EPA-660/3-73-005
Ecological Research Series
OXIDATION OF ORGANIC MATTER
IN SEDIMENTS
I
55
\
LU
C3
Office of Research and Development
U.S. Environmental Protection Agency
Washington, D.C. 20460
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and
Monitoring, Environmental Protection Agency, have
been grouped into five series. These five broad
categories were established to facilitate further
development and application of environmental
technology. Elimination of traditional grouping
was consciously planned to foster technology
transfer and a maximum interface in related
fields. The five series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
1. Environmental Monitoring
5. Socioeconomic Environmental Studies
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,
transport, and pathway studies to determine the
fate of pollutants and their effects. This work
provides the technical basis for setting standards
to minimize undesirable changes in living
organisms in the aquatic, terrestrial and
atmospheric environments.
EPA REVIEW NOTICE
This report has been reviewed by the Office of Research and
Development, EPA, and approved for publication. Approval
does not signify that the contents necessarily reflect the
views and policies of the Environmental Protection Agency,
nor does mention of trade names or commercial products
constitute endorsement or recommendation for use.
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EPA-660/3-73-005
September 1973
OXIDATION OF ORGANIC MATTER IN SEDIMENTS
By
Mario M. Pamatmat
Auburn University
Auburn, Alabama
R. Stephen Jones
Herbert Sanborn
Ashok Bhagwat
University of Washington
Seattle, Washington
Project 16070 EKZ
Program Element 1B1025
Project Officer
Dr. Milton H. Feldman
Pacific Northwest Environmental Research Laboratory
National Environmental Research Center
Corvallis, Oregon 97330
Prepared for
OFFICE OF RESEARCH AND DEVELOPMENT
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 $1 30
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ABSTRACT
A suitable sampler for taking undisturbed sediment samples was developed.
Techniques were worked out for measuring (a) oxygen uptake by intact
sediment cores, (b) dehydrogenase activity of sediment bacteria, and (c)
their actual metabolic heat release. Dehydrogenase activity as a relative
measure of anaerobic metabolism was calibrated by direct calorimetry for
use in determining natural rates of sediment metabolism. The concentra-
tion of reduced end products of anaerobic metabolism was determined by
an iodometric and dichromate method. Laboratory experiments were
conducted to determine the equivalents between rates of oxygen consumption
on the one hand and loss of organic carbon of sediments and liberation of
nutrient salts, e.g. nitrates, phosphates, silicates, and ammonia, on the
other. Seasonal measurements of oxygen consumption at 33 stations in
Puget Sound provided benchmark information for an area that may be subject
to worsening conditions due to the impact of increasing human population.
In situ oxygen uptake by the sediment can be estimated by shipboard
measurements with sufficient accuracy. The original working hypothesis,
however, that total oxygen uptake represents a measure of total metabolism,
aerobic plus anaerobic, in the sediment column appears erroneous, at
least in organically lich sediment. The rate of total oxygen uptake by
intact cores represents aerobic plus part of the anaerobic metabolism in
a surface layer of indeterminate thickness. At present the only practical
way to estimate total aerobic and total anaerobic metabolism in sediments
is to combine the rates of respiratory oxygen uptake by undisturbed
sediment cores with estimates of anaerobic metabolism derived from
dehydrogenase assay of subsurface sediment layers.
The rate of oxygen uptake by the sediment, however, remains a useful
index of equilibrium conditions among the various factors that affect this
rate: oxygen tension, temperature, salinity, turbulence, available
metabolizable energy, size and composition of the community, compactness
and porosity of sediments and perhaps more. As sedimentation rate of
oxidizable organic matter increases, e.g. in cases of organic pollution
and eutrophication, anaerobic metabolism becomes a relatively more
important process in the mineralization of organic matter in sediments.
In this situation, the estimation of anaerobic metabolism by the
dehydrogenase assay technique is particularly desirable.
This report was submitted in fulfillment of project 16070 EKZ under the
partial sponsorship of the Environmental Protection Agency.
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CONTENTS
Page
Abstract i
List of Figures iii
List of Tables v
Acknowledgments vii
Sections
I Conclusions 1
II Recommendations 3
III Introduction 5
IV Methods 10
V Results 20
VI Discussion 76
VII References 81
VIII Appendices 85
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FIGURES
No.
1 The multiple corer shown in different stages of opera- 11
tion.
2 Cross-section of microcalorimeter and environmental- 16
temperature-control system.
3 Chart of Lake Washington showing 10-m isobaths and 18
location of stations.
4 Chart of Puget Sound showing location of 33 stations. 25
5 Total oxygen uptake versus temperature at various sta- 27
tions in January.
6 Total oxygen uptake versus temperature at various 28
stations in July.
7 Predicted rates of total oxygen uptake and chemical 31
oxidation at station 1 as functions of temperature and
season.
8 Predicted rates of total oxygen uptake and chemical 32
oxidation at station 23 as functions of temperature and
season.
9 Seasonal cycle of in situ total oxygen consumption, 33
chemical oxidation, respiration, oxygen tension,
salinity, and temperature at station 1.
10 Seasonal cycle of in situ total oxygen consumption, 34
chemical oxidation, respiration, oxygen tension,
salinity; and temperature at station 23.
11 Rates of total oxygen consumption at 10°C in January and 36
July versus organic carbon content of the sediment.
12 Calibration line for the microcalorimeter at 9.9 C and 45
19.5°C
iv
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No. Page
13 Dehydrogenase activity as a function of sodium 49
citrate concentration.
14 Dehydrogenase activity at 0. 04 M sodium citrate as 50
compared to dehydrogenase activities at 0.20 M sodium
citrate and without added substrate.
15 Effect of substrate concentration on the absorbance of 51
mercuric-chloride-treated blanks.
16 Dehydrogenase activity as a function of amount of sediment 52
of two different levels of metabolic activity.
17 Dehydrogenase activity as a function of the rate of 54
metabolic heat release.
18 Vertical distribution of dehydrogenase activity at six 56
stations in Lake Washington in October 1972.
19 Vertical distribution of metabolic heat release at six 58
stations in Lake Washington in October 1972.
20 Concentration of reduced substances in Puget Sound 61
versus depth of sediment layer.
21 Concentration of reduced substances versus depth 62
of sediment layer off the Oregon-Washington coast.
22 Vertical distribution of the concentration of reduced 63
substances in Lake Washington sediments.
23 Rates of inorganic chemical oxidation of undisturbed 65
sediment cores versus average concentration of reduced
substances in the 0-1, 1-2, and 3-4 cm layers.
24 Effect of tidal current during ebb tide on the gradient of 70
oxygen concentration above the bottom at the entrance to
Port Madison (122°30.2'W, 47O43.5'N, 104 m depth).
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TABLES
No.
1 Depth and measured parameters of the sediment at 21
different stations in Puget Sound.
2 Seasonal changes in dissolved oxygen (ml/liter), tempera- 22
ture (°C), and salinity (°/oo) of bottom water at different
stations in Puget Sound.
3 Analysis of variance to determine effect of stations, 30
temperature, and season on rates of total oxygen uptake
at 23 stations.
4 Analysis of variance to determine effects of stations, 37
temperature, and season on rates of oxygen consump-
tion by abiotic chemical oxidation at 23 stations.
5 Total oxygen uptake, inorganic chemical oxidation, and 40
respiration at stations 24 to 33 in Puget Sound.
6 Dissolved oxygen, temperature, salinity, total carbon 42
and sand fraction at stations 24 to 33 in Puget Sound.
7 Relative levels of dehydrogenase activity resulting from 47
the use of the same concentration of different substrates
added to replicate sediment samples from Lake Washington.
8 Comparisons of dehydrogenase activity converted to 59
metabolic heat release by means of the regression equa-
tion with rates of total oxygen uptake and inorganic
chemical oxidation converted to calories per core per hr
by means of the factor 4.8 calories equal 1 ml of oxygen
consumed.
9 Total oxygen uptake, inorganic chemical oxidation, and 68
respiration in Clam Bay at the site of the fish rearing
pens.
10 Oxygen debt of the sediment in Clam Bay at the site of the 72
fish rearing pens.
11 Oxygen debt (ml 02/ml sediment) of the sediment in 73
Clam Bay at the site of the fish rearing pens in
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No. PEE
February.
12 Rates of total oxygen uptake, inorganic chemical 74
oxidation and respiration (ml 02 g "^hr"1) per cent
organic carbon, and nutrient concentration of overlying
water (u g-at 1 ) during a 43-day experiment.
vii
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ACKNOWLEDGEMENTS
The financial support by the U. S. Environmental Protection Agency and the
scientific interest in and endorsements of the project by Dr. Milton H.
Feldman are gratefully acknowledged.
We appreciate the shipboard assistance by Skippers G. Drewry, M. Popp,
C. Short, and T. Styron of the Department of Oceanography's research
vessels HOH and ONAK.
viii
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SECTION I
CONCLUSIONS
In situ oxygen uptake by the sediment can be estimated by shipboard measure-
ments with sufficient accuracy.
The rate of total oxygen uptake by the sediment represents the sum of aerobic -A
plus anaerobic metabolism in a surface layer only of indeterminate thick-
ness.
In situ rates of anaerobic metabolism in the sediment column can be determined
by means of a TTC method of total dehydrogenase assay.
In Lake Washington, which has had a long and well-documented history of
eutrophication and deposition of sewage effluent, anaerobic metabolism by ~fc
bacteria alone in the sediment column far exceeds total metabolism as
estimated by the rate of total oxygen uptake by undisturbed cores.
As the rate of sedimentation of organic matter increases, e.g. as a ^
consequence of eutrophication or organic pollution, anaerobic metabolism
becomes a relatively more important process in the mineralization of
organic matter in sediments.
Benchmark measurements of oxygen consumption by the sediment are
useful indices of equilibrium conditions among the various factors that
affect the rate of uptake, such as oxygen tension, temperature, salinity,
turbulence, available metabolizable energy, size and composition of the
community.
Benthic community metabolism decreases with decreasing supply of 71
oxidizable organic matter.
Accumulated reduced products of anaerobic metabolism may be measured
by a dichromate oxidation technique.
Reduced end products of anaerobic metabolism near the sediment surface ^
are in a state of dynamic equilibrium between the rate of formation and
the rate of oxidation.
*&•
Below a few centimeters reduced end products of anaerobic metabolism
are no longer effectively oxidized; they accumulate, as shown by an
increase in concentration with depth of sediment layer.
If sediment layers can be dated, such that the time they are removed from
1
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the surface zone of oxidation can be ascertained, then total anaerobic
metabolism from that time can be determined from the concentration
of reduced substances in that layer.
There is no easy single method for measuring total benthic community
metabolism in the sediment column. Direct calorimetry may be the only
means of measuring aerobic plus anaerobic metabolism of undisturbed
sediment cores, but it does not look promising for field studies.
At present the only practical way to estimate total aerobic and anaerobic
metabolism in sediments is to combine the rate of respiratory oxygen
uptake by undisturbed sediment cores with estimates of anaerobic
metabolism derived from dehydrogenase assay of subsurface sediment
layers.
It is extremely difficult to quantify the effect of each variable on benthic
community metabolism (e.g. total oxygen uptake) because of factor
interactions.
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SECTION II
RECOMMENDATIONS
The described research has shown the usefulness and limitations of benchmark
information on the rates of oxygen consumption by the sediment. The rate of
oxygen uptake is a characteristic parameter of an area and indicative of
equilibrium conditions. Thus, where conditions that affect the rate of uptake,
e.g. supply of organic matter to the bottom as a result of pollution or
eutrophication, is expected to change, the effect of such changes can be
assessed in terms of changes in benthic oxygen consumption. The rate of
uptake, however, is affected by many other factors, such as oxygen tension,
temperature, salinity, composition of the community, and factor interactions.
For example, acclimatization to seasonal or long-term temperature changes
by some communities and varying degrees of acclimatization make it
difficult to quantify the effect of temperature. In spite of this difficulty,
it is recommended that areas susceptible to organic pollution be subjected
to a study of benthic community metabolism. In addition to usual measure-
ments of benthic oxygen uptake, the following routine field measurements
are also recommended:
1) Anaerobic metabolism in the sediment column to be determined
by its dehydrogenase activity;
2) the concentration of total reduced substances in the sediment
column.
These latter measurements are especially relevant and particularly imperative
in areas designated for dumping of sewage sludge such as the New York Bight,
because they would indicate the degradation of quickly buried organic matter
that-the rates of oxygen uptake alone would not reveal. To better understand
the degradation of organic matter in bottom deposits, more work is called for
on the following problems and I suggest that these problems are in the interest
of the Environmental Protection Agency:
1) Development of a chemical method for the determination of an-
aerobic metabolism of benthic macrofauna, meiofauna, and
microfauna.
2) The quantitative relationship between the level of anaerobic
metabolism in sediments and the resulting accumulation of
total reduced substances.
3) The relationship under a wide range of conditions between
measures of dehydrogenase activity and actual rates of metabolic
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heat release of undisturbed sediments.
4) The measurement of the rate of sedimentation of particulate
organic matter in different areas and its relationship to the
level of benthic community metabolism.
5) Long-term experiments on the quantitative relationship between
anaerobic metabolism and the loss of organic carbon in sediments.
6) The carbon equivalent of total humic fraction of organic matter in
sediments and the relative oxidizability of organic matter containing
various fractions of humic substances.
7) Laboratory experiments on temperature acclimation by benthic
communities.
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SECTION III
INTRODUCTION
The general cycle of elements and their compounds, the biological, physical,
and chemical processes involved therein, and the concomitant flow of energy
through terrestrial and aquatic ecosystems are generally understood. The
biggest gaps in our knowledge concern the rates of the various processes and
the quantitative effect of conditions in nature that affect these rates. It is
now widely recognized that these gaps are critical in considering many aspects
of man's use and management of the earth's natural resources, e.g.
understanding of biological productivity, decisions concerning domestic
and industrial use of waters in streams, lakes, estuaries, and the open
ocean, shoreline management, barging of wastes to the oceans, etc. One -X
of the least understood processes takes place in the basins of bodies of
water, after organic and inorganic materials have settled on the bottom
or been taken up by the sediment. These include the degradation and
mineralization of organic matter and the exchange of substances and by-
products of metabolism between the sediment and the overlying water.
The oxidation of organic matter in the sediment may be measured by a
number of ways: by the rate of decrease in organic matter content during
incubation, by the rate of evolution of carbon dioxide, by different
measures of microbial activity such as uptake of C-14 in labeled organic
substrates, enzyme activity, such as dehydrogenase or oxidase, etc.,
by direct calorimetry and by the rate of oxygen consumption. We chose to
develop the latter technique by virtue of its directness, its sensitivity,
the state of the art in oxygen measuring techniques, its natural involvement
in in situ processes, and its applicability to all aerobes. The interpretation^,
of oxygen consumption by the sediment is, however, far from being
straightforward.
RATIONALE OF DESCRIBED WORK
In situ Measurements-In view of envisioned difficulties and likely sources of
errors in the measurement of oxygen uptake by the sediment, Pamatmat
and Fenton (1967) developed an instrument system for making in situ
measurements to 180 m depth. A disadvantage, besides that of cost, became
evident when results showed no correlation with measured parameters of
the sediment but indicated a significant correlation with bottom water
temperature and a seasonal fluctuation in oxygen consumption rate (Pamatmat
and Banse 1968). The in situ method does not easily allow any manipulation
of water temperature which was necessary if the effect of temperature was
to be isolated from other possible seasonal effects. The problem demanded
shipboard measurements of oxygen uptake which could be done under
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controlled temperature, i:6.-, the sam6 temperatures throughout the year.
Development M a. jjiijpboaM MethOd^A shipboard method requires first Of
all that representative samples Of the benthie community (sediment plus
inhabitants)/ be ^brought aboirdrthe ship Undistefbed; No satisfactory
sampling gear existed at the time arid it was necessary to invent one.
A multiple coring device (^amatmat 19*71a) resulted from this effort and
was subsequently used for taking samples in Puget Sound in all kinds of
sediment, offshore from the continental shelf with hard-packed sand to
the deep Sea With soft oozd, the Aleutian Trench, and in Lake Washington
with its/gyttja type of sedimeht/ 'the coririg device is described in greater
detail together with later modifications in this report, totalled plans for
its construction are to be found in the Appendix.
comparisons of shipboard measurements of oxygen uptake by sediment cores
with in,. sitti measurements have shown that at the same teiriperature the
shipboard method yields the Same e_stimate of total uptake and inorganic
chemical oxidation as the in Situ technique (Pamatniat i971a, 1971b).
Significance of Reduced Substatt.Gjg in $6diments-'>rhe total oxygen uptake by
the seabed is partly due i to respiratory uptake by aerobic organisms and
partly dtie to abiotic 'chemical .oxidation of reduced substances like fef rous >
mangaiibus, sulfides, et6. in 6rder to understand differences in the rate
of abiotic dhemiBal uptake, it is hecessary to measure the concentration of
these reduced substances. A technique for theif estimation was therefore
developed. •••'-',': '/:'- ;'"' '•• .^ "- •^'•••'..;'\ " '-;' "''''' ' ' ': : : '••-.- '•''.'•
Humic Acid .Content olgedt^entsftt is well knOwn that only a fraction of the
organic matter in, Sediments is metaboli^able; the refractory portion of
organic matter has been called humus (Wa^ksmah 1933). onie might expect
that the difference between total organic matter and the humus f rabtion
would therefore represent the amount 'of oxidizable. organic matter, 'This
would be a better naeastir^ of available food to the benthic community.
Effect of Sediments oft Oxygen Tension of ^Bottom Water -The amount of '
reduced substances in the sediment is proportional to its oxygen debt
(Pamatmat l&71b); Mence, one would Expect that the spreading of such
substances in the water -would affect' the cixygen tension. . Observations via
underwater television r'eyealed that the concentration of Suspended particles
in bottom water increased with intettsity of tidal curirents (Pamatmat l97lb) ,
Hence, we attempted. to show1 that resuspenslbn. of -bottom deposits lowers -
the oxygen tension of^ the ;water.. ' ' . '-;•=-,•' V^>:'-< .' , , ••••'• ..-''• •,-•• .'.'••••
pehydrogeMse ! ASsay. for Measuri^^ A^ •
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The problem of clarifying the quantitative relationship between the rate of
abiotic oxygen consumption and anaerobic metabolism in the sediment
requires the direct measurement of anaerobic metabolism. An imposing
difficulty in measuring anaerobic benthic metabolism is the diversity of
metabolic types of heterotrophic bacteria, which could include various
fermenters, nitrate reducers, denitrifiers, sulfate reducers, and methane
bacteria, in addition to anaerobic macrofauna, meiofauna, and microfauna.
Quantitative chemical analysis of metabolic end products would not be
practicable.
We turned to a method of dehydrogenase assay (Lenhard 1956) which
measures the rate of hydrogen production during intermediary metabolism
by means of triphenyltetrazolium chloride (TTC) which, in the absence of
oxygen, reacts with hydrogen to form a red-colored compound,
triphenylformazan (TPF), whose amount is measured colorimetrically.
The concentration of formazan produced is a function of incubation time,
pH, temperature, kind of substrate added if any, substrate concentration,
plus the population density of microorganisms. The relative measure of
metabolic activity was calibrated by direct calorimetry to enable its use
in determining the actual rate of metabolism under natural field conditions.
Direct Calorimetry -The measurement of metabolism by direct calorimetry
circumvents the complexities of dealing with mixed metabolic types
(Forrest et al. 1961). ZoBell et al. (1953) observed that sediment
bacteria liberated enough heat to raise the temperature of organically rich
sediment. Ordinarily; however, very sensitive and expensive micro-
calorimeters (Calvet and Prat 1963) would be required to detect the heat
output by sediment bacteria. We describe here our experience with a
gradient type of microcalorimeter and assess the present outlook for its
use in ecological studies. The microcalorimeter would be indispensable
for calibrating such a chemical method as the dehydrogenase assay.
Laboratory Experiments on Organic Matter Oxidation-In addition to an
overall measure of benthic community metabolism, it is equally desirable
to know the concomitant loss of organic carbon and recycling of nutrient
salts during the mineralization of organic matter. The possibility of
determining these equivalents was examined in a long-term experiment
involving the periodic measurement of oxygen consumption, the carbon
content of the sediment, and the nutrient concentration of the overlying
water.
Sediment and Other Environmental Parameters-Relying on the usual
approach of attempting to explain differences in benthic community
metabolism between places by searching for correlations with possible,
known, and suspected factors we made the following measurements:
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total carbon, organic carbon, organic nitrogen, sand-silt-clay fractions of
the sediment, biomass of macrofauna, temperature, salinity, oxygen
concentration of bottom water, and depth of station. Later on, when
techniques had been developed, the concentration of total reduced substances
was also routinely determined. Humic acid content of sediments was
measured in a few samples. All these measurements and analytical
determinations were of value in a negative way: they served to emphasize
the fact that the rate of benthic oxygen consumption although affected
by some of them was also greatly influenced by other factors.
Unplanned Field Experiment-By the end of the first year of research, it
was evident that a major factor influencing the rate of oxygen uptake by the
sediment was the rate of supply of oxidizable organic matter to the bottom.
Just then, a project on pen-rearing of salmonids was started in Clam Bay.
The aquaculture operation called for artificial feeding of the fish with
formulated pellets. An undetermined quantity of uneaten food plus fish feces
settled to the sediment ~ a clear case of increased rate of supply of organic
matter to the bottom. Rates of oxygen uptake were measured along a
transect of stations running across the floating fish pens. The measure-
ments were repeated after the fish were harvested and the floating pens
had been removed.
Benchmark Survey of 33 Stations in Puget Sound-Even before we were certain
that the rate of total oxygen uptake did not represent total metabolism in the
sediment column, it became apparent that the rate of oxygen uptake was
characteristic of each area studied and probably was a useful overall
indicator of equilibrium conditions among all the prevailing factors at the
time of measurement. It was then imagined that measurements over a
wide area of Puget Sound would be of value in later assessing possible
eutrophication of the estuary as a result of direct discharge of sewage
sludge or indirectly through some physical modification of the body of water.
OBJECTIVES
1) To develop a technique suitable for routine measurements of benthic
community metabolism in large-scale surveys.
2) To develop and adapt analytical methods to the study of sediment parameters
which may lead to better understanding of decomposition of organic matter
in sediments.
3) To evaluate the rate of oxygen uptake by the sediment as a measure of
benthic community metabolism.
4) To perform a base-line survey of benthic oxygen consumption in Puget
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Sound.
5) To develop a method suitable for routine measurements of anaerobic
metabolism in sediments.
6) To perform laboratory experiments that would clarify certain aspects
of benthic community metabolism and material exchange between the
sediment and the overlying water.
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SECTION IV
METHODS
CONSTRUCTION OF CORER
The need for an appropriate sampler is discussed by Pamatmat (1971a) who
describes a multiple coring device that seemed to meet necessary require-
ments of the problem on hand. Detailed plans for the construction of such
a gear are presented in the Appendix.
A full description of the corer's principle of operation is as follows:
While suspended from the winch cable during descent, the cylinder (A,
Fig. la) is full of water. As soon as the frame has settled on the bottom
and the cable becomes slack, the weights (B) force the piston (C) to
descend at a slow speed as regulated by the size of the opening on the
top of the cylinder through which water escapes. The coring tubes (D) at
full penetration are shown in Fig. Tib. When the corer is being pulled off
the bottom, water is sucked in through the same opening and a half-inch
check valve on top to refill the cylinder. When the coring tubes are again
all the way up (Fig. Ic), the external core catchers (E) flip underneath to
seal them. The principle of operation is essentially similar to that of
Craib (1965), which is, however, a single-barreled corer with a more
complicated core catcher. A Niskin bottle (F) attached to the frame is
automatically tripped when a line attached to a descending weight stand
pulls a pin (G) that releases the messenger (H).
The corer is presently used with fiberglass-reinforced epoxy tubes ("green
thread" or "red thread" pipe made by A. O. Smith, Little Rock, Arkansas)
of 5.7-cm inside diameter. They are impermeable to dissolve oxygen and are
of uniform bore. The only objectionable feature of the material is its
opacity; but the sediment surface is seen clearly from the top and, when
held against a light, the wall is translucent enough to allow a check for the
presence of unwanted air bubbles. "Red thread" pipe is thinner, more
translucent, and cheaper.
The tube is attached to the corer by means of a polyvinyl-chloride (PVC)
plastic coupling (Fig. Id). A tight seal is provided by an inside O-ring.
The coring tube is secured to the coupling by means of three set screws
with wing nuts for quick detachment afterwards.
The guides (I) for the tubes serve to seat the core catchers firmly out of the
10
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Figure 1. The multiple corer (a) ready to be lowered, (b) when the piston is
fully extended and the coring tubes are in full penetration, (c)
when the piston and coring tubes are retracted, and with an
external core catcher sealing the bottom of each tube, (d) Close-up
of PVC coupler to which a coring tube is secured by set screws.
11
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way initially. Each catcher, a solid rubber ball (E), is held out of the way
of the tube by a taut stainless steel wire (J) hooked to the lower end of the
piston. As the piston descends, the previously slack upper.section of the
wire (K), which is secured to the bottom plate of the cylinder, gets taut
and pulls the now slack lower section (J) off a hook (L). The total length
of the wire provides enough slack for the rubber ball to flip into place
when the tube is fully retracted into the guide (Fig. lc). Rubber tubings
(M) pull the ball to center and up tight against,the lip of the tube. The lower
end of the coring tube is ideally flush with the edge of the guide; if it extends
too far below, the ball may not flip into place; but if it is too far up, the
ball may not seal tightly.
To insure good flushing of the coring tube during descent, the ball valve (N),
may be suspended by a clip attached to an eye (O) directly above each
weight stand. As the piston continues to descend after the tubes are several
inches into the sediment, the ball is pulled off the clip by a retaining rod
passing through the weight stand. On the ship's deck, the piston is prevented
from descending by means of pins (R).
One later improvement consists of additional weights (100 kg or more) on the
frame. The greater weight of the frame speeds up the retraction of the
piston which must be all the way in before the external core catchers can
seal the bottom of the coring tubes; if the frame is top light, as it was before
adding the additional weights, the gear is some distance above the bottom
during retrieval before the piston is fully drawn back into its cylinder. It
is also thought that lowering the heavier core sampler at high speeds would
be possible without causing it to "plane". The device has been lowered at
speeds of up to 120 m/min.
A later modification involved relocating the drain holes around the rubber ball
that seals the top of the core. In their former location, enough turbulence
was evidently generated when raising the corer at high speed; some flushing
of the enclosed water above the sediment took place.
SHIPBOARD PROCEDURE FOR MEASURING OXYGEN UPTAKE
The method has already been described in detail (Pamatmat 1971a). Briefly,
as soon as the corer is aboard, the cores are transferred to a constant
temperature bath. Each sample is sealed with an oxygen electrode and
stirrer and the concentration of dissolved oxygen in the water is,monitored
for a sufficiently long time until a steady decline in oxygen tension is noted.
Then some of the cores,are poisoned with formaldehyde; the residual
uptake indicated by another steady but .slower rate of decrease in oxygen
tension is considered to be inorganic chemical oxidation. At the end of the
procedure, which may take up to several hours depending upon the rate of
12
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uptake, the water is measured to the nearest milliliter.
Since the rate of oxygen consumption is dependent on oxygen concentration
(Pamatmat 1971a) it is essential that the rate of inorganic chemical
oxidation and rate of total oxygen uptake be determined at the same
concentration, which should be that of the bottom water. To ensure that
there is no significant decrease in the oxygen tension during the
experiment, the volume of enclosed water above the core should be
adjusted to the expected rate of uptake. By using a millivolt stripchart
recorder with zero suppression, it is possible to determine the rate of
uptake accurately from changes in oxygen tension of about 5% of the
original concentration. If the level of metabolic activity is quite high
and the volume of water is relatively small, the rate of decline of oxygen
tension will be so rapid that by the time the rate of chemical oxidation is
being determined there will have been a large difference in oxygen tension
from the initial concentration.
EFFECT OF TEMPERATURE ON THE RATE OF OXYGEN CONSUMPTION
In earlier shipboard measurements in Puget Sound (Pamatmat 1971b) the
effect of temperature was determined by placing a set of replicate cores in
a bath at 5 C, another set at 10 C, and still another set at 15°C. This
requires a large number of replicate cores. In a later work (Pamatmat,
in press), the rate of uptake was determined of the same set of cores at
two temperatures, e.g. at 3 and 8°C, or at 8 and 13°C, but never at 3
and 13°C.
TOTAL REDUCED SUBSTANCES IN THE SEDIMENT
In earlier studies an iodometric method was used to determine total reduced
substances, but problems are associated with the use of iodine as oxidant
(Pamatmat 1971b). The use of potassium dichromate appears to eliminate
the problems (Pamatmat, in press). Comparisons with the actual oxygen
uptake by poisoned sediment shows a highly significant correlation between
oxygen debt and the milliequivalents of dichromate needed to completely
oxidize the reduced substances.
The procedure consists of adding 1 ml of 10 N H2SO and 10 ml of 0. 01 N
(0.02 N with highly reduced sediments) K^C^Oy to 50 ml of distilled water.
The solution, in a bottle train, is stripped of dissolved oxygen by bubbling
with nitrogen gas for about 5 min. Sediment samples are added to the
solution with a special sampler (Pamatmat 1971b) to avoid exposure to air;
the mixture is stirred continuously with a magnetic stirrer for 5 min and
allowed to settle for about 15 min. To a 20-ml aliquot of the supernatant,
1 ml saturated KI is added. After the mixture has been allowed to stand
13
-------
for 5 min the liberated iodine is titrated with thiosulfate. The total reduced
substances determined by this method apparently do not include organic
carbon as highly organic sediment samples that had been oven-dried at
90°C and.finely ground.did :not reduce dichrornate by*this procedure. .It is
possible, however, that in the drying, process volatile organics that would-
have reduced the dichromate had been lost; in-'any case, their amount is
considered negligible'in comparison with the total inorganic reduced
substances;
HUMIC ACID CONTENT OF/SEDIMENTS
This was determined strictly according to the procedure of Lenhard et al.
(1962). For blanks we used ground dried mud that had been combusted
in a furnace at 500°C. Less than 1 g of dry sediment is refluxed with 100
ml of 10% v/v HC1 for 2 hr to destroy carbonates'and hydrolyze non-humic
organic matter. The sample isJfiltered,, the insoluble fraction is washed
free of chloride and heated with 0.5% w/v NaOH for 2 hr on a boiling water
bath. After cooling, the extract is made up to 250 ml and centrifuged for
10 min at 3800 rpm. The optical density of the extract is measured in a
1-cm cell at 436 nm with a Beckman spectrophotometer.
DEHYDROGENASE ACTIVITY, OF SEDIMENT BACTERIA.
During a: cruise in July, following preliminary tests in the laboratory,
dehydrogenase activity-of sediment bacteria-was determined by-the following
procedure:'. Sediment samples were incubated with 5 ml Tris buffer and 5 ml
of triphenyitetrazolium-chloride-glucoser solution at 37°C in a water bath for
one hr..' The-reaction was stopped and the formazan simultaneously
extracted, with absolute iethanol. >The extract was centrifuged before
reading its.optical density at 483 nm. The results seemed to show
significant differences between stations and between layers of a sediment
core. Then it was decided that the effect of incubating at 37°C was difficult
to assess and the incubation had better be done closer to environmental
temperature.
The method finally worked out is an adaptation-of the method of Lenhard et al.
(1965) which was .modified according to results, of laboratory experiments with,
the use of different.substrates and incubation at 10°C.. On the basis of those
findings* the,following concentrations of reagents and procedure were
established: (1) TTC solution -^ Ig of 2, 3-5 triphenyltetrazolium-chloride
in 100 ml of distilled water;-(2) Tris buffer ~ 6. 037;g of tris.(hydroxymethyl)
aminomethane plus 20 ml of 1.0 N HCl in 1 liter of distilled water, pH
adjusted to 8. 4; (3) sodium.citrate solution-T: 74.g of .sodium citrate in 1..Q
liter of distilled-water.; ;(4):saturated;mercuric chloride (HgC1.2) solution; (5)
absolute ethyl alcohol.
14
-------
To each graduated 50-ml Erlenmeyer flask are added 3 ml of Tris bugger,
3 ml of sodium citrate solution, 2 ml of TTC, and 2 ml of sediment. The
total volume is made up to 20 ml with distilled water, the flask is swirled
a few times, and then allowed to stand undisturbed in the dark for 3 hr
at 10 C, Triplicate samples are run. For blanks, duplicate samples are
prepared similarly except for the addition of 1 ml saturated HgCl2- After
3 hr the activity is stopped with 20 ml of absolute alcohol, which also
extracts the formazan. The mixture is shaken ©very 15 min for 1 hr to
complete the formazan extraction. The mixture is centrifuged and the
clear supernatant is read at 483 nm in a spectrpphotometer. The sediment
is dried overnight at 90°C and the optical density of each sample is
normalized to 1 g of dried sediment. The difference between the untreated
and the HgCl2-treated samples represents the dehydrogenase activity of
the sediment.
DIRECT CALORIMETRY
The instrument used is a gradient or conduction type of calorimeter (Evans
1969; Hammel and Hardy 1963; Benzinger and Kitzinger 1963) with 12,000
copper-constantan junctions. Fig, 2 shows a cross-section of the system
arrangement as it was finally used during the experiments. The calorimeter
is buried in sand in the stainless steel vacuum Dewar vessel; the sand
serves as the heat sink. The temperature control system qonsists of the
closed 0. ll-nr* double-walled tank connected to the 0- 19-m^, floor-model,
externally circulating temperature bath. The Forma bath used has a rated
pumping rate of 38 liters/min. The temperature in the bath was constant
to within 0. 02°C with no drift. During the experiments the water bath was
set at 10°C. The calorimeter was electrically calibrated as described by
Berger (1969).
The procedure for direct calorimetry of sediment samples was as follows:
The sediment sample is packed inside a 60-ml, screw-capped culture tube.
This tube is wrapped in plastic and immersed in the water bath. After
temperature equilibration, the dry culture tube is quickly transferred to the
calorimeter. After the calorimeter restabilizes (at least 12 hr) the sample
is removed. The empty calorimeter restabilizes much more rapidly (4 hr)
and the decrease in thermal emf following removal of the sample represents
the rate of metabolic heat release.
In calibrating the dehydrogenase assay by direct calorimetry, the assay was
performed immediately or within a few hours after the sample was removed
from the calorimeter.
15
-------
POLYURETHANE
FOAM PLUG
_ S' x POLYURETHANE
^ / FOAM
AIR SPACE
STYROFOAM
PLUG
CALORIMETER
DEWAR VESSEL
AIR SPACE
Figure 2. Cross-section of microcalorimeter and environmental-temperature-
control system.
16
-------
COMPARISON BETWEEN OXYGEN UPTAKE BY CHEMICAL OXIDATION AND
ANAEROBIC METABOLISM IN THE SEDIMENT COLUMN AS DETERMINED
BY DEHYDROGENASE ASSAY
Samples were taken from 19 stations (Fig. 3) in Lake Washington with the
multiple corer. Oxygen uptake by the intact cores from all stations was
measured immediately at 10°C while replicate cores from five stations were
sectioned and some layers were assayed for dehydrogenase activity on
board ship. The core from a sixth station (station 19) was stored at 10°C
in the dark for one week before it was assayed for dehydrogenase activity.
Each layer, except the already liquid 0-1 cm layer, was mixed with a small
amount of distilled water to facilitate homogenization and measurement of
replicate samples. These layers also were analyzed for concentration of
reduced substances by the dichromate oxidation procedure.
LABORATORY EXPERIMENTS
A preliminary experiment to work out a method for determining the relation-
ship between oxygen uptake, loss of organic carbon from the sediment, and
liberation of nutrients was conducted over a two-month period. In order to
eliminate any complication with anaerobic metabolism and anoxic conditions
in the sediment, a small amount of sediment was allowed to settle in a thin
layer on the bottom of a 250-ml Erlenmeyer flask. A large batch of
sediment was mixed well into an homogeneous paste; all discernible large
particles, e.g. broken shells, macrofauna, gravel, were removed. Then
2 ml of the slurry was distributed into each of 20 replicate flasks which were
slowly filled with Millipore-filtered sea water and allowed to stand until the
water had cleared completely, except for two randomly picked flasks whose
oxygen uptake was determined within a few hours. The rest were kept in a
cold room at 9. 5°C and bubbled with air which had been bubbled through a
larger flask of water. On designated dates replicate flasks were picked at
random for determination of oxygen uptake, both total and residual uptake
after poisoning with HgCl2, the nutrient content of the water, and the
organic carbon content of the sediment.
GRADIENT OF DISSOLVED OXYGEN ABOVE THE SEDIMENT
During previous cruises to a station at the entrance to Port Madison (Fig. 4),
an increase in suspended sediment in the bottom water during ebb tide was
observed with underwater television. This resuspension of sediment could
increase jn_situ benthic oxygen uptake, which could be reflected by a tidal
change in the vertical distribution of oxygen in the bottom water. Electrodes,
each equipped with a magnetic stirrer, were mounted on a vertical angle
iron at 3, 33, and 93 cm above a broad plastic plate attached to the bottom
end of the angle iron. The whole mount was loosely fixed to a tripod (the
same one used in the in situ experiments on benthic metabolism and
described by Pamatmat and Fenton, 1968) so that when the tripod landed on the
17
-------
LAKE WASHINGTON
-45
-35'
4/°|-30
19'
15'
10'
Figure 3. Chart of Lake Washington showing IQ-m isobaths and location of
stations.
18
-------
bottom, the plastic plate rested on the sediment surface and as the tripod
settled into the soft bottom, the vertical angle iron was free to slide up.
Hence the lowermost electrode was positioned as close to the bottom as
possible without getting buried. The three electrodes were monitored
continuously with a multipoint recorder.
SEDIMENT AND WATER ANALYSES
The sediment size analysis was done according to standard geological methods
(Krumbein and Pettijohn 1938). The determinations of total and organic carbon
were made with the LECO analyzer (Laboratory Equipment Co., Michigan).
Organic nitrogen was determined by the COLEMAN analyzer (Coleman
Instrument Co, , Illinois).
Five-cm layers of the sediment cores (0 to 5 cm, etc.) were squeezed with
Reeburgh's (1967) device, and the interstitial water samples were analyzed
for dissolved phosphate, nitrate, silicate, and ammonia with an
AUTOANALYZER using an adaptation of the method of Armstrong et al.
(1967) for nitrate and silicate, by the method of Murphy and Riley (1962) for
phosphate, and by Koroleff's (1970) method for ammonia. When nutrient
values were very high, the samples were diluted with filtered sea water of
known nutrient concentration and re-analyzed.
Dissolved nutrients in all other samples were determined according to the
foregoing methods. All samples were frozen after addition of a few drops
of saturated mercuric chloride (HgCl2) and stored in the freezer for up to
three months before they could be analyzed.
19
-------
SECTION V
RESULTS
ENVIRONMENTAL PARAMETERS
The location of 33 stations in Puget Sound are shown in Fig, 4. All the
measured parameters at the first 23 stations are shown in Tables 1 and 2.
The variations in depths of stations with each cruise, corrected to mean
lower low water when greater than 3 m, are perhaps indicative of
navigational error, which is estimated to be a radius to 200 m. Some
stations are located on a slope (station 12), or at the bottom of a small
depression (stations 15 and 19), while others are on relatively broad flat
bottom (stations 8 and 9).
The mean organic carbon content of the sediment in the 23 stations during
5 cruises ranged from 0. 4 to 3.7% of dry sediment. Carbonate-carbon
was present in a significant amount only at station 13; at all other stations
organic carbon was not significantly lower than total carbon values. There
was no detectable seasonal change in the organic carbon content of the
sediment surface, nor was there any difference between the organic carbon
content of the 0-1 and 5-6 cm layers. Organic carbon and organic nitrogen
are strongly correlated (r = 0.97) with the following regression equation:
% organic nitrogen = 0.105 x % organic carbon - 0. 002%,
which signifies essentially a C:N ratio of 10.
A significant correlation (r = 0.89, d.f. = 21) was found between organic
carbon and percent silt plus clay (and therefore significant negative
correlation between organic carbon and percent sand), but there is considerable
variability indicating; heterogeneity of the samples or their sources in terms
of other factors. There are deep as well as shallow sandy stations so that
the sediment type is not strictly a function of depth.
At nearly all stations, salinity dropped from October 1969 to January 1970
and still further to the lowest values in April; then it increased again until
October 1970, The bottom water was significantly more saline in October
1970 than in October 1969. The seasonal change is small and is presently
considered of negligible importance as a physiological factor in benthic
community metabolism.
With few exceptions, temperature was lowest in January and highest in July.
20
-------
Table 1. DEPTH AND MEASURED PARAMETERS OF THE SEDIMENT AT
DIFFERENT STATIONS
Station
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
Depth
range
7-11
10-13
25-29
45-55
28-33
25-29
74-76
14-14
204-209
187-189
250-252
181-221
10-13
10-12
36-41
31-35
206-229
187-204
259-299
221-224
204-241
118-121
177-182
Totaia
c arbon
2,85
2.95
1.90
1.30
0.80
2.60
2.45
2.40
2.50
2.45
2.00
0,20
4,45
3.70
2.65
0.90
2.15
2.10
1.55
0.40
1.95
2.40
2,55
i
Organic3-
carbon
2.65
2.95
1.90
1.30
0.65
2.60
2.30
2.40
2.50
2.45
1.85
0.20
2.80
3.70
2.50
Carbonatea
carbon
0.20
0
0
0
0.15
0
0
0
0
0
0.15
%Sand
10
5
87
41
63
5
3
14
4
13
53
0 ! 96
1.65
0
0.15
0.90 JO
1.85
1.85
1.40
0.40
1.85
2.25
2.35
0,30
0.25
0.15
0
0.10
0.15
0.20
7
17
54
4
9
41
87
10
2
2
-._ .--*._ — _
1
%Silt
68
78
8
47
28
70
70
68
68
61
34
2
73
64
39
70
66
48
9
67
68
67
%Clay
23
17
5
12
9
25
27
17
28
26
13
2
20
19
7
26
25
11
4
23
29
31
aAverage percent of dry sediment.
21
-------
Table 2. SEASONAL CHANGES IN DISSOLVED OXYGEN tail/tfK?.r), TEMPERA'
TUBE (°C) AND SALINITY (°/oo) OF BOTTOM VVAT.r'iJ A'I THE DIF-
FERENT STATIONS.
Station
1
2
3
4
5
6
r?
t
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
Oct
4.50
4.20
4.25
4.00
4.40
3.75
3.90
3.90
3.55
3.95
3.95
4.20
5.50
3.90
3.95
3.90
3.95
3.95
3.70
3.00
Dissolvec
Jan
6.55
5.95
5.90
5.20
6.15
5,55
5.25
5.15
5.80
5.50
6.00
6.10
5.20
5.30
6, 10
5.20
5,60
2.70
-(. 20
1 oxygen
Apr
7.15
6.65
5.85
5.95
5.40
5.50
5.45
5 . 45
5.45
6,30
6.60
6.25
5.50
5.45
5, 60
5.35
5.10
1.60
2.55
Jul
6.50
4.35
5.85
5.45
6.15
4.70
4.55
4.65
4.60
4.65
4.60
5.75
5.80
6.60
5.80
4.50
4.65
6.30
4.80
4.10
4.60
2.65
':! ., u l )
Oct
5.55
4.85
4.95
5.10
4.65
4.45
3.95
4.95
4.50
4.35
4.80
4.65
5.05
4.95
4.40
4.75
4.20
4.65
4.75
4.25
3.35
3.80
22
-------
Table 2 (continued). SEASONAL CHANGES IN DISSOLVED OXYGEN (ml/liter),
TEMPERATURE (°C) AND SALINITY (%o) OF BOTTOM
WATER AT THE DIFFERENT STATIONS.
Station
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
Temperature
Oct
14.60
13.28
13.12
12.40
12.39
13.26
12.10
11.94
11.04
10.73
10.54
11.50
12.97
12.37
10.41
10.36
10.36
10.44
10.43
11.04
Jan
7.74
8.22
8.66
8.61
8.46
8.82
8.54
8.32
8.46
8.30
8.05
8.26
8.54
8.28
8.20
8.76
8.30
8.84
9.44
Apr
9.23
8.92
8.75
8.67
8.68
8.88
8.47
8.47
8.46
9.24
9.64
8.94
8.69
8.45
8.46
8.40
8.40
9.23
9.60
Jul
15.62
13.49
13.29
12.04
12.85
12.18
11.57
12.40
10.00
10.56
10.72
12.29
13.84
15.39
12.46
11.14
10.74
10.66
10.77
10.88
10.67
9.08
9.18
Oct
12.56
12.44
12.29
11.93
11.92
12.33
11.82
11.58
10.72
10.20
11.15
11.92
11.45
11.30
11.04
10.49
10.62
9.94
9.95
9.92
10.76
10.14
-------
Table 2. (continued) SEASONAL CHANGES IN DISSOLVED OXYGEN (ml/liter),
TEMPERATURE (°C) AND SALINITY (°/oo) OF BOTTOM
WATER AT THE DIFFERENT STATIONS.
Station
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
Salinity
Oct
28.99
29.65
29.91
29.83
29.59
30.07
30.15
'30.66
30.65
30.69
30.38
29.76
29.50
30.79
30.85
30.83
30.70
30.78
30.69
Jan
28.40
29.06
29.60
29.29
29.32
30.12
30.16
30.12
29.47
28.94
29.68
30.16
30.24
29.86
30.20
30.40
30.47
Apr
27.33
28.26
28.64
28.74
28.88
29.03
29.94
29.95
30.02
28.76
28.58
28.97
29.36
30.10
30.08
30.25
30.36
29.97
30.37
Jul
29.14
29.27
29.44
29.57
29.61
29.45
29.60
29.63
30.22
30.39
30.46
29.80
29.76
29.45
29.56
30.00
30.51
30.51
29.80
30.49
30.48
30.08
30.16
Oct
29.80
30.09
30.18
30.36
30.36
30.30
30.48
30.57
30.86
31.11
30.63
30.45
30.15
30.44
30.79
30.96
30.86
31.29
31.24
31.29
30.91
31.04
24
-------
Figure 4. Chart of Puget Sound showing location of 33 stations. X marks the
location of station where the effect of scouring on oxygen tension of
bottom water was observed.
25
-------
In October 1969, it was significantly higher than in October 1970. The effect
of temperature is discussed more fully later.
There was some variation in the seasonal cycle of oxygen concentration in the
different stations, some stations having the highest concentrations in January
and others in April, but in general the values dropped in July and October.
The oxygen tension of the bottom water in October 1970 was significantly
higher than in October 1969; this may be related to the significantly lower
temperatures in October 1970. The shallowest stations had the highest oxygen
tension, station 1 becoming supersaturated in April. Stations 22 and 23,
being located behind threshold sills, had the lowest oxygen tension and
exhibited a reverse seasonal'cycle. The oxygen data indicate flushing of the
bottom water at both stations between April and July and again between
July and October. The influence of the seasonal cycle in oxygen tension of
the bottom water on benthic oxygen consumption is discussed later.
TOTAL OXYGEN CONSUMPTION
The rates of measured total oxygen uptake in Puget Sound during the year
ranged from 4 to 56 ml 02/m2 per hr. The average rates at each of the
stations where measurements were made at three temperatures are shown
for January and July (Figs. 5 and 6). There is a significant regression of
standard deviations of the rates of uptake versus the means (P <0.001) as the
following regression equation shows:
standard deviation = (0.147 x mean rate of uptake) + 0.160.
The variability between replicate measurements at each temperature increased
with increasing rates but remained reasonably small. However, the data
show a fairly high variability in the apparent effect of temperature on the rates
at each station. In January, for example, stations 4 and 7 show no
significant effect between 5 and 10°C but show a greater temperature coefficient
between 10 and 15°C than the other stations. Ordinarily this type of data is
considered as evidence for temperature adaptation between 5 and 10°C and
lack of adaptation between 10 and 15°C, e.g. the findings of Duff and Teal
(1965) regarding the rates of gas exchange by the intertidal sediment at
different latitudes. While there may be certain interesting differences
between stations that would account for apparent differences in their QlO's of
oxygen consumption, these differences may be attributed to chance until they
can be verified. The seasonal change is evident from the increases in the
elevation of the various curves from January to July.
Because of the significant regression of standard deviations on the means, an
analysis of variance to determine any significant differences between stations,
seasons, and temperatures was performed after logarithmic transformation
of the data. The results of the analysis show significant differences between
26
-------
24
20
CD
GL
CM 16
E
UJ
CL
=) 8
<
O
10
TEMPERATURE (°C )
15
Figure 5. Total oxygen uptake versus temperature at various stations in
January.
27
-------
!0
TEMPERATURE (°C )
15
Figure 6. Total oxygen uptake versus temperature at various stations in
July.
28
-------
stations, seasons, and temperatures (Table 3). One output of computer
program BMDX 64 (general linear hypothesis, version of July 27, 1965,
Health Sciences Computing Facility, University of California at Los Angeles)
which was used in the analysis of variance is a matrix of predicted rates
of uptake for each station, each season, and each temperature (Appendix). These
predicted rates are summarized for station 1 (Fig. 7) and station 23
(Fig. 8), which have the highest and lowest rates, respectively. The 95%
confidence limits are predicted rates multiplied or divided by 1.17.
In effect, the computer program has extracted for all the stations together the
average trend of the seasonal cycle and of the relationship between uptake rates
and increasing temperature; hence, only the magnitude of the changes differs
between stations. The calculated QIQ'S are 2.3 between 5 and 10°C and 1.5
between 10 and 15°C. The seasonal trend as shown in Figs. 7 and 8 is an
increase from January to April to a maximum in July and a decrease to
October to a minimum in January. The rates were the same in October 1969
and October 1970 in all stations except 4 and 6, where the rates were
significantly lower and higher, respectively, in 1970. Nevertheless, station
6 still showed a decrease from July to October 1970. Note that the seasonal
cycle described is of the rates versus temperature curves. From these four
seasonal curves for each station, the in situ rates could be read against the
actual temperature of the bottom water each season, resulting in the actual
seasonal cycle in situ in each station as depicted in Figs. 9 and 10 for
stations 1 and 23. The effect of temperature was to increase the amplitude
of the cycle in total uptake.
From the results of the long-term experiments on partitioning (Pamatmat
1971), a positive effect of higher oxygen tension of the water on the rates
during January and April is to be expected, i.e., if the experiments in
January and April had been conducted at the same oxygen tension as in. July
and October, the rates would have been even lower than they were. However,
the effect of oxygen tension on the rate of uptake has not been adequately
studied. All cores showed steady rates of uptake for at least 2 hr, during
which period the oxygen tension dropped by several tenths of a ml/liter.
Over longer periods the rates declined slowly, evidently depending on the
relative rates of respiration and chemical oxidation. The effect of the
1-2 ml/liter difference in oxygen tension between summer and winter can
be quite large.
The significant differences between stations in total uptake still cannot be
explained fully in terms of oxygen tension, temperature, or sediment properties.
There are suggestive trends of decreasing rates with depths of stations and
increasing sand fraction, and of increasing rates with increasing carbon content
of the sediment or increasing silt and clay fraction; however, none of these
correlations is significant, indicating the influence of one or more factors not
related to these measured parameters.
29
-------
Table 3. ANALYSIS OF VARIANCE ON MEASUREMENTS OF TOTAL UPTAKE
(LOGARITHMICALLY TRANSFORMED DATA)
Source
Mean
Temperature
Season
Station
Error
Sum of Squares
1942.83
28.79
8.69
38.42
40.97
Degrees of
Freedom
1
2
3
22
558
Mean Square
1942.83
14.40
2.90
1.75
0.073
Variance
Ratio (F)
26462.57
196. 07a
39.47a
23.79a
aP<0.01
30
-------
CD
Q.
CM
30
— 26
22
o
o
z
Id
CD
X
O
o
l-
Q- 20
S
CO
16
12 —
OCT , TOTAL UPTAKE
JAN.
APR
-A- JUL
m- OCT., CHEMICAL OXIDATION
"T_ JAN.
••- APR.
•A- JUL.
10
TEMPERATURE (°C 1
15
Figure 7. Predicted rates of total oxygen uptake and chemical oxidation at
station 1 as functions of temperature and season. The differences
in elevation between regression lines represent seasonal changes
independent of temperature effect.
31
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_£
H
9 10
h-
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2
UJ
§ 2
OCT., TOTAL UPTAKE
JAN. " "
APR.
JUL.
OCT., CHEMICAL OXIDATION
JAN.
TEMPERATURE
Figure 8. Predicted rates of total oxygen uptake and chemical oxidation at
station 23 as functions of temperature and season. The differences
in elevation between regression lines represent seasonal changes
independent of temperature effect.
32
-------
32
28
24
O)
Q.
20
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2
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=)
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— TEMPERATURE
>~ SALINITY
- OXYGEN
~ IN SITU TOTAL UPTAKE
IN SITU CHEMICAL .OXIDATION
IN SITU RESPIRATION
OCT
JAN
APR
JULY
OCT
1969
1970
Figure 9. Seasonal cycle of in situ total oxygen consumption, chemical
oxidation, respiration, oxygen tension, salinity, and temper-
ature at station 1.
33
-------
O)
Q.
9 8
TEMPERATURE
SALINITY
OXYGEN
IN SITU TOTAL UPTAKE
IN SITU CHEMICAL OXIDATION
IN SITU RESPIRATION
cc
z> "—
d
I- 2:
a:
u
— o
o
OJ
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31 5
JULY
OCT
1969
1970
Figure 10. Seasonal cycle of in situ total oxygen consumption, chemical
oxidation, respiration, oxygen tension, salinity, and tempera-
ture at station 23.
34
-------
The situation in Puget Sound is depicted in Fig. 11. In January at 10°C,
total oxygen uptake was almost significantly correlated with organic carbon
concentration (r = 0.53 versus r = 0.57 at P0g 05). Part of the variability
here is probably due to differences between stations in the oxygen concentra-
tion of the bottom water (Table 2). An eye-fitted regression line for the
January data would have a high positive Y-intercept and a rather flat slope.
Since it is reasonable to assume that in the complete absence of organic
matter in the sediment there cannot be metabolic activity, the regression line
should pass through the origin. As for the slope, there is no theoretical basiE
for predicting it although the indicated low increments of oxygen consumption
with increasing organic matter content of the sediment conform with the now
common notion that much of the organic matter in the sediment is not only
nonliving but refractory to biochemical oxidation.
The same scatter plot for July at 10°C shows a much increased variability.
At the same temperature, but generally lower oxygen tension, the rates
were almost invariably higher in July; if the experiments in July had been
run at the same oxygen tension as those in January, the rates would have been
even higher. It is interesting that the shallow stations (arbitrarily designated
as those less than 100 m deep) increased by an average of 9 ml/m per hr
from January to July, while the deep stations on the average increased by only
3 ml/m2 per hr. This difference between shallow and deep stations is
statistically significant (P<0.01). As will be discussed later, the increased
scatter in July may be attributed to different rates of supply of organic matter
to the various stations.
INORGANIC CHEMICAL OXIDATION
What was said about variability in the measurements of total uptake may also
be said about the measurements of chemical oxidation and the effect of
temperature. However, unlike the rates of total oxygen uptake, the standard
deviations in the rates of chemical oxidation are much smaller and are
independent of the means. The average standard deviation is 1.01 ml/m
per hr. The analysis of variance was therefore done on the untransformed
data, giving the results shown in Table 4. There are highly significant
differences between stations, seasons, and temperatures. The predicted
rates are shown in Figs. 7 and 8 for stations 1 and 23. The 95% confidence
limits are predicted values ±1.9 ml/m2 per hr. It is clear that chemical
oxidation is very much less variable than total oxygen consumption.
The curves of chemical oxidation versus temperature show that the rela-
tionship cannot be described in terms of the usual temperature coefficient
of metabolic rates. The curves are parallel and would indicate decreasing
Q-. Q'S with increasing elevation. In fact the relationship shows that chemical
oxidation is a linear function of temperature. It appears to increase by 0.08
35
-------
28
24
20
CD
Q.
CM
e
16
h-
o
8
0
6
II
,14
TOTAL UPTAKE AT 10 °C IN JANUARY
TOTAL UPTAKE AT 10 °C IN JULY
0
1234
ORGANIC CARBON (% of dry sediment)
\
7
/-J
13
Figure 11. Rates of total oxygen consumption at 10°C in January and July
versus organic carbon content of the sediment.
36
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Table 4. ANALYSIS OF VARIANCE ON MEASUREMENTS OF CHEMICAL
OXIDATION (UNTRANSFORMED DATA)
,1... — —~ — .-. ...
Source
Mean
Temperature
Season
Station
Error
Sum of Squares
10195.50
623.35
76.47
1008.64
1085.26
Degrees of
Freedom
1
2
3
22
301
I
Mean Square
10195.50
311.68
25.49
45.85
3.6055
Variance
Ratio (F)
2827.75
86.44a
7.07a
12.72a
0.01
37
-------
ml 02/in2 per hr for each degree centigrade increase in temperature from
5 to 15°C. The unusual temperature effect on chemical oxidation rates may
be attributed to the fact that the chemical reactions take place within the
sediment and are controlled by diffusion as well.
The seasonal cycle is similar to that of total uptake, increasing from January
through April to a maximum in July and decreasing through October to a
minimum in January. The seasonal cycle is likewise enhanced by the
temperature cycle (Figs. 9 and 10),
The significant differences between stations will be discussed in connection
with the concentration of reduced substances in the sediment.
RESPIRATION
The greater portion of variability in total oxygen uptake is obviously variability
in respiration. The rate versus temperature curves of the different stations
are even more variable than those of total uptake. The analysis of variance
for respiration could have been done on the actual differences between measured
total uptake and its corresponding chemical oxidation. Instead, since there were
many values of total uptake without corresponding values of chemical oxidation,
the analysis of variance was performed on the differences between actual
total uptake and predicted chemical oxidation corresponding to that station,
temperature, and season. This is justified by the accuracy of the predicted
values of chemical oxidation. The analysis of variance was done on log-
transformed data since their standard deviations have a significant regression
on the means, with the following equation:
standard deviation = (0.211 x mean) + 0.877
The analysis of variance showed significant differences between stations,
seasons, and temperatures. The seasonal trend is the same as that shown
by both total uptake and chemical oxidation. The seasonal cycle of in situ
respiration is shown as the difference between the total uptake and chemical
oxidation curves (Figs. 9 and 10). There is a highly significant correlation
between respiration and inorganic chemical oxidation at 5OC (r = 0.34,
d.f. = 34), 10°C (r = 0.47; d.f. = 74), and 15°C (r = 0.56; d.f. = 43).
However, there were certain cruises whose results (grouped separately
into the three temperatures) were not significantly correlated.
The much greater variability in respiration suggests that possibly the 5.7-cm
corer is less than the optimum size for sampling the aerobic organisms.
Since the 27~cm diameter bell jars do not show any less variability in their
estimates of oxygen consumption than the cores, it is presumed that the two
methods have different sources of variability (Pamatmat 1971).
38
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OXYGEN UPTAKE AT OTHER STATIONS
Ten more stations (stations 24-33, Fig. 4) were studied seasonally in 1971-72,
but all measurements were done at 10°C only. The data are presented in
Table 5 while dissolved oxygen, temperature, salinity, total carbon, and sand
fraction are given in Table 6. They clearly show a seasonal cycle except that
stations 29 and 30 in the Snohomish river remained high in oxygen uptake from
September to February and showed a sharp drop in metabolic activity from
February to July, while station 27 at the mouth of the Puyallup river showed
a dramatic increase from February to July 1972 to an average value much
greater than that of July 1971. None of the environmental parameters appear
to explain these changes (see HUMIC ACID CONTENT OF SEDIMENTS).
Whereas most stations with soft sediment show that aerobic respiration is
smaller, sometimes much smaller, than the rate of chemical oxygen uptake,
station 29 indicates that sandy sediment in shallow water that is washed by
currents have relatively much higher aerobic respiration and correspondingly
low concentrations of reduced substances. In inland and estuarine waters,
however, this will be the exception. They are to be observed in shallow coastal
areas exposed to surf and tidal currents (Smith 1970; Smith et al. 1971),
DIRECT MICROCALORIMETRY
The results of the electrical calibration of the calorimeter at 10 and 20°C is
shown in Fig. 12. The thermal emf is proportional to power or rate of heat
production. Furthermore, the calibration factor of 0.62 x 10~6 cal/sec per
microvolt is independent of temperature.
At low rates of heat production the high sensitivity of the calorimeter was not
enough to produce reliable results at all times. It is subject to baseline
instability as it is affected by erratic changes in room temperature. The
environmental temperature control system was inadequate in insulating the
calorimeter from ambient temperature drifts of more than 2 or 3°C. Under
this condition, the calorimeter exhibited long-term baseline fluctuations of
as much as -10 microvolts. Furthermore, although an empty calorimeter
under constant temperature should show zero emf., the present calorimeter in
its present set-up always showed a baseline above zero, anywhere from a
few tens to a few hundreds of microvolts.
The calorimeter was designed to measure low but steady levels of metabolic
activity. The original plan was to place a whole sediment core (5.7 cm in
diameter and up to 30 cm long) in Its original coring tube. It soon became
evident that such a large thermal mass unduly prolonged equilibration time
to an impractical number of days. In short, the instrument could not be
used for the purpose that it was designed.
39
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Taolo 5, .HATES OF TOTAL OXYGEN UPTAKE, INORGANIC CHEMICAL OXIDATION, AND RESPIRATION
(ml/m2 per hr) AT STATIONS 24 TO 33 IN PUGET SOUND.
1
Station j
24
25
26
27
28
Total Uptake
Chemical oxidation
Respiration
Total uptake
Chemical oxidation
Respiration
Total uptake
Chemical oxidation
Respiration
Total uptake
Chemical oxidation
Respiration
Total uptake
Chemical oxidation
Respiration
July
14.5,12.4,14.8,
10.3,15.1
7.0
7.5
21.2,23.4,20.3,
13.4,12.5
10.3
10.9
42.2,44.8,59.5,
38.1,41.3
19.4
22.8
13.7,11.8,13.4,
11.5
7.4
6.0
16.7,13.9,21.2,
22.2
9.4,9.1
7.3,13.1
September
February
14.2,7.9,8.0,
13.9
9.1,7.0
5.1,1.0
14.5,14.6,12.8
15.0
6.1,9.8
8.4,5.2
25.9,33.2,23.3,
26.4
15.7,13.1
10.2,10.2
11.5,12.1,11.7
13.8
6.6,5.8
5.5,5.9
10.1,8.7,7.5,
5.6
5.3,3.4
3.4,4.1
July
16.7,15.5,12.2,
13.5
6.8,7.7
9.9,5.8
13.4,14.3,15.8
14.4
7.7,7.0
5.7,8.8
47.4,31.7,22.2
22.2
9.5
56.5,37.7,48.6
31.0
18.1,15.3
38.4,22.4
16.8,16.0,15.8
17.6
11.2
5.6
*>•
o
-------
Table 5. (continued) RATES OF TOTAL OXYGEN UPTAKE, INORGANIC CHEMICAL OXIDATION, AND
RESPIRATION (ml/m2 per hr) AT STATIONS 24 TO 33 IN PUGET SOUND.
Station
29
30
31
32
33
Total uptake
Chemical oxidation
Respiration
Total uptake
Chemical oxidation
Respiration
Total uptake
Chemical oxidation
Respiration
Total uptake
Chemical oxidation
Respiration
Total uptake
Chemical oxidation
Respiration
Julv
September
61.2,49.4,48.2
12.3,15.2
48.9,34.2
23.0,23.0
19.4
3.6
7.2,10.3,7.7
7.6
0
11.6,8.6
5.4
6.2
13.2,12.9,7.6
8.6
4.6
February
58.2,86.5,57.9
61.7,
5.0,15.6
52.9,46.1
28.2,30.5,39.5
30.6
7.5,6.9
23.0,32.6
8.1,16.5,13.7
5.9
7.9.9.8
8.6,3.9
7.0,8.5,7.7,4.9
5.9,6.6
2.6,1.1
10.8,12.2,9.9
14.1,
7.8,10.6
July
29.6,37.9,31.1
10.3,16.0
19.3,21.9
6.9,5.7,8.9
7.7
5.5
0.2
8.6,6.5,6.0
6.4
3.8
2.2
13.4,14.1,10.5
15.0
4.0,4.1
6.5,10.9
13.3,12.3,13.6
14.5
8.1,7.8
4.4,3.5 4.2,6.7
I
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Table 6. DISSOLVED OXYGEN (ml/liter) TEMPERATURE (°C), SALINITY (0/00) TOTAL CARBON (%) AND
SAND FRACTION (%), AT STATIONS 24 TO 33 IN PUGET SOUND.
Station
24
25
26
27
Dissolved oxygen
Temperature
Salinity
Total carbon
Sand f raction
Dissolved oxygen
Temperature
Salinity
Total carbon
Sand fraction
Dissolved oxygen
Temperature
Salinity
Total carbon
Sand fraction
Dissolved oxygen
Temperature
Salinity
Total carbon
Sand fraction
July
6.15
10.18
28.34
1.60
37
4.90
9.47
29.88
2.15
15
5.60
9.74
29.15
0.97
57
4.80
8.97
29.85
1.91
15
September
February
5.50
6.91
29.06
0.89
59
6.00
6.70
29.66
2.54
14
7.40
7.12
29.31
1.59
47
6.80
7.36
29.76
0.99
20
July
5.50
10.53
28.38
1.80
35
4.45
9.33
29.66
2.19
21
5.20
9.99
28.91
0.96
66
4.55
9.16
29.58
1.32
17
-------
Table 6. (continued) DISSOLVED OXYGEN (ml/liter) TEMPERATURE (°C), SALINITY (0/00) TOTAL CARBON
(%) AND SAND FRACTION (%), AT STATIONS 24 TO 33 IN PUGET SOUND.
Station
28
29
30
31
Dissolved oxygen
Temperature
Salinity
Total carbon
Sand fraction
Dissolved oxygen
Temperature
Salinity
Total carbon
Sand fraction
Dissolved oxygen
Temperature
Salinity
Total carbon
Sand fraction
Dissolved oxygen
Temperature
Salinity
Total carbon
Sand fraction
July
12.18
28.58
0.03
96
September
4.35
13.59
20.96
0.85
5.55
14.04
6.00
2.50
8.66
29.99
2.99
February
8,65
6.52
28.01
0.45
8.90
3.90
2.12
1.16
85
6.15
3.74
0
0.29
96
3.10
9.14
80,30
3.95
5
July
5.75
11.06
26.86
0.41
53
7.00
13.16
11.54
1.52
63
8.00
10.76
0
0.14
100
3.80
8.28
29.82
2.67
3
-------
Table 6. (continued) DISSOLVED OXYGEN (ml/liter) TEMPERATURE (°C), SALINITY (0/00) TOTAL CARBON
(%) AND SAND FRACTION (%), AT STATIONS 24 TO 33 IN PUGET SOUND.
Station
32
33
Dissolved oxygen
Temperature
Salinity
Total carbon
Sand fraction
Dissolved oxygen
Temperature
Salinity
Total carbon
Sand fraction
___July_
September
0.90
8.97
29.85
0.70
8.53
28.48
February
3.15
9.49
30.34
2.19
4
2.85
9.14
29.80
3.94
9
July
3.50
7.76
29.35
2.25
3
2.60
8.34
28.66
3.88
10
-------
u_
S
L±J
CC
LJ
CALORIMETER AT 9.9 °C 3 29 x I04 OHM
RESISTANCE HEATER SUSPENDED IN AIR _
CALORIMETER AT 19.5 °C 1200 OHM RESISTANCE
0 HEATER IMMERSED IN WATER
CALORIMETER AT 19.5 °C 329x10 OHM
RESISTANCE HEATER SUSPENDED IN AIR _J
CALIBRATION CONSTANT: 0.6 2^t col /sec /u v
2 -
I -
20
q (ca /sec) x 10'
Figure 12. Calibration line for the microcalorimeter at 9.9°C and 19.5°C.
45
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The most reliable measurement of metabolic heat release is obtained with
small samples suspended in the middle by a thin monofilament and which may
be left inside until the signal emf is steady. This takes about 12 hr. Then
the sample is quickly removed. The empty calorimeter re-stabilizes much
more rapidly (its signal is within 5% of the total change in 3 hr) and the
decrease in thermal emf following removal of the sample represents the
rate of metabolic heat release.
DEHYDROGENASE ACTIVITY OF PUGET SOUND SEDIMENTS
Any dehydrogenase assay technique by itself yields only relative measures
of metabolic activity. The results from Puget Sound (Table 2, Appendix)
merely indicate that there is fairly active anaerobic metabolism in station
33, the level of activity is generally detectable in most places, and very low
or not significantly different from zero in some.
Other tests performed with Puget Sound sediment at 37°C indicated that
aerating, grinding, autoclaving, and treating the sediment sample with
lysozyme resulted in a reduction or complete cessation of apparent metabolic
activity. The effect of aeration points to a deleterious effect of oxygen and
therefore the apparent activity without aeration must be anaerobic. The
effect of grinding, autoclaving, and addition of lysozyme means that the
production of formazan must be the result of live metabolic activity and not
of a chemical reaction.
The results of the assay at 37°C may or may not be correlated with the natural
activity at much lower temperatures, i.e., the enhancement of activity at
37<>c may or may not be in direct proportion to the natural activity. The thermal
shock of a sudden temperature change of 20 or 30°C is difficult to assess. Hence,
incubation of samples at or close to in situ temperatures would be desirable.
Puget Sound samples incubated at 10°C with glucose for as long as 6 hr, how-
ever, did not produce any detectable formazan.
DEHYDROGENASE ACTIVITY OF LAKE WASHINGTON SEDIMENTS
The following results were obtained with Lake Washington sediments during
the course of our investigation to increase the sensitivity of the assay so that
positive results may be obtained at 10°C.
Selection of Substrate - Besides glucose, we tried citrate, rnalate, lactate,
succinate, and pyruvate. Sodium citrate gave the highest activity during a
3-hr incubation at 10°C (Table 7); glucose yielded on the average only 39%
of the activity due to citrate. This is in contrast to the findings of Lenhard
et al. (1965) that glucose was more effective than the sodium salts of
lactic, citric, succinic, and glutamic acids. Evidently there is a question
46
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Table 7. RELATIVE LEVELS OF DEHYDROGENASE ACTIVITY RESULTING
FROM THE USE OF THE SAME CONCENTRATION OF DIFFERENT
SUBSTRATES ADDED TO REPLICATE SEDIMENT SAMPLES FROM
LAKE WASHINGTON
Substrate
Sodium citrate
Sodium malate
Sodium lactate
Sodium succinate
Sodium pyruvate
Glucose
Dehydrogenase activity (absorbance/gram dry sediment)
Replicates
1.17, 1.18, 1.23
0.89, 0.89, 0.93
0.66, 0.77, 0.70
0.57, 0.57, 0.58
0.01, 0.02, 0.01
0.45, 0.46, 0.48
Average
1.19
0.90
0.71
0.57
0.01
0.46
47
-------
of dehydrogenase specificity, which in Lake Washington sediments differs from
that in activated sludge.
Dehydrogenase activity increases with increasing concentration of sodium
citrate, reaching a peak at 0.15 M and declining at a higher concentration
(Fig. 13). The effect of additional substrate appears to be to enhance
or amplify the activity in direct proportion to the natural dehydrogenase
activity. Fig. 14 shows that at a final concentration of 0. 04 M the measured
activity is on the average 5.3 times that of replicate samples without any
substrate, while at 0.20 M it is 1.4 times the activity at 0.04 M.
Sodium citrate has an unexplained effect on the absorbance of the HgC^-treated
blanks (Fig. 15). The blanks with 0. 04 M citrate have about 43% higher
absorbance than replicate blanks with 0. 20 M citrate or without substrate,
and there appears to be no difference between the two latter treatment replicates.
Amount of Sediment - The measured activity increases but the increment of
activity decreases with increasing amount of sediment (Fig. 16), which may
be an indication of nutrient limitation with increasing amount of sediment.
Therefore, normalizing the result to unit weight of dry sediment is computa-
tionally inadequate. To minimize error, we have tried using as close to
0.3 g dry sediment as possible but the final weights still varied from 0.1 to
0. 5 g; this wide range may be a source of variability in the final results.
Obviously the amount of sediment should be limited to a quantity within which
the activity is proportional to quantity. If the problem is caused by nutrient
limitation, it could be solved by periodic stirring of the sediment during
incubation, provided that this does not oxygenate the sample.
Exclusion of Oxygen - The presence of oxygen causes an underestimate of
dehydrogenase activity. We have tried guarding against the presence of
oxygen during incubation by flushing and sealing the incubation flask with
nitrogen. We have not detected a significant difference between nitrogen-
flushed and unflushed replicates. Evidently the concentration of reduced
substances in the sediment is enough to consume oxygen in solution and keep
the settled sediment anoxic during the course of the reaction. The formation
of formazan itself is prima faci,e evidence of anaerobic metabolism; no
dehydrogenase activity is detected when the sample is aerated.
Blanks - Lenhard et al. (1965) and others have used as their blanks the
reaction mixture minus TTC. We were concerned, however, with the
possibility of a chemical reduction of TTC since sometimes we are dealing with
highly reduced sediments. Although Casida et al. (1964) have shown that
chemical reduction of TTC appears to occur only at much higher temperatures
(above 65°C), we think that an appropriate blank is one that contains TTC
48
-------
2.0
.05
.10 .15 .20 .25
MOLAR CONCENTRATION
.30
Figure 13. Dehydrogenase activity as a function of sodium citrate concentration.
49
-------
CD
P CL M
& CD 0
&"• P
1?
o Kr
S- CD
ro P
P CO
&8
II
ty P
o i-s
S CD
0.04M SODIUM CITRATE VS
NO ADDED SUBSTRATE
0.20 M VS 0.04 M SODIUM CITRATE
DEHYDROGENASE ACTIVITY (dbsorbance g" dry sediment)
-------
1.2
1.0
o>
en
0.8
o>
c 0.6
o
-Q
o 0.4
GO
0
NO SUBSTRATE VS .0375 M SODIUM CITRATE
0.2 M SODIUM CITRATE VS .0375 M SODIUM CITRATE
0.5 1.0
BLANK (absorbance g~! dry sediment
Figure 15. Effect of substrate concentration on the absorbance of mercuric
chloride-treated blanks.
51
-------
Ui
N>
ct>
I—'
05
CD ^<
i-S &
CD >-i
P O
f- Cfq
h— > CD
CD S3
-
H
>
H
O
UJ
z
UJ
o
QC.
Q
I
UJ
Q
1.0
0.5
O
0.5
1.0
1.5
2.0
DRY SEDIMENT (g)
o
-------
but has been poisoned to kill any dehydrogenase activity. Mercuric chloride
has been shown to be the most effective agent in stopping dehydrogenase
activity (Lenhard 1965). We obtained the same blanks with HgCl2-treated
and autoclaved samples.
MICROCALORIMETRIC CALIBRATION OF DEHYDROGENASE ACTIVITY
The significant regression of dehydrogenase activity on the actual rate of
metabolic heat release (Fig. 17) signifies a fairly high degree of
functional relationship between the two measures of community metabolism.
The sediment samples from Lake Washington had metabolic rates of 3-17mcal
g- hr- , corresponding with relative dehydrogenase activities giving ab-
sorbances of 0. 27-0. 86 g- . In this range, the regression of dehydrogenase
activity on metabolic heat release is
Y = 0.037 + 0.046 X
where Y is dehydrogenase activity in absorbance units per gram and X is
metabolic heat release in millicalories per gram per hour. The regression
coefficient is significant at P < 0. 001 with 95% confidence limits of 0. 039-
0. 053. The Y-intercept is not significantly different from zero; this may
signify that purely chemical exothermic side reactions in the sediment, e.g.
neutralization of organic acids (Forrest et al. 1961) are negligible.
To obtain samples with even higher rates of metabolic heat release, two
sediment samples from Lake Washington were treated with dried ground
zooplankton and glucose and allowed to ferment for two days. These samples
were then placed in culture tubes for calorimetry measurements. After
their removal from the calorimeter, their dehydrogenase activity was
immediately assayed. The regression line with these samples included with
the untreated samples is given by the equation
Y = 0.166 + 0.030 X:
The regression is significant at P<0. 001 with 95% confidence limits of
0. 025-0. 035. The Y-intercept is significantly greater than zero. Further-
more, there is a significant difference between the slopes of the two
regressions. The difference between the two regressions may be attributed
to the effect of the added substrate and consequent increase in exogenous
metabolism. When removed from the calorimeter the culture tubes containing
the treated samples were under internal pressure due to the liberated gaseous
fermentation products. The bacterial populations in the treated samples may
have been in a logarithmic phase of growth whereas those in the untreated
samples may have been closer to steady state. Although both regressions
indicate that the enzyme activity measured in 3 hr is proportional to the
53
-------
OQ
CD
h-'
-Q
CD CD
V-' O
CD Oq
ja CD
ra !3
CD P3
OQ
CD
O
<-t-
i-<-
<
S
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a
CD
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to
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toJ
UJ
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Y-0.037+ 0.046 X
Y= 0.166 4- 0.030X
METABOLIC HEAT RELEASE (m cal g hr"1 )
-------
undisturbed rate of anaerobic heat release by the microbial community, it
appears that the relationship between dehydrogenase activity and metabolic
heat release may vary according to the microbial population's growth
phase.
The first regression equation was used to estimate the natural anaerobic
metabolism of sediments in Lake Washington.
DISTRIBUTION OF DEHYDROGENASE ACTIVITY IN LAKE WASHINGTON
The relative measures of dehydrogenase activity at the six stations are
shown in Fig. 18. The core from station 2 was as long as those from the
other stations but was assayed to 5 cm only. There is a trend of decreasing
activity per unit weight of sediment with increasing depth of sediment layer.
There are significant differences between stations and between layers at
each station. There is only a poor correlation between dehydrogenase activity
and percentage carbon of the sediment (r = 0.42, d.f. = 16). It is interesting
that when the sediment samples to which zooplankton and glucose had been
added are included in the correlation test, r worsened to -0. 21 with d.f. =
18. This suggests that when an amount of readily oxidizable organic matter
(although insignificant relative to the large amount of old organic matter)
becomes available there is a rapid increase in dehydrogenase activity.
There is a highly significant correlation between dehydrogenase activity and
ultimate oxygen demand of the sediments (Lenhard et al. 1962; Edwards and
Rolley 1965). Ford et al. (1966) and Stevenson (1959) observed good
correlations between dehydrogenase activity and the oxygen consumption of
replicate samples, which indicate that the organisms were able to use either
oxygen or TTC as hydrogen acceptors, though not necessarily with equal
efficiency. On the other hand, Edwards and Rolley (1965) found no
correlation between the rate of total oxygen consumption by intact sediment cores
and dehydrogenase activity; it is not clear how the sediment samples were
taken for dehydrogenase assay, but presumably they were from the top 2-em
layer of the sediment cores. The discrepancy between the findings of
Edwards and Rolley (1965) and those of Ford et al. (1966) and Stevenson
(1959) may be the result of differences in metabolic types of the samples or
in the techniques by which oxygen uptake was measured.
In experiments with decomposition of flax residues in soils, Stevenson (1959)
found that during the first three days there was an increase in enzyme activity
that was not correlated with an increase in bacterial numbers; however,
over the long run the change in enzyme activity paralleled the change in
bacterial numbers. Casida et al. (1964) also noted that dehydrogenase
activity increases with increase in total bacterial numbers in organically
treated soils. When dehydrogenase activities of different untreated soils
were analyzed, however, Stevenson found no relationship with total number
55
-------
0
DEHYDROGENASE ACTIVITY (absorbance g~' dry sediment)
1.0 1.5 2.0 2.5 3.0 3.5 40 45 55
Figure 18, Vertical distribution of dehydrogenase activity at six stations in
Lake Washington in October 1972. The horizontal bars denoting
^ one standard deviation have been displaced where they would
have overlapped.
-------
of bacteria, and Casida et al. found no relationship with Gram-negative bacteria,
fungi, or actinomycetes. Evidently different soils harbor microbial species of
different metabolic types in sufficient variety to explain Stevenson's findings.
The lack of correlation may have resulted more from differential success with
different species in plate cultures than from inherent differences in
dehydrogenase activities in different species.
METABOLIC HEAT RELEASE OF LAKE WASHINGTON SEDIMENTS
The relative measures of dehydrogenase activity at the six stations were
converted to rates of metabolic heat release (in millicalories per gram per
hour) according to the regression equation presented earlier. From the
measured dry weight of each one-cm layer, units of millicalories per gram
per hour were converted to millicalories per layer per hour (Fig. 19). The
rate of total heat production by each core was obtained by integrating each
curve.
As a result of compaction, sediment weight per layer increases with depth;
hence, the actual contribution of deeper layers to total metabolism is greater
than is indicated by the decreasing trend of activity per unit weight of sediment.
Possibly activity per unit weight of sediment decreases with depth because of
decreasing available metabolizable energy, but activity per layer is
maintained somewhat because of increasing surface area with depth.
METABOLIC HEAT RELEASE VERSUS OXYGEN UPTAKE
The rates of total oxygen consumption by the intact cores and the residual
oxygen uptake or abiotic chemical oxidation after poisoning of the overlying
water were converted to rates of heat release by using the factor of 4.8
cal liberated per milliliter G£ uptake.
The bottom water temperatures at stations 2, 3, 4, 9, 14, and 19 were 8.2,
8.4, 8.3, 7.9, 8.0, and!3.3°C, respectively. Hence, the in situ metabolic
activity may be expected to be slightly less at the first five stations and more
at station 19 than the estimated activity at 10°C.
Table 8 shows the results of the two methods of estimating benthic community
metabolism. Note that the estimated dehydrogenase activity of the core from
station 2 would have been higher if the entire core had been assayed. The
estimate by the dehydrogenase method may of may not include the metabolic
activity of some aerobes. In the top 1-cm layer, for example, where aerobic
microorganisms would be found in nature, their metabolism would be included
if they were able to use TTC as hydrogen acceptor in the absence of oxygen.
Rarely were Chironomus larvae seen in the sediment, but when they were
present we do not know whether their metabolism was included in the enzyme
assay.
57
-------
0
4 -
e
o
Q_
UJ
Q
METABOLIC HEAT RELEASE (m cal layer"1 hr~')
40 80 120 160 200 240 280 320 360 400
32
Figure 19. Vertical distribution of metabolic heat release at six stations in
Lake Washington in October 1972. The horizontal bars denoting
- one standard deviation have been displaced where they would
have overlapped.
58
-------
Table 8. COMPARISONS OF DEHYDROGENASE ACTIVITY CONVERTED TO RATE OF METABOLIC HEAT
RELFASE (IN CALORIES PER CORE PER HOUR) WITH TOTAL OXYGEN UPTAKE AND CHEM-
ICAL OXIDATION CONVERTED TO METABOLIC HEAT RELEASE (IN CALORIES PER CORE PER
HOUR) BY USING THE FACTOR 1 ml 00 = 4.8 cal
Ol
CD
Station
2
3
4
9
14
19
Core
length (cm)
6
32
21
21
21
16
Total Or> uptake
0.27, 0.34, 0.26,
0.16, 0.16
0.38, 0,42, 0.38,
0.31
0.41, 0.45, 0.28,
0.33, 0.38
0.35, 0.28, 0.22,
0.19, 0.17
0.45, 0.39, 0.36,
0,36, 0.32, 0.31
0.27, 0.24, 0.19
0.19
0.365 0.36, 0.28,
0.25
Chemical oxidation
0. 10, 0.11, 0.12
0.24, 0.26
0.18, 0.18, 0.20
0.12, 0.13
0.11, 0.08, 0.11,
0.10, 0.11
0.21, 0.23, 0.24
Dehydrogenase activity
Range ! Average
0.37-0.54
0.88-1.6
2.8-3.6
1.7-2.8
3.4-4.7
0.39-0.46
0.39
1.3
3.0
2.1
4.0
0.41
-------
With the exception of station 19, the estimate of benthic anaerobic metabolism
by the dehydrogenase method greatly exceeds the estimate of benthic aerobic
plus anaerobic metabolism by the total oxygen uptake and even more greatly
exceeds the estimate of anaerobic metabolism by chemical oxygen consumption.
Even if the estimate of anaerobic metabolism by the former method is reduced
by the metabolism of the top 1-cm layer, which may have included some
aerobic respiration, a large discrepancy would remain. The discrepancy
would be larger still if possible anaerobic metabolism by macrofauna is
included. It is clear that the rate of total oxygen uptake does not give an
accurate estimate of the total benthic metabolism in the sediment column.
It is equally clear that the rate of total oxygen uptake by the sediment surface
is equivalent to the rate of dehydrogenase activity in a thinner upper layer.
We do not know if the week-long storage of the core from station 19 has
anything to do with the closer agreement between the two methods for that
core. This core was also shorter than the others except the one from
station 2. The results from station 19, however, indicate that where there
is a rapid decline in anaerobic metabolism with depth the rate of total
oxygen uptake will be a close estimate of total metabolism in the sediment.
DISTRIBUTION OF TOTAL REDUCED SUBSTANCES
The concentration of reduced substances in the sediment at the first 23 stations
in Puget Sound (determined iodometrically) increases linearly with the
logarithm of depth of the sediment layer (Fig. 20). Off the coast of Oregon
and Washington the concentration of reduced substances (determined by
dichromate oxidation), expressed as oxygen debt (Fig. 21), also increases
with depth. In Lake Washington, the concentration of reduced substances,
also determined by dichromate oxidation, likewise increases with depth
(Fig. 22) but only to about 11 to 16 cm and decreases below these layers.
The most reasonable explanation for increasing concentrations of reduced
substances with depth is that these products of anaerobic metabolism are
no longer effectively oxidized and begin to accumulate when buried under a
thickening layer of surface deposit. There is no correlation between the
concentration of reduced substances in sediment layers below 4 cm and the
rate of oxygen uptake by chemical oxidation.
The concentration of reduced substances is not correlated with the organic
carbon content of the sediment. There are, on the average, smaller concen-
trations in deepwater stations than in shallower stations (Fig. 20).
Offshore, the abyssal plains sediments contain less reduced substances
than the sandy shelf sediments although the latter contain smaller organic
carbon. There is increasing concentration of reduced substances with
increasing mud content which increases with increasing depth to the
60
-------
-0.04
0.4
E
u
0.6
0.8
1.0
2.0
LL!
Q
4.0
6.0
8.0
1 0.0
20.O
REDUCED SUBSTANCES(meq/m! sediment)
-0.02 0.0 0.02 0.04 0.06
> 100 m __<
51 -100m .
< 50 m .
Figure 20. Concentration of reduced substances in Puget Sound versus depth
of sediment layer.
61
-------
OXYGEN DEBT (ml 02 /m I Sediment)
O.I 0.2 0.3 0.4
CD
CO
8 g
•?&.
ct> o
O CD
£3 a
I C
-------
0
TOTAL REDUCED SUBSTANCES (meq mr'xICT2)
0 2 4 6 8 10 12 14 16 18
10
o 15
Qu
UJ 20
Q
25
30
35
1 I I T
I I I I I I
I 1
Figure 22. Vertical distribution of the concentration of reduced substances in
Lake Washington sediments.
63
-------
continental slope.
Lake Washington sediments contain higher concentrations of reduced substances
than Puget Sound or offshore sediments; they also contain much more organic
matter (Shapiro et al. 1971), presumably as a result of highly eutrophic
conditions and the discharge of sewage, both raw and treated, over a period
of four decades. The history of eutrophication of Lake Washington has been
studied and well documented by Dr. W. T. Edmondson and his students
(Edmondson 1969a, 1969b, 1970). Sediment below 11 cm which contains
lower concentrations may have been deposited before the onset of the period
of eutrophication of the lake. It is interesting that the concentration of organic
matter, although quite variable, has a decreasing trend with sediment depth
(Shapiro et al. 1971). The decrease may be attributed to long-term
mineralization through anaerobic metabolism, which in turn produced the
accumulated reduced end products.
The foregoing indicate that although the concentration of reduced substances
might be expected to increase with greatly increasing organic matter content,
it is not the concentration of total organic matter but the metabolizable
fraction that determines the extent of anaerobic metabolism. Much of the
organic matter in sediments is resistant to biochemical oxidation (Waksman,
1933) for perhaps various reasons which are not fully understood. Volkmann
and Oppenheimer (1962), who studied the decomposability of organic carbon
in different sediments of a shallow marine lagoon, found that organic matter
in coarse sediments decomposed more readily than that in fine sediments.
The difference in organic matter composition between the sediment types,
and compaction and adsorptive capacity of clay minerals, were given as
possible reasons. From our results there appears to be a greater fraction
of refractory organic matter in deep-water sediment than in shallow-water
sediment.
CHEMICAL OXIDATION VERSUS REDUCED SUBSTANCES
Whereas the concentration of reduced substances in deeper layers is not
correlated with the rate of chemical oxidation, the concentration in the
upper four centimeters is (Fig. 23). This relationship, together with the
seasonal cycle of chemical oxidation (Figs. 9 and 10), indicates a dynamic
equilibrium between anaerobic metabolism in the surface 4-cm layer, the
resulting formation of reduced by-products, and their consequent chemical
oxidation.
It appears then that reduced substances below about 4 cm are accumulating
while in the upper 4 cm they may be approximating steady-state condition,
except when the flux of fresh, more readily oxidizable organic matter
increases during the summer; then, anaerobic metabolism in the immediate
subsurface layers increases, resulting in higher concentrations of reduced
64
-------
0)
Q.
-------
substances, and therefore in higher rates of chemical oxygen consumption.
DEHYDROGENASE ACTIVITY VERSUS REDUCED SUBSTANCES
The absorbance of duplicate HgCl^-treated blanks shows little variability.
The coefficient of variation is usually less than 5% but occasional large
coefficients of up to 20% raised the average coefficient to 5.2%. There is
no correlation between the absorbance of the blanks and the concentration
of reduced substances (r = 0.043, d.f. = 23); hence reduced substances
apparently did not reduce TTC to formazan. The absorbance of the blank
is probably largely due to phytoplankton pigments and their degradation products
as evidenced by the greenish-yellow color of the ethanol extract.
The absorbance of triplicate reaction mixtures shows greater variability than
that of the blanks, with an average coefficient of variation of 19%. As
explained previously, part of the variability may have been due to differences
in sediment weights in replicate samples, although occasional large variations
within replicates could not be explained by this cause. There is a
significant increase in the variance with increasing level of dehydrogenase
activity (r = 0.52, d.f. = 31).
There is no correlation between dehydrogenase activity and concentration of
reduced substances (r = 0.024, d.f. = 23). Effenberger (1966), however,
noted a negative correlation between dehydrogenase activity and the redox
potential of activated sludge. As the E, decreased from about +190 to+50
mv the dehydrogenase activity (expressed as relative units) increased from
about 120 to 280. Since the effect of anaerobic metabolism is known to be
a decrease in E^ of the medium, the lower the E^ (in this experiment
approaching some steady-state condition) the greater the metabolic activity
and hence the higher the measured dehydrogenase activity. This may seem
to contradict our result, indicating no significant correlation between
dehydrogenase activity and concentration of reduced substances in Lake
Washington. The discrepancy is explained by the fact that the concentration
of reduced substances in the sediment is a function of time as well. Deeper
sediments have been anaerobic for a longer time than surface sediments and
are no longer being oxidized by dissolved oxygen diffusing into the sediment;
while surface sediments, especially when being turned over by macrofauna,
are still periodically aerobic. Under steady-state conditions, and where
no other factors such as differences in sediment compaction and oxygen tension
of the overlying water prevail, one might expect a correlation between the
concentration of reduced substances in the surface layer and its dehydrogenase
activity.
HUMIC ACID CONTENT OF SEDIMENTS
The humic acid content of samples from various stations has been measured in
66
-------
relative units only, namely, absorbance per gram of dry sediment (Table 9).
Assuming that humic acid solution conforms to Beer's law, the ratio of this
absorbance unit to per cent organic carbon in the different samples represents a
relative measure of the total organic carbon proportion of humic acid in the various
samples. The ratios vary by a factor of about 6 at most, e.g. the organic
matter in station 29 in Snohomish River consists of proportionately little
humic acid as compared to station 1 in Lake Washington. The latter station
contains much more organic matter than station 29 and therefore also
contains greater absolute quantities of both humic substances and oxidizable
carbon.
It may be significant that the three samples that showed the smallest ratios
were from stations 29 and 30 in Snohomish River and from Clam Bay, which
all showed unusually high rates of oxygen consumption that winter (Table 9).
Their rates of oxygen consumption declined the following July (when Puget
Sound stations normally exhibit a rise in benthic metabolism) when the
sediment smaples showed proportionately greater content of humic acid. With
the exception of these three stations, the others showed only slight differences
between February and July and between stations. Furthermore, there appears
to be only slight and inconsistent differences between the 0-1 and 5-6 cm layers
of sediment in Puget Sound.
Recent sediments from various geographical areas contain widely different
proportions (4-68%) of organic carbon as humic substances (Nissenbaum and
Kaplan, 1972). How these varying proportions of humic acids affect benthic
community metabolism is further complicated by the evidence that humic
substances originate in situ as well as from terrestrial sources (op. cit.)
In any case, our results point to a need for more thorough investigation of
humic acids in connection with research on benthic community metabolism.
It would also be desirable to establish the quantitative relationship between
absorbance and the concentration of humic acid in solution.
EFFECT OF TIDAL CURRENTS ON BENTHIC OXYGEN UPTAKE
The drop in oxygen concentration of bottom water during ebb tide (Fig. 24)
probably represents at least partly the effect of oxygen consumption by
resuspended sediment, although part of it could have been due to advection.
The horizontal mass movement of surface sediment by tidal current at
the same location has been observed on other occasions.
Just how important resuspension of sediment is in the total benthic oxygen
uptake in Puget Sound is difficult to say until we know how widespread this
type of periodic distrubance is. The oxygen tension does not drop by much,
but obviously the annual oxygen uptake by the bottom would be underestimated
if those periods of disturbance are not taken into consideration.
67
-------
Table 9. RELATIONSHIP BETWEEN HUMIC SUBSTANCES (absorbance/g dry sediment) AND ORGANIC
CARBON (per cent of dry sediment) IN DIFFERENT SEDIMENT LAYERS AND DURING
DIFFERENT SEASONS
Location
Puget Sound
Station
1
5
6
8
10
21
23
Absorbance per g
of dry sediment
0-1 cm layer
0.40
0.19
0.44
0.47
0.33
0.50
5-6 cm layer
0.40
0.21
0.19
0.46
0.47
0.35
0.52
Organic Carbon
per cent of dry sediment
0-1 cm layer
2.94
1.09
0.89
2.54
2.54
1.64
2.68
5-6 cm layer
2.58
1.20
1.06
2.45
2.49
1.64
2.68
A:B Ratio
0-1 cm layer
0.14
0.17
0.17
0.18
0.20
0.19
5-6 cm layer
0.16
0.18
0.18
0.19
0.19
0.21
0.19
-------
Table 9. RELATIONSHIP BETWEEN HUMIC SUBSTANCES (absorbance/g dry sediment) AND ORGANIC
CARBON (per cent of dry sediment) IN DIFFERENT SEDIMENT LAYERS AND DURING
DIFFERENT SEASONS
(continuation)
Location
Puget Sound
Snohomish
River
Lake
Wa shington
Blanks^
Station
24
25
26
27
28
31
32
33
Clam Bay
29
30
1
8
9
Absorbance per g
of dry sediment
February '72
0.10
0.42
0.17
0.14
0.054
0.74
0.42
0.76
0.067
0.039
0.015
1.54
0.79
0.78
0.020
0.028
July '72
0.25
0.35
0.20
0.047
0.47
0.44
0.72
0.033
0.18
0.018
2.62
0.73
0.78
Organic Carbon
per cent of dry sediment
February '72
0.89
2.54
1.59
0.99
0.45
3.95
2.20
3.94
1.02
1.17
0.29
7.55
6.29
6.79
0.0
0.0
July '72
1.80
2.19-
0.96
1.32
0.41
2.67
2.25
3.88
0.24
1.53
0.15
14.06
6.01
6.22
A:B Ratio
February '72
0.11
0.17
0.11
0.14
0.12
0.19
0.19
0.19
0,066
0.033
0.052
0.20
0,13
0.11
July '72
0.14
0.16
0.15
0.11
0.18
0.20
0.19
0.14
0.12
0.12
0.19
0.12
0.13
a)Sediment combusted for 2-3 hr at 500°C
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TIME (hours)
0600
0800
-------
EFFECT OF INCREASED ORGANIC SUPPLY TO THE BOTTOM
Directly underneath one of the floating fish pens in Clam Bay the rate of
total oxygen uptake averaged 125 ml/m2 per hr, with respiratory uptake
being 86 and chemical oxidation 44 ml/m2 per hr. The rate of total uptake
decreased rapidly to the north and south away from the pens (Table 10).
The increased rate of oxygen uptake appears to be confined to the immediate
vicinity of the floating pens, although there may have been a slight influence
as far as 15 m to the south.
The oxygen debt of the sediment underneath one of the pens is also about twice
as high as those to the north and south (Table 11). The increase in reduced
substances extended to the 3-4 cm layer. The sediment smelled of H2S.
The fish were harvested in May and the pens were removed. During a
subsequent cruise in July, the rate of oxygen uptake had dropped to 24 ml/m2
per hr while oxygen debt had decreased to 0. 05 meq per ml of sediment. There
was no evidence of the highly polluted condition just a few months earlier,
except for the persistent absence of macrofauna. Until late summer there
were no infauna found; the only benthos to be seen were amphipods on the
sediment surface (Mahnken, personal communication).
At least part of the reason for the accumulation of organic matter underneath
the pens is believed to be the hindrance of water circulation by the nets.
During extreme low tides the bottom of the net would scrape the sediment
surface. It appears that once the nets were removed, tidal currents swept
away lighter surface deposits and the increased turbulence of the overlying
water enhanced the diffusion of dissolved oxygen into and of reduced
substances out of the sediment.
RELATIONSHIP BETWEEN OXYGEN UPTAKE, CARBON OXIDATION, AND
NUTRIENT RELEASE BY THE SEDIMENT
The rates of oxygen uptake, organic carbon content of the sediment, and
nutrient concentrations of the overlying water of individual samples after
increasing periods of incubation are shown in Table 12. The initial rates of
oxygen uptake, measured when the water was still turbid although most of
the sediment had settled to the bottom, show the high rates of oxygen consump-
tion by disturbed sediment. Two days later the rates of uptake appear to have
stabilized roughly to a constant rate until the end of the experiment. During
this time the organic carbon showed a statistically significant decrease
(P <0.05) which averaged about 1.4 mgC/g dry sediment per month. Assuming
that respiratory oxygen consumption averaged 2.7 ul/g dry sediment per hr,
the respiratory quotient would be about 1.3. This value could mean that not
71
-------
Table 10. RATES OF TOTAL OXYGEN UPTAKE, INORGANIC CHEMICAL
OXIDATION, AND RESPIRATION (ml/m2 per hr) AT 10°C IN
CLAM BAY AT THE SITE OF THE FISH REARING PENS IN
FEBRUARY
Station
1
2
3
4
5
6
7
8
9
Distance from
fish pens (meters)
15 south
30 south
45 south
75 south
15 north
30 north
45 north
75 north
underneath
Total
Uptake
36,42,31
22,21
14,10,11
30,12,14
11,14
24,14
17,17,20
18,19,10,19
130,120
Chemical
Oxidation
22
13
10
13
9
14
11
12,7
44
Respiration
14
8
4
17
5
5
9
6,3
86
72
-------
Table 11. OXYGEN DEBT (ml 02/ml sediment) OF THE SEDIMENT IN CLAM
BAY AT THE SITE OF THE FISH REARING PENS IN FEBRUARY
Station
1
2
3
4
5
6
7
8
9
Distance from
fish pens meters
15 south
30 south
45 south
75 south
15 north
30 north
45 north
75 north
underneath
Sediment Layer
0-1 cm
0.20,0.23
0.24,0.27,
0.24
0.15,0.12,
0.07
0.09,0.09
0.24,0.28
0.18
0.10,0.08
0.11
0.05,0.04
0.04,0.03
0.51,0.63
1-2 cm
0.28,0.21
0.20,0.17
0.12,0.12
0.08,0.10
0.27,0.26
0.08,0.09
0.08,0.09
0.24,0.13
0.63,0.50
3-4 cm
0.25,0.27
0.15,0.17,
0.17
0.13,0.14
0.08,0.08
0.23,0.21
0.20,0.14,
0.18
0.25,0.12,
0.09
0.11,0.14,
0.16
0.51,0.58
73
-------
Table 12. RATES OF TOTAL OXYGEN UPTAKE, INORGANIC CHEMICAL OXIDATION AND RESPIRATION
(ml 02 g -1hr~1)PER CENT ORGANIC CARBON, AND NUTRIENT CONCENTRATION OF
,-1
OVERLYING WATER (/ig-at 1 ) DURING A 43-DAY EXPERIMENT
Time
Hours
0
21
44
68
140
380
572
741
1030
Total
Uptake
30
11
7.5
7.2
5,9
7.2
3.3
6.4
5.1
Chemical
Oxidation
18
7.9
5.5
4.8
5.9
4.6
0.9
2.9
2.0
Respir-
ation
12
3.1
2.0
2.4
0
2.6
2.4
3.5
3.1
Per Cent
Carbon
2.18
2.09
2.01
2.03
2.06
1.89
1.96
1.94
1.85
sio4
50
110
250
145
235
355
370
400
— — -»
N03
29
31
49
31
46
70
60
88
— —
NH3
0.2
8.1
3.0
2.6
0.0
0.1
0.0
— — —
P04
1.9
2.8
3.0
3.4
5.3
10.2
11.9
13.3
18.6
-------
all of the organic carbon loss from the sediment was the result of complete
oxidation.
The nutrient values show that ammonia disappeared completely while
silicate, nitrate, and phosphate increased with time. On the average, the
sediment released silicate, nitrate, and phosphate at the rates of 0.05,
0.008, and 0.002 microgram-atoms/g of dry sediment per hr, respectively.
The released ammonia may have been nitrified to nitrate. These amounts
were released during respiratory oxygen uptake of 2.7 microliters/g dry
sediment per hr.
This preliminary experiment shows a promising approach of establishing
relationships between metabolism, decomposition of organic carbon, and
nutrient release by the sediment under various conditions.
75
-------
SECTION VI
DISCUSSION
TOTAL OXYGEN UPTAKE AS A MEASURE OF COMMUNITY METABOLISM
IN THE SEDIMENT COLUMN
Total benthic metabolism is a complex of aerobic and anaerobic activity. In
order for the rate of total oxygen uptake to be a measure of total metabolism
the rate of chemical oxidation of reduced substances must be in equilibrium
with the rate of formation of these substances by anaerobic metabolism.
Although there is evidence that such an equilibrium may exist in the surface
few-centimeters layer, the increase with sediment depth of concentration of
reduced substances indicates an accumulation in the deeper layers, i.e.,
these are not being oxidized and therefore the rate of oxygen uptake by chemical
oxidation is underestimating anaerobic metabolism in deeper layers. This
underestimation is also indicated by the fact that the rate of oxygen consumption
by intact sediment cores is independent of the length of the cores beyond a few
centimeters (Edwards and Rolley, 1965).
A direct comparison between the rate of oxygen uptake by chemical oxidation
and integrated anaerobic metabolism from dehydrogenase assay of sediment cores
from Lake Washington indicates by how much anaerobic metabolism in the
sediment column may be underestimated. The large discrepancy between the
two measures in Lake Washington may be attributed to the increasing relative
importance of anaerobic metabolism with increasingly organic soft sediments
as a result of organic pollution or eutrophication. Where anaerobic metabolism
is relatively less important then the rate of inorganic chemical oxidation
should be a better estimate of total anaerobic metabolism and the rate of total
oxygen uptake should accordingly be a better estimate of total metabolism in
the sediment column than the results from Lake Washington indicate.
In ecological investigations, we are interested in the quantitative role of the
benthos in the annual cycle of materials and energy flow through the ecosystem;
that is, we want to know how much of the annual net primary production
settles to the bottom and how much of this is mineralized during that year by
the benthos. Obviously, if the benthic community in the deeper sediment
layers is metabolizing long-buried organic matter, its metabolic activity is
not related to the annual sedimentation of organic matter and the energy flow
through the rest of the ecosystem. It may be that the true measure of the
benthic community's impact on the annually sedimented organic matter is
reasonably estimated by the rate of total oxygen consumption by the sediment
surface. It would be desirable to obtain direct measurements of sedimentation
rates; these plus information on the average annual leftover organic matter
in the bottom should allow us to place an upper limit to the annual extent of
76
-------
degradation of the current year's sedimented organic matter. The limiting
values hopefully will agree with the estimated annual oxygen consumption by
the sediment. The role of burrowing macrofauna that feed on the sediment
surface in mixing and transporting newly sedimented organic material below
the surface should be investigated also.
Even if the rate of total oxygen uptake by intact sediment surface represents
total metabolism by the aerobic and anaerobic organisms in a surface layer
only of indeterminate thickness (a few centimeters in any case), the measure
is still probably a useful characteristic parameter of an area; it would seem
to be an index of equilibrium conditions among the various factors that
affect the rate of uptake, e.g. oxygen tension, temperature, salinity,
turbulence, available metabolizable energy, size and composition of the
community, compaction and porosity of the sediment, and maybe more. It
would be desirable to unravel the quantitative effects of these various factors,
but in the absence of such knowledge it is still useful to have a total measure
of their combined effects.
There seems to be no easy single method to measure accurately total benthic
community metabolism in the sediment column. Direct calorimetry may be
the only means of measuring aerobic plus anaerobic metabolism of undisturbed
sediment cores, but the outlook for its use in field studies does not look
promising. At present the practical way to estimate total aerobic and anaerobic
metabolism in sediments will be to combine the rate of respiratory oxygen uptake
by undisturbed sediment cores with estimates of anaerobic metabolism derived
from dehydrogenase assay of subsurface sediment layers.
GENERAL APPLICABILITY OF THE TTC METHOD FOR MEASURING NATURAL
RATES OF ANAEROBIC METABOLISM
A number of questions may be raised about the method of using TTC plus
substrate to measure anaerobic metabolism. If TTC is a more efficient
hydrogen acceptor than the natural hydrogen acceptors available to the
organisms, it may actually stimulate dehydrogenase activity. The added
substrate undoubtedly raises the natural level of metabolic activity. Hence,
the apparent dehydrogenase activity measured is certainly not the natural
level of metabolic activity in situ, even if all other physical and chemical
conditions in nature are maintained. If TTC alone without additional substrate
is used, the measured activity may still be higher than in_situ rates.
By comparing any dehydrogenase method with direct calorimetry, the above
questions become immaterial; the effect of TTC and added substrates are in
essence systematic errors that are taken care of by the comparison. If the
method is to be of wide applicability, however, it is essential to verify that
the magnitude of the systematic error remains the same for different microbial
77
-------
communities. The discrepancy between our results and those of Lenhard et al.
(1965) regarding the relative effects of added glucose and sodium citrate
presupposes nonuniformity of the systematic error due to added substrate.
We have shown, however, a direct proportionality between the results with
two concentrations of sodium citrate and with no added substrate. There
should be a significant regression of measured dehydrogenase activity
without added substrate and metabolic heat release. If the systematic error
due to the effect of TTC alone is the same on all microbial organisms, the
only problem would be one of low sensitivity in some if not most places. We
have not investigated the effect of much longer incubation time without
additional substrate on the rate of formazan production. As mentioned
earlier, Farkas (1966) has placed a limit of 3 hr on incubation time, but
it is not clear why he should find this necessary. Obviously, incubation time
may be extended for as long as the rate of formazan production remains
constant.
In any case, the regression showing a high degree of functional relationship
between dehydrogenase activity and metabolic heat release under one set
of conditions (10°C, 3-hr incubation, 0.1% TTC, 0.04 M sodium citrate)
warrants further investigation of the relationship under a wider range of
conditions. It seems particularly imperative to test the relationship at
different temperatures and with the dehydrogenase method carried out at
the same or higher temperatures than the calorimetry. With this information
it may be possible to obtain positive results with any dehydrogenase method
and by comparison with a calibrated method determine the equivalent metabolic
heat release in nature.
The dehydrogenase activity of some Metazoa has been shown to be corrleated
with their aerobic metabolism (Curl and Sandberg 1961; Packard and Taylor
1968), but the assay is much more difficult than the measurement of oxygen
consumption and therefore the latter remains the preferred method of
measuring aerobic metabolism. A dehydrogenase assay, however, could
be a very useful measure of anaerobic metabolism by metazoans and deserves
to be developed.
The metabolism of heterotrophic sediment bacteria has also been assessed in
terms of their uptake of radioactive glucose or acetate (Wood 1970; Harrison
et al. 1971; Sorokin and Kadota 1972). Robbie (1969) discusses the
significance and limitations of the method. Such a method also might be
compared with direct calorimetry. Since the measurement of dehydrogenase
activity requires less expensive equipment and materials, it would seem
preferable to the measurement of the rate of uptake of a specific substrate.
78
-------
EFFECT OF TEMPERATURE ON BENTHIC COMMUNITY METABOLISM
There is a problem about comparing benthic community metabolism in various
areas that differ in temperature. For example, when comparing deep-sea
benthic metabolism at 2°C with shallow sub-tidal or intertidal metabolism at
15°C or greater in summer, we do not know how much of the difference in
metabolic rate is due to the temperature difference and how much is due to
other factors. The difference in benthic community metabolism between
intertidal at 2°C in winter and the deep-sea at 2°C would be due exclusively
to factors other than temperature, but it will be most unusual for natural
benthic metabolism to be measured at exactly the same temperature in
different places.
Any comparison without consideration of temperature differences tacitly
assumes that each benthic community is completely acclimatized to
temperature at the time of measurement. This assumption may be correct
for areas of more or less constant temperature throughout the year but it
may not be reasonable for those areas subject to a pronounced seasonal
temperature cycle in view of the different degrees of temperature compensa-
tion exhibited by different species (Precht, 1958, Prosser, 1958). In the
latter areas, it would be desirable to partition the seasonal difference in
metabolic rate between the temperature effect and the other factors.
The technique employed in the present study of measuring the rates of oxygen
uptake at 5, 10, and 15°C throughout the year resulted in acute metabolism-
versus-temperature curves, oxygen consumption rates at the same tempera-
tures throughout Puget Sound, and also rates at prevailing bottom-water
temperatures at the time of measurement in the different stations. These
stations show differences in metabolism-versus-temperature curves
indicating differences in temperature adaptation. Where seasonal temperature
fluctuations and temperature differences between study sites are no more
than 5°C or so, such as in subtidal Puget Sound, this technique may be all
right, but where these are much greater there may be the additional problem
of dealing with cold-adapted communities at one time or place and warm-
adapted communities at another time or place. The cold-adapted community
would be subjected to a large acute temperature rise while the warm-adapted
community would be subjected to a large acute temperature lowering. It
may be doubtful that their respective metabolism at the same intermediate
temperature is devoid of any temperature effect. In such a case it would
seem better to measure oxygen consumption rates after acclimation to the
same temperature but this would take days and the storage time would then
introduce another unknown factor,,
79
-------
More experiments are clearly needed to understand the differences in
benthic metabolism between areas of different temperatures. The effect
of temperature on metabolism may be further complicated by an interaction
with pressure at great depths (Jannasch et al., 1971).
80
-------
SECTION VII
REFERENCES
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measurement of upwelling and subsequent biological processes by means of
the Technicon AutoAnalyzerR and associated equipment. Deep-Sea Res.
14:381-389, 1967.
Benzinger, T. H. and C. Kitzinger. Gradient layer calorimetry and human
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industry, vol. 3, part 3, Biology and medicine, J. D. Hardy(ed.). N. Y.,
Reinhold Publ. Comp. , 1963. p. 87-109.
Berger, R. L. Calibration and test reactions for microcalorimetry. In:
Biochemical Microcalorimetry, H. D. Brown(ed.) N. Y. , Academic Press,
1969. p. 221-234.
Calvet, E. and H. Prat. Recent progress in microcalorimetry. Edited and
translated by H. A. Skinner. N. Y. , The MacMillan Co. , 177 pp.
Casida, L. E. , Jr. , D. A. Klein, and T. Santoro. Soil dehydrogenase
activity. Soil Science. 98:371-376, 1964.
Craib, J. S. A sampler for taking short undisturbed marine cores. J. Cons.
perm. int.Explor. Mer. 30:34-39, 1965.
Curl, H. , Jr. and J. Sandberg. The measurement of dehydrogenase activity
in marine organisms. J. Mar. Res. 19:123-138, 1961.
Duff, S. and J. M. Teal. Temperature change and gas exchange in Nova Scotia
and Georgia salt-marsh muds. Limnol. Oceanogr. 10:67-73, 1965.
Edmondson, W. T. Cultural eutrophication with special reference to Lake
Washington. Commun. Int. Ass. Theor. Appl. Limnol. 17:19-32, 1969.
Edmondson, W. T. Eutrophication in North America- In: Eutrophication:
causes, consequences, correctives. Nat. Acad. Sci. , Wash. , D. C.
1969. p. 124-149.
Edmondson, W. T. Phosphorus, nitrogen, and algae in Lake Washington after
diversion of sewage. Science. 169:690-691, 1970.
Edmondson, W. T. and D. E. Allison. Recording densitometry of
X-radiographs for the study of cryptic laminations in the sediment of Lake
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Washington. Limnol. Oceanogr. 14:317-326, 1970.
Edwards, R. W. and H. L. J. Rolley. Oxygen consumption of river muds.
J. Ecol. 53:1-19, 1965.
Effenberger, M. Formal discussions. In: W. Bucksteeg. 1966. Determin-
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Evans, W. J. The conduction-type microcalorimeter. In: Biochemical
Microcalorimetry, H. D. Brown(ed-), N. Y. Academic Press, 1969. p.257-273.
Farkas, P. Formal discussions. In: W. Bucksteeg. 1966. Determination
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Ford, D. L., J. T. Yang, and W. W. Eckenfelder. Dehydrogenase enzyme
as a parameter of activited sludge activities. Proc. 21st Industrial Waste
Conference, Purdue University, 1966. p. 534-543.
Forrest, W. W. , D. J. Walker, and M. F. Hopgood. Enthalpy changes
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1961.
Harrison, M. J. , R. T. Wright, and R. Y. Morita. Method for measuring
mineralization in lake sediments. Applied Microbiol. 21:698-702, 1971.
Robbie, J. E. A method for studying heterotrophic bacteria. In:
A manual on methods for measuring primary production in aquatic environments,
including a chapter on bacteria. R. A. Vollenweider (ed.). IBP Handbook
No. 12. Oxford, Blackwell Scientific Publications, 1969. p. 146-151.
Koroleff, F. Direct determination of ammonia in natural waters as
indophenol blue. Information on techniques and methods for sea water
analysis. Interlab. Rep. No. 3, Int. Coun. Explor. Sea., 1970.
p. 19-22.
Krumbeiii, W. C. and F. J. Pettijohn. Manual of sedimentary petrography.
N. Y.. , Appleton-Century-Crofts, Inc. 1938. 549 p.
Kleckner, J. F. The role of the bottom fauna in mixing lake sediments. Master's
Thesis. Seattle, Univ. Wash. , 1967. 61 p.
Lenhard, G. Die Dehydrogenaseaktivitat des Bodens als Mass fur die
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Mikroorganismentatigkeit im Boden. Zeit Pflanzenernahrung, Bungling,
Bodenkunde. 73:1-11, 1956.
Lenhard, G. Dehydrogenase activity as criterion for the determination of
toxic effects on biological purification systems. Hydrobiologia: 25:1-8,
1965.
Lenhard, G. , W. R. Ross, and A. du Plooy. A study of methods for the
classification of bottom deposits of natural waters. Hydrobiologia, 20:223-240,
1962.
Lenhard, G. , L. D. Nourse and H. M. Schwartz. The measurement of
dehydrogenase activity of activated sludge. In: Adv. Water Pollution Res. ,
J. K. Baars (ed.). Proc. 2nd Int. Conf. , Tokyo., Vol. 2. London,
Pergamon Press, 1975. p. 105-127.
Murphy, J. and J. P. Riley. A modified single solution method for the
determination of phosphate in natural waters. Analyt. Chim. Acta.
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Nissenbaum, A. and I. R. Kaplan. Chemical and isotopic evidence for the
in situ origin of marine humic substances. LimnoL Oceanogr. 17:570-582,
1972.
Packard, T. T. and P. B. Taylor. The relationship between succinate
dehydrogenase activity and oxygen consumption in the brine shrimp Artemia
salina. Limnol. Oceanogr. 13:552-555, 1968.
Pamatmat, M. M. Oxygen consumption by the seabed. IV, Shipboard and
laboratory experiments. Limnol. Oceanogr. , 16:536-550, 1971.
Pamatmat, M. M. Oxygen consumption by the seabed. VI. Seasonal cycle
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Hydrobiol. 56:675-699, 1971.
Pamatmat, M. M. Benthic community metabolism on the continental terrace
and in the deep sea in the North Pacific. Int. Revue ges. Hydrobiol. In
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Pamatmat, M. M. and D. Fenton. An instrument for measuring subtidal
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84
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SECTION VIII
APPENDICES
Page
Table 1. Computer output of predicted rates from analysis 86
of variance of total oxygen uptake, inorganic
chemical oxidation, and respiration (ml oxygen
nT^r"1) during four seasons at 5, 10, and 15°C.
2. Dehydrogenase activity of sediments following 89
incubation of samples with glucose at 37°C for
one hour.
Figure 1. Top view of multiple corer showing four cross braces 90
on top welded to a center piece of 3-inch long cylinder
which holds the upper end of the dashpot in place.
2. Side view of multiple corer showing cylinder support. 91
3. Sectional view across the lower end showing the 92
guides for each of the coring tubes.
4. Coring tube guide showing the split lower section that 93
is spread out to stretch the rubber tubing away from
the path of the coring tube and the curved overhang
to keep the core catcher in proper position away from
the path of the coring tube.
5. Diagonal section showing two opposite coring tubes and 94
their weight stands.
6. Piston and rod assembly. 95
7. Dashpot showing the bottom end which is bolted to 96
steel plate center piece.
8. Bottom cylinder mounting plate. 97
9. Weight stand assembly. 98
10. Lower end of the weight stand into which is threaded 99
a PVC coupling piece.
85
-------
Page
Figure 11. Coupling piece that holds a 15-inch long coring 100
tube in place.
Annotated Bibliography 101
85a
-------
Table 1. COMPUTER OUTPUT OF PREDICTED RATES FROM ANALYSIS OF VARIANCE OF TOTAL OXYGEN
UPTAKE, INORGANIC CHEMICAL OXIDATION, AND RESPIRATION (ml oxygen m"2^"1)
DURING FOUR SEASONS AT 5, 10, AND 15°C
Stations Oct.
1 12.9
2 9.4
3 10.0
4 10.1
5 7.3
6 7.4
7 8.3
8 11.4
9 6.5
10 6.4
11 7.4
12 7.3
13 10.0
14 10.7
15 8.3
16 8.9
17 7.5
18 6.7
19 8.6
20 7.1
21 7.7
22 5.4
23 4.2
Jan.
11.1
8.1
8.6
8.7
6.2
6.4
7.1
9.8
5.5
5.5
6.3
6.3
8.6
9.2
7.2
7.7
6.4
5.7
7.4
6.1
6.6
4.7
3.6
5°C
Apr.
12.6
9.2
9.8
9.9
7.1
7.3
8.2
11.2
6.3
6.3
7.2
7.2
9.8
10.5
8.2
8.8
7.3
6.6
8.4
7.0
7.6
5.3
4.1
TOTAL
Jul.
15.9
11.6
12.3
12.5
9.0
9.2
10.3
14.1
8.0
7.9
9.1
9.0
12.4
13.2
10.3
11.1
9.2
8.3
10.6
8.8
9.6
6.7
5.2
Oct.
19.5
14.2
15.1
15.3
11.0
11.2
12.6
17.3
9.8
9.7
11.2
11.1
15.3
16.2
12.7
13.6
11.3
10.2
13.0
10.8
11.8
8.3
6.4
OXYGEN UPTAKE*
Jan.
16.8
12.3
13.0
13.2
9.5
9.7
10.9
14.9
8.4
8.4
9.6
9.5
13.1
13.9
10.9
11.6
9.7
8.7
11.2
9.3
10.1
7.1
5.5
10°C
Anr.
19.2
14.0
14.9
15.1
10.8
11.0
12.4
17.0
9.6
9.6
11.0
10.9
15.0
15.9
12.4
13.3
11.1
10.0
12.8
10.6
11.6
8.1
6.2
Jul.
24.2
17.6
18.8
19.0
13.7
13.9
15.7
21.5
12.2
12.1
13.9
13.8
18.9
20.1
15.7
16.8
14.0
12.6
16.1
13.3
14.6
10.3
7.9
Oct.
24.1
17.6
18.7
19.0
13.6
13.9
15.6
21.4
12.1
12.0
13.9
13.7-
18.9
20.0
15.7
16.8
14.0
12.5
16.1
13.3
14.5
10.2
7.9
Jan.
20.7
15.1
16.1
16.3
11.7
11.9
13.4
18.4
10.4
10.4
11.9
11.8
16.2
17.2
13.5
14.4
12.1
10.8
13.8
11.4
12.5
8.8
6.8
15UC
Apr.
23.7
17.3
18.4
18.6
13.4
13.6
15.4
21.0
11.9
11.8
13.6
13.5
18.5
19.7
15.4
16.5
13.8
12.3
15.8
13.1
14.3
10.0
7.7
Jul.
29.9
21.8
23.2
23.5
16.9
17.2
19.4
26.5
15.0
14.9
17.2
17.0
23.4
24.8
19.4
20.8
17.4
15.5
20.0
16.5
18.0
12.7
9.7
00
*The 95% confidence limits of the rate at each station are the predicted rate multiplied and divided by 1.17.
-------
Table 1. (continued) COMPUTER OUTPUT OF PREDICTED RATES FROM ANALYSIS OF VARIANCE OF TOTAL
OXYGEN UPTAKE, INORGANIC CHEMICAL OXIDATION, AND RESPIRATION (ml
oxygen m~2hr~1) DURING FOUR SEASONS AT 5, 10, AND 15°C
00
INORGANIC CHEMICAL OXIDATION*
*— ' ' ' _
5°c io°c i5uc
Stations
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
Oct.
8.8
6.8
9.9
6.2
5.4
6.9
5.5
9.0
4.5
5.7
5.5
3.8
8.4
5.6
6.5
6.5
4.1
4.6
5.8
4.3
5.2
2.0
3.2
Jan.
8.0
6.0
9.1
5.5
4.7
6.1
4.7
8.2
3.7
5.0
4.7
3.0
7.6
4.9
5.7
5.7
3.3
3.9
5.0
3.5
4.5
1.2
2.4
Apr.
816
6.6
9.7
6.0
5.2
6.7
5.3
8.8
4.2
5.5
5.3
3.5
8.2
5.4
6.2
6.2
3.9
4.4
5.6
4.0
5.0
1.8
2.9
Jul.
9.5
7.5
10.6
6.9
6.1
7.6
6.2
9.7
5.1
6.4
6.2
4.4
9.1
6.3
7.1
7.2
4.8
5.3
6.5
4.9
5.9
2.7
3.8
Oct.
10.8
8.7
11.9
8.2
7.4
8.9
7.4
11.0
6.4
7.7
7.5
5.7
10.4
7.6
8.4
8.4
6.1
6.6
7.8
6.2
7.2
4.0
5.1
Jan.
10.0
8.0
11.1
7.4
6.6
8.1
6.7
10.2
5.6
6.9
6.7
4.9
9.6
6.8
7.6
7.6
5.3
5.8
7.0
5.4
6.4
3.2
4.3
Apr.
10.5
8.5
11.6
8.0
7.1
8.6
7.2
10.7
6.2
7.5
7.2
5.5
10.1
7.3
8.2
8.2
5.8
6.3
7.5
6.0
7.0
3.7
4.9
Jul.
11.4
9.4
12.5
8.9
8.0
9.5
8.1
11.6
7.1
8.4
8.1
6.4
11.0
8.3
9.1
9.1
6.7
7.3
8.4
6.9
7.9
4.6
5.8
Oct.
12.9
10.9
14.0
10.4
9.5
11.0
9.6
13.1
8.6
9.9
9.6
7.9
12.5
9.8
10.6
10.6
8.2
8.8
9.9
8.4
9.4
6.1
7.3
Jan.
12.2
10.1
13.2
9.6
8.8
10.3
8.8
12.4
7.8
9.1
8.9
7.1
11.8
9.0
9.8
9.8
7.5
8.0
9.2
7.6
8.6
5.4
6.5
Apr.
12.7
10.7
13.8
10.1
9.3
10.8
9.4
12.9
8.3
9.6
9.4
7.7
12.3
9.5
10.3
10.4
8.0
8.5
9.7
8.1
9.1
5.9
7.0
Jul.
13.6
11.6
14.7
11.0
10.2
11.7
10.3
13.8
9.3
10.5
10.3
8.6
13.2
10.4
11.3
11.3
8.9
9.4
10.6
9.0
10.0
6.8
8.0
*The 95% conficence limits of the rate at each station are the predicted value - 1.9 ml
-2
-1
-------
Table I. (continued) COMPUTER OUTPUT OF PREDICTED RATES FROM ANALYSIS OF VARIANCE OF TOTAL
ORGANIC CHEMICAL OXIDATION, AND RESPffi
) DURING FOUR SEASONS AT 5, 10, AND 15°C
OXYGEN UPTAKE, INORGANIC CHEMICAL OXIDATION, AND RESPIRATION (ml
oxygen m T
-'hr
00
oo
RESPIRATION
5°C
Stations
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
Oct.
4.4
2.6
1.4
3.5
1.6
0.9
2.5
2.9
1.4
1.0
1.6
2.6
1.8
4.3
1.9
2.3
2.6
1.6
2.8
2.0
2.2
1.9
0.3
Jan.
3.6
2.1
1.0
2.8
1.3
0.6
2.0
2.3
1.1
0.7
1.2
2.1
1.4
3.5
1.5
1.8
2.1
1.3
2.3
1.5
1.7
1.5
0.1
Apr.
4.7
2.8
1.5
3.7
1.8
1.0
2.7
3.1
1.5
1.1
1.7
2.8
1.9
4.5
2.1
2.4
2.8
1.8
3.0
2.1
2.3
2.1
0.3
Jul.
6.7
4.2
2.4
5.4
2.8
1.7
4.0
4.5
2.4
1.9
2.7
4.1
3.0
6.5
3.2
3.7
4.2
2.8
4.5
3.2
3.5
3.2
0.8
Oct.
8.1
5.1
3.1
6.6
3.5
2.2
4.9
5.6
3.1
2.4
3.4
5.1
3.8
7.9
4.0
4.6
5.2
3.5
5.5
4.0
4.4
4.0
1.2
10°C 15UC
Jan.
6.8
4.2
2.5
5.5
2.8
1.7
4.1
4.6
2.5
1.9
2.8
4.2
3.1
6.6
3.3
3.8
4.3
2.8
4.6
3.3
3.6
3.2
0.8
Apr.
8.6
5.5
3.3
7.0
3.7
2.4
5.2
5.9
3.3
2.6
3.7
5.4
4.0
8.4
4.2
4.8
5.5
3.7
5.9
4.3
4.6
4.2
1.3
Jul.
12.0
7.7
4.8
9.9
5.4
3.6
7.4
8.3
4.8
3.8
5.3
7.6
5.8
11.7
6.1
6.9
7.8
5.4
8.3
6.1
6.6
6.1
2.1
Oct.
8.3
5.3
3.2
6.8
3.6
2.3
5.1
5.7
3.2
2.5
3.5
5.2
3.9
8.1
4.1
4.7
5.3
3.6
5.7
4.1
4.5
4.1
1.2
Jan.
7.0
4.4
2.5
5.6
2.9
1.8
4.2
4.7
2.6
2.0
2.9
4.3
3.1
6.8
3.3
3.9
4.4
2.9
4.7
3.4
3.7
3.3
0.9
Apr.
8.8
5.6
3.4
7.2
3.8
2.5
5.4
6.0
3.4
2.6
3.8
5.5
4.1
8.6
4.3
5.0
5.6
3.8
6.0
4.4
4.8
4.3
1.3
Jul.
12.3
7.9
4.9
10.1
5.5
3.7
7.6
8.5
5.0
3.9
5.4
7.8
5.9
12.0
6.2
7.1
8.0
5.5
8.5
6.3
6.8
6.2
2.2
-------
Table 2. DEHYDROGENASE ACTIVITY OF SEDIMENTS FOLLOWING
INCUBATION OF SAMPLES WITH GLUCOSE AT 37°C
FOR ONE HOUR
Location
Puget Sound
Lake Washington
Station
24
25
26
27
28
29
30
31
Sediment
layer
0-1
1-2
5-6
0-1
1-2
5 »6
0-1
1-2
5-6
0-1
1-2
5-6
0-1
1-2
5-6
Absorbance/ml
centrifuged sediment
0
0
0
0.01
0
0.01
0.01
0.02
0.03
0
0.01
0.01
0.02
0.02
0.02
0-1 0.06
1-2 0.03
5-6 0.01
0-1
1-2
5-6
0-1
1-2
j 5-6
32
33
1
8
9
0-1
1-2
5-6
0-1
1-2
5-6
0-1
1-2
5-6
0-1
102
5-6
0-1
1-2
5-6
0
0
0
0
0
0
0
0
0.05
0.06
0.13
0.20
0.02
0.03
o.oe
0.12
0.09
0.06
0.12
0.20
0.08
89
-------
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ANNOTATED BIBLIOGRAPHY OF UW CONTRIBUTIONS
RESULTING FROM THIS AND RELATED PROJECTS
Pamatmat, M. M. A continuous-flow apparatus for measuring the metabolism
of benthic communities. Limnol. Oceanogr. 10:486-489, 1965.
Continuous water flow per se over the sediment surface is not absolutely
essential for estimating the rate of benthic oxygen uptake. Practically similar
results are obtained by short-term measurements with bell jars. The impor-
tant point is to minimize underestimation which would result from the develop-
ment of a steep oxygen gradient next to the sediment surface if the experiment
runs too long and the enclosed water is not stirred periodically.
Pamatmat, M. M. Ecology and metabolism of a benthic community on an
intertidal sandflat. Int. Revue ges. Hydrobiol. 53:211-298, 1968.
The highest rates of benthic oxygen uptake have been measured in communities
with high primary productivity and rapid sedimentation of organic matter,
indicating that community metabolism is normally food limited and governed
by its rate of supply. There is a pronounced seasonal cycle of community
metabolism in False Bay which may be caused partly by temperature effect on
metabolism and partly by a seasonal fluctuation in the size of the community.
A study of metabolic temperature adaptation on the community level would appear
to be useful.
Pamatmat, M. M. and D. Fenton. An instrument for measuring subtidal benthic
metabolism in situ. Limnol. Oceanogr. 13:537-540, 1968.
In view of many possible sources of error in shipboard measurements, in. situ
measurements of benthic oxygen uptake are called for, if only to check initial
results of a shipboard method. The in situ instrument was used to depths of
180 m and gave higher estimates of oxygen uptake than measurements with
sediment cores aboard ship. The shipboard method was presumed to be in
error and work was continued to improve it. This is supposed to be the first
paper of a series on oxygen consumption by the seabed.
Pamatmat, M. M. and K. Banse. Oxygen consumption by the seabed. II.
In situ measurements to 180 m depth. Limnol. Oceanogr. 14:250-259,1969.
A summer increase in the rate of oxygen consumption by the sediment in Puget
Sound was partly explained by the rise in temperature. Unexplained variability
was attributed to the increased activity of small organisms following an increase
in the supply of oxidizable organic matter from the plankton. There appears to
be a need for understanding the seasonal response of the benthic community to
temperature, i.e. whether the community adapts, and to what degree it adapts,
101
-------
to seasonal temperature cycle. This study would require shipboard experi-
ments.
Pamatmat, M. M. Oxygen consumption by the seabed. IV. Shipboard and
laboratory experiments. Limnol. Oceanogr. 16:536-550, 1971.
High initial and operational cost of the in situ instrument, difficulties with and
shortcomings of the in situ method made a shipboard method look attractive.
Further work on the development of a shipboard method uncovered sources of
error. Finally a shipboard method for measuring the oxygen uptake of
undisturbed cores of sediment with their overlying water and intact mud-water
interface gave the same results as in situ measurements at 22 m depth.
Hydrostatic pressures up to 18 atm showed no effect on total oxygen uptake of
cores from 180 m depth. Partitioning experiments with intact cores show ed
that oxygen uptake by inorganic chemical oxidation is often greater than
respiratory consumption. Rates of oxygen uptake off Peru and the coast of
Washington and Oregon are given. An attempt to estimate anaerobic metabolism
in the sediment by measuring rates of denitrification in different sediment layers
was successful in detecting denitrification activity but it was not possible to
determine the true level of undisturbed denitrification rates.
Pamatmat, M. M. Oxygen consumption by the seabed. VI. Seasonal cycle
of chemical oxidation and respiration in Puget Sound. Int. Revue ges.
,Hydrobiol. 56:769-793, 1971.
Baseline study of benthic oxygen uptake in Puget Sound. Presents more evidence
that benthic community metabolism is forced by the flux of oxidizable organic
matter to the bottom. There is a seasonal cycle of oxygen uptake independent
of the temperature cycle but the temperature cycle enhances the seasonal cycle
of oxygen uptake which is highest in July, decreases through October to a low
in January and increases from January through April to a high again the
following July.
A correlation was shown between the concentration of reduced substances in the
upper 4 cm of sediment and the rate of oxygen uptake by inorganic chemical
oxidation. Since some of these reduced substances are metabolic by-products
of anaerobic metabolism, it appears that the seasonal cycle of inorganic chemical
oxidation may be indicative of a seasonal cycle of anaerobic metabolism. The
concentration of reduced substances in the surface layer of sediments appears to
be in dynamic equilibrium; deeper in the sediment the concentration increases
with depth, indicating an accumulation. This, together with the observation by
others that benthic oxygen uptake is independent of the length of the sediment core
beyond a few centimeters deep means that the rate of chemical oxidation under-
estimates the anaerobic metabolism in the sediment column.
Pamatmat, M. M. Benthic community metabolism on the continental terrace
102
-------
and in the deep sea in the North Pacific. Int. Revue ges. Hydrobiol., In
press.
The rates of total oxygen uptake, residual oxygen consumption after poisoning
with formaldehyde, and respiration of undisturbed sediment cores from the
continental terrace, the abyssal plains, and the Aleutian Trench of the North
Pacific were measured aboard ship. The rates of total oxygen uptake decreased
with increasing depth of water. Respiratory uptake decreased with increasing
depth to undetectable levels in the deep sea except at stations on the Aleutian
Trench and relatively close to the Washington coast where there was additional
evidence of anaerobic metabolism, especially of denitrification or nitrate
reduction. In most of the North Pacific deep-sea stations, interstitial water
appears to contain dissolved oxygen, there was no accumulation of reduced
by-products of anaerobic metabolism and there was no evidence of denitrification
or nitrate reduction; hence metabolic activity appears to be only aerobic. Even
though the shipboard technique was not sensitive enough to detect aerobic
respiration in most deep-sea core samples, significant metabolic activity in the
sediment has taken place as indicated by the nutrient enrichment of interstitial
water relative to the bottom water overlying the sediment. These observations
emphasize the controlling importance of the rate of supply of oxidizable organic
matter to the bottom in the metabolic activity of the benthos.
Pamatmat, M. M. Anaerobic metabolism in Lake Washington sediments.
Limnol. Oceanogr. In press.
A dehydrogenase assay technique which gives relative estimates of benthic anaerobic
metabolism, primarily of sediment microorganisms, was calibrated by direct
microcalorimetry in order to be able to estimate the natural anaerobic metabolic
activity in the sediment column. Dehydrogeanse activity, which generally
decreased with depth of sediment layer, was detectable to 31 cm. The integrated
metabolic heat release based on the measured dehydrogenase activity was
invariably greater than the metabolic heat release calculated from the rates of
oxygen uptake. Thus, it appears that the rate of total oxygen uptake by the
sediment surface underestimates benthic community metabolism, in the sediment
column, The magnitude of the underestimation should be expected to be large
in organic-rich sediments and may be negligible in oligotrophic waters.
Banse, K. , F. H. Nichols, and D. R. May. Oxygen consumption by the seabed.
III. On the role of the macrofauna at three stations. Vie et Milieu. Suppl.
22:31-52, 1971.
Exemplifies a computational method of determining the fraction of benthic
community metabolism that is due to the macrofauna. Weaknesses in the
approach involve the usual problem of sampling variability, sampling
insufficiency, and the questionable applicability of respiration data from
unrealistic experiments on the effects of temperature and oxygen tension on the
103
-------
rates of oxygen consumption of infauna that are removed from the sediment.
With these possible sources of error in mind, the total macrofauna are
estimated to utilize 20 to 40 per cent of the oxygen consumed by the benthic
community.
May, D. R. The effects of oxygen concentration and anoxia on respiration
of Abarenicola pacifica and Lumbrineris zonata (Polychaeta). Biol. Bull.
142:71-83, 1972.
This paper is the fifth in the series on oxygen consumption by the seabed. It
considers further the problem of realistically estimating the true rates of
oxygen consumption by infauna in nature as distinct fromtheir respiration
under laboratory conditions. L. zonata appears able to regulate its
respiratory rate down to an oxygen concentration of about 2 ml 02/1 while
A. pacifica's respiration is a linear function of oxygen concentration at all
concentrations up to 7 ml 02/1. Both species exhibit an ability to survive anoxia
for several days. It is important to know the actual availability of oxygenated
water to burrowing forms.
Nichols, F. H. Carbon and energy flow through populations of a numerically
dominant macroinvertebrate, Pectinaria californiensis Hartman, in Puget
Sound, Washington, with reference to larger, rarer coexisting species. Ph. D.
Thesis, Univ. Wash., Seattle. 164pp. 1972.
A thorough study of a single species' role and importance in the community at
several stations in Puget Sound. The author concludes that the small but
abundant P. californiensis contributes more greatly to the metabolic processes
and to the food-chain dynamics of the seabed of Puget Sound than do the larger
but rarer echinoderms, Brisaster latifrons and Molpadia intermedia. More
data are presented and discussed relative to the problem of estimating the
fraction of benthic community metabolism that is due to the macrofauna.
104
-US. GOVERNMENT PRINTING OFFICE 1973 545-31Z/159 1-j
-------
1
V
5
Accession Number
V
r\ Subject Field & Group
05A
SELECTED WATER RESOURCES ABSTRACTS
INPUT TRANSACTION FORM
Organization
Department of Oceanos:raT>hY
University of Washington
Seattle, Washington
Title
Oxidation of Organic Matter in Sediments
10
Author(s)
Mario M0 Pamatraat
S. Stephen Jones
Herbert Santa or n
Ash ok Biiagwat
16
Project Designation
16070 EKZ
21
Not.
'g
U.S. Environmental Protection Agency Report
EPA-660/3-73-005, September 1973.
22
Citation
23
Descriptors (Starred First)
Acclimatization, anaerobic condition, benthos, bio-degradation, biochemical
oxygen demand, bottom sediments, humic acids, oxidation, oxygen demand,
organic matter, Pacific Northwest U.S., water pollution effects
25
Identifiers (Starred First)
Anaerobic metabolism, benthic community metabolism, benthic oxygen uptake,
dehydrogenase activity of sediments, oxygen debt, metabolic heat release,
microcalorimetry, Lake Washington, Puget Sound, Sediment nutrient release
07 Abstrac.
Techniques were developed for sampling undisturbed sediment interface, and mea-
suring oxygen uptake by intact sediment cores, dehydrogenase activity of sediment
bacteria, and metabolic heat release by benthic organisms, Lehydrogenaee activity,
a relative measure of anaerobic metabolism, was calibrated by direct microcalori-
metry to provide estimates of actual metabolism under field conditions. The oxygen
debt of sediments was determined by a dichromate method. Laboratory experiments
were conducted to determine the relationship between oxygen uptake, loss of carbon,
and release of silicate, nitrate, ammonia, and phosphate by sediments. The oxygen
consumption at 33 stations in Puget Sound was measured each season to provide base-
line data for this estuary. The original working hypothesis, that total oxygen up-
take represents a measure of total metabolism in the sediment column appears erron-
eous, at least in organically rich sediment where anaerobic metabolism may greatly
exceed aerobic metabolism. As sedimentation rate of oxidizable organic matter
increases, as in cases of organic pollution and eutrophication, anaerobic metabolism
becomes an important process that is measurable by dehydrogenaee assay. In less
organic sediments, the rate of oxygen uptake may be a fair estimate of total
metabolism. Furthermore, it is a useful index of equilibrium conditions among the
various factors that effect the rate of oxygen uptake, e.g. oxygen tension, temp-
erature, turbulence, available metabolizable energy, composition of community8 etc.
Abstractor Mario M. Pamatmat
Institution
Dept. of Fisheries, Auburn University, Auburn, Ala
WR;102 (REV, JULY 1969)
WRSI C
S F M n WITH COPY CDF DOCUMENT, TO; WATER RESOURCES SCIENTIFIC INFORMATION CENTER
1 T U.S. DEPARTMENT OF THE INTERIOR
WASHINGTON, D. C. 20240
r.pni 1 B70.-3 BO-930
------- |