The survival of sewage bacteria at various ocean depths

CEL THN = BAS

Technical Note N=-1396

THE SURVIVAL OF SEWAGE BACTERIA AT VARIOUS OCEAN DEPTHS

By

H. P. Vind, J. S. Muraoka, and C. W. Mathews

July 1975

Sponsored by

DIRECTOR OF NAVY LABORATORIES

Approved for public release; distribution unlimited.

CIVIL ENGINEERING LABORATORY
Naval Construction Battalion Center
Port Hueneme, California 93043

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REPORT DOCUMENTATION PAGE BEFORE COMPLETING FORM
T. REPORT NUMBER 2. GOVT ACCESSION NO| 3. RECIPIENT'S CATALOG NUMBER
TN-1396 DN344018
4. TITLE (and Subtitle) 5. TYPE OF REPORT & PERIOD COVERED
THE SURVIVAL OF SEWAGE BACTERIA AT Final; June 1973—June 1974
VARIOUS OCEAN DEPTHS 6. PERFORMING ORG. REPORT NUMBER
7, AUTHOR(S) TS. CONTRACT OR GRANT NUMBER(S)

H. P. Vind, J. S. Muraoka, and C. W. Mathews

9. PERFORMING ORGANIZATION NAME AND ADDRESS 10. PROGRAM ELEMENT, PROJECT, TASK
AREA & WORK UNIT NUMBERS

Ret gene conceicer , 61152N; ZR-00001,
av nN Cc 1
< : ZR-031-02; Z-ROOO-01-140
Port Hueneme, California 93043 g
11. CONTROLLING OFFICE NAME AND ADDRESS 12. REPORT DATE
: July 1975
Director of Navy Laboratories IEwENUMEGRIGRIBASES
Washington, DC 20376 20
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18. SUPPLEMENTARY NOTES

19. KEY WORDS (Continue on reverse side if necessary and identify by block number)

Ocean, seawater, sunlight, survival, hydrostatic pressure, sewage bacteria, ocean pollution,
E. coli.

20. ABSTRACT (Continue on reverse side if necessary and identify by block number)

Sewage outfalls in the ocean are usually relatively close to shore at depths of 200
feet or less. An investigation was undertaken to ascertain if Escherichia coli, the principal
species of bacteria in sewage, would survive for shorter or longer periods if the sewage
were discharged at depths of 1,000 feet or so, where there is no light, and where the
pressure is greater and the temperature is lower. Cultures of the Seattle strain of E. coli
in autoclaved seawater were placed in 25-ml bags made of dialyzing tubing. Some of the

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20. Continued

bags were suspended near the surface of the ocean, some at depths of 200 and some at
depths of 1,000 feet. Some of the bags were suspended in opaque containers to protect
them from sunlight; others were suspended in translucent containers. All of the E. coli
cultures exposed near the surface of the ocean in translucent containers died in approx-
imately 4 hours. Those suspended near the surface in opaque containers survived for
periods of an estimated 2 weeks. Cultures of E. coli suspended in either translucent or
opaque containers at depths of 200 and 1,000 feet (where little or no light penetrates)
also survived for periods of an estimated 2 weeks, with only slight differences in the
mortality rates at these two depths. If the sewage were discharged at a depth of 1,000
feet, there would be no danger of contaminating surface waters because the cold deep
water does not mix with the warmer surface waters. If the sewage were discharged at

a depth of 200 feet, there would probably also be no danger of contaminating surface
waters unless the thermocline was deeper than that. If the sewage were discharged at
shallow depths, there would be contamination of surface waters; but at least one species
of the contaminating microorganisms would probably survive for only a few hours in
sunlight.

Library Card
Civil Engineering Laboratory
THE SURVIVAL OF SEWAGE BACTERIA AT VARIOUS
OCEAN DEPTHS (Final), by H. P. Vind, J. S. Muraoka, and
C. W. Mathews
TN-1396 20 p. illus July 1975 Unclassified

1. Sewage bacteria—survival 2. Escherichia coli I. Z-ROOO-01-140

Sewage outfalls in the ocean are usually relatively close to shore at depths of 200 feet or
less. An investigation was undertaken to ascertain if Escherichia coli, the principal species of
bacteria in sewage, would survive for shorter or longer periods if the sewage were discharged at
depths of 1,000 feet or so, where there is no light, and where the pressure is greater and the
temperature is lower. Cultures of the Seattle strain of E. coli in autoclaved seawater were
placed in 25-ml bags made of dialyzing tubing. Some of the bags were suspended near the
surface of the ocean, some at depths of 200 and some at depths of 1,000 feet. It was found
that if the sewage were discharged at a depth of 1,000 feet, there would be no danger of con-
taminating surface waters because the cold deep water does not mix with the warmer surface
waters; if the sewage were discharged at a depth of 200 feet, there would probably also be no
danger of contaminating surface waters unless the thermocline was deeper than that; if the
sewage were discharged at shallow depths, there would be contamination of surface waters,
but at least one species of the contaminating microorganisms would probably survive for only
a few hours in sunlight.

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CONTENTS

INTRODUCTION... .

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Oem IEQDOSUIFAs oo 6 6 6 0 ooo

Bacterial Cultures. .

Bacterial Counts. ....

EXPERIMENTAL RESULTS - 5 . = . «© « = «= « «

Effect of Sea Salts on the Survival of E. coli. .

Effect of Ocean Depth on Survival of E. coli.

Effect of Sunlight on the Survival of E. coli.

Sunlight Effect on E. coli in Glass and in
Polyethylene) = 2 2 = ~

Effect of Sunlight on Seawater

DISCUSSION OF RESULTS

CONCLUSIONS .. .

INGINOMLEDEMINITS, 6 56 6 56 50 6086000050000

REFERENCES.

Figure

Figure

Figure

Figure

Figure

LIST OF ILLUSTRATIONS
Diagram of ocean exposure station .

Lowering steel drums from which sample line will
be suspended in the ocean.

Weight for holding sample line taut in the ocean .

Winch for lifting and dropping sample line and
anchor in the ocean.

Serial dilution of water samples for bacterial
counts.

Page

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Figure

Figure

Figure

Figure

Figure

Figure

Figure

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tablet

Table 2.

LIST OF ILLUSTRATIONS

continued

Filtering bacteria from diluted fraction of water

Sempll@ 6 6 6 6 6 6 0 6

° e ° e ° ° ° e e ° ° . . ° ° .

Dialyzer bags filled with cultures of E. coli

in seawater. ....

Translucent and opaque
water samples.

cages for holding bagged

Emptying dialyzer bag into sterile test tube .....

Bacterial colonies on
120-hour samples from
GIEPNEN 6 6 6 5 Oo

Bacterial colonies on
120-hour samples from

Bacterial colonies on
120-hour samples from
depth .

Bacterial colonies on
120-hour samples from

duplicate sets of membranes for
translucent cage at 200-foot

duplicate sets of membranes for
opaque cage at 200-foot depth .

duplicate sets of membranes for
translucent cage at 1,000-foot

. ° . . .

duplicate sets of membranes for
opaque cage at 1,000-foot depth

LIST OF TABLES

Swigehveul (ue Wo Collbt Ele SeEIG 696 56 56 6 6 6.0 6 6

Effect of Sunlight on Survival of E. coli ......

vi

Page

INTRODUCTION

The disposal of sewage in the sea and adjoining estuaries is wide-
spread. The outfalls are usually relatively close to shore and the sewage
is discharged at depths of 200 feet or less. The purpose of the study
undertaken by the Civil Engineering Laboratory (CEL), Port Hueneme, CA,
and described in this technical note is to determine whether sewage
bacteria would survive for longer or shorter periods if the sewage were
discharged at greater depths, where there is less light, where the
pressure is greater and where the temperature is lower. Bacteria of the
species Escherichia coli, the most abundant bacterial species in human
wastes, were employed in the study as representative sewage bacteria.

METHODS AND MATERIALS
Ocean Exposure

Small containers of sewage bacteria were exposed in the ocean by a
team of Navy divers who had had extensive experience in conducting ocean-
ographic experiments. A modified aluminum LCM-8 was employed in the
undertaking.

The exposure site was approximately 3 miles offshore, 5 miles
southeast of Point Mugu, California. The water depth was 1,300 feet.
Ocean exposure stations were established near the surface of the ocean
and at depths of approximately 200 feet and 1,000 feet. The exposure
stations were simply tethering hooks or loops for suspending samples at
various positions on a 1,000-foot line (Figure 1). The line was supported
from the surface by three steel drums serving as buoys (Figure 2). A
500-pound weight at the end of the line held it taut in the water
(Figure 3). The assembly was prevented from drifting by a separate 600-
foot-long line attached to the 500-pound weight on one end and to an
ocean-bottom anchor on the other. Powerful winches (Figure 4) were used
to raise and lower the assembly when samples were removed or placed in
the ocean.

Bacterial Cultures

Pure cultures of Seattle strain E. coli as supplied by Roche
Diagnostics, Division of Hoffman-LaRoche, Inc., Nutley, NJ 07110, were
employed in all of the experiments. The bacteria were supplied as
BACT-CHEK discs, which are composed of dried bacterial cells and have a
diameter of approximately 4 millimeters and a thickness of approximately
1/2 mm. Each disc contains 100,000 to 10 million viable microorganisms.

Bacterial Counts

Coliform counts were made by a widely used modification [1] of the
Standard Total Coliform Membrane Filter Procedure [2]. The counts were
made with field monitoring equipment developed and supplied by the
Millipore Corporation, Bedford, MA. The equipment included a syringe with
two one-way valves. With the syringe, water samples were drawn through
membrane filters having pores sufficiently small (0.45 millimicron) to
retain most microorganisms. The membrane filters were mounted in
*“Millipore Field Monitors’’ which are sterile, disposable, plastic
devices, serving both as filter holders and culture chambers. The
membranes are supported on absorbent pads for retaining culture medium.

The water samples to be counted were. serially diluted (Figure 5)
in test tubes, each containing 9 ml of an autoclaved 3:1 mixture of
distilled water and seawater. The diluted fractions of the samples were
then drawn through the membrane filters contained in the Millipore Field
Monitors (Figure 6). In this step, all of the bacteria in the diluted
fractions were deposited on the surfaces of the membrane filters. The
absorbent pads on which the membrane filters were mounted were each
moistened with 0.8 ml of sterile ‘‘MF-Endo Broth,’’ a proprietary medium
prepared specifically for the isolation and identification of coliform
bacteria. The monitors were then closed and incubated at 35°C for 24
hours.

Each coliform bacterium on the surface of the membrane filters
multiplies many times in the course of 24 hours and ultimately forms
a macroscopically visible aggregate or colony. Most other bacteria species
do not multiply on the selective MF-Endo Broth. The number of colonies
which develop is assumed to be the same as the number of coliform bacteria
in the serially diluted fraction of the water sample that was filtered.
Because most samples were counted in duplicate and several dilutions
were made of each, several estimates were obtained of the number of
microorganisms in a unit volume of each sample. When the results were
averaged, greatest reliance was placed on plate counts in the range of
20 through 80.

EXPERIMENTAL RESULTS
Effect of Sea Salts on the Survival of E. coli

In preparation for experiments which were to be conducted at sea,
several preliminary tests or exercises were conducted in the laboratory.

In the first exercise, one BACT-CHEK disc was placed in a test tube
containing a 3:1 mixture of distilled water and seawater. The test tube
was incubated for 2 hours at 35°C. At the end of that time the disc was
sufficiently soft to disintegrate when the test tube was shaken. The
bacteria were then uniformly distributed throughout the tube. The
contents of the tube was added to a 1-liter flask filled with filtered
autoclaved seawater. Bacterial counts were made at various intervals and
the following counts were obtained:

Time (hours) Count (per ml)

0 2,000
1 2,000
3 2,700
24 300

The exercise was repeated with the following results:

Time (hours) Count (per ml)
0 2,100
22 800
25 900
91 400
139 300

In a similar test, a BACT-CHEK disc was incubated in a test tube of
water at 35 C for 24 hours, instead of for 2 hours, before it was added
to a flask containing 1 liter of filtered autoclaved seawater. Immediately
after mixing, the bacterial population of the seawater was approximately
200,000 per ml instead of approximately 2,000, as was the case when the
discs were incubated for 2 hours.

In a final laboratory exercise, the effect of added nutrient on E.
coli populations was tested. Ten-ml quantities of a freshly prepared E.
coli culture were added to individual 250-ml flasks of filtered seawater
and to flasks of seawater containing added tryptic soy broth (a proprietary
mixture of nutrients and salts sold by Difco Laboratories, Detroit, MI
for preparing culture media for microorganisms). The following coliform
counts per ml were obtained.

Count in Tryptic

Time Count in Filtered Soy Broth and
(hours) Seawater Seawater
0 45,000 35, 000
26 4,000 25 million

Effect of Ocean Depth on Survival of E. coli

Comparisons were made of the numbers of E. coli surviving in seawater
cultures exposed for various periods of time at various depths in the
ocean. The cultures included E. coli suspended in filtered autoclaved
seawater and in an autoclaved mixture of seawater and human feces. Some
of the cultures were contained in 25-ml bags made of cellulose acetate
dialyzer tubing tied at each end (Figure 7). The dialyzer bags permitted
water soluble substances to diffuse freely between the bags and the
ocean but retained the cultures of E. coli in the bags and prevented the

entry of microorganisms and proteins from the ocean. Because of the
possibility that the dialyzer bags might rupture, pliable, translucent,
polyethylene bottles were also employed as containers for some of the E.
coli cultures. Although the bottles were essentially impermeable, they
did permit the cultures to be exposed to ocean pressures and temperatures.

Synthetic sewage water was prepared for the experiment by adding
approximately 10 grams (wet weight) of human feces to a 2-liter flask
of seawater. The flask was autoclaved for 20 minutes at 15-psi steam
pressure. A 2-liter flask of filtered seawater was autoclaved at the
same time. Both flasks were inoculated with E. coli in the usual
manner. Two of the BACT-CHEK discs were used in the preparation of each
of the two cultures.

Some of the seawater culture was distributed into the dialyzer bags
and some into 25-ml polyethylene bottles. The synthetic sewage culture
was distributed only into the polyethylene bottles.

The dialyzer bags and the 25-ml polyethylene bottles containing the
E. coli cultures were placed in wide-mouthed, 500-ml polyethylene bottles
in which numerous 1/4-inch round ventilation holes had been drilled. The
larger bottles served as cages for che smaller containers. They were
securely fastened to the anchored nylon line and suspended at various
depths in the ocean.

Bacterial counts were made after various time intervals. Counts
were also made on the remains of the original cultures which were
maintained in the laboratory at room temperature. The results of the
experiment are summarized in Table 1.

Effect of Sunlight on the Survival of E. coli

An investigation was made of the effect of sunlight on the survival
of E. coli. Cultures of the microorganisms were exposed in opaque and
in translucent containers at the ocean surface and at depths of 200 and
1,000 feet. Comparisons were then made of the numbers of microorganisms
surviving in the opaque and translucent containers. The experiment was
conducted in the following manner.

A culture containing 100,000 E. coli per ml was distributed into 54
dialyzer bags with a capacity of approximately 25 ml each. Six of the
bags were retained in the dark in the laboratory for control tests,
three being maintained at room temperature and three at 3 C. The other
48 bags were divided into pairs and distributed into 24 cages made of
polyethylene bottles in which numerous 1/4-inch round ventilation holes
had been drilled. Twelve of the cages had been made opaque by a covering
of electrician’s black tape; the other twelve remained translucent
(Figure 8).

The following day, two of the opaque cages and two of the translucent
cages containing dialyzer bags of E. coli culture were exposed near the
ocean surface for periods of 1 and 4 hours. The exposures were made by
simply fastening the cages on the end of a length of nylon parachute
cord and lowering them over the side of the ship. The bottles floated
and remained on the surface. The third of the opaque and the third of
the translucent cages were not exposed in the ocean and were employed as
zero hour controls.

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The other 18 cages were attached to the nylon line anchored in the
ocean. Three each of the opaque and translucent cages were suspended
from the line at a depth of only a few feet; three each, at a depth of
200 feet; and three each, at a depth of 1,000 feet. One opaque and one
translucent cage was removed from each depth at the end of 24, 48, and
120 hours.

The bags were removed from the cages and samples for bacterial
counts were obtained by piercing the bags (Figure 9). Figures 10 to 13
show the colonies which developed on the field monitor plates for the
final or 120-hour samples after the series of dilutions of 10 ml of the
contents of the bags. The results of the experiment are summarized in
Table 2.

Sunlight Effect on E. coli in Glass and in Polyethylene

A comparison was made of the survival of E. coli exposed to sunlight
in containers made of pyrex glass and containers made of polyethylene.
The shorter wavelengths of ultraviolet light are less able to penetrate
the glass than the plastic containers.

A culture was prepared by inoculating 1-1/2 liters of autoclaved
seawater containing 0.5 gram of ‘‘Eugenbroth’’ (a proprietary mixture
of nutrients and salts sold by the Division of Bio Quest, Cockeysville,
Maryland for preparing bacteriological media with E. coli). The culture
was incubated for 24 hours at 32 C, at which time growth of E. coli was
sufficient to cause the medium to be cloudy. One hundred ml of culture
was added to each of three small polyethylene bottles and three pyrex
glass flasks. The six containers were placed outdoors in the sunlight
for 6 hours. During the first 2 hours, the sun was obscured by fog and
low-lying clouds. During the next 2 hours the cloud cover disappeared,
and the sunlight was very bright for the last 2 hours of the exposure.
Periodically, bacterial counts were made of the contents of the 6 flasks.
The following values were obtained for the counts per ml:

Containers O hr 72 lone 6 hr
2 6 6

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Glass 24 x 10 28 x 10 <10

Effect of Sunlight on Seawater

An experiment was conducted to ascertain if sunlight altered the
chemical composition of seawater in such a way as to make it unsuitable
for the growth of E. coli. Four Erlenmeyer flasks, each containing 100
mg of Eugenbroth preparation and 400 ml of seawater, were autoclaved at
15 psi for 20 minutes. Two of them were then exposed to sunlight for
one complete day and the other two were stored in the dark. All four
flasks were then inoculated with 1-ml portions of a vigorous E. coli
culture, and all four were incubated in the dark for 24 hours. Counts
of E. coli were then made, and the following results were obtained:

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E. coli in medium preexposed to sunlight, 19 x 10° per ml.
E. coli in medium protected from sunlight, 14 x 10 per ml.

DISCUSSION OF RESULTS

Numerous investigators have studied the effects of seawater, sunlight,
pressure, or ocean exposure on the survival of sewage bacteria. So many
of their results are conflicting [3] that no attempt is made to compare
the results of the study reported here with the results obtained by
other investigators.

E. coli cells gradually died off in filtered autoclaved seawater.
Apparently they died from lack of nutrients rather than from any harm
caused by the sea salts, because they flourished and multiplied rapidly
in seawater containing added nutrients such as tryptic soy broth or
human feces. It appears that the filtered seawater contained insufficient
nutrients for the microorganisms to multiply. The BACT-CHEK discs
themselves may have contained some nutrients because the discs produced
cultures with a hundredfold greater E. coli population when they were
incubated in test tubes of water for 24 hours than when incubated for 2
hours.

In both experiments performed at sea, the counts for samples
exposed at 1,000 feet tended to be higher than the counts for samples
exposed at 200 feet. The differences were not great, however, and were
surely within the range of possible experimental error. In both experiments
striking differences were noted in the counts on the samples exposed at
200 or 1,000 feet and those exposed near the surface. Unless protected
from light, the bacteria died very rapidly at the surface. There were
no great differences in the mortality rates of E. coli in filtered
seawater maintained in the laboratory at room temperature, in the
refrigerator, or in the ocean at depths of 200 or 1,000 feet. In all
instances, there were probably insufficient nutrients; and the bacteria
died off at rates which would leave few or none living in 1 or 2 weeks.

Survival times might have varied more had the media contained more
nutrients. It is likely that the populations of bacteria would have
increased initially. The nutrients would probably have been used up
more rapidly at some temperatures than at others, and the subsequent
decline in numbers might then have been more markedly influenced by
temperature.

Since the E. coli in this experiment were enclosed in dialyzer bags,
it is possible that the bacteria would die even more rapidly if
discharged directly into the ocean. It seems unlikely that they would
have survived for a longer period of time, unless they were protected in some
manner, such as inside a grease ball or inside a fecal mass.

During the summer in temperate latitudes, and year around in lower
latitudes, near-shore surface waters circulate and mix as a layer.

Water below the thermocline, which develops in these waters at depths of
100 feet or so, mixes only slightly with the water above the thermocline

[4]. Under these conditions, sewage discharged at a depth of 1,000 feet
would be less likely to contaminate surface waters than would sewage
discharged near or above the thermocline. Bacterial survival time would
be a less important consideration at the greater depth, so far as man is
concerned.

The brief survival period of E. coli cells in containers floated on
the surface of the ocean and exposed to sunlight (0-, 1-, and 4-hour
samples, Table 2) was not surprising. It is well known that sunlight
kills E. coli in shallow dishes of water [5,6].

The apparent germicidal properties of sunlight on the samples
exposed several feet below the ocean surface for 24 and 48 hours (Tables
1 and 2) was somewhat unexpected. These samples were tethered to the
nylon line at a distance of approximately 10 feet from the surface. The
short cord by which they were attached to the line may have permitted
the samples to rise a few feet but not to the surface. The samples, no
doubt, oscillated in depth with the waves; but, in any event, the sunlight
had to penetrate several feet of water to reach the E. coli cultures.

It is generally believed that, at the most, the germicidal radiation
in sunlight penetrates 1 meter in seawater [5]. Zobel [6] gives 3
meters as the limiting depth for the penetration of abiotic radiation,
but he concludes that at greater depths than 10 to 20 centimeters the
radiation is too feeble to sterilize seawater.

In the last of the sunlight experiments at CEL, germicidal radiations
were able to penetrate both pyrex glass and polyethylene plastic. Pyrex
glass is opaque to wavelengths shorter than 2,800 Angstroms. Its trans-
parency gradually increases with longer wavelengths, and it transmits
greater than 90% of radiation from 3,600 to 7,000 Angstroms [7].
Polyethylene is opaque to wavelengths shorter than 2,270 Angstroms. Its
translucency gradually increases with longer wavelengths, and it transmits
a relatively high percentage of ultraviolet and visible radiation longer
than 2,800 Angstroms [8]. Finally, that part of the sun’s radiation
that is composed of wavelengths shorter than 2,920 Angstroms is cut off
completely by the atmospheric ozone layer and by oxygen [9]. The
results of the CEL experiment indicate that the intensity of sunlight
radiation with wavelengths greater than 2,920 Angstroms must be great
enough to kill E. coli cells in seawater.

An extensive investigation by Lukiesh [7] at General Electric
Laboratories, Cleveland, OH, indicated that the maximum germicidal
effectiveness in the killing of E. coli in shallow dishes of water is
exhibited by radiant energy with wavelengths of 2,537 to 2,575 Angstroms.
However, with high enough intensities and prolonged exposures, all wave-
lengths in the ultraviolet, and even in the visible spectrum, were
germicidal. At a wavelength of 4,000 Angstroms, 10,000 times as much
radiant energy, and at a wavelength of 7,000 Angstroms, 100,000 times as
much radiant energy were required to kill E. coli cells as was required
at a wavelength of 2,540 Angstroms. Hence, even though surface sunlight
contains no radiation shorter than 2,920 Angstroms, it is germicidal,
apparently because it contains some ultraviolet radiation with wavelengths

from 2,920 to 4,000 Angstroms and very intense visible radiation from
5,000 to 6,000 Angstroms. The intensity of the ultraviolet radiation in
sunlight varies considerably from day-to-day and hour-to-hour.

The transmission of ultraviolet light in seawater also varies
considerably. The concentrations of microorganisms, nitrate ions,
sediments, and organic matter all help to determine the transparency of
seawater to ultraviolet radiation [10]. As depth increases, the intensity
of ultraviolet light decreases. The shorter the wavelength, the greater
is the decrease in intensity with depth. In consequence, the average
wavelength of sunlight radiation increases with depth of penetration.

If there were radiations in sunlight with a wavelength of 2,540
Angstroms, the most highly germicidal wavelength, they would penetrate
no more than 1/2 meter of seawater. Ultraviolet radiation with a wavelength
of 2,920 Angstroms, the shortest in sunlight, penetrates no more than
approximately 3 meters of seawater [6]. Ultraviolet sunlight with a
wavelength of 4,000 Angstroms may penetrate 10 to 20 meters, and visible
light may penetrate to a depth of 100 meters [8].

The maximum depth at which the detrimental effects of sunlight are
great enough to overcome the ability of E. coli cells to resist and
multiply has not been precisely determined. Beyond the depth of sunlight
penetration, E. coli cells survived for nearly 2 weeks in seawater
containing insufficient nutrients for growth. Whether they would survive
for shorter periods when exposed to sunlight radiation penetrating
to depths of 10, 25, 50, or 100 feet is not known. This information is
part of that needed for a thorough assessment of the hazards of disposing
of sewage at sea.

CONCLUSIONS

Escherichia coli, the principal bacterial species of sewage, is
very sensitive to sunlight. Unless protected inside a grease ball or a
fecal mass, microorganisms of this species in sewage discharged near the
ocean surface would surely perish within a few days. There would be
relatively small differences in the mortality rates of E. coli in sewage
discharged at ocean depths of 200 and 1,000 feet. Very little sunlight
penetrates to these depths, and sewage bacteria of this species would
survive for an estimated week or two. If the sewage were discharged at
a depth of 1,000 feet, there would be no danger of contaminating surface
waters because the cold deep water does not mix with the warmer surface
waters. If the sewage were discharged at a depth of 200 feet, there
would probably also be no danger of contaminating surface waters unless
the thermocline was deeper than that.

ACKNOWLEDGMENTS

LT Anthony M. Parisi and LTJG James E. Halwachs designed the assembly
for anchoring the water samples in position in the ocean; and they were
in command of the diving vessel and all operations at sea. Members of
the crew included Navy Divers Larry Hecht, Larry Wenban, Donald Forster,
Larry Stowers, Joe Hierholzer, and Robert Hurt. Mr. Kirk Kingsbury of
the CEL Riggers and Mechanics Shop operated the drum winches used to
raise and lower the ocean-anchored lines. The invaluable contribution
of these men is greatly appreciated.

cluster of
floating
steel drums

~ ~~ ocean surface

tethering loop
10-foot depth

tetheringlocp .
200-foot depth

1,300 feet

tethering loop
1,000-foot depth

Sa

SS —_ OS

500 |b weight
600 feet

ocean bottom

Figure 1. Diagram of ocean exposure station.

12

anchor

Figure 7. Lowering steel drums from which sample
line will be suspended in the ocean.

Figure 3. Weight for holding sample
line taut in the ocean.

13

SN Be
,

Figure 4. Winch for lifting and dropping sample line
and anchor in the ocean.

Figure 5. Serial dilution of
samples for bacte- -
rial counts. oy ie oa roe

el

14

Figure 6. Filtering bacteria from diluted fraction
of water sample.

Figure 7. Dialyzer bags filled with cultures of
E. coli in seawater.

15

Figure 9.

Emptying dialyzer
bag into sterile
test tube.

16

Figure 8.

Translucent and
Opaque cages for
holding bagged
water samples.

eo

Dilutions: 1

V-CC-120

Figure 10. Bacterial colonies on duplicate sets of
membranes for 120-hour samples from
translucent cage at 200-foot depth.

ae

Dilutions: 1 2 3

VI-CC=120

Figure 11. Bacterial colonies on duplicate sets of
membranes for 120-hour samples from opaque
cage at 200-foot depth.

17

Dilutions: 1

Figure 12. Bacterial colonies on duplicate sets of membranes
for 120-hour samples from translucent cage at
1,000-foot depth.

Dilutions:

VI-K-120

Figure 13. Bacterial colonies on duplicate sets of membranes
for 120-hour samples from opaque cage at 1,000-foot
depth.

18

REFERENCES

1. Millipore Corp., Bedford, MA. Application Manual AM302, Biological
Analysis of Water and Wastewater. 1973. pp 52-56.

2. American Public Health Association and the American Water Works
Association Water Pollution Control Federation. Standard Methods for
the Examination of Water and Wastewater. 13th Edition, 1971. pp 679-683.

3. Isadore Nusbaum and Richard M. Garver. ‘‘*Survival of coliform organisms
in Pacific Ocean coastal waters,’’ Sewage and Industrial Wastes, Vol
27, Dec 1955, pp 1383-1390.

4. H. W. Harvey. The chemistry and fertility of sea waters. Cambridge,
The University Press, 1963, pp 16-18, 100.

5. Y. Yoshpe-Purer and H. I. Shuval. ‘‘Salmonella and Bacterial Indicator
Organisms in Polluted Coastal Water and their Hygienic Significance, ’’

in, Marine Pollution and Sea Life, by Maria Ruivio. Published by arrange-
ment with Food and Agricultural Organization of the United Nations. London
Fishing News Books, LTD, 1972, pp 574-580.

6. Claude E. Zobel and George F. McEven. ‘‘The lethal action of sunlight
upon bacteria in seawater,’’ Biological Bulletin, Vol. 68, No. 1, Feb
1935, pp 93-100.

7. Mathew Luckiesh. Applications of germicidal, erythemal and infrared
energy. New York, NY. D. Van Nostrand Company, Inc., 1946, pp 107-136.

8. H. B. Klevens and J. R. Platt. ‘‘Ultraviolet transmission limits of
some liquids and solids,’’ Journal of the American Chemical Society,
Vol. 69, part II, Dec 1947, pp 3055-3062.

9. Gerhard Neumann and Willard J. Pierson. Principles of physical
oceanography. Englewood Cliffs, NJ, Prentice Hall, Inc., 1966, pp 53-55.

10. J. Duclaux and P. Jeantet, ‘‘Transparence of natural waters to
ultra-violet rays,’’ Comptes Rendus Hebdomadaives des Seances de 1’Academie
des Sciences. vol 181, 1925, pp 630-1. (Abstract in Chemical Abstracts,
vol 20, 1926, p 251.)

19

DISTRIBUTION LIST

SNDL No. of Total
Code Activities Copies
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Engineering Field Divisions and
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- 344 346 CEL Special Distribution List No. 9 for

persons and activities interested in
reports on Environmental Engineering
(R&D for pollution control)

20