shaik umme salma,college of fisheries,mangalore, usalmashaik@gmail.com credit seminar

1. RESPONSE OF AQUATIC FERN, AZOLLA TO
WATER CONTAMINATION
MAJOR ADVISOR: Dr. A.T. RAMACHANDRA NAIK
Shaik Umme Salma
MFK 1710
DEPARTMENT OF AQUATIC ENVIRONMENT
MANAGEMENT

2. INTRODUCTION
RESEARCH PAPERS
MATERIAL AND METHODS
RESULTS AND DISCUSSIONS
CONCLUSION
O
U
T
L
I
N
E

3. • Azolla
• Fresh water - Moist soils, Ditches marshy ponds - Tropical belts .
• 3–10 days.
• Heavy metals.
• Important species are:
Azolla pinnata.
Azolla nilotica.
Azolla mexicana.
INTRODUCTION

4. SCIENTIFIC CLASSIFICATION
Kingdom : Plantae
Division : Pteridophyta
Class : Polypodiopsida/
Pteridopsida
Order : Salvinales
Family : Salvinaceae
Genus : Azolla

5. STRUCTURE OF AZOLLA
• Typically triangular
• Length : 1.5 to 3.0 cm
• Breadth : 1 to 2 cm
• Dorsal lobe : (Anabaena azollae ) - “Live Nitrogen Manufacturing
Factory”

6. ECONOMIC VALUE
Nitrogen : 5.0 %
Phosphorus : 0.5 %
Potassium : 2.0-4.5%
Calcium : 0.1-1.0%
Magnesium : 0.65 %
Manganese : 0.16 %
Iron : 0.26 %
Crude Fat : 3.0-3.3 %
Sugar : 3.4-3.5 %
Starch : 6.5 %
Chlorophyll : 0.34-0.55 %
Ash : 10.0 %

7. Water Soil pH Light
Relative Humidity & Temperature Nutrition
REQUIREMENTS FOR AZOLLA GROWTH

8. • Heavy metals (Hg and Ni), Petroleum hydrocarbons and pesticides.
Consequences:
Methods to reduce Pollution:
• Soil contamination(metal), methods such as:
1. Excavation
2. Thermal treatment
3. Acid leaching
4. Electro reclamation
Pollution

9. Water treatments:
1. Sedimentation,
2. Thermal treatment with mechanical filtration
• Phytoremediation:
• Greek - plant,
• Latin -restoring balance

10. PHYTOREMEDIATION
Phytoextraction
Phytostabilization
Phytovolatization
Rhizofiltration
Phytotransformation
Phytostimulation

11. Phytoextraction

12. Phytostimulation Phytovolatization

13. Phytostabilization Phytotransformation

14. Rhizofiltration

15. The use of Azolla in rice fields promotes changes in the physical, chemical, and
biological properties of the soil as well as the soil-water interface, favoring rice
production. (Cheng et al., 2010,2015)
The excessive and indiscriminate use of antibiotics in farm practices (mainly as feed
additives) is the major source of these compounds to the surrounding environment, and
aquatic systems are natural sinks for antibiotic runoffs from agricultural lands.
(Gomes et al., 2017)
Azolla has been shown to be useful in wastewater treatment due to its ability to take
up contaminants such as heavy metals and antibiotics (i.e., penicillin).
(Antunes et al., Malakootian et al., 2015)

16. The potential environmental risks of antibiotics have attracted significant
attention and some of them are very resistant to abiotic and biotic degradation
(Girardi et al., 2011; Nie et al., 2013)
Among the antibiotics, fluoroquinolones such as ciprofloxacin are widely used
in both human and veterinary medicines and milligram levels of ciprofloxacin
have been observed in the environment which is extremely worrisome, as at
those levels, it has been found to be toxic to numerous organisms, including
algae and plants
(Migliore et al., 2003; Golet et al., 2003; Martínez-Carballo et al.,
2007; Robinson et al., 2005; Martins et al., 2012; Gomes et al., 2017 )

17. Some antibiotics, such as erythromycin, were observed to be very toxic to
Anabaena, killing that cyanobacteria e resulting in Azolla plants without their N-
symbionts (Forni et al., 1991)
I
The particular case of antibiotics, their removal by sewage treatment plants is not
effective , leaving phytoremediation as an emerging technology for
decontamination. (Petrovic et al., 2005; Karthikeyan and Meyer, 2006
),

19. PAPER - 1
IF 1.7

20. Significance
This study will be useful in the establishment of Azolla as a potential Cipro-
phytoremediation species.
Additionally, it is important to understand if water contamination by Cipro will
result in N-fixing losses by Azolla, which would imply economic impacts in
terms of its use as a biofertilizer
(Ventura and Wantanbe, 1993; Wagner, 1997)

21. • To investigate the ability of the aquatic fern Azolla to take up
ciprofloxacin (Cipro)
• Effects of that antibiotic on the N-fixing process in plants grown in
medium deprived (-N) or provided (+N) with nitrogen (N).

22. MATERIALS and METHODS

23. Fundacao Zoo-Botanica
+N; -N
Prior to the initiation of the treatments, the plants were
acclimated for 15 days in +N or -N CHU 10 medium
Temperature 20 ± 2 ℃ under 12-h
photoperiod
0
0.75
1.05
2.25
3.05
Analytical-grade ciprofloxacin (purity > 98%) was
purchased from Sigma-Aldrich (Brazil)
The effects of Cipro on Azolla, plants were investigated
after 5 days of exposure

24. Numbers of heterocysts
Heterocysts/vegetative cell ratio was
evaluated by quantification of both cell
types using an optical microscope at a
magnification of 400×
Leaves of Azolla fronds per replicate were pooled
together and lightly macerated material was
then fixed with lugol acetic solution for
later quantification
All mature heterocysts and vegetative cells were
quantified in each field of a slide in at least 40
fields, for a total of at least 800 cells/slide

25. Nitrogenase assays were performed using the acetylene (C2H2) reduction technique
Peters and Mayne(1974)
Nitrogenase activity and nitrogen metabolism
5 fronts were incubated for 60 min in calibrated 15 ml flasks -5 ml of the test
growth media & 10% C2H2
The gas phase was analyzed for C2H2 by gas chromatography using a Perkin Elmer F17
gas chromatograph equipped with a flame ionization detector and a Porapak® T80/100
column running at 100 ℃, with N 2 as the carrier gas
Duplicate samples were assayed from each flask at each time interval (0 or 60 min);
four flasks were employed for each specific treatment

26. 500 µl extraction
solution
0.1 M phenol
170 µM sodium
nitroprrusside
0.125M NaoH,
0.15 M Na2HPO4.
12 H2O,
3% NaOCl
500µl ( 625nm)
0.1 g plant tissue

27. Ground sample @ 0.5 gm
After acid hydrolysis with 50 ml of 6 N HCl for 24 h at 110℃
20 l was then injected in reverse-phase HPLC (Model 23250,
ISCO, USA)
TheMP-A was 40 mM NaH2PO4, adjusted to pH 7.8 with NaOH,
while MP- B was acetonitrile/methanol/water (45/ 45/10 v/v/v)
Separation @flow rate of 2 ml/min with a gradient program of 1.9
min at 0% # 16.3-min step that raised eluent B to 53%.
Washing with 100% B and equilibration at 0% B was then
performed in a total analysis time of 26 min.
µ

28. Photosynthetic responses and hydrogen peroxide concentrations
PAM fluorometer (model PAM- 2500, Walz, Effeltrich, Germany) was used to study the effects of
Cipro on photosynthesis
After dark adaptation (15 min), chlorophyll fluorescence was evaluate in fronds
0, 32, 43, 61, 87, 131, 190, 284, 416, 619,912 µmol photons m-2 s-1
Relative rate of electron transport through PSII (ETR)
(Krall& Edwards, 1992)
Maximal photochemical efficiency of PSII
(Kitajima and Butler,1975)
Photochemical quenching (qP)
(Van Kooten and Snel, 1990)Hydrogen peroxide (H2O2) concentrations Velikova et al. (2000)

29. JMP - 7.0
STATISTICAL
ANALYSIS
ANOVA
Shapiro-Wilk
Bartlett

30. RESULTS and DISCUSSION

31. Ciprofloxacin concentrations in Azolla
0
20
40
60
80
100
120
140
160
180
Ciprofloxacin concentrations in Azolla
0 0.75 1.05 2.25 3.05
cipro(µgg-1DW)
Cipro (mg -1 )
-N
+N

32. Contaminant concentrations ≥100 ppm in dry mass has been used to
classify plants as hyperaccumulators
(Baker et al., 2000)
≥100 ppmshould be attained in in situ studies, as light, temperature, pH
conditions, and chemical interactions in nature can influence xenobiotic take
up by plant (Chalifour and Juneau, 2011; Gomes et al., 2016)

33. 0
2
4
6
8
10
12
14
Heterocysts/vegetative cell ratios
0 0.75
Cipro (mg -1 )
1.05
2.25 3.05
Heterocysts/vegetativecellratios
-N
+N

34. The nitrogen-fixing capacity of each leaf of Azolla is related to the heterocyst
frequency of the symbiont it hosts
(Watanabe, 1982)

35. Nitrogenous activities
0
5
10
15
20
25
30
35
nitrogenous(nmolC2H4g-1FW)
0 0.75 1.05 2.25 3.05
Cipro (mg -1 )
-N
+N

36. Total nitrogen
0
0.5
1
1.5
2
2.5
3
3.5
0
0.75 1.05 2.25 3.05Cipro (mg -1 )
Totalnitrogen(µgg-1DW)
-N +N

37. Ammonium concentrations
0
2
4
6
8
10
12
14
16
18
20
0 0.75 1.05 2.25 3.05Cipro (mg -1 )
NH4
+nmolg-1FW)
-N
+N
Similar results were observed by Forni et al., 1991 in Azolla-Anabaena
treated with the antibiotic novobiocin.

39. By evaluating the effects of Cipro on plants supplied with, or deprived of N we
observed that the antibiotic is not only toxic to Anabaena, but apparently also
toxic to Azolla, as negative effects of Cipro were noted even in +N plants.
(Gomes et al., 2017)
Proline accumulation also attested to oxidative stress induced by the antibiotic,
as that amino acid is a common protective-response of plants to stress conditions
and its biosynthesis is stimulated by increased cellular-ROS concentrations.
(Hayat et al., 2012;Soshinkova et al., 2013)

40. Electron transport through PSII (ETR)
0
2
4
6
8
10
12
0 0.75
1.05 2.25 3.05
Cipro (mg -1 )
ETR
-N
+N

41. Deleterious effects of Cipro on photosynthesis have been observed and this was
initially attributed to its ability to disrupt electron flow in the chloroplast electron
transport chain (ETC) as it is structurally similar to known inhibitors of the
oxidizing site of PSII (Aristilde et al., 2008: 2010; Gomes et al., 2017)
Instead of competing for electrons with electron carriers in thylakoids, Cipro would
directly bind to PSII, interfering with energy transfer from excited antenna
chlorophyll molecules to the reaction center (Aristilde et al., 2010)
Interference of Cipro with photosynthesis was due to changes in the PSII integrity
driven by H2O2 accumulation that resulted from interference with the
mitochondrial ETC by Cipro (Gomes et al., 2017)

42. Hydrogen peroxide (H2O2) concentrations
0
0.5
1
1.5
2
2.5
3
0 0.75 1.05 2.25 3.05
Cipro (mg -1 )
H2O2µgg-1DW
-N +N

43. Among the deleterious effects of ROS on photosynthesis, is that they can
disturb the photochemistry of that process by suppressing repair and assembly
of the D1-protein (a PSII associated protein)
(Takahashi and Murata, 2008)
Interference with energy metabolism (photosynthesis and respiration) will result
in depressed reducing power generation, such as NADPH and NADH, which are
products of linear electron transport in chloroplasts and mitochondria
(Murchie and Lawson, 2013)

44. Maximal photochemical efficiency
(FV/FM)
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
0 0.75 1.05 2.25 3.05Cipro (mg -1 )
(FV/FM)
Treating plants with Cipro also reduced the maximum photochemical
efficiency of PSII (FV /FM ) , which serves as a proxy for PSII integrity
(Walter et al., 2003)
-N
+N

45. photochemical quenching (qP)
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
0 0.75 1.05 2.25 3.05
Cipro (mg -1 )
(qP)
-N
+N

46. Under controlled conditions , qP has been directly correlated with ATP and
NADPH production rates
(Genty et al., 1990; Murchie and Lawson, 2013)
Cipro was observed to decrease the activities of mitochondrial Complexes I
to IV, resulting in reduced NADH production
(Gomes et al., 2017)

47. • The observed reduction in N-fixing ability of Azolla-Anabaena was
considerable, and may result in less N availability in plantations, losses in
yields, and impact on the use of the nitrogen-fixing Azolla for biofertilization
• Shown that this fern is a potential candidate for Ciprobioremediation, since
plants could survive under high concentrations of Cipro and could accumulate
high quantities of this antibiotics in their tissues.

48. PAPER-II
IF-4.1

49. The Bioremediation has been proven successful in numerous applications
especially treating petroleum contaminated soils.
Petrochemical plants generate an aqueous effluent containing various
conventional pollutants as well as specific petrochemicals and intermediates.

50. To investigate the metal and fluoride uptake capacity from the
wastewater of Oil and Petroleum refining industry at Vadodara.
To determining its phytoremediation capacity.

51. According to the Science News references fluorides and heavy metals are
found to be having toxic effects on fishes and human beings even below the
permissible limit of CPCB CPCB report.,2008
The bioremediation has been proven successful in numerous applications
especially treating petroleum contaminated soils
Carmargo., 2003
Environment becoming more reducing, result in decrease in availability of
heavy metals, or metals in general to plants
Misra et al., 1991; Nagajyoti et al., 2010

52. MATERIALS and METHODS

53. 1ml/ml
0.5ml/ml
physico-chemical parameters

54. Digestion and analysis of elements in plant tissue and in the
wastewater
Digestion and analysis of elements in plant tissue and in the
wastewater
Concentrations of metals present in the solutions (for both
wastewater and plant tissue) were measured
using AAS.
Preparation of the test plant materials for elemental analysis
was done by wet-digestion method using con. HNO3
Initial metal concentration & remaining metal concentration in
the solution after the treatment period (7days) was taken to be
metals bound to the plant

55. Percent removal efficiency = inlet pollutants - outlet pollutants / inlet pollutants ×100
The amount of metal ions per unit of the plant material(biosorbent)
q = [(C0 – C1) V]/M
BCF = Concentration of metal in plant tissue / Initial
Concentration of metal in external solution
Demirbas et
al., 2004
Deval et al.,
2012
Zayed et al.,
1998

56. Wilcoxson signed ranked test .
SPSS version 19.0

57. RESULTS AND DISCUSSION

58. 0
1
2
3
4
5
6
CONCENTRTIONOFIONSARE
EXPRESSESINMG/L initial concentration
limiting value
1ml/ml
0.5ml/ml
Initial concentrations of ions in the effluent, con. after treatment in 1ml/ml medium, 0.5ml/ml medium are
expressed in each bar as mean ± S.E (n=3) and the permissible limit of each pollutant
F Cr Hcr Pb Zn CuNi Cd Fe
Heavy metals

59. PH
• 8.2
TDS
• 19mg/lt
BOD
• 81 mg/lt
COD
• 892mg/lt
EC
• 1.393dS/m

61. IONS
%
Absorption
in 1ml/ml
%
Absorption
in 0.5ml/ml
BCF in
1ml/ml
BCF in
0.5ml/ml
Absorption
in mg/ gm
of tissue in
1ml/ml
Absorption
in mg/ gm
of tissue in
0.5ml/ml
Cu 88.627451 80.392157 0.8862745 0.8039216 0.0598675 0.0262821
Cr 73.298429 68.586387 0.7329843 0.6858639 0.1854305 0.0839744
Hcr 77.777778 100 0.7777778 1 0.0092715 0.0576923
Pd 57.142857 42.857143 0.5714286 0.4285714 0.005298 0.0019231

62. Hexavalent chromium, chromium and fluoride have an anagonistic effect on
the uptake of other metal ions by competing with their carrier proteins or by
some other way of interactions
Shanker et al., 2005; Lorestani et al., 2011
Lead, copper and cadmium was also found to be having an interfering effect on
each other in their absorption capacity by plants
Salim et al., 1993
Lead was absorbed more than 45-50% from the medium it may inhibit the
activity of enzymes at cellular level by reacting with their sulfhydril groups,
cause water imbalance, alterations in membrane permeability, disturbs
mineral nutrition and thus may affect the uptake of other ions
Sharma et al., 2005;Nagajyoti et al., 2010

63. Ions %
Absorption
in 1ml/ml
%
Absorption
in 0.5ml/ml
BCF in
1ml/ml
BCF in
0.5ml/ml
Absorption
in mg/ gm
of tissue in
1ml/ml
Absorption
in mg/ gm
of tissue in
0.5ml/ml
Zn 23.728814 0 0.2372881 0.2625 0.018543 0.0134615
Ni 37.037037 0 0.3703704 0.0357143 0.013245 0.000641
Fe 10.47619 35.238095 0.1047619 0.352381 0.0291391 0.047357
F 4.6153846 0 0.0461538 0 0.0039735 0
Cd 36.666667 20 0.3666667 0.2 0.001457 0.0003846

64. Cd was reported to interfere with the uptake, transport and use of several
elements (Ca, Mg, P and K) and water by plants
Das et al., 1997; Nagajyoti et al., 2010
No synergistic/antagonistic effect was noted for the multiple metal
experiments, in terms of metal removal for lead and nickel
Axtell et al., 2003
Though the concentration of Zinc in the initial concentration of pure
wastewater was more than copper but still copper was found to be absorbed
of about 88% and 80% from the pure and diluted wastewater
Ebbs & Kochian 1997

65. (F) concentration as low as 0.5 mg/l can adversely affect invertebrates
and fishes
Carmargo., 2003
Even at low ambient concentration fluoride can cause a no. of
physiological and biochemical changes in plants without visible sign of
injury Bhardwaj.,2010
The absorption capacity of a plant thus is not only the outcome of the
combination of metal ions present in the solution or media together with other
influencing factors like PH, BOD, COD, TDS, EC etc. but also the type of the
plant growing in it
Yamamoto., 1987

66. Ions %
Absorption
in 1ml/ml
%
Absorption
in 0.5ml/ml
BCF in
1ml/ml
BCF in
0.5ml/ml
Absorption
in mg/ gm
of tissue in
1ml/ml
Absorption
in mg/ gm
of tissue in
0.5ml/ml
Cr 73.298429 68.586387 0.7329843 0.6858639 0.1854305 0.0839744
Fe 10.47619 35.238095 0.1047619 0.352381 0.0291391 0.0291391
The concentration of chromium and iron are high in comparision to other metal
ions in the medium which might be the main interacting factor affecting the
absorption of other ions.

67. BCF of chromium, hexavalent chromium and copper indicates its ability to
phytoremedify these metal ions from the multimetal solution
Lorestani et al.,2011
The percentage absorption of metal ions was found to be in the order of
Cu> HCr> Cr> Pb> Ni> Cd> Zn> Fe> F in pure wastewater (1ml/ml)
HCr> Cu> Cr> Pb> Fe> Cd> Ni=Zn= F in the diluted medium ( 0.5ml/ml).
No significant difference in the % absorption in the different
concentrations of the wastewater shows no effect of dilution on the
absorption capacity of ions by the test plant.

68. • Significant difference between the initial and final concentrations of the
wastewater with respect to metal ions and fluorides after the treatment with
Azolla pinnata var. imbricata and BCF
• Chromium
• Hexavalent chromium &
• Copper
• Indicates its ability to phytoremedify these metal ions from the multimetal
solution as well as pollution monitoring of heavy metals and fluorides.
ONE-1

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