Open Journal of Metal, 2013, 3, 8-20
http://dx.doi.org/10.4236/ojmetal.2013.32A1002 Published Online July 2013 (http://www.scirp.org/journal/ojmetal)
Distribution, Enrichment and Accumulation of Heavy
Metals in Soil and Trigonella foenum-graecum L.
(Fenugreek) after Fertigation with Paper Mill Effluent
Vinod Kumar*, Ashok Kumar Chopra
Department of Zoology and Environmental Science, Gurukula Kangri University, Haridwar, India
Received April 30, 2013; revised June 1, 2013; accepted June 10, 2013
Copyright © 2013 Vinod Kumar, Ashok Kumar Chopra. This is an open access article distributed under the Creative Commons At-
tribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is prop-
erly cited.
ABSTRACT
The aim of the study was to investigate distribution, enrichment and accumulation of heavy metals in soil and
Trigonella foenum-graecum (var. Pusa Early Bunching) after fertigation with paper mill effluent. Doses of paper mill
effluent viz. 5%, 10%, 25%, 50%, 75% and 100% were used for fertigation of T. foenum-graecum along with bore well
water (control). The results revealed that paper mill effluent had significant (P < 0.05) effect on EC, pH, OC, Na+, K+,
Ca2+, Mg2+, Fe2+, TKN, 3
4
PO

, , Cd, Cr, Cu, Mn and Zn of the soil in both seasons. Insignificant (P > 0.05)
changes in WHC and bulk density of the soil were observed after irrigation with paper mill effluent. The agronomical
performance of T. foenum-graecum was increased from 5% to 25% concentration and decreased from 50% to 100%
concentration of paper mill effluent as compared to control in both seasons. The heavy metals concentration was in-
creased in T. foenum-graecum from 5% to 100% concentrations of paper mill effluent in both seasons. Biochemical
components like crude proteins, crude fiber and crude carbohydrates were found maximum with 25% paper mill efflu-
ent in both seasons. The enrichment factor (Ef) of various heavy metals was in order of Cd > Mn > Cr > Cu > Zn > Fe
for soil and Mn > Cu > Cr > Cd > Zn > Fe for T. foenum-graecum plants after fertigation with paper mill effluent.
Therefore, paper mill effluent can be used as a biofertigant after appropriate dilution to improve yield of T. foenum-
graecum.
2
4
SO 
Keywords: Trigonella foenum -graecum; Agronomical Characteristics; Enrichment Factor; Fertigation; Heavy Metals;
Paper Mill Effluent
1. Introduction
Industrial or domestic effluent is mostly used for the fer-
tigation of agricultural crops, mainly in urban and peri-
urban regions, due to its easy availability, disposal prob-
lems and scarcity of fresh water [1,2]. Irrigation with
effluents is known to contribute significantly to the heavy
metals content of soil as well as crop plants [3-5]. Heavy
metals are very harmful because of their non-biode-
gradable nature, long biological half-lives and their po-
tential to accumulate in different body parts [6-8]. Most
of the heavy metals are extremely toxic because of their
solubility in water [3,9,10]. Wastewater contains sub-
stantial amounts of toxic heavy metals, which create
problems [6,11-13]. Excessive accumulation of heavy
metals in agricultural soils through wastewater irrigation,
may not only result in soil contamination, but also affect
food quality and safety [8,14-16].
Heavy metals accumulation in agricultural soils is of
increasing worldwide concern and particularly in India
with the rapid development of industrialization and ur-
banization [17-19]. Heavy metals are easily accumu-
lated in the edible parts of leafy vegetables, as compared
to grain or fruit crops [20,21]. Vegetables take up heavy
metals and accumulate them in their edible and inedible
parts in quantities high enough to cause clinical problems
both to animals and human beings consuming these me-
tal-rich plants [8,22]. A number of serious health prob-
lems can develop as a result of excessive uptake of die-
tary heavy metals [19,23-25]. Industrial effluent is most-
ly used for the fertigation of agricultural crops, mainly in
*Corresponding author.
C
opyright © 2013 SciRes. OJMetal

V. KUMAR, A. K. CHOPRA 9
urban and periurban regions, due to its easy availability,
disposal problems and scarcity of fresh water [26,27].
Long term irrigation with effluents is known to contrib-
ute significantly to the heavy metals content of soil and
increase the chances of their entrance in food chain, and
this ultimately causes significant geoaccumulation, bio-
accumulation and biomagnifications [13,28].
India has 666 pulp and paper mills, out of which 632
mills are agro-residue based mills [29,30]. They generate
a huge amount of wastewater (black liquor) having high
biological oxygen demand (BOD) and chemical oxygen
demand (COD) values [13,31]. Fenugreek is used as ve-
getables as well as pulse [32]. The leaves and young pods
are used as vegetables and the seeds as condiments. It has
also some medicinal value. It prevents constipation re-
moves indigestion stimulates the spleen and is appetiz-
ing and diuretic. The leaves are quite rich in protein min-
erals and Vitamin C [32].
Irrigation of crops with effluents is a very common
practice in India due to scarcity of irrigation water [33,
34]. The effect of irrigation with effluents is also studied
in many crops to observe the concentration of accumu-
lated metals to which human beings are exposed [5,35,
36]. Heavy metals are easily accumulated in the edible
parts of leafy vegetables, as compared to grain or fruit
crops [4,7]. Vegetables take up heavy metals and accu-
mulate them in their edible and inedible parts in quanti-
ties high enough to cause clinical problems both to ani-
mals and human beings consuming these metal-rich
plants [16,21]. A number of serious health problems can
develop as a result of excessive uptake of dietary heavy
metals [12,37].
In recent years, many studies have carried out about
effluents quality and its effect on soil and agricultural
crops [7,15,38-40]. The researches indicated paper mill
industries not only led to accumulation of toxic elements
in soil environment, but also increased the risk of accu-
mulation in crop plants [20,35,37]. The present study was
conducted with an aim to study the distribution and ac-
cumulation of heavy metals in soil and potential of the
commonly grown leafy vegetable Trigonella foenum-
graecum L. (Fenugreek) after fertigation with paper mill
effluent.
2. Materials and Methods
2.1. Experimental Design
A field study was conducted at the Experimental Garden
of the Department of Zoology and Environmental Sci-
ences, Faculty of Life Sciences, Gurukula Kangri Uni-
versity Haridwar, India (29˚55'10.81''N and 78˚07'08.12''E).
The crop was cultivated in the summer and winter sea-
sons during the year 2010 and 2011. Seven plots (each
plot had an area of 9 m2) were selected for seven treat-
ments of paper mill effluent viz. 0% (control), 10%, 25%,
50%, 75% and 100% for the cultivation of T. foe-
num-graecum. The seven treatments were placed within
seven blocks in a randomized complete block design.
2.2. Effluent Collection and Analysis
The effluent samples were collected from the Uttranchal
Pulp & Paper Mills (P) Ltd. Haridwar (29˚46'4''N
77˚50'47''E), which produces paper from agricultural
waste or residues. Effluent was collected from a settling
tank installed on the campus, by the paper mill, to reduce
biological oxygen demand (BOD) and solids. The efflux-
ents were collected in plastic container, and were brought
to the laboratory and analyzed for total dissolved solids
(TDS), pH, electrical conductivity (EC), dissolved oxy-
gen (DO), BOD, COD, chlorides (Cl−), bicarbonates
(HCO3−), carbonates


2
3
CO

, sodium (Na+), potassium
(K+), calcium (Ca2+), magnesium (Mg2+), total Kjeldahl
nitrogen (TKN), nitrate


2
3

NO , phosphate


3
4
PO

,
sulphate


2
4
SO

, cadmium (Cd), chromium (Cr), copper
(Cu), iron (Fe), manganese (Mn), zinc (Zn), standard
plate count (SPC) and most probable number (MPN)
following standard methods [41,42] and used as fertigant.
2.3. Sowing of Seeds and Irrigation Pattern
Seed of T. foenum-graecum were sown at the end of
February 2010 and 2011 for the summer crop and at the
end of October 2010 and 2011 for the winter season crop.
Seed of T. foenum-graecum, cv. Pusa Early Bunching,
were procured from ICAR, Pusa, New Delhi, and steril-
ized with 0.01% mercuric chloride and soaked in water
for 12 hrs. Seeds were sown in 10 rows with a distance of
30.0 cm between rows, while distance between the seeds
was 15 cm. The thinning was done manually after 15
days of germination to maintain the desired plant spacing
and to avoid competition between plants. The plants in
each plot were fertigated twice in a month with 50 gal-
lons of paper mill effluent with 5%, 10%, 25%, 50%,
75% and 100% along with bore well water as the control.
2.4. Irrigation Pattern, Soil Sampling and
Analysis
The plants in each plot were fertigated twice in a month
with 50 gallons of paper mill effluent concentrations 5%,
10%, 25%, 50%, 75%, 100% and bore well water as the
control. The soil was analyzed prior to planting and after
harvest for various physico-chemical parameters like soil
texture, bulk density (BD), water holding capacity
(WHC), EC, pH, OC, Na+, K+, Ca2+, Fe2+, Mg2+, 3
4
PO

,
2
4
SO

, TKN, Cd, Cr, Cu, Mn and Zn determined follow-
ing standard methods [42].

V. KUMAR, A. K. CHOPRA
10
2.5. Study of Crop Parameters
The agronomic parameters of T. foenum-graecum at dif-
ferent stages (0 - 120 days) were determined following
standard methods for seed germination, plant height, root
length, number of flowers, number of fruits, fruits length
and crop yield [43]; dry weight [44]; chlorophyll content
[45]; relative toxicity (RT) [46], Leaf Area Index (LAI)
[47] and harvest index (HI) [48]. The nutrient quality of
T. foenum-graecum was determined by using the follow-
ing parameters; crude protein, crude fiber and the total
carbohydrate in dry matter were determined by standard
methods [49].
2.6. Extraction of Metals and Their Analysis
For metal analysis a 5 - 10 ml sample of paper mill ef-
fluent, and 0.5 - 1.0 g of air dried soil or plants were di-
gested in tubes with 3 ml of conc. HNO3 digested in an
electrically heated block for 1 hr at 145˚C. To this mix 4
ml of HClO4 was added and heated to 240˚C for 1 hr.
The mix was cooled and filtered through Whatman # 42
filter paper and made to 50 ml and used for analysis.
Metals were analyzed using an Atomic absorption spec-
trophotometer (PerkinElmer, Analyst 800 AAS, GenTech
Scientific Inc., Arcade, NY) following standard methods
[41,42]. The enrichment factor (Ef) for metals accumu-
lated in paper mill effluent irrigated soil and T. foenum-
graecum was calculated following methods [22].
2.7. Data Analysis
Data were analyzed with SPSS (ver. 14.0, SPSS Inc.,
Chicago, Ill.). Data were subjected to one-way ANOVA.
Mean standard deviation and coefficient of correlation
(r-value) of soil and crop parameters with effluent con-
centrations were calculated with MS Excel (ver. 2003,
Microsoft Redmond Campus, Redmond, WA) and
graphs produced with Sigma plot (ver. 12.3, Systat Soft-
ware, Inc., Chicago, IL).
3. Results and Discussion
3.1. Characteristics of Paper Mill Effluent
Values of physico-chemical and microbiological parame-
ters varied over paper mill effluent concentration (Table
1). The paper mill effluent was alkaline i.e. pH 8.74. The
alkaline nature of the paper mill effluent might be due to
presence of high concentrations of alkalis used in pulping.
The BOD, COD, Cl−, Ca2+, Fe2+, TKN, , MPN and
SPC were above the prescribed limits of the Indian Irri-
gation Standards [50]. High BOD and COD might be due
to presence of high utilizable organic matter and rapid
consumption of dissolved inorganic materials. The higher
bacterial load (SPC and MPN) in paper mill effluent
might be due to presence of more dissolved solids and
organic matter in effluent as earlier reported by Kumar,
2010. The TKN,
2
4
SO 
3
4
PO

, K+, Ca2+ and Mg2+ in effluent
were higher than the prescribed standards (Table 1).
In the present study, the content of BOD, COD, TKN,
Cl−, 2
4
SO

and 3
4
PO

were more in paper mill effluent
then the content of BOD (668.56 mg·L−1), COD (1026.48
mg· L −1), total nitrogen (42.34 mg·L−1), chlorides (426.75
mg· L −1), sulphate (675.82 mg·L−1) and phosphate (51.30
mg· L −1) in paper mill effluent reported by Patterson et al.
[51]. In the case of metals, the contents of Cd, Cr Cu, Fe,
Mn and Zn were higher than permissible limits for in-
dustrial effluent [50]. The content of these metals in pa-
per mill effluent were also higher then the content of Cd
(9.36 mg·L−1), Cr (16.46 mg·L−1) Cu (10.52 mg·L−1) and
Zn (10.64 mg·L−1), in paper mill effluent reported by
Singh et al. [52].
3.2. Effect of Paper Mill Effluent on
Characteristics of Soil
Physico-chemical characteristics of the soil characteris-
tics changed due to irrigation with paper mill effluent. At
harvest (120 days after sowing) there was no significant
change in the soil texture (loamy; 40% sand: 40% silt:
20% clay). WHC and BD were insignificantly (P > 0.05)
affected by different concentrations of paper mill effluent
in both the cultivated seasons (Table 2). Season, paper
mill effluent concentration and the their interaction af-
fected OC, TKN, all cations like Na+, K+, Ca2+, Mg2+,
Fe2+, anions 3
4
PO

and and metals Cd, Cr, Cu,
Mn and Zn of the soil (Tables 2-4). It has also been ob-
served that effluent irrigation generally adds OC, Na+,
Ca2+, K+, Mg2+,
2
4
SO 
3
4
PO

, 3, Cl−, Zn, Cd, Cr, Cu, Ni
and Mn to the soil [29,51]. WHC and BD were reduced
from their initial (control) values 43.12% and 1.41
gm·cm−3 to 42.34% and 1.40 gm·cm−3 respectively with
100% paper mill effluent concentration. The pH of the
soil was turned alkaline to more alkaline (8.89 and 8.98)
after irrigation with 100% paper mill effluent in both
seasons (Table 5). The change in soil pH and reduction
in WHC and BD after paper mill effluent irrigation have
also been observed earlier by
HCO
Kumar and Chopra [29,31].
Paper mill effluent had significant (P < 0.01) effect on
EC, pH, OC, Na+, K+, Ca2+, Mg2+, TKN, 3
4
PO

, 2
4
SO

,
Fe, Zn, Cu, and Mn of the soil in both seasons (Table 5).
In the present study, more irrigation of T. foenum-
graecum considerably increased the content of OC, Na+,
K+, Ca2+, Mg2+, Fe2+, TKN, , Zn, Cd, Cu,
Mn and Cr in soil. Soil pH was affected by the 50%, 75%
and 100% paper mill effluent concentrations (Table 5).
The 25% to 100% paper mill effluent concentrations sig-
nificantly (P < 0.05) affected EC, OC, TKN, Na+, K+,
Ca2+, Mg2+, Fe2+,
3
4
PO 2
4
SO 
3
4
PO

, , Cu, Cr, Cd, Mn and Zn
in T. foenum-graecum cultivated soil in both seasons (Ta-
bles 5 and 6). Irrigation with 100% paper mill effluent
2
4

SO

V. KUMAR, A. K. CHOPRA
11
Table 1. Physico-chemical and microbiological characteristics of paper mill effluent (PME).
Effluent concentration (%)
Parameter
0 (BWW)a 5 10 25 50 75 100
BISb for
irrigation water
TDS (mg·L−1) 245.67 1468.50 1896.80 2412.37 2688.65 2998.77 4086.00 1900
EC (dS· m−1) 0.32 2.14 2.92 3.98 4.18 4.96 6.63 -
pH 7.58 7.68 7.79 7.86 7.97 8.24 8.74 5.5-9.0
DO (mg·L−1) 8.48 5.34 4.76 3.98 2.45 1.74 Nil -
BOD (mg·L−1) 3.76 78.90 168.56 342.43 632.48 964.57 1256.84 100
COD (mg·L−1) 5.84 172.34 324.66 842.78 1556.76 2256.92 2976.40 250
Cl− (mg·L−1) 62.45 83.66 142.70 264.60 512.45 785.54 970.50 500
3
HCO (mg·L−1) 264.70 293.64 312.44 424.88 879.90 996.45 1034.56 -
2
3
CO  (mg·L−1) 110.88 128.45 262.70 368.97 670.44 825.60 934.65 -
Na+ (mg·L−1) 8.34 32.44 65.86 142.34 294.30 388.55 507.32 -
K+ (mg·L−1) 4.76 18.61 39.44 84.50 172.80 234.72 287.34 -
Ca2+ (mg·L−1) 29.60 68.90 127.78 264.47 448.90 593.90 798.30 200
Mg2+ (mg·L−1) 12.78 24.56 50.76 112.30 182.56 258.45 326.72 -
TKN (mg·L−1) 19.10 38.60 78.20 104.76 209.33 312.44 432.65 100
2
3
NO  (mg·L−1) 34.56 58.40 119.34 198.34 267.88 487.20 562.34 100
3
4
PO  (mg·L−1) 0.06 10.56 22.12 56.70 115.50 185.42 234.50 -
2
4
SO  (mg·L−1) 78.90 134.80 202.50 422.41 845.68 1460.20 1696.40 1000
Fe2+ (mg·L−1) 0.42 1.19 2.42 4.98 10.04 15.78 20.12 1.0
Cd (mg·L−1) BDLc 0.72 1.43 2.98 5.44 7.24 10.90 15
Cr (mg·L−1) BDL 1.32 2.68 5.67 10.34 16.78 21.34 2.00
Cu (mg·L−1) BDL 1.19 2.39 5.93 11.23 18.43 22.49 3.00
Mn (mg·L−1) 0.02 0.72 1.45 4.26 7.70 10.61 15.45 1.00
Zn (mg·L−1) 0.04 0.60 1.22 3.12 6.26 8.42 12.56 2.00
SPC (SPC mL−1) 4.8 × 101 6.8 × 105 5.3 × 106 7.3 × 108 8.6 × 109 9.2 × 1010 9.7 × 1013 10000
MPN (MPN 100 mL−1) 3.6 × 101 4.7 × 104 6.4 × 105 8.1 × 106 5.7 × 108 6.8 × 109 6.2 × 1011 5000
aBWW = bore well water; bBIS = bureau of Indian standard; cBDL = below detection limit; Least squares means analysis.
Table 2. ANOVA for effect of paper mill effluent on soil
characteristics. Table 4. ANOVA for effect of paper mill effluent on con- centrations of metals.
Source WHC BDEC pH OCTKN
Season (S) ns ns ns ns * *
PME concentration (C) ns ns ** * ** **
Interaction S × C ns ns * * ** **
Source Cd Cr Cu Mn Zn
Season (S) * * * ns *
PME concentration (C)** ** ** * **
Interaction S × C ** ** ** ** **
ns, *, **Non-significant or significant at P ≤ 0.05 or P ≤ 0.01, ANOVA.
ns, *, **Non-significant or significant at P ≤ 0.05 or P ≤ 0.01, ANOVA.
had the most reduction in WHC, BD; and increase in EC,
OC, Na+, K+, Ca2+, Mg2+, Fe2+, TKN, ,
3
4
PO 2
4
SO

, Cd,
Cr, Cu, Mn and Zn in both seasons (Tables 5 and 6). The
findings were very much in accordance with Patterson et
al. [51].
Table 3. ANOVA for effect of paper mill effluent on con-
centrations of cations and anions.
Source Na+ K
+ Ca2+ Mg2+ Fe2+ 3
4
PO

2
4
SO

Season (S) * * * * * * *
PME concentration (C) ** * * * ** ** **
Interaction S × C ** ** ** ** ** ** **
Total average organic matter content in the soil irri-
gated with effluent was higher than the soil irrigated with
bore well water. The more organic matter in effluent ir-
rigated soil might be due to the high organic nature of the
*, **Significant at P ≤ 0.05 or P ≤ 0.01, ANOVA.

V. KUMAR, A. K. CHOPRA
12
Table 5. Effects of paper mill effluent concentration and season interaction on physico-chemical characteristics of a loamy
soil before and after irrigation of T. foenum-graecum in both seasons.
Season × %PME EC (dS·m−1) pH OC (mg·L−1) Na+ (mg·L−1) K+ (mg·L−1) Ca2+ (mg·L−1) Mg2+ (mg·L−1)
0 2.17 7.67 0.42 19.34 165.40 18.54 1.82
5 2.64ns 8.17ns 1.69* 26.44ns 175.39ns 39.20ns 4.34ns
10 2.76ns 8.32ns 3.77* 28.93* 184.69ns 47.80ns 7.89ns
25 2.96* 8.41ns 5.76** 33.42* 196.86* 87.45* 11.30*
50 3.02* 8.57* 6.87** 39.67* 218.73* 129.50* 14.42*
75 3.15* 8.82* 8.45** 44.54** 234.40** 152.67* 18.55*
Winter
100 3.27** 8.89* 10.12** 50.72** 239.86** 175.68** 25.90*
0 2.18 7.69 0.44 19.88 165.70 18.89 1.83
5 2.78ns 8.22ns 1.74* 29.60ns 182.20ns 42.57ns 4.54ns
10 2.94ns 8.36ns 4.86* 31.87* 206.77ns 50.56ns 9.05ns
25 3.07* 8.47ns 6.75** 36.75* 214.79* 92.55* 13.24*
50 316* 8.69* 8.98** 42.32* 227.56* 135.65* 16.34*
75 3.26* 8.87* 9.78** 47.50** 239.54** 161.34* 20.11*
Summer
100 3.32** 8.98* 11.56** 54.66** 248.70** 180.40** 28.76*
ns, *, **Non-significant or significant at P < 0.05 or P < 0.01, Least Squares Means analysis.
Table 6. Effects of paper mill effluent concentration and season interaction on physico-chemical characteristics of a loamy
soil before and after irrigation of T. foenum-graecum in both seasons.
Season × %PME TKN
(mg·L−1)
3
4
PO 
(mg·L−1)
2
4
SO 
(mg·L−1)
Fe2+
(mg·L−1)
Cd
(mg·L−1)
Cr
(mg·L−1)
Cu
(mg·L−1)
Mn
(mg·L−1)
Zn
(mg·L−1)
0 42.23 55.70 78.90 2.86 0.76 0.87 2.12 0.46 1.11
5 62.96ns 72.76ns 80.77ns 4.09* 2.30* 1.92* 3.88* 1.29* 1.90*
10 74.50** 82.55ns 92.30ns 5.04* 2.89* 3.02* 4.89* 1.43* 2.54*
25 145.76** 102.20* 103.54* 7.10** 3.94** 5.12** 6.10** 2.07** 2.78**
50 217.80** 118.60** 127.77* 8.78** 5.14** 6.09** 7.21** 2.96** 3.98**
75 278.56** 128.77** 138.90** 9.23** 6.21** 6.88** 8.44** 3.62** 4.32**
Winter
100 304.66** 138.79** 147.84** 11.20** 7.34** 7.93** 9.87** 4.35** 5.11**
0 42.88 56.12 78.98 2.86 0.78 0.88 2.13 0.48 1.13
5 68.87ns 78.92ns 86.60ns 4.20* 2.67* 2.11* 3.98* 1.42* 1.98*
10 82.45** 88.96* 97.56* 5.60* 3.43* 3.25* 5.11* 1.96* 2.78*
25 153.60** 108.84* 110.24* 7.44** 4.56** 5.60** 6.34** 2.12** 3.64**
50 224.78** 124.69** 134.80* 9.32** 5.78** 6.87** 7.50** 3.05** 4.12**
75 286.80** 134.56** 146.45** 10.94** 6.54** 7.45** 8.56** 3.77** 4.78**
Summer
100 312.87** 145.60** 156.70** 12.67** 7.80** 8.32** 10.33** 4.56** 5.67**
ns,*, **Non-significant or significant at P < 0.01; Least Squares Means analysis.
effluent. Kumar [29] found the organic content in the soil
irrigated with paper mill effluent to be higher than in the
soil irrigated with bore well water. Average values of
TKN, and K+ in the soil irrigated with effluent
were found to be higher than in soil irrigated with bore
well water. The high amount of TKN, and K+ in
the soil was due to irrigation with TKN,
3
4
PO 
3
4
PO 
PO3
4

and K+
rich paper mill effluent. The content of Na+ and 2
4
SO

was higher in the soil irrigated with paper mill effluent
indicating a link between soil Na+ and and higher
EC in the paper mill effluent.
2
4
SO 
The soil parameters, EC, OC, Na+, K+, Ca2+, Mg2+,
Fe2+, TKN, 3
4
PO

, 2
4
SO

, Zn, Cd, Cu, Mn and Cr posi-
tively correlated with paper mill effluent concentration in
both seasons (Table 7). The enrichment factor (Ef) of the
metals indicated that Cd was highest while Fe was lowest
in both seasons after irrigation with 100% paper mill
effluent. The Ef of metals were in the order of Cd > Mn

V. KUMAR, A. K. CHOPRA 13
Table 7. Coefficient of correlation (r) between paper mill
effluent and soil characteristic s in both seasons.
Paper mill effluent/soil characteristics Season r-value
Winter −0.97
Paper mill effluent versus soil WHC
Summer −0.98
Winter −0.95
Paper mill effluent versus soil BD
Summer −0.96
Winter +0.87
Paper mill effluent versus soil EC
Summer +0.88
Winter +0.90
Paper mill effluent versus soil pH
Summer +0.91
Winter +0.97
Paper mill effluent versus soil OC
Summer +0.98
Winter +0.95
Paper mill effluent versus soil Na+
Summer +0.96
Winter +0.96
Paper mill effluent versus soil K+
Summer +0.97
Winter +0.91
Paper mill effluent versus soil Ca2+
Summer +0.92
Winter +0.96
Paper mill effluent versus soil Mg2+
Summer +0.97
Winter +0.98
Paper mill effluent versus soil TKN
Summer +0.99
Winter +0.95
Paper mill effluent versus soil
3
4
PO 
Summer +0.94
Winter +0.99
Paper mill effluent versus soil
2
4
SO 
Summer +0.99
Winter +0.97
Paper mill effluent versus soil Fe2+
Summer +0.98
Winter +0.97
Paper mill effluent versus soil Cd
Summer +0.96
Winter +0.98
Paper mill effluent versus soil Cr
Summer +0.97
Winter +0.98
Paper mill effluent versus soil Cu
Summer +0.99
Winter +0.94
Paper mill effluent versus soil Mn
Summer +0.95
Winter +0.96
Paper mill effluent versus soil Zn
Summer +0.97
> Cr > Cu > Zn > Fe after irrigation with paper mill ef-
fluent in both seasons (Figure 1). The concentrations of
metals were higher in soil irrigated with effluent than in
0
2
4
6
8
10
12
14
CdCrCu Fe Mn Zn
Metals
En r ich ment factor (Ef)
Ef in wi nt er seas o n
Ef in s ummer s eason
Figure 1. Enrichment factor of the metals in soil after ferti-
gation with paper mill effluent. Error bars are standard
error of the mean.
soil irrigated with control water. Thus, fertigation with
distillery effluent increased nutrients as well as metals
content in soil. Enrichment of various metals was also
observed by Fazeli et al. [4] in soil after paper mill ef-
fluent irrigation.
3.3. Effect of Paper Mill Effluent on Seed
Germination of T. foenum-graecum
At 0 - 15 days after sowing, the maximum seed germina-
tion (98% and 96%) was for with control and the least
(87% and 86%) was due to treatment with 100% paper
mill effluent (Figure 2). Germination was negatively
correlated (r = −0.96 and r = −0.97) with paper mill ef-
fluent concentrations in both seasons. The ANOVA in-
dicated that season had no significant (P > 0.05) effect on
seed germination and relative toxicity. Paper mill efflu-
ent concentration and their interaction with season af-
fected seed germination of T. foenum-graecum, but not
relative toxicity (Table 8).
The maximum relative toxicity (110.34% and 113.95%)
of paper mill effluent against germination was for the
100% paper mill effluent (Figure 3) and it was positively
correlated (r = +0.50 and r = +0.52) with paper mill ef-
fluent concentrations in both seasons. The findings are
very much in accordance with Medhi et al. [53] reported
that the germination of green gram (Brassica campes-
tris L. and Pisum sativum L.) was decreased as concen-
tration of the paper mill effluent increased from 0 to
100%. The findings were also supported by Reddy and
Borse [32].
In the present investigation, the higher concentration
of paper mill effluent did not support seed germination.
The higher concentration of paper mill effluent lowered
germination of T. foenum-graecum likely due to presence
of high salt content in the effluent at these concentrations.
Seed take up water during germination and hydrolyse
stored food material and to activate enzymatic systems.
During germination salts can inhibit seed germination.

V. KUMAR, A. K. CHOPRA
14
Table 8. ANOVA for effect of paper mill effluent on germination and vegetative growth of T. foenum-graecum.
LAISource Seed germination Relative toxicity Plant heightRoot lengthDry weight Chlorophyll content
S eason (S)ns ns ns ns ns ns ns
PME concentration (C) * ns * ns ns * ns
Interaction S × C * ns * ns ns * ns
ns, *Nificant at P ≤ 5, ANOVA. on-significant or sign0.0
0
20
40
60
80
100
120
140
0510 2550 75100
Paper mil l efflue nt concentra tions (%)
Seed germination (%)
Germin at i on in su mmer seaso n
Germination in winter season
Figure 2. Seed germination of T. foenum-graecum after fer-
tigation with paper mill effluent. Error bars are standard
error of the mean.
0
20
40
60
80
100
120
140
160
0510 25 5075100
P aper mill effl uent co n c entr atio n (%)
R ela tive toxic ity (%)
Relative tox icity in summe r season
Relative to xicity in wi nter sea son
Figure 3. Relative toxicity of paper mill effluent against
ion of seed germination by
3.4. Effect of Paper Mill Effluent on Vegetative
and 8.67 cm), dry
owered the plant
seed germination of T. foenum-graecum. Error bars are
standard error of the mean.
he mechanism of inhibitT
NaCl may be related to radical emergence due to insuffi-
cient water absorption, or to toxic effects on the embryo.
Seed that absorb an insufficient amount of water can ac-
cumulated a large amount of Cl− when the osmotic pres-
sure of the substrate is increased by salt concentration,
and as a result, the seeds emerged slowly, and at higher
concentrations do not germinate [3,51]. High concentra-
tions are usually most damaging to young plants but not
necessarily at germination, although high salt concentra-
tion can slow germination by several days, or completely
inhibit it. Because soluble salts move readily with water,
evaporation moves salts to the soil surface where they
accumulate and harden the soil surface delays germina-
tion [30,52].
Growth of T. foenum-graecum
Vegetative growth at 45 days after sowing was affected
in both seasons (Table 8). Average plant height (24.25
29.45 cm), root length (7.89 and
weight (1.42 and 1.47 g), chlorophyll content (3.48 and
3.52 mg./g.f.wt) and LAI/plant (3.31 and 3.34) of T. foe-
num-graecum were observed with control while plant
height (30.86 and 32.75 cm), root length (10.36 and
11.48 cm), dry weight (1.67 and 1.72 g), chlorophyll
content (3.61 and 3.65 mg./g.f.wt) and LAI/plant (3.36
and 3.39) of T. foenum-graecum were noted with 100%
paper mill effluent in both seasons.
Maximum plant height (36.75 and 39.89 cm), root
length (11.78 and 12.14 cm), dry weight (1.87 and 1.92
g), chlorophyll content (3.86 and 3.92 mg./g.f.wt) and
LAI/plant (3.56 and 3.64) of T. foenum-graecum were
due to treatment with the 25% concentration of paper
mill effluent in both seasons. The findings were also sup-
ported by Reddy and Borse [32]. The ANOVA indicated
that paper mill effluent concentration significantly (P <
0.05) affected plant height, and chlorophyll content of T.
foenum-graecum (Table 8). Season had no effect on
plant height, root length, dry weight and LAI of T. foe-
num-graecum. The interaction of season and paper mill
effluent concentrations only affected plant height and
chlorophyll content of T. foenum-graecum (Table 8).
Plant height, root length, dry weight, chlorophyll con-
tent and LAI/plant of T. foenum-graecum were positively
correlated with paper mill effluent concentrations in both
seasons (Table 9). Redd y and Borse [32] reported the
maximum chlorophyll content in T. foenum-graecum at
25% concentration of distillery effluent. Medhi et al. [53]
reported that paper mill effluent irrigation increase chlo-
rophyll and protein contents in Indian mustard plants
(Brassica campestris L.) at the 25 and 50% paper mill
effluent concentrations followed by a decrease at 75 and
100% paper mill effluent. The findings were also sup-
ported by Reddy and Borse [32] who reported that the
growth of T. foenum-graecum (L.) decreased when con-
centration of paper mill increased.
Vegetative growth of T. foenum-graecum was de-
creased at higher concentrations of paper mill effluent. It
is likely due to that higher salt content in the higher paper
mill effluent concentrations, which l

V. KUMAR, A. K. CHOPRA 15
he
of flowers decreased as paper mill effluent
36.0 paper mill effluent in both
or flower production, or flower abortion. Maxi-
m
after showing) the most
(102f T. foenum-graecum were
ight, root length, dry weight, chlorophyll content and
LAI/plant of T. foenum-graecum. Vegetative growth is
associated with development of new shoots, twigs, leaves
and leaf area. Plant height, root length, dry weight and
LAI/plant of T. foenum-graecum were higher at 25% of
paper mill effluent it may be due to maximum uptake of
nitrogen, phosphorus and potassium by plants. The im-
provement of vegetative growth may be attributed to the
role of potassium in nutrient and sugar translocation in
plants and turgor pressure in plant cells. It is also in-
volved in cell enlargement and in triggering young tissue
or mersitematic growth [29,32]. Chlorophyll content was
higher due to use of 25% paper mill effluent in both sea-
sons, and is likely due to Fe, Mg and Mn contents in the
paper mill effluent, which are associated with chloro-
phyll synthesis [45]. The 25% paper mill effluent con-
centration contains optimum contents of nutrients re-
quired for maximum vegetative growth of T. foenum-
graecum.
3.5. Effect of Paper Mill Effluent on Flowering of
T. foenum-graecum
Numbers
concentration decreased (Table 9). At flowering stage
(60 days after sowing) the maximum flowers (33.00 and
0) was noted with 25%
seasons. Numbers of flowers/plant 23.00 and 25.00 were
with control and 27.00 to 29.00 with 100% paper mill
effluent in both seasons. Season, paper mill effluent con-
centration and interaction of season and paper mill ef-
fluent concentration had no significant (P > 0.05) effect
on number of flowers and number of fruits/plant (Table
10).
Nitrogen and phosphorus are essential for flowering.
Too much nitrogen can delay, or prevent, flowering
while phosphorus deficiency is sometimes associated
with po
um flowering was with the 25% paper mill effluent; it
might be due to that this concentration contains sufficient
nitrogen and phosphorus. Furthermore, P and K prevent
flower abortion so pod formation occurs [29,31,54].
Flowering of T. foenum-graecum was lower at higher
concentrations of paper mill effluent. This is likely due to
increased content of metals in the soil, which inhibits up-
take of P and K by plants at higher paper mill effluent
concentrations [29,31,32,54].
3.6. Effect of Paper Mill Effluent on Maturity of
T. foenum-graecum
At maturity stage (120 days
fruits/plants (31.00 and 34.00), fruit length (9.36 cm and
9.76 cm) yield/plant (19.14 and 22.39 g), and HI
3.52 and 1166.14%) o
Table 9. Coefficient of correlation (r) between paper mill
effluent and T. foenum-graecum in both seasons.
Paper mill effluent/T. foenum-graecum Season r-value
Winter +0.67
Paper mill effluent versus shoot length Summer +0.66
Paper mill effluent versus dry weight
Paper mill effluent versus chlorophyll content
Paper mill effluent versus LAI
Paper mill effluent versus no. of flowers/plant
Paper mill effluent versus no. of fruits
Paper mill effluent versus fruit length
Paper mill effluent versus crop yield/plant
Paper mill effluent versus harvest index
Paper mill effluent versus Cd
Paper mill effluent versus Cr Su +0.
Paper mill effluent versus Cu
Paper mill effluent versus Mn
Paper mill effluent versus Zn
Winter +0.18
Paper mill effluent versus root length Summer +0.17
Winter +0.27
Summer +0.28
Winter +0.29
Summer +0.30
Winter +0.47
Summer +0.49
Winter +0.48
Summer +0.47
Winter +0.58
Summer +0.57
Winter +0.48
Summer +0.49
Winter +0.14
Summer +0.13
Winter +0.47
Summer +0.43
Winter +0.98
Summer +0.99
Winter +0.97
mmer96
Winter +0.98
Summer +0.99
Winter +0.99
Summer +0.98
Winter +0.96
Summer +0.97
Table 10. ANOVA for effect of paper mnt -
ering and matuum-gr
Source No. of
flowers/plant
No. of
fruits
Fruit
length yield/plant index (HI)
ill efflueon flow
rity stage of T. foenaecum.
Crop Harvest
Season (S) ns ns ns ns ns
PME
concentration (C)
Interaction S × C
ns
ns
ns
ns
ns ns
ns
ns
ns ns
ns, non-ant.
r mill effluent in both seasons. Num-
lanrop d/pt anarvestx
um-graecum were positively correlated
signific
with the 25% pape
bers of fruits/pt, cyielland h inde
(HI) of T. foen

V. KUMAR, A. K. CHOPRA
16
with paper mill effluent concentrations in both seasons
d
yi
ons
n and
the interactiooncen-
(Table 9). Numbers of fruits/plant, crop yield/plant and
harvest index (HI) of T. foenum-graecum were not af-
fected by season, paper mill effluent concentration and
their interaction (Table 10). The number of fruits/plants
(21.00 and 23.00), fruit length (6.10 cm and 6.32 cm)
yield/plant (12.45 and 13.52 g), and HI (876.76 and
919.72%) of T. foenum-graecum were with the control
while with 100% paper mill effluent the fruits/plants
(25.00 and 27.00), fruit length (8.24 cm and 8.29 cm)
yield/plant (15.42 and 16.34 g), and HI (923.35% and
950.00%) of T. foenum-graecum were in both seasons.
The role of K, Fe, Mg and Mn at maturity is important
and associated with synthesis of chlorophyll, and en-
hances formation of fruits at harvest [29,52]. The K, Fe,
Mg and Mn contents could benefit pod formation an
eld of as it does for fenugreek (T. foenum-graecum L.)
as reported by Reddy and Borse [32]. The 25% paper
mill effluent favored fruits formation and crop yield of T.
foenum-graecum. This is likely due to presence of K, Fe,
Mg and Mn contents in 25% paper mill effluent; higher
paper mill effluent concentrations lowered fruits forma-
tion and crop yield of T. foenum-graecum .
3.7. Effect on Biochemical Constituents and
Metals in T. foenum-graecum
The content of various metals were positively correlated
with concentrations of paper mill effluent in both seas
(Table 9). Season, paper mill effluent concentratio
n of season and paper mill effluent c
tration affected all the biochemical constituents like
crude fiber, and crude carbohydrates, and metals like Cd,
Cr, Cu, Mn and Zn in T. foenum-graecum (Table 11).
Maximum crude proteins, crude fiber and crude carbo-
hydrates were recorded with 25% paper mill effluent
concentrations in both seasons (Figures 4-6). Content of
crude proteins (r = +0.45 and r = +0.47), crude fiber (r =
+0.37 and r = +43) and crude carbohydrates (r = +0.59
and r = +0.61) were noted positively correlated with pa-
per mill effluent concentration in both seasons. The 25%,
50%, 75% and 100% paper mill effluent concentrations
affected Cd, Cr, Cu, Fe, Mn and Zn contents in T. foe-
Table 11. ANOVA for effect of paper mill effluent on flow-
ering and maturity stage of T. foenum-graecum.
Source Cd Cr Cu Mn ZnCrude Crude Crude
proteins fiber carbohydrates
Season (S) * * * ns * * * *
PME
concentration
(C)
** ** ** ** ** ** ** **
Interaction
S × C
** ** ** ** ** ** ** **
0
10
20
30
C ru de proteins i n summer season
40
50
60
ns, *, **Non-significant or significant at P ≤ 0.05 or P ≤ 0.01, ANOVA.
0510 25 50 75100
Pape r mill effluent concentration (%)
Crudeeins (%) prot
Crude pr ot e i nwr so ns i n inteeas
Figure 4. Crude proteins in T. foenum-graecum after ferti-
gation with paper mill effluent. Error bars are standard
error of the mean.
0
10
20
30
40
50
60
70
80
iber (%)
90
100
0510 25 50 75100
Paper mill effluent concentration (%)
Crude f
Crude fiber i n summer season
Crude fiber in winter season
Figure 5. Crude fiber in T. foenum-graecum after fertiga-
tion with paper mill effluent. Error bars are standard error
of the mean.
0
10
20
30
40
50
60
hydr ates (%)
70
0510255075100
Pape r mill effluent concentration (%)
Crude car b o
Crude carbohydrates in summer season
Crude c arbohydrates in wi nter season
Figure 6. Crude carbohydrates in T. foenum-graecum after
fertigation with paper mill effluent. Error bars are stan-
dard error of the mean.
num-graecum. Increased irrigation frequency could lead
to increases of metals in tissues. The Cd, Cr, Cu, Fe, M
observed by Fazeli et al. [4] in
n
and Zn contents in T. foenum-graecum was highest with
100% paper mill effluent (Figures 7, 8). Enrichment of
various metals was also
paddy crops after paper mill effluent irrigation. The find-
ings are very much in accordance with Pathak et al.
[9,10].
The enrichment factor (Ef) was affected in both sea-

V. KUMAR, A. K. CHOPRA 17
Figure 7. Content of Cd, Cr and Cu in T. foenum-graecum
after fertigation with paper mill effluent. Error bars are
standard error of the mean.
Figure 8. Content of Fe, Mn and Zn in T. foenum-graecum
after fertigation with paper mill effluent. Error bars are
standard error of the mean.
sons (Figure 9). The Ef of various metals was in order of
Mn > Cu > Cr > Cd > Zn > Fe in T. foenum-graecum
both seasons. The metals contents were higher at
.fertigation improved the soil
affected the growth of T. foenum-
easons. The most agronomical growth
raec was
after irrigation with paper mill effluent (Figure 9). The
highest enrichment factor was for Mn; the least was for
Fe in T. foenum-graecum with 100% paper mill effluent
in
higher paper mill effluent concentration, and likely in-
hibited growth of T. foenum-graecum. The 25% paper
mill effluent favored vegetative growth, flowering and
maturity of T. foenum-graecum. This is likely due to op-
timal uptake of these micronutrients by crop plants,
which supports various biochemical and physiological
processes.
4. Conclusion
The present investigation concluded that, paper mill ef-
fluent fertigation increased EC, pH, OC, Na+, K+, Ca2+,
Mg2+, TKN, 3 PO , 2 SO , Fe, Zn, Cu, and Mn of the
soil in both s
4
easons
4
Thus,
nutrient status and
graecum in both s
of T. foenum-graecum was observed with 25% concen-
tration of paper mill effluent in both seasons. The growth
of T. foenum-gum inhibited at higher concentra-
0
2
4
6
8
10
12
14
16
hme n t factor (Ef)
Cd Cr Cu FeMn Zn
Metals
Enric
Ef i n sum mer season
E f in winter season
Figure 9. Enrichment factor of various metals in T. foe-
num-graecum after fertigation with paper mill effluent. Error
bars are standard error of the mean.
tions (50% to 100%), it might be due to the presence of
more content of heavy metals at these concentration
num-graecum plants
fter fertigation with paper mill effluent. Among both
cessing Zone (DEPZ), Bangladesh: Implication of Sea-
sonal Variation and Indices,” Applied Sciences, Vol. 2,
No. 3, 2012, papp2030584
s.
The enrichment factor (Ef) of various heavy metals was
in order of Cd > Mn > Cr > Cu > Zn > Fe for soil and Mn
> Cu > Cr > Cd > Zn > Fe for T. foe
a
seasons, maximum agronomical performance of T. foe-
num-graecum was noted in winter season. The effluent
has potentiality for its use as biofertigant in the form of
plant nutrients needed by T. foenum-graecum crop plant.
Therefore, it can be used as agro-based biofertigant after
its appropriate dilution for irrigation purposes for the
maximum yield of this crop. Further studies on the agro-
nomic growth and changes in biochemical composition
of T. foenum-graecum after paper mill effluent irrigation
are required.
5. Acknowledgements
The University Grants Commission, New Delhi, India is
acknowledged for providing the financial support in the
form of UGC research fellowship to the corresponding
author.
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