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Review

Phytotoxicity Testing of Atmospheric Polycyclic Aromatic Hydrocarbons

by
Selenge Tumurbaatar
,
Nora Kováts
* and
Katalin Hubai
Centre for Natural Sciences, Affiliation University of Pannonia, P.O. Box 158, 8200 Veszprém, Hungary
*
Author to whom correspondence should be addressed.
Atmosphere 2024, 15(9), 1143; https://doi.org/10.3390/atmos15091143
Submission received: 12 September 2024 / Accepted: 15 September 2024 / Published: 23 September 2024
(This article belongs to the Special Issue Toxicity of Persistent Organic Pollutants and Microplastics in Air)
Browse Figure
Versions Notes

Abstract

:
Atmospheric polycyclic aromatic hydrocarbons (PAHs) have well-known phytotoxicity on higher plants. However, while numerous bioindication studies have been targeted on how different symptoms indicate the deleterious effects of PAHs in the field, laboratory-scale phytotoxicity tests are much rarer. While ecotoxicity tests might rely on the very same end-points as bioindication studies, they have to comply with quality assurance criteria, repeatability being the most important. As such, proper reporting involves the description of the test compound, experimental design and conditions, test organism used, and end-points measured. The recent review intends to give an overview of studies available in the literature complying with these requirements. PAHs occur in the atmosphere both in gaseous form and bound to particles. As plants are exposed to both phases, test protocols available represent different exposure pathways, fumigation chambers vs. direct foliar treatment. Reported studies, therefore, are grouped based on the exposure route they intend to simulate.

1. Introduction

1.1. PAHs in the Atmosphere

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous organic pollutants with two or more fused benzene rings. They can be released via natural processes such as volcanic eruptions; however, anthropogenic activities such as biomass burning and coal and petroleum combustion provide the main sources [1].
In the atmosphere, they can occur in gaseous form (typically low molecular weight PAHs consisting of two-four aromatic fused rings) or bound to particles (higher molecular weight PAHs with more than four rings) [2].
Atmospheric particulate matter (PM) is grouped as coarse, fine, and ultrafine particles (UFPs) with aerodynamic diameters of 2.5 to 10 μm (PM10), <2.5 μm (PM2.5), and <0.1 μm (PM0.1), respectively. The smaller, the worse: particles with decreasing diameter have increased relative surface area, and in general, increased surface area results in a higher potential to bind potentially toxic chemicals [3].
It was experimentally shown that the PAH content of fine particulate matter is significantly higher than that of coarse PM [4]. In addition, the potential hazard of fine particles is increased by their availability. While the lifetime of larger particles (PM10) ranges from hours to days, that of the smaller size particles (PM2.5 and below) can range from days to weeks [5].

1.2. Exposure Pathways and Uptake Mechanisms

Foliar uptake is considered the major PAH exposure pathway for higher plants [6] (higher plants include the following taxa: Pteridophytes, Gymnosperms, and Angiosperms).
The uptake of gaseous PAHs can occur directly through the stomata [7] or via diffusion through the wax and cuticular lamellae into the interior parts of the leaves [8]. During dry deposition, particles settle on plant surfaces. Particle phase uptake can also occur directly via stomata [9] as atmospheric particulate matter was reported to pass through these openings, carrying potentially toxic compounds [10]. On the surface of the leaves, PAHs can also desorb from deposited particles and are collected in the cuticular wax [11].
In addition to the direct deposition of particles on soil or plant surfaces, both gas and particle phase PAHs can be washed out by rain or snow defined as wet deposition [12]. Wet deposition provides an important pathway for PAH removal from the atmosphere; however, it depends on the precipitation amount, showing seasonal patterns [13]. The process has been proven efficient for all particle-associated PAHs [14].
Comparing the uptake of gaseous vs. particle-bound PAHs, accumulation occurs at a higher rate for gas-phase PAHs [15]. Wang et al. experimentally showed that foliar uptake of LMW PAHs occurred via gaseous absorption [16].
Atmospheric PAHs can also be transported to the soil, providing an additional air/soil/root exposure route for plants [17]. Transport can also occur after dry or wet depositions. Uptake from the soil, however, mainly occurs for LMW PAHs, the soil/root/aerial part transfer being significantly lower for HMW PAHs [18]. According to Jia et al., gas-phase absorption contributed to 90.6% while uptake from soil to 9.4% of PAH accumulation in different leafy vegetables [19].
Possible exposure routes for both LMW and HMW PAHs are illustrated in Figure 1.

2. Toxic Effects

PAHs have been reported to have phytotoxic effects on higher plants [20,21]. Exposed plants have been found to exhibit a wide variety of responses, varying from biochemical markers to community-level symptoms. In general, growth impairment can be considered as an ultimate symptom [22] most often measured as biomass reduction in comparison to the control. Protocols measure either shoot dry weight or fresh weight [23], cutting plants at soil level [24].
Species-specific biomass reduction was reported by Storch-Böhm et al. when the sensitivity of higher plants was assessed following exposure to diesel engine exhaust [25]. A decrease in biomass can be explained by the impairment of photosynthetic processes [6,26]. PAHs of different molecular weights were found to inhibit PS II photochemistry in the study by Jajoo et al. [27].
Physiological and morphological traits involve relatively easy-to-measure symptoms that are considered reliable markers of environmental stress [28]. Chlorosis and necrosis reflecting direct damage were associated with air pollution in the 1940s–1950s [29,30].
Photosynthetic activity has also been a widely applied indicator of environmental stress, including air pollution. PAHs were found to affect both light and dark reactions by causing, among others, loss of photosynthetic pigments, reduction in PhotosystemI (PSI) and PhotosystemII (PSII) activity, a decline in stomatal conductance or, ultimately, reducing the net photosynthetic rate [31]. A decrease in the concentrations of photosynthetic pigments chlorophyll a and b was reported by Rabe and Kreeb [32], and most studies continue to use these easy-to-measure indicators. Chlorophylls and carotenoids are extracted by applying polar solvents such as acetone, methanol, or ethanol. Extracts are clarified, and the concentration of photosynthetic pigments is measured by UV-VIS spectrophotometry [33]. Chlorophyll a fluorescence measurement is a generally applied tool to evaluate the effects of PAHs on PSII activity [34]. Arikan et al. [35] detected the suppressions of stomatal conductance, carbon assimilation, and PSII photochemistry in lettuce (Lactuca sativa) exposed to PAH treatments. Additional biochemical markers include the measurement of foliar protein concentrations [36]. Reduced levels will reflect the inhibitory effect of air pollutants on protein synthesis [37].
One of the main mechanisms responsible for the phytotoxic effects is the production of reactive oxygen species (ROS), such as hydrogen peroxide (H2O2) and superoxide anion (O•2−). Wu et al. investigated the chemical composition and toxicity of fine particulate matter emitted from the combustion of petrol and diesel fuels [38]. A significant correlation was established between intracellular ROS generation and PAHs.
Oxidative stress influences different metabolic pathways and, ultimately, growth processes [39]. Cellular H2O2 was reported to have direct damage on cellular lipids and proteins [40], as well as on thylakoids, resulting in impaired photochemistry [41]. Plants’ defense mechanisms involve the activation of antioxidant enzymes such as ascorbate peroxidase (APX), catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD) [42]. These enzymes play a key role in dismutation or ROS detoxification [43].
As the first step, SOD converts O•2− into less toxic H2O2 and molecular oxygen (O2) [44]. Peroxidases (APX, POD) convert H2O2 into H2O and O2, detoxifying this compound (peroxidases have been given their name as they usually decompose peroxides) [45].
Liu et al. experimentally showed that treatment with phenanthrene, a three-ringed PAH, resulted in increased levels of H2O2 in Arabidopsis thaliana seedlings [46]. Simultaneously, increased POD activity was observed as H2O2 was reduced to H2O by peroxidase (POD).

3. Bioindication vs. Ecotoxicology

Traditionally, ecotoxicological tests are classified as one group of bioindication studies, provided they ‘quantitatively determine ecological effects’ [47]. It should be noted that in most field bioindication studies, neither the exact exposure nor the concentration of the contaminant is known. Moreover, symptoms might develop due to synergistic effects [48]. While these symptoms are recorded, no clear dose–effect or, more precisely, concentration–effect relationships can be established as environmental concentrations of the pollutant(s) are not known. Doucette et al. [49] suggest that the diversity of experimental approaches and lack of basic data, such as exposure or plant growth conditions, make inter-study comparisons inappropriate.
Ecotoxicological tests (or bioassays), on the contrary, rely on the principle of causality; that is, a clear causal relationship needs to be determined between the concentration(s) applied and the ecological effects assessed and quantified. These tests might use the very same end-points as field bioindication studies but in a controlled environment and in a reproducible way. End-point refers to measured responses given to the treatment during the test [50]. In order to ensure reproducibility, the protocol used throughout the test clearly describes all necessary steps, including sample preparation, concentrations applied, source and treatment of test organisms, and environmental conditions necessary for performing the tests; also, the duration of exposure is clearly determined. Experimental design should be clearly described or, even better, be illustrated by a diagram [51]. Assuming this information is provided, tests can be repeated if clarifications are needed. Following the same protocol, different pollutants can be tested on the same test organism, or the effects of the same contaminant can be assessed on different test organisms. Repeatability is one of the most important quality assurance criteria in ecotoxicity testing for rendering a test acceptable [52].
As such, the aim of this study is to review existing applications where higher plants were used in controlled ecotoxicological tests for assessing the impact of atmospheric PAHs following different exposure pathways. Specific criteria for inclusion were that studies comply with the requirements of reporting phytotoxicity test results. The checklist suggested by Hanson et al. [53] was used as a reference (Table 1).
Based on the checklist presented above, available phytotoxicity studies were carefully evaluated. Studies dealing with soil contamination were directly excluded. Literature reporting reproducible experiments was selected and processed further. Reported tests fulfilled the following criteria: (1). Test compound (individual PAHs or complex sample) is specified. Sample preparation is described, concentration series applied is given. (2). Test organism explicitly defined. Taxonomic description, source, plant compartment used (e.g., whole plant, roots/shoots, cell culture, etc.). The age of the test organisms used is given. Culturing is reported. (3). Mode of treatment is described. (4). Duration(s) of exposure are defined. (5). Exact description of end-points applied. Measuring techniques are (a) explicitly described or (b) properly referenced. (6). Results are quantified and provided.
It needs to be emphasized why reproducibility is a matter of key importance. Of several hundreds of PAHs, 16 compounds are defined as having the highest environmental relevance by the Environmental Protection Agency of the United States [54]. As such, reported test protocols should be further used to assess the potential toxicity of additional PAHs not included in the original study. In addition, higher plants reportedly have different sensitivity to organic pollutants, including PAHs, so their tolerance to air pollution might vary. Not only does species-specific sensitivity matter [55], tolerance might depend on several factors, the age of the organism being one of the most important ones [56]. As such, experiments conducted on a single PAH using a limited number of test species should be comparable in order to gain broad information on PAHs’ phytotoxicity on higher plants.

4. Reported Tests

As mentioned previously, PAHs can occur in the air in the gaseous phase and bound to particles, separating not only different PAH species but uptake mechanisms as well. Test systems available simulate the uptake of PAHs via different pathways. They are discussed separately.

4.1. Fumigation Chambers

As early as 1952, Haagen-Smit et al. used a fumigation chamber to study the effect of the ozonation products of hydrocarbons on selected plants such as spinach, sugar beets, endive, oats, and alfalfa [57]. Since then, experimental chambers have long been in use [58,59]. Shann and Adriano [60] designed a simple chamber to assess the effect of simulated aerosols.
Simulated diesel exhaust emissions were tested in a solardome fumigation facility [55]. In this study, 12 native European herbaceous species such as great plantain (Plantago major), black knapweed (Centaurea nigra), sorrel (Rumex acetosa), autumn hawkbit (Leontodon autumnalis), common bird’s-foot trefoil (Lotus corniculatus), white clover (Trifolium repens), perennial ryegrass (Lolium perenne), common sowthistle (Sonchus oleraceus), white goosefoot (Chenopodium album), Oxford ragwort (Senecio squalidus), annual meadow grass (Poa annua), and Timothy grass (Phleum pratense) were exposed for a prolonged exposure, varying between 12 and 45 weeks. Phytotoxicity was assessed based on numerous end-points, such as growth impairment, flower development, leaf senescence, and leaf surface wax characteristics. Some end-points, e.g., growth impairment, showed strong species-specific sensitivity.
The same solardome fumigation facility was used in the following study [56], involving the same batch of species. Symptoms were, in general, strongly species-specific; some species, e.g., suffered from growth impairment resulting in biomass reduction, while others showed an increase in the above-ground biomass. The photosynthetic rate of four species was assessed, including S. oleraceus, which showed an increased rate. As the study used prolonged exposure, it was possible to compare the sensitivity of test plants in different age groups. Older plants proved less sensitive than younger plants in most cases.
Viskari et al. [61] used a fumigation chamber to study the effects of motor vehicle exhaust gas on Norway spruce seedlings (Picea abies (L.) Karst) and plant–insect interactions of spruce shoot aphid (Cinara pilicornis Hartig). While one of the main objectives of the study was to assess how this interaction could be affected, no change in aphid performance was detected. On the other hand, biochemical end-points were sensitive indicators of the stress, resulting in increased concentrations of proline, glutamine, threonine, aspartic acid, glycine, and phenylalanine and decreased concentrations of arginine, serine, alanine, and glycine in young needles.
Paull et al. [62] used hybrid diesel fuel candles, and the phytotoxic effects of the generated PM on eight commonly cultivated green wall plants were studied under glasshouse conditions. The battery of test plants involved spider plant (Chlorophytum comosum variegatum), fire flash (C. orchidastrum), fiddleleaf fig (Ficus lyrata), goldfish plant (Nematanthus glabra), lemon button fern (Nephrolepis cordifolia duffii), Boston fern (N. exaltata bostoniensis), Australian ivy palm (Schefflera amate), and dwarf umbrella tree (S. arboricola). Plant sensitivity to different end-points (such as leaf area, leaf pH, relative water content, chlorophyll content, stomatal conductance, and Maximum Quantum Efficiency of Photosystem II) varied species by species; however, stomatal conductance proved an ultimate response as it significantly decreased in all test plant species.
Desalme et al. constructed an experimental chamber where air was enriched with phenanthrene (PHE) by passing through an evaporator filled with PHE pills, applying a concentration of 150 µg/m3 with an exposure of one month [63]. In this way, PHE was transferred from the air both to the leaves and to the soil surface. While accumulation of PHE was measured in both plant species tested (red clover, Trifolium pratense L. and ryegrass, Lolium perenne L.), the two species showed different sensitivity. Ryegrass was much less sensitive, and no biomass reduction was experienced, but PHE exposure elucidated a biomass reduction of around 15% in clover. In another study by Desalme et al. [6], PHE was applied in a concentration of160 µg/m3 for one month; the test species was clover in this case. PHE was found to induce a reduction in plant growth parameters such as growth rate and net assimilation rate. In addition, a lower carbon assimilation rate and chlorophyll content were experienced, which might have explained the overall reduction in biomass in exposed plants.
A gas exposure system was used in the work of Slaski et al. [64] to study the effects of naphthalene and creosote (a substance containing 90% PAHs) on five crop species: canola, barley, field pea, alfalfa, and lettuce. As the measure of toxicity, chlorophyll fluorescence was measured daily using a portable screening chlorophyll fluorometer, while chlorophyll content in leaves was measured at the end of the experiment.

4.2. Direct Foliar Treatment

In order to assess wet or dry depositions, different treatment methods have been used. As wet deposition implies that either gaseous or particle-bound PAHs are washed out by precipitation, treatment methods need to ensure foliar contact with aqueous media. To simulate foliar uptake, Kummerová and Vánová [65] put freshly cut leaves of pea plants (Pisum sativum L.) in increasing concentrations of fluoranthene (FLT) (0.1, 1, and 10 mg/L). Acute exposure was simulated using 12-, 24-, and 48-h exposure. Chlorophyll fluorescence parameters (F0, FV/FM, and ΦII) were followed. Results suggested an overall toxic effect of FLT, as chlorophyll fluorescence showed a clear concentration–effect pattern, increasing with increasing concentration of the sample (0.1, 1, and 10 mg/L). One possible explanation for the increase in chlorophyll fluorescence was the destruction of the photosynthetic pigment molecules.
In an in vitro study, apical segments of pea plants (Pisum sativum L., cv. Garde) were exposed to FLT by immersing test plants in different FLT concentrations [66]. In addition to growth parameters, levels of phytohormones such as abscisic acid (ABA) and cytokinins were assessed and proved to be sensitive indicators of plant stress. ABA showed a concentration–effect response to FLT, demonstrating its good indicator potential to assess PAH toxicity [67]. FLT was found to have a negative effect on growth, Chl a, Chl b, and carotenoids at higher concentrations (1.0 and 5.0 mg/L).
In a similar study, pea leaves were immersed in different concentrations of naphthalene (NAP), phenanthrene (PHE), and fluoranthene (FLT) to assess inhibitory activity on the photochemical activity of photosystem II (PSII) [68], using exposure times of 0.5–72 h. Inhibition appeared in a concentration-dependent manner; however, exposure also influenced the magnitude of the effect. During short-time exposure, NAP triggered the strongest response as this compound has the highest water solubility. On the other hand, during the long run, PHE and FLT showed higher toxicity. They have lower water solubility and can accumulate in the thylakoids. Based on electron microscopy images, the study revealed that accumulation of PAHs led to distortion of thylakoid membranes, which in turn influenced the photochemical activity of PS II.
The first fully expanded true leaves of cucumber test plants were immersed for 4 h in a solution of 0.2, 1, 5, and 10 mg/L PHE in the study by Jin et al. [69]. Results demonstrated the direct inhibition of the photosynthetic electron transport and Rubisco carboxylation activity. Also, accumulation of ROS was shown, resulting in net increases in D1 protein degradation, which in turn caused PSII photoinhibition. Negative effects occurred at 1 mg/L PHE concentration and above. The study highlighted the mechanisms via which PHE interferes with photosynthetic processes.
Babula et al. [70] used an experimental plant cell model, tobacco BY-2 cell suspension culture, to study the cytotoxic effect of fluoranthene. The study revealed FLT accumulation in lipophilic cell compartments, especially in biomembranes. Production of ROS was also found in these compartments, leading to the damage of biomembranes partially by disrupting their semipermeability.
Oguntimehin et al. [71] applied a growth chamber to study the effect of two PAHs, fluoranthene (FLU) and phenanthrene (PHE), on the evergreen conifer Japanese red pine (Pinus densiflora Sieb. et Zucc.), with an exposure of 3 months. The test chambers were covered with transparent ethylene–tetrafluoroethylene copolymer film to ensure over 95% light transmission. Treatment was conducted by spraying the foliage with solutions containing these PAHs (10 mM each). The PAHs were found to have negative effects on net photosynthesis, stomatal conductance, initial chlorophyll fluorescence, the contents of total chlorophyll, and ribulose 1,5-bisphosphate carboxylase. The effects of FLU proved to be more significant. In another study, the negative effects of FLU were assessed on Japanese red pine [72]. Two concentrations were applied, 5 and 10 mM FLU, using 5-, 10-, and 14-day exposure. Symptoms were dependent on the dose of FLU application as well as on the length of exposure. While no negative effect was detected after 5-day exposure, treatment with 10 μM FLU triggered negative changes in gas exchange during a 10-day exposure period. Finally, using 14-day exposure, changes in chlorophyll fluorescence and needle dry mass were detected. In both studies, the addition of mannitol, which is a reactive oxygen scavenger, mitigated the negative effects of PAHs.
The same experimental design was used to assess the effect of fluoranthene (FLT) on cherry tomato plants (Lycopersicon esculentum Mill) [73]. Plants were sprayed with FLT and a mixture of FLT and mannitol solutions for 30 days using 10 μM FLT concentration considered environmentally relevant. Measured end-points such as stomatal conductance, Chl a, Chl b, and the total chlorophyll contents of the tomato leaves were significantly reduced after the treatment. In the dark, the photosynthetic rate was approximately 37% lower in the treated plants, and the photochemical efficiency of PSII also decreased. In addition, visible injury symptoms on the leaves were detected. Mannitol, a reactive oxygen scavenger, mitigated FLT phytotoxicity, also supporting that FLT causes phytotoxic effects via ROS production. Using the same chamber conditions, Sunpatiens (Impatiens spp.) were exposed separately or in combination to fluoranthene (10 lM), ozone gas (130 ppb), and sulphuric acid mists (pH 3) for 21 d [74]. FLT and acid mist both seemed to have the same negative impact on chlorophyll fluorescence and gas exchange and had a synergistic effect when applied in combination. On the other hand, similarly to previous studies, mannitol seemed to mitigate these effects. In another study, FLT enhanced the harmful effects of acid mist when applied simultaneously on juvenile Japanese red pine (P. densiflora Sieb. et Zucc.) [75].
A similar experimental setup was followed in the study of Khpalwak et al. when individual and combined effects of FLT and PHE were investigated on marigold (Calendula officinalis L.) [76]. FLT and PHE were applied in the concentration of 10 μM, and treatment via fumigation was conducted for 40 days. FLT treatment resulted in a significant decrease in stomatal conductance, internal carbon dioxide concentration, and chlorophyll a and b, as well as total chlorophyll. Similarly to the previous experiment, the mitigating effect of mannitol was assessed by providing combined treatments with these PAHs and mannitol. Mannitol successfully reduced the phytotoxic effect of FLT, as in the combined treatment, these parameters were close to the control. The study also demonstrated that FLT had higher phytotoxicity than PHE. It should also be noted that these growth chambers operated under field conditions, representing real-world environmental conditions.
In a study by Ahammed et al. [77], foliar treatment with PHE was applied for 14 d on different plant species, such as pakchoi (Brassica rapa cv. chinensis), cucumber (Cucumis sativus cv. Jinyan4), flowering Chinese cabbage (Brassica campestris L. ssp. chinensis var. purpurea Bailey), tomato (Solanum lycopersicum L. cv.Hezuo903), and lettuce (Lactuca sativa L.). The concentrations applied were 30, 100, and 300 mM PHE. Phytotoxicity was assessed based on a wide range of end-points such as growth, chlorophyll contents, and antioxidant enzymes, including superoxide dismutase, guaicol peroxidase, catalase, ascorbate peroxidase, and glutathione reductase. The decrease in growth, photosynthesis, and chlorophyll contents showed a concentration-dependent pattern. Antioxidant enzymes showed even more sensitive responses. However, in higher concentrations, a decrease in the antioxidant enzymes was experienced, indicating severe damage to plants. Species-specific sensitivity was also described, with pakchoi showing the highest and flowering Chinese cabbage with the lowest sensitivity.
In a following study [78], PHE and a bioactive plant steroid, 24-epibrassinolide (EBR), were simultaneously applied to tomato plants. First, the foliar parts were sprayed with 0.1 mM 24-epibrassinolide which was followed 24 h later by the spraying of 100 mM PHE. Treatments were repeated. The study demonstrated the protective effect of EBR, primarily via the enhancement of antioxidant and detoxification enzymes. It should also be noted that without EBR treatment, PHE stress resulted in an abnormal thylakoid structure. Gene expression assays revealed that the addition of EBR played a significant role in the expression of antioxidant genes.
The above-mentioned studies applied naphthalene (NAP), phenanthrene (PHE), and fluoranthene (FLT). FLT is considered a model PAH in phytotoxicity studies [70]. NAP, PHE, and FLT are lighter (2–3 ring PAHs) that have been found to be dominant in a wide range of atmospheric samples [79]. They are less toxic than HMW PAHs; however, they are also ready to react with other pollutants in the atmosphere, thereby forming more toxic derivatives [80].
Phytotoxicity of 6 PAHs—ANT, B(a)P, B(a)A, FLU, PHE, and PYR—were compared in the experiment by Huang et al. [81]. Test plants—Brassica napus (canola) and Cucumis sativus (cucumber)—were sprayed with samples containing these PAHs at 2, 4, or 8 mg/L concentration; treatments were conducted 4 times daily for 20 days. Phytotoxicity was evaluated based on shoot biomass reduction, number of chlorotic lesions, and inhibition of photosynthesis as measured by chlorophyll fluorescence. Comparing the effect of different PAHs, B(a)P exerted the highest phytotoxicity, causing the highest growth inhibition, and also, the number of chlorotic lesions was the highest in the case of this PAH.
In order to simulate wet deposition and to assess the effect of water-soluble components of whole aerosol samples, the No. 227 OECD Guideline for the Testing of Chemicals: Terrestrial Plant Test: Vegetative Vigour Test [23] (hereinafter referred to as ‘Guideline’) was adopted [82]. The Guideline was originally designed to evaluate the phytotoxicity of general chemicals, biocides, and crop protection products [83,84]. The principle of the test protocol is that aqueous samples are sprayed on the above-ground parts of test plants when the plants reach the 2 rather than the 4 true-leaf stage.
Aqueous extract prepared from diesel emission was used to compare the sensitivity of different, common European roadside plants including wild parsnip (Pastinaca sativa L.), common daisy (Bellis perennis L.), common thistle (Cirsium arvense (L.) Scop.), common sowthistle (Sonchus oleraceus L.), common dandelion (Taraxacum officinale F.H. Wigg.), soapwort (Saponaria officinalis L.), goosefoot (Chenopodium album L.), white clover (Trifolium repens L.), Mediterranean sage (Salvia aethiopis L.), hoary plantain (Plantago media L.), common sorrel (Rumex acetosa L.), meadow buttercup (Ranunculus acris L.), and wood avens (Geum urbanum L.) [85]. The Guideline basically assesses phytotoxicity relying on biomass measurements and visible symptoms. These end-points were supplemented with biochemical markers such as photosynthetic pigments and stress enzymes. Species used in the study showed different sensitivity, with soapwort having the highest response. Comparing different end-points, biomass was the most responsive parameter.
A similar diesel exhaust sample was applied in the study by Hubai et al. to evaluate if essential oil production can be used as a measure of chemical stress [86]. Results showed that traffic-related PAH emissions significantly influenced linalool production in sweet basil (Ocimum basilicum L.).
An overview of these tests is given in Table 2, indicating the component(s) tested and plant species used.

4.3. Phytotoxicity Testing of Photomodified PAHs

Toxicity with and without photomodification of fluoranthene (FLT) was studied by using short-time exposure (12, 24, and 48 h) in the above-mentioned study by Kummerová and Vánová [65]. Results suggested enhanced toxicity of phFLT in comparison to FLT. A similar study was conducted by Kummerová et al. [87]. Test compounds were applied at 0.05, 0.5, and 5 µM concentrations. During the test, the first germination rate was assessed by placing seeds on filter paper soaked with each experimental solution; seedlings were later further cultivated for 25 days. The phytotoxicity of FLT and phFLT was compared based on the shoot and root length of seedlings. All applied concentrations of both FLT and phFLT had an inhibitory effect as the length of roots and shoots was reduced. However, a significant decrease in root growth was measured only in the case of the highest concentrations of both compounds, 5 µM. The photomodified form had a significant effect on shoot length, while in the case of FLT, the decrease was non-significant.
Photoinduced toxicity of two PAHs, anthracene (ANT) and benzo[a]pyrene (B(a)P)), was also observed in the case of terrestrial plants, Brassica napus (canola) and Cucumis sativus (cucumber), after foliar application [81]. Shoot biomass and chlorophyll content reduction were the most significant symptoms.

4.4. Uptake via Roots

As atmospheric deposition affects plants directly and indirectly via soil contamination and following uptake via the roots, a significant portion of the studies have been targeted to define the potential exposure pathways between plants and potential airborne contaminants. Zuo et al. [88] designed an airtight two-chamber exposure device, where the upper and the bottom chambers of the device were separated by an aluminum foil, making possible separate exposure of above-ground parts and roots. Five PAH compounds were tested on maize, such as naphthalene (NAP), acenaphthylene (ACY), acenaphthene (ACE), fluorene (FLU), and phenanthrene (PHE). While positive correlations were established between air and aerial tissue concentrations of the PAH species applied, translocation from the roots to the leaves was practically negligible in the study.
In a study by Wieczorek and Wieczorek [89], ANT was applied separately to the leaves of vegetable plants (Lactuca sativa L. and Raphanus sativus L.) by spraying and to the soil surface. Both foliar and soil application resulted in the reduction of plant biomass, but only foliar application elucidated the decrease in the photosynthetic rate. While in the case of foliar application, ANT was accumulated only in the leaves, translocation from the roots to other parts of the plants was observed after the treatment of the substrate. Another study by Wieczorek et al. [90] also supported the low rate of translocation of tested PAHs, anthracene, and benzo[k]fluoranthene from the above-ground parts of plants to the roots.
In the study by Daresta et al. [91], tomato (Solanum lycopersicum L.) plants were grown on PM10 collected on quartz fiber filters. In comparison to the control, significant differences were experienced in the growth of the root apparatus. ROS concentrations were measured in both the control and the treated roots, and ROS values obtained for the latter were significantly higher, suggesting that PM was able to trigger ROS production. In addition, a deleterious effect of PM10 was established on photosynthetic activity, as Chl a, Chl b, and carotenoid content showed a decrease in the treated samples. Mondal et al. [92] tested pant germination on black carbon (BC) samples collected from diesel-powered car emission pipes. Germination success was negatively affected by the treatment; also, morphological symptoms such as abnormal structure of stomata were detected in young plants. Biochemical markers were also used, such as soluble sugar, proline content, plant pigment of chlorophyll, and protein. Proline and protein content proved to be sensitive end-points, showing a significant positive correlation with total chlorophyll.

4.5. Phytotoxic Effects on Freshwater Species

Freshwater systems will most possibly receive atmospheric PAH contamination via wet and dry depositions [93]. In ecotoxicity testing, freshwater higher plants are represented by duckweed (Lemna) species. Lemna sp. has long been used in the evaluation of freshwater contaminants [94]. In order to harmonize laboratory work, international standards apply [95,96]. National standards are also available [97,98,99].
In the international context, L. minor [100] and L. gibba [101] are the generally used species. The principle of the assay is quite simple. Test organisms are immersed in the contaminated sample, and growth inhibition is evaluated after 1 week of exposure.
The Lemna assay has been used to evaluate the toxic potential of photomodified PAH species. Two separate studies conducted on L. gibba as a test organism revealed that UV radiation significantly enhances the phytotoxicity of different PAHs, such as ANT, PHE, and B(a)P [102], as well as NAP, FLU, and PYR [103]. Higher ecotoxic potential might have developed due to the appearance of photo-oxidized compounds. It was also reported that natural sunlight also causes photomodification in these PAHs, resulting in an increased toxic effect [104].

5. Conclusions and Future Directions

Phytotoxicity testing protocols imply that strict quality assurance criteria are met. Correct reporting on materials used (samples and chemicals, as well as test organisms) and methodology followed (including the practical step-by-step description of the testing process, test duration, and conditions needed during the experiments) are necessary for the reproducibility and repeatability of the test. These quality assurance criteria ensure that similar tests can be conducted where the performance or sensitivity of different test organisms to the identical pollutant can be compared or vice versa, and the sensitivity of the same organisms can be assessed toward a range of possible toxicants. Standard or quasi-standard test protocols will also prove a proper tool to extend studies using different (e.g., prolonged) exposure times or different environmental conditions.
In the reported studies, a relatively limited range of PAHs and limited number of test organisms have been addressed. Since at least 16 priority PAHs have been defined, reproducibility of these studies is necessary if investigations are to be extended to assess the phytotoxicity of other compounds. Also, the emission of PAHs is causing an ever-increasing environmental threat, influencing higher plants both in natural and man-made habitats. In order to assess the possible impact on a wider range of species than addressed by these studies and to compare their sensitivities, well-defined protocols are needed.
In addition to fulfilling quality assurance criteria, ecotoxicological tests build a concentration–response relationship where the effects of selected compound(s) are assessed on pre-defined end-points. Studies reported in this review were targeted not only to quantify the potential damage, but mechanisms of phytotoxicity could be clarified, clearly demonstrating the role of PAHs in ROS production. Also, the concentration–effect relationship between PAH pollution and damage to photosynthetic processes was described.
However, in comparison with field biomonitoring studies, it should be emphasized that the number of lab-scale phytotoxicity studies is rather low. As both the range of tested species and evaluated atmospheric samples are limited, definitely more ecotoxicological testing is needed to make extrapolations to more complex systems possible.

Author Contributions

Conceptualization, S.T., N.K. and K.H.; methodology, S.T.; resources, N.K.; writing—original draft preparation, S.T. and N.K.; writing—review and editing, N.K.; visualization, K.H.; supervision, N.K.; project administration, N.K.; funding acquisition, N.K. and K.H. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the NKFIH-872 project ‘Establishment of a National Multidisciplinary Laboratory for Climate Change’.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

No new data were created or analyzed in this study. Data sharing is not applicable to this article.

Acknowledgments

Selenge Tumurbaatar was supported by the Stipendium Hungaricum scholarship program, financed by the Tempus Public Foundation.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

PM—particulate matter; PAH—polycyclic aromatic hydrocarbon; LMW—low molecular weight; HMW—high molecular weight; ABA—abscisic acid; naphthalene—NAP; acenaphthylene—ACY, acenaphthene—ACE, fluorene—FLU; phenanthrene—PHE; anthracene—ANT; fluoranthene—FLT; pyrene—PYR; benzo[a]anthracene—B(a)A; benzo[a]pyrene—B(a)P; ROS—reactive oxygen species; POD—peroxidase; PSI—PhotosystemI; PSII—PhotosystemII.

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Atmosphere 15 01143 g001
Figure 1. Schematic diagram showing potential exposure routes for LMW and HMW PAHs.
Figure 1. Schematic diagram showing potential exposure routes for LMW and HMW PAHs.
Atmosphere 15 01143 g001
Table 1. Checklist for studies fulfilling minimum criteria for proper phytotoxicity test reporting (modified after Hanson et al. [53].
Table 1. Checklist for studies fulfilling minimum criteria for proper phytotoxicity test reporting (modified after Hanson et al. [53].
Basic Information
1. Test compound
 Name
 Supplier
 Purity
 In case of complex samples, source, and collection method
2. Experimental design and conditions
 Description of treatment method
 Exposure levels/concentrations tested
 Number of replicates
 Number and type of controls
 General test conditions
 Duration of exposure
3. Test organism
 Taxonomic name (including varieties if applicable)
 Source
 Cultivation, conditioning
 Age
 Control performance criteria
4. End-points
 End-points clearly defined and quantified
 End-points statistically evaluated
Table 2. Overview of tests using direct foliar treatment.
Table 2. Overview of tests using direct foliar treatment.
ComponentTest OrganismTreatment MethodReference
FLTpea (Pisum sativum L.)ImmersionKummerová and Vánová 2007 [65]
Váňová 2009 [66]
Vánová et al., 2009 [67]
NAP, PHE, FLTpea (Pisum sativum L.)ImmersionKreslavski et al., 2017 [68]
PHEcucumber (Cucumis sativus L.)ImmersionJin et al., 2017 [69]
FLTtobacco BY-2 cell cultureSuspension cultureBabula et al., 2012 [70]
FLU, PHEJapanese red pine (Pinus densiflora Sieb. et Zucc.)SprayingOguntimehin et al., 2008 [71]
FLUJapanese red pine (Pinus densiflora Sieb. et Zucc.)SprayingOguntimehin and Sakugawa 2008 [72]
FLTcherry tomato (Lycopersicon esculentum Mill)SprayingOguntimehin et al., 2010a [73]
FLTsunpatiens (Impatiens spp.)Spraying Oguntimehin et al., 2010b [74]
FLTJapanese red pine (Pinus densiflora Sieb. et Zucc.)SprayingOguntimehin et al., 2013 [75]
FLT, PHEpot marigold (Calendula officinalis L.)SprayingKhpalwak et al., 2018 [76]
PHEpakchoi (Brassica rapa cv. chinensis),
cucumber (Cucumis sativus cv. Jinyan4),
flowering Chinese cabbage (Brassica campestris L. ssp. chinensis var. purpurea Bailey),
tomato (Solanum lycopersicum L. cv. Hezuo903)
lettuce (Lactuca sativa L.). 		Spraying Ahammed et al., 2012 [77]
PHEtomato (Solanum lycopersicum L. cv. Hezuo903)SprayingAhammed et al., 2015 [78]
ANT, B(a)P, B(a)A, FLU, PHE, PYRcanola (Brassica napus L.), cucumber
(Cucumis sativus L.)		SprayingHuang et al., 1996 [81]
Urban PM extractcucumber (Cucumis sativus L.)SprayingKováts et al., 2017 [82]
Diesel emission extractwild parsnip (Pastinaca sativa L.)
common daisy (Bellis perennis L.)
common thistle (Cirsium arvense (L.) Scop.)
common sowthistle (Sonchus oleraceus L.)
common dandelion (Taraxacum officinale F.H. Wigg.)
soapwort (Saponaria officinalis L.)
goosefoot (Chenopodium album L.)
white clover (Trifolium repens L.)
Mediterranean sage (Salvia aethiopis L.)
hoary plantain (Plantago media L.)
common sorrel (Rumex acetosa L.)
meadow buttercup (Ranunculus acris L.)
wood avens (Geum urbanum L.)		SprayingKováts et al., 2021 [85]
Diesel emission extractsweet basil (Ocimum basilicum L.)SprayingHubai et al., 2021 [86]
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Tumurbaatar, S.; Kováts, N.; Hubai, K. Phytotoxicity Testing of Atmospheric Polycyclic Aromatic Hydrocarbons. Atmosphere 2024, 15, 1143. https://doi.org/10.3390/atmos15091143

AMA Style

Tumurbaatar S, Kováts N, Hubai K. Phytotoxicity Testing of Atmospheric Polycyclic Aromatic Hydrocarbons. Atmosphere. 2024; 15(9):1143. https://doi.org/10.3390/atmos15091143

Chicago/Turabian Style

Tumurbaatar, Selenge, Nora Kováts, and Katalin Hubai. 2024. "Phytotoxicity Testing of Atmospheric Polycyclic Aromatic Hydrocarbons" Atmosphere 15, no. 9: 1143. https://doi.org/10.3390/atmos15091143

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