EPA-R3-73-024
APRIL 1973                      Ecological Research Series
Phosphorus Release

from Lake Sediments

                                 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
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was  consciously  planned  to  foster   technology
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   1.  Environmental Health Effects Research
   2.  Environmental Protection Technology
   3.  Ecological Research
   4.  Environmental Monitoring
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This report has been assigned  to  the  ECOLOGICAL
RESEARCH  series.   This series describes research
on the effects of pollution on humans,  plant  and
animal   species,  and  materials.   Problems  are
assessed   for   their   long-   and    short-term
influences.    Investigations  include  formation,
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to  minimize   undesirable   changes   in   living
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                                                      EPA-R3-73-024
                                                      April 1973
       PHOSPHORUS RELEASE FROM LAKE  SEDIMENTS
                           By

                     R. E.  Wildung
                     R. L.  Schmidt
                   Project  16010 DUA
                    Project  Officer

                   Charles F.  Powers
      National Eutrophication Research  Program
           Environmental Protection Agency
       National Environmental Research  Center
                Corvallis, Oregon 97330
                     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 $2.35 domestic postpaid or $2.00 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 reviews 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
The principal objectives of these investigations were to
chemically define the major forms of phosphorus (P) in sedi-
ments of Upper Klamath Lake, Oregon, determine the potential
for release of P from the sediment as influenced by water and
sediment composition and environmental parameters, and establish
the relationship between P release and algal  growth.

Methods were developed for the characterization of inorganic
and organic P components and for measurement of P release from
sediments and subsequent availability to algae.  Sediment
characterization was extended to other lake systems including
Shagawa Lake in Minnesota, Agency and Diamond Lakes in Oregon
and Lake Erie.

Sediments of Upper Klamath Lake, although differing in their
ability to release P, exhibited seasonal changes in P concen-
tration.  These changes were most pronounced in the inorganic
P fraction and in a bay which received agricultural runoff and
initially contained relatively large quantities of P in the
sediment interstitial water.  Release of and resorption of P
associated with the solid phase occurred.  Release appeared to
be largely from non-occluded Fe-P whereas resorption was primarily
in the form on non-occluded Al-P.  The rate and extent of P
release, described by regression models, was related to sediment
composition.  Release was accelerated by temperature and the
presence of a P sink such as an anion exchange resin.  Evidence
obtained in the field suggested that actively reproducing phyto-
pi ankton may also serve as a P sink increasing P release.  Algal
growth response to P released from sediments during dialysis was
approximately equivalent to the response to orthophosphate.

This report was submitted in fulfillment of Contract 14-12-508
under the sponsorship of the Office of Research and Monitoring,
Environmental Protection Agency.

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                          CONTENTS


Section                                                        Page

   I      Summary and Conclusions 	    1

            Development of Methods for Measurement of
            Sediment Phosphorus 	    1

            Phosphorus Status of Sediments as Related to
            Changes in Limnological  Conditions   	    2
                        «
            Laboratory Studies of the Potential  for P
            Release From Sediments  	    3

  II      Recommendations 	    7

 III      Introduction  	    9

  IV      Phosphorus Status of Lake Sediments as Related
          to Changes in Limnological Conditions  	   11

            A.  Total, Inorganic and Organic Phosphorus ...   11

            B.  Phosphorus Mineral and Organic Components  .  .   28

   V      Potential for Release of Phosphorus From
          Sediments - Laboratory Investigations  	   59

            A.  Laboratory Methods for Measurement of
                Phosphorus Release and Algal Growth
                Potential	   59

                Membrane Dialysis System  	   60

                Filter Dialysis System  	   60

                Direct Equilibration with an Anion Exchange
                Resin	   64

                Estimation of Algal  Growth Potential  ....   65

            B.  Factors Influencing the Release and Algal
                Growth Potential  of Sediment Phosphorus ...   78

                Sediment Physical  Parameters and
                Temperature	   78

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                          CONTENTS
Section                                                        Page
                Sediment Chemical Composition and
                Temperature	   87
                Sediment Inorganic Phosphorus Equilibria
                and Long-Term Incubation  	   96
                The Presence of a Phosphorus Sink	115
                                         *•
                Direct Equilibration with an Anion
                Exchange Resin  	  131
                Algal  Growth in the Dialysis Cell	133
  VI      Acknowledgements   	  143
 VII      References	145
VIII      Appendix	153
            A.   Shagawa  Lake,  Minnesota 	  153
            B.   Lake Erie	175
            C.   Agency Lake, Oregon 	  175
            D.   Diamond  Lake,  Oregon  	  175
                            v^

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                          FIGURES
Number                                                        Page

 1.    Location of sampling sites on Upper Klamath
       Lake, Oregon	   12

 2.    Comparison of methods for determination of total
       sediment phosphorus contents 	   20

 3.    Seasonal changes in total, inorganic and organic
       sediment phosphorus concentrations at three
       sampling locations on Upper Klamath Lake 	   22

 4.    Correlation of seasonal  changes in sediment and
       lake water phosphorus, carbon and nitrogen 	   23

 5.    Recovery of inositol hexaphosphate from an anion
       exchange resin by gradient elution with increasing
       concentrations of acid	   36

 6.    Recovery of inositol hexaphosphate from an anion
       exchange resin by elution with acid at two
       concentration levels 	   37

 7-    Recovery of inositol hexaphosphate from an anion
       exchange resin by elution with acid at two
       concentration levels 	   38

 8a.    Monthly distribution of phosphorus in inorganic
       phosphorus fractions of the sediments of Howard
       Bay, Upper Klamath Lake, Oregon  	   41

 8b.    Monthly distribution of phosphorus in inorganic
       phosphorus fractions of the sediments of Howard Bay,
       Upper Kalamth Lake, Oregon 	   42

 9a.    Monthly distribution of phosphorus in inorganic
       phosphorus fractions of the sediments near Buck
       Island, Upper Klamath Lake, Oregon 	   43

 9b.    Monthly distribution of phosphorus in inorganic
       phosphorus fractions of the sediments near Buck
       Island, Upper Klamath Lake, Oregon 	   44

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                          FIGURES


Number                                                        Pa9e

lOa.   Monthly distribution of phosphorus in inorganic
       phosphorus fractions of the sediments of Pelican
       Marina, Upper Klamath Lake, Oregon 	   45

lOb.   Monthly distribution of phosphorus in inorganic
       phosphorus fractions of the sediments of Pelican
       Marina, Upper Klamath Lake, Oregon .... 	   46

11.    Sediment dialysis apparatus  ... 	   61

12.    Algal cell concentration (tentative coulter counts)
       as a function of solution optical density  	   74

13.    Solution optical density as a function of algal
       dry weight	   75

14.    Standard bioassay growth curves (0.00 to 0.08 yg
       phosphorus/ml of culture media)  	   76

15.    Standard bioassay growth curves (0.1  to 0.6 yg
       phosphorus/ml of culture media)  .	   77

16.    Standard curve of growth response (log scale) with
       increasing levels of phosphorus utilizing the
       revised algal bioassay procedure 	   79

17.    Concentration of total  and inorganic  phosphorus in
       the external  water during incubation  of lake sediments
       (membrane dialysis system) over a range of experimental
       parameters (treatments  1, 2, 3; Table 21)	   83

18.    Concentration of total  and inorganic  phosphorus in the
       external  water during the incubation  of lake sediments
       (membrane dialysis system) over a range of experi-
       mental  parameters (treatment 4, Table 21)  	   84

19.    Concentration of total  and inorganic  phosphorus in the
       external  water during the incubation  of lake sediments
       (membrane dialysis system) over a range of experimental
       parameters (treatments  5, 6; Table 21)	   85

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                          FIGURES
Number                                                        Page

20.    Concentration of total and inorganic phosphorus in the
       external water during incubation of lake sediments
       (membrane dialysis system) over a range of experimental
       parameters (treatments 7,8; Table 21) 	   86

21.    Concentration of total solution phosphorus in the
       external water during incubation at 10°C of Upper
       Klamath and Agency Lake sediments in a dialysis
       cell	   94

22.    Concentration of total solution phosphorus in the
       external water during incubation at 23°C of Upper
       Klamath and Agency Lake sediments in a dialysis
       cell	   95

23.    Algal growth response in dialysates (Table 27) of
       lake sediments	   98

24.    Sediment phosphorus release as a function of
       incubation time in dialysis systems (Table 29) ....  106

25.    Regressions of sediment phosphorus release in
       dialysis systems with and without resin in the
       water half-cell (Table 42)	124

26.    Comparison of phosphorus contents in inorganic
       phosphorus fractions of Howard Bay sediments after
       incubation in the dialysis system in the presence
       and absence of an anion exchange resin 	  128

27.    Comparison of phosphorus contents in inorganic
       phosphorus fractions of Buck Island sediments after
       incubation in the dialysis system in the presence
       and absence of an anion exchange resin 	  129

28.    Comparison of phosphorus contents in inorganic
       phosphorus fractions of Pelican Marina sediments
       after incubation in the dialysis system in the
       presence and absence of an anion exchange resin  ...  130

29.    Release of phosphorus from Howard Bay sediments
       to an anion exchange resin in the direct equili-
       bration system (Table 51)	134

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                          FIGURES
Number                                                        Page

30.    Release of phosphorus from Buck Island and
       Pelican Marina sediments to an anion exchange
       resin in the direct equilibration system
       (Table 51)	135

31.    Grid system - Shagawa Lake, Minnesota  	  154

32.    Representative X-ray diffractograms of chemically
       treated sediments from Shagawa Lake  	  157

33.    Correlations of water depth,  total, inorganic and
       organic phosphorus, total  carbon  and total  nitrogen
       contents of sediment samples  from Shagawa Lake ....  163

34.    Grid system - Diamond Lake, Oregon	182

35.    Representative X-ray diffractograms of chemically-
       treated sediments from Diamond Lake	184
                            x

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                           TABLES
Number                                                        Page

   1    Description of sampling sites on Upper Klamath
       Lake, Oregon	   14

   2    Depth and chemical properties of sediments,
       Upper Klamath Lake, Oregon 	   17

   3   Sampling and analytical variability associated
       with the determination of total  and inorganic
       phosphorus in lake sediments	   18

   4   Seasonal changes in sediment organic carbon and
       total nitrogen	   25

   5   Seasonal changes in elemental composition, pH and
       temperature of surface water 	   27

   6   Serial extraction procedure employed for the
       fractionation of sediment inorganic phosphorus ....   31

   7   Serial extraction procedure employed for fractionation
       of the fulvic acid (NaOH-HCl soluble) components of
       lake sediments after concentration on activated
       carbon  ..... 	   32

   8   Distribution and recovery of organic phosphorus
       in organic extractives and residual components of
       Howard Bay sediments	   35

   9   Distribution of phosphorus in inorganic phosphorus
       fractions of Upper Klamath Lake sediments  	   40

  10   Summary of the regression of individual extractant
       phosphorus concentrations against total inorganic
       sediment phosphorus for all Upper Klamath Lake
       locations sampled during a single season 	  .   49

  11    Seasonal distribution of organic carbon in the
       benzene-ethanol, humic acid and fulvic acid fractions
       of lake sediments	   51

  12.  Seasonal distributions of organic carbon in components
       of the fulvic acid fraction of lake sediments  ....   53
                              x^

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                           TABLES
Number                                                        Pa9e

  13   Seasonal  distribution  of organic  phosphorus  in
       benzene-ethanol,  humic acid and fulvic  acid  fractions
       of lake sediments	    55

  14   Seasonal  distribution  of organic  phosphorus  in
       components  of the fulvic acid  fraction  of lake
       sediments	•	    56

  15   Equilibration of  phosphorus between  water cells
       separated by a 0.45  p  filter in the  dialysis
       system	    63

  16   Removal  of  orthophosphate phosphorus  from solution
       by an  anion exchange resin	    66

  17   Acid displacement of orthophosphate  phosphorus
       from an anion exchange resin	    67

  18   Experimental  design employed in investigations of
       direct uptake of  phosphorus from  moist  sediments  by
       different quantities of an  anion  exchange  resin  in a
       direct equilibration system .  	    68

  19   Uptake of phosphorus from moist sediments  by  an
       anion  exchange resin as  influenced by resin
       concentration in  a direct equilibration system ....    69

  20   Summary of  regressions  of phosphorus  uptake by
       resin  systems against  log of time	    72

  21    Factorial design  employed in investigations of
       phosphorus  release to  solution  (membrane  dialysis
       system)	    80

  22   Quantities  of sediment  and  water employed  to
       obtain  experimental parameters  in phosphorus  release
       studies (membrane dialysis  system) 	  .....    81

  23    Influence of  incubation  temperature on  phosphorus
       release from  sediments  (membrane dialysis  system)  .  .    88
                            x^^

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                           TABLES
Number                                                        Page

  24   Influence of sediment:interstitial  water ratio on
       phosphorus release from sediments (membrane dialysis
       system)  	    89

  25   Influence of sediment:external  ratio on phosphorus
       release from sediments (membrane dialysis system)  . .    90

  26   Experimental design employed in investigations of
       the influence of temperature and lake sediment
       composition on phosphorus release to solution  ....    91

  27   Concentrations of total  phosphorus  and inorganic
       phosphorus in the external  water after incubation of
       sediments for 45 days in a  dialysis cell	    93

  28   Prediction, using a multiple regression model, of
       the influence of temperature and lake sediment
       composition on the release  of sediment phosphorus
       to solution	    97

  29   Experimental design employed in investigations of
       sediment phosphorus release to  solution  	   100

  30   Sediment interstitial water composition prior to
       dialysis	102

  31   Concentrations of total  phosphorus  and inorganic
       phosphorus in the external  water during incubation
       of replicate Howard Bay  sediments in dialysis
       cells	103

  32   Concentrations of total  phosphorus  and inorganic
       phosphorus in the external  water during incubation
       of Buck Island sediments  in a dialysis cell   	104

  33   Concentrations o/f total  phosphorus  and inorganic
       phosphorus in the external  water during incubation
       of Pelican Marina sediments in  a dialysis cell  ....   105

  34   Fraction of sediment total  and  inorganic  phosphorus
       released to the external  water  during incubation
       (90 days)  of Upper Klamath  Lake sediments in  dialysis
       cells	108

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                           TABLES


Number                                                        Pac(e_

  35   Relative  changes  in  total phosphorus  and  in  inorganic
       phosphorus  fractions  of  Howard  Bay sediments  on  long-
       term incubation  in the dialysis  system  	   109

  36   Relative  changes  in  total phosphorus  and  in  inorganic
       phosphorus  fractions  of  Buck  Island sediments  on
       long-term incubation  in  the dialysis  system   	   110

  37   Relative  changes  in  total phosphorus  and  in  inorganic
       phosphorus  fractions  of  Pelican  Marina  sediments  on
       long-term incubation  in  the dialysis  system   	   Ill

  38   Equilibration  of  solution 32P with lake sediments
       in the  dialysis system	113

  39   Distribution of  32P  in sediment  extracts  after
       equilibration  (30 days)  with water containing  32P  in
       a dialysis  system 	   114

  40   Algal growth in sediment dialysates   	   116

  41    Algal growth in standard media amended with
       sediment  dialysates   	   117

  42   Experimental design employed in  investigations of
       sediment  phosphorus released to  solution  containing
       anion exchange resin	118

  43    Total phosphorus  and  inorganic phosphorus
       (cumulative) released to solution during  incubation
       of replicate Howard Bay  sediments in  the  presence
       and  absence of an anion  exchange resin  	   120

  44    Total phosphorus  and  inorganic phosphorus
       (cumulative) released to solution during  incubation
       of Buck Island sediments in the  presence  and
       absence of an anion exchange resin ..........   121

  45    Total phosphorus  and  inorganic phosphorus
       (cumulative) released to solution during  incubation
       of Pelican Marina sediments in the presence and
       absence of an anion exchange resin	 .   122
                             x^v

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                           TABLES
Number                                                        Page

  56   Distribution of carbon and nitrogen in Shagawa
       Lake sediments	159

  57   Summary of the concentrations of carbon,  nitrogen
       and phosphorus in sediments of Shagawa Lake	160

  58   Distribution of total  carbon, nitrogen and
       phosphorus in Shagawa Lake sampling locations
       west and east of the sewage outfall  	162

  59   Geographical distribution of total  phosphorus in
       sediments of Shagawa Lake	164

  60   Multiple regression model to predict total sediment
       carbon and phosphorus contents of Shagawa Lake
       sediments	166

  61   Categorization of total phosphorus  concentrations
       in the sediments of Shagawa Lake	168

  62   Sampling and analytical variability associated with
       the determination of the forms of phosphorus in
       sediments (A6d) of Shagawa Lake  	   169

  63   Sampling and analytical variability associated with
       the determination of the forms of phosphorus in
       sediments (B13d) of Shagawa Lake 	   170

  64   Sampling and analytical variability associated with
       the determination of the forms of phosphorus in
       sediments (B15d) of Shagawa Lake 	   171

  65   Sampling and analytical variability associated with
       the determination of the forms of phosphorus in
       sediments (Clld) of Shagawa Lake 	   172

  66   Sampling and analytical variability associated with
       the determination of the forms of phosphorus in
       sediments (D8c) of Shagawa Lake  	   173

  67   Sampling and analytical variability associated with
       the determination of the forms of phosphorus in
       sediments (F5a) of Shagawa Lake  	   174

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                           TABLES


Number                                                        Page

  68   Description  of sampling sites  on  Lake Erie	176

  69   Distribution of organic and  inorganic phosphorus
       in Lake Erie sediments  as  determined  by  high
       temperature  ignition  methods  	  177

  70   Distribution of phosphorus in  inorganic  phosphorus
       fractions  of Lake  Erie  sediments	178

  71    Description  of sampling site on Agency Lake,
       Oregon	179

  72    Distribution of organic and inorganic phosphorus
       in Agency  Lake sediments as determined by  high
       temperature  ignition  methods 	  180

  73    Distribution  of phosphorus in  inorganic  phosphorus
       fractions  of Agency Lake sediments  	  181

  74    Total and  inorganic phosphorus content of
       Diamond Lake  sediments		185
                           xv^

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                         SECTION I

                  SUMMARY AND CONCLUSIONS

The principal objectives of these investigations were to
(i) chemically define the predominant inorganic and organic
forms of P in sediments in Upper Klamath Lake, Oregon,
(ii) determine the potential for release of soluble P from
the solid phase with changes in temperature, pH and water
and sediment composition, (Hi] establish the relationship
between P release and algal growth and (i-o) develop suitable
methods for accomplishing the above.  These methods were also
applied to Shagawa Lake in Minnesota, Agency Lake and Diamond
Lake in Oregon and Lake Erie (summarized in the Appendix).

The Upper Klamath Lake studies were undertaken in several
phases based on the original objectives.  These included
(i) development of methods for measurement of total, inorganic
and organic sediment P and for characterization of sediment
P mineral and organic components (Section IV A, B), (ii) exami-
nation of sediments that Changed with limnological conditions
to determine the most labile P mineral or organic components
(Section IV B), and (Hi) laboratory studies to develop
methods for measurement of the potential for sediment P release
and to verify the changes in sediment chemistry postulated
from analysis of field samples (Section V).

DEVELOPMENT OF METHODS1 FOR MEASUREMENT OF SEDIMENT PHOSPHORUS

In initial testing of methods for measurement of the forms of
sediment P, total sediment P determined by Na2C03 fusion,
HCl-NaOH extraction and high temperature ignition methods
generally agreed within 8%, approximating field sampling
variation.  However, high temperature ignition methods con.-
sistently underestimated total P compared to the other methods,
evidently due to the presence of P in mineral-occluded forms
which were not subject to extraction after ignition.

Estimates of inorganic sediment P using H2S01* extraction
and the summation of inorganic P fractions representing various
forms of Al  and Fe-P measured by serial extraction of the
sediments were not significantly different.  For routine
measurements of inorganic P potentially available to solution
in Upper Klamath Lake systems, the simpler H2S04 extraction
method was employed.  In cases in which more definitive infor-
mation was required regarding the forms of inorganic P present,
the serial extraction scheme was employed.

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The sediment organic material  was fractionated into benzene-
ethanol soluble, humate (alkali  soluble, acid insoluble) and
fulvate (alkali soluble, acid soluble)  components.   The fulvate
fraction was further characterized by separation into 5 fractions
based on extractability after concentration on activated
C.  A method was developed for measurement of inositol  hexa-
phosphate in sediment extractives.

Organic C in benzene-ethanol, humate and fulvate fractions
ranged from 65.0 to 83.2% of total sediment organic C contents
Organic P in these fractions  ranged from 36.8 to 51.2% of
sediment organic P content.  Little organic P was subject
to hydrolysis by the solvents employed  and the nonextractable
fraction must be considered quite stable chemically and micro-
biologically, contributing little to P  release from sediments.
Inositol hexaphosphate was not detectable (< 10 yg/g) in Upper
Klamath Lake sediments.

PHOSPHORUS STATUS OF SEDIMENTS AS RELATED TO CHANGES IN
LIMNOLOGICAL CONDITIONS

Limnological conditions and their relationship to changes
in sediment P status differed with lake location.  Seasonal
changes in sediment composition  were most pronounced at a lake
location which received agricultural runoff waters  and where
maximum biological activity occurred as reflected in increased
surface water organic C and N, turbidity and phytoplankton
growth.  At this location, total sediment P decreased sharply
during the late spring and early summer corresponding to the
period of exponential growth  of the lake phytoplankton population
During the late summer and early fall when plankton growth
entered the stationary and declination  phases, sediment organic
C, total N, and P increased,  evidently as a result of the
deposition of detritus containing these elements.  Combining
data from all locations, changes in sediment inorganic P were
negatively correlated to surface water organic C.  Inorganic
P appeared to account for most of the variation in total P.
However, some mineralization  of organic C and N occurred, and
changes in total sediment P were highly correlated to changes
in sediment organic C and N.

Sediments  exhibiting seasonal  changes in distribution of
total, inorganic and organic  P during a single season were
further examined to determine if observed changes could be
attributed to specific inorganic and organic chemical fractions.
In general it appeared that P soluble in 0.1 N_ NaOH, taken as
non-occluded Fe-P, accounted  for most of the reduction in

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inorganic P, whereas increases in inorganic P were due primarily
to increases in NH^F-soluble P taken as non-occluded Al-P-
Considering data from all lake locations during the year, P
soluble in chemical extractions taken to represent forms of Al-P,
Fe-P and Fe and Al oxide occluded P accosted for approximately
85% of the changes occurring throughout a 1-year sampling period.

Measurement of the distribution of organic C in sediment
organic extracts from a single location sampled seasonally
indicated that the major portion of extractable organic
C was in the fulvic acid fraction.  The major portion of fulvic
acid C was present in higher molecular weight substances.
The C contents of the fulvate and humate fractions, relatively
resistant to microbial attack, were not related to changes
in sediment organic C which took place during the monitoring
period.  However, organic C in the benzene-ethanol fraction
varied with changes in sediment organic C.  Organic materials
likely contained in this fraction, i.e. lipids, fats and waxes
derived from fresh or relatively undecomposed organic detritus
should serve as available energy sources for the sediment
microfloral population under conditions of increased temperature
in the spring.  As phytoplankton growth in surface waters
declines in late summer, the rate of deposition of these
materials may exceed the rate of decomposition resulting
in an increase in sediment concentration.

Because approximately 50% of the sediment organic P consisted
of highly stable materials not soluble in the solvents employed,
changes in sediment organic P would not be attributed definitively
to a single fraction.  However, it is noteworthy that as in
the case of organic C, the fulvate fraction contained the
highest concentration of the recovered sediment organic
P.  In contrast to sediment organic C, the largest proportion
of fulvic acid organic P was present in water soluble fractions
of relatively low molecular weight.  Water soluble, and likely
the most labile, fulvate fractions and the benzene-ethanol
fractions varied on an absolute basis with seasonal changes
in sediment organic P.  If it is necessary to predict changes
in sediment organic P it would appear that these fractions
would be a useful point of departure for future research.

LABORATORY STUDIES OF THE POTENTIAL FOR P RELEASE FROM SEDIMENTS

Although gross changes in sediment P composition occurred
at one location during the field phase of the program, it
was recognized from the onset of the program that, due to the
high concentration of P in the sediment relative to the lake

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water, gross changes in sediment P status  need not occur in
order for sediments to significantly influence water,composition.
Therefore laboratory studies  were undertaken to define more
precisely the rate and extent of P release from sediments.
Several methods were developed and preliminarily tested
with respect to their ability to allow (i) separate manipulation
of sediment and water parameters (ii)  measurement of the kinetics
of P release as well as the potential  for  P release and
(Hi] evaluation of the algal growth potential of released
P.  Prototypic methods .included a dialysis method involving,  s
equilibration of sediment in  cellulose acetate membrane tubing
with water, a dialysis method in which sediment and water
in glass half-cells were partitioned with  a 0.4S y filter
and a sediment-resin equilibration system  (Section V A).  These
tests were evaluated as to optimum conditions for P release
and utilized in subsequent studies of the  factors influencing
the P status of lake sediment (Section V B).

Field studies indicated that  inorganic P fractions were
primarily responsible for seasonal changes in sediment P content.
These changes were related to biological action by the surface
waters and at one location the surface water temperature.
In the laboratory studies emphasis was therefore accorded
the effects of P sinks in the forms of an  anion exchange
resin and algal growth, and on temperature as these may
have effected changes in sediment inorganic P.  Simple and
multiple regression models were devised which described
the rate of P release from sediments as a  function of time, temper-
ature and sediment composition.  The results of the release
studies were related to algal growth potential using the EPA
provisional bioassay.

Several general conclusions may be drawn from the P release
studies using the filter dialysis system.   In general, the quantity
of P released to solution increased with incubation time to 90
days.  Inorganic P generally  constituted the major portion
of P released to solution.  Differences in the rate and extent
of P release in the dialysis  systems reflected differences in
total sediment P and interstitial water P  concentration.  However,
if P present in the interstitial waters was subtracted from
total P release in the dialysis system, it could be concluded
that similar quantities of P, amounting to approximately
12% of total inorganic P, were released from the solid phase
of sediments from all locations.

As in the case of sediment sampled during  the field phase,
chemical  extracts representing forms of Al-P and Fe-P appeared to

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account for most of the variation in sediment inorganic P
during laboratory incubation,  Reductions in 0.1  M[ NaOH-
extractable P, representing non^pccluded Fe-P forms, were most
consistently responsible for decreases in sediment inorganic
P.  Increases in inorganic P, when observed, were generally
associated with increases, in th"e NH^F extract representing non-
occluded Al-P forms.  Experiments designed to measure sorption
of 32P spiked into the water half-cell during sediment dialysis
tended to substantiate this phenomenon.  The major portion
of 32P sorbed was in the NHi+F fraction.  Temperature had
a marked effect on P release to solution with the maximum
P release to solution occurring at the higher temperature
level.  Algal growth on standard media containing only P released
from the sediments was equivalent to orthophosphate-P.

Direct equili-bration of the sediments with anion exchange resin
markedly increased P release relative to sediments not containing
resin.  The presence of a P sink therefore had a substantial
influence on the quantity of P released to solution indicating
that under the proper circumstances biological growth might
function in an analogous manner.  Approximately 33% of total
sediment P was released to the resin from the sediment which
exhibited maximum changes in the field compared to < 15% for
other lake locations which varied less on a seasonal basis.
This relatively simple equilibration method resulted in the
release of considerably more P from the sediments than other
methods and the quantity released approximated maximum seasonal
variation for the sediments studied.  This technique therefore
would appear amenable to measurement of the potential for maximum
P release to solution and useful in evaluating the influence
of this parameter for comprehensive lake management.
                                5

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                         SECTION II

                      RECOMMENDATIONS

This program was limited primarily to basic studies of the
chemical nature of lake sediment P and the potential for P
release from sediments.  Although development of remedial
measures for stabilization of sediment P was not within the
scope of this program, the background information on Upper
Klamath Lake sediments presented here may be used in evaluation
of the necessity and general types of control measures.  In
addition, the experimental techniques and procedures that were
established should prove useful in future evaluation of lake
systems.

In lake management, the sediments must be viewed in the
perspective of the total lake system.  The potential for
release of P from sediments may or may not be significant
relative to other factors influencing eutrophic process in
a particular lake system.  An indication of this significance
may be obtained by using lake nutrient budgets in which the
distribution of P in the various ecological compartments is
determined.  The inorganic P is the most labile fraction.  If
the inorganic P in sediments, assuming that it was all released,
is of sufficient magnitude to influence remedial procedures,
the potential for P release and its effects should be considered.

It is likely that measures can be developed which limit P input
into a particular lake system.  The principal problem thus
becomes that of estimating the potential P release from sediments
over the long term.  With this knowledge, decisions can be made
as to the efficacy of treatment of input waters and, subsequently,
treatment of sediments as necessary.  To obtain this information
it is necessary to have enough samples to satisfy statistical
criteria, estimate potential P release under conditions optimum
for release and estimate the potential for increased biological
growth resulting from release.  This information can then be
used in conjunction with a nutrient budget to establish the
necessity for treatment.

Tha results of these investigations indicate that sediments
within a lake system are not necessarily uniform^with respect
to P composition or ability to release P.  The re fore^a__sui table
sampling scheme must be devised.  This may be accomplisliedTas
described in the Appendix of this report.  In general, analyses
of sediments taken from lake transects may be categorized as to
P concentration and permanent sample sites, identified using a

-------
 grid  system,  randomly selected from these categories.   Evidence,
 presented  in  this report indicates that seasonal sampling at these
 sites  may  be  warranted to accurately bracket sediment P concentra-
 tions.

 Several  relatively simple methods were devised to estimate the
 potential  for P release from sediments.  The sediment-anion exchange
 resin  equilibration method gave maximum release.  These data should
 be evaluated  for individual lake systems in terms of the potential
 influence  on  P concentration of lake waters and on biological growth
 over the long term.  Long-term effects should be considered because
 orthophosphate concentration of lake waters may remain quite low
 yet support algal  growth.  The present investigations indicate
 that for practical purposes P released may be considered to have
 the algal  growth potential  of orthophosphate P.

 In the case of Upper Klamath Lake sediments, chemical treatments
 which will minimize release of non-occluded Fe-P should be initially
 evaluated.  These  may include treatments which result in maximal
 sediment pH and aeration, minimizing Fe-P solubility.  Since Al-P
 appears to be largely responsible for sorption of P, a treatment
which also increased the concentrations of sediment Al would appear
desirable.  Colloidal  slurries of Al  hydroxide or silicate may
serve multiple purposes  in  this regard.  The dialysis system
utilized in these  investigations  may find unique application in
testing potential  remedial  procedures.   The lake water may be
individually treated and the influence  on P release from the
sediments evaluated.

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                        SECTION III

                        INTRODUCTION

Eutrophication, the process of water enrichment, is of
considerable national concern.  Increases in the quantities
of nutrients disposed to the nation's waters by municipal,
industrial and agricultural activities have been accompanied
by an increase in the requirements of a growing population
for water of high quality.  Thus, although eutrophication is
a natural process normally accompanying lake aging, dramatic
changes in national priorities and needs require that direct
action be taken to reverse the eutrophic process in many
waters.

One of the most important results of eutrophication is the develop-
ment of undesirable biological populations.  The normal  N
concentrations of most lakes are sufficient to support the
vigorous biological growth observed in eutrophic lakes,
but it is likely that the small quantities of naturally  available
P are not.  Thus, although the relative importance of nutrients
required for phytoplankton growth is a subject of recent
controversy, the supply of P to the lake and P availability
to the biota may be prime factors controlling the rate and
extent of eutrophication.

The sources of P to waters are varied and include industrial
and municipal wastes, surface runoff, and return from irrigation
and ground water.  Reactions which may deplete the supply
of P in fresh waters are equally complex and include uptake
by the aquatic biota, precipitation, and sorption by suspended
particles and bottom sediments.  These depletion reactions
may be in part reversible, thus functioning as future sources
of P to the lake.

The possibility that bottom sediments may function as a  reservoir
of available nutrients to eutrophic lake waters has long
been acknowledged but, at the onset of the program, little
definitive information on the rate and extent of P release and
the factors influencing release was available.  This information
is essential because sediments may have the capacity to  resupply
nutrients removed from lake water, or streams influent into
lake water, by newly developed chemical treatment processes.

The exchange of P between sediments and the overlying water
appears  to be dependent upon a number of chemical, physical
and biological  parameters as summarized by Lee (1970).  Laboratory

-------
studies (Gerhold and Thompson 1969;  Golterman  et al.  1969)  have
shown that sediments may serve as  a  sole source  of P  to algae
which function as a biological sink  for P in water resulting
in the dissolution of phosphate minerals.

In order to provide insight into the effects of  environmental
factors on the rate of P release to  solution and subsequent-
availability to algae, a study was designed to determine
(i) the predominant inorganic and  organic forms  of P  in sediments
of the Upper Klamath Lake,  Oregon, (ii] the rate of release
of soluble P from the solid phase  with  changes in environmental
parameters of temperature,  pH, and water and sediment composition
and (Ui) the relationship  of P release to algal growth requirements,

Initially the program was conceived  as  primarily a laboratory
study.  However because of  the concurrent EPA  program dealing
with the water quality of Upper Klamath Lake,,the principal
portion of the research effort during the first  two years  (1969,
1970) of the program was designed  to determine if detectable
quantities of P were released from sediments.  This release was
detected by monitoring changes in  the concentration of total
sediment P, inorganic P and organic  P on a seasonal basis
and correlating these changes with limnological  changes which  ,
may have affected the release of P.   Inherent  in these studies
was the development of statistically suitable  methods for      . -,
lake sediment sampling and  methods for the characterization
and measurement of sediment P components.  Subsequently,
laboratory methods were developed  for measurement of  P release
to solution and P availabili;ty to  algae.  In all investigations
emphasis was placed on identification of the labile forms
of sediment P and upon estimation  of the maximim potential
for P release.   Thus, a background  of information was provided
to evaluate the necessity for specific chemical  control    :
measures designed to stabilize labile sediment P forms'.

With the development of satisfactory methods for sediment
P determination, these methods were  applied to the characterization
of sediments from Shagawa Lake, Minnesota; Agency Lake, Oregon;
Diamond Lake, Oregon and Lake Erie to provide  a  basis for
future EPA nutrient modeling efforts.  The extension  of
research to these lake systems allowed broader development
and testing of statistical  methods for sediment  sampling
and chemical methods of P measurement.   These  data are included
in the Appendix section of  this report.  However, Upper Klamath
Lake received principal research emphasis particularly in
terms of determination of the factors influencing P release,
and it is this lake system  which will be discussed in detail
in the text of this report.
                               10

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                         SECTION IV

       PHOSPHORUS STATUS OF LAKE SEDIMENTS AS RELATED
           TO CHANGES  IN LIMNOLOGICAL CONDITIONS

Because little was known of the forms of P present in lake
sediments or of the availability of sediment P to biota in the
overlying waters, an initial study was undertaken to develop
methods for measurement of total sediment P and for character-
ization of sediment P  mineral and organic components.  These
methods were subsequently applied to sediments from Upper
Klamath Lake sampled on a seasonal basis to determine (i] if
detectable quantities  of P were released or taken up by sediments,
(ii) the fraction of sediment P responsible for release
or uptake and (Hi] environmental parameters influencing
sediment P transformations.  Total, inorganic and organic
sediment P were monitored over1 a two-year period.  These
results are summarized in Part A of this section.

Sediments exhibiting marked changes in P with changes in limno-
logical conditions were analyzed in more detail to determine the
most labile P mineral  or organic P components.  Identification
of components most responsive to changes in limnological conditions
may allow the development of specific chemical treatments
to stabilize sediment  P.  These results are summarized in Part
B of this section.

A.  TOTAL, INORGANIC AND ORGANIC PHOSPHORUS

This study was designed to (i) evaluate several techniques
used for characterizing sediment P, (ii] determine if seasonal
changes in composition of lake sediments necessitated seasonal
sampling to obtain representative data, (Hi] determine if
detectable quantities of P were released from sediments by
monitoring changes in the concentration of total  sediment P,
inorganic P and organic P on a seasonal basis and (iv} correlate
these changes with limnological changes which may have affected
the release of P.

                   Methods and Materials
Sediment Sampling and Characterization

Sediment samples were taken to an approximate depth of 10 cm
with an Ekman dredge from three locations on Upper Klamath
Lake in south-central Oregon (Figure 1) during the 1969 and
1970 seasons.  The sampling locations were selected on the bases
                              11

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                                     WILLIAMSON
                                       RIVER
             UPPER
            KLAMATH
             LAKE
FIGURE 1.  LOCATION OF  SAMPLING SITES ON UPPER  KLAMATH  LAKF
           OREGON.                                          h'
                                  12

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of representative locations and water depth.   The sediments
from sites designated Howard;Bay, Buck Island and Pelican
Marina were sampled at approximately monthly  intervals during
the period of maximum lake biological activity (May to
September); fall and winter samplings were less frequent
(Table 1).  The samples were packed and transported in ice and
then placed under refrigeration at 4 C in the laboratory.
Subsamples of the moist sediments were analyzed directly after
remixing and drying at 60 C for 48 hours.

The sediments were initially characterized as to mineral
composition (Whittig, 1965) and cation exchange capacity
(Hajek and Wildung, 1969).  Sediment total solids, pH and
carbonate content were routinely measured on  each sample.

Sediment organic C and N monitored during the 1969 season
were determined, respectively, by, microcombustion techniques
(Hajek and Wildung, 1969) and by the Duriias method (Welcher,
1966).

To determine variability associated with sediment sampling
at the selected sites a total  of 8-10 sediment samples,
representing 2-3 dredge loadings per sample,  were taken at
each site in April, 1970 and subsampled in duplicate for analyses
of total and inorganic, P.

Water Analyses

The Environmental Protection Agency maintained a concommitant
program of lake water analyses during 1969.  Parameters measured
in lake water pertinent to the present studies included
total N, NH3-N, N03-N, total and organic C, turbidity (Secchi
disc), pH and temperature (Gahler, 1969).

Phosphorus Analyses

Preliminary investigations were conducted to  determine the
applicability of several analytical techniques for total P and
inorganic P to lake sediment systems.  These  investigations
were continued during the course of the study to include sediment
samples differing in chemical  characteristics with time.
Means of P values determined by each method were compared
for 5 sediment samples from each of 3 lake locations (n = 15).
For these purposes, total P in the sediments  was determined
by Na2C03 fusion (Jackson, 1958), high temperature ignition
(Walker and Adams, 1958) and extraction (Mehta et al., 1954)
techniques.  Inorganic P solubilized by these methods was analyzed
                                13

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Table 1.   Description  of  sampling  sites on Upper  Klamath  Lake, Oregon.
Sample
designation
H-l
H-2
H-3
H-4
H-5
H-6
H-7
H-8
H-9
H-10
H-ll
H-12
H-13
B-l
B-2
B-3
B-4
B-5
B-6
B-7
B-8
B-9
B-10
B-ll
B-12
B-13
Date of
collection Location
5-7-69 42° 18.5'N, 121° 5p.7'W
6_3_69 (Howard Bay)
7-15-69
8-28-69
10-21-69
1-13-70
3-25-70
4-27-70
6-3-70
7-7-70
8-11-70
9-17-70
4-13-71
5-7-69 42° 17.5'N, 121° 50.7'W
6-3-69 (Buck Island)
7-15-69
8-28-69
10-21-69
2-24-70
3-25-70
4-27-70
6-3-70
7-7-70
8-11-70
9-17-70
4-13-71
                                  14

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Table 1.  (Cpnt.)
Sample
designation
P-l
P-2
P-3
P-4
P-5
P-6
P-7
P-8
P-9
P-10
P-ll
P-12
P-13
P-14
P-l-5
Date of
collection Location
5-7-69 42° 14.3'N, 121° 48.5'W
6-3-69 (Pelican Marina)
7-15-69
8-28-69
10-21-69
1-13-70
2-24-70
3-25-70
4-27-70
6-3-70
7-7-70
8-11-70
9-17-70
12-10-70
4-13-71
                                   15

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by the method of Fogg and Wilkinson  (1958).

Sediment inorganic P concentrations  were estimated by H2S04
extraction (Walker and Adams,  1958), and by  summation of non-
occluded inorganic P fractions (Williams, et al.,  1967).

Routine measurements of total  P and  inorganic P  reported
herein were conducted using the high temperature  ignition
and HzSOit extraction methods,  respectively (Walker and Adams,
1958).  Organic P was taken as the difference between total
and inorganic P.

                   Results and Discussion

Upper Klamath Lake has a surface area of 31,000  ha and a mean
depth of 2.5 m.  It does not receive municipal sewage, and
nutrients in drainage from cultural  activities are estimated
(Miller and Tash, 1967) to account for less  than  25% of the
total nutrient input into the  lake system.  However, the
lake is eutrophic, apparently  as a result of natural aging
processes.  A nuisance bloom of blue-green algae,  primarily
Apham'zomenon flos-aque has occurred for many years.  The time
and species distribution of nuisance blooms  vary  from year to
year, but follow a general pattern of a spring bloom of diatoms
and an early summer bloom of blue-green algae.

The sediments of Upper Klamath Lake  are neutral  to slightly
acidic, noncalcareous and silty clay in texture  (Table 2).  Major
clay mineral components are chlorite, vermiculite  and mica.
The cation-exchange capacity of the  sediments ranged from
33 to 55 me/lOOg with differences likely resulting from differences
in clay content.  The C, N and P contents of the  Howard Bay
and Pelican Marina sediments varied  significantly  with season
and will be separately discussed in  a following  section.

Estimation of Sampling and Analytical Variability

The combined effects of sampling and analytical  variability
of P at the selected sampling  sites  are outlined in Table 3.
The standard deviations (n = 8-10) for this  sampling interval
were taken as indicative of sampling and analytical variation
throughout the season.  Variations in total  and  inorganic
P of replicate samples taken throughout the  year approximated
those obtained in this study.   Sediment P values  which differed
by more than one standard deviation  were therefore taken
as real  differences.
                              16

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Table 2.  Depth and chemical properties of sediments, Upper
          Klamath Lake, Oregon.
           Overlying             C03-C       Cation     Major clay
 Sample     water                (CaCOs      exchange   mineral
Location    depth         pH     equivalent) capacity   components
             m                      mg/g     me./100g

Howard Bay   2.5       6.4-6.9     <0.5        33

Buck Island  2.7       6.2-6.9     <0.5        36       chlorite
                                                        vermiculitfe
                                                        mica
Peli can
 Marina      3.4       6.1-7.3      <0.5       55
                             17

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table 3.  Sampling and analytical  variability associated with the
          determination of total  and inorganic phosphorus in lake
          sediments.
. , i , . . - - — 	 . — j 	 : — L— 	
Total P
Standard
Mean Deviation
yg/g
1033 48.1
629 26.7
662 17.5
Inorganic P
Coefficient Standard
of Variation Mean Deviation
% yg/g
Howard Bay
4.7 574 52.7
Buck Island
4.3 295 28.7
Pelican Marina
2.6 348 11.9

Coefficient
of Variation
•35
9.2
9.8
3.4

 Based on oven  dry (60  C) weight.
?
'Mfeans of 8-10  samples  analyzed  in duplicate at each  location.
                              18

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Evaluation of Methods for P Analyses

Preliminary studies in Upper Klamath Lake sediment systems
indicated a relatively close agreement between the Na2C03 fusion
and the HCl-NaOH extraction methods for total P (Figure 2).  The
ignition method resulted in significantly lower P values than
either the fusion or extraction methods [p (probability) < 0.01].
The ignition method consistently gave results for total P that
were about 8% lower than results from Na2C03 fusion or extraction
methods.  A similar phenomenon was observed in ancillary studies
with lakes from other regions.

Estimates of total P obtained by summing (i) the quantity
of residual occluded P solubilized by Na2C03 fusion after serial
extraction of inorganic P (Williams et al., 1967) with (ii) the
total P measured by high temperature ignition were not significantly
different than total sediment P as measured by Na2C03 fusion
methods (Figure 2).  This suggests that a portion of the
inorganic P was present in mineral occluded forms which were
not subject to extraction after ignition.  Hdwever, the
possibility that organic P was volatilized during ignition
would also account for the observed low total P values using
the ignition method.  To investigate this possibility, sediment
samples were ignited and the ash remaining after ignition
subjected to Na2C03 fusion.  Ignition prior to fusion did
not reduce measured total P content relative to Na2C03 fusion
only-  Apparently no P was lost through volatilization on
ignition, and low recovery by the ignition method may have
resulted from the lack of H2S04 extractability of naturally
occluded minerals or the effect of ignition on the extractability
of sediment P as suggested by Sommers et al., (1970).  These
investigations were concerned with comparative changes in lake
sediments and differences between the Na2C03 fusion and ignition
methods were relatively small and consistent.  The simpler
ignition method was therefore used for routine analyses of total
P.

Estimates of inorganic P in sediment samples from all locations
throughout the 1969 season by (i] H2SOi4 extraction (Walker
and Adams, 1958) and inorganic P fractions (Williams et al.,
1967) were not significantly different (p < 0.05).  It was
therefore concluded that for routine measurements of inorganic
P potentially available to solution in these systems the
simpler H2SOit extraction was the method of choice.
                             19

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    700
en
    600
o
o

-------
Seasonal Changes in Sediment Composition

Seasonal changes in total, inorganic and organic sediment
P concentrations at the three sampling locations on Upper
Klamath Lake during 1969 and 1970 are summarized in Figure
3.  Total P ranged from 413 to 1070 yg/g for Buck Island,
521 to 629 yg/g for Pelican Marina and 437 to 707 yg/g for
Howard Bay.  Thus, the greatest variability in total P occurred
at the Howard Bay location with less pronounced changes at
;the Pelican Marina location.  Sediments at the Buck Island
'location did not exhibit large seasonal variations.

Total sediment P at the Howard Bay location decreased markedly
from  May to June and  July, 1969.  By August, the total P content
of the Howard Bay sediment had increased to levels above
.those observed earlier and August levels were apparently maintained
through March, 1970.  In 'April , 1970, a marked reduction
in total P to the low levels observed in 1969 occurred.  Analyses
during the remainder  of. the summer of 1970 indicated a return
of total P to previous high levels followed by a depression
in P  content in August an:d an increase to 1070 yg/g in September.
Total P in April, 1971 (not shown) was 1033 yg/g indicating
an overall increase in sediment P from the spring of 1969 to
the spring ,of 1971,. "\In contrast to the other locations,
Howard Bay annually receives land runoff waters as a result
of fall flooding of adjacent agricultural lands for rodent
control-  The lands are drained into the bay in the spring
prior to planting.  Total P in sediments of Pelican Marina reached
a minimum in July. 1969 and increased to a maximum in January,
1970, with "an overall increase in total P between June, 1969
and April , 1971 amounting to 110 yg/g.

Changes in inorganic  sediment P (Figure 4) were closely correlated
[r (correlation coefficient) = 0.960] to total sediment P at
all locations.  If it is assumed that total and inorganic
P are statistically independent and have equal variances,
the r value for total and inorganic P may be expected to approximate
0.707.  On this basis it is possible to test the significance
of r  (Snedecor, 1956).  The r value for total and inorganic
P is  significant (p < 0.01) indicating the inorganic P fraction
was responsive to changes in lake conditions which affected
total sediment P content.  In fact, changes in inorganic
P accounted for a major portion of the changes in total sediment
P.  In the case of the sediments exhibiting the most marked
change in total P, (Howard Bay from May to June 1969) inorganic
P accounted for &0% of the reduction in total P.
                             21

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    1000
Ol


 t\
is:
o
t-H
I—
<

h-
2=
LU
O
•zz
o
o
"Z.
UJ
s:
I—1
Q
UJ
00
    400  -
200
  0


600



400


200


  0



600


400


200


  0
                                             0 TOTAL  P

                                             A ORGANIC  P

                                             0 INORGANIC
                       1
                     1  1
                                1  1
                                      I  I
                                             ,
             BUCK  ISLAND
        >  i
             PELICAN  MARINA
                                    p— Q— 0**
                   ,  ,  !  ,  ,   1  ,  ,  1  ,  .  1   ,
   JUNE
               SEPT'   DEC   MAR   JUNE  SEPT

               1969                1979

                     TIME,  MONTHS
  FIGURE  3.   SEASONAL CHANGES IN TOTAL, INORGANIC  AND  ORGANIC
             SEDIMENT PHOSPHORUS CONCENTRATIONS  AT  THREE
             SAMPLING LOCATIONS ON UPPER KLAMATH LAKE.
                              22

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    Q
    LU
    C/1
               TOTAL
                 P
             INORGANIC
                 P
ORGANIC
   C
               TOTAL
                 N
              ORGANIC
                 C
               TOTAL
FIGURE 4,   CORRELATION OF SEASONAL CHANGES IN SEDIMENT AND
           LAKE WATER PHOSPHORUS, CARBON AND NITROGEN.
           TRIPLE, DOUBLE AND SINGLE ASTERISKS DESIGNATE
           SIGNIFICANCE (n=13) AT THE p<0.001, <0.01,  AND
           <0.05 LEVELS RESPECTIVELY.
                                 23

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Although changes in inorganic P appeared to account for most
Of the changes in total P, changes in sediment organic C during
the 1969 season (Table 4) closely paralleled changes in total  P
(Figure 3) at Howard Bay and Pelican Marina sites with little
change occurring at the Buck Island location.  Combining
data from all locations, sediment organic C was correlated (r
•= 0.829; p < 0.001) to total P during the 1969 season.  Changes
in organic C were most pronounced at the Howard Bay location.
A fraction of sediment organic C was apparently mineralized
during the spring, decreasing at Howard Bay from May to June,
1969 (Table 4).  A portion of this organic material was evidently
organic P, changes in which were significantly related (r = 0.880;
p < 0.05) to changes in organic C for the Howard Bay sediments.
Organic C increased from July to October 1969, likely due to the
deposition of plankton detritus.

the C:N ratio of the sediments at all locations was quite
constant throughout the season ranging from 6.1 to 7.1 (Table
4).  Utilizing combined data from all sample sites during
the 1969 season, changes in total sediment N were correlated
to organic C (r = 0.893; p < 0.001) and to total P (r = 0.746;
p < 0.001).  The correlation of total N to organic C for all
sample sites was due primarily to the close relationship
between seasonal changes in sediment concentrations of these
elements at the Howard Bay and Pelican Marina sites.  Reductions
in the concentrations of sediment C during the spring and early
summer at Howard Bay were accompanied by proportionate reductions
in sediment N as might be expected if sediment biota were
active in the mineralization of C.  Increases in sediment
N occurred during the late summer and fall, apparently resulting,
analagous to C, from the deposition of plankton detritus.
The C:N ratio of the sediments was higher than might be
expected if they consisted solely of fresh phytoplankton tissue
(C:N ratio approximately 5:1).  A portion of the N in the
tissue must therefore have been mineralized during settling
or on deposition in the sediments.

Influence of Limnological Conditions on Sediment Composition

Biglogical Actlyity.  Decreases in total P and inorganic
P in Howard Bay sediments in the early summer of 1969 (Figure  3).
corresponded to the period of exponential growth of Aphanizomenpji
flos-aque, the predominant blue-green algae in the laRe~~
system.  Secchi disc measurements in Howard Bay during June
and July, 1969 were 34 and 10 cm, respectively, compared
with 70 cm or greater during August and September indicating
increased water turbidity and algal growth during the early summer.
                             24

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 Table 4.   Seasonal  changes in sediment organic carbon and total nitrogen,
Sampling
date*


5-7
6-3
7-15
8-28
10-21

5-7
6-3
7-15
8-28
10-21


5-7
6-3
7-15
8-28
10-21
Sedi merit
Carbon


7.9
6.4
6.6
7.7
8.6

6.9
6.8
6.5
6.3
6.7
i

6.3
6.9
6.3
6.4
6.8
concentration of
Nitrogen
01
Howard Bay
1.12
0.98
0.94
1.09
1.28
Buck Island
1.06
1.12
1.02
0.92
0.95

Pel "lean Marina
0.91
1.00
0.95
0.93
0.97
Ratio of
Carbon/Nitrogen
, i

7.1
6.5
7.0
7.1
6.7

6.5
6.1
6.4
6.9
7.1


6.9
6,9
6.6
6,8
7.0
1969
                               25

-------
 In June and July, Secchi  disc readings at other lake locations
 were in excess of 70 cm suggesting less phytoplankton growth
 at these sites relative to Howard Bay.  An Aphanizomenon
 bloom of similar magnitude occurred at the same time in 1968,
 but measurements of sediment nutrient levels were not initiated
 until 1969.  The Aphanizomenon bloom in 1970 was not of the
 same magnitude as observed in 1969 with Secchi  disc measurements
 at the Howard Bay location in June and July, 1970 of 140 and
 200 cm, respectively.  Reductions in sediment P (Figure 3) in
 April, 1970 and August, 1970 coincided with an  extensive increase
 in diatom numbers in April and a delayed Aphanizomenon bloom
 in August.  Considerable  quantities of sediment P were thus
 released at times of maximum biological growth  in Howard Bay.
 These losses from the sediments were not reflected in increased
 concentrations of P in the waters.

 Phytoplankton use of P in Howard Bay evidently  provided a
 substantial biological sink that reduces the solution concentration
 of orthophosphate and decreases total sediment  P.  Concurrently,
 N and organic C were also released from the sediment.  These
 effects were not as pronounced at the other sample locations
 perhaps due to relatively less phytoplankton growth in waters
 at these locations as exhibited by lower turbidities during
 this period.  Sediment interstitial water concentrations
 of orthophosphate at Howard Bay in April 1969 were higher
 (7-1 mg/liter) than other lak|e locations (< 0.5 mg/liter) perhaps
 as a result of agricultural runoff to the bay in the early
 spring.  This additional  P, if available to the biota, may
 have provided the impetus for the increased growth which
 results in sediment P release in the early summer.  Increases
 in total  P, organic C and N from August to January, 1969
 apparently resulted from  the deposition on the  sediment and
 partial mineralization of large quantities of dead algal
 tissue as the algal population declined.

 Surface Water Parameters.  An examination of the chemical
 composition of the lake water (Table 5) during  the 1969 season
 provides  further insight  into the lake phenomena influencing
 sediment  P, C and N status.  During this period the concen-
 tration of soluble orthophosphate in the water  of Howard
 Bay remained at < 0.01 mg/liter.  However, organic C contents,
 a measure of biological activity, increased from 4 mg/liter
 in May to 10 and 23 mg/liter in June and July and then decreased  '
to 7 mg/liter in August.   Organic C contents of lake waters
 from the  other sampling locations did-not exceed 9 mg/liter
over the  same period.  Organic C in the lake waters was nega-
tively correlated (r = -0.563; p < 0.05) with sediment inorganic
 P when data from all  locations was combined. This is further
                               26

-------
           Table  5.   Seasonal  changes  in  elemental  composition,  pH  and  temperature  of surface waters.
ro
Sampling*
date


5-7
6-3
7-15
8-28
10-21

5-7
6-3
7-15
8-28
10-21

5-7
6-3
7-15
8-28
10-21


total



0.8
5.3
14.6
3.3
3.2

0.8
1.4
2.0
2.3
2.6

0.6
1.6
2.8
2.9
3.8

Nitrogen
ammonium



0.05
2.70
11.00
1.30
0.70

0.06
0.47
0.08
0.40
0.07

0.03
0.52
0.23
0.72
0.04
Carbon
nitrate



<0.01
0.01
0.09
0.04
<0.01

<0.01
<0.01
<0.01
<0.01
<0.01

<0.01
<0.01
<0.01
<0.01
<0.01
total


Howard Bay
14
35
35
25
25
Buck Island
14
18
19
22
22
Pelican Marina
14
21
22
22
22
organic



4
10
23
7
7

4
5
9
7
7

4
4
7
6
6
Ortjiophosphate



<0.01
<0.01
<0.01
<0.01
<0.01

<0.01
<0.01
<0.01
<0.01
<0.01

<0.01
<0.01
<0.01
<0.01
<0.01
pH
units

7.0
9.5
8.4
9.4
8.3

8.5
9.2
9.4
9.7
8.8

6.4
9.6
9.6
9.7
8.3
Tempe ra
ture
°C

13.2
19.0
19.2
18.3
8.5

13.5
18.5
19.0
17.6
8.5

13.4
18.3
18.5
18.9
8.5
         1969 season

-------
evidence that the lake sediments served as''reservoir of inorganic
P to support biological growth in the lake waters.

Paralleling organic C, the marked increases in total N, NH3-N and
N03-N (Table 5) that occurred in the waters of Howard Bay during
the early summer reflect increased production of biological
tissue and increased biological  activity.  A decrease in
N forms occurred from July to August.  Thus the C and N analyses
tend to substantiate turbidity measurements and visual observations
of heavy phytoplankton growth in Howard Bay relative to the
other locations in June and July.  At the Buck Island and
Pelican Marina locations, total  N in water increased gradually
from May to October (Table 5).  Combining water data from
all lake locations, total N was  highly correlated to organic
C (r = 0.972; p < 0.001).  As in the case of Howard Bay,
the NH3-H contents at Buck Island and Pelican Marina were
highest during the late spring and summer months with minimum
levels occurring in May and October.  However, the magnitudes
of the changes were much less than for the Howard Bay location.
In contrast to the waters of Howard Bay, N03-N did not exceed
0.01 mg/liter throughout the season.

At all lake locations surface water pH (Table 5) was higher during
the summer than the spring and fall reflecting increased
biological utilization of C02 during the summer.  The water
pH was not, however, significantly correlated to changes'
in sediment P, N or C.

Water temperature (May, 1969 to  January, 1970) was negatively
correlated (r = -0.755; p < 0.05) to total sediment P in Howard
Bay sediments (Figure 3, Table 5).  That is, sediment P concen-
trations tended to decrease with increased temperature and
increase with decreased temperature.  Changes in water temperatures
were approximately equivalent at other locations but these changes
were not related to release of P from the sediment.  Thus, temper-
ature alone cannot explain the variations in sediment total P
observed at the Howard Bay location but rather appeared
to play an indirect role in influencing biological growth.

B.  PHOSPHORUS MINERAL AND ORGANIC COMPONENTS

The concentrations of total P in the sediments of Western Lake
Erie have been recently shown (Skoch, 1968) to change greatly
on a seasonal  basis and the changes appear to be related to changes
in total  Fe and C.  In concurrent investigations (Section IV,
A) dealing with the relationship of sediment P to water quality
in Upper Klamath Lake, significant seasonal changes in total
                              28

-------
sediment P were also observed.  In the Upper Klamath Lake studies,
variations in total sediment P were correlated with changes in
sediment inorganic P and with organic C,  Inorganic P appeared
to account for a major portion of the variation in total sediment
P.  These effects were particularly pronounced at the Howard
Bay location where sediments exhibited a marked reduction
in total P and inorganic P during the early summer, evidently
releasing P in response to the utilization of orthophosphate
by a heavy bloom of blue-green algae.  Increases in sediment P
and organic C followed cessation of the algal bloom apparently
as a result of the deposition of algal detritus.  Thus,
in addition to a planned examination of the changes in sediment
P mineral components on a seasonal basis we had an opportunity to
investigate the chemical changes in the sediment inorganic
and organic P fractions when occurred in the field in response
to biological demands and upon deposition of organic detritus
following the cessation of phytoplankton growth in the late summer
and fall.

This investigation was designed to determine (i] the relative
changes  in concentration of chemically defined P mineral
fractions responsible for seasonal changes in sediment inorganic
P and  (ii] the distribution of C and P in the benzene-ethanol
soluble, humate and fulvate components of the sediment organic
fraction during phytoplankton growth and upon deposition
of phytoplankton detritus.

                   Materials and Methods

Sediments were sampled periodically during the 1969 and
1970 seasons from three locations (Howard Bay, Buck Island
and Pelican Marina) on Upper Klamath Lake as previously described.
Sediments exhibiting marked seasonal changes in P distribution
in inorganic and organic P fractions during the first year
were further examined to determine if observed changes in
total  P status could be attributed to specific inorganic and
organic chemical fractions.

Phosphorus Mineral Components

Total  P was determined by Na2C03 fusion (Jackson, 1958).  The
inorganic P fraction of sediments sampled from all locations
during May, June, July, August and October, 1969 were characterized
using  the serial extraction techniques (Table 6) of Chang
and Jackson (1957) as modified by Williams, et al. (1967).  The
method was further modified (Results and Discussion Section)
to obtain complete recovery of added P in the reductant-soluble
                              29

-------
P fraction.  Inorganic P was  estimated by H^SO^ extraction
(Walker and Adams, 1958) and  summation of non-occluded inorganic
P fractions 1-6 (Table 6).   Inorganic P values obtained by these
methods were not significantly different for these sediments
(Section IV, A).

Organic Phosphorus Components

To provide insight into the chemical  changes which may have
occurred concommitantly with  changes  in sediment concentrations
of total P and C, the sediments of Howard Bay, sampled in
May, June, July and August (Table 1), were fractionated into
benzene-ethanol (1:1) soluble, NaOH soluble-HCl insoluble
(designated humic acid), and  NaOH-HCl soluble (designated
fulvic acid) components as described by Wildung et al. (1965,
1971).  The yields of organic matter, total  P and organic
C were measured for each extract.  The fulvic acid components
were further characterized as described for soil systems
by Forsyth (1947) and Stevenson (1965a) by concentration
from aqueous solution on activated C and serial dissolution
of the adsorbed material with acetone (90%), distilled H20 and
NaOH (0.5 M), yielding the fractions  described in Table 7.

Techniques were developed to  measure  sediment levels of inositol
hexaphosphate, believed to constitute the bulk of the organic
phosphorus in soils.  The method described by Caldwell and
Black  (1958) and Stevenson (1965) for isolation of inositol
hexaphosphate from soils was  initially evaluated for application
to sediment systems.  In general, the method involved successive
extractions of soils with HC1 (concentrated) and NaOH (0.5
N) to remove separate inositol hexaphosphates.

Organic contaminants were removed from the NaOH extract by precipi-
tation at pH 1.0 or less and  centrifugation.  The HC1 and purified
NaOH extracts were combined and the Fe salt of inositol hexaphosphate
was precipitated by titration (NaOH)  to pH 1.8.  The precipitate
was dissolved in 0.85 N_ HC1 and the solution eluted through
a glass column containing an  anion exchange resin.  The
inositol hexaphosphate and lower phosphate esters of inositol
and inorganic P were removed  by elution with increased concentrations
of acid.  Direct application  of the method published for
soils resulted in incomplete  recoveries of inostiol hexaphosphate
from sediments and therefore  several  modifications were employed
as outlined in the Results and Discussion Section.
                               30

-------
      6.  Serial extraction procedure employed for the fractionation
          of sediment inorganic phosphorus!.
Treatment
1
                Treatment
Time
Probable major mineral
    P extractives

1
2
3
4



NH4C1 (0.5M)
NH4F (0.5M, pH 8.2)
NaOH (0.1 M_)
Na9S,0. (0.1M)
L. L* T1
Na3C6H5°2 ' 2H2°
hr
0.5
24
17
0.3



Readily soluble P
Al-P
Fe-P
Fe, Al -oxide occl
Fe-P, Al-P





uded


             (0.3M, pH 7.3)
5 NaOH (l.OM)
6 HC1 (0.5M),(1.0M)
7 Ignition at 550 C
followed by HC1 (l.OM)
8 Na2C03 fusion
followed by HC1 (9.0M)
17
1,5
1
16
0.3
0.5
Fe, Al -oxide occluded
(Al, Fe)-P
Ca-P
Residual
organic P
Residual
inorganic P

 after Williams et al.  (1967)
                               31

-------
Table 7.   Serial  extraction procedure employed for fractionation of
          the fulvic acid (NaOH-HCl  soluble)  components of lake
          sediments  after concentration  on activated carbon.
     '                 "   ""'  T~         Probable organic major
Fraction             Treatment               extractives


  A               Aqueous  elutant         Low molecular weight, fLO
                  from activated         soluble  organic  compounas
                  C                      (simple  sugars,  purine
                                         bases, ami no acids)
  8               Acetone  (90%)           Tannins
  C               Distilled  H20           Polysaccharides

  D               NaOH (0.51^)             Pentose  sugars,  nonprotein
                                         nitrogen,  organophosphorus
  E               Activated  C             Unknown
                  residual
  Traction B may be  purified  by evaporation  of  acetone,  dissolution
   of the residue in  ethanol and repeated  precipitation  from ethanol
   using ether;  fraction  C may be  purified  by repeated precipitation
   from aqueous  solutions using acetone.
                              32

-------
                   Results and Discussion

Evaluation of Analytical Techniques

The development of suitable methodology for the measurement
of P compounds in bottom sediments is complicated, as in the
case of soils, by a lack of knowledge of the chemical forms
in which the element may be present.  In the absence of this
knowledge, it is not possible to precisely determine recoveries
on extraction or the degree to which the compound may be altered
by the extraction procedure.  Therefore, for the purposes
of this investigation recoveries of total inorganic P and
chemically extracted P components were measured using orthophosphate
amendments and elemental balances, i.e. the recovery of C or
P in individual fractions as a function of total P or total
C.

In general, the Williams et al. (1967) modification of the Chang
and Jackson (1958) method gave satisfactory (>90%) recoveries
of added inorganic P in lake sediments.  However, quantities
of reductant-soluble P  (Treatment 4, Table 6) approached
minimum detectable limits and use of the Watanabe and Olsen
(1962) procedure for measurement of reductant-solubilized
P as recommended by Williams et al., (1967) consistently resulted
in incomplete recovery of P added at low (<50 yg/g) concentration
levels.  Alternatively, use of H2Q2 as an oxidant as recommended
by Chang and Jackson (1958) was time consuming and complicated
by concentrations of 0.5 ppm P in reagent grade H202.  A new
procedure was therefore developed which involved drying of the
extract (70 C), ignition (600 C) or digestion (HClOiJ, dissolution
of the ignited residue in H2S04 (2 H) and colon metric analyses
of P by the method of Fogg and Wilkinson (1958).  The modification
allowed recoveries of added P in excess of 90% and detection
of 4 yg P/g of sediment.

It should be emphasized that the entire P mineral fractionation
scheme is based on known solubilities of model compounds
and that dissolution of discrete mineral forms by each solvent
is not possible in the complex lake sediment systems.  The
results should therefore be used only as an indication of the
mineral types which may be present.  However for development
of chemical control measures it should prove useful to know the
relative chemical solubilities of the mineral components.
Due to the extensive effort required to complete the P mineral
fractionation, analyses were limited to samples from the
1969 season when much of the seasonal variation occurred.
                               33

-------
Recovery of organic C in the benzene-ethanol ,  humate and
fulvate fractions ranged from 65.0 to 83.2% of the total sediment
organic C content.  Recovery of organic P in these fractions
ranged from 36.8 to 51.2% of sediment organic  P content.
The unrecovered C and P were largely present in the sediment
as residuals not soluble in the solvents employed (Table
8).  Recoveries of extracted sediment organic  P, organic
P present as residuals in the silica fraction  removed by precipi-
tation during the extraction, and as residuals in the sediments
ranged from 89.0 to 101.8% of total  organic P.  Thus, little
organic P was subject to hydrolysis  by the solvents employed
and a relatively large fraction of the sediment organic
P was nonextractable.  This latter fraction is likely quite
stable and would contribute little to P release from sediments.

When published methods for determining inositol hexaphosphate
in soils were applied to soils, insufficient inositol  hexa-
phosphate was recovered for reliable quantitative analysis.
Incomplete recoveries of inositol  hexaphosphate during separation
on the anion exchange resins, chemical purification, and
extraction from lake sediments cummulatively reduced total
recovery to levels incompatable with quantitative analysis.
Therefore, new methods and ways to improve the analysis at
each of these stages were investigated.

The recovery of a known quantity of inositol  hexaphosphate
from an anion exchange resin (Smith  and Clark, 1952) by
gradient elution with increased concentration  of HC1 is
outlined in Figure 5.  It is evident that complete recovery
of inositol hexaphosphate from the column may  be obtained
by this method.  However, large volumes of HC1 are required,
over a relatively long time period (flow rate  1 ml/minute) to
obtain a single analysis.  In order to reduce  the analytical
time required, recovery was measured in a comparable manner
using the method of Caldwell" and Black (1958).  This method
involved elution of the inositol hexaphosphate with 1.4 N^ HC1
only after removal  of inorganic P from the column using 0.85
N^ HC1.   The method allowed complete  recovery of inositol
hexaphosphate from the column without use of large volumes
of eluent, (Figure 6).

Amendments -of the combined HC1 and NaOH sediment extracts,
after chemical  purification, with a  known quantity of inositol
hexaphosphate followed by column separation of the compound
(Caldwell  and Black, 1958) resulted  in over 95% recovery
of P added as inositol hexaphosphate after elution with 1500 ml
of 1.4 N^ HC1  (Figure 7).  Thus, the  presence of sediment
                               34

-------
        Table 8.   Distribution  and  recovery of organic  phosphorus  in  organic  extractives  and  residual
                   components  of Howard  Bay sediments.
oo
en

Samp! e 2
designation

H-l
H-2
H-3
H-4

Silica
fraction

43
54
45
91
Organic P
Sediment
residual
n / rt
97
96
88
116

Sum of
organic
extractive

282
276
258
348

Untreated
-sediment
s6

317
271
290
382

Recovery

-------
to
              60
              50
              40
              30
              20
              10
-  3.0
- 1.0
                        1
                                O TOTAL P
                                U FRACTION OF TOTAL P ELUTF.D
                                A fld CONCENTRATI-ON
                                                                                100
                                                               80
                                                                                60
                                                                                40
                                                                                   LU
                                                                                   Q.
     1000    2000
                                      3000    4000     5000
                                            HC1,  mis
6000     7000
 FIGURE 5.   RECOVERY OF INOSITOL  HEXAPHOSPHATE  FROM  AN ANION EXCHANGE  RESIN BY GRADIENT
             ELUTION WITH INCREASING CONCENTRATIONS  OF ACID.

-------
      50
      40
  
-------
   Oi
   O
       25
       20
       15
       10
1
                          —I   -i    i     i    i     r-

                           O TOTAL P
                           n FRACTION OF  TOTAL P ELUTED
                           • INORGANIC P
                           • FRACTION OF  INORGANIC
                             P ELUTED

                             /
                                    a*

                        I n unn
                               i
                  500      1000
            HC1 ,  mis 0.85 N
500      1000
 HC1 , mis 1.40 N
                                                      — n
                                                 100
                                                            80
                                                            60
                                                            40
                                                            20
                                                               CD
                                                               o;
                                                               o
                                                               o
                                                               z:
                                                               <
                                            1500
FIGURE  7.   RECOVERY  OF INOSITOL  HEXAPHOSPHATE FROM  SEDIMENT
            EXTRACTS  USING ANION  EXCHANGE  CHROMATOGRAPHY .
                                38

-------
extractives caused in a marked increase in elutant required
to remove P, likely as inositol hexaphosphate from the column.
The P added as inorganic P was quantitatively removed (recovery
95.6% from the column by prior elution with 0.85 ^ HC1).  The
technique proved highly replicable and consistently allowed
over 95% recovery of P added as inositol hexaphosphate.  Amendment
of sediments with inositol hexaphosphate followed by chemical
extraction, chemical purification and column separation
resulted in similarly high recoveries of added inositol hexa-
phosphate phosphorus.  Thus, analyses of sediments for inositol
hexaphosphate may now be undertaken with reasonable confidence.
It should be noted, however, that further methodological refine-
ment is required.  To insure the identity of separated inositol
hexaphosphate in sediment extractives, the C:P ratio of the
material eluted from the column at the inositol hexaphosphate
position on the chromatograph must be determined.  In addition,
recoveries of inositol hexaphosphate from the anion exchange
resin were occasionally reduced by an amorphorus A1(OH)3
precipitate which formed during chemical purification and
precipitation of the inositol hexaphosphate Fe salt at pH 1.8.

Seasonal Changes in Sediment Inorganic P Components

The distribution of P in the principal P mineral fractions
Of Upper Klamath Lake is summarized in Table 9 and further
illustrated in Figures 8, 9 and 10.  These data help evaluate
(•£) the potential of procedural artifacts created by the
influence of chemical treatments on subsequent measurements
during serial extraction and (ii) the effect of changes
in concentrations of individual minerals or groups of minerals
on changes in total inorganic P observed throughout the
year.  The latter objective was:deemed particularly important
to the identification of minerals significant in supplying
P for phytoplankton growth.

The concentrations of P in the NH^F and residual organic
P fractions of Howard Bay (Table 9; Figure 8) increased markedly
from spring to fall whereas the NaOH (0.1 M) fractions exhibited
a marked decrease from May to June followed by a slight
increase to October.

As in the case of Howard Bay, the Buck Island sediments (Table
9; Figure 9) exhibited an increase in residual organic P with
time, but this was not accompanied by an increase in the NH^F-P.
The extractable P in the NaOH (0.1 M) fraction of Buck Island
sediments also decreased with time.  This decrease was accompanied
by a decrease from May to June of Na2S2OLf - Na3C5H502 and
NaOH (1.0 M) P forms.


                               39

-------
  Table 9
.   Distribution of phosphorus in inorganic phosphorus  fractions  of  Upper  Klamath  Lake  ^ediments.
Sample   ,                                        P extracted^ with
designation1 NfyCl
(0.5M)
H-l
B-l
P-1
H-2
B-2
P-2
H-3
B-3
P-3
H-4
B-4
P-4
H-5
B-5
P-5
5
6
8
5
5
5
13
6
4
7
10
4
10
2
2
NH4F
(0.5M)
30
47
41
67
54
55
S2
45
50
107
50
38
115
39
47
NaOH NaS204 (O.IM) NaOH
(0.1M) NasC6H502 (0.3M) (l.OM)
in
131
109
25
59
54
28
47
17
37
23
26
67
25
26
72
109
36
56
44
:
22
39
35
^46
57
40
66
40
37
— vg/g 	
20
25
17
22
7
5
16
20
21
31
29
32
32
27
27

(0.5M)
21
22
43
18
21
23
18
23
43
16
26
_33
23
26
44
HC1
(l.OM)
0
0
0
15
8
17
6
6
TO
7
7
8
9
9
10
K
Total HI
21
22
43
33
29
40
24
29
53
23
33
41
32
35
54
jnition- N 33003
:i (KOM) fusion
HC1 (9.0M)
2
3
8
54
54
30
44
45
44
fr9
61
59
96
65
60
40
67
90
106
70
86
55
55
66
54
55
55
95
60
64
  'Table 1
  -Table 6

-------
en
^-i
en
 
-------
en
3.
et
o;
o
o
o
LjJ
co
 40


 20


  0


 40


 20


  0

100



 50



  0


100



 50
             NaOH(l.OM)
HC1
IGNITION
             FUSION
                                  1
                            J      A

                             TIME, MONTHS
 FIGURE 8b.  MONTHLY  DISTRIBUTION  OF  PHOSPHORUS  IN  INORGANIC
             PHOSPHORUS FRACTIONS  OF  THE SEDIMENTS  OF  HOWARD
             BAY,  UPPER KLAMATH  LAKE,  OREGON.
                                42

-------
    20
 C7>
 3.
    100
     50
S   100
o
UJ
GO
 50



  0


100



 50
           NH4C1
           NH4F
           Na)S)0,-Na,C,Ht-0
                    J D 0
                           I
                              I
                           J      A
                           TIME, MONTHS
FIGURE 9a.
         MONTHLY DISTRIBUTION  OF PHOSPHORUS  IN  INORGANIC
         PHOSPHORUS FRACTIONS  OF THE SEDIMENTS  NEAR  BUCK
         ISLAND, UPPER  KLAMATH LAKE, OREGON.
                               43

-------
Ol


01
o
t—t
I—
•=c
      40



      20



       0



      40



      20



       0
  LU
  O

  o  100
  o
    50



     0



   100



    50
           NaOH(l.OM)
           HC1
             IGNITION
             FUSION
                              '     A   '

                             TIME, MONTHS
FIGURE 9b.  MONTHLY  DISTRIBUTION  OF  PHOSPHORUS IN  INORGANIC
            PHOSPHORUS FRACTIONS  OF  THE SEDIMENTS  NEAR  BUCK
            ISLAND,  UPPER KLAMATH  LAKE, OREGON.
                                  44

-------
CD

cn
o


D-
     20


     0



    100



     50
    100
     50
    100
    50
          NH4C1
          NH4F
           NaOH(O.lM)
                                  \
                                        1
                           J       A       S
                            TIME,  MONTHS
                                                   •o
 FIGURE  lOa.
              MONTHLY DISTRIBUTION  OF  PHOSPHORUS IN INORGANIC
              PHOSPHORUS FRACTIONS  OF  THE SEDIMENTS OF  PELICAN
              MARINA, UPPER KLAMATH  LAKE, OREGON.
                                   45

-------
en

en
o
(—
<
o

Q-
Q
LLj
(SI
 40


 20


  0


 40


 20


  0

100



 50
    100
    50
             NaOH(l.OM)
             HC1
             IGNITION
             FUSION
                                         _L
                           J       A       S
                            TIME,  MONTHS
 FIGURE lOb
           MONTHLY DISTRIBUTION  OF PHOSPHORUS  IN  INORGANIC
           PHOSPHORUS FRACTIONS  OF THE SEDIMENTS  OF PELICAN
           MARINA, UPPER KLAMATH LAKE, OREGON.
                                   46

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The Pelican Marina sediments increased in NaOH (0.1 M)-P and
residual organic P from May to October.  As in the case
of the other sediments, NaOH (0.1 M)-P decreased over this
time period.

Two change's tn "mineral P over the season common to all sediment
locations were (i) an increase in residual organic P and
(H] a marked decrease in NaOH (0.1 M) soluble P.  Howard
Bay sediments, which showed the largest seasonal  variation in
total and inorganic P, exhibited marked increases in NH^F-P
but this was not common to sediments from other locations.

Increases. In, residual,, organic P in the cases of Howard Bay and
Buck Island sediments appeared to reflect overall increases
in sediment total, organic P (Figure 3).  However in the
Pelican Marina sedimerits total organic matter P decreased,
yet residual organic P increased.  The increase in residual
organic P was accompanied by a decrease in the next fraction,
i.e. the residual inorganic P fraction.  Perhaps this results
from incomplete solubilization of residual organic P which
was subsequently measured on fusion. . It should also be noted
that organic matter is solubilized by NaOH, the solvent
used for extraction of various forms of Fe-P.  Decreases
in the Fe-P fraction may have been due in part to incomplete
extraction of organic P which later was recovered in the
residual organic P fractions. "However,, increases in the
residual organic P fraction could not account entirely for the
marked decreases in the Fe-P fraction during the season
and this decrease must therefore be considered real in part.

The sediments of Howard Bay (Figure 3) exhibited the greatest
changes in inorganic P during the 1969 and 1970,seasons.  An
initial reduction in sediment total and inorganic P early
in the spring followed by increases in total and inorganic
P in the summer and fall have been previously attributed
(Section IV, A) to biological uptake of P early in the season
followed by the deposition and partial mineralization of
biological tissue later in the season.  Evaluation of the
changes in P mineral components for Howard Bay over this
period indicates that the principal minerals resulting in a
decrease of sediment P were in the NaOH (0.1 M) or Fe-P fraction
The P in this fraction gradually increased during late summer
and fall, but increases in inorganic P during this period
were due primarily to increases in minerals in NH4F or Al-
P fraction.   Minerals in the Fe-P fraction appeared to supply
P to solution during biological :growth with a reduction
in the Fe-P fraction (Figure 8) amounting to 86 yg P/g of
                              47

-------
sediment from May to June (HI and H2, respectively; Table
9) compared to a total reduction in inorganic P (Treatments i-
6; Tables 6, 9) of 60 yg P/g (Treatments 1-6 have been taken,
Section IV, as representing the labile inorganic P fraction).
The differences between 86 yg P/g and 60 pg P/g appeared
to be accounted for by relative increases in the NH4F (Al-
P) and HC1 (Ca-P) fractions.

Minerals in the NH4F fraction accounted for a major portion of
the increase in inorganic P from July to October with smaller
contributions from the various Fe-P fractions.

It is difficult, on the basis of individual sample site
comparisons, to estimate the extent to which changes in
the inorganic P fractions contributed to changes in the total
inorganic P fraction for all sediment locations.  In order
to obtain a gross estimate of this effect, a regression analysis
of P concentrations in individual extracts against total
inorganic P was employed.  It should be noted, however, that
the results are indicative of trend only since the inorganic
P components are inherently related to total inorganic P of which
they are a fraction.  To undertake the regression analyses,
several assumptions were made:  (i) changes in the Al-P, Fe-
P and Fe, Al-oxide occluded Fe-P, Al-P fractions (Treatments
2, 3, 4; Table 6) which are of the largest magnitude would have
the greatest effect on total inorganic P and (ii} the inorganic
P treatments are independent statistically and have equal
variances.  With these assumptions it  is possible to state that
total inorganic P regressed against the sum of 3 fractions will
have an expected r value of 0.707.  Hence the degree that the
r value exceed this "expected" value is a measure of the
extent of correlation to total relative to the remaining
3 fractions.  The results are summarized in Table 10.  Based
on the above assumptions, Treatments 2, 3, 4, representing
forms of Al-P, Fe-P and Fe and Al oxide occluded P accounted
for approximately 85% of the variance in total inorganic
P occurring at all  sampling locations throughout t,he year.

Distribution of C and P in Sediment Organic Fractions

Although the physicochemical properties of the sediment
organic matter are likely altered upon extraction, particularly
when alkali  is employed as an extractant (Bremner, 1950), initial
definition of sediment P components on the basis of solubility
may prove useful  in designing remedial techniques to minimize
release of P from sediments.  In addition, changes in the concentra-
tion of those components which may occur in response to changes in
                               48

-------
 Table 10.   Summary of the regression of individual  extractant phosphorus  concentrations  against
            total inorganic sediment phosphorus for  all  Upper Klamath  Lake locations  sampled
            during a single season.
Extractant
Degrees
of
freedom
Regression
Intercept coefficient
Standard error
of regression
coefficient
Correlation
coefficient
Variation of
total inorganic
P attributable to
changes in
extractant P
Sum of Treat-
ments 2,3,4
(Table 6)
13
130
1.003
0.120
0.923
                                                      1
85.2
 Significant at the p < 0.05 level

-------
limnological conditions may provide insight into the chemical
nature of the labile sediment organic components.

As in the case of the inorganic P fractional on, the organic
extractions and analysis are highly time-consuming and therefore
the number of samples which could be analyzed  was limited.
Sediments from Howard Bay were selected for this phase of the
study because changes in the sediment organic  P content occurred
concommitantly with changes in limnological conditions.

Carbon.  The distribution of organic C in the  benzene-ethanol,
humic acid and fulvic acid fractions of Howard Bay sediments
during the monitoring period ranged from 6.9 to 11.5%, 7.8
to 10.3% and 49.8 to 66.0%, respectively (Table 11).  Thus,
the bulk of the recovered organic C was in the fulvic acid
fraction and therefore soluble in both alkali  and acid.  Changes
in the concentrations of C in this fraction with time were,
however, not related to changes in sediment organic C.

The concentrations of C in the benzene-ethanol fractions decreased
from May to June and subsequently increased from July to August
corresponding to similar reductions in sediment organic
C and accounting, respectively, for 20.3% and  38.0% of the
reduction in sediment organic C from May to June and the increase
in sediment organic C from July to August (Table 4).  Organic
materials likely contained in this fraction, i.e. lipids,
fats and waxes derived from fresh or relatively undecomposed
organic detritus, should serve as relatively available energy
sources for the sediment microfloral population under the
conditions of increased temperatures in the spring.  As phyto-
plankton growth declines in late summer, the rate of deposition
of these materials may exceed the rate of decomposition resulting
in an increase in sediment concentration.

Humic acid C (Table 11) was quite stable during the year
accounting for a relatively constant proportion of total sediment
C.  If, analagous to soil humic acid, sediment humates consist
of polymers of aromatic compounds, microbial stability of this
fraction may be expected.

The major portion of fulvic acid C (Table 11,  12) was present
in subfractions B, C, and D (Table 7).  Investigations with
a diverse series of soils including a peat (Forsyth, 1947) indicated
that in soil  systems only a small fraction of  the organic
matter in the fulvic acid fraction was eluted  through charcoal
with water (Fraction A, Table 7).  Most of the organic matter
consisted of more complex substances which were present in
                               50

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        Table 11.   Seasonal  distribution of organic carbon  in  the  benzene-ethanol,  humic  acid and fulvic
                   acid fractions  of lake sediments.
en
Sample -,
designation

H-l
H-2
H-3
H-4
Sediment
organic C
content
mg/g
79.30
63.80
65.60
77.30


Benzene-ethanol
fraction
Absolute
mg/g
7.88
4.73
4.50
8.94
Fraction
of total
%
9.9
7.4
6.9
11.5
Organic C
2 in
Humic acid
fraction
Absolute
mg/g
7.24
4.99
6.77
6.80
Fraction
of total
%
9.0
7.8
10.3
8.8


Fulvic acid
fraction
Absolute
mg/g
33.50
31.80
43.40
43.60
Fraction
of total
%
57.4
49.8
66.0
56.4
Total organic C
recovered
Absolute
mg/g
48.62
41.52
54.57
59.34
Fraction
of total
%
76.4
65.0
83.2
76.7
r
        'Table 1

        "Based on oven-dry (60  C)  weight

-------
subsequent fractions.  By analogy to soil  systems (Forsyth,
1947), sub-fractions B, C, and D likely contained materials
of relatively high molecular weight including phenolic glycosides
(B), polysaccharides (C) and mixtures of N compounds and
pentose sugars.

Fraction D contained the largest quantities of C throughout
the season and increased from < 40% of the fulvic acid C in
May and June to over 60% in July and August (Table 12).  In
characterizing subfraction D components isolated from soil,
Forsyth (1947) employed simple dialysis followed by electrodialysis
to remove the alkali and concentrate the extract.  The bulk
of the material formed at the anode.  The  material did not
give positive protein tests but contained  the largest quantity
of N and considerable quantities of P.

Phosphorus.  As in the case of organic C,  the fulvic acid
fraction contained most of the recovered organic P (Table
13) ranging from 26.2 to 32.5% of sediment organic P.  Benzene-
ethanol soluble P ranged from 2.9 to 4.1% of organic P and varied
directly with it.  Humic acid organic P accounted for 7.8
to 12% of the sediment organic P.  Although there was a marked
decrease in humic acid P concentration from May to June
as in the case of the other extractants, humate P remained
constant from June to August.  Taken together, organic P in
the fulvate, benzene-ethanol and humate fractions accounted
for 35% of the reduction in organic P from May to June and 20%
of the increase from July to August with the unrecovered
fraction (Table 8) accounting for 65% of the decrease from
May to June and organic P in the silica and sediment residual
fractions (Table 8) accounting for much of the increase
from July to August.

In general, changes in sediment organic P  could not be attributed
to changes in organic P concentration in any single fraction.
Benzene-ethanol, fulvate and humate fractions together accounted
for less than 50% of the total organic P.   Approximately
50% of the sediment organic P consisted of organic P in
the silica fraction or as nonextractable sediment residual
(Table 8).  The organic P in the silica fraction likely consisted
of silica occluded organic matter from both the fulvic and
humic fractions.  The unextractable organic P residual in
the sediment may have been present as highly stable mineral
organic P complexes.

In contrast to sediment organic C (Table 12) and soil organic
matter (Forsyth, 1947), which were present in highest concentra-
tions  in fulvic acid subfractions B, C and D,  the largest
                                52

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        Table 12.   Seasonal  distributions of organic carbon in components of the fulvic acid fraction
                   of lake sediments.
en
CO
Sample ,
designation
H-l
H-2
H-3
H-4
Fulvic acid
organic carbon
mg/g
45.49
31.75
43.26
43.63
2 3
Distribution of organic carbon in fractions '
A

6.1
5.3
1.8
3.9
B
	 
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 proportion of the fulvic acid organic P (Table 14) was
 present  in subfraction A which contained water soluble materials
 likely of relatively low molecular weight.  Only organic
 P  in the water soluble and consequently the most labile sub-
 fractions A and C varied on an absolute basis with seasonal
 changes  in sediment organic P (Table 13).  If it is necessary
 to  predict changes in sediment organic P it would appear that
 these chemical fractions would be a useful point of departure
 for future research.

 Isomers  of inositol hexaphosphate are believed to constitute
 the bulk of the organic P in soils.  It is likely that inositol
 hexaphosphate present in soils is derived largely from terrestrial
 plants which contain considerable quantities of the substance
 as  the Ca-Mg salt, phytin.  It is also known that certain
 soil microorganisms may synthesize inositol hexaphosphate
 but little information is available regarding the quantity
 of  inositol hexaphosphate formed in this manner in soils
 or  sediments.

 Phytoplankton, the likely principal source of organic P to
 sediments of eutrophic lakes contains only small  quantities
 of  inositol hexaphosphate.  Thus if inositol  hexaphosphate
 is  present in sediments in significant quantities it must
 result from microbial synthesis  or be of terrestrial  origin.
 If  of terrestrial  origin, it may serve as a useful indicator
 of  terrestrial sources of organic P.

 At  soil or sediment pH values between pH 1.5 and approximately
 pH  4.0,  inositol  hexaphosphate may be expected to be relatively
 insoluble as Fe or Al salts.  Above the pH range of 5.5
 to  6.5 inositol  hexaphosphate is likely insoluble as  Ca or
 Mg  salts (Jackman  and Black, 1951).  At pH values between
 4.0 and approximately 6.5 finite solubility of inositol
 hexaphosphate, and subsequent release of the material to solution
 from the solid phase, may be expected.  The extent of release
 will be highly dependent upon water and sediment properties.
 If  inositol  hexaphosphate represents a significant portion
 of  the sediment organic P fraction, remedial  measures based
 on  the alteration  of sediment pH or sediment concentration
 of  Fe-Al  or Ca-Mg  might be successful  in reducing the release
 of  inositol  hexaphosphate to solution.  The factors influencing
 the mineralization of inositol  hexaphosphate in sediments
 would  also be  deserved of detailed attention.   In the present
 studies  a suitable method was developed for estimation of
 inositol  hexaphosphate in sediments.  Inositol  hexaphosphate
was not present  in detectable (< 10 yg/g of sediment) quantities
                               54

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       Table 13.   Seasonal distribution of organic phosphorus in benzene-ethanol, humic acid and fulvic
                  acid fractions of lake sediments.
en
en




Benzene-ethanol
fraction

Sample o
designation

H-l
H-2
H-3
H-4
Sediment
organic P
content
ug/g
317
271
290
382


Absolute
pg/g
13
10
9
n
Fraction of
total
organic P
%
4.1
3.7
3.1
2.9
Organic P
f-ulvi
recovered in
c acid
fraction


Absolute
yg/g
89
88
84
100
Fraction of
total
organic P
%
28.1
32.5
29.0
26.2

Humic acid
f racti on


Absolute
ug/g
40
28
32
30
Fraction of
total
organic P
%
12.6
10.3
11.0
7.9

      Based on oven-dry (60  C) weight


     'Table 1

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en
cr>
            Table 14.  Seasonal distribution of organic phosphorus in components of the fulvic
                       acid fraction of lake sediments.
Sample
designation

H-l
H-2
H-3
H-4

1 Table 1
2
Based on oven-dry
3Table 7
Fulvic acid
organic P
content

yg/g
89
88
84
100


(60 C) weight

2 3
Distribution of organic C in fractions '
A B C D E
o/
49.4 18.0 13.5 7.9 11.2
36.4 11.4 3.4 9.1 39.7
31.0 20.2 7.1 13.1 28.6
36.0 9.0 17.0 12.0 26.0





-------
in Upper Klamath Lake sediments.   Future investigations  might
be designed to determine its concentration in a broad range
of sediments and its importance as an indicator of terrestrial
contamination or as a supplier of P to solution.
                               57

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                         SECTION V

            POTENTIAL FOR RELEASE OF PHOSPHORUS
         FROM SEDIMENTS -- LABORATORY INVESTIGATIONS

The P status of Upper Klamath Lake sediments changed markedly
at the Howard Bay location as limnological conditions changed
with the seasons.  Less pronounced changes occurred at the
Buck Island and Pelican Marina sampling locations than at
Howard Bay.  These changes in P status provided impetus for
extension of the field studies to the laboratory.  However,
it was also recognized that due to the high concentration
of P in the sediment relative to the water, gross changes
in sediment P status need not occur in order for sediments
to significantly influence water composition.  Therefore
laboratory studies were undertaken to more precisely define
the rate and extent of P release from sediments.  Inherent in
this objective was the development of suitable methodology
for the incubation and study of sediments in the laboratory.
The laboratory investigations may be divided into two sections
dealing with (i) development of methods for measurement of P
release and algal growth potential and (H) measurement
of factors influencing the release and algal growth potential
of sediment P.

A.  LABORATORY METHODS FOR MEASUREMENT OF PHOSPHORUS RELEASE
    AND ALGAL GROWTH POTENTIAL

Initially emphasis was placed on development of laboratory
techniques for measurement of P release which allowed the
manipulation of the parameters of sediment and water composition,
pH, aeration and temperature.  A dialysis system appeared
to offer the greatest versatility in that the sediment and
water could be partitioned, subjected to individual treatment
and the effect on P equilibria measured.

Several prototypic methods were developed and evaluated
as to efficacy of measurement of the potential P release
from sediments and of the influence of environmental parameters
on P release.   These included (i) a dialysis method involving
the equilibration of sediment in a cellulose acetate membrane
with distilled water, (i-i) a dialysis method in which sediment
and water in glass half-cells were partitioned with a 0.45 y
filter and (Hi] a sediment-resin system based on the soils
method of Cooke (1966) in which P was taken up directly by anion
exchange resin placed in the moist sediment.
                             59

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                  Membrane Dialysis System

As an initial test of the dialysis  concept for the measurement
of P sediment-water equilibria, a cellulose acetate membrane
system was designed.

Materials and Methods

Moist Howard Bay sediment samples were sealed in cellulose
acetate dialysis tubing (4.1  cm in  dia) and immersed in
distilled water (600 ml).  Aliquots (15 ml) were removed
from the water outside the membrane at periodic intervals
and analyzed for orthophosphate.   After sampling, a volume
of water equal to the aliquot removed was  re-added to maintain
a constant volume.  The treatments  were maintained in the
dark at 23 + 2 C.

Results and Discussion

The release of P from Howard  Bay sediments amounted to 15.6 yg
P/g of moist sediment.  Release represented a small fraction
of total inorganic P.  However P release amounted to approximately
three times the quantity of readily extractable (NH^Cl) P for this
sediment.  Thus, P dissolution occurred.  A second, more
extensive experiment was therefore  undertaken utilizing this
system.  The results of the second  experiment are reported
in Section V, B.  Those studies indicated  that the cellulose
acetate was visually degraded after approximately 15-20 days
and long-term incubation or precise control of pore size
were not possible with this system.

                  Filter Dialysis System

To overcome problems of membrane decomposition and provide
more precise control of membrane pore size, an alternative
to the cellulose membrane technique was devised.  The new apparatus
(Figure 11), consisted of two pyrex glass  half-cells separated
by a membrane filter supported on two fritted glass filters
(coarse pore size) incorporated into each  half-cell.  The half-
cells, one containing sediments and^the other containing
water, were bound together under spring tension and gently
shaken.  The water cells were periodically analyzed for
total  and inorganic P.  Aliquots removed for analysis were
replaced with a fresh increment of distilled water.  In later
experiments the entire water  volume was removed from the
half-cell  and replaced with distilled water.
                               60

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                                  HALF-CELL  (WATER]
                                   FRITTED DISC
                                  MEMBRANE FILTER
                                   FRITTED DISC
                                   HALF-CELL (SEDIMENT;
FIGURE  11.  SEDIMENT  DIALYSIS APPARATUS
                      61

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 The  new dialysis system was evaluated as to the rate of diffusion
 of P across the fritted glass filter and the effect on diffusion
 rate of restriction of particle size to 0.45 y.   In later
 experiments described in Section V, B, the extent of resorption
 of P from the water cells by the sediments and the influence
 of composition (distilled water vs. dilute salt media) on
 diffusion rate were evaluated.

 Materials and Methods
 To  determine the rate of P diffusion across the 0.45 y membrane
 used  in the dialysis system, P (20.38 yg/ml) was added to one
 half-cell, normally employed for sediments but containing water.
 The rate of diffusion into the second half-cell, containing
 distilled water only, was monitored.  The results were expressed
 as  a  percentage of complete diffusion (an equilibrium concentration
 of  50% of the P added to the "sediment" half-cell).  To determine
 the extent of diffusion across the fritted disk alone, the
 above experiment was repeated in the absence of the filter.

 Results and Discussion

 Diffusion of P through the 0.45 y filter separating the
 dialysis cells was relatively slow:  only 24.1% after 20 days
 (Table 15).  Thus, diffusion must be considered a factor limiting
 the rate of sediment P release determined using the dialysis
 systems.  Removal  of the 0.45 y filter leaving only the
 fritted discs separating the cells resulted in complete equili-
 bration in 48 hours.   Thus,  diffusion rate would not be
 limiting in experiments in which P release could be measured
 in the absence of the filter, but sterility of the water half-
 cell could not be  maintained.

 Use of the filter  dialysis system has several  advantages
 compared to the cellulose acetate dialysis tubing employed
 previously.   For examples, (i)  the filter is not biodegradable
 under the conditions  employed and (ii] the filter pore size
 is precisely defined  and may be changed to allow accurate
measurements  of the type and extent of particulate diffusion
 from the sediment,  (Hi] sediments and waters  may be initially
treated separately  as  required  allowing evaluation of a broad
range  of parameters and (iv]  the water half-cell  may be maintained
in a sterile  condition  while dialyzing against a non-sterile
sediment provided  the  filter pore size is < 0.45 y.
                            62

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Table 15.  Equilibration of phosphorus between water cells separated by a
           0.45 y filter in the dialysis system.
Incubation
time
days
1
2
3
4
5
6
7
8
10
14
20
P diffused through membrane
% of complete diffusion^
2.0
4.9
7.3
9.7
11.4
14.0
17.0
18.4
21.7
22.3
24.1

 Initial concentration in P half-cell 20.38 yg/ml

 Complete diffusion was taken as an equilibrium concentration of 50% of the P
 added
                                     63

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       Direct Equilibration With an Anlon Exchange Resin

 To devise a simple technique which may be useful on a routine
 basis  for estimation of the potential for P release from
 sediments, a method previously reported for the evaluation
 of the kinetics of P release from soils (Cooke, 1966) was modified
 and applied to sediment systems.  The modified method simply
 involved direct equilibration of moist sediments with an anion
 exchange resin, separation of the resin from the sediment
 sample at regular time intervals and displacement and measurement
 of the P on the resin.  The technique offers the advantages
 of (i] simplicity, (ii) measurement of the kinetics of P release
 and (Hi] measurement of potential release since a P sink
 is provided shifting sediment-solution P equilibrium in
 the direction of the solution phase.  However, in order
 to use the method, the optimum conditions for measurement
 had to be determined.  Preliminary tests were therefore conducted
 to determine (i) the best method for incubating and subsequently
 separating the resin from the sediment, (ii) the efficiency
 of P uptake by the resin, and (Hi,} the optimum moisture
 to sediment and resin to sediment ratios.  After initial
 testing, an evaluation was made of the release of P in the
 new system as related to release in the dialysis system, in
 the presence and absence of anion exchange resin and to algal
 growth potential.  The results are recorded in Section V, B.

 Materials and Methods
To assure the suitability of the anion exchange resin selected
for release studies, an experiment was designed to test
the efficiency of orthophosphate P uptake and release.  Resin
solutions at concentrations of 1-5 g/100 ml  were spiked with
123 to 12280 yg P and equilibrated for 17 hr.  At the end of
the equilibration period, the concentration  of P remaining
in solution was determined and the resin removed.  The P on
the resin was exchanged by equilibration with 100 ml of HC1 (0.1
to 1.0 N) and an aliquot analyzed for orthophosphate P as previously
described.

To facilitate separation of the resin from the sediments,
small  capsules (1.2 cm x 2.0 cm) containing  resin (1.0 g) were
mixed  with sediments (27.5 g wet weight, 2.5 g dry weight)
and 25 ml of distilled H20 in polypropylene  bottles.  The
system was then equilibrated (minimum 2 hr)  with 50 ml of HC1
(0.1  N),  a satisfactory method as determined above, and
the equilibration solution analyzed for P.
                                64

-------
As a list of the influence of resin sediment ratio in the
batch equilibration system, various concentrations of resins
were mixed with Howard Bay sediment (27.5 g wet weight, 2.5
g dry weight) and 25 ml of distilled water in polypropylene
bottles and equilibrated with gentle shaking.  Periodically
the sediment was decanted and the resin washed with distilled
water.  The washed resin was subsequently equilibrated (minimum
2 hr) with 50 ml of HCL (0.1 N) and the equilibration solution
analyzed for P.

Results and Discussion

The suitability of the anion exchange resin for uptake of P from
solution is exemplified by the data presented in Table 16.
Complete removal of P from solution was obtained for water samples
of concentrations many-fold higher than normally measured
in dialysis systems (< 612 yg P).  The P may be quantitatively
displaced with acid and subsequently analyzed (Table 17).

Optimum release after 15 days incubation was attained at resin:
sediment ratios 1:2 and 1:1 (Treatments 3 and 4, respectively,
Tables 18, 19).  Reduced release at a 2:1 ratio may have
been due in part to a resin effect on solution pH (Table
19).  Extension of incubation time to 30 days (Treatments
3, 4, and 5) resulted in additional release but at a reduced
rate.  The rate of release could be described by regression
of the P released against the log of time (Table 20).

Measurements of total  P in solution indicated that a small fraction
(likely organic P) of the P released was not retained by the
resin.  The maximum quantities of total P in solution during
the first days of incubation were approximately equivalent
for all treatments.  However, during the latter phases of
incubation total solution P decreased in systems not containing
resin.  This decrease was accompanied by a reduction in
solution pH which was  not observed in the resin systems.  The
resin evidently served as a buffer maintaining a relatively
constant pH.

            Estimation of Algal Growth Potential

A preliminary test of the EPA Provisional method (1969) for
estimation of algal growth potential  was undertaken to establish
(i) the most suitable  method of algal  growth measurement
and (-Li) a standard growth curve.   A revised version of
the Algal  Assay Procedure was issued in August, 1971.  This
method was subsequently evaluated in a manner similar to that
described below.  A standard curve for the new media is included.
                               65

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Table 16.  Removal  of orthophosphate pKosphorus from solution
           by an anion exchange resin.
Fate of P after equilibration
Total P
added
pg
123
306
612
612
612
612
1228
3057
6114
12280
12280
12280
Quantity
of resin
g
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
2.5
5.0
P removal
from solution
ug
123
303
597
591
592
592
1133
2558
4496
8113
9482
10430
Fraction
removed
%
100.0
99.0
97.5
96.6
96.7
96.7
92.3
83.7
73.5
66.1
77.2
84.9
                             66

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Table 17.  Acid displacement of orthophosphate pKosphorus from an
           anion exchange resin.
Acid
concentration
me . /ml
0.1
0.2
0.5
1.0
Total P
on resin
yg
597
591
592
592
Quantity
of resin
g
1.0
1.0
1.0
1.0
P displaced
to solution
yg
582
601
591
590
Fraction
displaced
%
98.3
101.7
99.8
99.7
                               67

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  Table 18.   Experimental  design  employed  in investigations of
             direct uptake of phosphorus from rnoist sediments
             by different  quantities  of an anion  exchange resin
             in a direct equilibration  system.

Treatment
number


1
2
3
4
5
6
Quantity of
Sediment1 '2
9, ,
r-..,- .
2.5
2.5
2.5
2.5
2.5
2.5

Resin


0.25
0.50
1,25
2.50
5-00
0.00

Resin:
sediment


1:10
1:5
1:2
1:1
2:1

]27.5 g wet weight  (9.05%  solids)
2
 After addition  of 25 mis distilled  HgO,  sediment  solution  initially
 a total  of 45 yg  total  P,  32 yg  inorganic  P.
                                68

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Table 19,  Uptake of phosphorus from,moist sediments  by an anion
   i        exchange resin as influenced by resin concentration in
           a direct equilibration system.
Incubation
time
Ha\/c
Udyb
i
2
4
7
11
15

1
2
4
7
11
15

1
2
4
7
11
15
18

Total


23
22
24
39
44
48

19
20
26
30
39
36

18
16
18
26
35
36
33
P in solution
P " Inorganic P

	 _ 	 ,. yg/g
Treatment 1
10
6
2
10
12
16
Treatment 2
10
6
5
12
12
12
Treatment 3
10
0
2
8
13
14
12
Total P on
resin


12
50
101
132
136
196

16
72
112
125
129
184

15
57
115
152
194
252
234

PH


6.6
6.8
6.7
6.8
6.6
6.8

6.6
6.6
6.6
6.6
6.6
6.7

6.4
6.4
6.4
6.4
6.4
6.6
6.4
                               69

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Table 19 (Continued).
Incubation
time
1 j — - ' •"
days
22
26
30

1
2
4
7
n
15
18
22
26
30

1
2
4
7

Total

32
36
34

18
15
17
24
30
24
34
29
34
26

12
11
15
14
P in solution
P Inorganic P
• • • 	 - — — . '' • .••;•• • ,..,- —
	 ug/g sediment 	
Treatment 3 (continued)
8
10
10
Treatment 4
8
2
3
8
16
10
16
14
17
12
Treatment 5
4
2
4
4
Total P on
resin

245
256
308

38
103
145
185
200
266
277
237
298
302

68
no
126
122

PH

6.6
6.8
6.8

6.2
6.2
6.0
6.0
6.0
6.2
6.1
6.2
6.6
6.6

6.0
5.5
5.7
5.6
                                 70

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Table 19 (Continued).
Incubation
time
A a \/c
Uayb
11
15
18
22
26
30

1
2
4
7
11
15
18
22
26
30


Total


26
21
18
12
18
21

23
26
34
36
33
28
40
40
42
44

P in solution
P Inorganic
___ _ __ _ iirt/n
yg/g
Treatment
16
8
7
6
12
12
Treatment
12
4
10
8
27
7
21
20
21
11

Total P on
P resin

5 (continued)
136
166
192
195
252
276
6











pH


5.6
5.8
5.8
6.0
6.2
6.4

7.0
6.9
6.9
6.8
6.8
6.8
6.8
6.8
7.0
7.0

 T
  Table 18
                               71

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          Table 20.   Summary  of regressions  of phosphorus uptake by resin systems against log of time.
TV>
Treatment Degrees of
No. freedom
1 4
2 4
3 8
4 8
5 8
Intercept
9.88
23.17
2.66
41.63
57.36
Regression
coefficient
143.16
122.80
189.56
172.88
114.68
Std. error
of regression
coefficient
14.74
15.38
11.00
12.61
20.22
Correlation
coefficient
.9792
.9703
,9872
.9792
.8952
      1
       Table 18
      "p < 0.001
       p < 0.01

-------
Methods and Materials

The algae Selenastrum capn'cornutum was employed as described
by the Joint Industry/Government Task Force on Eutrophication
(1969) to assay the availability of P released to solution
from sediments.

To evaluate optical density (600 my) as a measure of algal
growth response, aliquots (0.5 ml) of the culture solutions
were taken during growth of the test organism and optical
density measurements correlated with tentative Coulter counts
and dry weight measurements at 7 incubation time intervals.
Each measurement was replicated 8 to 10 times.

Standard growth curves were established over the concentration
range 0.01 to 0.6 mgP/liter by amendment of the standard
culture media with solutions of known P (KH2P04) concentration.
A standard curve for the revised method (National Eutrophication
Research Program, 1971) utilizing maximum cell concentrations
as an index of P concentrations in the media was developed
as described above.

Results and Discussion

Optical densities of the culture media during growth of
the test organism were linearly correlated with tentative
Coulter counts between 60 x 108 and 10 x 109 cells/ml (Figure 12),
and dry weight Figure 13).   The convenient optical density
measurement was utilized as an index of algal growth in subsequent
studies with this media.

The growth responses of the test organism to P over the
concentration ranges 0.00 to 0.08 yg/ml and 0.10 to 0.60 yg/ml
are illustrated in Figure 14 and 15, respectively.  The
assay methods appeared sensitive to 0.04 yg/ml with the
best response occurring above 0.10 yg/ml.

The revised version of the provisional bioassay method (Algal
Assay Procedure Bottle Test, 1971) utilized a nutrient media
of lower concentration than the original version and the
direct counting method proved to be the most practical means
of evaluating growth.  The new media was utilized in the
latter experiments described in Section V, B.  With the
new media, the maximum specific growth rate (ymax) did not
serve as an index of P concentration in solution.  In fact
the data indicated that ymax decreased as P concentrations
increased (0.05 to 0.25 yg/ml).  This apparently reflected
                              73

-------
    10   -
oo
o
O
o
o
               0.05     0.10       0.15       0.20

                    OPTICAL DENSITY  (600  my)
                                           0.25
FIGURE  12.
ALGAL CELL CONCENTRATION (TENTATIVE COULTER

COUNTS)  AS A FUNCTION OF SOLUTION OPTICAL DENSITY
                             74

-------
          0.5
                                                  O  _
      o
      o
      WD
          0.4
en
      1-4   01
      ,r>   V • O
      t/o
          0,2
      D.

      O
          0. 1
                         60
                                      I
             I
I
120         180         240

    DRY WEIGHT, mg/mer
          300
360
       FIGURE 13. SOLUTION  OPTICAL  DENSITY AS A FUNCTION OF ALGAL DRY  WEIGHT,

-------
    0.10 -
    0.08 -
o
o
IO
    0.06  -
Ou
o
    0.04  -
    0.02  -
                                               MEDIA P
                                            CONCENTRATION
                                                yg/ml
                             120        180


                       INCUBATION TIME, HOURS
240
  FIGURE 14.  STANDARD BIOASSAY GROWTH  CURVES  (0.00 TO  0.08 yg
              PHOSPHORUS/ml  OF CULTURE  MEDIA).
                              76

-------
         0.4  r
      o
      o
      to
      Q_

      O
                                      0.1  yg/ml
                     60       120
180      240
                      INCUBATION TIME, HOURS
FIGURE 15. STANDARD.BIQASSAY GROWTH CURVES (0.1,TO 0.6 U9
           PHOSPHORU$/ml OF CULTURE MEDIA).
                           77

-------
the extent of biomass  measured.   The  relative growth rate
increased with decreased biomass.   Maximum cell  counts were
proportional  to P concentrations  (Figure 16), and these
data will be  utilized  in interpretations,  although for
comparative purposes,  both  ymax and maximum cell  concentrations
will be reported.

B.  FACTORS INFLUENCING THE RELEASE AND ALGAL GROWTH
    POTENTIAL OF SEDIMENT PHOSPHORUS

Studies of the Upper Klamath Lake sediments in the field
(Section IV)  indicated that changes in the inorganic P fraction
were primarily responsible  for changes in  sediment P status.
These changes were negatively correlated as to changes in
surface water organic  C and, at the Howard Bay location,
to surface water temperature. Thus,  in the laboratory studies
emphasis was  placed on the  effects of a P  sink in the form
of an anion exchange resin  and algal  growth and on temperature
as these may  have effected  changes in sediment inorganic
P.  In conjunction with the development of methods for measurement
of P release  and to provide a basis for a  broader application
of the results of these studies,  the  influence of sediment
and water chemical and physical characteristics and water
composition on P release were also evaluated.  Where feasible
the results of all release  studies were related to algal
growth potential.

        Sediment Physical Parameters  and Temperature

This experiment was designed as an initial test, using a cellulose
acetate membrane dialysis system, to  bracket the influence
of sediment:interstital water ratio,  sediment:external water
ratio and temperature  on P  release to solution from Upper
Klamath Lake  sediments.

Materials and Methods

A preliminary study (Table  21) was undertaken to test the
membrane dialysis system and to  evaluate the influence of the
above mentioned parameters  on P  release to solution utilizing
Howard Bay sediments.   Experimental details are given in Table
22.

Moist sediment samples were sealed in cellulose acetate dialysis
tubing, immersed in distilled water,  incubated at 10 C or 23
C, and sampled as previously described in Section V, A.
                              78

-------
    10
o
t—(
I—
<
LLJ
O
    10'
                    LOG y=2.063x+5.893

                    (r=0.797, ri=18, p<0.001)
                  I
I
I
                0.05        0.10      0.15      0.20

                        MEDIA P  CONCENTRATION yg/ml
                             0.25
  FIGURE 16. STANDARD CURVE  OF  GROWTH  RESPONSE  (LOG  SCALE)
             WITH  INCREASING  LEVELS  OF  PHOSPHORUS  UTILIZING
             THE REVISED ALGAL  BIOASSAY  PROCEDURE.
                               79

-------
Table 21.  Factorial  design employed in investigations of phosphorus
           release to solution (membrane dialysis system).
        Independent Variable
 Number
Description
1
2
3
Treatment
Number
sediment :external water ratio
sediment interstitial water ratio
Temperature
1:10 1
1:5 1
10 C
Treatment design for independent variable
:20!
:10
25 C
numbers
                  1                     2                     3
   1*
   2
   3*
   4
   5*
   6
   7*
   8
  duplicated
                                80

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00
       Table 22.  Quantities of sediment and water employed to obtain experimental parameters  in
                  phosphorus release studies (membrane dialysis system).
Treatment
Number

la
Ib
2
3a
3b
4
5a
5b
6
7a
7b
8
Sediment
g
18.4
19.5
19.1
19.9
20.8
18.7
17.9
20.8
19.3
18.0
20.1
19.0
3
Interstitial
Water
ml
101
102
104
208
208
102
179
208
193
95
107
190
Sediment:
Interstitial
Water Ratio

5.5
5.2
5.5
10.4
10.0
5.5
10.0
10.0
10.0
5.3
5.3
10.0
4
External
Water
ml
184
195
382
219
208
186
358
416
193
326
402
380
Sediment:
External
Water Ratio

10.0
10.0
20.0
11.0
10.0
10.0
20.0
20.0
10.0
18.1
20.0
20.0
       1
        Table 21

       'Oven dry  (60 C)weight

        Lake Water
       i
        Distilled water

-------
The total  and inorganic  P  contents  of water external  to the
sediment were measured at  periodic  intervals.

Results and Discussion

The results of the P release  studies  are  reported for 21-day
incubation periods.   Limitation  of  incubation  time to 21  days
for several treatments was  necessitated by  the microbial
decomposition of the cellulose acetate membrane.   To  overcome
problems of membrane pore  size,  an  alternative to the cellulose
acetate membrane in  dialysis  studies  was  designed. The
new apparatus will be discussed  in  subsequent  sections.

In the P release studies  using the  cellulose membrane, the
pH of the systems remained  relatively constant throughout
the incubation periods ranging from pH 6.0  to  6.9 for all
treatments except for a  temporary reduction of approximately
1  pH unit from 2 to  4 days  of incubation  in Treatments 4  and
5.  During this period P was  rapidly  released  from the sediments.
The P release continued  at  an accelerated rate to 10  days
of incubation although pH  returned  to initial  levels.

The concentrations of P  in  the external water  during  the
entire period of incubation are  shown in  Figures  17,  18,
19, and 20.  The influences of the  incubation  parameters
of temperature, sediment:interstitial  water ratio and
sediment:external water  ratio are presented in Tables 23, 24
and 25 respectively, which  give  the summation  of  treatment
effects for total P  in the  external water-   The results may
be summarized as follows:   (i) both inorganic  and organic
P were present in the non-sterile external  solution within
24 hours of incubation,  (H)  P release at 10 C was less
than 30% of that released  at  23  C,  (Hi)  maximum  P release
occurred in systems  in which  the external water:interstitial
water ratio was 2:1  as compared  to  1:1 or 4:1, (iv) under
optimum conditions,  P release amounted to several times
the quantity of readily  exchangeable  (NH^Cl) P.

The limiting pore diameter  of the permeable membrane  was
25-40 A precluding the diffusion of microbial  cells and suggesting
that organic P present in  the external water during the
initial 1-2 days of  incubation diffused from the  sediment
as it is unlikely that significant  microfloral populations
developed in the water during this  period.   After approximately
15 days, the membrane was  observed  to be  decomposing, therefore,
the extent of degradation  of  the membrane during  incubation
is unknown and limiting  pore  diameter after the initial phases
                              82

-------
       8

       8
    O)

    O)
    OO
    LU
    GO
    GO
    et
    O
I    '    I    '     I
 TREATMENT 1
                O INORGANIC  P
                D TOTAL  P
                            TREATMENT 2
                O INORGANIC  P
                D TOTAL  P
                            TREATMENT 3
                0 INORGANIC  P
                n TOTAL  P
                  4        8       12       16

                      INCUBATION  TIME,  DAYS
FIGURE 17.  CONCENTRATION OF TOTAL AND INORGANIC PHOSPHORUS  II
           THE EXTERNAL WATER DURING INCUBATION OF LAKE
           SEDIMENTS (MEMBRANE DIALYSIS SYSTEM) OVER A RANGE
           OF EXPERIMENTAL PARAMETERS (TREATMENTS 1,2,3;
           TABLE 21 ).
                              83

-------
  Cr>

  ? 20
     16
  o
  p:  12
  UJ
  	I
  LU
  LU  4
                          TREATMENT 4
O INORGANIC P
D TOTAL P
  ID
  O
            8       12      1C

          INCUBATION TIME, DAYS
20
FIGURE 18.  CONCENTRATION OF TOTAL AND INORGANIC PHOSPHORUS
           IN THE EXTERNAL WATER DURING THE INCUBATION OF
           LAKE SEDIMENTS (MEMBRANE DIALYSIS SYSTEM) OVER A
           RANGE OF EXPERIMENTAL PARAMETERS (TREATMENT 4,
           TABLE 21).
                                 84

-------
      16
   en

   en
      12
       8
   O
   CtL
   LU
   CO
       0
                            TREATMENT 5
                                   O INORGANIC P
                                   D TOTAL P
                            TREATMENT 6
                   O INORGANIC P

                   a TOTAL P
                         8       12      16

                       INCUBATION TIME, DAYS
20
FIGURE 19. CONCENTRATION OF TOTAL AND INORGANIC PHOSPHORUS  II
           THE EXTERNAL WATER DURING INCUBATION OF LAKE
           SEDIMENTS (MEMBRANE DIALYSIS SYSTEM) OVER A RANGE
           OF EXPERIMENTAL PARAMETERS (TREATMENTS 5,6;
           TABLE 21 ).
                                  85

-------
 o>
  CT)
  3.
 o
 a:
Lu
O

LU
GO

-------
of incubation can not be precisely defined.

       Sediment Chemical Composition and Temperature

Investigations were designed to (i) measure  the rate and
extent of P release to solution as a function of sediment
composition and temperature, (ii]  develop a  multiple regression
model, incorporating variables selected on the basis of (•£)  above,
for the prediction of the extent of P release at given time
intervals and (Hi] estimate the algal growth potential of released
P utilizing the EPA Provisional Bioassay.

Materials and Methods
P release from moist Upper Klamath and Agency Lake sediment
samples was determined using the filter dialysis system.
The filter dialysis system was designed as an alternative
to the cellulose membrane dialysis system and has been previously
described (Section V, A).

For the release experiment, the sediments (Table 26), partitioned
from distilled water by a 0.45 y filter, were incubated
in the dark at 10 C and 23 C for a period of 45 days and total
and inorganic P released to the external water were measured
at periodic intervals.

To determine sediment and experimental parameters influencing
the rate and extent of P release to solution, a multiple
regression analysis of the experimental results was undertaken.
The following variables were considered for their relative
influence on 9, 16 and 31 day cummulative P release values:
(i) sample weight (g), (ii] weight of sediment (g), (Hi) water
volume in water cell (ml), (iv) water volume in sediment
cell (ml), (y) total P (yg/g), (vi) inorganic P (yg/g), (vii)
organic P (yg/g, %), (v-iii) temperature (C), (ix] sediment
solids (g/100 ml),  (x) ratio of water volume in sediment
cell to volume in water cell, (xi] interactions, i.e. temperature
and organic P and temperature and inorganic P.  After considering
possible combinations of these variables on the computer,
a model was selected which best described or predicted P release
at given time intervals.

The algae Selenastrum capricornutum was employed as previously
described (Section  V, A) to assay the availability of P released
to solution from sediments.

To test the bioassay method, dialysis cell solutions from
Treatments 1 through 4 (Table 26) were employed.  The solutions
                               87

-------
Table 23.  Influence of incubation temperature on phosphorus release
           from sediments Cmembrane dialysis system).


Comparable
treatment^


la
Ib
4
2
7a
7b
3a
3b
6
5a
5b
8


10 C
(A)


41.8
22.6

10.0


13.9
15.2



25.2
Summation of external
water P contents2 »3 at
23 C
(B)




80.0

43.0
20.1


44.2
117
71.0



A
B



0.40


0.32


0.33


0.27

 'Table 21

2
 Total P measurement; 21 days of incubation


 Duplicates averaged
                                88

-------
Table 24.  Influence of sediment: interstitial water ratio on
           phosphorus release from sediments (membrane dialysis
           system).



Comparable
treatment^

la
Ib
3a
3b
2
8
4
6
5a
5b
7a
7b



1:10
(A)



13.9
15.2

25.2

44.2
117
71.0


Summation of external
water P contents2>3 at
sediment: interstitial
water ratios of
1:5
(B)

41.8
22.6


10.0

80.0



43.0
20.1



A
B



0.45

2.52

0.55



2.98

]Table 21

 Total P measurement; 21 days of incubation

 Duplicates averaged
                               89

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 Table  25.   Influence of sediment:external water  ratio on phosphorus
            release from sediments  (membrane  dialysis  system).



Comparable
treatment^

la
Ib
2
3a
3b
8
4
7a
7b
5a
5b
6



1:20
(A)



10.0


25.2

43.0
20.1
117
71.0
Summation of external
water P contents2'^ at
sediment:external
water ratios of
1:10
(B)

41.8
22.6

13.9
15.2

80.0



44.2



A
B


0.31


1.75



0.39

2.14
t
 Table 21
2
 Total P measurement;  21  days of incubation

 Duplicates averaged
                               90

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Table 26.  Experimental design employed in investigations of the influence  of temperature  and
           lake sediment composition on phosphorus release to solution.


Temperature
C
10
23
10
23
10
23
10
23
Sediment
sample
designation^

H-2
H-2
A
A
H-4
H-4
P-4
P-4
Sediment composition
Wet
wei ght
g
322
244
383
340
372
336
246
315
Total
solids
%
7.7
7.7
13.7
13.7
8.1
8.1
9.2
9.2
Total
P

	 uy/
417
417
709
709
823
823
507
507
Inorganic
P

g
200
200
449
449
441
441
247
247
Organic
P

271
271
260
260
382
382
260
260
Treatment
number

la
Ib
2a
2b
3a
3b
4a
4b

 Table  1, A represents Agency Lake

-------
were removed after 45 days of incubation.  The P concentrations
of the solutions utilized are given in Table 27.  The solutions
were assayed directly by inoculation with the test organism
and measurement of its growth rate without further addition
of nutrients.

Results and Discussion

The quantities of total  P, inorganic P and organic P released
to solution during incubation of lake sediments at 10 and
23 C are illustrated in  Figure 21  and 22.  In general., the
rate of P release to solution increased with time.  Inorganic
P generally constituted  the major portion of P released to 31
days of incubation.   However, at 45 days of incubation, the
contribution of organic  P to total  P had increased amounting
to 30-80% of the total P detected.   Temperature had a marked
effect on P release  to solution, with the maximum P release
occurring at the high temperature level.  After 45 days
of incubation, organic P constituted a larger fraction of total
P at the low temperature values.  Relatively large quantitites
of P were released from  sediment H-4 (Treatments 3a and b) at
both temperature levels.  Sediment  H-4 contained higher concentra-
tions of total P and organic P than the other sediments
investigated.

Multiple correlation, based on the  data described in Tables
26 and 27, was employed  to develop  a model  for the description
of P release in these systems.  Consideration of the possible
sediment and experimental  parameters influencing release
using computer simulation techniques resulted in the selection
of several influential variables which were incorporated
into the following model:

     Y = B0 + BiXj + B?X^ + B3(Xx  • XJ + B4X5

where Y = total solution P at 16 or 31 days of incubation

     BQ5 B1? B2, B3, and B^ = partial  regression coefficients

     Xi = incubation temperature

     X^. = organic phosphorus (yg/100 g)

     X5 = total solids (g/100 ml)

     Xi-Xi,. = temperature,  organic phosphorus interaction
                              92

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Table 27.  Concentrations of total phosphorus) and inorganic phosphorus
           in the external water after incubation of sediments for
           45 days in a dialysis cell.
Treatment
Number^

la
Ib
2a
2b
3a
3b
4a
4b

Total

0.23
0.64
0.01
0.45
1.66
6.08
0.19
0.58
Concentration in solution of
P Inorganic P

0.14
0.35
0.00
0.17
1.00
3.87
0.08
0.30
                              93

-------
Q
U-l
GO
CD
^
o.
en
3.
a:
CL
     100

      50
      10
    0.5
    0.1

    0.05
    0.01
V  TREATMENT  la
A  TREATMENT  2a
•  TREATMENT  3a
*  TREATMENT  4a
          J_
I
I
                   10         20
                        INCUBATION
                         30
                     TIME, DAYS
                    40
                    50
  FIGURE  21.  CONCENTRATION OF TOTAL SOLUTION  PHOSPHORUS  IN  THE
             EXTERNAL WATER DURING INCUBATION AT  10  C  OF
             UPPER KLAMATH AND AGENCY LAKE SEDIMENTS IN  A
             DIALYSIS CELL,
                                  94

-------
UJ
CO
CD

D-
Ol
3.
UJ
    100


     50
     10
   0.5
   0.1
   0.05
   0.01
0  TREATMENT  Ib

D  TREATMENT  2b

•  TREATMENT  3b

A  TREATMENT  4b
                  10       20          30

                         INCUBATION  TIME,  DAYS
           40
50
  FIGURE 22. CONCENTRATION OF TOTAL  SOLUTION  PHOSPHORUS  IN  THE
             EXTERNAL WATER DURING  INCUBATION  AT  23  C  OF  UPPER
             KLAMATH AND AGENCY  LAKE  SEDIMENTS IN A  DIALYSIS
             CELL,
                                  95

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The ability of the model  t6 predict P release to solution
in the dialysis systems after 16 and 31  days incubation is
outlined in Table 28.   At both time intervals the multiple
regression coefficient and residual variance values indicated
excellent correlation  with good predictive capability and low
inherent variance.

Of the solutions subjected to bioassay (Table 27), growth
of the test organism occurred only in solutions from Treatments
3a and b (Figure 23) which contained 1.00  and 3.87 yg p/ml.
Treatments 3a and b exhibited optical densities of 0.237
and 0.222 respectively after 436 hr of incubation corresponding
to an inorganic P solution concentration,  determined from total
growth in the bioassay method, of approximately 0.25 yg/ml
(Figure 15).  The growth  rate of organisms in solutions from
Treatments 3a and b was slower with growth approaching the
stationary phases after approximately 240  hours of incubation
compared to solutions  containing a complete nutrient supply
(Figure 14, 15) which  generally attained the stationary phase
at 60 to 150 hours.  The  solutions were  assayed without additional
essential nutrients.  Available nutrient supplies in the
dialysate had to originate firom the sediment.  The different
response of the test organisms in solutions where growth
occurred compared to the  standard curves (Figure 14, 15) reflects
a deficiency of nutrients other than P which was not limiting.

Sediment Inorganic Phosphorus Equilibria and Long-Term Incubation

The objectives of this investigation were  to determine (i) the
rate and extent of P release from sediments in the dialysis
system over a 90 day incubation period,  (ii) changes in
sediment mineral components which occurred on incubation
(iii] the extent of sediment sorption of 32P spiked into the
water half-cell, i.e.  the equilibrium distribution of P during
release, (iv] the sediment mineral component into which the
sorbed 32P was incorporated and (v) the  algal growth potential
of released P.

Materials and Methods
Sediments taken in April, 1971, from the Howard Bay, Buck Island
and Pelican Marina locations on Upper Klamath Lake were
incubated in the dark at 25 C in a dialysis system containing
distilled water or a dilute P solution.   In the present studies,
the dialysis cells were gently shaken as opposed to the
static systems employed previously.   In  addition to distilled
water treatments, replicate water half-cells were spiked with a
                            96

-------
Table 28.  Prediction, using a multiple regression model, of the
           influence of temperature and laice sediment composition
           on the release of sediment phosphorus to solution.
Treatment
number
la
Ib
2a
2b
3a
3b
4a
4b
Multiple correl
coefficient
Total P
Observed

After
0.20
2.15
0.06
0.07
2.23
19.77
0.33
2.71
ation 0.986
release to solution
Predicted

ug/g
16 days incubation
0.58
3.04
-0.21
0.75
2.20
19.69
0.26
1.22

Residual variance 1.23
la
Ib
2a
2b
3a
3b
4a
4b
After
0.29
8.75
0.04
2.07
12.53
65.29
0.44
5.66
31 days incubation
1.35
9.52
-0.53
3.23
12.43
65.22
0.05
3.80
       Multiple correlation 0.998
         coefficient

       Residual variance    2.32
                               97

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      O
      o
CO     LiJ
      D-
      O
           0.2
          0.15
          0.10
0.05
                        1         T
                                                           O  TREATMENT  3a

                                                           •  TREATMENT  3b
                                  I	I
                                                   I	I
60       120
                                          180       240       300

                                         INCUBATION TIME,  HOURS
                                                            360       420
    FIGURE 23. ALGAL GROWTH RESPONSE  IN DIALYSATES  (TABLE  27)  OF  LAKE  SEDIMENTS,

-------
solution containing 1 ppm P as KH2P04 and 32P to determine
if resorption occurred.  The experimental design is outlined
in Table 29.  Dialysates from Treatments 1, 2, 3, and 7 were
analyzed periodically for pH, total and inorganic P.  Up to
41 days of incubation, aliquots of the solution were removed
for analysis.  After 41 days the dialysates were drained
completely and the cells refilled with water.  This procedure
was continued in all subsequent experiments.  Dialysates from
Treatments 4, 5, 6, and 8 were analyzed at the end of a 30
day incubation period for total P and 32P in solution.

Sediment interstitial waters were separated by gravity filtration
over a Whatman Number 40 filter paper.  To remove suspended
solids, the filtrate was passed through a 0.45 p filter.
Separated interstitial waters were subsequently analyzed for
total and inorganic P.

The sediments not receiving the 32P spike (Treatments 1,
2, 3, and 7; Table 29) were analyzed at the end of a 90 day
incubation period for total, inorganic and organic P and for
the distribution of P in P mineral fractions.  Sediments equilibrated
with 32P (Treatments 4,5,6 and 8; Table 29) were analyzed
in a similar manner at the end of the 30 day incubation period
except that the sediment P mineral extracts were also analyzed
for 32P.

The determination of 32P in sediment fusion extracts, acid
extracts of inorganic P, and P fractionation extracts was accom-
plished using a liquid scintillation cocktail consisting
of an aqueous mixture of approximately 460 g/£ toluene, 230 g/a
Triton X-100, 4 g/£ POPOP and 11 g/£ Ca(OH)2.

An aliquot (1 ml) of each solution to be counted was pipetted
into a scintillation vial.  A second aliquot (10 ml) of
scintillation cocktail and sufficient distilled water to bring
to volume (20 ml) were added.  All counting was related
to the 32P content of the initial  solution added to the
water dialysis cells.  Standards for each extract were prepared
by pipetting a standard 32P solution (1 ml) and an analogous
extract (1 ml) containing no 32P into a scintillation vial
and adding scintillation cocktail  and distilled H20.  Blanks
for background were similarly prepared, omitting the 32P solution.

To assay the growth potential of the nutrients released by the
sediments, dialysate solutions from Treatments 1, 2, 3 and
7 (Table 29) sampled after 41 days of incubation, were inoculated
with the algae Selenastrum capricornutum and incubated as
                                99

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       Table 29.   Experimental  design employed in  investigations  of sediment phosphorus release to solution.
o
o

Treatment
number

1 .
2
3
4
5
6
7
8

Sediment
sample ,
designation

H-13
B-T3
P-15
H-13
B-13
P-15
H-13
H-13


Wet
weight

481
492
480
469
478
457
456
438

Sediment
Total
solids
°/
10
9.1
9.1
10.2
9.1
9.1
10.2
9.1
9.1

; Compos iti or
Total
P

	 ug/g-
1033
629
662
1033
:629
662
1033
1033.

i
Inorganic
P

574
295
348
574
295
348
574
574


2
Initial water P content

vy/m i
0.0
0.0
0.0
1.0
1.0
1.0
0.0
1.0

       'Table  1


       7                                          32
       Water  cells  amended  with  P  also contain  P   spike

-------
described by National Eutrophication Research Program (1971).
This revised version of the method described in Section
V, A was utilized for bioassays in all subsequent experiments.
In addition to direct measurements on the dialysates, aliquots
of the dialysates spiked into the recommended growth media were
also evaluated to determine standard growth potential.   Algal
biomass was estimated by direct microscopic counting (Neubauer
counting chamber).

Results and Discussion

The sediment interstitial water compositions prior to dialysis
are given in Table 30.  The Howard Bay sediment which exhibited
highest total P values contained 66.0 yg total  P/g of sediment
in the interstitial water whereas interstitial  waters of Buck
Island and Pelican Marina sediments contained less than
1.0 yg P/g of sediment.

The results of long-term dialysis of Upper Klamath Lake
sediments are summarized in Tables 31-33.  Linear regressions
describing sediment release are illustrated in Figure 24.

Differences in the rate and extent of P release in the  dialysis
systems reflected differences in total sediment P (Table
29) and interstitial water P concentration.  After 30 days
of dialysis, the Howard Bay, Buck Island and Pelican Marina
sediments had released P amounting to 45.8, 8.4 and 9.9 yg P/g
of sediment, respectively.  Howard Bay sediments, containing
high concentration? of P in the interstitial waters relative
to the other sediments, released greater quantities of P to solution,
and total P released was less than that present in the  interstitial
waters.  However, the Buck Island and Pelican Marina sediments
released at least a factor of 8 or more P to solution than
was present in the interstitial waters indicating that  P was
released from the solid phase.  Inorganic P accounted for
most of the P present in the water cell.  After 90 days
of incubation, the release rate was nearly constant or  had
decreased and total P release amounted to 143.0, 39.9 and 40.7 yg
P/g for sediments of Howard Bay, Buck Island, and Pelican
Marina, respectively.  Thus, as after 30 days incubation,
maximum total release occurred in Howard Bay sediments,
however, this amounted to only approximately 2 times the
quantity originally present in the interstitial waters.
P release from the other sediments was a factor of at least
40 greater than that present in the interstitial waters,
and thereby originated primarily from the solid phase.   Over
95% of the P in the external  water was inorganic P.  Thus, soluble
                             101

-------
Table 30.  Sediment interstitial water composition  prior to dialysis,
Sediment
samples                         Interstitial  water total P content
                                ug/mlyg/g1
Howard Bay                        6                  66.0
Buck Island                       <0.1               <1.0
Pelican Marina                    <0.1               <1.0
 Based on oven  dry (60  C)  weight
                           102

-------
Table 31.  Concentrations of total phosphorus and inorganic phosphorus
           in the .external water during incubation of replicate Howard
           Bay sediments in dialysis cells (Treatments 1 and 7)J
Incubatio
time

3
6
9
13
16
20
23
27
30
41
57
66
90
pH
n Treatment
1

6.4
6.3
6.5
6.6
6.6
6.6
6.7
6.6
6.5
6.9
6.7
6.5
6.5
7

6.3
6.3
6.5
6.7
6.7
6.6
6.7
6.6
6.5
6.9
6.6
6.4
6.5
Total P
Treatment
1


1.2
5.9
10.6
15.4
20.2
28.0
35.1
42.0
45.8
64.1
94.9
112.4
143.0
7
•-yg P/g
2.4
5.9
13.2
17.5
21.5
29.4
33.2
37.8
42.9
62.7
93.5
112.5
148.8
Inorganic P
Treatment
1

seuimen t 	
1.3
3.7
7.9
14.6
19.3
27.0
33.5
39.8
44.4
61.5
92.1
109.1
138.9
7


2.1
5.8
12.5
17.5
21.1
27.6
31.7
36.7
41.1
58.4
89.2
108.0
144.5

 Table 29
                             103

-------
    Table 32.   Concentrations  of  total  phosphorus  and  inorganic  phosphorus
               in  the  external water  during  incubation  of  Buck  Island
               sediments  in  a  dialysis  cell  (Treatment  2) J
Incubation
time
j
days
3
6
9
13
16
20
23
27
30
41
57
66
90
PH

6.3
6.1
6.4
6.8
6.6
6.6
6.9
6.6
6.5
7.3
6.7
i
6.3
6.4
Total P

0.4
0.7
1.6
2.0
3.4
4.9
5.9
7.4
8.4
12.8
19.4
27.0
39.9
Inorganic P

<0.1
0.1
0.9
1.7
3.1
4.6
5.5
6.6
7.6
11.5
17.8
25.1
38.0
'Table  29
                                 104

-------
Table 33.  Concentrations  of total phosphorus and inorganic phosphorus
           in the external water during incubation of Pelican Marina
           sediments in a dialysis cell (Treatment 3).'
Incubation
time
davs
3
6
9
13
16
20
23
27
30
41
57
66
90
PH

6.3
6.1
6.4
6.6
6.5
6.6
6.7
6.6
6.4
7.0
6.5
6.3
6.3
Total P


0,4
1.5
2.0
3.4
5.6
6.8
8.9
9.9
14.6
26.8
31.4
40.7
Inorganic P
impnl"

<0.1
0.8
1.8
2.9
4.7
6.2
7.8
9.2
13.1
25.3
29.7
39.1
  'Table  29
                              105

-------
LU
GO
o
Di
   150
   100
    50
    40
o:

^   30
    20
    10
              20
                                  HOWARD BAY
                                  O CELL 1
                                    y=1.696x-4.712
                                    (r=0.998, n=14
                              p<0.00l)
             D CELL  7
               y=1.716x-5.143
               (r=0.999, n=13, p<0.001)
                 BUCK ISLAND (CELL 2)
                 y=0.451x-3.609
                 (r=0.989, n=13, p<0.001)
                 PELICAN MARINA ( CELL 3)
                 y=0.501x-3.804
                 (r=0.993, n=13, p=<0.001)
40      60

TIME,  DAYS
                                     80
100
  FIGURE  24.  SEDIMENT  PHOSPHORUS RELEASE AS A FUNCTION OF
              INCUBATION  TIME IN DIALYSIS SYSTEMS(TABLE 29)
                             106

-------
organic P compounds were not diffused through the 0.45 y filter
and the external water cell remained largely sterile although
aseptic techniques were not employed.

Approximately 6% and 12% of the total and inorganic P fractions
of Buck Island and Pelican Marina sediments were released
to solution during incubation (Table 34).  Larger percentages
were released from Howard Bay sediments amounting to approximately
14 and 25% for total and inorganic P, respectively.  However,
if the P present in the interstitial waters is subtracted
(6.1% of total P in the case of Howard Bay sediments; Tables
29, 30) it may be concluded that similar quantities of P were
released from the solid phase of sediments from all locations.
Thus, whereas more P is available for algal growth at Howard
Bay the contribution of the solid phase to release is approximately
equivalent to the other locations.

It is noteworthy that Treatments 1 and 7 (Table 29), replicates,
were quite comparable during the entire course of the study
providing substantiation for the precision of the methods
employed.

The relative changes in sediment total P and in P mineral components
during long-term incubation in the dialysis system are summarized
in Tables 35-37.  It should be recognized that procedural
errors in the mineral fractionation scheme may bracket relatively
small differences in sediment P effected by P release to solution.
However, these studies were conducted to establish if trends
in mineral P dissolution accompanied release.  The following
comparisons were undertaken for that purpose.

Sediments from all locations exhibited reductions in total
sediment P.  In the case of Howard Bay, reductions in total
sediment P closely approximated that P released to solution
(Table 31).  The agreement was not as close for Buck Island and
Pelican Marina sediments (Tables 32 and 33) but differences
were within procedural error.  Changes in inorganic P measured
in Treatments 1-8 accounted for most of the reduction in sediment
P on incubation for Howard Bay and Buck Island sediments (Table
35 and 36).  However, Pelican Marina sediments (Table 37) exhibited
an increase in inorganic P measured in this manner.  In absolute
terms, the readily exchangeable (NH^Cl) fraction was reduced
to less than detectable levels in all cases.  Most of the
reductions in Howard Bay and Buck Island sediments occurred
in the NH4F and NaOH (0.1  M) fractions.  Pelican Marina sediments
exhibited a reduction only in the NaOH (0.1 M) fraction with
other fractions exhibiting increases.  In terms of overall
                          107

-------
Table 34.  Fraction of sediment total  and inorganic phosphorus
           released to the external  water during incubation
           (90 days) of Upper Klamath Lake sediments in dialysis
           cells.
                              Concentrations  of P in external  water
-, Sediment
Treatment sample
1
7
2
3
Howard Bay
Howard Bay
Buck Island
Pelican
Marina
Total P
i rt
Inorganic P
//i .
	 yg/y 	 	
143.0 138,9
148.8
39.9
40.7
144.5
38.0
39.1
Fraction of
Total P
	 .u_0/-
13.8
14.4
6.3
6.1
sediment'
Inorganic P

24.2
25.2
12.9
11.2

 'Table 29
                            108

-------
Table 35.  Relative changes in total phosphorus and in inorganic
           phosphorus fractions of Howard Bay sediments on long-
           term incubation in the dialysis system.
Chemical ,
treatment



1 NH4C1
2 NH4F
3 NaOH (0.1M)
4 Na2S204-Na3CgH5(
5 NaOH (l.OM)
6 HC1
7 Ignition
8 Fusion
Total Treatments
1-8
Total Sediment P2


Before
incubation


6
213
142
)2 62
29
24
0
56
532

1033

Sediment inorganic P
After 90 day
incubation

	 yg/g 	
1
148
87
43
87
20
0
42
426

886



Alteration


-5
-65
-55
-19
+58
-4
0
-14
-106

-147

T
 Table 6

'Na2C03 fusion
                              109

-------
  Table 36.   Relative  changes  in  total  phosphorus  and  in inorganic
             phosphorus  fractions of  Buck  Island sediments  on long-
             term incubation  in the dialysis  system.
Chemical ,
treatment

1 NH4C1
2 NH4F
3 NaOH (0.1M)
4 Na S 0 -
Na3C6H5°2
5 NaOH (l.OM)
6 HC1
7 Ignition
8 Fusion
Total Treatments
1-8
Total Sediment P^

Before
incubation

1
71
86
34
78
24
0
47
341
606
Sediment inorganic P
After 90 day
incubation

0
53
70
32
83
22
0
32
293
553

Alteration

-1
-18
-16
-2
+5
-2
0
-15
-48
-53
'Table 6
        fusion
                              110

-------
   Table  37.   Relative  changes  in  total  phosphorus  and  in  inorganic
               phosphorus  fractions  of Pelican  Marina  sediments  on
               long-term incubation  in the  dialysis  system.
Chemical -,
treatment

1 NH4C1
2 NH4F
3 NaOH (0.1M)
4 Na2S2°4~
Ncl o^C ''r-Urt
«5 D D £
5 NaOH (l.OM)
6 HC1
7 Ignition
8 Fusion
Total Treatments
1-8
Total Sediment P2

Before
incubation

1
35
101

30
52
46
0
61
326
693
Sediment inorganic P
After 90 day
incubation

0
85
69

46
88
52
0
52
392
614

Alteration

-1
+50
-32

+16
+36
+6
0
-9
+66
-79
T
 Table 6

"Na2C03 fusion
                                111

-------
effects after 90 days incubation the NK4F (Al-P) and NaOH (0.1
M) (non-occluded Fe-P) fractions appeared to be responsible
for most of the decreases in inorganic P observed in Howard Bay
and Buck Island sediments.  Pelican Marina sediments exhibited
decreases in the NaOH (0.1 M) fraction as well but this decrease
was more than compensated for by increases in the other fractions.
It appeared that organic P (in this case taken as the difference
between total P and P in fractions 1-8) was mineralized
in both Howard Bay and Pelican Marina sediments.  In the case
of Howard Bay sediments, a portion of this P was released, whereas
in the Pelican Marina sediments the P was stabilized in
mineral forms.  Most of the reductions in Buck Island sediments
was due to reductions in mineral P.

Comparing the results of these analyses with changes observed
in samples taken from the field (Section IV, B), it is noteworthy
that in both cases the P in the NH^F, NaOH (0.1 M_) and Na2S204-
Na3C6H502 extracts representing forms of Al-P and Fe-P (Treatments
2, 3 and 4; Table 6) appeared to account for most of the variation
in inorganic P.  Reductions in NaOH (0.1 M) extractable P,
representing non-occluded Fe-P forms, were most consistently
responsible for decreases in sediment inorganic P.  Increases
in inorganic P when observed were generally associated with
increases in the NH^F extract representing non-occluded
Al-forms.

The extent of resorption of P during dialysis may be estimated
from 32P equilibration studies.  At the end of 30 days,
resorption by sediments of 32P spiked into the water half-
cell  ranged from 8.5 to 26.3% (Table 38).  Treatments 4 and
8, replicates, were not comparable.  Experiments have shown
(Section V, A) that the rate of P interchange between the
cells is a function of diffusion rate which is reduced by the
presence of the 0.45 y filter.  Both release and diffusion
were  restricted in Treatment 4 compared to Treatment 8 likely
by a  bubble of air adjacent to the filter.  However, the
data  indicate that, as expected, P release was the dominant
reaction in these systems.

The major quantity of P sorbed was associated with the NHuCl (readily
exchangeable) and NH4F (Al-P) fractions (Table 39).  The ^2P
not recovered may be present in part as organic forms.  These
experiments tend to substantiate results from both laboratory
incubation  and field studies which indicate that increases
in inorganic P may be generally accounted for by increases
in the NH4F fraction.
                            112

-------
Table 38.  Equilibration of solution 32P with  lake  sediments  in  the dialysis system.
Treatment
number
4
5
6
8
1 Table 29
<-T-.U1.~ 1
2
Sediment sample
designation
H-13
B-13
P-15
H-13


Total P
Initial
A

458
472
511
487


in solution
Final
B

-yg 	
1178
682
704
1899


Released
(A-B)

720
210
193
1412


P in solution
Initial Final
C D
rr\m( YTO7
8.38 7.67
8.64 6.37
9.35 7.48
8.91 6.89


Sorbed
(C-D)
\_ 	
i 	
0.71
2.27
1.87
2.02


°°

8.5
26.3
20.0
22.7



-------
                              32
   Table  39.   Distribution of   P  in sediment  extracts  after

              equilibration  (30 days) with water  containing

              32p  in  a  dialysis system.
                                                            2
                  .                        Dialysis Treatment

Chemical Treatment                      4         56
                                                     0/3
                                                    "10
NH4C1 (0.5M)                             10.2      1.4     1.0      9.6



NH.F (0.5N[, pH 8.2)                      58.2     65.5    67.9     37.7



NaOH (0.1M)                              0.0      0.0     0.0      0.0



Na2$204 (0.1M)                           0.0      8.8     5.9      5.5




Na3C6H5°2 f0'3^' pH  7'3)



NaOH (l.Oh)                              0.0      0.0     0.0      3.0



HCL (1-OM)                               8.8      5.9    17.0      1.2







TOTAL                                   77.2     81.7    91.8     57.0






]Table 6                                                            ~~



2Table 29


3                    32
 Percentage of total    P  initially sorbed by sediment
                                114

-------
Dialysates from Treatments 1, 2, 3 and 7 (Table 29) sampled
after 41 days of incubation and containing 0.7 to 3.4 yg inorganic
P/ml supported algal growth (Table 40).  As a preliminary
test, these solutions were assayed without additional essential
nutrients.  Nutrients available for growth therefore had
to originate from the sediment.  Growth in the dialysates
was preceded by a longer lag phase than in the standard media
likely reflecting a deficiency of nutrients other than P which
was not limiting.  It is noteworthy that considerable algal
growth occurred on the unamended sediment dialysates substantiating
the importance of sediments as sources of nutrients other than
P.  A distilled water blank containing only the algal inoculum
(washed four times with distilled water) initially supported
some algal growth indicating that small quantities of nutrients
were retained on the cell surface.

The algal growth responses of dialysates (Table 41) calculated
from the maximum cell concentration using the standard curve
previously established (Figure 16) agreed reasonably well
with determined inorganic P levels.  The algal growth potential
of P in the dialysates therefore appeared equivalent to orthophosphate-
P.  Growth in the dialysates was likely limited by the absence
of nutrients present in the standard growth media.

             The Presence of a Phosphorus Sink

To provide a sink for P released from sediments analogous
to the effects of biological growth, the water half-cells in
the dialysis units were amended with an anion exchange resin.
The effect of the resin on the rate of P release was evaluated
by monitoring both resin and solution P concentrations during
incubation.  These experiments were continued for 30 days and
terminated.  The sediments from control and resin-amended
systems were fractionated into P mineral fractions after
incubation to determine if incubation effected detectable
changes in sediment P mineral status.  Algal growth potentials
of the solutions from the controls, not containing resin,
were subsequently determined and the results compared with the
resin systems.

Materials and Methods
Sediments taken in April, 1971, from Howard Bay, Buck Island,
and Pelican Marina locations on Upper Klamath Lake were incubated
in the dark at 25 C in a dialysis system containing distilled
H20 as previously described (Section V, A).  The experimental
design is outlined in Table 42.  Periodically, the water cells
                          115

-------
  Table 40.  Algal  growth in sediment dialysates.
Treatment

1
2
3
7
Standard Media
Blank
P 3
Concentration
yg/ml
3.36
0.67
0.86
2.94
0.19
0.0
Maximum specific
growth rate^
vi max
2.14
1.53
1.75
1.72
1.26
1.17
Maximum cell
concentration
cells/ml
2.8 x 106
3.5 x 106
2.4 x 106
3.0 x 106
1.9 x 106
1.0 x 104
Vable 29
2        In (X2/X])
 ymax ~  ~~
                    days
                        "
 where x,, = biomass  concentration  at end  of  selected time  interval

       x-| = biomass  concentration  at beginning  of selected time  interval

    t2-t| = elapsed  time  (days)  between selected  determinations  of biomass

Sediment dialysates  only;  additional  nutrients  were  not  added
                             116

-------
  Table 41.  Algal growth  in standard media mended with sediment
             dialysates.
Treatment

1
2
3
7
]Table 29
2 In (x2/x1
hnax to-ti
P
Concentration
in media
yg/ml
0.07
0.05
0.03
0.06
i> .,
days
Maximum specific
growth rate^
M max
1.37
1.48
1.74
1.43

Maximum cell
concentration
cells/ml
1.2 x 106
7 x 105
9.5 x 105
9.7 x 105

where x~ = biomass concentration at end of selected time interval


      x, = biomass concentration at beginning of selected time  interval

   t-tn = elapsed time (days) between selected determinations  of biomass,
                               117

-------
       Table 42.  Experimental design employed in investigations of sediment phosphorus released to
                  solution containing anion exchange resin.
00
Treatment
number
1

2
3
4
5
6
7
8
Sediment
sample -,
designation
H-13

H-13
B-13
P-15
H-13
H-13
B-13
P-15
Sediment Composition
Wet
weight
g
485

508
500
461
470
455
477
376
Total
solids
%
9.1

9.1
9.1
10.4
9.1
9.1
9.1
10.4
Total
P

1033

1033
629
662
1033
1033
629
662
Inorganic
P Water cell

-yg/g 	
574 Anion exchange
resin
574
295
348
574 Distilled H20 only
574
295
348
1 Table 1

-------
were analyzed for P released from the sediment.  The water half-
cell containing the resin was evacuated using a vacuum siphon
and the resin collected in a column over a screen (100 mesh).
Total P in the solution was determined following HC10.4 digestion.
The cell was refilled with fresh water and resin.  The P on
the resin removed from the cell was extracted (0.1 N_ HC1) and
analyzed.  Sediment P mineral fractionations and algal growth
potential measurements were conducted as described in Sections
IV, B and V, A, respectively.

Results and Discussion

The presence of the anion exchange resin in the water half-
cell did not increase P release from Upper Klamath Lake
sediments over a 30 day incubation period (Tables 43, 44, 45;
Figure 25).  In fact, after 30 days, total P in solution
plus P on the resin was slightly less than total P released
to solution in treatments containing water alone.  Differences
in solution pH likely resulting from sorption of the hydroxyl
ion by the resin may have influenced the P release rate at
the sediment-water interface.  Higher water pH values, most
closely approximating the theoretical pH for maximum solubility
of Al and Fe phosphates (Lindsay and Moreno, 1960) occurred
in the absence of the resins.  However, sediment pH (Table 46)
remained unchanged after incubation for 30 days.

It appears that the presence of the biota in the water cell would
not result in additional sediment P release through uptake
and retention of P from solution, however it is not possible
to estimate the effect of different water pH values on P release
in these systems.

It is noteworthy that the maximum rate of P release (Figure 25)
occurred for Howard Bay sediment which contained relatively
large quantities of P in the interstitial waters (Table 30).  The
rate was most rapid during the first 7 days of incubation.
All interstitial water P was not released from Howard Bay sediments
exposed to resin.  However, P was released gradually to 20 days
in Buck Island and Pelican Marina sediments amounting to many
times the interstitial water P after 30 days.

The changes in the quantities of sediment total P and inorganic
P in sediment P mineral fractions after incubation for 30 days
in the presence and absence of an anion exchange resin are
given in Tables 47-49.  Resin and distilled water treatments
were quite similar in total inorganic P as might be expected
from the measurements of P released to solution.  However,
                             119

-------
         Table 43.   Total  phosphorus and inorganic phosphorus  (cumulative)  released  to  solution  during
                    incubation of replicate Howard Bay sediments  in  the  presence  (Treatment  1,2) and
                    absence (Treatment 5,6) of an anion exchange  resinJ
ro
o
Incubation
time
days
2
5
8
12
15
19
22
26
30
Solution pH
Treatments
1

5.9
5.6
5.8
5.7
4.8
5,6
5.1
5.3
5.2
2

~ till 1 tb'
4.4
5.6
5.6
5.4
5.6
4.5
4.2
4.2
4.2
5

6.2
6.4
6.4
6.1
6.4
6.2
6.2
6.2
6.1
6

6.2
6.5
6.4
6.2
6.4
6.3
6.3
6.3
6.2
Total P in solution
Treatments
1

0.0
0.5
0.7
1.6
2.5
3.9
4.8
5.6
6.2
2 5

0.0 1.1
0.3 15.8
0.5 31.5
1.7 38.7
2.4 45.3
3.7 52.5
4.4 57.6
5.4 63.9
6.1 69.6
6
_
0.5
17.6
29.1
37.5
42.7
49.8
55.0
61.0
67.4
Pon resin Inorganic P in solution
Treatments Treatments
1
P/g sedi
3.2
9.9
15.1
22.8
27.1
34.2
36.8
42.1
48.3
2

1.6
9.1
14.4
23.1
27.4
33.3
37.4
43.1
49.4
5

0.9
15.5
29.1
36.7
42.9
49.8
55.0
61.1
66.5
6

0.4
17.3
27.0
34.9
40.0
46.6
51.8
57.6
63.6
   1
    Table 42

-------
Table 44.  Total phosphorus and inorganic phosphorus (cumulative)
           released to solution during incubation of Buck Island
           sediments in the presence (Treatment 3) and absence
           (Treatment 7) of an anion exchange resin.'
Solution
PH
Incubation Treatments
time
days
2
5
8
12
15
19
22
26
30
T
— units--
4.4
5.8
4.5
4.2
4.5
4.1
4.1
4.0
4.1
7

6.1
6.5
6.5
6.2
6.4
6.2
3.6
6.0
6.4
Total P
in solution
Treatment
3

0.0
0.0
0.2
0.5
1.0
1.7
2.0
2.2
2.4
7

0.0
1.6
3.5
5.0
6.8
9.8
12.7
15.6
18.1
P on resin
Treatment
3

0.3
1.1
1.5
2.7
3.7
5.2
6.1
7.5
9.0
Inorgar ic P
in solution
Treatment
7

0.0
1.5
3.1
4.5
6.1
8.6
11.3
14.1
16.1
 Table 42
                              121

-------
Table 45.   Total  phosphorus and inorganic phosphorus (cumulative)
           released to solution during incubation of Pelican Marina
           sediments in the presence (Treatment 4) and absence
           (Treatment 8} of an anion exchange resin.'
Solution
PH
Incubation Treatment
time
days
2
5
8
12
15
19
22
26
30
4
	 um
4.1
5.4
4.3
4.2
4.4
4.2
4.1
4.2
4.0
8
fc
us —
6.0
6.5
6.4
6.2
6.5
6.3
6.2
6.2
6.1
Total P
in solution
Treatment
4

0.0
0.0
0.2
0.5
0.8
1.3
1.4
1.8
2.1
8

0.0
0.6
0.8
2.4
4.2
6.7
8.3
10.1
13.4
Inorganic P
P on resin in solution
Treatment Treatment
4

0.1
0.4
0.8
2.5
3.4
4.9
5.8
7.3
9.4
8

0.0
0.4
0.5
1.9
3.6
5.7
7.3
8.8
11.1

 'Table 42
                            122

-------
Table 46.  Sediment pH after equilibration (30 days) in dialysis systems,
1
Treatment
1
2
3
4
5
6
7
8

Sediment pH
6.6
6.6
6.5
6.4
6.5
6.8
6.5
6.6
]Table 1
                             123

-------
          80 -
          60 -
          40 -
          20 -
1 (RESIN
996,  n=9
2 (RESIN
996.  n=9
5 (WATER
971,  n=9
6 (WATER
973,  n=9
 y=1.577x+2
 p<0.001
 y=1.660x+0.90
 p<0.001)
ONLY)  y=2
 p<0.001)
ONLY)  y=2
 p<0.001)
                                   15      20

                                 IME, DAYS
          20

        CT)

        5 15


        LU

        £ 10
        UJ
        	I
        LU

        "*  5



           0
                   ffi CELL 3 (RESIN) y = 0.314x-0 . 72
                     (r=0,996,  n=9, p<0.001)
                   V CELL 4 (RESIN) y=0.336x1.35
                     (r=0.991,  n=9, p<0.001)
                   O CELL 7 (WATER ONLY)  y = 0.656x-2 . 0 1
                     (r=0.995,  n=9, p<0.001)
                   V CELL 8 (WATER ONLY)  y = 0.486x-2 , 33
                     (r=0.984,  n=9, p<0.001)
                                   15      20
                                TIME, DAYS
                          25
                         30
FIGURE  25.  REGRESSIONS OF  SEDIMENT PHOSPHORUS RELEASE  IN
             DIALYSIS  SYSTEMS WITH AND  WITHOUT RESIN IN  THE
             WATER  HALF-CELL (TABLE 42).
                                  124

-------
Table 47.  Comparison of the distribution of phosphorus in Howard
           Bay sediment inorganic phosphorus fractions after
           incubation (30 days) in dialysis system in the presence
           and absence of an anion exchange resin.
Chemical
Treatment
NH4C1
NH4F
NaOH (0.1M)
Na2S204- Na3C6H502
NaOH (l.OM)
HC1
Ignition
Fusion
Treatment 1-8
Total Sediment P2
Sediment Inorgani
Initi

6
213
142
62
29
24
0
56
532
1033
With
al Res i n

My/ Q 	
4
152
56
85
59
22
0
40
418
913
c P
Without
Resin

0
156
68
74
85
24
0
37
443
885
      T
       Table 6
      2Na2C03  fusion
                              125

-------
Table 48.   Comparison of the  distribution  of phosphorus  in Buck
           Island sediment inorganic  phosphorus  fraction after
           incubation (30 days)  in  dialysis  systems  in the
           presence and  absence  of  an anion  exchange resin.
2
Chemical
Treatment
NH4C1
NH4F
NaOH (0.1M)
Na2S2°4"
NaOH (l.OM)
HC1
Ignition
Fusion
Treatment 1-8
Total Sediment P2
]Table 6
/•*,
^Na0C?-- fusion
2 o
With
Initial Resin
	 yg/g—
1 1
71 42
86 42
34 52
78 42
24 21
0 0
47 28
341 228
606 564


Without
Res i n
0
42
52
40
66
8
0
29
247
567


                               126

-------
Table 49.  Comparison of the distribution of phosphorus in Pelican
           Marina sediment inorganic phosphorus fractions after
           incubation (30 days) in dialysis systems in the presence
           and absence of an anion exchange resin.
Chemical
Treatment

NH4C1
NH4F
NaOH (0."W)
Na,C,HcO,
o b b c
NaOH (l.OM)
HC1
Ignition
Fusion
Total
Total Sediment P2

]Tab1e 6
Initial

1
35
101
30
52
46
0
61
326
693


With
Resin

	 uy/g-
1
40
46
48
54
44
0
48
281
632


Without
Resin

0
44
61
38
65
45
0
45
298
636


      Na2C03 fusion
                                127

-------
   300
   200
en

en
z:
o
CJ>
z
o
   100
   0  0
   300
o
LU
1/5
   200
   100
             NH4F
           0123

            IN NaOH
0123

 1M NaOH
                                       012
   D       H
  TOTAL  P  INORGANIC P
 0123

 IGNITION
0123

 FUSION
 FIGURE 26. COMPARISON  OF  PHOSPHORUS CONTENTS  IN  INORGANIC
            PHOSPHORUS  FRACTIONS OF HOWARD BAY SEDIMENTS
            AFTER  INCUBATION  IN THE DIALYSIS SYSTEM  IN  THE
            PRESENCE  AND  ABSENCE OF AN ANION EXCHANGE  RESIN
            (COLUMNS  1,  2,  AND 3 DESIGNATE VALUES  OBTAINED
            AFTER  INCUBATION,  RESPECTIVELY, FOR  90 DAYS -
            DISTILLED WATER ONLY, 30 DAYS - RESIN, 30  DAYS  -
            DISTILLED WATER ONLY).
                             128

-------
    200
 CD
 o
 K-1
 I—
 <
 O
 o
 o
    100
    200
 O
 UJ
 GO
    100
0  1  2

  NH4F
             0123

               IN  NaOH
0123
0.1M NaOH
                                             01
                                  TOTAL  P   INORGANIC  P
                0123

                 IGNITION
               0123

                 FUSION
FIGURE 27  COMPARISON OF  PHOSPHORUS  CONTENTS  IN  INORGANIC
nbUKt LI. PHOSPHORUS P.RACJIONS  OF  BUCK  ISLAND  SEDIMENTS
           AFTER  INCUBATION  IN THE  DIALYSIS  SYSTEM  IN  THE
           PRESENCE AND ABSENCE  OF  AN  ANION  EXCHANGE  RESIN
           (COLUMNS 1, 2, AND 3  DESIGNATE  VALUES  OBTAINED
           AFTER  INCUBATION, RESPECTIVELY,  FOR  90 DAYS -
           DISTILLED WATER ONLY,  30  DAYS  -  RESIN, 30  DAYS
           DISTILLED WATER ONLY).
                               129

-------
    200
CD

CD
    100
UJ
CJ
O
CJ
    200
o
LU
CO
    100
0123

   NH4F
0123       0123

 O.lMNaOH    Na2S2°4~Na3C6H5°2
                   D       H
                 TOTAL  P   INORGANIC P
             0123

              IN NaOH
                0123

                 IGNITION
                0123

                  FUSION
FIGURE 28. COMPARISON OF PHOSPHORUS CONTENTS  IN  INORGANIC
           PHOSPHORUS FRACTIONS OF PELICAN  MARINA SEDIMENTS
           AFTER INCUBATION  IN THE DIALYSIS SYSTEM IN THE
           PRESENCE AND ABSENCE OF AN  ANION EXCHANGE RESIN
           (COLUMNS 1, 2, AND 3 DESIGNATE  VALUES OBTAINED
           AFTER INCUBATION, RESPECTIVELY,  FOR  90 DAYS -
           DISTILLED WATER ONLY,  30 DAYS  -  RESIN, 30 DAYS -
           DISTILLED WATER ONLY).
                                130

-------
regardless of treatment, incubation effected changes in
P mineral distribution.  The inorganic P extractable by NH4F
was markedly reduced in the cases of Howard Bay and Buck  Island
and P in the NaOH  (0.1 M) fractions was reduced in all sediments.
In contrast, P extractable by Na2S20it-Na3C6H502 increased
on incubation for  all sediments.   Evidently, Al and ferric
Fe were in part released and in part converted to more reduced
forms of Fe on incubation under anaerobic conditions.  These
results are illustrated in Figure 26, 27 and 28.  For comparative
purposes, values obtained after incubation of the sediments
for 90 days with distilled water (Tables 31-33) are also
illustrated.  It is apparent that in the case of these fractions,
incubation at 30 days in the dialysis system was sufficient
to reduce sediment P to near minimum levels and further incubation
to 90 days did not result in additional release of P.

The algal growth potential of sediment dialysates spiked into
standard media are given in Table 50.  As in previous measurements
the ymax of dialysates was not proportional to P concentration.
Solution P concentrations between 0.08 and 0.11 yg/ml (Treatments
5, 6 and 7) supported approximately equivalent maximum growth
whereas significantly less growth was measured in the dialysate
containing 0.04 yg/ml (Treatment 8).

     Direct Equilibration With an Anion Exchange Resin

The development of a method for the assay of the potential
for P release from sediments using direct sediment equilibration
with an anion exchange resin was described in Section V, A.  The
application of this method to the sediments of Upper Klamath
Lake is described  in this section.  The results are related
to release in the dialysis system.

Materials and Methods
Using the system described in Section V, A, sediments from
Howard Bay, Buck Island and Pelican Marina (2.5 g dry weight)
taken in April, 1971, were incubated in the dark at 25 C with
1.25 g resin.  The results for 30 days of incubation are
summarized.

Results and Discussion

In contrast to dialysis systems (Tables 43-45) sediments
incubated with resin in the direct equilibration systems exhibited
considerably more release than sediments not receiving resin
(Table 51).  In fact, Howard Bay sediments released approximately
                              131

-------
   Table 50.   Algal  growth in standard media amended with sediment
              dialysates.
Treatment

5
6
7
8
P concentration
in media
yg/ml
0.10
0.11
0.08
0.04
Maximum specific
growth rate^
ymax
1.46
1.35
1.56
1.60
Maximum cell
concentration
cells/ml
1.3 x 1C6
1.2 x 106
1.5 x 106
5.3 x 105





able 42
 max
                         ,
 where x^ = cell  concentration  at end  of selected time  interval
       x.]  = cell  concentration  at beginning  of selected time interval
  t2 - t-j  = elapsed time  (days)  between  selected  determinations  of biomass
                                132

-------
33% of the total sediment P during a 30 day incubation with
the resin (Figure 29).  The release of P from Buck. Island
and Pelican Marina sediments, over a 35-day incubation period
amounted, respectively, to 84 and 50 yg/g of sediment in
the presence of the anion exchange resin (Figure 30).  In systems
which did not contain resin, release of total P amounted
to less than 15% of the P contained on the resin.  Thus,
in contrast to dialysis systems, the presence of a P sink
has a substantial influence on the quantity of P released
to solution indicating that a biological sink in surface waters
may have similar effects.  The influence would likely be
considerably less pronounced due to a lack of direct contact
with sediment interstitial waters.

The presence of an anion exchange resin tended to buffer
the sediments at a relatively constant pH, whereas the pH of
Buck Island and Pelican Marina sediments was considerably
lower in the absence of resin after approximately 7 days
of incubation.  The lowering of pH would likely tend to increase
P release and therefore minimize release differences between
treatments.

Howard Bay sediments released 4-6 times as much P to the
resin as the other sediments.  Thus, both dialysis and direct
equilibration systems substantiate field observations of the
labile nature of P in Howard Bay sediments relative to sediments
from other locations.  The data indicate that the lack of resin
effect in the dialysis systems resulted from a limitation
on water interchange and P diffusion through the 0.45 u filter
or reduced uptake rate resulting from lower P concentrations
in solution.

             Algal Growth in the Dialysis Cell

It has been previously shown that direct incubation of sediments
with a P sink in the form of an anion exchange resin increases
the rate and extent of sediment P release (Table 51).  Furthermore,
dialysates from the sediment-distilled water dialysis systems
will support algal growth at rates comparable to those in standard
media (Table 40, 41).  This experiment was designed as a preliminary
test to determine (i] if algae could be cultured directly
in the dialysis system, (ii] specific growth rates of algae
under these conditions and (Hi] the effect on P release of
a P sink in the form of actively reproducing algal cells.
                              133

-------
        320
     en

     cn
        240
     00
     UJ
     CtL

     O
     I—




     £  160

     UJ
         80
     Q
     LU
             T
              D
                               y=189.56x+2.66

                               (r= 0.987, n = 20,  p<0.00i;
                        10
20
30
                                DAYS
FIGURE 29.  RELEASE OF PHOSPHORUS FROM HOWARD  BAY SEDIMENTS
            TO  AN  ANION EXCHANGE  RESIN IN THE  DIRECT
            EQUILIBRATION SYSTEM  (TABLE 51).
                                134

-------
    80
    60
 ^  40

 3.
    20
 O
 I—

 Q
    80
Q   60
LU
oo
    40
    20
             T	1	1	—I

              TREATMENT 3, BUCK ISLAND
                                                         O  ~
                             8
         X
     VJ
,x
             y=2.731x-4.16
             (r=0.982,  n=20,  p<0.00
       O O
           8
          TREATMENT 5,  PELICAN MARINA
                B
           B i
    y=2.428x-0.66
    (r=0.874, n=20,  p<0.001)

I	I	I	I
                    10      15      20

                            TIME, DAYS
              25
                                 30
35
FIGURE 30.  RELEASE OF PHOSPHORUS  FROM BUCK ISLAND AND  PELICAN
            MARINA SEDIMENTS TO  AN ANION EXCHANGE RESIN  IN  THE
            DIRECT EQUILIBRATION SYSTEM (TABLE 51).
                                 135

-------
Table 51.  Release of phosphorus  from moist Howard Bay (Treatments 1
           and 2), Buck Island (Treatments 3 and 4 and Pelican Marina
           (Treatments 5 and 6) sediments in the presence (Treatments
           1, 3 and 5) and absence (Treatments  2, 4 and 6)  of anion
           exchange resin (direct equilibration system).
Incubation
time
Ha we
aciys
i
2
4
7
11
15
18
22
26
30

1
2
4
7
11
15
18
22
26
30

Total


18
16
18
26
35
36
33
32
36
34

23
26
34
36
33
28
40
40
42
44
P in solution
P Inorganic

9/9
Treatment 1
10
0
2
8
13
14
12
8
10
10
Treatment 2
12
4
10
8
27
7
21
20
21
11
Total P
P on resin

o c U 1 1 1 1C 1 1 U — — — — — —
15
57
115
152
194
252
234
245
256
308












PH


6.4
6.4
6.4
6.4
6.4
6.6
6.4
6.6
6.8
6.8

7.0
6.9
6.9
6.8
6.8
6.8
6.8
6.8
7.0
7.0
                           136

-------
Table 51 (Continued).
Incubation
„ ti me
Ha \/<;
UQjr O
1
2
4
7
11
16
21
25
30
35

0
1
2
4
7
11
16
21
25
30
35

Total


6
12
12
14
17
14
14
16
15
20

8
8
11
10
7
16
12
6
9
4
6
P in solution
P Inorganic P

Treatment 3
-
-
-
1
1
4
2
2
4
2
Treatment 4
0
-
-
-
-
1
4
1
2
2
3
Total P
on resin


1
1
2
16
22
50
44
68
86
84













pH


6.4
5.8
6.1
5.7
5.6
6.2
6.0
6.2
6.3
6.3

7.2
6.8
6.3
6.3
5.6
5.6
5.7
5.0
4.8
4.3
4.3
                               137

-------
Table 51 (Continued).
Incubation
time
Have
Uayb
i
2
4
7
11
16
21
25
30
35

0
1
2
4
7
11
16
21
25
30
35

Total


4
8
7
10
9
14
12
14
15
18

9
6
6
15
12
8
7
9
5
4
6
P in solution
P Inorganic P

Treatment 5
-
1
1
2
1
8
2
5
4
5
Treatment 6
4
-
1
1
1
1
4
2
2
1
2
Total P
on resin


2
1
3
12
15
58
56
72
94
50













PH


6.2
5.7
6.2 ,
5.5
6.0
6.2
6.0
6.2
6.3
6.3

7.4
6.6
6.1
6.4
5.6
5.8
6.1
5.6
5.2
5.0
4.8
                            138

-------
Materials and Methods

Dialysis cells were prepared in the manner described previously
(Section V, A) using sediments from1 Howard Bay, Buck Island
and Pelican Marina.  However the membrane filter between
the half-cells was omitted since previous work had shown that
the 0.45 y membrane filter had a restrictive effect on P diffusion
between cells.  Standard media less P (300 mis) was added
to the water half-cells and inoculated with Selenastrum
capricornutum (103 cells/ml).   Uninoculated dialysis cells were
also prepared.  The experimental design is outlined in Table
52.  No effort was made to maintain asepsis as the omission
of the membrane filter allowed the  passage of microorganisms
between the cells.
                                t
                                i
The cells were incubated in a vertical position under light
at 25 C with gentle gyration (120 rpm).  During the first
week, aliquots (30 ml) were removed daily for P determination
and algal cell count.  Sufficient media was added to the upper
cell to maintain constant volume.   On the seventh day, the
upper cell was drained and fresh media added.  The dialysis
cells which had been originally inoculated were then reinoculated
using the drained media (5 ml).

Total P and inorganic P were determined as previously described
(Section IV, A).  Algal growth was  measured by the direct cell
count technique.

Results and Discussion

The dialysis cells developed a visible algal growth after
4 days of incubation with or without previous inoculation
with Selenastrum.  Subsequent microscopic examination of the
media revealed no growth of Selenastrum occurred but rather
another species of algae, tentatively identified as Chloroella
sp., was present.  The omission of  the 0.45 y membrane filter
may have allowed the passage of Chlorella autospores from
the sediment into the media and the subsequent growth of
the algae as P was released.

The algal growth parameters of the  Chlorella sp. are given
in Table 53.  The maximum specific  growth rates, ymax» and
total cell counts at 7 days for Howard Bay sediment are higher
than the parameters in previous experiments in which Selenastrum
was cultured in the absence of sediments.  However Selenastrum
was cultured in media dilutions of  aliquots from the dialysis
systems with inorganic P concentrations of 0.06 to 0.11 yg/ml
                              139

-------
Table 52.  Experimental design employed in investigations of sediment phosphorus  release  to
           inoculated algae growth media.

Treatment
number

1
2
3
4
5
6
7
8
Sediment
sample ,
designation

H-13
H-13
B-13
P-15
H-13
H-13
B-13
P-15

Wet
weight

485
508
500
461
470
455
477
376
Sediment
Total
solids
V
h
9.1
9.1
9.1
10.4
9.1
9.1
9.1
10.4
Composition
Total
P
\ \C\ 1 C\
	 yg/ g-
1033
1033
629
662
1033
1033
629
662

Inorganic
P Water Cell

574 Media and Inoculum
574
295
348
574 Media only
574
295
348

-------
 Table 53.  Growth of Chlbrella sp. cultured in the dialysis system
utilizing
Sediment
sample
designation

Howard Bay



Buck Island
Pelican Marina

sediments as a
Treatment
number

1
2
5
6
3
4
8
source of phosphorus
2
Specific growth
rate
ynax
3.045
2.303
2.862
2.731
1.712
2.429
1.992

Cel
7
eel
6.5
9.2
9.4
8.1
3.6
7.5
1.1

1 counts,
days
Is/ml
x 106
x 106
x 106
x 106
x 105
x 105
x 105
]Table 52
2
Calculated as previously described (table 40)
                                   141

-------
for Howard Bay sediment and 0.03 to 0.08 yg/ml  for Buck
Island and Pelican Marina sediments.   The total  P concentrations
for this experiment ranged from 1  to 2.5 yg/ml  during the
period of maximum growth for Howard Bay sediment and from
0.08 to 0.24 for Buck Island and Pelican Marina sediments.
The 16-20 fold increase in P concentration for the Howard
Bay sediment dialysates and the use of a native organism
likely account for the increase in growth parameters shown
in this experiment.

After 7 days, total P released from the sediments to the water
half-cell, determined by including digested algal tissue, amounted
to 17.5, 0.7 and 0.7 yg/g of Howard Bay, Buck Island and Pelican
Marina sediments, respectively.  Reinoculation  at 7 days
followed by another 7 day incubation  period resulted in release
of an additional 4 yg P/g of Howard Bay sediments after a total
of 12 days.   However no additional P was lost after 12 days
incubation,  and no further P release occurred with the Buck
Island and Pelican Marina sediments during the  second 7 day
incubation period.

The P release rate in the vertical dialysis systems without
the 0.45 y filter and containing the algal culture was quite
similar to release in horizontal  systems containing the
filter and anion exchange resin (Figure 25).   It appears
that when the dialysis cells are in the vertical positions,
diffusion of P through the membrane is not a  limiting factor
as suggested by earlier experiments with water  in each half-
cell.  An experiment similar to that described  in this section
but incorporating a 0.45 y filter partition between sediments
and water to reduce the possibility of sediment biological
contamination of the water half-cell  would therefore be in
order.

It is evident that in these systems,  sediments  supply ample
P for biological growth.  This method with suitable modifications
may serve in routine assessments  of the potential for P release
from sediments and subsequent availability of released P to
algae.
                             142

-------
                         SECTION VI

                      ACKNOWLEDGEMENTS

Technical contributions to these studies were made by R. C.
Routson, J. W. Blaylock, M. P. Fujihara and J. L. Armantrout,
   M. Thomas, R. J. Olson and R. D. Gilbert of BatteTie-
              by A. R. Gahler and W. D. Sanville of the
              Protection Agency.  Their assistance is sincerely
J.
Northwest and
Environmental
appreciated.
The support of the project by the Office of Research and
Monitoring, Environmental Protection Agency and the guidance
provided by A. R. Gahler and C. F. Powers, Project Officers,
are gratefully acknowledged.
                             143

-------
                        SECTION VII

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-------
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-------
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                            151

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                         SECTION VIII

                          APPENDIX

CHEMICAL CHARACTERISTICS OF SEDIMENTS FROM SEVERAL LAKE SYSTEMS

                A.  SHAGAWA LAKE, MINNESOTA

This section describes the results of efforts to compile
and statistically analyze, through correlation and mathematical
modeling, data obtained on the C, N and P contents of sediments
taken in September, 1970 from Shagawa Lake, Minnesota.  The
primary goals of these studies were to (i] characterize the
sediments as to mineralogical composition, (ii) establish
mean variation in total lake sediment C, N and P contents
and (ii-i] determine if mathematical relationships between
the elements existed which would provide insight into their
distribution, deposition, or transformations in the lake
system.  Inherent in these investigations was the development
of background information to assist in selection of lake
sediment sample sites for future investigations of P release.
Sample sites on Shagawa Lake are identified by location
on a lake grid system (Figure 31).

                    Materials and Methods

Analyses of the mineral composition of the intact sediments
were conducted using X-ray diffraction techniques.  Following
a series of ion saturation and heat treatments (Whitlig, 1965),
sediment samples were oriented on glass slides, glycerated
and analyzed in a Norelco X-ray diffractometer using Cu-K shell
a radiation and a Ni filter (Kittrick and Hope, 1963).

The methods of analyses for sediment P concentrations have been
previously described (Section IV).  The concentrations of total
C and N in the sediments were provided by EPA personnel
at the Pacific Northwest Laboratory, Corvallis.

Total  S was determined by Na-CO., fusion techniques essentially
as recommended by the Association of Official  Agricultural
Chemists (1955).
                           153

-------
a
c
b
d
                                         G    H
FIGURE 31.  GRID SYSTEM - SHAGAWA LAKE, MINNESOTA
                          154

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                   Results and Discussion

The minera"logical  compositions of Shagawa Lake sediments,
as determined by X-ray diffraction techniques, are given in
Table 54.  Sediment samples from 6 locations throughout the
lake were similar  in mineral composition, and no significant
mineralogical differences were observed between samples.
Kaolinite may have been present, but only in trace quantities.
The diffractograms (Figure 32) indicate the presence of expanding
lattice materials, however the apparent tendency to collapse
under K saturation suggests the presence instead of weathered
mica (beidellite-like material).  The samples were not separated
according to particle size and therefore it was not possible
to determine definitively if montmorillonite, a clay-sized
(<2 micron) mineral, was present.  In general, the sediments
appear to consist  primarily of finely-divided granitic materials
derived from glacial deposited parent materials.

Total S in Shagawa Lake sediments (Table 55) ranged from 0.28%
(Clld) to 0.95% (B15d).  The concentration of S was generally
related to X-ray diffraction response at the diffraction
angle attributed to gypsum, however due to differences in crystalinity,
response and amorphous background these comparisons cannot
be considered quantitative.

Highest S concentrations were in sediments taken from deeper
locations, i.e. A6d, B15d and D8c.  It might be expected,
however that S at  these lower depths would be present primarily
in reduced forms rather than as gypsum.

Mean C, N and P contents for Shagawa sediments sampled at 31 locations
on Shagawa Lake are given in Tables 56, 57.  Due to the
magnitude of the differences in total  C and N and P, C and
N contents are expressed as percentages whereas P contents
are expressed on a yg/g basis.  Considering the wide range
in sample site locations within the lake, elemental  composition
of the sediments did not deviate widely from the mean.  In terms
of the total concentration, maximum variation occurred in
the case of N (x, 1.20; Sd, 0.3).

The sewage effluent (grid location E10, Figure 31)  from the
town of Ely, Minnesota is likely the principal  source of the
excessive quantities  of nutrients  entering Shagawa  Lake.
However, little is  known of the distribution or fate of these
materials upon entrance into the lake  system.   To provide
insight into the location of the effluent in the lake, data
on elemental distribution in sediments  was statistically
                            155

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      Table 54.  Mineralogical composition of the sediments of Shagawa
                 Lake.
                                    1                       2
Mineral type,  relative concentration  and diffraction angle


Abundant (>20%)          Present (10-20%)                   Trace (5-10%)


quartz (27)              gypsum (21.0,  11.5)                amphibole (10.5)

                        chlorite  (25.5,  23.5,  12.5,
                                   6.5)

                        feldspar  (28.0,  22.5)

                        mica (27)

                        amorphous  (background)
 Concentration defined approximately by comparison of relative height of
 diffraction maxima for minerals  of known  crystalinity and response.
2
 Diffraction angle  (20)  on  K saturation,  air dried.
                                       156

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CD
>—i
UJ
                Mg-SATURATED GLYCEROL
                             SOLVATED
             K-SATURATED,  AIR DRIED
                        K-SATURATED,  HEATED   500UC)
                       l
30      26
                   22       18       14       10

                    DIFFRACTION ANGLE (20)
FIGURE 32  REPRESENTATIVE  X-RAY  DIFFRACTOGRAMS  OF  CHEMICALLY
           TREATED  SEDIMENTS  FROM  SHAGAWA  LAKE.
                              157

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   Table  55.  Total sulfur concentration in sediments of Shagawa
             Lake, as related to relative gypsum concentration.
Sample                       Sulfur                     Relative  height
designation                  content                    height of X-ray
                                                        diffraction maxima
   A6d                        0.77                            +++

   B13d                       0.35                             ++

   B15d                       0.95                             ++

   Clld                       0.28                              +

   D8c                        0.47                             ++

   F5a                        0.39                             ++
                                 158

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Table 56.  Distribution of carbon and nitrogen in Shagawa
           Lake sediments.
Sample Site
Grid Location

A5c
A6d
AlOd
A17b
Bib
B2c
B3c
B5c
BlOc
B13b
B13d
B15a
B15b
C3a
C8b

Water
Depth

5
12
4.5
5
4
4
2
6
6
5
5
6.5
12
3
8

Content
C
- -°/
14.6
14.2
9.7
8.1
13.6
2.6
28.1
15.1
7.9
10.6
9.7
10.7
10.9
17.4
12.9

of
N

1.5
1.5
1.0
1.0
1.3
.4
1.7
1.5
.9
1.3
1.0
1.1
1.3
1.5
1.5

Sample Site
Grid Location

C8d
did
C12d
C13b
C15b
D8b
D8c
Dlla
E5b
E6c
E7c
El Ob
F5a
F9a
G4c
G5a,
Water
Depth

11
6
6
5.5
10
8
12
5.5
6
6
6
6
6
5.5
3
6
Content
C
	 °i 	
13.6
10.2
9.8
1.3
11.2
13.1
13.2
10.0
16.0
14.1
13.0
11.8
13.4
9.6
16.5
15.0
of
N

1.5
1.0
1.0
.2
1.4
1.3
1.5
1.2
1.6
1.5
1.5
1.1
1.3
.9
1.2
1.5

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        Table 57.  Summary of the concentrations of carbon, nitrogen and phosphorus in sediments of
                   Shagawa Lake.
01
o
Variable1
Total C(%)
Total N(%)
Total P-tvg/g)
Inorganic P
absolute (ug/g)
fraction of total P(%)
Organic P
absolute (pg/g)
fraction of total P(%)
Mean
(x)
12.20
U20
1231

691
56.8

541
43.2
Standard
deviation
(Sd)
4r6
0,3
238

160
10. 3

169
10.9
95% confidence
value
(95% CV)
1.70
0.10
88

59
3.9

61
3.9

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analyzed on the basis of geographical location.  In interpretations,
particular emphasis was placed on the sediment distribution
of elements in relation to the sewage outfall.

Although the direction and rate of intermediary current flows
are unknown, the general direction of lake current is likely
from west to east,  i.e., from the mouth of the Burntside
River at the west end of the lake to the outlet of the Shagawa
River at the east end of the lake.  Thus, it might be expected
that the sewage effluent located on the south lake shore about
midway on an east-west transect would accumulate in the
sediments east of the outfall.  Significant (p <0.01) locational
differences in the  sediment concentration of C and N exist
(Table 58).  However, mean total C and N are approximately
50% higher in locations west or "upcurrent" from the sewage
outfall.  Total P was not significantly different between
locations.  Thus, assuming the C and N are not lost before
deposition on the sediments, presumptive evidence suggests
that the general depositional pattern of C and N originating
from the sewage effluent, to the west, is perhaps reflecting
current effects different from the expected.  Furthermore,
assuming that the sediment samples were representative,
either P is not subject to similar mechanisms of transport
and deposition or lake sediment (or water) transformations
take place which tend to normalize the content of sediment
P.  A more detailed evaluation of total sediment P on the
basis of lake location is presented in Table 59.   Analyses
of variance showed no significant locational effects.   However,
total P is highest in grid locations immediately north of the
sewage outfall (grid locations A-E).

As in the case of sediment elemental distribution, the transformations
of C, N and P in lake sediments are difficult to infer from
direct analyses.  However, some insight into gross changes,
important to remedial measures, may be obtained.   Linear intercorre-
lations of water depth, sediment total  P, inorganic P, organic
P and total C and N are given in Figure 33.   Significant
correlations existed between total P and inorganic P,  independent
measurements.  As may be expected, organic P, taken as the
difference between total and inorganic P, also correlated with
total P.  Significant correlations between organic P,  total
C and total N provide evidence that either the organic or total
P measurement serves as a valid measure of P in  organic
combinations since a relatively constant ratio of these
elements may be expected in biological  tissue and in  the
sediment organic phase.  It should be noted, however,  that
principal  correlations are between C and N and correlation
of P with  these variables is  comparatively low.   In general,
                          161

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         Table 58.   Distribution  of total  carbon,  nitrogen  and  phosphorus  in Shagawa Lake sampling
                    locations  west (grid sections  1-9)  and  east (grid  sections  10-18) of the sewage
                    outfall.!
01
no
East location
(A)
Variable

Total C
Total N
Total P
Standard
Mean deviation

9.4
1.0
0.128
	 V 	
2.7
0.3
0.031
Number
values

13
13
13
West location
(B)
of Standard
Mean deviation

14
1
0

.2
.4
.120
°i
4.7
0.3
0.017
Number of
values

18
18
18
Location differences
(A-B)
Mean
difference

4
0
-0

.8
.4
.009
Standard
error of
difference
	 ol 	
1.5
0.1
0.009
T
value

3.32
3.!2
1.0

       Sewage effluent located at  grid  section  E10  (Fig.  31).

       Significant at the 0.01 probability  level.

-------
               TOTAL  P
             INORGANIC  P
              ORGANIC  P
               TOTAL  C
               TOTAL  N
             WATER  DEPTH
FIGURE 33.  CORRELATIONS OF WATER DEPTH,  TOTAL,  INORGANIC,
           AND ORGANIC PHOSPHORUS;  TOTAL CARBON;  AND TOTAL
           NITROGEN CONTENTS OF SEDIMENT  SAMPLES FROM
           SHAGAWA LAKE.   ASTERISK DESIGNATES p <0.05.
                               163

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        Table  59.  Geographical distribution of total phosphorus in sediments of Shagawa Lake.
en
Grid-, 7
section '
North-south
A
B
C
D
E
F
G
East-west
3
5
6
8
10
11
13
15

Figure 31.
Number of
sample
sites

4
9
7
3
4
2
2
2
5
2
4
3
2
3
3


Mean
of total
P

1240
1260
1260
1350
1180
1060
1070
1130
1200
1150
1390
1020
1380
1220
1430


Standard
deviation
(Sd)
— yg/g 	
100
300
300
30
200
-
-
-
100
-
100
200
-
600
200
-t .-

               o
                Grid sections with a single value were excluded.

-------
sediment P transformations which influence the concentration
of one parameter may also be expected to have an influence
on the correlated parameters.

A linear correlation between sediment total P and water depth
indicates a significant relationship between these two variables.
The correlation must be considered tenuous, due to the relatively
small variation from the mean in sediment total P concentrations
(Table 57).  It would appear, however, that water depth must
be considered in sampling programs designed to determine
the quantity and distribution of sediment P and in interpretation
Of chemical factors influencing P release to solution for both
physical and chemical phenomena may be influenced by depth
of the overlying water.

As an additional guide to the determination of factors influencing
elemental transformations in the sediments, a multiple regression
model was devised to determine the parameters important to the
prediction of total C and total P.  All measured parameters
were considered except in the case of the prediction of total
P for which inorganic P and organic P were omitted.  The final
results are summarized in Table 60.

Variation in total sediment C could be predicted (Rz = 82%)
using the coefficients of water depth and pediment N content.
Thus, total C may be expected to be markedly influenced
by these variables.  A portion of total P variation (Rz = 38%)
could be predicted using the coefficients of total sediment
N and C content.  Water depth did not significantly influence
the prediction of total P as might be expected from the
simple correlation between these two variables (Figure 33).
Multiple regression analysis involves the utilization of several
parameters which are interrelated and inclusion of N content
into the equation negated water depth as a useful  parameter.
Apparently, variation in N accounted for a major portion
of the variation attributed to water depth in simple correlations.
It is likely that a water depth-N content interaction term
would improve the regression coefficient, however interactions
were not considered in this model.

In brief summary, the results indicate that sediment concentrations
of C, N and P are not highly variable.  Variation  in total
C, N and P that exists appears at least partially  related
to sample location and water depth.  Furthermore,  factors which
influence the concentration of C strongly influence N, and
to a lesser extent, P-
                          165

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CT>
        Table 60.  Multiple regression model to predict total  sediment carbon  and  phosphorus contents
                   of Shagawa Lake sediments.                                               ^


Dependent
variable




Total carbon

Total phosphorus




123
Intercept Parameter coefficients ' *
Depth N C
content content
m __ _ °/

0.461 -0.607 12.638
10.150 tl.140
0.077 0,079 -0.0042
tO. 019 +0.0010

Variation in
Multiple total C
correlation attributable
coefficient to parameters
•)
(R) (RZ)

%_
_.___ — _ 	 	
0.90 82

0.62 38


       Includes  standard  error (SE).

      2
       Total  phosphorus parameter not  significant in  prediction  of total  carbon.


       Multiple  correlation  coefficient for  total phosphorus  model  is  0.88 if inorganic phosphorus
       parameter included.

-------
For future investigations designed to estimate the potential
of the sediments to release P to solution prior to and after
sewage treatment, sediment samples must be taken which are
representative of the lake system within reasonable limits
of error.  To accomplish this, error due to sampling variation
must be known.  Furthermore, available information on lake
characteristics must be utilized to select sediment samples
which are most representative of all the sediments so that
the results may be extrapolated to the total lake system.  Based
on these requirements and the results of statistical analyses
outlined above it was recommended that the following samples
of Shagawa lake sediments be taken:  sample locations (Figure 31)
Bib; B5c, B15b, C8b, C12d and E6c.

Except for sample location B15b, locations were randomly
selected from categories (Table 61) based on total sediment
P concentrations.  The coefficient of variation for the
categories of sediment total P concentration outlined in Table
61 were approximately 5% compared to a coefficient of variation
for total P for all sample locations of approximately 20%.  Thus,
a stratified random sample design was employed to select the
sample locations.  A single random sample was selected for
each category.  Sample location B15b was added to provide
representation for the sediments in the deep section at the
east end of the lake.  Much of the deviation from the mean
water depths and C, N and P contents are represented by the
sample locations shown.

To achieve a coefficient of variation of <10% for each sample
location, it was estimated that 6 replicate samples must
be taken through the ice at each location.

The Shagawa Lake sediment samples were subsequently taken
in March, 1971 at locations A6d, B13d, B15d, Clld, D8c, and
F5a (Figure 31).  A total of 6 sediment samples from each
location, representing 2-3 dredge loadings per sample, were
individually mixed and subsampled in duplicate for analyses
of total solids, total P and inorganic P-

The coefficients of variation for total solids, total  P (NazCO.j
fusion) and inorganic P (HZSOH extraction) ranged from 2.8
to 6.9%, 3.9 to 13.2% and 4.2 to 19.2%, respectively (Tables
62-67)-  Samples F5a and B13d exhibited the low and high
values, respectively, for each of the analyses conducted.
Sample F5a was located in a relatively shallow area in which
there was little chance for contamination by sewage or mine
tailings, whereas the B13d location was deep by comparison
                           167

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Table 61.  Categorization of total phosphorus concentrations in the
           sediments of Shagawa Lake.
Categories consisting of sediments with
total P Cug/g) in the range Of
900-1000
Site
Bib
B2c
B3c
BlOc
El Ob
F5a
F9a
G4c


Value
1024
923
1048
915
942
1074
1043
982


1100-1300
Site
A5c
A6d
AlOd
C3a
C8d
E5b
E6c
G5a


Value
1218
1130
1227
1208
1291
1211
1176
1154


1300-1500 1500-1700
Site
A17b
B5c
B15a
Clld
C12d
C15b
D8b
D8c
Dlla
E7c
Value Site
1368 B13b
1346 B13d
1303 B15d
1464 C8b
1456
1335
1368
1381
1315
1398
Value
1577
1550
1660
1535






Mean
(x)      994             1202             1374             1581
Standard
deviation
(Sd)      62               49               54               56
Coefficient
of variation
(Cv)       6%               4%               4%               4%
 Figure 31
                             168

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Table 62.  Sampling and analytical  variability associated with  the determination  of the  forms  of
           phosphorus in sediments  (A6d)  of Shagawa Lake.
Sample
designation
Total soli
repli
cates
ds
Total P
mean
replicates
mean
Inorganic P
replicates
mean

-------
      Table 63.  Sampling and analytical variability associated with the determination of the forms of
                 phosphorus in sediments (B13d) of Shagawa Lake.
o
Sample
designation
1
2
3
4
5
6
MEAN
Standard
Deviation
Coefficient
of Variation
Total
repli

12.89
11.61
13.03
12.72
14.67
13.15
13.01
.98
7.6%
solids
cates

13.
12.
12.
12.
14.
13.
13.
4
V
h 	 -'
49
28
61
85
72
21
19
86
6.5%


mean

13
11
12
12
14
13
13


.19
.94
.82
.78
.70
.18
.10
.91
6.9%

repli

1376
1766
1363
1451
1130
1335
1404
208
14.8%
Total P
cates

1276
1526
1529
1324
1053
1324
1339
177
13.2%

mean

	 ug/g-
1326
1646
1446
1388
1092
1330
1371
181
13.2%

Inorganic
replicates

809
1182
786
850
626:
769
837
185
22.1%

746
969
941
782
569
744
792
...447
18.5%
P
mean

778
1076
864
816
598
756
815
157
19.2%

-------
Table 64.  Sampling and analytical variability associated with the determination of the forms of
           phosphorus in sediments (B15d) of Shagawa Lake.
Sample
designation
1
2
3
4
5
6
MEAN
Standard
Deviation
Coefficient
of Variation
Total solids
replicates

10.
9.
11.
9.
9.
10.
10.
B
7.

48
80
69
76
87
08
28
74
,2%
01
	 £ 	
10.47
8.47
10.25
11.47
9.98
10.12
10.18
.88
8.6%
mean

10
9
10
10
9
10
10

5

.48
.27
.99
.62
.92
.10
.23
.60
.9%
Total P
replicates

2091
1731
1676
2490
1766
1749
1917
317
16.5%

2093
1674
1969
2108
2070
2070
1997
166
8.3%
mean
,,n 1
Inorganic P
replicates

	 ~yy/a
2092 1523
1702
1822
2299
1918
1910
1957
211
10.8%
1106
1135
1718
1387
1390
1376
232
16.9%

1408
1040
1285
1376
1407
1340
1309
140
10.7%
mean

1466
1073
1210
1547
1397
1365
1343
174
12.9%

-------
ro
       Table  65.   Sampling  and  analytical  variability associated  with  the  determination  of  the  forms
                  of phosphorus in  sediments  (Clld)  of Shagawa  Lake.
Sample
designati on
1
2
3
4
5
6
MEAN
Standard
Deviation
Coefficient
of Variation
Total solids
replicates

12.
11.
11.
11.
11.
11.
11.
.
3.

44
87
71
45
32
46
71
41
5%

12.93
12.00
11.91
12.11
11.40
11.26
11.94
.59
5.0%
mean

12
11
11
11
11
Tl
11

4

.68
.94
.81
.78
.36
.36
.82
.49
.1%
Total P
replicates

1350
1510
1364
1651
1614
1624
1519
134
8.8%

1319
1429
1414
1565
1623
1625
1496
127
8.5%
mean

y/ 9
1334
1470
1388
1608
1618
1624
1507
128
8.5%
Inorganic P
repli

787
847
769
962
934
981
880
.*?•
10.4%
cates

748
843
802
937
970
964
877
,93
10.6%
mean

768
845
786
950
952
972
879
• 91
10.3%

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GO
    Table 66.  Sampling and analytical variability associated with the determination of the forms
               of phosphorus in sediments (D8c) of Shagawa Lake.
Sample
designation
1
2
3
4
5
6
MEAN
Standard
Deviation
Coefficient
of Variation
Total solids
replicates
9.01
9.02
8.87
9.76
9.14
9.35
9.20
.32
3.4%
8
8
8
9
8
8
8

2
.64
.57
.58
.22
.61
.65
.71
.25
.9%
mean
3.
8.
8.
9.
8.
9.
8.
B
3.
82
82
72
49
88
00
96
28
1%
Total P
replicates
4555
4811
4330
3283
4595
3961
4256
557
13.1%
4144
4703
3981
3110
4378
4019
4056
535
13.2%
mean
— pg/g—
4350
4757
4156
3196
4486
3990
4156
540
13.0%
Inorganic
repli
3556
3681
3426
2469
3790
3177
3350
481
14.4%
cates
3105
3508
3078
2331
3543
3212
3130
439
14.0%
P
mean
3330
3594
3252
2400
3666
3194
3239
452
14.0%

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Table 67,  Sampling and analytical variability associated with the determination of the forms of
           phosphorus in sediments (F5a) of Shagawa Lake.
Sample
designation
1
2
3
4
5
6
MEAN
Standard
Deviation
Coefficient
of Variation
Total solids
replicates

9.47
9.69
9.40
9.73
10.00
10.28
9.76
.33
3.4%

9
9
9
9
10
9
9

2
of
.25
.84
.64
.98
.04
.80
.76
.29
.9%
mean

9.
9.
9.
9.
10.
10.
9.
.
2.

36
76
52
86
02
04
76
27
8%
Total P
replicates

1277
1163
1175
1191
1164
1175
1191
43
3.6%

1266
1121
1134
1175
1176
1186
1176
51
4.3%
mean

1271
1142
1154
1183
1170
1180
1183
46
3.9%
Inorganic
replicates

j- - - -
603
583
564
568
569
544
572
20
3.5%

618
530
530
542
537
573
555
35
fe.3%
P
mean

611
556
547
555
553
558
563
24
4.2%

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and was mottled  (red and gray) in appearance reflecting unusual
genesis or contamination perhaps by mine tailings.  Highest
total P values were obtained for two deeper locations in
the lake  (B15d,  D8c).  Unusually high total P values  (mean
4156 vg/g) were  obtained for location D8c which was the
deep hole in closest proximity to the sewage outfall.

                        B.  LAKE ERIE

This section summarizes the results of chemical measurements
of total, inorganic and organic P and P in inorganic  P fractions
for sediments from Lake Erie.

                   Materials and Methods
Sediment sampling sites on Lake Erie are described in Table
68.

Methods employed for the determination of the forms of sediment
inorganic P are described in Section IV, A.

                  Results and Discussion

The results are outlined in Tables 69 and 70.

                  C.  AGENCY LAKE, OREGON

This section summarizes the results of chemical  measurements
of total, inorganic and organic P and P in inorganic P fractions
for sediment from Agency Lake, Oregon.

                   Materials and Methods
Sediment sampling sites on Agency Lake are described in
Table 71.

Methods employed for the determination of the forms of sediment
inorganic P are described in Section IV, A.

                   Results and Discussion

The results are outlined in Tables 72 and 73.

                  D.  DIAMOND LAKE, OREGON

This section outlines the results of investigations to determine
(i] the mineralogical composition and (H) the total and
inorganic P contents for the sediments of Diamond Lake, Oregon.
                           175

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 Table 68.   Description  of  sampling  sites  on  Lake  Erie.
  Sample
designation
 Date of
col lection
  Approximate
sampling depth
Sediment Water  Location
  6-25-1

  1-16-2

  I-23-la

  J-21-1

  K-28-1

  I-23-lb
7-18-69

7-15-69

7-15-69

7-16-69

7-17-69

9-23-69
  cm       m

0-10     22.8   4TS '51.2'N, 81° 26.0'W

0-10     21.3   42° 01.TN, 82° 11.5'W

0-10     25.3   42° 02.6'N, 816 37.5'W

0-10     24.0   42° 09.9'N, 81° 45.TW

0-10     23.4   42° 14.TN, 81° 15.3'W

0-10     25.3   42° 02.6'N, 81° 37.5'W
                               176

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  table  69.   Distribution of organic arid inorganic phosphorus in Lake
              Erie se'dlmfcritS as' detierttiiried by high temperature ignition
              methods.
Sample ,
designation

G-25-1
1-16-2
U23-ld
J-21-1
K-28-1
I*23-1b
total P2
ng/g
751
591
1238
713
782
713
inorganic P 	
abSQluti fraction
6f total
.. „ ,.. , .. ,/• ..... ,...!'. • • ,L.'_l!r.' ..!',. 	
^/g
541
782
906
528
625
524
%
72.0
78.9
73,2
74.1
79.9
73.4
,, Organi
absolute
ug/g
210
209
332
185
157
190
c P

fraction
of total
(V
11
28.0
21.1
26.8
25.9
20.1
26.6







T
 table 68
                                  177

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       Table  70.   Distribution  of phosphorus  in  inorganic  phosphorus  fractions of Lake Erie sediments.
___^ _ P extracted with

NHC1   NHF    ™    NaS°   ^    NaOH             HC1
       Sample    ,    4        4               a24         a                              Ignition-    23

     designation1  (0.5M)   (0.5M)   (0.1M)   NaoC,H,-0       (0.3M)   (0.5M)   (l.OM)  Total   HCl(l.OM)   HC1(9.0M)
                                       _ 3  U 0 ~ _ "~~ _ _ ~ _ ~ _    _ _


      1-23-1         4       238       2        278          42       350     49      399       130         93
oo

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Table 71.  Description of sampling site on Agency  Lake,  Oregon.
Sample
designation
A-1
A-2
A-3
A-4
Date of
collection
6-3-69
7-15-69
9-8-69
10-24-69
Approximate
sampling depth
Sediment Water
cm
0-10
0-10
0-10
0-10
1
1
1
1
m
.8
.8
.8
.8
Locati
42 *
42 o
42°
42°
31
31
31
31
on
.5'N,
. 5 ' N ,
,5'N,
.5'N,

121°
121°
121°
121°
58.
58.
58.
58.
5
5
5
5
'W
'W
'W
'W
                              179

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Table 72.   Distribution of organic and inorganic phosphorus in Agency
           Lake sediments  as  determined by high temperature ignition
           methods.
Sample ,
designation


A-l
A-2
A-3
A-4
Total P2

ug/g
475
725
426
675
Inonjanic P
absolute

ug/g
219
478
192
424
fraction
of total
%
46.1
65.9
45.1
62.8
Organic P
absolute

ug/g
256
247
234
252
fraction
of total
la
53.9
34.1
54.9
J7.2

    Table 71
                                180

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     Table 73.  Distribution of phosphorus in inorganic phosphorus fractions of Agency Lake sediments.
CO


Sample . NH4C1
designation (0.5M)


A-l <5
A-2 5
A-3 6
A-4 <5

Vable 71


NH4F
(0.5M)


71
51
62
49




NaOH F
(O.IM) r


40
6
41
60




teS204(0.
te3C6H502


55
48
96
67


2
P extracted wi

1M) NaOH
,(0.3M) (l.OM)

	 ug/g 	 	
7
21
36
38


th


(0.5M)


44
51
114
185




HC1
(l.OM)


9
14
21
29





Total HCld.OM


53 61
65 45
135 51
215 52



Na0CO,
2 3
fusion
) HC1(9.0M)


92
60
78
100



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FIGURE 34.  GRID SYSTEM - DIAMOND LAKE, OREGON
                           182

-------
                                     on
                   Materials and Methods

The Diamond Lake sediment samples (Ale, B2a, B5a, C3d, C6a,
D7a) were taken in July, 1971 and designated according to locati
on the EPA grid system (Figure 34).

Analyses of the mineral composition of the intact sediments
were conducted using X-ray diffraction techniques (Section
VI, A).

Total sediment P was determined by NazCO.j fusion and inorganic
P was estimated by HZS04 extraction as described in Section
IV.  Organic P was taken as the difference between total
and inorganic P.

                   Results and Discussion

Representative X-ray diffractograms of Diamond Lake sediments
are given in Figure 35.  Diffraction maxima did not differ
significantly with sampling location.  The sediments contained
relatively low concentrations of crystalline minerals as  reflected
in the near absence of sharp diffraction maxima.  This is in
accordance with previous observations that the volume of discrete
crystals in ash deposits close to Crater Lake is less than 1%
(Williams, 1942).  The maxima at a diffraction angle of 28 (3.2
A spacing) are due to residual KC1 after treatment.  All  samples
exhibited broad diffraction maxima at diffraction angles  (Figure
36) of 22 and 10 (4 and 10 A spacings, respectively).  The broad
maxima are an indication of a relatively high percentage
of amorphous material.  Saturation with K tended to sharpen
the 10 A maxima, perhaps as the result of the collapse of small
quantities of expandable material.  In several samples,
heating to 500 C reduced the height of the 10 A peak, indicating
the presence of endellite.  Endellite is a hydrated halloysite
mineral known to be present in some volcanic soils.  In general,
the mineral fraction of the Diamond Lake sediments appears
to be derived largely from pumice probably originating from
the volcanic eruption of Mt. Mazarna.

Total P contents (Table 74) of Diamond Lake sediments ranged
from 510 yg/g (Ale) to 994 yg/g (B5a).  Although no precise
pattern of P distribution with sediment location was apparent,
higher P values were generally associated with the northern,
deeper portions of the lake.  A notable exception was the
relatively high P value at the mouth of Camp creek (D7a).
183

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LU
o:
                            I—'—I—'—I—'—I    ^
                            Mg-SATURATED, GLYCEROL-SOLVATED
               K SATURATED, AIR-DRIED
           K SATURATED, HEATED  (50CTC)
                               _L
  30
26
22      18       14      10

   DIFFRACTION ANGLE (20)
   FIGURE 35.  REPRESENTATIVE X-RAY D I FFRACTOGRAMS OF CHEMICALLY
              TREATED SEDIMENTS FROM DIAMOND LAKE,
                               184

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        Table 74.  Total and  inorganic  phosphorus  content  of Diamond Lake sediments.
CO
en
Sample
designation

Ale
B2a
34a
B5a
B6a
B7a
C2c
C3d
C4b
C5a
C6a
C7a
D5a
D7a
Total solids
%
8.41
6.92
5.68
6.05
8.27
15.12
6.30
5.70
7.51
7.95
8.48
9.32
8.50
23.84
Total
ug/g
510
826
713
994
485
460
759
847
589
483
462
655
466
974
Inorganic
P Absolute
pg/g
131
302
320
472
99
166
303
368
210
119
106
230
no
585
P
Fraction
of total
V
.•o
25.7
36.6
44.9
47.5
20.4
36.1
39.9
43.4
35.7
24.6
22.9
35.1
23.6
60.1
Organic
Absolute
ug/g
378
524
392
522
386
294
456
480
380
364
356
424
356
388
P
Fraction
of total
%
74.3
63.4
55.1
52.5
79.6
63.9
60.1
56.5
64.3
75.4
77.1
64.9
76.4
39.9
       Based on oven dry  (50 C) weight

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 SELECTED WATER
 RESOURCES ABSTRACTS

 INPUT TRANSACTION FORM
/, Re (tort tf a.
                                                                •',5.' • Report Date
        PHOSPHORUS RELEASE FROM LAKE  SEDIMENTS
 7.  Aufhorfs)

    Wlldung, Raymond E. and Schmidt, Ronald L.
    Battelle,Pacific Northwest Laboratories



 12.  Sp-''nsorin:r Orgaai+afion •,', ,   •'        'v*
      Environmental Protection Agency report
      number, EPA-R3-73-024,  April 1973.
                                                                    RepoftNo.
                    / i .  C\.i,iti''icif Grant No.

                       14-12-508
                                rt and.
                      .Period Cbveted •
           :  Investigations were undertaken to characterize  the major  inorganic  and
organic forms of phosphorus in sediments of Upper  Klamath  Lake, Oregon,  determine the
potential for release of phosphorus from the sediment  as influenced  by water  and  sedi-
ment composition and environmental parameters, and establish  the relationship between
phosphorus release and algal growth.  Sediment characterization was  extended  to other
lake systems including Shagawa Lake in Minnesota,  Agency and  Diamond Lakes  in Oregon
and Lake Erie.  Sediments of Upper Klamath Lake, although  differing  in their  ability
to release phosphorus, exhibited seasonal changes  in phosphorus concentration.  These
changes were most pronounced in the inorganic phosphorus fraction  and in a  bay which
received agricultural runoff and initially contained relatively large quantities  of
phosphorus in the sediment interstitial water.  Release of and resorption of  phosphorus
associated with the solid phase occurred.  Release appeared to be  largely from non-
occluded iron forms of phosphorus whereas resorption was primarily in the form of non-
occluded aluminum forms of phosphorus.  The rate and extent of phosphorus release, de-
scribed by regression models, was related to sediment  composition.   Release was accel-
erated by increased temperature and the presence of a  phosphorus sink such  as an  anion
exchange resin in laboratory studies or actively reproducing  phytoplankton  in field
studies.  Algal growth response to phosphorus released from sediments during  dialysis
was approximately equivalent to the response to orthophosphate.
(Wilduna-Battelle)	
 17a. Descriptors
     *Water Quality, *Eutrophication, *Pbosphorus,  *Inorganic  and  Organic  Compounds,
     *Sediments, Algae, Carbon, Nitrogen,  Sediment—Water  Interface,  Mineralogy
 l?b. Identifier
     *Upper Klamath Lake, Oregon,  *Agency Lake,  Oregon,   *Diamond  Lake,  Oregon,
     *Shagawa Lake, Minnesota,  "take  Erie
M A-.'i,<<"li>Y ••/£• Security Class,
' (Repor.)
>0. Se'.nrityChss.
' ' (Page)
21. Wo. Of ,
Pages • ,
,.'i. ,ipjrj-s
Send To:
WATER RESOURCES SCIENTIFIC INFORMATION CENTER
U.S. DEPARTMENT OF THE INTERIOR
WASHINGTON. D. C. 2O24O
R. E. Wildung 1 ;,,, ., Battell e, Pacific Northwest Laboratories

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