TISSUE ANALYSIS AS A MEASURE OF NUTRIENT AVAILABILITY FOR THE GROWTH OF ANGIOSPERM AQUATIC PLANT!? G. C. Gerlof-j and P. H. Krombhok Department

of Botany,

University

of Wisconsin,

Madison

53706

ABSTRACT A tissue analysis technique was used to evaluate nitrogen and phosphorus supplies in natural waters for the growth of angiosperm aquatic plants. Tissue content of nitrogen and phosphorus was employed as an index of element availability in lakes from which plants were collected. This required establishment in the laboratory of the critical level for each element, that is, the minimum tissue content associated with maximum growth. To establish critical levels, a system was developed for culturing algae-free plants in a synthetic nutrient medium. The critical nitrogen content for the several species studied was approximately 1.3%; the comparable phosphorus value was 0.13%. The nitrogen and phosphorus contents of 13 species of aquatic plants collected during the summer from nine lakes were compared with the critical concentrations. In nine samples obtained mostly during periods of heaviest plant growth, the phosphorus content was at or below the critical level; in no sample was the nitrogen content less than the critical concentration. The results indicated that in all but one of the lakes, phosphorus supply was more likely to limit higher aquatic plant growth than was nitrogen. The primary importance of this study is as an initial step in the development of a satisfactory technique for evaluating nutrient supplies in natural waters.

INTRODUCTION

Problems arising from the accelerated pollution of lakes and streams have emphasized the urgent need for procedures by which the availability of nutrients for the growth of aquatic plants in natural environments can be correctly evaluated. These procedures would be of great value to aquatic biologists and sanitary engineers forced to make decisions on the effects of specific pollution sources on the nuisance conditions resulting from growths of algae and higher aquatic plants in lakes and streams ( Lea, Rohlich, and Katz 1954). Plant nutritionists for many years have been concerned with the evaluation of nutrient supplies for the field culture of agricultural and horticultural crops. In some cases, soil chemical extraction procedures and tests which approximate the capacities of crop plants to remove nutrients from soils have been developed and applied. l This work was made possible by a grant to the University of Wisconsin from the Soap and Detergent Association. The assistance of Steven Curtis and Gerald Lower in analyzing tissue samples and of Philip Doepke and William Hauser in collecting plants is gratefully acknowledged.

The numerous problems associated with these chemical tests have led, however, in recent years to the wide application (Lundegardh 1951; Reuther 1961) of a technique commonly designated as tissue anal ysis. In this approach, the concentration of an element in a plant is considered a reliable indicator of the availability of that element in the environment in which the plant grew. The concentration of any element in a plant may, of course, vary over a relatively wide range. Application of the tissue analysis technique requires the establishment of a critical level or concentration for each element, that is, the minimum tissue content in a particular species that is necessary for maximum growth. When growth requirements other than nutrients are adequate, tissue contents below a critical concentration are associated with deficiencies of that element resulting in less than maximum yields; tissue contents above a critical concentration have no effect on yields and for this reason are referred to as luxury consumption. The critical concentration for a particular element must be carefully established in controlled experiments in

529

530 TABLE

solution

G. C. GERLOFF

AND

1. Composition of a modified Hoagland’s used for the culture of angiosperm aquatic plants

Salt

KNO,

0.5 M stock solution in l-liter final solution (ml)

2.0 2.0

MgSOc7H,O

0.8

KH,POd KCl* H,BO,* MnS04*HzO* ZnSO,*7HzO* CuSOa- 5HzO* (NH&Mo70zc4HzO* Fe* EDTA

0.4 -

Element in final solution (mm)

N -42 K -47 Ca -40 P - 6.2 S -12.8 Mg9.6 Cl B MnZn Cu MOFe -

1.77 0.27 0.27 0.13 0.03 0.01 0.40

* Trace element stock solutions were prepared at 1,000 X the concentration of the final solution. One ml of each stock solution was added to the final culture medium.

which yield can be related to varying tissue concentrations of that element. Tissue analysis seems particularly adaptable to evaluations of the availability of mineral elements for the growth of aquatic organisms in lakes and streams. Efforts to develop the alternative approach of relating nutrient concentrations in the water to potential for supporting plant growth have been reviewed by Fogg ( 1965). Several factors complicate this approach, for example, the difficulty of analyzing for the very low concentrations of elements in natural waters, the problem of distinguishing between available and nonavailable forms of an element, and the fact that the volume of water from which an organism withdraws a nutrient element as well as the concentration of the element determines the total available supply. In some cases, the independent shifting and movement of water and organisms make it difficult to obtain water samples that reliably represent the substrates from which specific plants absorbed nutrients. Because of these problems, Gerloff and Skoog (1954, 1957) used a tissue analysis technique to evaluate nutrient availability in Wisconsin lakes for the growth of the bloom-producing blue-green alga Microcystis aeruginosa.

P. H.

KROMBHOLZ

The data to be presented were obtained in attempts to apply a tissue analysis procedure in evaluating nitrogen and phosphorus availability to angiosperm aquatic plants in lakes and streams. This required establishment of critical nitrogen and phosphorus concentrations in several common aquatic species under laboratory conditions. To accomplish this, a system was developed for growing algae-free cultures of these organisms in synthetic nutrient media. The established critical levels then were compared with the nitrogen and phosphorus contents of a number of higher aquatic plants collected at intervals throughout the growing season from Wisconsin lakes varying considerably in general fertility and productivity. EXPERIMENTAL

PROCEDURE

Algae-free cultures in a synthetic nutrient medium There are relatively few reports (Bourn 1932) of successful attempts to grow higher aquatic plants in synthetic nutrient media. Because this was essential for the establishment of critical nitrogen and phosphorus levels, modifications of one of the Hoagland solutions ( Hoagland and Snyder 1933; Johnson et al. 1957)) the media most commonly used in nutrient culture experiments with crop plants, were compared for the culture of several aquatic species. The most satisfactory modification, used in all experiments to be reported, is presented in Table 1. This medium contains only onefifth the concentrations of the major essential elements present in the original Hoagland’s solution but the full-strength concentrations of the trace elements. Iron was added to the cultures as a mole : mole complex with EDTA and usually at an initial concentration of 0.4 ppm. After the plants were well established in a series of cultures, another 0.4 ppm of iron was added. The Fe-EDTA complex was autoclaved separately from other constituents of the medium. In attempting to use this medium in actual experiments, it was immediately obvi-

TISSUE

ANALYSIS

TO

DETERMINE

ous that algae-free cultures of the experimental plants were essential. Otherwise, the abundant algae growth made it impossible to interpret the results in terms of the nutritional requirements of the higher aquatic species. The most satisfactory technique for obtaining algae-free plants was to immerse the appropriate seeds in X-strength Clorox (equivalent to 1.05% hypochlorite ) for 15 min. The seeds then were rinsed in sterile distilled water, and the seed coats were cut with a sterile razor blade. It has been reported that the seeds of many aquatic plants are dormant immediately after release in the autumn, and that a cold treatment of 5-7 months is necessary to break this dormancy (Muenscher 1936). In the present study, the seeds sprouted readily after the seed coat was chipped. Seeds were sprouted in absolute darkness to reduce further growth of any algae not killed by the Clorox. The seedlings were separated from the seed coat, were rinsed several times in sterile distilled water, and then were placed in autoclaved nutrient solution. With this technique, the following species (Fassett 1957) were obtained in algae-free culture: Cerutophyllum demersum, Vallisneriu americanu, Hetwanthera dubia, E lodea occidentalis, Najas f lexilis, and Zannicheliu palustrb. All of these organisms except IV. flexilis and C. demersum grew well in the nutrient medium (Table 1). Further work must be carried out to determine the reason for the less than optimum growth of the latter two plants. The relatively high concentrations of the trace elements suggest a toxicity from one or more elements in this group. In any case, further modifications of this medium are suggested before it is used for plants and culture conditions different from those of the present study. Experiments

to establish critical levels

The technique used to establish nitrogen and phosphorus critical levels involved growth of a particular species in a series of duplicated cultures similar in all respects except for various concentrations of either nitrogen or phosphorus. The cultures con-

NUTRIENT

AVAILABILITY

531

sisted of 3-liter Florence flasks containing 2 liters of nutrient medium. The flasks were stoppered and closed except for aeration and exhaust tubes provided with cotton filters that passed through the stoppers. Each complete assembly was sterilized by autoclaving for 20 min. Air enriched with COs (0.5 to 1.0% CO,) was continually bubbled into all cultures, with the rate of flow adjusted to provide a pH of 6.0 to 6.5 in freshly inoculated media. All cultures were kept in a constant environment room maintained at approximately 25C under a light intensity of 5,400-6,500 lux. The cultures in a particular experiment were inoculated with small sections, approximately equal in weight, of the appropriate species removed from continuously maintained stock cultures. There was no sand or other rooting medium in the culture flasks. As growth developed in a series of cultures containing various concentrations of nitrogen or phosphorus, the supply of the element varied was progressively exhausted at the lowest concentrations, and the concentrations of the element in the plants represented growth-limiting values. With higher external concentrations, plant contents represented various degrees of luxury consumption. To establish critical nitrogen and phosphorus values, the plants were harvested when a range of growth and of nitrogen or phosphorus concentration in the plant tissues was represented; oven-dry yields were determined; plant tissues were analyzed for either total nitrogen or phosphorus, and the yield and tissue content data were plotted. Harvested plants were dried in a forced-draft oven at 65-706. Nitrogen analyses were by a semimicroKjeldahl procedure; phosphorus determinations were by a vanadomolybdate (yellow complex) procedure following wet-ashing of the tissues in a HNO,-H,SO,-HClO, mixture. Field collection of samples Plant sampIes to be analyzed for total nitrogen and phosphorus were collected from a number of Wisconsin lakes. The shallow root systems as well as the shoots

532

G. C. GERLOFF

AND P. I-1. KROMBHOLZ

I 1.0 NITROGEN

2.0 CONTENT

OF

3.0 TISSUE

IN PER

4.0 CENT

FIG. 1. Relationship between the oven-dry weight and the nitrogen content of Vallisneria americana when cultured in a nutrient medium containing various concentrations of nitrogen,

were obtained in sampling V. americana, Eriocaulon septangulare, and Lob&a dortmania, plants that send up leaves from a crown. All the other species produce stems bearing leaves, and the root systems and underground stems of these plants were not included in tissue samples. Collections were made to water depths of approximately 1.5 m, using a rake to pull up the plants. In successive samplings of a particular lake, samples were obtained from approximately the same area at each date. Only plants that appeared healthy and vigorous were taken. Immediately alter sampling, dead leaves, mud, and other debris were carefully removed from the plants by hand washing and by spraying with a high velocity jet of water. Plants coated with calcium precipitates were rinsed in 2.0-3.0% HCl for about 1 min and then in freshwater. After free water was removed by blotting with gauze towels, the plants were dried at room temperature before the final drying at 6570C in a forced-draft oven. Before analyses, all tissues were ground in a micro-Wiley mill, and samples were stored in tightly-stoppercd bottles.

I 0.1

I 0.2

t 0.3

PHOSPHORUS

I 0.4 (iONTENT

I 0.5

I 0.6 OF TISSUE

I 0.7

I 0.8

,

IN PER CENT

FIG. 2. Relationship between the oven-dry weight and the phosphorus content of Vallisneriu americana when cukured in nutrient media containing various concentrations of phosphorus.

dentalis, V. americana, C. demersum, and H. dubia. Examples of the graphs of yield vs. tissue content from which critical values were determined arc: presented in Figs. 1 and 2, plotted from I’. americana data. The detailed data from the nitrogen experiment also are presented ir Table 2 to permit an evaluation of the agreement between duplicate samples and to :?rovide a basis for calculating recovery in the plant tissues of the nitrogen added to the culture medium. The curves in Figs. 1 and 2 show that the nitrogen and p:hosphorus contents in V. americanu varied over a wide range, from 0.78 to 4.28% nitrogen and from 0.10 to 0.70% phosphoru:;, and that below approximately 1.3% nitrogen and 0.13% phosphorus, the plant content of either element was closely correlated with plant yield. The critical nitrogen concentration for V. americana seems, therefore, to be approximately 1.3% and the critical phosphorus concentration 0.13%. Tissue contents above these values represent 1u:;ury uptake which is without effect on plant yield. The critical concentrations for the other species studied were approximately the same as the values for V. americana. This consistency does not in general correlate EXPERIMENTAL RESULTS with results from agricultural and horticultural species. There is, however, likely to Establishing critical nitrogen and be less variation in the amount of woody, phosphorus concentrations constituents in aquatic nonprotoplasmic species than in crop plants. This would Experiments were carried out to establish the critical levels of nitrogen and phos- minimize species variations in critical conphorus in the following species: E. OCC~- tents.

TISSUE

TABLE 2.

N content of medium (ppm)

84 42 21 10.5 4.2 2.1

ANALYSIS

TO

DETERMINE

NUTRIENT

533

AVAILABILITY

Plant yield, plant nitrogen content, and the degree of nitrogen recocery when Vallisneria americana was cultured in solutions of various NOs-N contents Oven-dry

plant-wt

(g)

1

2

Avg

2.18 2.45 2.36 1.14 0.68 0.48

1.89 2.13 2.29 1.14 0.71 0.47

2.04 2.29 2.32 1.14 0.70 0.48

Tissue

1

4.34 2.57 1.30 1.32 0.88 0.75

It is apparent from Table 2 that there was reasonably good agreement between the duplicate values for weight determinations and total nitrogen analyses, particularly in treatments that resulted in tissue contents at or below the critical values. Throughout the experiments, the results were often quite inconsistent in cultures that contained high concentrations of nitrogen or phosphorus, due to uneven initiation of growth of the inoculum. The effects of these differences would be minimized in cultures in which the supply of an element was exhausted partway through the growth period. In cultures containing 21 ppm or less nitrogen, most of the element added to the medium was recovered in the plants (Table 2). At the highest external concentrations, there was far more nitrogen in the medium than the plants absorbed, even with considerable luxury consumption. Nitrogen and phosphorus contents of plunts collected from lakes Following establishment of the nitrogen and phosphorus critical concentrations, an attempt was made to use this information in evaluating nitrogen and phosphorus availability in Wisconsin lakes. The critical concentrations were compared with the nitrogen and phosphorus contents of various plants collected from nine Wisconsin lakes at four intervals during the summer. The lakes were selected to represent a range of fertility, as indicated by water hardness and by general observations on productivity. Sampling was not limited to the species cultured in the laboratory, and the same species could not be obtained in all lakes

content

of N

(% )

2

Avg

4.22 3.04 1.35 1.33 0.86 0.80

4.28 2.80 1.33 1.33 0.87 0.78

N added cultures (mg)

168 84 42 21 8.4 4.2

to

N recovered in plants (mg)

87 64 31 15 6.1 3.7

or on successive sampling dates. Examples of the results of the collections and analyses from two of the lakes are presented in Tables 3 and 4. The analyses show that the nitrogen and phosphorus contents of different species from the same lake on a particular date were by no means the same. In Lake Mendota for example, on 18 August 1964 the nitrogen and phosphorus concentrations in Myriophyllum spp. were 2.63 and 0.35%, respectively, while in Potamogeton xosteriformis the concentrations were 3.65 and 0.59%. Similar variations would probably be encountered in a mixture of land plants from the same site and undoubtedly reflect differences both in plant capacity to accumulate the two elements and in growth habit. The variations also suggest that comparisons of lake fertility through tissue analysis should be based on comparisons of the analyses of samples of the same species. Nitrogen and phosphorus values from early in the season, obtained when the water is relatively rich in nutrients and before the development of heavy plant growth, should in general reflect the basic fertility of lakes while the midseason values, obtained when plant growth is heaviest, should indicate whether the supply of a specific element had been sufficiently reduced to become limiting for optimum growth of particular species. The average figures for the samples obtained on the first sampling date, show that in general the nitrogen and phosphorus contents of the plants did correlate well with the recognized fertility of the two lakes. Lake Mendota in southern Wisconsin is a highly pro-

534 TABLE

G. C. GERLOFF

AND

P. H.

KROMBHOLZ

3. The total nitrogen and phosphorus contents of samples of higher aquatic plants collected at interoals during the growing season from Lake Mendota, a highly 29 June Plant species

%N

Ceratophyllum demersum Heteranthera dubia Myriophyllum spp. Potamogeton richardsonii Potamogeton zosteriformis Vatlisn4ha americana * Methyl

orange

alkalinity-140

4.43 3.79

2.72 -

3.65 3.85 ppm;

1964

22 July

1964

18 Aug

fertile 1964

%P

%N

%P

%N

%P

0.75 0.55 0.41

2.11 3.24 2.42 3.73 3.70 2.88

0.51 0.69 0.35 0.45 0.35 0.42

2.17 2.32 2.63 3.24 3.65 2.34

0.56 0.51 0.35 0.32 0.59 0.43

mmhos;

and

(Poff

-

0.42 0.42

conductance-281

ductive lake, which is reflected in average nitrogen and phosphorus contents of 3.69 and 0.51%, respectively, on the initial sampling date (29 June 1964). Comparable values for plant samples from Lake Nebish, a relatively infertile lake, were only 2.24 and 0.21%. The very high fertility of Lake Mendota was again apparent in the July, August, and September samplings. In no case was either the nitrogen or phosphorus content of a sample below the suggested critical concentrations of 1.3% nitrogen and 0.13% phosphorus; in fact, the values far exceeded these concentrations. A comparison of the degree to which nitrogen and phosphorus contents exceed the critical values in the two species lowest in nitrogen (C. demersum on 22 July 1964 and V. americanu on 14 Sept 1964) suggests that under the pressure of further growth, nitrogen would be more likely to become limiting than would phosphorus. The low fertility of Lake Nebish correlates with the fact that in five of the 15 plant samples from that body of water, the phosphorus content was at or below the critical level and in several other samples the values were close to the critical figure. The nitrogen content was never less than 1.3%, and in only one species, L. dortmaniu, was it below 2.0%. This suggests that in Lake Nebish phosphorus supply is more likely to limit plant growth than is nitrogen suPPlY* The above data should not be interpreted to suggest that growth-limiting tissue concentrations would be observed only in the

pH-8.7

and

Threinen

lake* 14 Sept

1964

%N

%P

3.41 2.67 2.77 2.59 3.37 1.98

0.71 0.58 0.41 0.23 0.44 0.37

1962).

least fertile lakes. This is determined by density of plant growth relative to nutrient supplies. Table 5 is a summary of the nitrogen and phosphorus analyses throughout the growing season of species which at one or more sampling dates had a nitrogen content below 1.5% or a phosphorus content below 0.15%, values approaching but slightly above the critical concentrations. In no case was a nitrogen content below the critical value of 1.3%, although in one sample of L. dortmaniu it was below 1.5%. The phosphorus content was 0.15% or below in 17 samples and 0.13% or less in eight. In 12 of the samples with phosphorus contents of 0.15% or less, the nitrogen content was 2.0% or greater. These limited data seem to support the viewpoint that (with the exception of Lake Mendota) in the lakes sampled phosphorus is more likely to become a limiting factor for higher plant growth than is nitrogen. This assumes that the critical nitrogen and phosphorus values for all species are approximately the same. This was true for the species studied in the laboratory, but those were species characteristic of fertile lakes. Whether the same critical values apply to species most often found in very infertile lakes, for example E. septangulare and L. dortmaniu, can only be determined by further investigation. DISCUSSION

The results of this investigation suggest that the technique of tissue analysis has considerable potential for assaying the nutrient status of lakes and streams. With

TISSUE TABLE

4.

ANALYSIS

TO

DETERMINE

535

AVAILABILITY

The total nitrogen and phosphorus contents of samples of higher aquatic plants collected at intervals during the growing season from Lake Nebbh, a relatively infertile lake* 24 June 1964

Plant species

Elodea spp. Eriocaulon septangulare Lobelia dortmunia Potamogeton epih ydrus * Methyl

NUTRIENT

orange alkalinity-17

%N

15 July 1964

ppm;

1 Sept 1964

%P

%N

%P

%N

%P

%N

%P

03 0.13 0.33

2.10 2.26 1.89 3.19

0.14 0.12 0.16 0.30

2.86 2.09 1.48 2.38

0.24 0.11 0.10 0.19

2.56 2.03 1.95 2.84

0.15 0.10 0.23 0.30

-

2.16 1.78 2.79

5 Aug 1964

conductance--34

mmhos;

further refinement, it should be possible to determine whether the supply of nitrogen, phosphorus, or some other element becomes limiting at any time du+ng the season for the optimum growth of’ particular species of angiosperm aquatic plants. Any inconsistencies and problems from this initial study should not discourage further development of the tissue analysis technique. Successful use of tissue analysis in agriculture and horticulture is based on hundreds of investigations, and the situations under study usually are much simpler than the lakes studied here. Also, in any particular application in agriculture and horticulture, the investigator is concerned only with a single species, both in the establishment of critical levels and in field sampling. Lakes contain many higher plant species and the differences in nutrient content associated with variation in root penetration into bottom muds and in capacity to absorb nutrients make interpretation of the results in terms of the general fertility of a lake difficult. In agricultural and horticultural application of tissue analysis, there has been a trend toward the establishment of index tissues, that is, the collection and analysis of plant parts that are more reliable indicators of the availability of specific nutrients than are entire plants (Ulrich and Berry 1959; Smith 1962). Re-export and reuse of some elements, changes in the proportion of woody to nonwoody tissues with aging, and the gradual decrease in some elements that is characteristic of the maturing of plants are factors that contribute to the effectiveness of using index tissues. Further study on the most reliable parts of aquatic

and pH-7.1

(Black,

Andrews,

and Threinen

1963).

plants to sample is recommended. HOWever, the general lack of woody tissues in aquatic plants may make sampling of entire plants more generally satisfactory than it is in work with terrestrial species. In all cases, the tissue sampled from aquatic plants must be from healthy and metabolically active parts. Otherwise, the loss of nutrients characteristic of dying and disintegrating tissues may erroneously be interpreted as tissue contents below critical levels. Disintegrating tissues should be detected by plant appearance and by low contents of a number of elements. It is of interest to compare the critical values established for angiosperm aquatic plants with those for other species. For example, 2.2% nitrogen and 0.12% phosphorus were considered the minimum concentrations for satisfactory citrus production ( Chapman 1961)) while approximately 1.0% nitrogen and 0.08 to 0.10% phosphorus were critical concentrations in spruce and pine seedlings ( Ingestad 1959, 1960). In a report of the phosphorus contents of 12 vegetable species showing symptoms of phosphorus deficiency ( Wallace 1961) , seven of the values were below O.lO%, and the highest value was only 0.15%. A total nitrogen content of 2.2% was suggested as a critical level for fruit trees of the northwestern United States (Woodbridge, Benson, and Batjer 1961) . The critical nitrogen level of 1.3% in the present studies is toward the lower limit of the crop plant values mentioned above, and the phosphorus content is about the average. In applying the tissue analysis technique for assaying nitrogen and phosphorus supplies in lakes for the growth of algae, Ger-

536

G. C. GERLOFF

AND

P. H.

KROMBHOLZ

TABLE 5. The nitrogen and phosphorus contents of aquatic plunts in which either the nitrogen or the phosphorus content was below the critical concentration at one or more sampling dates during the 1964 growing season Species

Elodeu spp. E riocaulon septang&are Lobelia dortmania Potamogeton amplifolius Potamogeton richardsonii Potamogeton robinsii

Lake

Trout Nebish Nebish Little Spider Weber Nebish Sparkling Little John

24,25

June

16, 17 July

6,7 Aug -~ %N %P

%N

%P

%N

%P

3.02 -

0.30 -

2.16 2.40 2.38 1.78 1.53

0.18 0.19 0.16 0.13 0.15

1.78 2.10 2.26 2.57 2.42 1.89 1.68 3.78

0.19 0.14 0.12 0.16 0.14 0.16 0.20 0.22

2.57 2.86 2.09 2.17 2.11 1.48 1.62 2.42

1.98 2.35 2.12

0.18 0.14 0.16

1.97 1.95 2.13

Trout Big Kitten Big Kitten

loff and Skoog (1954, 1957) found the critical nitrogen content for the blue-green alga M. aeruginosa to be 4.0% and the critical phosphorus content only 0.12%. From a comparison of these values with nitrogen and phosphorus contents of algae from blooms of the same organisms in lakes, it was concluded that nitrogen supply was more likely to become growth-limiting than was phosphorus. This conclusion contrasts with the present data, which suggest phosphorus as a primary limiting factor for aquatic angiosperms. However, the high critical nitrogen content of the M. aeruginosa should be noted. This could well be responsible for the difference in the limiting factor for the two types of organisms. One feature of applying the tissue analysis technique to aquatic plants that contrasts sharply with its use on crop plants is the inability to verify the validity of the technique by correlating tissue contents below the critical levels with actual decreases in plant growth and yield in the field. This is readily accomplished with crop plants, but undoubtedly will remain difficult with aquatic plants. General observations of plant growth in particular bodies of water and the effects of adding supplemental quantities of nitrogen or phosphorus to waters low in these elements could be of some help. However, it may be necessary,

2, 3 Sept %N

%P

0.33 0.24 0.11 0.11 0.27 0.10 0.15 0.14

2.11 2.56 2.03 2.23 1.98 1.95 1.85 2.78

0.12 0.15 0.10 0.16 0.20 0.23 0.17 0.20

0.14 0.20 0.15

2.69 2.14 2.36

0.17 0.20 0.13

in general, to assume that, as demonstrated repeatedly with crop plants, values below the critical contents established in the laboratory do correlate with decreased plant yields in the field. The large variations in nitrogen and phosphorus contents of various species collected from the same lake on the same date justify a final note of caution against generalizing that nitrogen or phosphorus is limiting for the growth of all species in a lake on the basis of below-the-critical-content values in one species. The marked differences in the rooting and growth habits of various species and variations in the extent to which nutrients are derived from the water phase could easily result in situations in which some species were adequately supplied with an element while others were not, even when they occurred quite close together. REFERENCES BLACK, J. J., L. M. ANDREWS, AND C. W. THREI1963. Surface water resources of Vilas NEN.

County. Wisconsin Conserv. Dept., Madison. 317 p. BOURN, W. S. 1932. Ecological and physiological studies on certain aquatic angiosperms. Contrib. Boyce Thompson Inst., 4: 425-496. CHAPMAN, H. D. 1961. The status of present criteria for the diagnosis of nutrient conditions in citrus, p. 75-106. In W. Reuther [ed.], Plant analysis and fertilizer problems. Publ. No. 8, AIBS. Washington, D.C.

TISSUE

ANALYSIS

TO

DETERMINE

FASSETT, N. C. 1957. A manual of aquatic Univ. Wisconsin Press, Madison. plants. 405 p. FOGG, G. E. 1965. Algal cultures and phytoUniv. Wisconsin Press, plankton ecology. Madison. 126 p. GERLOFF, G. C., AND F. SKOOG. 1954. Cell contents of nitrogen and phosphorus as a measure of their availability for growth of Microcystis aeruginosa. Ecology, 35: 348-353. -, AND -. 1957. Nitrogen as a limiting factor for the growth of Microcystis aeruginosa in southern Wisconsin lakes. Ecology, 38 : 556-561. HOAGLAND, D. R., AND W. C. SNYDER. 1933. Nutrition of strawberry plants under controlled conditions: (a) Effects of deficiencies of boron and certain other elements: ( b ) Susceptibility to injury from sodium salts. Proc. Am. Sot. Hort. Sci., 30: 288-294. INGESTAD, T. 1959. Studies on the nutrition of forest tree seedlings. II. Mineral nutrition of spruce. Physiol. Plantarum, 12 : 568593. -. 1960. Studies on the nutrition of forest tree seedlings. III. Mineral nutrition of pine. Physiol. Plantarum, 13 : 513-533. JOHNSON, C. M., P. R. STOUT, T. C. BROYEX, AND A. B. CARLTON. 1957. Comparative chlorine requirements of different plant species. Plant Soil, 8: 337-353.

NUTRIENT

AVAILABILITY

537

LEA, W. L., G. A. ROHLICH, AND W. J. KATZ. 1954. Removal of phosphates from treated Sewage Ind. Wastes, 26: 261-275. sewage. 1951. Leaf analysis. [Transl. LUNDEGARDH, H. by R. L. Mitchell,] Hilger and Watts, Ltd., London. 176 p. MUENSCHER, W. C. L. 1936. Storage and germination of seeds of aquatic plants. Cornell Univ. Agr. Expt. Sta. Bull. No. 652. 17 p. POFF, R. J., AND C. W. THREINEN. 1962. Surface water resources of Dane County. Wisconsin Conserv. Dept., Madison. 61 p. REUTHER, W. [ea.] 1961. Plant analysis and fertilizer problems. Publ. No. 8, AIBS. Washington, D.C. 454 p. SMITH, P. F. 1962. Mineral analysis of plant tissues. Ann. Rev. Plant Physiol., 13: 81-108. ULRICH, A., AND W. L. BERRY. 1961. Critical phosphorus levels for lima bean growth. Plant Physiol., 36 : 626-632. WALLACE, T. 1961. The diagnosis of mineral deficiencies in plants by visual symptoms. Chem. Publ. Co., New York. 125 p. WOODBRIDGE, C. G., N. R. BENSON, AND L. P. BATJER. 1961. Nutrition of fruit trees in the semiarid regions of the Pacific Northwest, p. 64-73. In W. Reuther [ea.], Plant analysis and fertilizer problems. Publ. No. 8, AIBS. Washington, D.C.