United States
Environmental Protection
Agency
Environmental Monitoring and Support EPA-600, 4-79-007
Laboratory January 1979
Cincinnati OH 45268
Research and Development
&EPA
New Approaches to the
Preservation of
Contaminants in
Water Samples
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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 MONITORING series.
This series describes research conducted to develop new or improved methods
and instrumentation for the identification and quantification of environmental
pollutants at the lowest conceivably significant concentrations. It also includes
studies to determine the ambient concentrations of pollutants in the environment
and/or the variance of pollutants as a function of time or meteorological factors.
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
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EPA-600/4-79-007
January 1979
NEW APPROACHES TO THE PRESERVATION
OF CONTAMINANTS IN WATER SAMPLES
by
J. Saxena and E. Nies
Center for Chemical Hazard Assessment
Syracuse Research Corporation
Syracuse, New York 13210
Grant No. R 804609010
Project Officer
Terry C. Covert/Guy Simes
Quality Assurance Branch '
Environmental Monitoring and Support Laboratory
Cincinnati, Ohio 45268
ENVIRONMENTAL MONITORING AND SUPPORT LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
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DISCLAIMER
This report has been reviewed by the Environmental Monitoring and Support
Laboratory, U.S. Environmental Protection Agency, and approved for publica-
tion. 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
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FOREWORD
Environmental measurements are required to determine the quality of
ambient water and the character of waste effluents. The Environmental Moni-
toring and Support Laboratory - Cincinnati conducts research to:
0 Develop and evaluate techniques to measure the presence and concen-
tration of physical, chemical and radiological pollutants in water,
wastewater, bottom sediments, and solid waste.
0 Investigate methods for the concentration, recovery, and identifi-
cation of viruses, bacteria, and other microbiological organisms in
water; and to determine the responses of aquatic organisms to water
quality.
0 Develop and operate an Agency-wide quality assurance program to
assure standardization and quality control of systems for monitor-
ing water and wastewater.
There is an ever-increasing interest to maintain sample integrity during
the sample collection-sample analyses cycle. Present preservation techniques
have limitations. Biological reactions taking place in a sample may cause a
breakdown of contaminants, a conversion of soluble constituents to organi-
cally bound material in cell structures, a solubilization of insoluble
material, or even the conversion of material into a gaseous phase which then
escapes from the sample. In view of the limitations of the preservation
methods currently used, this report looks at the preservation possibilities
in the biological control of microorganisms through the use of enzymes and
antibiotics.
Dwight G. Ballinger, Director
Environmental Monitoring and Support
Laboratory - Cincinnati
iii
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ABSTRACT
The potential of antibiotics, chemical biocides and lytic enzymes in
preserving nitrogen and phosphorus series of nutrients, biological oxygen
demand, and oil and grease in water and wastewaters was studied. Preliminary
selection of agents for sample preservation was based on their ability to
inhibit cell growth and oxygen utilization in primary and secondary effluents
and natural water samples. The drugs which exhibited potential based on this
criteria were: chlorhexidine, vantocil IB, polymyxin B + neomycin + strep-
tomycin (PNS), erythromycin 4- polymyxin B + neomycin (EPN), erythromycin +
polymyxin B + streptomycin (EPS), polymyxin B + chloramphenical + neomycin
(PCN) and gentamycin + amikacin + polymyxin B (GAP). The effective concen-
tration range was 100 - 200 yg/ml for primary effluents, 50 - 100 yg/ml for
secondary effluents, and 10 - 50 Mg/ml for fresh waters. Lysozmyme with or
without ethylenediamine tetraacetic acid or Tris failed to control micro-
organisms in wastewater samples. Sodium sulfide, which has been used to slow
the growth rate of bacteria in suspension, was ineffective as well.
Chlorhexidine and vantocil IB stabilized nitrate and nitrite in fresh
water and relatively clean secondary effluents. Other antibiotics were
unsuccessful in preservation of these nitrogen forms. Antibiotic mixtures
with erythromycin as one component interfered with NOg determination.
Presence of antibiotics caused interference in determination of inorganic
phosphate by vanadophosphoric or ascorbic acid methods.
The antimicrobial agents tested did not offer a practical solution to
the preservation of BOD in wastewaters due to the fact that the agents added
for preservation inhibited the activity of the seed even after the dilutions
required for BOD determination. Efforts to selectively remove and/or
inactivate the test antimicrobial agent prior to BOD determination by boiling
the sample in a sealed ampoule, or pH adjustment, were unsuccessful. Other
approaches tested including preacclimation of seed to the test antimicrobial
agent and the use of lower concentrations of the preservation also failed to
relieve the inhibition.
Oil and grease levels were stabilized by GAP and PCN for up to 2 weeks
in relatively clean waters only. Chlorhexidine and vantocil IB effectively
stabilized pH and dissolved oxygen in natural water samples but were not
effective in municipal wastewaters.
The results demonstrate that antibiotics offer a viable alternative to
conventional methods for preservation of fresh water samples but not for
preservation of sewage effluents.
iv
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This report is submitted in fulfillment of Grant R 804609 by Syracuse
Research Corporation under the sponsorship of the U.S. Environmental
Protection Agency. This report covers a period from 9-1-1976 to 11-30-1978
and work was completed as of 7-1-1978.
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CONTENTS
Foreword ill
Abstract iv
Figures viii
Tables -. ix
Abbreviations x
Acknowledgment . . . ; xi
I. Introduction ..... 1
II. General Conclusions ......... 3
III. Recommendations ..... 4
IV. Literature Search 5
V- Background 6
Stability of water pollutants during storage and/or
Transportation 6
Available methods of preservation of water samples
and their limitations 8
VI. Preliminary Selection of Preservation Agents based
on Their Antimicrobial Action in Waters and Wastewaters ... 11
VII. Evaluation of Selected Treatments as Preservatives of
Nitrogen-Forms in Water and .Wastewater . . 23
VIII. Evaluation of Selected Treatments as Preservatives of
Phosphates in Water 30
IX. Evaluation of Selected Treatments as Preservatives of
BOD in Wastewater 34
X. Evaluation of Selected Treatments as Preservatives of
Oil and Grease in Wastewater 38
XI. Evaluation of Selected Treatments as Preservatives of
pH in Water 40
References 41
vii
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FIGURES
Number Page
>; 1 Effect of test agents on microbial population in
primary municipal effluents collected on two different dates 16
2 Effect of test agents on microbial population in
secondary effluent from trickling filter process collected
on two different dates 17
3 Effect of test agents on microbial population in secondary
effluent from activated sludge process 18
4 Dissolved oxygen as affected by the presence of test
agents in primary and secondary municipal wastewater
effluents 19
5 Effect of test agents on microbial population in
natural waters 21
6 Dissolved oxygen as affected by the presence of test
preservation agents in natural water samples fortified
with glucose-glutamic acid (150 rng/i each) 22
7 Preservation of nitrate and nitrite in spiked natural
;. water samples by vantocil and ehlorhexidine. •..,,,,., 25
8 Preservation of nitrate and nitrite in spiked natural
water samples by antibiotic combinations GAP and PNS .... 26
9 Preservation of nitrate and nitrite in secondary effluent
from activated sludge process ... 28
10 Influence of antibiotics addition on the levels of
orthophosphate in spiked fresh waters 33
11 Potential of antimicrobial agents for preservation of oil
and grease in the secondary effluent from activated
sludge process 39
viii
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TABLES
Number Page
1 Examples of the reactions which may be responsible for
contaminant concentration changes in water samples .... 7
2 Examples of preservation techniques currently in use . . . 9
3 Chemical nature and source of the antimicrobial agents
studied 13
4 Antimicrobial agents tested for their effect on
microbial population in waters- and wastewater samples . . 14
5 Influence of the presence of preservatives on analytical
determination of nitrate and nitrite 27
6 Effect of the addition of test sample preservation agents
on orthophosphate determination by ascorbic acid method . . 32
7 Stabilization of pH by antimicrobial drugs in fresh water
samples fortified with glucose-glutamic acid (150 mg/£ ea). 40
ix
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ABBREVIATIONS AND SYMBOLS
yg/ml micrograms/milliliter
mg/£ milligrams/liter
ppm parts per million
BOD Biochemical Oxygen Demand
COD Chemical Oxygen Demand
TKN Total Kjeldahl Nitrogen
D.O. Dissolved Oxygen
APHA American Public Health Association
Tris Tris(hydroxymethyl)aminomethane
EDTA Ethylenediaminetetracetic acid
x
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ACKNOWLEDGEMENT
Syracuse Research Corporation wishes to acknowledge the assistance of
the staff of Meadowbrook-Limestone Wastewater Treatment Plant, Manlius,
N.Y., and Wetzel Road Wastewater Treatment Plant, Liverpool, N.Y., for their
assistance and cooperation in sampling. Antibiotics donated by Bristol
Laboratories, Hoffmann-LaRoche, Lederle Laboratories and Imperial Chemical
Industries are also appreciated.
Cost sharing by the American Petroleum Institute at the level of 5% is
gratefully acknowledged. API Project Officer: Dr. Geraldine V. Cox.
xi
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SECTION 1
INTRODUCTION
Increased effort to prevent water pollution has led to higher demands for
analysis of waste before discharge, and receiving waters. For example,
analysis of municipal wastewaters for selected constituents and parameters
is required by regulatory agencies for NPDES permit program. The number of
analyses that can be performed at the sampling site are very limited, and
samples must be transported or shipped to a laboratory for detailed chemical
analysis.
A sample of polluted water is an extremely complex and dynamic system
containing many chemical and biological entities. Since the majority of the
water monitoring sites are at remote locations where "grab" samples are taken
for further chemical analyses, the microbial utilization or transformation of
some chemical constituents in a water sample enroute to, or in storage at, a
laboratory may be a source of considerable error, especially in trace
analysis of pollutants. In order to have correct information on the concen-
tration of contaminants in the water samples, it is necessary to stop the
changes that take place in the samples after their collection.
The preservation methods available at present - pH control, chemical
addition, refrigeration, and freezing have many limitations, either mechanical
or chemical. These methods are based on some of the very early work on
samples preservation. For instance, the recommended method to stabilize
phenols is a modification of the work performed over 30 years ago (Ettinger
£t_ral_., 1943). The basic problem is that many of these preservation systems
do not work well. Certain chemical preservatives interfere with analytical
methods or may significantly alter the chemical composition of the sample.
Preservation of samples by refrigeration or freezing is impractical and
expensive. When HgCl2 is used as an antimicrobial agent, the disposal of the
mercury containing sample is a recognized problem.
In view of the limitations of the preservation methods currently in use,
this investigation was undertaken to test the potential of antibiotics,
chemical biocides, and lytic enzymes for preserving water samples. Such
untried systems may offer a distinct improvement over existing procedures.
Since many alterations in water and wastewaters samples are produced by
microbial activity, efforts in this study were directed towards controlling
these alterations as opposed to chemical and physical alterations and losses.
The study was divided into two phases. The first phase examined the
effect of numerous antimicrobial agents on the microbial population in waters
of varying purity with the objective of selecting agents with potential for
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sample preservation, and the determination of effective concentration ranges
for such agents. The second phase involved studying the preservation of
contaminants and sample parameters by the antimicrobial agents selected in
phase 1.
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SECTION II
GENERAL CONCLUSIONS
Antibiotics and biocides tested demonstrate potential for stabilization
of sample constituents and parameters in fresh waters. However, their
utilization for preservation of sewage samples is unlikely because of the
diversity of microorganisms present.
Chlorhexidine and vantocil IB can be used to stabilize nitrate and
nitrite in fresh waters for at least 16 days at room temperature. They can
not be used for preservation of phosphates because of interference with the
analytical method for phosphates.
Preservation of water and wastewater samples with antimicrobial agents
for BOD is not feasible because of the interference caused by the preservatives
in biological activity during measurement. At lower concentrations, the anti-
microbial agents used tend to be utilized as carbon sources by microorganisms
resulting in erroneous BOD values. Oil and grease can be statilized for up
to two weeks in relatively clean effluents with the addition of GAP and PCN.
The study has not led to a suitable and practical method of water and
wastewater sample preservation and efforts in this direction should be
continued.
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SECTION III
RECOMMENDATIONS
A preservative must satisfy many requirements before it can be regarded
as suitable for water and wastewater sample preservation. General requirements
which must be met are (i) it should be more effective than the methods
presently available, (ii) it should stabilize a large number of sample con-
stituents/parameters, (iii) it should offer no or minimal interference with
the analytical procedure, and (iv) it should be safe to use and pollution-free.
Apparently, the process of finding a suitable preservation method is expected
to be slow and time consuming.
The studies undertaken have not led to a practical solution to
problem of water and wastewater samples preservation, and hence the search for
new methods of sample preservation must be continued. The approaches which
have not been successful in our studies should provide clues to .those methods
which will be successful.
It is recommended that research be pursued in the following avenues :
1) Preservation of sewage effluents should be attempted with other untried
antimicrobial agents. For hard to preserve sample constituents/parameters,
the antibiotic action should be used in conjunction with other methods e.g.
sample storage at 4°C, preheating of the sample, pH adjustment, etc.
2) Efforts should be directed towards identifying the species of micro-
organisms which show resistance to antimicrobial agents. This will help in
devising methods to control them. A recent paper by Kelch and Lee (1978)
entitled "Antibiotic resistance pattern of gram negative bacteria isolated
from environmental sources" provides some information in this direction.
3) Antibiotics and biocides show promise for preservation of fresh
water samples and should be investigated further.
4) The use of antimicrobial agents for preservation of BOD in water and
wastewater samples does not appear feasible and it is recommended that other
approaches be explored. Examples of approaches which could be tried are:
gamma irradiation, quick heating by microwave, etc.
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SECTION IV
LITERATURE SEARCH
During the initial phase of the project, a literature search was con-
ducted to obtain relevant references relating to the general subject of water
sample preservation. Papers were gathered with the aid of computerized
and manual searches. The key words selected were: water storage,
preservation, contaminants, water sample, urine, stability, oil and grease,
total organic carbon, chemical oxygen demand, biplogical oxygen demand,
nitrate, phosphate and phenol. Back issues of the Journal of Antimicrobial
Agents and Chemotherarpy were searched to collect information regarding
recently developed antimicrobial agents. The result of .the review work is
reflected by the different pertinent references cited in various sections
of the report..
NTIS and SSIE data bases were searched in order to obtain information
concerning other on-going or recently completed projects in the area of
water sample preservation. The search revealed no relevant projects.
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SECTION V
BACKGROUND
This section presents information concerning alteration of water and
wastewater composition during storage and/or transportation, and the methods
available to prevent these changes. A general description is given here
to form a basis for the study undertaken. Information pertinent to the
stabilization of specific sample constituents and parameters studied is
presented in respective sections later in the report.
STABILITY OF WATER POLLUTANTS DURING STORAGE AND/OR TRANSPORTATION
The stability of the water samples in transit from the sampling site
to the laboratory, or during storage, has received very little attention.
Alteration in the concentration of contaminants in the water samples can
generally be attributed to (i) biological transformation, (ii) alteration
by chemical agents (hydrolysis, oxidation, etc.) and (iii) physical losses
(adsorption, volatilization, etc.).
The available information suggests that biological agents, par-
ticularly bacteria, play a dominant role in causing these changes. It has
become well established that, when fresh water or seawater is stored in
glass containers, bacteria multiply rapidly to numbers which are often far
in excess of those found under natural conditions (Franklands, 1894; Whipple
1901; ZoBell and Grant, 1943; ZoBell, 1946; Taylor and Collins, 1949). The
growth of bacteria in waters transferred to containers is rapid, and maximum
numbers are usually found on the second or third day after filling (ZoBell
and Stadler, 1940). This growth is apparently at the expense of the con-
taminants present in the water samples. The biological transformation
reactions summarized in Table 1 are examples of the biological influence on
sample composition. Although all the changes listed in Table 1 have not
been directly shown to occur in water samples, such alterations have been
noted in the environment. It is likely that the microorganisms responsible
for these conversions will also be present in natural waters.
Phenol in moderate concentration has been reported to be degraded by
microorganisms very rapidly (Harlow, 1939, Ettinger et al., 1943). The
biochemical activity has been reported at pH values ranging from low of 2
(Ruchhoft et al., 1940) to high of 13 (Theriault and McNamee, 1930). A
positive correlation between the loss of phenolic compounds and micro-
biological activity in wastewaters has been noted by Carter and Huston (1978).
Ettinger e± al. (1943) reported over a 90% loss of the phenol content of a
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TABLE 1. EXAMPLES OF THE REACTIONS WHICH MAY BE RESPONSIBLE
FOR CONTAMINANT CONCENTRATION CHANGES IN WATER SAMPLES
Pollutant
Example of microorganisms ' Chemical or physical
which may be responsible alterations affecting
for pollutant concentra- pollutant concentration
tion changes *
Nature of changes produced
Reference
Inorganic nitrogen
(Ammonia, nitrate
& nitrite)
Sulfide & sulfate
Sulfide
Organic nitrogen
Phosphate
Herbicides
Phenylureas
Fhenylcarbamates
Chlorinated aliphatic
acids
Phenoxyalkonoic acids
Phenoxyalkonoic acid
esters
Insecticides
Dieldrin
Detergents
Alkyl benzene
sulfonate
Hydrocarbons
Other aliphatic
and aromatic
compounds
(including
phenolics)
Bacillus mycoides. Proteus
vulgaris , IS. Coli
Nitroaomonas
Nltrobacter
Proteus vulgaris
Thiobacilli
Desul f o vibrio
acid pH
Bacillus mycoides ,
Proteus vulgaris, £. Coli
all microorganisms
Xanthomonas sp.
Sarcina sp.
Bacillus sp.
Pseudomonas sp.
Pseudomonas sp.
Plavobacterium sp.
Aj>robacterium sp.
Acromobacter sp.
Arthrobacter sp.
Nocardia, Flavobacterium sp.
Hydrolysis
Pseudomonas sp.
Bacillus sp.
adsorption on
Pseudomonas sp.
Pseudomonas sp . , Micro-
rrWus sp.. Mycobaterlum sp.,
Nocardia
Acetobacter sp.
Pseudomonas sp.
nitrogenous organic matter
•*• Amino acids -*• NHj
NHif •> NO^
NOj -»- NOa
cystelne
HjS -*• S* -»• 8203 -*• SOij
S0~ -*• H2St
H2S+
organic nitrogenous
compounds -*• NH3
Inorganic phosphate -«-*•
organic phosphate
Used as carbon source
Used as carbon source
Used as carbon source
Used as carbon source
Ester -* acid
Unidentified
container wall
Alkyl benzene sulfonate
Inorganic sulfur compounds,
tertiary butyl alcohol,
phenol, benzole acid etc.
Several hydrocarbons
serve as source of carbon
for growth
Most are used as carbon
source
Thimann, 1963
Aleem, 1970
Aleem, 1970
Thimann, 1963
Saxena, 1970
Thimann, 1963
Thimann, 1963
Thimann, 1963
Kearney, 1966
Kearney, 1966
Kearney, 1966
Kearney, 1966;
Goerlitz and
Lamar, 1967
Goerlitz and
tamar, 1967
Henzla, 1969
Weil and Quentin,
1970
Horvath and Koft,
1972
McKenna and
Kalllo, 1965
Heukelekian and
Dondero, 1963;
Stanier and
Doudoroff, 1970;
Thimann , 196 3 .
* Although these microorganisms have been Isolated mostly from soil, similar microorganisms can be expected to be present in the aquatic
environment, since the bacterial population of water depends predominantly on the extent and character of its contact with earth's
surface.
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Mahoning River sample when stored as collected, A reduction in the phenol
content of the river water sample was also observed during storage at 2°C;
under these conditions nearly 90% of the initial phenol present was lost in
11 days. Rapid breakdown of monochlorophenols in polluted surface waters has
been reported by Ettinger et al. (1950). The change in concentration of some
chlorinated aliphatic hydrocarbons (chloroform, 1,1,2-trichloroethane, and
1,1,2,2-tetrachloroethane) in seawater and sterilized fresh water was examined
by Jensen and Rosenberg (1975). Four different systems were used: (1) daylight,
open system, (2) daylight, closed system, (3) darkness, closed system, (4)
darkness, 2 atm. closed system. The disappearance of the compounds was
greatest in the open aquaria, indicating that loss due to evaporation was
greater than other degradation losses. The decrease in certain chlorinated
aliphatic hydrocarbons was also attributed to biological degradation.
Dokiya et^ &l_. (1974) noted that organomercurial compounds were lost from
natural marine water in 15 days, but not from distilled water. This suggested
that perhaps microorganisms present in the marine water may have been respon-
sible for the loss. Swisher et al. (1973) have reported biodegradation of
nitrolotriacetate-metal chelates in river water upon incubation.
The studies described above clearly show that a considerable change in
the composition of a water sample may take place during storage and trans-
portation. The instability of some chemical constituents in water samples
may be a considerable source of error in their analysis.
AVAILABLE METHODS OF PRESERVATION OF WATER SAMPLES, AND THEIR LIMITATIONS
Most water samples for organic and inorganic analysis must be protected
from changes in composition from the time of collection to the time they are
analyzed in the laboratory (i.e. during transport and storage of the sample).
The methods currently used by the Environmental Protection Agency to stabilize
contaminants in water samples are pH control, chemical addition and refriger-
ation (U.S.E.P.A., 1974). These methods are intended to control bio-
logical activity, retard hydrolysis and complex formation, and reduce vola-
tility of constituents. Refrigeration is the most acceptable method for
controlling microorganisms since it inhibits their growth without otherwise
altering the sample. However, the technique is difficult to use in field
settings. Sulfuric acid has generally been used as a bacterial inhibitor in
samples for COD, oil and grease, organic carbon, etc. Presently, a combina-
tion of refrigeration and sulfuric acid is used to preserve oil and grease in
water samples. The type of preservation methods as they relate to the various
water parameters are presented in Table 2.
Antibiotics and biocides as substitutes for the conventional methods of
preserving water and wastewater samples have received little attention, even
though they offer many advantages over the existing methods. For example,
unlike HgCl2, antibiotics are pollution free and safe to handle. Berg et al.
(1966) reported stabilizing COD in primary effluents by heating the effluent
to 80°C and subsequent addition of polymyxin B. Heating alone failed to
control microbial population in these samples. The potential of antibiotics
and biocides for inhibiton of microbial activity in samples is also
i
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TABLE 2. EXAMPLES OF PRESERVATION TECHNIQUES CURRENTLY IN USE
(U.S.E.P.A., 1974, Huibregtse and Moser, 1976)
Parameter
Preservative
Maximum
holding period
Acidity-Alkalinity
Biochemical Oxygen Demand
Chemical Oxygen Demand
Chloride
Color
Cyanide
Dissolved Oxygen
Herbicides
Metals, Total
Nitrogen, Ammonia
Nitrogen, Kjeldahl
Nitrogen, Nitrate
Nitrogen, Nitrite
Oil and Grease
Organic Carbon
PH
Phenolics
Surfactants
Total Phosphorus
Refrigeration at 4°C
Refrigeration at 4°C
H2S04 to pH <2
None required
Refrigeration at 4°C
NaOH to pH 12
Determine on site
Acidify samples to pH 4.0
5 ml HNO per liter
Cool, 4°C
H2S04 to pH <2
Cool, 4°C
H.SO, to pH <2
Cool, 4°C
H2S04 to pH <2
Cool, 4°C
H2S04 to pH <2, Cool, 4°C
H2S04 to pH <2
Determine on site
1.0 g CuSO,/£ +
Cool, 4°C 4
Cool, 4°C
Cool, 4°C
to pH <4
24 hours
6 hours
7 days
7 days
24 hours
24 hours
No holding
6 months
24 hours
24 hours
24 hours
24 hours
24 hours
24 hours
No holding
24 hours
24 hours
24 hours
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revealed by the studies undertaken by Collins et al. (1976)^ These authors
reported that nitrification in aquatic recirculation systems could be effec-
tively inhibited by erythromycin or 2-chloro-6-(trichloromethyl)pyridine.
The diversity of the microorganisms present in the water samples makes
complete and unequivocal preservation rather difficult. Unlike the sample
compositional changes brought about by the presence of microorganisms, the
changes caused by certain physical and chemical agents can father,easily be
controlled. For example, by tightly stoppering the water sample, the losses
due to volatilization can be reduced. By rinsing the sample bottle with the
extraction solvent, the compound can be desorbed from the walls of the con-
tainer (Breidenback j2t al., 1964). On the other hand,;the methods available
currently to prevent microbial activity in the samples are not very effective.
Certain bacterial acidophiles will continue to grow in samples even if the pH
is less than 2.0. Addition of 40 mg/£. HgCl?, the recommended concentration
for preservation, is often not completely effective in eliminating microbial
growth in highly polluted samples, yet higher concentrations would present
both environmental and health hazards (Huibregtse and Moser, 1976; Zobell and
Brown, 1944). Storage at 4°C is not possible for long since microbiai
multiplication and metabolism will continue at this temperature, although at
a slower rate than at ambient temperatures.
Many of the preservation systems produce extensive changes in the sample
which may preclude certain analytical determinations. Availability of suit-
able method(s) of sample preservation is becoming increasingly important as
more and more organic contaminants are being,detected and monitored in
water samples. Preservation of samples by refrigeration is free from many
limitations but is expensive and difficult to effect in the field.
In view of the limitations of the preservation methods currently in
use, the proposed investigation was intended to examine the potential of
antibiotics, chemical biocides and lytic enzymes in sample preservation.
10
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SECTION VI
PRELIMINARY SELECTION OF PRESERVATION AGENTS BASED ON THEIR ANTIMICROBIAL
ACTION IN WATERS AND WASTEWATERS
The proposed preservation approaches were first screened by studying
their effect on microorganisms in waters of varying degree of purity.
Evaluation based on the effect on microbial growth and/or metabolic activity
is simple and less time consuming than measurement of sample parameters/
constituents and hence can be applied to a large number of test agents.
Moreover, it is a valid approach due to a direct correlation between micro-
biological activity and sample deterioration. The preservation systems which
effectively controlled microbial growth and metabolic activity were pursued
further. The effective concentration range of the preservants was also
determined from these studies.
SAMPLES AND SAMPLING PROCEDURE
Tests were performed utilizing waters of varying degree of purity. Fresh
waters with low levels of contamination were considered representative of rela-
tively pure water, secondary effluents from activated sludge and the trickling
filter process as moderately polluted water, and primary effluent as grossly
polluted water.
Fresh water samples were obtained from two sources - Keuka Lake in
Hammondsport, N.Y., and Chittenango Creek in Chittenango, N.Y. Keuka Lake
samples were obtained from the "raw water" tap which serves as a source of
water for the Dept. of Public Works in the Village of Hammondsport. The lake
receives agricultural run-off and winery waste. Chittenango Creek samples
were obtained from a location 2.5 miles south of the Village of Chittenango.
The creek is a tributary of Oneida Lake and drains an area of 314 sq. miles.
It receives discharge from a domestic wastewater treatment plant.
Secondary effluents used in these studies were obtained from (a) Wetzel
Road Sewage Treatment Plant, Liverpool, N.Y., which uses trickling filters
as a secondary treatment process, and (b) Meadowbrook-Limestone Wastewater
Treatment Plant, Manlius, N.Y., which uses activated sludge as a secondary
treatment process. Samples were obtained from the overflow weirs of the
clarifiers. Primary effluent samples were obtained from the overflow weirs
of the primary clarifier from Wetzel Road plant only.
Fresh samples were collected for each study and brought back to the
laboratory within one hour. Tests revealed that storage of sample by
freezing for use in later experiments was not feasible, since it resulted in
11
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increased sensitivity of microorganisms towards drugs. Samples were brought
to room temperature by warming in a water bath prior to use.
CHEMICAL AGENTS TESTED
Test compounds evaluated for their antimicrobial potential included anti-
biotics, chemical biocides and lytic enzymes. Sodium sulfide, a product
found to be effective in rendering bacteria dormant in liquid suspension for
indefinite periods (Horsfall and Gilbert, 1976) was also included in the study.
The chemical nature and source of the antimicrobial agents tested are given
in Table 3. Antibiotics were tested in combinations which were made based
on information in the literature concerning the type of microorganisms con-
trolled by specific antibiotics (Table 4). The objective was to achieve a
broad spectrum mixture. The failure of single antibiotics to control micro-
organisms in water and wastewater samples revealed by preliminary experiments,
prompted utilization of this approach. Stock solutions of antimicrobial
agents were prepared in sterile distilled water and stored in refrigerator.
Solutions of antimicrobial agents which were known to be unstable under these
conditions were prepared fresh for each experiment.
EXPERIMENTAL PROCEDURE
The water samples under investigation were homogenized by shaking and
aliquoted into BOD bottles containing a predetermined volume of the test
preservative solution. Untreated samples served as control. The bottles
were incubated at room temperature. At specific time points, dissolved oxygen
was measured by immersing a BOD probe in the bottles and 1 ml aliquots were
withdrawn for standard plate count. Media preparation, dilution, plating
and counting was performed according to the procedure described in APHA
Standard Methods (APHA, 1975). Plate counts were done in triplicate and
the results expressed as an average of the three measurements. In some
experiments the results are expressed as less than a fixed number of colonies/
ml, for example <10 . This fixed number represents the lowest dilution of
sample plated and was chosen in such a way as to sufficiently dilute the
antibiotics to remove any growth inhibition yet enable enough colony growth
on a plate to make the count statistically significant. The time course for
most experiments described in this section was 4-6 days. This incubation
period was considered adequate for the following reasons: (i) microbial
growth was complete during this time in untreated samples, therefore, the
effect of drug treatment on all phases of the growth cycle could be studied,
(ii) in instances where microorganisms overcame the inhibitory effect of
drugs, this phenomenon occurred between 24-48 hours and hence the incubation
period was sufficient to allow us to note the ineffectiveness of the drug,
and (iii) the aim in examining the antimicrobial action of drugs was only to
make a preliminary selection for detailed sample preservation studies.
12
-------
TABLE 3. CHEMICAL NATURE AND SOURCE OF THE ANTIMICROBIAL AGENTS STUDIED
Antibiotics - common name
Chemical nature
Source
.Amikacin
Amoxicillin
'Ampicillin
BL-5786
Chloramphenicol
Chldrtetiracycline
hydrochloride
Polymyxin E
Ery thrpmy e.in
Ge,ntamicln .
Mecillinam
Mine cy dine
Neomycin Sulfate
Oxytetracycline dihydrate
Polymyxin B Sulfate
Streptomycin
Tetracycline
Ticarcilliri
Biocides
Cetrimide,
Chlo rhexldine
semisytithe tic aminoglycoside
antibiotic
3-lactam antibiotic
modified penicillin antibiotic
semisyrithetic cephalosporin
tetracycline antibiotic
polypeptide antibiotic
macrolide antibiotic
aminoglycoside antibiotic
3-lactam antibiotic
tetracycline analog
aminoglycoside, antibiotic
tetracycline antibiotic
polypeptide antibiotic
aminoglycoside antibiotic
tetracycline antibiotic
cationic antiseptic .
(cetyltrimethyl ammonium biomlde)
substituted biguanlde
substituted biguanide
Bristol Labs
Bercham Lab.
Bristol Labs
Bristol labs
Sigma Chem. Co.
Sigma Chem. Co.
Sigma Chem. Co.
Sigma Chem. Co.
Sigma Chem. Co.
Hoffmann-LaRoche,
Inc.
Lederle Labs
Sigma Chem. Co.
Sigma Chem. Co.
Sigma Chem. Co.
Sigma Chem. Co.
Sigma Chem. Co.
Bercham Lab
Sigma Chem. Co.
Id, Ltd.
ICI Ltd.
Fisher Scientific
Vantocil IB
Positive Controls
Mercuric 'Chloride (HgCl2>
Enzymes
Lysozyme Muramidase-enzyme isolated frpm ICN
chicken egg white
Miscellaneous agents
Sodium Sulfide Fisher Scientific
* From Korzybski et al., 1967; Lorian, 1966.
13
-------
TABLE 4. ANTIMICROBIAL AGENTS TESTED FOR THEIR EFFECT ON
MICROBIAL POPULATION IN WATERS- AND WASTEWATER SAMPLES
Test agent
Referred to in
the text as *
Test concentration
range (yg/ml each
drug)
Chlorhexidine Chlorhexidine
Vantocil IB Vantocil
Amikacin + Gentamicin —
Amikacin + Polymyxin —
Amoxicillin + Mecillinam
BL-S786 •¥ Neomycin
Neomycin + Col is tin
Polymyxin + Neomycin PN
Polymyxin + Gentamicin —
Polymyxin + Ticarcillin
Amikacin + Amoxicillin + Polymyxin
Ampicillin + Mecillinam + Neomycin
Ampicillin + Streptomycin + Neomycin —
Gentamicin + Amikacin + Polymyxin GAP
Neomycin + Ticarcillin 4- Polymyxin
Neomycin + Streptomycin + Colistin
Neomycin + Mecillinam + Amoxicillin
Polymyxin + Neomycin + Streptomycin PNS
Polymyxin + Neomycin + Ampicillin PNA
Erythromycin + Polymyxin + Neomycin EPN
Erythromycin + Polymyxin + Streptomycin EPS
Polymyxin + Chloramphenicol + Neomycin PCN
50 -
50 -
100 -
100 -
100 -
100 -
100 -
100 -
50 -
100 -
100 -
100 -
100 -
100 -
100 -
100 -
100
50 -
100 -
100 -
100 -
100 -
200
100
200
200
200
200
200
500
100
200
200
200
200
200
200
200
100
200
200
200
200
Abbreviations are given only for those drugs which are repeated in the
text.
14
-------
RESULT AND DISCUSSION
Effect of Drugs on Microorganisms in Sewage Samples
The effect of selected drugs on viable cell count and oxygen utilization
in primary and secondary effluents stored in glass bottles is shown in
Figs. 1-4. Data is not shown for the drugs which, were without effect. These
drugs were amikacin + gentamycin, amikacin + polymyxin, amoxicillin +
mecillinam, BL-S786 + neomycin, neomycin + colistin, polymyxin + gentamycin,
polymyxin + streptomycin 4- neomycin, neomycin + tricarcillin + polymyxin,
neomycin + streptomycin + colistin, and neomycin + mecillinam + amoxicillin.
Complete control of microorganisms*in primary effluents could not be achieved
with any of the drugs tested except for chlorhexidine in one sample. Vantocil,
EPN and EPS partially inhibited microorganisms in these samples. Oxygen
utilization data shown in Fig. 4 corroborated these findings to some extent.
It was surprising that PN and PNS caused partial inhibition of oxygen utiliza-
tion, although they failed to inhibit growth. Some inconsistencies in the
results were noted between the effluent samples collected on different dates.
For example, EPN controlled cell numbers in effluents collected on 3-29-1978
but not in those collected on 6-27-1978. The failure of the drug may be linked
to higher initial cell populations in the latter sample, or other differences
between the two samples.
In secondary trickling filter effluents, biocides, Vantocil IB and chlor-
hexidine proved very effective in controlling microbial population and the
oxygen utilization data confirmed their effectiveness (Fig. 4). Antibiotic
combinations - PCN, EPN, PNA, EPS and PNS - succeeded in keeping the cell
population low for 24 hours, but growth resumed afterwards. These biocides
and antibiotic combinations at 100 yg/ml (each antibiotic) were without effect
in the secondary effluents from activated sludge process. Strong inhibition
of microbial growth was noted with some drugs in these effluents when con-
centration was doubled (Fig. 3).
•Studies were performed with antibiotic combinations - EPN, EPS and PN
(100 vg/ml each antibiotic) - to determine if divided applications of the
antibiotics over a period of time were more effective than a single applica-
tion in the beginning. The results showed no significant increase in effec-
tiveness of drugs when one-half the amount of the drug was added in the
beginning and the remaining half at 24 or 48 hours.
Commercially available egg white lysazyme failed to control microorganisms
in wastewater samples. In fact, the presence of lysozyme caused stimulation
of growth which may be linked to the ability of some microorganisms to
utilize lysozyme as a nutrient source. The predominance of Gram-negative
Enterobacteria in sewage effluents may have been responsible for the ineffec-
tiveness of lysozyme. Addition of EDTA and/or Tris, a treatment which is
* The term "microorganisms" is used here to describe all the organisms
capable of forming a colony on standard plate count agar.
15
-------
tr.
UJ
ffi
1 i •
I A SAMPLE COLLECTED 3-29-1977
10" -
104 r
<103
CONTROL
CHLORHEXIDINE {100 pg/ml)
1 2 3 4
STORAGE TIME (DAYS)
PN(500Wg/ml EACH)
EPN ( 200^g/ml EACH )
PNS( 100^g/ml EACH)
VANTOCIL IB (100 Aig/ml)
1 2.3 4
STORAGE TIME ( DAYS)
1234
STORAGE TIME (DAYS I
Figure 1, Effect of test agents on microbial population in primary
municipal effluents collected at two different dates.
-------
. SAMPLE COLLECTED 3 - 29 - 1977
B. SAMPLE COLLECTED 3-29-1977
PCN (100 jig/rnl EACH )
GAPMOOiVml EACH)
10*
<103
CHLORHEXIDINE ( 50 j/g/ml )
VANTOCILIBdOOfig/ral)
12345
STORAGE PERIOD (DAYS)
SAMPLE COLLECTED 6-27 - 1977
0123 5
STORAGE PERIOD ( DAYS)
D. SAMPLE COLLECTED 6-27 - 1977
t£
ui
to
<103
EPS (100 fig/ml EACH)
PNS( 100fig/ml EACH)
PNA( 100/jg/ml EACH)
10" r
<103
12345 012345
STORAGE PERIOD ( DAYS) STORAGE PERIOD (DAYS)
Figure 2. Effect of test agents on microbial population in secondary
effluent from trickling filter process collected at two
different dates.
17
-------
oc
UJ
DO
UJ
o
SAMPLE COLLECTED 4-18-1977 "
EPN(200^9/ml EACH)
PNS( 100M/ml EACH)
CONTROL
PCN (200 yjg/ml EACH )
GAP(200jug/ml EACH)
PNA( 200 ng/ml EACH)
2345
STORAGE PERIOD ( DAYS)
CHLORHEXIDINE ( 200 M9/ml EACH )
EPS (200 Aig/ml EACH )
I . f . I
6
Figure 3. Effect of test agents on microbial population in
secondary effluent from activated sludge process.
18
-------
CHLOHHEXIDINE (100 ug/ml)
VANTOCIL 18(100 ng
EPNI200ng/ml EACH)
EPS!200jig/ml EACH)
PNS( 100fig/ml EACH)
PN(BOO(ij/rnl EACH)
B. SECONDARY EFFLUENT FROM TRICKLING
FILTER PROCESS
SAMPLE COLLECTED 3-29-1977
2 3 4 B
STORAGE TIME (DAYS I
' | ' I I | ' I ' I ' I ' I '
C. SECONDARY EFFLUENT FROM ACTIVATED
SLUDGE PROCESS
SAMPLE COLLECTED 4 -18-1977
VANTOCIL IBHOOfig/ml)
CHLORHEXIDINE1100fig/ml I
PNS1100jig/ml EACH)
1
GAP! 100(ig/ml EACH)
PNA( 100 fig/ml EACH I
EPNOOOfig/mlEACHI
EPS! 100ng/ml EACH)
PCN( 100(<9/ml EACH)
CONTROL
1,1,
23456
STORAGE TIME (DAYS)
PCNIZOOpg/mlEACH)
CHLORHEXIOINE (100 ug/ml I
PNA1200 fig/ml EACH I
EPS!200(ig/ml EACH)
EPNI 200 fig/ml)
GAP (200 tig/ml I
PNS(100fij/ml EACH)
CONTROL ~]
i I i I
2 3 4 S
STORAGE TIME < DAYS)
Figure 4. Dissolved oxygen as affected by the presence of test agents
in primary and secondary municipal wastewater effluents.
-------
known to enhance the susceptibility of microorganisms to lysozyme attack,
was also without effect. Sodium sulfide in concentrations as high as
1000 ppm showed no significant effect on viable cell count. Oxygen con-
centration in the samples treated with sodium sulfide decreased rapidly.
This may be linked to the strong reducing action of sodium sulfide.
Effect of Drugs on Microorganisms in Fresh Water Samples
All the drugs examined showed a positive effect in inactivating micro-
organisms in fresh water samples during storage (Fig. 5). Unlike the treated
samples, control samples showed substantial increases in cell numbers. The
drugs were effective at concentrations lower than those needed to control
microorganisms in sewage effluents. The antibiotics and biocides became less
effective when the waters were spiked with a carbon source (glucose-glutamic
acid 150 mg/£ each) to simulate heavily polluted fresh waters (Fig. 5D).
Vantocil was the only drug among those studied which resembled HgCl- in bac-
teriocidal action under these conditions, although both vantocil and chlor-
hexidine were effective in preventing oxygen utilization (Fig. 6).
CONCLUSION
Sewage effluents contain a wide range of bacteria, as well as a large
population of bacteria (Taber, 1976 and Dias & Bhat, 1964). Natural water,
unless heavily polluted by wastewater discharge or run-off, would not support
such large populations of bacteria but may contain a wide variety of bacteria.
Any antibiotics selected as possible preservatives should therefore meet
several requirements:
1. Antibiotics should be broad spectrum in nature, i.e. effective
against a broad range of microorganisms.
2. They should be sufficiently water soluble and stable to pH and
temperature variations and light.
3. Antibiotics chosen should be such that microorganisms do not
develop rapid resistance.
The amount of antibiotic that can be added to a sample is limited by the
solubility of that antibiotic and by the prospect that too much antibiotic
may interfere with any chemical analysis. From the results of the experiments
presented in this section, it appears that vantocil IB and chlorhexidine are
the best routes to explore. The antibiotic combinations: PNS, EPN, EPS, PCN,
GAP and to some extent PN and PNS also show potential for water and wastewater
sample preservation, since these drugs controlled microorganisms in many
samples but not in all. The drugs selected here were evaluated for their
ability to stabilize sample constituents/parameters.
20
-------
A. KEUKA LAKE WATER
B. CHITTENANGO CREEK WATER
C. CHITTENANGO CREEK WATER
1*0
10« F—r
1 ' I 'I ' I ~
SAMPLE COLLECTED 2-28-1977
106 -
0 103 r
PNSI50lit/ml EACH)
PNA(50.«g/ml€ACH)
EPN (BO'/ag/ml EACH )
EPSIBOps/lmlEACH)
CHLORHEXID1NE (10 |ij/ml)
P6N (5(M|/ni) EACH )
GAP (50eg/ml EACH)
PN( 100/ig/rol EACH)
7 I i I . 1 .
<102
D . CHITTENANGO CREEK WATER
SPIKED WITH CARBON SOURCE
12 3 4
STORAGE TIME ( DAYS)
1 1 1
SAMPLE COLLECTED 8-1-1977
SAMPLE COLLECTED 7-19- 1977
GAP(50pg/ml EACH)
PNS 150 eg/ml EACH I
CHLORHEXIDINEI BO ng/ml)
VANTOCILIB(50^9/ml)
HgCI2(40mB/l)
H2SO4(2mlconc./l)
GAP(50fig/ml EACH)
PNSI 50 jig/ml EACH)
PCN(50*cg/ml EACH)
i ,:2 3
STORAGE TIME (DAYS)
2468
STORAGE TIME (DAYS)
2 4
STORAGE TIME ( DAYS)
10
Figure 5. Effect of test agents on microbial population in natural waters.
-------
120
I ' I
N>
NJ
CHITTENANGO CREEK WATER
SAMPLE COLLECTED 11-16-1977 -
HgCI2(40M9/ml)
CHLORHEXIDINE (50 jug/ml)
VATOCIL(50/ig/ml)
120
110
GAP{50mg/ml EACH)
PNS(50 M9/ml EACH)
23456
STORAGE TIME{DAYS)
CHITTENANGO CREEK WATER
SAMPLE COLLECTED 11 -1 -1977
23456
STORAGE TIME ( DAYS)
Figure 6. Dissolved oxygen as affected by the presence of test preservation
agents in natural water samples fortified with glucose-glutamic
acid (150 mg/£ each).
-------
SECTION VII
EVALUATION OF SELECTED TREATMENTS AS PRESERVATIVES
OF NITROGEN-FORMS IN WATER AND WASTEWATER
Nitrogen is present in water in several forms - organic nitrogen,
ammonia, nitrate and nitrite. Since microorganisms present in water are
capable of utilizing and/or causing interconversion of these forms, it is
necessary to add an effective preservative to the sample at the time of
collection. Howe and Holley (1969) reported the following changes in the
untreated samples of surface water and settled sewage: (a),NH3 concentration
increased during early periods of storage but decreased to zero after 20 days
at room temperature, (b) Organic nitrogen decreased rapidly within the first
five days then stabilized. (3) NO- increased for the first few days, later
decreased to zero, with a concomitant increase in NO-. Similar changes in
nitrogen-forms in water samples during storage at room temperature were noted
by others (Hellwig, 1964, 1967).
No ideal preservative has yet been found which can be used to stabilize
all nitrogen parameters without interference with analytical methods. Sul-
furic acid is not a practical preservative when either NO. or NO, is to be
examined, since acid catalyzes the conversion of NO- to NO- (Brezonik & Lee,
1966; Howe & Holley, 1969; Jenkins, 1968). Even preservation by HgCl- has
its limitations. At the recommended concentration of 40 yg/ml, HgCl- does
not preserve highly polluted samples (Hellwig, 1967). Higher concentrations
are not recommended because of interference with the distillation step in
the analysis for NH, (Hellwig, 1967; Huibregtse, 1976; and Howe & Holley,
1969). If NO- or NO, is not a parameter to be examined, then acidifying the
samples to a pH of 2.0 with concentrated sulfuric acid would be a preferred
method because of the health and environmental hazards linked with the use
of HgCl-. Freezing of the samples stabilizes N02> NH3> NO., and Kjeldahl
nitrogen but often immediate freezing of samples is impractical or impossible
and. there may be a delay of anywhere from several hours to several days
before freezing (Marvin & Proctor, 1965; Proctor, 1962; Degobbis, 1973; and
Thayer, 1970).
This section presents the results of experiments designed to test the
effectiveness of antibiotics and biocides in preserving NO^ and N03 levels
in natural water and effluent samples. The objective was to find a suitable
replacement to the currently used preservatives, which would be safe to use
and would not interfere with any of the nitrogen-series analyses.
23
-------
EXPERIMENTAL PROCEDURE
Primary and secondary effluents and fresh waters were collected as des-
cribed earlier. Samples were aliquoted in 59 ml volumes in glass screw top
bottles (capacity 60 ml). Following addition of 1 ml solution of the test
drug, the bottles were*1 stored at rbom temperature. Whole samples were
sacrificed at designated intervals for'analysis'of nitrogen forms. Sample
stored without any preservative served as a control. Several effluents,
particularly primary effluents, had to be passed through a prefilter and a
membrane filter {0.45 nin) prior to use in the preservation studies.
Nitrate was Determined by the Brucine method'and riitrite by diazotization
method (USEPA, 1974)* Ammonia was determined with an Orion ammonia electrode,
Model 95-10, according to-instructions in the'accompanying instruction manual.
Absorbance values were determined with a Bausch and Lomb Spectrbriic 20.
RESULTS AND DISCUSSION
Initial experiments "Nffere7 performed to determine which drugs, if any,
would interfere with the chemical determination of nitrogen compounds in
water. Standard solutions of nutrients were prepared and divided into two
portions. ' Test preservative was added to one portion while the other served
as control. Nutrient analyses were performed on the two samples and the
values obtained were,compared^ The results are presented in Table 4. Several
antibiotic combinations interfered with the Brucine determination of NO,,
especially combinations containing erythromycin or ampicillin. None of the
combinations tested interfered with N0_ determination by the diazotization
method. Although no specific testing was done for interference with NH,
determination, none was anticipated if the" 'probe method of determination was
used. The antibiotic combinations and biocides which showed no/or minimal
interference were used for further studies.
Fresh waters collected from Chittenango Creek showed measurable concen-
trations of N03 (0.30 - 0.87 mg/Jl) but very low concentrations of N02
(^0.014 mg/£). The samples were therefore spiked with;nitrite (0.017 mg/JO
prior to use. Evaluation of the prospective preservation agents could
not be pursued with these waters because the cbriceritration of nitrate and
nitrite did not vary significantly upon storage even in the unpreserved
samples. The problem was overcome by fortifying the samples with a carbon
source (glucose-glutamic acid, 150 mg/£ each) to stimulate microbial activity.
The addition of an exogenous carbon source induced a rapid decrease in the *
levels of NO and NO- (Figs. 7 and 8). Chlorhexidine'arid vantocil (50 jig/ml)
stabilized NO- and NO- in these samples for up to 30 days; PNS and GAP f '
(50 yg/ml eacn antibiotic) could stabilize these nutrients only for 3 days.
Increasing the drug concentration to 100 yg/ml of each antibiotic did not
show a significant improvement in their preservation potential. Effort to
retest these drugs on another fresh water source (Onondaga Lake water) failed
because of the presence of a substance in this water which interfered with
nitrate measurement. Onondaga Lake is heavily contaminated with industrial
and domestic waste and therefore the interference noted during analytical
measurement is not surprising.
24
-------
1.0
I I I ' I I I I I I I I I I I I I I I I I I
CHITTENANGO CREEK WATER
SAMPLE COLLECTED 11-16-1977
VANTOCILIB(50,ug/ml)
HgCI2(40M9/ml) ^ A
CHLORHEXIDINE (50^g/ml}
15 20 25
PERIOD OF STORAGE(DAYS)
1 1 I T
I •
' '
CHITTENANGO CREEK WATER
SAMPLE COLLECTED 11 -16 -1977
VANTOCIL IB(50fig/ml)
HgCI2 (40Mg/ml)
CHLORHEXIDINE (50;ug/ml)
I I I I
UNTREATED
——
I I I I
15 20 25
PERIOD OF STORAGE(DAYS)
30
35
Figure 7. Preservation of nitrate and nitrite in spiked natural water
samples by vantocil and chlorhexidine. Samples were fortified
with glucose-glutamic acid (150 mg/£ each) to induce changes
in the levels of nitrate and nitrite.
25
-------
0.8
l I l \
CHITTENANGO CREEK WATER
SAMPLE COLLECTED 10-10-1977
HgCI2(40M9/ml) !
*^^ "^™ *^* ^K -— A
—A
GAP(50fig/ml EACH)
PNS(50M9/ml EACH)
O
UNTREATED
0.0'
I I
I I
468
PERIOD OF STORAGE(DAYS)
TO
12
0.10
I I I I | I I I I | I l l I | I I I I
CHITTENANGO CREEK WATER
SAMPLE COLLECTED 10-10-1977
6 HgCI2(40Mg/ml)
~~"~—~ \
PNS(50ug/mlEACH)
GAP(50ug/mlEACH)
I I I I I I I I I I
468
PERIOD OF STORAGE(DAYS)
Figure 8. Preservation of nitrate and nitrite in spiked natural water
samples by antibiotic combinations GAP and PNS. Samples
were fortified with glucose-glutamic acid (150 mg/£ each)
to induce changes in the levels of nitrate and nitrite.
26
-------
TABLE 5. INFLUENCE OF THE PRESENCE OF PRESERVATIVES
ON ANALYTICAL DETERMINATION OF NITRATE AND NITRITE
Concentration Analytical-Interference
Treatment (yg/ml) N02 . NO,
Chlorhexidine 50 - 100
PNS 100
EPN 100 - 200 - +
PCN 100
GAP 100
PN 500*
PNA 100, - +
EPS 200 - +
Vantocil 100
+ Interference.
•>- No interference.
Storage of unpreserved sewage, samples from activated sludge process at
room temperature was accompanied with increase in N0_ and N0_ levels which
continued up to 14 days (Figure 9) . The levels of NO- remained unchanged
afterward, while the level of N0« sharply declined., These nutrients were
stabilized in the samples to which chlorhexidine or vantocil had been added
at 100 yg/ml. In primary effluents, these drugs failed to Stabilize N03
concentrations while the results with regard to N02 stabilization were
inconclusive. PNS, the only antibiotic combination evaluated for sample
preservation potential in trickling filter effluent, proved to be ineffective.
Vantocil showed potential in stabilizing changes in NH*3 concentrations in
effluent samples, however, confirmation of these findings is necessary.
CONCLUSION
Chlorhexidine and vantocil IB show promise as preservatives of N0£ and
NO levels in fresh water, and relatively clean sewage effluent samples.
They offer no interference with the analytical determination of N0» and NO .
The biocides are effective for at least 28 days of storage at room temperature.
If these treatments are combined with storage at 4°C, holding times might
be extended.
27
-------
2.t
1.7
01
0 9
0.5
I I I I I I I I I | I I I I
- A
~ SAMPLE COLLECTED
- 12-13-1977
nijii i ijiiIT
UNTREATED
VANTOCIL IB (100/jg/ml)
CHLORHEXIDINE (100//g/ml)
HgCI2<40Mg/ml)
I , , . , I
I I I I I I I I I I I I I I I I I I I I I
I I ... I
I I I i I
10 15 20 25
PERIOD OF STORAGE(DAYS)
30 35
0.19
0.15 —
0.11 —
0.07
0.03
I . i . I . i l I I i . | I l I I
SAMPLE COLLECTED 12-13-1977
VANTOCIL IB (100^g/ml)
CHLORHEXIDINE (100M9/ml)
HgCI, (40/jg/ml)
10 15 20 25
PERIOD OF STORAGE (DAYS)
Figure 9. Preservation of nitrate and nitrite secondary effluent
from activated sludge process.
28
-------
Antibiotics and biocides at the concentration range of 100-200 pg/ml fail
to preserve highly polluted samples (e.g. primary effluent). Use of concen-
trations higher than these is impractical because of solubility problems and
possible interference with analytical determinations.
29
-------
SECTION VIII
EVALUATION OF SELECTED TREATMENTS AS PRESERVATIVES OF PHOSPHATE IN WATER
INTRODUCTION
The measurement of phosphate is an integral part of water and wastewater
analysis since high levels of phosphate can seriously disrupt the ecology of
water systems. In freshwater ecosystems, an increase in N and P compounds
can accelerate the complex series of interactions which govern the growth of
plant, animal, and microbial populations. Increase in concentration of
nutrients can result in the increased growth of littoral vegetation, develop-
ment of algal blooms, and deoxygenation of hypolimnic waters (Keeney, et al.,
1971).
If samples are stored without treatment, marked changes in the level of
inorganic and organic phosphate can occur due to microbial metabolism, and/or
adsorption on detritus and sample bottle walls (Gilmartin, 1967). The level
of inorganic phosphate may increase by enzymatic decomposition of organic
phosphates or it may decrease as bacteria, in the process of multiplication,
convert inorganic phosphate to organic phosphate cellular compounds and
components (Murphy & Riley, 1956).
Phosphate preservation involves many variables, including size and
material of sample container, volume and type of sample material, filtration,
length and temperature of storage, concentration and form of preservative,
concentration range of phosphate studied, and the methods of analysis
employed (Jenkins, 1967). Numerous chemical preservatives have been tested,
including chloroform, toluene, 1,2 dichloroethane, sodium fluoride, aluminum
hydroxide, thymol, formalin, mercuric chloride, and sulfuric acid (Hellwig,
1964; Heron, 1962; Gilmartin, 1967; Jenkins, 1967; and Murphy & Riley, 1956).
Addition of HgCl, or H2SO, along with cooling to 4°C is the method currently
recommended by EPA for preservation of various forms of phosphate in water
and wastewater samples for up to 24 hours (U.S.EPA, 1974). H^SO, is not
recommended for preservation of condensed phosphates due to their instability
in acidic samples. HgCl2 may cause analytical interference when the chloride
level of the sample is low.
Algae can contain large quantities of phosphorus and their death by the
added preservative can result in release of significant quantities of phos-
phorus in the sample. To overcome this problem, Fitzgerald and Faust (1967)
recommended that samples intended for preservation by methods other than
refrigeration be filtered to remove algae.
30
-------
The review of literature presented above shows that there is need for
developing more effective and safer method for stabilizing various forms of
phosphates in water and wastewater samples. This section presents the
results of several experiments designed to test the effectiveness of anti-
biotics and chemical biocides in the preservation of phosphates.
MATERIALS AND PROCEDURE
Fresh water and wastewater samples were collected as described in Section
VI. Experimental procedure for phosphate preservation studies was similar
to that described earlier for preservation of nitrogen compounds. Fresh
waters were spiked with potassium dihydrogen phosphate to give 0.1 mg P/2.
prior to use in preservation studies. Effluent samples which contained large
amounts of suspended material were filtered through a glass fibre prefilter
and 0.45y filter. Membrane filters were prewashed by soaking overnight in
distilled water (APHA, 1975).
Phosphate was determined using the EPA recommended Ascorbic Acid Method
(U.S. EPA, 1974). Absorbance values were determined with Bausch and Lomb
Spectronic 20.
RESULTS
Initial studies were performed to determine which antibiotics/chemical
biocides would interfere with the chemical determination of phosphate by the
Ascorbic Acid and Vanadophosphoric Acid Methods. A standard solution of
orthophosphate (0*2 mg P/£) was divided into two portions - test preservative
was added to one half, the remainder was without any additions. Analyses
were performed on the two samples and the values obtained were compared.
Orthophosphate could not be determined in the presence of EPS or vantocil
by the Ascorbic Acid Method (Table 6). The latter biocide resulted in the
formation of a white precipitate which precluded absorbance measurement.
Other antibiotics presented no interference in the concentration range tested.
All the antibiotics/chemical biocides listed in Table 6 interfered With
orthophosphate determination by Vanadophosphoric Acid Method (APHA, 1975).
A precipitate was formed upon the addition of color reagent which precluded
making absorbance measurements. Interference caused by the presence of PNA,
EPS and vantocil persisted even after the persulfate digestion of samples
required for total phosphate determination.
Preservation studies with fresh waters were conducted using samples
obtained from Chittenango Creek. Spiked untreated samples during 10 day
storage at room temperature failed to show marked changes in orthophosphate-
and total phosphate concentrations. Changes were induced by addition of
glucose-glutamic acid (150 mg/S, each) to the samples (Fig. 10). Orthophos-
phate levels could not be stabilized by GAP or PNS (50 yg/ml each antibiotic),
or by HgCl . No improvement in preservation ability was seen upon increasing
31
-------
the antibiotic concentration to 100 yg/ml for each antibiotic. Ghlorhexidine
interfered with orthophosphate analysis in many fresh water- samples and
hence could not be studied.
Difficulties were encountered in preservation of phosphates in sewage
samples. Many antibiotics/chemical biocides which presented no interference
in phosphate determinations in spiked distilled water caused severe inter-
ference in sewage samples. The intensity of interference was•somewhat
dependent upon the quality of the samples. For example, interference was
most intense and occurred frequently in primary effluent samples. Filtration
of sample through Q.45vi filters prior to analysis failed to remove the
interfering material.
TABLE 6. EFFECT OF THE ADDITION OF TEST SAMPLE PRESERVATION AGENT
ON ORTHOPHOSPHATE DETERMINATION BY ASCORBIC ACID METHOD
Antibiotic/chemical Concentration
biocide added (yg/ml each) Effect noted
PNS
PNA
EPS
EPN
PN
GAP
PCN
Vantocil IB
Chlorhexidine
50 - 100
100
100 - 200
50 - 100
500
50 - 100
50 - 100
50 - 100 '
100
-
*
+
-
*
-
-
+
-
- = No interference, + = Interference, *,= Not determined.
CONCLUSION
The antibiotics and chemical biocides tested do not offer a solution to
the problem of phosphate preservation in water and wastewater samples. Most
test agents interfered with phosphate analysis and those which did not
interfere were ineffective in preserving samples for any reasonable length
of time. Combining preservation action of antibiotics/chemical biocides
with storage at 4°C may offer hope for preserving these samples.
32
-------
\ \
CHITTENANGO CREEK WATER
SAMPLE COLLECTED 10-10-1977
\HgCI2(40Mg/ml)
PNS(50Mg/ml EACH)
GAP {50 M9/ml EACH
I i i I I i I I I I I I
468
PERIOD OF STORAGE ( DAYS)
Figure 10. Influence of antibiotics additions on the levels of orthophosphate in spiked
fresh waters. The samples were fortified with glucose-glutamic acid
(150 mg/H each) to induce changes in phosphate concentration.
-------
SECTION IX
EVALUATION OF SELECTED TREATMENTS AS PRESERVATIVES OF BOD IN WASTEWATER
Biochemical oxygen demand (BOD) has been used as a measure of the presence
of organic materials in aqueous solution and, subsequently, as a measurement
of water quality. BOD determination reflects the presence of materials in
water which will be oxidized biologically and has special importance in
determining the quantity of materials discharged into receiving water (Stack,
1971). Although the test has limitations (for example, the length of time
required for testing), it is one of the standard tests for evaluation of water
quality, especially effluent quality, and most discharge permits require BOD
monitoring to meet permit standards.
There is no real preservation technique for BOD. Both EPA and the APHA
recommend utilization of the sample as soon as possible with a maximum holding
time of 6 hours at 4°C. The BOD in unpreserved samples can change dramatically
in very little time depending upon the quality of the water and the holding
conditions. BOD can decrease 20-40% in sewage samples during the first 6-8
hours after collection and as much as 90% after a 3-day storage at room tem-
perature (Phillips & Hatfield, 1941). The rate of reduction varies according
to the storage temperature, the lower the temperature the slower the rate,
and the sealed samples lose less BOD than those exposed to air (Phillips &
Hatfield, 1941; Loehr & Brown, 1967).
The biological nature of the test makes it difficult to find a suitable
preservative. Nothing can be added which will be toxic to the seed employed
in the actual test, since this will result in low BOD values. Likewise,
nothing can be used which will add to BOD values by serving as a carbon source.
Temperature control has received the most attention and the results of testing
indicate that chilling to near 0°C will keep BOD stable for approximately
6 days while freezing samples (-5°C) will stabilize BOD for up to 6 months
(Loehr & Bergeron, 1967; Fogarty & Reeder, 1964; and Morgan & Clark, 1964).
Chilling or freezing must be initiated as soon as possible, either during or
soon after collection. However, immediate chilling or freezing of samples
may not be practical in a field situation, especially if large volumes of
sample need to be collected.
In view of the fact that there are currently no suitable methods for BOD
preservation, studies were undertaken to test the feasibility of using anti-
biotics and chemical biocides as preservatives of BOD in wastewater samples.
Since BOD analysis is almost always preceded by dilution of the samples, it
was assumed that dilution would relieve the antimicrobial action of the
preservative, thereby making BOD measurement possible. In addition, several
34
-------
antibiotics which are known to be bacteriostatic as opposed to bacteriocidal
were also examined for their ability to preserve BOD. Such antibiotics do
not irreversibly damage microorganisms, and therefore they may be more
suited for this purpose.
MATERIALS AND PROCEDURE
These studies were carried out using secondary effluents from the
Meadowbrook-Limestone Wastewater Treatment Plant. Samples were collected and
brought to the laboratory as described in Section VI. BOD's of these waste-
waters ranged between 3.45 - 19.1 ppm during the study period. Samples were
stored in glass bottles at room temperature with or without the addition of
preservative. Whole samples were sacrificed for BOD measurement at desired
intervals. Experiments were performed in triplicate and results are
expressed as averages.
BOD determinations were performed according to the procedure in Standard
Methods for the Examination of Water and Wastes, 14th ed. Settled sewage
was used as seed and added to dilution water at a concentration of 2 ml/I. A
standard solution containing 150 mg/£ each of glucose and glutamic acid was
included with each BOD study as a check for seed viability and experimental
procedure as recommended by APHA. Dissolved oxygen was measured with a YSI
Model 54ARC Dissolved Oxygen Meter with BOD probe and attached stirrer.
RESULTS AND DISCUSSION
Presence of antimicrobial agents in the samples intended for BOD analysis
can present the following problems: (i) sample dilution for BOD determination
may not be enough to overcome antimicrobial action of the drug; this will
result in inhibition of the seed activity and consequently in low BOD values;
(ii) the antimicrobial agents may serve as an additional carbon source
leading to higher BOD values for treated samples. It was essential, therefore,
to study the interference of such agents with BOD anal>sis prior to evaluating
their preservation potential.
Test preservatives were added to freshly collected wastewater samples
and 5-day BOD was determined following the appropriate dilutions (1:5 to 1:20).
The results were compared with the BOD of untreated samples. The antimicro-
bial drugs studied included chlorhexidine, vantoeil, PNS, and GAP. These
drugs in the concentration range 10 - 100 yg/ml (before dilution for BOD)
severely inhibited microbial metabolism, which led to very low or no biolog-
ical oxygen demand. Preacclimation of the seed with test drugs failed to
relieve the inhibition since the BOD remained between 0-6% of the control
values. Of special interest was the finding that chlorhexidine caused
complete inhibition of oxygen utilization at 10-20 yg/ml but only partial
inhibition of oxygen utilization at 30-100 yg/ml. Oxygen depletion at
higher concentration but none at lower concentration is difficult to explain.
A possible explanation may be that a higher concentration of the drug induces
appropriate enzymes which enable the microorganisms present to utilize the
drug as a carbon source. The severe interference of these antimicrobial agents
with BOD analysis precluded their utilization in preservation of samples for BOD.
35
-------
Elimination' or inactivation of the antibiotic prior to BOD analysis was
explored as a possible, solution to the problem. Studies with PNS (50-100 yg/ml)
showed that boiling the drug solution in a sealed container for up to 40 minutes
failed to affect the potency of the drug. The drug was stable to alkaline and
acid conditions since pH adjustment was also without any effect. Autoclaving
caused a significant decrease in the BOD,- of untreated samples and was, there-
fore, not given further consideration.
It appeared likely that drugs which are bacterlostatic as opposed to
bacteriocidal, may* offer a better chance for stabilizing BOD samples.', Two
bacteriostatic antibiotics - chloramphenicol and erythromycin - were studied
for their preservation potential.' ' Chloramphenicol did not significantly
affect BOB,, values when added, to\the wastewaters at concentrations up ,to
10 ygVml. Higher drug concentrations gave lower BOD_ values suggesting
inhibitory effect of the drug. Erythromycin at similar concentrations
(10 - 50 yg/ml) caused complete inhibition of oxygen depletion. Chloramphen-
icol at concentrations lower than 10 yg/ml failed to prevent microbial growth
in wastewaters during storage and therefore was considered of little value
as a sample preservation agent; Furthermore, utilization of this drug by
microorganisms as a carbon source at these concentrations also presented a
problem. BOD_ of a pure antibiotic solution (10 yg/ml before BOD dilution)
varied from time to time and ranged between 1..6 - 5.0 ppm
BOD preservation could not be achieved even by storage of chloramphenicol
(<10 yg/ml) treated samples at 4°C. Combining antibiotic action with adjust-
ment of the sample to a nonphysiological pH (pH 4.0 or 10.)) was not considered
for BOD_ preservation because pH adjustment by itself caused a significant
lowering of the BOD_ of the samples.
CONCLUSION
The biological nature of the BOD test makes it very difficult to find
a suitable method for BOD preservation. Chemical additives cannot be used
since they may prove to be toxic to microorganisms even after the necessary
dilution for BOD analysis. Any additive that can be metabolized by microbes
cannot be used since it will contribute to BOD of the sample.
No suitable method for preservation of samples for BOD analysis is
currently available. EPA recommends storage of samples by refrigeration but
only for a maximum period of 6 hours. Freezing is a generally recognized
method for long term preservation (Loehr & Bergeron, 1967; Fogarty and, Reeder,
1964;, and others). Freezing reduces the microbial activity to a minimum
without irreversibly damaging the microorganisms. It was considered feasible
to achieve the same objective by addition of bacteriostatic drugs. At the
time of BOD analysis, it was hoped that sample dilution would relieve the
static effect, thereby allowing interference-free BOD analysis.
36
-------
In this study, antimicrobial drugs could not be successfully used to
preserve samples intended for BOD analysis. The problem was related to the
biological nature of the BOD test. The drugs tested interfered with BOD
analysis by inhibiting seed activity. Acclimation of seed to the drug did
not alleviate the problem. Lower concentrations of the drugs failed to
preserve samples. In some instances they served as carbon sources and led
to erroneous BOD,, values. Selective elimination or inactivation of the
drug prior to BOD,, analysis could not be achieved by pH adjustment or
heating of the treated sample.
37
\
-------
SECTION X
EVALUATION OF SELECTED TREATMENTS AS PRESERVATIVES
OF OIL AND GREASE IN WASTEWATER
INTRODUCTION
Oil and grease may be present in water due to the decomposition of plank-
tonic and/or higher forms of aquatic life, and from pollution resulting from
industrial and domestic wastes (Hach Chemical Co., 1975). Since many of
these hydrocarbons can be utilized by bacteria, it is essential that samples
to.be analyzed for oil and grease be stored properly. The accepted procedure
is to acidify samples to pH <2 and store at 4°C. Holding time is approxi-
mately 24 hours (U.S. EPA, 1974). We examined the possibility of long-term
preservation of oil and grease samples by addition of antibiotics and chemical
biocides.
MATERIALS AND PROCESURES
Effluent samples were collected from the overflow weirs of clarifiers
at Wetzel Road and Meadowbrook-Limestone Treatment Plants as described in
Section VII. Samples were brought to room temperature prior to use. One
liter volumes were distributed into 1 liter, glass-stoppered bottles.
Following the addition of the desired preservatives they were stored at room
temperature. Whole samples were sacrificed for analysis. Oil and grease
content was determined by extraction with petroleum ether (APHA, 1971).
RESULTS AND DISCUSSION
Preliminary experiments were performed to determine which antibiotics
would interfere with oil and grease analysis. Effluent samples were divided
into two portions. One remained without any additions, test drugs (100-200
yg/ml each antibiotic) were added to the other. Oil and grease determinations
in the two samples revealed no interference by the drugs tested - chlorhexidine,
vantocil, GAP, PNS, EPN, and PCN.
PCN and GAP were found to be effective in preserving oil and grease in
secondary effluents obtained from activated sludge process for 8-12 days
(Fig. 11). Other drugs exhibited no effect on the changes occurring in oil
and grease content during sample storage. PCN and GAP could not stabilize
oil and grease in primary effluents or in secondary effluent obtained from
trickling filter process. The results show that antibiotics can effectively
stabilize oil and grease in relatively clean effluents only. If the antibio-
tics are used along with storage at 4°C, it may be possible to stabilize oil
and grease in heavily polluted samples, and perhaps also to extend the holding
period.
38
-------
CO
VD
120
110
100
90
80
70
g 60
a
1 50
40
30
20
10
0
- A
I ' I ' T ^ I ' I ' I ' T^
SAMPLE COLLECTED 2-14-1977 -
PNS( 100ng/rnl EACH)
GAPdOOng/ml EACH)'
5 6 7 8 9 10 11
STORAGE TIME (DAYS)
12 13 14 15
I ' I ' I ' I ' I
SAMPLE COLLECTED 2 -
' I ' I
14 -1977
A PCN (100 jug/ml EACH )
CONTROL
EPN( 100(is/ml EACH)
5 6 7 8 9 10 11
STORAGE TIME (DAYS)
I . I
12 13 14 15
Figure 11. Potential of antimicrobial agents for preservation of oil and grease
in the secondary effluent from activated sludge process.
-------
SECTION XI
EVALUATION OF SELECTED TREATMENTS AS PRESERVATIVES OF pH IN WATER
As microbial metabolism proceeds in unpreserved aqueous samples, the pH
of the samples changes. For example, a low level of dissolved oxygen may
trigger anaerobic respiration and subsequent acid production, or the decompos-
ition ,of nitrogenous compounds may result in an increase in pH (Loehr and
Bergeron, 1967; Hellwig, 1964). Generally there is no recognized need for
stabilizing pH changes since measurements can be made in the field with the
help of a portable pH meter or pH kits. In view of the fact that many of the
drugs tested in our studies effectively controlled microbial growth and oxygen
utilization in water and sewage samples, it was of interest to determine if
these agents would simultaneously.stabilize pH changes.
Fresh waters collected from Chittenango Creek upon storage showed a pH
change no more than 0.2 pH units over the 10-day test period. To accelerate
a change in pH in these samples, glucose-glutamic acid was added (150 mg/£ each)
In the fortified samples, pH decreased over the 30-day period at room tem-
perature while it remained unchanged in ehlorhexidine and vantocil treated
samples (Table 7). The effectiveness of these drugs in stabilizing pH
changes was also observed with Onondaga Lake water. PNS and GAP could not
stabilize pH for longer than 6 days at a concentration of 100 yg/ml. Addition
of mercuric chloride caused an immediate decrease in pH of some samples
(Table 7) and is therefore unsuitable as a preservation agent for pH.
Stabilization of pH in sewage effluents could not be achieved to a significant
extent by any of these drugs.
TABLE 7. STABILIZATION OF pH BY ANTIMICROBIAL DRUGS IN FRESH WATER
SAMPLES FORTIFIED WITH GLUCOSE-GLUTAMIC ACID (150 mg/£ each).
SAMPLES COLLECTED FROM CHITTENANGO CREEK, DATE 11-16-77.
Test drug and " Sample pH at days shown
concentration , 0 1 2 5 12 16 26 30
Untreated 8.0 8.1 7.1 6.3 6.1 6.4 6.4 6.6
Chlorhexidine,
50 yg/ml 8.1 8.1 8.1 8.2 8.1 8.2 8.1 8.2
Vantocil IB,
50 yg/ml 8.1 8.1 8.1 8.1 8.1 8.2 8.1 8.2
HgCl2, 50 yg/ml 7.4 7.4 7.5 7.4 7.4 7.5 7.6 7.8
40
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Oxygen Uptake of Lake Bacteria. J. Bacteriol. 39:307.
45
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing/
1. REPORT NO.
EPA-600/4-79-007
3. RECIPIENT'S ACCESSION NO.
4. TITLE ANDSUBTITLE
New Approaches to the Preservation of Contaminants
in Water Samples
5. REPORT DATE
January 1979 issuing date
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
J. Saxena and E. Nies
8. PERFORMING ORGANIZATION REPORT NO
TR-78-568
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Center for Chemical Hazard Assessment
Syracuse Research Corporation
Syracuse, New York 13210
10. PROGRAM ELEMENT NO.
11. CONTRACT/GRANT NO.
R 804609010
12. SPONSORING AGENCY NAME AND ADDRESS
Environmental Monitoring and Support Lab,
Office.of Research and Development
U.S. Environmental Protection Agency
Cincinnati. Ohio 45268
- Cinn, OH
13. TYPE OF REPORT AND PERIOD COVERED
Final 9/1/76-8/31/78
14. SPONSORING AGENCY CODE
I
EPA/600/06
15. SUPPLEMENTARY NOTES
16. ABSTRACT
The potential of antibiotics, chemical biocides and lytic enzymes in preserving
nutrients, biological oxygen demand and oil and grease in water and sewage effluents
was studied. Preliminary studies concerning the effect of drugs on cell growth and
oxygen utilization in samples stored at room temperature led to the selection of
chlorhexidine, vantocil and many combinations each containing three antibiotics from
among polymyxin B, neomycin, erythromycin, streptomycin and chloramphenicol. The
effective concentration range was ^50 pg/ml each antibiotic for clean waters and as
high as 200 yg/ml each antibiotic for heavily polluted water (e.g. primary effluents).
Chlorhexidine and vantocil IB stabilized nitrate and nitrite in fresh waters and
relatively clean secondary effluents only. Presence of antibiotics caused inter-
ference in determination of orthophosphate. The antimicrobial agents tested
interfered with BOD determination by causing inhibition of oxygen depletion and there-
fore were of no value for preservation of this parameter. Efforts to selectively
remove and/or inactivate the drug before BOD determination were unsuccessful. Oil
and grease levels were stabilized by antibiotics for up to two weeks in relatively
clean waters only. The results demonstrate that antibiotics offer a viable alternative
to conventional methods for preservation of some constituents and parameters in fresh
water samples but not in sewage effluents.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.IDENTIFIERS/OPEN ENDED TERMS
COSATI Meld/Group
Antibiotics
Enzymes
Water
Wastewater
Sample preservation
Stabilization of
parameters/constituents
Antimicrobial drugs
Nutrients, Biological
oxygen demand, Oil and
grease, Water & wastewater
analysis.
68D
8. DISTRIBUTION STATEMENT
Release to public
19. SECURITY CLASS (ThisReport)
Unclassified
20. SECURITY CLASS (This page)
Unclassified
21. NO. OF PAGtS
58
22. PRICE
EPA Form 2220-1 (9-73)
46
6USGPO: 1979 — 657-060/1578
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