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 HgClo-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-.7mcal
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


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 (HgClg) and stored in the freezer for up to
three months before they could be analyzed.

19


RESEARCH REPORTING SERIES

Research reports of the Office of Research and
Monitoring, Environmental Protection Agency, have
been grouped into five series. These five broad
categories were established to facilitate further
development and application of environmental
technology. Elimination of traditional grouping
was consciously planned to foster technology
transfer and a maximum interface in related
fields. The five series are:

1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research

4. Environmental Monitoring

5. Socioeconomic Environmental Studies

This report has been assigned to the 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 formaticn,
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.


OXYGEN DEBT (ml 05/ml Sediment)

Ww
w a
Ga oa
x= Zz 4 2
o oad na =
2) ao <
a2 >» a> @
= Z—£oa_Fa ? 4
° zS3a0902z20GQaQ0 4,0
wa2 Pu 2F2Dq2
ZHzPSZZFzAHSZ
= oa oO
— bE
Z2e@eozddga
g7 eg ss
| i i L | | I ! {
o Nn + © o ogo 4 rs ge @ 8

(wo) Y3AV1 LNAWIGSS

Figure 21. Concentration of reduced substances versus depth of sediment
layer off the Oregon-Washington coast.

62


Table 11. OXYGEN DEBT (ml 0,/ml sediment) OF THE SEDIMENT IN CLAM
BAY AT THE SITE OF THE FISH REARING PENS IN FEBRUARY

Distance from Sediment Layer
Station fish pens meters 0-1 cm 1-2 cm 3-4 cm
1 15 south 0. 20,0. 23 0.28,0.21 |0.25,0.27
2 30 south 0.24,0.27, | 0.20,0.17 |0.15,0.17,
0.24 0.17
3 45 south 0.15,0.12, | 0.12,0.12 |0.13,0.14
0.07
4 75 south 0.09,0.09 | 0.08,0.10 0.08, 0.08
5 15 north 0.24,0.28 | 0.27,0.26 {(0.23,0.21
0.18
6 30 north 0.10,0.08 | 0.08,0.09 |0.20,0.14,
0.11 0.18
7 45 north 0.05,0.04 | 0.08,0.09 |0.25,0,12,
0.09
8 75 north 0.04,0.03 | 0.24,0.13 |0.11,0,14,
0.16
9 underneath 0.51,0.63 0.63,0.50 §0.51,0.58

73


5)

6)

Sound.

To develop a method suitable for routine measurements of anaerobic
metabolism in sediments.

To perform laboratory experiments that would clarify certain aspects
of benthic community metabolism and material exchange between the
sediment and the overlying water.


89

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

| Absorbance per g Organic Carbon —=—
of dry sediment per cent of dry sediment A:B Ratio
Location Station 0-T cm layer[ 5-6 cm layer 0-T'cm layer| 5-6 cm layer 0-T cmTayer [5-6 cm layer
Puget Sound 1 0.40 0.40 2.94 2.58 0.14 0.16
5 0.19 0.21 1.09 1.20 0.17 0.18
6 0.19 0.89 1.06 0.18
8 0.44 0.46 2.54 2.45 0.17 0.19
10 0.47 0.47 2.54 2.49 0.18 0.19
21 0.33 0.35 1.64 1.64 0.20 0.21
23 0.50 0.52 2.68 2.68 0.19 0.19



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 05/m? 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 Q10'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


Pili jel>
ra
b-LP}--4— y
EEG
& tof BY
sf 7 #3
~~
, dys r
eM SS
RY BR
K i a
eof -$ 2)
FHe ss
Ay Ae

YER.

Figure 7. Detailed drawings of the dashpot showing the bottom end which is
bolted to steel plate center piece.

96


32

28

OXYGEN CONSUMPTION (mi/m?2 per hr )

| ~O- TEMPERATURE

L_ ~&TM~ SALINITY

-®- OXYGEN

[ ~~ IN SITU TOTAL UPTAKE

24h —- IN SITU CHEMICAL OXIDATION

—&—- IN SITU

1 I T

RESPIRATION

Figure 9.

1969 1970

Seasonal cycle of in situ total oxygen consumption, chemical
oxidation, respiration, oxygen tension, salinity, and temper-
ature at station 1.

33

TEMPERATURE (°C)

a

B

2 SALINITY (%o)

CONCENTRATION (mi/1)

O2


DEPTH (cm)

24

26

28

30

32

METABOLIC HEAT RELEASE (m col layer”! hr7')
40 80 120 160 200 240 280 320 360 400

STATION No. 20 —-4—
3 —O—
4 —o—
9 =-a--
14 —-@-—
19 --e-=

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



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 Q'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 Aprii 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 mi/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


(cm)

DEPTH

REDUCED SUBSTANCES (meq/ml sediment)

-0.04 -0.02 0.0 0.02 0.04 0.06
0.4
TT PT
o6 - >100om _
0.8 -— 51 -100m _]
LOR < 50m _
2.0 -- =
4.0 om
6.0 F—- _
8.0 -—- +
10.0 F—- _
is]
20.0 -- 7
| 4 |
Figure 20. Concentration of reduced substances in Puget Sound versus depth

of sediment layer.

61


A SO fe
-

Colder Moonting Plale Jy” Stee! Plate
/ Reguned

Figure 8. Detailed drawing of the bottom cylinder mounting plate.

97


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. ‘he 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 38°C. Under
this condition, the calorimeter exhibited long-term baseline fluctuations of
ag much as +10 microvolts. Furthermore, altbough 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 ievels of metabolic
activity. The original plan was to place a whole sediment core (5.7 em in
diameter and up to 30 em 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 couid not be
used for the purpose that it was designed.

39


SECTION VII

REFERENCES

Armstrong, F. A. J., C. R. Stearns, and J. D. H. Strickland. The
measurement of upwelling and subsequent biological processes by means of
the Technicon AutoAnalyzer® and associated equipment. Deep-Sea Res.
14:381-389, 1967.

Benzinger, T. H. and C. Kitzinger. Gradient layer calorimetry and human
calorimetry. In: Temperature, its measurement and control in science and
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. andJ. 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

81


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.


CONTENTS

Abstract
List of Figures

List of Tables

Acknowledgments

Sections

I Conclusions

TL Recommendations
I Introduction

IV Methods

Vv Results
VI Discussion
Vil References
Vil Appendices

Ait

Page

ili

vii

10

20

76

81

85

o4aat

act fo


TOTAL UPTAKE (mt! 0, /m? per hr )

28

24

ol4

x 23

x TOTAL UPTAKE AT 10 °C IN JANUARY
e TOTAL UPTAKE AT 10 °C IN JULY

i | | |

| 2 3 4 5
ORGANIC CARBON (% of dry sediment)

Figure 11. Rates of total oxygen consumption at 10°C in January and July

versus organic carbon content of the sediment.

36


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


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


EPA -660/3-73-005

Ecological Research Series



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


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 zich 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.

ii


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 je 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


Table 2. (continued) SEASONAL CHANGES IN DISSOLVED OXYGEN (ml/liter),
TEMPERATURE (°C) AND SALINITY (°/oo) OF BOTTOM

WATER AT THE DIFFERENT STATIONS.

Salinity

Station Oct Jan Apr Jul Oct
1 28.99 28.40 27.33 29,14 29.80
2 29.06 28.26 29.27 30.09
3 29.65 29.44 30.18
4 29.91 29.60 28.64 29.57 30.36
5 29.83 29.29 28.74 29.61 30.36
6 29.59 29.32 29,45 30.30
7 30.07 28.88 29.60 30.48
8 30.15 29.03 29.63 30.57
9 30.66 30.12 29.94 30.22 30.86

10 30.65 30.16 29.95 30.39
11 30.69 30,12 30.02 30.46 31.11
12 30.38 29.80 30.63
13 29.76 29.47 28.76 29.76 30.45
14 29.50 28.94 28.58 29.45 30.15
15 29.68 28.97 29.56 30.44
16 29.36 30.00 30.79
17 30.79 30.16 30.10 30.51 30.96
18 30.85 30.24 30.08 30.51 30.86
19 30.83 29.86 30.25 29.80 31.29
20 30.70 30.36 30.49 31.24
21 30.78 30.20 30.48 31. 29
22 30.69 30.40 29.97 30.08 30.91
23 30.47 30.37 30.16 31.04

24


Mikroorganismentatigkeit im Boden. Zeit Pflanzenernahrung, Dungung,
Bodenkunde. 73:1-11, 1956.

Lenhard, G. Dehydrogenase activity as criterion for the determination of
toxic effects on biological purification sy stems. 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. andJ. P. Riley. A modified single solution method for the
determination of phosphate in natural waters. Analyt. Chim. Acta.
27:31-36, 1962.

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
of chemical oxidation and respiration in Puget Sound. Int. Revue ges.
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
press, 1973.

Pamatmat, M. M. Anaerobic metabolism in Lake Washington sediments.
Limnol. Oceanogr. In press, 1973.

Pamatmat, M. M. andK. Banse. Oxygen consumption by the seabed. Il.
In situ measurements to a depth of 180 m. Limnol, Oceanogr. 14:250~259,

1968.
83


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), Hobbie (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


controlled temperature, 1 e. 4 the game temperatures 2 throughout 1 the year. :

Development of: a. Shipboard Method-A shipboard method requires first of
all that representative samples of the benthic comrtunity (sediment plus
inhabitants)-be-brought aboard the ship undisturbed: ..No satisfactory
sampling gear existed at the timé and it was hecessary to invent one.

A multiple corthg devide (Pamatmat 1971a) resulted from this effort and
was subsequently used for taking samples in Puget. Sound in all kinds of
- sediment, offshore from the coritinental shelf with hard-packed sand to
the deep sea with soft ooze, the Aleutian Trench, and in Lake Washington
with its.gyttja type of sediment. The coririg device is des¢ribed in greater
detail together with later modifications in this report. Detailed plans for
its construction are to be found in thea ppendix. . ,

Compatisons of shipboard iheasuréiignts of oxygen uptake by sadivnent cores
with in situ measurements have shown that at the same teniperature the

* shipboard method yields the sate estimate of total uptake and inorganic
chemical oxidation as the in situ. situ technique (Pamatmat 197la, 1971b).

Significance of Reduoed Substances’ in Sediments-The total oxygen uptake by
the seabed is partly due to respiratory tiptake by aerobic organisms and :
partly ‘die to.abiotic chemical oxidation of reduced substances like ferrous, .
manganous,: sulfides, et: In order to understand differences in the rate ~
of abiotic chemiéal uptake, it is hecessary to méagure the. concentration of
these reduced substancés. A technique tot thelr’ estimation was therefore
developed. wy . poe a : Det

Humic “acid Content of Sedtsente-It 4 is well known that only a fraction: of f the
organic matter-in sediments is metabolizable; the refractory portion of
organic matter has beén called humus (Wdksman 1933). One might expect
that the difference between total organic matter and the humus fraction
would therefore représerit the. amount of oxidizable organic matter, This
would be a batter medgure of ‘available food to the benthic community.

Effect of Setiments on Oxygen Tension of, Bottom Water ~The amount of °
reduced substances in the sediment is proportional to its oxygen debt
(Pamatmat 1971b); Hence, ohe would éxpect.that: the spreading of such .
substahces in the water-would affect the oxygén tension. Observations via
underwater television revealed that the concentration of suspended particles _
in bottom water incréased-with intensity: of tidal currents (Pamatmat 1971b). .
Hence, we attempted.to stow: that ‘Fesuspension of -bottom: eboaits le lowers. .

the oxygen tension of the water, .

Dehydrogenase Asay for Measining Angerabi Meiabolism in. 1 Sediments

ag


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 HgClo-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 facie 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


DEHYDROGENASE ACTIVITY OF SEDIMENTS FOLLOWING

Table 2.

INCUBATION OF SAMPLES WITH GLUCOSE AT 37°C

FOR ONE HOUR

2
4
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89


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)
Absorbance per g Organic Carbon
of dry sediment per cent of dry sediment A:B Ratio
Location Station February '72 July '72 February '72 July '72 February '72 | July '72
Puget Sound 24 0.10 0.25 0.89 1.80 0.11 0.14
25 0.42 0.35 2,54 2.19 0.17 0.16
26 0.17 1.59 0.96 0.11
27 0.14 0.20 0.99 1.32 0.14 0.15
28 0.054 0.047 0.45 0.41 0.12 0.11
31 0.74 0.47 3.95 2.67 0.19 0.18
32 0.42 0.44 2,20 2.25 0.19 0.20
33 0.76 0.72 3.94 3.88 0.19 0.19
Clam Bay 0.067 0,033 1.02 0.24 0. 066 0.14
Snohomish 29 0.039 0.18 1.17 1.53 0.033 0.12
River 30 0.015 0.018 0.29 0.15 0.052 0.12
Lake 1 1.54 2.62 7.55 14.06 0.20 0.19
Washington} 8 0.79 0.73 6.29 6.01 0.13 0.12
9 0.78 0.78 6.79 6.22 0.14 0.13
Blanks®) 0.020 0.0
0.028 0.0

) Sediment combusted for 2-3 hr at 500°C


Table 3. ANALYSIS OF VARIANCE ON MEASUREMENTS OF TOTAL UPTAKE
(LOGARITHMICALLY TRANSFORMED DATA)

Degrees of Variance
Source Sum of Squares Freedom Mean Square | Ratio (F)
Mean 1942.83 1 1942.83 26462. 57
Temperature| 28.79 2 14.40 196.07°
Season 8.69 3 2.90 39.47
Station 38.42 22 1.75 23.79%
Error 40.97 558 0.073

aP<0,01

30


ab

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 July September February July

24 Dissolved oxygen 6.15 5.50 5.50
Temperature 10.18 6.91 10.53
Salinity 28.34 29.06 28.38
Total carbon 1.60 0.89 1.80

Sand fraction 37 59 35
25 Dissolved oxygen 4,90 6.00 4.45
Temperature 9.47 6.70 9.33
Salinity 29.88 29.66 29.66
Total carbon 2.15 2.54 2.19

Sand fraction 15 14 yal
26 Dissolved oxygen 5.60 7.40 5.20
Temperature 9.74 7.12 9.99
Salinity 29.15 29.31 28.91
Total carbon 0.97 1.59 0.96

Sand fraction 57 AT 66
27 Dissolved oxygen 4.80 6.80 4.55
Temperature 8.97 7.36 9.16
Salinity 29.85 29.76 29.58
Total carbon 1.91 0.99 1.32

Sand fraction 15 20 17



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

Dehydrogenase activity (absorbance/gram dry sediment)

Substrate Replicates Average
Sodium citrate 1.17, 1.18, 1.23 1,19
Sodium malate 0.89, 0.89, 0.93 0.90
Sodium lactate 0.66, 0.77, 0.70 0.71
Sodium succinate 0.57, 0.57, 0.58 0.57
Sodium pyruvate 0.01, 0.02, 0.01 0.01
Glucose 0.45, 0.46, 0.48 0.46

47


ey

Table 6. (continued) DISSOLVED OXYGEN (mi/liter) TEMPERATURE (°C), SALINITY (0/00) TOTAL CARBON
(%) AND SAND FRACTION (%), AT STA'TIONS 24 TO 33 IN PUGET SOUND.

Station duly September Pebruary_ ; ; July

28 Dissolved oxygen 8.65 5.75
Temperature 12.18 6.52 11.06
Salinity 28.58 28.01 26.86
Total carbon 0.03 0.45 0.41
Sand fraction 96 53

29 Dissolved oxygen 4.35 8.90 7.00
Temperature 13.59 3.90 13.16
Salinity 20.96 2.12 11.54
Total carbon 0.85 1.16 1.52
Sand fraction 85 63

30 Disscived oxygen 5.55 6.15 8.00
Temperature 14.04 3.74 10.76
Salinity 6.00 0 0
Total carbon 0.29 0.14
Sand fraction 96 100

31 Dissolved oxygen 2.50 3.10 3.80
Temperature 8.66 9.14 8.28
Salinity 29.99 30,30 29.82
Total carbon 2.99 3.95 2.67
Sand fraction 5 3



Table 10. RATES OF TOTAL OXYGEN UPTAKE, INORGANIC CHEMICAL
OXIDATION, AND RESPIRATION (ml/m* per hr) AT 10°C IN
CLAM BAY AT THE SITE OF THE FISH REARING PENS IN

FEBRUARY
Distance from Total Chemical
Station fish pens (meters) Uptake Oxidation | Respiration
1 15 south 36,42,31 22 14
2 30 south 22,21 13 8
3 45 south 14,10,11 10 4
4 75 south 30,12,14 13 17
5 15 north 11,14 9 5
6 30 north 24,14 14 5
7 45 north 17,17,20 1 9
8 75 north 18,19,10,19 12,7 6,3
9 underneath 130,120 44 86

72


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-
ation of sludge activity - a possibility of controlling activated sludge plants.
Proc. 3rd Int. Conf. Water Pollution Research, Munich, 1966, p. 94-97.

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
of sludge activity - a possibility of controlling activated sludge plants. Proc.
3rd Int. Conf. Water Pollution Research, Munich, 1966. p. 98-102.

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
associated with the lactic fermentation of glucose. J. Bacteriol. 82:685-690,
1961.

Harrison, M. J., R. T. Wright, and R. Y. Morita. Method for measuring
mineralization in lake sediments. Applied Microbiol. 21:698-702, 1971.

Hobbie, 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.

Krumbein, 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. 61p.

Lenbard, G. Die Dehydrogenaseaktivitat des Bodens als Mass flr die

82


Table 4. ANALYSIS OF VARIANCE ON MEASUREMENTS OF CHEMICAL
OXIDATION (UNTRANSFORMED DATA)

Degrees of Variance

Source Bum of Squares Freedom Mean Square Ratio (F)
Mean 10195.50 1 10195. 50 2827.75
Temperature 623.35 2 311.68 86.447
Season 76.47 3 25.49 7.07%
Station 1008.64 22 45.85 12.722
Error 1085. 26 301 3.6055
®p < 0.01

37


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


66

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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


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, malate, 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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weight stands.

94


SECTION V

RESULTS

ENVIRONMENTAL PARAMETERS

The location of 33 stations in Puget Sound are shown in Fig. 4. Ail the
measured parameters at the first 23 stations are shown in Tables | 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


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98


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Figure 6. Total oxygen uptake versus temperature at various stations in
July.

28



2.0

LS

(g)

1.0

DRY SEDIMENT

05

i i

) 6 °

7~ fo}
(Q0U0G10SqD) ALIAILLOW 3SVN39OYNOAH3G

14

Figure 16. Dehydrogenase activity as a function of amount of sediment of two
different levels of metabolic activity.

52


1 | Accession Number 2 | Sublect Fietd & Group

OSA SELECTED WATER RESOURCES ABSTRACTS
Ww INPUT TRANSACTION FORM
0:

ganization
Department of Oceanography
University of Washington
Seattle, Washington

6 | tHe

—_ Oxidation of Organic Matter in Sediments

10 | Authors) 16 | Project Designation

—— Mario Mo Pamatmat 16070 EKZ
R. Stephen Jones D1] Noe .
Herbert Sanborn U.S. Environmental Protection Agency Report
Ashok Bhagwat EPA-660/3-73-005, September 1973.

22 Citation

23 | Descriptors (Starred First)

Acelimatization, anaerobic condition, benthos, biodegradation, biochemical
oxygen demand, bottom sediments, humic acids, oxidation, oxygen cemand,
organic matter, Pacific Northwest U.S., water pollution effects

25 | [aentifiers (Starred Fiest)

—— 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

97 | Absirac._ ; ; - ;

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. Dehydrogenase 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-
teke 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 metaboliam
becomes an important process that is measurable by dehydrogenase assay. In lesa
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 community, ets.

Abstractor wanio M. Pamatmat Tnstitationn a nt, of Fisheries, Auburn University, Auburn, Ala

Wait02 (REV. JULY 1960) SEND, WITH COPY @F DOCUMENT, TO: WATER RESOURCES SCIENTFIC INFORMATION CENTER
wRaic ? Uis, DEPARTMENT OF THE INTERIOR
WASHINGTON, D.C, 20240

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Figure 24. Effect of tidal current during ebb tide on the gradient of oxygen
concentration above the bottom at the entrance to Port Madison
(122930.2'W, 47°43.5'N, 104 m depth). The calibration
constants for the electrodes at the 3-cm, 33-cm, and 93-cm
levels were 1.02, 1.02, and 0.93 ml 0,/liter per mv,
respectively.

70


Table 2. SEASONAL CHANGES IN DISSOLVED OXYGEN (n)/ /iitar). TEMPERA:
TURE (°C) AND SALINITY (°/o0) OF BOTTOM WATi a AU LAH DER
FERENT STATIONS.

Dissolved oxygen

Station Oct Jan Apr Jul Oct
1 4,50 6.55 7.15 6.50 5.55
2 4.20 5.95 6.65 4.35 4.85
3 4,25 5.85 4,95
4 4,00 5.90 5.85 5.45 5.10
5 4.40 5.20 5.95 6.15 4.65
6 3,75 6.15 4.70 4,45
a 3.90 5,55 5.40 4.55 3.95
8 3.90 5.50 4,65 4,95
9 3.55 5.25 5.45 4.60 4.50

10 3.95 5.16 5.45 4.65
ii 3.95 5.80 5.45 4,60 4,35
12 5.75 4.80
13 4,20 5.50 6.30 5.80 4,65
14 5.50 6.00 6.60 6.60 5.05
15 3.90 6.10 6.25 5.80 4,95
16 5.20 5.50 4,50 4.40
17 3.95 5.30 5.45 4.65 4.75
18 3.90 6.10 5. 60 6.30 4.20
19 3.95 5.20 5.35 4.80 4.65
20 3.95 5.10 4.10 4,75
21 3.70 5.60 4,60 4,25
22 3.00 2.70 1.60 2.65 3.35
23 4, 20 2,55 oa} 3.80

22


(continued) COMPUTER OUTPUT OF PREDICTED RATES FROM ANALYSIS OF VARIANCE OF TOTAL

Table 1.

, INORGANIC CHEMICAL OXIDATION, AND RESPIRATION (ml

) DURING FOUR SEASONS AT 5, 10, AND 15°C

OXYGEN UPTAKE
oxygen m72hr7

INORGANIC CHEMICAL OXIDATION*

T5°C

10°C

5OC

Jan. Apr. Jul. Oct. Jan. Apr. Jul. Oct. Jan. Apr. Jul,

Oct.

Stations

9.5 10.8 10.0 10.5 11.4 12.9 12.2 12.7 13.6

7.5
10.6

8.6
6.6

8.0

8.8
6.8

9.9

8.5 9.4 10.9 10.1 10.7 11.6
12.5

11.6

8.0

11.

6.0

13.2 13.8 14.7

14,0

11.9

9.7

1
5.5

10.1 11.0

9.6
8.8
10.3

10.4

8.9
8.0
9.5

8.1
11

8.0

7.4
6.6

8.2

6.9
6.1
7.

6.0
5.2

6.7

6.2

10.2

9.3

9.5
11.0

a1
8.6

7

7.4
8.9

4.7

5.4
6.9

11.7

10.8

1
6.7
10.2

6.1

10.3
13.8

9.4
12.9

8.8
12.4

9.6
13.1

7.2
10.7

TA
11.0

6.2
9.7

5.3
8.8

4.2

8.2

4.7
3.

5.5
9.0

87

9.3
10.5

8.3
9.6

7.8
9.1

8.6
3.9

6.2 7.

7.5
7.2

5.6
5.5
10.1

6.4
7.7

7

4.5

8.4
8.1

5.0 5.5 6.4 6.9
5.3 6.2

4.7

5.7

10
11

10.3

9.4
7.7
12.3

9.6

6.7
4.9

7.5

iro)

8.6
13.2

7.1
11.8

7.9

12.5

6.4
11.0

3.5 4,4 5.7
8. 10.4

3.0

3.8

12
13

9.6
6.8
7

9.1

2

7.6

8.4

10.4

9.5
10.3

9.0
9.8

9.8
10.6

8.3

7.3

7.6
8.4

8

6.3
7.1

5.6 4.9

14
15
16
17
18
19

11.3

9.1

6.2
6.2

5.7

6.5
6.5

9.8 10.4 11.3

10.6

1
6.7

7.6
5.3

7.2
4.8

5.7
3.3
3.9

8.9

8.0
8.5
9.7

7.5
8.0

9.2

8.2

5.8

6.3

6.1

3.9

4.1

9.4
10.6

8.8

7.3
8.4

5.8
7.0
6.9

6.6
7.8

6.2
7.2

5.3

4.4

4.6

9.9

7.5

6.5
4.9

5.6
4.0

5.0

5.8

9.0
10.0

1

8.
9.1

7.6

8.6

8.4
9.4
6.1

6.0
7.0

3.7

5.4

3.5
4.5

4.3

20
21

9
4.6

5.9
2.7
3.8

5.0

5.2
2.0

5.9 6.8

5.4
6.5

3.2
4.3

1.8 4.0
5.1

2.9

1.2

22
23

8.0

7.0

7.3

5.8

4.9

2.4

3.2

*The 95% conficence limits of the rate at each station are the predicted value + 1.9 ml 0g m7? pr “4


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,
é.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

3


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/; ‘m? 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 HS.

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

7


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

TT


12

February.

Rates of total oxygen uptake, inorganic chemical
oxidation and respiration (ml 09 g -lpgr-l) per cent
organic carbon, and nutrient concentration of overlying
water (u g-at 17}, during a 43-day experiment.

vil

74


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 from their 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. 164 pp. 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 1:73 S40-31e/159 es


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.

15


e se
24
3°
a
| ay
i ai
38
y
A

Figure 11. Detailed drawing of a PVC coupling piece that holds a 15-inch
long coring tube in place.

100

4 Reguired


Pag e

Figure 11. Coupling piece that holds a 15-inch long coring 100
tube in place.

Annotated Bibliography 101

85a


20
METABOLIC HEAT RELEASE (m cal g! hr! )

0.037 + 0.046 X
0.166 + 0.030 X

—e— Y
—o— Y

| i t L 1 No
oO [o} o fo} wo fo}
N Nn - oO

(juawipes kip 6 aounquosqo) ALIALLOV ASVN390NCAH3G

Figure 17. Dehydrogenase activity as a function of the rate of metabolic
heat release.

54


2.0

15

10

0.20 M VS 0.04M SODIUM CITRATE
DEHYDROGENASE ACTIVITY (absorbance gq! dry sediment )

0.04M SODIUM CITRATE VS.
NO ADDED SUBSTRATE

0.5

1 1
2 © °o 0 ° fre) °
“ a A a = ro)

(uawipas Aup 1-6 g0uDqsOSqD) ALIAILOV ASVNA9OYGAH]SG

oO

Figure 14, Dehydrogenase activity at 0.04 M sodium citrate as compared to
dehydrogenase activities at 0.20 M sodium citrate and without
added substrate.

50


way initially. Each catcher, a solid rubber ball (F), 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. 1c). 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 too 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 197la). 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 stéady decline in oxygen tension is noted.
Then some of the’ cores care 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


Py

Table 5. (continued) RATES OF TOTAL OXYGEN UPTAKE, INORGANIC CHEMICAL OXIDATION, AND
RESPIRATION (ml/m? per hr) AT STATIONS 24 TO 33 IN PUGET SOUND.

Station July September February July
29 Total uptake 61.2,49.4, 48.2 58.2,86.5,57.9 29.6,37.9,31.1
61.7,
Chemical oxidation 12.3,15.2 5.0,15.6 10.3,16.0
Respiration 48.9,34.2 52.9,46.1 19.3,21.9
30 Total uptake 23.0, 23.0 28. 2,30.5,39.5 6.9,5.7,8.9
30.6 7.7
Chemical oxidation 19.4 7.5,6.9 5.5
Respiration 3.6 23.0,32.6 0.2
3k Total uptake 7.2,10.3,7.7 8.1,16.5,13.7 8.6,6.5,6.0
5.9 6.4
Chemical oxidation 7.6 7.9.9.8 3.8
Respiration 0 8.6,3.9 2.2
32 Total uptake 11.6,8.6 7.0,8.5,7.7,4.9 13.4,14.1,10.5
15.0
Chemical oxidation 5.4 5.9,6.6 4.0,4.1
Respiration 6.2 2.6,1.1 6.5,10.9
33 Total uptake 13,2,12.9,7.6 10.8,12.2,9.9 13.3,12.3,13.6
14.1, 14.5
Chemical oxidation 8.6 7.8,10.6 8.1,7.8
Respiration 4.6 4.4,3.5 4.2,6,7


THERMAL EMF (mv)

CALORIMETER AT 9.9°C 3.29 x104 OHM
RESISTANCE HEATER SUSPENDED IN AIR = 4

CALORIMETER AT 19.5 °C 1200 OHM RESISTANCE,
© “HEATER IMMERSED IN WATER

» CALORIMETER AT 19.5 °C 329x107 OHM
RESISTANCE HEATER SUSPENDED IN AIR 4

CALIBRATION CONSTANT: 062 weal /sec/ur

Figure 12.

10 20 30 40 50
q (cal/sec) x 104

Calibration line for the microcalorimeter at 9.9°C and 19.5°C.

45


PL

Table 12. RATES OF TOTAL OXYGEN UPTAKE, INORGANIC CHEMICAL OXIDATION AND RESPIRATION
(ml 09 g -lnr7))pER CENT ORGANIC CARBON, AND NUTRIENT CONCENTRATION OF
OVERLYING WATER (pg~at 1") DURING A 43-DAY EXPERIMENT

Time Total Chemical Respir- | Per Cent
Hours Uptake Oxidation ation Carbon Si04 NOg NHg P04
0 30 18 12 2.18 50 29 0.2 1.9
21 i 7.9 3.1 2.09 110 31 8.1 2.8
44 7.5 5.5 2.0 2.01 250 49 3.0 3.0
68 7.2 4.8 2.4 2.03 145 31 2.6 3.4
140 5.9 5.9 0 2.06 235 46 0.0 5.3
380 7.2 4.6 2.6 1.89 355 70 O.1 10.2
572 3.3 0.9 2.4 1.96 370 60 --- 11.9
TAL 6.4 2.9 3.5 1.94 400 88 0.0 13.3
1030 5.1 2.0 3.1 1.85 — -- --- 18.6



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:

7


N
°

a
T
L

DEHYDROGENASE ACTIVITY
(absorbance g@! dry sediment )
a 5

fo) L i l
ie) 05 10 AS 20 .25 30

MOLAR CONCENTRATION

Figure 13. Dehydrogenase activity as a function of sodium citrate concentration.

49


10

11

12

FIGURES

The multiple corer shown in different stages of opera-
tion.

Cross-section of microcalorimeter and environmental-
temperature-control system.

Chart of Lake Washington showing 10-m isobaths and
location of stations.

Chart of Puget Sound showing location of 33 stations.

Total oxygen uptake versus temperature at various sta-
tions in January.

Total oxygen uptake versus temperature at various
stations in July.

Predicted rates of total oxygen uptake and chemical
oxidation at station 1 as functions of temperature and
season.

Predicted rates of total oxygen uptake and chemical
oxidation at station 23 as functions of temperature and
season.

Seasonal cycle of in situ total oxygen consumption,
chemical oxidation, respiration, oxygen tension,
salinity, and temperature at station 1.

Seasonal cycle of in situ total oxygen consumption,
chemical oxidation, respiration, oxygen tension,

salinity, and temperature at station 23.

Rates of total oxygen consumption at 10°C in January and
July versus organic carbon content of the sediment.

Calibration line for the microcalorimeter at 9. 9°C and
19.5°C

iv

Page
11

16

18

25

27

28

31

32

33

34

36

45


substances, and therefore in higher rates of chemical oxygen consumption.
DEHYDROGENASE ACTIVITY VERSUS REDUCED SUBSTANCES

The absorbance of duplicate HgC19-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 Ey of the medium, the lower the Ej, (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


Table 1. DEPTH AND MEASURED PARAMETERS OF THE SEDIMENT AT
DIFFERENT STATIONS

Depth | Totalal” Organic? | Carbonate’

Statior} range | carbon} carbon carbon Sand | %Silt} Clay
1 7-11 2,85 2.65 0.20 10 68 23
2 10-13 2.95 2.95 0 5 78 17
3 25-29 1.90 1.90 0 87 8 5
4 45-55 1.30 1.30 0 41 47 12
5 28-33 0.80 0.65 0.15 63 28 9
6 25-29 2.60 2.60 0 5 70 25
7 74-76 2.45 2.30 0 3 70 27
8 14-14 2.40 2.40 ) 14 68 17
9} 204-209; 2.50 2.50 0 4 68 28

10 | 187-189; 2.45 2.45 0 13 61] 26
11 | 250-252) 2.00 1.85 0.15 53 34 13
12 | 181-221; 0.20 0. 20 0 96 2 2
13 10-13 4,45 2.80 1.65

14 10-12 3.70 3.70 0 7 73 20
15 36-41 2.65 2.50 0.15 17 64 19
16 31-35 9.90 0.90 i) 54 39 7
17 | 206-229; 2.15 1.85 0.30 4 70 26
18 | 187-204) 2.10 1.85 0,25 9 66 25
19 | 259-299} 1.55 1.40 6.15 41 48 11
20 | 221-224] 0.40 0.40 ) 87 9 4
21 | 204-241) 1.95 1.85 9.10 10 67, 23
22 | 118-121] 2.40 2.25 6.15 2 68 29
23) 177-182) 2.55 2.35 0.20 2 67 31

anak. wee ee

@average percent of dry sediment.

21


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


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 02 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 13.3°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


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 dichromate by:this procedure.. .It is
possible, however, that in the drying. process volatile organics that. would:
have reduced the dichromate had been lost; invahy 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 isifiltered,. 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 ina
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 triphenyltetrazolium-chloride-glucose: solution at-37°C in a water bath for
one hr.: The-reaction was stopped and the formazan simultaneously
extracted. with absoluteiethanol. » The extract was centrifuged before

reading its. optical density at-483nm.- 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 andincubation at 10°C... Qn the basis of those
findings; the. following concentrations of reagents and procedure were
established: (1) TTC. solution. ~- 1-g 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 HC1 in 1 liter of distilled water, pH
adjusted to 8.4; (3) sodium.citrate solution —-.74-g¢ of sodium citrate in 1.0,
liter of distilled: water; -(4): saturated:mercuric chloride (HgClg) solution; (5)
absolute ethyl alcohol.

14


oct’ TOTAL UPTAKE | i

JAN. 4 7 4
APR" "

vuL “ 4
OCT., CHEMICAL OXIDATION a

JAN " ”
APR
JUL

20

OXYGEN CONSUMPTION (mi/m@per hr)

16 4
—_—A
-7— —3
er
= -T=j=7TY¥ 4
y’ ey Cee ar ae
ee aan
a ae DS 4
— —_
eee o-
_ wa 4
8 i | aI
5 10 15

TEMPERATURE (°C)

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


iN

°

°
@
T

0.6

°
b
T

BLANK (absorbance g! dry sediment)
Oo
nN
T

T

4 NO SUBSTRATE VS 0375 M SODIUM CITRATE

4a

1

7 4 0.2M SODIUM CITRATE VS .0375M SODIUM CITRATE 47

1

Figure 15.

0.5

BLANK (absorbance gq’ dry sediment )

1.0

Effect of substrate concentration on the absorbance of mercuric-

chloride~-treated blanks.

51


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 EX PERIMENTS

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 pericd. 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 HgClg, 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
inerease in 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
game 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


Pamatmat, M. M. andD. Fenton. An instrument for measuring subtidal
benthic metabolism in situ. Limnol. Oceanogr. 13:537-540, 1968.

Precht, H. Concepts of the temperature adaptation of unchanging reaction
sy stems of cold-blooded animals. In: Physiological adaptation, C. L.
Prosser (ed.). Amer. Physiol. Soc., Wash. D.C, 1958. p- 50-78.

Prosser, C. L. General summary: the nature of physiological adaptation.
In: Physiological adaptation, C. L. Prosser (ed.). Amer. Physiol. Soc.,
Wash., D. C. 1958. p. 167-180.

Reeburgh, W. S. An improved interstitial water sampler. Limnol. Oceanogr.
12:163-165, 1967.

Shapiro, J., W. T. Edmondson, and D. E. Allison. Changes in the chemical
composition of sediments of Lake Washington, 1958-1970. Limnol. Oceanogr.
16:437-452, 1971.

Smith, K. L., Jr. Structural and functional aspects of a sublittoral community.
Ph. D. Thesis. Athens, Univ. Georgia, 1971. 160 p.

Smith, K. L., Jr., K. A. Burns, andJ. M. Teal. In situ respiration of
benthic communities in Castle Harbor, Bermuda. Marine Biology. 12:196-199,
1972.

Sorokin, Y. I., and H. Kadota (eds.). Techniques for the assessment of
microbial production and decomposition in fresh waters. IBP Handbook No.
23. Oxford, Blackwell Scientific Publ., 1972.

Stevenson, I. L. Dehydrogenase activity in soils. Can. J. Microbiol.
5:229-235, 1959.

Volkmann, C. M. and C. H. Oppenheimer. The microbial decomposition
of organic carbon in surface sediments of marine bays of central Texas gulf
coast. Publ. Inst. Mar. Sci., Univ. Texas. 8:80-96, 1962.

Waksman, S. A. On the distribution of organic matter in the sea bottom and
the chemical nature and origin of marine humus. Soil Sci. 36:125-147, 1933.

Wood, L. W. The role of estuarian sediment microorganisms in the uptake

of organic solutes. Ph. D. Thesis. Raleigh, North Carolina State Univ. ,
1970. 75 p.

ZoBell, C. E., F. D. Sisler, and C. H. Oppenheimer. Evidence of biochem-
ical heating in Lake Mead mud. J. Sed. Petrol. 23:13-17, 1953.

84


ml O5/mn? perv 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 Janusry. The seasonal eycle is likewise enhanced by the
ternperature 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 cespiration. 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 50C (r = 0.34,

df. = 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


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 hetween 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 he 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.

73


DEPTH (cm)

DEHYDROGENASE ACTIVITY (absorbance g7' dry sediment )

ine) L5 2.0 25 3.0 35 40 4.5 5.0 5.5

° 0.5
ie}

4
STATION No. 20 ———= 7

30

22 4 —o 4
ions ated

24 140 =O = 4
19

26 7

28 ;- 4

SO, +

32 i i ! 4 i I | i l 1

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
have overlapped.

where they would


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


lage]
448)

48°

45"
NAUTICAL MILES
a7 faz"
30 30
Ist is’
15" 123° 45! 122°30! \5° 122°

Figure 4. Chart of Puget Sound showing location of 33 stations. KX marks the
location of station where the effect of scouring on oxygen tension of
bottom water was observed.

25


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 4%
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 *
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. asa +
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 +
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


OP

HATES OF TOTAL OXYGEN UPTAKE, INORGANIC CHEMICAL OXIDATION, AND RESPIRATION
(ml/m? per hr) AT STATIONS 24 TO 33 IN PUGET SOUND.

Station _ July September February July
24 Total Uptake 14.5,12.4,14.8, 14.2,7.9,8.0, 16.7,15.5,12.2,
10.3,15.1 13.9 13.5
Chemical oxidation | 7.0 9.1,7.0 6.8,7.7
Respiration 7.5 5.1,1.0 9.9,5.8

26

27

Total uptake

Chemical oxidation
Respiration

Total uptake

Chemical oxidation
Respiration

Total uptake

Chemical oxidation
Respiration

Total uptake

Chemical oxidation
Respiration

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,

14.5,14.6,12.8
15.

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

13.4,14.3,15.8
14.
+7,7.0

-7,8.8

an

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



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 ONAR.

viil


o nN

OXYGEN CONSUMPTION (mism® per fr)
b

Att
T T T LS
—O— TEMPERATURE wt
[~ —4— SALINITY 4 « EG
- E2z
-° OXYGEN EzS
[- —S— _INSITU TOTAL UPTAKE 4a 2 °
—O-—- IN SITU CHEMICAL OXIDATION a’o
[- —@— IN SITU RESPIRATION a4 2 315
i
10 4
~ _ 293
‘e-
[- _- a, 48 2
i a----- a— -_ + {
L | | 27
OcT JAN APR 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


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 B°C, another set at 10°C, and still another set at 15°C. This
requires a large number of replicate cores. Ina 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°, 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 HgSO, and 10 ml of 0.01 N
(0.02 N with highly reduced sediments) KgCrg0z7 to 40 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


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


14

15

16

17

18

19

20

21

22

23

24

Dehydrogenase activity as a function of sodium
citrate concentration.

Dehydrogenase activity at 0.04 M sodium citrate as
compared to dehydrogenase activities at 0.20 M sodium
citrate and without added substrate.

Effect of substrate concentration on the absorbance of
mercuric~chloride-treated blanks.

Dehydrogenase activity as a function of amount of sediment

of two different levels of metabolic activity.

Dehydrogenase activity as a function of the rate of
metabolic heat release.

Vertical distribution of dehydrogenase activity at six
stations in Lake Washington in October 1972.

Vertical distribution of metabolic heat release at six
stations in Lake Washington in October 1972.

Concentration of reduced substances in Puget Sound
versus depth of sediment layer.

Concentration of reduced substances versus depth
of sediment layer off the Oregon-Washington coast.

Vertical distribution of the concentration of reduced
substances in Lake Washington sediments.

Rates of inorganic chemical oxidation of undisturbed

sediment cores versus average concentration of reduced

substances in the 0-1, 1-2, and 3-4 cm layers.

Effect of tidal current during ebb tide on the gradient of

oxygen concentration above the bottom at the entrance to

Port Madison (122°30.2'wW, 47°43.5'N, 104 m depth).

v

49

50

51

52

54

56

58

61

62

63

65

70


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.
Ill. 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


DEPTH (cm)

TOTAL REDUCED SUBSTANCES (meq mi!x1072)

ie) 2 4 6 8 ire) 12 14 16 18
0 T T T
5 4
10 4
15 4
20 4
STATION No. 2 —~—
25 3-~oO— 4
4-—-0—
9 --&n =
30- 4
14 ~-@--
19 --e-—
ggL Lb bt
Figure 22. Vertical distribution of the concentration of reduced substances in

Lake Washington sediments.

63


LAKE WASHINGTON
4 bas"
4 -
7 3 MSUANITA GAY
4 were | ao
union ext NUEBSTER // 7
1 BELLEVUE r
i
12
4 . Las:
14 ae
15 AQ
18
4 16 L
o, |
; Feron STP 80
a2e
LL?
19 s* 10!

Figure 3. Chart of Lake Washington showing 10-m isobaths and location of
stations.

18


6

7

heat release of undisturbed sediments.

The measurement of the rate of sedimentation of particulate
organic matter in different areas and its relationship to the
level of benthic community metabolism.

Long-term experiments on the quantitative relationship between
anaerobic metabolism and the loss of organic carbon in sediments.

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,

Laboratory experiments on temperature acclimation by benthic
communities.


Table 2 (continued). SEASONAL CHANGES IN DISSOLVED OXYGEN (ml/liter),
TEMPFRATURE (°C) AND SALINITY @/oo) OF BOTTOM

WATER AT THE DIFFERENT STATIONS,

Temperature

Station Oct Jan Apr Jul Oct
1 14.60 7.74 9.23 15.62 12.56
2 13, 28 8.22 8.92 13.49 12.44
3 13.12 13.29 12.29
4 12.40 8.66 8.75 12.04 11.93
5 12.39 8.61 8.67 12.85 11.92
6 13.26 8.46 12.18 12.33
7 12.10 8.82 8.68 11.57 11.82
8 11.94 8.88 12.40 11.58
9 11.04 8.54 8.47 10.00 10.72

10 10.73 8.32 8.47 10.56
11 10.54 8.46 8.46 10.72 10. 20
12 11.50 12.29 11.15
13 8.30 9.24 13.84 11,92
14 12.97 8.05 9.64 15.39 11.45
15 12.37 8.26 8.94 12.46 11.30
16 8.54 8.69 11.14 11.04
17 10.41 8.28 8.45 10.74 10.49
18 10.36 8.20 8.46 10.66 10.62
19 10.36 8.76 8.40 10.77 9.94
20 10.44 8.40 10.88 9.95
21 10.43 8.30 10.67 9,92
22 11.04 8.84 9.23 9.08 10.76
23 9. 44 9.60 9.18 10.14

we
oO


ple

6"dlig

a
Ay steel

7 oF 7

Figure 2. Side view of multiple corer showing cylinder support.

91


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

m72hr7ly 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 Daghpot 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


SECTION IIT

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
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 x
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

5


wy

FID Tube,
Lora as si

alt

weld 7

Figure 1. Top view of multiple corer showing four cross braces on top welded
to a center piece of 3-inch long cylinder which holds the upper end
of the dashpot in place. The four cylinder supports are welded to a
center piece of 1/4-inch steel plate onto which the bottom of the
dashpot is bolted.

90


56

“9 omg

“A[quiesse por pus uoisid jo Sutmearp potiejed

Pstoo and Rod
ANd! To be -O1L" hess Haw ”
TD. of Brass Take na / Ke
S| / on
NSE
7 fo | bi Evel View
Stainhas ‘
Y
NG ¥e 4
— 34"
v Heke



6g

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 9, = 4.8 cal

Core Dehy drogenase activi!
Station length (cm) Total 09 uptake Chemical oxidation Range Average
2 6 0.27, 0.34, 0.26, 0.10, 0.11, 0.12 0.37-0.54 0.39
0.16, 0.16
3 32 0.38, 0.42, 0.38, 0.24, 0.26 0.88-1.6 1.3
0.321
4 21 0.41, 0.45, 0.28, 0,18, 0.18, 0.20 2,8-3.6 3.0
0.33, 0.38
9 21 0.35, 0.28, 0.22, 0.12, 0.13 1.72.8 2.1
0.19, 0.17 -
14 21 0.45, 0.39, 0.36, 0.11, 0.08, 0.11, 3.4-4.7 4.0
0.36, 0.32, 0.31, 0.10, 0.11
0.27, 0.24, 0.19]
0.19
19 16 0.36, 0.36, 0.28, 0.21, 0.23, 0,24 0.39-0. 46 0.41
0.25



I T T T I I To! T
°
- 4
e
a 4
°
° =
_~ e
r LL °
5 10 ° °. 4
aL ° °
~ e e :
S ° ° °
= 8fr- e 4
E e
> e
Sob ° 4
= °
Kb
< ee °
a 6k ° 4
3 e oe
=)
a — 4
iS)
= e
w 4b e _
x=
1S)
2 4
t- on
Py a a
-.04 -.02 ce) -02 04
REDUCED SUBSTANCES (mea/mi of sediment)
Figure 23. Rates of inorganic chemical oxidation of undisturbed sediment cores

versus average concentration of reduced substances in the 0-1,
1-2, and 3-4 cm layers. The correlation was significant between
rate of uptake and concentration of reduced substances in any of
the three layers, but rate of chemical oxidation did not correlate
with concentration of reduced substances in the deeper layers.

65


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. 1a) 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. tb. 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. 1c), 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. 1d). 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


4

Figure 3. Sectional view across the lower end showing the guides for each

L ie
e 33
8 xB
Oo Ag
: =
Re

Welok
7 places

8
"def Hows

Ap ins

45,"

of the coring tube s.

92

z


To each graduated 50-ml Frlenmeyer 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 HgCly. After
3 hr the activity is stopped with 20 ml of absolute alcohol, which also
extracts the formazan. The mixture is shaken every 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 spectrophotometer. 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 HgClo-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 consists of the
closed 0.11-m double-walled tank connected to the 0. 19-m3, 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 aftér the sample was removed
from the calorimeter.

15


vr

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 duly September February July

32 Dissolved oxygen 0.90 3.15 3.56
Temperature 8.97 9.49 7.76
Satinity 29.85 30.34 29.35
Total carbon 2.19 2.25
Sand fraction 4 3

33 Dissolved oxygen 0.70 2.85 2.60
Temperature 8.53 9.14 8.34
Salinity 28.48 29.80 28.66
Total carbon 3.94 3.88
Sand fraction 9 10



(continued) COMPUTER OUTPUT OF PREDICTED RATES FROM ANALYSIS OF VARIANCE OF TOTAL

Table 1.

C CHEMICAL OXIDATION, AND RESPIRATION (ml

) DURING FOUR SEASONS AT 5, 10, AND 15°C

t
I

INORGAN

oxygen mTM 2hy

OXYGEN UPTAKE

RESPIRATION

15°C

10CC

BOC

Jan. Apr. Jul. Oct. Jan. Apr. Jul. Oct. Jan. Apr. Jul,

Oct.

Stations

12.3

8.8

7.

12.0

8.6

3 4.7 6.7 1 6.8

4.

7.9

5

5.3
3.2

7.7
4.8
6.8

4.2

4,2

2.8

2.6

3.4
7.2
3.8

3.3
7

2.5

5.5
2.8

2.4

1.5
3.7
1.8
1.0
2.7

1.0
2.8

1.4

1o.1

5.

9.9
5.4

6.6
3

5

5.5

3.6 2.9

2.3

3.7

2.8

1.3
0.6
2.0
2.3

1,

7
7.6

2.5

1.8
4,2

3.6
7.4
8.3
4.8

2.4
5.

1.7
4.1

2
4.9

1.7
4.0
4.5

0.9
2.5
2.9
1
1

al

5.7
3.2

88

6.0
3

4.7

5.9
3.3

4.6

5.6

1
1.5

5.0
3.9
5.4
7.8

2.6

2.5
1.9
2.8
4.2

1

2.4
3.4

5.1

2.4
1.9
2.7

4.1L

1

1.
0.7

2.6
3.8

2.0
2.

2.5
3.5
5.2

3.8

2.6
3.7

1
1.7
2.8
1.9

4.5

10
11

5.3
7.

1.

1.6
2.6
1.8
4,3

5.5

4.3

5.4
4.0

1
1.4
3.5

12
13

5.9
12.0

4.1

3.1
6.8

3.9
8.1

5.8

11.7

3.1
6.6

3.8

3.0
6.5

8.4
4,2

7.9
4.0
4.6

14
15
16
17
18
19
20
21

4.3 6.2

5

3.3

4.1

6.1
6.9

3.3

3.2

5 2.1
3

1.8

1.9

TA

3.9
4.4

4.7

4.8

3.8
4.3

2.4
2.8

2.3

8.0
5.5

5.6
3.8

5.3

7.8
5.4
8.3

5.5
3.7

5.2
3.5

4.2
2.8

2.6
1.6

2.8

2.9
4.7

3.6
5.7

2.8

1.8
3.0

2.1

1.3
2.3
1.5
1.7

6.0

5.5 4.6 5.9

4.5
3

4.4
4.8

3.4
3.7

4.3 6.1 4.1
4.6

3.3

4.0

6.6 4.5

6.1

4.4 3.6
3.2
0.8

3.5

2.3

2.2

6.2

4.3

3.3
0.9

4.1

4.2

4,0

22
0.8

2.1
0.3

1.5
O.1

1.9
0.3

22
23

2.2

1.3

1.2

2.1

1.3

1.2



24

20

TOTAL UPTAKE (ml 0,/m? per hr )

12h
L
ak
ak
L | L
c 15

10
TEMPERATURE (°C )

Figure 5. Total oxygen uptake versus temperature at various stations in
January.

27


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


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-cm
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


TABLES
No. Page

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

vi


COMPUTER OUTPUT OF PREDICTED RATES FROM ANALYSIS OF VARIANCE OF TOTAL OXYGEN

Table 1.

UPTAKE, INORGANIC CHEMICAL OXIDATION, AND RESPIRATION (ml oxygen m7?hr7})

DURING FOUR SEASONS AT 5, 10, AND 15°C

86

ot
Bl aannwantnoanotatavnononh
SGrigaanaeraqscwutrravacrucscsana
NANNNTH A ANH RH AH NNT NHN NSA
ut
KS re OAK HHA KODAK aAAKH AHO
° AGHA AA ANH AAA AA AAA AAA
te
4
gh rd damn att tt aor nint a oo tio ©
A swsdtt dd oSHHA OKAY NSH HANDS
ee
Bl HOKroOMOHHSCAKARSCrAOHHANAD
Of Fr dana wHiNNannaoSuSetnNoartor
CNS Oe ee ee Se ee
3 NODOHFAEFNAHADARHAEFDOHHMONDA
SJ ¥raaganiwdAnNntanndscudstincardor
Nett AH A ANA TAN HA ANN AAA AHA aS
*
fy
Ade
<j [HP NOBHDSOHOOHVSORORYMHOMOHOAAN
St} avivincddr a dich bAKH SASH HOS
aS SSS8Sa0 85 HSSssanaaaa
=)
zim
5 gl amonnraattonnager nani
SP [5l cana cdactaodaadganscdiagandacrnu
x“ aon are dann Lal baal
°
A .
< ast WONHMONOMAEFAHANANArFONMNNDODAOM WH
5 OC] SHwBvVHHAK AD BDHHKEOAKDHSONSHHOS
& en oe oe ie oe oe So ee
| POMMONMHORHOHTAMHNMDOHDONA
5, BSANND ASH Hr BAANAHSHAKBSHDOEH
aaa ae dada a
| CNWAAMRANNANNDNNDAMOHOOAM
al ARGarraoddodrradcdorcaornrud
iS) 4 a a
io
te
S| dewvedsscrasunwsdeaodrrdurddsdia
Bg] RYOn AK NH NOTH MOnMAKHROoNEIWA
NOoorranvoorroda bf
of sess d ssuaaordsarrud
n
g
Buena tnoronascHnantynornnagonan
4 Meee RR AT HHH NNN AN

*The 95% confidence limits of the rate at each station are the predicted rate multiplied and divided by 1.17.


—O- OCT, TOTAL UPTAKE
| ee JAN. ”

O- APR ” ”
[mee JUL " “

10 f= OCT, CHEMICAL OXIDATION
-y— JAN " “ em

OXYGEN CONSUMPTION (mI/ m? per hr)
\
|

TEMPERATURE (°C)

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


. >}
le AO ey BX
3, Sam
63 3a Nel Ge
$3 . + feLe i v
= a) SN
e at i 3
48 ~ S
se L
“a
3a
a
eS
XM
3
a)

Figure 4. Detailed drawing of a coring tube guide showing the split lower
section that 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.

93


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 Po, 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 basis
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/m? 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/m? 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
Qio'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