Chapter 5
LABORATORY BIOASSAY FOR ASSESSING
ALGAL GROWTH POTENTIAL OF STREAM
WATER
5.1
Of
some
15
Introduction
elements
needed
for
growth
and
metabolism of algae, the supply of nitrogen and phosphorus
is
frequently
concentrations
accelerate
problems
growth
limiting
of
algal
nitrogen
natural
and
growth and cause
in wa terbod ies.
generally
in
occurs
waters.
Higher
phosphorus,
however,
serious eutrophication
Phosphorus 1 imitation of algal
in
limitation is common in seas.
lakes,
whereas
nitrogen
Similar informations about
lot ic sys terns are scarce, although 1 imi ted efforts on the
temperate
nitrogen
streams
limiting
of
north America
conditions
showed
(Stockner
phosphorus or
& Shortreed 1978,
70
Elwood et al. 1981, Grimm et al. 1981).
So far, only one
report has come from the tropical region showing phosphorus
limitation of algal growth in a Costa Rican stream (Pringle
et al. 1986).
Micronutrient deficient conditions have also
been reported in laboratory and natural streams (Wuhrmann
&
Eichenberger 1975, Pringle et al. 1986).
This
algal
chapter
growth
bioassays
in
were
caericornutum
identifies
two
nutrient
deforested
performed
with
limitation
streams.
the
test
of
Laboratory
alga Selenastrum
due to its sensitive reaction to nutrients
/
and toxicants (US EPA 1971).
5.2
Sites
A,
B and
Study Area
C were
selected
for
the
study.
Details are given in Chapter 2.
, I
•
The
March
Materials and Methods
5.3
1989
experiments
and
June
were
1989,
conducted
respectively
winter, spring and rainy seasons.
field.
October
1988.
falling 'under
Flow was measured in the
Stream water was collected in polyethylene bottles
and analysed for various nutrients
chemical
in
parameters
as
already
and
other
described· in
physicoChapter
3.
Except flow rate (n=9), all measurements were carried out
i n t r ip 1 i c a t e •
71
Algal
assay
procedure-bottle
test
was
used
determine nutrient limiting condition (US EPA 1971).
test alga S.
capricornutum was
obtained from Dr.
to
The
Olav M.
Skulberg, Norwegian Institute of Water Research, Oslo.
The
synthetic algal nutrient medium {US EPA 1971) was used for
the maintenance of stock cultures at 24±1°C in a 14 h light
(PAR
7 0 }lm o 1 • m
nutrient
Sodium
medium
nitrate
magnesium
chloride
-2
•s
-1
10
contains
25.5,
chloride
4.4
and
)
and
the
h
dark
following
dipotassium
5.7,
sulphate
bicarbonate
The
salts
hydrogen
magnesium
sodium
cycle.
15.0.
algal
(mg.l -1 ):
phosphate
14.7,
1.0,
calcium
Other
salts
added to the the nutrient medium are (,.og.l- 1 ): boric acid
185.5,
manganese
cobalt
chloride
molybdate 7.2,
chloride
copper
0. 7'
ferric
264.2,
with
various
phosphorus
(P0 -P),
4
chloride
chloride
32.7,
0.009,
sodium
chloride 96.0, disodium EDTA 300.0.
The test waters were filtered,
spiked
zinc
autoclaved and aspetically
concentrations
and
combinations .(see Table
trace
5.1).
The
of
nitrogen
elements
in
(N0 3 -N),
different
concentration of
trace
elements was as used in the synthetic medium (US EPA 1971).
Experiments were carried out in 38 x 150 mm culture tubes
containing
spikes.
10 ml
S.
of
test waters
capricornutum
with
or
without
various
was
inoculated in each culture
tube at an initial density of 10 3 cells .ml -l. The culture
tubes were incubated under conditions used for maintaining
the stock cultures. The tubes were hand-shaken. twice daily
to
resuspend
the
cells.
The maximum standing crop
(algal
cell count on the 14th day) was measured with the help of a
hemocytometer.
Table 5.1.
Experimental design for
No.
.:..1 bioassay.
Treatment
-----------------------------------------------------------------------1.
Stream water (control)
/
2.
Stream water+ 1.0 mg.l- 1
N
3.
Stream water + 0.5 mg. 1- 1
N
4.
Stream water + 0.05 mg. 1- 1
5.
Stream water + 0.025 mg. 1- 1
p
6.
Stream water + 0.5 mg. 1-1
N
+ 0.05 mg. 1-1
p
7.
Stream water + 1 . 0 mg. 1-1
N
+ 0.05 mg. ,-1
p
8.
Stream water + 0.5 mg. 1- 1
N
+ 0.025 mg. ,-1
9.
Stream water + trace elements
•
P
1 c.
Stream water + trace elements + 0.5 mg. 1- 1
11 •
Str~am water+ trace elements+ 0.05 mg.l- 1
12 •
Stream water + trace elements + 0.5 mg. 1- 1
13.
Full strength medium
p
N
P
N + 0.05 mg. 1- 1
P
72
Cell count data were log-transformed and subjected
to
Least
ANOVA.
calculated
to
significant
compare
treatments (Snedecor
the
The
algal
& Cochran
5.4
important
difference
(P
biomass
~
0.05)
for
was
various
1976).
Results
physico-chemical
characteristics
of
stream water at different sites are illustrated in Figs.
3.1 to 3.5.
Although the physico-chemical
stream water have already been dis.cussed in
brief description for
periods,
1989
during
given hereafter.
nutrients
October
properties of
Chapte~
3, a
1988, March 1989 and June
which bioassays were
conducted,
is
Velocity, conductivity, and the level .of
exhibited
considerable
variations.
Marked
fluctuation in velocity were observed, with lowest value in
March 1988 (10-33 cm.s- 1 ) and highest in June 1989,(62-112
cm.s
-1
).
The pH was much more closely
co~fined
over space
and time, but conductivity showed an inverse relation with
flow.
Ammonia level was
1989 and October 1988.
maximum in October 1988.
total
phosphorus ,showed
more during June 1989 than March
Nitrate-nitrogen concentration was
Soluble-reactive phosphorus and
high
values
in
October
1988.
Changes in dissolved silica were not pronounced, except at
site A.
The concentrations of calcium and magnesium were
low in October 1988 at all stations.
The number of taxa (Figs. 3.10 and 4.1) representing
73
the epilithon and the epipelon respectively was highest at
site C except during October 1988 when site B showed the
highest.
During June 1989 minimum number of taxa occurred
in the epipelon at sites A and B, and high flow rate made
epilithic
flora
difficult
to
study.
Maximum number
of
species encountered during March 1989 and dominant species,
of
most
are
which
illustrated in Figs.
members
of
Bacillariophyta,
are
to 3.8 for the epilithon and in
3.6
Tables 4.3 to 4.5 for the epipelon.
Figures
5.1,
and
5.2
5.3
show
that
the
ma~imum
standing crop of alga was obtained by phosphorus addition to
Phosphorus in combination with nitrogen
the test waters.
yielded better results.
Phosphorus supplementation to test
waters yielded higher biomass at 0.05 mg.l- 1 than at 0.025
mg .1
-1
, except at site A in October 1988 and at site C in
March 1989.
stream
Amongst the various treatments, enrichment of
water
with
mg.l -1
10
nitrogen
and
0.05
phosphorus increased the standing crop maximally.
and
mg.l -1
Nitrate
trace element addition did not elicit marked effect.
None of the treatments levelled the results shown by the
full strength medium.
5.5
The low
le\~ls
of nitrogen, phosphorus, calcium and
magnesium suggests that
softwa ter by nature.
Discussion
the streams are oligotrophic and
Ammonia-nitrogen was at an extremely
I
7
Oct 88
I
6-
r-
r--
,.--
r-
-
rr~
5-
4
r-
-
rr---
~~
7I
E
....
6-
I
r-
c
0
0\
0
..-r-
5
-
~
u
r-
r-
r-
.0
E
::;)
-
r-
~
Mar 89
4
rr-
,-
l
~~
7-
I
Jun 89
6-
r-
1
r-
r-
..--
r-
r-
-
11
12
r-
5,_
rrr-
r-
4
~~
2
3
4
5
6
7
8
n
9
10
13
' Treatments
Fig.S.l. Cell yield of Selenastrum capricornutum on the 14th day
in test water collected from site A and supplemented
with N, P and trace elements.
Treatments have been
explained in Table 5.1. Vertical bars show LSD (P<
0. 05).
7
1
oct 'as
6
.......--
....-
rr-
r-
r-
....-
5r-
r--
,--
r-
4
·~E
,--
~;._J
7I
Mar 89
r-
:r.
~
u
0
r-
4
7
....--
,....
...-
rr-
r-
-
5
0
Ol
r-
.--
r-
r-
-:::;..
.J
-.
Jun '89
l:
,--
,..-
r-
r-
r-
-
rr-
5
r-
4
...-
-
1. 2
3
r-
r-
~;..
J
4
5
6
7
8
9
10 11
12
13
Treatments
Fig. 5.2. Biomass yield
of S. capricornutum on the 14th day in
test water of sfte B enriched with N,P and trace
elements.
Treatment as in table 5 .1.
Vertical bars
show LSD (P
0.05).
<
7_
0-:t '88
6_
1
r-
..--
5_
,--
E
.....
7_
I
E
~
Mar 89
~
..0
..--
I
6_
r-
::J
,..-
r-
c
b)
u
5_
0
en
0
r-
..--
I
Jun B9
6_
..--
:r.
5_
..--
2
3
4
5
6
7
8
9
10 11
12 13
Treatments
Fig. 5.3. Cell yield of §_. capricornutum on the 14th day water
collected from Site C and enriched with N, P and trace
elements.
Treatments as in Table 5.1.
Vertical bars
show LSD (P < 0.05).
74
low level because the streams are aerated without excessive
organic loading.
The high N : P atomic ratio suggests the
streams to be phosphorus deficient.
supported
this
increased
water.
from
by
contention
phosphorus
The bioassay results
because
algal
supplementation
growth
to
the
was
stream
Phosphorus-limiting conditions have been reported
some
temperate
& Bowers
Pringle
streams
also
(Peterson et al.
1983,
1984).
The total number of species encountered during the
bioassay
period
is
(O'Quinn
& Sullivan
Depauperate algal
much
less
1983;
flora
of
than
the
Rushforth
previous
&
Squires
reports
1986).
streams has been ascribed to·
nutrient deficiency (Chessman 1986) or low pH (Keithan et'
al.
Phosphorus-limiting
1988).
condition
seems
to
be
responsible for the diminutive algal flora in the present
study. Furthermore,
seem
to
diatoms
have
in the present work, low pH does not
exerted
because
most
a major
of
the
influence
taxa
particularly on
encountered have
been
previously reported at pH 7, with best development above 7
(Lowe 1974).
The biomass (2.1-11.4 mg.m- 2 ) was much lower than in
a softwater stream studied by Marker (1976).
biomass values ranging from below 10 mg.m
mg.m -2
in summer.
community
accural
by
-2
He obtained
in
{
~to
50
Year-long domination of the epilithic
diatoms
may
explain
the
low
chlorophyll !
in the selected streams (see La Perriere et al.
75
1989).
Soluble reactive phosphorus saturates the growth of
algae at various
>
levels:
7 j\lg.l- 1 in filamentous algae
(Seeley 1986), and C::::. 4 .,.ug.l -1 in case of diatoms ( Bothwell
1985).
}Jg .1
-1
site
The range of SRP in our case was far lower (0.3-1.7
),
3
except at
sites 1 and
Jones et al.
in March 1989.
higher
chlorophyll
phosphorus
a
2 in October 1988 and at
for
concentrations,
Missouri
whereas
( 1984) demonstrated
streams
&
Krewer
with
Holm
low
(1982)
reported positive relation between chlorophyll a and total
dissolved phosphorus in artificial streams.
Increased
biomass
with
phosphorus
addition
in
bioassy experiments suggests that phosphorus deficiency is
limiting algal productivity in the selected streams.
consequence,
reach
the
the
standing crop of stream algae
nuisance
level
(100-150
mg
As a
is yet
chlorophyll
to
a'.m- 2
I
Welch et al.
level
of
streams
1988).
nutrients,
is
further
It may, however, be attained if the
particularly
increased
due
phosphorus,
to
in
these
intensification
of
disturbances in the catchments.
Though
1abora tory
bioassays have
proved
useful
in
defining the nutrient limited algal growth under controlled
conditions,
the
the
laboratory
in situ conditions cannot be simulated in·
and
the
factors cannot be studied.
interactive
effect
of
various