Environmental Risk Assessment of Heavy Metals by Exploring Chemical Fractions, Leachability, Bioavailability in Road Dusts from Steel-Industrial City (Anshan), Northeastern China


 This study evaluated chemical fractions, potential leachability, and bio-accessibility of heavy metals (Cr, Cu, Cd, Ni, Pb, and Zn) in road dusts from the steel-industrial city (Anshan), Northeastern China. Chemical fractions of heavy metals were determined using Tessier sequential extraction method. The environmental risk assessment was evaluated using short-term extraction tests: TCLP, PBET, and CaCl2. Sequential extraction analysis reveals that Cr and Ni primarily existed in residual form. The non-residual fraction of heavy metals decreased in the order of Zn (average 57.78%)> Cu (39.16%)> Pb (30.73%)≈ Cd (30.67%)> Ni (19.06 %)> Cr (8.7%%). The results showed that Cd, Cu, Zn and Pb, which were extremely concentrated in potentially mobile fractions, had highly potential environmental risks. The mobility of Cd and Zn was usually higher than those of Cr, Cu, Pb and Ni, which means that Cd and Zn have higher hazardous to ecosystem. The order of bioavailability identified by PBET method was generally Zn>Cd>Pb>Ni>Cu>Cr. There was a significantly relationship between PBET, TCLP-test and bioavailable parts (F1+F2+F3+F4, SUM4) of sequential extraction, respectively. Multiple linear regression analysis indicated that toxicity and bioavailability of heavy metals were not only depended on RDs properties, but also lied on the total heavy metals.


Introduction
Road dusts (RDs) is an important heavy metal carrier immigrating on the urban surface mainly through atmospheric sedimentation and human emissions. RDs particles were regarded as an indicator on effecting air quality and human health, such as providing air particle materials (e.g. PM 2.5 and PM 10 Lee et al. (2013) reported the study of heavy metals (As, Cd, Cu, Pb, Sb and Zn) in RDs and demonstrated that anthropogenic inputs contributed much more than originating from earth's crust. RDs particles were much ne solid phase and almost ubiquitous, which can be easily oated and transported by wind, possibility entrancing the human body by ingestion, dermal contact, or breathing. Thus, these heavy metals in RDs particles were the potential hazards to environment safety and human health.
on trace metal mobility, as well as on their availability or toxicity, in comparison with the total element content. In the present study, the chemical speciation of heavy metals (Cr, Cd, Cu, Ni, Pb and Zn) in road dust samples was studied by using three different leaching tests. The chemical speciation was also done to investigate the potential mobility and bioavailability. Thus, the objectives of this research were to 1) access the distribution and environmental risks of heavy metals using sequential chemical extraction; 2) to correlate metals mobility by using different short-term batch extraction procedures; 3) to evaluate the effect of road dust properties on the leachability of metals; and 4) to explore the mechanisms of heavy metals dissolution in RDs.

Road dust samples
Eighty-nine road dust samples were collected from different areas of Anshan city, Liaoning Province, China. The road dust samples were collected by gently sweeping using clean plastic dustpans and brushes. Extraneous matter such as small pieces of brick, paving stone, leaves and other debris were removed. Then, the samples were dried in an oven at 40°C for 3 days, sieved to less than 250μm size, and stored in polypropylene bottles. The detail information on study area can be found in our previous research (Xiao et al. 2015).
The basic properties of RDs samples were determined by standard procedures (Zhang and Gong 2012

XRD
X-ray diffraction (XRD) was used to identify the crystalline phase in RDs particles. X-ray diffraction analysis was performed on nonoriented powder RDs samples using Cu K radiation (45 kV, 300mA) on a Rigaku X-ray diffractometer. The diffraction pattern was recorded from 10° to 80° 2θ at a speed of 0.2° 2θ/min.

Selective sequential extraction procedure
The sequential chemical extraction process was performed according to the method proposed by Tessier et al. (1979) Physiologically based extraction test (PBET) extractable content was obtained according to Ruby et al. (1996). PBET extraction was conducted by weighing 1.0 g dust into polypropylene bottles, adding 100 ml PBET (1L PBET solution contained 1.25 g pepsin, 0.5 g citrate, 0.5 g malate, 0.42 ml lactic acid, and 0.5 ml acetic acid, pH = 2.5) solution, following 1 h shaking (100rpm) at water bath (37°C). The mixture was centrifuged, and the supernatant was ltered through with the 0.45μm lter paper.
Metal concentrations were determined by ICP-AES (ICP-AES, ICAP6300DUO, Thermo Electron Corporation). Standard reference material (GBW07401) was periodically used for quality assurance of the RDs heavy metal measurement.
The leachability of heavy metals in RDs samples was calculated by the following equation.
The leachability of metals in RDs (%) = [(C metal , mg/L)(V, L)/(Q metal , mg/g)(m, g)]×100%, where C is the metal concentration in the extractable solution and Q element is the metal content in the RDs.

Data analysis
Descriptive statistics (mean and standard deviation) of RDs properties were performed applying Microsoft Excel for Windows 10. Statistical analyses were conducted using SPSS 16.0 for Windows (SPSS Inc.) and plotted by Origin Version 8 (OriginLab Corporation).The Spearman correlation coe cient, r, was used to measure the relationship between two quantitative variables. All results were expressed as averages of three replicates with standard deviation.

Basic properties of RDs
The basic properties of RDs samples were shown in  (Xiao et al., 2020). These basic properties of RDs in high pH, TC, and Fe oxides content might affect the chemical fraction and bioavailability of heavy metals.
The XRD patterns of selected representative RDs samples are displayed in Fig.1. X-ray diffraction pattern con rmed that quartz, calcite, albite, kaolinite, chlorite, muscovite, hematite, and magnetite are the seven main kinds of minerals in RDs particles. A small amount of cronstedtite and sanidine could also be found in the RDs particles. Upon semi-quantitatively analyzing the intensity of each mineral in XRD patterns, it con rmed that hematite and magnetite are generally the dominant iron species in RDs particles. The high Fe oxides in RDs could aggravate the heavy metal environment risk due to their lattice structure and adsorb heavy metals on their surface. In our previous study, iron particles, regarding as the carries of heavy metal, can be used as an indirect indicator to evaluate the heavy metals contamination and identi cation of pollution source (Zong et al. 2017).

Chemical fractions of heavy metals
The chemical fraction of heavy metals in RDs samples is showed in Fig.2 was the second most important fraction, followed by the carbonate fraction (10.08%) and the reducible fraction (6.51%). The exchangeable fraction was found to be the least contributor for Pb, accounting for 0.03%. It suggested that Pb was primarily occluded in Fe-Mn oxides forms, besides the residual fraction. This result is in line with the previous results that the high a nity of Pb for Fe oxides makes it possible for them to act as long-term sinks for Pb (Banat et al. 2005).
Zinc: The residual fraction of Zn accounted for 24.25-54.93%, and the exchangeable form was 0.02-0.93%. The dominating chemical form for Zn was the residual fraction (42.22%), and the carbonate (28.20%) was of the secondary important. A proportion of Zn was found to be oxidizable (19.37%) and reducible (9.98%), and the exchangeable fraction made up the smallest proportion (0.22%). High carbonate fraction was observed in Fig.2. It indicated that Zn was strongly associated with carbonate in RDs particles. Lee et al (2015) reported that Pb in carbonate phase (ZnCO 3 ) was observed through scanning electron microscope (SEM) equipped with energy dispersive X-ray spectroscopy (EDS). The sum unstable fractions of carbonate, oxidizable and reducible accounted for a high amount (57.55%). The result was in accordance with Świetlik et al. (2015), who proposed an assumption that a direction of changes in Zn distribution pattern was determined under sequence environment conditions changes.
Nickel: Ni was generally found in its residual form, accounting for average 80.94%. The exchangeable fraction, the concentration associated with the carbonate, with the Fe-Mn oxides and organic matter represent 0.17, 4.05, 6.27, and 8.57% of the total content, respectively.
Chromium: The Cr in RDs samples particles was associated predominantly with the forms of residual forms (91.43%), whereas the exchangeable, carbonate and organic-bound fractions were almost negligible (<5%). In this study, Ni and Cr were generally found in their residual fraction, with an average value of 80.94% and 91.43%, respectively. In summary, the percentages of Fe-Mn oxide fraction of Cd, Pb, and Zn were higher than the other three metals. The proportion of residual Cr and Ni were higher than the other metals. Cu had higher organic-bound forms.
The relatively bioavailability of metals decreased in the order: exchangeable>bound to carbonate>bound to Fe-Mn oxide>bound to organic>residual fractions, and the top four fractions represented high mobility and potential bioavailability. In particular, the exchangeable fraction was considered readily mobile and bioavailable in the environment. Fraction of exchangeable is the easiest adsorbed by organisms of all the ve fractions. Bound to carbonate can dissolved easily by water and adsorbed by organisms. Bound to Fe-Mn oxide can be released to solution when Eh or pH has changed. Otherwise bound to organic matter is not easily adsorbed by organisms, and residual fraction is invalidation to organisms. Except the resident fraction, the other four fractions of heavy metals can be leached, which were be regarded as the potential risk forms. In summary, the sum of top four fractions accounted for 30.67% for Cd, 8.7% for Cr, 39.16% for Cu, 19.06 for Ni, 30.73% for Pb and 57.78% for Zn, respectively. The result implied the high mobility and potential bioavailability in the following order: Cr<Ni<Cd<Pb<Cu<Zn. Thus, the chemical speciation except residual fraction with relatively stable heavy metals can be used an index of heavy metals active state. Clearly, the sum four fractions (F1+F2+F3+F4) of Cd, Cu, Pb and Zn almost accounted more than 30% of total heavy metals, meaning that these four elements have high available concentrations, especially for the Zn element. This result shows that highly mobile metals and readily extractable fractions pose a high potential risk to the environment. The result was agreed with our previous study that heavy metals in RDs were moderate to high polluted and huge potential ecological risk (Xiao et al., 2020). That's may be the reason why high availability and mobility of the trace metal Cd, Cu, Pb and Zn named the "urban heavy metal" (De Miguel et al. 1997). The elements Cr and Ni are predominantly in resident fraction, indicating that the Cr and Ni have low potential toxic. In summary, the metals Cd, Cu, Pb and Zn were primarily associated with non-residual fraction and thus were classi ed as potentially mobile elements. The Ni and Cr were classi ed as immobile elements because they were strongly bound to the residual fraction.

Risk assessment of heavy metals in RDs
The result of TCLP, EDTA, and 0.1M CaCl 2 extractable content heavy metals in RDs samples was displayed in Fig.3 In addition, the concentrations of CaCl 2 -, TCLP-, and PBET extractable heavy metals are calculated as leachability ratio to evaluate heavy metals mobility, leachability, and bioaccessibility (Fig. 4). The extraction percentage by PBET, TCLP and CaCl 2 represents human bioaccessibility, toxicity and mobility proportion of heavy metals in RDs particles. The mean PBET extractability is 13.76%, 0.63%, 2.35%, 4.00%, 4.22% and 30.91% for Cd, Cr, Cu, Ni, Pb and Zn, respectively. The Cr exhibits the lowest leachability among the investigated metals, which agreed with the fact that Cr is dominant in the residual fraction. Otherwise, Cd and Zn have higher PBET leachability values, indicating higher potential risks to the environment and human health. In summary, the average leachability of heavy metals follows the order Zn>Cd>Pb>Ni>Cu>Cr. The mean TCLP extractability is 5.72%, 0.12%, 0.57%, 3.00%, 1.04%, and 15.05% for Cd, Cr, Cu, Ni, Pb and Zn, respectively. The highest leachability was observed in Zn, followed by Cd. The leachability of Cr, Cu and Pb was almost less than 1%, indicating that these three metals are hard removed from RDs particles. It also means relative low hazardous for environment. The average CaCl 2 extractability is 0.64% for Cd, 0.01% for Cr, 0.35% for Cu, 0.19% for Ni, 0.11% for Pb and 0.11% for Zn, respectively.
There are great differences in the bioavailability of the different metals. The metal Zn and Cd have higher bioavailability, while relative lower values are recorded for Cr and Ni. Similarly, the bioavailability estimated by PBET extraction is much higher than the leachability estimated by TCLP and CaCl 2 extraction in each corresponding heavy metal element. RDs particles are small and ubiquity, which can easily be uptake by food ingestion, dermal contact, or breathing. Therefore, the high leachability of heavy metals in RDs particles indicated high potential hazardous to the human health and environment safe. Overall, the bioavailability of Cr and Ni is low, indicating that Cr and Ni have low potential hazard, which is in accordance to the chemical results that Cr and Ni are main in residual fraction. However, the urban metals of Cd, Cu, Pb and Zn containing high bioavailability in RDs particles should be taken into account their huge potential toxicity and mobility.

Relationship between availability and chemical fractions
As mentioned above, the available, mobility and toxicity of heavy metals bound in RDs are largely depended on their chemical speciation. Better to understand the chemical fraction contributions to bioaccessible heavy metals, a relationship between availability and different chemical fractions, including sum of the rst two fractions (F1+F2, SUM2), the rst three fractions (F1+F2+F3, SUM3) and the rst four fractions (F1+F2+F3+F4, SUM4), was carried out in table 2. The residual fraction (F5) was not included due to its least bioavailability. Except Cu, there is a signi cantly positive relationship between SUM2, SUM3, and SUM4 and the concentration of TCLP-and PBETextractable metals. However, there was almost no signi cant relationship between SUM2, SUM3, and SUM4 and the concentration of CaCl 2 -extractable metals. In addition, there is a signi cant relationship between CaCl 2 -extraction and SUM3 and SUM4 for Zn, respectively. For Pb, the correlation coe cient was extremely high between PBET-extractable and chemical fractions, with R 2 0.888 for SUM2, 0.929 for SUM3 and 0.932 for SUM4, respectively. It suggested bioaccessibility Pb mainly came from SUM4. Similar founding was also observed in TCLP-extractable Pb. For Zn, the highest correlation coe cient was observed between PBET-extractable and SUM4, while the satisfactory correlation of R 2 (0.614) was found between TCLP-extractable solution and SUM2, followed by SUM3 with R 2 0.517. The results suggested that TCLP-extractable Zn mainly originated from Zn in exchangeable and carbonate bound, while PBET-extractable Zn came from Zn in SUM4. Comparison with the chemical fractions and single extractable method, the PBET has much more ability to dissolve heavy metals in RDs particles.
In general, the relationship between chemical speciation and extractable tests can re ect the potential ecological risk of heavy metals in RDs, which can be better to re ect the bioavailability of heavy metals in RDs particles. The similar result was found by Li et al. (2015a), who found that the variation in Pb bioaccessibility should be explained by Pb in SUM2 and SUM3, respectively. Results imply that mobility or bioavailability of heavy metals is highly correlated to their chemical fractions in RDs particles. In a word, chemical fractions of heavy metals in RDs particles controlled their dissolution and toxicity.

Environment signi cance
Bioavailability and leachability of heavy metals could be controlled by many RDs properties, such as the total contents, pH, Ec, PSD, organic carbon, mineral phases, and so on. Table 2  showing high correlation between total contents and extractable ones. The total content of heavy metals in RDs was as a source pool that could be the one of main factors controlling the leachability of heavy metals. In addition, there was one point should be pointed out that the Pb regression equations by CaCl 2 could not be built. It might be the high average pH value (average 8.44) of RDs particles, limiting element Pb dissolving from the solid phase surface. Exchangeable Pb element is also hard to remove from the RDs samples at the high pH situation. According to the result of stepwise multiple linear regression analysis, the main factors in limiting heavy metals dissolving from RDs were the parameters of pH, organic carbon, carbonate, and the corresponding metal element. Compared with the regression equations of three extractable methods, PBET method was the best choice in high correlation coe cient to predict the relationship between the heavy metals concentrations and extractable concentration. The similar results were found by Li et al. (2013) and Rodrigues et al. (2013), who both found the extraction of PBET could well predict the Cu, Zn and Pb bioaccessibility. The association of Cu, Pb, and Zn in sediment particles has already been observed and these elements have been identi ed as typical "urban" metals by some authors (De Miguel et al. 1997).
It suggested that RDs properties, besides the total heavy metals, did in uence heavy metals bioavailability ( contain higher mobility and can be more harmful to the environment. In addition, the proportion F2+F3 of Zn and Pb in RDs is relative high, with the average value of 57.58% and 28.19%, respectively. It indicates that Zn and Pb can be released easily when oxidation conditions are changed into slightly reducing conditions. This is in agreement with our previous founding for urban top soil (Zong et al. 2016a). In case of Cu, the chemical fraction predominated in F4 fraction. It suggested that Cu can leach into the surrounding environment in strong oxidization or reduction conditions.
In order to evaluate the heavy metal mobility, the mobility index K was de ned, which was calculated on the basis of the ratio of while Cr is the least mobile metal with a low risk for urban environment ecosystem. Based on the K value, the metal Zn, Cd, and Pb can be classi ed as highly mobile elements group, and Ni and Cr are classi ed as immobile elements one. Obviously, the chemical fractions of highly mobile elements group were high in F1 and F2, while the proportion of F5 in the immobile element group is overwhelming (more than 80%). In particular, the Cu is classi ed as less mobile element group because of its predominantly chemical fraction in the organicbound. It should be pointed out that the highly mobile heavy metals had greater environment ecosystem and human health risks.
Meanwhile, the less mobile element Cu also has potentially risk depending on the strong oxidizing or strong reducing environment conditions. The immobile elements have relative low environment risk due to these elements xed within their crystal structure.

Conclusions
average percentage distribution for Pb tended to increase in the order: F5>F3>F2>F4>F1. Cr and Ni contained high percentage in residual fraction, and were classi ed as immobile elements. The metals Cd, Cu, Pb and Zn were primarily associated with non-residual fraction and classi ed as potentially mobile elements.