EPA-R1-73-001

                      Environmental Health Effects Research Series
rebruary 1973
Plant Analysis for Nutrient

Assay of Natural Waters
                                    Office of Research and Monitoring



                                    U.S. Environmental Protection Agency


                                    Washington, D.C. 20460

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            RESEARCH REPORTING SERIES
Research reports of the  Office  of  Research  and
Monitoring,  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 ENVIRONMENTAL
HEALTH  EFFECTS  RESEARCH  series.   This   series
describes  projects  and  studies  relating to the
tolerances of man for  unhealthful  substances  or
conditions.   This work is generally assessed from
a medical viewpoint,  including  physiological  or
psychological  studies.  In addition to toxicology
and  other  medical  specialities,   study   areas
include   biomedical  instrumentation  and  health
research techniques utilizing animals - but always
with  intended   application   to   human   health
measures.

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                                                          EPA-R1-73-001
                                                          February 1973
               PLANT ANALYSIS FOR NUTRIENT

                 ASSAY  OF NATURAL WATERS
                            By

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

                    Project 18040 DGI

                     Project Officer

                     Dr. Gary Glass
           National Water Quality Laboratory
                   6201 Congdon Blvd,
                 Duluth, Minnesota 55804


                      Prepared for

           OFFICE OF RESEARCH AND MONITORING
         U.S.  ENVIRONMENTAL PROTECTION AGENCY
                 WASHINGTON,  D.C. 20460
For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20402
            Price 95 cents domestic postpaid or 70 cents GPO Bookstore

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                   EPA Review Notice

This report has been reviewed by the Environmental
Protection Agency 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,

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                       ABSTRACT

Plant analysis was developed as a relatively simple
procedure for evaluating nutrient supplies and growth-
limiting nutrients for nuisance macrophytes in lakes and
streams.  Plant analysis requires establishing in index
segments of the macrophytes the critical concentration
(minimum plant concentration for maximum yield) of each
essential nutrient likely to limit growth.  Critical
concentrations for nitrogen, phosphorus, sulfur, calcium,
magnesium, potassium, iron, manganese, zinc, boron, and
molyldenum were established in appropriate index segments
of Elodea occidentalis.  The copper critical concentration
was estimated.  Critical concentrations for nitrogen,
phosphorus, and several other elements were established in
Ceratophyllum demursum.

To evaluate plant analysis, samples of Elodea and Cerato-
phyllum were routinely collected from Wisconsin lakes,
analyzed for essential nutrients, and the analyses were
compared with the critical concentrations for indications
of nutrient deficiency.  A growth-limiting role of an
element in a lake was indicated by plant concentrations
below the critical level.  Nitrogen, phosphorus, calcium,
and copper were at or close to critical levels in one or
more lakes.  Neither phosphorus nor nitrogen seemed to be
a general growth-limiting nutrient in the lakes sampled.
The most unexpected result was an indication of copper
deficiency in several lakes.

From the extensive nutritional experiments to establish
critical element concentrations, a synthetic nutrient
medium for general macrophyte culture was developed.

This report was submitted in fulfillment of Project No.
18040 DGI under the sponsorship of the Water Quality
Office, Environmental Protection Agency.
                          111

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                       CONTENTS

Section                                                Page

    I   Conclusions                                      1

   II   Recommendations                                  3

  III   Introduction                                     5

   IV   Development of Plant Analysis as a Nutrient
        Assay Technique                                  9

    V   Assay of Nutrients in Lakes by Plant Analysis   25

   VI   Development of Macrophyte Nutrient Culture
        Solution                                        39

  VII   Acknowledgments                                 51

 VIII   References                                      53

   IX   Appendix                                        57
                           v

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                       FIGURES

                                                      Page
                                                      	 -* —

1    Relationship between plant growth and the
     concentration of an essential element in the
     plant                                              6

2    Yield of Elodea as related to total nitrogen
     concentrations in index segments                  15

3    Growth of Elodea as length increase under
     adequate and inadequate nitrogen supply           17

4    Effect of light intensity on Elodea growth        48

5    Effect of temperature on Elodea growth            49
                       VI

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                        TABLES

No.                                                    Page

 1    Composition of macrophyte nutrient medium         ID

 2    Yield of and total nitrogen concentration in
      index segments of Elodea grown in solutions
      differing in nitrogen content                     14

 3    Total nitrogen concentration in nitrogen-
      deficient Elodea segments                         17

 4    Yield and total phosphorus concentration in
      index segments of Elodea grown at various
      phosphorus concentrations                         19

 5    Phosphorus concentration in phosphorus-deficient
      Elodea segments                                   20

 6    Critical concentrations of essential elements
      in index segments of Elodea                       21

 7    Critical concentrations of essential elements
      in index segments of C era t ophy Hum                22

 8    Essential element concentrations in Elodea
      from Little John Lake                             28

 9    Essential element concentrations in Elodea
      from Salsich Lake                                 29

10    Concentrations of growth-limiting elements in
      Elodea from Wisconsin lakes sampled in late
      July                                              32

11    Essential element concentrations in Ceratophyllum
      from Lake Mendota                                 33

12    Nitrogen and phosphorus concentrations in
      macrophytes from nutrient assay baskets           35

13    Nutrient solution recommended for macrophyte
      culture                                           41

14    Toxicity of copper to Ceratophyllum               43
                           VII

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15    Comparison of iron sources for the growth of
      Ceratophyllum                                     44

16    Comparison of macrophyte growth in various
      nutrient solutions                                46
                         Vlll

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                       SECTION I

                      CONCLUSIONS

The primary conclusions from studies on this project
can be summarized as follows:

1.  Plant analysis is a relatively simple and useful
    procedure for evaluating nutrient supplies and growth-
    limiting nutrients for nuisance macrophytes, and
    probably for other obnoxious plants as well.

2.  Plant analysis is most reliable as a diagnostic
    technique when based on the index segments established
    in these studies  (the first and second one-inch
    segments of stems and laterals) rather than on entire
    plants.

3.  Establishing critical concentrations for most of the
    essential elements in Elodea occidentalis makes it
    possible to use this species as a general bioassay
    organism in evaluating whether any of these elements
    become  growth-limiting in lakes or streams.

4.  Evaluations of nutrient supplies in  Wisconsin lakes
    indicated neither nitrogen nor phosphorus was a
    general limiting nutrient in the lakes sampled.

5.  The element most likely to limit macrophyte growth
    varied in different lakes.  Evidence was obtained that
    nitrogen, phosphorus, calcium, and copper were limiting,
    or were close to growth-limiting, in different lakes.

6.  Although confirming evidence is required, the results
    indicated copper deficiency is common in soft-water,
    infertile northern Wisconsin lakes.

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                      SECTION II

                    RECOMMENDATIONS

Simple, reliable techniques are urgently needed for nutrient
assay in lakes and streams.  This project was an initial
effort to develop and apply plant analysis as a nutrient
assay.  The results suggest plant analysis is a promising
technique.  However, additional work is recommended to
verify this promise and to further refine the technique.

Critical concentrations and index segments should be
established in macrophytes other than the two species used
in the work on this project.  It would be desirable to
know if the critical concentrations for Elodea occidentalis
and Ceratophylum demursum were generally applicable to
macrophytes.  It also seems desirable to develop plant
analysis using aquatic organisms other than macrophytes.
A filamentous, green alga which is responsible for
nuisance conditions seems a suitable possibility.

Determining whether analysis for the total concentration
of an element or for an extracted fraction more reliably
correlates with yield also would be desirable.  In
agricultural applications, for some elements and in some
species extracted fractions have been found more reliable
than total concentrations.

Further evaluations of the plant analysis technique should
be made using the organisms and critical concentrations
established on this project.  This would involve additional
samplings of Elodea and Geratophylum from Wisconsin lakes
differing in fertility.  Further tests with the assay
species isolated in floating, porous baskets and sampled
through the summer, as initiated on this project, also
seem highly desirable.  An effort should be made to obtain
samples of natural populations of macrophytes from lakes
in other parts of the United States in which deficiencies
of specific elements have been indicated.  Analyses would
be compared with critical concentrations established on
this project to verify the suggested deficiencies and the
usefulness of the plant analysis technique.

Results on this project suggested macrophytes in some
northern Wisconsin lakes are copper deficient.  Copper
becomes growth limiting.  This must be verified.  If
true, the copper requirements of other nuisance aquatic
plants should be studied.

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Plant analysis is one of several techniques suggested or
in use for nutrient assay in natural waters.  This seems
a highly appropriate time to evaluate these various tech-
niques and to compare them with plant analysis.  Chemical
analyses of water samples/ 1*C uptake following nutrient
enrichment, the Provisional Algal Assay Procedure, and
enzyme assays such as the measure of phosphorus deficiency
in plants by phosphatase activity are among the techniques
that should be compared.

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                      SECTION III

                     INTRODUCTION

Aquatic plants, both algae and angiosperms  (macrophytes),
are responsible for nuisance conditions when excessive
growths of the organisms develop in polluted lakes and
streams.  One approach to the control of these undesir-
able growths is to reduce supplies of an essential
nutrient element to growth-limiting levels.

Considerable evidence indicates that nitrogen and phos-
phorus are most likely to become limiting for plant
growth in aquatic environments, and most investigators
favor phosphorus as the primary limiting nutrient.
However, evidence obtained by Gerloff and Skoog  (1957)
suggested that nitrogen rather than phosphorus limited
growth of the blue-green alga Microcystis aeruginosa in
several southern Wisconsin lakes.  In a recent report,
 (Ryther and Dunstan, 1971) nitrogen was considered to
become limiting for algae in coastal marine waters of
northeastern United States.  Data obtained by Goldman  (1960,
1964) indicated that in some lakes trace element supplies
limit aquatic plant growth.

Reliable assays of nutrient supplies in lakes and streams
relative to plant needs would be highly useful to engineers,
water chemists, biologists, and others who must predict
and assess the effectiveness of suggested pollution control
measures.  Various procedures have been proposed and used
for nutrient assay, for example chemical analyses of water
samples and enrichment bioassays involving growth or
photosynthesis of aquatic organisms following additions
to water samples of nutrient elements that might be
growth limiting  (Gerloff, 1969).  There are problems
associated with both approaches and neither has been
developed to a degree that has led to acceptance as a
general diagnostic procedure.  For example, it is difficult
to interpret water analyses in terms of concentrations at
which specific elements become growth limiting  (Lee, 1969;
Lund, 1969).  Enrichment assays, in addition, are subject
to errors associated with extending data obtained with
isolated samples to evaluations of nutrient supplies in
large bodies of water  (Gerloff, 1969).  As an alternative,
a bioassay based on plant rather than water samples has
been developed  (Fitzgerald, 1969).  The amount of ortho-
phosphate extracted from plant samples with boiling water
and the uptake of NEU-N in the dark correlate with the

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adequacy of phosphorus and nitrogen  nutrition,  respectively/
of the plants sampled.

Plant or tissue analysis is a technique  for  nutrient assay
that has found widespread application  in assessing the
nutrient status of soils for the production  of  agricultural
and horticultural crops  (Bould, et al.,  1960; Reuther, 1961;
Smith, 1962; Chapman, 1966).  Plant  analysis is based on
the observation that the concentration of any essential
element in a plant can vary over a considerable range,
and that a primary factor determining  the concentration
in a healthy plant is the availability of the element in
the environment.  The essential relations on which tissue
analysis is based are shown in Figure  1  (Ulrich, 1961).
    Ul
    >
                              ADEQUATE  ZONE
                   CRITICAL CONCENTRATION
              DEFICIENT
                ZONE
                  CONCENTRATION  IN  TISSUE
Figure  1.   Diagram of the relationship between plant yield and
   the concentration of an essential element in specific plant
   parts (From Ulrich, 1961).

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The nearly vertical part of the hypothetical curve is
termed the "Deficient Zone"; here, plant yield is in-
creasing markedly, but the concentration in the organism
is changing very little.  In the horizontal part of the
curve, organism concentration of the element is increasing,
but yield is not; this is the "Adequate Zone", or, more
commonly, the "Zone of Luxury Consumption".  The "Transition
Zone" is that part of the curve between the zones of
deficiency and adequacy.  Successful application of tissue
analysis depends on establishing for a species the critical
concentration for each element of interest.  The critical
concentration is that content which is just inadequate
for maximum growth.

Application of the plant analysis technique in evaluating
nutrient supplies for aquatic plants would require
establishing in laboratory experiments the critical
concentration for each potentially growth-limiting essential
element in the plant species of interest.  The same species
then would be collected from lakes and streams, analyzed
for various elements, and the concentrations compared with
the critical levels.  If a plant from the field contains
less than the critical concentration of an element, the
supply of that element was limiting growth in the environ-
ment from which the plant was collected.  More growth would
result if greater amounts of the nutrient could be absorbed.

The plant analysis technique seems particularly applicable
to evaluating nutrient supplies in aquatic environments
because of the difficulties in obtaining representative
water samples and in interpreting concentrations of
elements in terms of potential for plant growth.  In
plant analysis, aquatic plants become the sampling devices
and the concentration of an element in plants reflects
all the factors which influenced availability of that
element in the environment from which the plants were
collected.

The work to be reported was concerned with  (1) development
of plant analysis as a simple procedure for evaluating
nutrient supplies in lakes and streams,  (2) testing of the
plant analysis technique in nutrient evaluation in
representative Wisconsin lakes, and  (3) development,
through laboratory studies, of an optimum culture medium
and general growth conditions for the culture of macro-
phytes.  The experimental work on this project will be
presented in three sections corresponding to the objectives
mentioned above.

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                      SECTION IV

     DEVELOPMENT OF PLANT ANALYSIS AS AN ASSAY OF

NUTRIENT SUPPLIES FOR THE. GROWTH OF NUISANCE MACROPHYTES

Development of plant analysis for nutrient assay in lakes
and streams required that the critical concentrations of
all of the essential elements likely to limit plant growth
would be established in  laboratory experiments for the
macrophytes on which the assay was to be based.  Elodea
6 cc identalis and CeratophyTlum demursum were selected as
the primary test organisms.  Both species are abundant
and troublesome in Wisconsin lakes.  There is a marked
morphological difference in the two organisms in that
Elodea produces abundant roots while Ceratophyllum
does not.  In the time available, critical concentrations
for all the essential elements except chlorine and copper
were established in Elodea occidentalis and for a number
of the elements in Ceratophy11urn demursum.


                EXPERIMENTAL PROCEDURES

The composition of the nutrient medium used in the critical
concentration experiments is indicated in Table 1.  This
is the same culture medium employed in earlier studies
 (Gerloff and Krombholtz, 1966) except that iron  (0.56 ppm)
was provided as a chelate of EDDHA  (ethylenediamine di- (a-
hydroxyphenylacetate) rather than as FeEDTA.  Algae-free
cultures" were essential.  Otherwise, the abundant algae
growth which developed made it impossible to interpret the
results in terms of the  nutritional requirements of the macro-
phytes.  The procedure for obtaining algae-free macrophytes
also was described earlier  (Gerloff and Krombholtz, 1966).

The most convenient culture vessels were three-liter Florence
flasks containing two liters of nutrient medium.  The flasks
were stoppered and closed except for aeration and exhaust
tubes provided with cotton filters in glass-tubing that
passed through the stoppers.  Each complete assembly was
sterilized by autoclaving.  The cultures were bubbled con-
tinuously with air filtered through cotton and activated
charcoal and then enriched to 1% CO2.  All cultures were
kept in a constant environment room or growth chambers
maintained at approximately 23°C and provided with artificial
light of 800-1700 foot candle intensity.  The cultures  in a

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Table 1.  Composition of a modified Hoagland's solution used
          for the culture of angiosperm aquatic plants.
      Salt
                    Cone.
   0.5 M
soln. in 1 1
final soln.
     (ml).
                                            Cone, in final
                                            solution (ppm)
KNO3

Ca(NO3)2'4H2O
                    0.5 M
KC1

H3B03

MnSGU-H20
(NH4)6M07024-4H20

FeEDDHA
   2.0

   2.0

   0.8

   0.4
                      **
N  - 42

K  - 47

Ca - 40    S - 12.8

P  -  6.2Mg-  9.6

Cl -  1.77

B  -  0.27

Mn -  0.27

Zn -  0.13

Cu -  0.03

Mo -  0.01

Fe -  0.56
 *  Trace element stock solutions were prepared at 1,00OX
    the concentration of the final solution.  One ml of each
    stock solution was added to each liter of the final
    culture medium.

**  Iron was provided as a chelate of ethylene di(o-hydroxy-
    phenylacetate); 0.56 ppm of Fe  was added when the cul-
    ture medium was prepared and 0.28 ppm 1-2 weeks into the
    culture period.
                          10

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particular experiment were inoculated with small sections,
approximately 2 inches in length, of the appropriate species
removed from continuously maintained stock cultures.
Culture periods of 4-5 weeks were necessary to obtain the
yields reported.

To develop macrophytes deficient in the essential trace
elements  (Mn, Zn, B, Cu, and Mo) it was necessary to use
standard procedures for reducing environmental contamina-
tion, that is in acid-washing culture and media containers,
double-distilling water used to prepare culture solutions,
and in purifying culture media salts  (Hewitt, 1966; Stout
and Arnon, 1939).

Inorganic analyses were by quantitative procedures in
general use for plant analysis.  Total nitrogen was
determined by a semi-micro Kjeldahl procedure.  Phosphorus
determinations were by a vanado-molybdate yellow-complex
procedure following dry ashing of oven-dried plant material
at 550°C  (Jackson, 1958).  Potassium analysis was by emission
flame photometry of 1 N ammonium acetate extracts of tissue
samples.  Tissues were"~prepared for iron analysis by a
combined wet-dry ashing procedure which permits ashing at
low temperatures.  Iron then was determined as a complex
with o-phenanthroline.  Following dry ashing and acid
solution of the residues, calcium, magnesium, zinc, manganese
and copper were determined by atomic absorption using a
Jarrell-Ash instrument.  Boron was determined as a curcumin
complex  (Johnson and Ulrich, 1959) and molybdenum as a
complex with thiocyanate following stannous reduction
 (Johnson and Arkeley, 1954).  Both analyses were on dry-
ashed tissues.  Analyses for sulfur were by turbidimetric
measurements of BaSCK precipitated in HNOa-HCICK digests of
plant tissues  (Blanchar, Rehm, and Caldwell, 1965).

To minimize contamination, samples analyzed for trace
elements and iron were ground with an agate mortar and
pestle.  When sample size permitted, analyses for the major
essential elements were on plant material ground in a
Wiley Mill equipped with a stainless steel screen.  Other-
wise the samples again were ground with a mortar and pestle.
In the initial work on this project, critical concentra-
tions were established and evaluated from analyses of
entire plants.  As in agricultural applications, this proved
unsatisfactory, particularly in the collection and analysis
of plants from lakes in which nutrient supplies were being
evaluated.  Large portions of plants too often were
unhealthy for reasons other than nutrient deficiency;
reduced light for example, and were low in nutrient

                          11

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concentrations as a result.  This could give a false
indication that a specific element was growth limiting.
Therefore, the critical concentration of each element was
established in a plant part (index segment) which more
accurately reflected environmental supply than did the
entire plant.

Two procedures were employed in determining the critical
concentration of nitrogen and phosphorus.  One approach
was similar to the procedure routinely used in agricul-
tural applications of plant analysis.  The plants were
grown in nutrient cultures similar in all respects except
for the concentration of the element under investigation.
Concentrations of that element varied from suboptimal to
above optimal.  The plants were harvested after a culture
period of approximately four weeks,  when ranges of growth
and of element concentration in the plants were represented-
All treatments were at least in duplicate and often in
triplicate.

To establish the most suitable index segment/ at harvest
plant material from each culture was divided into three
or four segments:  the terminal one inch of growth on
main branches and laterals, the second inch of growth,
the third inch of growth (in some cases), and the remainder
of the plants.  Samples were oven-dried to constant
weight at 65-70°C, and analyzed for the element under
study.  A critical concentration was established from
curves relating average oven-dry yield and tissue content
of the element under study in the plant segments from
each treatment of an experiment.  The first one-inch
portion cut from all main shoots and laterals was found
to be the most suitable index segment for elements which
are relatively immobile in plants, that is, they are not
re-exported from older to younger tissues.  The second
one-inch was the index segment for mobile elements.

A second procedure was developed for establishing critical
concentrations in Elodea.  Individual plants were grown in
34 x 20 x 5 cm Pyrex trays containing 1200 ml of the •
appropriate nutrient medium.  Each tray was covered with a
glass plate which was sealed to the tray with Apiezon
sealing compound  (Associated Electrical Industries, Ltd.)
after the culture assembly had been autoclaved and inoculated.
This was necessary to minimize algae contamination.  Two
                          12

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holes were drilled in each glass cover.  Surgical rubber
tubing inserted through one hole brought air  (filtered and
1.0 percent C02 enriched) into the culture.  Four capillary
air outlets provided vigorous aeration of the nutrient
medium.  The second hole was plugged with cotton and
permitted air 'to escape.  The inoculum for a tray was a
1-1/2 - 2 inch terminal segment of an Elodea main shoot
or lateral.

In a specific experiment, some trays contained the com-
plete medium; others contained amounts of nitrogen or phos-
phorus which would support a maximum rate of growth only
for a limited period.  Plant growth in the trays was followed
by daily measurements of the total length of the plants,
both main and lateral shoots.  These measurements were made
without removing the cover from a tray.  From continuous
plots of growth in the deficient and the complete cultures,
the point at which the nitrogen or phosphorus content of
plants in the deficient cultures had been sufficiently
reduced to affect the rate of growth could be determined.
At this point, plants in all trays were harvested, divided
into various segments, oven-dried, and analyzed.
                        RESULTS

Critical Nitrogen Concentration in Elodea

The data in Table 2 and the curves in Figure 2 are from an
experiment to establish the critical level of nitrogen in
the most suitable index segment of Elodea occidentalis.
The data on total plant yields in Table 2 show that growth
was limited by the nitrogen supply in solutions containing
10.5 and 14.0 ppm nitrogen and reached a maximum of 2.34 g
at 21.0 ppm nitrogen.  The large increase in yield between
14.0 and 21.0 ppm suggests that the critical concentration
is approximately the amount of nitrogen in plants of the
21.0 ppm nitrogen cultures.  The nitrogen contents of
plants from the 31.5 and 42.0 ppm treatments represent
luxury consumption of nitrogen, that is, increasing
nitrogen content which did not result in further yield
increases.

The second one-inch segment of the main shoots and laterals
was selected as the most satisfactory index part for nitro-
gen.  The possible re-export of nitrogen from older to
younger tissues under conditions of nitrogen deficiency,

                           13

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Table 2.  The total nitrogen content of the first and second
          one-inch sections of stems and branches of Elodea
          o cc i denta1is after culturing in solutions
          differing in nitrogen content to establish the
          critical nitrogen concentration in an index
          segment.
            Oven-dry plant wt.
                 (g/2 1)
Tissue N content
N content
of medium
/ /^ \ Jr XCI.I.L I"
"' 1 2 Ave. segment
10.5 1.583 1.575 1.579 1st
2nd
3rd
14.0 1.694 1.928 1.833 1st
2nd
3rd
21.0 2.428 2.252 2.340 1st
2nd
3rd
31.5 1.933 2.371 2.152 1st
2nd
3rd
42.0 1.861 1.931 1.896 1st
2nd
3rd
1"
1"
1"
1"
1"
1"
1"
1"
1"
1"
1"
1"
1"
1"
1"
1
1.
1.
1.
2.
1.
1.
2.
1.
1.
4.
3.
2.
5.
4.
3.

45
14
07
04
52
37
13
63
36
10
19
54
56
38
45
2
1.
1.
1.
1.
1.
1.
2.
1.
1.
3.
2.
2.
5.
4.
3.
Ave.
49
15
09
75
35
09
05
55
41
21
45
07
35
26
31
1.
1.
1.
1.
1.
1.
2.
1.
1.
3.
2.
2.
5.
4.
3.
47
14
08
89
43
23
09
59
39
67
82
31
45
32
38
                          14

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   <\J
      2.5
    I
    C
    o>
    >  1.5
    O
                                                    l" segment
                                     2nd I" segment
               1.0
2.0
       3.0
4.0
5.0
6.0
                       Tissue content  of  N (%)
Figure 2.  The relationship  between yield and total nitrogen
  content of the first  and second  one-inch terminal segments
  of Elodea occidentalis  grown  in  solutions of varying
  nitrogen content.
and  also  as  a result of senescence and non-nutritional
abnormalities in the older tissues made the terminal one-
inch segment an unsatisfactory index region.  This was
supported by unreported results which showed that in plants
which had been very deficient in nitrogen for a consider-
able period, the nitrogen content of terminal one-inch
segments  was actually above the critical level, apparently
due  to re-export from extensive regions of older, dying
tissues.
                          15

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A relatively constant concentration of an element through-
out the range of yield response to that element is a
desirable feature of an index region and was another
factor considered in the selection of the second one-
inch segment for nitrogen assay.  The nitrogen content
of the third one-inch segment also varied only slightly
(Table 2).  However, this segment was rejected as an index
region because a large proportion of the lateral brances
on Elodea occidental!s were less than three inches in length.
In the collection of field samples, it would probably be
difficult to obtain sufficient tissue for analysis from
the third one-inch segment.

On the basis of the above considerations, the critical
nitrogen concentration for Elodea occidental!s was esta-
blished as 1.60 percent in the second one-inch segment.
Yield and nitrogen values for duplicate cultures are
included in Table 2 to indicate the variation between
replicates in experiments of this stype.  The nitrogen
concentration in the second one inch varied over a consider-
able range, from 1.14 to 4.32 percent.

The results in Table 3 and Figure 3 are from an experi-
ment to establish the critical concentration of nitrogen
for Elodea occidentalis using the tray procedure.  In
one set of trays, the N03-N concentration in the nutrient
medium was 42 ppm; in another set, the concentration was
only 4.2 ppm.  The total nitrogen concentration in the
second one-inch segment of nitrogen deficient plants from
the low nitrogen trays at harvest was 1.23 percent.  This
is slightly below the 1.60 percent critical concentra-
tion established by the batch procedure.  However, as
indicated in Figure 3, the Elodea plants probably were
deficient in nitrogen several days before a decision to
harvest the plants seemed justified.  The dry weight of
the low nitrogen culture plants was only 393 mg per tray
in comparison with 504 mg under high nitrogen.  The 14.0
ppm external nitrogen treatment in Table 2 represents a
comparable relative yield decrease due to nitrogen
deficiency.  The nitrogen concentration in the second
one-inch segment from that treatment was 1.43 percent
which is in reasonable agreement with the 1.23 percent
value from the tray experiment.
                           16

-------
Table  3.   The total nitrogen content of various segments of
           Elodea occidentalis harvested when nitrogen
           deficiency had  reduced the rate of growth as
           indicated by  length increase.

Nfin"n •§- f*Y\ 4~
of medium
(mg/1)
4.2
42.0

PI +• -t-
at harvest
(mg)
393
504
Tissue

1st
1"
1.61
5.85
content
/ Q,' \
\ "O /
2nd
1"
1.23
4.34
of N

Remainder
of plant
1.69
3.01
           120
           100
         E
         o
         o
         o>
         0)
         •o
         o
            80
            60
         _  40


         I

            20
                                           Plants harvested
                                        '— 42.0 mg N/L
                                             Plants harvested
                                         — 4.2 mg N/L
                               12
16
20
                          Days  of growth
Figure  3.   Daily increase  in total length of  stems and
  lateral  branches of Elodea occidentalis grown in tray
  cultures under high and  low levels of

                            17

-------
Critical Phosphorus Concentration in Elodea

Yield and tissue phosphorus concentrations of Elodea
grown in nutrient media varying in phosphorus content are
presented in Table 4.  As with nitrogen, the youngest
tissues, that is the terminal one-inch of growth, had the
highest phosphorus concentration.  This was anticipated
because both phosphorus and nitrogen are readily re-
exported from younger to older tissues.  The second one-
inch segment was considered the most satisfactory index
segment in which to establish the critical phosphorus
concentration.

i:ry-weight yields did not level off at a constant maximum
as sharply as they did in the nitrogen experiments.  There-
fore, it was more difficult to establish the critical
phosphorus content with certainty.  When data of Table 4
were graphed, there was a marked decrease in the yield
response per unit of phosphorus made available to the
plants at tissue concentrations above approximately 0.14
percent phosphorus in the second one-inch segment.  This
was considered the critical phosphorus concentration in
that segment.  The 0.14% value is supported by the data in
Table 5 obtained from an experiment in which the critical
level of phosphorus was established using the tray
procedure.  At the time of harvest, the plants 'were
severely phosphorus deficient as indicated by the small
amount of growth in the low phosphorus treatment.  The
phosphorus concentration in the index tissue of these
severely deficient plants, 0.12 percent, was slightly
lower than the comparable value obtained with the batch
procedure.  In an earlier study  (Gerloff and Krombholz,
1966), the critical phosphorus content for Elodea occidentalis
on a whole plant basis was 0.14 percent.  It is not sur-
prising that the values based on the entire plant and the
second one-inch segment should be the same, because
analysis of entire plants represents an average of younger
tissues with a higher phosphorus concentration than the
second one-inch segment and older tissues with a lower
concentration.
                           18

-------
Table 4.  The total phosphorus content of various portions
          of Elodea occidentails after culturing in solutions
          differing in phosphorus content to establish the
          critical phosphorus concentration in an index
          segment.
P content Ave. oven-dry
of medium plant wt.* Plant
(mg/1) (g/21) segment
0.76 1.383 1st 1"
2nd 1"
3rd 1"
Remainder
1.16 1.730 1st 1"
2nd 1"
3rd 1"
Remainder
1.55 1.984 1st 1"
2nd 1"
3rd 1"
Remainder
3.1 2.329 1st 1"
2nd 1"
3rd 1"
Remainder
6.2 2.788 1st 1"
2nd 1"
3rd 1"
Remainder
Tissue
P content
(%)
0.10
0.06
0.06
0.09
0.14
0.08
0.07
0.10
0.16
0.10
0.11
O.il
0.25
0.18
0.17
0.17
0.39
0.35
0.33
0.31
 *  Averages of duplicate cultures,
                          19

-------
Table 5.  The total phosphorus content of various segments
          of Elodea bccldentalis harvested when phosphorus
          deficiency had reduced the rate of growth as
          indicated by length increase.
                               .Tis.s.ue. content of. P, .(.%.)
C (M-VJU L.CI1L.
of medium
(mg/1)


0.2
6.2
e JLO.U \- wu.
at harvest
(mg)
142
625.
1st
1"
0.12
0.95
2nd
1"
0.12
0.75
3rd
1"
0.09
0.71
Remainder
of plant
*
0.78
   Insufficient tissue for analysis
    Critical Concentrations of Additional Elements

The results of experiments to establish the critical
concentrations of elements other than nitrogen and phos-
phorus in index segments of Elodea are summarized in
Table 6.  As anticipated/ the critical concentrations for
the trace elements were much lower than for the major
essential elements.  Values ranged from 8 ppm for zinc
to only 0.15 ppm for molybdenum.  The total range of
concentration of each element also is indicated in Table
6.  It is apparent that the concentrations of the essential
elements in Elodea are not fixed values but vary over a
considerable range.

Critical concentrations were not established for the trace
elements chlorine and copper.  Because of the low plant
requirements for chlorine and the large quantities in
rainfall as a result of atmospheric contamination, it is
unlikely chlorine would be a limiting factor in macro-
phyte growth under field conditions.  This is not true of
copper.  As established in Section V of this report,
there is a strong possibility that copper supply is
growth limiting in some northern Wisconsin lakes.
Unfortunately, in spite of elaborate purification efforts,
several experiments to establish a critical copper con-
centration in Elodea were unsuccessful.
                          20

-------
Table 6.  Critical concentrations and the range of
          concentrations of essential nutrient
          elements in index segments of Elodea bccidentalis

Element
N
P
S
Ca
Mg
K
Fe
Mn
Zn
Mo
B
Index
segment
2nd 1"
2nd 1"
2nd 1"
1st 1"
2nd 1"
2nd 1"
1st 1"
1st 1"
2nd 1"
1st 1"
1st 1"
Critical
cone.
1.60%
0.14%
0.08%
0.28%
0.10%
0.80%
60 ppm
4 . 0 ppm
8 . 0 ppm
0 . 15 ppm
1 . 3 ppm
Range in
cone.
1.14- 4.32%
0.06-0.35%
0.06-0.46%
0.17-0.62%
0.06-0.19%
0.25-2.51%
40-219 ppm
2.2 -16.7 ppm
3.6 -34.4 ppm
0.04-6.4 ppm
0.3 -11.2 ppm
       Critical Concentrations in Ceratophyllum

Although Elodea occidental!s was selected as the primary
organism on which to base the plant analysis assay, the
critical concentrations of nitrogen, phosphorus, and
several other elements were established in Ceratophyllum
demursum.  These are presented in Table 7.

The critical nitrogen concentration in the second one-inch
segment of Cer atophy1lum was 1.30 percent.  This value
is in agreement with the critical concentration reported
for entire Ceratophyllum demursum plants in a previous
                           21

-------
Table 7.  Critical concentrations and the range of
          concentrations of several essential nutrient
          elements in index segments of Ceratophy1lum
          demursum.
Element
N
P
Ca
Mg
K
B
Index
segment
2nd
2nd
1st
2nd
2nd
1st
1"
1"
1"
1"
1"
1"
Critical
cone.
1.
0.
0.
0.
1.
2.
30 %
10 %
22 %
18 %
70 %
8 ppm
1.
0.
0.
0.
1.
1.
Range
cone.
00 -
09 -
11 -
11 -
58 -
49 -
2.
0.
0.
0.
5.
12
in
42 %
41 %
47 %
40 %
10 %
. 54ppm
study (Gerloff and Krombolz, 1966}„  The critical phos-
phorus concentration in the second one-inch was 0.10 per-
cent.  A comparison of Tables 6 and 7 shows that the
critical nitrogen and phosphorus concentrations were
somewhat lower in CeratophyHum than in Elodea.  This
probably is related to a higher stem to leaf ratio in
Cer a tophy Hum.

The greatest difference in critical concentrations in the
two species was in potassium.  The critical potassium
concentration in Elodea was only 0.80%; in Ceratophyllum,
it was 1.70% or slightly more than double the Elodea.
value.  Only further experimentation can determine if
this difference is significant in terms of growth and
distribution of the plants in lakes and streams.
                           22

-------
                         DISCUSSION

Two aspects of this study seem critical in developing
plant analysis as a practical nutrient assay for aquatic
environments.  First, critical concentrations were
established in Elodea occ ideritalis for most of the essential
elements rather than just nitrogen and phosphorus.  This
greatly increases the usefulness  of the technique.  It
also has made this initial effort with aquatic environ-
ments one of the most complete studies of plant analysis
with any species.  Secondly, it is considered important
that the technique has been based on critical concentra-
tions in index segments.  This should minimize errors
resulting from sampling either terminal tissues, which
might be disproportionately high in some elements even
under deficiency conditions, or older senescent tissues,
which could have a low content of the elements even under
adequate nutrient supplies.

Interpretation of the plant yield-tissue content response
curves is subjective to some degree.  The curves often
do not agree with the theoretically ideal curve (Ulrich,
1952).  There has also been some disagreement among
investigators on the point on the response curve at which
the critical level should be established.  The approach
in this study was to consider the critical concentration
the tissue content at a point approximately 5 percent
below the maximum yield represented by the leveling off
of the response curve.  This conforms with the inter-
pretation of numerous investigators when applying the
tissue analysis technique to the culture of agricultural
and horticultural crops.

The reproducibility and reliability of the reported
critical contents are of some concern.  The degree of
variation in yield and in tissue content of nitrogen in
duplicate cultures of the same experiment was indicated
in Table 2.  This is typical of the best experiments in
this study.  Better agreement in replicated cultures
cannot be expected as long as the plants are propagated
vegetatively.  Even with the most careful selection of
plant segments when subculturing, it is impossible to
obtain uniform initiation of growth from terminal and
lateral buds.

The results from this and a related study  (Gerloff and
Krombholz, 1966) allow comparison of critical values for
the same element established in separate experiments and
by different techniques.  In a particular species it

                           23

-------
seems possible to reproduce the nitrogen critical level to
+0.1 percent and the phosphorus level to + 0.01 percent.
This suggests the desirability of reporting the critical
percentage as a range rather than a specific value.
However, the common practice in agricultural applications
has been to report specific values.  That practice has
been continued here.  The degree of reliability indicated
above must be recognized and understood in using the
critical concentrations.  The values reported here seem
fully as reliable as those reported for crop species.
                          24

-------
                       SECTION V

         EVALUATION OF PLANT ANALYSIS  FOR THE

    ASSAY OF NUTRIENT SUPPLIES IN WISCONSIN  LAKES

After critical  concentrations of the essential  elements  in
Elodea occidental is and  Ceratophylluni  demursum  were
established in  laboratory  experiments,  plant analysis was
used to evaluate nutrient  supplies  and growth-limiting
nutrients in Wisconsin lakes.  This involved collecting
samples of the  first and second one-inch  segments  of the
Elodea and Ce r atophyllum from lakes known to vary  consider-
ably in fertility.  Samples were obtained at intervals
throughout the  growing season, were analyzed for the
essential elements, and  the values  were compared with the
critical concentrations.  Concentrations  of  an  element
consistently above the critical level  were interpreted
to  indicate the element  was in abundant supply  and not
limiting plant  growth; a concentration at or below the
critical level  indicated supplies of that element  had
become limiting at the time of sampling.   In this  case/
restriction of  entry'of  the growth-limiting  element-into
the specific body of water could be expected to reduce
growths of nuisance macrophytes.  Conversely, additions  of
the element probably would increase macrophyte  growth.
                 EXPERIMENTAL PROCEDURE

 During  the  summer of  1970,  samples  of Elodea occidentalis,
 the  primary assay organism,  were routinely collected  from
 9  Wisconsin lakes.  All of  the lakes sampled except one
 are  located in or near Vilas County in  northern Wisconsin
 in non-agricultural areas.   They are relatively infertile.
 Water hardness varied from  14 to 65; the pH of  the water
 was  in  the  range of 7.1 to  8.0 (Black,  Andrews, and
 Threinen, 1963).

 Plant samples  collected from lakes  were lifted  from the
"water with  a garden rake and taken  to the laboratory  while
 immersed  in lake water.  Within several hours,  sufficient
 first and second inch segments to represent 3-4 g of  oven-
 dry  material were cut from  the main branches and laterals
 of healthy,  green plants.   After repeated rinsing in
 lake water  to  eliminate debris, the samples were soaked
 30-45 seconds  in 0.2N HC1,  rinsed with  lake water,


                           25

-------
rinsed in distilled water, placed in nylon bags/ and dried
to constant weight at 65-70°C.

Total nitrogen analyses on all field samples were by a
semiraicro Kjeldahl procedure.  In addition, all samples
were analyzed for 10 elements by the Jarrell-Ash Multichannel
Emission Spectrometer at the Wisconsin Alumni Research
Foundation Laboratories.  In all lakes in which there were
indications from the emission spectrometer analyses that
a specific element had become limiting/ or was close to
limiting, for plant growth, analyses were verified by the
quantitative procedures used in establishing critical
concentrations in the laboratory.  This included analyses
for calcium, phosphorus, and copper.

Samples analyzed by the Jarrell-Ash Spectrometer were
ground in a Wiley Mill equipped with a stainless steel
screen.  Samples analyzed by conventional quantitative
procedures were ground with an agate mortar and pestle to
minimize trace element contamination.

Some additional samples were obtained from natural macro-
phyte populations in Wisconsin lakes during the summer of
1971.  However, emphasis was shifted to another use of
the plant analysis technique.  The Elodea bioassay
organism was placed in porous/ inert containers near the
surface in lakes in which nutrient supplies were to be
evaluated.  Samples of the first and second one-inch index
segments were to be removed periodically, analyzed for
elements suspected to limit growth, and evaluated by
comparisons with the critical concentrations.  The
anticipated advantage of this procedure was that the
assays would primarily reflect nutrient availability in
the water layer and would be relatively uninfluenced by
the direct abosrption of nutrients from sediments.

Cylindrical baskets 8-1/2 inches in diameter and 12
inches long were constructed of 1/4 inch mesh polyethylene
screen.  The cylinders were closed at the ends with the
polyethylene mesh and were reinforced with plastic rings
1/4 inch wide.  After the baskets were anchored in
relatively sheltered areas of lakes in the vicinity of
macrophyte beds where the water was no more than 5 feet
deep, an inoculum of laboratory-grown Elodea or
Ceratophyllum was placed in each.  The baskets were
floated near the water surface by two empty, sealed
one-gallon polyethylene bottles.  One basket of each
of the two macrophytes was placed in 5 northern
Wisconsin lakes.

                          26

-------
                        RESULTS

Because of the large amount of data, only analyses of the
macrophytes from representative lakes are presented and
discussed in this section.  Complete analytical data on
the samples from the additional lakes are presented in
the Appendix.
Samples of 1970

The analyses of Elodea from one of the most fertile
northern Wisconsin lakes sampled  (Little John) and one of
the least fertile  (Salsich) are presented in Tables 8
and 9.

Concentrations of nitrogen and phosphorus in the lake
plants are of particular interest because these elements
most frequently have been considered  limiting for aquatic
plant growth.  In all Elodea  samples  from Little John
Lake, the nitrogen and phosphorus concentrations were well
above the critical concentrations of  1.60% and 0.14% in
the second one-inch segment.  The lowest phosphorus
concentration was 0.40% on July 27 indicating very ade-
quate supplies of that element all during the growing
season.

The lowest concentrations of  elements in general were
observed in samples from July 27.  This sampling apparently
was in the period of most rapid plant growth and greatest
pressure on nutrient supplies.  However, even in the July
27 samples, the concentrations of all elements, except
nitrogen were at least double the critical concentrations
reported in Table 6.

The nitrogen and phosphorus analyses  of Elodea samples
from Salsich Lake contrast with the results from Little
John.  The nitrogen concentrations were in general slightly
higher in the Salsich samples, but the phosphorus con-
centrations were considerably lower.  For example, in the
second one-inch segment from  July 28  the phosphorus con-
centration was only 0.18% and in the  August 19 sample
0.15%.  Comparable phosphorus values  in the Little John
samples were 0.40 and 0.54%.  The low values for Lake
Salsich plants, which were verified with a standard
colorimetric procedure, approach the  critical concentration
of 0.14%, and indicate that in this lake phosphorus sup-
plies were close to limiting  for Elodea growth.  Further
additions of phosphorus could be expected to increase the

                          27

-------
      Table 8.   Essential element content of  first  and  second one-inch  segments  of
                Elodea occidentalis  collected from  Little  John Lake during  the
to
00
summer of 1970.
Date
Sampled
June 16
July 6
July 27
Aug. 18
Sep. 8
Segment
1"
2"
1"
2"
1"
2"
1"
2"
1"
2"

N
3.50
3.35
3.05
2.66
2.81
2.22
3.58
2.84
3.49
3.10

P
0.65
0.70
0.54
0.54
0.47
0.40
0.65
0.54
0.80
0.68
%
K
2.30
2.30
2.75
2.55
2.45
2.45
2.80
3.25
2.55
3.45

Ca
0.52
0.85
0.54
0.91
0.68
1.07
1.09
1.55
1.09
1.17

Mg
0.22
0.23
0.22
0.19
0.21
0.19
0.25
0.20
0.24
0.19

Fe
>1000
>1000
795
>1000
665
855
905
820
760
625

B
21.1
24.9
13.6
14.9
13.5
16.6
14.9
13.3
13.5
12.5
Ppm
Cu
1.3
2.1
1.5
2.6
2.2
3.4
1.8
3.6

Mn
404
>410
>410
>410
386
>410
254
>410
360
>410

Zn
46
35
71
86
56
74
95
89
66
81
      Except for N, all analyses were made with a Jarrell-Ash direct-reading,
      computer-programmed emission spectrometer.

-------
N)
10
      Table 9.   Essential element content of first and second one-inch segments of
                Elodea occidentalis collected from Salsich Lake during the summer
of 1970.
Date
sampled
July 7
July 28
Aug. 19
Sep. 10
Segment
*
1"
2"
1"
2"
1"
2"
1"
2"

N
3.78
3.47
3.55
3.14
3.50
3.02
3.47
2.83

P
0.35
0.21
0.25
0.18
0.33
0.15
0.40
0.40
%
K
2.20
2.80
2.20
2.65
2.65
2.80
2.35
2.10

Ca
0.48
0.64
0.42
0.66
0.50
0.62
0.64
0.91

Mg
0.21
0.17
0.19
0.16
0.20
0.16
0.18
0.19

Fe
555
445
450
480
545
430
485
580

B
12.1
13.1
12.4
13.5
11.5
8.8
8.4
12.5
Ppm
Cu
6.2
4.3
5.0
3.8
4.7
1.8
3.1
4.0

Zn
197
182
160
224
162
156
177
177

Mn
98
150
116
190
124
185
137
225
      Except for N, all analyses were made with a Jarrell-Ash direct-reading,
      computer-programmed emission spectrometer.

-------
presently sparse weed beds in Salsich Lake.

Because of inadequate plant tissue, it was impossible to
run molybdenum analyses on all samples from the 9 lakes.
The range of molybdenum in 7 samples analyzed was 0.50 to
0.73 ppm, in the terminal one-inch, with an average of
0.57 ppm.  Even the lowest concentration was 3 to 4x the
critical concentration of 0.15 ppm presented in Table 6,
thus eliminating the need to consider molybdenum as a
possible growth-limiting factor.

The copper analyses reported in Tables 8 and 9 are of
interest because some are sufficiently low to suggest Cu
deficiency.  This cannot be verified until the copper
critical concentration is firmly established in laboratory
experiments.  However, comparisons with critical concentra-
tions determined for agricultural and horticultural
terrestrial species support the indicated copper deficiency.
For example, a critical copper concentration of 5 ppm
has been reported for corn, alfalfa, and soybeans and 7 ppm
for alfalfa (Melstead, Motto, and Peck, 1969).  Less than
4 ppm Cu in the leaves was associated with copper deficiency
in citrus  (Chapman, 1961).  Several of the copper con-
centrations in Elodea from Salsich and all of the values
from Little John were less than 4 ppm.  For a number of
samples the copper data reported in Tables 8 and 9 were
checked by atomic absorption analysis.  The atomic absorp-
tion values were consistently higher.  However, the copper
contents obtained by atomic absorption still were below
the 4-7 ppm critical concentrations for economic crops and
confirm that either Elodea was copper deficient in Little
John Lake, or that it has an unusually low requirement
for that element.

Although the critical copper concentration has not as
yet been determined, it has been possible to grow Elodea
severly deficient in copper.  Yields from cultures to
which copper was not added were only 25 percent (0.909 g)
of yields  (3.636 g) with copper added.  The copper
concentration in the first two inches of the copper
deficient plants was 1.57 ppm; in normal plants, it was
17.7 ppm.  From experience with the other trace element
cations, it can be anticipated that the critical copper
concentration will be at least double the 1.57 ppm in the
deficient plants.  This supports the view that copper
supplies did become growth-limiting in some of the lakes
sampled.
                          30

-------
In Table 10, data are presented for samples taken from 8
lakes during the late July period of maximum stress on
nutrient supplies.  Analyses are reported only for the
four elements which critical concentration comparisons
indicated might limit plant growth.  When the Jarrell-
Ash data were verified by standard quantitative procedures,
data obtained by both types of analyses are presented.

Comparisons with analyses of samples from other lakes
emphasize that the phosphorus concentration in plants from
Salsich Lake  (0.18%) was relatively low.  Whitney Lake
also seems close to phosphorus deficiency for Elodea
growth, as indicated by the 0.17% value for the index
segment.  The data on Clear Lake are of interest because
the phosphorus concentration was relatively high at
0.24% but the nitrogen concentration of 1.94% in the second
one-inch was the lowest of any sample, and only slightly
above the 1.60% critical value.  The 2.70% and 0.29%
concentrations of nitrogen and phosphorus, respectively,
in the Erickson Lake samples were high, but the 0.28%
calcium concentration in the terminal one-inch index
segment was at the critical level.  It is not surprising
that calcium might be a limiting factor in soft water
lakes and that species with a higher requirement for
calcium than Elodea, or less capacity to absorb calcium
from the environment, might be eliminated from soft water
lakes such as Erickson.

Probably the most consistently low values in Table 10 are
to be observed in the copper data.  In Elodea from 5 of
the 8 lakes copper concentrations were low enough to
suggest a growth-limiting role of that element.

In contrast to the other 6 lakes, Allequash and Little
Spider Lakes seem relatively well supplied with all the
essential nutrient elements.

The data in Table 11 are from analyses of Ceratophyllum
demursum index segments from Lake Mendota at Madison,
Wisconsin.  Mendota is an extremely fertile, hard-water
lake which has extensive beds of macrophytes and trouble-
some algae blooms.  The unusual fertility of Lake Mendota
is reflected in the very high concentrations of nutrient
elements in the samples.  For example, the average con-
centration of nitrogen in the second one-inch segment was
3.75%; the phosphorus concentration was 0.65%.  These
values are well above comparable values in plants from the
northern Wisconsin lakes and are approximately 3x and 6x

                          31

-------
Table 10.  Concentrations of potentially growth-limiting elements in Elodea
           occidentalis collected from northern Wisconsin lakes during late
           July, 1971.
Concentration of
Lake
sampled
Allequash
Clear
Whitney
Erickson
Big Kitten
Salsich
Little John
Little Spider
Date
sampled
July
July
July
July
July
July
July
July
27
29
29
28
29
28
27
28
Methyl
orange
a Ik . *
39
33
22
24
65
14
45

Plant
segment
1"
2"
1"
2"
1"
2"
1"
2"
1"
2"
1"
2"
1"
2"
1"
2"
N

3.
3.
2.
1.
2.
2.
3.
2.
2.
2.
3.
3.
2.
2.
3.
2.


53
19
41
94
34
02
41
70
71
16
55
14
81
22
36
59

JAS**
0.34
0.28
0.42
0.28
0.23
0.21
0.47
0.29
0.42
0.40
0.25
0.18
0.47
0.40
0.44
0.28
P
Std.**
0.31
0.22
0.34
0.24
0.21
0.17
0.41
0.28
0.39
0.34
0.26
0.18
0.35
0.25
0.37
0.27
Ca
JAS
0.42
0.61
0.69
0.66
0.68
0.89
0.28
0.44
1.61
1.51
0.42
0.66
0.68
1.07
0.52
0.61
Cu
JAS
3.8
3.4
2.2
1.8
2.2
1.8
3.8
2.2
2.2
5.0
3.8
2.2
3.8
2.2
Std.
6.5
7.0



3.9
2.7

2.4
3.3
6.5
4.5
 * Values for methyl orange alkalinity were reported by the Wisconsin Department
   of Natural Resources.

** JAS indicates analyses made with a Jarrell-Ash direct-reading, computer-pro-
   grammed emission spectrometer; Std. refers to analyses by standard colori-
   metric, emission flame photometer, and atomic absorption procedures.

-------
     Table  11.  Essential  element  content  of  first  and  second  one-inch segments  of Ceratophyllum
OJ
CO

Date
sampled
June 18
July 20
Aug. 8
Sept . 1
demursum

collected from Lake Mendota during the summer of
1970.



% Ppm
Segment
1"
2"
1"
2"
1"
2"
1"
2"
N
3.75
2.90
5.40
4.35
4.28
4.21
4.14
3.55
P
0.49
0.39
0.92
0.80
0.70
0.61
0.77
0.80
S K
4.25
3.45
4.45
4.25
4.80
4.70
0.32 3.10
0.40 3.95
Ca
0.48
0.52
0.45
0.47
0.31
0.55
0.50
0.71
Mg
0.81
0.98
0.69
0.68
0.70
0.69
0.88
0.89
Fe
460
540
920
890
495
565
>999
930
B
15.4
19.5
16.6
18.8
12.7
13.3
16.4
16.8
Cu
3.9
3.7
12.3
9.8
12.8
7.6
14.2
10.8
Zn
101
121
163
203
70
70
117
131
Mn
>1000
>1000
>1000
>1000
247
>1000
388
>1000
Mo
0.42



      Except for N,  all  analyses were made with a Jarrell-Ash  direct-reading,  computer-programmed
      emission spectrometer.

-------
the critical concentrations for nitrogen and phosphorus,
respectively.  Concentrations of all the trace elements
also were relatively high.  The data suggest it would be
necessary to remove considerable quantities of nitrogen,
phosphorus, or any other essential nutrient, from pollution
sources entering Lake Mendota to significantly reduce
nuisance macrophyte growths.

Samples of 1971

In Table 12, data are presented from the analyses of
Elodea and Ceratophyllum which had been in the assay
baskets for approximately two months, from late June until
early September.  Samples also were collected in late
July from the natural populations in these lakes and
analyzed.  Unfortunately, both species were not present
in all 5 lakes.  The concentrations of nitrogen and
phosphorus were well above the critical concentrations of
1.60% nitrogen and 0.14% phosphorus in Elodea and 1.30%
and 0.10% in Ceratophyllum in all samples from the natural
populations.  There was no indication either nitrogen or
phosphorus was growth limiting in early July.

Comparisons of nitrogen and phosphorus concentrations
in natural populations of the two species obtained from
the same lake  (Clear or Little John) are of interest.
Elodea produces roots which presumably are imbedded in
bottom sediments while Ceratophy11um has almost no roots.
Elodea, therefore, obtains nutrients from both the mud
and wat.er; Ceratophyllum must rely on the water.  Nitrogen
concentrations were much loiffer in Ceratophyllum than in
Elodea in both Clear and Little John lakes suggesting
the advantage of roots in drawing on bottom sediment
nitrogen supplies.  There was little difference in the
phosphorus concentrations in the two species, indicating
an unusual capacity of Ceratophy1lum to absorb phosphorus
from the very low concentrations in the water.  The col-
lections of 1970 indicated Allequash to be a fertile lake.
This correlates with the lack of a difference in nitrogen
concentrations in Elodea and Cera tophy11um from this lake.

Unfortunately, the Elodea and C er atophy11um inoculated
into the baskets grew very poorly.  There was hardly
enough tissue for nitrogen and phosphorus analyses by
early September when the baskets were removed from the
lakes.  Whether this was due to the shock of transfer of
laboratory-grown plants to the lake environments, to
placement of the plants too near the surface, or to other

                          34

-------
      Table 12.   Nitrogen and phosphorus analyses of index segments of Elodea
u>
Ul
occidentalis and Ceratqphyllum demursum from natural populations
and
Lake
Nebish
Clear
Allequash
Little John
Salsich
assay baskets
Natural pop
Elodea (%)
N P
4.11 0.32
2.68 0.37
2.65 0.21
2.30 0.27
— —
in northern Wisconsin lakes.


's. 7-31-71 Assay baskets 9-11-71
Ceratophyllum(%) Elodea (%)
N P N P
1.20 0,08
1.69 0.28 1.58 0.11
3.00 0.24 1.57 0.26
1.73 0.31 1.04 0.12
1.25 0.09
Cer atophyllum (
N P
1.90 0.
1.64 0.
— _
1.83 0.
1.50 0.
%)

24
19
-
24
15

-------
reasons must be determined in future studies.  The low
concentrations of both nitrogen and phosphorus in the Elodea
suggests this was due to poor physiological condition
induced by factors other than nutrient deficiencies.  In
the CeratophylTum samples, nitrogen was proportionally
closer to the critical concentration (1.30%) than was
phosphorus (0.10%).

The similarity in nitrogen and phosphorus concentrations
in the Ceratophyllum from the natural populations and the
baskets suggests the absence of roots might make Cerato-
phyllum a more suitable assay organism than Elodea for
evaluating nutrient supplies for all non-rooted green
plants including algae.
                      DISCUSSION

The results obtained suggest plant analysis can be a
useful relatively simple procedure for nutrient assay in
aquatic environments, and that it is a procedure which
offers several advantages.  Plant analysis minimizes the
difficulties associated with obtaining representative
samples of the aquatic environment.  In this procedure,
plants become the sampling device, and a single analytical
value provides an integrated expression of all the factors
which affected the availability of an element in the micro-
environments to which the plants were exposed during
growth.  Chemical analyses of water samples must be inter-
preted in terms of fractions available and unavailable to
plants and concentrations and quantities which become
limiting for growth.  These problems are reduced in the
plant analysis procedure, because plant analysis is based
on values which have been correlated with plant growth and
yield responses.

Two aspects of the field data seem worthy of comment.
First, although nitrogen and phosphorus are the elements of
most interest in practical pollution control, the data
indicated that neither element was a general limiting
nutrient for Elodea in the lakes sampled.  In some lakes,
nitrogen or phosphorus was near the critical concentration.
In these ecosystems, reduction in available supplies of
nitrogen or phosphorus, whichever was limiting, probably,
would reduce macrophyte populations; also, further
eutrophication with these elements would accentuate
nuisance growths.  A second point of interest, and

                          36

-------
obviously in need of verification, is the suggested copper
deficiency in northern Wisconsin lakes.  This focuses
attention on the importance of trace elements in the field
nutrition of aquatic plants.  Copper deficiency also
would be of some practical importance, because if that
element controls plant growth in certain lakes, limited
reductions in nitrogen or phosphorus pollution might not
improvd nuisance conditions in those lakes to the degree
anticipated.

Although the results presented suggest plant analysis is
a useful bioassay, the need for additional studies to refine
the technique is emphasized.  Hundreds of investigations
have provided the information necessary to firmly establish
plant analysis as a procedure for nutrient assays in soils.
Undoubtedly, there will be unique aspects in applications
of  plant analysis to aquatic plants and environments.
Further investigations of the most suitable index segments,
more precise establishment of critical concentrations,
and the determination of critical concentrations for
other macrophytes and algae seem worthy of immediate
study.
                           37

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                      SECTION VI

         OPTIMUM NUTRIENT SOLUTION AND GROWTH

        CONDITIONS FOR THE LABORATORY CULTURE OF

                 NUISANCE MACROPHYTES

Successful laboratory studies of the nutrition and
physiology of any plant are facilitated by the avail-
ability of a suitable nutrient culture medium and know-
ledge of optimum growth conditions.  A satisfactory
culture medium is one in which essential elements do not
become growth limiting during extended culture periods,
total salt concentration and concentrations of heavy metals
are below toxic levels, and the pH remains within a suit-
able range as plants absorb nutrients from the medium.

Several procedures have been used in formulating nutrient
media, including approximation of proportions of the
essential elements in soil extracts, determining the amounts
of elements taken up from nutrient solutions, and by
measuring responses to systematic variations in the con-
centrations of individual elements in a medium.

Relatively little laboratory work has been carried out in
which macrophytes have been cultured in synthetic media
 (Bourn, 1932).  In studies prior to this project, it had
been established that macrophytes made reasonably satis-
factory growth in a modified Hoaglands solution  (Gerloff
and Krombholz, 1966), a medium used for many years in
laboratory and greenhouse studies with crop plants.  The
modified Hoagland's medium was used in the studies pre-
sented in Section IV of this report.  Systematic varia-
tions of the concentrations of each essential element  in
these experiments made it possible to estimate the mini-
mum concentration of each element in a culture medium
for optimum growth of the macrophytes under study.  The
formulation of a culture medium based on these optimum
concentrations was suggested.  In Section VI experiments
will be discussed in which this medium was systematically
varied to develop a general macrophyte nutrient culture
solution.  Results also are presented from several studies
to determine optimum conditions of temperature and light
for macrophytes.
                           39

-------
                EXPERIMENTAL PROCEDURES

The general culture conditions and harvesting procedures
employed were described in Section IV.

Several chelates of iron were compared as an iron source/
primarily.EDTA  (ethylenedinitrilo)-tetraacetic acid and
EDDHA  ethylenediamine di-(o-hydroxyphenylacetate)
The EDDHA was obtained from Geigy Chemical Co. as an 86.3
percent pure salt.  Iron chelates were prepared as a
0.01 M stock solution of FeEDTA  (2.78 g FeSCV 7H2O and 3.72
g Na2EDTA)  and as a 0.01 M stock solution of FeEDDHA (140
mg KOH, 278 mg FeS0lt*7H2O, and 424 mg EDDHA),
                        RESULTS

The composition of the macrophyte culture medium developed
on this project is presented in Table 13.
Concentrations of Elements

The initial step in formulating the macrophyte medium was
to determine from the critical concentration experiments
in Section IV the minimum concentration of each essential
element which was just adequate to produce maximum growth
of Elodea in a 4-week period in two liters of solution.
These values were compared with the amounts of the elements
necessary to maintain the critical concentration in 5.0 g
of oven-dry growth of Elodea and Ceratophyllum.  From these
comparisons some upward adjustments in element concentra-
tions seemed necessary, particularly in the three major
element cations, calcium, magnesium, and potassium.  As
a result, the concentrations of the essential elements
in the solution of Table 13 are approximately double the
minimum concentrations for maximum yield derived from
critical concentration experiments.

Providing each element at the lowest but adequate con-
centration was considered desirable to reduce the
possibility of adverse effects on growth due to high salt
concentrations and toxic trace element concentrations.
Minimum deviation from the low concentrations characteristic
of lakes and streams also seemed desirable.
                          40

-------
Table 13.  Composition of medium recommended for macrophyte  culture
Salt
KN03
Ca(NO3) 2'4H2O
MgS(H-7H2O
KH2PCH
NaNO3

KC1
H3B03
MnSCK-H2O
ZnSCU- 7H20
CuSCH- 5H2O
(NHh)6M07p21t' 4H20
*FeEDDHA
Stock sol'n,
wt. /liter
10.10 g
37.76 g
14.79 g
5.44 g
12.75 g

746 mg
155 mg
169 mg
115 mg
12.5 mg
3.7 mg
	
Ml stock/liter
final solution
5
5
5
5
5

1
1
1
1
1
1
10
Element in final
solution, ppm
N -
K -
Ca -
Mg -
S -
P -
Cl -
B -
Mn -
Zn -
Cu -
Mo -
Fe -
39.9
27.3
32.0
7.2
9.6
6.2
0.354
0.027
0.054
0.026
0.003
0.002
0.56
*  FeEDDHA solution is prepared by dissolving 140 mg KOH in 400 ml double dis-
   tilled water, adding 424 mg 85% pure EDDHA, and stirring until dissolved.
   Finally 278 mg FeSCK '7H2O dissolved in 500 ml double distilled water is
   added and the solution is diluted to 1 liter.

-------
Chemical Salts

Solubility and effect on the pH of the medium throughout
the growth period are primary factors in the selection
of salts to provide essential elements in a nutrient
medium.  The four salts Ca (NO3) 2 • 4H2O, KN03, MgSO^.TH^O,
and KH2P0lt have been widely used in plant culture media,
including the modified Hoagland's solution from which the
macrophyte medium was derived.  The NaNOa in the recom-
mended medium is to provide additional nitrogen without
increasing the concentration of another essential element.

As will be apparent in data to be presented/ the pH of
the macrophyte solution immediately after preparation and
autoclaving was approximately 5.0, and after equilibrium
with the 1% CO 2 in air mixture it was 4.9.  As plants
absorbed ions , the pH of the medium increased so that at
harvest it correlated to a degree with plant yields.  In
general, pH values at harvest were in the range of 6.0 to
7 . 5 for Elodea and Ceratophyllum and somewhat higher for
Myriophyllum, as high as 10.0.  Differences in yield and
absorption of ions account for these variations.
Trace Element Concentrations

The primary difference in the culture medium used in the
critical concentration experiments of Section IV and the
medium derived from those experiments is the concentrations
of the trace elements which were reduced to 1/5 to 1/10
the concentrations in the original medium.  This seems
desirable because, except for chlorine, the range in
concentration between adequacy and toxicity of the trace
elements can be narrow as illustrated in the data of
Table 14 showing the response of Elodea to copper.  The
average yield from triplicate cultures provided with 0.006
ppm copper was 1.352 g; with 0.03 ppm copper, yield was
reduced to 1.098 g; and with 0.15 ppm, yield was only
0.531 g.  The adverse effects of concentrations of trace
elements close to the toxicity level probably are primarily
on the inoculum and on the initiation of growth in a
culture.  If the concentration of an element is not so
high that the inoculum is killed, the amount of the ele-
ment per unit of material is reduced as growth develops
and initial toxicity effects in a culture no longer will
be evident.
                          42

-------
Table 14.  Toxicity of copper  to Elodea occiden'falls,
Cu in
culture
soln. , ppm
0.0
0.003
0.006
0.03
0.15
Yield of
Elodea,
g/2i.*
1.325
1.399
1.352
1.098
0.531
       *   Average of triplicate cultures,
 Iron Source
 Maintenance of adequate available iron is a critical
 factor in successful growth of plants in aerated,  liquid,
 culture media.  The low solubilities of iron precipitates
 which form at the slightly acid and alkaline pH values  of
 nutrient media make inorganic iron sources, in general,
 unsatisfactory.  As a result, chelating agents have been
 employed for a number of years to maintain iron avail-
 ability.  Ferric citrate and particularly FeEDTA have been
 effective in many culture media.  Because of a capacity
 to maintain iron in a soluble form at higher pH values
 than does EDTA, EDDHA has recently been the iron source
 in several culture media  (Wallace, 1966).

 The importance of the iron source in the culture of
 macrophytes is indicated in Table 15.  Ferric citrate was
 completely unsatisfactory as a source of iron for
 Ceratophyllum demursum.  FeEDTA was far superior to ferric
 citrate but was less satisfactory than FeEDDHA.  Because
 of the increase in the pH of the culture medium associated
 with autoclaving, it seemed desirable to autoclave the
 iron source separately and add it to culture media after
 autoclaving.  However, the data indicate separate auto-
 claving was not beneficial.
                           43

-------
Table 15.  Comparison of several iron sources for the
           growth of Ceratophyllum demursum in a synthetic
           culture medium(0.56 ppm Fe provided from each
           Fe source).
       Fe                 Oven-dry plant       pH of medium
     source                 wt  (g/1)*           at harvest
Fe citrate, auto-
claved separately
FeEDTA , auto-
claved separately
FeEDTA, autoclaved
with the medium
FeEDDHA, auto-
claved separately
FeEDDHA, autoclaved
with the medium
0.008
0.833
0.843
1.315
1.373
5.72
7.00
6.95
7.14
7.10
*  Each value is an average of results from duplicate
   cultures.
From experience on this project, it is difficult to
generalize on the response of macrophytes to a specific
concentration or source of iron.  Even the addition of
0.56 ppm iron as FeEDDHA has not always provided adequate
iron.  Also, FeEDTA sometimes is as suitable an iron
source as is the EDDHA complex.  The most satisfactory
results have been obtained by adding 0.56 ppm iron as
FeEDDHA when the culture medium is prepared and then a
second and often a third increment of 0.28 ppm iron after
growth is initiated.  The supplemental iron is added
aseptically when the red color of the FeEDDHA complex
has nearly disappeared, usually 7 to 10 days into the
culture period.  A third addition may be necessary if the
red color again disappears.
                          44

-------
Yield Comparisons

As indicated in Table 16, evaluations of the recommended
macrophyte medium were possible through comparisons of
plant yields in that medium, in modifications of it, and
in the original medium.

For Elodea, the recommended medium  (x macrophyte) produced
as much yield as the original  solution in  two experiments.
Dilution of the recommended medium  (x/2 macrophyte) re-
duced the higher yield.  Apparently  the supply of one or
several elements had become limiting.  Doubling the concen-
trations of all constituents  (2x macrophyte) reduced yield
slightly.

The proposed macrophyte  solution was also  equal to, or
somewhat superior to, the original medium  in the culture
of Myriophy1lum.  Dilution to  half-strength did not reduce
yields with MyriophyHum, probably because its require-
ments for nitrogen  and phosphorus are less than the re-
quirements of Elodea.

The recommended medium has not been  quite  as effective for
the culture of Ceratophyllum as was  the original medium.
This is verified by data from  two experiments reported
in Table 16.  The reason is unclear. Ceratophyllum does
have a higher potassium  critical concentration than Elodea.
However, increased  solution concentrations of potassium
did not increase yields.  Higher concentrations of indivi-
dual trace elements also were  without effect.  Cerato-
phyllum demursum in general has been a less satisfactory
experimental organism than Elodea occidentalis or Myrio-
phyllum spicatum, primarily because  of sudden leaf drop
from the stems and  branches after several  weeks of culture.
This accounts for the relatively low yields of Cerato-
phyllum in Table 16.

Probably the most unexpected result  in Table 16 was the
positive response of Elodea and Myriophy1lum to the addi-
tion of Na2CO3  (120 mg per liter) to the medium.  The
effect was particularly  evident as more rapid growth in
the early  stages of the  cultures.  It is understandable,
therefore, that the response of various species to Na2C03
and of the same species  in different experiments might
vary.  Macrophyte yields were  not increased by the addi-
tion of Na2SiO3«5H2O.  This suggests the beneficial effect
of Na2CO3 was on carbonate-bicarbonate ratios in the medium
rather than on pH.
                          45

-------
Table 16.  Comparisons of the growth of three macrophyte species in an initial culture medium and in modifi-
           cations of a newly developed medium.
Culture
medium
Original
X/2 Macrophyte
X Macrophyte
2X Macrophyte
X Macrophyte
plus Na2C03
Elodea occidentals

Exp.
Yield
Cg/21)
2.732
2.722
2.535
1.897
3.464
1
Final
PH
6.81
6.08
7.03
8.17
7.47
Exp. 2
Yield Final
(g/21) pH
3.251
2.952
3.507
__
4.179 7.41
Myriophyllum spicatum
Exp.
Yield
Cg/21)
3.514
4.656
4.796
4.141
4.787
1
Final
pH
9.73
7.71
9.59
9.72
9.77
Exp.
Yield
Cg/21)
4.594
--
4.424
--
5.095
2
Final
pH
10.0

10.13

10.24
Ceratophyllum demursum

Exp. 1
Yield Final
Cg/21) pH
1.790
--
1.433
--
--
Exp.
Yield
Cg/21)
1.576
0.941
1.205
1.405
0.922
2
Final
pH
6.94
6.67
6.79
6.61
6.88

-------
Optimum Light

Macrophyte growth could be affected by the intensity,
quality, and photoperiod of the available light.  Because
they grow submerged in water, macrophytes might respond
to these light factors quite differently than do terres-
trial species.

There were only limited opportunities for varying light
quality in the artificially lighted growth chambers used
in these studies.  Some control was possible in the
selection of fluorescent bulbs, which provided most of
the light, and in the number of incandescent bulbs used
to supplement radiation of long wavelengths.  In the few
variations attempted, there were no indications that any
combination was superior to the ratio of fluorescent to
incandescent light provided in the Sherer Growth Cabinets,
Model CEL 25-7 HL, used in the current studies.  Each
chamber contained 6, cool-white, 4-foot fluorescent bulbs
and 12, 25 watt incandescent bulbs.  These are ratios
considered suitable for crop plant growth under artificial
light.  There also was no indication that a light-dark
cycle in each 24-hour period was superior to continuous
light.

The effect of variations in light intensity on the growth
of Elodea was investigated in an experiment summarized in
Figure 4.  The experimental setup was similar to that
described in Section IV for establishing nutrient critical
concentrations by the tray procedure.  This procedure was
used, because it permitted rapid evaluations of macrophyte
response to light.  One set of trays was maintained at
approximately 1700 ft candles throughout the culture period;
another set was exposed to different light intensities
every several days during the culture period.

The results in Figure 4 compare the percent increase in
length of Elodea in control trays exposed to continuous
light of 1700 ft candles and in trays exposed to different
light intensities at various stages of the culture period.
The data indicate Elodea was not damaged by light intensities
as high as 2600 ft candles; in fact, growth was slightly
better at 2600 ft candles than at 1700.  When light inten-
sity was reduced to 710, 390, and 110 ft candles, growth
was less than at 1700 ft candles and progressively less
with each decrease in intensity.
                           47

-------
     50
     40
g
^30
O
0>
C 20
£ 10
3

»


-
-

—

0



^^m
0
§


—




O






^M«





1







^^m




O






^VB





O
O
•••












O
^^






—





0
g







            Light  Intensity  (ft. candles)
Figure 4.  The growth of Elodea occidentails,  as length in-
  crease, during periods of exposure to various light
  intensities.
The limitations of using length increase as a criterion
of growth must be recognized.  Nevertheless/ from the
data obtained, maintenance of a specific light intensity
does not seem critical in macrophyte culture.  Elodea
made reasonably good growth over the range of light
likely to be available in artificially lighted growth
chambers.  On this project, light intensities were main-
tained in the range of 800-1700 ft candles.

As with terrestrial species, light intensity had a marked
effect on plant morphology and color.  At the lower light
intensities, the Elodea was unusually dark green in
color and the leaves were thinner and the internodes
longer than in the plants grown at 1700 ft candles.
                          48

-------
Optimum Temperature

The temperature of 23°C  used for macrophyte culture on
this project was established from the data presented
in Figure 5.  The growth of  ETodea,  as length increase
                                          r*- Constant 23°C
                                        —Control
                                        —Variable
              23*
29° '  32
                                            * '
                Variable temperature (°C)
 Figure  5.   The  growth of Elodea occidentalism  as length in-
   crease/  during exposure to various temperatures.
was  compared in a set of trays maintained at 23°C and
in trays  exposed to temperatures varying from 20 to
32°C during different phases of the culture period.

Growth was  slightly less at 20°C than at 23°.  There were
were only slight differences in growth at 23°C and
temperatures up to. 32°C.  It seems, therefore, that Elodea
is not highly sensitive to temperatures within the 20 to
30°C range.
                           49

-------
C0'2 Concentration

For many years, it has been common practice to bubble
CO2-enriched air through algae cultures.  For unicellular
green algae/ 5% COa has been widely used.  There was
reason, therefore, to question that the recommended
1% was the optimum CO2 level for macrophytes under the
culture conditions employed.  In experiments which will
not be reported in detail, COa concentrations less than
0.4% resulted in a reduced rate of Elodea growth.  The
rate of growth was, however, not increased by raising the
COa concentration above 0.4%.  The maximum concentration
tested was 2.0% which was not inhibitory to the Elodea.
                      DISCUSSION

The nutrient medium for macrophytes developed in this
study was demonstrated to produce excellent growth of
several species.  It is anticipated the medium will be
equally suitable for other macrophytes.  This optimism
is based on general experience with plant culture media.
Once the general features of a nutrient medium are established,
it usually is satisfactory for many species.  Species with-
in a major taxonomic group are not sensitive to minor
variations in nutrient solution composition.

Establishing techniques and conditions necessary for
laboratory culture of macrophytes should stimulate
basic studies on the neglected area of the nutrition
and physiology of these organisms.  This background
information is critical for understanding the behaviour,
distribution, and ecology of the macrophytes.  Laboratory
studies can be particularly useful in providing the basic
data on macrophyte nutritional requirements needed in
developing measures to control nuisance growths of these
organisms.
                          50

-------
                      SECTION VII

                    ACKNOWLEDGMENTS
The assistance of Kathy Fishbeck, Ann Mickle, Marcia
Wuenscher, Frann Hutchison, Brian Marcks, and Dean Radtke
in various aspects of the work reported is gratefully
acknowledged.

This work was supported and financed by a grant from the
Water Quality Office of the Environmental Protection
Agency.  Appreciation is expressed for the encouragement
and help provided by the Grant Project Officer, Dr. Gary
Glass of the National Water Quality Laboratory, Duluth,
Minnesota.  His sincere interest in the work extended his
role well beyond that of administrator of funds.
                           51

-------
                     SECTION VIII

                      REFERENCES
Black, J. J., L. M. Andrews, and C. W. Threinen.  Surface
   water resources of Vilas County.  Wisconsin Conservation
   Department.  1963.

Blanchar, R. W. , G. Rehm, and A. C. Caldwell.  Sulfur in
   plant materials by digestion with nitric and perchloric
   acids.  Soil Sci. Soc. Amer. Proc. 29: 71-72.  1965.

Bould,C.E., E.G. Bradfield, and G. M. Clarke.  Leaf analysis
   as a guide to the nutrition of fruit crops.  I.  General
   principles,  sampling techniques, and analytical methods.
   J. Sci. Food Agr. 11: 229-242.  1960.

Bourn, W. S.  Ecological and physiological studies on certain
   aquatic angiosperms.  Contrib. Boyce Thompson Inst. Plant
   Res. 4: 425-496.  1932.

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.I.B.S. Washington, D.C.  1961.

Chapman, H. D.  Diagnosis criteria for crops and soils.
   Univ. of Calif. Div. of Agricultural Sciences.   1966.

Fitzgerald, George P.  Field and laboratory evaluations of
   bioassays for nitrogen and phosphorus with algae and
   aquatic weeds.  Limnol. Oceanogr. 14: 206-212.   1969.

Gerloff, G. C.  and F. Skoog.  Nitrogen as a limiting factor
   for the growth of Micro cystis aeruginosa in southern
   Wisconsin lakes.  Ecology 38: 556-561.  1957.

Gerloff, G. C.  Evaluating nutrient supplies for the growth
   of aquatic plants in natural waters.   (In) Eutrophication:
   Causes, Consequences, Correctives.  Proc. Symp. Nat. Acad.
   Sci., Washington, D.C.  1969.

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.

Goldman, C. R.  Molybdenum as a factor limiting primary
   productivity in Castle Lake, California.  Science 132:
   1016-1017.   1960.
                            53

-------
Goldman, C. R.  Primary productivity and micronutrient
   limiting factors in some North American and New Zealand
   lakes.  Verh. Intern. Verein. Limnol. 15: 365-374.  1964.

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 Mailing, Madistone, Kent.  1966.

Jackson, M. L.  Soil Chemical Analysis.  Prentice-Hall, Inc.
   Englewood Cliffs, N. J.  1958.

Johnson, C. M. and T. H. Arkeley.  Determination of molybdenum
   in plant tissue.  Analytical Chem. 26: 572-573.  1954.

Johnson, C. M. and A. Ulrich.  Analytical methods for use in
   plant analysis.  Exp. Bulletin 766.  California Agricultural
   Experiment Station.  Berkeley, Calif.  1959.

Lee, G. F.  Analytical chemistry of plant nutrients.  (In)
   Eutrophication:  Causes, Consequences, Correctives.  Proc.
   Symp. Nat. Acad. Sci., Washington, D.C.  1969.

Lund, J. W. G.  Phytoplankton.   (In) Eutrophication:  Causes,
   Consequences, Correctives.  Proc. Symp. Nat. Acad. Sci.,
   Washington, D.C.  1969.

Melstead, S. W., H. L. Motto, and T. R. Peck.  Critical
   plant nutrient composition values useful in interpreting
   plant analysis data.  Agron. Jour. 61: 17-20.  1969.

Ryther, J. H. and W. M. Dunstan.  Nitrogen, phosphorus, and
   eutrophication in the coastal marine environment.  Science
   171: 1008-1013.  1971.

Reuther, W.  (ed.) Plant analysis and fertilizer problems.
   Publ. No. 8, A.I.B.S.  Washington, D.C.  1961.

Smith, P. F.  Mineral analysis of plant tissues.  Ann. Rev.
   Plant Phys. 13: 81-108.  1962.

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.

Ulrich, A.  Physiological bases for assessing the nutritional
   requirements of plants.  Ann. Rev. Plant Phys. 3: 207-
   228.  1952.

                          54

-------
Ulrich, A.  Plant analysis in sugar beet nutrition.   (In)
   W. Reuther  (ed.)> Plant Analysis and Fertilizer Problems,
   Publication 8, Amer. Inst. Biol. Sci., Washington, D. C.
   1961.

Wallace, A.  Current Topics in Plant Nutrition.  Department
   of Agricultural sciences  (Plant Nutrition), U.C.L.A.,
   Los Angeles, Calif.  1961.
                          55

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                      SECTION IX

                       APPENDIX

The concentrations of essential elements in first and second
one-inch segments of Elodea oc c i dentalls and Cera tophy Hum
demursum collected at intervals from various Wisconsin lakes
during 1970.

Table                                             Page
  A         Allequash Lake/ Elodea                 58

  B         Clear Lake, Elodea                     59

  C         Little Spider Lake, Elodea             60

  D         Erickson Lake, Elodea                  61

  E         Whitney Lake, Elodea                   62

  F         Nebisch Lake, Elodea                   63

  G         Big  Kitten Lake, Elodea                64

  H         Big  Kitten Lake, Ceratophyllum         65

  I         Little John Lake, Ceratophyllum        66
                            57

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A.  Allequash Lake, Elodea.
Date
Sampled

June 11
01
00

July 6

July 27

Aug. 18
Segment
1"
2"
1"
2"
1"
2"
1"
2"
N
P
S K
JAS Std .
3.71 0
3.55 0
3.42 0
2.91 0
3.53 0
3.19 0
2.94 0
2.82 0
.47
.35
.31
.24
.34 0.31
.28 0.22
.42
.35
2.10
2.20
1.90
1.79
2.00
1.90
0.26 2.20
0.29 2.30
Ca
JAS Std.
0.47
0.57
0.42
0.59
0.42 0.41
0.61 0.54
0.55
0.80
Mg
JAS Std.
0.22
0.18
0.18
0.19
0.23 0.18
0.21 0.14
0.20
0.22
Fe
XLOOO
>1000
610
775
745
XLOOO
XLOOO
950
B
18.1
16.6
11.6
15.4
15.9
18.8
14.4
17.0
Ppm
Cti
JAS Std.
4.7
4.2
2.3
2.6
3.8 6.5
3.4 7.0
5.7
6.1
Zn
123
128
87
101
90
118
64
88
Mn Mo
70
103
116 0.58
193
83
168
93
158

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      B.  Clear  Lake,  Elodea.
Ul
Date
Sampled

June 18


July 8


July 29


Aug. 20

Segment
1"

2"
1"

2"
1"

2"
1"

2"
N P
JAS Std.
3.27 0.56 0.39

2.67 0.51 0.32
2.47 0.47 0.28

2.28 0.33 0.24
2.41 0.42 0.30

1.94 0.28 0.24
2.62 0.42 0.27

2.03 0.38 0.27
,S
K
Ca
JAS Std.
2

2
2

2
0.30 2

0.28 2
2

2
.10 0

.65 0
.10 0

.35 0
.00 0

.35 0
.30 0

.75 1
.31

.44
.68

.73
.69 0.65

.66 0.80
.85

.05
Mg
JAS Std.
0.21

0.20
0.17

0.17
0.21 0.15

0.16 0.14
0.17

0.17
Fe
995

>1000
>1000

>1000
715

665
750

555
B
17.5

21.6
16.0

14.3
15,9

14.8
13.8

11.3
Ppm
Cu Zn
5.9 77

5.5 95
4.3 106

2.3 90
2.2 131

1.8 103
2.2 89

<1.0 93
Mn
1000

1000
1000

1000
356

398
392

1000
Mo
0.54


0.73









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C.  Little Spider Lake, Elodea.

Date
Sampled Segment
1"
June 15
2"
en
o
1"
July 7
2"
1"

N



P

S
-o
K
JAS Std.
3.59

2.78
3.75
3.08
3.36
0

0
0
0
0
.47

.30
.42
.28
.44 0.37
2

2
2
2
0.29 2
.45

.80
.20
.65
.65

Ca
JAS Std.
0.44

0.66
0.44
0.62
0.52 0.55

Mg
JAS Std.
0.28

0.29
0.26
0.25
0.31 0.25

Fe
740

775
610
625
390

B
18.8

19.9
14.3
13.1
17.0
Ppm
Cu
JAS Std.
2.1

<1.0
2.3
<1.0
3.8 6.5

Zn Mn
64 170

80 268
79 215
80 290
127 165
July 28
           2"     2.59 0.28 0.27 0.31 2.65 0.61 0.64 0.28 0.24  400 16.6  2.2 4.5 105 259

           1"     2.64 0.32           2.75 0.68      0.32       425 14.4 <1.0     108 198
Aug. 19
           2"     2.29 0.23           2.90 0.85      0.30       345 13.8 <1.0      89 286

-------
D.  Erickson Lake, Elodea.
                   N
                     K
                                   Ca
 Date
Sampled  Segment
                  Mg
Fe
    Ppm

B   Cu   Zn  Mn  Mo
     JAS  Std.
                                JAS  Std. JAS  Std.
June 16
July 9
July 28
Aug. 19
Sept. 8
1"

2"

1"

2"

1"

2"

1"

2"

1"

2"
2.80 0.36 0.33 0.24       >1000 17.0 7.9  99  96 0.59

3.10 0.37 0.36 0.18 0.16  >1000 18.1 4.1  77 121
3.96 0.54

3.40 0.39

3.70 0.44           2.35 0.32 0.28 0.21

2.94 0.23           2.80 0.61 0.37 0.17 0.15

3.41 0.47 0.41 0.27 2,75 0.28 0.28 0.22 0.18

2.70 0.29 0.28 0.28 2.75 0.44 0.40 0.18 0.14

3.28 0.49           2.55 0.44 0.42 0.21

2.59 0.34           2.75 0.59 0.52 0.18 0.15
                            695 11.4 2.6 114  80

                            670 12.1 2.6 161 109

                            465 13.1 3.8  97  65

                            440 15.9 2.2 145  98

                            730 12.7 4.7 100  80

                            450 12.7 2.2  78  65

                            605 10.7 3.1  80 114
2.89 0.42           1.20 0.69 0.56 0.18

2.15 0.29           1.54 0.78 0.72 0.19 0.16    365  9.4 1.5  79 116

-------
     E.  Whitney Lake, Elodea.
                         N
                              K
        Ca
      Date
     Sampled  Segment
Mg
Fe
   Ppm


B   Cu   Zn  Mn  Mo
             JAS  Std.
     JAS  Std  JAS  Std.
Cfi
to
     June 18
     July 8
     July 29
     Aug. 20
     Sept. 9
1"     2.88  0.38


2"     2.23  0.34


1"     2.66  0.28            1.85 0.61      0.16


2"     2.18  0.20            2.20 0.82      0.18


1"     2.34  0.23 0.21  0.32 1.42 0.68 0.74 0.15 0.10


2"     2.02  0.21 0.17  0.42 1.76 0.89 1.02 0.17 0.15


1"     3.09  0.37            2.30 0.57      0.20


2"     2.52  0.33            2.65 0.87      0.19


1"     3.50  0.49            2.30 0.44      0.20


2"     2.98  0.39            2.65 0.64      0.18
1.63 0.32 0.32 0.17 OU4  >1000 19.1 2.5  75  63 0.53


1.77 0.44 0.42 0.17 0.14  >1000 22.5 3.7  97  93


                            805 12.1 2.3  86 100 0.53


                            765 13.6 2.6 112 150


                            999 15.4 2.2 125 137


                            790 11.1 1.8 141 155


                            965 11.7 2.2 158  93


                            965 14.4 2.2 174 126


                            830 10.2 2.6 115  65


                            890 13.K1.0 109 103

-------
      F.   Nebisch Lake,  Elodea
%
N P K

Sampled Segment JAS Std.
Ppm
Ca Mg Fe B Cu



Zn Mn


                    1"      3.76   0.75   0.44   2.65   0»42   0.22    660   11.6   8.1   135   119
      Sept.  10
2                   2"      4.41   0.56   0.38   2,80   0.61   0.19    580   10.7   5.0   123   160

-------
G.  Big Kitten Lake, Elodea
                 N
                              K
                                 Ca
 Date
Sampled Segment
                       Mg
Fe   B
 Ppm

Cu     Zn  Mn
Mo
           JAS  Std .
JAS  Std. JAS  Std. JAS  Std.
                                                            JAS Std
June 18
July 8
July 29
Aug. 20
Sept. 9
1"

2"

1"

2"

1"

2"

1"

2"

1"

2"
3.11 0.49 0.38      2.55 2.97 0.87 0.81 0.23 0.18 685 15.4  2.5 5.3  67 >1000 0.50

2.74 0.44 0.27      2.80 3.15 1.11 0.90 0.22 0.16 595 17.5  2.5     133 >1000

3.840.560.50      2.45      0.66 0.73 0.22 0.22 -305 10.9  2.34.5  89   223

3.13 0.44 0.35      2.90 3.09 0.89 0.83 0.19 0.18 350 12.7  1.0      87   356

2.71 0.42 0.39 0.29 2.45 2.28 1.61 2.24 0.22 0.21 260 12.0 <1.0 3.9  74   313

2.16 0.40 0.34      2.45      1.51 2.61 0.23 0.22 225 13.5  2.2 2.7  78   348

2.62 0.38 0.34      2.30 2.50 1.69 1.80 0.23      400 11.6 <1.0 1.8  55   240

2.13 0.34 0.33      2.45 2.54 2.03 2.59 0.25      295 12.2 <1.0      43   290

2.54 0.44 0.34      1.85 2.15 2.41 2.09 0.22      260  5.3 <1.0 1.9  64   348

2.35 0.51 0.35      2.00 2.01 2.01 3.51 0.23      180  7.1 <1.0      58   333

-------
H.  Big Kitten Lake, Ceratophyllum
                                        %                                          Ppm

                   N       PS       K        Ca        Mg       Fe  B      Cu    Zn  Mn
 Date                  	       	            —=	
Sampled  Segment       JAS  Std.      JAS  Std. JAS  Std. JAS  Std.            JAS Std.

           1"     3.48 0.42 0.36 0.37 4.05 4.62 0.15 0.27 0.61 0.66  200 17.5 <1.0 3.1 63 >1000
July 29
           2"     2.48 0.23 0.23      4.35      0.28      0.64       245 17.5 <1.0 2.7 87 >1000

           1"     2.45 0.28 0.28      3.45      0.29 0.68 0.83       255 13.8 <1.0 2.2 33   368
Aug. 20
           2"     1.86 0.19 0.19      3.45 4.19 0.37 0.35 0.72 0.83  345 14.4 <1.0     24 >1000

           1"     2.48 0.42 0.27 0.27 3.45 4.14 0.32 0.29 0.72       140 14.6  1.5 2.9 44   374
Sept. 9
           2"     1.81 0.29 0.19 0.26 3.65 4.03 0.31 0.28 0.78       150 14.6 <1.0     39 >1000

-------
I.   Little John Lake, Ceratophyllum
                                                                              Ppm
                 N
K
Ca
Mg
Fe
Cu   Zn   Mn
UctLC
Sampled Segment


y\
3\


fr
p
w
H
9
a
g
H
3
|
9
o
o
3
H1
vD
•^j
1
Ul
r
1"
June 10
2"
1"
July 6
2"

1"
July 27
2"


1"
Aug. 18
2"


1"
Sept. 8
2"



3.48

2.84
2.73

2.35

2.14

1.86


2.27

1.96


3.00

2.35



JAS Std.
0.58

0.56 0.35
0.51 0.38

0.51 0.33

0.30 0.24

0.30 0.20


0.34 0.28

0.30 0.21


0.75 0.49

0.61 0.35



JAS Std.
3

2
3

2

0.19 3

0.20 2


2

2


0.29 3

0.28 3



.00

.45 2.74
.65 3.69

.90 3.50

.10 3.59

.75 3.66


.65 3.44

.75 2.96


.25 4.23

.00 3.96



JAS
0.24

0.42
0.10

0.21

0.13

0.17


0.14

0.16


0.23

0.24



Std. JAS Std.
0.

0.33 0.
0.19 0.

0.25 0.

0.21 0.

0.24 0.


0.26 0.

0.23 0.


0.27 0.

0.28 0.



70

85
64

73 0.77

64 0.56

78 0.72


50

64 0.62


80

78



850 17

>1000 28
415 14

520 16

525 13

660 13


685 13

540 12


925 18

>1000 18



JAS
.5 3.0

.3 5.6
.9 1.45

.3 1.00

.5 <1.0

.5 <1.0


.8 1.4

.7 <1.0


.9 1.5

.9 1.5



Std
79 >1000

4.4 96 >1000
4.1 49 >1000

49 >1000

1.0 32 >1000

1.5 41 XLOOO


3.8 28 >1000

2.8 30 >1000


2.0 49 >1000

46 >1000




-------
  SELECTED WATER
  RESOURCES ABSTRACTS
  INPUT TRANSACTION FORM
         1. Report No,
  4. Title

 Plant Analysis for Nutrient Assay of Natural Waters
  7. AuthoT(s)

 Gerloff, Gerald  C.
  9. Organization
 Department of  Botany and Institute  of Plant Development
 University of  Wisconsin - Madison


  12. Sponsoring Organization

  15. Supplementary Notes

                Environmental Protection Agency report number
 	       EPA-R1-73-001. February 1973.	
             3. Accession No.

             w

             5. Report Date
             6.
             8. Performing Organization
               Report No.
            10. Project No.
              18040 DGI
                             11. Contract/Grant No.
                              & Type of Report and
                                Period Covered"
  16.  Abstract

 Plant analysis has been developed as a procedure for evaluating nutrient  supplies and
 growth-limiting nutrients for nuisance macrophytes in lakes and streams.   Plant
 analysis is based on establishing in index segments the critical concentration
 (minimum plant concentration for maximum yield)  of each essential nutrient element
 likely to limit growth of nuisance macrophytes.   Critical concentrations  for nitrogen,
 phosphorus, sulfur,  calcium, magnesium,  potassium, iron, manganese,  zinc,  boron,  and
 molybdenum were established in appropriate index segments of Elodea  occidentalis.  The
 critical copper concentration was estimated.   Critical concentrations  for several
 elements also were established in Ceratophyllum demursumu

 To evaluate plant analysis as an assay procedure, index segments of  Elodea and
 Ceratophyllum were routinely collected from Wisconsin lakes, analyzed,  and the analyses
 were compared with the critical concentrations for indications of nutrient deficiency.
 Nitrogen, phosphorus, calcium, and copper  were at or close to critical  levels in one
 or more lakes.  Neither nitrogen nor phosphorus seemed  to be a general growth-
 limiting nutrient in the lakes sampled.
  17a. Descriptors
 Nutrient Requirements, Deficient  Elements, Mineral Needs,  Eutrophication, Aquatic
 Weeds, Bioassay,  Fertility.
  Hb. Identifiers
 Wisconsin  Lakes
 Macrophytes
  17c. CO WRR Field & Group
  18.  Availability
19. Security Class*
                          20. Security Class.
     21. No. of
Send To:
     22. Price
WATER RESOURCES SCIENTIFIC INFORMATION CENTER
U.S. DEPARTMENT OF THE INTERIOR
WASHINGTON, D. C. 20240
  Abstractor   G.  C.  Gerloff
^institution  University of Wisconsin-Madison
WRSIC 102 (REV IUNE 1971)
                                            SPO 913.261

-------