Phytoremediation Using Bamboo to Reduce the Risk of Chromium Exposure from a Contaminated Tannery Site in Kenya

Background. This study examines an intervention strategy to reduce the risk of chromium (Cr) exposure. It follows a previous Cr exposure investigation, which revealed that large volumes of Cr-contaminated waste were burnt on site. The study site had a long history of land-based waste disposal since 1994. Objective. The potential for phytoremediation using bamboo species to restore Cr-contaminated soil was evaluated. Methods. Chromium levels and physico-chemical properties of the tannery and control soils were analyzed before transplanting six different bamboo species. Translocation, bio-concentration and bioaccumulation factors of the species were assessed for phytoremediation capabilities. Results. Chromium levels in the tannery soils ranged from 1337.0 to 3398.0 mg/kg dw. The chromium levels were significantly higher (P < 0.05) than those of the control soils (0.20 to 2.34 mg/kg dw) and markedly exceeded the recommended limit of 100 mg/kg dw. The physicochemical properties of the tannery soils were also significantly varied (P < 0.05) compared to the control soils. In all cases, the species grown in the tannery soils were tolerant to a wide range of prevailing conditions. All of the bamboo species in the present study had a 100% survival rate in the tannery soils, except for D. birmanicus, which had a survival rate of 83.3%. Moreover, growth performance of the species in the tannery and control soils as evaluated by height and clump diameters did not vary significantly (P > 0.05). However, Cr levels in the tannery differed significantly (P < 0.05) among the species and rhizosphere soils. D. asper, B. vulgaris, D. membranaceus and B. blumeana had a bio-concentration factor (BCF) > 1 and translocation factor (TF) < 1, indicating that they are suitable for phytostablization. On the contrary, B. bambos had a bioaccumulation factor (BAF) < 1 and TF > 1, indicating potential for phytoextraction, while D. birmanicus showed no potential for phytoextraction or phytostabilization. Conclusions. The present study identified D. asper, B. vulgaris, D. membranaceus and B. blumeana as suitable for restoration of Cr-contaminated tannery sites. Close monitoring of toxic metals is necessary during application of these species. Further studies are also recommended using a wide variety of bamboo species to optimize their application in phytoremediation.


Introduction
Leather processing is one of the most important sources of chromium (Cr) exposure. 1-8 This is because basic chromium sulfate is not fully utilized during the tanning process. Its uptake is estimated to vary between 55 and 70%. 1-4 The residual amount usually escapes in aqueous effluent. Major exposure pathways include uncontrolled disposal of tannery wastes that result in degradation of the environment over a period of time. 1-4 Land-based disposal of Crcontaminated sludge from a poorly managed effluent treatment plant is the most significant source of exposure. 1-3 For instance, in India, between 2000 and 3000 tons of Cr is discharged into the environment annually from tanneries. 1, 4 Indiscriminate release of Cr in soil and water resources is therefore a serious public health concern. 1-8 Recent studies in tanneries by Were et al. 3 found that huge volumes of waste are dumped in open fields. The dry solid wastes are burned, thereby contributing to elevated levels of airborne Cr within the tannery and its environs. Under those conditions there is a possibility of oxidation of trivalent chromium (Cr (III)) which is less harmful than the more toxic hexavalent chromium (Cr (VI)). 8 Studies have further indicated that Cr in soil usually presents a combination of both Cr(III) and Cr(VI) as it Research undergoes a series of transformations such as oxidation, sorption, precipitation and dissolution. 7, 8 Oxidants such as dissolved oxygen and manganese dioxide (MnO2) are capable of converting Cr(III) to hexavalent Cr. 8 Several investigations have detected considerable levels of toxic Cr(VI) in the surface water as well as groundwater. 1,4,8 Moreover, at a higher pH value, Cr (VI) is more bio-available than Cr (III). It is in this form that Cr is highly oxidizing, soluble, and mobile and poses the greatest risk to human health and the environment. 3,4,8 Hexavalent chromium is a powerful epithelial irritant and is also considered to be a human carcinogen. [1][2][3][4][5][6][7][8] The common practice of dumping of Cr-contaminated waste in open fields and the subsequent discharge of Cr into water resources requires stricter controls. 1-8 There are several techniques that have previously been applied as a strategy to clean up sites contaminated with heavy metals, including remediation by use of chemical and thermal methods. 6,9 Most of these methods pose technical difficulties in attaining optimum results and require large financial investments. 6 Furthermore, the usual methods of excavation and subsequent disposal of contaminated waste to dumpsites have the potential of spreading and shifting these contaminants to other locations. 1,3,9 Phytoremediation is an economically feasible method that uses plants, which have exceptional metalaccumulating capabilities, to restore contaminated sites. 1,6,11, 12 The method is less disruptive to the environment and more acceptable to surrounding communities. 6,9 There are a large number of plants that contribute towards heavy metal removal and demonstrate good potential for application in a remediation progam. 8,9 These plants have unique and selective metal uptake abilities where they bioaccumulate, translocate or degrade contaminants. 6,9,11, 12 Bamboo species usually thrive in toxic environments with minimal maintenance and produce a large amount of biomass. 10- 12 They are usually harvested from two years onward for their diverse applications. 10- 12 In this study, six bamboo species were selected for their heavy metals accumulation and translocation potential to restore Cr-contaminated tannery soil. The establishment of bamboo species on the contaminated site is expected to stabilize the soil with extensive root systems and prevent wind and soil erosion. This will prevent the risk of spreading Cr to other areas; thereby reducing associated adverse health effects. [1][2][3][4][5][6][7] This study is an intervention strategy to reduce the risk of Cr exposure, following a previous Cr exposure assessment, which revealed large volumes of Cr-contaminated tannery waste that was subsequently burnt in the open, contributing to elevated levels of Cr exposure. 3 The tannery workers were found to have significantly reduced lung function, as well as respiratory and dermatological complaints associated with high levels of Cr exposure. 3 The study is therefore part of comprehensive ongoing research activities aimed at reducing the risk of Cr exposure in tanneries.

Methods
The study began in January 2015 and ended in January 2017.

Site Description
The study area was a Cr-contaminated site within the selected tannery, on an acre of land in close proximity to residential areas. 3 Its geographical coordinates are 1° 17' 0" South, 36° 42' 0" East. The site has had a long history of land-based disposal of waste from the tannery since 1994. On average, the annual consumption of chromium sulphate in the tannery is about ten tons. 3 Chromium sulphate utilization during the tanning process was estimated to be at 60%, suggesting that over 4 tons of Cr are discharged on a yearly basis into aqueous effluent. 3 Although the tannery has an effluent treatment plant, it is poorly managed, and the plant is located about 50 m away from the Nairobi River and adjacent to agricultural land. 3 It is therefore a major potential source of Cr migration into soil and water resources. 1-7 Table 1 presents a summary of activities within the tannery that contribute to Cr exposure.
The general topography of the study area is flat, windy, and characterized by low rainfall with well-drained sandyloamy soils suitable for irrigation of food crops, mainly vegetables and maize. 3 There are several scattered boreholes in the area, contributing to the possibility of Cr leaching into the

Research
water. Furthermore, tannery washings are channeled to a nearby farm and the rest could drain into the Nairobi River. 3 It is also plausible that Cr-containing leather dust and fumes are blown and settled as depositions. 3 During rainy seasons, these depositions are then washed by surface runoff, spreading the contaminants further. 3 Use of Cr-contaminated sludge as manure was further observed to be a common practice in the area, and could result in severe cases of contamination of soils, as well as water resources. There are several pathways through which Cr could migrate and accumulate along the food chain. 1-7 The present study investigated the applicability of bamboo species in controlling the leaching and spreading of Cr from contaminated sites to surrounding areas.

Sampling of Bamboo Rooted Cuttings
A total of eighty-four (N = 84) three-month-old rooted cuttings of six different bamboo species: Bambusa blumeana, Bambusa bambos, Bambusa vulgaris, Dendrocalamus asper, Dendrocalamus birmanicus and Dendrocalamus membranaceus were selected for phytoremediation. The rooted cuttings of 30 cm in height were nurtured in black polythene pots with Cr-free soil by the Kenya Forestry Research Institute. These species were selected on the basis of their homogeneity from a larger population by considering their shoot quality and adaptability to being established in the prevailing environmental conditions. 10, 12 Their potential for accumulating heavy metals in order to restore the contaminated sites was also evaluated. 11,12

Sampling of Soils and Transplanting of Bamboo Rooted Cuttings
The tannery site was secured prior to transplanting of the bamboo species. Seventy-two (N = 72) holes of approximately 60 cm in diameter and 30 cm in depth sufficient to accommodate the bamboo rooted cuttings, were dug at each of the sampling points. 11 Holes were spaced 2 m apart in order for the bamboo species to cover the area that was under direct influence of land-based disposal of Cr contaminated waste. Following the same procedure, twelve (N = 12) holes were dug in a garden, which served as control samples. Rooted cuttings of each of the 6 selected bamboo species were then transplanted in a row and watered with an equal amount of Cr-free water. Each hole was re-filled with the same soil, which was also collected in triplicates in a clean polythene bag for determination of Cr level and physicochemical properties. Each soil sample was considered as a representative of growing media for the corresponding bamboo species that was planted. The Research bamboo plant was then separated into respective organs composed of roots and shoots (stems and leaves). They were then washed in water and rinsed thoroughly with deionized and distilled water in order to remove soil particles and debris. 15, 16 The plant materials were subsequently chopped into small pieces using a stainless-steel knife. The samples were dried at 70°C to a constant weight. They were then milled in a cyclone mill to a particle size of 0.3 mm. 9, 15 The samples were packed in zip lock polythene bags, and stored in desiccators prior to chemical digestion.

Table 1 -Description of Major Activities in the Tannery Associated with Chromium Contamination of the Tannery Site
Similarly, soil samples were air-dried at 70°C until a constant weight was attained. Any clods and clumps were removed and samples were mixed homogeneously. 15, 16 The samples were then sieved through a 2-mm sieve to remove coarse particles prior to chemical digestion.

Chemical Digestion of Plant Materials and Soil Samples
A sample of 0.5000 g of air-dried pulverized plant materials were digested using 7 mL of concentrated nitric acid (HNO3): 1 mL hydrochloric acid (HCI) (7:1) in a fluorocarbon polymer (PFA/TFM) closed system microwave. The vessel liner was equipped with an extraction fume system (Anton Paar, Australia). The microwave unit was equipped with a quartz power system (1400 W). After cooling the vessel, the clear liquid was diluted to 50 mL in acid-washed vials.
Dried ground soil samples of 1.5 g were transferred to the 100 mL digesting tubes. This was followed by addition of aqua regia, a mixture of 14 mL concentrated HNO 3 and HCl, 70% (Fisher Scientific, UK) in a ratio of 1:3. The tubes were covered by a funnel and digestion at 140°C was carried out in a fume chamber using a digestion block (Gerhardt, Germany). This was heated until about 4 mL was left in the tube. The procedure was repeated by adding a further 14 mL of aqua regia and allowed to evaporate to a volume of about 4 mL. After cooling, the solution was filtered through Sartorius membrane filters (3 hw). The filtrate was then made up to a volume of 25 mL with de-ionized and distilled water prior to analysis of total Cr. Laboratory blanks were prepared in the same way by having all the components added during the digestion process without the corresponding plant materials or soil samples. All the digested samples including the laboratory blanks were then taken for the spectroscopic analysis to determine the levels of total Cr.

Analysis of Total Chromium Levels
The plant materials, soil and blank samples were analyzed for levels of total Cr instead of Cr(VI) using inductively coupled plasma optical emission spectrometry (ICP-OES). This is due to the interconversion of Cr(VI) and (III) during sample preparation and chemical analysis, which poses an analytical challenge for the determination of Cr(IV) levels. 3. 8 Total Cr levels were therefore determined in the soils before transplanting bamboo species, and those of the corresponding rhizosphere soils, roots and shoots after two years of growth period. The levels were expressed in milligram per kilogram of dry weight (mg/kg dw) of the respective samples.

Quality Assurance and Control
Samples were analyzed using adequate quality assurances and controls (QA/QC) to determine the reliability and accuracy of the results. Precautions were taken to avoid external contamination of the samples. All reagents used throughout the analytical procedure were of high purity analytical grade. Glassware was soaked in 0.5% (v/v) of HNO 3 and species were thereafter rain fed and maintained in a natural environment.

Determination of Physico-chemical Properties of the Soil
Soil samples that were collected from the sampling points prior to transplanting bamboo species in the tannery and control sites were assessed for Cr content as well as physico-chemical properties. The properties included the pH, electrical conductivity (EC), moisture and organic matter content. 13 The pH and EC were determined by vigorous mixing of 10 g of soil and 50 mL of water. A deionized water ratio (w/v) of 1:5 allowed soluble salts to dissolve and the ionic exchange to reach equilibrium. Then the pH and EC were determined using digital meters with a combination of a pH electrode and a 1-cm platinum conductivity cell. Moisture content was calculated by the mass difference before and after drying of soils samples at 105°C to a constant mass. 13 The organic matter was expressed as a percentage (%) of carbon and calculated using the Walkley-Black potassium dichromate wet oxidation by the titration method. 14 Particle size distributions were analyzed using dry sieving techniques. 13

Growth Performance of Bamboo Species and Sample Preparation
Height and clump diameter of the bamboo species were measured using a clinometer and diameter tape, respectively to evaluate growth performance after two years of transplanting. 10- 12 The clump diameter was determined by measuring the distance covered by a cluster or group of stems of bamboo growing from a common underground rhizome system. Subsequently, the whole bamboo plant and corresponding soil samples at 0 -30 cm depth of rooting zone were carefully collected from each sampling point. Each Research rinsed several times with distilled and de-ionized water prior to use.  ), which was acceptable when r 2 > 0.995. The validity of the method was further ascertained by cross method checks and replication analysis. All samples were analyzed in triplicates and the average of the determinations was taken when the relative standard deviation was less than 5% to establish reliability of the results.

Statistical Analysis
The samples were analyzed in Were, Wafula, Wairungu   Research triplicates and the data obtained was then reported as mean ± standard error (SE) using the Statistical Package for the Social Sciences (SPSS) program (version 17.0, SPSS Inc, Chicago, Illinois). Independent Student's t-test was used to compare parameters from the control and tannery site. Statistical analysis of variance (one-way and twoway analysis of variance (ANOVA)) at P < 0.05 was used to compare variables between and within the groups.

Accumulation and Translocation of Chromium
The capability of six different bamboo species to take up Cr from tannery contaminated soils was evaluated by determining the bioconcentration factor (BCF), translocation factor (TF) and bioaccumulation factor (BAF). The BCF was defined as the ratio of Cr levels in the roots to that of the rhizosphere soil of the bamboo species, while TF was the ratio of levels of Cr in the shoots to that of the roots of bamboo species. The BAF was calculated as the ratio of levels of Cr in the shoots over that of the rhizosphere soil of the bamboo species. Bamboo species with a high BAF value (BAF > 1) are suitable for phytoextraction, while those with BCF (BCF > 1) and low TF (TF < 1) have the potential for accumulation of Cr in the roots, also known as phytostabilization. It should also be noted that a TF > 1 indicates that the species has the ability to translocate Cr from the roots to the aerial parts. 8 With regard to physico-chemical parameters of the soils, the pH values for the tannery soils (N = 70) varied from slightly acidic to alkaline (6.70 -8.60), but the mean values for the six bamboo species were not significantly different (P > 0.05). On the contrary, the pH values for the control soils ranged from slightly acidic to neutral (6.10 -7.00). Furthermore, the tannery soils had significantly higher (P < 0.05) electrical conductivity (EC) than the control soils.   Table 3. However, no significant interaction (P > 0.05) was found between bamboo species and treatment type.  Moreover, the levels of Cr translocated in the aerial parts of the bamboo species in the garden soils varied slightly from 0.23 -0.98 mg/kg dw, although the differences in the levels were not significant (P > 0.05). Table 5 presents the results of a two-way ANOVA on the effect of bamboo species and treatment type on the dependent variables. There was a significant (P < 0.05) influence between bamboo species and treatment type on levels of Cr in the rhizosphere soil, roots and shoots, while clump diameter did not show significant interaction (P > 0.05) between bamboo species and type of treatment.   Table 5 Effects of Bamboo Species and Treatment Type on the Dependent Variable. All Two Years of Growth.

Table 4 -Summary of Growth Performance and Chromium Levels in Rhizosphere Soils, Roots and Shoots of Bamboo Species-Two Years of Growth Independent Student's t-test compares parameters of the tannery and control soils; P-value < 0.001 indicates a significant difference between the control and tannery variables; mean values followed by the same small letter within the same row do not differ significantly (One -Way ANOVA, SNK-test, α = 0.05) Abbreviations: SE, standard error
Two-Way ANOVA, (P < 0.05) indicates significant interaction between bamboo species and treatment type.

Discussion
The present study found that levels of Cr in the soils were greatly varied and dumping of Cr-bearing waste from the tannery was a major source of Cr exposure. The levels of Cr in the tannery soils ranged from 1337.0 -3398.0 mg/kg dry weight (dw) and were significantly higher (P < 0.05) than those of the garden soils (0.12 -2.15 mg/kg dw). The former levels markedly exceeded the recommended limit of 100 mg/kg dw. 2, 15 This result is not surprising as the tannery site has been under the strong influence of land-based disposal of Cr-containing waste since 1994. The presence of higher levels of Cr in the tannery soils is an indication of the risk of exposure to humans as well as the environment. 1-8,15, 16 The residence time of Cr in soils is estimated to vary between 1000 and 10,000 years. 2 The contaminated site in this case acts as a secondary pollution source for Cr, which is under the influence of several factors such as surface runoff, wind and soil erosion that is capable of spreading Cr contaminants to other areas. These findings are in agreement with those of Stępniewska and Bucior 15 who reported significant levels of Cr contamination in soils, water and plants that were in close proximity to a tannery waste lagoon. As previously mentioned, most of the tanneries are located in ecologically fragile zones in the vicinities of residential areas, water sources and agricultural land. 1-3 Quite a number of studies have revealed that toxicities of Cr are dependent on its speciation, and Cr(VI) is highly toxic, oxidizing, and more mobile and soluble than Cr (III). 3,4,8, 16 Similarly, levels of Cr that are bioavailable in soils are strongly linked to pH as a function of solubility and electrical conductivity among other physico-chemical properties of the soils. 8 Trivalent Cr is easily converted to Cr(VI) when the pH value is greater than 6.0. 8, 16 In the present study, the pH values varied from 6.1 -7.0 and 6.7 -8.6 for the control and tannery site, respectively. The prevailing conditions of the soils therefore seemed to favor the transformation of Cr(III) to Cr(VI). However, the actual mechanism involved in accumulation and translocation of Cr in plants as well as the associated soil chemistry is complex given that Cr(III) is nonessential in plants. 8 It should be emphasized that the availability of Cr(III) is a health risk due to possible conversion to Cr(VI). Symptoms of Were, Wafula, Wairungu Overall, the species were healthy and the average growth performance evaluated by height and clump diameter between those grown on the tannery and control soils did not show a significant difference (P > 0.05).
The present study also revealed a notable reduction of Cr levels from the tannery soils after a two year growth period for the bamboo species. High variabilities in the levels of Cr accumulated and translocated in various parts of the bamboo species as well as in their corresponding rhizosphere soils were also observed. The significantly high (P < 0.05) levels of Cr in the roots and shoots were therefore derivatives of Cr from the tannery soils. However, it is difficult to interpret these results and assess the actual amount of Cr that was bioavailable in the soils for uptake by the bamboo species. 8, 16 This is due to the complexity of soil chemistry that involves speciation of Cr and mechanisms for Cr uptake. 8 Nonetheless, the differences in accumulation and translocation of Cr as well as growth performance among diverse bamboo species are very useful data for the selection of suitable species for restoration of Cr-contaminated tannery sites. There are more than 15  It is worth noting that the four species are capable of attaining a height of 30 m after five years of growth. 10-11 In particular, B. vulgaris grown in the tannery soils for two years had an Were, Wafula, Wairungu Research average height of 3.9±0.1 m ranging from 2.8 -6.5 m, and a relatively low mean level of 21.3±3.2 mg/kg dw Cr in the aerial parts. Moreover, the shoots of B. vulgaris are edible and may not pose a risk to animals. Soil removal due to leaf fall and disposal of biomass are also not necessary. 8, 18 The cost and degree of disruption is therefore minimal and at the same time the ecosystem restoration is enhanced. 9 The species is effective in preventing migration of Cr from a highly contaminated site to fragile ecological zones. 11- 12 The main concern is however accumulation of Cr in the roots and possible release to the environment. Longterm maintenance of the species is therefore important for sustainability of the phytoremediation program.
On the other hand, caution should also be taken to prevent the other species from transferring Cr into the food chain through translocation of elevated levels of Cr to the aerial parts. Further research on these species is highly recommended to optimize phytoremediation techniques in restoration of several contaminated sites as there are a number of native and exotic bamboo species in existence in Kenya. 10-12 There are several limitations to the present study. In some cases, large amounts of fallen leaves, bearing Cr as shown in Figures 1 to 4 were not removed, which could have contributed to the Cr burden in the rhizosphere soils upon decomposition. Furthermore, total Cr was quantified in place of Cr(VI), as a result of analytical challenges associated with interconversion of Cr(III) and (VI). In addition, height and clump diameter were used in this study to estimate growth performance of bamboo species as an important factor in phytostabilization strategies, although determination of fresh and dry weight are regarded as the best biometric Were, Wafula, Wairungu