Determination of Total Chromium and Chromium Species in Kombolcha Tannery Wastewater , Surrounding Soil , and Lettuce Plant Samples , South Wollo , Ethiopia

This research paper deals with the determination of total chromium (total Cr), Cr(III), and Cr(VI) in Kombolcha leather industrial wastewater and the surrounding (soil and lettuce plant) samples where the wastewater flows. The levels of total Cr, Cr(VI), and Cr(III) in wastewater, soil, and lettuce plant samples were determined by FAAS, UV/Vis spectrophotometer, and difference (Cr(VI) from total Cr), respectively. Among all samples taken, the maximum amounts of total Cr, Cr(III), and Cr(VI) were obtained at the discharging point and the minimum amounts of total Cr and Cr(III) were found downstream (400 m from the junction) of Kombolcha leather industrial wastewater. On the other hand, the minimum concentration of Cr(VI) was obtained in lettuce plant sample. The amounts of total Cr in all samples except soil sample were above the permissible limit as set by WHO/FAO. The concentrations of Cr(III) in all wastewater samples were above the permissible level, whereas the concentration of Cr(VI) in wastewater was above the permissible level except 400 m from the junction. The result showed that a remarkable elimination of total Cr and Cr species has not been achieved by this leather industry as its level was not much decreased when entered into the water systems. Therefore, effective treatment methods should be applied to the wastewater for the wellbeing of the surroundings.


Introduction
The release of huge amount of industrial toxic waste products to the environment has detrimental effects on humans and animals [1].Waste toxicity and postdisposal behavior, poor planning, poor treatment processes, improper disposal, and poor management of disposal sites are the main reasons of serious contamination problems at industrial and hazardous waste disposal areas [2,3].Electroplating and leather industries are the major cause for high amount of chromium in the biosphere.This huge quantity of chromium salts discharged as tannery waste has raised several ecological concerns [4].
Chromium occurs in the environment in different forms under various chemical, physical, and morphological conditions.Under these conditions, Cr shows oxidation states from 0 to 6 in the environment.Among those oxidation states, only Cr(III) and Cr(VI) are stable [5][6][7].The total mean concentration of chromium ranges from 7 to 150 ppm [8].However, its content and distribution in the soil mainly depend on the type of the soil's mother rock.The presence of additional amounts of Cr in the soil is caused by human activity.Chromium takes place as Cr(III) cation only in a strongly acidic and reducing medium, while Cr(VI) occurs in a strongly basic and oxidizing medium as CrO 4 2− anion [9][10][11][12].Because of the tendency of Cr(III) to bind to cell walls of plants, it is mainly concentrated in roots [13].Cr(VI) is the most available form of Cr species and very unstable form under normal soil conditions.However, its availability depends on soils properties and especially on soil texture and pH.Contents of Cr in plants have recently received much attention due to the knowledge of this essential micronutrient in human metabolic processes and its carcinogenic effects.Thus, an adequate rate of nutritional Cr has become an important issue [14].
Kombolcha is located 252 km from the capital of Addis Ababa, Ethiopia.Kombolcha tannery was built in 1967 in Kombolcha town, South Wollo Administrative Zone, Amhara regional state.The town has a latitude and longitude of  The processing wastes are treated with only a classical method of waste treatment mechanism before being discharged to the surroundings.The classical methods are adsorption of waste by plants as it passes on the watersheds.No work has been done ever before on this leather industrial waste.This industry is located at the riverside of Luyelye and discharges its waste to this river.This river water also enters into a big river Borkena.This may potentially risk the lives of humans and animals that are directly or indirectly dependent on these river waters.Therefore, the purpose of this research is to determine the concentrations of total Cr and Cr(VI) in wastewater, soil, and lettuce plant samples using FAAS and UV/Vis spectrophotometer whereas the concentration of Cr(III) was done by difference (concentration of Cr(VI) from total Cr).

Sampling Site.
The study was carried out at Kombolcha town which is found in Amhara region of South Wollo Zone, Ethiopia.The required wastewater, soil, and plant samples were collected from the areas closer to the leather industry.

Sample Collection and Preparation
2.2.1.Water Sampling.The wastewater samples were collected from four different places: at discharging point (KW1, Figure 1), at junction (KW2), and after the junction at a distance of 200 m (KW3) and 400 m from the junction or downstream (KW4), where KW is Kombolcha wastewater.Sampling time was based on the discharging time of the tannery wastes from the industry.Then, the samples were collected at 6 : 30 AM in the morning using 500 mL high density polyethylene bottles and preserved with 2 mL of HNO 3 .Then, the samples were immediately transported to University of Gondar chemistry laboratory by using an ice bag and finally the wastewater samples were filtered and ready for digestion.

Plant Sampling.
Fresh lettuce plant samples were collected from the farmland of the areas nearly 2.5 km far from Kombolcha leather industry.To make the samples representative three subsamples (1 kg) from each sampling site were taken from the irrigated farmland.This farmland was randomly chosen from the three triangular corners of the area.One kilogram of fresh plant samples was collected from the farmland and put in labeled clean polyethylene plastic bags and brought to the laboratory by an ice bag.The sampling sites of plants and soils are represented in Figure 2.

Soil Sampling.
The soil samples were collected exactly at the same place as the plant samples were grown (15-20 cm depth).Sampling was done similar to the plant.Halfkilogram soil samples were collected from the farmland and put in labeled clean polyethylene plastic bags and brought to the laboratory by an ice bag.The soil samples were mixed together and homogenized to form one composite sample.Then the composite sample was air dried for three days and sieved through a 2-mm sieve to remove large debris, stones, and gravels.Finally, the sieved samples were ground using a mortar and pestle to pass a 500 m sieve and ready for digestion.

Validation of the Method.
The metal concentration and speciation were determined at optimum conditions of ratio of acids mixtures, time of digestion, and temperature by varying one condition and keeping others constant.Then, the determination of the concentration of total Cr, Cr(III), and Cr(VI) was done using the optimized parameters.The spiking experiments were analyzed by using standard reference materials.The validations of the method were done by adding 1 mL of reference standard material to wastewater, lettuce, and soil samples.The digestion was carried out side by side with the sample digestion.For the case of Cr(VI) on the above spiked samples 1 mL of prepared diphenyl carbazide solution was added and a red violet color was immediately developed.
From the absorbance reading the amount of Cr(VI) was obtained using calibration curve.

Digestion of Lettuce Plant and Soil Samples.
Applying the optimized condition, 0.5 g of air dried and homogenized lettuce plant and soil samples were transferred into 250 mL different round bottom flasks separately.Then, 7 mL of a mixture of HNO 3 (69-72%), HClO 4 (70%), and H 2 O 2 (30%) with a volume ratio of 4 : 2 : 1 (v/v) was added to the lettuce plant sample, whereas 6 mL of modified aqua-regia (3 : 1 : 0.5) ratio of HCl (36%), HNO 3 , and H 2 O 2 was added to the soil sample.Then, the mixture was digested on a Kjeldahl digestion apparatus fitting the flask to a reflux condenser.The optimum temperature and digestion time for lettuce sample were 270 ∘ C and 2 hr and for soil sample were 270 ∘ C and 3 hr.The digested samples were allowed to cool to room temperature for 10 min.To these cooled solutions 15 mL of distilled water was added to dilute the acid concentration.Then, the solutions were filtered with Whatman filter paper (110 mm, diameter) into 50 mL volumetric flask.The round bottom flask was rinsed subsequently with 5 mL distilled water until the total volume reached around 45 mL.To this final solution 2% HNO 3 was added and filled to the mark.The digested solutions were carried out in triplicate for both samples.Digestions of reagent blanks for both samples were also performed in parallel with the samples.The digested samples were kept in a refrigerator for 12 hr.Then, the amount of total Cr in the sample solutions was determined [15].

Digestion of Wastewater
Samples.50 mL of each of the filtered water samples was transferred into different 250 mL round bottom flasks.To this sample 10 mL of aqua-regia (3 : 1) ratio of 37% HCl to (69-72%) HNO 3 and 2 mL of H 2 O 2 (30%) were added and the mixture was digested on a Kjeldahl block digestion apparatus fitting the flask to a reflux condenser at the optimized conditions (with programmed temperature at 300 ∘ C for 2 hr until 10 mL of the water sample remains).The next steps were similar to the steps applied in plant and soil samples.The digested samples were kept in a refrigerator for 12 hr, until the levels of the total Cr and Cr(VI) were determined [16].

Analysis of Wastewater, Plant, and Soil Samples for
Total Cr and Cr(VI) Levels.The instruments were calibrated using four series of working standards for FAAS (BUCK SCIENTIFIC, MODEL 210 VGP, USA) and five for UV/Vis spectrophotometer (Intech-Model-I-290, USA).The working standard solutions of Cr were prepared freshly from 1000 ppm stock solution (Buck Scientific, 99.99%) by diluting the intermediated standard solution (25 mg/L).The instrument was calibrated using five series of working standards for UV/Vis spectrophotometer.A solution of 1 mL of diphenyl carbazide was added to each sample and working standard solution.Similarly, the pH of the spiked sample solution was adjusted to the desired value (pH = 1) at which the recovery of Cr(VI) is the highest.Then the equilibrium concentration of Cr(VI) in the organic phase was determined spectrophotometrically by measuring the absorbance of Cr(VI)-DPC (diphenyl carbazide) complex at 540 nm [17].

Results and Discussion
3.1.Recovery of the Method.As shown in Tables 1 and 2, the results of percentage recoveries of total Cr and Cr(VI) for wastewater, lettuce plant, and soil samples were within the acceptable range [18].The average percent recoveries were 98.1 ± 6.3 for wastewater, 94.1 ± 2.81 for lettuce plant, and 94.6 ± 4.1 for soil samples using FAAS.Therefore, this verifies that the optimized digestion procedure was valid for wastewater, lettuce plant, and soil sample analysis.The average percent recoveries of Cr(VI) for wastewater, lettuce plant, and soil samples were 94.64 ± 3.34, 91.00 ± 2.81, and 92.40±7.63,respectively.This verifies that the procedures were valid for wastewater, lettuce plant, and soil samples analysis.

Determination of Total Cr, Cr(VI), and Cr(III).
In this study, the total Cr concentrations in wastewater, soil, and plant samples at different sites of the industry were determined by FAAS.Significant amount of total Cr was released at the discharging point.The main source of total Cr in the nearby water system comes from the tanning effluent.This is because even after the wastewater joins the river water the concentration of total Cr was decreasing slightly.The effluent was much concentrated with total Cr at the discharging point and decreased as it flows down from the discharging point.
In the analysis of Cr(VI) using UV/Vis, a sharp peak was observed at wavelength of 540 nm due to absorption of light by Cr(VI)-DPC complex.The influences of pH on the signals of Cr(VI) in all samples were obtained in the range of pH 1 to pH 4. The results showed that the highest absorbance was obtained at pH 1.Therefore, pH 1 was chosen as the optimum pH value for all measurements.The high load of green coloration of the sample was due to the presence of high Cr(III) aqua complex.The concentration of Cr(III) was calculated by the difference of Cr(VI) concentration from the total Cr content.The distribution of total Cr, Cr(VI), and Cr(III) concentrations in the wastewater, soil, and lettuce plant samples at different sites and different distances is presented in Table 3.
The maximum and minimum concentrations of total Cr, Cr(III), and Cr(VI) were 823.87 ± 13.62 and 1.24 ± 0.4821; 821.86 ± 13.569 and 1.207 ± 0.4772; and 2.014 ± 0.051 and 0.033 ± 0.0049, respectively.The maximum concentrations were obtained at the discharging point of wastewater sample while the minimum was found at the downstream water.The reason for the maximum value at the discharging point was due to the use of high amount of chrome salt.Besides the concentrations of total Cr, Cr(VI), and Cr(III) were dramatically decreased from the discharging point to the junction point.These might be due to adsorption of wastes by various plants in the watershed and dilution of the waste with river water [19].
From the total Cr concentration it was found that Cr(VI) accounts for 0.24-2.66%,while Cr(III) accounts for 97.34-99.76% of the pollution status of the wastewater.The reason for the increased Cr(VI) concentration in the wastewater compared to its total Cr concentration might be due to the pH (acidity) of the waste.The pH of the wastewater samples measured was found to be 3.6 to 4.7 before and 5 to 5.8 after junction.This showed that the wastewater was acidic which contributes to the increased accumulation of Cr(VI) in the study areas.It is because Cr(VI) is more stable and exists in much proportion in the acidic medium as HCrO 4 − (1 < pH < 7).Similarly, the concentration of Cr(VI) was found to be 0.075% in soil and 0.73-1.22% in plant samples.As a result, Cr(III) is widespread in all samples compared with Cr(VI) which is consistent with the one described by Onchoke and Sasu [16].

The Potential Pollution Effect of the Kombolcha Tannery
Wastewater.The determinations of the levels of total Cr, Cr(VI), and Cr(III) in the wastewater, soil, and plant samples around the area of the leather industry were performed to assess the influence of the tannery effluent on the surrounding environment.The levels of both Cr(III) and Cr(VI) were decreased from discharge point to downstream wastewater samples as shown in Figures 3 and 4. The distribution of both Cr(III) and Cr(VI) in the wastewaters follows the order KW1>KW2>KW3>KW4.The concentrations of Cr(VI) in plant and soil samples were much lower than Cr(III).This is due to the fact that chrome (Cr(III) salts) is used which contains about 25% Cr 2 O 3 with 33% basicity [20][21][22].Basic chrome sulphate liquor is also used which is prepared by reducing the Na/K dichromate in the presence of H 2 SO 4 [23] and also in addition to this in the soil Cr(VI) interacted with organic matters and reduced into Cr(III).As a result, the concentration of Cr(III) is extremely higher than that of Cr(VI).
ANOVA also showed that mean concentrations of total Cr and chromium species at the discharging point in wastewater sample were statistically significant ( = 0.05) when compared with other samples (from junction to downstream wastewater, soil, and plant).However, the post hoc descriptive analysis showed that the mean concentrations of total Cr and chromium species had no significant differences among all samples except KW1.
The results obtained in this research were compared with other reported values.The amounts of total Cr at the discharging point of Kombolcha tannery wastewater were higher than the values described by Homa et al. (19.126-39.696mg/L) [15].The concentration of Cr(III) is also extremely higher than the reported value by Homa et al. (19.126-39.374mg/L) [15].Moreover, the amount of Cr(VI) obtained in this study at the discharging point was higher than that of Homa et al. (0-0.322mg/L) [15].In the downstream case, all chromium species were lower than other reported values.The results of this study were also compared with the maximum permissible limit (0.1 mg/L) set by WHO.The total Cr values in all wastewater samples were higher than the maximum permissible limit (0.1 mg/L) set by WHO/FAO.
The value of total Cr (22.6 mg/kg) in the soil sample of this study was much lower than the values reported in Homa et al. (254.25-1581.66mg/kg) [15] as well as the permissible limit (100 mg/kg) by WHO and FAO.The total Cr, Cr(III), and Cr(VI) which were obtained in the lettuce plant samples were also lower than those reported by Homa et al. (7.4163-10.473, 7.3913-10.36,and 0.025-0.113mg/kg, resp.)[15], but total Cr was higher than the permissible level (1.3 mg/kg) by WHO and FAO.
Generally, chromium and chromium species released to the environment have detrimental effects on human and animal hygiene and also are not easily degraded or removed from the environment.Some of them are gastric cancer, disability, genetic modification, and kidney and liver problems [24][25][26].This study shows that huge amounts of chromium and chromium species were released to the surrounding river water, irrigated land, and plants.Even if the river water dilutes the waste, it does not mean that it is harmless.This is because it is accumulated in the environment which is due to its interaction with different substances and found either as a precipitate or in the food chain.

Conclusions
The levels of total Cr, Cr(VI), and Cr(III) in tannery wastewater, soil, and lettuce plant samples were determined by FAAS and UV/Vis and through difference, respectively.The total Cr and Cr species determined in the sample collected from the discharging point were much more concentrated than the downstream sample (at a distance of 400 m from the junction point).The concentration of Cr decreases from discharge point to downstream sample.The decrease in concentration was due to adsorption by various plants in the watershed classical treatment method and dilution as the wastewater joins the river.The amounts of total Cr and Cr species were beyond the permissible limit set by WHO and FAO.The amount of total Cr, Cr(VI), and Cr(III) which was entering into the river was actually not too much as compared to its level in the discharge point of the specified site.But, the result showed that a remarkable elimination of total Cr, Cr(VI), and Cr(III) has not been achieved by this leather industry as its level was not much decreased when entered into the water systems.Since the discharged chromium wastes entered to the nearby Kete River this in turn caused the contamination of the big river Borkena.As a result, this contamination can increase the accumulation of chromium in the river water and the surroundings and bring a potential adverse impact to the population around it.Therefore, effective treatment methods should be applied to the wastewater to protect the ecosystem.

Figure 1 :
Figure 1: Wastewater at the discharging point.

Figure 2 :
Figure 2: The place where the plant and soil samples were taken.

2. 7 .
Statistical Analysis.Statistical analyses were carried out by one-way ANOVA of Tamhane and LSD comparison post hoc tests.Significant differences were accepted at  < 0.05.The analyses were performed using SPSS Statistics software (version 16.0 for Windows 7).

Figure 3 :
Figure 3: Total Cr concentration in wastewater ((a) = average concentration of all wastewater samples; (b) = average concentration from junction to downstream wastewater samples).

Figure 4 :
Figure 4: Comparison of the concentration of Cr(VI) and Cr(III) in the wastewaters ((a) = average concentration of all wastewater samples; (b) = average concentration from junction to downstream wastewater samples).

Table 1 :
Recovery test of total Cr by FAAS for tannery wastewater, soil, and plant samples.

Table 2 :
Recovery test of Cr(VI) using UV analysis for tannery wastewater, soil, and plant samples.

Table 3 :
Distribution of total Cr, Cr(VI), and Cr(III) concentration (mean ± SD; mg/L for wastewater samples and g/g for soil and plant samples) in different sites of Kombolcha leather industry.
a = mg/L for wastewater samples; b = g/kg for soil and plant samples.