SEPA
United States
Environmental Protection
Agency
Health Effects Research
Laboratory
Cincinnati OH 45268
EPA-600/1-79-029
August 1979
Research and Development
Determination of
Breeding Sites of
Nematodes in a
Municipal Drinking
Water Facility
-------
RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into nine series. These nine broad cate-
gories were established to facilitate further development and application of en-
vironmental technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields.
The nine series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4, Environmental Monitoring
5. Socioeconomic Environmental Studies
6. Scientific and Technical Assessment Reports (STAR)
7. Interagency Energy-Environment Research and Development
8. "Special" Reports
9. Miscellaneous Reports
This report has been assigned to the ENVIRONMENTAL HEALTH EFFECTS RE-
SEARCH series. This series describes projects and studies relating to the toler-
ances of man for unhealthful substances or conditions. This work is generally
assessed from a medical viewpoint, including physiological or psychological
studies. In addition to toxicology and other medical specialities, study areas in-
clude biomedical instrumentation and health research techniques utilizing ani-
mals — but always with intended application to human health measures
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
-------
EPA-600/1-79-029
August 1979
DETERMINATION OF BREEDING SITES
OF NEMATODES IN A
MUNICIPAL DRINKING WATER FACILITY
by
Averett S. Tombes
A. Ray Abernathy
Clemson University
Clemson, South Carolina 29631
Grant No. R804292010
Project Officer
Elmer Akin
Viral Diseases Group
Health Effects Research Laboratory
Cincinnati, Ohio 45268
HEALTH EFFECTS RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
-------
DISCLAIMER
This report has been reviewed by the Health Effects Research Laboratory,
U.S. Environmental Protection Agency, and approved for publication. Approval
does not signify that the contents necessarily reflect the views and policies
of the U.S. Environmental Protection Agency, nor does mention of trade names
or commercial products constitute endorsement or recommendation for use.
ii
-------
FOREWORD
The U.S. Environmental Protection Agency was created because of
increasing public and government concern about the dangers of pollution
to the health and welfare of the American people. Noxious air, foul
water, and spoiled land are tragic testimony to the deterioration of our
natural environment. The complexity of that environment and the interplay
between its components require a concentrated and integrated attack on
the problem.
Research and development is that necessary first step in problem
solution and it involves defining the problem, measuring its impact, and
searching for solutions. The primary mission of the Health Effects
Research Laboratory in Cincinnati (HERL) is to provide a sound health
effects data base in support of the regulatory activities of the EPA.
To this end, HERL conducts a research program to identify, characterize,
and quantitate harmful effects of pollutants that may result from
exposure to chemical, physical or biological agents found in the environ-
ment. In addition to valuable health information generated by these
activities, new research techniques and methods are being developed that
contribute to a better understanding of human biochemical and physiological
functions, and how these functions are altered by low level insults.
This report provides an assessment of the origin and occurrence of
nematodes in treated drinking water. The results of this investigation
indicated that nematodes could enter treated water if they were present
in significant numbers in the source water. No health hazard has been
demonstrated from the ingestion of low numbers of these organisms.
However, the possibility that nematodes could harbor pathogenic micro-
organisms if the nematodes originated from heavily polluted environments
lends additional support to the premise that water sources used to
produce drinking water should be of the highest quality possible.
R.-drGarner
Director
Health Effects Research Laboratory
iii
-------
ABSTRACT
The source of nematodes in finished water has been clearly demonstrated
to be in the raw water and not in the sand filter or another part of the
water treatment facility. The benthic layer of the rivers and lake provides
a supportive environment for a large nematode population which is suspended
in the water column by the scouring action of increased streamflow following
heavy rains. Thus a direct relationship exists between nematode density in
finished water and rainfall.
Continual sources of nematodes for the rivers and lake, in addition to
adjacent agriculture land, are a sewage lagoon and sanitary landfill, both
of which flow into the river. Three genera of nematodes which appear in the
lagoon effluent also appear in the finished water.
An improved method for the detection of nematodes in water was developed
whereby nematodes could be extracted and concentrated onto a 12-mm nucleo-
pore membrane and identified by scanning electron microscopy.
This report was submitted in fulfillment of R804292010 by Clemson
University under the sponsorship of the U.S. Environmental Protection Agency.
This report covers the period February 9, 1976, to August 15, 1977, and work
was completed as of August 15, 1977.
iv
-------
CONTENTS
Disclaimer ii
Foreword ill
Abstract iv
Contents v
Figures vii
Tables vili
Acknowledgments ix
1. Introduction 1
Objectives of study 1
Summary of previous 1
2. Conclusions 3
Extraction, concentration and detection
of nematodes in finished water 3
Nematode density and annual cycle 3
Nematode removal within treatment facility 3
Nematode concentration in sand filter 4
Nematode density and rainfall A
Nematode density and temperature 4
Density of nematodes in sewage lagoon
effluent, river and lake 5
Diversity of nematodes in sewage effluent,
river, lake and finished water 5
Summary conclusions 5
3. Recommendations 7
4. Materials and Methods 8
Samples from finished water 8
Samples from river, lake, and sewage lagoon
effluent 15
Samples from sand filter 15
Generic determination 16
5. Results and Discussion 17
Extraction, concentration and detection
of nematodes in finished water. 17
Nematode densities and annual cycle 17
Nematode removal within treatment facility 20
Nematode concentration in sand filter 20
Nematode density and rainfall 24
Nematode density and temperature 26
Density of nematodes in sewage lagoon
effluent, river and lake 26
-------
Page
Diversity of nematodes in sewage effluent
river, lake and finished water 31
References 34
vi
-------
FIGURES
Number Page
1. Watershed study area In Easley South Carolina 9
2. Apparatus for extraction of particulate matter from
finished water 10
3. Farticulate matter being washed from sieve onto 12-mm membrane
in Swinney filter 10
4. Particulate matter being washed from 47-mm membrane onto 12-mm 12
membrane
5. Swinney filter opened and 12-mm membrane being placed onto
SEM stub 12
6. Two nematodes on 3-Vm pore membrane in SEM 14
7. High magnification of anterior region of nematode 14
8. Nematode density in finished water for 54 weeks during
1976-77 22
9. Nematode density in finished water and the average rainfall
in the watershed area for 54 weeks during 1976-1977. ... 25
10. Nematode density in finished water and the streamflow for
54 weeks during 1976-1977 27
11. Nematode density in finished water and the turbidity of
the lake water for 54 weeks during 1976-1977 28
12. Relationship between nematode concentration in finished
water and water temperature 29
vii
-------
TABLES
Number Page
1. A comparison of the two detection methods for determining
nematode length and population density in duplicate water
samples 18
2. Nematode density in finished water collected through three
sieves 19
3. Length distribution of nematodes collected from 100 gallons
of finished water on twelve separate dates 21
A. Nematode concentration at three locations in the treatment
plant during dry and rainy periods 23
5. Density of nematodes at various sites along the Saluda
River during May and June 1977 30
6. Genera and the frequency of observations from four locations
along the Saluda River basin 32
viii
-------
ACKNOWLEDGEMENTS
The cooperation received from the personnel of the City of Easley's
Saluda Lake water treatment facility during the entire course of this
study is greatly acknowledged.
Field and laboratory assistance received from the following graduate
and undergraduate students is acknowledged: Thomas Malone, William
Nicholas, Suzanne Ulmer, David Welsh, and David Yonge. Technical assistance
was received at different stages in this study from the following: Joan
Hudson, Karen Sindar and Nelwin Stone. Meteorological data was obtained
through the courtesy of Alex Kish. All of the above were associated with
Clemson University at the time of their connection with this project.
ix
-------
SECTION 1
INTRODUCTION
OBJECTIVES OF STUDY
The overall objective of this study was to determine the source of
nematodes that frequently are found in finished potable water. Two
hypotheses were that nematodes breed in the sand filter of treatment
facilities; and that nematodes enter water treatment facilities in raw
surface water and are not completely removed. Both hypotheses were tested
at the modern water treatment facility serving Easley, South Carolina.
A secondary objective was to improve the current methodology for
extracting, concentrating, and detecting nematodes in drinking water.
SUMMARY OF PREVIOUS WORK
The presence of nematodes in water treatment systems and their potential
effects on water quality was reported as early as 1918 when Cobb noted their
presence in slow sand filters (1). Cobb's early research has since been
followed up by numerous investigators who have shown that nematodes exist
in many public water supplies. In the summer of 1953, Kelly observed
nematodes in the effluents from slow sand filters of the Norwich, England,
water system. Kelly proposed the use of a microstrainer fabric, 23-um
pore size, to remove the nematodes and similar microorganisms (2). During
the development of a procedure for detecting the Entameba histolytica cyst
in water samples, nematodes were also found in a water supply whose source
was the Ohio River (3). This discovery prompted a nationwide survey which
eventually confirmed the presence of nematode populations in 16 of 22
public water supplies examined (4).
Questions that need to be addressed concerning nematodes in drinking
water are: what are the breeding sites of the nematodes, do they pose a
public health problem, and what is the effectiveness of the treatment
facility in removing nematodes from raw water.
The sources, or breeding sites, of nematodes have been an obvious
point of concern, and Chang and co-workers have discussed two possibilities:
first, that the nematodes are of raw water origin (5) or second, that they
may be breeding within the treatment plant. It is the ambiguity of this
literature more than any other item that has served as the genesis for
this particular study.
-------
At present, no problem other than one of aesthetics has been shown to
be associated with nematodes in public water supplies. However, some
investigators have suggested potential problems more serious in nature, such
as disease transmission. Nematodes are among the many invertebrates which
serve to decompose sewage in treatment plants. From these plants nematodes
are discharged into waterways which may serve as supply sources for public
water. Nematodes have been shown to ingest jSalmonella and Shigella bacteria
and small amounts of Coxsachie and Echo viruses which remained completely
protected within the nematode gut even when 90% of the nematodes in the
study were immobilized by a 95-100 ppm dosage of chlorine (6). Chang also
showed that Salmonella in the nematode gut survived a 10-ppm free chlorine
dosage for 15 minutes and remained viable when defecated by the nematode
onto a suitable medium; this example indicates that it is possible for a
nematode to harbor pathogenic micro-organisms, protect them through chlorine
treatment, and then release them to the finished water.
With these observations in mind, we decided to conduct a study which
would answer with greater certainty the questions concerning the nematodes1
origin.
-------
SECTION 2
CONCLUSIONS
EXTRACTION, CONCENTRATION, AND DETECTION OF NEMATODES IN FINISHED WATER
The procedure developed for this study was field- and laboratory-tested
for over a year and has been found to provide data rapidly and with a high
degree of reproducibility. This procedure has been compared to the current
standard optical microscopic technique and has been shown to be approximately
four times as sensitive.
Conclusion
The new nematode collection and detection method is more accurate than
the optical technique and has been used extensively during this study.
NEMATODE DENSITY AND ANNUAL CYCLE
After monitoring the nematode density in the finished water of the
Easley city plant every two weeks for twelve consecutive months, we found
that there was no monthly or annual cycle in the population dynamics.
Density varied between 0.5 and 1.5 nematodes per gallon for most of the
year, with variations occurring as a result of environmental conditions
but not as a consequence of breeding within the sand filters in either the
fall or winter.
Conclusion
There is no evidence of a monthly or annual cycle in the population
density of nematodes in the finished water.
NEMATODE REMOVAL WITHIN TREATMENT FACILITIES
The search for nematodes in the river and lake showed high densities
and similar genera were present in both the raw and finished water.
Examination of water samples taken at the beginning, midway through, and
at the end of the water treatment process showed that between 90-98% of the
nematodes were removed from the water. Two to ten percent of the population
passed through to the finished water unharmed as worms.
Conclusion
The water treatment facility removes between 90-98% of the nematodes,
leaving between 2-10% in the finished water.
-------
NEMATODE CONCENTRATION IN SAND FILTER
One sand filter was carefully dissected, and samples of water, anthra-
cite, sand, and gravel were removed for examination. A low density of
average-sized nematodes was found scattered randomly throughout the filter
with no concentration of worms in corners or other relatively undisturbed
areas. The density of nematodes at any location in the filter (approximately
one per gallon) was not greater than that expected to be present in a
similar volume of water passing through the filter.
Conclusion
Nematodes are not breeding within the sand filter.
NEMATODE DENSITY AND RAINFALL
During September and October 1976 a positive correlation was observed
to exist between the density of nematodes in both the raw and finished
water and the local rainfall, streamflow, and lake turbidity. This relation-
ship was repeated on a small scale following a moderate rainfall in early
December and on a larger scale following a major spring rain in April 1977.
Conclusion
Nematodes from the benthos are brought into the water column of a river
and lake by the scouring of the river bed following a heavy rain.
NEMATODE DENSITY AND TEMPERATURE
The only time during the 12 months when no nematodes were detected was
in late January and early February 1977. This was during an exceptionally
cold winter when the water temperature reached new lows and a layer of ice
covered much of the lake. At such low temperatures apparently nematode
motility is reduced below the level required to keep the animals suspended
in the water column. They most likely sink to the bottom, remaining
relatively motionless until the water temperature increases, whereupon
muscle activity returns, motility is regained, and some nematodes again
become distributed throughout the water column. They subsequently enter
the water intake pipe with the lake water.
This period of extreme cold provided a rather unique condition in this
geographic area in which to test the effects of an uncommonly low water tem-
perature on the population density of nematodes in the finished water.
Conclusion
There is a water temperature below which nematodes apparently lose
muscular activity and settle away from the water column; thereby water is
free of nematodes.
-------
DENSITY OF NEMATODES IN SEWAGE LAGOON EFFLUENT, RIVER, AND LAKE
Through aerial photography and ground observation a stream was located
a quarter mile in length, entering the Saluda River one mile upstream from
the treatment plant and originating in the vicinity of a recently closed
sanitary landfill. Eight miles upstream is an aerated sewage lagoon with
its effluent flowing into the river. Bordering the lake and river are
cattle and swine pastures from which runoff flows directly into the body of
water. Less than 60 nematodes per gallon were detected entering the river
from the sanitary landfill drainage and between 50-1500 nematodes per
gallon were detected entering the north arm of the Saluda River from the
sewage lagoon.
Conclusion
Organic material apparently enters the river from several sources, thus
providing an ideal benthic breeding habitat for nematodes which have
entered from waste treatment facilities or from adjacent farm land.
DIVERSITY OF NEMATODES IN SEWAGE EFFLUENT, RIVER, LAKE, AND FINISHED WATER
Among the thirteen genera of nematodes found in sewage effluent enter-
ing the river, three (Butlerius, Diplogaster and Rhabditis) comprised 80%
of the total. Downstream and below the effluent of the stream draining
the sanitary landfill, thirteen genera were again found. However, only
five of those entering from the sewage lagoon were present here and only
two of the three dominant genera, Diplogaster and Rhabditis, were found.
In the lake at the level of the intake pipe, five genera were recovered with
no representatives from the thirteen in the lagoon effluent. Finally, in
the finished water ten genera were identified; three were also present in
the sewage effluent, but Rhabditis was the only sewage dominant genus also
recovered from the finished water.
Conclusion
Rhabditis is the only dominant nematode genus entering the river from
the sewage lagoon which is also found to be present in finished water.
SUMMARY CONCLUSION
A simple and very fundamental biological principle has been observed
concerning the source of nematodes in finished water: as an environment is
changed, so will there be a change in organisms and their densities. Bio-
logists know that if a river becomes polluted with organic substances which
could serve as nutrients for saprophagous organisms, these organisms
will be supported and their population will increase. As the Saluda River
has become polluted, the nematode population has increased in the nutrient-
rich benthic layer. Following a moderate to heavy rain, when their habitat
is disturbed by the scouring action of an increased water flow, the con-
centration of nematodes in the water column may increase dramatically. The
-------
water treatment facility is capable of removing a high percentage of nema-
todes, but 2-10% of the worms pass through the final sand filter. If in the
reservoir the concentration of the worms increases by an order of magnitude,
such as following a moderately heavy rain, so will the number in the
finished water be increased by an equal amount. Thus, one approach to
removing nematodes from finished water is to correct a fundamental error,
that of allowing the high nutrient pollution to flow into source waters.
-------
SECTION 3
RECOMMENDATIONS
1. Our principal recommendation is that the sources of nutrients
flowing into rivers and lakes must be controlled so that the growth of
nematodes is not encouraged by a continual enrichment of the food supply
in their habitat.
2. The filtering efficiency of water treatment facilities and specific-
ally of sand filters relative to the removal of nematodes should be deter-
mined for a variety of municipal filters and water qualities.
3. A detailed study should be conducted to determine the composition
of organisms, both protozoa and metazoa, in effluent from aerated sewage
lagoons and in drainage from sanitary landfills. Theoretically the effluent
should be clean, but that is clearly not what we found in this study. To
our knowledge there are no data on this particular aspect of effluent from
both of these waste disposal systems.
4. Increased consciousness should be exercised by state and federal
agencies concerning the proximity of waste treatment effluent to the intake
of municipal water supplies.
5. The technique developed for nematode detection should now be
evaluated for the extraction, concentration, and identification of water-
borne protozoa i.e. Giardia.
6. The possibility that Anonchus, Butlerius, Diplogaster, and
Rhabditis nematodes from sewage lagoons could be carriers of bacteria and
viruses should be investigated.
-------
SECTION 4
MATERIALS AND METHODS
For this study we chose a municipality which supported a modern water
treatment facility, obtained its water from an adjacent reservoir, and
showed a consistent nematode population (largely of the family Rhabditidae)
in its finished product. The reservoir received water from one major river
which had several tributaries. Organic effluent entered the river system
from an aerated sewage lagoon and by drainage from a recently closed sani-
tary landfill. The lagoon was approximately eight miles and the landfill
one mile upstream from the reservoir (Figure 1).
Nematode density determinations were made on water samples collected
from sewage lagoon effluent, several areas along a river and reservoir,
water treatment plant intake, and effluent from settling tanks and sand
filters. The basic method developed and used in the 12-month survey of
finished water is presented in detail. Modifications to this procedure as
required for other water sources are given in the appropriate sections.
SAMPLES FROM FINISHED WATER
Water filtration
All samples were taken within the laboratory of a modern municipal
water treatment facility which processes approximately four million gallons
of water each day. A series of three eight-inch diameter, two-inch deep,
US Standard sieves (Tayler, Inc.) was placed on an eight-inch steel funnel
supported by a ring stand near the water tap. The first sieve had a 25-ym
pore screen (#500 mesh), followed by two sieves, each with screens of
greater pore sizes which served as supports for eight-inch inserts (secured
to the frame with opaque bathtub caulk) of polyetheylene woven material
(Tetko, Inc.). The second sieve had an insert with 20-ym pores (HD7-20)
and the third, an insert with 10-ym pores (HD7-10 Super). Every fourth
gallon of water filtered through sieves (25-, 20-, and 10-ym pores) was
passed through a standard parabolic, steel filtration funnel with a 47-mm
diameter membrane filtration unit, coupled to a 1-gallon vacuum flask. A
47-mm, UniporeR polycarbonate, 3-ym pore membrane (Bio-Rad Laboratories)
was placed on the filtration unit through which the sieved water passed.
The filtration apparatus for the collection of nematodes is shown in
Figure 2. A more detailed description of the procedure follows.
A municipal water meter (Neptune, Inc.) is attached to the sample faucet
and adjusted to a flow rate of approximately 1.5 gpm. Water is run through
-------
A/
TABLE ROCK
RESERVOIR
AERATED
' SEWAGE
NORTH
SALUDA
RESERVOIR
LAGOON
s, , SANITARY
LAND FILL
6.7,8-iS SALUDA
LAKE
O
3
_l
CO
10 Miles
Figure 1
Watershed study area in Easley, South Carolina.
-------
Figure 2
Apparatus for the extraction of
particulate matter from
drinking water.
Figure 3
Particulate matter being washed
from sieve onto 12-mm membrane
in Swinney filter.
10
-------
the meter and 3/4-inch i.d. plastic tubing for a sufficient period of time
to clean the line of rust. The meter reading is recorded, and tubing from
the flow meter is placed over the top of the first sieve and secured in that
position. At this flow rate no accumulation of water should occur at the
24-ym pore sieve. There is usually a shallow head of water on the second
and third sieves, and frequent flow rate adjustments may be necessary to
prevent excessive accumulation and overflow. After three gallons have
passed through the sieves, the support funnel, and into the sink, the
plastic drain hose is quickly moved to the parabolic funnel for filtration
through the 47-mm diameter, 3-ym pore polycarbonate membrane. As sieved
water begins to fill the funnel, the vacuum pump is turned on to 15 psi to
facilitate filtration. When the filtered water approached the one-gallon
mark on the vacuum flask, the drain hose is removed from the parabolic
funnel and sieved water is directed into the sink. The pump is then turned
off, the funnel removed carefully, the flask emptied and the funnel replaced.
The sequence of passing one gallon of sieved water through the membrane for
each four gallons of sample water filtered through the three sieves is
repeated. Ideally, one hundred gallons of water are processed in this
manner, 25 of which will have been sieved and also filtered through the
membrane.
Optical Microscopy
At this point, one of two methods for the further concentration and
identification of nematodes can be employed. The filtered material from
each sieve and membrane can be processed wither for optical or scanning
electron microscopy. The optical procedure will be discussed first. If
there is a small amount of material remaining on each sieve the residue can
be transferred to a 13-mm diameter, 3-ym pore membrane in a Swinney (Fisher
Scientific) filter; however, if there is a large amount the transfer must be
made to a separate 47-mm diameter membrane. The latter is accomplished by
repeatedly flushing each tilted sieve with distilled water and collecting
the wash in the parabolic funnel with a 47-mm diameter, 3-ym pore membrane.
For a small accumulation of material the smaller 13-mm membrane is employed.
For this, a one-hole rubber stopper with short tubing inserted is placed
onto the top of the vacuum flask. One end of a Swinney filter holder is
attached to the plastic tubing and a 20-cc syringe is secured to the other
end of the Swinney device. A 3-ym pore polycarbonate membrane is placed
within the Swinney and a 3-4 inch diameter plastic funnel is positioned in
the neck of the syringe (Figure 3). The transfer is conducted as described
above by flushing each of the three sieves and the 47-mm diameter membrane
(Figure 4) with distilled water and collecting the material on four separate
13-mm membranes. After the transfer of filtered material to the 3-ym pore
membranes is complete, each membrane is placed in a Syracuse watch glass,
or similar shallow disk, with a few milliliters of distilled water. The
surface of the membrane and the surrounding water are then examined under
a stereoscopic microscope for a total nematode count. Selected worms can be
removed to a slide, covered with a coverslip, and then identified with
bright field or phase optics.
11
-------
Figure 4
Particulate matter being washed
from 47-mm membrane onto
12-mm membrane.
Figure 5
Swinney filter opened and 12-mm
membrane being placed onto
SEM stub.
-------
Scanning Electron Microscopy
If scanning electron microscopy (SEM) is desired for the detection of
nematodes, the residue on each of the three sieves and on the 47-mm membrane
must be transferred to 13-mm diameter, 3-ym pore membranes. This is only
practical if there is a small accumulation of material on the sieves and
membrane. The procedure will be the same as discussed above and the trans-
fer must be carefully and completely made to the smaller membranes (Figures
3 and 4). As the last few milliliters of wash are drawn through the
Swinney apparatus, 10-ml of 2% glutaraldehyde are added to the syringe and
3-ml filtered through the Swinney holder. The syringe and Swinney holder
are removed together from the short plastic tubing and positioned upright
for one hour. This will fix the nematodes so they will not become exces-
sively distorted on air drying. After the nematodes have been in glutaralde-
hyde for one hour, each syringe is placed back on the plastic tubing and the
fixative is replaced with 50% ethanol. The dehydration is continued, allow-
ing each increasing concentration of alcohol (75%, 95%, 100%) to remain in
the syringe for 10 minutes. After the second rinse of absolute ethanol has
been pulled through the syringe the Swinney holder is removed from the
syringe, opened and the membrane removed and placed in a protected area to
dry (Figure 5). The particulate matter does not adhere strongly to the
membrane and may be lost by any sudden movement of air current. Within ten
minutes the membrane is secured to an SEM stub with double stick tape.
After metal coating the membrane is examined at approximately 200 X for
the detection, identification, counting and sizing of the nematodes. The
time required to examine each stub varies from 30-60 minutes, depending
on the amount of extraneous material present and the concentration of
nematodes. At this magnification, the nematodes are usually identifiable
by their characteristic shape (Figure 6). If positive identification is
difficult at 200 X, magnification is increased to a power where the cephalic
region and cuticular patterns can be seen (Figure 7).
Nematode Densities Calculated
To obtain the total number of nematodes per 100 gallons of sample, from
either the optical or scanning electron microscope, the following method is
employed:
(a) Number of nematodes present on 25-ym sieve = A
(b) Number of nematodes present on 30-um sieve = B
(c) Number of nematodes present on 10-ym sieve = C
(d) Number of nematodes present on 3-ym membrane = D
A + B + C +(4 x D) = E = Number of nematodes/100 gallons
The number of nematodes on the 3-ym membrane is multiplied by a factor of
four because only 25 gallons are filtered through the membrane, and a total
of 100 gallons is filtered through each sieve. On occasions, we have not
been able to process 100 gallons because of excessive inorganic (rust) or
organic (algae) material in the drinking water; a fraction of the desired
volume, as recorded on the flow meter, will suffice in the calculations.
13
-------
. \
.
Figure 6
Three nematodes on
3-prn pore membrane in SEM.
Figure 7
High magnification of anterior
region of nematode showing
annular ridges and mouth.
1A
-------
Comparative Study
To compare the effectiveness of the two procedures and also to test the
reproducibility of samples taken in series, the following study was conduct-
ed. On alternate days over a ten-day period, two separate samples of drink-
ing water, were taken one hour apart from the water treatment facility
laboratory. The first was processed for optical and the second for scanning
electron microscopy. Examinations of the collected material were conducted
by separate individuals and data were not compared until after five examin-
ations had been completed.
SAMPLES FROM RIVER, LAKE AND SEWAGE LAGOON EFFLUENT
A modification of the centrifugal-floatation technique (7) was used to
determine the density of nematodes in water samples taken from eight sites
along the Saluda River (Figure 1). On each of six collection dates in May
and June of 1977, multiple one-gallon samples were taken from the following
locations: 1) fifty feet above the sewage lagoon effluent on the North
Saluda, 2) at the mouth of the sewage lagoon effluent, 3) from the South
Saluda River, 4) above the effluent from the sanitary landfill, 5) effluent
from the landfill, and 6) lake water at the surface, 2 m, and 3 m from the
bottom. In the laboratory each one-gallon sample was shaken for one minute
and a 320 ml sample was divided equally among eight 50 ml polycarbonate
centrifuge tubes. The tubes were balanced and centrifuged at 200 x g
for five minutes. The supernatant from each tube was then filtered through
a single 47-mm, 3-micra Unipore polycarbonate membrane in a parabella
funnel attached to vacuum flask. A stream of distilled water washed material
from the membrane into a Syracuse watch glass which was then examined under
the dissecting scope.
The residue in each centrifuge tube was resuspended thoroughly in a
sugar-water solution (484 g of sugar per liter of distilled water gives a
solution with specific gravity of 1.18) and centrifuged as before. The
supernatant of each tube was then poured into a 600 ml beaker with 300 ml
of tap water, stirred and allowed to settle for about 15 minutes to dilute
the syrup and to allow the nematodes to recover from any deleterious
osmotic effects of the syrup. The sugar solution centrifugation may be
repeated if necessary if the nematode concentration is greater than 100
nematodes/per gallon, as is usually the case with sewage lagoon effluents.
After 15 minutes the supernatant and tap water were filtered through a
47-mm, 3-micra pore membrane as above. The material on the membrane was
again washed with distilled water into a Syracuse watch glass and examined
under a dissecting microscope or transferred to a 13-mm membrane for SEM
examination.
SAMPLES FROM SAND FILTER
To ascertain the importance of the sand filter as a nematode-breeding
area, random samples of sand, anthracite, and gravel were taken from the
filter just before backwashing, and water samples were taken from the back-
wash effluent. Also the sediment or floe on the bottom of the sedimentation
tanks was sampled. Nematodes can be separated and extracted from inert
15
-------
debris by their motility. Requirements for the technique (8) were a funnel
with a piece of rubber tubing attached and closed by a clamp. The funnel
was placed on a support and contained the sample on a piece of tissue
supported by a wire screen. The funnel was then filled with water until
the tissue was submerged. Active nematodes passed through the tissue and
collected in the funnel stem. The nematodes were then collected and concen-
trated in accord with the above procedures for optical or scanning micro-
scopy.
GENERIC DETERMINATION
The procedure used to collect samples at four locations for the deter-
mination of generic diversity of nematodes was as follows. The aerated
sewage lagoon effluent samples were collected in one-gallon plastic bottles
directly from the effluent pipe that empties into the river. The Saluda
River samples were collected at a depth of 1/2 m at a point several kilo-
meters below the entrance into the river of the stream draining the
sanitary landfill (Figure 1). Lake samples were collected at a depth of
1 m within 2 m of the intake for the water treatment facility. The techni-
que for collecting nematodes from finished water was presented earlier.
Extraction of nematodes from sewage effluent, river, and lake samples
was most often accomplished by letting the one-gallon sample of water
settle for 24 hours. A 10-ml portion of the bottom layer was then taken
with a pipette and placed in a Syracuse watch glass. Two alternate methods
were used to a lesser extent. The first involved a steep-sided wine bottle
in which the one gallon sample was placed. The neck of the bottle was
tightly fitted with an 8 inch rubber hose, with a hose clamp attached to the
end to insure water tightness. The bottle was inverted and allowed to stand
for 24 hours. A second clamp was placed four inches above the bottom clamp,
and a 10-ml sample was collected in a watch glass from the tubing by
removing the bottom clamp.
The second method, employed only for river and lake samples, had 25
gallons of water poured through a series of our eight-inch sieves of 500,
106, 53 and 25 micra mesh pores. The trapped sediment on the last two
sieves was then washed into a 25-ml sample vial with distilled water and
taken to the laboratory for examination in a watch glass.
The liquid in the Syracuse watch glass was examined under a stereo
microscope using indirect fluorescent lighting. The nematodes were pre-
served by both infiltration with glycerin and preservation in 5% formalin.
Identification of the nematodes was accomplished under 400 X or 1000 X
phase contrast optics with the aid of two taxonomic keys (9, 10).
16
-------
SECTION 5
RESULTS AND DISCUSSION
EXTRACTION, CONCENTRATION, AND DETECTION OF NEMATODES IN FINISHED WATER
Through the use of the prescribed series of sieves and membranes, an
assortment of small plants and animals can be efficiently removed from
drinking water. The extracting and concentrating procedures are simple,
can be completed within a two-hour period, and utilize equipment costing
less than five hundred dollars.
The effectiveness of the SEM method for detection, when compared to the
optical procedure, is indicated in Table 1. The SEM was four times more
effective, with a total of 167 worms identified, than the stereoscopic
microscope with 40 observed from duplicate water samples. The nematodes
observed optically from each sieve and membrane were larger than those
measured in the SEM. The smallest nematodes recorded were 75um by 3pm,
a measurement which is close to the dimensions of a newly hatched larva,
and these were observed only with the SEM. The accuracy of the extraction
and concentration procedures when coupled with the SEM for detection is
difficult to determine. However, based on limited recovery studies with
known numbers of laboratory-reared nematodes of different sizes, we believe
that the recovery is approximately 75%. When the 3-ym pore membrane is
changed to a 1-ym pore, the rate of water flow is greatly reduced and
retention of particulates is increased. When the pore size is increased to
5-um, fewer nematodes are retained but the rate of water flow is enhanced.
Membranes with 3-ym pores were thus judged to be the most desirable.
The effectiveness of these procedures when coupled with stereoscopic
microscopy is considerably less than with the SEM. The limited magnifica-
tion of the optical system, the depth of the water in the Syracuse watch
glass, and the arrangement and type of lighting for visualizing the nema-
todes are all features which limit the detection of the smaller nematodes
with optical microscopy.
NEMATODE DENSITIES AND ANNUAL CYCLE
The data in Table 2 present the number of nematodes collected from
finished water samples for each collection day, beginning June 18 and
ending July 3. It was during this preliminary study that the procedure
included only the three eight-inch sieves and not the 3-ym pore membrane.
This data, when compared to the subsequent results obtained with the use of
the 5-ym membrane, gives an indication of the ability of the nematodes to
pass through a 10-ym sieve and also suggests that the majority of the nema-
todes were motile.
17
-------
Table 1. A COMPARISON OF THE TWO DETECTION METHODS FOR DETERMINING NEMATODE LENGTH AND POPULATION
DENSITY IN DUPLICATE WATER SAMPLES
oo
Stereoscopic Microscopy
Filters
Total nematodes
from 5 samples
(500 gal)
Average
nematode
length
Scanning Electron Microscopy (SEM)
Total Nematodes Average
from 5 samples nematode
(500 gal) length
25 microns
20
10
3
13
6
4
17
485 microns
. 333
315
405
34
20
9
104
436 microns
294
305
332
Total nematodes
40
167
-------
Table 2. NEMATODE DENSITY IN FINISHED WATER COLLECTED THROUGH THREE
SIEVES, THE SMALLEST WITH A 10-UM PORE SIZE
Date of sample Nematodes per gallon
6-18-76 0.51
6-21-76 1.67
6-22-76 0.89
6-23-76 0.40
6-25-76 0.14
6-28-76 0.28
7-1-76 0.27
7-3-76 0.18
19
-------
During the months of July, August, and early September the length of
all nematodes collected was determined by measuring each worm on the video
screen of the SEM, and that distribution is presented in Table 3. Approxi-
mately 70% of all nematodes collected were between 100 and 300 micra in
length, indicative of a larval population. Only 15% of the nematodes were
less than 100 micra in length. Previous investigators have used dissecting
microscopes to confirm the presence of nematodes, and such detection is
difficult when the length of the worm is less than 100 micra, especially if
the nematode is nonmotile. Identification problems due to size and non-
motility were eliminated with the SEM.
The major period of collection and enumeration extended for 12 months
from July 1976. The data in nematodes per gallon of finished water are
shown in Figure 8. There is clearly a base line of approximately one per
gallon with three sharp deviations: early October, early December, and
middle April. These peaks are believed to be a consequence of an environ-
mental perturbation, which will be discussed later, and they are not a con-
sequence of a reproductive cycle which we anticipated finding.
NEMATODE REMOVAL WITHIN TREATMENT FACILITY
By determining the nematode density in (a) raw water entering the treat-
ment plant, (b) the influent to the sand filter, and (c) the finished water,
the effectiveness of the treatment process on nematode removel could be
evaluated. On each of five collection dates during periods of low rainfall
the three samples were taken within a three-hour period and the concentra-
tion at each location is expressed in Table 4 as the average of the five
samples. For these samples the treatment process removed 98% of the nema-
todes entering the system. These results are similar to those reported by
Gupta (11) who showed that 99% of the nematodes could be removed by coagula-
tion, sedimentation, and filtration, and to Peterson et al. (12) who noted
removal of 98% nonmotile and 25% motile nematodes by a similar process.
There was only one sampling date following a period of heavy rain, and
the highest concentration of nematodes in the finished water, 15 per gallon,
was found on this occasion. Removal efficiency by the treatment processes
apparently decreased when the nematode concentration in the water entering
the plant was increased because of the heavy rainfall. The entire treat-
ment process removed only 91% of the nematodes (Table 4).
These data also provide evidence that the source of nematodes in the
finished water is the raw water and not an area within the treatment plant.
Increases in the concentration in the raw water were always accompanied by
increases in the influent to the sand filter and in the finished water.
NEMATODE CONCENTRATION IN SAND FILTER
The examination of component parts of the sand filter for nematodes re-
sulted in very few positive samples, approximately one nematode per gallon.
If gravid worms had been present in the recesses of the filter where they
were protected from the turbulence of back-washing and capable of some
sustained production of larvae, both adult and juvenile worms would have
20
-------
Table 3. LENGTH DISTRIBUTION OF NEMATODES FROM 100-GALLON SAMPLES OF FINISHED WATER COLLECTED
ON 12 SEPARATE DATES
LENGTH DISTRIBUTION (MICRONS)
Date of Sample
7-10-76
7-13-76
7-19-76
7-22-76
7-24-76
7-25-76
7-26-76
8-13-76
8-18-76
8-27-76
9-3-76
9-14-76
TOTAL
<100
32
24
17
43
12
16
20
4
24
16
1
4
213
100-
199
79
88
58
81
57
37
85
28
31
45
15
16
621
200-
299
18
24
27
23
28
25
41
29
37
25
20
31
328
300-
399
11
9
3
1
15
1
10
6
6
29
11
12
114
400-
499
3
0
2
1
2
0
1
2
16
6
3
4
40
500-
599
0
0
0
1
0
0
0
0
12
5
1
2
21
600-
699
0
0
1
0
0
1
0
0
4
2
0
0
8
700-
799
0
0
0
0
0
0
0
0
0
0
0
1
1
800- goo-
goo 1000
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
1
Total
143
145
108
150
115
80
157
69
130
129
51
70
1347
-------
* - NEMflTOOE CONCENTRRTION
l oo S'.OO
JUL flUG
a'. 00 13.00
SEPT
17.00 21.00 25.00 29.00
WEEKS
OCT NOV DEC JflN FEB
39.00 37.00 Ul.OOufe.OOU9.00
MflR flPR MflT JUN JUL
Figure 8
Nematode density in finished water for 54 weeks
during 1976-77.
22
-------
Table 4. NEMATODE CONCENTRATION AT THREE LOCATIONS IN THE TREATMENT PLANT
DURING DRY AND RAINY PERIODS
Location
Negligible rainfall
Heavy rainfall
Number of Percent reduction Number of Percent reduction
nematodes of numbers in nematodes of numbers in
present raw water present raw water
Raw water 24
Sand filter
influent 3.5
Finished water 0.5
85
98
158
64
15
40
91
23
-------
been identified. These few nematodes that were observed were similar in
size to those found in the influent to the sand filter and in the finished
water.
In the face of these observations, the conclusion of Section B, that
the nematodes are not breeding in the sand filter, is supported. It was
also determined that the careful and complete sampling of a second filter,
with all of the associated technical problems for the treatment plant per-
sonnel, would not be necessary.
NEMATODE DENSITY AND RAINFALL
The effects of rainfall were first noted in results of samples collected
over the 16-day period by the preliminary procedure utilizing only sieves
(Table 2). The first sample of the study contained 0.51 nematodes per gallon.
The rainfall for that sample date was measured at 0.66 inches per day. The
concentration in the sample two days later increased to 1.67 nematodes per
gallon with rainfall recorded at 0.83 inches per day. As rainfall decreased
during the following days, nematode concentrations also tended to decrease.
In support of the above observation, increases in nematode concentra-
tions in the finished water after periods of rainfall were also found in
samples collected by the finalized procedure (Figure 9). For week 12, during
the middle of September, no rainfall was recorded and the concentration of
nematodes was 0.86 per gallon. During the following week the concentration
increased to 6.53 nematodes per gallon with a rainfall of 0.71 inches per
day. The sample for week 13 resulted in a concentration of 15.0 nematodes
per gallon with an accompanying rainfall of 2.17 inches per day. The next
week no rainfall was recorded, and the concentration dropped to a low 0.51
per gallon.
Rainfall may increase the concentration of nematodes in the finished
water in two ways. First, Englebrech, et_ _al., noted significant increases
in the nematode population of a stream during periods of high runoff (13).
Nematodes are known to be abundant in the top few inches of soil; conse-
quently, rainfall with intensities large enough to cause runoff probably
flushes the nematodes from the soil into a receiving stream. These nematodes
are then carried by the stream to the intake of the water treatment facility
and eventually are observable in the finished water. The land in the area of
Saluda River and Saluda Lake is hilly and used for farming. Although we did
not conduct surveys, nematodes in fields and pastures should be abundant and
are probably flushed into the river by heavy rainfall. The increase in
nematode concentrations in the finished water for week 14 may partially be
the result of runoff. Prior to this sample which resulted in a total of 15
nematodes per gallon, the highest intensity of rainfall was recorded. At
one rainfall station, 4.78 inches of rain fell in a 24-hour period. Such
information suggests that runoff very probably contributes to the nematode
population in the raw water and also to the concentration in the finished
water.
Secondly, Baliga &t^ atl., found that the concentration of nematodes in
the benthos of a stream decreased following high flows in the stream (7).
24
-------
PS
* - NEMfiTOOE CONCENTRHTION
m - RfllNFRLL
°1 00 5'. 00
JUL HUG
LOO J is.oo" * 17.00
SEPT OCT
'.oo sfe.oo iSToo
WEEKS
NOV DEC JRN FEB
-T • • • • •• I • • • I »-tP"
33.00 37.00 VI.00 «S.OO 19.00
MflR RPR HflY JUN JUL
Figure 9
Nematode density in finished water and the average rainfall
in the watershed area for 54 weeks during 1976-77.
25
-------
The drop in density, they concluded, was as a consequence of scouring the
nematodes from the benthic area. In our study, high concentrations of nem-
atodes were found in the finished water during periods of increased stream-
flow of the Saluda River. Such increased water flow very probably moved the
nematodes from the benthos into the water column and carried them to the lake
and subsequently to the intake pipe of the water treatment plant. Stream-
flow measurements were obtained from a United States Geologic Survey
(U.S.G.S.) station located about 1.2 miles downstream from the water plant.
The relationship between stream flow and nematode concentrations in the
finished water is shown in Figure 10.
Incidental to this rather strong relationship between the forces
associated with streamflow and the movement of nematodes from the benthic
area into the water column is the change in turbidity of the river. Thus,
a direct correlation exists between the lake turbidity monitored at the
water treatment facility and density of nematodes in finished water, as is
shown in Figure 11.
NEMATODE DENSITY AND TEMPERATURE
Previous investigations (7) have indicated that nematode concentrations
in a stream benthos are higher during the cooler months and lower during
warmer months. Similar observations were recorded in this study which
indicates that nematode concentration in the finished water generally
decreases with decreasing water temperature. When density data from July to
January, except for samples during rainy periods, are plotted against water
temperature a relationship between nematodes in the finished water and the
temperature of the finished water is detected and is presented in Figure 12.
The concentrations in water are highest at a temperature of 23-26°C and the
lowest at a temperature of 4°C. Motility decreases as the temperature de-
creases and the less motile worms are removed more effectively by filtration
(11). This results in a decrease in the nematode concentration of the
finished water.
This relationship is greatly strengthened by the observation that no
nematodes could be detected in the finished water during the period in
January when the water temperature went below 4°C and ice was on the lake.
DENSITY OF NEMATODES IN SEWAGE LAGOON EFFLUENT, RIVER AND LAKE
There is an increase in the density of nematodes as the river flows
toward the lake (Table 5). The South Saluda does not receive the agricul-
tural, industrual, or municipal drainage that flows into the North Saluda
and this is apparent in a lower average nematode count. The density of
worms from the lagoon effluent is artificially low after the third sample
collection because an increased level of chlorine is added to the effluent.
The presence of chlorine gas was noticeably greater after a conversation with
the plant operator when the high nematode density in the previous collection
was mentioned.
After the North and South Saluda rivers merge the average density just
above the landfill effluent was 18 nematodes per gallon. Twelve per gallon
26
-------
- NEHfl73DE CONCENTRRTION
- STREflMFLOW
°i 00 5'. 00
JUL RUG
9'.00 13.00 17.00 21.00
SEPT OCI NOV
25.00
WEEKS
DEC JfiN FEB MHR RPR MflY
JUN
JUL
Figure 10
Nematode density in finished water and the streamflow
for 54 weeks during 1976-77.
27
-------
00
55"1"1
mo
is.
* - NEMflTBOE CONCENTRRTION
m - TURBIDITT
"l.OO 5'. 00
JUL flUG
.00
13.00 17.00
21.00
25.00
WEEKS
SEPT OCT N0V DEC JfiN
29.00 33.00 37.00 It'l . 00
FEB MflR flPR MflY
19.00
JUN JUL
Figure 11
Nematode density in finished water and the turbidity
of the lake water for 54 weeks during 1976-77.
28
-------
1.2
1.0
0.8
o
\
o
0.4
LJ
0.2
8° 12° 16° 20°
WATER TEMPERATURE °C
28'
Figure 12
Relationship between nematode concentration
in finished water and water temperature.
29
-------
Table 5. DENSITY OF NEMATODES AT VARIOUS SITES ALONG THE SALUDA RIVER
DURING MAY AND JUNE 1977
Number of nematodes per gallon
Location
South Saluda River
North Saluda
Sewage lagoon
Saluda River
River
effluent
Sanitary landfill effluent
Lake Saluda,
Lake Saluda,
Lake Saluda,
surface
2 m from bottom
1 m from bottom
5-6
0
0
378
35
35
12
35
24
5-11
12
12
1,488
12
24
12
35
12
5-20
0
12
473
12
35
12
0
35
6-2
0
24
71
0
24
0
0
0
6-17
12
12
159
12
0
0
0
0
6-27
0
0
59
35
59
0
24
35
Average
4
10
438
18
30
6
16
18
30
-------
is the mean average for the samples from the entire river system if the two
effluent figures are not counted. In several samples, and especially on
May 11, the nematode population in the Saluda River was the same as that in
either of its forks before the sewage effluent entered the river. The
sewage effluent, then, appeared to be effectively diluted by the river
drainage system. Along the eight-mile course downstream the nematodes were
probably well dispersed through the water or eventually settled out, espe-
cially during dry periods with no runoff and low flow rate.
The same relationship was found to be true of the effluent from the
sanitary landfill. Although the concentration of nematodes in the drainage
stream was slightly higher than that in the river, by the time the water
reached the lake the density had dropped to its previous level.
The density of nematodes in the lake was stratified with increasing
numbers toward the bottom; however, no benthic samples were taken. The
intake to the water treatment plant is located about 1.5 m from the bottom
of the lake, and it is near that level that the greatest concentration of
nematodes was found in the water column.
The summer months of June and July 1977 were extremely dry. With no
rainfall and with a resulting minimal streamflow the population density of
the river was very low, and in some cases no nematodes were found.
DIVERSITY OF NEMATODES IN SEWAGE EFFLUENT, RIVER, LAKE AND FINISHED WATER
The nematode genera and the frequency of their appearance in samples
from four collection sites are given in Table 6. The water samples were
taken and generic determinations made during the summer of 1977. Eighty
percent of the nematodes in the lagoon effluent represented three genera:
Butlerius_. Diplogaster and RhabdjLtis; ten genera composed the remaining
twenty percent. The three major genera have been associated by other
authors with waste treatment ecosystems (1, 7, 14, 15).
The river samples provided a wide variety of nematodes, thirteen
genera, but without one genus or a group of genera dominating. Of the three
major genera in the sewage effluent Butlerius was the only one not found to
be present in the river. Of the thirteen genera in the river five were
observed in the sewage effluent: Anonchus» Diplogaster, Monhystrella,
Rhabditis and Tobrilus.
The Lake Saluda sample, taken at a depth of 1 m but 2 m above the
intake to the water treatment facility, contained only five genera in a
sample of ten nematodes. Dorylaimoides was the predominant genus. It is
most interesting that none of the five had been observed in the sewage
effluent or in the river sample and none appear on the list of nematodes
from finished water.
In finished water seventeen nematodes were identified and were found to
represent ten genera. Three genera, Anonchus, Nothotytenchus and Rhabditis
were on the list of genera in the sewage lagoon effluent, and together they
31
-------
Table 6. GENERA OF NEMATODES AND FREQUENCY OF THEIR OBSERVATIONS FROM FOUR LOCATIONS ALONG THE
SALUDA RIVER BASIN
to
Sewage lagoon effluent
Acrobeloides
Allionema
Anonchus
Butlerius
Cephalobus
Diplogaster
Diploscapter
Monhystrella
Mononchoides
No tho ty t euchus
Plectus
Rhabditis
Tobrilus
1
1
1
23
1
12
1
2
3
1
1
31
4
Saluda River
Actinolaimus
Alaimus
Anonchus
Desmolaimus
Diplogaster
Domorganus
Monhystrella
Paractinolaimus
Rhabditis
Rhabdolaimus
Teratocephalus
Tobrilus
Tylencho laimus
1
1
1
1
1
1
2
1
2
1
2
1
1
Lake Saluda
P_g_ry_l_ajmqide_s_
Monochromadora
Mononchus
Oncholaimus
Prochromadorella
1
2
2
1
1
Finished Water
Anaplectus
Anonchus
Lep to laimus
Leptonchus
Microlaimus
Mylonchulus
Nothotytenchus
Prodesmodora
Rhabditis
Rhabdolaimus
1
1
2
1
1
2
5
1
1
2
-------
represent 41% of the finished water nematode population. Of those three,
Anonchus and Rhabditis were also present in the Saluda River.
Therefore, of the thirteen genera in the sewage lagoon effluent, five
were identified in the river approximately six miles downstream and two
(Anonchus and Rhabditis) of these five were identified in the finished water.
It is interesting that none of the five genera identified from Lake Saluda
appeared at any other area examined.
Even though the same nematode genera were found in finished water as
are known to enter the river-reservoir system from a sewage lagoon eight
miles upstream, it certainly does not provide conclusive evidence that the
nematodes are from the same source. It merely suggests that they may be
from the same source. Attempts to tag large numbers of nematodes entering
the river from the lagoon with a fluorescent marker and recapturing'the
nematodes at points downstream were begun, but technical problems were not
solved before the project was terminated.
33
-------
REFERENCES
1. Cobb, N.A. Filter-bed nemas: Nematodes of the slow sand filter-beds of
American cities. Contrib. Sci. Nemato. 7:189-212, 1918.
2. Kelley, S.N. Infestation of the Norwich, England, water system. J. Amer.
Water Work Assoc. 47:330-334, 1955.
3. Chang, S.L. and P.W. Kabler. Detection of cysts of Endamoeba histolytica
in tap water by the use of membrane filter technique. Amer. J. Hyg.
64:170, 1956.
4. Chang, S.L., R.L. Woodward and P.W. Kabler. Survey of free-living
nematodes and amebas in municipal supplies. J. Amer. Water Works
Assoc. 52:613-618. 1960.
5. Chang, S.L., J.H. Austin, H.W. Poston and R.L. Woodward. Occurrence of a
nematode worm in a city water supply. J. Amer. Water Works Assoc.
51:671-676, 1959.
6. Chang, S.L., G. Berg, N.A. Clark and P.W. Kabler. Survival, and
protection against chlorination, of human enteric pathogens in free-
living nematodes isolated from water supplies. Amer. J. Trop. Med.
Hyg. 9:136-142, 1960.
7. Baliga, K.Y., J.H. Austin, and R.S. Engelbrecht. Occurrence of nematodes
in benthic deposits. Water Research. 3:979-993. 1969.
8. Baermann, G. Eine einfache methode zur Affindung von Ankylostomum
(Nematoden) Larven in Erdproben. Geneesk. Tijdschur. Ned. - Indie.
57:131-137. 1917.
9. Ferris, V.R., L.M. Ferris, and J.P. Tjepkema. Genera of freshwater
nematodes (Nematoda) of Eastern North America. Dept. of Entomology
Bulletin, Purdue Univ. West Lafayette, Indiana. 1976.
10. Goodey, T. Soil and Freshwater Nematodes. Butler and Tanner Ltd.
London. 1963. 318 pp.
11. Gupta, M.K. Motility control for the removal of nematodes. M.S. Thesis,
University of Illinois, Champaign-Urbana, 111. 1971. 56 pp.
12. Peterson, R.L., R.S. Engelbrecht and J.H. Austin. Free-living nematode
removal by rapid sand filters. Jour. Sanitary Eng. Div., ASCE.
92:229, 1966.
34
-------
13. Engelbrecht, R.S., R.I. Dick and M.R. Matteson. Factors influencing
free-living nematodes in water supplies. Dept. of Civil Engineering
Bulletin, Univ. of Illinois, Champaign-Urbana, 111. 1963.
14. Austin, J.H. Colloquim on the Genus Diplogaster (senso loto) and
ecology of nematodes in waste treatment and surface waters. School
of Engineering Bulletin, Univ. of Fla., Gainesville. 1964.
15. Nicholas, W.L. The Biology of Free-living Nematodes. Oxford Univ.
Press, London. 1975. 219 pp.
35
-------
TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing}
i. REPORT NO.
EPA-600/1-79-029
3. RECIPIENT'S ACCESSION NO.
4. TITLE AND SUBTITLE
Determination of Breeding Sites of Nematodes in a
Municipal Drinking Water Facility
S. REPORT DATE
August 1979 issuing date
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
Averett S. Tombes and A. Ray Abernathy
8. PERFORMING ORGANIZATION REPORT NO.
. PERFORMING ORGANIZATION NAME AND ADDRESS
Department of Biology
Clemson University
Clemson, South Carolina 29631
10. PROGRAM ELEMENT NO.
1CC614
11.««NTRAST/GRANT NO.
R804292010
12. SPONSORING AGENCY NAME AND ADDRESS
Health Effects Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati. Ohio 45268
13. TYPE OF REPORT AND PERIOD COVERED
Final; 2/9/76 - 8/9/77
14. SPONSORING AGENCY CODE
600/10
1 57 SUPPLEMENTARY NOTES
Portions of this work also published in: (1) Scanning Electron Microscopy. Vol. 2,
1978 and (2) Water Research, (In press).
16. ABSTRACT
The question concerning the source of nematodes in finished water has been answered
by clearly demonstrating that these invertebrates do not breed in the sand filter
or another part of the water treatment facility but in the raw water source. The
benthic layer of the rivers and lake provides a supportive environment for a large
nematode population which is suspended in the water column following heavy rains by
the scouring action of increased streamflow. Thus a direct relationship exists
between nematode density in finished water and rainfall.
Continual sources of nematodes for the rivers and lake, in addition to adjacent
agriculture land, are a sewage lagoon and sanitary land fill, both of which flow
into the river. Two genera of nematodes which appear in the lagoon effluent also
appear in the finished water.
An improved method for the detection of nematodes in water was developed whereby
nematodes could be extracted and concentrated onto a 12 mm nucleopore membrane and
identified by scanning electron microscopy.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS C. COSATI Field/Group
Nematoda, Water Treatment, potable water,
water supply, microbiology
Scanning Electron Micro-
scopy, Sand filtration,
nematode densities
57K
18. DISTRIBUTION STATEMENT
Release to public
19. SECURITY CLASS (ThisReport)
Unclassified
21. NO. OF PAGES
46
20. SECURITY CLASS (Thispage)
Unclassified
22. PRICE
EPA Form 2220—1 (Rev. 4—77) PREVIOUS EDITION is OBSOLETE
36
« U.S. GOVCMIMCNTPIIIimilGOFFICE. 1979 -657-060/5392
------- |