Bull Environ Contam Toxicol (2015) 95:687–693
DOI 10.1007/s00128-015-1600-y
Morphological, Physiological and Biochemical Impact of Ink
Industry Effluent on Germination of Maize (Zea mays), Barley
(Hordeum vulgare) and Sorghum (Sorghum bicolor)
Chaâbene Zayneb1 • Khanous Lamia2 • Ellouze Olfa3 • Jebahi Naı̈ma1 •
C. Douglas Grubb4 • Khemakhem Bassem1 • Mejdoub Hafedh1 • Elleuch Amine1
Received: 7 July 2014 / Accepted: 8 July 2015 / Published online: 4 September 2015
Ó Springer Science+Business Media New York 2015
Abstract The present study focuses on effects of
untreated and treated ink industry wastewater on germi-
nation of maize, barley and sorghum. Wastewater had a
high chemical oxygen demand (COD) and metal content
compared to treated effluent. Germination decreased with
increasing COD concentration. Speed of germination also
followed the same trend, except for maize seeds exposed to
untreated effluent (E), which germinated slightly faster
than controls. These alterations of seedling development
were mirrored by changes in soluble protein content. E
exerted a positive effect on soluble protein content and
maximum levels occurred after 10 days with treated
effluent using coagulation/flocculation (TEc/f) process and
treated effluent using combined process (coagulation/floc-
culation/biosorption) (TEc/f/b). Likewise, activity of
a-amylase was influenced by effluent composition. Its
expression depended on the species, exposure time and
applied treatment. Nevertheless, current results indicated
TEc/f/b had no observable toxic effects on germination and
could be a beneficial alternative resource to irrigation
water.
& Elleuch Amine
amineelleuch@hotmail.com
1 quantities of wastewater are packaging and printing. The
Laboratoire des Biotechnologies Végétales Appliquées à
l’Amélioration des Cultures, Faculté des Science de Sfax,
Université de Sfax, BP 1171, 3000 Sfax, Tunisia
2
Unité de Toxicologie-Microbiologie Environnementale et
Santé/Faculté des Science de Sfax, Université de Sfax,
BP 1171, 3000 Sfax, Tunisia
3
Unité de Bioconversion des Enzymes/Ecole National des
Ingénieurs de Sfax, Université de Sfax, Sfax, Tunisia
4
Leibniz Institute of Plant Biochemistry, Halle, Germany
Keywords Effluent treatment  Seedling mortality 
Speed of germination  Soluble protein  a-amylase
As a result of water supply and demand imbalances,
wastewater reclamation and reuse has become an important
element in water resource planning in arid and semi-arid
regions (Abedi-Koupai and Bakhtiarifar 2003). In recent
years, a number of Mediterranean countries such as Tuni-
sia, have practiced various water conservation methods
especially in agriculture, the largest water consumer. For
this reason, countries in this area are greatly concerned
with water-saving measures and with upgrading and
streamlining management (Neubert and Benabdallah
2003). Untreated wastewater is widely used in agriculture
and can introduce a large amount of organic and inorganic
contaminants, including metals, into agricultural lands
(Hussain et al. 2001). However, the use of non-conven-
tional water resources, such as treated wastewater, could be
of benefit to arid countries. Costs of treatment can be
prohibitive (Al-Lahlam et al. 2003). Irrigation of agricul-
tural fields using treated wastewater (TE) under controlled
conditions is being extensively practiced in countries faced
with water shortage problems, but the safety of this prac-
tice is largely unexamined.
Among industrial activities that discharge significant
latter is an example of industries posing increasing envi-
ronmental problems. The printing industry has evolved to
adapt to market requirements, with an annual rate of growth
exceeding 93 %. Ink production, in particular, is among
industries that contribute most to water pollution (Patil et al.
2010). This pollution is driven by effluents that may contain
a wide variety of pollutants (dyes, pigments, solvents, met-
als, etc.) (Ma and Xia 2009). Effluent toxicity is related to its
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composition, particularly the presence of recalcitrant
chemicals with carcinogenic and mutagenic effects (He et al.
2013). Hence, this wastewater cannot be directly discharged
into ecosystems without treatment, not only due to its
deleterious effect on human health and the environment, but
also aesthetically due to the visibility of pigments, even at
low concentrations (Ding et al. 2011). Previous research has
demonstrated the efficiency of a combined process (coagu-
lation/flocculation and biosorption) for treatment. Reuse of
treated printing ink wastewater for agriculture could poten-
tially be part of the solution to problems of water scarcity.
However, even treated effluent may have an effect on plant
development and growth. Metal accumulation in plant tissue
can decrease the biomass, as well as chlorophyll and protein
concentrations in leaves and stems (Manios et al. 2002).
This study aimed to evaluate the potential effects of
untreated and treated printing ink effluent when used to
irrigate maize (Zea mays), barley (Hordeum vulgare) and
sorghum (Sorghum bicolor) seeds for germination. To
obtain a more complete picture of effluent effects, various
morphological, physiological and biochemical parameters
were monitored in detail.
Materials and Methods
Wastewater was collected from a Tunisian company spe-
cializing in manufacturing custom corrugated packaging
and paper processing and manufacturing bags and envel-
opes. Effluent from print washing machines was collected.
Wastewater samples were taken from the inlet and outlet
station. Different types of wastewater have been used in
this study: untreated effluent (E); treated effluent using
coagulation/flocculation process (TEc/f) and treated effluent
using combined process (coagulation/flocculation/biosorp-
tion) (TEc/f/b) (Table 1).
Mineral contents of effluent samples were also deter-
mined. Each sample was digested with nitric acid 1 %
HNO3 (v/v) and filtered. The filtrate was collected in a
flask, diluted and directly analyzed using atomic absorption
spectrophotometry (Perkin Elmer-Model 2380) to deter-
mine metal concentrations in mg/L (Joseph et al. 2006).
Seeds of maize (Z. mays), barley (H. vulgare) and sor-
ghum (S. bicolor) were surface-sterilized with 15 %
sodium hypochlorite followed with repeated washing with
sterilized distilled water. Uniform sized seeds (10) were
placed on filter paper in each petri dish and 10 mL of each
solution was used. Effluents were applied every alternate
day. Petri dishes were maintained at 25 ± 1°C with a 16 h
period of light in a growth chamber after 2 days of dark-
ness. This in vitro germination was designed with three
replicates and all parameters were recorded every 2 days of
growth for 15 days.
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Bull Environ Contam Toxicol (2015) 95:687–693
Seed germination was recorded and total germination
was calculated and expressed in percentage (GP = seeds
germinated/total seeds 9 100). The speed of germination
(SG), mean daily germination (MDG) and the peak value
(PV) were measured according to Czabator (1962). Mean
germination time (MGT) was calculated following the
formula given by Ellis and Roberts (1981). However,
germination value was calculated following the formula
given by Osman (2004). The formula used to calculate
seedling mortality (SLM) was as follows: SLM = (number
of non-germinated seeds/number of days) 9 100.
Germination index (GI) was calculated following the
formula:
NE  LE
GI % ¼  100
NC  LC
where NE is the number of germinated seeds watered by
the effluent, NC is the number of germinated seeds in the
control, LE is the average length of the radical of germi-
nated seeds watered by the effluent, and LC is the average
length of the radical of germinated seeds in the control.
To determine soluble protein content, samples were
prepared by homogenizing the fresh tissue with a mortar
and pestle in a potassium phosphate buffer 0.1 M, pH 7
(1.5 mL: 0.5 g fresh weight) to extract soluble protein.
Homogenate was centrifuged at 13,000g for 15 min. All
operations were performed at 4°C. Protein concentration
determination was carried out spectrophotometrically
according to the method of Bradford (1976), using bovine
serum albumin as a standard.
The amount of reducing sugars released by the action of
amylases on starch was measured at 37°C and pH 7 for
10 min. The reaction mixture contained 0.5 % (w/v) starch
in 25 mM phosphate buffer (pH = 7) and the enzyme
solution in a final volume of 1 mL. The concentration of
reducing sugar was determined by the dinitrosalicylic acid
(DNS) method (Miller 1959). One unit of amylase was
defined as the amount of enzyme required to produce
reducing sugars equivalent to 1 lmol glucose/min.
Data in all experiments were statistically analyzed for
the calculation of standard error (S.E.). Student’s t test was
administered for testing hypotheses with the help of Sigma
Stat 2.0. Statistically significant differences at the p \ 0.05
level are indicated.
Results and Discussion
Physicochemical and biological constituent analyses of ink
wastewater results obtained before and after treatment
(Table 1) revealing that untreated effluent had a high COD
and metals concentrations (Cu, Ni and Pb) compared to
treated effluent. Treatment of wastewater using TEc/f or
Bull Environ Contam Toxicol (2015) 95:687–693 689

Table 1 Average physicochemical characteristics and metals concentration of treated and untreated ink wastewater in Tunisia
Parameters E TEc/f TEc/f/b WHO/FAO guideline WHO/FAO guideline
Irrigation water In plants (mg/kg)

pH 7.79 ± 0.79 7.25 7.25 6.5–8.4 –

COD (mg/L) 38,595 ± 20,926 656 388 90 –
NTU 3331 936 355 B5 –
RE-Tur (%) 98 44 44 – –
TMF (109 CFU/mL) 154 75 27 \200 –
Fungi Absent Absent Absent – –
Zinc (Zn) (mg/L) 42.86 2.32 0.49 2.0 60
Lead (Pb) (mg/L) 4.12 0.22 0.009 5.0 0.3
Sodium (Na) (mg/L) 900.5 195.84 127.41 23 25–150
Potassium (K) (mg/L) 72.9 6.92 10.17 39.1 15–50
Magnesium (Mg) (mg/L) 317.87 3.41 3.1 12.2 10–35
Nickel (Ni) (mg/L) 3.52 0.3 0.03 0.20 0.02–50
Iron (Fe) (mg/L) 6.73 0.24 0.16 5.0 400–500
Copper (Cu) (mg/L) 5.04 1.46 0 0.20 2.5
Cadmium (Cd) (mg/L) bd bd bd 0.01 \2.4
E untreated effluent, TEc/f treated effluent using coagulation/flocculation process, TEc/f/b treated effluent using combined process (coagulation/
flocculation/biosorption), COD chemical oxygen demand, NTU turbidity, Nephelometric Turbidity Unit, Re-Tur turbidity removal, TMF total
mesophilic flora, bd below detection

TEc/f/b removed about 90 % of organic matter, improving
treated water (NTU = 3331 in the E; NTU = 355 in the
TEc/f/b; Imen et al. 2012). In addition, analysis of the
effluent mineral content (Table 1) showed that except for
Pb and Cd, all measured elements in treated effluents were
below the permissible levels of metals in irrigation water as
per WHO (2006).
Combined treatment removed almost 100 % of some
metals, such Cu, Pb and Ni. There was no significant dif-
ference in the Mg, K and Fe concentration between TEc/f
and TEc/f/b. However, the concentration of Na in all tested
samples was far above recommended levels. Sodium tox-
icity may cause both direct and indirect damage to many
plants and reduce the availability of some micronutrients,
e.g. Fe and P.
Percent germination in all examined seeds irrigated with
untreated wastewater was much lower than the control
(distilled water; Fig. 1). The use of E significantly
(p \ 0.05) decreased germination by about 50 % for maize
and sorghum and about 75 % for barley. The presence of
toxic substances (organic and inorganic) in effluent might
be the origin of this inhibition (Table 1). Indeed, for barley
and sorghum, the rate of germination inhibition reached
levels similar to those observed for cabbage treated with
monosodium glutamate (MSG) wastewater (Liu et al.
2007). According to those authors, low dissolved oxygen
linked with high COD and TSS in effluent negatively
affected the germination, and later growth, of seedlings.
However, this same study found no such relationship for
tomato seeds treated with MSG wastewater (Liu et al.
2007). Secondly, metal content of effluents may adversely
affect the germination process. Metals can bind to func-
tionally important domains of biomolecules and thereby
inactivate them (Sarita et al. 2007). Moreover, Nagda et al.
(2006) found that, at higher concentrations of industrial
effluent, seed germination efficiency decreases. These
authors suggested osmotic stress caused by effluent with
higher concentrations of total salts makes imbibition more
difficult and retards germination.
The combined treatment process seems to be the best
method for water detoxification. It removed the bulk of
organic constituents and was especially effective at
decreasing metals such as Cu, Pb and Ni. Consequently,
TEc/f/b had no quantitative effects on germination. For
example, barley germinated at a rate of 99 % under TEc/f/b
treatment compared to 90 % and 22 % of seeds irrigated
with TEc/f and wastewater, respectively. These results
conform to the work of Fendri et al. (2012), who showed
biological treatment completely removed factors inhibiting
the germination of oat seeds.
Interestingly, while the germination of sorghum and
barley were almost unaffected by contaminants remaining
in TEc/f, maize seeds were actually more sensitive to this
partially treated effluent than to the raw effluent (Fig. 1).
Thus the suitability of wastewater treatment regimen
depends on the crop to be irrigated. Besides germination,
considerable variations were also recorded in different
physiological parameters that can provide important

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690 Bull Environ Contam Toxicol (2015) 95:687–693

Fig. 1 Maize, barley and sorghum germination percentage with effluent, TEc/f treated effluent using coagulation/flocculation process,
treated and untreated ink wastewater. Data are mean ± SD of three TEc/f/b treated effluent using combined process (coagulation/floccu-
independent experiments. An asterisk indicates a significant mean lation/biosorption)
difference from control at p \ 0.05. W distilled water, E untreated

information on effects of effluent on seeds. In fact, results
illustrated in Table 2 showed variability in the behavior of
plant species tested during germination. The speed germi-
nation (SG) was strongly reduced in both barley and sor-
ghum, but was almost unaffected in maize, except by TEc/f,
providing further evidence that this partially treated efflu-
ent contains something to which maize is especially
sensitive.
Similarly, previous research showed that Brassica napus
plants irrigated with higher concentrations of distillery
effluents had lower germination rates (Piyush and Anu-
radha 2011). Speed of germination also followed the same
trend. It was found to be maximal in the lower E concen-
tration (E20%) and minimum at high E concentration
(E100%) (Piyush and Anuradha 2011). Percent inhibition
was maximum in E100% (19.4 %) and minimum in E20%
which can be attributed to the increase in salt content with
increasing concentrations of effluent. The complex chem-
ical nature of toxicity induced by the latter was confirmed
by the augmentation of SLM following inhibition of ger-
mination. In contrast, the work of Ibrahim et al. (2012)
performed on maize seeds showed wastewater from food
processing industries contributed significantly to improve
germination in this species. Even if the capacity of ger-
mination was not affected by the various types of treated
water, MGT and MDG varied from the minimum for TEc/f
to the maximum for TEc/f/b, and the differences across
treatments were highly significant in maize and barley. It

Table 2 Wastewater effects on different physiological parameters of seed germination in Tunisia

Species SG (s/d) MDG (s/d) MTG (d) PV (s/d) SLM (%) GI (%)

Maize W 0.62 ± 0.16 0.52 ± 0.12 0.76 ± 0.06 0.31 ± 0.03 53.33 ± 15.28 100
E 0.69 ± 0.13 0.53 ± 0.11 0.84 ± 0.14 0.3 ± 0.05 73.33 ± 11.55 9 ± 0.14
TEc/f 0.3 ± 0.05 0.25 ± 0.13 0.48 ± 0.2 0.88 ± 0.96 90 ± 0 1 ± 0.03
TEc/f/b 0.71 ± 0.23 0.52 ± 0.17 0.73 ± 0.25 0.74 ± 0.65 56.66 ± 11.55 84.74 ± 1
Barley W 4.49 ± 0.33 2.41 ± 0.95 5.17 ± 3.11 2.83 ± 0.76 0±0 100 ± 1.66
E 0.37 ± 0.03 1 ± 1.32 0.6 ± 0.52 0.23 ± 0.2 76.67 ± 20.81 38.6 ± 0.3
TEc/f 2.92 ± 0.21 1.22 ± 0.21 2.95 ± 0.34 0.83 ± 0.57 11.54 ± 6.67 48.1 ± 2
TEc/f/b 3.63 ± 0.89 2.44 ± 0.96 2.38 ± 1.75 1.33 ± 0.58 13.33 ± 5.77 62.21 ± 5.7
Sorghum W 3.06 ± 0.63 1.61 ± 0.67 2.3 ± 2.1 3.68 ± 3 16.66 ± 15.2 100
E 0.68 ± 1.02 0.65 ± 0.5 0.52 ± 0.46 1.25 ± 0.19 33.33 ± 4 0
TEc/f 2.73 ± 0.27 1.5 ± 0.5 2.62 ± 2.35 2.39 ± 1.7 23.33 ± 20.8 52.37 ± 6
TEc/f/b 2.71 ± 0.56 1.47 ± 0.13 3.26 ± 2.9 3.41 ± 2.65 26.66 ± 21 387.3 ± 40
GP germination percentage, SG speed of germination, MGT mean germination time, MDG mean daily germination, SLM seedling mortality, PV
peak value, GI germination index

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Bull Environ Contam Toxicol (2015) 95:687–693
was also observed that PV of germination for treated water
was found to be maximal in barley and sorghum irrigated
seeds with TEc/f/b and minimal in E. The behavior of B.
napus seeds treated with distillery effluent followed the
same trend (Piyush and Anuradha 2011).
The GI for barley and sorghum varied from 100 % to
38 % and from 100 % to 0 %, respectively, in distilled
water (W) and E (Table 2). Similarly, increasing concen-
tration of anionic surfactants in wastewater induced
diminution of GI for Triticum aestivum and Secale cereal
(Ewa and Maciej 2007). The same results were recorded in
B. napus seeds treated with different distillery effluent
concentrations (Piyush and Anuradha 2011). According to
these authors, GI decline may be attributed to the toxicity
caused by increasing amount of various organic and inor-
ganic compounds present in higher wastewater concentra-
tions. Similar observations were made by Padhan and Sahu
(1999) while working on the effect of rice mill effluent on
seed germination. They found a decrease in the values of
GI in seeds treated with 60 % and 75 % effluent concen-
trations. However, relating to the low values of germina-
tion percentage, GI results obtained for maize seeds treated
with TEc/f (1 %) were less than the E (9 %). The current
study observed that TEc/f/b had no observable phytotoxic
effect on any of the test species.
The alteration of germination can be explained in part
by a modification in water absorption (a major factor for
seed germination). In fact, it may perturb mobilization of
crop reserves. In the present work, the biochemical
response of treated seeds was investigated by soluble
protein content changes under the various treatments
(Fig. 2).
Independent of water type, similar trends in soluble
protein levels over time for all seeds were evident. Indeed,
in the presence of E, protein levels seem to be higher
Fig. 2 Effects of various
effluents on soluble protein
content of maize, barley and
sorghum seedlings during
15 days (d) of exposure. Data
are mean ± SD of three
independent experiments. An
asterisk indicates a significant
mean difference from control at
p \ 0.05. W distilled water,
E untreated effluent, TEc/f
treated effluent using
coagulation/flocculation
process, TEc/f/b treated effluent
using combined process
(coagulation/flocculation/
biosorption)
691
compared to other types of water. Maximum protein con-
tent in major species occurred after 10 days of treatment
with TEc/f and TEc/f/b, followed by a drop at the 15th day.
These results may be due to an inhibition of proteolytic
activity in the presence of E. On the other hand, application
of a hybrid wastewater treatment may protect proteases,
since protein content in this treatment was lower than that
seen with raw effluent and similar to the control. The
positive effect of E on soluble protein content could be an
effect of the nutrients existing in untreated wastewater.
In addition to organic matter, most of the metals present
in the waters are necessary, in small amounts, for plant
growth. They may help seeds to develop by providing
easily absorbed micronutrients (Tolra et al. 1996). In this
experiment, essential elements such as K, Mg, and Zn were
diluted in the ink wastewater. Also, the nitrogenous com-
pound NO plays a key role in seed germination (Wong
et al. 1997). The toxicity of metals absorbed by plants can
be compensated by the accelerated growth of the plants due
to nitrogen availability (Tolra et al. 1996).
Over the course of the experiment, the amount of metals
added continually to the petri dish exceeded that necessary
for plant germination and growth. The parallel increase in
nitrogen could no longer counterbalance the toxicity, and
soluble protein concentration at the 15th day therefore
decreased. Extended exposure periods may subject seeds to
supplementary stress due to space limitations, such as the
accumulation of secondary metabolites or waste products,
contributing to a reduction in protein content. Such effects
have been reported by Sharma and Gaur (1995) when
working with Lemma polyrrhiza plants and Kevresan et al.
(2001) when working with Pisum sativum.
Among the widely expressed proteins during germina-
tion are hydrolases, necessary for the release of nutrients
from reserves and their transport to embryonic tissues. The
123
692
current study was interested in examining the effect of
treated and untreated wastewater toxicity on the amylolytic
activity, an indicator of the gibberellic acid (GA) level in
germinating seeds (Beri and Gupta 2007). GA is a classical
plant hormone that mediates cell elongation, seed germi-
nation, flowering and other physiological responses
(Richards et al. 2001). It also induces synthesis of alpha-
amylase activity.
Figure 3 provides treatment effects on the activity of
amylase between the studied species. The variability of
organic composition of the water used during this study
affected the expression of the enzyme and their activities in
metabolic pathways during the process of germination. The
application of E during germination caused amylolytic
activity disturbances in tested seeds (Fig. 3). However, the
irrigation of maize and barley seeds with untreated
wastewater after 15 days resulted in an increase in amylase
activity (Fig. 3a, b). Bravo and Vieira (1970), who studied
the effect of osmotic stress on Gossypium plants, explained
this rise in enzyme activity after decrease in the 10th day
by the loss of amylases inhibitors expressed under stress, or
Fig. 3 Effects of various effluents on a-amylase activity of maize (a),
barley (b) and sorghum (c) germinated seeds. Data are mean ± SD of
three independent experiments. W distilled water, E untreated
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Bull Environ Contam Toxicol (2015) 95:687–693
by the production of new precursors of the substrate
adapted to stressful conditions. In contrast, for sorghum,
this phenomenon was not observed. Similarly, Fendri et al.
(2012) demonstrated that a-amylolitic activity in Avena
sativa seeds treated with domestic wastewater continued to
decline over the entire stress period. According to these
authors, it might be due to the high salt load and the
presence of metals capable of generating free radicals
affecting various physiological and biochemical processes
in germination.
The use of treated ink industry wastewater using the
combined process of coagulation/flocculation/biosorption
(TEc/f/b) in plant nourishment could be a beneficial alter-
native resource to fresh water irrigation. On the basis of
overall performance as exhibited by three crops (maize,
barley and sorghum) when subjected to the raw effluent, it
is clear that ink wastewater is toxic for germination and
plant development. Thus, E cannot be used directly for
irrigation purposes in agriculture. It is also suggested that
treatment of this water is necessary to minimize pollution
effects before it is discharged to the land.
effluent, TEc/f treated effluent using coagulation/flocculation process,
TEc/f/b treated effluent using combined process (coagulation/floccu-
lation/biosorption)
Bull Environ Contam Toxicol (2015) 95:687–693
Acknowledgments This work was supported by grants from the
Tunisian Ministry of Higher Education and Scientific Research. It was
accomplished at the Faculty of Sciences of Sfax. Authors gratefully
acknowledge the support of Mohamed Ayadi for technical assistance.
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