Impact of treated municipal wastewater irrigation on turnip (Brassica rapa)

Abstract

The effect of treated municipal wastewater on the roots and the leaves of turnip was studied to compare the 50% and 100% wastewater of 34 ml/d Sewage Treatment Plant (STP) with different doses of potassic fertilizers. Turnip (Brassica rapa) was used as a test plant. A pot experiment was conducted, using a factorial randomized block design to investigate the growth and translocation of heavy metals to the leaves and the roots of turnip. The concentration of heavy metal in wastewater used for irrigation was within the limits. However, the concentration in the plant parts showed a significant rise due to continuous use of wastewater. The concentration of heavy metals in leaves and roots was at excessive levels at 40 and 55 days after sowing (DAS), while at 70 DAS, metal concentration was comparatively low. The range of heavy metals in wastewater irrigated plants was Cd = 1–16.3, Ni = 0–136, Fe = 263–1197, Cu = 0–18, Mn = 37–125, and Zn = 42–141 mg/kg. Concentration of heavy metals in plants was found in the order of Fe>Zn>Ni>Mn>Cu>Cd.

Keywords:

• treated municipal wastewater
• wastewater irrigation
• heavy metals
• turnip

1. Introduction

The demand for water by agriculture has increased during recent decades as a result of growing populations and altered precipitation patterns (Forslund et al. 2010). A number of governmental authorities have turned their attention to the utilization of the secondary or tertiary treated effluent in order to alleviate water scarcity. Consequently, treated wastewater reuse for irrigation purposes is being widely implemented (Fatta-Kassinos et al. 2011) in many countries for recycling plant nutrients (Kalavrouziotis et al. 2012). The wastewater constitutes a significant plant nutrient source for soils of low fertility. The municipal wastewater could also be used for crop irrigation with minimum environmental consequences if it does not contain high levels of heavy metals. The treated municipal wastewater is rich in plant nutrients (N, P, K, S, etc.,) and is of considerable value when compared with an equivalent cost of fertilizers. It usually has low levels of heavy metals (Pb, Cd, Ni, Co, Cr, etc.,) (Kalavrouziotis et al. 2010). Wastewater may also contain increased K and S levels (Rattan et al. 2005) and may also contribute to the accumulation of organic matter up to 59% (Kalavrouziotis et al. 2008). The use of treated wastewater also provides a low-cost method for its disposal and conservation of this water through its recharge to groundwater. However, apart from these benefits, wastewater pollution by trace metals and toxic organic contaminants must also be taken into account. This wastewater is not only a source of irrigation water, but it is also a carrier of significant quantities of macro and micronutrients and organic matter.

Use of untreated or improperly treated wastewater in irrigation has been shown to affect soils, crops, food quality, safety (Khan et al. 2008), and the management of water (Batarseh et al. 2011). Industrial and domestic effluents are either used or disposed off on land for irrigation purposes that create both opportunities and problems. The main concerns are risk due to pathogens, heavy metals, and other chemicals that may be present in the wastewater (Nair et al. 2008). This practice can severely harm human health and the environment mainly due to not only the associated pathogens but also heavy metals and other undesirable constituents, depending on the source (Qadir et al. 2010). Heavy metals present in wastewater may be toxic to plants and disturb a wide range of biochemical and physiological processes, such as photosynthesis, pigment synthesis, protein metabolism, and membrane integrity if taken up at excessive levels (Yang et al. 2008). Nutrients, supplied via treated wastewater, may either favor plant growth by synergistically interacting with another element, or it may negatively affect, by antagonizing with it, i.e. by reducing its concentration, thus unfavorably affecting plant growth (Kalavrouziotis et al. 2008, Kalavrouziotis & Koukoulakis 2009). Numerous interactions occur in plants; their number and intensity may increase under the effect of various inputs such as the treated wastewater and fertilizers (Kalavrouziotis & Koukoulakis 2010). It was also found that the treated wastewater could act as a factor that intensified some of the interactions between heavy metals as well as macro and microelements in the soil and plants. These interactions could increase or decrease the level of the interacting heavy metals, depending on whether they were synergistic or antagonistic. Also, they could take place in the plant, in its various organs, i.e. roots, leaves, and heads or sprouts, thereby, contributing to the spatial distribution of the heavy metals in the plant – an issue of great importance, as it is related to human health (Kalavrouziotis et al. 2009). The toxicity due to heavy metals depends on the total concentration as well as the form or the species in which these are present in the soil, water, or wastewater. The ability of plants to accumulate trace elements in their edible parts varies between plant species and among genotypes within the species. Thus there are genetic controls over the trace elements concentrations found in edible portions of higher plants (Midrar-ul-Haq et al. 2005). The objective of the current study was to investigate the effect of 34 mL/d wastewater (STP located at Noida) on the growth as well as accumulation of heavy metals in turnip (Brassica rapa).

2. Materials and method

2.1. Preparation of experimental pots

Experiment was conducted in 72 pots of 25 cm diameter during the rabi season of 2008–2009. Pots were filled with 5 kg soil and mixed with 2% organic manure. Sufficient quantity of tap water was added to each pot to provide necessary moisture for germination. Nitrogen, phosphorus, potassium (NPK) doses were applied 24 hours in advance to avoid seed injury. In the month of September, 10 seeds of turnip (Brassica rapa) were sown at equal distances in each pot. The pots were then transferred to a field. Seeds germinated within 10 days afterward, leaving the healthiest one in each pot, and the other plants were removed. Sampling was done 40, 55, and 70 days after sowing (DAS) for growth parameters and heavy metals accumulation. There were eight sets of pots, and nine pots were maintained at each condition. The pots were then divided into two sets. One set was daily watered with 100 ml of treated wastewater (100% ww) while the other set was watered with a mixture of 50 ml wastewater and 50 ml of tap water (designated as 50% ww). The aim of this experiment was to study the effect of 50% and 100% wastewater with uniform basal dose of nitrogen (N₅₀) and phosphorous (P₂₅) and varying amount of potassium (K₀, K_12.5, K₂₅, and K₅₀). The scheme of treatments of wastewater and fertilizer doses applied in the experiment is given in Table 1.

Table 1. Scheme of applied treatments in experiment.

	Irrigation water treatments
Fertilizer treatments (Kg NKP ha^−1)	50% ww	100% ww	Remarks (NKP kg/ha)
N0P0K0 (Control)	+	+	No fertilizer
N50P25K12.5	+	+	(50 + 25 + 12.5)
N50P25K25	+	+	(50 + 25 + 25)
N50P25K50	+	+	(50 + 25 + 50)

2.2. Wastewater and soil sample analysis

Wastewater samples were collected from Upflow Anaerobic Sludge Blanket (UASB) based in 34 mL/d Sewage Treatment Plant located at Noida (UP), India. Physicochemical parameters and heavy metals (Cd, Ni, Fe, Cu, Mn, and Zn) of treated effluent were analyzed using standard methods (APHA 1998). Measurements were made in triplicate. Equipment/instruments/methods listed in Table 2 were used for parametric determinations. Analysis results are given in Tables 3 and 4. For coliform analysis, samples were collected in sterilized bottles. Bulk of the samples were collected in plastic bottles and transported in an icebox. Procedures listed in Standard Methods (APHA 1998) were followed for sample collection, preservation, transportation, and analysis. Samples for heavy metal analysis were collected in plastic bottles containing 2 ml HNO₃ to maintain pH<2.0 and to prevent the precipitation of metals. Acidified samples (350 ml) were digested with HNO₃ and filtered. The filtrate was aspirated into a Atomic Absorption Spectrophotometer (Model GBC Avanta M) for the analysis of Cd, Ni, Fe, Cu, Mn, and Zn.

Table 2. Instruments and methods used in experimentation.

Parameter	Technique/instrument model	Parameters	Technique/instrument, model used for soil analysis
pH	pH meter, cyberscan 5–10 pH	Soil texture	US Department of Agriculture soil textural triangle (Arora, 2000)
Turbidity	HACH, 2100 AN turbidity meter	Cation exchange capacity (CEC; meq 100 g^−1 soil)	Ganguly, 1951
Conductivity	Conductivity meter	pH	pH meter, Behera, 2006
DO	DO meter, HQ 10 Hach portable LDO^™ & aqualytic OX 24, Germany	Organic carbon (%)	Walkley and Black, 1934
BOD	BOD pressure sensor and inductive stirring system, aqualytic GmBH & Co., Germany	Electric conductivity (EC) (µmhos/cm)	Conductivity meter (Bear 1964)
COD	COD analysis system, (1) Hach COD reactor model 45600(2) Spectrophotometer, DR/4000	Total nitrogen (mg/kg)	Hach Manual (1999)
Hardness	Titration with 0.01M ethylene di amine tetra acetic acid (EDTA), using Erichrome Black T (EBT) indicator and ammonia buffer	Total phosphorus (mg/kg)	Spectrophotometer, DR/4000 (Bear, 1964)
Alkalinity	Titration with N/50 H2SO4, using bromocresol green indicator	Total potassium (mg/kg)	Flame photometer (Bear, 1964)
Chloride	Argentometric method	Heavy metals (mg/kg)	Hach Manual (1999), AAS (model GBC Avanta M)
sulfate	Turbiditymetric method
Sodium	Microprocessor-based flame photometer
Potassium	Flame photometer
Calcium	Titration with 0.01M EDTA, murexide indicator
Total Coliform	Multiple tube fermentation technique autoclave
Total dissolved solids (TDS)	Evaporation at 105°C
Phosphate	Spectrophotometer, DR/4000
Ammonical nitrogen	Spectrophotometer, DR/4000
Nitrate nitrogen	Spectrophotometer, DR/4000
Heavy metals	Atomic absorption spectrophotometer

Table 3. Characteristics of the treated municipal wastewater (TMWW) and soil used for the irrigation of turnip (Brassica rapa).

Parameters	Wastewater* (range)	WHO^a/Pescod^b/FAO^c limits of water quality for irrigation	Parameters	Soil (range)
pH	7.24–7.50	6.5–8.4^c	pH	7.80–8.12
EC (µmhos/cm at 25°C)	827–858	0.25–3.0^c	Soil texture	Sandy loam
TDS (mg/L)	400–738	<2000^c	Total potassium (mg/kg)	50–60
BOD (mg/L)	35–46	100^b	Total phosphorus (mg/kg)	220–240
COD (mg/L)	120–155	250^b	Total nitrogen (mg/kg)	156–230
DO (mg/L)	0–0	–	Organic carbon (%)	0.75–0.90
Calcium as Ca (mg/L)	116–119	<400^c
Magnesium as Mg (mg/L)*	62–65	<61^c
Potassium as K (mg/L)*	25–30	<2.0^c
Sodium as Na (mg/L)	335–350	<460^c
Total hardness as CaCO3 (mg/L)	520–530	–
Total alkalinity as CaCO3 (mg/L)	480–510	<610^c
Chloride as Cl^− (mg/L)*	525–535	<350^c
sulfate as SO4^−2 (mg/L)	165–175	–
Phosphate as PO4-P (mg/L)*	5.8–10.6	<2.0^c
Nitrate nitrogen as NO3–N (mg/L)	0.4–3.2	<10.0^c
Ammonical nitrogen as NH4–N (mg/L)*	53–70	<5.0^c
Total coliform (MPN/100 mL)	20×10^4−93×10^5	–
Feacal coliform (MPN/100 mL)*	20×10^4−93×10^5	1000^a

* Values crossed the limits of WHO^a (2006), Pescod^b (1992), and Ayers and Westcot FAO^c (1994) for irrigation water.

Samples of the soils were collected from the experimental pots prior to the experiment and addition of NPK and analyzed for pH, soil texture, total N, P, K, and heavy metals (Cd, Ni, Fe, Cu, Mn, and Zn). Soil samples were air-dried, ground, and passed through 2 mm sieve and analyzed by means of methods internationally accepted. Results are given in Tables 3 and 4. Measurements were made in triplicate. Equipment/instruments/methods listed in Table 2 were used for parametric determinations.

Table 4. Heavy metals in wastewater soil and plant parts, critical plant concentration, and permissible limits of Indian standards of heavy metals.

	Range of heavy metal concentration in wastewater and soil sample		Permissible limits of Indian standards of heavy metals (Pescod 1992; Awasthi 2000)		Range of heavy metals in leaf and root of turnip (mg/kg DW)
Heavy metals	Wastewater^a (mg/L)	Soil^b (mg/kg)	Water (mg/L)	Soil (mg/kg)	Leaf	Root	Critical plant concentration (mg Kg^−1) given by Kabata-Pendias and Pendias (1992)
Cd	0.006–0.015	12–15	0.01	3–6	1.0–4.7	2.0–16.0	5–30
Ni	0.011–0.016	118–162	0.2	75–150	0–99	17–136	20–100
Fe	0.201–0.271	14400–15800	–	–	445–1197	292–1107	–
Cu	0.022–0.031	18–26	0.2	135–270	0–12.6	0–18	300–500
Mn	0.072–0.148	300–350	0.2	–	61–125	36–130	100–400
Zn	0.087–0.115	147–195	2.0	300–600	58–121	42–141	5–30

^a Results of analysis of triplicate sets samples collected every month for three months (nine samples).

^b Results of analysis of triplicate samples collected before sowing.

2.3. Plant sample analysis

Three replicates of plant samples from each treatment were collected and gently washed with running tap water to remove the soil particles attached to the plant surfaces and, finally rinsed with deionised water. Subsequently, plant measurements were made. The plant growth was analyzed for root diameter, number of leaves, plant height, plant fresh weight, and plant dry weight. For heavy metal analysis, three replicates of plant samples (leaves and roots) were oven-dried at 70°C until a constant weight. Dried samples were crushed, digested, and then analyzed for Cd, Ni, Fe, Cu, Mn, and Zn by atomic absorption spectrophotometer (Model GBC Avanta M) as per the procedure given in Hach Manual (1999).

2.4. Statistical analysis

The data were statistically analyzed according to Panse and Sukhtame (1985). The ‘F’ test was applied to assess the significance of data at 5% level of probability (P≤0.05). The model of analysis of variance (ANOVA) and critical difference (CD) was also calculated to compare the mean values of various treatments.

3. Results and discussion

3.1. Physicochemical characteristics of wastewater and soil samples

The quality of wastewater was assessed with respect to various physicochemical properties and some of the heavy metals (Tables 2 and 3). The concentration of almost all the elements analyzed, especially the essential macro and micro nutrients, was within the permissible limits FAO/WHO/Pescod. It was further observed that wastewater also contained total coliform and fecal coliform in the range of 21×10⁴–93×10⁶. These values were much higher than the permissible limits (i.e. 1000 MPN/100 mL) specified by WHO for unrestricted irrigation (WHO 2006).

The texture of the soil was sandy loam. This has great influence on root growth and its ability to absorb water and nutrients in quantities sufficient for optimum growth which is most suited for root vegetables. The organic matter content was within the range of 0.75–0.90% as high organic carbon means immobilization of many metal ions. The presence of organic matter is important as it is also a source of plant nutrients in addition to its role to provide organic colloids of soil. Therefore, it increases the ion exchange capacity, water holding capacity, and soil fertility as it regulates the soil water and air supply, which in turn control the rate at which nutrients are absorbed by the roots. Values of soil pH fall in a range of 7.80–8.12 and the uptake of various plant nutrients is pH dependent. However, the soil contained 220–240 mg/kg P, 50–60 mg/kg K, and 156–230 mg/kg N, which may be an additional source of these macronutrients (Table 3). It may be pointed out that some of the factors known to affect the solubility and availability of nutrients include CEC, soil texture, pH, and organic matter content (Haghiri 1974; Williams et al. 1980; Verloo & Eeckhout 1990). On the basis of these characteristics, soil may be termed as suitable for crop cultivation except the pH as pointed out above.

3.2. Growth of turnip plant

Growth of the plant measured as plant fresh weight, root fresh weight, leaf fresh weight, plant height, leaf number, and root diameter is shown through Figure 1(a–f). CD at 5% are given in Table 5. Irrigation with 100% wastewater proved less effective than 50% wastewater. Fifty percent ww proved effective in experiment for enhancing the growth parameters over 100% ww because without dilution, it proved harmful due to excessive ion contents. Thus the combination of 50% ww N₅₀P₂₅K₂₅ recorded the maximum plant growth, which was also at par with 50% wwN₅₀P₂₅K_12.5 showing the economy of K fertilizer, while higher K dose (K₅₀) with 50% wastewater as well as with 100% wastewater was excessive as it decreased the plant growth. Fresh weights of the plants, roots, and root diameter increased with increase in growth period (Figure 1a, b, and f). Plant height increased marginally (Figure 1d) with the advancement of age of the plant. However, number of leaves (Figure 1e) did not show any trend. The increase in leaf number from 40 to 55 days followed by a decline in some cases was observed, which may be due to the senescence in older leaves at the last stage of growth. Fresh weight of plant, roots, leaves, and root diameter increased up to harvest stage (70 DAS) of sampling. Among the various potassium levels K_12.5 with 50% wastewater proved optimum for most of the growth characteristics shown in Figure 1(a–f). Higher K dose (K₅₀) was excessive, as it decreased the plant growth. Potassium has many roles to play because it is an activator of many enzymes that are essential for photosynthesis and respiration, and it also activates enzymes needed to form starch and proteins. This element is also a major contributor to the osmotic potential of cells and, therefore, to their turgor pressures an important feature of root crops. It may also be mentioned that K also promotes translocation of sugars from the leaves to the tubers of potatoes because of which starch content of the tubers is high with well-supplied K (Lachover & Arnon 1966).

Figure 1. Growth parameters at 40, 55, and 70 DAS.

Table 5. CD at 5% at 40, 55, and 70 DAS of plant fresh weight, root fresh weight, leaf fresh weight and plant height, leaf number, and root diameter.

	Plant fresh weight			Root fresh weight			Leaf fresh weight			Plant height			Leaf number			Root diameter
CD at 5%	40	55	70	40	55	70	40	55	70	40	55	70	40	55	70	40	55	70
Water	1.22	1.47	4.65	0.187	0.725	3.11	0.531	1.50	1.62	0.547	0.380	0.629	0.067	0.330	NS	0.057	NS	0.241
Treatment	1.73	2.08	6.58	0.264	1.025	4.40	0.751	2.13	2.29	0.774	0.538	0.889	0.094	0.467	0.50	0.081	0.175	0.341
Interaction	2.44	2.95	9.30	0.373	1.450	6.23	1.061	3.01	3.23	1.094	0.761	1.257	0.133	0.660	NS	0.114	0.247	0.483

3.3. Distribution of heavy metals in plant parts

The concentration of heavy metals in leaves and roots of turnip at 40, 55, and 70 DAS is shown in Figure 2 (a–f). The range of heavy metal concentrations (mg/kg) in plant parts harvested on different days and a comparison with standards given by Kabata-Pendias and Pendias (1992) is tabulated in Table 4. CD of Cd, Ni, Fe, Cu, Mn, and Zn is tabulated in Tables 6 and 7. The range of heavy metals in wastewater-irrigated plants was Cd=1–16.3, Ni=0–136, Fe=263–1197, Cu=0–18, Mn=37–125, and Zn=42–141mg/kg. In our study, heavy metal concentration was in the order of Fe>Zn>Ni>Mn>Cu>Cd. Grytsyuk et al. (2006), Zheng et al. (2007), Arora et al. (2008), and Khan et al. (2003) reported more or less a similar trend while working on various vegetables, including turnip. In our findings, Fe content was maximum because of its high concentration in soil and wastewater compared to other elements. Like Fe, chelated Zn is important for its transport to root surface for uptake, and its availability is predominantly governed by the pH and adsorbed Zn on soil colloids. It may also be observed that primary and secondary minerals that dissolve initially provide Zn to the soil solution for its availability which may also be responsible for comparatively higher Zn content. Zn was followed by Ni may be due to use of sewage sludge and result in higher levels of Ni. In addition, it is also readily taken up by most plant species (Havlin et al. 1999). It was followed by Mn. In general, higher concentration of Mn may be because it is easily taken up by the plants when it occurs in soluble form. Lower concentration of Cd after Cu may be due to their greater dependability on solubility and soil pH. Most of the heavy metals decreased with the advancement of age in edible parts of the plant. This decreasing trend can be ascribed to the exponential increase in growth, and as a result of dilution with growth effect, even higher quantities of elements appear to be less when expressed on per unit basis (Moorby & Besford 1983). Ni content in leaf and Cu content in root were increased with growth which may be because of the selective properties of ion absorption in plants (Devlin & Witham 1986). However, even on using the same species, the uptake by plants does not necessarily correlate with the bioavailable metal concentration in the soil or the total metal concentration. This is probably due to the genotype with inherent capability of different metal uptakes, as pointed out above, as in the case of Ni and Cu. It may also be added that plants also differ in their transport of ions, resulting in differences in concentration in plant parts (Greger 2004), as observed in the present study also, where the heavy metals fluctuated between the root and the leaf, depending upon the growth stage, availability of individual element, and also their interactions. In many cases, heavy metal concentration was more in plants grown under 50% ww than the 100% ww in case of leaf and root. This was not surprising because metal uptake may differ in relation to external concentration and genotypes. It may also be pointed out that uptake is not linear in relation to increase in wastewater concentration in many cases, which may be because metals are bound in the tissues, causing saturation which is governed by the rate at which the metal is conducted away. Therefore, the uptake efficiency is more at a low concentration which was observed in solution culture (Greger et al. 1991) as well as in soil (Greger 1997) for Cd. It may be because of low metal concentration per absorption area giving low competition between the ions at the uptake sites while the opposite occurs at high concentration.

Figure 2. Cd, Ni, Fe, Cu, Mn, and Zn in leaf and root at different stages of growth.

Table 6. CD at 5% at 40, 55, and 70 DAS for Cd, Ni, and Fe.

	Cadmium (mg/kg)						Nickel (mg/kg)						Iron (mg/kg)
	Leaf			Root			Leaf			Root			Leaf			Root
CD at 5%	40	55	70	40	55	70	40	55	70	40	55	70	40	55	70	40	55	70
Water	0.165	0.076	0.138	0.448	0.114	0.048	0.429	3.22	0.679	3.79	0.95	2.84	42.8	25.4	28.5	31.1	8.18	12.5
Treatment	0.233	0.107	0.195	0.634	0.162	0.068	0.606	4.55	0.960	5.36	1.34	4.02	60.6	35.9	40.3	44.0	11.6	17.7
Interaction	0.330	0.151	0.276	0.897	0.229	0.096	0.858	6.44	1.36	7.58	1.89	5.69	85.7	50.8	57.0	62.2	16.4	25.0

Table 7. CD at 5% at 40, 55, and 70 DAS for Cu, Mn, and Zn.

	Copper (mg/kg)						Manganese (mg/kg)						Zinc (mg/kg)
	Leaf			Root			Leaf			Root			Leaf			Root
CD at 5%	40	55	70	40	55	70	40	55	70	40	55	70	40	55	70	40	55	70
Water	0.445	0.277	0.037	0.367	0.205	0.507	2.40	4.59	3.62	1.12	1.92	1.94	3.77	3.11	3.30	4.33	3.39	2.94
Treatment	0.630	0.392	0.052	0.532	0.289	0.717	3.39	6.50	5.12	1.58	2.72	2.75	NS	4.39	4.67	6.12	4.80	4.15
Interaction	0.890	0.554	0.073	0.752	0.409	1.041	4.80	9.19	7.24	2.24	3.84	3.89	NS	6.21	6.60	8.66	6.79	5.87

CD, critical difference; DAS, days after sowing.

3.3.1. Cadmium accumulation in plant parts

In root at 40 and 55 DAS, 50% ww showed maximum concentration, while at 70 DAS, it was 100% ww. At 40 DAS, 50% wwK₅₀N₅₀P₂₅ accumulated higher Cd content, and it was critically different with other interactions, It was followed by 100% ww K₅₀N₅₀P_25, 100% ww K₂₅N₅₀P_25, and 100% ww K_12.5N₅₀P_25. At 55 DAS, 50% ww K₂₅N₅₀P₂₅ accumulated more and had similar effect with 50% ww K₅₀N₅₀P₂₅ followed by 50% ww K_12.5N₅₀P_25, and the last treatment had similar effect with 100% ww K_12.5N₅₀P_25, 100% ww K₅₀N₅₀P_25, and 50% ww K₀N₀P_0, while at 70 DAS, the combination 100% ww K₅₀N₅₀P₂₅ recorded maximum effect, and it was followed by 50% ww K_12.5N₅₀P₂₅ and 100% ww K₂₅N₅₀P_25. In case of leaf, 50% ww gave more value. Among the K doses, K_12.5 at 40 and 55 DAS, while K₂₅ at 70 DAS, showed maximum heavy metal content. Among the interactions of wastewater and potassium, 50% ww K_12.5N₅₀P₂₅ accumulated higher Cd and was at par with 100% ww K₅₀N₅₀P₂₅. At 55 DAS, 50% ww K_12.5N₅₀P₂₅ had more, and it was critically different with other treatments, while at 70 DAS, 100% ww K_12.5N₅₀P₂₅ accumulated more Cd content and was equally effective with 50% ww K₂₅N₅₀P_25, 50% ww K₅₀N₅₀P_25, and the last treatments was at par with 50% ww. Cd concentration was more in root at 40 and 55 DAS, while at 70 DAS, it was more or less similar in both organs (Figure 2a). Its concentration generally increased with K doses in root as well as in leaf and generally decreased with growth. Cd concentration was increased with K doses in case of root, and Cd concentration was more in the root than the leaf, which was also in agreement with the work of Demirezen and Aksoy (2004) and Kalavrouziotis et al. (2008). On the contrary, at harvest stage, Cd was surprisingly below the toxic levels. It was obviously due to the increased root fresh weight, increased root diameter, and plant fresh weight at this stage, which was supposed to be responsible for its dilution, although the total Cd content could have remained unchanged. It may be relevant here to point out the work of Chaney and Hornick (1977), who have reviewed the work on Cd and reported much difference in the ability to absorb it, where lowest concentration was observed in rice, sudan grass, and clover and higher in spinach and turnip. On the contrary, Cd in leaf was below the toxic levels at last stage of sampling, as it was generally retained by the roots instead of transporting it toward the shoot.

3.3.2. Nickel accumulation in plant parts

The concentration of Ni generally increased with K doses in both leaf and root. Its concentration increased at 55 DAS, while it decreased at the harvest stage. Ni content was more in root at 40 and 70 DAS, while at 55 DAS, it was more in leaf. In case of root, 50% ww showed higher concentration at all the three stages, and among the K doses, K₅₀ at 40 DAS, while at 55 and 70 DAS, K_12.5 accumulated more. In case of interactions at 40 DAS, 50% ww K₅₀N₅₀P₂₅ had maximum Ni, which was critically different with other treatments. At 55 DAS, the interaction of 100% ww K_12.5N₅₀P₂₅ accumulated higher content and was statistically similar with 50% ww K_12.5N₅₀P₂₅. At 70 DAS, 50% ww K_12.5N₅₀P₂₅ proved more effective and was critically different with other treatments, followed by 50% ww K₅₀N₅₀P₂₅ and 50% ww K₂₅N₅₀P₂₅. In case of leaf, 100% ww accumulated maximum Ni content and K₅₀ recorded more Ni at 40 and 70 DAS, while at 55 DAS, it was K_12.5. The combination of 100% ww K₅₀N₅₀P₂₅ accumulated maximum Ni and was critically different with other interactions followed by 50% ww K₅₀N₅₀P₂₅, 50% ww K₂₅N₅₀P₂₅, 100% ww K₂₅N₅₀P₂₅, and 50%ww K₀N₀P₀. While at 55 DAS, 100% ww K₅₀N₅₀P₂₅ accumulated more, and it was statistically at par with 100% ww K_12.5N₅₀P₂₅ and 100% ww K₂₅N₅₀P₂₅. At 70 DAS, 100% ww K₅₀N₅₀P₂₅ proved more effective, and it was followed by 50% ww K₅₀N₅₀P₂₅. Its concentration was more in root at 40 and 70 DAS, while at 55 DAS, it was more in leaf (Figure 2b). It may be because of its easy mobility in plant parts, therefore, after initial growth up to 40 DAS, where its concentration was higher, it was transported more toward the leaves at 55 DAS, stage. However, at harvest stage, due to senescence in some of the older leaves, it was transported back toward the root. In our findings, Ni content ranges from 0 to 136 mg/kg, while Shacklette (1980) reported that the Ni content in vegetables ranges from 0.2 to 3.7 ppm (DW). In our study, data indicated that metal accumulated by turnip plants was largely retained in roots as indicated by translocation factor (TF) values which were <1 at 40 and 70 DAS, while at 55 DAS, and TF value in general was >1.

3.3.3. Iron accumulation in plant parts

In root, Fe content decreased at the second stage and again increased at the harvest stage. While in case of leaf, it generally decreased with growth. At 55 and 70 DAS, its concentration was more or less similar. Fe content was more in leaf than in root. Among the fertilizer doses at 40 DAS, K₅₀ showed maximum Fe content, while at 70 DAS, K_12.5 accumulated more. Among the interactions, 100% ww K₅₀N₅₀P₂₅ gave higher Fe content, and it was critically different with other treatments. At the second stage, 100% ww K₀N₀P₀ gave more Fe, and it was statistically equal with 50% ww K₂₅N₅₀P₂₅, and the last treatment was equaled by 50% ww K₅₀N₅₀P₂₅ and 100% ww K_12.5N₅₀P₂₅. While at 70 DAS, 50% ww K_12.5N₅₀P₂₅ accumulated maximum Fe and was equal in effect with 100% ww K_12.5N₅₀P₂₅ and 50% ww K₀N₀P₀. In case of leaf, at 40 DAS, 100% ww proved effective, while at 55 and 70 DAS, 50% ww registered higher Fe content (Figure 2c). At 40 and 70 DAS, K₅₀ accumulated more, while at 55 DAS, K₂₅ gave more in leaf. At 40 DAS, interaction of 100% ww K₅₀N₅₀P₂₅ registered maximum Fe content, and it was critically different with other combinations followed by 50% ww K_12.5N₅₀P₂₅, 100% ww K_12.5N₅₀P_25, and 100% ww K₂₅N₅₀P_25, and the last was at par with 50% ww K₂₅N₅₀P_25, 100% ww K₀N₀P₀, and 50% ww K₅₀N₅₀P₂₅. At the second stage, 50% ww K₂₅N₅₀P₂₅ was critically different with the others, and it was followed by 50% ww K_12.5N₅₀P_25, 100% ww K₂₅N₅₀P₂₅, and 50% ww K₅₀N₅₀P₂₅. While at 70 DAS, 50% ww K₅₀N₅₀P₂₅ accumulated more Fe. It differed with other interactions and followed by 100% ww K_12.5N₅₀P₂₅, which was statistically similar with 50% ww K₂₅N₅₀P_25, 50% ww K_12.5N₅₀P₂₅, 50% ww K₀N₀P₀, and 100% ww K₂₅N₅₀P₂₅. In our results, Fe contents increases under K fertilizers, and the increase may be because of their roles in enhancing the root growth and thereby the surface area for its greater absorption (Tisdele et al. 1995). The TF values were generally >1 and shows more accumulation in the leaf than the root. About 75% of total leaf Fe is associated with chloroplast and up to 90% of Fe leaves occur with lipoprotein of chloroplast and mitochondria membrane, therefore, its presence in larger amount in leaves was understandable (Havlin et al. 1999).

3.3.4. Copper accumulation in plant parts

In root, its concentration generally decreased with growth, and at 40 and 70 DAS, generally decreased with K doses, while at 55 DAS, it increased in some cases. In case of leaf, its concentration decreased with growth (Figure 2d). Concentration of Cu increased with K doses at 40 DAS, while at 55 DAS, it generally decreased. Cu concentration was more in the root than in the leaf at 55 and 70 DAS. In the root, 50% ww was at 40 DAS, while at 55 and 70 DAS, 100% ww showed maximum Cu content. Among the potassium doses at 55 DAS, K₂₅ had more, while at 40 and 70 DAS, it was in K_12.5. Among the interactions, 50% ww K_12.5N₅₀P₂₅ accumulated maximum Cu content and was followed by 50% ww K₂₅N₅₀P₂₅ and was critically different with the other treatments. At the later stage, 50% ww K₂₅N₅₀P₂₅ proved more effective, and it was critically different with other combinations followed by 50% ww K_12.5N₅₀P₂₅. The last combination was at par with 100% ww K₅₀N₅₀P_25, while at 70 DAS, 100% ww K_12.5N₅₀P₂₅ gave more Cu content. In the case of leaf at 40 and 70 DAS, 100% ww accumulated higher Cu, while at 55 DAS, it was 50% ww. Among the potassium doses, K₅₀ accumulated maximum Cu content at 40 DAS, while at 55 and 70 DAS, it was K_12.5. Among the interactions, 100% ww K₂₅N₅₀P₂₅ by giving an increase of 43.55% higher Cu content and was statistically similar with 100% ww K₅₀N₅₀P₂₅. The last treatment was at par with 100% ww K_12.5N₅₀P₂₅. At the second stage, 50% ww K_12.5N₅₀P₂₅ registered more Cu in the leaf, and it was critically different with other treatments. At 70 DAS, 100% ww K_12.5N₅₀P₂₅ accumulated maximum Cu content, followed by 100% ww K₂₅N₅₀P_25, 100% ww K₅₀N₅₀P₂₅, and 100% ww K₀N₀P₀. While in the case of K, its concentration was generally decreased with increase in doses due to its interaction with K, where potassic fertilizers can also induce Cu deficiency (Tisdele et al. 1995). The TF value was generally <1 at 55 and 70 DAS, as the Cu mobility within plant tissues strongly depends on the level of Cu supply, and it has low mobility relative to other elements in plants. In addition, the distribution of Cu within plants is highly variable as within roots, and Cu is associated mainly with cell walls and is largely immobile. The majority of the plant species, therefore, can accumulate more Cu especially in the root as observed at the later two stages, while at an early stage, TF value was >1. The highest concentrations of Cu in shoots are always in phases of intensive growth which was evident in our study at 40 days.

3.3.5. Manganese accumulation in plant parts

At the first stage, its concentration was more or less similar in root and leaf, but at 55 and 70 DAS, it was more in leaf. Its concentration generally increased with K doses in root and leaf at 40 DAS, while at 55 and 70 DAS, it generally decreased in the root. Mn concentration also decreased with growth, while at 55 and 70 DAS, it was more or less similar in root (Figure 2e). In the leaf at 40 and 70 DAS, its content was more or less similar, while at 55 DAS, it showed higher values. At 40 DAS, 50% ww, while at 55 and 70 DAS, 100% ww accumulated higher Mn content in root. At 40 DAS, K₅₀ gave higher content, while at 70 DAS, K_12.5 accumulated more. Among the interactions at 40 DAS, 50% ww K₅₀N₅₀P₂₅ had more Mn content, and it was critically different with other combinations followed by 100% ww K₂₅N₅₀P₂₅ and 100% ww K₅₀N₅₀P₂₅. At 55 DAS, 100% ww K₀N₀P₀ registered more content and was critically different with others, followed by 50% ww K₅₀N₅₀P₂₅. At 70 DAS, 100% ww K₀N₀P₀ gave the maximum value, and it was equally effective with 100% ww K_12.5N₅₀P₂₅ and 100% ww K₂₅N₅₀P₂₅. The last treatment was at par with 50% ww K_12.5N₅₀P₂₅ which in turn was at par with 50% ww K₂₅ N₅₀P₂₅ and 100% ww K₅₀N₅₀P₂₅. In case of leaf, 50% ww gave more Mn content by giving an increase over 100%ww at all the sampling stages. Among the potassium doses at 40 DAS, K₅₀ recorded higher Mn content which was equally effective with K₂₅. At 55 DAS, K_12.5 was equal to K_25, in effect of Mn accumulation. While at 70 DAS, K₂₅ accumulated higher Mn and was equally effective with K₅₀. Among the interactions, 50% ww K₅₀N₅₀P₂₅ accumulated maximum Mn and was statistically similar with 100% ww K₂₅N₅₀P₂₅. At 55 DAS, it was 50% ww K₂₅N₅₀P_25, which was statistically similar with 100% ww K_12.5N₅₀P₂₅ and 50%wwK_12.5N₅₀P_25. At the last stage, 50% ww K₀N₀P₀ accumulated more Mn and was equally effective with 50% ww K₅₀N₅₀P₂₅ and 100% ww K₂₅N₅₀P₂₅. It may also be added that differences in Mn content under different concentrations of wastewater were due to differential absorption by the root (Bowen 1979) It may be pointed out that potassic fertilizer has a great role in Mn and its uptake which was reflected during the observations in the experiment, and it can be so strong that it produces toxicity in sensitive crops (Tisdele et al. 1995). The TF values in general were >1, although it is reverse in some cases. It may be because Mn concentration fluctuates greatly within the plant parts and within the vegetative period. It is not only an effect of the plant characteristics but also of the pool of available Mn which is controlled basically by soil properties. Although it is relatively immobile like iron, its mobility varies depending upon its supply.

3.3.6. Zinc accumulation in plant parts

In leaf, Zn concentration generally increased with K doses at all the three stages. Its concentration was more or less similar in leaf and root. The concentration generally decreased with growth. In root, at 40 and 55 DAS, 50% ww was responsible for maximum Zn content, and at 70 DAS, it was 100% ww. At 40 DAS, K₅₀ registered maximum content at 55 DAS, and K_12.5 accumulated more Zn and was statistically equal to K₅₀. While at 70 DAS, K_12.5 was statistically at par with other K doses (Figure 2f). Among the interactions, Zn content was more in 50% ww K₅₀N₅₀P₂₅, and it was statistically similar with other interactions, followed by 100% ww K₅₀N₅₀P₂₅, 100% ww K₂₅N₅₀P₂₅, and 50% ww K₂₅N₅₀P₂₅. The last treatment was at par with 100% ww K_12.5N₅₀P₂₅ and 100% ww K₀N₀P₀. At 55 DAS, 50% ww K_12.5N₅₀P₂₅ had more Zn content, and it was statistically similar with 100% ww K_12.5N₅₀P₂₅, 50% ww K₂₅N₅₀P₂₅, 100% ww K₅₀N₅₀P_25, 50% ww K₀N₀P₀, and 50% ww K₅₀N₅₀P₂₅. While at the last stage, 100% ww K₅₀N₅₀P₂₅ gave more value, which was also statistically equal to 100% ww K_12.5N₅₀P₂₅ and 100% ww K₂₅N₅₀P₂₅. In case of leaf, at 40 DAS, it was 100% ww, whereas at 55 and 70 DAS, 50% ww accumulated higher Zn content. At 55 and 70 DAS, K₅₀ was also critically different with other treatments. At 55 DAS, interaction of 50% ww K₅₀N₅₀P₂₅ registered higher Zn content, followed by 50% ww K_12.5N₅₀P₂₅ and 100% ww K₂₅N₅₀P₂₅. The last treatment was also at par with 100% ww K₅₀N₅₀P₂₅ and 100% ww K_12.5N₅₀P_25, while at the last sampling, it was 50% ww K₅₀N₅₀P₂₅. It was also equal in effect with 50% ww K₂₅N₅₀P₂₅, 50% ww K₀N₀P₀, and 50% ww K_12.5N₅₀P₂₅. The TF values were generally <1. Therefore, the roots often contained much more Zn than the tops, particularly when plants are grown in Zn-rich conditions. With luxury levels of soil Zn, this element may be translocated from the roots and accumulated by the tops of the plant. Therefore, the rate of Zn absorption differs greatly among both plant species and growth medium.

4. Conclusion

The following conclusions could be drawn in relation to the evaluation of the treated municipal wastewater on turnip plants.

Heavy metals Cd, Ni, Fe, Cu, Mn, and Zn were within the permissible limits for irrigation water. The microbiological examination of the wastewater revealed the presence of some pathogenic microorganisms, therefore, the growers may be warned to be careful during irrigation operation.

Irrigation with treated effluent increased the concentration of heavy metals in the plant parts. The rate of accumulation of heavy metals in leaves and roots is observed to be depended on the relative efficiency or transfer factor of these metal ions from soil to roots and leaves.

Wastewater irrigation increased the growth parameters, plant fresh weight, root fresh weight, leaf fresh weight, and root diameter increased with advancement of the plant. Further, concentration of heavy metal reduced with growth of biomass of root and leaves. In general, accumulation is observed more in roots than in leaves, and the dilution increased rapidly after 40 DAS. Therefore, the early stage of harvesting may lead to more heavy metal contamination, and, therefore, a poor food quality and higher health risk, specially, when used in food intake for the long term.

Fifty percent ww proved best over 100% ww, therefore, it can be used after dilution. The combination of 50% ww K₂₅ proved beneficial in enhancing the growth which was at par with 50% ww K_12.5, indicating the usefulness of wastewater where 12.5 kg K/ha could be saved.

The levels of Cd, Ni, and Zn in the leaves and roots were found to be more than the toxic limits given by Kabata-Pendias and Pendias (1992). In leaf, Cd, Cu, and Mn concentration was below the toxic level. Heavy metal concentration was found in the order of Fe>Zn>Ni>Mn>Cu>Cd. The results show that there is a risk associated with consumption of turnip, therefore, it is not advisable to consume turnip cultivated under this water. However, more work is needed in this respect to accurately quantify the seriousness and severity of pollution.