<h1>Effects of road dust on growthcharacteristics of <em>Sophora japonica</em> L. seedlings</h1><h2>Abstract:</h2><p>Road dust is one of the most common pollutants and causes a series of negative effects on plant physiology. Dust impacts on plants can be regarded as a combination of dust load, dust composition and dust grainsize impacts on plants, however, it is lack of integraldust effects studies that from those three aspects. In our study, <em>S. japonica </em>seedlings were artificially dusted by road dust collected from the road surface of Beijing to study the impacts of the road dust on nitrogen allocation, biomass allocation and photosynthetic pigments from aspects of dust composition, dust load and dust grainsize. The results showed that, most growth characteristics <em>S. japonica </em>seedlings were influenced by dust composition and dust load. Leaf N, root-shoot ratio and chlorophyll a/b were significantly affected by dust composition and dust load; leaf C/N, shoot biomass, total chlorophyll and carotenoid were significantly affected by dust load; stem N and stem C/N were significantly affected by dust composition; dust grainsize did not affect any growth characteristic alone. Road dust influenced growth characteristics more seriously than loam. Higher dust load could make the differences between road dust and loam treatments greater. Dust elements well related to the shoot N, shoot C/N and root-shoot ratio of <em>S. japonica </em>seedlings. Relevant knowledge would benefit the management of urban greenspace.</p><p><strong>Keywords:</strong></p><p>Dust composition; dust load; dust grainsize; nitrogen; biomass; pigment.</p><h2>1 Introduction</h2><p>Road dust is one of the most common pollutants in cities and consists of particles from atmospheric and vehicular-related deposition, also materials produced by soil erosion and pavement abrasion(<a href="#_ENREF_28">Tang et al., 2013</a>). Road dust can cause a series of negative effects on plant physiology, e g. reducing diffusive resistance(<a href="#_ENREF_29">Thompson et al., 1984</a>), blocking the stomata (<a href="#_ENREF_7">Farmer, 1993</a>), increasing absorption of insolation(<a href="#_ENREF_5">Eller, 1977</a>), lowering pigment contents (<a href="#_ENREF_22">Prajapati and Tripathi, 2008</a>; <a href="#_ENREF_23">Prusty et al., 2005</a>) and restraining photosynthesis (<a href="#_ENREF_3">Chaturvedi et al., 2013</a>). However, quantified studies of road dust effects were much fewer than those qualitative ones.</p><p>Dust impacts on plants can be regarded as a combination of dust load, dust composition and dust grainsize impacts on plants. Dust load effect studies were more than dust composition and dust grainsize effect studies. In previous artificial dusting studies, several dust load treatments were compared (<a href="#_ENREF_1">Armbrust, 1986</a>; <a href="#_ENREF_2">Charlesworth et al., 2003</a>; <a href="#_ENREF_11">Kuki et al., 2009</a>; <a href="#_ENREF_26">Shukla et al., 1990</a>), concerning loam, fly ash, iron dust and cement dust. As for the dust composition effects on plants, in ore dust studies, ore element contents in dust and plants were often linked together(<a href="#_ENREF_12">Kuki et al., 2008</a>; <a href="#_ENREF_18">Neves et al., 2009</a>). However, dusts of different composition lacked of comparison in the way of affecting plants. Studies of grainsize effects on plants were rarely seen. Stomatal conductance of plants dusted by carbon black of different grainsize were compared(<a href="#_ENREF_9">Hirano et al., 1995</a>).When dust grainsize was larger than the stomata opening, particles piled up on the pore, if smaller they clogged the stomata and affected gas exchange, thus affected growth of the plants(<a href="#_ENREF_24">Rai et al., 2010</a>). It is lack of integraldust effects studies that from aspects of dust load, dust composition and dust grainsize.</p><p><em>Sophora japonica</em> L. is a native tree species that is widely distributed in North China. It is the dominate street tree species planted in Beijing andaccounts for 81% of the street trees in urban areas of Beijing (<a href="#_ENREF_32">Zheng and Zhang, 2011</a>). Its growth plays an important role in adjusting the air quality and keeping the health of the urban environment. In our study, <em>S. japonica </em>seedlings were artificially dusted by road dust collected from the road surface of Beijing to study the impacts of the road dust on nitrogen allocation, biomass allocation and photosynthetic pigments from aspects of dust composition, dust load and dust grainsize.</p><p>Road dust carries high concentration of heavy metals, such as Cd, Cr, Cu, Pb and Zn (<a href="#_ENREF_2">Charlesworth et al., 2003</a>; <a href="#_ENREF_28">Tang et al., 2013</a>), which are different from those natural dusts such as loam in composition. To highlight the dust composition effects of road dust, we used loam from unpolluted rural area as a comparison. Loam is often used to study the physical effects of dust on plants(<a href="#_ENREF_1">Armbrust, 1986</a>; <a href="#_ENREF_9">Hirano et al., 1995</a>). Road dust and loam were set as two dust composition treatments in our study.As far as dust load effects, dust loads of street tree <em>S. japonica</em> in our previous study and an extremely high dust load situation were simulated. As for dust grainsize, dry sieving method was used to divide dusts into fine and coarse grainsize. We suppose that dust effects on growth characteristics of <em>S. japonica </em>seedlings would enhance with the higher dust load as well as finer dust grainsize. The purposes of our work were todetect: 1. Whether dust load, dust composition and dust grainsize of dust all impact on growth characteristics? Which characteristics do they impact? 2. Are there differences between road dust and loam effects on growth characteristics? Whether dust elements relate to growth characteristics?</p><h2>2 Materials and methods</h2><h2>2.1 Plant experimental material</h2><p>Seed germination was performed under greenhouse conditions at the Beijing Forestry University Forest Science Co,Ltd., in April 2012. <em>S. japonica </em>seedlings were obtained by the germination of seeds provided by the nursery of Qingyuan County in Hebei Province. The seeds were soaked in 85–90°C water for 24hours and then germinated in nursery trays when the seeds swelled. If the seeds did not swell properly, we repeated the soaking once or twice. Then, 20 days after emergence, the seedlings were transferred to plasticpots containing cultivation soil (sand + peat + vermiculite in a proportion of 2:2:1). Each pot contained one plant. In August 2012, seedling heights were 30–50cm, and the crown widths were about 30cm.</p><h2>2.2 Collection of road dust</h2><p>The road dust used in the experiment was collected from the surface of some busy traffic streets between the 2nd Ring and 3rd Ring Roads of Beijing. Loam used in this experiment was collected from mountainous area far from traffic in Miyun county of Beijing. The road dust and loam were sieved with nylon fine screen to coarse particles (53 ?m <diam <106?m) and fine particles (diam <53?m).</p><h2>2.3 Maximum dust load experiment and treatments</h2><p>The seedlings were irrigated and their leaves were washed three days before artificial dusting. We placed plastic wrap on the soil surface to avoid thedust retention.Road dust was applied by using a dust sprayer until the leaf surface could not accommodate any more dust. The maximum applied dust (A<sub>m</sub><sub>ax</sub>) was then recorded. Leaves of 10 replications were collected to count the maximum dust load (D<sub>m</sub><sub>ax</sub>). The result ofthe maximum dust load experiment is shown in Table2, and the average of the maximum dust load D <sub>max</sub> was 16.97 g·m<sup>-2</sup>, corresponding to the maximum applied dust of 73.08 g/pot.</p><p>In October 2012, the dusting experiment was conducted including nine treatments, and each treatment had nine replicate seedlings. In our previous study, the dust load of <em>S. japonica</em> in streetswas significantly higher than that of <em>S. japonica</em> in parks, and the dust load of street tree <em>S. japonica </em>had a maximum value of 2.812 g·m<sup>-2</sup> and an average value of 0.683 g·m<sup>-2</sup> (<a href="#_ENREF_4">Dai et al., 2012</a>). Therefore, we regarded 2.812 g·m<sup>-2</sup> as low dust load treatment. We defined the maximum dust load 16.97 g·m<sup>-2</sup>as high dust loadtreatment. We took A<sub>0</sub> treatment with no dust as contrast.The dust load amounts of treatments are shown in Table3.</p><p>The seedlings were irrigated and the leaves were washed three days before dusting as then treated and the amount of dust shown in Table 3. The seedlings were kept stable for 21 days and the irrigation water was only applied to the soil surface directly by tubule. Immediately after dusting, three seedlings per treatment were cut down to test their actual dust load. Whole leaves from each seedling were washed in distilled water and ultrasonic oscillated for 3 min, and then vacuum filtered through pre-weighed filter membrane(?=0.45µm)(<a href="#_ENREF_8">FreerSmith et al., 1997</a>).</p><h2>2.4 Biomass, nitrogen and pigments measurement</h2><p>Three seedlings from each treatment were excavated and washed in water. The roots, stems, and leaves were separated and oven dried at 80°Cfor at least 24 hours to obtain the dry weight biomass. Then dried roots, stems and leaves were powdered by ball mill. Nitrogen contents were tested by Elementar Vario EL III using dry combustion method. The second, third, and fourth leaves from the top of each sprig were collected and put into liquid nitrogen using three seedlings foreach treatment. Fresh leaves were extracted with 95% ethanol and the absorbance of the leaf pigment extract was measured separately at 470, 649, and 665 nm. Chlorophyll and carotenoid were calculated according to the specific absorption coefficients of Lichtenthaler (<a href="#_ENREF_15">Lichtenthaler, 1987</a>).</p><h2>2.5 Statistical analyses</h2><p>Comparisons between three treatments were done by the LSD test of one-way ANOVA. Comparisons between two treatments were done by the independent T test. Univariate GLM was used to analysis the effects of three dust treatments and their interactions. All of the above tests were done by SPSS 15.0.</p><h2>3 Results</h2><h2>3.1 Effects of dust composition, dust load and grain size on carbon and nitrogen allocation</h2><p>Based on the Multivariate ANOVA results (Table 4), dust composition and dust load significantly affected leaf N. Dust composition significantly affected stem N and stem C/N. Dust load significantly affected leaf C/N. The interaction of dust composition and dust load affected root N. The interaction of all three dust attributes affected root N and root C/N. As shown in Figure 1, leaf N of A8 treatment was significantly lower than A0. Stem N of A2, A4, A5 and A6 were significantly lower than A0. Root N of A7 was significantly higher than A3. Leaf C/N of A8 treatment was significantly higher than A0, and that of A7 was significantly higher than A3. Stem C/N of A2 was significantly higher than A0 and A1, and that of A8 was significantly higher than A0. In general, leaf N and stem N decreased while leaf C/N and stem C/N increased after dusted. Root N and root C/N had no obvious trend. Stem N of road dust treatment (A2) was significantly lower than that of corresponding loam treatment (A1), while stem C/N of road dust treatment (A2) was significantly higher than that of corresponding loam treatment (A1). High load treatment (A7) had significant higher leaf C/N than corresponding low load treatment (A3).</p><h2>3.2 Effects of dust composition, dust load and grain size on biomass allocation</h2><p>According to the Multivariate ANOVA results (Table 5), shoot biomass and root-shoot ratio were significantly affected by dust load. Root-shoot ratio was significantly affected by dust composition meantime. As shown in Figure 2, shoot biomass decreased except in A1 treatment. Shoot biomass of A5 was significantly lower than A1. Root biomass decreased in A3, A4, A5, A6 and A7. Root biomass of A5 was significantly lower than A1, and that of A8 was higher than A7 significantly. Total biomass decreased except in A1 treatment. Total biomass of A5 was significantly lower than A1. Root-shoot ratio increased in A4, A5, A6, A7 and A8. In general, shoot biomass, root biomass and total biomass had a trend of decline after dusted, while root-shoot ratio had a trend of increase. High load treatment (A5) had significant lower shoot biomass, root biomass and total biomass than corresponding low load treatment(A1). Root biomass of road dust treatment (A8) was significantly higher than corresponding loam treatment(A7).</p><h2>3.3 Effects of dust composition, dust load and grain size on photosynthetic pigments</h2><p>According to the Multivariate ANOVA results (Table 6), chlorophyll a/b, total chlorophyll and carotenoidwere significantly affected by dust load. Chlorophyll a/b was significantly affected by dust composition meantime. Carotenoid was significantly affected by the interaction of dust load and dust grainsize. As shown in Figure 3, chlorophyll a/b of A6 and A8 treatments were significantly lower than A0. Total chlorophyll of A7 and A8 were significantly lower than A0. Carotenoid of A6 was significantly higher than A0. In general, dust treatments decreased Chlorophyll a/b, total chlorophyll and carotenoid. Significant differences were all appeared in high load treatments.</p><h2>3.4 Relationship between dust elements and growth characteristics</h2><p>Metal and nutrition elements in dust caused a dust composition difference between road dust and loam (Table 1), which well correlated to growth characteristics (Table 7). Leaf N was negatively correlated with Cu, Zn, Cr, Ni, P and C contents of dust. Stem N was negatively correlated with all elements except K contents in dust, while stem C/N was positively correlated with all elements except K contents in dust. Leaf C/N was positively correlated with Cr, Ni, P and C contents of dust. Root-shoot ratio was positively correlated with Cu, Cr, Ni, P and C contents of dust. Pigments were not correlated with dust elements. To sum up, metals, P, N and C contents in dust had negative effects on shoot N, but had positive effects on shoot C/N and root-shoot ratio. Higher content of N in road dust did not make the shoot or root N of road dust treatments higher.</p><p>As shown in Table 8, leaf N positively correlated with chlorophyll a/b and total chlorophyll, but negatively correlated with leaf C/N, stem C/N and root-shoot ratio. Stem N positively correlated with chlorophyll a/b, but negatively correlated with leaf C/N, stem C/N and root-shoot ratio. Root N negatively correlated with root C/N. Higher root-shoot ratio and lower pigments in road dust treatments corresponded to lower shoot N and higher shoot C/N.</p><h2>4 Discussion</h2><h2>4.1 Growth characteristics were affected by dust</h2><p>In our study, most growth characteristics were influenced by dust. Shoot biomass, root biomass, total biomass, leaf N, stem N, chlorophyll a/b, total chlorophyll and carotenoid of <em>S</em><em>. </em><em>japonica</em>seedlings had a trend of decline after dusted, while root-shoot ratio, leaf C/N, stem C/N had a trend of increase. Higher root-shoot ratio and lower pigments in road dust treatments corresponded to lower shoot N and higher shoot C/N. Plants allocate relatively less biomass to leaves and more to their roots when nitrogen is in short supply(<a href="#_ENREF_13">Lambers et al., 1998</a>). N plays a crucial role in photosynthetic production and total leaf N had a linear correlation with chlorophyll content (<a href="#_ENREF_6">Evans, 1989</a>; <a href="#_ENREF_31">Watari et al., 2012</a>). Our consequence was consistent with those studies.</p><p>Decrease of N content, biomass and pigments were found in previous researches, while C/N, root biomass and root-shoot ratio were not involved. Leaf N of street trees were significantly lower than those trees in clean area(<a href="#_ENREF_3">Chaturvedi et al., 2013</a>). Simulated dusting made leaf and stem N of cotton decrease (<a href="#_ENREF_14">Li, 2005</a>). Cement dust caused a decline on needle N content of <em>Pinus sylvestris </em>(<a href="#_ENREF_17">Mandre, 2009</a>) and N content of wheat (<a href="#_ENREF_20">Pandey and Sanjeev, 1996</a>). Cement dust also decreased dry mass of <em>Picea abies </em>(<a href="#_ENREF_19">Ots et al., 2011</a>) and biomass of tomato and radish (<a href="#_ENREF_16">Magray et al., 2013</a>). Dust on street trees made the photosynthetic pigments decreased (<a href="#_ENREF_22">Prajapati and Tripathi, 2008</a>; <a href="#_ENREF_23">Prusty et al., 2005</a>).</p><p>Nitrogen is an important nutrient element that closely relates to growing developmentand organ formation of plants. Decrease of leaf N and stem N in our study contribute to the increase of leaf C/N and stem C/N. Moreover, the seedlings were very young, so root biomass took up a large proportion of total biomass. Reduction in shoot biomass did not cause a very significant decrease on total biomass, but contribute to a increase on root-shoot ratio. Rising of root-shoot ratio means <em>S. japonica</em> seedlings allocate more biomass to root than to crown when stressed by dust.</p><h2>4.2 Dust load impacts leaf N, leaf C/N, shoot biomass, root-shoot ratio, chlorophyll a/b, total chlorophyll and carotenoid</h2><p>In our study, dust load significantly affected leaf N, leaf C/N, shoot biomass, root-shoot ratio, chlorophyll a/b, total chlorophyll and carotenoid. High load treatments tended to have lower leaf N, shoot biomass and pigments than those of low load treatments, as well as higher leaf C/N and root-shoot ratio.</p><p>Dust load impacts on shoot biomass and pigments were studied in previous researches, but N content, C/N and root-shoot ratio were not studied before. Biomass of wheat and <em>S.tomentosa</em> increased and then deceased with dust load (<a href="#_ENREF_11">Kuki et al., 2009</a>; <a href="#_ENREF_27">Singh and Siddiqui, 2003</a>). Biomass and pigments of cotton fluctuated and then decreased with a series of rising dust load (<a href="#_ENREF_1">Armbrust, 1986</a>). Pigments of wheat increased and then deceased with the dust loads of fly ash(<a href="#_ENREF_27">Singh and Siddiqui, 2003</a>). In other words, shoot biomass and pigments of plants may increase or decrease at lower dust load, but would surely decrease at very high dust load, which was consistent with our studies. High dust load indicated the maximum dust load of <em>S. japonica</em> and low dust load indicated the maximum value of street tree <em>S. japonica</em>. Most growth characteristic differed in those two dust loads.</p><h2>4.3 Dust composition impacts leaf N, stem N, stem C/N, root-shoot ratio and chlorophyll a/b</h2><p>In our study, dust composition significantly affected leaf N, stem N, stem C/N, root-shoot ratio and chlorophyll a/b. Road dust treatments had lower leaf N, stem N, and chlorophyll a/b than those of loam treatments, as well as higher stem C/N and root-shoot ratio.</p><p>Previous studies on road dust effects were concentrated on pigments, gas exchange and chlorophyll fluorescence(<a href="#_ENREF_3">Chaturvedi et al., 2013</a>; <a href="#_ENREF_22">Prajapati and Tripathi, 2008</a>; <a href="#_ENREF_23">Prusty et al., 2005</a>). Loam effects on photosynthesis and biomass were ever studied(<a href="#_ENREF_1">Armbrust, 1986</a>; <a href="#_ENREF_9">Hirano et al., 1995</a>). However, effects of road dust and loam on growth characteristics were not compared before. As two kinds of dusts that have great differences in composition, road dust contains much higher contents of Cu?Zn?Cr?Pb?Ni?P?N?C compared to loam. In our correlation analysis, heavy metals, P, N and C contents in dust had negative effects on shoot N, but had positive effects on shoot C/N and root-shoot ratio. Stress of heavy metals like Pb can reduce the N absorption of plants, because excess heavy metal disturbs plants’ cellular metabolism, and thus influence the root absorption of necessary nutrient elements(<a href="#_ENREF_10">Kang, 2012</a>; <a href="#_ENREF_25">Sharma and Dubey, 2005</a>). Trace elements in PM can cause increasing concentration of trace elements in plant shoots by covering foliage (<a href="#_ENREF_21">Pavlik et al., 2012</a>; <a href="#_ENREF_30">Uzu et al., 2010</a>). Whether heavy metals absorbed by leaf surface could disturb the cell metabolism is worth exploring in future. Higher content of N in road dust did not make the shoot or root N of road dust treatments higher.</p><h2>4.4 Combination of dust load, dust composition and dust grainsize</h2><p>In our study, leaf N, root-shoot ratio and chlorophyll a/b were significantly affected by dust composition and dust load; leaf C/N, shoot biomass, total chlorophyll and carotenoid were significantly affected by dust load; stem N and stem C/N were significantly affected by dust composition; dust grainsize did not affect any growth characteristic alone. Combination of dust composition and dust load was obvious. High dust load influenced growth characteristics more seriously than low dust load; road dust influenced growth characteristics more seriously than loam. Higher dust load could make the differences between road dust and loam treatments greater. Two dust grainsize in our study may not be far enough to present different effects on growth characteristics. However, for leaf N, stem N, leaf C/N, stem C/N, root-shoot ratio, chlorophyll a/b and carotenoid, the most strongly affected treatment was the high load fine road dust(A8), which meant the combination of high dust load, road dust and fine grainsize could affect growth characteristics most seriously.</p><h2>5 Conclusion</h2><p>Most growth characteristics <em>S. japonica </em>seedlings were influenced by dust composition and dust load. Leaf N, root-shoot ratio and chlorophyll a/b were significantly affected by dust composition and dust load; leaf C/N, shoot biomass, total chlorophyll and carotenoid were significantly affected by dust load; stem N and stem C/N were significantly affected by dust composition; dust grainsize did not affect any growth characteristic alone. Road dust influenced growth characteristics more seriously than loam. Higher dust load could make the differences between road dust and loam treatments greater. Dust elements well related to the shoot N, shoot C/N and root-shoot ratio of <em>S. japonica </em>seedlings.</p><h2>Cover letter</h2><p>Dear Editor:</p><p>I would hereby like to submit our manuscript, titled <em>“Effects of </em><em>road dust </em><em>on </em><em>growth</em><em>characteristics </em><em>of</em><em> Sophora japonica L.</em><em> seedlings</em><em>”</em>,to be considered for possible publication in <em>Journal of Environmental Science</em>.</p><p>Road dust is one of the most common pollutants and causes a series of negative effects on plant physiology.Road dust impacts on plants can be regarded as a combination of dust load, dust composition and dust grainsize impacts on plants. Dust composition and dust grainsize effects on plants were rarely studied previously. Moreover, it is lack of integral dust effects studies that from all those three aspects. In our study, <em>S. japonica </em>seedlings were artificially dusted by road dust collected from the road surface of Beijing to study the impacts of the road dust on nitrogen allocation, biomass allocation and photosynthetic pigments from aspects of dust composition, dust load and dust grainsize. Road dust carries high concentration of heavy metals, such as Cd, Cr, Cu, Pb and Zn, which are different from those natural dusts such as loam in composition. To highlight the dust composition effects of road dust, we used loam from unpolluted rural area as a comparison.</p><p>Our paper will be of broad interest to the readers of <em>Journal of Environmental Science</em>, as it is one of the first to compare the dust compositon, dust load and dust grainsize effects on growth characteristics of plants. Our results highlight the differences between road dust and loam effects as dust composition differences and relationships between dust elements and growth characteristics approved it.</p><p>The manuscript has been revised and approved by all co-authors. No part of this paper has been published or submitted elsewhere. As we are non-native English speakers, an editor at EditorWorld (https://www.editorworld.com/) corrected the grammar and focused on enhancing fluency, clarity, and flow. Below, I have suggested the names of five potential reviewers with appropriate backgrounds to review this paper.</p><p>Thank you very much for considering our manuscript. Please do not hesitate to contact me if you require any additional information.</p>