ISTVÁNOVICS, VERA, KURT PETTERSSON, DON PIERSON, AND

Notes
890
Limnol. Oceanogr., 37(4), 1992, 890-900
0 1992, by the American Society of Limnology
and Oceanography,
Inc.
Evaluation of phosphorus deficiency indicators for summer
phytoplankton in Lake Erken
Abstract-Short-term changes in nutrient status of summer phytoplankton were studied in
Lake Erken in 1988. The P deficiency index (PDI),
which is defined as the rate of light-saturated photosynthesis (P,,,,) divided by the maximum phosphate uptake velocity (I’,,,), was a superior indicator of P deficiency, despite the fact that
dramatic shifts in nutrient status did not occur
during the study period. Indicators containing
physiological rates (orthophosphate turnover
time, TT; Chl-specific total alkaline phosphatase
activity, TAPAB; and Chl-specific maximal phosphate uptake velocity, VBmaX)
were also sensitive
enough to show changes in the P status of algae.
In contrast, indicators based on the chemical
composition of the particulate matter (PN: PP,
Chl-specific surplus P content, SPS)failed to show
short-term variations in algal nutritional status. Correlations were found between PDI and
TAPAB, PDI and TT, as well as PDI and turnover
time of surplus P (TSP). PDI ranges previously
suggested to indicate different degrees of algal P
deficiency were found to describe P deficiency
adequately in Lake Erken.
Several approaches have been used to
identify limiting nutrients in aquatic ecosystems. The chemical composition of particulate matter and biomass-specific physiological rates have been widely suggested
as potential indicators of algal nutrient status. In spite of numerous recent attempts to
find suitable metabolic indicators, a clear
guide to the assessment of nutrient limitation among natural plankton communities
has not been produced (Healey and Hendzel
1980; White et al. 1985; Vadstein et al.
1988).
When biomass-specific rates are used, two
basic problems arise. First, the relative conAcknowledgments
This work was carried out at the Erken Laboratory
of Uppsala University. Vera Istvinovics visited the
laboratory as a guest researcher on a fellowship from
Uppsala University. We are indebted for comments
by two anonymous referees. For technical and analytical work we thank Ineke Barten, Rima Abu Middain,
and Ulf Lindqvist.
Financial support was given by the Swedish Natural
Science Research Council.
tribution
of algae and bacteria to many
physiological rates varies both seasonally
and among lakes. Examples include orthophosphate turnover times (Lean and Pick
1981; Lean et al. 1983), alkaline phosphatase activity (Jansson et al. 1988), and NH,+
enhancement of dark CO2 fixation (White
et al. 1985). Second, estimators of algal biomass need to be corrected for different
sources of interference (chlorophyll degradation products; detrital, bacterial, and zooplankton contributions to particulate C), either by measuring the interfering substances
directly or by ensuring that more-or-less
valid conversion factors are used. A promising candidate for an algal nutrient-deficiency indicator should therefore include
two physiological rates: one indicative of
potential algal demand for the nutrient and
another that measures actual algal capacity.
Until now only one such indicator has been
proposed: the P deficiency index (PDI) of
Lean and Pick (198 1). PDI relates the optimal rate of photosynthesis (Popt, pg C liter-l h-l) to the maximal phosphate uptake
velocity ( V,,,, pg P liter-l h-l). Popt is an
estimate of the maximal potential for algal
growth and will therefore also estimate
maximal potential P demand. Being an estimate of photosynthesis, it will be free of
both bacterial and detrital interference. V,,,
is a measure of the maximal rate of P uptake
at saturating phosphate concentrations. As
phytoplankton
dominate P uptake under
these conditions (Lean et al. 1983), V,,, will
be indicative of algal P demand and also be
free of bacterial and detrital interference.
Surprisingly, PDI has been sparsely used
given its potential merits (Lean et al. 1983;
Istvanovics and Herodek 1985); it has been
compared to other indicators of algal P status only in Lake Ontario (Lean et al. 1987).
The nutrient status of spring and summer
phytoplankton has been studied thoroughly
in Lake Erken on the basis of several indicators of nutrient deficiency (Pettersson
Notes
1980,1985a). Here we have determined PDI
and compared it with various other nutrient-deficiency indicators. Because we followed short-term changes in nutrient conditions during succession of the summer
phytoplankton,
sensitivity of PDI to less
dramatic nutritional
changes than in the
Lake Ontario study could be tested (Lean
et al. 1987). As PDI is exempt from bacterial and detrital interferences that affect
other measurements of P deficiency we begin with the a priori assumption that PDI
is a superior estimate of P deficiency. Here
we examine the relationship between PDI
and other commonly used indicators of algal P status to confirm the value of PDI as
an indicator of P deficiency and to examine
the shortcomings of other indicators. We do
so first by correlation analysis using data for
2 yr with different sequences of thermal development: phytoplankton
succession, and
nutrient deficiency (Pierson et al. in press).
Second, we examine temporal variations in
PDI and the other indicators of P deficiency
during 1988, when our sampling was most
intensive. Such an analysis allows us to examine in detail the factors responsible for
inconsistencies in the correlation analysis.
We took water samples at a buoy situated
700 m offshore above the deepest point of
the lake (z,,, = 21 m). Either a Ruttner
sampler or a pump was used to collect water
which was then placed in 2-liter, opaque
plastic bottles. Sampling began at about 1000
hours on each occasion, and samples were
collected twice weekly between 10 June and
12 September 1988. In 1989 the sampling
was performed in a similar way, but only
once a week.
The concentration of Chl a was measured
fluorometrically
and corrected for degradation products according to the method of
Strickland and Parsons (1972). Soluble reactive P (SRP) was determined according to
Murphy and Riley (1962), and surplus P
(SP) according to Fitzgerald and Nelson
(1966) with minor modifications
(Pettersson 1980). Particulate P (PP) and total P
(TP) were measured after persulfate oxidation in an autoclave (Menzel and Corwin
1965). Particulate N (PN) was determined
with a CHN analyzer (Carlo-Erba) after filtration onto precombusted (4 h, 550°C)
891
Whatman GF/F glass-fiber filters. NH4+,
NOz-, and N03- were measured according
to standard methods (Ahlgren and Ahlgren
197 8). Total alkaline phosphatase activity
(TAPA) was determined with a Turner filter
fluorometer
using 4-methylumbelliferylphosphate as a fluorogenic substrate (Pettersson 1980).
Photosynthesis
vs. irradiance relationships were obtained by a small-volume,
short (20 ml for 20 min) 14C incubation
method as described by Pierson et al. (in
press). Bacterial production was measured
by [3H]thymidine
incorporation
into cold
TCA-insoluble
material and DNA (Bell et
al. 1983). In the epilimnion of Lake Erken,
85% of the thymidine incorporated
into
macromolecules was in DNA (Bell et al. unpubl.).
In order to determine phosphate uptake
kinetics, we added carrier-free H332P04 and
different phosphate enrichments (0.5-8 .O pg
P liter-‘) simultaneously
to duplicate or
triplicate 200-ml samples that were then incubated at in situ temperatures. Isotope uptake was stopped at appropriate time intervals by transferring -20 ml of subsample
into scintillation
vials containing
50 ~1
KH2PO4 (final concn -500 pg P liter-‘).
Falkner et al. (1980) found this method satisfactory in stopping uptake of 32P tracer by
blue-greens, and no difference has been
found in Lake Erken when 32P uptake was
terminated by immediate filtration or cold
phosphate addition. Subsamples were kept
at 5°C in the dark until filtration through
0.2- or 0.45~pm membrane filters (usually
within 4 h). Radioactivity
of 5-ml filtrates
was measured as Cherenkov radiation with
internal standards.
Before 32P and 14C uptake experiments,
the water was prefiltered through a 200~pm
plankton net to remove larger zooplankton.
Prefiltered
and unfiltered
samples were
compared in August when Gloeotrichia
echinulata became dominant because this
large alga does not pass the net.
Phosphate uptake was described by the
simple two-compartment
model. The natural logarithm of the percentage radioactivity in the filtrates was plotted as a function
of time and the rate constant determined
during the period when the relationship was
Notes
892
locity, v, is equal to 0.5 V,,,, and S (pg P
liter-l) the ambient orthophosphate
concentration in the water.
Here Chl a is used as a measure of phytoplankton biomass in order to minimize
interference from bacterial and detrital C
when estimating that biomass. Indicators of
P deficiency are scaled to Chl a and then
termed specific rates or specific variables.
In 1989, as a result of shorter and less
stable thermal stratification and higher hyPDI
polimnetic temperatures, there was greater
h3 c tig W’l
SRP transport to the epilimnion
than in
10
1988. Consequently, the algae were less P
1
B
deficient in 1989 than in 1988 (Pettersson
et al. 1990; Pierson et al. in press).
Lean et al. (1987) found a clear negative
correlation between TAPA and PDI in Lake
Ontario. Because most of the alkaline phosphatase activity was not algal in this lake
(Pick 1987), the correlation became worse
when Chl-specific TAPAB was used (Lean
0.01
et al. 1987). In contrast to Lake Ontario,
10
1
100
1000
specific TAPAB is a good indicator of algal
0.1
PDI
P deficiency in Lake Erken (Pettersson 1980,
h.ec hzwll
1985a). In Lake Erken, size-fractionation
Fig. 1. Relationships between PDI and specific
studies have shown that -80% of the total
TAPAB (A) and TAPA (B) in the upper epilimnion
phosphatase activity originates from or is
(0.5-3 m). In panel B the continuous line shows the
associated with algae, and TAPAB drops
regression between PDI and TAPA [log,,TAPA
=
to a minimum
when enzymatically
de-0.227(log,,PDI)
+ 0.132; n = 32, r = -0.441, the
dashed line shows the same regression in Lake Ontario
termined orthophosphate
concentration
presented by Lean et al. (1987), and the dotted line
exceeds 1 pg P liter-l (Pettersson 1979).
shows the regression between PDI and specific TAPAB
Therefore,
in the epilimnion of Lake Erken
[log,,TAPAB = -0.384(log,,PDI)
- 0.214; n = 32, r
there was a better negative correlation be= -0.601.
tween specific TAPAB and PDI than in Lake
Ontario (Fig. 1). The 1989 data showed a
linear. Turnover times of orthophosphate
similar relationship as that found in sumin the water (TT, min) were calculated as mer 1988, although during the much larger
reciprocals of the uptake rate constants.
bloom of G. echinulata some high specific
Dependence of TT on phosphate concenTAPAB values were measured when PDI
tration at different phosphate additions (A, indicated low P deficiency.
pg P liter-l) was described by the formula
Specific TAPAB averaged O-50+0.24
derived from the Michaelis-Menten
equa- nmol (pg Chl a)-’ mine1 when PDI showed
tion:
extreme P deficiency (< lo), decreased to
0.16 + 0.09 when P deficiency was moderate
(PDI between 10 and 30), and dropped to
0.11+0.04 when PDI > 30 were indicative
of low P deficiency (Table 1). Specific SPB
~0.2 pg P (Chl a)-’ indicates P deficiency
where V,,, (pg P liter-l h-l) is the maximal
of algae (Fitzgerald and Nelson 1966; Petphosphate uptake velocity at saturating
tersson 1980; Vadstein et al. 1988). The
phosphate concentrations, K (pg P liter-‘)
much higher values in Lake Erken during
the apparent half-saturation
constant (the
the entire summer 1988 (Table 1) could be
phosphate concentration where uptake ve-
I0
l
I
.
.““I
’
. ..‘.I
.
’ ’ ‘.--Y
1988
1989
- ’ . “I
Notes
Table 1. Comparison
significant (>0.05).
of indicators of P deficiency. P values: *- ~0.05; **-
Degree of P deficiency
~,a, [I% p
(pg Chl a)-l h-‘1
Mean + SD (n),
this study
Range
Moderate
(10-30)
Extreme
(<lo)
Variable
TAPAE [nmol
kg Chl a)-* min-‘1
Mean + SD (n),
this study
Range
TT (min)
Mean f SD (n),
this study
Range
893
~0.01; ***-
<O.OOl; NS-not
on the basis of PDIt
(20)
(3KO)
0.2-0.7$
0.539+0.246***(5)
0.336-0.897
<60§
0.205+0.160*(14)
0.058-0.68 1
0.154+0.137(19)
0.03 l-O.526
0.150+0.139(3)
0.055-0.3 10
44+49*(7)
6-141
96*85**(14)
14-258
437+417(12)
48-l ,200
2,558+272(3)
> 2,300
0.147&0.072***(14)
0.040-0.305
0.057 k0.026NS( 12)
0.026-o. 107
0.026+0.009(3)
0.016-0.033
1.20*0.72(5)
0.19-1.88
0.80a0.77(16)
0.11-2.81
0.51+0.31(19)
0.01-1.07
0.62+0.23(3)
0.41-O-87
20-3Oll
1O-2017
10.4+2.8(5)
8.3-13.8
11.7&3.0(15)
4.5-l 6.4
0.2-3 )I
1.221+1.099***(7)
0.381-3.197
WE [PLgp
(a Chl WI
Mean + SD (n),
this study
Range
PN:PP
[Il.gN OLgW’l
Mean + SD (n),
this study
Range
<0.2#
t Lean and Pick 1981.
* Pettersson 1980; Jansson et al. 1988.
0 Lean et al. 1983.
1)Istvtiovics
and Herodek 1985.
# Fitzgerald and Nelson 1966; Pettersson
ll Healey and Hendzel 1980.
1980; Vadstein
<loll
14.1-+4.4(15)
8.2-23.1
9.8?3.8(3)
5.8-13.3
et al. 1988.
due to significant bacterial interference in
early summer and phosphorus-rich
G.
echinulata colonies later on. Thus, specific
SPB did not indicate levels of P deficiency
during June and July that were consistent
with PDI and other P deficiency indicators.
Orthophosphate turnover time (TT) depends on both the apparent half-saturation
constant (K) and the transport capacity
( Vmax)of the plankton [mathematically
TT
= (K + s)/ Vmax].As smaller organisms have
lower half-saturation constants than larger
organisms, the picoplankton, including bacteria, most strongly influence TT values
measured at the ambient orthophosphate
concentration (Lean et al. 1983, 1987). V,,,
on the other hand, is the uptake velocity at
saturating phosphate concentrations, where
larger size classes dominate uptake (Lean et
al. 1987; Istvanovics et al. 1990). Although
TT and PDI are influenced by different fac-
tors, Lean et al. (1987) found a strong positive correlation between the two variables
in Lake Ontario. A similar relationship was
also observed in Lake Erken. In 1989 both
TT and PDI were usually higher than in
1988, but the data fit the same relationship
in both years (Fig. 2A). In spite of a considerable overlap among TT ranges under
extreme, moderate, and low P deficiencies
as indicated by the PDI values, the averages
increased rapidly (Table 1).
White et al. (1982, 1985) found that much
of the variation of TT in Canadian and New
Zealand lakes could be explained by changes
in PN : PP ratios and SRP concentrations.
The shortest turnover times were associated
with high PN : PP ratios and low SRP concentrations. Dependence of TT on PN : PP
ratios would explain why TT can be used
as a phosphorus deficiency indicator in spite
of bacterial influence (affinity, K): a high
Notes
894
10000 =
fJB
0
1000 _
Is
s
E
0
0
100 _
0
O0
0
0
0
0
O-a
0
.
0: fl
0.
0
0
-*
l+
11
10
15
20
PN:PP [pg N (ug P)-’ ]
Fig. 2. Relationships between orthophosphate TT
in the water and PDI (A) and between PN: PP ratios
of the seston and TT (B) in the upper epilimnion (0.S
3 m). In panel A the continuous line shows the regression in Lake Erken [log,,PDI = 0.620(log,,TT) +
0.084; n = 36, r = 0.811, and the dashed line shows
the same regression in Lake Ontario presented by Lean
et al. (1987).
PN : PP ratio also indicates a dominance of
P-poor phytoplankton
in seston. Such relationships were not observed, however, in
Lake Erken. The dependence of TT evidently resulted from phytoplankton succession that led to dominance of blue-greens.
The highest PN : PP ratios were measured
during the cyanophyte bloom as a result of
N2 fixation. At this time PDI values were
high and TT values increased. In 1989 PN :
PP ratios were generally lower, whereas TT
values were higher than in 1988. When data
from both years were plotted together, a
slight inverse tendency between PN : PP ratios and TT could be seen (Fig. 2B).
Currie et al. (1986) showed that partitioning of orthophosphate uptake between
bacteria and algae covaried with TT in sev-
era1 lakes. They concluded that the partitioning in favor of bacteria is principally a
function of the severity of P deficiency. The
relationship between PDI and TT presented
here (Fig. 2A) and by Lean et al. (1987)
supports this conclusion. Thus a high degree
of P deficiency (as indicated by a low PDI
value) would lead to dominance by bacteria
of P uptake (lower K) and a faster turnover
(lower TT). Currie et al. (1986), however,
explained this relationship as increasing orthophosphate
concentrations
under decreasing P deficiency. This explanation does
not apply in Lake Erken, where epilimnetic
SRP concentrations showed a tendency to
decrease and enzymatically
measured orthophosphate was undetectable during periods when TT showed drastic increases. We
conclude that it is the rate of orthophosphate supply controlled by planktonic interactions (the rate of phosphate regeneration) and by phosphate inputs from the
hypolimnion and sediments that are the key
factors.
Average Chl-specific vB,,, decreased rapidly as the P status of the phytoplankton
improved (Table 1). Specific p,,,
shares
with PDI freedom from bacterial interference, but detritus might affect the chlorophyll measurements and thus bias the results.
The SP: I’,,, ratio provides an estimate
of algal SP turnover (TsP). Stored P in algae
has been shown to be one of the most mobile
intracellular P pools (Fitzgerald and Nelson
1966; Pettersson 1980; White et al. 1985;
Vadstein et al. 1988). Although some of the
variation in SP can be attributed to changes
in bacterial abundance, V,,, varied by a
larger extent and therefore had a dominant
influence on TSP. TSP ranged between 0.38
and 3 1 h and was positively correlated with
PDI (Fig. 3A).
Turnover time of algal C (TPC = PC : P,,J
was also estimated (after subtracting from
the PC values an estimated 200 pg liter-’
of detrital C) and found to be weakly inversely correlated with PDI (Fig. 3B). Variation of T,, was relatively small ( 1 l-59 h)
over a large range of PDI values compared
to the variations seen in TSP. The consistency of Tpc suggests that P,,, is a temporarily stable measure of potential P demand
895
Notes
-60
0.1 i
0.1
1
10
100
1000
PDI bg C Ws PI-’ 1
1000 ?
i B
2
loo-
z
b
10 _
.
0
*I
0.1
1
10
100
1000
PDI hs C (vg PI-* 1
Fig. 3. Relationships between PDI and T,, (A) and
T,, (B) in the upper epilimnion (0.5-3 m). Regressions:
= 0.750(log,,PDI)
- 0.391; y1= 34,
panel A-log,,T,,
r = 0.72; panel B-log,,T,,
= -O.l98(log,,PDI)
+
1.707; n = 35, r = -0.43.
and shows that most variability in PDI is a
result of variations in P deficiency (I’,,,).
The relationships between PDI and tumover of either algal C or P are expected,
because PDI should indicate ability of P-depleted plankton to take up phosphate in excess of their growth requirements (Lean and
Pick 198 1). P-deficient algae will have lower
P,,, and higher V,,, in comparison to P-sufficient ones, because a larger portion of their
energy store (ATP) is used for phosphate
uptake and less is available for C fixation.
Therefore P deficiency leads to longer tumover time of algal C and shorter turnover
of algal P.
The above discussion shows a general
correspondence between PDI and other indicators of nutrient deficiency. But will the
methods agree well enough to characterize
short-term changes in nutrient status of the
Jun
Jul
A%
sep
Fig. 4. Biomass (A) and production (B) of phytoplankton and bacterioplankton in the upper epilimnion
(0.5-3 m). Bacterial cell counts were converted to carbon assuming 2.2 x lo-’ Fg C prnd3 C content, and
the conversion factor for thymidine incorporation was
2 x lOI cells (pm01 thymidine))’
(Bell et al. 1983).
Vertical lines separate the four periods with different
degrees of P deficiency (see text).
plankton? During 1988 nutrient status of
the phytoplankton
changed and four periods could be distinguished. Because external nutrient loadings are very low during
summer (~20 pg P m-* d-l and 800 pg N
m-* d-l; Pettersson 19853), any changes in
nutrient deficiency must be related to processes in the lake. Similar periods could be
distinguished
on the basis of seasonal
changes in planktonic structure and interactions (Bell et al. unpubl.) as well as of
epilimnetic nutrient loading associated with
changes in thermal stratification (Pierson et
al. in press).
From 10 to 28 June (period 1) algal biomass and production were low (Fig. 4), with
cryptophytes and chrysophytes dominating
(75% of algal biomass). Concentrations of
Notes
896
80
I
I
I
Y
I
0
-
Jy
:
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
B
1
‘Idi
0
I
I
I
iI
I
I
I
0
II ,
-
-5 0.8
E
r( 0.6
# '
4s
bU
E 0.2
$3 s 0.0
0.4 I
0
15
10
5
0
Jun
Jul
Aug
Sep
Fig. 5. Nutrients in the upper epilimnion (0.5-3 m)
and the hypolimnion (15 m). Vertical lines separate
the four periods with different degrees of P deficiency
(see text).
SRP and dissolved inorganic N (DIN =
N03- + N02- + NH,+) were relatively high
but decreased through the period (Fig. 5A,B).
PDI values between 1 and 25 (mean:
8.4 + 9.7; Fig. 6A) indicated extreme P deficiency, and this conclusion was supported
by other indicators of nutritional status. Or-
Jlln
Jul
Aug
Sep
Fig. 6. Indicators of P deficiency in the upper epilimnion (OS-3 m). Vertical lines separate the four periods with different degrees of P deficiency (see text).
thophosphate TT below 60 min (Fig. 7A)
indicated high P demand according to Lean
et al. (1983). Specific TAPAB ranged between 0.4 and 0.8 nmol (hg Chl a)-’ min-l
(mean 0.5750.23; Fig. 6C), also indicating
P deficiency. Specific pma, varied from 0.1
to3.2pgP(pgChla)-‘h-*
(mean 1.1kl.l;
Fig. 6B), a range typical for the extremely
Notes
897
P-deficient phytoplankton
of Lake Balaton
[0.2-3 pg P @g Chl a)-’ h-l; Istvanovics and
Herodek 19851.
Specific SPB values were 0.5-2.0 pg P (pg
Chl a)-’ (mean 1.19 kO.59) in Lake Erken
during period 1 (Fig. 6D). These high SPB
values were not consistent with the other P
deficiency indicators mentioned above and
could be explained by the influence of bacteria and flagellates on SP concentrations.
In May and early June 1989,60-97% (mean
80%) of SP was found in these organisms.
Bell et al. (unpubl.) estimated that phagotrophy provided -70% of the P requirement of the flagellates in June 1988 and
therefore concluded that they were not limited by P. Extreme P deficiency of phytoplankton during period 1 could be explained
by strong competition between larger algae
(> 12 pm) and bacteria for phosphate.
From 1 to 22 July (period 2) phytoplankton production and biomass increased exponentially (Fig. 4) and larger species (As-
terionella formosa, Ceratium hirundinella)
became dominant. PDI values between 15
and 45 (mean 24.4+ 10.6; Fig. 6A) indicated
that phytoplankton
were moderately P deficient. In accordance with this conclusion,
specific TAPAB decreased to < 0.3 nmol (pg
Chl a)-’ min-’ (mean 0.18kO.14; Fig. 6C)
and specific pm=, to ~0.3 pg P (pg Chl a)-’
h-l (mean 0.20+0.07; Fig. 6B).
Various processes could contribute to the
improvement of algal P status during period
2. First, there was a temperature-dependent
increase in the orthophosphate concentration in the hypolimnion
from 28 June onward (Fig. 5A; Pettersson et al. 1990). Some
of this phosphate entered the epilimnion and
resulted in an increase of the total P (TP)
concentration (Fig. 5C). On the basis of the
TP increase and a mean epilimnion depth
of 6 m (Pierson et al. in press), the net transport of P from the hypolimnion
was estimated to be 2 mg P me2 d-l. Epilimnetic
SRP concentrations remained low during
period 2 (Fig. 5A), but a continuous increase
in the epilimnetic K + S values (Fig. 7B)
can in part be attributed to an increase in
S, the orthophosphate concentration due to
internal loading. Second, P recycling within
the epilimnion caused by grazing of cladocerans on bacterioplankton
(Bell et al. un-
0.01
Jlln
-I
Jul
Aug
Sep,
Fig. 7. Parameters of phosphate uptake kinetics in
the upper epilimnion (OS-3 m), the metalimnion, and
the hypolimnion (15 m). Vertical lines separatethe four
periods with different degreesof P deficiency (seetext).
publ.) may have eased P deficiency of phytoplankton.
Integrated algal production ranged from
0.6 to 0.8 g C m-2 d-l in period 2 (Pierson
et al. in press). If we take into account the
average P cell quota of the > 12-pm fraction
in May-July
1989 [lo pg P (mg C)-‘1, P
demand of the phytoplankton
could be 68 mg P mW2 d-l. Therefore a combination
of internal P loading and enhanced P regeneration would be needed to sustain high
primary production, as well as to ease the
P deficiency of phytoplankton.
From 26 July to 5 August (period 3) the
epilimnion deepened from 8 to 11 m. As
the deepest 40% of the lake basin (> 10.5
m) contains P-rich accumulation
sedi-
898
Notes
ments, this deepening resulted in increased both epilimnetic SRP and DIN concentraphosphate inputs to the epilimnion and a tions (Fig. 5A,B).
rise in SRP concentration (Pierson et al. in
Physiological rates were more sensitive to
press; Fig. 5A). Even though DIN concenP deficiency than were indicators based on
tration was exceptionally high in 1988 (cf. chemical composition
of the particulate
Bostrom 198 1) and exceeded 20 pg N liter-l
matter (Table 1). Although TT is strongly
even during period 3 (Fig. 5B), a relative
affected by bacteria, and TAPA may be inshortage of NH4+ as well as improved phos- fluenced to varying degrees by factors other
phate availability
could provide competithan microorganism nutritional status (Pettive advantage to N,-fixing species (Pet- tersson 1980; Pick 1987; Jansson et al.
tersson et al. 1990). N,-fixing blue-greens
1988), both TT and specific TAPAB showed
(Anabaena spp. and G. echinulata) ap- statistically significant differences when PDI
peared in the phytoplankton, and G. echinu- indicated different degrees of P deficiency.
Zata formed a large bloom in early August PDI was a superior indicator, however, of
(Fig. 4). PDI varied from 28 to 50 (mean short-term, moderate changes in P deficien38 k 15; Fig. 6A) indicating low P deficiency. As was emphasized by Lean and Pick
cy. In agreement with this conclusion, TT (198 1) and Lean et al. (1987), the advantage
exceeded 100 min (mean 180 & 110; Fig. 7A), of relating two algal dominated rates (as in
and specific VB,,, averaged at 0.08 -to.03
the PDI) is that the ratio will be automatically scaled for temperature and biomass
pg P (pg Chl a)-’ h-l (Fig. 6B).
As phosphatases are constitutive in blue- differences and will be free of detrital or
greens (Pettersson 1980; Jansson et al. 1988), bacterial interference. Similar to PDI, VB,,,
was also a sensitive indicator of P deficiency
a small peak of specific TAPAB not indicative of P deficiency was observed during
(Table 1).
this period (Fig. 6C). Specific SPB showed
Throughout the study, epilimnetic PN:
wide variations between 0.01 and 1.4 pg P PP ratios were fairly constant in the interval
(pg Chl a)-’ (mean 0.49kO.5 1; Fig. 6D) due 8 : 1 to 20 : 1 (Fig. 5D), and PC : PN ratios
to increased transport of P-rich G. echinu- showed no marked deviations from the raZata colonies from the accumulation sedi- tio of 6 : 1 (by weight; Pettersson et al. 1990).
ments to the epilimnion. This recruitment
These PN : PP and PC : PN ratios are close
represented a significant P input (Pettersson
to the values characteristic of nutrient-sufet al. 1990). Temporal variability in specific ficient phytoplankton (Healey and Hendzel
SPB would be expected because this alga uses 1980) and suggest more or less balanced
its internal P stores for growth, with negli- supplies of both nutrients. We suggest that
gible phosphate uptake in the epilimnion
the coexistence of P-rich and P-deficient
(Pettersson et al. 1990; Istvanovics et al. species could also result in the relative stability of these ratios and apparent nutrient
1990).
After 9 August (period 4) collapse of the sufficiency (Pettersson et al. 1990). Smaller
cyanophyte bloom was followed by another
organisms could influence the PN : PP ratios
bloom of diatoms and cryptophytes in late in period 1, when nearly optimal PN : PP
August (Fig. 4). At this time PDI indicated
ratios could result from the presence of both
moderate-to-low P deficiency (14-72, mean P-rich bacteria and flagellates with low PN :
42+22; Fig. 6A). TT exceeded 100 min PP ratios and extremely P-deficient algae
(mean 200& 70; Fig. 7A), whereas specific with high PN : PP ratios. Therefore PN : PP
VBmax and specific TAPAB were low ratios and specific SPB may not be sensitive
[0.08+0.06
pg P (pg Chl a)-’ h-l and enough to indicate small, short-term shifts
re- in phytoplankton
0.12kO.13 n mol @g Chl a)-’ minl,
nutrient status (Table 1)
spectively; Fig. 6B,C].
without prior separation of planktonic comPeriod 4 coincided with the complete de- ponents.
terioration of thermal stratification, which
The PDI, which was introduced by Lean
further increased nutrient inputs to the epi- and Pick (198 1) as an indicator of P defilimnion. Nutrients in excess of algal re- ciency, has been tested in only a limited
quirements caused measurable increase in number of lakes, but has proved to be a
Notes
reasonable indicator in each case (Lake Erie:
Lean et al. 1983; Lake Balaton: Istvanovics
and Herodek 1985; Lake Ontario: Lean et
al. 1987). PDI was shown in this study to
be sensitive enough to indicate relatively
small changes of algal nutrient status in Lake
Erken and was correlated with other indicaters of P deficiency. On the basis of the
available information, PDI seems to behave
reliably as an indicator of P deficiency.
Vera Istvhovics
Balaton Limnological Research Institute
H-8237 Tihany, Hungary
Kurt Pettersson
Erken Laboratory, Institute of Limnology
Uppsala University
S-761 00 Norrtalje, PL 4200, Sweden
Don Pierson
Division of Hydrology
Uppsala University
S-753 09 Uppsala
Russell Bell
Institute of Limnology
Uppsala University
S-75 1 22 Uppsala
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Submitted: 27 July 1989
Accepted: 15 July 1991
Revised: 5 November 1991
Limnol. Oceanogr., 37(4), 1992, 900-907
0 1992, by the American
Society of Limnology
and Oceanography,
Inc.
A rapid chromatographic method for recovery of l 5N02- and N03- produced
by nitrification in aqueous samples
Abstract -The sensitivity and comparative
simplicity of 15Nstable isotopic tracer techniques
has been used to quantify rates of nitrification in
aquatic systems. However, the most commonly
used method for recovery of inorganic oxidized
nitrogen compounds from aqueous samples,
which is based on liquid-liquid partitioning, is
time consuming and contamination prone. We
describe a solid-phase rapid chromatographic
method for recovery of ISNO,- and NO,- produced by nitrification in aqueous samples. Compared to liquid-liquid partitioning, the advantages are significantly reduced processing time and
reduced potential for contamination. Typical results are presented for the tidal, freshwater reaches of the James River estuary.
Nitrification
is an important nitrogen cycling process in marine and freshwater environments. The availability of 15N stable
isotopes as tracers and the comparatively
inexpensive l SN emission spectrometer has
facilitated experimental approaches for the
direct measurement of nitrification
in various aqueous environments (Harrison 1983;
Enoksson 1986). However, the most widely
used method (Olson 198 1) for conversion
of N02- and N03- produced through oxidation of 15NH,+ to a chemical form recovAcknowledgments
Thanks to Joseph N. Boyer for his initial development work with 15N methodology.
Funding for this work was provided in part by the
Richmond Regional Planning District Commission,
2201 West Broad St., Richmond, Virginia 23220, and
NOAA SeaGrant Project R/CM-l 3, Virginia SeaGrant
Program.
Contribution 172 1 from the Virginia Institute of Marine Science.
erable for isotopic analysis remains time
consuming, costly, and susceptible to contamination. We describe a modification of
the existing method that uses solid-phase
column chromatography
to eliminate repetitive and time-consuming
liquid-liquid
extractions and to minimize the potential
for isotopic dilution resulting from N contamination.
This method has been used to quantify
nitrification rates in a tidal freshwater river
and estuarine waters by measuring changes
in abundance of the stable isotope 15N in
N03- -N02- pools over a time course begun
after adding 15NH4+ at tracer concentrations. To determine enrichment
in the
N03--N02pool it was necessary to convert these oxidized compounds to a form
suitable for analysis by emission spectrometry. Previously we had used the recovery
method described by Olson (198 1) that requires chemical complexation
of sample
N02- with aniline sulfate under acidic conditions. This reaction yields a diazonium
salt that is condensed with alkaline P-naphthol to form a base-soluble azo dye. The dye
is acidified, reducing its solubility in water,
and recovered through solute partitioning
by repeated extractions from the aqueous
phase into a nonpolar solvent (Olson 198 1).
Next, the dye is concentrated by solvent
evaporation and transferred to an emission
tube. Typically, a high vacuum train is used
to remove atmospheric N2 and other interfering gases, the tubes sealed, and the organic N converted to N2 by a micro-Dumas
combustion process (Fiedler and Proksch