Assessment of Cu, Pb and Zn content in selected species of grasses and in the soil of the roadside embankment

Abstract It was assumed in the study that heavy metals occurring in soils and the air accumulate in grasses constituting the main species used in the turfing of soil in road verges and embankments along traffic routes and in other parts of urbanized areas. The aim of the present study was to assess the bioaccumulation of Cu, Pb, and Zn in three selected lawn cultivars of five grass species and in the soil of the roadside green belt in terms of soil properties and heavy metal uptake by plants in the aspect of determining their usefulness in protecting the soils from contamination caused by motor vehicle traffic. Samples of the plant material and soil were collected for chemical analysis in the autumn of 2018 (October) on the embankment along National Road No. 17 between Piaski and Łopiennik (Poland), where 15 lawn cultivars of five grass species had been sown 2 years earlier. During the study, Cu, Pb, and Zn levels were determined in the aboveground biomass of the grasses under study and in the soil beneath these grasses (the 0–20 cm layer). All the grass species under study can thus be regarded as accumulators of Cu and Zn because the levels of these elements in the aboveground biomass of the grasses were higher than in the soil beneath these grasses. The present study demonstrates that the grasses can accumulate a large amount of Cu and Zn from soils and transfer it to the aboveground biomass. Tested species of grasses are not a higher bioaccumulators for Pb. The best grass species for the sowing of roadsides embankment, with the highest BCF values for the studied metals, is Lolium perenne (Taya variety).

The plants' intake of Zn from the soil is limited and depends on the pH, the physicochemical properties of the soil, and the activity of microorganisms in the rhizosphere. Zn is absorbed by the roots mostly as a divalent cation (Zn 2+ ) (Gupta, Ram, & Kumar, 2016).
The factors which control Zn mobility in the soil are very similar to those listed for Cu, but, unlike other heavy metals, Zn occurs in the soil very frequently in easily soluble forms. The Zn absorption rate differs considerably between plant species. According to Kabata-Pendias and Pendias (2001), Zn is accumulated the most in the aboveground parts of plants in ecosystems where this pollutant occurs in the air. On the other hand, the solubility and availability of Zn for plants is negatively correlated with the saturation of the soil with Ca and P compounds. According to the investigations by Kabata-Pendias and Pendias (1999), in the case of a high pH (7.2-7.8), Zn intake by barley is closely correlated with Zn levels in the soil. It is believed that Zn stimulates the plants' resistance to dry and hot weather as well as bacterial and fungal diseases. On the other hand, the toxicity of Zn depends on the species, genotype, and growth stage. Rout and Das (2003) demonstrated an inhibitory effect of Zn on Cu where the intake of one element inhibited the intake of the other. This may indicate the same mechanisms of the absorption of both metals.
Although Pb is regarded as a metal with the lowest bioavailability to plants, large amounts of it accumulate in plants (mainly the roots).
According to Iskandar (2001), the pH of the soil has little influence on the availability of Pb. Other researchers believe that Pb is easily absorbed by plant leaves because stomata leaves are larger than lead particle size (Farid, Shams Farooq, & Khan, 2017). Hamdy, Al Obiady, and Al Mashhady (2015) found Pb occurring in the air and settling on the leaves of plants to be a considerable source of this metal in the aboveground parts of plants. They calculated that up to 95% of the total Pb content in the plants can originate from the Pb suspended in the air. Pb occurring in the soil is bound with carboxylic functional groups of uronic acid contained in the mucus of root cells, or by means of polysaccharides on the surface of the rhizodermis (Seregin & Ivanov, 2001). Studies show that some grass species, such as Festuca rubra (Ginn, Szymanowski, & Fein, 2008) or Paspalum notatum (Araújo, Lemos, Ferreira, Freitas, & Nogueira, 2007), are able to absorb Pb through their root system. Pb can also be absorbed by the roots by means of passive intake resulting from the difference of concentration levels. It should be remembered, however, that the rhizodermis of the roots prevents Pb from traveling to the aboveground parts of the plants. The biggest amounts of Pb accumulate in the tips of the roots because young root cells have thin cell walls (Hamdy et al., 2015); Seregin, Shpigun, & Ivanov, 2004).

Due to the fact that since 2005 Research Centre For Cultivar
Testing in Poland has not been conducting the assessment of the utility value of lawn grass varieties, therefore the sensitivity of these varieties to unfavorable habitat conditions is not known. During this time, there has been a huge progress in breeding lawn grass varieties, both Polish and foreign, which may differ from sensitivity to stressful environmental factors and their suitability for phytoremediation and sodding of contaminated areas.
It was assumed in the study that heavy metals occurring in soils and the air accumulate in grasses constituting the main species used in the turfing of soil in road verges and embankments along traffic routes and in other parts of urbanized areas. The aim of the present study was to assess the bioaccumulation of Cu, Pb, and Zn in three selected lawn cultivars of five grass species and in the soil of the roadside green belt in terms of soil properties and heavy metal uptake by plants in the aspect of determining their usefulness in protecting the soils from contamination caused by motor vehicle traffic.  (Domański, 1998) and IHAR (Prończuk, Prończuk, & Żyłka, 1997), on both sides of the road (Figure 1). The plots were located about 20 cm from the side of the road. The species selected for investigation are the most frequent and the largest components of grass ecosystems of roadsides (Harkot, Wyłupek, & Czarnecki, 2005;Stawicka, 2003).

| MATERIAL AND ME THODS
The samples of the plant material (aboveground biomass of each species) and soil material were collected for laboratory analysis at about 0.5 m from the edge of the plot, in three repetitions. There were collected nine representative samples of plants and soil (three samples from each plot) per variety, so 27 samples per species (three variety). Soil was sampled from the 0-20 cm layer using a soil probe (Egner's sampler), at the same spots where the plant material was sampled. The soil and plant samples were hidden in hermetic containers (string bags) immediately after collection. Then, the plant samples were washed for 10 s with distilled water, dried, and hidden in airtight containers.
The granulometric composition was determined using the Casagrande areometric method in Prószyński's modification according to PN-R-04032:01.12.10. It consists in measuring the density of soil suspension during particle sedimentation using a soil hydrometer, at intervals needed for subsequent fractions to fall.
Soil pH was determined using a glass electrode in a soil suspension in a 1 mol/L KCl solution according to PN-ISO 10390:1997. Soil salinity was measured using a conductometric method according to KQ/PS-50 ver. 03 from 01.10. 2010. The soil weighed 10 g was shaken in 25 ml distilled water for 1 hr. Then, the conductivity was determined using an Elmetron conductometer. Extraction of P and  (Bech et al., 2012;Wu et al., 2011;Zacchini et al., 2009).
The results of the analysis of heavy metal levels in the plant and soil material were processed statistically in SAS v. 9.1 software by means of variance analysis. Analysis of variance was used to assess differences between means of examined variables for selected grass species and their varieties. Mixed models were used, in which the species (or variety) was a fixed factor, while the plot was used a random factor. Multiple comparison Tukey's test was used to verify the significance of differences between the means assessed. Spearman's rank-order correlation coefficient was used to determine the correlation between variables. In statistical analysis, significance level α = .05 was assumed. The control object consisted of three control plots on each of them three measurements were taken (this is to assess the variability between plots so that the experiment is equally founded).
The soil samples analyzed had an alkaline pH, and the pH levels did not vary significantly between sites except for soil samples collected from beneath F. ovina and L. perenne, where the pH was significantly the highest in comparison to the other species and control soil. Salinity was higher in F. arundinacea, F. ovina, and F. rubra, while lower in L. perenne and P. pratensis compared to control soil. The Mg level was significantly lower in samples of soil from beneath F. ovina and F. rubra. Compared to control soil, higher P level were found in the soil samples collected from Zn, and Pb levels was found in the soil from beneath the Natara cultivar. Among the P. pratensis cultivars, the highest Cu and Zn levels was determined in the soil from beneath the Bila cultivar.
The highest Pb level was found in the soil from beneath the Alicja cultivar ( Table 2).
The analyses showed that the lowest Cu, Zn, and Pb levels (p < .05) occurred in the aboveground biomass of P. pratensis (Table 3). The highest significant Cu level was found in L. perenne, Zn-in F. rubra, and Pb-in F. arundinacea. Within the F. arundinacea species, the Cu, Zn, and Pb levels were recorded in the Romina cultivar. Within the F. rubra species, the highest Cu, Zn, and Pb levels were recorded in the leaves of the Nista cultivar. Within the F. ovina species, the highest Cu, Zn, and Pb levels were found in the Tomika cultivar. Within the Lolium Perenne species, the lowest Cu and Pb levels were found in the Nira cultivar, and the highest Cu levels were found in the Natara cultivar, Zn-in Nira, and Pb-in Taya. Among the cultivars of P. pratensis, the highest levels of Cu and Zn were determined in the Bila and Alicja cultivar, and of Pb-in the Ani cultivar (Table 3, Table S2).
In all the analyzed soil samples, no significant correlations were found between the soil pH and levels of the heavy metals studied (  (Table 4).
In all the analyzed samples of the plant material, no significant correlations were found between the soil pH and salinity on the one hand and heavy metal levels on the other (Table 5). The analysis of the aboveground parts of F. ovina showed that Cu and Zn levels in the leaves were rising with decreasing P level, and Pb level in the leaves increased with decreasing K levels. A relationship was found in F. rubra between an increase in P levels in the soil and decreased Zn and Pb levels in the plants as well as between increased K levels in the soil and increased Cu, Pb, and Zn levels in the aboveground parts of F. rubra. Analyses also showed that Pb levels in the aboveground parts of L. perenne increased with increasing Mg levels in the soil. In the case of the other species, no significant relationships (no correlations) were found between macro-element levels and the accumulation of heavy metals in the plants (Table 5).
Spearman's rank-order correlation analysis showed that Cu and  x,y,z -designation of groups of homogeneous varieties within the species at the significance level α = .05.
Bold -average value for species.
relationships were found between heavy metal levels and their accumulation in the plants (Table 6).
Assessing the degree of bioconcentration of heavy metals, the highest BCF for the Pb, Zn, and Cu was calculated for L. perenne and F. ovina, and the lowest for P. pratensis. The tested species showed a high BCF for Cu-above 1. While BCF for Zn was higher than 1 in most of the tested varieties except Asterix (F. arundinacea), Tenis (F. ovina), Natara (Lolium prerenne), Ani and Bila (P. pratensis). The lowest BCF was calculated for Pb, around 0.2 (Table 7).

| D ISCUSS I ON
The investigations showed that fine-grained gravel predominated (73%), while sand accounted for 9% and fine dust for 8% in the granulometric composition of the top layer of soil (0-20 cm). Thus, soil water deficit can occur in years with small precipitation volumes. This is because the size of granulometric fractions is closely correlated with the physicochemical properties of the soil such as hygroscopicity and coefficient of wilting which reach their maximum levels in the colloidal clay fraction (Paluszek, 2011 Note: a-d -designation of homogeneous species groups at the significance level α = .05.
x,y,z -designation of groups of homogeneous varieties within the species at the significance level α = .05.
Bold -average value for species.

TA B L E 3
The content of Cu, Zn, and Pb in grass of the examined species of grasses (in mg k −1 d.w.) leaves and their assimilation by the plants (Chojnacka et al., 2005;Yan et al., 2012). In their studies of heavy metal content in grasses Onder, Dursun, Gezgin, and Demirbas (2007)

also found that Cu and
Zn levels in the grasses were higher than in the soil. On the other hand, Amusan, Bada, and Salami (2009) and Wei, Ge, Chu, and Feng (2015) found that heavy metal levels in the grasses were considerably lower than in the soil. Also in the study by Onder et al. (2007), Pb levels in the aboveground parts of grasses were lower than in

TA B L E 4
Relationship between pH value, salinity and content of macroelements and accumulation of heavy metals in soil (for p < .05) the soil. The levels of heavy metals in the analyzed grasses can be arranged in the following order: Zn > Cu > Pb. The same order of the accumulation of heavy metals by grasses was found in studies by Dinelli and Lombini (1996), Puschenreiter and Horak (2000), and Boularbah et al. (2006). a BCF higher than 1. The term hyperaccumulator was first used by Brooks, Lee, Reeves, and Jaffre (1977) and was originally used to define plants containing more than 1,000 ng/g(ppm) nickel in dry tissue. Kramer (2010) stated in his review that hyperaccumulation concentration criterion for Cu and Pb are >1,000 µg/g, while for Zn > 10,000 µg/g. Our research has shown that all tested grass species were good accumulators of Cu, while moderate accumulators for Pb and Zn. In turn, Kumar, Ahirwal, Maiti, and Das (2015) found that Saccharum munja and Cynodon dactylon were good Pb and Zn hyperaccumulators. Based on studies by Gupta et al. (2009), andCenkci et al. (2010), we know about the interactions between Pb and Zn. Lead has a strong affinity with sulfhydryl groups (-SH) occurring in proteins forming part of metalloid enzymes, as a result of which these enzymes do not bind microelements such as Fe or Zn (Gupta et al., 2009). The possibility of easy availability and movement of metals between root and shoot is usually determined by correlating the metal concentration in different plant parts (Pandey, Singh, Singh, & Singh, 2012). To assess the negative and positive translocation and allocation of metals, correlation coefficient is an important statistical tool to determine the phytoavailability of different metals in soils. Soil chemical composition and the plant species are the two most important factors which determine the phytoavailability of heavy metals (Kumar et al., 2015). Our study did not reveal any significant correlations between the soil pH and levels of the heavy metals studied. This is consistent with the previously published data that does not show any correlation between the soil pH and levels of heavy metals in soil (Conesa, Faz, & Arnaldos, 2006). Puschenreiter and Horak (2000) (2000) and Zhu, Smith, and Smith (2001) who demonstrated an excessive accumulation of P in the shoots of cereals during a deficiency of Zn in the soil.
Cu, Zn, and Pb levels in the aboveground biomass of the grasses did not exceed the permissible levels of these elements in plants (Boularbah et al., 2006;Kabata-Pendias & Pendias, 2001). According to Kramer (2010), critical toxicity level for Cu is 20-30 μg/g, for Pb-0.6-28 μg/g, and for Zn-100-300 μg/g. The investigations did not find any significant correlations between Cu levels in the soils and

| CON CLUS IONS
All the grass species under study can thus be regarded as accumulators of Cu and Zn because the levels of these elements in the aboveground biomass of the grasses were higher than in the soil beneath these grasses. The present study demonstrates that the grasses can accumulate a large amount of Cu and Zn from soils and transfer it to the aboveground biomass. Tested species of grasses are not a higher bioaccumulators for Pb. The best grass species for the sowing of roadsides embankment, with the highest BCF values for the studied metals, is L. perenne (Taya variety).

ACK N OWLED G M ENT
The research was financed by the Ministry of Science and Higher Education for the Dissemination of Science (766/P-DUN/2019).

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data are available in the manuscript.