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THE EFFECTS OF CALCIUM SALTS ON THE
GROWTH AND UPTAKE OF PIIOSPIIORUS
BY CERATGPI‘IYLLUM DEMERSUM

flies}: Io:- IIw Degree OI M. S.
MICHIGAN STATE UNIVERSITY

Ted Randall Batterson
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(x If? ABSTRACT

THE EFFECTS OF CALCIUM SALTS ON THE GROWTH AND

UPTAKE OF PHOSPHORUS BY CERATOPHYLLUM DEMERSUM

 

By

Ted Randall Batterson

Certain non-toxic substances have been proposed for phosphorus
inactivation as a means of lake rehabilitation. Calcium salts are
thought to be such substances.

The calcium salts (CaCO CaSOh and both combined in a 1:1 ratio)

3.
were dosed at O, 7, lb and 21 ppm in triplicate for each of twelve
treatment combinations. The effects these doses had on the aquatic
macrophyte, Cbratophyllum demersum were measured.

The plant showed no response to these dosages, in either
productivity or tissue concentrations of phosphorus. Thus, these

salts, at the specified dosage levels, would be ineffective in

controlling this weed.

THE EFFECTS OF CALCIUM SALTS ON THE GROWTH AND

UPTAKE OF PHOSPHORUS BY CERATOPHYLLUM DEMERSUM

 

By

Ted Randall Batterson

A THESIS
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
MASTER OF SCIENCE

Department of Fisheries and Wildlife

1975

ACKNOWLEDGMENTS

I would like to express my sincere thanks to Professor
Clarence D. McNabb for affording me with an experience I would
not have had otherwise and helping me to expand myself, both as
a person and a student.

Thanks is extended to Professors Kenneth W. Cummins and
Niles R. Kevern for serving on my graduate committee. Special
acknowledgment is due Professor Cummins for providing invaluable
assistance and time throughout this endeavor.

My fellow graduate students, Louis A. Helfrich, Diana L. Weigmann
and Robert P. Glandon, deserve thanks and especially James R. Plautz
who gave so much of his time and effort to this project.

My deepest appreciation goes to my wife, Kathleen and daughter,
Cassandra, for without their support this undertaking would not have
been made.

This study was supported in part by funds provided by the
United States Environmental Protection Agency Training Grant (T-900331)
and the Michigan Agricultural Experiment Station at Michigan State

University.

ii

TABLE OF CONTENTS

LIST OF TABLES

LIST OF FIGURES . . .

INTRODUCTION

METHODS .

RESULTS .

DISCUSSION

CONCLUSION

LITERATURE CITED

iii

10
lh
16

17

Number

LIST OF TABLES

Treatment combinations and experimental unit
numbers.

Overall means for those parameters not showing
significant differences.

iv

Number

LIST OF FIGURES

Page
The response of ambient orthophosphate to 12
increasing dosage with treatment compounds
(CaCO , CaCO :CaSOh, CaSO ). Means i one
standard errgr are shown.
The rate of growth of CeratophyZZum demersum 13

in all enclosures over the experimental
interval. Means i one standard error are
shown. Biomass doubling time was approximately
16 days .

INTRODUCTION

Limnologists are frequently asked to recommend strategies for
lake rehabilitation. Approaches have been many and varied and as
Dunst, et al. (l97h) concluded, it is highly dependent upon the
specific situation in question.

One viable means, that has some universality, is nutrient
depletion of phosphorus. The reason for this attack on phosphorus
is because it is a "key" nutrient (Lee, 1973); that is, a nutrient
that can be controlled with current technological and financial
resources and that may not be limiting at that particular point in
time, but can be made limiting through specified control efforts.
It has been shown that biological productivity is directly coupled
to phosphorus cycling in relation to allochthonous inputs (Wetzel,
1975). Ideally, if one could lower the loading rate, productivity
would decline. Many times this is an infeasible approach, since a
diffuse source of phosphorus is enriching the lake. In certain of
these cases, phosphorus might be most readily removed from the system
by inactivation once it is within the lake basin.

Extrapolation from successful waste treatment technology would
indicate that lime (CaO, slaked or Ca(OH)2, unslaked) (Owen, 1953;
Buzzell and Sawyer, 1967; and Schmid and McKinney, 1969), alum
(Al2(SOh)3-th20) (Malhotra, et aZ., 196A), ferrous and ferric sulfate,

cupric sulfate and sodium aluminate (NaAlO ) (Lea, et al., 195h;

3

Albertson and Sherwood, 1969) are all chemicals that provide feasible
means of reducing phosphorus concentrations in water. However, all
but alum, have some inherent problems associated with in situ
applications at dosage levels that would have a significant effect.
Drastic pH alterations due to the addition of lime, toxicity of
cupric sulfate and cost of sodium aluminate are examples.

In May of 1970, Peterson, et a1. (1973) implemented waste
treatment technology by applying 200 mg/l alum to Horseshoe Lake,
Wisconsin. The results were an overall improvement in water quality
reflected by a decrease in total phosphorus concentrations, improved
dissolved oxygen levels ostensibly associated with reduced autochthonous
production and increased Secchi disc readings.

At about this same time another non—toxic phosphorus inactivating
chemical was being employed by Laing (l97h) for lake restoration.
Technical information regarding this material was scant during this
period of patent development. It was reported to be a calcium based
compound that was dosed at approximately 15 mg/l. Its remedial effects
were purported to be lowered phosphorus concentrations and concomitant
reductions in algal and macrophytic productivities. These reported
responses provided the impetus for this research.

A priori, assuming the compound was either CaCO (calcite) or

3
CaSOh-2H20 (gypsum), Laing's dosage levels would be ineffective in
producing a noticeable phosphorus reduction. This observation is
based on values obtained from using equations from Stumm and Morgan
(1970). With specified temperatures, pH and solubility products

effective treatment would require at least a magnitude of difference

between the alkalinity level and dosage level of those salts to have a

significant lowering of phosphorus due to hydroxyapatite formation
and precipitation. What Stumm and Morgan and other theoretical
equations fail to take into consideration are the complex array of
interactions that come into play in natural aquatic environs. The
theoretical considerations are based on straight stoichiometric
relations of one or two species within a container of distilled
water. Thus a multitude of interactions and synergisms that are
inherent in the natural waters are ignored. Things such as multiple
chemical species, coprecipitation, dissolved organics, adsorption,
absorption and flocculation are a few. As Otsuki and Wetzel (1972)
showed, coprecipitation of phosphates with carbonates can greatly
increase the amount of phosphorus inactivation. Thus, there might

be some beneficial effects of dosing CaCO and CaSOh-2H20 at low

3
levels. The efficacy of these dosing rates over time, is dependent

on the reducing strength of the environment in which resulting
compounds settle and their resultant availability to rooted aquatic
macrophytes. The work of Solski cited in Wetzel (1975) has shown

that plants are effective phosphorus pumps and can recycle the element
quite appreciably. This has been further substantiated by the work

of McRoy, et al. (1972) on eelgrass (Zostera marina L.).

The hypothesis then to be tested in this study is: do calcium
salts (carbonate and sulfate), at dosage levels of maximally 21 mg/l,
lower productivity or effect the phosphorus tissue concentration of
an aquatic macrophyte. Ceratophyllum dkmersum was chosen as the test

organism because it is a non-rooted plant, thus eliminating the need

for monitoring the sediments, and from the work of McNabb, et a1. (1972)

and McNabb and Tierney (1972), C. demersum was shown to respond
proportionally to available phosphorus as reflected in tissue

concentrations of that element.

METHODS

For stringent statistical analysis a factorial experiment was
chosen that was of a split-plot, repeat measurement design. The
general model for the three fixed factors, A (compound type), B (dosage
level) and C (time) was taken from Gill and Hafs (1971).

The experiment was undertaken in the fourth cell of the system
of waste stabilization ponds at Belding, Michigan described by
Bulthuis (1973). A roped enclosure, approximately twenty feet square,
was tethered off in the middle of the pond, free of any macrophytic
growths.

Thirty-six experimental units (3 replications of each of the
twelve treatment combinations) that consisted of h-mill polyethylene
bag (89.5h cm X l6h.h7 cm) were suspended from a 50 cm diameter,
doughnut shaped styrofoam float. Each unit was filled with 2h0
liters of ambient water. Placed inside of the poly-bag and suspended
near mid-depth were two nylon-mesh bags holding Ceratophyllum démersum.
Each unit was covered with a plexiglass plate and allowed to equilibrate
for h8 hours before treatment.

Cératophyllum demersum that was placed within the nylon bags
was obtained from the experimental pond and subjected to a series of
rinses in pond-water to remove invertebrate organisms and Lemna minor.
Terminal portions, 18 cm in length, were selected, shaken, blotted and
wet-weighed into 30 gram portions. These were placed within the

nylon-mesh bags, which were large enough to facilitate a quadrupling

in biomass without crowding effects. Eighty—five wet-weighed plant
packets were made, of which 13 were randomly selected and brought
back to the laboratory for analysis. The remaining 72 were randomly
assigned, two at a time, to the poly-bag units. Table 1 depicts the
treatment combinations. Each of the twelve treatment combinations

were triplicated.

Table 1. Treatment combinations and experimental unit numbers.

 

FACTOR A
(COMPOUND TYPE)
All CaCO3 CaCO3:CaLSO,4 All CaSOh
(1:1)

 

0 mg/l ------- Controls --------
Experimental units 1-9

FACTOR B 7 mg/l 10—12 13-15 16-18
(DOSAGE LEVEL)
1h mg/l 19—21 22—2h 25—27

21 mg/l 28-30 31-33 3h-36

 

At the time of treatment, the compounds were weighed and placed
in individually labeled plastic bags for transport to the field. The
covers were removed from the experimental units, water withdrawn from
the unit and mixed with a randomly selected treatment compound to form
a slurry. This slurry was then slowly introduced into the unit with
stirring, after which the units were covered and labeled as to
treatment combination. Though no compound was added to bags 1 through

9, they were subjected to the same mechanical treatment.

A sampling device was constructed that would allow sampling
from a unit with minimum disturbance to the water column. It consisted
of a board (56 cm X 9 cm) with three holes in it that would receive
PVC tubing of equal diameter but differing lengths. The lengths were
15 cm, 53 cm, and 102 cm and labeled respectively, surface, middle
and bottom. Poly-tubing was used to connect each PVC tube to the
import side of a small electric pump. The export side had a tube that
transported the water to an appropriately labeled sample bottle.

For each sampling time, enough water per depth per bag was
withdrawn to allow for the following analyses in the laboratory:
total alkalinity, phenolphthalein alkalinity, total hardness, ortho—
phosphate and total phosphate. One of the mesh bags containing
C. demersum was removed, the contents rinsed in pond-water, placed
in labeled poly—bags and transported back to the laboratory. Dry
weights, ash-free dry weights and tissue concentrations of phosphorus
were obtained from these. In situ measurements of temperature and
pH were made.

The chemical analyses for total alkalinity, phenolphthalein
alkalinity, total hardness, orthophosphate and total phosphate were
performed to specifications outlined in Standard Methods for the
Examination of Water and Wastewater (Anon., 1973).

Total alkalinity was measured with bromcresol green-methyl red
indicator (endpoint = pH h.5), while phenolphthalein alkalinity was
measured at an endpoint of pH 8.3. Total hardness was analyzed with
HexaVerR Hardness Titrant and ManVerR Hardness Indicator—2, prepared
by Hach Chemical Company. These measurements were expressed as mg/l

(ppm) CaCO3.

Phosphorus analyses were done by colorimetry using the l-ascorbic
acid-single reagent method with absorbancies read at 880 nm on a
Bausch and Lomb Spectronic-20. Matched cuvettes having a light pathway
length of 1.5 cm were used. Values were determined by comparison to
standard curves and expressed as ppm POM—P. The data points comprising
a standard curve were based on known concentrations of potassium
dihydrogen phosphate (KHEPOA) that were subjected to the same procedures
and dilutions as the samples.

Total phosphorus was measured after digestion, with persulfate
oxidation carried out in 125 ml Erlenmeyer flask, the contents of
which were brought to boiling on a hot plate. Following cooling and
neutralization (pH 7.3), each sample was rediluted to the original
volume of 100 ml. The orthophosphate values represent that faction
of the total phosphate which will react with the combined reagent
after 10 minutes following its addition to the sample.

Well washed plant material was returned from the field in poly-
bags, placed in a forced draft drying oven and dried at 800C until a
constant weight was obtained (circa h8 hours). The entire sample
was then homogenized in a micro-Wiley mill using a hO mesh screen
and placed back into the drying oven. Ash—free dry weights were
obtained by taking a 100 mg sub-sample and igniting at SSOOC in a
muffle furnace for h hours. Tissue phosphorus was determined by
taking a 500 mg sub-sample and wet ashing with 10 ml of l to 1 HNO3
and HCth acid mixture in a Bethge distillation apparatus. The residue
was diluted and neutralized and then colorimetrically analyzed, using

the l—ascorbic acid—single reagent method.

Glassware for the phosphorus analyses was cleaned with sulfuric
acid-dichromate solution, followed by a wash and rinse in hot, 30%
(v/v) HCl and finalized by triple rinses in deionized distilled water.
All other glassware, except the Bethge distillation apparatus, was
cleaned by the same procedure, but deleting the hot HCl bath. The
Bethge distillation apparatus was cleaned by soaking in a l to l HNO3
and HOH bath overnight and then tripled rinsed with deionized distilled

water.

RESULTS

Appropriate statistical tests were derived from the general
model. These tests were based on mean squares of the appropriate
factors, interactions and error terms. Initially, due to the factorial
nature of the experiment, significant interactions were examined
(Snedecor and Cochran, 1967).

The data that were collected can be divided into two components;
those that were chemically related and those that have biological
significance. There were no measurable differences (at alpha = 0.05)
between surface, middle and bottom waters for any of the chemical
components. Thus, those data for the experimental units were pooled.
Table 2 represents those chemical parameters which were tested for
mean factor differences and found not to be significantly different
(at alpha = 0.05). They are therefore expressed as overall means i
standard error.

Table 2. Overall means for those parameters not showing significant
differences.

 

 

Parameter Mean i Standard error
Total hardness (mg/l CaCO3) 199.72 i 2.1M
Phenolphthalein alkalinity (mg/l CaCO3) 25.60 i 1.01
Total alkalinity (mg/l CaC03) 156.62 : 2.03
Orthophosphate (mg/l POh-P) 1.017 t 0.0h7
Temperature 22.30 i 0.30

pH 8.90

 

lO

11

For each factor, appropriate statistical tests were utilized.
For factor A (compound type), the means were tested by Tukey's HSD
(Honestly Significant Difference) method. Factor B (dosage level)
means were compared by Dunnett‘s test and factor C (time) means were
compared using Student's—t test. In the case of orthophosphate,
though there were no significant differences, there was an apparent
depression of that substance with increasing dosage levels (Figure 1).

Total phosphorus presented a different case, for in that instance,
there was a significant interaction (at alpha = 0.05) between the
dosage level and time. Therefore, treatment combination means (BC
means) were analyzed using Dunnett's test. Since there were no
differences between compound types (factor A), the BC means were
averaged over all compound types. For time-l (Tl) there were no

significant differences, but T had significant differences (at

2
alpha = 0.05) between the controls (0 ppm) and dosage levels of IA
and 21 ppm.

The biological parameters were derived from the response of
Cératophyllum demersum. The ash-free dry weights increased linearly
over time (Figure 2). A doubling of biomass took approximately 16
days. The percent tissue concentration of phosphorus, based on

previously sited statistical analyses, showed no differences between

compound types or dosage levels, and had an overall mean of 2.1%.

l2

 

 

 

 

 

1.3w 7'
1.22-‘A
.I I
1.1'-
Soluble
PO4-P
mQ/L 1.0- f
0.9-
0.8-I f I l I
C) '7 144 :21

Treatment Compounds
mg/ L
Figure 1. The response of ambient orthophosphate to increasing dosage

with treatment compounds (CaCO , CaCO :CaSO , CaSO ).
3 A 14
Means i one standard error are shown.

.1
4.0“
3.0-
Ash-Free
Dry Welght ‘
in Grams
2.0-

 

1.0-

13

Y=1.8174 4' 0.1108X

r2:0.9968

 

I | ' I

16 24

Time In Days

Figure 2. The rate of growth of Ceratophyllum démersum in all
enclosures over the experimental interval. Means i one

standard error are shown.

approximately 16 days.

Biomass doubling time was

DISCUSSION

The hypothesis to which this study was directed would have to
be rejected on the basis of the data that were obtained. Productivity
of the aquatic macrophyte was not reduced. It displayed a doubling
time close to the minima reported in McNabb and Tierney (1972).
Regarding orthophosphate, which showed no significant differences,
McNabb and Tierney's (1972) predictive response is correct, for there
was no concomitant difference in the percent tissue phosphorus. The
total phosphorus which showed a significant decline, is of no
consequence, since this is an unavailable resource to the plant
(Sculthorpe, 1967). Therefore, no matter what was transpiring in
the ambient waters in relation to phosphorus, the plant responded
as if it was constant.

Since the onset and subsequent completion of this experiment,
Laing and Adams (1975) have revealed the various components of

their chemical, which on a percent weight basis are the following:

Active ingredients

Calcium sulfate . . . . 80.0%
Aluminum sulfate . . . 17.0%
Boric acid . . . . . . 1.0%
Inert ingredients . . . . . 2.0%

Taking Laing's average dose as 15 ppm and using 17% by weight as
aluminum sulfate, the dosage level of that compound is 2.55 Ppm. If

this is expressed as a percentage of the value (200 ppm aluminum sulfate)

1h

15

that was used on Horseshoe Lake, Wisconsin (Peterson, et aZ., 1973)

for a significant treatment effect, namely 1.3%, one sees that this
component of Laing's compound would have little impact on phosphorus
reduction. Essentially then the compound is functionally calcium
sulfate. This would facilitate direct comparison between his data

and mine. Laing (l97h), Trent and McArthur (197A) and Laing and Adams
(1975) each talk of the efficacy of this compound, maximally dosed at

20 ppm, in reducing productivity by means of phosphorus inactivation.
Laing and Adams (1975) mention cases in which there is immediate die-off
of Cbratophyllum demersum after treatment with only 10 ppm of their
compound. Their proposal that this is due to phosphorus inactivation

is highly improbable based on my findings. The only possible explanation
for this discrepancy, is that the compound does not lower productivity
by phosphorus inactivation but by some other, as yet, undetermined

mechanism.

CONCLUSION

The hypothesis that calcium salts (carbonate and sulfate), at
maximal dosage levels of 21 ppm, lower productivity of Ceratophyllum
demersum has been rejected on the basis of the data obtained. Not
only was there not a reduction in productivity, but the percent tissue
phosphorus concentrations were unaltered. The data indicate that
Laing's compound can not be used as an effective means of lake

rehabilitation.

16

LITERATURE CITED

LITERATURE CITED

Albertson, O. E. and R. J. Sherwood. 1969. Phosphate extraction
process. Jour. Water Poll. Control Fed. u1(8): 1h67-1h78.

Anon. 1973. Standard methods for the examination of water and
wastewater, 13th edition. American Public Health Association,
Washington, D. C. 87h pp.

Bulthuis, D. A. 1973. Metal cycling in macrophyte dominated waste—
water ponds. M.S. Thesis, Mich. State Univ., East Lansing h882h.

33 pp.

Buzzell, J. C., Jr. and C. N. Sawyer. 1967. Removal of algal
nutrients from raw wastewater with lime. Jour. Water Poll.
Control Fed. hl(7): 1259-1276.

Dunst, Russell C. et al. 197k. Survey of lake rehabilitation
techniques and experiences. Tech. Bull. No. 75. Wisconsin
Department of Natural Resources, Madison, Wisc. 179 PP.

Gill, J. L. and H. D. Hafs. 1971. Analysis of repeated measurements
of animals. Jour. Animal Sci. 33(2): 331—336.

Laing, R. L. 197A. A non-toxic lake management program. Hyacinth
Control Jour. 12: h1-h3.

Laing, R. L. and A. Michael Adams. 1975. A study of the efficacy
of Clean—Flo Lake Cleanser (TM) in controlling aquatic plants
in three aerated Minnesota lakes. Clean-Flo Laboratories, Inc.
Hopkins, Minn. Mimeo. 67 pp.

Lea, W. L., G. A. Rohlich and W. J. Katz. 195A. Removal of phosphates
from treated sewage. Sewage and Industrial Wastes 26(3): 261-275.

Lee, G. Fred. 1973. Role of phosphorus in eutrophication and diffuse
source control. Water Research, Vol. 7. Pergamon Press, England.
pp. 111—128.

Malhotra, S. K., G. F. Lee and G. A. Rohlich. 196M. Nutrient removal
from secondary effluent by alum flocculation and lime precipita-

tion. Inter. Jour. Air and Water Poll., Vol. 8. Pergamon Press,
England. pp. h87—500.

17

l8

McNabb, C. D. and D. P. Tierney. 1972. Growth and mineral accumula-
tion of submerged vascular hydrophytes in pleioeutrophic environs.
Tech. Report No. 26. Inst. Wat. Res., Mich. State Univ., East
Lansing h882h. 33 pp.

McNabb, C. D., D. P. Tierney and S. R. Kosek. 1972. The uptake of
phosphorus by Garatophyllum demersum growing in wastewater.
Tech. Report No. 2h. Inst. Wat. Res., Mich. State Univ., East
Lansing h882h. 23 pp.

McRoy, C. Peter, Robert J. Barsdate and Mary Nebert. 1972. Phosphorus
cycling in an eelgrass (Zostera marina L.) ecosystem. Limnol.
Oceanogr. 17(1): 58-67.

Otsuki, A. and R. G. Wetzel. 1972. Coprecipitation of phosphate
with carbonates in a marl lake. Limnol. Oceanogr. 17(5): 763-

767.

Owen, Richard. 1953. Removal of phosphorus from sewage plant effluent
with lime. Sewage and Industrial Wastes 25(5): SUB-556.

Peterson, J. P., J. P. Wall, T. L. Wirth and S. M. Born. 1973.
Eutrophication control: nutrient inactivation by chemical
precipitation at Horseshoe Lake, Wisconsin. Tech. Bull. No. 62.
Wisconsin Department of Natural Resources, Madison, Wisc. 19 pp.

Schmid, L. A. and R. E. McKinney. 1969. Phosphate removal by a lime-
biological treatment scheme. Jour. Water Poll. Control Fed.

h1(7): 1259-1276.

Sculthorpe, C. D. 1967. The biology of aquatic vascular plants.
Edward Arnold, Ltd., London. 610 pp.

Snedecor, G. W. and W. G. Cochran. 1967. Statistical methods, 6th
edition. Iowa State Univ. Press, Ames, Iowa. 53% pp.

Stumm, W. and J. J. Morgan. 1970. Aquatic chemistry. Wiley-
Interscience, New York. 583 pp.

Trent, Lowell and il McArthur. 197A. Results of testing Clean—Flo

Lake Cleanser TM in Florida lakes. Hyacinth Control Jour. 12:
uu-us.

Wetzel, Robert G. 1975. Limnology. W. B. Saunders, Co., Philadelphia.
Th3 PP-

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