low Cu content. Based on comparisons with critical concentrations in
plants in which Cu nutrition has been studied, Cu concentrations in the
macrophytes were low enough to suggest Cu was at times a growth-
limiting nutrient in the lakes sampled (Gerloff, 1973).

Much effort has been directed to studying the Cu nutrition and estab-
lishing the Cu critical concentration in several macrophytes, particu-
larly Elodea occidentalis. This work required elaborate purification
of the nutrient culture media and the environment by standard tech-
niques employed in trace element research (Hewitt, 1966). The very
small amounts of Cu present as contamination in average environments
can provide sufficient Cu to meet the needs of plants.

Data from experiments on the Cu requirements of several organisms are
presented in Table 4. Tomato was included in the experiments because
yields with this species for which a Cu requirement is well established
served as a bioassay for the effectiveness of the Cu purification pro-
cedures. The technique employed was obviously adequate, as indicated
by an average yield in triplicate tomato cultures of only 0.65 g.

These plants showed symptoms of extreme Cu deficiency. The purified
nutrient salts, double-distilled water, and general technique used in
the tomato cultures were employed in preparing deficiency cultures of

Elodea occidentalis, Myriophyllum spicatum, and the green alga Drapar-

naldia plumosa. A number of experiments with Elodea were negative,
that is Cu deficiency was not established. However, in the experiment
reported average yields in control cultures were 52% more than in
minus Cu cultures. Copper deficiency definitely was established. The
very low Cu requirement of Elodea is indicated by the much greater
yield of Elodea (2.99 g) over tomato (0.65 g) when grown in the same
volume (2-liters) of nutrient solution. Copper deficiency was not
established in Myriophyllum and Draparnaldia even when yields per unit

of culture medium were greater than with Elodea. The average growth
of Myriophyllum in minus Cu cultures was 8.04 g and in Elodea cultures
only 2.99 g. These results suggest Myriophyllum and Draparnaldia have

even lower Cu requirements than Elodea, or, although it seems very

10


6.0b °
e
5.0F
a
xg
es 40
K e
=
> 3.0-F
a
a
1
r4
> 20b
rion e
L it i I
0.1 05 1.0 15

TISSUE CONTENT OF K(%)

Figure 3. The relationship between yield and total potassium content
of the second one-inch terminal segments of Myriophyllum spicatum
grown in solutions of varying potassium content.

of sampling. The lowest N concentration in second one-inch segments
from Site 1 was 2.72% in the July 3, 1973, sample; the lowest in
Site 2 samples was 1.85% in the August 10, 1973, sampling. The
average N concentration in samples from both sites was 2.78% or

3.7x the critical concentration.

The lowest P concentration in second one-inch segments from Site 1
was 0.26% and from Site 2, 0.14%. These minimum values are respect-

ively 4x and 2x the 0.07% critical P concentration.

In contrast to N and P there were definite indications that K was
a limiting nutrient for Myriophyllum growth in Lake Wingra. At both
sites the K concentration in 8 of 9 samples of second one-inch

31


rooted, submerged macrophyte were also collected and analyzed.
EXPERIMENTAL PROCEDURES

Myriophyllum spicatum was isolated and cultured in synthetic media
by the general techniques previously described (Gerloff, 1973).

The composition of the nutrient solution employed is presented in
Table 11. This is a slight modification of a nutrient solution
described earlier (Gerloff and Krombholz, 1966). The trace elements
are in the concentrations presented in a modified Hoagland's solution
for higher plants (Johnson et al., 1957). Iron was provided as a
mole:mole complex with EDDHA (ethylene-diamine di-[o-hydroxypheny1l-
acetate]). Additional Fe-EDDHA complex was added to the cultures
whenever the pink color resulting from the complex disappeared. All

nutrient media were autoclaved before use.

To establish N, P, and K critical concentrations in Myriophyllun,
the plants were grown for 4 to 6 weeks in a series of triplicated
cultures similar in all respects except for the element in question.
Each flask was inoculated with two two-inch shoot tips.

The cultures were continued until there was a marked growth difference
between those containing the lowest and highest levels of the element
varied and also a noticeable difference at the intermediate levels.
Upon harvest, the plants of each flask were dissected into three cate-
gories: first one-inch segment from the shoot growing tip, second one-
inch segment from the growing tip, and remaining plant parts. Roots
and any flowers were put in the remainder category. The first one-inch
segment was measured by drawing the leaves on the tip forward and
measuring from the tips of the leaves. The second one-inch was
measured as an inch segment of stem. After harvest, the plants were
dried for 48 hours in a forced-draft oven at 60-65°C, weighed, ground
in an agate mortar and pestle, and analyzed. Nitrogen analyses were
by a semi-micro Kjeldahl procedure; P determinations were by a vanado-
molybdate (yellow complex) colorimetric procedure following dry-ashing;
and K analyses were by flame emission with a Coleman flame photometer
after extraction of the tissue with 1 N ammonium acetate.

27


SECTION VII
COMPETITION FOR GROWTH-LIMITING AMOUNTS OF NUTRIENTS
MADE AVAILABLE AT VERY LOW CONCENTRATIONS

IN MIXED CULTURES OF AQUATIC PLANTS

Nuisance conditions in lakes, even in one general area, are not

caused by the same species of algae and macrophytes. Hard water lakes
are characterized by different species than are soft water lakes;
blue-green algae often become the dominant organisms following eutro-
phication; and Myriophyllum spicatum frequently is the primary macro-
phyte after its introduction into Midwestern lakes. Differing responses
to nutrient concentrations must be one factor determining aquatic plant
distribution. The limited information available on this point has

been obtained primarily through attempts to correlate nutrient con-
centrations in lakes with relative organism abundance.

A primary aspect of this project has been to develop laboratory pro-
cedures which would permit comparisons of the capacities of aquatic
plants to compete for nutrients that were made available at very low
concentrations characteristic of lakes and streams. Tests have been
made with three different procedures and techniques. One procedure
involved growing two or more aquatic plants in the same culture flask
with adequate supplies of all essential elements except one. The
organisms were forced to compete for that element by making small
additions to the culture at intervals during the growth period.

The total amount of the element provided was less than adequate for
optimum growth of both organisms.

A second technique involved solution-replacement cultures in which
plants were confined to a culture flask in which the nutrient solution
containing a specific element at a low concentration-was frequently
replaced. The total amount of the element made available to the plants
was more than required for maximum growth. The goal with this tech-
nique was to establish the borderline concentration of the element

36


growth are recommended rather than the procedure which measures
1

only rate of uptake.


loo

8
T
e

mg P ABSORBED/gm DRY WT/20 MIN x 103

iT L L it 1 1

0.5 10 15 2.0 25 3.0
EXTERNAL P CONCENTRATION (ppm)

Figure 9. Relationship of rate of phosphorus uptake to external
KH2PO4 concentration in Anabaena sp. The concentration of CaCl2
was 0.5 mM.

between species regarding absorption rates was not constant from
one external concentration to another.

When double-reciprocal plots of absorption against external con-
centration are made (Lineweaver and Burk, 1934) for Elodea occiden-
talis shoots and Draparnaldia plumosa, the resulting lines are
curvilinear, not straight, as shown in Figure 10 and 11. The
plots for all eight species tested were curvilinear which suggests
that more than one first-order reaction is involved in P absorption
(Hagen and Hopkins, 1955).

Figures 12 and 13 represent plots of P absorption against absorption

64

35


comparative studies with the various organisms observed to dominate
in lakes or streams differing in fertility and nutrient composition.
For example, several species of blue-green algae produce heavy blooms
in eutrophic Wisconsin lakes and the green alga Cladophora is very
abundant in areas of the Great Lakes following pollution.

The results obtained also are of interest in indicating that elements
other than N and P may be of key importance in nutritional ecology.
This is supported by the marked difference in response of Elodea

and Draparnaldia to growth-limiting supplies of K and of Ca.

46


was 0.13%. This is close to the critical concentration of 0.10%.

The Mg concentration in Draparnaldia was 0.10%, well below the critical
concentration of 0.20%.

Two experiments were carried out in which two macrophytes (Elodea

occidentalis and Myriophyllum spicatum) and the Draparnaldia were grown
in mixed cultures. The data in Table 19 show the results from competi-

tion for a limited P supply, made available at 0.075 ppm. Again a
somewhat superior competitive capacity of Draparnaldia is indicated
because it represented 40% of the total yield. Myriophyllum (37% of
the total yield) was somewhat more aggressive than Elodea (23% of
the total).

The very low aggressiveness of Draparnaldia in K uptake at 0.39 ppm
is supported by data in Table 20. Draparnaldia represented only 12%
of the total yield. Myriophyllum was somewhat more successful in
growth under low K than was Elodea. The former produced 53% of the
total yield; Elodea represented only 35% of the total.

DISCUSSION

The primary goal of the studies reported was to demonstrate that

the technique employed can be useful in characterizing the capacities
of aquatic plants to compete for nutrients at the low concentrations
found in lakes and streams. The validity of the technique seems
justified by the results obtained, particularly the marked changes

in the dominant organism when the organisms competed for different
elements. For example, the green alga Draparnaldia was somewhat

more successful than Elodea in competition for N and P at low con-
centrations. However, Draparnaldia was extremely ineffective in
competition for K at 0.39 ppm. It made almost no growth. In contrast,
Elodea made almost no growth when Ca was made available at a concentra-
tion of only 0.80 ppm.

Hopefully data obtained by the technique used can be applied in
evaluating the role of nutrient supply and inorganic pollution in
determining nuisance plant distribution. This of course will require

44


os

Table 21. RESPONSE OF Elodea occidentalis TO VERY LOW CONCENTRATIONS OF ESSENTIAL
ELEMENTS MADE AVAILABLE IN NUTRIENT SOLUTION REPLACEMENT CULTURES
Elodea yield Element in Elodea Added
Element Soln. { Element element
Element conc. volume added Dry wt. Relative Conc. Wt. absorbed
varied (ppm) Q) (mg) (mg) (%) (%) (mg) (%) t-test
P 1.50 64. 96.00 528 100 1.11 5.86 6.1
0.03 64. 1.92 418 79 0.17 0.71 37.0 <.05
P 1.50 77. 115.50 331 100 1.20 3.97 3.4
0.02 77. 1.54 171 52 0.18 0.31 20.0 <.01
N 8.40 30. 252.00 409 100 4.25 17.38 6.9
0.30 30. 9.00 273 67 1.40 3,82 42.5 <.01
Ca 8.00 48. 384.00 698 100 0.58 4.05 11
0.37 48. 17.76 454 65 0.20 0.91 5.1 <.001
Ca 8.00 35. 280.00 318 100 0.56 1.78 0.6
0.17 35. 5.95 254 80 0.17 0.43 7.2 <.2
Ca 8.00 32. 256.00 254 100 0.52 1,32 0.5
0.035 32. 1.12 46 18 0.14 0.06 5.8 <.001
K 9.75 52. 507.00 482 100 4.03 19.28 3.8
0.10 52. 5.20 462 96 1.28 5.91 113.7 >.50
aThe 54 mg inoculum (net weight) containing 5% K added 2.7 mg K to the experiment so the total uptake

was closer to 85%.



Table 22. COMPOSITION OF THE SOLUTION USED FOR
THE CULTURE OF ALGAE AND Lemna minor

Salt per 1. soln. Element in soln.

Salt (mg) (ppm)
NaNO; 496 N - 81.7
K2HPOy 39 K -17.5
MgSO, °7H20 75 Ca- 9.8
CaClo 27 P - 6.9
NagSi03 *5SH20 43 S - 9.8
Na2CO3 20 Mg - 7.5
EDTA 1
Fe citrate 6 Fe - 1.12
Citric acid
Kc1? Cl - 0.35
H3B03" B - 0.054
MnSO, *H20* Mn - 0.054
ZnSO 4* 7207 Zn - 0.026
CuSO, *SH20* Cu - 0.006
(NHy) ¢Mo402 4 °4H20* Mo - 0.002
coC12?

8Trace element stock solutions were prepared at 5000x the concentration
of the final solution. 1.2 ml of a solution of equal volumes of each
of the trace element stock solutions was added to each liter of the
final culture mediun.

cocis was added only to media for blue-green algae at a Co concentra-
tion of 0.001 ppm.

54


cy

Table 16. GROWTH AND K CONTENT OF Elodea occidentalis AND Draparnaldia plumosa
WHEN COMPETING FOR A GROWTH-LIMITING SUPPLY OF K IN A MIXED CULTURE
Elodea Draparnaldia
K Yield (g) Prop. of} K content (%) Yield (g) Prop. of | K content (%) |Mixed culture.
conc. yield yield yield
(ppm) | Alone | +Drap. (%) Alone} +Drap. | Alone | +Elodea (%) Alone |+Elodea (g)
39 2.19 | 1.53 62 3.98] 4.38 1.46 | 0.93 38 4.23 | 2.02 2.46
0.39) 1.93] 1.73 97 0.86} 0.97 1.41 | 0.05 3 0.67 | 1.08 1.78
Table 17. GROWTH AND Ca CONTENT OF Elodea occidentalis AND Draparnaldia plumosa
WHEN COMPETING FOR A GROWTH-LIMITING SUPPLY OF Ca IN A MIXED CULTURE
Elodea Draparnaldia
Ca Yield (g) Prop. of{ Ca content (% Yield (g) Prop. of| Ca content (%) | Mixed culture
conc. yield yield yield
(ppm) | Alone | +Drap. (%) Alone | +Drap.{| Alone| +Elodea (%) Alone |+Elodea (g)
32 1.90} 1.59 69 0.78} 0.71 1.48] 0.72 31 0.20 } 1.49 2.31
0.8 0.09 | 0.08 5 0.46{ 0.51 1.28) 1.52 95 0.05 | 0.04 1.60



SECTION V
COMPARISONS OF PROCEDURES FOR ASSAYING NUTRIENT

AVAILABILITY IN AQUATIC ENVIRONMENTS

The availability of assay procedures which correctly measure available
nutrients and growth-limiting nutrients for aquatic plants is impor-
tant in reducing nuisance conditions resulting from heavy growths of
these plants. Chemical analysis of water samples probably has been
the most used nutrient assay. As an aid in interpreting chemical
analyses, critical concentrations of soluble N and P for nuisance
bloom development have been proposed, for example by Sawyer (1947)
and Vollenweider (1968). These values have been widely used in
pollution control efforts. However, as indicated in a review of
nutrient assay procedures (Gerloff, 1969), there are problems in
obtaining representative water samples for chemical analysis and in
evaluating the results.

As an alternative to water analysis, various bioassay procedures have
been developed. These include nutrient enrichment cultures, ranging
in scale from laboratory flask cultures to field experiments involving
large polyethylene bags containing hundreds of liters of water
(Schelske and Stoermer, 1971; Powers et al., 1972) and to studies with
entire lakes (Schindler, 1974). The technique of plant or tissue
analysis uses variations in the concentrations of elements in plants
to reflect availability of the elements in the environments in which
the plants grow. Fitzgerald (1969) developed bioassays which evaluate
environmental P supplies in terms of P extracted from plants during
one hour of boiling in water and of N by the rate of NHy-N uptake

in the dark.

As a step in the development of plant analysis as an assay of nutrient
supplies for nuisance macrophyte growth, it seemed highly desirable to

compare plant analysis with other assays by evalv

ating available
nutrients in a series of lakes. The procedures selected for comparison

14


Table 12. _ NITROGEN, PHOSPHORUS, AND POTASSIUM CONCENTRATIONS
(OVEN-DRY BASIS) IN SECOND ONE-INCH SEGMENTS OF
Myriophyllum spicatum SHOOTS COLLECTED FROM LAKE WINGRA

Site 1 Site 2
Date
Year sampled SN % P %K %N %P %K

1971 July 26 2.81 0.29 0.27 2.52 0.18 0.23

Aug. 19 2.92 0.29 0.24 1.99 0.14 0.19

1972 June 15 3.53 0.40 0.19 2.30 0.17 0.46
June 29 3.96 0.45 0.20 2.10 0.16 0.19
July 27 3.78 0.44 0.27 2.21 0.18 0.26
Aug. 10 4.30 0.61 0.42 1.85 0.18 0.31

Aug. 31 3.43 0.33 0.30 2.43 0.25 0.30

1973 July 3 2.72 0.26 0.20 2.23 0.20 0.22

Aug. 6 3.11 0.44 0.30 1.89 0.21 0.25

segments was below the 0.35% critical concentration. The lowest
K concentration at each site was 0.19%; the average K concentration
in the 18 samples was only 0.24%. This suggests severe K deficiency.

Analysis of a number of samples of Ceratophyllum demersum collected
at the same sites as the Myriophyllum, are presented in Table 13.
The critical concentrations for Ceratophyllum, based on second one-
inch index segments, are higher than those for Myriophyllum. They
are 1.30% N, 0.10% P, and 1.70% K (Gerloff, 1973). Comparisons

of these values and the analyses of the Lake Wingra samples indicate

32


300

N
a
fo}

nN
°
°

a
fe)

3
fo)

a
fo}

mg P ABSORBED/gm DRY WT./20 MIN x 103

er

1 1 1 1 1 1
0.5 1.0 1.5 2.0 2.5 3.0 3.5

EXTERNAL P CONCENTRATION(ppm)

Figure 8. Relationship of rate of phosphorus uptake to external KH2PO,
concentration in Draparnaldia plumosa. The concentration of CaCl2
was 0.5 mM.

In most species, rate of P absorption continued to increase even
at the highest concentrations. Absorption did reach a maximum in
Lemna minor, Anabaena sp., and Microcystis aeruginosa. Highest

absorption rates occurred with Draparnaldia plumosa and Stigeoclonium

tenue, the two species of green algae tested. The relationship

63


CONTENTS

Sections

I Conclusions

II Recommendations

III Introduction

IV Critical Concentrations of Essential Elements in
Various Aquatic Plants

Vv Comparisons of Procedures for Assaying Nutrient
Availability in Aquatic Environments

VI Potassium as a Growth-Limiting Nutrient for
Myriophyllum spicatum in a Eutrophic Lake

VII Competition for Growth-Limiting Amounts of Nutrients
Made Available at Very Low Concentrations in Mixed
Cultures of Aquatic Plants

VIII Growth of Elodea occidentalis at Low Concentrations
of Inorganic Nutrients Made Available in Solution-
Replacement Cultures

Ix Comparisons of Rates of Phosphorus and Rubidium
Uptake by Several Macrophytes and Algae

x References

Page

14

26

36

47

53

76


20-

1
mg P
ABSORBED

ab
L
L
L

10 15 20 2 30 35

Figure 11. Double-reciprocal plot of rate of phosphorus uptake in
Draparnaldia plumosa in relation to external concentration.

affinity site and operates at low external P concentrations, although
both operate simultaneously. From the straight line components,
values for Vmax and Km for mechanism "a" and mechanism "b" were
calculated (Table 24).

66


125 -

100

75
'
mg P
ABSORBED

sO

25

P CONC.

Figure 10. Double-reciprocal plot of rate of phosphorus uptake in
excised shoots of Elodea occidentalis in relation to external
concentration.

over P concentration, as originally done by Hofstee (1952) for
enzymatic reactions. As in the double-reciprocal plots, the resulting
lines are curvilinear rather than straight. They were curvilinear
for all eight species. This also suggests that more than one first-

Order reaction is involved in P absorption.

Using graphical methods, these curvilinear plots were resolved into
their two straight line components, mechanism "a" and mechanism
"p". Mechanism "a" is a low affinity site and operates principally at

high external P concentrations, while mechanism "b" is a high

65


400

300

200 fF

Lieto}

mg P ABSORBED/gm DRY WT./20 MINUTES x 103

1 1 1 @

5 10 15 20
mg P ABSORBED
P CONC.

Figure 13. Relationship between rate of phosphorus uptake and
phosphorus uptake/external phosphorus concentration for Draparnaldia
plumosa.

68


The results obtained do not permit a conclusion that one procedure

is more reliable than the others. This was not an initial goal of the
study. However, as a general observation it can be stated that bio-
assays in which plants growing in natural environments are the sampling
devices and can reflect all factors which affect nutrient availability
over a period of time seem to offer advantages over assays based on
water samples taken infrequently. Then the rate of replacement of
utilized nutrients is not adequately considered. This replacement

may be from organism decay, release from bottom sediments, N fixation,
and nutrient inflow. The reliability of the plant analysis assay

was supported by agreement of the 1973 data and data from 1971

using the same technique in these lakes. Of the lakes sampled,

in both years Whitney Lake was indicated to be most deficient in P;
Clear Lake was most deficient in N; and Erickson Lake was indicated

as low in Ca.

25


Table 4. GROWTH RESPONSE OF SEVERAL PLANT SPECIES WHEN CULTURED
IN NUTRIENT MEDIA AND ENVIRONMENTS DEFICIENT IN COPPER

Ave. oven-dry Ave. Cu conc. in
Treatment Organism yield, g/21 plants, ppm
-Cu Elodea 2.99 0.59
+Cu occidentalis 4.43 1.47
-Cu Myriophyllum 8.04 _
+Cu spicatum 7.72 _
~-Cu Draparnaldia 3.40 _
+Cu plumosa 3.15 _
-Cu Tomato 0.65 _
+Cu 4.80 _

3oven-dry weight yields for Elodea, Myriophyllum, and Draparnaldia are
the averages of 5 replicates in each treatment; tomato weights are the
averages of 3 replicates.

ll


Chemical Analyses of Water Samples

The data in Table 6 are from chemical analyses for several N and P
fractions in aliquots of the lake samples which also were analyzed by
the PAAP test. The samples were analyzed by the Water Chemistry
Laboratory of the Department of Natural Resources at Delafield,
Wisconsin. Following collection and prior to analysis, the samples
were preserved by adding mercuric chloride as directed by the Water
Chemistry Laboratory. Because some analyses were reported as below
the limit of detection, data could not be averaged and are presented

for both the July and August sampling dates.

The POy-P, NO3-N, and NH3-N data are of interest in relation to
indications that N or P supplies were limiting plant growth. Concen-
trations of 0.015 ppm inorganic P and 0.30 ppm inorganic N were
suggested as critical water concentrations above which nuisance algae
blooms could be expected (Sawyer, 1948). These were values for early
summer prior to heavy demands on nutrients. The data in Table 6 are
from later in the summer. Nevertheless, it is of interest that in all
lakes PO,-P values in the July samples were above the 0.015 ppm
Sawyer critical concentration. In the August samples, P0,-P concen-
trations in four lakes were below 0.015 ppm. In contrast, values for
NO3-N were below 0.30 ppm in every sample at both sampling dates. The
average NO3-N concentration in the July samples was only 0.05 ppm. All
NH3-N concentrations were reported as less than 0.03 ppm so seem
insignificant.

When the ratios of NO3-N:PO,-P in the lake water samples are compared
with the 12:1 ratio of the critical plant tissue concentrations for

N and P in Elodea (1.60% N and 0.14% P), it appears that N is more
limiting for growth than P. The average NO3-N:P0,-P ratio in the
July water samples was 1.4:1 and in the August samples 9.6:1.

17


2 and 3 is in their application to nutrient assay by the plant
analysis procedure. Unusually high or low critical concentrations also
are of interest in indicating high or low nutrient requirements of
specific organisms that may be of ecological importance. The critical
concentration of N in the blue-green alga Microcystis aeruginosa was
4.0%. This value was nearly twice the second highest critical concen-
tration of 2.30% in the green alga Draparnaldia plumosa. The critical
P concentration in Microcystis was 0.12%, a value close to or even
below the critical concentrations in most green algae and macrophytes.
Relatively large quantities of N must be available for Microcystis
growth in lakes and streams. The low Ca critical concentrations in

algae, the very low Cu requirements of macrophytes, and the wide varia-
tions in K critical concentrations also are of interest. The range
in K values suggests this element may play a more significant role in

aquatic plant distribution than is presently recognized.

The most consistent duplication in critical concentrations in various
experiments was obtained with Lemna minor. Lemna also is relatively
easy to grow in synthetic culture media and to quantitatively remove
from solutions at harvest. When collected in the field, Lemna should
be easier to free of contaminating organisms and debris than would
algae. In addition, Lemna provides more uniform tissue than do macro-
Phytes such as Elodea. These features suggest Lemna might be a suit-
able organism to confine in porous baskets in lakes and to sample
periodically for nutrient evaluation by plant analysis (Gerloff, 1973).

13


SECTION VI
POTASSIUM AS A GROWTH-LIMITING NUTRIENT FOR

Myriophyllum spicatum IN A EUTROPHIC LAKE

Lake Wingra, located within the city limits of Madison, Wisconsin,
is a small (140 hectares) and shallow (maximum depth, 6.4 meters)
lake with a severe aquatic weed problem. The lake is primarily
spring-fed and the southern shore, which is undisturbed woods, is
included in the University of Wisconsin Arboretum. The northern
shore is dominated by recreational and residential areas (Bauman,
Hasler, Koone, and Teraguchi, 1973). The dominant vegetation in
the lake is Myriophyllum spicatum (relative frequency 64%), a
submerged, rooted angiosperm that has been introduced from Europe
(Nichols, 1971).

The problem of accelerated lake eutrophication has stimulated the
recent proliferation of techniques for assessing the nutrient status
of natural waters and of investigations of the movement of mineral
elements through aquatic ecosystems. Most of these studies have
concentrated on N and P, and in many cases one or the other has been
found to be limiting plant growth. Lake Wingra has been the site of
one such study as part of the US/IBP Eastern Deciduous Forest Biome
Project. One phase of the general project of modeling physical,
chemical, and biological processes in the Lake Wingra Basin focused
primarily on the movement of P through the ecosystem.

This paper presents data from application of the bioassay known as
plant analysis (Gerloff and Krombholz, 1966) in evaluating the
availability of three major nutrient elements, N, P, and K, for
plant growth in Lake Wingra and in determining if one of these
elements became limiting for plant growth. This study was stimulated
by the extensive work on Lake Wingra in the IBP program, but was not
a part of that project. In addition to the samples of Myriophyllum
spicatum, samples of Ceratophyllum demersum, a less abundant, weakly-

26


Table 1.

CRITICAL CONCENTRATIONS AND THE RANGES IN CONCENTRATIONS OF

VARIOUS ESSENTIAL ELEMENTS IN SEVERAL AQUATIC ANGIOSPERMS

Elodea occidentalis

Ceratophyllum demersum

Myriophyllum spicatum

Index Critical Range in Critical Range in Critical Range in

Element segment conc. conc. conc. conc. cone. conc.

N 2nd 1" 1.60% 1.14-4,32% 1.30% 1,00-2.42% 0.75% 0.61-1.58%

P 2nd 1" 0.14 0.06-0.35 0.10 0.09-0.41 0.07 0.04-0.18

s 2nd 1" 0.08 0.06-0.46 _ _ _ _

Ca Ist 1" 0.28 0.17-0.62 0.22 0.11-0.47 _ _

Mg 2nd 1" 0.10 0.06-0.19 0.18 0.11-0.40 _ _

K 2nd 1" 0.80 0.25-2.51 1.70 1.58-5.10 0.35 0.30-1.13

Fe Ist 1" 60 ppm 40-219 ppm _ _ _ _

Mn 1st 1" 4.0 2.2-16.7 _ _ —_ _—-

Zn 2nd 1" 8.0 3.6-34.4 _ _ 1.8 Ppm} 1.6-6.9 ppm

Mo 1st 1" 0.15 0.04-6.4 —_ _— —_ —

B 1st 1" 1.3 0.3-11.2 5.0 ppm} 2.3-11.9 ppm _ _



100

a N
° a

mg P ABSORBED/gm DRY WT./20 MINUTES x 103
Nn
a

_ ! le n
0. 0.2 03 0.4 0.5

mg P ABSORBED
P CONC.

Figure 12. Relationship between rate of phosphorus uptake and

phosphorus uptake/external phosphorus concentration for excised
shoots of Elodea occidentalis.

67


almost complete recovery of K supplied at only 0.10 ppm. Less
.efficient mechanisms for NO3 and Ca uptake were indicated by sharply
reduced yield with NO3-N made available at 0.30 ppm and Ca at 0.37
ppm.

The data obtained seem of considerable interest in relation to the
interpretation of chemical analyses of water samples. If nutrient
concentrations in lakes and streams are reduced to the concentrations
which gave decreased yields in these experiments, it is apparent

that nuisance plant growths could be reduced for two reasons. First,
amounts of the elements required for specific amounts of growth

might not be available. Secondly, the concentrations might be reduced
to levels at which rates of absorption could not keep pace with needs
for growth, even though absolute amounts of the elements seemed
adequate. In other words, it cannot be assumed that each unit of a
nutrient is equally effective in promoting plant growth over the
entire range of concentration present in lakes and streams. Data
obtained by the procedure described would be useful in indicating
concentrations at which this decreased effectiveness occurs for
various elements. For example, the results correlate with the 0.30 ppm
soluble N and 0.015 ppm P water concentrations at the start of the
growing season considered critical for nuisance algae bloom production
(Sawyer, 1947).

Data were obtained only with Elodea in this study. Application of
this information to questions and problems relating to the importance
of specific nutrients in controlling Elodea distribution in lakes
must await comparative studies on the growth of other species of
macrophytes and algae at very low concentrations of various elements.
Application to nutritional ecology should be a major use of the

information obtained.

The capacity of Elodea to obtain adequate K when made available at

0.10 ppm (considerably lower than concentrations in infertile lakes)
correlates with the extreme aggressiveness of Elodea in K uptake

when competing with a green alga for limited K in experiments described

in Section VII.
52


ACKNOWLEDGMENTS

The assistance of Frann Hutchison, Vic Muth, Ann Mickle, John Devereux,

Leslie Pratt, and Marc Hanson in various aspects of the work reported
is gratefully acknowledged.

Appreciation also is expressed for the assistance and patience of
the Grant Project Officer, Mr.. William E. Miller, National
Environmental Research Center, Corvallis, Oregon.

viii


£7

Table 10. TRACE ELEMENT CONCENTRATIONS IN INDEX SEGMENTS OF Elodea occidentalis
COLLECTED FROM NORTHERN WISCONSIN LAKES DURING JULY AND AUGUST

Concentration
(ppm)
Fe Mn cu B Zn
Lake sampled I II }Ave. I II }Ave.} I II |Ave. I II] Ave. I | II | Ave.
Little John 1020 | -- {1020 378 | -- |378 | 2.5 ~~ {2.5 10.0 | -- {10.0 21|-- | 21
Clear 376 |461 | 419 |>800 | 845 | -- | 3.7 | 6.3 [5.0 9.3 {15.5 | 12.4 40 | 30 | 35
Whitney 280 {161 | 221 108 | 79 | 94} 2.6} 4.3 43.5 9.5 }10.3] 9.9 54} 46} 50
Allequash 763 | 356 | 560 457 | 287 }372 | 1.9 } 7.8 |4.9 12.4 |14.2 | 13.3 45 | 80] 63
Salsich 499 }383 } 441 37 | 46) 42] 2.6] 5.1 |3.9 9.8 }14.9 | 12.4 64|52 | 58
Erickson 442 1568 | 505 72 | 94) 83 | 9.5 | 5.4 7.5 12.3 [16.7] 14.5 | 114/47) 81

i
The index segment for Fe, Mn, and B was the first one-inch of main stems and laterals; for Cu and Zn,
the second one-inch.

The critical concentrations are 60.0 ppm for Fe, 4.0 for Mn, 0.8 for Cu, 1.3 for B, and 8.0 ppm for Zn.


o @ 3 b

3 3 ro) 8

T T — T
\

BY
°
T

mg P ABSORBED/ gm DRY WT, x 104

20 %---X shoots
@—e roots

C 1 L L 1 1 !

5 10 15 20 25 30

TIME (MINUTES)

Figure 4. Relationship of phosphorus uptake to time in excised roots
and shoots of Elodea occidentalis. Substrate concentration was
1 x 107° M KHoPO, with CaCiz at 0.5 mM.

Time Experiments

The quantities of P absorbed by Elodea occidentalis roots and shoots
are plotted against time in Figure 4 for time intervals between 1 and
30 minutes. The external P concentration was 0.031 ppm. The plot
shows that steady-state conditions occurred for both the roots and
shoots over a period of 30 minutes. The shoots showed a slightly
higher rate of P uptake.

59


50F

40-

30F

20F e

mg P ABSORBED/gm DRY WT./20 MIN x103

1 1 1 i ! J a]
0.5 10 1S 20 25 3.0 35

EXTERNAL P CONCENTRATION (ppm)

Figure 6. Relationship of rate of phosphorus uptake to external KH2PO,
concentration in excised roots of Elodea occidentalis. The
concentration of CaClo was 0.5 mTM.

was 0.5 mM. Steady-state conditions occurred over the full 180
minutes for the roots, but only over the first 60 minutes for the
shoots. Plots for Myriophyllum spicatum and Ceratophyllum demersum
shoots resembled the plot for Elodea occidentalis shoots.

On the basis of these time experiments, it was concluded that steady-
state conditions would occur, for the species examined, over at

61


mg P ABSORBED /gm DRY WT./20 MIN x 10%

1 L 1 1 1 1 1
0.5 Ke} 15 2.0 25 3.0 35

EXTERNAL P CONCENTRATION (ppm)

Figure 7. Relationship of rate of phosphorus uptake to external
KH2P0, concentration in excised shoots of Elodea occidentalis.
The concentration of CaCl2 was 0.5 mM.

least 20 minutes in experiments involving either P or Rb. Uptake

periods of 20 minutes were used in all subsequent experiments.

Concentration Experiments - P

Rates of P uptake over a 0.00031 to 3.1 ppm concentration range

were measured for all 8 species. Data for Elodea occidentalis

roots and shoots, Draparnaldia plumosa, and Anabaena sp. are presented
in Figures 6 through 9.

62


There was no uptake of NH4-N by any Elodea sample. Thus, Elodea from
every lake was indicated to be adequately supplied with N, Also every
extractable PO,-P value was above the 50 to 70 ug/100 mg value
indicative of P deficiency. Most values were close to or above the
190 to 200 yg/100 mg dry tissue associated with adequate P. The
average value was 181. The P0,-P extraction tests did indicate that
Whitney Lake contained much less available P than the other lakes.
The average extractable P value for Whitney was 94, only 47% of the
average for the other five lakes.

Nutrient Bioassay by Plant Analysis

The data in Table 8 are the results of total N and P analyses of
second one-inch index segments of Elodea collected from the various
lakes during July and August. These analyses were evaluated for
indications that N or P had become growth-limiting by comparisons
with 1.60% N and 0.14% P established critical concentrations (Gerloff
and Fishbeck, 1973).

No sample was below the critical N concentration of 1.60%. However,
the August Elodea sample from Clear Lake was close to the critical
concentration indicating borderline N deficiency. In contrast, the
average P concentration in Elodea from Clear Lake was equal to the
highest value from any lake. The analyses of samples from Whitney
showed that P had become limiting for Elodea growth in that body

of water. Phosphorus concentrations in samples from both dates

were below the 0.14% critical concentration. Average P concentrations
in samples from all other lakes were approximately double the critical
concentration and varied only within the range of 0.25 to 0.27%.

Analyses for the concentrations of major element cations in appro-
priate index segments are presented in Table 9. In earlier work, the
critical concentration for Ca in Elodea was established at 0.28%;

for Mg, 0.10%; and for K, 0.80%.

Potassium concentrations in the Elodea averaged 2.33% with the lowest

value at 1.70%. All K concentrations were at least double the

20


|

mmoles Rb e

ABSORBED
x 103

lor

5 10 15 20
|
Rb CONC.

Figure 16. Double-reciprocal plot of rubidium uptake in excised
roots of Elodea occidentalis.

experiments, a straight line also was obtained for Ceratophyllum
demersum shoots and a curvilinear line for Myriophyllum spicatum

shoots and the alga Draparnaldia plumosa. These somewhat limited
data suggest that, within the range of concentrations tested, one

72


13

14

15

16

17

Relationship Between Rate of Phosphorus Uptake and
Phosphorus Uptake/External Phosphorus Concentration

for Draparnaldia plumosa

Two Possible Relationships Between Rate of Rubidium
Uptake and External RbC1 Concentration in Excised
Roots of Elodea occidentalis

Relationship of Rate of Rubidium Uptake to External
RbC1 Concentration in Excised Shoots of Elodea
occidentalis

Double-Reciprocal Plot of Rubidium Uptake in Excised
Roots of Elodea occidentalis

Double-Reciprocal Plot of Rubidium Uptake in Excised
Shoots of Elodea occidentalis

68

70

71

72

73


10

11

12

FIGURES

The Relationship Between Yield and Total Nitrogen
Content of the Second One-Inch Segments of

Myriophyllum spicatum

The Relationship Between Yield and Total Phosphorus
Content of the Second One-Inch Segments of

Myriophyllum spicatum

The Relationship Between Yield and Total Potassium
Content of the Second One-Inch Segments of

Myriophyllum spicatum

Relationship of Phosphorus Uptake to Time in Excised
Roots and Shoots of Elodea occidentalis

Relationship of Rubidium Uptake to Time in Excised
Roots and Shoots of Elodea occidentalis

Relationship of Rate of Phosphorus Uptake to External
KH2P0, Concentration in Excised Roots of Elodea
occidentalis

Relationship of Rate of Phosphorus Uptake to External
KH2PO, Concentration in Excised Shoots of Elodea
occidentalis

Relationship of Rate of Phosphorus Uptake to External
KH2P0, Concentration in Draparnaldia plumosa

Relationship of Rate of Phosphorus Uptake to External
KH2PO, Concentration in Anabaena sp.

Double-Reciprocal Plot of Rate of Phosphorus Uptake in
Excised Shoots of Elodea occidentalis in Relation to
External Concentration

Double-Reciprocal Plot of Rate of Phosphorus Uptake in

Draparnaldia plumosa in Relation to External
Concentration

Relationship Between Rate of Phosphorus Uptake and

Phosphorus Uptake/External Phosphorus Concentration
for Excised Shoots of Elodea occidentalis

iv

Page

29

30

31

59

60

61

62

63

64

65

66

67


Table 2. CRITICAL N AND P CONCENTRATIONS AND THE RANGES IN
N AND P CONCENTRATIONS IN Lemna minor (DUCKWEED)

Critical conc. Range in conc.
- Element (%) (%)
N 0.90 0. 40-3. 32
P 0.08 0.03-0.73

Table 3. CRITICAL CONCENTRATIONS OF VARIOUS ESSENTIAL
ELEMENTS IN SEVERAL GREEN AND BLUE-GREEN ALGAE

%

Species N P Ca Mg K
Chlorella pyrenoidosaTM - - 0.00 0.15 0.40
Scenedesmus quadricanda* - - 0.06 0.05 0.25
Draparnaldia plumosa? 2.30 0.18 0.03 0.20 2.40
Stigeoclonium tenue” - - 0.03 | 0.25 | 1.90
Microcystis aeruginosa” 4.0 0.12 0.04 0.30 0.50
Nostoc muscorum® - - 0.02 0.25 0.80

aunicellular green algae

beat amentous green algae

“blue- green algae


14

15

16

17

18

19

20

21

22

23

24

Growth and Total N Content of Elodea occidentalis and
Draparnaldia plumosa Competing for a Growth-Limiting
Supply of N in a Mixed Culture

Growth and P Content of Elodea occidentalis and
Draparnaldia plumosa Competing for a Growth-Limiting
Supply of P in a Mixed Culture

Growth and K Content of Elodea occidentalis and

Draparnaldia plumosa When Competing for a Growth-Limiting

Supply of K in a Mixed Culture

Growth and Ca Content of Elodea occidentalis and

Draparnaldia plumosa When Competing for a Growth-Limiting

Supply of Ca in a Mixed Culture

Growth and Mg Content of Elodea occidentalis and

Draparnaldia plumosa When Competing for a Growth-Limiting

Supply of Mg in a Mixed Culture

Growth of Myriophyllum spicatum, Elodea occidentalis,
and Draparnaldia plumosa When Competing for a Growth-
Limiting Supply of P in a Mixed Culture

Growth of Myriophyllum spicatum, Elodea occidentalis,
and Draparnaldia plumosa When Competing for a Growth-
Limiting Supply of K in a Mixed Culture

Response of Elodea occidentalis to Very Low Concentra-
tions of Essential Elements Made Available in Nutrient
Solution Replacement Cultures

Composition of the Solution Used for the Culture
of Algae and Lemna minor

Comparison of K and Rb Uptake Rates from External
Concentrations of 0.5 mM KCl, 0.5 mM RbCl, 0.01 mTM
KCl, and 0.01 mM RbC1

The Apparent Vmax and Km of the Two Carriers Involved
in P Uptake in Eight Species of Aquatic Plants

vii

40

40

42

42

43

45

45

sO

54

58

69


largely because of the lack of direct experimental evidence -for ion
absorption by the roots of aquatic macrophytes (Sculthorpe, 1967).
Most of the studies on ion absorption in aquatic macrophytes have
been with shoots and leaves. Recent work in several laboratories
on both freshwater (Bristowe and Whitcombe, 1971; DeMarte and Hartman,
1974) and marine (McRoy and Barsdate, 1970) angiosperms has shown
that the roots of some aquatic macrophytes do absorb ions and that
these ions are then translocated to the shoot. The results in
Figures 4, 5, 6, and 14 indicate that roots of Elodea occidentalis
absorb both P and Rb. At low P and Rb concentrations, under aerobic
conditions, the rates for both roots and shoots are comparable, but
at high P concentrations, rates for the shoots are higher than for
the roots. At least under aerobic conditions, the relative contri-
bution of the roots and shoots to the P or K nutrition of a whole
plant would depend on the root/shoot ratio of the plant and on the
ion concentrations in the substrate and surrounding water.

Kinetic studies of ion uptake by roots have led to the concept that
for many nutrient elements two apparent sites or mechanisms are involved
in active ion uptake (Epstein, 1972). Most of the studies have been
with crop plants, such as barley and corn; very little work has been
with aquatic macrophytes, although dual mechanisms for K (Jeschke,
1970), POx, and SO, (Jeschke and Simonis, 1965) have been reported
in Elodea densa leaves. The results in the present experiments
indicate that two mechanisms are involved in P absorption and are
similar to those found in barley (Hagen and Hopkins, 1955), corn
(Carter and Lathwell, 1967), alfalfa and millet (Noggle and Fried,
1960). In all cases, the two mechanisms operate simultaneously,
although one mechanism (site b) has a higher affinity for P than the
other. Both Vmax, and Vmax, were higher for the aquatic plants than
for barley (Hagen and Hopkins, 1955) and corn (Carter and Lathwell,
1967) roots and both Km, and Kn, were lower in the aquatic plants.
The lower the Km value, the higher the affinity of the ion carrier
for the ion, so both uptake mechanisms have a higher affinity in the
aquatic plants for P than their counterparts in barley and com.

74


Gerloff, G. C. Evaluating Nutrient Supplies for the Growth of
Aquatic Plants in Natural Waters. In: Eutrophication: Causes,
Consequences, Correctives. Proc. Symp. Nat. Sci., Washington, D. C.,
1969.

Gerloff, G. C. Plant Analysis for Nutrient Assay of Natural Waters.
EPA Environ. Health Res. Series, EPA-R1-73-001. 1973. 66 p.

Gerloff, G. C. and K. A. Fishbeck. Plant Content of Elements as a
Bioassay of Nutrient Availability in Lakes and Streams. In:
Bioassay Techniques andEnvironmental Chemistry. Ann Arbor Science
Publishers, Inc., 1973. p. 159-176.

Gerloff, G. C. and P. H. Krombholz. Tissue Analysis as a Measure of
Nutrient Availability for the Growth of Angiosperm Aquatic Plants.
Limnol. Oceanogr. 11:529-537, 1966.

Gerloff, G. C. and F. Skoog. Nitrogen as a Limiting Factor for the
Growth of Microcystis aeruginosa in Southern Wisconsin Lakes.
Ecology. 38:556-561, 1957.

Hagen, C. E. and H. T. Hopkins. Ionic Species in Orthophosphate
Absorption by Barley Roots. Pl. Phys. 30:193-199, 1955.

Hewitt, E. J. Sand and Water Culture Methods Used in the Study
of Plant Nutrition. Tech. Commun. No. 22 of the Commonwealth
Bureau of Horticulture and Plantation Crops, East Malling,
Madistone, Kent. Second Edition. 1966.

Hoagland, D. R. and W. C. Snyder. Nutrition of Strawberry Plants
Under Controlled Conditions: (a) Effects of Deficiencies of Boron
and Certain Other Elements. (b) Susceptibility to Injury from
Sodium Salts. Proc. Am. Soc. Hort. Sci. 30:288-294, 1933.

Hofstee, B. H. J. On the Evaluation of the Constants Vmax and Km
in Enzyme Reactions. Science. 116:329-331, 1952.

Hughes, E. 0., P. R. Gorham, and V. A. Zehnder. Toxicity of a
Unialgal Culture of Microcystis aeruginosa. Canadian Jour.
Microbiol. 4:225, 1958.

Jeschke, W. D. Der Influx von Kaliumionen bie Blattern von Elodea
densa, Abhangigdeit vom Licht, von der Kaliumkonzentration und
von der Temperatur. Planta. 91:111-128, 1970.

Jeschke, W. D. and W. Simonis. Uber die Aufnahme von Phosphat-und
Sulfationen durch Blattern von Elodea densa und ihre Buinflussung
durch Licht Temperatur und Aussenkonzentration. Planta. 67:6-32,
1965.

77


were: (1) the Provisional Algal Assay Procedure (PAAP Test), (2) the
Fitzgerald bioassay for available P and N, (3) chemical analyses of
water samples, (4) plant analysis based on samples from natural
populations in the lakes, and (5) plant analysis based on samples
from a bioassay macrophyte introduced into the lakes. Data will be

presented and discussed in relation to each assay.
EXPERIMENTAL PROCEDURES AND RESULTS

Six northern Wisconsin lakes were selected for sampling. Previous

tests (Gerloff and Fishbeck, 1973) showed these lakes varied consid-
erably in general fertility and that some of the lakes were charact-
erized by deficiencies of specific elements. Samples for the assays

were collected during the summer of 1973.

Provisional Algal Assay Procedure

In Table 5 average values are presented from the PAAP test on samples
of water collected from six lakes during two periods in July and
August when aquatic plant growth and demands for nutrients were at

a maximum. The green alga Selenastrum capricornutum was the assay

organism.

A primary goal of this study was to compare the elements which would
be indicated as principal growth-limiting nutrients in lakes

sampled by various procedures. In all six lakes the PAAP test showed
P to be in least abundant supply for the growth of Selenastrum.

There was sufficient P in the lake water samples for an average of
1.66% of maximum growth (range of 1.09 to 2.31%), that is, 1.66%

of growth with N, P, Fe and Cu added to the water. There was
sufficient N in the water for an average of 29.6% of maximum growth
(range of 14.6 to 46.6%).

Unexpectedly, yields actually were less when all essential elements
were provided than when only N, P, Fe and Cu were added. Additions
of all the elements apparently resulted in heavy metal toxicity or
an unfavorable pH or nutrient balance.

15


EPA-660/3-75-027
JUNE 1975

Ecological Research Series



Johnson, C. M., P. R. Stout, T. C. Broyer, and A. B. Carlton.
Comparative Chlorine Requirements of Different Plant Species.
Plant and Soil. 8:337-353, 1957.

Lineweaver, H. and D. Burk. The Determination of Enzyme Dissociation
Constants. J. Amer. Chem. Soc. 56:658-666, 1934.

McRoy, C. P. and R. J. Barsdate. Phosphate Absorption in Eelgrass.
Limnol. Oceanogr. 15:6-13, 1970.

Michaelis, L. and M. L. Menten. Die Kinetik der Invertin wirkung.
Biochem. Z. 449:333-369, 1913.

Nichols, S. A. The Distribution and Control of Macrophyte Biomass
in Lake Wingra. UW Water Resources Center, Final Completion Report,
OWRR B-019-WIS. 111 p. 1971.

Noggle, J. C. and M. Fried. A Kinetic Analysis of Phosphate Absorption
by Excised Roots of Millet, Barley, and Alfalfa. Soil Sci. Soc.
Proc.. 24:33-35, 1960.

Powers, C. F., D. W. Schults, K. W. Malueg, R. M. Brice, and M. D.
Schuldt. Algal Responses to Nutrient Additions in Natural Waters.
II. Field Experiments. In: Nutrients and Eutrophication Special
Symposia, Vol. I, 1972. p. 141-154.

Rains, D. W. Kinetics and Energetics of Light-enhanced Potassium
Absorption by Corn Leaf Tissue. Pl. Phys. 43:394-400, 1968.

Sawyer, C. N. Fertilization of Lakes by Agricultural and Urban
Drainage. J. New England Water Works Assoc. 61:109-127, 1947.

Schilske, C. L. and E. F. Stoermer. Eutrophication, Silica Depletion,
and Predicted Changes in Algal Quality in Lake Michigan. Science.
173:423-424, July 1971.

Schindler, D. W. Eutrophication and Recovery in Experimental Lakes:
Implications for Lake Management. Science. 184:897-898, 1974.

Sculthorpe, C. D. The Biology of Aquatic Vascular Plants. London,
Edward Arnold Ltd., 1967. 610 p.

Stout, P. R. and D. I. Arnon. Experimental Methods for the Study of
the Role of Copper, Manganese and Zinc in the Nutrition of Higher
Plants. Amer. Jour. Bot. 26:144-149, 1939.

Vollenweider, R. A. Scientific Fundamentals of the Eutrophication
of Lakes and Flowing Waters, with Particular Reference to Nitrogen
and Phosphorus as Factors in Eutrophication. Organization for
Economic Cooperation and Development, Paris. 1968. 159 p.

78


The total amount of an element made available in deficient cultures
was approximately 50% of the amount considered necessary to maintain
the critical concentration in the anticipated plant yield. The
frequency of addition of the element increased as the growth period
progressed. For example, in the N experiment a N addition equivalent
to 0.30 ppm in two-liters of solution was made each 24 hours of the
first 5 days of the culture period; two additions were made each

24 hours during the last 6 days.

At the termination of an experiment, the plants were carefully
separated. Elodea and Myriophyllum were lifted from the culture
medium, and adhering algae were carefully washed off to be combined

with the algae from the culture medium. Draparnaldia was recovered
from the culture solution by filtration through 270-mesh bolting
cloth held in a Biichner funnel. After drying at 65-70°C and weighing,
the plants were analyzed. Nitrogen determinations were by a semi-
micro Kjeldahl procedure, P by a stannous-reduced phosphomolybdate
yellow-color procedure on dry-ashed residues, Ca by atomic absorption
analysis of dry-ashed tissues, and K by atomic emission following
extraction with N NHsAc.

RESULTS

The relative capacities of two species to compete for an element in
the experiments reported are indicated by (1) the percentage of the
total yield represented by each species when grown at a growth-
limiting supply of the element and (2) a comparison of the proportion
of growth represented by each species under adequate and less than
adequate quantities of the element. Actual reduction of the supply
of an element to a growth-limiting concentration in an experiment

is evident from reduced combined yields of the organisms at the low
element concentration and also element concentrations in the harvested
plants close to or below recognized critical concentration. It was
important that differences in growth rate were not primarily responsi-
ble for yield differences of the mixed-culture organisms. Marked
differences in growth rates would be apparent in comparisons of yields

38


N and P supplies remain adequate. The need for caution in generalizing

that P is a primary growth-limiting nutrient in all aquatic ecosystems
is indicated.

4. The macrophyte Elodea occidentalis and the filamentous green
alga Draparnaldia plumosa differ markedly in their capacities to
compete for specific nutrients. For example, Elodea is much more
effective than Draparnaldia in obtaining K; in contrast, Elodea
is very ineffective in competing for Ca at low concentrations.

5. At N and P concentrations approximating critical water concentra~
tions for nuisance algae blooms, Elodea is unable to produce maximum
yield even though adequate total amounts of the elements are available.
For example, Elodea yield was much reduced at a maintained concentra-
tion of 0.30 ppm NO3-N and 0.02-0.03 ppm P.

6. Vmax and Km values for P uptake, calculated from rates of
uptake at various external concentrations, differ considerably among
algae and macrophytes. Km values are similar for roots and shoots
of Elodea occidentalis. Two mechanisms or carriers for P uptake,
which are effective at different external concentrations, seem to
function in P uptake by both algae and macrophytes.

The results with the last three techniques demonstrated their
potential for obtaining data to explain nuisance aquatic plant
distribution in relation.to lake fertility and pollution,


SECTION I

CONCLUSIONS

There were two primary aspects to the studies reported. One was
concerned with further refinement and testing of plant analysis as
a bioassay of nutrient supplies in lakes and streams, The second
involved the development and testing of procedures for evaluating
relative capacities of aquatic plants to compete for and absorb
nutrients at the low concentrations characteristic of lakes and

streams.
The primary conclusions can be summarized as follows:

1. Plant analysis and other commonly used assay procedures do not
provide results that agree when employed in evaluating nutrient
supplies and growth-limiting nutrients in lakes. Even assays by

the Provisional Algal Assay Procedure and chemical analysis on
aliquots from the same water sample do not agree. The plant analysis
bioassay seems to offer several advantages over the other procedures
tested.

2. The critical concentrations for a number of elements are now
established in a variety of aquatic plants, including macrophytes and
algae. In addition to providing essential standard values for
bioassays by plant analysis, the critical concentrations indicate
interesting variations in the nutritional requirements of the organisms.
The data also show that the critical concentration for an element can
vary considerably among different organisms, so much so that the same
critical values cannot be generally applied even within one taxonomic
group. Specific critical concentrations should be established for

each species of interest.

3. During the summer months, K supply, as indicated by plant analysis,
becomes growth-limiting for the macrophytes Myriophyllum spicatum and
Ceratophylium demersum in a eutrophic southern Wisconsin lake;



at 0.37 ppm and 0.17 ppm Ca. There was almost no Elodea growth at
0.035 ppm. Plants that did grow were nearly black in color. At

0.37 ppm Ca, only 5.1% of the Ca made available in the 48-liters of
nutrient solution was absorbed by the Elodea. The ineffective
absorption of Ca at concentrations below approximately 0.50 ppm
suggests that in lakes with very soft water, Ca supply may be a factor
in determining which species are abundant.

The critical Ca concentration in the terminal one-inch of Elodea
is 0.28%. In plants grown at 0.37 ppm Ca and less, even whole plant
analyses (0.14-0.20%) were well below the critical concentration.

The absorption by Elodea of K made available at low concentrations
contrasted sharply with the absorption of Ca at comparable concentra-
tions. Even at 0.10 ppm K, there was no decrease in yield.
Furthermore, the 1.28% K concentration in the Elodea was far above
the 0.80% critical concentration, indicating the plants were absorbing
sufficient K for normal growth. At 0.10 ppm, Elodea so effectively
removed K from the nutrient solution that the indicated recovery was
greater than the amount supplied. The additional K probably was
derived from inoculum Elodea and from impurities in the large volumes

of nutrient solution.
DISCUSSION

This study was primarily concerned with developing a technique for
evaluating capacities to absorb nutrients at concentrations comparable
to those in natural waters. The technique developed seemed very
satisfactory for such studies with macrophytes and definitely superior
to several types of continuous-flow cultures tested earlier on this
project. The procedure would have to be modified to be effective with
algae. In the apparatus used with Elodea, algae would be washed

from culture vessels each time the nutrient solution was replaced.

Providing adequate total amounts of an element at low concentrations
is a critical aspect of the procedure developed. The K results
verify this was accomplished. There was no reduction in yield and

$1


DISCUSSION

This study is unique in indicating that K rather than N or P became
limiting for the growth of a nuisance aquatic plant in a eutrophic
lake. This probably is the first evidence for K deficiency in plants
in natural aquatic environments. The occurrence of K deficiency

in Lake Wingra Myriophyllum supports the suggestion (Gerloff, 1968)
that it is incorrect to assume that P is the primary growth-limiting
nutrient in all lakes. As in terrestrial environments, the principal
growth-limiting nutrient in aquatic environments most often will be

P or N but in specific environments it may be any other major element
or even a trace element.

One procedure for reducing nuisance plant growths in lakes is to
lower the input of a particular element, or elements, especially the
element considered most likely to limit plant growth. One of the
easier elements to control is P. The present study shows that
reduced entry of K into Lake Wingra would have a greater effect on
the standing crop of Myriophyllum than would reduced P input. This
is not to advocate discontinuance of efforts to reduce P input, but
to recognize that the results of such an effort may not have the
expected effects in reducing Myriophyllum biomass.

Several aspects of this study relate to the practical application
of plant analysis as a nutrient bioassay. First, the critical con-
centrations of N, P, and K were much lower in Myriophyllum spicatum
than in other macrophytes in which critical concentrations have
been established. Therefore, critical concentrations must be deter-
mined for each species of interest. Secondly, the data obtained
demonstrated that caution should be used in generalizing that an
element is limiting the growth of all species in a lake from below-
the-critical-concentration values in one species. The K supply was
indicated as severely limiting for growth of Myriophyllum but only
on the borderline of limiting for Ceratophyllum. Thirdly, the
results show that differences in nutrient availability within a
lake can be demonstrated with plant analysis. As shown in Tables

34


Rate of ion uptake is one primary quantitative nutritional difference
between plant species. The results obtained indicate that the P and

Rb uptake rates of the various aquatic plants and plant parts do differ.
On the basis of these differences, certain species might be predicted
to outcompete others in nutrient deficient environments. For example,
based on uptake rates at 0.0031 ppm P, under conditions of low P

supply Draparnaldia plumosa would be expected to outcompete the

other seven species tested. Myriophyllum spicatum would be the least
effective in competition for P. The indicated uptake rates for all

eight organisms at 0.0031 ppm P was: Draparnaldia > Stigeoclonium >

Elodea > Microcystis > Anabaena > Ceratophyllum > Lemna > Myriophyllum.

At an external concentration of 0.05 mM Rb, the relative uptake rates

for the four organisms tested were: Draparnaldia > Ceratophyllum >

Elodea > Myriophyllum. This relationship also would be the predicted
outcome of competition between these organisms under limited K supply.
The Rb uptake rate for Ceratophyllum demersum was 8x greater than for

Myriophyllum spicatum. This might explain the much higher concentra-
tions of K in Ceratophyllum than in Myriophyllum collected from Lake

Wingra (Section VI). The higher Rb uptake rate in Draparnaldia than in
Elodea does not, however, correlate with the much greater aggressive-
ness of Elodea when competing with Draparnaldia for a growth-limiting
K supply as described in Section VII. Further stidy will be required
to establish which procedure more accurately reflects competition for
nutrients under field conditions. Until confirming information is
available, there seems justification for attaching greater reliability
to a procedure in which effective competition for an element is
actually reflected in plant yield.

75


critical concentration, so there was no indication that this element
became growth-limiting. In contrast, Mg concentrations were only
slightly above the critical value in all six lakes and Ca concentra-
tions were close to or below the critical concentration in two lakes
(Salsich and Erickson). The Ca concentrations in the Elodea correlate
well with the alkalinity values for the lakes reported in Table 6. The
highest Ca concentration (1.25%) was in plants from Little John Lake
which also had the hardest water (alkalinity of 46); the lowest Ca
concentration (0.20%) was from Salsich which had the softest water
(alkalinity of 12). The Ca concentration in the Lake Whitney samples
was relatively high even though the alkalinity was only 20. This
probably is because P was the limiting element in that lake.

Data from analyses of the Elodea samples for Fe, Mn, Cu, B, and Zn
are presented in Table 10. All values are above established critical
concentrations indicating that none of these elements was limiting
Elodea growth. In comparison with critical Cu concentrations in
terrestrial species (4-7 ppm) some of the Cu values for Elodea are
very low. In earlier work similar low Cu concentrations were inter-
preted as indicating Cu deficiency in Elodea. Additional studies have
shown Elodea has an extremely low Cu requirement in comparison with
other angiosperm plants. Therefore, it is doubtful the low concentra-
tions in the Elodea indicate a growth-limiting role of that element.
Copper might be critical, however, for other aquatic plants.

Plant Analysis of Introduced Elodea

A second application of plant analysis in this study was to introduce
Elodea into the lakes in such a way that it could be routinely sampled
and analyzed to indicate nutrient supplies. The Elodea was floated
near the water surface in plastic-mesh, porous cylinders as described
in earlier work (Gerloff, 1973). At the surface, Elodea would
directly derive nutrients only from the water and not from both the
water and the bottom muds. It was anticipated that this would result
in a more suitable general assay for nutrients in lake water than
would sampling macrophytes rooted in bottom muds.

22


of the two organisms when each was grown separately and not competing

for a nutrient.

The data in Table 14 indicate relatively little difference in the
efficiency of NO3-N utilization by Elodea and Draparnaldia. At the
low concentration of NO3-N (0.30 ppm), yields were approximately
equal (47% vs. 53% for Elodea and Draparnaldia respectively). The
change in the proportion of total yield represented by each species

at the two NO3 concentrations suggests somewhat greater efficiency
in utilization by Draparnaldia. Yield represented by Draparnaldia,
when the organisms were grown together, increased from 36% at

33.6 ppm N to 53% at 0.30 ppm.

When grown alone, Elodea yield decreased markedly at the lower NO3
concentration (from 1.07 to 0.37 g) while Draparnaldia yield decreased
only slightly from 0.49 to 0.41 g. Total yield in the mixed cultures
was only about one-half the yield at high NO3 (0.60 vs. 1.25 g).

The data in Table 15 are from an experiment involving competition
for a limited P supply. Again yields were about equal when the two
organisms were grown in competition at the 0.075 ppm concentration
of P (54% for Elodea and 46% for Draparnaldia). However, comparisons
of yields at high and low P suggest a superior competitive capacity
of Draparnaldia. The proportion of total yield represented by Elodea
decreased from 89 to 54% while the yield represented by Draparnaldia
increased from 11 to 46%. When cultured separately, Elodea yield
decreased from 1.71 g to 0.81 g at the lower P concentrations while
yield of Draparnaldia remained constant at 0.67 g. Total yield of
the two organisms grown in combination was 1.48 g at 6.2 ppm P and
only 1.03 g at 0.075 ppm. Under low P, the P concentration in

each organism was at or close to its respective critical concentra-
tion of 0.14% for Elodea and 0.18% for Draparnaldia, thus indicating
P deficient plants.

39


Table 8. TOTAL N AND P CONCENTRATIONS IN SECOND ONE-INCH
INDEX SEGMENTS OF Elodea occidentalis COLLECTED
FROM NORTHERN WISCONSIN LAKES

Total N (% dry wt.) Total P (% dry wt.)
Lake sampled I II Ave. I II Ave.
Little John 1.78 2.11 1.95 0.23 0.27 0.25
Clear 1.96 1.65 1.81 0.31 0.23 0.27
Whitney 2.18 2.15 2.17 0.11 0.14 0.13
Allequash 2.16 2.34 2.25 Q.20 0.33 0.27
Salsich 3.02 3.57 3.30 0.21 0.29 0.25
Erickson 3.10 3.16 3.13 0.21 0.30 0.26

I, samples collected July 20-21; II, samples collected August 15-16.

Table 9. MAJOR ELEMENT CATION CONCENTRATIONS IN INDEX SEGMENTS OF
Elodea occidentalis COLLECTED FROM NORTHERN WISCONSIN LAKES

Ca cone. (%) Mg conc. (%) K conc. (%)
Lake sampled I II | Ave. I II | Ave. I II | Ave.
Little John 1.25 -- 1.25 | 0.10 }0.12] 0.11 2.731 1.70 | 2.22
Clear 0.67} 0.83} 0.75 | 0.11 } 0.11] 0.11 2.38} 1.75] 2.07
Whitney 0.68} 0.62} 0.65 | 0.11 | 0.12 | 0.12 1.89 | 2.21 | 2.05
Allequash 0.73] 0.86} 0.80 | 0.11 | 0.13] 0.12 2.01 | 2.17 | 2.09
Salsich 0.20 | 0.31} 0.26 | 0.11 | 0.12] 0.12 2.44 | 3.24] 2.34
Erickson 0.29 | 0.36] 0.33 | 0.11 {0.13 | 0.12 2.31 | 3.08 | 2.70

The index segment for Ca was the first one-inch of the main stems and
laterals; for K and Mg, the second one-inch was used.

21


Table 23. COMPARISON OF K AND Rb UPTAKE RATES FROM
EXTERNAL CONCENTRATIONS OF 0.5 mM KCl,
0.5 mM RbCl, 0.01 mM KC1, AND 0.01 mM RbC1

mM cation absorbed/g dry
tissue wt/20 min (x10")
From From From From
0.5 mTM 0.5 mM 0.01 mM 0.01 mTM
Kcl RbC1 KCl RbCl
Plant (K,mM) (Rb ,mM) (K,mM) (Rb ,mM)
Myriophyllum shoots 150.47 57.82 0.69 0.74
Ceratophyllum shoots 142.14 167.73 7.72 5.80
Elodea roots 66.00 36.53 1.21 0.50
Elodea shoots 350.54 315.46 2.22 2.83

All solutions were labeled with ®*Rb; the concentration of CaCl2 was
0.5 mM.

In three cases, Myriophyllum spicatum shoots at an external cation
concentration of 0.5 mM and Elodea occidentalis roots at an external
cation concentration of both 0.5 mM and 0.01 mM, Rb and K were not
absorbed at the same rate. Results for Rb and K were comparable in
all of the other trials. Because Rb could not be used as a tracer for
K in all cases, experiments varying either time or external concentra-
tion were run with RbCl in the external solution rather than KCl.

The uptake patterns obtained using RbCl can be applied, in most cases,
to the uptake of K.

58


10

11

12

13

TABLES

Critical Concentrations and the Ranges in Concentrations
of Various Essential Elements in Several Aquatic
Angiosperms

Critical N and P Concentrations and the Ranges in N and P
Concentrations in Lemna minor (Duckweed)

Critical Concentrations of Various Essential Elements in
Several Green and Blue-Green Algae

Growth Response of Several Plant Species When Cultured
in Nutrient Media and Environments Deficient in Copper

Bioassay for Growth-Limiting Nutrients in Six Northern
Wisconsin Lakes by the Provisional Algal Assay Procedure

Chemical Analyses for N and P Fractions in Water Samples
from Six Northern Wisconsin Lakes

Bioassays by the Fitzgerald Tests for P (Hot Water
Extractable) and N (NH,-N Uptake in the Dark) in Elodea
occidentalis Collected from Northern Wisconsin Lakes

Total N and P Concentrations in Second One-Inch Index
Segments of Elodea occidentalis Collected from Northern
Wisconsin Lakes

Major Element Cation Concentrations in Index Segments of
Elodea occidentalis Collected from Northern Wisconsin Lakes

Trace Element Concentrations in Index Segments of Elodea
occidentalis Collected from Northern Wisconsin Lakes
During July and August

Composition of a Modified Gerloff and Krombholz Solution
Used for the Culture of Angiosperm Aquatic Plants

Nitrogen, Phosphorus, and Potassium Concentrations (Oven-
Dry Basis) in Second One-Inch Segments of Myriophyllum
spicatum Shoots Collected from Lake Wingra

Nitrogen, Phosphorus, and Potassium Concentrations (Oven-

Dry Basis) in Second One-Inth:Segments of Ceratophyllum
demersum Shoots Collected from Lake Wingra

vi

11

16

18

19

21

21

23

28

32

33


unlikely, that Cu is not required by these plants.

Analyses for Cu in the deficient plants have been difficult because

of the extremely low concentrations. Some data on Elodea obtained
with a solvent-extraction Atomic Absorption procedure are presented.
The Cu concentration in the deficient Elodea was consistently less than
a ppm and averaged only 0.59 ppm. This is far below the critical Cu
concentration in agricultural crop plants which usually is within the
range of 3 to 8 ppm (Chapman, 1966).

The conclusion from the results presented is that the very low Cu
concentrations in macrophytes from northern Wisconsin lakes probably
do not reflect deficiency and a growth-limiting role of that element,
but rather very low requirements for Cu. The physiological basis for
this unusually low Cu requirement is of considerable interest. The
low Cu requirement also may be a factor in adaptation of these plants
to low Cu environments in northern Wisconsin lakes.

DISCUSSION

An obvious question about the application of the plant analysis tech-
nique is whether the same critical concentrations are applicable to
all species in one taxonomic group, for example among aquatic macro-
phytes or green algae. It was suggested that the same N and P critical
concentrations could be generally used among macrophytes (Gerloff,
1969). However, the data on macrophytes in Table 1 indicate that this
is not true. The critical concentrations for N and P are slightly
lower in Ceratophyllum demersum than in Elodea occidentalis and are
much lower in Myriophyllum spicatum, approximately 50% of the values

in Elodea. Values for K also varied widely among the macrophytes,

from 0.35% in Myriophyllum ‘to 1.70% in Ceratophyllum. These results
establish that, as with agricultural and horticultural species, the

use of plant analysis to assay nutrient supplies to aquatic plants
requires that critical concentrations must be established for each
species of interest.

The primary value of summarizing critical concentrations in Tables 1,

12


SECTION X

REFERENCES

,

Andrew, C. S. A Kinetic Study of Phosphate Absorption by Excised
Roots of Stylosanthes lumilis, Phaseolus lathyroides, Desmodium
uncinatum, Medicago sativa, and Hordeum vulgare. Aust. J. Agr.
Res. 17:611-624, 1966.

Bauman, P. C., A. D. Hasler, J. F. Koonce, and M. Teraguchi.
Biological Investigations of Lake Wingra. EPA Ecol. Res.,
EPA-R3-73-044. 1973. 118 p.

Bertramson, B. R. Phosphorus Analysis of Plant Material. Pl.
Phys. 17:447-454, 1942.

Bristow, J. M. and M. Whitcombe. The Role of Roots in the
Nutrition of Aquatic Vascular Plants. Amer. J. Bot. 58:8-13, 1971.

Carter, 0. G. and D. J. Lathwell. Effects of Temperature on
Orthophosphate Absorption by Excised Corn Roots. Pl. Phys.
42:1407-1412, 1967.

Chapman, H. D. The Status of Present Criteria for the Diagnosis
of Nutrient Conditions in Citrus. In: Colloquium on Plant
Analysis and Fertilizer Problems, Publ. No. 8, A.1.B.S.,
Washington, D. C., 1961.

Chapman, H. D. Diagnosis Criteria for Crops and Soils. Univ.
of California Div. of Agricultural Sciences. 1966.

De Marte, J. A. and R. T. Hartman. Studies on the Absorption of
32p 59Fe and “5Ca by Water-milfoil (Myriophyllum exalbescens
Fernald), Ecology. 55:188-194, 1974.

Epstein, E. The Essential Role of Calcium in Selective Cation
Transport by Plant Cells. Pl. Phys. 36:437-444, 1961.

Epstein, E. Mineral Nutrition of Plants: Principles and Perspectives.
New York, John Wiley and Sons, Inc., 1972. 412 p.

Epstein, E. and C. E. Hagen. A Kinetic Study of the Absorption
of Alkali Cations by Barley Roots. Pl. Phys. 27:457-474, 1952.

Fitzgerald, George P. Field and Laboratory Evaluations of Bioassays

for Nitrogen and Phosphorus with Algae and Aquatic Weeds. Limnol.
Oceanogr. 14:206-212, 1969.

76


length of time between drainings, any desired volume of solution

could be provided.

All apparatus was thoroughly cleansed and sterilized prior to the
initiation of an experiment. However, the large volumes of solution
required made it impractical to sterilize the nutrient medium.
Contamination did not prove to be a problem if the solutions were
replaced at least once a day. When the culture vessels were drained,
suspended contaminants as well as depleted medium were rapidly carried

away .

The Elodea cultures were exposed to continuous fluorescent-tungsten
light of approximately 900 foot-candles at approximately 25°C.
Cultures were aerated with a 1% COg-air mixture. Harvested plants
were dried at 65-70°C. Analysis for N, P, K and Ca were by the

methods indicated in Section VII.
RESULTS

The results in Table 21 show that at 0.02 and 0.03 ppm P and at 0.30
ppm NO3-N yields of Elodea were sharply and significantly reduced in
comparison with yields in the control medium. Yield at 0.02 ppm

P was reduced to 52% and at 0.30 ppm NO3-N to 67% of control yields.
The N and P analytical data support the conclusion that Elodea growth
was limited by deficiencies of these two elements. Concentrations
were very close to the critical values of 1.60% N in the second one-
inch segment and 0.14% P in the same segment. Because of inadequate
plant material, the analyses reported are of whole plants rather than

of second one-inch segments.

The concentrations of Ca and K in infertile, soft water lakes can

serve as reference values in evaluating the growth of Elodea at low
concentrations of those two elements. In northern Wisconsin lakes
with alkalinity values of only 4.0-6.0, Ca concentrations were as low
as 0.5-1.5 ppm; K concentrations were 0.5 to 1.0 ppm. In an experiment
not reported, yield was not significantly decreased when the Ca concen-
tration was reduced to 0.80 ppm. However, yield was decreased sharply

49


2.0 -

i
mmoles Rb
ABSORBED

x 10-5

! 1 J 1
5.0 10.0 15.0 20.0

!
Rb CONC.

Figure 17. Double-reciprocal plot of rubidium uptake in excised
shoots of Elodea occidentalis.

mechanism is involved in Rb and K uptake by Elodea and Ceratophyllium

shoots and two or more mechanisms in the other species. Data were
considered insufficient to compute Vmax and Km values.

DISCUSSION

Whether submerged aquatic plants absorb ions mainly from the substrate
or from the surrounding water has long been a topic of controversy,

73


TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)

1, REPORT NO. 2. 3. RECIPIENT'S ACCESSION*NO.
EPA-660/3-75-027
4, TITLE AND SUBTITLE 5. REPORT DATE

Nutritional Ecology of Nuisance Aquatic Plants 5 PERFORMING ORGANIZATION CODE

7. AUTHOR(S) . PERFORMING ORGANIZATION REPORT NO.
Gerald C, Gerloff
9. PERFORMING ORG ANIZATION NAME AND ADDRESS 10, PROGRAM ELEMENT NO.

Department of Botany

: : . : : 11. CONTRACT/GRANT NO.
University of Wisconsin-Madison

Madison, Wisconsin 53706 R-800504

12, SPONSORING AGENCY NAME AND ADDRESS 13, TYPE OF REPORT AND PERIOD COVERED
National Environmental Research Center Finalew 9/28/72 to 10/31/74
Office of Research and Development 14. SPONSORING AGENCY CODE

U,S, Environmental Protection Agency
Corvallis, Oregon 97330

15, SUPPLEMENTARY NOTES

[16. ABSTRACT

Plant analysis was compared with other techniques in assays for available and
growth-limiting nutrients in northern Wisconsin lakes. The data were in poor
agreement. To further develop plant analysis, critical concentrations of a number
of elements were established in various macrophytes and algae. Critical concentra-
tions varied markedly in different organisms, The plant analysis bioassay indicated
K supply, rather than N or P, became limiting for macrophyte growth in a eutrophic
lake.

Three procedures were developed to evaluate the capacities of macrophytes and algae
to compete for nutrients at the low concentrations in lakes. These procedures
involved (1) competition between several organisms in the same culture for a
growth-limiting amount of a nutrient, (2) nutrient replacement in cultures

to establish the borderline concentration at which an organism failed to make
maximum growth even though the total nutrient supply was adequate, and (3) measure-
ment of rates of nutrient uptake and calculation of Vmax and Km values. The
competitive and uptake capacities of various aquatic plants for a specific

element differed markedly,

h7. KEY WORDS AND DOCUMENT ANALYSIS
fa. DESCRIPTORS. b.IDENTIFIERS/OPEN ENDED TERMS |c. COSATI Field/Group
Bioassays Critical Concentrations
Competition Limiting Nutrients
Deficient Elements Nuisance Aquatic Plants
Nuisance Algae Plant Analysis
18, DISTRIBUTION STATEMENT 719. SECURITY CLASS (This Report} 21. NO. OF PAGES
Release unlimited 30. SECURITY CLASS (This page] 22. PRICE
=

EPA Form 2220-1 (9-73)
U.S. GOVERNMENT PRINTING OFFICE: 1975~698-710 1166 REGION 10



solutions by centrifugation.
THEORY
Phosphorus absorption, according to Hagen and Hopkins (1955), and

cation absorption, according to Epstein and Hagen (1952), can be
expressed by the following equations:

ki kg
R+M = RM =~ REM (1)
kp ky

where R is a metabolically produced carrier, M is the ion being
transported into the cell, RM is the carrier-ion complex, and k is
the rate constant for each reaction. For the absorption of both P
and K, ky is negligible. These reactions are analogous to the
Michaelis-Menten (1913) theory of enzymatic reactions. An equation
expressing the relationships in equation 1 is

(Vmax - v) [M]/v = Km (2)

where Vmax is the maximuin rate of absorption at infinite substrate
concentration, v is the observed rate of absorption at ion concentra-
tion M, and Km is the Michaelis constant or the ion concentration at
(3s) Vmax.

Equation 2, as observed by Lineweaver and Burk (1934), can be written
in a linear form:

1/v = Km/(Vmax[M]) + 1/Vmax (3)

56,


>
a
T

-
fe)
T

OVEN-DRY WT.(g/2 2)
o
oa
T

n i | i

0.6 1.0 5 2.0
TISSUE CONTENT OF N(%)

Figure 1. The relationship between yield and total nitrogen content
of the second one-inch terminal segments of Myriophyllum spicatum
grown in solutions of varying nitrogen content.

RESULTS

Figures 1, 2, and 3 present the curves from which critical concentra-
tions of N, P, and K were established in Myriophyllum spicatum.
Because of the mobility of N, P, and K in plants (Gerloff, 1973),
second one-inch sections were selected as index segments and critical
concentrations were based on analyses of those segments. The data
show that concentrations of each element varied over a wide range,

N from 0.61 to 1.58%, P from 0.04 to 0.18%, and K from 0.30 to 1.13%.
The critical concentrations were established as 0.75% N, 0.07% P,

and 0.35% K. These values are the concentrations associated with
yields about 5% below the maximum. Element concentration above the

29


Table 24. THE APPARENT Vmax AND Km OF THE TWO CARRIERS INVOLVED

IN P UPTAKE IN EIGHT SPECIES OF AQUATIC PLANTS

Vmax, Vmax, km, Kn,
mg P abs./g dry
Species wt./20 min (x10°) ppm

Elodea occidentalis roots 34 8 1.12 -012
Elodea occidentalis shoots 55 45 1.29 -011
Ceratophyllum demersum 67 1 0.37 0.0023
Myriophyllum spicatum 43 1 1.15 -00045
Lemna minor 60 30 1.34 0.123
Draparnaldia plumosa 330 70 1.65 0.0036
Stigeoclonium tenue 100 70 0.27 0.015
Microcystis aeruginosa 185 2 2.47 -004
Anabaena sp. 100 10 2.07 0.024

69


6.0-

5.0-

OVEN-DRY WT. (9/22)
>
(2)
T

1 1 1 I 1 1
0.01 0.05 0.10 0.15 0.20
TISSUE CONTENT OF P (%)

Figure 2. The relationship between yield and total phosphorus content
of the second one-inch terminal segments of Myriophyllum spicatum
grown in solutions of varying phosphorus content.

critical levels represented luxury consumption that did not produce
further yield increases. In fact, the highest plant concentrations
of N and P were slightly inhibitory.

Following establishment of N, P, and K critical concentrations in
Myriophyllum, nutrient availability in Lake Wingra was evaluated

by comparisons of the analyses of index segments collected from

two sites in the lake with the critical concentrations. The analytical
data are presented in Table 12.

There were no indications that supplies of eithe#'N or P became
limiting for Myriophyllum growtk ‘at any time during the three years

30


sv

Table 19. GROWTH OF Myriophyllum spicatum, Elodea occidentalis, AND Draparnaldia plumosa
WHEN COMPETING FOR A GROWTH-LIMITING SUPPLY OF P IN A MIXED CULTURE

Myriophyllum Elodea Draparnaldia
Mixed
P Yield (g) Prop. of Yield (g) Prop. of Yield (g) Prop. of | culture
conc. yield yield yield yield
(ppm) | Alone | +E1. & Drapj  (%) Alone | +Myr. & Drap (%) Alone |+El. § Myr. (%) (g)
6.2 1.06 0.73 33 1.07 0.50 23 0.79 0.95 44 2.18
0.075} 0.86 0.52 37 0.99 0.32 23 0.61 0.56 40 1.40

Table 20. GROWTH OF Myriophyllum spicatum, Elodea occidentalis, AND Draparnaldia plumosa
WHEN COMPETING FOR A GROWTH-LIMITING SUPPLY OF K IN A MIXED CULTURE

Myriophyl lum Elodea Draparnaldia
Mixed
K Yield (g) Prop. of Yield (g) Prop. of Yield (g) Prop. of |culture
conc. yield yield |---| yield yield
(ppm) | Alone | +E1. & Drap (%) Alone | +Myr. & Drap (%) Alone |+El. § Myr. (%) (g)
39 1.55 0.96 51 1.50 0.59 31 0.71 0.35 18 1.90
0.39] 1.25 0.91 53 1.39 0.61 35 0.74 0.21 12 1.73



8T

Table 6. CHEMICAL ANALYSES FOR N AND P FRACTIONS IN WATER SAMPLES FROM SIX NORTHERN WISCONSIN LAKES

Concentration
(ppm)
POs -P Total P NO3 -N NH3 -N NO2 -N Total N

Lake Ave. | Ave,
sampled I II I II I II I Il I II I II | alk.}| pH
Little

John 0.043 }0.016 |0.08 |0.03 } 0.04 }0.25 }<0.03 |<0.03 | 0.006 | 0.002 |0.91 | 0.83} 46 | 7.6
Clear 0.023 ]0.009 | 0.05 |0.03 | 0.07 |0.11 }<0.03 }<0.03 | 0.002 } 0.002 |0.84}0.64} 33 | 7.3
Whitney 0.016 | 0.013 | 0.07 |0.01 | 0.04 |0.11 |<0.03 |<0.03 |<0.002 |<0.002 {1.03} 0.81] 20 | 7.2
Allequash | 0.037 }0.017 | 0.08 | 0.03 | 0.08 | 0.04 |<0.03 |<0.03 | 0.002 } 0.002 |1.13]0.71} 34 | 7.5
Salsich 0.067 }0.009 | 0.04 | 0.01 | <0.04 | 0.09 |<0.03 |<0.03 ] 0.002 | 0.000 }0.85]0.57| 12 | 7.0
Erickson {0.038 {0.005 | 0.04 | 0.02 | 0.05 | 0.06 |<0.03 | 0.04 | 0.002 |<0.002 |0.97]0.65) 23 | 7.2

I, from July

20-21 sampling period; II, from August 15-16 sampl

ng.


was inadequate to produce yields equal to those in the control solution.

It was essential that the total amount of an element available

to Elodea should be considerably in excess of the amount needed for
the yield produced. At the lowest element concentrations studied,
this required large volumes of solution. As a result, the lowest
concentrations tested were determined by the solution volumes that
could be provided conveniently.

To calculate the total amount of an element to be provided at low
concentrations, it was recognized that about 500 mg of dry-weight
Elodea would be produced in the 1l-day experimental periods. On
the basis of established Elodea critical concentrations an amount
of the element was added to the cultures each day which would support
a minimum of 100 mg of growth. For example, the Elodea critical P
concentration is approximately 0.15%. To provide the required

0.15 mg of P at a concentration of 0.02 ppm it was necessary to

add at least 8-liters of culture medium every 24 hours. Growth

the last day of an li-day period was calculated to be no more than
80 mg, if growth were continuous and exponential. Therefore, on that
day the P made available to the Elodea was only 20% in excess of the
requirement. On other days, additions of P relative to requirements

were much greater.

Two types of culture apparatus were employed. The first experiments
involved continuous-flow cultures using large test tubes as culture
vessels. Sterile medium was pumped or allowed to flow through the
culture vessels at a controlled and constant rate as in a chemostat.
A more satisfactory apparatus, and the apparatus in which most of
the experiments were carried out, utilized 6-liter Florence flasks
fitted with bottom drains as culture vessels. At intervals controlled
by a time clock, valves on the outlets opened and emptied the flask
contents, except for the Elodea, and about 100 ml of culture medium.
As soon as the vessels drained, the valves closed and fresh medium
from pyrex carboys flowed into the flasks to a level controlled

by a pyrex float valve. By adjusting the level of solution and the

48


If the process of absorption at a steady state involves a single
first-order reaction, i.e., a single carrier mechanism, then a
double-reciprocal plot of v and [M] will result in a straight line
(Hagen and Hopkins, 1955; Hofstee, 1952). If more than one first-
order reaction is involved, the double-reciprocal plot is a curvilinear

line.

Equation 2 can be written in a second linear form (Hofstee, 1952):

Vmax = v + (v/[M]) *Km (4)

Again, if only one first-order reaction is involved, the resulting

plot of v against v/[M] is a straight line, but a curvilinear line
results if two or more first-order reactions are involved. The Vmax-

of all reactions combined is given by the intercept with the ordinate,
and the reaction components may be obtained graphically (Hofstee, 1952),
so that Vmax and Km for each component may be calculated.

RESULTS

Check of Rb Labeling

Measurement of K absorption using radioactive tracers is difficult
because the half-life of *?K is very short (12.4 hours). In several
systems, the absorption rate and mechanism of Rb uptake have been
found to be identical to that of K (Epstein, 1961; Rains, 1968),

and consequently, °*Rb, with a half-life of 18.77 days, has been used
as a tracer for K. In some systems, however, the uptake rates

have been shown to be different (Jeschke, 1970), so a check of the
validity of using °*Rb as a tracer for K was made. For each species,
cation uptake was measured in four different external solutions:

0.5 mM KC1, 0.5 mM RbCl, 0.01 mM KCl, and 0.01 mM RbCl. All solutions
were labeled with °°Rb. Results were calculated as mM cations
absorbed and are presented in Table 23.

57


ABSTRACT

Plant analysis was compared with other frequently used techniques in
assays for available nutrients and growth-limiting nutrients in several
northern Wisconsin lakes, Data from the different procedures were in
poor agreement. The plant analysis bioassay suggested that elements
other than P limited plant growth in some of the lakes studied.

As a further development of plant analysis, critical concentrations
of a number of elements were established in various macrophytes and
algae. The data showed that the critical concentrations for an
element can vary greatly in different organisms, so much so that
specific critical concentrations must be established for each
aquatic species utilized in plant analysis bioassays.

The plant analysis bioassay indicated that K supply, rather than N
or P, became limiting for the growth of the macrophytes Myriophy1lum
spicatum and Ceratophyllum demersum in a shallow eutrophic lake.

Three procedures were developed and tested for evaluating the
capacities of macrophytes and algae to compete for nutrients at
the low concentrations characteristic of lakes. These procedures
involved (1) competition among several organisms in the same
culture for a growth-limiting amount of a nutrient, (2) nutrient
replacement in cultures to establish the borderline concentration
at which an organism failed to make maximum growth even though the
total nutrient supply was adequate, and (3) measurement of rates
of nutrient uptake and calculation of Vmax and Km values in terms of
Michaelis-Menten kinetics. The competitive and uptake capacities
of various aquatic plants for a specific element differed markedly.

This report was submitted in fulfillment of Grant R-800504 by
Gerald C. Gerloff under the sponsorship of the Environmental
Protection Agency. Work was completed as of October 31, 1974.

ii


under study at which the plants in culture could not absorb enough

of the element to produce maximum growth.

A third approach was to establish the rates of uptake of key elements
at various external concentrations according to Michaelis-Menten
kinetics. Uptake by various species then would be calculated as

Km and Vmax values and comparisons of accumulation capacities of the
species would be made. This technique perhaps would give different
results than the first two because the rate of uptake measured in
short-term experiments might not be associated with optimum utiliza-

tion of an element in plant growth.

Results with these three procedures will be presented in Sections
VII, VIII and IX of this report.

EXPERIMENTAL PROCEDURES

The experimental organisms in the mixed cultures were two macrophytes,

Elodea occidentalis and Myriophyllum spicatum, and a filamentous green

alga, Draparnaldia plumosa. It was essential that the organisms
employed could be readily separated at the termination of an experiment
when dry-weight yields were determined. It also seemed desirable that
the organisms should grow at about the same rate so that one species
would not dominate a culture. Because growth of macrophytes is slowly
initiated in freshly inoculated cultures, macrophytes were inoculated
into mixed cultures 5 days prior to Draparnaldia.

Plants were grown in two-liters of medium in three-liter Florence
flasks. The culture medium was the solution described in Table 11
with the trace element concentrations slightly modified. This solu-
tion proved a satisfactory medium for both the Draparnaldia and the
macrophytes. Other culture conditions were as described earlier
(Gerloff and Krombholz, 1966). To insure that the desired low con-
centrations of an element would not be exceeded, the growth-limiting
element in a specific experiment was added to cultures at intervals
during the growth period. All treatments were in triplicate.

37


12 and 13, N and P concentrations in index segments were consistently

lower at Site 2 than Site 1.

Because use of plant analysis in aquatic environments is relatively
new and because of unexpected indications of K deficiency, several
critical steps in the plant analysis procedure were repeated. The
critical concentrations for N, P, and K were verified in different
experiments and the K analyses were checked by the Wisconsin Alumni
Research Foundation Laboratories with a Jarrell-Ash Multichannel
Spectrometer. It also was considered possible that the second one-
inch segments analyzed for K were not as carefully measured as required.
However, this would not have resulted in major differences in the
results, because the critical concentration in the first one-inch
segment was only 0.05% (0.35% vs. 0.40%) greater than the second
one-inch. Nevertheless, it would be desirable to verify by other
diagnostic techniques the primary conclusion of this study that

K supply limited growth of Myriophyllum spicatum in Lake Wingra.

35


300F x
+ weer
2 _e-o7
x ee_o77
E iz x
= /
> 200+ ,
s y
6 /
§ /
_ 7
& /
ro} 4
8 100 F 7X
x
4
/
Fd /
H / %---X shoots
2 @—e roots
€

it 1 1 _L 1 it

20 40 60 80 100 120 140 160 180
TIME (MINUTES)

Figure 5. Relationship of rubidium uptake to time in excised roots
and shoots of Elodea occidentalis. Substrate concentration was
0.5 mM RbCl with CaCl2 at 0.5 mM.

A time plot for Ceratophyllum demersum shoots also was run but not
reported, Steady-state conditions occurred over the first 20 minutes,
but the uptake rate fell off at 30 minutes. A time plot for Stigeoclo-
nium tenue showed steady-state conditions over a period of 60 minutes.
Stigeoclonium showed an overall higher rate of P uptake than either

of the macrophytes.

The plot of time against rate of Rb absorption for Elodea occidentalis
roots and shoots is shown in Figure 5. The external RbC1 concentration

60


SECTION III

INTRODUCTION

The nutritional requirements of most autotrophic plants, including
fresh-water algae and macrophytes, are relatively simple. In the
absence of toxic conditions, green plants make normal growth upon
exposure to suitable temperature, adequate light, water, and inor-
ganic nutrients. External supplies of vitamins, amino acids, and an
organic energy source need not be provided. The inorganic nutrients,
therefore, become of critical importance in the control of nuisance
plants in aquatic environments and in understanding their distribution
in various bodies of water.

This project has been concerned primarily with two aspects of the
nutritional ecology of nuisance aquatic plants. One aspect was

the further development of plant analysis as a reliable bioassay
for nutrient supplies and for growth-limiting nutrients in aquatic
environments. The other dealt with the development of techniques
and the accumulation of data for evaluating the importance of
inorganic nutrients in the occurrence of nuisance plants in specific
lakes and streams. Both of these aspects relate to possible control
of nuisance plants in aquatic ecosystems. For clarity in presentation,
each aspect of these studies will be presented in a separate

section of this report.


SECTION VIII
GROWTH OF Elodea occidentalis AT LOW CONCENTRATIONS OF

INORGANIC NUTRIENTS MADE AVAILABLE IN SOLUTION-REPLACEMENT CULTURES

When grown in the laboratory, aquatic plants usually are cultured

in small volumes of nutrient medium containing much higher concentra-
tions of the essential elements than normally are present in lakes
and streams. The high concentrations are necessary to provide
amounts of the elements for high rates of yield from small volumes
of solution. Data on the responses of aquatic organisms to element
concentrations obtained under these conditions are rarely applicable

to non-laboratory conditions.

Responses of organisms grown in the laboratory to the low concentra-
tions of N, P, and other elements characteristic of natural waters
can be measured by several techniques. These include continuous-
flow cultures, the use of very large volumes of culture medium,

and the frequent addition of a specific element to a culture to
maintain a nearly constant, low concentration. The work to be
reported was concerned with establishing the borderline concentration
of N, P and each of the major element cations at which maximum rate
of growth no longer could be maintained even though the total amount
of an element made available to the Elodea was more than adequate.

EXPERIMENTAL PROCEDURES

Because of the large volumes of nutrient solution required, only

two treatments, which were replicated, could be included in each
experiment. In every experiment, one treatment always was the same
control solution consisting of the medium described in Table 11

at one-quarter strength. The second treatment was the same as the
control except one element was provided at a much lower conceritration.
Experiments were repeated with a progressively lower concentration

of the element under study made available in each successive experiment

until a concentration was established at which rate of absorption

47


SECTION IX
COMPARISONS OF RATES OF PHOSPHORUS AND RUBIDIUM UPTAKE

BY SEVERAL MACROPHYTES AND ALGAE

This is a study to determine and compare the rates of P and K uptake
in eight species of aquatic plants, to compare the uptake rates of
Elodea occidentalis roots and shoots, and to predict the outcome of
competition between species grown at low P or K levels from a compari-

son of uptake rates at those levels.

MATERIALS AND METHODS

Kinetic studies of PO, absorption were carried out with four
species of aquatic flowering plants found in Wisconsin lakes: Elodea

occidentalis, Ceratophyllum demersum, Myriophyllum spicatum, and Lemna

minor. Four species of algae were also studied: two filamentous

green algae, Draparnaldia plumosa and Stigeoclonium tenue, and two

blue-green algae, Microcystis aeruginosa and Anabaena sp. Kinetic
studies of Rb absorption involved the Elodea, Ceratophyllum, Myrio-

phylium, and Draparnaldia.

The macrophytes to be studied were cultured under continuous light of
S500 f.c. in the nutrient solution described in Table 11. The four
species of algae and the Lemna minor were grown (Table 22) in a modi-
fied Hughes, Gorham, and Zehnder medium (1958) under continuous

light of 200 f.c. All species were grown at approximately 23°C and
all except the Lemna and the algae were continuously aerated with a
1% CO2 in air mixture. The plants were grown between 3 and 4 weeks,
until enough material was present for an experiment. At the time
the experiments were run, concentrations of P and K in all species

were in excess of their respective critical levels.

For the experiments involving the macrophytes, except Lemna minor,
the terminal two-inch segments of either roots or shoots were excised.
Whole plants of Lemna were used. After being gently blotted on dry

53


data on critical concentrations in algae and macrophytes obtained by
the Principal Investigator over a period of several years. Some of the

data were reported previously.
RESULTS

Table 1 includes data on the critical concentrations for various essen-
tial elements and the range of concentrations of those elements in
index segments of several aquatic angiosperms. Index segments were the
first or second one-inch segments cut from the main shoot and laterals.
The data on Myriophyllum spicatum, a serious nuisance in Wisconsin
lakes, have not been reported previously.

Undoubtedly data on N and P critical concentrations are of most general
interest because of their suspected critical roles in eutrophication
and promotion of nuisance plant growths. For this reason, critical
concentrations of N and P were established for all three macrophytes
studied. It would have been highly desirable to determine the critical
concentrations for all of the additional essential elements in each
species. Unfortunately, these experiments require much time and effort

and have not been possible.

The data in Table 2 are for Lemna minor (duckweed). It is an aquatic
angiosperm but was not included in Table 1 because with this small
organism entire plants rather than index segments were analyzed.

Table 3 presents critical concentrations in several green and blue-
green algae. It should be noted that in the original presentation of

the Microcystis aeruginosa data it was recognized that the mucilag-

inous sheath surrounding cells of this organism could complicate use
of plant analysis with this species (Gerloff and Skoog, 1957). A

procedure was developed for adjusting the critical concentrations to
account for variations in the amount of sheath associated with Micro-

cystis obtained from various sources.

One of the most interesting features of the collection and chemical
analysis of macrophyte samples from relatively infertile northern
Wisconsin lakes in an earlier aspect of this project was their very


EPA-660/3-75-027
JUNE 1975

NUTRITIONAL ECOLOGY OF

NUISANCE AQUATIC PLANTS

by

Gerald C. Gerloff
Department of Botany and
Institute of Plant Development
University of Wisconsin
Madison, Wisconsin 53706

Grant R-800504
Program Element 1BA031
ROAP/Task No. 21AJF/005

Project Officer

William E. Miller
Pacific Northwest Environmental Research Laboratory
National Environmental Research Center
Corvallis, Oregon 97330

NATIONAL ENVIRONMENTAL RESEARCH CENTER
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY

CORVALLIS, OREGON 97330

For sale by the Superintendent of Documents
Printing Office, Washington, D.C.

5. Government
16402



Table 11. COMPOSITION OF A MODIFIED GERLOFF AND KROMBHOLZ SOLUTION
USED FOR THE CULTURE OF ANGIOSPERM AQUATIC PLANTS

1.0 M stock solution Element
per 1 final solution in final solution

Salt (m1) (ppm)

KNO3 0.8 N - 33.6
Ca(NO3) 2° 4H20 0.8 K 39.0
MgSO, ° 7H20 0.4 Ca 32.0
KH2PO4 0.2 Po - 6.2
S - 12.8

Mg - 9.6
Kc1* cl- 1.77
H3B0 3% B 0.27
MnSO,,°H207 Mn - 0.27
ZnSO 4° 7H207 Zn - 0.13
CuSO, *5H,07 Cu - 0.03
(NHy) 6Mo7024*4H20° Mo - 0.01
Fe*EDDHA Fe - 0.56

4Trace element stock solutions were prepared at 1000x the concentration
of the final solution. One ml of each stock solution was added to
each liter of the final culture medium.

28


a
°
1

a
fe)
T

ey
°
T

Nn
e]

6

mmoles Rb ABSORBED/gm DRY WT./20 MIN x10*
3
T

1 n n 1 1 n n 1 n
0! 0.2 03 0.4 0s 0.6 O07 0.8 0.9

EXTERNAL Rb CONCENTRATION (mM)

Figure 14. Two possible relationships between rate of rubidium
uptake and external RbCl concentration in excised roots of Elodea
occidentalis. The concentration of CaCl2 was 0.5 mM.

Figures 14 and 15 show the relationship between external Rb con-
centration and rate of Rb absorption in Elodea occidentalis roots
and shoots. The external Rb concentration varied from 0.002 to
1.0 mTM.

70-


Table 5, BIOASSAY FOR GROWTH-LIMITING NUTRIENTS IN SIX NORTHERN
WISCONSIN LAKES BY THE PROVISIONAL ALGAL ASSAY PROCEDURE

Algae growth as % of maximum
(N, P, Fe, Cu) with addition of

(-N) | (-P) | (-Cu)
Lake sampled | None | P,Fe | N,Fe | N,P,Fe | N,P,Fe,Cu | All essential
Little John 1.14 | 14.9 | 1.63 | 103.8 100.0 52.4
Clear 1.00 | 14.6 | 1.09 85.1 100.0 51.7
Whitney 1.08 | 43.1 | 1.75 55.9 100.0 59.3
Allequash 2.20 46.6 2.31 94.4 100.0 49.3
Salsich 1.02 38.2 1.79 69.1 100.0 85.8
Erickson 0.69 | 20.0 | 1.38 60.5 100.0 50.3

Most values are averages from analyses on water samples collected during
two sampling periods, July 20 and 21, and August 15 and 16, 1973.

Triplicate samples were run on each treatment of each water sample from
a sampling date.

The average dry weight yields of Selenastrum in the cultures represented
by a relative growth of 100.0 (N, P, Fe, and Cu added) were 32.5 mg/1
for Little John Lake, 35.0 for Clear Lake, 29.7 for Whitney, 26.8 for
Allequash, 24.6 for Salsich, and 36.2 for Erickson.

16


RESEARCH REPORTING SERIES

Research reports of the Office of Research and Development,

U.S. Environmental Protection Agency, have been grouped into
five series. These five broad categories were established to
facilitate further development and application of environmental
technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in
related fields. The five series are:

1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research

4. Environmental Monitoring

5. Socioeconomic Environmental Studies

This report has been assigned to the ECOLOGICAL RESEARCH STUDIES
series. This series describes research on the effects of pollution

on humans, plant and animal species, and materials. Problems

are assessed for their long- and short-term influences. Investigations
include formation, transport, and pathway studies to determine

the fate of pollutants and their effects. This work provides

the technical basis for setting standards to minimize undesirable
changes in living organisms in the aquatic, terrestrial and atmospheric
environments.

This report has been reviewed by the Office of Research and
Development, EPA, and approved for publication. Approval does
not signify that the contents necessarily reflect the views and
policies of the Environmental Protection Agency, nor does mention
of trade names or commercial products constitute endorsement or
recommendation for use.


ov

Table 14.

GROWTH AND TOTAL N CONTENT OF Elodea occidentalis AND Draparnaldia plumosa

WHEN COMPETING FOR A GROWTH-LIMITING SUPPLY OF N IN A MIXED CULTURE

Elodea Draparnaldia
NO3-N Yield (g) Prop. of | N content (%) Yield (g) Prop. of| N content (%) | Mixed culture
conc. yield yield. yield
(ppm) | Alone | +Drap. (%) Alone | +Drap. | Alone | +Elodea,| (%) Alone | +Elodea (g)
33.6 1.07] 0.80 64 4.44} 3.85 0.49) 0.45 36 6.83} 5.07 1.25
0.30] 0.37] 0.28 47 2.68; 1.88 0.41 | 0.32 53 1.70} 1.51 0.60

All yields are average oven-dry weights from triplicate cultures.

Table 15, GROWTH AND P CONTENT OF Elodea occidentalis AND Draparnaldia plumosa
WHEN COMPETING FOR A GROWTH-LIMITING SUPPLY OF P IN A MIXED CULTURE
Elodea Draparnaldia
P Yield (g) Prop. of | P content (%) Yield (g) Prop. of | P content (%) | Mixed culture
conc. yield yield yield
(ppm) | Alone | +Drap. (%) Alone | +Drap. | Alone | +Elodea (%) Alone | +Elodea (g)
6.2 1.71 1.32 89 0.72} 0.62 0.67} 0.16 11 1.30 1,29 1.48
0.075} 0.81] 0.56 54 0.21} 0.12 0.67] 0.47 46 0.18] 0.16 1.03



Results with the assay baskets were disappointing. Neither Elodea
brought from the laboratory as inoculum nor Elodea picked from local
populations grew well enough in the baskets to justify presenting the
limited analytical data obtained. Elodea from lake populations seemed
to grow somewhat better than laboratory-cultured plants. Problems
with this technique must be solved before it can be considered a
successful application of the plant analysis bioassay.

DISCUSSION

The point of primary interest in this study is the degree of agreement
in predictions of growth-limiting nutrients by the various procedures.
On this point, two general observations seem justified. First, there
was satisfactory agreement among the three bioassays in evaluating N
and P supplies. All three procedures indicated P was more likely to
become a growth-limiting nutrient in the lakes than was N. This was
true even though the PAAP test is based on water samples only while
the Fitzgerald tests and plant analysis involve Elodea which can
absorb nutrients both from water and mud substrates. The Fitzgerald
tests and plant analysis agreed in indicating that in all the lakes
Elodea was adequately supplied with N. The two tests also agreed in
indicating that P supply was least adequate for Elodea in Whitney Lake.

A second general point was that the conclusions from the bioassays
and chemical analyses of water samples did not agree. Chemical anal-
yses indicated N supplies would more readily become limiting than
would P; the bioassays indicated P would first be in the limiting
role. This difference could not be due only to the absorption by
Elodea nutrients from both water and bottom muds because the PAAP
test is a bioassay employing aliquots of the water samples used in
chemical analyses. The most obvious explanation is, of course, that
the base values against which the chemical analyses were evaluated
are not appropriate. Relatively small differences in the base values
in any of the tests could drastically modify the conclusions derived
from the tests.

24


Table 13. NITROGEN, PHOSPHORUS, AND POTASSIUM CONCENTRATIONS
(OVEN-DRY BASIS) IN SECOND ONE-INCH SEGMENTS OF
Ceratophyllum demersum SHOOTS COLLECTED FROM LAKE WINGRA

Site 1 Site 2

Date
Year sampled %N

oe
nw

%K %N %P

oe
~~

1971 July 26 3.27 0.32 2.49 -- -- --

Aug. 19 2.00 0.21 2.11 -- -- --

1972 June 15 3.19 0.36 3.57 -- -- --
June 29 3.71 0.50 2.70 -- -- --
July 27 3.05 0.29 2.01 -- -- --
Aug. 10 2.90 0.32 2.45 2.48 0.18 2.55

Aug. 31 2.55 0.36 3.10 1.88 0.16 1.38

1973 July 3 2.75 0.39 3.27 2.41 0.31 2.80

Aug. 6 2.51 0.39 2.37 2.02 0.18 2.14

that N and P supplies did not approach limiting levels. The lowest
N concentration was 1.88% (August 31, 1972), and the average was
2.67%. The lowest P concentration was 0.16% (August 31, 1972)
while the average concentration was 0.30%.

In contrast to the Myriophyllum data, the K concentration in Cerato-
phyllum -dropped below the 1.70% critical concentration in only one
sample. That was to 1.38% on August 31, 1972. The average K
concentration was 2.53%, or 1.48x the critical concentration. Even
though K was less limiting for Ceratophyllum than for Myriophyllun,

the analyses indicated K would more likely limit Ceratophyllum growth
than would N or P.

33


Fitzgerald Tests for Available N and P

The data in Table 7 are the results of bioassays for available N and

P in lake water samples by the Fitzgerald tests. One problem in

using the Fitzgerald assays is the lack of definite standards against
which to evaluate analyses of particular species collected from lakes.
In a publication describing the procedures (Fitzgerald, 1969), it is
suggested that, in general, uptake rates in excess of 15 ug NHy-N/10 mg
dry tissue/hr indicate N deficient plants. Extractable P0O,-P values

of 190 to 200 ug/100 mg dry tissue indicate plants adequately supplied
with P; values of 50 to 70 g/100 mg suggest P deficient plants. A
laboratory calibration trial with Elodea on this project indicated

the 50 to 70 yg/100 mg P values were slightly high for that organism.
Borderline values for Elodea of 25 to 30 ug/100 mg seemed more suitable.

Table 7. BIOASSAYS BY THE FITZGERALD TESTS FOR P (HOT WATER
EXTRACTABLE) AND N (NH,-N UPTAKE IN THE DARK) IN Elodea
occidentalis COLLECTED FROM NORTHERN WISCONSIN LAKES

P extracted NHy-N uptake
Lake sampled (ug/100 mg dry Elodea) (ug/hr/10 mg dry Elodea)
Little John 186 None
Clear 146 None
Whitney 94 None
Allequash 293 None
Salsich 193 None
Erickson 176 None

Values presented are averages for samples collected during two periods,
July 20-22, 1973, and August 15-19, 1973.

19


SECTION II

RECOMMENDATIONS

The development of plans for the control of nuisance plant growths in
lakes and streams optimally requires (1) knowledge of the qualitative
and quantitative nutritional requirements of the organisms involved,
(2) recognition of unusual nutritional requirements of specific
organisms, (3) knowledge of the capacities of the organisms to

compete in obtaining essential growth requirements from the environ-
ment, and (4) the availability of procedures for accurate evaluation of
nutrient supplies in natural waters. This project has contributed to
several of these requirements. The primary recommendations on the

use and further development of the information obtained are:

1, Plant analysis is a reliable bioassay and should be more widely
used in evaluating nutrient supplies and growth-limiting nutrients

in aquatic environments.

2. Plant analysis should be further developed and refined as an
assay procedure. This should include establishing critical concen-
trations of various elements in additional nuisance aquatic plants

and comparisons of plant analysis with other assay techniques.

3. The possibility that inorganic nutrients other than N and P can
be critical in nuisance plant growth should be more widely recognized

and tested for.

4. The techniques developed on this project for comparing aggressive-
ness and competitiveness in nutrient uptake at lake concentrations
should be applied to additional organisms, particularly major nuisance
aquatic plants. The data obtained should contribute to explanations
of the distribution of these plants in relation to lake fertility

and pollution.

5. For evaluating competitiveness in nutrient uptake, the two pro-
cedures from this project which reflect uptake that results in plant


cheesecloth, approximately 0.4 g samples of plant material were
weighed out and transferred to 60 ml of 0.5 mM CaCl2 solution (Epstein,
1961) contained in 100 ml beakers. The beakers were then placed in

a water bath maintained at 30°C for 15 minutes. Immediately before
being placed in experimental solutions, plant samples were rinsed
with two portions of 0.5 mM CaClz at 30°C. At the start of an
absorption period, the material was removed from the second rinse
solution and placed in experimental solutions, which contained 0.5 mM
CaClz and were suitably labeled with either °"P or °®Rb. Solutions
were continuously aerated during the uptake period. The ratio of
solution volume to time (100 ml per 10 minute absorption period) was
kept constant for all experiments (Andrew, 1966). The absorption
period used when external concentrations were varied was 20 minutes.

At the end of the absorption period, experimental solutions were
either sucked or poured off rapidly and the plant material was rinsed
five times with an unlabeled KH2P0,4 or KCl solution. The concentra-
tion of the rinse solution was at least ten times that of the labeled
experimental solution and contained 0.5 mM CaCl2. Plant material
remained in the final rinse for 15 minutes, was then placed in
aluminum bags and dried in a forced-draft oven at 60°C. After at

least 48 hours, the dry weight of the material was determined.

Plant material being assayed for 32p was ashed in a muffle furnace
for 6 hours at 600°C after being pretreated as outlined by Bertramson
(1942) for total P analysis. Material assayed for ®6Rb was ashed
for 4 hours at 500°C. The ash, in both cases, was dissolved in

2 N HCl and quantitatively transferred to 10 ml volumetric flasks.

Two aliquots of each sample were counted in a Packard Tri-Carb
liquid scintillation counter. Each P sample was counted for 1 minute
and each Rb sample for 10 minutes using the °H channel. No scintilla-

tion solution was used.

The procedures in experiments involving algae were identical to the
above except that green algae samples were removed from all solutions

by filtration and blue-green algae samples were separated from

55


8

100

mmoles Rb ABSORBED/gm DRY WT/20 MIN x 104

1 1 1 1 1 1 1 1 —L

O.l 0.2 0.3 0.4 0.5 06 0.7 08 09g
EXTERNAL Rb CONCENTRATION (mM)

Figure 15. Relationship of rate of rubidium uptake to external RbCl
concentration in excised shoots of Elodea occidentalis. The
concentration of CaCl2 was 0.5 mM.

When double-reciprocal plots of Rb absorption rate against external
concentration were made, as shown in Figures 16 and 17, for roots
and shoots of Elodea occidentalis the resulting line for the roots

was curvilinear; for the shoots, the line was straight. In unreported

71


In contrast to its effectiveness in N and P uptake, Draparnaldia

was very ineffective in competition for a limited K supply (Table 16).
“ At 0.39 ppm K, only 3% of the yield in mixed cultures was represented

by the alga. Under an adequate K supply, Draparnaldia did compete

quite effectively with Elodea producing 38% of the yield in a mixed

culture.

The analytical data also support Draparnaldia's inefficiency in K
uptake. The K critical concentration in Elodea has been established
as 0.80%; in Draparnaldia, as 2.40%. When grown alone with adequate
K, the K concentration in Draparnaldia was 4.23%, well above the
critical concentration. However, in competition with Elodea the K
concentration was slightly below the critical concentration at 2.02%.
Under growth-limiting K, the K concentration in Elodea remained
slightly above the critical concentration at 0.97% in competition
with Draparnaldia. However, the K concentration in Draparnaldia

was far below the critical concentration at only 1.08%.

The data in Table 17 show that the relative efficiency of the two
organisms in Ca uptake was the reverse of effectiveness in K uptake.
Elodea was completely ineffective in obtaining adequate Ca at 0.8 ppm.
In the mixed culture, only 5% of the yield was represented by Elodea.
Under adequate Ca, 69% of the yield was Elodea. The very poor perfor-
mance of Elodea under low Ca seems due to a general inability to
absorb Ca at low concentrations. Even when grown alone, there was
almost no growth at 0.8 ppm Ca. In contrast, in the previous
experiment Draparnaldia grew very well at 0.39 ppm K in the absence
of competition from Elodea.

The results on competition for Mg are quite comparable to the N and

P data (Table 18). At the growth-limiting supply of Mg (0.2 ppm),
yields of the two organisms were about equal (59% and 41% for Elodea
and Draparnaldia, respectively). The increase in the proportion of
the yield represented by Draparnaldia between the high and low Mg
supply (15 to 41%) suggests Draparnaldia is somewhat more competitive.
The Mg concentration in Elodea grown in competition for 0.2 ppm Mg

41


£y

Table 18. GROWTH AND Mg CONTENT OF Elodea occidentalis AND Draparnaldia plumosa
WHEN COMPETING FOR A GROWTH-LIMITING SUPPLY OF Mg IN: A MIXED CULTURE
Elodea Draparnaldia
Yield (g) Mg content (%)} Yield (g) Mg content (%)
Mg Prop. of Prop. of Mixed culture
conc. yield yield yield
(ppm) | Alone | +Drap. (%) Alone | +Drap. }Alone |+Elodea (%) Alone | +Elodea (g)
9.6 2.81] 2.91 85 0.69) 0.63 1.62 | 0.50 15 0.20; 0.19 3.41
0.2 1.93} 1.09 59 0.13} 0.13 1.45 | 0.77 41 0.11} 0.11 1.86



SECTION IV
CRITICAL CONCENTRATIONS OF ESSENTIAL

ELEMENTS IN VARIOUS AQUATIC PLANTS

Plant analysis is a technique for evaluating inorganic nutrient avail-
ability and growth-limiting nutrients in the environment from the con-
centrations of those nutrients in plants. The key point in applying
the plant analysis technique is to establish the critical concentration
of each element of interest in the species under study. The critical
concentration, usually established in laboratory experiments, is the
minimum concentration of an element, or more often slightly less than
the minimum concentration, in a plant which will permit maximum plant
yield and growth. Often critical concentrations are determined in
index segments rather than entire plants. Nutrient concentrations in
index segments more accurately reflect environmental availability of
an element than do nutrient concentrations in the entire plant.

If the concentration of an element in a field sample of an aquatic
plant, or in the index segment, is above the established critical con-
centration, the plant was adequately supplied with the element in the
environment from which it was collected. If the concentration is below
the critical level, supply of that element had become limiting for
optimum plant growth.

Development and application of the plant analysis technique in assaying
nutrient supplies in aquatic environments has been a primary interest
of the Principal Investigator for a number of years. Examples of the
use of plant analysis and discussions of possible advantages of that
technique over other assay procedures, such as chemical analysis of
water samples, have been presented by Gerloff and Krombholz (1966) ;
Gerloff (1969, 1973); and Gerloff and Fishbeck (1973). A number of
critical concentrations were established in algae and macrophytes on
this project and will be reported. Because of possible use in pollu-
tion control efforts, it also seemed desirable to summarize all the