22
3. Material and Methods
3.1. Sample collection
The locations of bed and core sediments
and suspended matter
collected from the Godavari River basin are
indicated
in the
basin map (Fig. 7). Additional information on sampling is given
in Table 2.
The first sampling was done in the month of May 1993. 10 litre
running
water
was
collected
in
locations in the river basin.
for 72 hours.
The supernatant was siphoned off.
scrapped out
sediments
were
bottles
from
the
collected
4
The remaining
and the dried suspended matter
watch
by
from
This water was kept undisturbed
water was evaporated at 40°C,
was
polythene
glass
scooping
and
upper
weighed.
ca.
6
The
bed
of
bed
cm
sediments lying under flowing water at a depth of about 50 cm.
The sediments were transferred immediately into a polythene bag.
The core samples were collected by manually inserting a PVC pipe
(ca. 4 cm) in the bed sediments. These cores were brought to the
lab and cut horizontally into 2 cm sections. The sediments were
dried
at
temperature
40°C
before
matter
samples
across
the
and
stored
further
were
river at
in
polythene
analysis.
collected
from
Raj ahmundry
Second
5
(site
bags
set
equally
13)
litre water was collected with Niskin bottle
below
of
point.
0.4511
Water was
filtered
in
the
field
in August
polycarbonate membrane filters
points
1994.
(Hydrobios,
through
4
°C
suspended
spaced
from approximately 1 meter below the water surface
0
5
Kiel)
from each
preweighed
(Schleicher and Schuell),
and the suspended matter on filter was poisoned by a few drops
of 3.3 gil mercuric chloride solution and then air dried. Later
74
72 E
84
82
80
78
76
THE GODAVARI RIVER AND ITS TRTBlJTARTES
Sampling locations
22
N
\~" '"
) .";"
~
OF "
BENGAL "
.
20
Bombay :
18
+ Bed sediments
Suspended matter
o Core sediments
~
Hyderabad
Arabian Sea
16N
a
5J
100
150
ZXl
kIn
Source: Der Grosse Bertelsmann Weltatlas. 1961
Fig, 7: Sampling locations in the Godavari River basin
24
Table 2: Sites and additional information on sampling
Site no.
Site
River
Sample
Date
Time
Someshwar
Godavari
I
05.05.93
07:00
Nanded
Godavari
I, II
06.05.93
10:30
3
Adilabad
Penganga
II
07.05.93
07:30
4
Soan
Godavari
II
07.05.93
14:30
5
Sangareddypet
Manjera
II
08.05.93
14: 15
6
Mancheryal
Godavari
II
09.05.93
14:30
7
Ashti
Wainganga
II
10.05.93
09:45
8
Kalesar
Godavari
I, II, III
11.05.93
06: 15
9
Nagram
Pranhita
II
11.05.93
06:45
10
Bhopalpatnam
Indravati
II
11.05.93
14:00
11
Jagdalpur
Indravati
II
12.05.93
08:00
12
Bhadrachalam
Godavari
II
13.05.93
05: 15
13
Rajahmundry
Godavari
I, II, III
13.05.93
15:00
13*
Rajahmundry
Godavari
I
05.08.94
11-15:30
14
Ravulpalam
Gautami Godavari
II, III
14.05.93
13:30
15
Yanam
Gautami Godavari
II, III
14.05.93
09:45
16
Pengonda
Vasishtha Godavari
II, III
14.05.93
14:30
17
Nursapuram
Vasishtha Godavari
II, III
14.05.93
16:30
.2
I
Suspended matter
II
Bed sediments
III Core sediments
*
Monsoon season
-
25
in the laboratory the filters were dried at 40°C. These filters
were weighed again for total
suspended matter estimation.
The
sediments were powdered with the help of pestle and mortar and
homogenised before
analysis
of
individually
performing any
suspended
for
all
matter
five
chemical
on
points,
analysis.
filter
and
then
Chemical
was
performed
this
data
was
averaged as monsoon season sample.
3.2. Sample analysis
3.2.1. Carbonate carbon
Carbonate
carbon
was
determined
with
Carmhograph
from
6
Wosthoff. Carbonate content is measured by monitoring change in
conductivity of sodium hydroxide solution through which carbon
dioxide,
generated as a
acid and carbonates,
bed
and
core
result
is passed.
sediments,
of
reaction between phosphoric
5-80 mg
respectively)
(suspended matter and
dried
and
homogenised
sediments were weighed in separate 50 ml Erlenmeyer flasks. As a
standard approximately 10 mg calcium carbonate was also weighed
in 4 flasks.
Approximately 10 ml 2N phosphoric acid was added,
and the mixture was heated to boil. The carbon dioxide generated
from
this
mixture
was
passed
through
18
ml
0.05
N
sodium
hydroxide solution. The change in conductivity of this solution
was compared with that generated by calcium carbonate standard.
Standard deviation in this technique was ±1%.
3.2.2. Total Carbon and Nitrogen
Total C and.N were determined with Nitrogen Analyser NA1500 from
Carlo-Erba.
Sample was weighed
in
tin
capsule
and
placed
in
autosampler of this instrument. The sample is flash combusted at
26
1020 °C in an oxidation column under oxygen current for 40 sec.
The combustion products (C02, N2' NOx, S02, H20) are transported
by constant flow of the carrier gas
contains
chromium
(III)
oxide
cobaltic oxide granules.
oxides
is
and
silver
In this column,
formation of nitrogen
contains
cupric-
copper metal in the form of coarse powder at 650°C.
In this
column,
removed.
oxygen is
elemental
interfering
cobaltous
sulphur
are
and
coated
and
compounds
inhibited
(He). The oxidation column
The
halogenated
reduction
,
column
removed and nitrogen oxides
nitrogen.
After
passing
through
are
these
reduced to
columns,
gas
mixture is directed through water filter (magnesium perchlorate)
and
then
detector
through
(TCD)
separation
measures
column.
the
A
thermal
difference
conductivity
between
conductivity of carrier gas and that of reference gas
30 mg
(1-5 mg for suspended matter)
thermal
(He). 10-
sample was weighed in tin
capsules; Sulphanilamide (C6HSN202S)
standard ca. 1 mg was also
weighed.
1
After
every
10
samples,
standard
Reproducibility of results in this method ±1% of
value.
Instrument Operating Conditions
Gas E"low
Carrier gas (He)
70 kPa,
SO ml/min
Reference gas (He)
70 kPa,
30 ml/min
Oxygen
70 kPa,
20 ml/min
Servo air
Detector
350 kPa
Thermal conductivity detector (TCD)
Filament temperature
Oxidation furnace
140°C
1020 °C
was
measured.
the absolute
27
Reduction furnace
650°C
Oven
60°C
Analytical cycle
400 sec
The organic carbon content
(Corg) was calculated as a difference
between total carbon and carbonate carbon.
3.2.3. Amino acids
Amino acids were determined with LKB Amino Acid Analyser (4151
from Pharmacia Biotech.
Alpha Plus)
Details of the methodology
has been given by Michaelis and Ittekkot
(1982).
In brief, acid
hydrolysed amino acids elute in accordance with their molecular
structure
buffers
and
with
charge
under
ascending
Phthaldialdehyde
pH.
the
influence
The
eluted
(OPA)/mercaptoethanol
of
sodium
amino
acids
citrate
form
which
complex,
0-
is
quantified by a fluorescence detector.
70-150 mg bed and core sediments (5-10 mg suspended matter) were
hydrolysed
under
argon
gas
atmosphere
(suprapure)
for 22 hrs. at 110°C. 2 ml supernatant was pipetted
out and evaporated to dryness 3-4 times
with
3
ml
6
N
HCl
(each time the residue
was dissolved into ca. 5 ml DDW) with a rotary evaporator until
free of acid. The residue was then dissolved into 1 ml dilution
buffer.
20-60
loading
capsule,
chromatography
/11
of
this
which
(GLC)
solution was
transferred
column of
the
injected in
sample
to
the
sample
gas
liquid
the amino acid analyser.
Total
amino acids were calculated as,the sum of individual amino acids
detected
and
quantified.
multiplied by 1.4
Hexosamine
(Muller et al.,
1986)
concentrations
were
in order to compen'sate
28
for the partial loss during hydrolysis. The analytical error in
this method was less than 10% (Fig. 7a).
instrument and operating conditions
GLC column
Ion-exchange
resin
Detector
Standard
Flow rate:
Buffer
Reagent
No. 138, 150x4.6 mm steel column
DC4-75, special 7~1 (cation exchange, Na form)
Fluorescence detector (FLD-6A from Shimadzu)
with 100 ~l flow through cell
.
®
AA standard solution AA-S-18 (Slgma ), and a
mixture of hexosamines and non-protein amino
acids.
19.1 ml/h
17.5 ml/h
Programme
Step
I
2
3
4
5
6
7
8
9
Time
(min.)
18
4
6
18
10
15
70
8
5
13
5
2.5
0.5
Buffer
Reagent
2
4
2
3
4
5
5
6
1
1
1
1
2
1
1
x
x
x
x
x
x
x
x
x
10
11
12
13
14
1
15*
1
* capsule loaded
x
x
x
x
x
Reagent (2 litre)
Chemicals
Potassium hydroxide
Boric acid
07 Phthaldialdehyde
Ethanol
Mercaptoethanol
Brij solution (30%)
Quantity
63.0 g
74.2 g
1.0 g
10.0 ml
5.0ml
6.0ml
29
G-ABA
66.00
B-A ~A
ASP
>
~
,-,,
'
MET
SER
tHR
64.00
GLU
~
i
~ ;
0
LEU
trR
~
-~
~
~
0
GLUAM
GMAM
I\ILE PHE
A
GLY
Vl ,
Standard .
HIS
ARG
ORN
62.00
LYS
< .)
~
NH3
I
a
~
60.00
r--.---Y
,u
1.1
~ I.J
l.L
"'-.Iu
, ...
"'W
"i
'~-V-"-"1~
90:00.0
60:00.0
30:00.0
L~
120:00.0
Time (min)
L19 1!
ASP
120.00
>:1".-..,
VAL
110.00
GLU
'-"'
~
Vl
~
!
100.00
.I
0
0..
-Vl
~
~
~
THR I
90.00
R
LEU
< .)
a
~
ilI
0
~
I
GIirY
SO.OO
70.00
60.00
/.1
30:00.0
60:00.0
9000.0
120:00.0
Time (min) .
Fig. 7a: Typical Amino acid chromatograms by 4151 Alpha Plus Amino Acid Analyser
30
ButTers
Buffer
(1 lit.)
pH
Chemicals
Quantity (g)
Sodium citrate
Sodium chloride
Isopropanol
14.79
1.10
84.91 ml
2.85
2
Sodium citrate
Boric acid
9.8
50.0mg
3.46
3
Sodium citrate
Boric acid
9.8
50.0 mg
4.65
4
Sodium citrate
5
Sodium citrate
Lithium chloride
Boric acid
10.45
8.48
50.0 mg
6
Sodium hydroxide
16
Sodium citrate
Sodium chloride
Isopropanol
14.79
1.10
84.91 ml
Dilution
buffer
6.15
9.8
10.30
2.20
The amino acids in the present work have been grouped as
follows:
Acidic AA
Asp, Glu
Basic AA
His, Orn, Lys, Arg
Neutral AA
Ser, Thr (hydroxy)
i
Gly, Ala (straight)
Val, lIe, Leu (branched)
Aromatic AA
Phe, Tyr
Non-protein AA
Orn, p-ala, y-aba
Sulphur cont'AA
Met
i
31
3.3. Biogeochemical Indicators
Amino acids and carbohydrates make 40-80% of the organic matter
associated with plants and animals,
and consequently constitute
a considerably large fraction of the initial organic input into
the aquatic sedimentary environment
They
and
hexosamines
are
among
(Degens and Mopper,
the
more
easily
1976).
degradable
constituents of organic matter and are preferentially degraded
during settling of organic particles in the water column
(e.g.
Handa and Tominaga, 1969; Lee and Cronin, 1984).
According to Wakeham et al.
(1984), the decrease in bulk protein
amino acids relative to bulk non-protein amino acids in a sample
may result from the preferential decomposition of protein amino
acids,
which are
easily digestible.
For
instance,
non-protein
amino acids, p-alanine (p-ala) and y-aminobutyric acid (y-aba)
in
sediments may be enzymatically decomposed products of aspartic
and glutamic acid (Lee and Cronin,
1982). Ratios of these amino
acids, i.e. Asp/p-ala and Glu/y-aba, have been used as indicators
of
the
degree
of
microbial
degradation
of
organic
matter,
whereby low ratios indicate relatively more microbially degraded
nature of organic matter
al.,
(Degens and Mopper,
1976;
Ittekkot et
1984). Some biological transformation reactions of organic
nitrogen compounds resulting in the formation of amino acids . (*
marks
the
non-protein
amino
acids)
are
as
follows
Cronin, 1982):
Aspartic acid
-C02
HOOC-CH2-CH(NH2)-COOH
--->
p-alanine*
H2N-CH2-CH2-COOH
~Lee
and
32
Glutamic acid
-C02
HOOC-CH2-CH2-CH(NH2)-COOH
Uracil
-NH3
(C4N2)H402
--->
y-aminobutyric acid*
--->
H2N-CH2-CH2-CH2-COOH
B-alanine*
H2N-CH2-CH2-COOH
Arg is known to be hydrolysed to Orn and urea by the action of
enzyme araginase
(Lehninger,
amino acids
and hexosamines
(AA)
1982). The ratio between the total
(HA)
indicates the nature of
organic matter in terms of their relative degree of -microbial
reworking
and
their
phyto-
and
zooplankton
sources.
The
low
AA/HA ratios, with HA being mostly Gluam, are a clear indication
of
large
amounts
of
chitinous
materials
(Degens
and
Mopper,
1976; Degens and Ittekkot, 1984).
Chitinous zooplankton and bacterial biomass may be distinguished
by their Gluam/Galam ratios. Gluam is the major constituent of
chitin
whereas
Galam
is
present
only
in
trace
amounts
in
zooplanktons (Muller et al., 1986). In- many bacterial cell walls
both HA are present (Wolla et al., 1984) and Gluam/Galam ratio
<
4 were measured within various bacterial species (Reistad, 1975;
Kandler, 1979)
Humus contains glucosamine primarily from fungal
and bacterial cell walls
various
(Parsons,
1981;
Stevenson,
soil orders Gluam/Galam ratio has been
1994)
found
In
to vary
from 2 - 6 (Stevenson, 1994).
The utility of amino acids and amino sugars as -biogeochemical
indicators
matter
has
for
source and decompositional
been
reported
1964; Henrich et al.,
for -sediments
pathways
(e. g.
1984; Montani et al.,
al., 1987; Seifert et al., 1990a, b).
of
Degens
organic
et
al.,
1982; Steinberg et
Individual amino acids in
33
sediment
trap
indicators
for
material
and
intensity
sediments
of
have
been
decomposition;
and
used
as
hexosamine
distributions have been used to determine organic matter sources
(e.g. Cowie and Hedges, 1984; Izdar et al.,
1987; Liebezeit and
Bodungen, 1987; Seifert et al., 1990a, b; Haake et al., 1992)
Another parameter,
weight
ratio
of
which
Corg
to
is
being used as
TN
(C/N)
a
indicator,
bulk
1S
parameter.
the
Since
proteins have a C/N ratio of about 3, organisms rich in protein
sho~
low C/N ratios (Muller, 1977). On an average phytoplanktons
have a C/N ratio of about 6 and terrestrial organic matter upto
36 (Redfield et al., 1963; Walsh et al., 1981; Ertel and Hedges,
1983). Higher plants are main contributors of organic matter in
terrestrial
environment.
They
contain
<20%
proteins
and
therefore show high C/N ratios (Muller, 1977). Wide range of C/N
ratio is also indicative of the extent to which organic matter
has been degraded,
Humus
compounds
matter of
and of depletion of its protein compounds.
account
river waters,
for
the
and are
greater
part
chemically
biological stability (Bordovskiy, 1965).
of
the
organic
typified by
high