Phytoremediation potential of Amaranthus hybridus L. (Caryophyllales: Amaranthaceae) on soil amended with brewery effluent

Toxicity of heavy metals above the normal threshold constituted a threat to humanity and biodiversity. Phytoremediation has become a novel and emerging technology of cleaning polluted sites through the use of plants. A study was carried out at the screen house located besides the academic building of the Federal University of Technology, Akure, Nigeria, to evaluate the phytoremediation potential of Amaranthus hybridus L. (Caryophyllales: Amaranthaceae) on a brewery effluent. The parameters investigated include chlorophyll content, the concentration of the metals in the plants, Bioconcentration Factor (BCF) and Translocation Factor (TF) was studied. Three different concentrations of brewery effluent were used at 50, 100 and 150 mL/5 kg of soil, respectively. The results of this study under controlled conditions indicate that effluent application increased chlorophyll content, reduced plant height and stem girth. Three heavy metals (iron, cadmium, and chromium) were detected in the shoots and leave of both plants after the experimental period. The translocation factor (less than 1) and bioaccumulation factors (greater than 1) were below the permissible limits hence indicating a possible bio-accumulator for the heavy metals investigated. Brewery effluent reduce the plant height but increase the leaf area of A. hybridus under high concentrations which possibly suggest an adaptive mechanism developed by the plant under stress.


Introduction
There has been an increasing concern with regard to the accumulation of heavy metals in the environment as they pose a threat to both human health and the natural environment (Abioye et al., 2009). This is due to the fact that unlike many substances, metals are not biodegradable and hence accumulate in the environment (Ghanbari- Bonjar and Leel, 2004).
Effluents are wastes produced from industries and they vary depending on the human activities that produce them. Production of these wastes is an integral part of industrial activities but unfortunately, our inability to anticipate the magnitude of undesirable consequences of uncoordinated release of effluents in our environment in connivance with industrial boom have resulted in wanton destruction of our ecosystem.
Higher plants can accumulate heavy metals in different concentrations, but significant differences in metal accumulation exist between and within plant populations. In this study Amaranthus hybridus L. (Caryophyllales: Amaranthaceae) was considered because it is a popular nutritious leafy vegetable crops, rich in proteins, vitamins and minerals, and consumed virtually in the whole continent of Africa, Asia and South America. Their quick growth and great biomass makes them some of the highest yielding leafy crops and therefore suitable for phytoremediation purpose.
The objective of this study therefore was to investigate the effect of brewery effluent on some soil chemical properties and growth of A. hybridus plant and to determine the phytoremediation potential of the plants using heavy metal uptake into roots and stem.

Experimental site
The screen house experiment was carried out in the academic building of the Federal University of Technology, Akure (Latitude (7° 16' N and Longitude, 15° 12' E), Ondo State, Nigeria.

Soil collection
Top soils (0-15 cm) were colleted from Aba Oyo Community of the Federal University of Technology, Akure. The soils were thoroughly mixed by a mechanical mixer and passed through 4 mm sieve to remove fibre and non soil particulate in the sample. The chemical and physical properties of the soils were determined prior to planting.

Seed and effluent collection
The seeds were obtained from International Institute of Tropical Agriclture, Ibadan. Brewery effluent was collected at the international brewery, Ilesha, Osun State, Nigeria.

Experimental design
The experiment was laid out in a completely randomized design (CRD) with five replicates. The brewery effluent was applied at the following rates of 0, 50, 100 and 150 mL concentration per 5 kg soil and later left for 2 weeks to allow for mineralization planting the seeds. The plants were treated with effluent twice a week.

Measurement of growth parameters
Growth parameters were observed and measured every 2 weeks after planting (WAP). This was done for 8 weeks after which the plants were harvested, dried in the oven at 105 °C for 48 h to a constant weight. The growth parameter measured were plant height, leaf area, stem girth, number of leaf and chlorophyll content of the fresh leaf. The chlorophyll content was determined according to the method of Arnon (1949).

Soil analysis
Soil analysis was carried out before and after the experiment. The soil pH was determined at soil:water ratio of 1:1 using glass electrode pH meter. The particle size analysis of the soil was by the method of Bach et al. (1958) as modified by Day and Wong (1965) whereas the organic carbon content of both soil and effluent were analysed by chromic acid wet oxidation method of Fosu et al. (2004). The nitrogen was determined by microkjeldal procedure as described by Fosu et al. (2004). The available phosphorus was extracted with Bray and Kurtz solution 1 (Bray and Kurtz, 1945). Exchangeable acidity was determined by the KCl extraction and titration methods of Maclean (1965). The total dissolved solids and suspended solids in the brewery effluent were analysed by the method of Ademoroti (1996). The alkalinity was determined by the method of Larson and Henley (1955). The electrical conductivity was determined using the model 230 HT CORNING conductivity meter. The Biological Oxygen Demand (BOD) and Chemical Oxygen demand (COD) were determined using the method of Ademoroti (1996).

Digestion of plant samples
The procedure according to Awofolu (2005) was used for digestion of plant sample. 0.5 g of sieved leaf samples were then weighed into 100 cm³ beaker. A mixture of 5 cm³ concentrated HNO3 and 2 cm³ HClO4 were added to dissolve the sample. The beaker was heated at moderate temperature of 110 °C on a hot plate for 1 h in a fume hood until the content was about 2 cm³. The digest was allowed to cool, filtered into 50 cm³ standard volumetric flask and mad e up to the mark with distilled deionized water. A serial dilution method was used to prepared the working standards and the concentrations of the metals in each sample digest were determined using atomic absorption spectrophotometer (Buck Model 210 VGP) equipped with a digital readout system.

Determination of the Bioconcentration Factor (BCF)
The Bioconcentration Factor (BCF) of metals was used to determine the quantity of heavy metals that is absorbed by the plant from the soil. This is an index of the ability of the plant to accumulate a particular metal with respect to its concentration in the soil and is calculated using the formula:

Determination of the movement of metals from roots to plants
To evaluate the potential of plants for phytoextraction, the translocation factor (TF) was used. This ratio is an indication of the ability of the plant to translocate metals from the roots to the aerial parts of the plant. It is represented by the ratio: TF = Metals that are accumulated by plants and largely stored in the roots of plants are indicated by TF values < 1 with values > 1 indicating that the metals are stored in the stems and leaves.

Data analysis
Data obtained were subjected to analysis of variance and means were separated using Turkey's B test.

Results
Effect of brewery effluent on the stem girth of A. hybridus is shown in Figure 1. There were significant differences on the effluent treated plants at 8 WAT and 10 WAT, respectively, under the control and 50 mL brewery effluent (BET). However, the BET does not have significant effect on the stem girth of A. hybridus at 4 WAT and 6 WAT, respectively.    Effect of BET on the leaf area of A. hybridus is presented in Figure 3. There were significant rapid increases in the leaf area of A. hybridus under the 150 mL BET at 6th, 8th and 10th WAT.
Fresh and dry weight of A. hybridus under different concentrations of brewery effluent is shown in Table 1. The dry weight of A. hybridus was observed to decrease gradually as the effluent treatment increseses.
Heavy metal accumulation of A. hybridus root ranged between 0.07 and 0.68 across the brewery effluent ( Table 2). The highest heavy metal accumulation of 0.68 was recorded in iron at 150ml BET while the least was observed in copper (0.07) under the control treatment. A. hybridus shoot metal accumulation showed that the chromium accumulation ranged between 0.20 and 0.48 across the BET. Also the highest metal accumulation was observed in iron (0.55). However, the least was noticeable in copper (0.05) under the control (Table 3).    Bioaccumulation factor (BF) of the heavy metals in A. hybridus under the BET is presented in Table 5. The BF of the heavy metals indicated that Fe ranged between 0.81 and 1.38 while Cu (1.45 and 1.59) and Cr (1.06 and 3.07). Cr accumulated more (1.45) at 150 mL BET the BF was less than 1 in Fe at 50 and 100 mL BET, respectively.
Translocation factor (TF) of heavy metals accumulated in Amaranth under BET is presented in Table 6. The TF of Cu is greater than 1 unlike Cr (0.50) and Fe (0.89) where their TF are less than 1 at 150 mL BET.

Discussion
Increase in soil pH at the highest effluent treatment level in this research agrees with report of Orhue et al. (2005) using rubber effluent as an amendment in an ultisol. The Nitrogen decreased in this research indicates that the use of N by microorganisms for metabolic activities did not in any way reduce the C/N ratio below that of control. Ghanbari-Bonjar and Lee (2004) using cassava effluent reported an increase in soil nitrate. The reduced available Phosphorus at 50, 100 and 150 mL effluent concentration treatment may be due to higher P uptake resulting from favourable soil pH range. The brewery effluent reduces the leaf area of the plant significantly under the 50 mL treatment. Findings from this research are in conformity with Yilmaz et al. (2012), who reported that leaves from plants growing in polluted area were significantly reduced than leaves from an unpolluted area. The increase in leaf area recorded under 150 mL brewery effluents could possibly be due to some adaptive mechanisms employed by some characters in the plant in order to withstand the effluent concentrations. Morphological and physiological responses are integral components of plants under stressed conditions (Adu-Gyamfi et al., 2012). The mechanisms of resistance to toxic chemicals have not been fully explored but could be suggestive of the ability of different plants to detoxify heavy metal or exclude them from the roots. Plant biomass (fresh and dry weight) of Amaranthus hybridus under brewery effluent decreased significantly (Khan et al., 2008). Heavy metals even at low concentrations may result in phytotoxicity by impairing a range of cellular activities and reducing the uptake of other essential nutrients (Khan et al., 2008). Plants respond to effluent stress and their uptake, translocation and accumulation of metabolites vary  depending on their genetic make-up and ecophysiology. There is a likelihood that Amaranthus has a mechanism to detoxify excess Chromium in the vacuole, thus concentration of Chromium in the tissue were all below the WHO/FAO permissible limit recommended in vegetables (WHO, 2007). Transfer factor (TF) of Cu demonstrated greater affinity for the metal by Amaranthus hybridus. Copper, an essential micronutrient is an essential cofactor for several oxidative stress related enzymes (Yilmaz et al., 2012). The bioaccumulation factor represents the ability of A. hybridus to extract heavy metals from the soil. BCF values of zero indicate limited movement from the soil to the plant. According to Ismail et al., 2013, for a plant to be considered for phytoremediation. it should have a few of the following traits to make its use feasible: Ability to extract, degrade or stabilize the contaminant, Tolerance to high levels or concentrations of the contaminant, Rapid growth rate and high biomass production, Cosmopolitan growth and ease for harvesting.
The observation of the growth of the plant over a 10 weeks period indicates that A. hybridus can tolerate high brewery effluent concentrations as indicated by the high BCF index .Metals that are accumulated by plants and largely stored in the roots of plants are indicated by translocation factor values less than 1. Values greater than one indicate translocation to the aerial parts of the plant. However, the ability of A. hybridus to be considered for phytoextraction has to be encouraged, as the Translocation Factor index indicates that only when the Cu is being translocated to the aerial parts of the plant. Findings by Hu et al., (2013) support Cu being concentrated in the roots and not translocated to the aerial parts of plants by determining the uptake and accumulation of Cu. Amaranthus hybridus clearly demonstrates that it can tolerate difficult soil conditions.
In this study, the morphological properties of plants show that brewery effluent application reduces plant height in A. hybridus but increased other morphological parameter such as number of leaves and leaf area. In agreement with the result of this research many researchers observed that at high concentration of numerous effluents such as textile, paper, marble, dairy and brewery along with high osmotic pressure, result in decreased germination . This work contradicts the work of many researchers who documented that under different concentrations of brewery effluent, textile and paper effluents etc. there is improved seedling lengths of various crops (D'Amore et al., 2005). But it has been stated that high amounts of heavy metals in soils hinder plant growth, physiological, metabolic processes and nutrient uptake. (Khan et al., 2008). Lombi and Gerzabek (1998) also documented reduction in a variety of growth parameters of soybean with the application of textile dying effluent at various concentrations. Sposito and Page (1984) found growth promoting effect of treated textile waste when applied at low concentration.
There was a reduction in fresh and dry weight with increase in brewery wastewater concentration. The reduction in fresh and dry weight of A. hybridus is in agreement with Kuo et al. (1983) who confirmed in their report of a reduction in total plant biomass in carbide waste polluted habitats. Similarly, John (2011) also reported the effect of carbide waste polluted soil on the biomass of tomato plants, in which the effects was attributed to reduction in cell expansion and photosynthesis which play a major role in reduction of fresh and dry weight. The result of reduction in fresh and dry weight obtained at relatively higher concentration of brewery wastewater can be explained by Lombi and Gerzabek (1998) that, it is possible that the toxicity of the brewery wastewater could have interfered with the plant metabolic activities leading to poor water absorbance and development.
This study revealed that A. hybridus is a hyperaccumulator of heavy metals (iron, chromium and copper). Three heavy metals were detected in the brewery waste water iron (Fe), chromium (Cr) and Copper (Co). These heavy metals were detected at relatively high concentration in root and shoot of A. hybridus which means that there was a translocation of heavy metal through root uptake which is supported by the work of Hiroaki (2013). These heavy metals were generally lower than suggested maximum tolerable limit (WHO, 1996), this is supported by the work of Basta et al. (2005) who performed an experiment with vegetables irrigated with waste water from different industries in Faisalabad and analyzed the presence of various heavy metals. They found that the concentration of Cadmium, Copper, Chromium, Lead and Zinc was less than the suggested maximum tolerable levels (0.01mg kg-1, 10.00 mg kg-1, 1.30 mg kg-1, 2mg kg-1 and 5.00 mg kg-1 respectively as reported by WHO, 1996). It is important to determine the metal contents of vegetables from health, food nutrition perspective and for crop yield technology point of view. Metal accumulation in edible portions of plants varies and depends on both soil composition and rate of uptake by each plant. For good health and optimum human performance, adequate intake of essential elements and nutrients is crucial (Kapaj, 2006). Toxic effluents released from various industries contain heavy metals in addition to nutrients, which have an effect on plant and soil in a number of ways (Kuo et al., 1983). The excess of metal ions may induce a series of biochemical and physiological changes in plants (John, 2011). Accumulation of toxic heavy metals in plant living cells results in various deficiencies, reduction of cell activities and inhibition of plant growth (Basta et al., 2005). They also result in chlorosis, reduced water and nutrient uptake; affect enzymatic action by exchanging metals ions with metaloenzymes, damage root tips and the enzymes (Khan et al., 2008).
The total chlorophyll content in each plant increased with increasing brewery effluent constituent. The consistency in chlorophyll content among the treatments indicates that the effluent is effective in promoting germination, growth, and chlorophyll and protein control. This observation collaborated with the views of Orhue et al. (2005) who reported that chlorophyll content of maize plant was positively enhanced by brewery effluent. This result indicates that brewery effluent stimulates the synthesis of chlorophyll and the synthesis is accelerated at low concentration of the effluent. Similar observations have been reported by Alexander (2006). The increase in chlorophyll content may be due to low level of heavy metals in the effluent and the availability of Fe/Mg for the synthesis of Chlorophyll. Keller et al. (2002) reported that excess concentration of some heavy metals such as Co, Cu, and Cr decrease chlorophyll concentration by inhibiting electron transport. While Cu has been reported by Sandmann et al. (1980) to decompose the chloroplast membrane of plant.

Conclusion
This study concludes that brewery effluent reduce the plant height but increase the leaf area of Amaranthus hybridus under high concentrations which possibly suggest an adaptive mechanism developed by the plant under stress. The mechanism of detoxification of plants growing in polluted areas should be explored.

Conflicts of interest
Authors declare that they have no conflicts of interests.