toxicity studies of various cell lines in other systems should be given greater focus to better understand the diseases caused in each system by biodiesel. After sufficient in vitro toxicity assessment data are available, in vivo toxicity assessments should be considered in the future to more accurately assess human health risks. It is worth mentioning that the current toxicity studies of alternative fuels focus on the toxicity of PM or PM extracts. The overall toxicity of all biodiesel emissions (gas phase and particle phase) need further research. In addition, more effective and realistic biological models need to be developed and used to better simulate the adverse effects of alternative fuels on the environment and human health. Simple, rapid, and cost-effective cytotoxicity assay systems should also be further developed to assess cytotoxicity in multiple ways.