Current Opinion in Agriculture
Curr. Opin. Agric.
2016 5(1), 5–8.
Effect of triclosan on germination and seedling growth
of mustard seeds
Rakesh Kumar Bhardwaja1, Vibha Bhardwaj*2
1
2
Crop Department, Dyal Singh College, Karnal-132001 India
Central Soil and Salinity Research Institute, Karnal-132001 India.
*
Corresponding author's E-mail: [email protected]
Abstract
Triclosan (TCS) is a widely used antimicrobial agent found in consumer products and is released to the environment at sub-ppb
concentrations following incomplete removal by wastewater treatment plant (WWTP) processing. TCS released to the environment is
acutely and chronically toxic to aquatic organisms. The study is aimed to determine the effects of domestic waste waters on seed
germination and seedling growth of some selected plants under laboratory conditions. The effect of effluent was compared to that of
control (distilled water). The results revealed that the two types of polluted water significantly affect germination, root & shoot
elongation and highly affected in domestic waste water and Triclosan containing waste water. As WWTPs discharge into aquatic habitats
and as constructed wetlands are of increasing interest for polishing wastewater for reuse purposes, understanding the effect of TCS on
wetland plants is necessary to understand impacts on wetland functions and services. We have studied the effect of TCS on germination
of four types of mustard seed germination Physical parameters: Length & weight of Root and Shoot of the germinated seed. Moreover
the Protein, Chlorophyll, Potassium, Magnesium and Calcium have also been quantitatively measured in the root and shoot of the
germinated seeds. The greening of the coleoptile was delayed with the increase in the effluent concentration. Percentage of germination,
viability, number of roots, shoot and root length, fresh weight, and dry weight of the rice seedlings showed an inverse relationship with
the effluent concentration.
Keywords: Sewage water, Triclosan , Mustard, Seed germination
Introduction
Increased population growth accompanied with lifestyle
changes, like the wider application of pharmaceutical and
personal care products (PPCPs) in everyday urban activities
including human and veterinary drugs, antibiotics, fragrances,
sunscreens, and antiseptics has changed the composition of
municipal waste water. As the demand for quality water
increases with escalating population growth, increase in
household waste water will also occur. Pharmaceutical and
personal care products belong to a relatively new group of
contaminants referred to as unrecognized or emerging
contaminants and have dominated urban waste water.
Municipal waste water treatment plants (WWTPs) are the
primary route of entry of PPCP’s to the environment.
Developed countries intensively promote and improve waste
water treatment systems, due to its potential use in agriculture,
which, however requires a detailed evaluation of water quality
effect on agricultural plants (Tal, 2006). To prevent the negative
impact of contaminated water on crops, it is important to
conduct continuous evaluation of water quality. It is familiar
that some plants are very sensitive to high content of pesticides,
heavy metals and organic substances in soil and water.
Proper treatment system in Indian industries is recommended to
meet the international standards, but presently less than 10% of
the waste water generated is treated and the rest of untreated
water is discharged into the nearby water bodies. The use of
industrial effluents for irrigation has emerged in the recent
past as an important way of utilizing waste water, taking the
advantage of the presence of considerable quantities of N, P,
K and Ca along with other essential nutrients (Niroula, 2003)
But there can be both beneficial and damaging effects of waste
water irrigation on crops including vegetables.
Therefore, it is necessary to study the impact of these effluents
on crop system before they are recommended for irrigation.
The present investigation was carried out to study the effect of
untreated effluents from s e w a g e s on seed germination of
mustard seeds. Mustard seeds are typically about 1 or 2 mm
in diameter may be colored from yellowish white to black. They
are important spices in many regional cuisines. The seeds can
come from three different plants: black mustard (Brassica
nigra) [RVB15-I], brown Indian mustard (B. juncea), [RVB15II], Yellow mustard [RVB15-III], and white mustard (B.
hirta/Sinapis alba) [RVB15-IV]. Generally the mustard seeds
take three to ten days to germinate if placed under the proper
conditions, which include a cold atmosphere and relatively
moist soil. Brown and black mustard seeds return higher yields
than their yellow counterparts.
Triclosan (TCS; 5-chloro-2-[2,4-dichlorophenoxy]phenol) is
an antimicrobial additive in a wide range of personal care
products and as such is typical of down-the-drain PPCPs.The
triclosan is one of the most frequently detected compounds in
the sewage analysis and is found to reveal toxic effects on
aquatic organisms (Degraeve et al. 1980; Kaiser et al. 1991).
The toxicity studies of TCS have shown that this compound is
associated with toxic effects (Orvos et al. 2002) especially to
aquatic flora. Triclocarban is commonly used (together with
triclosan) as an antifungal and antibacterial agent. Although
relatively little data exists about the toxicity of TCC.
Antimicrobial compound triclocarban (TCC) is also prevalent in
aquatic environments and co-occurs with triclosan. Triclocarban
Ma & Tyro
(TCC) is commonly used in bar soaps, while triclosan (TCS) is
used in wide range of products like toothpaste, soaps,
deodorants and cosmetics (Halden et al. 2005). TCS, has very
low solubility and to conduct toxicity assays of these
compounds preparation of stock solution in organic solvents is
also required. Selection of the right solvent and its
concentration is very important to detect the toxicity effects of
the test substance alone.
To look at the effects of carrier solvents on seed germination
and seedling growth of wetland plants. Seed germination and
seedling growth are commonly used tests in standard test
guidelines for phytotoxicity test of terrestrial plants. This study
will identify the best solvent and concentrations to be used in
toxicity assays of various pollutants on wetland plants. To look
at bioaccumulation patterns of the antimicrobial compounds
triclosan (TCS), triclocarban (TCC) and methyl-triclosan
(MTCS) (metabolite of triclosan) on wetland plants exposed to
WWTP’s effluent.
Material and methods
The sewage samples were collected from the inlet of Sewage
Treatment Plant Karnal two times a day at 10 am and 5 pm. The
collected sewage samples were filtered through Whatman paper
No.1 and kept at 25°C. Certified seeds of four selected plants
were purchased from the local market. Selected metal-tolerant
plants included mustard greens (Brassica juncea L.). The
healthy and uniform seeds of black mustard (Brassica nigra) [
RVB15-I], brown Indian mustard (B. juncea), [RVB15-II],
Yellow mustard [RVB15-III], and white mustard (B.
hirta/Sinapis alba) [RVB15-IV], were selected and surface
sterilized with 0.1% HgCl2 and thoroughly washed with
distilled water to avoid surface contamination and 20 seeds
were placed equidistantly on soaked filter paper in Petri dishes.
The seeds were irrigated with equal quantity of different
effluent samples and the seeds irrigated with distilled water
were taken as control. For each species 5 Petri dishes were
taken as replica for each treatment. Then Petri dishes were
placed in growth chamber at 30°C for 3 days in the dark.
Fig 1. Growth of mustard seeds in (a) Triclosan, (b) Sewage water
containing Triclosan, (c) in control , (d) Comparison in triclosan and
control .
6
Germinated seeds of were counted after 48 hours for
percentage germination. The root length, shoot length,
number of lateral roots were recorded after 7 days and dry
weight of seedlings was taken after keeping them in hot air
oven at 80°C for 24 hours. The moisture content was obtained
by the difference between the fresh weight and dry weight.
Moreover the potassium, Calcium Magnesium, Chlorophyll and
protein contents were measured following the standard
procedure. Data obtained was analyzed for variance (ANOVA).
Also, the data normality of data and existence of layouts
assessed through common normality tests such as one-sample
Kolmogorov-Smirnov test.
Results and discussion
Germination, root elongation and shoot length are the most
authoritative parameters that indicate changes in environmental
and the sensitivity of plants to certain contaminants in water
depends on the concentration and type of pollutants (WHO
2002, WHO 2003, Lin et al. 2010). The results of root and
sooth length have been reported in Table No. 1 while the fresh
and dry weight of root and shoot in Table 2. Root length and
Shoot length of germinated mustard as presented in Table 1,
was significantly higher in control being maximum in 9.8 cm
for RVB15 –IV compared to sewage water being lowest 2.8 cm
RVB15-I , (F =2.462*, p<0.05). The figure clearly shows that
no seed germination was observed in presence of triclosan.
Significantly it can be observed from the data presented in
Table 2 that the fresh weight of root and Shoot is higher in
control rather than in sewage water while same pattern is
observed for dry weight, Table 3 show the level of chlorophyll
measured. The level of Minerals Potassium Calcium,
Magmisium measured in shoot and roots are reported in Table 4
and 6 while the total protein content have been reported in
Table 7.
The results of One way ANOVA indicate that differences
between germination of mustard depending on the water quality
were not significant (F=0.603NS, p > 0.08). However one
should bear in mind that the minimal germination for mustard
seed, defined by Regulation cited above, is 75%, and was not
achieved in sewage water containing troiclosan. These results
suggest that sewage water is not appropriate medium for the
cultivation of mustard.
Germination assessment of different plant species in distilled
and sewage revealed unexpected results. Results showed that
87% germination in control condition while in sewage
significantly decreased germination percentage by 58 % and
triclosan reduced germination percentage by 96%).
Our data supports the possible enrichment of antibiotic-resistant
microorganisms in soil microbial communities after exposure to
triclosan-enriched greywater. Short-term irrigation of soils with
sewage water supplemented with triclosan was shown to have
impacts on both the presence of triclosan and antibioticresistant microorganisms in soil as well as on the structure of
microbial communities present in the soil. Our results show
similarity with the results of Bazai et al. (2006) who suggested
that plumule length is decreasing in higher concentration of
polluted water. There was no relationship developed with
increasing effluents and weight parameter. Fresh weight and dry
weight were decreasing after application of both types of
polluted water as compared to control conditions.
7
Analysis of organic manure and nano-fertilizers on sunflower
S.NO
Genotypes
1
2
3
4
RVB15-1
RVB15-II
RVB15-III
RVB15-IV
S.NO
Genotypes
1
2
3
4
RVB15-1
RVB15-II
RVB15-III
RVB15-IV
S.NO
1
2
3
4
S.N
O
1
2
3
4
S.N
O
Table 1. Root length and shoot length (cm)
Control
In sewage Sample collected
Averaged
Averaged
Averaged
Averaged
Root Length
Shoot Length
Root Length
Shoot Length
(cm)
(cm)
(cm)
(cm)
5.1
3.08
2.8
3.8
5.9
4.5
3.9
4.1
5.3
3.64
3.5
4.6
9.8
5.1
3.3
3.7
Root
Fresh
Weight
Table 2. Root and shoot fresh and dry weight (mg)
Control
In sewage Sample collected at !0 am
Shoot
Root
Shoot Dry
Root Fresh
Shoot
Root Dry
Shoot Dry
Fresh
Dry
weight
Weight
Fresh
weight
weight
weight
weight
weight
0.0425
0.492
0.435
0.1055
0.0704
0.1273
0.0635
0.1335
S.NO
0.0038
0.0060
0.0039
0.0046
0.0213
0.0121
0.0325
0.0122
0.0645
0.0724
0.0687
0.0564
0.0013
0.0006
0.0019
0.0023
0.0065
0.0087
0.0132
0.0046
Table 3. Chlorophyll content (100 mg sample (leaves) and 5ml extraction volume)
Control
In sewage Sample collected at
In sewage sample collected
10 am
at 5 pm
Total Chlorophyll g/Lt
Total Chlorophyll g/Lt
Total Chlorophyll g/Lt
RVB15-1
0.000607
0.000799
0.000883
RVB15-II
0.000580
0.000787
0.000819
RVB15-III
0.000547
0.000819
0.001068
RVB15-IV
0.000537
0.000678
0.000742
Table 4. Potassium content in shoot and root
Root K (PPM)
Genotypes
RVB15-1
RVB15-II
RVB15-III
RVB15-IV
Genotypes
In Control
sewage Sample
collected at 10 am
sewage Sample
collected at 5
pm
In
Control
7.0
13.9
11.0
8.4
3.1
7.0
2.5
8.4
4.2
6.8
2.3
2.1
26.5
27.6
24.3
37.3
Table 5. Calcium content in shoot and root
Root Ca (PPM)
RVB15-1
RVB15-II
RVB15-III
RVB15-IV
Genotypes
0.6935
1.540
0.8241
0.7543
RVB15-1
RVB15-II
RVB15-III
RVB15-IV
In sewage
Sample
collected at
!0 am
0.2185
0.1262
0.3562
0.4985
In sewage
Sample
collected at 5
pm
0.3165
0.2250
0.4421
0.0500
In
Control
0.7356
0.9829
4.174
1.727
Table 6. Magnesium content in shoot and root
Root Mg (PPM)
In
Control
1
2
3
4
0.0027
0.0030
0.0035
0.0041
Genotypes
In
Control
1
2
3
4
In water containing Triclosan
Averaged
Averaged
Root Length
Shoot Length
(cm)
(cm)
0
0
0
0
0
0
0
0
0.4803
0.4985
0.4562
0.3936
In sewage
Sample
collected at
10 am
0.3255
0.1101
0.090
0.365
In sewage
Sample
collected at 5
pm
0.3912
0.2211
0.2813
0.1060
In
Control
0.8722
0.8716
2.582
1.212
Shoot K (PPM)
In sewage
Sample
collected at
10 am
20.4
15.6
22.1
18.5
In sewage
Sample
collected at 5
pm
15.1
18.5
7.3
17.7
Shoot Ca (PPM)
In sewage
Sample
collected at
!0 am
0.2351
0.1173
0.1283
0.721
In sewage
Sample
collected at 5
pm
1.018
0.3281
1.126
0.9823
Shoot Mg (PPM)
In sewage
Sample
collected at
10 am
0.7812
0.4421
1.8712
0.9236
In sewage
Sample
collected at 5
pm
0.8131
1.006
0.8120
0.1213
Ma & Tyro
S.NO
Genotypes
1
2
3
4
RVB15-1
RVB15-II
RVB15-III
RVB15-IV
Table 7. Protein content in shoot and root
Protein mg/ml
In
In sewage
In sewage
In Control
Control
Sample
Sample
collected at collected at
10 am
5 pm
67.200
40.860
30.392
0.199
50.653
45.588
47.952
0.150
72.603
33.769
45.925
0.215
42.886
43.562
41.873
0.127
Powel et al. (1996) suggested due to pollution stress the fresh
weight of seedling reduced. The statistical analysis of data
shows that most of the seeds germination was affected by
treatments with both domestic waste water treatments and
pharmaceutical industrial waste water treatments.
Conclusions
This study demonstrated that application of triclosan treatment
increase some important traits of mustard seeds in germination
and seedling growth. Percentage of germination, viability,
number of roots, shoot and root length, fresh weight, and dry
weight of the rice seedlings showed an inverse relationship with
the effluent concentration.
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