- EPA-600/2-84-116
June "1984
EVALUATION OF URBAN RUNOFF
AND
COMBINED SEWER OVERFLOW MUTAGENICITY
by
Stuart J. Spiegel
Edwin C. Tifft, Jr.
Cornelius 3. Murphy, Jr.
O'Brien & Gere Engineers, Inc.
Syracuse, New York 13221
and
Randy R. Ott
-Department-of Drainage-and-Sanitation-
County of Onondaga
North Syracuse, Hew-York -13212
CR-306640
Project Of-ficiers-
Richard Field
Robert Turkeltaub
Storm and Combined Sewer Program
Wastewater Research Division
Mtraicipal Environmental Research Laboratory (Cincinnati)
Edison, New Jersey 08837
MUNICIPAL ENVIRONMENTAL RESEARCH LABORATORT
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
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(I'kati
TECHNICAL REPORT DATA
s on tin rc> i «( tr}on completing)
i REPORT NO
EPA-600/2-84-11G
3 RECIPIENTS ACCesSIOI» NO
4 TITLE AND SUBTITLE
"Evaluation of Urban Runoff and Combined Sewer
Overflow Mutagenicity"
5 ftPORT DATE
June 1984
6 PERFORMING OHGAN:ZATION CODE
I? AUT"°«(S1stuart J. Spiegel, Edvsin C. Tifft,.Jr.,
"Cornelius B. Murphy, Jr., and Randy R. Ott*
8 PERFORMING ORGANIZATION BliPORT NO.
9 PERFORMING
OflGANIZATlOU NAME AND ADDRESS
rien & Gere Engineers, Inc.
10 PROGRAM-fcLEMENT NO.
1304 Buckley Road, Syracuse, New York 13221 .
*County of Onondaga
125 Elwood Davis Ro?d, North Syracuse, NY 132.12
11. CONTRACT/GRANT NO.
CR-806640
12 SPONSORING AGENCY NAME AND ADORESS
Municipal Environmental research Laboratory - Gin.,OH
Office of Research and Devel-opment-
U.S. Environmental Protection Agency
Cincinnati, Ohio 45268 ' "
13. TYPE OF REPORT AND PERIOD COVERED
Final Report - 4/80 - 1/82
14. SPONSORING AGENCY CODE
EPA/600/14
,5. SUPPLEMENTARY NOTES
gram, Edison, NJ 08837 - Com!. (201t 321-E674; TTS
Chief, Storm ^.-CombinediSewer Pro-
340-6674
IS. ABSTRACT
The introduction of potential rnutagens to the.huraan.environment may serve to-increase-the-rate-Qf zcon-
tact with substances that contribute to cancer incidence in the-general population The prinarv-purpose-
of-this study was to evaluate combined sewer overflows and.urban runoff for the presence ofj:heiiikal_3utai_
gens. The Ames Salmone*la/microsome rutagenicity-te"s1Thas~eniployed as~a~~general biological effects test
for the qualitative detection of mutagens 'n the sanitary environment, including rain_unban_eunof.f, sarvi-
tary-wastewater, combined sewer overflows, sewage treatment plant effluent, and recevving-wat-erSv^-The -
Anes-test~is a relatively sensitive and simple bacterial test for detecting chemical mutagens-. Its ad-
vantages over long-term animal tests are speed, ease, and relative low cost. The test employs previously
nutated Salmonella typhimurium LT2 bacterial strains which have a tendency to undergo a sjb-equent mi.--
tat'on back to their natural state when exposed to mutagenic.compounds»
- Nineteen samples elicited a detectable response to one or more of the five Salmonella typhimurium test
strains, with or without metabolic activation. Mine of these samples (47S) were of urban runoff in-the
project area, metropolitan Syracuse (Onondaga County) N.Y., Including 17 of 30 detectable responses (57S)
Five of the samples (26J) were from combined sewer overflows (7.of 30 detectable rujponses, or 23X).
/ —
The results indicated that substances present in urban "runoff which produce a detectable response-in the
-Ames test may be diluted or inactivated in combination with sanitary sewaqe to form combined «wage, since
fewer responses were detected, in combined sewer overflow than in urban runoff. . ....
,, T!!iSn«f??rt is in Part1al fulfillment of the U.S. .Environmental Protection Agency-Cooperative -Agreement
ito. CK-806o40. i
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
jb IDENTIrlERS/OPEN ENDED TERMS C. COSAT1
IB DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
19 SECURITY CLASS (ThltReport}
UNCLASSIFIED
21. NO. OF PAGES
133
20 SECURITY CLASS (Thispage)
UNCLASSIFIED
22. PR;CE
EPA Form 222b-t (9-73)
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DISCLAIMER
."The information in this document has been funded wholly or in part by
the United States Environmental Protection Agency under assistance agreement
number CR-806640 to O'Brien & Gere Engineers, Inc.' It has been subject to
the Agency's peer and administrative review, and it has been approved for pub-
lication as an EPA document. Mention of trade names or commercial products
-does-not constitute-endorsement or recommendation for use."
ii
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FOREWORD
The U.S. Environmental Protection Agency was 'created because of
increasing public and governmental concern about the -dangers of pollution—to
the health and welfare of the American people. Noxious air, foul water, and
spoiled land are tragic tes'tiaony to tha deterioration of our natural environ-
ment. The1 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
arid it involves defining the problem, measuring its impact, and searching for
solutions. The Municipal Environmental Research Laboratory develops.new,_and.
improved technology and systems for the prevention, treatment, and management
^f wastewater and solid and hazardous waste pollutant discharges from munic-
ipal and community sources, for the preservation and treatment- of public
drinking water supplies and to minimize the adverse economic, social, health,
and aesthetic effects of pollution. This publication is one of the pro'ducts-
of that research; a most vital communications link between—the ..esec 'cher- and-
the user community.
Studies of the, impact-of urban runoff and combined .sewer ov.erflo.ws .on. the- -
environment "have been limited to water quality and short term public healthl
-(microbia-l—pathogen-)- -±ssras-. The—po-trenti-ai f err -iorrg—term—b±oi'o^g±cai—impacts—
from these -sources has gone largely without notice nor investigation. This
report -pxesents, the results-of a screening study-into-this-.question.
Franci-s T, Mayo •
"Director . . .
Municipal Environmental
Research Laboratory- " '
iii
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ABSTRACT
The introduction of potential mutagens to the human environment may serve
to increase the rate of contact with substances that contribute to cancer
incidence in the general population. The primary purpose of this study was to-
evaluate combined sewer overflows and urban runoff for the 'presence of
chemical mutagen-s. The Ames Salmonella /microsome mutagenicity test was
employed as a general biological effects test for the qualitative detection of
mutagens in the sanitary environment, including rain, urban runoff, sanitary
wastewater, combined sewer overflows, sewage treatment plant effluent, and
receiving waters. .The Ames- test -is -a relatively-sensitive- and* simple~bacterial
test for detecti.ig chemical mutagens. Its advantages over long-term animal
tests are speed, ease, and relative low cost. The test employs previously
mutated Salmonella typhimurium LT2 bacterial strains which have a tendency to
undergo a subsequent mutation back to their natural state when exposed to
mutagenic compounds.
Nineteen samples -elicited a detectable— response -to one or more of "
five Salmciella typhimurium test strains, with or without me,taboH:c--acti— -
vation. Nii.e of these samples (47%) were of urban runoff in the project area,
metropolitan Syra^jse (Onondaga County) , ,N.Y. , including 17 of 30 detectabie-
responsas (57%). Five of the samples (26%) were from combined sewer_ovex£lo.vs-
(T of 30 detectable responses, or 23%).
The results indicated thac substances present in urban runoff which
produce a detectable response in the Ames test may be diluted or inactivated
in combination with sanitary sewage to form combined sewage, since fewer
responses were detected in CSO than in urban runoff.
Due to budget constraints, it was not possible to conduct an in depth
investigation. A complete study should include duplicate analyses and '
extraction of samples at pH 3 and 12, as well as ambient pH".
This -reportr-rs "in"f ulf illment of the'TTI'S . Environmental Protection Agency
(EPA) Grant R806640, The period of study covered by this report is from April
1980 to January 198?.
iv
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•COH-TENTS-
Abstract ....." . iv
Figures vi
Tables , .^vi±
Abbreviations viii
-Acknowledgments ......... is
1. Introduction > -«~. -. -. 1
2. Conclusions ......... .._. _.—»- . ^ . .- ~3^
"3~. Recotomendations -,.-»» ^ .... > . . 4
4.' Background . . . . -_-..—,—,-.—. -.-.—r~f~—r~v . . . . . . 5~
History of Combined-Severs . ."• 5 .
Qualitative Aapaces of Urban Stormwater Runoff".... 7
Polluted Precipitation . . TO
Ames Test 10~
Zroject At oa Location ...13
Project Background 13
5: Materials 5
Ames Test 5
Chemical Analyses . * .....-..-....„ 19 _.
6. Procedures '. . . » 20
Plan of SCu&y . . . ... ^ . ... .2Q
Sample Collection 2Q
Data Management ................... ^_30
Chemical Analysis „ ,- 32
Mutagenicity Testing „ . . 32
7. Results .. 46
Chemical Analyses ' ". » . 46-
Mutagenicity Testing ^ ; . . 4"6"
"8. Discussion . - ^ ...•..._ 54
Interpretation of Results 54
Extractions 66
Falsa Positive and False Negative Results 67
9. .Summary . . - '69
References 70
Appendices . 76
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FIGURES
Number . Page
1. ifypical Combined Sever ;..... ._ . .- . . 6
2. Location of Onondaga County in New York State 14
3. Onondaga County, New York • 15
'4. Location of Project CSO Sites ' 23
'5. "Sample Collection Sitej: Drainage Area 004
i&d Drainage Area 005 .~25"
6. Sample Collection Sites: Drainage Area 005
and Drainage Area 01-9- , . . r . -.—.- ^- -*—26-
7. Sample Collection Sites: Drainage Area 037 27
8. sample Collection Sites: Drainage Area 043 ....... . -. 28
9. Field Data Check Sheet v . "31
10. Concentrated Sample Extracts 38
11. Range of Sample Test Coccentrations and Dosages » 4T
12. Response Curve for Sample Number* 95^74 • ~ ..... 56
13. "Response'Curve—"or Sample Number 95475 '..... =5?r
-14. -Response~Curve-for Sample, Number 9547-6- -'. .;—.._". ,. _^ .... ^5&.
15. Response-Curve for Sample Number 95477 ....59 "
16. Response Curve for Sample Number 94491 ". . ." „ . 60
17. . Response Curve for Sample Number 95336- ... ...... 61
18. Response Curve for Sample Number 95368 .... ".".". '." ". . 62
19. .. Response Curve for Sample Number 95739 ..63
20. Response- drrve- for Sample Ntnnber 95871 ......... •,.64
vi
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.TABLES
Number Page
1 Pollutant Concentrations in Stormwater Runoff 8
2 . Pollutant Concentrations in Combined Sewer Overflows ... 9
i3 Percentages of Scream Loads of Chemicals
Resulting from Direct Atmospheric Washout . . II
4 VogcI-Bonner Minimal Salts Base Medium • . . 18
5 S-9 -Reaction Mixture 18
_6— -Characterization of CSO -Site-Drainage-Areas-" in-
Syracuse, New York 22
7 Characterization of CSO Site Drainage Areas in
Rochester, New York and Washington, D.C 29-
3 Rain Data for Storm Events, Syracuse, New York . .'. . . .30
9 Project Sample and Data Management Identification
Scheme '.. . . .33-
10- Resuits-of Spot Teats: "Sample ToxLcity :3T
11 Comparison of Spontaneous Reversion Rates for
Bacterial Test Strains - 44
12 Summary of Chemical Analyses for.Urban Runoff . "
' _and_Rain. ..--.»- -, 47~
13 Summary of .Chemical Analyses for CSO Samples 48
14 Summary of Chemical Analyses for_Dry_Weather
Sanitary Samples ^ 49
15 ' Summary Table of Phase I Mutagenicit} Results 51
16 Summary of Detectable Responses, Phase I Analyses ..... 55
17 Phase II Saitple Verification - MARs 66
vii
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ABBREVIATIONS
Ames Test
BOD
CD
CR
CSO
CU
°C
DCM
DMSO
DNA
EPA
G-6-P
km
1
MAR
Metro
ml
mM •
MMS-
MNNG-
N
NADF
-O&G
ONA
ONE
PB
TOC
.TPS
U
Pi
Ames Salmonella'/ml'crosome~mutctgen(ri-ty~te£
five day biochemical oxygen demand
cadmium
chromium
combined sewer overflow
copper
degrees Celsius
dichHorome'-hane (mefchylene chloride)
dimethyl sulfoxide (methyl sulfoxide)
deoxyribonucleic acid
U.S. Enviro imental Protection Agency
glucose-6-phosphate
kilometer
liter
mutagenic activity ratio
Metropolitan Syracuse Treatment Plant
milligrams per liter
millxliter
millimolar : *_
methyl methanesulfonate
N-methyl-N'-nitro-N-nitrosoguanidine
Normal
nicotinamide adenine dinucleotide phosphate
oil and grease-
Oxoid. Nutrient Broth No» 2 plus 1.5X 23ar
Oxoid Nuti ent Broth No. 2
lead
total organic carbon
total suspended solids
micron
microliter " - -
viii
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-ACKNOWTEDGE4ENTS
The authors would like Co thank joi Mastriano and Sam Rizzo of the
Onondaga County Department of Drainage and Sanitation for their assistance in
the collection of samples, the engineering staff of O'Brien -A—Gere—£or~their
assistance in the site selection process, and the j->.rsonnei of the O'Brien *
Gere laboratory for their performance of all physical and chemicai sample
analyses. Robert Turkeltaub, the project officer, provided" valuable direction
for the project. The support of the Storm and Combined Sewer Section, Edison,.
-New- Jersey; of the OSEPA Municipal Environmental" Research Laboratory,
Cincinnati, Ohio; and of Richard Field, Chief, Storm and Combined Sewer
Section, USEPA, was appreciated. Francis Brezenski, Robert-Davis~and~Barbara
Archdeacon of the USEPA Region II Environmental Services Division, Edison, New
Jersey, and their staff were extremely helpful in their analysis -of split
samples and the review of this project report-
Assistance to the. study was provided* by Dr. 1. Ernest faemphlll,
Department of Biology,,^.yjracuse-Uni?6rs±tyT"Syracu3e, HI".
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SECTION 1
INTRODUCTION
t
'Itj has only been since 1964 that urban runoff arid combinsd sewer over-
flows (CSO) have been identified as contributors of pollution to receiving
streams. It. was not until nearly ten years later that urban runoff and CSC
were implicated as major sources of parciculates, nutrients and mlcrobial
loading-to urban waterways. -In—recent^years, almost any potential pollutant
to be found in the urban environment can be traced to receiving streams via
this transport pathway (37).
In order to decrease surface water pollution from these, sources, the U.S.
Environmental Protection Agency (EPA) has • engaged studies and provided funds
for the design and construction of prototype CSO treatment .facilities.—These-
_facilities -are primarily designed' ~to utilize seme form of solids removal-
followed by disinfection.
However, past studies which have served to characterize urban runoff_and_
CSO, and identify constituent .pollutants^-have.-not -conclusive-ly-deal-t-wttbrthe '
•moot recent concern to arise in pollution control, that of the fat&-ard—effectr
of organic chemicals in these systems. Although potentially toxics-mitagenic,
carcinogenic or teratogenic chemicals are readily identifiable in the.-urban-
environment , there has been little attempt to determine their presence and.
concentrations in urban runoff and CSO. There are several reasons for this-
omission. Such studies would require the analysis of long lists of compounds,
as in priority pollutant analysis, vad these lists are constantly expanding in
length. There are technological difficulties in the analysis of such; com-
pounds. The detection limits of available instrumentation may -a-iC.be-suffi-
ciently low to detect Binute concentrationo of contaminants ac levels which-—
-may still- pose—a potential health hazard. The_.cost- of -analyzing—a—singie—
sample under these conditions is high; the analytical cost of sufficient
samples for a long list of compounds to provide statistically significant
amounts of data is staggering. Further, the result? of such determinations
still would not provide any biohasard risk index - any measure of the biolog-
ical effects of these compounds "whan" in their environmental medium, par-
ticularly on the human system, and especially, any syn«rgistic or multiplica-
tive effects of such substances. • •
At this time, most treatment systems for CSO "are still in the- initial
planning or evaluation phases. While there has been general agreement that -
treatment should consist of solids removal and disinfection, there has been
little evaluation o£ the need for additional levels of treatment. Due to the
highly variable nature and occurrence of urban runoff and CSO, it is doubtful
if the requirement for advanced treatment can be ascertained solely by
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characterization of a list of specific chemicals. Instead, general infor-
mation describing the potential biohazard risk of these systems would be a
valuable criterion for the determination of the need for advanced treatment.
' It is upon this premise that this study has been based, that urban runoff
may substantially contribute substances of potential risk to the human
-environment and that a preliminary analytical survey- utilizing "Che- "Ames
Salmoaella/micrcsome txutagenicity test might serve to outline the presence and
scope or that potential.
To this end, samples were taken of" environmental contributors ~t& the -CSO~
system. These included rain, urban runoff, dry weather sanitary flows, and
CSO, as well as influent and effluent from .the Metropolitan Syracuse Treatment
Plane, (Metro), and samples from Ouondaga Lake, the .ultimate—receptor— of the
overflows. Samples were tested by the Ames test and subjected to analysis for
other chemical parameters, Including total suspended solids (TSS)',- total
organic carbon (TOO), five day biochemical oxygen demand (BOD), oil and grease
(O&G)-,- and the heavy matals cadmium (CD), chromium (CR), lead"(PB)7-and copper
(CtT). The results of the Ames test were compared to the baseline chemical
analyses to determine the presence of any correlation which might assist in
-the identification of- the .source of mutagenic -activity-.
Since inorganics, especially metals, are removed in the sample extraction-
procedure, the primary purpose of heavy metal anrlysis is- for-the charac-
terization—of—the—samples, and evaluation as to whether- die concentrations
'were typical or atypical of the sample type.
• JEhis-study' was— completed in two phases. Phase T involved"the collection.
and" analysis of 74 -sunples, -during- 1980,- -Based—on—the—results—of—-those-
analyscs, modifications were made to some of the procedures. Phase II •
involved the collection and analysis of an additional eleven ^3anples_in 1981
from those drainage areas which elicited detectable- responses- In the 1980
Phase I effort. ' '
" The -procedures and results of this study will be described in later -
sections of this report. . . .. .
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SECTION 2
CONCLUSIONS
1. Nineteen samples may be interpreted to have induced a -detectable
mutagenlc response fron one or more of che test strains, with or without
metabolic activation. Nine at these samples were of urban, runoff. Some
samples required a two hundred-fold concentration for detection of this
activity. Of a total of 30 detectable responses, 17 (577) were found in
urban runoff, while 7 (23Z) were found in combined, sewer-overflows^
2. ?he samples eliciting a detectable response did not appear correlated to
levels of any of the chemical parameters measured, nor to any particular
CSO site. The experimental design appeared too limited in terms of
number of samples and the chemical parameters measured to obtain corre-
lations between detectable mutagenic activity and chemical character-
istics of samples or sample types (CSO, urban runoff, etc.*).
3. Substances present in urban runoff which produce a detectable response in
the Aires test may be diluted or inactivated in combination with -sanitary—
sewage when it forms combined sewage, since fewer responses were detected—
* in-.CSO fhan in-urban runoff.
V
4. This study is indicative of-the-need-for~a"more comprehensive survey-of a
similar nature. However, problems encountered in analytical methodology,
sample toxicity, and the inherent chemical variability of CSO and urban-
runoff pose difficulties in preparing definitive conclusions from this
• preliminary survey which can assist in policy decisions.
5. Use of the spot test as a preliminary step in the analysis of_ polluted ••
•aquatic samples by the Ames Salmonella/microsome mutagenicity test may be
.precluded by sample toxicity at ambient-concentrations. .
6. Dichloromethane (mothylene chloride; may sarve as both a disinfectant and
extractant. However, should extraction and/or concentration' not be
required in other aquatic environmental samples, a more suitable disin-
fection procedure may be necessary.
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-SECTION 3
- -RECOMMENDATIONS
1. An evaluation should be performed to determine an optimum method of test
sample disinfection for those samples with high levels of particulates,
oils and microbial organisms, such as those analyzed by toe Ames test in-
- this study.
2. The extraction procedure recommended for this project should be evaluated
as to its sensitivity in recovering known mutagens. -This-would"-establish
a detection limit threshold for the!assay under these analytical condi-
tions and assist in risk evaluations based on the review of analytical
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SECriON 4-
BACKGROUND
HISTORY OF COMBIBED SEWESS
! ,
Beginning around the middle of the 19th Century, -sewers-were- constructed
in urbpn areas of the U.S., originally to conduct atonnwater runoff to the
nearest watercourse. Sanitary wastes were disposed of separately at-Indi-
vidual residences. Later, as population increased, health problems developed
as a result, of-waste disposal at so many -Individual"locations.
A convenient method was needed to eliminate sanitary wastes from, the—
urban environment. Existing storm sewers were availaoie -sad were "frequently .
adapted to transport sanitary wastes away from the community. Sewers which-'
carried both stormwater and sanitary wastes were designated combined- sewers.
Nuisance conditions in the watercourses -resulted, which led* to the-construc-
tion.of-interceptor-sewera,- to- "intercept" dry-weather sanitary 'fTows-iipstreaai-
frcm- receiving -watercourses'. Howevez» these intercepting sewers were- -not
designed- with-sufficient—capacity" to carry all- sanitary wastes and_storcwatet-
during, wet wear her. periods. In general, the interceptors were designed.-to-
tarry- only 2 to 3 times the average-dry-weather— flow—from~tr±btttary~drfinage
areas,. Thus, when storms occurred, runoff reaching the collection system
resulted in flows in the trunk sewers in excess of Jihe—carrying—capacity--of
-the interceptors, creating the potential for surcharging of. sewers* flooding~
of-streets and private basements, and resulting in public health dangers.*. In-
order to relieve the trunk sewers of excess flow, overflow pipes were
installed at frequent intervals along the interceptors which discharged a
combination of excess stormwater and sanitary wastes. (Figure 1). _In..
hydraulically limited circumstances,. such .as underdesigned-pipe-capacitifts or
blocked sewers, undiluted sanitary sewage was discharged du'clTig^dry-weather
periods as well (24).
As urban centers continued to grow and discharges of CSO wastewaters to
receiving waters increased, the natural assimilation capacity of many streams-
was exceeded an<< serious water quality problems resulted. Since -1950,
emphasis in newly sewered areas has been placed on construction- of separate_
sanitary- -and storm severs. By building separate sewers, excessive "flows to
the waste treatment plant during wet weather periods are avoided and the
entire sanitary flow is treated without direct discharge to and - subsequent
degradation of the receiving water. Stormwatsr runoff in separately-sewered-
areas is conducted by the scorm sewers directly to receiving waters. This
does not alleviate pollution as a result of" the stormwater contamination
during urban runoff in these cases.
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Figure 1. Typical Combine1 Sewer
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"Prior to 1964, when the U.S. Public Health Service published its study
"Pollutional Effects of Storm Wafers and Overflows from Combined Sewer
System*, i .A Preliminary AupraJsal", surface runoff from rainfall events was
consideret| to be relatively clean and "he discharge of runoff wacer from a
storm sewer was believed to have littlt effect on the water quality of the-
receiving stream. Since 1964* federally supported soidies have- shown that
surface runoff .contains -high—concentrations-- of--pol-lutantsr Rain reiaoves
pollutants from yards, sidewalks and streets. These pollutants may include
oxides of sulfur and nitrogei, dust, hydrocarbons, dirt, common street litter.
deicing chemicals, organic matter, pesticides from lawns, eroded nonorganic
materials, traffic residue (heavy metals, lead, petroleum compounds-,- rubber)
and animal droppings. Illegal cross-connections between overloaded sanitary
steers and storm severs contribute untreated municipal sewage to the storm-
vatsr flows.
Untreated discharges from combined sewers have proven to be_a_signi£icant.
source of pollution in terms of impact upon receiving water quality. During
periods of extended dry weather with only sanitary waste flowing in the
•combined sewar cystum, solids tend to settle, tad accumulate- on - the- bottom
-(invert) of the pipes. The first significant rainfall event .following the dry
period causes r. surge of flow ir the sewer which flushes' this residue from the
system. The pollutional cont.:lbutioa of this flush can be' significant.
Studies conducted in Buffalo, lt>su York have shown that 20 Co 30-percent-of tL-e-
amnial sanitary waste discharged .into -the combined sewer are- settled:
eventually flushed frois '^lie system-with—a—stora-<—gene-ratcd—f-i-ctf—suTge
The flow surge and associated flush of settled waste is referred— to^jas the
"first-flush". A first flush discharge -to--the receiving water—can—produce a
shock load deleterious to aquatic life.
^QUALITATIVE. ASEECIS.-O? URBAN- ST-ORMWATER RfcflOFF- ' ~ —
Little data was available relative to direct urban stormwater runoff "in
Onondaga County prior to this study. Therefore, the eytent of the contribu-
tion of pollutants in the stormwater runoff to the CSO system cannot be fully
evaluated-. However, sfudieo conducted in othtr parts of the iJnited -States
have indicated that large quantities of relatively contaminated water drains
to- watercourses with no form x»f treatment to reduce the pollutant load.
Various stuiies have indicated, -that -total suapended 'solids and—settleable
—solids—concentrations were frequentrly—highei: ^n urban storawater runoff than
In combined sewer overflows. Table 1 presents tha pollutant characteristics
of stormwater ruaoff in several cities iix. the U.S., while Table 2 presents
pollatant character!.dtics of some combined sewer-overflows in the U.S. ^33).
These tables indicate that additional work to reduce direct stornwater—runoff
say be necessary, afte? CSO abatement is achieved, to assure that receiving
vater quality standards ar. maintained.
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00
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O.JO
...
20 111 | 1.41 1.11 0.62 0.4(
7-56 48-170 0.57-2.6s 4.12-S.I 0.3J-1.00 0.15-100
> . i
4 . it..
UM
S. 15
—
0.46
0.17
0.24
...
0.75
0.15
O.tS-75
J
r«c«i
col'xor»a*
M06
...
230
20.100
^0.100
420
Jl.500
2jo-b.ooo
i
r i
Crguili=t/)00 ail.
-------
TA8U 2. FOLUIIAMT CONCENTRATIONS IN COHPIHEO SIKH OVERFIMS.
City
DM Hotnai, IOM
HI>Mufc», Ml icon* In
thi» York City. Hex York
Monton Cr4«k
Spring Cr4ek
R«clf», tiliconiln
Bocho»t«r, Hew VorK
Av«r«u* (nut tMlgtittd)
fi.r.9.
TSS VSS
411 117
121 109
JOG 182
J47
i
5S) 154
27J
370 140
773-5S1 109-182
Av«r»(n
6005
£4
59
222
111
' 158
til
pollutant cow.nt
Kjalitahl
COD nitrogen
i..
264
Ml
358
...
}"
59-222 2C4-401
4.»
16.*
2.C
J.8
2.6-4.
rtttooi, v)f\
tot.l
nltrogtn
4.i
*.J
—
—
>
t--
9.1
9 4.J-16.6
FtWf
phorul
1.8*
1.2}
...
.
2.78
1.95
1.t3-2.7«
Ortlw-
, phogphit* Loftd
t.JI
0.86
0.60
—
0.92
0.88 0.14
1.00 0.3f
O.I&M.3I 0.14-0.60
i
,.cfl ^
col 1 1 oj- •»
—
..t
"f
--P
201,000
1,140,000
$70,000
201.000-140,000
-------
The United data presented in Table 1 indicate that pollutant loadings
from- urban- stomwater runoff could result in serious deleterious effects on
the surface waters to which they discharge. The impacts may be localized in
nature, or could extend for long distances and for long periods of time after
the storm event. To date, little information is available to define the
extent of the problem, if r.ny, in Onondaga County. The limited data generated
-as—part of this study, which will be presented in later sections of this
report, do indicate similar potential-water-quality-impacts to those inplied
"by Table 1. The various studies from which Table 1 were developed indicate
that this source of pollution should not be neglected.
POLLUTED -PRECIPITATION
Studies in recent years have indicated -that precipitation may be the
vehicle for significant contamination of surface waters with pollutants from
industrial 'and urban sources. This contamination may originate hundreds of
miles from ita eventual inpact, transported by regional weather—patterns.
Atmospheric washout of pollutants extends far beyond acidic components which
contribute to the depressed pH characteristic of rainfall in the Northeast.
The term "acid rain" may well serve as an umbrella for a who IB -gamut of
atmospheric pollutant washout' problems caused by contamination of surface
waters by particulataa, oxidants, and a variety of substances, many probably
unidentified in precipitation ac this time (Table 3) (13).
In~ Gnondaga County, the washout -of atmospheric -pollutants "has not been _
investigated so that the extent_o£_their— ef-fecc—i-sr-iiaksoTrerr~~¥ith~e^tensive"~
industrial development in western New York, ~ -coupled with regional—weather-
patterns, the potential for this type of pollution exists although its
importance as compared to other quality issues may be slight: However,
because of the relatively novel aspect of the entire washout concept,- the
potential for serious pollutant problems-should not be discounted.
AMES TEST
The contribution of pollutional loadings to receiving streams- from CSO,
stormsater runoff and atmospheric washout is composed of a wide variety of
compounds. The impacts to the environment. ' and to public health of these
compounds may be as individual species; or synergistic. Until recently,, the
emphasis in environmental surveillance^ was-on the quantification of specific-
contaminants with little or co—concern given to their actual impact. It is
now generally accepted that environmental factors may alter a substance
sufficiently to affect its identification; therefore, it would not be
unreasonable to espect the impact on biological • systems also to be. altered.
Therefore, the use of biological effects tests which correlate to "in situ
impacts-are now being used in a wide array o.f environmental applications.
10
-------
TABLE 3. PERCENTAGES OF STREAM LOADS t)F CHEMICALS-
RESULTING FROM BISECT ATMOSPHERIC WASHOUT (1).
—Nor-thea&tern—U-.-S-.
Substance 1963-68
chloride - 30-60
cklcium * 20
sodium ' 20-30
magnesium -_20-.50
potassium 70-80
sulfate 90
t
nitrogen. . 120-ISO —
—The- Anear-Salmonella/mammalian-microsome mutagenicity test was dev&loped-
-by—Dr.--Bruce—N. -Ames-and -co-workers of "the "University of" -California at
Berkeley in *he -early 1970's (2). Originally developed dtftlng. studies of how
1>acterial .genes are_-switched on- and -off in -response to—the—presence ~of the
amlno acid histidine .in.the growth medium and of the effect of-mutations which"
perturbed this control mechanism, it has become a very sensitive and simple
bacterial test for detecting chemical mutagens. Its advantages over other
tests are speed, ease-and relative low cost. Thus, the bacterial mutagenesis
test provides a mechanise whereby large numbers of -chemicals can be screened
for mutagenic activity in a relatively short time (5).
The Ames test, relies on the tendency of- some previously— m&tated-bacterial
cells to—undergo—a—subsequent -mutation—frarck—to "their natural~state (revert)"
when exposed to certain mutagenic chemicals. The compounds are tested on
petri plates with Salmonella typfalmurium bacteria, LT2 striin. Five primary
tester atrains have been developed, after a screening process involving
hundreds of mutants, which may be reverted from—a—histidine—amino -aeid-
requirement (histidine auxotrophy) to nutritional normality (prototrophy) by a
variety of mutagens, with sensitivity and specificity (41).
Normally, Gram negative bacteria have a rather impermeable -envelope that
reduces the. penetration of many chemicals. Ames introduced, a—mutation -which
gives rise to envelope defects, resulting in a greater' sensitivity of the
organism to DNA-damaging agents, and also introduced a plasmid (foreign
genetic element) which nakes DNA replication more error prone - the test-
chemical damages the bacterial DNA which can no longer be repaired (41>. This
results in the creation of a strain in which a few molecules of a mutagen may
11
-------
create a DNA. lesion, and each, lesion- may cause a -mutation. Some of these
mutations will be such that the internal ability to supply histidine is
restored. The number of these chemically induced revertants Is. compared to.
the background (spontaneous) reversion rate to determine the mutagenic
strength of the test compound (5) .
Several workers discovered that many substances must be- converted" "by
enzymes in liver or other tissues to an active Celectrophilic) . form which is
the true mutagen (17,21,23). Ames added homogenates of rat liver to the petri
plates, incorporating an important in vivo aspect of mammalian metabolism into
the in vitro test (5,12). In fact, although the sensitivity may be affected.
it appears that any mammalian liver may be used to prepare the microsomal
fraction, known as the ,. S-9 fraction, as veil as other tissue fractions
(5,12,16,42,43). , I ,
About 85% of the known carcinogens subjected to the Ames test have been
detected as mutagens. Less than 102 of non-carcinogens have been found to be
mutagenic in the test (28,38,40754,55).. This affirms the desirability of
using this rapid, economical test system as a screening technique to identify
those pottntially dangerous substances among the thousands of chemicals and
mixtures found in the human environment.
The Ames test can be used to evaluate the mutagenic potential of mixed or
undefined solutions and pure compounds. It has been used, to screen drinking..
water (10,22), municipal (56) and industrial (18) wastewaters, industrial
products and a wide variety of other substances, such as: -
foods and food extracts
saccharin (6) •
caffein (8)
cooked beef (11)
textile dyes (20)
fly ash (32)
photocopies (35)
cosmetics (45)
crankcase oils (52)
pesticides and herbici-iea- (60)
aniaal feed additives (49) •
-air~enissions-and -pollutants -(64)
plant extracts (27)
algal by-products (24)
cigarette smoke condensates (30)
fungal toxins (68)
surface waters" (53)
recycled waters (25) - ' -
12
-------
PROJECt AREA LOCATION
Onopdaga County is located near the geographical center of New York State
(Figure 2).' The county is about 34 miles (55 km) from north to south, 30
miles (48 km) vide in an east-west direction, with a land area of 793.5 square
-miles (2063 square kilometers), (9>. With a population o£ 474,703—(1975 -est.)
(?), the county ranks as the tenth most populous in the state,.
Syracuse, the county seat, is centrally located (Figure 3) and has a
population (1975 est.) -•>£ 183,334 (9)' living in a land area of -approximately
25.0-square miles (65 a-mare kilometers).
•f
PROJECT BACKGROUND
In the past decade, Onondaga County, New York, has administered a series
of studies and construction projects whose objective has been to improve the
level cf water quality in Onondaga Lake, its tributaries—and—downstream
surface waters. The major sources of pollution were identified as industrial
waste discharge, insufficiently treated municipal wastewater, and combined
-sewer—overflows» An industrial, waste program.,was_-initiated_in_the_county Jja.
1973 to reduce liquid waste sources, quantities, composition and disposition.
Municipal wastewater treatment hss been upgraded in the Syracuse Metropolitan
area, particularly with the completion of the .recent .-expansion- -of the
^Metropolitan Syracuse Sewage Treatment^ Plants Finally, in—IS79^—preliminary
design plans were prepared and submitted for structural and" non-structural
improvements to the-Metropolitan Syracuse~Sewera^e~Sys"tem~:fcnarf~would decrease"
the—quantity aad upgrade the quality of CSO. discharges. -Presently,. the
program is in its preliminary phase involving the application of* best manage-
ment practices to the system. In addition to the generation of baseline data
in -regard to the Ames test, this study may be useful in the determination-of
the necessity of advanced treatment of CSO discharges to prevent the introduc-
tion of mutagenic substances to the aquatic environment.
13
-------
CANADA
VEBMONT
Figure 2. Location of Onondaga County in New York State.
14
-------
Figure 3. Onondaga County, Nev; York.
15
-------
SECTION 5
-MAT-EMAfcS-
AMES- TEST
General . . ~ " '
procedures employed in the analytical phase of the project .were-derived
from three sources:
-A)- a-form—instruction sheet ("Supplement to the"Methods" Paper", June
1979) supplied with the test strains from the laboratory of Dr.
Bruce Ames (3);
i
b) an "Anas Test Information Package" prepared by the EPA 457}j- and"
cJF a primary methodology journal article-C5H
The- analytical procedures as outlined—in—this—section were forwarded -to the
Project-Officer "for review prior to use in the project,' -Recommendations made
on- the .basis—of this- review were incorporated into the ..test—uetbodo-logy—and
Are- ref erenced_as-appl-ieab-le;
Test Strains . ' ..'...
For analyses performed under Phase I, the wild-type Salmonella
typhimurium LTZ and five histidine dependent test strains were obtained from
Dr. Bruce N. .Ames, Department of Biochemistry, University of California,
Berkeley: TA98, TA10Q, TA1535, TA1537, TA1538. For Phase- lX,~-copies-of~the
five bacterial test strains were obtained from the USEPA Region, II Environ-
mental Services, Division, Edison, KJ. Strains were grown -on Oxold Nutrient-
Broth Uo. 2 plates (Oxoid Ltd.,_ Basingstoka-, England)' with._l .52—agar— £BBL, -
-Cocfceysvir le, MD) (hereafter-known- as ONA) (3). Colonies were-picked for
overnight cultures as required, grown in Oxoid Nutrient Broth No. 2 (hereafter
known as ONB). Eetri plates used were Falcon Muta-Assay Dishes -(Falcon
Ldbware, Oxnard, CA) (Cat. No. 1029), sterilized without ^ethylene-oxide-, which
may cause s. mutageni~ response in the- test (3-). '
Solutions • ' „" -- "
The minimal salts medium was" prepared as by Yogel- and Bonner (67-) -except
at a strength 01 10X rather than SOX (Table 4"); color changes noted in the JOX
medium when autoclaved were not evident in the 10X preparation. When
diluted to normal strength, agar was added at a final concentration of
16
-------
l.SZ/liter. A supplemental glucose solution was autoclaved separately such
that the final medium would contain 52 glucose (Sigma Chemical Co., St. Louis,
MO.; cat. no. G-5000). . -
i ' «
, Top agar contained 0.5% NaCl (Mal.linckrodt) and 0.62 agar, autoclaved and
stored in 100 ml portions in milk dilution bottles (5,57). The ^top-agar -wa-s
melted in a water.-ha.th -as-needed-prior—to-use-.
The 0.5 mM (loillimolar) L-histidine and 0...5 mM biotin- (both' Sigma.}-
solutions were autoclaved and stored refrigerated for later use (5,57).
" • The S-9 reaction mixture was prepared as indicated (Table 5)(5,57). The
salts solution and phosphate buffer were prepared and autoclaved for later
usa. NADP -and G-6-P were prepared fresh; both solutions were filter ster-
ilized (0.45u pore size membrane filter; millipore Corp.. Bedford, HA) and
prepared using preautoclaved distilled, deionized water. Any. excess_aolutlon
was refrigerated and used within one week. Commercial S-9 extract was stored
frozen and hand-thawed prior to use. The reaction mixture was stored on ice
prior to and during use.
Standards and spikes were prepared from either benzo(a)pyrene
(3»4-benzpyrene) (hereafter known as BaP) (Sigca, cat. no. B-3500)-, methyl
methanesulfonate (hereafter known as MMSX, (Eastman Kodak Co_._,_R0chester-,-~NJQ ,~
. or N-methyl-Nf-nitro-N-nitrosoguanidine (hereafter known as MNNG),==(Aldr±chi-
Chemical Co., Inc., Milwaukee, WI). .
. Other chemicals "required included ampicillin (Sigma, cat. -nor~A-6140)7
dimethyl-sulfoside (hereafter known as DMSO) (Mallinckrodt, spectrophotometric
grade); dichloromethane (hereafter known as DCH) acetone and hexane (all
Mallinckrodt ^ nanograde); -and--crystal violet (Q-.012 solution') "(Matheson
Coleman & Bell, Cincinnati, OH). ~
Glassware
All pipettes, beakers, flasks/, test tubes, dilution bottles, .sample
containers and sample storage vials were glass. No plasticware was used in.
contact with sample aliquots to'be used for the Ames test.
-Glassware-utilized in--the-extractiorr—of—raw samples was soap and water
washed, Chremerge (chromic acid-sulfurlc acid solution) soaked overnight,
water rinsed and finally, rinsed three times in acetone followed by -three
hexane rinses. -
* »
Bottles used for the collection and storage of samples were one gallon
(3.8 1) amber glass obtained from Empire State Bottle Co: of Syracuse, Inc.
(Syracuse, NT). These bottles and the sample extract storage vials required
their caps to be foil lined prior to use.- Both were-DCM rinsed-prior-to-use;
the vials were also autoclaved.
17
-------
TABLE 4. VOGEL-BONNER MINIMAL SALTS BASE" MEDIUM (67) .
Compound
MgS04'7H20.
citric acid
K.HP04 anhydrous
NaNH4HPQ4'4H20
distilled, deionized water to 1 1.
i /
Amount (g)
2
20
100
• 35 .
All chemicals were Mallinckrodt, Inc. (Paris, K!) analytical reagent
grade.
5. S-9 REACTION MIXTURE.
Compound
Aroclor- 1254-Induced Rat
Liver S-9
0.1M- NADP3
l.OM G-6-Pb
0.4M- MgCl2 + 1.65M KC1 (salts)
phosphate buffer
sterile H20
Total
Amount (ml)
1.0
0.4
0.05
0.2
5.0
3.35
10.
' Source
Litton Bionetics,
Kensington, MD
(Cat i So _8360-01)-
Sigma (cat. No. N-0505>
Sigma (cat. No. G-7879)
Both Mallinckrodtr
" Both Mallinckrodt
- distilled, deionized
beta-nicotinsmide adenine dinucleotide phosphate, monosodiua salt
D-glucose-6-phosphate, monosodium salt
C 0.4M NaH PO *H20 + 0.4M Na2HF04*7H20, pH 7.4
18
-------
CHEMICAL ANALYSES
All chemicals -used for analyses other than ulie Ames .test were reagent
/grade, unless otherwise specified ta the procedures.
19
-------
SECTION 6.
-PROCEDURES"
PLAN OF STUDY
I
• - A general plan of study (Detailed Project Work Plan: Urban Runoff ard
Combined Sever Overflow Mutagenicity, May, 1980) was prepared for this. j. -reject
in conjunction with the Project Officer. The plan of study set forth project
objectives* outlined procedural protocols, and delineated problems which might
t« encountered. Due'to the known variability-in-the chemical—composition of"
CSO and urban runoff,, tha inherent difficulties in working with a biological
system* and the aspects involved is. performing a noval but limited survey, it
was anticipated that analytical and other general problems- -would-be—encoun-
tered which would be difficult to overcome within the 'original framework of
the plan of study. For this reason, the plan of study was prepared to be
flexible, with the success of each phase governing later options. As antic-
ipated, several modifications to_the_plaa of--study* were made to-accommodate
theae experimental difficulties. This report reflects jchose- modifications
which alter the letter, but not the spirit of the original project. The major
limiting factors were the prolonged, and severe drought in Onondaga-County, "in'
the suEicer of 1980 and the general absence of a high -level— of—mutsgenic—
activity, as compared to studies in other aquatic environmental—systems-
do,18,22,25,53,56).
As a result or the 1980 study, additional sample collection and analysis
were performed in 1981 for verification.of initial conclusions based on 1980.
data.
SAMPLE COLLECTION
t _ ,
Provisions were made in the original plan of study, for the collection of
seven types of samples, in Onondaga County (Syracuse): , -....-
a. combined sewe.r overflows,
b. dry weather sanitary flow,
c. urban runoff,
d. influent and effluent to Metrot
e. Onondaga Lake, and
. f. rain
Tha project plan of study required the selection and collection, of one
CSO sample during four wet weather events front each of six CSO discharges. -,
The objective was to select six drainage areas to be examined, each with
divergent characteristics of size, land use, population density and/or percent
20
-------
iopcrviousness. Selection was made based upon previous studies petfonaed iu
ihe S/racuse are.1 "o characterize CSO sites '48).
• The physical characteristics of the six CSO sites which were selected ire
presented in Table 6 and are summarized as follows:
t
(a) site 005 - a small drainage area with, a high percentage of"
impervious surface (86*) and predominantly commercial land use;
-however-, -Metre historical" chemical data was. available from
previous studies to characterize thi? area;
(b) site 019 - a large drainage area with mixed land use;
(c) site 027 - the only drainage area, which includes a significant
• amount of industrial land use;
/
(d) site C37 - a drainage area, with a high percentage of impervious
surface aiul mixed land use;
(e) site 043 - a large, high uensity residential area; and
Xf) site 046 - the onlv dr? Inage area.- which is primarily open
space.
In a-Jdition, two alternate -sites-were -chosen in the event that diffi-
culties arose at any time in- the collection of samples from the primary, sites,.
These alternates w,ere: • -~ - '~ '• ~~ '
(a) site 004 - This drainage area is a large basin characterized h;
mixed residential/commercial land use; and
•(-b) site- 074 - a drainage area with p-edominantl*,
commercial/industrial land use.
The locations of the project CSO sites may fce found on Figure 4.
The sample collection sites were chosen using three selection criteria:
(a) uniqueness of drainage area characteristics - drainage areas with
-percent imperviousness, -population density,. siz.e_and—land— use-mix
-representative of several others- in th* city were -evaluated for
selection. In addition, drainage areas which were unique in the city
(i.e. predominantly open-space, such as site 046) were selected.
Cb_l ease and safety of santpie- collection - where several sites of
-similar physical characteristics could have been selected,. ease of
sample collection was considered for the preservation of sample
integrity. In eddition, both receiving streams traverse highly
trafficked urban areas where sample collection would, be. performed
•through manholes in streets. To ensure the safety of personnel,
accessibility during high traffic periods was considered.
•21
-------
1
I
OvfrflM Collector »iMfJ f«rcint
Nudur S»»t
Ar*<
»* , (*cr*t) • lufHirvlou*
oos wis ii. t *s.8
01* 4<
M7.2 4 .
i
OJ7 MIS ISI.2 M.J
0)7 MIS J9-7 SO.O
CM MIS 270 « 4S.1
MC MIS J5.1 It. »
004 IBIS J5J.J 42.0
074 HIS 74.2 40,0
' .Av«r»o» Smlttry Und
| Ar«»
i ) , Land S«OM l^crai) tit*
.OOS II.C t
.OOS M^.O I.J
.010 SIO.l 1.1.1.4
.OOS ».0 1.2.J
i
1 .OOS . U«1.4 1
1
j .02} 2S.1 1,5
.070 242.9 t.>
.01* - 1.4
i , '
i
Pope! -»i 1^1
(O.n.Hy/^r.f
•
IJ.t.
44.7
47.4
71.7
23.1
19.9
-
I
collector tyitc*i IBIS - Htrbor 8,-cwk InHrc.ptlnj St»«r, MIS- Kiln tnurc.ptln) Swrtf
rwvaff «r0«t and ttnlt
to pirtUI «cwor wpir
try
-------
&. .'...;
Fl'9Ure *•• Cation of Project
23
CSO Sites
-------
fc) available baseline data - physical and chemical analyses of CSOs in
• the Syracuse area have been performed during previous charac-
terization studies. These data could serve as a baseline* to which
this study could be compared. However, this criterion was. con-
sidered least important in site selection.
CSO samples were collected during four wet weather events in glass
-containers, lowered from manholes tiJrectly above the overflow .pipe. Sufficient
sample- was transferred to fill an amber glass bottle for Ames testing,. a_50_ml_
plastic container with nitric acid preservative far TOG and heavy metals
analysisr and a plastic half gallon .container" for other, chemical/physical
analyses*. This sacple storage and preservation"procedure was followed for ail
sastples. Glass containers were used for the collection and storage of Ames
samples- since- it was unknown what effect plastic would have on the samples.
Sanitary sewage samples were collected from the combined sewer either
before or after a storm event, as appropriate to the collection schedule, in
the same manner as for tha CSO-samples.
Urban runoff samples uere collected from -catchbasina in their corre-
sponding CSO sites. Where necessary, curbside runoff was used. Figures 5
through""8 depict the sample collection points.
Samples were collected from the north and south basins of Onondaga Lake
at the 9 meter level, the lower limit of the epilimnion (44). - -
Sain, samples were collected at the 0'Brien^_(^e_Jy^a^uj^^o_f^ice:;/in_
36-inch diameter stainless steel pans pre-rlnsed with DCM. These pans were
placed-away- from buildings, cars, trees and pavement to minimize contamination
from extraneous sources. Rain from two perns was combined until sufficient
sample vas collected for analytical purposes. The pH of this sample was taken
immediately; the sample was then dispensed as previously .described.
Additionally, three CSO samples were collected in Washington, D..C. „ one
each at three separate sites. Due to difficulties in obtaining manual grab
samples at these sites, collection was facilitated by the use of an automatic
sampler. Also, a CSO'and a -runoff sample each-were taken from a site.in
Rochester, NY (site 007-Haplewood Avenue) during two storm events (a total of
two CSO and two runoff samples).. Table 7 presents a characterizatioa-of all
of these CSO sites.
24
-------
Figure 5. Sample Collection Sites: (above) Drainage Area 004, lookin'g-south;
(•below) Drainage Area 005, looking south - concrete encasement
crossing center of picture contains Harbor Brook
25
-------
*; -"JSSisk ~ ~- - 1 .TV .1'-•-.".--•:
Figure 6. Sample Collection Sites: (above) Drainage Area 005, looking west;
(below) Drainage Area 019, looking north
26
-------
Figure 7. Sample Collection Sites: (above) Drainage Area Q37, looking
northeast; (below) Drainage Area 037, CSO discharge
27
-------
Figure 8. Sample Collection Sites: Drainage-Area 043, CSa discharge
28 ' -
-------
TABLE 7. CHARACTERIZATION OF CSO DRAINAGE. AREAS IN ROCHESTER,
' NY AND WASHINGTON, D.C.
Site
Location
Rochester
Washington
.Washington
Washington
Site
Number
00?
'024
034
038 * .
. Area
(Acres)
729
4235
555
320
Perc-int
Impervious
- 38
60
65 -
55
Land
Use*
r,z
3,4
-3,4
3,4
,
-land use-: 1- single family residential
2--multi-family-residential
3- commercial • v '.
4- industrial
i . —
Sample collection in Syracuse was performed by personnel from the
Onondaga Gounty Department of Drainage and Sanitation, luitially, the
criterion described in the plan of study for frequency of wet weather sample
-collection~was~that storms would'be saapled~*lf they were separated~~v>y- a'"four
day interval from any previous wet weather event. In Syracnsa, norma-EL-y—a~
rainy city, this was not considered to present a - problem. However-, the
Northeast experienced an extremely dry year in 1980 and the first-half—of-
1981,°. producing, droaght conditions in the—region.... This iack_af_pj^cipita--
tion» coupled with concent for the safety of sample collection par_s.onnel_i£
it became necessary to work at night in highly trafficked streets, resulted in
plans to reduce the sampling interval, if necess.-ry. However, due to a
continved lack of precipitatior, wet weather samples were collected within 7
days o; each other in five of six instances (Table 8). Sampling dates and
times are presented in the tabulation in Appendix A. Rain
-------
XABLE 8, RAIN--DATA FOR STORM EVENTS, SYRACUSE,
Date
7-25-80.
-9=17-30 —
10-13-80
10-20-80 f
9-2-81 /
SH'4-8-1
1
Rainfall
Start
0500
1500
0400
17CO
(
0700
-07t)0-
Duration
StOTJ
•1400
1700
2200
2400
1000
(10-3-81)
0300
(10-5-81)
Volume Antecedent
(inch-2 ) Dry Days
0.8-7
0.27 -
0.07
. 0.12
*
0.83
Oi40
•
'2
2
3"
2
8
__L
Sample collection in Rochester, NY and Washington, D.C. was-performed-by-
O'Brien 4 Gere personnel. All samples vere transported.. to_tlie_=Syracuse.~
laboratory by air as soon as practical,- usually -within 3 hours-, where -theyj"
were • assigned a laboratory, accessions—number-,- -Sample portions for
chemical-/physical analysis were refrigerated; the por.tiom-.forL-Amesr-trestingswas;
treated^as discussed" elsewhere in this section. • .
DATA MANAGEMENT . .
A computerized system for the identification of samples-and-manageaienc'of '"
data generated by any project has been developed. For this project, it
included:...
a. field identification -_ .a field check sheet (Figure 9-> -was-^nrepaxed
for sample collection personnel which contained . all. pertinent
identification information for any sample. A sheet was prepared for
each—sample- and-accompanied -the-appropriate -sample containers to the
-laboratory.
b. sample identification - as each sample enters the laboratory., it was
assigned a unique, five-digit accession number found, on- -a. sample
Identification-ticket. This identifier^permits-the-"discrete-organi-
zation of all information and data relating to that sample, whether
for analytical identification purposes, reference in paper-copy
records and correspondence, or complete storage ard recall.
30
-------
-2-
i Qcso
2 Sanitary
Flow
Runoff
Temporary No.
Sample Data
Sample Time
SAWU 1.0. CHECKLIST
(1) SYRACUSE CSO § URBAN MUTAGENICITY STUDY
S1t« Htsaoer .
Sltt Number
Site Huabtr
Qefflu«nt
S ^jonondaga Lake Oeptn
6 C3Erl8 Bo"l«v«rd Stora Soar
7 ftaln Conwnts:
wet weather-
dry WMther> b*fon storm-
dry Mather, after storm
before storm
stonn"
3 F"3 after storm
Comcnts:
_/,|TT»et weather
* Pi-dry weather, before
u-^storm
dry weather, rafter
stora
Figure 9. Field Data "Check Sheet.
. 31
-------
c. data organization - in a preliminary planning phase of any
.analytical investigation involving the O'Brien & Gere laboratory, a
computer codification format must be established which will later
serve as the basis for data storage, recovery and identification.
This format is characterized by the categorization of samples, with
-any type of identification permissible for the classification. The
.chosen coding scheme for identification having been established, the
description of an individual sample may be recorded in appropriate
identification fields on the staple ticket. There are seven
distinct identification fields, four of which carry variable options
and contain 9999 sub categories, while a fifth contains 999 subcat-
egories. These permit several hundred options for the segregation
of data and increase the ease of sorting edited data f jr evaluation.
For this s idy, a scheme was developed 'for the classification of project
samples (Table '») . This scheme permitted the segregation of data by the city
sampled and the sample source.
CHEMICAL ANALYSIS -
- All chemical analyses were performed -by approved- procedures— (-1 ,-44-) . The
pH of all samples was measured from an unpreserved aliquot of sample immedi-
ately nipou arrival in the laboratory with a Corning Model 12 pH Meter (Corning
Scientific Instruments, Corning, NY). TOC of nitric, acid preserved—samples
was-determined on a Beckmatc Model- 915- 'Total "Organic Carbon Analyzer ~(Beckman
-Scientific Products, Fullerton, C6). Heavy metal ana3.ysia was
Varian AA-5 75 " Series Atomic Ab'sjrptibn Spectropfiotometer - (Varian Techtr on,
Springvale, Australia) . '
MOTAGENICITY TESTING
General
As mentioned previously, three sources served as the pr^'jaary references
for -mutagenicity testing procedures (3,5,57). The procedures used were
largely those of Robnett (57); modifications from ether • sources were- subject
to -review and approval by the EPA. Some additional modifications were sug-
gested by the EPA (19,65) and. were Incorporated into the- projecr-j_:they_are
discussed as appropriate.
Sample Preservation and Disinfection
One of the limitations of the Ames test is the requirement that samples
be sterile prior to their application in the 'test. Besides the obvious fs.ct
that—any- bacterial contamination would interfere with the growth and scoring
(counting) of colonies produced by the- test strains, metabolic by-products
from microbial contaminants could interact with the test substaz.ce to aroduce
false results.
Since -samples might arrive in the laboratory at "any time of the day or
night, and immediate extraction would be impractical, it was necessary to
develop a procedure -to disinfect samples upon their arrival to reduce
32
-------
TABU ». HtOJECf SAHH£
CO
CO
IOM - City
IM2 - S»
1
«pU siirc* '
IM) • location
OH- Octcrlftlor
I - Syracut*
t -cso
2 • Sanitary flcn
1 - Runoff
4 » Hftro
5 - Onondtda Uk« ,
£ - CrU OQ'ul.v.fd StoU S«»r
7 - 8«l»
SIU «k-bcr
lit* Nurfitr
Sit* Hi»b»r
I » IMIuMt
2 > «fflu»ot
2 * dry metlur,
b«for« »tor«
3 M dry voatSer,
•U»r ttuin
I • befor* ltor«
2 • during itorm
] *• alter ttoro
1 - «t «.«thor
^ ^ dry Mbatlwr^
t Iroforfc ttor«
1 V dry M«tlier,
aftor ktora
1 • wat H«attter
2 | dry malhor,
I before atorai
1 ^ dry
I
af(er itorai
aouth 2 - north p*pth (Mtara)
bafln b««lrt
-------
TWLE 9. PROJECT SAXPU NO DATA nWACOENT IDCHTIF4CAIIW SCHO*.
IDfl - CUV
2 » Ibslilngton, D.C.
) - Otic*, NY
* » Rocrw.t.r. NV
« f
t
ID12 « S«pt. Sourco
t -cso
\
2 • Sin It try flu
1 - Runoff
1 - CSO
2 » SftnlUry Flow
3 - Runoff
1 « CSO
2 - Stnlttry flw
} - Runoff
IDI1 - Uoitlon
SIU NudMf
Sit* Mu*.r
SIU NuatiMr
Sit* Huab«r
t
Sit* Mt»b*r
Sit* NuWxr
SIU t*3*.r
f
' SIU Hux6*r
SIU NuX»r
10 Ik - Ooicrlptor
t • Mt twttUr
2 M dry wc«t^»erf
1 •* b«for* »ior»
')•
1 <• ««t M4tlNir
2 » dry twitlxr,
bofor* »torm
I
1 * t.foro fttorn
i
i • net HMthor
2 • dry mtttwr.
U(or» itom
i
t • b.for* *turn
1 I
-------
microbial degradatioa of substances in the sample over time. Normal oxidant
disinfectants (chlorine, chlorine dioxide, other halogen compounds., .ozone) are
known to alter chemical species, causing the formation of "tutagenic compounds
from uoh-mutagens; autoclaving the sample could descroy heat labile (sensi-
tive) organic mutagens, or cause their volatilization; -filtratioc. could remove
mutagenic organics adsorbed to participates or oil and grease globules; and
irradiation with ultra-violet might alter the structure of target compounds,
since many known organic carcinogens and mutagens are DV-absorptive. There-
fore, an alternative was required which would not physically or chemically
_alter_the._compounds_present_in the_jenvironmental-.sample..
Overbye and Margolin (50) used l-.O ml of chloroform to disinfect 250 ml
of raw t star sample from the Arthur Kill (New York City). Since chloroform, a
known carcinogen, is one of the few carcinogenic substances which is not
mutagenic in the Ames test (62), its presence could not cause any false
positive results. Chloroform, heavier than water, would sink in globules to
the bottom of the sample container after agitation and disinfection. Aliquots
of disinfected, raw sample could then be removed for testing from the top of
the sample container without removing any of the chloroform disinfectant.
The chloroform procedure was evaluated 'for use in this study. Samples of
wastewater influent to the Metropolitan Syracuse Treatment Plant were col-
lected; these were used in the disinfection: evaluation -and- considered—to be
representative of the most miczobially contaminated environmental sample
likely to be encountered during the project. However, the chloroform disin-
fection procedure was found not to be of value for three reasons:
a. at the ratio "of chloro£orm_suggested_(5.Q.X-4n_iO&ji^^
fection was not • achieved. Th^ concentration. of_ chloroform^ was
increased without improved results. Samples, containing...varying
amounts of chloroform were rhs^en at 0, 10 and 20 minutes, then
allowed to settle. Aliquots of 1.0 ml were removed from the top of
.cash sample container,- spread -plate inoculated—onto-OKA—plates-and
incubated at 37°C for 24 hours. Disinfection was not achieved. No
specific reason for this lack of disinfection efficiency, is evident,
nor was further investigation of the subject conducted.
*
b. spot tests performed on some raw test 'sample's did' not - elicit
positive results, Indicating a lack of mutageaicity of the raw
sample. An explanation of 'the spot test, a rapid screening method
for mutagenieity., is,presented in a following,section.
c. chloroform might be acting as ''an extractant in the disinfection
procedure, thereby removing mutagenic organics from the aqueous
layer. These organics would then be contained in the heavier
chloroform phase at the bottom of the flask. This ^process could
have resulted in a false negative spot test result for test samples
when test sample was removed from the upper aqueous phase in the
flask. Since the efficiency of chloroform as an extractant in this
test has not been demonstrated, nhe recommended extractant,
35
-------
dichloromethane (57), was used. Extensive testing, beyond the scope
of this project, would have been required "to valiv>cte chloroform as-
a Suitable extract ant.
The" EPA 'recommended extraction procedure (57) (44 FR 69541) involves the
use of dichloromethane (methylene chloride) . DCM was evaluated as a dis-
infectant in the same manner as chloroform; the results were .found to be
favorable.
The procedure used was the addition of 100 ml of DCM to 2 1 of sample in
the glass sample container. The -sample was shaken vigorously Joe two minutes*
This served to disinfect the sample and provide the first of three extrac-
tions. The sample bottle was vented to release vapor pressure,, then recapped.
/ ' • " '*
Extraction and Concentration
The extraction and concentration procedure used for this project was that"
recoimnended by uha EPA (57.) with minor modifications. The extraction pro-
cedure was as follcvs:
i
a. (if the sample were neutralized and disinfected as. described_in .the.
preceding stction, this procedure could proceed directly to -step
(c).) The sample was shaken veil; 2 1 was measured into a separatory
funnel -following neutralization with either 6N NaOH or 6K: HCl',-.as
Generally^-neutralization was _nojt_iequired_since most
samples were- close to- pH 7 .
b. 60 ml of DCM -was -added first to. the ' graduated cylinder'
measure the sample, swirled to rinse the sides, then transferred to
the sample bottle; it was capped and shaken 30 seconds* to" rinse, the.
bottle walls. This solvent was transferred, to the separatory.
funnel. - - .
c. the funnel was shaken for 2 minutes with periodic venting to release
vapor pressure. The organic (bottom) layer was allowed to -separate
for a ^ninlmnn of 10 minutes. The DCM layer was collected in a. 250
ml flaak; any emulsion was reserved- in the separatory funnel-wish
the aqueous phase.
d. -steps-(b)- and (c)-were repeated-an additional— two—times
extract being co'Hected in the same "flask each" time . "following the
third extraction, any remaining emulsion was taken into the flask.
e. the combined extract in the flask was dried with anhydrous -sodium
-sulfate, (Na_SO,>, -adding sufficient quantities--for— the-salt-to-flow-
freely. Since the emulsions formed generally were, extensive and
would have clogged a Ha. SO, column, but may have contained target.
compounds,, the salt was added directly to the flask containing, the
combined extract.
36
-------
f. the extract was transferred into a 500 ml Kuderna Danish flask. The
drying flask -was rinsed with three 5 ml aliquots of DCM, each rinse
being combined with the extract in Che Kuderna Danish flask.
g. the Kuderna Danish flask was topped by a Snyder column and the
extract reduced to 1-5 ml in a water bath.
h. the extract was transferred to a 1.0 ml graduated concentration tube
, and placed in a Routes Tube Heater (Routes Scientific, Vine land,
¥J)\ -As the volume was reduced, any remaining extract was added to
.the. _tube4 the- .extract—was- not—permitted- -to reduce—to—dryness-.-
Finally, ths 250 ml flask .as rinsed three times, each with 1-2 ml
of DCM; these rinses were added to the concentration tube and
reduced ,in the same manner. The nitrogen (N_) • blowdown step in the
recommenced procedure (57) was not employed Since it was found that
the 2-step Kuderna-Danish/cube heater concentration readily reduced
the extract to the desired volume. The additional glassware
transfer which the N_ step may have required could have increased
the risk of contamination, or loss of sample.
i. the remaining extract was transferred to a storage vial'. The
graduated tube vaa rinsed three times with DHSO, and brought to a
final volum* In the vial of 10 ml. This resulted in an effective
1 concentration factor for this extract of 200 times over the original
2 liter sanpla. The resultant extracts a?e depicted in Figure 10.
A water blank and two spiked water samples wetre- Included in -each-batch-of
sample extracted. One spika was a mutagen active without microsomajU-activa^
tion, the other with activation.
Spot Tests
Spot tests (5) -were performed on the first 32 samples extracted"X25~
samples, 3 blanks, 4 spikes) using three—of the- test strains (TA$8—TMOOv
TA1537). The spot test is the simplest method of testing compounds--for-
mutagenlcity and may be used for the rapid screening of large numbers of
compounds in a short period of time. A drop of test compound is-- placsd-
dlrectly on the agar surface. As the compound diffuses through the medium, an
Indication of mutagenlcity, toxicity, and necessity of metabolic activation
can be obtained. In this instance, aliquots of 32 of the test samples were
applied as approximately 50 ul of the raw concentrate (2 pipet drops),. Platesr
were Incubated for 48 hours at 37°C. Of these- -samples, sixteen -W2re-tox±e--ta
-one-or more-of the-test strains-(Table~10) j-w±th—12~samples-toxic-to~TAS87~six
samples toxic to TA100, and only three toxic to. TA 1537. However, none—of
these samples exhibited any positive mutagenlcity.
Due to the difficulty of obtaining any meaningful preliminary. results
using the spot test with these samples, it was dropped from -the procedure.
37
-------
Figure 10. Concentrated Sample Extracts"
38
-------
-I
TABLE 10. RESULTS OF SPOT TESTS: SAMPLE TOXICITY (+ indicates toxicitj/.
Sample Hurcber TA 98 TA 100 TA 1537
94251 +
94252 +
94253 . + + '
94254 . +
f 94255 +
94256 + '
S4482
94490 + . +
94431 +
94492- * +
94493 *
94494 + " „ J
94495
94496
94497
94498-
-94*99-
~ 94SOO " - - „
"•=94501 - ' • •
--94507- ->- - -*-
-94508
-94635-
S4636 -- ... . ..-..t"_
94660 +
" 94661 * +
34663
94765
94766
-94780-
94781
94782
39
-------
Ames- Testing
Detailed procedural descriptions of the Ames test are available elsewhere
(3,5,57). This section will only highlight the procedures used in this
study. < - -
Tor Thase I", two 10-fold "aerial dilutions were ftepared ironL the. sample
extract/concentrate, using sterile distilled, deionized water as the diluent.
These dilutions resulted in three concentrations (57)~ for application-^in—tfae^
teat: 200X (the extract), 20X (the first 10-fold dilution), and 2X (the second
10-fold serial dilution). A dosage of TOO ul of each of these- concentrations-
was used, each applied in duplicate. The 100 ul dosage of the most concen-
trated -form of the sample was,, the strongest application used for the deter-
mination of a positive response; this can be defined as representing the
analysis and evaluation of the worst case available in the form of a highly
concentrated 'sample extract. Initial attempts with the application of 500 ul
of sample resulted: in toxicity to test strains in too many cases for general
applicability. • • •
A 100 ul portion of the appropriate tester strain was added to .1 test
tube containing 3.0 ml of melted top agar, followed by sample. A- 300 ul
aliquot of the S-9 reaction mixture was added last, as appropriate, just prior
to Sizing of the tube's contents and pouring them into the petri plate con-
taining m^n^tiifli medium.
.• *
A preliminary evaluation of the methodology indicated some • difficulty-ia-
spreading the sample-top agar Mature evenly over the medium in the petri dish
due to the quality of the concentrated -sample. Therefore, the amount—of—tog
agar was- increased-from- the--recommended_.2.0 ml (5,57)^ to 3.0 ml? the-concen—
tration of histidine and biotin in the top agar was kept the same. It was
expected that this increase in histidine-biotin/plate might result in an
increase in spontaneous revertants due to the availability of a greater amount"
of histidine and biotin per plate. However, by using this method for-all
controls, internal consistency is maintained. Spontaneous revertant levels
are available elsewhere in this section. They demonstrate the improved-
spontaneous revertant levels obtained with this procedure.
• Figure 11 presents the full range of concentrations and dosages used for
each sample during Phase I; concentrations and dosages used during -Phase II
wAlLbe-.descrlbed..in subsequent sections. „
Plates were wrapped in plastic bags prior to 'incubation. This- reduced
moisture loss. Also, the risk of contaminating surrounding plates from those-
of one sample which may contain a volatile mutagen was reduced. However, the
Salmonella strains generally do not respond to volatile compounds unless—the
tests are conducted individually in sealed desiccators (62). ~
The platestwere incubated at 37°C for 48 hours; revertant colonies were
then counted on a Darkfield Quebec Colony Counter (American Instrument Co'. >
Silver Spring, MD). All plates were reviewed -under- a Bausch & Lomb- Dissecting
Stereoscope (20X) (Bausch & Lomb, Rochester, NT) to detect any possible effect
of the sample on the level of background growth.
40
-------
WITHOUT ACTIVATION
f
TESTER
STRAINS
(100 ul)
t
TAI538
TAI537
TAI535
TAIOO
TA98
O
O
O
0
O
b
O
O
0
O
o
o
o
o
o
o
o
o
o
o
J
o
I
o
p
0
0
o
0
o
o
o
200X 20X 2X
o o
o o
o o
o o
200 X
o o
o o
o o
o o
2OX
WITH ACTIVATION
(50O u I S-9 Reaction Mixture)
O O. O O O O TJU538
O O TAI537
O O TAS53S
O O TAIOO
O O TA98
TESTER
STRAINS
(100 ui)
2X
SAMPLE CONCENTRATIONS
(ICO ul Dosage)
SAMPLE CONCENTRATIONS
(lOOul Dosage) -
f 11, Range of Sample Test Concentrations and dosages.
-------
Controls
An extensive range of controls =was prepared each day that samples were
analyzed (5,57). These Included:
i a. spontaneous revertants - each bacterial test strain undergoes some,
spontaneous reversion. These rates are different for each strain.
-Although— this— rate- varies— somewhat— from— one— laboratory — 10— another*,
it should be relatively consistent within a laboratory and roughly
comparable from one laboratory to another. Triplicate plates— of
each strain with and without activation were prepared for each day .
of analyses. A discussion of spontaneous revertant values for thia.
$tudy ' follows this section, •
t. one plate of each test strain was prepared to test growth on
nutrient agar. This verifies the ^lability of each culture.
' c. The test strains all carry a deletion that covers- genes— involved- -in
the synthesis of the vitamin biotin and the am1.no acid histidine, so
l^hbt the organisms cannot synthesize these- compounds arid" "require-
them in the medium. One plate of each test "strain was prepared to
test growth on minimal medium without histidine and biocin. Only
the wild type LT2 strain should grow on this medium.
d. all solutions, were tested for sterility on ONA- plates. These-
included .the. tQp_agar^_the_^op^agazuHith- -histidine- and biotin, DKSO-,
. nutrient- agar, minimal medium agar, phosphate buffer^. -S-9-.reactixm~
xture, salt, solution, d^ttilled water, and standard solutions.
e. deep -rough (rfa) character - all of the strains have a rfa, or deep
•rough, mutation that eliminates -the-polysacchar±de"~side~chain~of~ the
lipopolysaccharide coating of- the bacterial surface,- -resulting in-
bacterial strains that are more permeable to test chemicals. To
-•—test for the presence of the rfa mutation, a crystal violet-
-Impregnated "fUter dink is placed on a nutrient agar plate which
already contains a top" agar culture, one plate for each strain. All
strains, except the LT2 wild type, should exhibit a zone of. growth
inhibition around the crystal violet disc, since the' •molecules )f
-the compound are_too. large to-enter-the— LT2 coat-.
~t. R factor - TA98 and TA100 contain an ampicillin-resistant
-------
Each of these aforementioned steps was taken each day Oat analyses wera
performed-; In addition, other quality control procedures were required.
These were:
a. extraction blanks - when a group of samples is prepared for
extraction, a bisnk consisting of 2 1 of distilled, ieiir-ized water
is extracted concurrently. Preliminary investigations for this
, - study have indicated that: this blank should not provoke a positive
response in the test.
b. extraction spikes - standard solutions c.; each of two mutagens is
1 added to two respective 2 1 porticns of distilled, deionized vater.
One mutagen is a compound Active withe ut metabolic activation, wblLi
the second is active upon activation with the. S-) liver preparation,
These spikes are extracted concurrently vith each group of sample
• - extractions. Initially, MNNG vas used as the uuactivated compound,
.'although this vas changed to MMS (varyls? concentrations froia 100 ul
'of a 10 ul/ml solution to 100 nl of HltS in a 2 1 blanic)-, when a.
problem was perceived in the analytical detection of the UHH6 in tb*
Ames test following extraction. The activated compound wa.« BaP
~ (varying concentrations -froa IOC to 500 ul of" a"~IOOO~ ug/id.
I solution).
'c. positive controls - positive controls ar? used to cest caa-^acterial
strains for autagenic propertied. This test wist ba performed each
time the strains are used. The same compounds used a? extraction
spikes were used as positive controls.
d. . duplicates - two samples, one «ach during the Pba&* I" u-TJf-
analyses, were split. Equivalent aliquots were extracts? concen-
trated and assayed at the' O'Brien & Gere laboratory iad~that c'~.
EPA— Region -II Envirosaienca!- Services— Division, -Edison,— Hew-Jersey—
Sample portions were transported to- the EPA in ref^igaraced
containero. A comparison of the split samples is _jtre&ent£d _ is
Section 7 (Results).
Spontaneous Revertac .a • " .
Spontaneous reversion rates are affected by the environment in tshich thu
strains are grown. Routine factors such as differences in brand and -batch
composition of media, atmospheric contaminants and -brands of
-------
lABU II. COWAKISOH Of SPONTANEOUS «VMS I OH RA1U fOi AACTCRIAL 1EST
T«»t Strain
1AM
IAIOO
,,11535
IA1S37
TAIS3*
• i. . •
1
21
65
12
*
C
1
,„ - . |
Ik 1
.
1 - i i
i i S iv v
1
JO-50 «j ' «} 13
1JO-100 Jl» 1M tit
| 10-35 21
•J-1S 11
> ' M IK
) S H 7
1
t
vi vii
W 15-75
17* $0 330
11 5-50
12 3-25
15-35 )j) > S1 S 2* 5-.
II - AM*. HcCtnn. «nd YiMftM, 19Ji (i).
Ill - CPA, Edltmi 7-27-tt. !
IV - (PA. Editon; i-6-t). | :
V - CPA ciiiturw. O'UrUn i C*r* l«bor«tor>. .
V: - IPA culiur.i, O'Brlon | Or. l.boritory. 120\ tlotlei-hUttdliw.
VII - tfc S«rri» .nd Slvilby. m» (15).
-------
of aucli revertants compiled from historical performance in the O'Brien & Gere
•Laboratory and art based on a minimum of 30 replicates. Based on -these
values, the revertant. rates -used in this--study -may have-been- somewhat low,
although they did exhibit internal consistency. For this reason, the cultures
vere used for all Phase I analyses.
As a result of these values,-however, the potential may have existed for
srme loss of sensitivity in the analytical results. Therefore, a second phase
of the study was initiated at the request of. the project- officer, -Samples-
were collected from two drainage areas which had exhibited detectable
-responses in- Phase- I analyals. "Copies of che five Salmonella tester strains
were obtained from the EPA Region II Environmental Services Laboratory at
Edison, NJ. Spontaneous revertant-values obtained from these .cultures by~EPA-
Edison are presented in Columns III and IV of Table 11, while Column V repre-
sents values obtained for these cultures in the O'Brien & Gere laboratory-.
Since the Column V valued were lower than the EPA values, the concentrations
of the histidine and biotin solutions were increased to 120Z, at the sug-
gestion of the EPA-Edison lab,-on the basis that autoclave sterilization night
be causing a breakdown of one of the two components. The - resultant spon-
taneous revertant values (Column_7I) .are-more.-comparable -to—those-=obtained-afr
EPA-Edison.
' de Serres and Shelby (15) reported the spontaneous revertant- values-
obtained from eight laboratories (Column VII). Based-on these values, the
spontaneous revertant values for the tester strains used in Phase I, while in
the low range, all are within a range considered acceptable. The authors
caution that^although. the reported- -range is—wide, the range—for-^any=tester-
strain- in one laboratory is narrower and consistent; these cr£te££a~sh~ou£<3r
govern acceptability of spontaneous revertant values.
45
-------
SECTION 7
-RESULTS-
CHEMICAL ANALYSES
Tables 12 through 14 summarize the chemical data obtained during this
study. A detailed tabulation of all chemical data is available in Appendix A.
Although the number of urban runoff samples collected in Syracuse was
limited to sixteen, a wide sample-to-sample variability is evident (Table 12).
It is also interesting to note that a comparison of the limited data from each
site finds values within the same approximate range. Rain analyses summarized
on the same table resulted in extremely low levels of the parameters measured^
including a low pH range. Additional pH measurements obtained"from precipita-
tion in Syracuse (O'Brien & Gere Engineers, Inc., unpublished -data^ indicates
that this low range of pH values is not .unusual* indicating a local acid
precipitation problem. * " "•
• Results of the chemical analy8is_ftf_GSO_CEable ..13-)—AncUsandAasy^sewage-
samples (Table 14) collected in this study -are comparable to—those—found- in
previous studies performed in Syracuse (48).. * Table 13 contains -data-obtained
from the chemical analysis of CSO during this study from samples, collected
concurrently with those for mutagenicity, as well as data obtained from the
same- sites during- CSQ-characterization- studies of 1976—1-977--{-in—parentheses-)-.
In urban runoff, BOD values were generally lower than those in CSO and sani-
tary waste, TSS was higher than in either CSO or sanitary -samples^,- and PB
concentrations were higher than in either of the other sample sources. OSG
values were generally higher in samples of sanitary waste, than in CSO or
runoff samples. •" ' : -"-'—
-K3TAGENICITY TESTING - - . ,. _ -• '- -
.Phase-I-
Criteria used in this study for the determination of a significant level
of mutageaicity were recommended by the EPA ,(19,65). The determination of
significance for each strain is based on the formula
MAR - E-CT
c
where
MAS. « mutagenis activity ratio
E « the number of induced revertants
46
-------
TAttU II. SUMMARY* Of OUMICAL A*M.ls£S fM URBAN BUNOff AW Ml)!, \fttiMt In Bg/l UnU»>
Otb.r.1 •• Noted.
1 , i ,
1 i
su* pit *
Huabor (Ungt .609. TOC
4 6.1-7.2 11 H
19 C.I-*.! It 90
27 «.2-«.9 i 14 51
37 6,8-9.0 JO 17
4J 7.0-7.9 40 11
4f* 7.3 20 4»
All6 " 6.1-6,9 16*17 76*17
Rachaiur- . i
Sjt. 7.1 32 29
k.ln 1.8-5,5 } 4
t
t
—~, U- ' — r^ ' r
1
t 1
iss CD at ps ! c
t
170 o.oi o.oi o.ai o
ii
44
750 O.OI 0,01 0.74 0.16
213 H *— ' : 1 —
• Oftly OM •M*fll<
' * ittndtrd dtvU
iv»l|nlil«
'r
-------
IMU 13. SUM/TO OF OUMICAL ANALYSES H* CSO SAHPU-S*. VllMt U (g/l IAtUi» OUwrafM NoUrf.
00
sit.
*
'»
27
37
43
46
« .
' All"
Viihlngton,
DC (All)
bd».t.r
•> Rutt*
«.5-7.3
(6.I-7.S)
6.8-9.1
3.7-7.5
(5.5-7.4)
6.4-7.1
(7.0-7.6)
7.0-7.2
(£.0-10.2)
7.4" I
(6.9-7.3)
1
3.7-9.1
6.5-6.«
7.1
BOD
SO
(62)
(-)
32
(«»)
116
02)
145
(107)
1»
(151) '
78*61
37
6
IOC
71
(121)
71
40
(128)
59
(18)
,162
(ill)
65
(161) i
1
44137
35
"
123
(530)
39}
143
(«0)
163
(5S)
355
(589)
1070
(444)
2751262
77
*
CO
<0.0(
(•)
•
-------
10
IM11E 1». MMMA8Y OF OlOtlCAL MWIYSES FOR DRY KEAIICft SAHIIA8Y SAMtfS. V.lu..
Othor.lio hoUd
Unl*>»
Sit.
Nu»b«r
»
19
n
IV
*)
t
«*
#'|fc
pit
5.)-7.5
C.4-I.I
7.4-7.7
6.t-7.0
•v
6.8-7 *
£.»
5.1-8.^ '
BCD. fOE IS9 CO
•*
III 111 K <0.0t
1)7 7C ft) 0.01
M 10
322 US
1)7 S*
SO 22
t
ujiuo tiisa
-
i6 l«.
- * lUn-Jtrd d«vi«tlaa.
-------
C ** the number of spontaneous revertants on
the -day that Che E revertants were
induced
j c » the historical rate of spontaneous
, reversions in the testing laboratory.
MAR values were calculated for all samples, with all five test
without and with S-9 liver activation. These are tabulated and presented in
Appendix B. A negative MAR results_when-E-is— less— than— C— for— a~ given— test
strain on a particular day; a zero value is obtained when E equals C, .and a
positive value results from E greater than C., although this-positive- value-may-
only be a fraction if E is only slightly greater than C. A sample is con-
sidered positive if it has a MAR of 2.5 or greater.
15 summarizes only those samples " and the particular strains (s)
with a MAR of 2.5 or greater. The MAR values presented are for 100 ul of a
200X sample concentrate (the highest concentration of any sample available -in —
this study and, therefore, the most likely sample dosage to . _induce-_mu-yigenic-
activity), with 500 ul" of S-9 reaction mixture and- 100 -nl-o£— the~apprtipriate-
test culture. Nineteen samples induced a detectable response in one or more
of -the test strains by this criterion.
Nine i of these nineteen samples which produced detectable responses were
of urban runoff, seven collected from Syracuse and two from Rochester, "NY", ~or
47Z of the runoff samples collected, while five of the^aamples-which-producejfc --
detectable responses (5 of 19, or 262X were- CSO^ -three- from -Ononda^a—Courtty
and two from Washington, B.C. . __^ ___ . _ - -•< — -
-Nin'e-of the—various samples listed in Table 15 as inducing ^ positive K&R
.were ..imtagen-ic -in— EA-1538— yet-~not- in" the more sensitive counterpart, TA9S.
There nave- been, indications that this is not art unusual test result^ that
TA1538"may show greater- sensitivity than TA98~with-some -chemicalar£I5}~.
No MAR of 2.5 or greater was obtained from the analysis of CSO from sites
4 and 46; sanitary wastewater from sites 37, 43 1 and 46; urban -runoff 'from
sites 27 and 46; influent -to the Metropolitan Treatment Plant; or CSO samples
from Rochester, N.Y. . .. • . . - •
Split Sample Analyses "
• During ?hanp. I-of—the study, a—sufficient— volume o"f~~OBG sample number
95864 Syracuse CSO from site 004, was collected so that one gallon could be
forwarded to the laboratory of the USEPA Region II Environmental Services
Division in Edison, NJ for concurrent analysis. The laboratory proce'dure used
is summarized in Appendix C.
50
-------
I»U IS. 5UH1WY IA8U Sf RUSC I HUIAGBUCIIV KSUUS,
' 1 I
K/W° HAS HMt MA* tMR
O'Brien IP CODE , TASO Ml 00 IA15JI TAIS37 UtSJO
*G«r« Htthout Wtb Without Blth Sfltlwut Wth Mlthout Mtti Wthout kith
»k«t.r City Siwre* Kxutloo 0>serl>tor . S-9 '$-» 5-» S-S S-» S-9 S-9 , S-9 S-S S-9
»*»92
953«,
9i3ia
15475
SS476
S5A77
9573S
MW
957*5
f587l
S5073
95*7*
9536S
M76S
9*76fi
attfij
9*66}
19
tl
n
4
19
27
4
J7
17
17
4}
4 2
„
24
34
7
7
1.9 2.8
f
».l
2.7
3.S
II .2 i.l
3.1 ' 4.1 2.6 1.4 2.5
1.0 l.C " ^.0 4.t
1.7
» 2.»
t.l 2.1
10.0
• ' • ' , . ' "t.l
2.6
!' 4.4
lt.2 _ ' 4.2
4.S
' , J.e
i : , .— . . . . . . 1 ,
MM - nuUgmlo tctlvlty r«tl»
-------
Tne Ames test was performed by the EPA on the first sample aliquot using
the Regional extraction procedure (57) "(Appendix C). The test was'conducted,
with, and without metabolic activation, using five volumes; of extract (1, 10,
100, 250, 500 ul), each in duplicate. The results of the test indicated a
positive nutagenic response TA1537 (pH 12 extract without metabolic activation
at 500 ul (MAR » 4.8). The experiment was repeated using six volumes of
extract (iQQ, 200, 300, 400, 500, 600, ul),"each in duplicate. The results of
this test indicated a .positive _mutagenic_response— with—the—bacterial—t-este-r-
straina TA1537 and TA1538 (pH 12 extract) without metabolic activation at
600 ul (TA1537, MAR - 2.9; TA1538, MAR.- 5.0).-
A second aliquot of the sample was extracted on the seventh day after
receipt, using the EPA Region V procedure (57). However, the Region V
criteria specifies that the sample should be extracted within the seven days
following collection. The test was conducted/ with and without metabolic
activation/ using five volumes of extract (100, 200, 300, 400, 500 ul), each
in duplicate. The results of the test indicated a negative mutagenic response
with the five bacterial tester strains. Possibly the results differed from
those obtained with the Region II criteria because the sample was held over
the recommended storage period.
The second aliquot was assayed at the O'Brien & Gere Laboratory -using the
analytical procedure discussed earlier. The test was conducted with and
without metabolic activation, using 100 ul of extract at three concentrations
(2X, 20X, 200X), each in duplicate. The results-of the. test-we're-negative for
all five bacterial tester «tra-fT|gT f>»»_B^mp<-Qnclegion -gs^-Chst^oJ—<±g=-EPA
laboratory. " -. " ,
Phase II
..During- Phase IX, the verification- phase of the—study, -a—similar split
sample was collected - O'Brien & Gere Engineers, Inc. (05G) sample number
29031, "wet weather influent to Metro, The EPA laboratory conducted -the
analysis by the Region V procedure as described in the preceding paragraphs
but only at ambient pH. A detectable response was found for .the test, strain
TA1538 without metabolic activation at 500 ul (MAS. -" 2.9)'- Bepetitioa-of the
experiment resulted in an apparent increased response as follows:
Volume (ul) .MAR
-400 -25.-S" '
450 • 36.6 "
~ ' 500 41.9
550 -7.2
600 , -
No explanation for this apparent increase in mutagenicity is readily evident.
52
-------
For the same sample, an MAR of 6.1 was obtained fir TA1538 with 500 ml of
the extract, and without metabolic activation. Open repetition-o£ the. test,
an H4R of 2.4 was found from the application of 500 ul of sample. 'iTierefo^e,
initially the--results- of the O'Brien •& Gere laboratory did indicate a
detect&bl'e mutagenic response in the split sample, as did the EPA lab;
however, this rasponse could not be confirmed, while tha± found by EPA-Edison
was. The initial responses obtained by both O'Brien & Gere (MAR - 6.1) and
that of EEA. (MAR » 2.9) from 500 ul/plate applied dosage are of similar
.magnitude.
-------
SECTION 8
DISCUSSION
INTERPRETATION OF RESULTS
To evaluate the data produced during this study, the results must be
compared with the mutagenic6" responses of known nutagens or carcinogens. An
Inference of relative risk can then be made for the test sample. Unfor-
tunately, such a comparison with other data reported In the literature, is
often not possible. Results may be reported in several forms:
/
(a) as revertants induced by test samples;
(b) as revertants Induced by test samples, less* the -spontaneous
' revertants for each bacterial test strain;
(c) with the dosage of test sample/plate-as umolas/plate.or_mg/-plate;-or
,»
(d) as raw analytical data, or as a calculated sample/ controX ratio.
It has been recommended that all journal articles . contain
revertant values In reports of the results of —studies- (15-)-. -Th±c — msy~be~
impractical where publishing costs are critical and page space is-atr— ar-
premium. The use of > Che mutagenic activity ratio (MAR) offers ja— means— of —
normalizing laboratory-to-laboratory variation in spontaneous revision levels-
while providing a stable index with which to compare results*
Phase I
The results of all applied dosages for samples which elicited ja.
detectable MAR (equal to or greater than 2.5) are presented in -.lsble_16v-
Three concentrations of 2X, 20X and 200X were applied at a dosage -of 100
ul/plate at -each- concentration. Nine- of the nineteen—samples -which— .'reduced —
-detectable responses (a positive MAS.) (Tatle 16) did so across the range .of.
all three concentrations; sis of these responses resulted from metabolic
activation. Four of these nine samples produced an MAR of 2.5 or greater- for
all three concentrations; these four responses are presented in Figures 12-15.
Tha four curves produced from the responses of these samples are generally
horizontal with little slope, "indicating that Jiese, samples did not exhibit an
increase in the mutagenic. response with an increase in the amount cf sample
applied. Generally, a response which increases with dosage or concentration
would be expected; however, a response such SB that exhibited by these- -samples-
is not unusual. The remaining five responses are presented in Figures 16-20.
Only sample number 95871 (Figure 20) exhibit*/ a logarithmic response curve.
54
-------
TABLL ib. SUMMARY OF DETECTABLE RESPONSES, PHASE I ANALYSES.
Sample
Number
i
94492
94492
95366
55368
-95475
9b476
-95477
95477
9449 \
94491
94491
94491
94491
95739
95739
95739
95739
94493
94497
-95745
95871
95871 •
95873
95474
95369
.94765
-54766
94766
94662
94663
Organism
TA98
TA100
TA1538
TA1538
TA1538
TA1538
TA100
TA1538
TA98
TA1537
TA1537
TA1538
TA1538
TA98
TA98
TA1537
TA1537
TA1535
TA98
TA1538
, TA100 -
. TA100
TA1538 -
TA1538 •
TA1538
TA1538
TA100
TA1537
TA1538
TA1538
S-9 Keaction
Mixture (ul)
500
—
500
500
500
- 500
500
500
500
.
'jOO
-
500
-
500
-
500
-
-
500
-
r.,m.
" -500--.
500
500
-
-
-
-
"
h
200X"
2.9
2.8
7.2 *
~9n
2.7
3.9
• 11.2
5.1
3.2
4.1
2.6
. 3.4
2.5
3.0
2.6
4,0
478
•"2.6 '
2.7
2.9 "
£8..
29.
10,
6.1
2.6
4.4
16...2.
4.2
4.5
3.8
nD
20X
0-.5
-ft. 3-
3:2
~670~
2.8-
4.0-
0.1
4,4>
0.2
.2.5
0.9
2.8
0.7
1.1
0.6
3.2
TT9~
-0.1
0.3
-(T.7
12.
J-J
.2.1
"5.6
1.3
"0.8
-0.5
-0.5
0.5
0.6
27
_
-
1.2
—273-
3.4
4.0
_
5.4,
-
-0.7
0.2
-
_
-
n.j8
-
_
.
-
1.7
w
-
-6.5-
.
-
~
_
•• -
. ^
-b-
- MAR = Mutagenic Activity Ratio
- concentration factor
55
-------
M.O-
12.0-
10.0-
8.0-
20-
Sample Number -95474
Organism - TAI530
r
zx
i 20X
EXTRACT
1 ill
i ! '
|riijure J2. Response tur^ fpj" Safn|)le Dumber
200X
-------
in
140-
120-
10.0-
80-
6.0-
40-
20'
2X
t
Sunipi* Numbor- 96475
0/snnism - TAI038
20X
II , w I,
EXTRACT coNC-rtMfflATioN
i ,
13. R6spc}>?k ruirye for fajnple Dumber 954^5.
200X
-------
in
CO
14.0-
12.0-
10.0-
QO-
6.0-
4.0-
2.0-
2X
Sompta Numlw - 9547J5
OffltuUim - TAI536
EXTRACt
20X
e 141 'Respon
fo^ Sample NfJiiJbe^ 95476,
200X
-------
in
10
14.0
120-
Sonr^te Number-95477
- TAI538
III
EXTRACT CONCENTRATION '
I
I I >
Figure 15. Response Cu'rve fo»i Sample Number 9^447,
eoox
-------
Ol
o
MO-
12.0-
100-
8.0-
60-
40-
2.0-
2K
t 'I
Figure 16. Response
Number -
- TAI538
I
20X '
CONCENTRATION
CUrve for Cample Number 94491.
IMP
200X
-------
140-
120-
IOX)-
8.0-
6.0-
2.0-
ni
-Ssanpto Number - 95366
- TAI530
2X
20X
f I
Figure ll. f:esfapns4 tur^e fcjf'Sample Number 95331$.
ioox
-------
O>
PO
KO-
12.0-
10.0-
8.0-
6.0-
4.0-
2.0-
2X
III
iili lit
Sompte Number-953&S
JCrflanlwn - TAI538
20X
EXTRACT CONCENTRATION
.Figure 18. Response Curve for Santple Number 95368j
! 1 i ! I
200X
-------
' * I
01
to
14.0-
12.0-
10.0-
8.0-
0.O-
4.0-
z.o-
Sompia Nufiibar ^-85739
Organism -r TAI537
20X
EXTl
' I
CONCTRATt^N
Figure 19. riespoii$e Cube for Sample Number 95739.
-------
70-O
12.0-
iao-
80-
6.O-
2.0-
Number - 95071
Organism - TAIOO
EXTRACT
mr*tj-r~*
(iONpENTRJVT
f
Figure 20. ^spoflste jfurve fojf^ Sample ^
-------
Response curves are presented in Appendix 0 for the other ten. -samples, .for
which a detectable response was observed in at least cne sample -concentration,.
Of the 30 detectable responses (responses from more than one test strain
for six of nineteen samples), ten. were from site OC4 (in three- samples), while-
four were from site 037 (also in three samples).- A more interesting observa-
tion is that -17 responses of the total of 30 (57%) were from 9 runoff samples-
(472 of the 19 samples from which detectable responses were obtained. CSO
samples produced 7 responses from 5 samples, 'while dry-weather sanitary-
samples elicited 4 responses from--3— samples,—-Therefore-,—urban—stormwater—
tunoff may be a contributor of potentially mutagenic substances to CSO and
receiving waters. However, since mutagenic activity is not. as-evident in the •
respective CSO samples as it is in the urban runoff, if is probable that the
concentrations of mutagenic substances in the runoff are diluted below the
detectable limits of this study as the runoff mixes with sanitary waste to
form CSO.
Phase II .
"For the verification phase of the study, an additional eleven samples
were collected in Syracuse in late summer of 1981. These samples included two
runoff and two CSO samples from both sites 004 and 037, two samples- of. .Metro-
influent, and a Tain sample. One gallon of each sample was extracted, with
the concentrated extract brought to a volume of 20 ml with DMSO, resulting in.
a concentrate of 189.25 times the original sample. Five dosages (500 ul, 400
ul, 300 ul, 200 ul, 100 ul) were applied. _ •
In two Instances, the samples did" elicit t detectable response. One
sample was CSO from site 004, while the other was Metro influent. The—CSO
sample (OBG No. 28524) had an MAR of 5.2 for tester strain1TA 1538-at-a-dosage -
-of 200 -ul;—higher—dosages resulted in a weaker response. The Metro influent
had an MAR of 6.1 for TA1538 at the highest applied dosage of 500 ul; lower
dosages.did not result in an-MAR-greater than-2.4.
Two samples produced toxieity: runoff sample site 004 (OBG No. 28835)
with -TA100 (no activation), Metro influent (OBG No. 28888) with TAlOO-
-------
TABLE 17. PHASE II SAMPLE VERIFICATION -.MARs.
OBG
Sample
Number
_2852-4_
28885
28888
S-9
Strain
TA1538~
TA100
TA100
TA100
Applied
Mix
- .
500 ul
Sample Dosage
500
-2-.2
• 1.4
' 1.6
1.3
(ul)
400
-1-.-8- -
1.2
1.5--
0.8
300
—079
1.3
1.4
0.5
29031 TA1538 - - '2.4 2.1 1.0
EXTSACTIONS
Tt "should be mentioned that the use of a liquid-liquid extraction and
subsequent concentration procedure for all samples in this study, ss recom-
mended in the EPA methodology) adds a bias to the results. The test extract
actually represents the DCM-extractable fraction, of the original—sample, a-
fraction which may contain a large portion- of the extractabie organic com-
pounds in the original environmental—saspl-s^—but— probsbiy-ncr£~233r-orgaictcET"
Other studies have used similar extraction procedures with concentration by
solvent reduction (36,51,61,63); resin -extraction' with solvent-desorption"and'
reduction also is used (10,22,56), a familiar technique in sample-clean-up for
organic chemical analysis. The choice of solvent fcr desorption and--the-
ability -of potential-ly unknown-mutagenic compounds in complex environmental
samples to be desorbed from resins are factors which m&y also bias results in
these procedures.
Both resin systems and filter sterilization (59) may preclude analysis of
organic mutagens adsorbed by particulates or absorbed to oil 'globules-. 'That
±s why a total sample disinfection/extraction/concentration procedure was used
{or this study in an attempt to retain as many potential organic mutagens as
possible -wicfain the limits of- -the DCM- procedure. However-,—many" potential
inorganic—mutagens, including heavy metals, were excluded by this procedure.
Other concentration, procedures utilized in other studies have included low
temperature, low pressure distillation (25) and diaZ.ysis (61).
In any system, the choice of solvent may be critical f or-another-reasoir:
some kaown mutagens have been found to lose considerable activity with time
spent in a solvent. It is unknown whether this is due to direct
:iutagen-solvent •'.nceractions or some other cause.
66
-------
I . .
FALSE POSITIVE AND FALSE NEGATIVE RESULTS
4
It .cannot- -be readily- determined whether- any results obtained" in this
study may be false positive or negative responses due to the implicit limita-
tions of the test.
Derailed discussions of factors influencing *ke Ames test to produce
false positive or false negative results are available elsewhere
(2,5,38,39,55). Several possible explanations exist for false positive
results in the Ames test which- have significance for highly -compiex-environ-
mental samples and, therefore, at least deserve mention here:
a. th«» bacterial tester strains contain enzymes capable of metabolizing
-the chemicals to be esced to active mutagenic forms, in- the- same
manner that the S-9 -it liver microsomal reaction mixture metabo-
! lizes pre-carcinogens to their active form. This unanticipated
! bacterial activation could generate, false positives-if—these—bac-
/ terial enzymes are not functional in mammals. .
b. the bacterial fester strains contain & mutation that deletes- the—
/ excision .repair system of th<: bacteria." -McGanfl—and-Amc3~C39)~~bave
I ' described the result of this as to ""quantitatively enhance the
sensitivity of the system rather than tc qualitatively alcer the
' . i response".
e. substances whici hava low mutagenic activity have been found-not to^
'be carcinogenic upon further testing in animal systems. This may be
-due—more— to—the— statistical- limitations of"~the animaT^testa~v5£cn
. - -make detection- of low activity diificult than to any iimitatiocr to
-the-Aaea—test?—Iorfact7~chi's'^sensitivity biT the Amea. .test majc make
it .particularly naeful for detecting low mutagettic activity "in
environmental jarjlee that would otherwise be-dlfficult—to^-det-ect-j—
By contract, the problem of false negatives may te of more concern .no.
-this-testr: . . .
a* tozicity of the test substance may cause bacterial cell death. This
*• ' toxicity may be specific for any. or all of the test strairs.
Generally_, the expression of. mutagenesis occurs at.levels below that
of toxicity. -Howevar,- the toxicity of the test substance-may^-not- be-
related to excessive DNA damage 01 mutagenic properties^.-but- -rather
to other rytotoxic properties (such as these of antibiotics).
T>~. technical inadequacies in the rat liver microsome activation- systear
may prevent detection of substances which require in vitro metabolic
activation. The rat liver system may not contain -the. -cofactors ior
activation of certain substances in the environmental -sample? this
has been" found-to be tiue of some pure compottnds.
c. test substances n?r -»-.dargo deactivation, either by biochemical'
compounds such as v/t«--«-;jae (58) and L-aacorbate .(Z&), or by the S-9
microsomal fraction (14;.
,.
67
-------
d. .although the tester strains have been made more sensitive to the
transport of test substances into the cell, there is'a molecular
size limitation to substances which come into contact with -the test .
,- organism. Since the test substance must enter the bacterial cell to
reach and alter the bacterial DNA, sufficiently large molecules are
' being excluded from the test. Should these large mole-vales be
• . . mut3.genic.j- they will not be-detected-, , -
—Any—of "these factors, or a combination of two or more, may be another
reason for very low or negative responses of the samples collected in this
study. Within these limitations, several general observations can be made as
a result of this study:
•^ f,
. . a." due to the drainage characteristics of the project area, and the
necessity of selecting sample collection sites which would provide
safe access with reasonable ease, industrial, commercial or mixed
land use characterized most of the sites. Ibis is not unusual since
./ / . / most e£. the- catchment areas~in-the~project area are characterized as
. r-uch. Diliiculties were encountered with sample collection from the
.'••', / ' one predominantly open space catchment area selected.
b. detectable responses were obtained with one or sore cf the test
: strains from nineteen samples. "cme of these samples -required
concentration up :o two hundred-fold to achieve this result^-
• ' > * °
• ' c, of 30' detectable responses in. 19 samples, 17 response's tSTJT were
from ? runoff samples. However, the substances responsible for_:thic-
..'_'. response may beco e diluted -or inactivated below the -detection.
« threshhoxd of the test. .by ^mixing- -with - sanitary -sewage—to—form
combined sewage.
t'
In-spite of these observations, i.i should be recognized that a sufficient
data base of Ames test results had not been generated by this study to reach
conclusions affecting policy decisions, standards or guidelines regarding_the
necessity of the advanced treatment of CSO or urban runoff. This study should
be conoidered indicative of the aeed for more comprehensive surveys of its
type if biological effects tests of thic, type are considered useful in the.
decisifn-making process. , - .."_.."'
68
-------
SECTION 9
SUMMARY
The only known causes of human cancer besides tne natural modification of
human genetic factors are radiation and chemicsls, eitbar natural (such as
aflatoxin) or synthetic. The relationship between unit age-trier and carcinogenic
activity of substances is not fully understood although sufficient empirical
evidence is available to indicate thac -many mutagens are -carcinogens (7,31,
39,4(6).
The introduction of potential nnitagens to the human.^^iivi.ronir.ent may serve
t£ increase the rate of contact with substances that contribute to cancer
incidence in the general population. The primary purpose of this study was
tne evaluation of mutagens in the sanitary environment, including— rain, urban
-rmoff, sanitary wastewater, combined se'~er overflows, sewage treatment plant
influent and offluent, and receiving waters. The study was limited in score
•so ":hat the results, if positive, may be indicative of mutagenic 'activity from
a particular source and its life cycle through the system*..
At present, the" U.S. Rny< rr>nfliPTxt-aT
extent to which it may be necessary to-, provide treatment --for— combined- sewer
-.overflows. The potential presence of a mutagenic component in-CSO-necessttates
a further reviow of the necessity of some form of advanced waste—treatment.
This study, conducted on samples collected -primarily— in— Sy-racu-re-p- NY-irr
conjunction with present, system evaluations and improvements, found clear,
strong mutagenic activity in several samples when analyzed -by the. -Ames test;
data was interpreted under criteria recommended by the EPA. The MAR (equal
•to or greater than 2.5) rule for assessment of mucagenecity is conservative.
and several samples exceeded this "value (2.5) -substantially indicating likeli-
-hood of dose response. Nineteen samples elicited a detectable response to one
or more of the five Salmonella typhimurium test strains,- (47%}— wet=e-of urban
runoff in the project area, including 17 of a total of 30 detectable responses
{MAR-equal to or greater than 2."5 ) T57%). Five of the_s.amp.les-.(26%0~were— f-rom
combined sewer overflows, consticuting 7-.- of the 30 detectable responses (23%).
However, resul-ts- indicate that detectable mutagenic activity may be present,
parcicularly in samples of urban runoff. Since the effects of chronic exposure
to low level carcinogens is not well know'n (the identification of threshhold
le.veJ.s- and their significance-), the- criteria for the interpretation and defini-
tion of low level mutagens is still -controversial, and- the concept of a re-
lationship of mutagenic activity in the Aires test to long teem animal carcino-
genicity tasting still draws positive and negative proponents, a more extensive
survey of this type should be considered.-"
69
-------
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7\
-------
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74
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75
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APPENDIX A
This appendix contains -the computer tabulation of all physical and
chemical data obtained for the samples collected in this study. Except for
pH, values listed are in mg/1. The key to. Jthe-computer ident±iica-tion-^brmat:
may "be found in Table 9.
76
-------
I Sift
INC.
LAeO«*rO*» DATA JtSTtH
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O'HUIrn i Ctat
INC.
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7-9
-------
APPErJDIX B
. The foilowins table contains MAR values for all samples. These- values
were obtained at a dosage of 100 ul of sample at a concentration of 2COX.
Values' at less concentrated applications are presented in Table 16 for those
samples vith an MAE equal to or greater than £.5 for one or more strains.
80
-------
Of)G
Sample
Number
94490
94491
94492
94493
94494
94495,
HAS |
Without HI
S9 S
TA98
t|i Without
4 S9
TAlOO
.0 , -O.Z 0.6
.2 3.2 1.2
.2 2.9 2.8
.9 0.5 1.3
.0 2.1 1.0
.C 0.9 1.3
94496 ,0 0,2 0.5
94497
94498
94499
>4255
94256
94182
94660
94500
94501
94G61
94652
91o63
9-J65
94766
2.7 0.8 1.0
0.9
1.6
1.0 ,-1.2
1.6 0.2
-0.5 -0.3 0,1
0
0.1
0 -tl.3
0 0
0 , -0.'2 ' 0,1
1,0
j,7
0.2
0.2
0.8
0.7
0
).8 -0.1
.0 ' 0.2
).7 , -0.5
I/O -0.1
1.0 0.2
i.S • - 0
1.6 16.2
With
S9
-0.2
0.2
0,1
0,3
0
0
-0.1
0.1
-0,2
0.2
0.1
-0.1
0
-0.1
0.9
0.4
0.2
0.8
0.3
0.4
b,
_. .
1 HA,
Without
i S!>tA1
[
1
1
j!
0
I
5
•c
C
(
0
.4
.6
.4
.6
.0
.2
.1
.7
.2
.4
.5
.3
.1
.3
0.2
0.7
-0.4
-0.3
0.5
0,2
t
! I*,
IT "!
h* '
o.?
o'.?r
0.2
-0.2
0.1
0.2
,-0.3
' 0
-0.5
-O.H
-0.3
0.3 '
0.1
0.3
0,2
0
-0»5
., 0,4
0.6
,0.7 I
ll,
HAR
lUthout Kith
S9 S9
TA1537
0.4
4.1
0.5
I.I
0,7
0.4
0,8
0.6
0.1
-1.3
-0.3
-0.9!
-0.2
G.2
0.5
2.4
-0.5
0.2
1.0
1.6
4.2
f
o.a
2.6
1.1
0.3
, 0.9
. -0.2
-0.7
0
0.1
0.1
0.2
-0.2
-0.1
0.9
1.9
1.8
0.6
1.4
2.3
1.3
I y
Without With
S9 S9
TA1S33
i
0.2
3.4
2.2
I.I
0.9
1.6
0 9
1.6
-0.3
.0
0.4
b
0.6
'1.3
-0.3
0.7
>1.0
4.5
3.9
4.4 '.
i<>;
0
• 2 5
1.6
0.5
2.3
1.2
-0.1
0.1
-0.1
0
0.5
b
0,6
1.3
1*9
1.2
0.7
0.7
l.J
0.5
b
-------
co
COG
Sample
(lurfccr
94847
95364
95365
S53C6
.3
0
0.1
0
0
0
0.2
-O.I
0.1
0.1
-0.1
b
0.1
-0.2
4
0
HAS i '
Without With
1 S9 59
TAI535 '
-1.2
-0.4
-0.3
-0.5
0.2
-0,3
-0.2
0.2
-0.4
0.2
0.6
0.5
-0.2
rO.2
1-0.2
0.2
0,2
•10.5
b
-0.6
0.3
-0.9
*
1.5
b
b
b
0
b
1
Ojl
-0.7
-0.1
-0.7
0.2
0.2
-0.3
-0.1
-0.3
-o.fc
-0.5
0.5
0,1
u
0.1
' 0.2
0 •.
-0.3'
b
0.2
-0.8
• -0.5
-0.1
0.8
b
-0.8
> b
-0.8
b
i!
Without Witt? Without Mlth
59 S9 59 S9
TA1537 7A1538
O.S 0
-0.5
-0.2 -6.4 0.2
0.2 0
0.8
a 0.6 0
O.S -0.3 ,F0
« o.:
1 0
0.8 0.3 0
a 0.3 •
b -l.Z b
1.2 -0.6 -0.3
1.0 OjJ
-0.8
4.0 4.'8 1.3
0.5 . ' -fll3 -0.3
-0.8 -0.4
1.5 -0.1
1.$ 0
. 0.5 -OJ
*
•
-0.3
' 0
0 -0.3 1.0
-0.5 b
-0.5 -0.3
0.2 O.J
a
0.3
0.3
-0.1 -0.4 0.3
« -0.2 a
0.2 -O.J
b b
tt -i.e
0.8 -1.'
-0.5 -i.;
1.5 -O.E
1.3
b
b
b
-0.2
b
0.6
1.1
1.9
7.2
2.2
9.1
2.6
1.5
-1.1
0.2
0.1
2.0
6.1
2.7
3.9
S.I
-3.5
-2.9
-3.2
-2.1
-2.3
-2.4
-3.0
2.0
b
b
b
-1.0
2.9
-------
HAR hAft MAR HAH
OBG
Sample
Hunter
?6253
95746
95740
95477
95815
95816
S5813
95814
96871
95870
95872
95869
95873
95817
95818
95822
95023
95865
95666
95067
95868
' - . to*
Hlthou't
S9'
With
S9 *
TA98
-0.8
-1.2
-1.2
-1.0
-1.0
-0 6
b
b
-0.3
-0.8
-0.8
-0.3
-0.4
hi
0.4
0
0.1
0.9
0,1
b
-0.7
-0.6
1-0.8 '
-1.0
rO-6 ,
a
•|0,4
-0.3 ,
b
-0.1 *
-0.3
-0.6
-I). 2
-0.7 •
0.3
0
0.3
-O.S
O.G
1.0
b'
0
•
I !
Without
59
TA100
-0.3
a
-0.5
b
a
-0.1
b
-0-2
68.
b
b
1.0
b
0.2
-0.2
0.3
0.5
0.7
0
0
0
! '
with
S9
Without With Without
S9 S9 S9
Hlth
S9
' TA1535 TA1537
' -
0.3
U.I
0.4
11.2
0.2
0.3
0.2
0.4
29
b
b
b
0.1
0
-0.1
0.2
0
0.5
-0.3 ,
-0.2
0.2
i
i
i i
Icily of raw ionceotrate (200X sample;
i
t
a 0,6 0.2
b 0 a
i -p. 5 -0.5
b -0.5, a
a ; a -0.8
b i a -1.2
-0.1 * b 2.0
b
h
-Oi6
-0.5
-0.6 .'
b
l.G
1.3
0.3
0.3
1.0
-0.4
0.3
-0.8
... .1
t '
ss appll
0.1 -0.3
0.3 -1.2
0 0
0.6 0.0
0,9 -0.5
b -0.4
0.8 b
0.2 0.5
-0.1 -0,8 .
I.O . 0,9
0.0 . -0.2
0.2 O.S
0.6 -0.8
0,1 -0.2
. , i ,.
1
(top ul); Icslser
0
b
-0,6
0.4
-1.0
-0.4
-0.9
-1.1
-0.3
-1.0
b
0,3
-0.3
-0.6
-0.6
-0.1
0.4
0.1
-0.1
0.4
0
<
1
MAR
Without
59
HUH
s?
TA1538 '
2.2
b
b
b
a
-0.2
b
b
-0.3
0.2
0
' -0.3 '
0
0.8
o.e
0.8
0.8
1.3
a
-0.4
a
* *.
1
-0.3
b.
-0.3
a
a
••1
-oJ
bi
1.2
-0.9
-0.9
10.3
-0.2
0.1
-0.4
0.4
0.2
O.S
-0.7
a
, J
concentrations did r.ot
sampljp toxic at all concentrations;
laboratory error '
-------
APPENDIX C
The following two flow charts summarize the extraction and "sat pro-
cedures used by the EPA Region II Environmental fsrvices Laboratory (Edison,
NJ) for'split sample analysis.
84
-------
AMES TEST EXTRACTION PROCEDURE FOfl ENVIRONMENTAL
• . WATER SAMPLES
, I GALLON SAVFt.5
BASE-NEUTRAL EXTRACTION- ADjUST TO pH 12 '
PLACE EACH ALIQUOT INTX 2L SEPaftATCRY
. it-' "« ' '< !
AOO
'.v ®»*K£rOR2MNUTi3
REPEAT
•TWICE
SEPARATE SaVE.ff ^NO WATER .-HASES
" ' AQUEOUS PH*.!E
^ +
' ACJO EXTRACTION -ADJUST pH TO 2
PUCE E4CH AUQJJOT 1\TC
FUNNELS
TWICE
SE/MATE SOLVENT A« WATEZLPHASSS
J -TH ^
MUEOUS PHASE $O>EWPH.i,cS
OtSCARO AOONaSX^TO DRY
COMBM! AUQUTIS
3MtSK TO Iptfll
tOWMTO 8ml-
SCVtHT PHA3H
"-> «
* &Q KaSO^ TO CRT-
*--. *
COMBIKS i'UOt. .'S
- ., -DAN6H 1
, BUD WWN TO 8 at
-I "• '- e
/ REWVE 2m. «"v< -
8LCrt OOUTN REUWNtfJG Smt TO 0.2 ml.
BRING UP TO A VOLUMS WITH DMSO OT
CONCENTRATION Of CAI6INM. SAMPLE
• • .
COHCENTRATE * CO X
TO 0.2. ml
P3INP JP Tv/AVOU,MEWPT.
or ioox
COXCENTRA7S * ISO X
85
-------
EXTRACTION PROCEDURE FOR
ENVIRONMENTAL WATER SAMPLES
I GALLON SAMPLE
(PREPARE 4-1 LITER AUQUQTSJ
BASE-NEUTRAL 'EXTRACTION
{ADJUST TO PH 121
IPIACE EACH I L SAMPLE INTO 3 U SSPftRATORY FUNNELS]
"f ADD 30 Ml/t.
| MIX FOR 5 MINUTES )
REPEAT TWICE
SOLVENT AND AOUEOUS PHASE
ATI/SOUS PHASE
FOR ACID EXTRACTION
1
ACID EXTRACTION
[ 'ADJUST TO pH Z J
PLACE EACH SIMPLE INTO
2 L SEPARATORY FUNKEUS
ADOS MI/L
|MIX FOR 5'MINUTES| REPEAT TWICE
[SEPERA7E SOLVENT AND AQUEOUS PHASE]
AQUEOUS PHASE
I DISCARD I
I
SOLVENT PHASE
T ADD Ha504-, FILTER
-
ROTOEVAPOSATE AT 44 «C
UNDER VACUUM TO DRYNESS
RESUSPEND EXTRACTED
MATERIALS IN OMSO
I
.SOLVENT >HASE_.
ACO NaS04 FILTER
ROTOEAftPORATE AT 44» C
UNDER VACUUM TO DRYNESS
RESUSPEND EXTRACTED
MATERIALS IN OMSO
86
-------
APPENDIX D
Dose Response Curves for Samples Without
Detectable Mutagenicity - Phase I •
87
-------
00
00
Number-94492
Organism - TA98 „
J40
12.0-
IOO-
8O-
60-
4.0
2.0-
200X
-------
00
VD
I4.Q
12.0-
- Number -94492
Organism - TAIOO
w/o S-9
200X
EXTRACT CONCENTRATION
-------
140-
ia.d-
100-
e.o-
6.0-
4O-
20-
Sompb Nombw -95366
- TA1538
2X
EXTRACT
n !
CON;EMTRATION
200X
-------
J4.0-
12.0-
100-
60-
6.0-
4.0-
2.0-
Sdmplo Numbar- 95368
Organism - TAI538
2X
20X i
CONCENTRATION
200X
I
-------
vo
ts>
140-
120-
100
ijiiiii
8.0-
6.0-
4.0-
Z.O-
?X
Sampt* Number - 95475
Organism - TAI538
EOX
EXTRACT
t
200X
-------
140-
12 fl-
IC 0-
8£>-
60-
40-
2.0-
Sontptt (Jumber - 95476
Organism - TAI538
2X
20X
•RAQT CJONCENTRfVTION
-------
HO
12 O- i
IOO-
8.0
6O
4O- -
2.0- -• T
200X
-------
pompta Nu«*>er-S5477
Organism - TAS533
w/SOO d S-9
14.0
13.0-
tao
8.0-
6.0-
4.0
2.0-
200X
-------
140
I2.O-
Sampto Number ~ 9443)
Organiim - TA98
w/500 ul 'S-9
2X
200X
-------
»40-i lit mi-
120-
vo
•M
20OX
-------
vo
00
SampteNumtw- 94491
Organism-TA1337 J
w/500 ul S-9 i
14 O
120- ->•--
;oo J
a.o- -
6.O---
4.0-
20- ••--
-------
VO
VO
14.0
I2O-
10.0-
8.O-
60-
, 4.O-
2.0-
Sompto Numbar - 94491
Oroonism - TAI538
-------
o
o
14.0
12.0-J
10.0-
80-
60-
4.0-
20-1
Sample Number -94491
Orgaptam - TAI536
w/SOO ul S -9
, t pox
EXTRACT, CONCENTRATION
200X
-------
14 P
12.0 J
Sample Number-96739
- TA98
Wo S-9
?OOX
-------
Somplo Number -95739
Organism - TA98
-w/500
-------
140
12.0
Numbw -93739
--TAS537-
2.0-
2X
200X
-------
Sctnplji Numwr-95739
Organism - TAIS37
w/500 ul Sc
140
120
IOO-
9.O-
6.0- r
4.0- ---
2.0J
2OOX
-------
"f
14 On
120-
IOO-
80-
<
60-
40-
20-
Sample Nombr- 94493
Ofeanl»m
t*/o S-9
2X
I"
I i
|20X
EXTRACT COW
200X
-------
I4.Q
I2.O
Sample Number -9444
Organism- TA98
w/o S-9
2OOX
-------
140
O '
Sompln Humber-93745
Of ganlsm -TA1538
w/500 ul S-9
200X t
-------
i o
00
70.0
69.O-J
5;
120-
10.0-
eo-i
6O-
4.0-
' 0.0-
2X,-
I
Sampto Number-95371
OrflonUro-TAlOO
200X t
"I
-------
300
S«npl« Numbcf-93871
Of flonlwn - TAIOO
w/SOOul S-9
200X
-------
14.0
120-
Sonnpla Number-95873
Orgonlttn- TAI339
w/500 ul S-9
-------
14,0-
12.6-
IQ.O-
80-
6.O-
4.0-
2.0-
Sojrpte Numbar -9?474
Orpopbm - TAI538
2X
p '.
200X
-------
Number-9536
Organism* TAI538
w/500 ul S-9
'200X
-------
Sampte Number-94T65 [I
Organism- TAI538
w/b S-9
200X
126-
100-
8O'
60-
4,0-
2P-
-------
160
200 X
-------
Sofiiplt -Number -94766
rL TAI337
w/b S-»
20QX
-------
HO
Sompl*
Organism- TAI538
w/o S-9
> r i1 i
- , • •|H
EXTRACT' CONCENTRATION
t I i' • •' I !
20OX
-------
Somplt Nunni>tl-946S
Organism - TAI330
S-9
20X '
EXtf?ACT CONCENTRATION
Yf 4 I
200X
-------
APPENDIX E
Dose Response Curves - Phase II Samples
Samples 28524 (CSO, Site 004) and -29031 (Metro'" influent) initially
—indicated—a -detectable—respt>n3eT~vhil-e--samples- 28885 —(runoff?—S±te-004-)—and
2888S (metro influent) initially exhibited toxicity to the test organisms.
The. samples were retested vith the- resultant dose responses as indicated in
the following four figures.
- 118 - '
-------
= Sample Number-28524
Organism - TAI538 '
w/o S-9
200-
"300 40O
APPLIED SAMPLE DOSAGE (ul)
500
119
-------
5
10-
**
r-
•T
+*-»*
pr
•h—
trr:
•***-
r=d
trr
, , ^_ — , ,
«d
TTT
I
bts
r~iUro_
••-••*
rrr
da
:rr
5==
£zz
**r
s
0
w
-=B
am
rg<
/o
cad
>*"*r
ipl
mi
S
SMI
er
sm
-<
s=;
i I • j ' "• 'I— — =
iuc
3
^
"Hrr
Jsfc
nb
TA
=
•tr-
*=
BT28
aoo
se
i=d
I— g«|
5
?=B
irr1
-B5
ag
^
ssa
^S:
'T« n
ss
~-
.*
200
300 ;4OO.
APPLIED* SAMPLE DOSAGE (ul)
,. -TSOO:
120
-------
3.0-
2.0-
<
LO-
Sample Number-2888&
Organism -TAIOO"f ,
-w/a S-9- --—
200
30O ' 400
APPtlED ~SAfrfPtE-DOSAGE~ (ul)
.5.0O
121
-------
3.0-
2.0-
i7 ; i ' • I' ' '=~
Sample Number-28888
Organism- TAlOO -1
w/!>00ul S-9 _ |
—ton
200
300 400
APPLIED -SAMPLE-QOSAGE- -tulj
500
122
-------
2.0-
1
1.0
ess-
SSEE
=
=====
=*~
1/i' ' ••"
Sample-N
Orgonism
w/o S-9
i
™=
~-~—
jmber-2S
-TA133S.
„',,;,,,
03h
n1;;;";;]
1 J,...i^--{i!i
200
3OO ' 4
------- |