MAY 1971
                             DISTRIBUTED BY:
                             National Technical Information Service
                             U. S. DEPARTMENT  OF  COMMERCE

                   1. Report No.
                     EPA-430/1 -74-007
                                                                3. Rupert Dane
                                                                   May 1974
4. T"le and Subtitle
   Water Quality Studies (161) training manual
7. Anthor(i)
   Charles E. Sponagle, training manual coordinator
                                                                8. Performing Organization Kept.
9. Performing Organization Name and Addreaa
   U. S. Environmental Protection Agency, OWPO
   Municipal Permits & Operations Division
   National Training Center,  Cincinnati, OH 45268
                                                                )0. Project/Task/Work Unit No.
                                                                11. Contract/Grant No.
12. Sponsoring Organization Name ant! Addreis
   Same as #9 above.
                                                                13. Type of Report tt Period
IS. Supplementary Notes
16. Abstracts
  Tfeis training manual consists of a series of outlines on various topics associated
   with the planning and conduct of stream monitoring and surveillance programs*  Thi
   manual is divided into eight sections: I - Sources of Pollutants/ from municipal and
   Industrial sources are considered; II - planning and Conducting Wafer Quality
   Surveys; III - Data Dandling and Jleporting; IV - Aquatic Biology; V - Chemistry;
   VI - Bacteriology; VII - Dissolved Oxygen and Dissolved 0xvgen Relationships; and
   Vin - (pther Copies,  specifically Aerial Reconnaissance in ^Pollution /Surveillance
   and certain aspects of courtroom procedures in connection with the enforcement
   of water pollution control regulations.,
17. Key Words and Document Analyaia.  17o. Descriptors
Aeration,  Aerial Photography,  Aerobic Conditions, Air-Water Interfaces, Algae,
Alkalinity, Ammonia,  Analytical Techniques, Anaerobic Conditions, Aquatic Environ-
ment, Aquatic Life,  Aquatic Plants, Aquatic Populations,  Aquatic Productivity,  ,
Bacteria,  Baseline Studies,  Basic Data Collections, Benthos,  Biochemical Oxygen
Demand, Biological  Communities, Biological Membranes, Bioassay, Chemicals,
Chemical Oxygen Demand, Chemical Reactions, Chemical Analysis, Chezy Equation,
Chlorides, Coliforms,  Control Systems,  Currents, Data Collections, Data Interpre-
tation,  Data Transmission, Degradation,  Discharge Measureaqmt, Discharge (Water),
                          Distribution, Domestic Wastes,  Ecological Distribution,
                                                    (see attached page)
I7«. COSATI Field/GroupQ6F, M, Q; 07B, C;08H;13B, H, K; 14B, D;17E;18G,H.
It. Availability Statement
Release to the Public
  ajt1 NTia-lI (R«V. i»-f»  ENDORSED BY ANSI AND UNESCO. .
19.. Security Class (This
                                                     20. Security
                                                                         21. No. of Pages
                                               THIS FORM MAY BE REPRODUCED
                                                                         USCOMM-OC >ia*-*74

Item 17. Continued
Environmental Control. Environmental ECCects, Essential Nut.rients. Estu~ine
Environment, Equations, Equipment. Eutrophication, Fertilizers. Filters. Fish,
Fish Behavior. Flow. Flow Measurement. Flow Rates, Flumes. Fluorenc.nce.
Fluorescent Dye. Frequency Analysis. Hardness. Heat pollution. Histograms,
Hydrologic Data. Impoundments, Industrial Wastes. Insecticides. Instrumentation~
Investigations, Irrigation, Irrigation ECfects, Irrigation Systems, Laboratory
Equipment. Laboratory Tests. Lakes. Law Enforcement. Lea.ching, Legal Aspects,
Lentic Environment. Limnology, Liquid Wastes.- Lotie Environment. Mannings
Equation. Mapping, Marshes. Mathematical Models. Membranes, Methodology,
Microbiology. Microorganisms. Monitoring. Municipal Wastes, Nitrites,
Nitrification, Nitrates, Nutrients, Nitrogen Compounds. Natural Streams. Odor.
Oil, On-Site Data Collections, On-Site Investigations. Open Channel Flow.
On-Site Tests. Organic Compounds, Oxygen Demand, Oxygen. Oxygen Sag.
Oxidation. Oxygen Requirements. Path of Pollutants, Persom}~l, Pesticides.
Phenols, Phosphates. Phosphorus Compounds. Photogrammetry. Photosynthetic
Oxygen. Pipe Flow. Planning. Pollutant IdentiCication, Pollutants, Power Plants.
Preservation. Quality Co~trol. Radar. Radioactivity Technicp.Jes. R.adioisotopes.
Rates. Reaeration. Remote Sensing. Return Flow. Saline Water. Samplers.
Sampling. Sedimentation, Sewage Bacteria. Sewage Effluents. Sewage Sludge.
Sewage Treatment, Slujge, Soil-Water-Plant Relationships, Statistical Methods.
Statistical Models, Statistics. Storm Runoff. Storm Water, Streamflow. Stream
Pollution. Surface Irrigation, Surface Runoff, Systematics. SurCactants. Surveys.
Technical Writing. Testing, Thermal Pollution, Toxicity, Tracers, Trophic
Level. Urban Runoff, Variability. Venturi Flumes. Waste Dilution. Waste Water
(Pollution), Wastes. Waste Water Treatment. Waste Treatment, Water Analysis,
Water Cooling, Water Flow, Water Level Recorders. Water Pollution, Water
Pollution Sources, Water Quality. Wa.ter Quality Control, Water Quality Standards,
Water Properties. Water Sampling, Water Temperature. Weirs.

Thli course Is designed for engineers, chemists,
biologists, bacteriologists, and other administrative
personnel responsible for the planning and conduct
of water pollution surveys.
        Water Program Operations
                May 1974

Throulh the Water Programs Operations Omce, Envlronmental
Protection Alency conducts programs of'research, technical
assiatance, enforcement, and technical tralninc for water
pollution control.
The objectives 01 the Training Program are to provide specialized
trUnin, in the field 01 water pollution control which will lead to
rapid application 01 new research findings through updating of
skWs of technical and professional personnel, and to train
new employees recruited from other proleseional or technical
areas in the special skills required. Increasing attention is
bein, liven to development of special courses provic!ing an
overview of the nature. causes, preventicll., and control of
water pollution.
SclenUsts, enlineers, and recognized authorities from other
A,_ncy pr0lI'&ms, from other govemment agencies, universities,
and iftdU8try supplement the training stall by serving as guest
18cturers. Most training is conducted in the lorm af short-term
ccurses of one or two weeks' duration. Subject matter Includes
se18cted practicalleatures of plant operation and design, and
water quality evaluation in field and laboratory. Specialized
aspects and recent developments of sanitary enlineering, chemistry,
aquatic biology, microbiology. and field and laboratory techniques
not prleralq available elsewhere, are included.
Tht! primary role and the responsibWty 01 the states in the
training of wastewater treatment plant operators are recognized.
Technical support of operator-training programs of the states is
available through technical consultations in the planning and
development of operator-training courses. Guest appearances
of instructors from the Environmental Protectim Age::J.cy, and
the loan of instructional materials such as lesson plans and
visual tra1n1n1 aids, may be available through special arrangement.
These tra1n1ng aids, including reference training manuals, may
be reproduced freely by the states for their own training programs.
Special catelories of training for personnel engag..d in treatment
plant operations may be developed and made avai1al.ble to the states
for their own further production and presentation.
A bulletin of courses is prepared and distributed periodically
b7 the National Training Center. The bulletin includes descriptions
ot courses, schedules, application blanks, and other appropriate
information. Organizations and interested individuals not on
the ma1linglist should request a copy from The National Training

Introductory Comments about the Training Manual
The manual accompaniu courau offered for
en,meare, ohemist., bio)ogists, bacteriolopsts,
and othen having responsibUitiu for planning
and workin, in water pollution surveys. The
scope, content, and levell\re intended
primarU~ for the worker who is reJatively
new in water quality surveys, and for the
more experienced worker who has such
specislized ruponsibilitlu that he has 11ttle
opportunity to gain a perspective with reprd
to the contributions made by various technical
spedalists in water quality studiu.
Course participants attend lectures, demon-
strations, and panel discu88ions. Field trips
to one or more local sites are included, where
student. participate actively in selected field
.urvey procedurell. In the laboratory, throu,h
demon.trations and limi~ed active participa-
tion, they pin in.i,ht into the nature of many
of the phas.. of required laboratory operations
and their interrelation.hips with field
Major subject are.. include:

Identifioation of pollution problems in the
aquatic environment in terms of .ourcell
of pollution, and .pedel pollution problems
in the ,~,raphic: ar.. in which the course
is pre.ented;
Elements of water quality surveys,
including pJanning, laboratory, field,
and reporting aspects. Special emphasis
is ,iven to stream surveys, with limited
attention to significant features of long-
term surveillance studies and special
aquatic lIituations such as lakes and
impoundments, coa.tal waters and t1da1
estuaries, and ground waters.
Technical subject areas include:
Oxy,en relationship;], DO, BOD, COD,
natural self-purification;
Chemical procedur",,, and evaluations in
pollution studies;
Aquatic orpnismil: significance,
applications, methods of measurements;
Bacterial pollutio~ indicators: significance,
applications, methods of measurements;
Related p~sical hsts and measurements.
The worker who desires more detailed
information on any of the specialized
technical subject matter in this course
should consider enrolJ.ment in a training
coune limited to the area of his special
interest. Several such courses are offered
by this program.
National Training Center
Water Program Operations
Environmental Protection Agency

,Outline Number
Pollutiona1 Characteri.tic. of Dome.tic Wa.tewaters
Sources and Effect. of Indu.tria1 Waste.
Influence of Land Runoff on Water QUiUty
Irrigation return Flow and Agricultural Runoff
Thermal Pollutional Overview
Water Quality Surveys:
Water Quality Survey.:
Flow Measurement
Organizing the Stream Survey
Organizing the Lake or Impoundment !1urvey
Tracing Natural Waters
Sampling in Water QUllity Studies
Sample Handling Field Through Laboratory
Collection and Handling of Samples for Bacteria
Water Quality Survey.: Role of Bacteriologist

Introduction to Statistics
Presentation of Data
Water Quality Surveys:
Bacteria Data

Water Quality Survey.:

The EP A Storet System
Pre.entation and Interpretation of
Preparation of Survey Reports
161. 4.74

The Aquatic Environment (Part 1 throulh 5)
The Identtltcation of Aquatic Orpni.ms
U8ini Benthic Biota in w.ater Quality Evaluation
Biolortcal Field Method.
Special Application. and Procedure. for 810a..ay
Alluatic Oriani.m. of Si,mf1cance in Pollution 9u1"Y8y.
Analytical Quality Control in EP A, WPO
Automatic Instrumentation for Wat.r Quality Mea.urements
Orpnic Analy..s in Pollution SuI'Y818 '
Interpretation of Chemical Data
BOD Tf!at Procedlares
lAboratory for BOD Teat
COD and COD/BOD Relation8hip.
Lab Procedure for Routine Level COD
00 Determtnat1or. by the Winkler Iodam.tric Tttr~tion
and Azide I\fodificatton
00 Determination by Electronic M...uremetlt
Phosphorou8 in the Aqueou. Environment
Laboratory Procedure for Nitrate Nitroren Modifed Brucine
Chemical Te.tl, Observations, and Mealurement, in 'Pield
Chemical Field Kit DescrLption
Bactertolofical Indicatators of Water Pollution
Examination of Water fi)r Col1formand Fecal StreptOCOCcu.8
Determination of Bacterial Pollution
Membrane Filter Laboratory and Field Procedures
Outline Number
Method 44


Oxygen Demand AnaLy8el
Effect of Some Variable I on the BOD Test
Mathemattcal Trea~ment of BOD Telt Relults
Dtuolved axy,en: Factors Affecting DO Concentration In Water
Oxygen Dynamici in Streams
Effect of Benthic Deposits on Dissolved Oxygen Resourcel
Effect of Aquatic Plants on Di8lo1ved OxYlen Relource.
Organic Enrichment and DO Relationships in Water
Aerial Reconnaislance in Pollution Surveillance
Case Preparation and Courtroom Procedure
Outline Number


Historically, water pollution activities cen-
tered around wastewaters originating from
private and municipal sources. Today, our
water pollution control activities recognize
many 8ources, including waltewaters
originatin, from industrial operations.
For the purpose of this discuuion, it is
appropriate that we define the nomenclature
used to classify various wastewaters which
are discharged to our streams. Many
agencies, associations, etc., have postulated
definitions of various wastewaters. It is not
the intention of this lesson to determine the
merits of the various definitions; however,
we should recognize that discrepancies do
exist. For our purpolles, wastewaters may
be classified as:
A Sanitary Wastewaters - waates which
originate in the home; this includes bath-
room, kitchen and laundry wastewaters.
B Domestic Wastewaters - wastewaters
orilinating from a residential community
with the normal amount of commercial
and service institution II (banks, shopping
centers, coin laundries, service stations,
hospitals, etc.). Here the major portion
of the wastewaters can be classified as
principally sanitary in nature.
C Municipal Wastewaters - wastewaters from
municipal complexes where a fair portion
(approximately 20'1'0 or more) of the waste-
wateu are from industrial operations with
the major portion being domestic sources.
D Industrial Wastewaters - wastewaters from
lndustrial operations where only a slight
percent of the wastewaters is from sanitary
faciUUes at the plant.
E Combined Wastewaters - a combination of
storm water runoff and sanitary, domestic,
WP. SUR. 22e. 3. 74
municipal, or industrial wastewaters.
Surface runoff may often constitute the
major portion of s:Jch wastewaters.
The terms "wastewater" and "sewage" often
are used interchangep,bly. "Wastewater" re-
fiects the changes in municipal discharges which
once were predomi:1&tely household and
commercial, but have changed to include a
larger fraction of indulltrial discharge.
Further, the word "sewage" is associated
with a certain "aromr." that prompted selec-
tion of a different t€rm with a more "cleaned-
up" connotation for professional and public
use. "Wastewater" ill the term accepted and
promoted by the Wc.tE'r Pollution Control
Federation. Its acceptance is increasing
with the term "sewage" becoming lesE
frequently ulled.
A The composition of various wastewaters
are of interest to all parties engaged in
water pollution control and water resources
development. Tt,e composition or
characteristics of any wastewater determine
its hazard to receiving waters and the
subsequent value of these wa'ters. The
engineer must have information on the
characteristics of a waste -in order to de-
sign and operate trE.atment facilities. The
industrialist should know the characteristicil
of the wastewater from his plant so he"may
better evaluate the effects of his plants'
wastes upon his neighbors and downstream
water users. In the forseeable future, an
industry's waste characteristics may be
one of the major yardsticks used to ascer-
tain whether or not that industry will be
allowed to locate in certain sections of the
country. Therefore, the importance of
defining or characterizinl any wastewater
becomes of significant concern.

PpllutiQAal Cb¥acteri8tiC8 of Dome8'tle \\Ute Water.
B FortUbately, dome.Uc WI.\.tewater. are
fairly uniform in colftp08iUon and vary
only .11ptly throulhoUt the country.
C FactOt'. that affect the characteri.tic. of
dOlBe.tie wa.tewater.: .
1 Comaawaity charaeten.Ue.

a Amount of avanable <:lrl'1&,e water

In area. where water u.e per oaplta
i. low or where there i. a hip con-
IUmpttVI! ule of water for dom..Uc
purponll (lawn .prinkliDt, etc.)
Ililbtly hi.her .tr.nfth wI.tewater.
may be expected-.
b Eeonoll'lic level.
In ,enel'll, hlper cOlt rnide.ts.l
areal contribute more volume of
wa.tewater due to hiper ula,.,
except pollibly in arid ar"l where
hi,h u.e may refiect con.umptive
u.e. luch 8S lawn aprink1in" etc.
Oarbap Find.... may add lip1f1-
cantly to the Itrenlth of the
2 COllecUon IYltem
a ' Separate lewer Iy.tem - lanitary
wa.tewaterl and .torm run-otf
carried in leparate leWer..
b ColI'Ibined sewer Iy.tem - one .ewer
used to carry both .anltary -.te-
water and Itorm ru.1-off.
e Condition of Iy.tem - a,e, Fad.I,
infiltration and ..flliration ratn,
d Deteation time in e.;,Uection .y.tem -
detention time or time 01 travel from
the pomt 01 collection to the point of
di.chal'p or treatment may .lp1li-
c8bt1y infiuencl! the characteri.tici
of the walte""'.r. Lon, timee of
travel may re.lIlt 1iI .eptiol.\y and
ruult in odor probleml.
3 Daily and hourly fiuc:tuationa in rate of
now will naturally affe~t the concentra-
tions that may be expected~
D Characteristici of Domelt1c: Wa.tewater
1 Table I illultrate. the constituents of
major concern normaJ.q: tOUlMl--in
domestic wastewatel'l aDd l1"es some
idea of the range of .::once.ations
that mipt be expectlld.
2 Wastewaters are uSl1811y characterized
under three major classification
a Pby.ical characterilti~
b Chemical chara~teri8-tie.
c Biololical characteristic.
3 Domeltic wastewater characteri.tic.-
Note - all values in 11'1,'1 except as
Four compo1umt. will be dilcu..ed becau.e
in mOlt ca.e. they are of prime COReern
from a pollution v1_poin~. The three
c081ponente of main interest are bacteria
(coliform), nutrients (includin4( biochemical
oxypn demand, nitropn, phoepborus) and
A Bacterial concentration8 - coliform I are
indicator orpai.ms u.ed to predict the
potI.~le pre.ence of pathorenic (die...)
orpniems. They are importaat from.
public- health .tandpoiat. It 4IIIuld be
noted that coUlorms are oa1)' 'I port-
ot the orpniems present.
B Nutrient.
1 Biochemical oxygen demaad (BOD) - by
definition a measure-of the amount of
0X1aen req1ll1red b)' Oi"pni.ms to

Pollutlonal Characteristics of Domeltic Waste Waters
Table 1.
Physkal cn.!lriJI,'tf'riRth-8
Color (ncm. ~rptid  Gra)' Gray Gray
Color (~rf'tif)  Gra,)'-hl.II 1\ n!ill kp~h Rlal,..kl~h
Odor (non- Beptk)  Mosty Musty Mosty
Odo~ (s'pllc'  Mosty - H2S H25 H2S
Tr mprratur(' w of .. ~50 - !IOo !")50 - 1100 :150 - 90°
Total S:Ol1dR*  4<;0 800 1200
l.'~tal yolaUlr soUds 250 425 80G
SL1sp('nderJ 8olid"  100 200 375
VolaUIE' 8l,Ulpendf'd BaUd. 75 1:10 200
S.ttl.abl. solids    
ChemIcal charac tf!'rl.Ucs
pH - (units)  6.5 7.5 8.0
Cl. 804, Cs, MIf. dr. '"   
Total nitrOIlf'n  15 40 60
OqillU\!r nllro~rn 10 25 40
J\MlT1onta nllroJ(rn  u,:; 1.0
Nitratf' nitroliwn  0.5 1.0
Total phosphatr - po.  15 .10
Tete I hartrria (~~~n:l) I > InlJ ":n , 108 100 ~ 108
Total l;oliCorm c~o~Nml) 1 J( lU'; :10" 1 06 100 X 106
n lorhf'm 11'81 oxyge-n 100  .:;!no  4<;0
dt>mand (S-day)  
. QuitE" varjahlf' dC'pc'IyHng on natural water qU81 it)' or rf'.ion.
..For the Crntr~1 States zonf'.
ItabUbe deil'adable organic material.
BOD i8 an indirect measure of the
amount of de,radable organic matter
present and a fairly good technique for
measuring the oxy,en demand of a
waite. Various modifications of the
BOD test are popular.
2 Nitrogen is a normal component of
lIanitary wastewater. Discharge of
nitrogen in the form of ammonia may
increase the burden upon the oxygen
rellourceB of strHms. Nitrates may
foster the growth of undesirable aquatic
3 Phosphorus is noiturally present in
sanitary wastewaters and with the
advent of synthetic detergents where
phosphateB are, an important ingredient
higher phosphorus concentrations are
now found in domestic wastewater than
in previous yea1'8.
C Suspended Solids. These are important
from an aesthetic vif!wpoint (floating
soUds. sludge banks. etc.) and
represent a source of organic
matter (BOD) in receiving waters.

PoUutioual Ch.racteri81ic. t1f .Id!m!.!.~te Wa.!!r,
B Waetewatera from Primary Treatment
Cone utue nt
Expected rante
of removal
Coliform I

Solidi - total luepended



Or praic - N
NH3 - N
N03 - N

Total pbolphate
50 - 60'"

35 - 651-

25 - 40'.
20 - 40%
.No chlorination.
C Walt~ater from Secondary Treatment
Expected range
of removal
SoUd. - total IUlpanded
Nitrogen "''''

Organk - N
N'H3 - N
N03 - N
Total ph.sphate ..
70 - 95%
70 - 90%

65 - 95%
65 - 80%
50 - 90'10
+ 200 - 500 %
20 - 60'.
, '" No chlarinetion.
"'. Removal primarUy associated with frelh
solidi removal and control 01 procell
Increalin, emphalil i. blinC ,lven to the
problem of ItOI'm water contamination due to
wa.h oft of accumulated lurtace reful.. This
cannot be ne,lected in the control of lurface
water quality.
----..~--~~- -.
(lee tablel "low),
Constituent Concentration I in ,Urban
Land Runoff at a CirlcinMti Sampling
Point, J&a1y 1962 through Sept. 1983'"
-.. .-- ._._---c-~~-_._~~~:
ParatMH"''' - -
.----.--. -- - -
30-1,000 11711
6.3-U i.5
1- ..
.-- ._---- ..~-
(118 caCo,)
, 2'.)-610

" 2.M
{NO !J.02-U.2
(uN) NU, I n.H.D

PnUut{nnA1 rhArA~+t=l1"'i.tf~. ~f nnm~.tf,.. \V!II-+. W!ltara
Comparison of Stc.rmwater Runoff
Loads and Sanitary Sewage Loads
(lbl yr I acre)'"
~:.~~.; '" - ~orlll
tr...}1 /flN..nitar)
--_.---- ----
H8 730 MO  \.10
VMS 100 :1f~1  H
con 240 !I(jO I OJ..
Bon :t! MO ! Ii
Po, 2.6 27 I ~I
Total nitro.~n 8.\1 fli I II
-N'   I 
--   -----
. Multiply by 1.12 to oblain kl/)',/hectar..
t lltormwat.,r runolf--B_d on _nUally
romple~ m'8Iu",lIM'nt of rainflill and ron...-
quent mnolf water quanti'y and 'Inality at the
.tudy .it.. durin. ~pl.mlK'r throllJlh Novom-
be, 19112, and March thronllh foIt'l,t.mhrr tll6:l,
proje..Wd to avorale alInulil r.,;nf,,11 III Cin-
~ Sanitary ""wa.....-3....d on IlOpulation
d.neity of II prnone/M" (22/h.rt8ff" and flow
of 100 IJId/rap rO...U m/dft)'/cap).
ANum.d rftw H1\'''' .I....nllho: 1'I~-2OI1
mill; VRI'I-130 mill; COIJ-3/iO mill; BOD
-:100 mill; 1'0,-10 l1li/1; Total N-JO mIll.
Pollution caused by wastewaters from urban
centers are still the major concern in the
United States since np.tural water quality is
most severely stressed at these locations.
Nutrient enrichment, plant and animal growth
tend to exceed natural stabl1zation capacity
of the surface water. Partially treated or
untreated wastewaters are primary consider-
ations. Secondary treated discharges fre-
quently are too rich in nutrients such as
nitrogen or phosphorus for discharge without
additional treatment. Storm water runoff
requires major con8ideration in the waste-
water renovation program.
Steel, E. W. Water Supply and Sewerage.
McGraw Hill. 1960.
Sawyer, C, N. Chemistry for Sanitary
Engineers. McGraw Hill. 1960.
Weibel, S. R., Anderson, R. J. and
Woodward, R. L. Urban Land Run-
off as a Factor in Stream Pollution.
Journal of the '/Vater Pollution Control
Federation. July 1964.
Hurwitz, E.. Beaudoin, R. and Walters, W.
Phosphates - Their Fate in a Sewage
Treatment Plant - Waterways System.
Water and Sewag~ Works Journal.
March 1965.
Levin, G. V.. and Shapiro, J. Metabolic
Uptake of Phosphorus by Wastewater
Organisms. Journal of the Water Pollu-
tion Control Federation. June 1965
Thil outline was prepared by Peter F.
Atkins, Jr., Sanitary Engineer, formerly
with the National Training Center, and
reviewed by F. P. Nixon. formerly Acting
Regional Training Officer, Northeast
Regional Training Cen~er, EPA, WPO,
Edilon Water Quality Relearch Laboratory,
Edison, N J 08817.
Delcriptors: Domestic Waites, Liquid
Waites, Municipal Wastel, Pollutants,
Sewage Effiuents, Sewage Treatment,
Storm Runoff, Storm Water, Surface
Runoff, Urban Runoff, Wastes, Waste
Treatment, Waste Water(Pollution), Waste
Water Treatment, Waterr Pollution, Water
Pollution Sources

A s a result of manufacturing every industry
produces some kinds and quantities of liquid-
borne waites. These may be detrimental to
quality of the r('ceiving waters in many ways:
toxicity, oxygen demands, sludge deposits,
surface films, thermal effects, etc. Since
agriculture is a form of production, the
effects of drainage from the land will be con-
sidered among the indultrial wastes.
Classification is one of the most important
steps in any water pollution control investi-
gation involving industrial waste discharges.
A There are essentially two approaches to
industrial waste classification:
Classification according to industry.
2 Classification according to the effects
the waste produces in the receiving
B By Industry
Under this scheme industrial wastes are
classified as those from the paper and
pulp industry, or antibiotic industry, or
citrus concentrate industry, etc.
1 As a whole, the manufacture of a
specific product will result in similar
liquid waste materials. Clauifying
wastewater by specific industries is
very valuable in that it allows for an
evaluation of the pollutionallollld being
discharged to III stream no matter where
the industry is located. In this regard,
calculated waste loadings should be
related to plant production, e. g., pounds
of BOD per ton of finished product.
WP. SUR. 21d. 3.74
2 This classific..ltion scheme is basNJ on
the assumptioT. that although manufac-
turing plants within an industry will
produce different quantities of waste,
the chemical com.tituents of the wastes
will be fairly uniform. Wastewaters
from various plants within the sa me
industry will have different effects on
water quality when discharged to
specific, unique receiving streams.
3 This classification gives no mdication
of interaction type effects produced by
more than one industry within a
specified area.
C By Effect
There are many classification sch£'m,'s
for industrial wastes based upon the ir
effects in the receiving water body. Thi"
type of classification has mort' advantages
for water quality DtlJdies than the previous
type, i. e., by industry. Many of the
analytical procedu:-es record the effects
of the indultrial waste (BOD, Bioassay,
etc. >, rather than the components.
Classification by effects is basC'd on the'
supposition that an industrial waste may be
characterized by a single predominant dfect.
In some cases, this may be true, but most
industrial wastes exhtbit multiple effects.
Classification according to one effect may be
unduly restrictive. H'otwithstanding thi"
problem, classification by effect appear" to
be the more popular "cherne because it is
more related to stream conditions and is
easily applied.

~ources and Effects of Industrial Wastes
- - --
A Oxygen Depleting Wastetl
ElfE'ct: Low DO levels, due to oxygen
de!11and. may alter the whole balance
of the biota of the receivi.~g stream.
The natural process of !el!-purtficaUon
may be delayed significantly because
of 'the lack of adequate oxygen resources.
The demand exerted may be either
chemical or biochemical.
2 T)'Pical Wastes: Examples of indu8trial
wastes having slgnitlcaDt oXYlLen demand
a Sultite waste liquors from pulp mills
b Canning plant wash efnuents
c Meat packing wastes
d Textile tlcourlng and dyeing efnuents
e Milk products wastes
FE'rmentation wastes
3 Measurement: Since the primary effect
of these materials II a reduction in dis-
solved oxygen. the BOD telt is commonly
used to determine the deoxypnation
potential at. the walt.. In conjunction
with thl BOD. the COD ~elt will yield
va!uable information.
4 Treatment: All of the methods for the
treatment of oxygen depleting wastes
involve satisfying all or part of the
oxygen demand before discharge to the
stream. This usually involves some
type of secondary treatment either
chemical or biological.
B Toxic Waites
I Effects: For the purpose of clauiflca-
tion, toxicity is considered a8 a direct
lethal effect on biological forms. as
contrasted to the indirect effects of
oxygen deficiency or smothering.
2 Typical Wastes: Among the many
wastes having toxic effects on
st~cam biota arE":
a Spent plating solutions containing
heavy metals and cyanides.
b Acid wAstes from pickling operations,
chemical manufActure. and mine
c Organic materials such as s,trang
phenolics. antibiotics. pbarmaceu-
ticala and petro-cherr.icaJ wastes.
and pesticides.
d Textile dyes
3 Measurement: Traditionally. toxic
wastes are evaluated by chemical
analysis of the waste to de'termine the
concentration of the offending compound.
Unfortunately, suitablt< analytical
methods are not available for some of
the newer, more conlplex materials.
The bioassay is rapidly aSS\tming a
key place in the evaluatlonot toxic
4 Treatment: Removal.:>f the toxic agent
is essential to adequate treatment.
Heavy metals are precipitated from
plating solutions. cyanide is oxidized
by chlorination. acidtJ are neutralized.
and organic compounds are oxidized
biochemically by the use of acclimatized
C Wastes Causing Physical Damage
I Effects: Certain industrial wastes
cause damale to the stream by physical
actions rather than chemical or bio-
chemical reaction. T\1ese materials
create an unfavorable l'nvironment
because of the specific physical proper-
ties which they possess.
2 Typical Wastes: Physical damage
may be associated with the following:

a Wastes from lumbering and mining
operations. which contribute large
amounts of silt, 3awdust. and other
insoluble deposits.
b Power plant disd.arges, rai..in~
the temperature of the stream in
the vicinity of the outfalL

c Petroleum refinery waite.. which
contribute 011. and other immiscible"
liquids, color, and sludge.
d Dyeing operations, which might
ponibly contribute large amounts
of color to receiving streaml.
3 Meaaurement: Wastes which create
insoluble deposits in the stream are
evaluated in terma of the aettleable
and total solids tests. Special tech-
niquea are required for other waates,
e. /I. . thermal, oil, and industrial
4 Treatment: As in the case of the other
typea of wastea, changing the properties
which cause damage ia the accepted
method of treatment. Settling basina
and ponds, coolinll systems, oil aep-
arators, and similar devicea satisfac-
torily remove the objectionable property.
D Wastes Producing Tastes and Odors
1 Effects: Many inaustrial effluents
contain substances which are capable
of imparting disagreeable tastes to
water supplies. Even very minute
amounts of these materials may cause
public reaction. Often the undesirable
tastes are increased by chlorination.
2 Typical Wastes: The following con-
tribute tastes and odors to water
a Petroleum and ~etro-chemical
waites, even when present in ex-
tremely small concentrations.
b By-product coke plants discharges
(phenolics) which are rendered very
objectionable by chlorination.
c Liquid wastes f~'om the manufacture
of synthetic rubber are often impli-
cated where off3nsive odorl are
3 Measurement: Be<:ause of the small
amounts of materials involved, special
concentrating techniques, such as the
Sources ~ Effects o~ Industrial Was!!!.
carbon filter, lire required. Identifica-
tion of the compounds by means of infra-
red spectroscopy and gas chromatography
are utilized. Threshold odor testing
&1110 yields val.!able information.
4 Treatment: Most of the compounds
which produce ustes and odors are
organic and lend themselves to bio-
logical treatrnerd. Certain wastes
(those from synthO'!tic rubber manufac-
turer for example) are resistant to
attack and require special treatment
E Waste Containing "Inorganic Dissolved
1 Many industrial effluents contribute
large amounts ot cations (Na, K, Ca,
MI, Fe) which may cause some dis-
tress to the stream biota.
2 Typical wastes: 'l'annery wastes and
irrigation waters.
3 Measurement: These wastes are
usually evaluated by chemical analysis
for the specific cation.
4 Treatment: chemical treatment, co-
agulation and ion exchange. Irrigation
waters pose a special problem as far
as treatment is r.oncerned and no ade-
quate treatment technique has been
F Radioactive Waste
I Effects: Contamination of water en-
vironment, der;truction of aquatic life.
2 Typical Wastes: nuclear reactor
cooling water, uranium ore mining
and refining.
3 Measurement and Treatment: Special
techniques due to the nature of the con-
taminant. Typel! ot treatment, ion
exchange. storage. coagulation and

SClu.rces and Effects of Industrial Wutes
G Corrosive Wastes
1 Effects: Highly acidic or basic wastes,
a1*houp also toxic, deserve special
attention as corrosive wastes. Such
wastes seriously dama,e piping systems
and may even corrode ships' hulls and
brid,e piers.
2 Typical Wastes: Examples of corrosive
wastes are:
a Spent picklin, solutions
b Alkali discharges produced dur~
manufacture of soap.
H Patl)o,enie Wastes

I Tile.. wastes may contein orl8'1iams
patho,.ldc to man or plants.
2 Typical Wastes

a Livestock production (cattle, poultry,
swine, laboratory animale)
b Tl.l\I1eri..
c Pharmaceutical man~"cture
d Food processing

3 Treatment: Disinfection with chlorine
or other active a,ent.
Many effects of industrial wastes may be
diminilthed or enhanced by climatololical
conditions. SOme of these to be evaluated
A Amount and Frequency of Rainfall
B Amount and Pattern of Run-off
C Stages and Pattern of Stream Flow
D Temperature Patterns - Stream and All'
E Sun~ight Patterns
F Wind Patterns
A Physical teatures of stream - crOBS-
sections: profUes, t\lrbulence, 'tc.
B Past poUution history of stream most im-
portant in evaluatinl preaent effect of any
new industrial waste.
Before any induatrial waste can be adequately
treated, sorne idea of the wastes character-
iatics must be -own. The most common
proceGllre is to -",lllate the wastewater in
light of the cend1t1oa of tbf! recelvu.. water
and the \I... to which u.e receiv.q water will
be put by downstream UHra. Tberelore,
approachiDI the problem based ~011 the detri-
mental etfects the wastewa.ter produces in the
stream and eUminaUng the causitive agent i.
the most logical method of atta.ck.
1 Eckentelder, W. W.. and O'CQIIIor, D. J.
Biolo.leal W.ste Trea.trnen(. Per-
gamon Pres., London. 1961.
2 Gurnham., C. F. Princip!.ea of bduatrial
Wa.te Treatment. Wi~ey Putilishing
Co., New York. 1955.
3 Nem.erow, N. L. Theories and PracUe.es
of Industrial Waste Treatment. Addison.
Wesley Publ1Q1ng Co. Reading Mass
1963. . .
4 Rudolf, W. Industrial Wutes, Their
Disposal and Treatrr.ent. Reinhold
Publishing Co. New York. 1953 (0 t
of print) . u

Sources and Effects of Industrial Wastes
This outline was prepared by P. F.
Atkins, Jr., Sanitary Engineer, formerly
with EP A Training Activities.
DescriDtors: Industrial Wastes, Liq uid
Wastes, Pollutants, Waste Treatment,
Waste Water (Pollution), Waste Water
Treatment, Wastes, Water Pollution,
Water Pollution Sources

I Earthlife as we know it requires large
quantities of water of the right quality, in the
right place, at the right time (SINGER, 1968).
A Each person haa high, average daily demands.
1 We each need to ingest about two quarts
of water per day.
2 To sustain the food chain from the soil
to the average person's stomach requires
2,500 gallons per day.
3 Adding the amount required to produce
hi. clothing increa.es the averaie per
capita water demanels to 3, 000 pllons
per day.
4 The production of c~'eature comforts
requires other prodigious amounts.
a Refining 1 gallon of 011 requires
18 i3-llon8 of wr.ter.
b Making a barrel of beer requires
300 pllons of water.
c Steam-powered generating plants use
191,700 gallon. f)f water for each ton
of coal that it b'lrns.
d Since it takes 60, 000 gallons of water
to produce a ton of paper, a large
paper mill need. as much water
(60,000 X 1, 000) as a city of 300, 000
persons (300, 000 X 200).
B The water supply of the Earth total. a
stallering 326, 000, 000 ~ mUes of
water (NACE, 1967).
1 Oceans, inland sess, glaciers, and
polar icecaps account for 99.37 percent
of this supply.
2 Ground water, vaoose water, and soil
moisture make, up 0.62 percent.
3 Fresh-water lakes and rivers have an
average instantaneous amount equal to
0.01 percent.
W. RE.lu.2a. 3. 74
4 The average, instantaneous, atmospheric
moisture, the source of all precipitation
upon the earth, amounts to only 0.001
percent of the earth's supply of moisture.
II Precipitation, extremely variable in
amount, but usually of high quality, is the
main source of the world's land runoff.
A In the United States. the average annual
precipitation is reJatively generous but it
ia unevenly distributed.
1 It averages abo",t 30 inches per year.
2 It varies from victually zero in the
arid interior desarts to more than
150 inches at the tops of some of the
high elevation mountain ranges of the
Pacific Northwast.
B Cyclonic storms extend across large areas.
1 Theae "genera." storms move from
welt to eaat across the United States.
2 These storms usually have fairly uniform
characteristics cut are subject to
marked orogrl&phic effects.
C Convection-type 8~or-ms tend to originate
in certain areas and to traverse somewhat
similar paths time after time.
1 Convection storms are relatively small,
short, and in~ense and are much more
common in some areas than in others.
2 Convection storms have peak intensities
for short periotls and these peaks
decrease al!l longer duration periods and
shorter frequencies (expected return
periods) of the storml!l are used.
3 An estimated rainfall intensity for
various duration »eriods and frequencies
may be ca1culatt!d from local r-ecords.

Influence of Land Runoff on WRter Quality
. To (10 this, the inteMitiee of all storms
on record must be listed in descending
order, for the duration p.eriod in
queeUon, and an ae~encanl order
number .eeigned to each Itorm,
Example 1. Rainfall intenelUel'! at CinclDnaU,
Ohio during different peak periods of l-year
and 50-year frequency (return period) etorms:
_loa Portooil
Int8Mitt.. (~p8r ~r)
Once per nu ce .. YMr.

3.'. ...0

3.11 I."

a. II 1."

1." '.10

1.01 1.10

0.81 1.'1

..005 O.U
Example 2. Rainfall intensities at Cincinnati.
Ohio durin, 5 and nO-minute periods of storms
with different frequencies (return periode):

'.11 0.81
4.81 0.11
...0 0.10
I.se 1.10
7.se 1."
.~. 2
Example 3. Five-minute rainfall rates. for
a 52-year record. in descending order and
their assigned, ascending order numbers:
Ordor ""mHr
RunaaU rate durin. tIhI moe' Imen.e
.-- poriod 01 .oh_",
n--I.cUl P.~' '-'I






I. .,
b Cbooee F, the frequency (return
period) d.slred and calculate, N from
the equation F . (M + l)/N
where F . frequency (return period)
in y..re,

M . yeare of record, and

N . order number,
Example 4. Determine N for a 10-year
frequency and 5-min\1te intensity for the
52-year record ueed in Example 3.
10. (52+1)/N; N. a 53/10.5,3
Interpolation from the table shown in
Example 3 usin, an N of 5.3 as calculated
In E:umple 4 ,ives an intenllity 01 6.43 inches
per hour.

Influence of Land R-moff on Water Quality
D Conta.mination of Rainfall is ullually
relatively low.
Example 5. Conetituent concentrations in a
O. 35-inch rainstorm that fell Ju)y 12. 1964
at Cincinnati. Ohio:
Constituent Concentration
pH 4.2 
A lIrallnity "'0.0 mgll
Total Solids 6.0 "
VolatUe Solids 3.6 "
Chemical OJtygen 3.3 "
Demand (COD)  

Nitrite Nitrogen
(N02 - N)

Nitrate Nitrogen
(NOS - N)

Ammonia Nitrogen
(NH3 - N)

Organic Nitrogen
(Org - N)

Total PO 4
0.0 mgll
0.26 "
... MUligrams per liter

E Rainfall frequency. duration. intensity. and
amount are used In various combinations
with other physical. chemical. and biological
data to provide the basis for a number of
engineering deeign purposee.
In The instantaneous volume of runoff draining
from the world's land surface at anyone time
is a relatively small amount of the world's
total water supply.
A Overland flow from an undieturbed rural
environment is ueually negligible.
1 HeavUy vegetated areas cushion raindrop
impact and keep the water clean.
2 Heavily vegetated areas have poroue soUs
and are able to ablorb clean water rapidly.
BLand uae can greatly increaee overland flow.
1 A good road eurface is impervioue to water.
2 Tillage practicII 
Influel1ge Qf Lpd Runoff on Water Quality
Example 6, Quality of drainale water resultini
from a rainetorm on one of the agricultural
waterehede under etudy by the USDA and PHS
at Coehoctoh', Ohio:
Kar-.. ... a. CO,)
ChamlulO117'" d....- ecoo)
Bloch.......1 ooq.... de_d (lIOII1
Total .on...
VolatUe eolld.
Bu_- 801....
Vo181Ue ...,-.t -.
HII.I.. ......... 1110. . '"
Hll..t. iii....... (1101 . HI
"lit"""''' lilt"'&.. eNIII . HI
Or.on1c II1t.o,..
Total "'''''''\1'''''' P04
OrIlla po 4
Tot.l coUlorm bact.ria
Fecal co1llor81 bacteria
Fecalltreptoeeae( bacteria
Total o.....c c.h1ortcl88
Ltndoao 1,....1>")
I, 4-D 1:111- _.111\1)
a The nitrate, phosphate, and fecal
coliform concentratione wen hi.h.
b Th-e total orpn1c clUoridee could be
of lIome concern.
2 lnten.ive ..mpling would be required in
order to determine tota1108de of COD-
s11tuente loet in the runoff.
3 Livestock f.edlote are trouble.ome in
some areae (SMITH, 1868).
a The Kaneas Board of Health ranks
large livestock feedlotll ae that State's
major pollution source.
b Some feedlots contain 10, 000 animale.
.80 II
10 uaIt.
T. T
100 18./1
'1. "

n8. ..

1M. "
511. If
183. "

58. ..
31. "

0.1 ..
'.1 It

0.4 ..
1.1 II
2.1 II
11.000. colant.. per 100 _1
340. 000
2... nanopam8 "1' Ut.1'
c Ten thousand catUe have the pollutl.o~
pot.nUal of 225,000 persona.
d Waetee f~om feedlots have caused
fl.eh kills and ruined Wr.ter SUppUes
of downstream communities.
D Urban development creates an artificial
environment with widely varying water
poUutioft potentiala.
1 Use and tr.tment of I.\rb&n landa and
watere varies w1de~.
a There are 11, &00 sewered CDmmu-
niU.s in the 50 United StateB.
b Theee communities r.nge in size
from l..s than 0,5 to about 4&4
squa..e milee.

Influence of Land Runoff on Water Quality
c Their a"rep.te area equall about
43.100 aquare mUea. or about 1.2
percent of the total area of the United
d Runoff coefficienta in urban areaa may
ranle from 0.05 for lawn., prdenl.
and parka. to over 0.90 for roof..
atreet.. and other aol1~-paved
e "Delian atorm" tributary areal to
Itorm lewer. are made up of com-
positel of theee typel of land uae.
f About 37 percent of a re.identla1,
11lht-commerclal type area il in
roofa, streets, and parking loti and
the remainder is in lawna. prdens,
and park..
I The entire surface area of lome
commercial urban areal may be
covered with imper-vious materials.
2 About 83 percent of the 11,500 aewered
U. S. communitiea (moltly the smaller
communitiel) have "aeparate" sswers
and the other 17 percent (mostly the
large and old communitiel) have
"combined" .ystems. or a mixture of
"aeparate" IIJ1d "combined" aystema,
with the mixture varying from almost
all "aeparate" to almolt all "combined".
a "Separate" lewer ayltema keep the
aanitary aewage separated from the
Itorm aewage eo they can be handled
b "Separate" system. have appealed to
small communities becau.e they could
use a amaller conduit for the lanitary
sewage if they uaed roadside ditches
for storm water runoff.
c "Combined" syatsms have a common
collector for both the lanitaryand
atorm water "ewage.
d During dry weather combined ayatems
divert all the aewa,e and the intercepted
draiDale water to the treatment plant.
e Durinl wet weather the volume of
mixture delivered for treatment may
be leveral times as large as the dry
weather volume.
f During heavy storms an untreated
volume of the mixture 100-200 times
larger thaI! that treated may overflow
to the recf/iving streams.
, For aome cities, "combined" systems
are .impler and cheaper and they
were often adequate years ago when
all or at leaat part of the flow could
be diacharged without treatment.
3 Quantity of atorm water runoff can be
eatimated by the "rational" method,
Q . CIA where:
C . runoff coefficient
I . rainfall intensity
A . draInage area
a The runQff coefficient (C) depends on
aurface conditions.
Example 7. Runoff coefficients for different
kind. of urban surfaces.
Kind of Urban Surface
Runoff Coefficient
0.70 - 0.95
0.60 - 0.85
0.55 0.80
0.5 - 0.7
Residential (apartments)

Reaidentlal (single and
double dwellings)


Undeveloped areas
0.25 - 0.6
0.05 - 0.25
b The rainfall intensity (I) is usually
expressed as inches per hour and
one inch of rainfall per hour is
equivalent to a runoff flow of 1.008
cubic feet of water per second per

Intluence of Land Runoff on Water Quality
c A "d.siwn" rainfall rate frequently
ull8d i9 that maximum which may be
expected in the particular return
period that has a duration period
equal to the concentration time of
the watershed point being considered.
Example 8. Using a 11:>-minute concentration
time, a 0.1:> runoff coefficient, and a rainfall
rate (tor a one-year return period and a
15-minute duration period) ot 2.55 inches per
hour (equivalent to 2.57 crs) the Zlow per acre
would be: (0.5) (2.57) (1) . 1.28 cubic teet
per lecond. In comparilon, a r.osidential
ar.. sewage. discharge at a population deneity
ot 10 per acre would be approximately
(10) (l00) 0/7.48) 0/86400) .0.00155 cuLic teet
per second. In tnis case, tne storm water
runotf amounts to 828 times tne ..wale flow
at thil peak runoff rate.
- "---- .--
4 Quality of drainage water trom a
residtOnUal, light-commercial area
differs if'eaUy in comparison to
drainaae from an agricultural area.
(Compare Example 9 with Example 8).
Examy-Ie ~ Constituent concentrations in
urban land runoff at Cincinnatf samplina
point, July througu Decemoer 1962 and
March throu&b September 1963 (WEIBEL,



Hareme.. (.a CaCO~1
Settleable 8011d8
VolatUe ..tUaable aol1da
Chemical oxnen da-d (COOl
Bioohem1c.l OXJ',ea tfemull (801)1
Nitrolen CU NI
Nitrite (NO,I
Nitrate (NO.)
Ammonia (NK.)
PO" (Total aoluble .a PO"I
Bacterial c01lD&a
Total Col1forma
Fecal ColUorma
"'_1 Straptooocei
3. 6
Rani. Mean
30-1000. 11111\8 170.
10-380.  81.
S.3-8.7  7.S
10-210 m,ll SII.
U-3OO.  83.
1-"8.  U.
28-UO.  78.
'-35. " 12.
5-1200.  210.
1-2110. " S'.
10-810. " 1111.
2-84  111.
0.011-0.2 " 0,05

0.1-1.5 0.4

. 0.1-1.11 0.8

0.2-4.8 1.7

0.07-4.3 " 0.8

Number of c01lllale. per 100
mU11l1tera ."ce.deII 1e
d..1F.t.d percent 01 ..mpl...
1I0f0 5O'J, 10f0
3, IlOO. &1,000. 410,000.
SOO. 10,1100. Tt,ooo.
4,1100. 20, aoo. 110, 000.

Influence of Land Runoff on Water Quality
Example 10. Compari80n of storm water
runoff loads. and 8anitary sewage loada.,
pounds per acre per year: (WEIBEL,
Constituent Storm Water Sanitary Sewage Ratio
Runoff (raw).. (Col. 1/Col.2)
Settleable Bolids 730, 540. 1. 35
Volatile 8ettleable 160. 360. 0.44
Chemical oxygen 240. 960. 0.25
demand (COD)   
Biochemical .:>xygen 33. 540. 0.06
demand (BOD)   
Phosphate (PO 4) 2.5 27. 0.09
Total Nitrogen 8.9 81. 0.11
... Period July 1962 throup September 1963, excluding January and February.
.. From a residential-1i8bt commercial urban area.
a The importance of the BOD load
carried by the storm water runoff
shown in Example 10 is probably
much greater than that shown by the
O. 06 ratio of 8torm water load to
raw sanitary sewage load.
b Sewage treatment would likely remove
90 percent of the raw sanitary sewage
c The 33 pounds per acre per year load
of BOD in the 8torm water runoff
would then be equivalent to 0.6 of the
54 pound8 per acre per year of BOD
load in the treated aewage effluent.
d The storm water runoff shown in
Example 10 i8 from a relatively
"clean" type of land U8e. Densely
populated and indu8trial urban areas
would probably produce more heavily
polluted storm water runoffs.
e Studies of stream water quality indicate
that coliform bacteria counts increase
as turbidity increases. (GELDREICH,
1966) (KUNKLE and MElMAN, 1968).
f Turbidity w.rtes almost directly with
streamflO'.'V. Therefore. much of the
bacterial load probably arises from
surface wash.
II Drainage rrom a non-urban area
such as the Lake Mendota watershed
can furnisn sufficient nutrients to
fertilize the water to the nuisance
aquatic growth-producing level.
(MACKENTHUN, 1965 p. 11)
h Some rural water supplies may
contain 500 milligrams of nitrate
nitrogen per ater. (MACKENTHUN.
1965 p. 11)
i On an agricultural watershed near
C08hocton, Ohio, two storms with
2,21 to 5. O~ inches of rainfall per
storm produced 6.600 to 76,300
gallons of runoff per acre.
(MACKENTHUN, 1965 p. 12)
j Phosphate (P04! in this runoff water
ranged from 0.115 to 0.42 pounds
per acre.

IDtluence of Land Runoff on Wa~er Quality
k Total nitroaen (N) ranaed from 0.20
to 6. 12 poundl per acre.
I The ule of pelUcide. hal inereand
agricultural producUon and improyed
mankind'i health and comfort,
(BOGAN, OKEY. and VARGAS. 1961)
(HOFFMAN. 1959)

m PelUcidea wUl have to be ulldwith
!norealinl care in order to avoid
tra.ic. lecondary.ffectl. however.
(WEBB. 1960) (WORRELL. 1960)
(OILETT. 1970)
n Pe.Ucidel already occupy a prominent
po.Uion among the 11.t of nlh-killin,
water pollutanta. (MCKEE and WOLF.
1963) (PRESSMAN, 1963) (PUBLIC
(BARRY. 1961)
o The compound a DDD, DDE, DDT,
chlordane. and endrin were found
rlaularly in samplel ot both water
and biota taken durinl the pelUcide
rnoaitorina ot the aquatic biota of the
Tule Lake National Wild..ife Refuae
and the occurrence of eodrin wa.
directly a..oclated with contaminated
irription return water lupplyinl( the
Retuae Lakel. (OODSIL and JOHNSON,
p Many ItutUe. .how that 80me herbioide
will appear in nearly all Itream.
Which now by or throUJh treated ar.....
(NORRIS. 19(7)
q A water treatment ot aoil-applied
picJoram peUet. for the control of
chaparral brulh relu1ted in the moye-
ment ot detectable amount. ot picloram
Jnto the .tream water. (DAVIS,
INGEBO. and PASI!:. 19(8)
I' The herbicide amitrole, aerially
Iprayed on 100 acre. 01 a headwater
Iub-baain of. Iteep, cl_rcut, forelt
waterahed in a IUYtcultural attempt
to control .almonberry, waa tound in
measurable amount. in IImpl.. ot
water near the down.tream edle ot
the eprayed area durin, and for 5 daYI
after Ipr&yina. (MARSTON, SCHULTS,
~HIROYAMA. .nd SNYDER, 1967)
5 "Combined" .ewer probleml may be
even more trOUble.ome than thoI.e of
I..rate Iyateme.
a Treatment plantl are leldom large
enouah to handle all the volume
produced during heavy Itorm..
b The quantiti.. and flow patterns are
comparable to separated Itorm water
flow I. However. ullually during
.torm period.. combined .y.tems
ean handle no more thsn 1.5 to 5
time. the normal dry weather flow.
c When the stormflowa exceed thia
amount (.ee the 826-fold Increase
ahown in Example B) .ome, most. or
aU of the flow muat overfiow to the
effluent rec.lvina atre&Inl.
d Durinl( the 1964 Chri.tmal floods
many of the .ewage treatment plant.
in the Pacific Northweat were com-
pletely inoperative. (MARSTON and
NIELSON, 1967)
e Ellewhere it hal been claimed that
al much a. 95 percent of the
lanitary ...,&,e ove..nO"NI during
.om. .torm..
f ".. ,.bout 1/3 of the city'. annual
production of .ewa,e .olids oyer-
nowed without treatment- although
only 2 to 3 percent of the lewage
volume actually ovedlowed. . . "

. Some "combined" l)'Stem. haye been
CUltom -tailored to ,rovide local
relief at certain erttlcal point8 by
diverting pert of thl! load to other
point. of overflow.
h Mo.t .torm .ewerl lil.nd certain parts
of moat combined IYlteme are of an
extremely localized nature.
i The number ot overfiow points varied
from 1 to <62 among 30 ciUe..
J Uttle IpecUic qualitative information
hal been determined :01' combined
8ewer overflows.

Influence of Land H.unoff on Water Quali,!-
Example 11. Quality of a combined sewer
pH 7.1 
Chloride 619. mgll
TotalloUdl 400. 
VoJatlle solids 144. "
Settleable solids 203. "
Sand 76. ,.
Dissolved oxygen 6.9 "
on and grease 33, "
Biochemical oxygen demand 59. "
IV Nearly all of the 17 percent of the United
States supply of water which is withdrawn,
used and returned to the waterways is polluted
to some degree. (SINGER.. 1968)
A This restricts its further use and con-
taminates the receivi,ng waters.
B The streams' self-purification processes
are unable to cope with today's massive
waste burdens and complex effluents.
C Effective waste management is needed to
protect clean waters and to upgrade polluted
waters so that they nlay be reused. The
land surface can be used as one method of
waste treatment and dlsposal.
1 The overlyinl{ soil and the underlying
geological strata may be thought of as
a three-dimensional, anisotropic,
chromatographic column capable of
adsorbing and removing solutes from a
percolating solution. (BAILEY, 1968)
a If the interactions between different
nitrogen and phoLphorus forms in the
effluent and soils and geological strata
are known, [t is possible to predict
the chromatographic behavior of the
"soil waste treatment system. "
b Using such information it would be
possible to set forth the criteria for
selecting sitel3 of "soil waste treat-
ment systems. "
c Similarly it would be possible to
indicate how those soils, lacking the
necessary critical properUes. can be
modified to SErve as "soil waste
treatment systems. "
2 An intensive study at Pennsylvania
State University has given much factual
information on the efficiency of the soil
ae a filtering medium (PENNYPACKER,
SaPPER, and KARDOS, 1967)
a Overland flow causes soil erosion and
washes silt and toxicants into the

b A void overland flow by increasing
infiltration. interflow and sub-flow.
(MARSTON and WHIPKEY. 1964).
Use sprinkler applications; disperse
and control ove; land flow s by the use
of grassed waterways.
1 Bailey, Gear ge W. Role of Soils and
Sediment in Wzter Pollution Control.
Part 1. Dept. o~ the Interior, FWPCA,
Southeast Water Laboratory. (1968)
2 Barry, E. M. Fish Kills by Chemicals.
Interstate Commission on the Potomac
River Basin. (1961)
3 Bogan" R. H., Okey, R. W. and Vargas,
D. J. Pesticides in Natural Waters.
Research 14:268 (1961)
4 Cot am, C. Pesticijes and Water Pollution
Proc. National Conference on Water
Pollution, PHS. (1960)
5 Davis, E.A., Ingeho, P.A., and Pase,
C.P. Effect of 2. Watershed Treatment
with Piclorarrt on Water Quality. USDA,
FS, Rocky Mountain Forest and Range
Experiment 5ta. Research. Note RM-I00.
( 1968)

Iq(luence of Land Runoff on Water Q1aality
6 Geldrelch, E. E. Sanitary Slgniflcance of
Fecal C01iforms in the Envlronment.
DI, FWPCA, Reaearch Serles IIWP-20-3.
7 Godsl1, Patrick J. and Johnson, W1ll1am C.
Residuu in Fish, WUdlife, and Estuaries.
Peaticid88 Monitoring Jou."'I1a11 (4):21-26.
Much 1966.
8 Hoffman, C.H. Are the Insecticide I
Required for Inlect Control Hazardoul
to Aquatic We? RATSEC, PHS, Biol.
Problem I in Water Pollution. Tech.
Report W 60-3, 61. (19~9)
9 Kunkle, Samuel H. and Meiman, James R.
San,pling Bacteria in a Mountain Stream,
Colorado State University. Hydrology
Paper 128. (1968)
10 Mackenthun, Kenneth M. Nitrogen IUld
Pholphorus in Water- -an Annotated
Sel,cted Bibliography of Their 81olo,ical
Effeets. HEW, PHS, DIV. of Water
Supply and Pollution Control. (1985)
11 Marston, Richard B. and Nielson, ~man J.
Watershed IUld River Basln Management
Re~ted to Pollution Problem. During
Hlgh Water. DI. FWPCA. PNWL. (1987)
12 Marston, Rlchard B.. Schultr, Donald W..
SMroyama, Tamotsu and Snyder, Larry V., 21
Amitrole Concentrations in Creek Waters
Downstream from an Aerally Sprayed
Astoria Watershed Sub-basin. DI. FWPCA.
PNWL. (Preliminary "eport) (1987) 22
13 Marston, Richard B. and Whipkey, R. Z,
Natural Waterworks, Ohio Woodlands.
2(3.):4-5. (1964)
14 McKee, Jack Edward and Wolf, Harold W.
Water Quality Criteria. California State
Water Quality Control Board. Pub. 3-A.
15 Nace, Raymond L. Are We Runnin, Out
of Water. DI. Geological Survey.
Clrc. '536. (1967)
16 Norris, Logan A. Chemin 1 Brulh Control
and Herbicide Residues 1:1 the Forest
Environment. Symposium Proceedings.
Oregon State University. (1967)

17 Pennypacker, S.P., Sopper, W.E. and
Kardos, L. T. Renovation of Waste-
water Effluent by Irrlpt10n of Foreet
Land. Journal Water Pollution Control
Fed. 39(2):285-296. (1967)
18 Preseman, Ralph. Pesticide..
California. State Water QuaUty Control
8oard, Sacramento, Calif. Pub. 113-A.
19 Public Health Service. Introductory Report,
The Columbla River Basin Project for
Water Supply and Water Quality
Management. Region IX, Portland,
Ore IOn. (1961)
20 Singer, Fred S. Water--The Dept. of the
Interior's 5Yltematic Management of a
Reaource. Remarks, Am. Aatronautical
Society, Laa Vegal, Nevada. News
Release, April 10, 19118.
Smith, T. R. Effect of A gricuhure on
Water Qlality. Remarks, Rotary Club,
Mt. Carmel, Illinois, February 5, 1968.

Webb, B. The Use of Herbicides in
Relation to Flah and Wildlife. Idaho
Wildlife Rev. 12:5, 11. (:960)
23 Weibel, S.R., Anderson, R.J. and
Woodward, R. L. Urb.-n Land Runoff
as a Factor in Stream Pollution.
Journal Water Pollution Control
Federation. 38(7)914-1124, 'July 1964.

Worrell, A. C. Pelts, Pesticides, and
People. American Forest Magazine,
88(7):39-81, July 1960.
GUette, James W., Editor. The
Biological Impact of Pesticides in the
Environment. Environmental Health
Sciences Center, Oregon University.
Certain portions of this outline contains
tralnlni material from prior outline by
Samuel R. Weibel.
Influence of Land Runoff on Water Quality
This outline was prepared by Dr. Richard
B. Marston, Water Quality Management
Specialist, Northwest Region, Portland,
Descriotors: Liquid Wastes, Pollutants,
Storm Runoff, Storm Water, Surface
Runoff, Urban Runoff, Wastes, Water
Pollution, Water Pollution Sources

Irrigation i6 a major use of water In the
United States, exceedin, that uaed for domes-
tic and indu6trial supply. ThiB ia largely a
6ituation which ex1Bts in western areas eince
over 90 percent of tbe irrigated land ie located
in the Vid regions _et of the Miuie.ippi
When water is utilized for the irrigation of
land, a portion of this water 18 returned to
surface or groundwater sources. Wah,r that
is appUed to the land ia known as "irrigation
water," and that which returns to the .tream
or lI'oundwater ia kn~wn aa "irrigation return
flow. "
Irrigation return flow Is a major Bource of
water pollution eince it affects the quality of
water for subsequent use6 8uch ae domeBtic,
induBtrial and irri,ation SUPp,IY;1 wildlife and
fisheriea, and recreation.:!, 4, ,9,11,12
Our objective in this lecture is to consider the
type of water problems which result from
irri,ation return flow and agricultural runoff
and the magnitude and significance of these
The problem of pollutbn from irrigation
becomea significant when we consider the
magnitude of the area under irrirtion and
the quantities of water involved.

A In 1980 it was estimated that there were
about 250 million acres under irrigation
in the world.
1 India and China lead the world with
approximately 70 million each.

2 The United States is third with over 36
million acrea.
a Over 32 miUion acres are in the 17
western etates which produce cropB
valued at over 3 billion dollars

B Irri,ated area has increased and continues
to increase at a rapid rate.
1 Irrigated acreage in the United States
hu increased tenfold from 1890 to 1956.
2 Overall increase during 2-year period
(1954 to 1958) of almost 5-1/2 million
3 It hall been predicted4 that by 1970, most
of the potential area of 51-1/2 million
acre. in the western etates will be under
A Water used for irrigation may be defined
in the followinIC waya:5, 8

1 The gross oV6rall supply required
2 That which Is withdrawn from the source
3 That which i8 delivered to the farms
4 That which is tran6pired or evaporated
from a cropped area (consumptive)
5 That which returns to surface or ground-
water sources (nonconsumptive use)
B Quantity of Irrigation Water Involved

1 In 1956 the t~tal quantity of water used
for irrigation in the 17 western .states .
was estimated to be 128 million acre-feet.'
2 By 1970 it is predicted that this3may in-
crease to 170 million acre-feet.
3 In 1980 total water used in the United 6
States was estimated to be 270,000 mgd
as follows (two significant figures):
a Rural
3,600 mgd
21,000 mgd
b Public supplies

c Self-suppJied
140,000 mgd
110,000 mgd
d Irrigation

1) Conveyance
25,000 mgd
2) Delivered to farms 85,000 mgd
C Quantity of Irrigation Return Flow
1 Irrigation is II. consumptive use of water
inasmuch as a.n average of about two-
thirds of the water diverted to the farm
is tost through evaporation from water
and land surfaces, and by transpiration
by plants (evapotran.piration).

lrriaation Return Flow and Aaricultural Runoff
2 An aver... 01 about one-th\rd 01 the
water diverted for irrigation returns
to surface streams or groundwater.
The \,-ctual quantity may vary from 20
to 80 percent of that applied, dependin,
on location, 8011 characteristic.,
climatic conditions, and m.n.,ement

3 Based on above figure., this totals
about 30,000 mid of return flow. If
this all occurs in a 3- to 4-month
groWing leason, then the da11y total il
more like 110,000 mid (about equal to
municipal and industrial waltte flow
for the same period).
4 This indicates quantitativel:r the magni-
tude of the irrigation return flow
A SourceB of Return Flow

1 The flow of water throu,h a typical
irri,ation project il shown by the flow
dia.ramll involvin, Burface and
,roundwater sources in FiJUre 1.
2 Water uled consumptively:

a ltvaporation from water and land
b Transpiration by plantE

3 Three sources contributing to return
a Overflow or wasta.e from canals
usually returnl directly to stream.

b Runoff ill the water applied in excelS
of the inriltration capacity of the land.
It may be high in turbidity due to
erosion and may cont..!n fertilizerl,
organic matter. and other contamin-
ants washed from the land.
o Se.p.,e or drain..,e is the water
which passes through the soil and
may either return to the Burlace
Btream or accumulate in the ground-
water strata. This portion ot the
return flow is significutly chanfed
in quality--eBpecially in minera
B Chan..s in Qu.lity
1 The mOBt significant chan,.s in
quality are:
. Ino....se in mineral cc;.ntent

b Increase in temperatur..~ turbidity,
color, and taste
c IncreaBe in nutrien" whiU promote
aquatic growth

d Presence of nitrat" inclmcentration
of health significance
e Potentially toxic quantitieB of
numerous peBticide materials.

2 Whether or not the aboye changes in
quality constitute a pollution problem
dependl on the specific SUUIltiOll and the
use to be made of the receiving water.
C Mineral Saltl (Salinity)
1 Return flows contain at IeQtlt three, and
often as hi,h as ten, timel the concen-
tration of mineral saltl as that of the
initlal irri,.tion water.

2 Loss of water through evaporation and
tranlpiraUon accounts for a major
portion of this increase sinc& the ..Its
are contained in a lelBer amount of
water. ThiB is illustratt!d in Figure 2.
3 The water 01 many weetern Bt...amll is
used several times for irrigation,
thereby increuing the opportunity for
further concentration of the salts. The
Rio Grand.. 1s a classic example of this
effect of irrigation on the mineral content
of a stream. The data of Table 1 show
thiB etfect at 7 sampl1n. points from
Otowi Bridge near Santa Fe to Fort
Quitman below El PaBo.
SALlJllTY 01' 1110 GRAIIDB PROM II.. sUllvno

Dli'I':"{I.....-ft. I
DIIMI"" ..M. '../U
DUltr'!'Rt =r
....... Non
, . I ,
I. U. 11. 140 "I ... "' ..,
110 iIIO flO ..u no aoa .. ~O
Source I
I Watar
. to

c...~.. ....,


~ Water
Irritatton Return Flow and A«ricultural Runoff
.. I.ep.&~ to Iroundwatel'
OVerflow (wa..ege)
I Eveoo...Uon --1 t
II Tran..piratlon
58.PAle to groundwater
Source I
~ Pump.d
. lateral.
I ,..,.. .".. I!
. Se.paBe to groundwater
Return Flow 1
~ Eveporation~ !

Transpiration =---.J
I Runo If
leturn Flow
\ \ I , I '
~,\\ 1'1/1,

Irr1aation Return now and Aaricultural Runoff
4 The growth of plants is adwraely
affected by high nlinity, therefore,
it is neceseary to {lu.h tt.e.e .alts
from the sol1 continually by the appli-
cation of water over and above the plant
requirements. This procedurejsl*nyrn
as maintaining a "salt balance", ,
which to be favorable requires that the
salt output from the project be equal to
or greater than the input. The smaller
volwne of water returning to the stream
must contain all the salte added with the
irri,ation water plus any which may
diseolve from the e011 particles.

5 Data for the Yakima River in Wuhinfton
are ehown in Table 2. The percentage
increase in salinity il about the same as
for the Rio Grande; however, the raDIe
of values is lower because of the low
concentration of salts in the initial
irrigation water.
Table 2

DI..ol"1I Selt.

Bampllnl Station
MUe8 Tra..1
Cle Elum
Roea Dam
Wapato Dam
"'Data from Wa.hIn,wn Pollution Control Comml..ion.
..M,jor ret\llln now. 8ftler above till. location. 3
6 Along with increased ealinity, there
also occur. an increaa. i'1 hardn.a..
The- effect of irrigation on total ud
permanent hardness in several
different areas is sho"n in Table 3.

A Iw.rdnes., value above 200 mgll as
CaCO is not satisfactory for domeatic
or mo\t in
element wal contained in the drainage
from an area to wI:1ich 25 m,ll wal

5 A studyll of the draina,e waters in
the Yakima Valley Ihowed that allot
the major nutrient elements increased
downstream due to drainage from
irri,ated areas.

G Nitratel
1 Nitrates in water euppliea are eigniticant
in that concentration in excess of 10
mill as N (40 mill as NOS) are dan-
gerous and may be latal it the water il
used in the feeding or intants.
2 Ammonia and organic nitrogen com-
pounds are oxidized by soU micro-
organilms to nitrates.
3 Nitratel are hi,h1;' .oluble and Will
move with the soi! water.
4 A portion is used by plants, but the
unu.ed portion may be contained in the
drainage water.

5 The data of Table 4 show how nitrates
are increased in the Oxnard Plain Area,
Ventura, California. The applied water
contained only :3 mill II NOS'

Nitrate .. NOS
Drain at Oxnard Road
Ditch near Pot't Hueneme
88. I
Ditch at Brld.e 520
ReYl10n SlOUCh
Ditch. Huenem. Road
Ditch ..ut cI W..t Road
Ditch .OUth 01 Nauman Road
Ditch on Plea. ant VI... Raid
.D8ta from C~lfornta Water Reoourcu Board for 0-
Plain Aro~ -
H Pesticides
1 Insecticides and herbicides applied to
plants and land may enter streams in
toxic quantities.
Irrigation Return Flow and Agricultural Runoff
2 Fillh kills have been reported that were
caused by persistent and toxic quantities
of these substr.nces.
S Many are known to persist for long
periods in the aoil.

4 It is reasonable to assume that some of
these may be washed from the soil along
with other mineral .alte.
A The Infiltration Processl
1 Infiltration is detined as the movement
of water through the soil surface into
the ground.
2 After entering ihe soil, it is referred to
as soil water (stored) or percolating
water (moving downward).

:3 Infiltration capacity is defined as the
maximum rate at which a given soil can
absorb precipit'tion as it falls.
4 If precipitation exceeds the' 'lfiltration
capacity, water accumulates on the
surface and rUlloff occurs.
5 Measurement of infiltration is difficult.
Indirect measurements are usually
made (preci pitation minus runoff).
6 Many physical tactors aftect and/or
control the infiltration process.
B Water Poll~tion Resulting From Agncultural
1 Fertilizers and agricultural lime
a Principal elements are: nitrogen,
phosphorus, p.:>tassium, calcium.
chloride, sul!!l.te. and magnesium.
b These reach Water courses in the
inorganic farm.

2 Animal waste problems are complicated
by concentrating large numbers of
animals in a smell area as in large
a Waste disposal presents a problem.

b One instance reported in which 75
cu. yd. of manure was dumped in a
river and approximately 1,000 fish
were killed over a distance of 3 miles
downstream. 12

lrriratlon Return Flow and A.lricultural Runoff
3 Pesticides are a source of great
concern in water pollution problems.
a Include insecticides, herbicides,
fungicides, growth regulators, etc.

b Chlorinated compounds including
DDT, endrin, dieldrin, toxaphene,
and others are mos t toxic to ftah
and are most commonly used. These
are de,raded slowly in the natural
c Organtc phosphorus compounds su ch
as parathion and malathion are
considered more toxic to warm-
blooded animals.
d Much more needs to be known in
order to control pollution from these

C Nutrient Content of Drainage WateralO

I Sylvesterl0 has reported on variations
in nutrient content from for.sted, urban,
and agricultural areas.
2 Streams from forested areal are lowest
in nutrient content.
a Lowest in soluble phosphate.

3 Urban street drainage was highest in
total Kjeldahl nitro,en.
4 Highest total phosphorus found in Bur-
face irrigation drains.

5 H~hest soluble phosphate and nitrate
found in subsurface irrigation return
flow drains.
1 Betson, Rogar P. What is Watershed
Runoff? Jour. Geophysical Res.
69:1541-52. 1964.
2 Cunningham, M. B.. Haney, P. D.,
Bendixen, T. W.. and Howard, C. S.
Effect of Irrigation Runoff on Surface
Water Supplies. Jour. AWWA.
45:1159-78. 1953.
3 Eldridge, E. F. Irrigation all a Source
of Water Pollution. JOlAr. WPCF.
35:614-25. 1963.

4 rlaigg, N. G. The Effect of Irrigation and
R.~urn Flow on Water Supplies.
Southwest Water Works Jour. 34:9-16.
5 MacKichan, K. A. Estimated Use of Water
in t" United States, 1955. Jour. AWWA
49:369-91. 1957.
6 Water Use in the United Stetes, 1960.
Jour. AWWA. 53:1211-15. 1961.
7 Scofield, S. C. Stream Pollution by
Irrigation Residues. Ind. and Enlr.
Chern. 24:1223-24. 1932.
8 Salt Balance in Irrigate~ Areas. Jour.
A,ri. Res. 61:17-39. 1940.
9 Silvey, J. K. G. Relation of Irrigation
Runoff to Tastes and Odors. Jour.
AWWA. 45:1179-86. 1953.
10 Sylvester, R. O. NutrIent Content or
Drainage Water from Forested, Urban,
and Agricultural Areas. Alpe and
Metropolitan Wastes, 1950 Seminar.
Robert A. Taft Sanitary Engr. Cente!',
Tech. Report W61-3. pp. M-87.

11 Seabloom, R. W. Quality and Signifi-
cam~e of Irri,ation Return Flow.
Jour. Irr. &. Drain. Div.. Proc. ASCE.
ffi3:1-27. 1983.
12 Webb, H. J. Water Pollutioa Resulting
from Agricultural Activit~s. Jour.
AWWA. 54:83-87. 1962.
13 Wilcox, L. V. Salinity Caused by
Irrigation. Jour. AWWA. 54:217-22.
14 Resch, W. F. Salt Ba!.ance and Leaching
Requirement in Irrigated Lands. USDA
Tech. Bul. 1290. 1963.
This outline was prepared by James P. Law,
Jr.. Research So11 Scientist, Robert S. Kerr
Water Research Center, Ada, Oklahoma.
Descriptors: Fertilizers, Irri,aUon, Irri-
gation Effects, Irrigation Systems. Leaching.
Liquid ,,",lIte8, PoUutants, Soil- Water- Plant
Relationships, Return Flow, Surface Irriga-
tion, Surface Runoff, Wastes, Water Pollu-
tion, Water PoUution Sources.

Thermal pollution has become an increasingly
common term in water management and pollu-
tion control circle's. The reason is that wasted
heat is perpetuating undesirable changes in
?ur aquatic environment, particularly evident
1Il severe cases. In addition, the overall
importance of temperature as a water quality
parameter has been recognized. We know
now that our waterways cannot be viewed or
treated a8 dumpgrounds for waste heat if we
deE/ire their continued beneficial usage.

In Up:ht of these factors, industry's accelerating
E'wission of h~&.ted wastes to the environment
poses hazards that have triggered an acute
awareness and concern on the part of the
public, the policy makers and industry itself.
Establishment of State-Federal water quality
standards bring the concern into sharp focus.
Adding to the problem is the increasing lead
time required for delivery of heavy power
generatin, equipment and other industrial
machinery, causing hurried management and
public policy decisions on plant siting, design
and equipment that will have long-ran,e impact
on the environment.
Thermal pollution covers both decreases and
increases in normal water temperature; how-
ever, almost all cases of thermal pollution
involve too much heat, rather than too little.

Many types of human activity can change the
normal temperature of water. Temperature
changes may be a secondary result. induced
by altering the environment (for example,
through road building or logging, by creating
impoundments, or by diverting flows for
irrigation). Or, water temperatures may be
changed directly by adding or taking away
heat. Our concern over changes in the normal
temperatures of water bodies is based pri-
marily on the effect on living organisms.
Temperature changes also induce slight
variations in physical and chemical prop-
~rties, thereby affecting such things as gas
solubility, particle settling rates, strati-
fication, and chemical rates of reaction.
These property changes are usually not too
important in themselves but, rather, are
significant because of their overall effect
on the ecology of a system.
IN. PPW. 7. 3.74
Biolollical effects of temperature on individual
orgamsms livinf in water influence all life
stages and processes -- reproduction, growth
and activity. MOl!t of the effects of increased
temperature stern from increased metabolism
and activity which require a greater consump-
tion of oxygen. U the oxygen supply Is not
sufficient, th~ organtem may be subjected to
severe stress. The toxicity of certain sub-
stances may also raise with temperature
increases. Such factors determine the
distribution and general well-being of a
given organtsr.l as well as his basic survival.

Temperature effects on all organisms in an
aquatic community are important because of
the interdependence of species. The overall
thermal environment determines the compo-
sition of species which will thrive. As tem-
peratures rise, le88 desirable species are
Ukely to become estabUshed. Continuing
producthity of proper food orpnisms is a
basic requirement for harvestable fish,
shellfish or other crops. An example of the
importanco of species composition was
observed after start-up of the Chalk Point
generating plant. When the water tempera-
ture rose due to the plant discharge, exten-
sive beds of Widgeon grass. Ruppi~, were
replaced by a species of Potamoge on. This
was important to the area because the RUP~~
had pr'!iviously been used as food by some
species of ducks and birds, but the species
which replaced it had very little food value
for such waterfowl.
Many Industries utiUze water for cooling and
thereby reject heat to the aquatic environment.
In 1984 about 50 billion gallons of water was
used for cooling and condensing purposes by
the power and manufacturing industries. This
was almost one-half of all water used in the
United States, or almost one-fifth of our
total fresh water supply. Power generating
facilities alone account for 800k of this cooling
w~ter use.
Because the power industry accounts for such
a large portion of cooling water usage, power
requirements offer a good correlation to
future waste heat I09.ds. PO'fler generation
has increased at a net rate of over 70/, annu-
ally, or, it has doubled every 10 years.
This rate of generation is expected to con-
tinue into the future. However, waste heat
rejection is expected to increase at least as
fast as power production! This is because

Thermal Pollution Overview
of le88 ppwer contribution from hydro sources.
a limiting efficiency being approached in
fossil-fueled plante. and lower efficiency of
the new nuclear-fueled plants. Hence. the
waste heat load from power plants, on a
national basi., win double before 1980 and
pos.ibly increase ninefold by the year 2000.

Wa.te heat from manufactl1rin, will aleo
increase in the future, but the rate of
increase will be le88 than that for power
and the total amount of waste ht=at involved
will be much l88e. Therefore, the primary
concern at present is the thermal pollution
potential of steam electric power plants,
althoup all industries must b~ considered.
The short term solution to waste heat prob-
lemll can be accomplished through utilization
ot coolinJ devices which offer a means of
di..patin, waete heat u.nder controlled condi-
tions, thereby preventing its undesirable
entrance to some water body. The ultimate
solution of the problem will be oased on new
methods of power generation which reject
substantially le89 heat to the environment,
and on L management approach which will
involve methods of using W&IIt;! energy for
beneficial or conatructive purposee.

The eetting of Water Quality Standarde has
done much to speed the development and ulle
af methods and machinery for waste heat
control. Standard or "cookbook" approaches
in applying control methodl are not yet Q
reality, however, so that each new power
plant siting brin,l its own unique problem.
Two factors which all large, new power
plants 40 have in common are the magnitude
and the quality of the waste heat load. It i8
the combination of low thermal quality and
hUJe quantity which increaaes the difficulty
and cost of heated effluent treatment or use.
It is important to emphast.z e the tremendous
a.mount of cooling water required to operate
II single, modern pt'wer plant becauM thil
factor, by itlelf, adds to the control problem.
Control measures, such all cooling ponds,
cooling towers, or dispersion and dilution
techniques would be much easier applied
to 100 cfs of effluent coolin, water than the
actual 1000, 1500 or even 2000 ch which
exhaust from plants of 1000 megawatts and
more. In most cases once-through coolina
is now totally unacceptable because rivers
and lakes cannot stand to have significant
pm-tions of tt,dr water warmed through
:W!~r plante without cauling serioul damage
tf> aquaUc life.
L' "
The efficiency of a power plant naturally
affects the amount of waste heat rejected,
which i8 the amount that must be dissipated
in any control method. Efficiency, nt, is
the electrical output divided oy the thermal
input times 100, for percent. For one kilo.
watt hour, which i. equal to 3413 BTU's,
3413 X 100

3413 + Waste Heat

The denominator of the efficiency equation
represents the heat required tQ produce one
kilowatt hour of electricity. It Is used as a
mealure of a plant'. effic:1ency, being pro-
portional to efriclenellt..If, and is caned
the plant "heat rate.

The heat rate 01. even the be lilt present day
fossil-fueled plants 18 about 8500 BTU's/KWH.
80 we see that, even at best, about 5000 BTU's
of heat mUlt be wasted for each KWH of
electricity that is prodllOed.
nt '"
We can evaluate more cloeely the amount of
heat rejected to coo11ng water from a plant,
knowing the efficiency or the heat rate and
by assuming some reasonable 101ges. For
fossn-fueled plants, lo..es account for
about 15"/, of the therma11nput, which i8 the
heat rate. About lorr, goes out the lItack;
5"/, is lost within the plant. So the heat that
must be rejected to cooling water, per KWH,
is equal to:

Heat to cooling water'" O. 85 X Heat
Rate - 3413
Hence, for a fossil-fueled plant at 40"/, effi-
ciency: (Typical new plant efficiency)


Heat to cooling water'" 0.85 (8533) - 3413
Heat Rate '"
'" 8533 BTU
. sbout 3800 BTUI KWH
For. nuclear plant, stack 10s8e8 are not
involved and the input 1018.,8 ar.e ~8tLl.~ted
to be 5"k of the input. Therefore,
Heat to cooling water'" O. 95 X Heat Rate -
So, for. nuclear plant at 33"/, efficiency,
(that's about the maximum for plants planned
to 1975):
Heat Rate '"
. 3:-1
10,340 BTU

.-~- ----_........~. --
Hea.t to cooling water a 0.95 (10,340) - 3413

a about 6400 BTU! KWH

We can see that nuclear plantl will not lelsen
the potential thermal pollution problem.
Inetead they will reject about 65% more
heat to coollng water than fOlln-fueled
Todayls technology offerl practical cooling
devices for dils1pating waste heat without
harm, methods which utilize direct air-
water contact depend primarily on evapora-
tion for heat transfer. Each pound of water
that is vaporized takE I about 1000 BTU's of
heat from the water remaining, thereby
lowering its temperature.

Co~ling ponds are the simple It method for
providing air-water contact for cooling
heated discharges. A few such ponds or
relervotrs are in use at the pre.ent time.
Cooling ponds can offer a cost advantage
if land is inexpensive, and they may offe~
recreational benefitl. For the IS realons
cooling ponds warrant further consideration
for future use.
The cooling towers which are al80 uled to
dissipate heat from a thermal di8charge
are not new. They have been uled exten-
sively in Europe for many years. In this
country their primary application has been
in small cooling water systems, although
Bome power plants 1n the Southw..t have
utilized towers for cooling.

There are many types of towers, but the
mogt common is the wet, or evaporative,
lype using either mechanical or natural
draft inducement. Mechanical dratt towers
depend upon fan I to create air movement
through the tower packing, which i8 the
heat transfer Bection of the tower. The
main advantage of tho mechanical draft
type of tower is that the air flow is rela-
tively constant, regardless of meteorologic
conditions. Therefore, tower location is
not a critical factor. AhlO, with controlled
air flow, close control of exit cold water
temperature may be achieved.
The principal disadvantage to mechanical
draft towers is their high operating and
maintenance cost, which are incurred
because of power raquirements and wear
io mechanical and electrical components.
A nother disadvantage is that the humid
exhaust is expelled relatively close to the
ground -- probably 60 or 70 feet high -- so
that fogging or recirculation problems may
Thermal PolllBion Overview
The cooling tower which is frequently
utilized In large power plant sYBtems at the
present time is the wet, natural draft hyper-
bolie type of tower. Over %0 of these art'
nO'll' in operation 0'0' in various stages of con-
struction in this country. Hyperbolic towers
derive their name from the profile of the
reinforced concrete shell. The largest
towers are almost 450 feet high and over
300 feet in diameter at the base. The
packing, or heat t!'ansfer section, if located
in or around the 10we1' 20 to 30 feet of the
concrete Ihell, so mC'st of the shell interior
is open space which functions aB a stack or
chimney. Warm water pauing through the
packing, heats the air and adds water vapor
to it. These proceases decrease the air
density and cause it to rise up the tower.
In operation, a continual upward air move-
ment in the tower 1s established.
The main reasons ;01' the increased popu-
larity of natural dril.ft towers as comparpd
to other types is their relatively low operating
and maintenance CO'lts. They do essentially
the same job as mechanical draft towers but
without the use of mechanical or electrical
components. !Illio, the great tower heights
expel moisture laden air at elevations where
they are not likely to result in ground fog or
recirculation problems.

Dry towers, those which do not have direct
air-water contact, are not in general use
for large power plant cooling. They are at
a disadvantage, both in terms of performance
and economics, when compared to their wet
One inherent limitation on dry towers is
that the theoretical limit of cooling is
governed by the dry bulb temperature. In
wet towers this limit is the wet bulb tem-
perature which is usually appreciable lower
than dry bulb. Because of heat transfer
limitations, dry towers require larger hest
transfer surfaces Iled therefore overall
the tower is relatively larger. ' .

The cost of using cooling towers as a methol
or thermal pollution control does not seem
prohibitive. For larJe power plant appli-
cations, for example, wet cooling towers
have increased production costs 1n the
neighborhood of 5 or 6 per cent. However.
in terms ot supply cost, or the cost to the
consumer, the increase amounts to about
one per cent, or slightly more. This Is
becauBe the increaBe in production cost is
dnuted before it gete to the consumer by
oth~r costs, including those for tram.miss!,)n,
fac11111es and equipment, and administratilzl

Thermal Pollution OVerview'
50 in terms of percentage. the cost of pre-
ventin, thermal pollut1e morE: of the energy,
thereby increasing efficiency and reducing
coo11na water requirements.

Other potential methods for large scale
power production include magneto hydro-
dynamics (MHD) and fuel cellll. These do
not require coolini water and hold promise
of efficiflncios ,reater than modern steam
There are additional new methods which are
be1nlinvestigated, but those mentioned leem
to hold the mOlt promise at this time.

Both old and new methodl of power produc-
tion reject enerlY in the form a' waste heat.
Weare hoping to turn this w..ute heat into
Maillet by using it for benencial purposes.
tThis involves a manaJ[ement a~proach and
the concept or total energy utiIUiation. To
implement such an approach, total community
eneriY needl must be analy.!!ed. more ef-
ficient methods of energy utilization must be
developed for each level of conawnption. and
constructive uses must be found for waste
heat. Investigations are .::urrently underway
for valid applications for waste heat in
agriculture, aquaculture or indu8try.

To 8ummarize briefly; there are many
real problems associated with waste heat --
its diepolal. effects and control. But
practical controle are ava1'able today.
Tomorrow we hope control wiU be replaced
by reduction and utilization of waste heat.
I Ronald R. Garton, "Biological Effects
a' Heated Water.' A Technical Seminar
on Thermal Pollution r:ponsored by
Southwest Region. FWPCA, San Francisco,
California. April 1969.

2 Industrial W ute Guide Cln Thermal
pouution, U:SUl, FW PCA, Paclue
Northwest Water Laboratory,
September 1969.
3 The Cost of Clean Water - Vol n
U~1J1. f'W YCA, January 19GB. ..'
U. S. GPO.
This outline was prepared by Frank H.
Rainwater, Chief. National Thermal
Pollution Re8earch Program, PNWWL,
Corvalli8. Oregon.
Descriptors: Heat Pollution. Liquid Wastes.
Pollutants. Pollution (Thermal) Power
~lant8, Thermal Pollution, Wa~te Water
(Pollution), Waate8. Water Cooling, Water
Pollution. Water Pollution SourceB
Water Temperature '

Organizing The Stream Survey
A Theoretical Approach
Thi8 discus8ion will pre.ent as a base,
the procedures for the conduct of an
ideal water quality 8urvey.
B Practical Limitation8
In practice, limitations of personnel,
facilities, time and money, always
will require compromise between the
ideal and the pOlIBible.
C Objectives of Quality Studies
The four ba8ic objectives behind the
almost limitless number of possible
rea80ns for water quality 8tudies are:
Determination of the natural water
quality of the stream.
Measurement, in a seleoted and
limited period of time, of the
existing erfects of wa.te. on water
quality and uses.
Procurement of data on waste loads,
water quality and stream charac-
teristics that will permit projection
of the data to describe probable
water quality and effects on uses
under a variety of conditions other
than thoBe thet prevailed during the
Determination of corrective meas-
ures needed to protect the stream
water quality for proper uses.
WP, SUR, Qf. 3. 74
Available Inforr.lation
Assemble and review all readily avail-
able maps, information, and reports
bearing on the stream under consid-
Problem and Objectives of Study
Define the problem requiring study
as completely as possible on the
basis of available information.
Tentative Plan of Study
Prepare a bdef preliminary plan of
study for guidance in the subsequent
field reconnaissance.
Include in the preliminary plan:
Location.s and strengths of known
sources of waste
Locations of areas of water use
and a list of legitimate water
Section of major stream and
locations of I.mportant tributaries
and unique features such as dams
and points of major diversions
Possible sampling stations
Sampling frequency and number of
samples at each station
8- 1

Water Qualitv Sur"fYs- - Oraanidnll 1'11<' Strea/n Survey
f Types of laboratory determinations
g Existing stn~am gauging stations and
additional points whel'e stream flow
data are needed.
h Other hydrological data needed and
possible means of procurement
i Potential laboratory locations
j Special supplies and equipment
k' Personnel requirements
1 Approximate cost of field operations
A Importance
This step is oCten neglected, but a small
advance party of two or three experienced
field men can obtain information and make
preparations that will save time and money
and avoid much confusion and possible
error when the subsequent field study
B Local Contacts
Contact local agencies and individuals who
use or have knowledge of the stream, and
assemble available maps. reports,
operating records. and verballnformation.
C Sources of Waste.
1 Sewage
a Obtain best available estimate of
sewerI'd population.
b Examine sewage treatment plant, if
any. and obtain copies of operating
records on volumes and characteristics
of sewage.
c Locate all points of discharge and
make any installations necessary for
sampling and gauging.
2 Industrial wastes
a Investigate and prepare flow diagram
of process with especial referencf'
to points of water use and waste
discharge, and qua:ltities of raw
materials and finished products.
b Obtain information on plant operating
schedule, with especial attention to
daily or seasonal ,-ariations. and to
any anticipated changes in process
or increase in proo'lction.
c Obtain data on water consumption.
numbers of plant employees by work
shifts. and disposal of domestic
d Investigate waste treatment facili-
ties, if any, and obtain operating
records on volumes and character-
istics of wastes.
I' Locate all points cf waste discharge.
trace origins in pla.nt ;:>rocess. and
make any installations necessary for
sampling and gauging.
f Collect samples for preliminary
examination in labol'atory to permit
selection of prope." analytical
D Stream
1 Observe entire reach of stream involved
by wading, walking bank. or boat.
2 Note especially points of waste discharge
and dispersion patterns, visual evidences
of pollution confluences a.nd mixing of
tributaries. stream flow characteristics,
approximate widths and depths. and
locations of prospective sampling
stations, and of wa.ter uses.
3 Collect bottom samples of biological
organisms and bottom deposits. and
observe evidences of ?lgae.

Water Quality Surveys. Organbtng The Stream Survey
a Bottom organisml serve as an index
of degree of pollution, and of length
of river that should be sampled.
b Bottom depolits indicate extent of
sludge deposits.
c Algae indicate, probable signiflcance
of photosynthesis and pouible need
for night sampling or light-dark
bottle technique for full evaluation
of dissolved oxygen variations.
4 Determine times of water travel between
pertinent points on stream.
a Knowledge of times of water travel
not only will permit the most intelli-
gent conduct of study, but allo is
eSlential to the complete analyais,
interpretation, &nd projection of
b Time and effort involved are a minor
portion of the totals devoted to the
stream study, and once established,
need not be repeated in subsequent
c Observations should be made at three
or more stream flows to develop a
time of trave~ versus stream dis-
charge curve, which will permit
interpolation for any desired stream
d Methods include: trac1n, an inherent
or added variaole stream constituent;
timing surface or submerged floats;
and determinin, stream cross sections
at selected intervals.
5 Select and identify sampling stations
a For DO evaluation on the majority of
streams, there should be: one station
just above each major waste source;
stations at about half day intervals for
two days time of travel below waste
lources; and stations at about one
day intervals for an additional three
to four days time of travel.
b For coliforn: evaluation, the stations
should be simllar to those for DO
except that the downstream stations
at one day intervals should extend
to a total of l()' to 20 days time of
water travel below the source of
c In very small or very turbulent
streams, natural purification may be
well advanced in as little as one to
two days time of water travel and
stations should be at much closer
intervals th...n sUlgested above.
d If tributary streams are important,
there should be one station near the
mouth of each tributary and one on
the main stream just above each
e Stations should be established as
close as practicable to points or
areas of important water uses.
fAt stations where wastes and tributary
waters are well mixed, one sampling
point near mid-channel usually is
g At stations wI-ere mixing is inadequate,
common practice is to sample at the
quarter points of the stream.
h Identify station& by adequate descrip-
tion and marking, and make any
Qecessary preparations for use by
Select best access routes to sampling
stations ana make a round-trip to
AtaHons to determine time required
for sampling.
E Preliminary Stream Samples
1 Collect one 01' more sets of stream
samples for preliminary examination.
a Dissolved oxygen results will be
useful in establishmg E'ampHng
stations and length of stream that
should be studied

Water quality SurVl'..Y.6 - Orli!anizlnll The Stream Survey
b Determinations of coliform organisms
and BOD wi!! auist in selection of
proper dilutions for subsequent use
when stuay is started.
c Determinations of oth.Jr constituents
will enable analyst to select most
acceptable laboratory procedures and
to determine sizes of sample portions
necessary for those analyses in which
concentrations of conlltltuents govern
choice of portion sizes.
F Laboratory Location
Examine potential laboratory locations in
relation to convenience to the Cield opera-
tions, adequacy of space and utilities,
storage space, and revisio;'1s or additional
facilities that will be needed.
G Miscellaneous
1 Locate local sources of supplies that
wlll be purchased during study.
2 Determine transportation routes and
achedule. for shipments of samples,
supplies. and materials.
3 Arranle for local personnel and special
fBcllltiea that wlll be used.
" Investigate rooming facWties conven-
ient to the field operations.
A Revision of Preliminary Plan
With the preliminary plan as a euide, and
with the knowledge gained from the pre-
liminary field operations it will be possible
to prepare an intelligent, workable plan
for the field study.
B Objectives of Study
Redefine, add to, or delete from, the
initial list of objectives, and prepare a
set of specific objectives which will
include provisions for 'all essential
answers to the problem at hand, but will
also eliminate needless expenditure, on
nonessential matters, of effort which
might better be devoted to the principal
C Period of Field Operations
1 The overall period selected for the
study should be at the time of year when
past experience indicate!! that stream-
flows usually are relatively stable.
2 For evaluation of DO depletion; a period
of warm weather and low IItreamflow
is desirable.
3 For evaluation of coliform contamination,
a period of intermediate flow may be
4 A period of intensive round-the-clock
sampling for a few days generally is
preferable to a period of a month of
sampling daily or on alternate days. but
a combination of both methods may be
better than either onp.
D Sampling
1 General
a If sources of wastes do not vary
si!l1\ificantly during period of study,
the wastes may bp. sampled in
advance of the stream study.

b Sampling procedures and preserva-
tion of samples should follow
"Standard Methods for the Examin-
ation of Water and Wastewater' and/or
procedures recorlmended by the
investigators agen::y.

2 Sewage
a Collect samples around-the-clock
at 15 minute to 1 hour intervals for
three to seven days.
b Composite sample portions in portion
to flow for three to four equal time
periods of each day.

Water Quality Surveys - Organizing The Stream Survey
:1 Indu!ltrial wastes
a In general. the better known the
process is. and the less variable
the waste discharge. the les8 de-
tailed need the 8ampling be.
b For well known processes with little
variation, equal sample portions
collected at one-half to one hour
intervals and composited for eight
hours or for a ,:omplete cycle of
operation on tltree to five days should
be adequate.
c For little known and variable pro-
cesses, detailed and prolonged
sampling may be neces8ary, with
samples collected at 5 to 15 minute
intervals, and comp08itt'd in pro-
portion to flow for six to 24 hour
periods for seven or more days or
complete cycles of operation.
4 Stream
a If streamflow, waste discharge, or
oxygen producUon by algae vary
widely throughout the day, sample
around-the-clock at 8everal stations
for at least one or two days to
establish the daily cycle of variation
in waste constituents and effects.
b In most streams, grab samples each
day or on alternate days for two weeks
to a month i8 adequate if conditions
remain stable.

c The total number of samples collected
from a single station during a field
study generally ranges between 15
and 20. In the absence of wide
variations in streamflow, 12 to 15
sets of samples usually yield
adequate, usa~le results.
d If sampling is not around the clock,
sample collection should be varied
throughout the day u much as is
feasible, and as a minimum. the
direction of the sampling tJllp should
be reversed on alternate days.

e When more than one point is sampled
at a station, individual field deter-
minations are made and a separatE'
bact~riological sample is taken at
each point, but samples for other
determinations may be combined in
a singlE' composite. Bacteriological
samples from two or more points at the
same statior. may be composited in the

E Gauging
1 Metfiods of waste and stream gauging are
covered 1ft another reference outline.

2 Generally stream discharge records
are obtained from existing U. S.
Geological Survey stations, or from
special stations established and rated
by them upon request.
3 Streamflow data lit each main stream
sampling station generally can be
computed with sufficient accuracy from
records for one or two main stream
gauging stations and one for each
major tributary.
F Laboratory Operaaons
1 Select principal determinations that
will measure pertinent waste con-
stituents and tteir effects, and
auxilliary determinations that will
contribute to interpretation of principal
2 Reject determinations that would be
"interesting" but would not contribute
to solution of the problem.
3 Generally useful principal determinations
common to n,any stream studies are
those for coliform bacteria, bottom
organisms, temperature. DO. and BOD.
and auxiliary determinations frequently
are for pH, alkalinity, and turbidity.

Other determinations must be selected
for special purposes and for special
type. of wastes.
5 Laboratory methods should follow
procedures recommended in "Standard
Methods for the Examination of Water
and Wastewater. "

6 Avoid excessive overload of the labor,,-
tory by working closely with the

Water QualitY Surve.Ys - Qr-'lanizinj[ .The Stream Survey
ls.boratory supervisor during the
planning stage and accepting his
estimate of the volume of work that
can be handled.
7 In establiab1ng the laboratory work
load, allow time for cslc'..1lation of
analytical results at the end of each day.
B Ship samples to the headquarters
laboratory for all determinations of
stable or preserved constituents that do
not have to be made in the field.

G Personnel
I The field crew should possells com-
petencies for the specific tasks involved,
or should be trained in advance if
2 Personnel needed commonly will include:
a Sanitary engineers
b Chemists
c Bacteriologists
d Biologists
e Sampling and laboratory aides
H Supplies and Equipment
I Prepare and use a check list of needed
field supplies and equipment,
2 All operations that can be performed
in advance at headquarters, such as
training. purchases. equipment repair.
and reagent preparation will save money
and valuable time in the field.
Cost Estimate
I Revise the initial estimate of tost of
field operations in light of the final
plan adopted.
2 Adjust the final plan tc.. fit the money
available for the study. or arrange
for the additional money needed.
A Briefing
I Assemble the field crew and explain
the problem, objectives, and details
of field operations,
2 Take at least key pereonnel on a tour
of pertinent physical features of the
3 Specify responsibilities of individuals
for the various phases of the operation.
B Communications
Arrange a system of communication by
which any member of the crew can be
contacted within a reasonable length of
C Records
1 A few simple field and laboratory
forms will serve to systematize the
maintenance of basic records.
2 Keep all permanent field and laboratory
records in bound volumes.
3 Encourage the recording of other than
routine observations by both sampling
and laboratory perlonnel,
4 Write down at once, and do not depend
on memory for all significant
D Revision of Operations
I Review all daily data accumulated at
the end of each day, and note especially
any omissions or unusual results.

._B"_-- -- ..-_._--~~
Water Quality Surveys - Organizing The Stream Survey
2 Un '.he basls of the daily review,
c<>nsirJer the need to:
a Add new deteI"JIinations if needed.
b Omll determinations that are not
showing significant results.
c Revise analytical methods.
d Change sampling stations or
scheduled timtos of sampling.
e Investigate ca".ses of apparently
abnormal or erroneous data.
3 Do not revise operations unless the
data or other new information indicates
changes are essential to achieve the
desired objectives.
A Analysis and Interpretation
The raw data are of only limited value
until submitted to analysis and inter-
pretation by an experienced person who
is familiar with the problem involved.
2 Methods of analysis and interpretation
constitute a major subject in them-
selves and are covered by other
3 No amount of statistical manipulation
of the data can produce sound con-
clusions unless the stream study is
soundly conceived, and carefully and
conscientiously executed.
A report should be prepared if the data
are to achieve their maximum usefulness,
since raw data in dead files only rarely
benefit anyone.
Ultimate Disposal
After use, the data should be filed, in-
dexed, and describ d so that they can
be available and understandable years
later to others not directly associated
with the study.
Maximum Usefulness
The results of a stream pollution study
yield their maximum benefit and return
their greatest satisfaction to those who
worked to obtain them only when the
results serve as a basis for actual cor-
rection of an abuse of stream water
Kittrell, F. W. A Practical Guide to
to Water Quality Studies of Streams.
USDI. FWPCA. CWR-5. 1969.
This outline was prepared by Francis W.
Kittrell, former Special Consultant,
National Field Investigations Center,
- - Cincinnati Office of Enforcement and
General Counsel, EP A, Cincinnati,
OH 45268
Descriptors: Baseline Studies, Data Col-
lections, InvestigatiC'ns, Hydrologic Data,
Methodology, Natural Streams, On- Site
Data Collections, On-Site Investigations Surveys,
Planning, Sampling, Stream Pollution, Wastes,
Water Analysis, Water Quality

Organizing The Lake or Impoundment Survey
Lake sampling and data collection are influenced
by physical features, chemical factors, and
biololical communities. ~~ features
include water temperature:tUrbidity, color,
water movement, li8ht penetration, wind
velocity and direction, bottom deposits, and
size, shape and dope of the lake baein.
Chemical facton include alkalinity, pH,
dluolved oxygen, free carbon dioxide,
hardness, nitrogen (organic, ammonia
nitrogen, nitrate nitrogen, and nitrite
nitrogen), phosphate (total and soluble), as
well as other specific elements that may be
of interest in a particular problem.
Biolo cal communities include the Uttoral
commun ty composed of rooted vegetation,
attached algae, fish, and a host of invertebrat..;
the limnetic community, principally fillh and
plankton; and the benthic community of midge
larvae, sludgeworms, fingernail clams, and
other bottom dwelling organisms.
A Development of Oblectives
B Thorough Field Investiption
C Precise Recommendetions Bued on
Factual Findin,s
D Prediction of water quality:
I If recommendations are not met
2 When recommendetions ar-e met
3 If recommendations are only partially met
E Demonstration to pl-ove authenticity of
A Deline specifically the problem; develop
W. RE. lk. 6a.3. 74
B Determine the types of samples necessary
to point to a solution to the problem.
C Delineate the sampll.ng points for each of
the selected types of samples.
D Select the time, and periodicity to sample.
E Judge the number of samples necessary to
complete the study.
I Define specifically the problem.
(Let us assume that the problem can
be defined as one of nuisance algal and
aquatic weed growths. nuisance midge
fly production and occasional shorell.ne
odon. )
2 Determine the types of samples
necessary to pol.nt to e solution to
the problem. These I.nclude:
a Flow measure=nents on streams
I.nfluent to ann discharging from the
lake or reservoir.
b Flow measurements on suspected
municipal and industrial waste sources
and from the receiving streams at
points of waste discharge.
c Water samples for selected chemical
determinations at flow measurement
points, from stream reaches known
to be unaffected by suspected waste
sources, froIl'. area streams carrying
natural land dral.nage only, from
precipitation dur.l.ng study period, and
from area shallow wells.
d Conduct a house to house survey of
shoreline dwellings to determine
method of waste disposal. number of
residences. and period of yearly
e Determl.ne {rom county agents the
types and amounts of fertilizers
applied to the drainage basin and the
application time for the crops

Orpnizir1i the Lake or Impoundment Survey
I Conduct a limnolopcal etudy 01 the
lake or impoundment:
1) Determine certain morphometric
characteriitici includin, area,
mean depth, maximum depth, area
of depth zonel, volume of depth
Itrata, ehorllenath, ar.. of
illandl, shore lenath 01 ieJande,
drainage ar.., aad rate of ruDOlf.
2) conect water lamplee lor eelected
chemical determinationl at
predetermined POintl, and vertically
within the water bod:1. Vertical
temperature and di'lolvedoxygen
determinations are necellary.
3) Collect water samples for
bacteriological determiDatiOn from
predetermined potnts, especially
tholeareall-Illbject to water-contact
recreationallports luch as
Iwl.rnming and skUng.
4) Conect plankton sample.. determine
percent predominant speciel
composition, esUmate avera,e
standing crop.
5) Map. identify. and eltimate.
abundance 01 the 8ubmerged
aquatic vegetation --population.
8) Determine avera,e ltanding crop
01 bottom launa and determine
percent compolitl.on of major
benthic forml.
T) Eltimate .tanding crop 01 filh...
determine predominant Ipeciee.
dtetermine relative iJ'OW'th,ratel.
3 Delineate the eamplir..g points for each
of the types 01 eamplf!s to be collected.
(Many times the limnologtcal samples
from the lake are collected from
imaginary transection lines. )

4 Select the time. 1"8011, and periodicity
of _ple collecttGnl.
5 Judie Ute n.mber of _",piee necessary
to- complete theltudy.
a To ensure -. -well-OJ'pntaed--aurvey
,and'laboratory-opcratt4». con-
lidenti..hould,be -gtven to
dUm.tt.g: the toUtl- number of
lampa.ea.ito-be collected during each
ptale of the st\tdy. Alorm J!uch as
the me that, followl - is often u.ed.
A DeterMine ,loeaUoR 01 .labc)'l'ator'y.
1 ,Field unit" or
2 --P,ermanent .tatioo
B Determine method of sample tlandling.
preservation if any, and traneportation
01 samples to laboratory;
C Select 8urvey teams and,assign duties.
1 , Flow measurement team
2 Stream ....mple collector.
3 Biological team
D Keep collected data cUt'rent, under.tandable,
and avaU.ble to-all partictpants.

Qrlaniz~ the Lake or Impoundment SUIYev

~.-i..a~;,i:?Z. thfo' Lake or Impoundment Survey

1 Coker, Robert E. Str.ams, Lakes, Ponds.
Th. University of North Caroltna Pren,
Chapel Hill, (1954), 337 pp.
2 Hutchin80n, G. E. A TreaUse on LlmnololY.
John Wiley and Sons, Inc., (1957),
1,015 pp.
3 Mackenthun, K. M. and Ingram, W. M. (1967)
Biological A 880ciated Problems in
Freshwater Environment., Their
IdentificaUon, InvesUp.Uon and Control.
United States Department of the Interior,
Federal Water Pollution Control
Admini8tration, 287 pp. (See Chapter 4
elp.cally. )
4 Needham, J. G. and Uoyd, J. T. The
Life of Inland Waten, Comstock
PUblishin, Company, Ithaca, New York
(1937 )
5 Reid, George K. EcolollY of Inland
Waters and Eltuaries. Reinhold
Publishin. Corporation, New York
(1961), 375 pp.
6 R uttner. F FundMmentals of LimnololY.
University of Toronto Pre.. (1953),
242 pp.
OI'.anllina the Lake or ,(mpounann::n. ""... ; :JY-
Standard Methods for the Examination
of Wa tel' and Wastewater. American
Public Health Alsociation, Latelt
8 Welch, P. S. Limnology. McGraw-Hill
Book Company, Inc. (1935), 471 pp.

9 Welch, P. S. Limnological Methods.
The Blakieton Company, Philadelphia,
(1948), 381 pp.
This outline was prepared by K. M.
Mackenthun, Formerly, Acting Chief, Bio-
10,lc&1 and Chemical Section, Technical
Advisory and Investi,ations Branch, Division
of Technical Services, Federal Water
Pollution Control Administration.
Descri ors: Be.seline Studies, Data
o eetions, Hydrologic Data, Impoundments,
Investigations, Lakes, Planning, On-Slte
Data Collections, On- Site Investigations,
Sampling, Surveys, Water Analysis,
Watl,r Quality

Flow measurements are among the more
important data collected during a water
quality survey. Such measurements are
used to interpret data variations, calculate
loadings, and expedite survey planning. If
the analysis of survay data involves estima-
tion of loads, the accurate measurement of
discharge assumes a level of importance equal
to that oflaboratory and analytical results.

In the following di.cu.aion, procedures for
meaaurement of atream flow and waste dis-
charge are deBcribed. Some of these pro-
cedures are used in loni-term, very detailed
water quality and 8UPP\Y studie.; others are
more autted to short-term pollution aurveya.
A Station Location
Four factors influence location of gauging
or flow measurement atatlona:
1 Survey objectives
2 Physical accessibility
3 Characteristics of the atream bed
4 Hydrologic effects
Survey objectives represent the major
influence on station location; depending
upon objectives, :lauging stations may be
located above and! or below confluences
and outfalls.
Physical acceuibll1ty determines the
ease and cost of installation and main-
tBnance of the station. The characteriaUcs
of the etream bed may greatly influence
the obtainable accuracy of measurement.
For instance, rocky bottoms greatly
reduce the accuracy of current metera.
Sedimentation in pools behind control
structures may influence stage-discharge
relationships. Hydrologic variations in
stream flow may cause washout or bypau
of the gauging sta.tlon. In the Southwest,
flash floodB have been known to wash out
or bypaSl! gau,ing stations by anumin,
different channels of flow.
IN. 00. 13a.3. 74
B Methodology
Choice of a specific measurement pro-
cedure is dependent upon at least three
1 The relation b"tween obtainable and
desired accuracy
2 Overall COBt of measurement
3 The quantity of flow to be measured
Ideally, dischargp meal\1Urements should
be reported to a specific degree of accuracy;
the ,auging procedure greatly influences
thi8 accuracy. The influence of overall
C08t on the gauging program is readily
apparent. Exteneive, detailed studies are
u8ually characterized by high costs for
automatic instrumentation and low personnel
coat; the opposite is u8ually true for less
detailed studies. The range of flows to be
mea8ured (within acceptable accuracy) is,
of course, not known prior to the survey.
However. experienced personnel usually
can make reasor.able estimates ot expected
flows from visu81 observations and other
data, and may r.ecommend appropriate
gau,ing procedures. In this regard,
experienced peu->nnel always should be

A Streams, Rivers, and Open Channels

I Current Meter

The current meter Is a device for
meaBuring the velocity of a flowing body
of water. The stream cross section is
divided into.. number of Bmaller Bections,
and the aver8gB velocity in each section
is determined. The discharge is then
found by Bumming the product8 of area
and velocity of each section.
2 Stage-discharge relationships

Large flows usually are measured by
development of and reference to a stage-
discharge curve; this procedure has long
been used by the U. S. Geological Survey.
Such ,augine Btations are composed of a
control structure located downstream of
the location Of measurement and Borne
type of water level indicator which iden-
tifieB the heirht of the water Burface
above a previously determined datum.
10 -I

Flow Measurement
Locatiop of the control structure so
that reliable measurements of flow
will be obtained at all river staps is
particularly important. The water level
may be continuously recorded by an
automatic rec order located in . wet
..U or may be Indicated directly on a
staff gaUie located at t:t. bank of the
river. Such stations must be calibrated
by measurement of now by velocity-
area methods (curpeI't meter) at all
expected sta,es of river flow.

3 Weirs
A weir may be defined as a dam or
impediment to flow, over which the
dl.8charre conforms to an equation.
The ed~e or top surface over which the
liquid flow. is called the weir cre.t.
The sheet of liquid falling over the weir
is caUed the nappl. The difference in
elevation between the crest and the
liquid eurlace at a .pec1tied location,
ulually a point upstre&ll1, is caUed the
_ir head. Head-discharge equations
baaed on precise installation require-
ment. have been developed for each
type of well'. We in .0 in.talled are
called standard weirs. Equations for
non-standard InstallaUons or unusual
types may be derived empiricaUy.

Weirs are simple, reli.ble measure-
ment devices and have been investigated
extensively in controUed experiments.
They are usually instaUed to obtain
continuous or semi-continuous records
of discharge. Limitations of weirs
include difficulty durin, installation,
potential siltation in the weir pond,
and a relatively hl,h head requirement.
O. " - 2.0 floet. Frequent errors in
weir InstallaUon include insufficient
attention to standard InstaUaUon re-
quirements and failure to ..lure com-
pletely free discharge of the nappe.
a Standard supprelsed rectan(Ular
Thl. type of weir Is enenUally a dam
placid acron a channel. The hel,ht
of the crest i8 80 controlled that con-
struction of the nappe in the vertical
direction is fully developed. Since
the end8 of the weir are coincident
with the sides of the channel lateral
contraction is impossible. This weir
requires a channel of rectanlfUlar
cross section, other special instal-
lation conditions, and il rarely used
In plant survey work. It il more
commonly used to measure the d18-
char,. of small IItreL'11S.
The .tandard equation for discharge
of a suppressed rectanlfUlar weir
(Pranci. equation) is:

Q . 3.33 LH3/2
Q . dtecharre. crs
L . len,th of the weir crest. feet
H '" weir head. feet
The performance of this type of weir
has been experimentally investigated
more intensively than that of other
weirs. At lealt six forms of the dis-
char,. equation are commonly
emplo1fld. The standard suppreued
-Iris 80metime... used when data
must be unuaual1y reliable.
b Standard contracted rectangular weir

The crest of this type of weir is
shaped like a rectangular notch.
The sid.. and level ed,e of the crest
are so removed from the sides and
bottom of the channel that contraction
of the nappe is fully developed in all
directions. This weir is commonly
used in both plant surveys and meas-
urement of streazr. diseharge.
The standard equation for discharge
of a contracted rectanf'ular weir
(corrected Franci. equation) i8

Q . 3.33 (L - O.2H)H3/2
Q . discharre. cr.
L . 1enlrth cf the 141_1 crest
eage, f.et
H . weir head. feet
O.2H . correction for end contractionl
al prop08ec1 by Francil

c Cipolletti weir
The CipOlletti weir is limilar to the
contracted rectt..nlfUlar weir except
that the sides of the weir notch are
inclined outward at a slope of 1
horillontal to 4 vertical. Discharge
throu,h a Cipolletti weir occurs as
though end contI actions were absent
and the standard equation does not
include a corresponding factor for

liCk- PligfJ
l'ep 0/ H is
1.. rod "Je rep
U6tter ~Cti01) rePort 1'oQ1.1
etllil llJe'L ~ by C'ecJ Ilt
. "JOcf . II dill. tho
to Pro erellt
The standard equation for discharge
throu,h a Cipolletti weir is

Q . 3.367 LH3/2
Q . discharge, ds
L . length of the level crest edge, feet
H . weir head, feet
The discharge of a Cipolletti
weir exceeds that ot' a suppressed
rectangular weir of equal crest
length by approximately 1 percent.

d Triangular weirs
The crest of a trlangular weir is
shaped like a V' notch with sides
equally inclined from the vertical.
The central anglC1 of the notch is
normally 60 or 90 degrees. Since
the triangular weir develops more
head at a giv~n discharge than does
a rectangular s~ape, it is especially
useful for measurement ot' small
or varying flow. It is preferred for
discharges less than 1 cfs, is a8
accurate as other shapes up to 10
cfs, and is commonly used in plant
Flow Measurement
The standard equation for discharge
of a 900 triangular W8ir (Cone
tormula) i8
Q ..
2. 49H2. 48
Q .
H .
discharge, cis
weir head, feet
Crest height and head
are measured to and from the point
of the notch, respectively.
e Accuracy and inRtallation
Quotations of weir accuracy express
the difference in performance between
two purportedly identical weirs and
do not indudv the effects of random
error in mealJurement of head. Weirs
installed acc\)rdini to the following
specifications should meuure dis-
charge wi thin:I: 5', of the values
observed when the previously cited
standard equations were developed.
1) The upstream face of the bulkhead
and/or weir plate shall be smooth
and in a vertical plane perpendicular
to the axis of the channel.
2) The crest edge shall be level, shall
have a square upstream corner,
and shall not exceed 0.08 in (2 mm)
in thickness. If the weir plate is
thicker than the prescribed crest
thickness the downstream corner
of the ere at shall be relieved by a
450 champfer.
3) The press'Jre under the nappe
shall be atmospheric. The maxi-
mum water- surface in the down-
stream coannel shall be at least
0.2 ft. below the weir crest.
Vents shall be provided at the
ends of standard suppressed weirs
to admit air to the space beneath
the nappe.
I" - 3

Flow Mea.ureme.
4) The approach charmel .hall be
.trallht and of uniform cro.s
88ction for a diltance above the
weir of 15 to 20 timee the maximum
head, or shall be so baffled that a
normal distribution of veloclUes
exiats in the flow approaching the
crest and the wetel' surface at the
point of head meaaurement is free
of dI.turbance.. The eras I.
sect tonal area of the approach
charmel shall be at leaat 6 times
the maximum area of the nappe at
the creat. '
5) The height of tbe cre.t above the
bottom of the approach cn.nnel
ahall be at leaat twice, and
preferably 3 Um..!a, the maximum
head 8I\d not lea. than 1 foot.
For the standard s'lpprea.ed weir
the crest hellht shall be 5 times
the maximum head. The nelaht of
triangular weln shall be meaaured
from the charmel bottom to the
point of the notciI.
6) There ahall be a clearance of at
l..at 3 times the maximum head
between the .ides of the channel
and the intenection of the maximum
water ,aurface with the aides of the
weir notch.
7) For atandard rectangular suppressed,
rectangular contracted, and
CipoUetti. wein the ma:dmum head
.haLl not exceed 1/3 the lenltb of
the level crest edle.
8) The head on the weir ahall be taken
as the difference in elevation
between the creet and the water
.urface at a point upatream a
di.tance of 4 to '0 time. the
maximum head or a minimum of
6 feet.
9) Ihe head used to c.ompute dis-
. .rle aball be the mean of at
_.t 10 aepal'&\e m..al1r.xnenta
~~ at equal illte"*14. 'the
-- rap of the I'Q8&prinl
dlwlce shall be 0..2 . 1. 5 f.et.

necapacitte. of w~ir. wb~ coofO~;
to .... .,.cUl.catbIDa .~"dl.cated
.. T". 1. '.
4 Par-.p flume
The Parshall flume 1s an open conatricted
0-.1 in which the rate of' now is
related to the upstream head or to the
dUference between upatream and down-
atream head.. It con.i.t. of an
entru08 ..cUon with convetpa,
vertioa1.wal1. and level flOor. a throat
section with parallel walls and floor
de~11.n1Di downatream, and an exU
section with diverging walls and floor
inclining downstream. Plan and
sectional views are shown in Figure 2.
Advantages of the Parshall flume include
a low head requirement. dep_dable
accuracy, large capacity ranle, and
self cleaning capabUi~y. It. primary
disadvantage i. the higo cost of
fabrication; this coat may be avoided
by use of a prefabricated flume. Use
of prefabricated flumes during plant
surveys Is becoming increaaingly
a Standard equatione
The dimensions of Parshall flumes
are epecified to Insure alreement
with standard equations. Table of
dimensions are available from
lIeveral .ources 3, 4. For flumes of
8 inch to 8 foot thrxt width the
fol1owinglltandard equations have
been developed.
1) 6 inch throat width
Q . 2.06 H 1.58
2) 9 inch throat width
Q . 3.07 H 1.53

Flow Measurement
Cn" Lullh CGoItn0t8d R.ataapIar. luppr....d R.ot&npla'. ClpoUoUlO 900 TrlanjlU1orO
(F..t) W.1r  W.1r  W.tr Weir 
 (~r,.-cl.1 Idl.char..-cl.J (dl.charre-cf.I (1II."h",.-of.1
 1&8... Min. Max. Min. Max. MID. Max. Min.
1.0 .&80 .288 .131 .291 .131 . ~01  
1. & 1." .n& 1.77 .4.7 1.79 .4U  
2.0 1.1' .584 3.6& .591 I." .1l1li  
I. & ..., .131 1.30 .7.. I.n .753  
1.0 8.11 .181 10.0 .193 10.1 .803  
I.' 11.8 1.03 U.I 1.04 15.0 1.08  
4.0 18.1 1.18 20.. 1.18 20.1 1.20  
4.. .... I." 27. & 1." 21.' l.n  
       8.&1 .046
5.0 18.' 1.48 10.' 1.48 10.8 I. II  
8.0 ".8 1.71 38.7 1.78 37.1 1."  
7.0 41.0 1.07 .... 1.08 n.3 2.11  
8.0 87.1 2.17 ea.8 2." 48.& 2.41  
8.0 11.1 2.87 n.o 2." 11.7 2.11  
10.0 ".1 1.87 81.1 2.18 ".0 I. OJ.  
1HZ 0.111, H~ 1.'''. H~ liS L
.. ..ctl..
"hr..t ..c,I..
..Wate, Jurf.c., .
. . . '.,-
':.:.,.': .:"
'0 - 5

F.", Mea.urement
3) 1 to 8 foot throat width
1 522W 0.026
Q . 4WH .
Q . free-flow di8charlle,
defined a. that condition
which e:llists when the
elevation of the down-
strean. water Artac.
a.bove the creat, H..., does
not exceed a pre.c~1bed
percentalle of the upstream
depth above the crest, H .
The pre8cribed percentaie
of subMergence i. 60
percent for 6 and 9 inch
flumes and 70 percent lor
1 to . foot fiumes
water surface ha. already begun
to decline. Table 2 indicate. the
total head requirements of .tandard
Parshall flumes. These lo.s.s
should be added to the normal
channel depth to determine the
elevation of the water surface at
the flume entrance. No head
10s8e8 are indicated for di8charge-
throat width combInation. for which
Ha is lees than 0.2 it. or lVeater
than 2/3 the sidewall depth in the
converlinl section.
c A ccuracy and instal1ation require-
A Parshall flume 11'111 me.sure
discharge within ¥ 5% of the
studard value it the followini
conditions are observed.
W . throat width, feet

Ha. upstream head above the
flume c.rest
1) The dimensione of the flume
shall conform to standard
The head required by a Parshall
flume is greater than (H -~)
becau.e Ha i. mea.urecfat a point
in the convergina sec.tion where th.
2) The downstream bead, ~, shall
not eaceed the recommenCied
percentage of the up.tream head,
b Head 108s
Dischar,e Head Los., F.et, In Flume 01 Indicated Width    I
 1 foot 2 feet 3 feet 4 feet 5 feet II feet 7 feet 8 leet 
0.5 .08        
1.0 .14 .09 .06      
2.5 .26 .16 .12 .10 .08 .07 .06 .05 
5.0 .42 .2'7 .10 .18 .13 .12 .10 .09 
10.0 .70 .45 .34 .27 -.22 .19 .17 .15 
30.0   .70 .56 .47 .40 .35 .30 
60.0     .68 .57 .49 .41 
H > 0.2,
. -
H < 2.0
a -

Flow Measurement
3) The upltream head .haU be
measured in a It Win, weU
connectea to the flume by a pipe
approximately 1-1/2 inchel in
4) The flume lhaU be inltalled in a
.trailbt channel with the centerline
of the flume parallel to the direction
of flow.
5) The flume lhall be so cbolen,
installed, or baffled that a normal
distribution of v.locm.. ailltl at
the flu... ..trance.
5 Tracsr material.
T.chniquea. materials, and inltrument.
are presently bein, refined to permit
accurate measurement of instantaneoul
or Iteady flow with several tracer
materials. Mea.urementl are made by
one of two methoell:
a Continuoul addition of tracer
b SIUI injection
With the first method, tracer is injected
into a stream at a continuou. and uniform
rate; with the second a sin,le dose of
tracer material i8 added. Both metho~
depend on ,ood transverse mbin, and
uniform dlsperclon throughout a stream.
The concentration ';)f tracer materialil
mealured downltream from the point
of addition. When continuoul addition
il employed, flow ;-atel are calculated
from the equation:

q . C . (Q + q) C
in which q . rate of tracer addition to
the stream at concentration, C, Q. the
Itream flow rate, and c . the relultina
concentration of the stream flow com-
bined with the tracer. For the slua
injection method
c ~ t
in which Q . tbs Itream dilcbarge,
S . the quantity of tracer added, c .
the weighted av..ra,e concentration of
tracer material during its passage p<
the sampling point, and ~t. the total
time of the samplin, period.
Disadvant&ges "f tracer methods include
m1xin., natural adlorption
and intederencllt, and bI,h equipment
6 Float.
Floats may be used to estimate the time
of travel between two points a known
di8tance apart. The velocity so obtained
may be multiplied by 0.85 to give the
average veloci;y in the vertical.
Knowing the mean velocity and the area
of the flowin, stream, the discharge
may be estimated. Floats should be
employed only when other methods are
B Pipes and Conduih
1 Weirs and ParehaU flumes
Weirs and ParsbaJ.1 flumes are commonly
instaUed in manholes and junction boxes
and at outfalls to measure flow in pipes.
AU conditions required for measurement
of open channel flow mu.t be observed.
2 Tracer materials
These methods are popular for
measurement of pipe flow because
they do not require installation of
equipment or modification of the flow.
These are espec1a~ convenient for
measurement of exfUtration and
3 Depth-.lope
If the depth of tne flowing stream and
the slope of th., sewer invert are known,
the di.charge may be computed by
means of any une of several formulas.
a Manning formula
Q . ~ A R2/3S1/2
Q . discharge, cfs

Flow Mea.urement
n . roulbn... co.f~icl8ftt
A . area of flow, ''l. ft.
R . hydrauUc racUu., ft.
. area divided by wette~perimeter, ft.
S . .lope, ft. per ft.
b Chezy formula
Q . CA"fRS
Q . di.charie, cf.
C . friction coefficient
A . ar.a of flow, Ii'l. ft.
R . hydrauUc radiu8, ft.
. area divided by wetted perimeter, ft.
s . .lope, ft. per ft.
.. Oplln eAd pipe flow
The following method. can be employed
w~en other more precie. mean. are not
practical. They can be employed,
hcfiever, only when th:lre 18 free di.-
char.. to the air.
a CoordiAate method
(FilUl'e. 3,4, and 5)

D1echarl' may be computed by the
foUowin, formula:

Q (IPm) . 180\ AX
,A . cr088 sectional ar.. of Uquid
in the pip. (.,. ft.)
X . di8tance betwe.n the end of the
pipe and the vertical puge in
ft., measured parallel to the
Y . vertical distance from wat.r
.urface at the end of the pipe
to theinter.ection of the water
lurfac. with the vertical gauge.
in ft.
- ll'
.. ~ ...11 ., ...Ii
........". .... .. LId
1.1" a. .."lie' L8 .....
1M , .... '.. ..,,,cat

nil - QlnMLL.
~~~ I
~~~.~ .
~~ '\\~~~~~,~ -

f'Dr ..... ,11'1e8:
Fleur. 3
Figure 4

Flow Meaaurement
-..." I"~
. ....
..-.. 18"
. en) ,. ~Uo' II It.....
Figure 5
b California pipe 11011' method
(Figure 6)
Thie method may be ueed only for
horizontal pipes having free die-
charge. If th!! pipe i8 not horizontal,
a connection must be made to one
that is. The horizontal length muet
be not les" than 6 times the diameter
of the pipe. Discharge may be com-
puted by the following formula:
Q (gpm) . T X W
where T . 3.900 (1 - !o) 1.88
W . d2.48
where a and d are measured in feet
~~ ~:t
tI :..:-_- --=:..---=-=
-- ---~
- . """""-' -~~"
.... ... "..,:'$..~
:"'.::::.:n.::;:I:, ':I:'-:-='~
Figure 6
C Head Measuring Devices
Seven! of the above gauging methods re-
quire the meaBUrement of water level in
order that di8cbarge may be determined.
Arr:f device used for thie purpose must be
referenced to some zero elevation. For
example, the zero elevation for weir
meaaurements ill the elevation of the weir
creat. The choice of method i8 dependent
upon the degree I'If accuracy and the type
of record de aired.
1 Hook gauge
The hook ga~ge meallure8 water eleva-
tion from a !ixed point. The hook is
dropped below the water surface and
then rai8ed until the point of the hook
just breaks the surface. This method
probably will give the most precise
reaultll whe!1 properly applied.
2 Staff gauge
The staff gauge is merely a graduated
scale placed in the water so that eleva-
tion may be read directly.
3 Plumb line
Thi8 method involvee measurement of
the distance from a fixed reference
point to the water eurface, by dropping
a plumb line until it just touches the
water surface.
4 Water level ~ecorder
Thie instrument is used when a continu.
ous record of water level is desired. A
float and counterweight are connected
by a steel tape which passes over a
pulley. The 110at should be placed in
a stilling well. A change in water level
causes the pulley to rotate which, through
a gearing system, moves a pen. The pen
traces water level on a chart which is
attached to a drum that is rotated by a
clock mecharu.sm. When properly in-
IItailed and maintained, the water level
recorder will provide an accurate,
continuous record.

Flow Me..urement
Certain portion. 01 thi. outUne contain.
trainini mater1allrom prior O\1tltn.. by
P.I!:. Len,don. A.E. aecher. and P.F.
Atkin.. Jr.
1 Planntn, and Mak1n, Indu"tr1al Wa.te
Survey. - Ohio River Vall.,. Water
Sanitation Commi..ion.
2 Stream Gaulin, Procedure. U. S. GeoJogical
SUFvey. Water Suppl1 Paper !JBB, (1943)

3 Kin,. H. W. Handbook of Hydraul1c..
4th Edition. McOraw-HtU. (19&4)
4 Water Mea.urement Manual. United
State. Department 01 the Interior
Bwe.u of Reclamation. (1987)
Thi. outUn. wa. prepared by F. P. N~on. "
lormerly Acting Regional TrainiJlg Officer,
Northeaat Relional Training Center. EPA,
WPO. Edl.8on Water Qt,a&Uty !\dearcl1
Laboratory. Ediaon. N J OIB 17
ne.criptor.: Chezy Equation. Di.oharge
Mea.urement. Di..r,. (Water), Flow.
Flow Mea.urement, Flow Rate.. Flume.,
Mannin,8 Equation, Open Channel Flow,
Pipe Flow, Streamfiow, Venturi Flume..
Water Flow, Water Level Recorders, Weirs

The validity of water pollution studies is de-
pendent upon an ability to describe the actual
time and space situation of a pollutant al it
blend8 with the natural receiving water. This
understanding is impo!"tant in order to predict
the 8ubsequent effects of the pollutant on the
receiving water and subsequent users or to
detect the source8 of pollution.
The nature of this blending is dependent upon
the physical and chemical propertie8 of both
the pollutant and the receiving water as well
as the mixing and flow characteristics of the
receiving channel, ba8in, or aquifer. Tracing
is an attempt to approximate the actual motion
and mixing of this blend a8 it move8 through
the channel, basin or aquifer.
Although there has been scant reference to
tracers in texts there has been rapidly in-
creasing number of technical articles de-
scribing their use in recent years. A com-
prehenaive compilation of early radioactive
tracer studies may be found in the report of
the "Time of Flow Studies, Ottawa River,
Lima, Ohio,,(l} one of the early attemptl to
compare various tracer techniques under
similar field conditions. Several reports
(see Bibliography) have discussed the use of
dyes during the €arly lG60's.
More refined and extensive tracer investiga-
tions may be expected a8 experience is gained
and a8 more complex studie8 ari8e. A water
quality agency shou\d develop competence in
at least one tracer h"chnique and would be
wile to be capable in some others as well.
Tracer applications are still in a highly
developmental sta,e. One finds many words
or phra8es to 8ugge8t Rimilar field findings.
WP. SUR.tr.lb.3. 74
In broad terms these measurements are con-
cerned with indications of masS movement,
blend mixing, and now direction. Basically,
tracers are used to determine:
A Flow Rates
1 In freshwater channels or lakes such
terms as passagE> time, time of travel,
time of flow, flow quantity or flow
volume are used.
2 In estuaries sllch terms as rate of re-
newal, flushing rate, drift velocity,
mass transfer or net (tidal) drift are
u8ed. The tidal fluctuation imposes a
need for consideration of flow reversal
3 In subsurface waters such terms as
recharge rate, now rate and residence
time are used. Related factors such
as basin capacity, porosity and per-
meability are determined.
B Flow Direction
1 In estuaries
a Direction of flow relative to tidal
phaees whi<'h do not necessarily
correspond to elack conditions.
b Obscure current circulation as in-
fluenced by the interlated factors of
tides, winds, Coriolis forces,
topography and density gradients.
2 In ground water
a Aquifer may flow contrary to initial
euperficial impression.
b Hydrostatic conditions may change
and reverse normal flow direction.

Tracina Natural Waten
C Mix1l1i Patterns
1 Short circuit effecta
a Eddying - prima1"ily the reault of
surface channel and basin
b Stratirication - primarily due to
temperature and denaUy d1ff,rences.
c Inter-connectiolls - between aquifers
in ,round water as well al solution
channels and open h'.dunl.
2 Distribution phenom.en..
a Diapersion of colloidal, soluble and
sUlpended subst.ncel
b D1ffusion of temperatures or galeous
Tracers are used in ma~ IUuationl,
A Treatment Plant Units
B Closed Conduits
C Open Channels
D Large Water Bodies
E Hydraulic Models
P' Subeurface Aquifers
G Subeurface Basins
Materials used for tracers include:
A Floats
1 Surface (wooden and plastic devices &s
well as fruit). Influenced by wind
action and debris.
2 Sub-surface "drogues". Apparent di-
rection must be carefully evaluated.
B Saltl
1 Common salt - hard to detect when less
than 1 mill.
2 Brackish and fresh_ter mixing in
estuaries and coastal aquifers.
3 Ammonium chloride. (See Table 1)
C Dye., auch as:
1 Rhodamine aeries - See section VI
2 pontacyl Brilliant Pink B - most stable
of fiuorescent dyes. Rather expenaive
3 Fluorescein - very ~nexpen8ive. Fluo-
re8ces very near natural stream back-
,round level.
4 Uranine - fiuoresce8 near stream
background level.
D Radioactive substances, such a8: (lee
Table Z) AEC must approve all experi-
ments. AEC has publillhed tables on
permilc1~le concentrations in unrestricted
1 Rubidium-86
2 Iodine-131

3 Tritium (83) gives b~~t overall perfor-
mance in subsurfacetal traoing.
4 Krypton-85 - used recently in gas
tralUlfer measurement in laboratory
and It ream studies. (12)
E Waete return characteristics; significant
built-in factors such as:
1 SUt - understandably dependent upon
velocity and obstruction..
2 Foam - ABS will foam at levele as low
as 1 - 5 mgll depending on hardness.

Traci~ Natural Water'"
Tracer Form
Chloride NaCI
Dextrose SUlar
Chloride NaCI
Dextrose Suear
Dextrole SlAlar
Ammonia Chloride
Boron H3803
Teat conditionl
Lab; Columns of &aDd aDd
sandy loam.
Field: 4. teet thick aquifer ot
sand and Iravel, 90 teet below
Field: Added in Cone. ot 10,000
ppm, ~OO ppm, 50 ppm, and 200
ppm respectively into a 4 foot
thick sand horizon at 2,100 feet
below aurtace in McKean
County. PeMsylvania.
Chloride most rapid. Fluores-
cein tar from satisfactory.
Fluorsscein considered best of
dyes. Chloride in the large conc.
reql!ired caused density currents. ,
Adsorption observed: dextrose -
little or none. boron slight
(cone. dropped 300/.) ammonia -
moderate, fluorescein - strong
(moved at rate about 1/10 t~t of
flood water). Greenburg(33 found
that oorax in Hanford fine sandy
loam penetrated only 2 feel.
'Na2B4007' 10H20 Field: Injection into input wells. Limit o)detection is reached
H3B03 Dilution factor "'6. aner 10 dilution.
Rhodamine B
Field: Water floodinll.
Field: Karst topo,raphy.
Dilution possible 105.
Dye not detected aner 6 X 106
Varill.tions resulting from sea-
sonal fiuctuations are useful to
study climate. (Also useful in
meteorology and glaciology).
Reproduced with permiasion of Iaotop.a. Inc.

Traci~ Natural Waten
Tracer Half-life Fonn Test conditions Remarks
H3 12.5 y. HTO Kar8t groundwater Useful results up to 30 km
   Chalk River soU, W\distur- Fluore8cein dye traveled only
   bed, but penetrated with about 3/4 as rut as tritium.
   driven piezometers. 
   Glaciofluvial sand and gravel, Chromium complex traveled
   various till BOils, and fis. as fast a8 trItium.
   llires and channell. 
saa 89.d Na2f!JJ ~ Chalk River soilllt waste Lags behind tritium indicating
  d1sposal site. some interaction with the soil.
    In soil containihg appreciable
    amounts of gypsum there ~Ould
    be exchanfe and 10s8 of S .
1131 8 d. Iodine, , feet thick aquifer of 8and Should be used 'With several ppm
  carrier free and ll'a'Yel, 90 feet below stable iodiDe carrier.
1131 8 d. KI Tracer compound8 u8ed with C060 complex tltr.suitable in this
Co60 5.2 y. EDTA - (Co) carriers KI, EDTA-(Na) and aquifer. KSCO (CN) appeared
  and K3Co(CN)6' Limel!ltone aqui- to be mOl!lt suitable of tracers
  K3 Co(CN) 6 fer consil!lted of some argil- tested in limestone aquifer.
  laceous material and marly 
   dolomite. Single-well pul8e 
Br82 36 h. NH,Br Aqulfel'8 in alluvium and per- Br82 satisfactory W\der these
  meable strata in calcareous conditions.
   marl series. 
Rb86 19 d. RbCI Groundwater direction at re- Tracer detectable 7 mile. from
   servoir site in Egypt. injection after 5 d.
1131 8 d. Nal Single pulse tracing at dam Detected very high subterranean
p32 14 d. NazHPO" 8ite Cor Jesenitz Reservoir weter velocity necessitating
  on Odrava River in sealing of porous layer.
1131 8 d. NaI Sinlle pulse tracing at dam Detennined direction and velo-
82 38 h. NaBr site on arges River in city of now of subsurface water.
Na 15 h. NaCI Roumania. 
  Reproduced with permission of Isotopes, Inc.

~] Acid - severe effect. upon total .tream
ecology. .If'
" Temperature - infra-r.d fllmir1i
techniql1e. reveal lOC t.mperatl1re
5 Li,nins - "Orzan" i. a commercial
product bued upon the.e non-de,radinc
6 Dye. - prevalent in textile wa.tse.
F Biota, various techniques po..ible with:
1 Mammals
2 Fish
3 Shellfish
Ideally a tracer should be:
A Biololically innocuG\ls to human and
aquatic life, with .~,ecial reference to
1 Acute toxicity
2 Long-term to'l(icity
3 Carcinogenic effects
" Genetic effects
B Stable or persistant despite the effeots of
1 Stream chemical conltituents
2 Bacteria
3 Sunlight
" Ad80rption
5 Temperature
6 Wind action
7 Inherent decay
Tracin.i Natural Waters
8 Channel obstructions
,9 Stratification
C Readily detectable either
1 Visually and/or
2 Instrumentally despite dilution or
background eff~cts;
D Representative: coincide with the real
waste and stream blend under study: this
involves misoibility and specific gravity
characteristics similar to the blend.
E Economic: this irlvolves careful evaluation
of the COlts for
1 Materials
2 Material preparation, and release -
ease of handling
3 Detection equipment
" Detection technique and recording -
convenience in operation
5 Stream deterioration
F Esthetically agreeable: inoffensive to:
1 Taste
2 Sight - conflict with easy detection
in widespread usp. for surface studies. )
A Rhodamine Dyes
1 A vaiIable from American Cyanamid,
DuPont, Allied Chemical and General
2 Price $4.95 per pound except
Rhodamine WT which is $10.00 per

Traeini Natural Waters
3 Specific l1'avity of 0.99 to 1. 13 depending
on the proportions of A leohol. Ethylene
Gl7col. Acetic Acid, and Water u.ed as
solvents in prepar1n. the .olutlon.
4 Solution. normally 1I1~, 2O'fo, 90%, 01"
40% dye by weight.
& pH 1. 0 to 9. O.
8 Peak wavelenlth of ad.orption at 525..&58~
and of fluorescence at 548-5851' are not
normall)' found in natural wate..s.
7 Detectable to O. 5 ~g/l with continuously
samplin. and recordin, equipment.
8 Visibly red to approximately 1.0 m,/l.
9 Rapid dispersion when dropped in water.
10 Subject to some de.tru~tlon by Uaht, but
much Ie.. than experienced with
11 D..troyed by a,ents such as h1Pochlor1te.
Rhodamine WT is more resistant.
12 Euentlally non-toxic.
B Measurinl! Equipment
1 Available from Turner Anoelat...
Beckman Inetruments. Inc.. American
In.trument Co.. Inc.
so two or more runs with temperatures
coverina the expected rar.ge of the study
water are deeirable. Also it is advl...ble
to make a caUbration with the study water.
This will check for backl1'ouod and any
difference in fluorelcent characteristics.
A few helpful hints are:
1 Pumpinl a known concentration sample.
from a plastic bag immersed in a
consant temperature water bath,
throulh the meter Is k convenient
calibration procedure. Continuous
dous1n. with a concentrated solution
into the bag will give ""rioue iDIItrument
readin.s through the detectable ranges.
2 Flush the sy.tem with methyl or butyl
alcohol solution before each calibration
to remove traces of dye.
3 Bubbles in the flow cuvett., win cause
erratic readin,s.
4 The fluorometer door is not li,ht tight.
so the inatrument sho'Jld be draped and
used out of intense light to prevent
erroneous hiah readin,s.
5 The .ample lines on a continuous flow
.ystem wiU tran.mit lil!ht tbrOUfh the
inatrument door cau.in, erroneous high
readin,s. Taping these t1abes with
black electrical tape for about one foot
willaUeviate problem.
2 Price as low as $1. 000 for photofluorometer D Stud7 Procedure.
complete or $500 for unit to mod1fya
3 A vailable for individua1 arab sample
analysis or continuous analysis of
..mple pumped through Instrument.
4 Automatic recorders avaU~ble.
"5 Will measure several dyes (fluore.cein,
rhodamine, etc.)
C Fluorometer Calfbration
The instrument .hould be calibrated before
and .ner a .tudy. The calibration i. a
function of temperature (about 2.3%/0 C)
1 Select objective. which then dictate:
a Lenrth of time to be studied
b Relea.. point
c Monitoring locations and acbedule8
2 Determine physical properties of
affected area.
a Probable net flows
b Total water volumes
c Probable water temperature

d Natural fluore8cent backcround
e Salinity
f Su.pended .ol1d.
3 E.t1mate required quant1t1e. at dye
4 Calibrate fiuorometers
5 Conduct mock r.m to te.t equipment
8 Public relation.
7 Relea.e and monitor dye
8 Proce.. and interpret data
9 Report
A MOlt Disper.ion Equation. are Normalized
Solutions of Fickl Second Law
c}t x ~x2
c . concentration, x . di.tance,
t . time, Dx . diffu.ion coefflcient
B Hydraulic Model Studie. of the Fate of
From instantaneoul releale compute
Iteady state noncon.ervative pollutant

C(xy) ./f(t) + i (t)
where f (t) repre.ent. concentration
function and, (t) . decay funcHon
C Coastal WatU Di.perlion Study, Hilo
Bay, Hawaii )
1 From instantaneous releale mea.ure
concentration profile durlna Ilack
Trada\a Natural Watt'18
:I Usina thelf! measurements and a two
dimensional dispersion model com-
pute D and D .
x Y
3 Two dimeWlional model
:I :I
~c D a c + D 3 c
3T. x ax2 y 3l
ox ()y
U . velocity in x direction
V . velocity in y direction
Normalized solution:
C .
a:'~y ''P - i [E - ~~+ ~ - :~]
D Channel Studies
1 From instantaneoul release compute
longitudinal dispersion coefficient
:I Mealure time-concentration profile
at liven atation
3 Thi. C va. t curve fits equation
C .
M (x - ut) 2
J. exp - 4 Dt
A (4wDO a
. Take log of both sides
t. (M\ (x - ut)2
Ct log \,A4irDJ - 4 Dt log e
II D can be determined from semi-log
1 (x - ut) 2
plot of Ct VS. t

Tr~~il),i Natural Waten
1 ~raub, C.P., Ludza.ek, F.J., Hagee,
G. R" and Goldin, A. S. Time of
Flow Studies. Ottawa River, Uma,
Ohio. Transaetionl, Amer. Geo-
physical Union, Vol. 39. pp '20-
426. 1958
Feuerstein, D. L., and Selleck, R. E.
Flourescent Tracers for Dilperaion
Meaaurements. Journal of the
Sanitary Eniineerin, Division, ASCE,
Vol. 89, No. SA4, Proe. Paper 3:188,
pp 1-21. August, 1963.
Buchanan, Tbomas J. ':'1me of Travel
of Soluble ContaminaDta in Streama.
Journal of the Sanitary Enlineerin,
DLVilion, ASCE, Vol. 90. No. SA3,
Proe. Paper 3932, pp 1~ 12. June, 1964. 12
4 Wriiht, R.R. and ColUn'l, M.R. Appli-
cation of Fluoreacent Tracing Tech-
niques to Hydrolo(l1e Studiea. Hydraulic
Engrs., USGS, Atlanta, GA.. Jour.
AWWA, Vol. 56:7'8. June, 196'.
5 Pritchard, D. W. and Carpenter, J. H.
Measurementa of Turbulent Diffusion
in Eatuarine and Inlhore Waters. Ches-
apeake Bay Institute, JOhnl Hopkin.
Univerlity. Contribution No. 53. 1960.
6 Ault, W.U.. and Hardeway, J.E. Surface
Tracing with Radioilotopel. IIotopics,
Ilotodel, Inc. Vol. :3 U. January, 1965.
DLachi8hin, A. N. Dye Dilperlion Studie s .
Jour. San. Engr. DLv., ASCE, Vol, 89,
No. SAl, Proc. Paper 3386, p. 29.
January, 1963.
8 O'Connell, R. L.. and Walter, C. M.
Hydraulic Model Teltl of Estuarial
Waste Diaperlion. Jour. San. Engr.
Div., ASCE, Vol. 89, No. SA 1, Proc.
Paper 3394, January, 1963. p. 51.
9 O'ConneU, R. L" and Walter, C. M. A
Study of Diaperlion in Hilo Bay,
Hawaii. Report pres:ared for the
U. S. Army Engineer District,
Honolulu, Hawaii. September, 1963.
10 Taivo,lDu, E. C., et a1. Tracer Measure-
mentl of Atmospheric Reaeration.
t. Laboratory Studtes. Prelented at
the Water Pollution Control Feder-
ation Conference, Bal Harbour,
Florida. September, 1964.

11 Cawley, W,A. and Rutledge, W. C.
Application of Radioactive Tracer
Techniques to Stream Improv4IDent.
Journal, San. Eng. Div., ASCE, Vol,
92, No. SAl, Proc. Paper 4640, p. I,
February, 1966.
Tllvoilou, E.C.. O'Connell, R.L"
Walter, C. M,. God.u. P. J., and
Loi.don, G. S. Tracer Mealurements
of Atmolpheric ReaeratiOll.-1, Labor-
atory Studi.l. Jour. WPCF, Vol, 37,
p. 1343.
13 U. S. Atomic Energy Commission, Re-
print from Federal Register, 17915,
Part 30.70, Dec. 17, 19114, p. 5.
l' American Cyanamid Company, A Dis-
cUllion of Techniques and Tracer
Dye. used in Water Trac1ag, Dyes
and TextUe Chemicals Department -
Bound Brook, NJ 08805
This outline was prepared by Dal. B. Parke,
Former Sanitary Enlineer, Hudson-Delaware
Ba.ins Office, EPA, WPO, Edison, NJ
.g;lcrr;.; Fluorescence,. Fluorescent
e, . at PoU\1tuts, Rad~oactivit:y
Technique., RadioilotOpes, Tracer.

Objective of Sampling
1 Water quality characteristics are not
\Ultform trom one body of water to
another, from plice to place in a
given body of water, or even from time
to time at a fixed location in a liven
body of water. A sampling program
8hould recognize 8uch variations and
provide a basis for interpretation of
their effects.
The purpo~e of collection of samples is
the accumuiation of data which can be
used to interpret the quality or condition
of the water under investigation. Ideally,
the sampling program should be so de-
.igned that a statistical confidence
Umit may be al!8ociated with each
element of data.
Water quality surveys are \Uldertaken
for a great variety of reasons. The
overall objec~ives of each survey greatly
influence the location of sampling
.tations, "ample type, scheduling of
.ample coll~ction8, and other factors.
This influence should always be kept in
mind during planning of the survey.
4 The samplini and testini program should
be estabU.hed in accordance with princi-
ples which will permit valid interpre-
The collection, handling, and testing
of each sample should be scheduled
and conducted in such a manner as
to aS8Ure that the re.ults will be
truly representative of the SOl rces
of the individual .amples at the time
and p)ace of collection;
The locations of sampling 8tations
and the schedule of sample collections
for the iotal sampling program should
WP. SUR. sg. lb. 3. N
be established In such a manner that
the stated Investigational objectives
will be met; and
Sampling should be sufficiently
repetitive OVE'r a period of time to
provide valid data about the condition
or quality of the water.
B Sample Variations
Interpretation of survey data is based on
recognition that variations will occur in
result8 from individual samples. While
it is beyond the !lcope of this discussion
to consider the implications of each in
detail, the following can be identified as
factors producing variations in data and
should be consiC:ered in planning the
samplinll program.
1 Apparent Variations
Variations of a statistical nature,
due to collection of samples from
the whole body of water, as con-
trasted with Poxamination of all the
water in the system.
Variations dU9 to inherent precision
of the analytical procedures.
Apparent variations are usually
amenable to statistical analysis.
:I True Differences
Variations of a cyclic nature
Diurnal variations, related to alter-
nating periods of slD1light and
Dirunal variations related to waste
discharges from communities.

Sa~.!nl in Water Qua!!!1. Studies
--- ------------------.--
Seasonal variations, related to
temperature and its subsequent
ertects on chemical and biolo.ical
proce!l8es and interrelat1olUlb1ps.
Variations due to t1dalinfillences,
in coastal and estuarine waters.
b Intermittent varia~ions
Dilution by rainfall and runoff.

Effects of irregular or intermittent
discharles of wastewater, such as
"slu.s" of industrial wastes.
Irrelular release of water from
impoundments, as fr?m power
c Continuine chanlu in water quality
Effects downstrean: from points of
continuous release of wastewater.
Effects of confluence with other
bodies of water.
Effects of pa8sa,e ?f the water
through or over polo,ical forma-
tions of such chemical or physical
nature as 11:> alter the charl!-cteristic8
of the water.
Continuing interactions of biolo,ical,
physical, and chemical factors in
the water, such al in the proce88 of
natural self-purification following
introduction of or,anic conte minant8
in a body of wa ter.
A The Influence o{ Survey Objectives
Much of the IIRmplin, desi,n will be
loverned by the stated purpose of the
water investigation. As an example of
how different objectives might mfiuence
lampling design, con8ider a watercourse
with points A and B located as indicated
in Figure 1.
-.---} .
Figure 1
Point A can be the point of discharge of
wastes from Community A. point B can
be any of several thin,s, such as an intake
of water treatment plant supplying Com-
munity B, or it might be the place where
the river crosles a polith:al boundary, or
It may be the place where the water is
subject to some legitimate use, such as
for fisheries or for recreational use.
I Assume that the objective of a water
quality investigation is to determine
whether desilnated standards of water
quali\y are met at a water plart intake
at POint B. In this case, the objective
only Is concerned with the quality of the
water as it is available at Point B.
Samplinl wUl be conducted only at
Point B.
2 Alternately, consider that there is a
recoll1ized unsatisfactory water quality
at Point B, and it is IIlleged that this
il due to discharges of inadequately
treated wastel, orilinaUna at Point A.
A88ume that the charge iB to investigate
this allegation.
In thi. case the Belected sampling Bites
will include at lealt three elements:
a At least one sampling site will be
located upstream from Point A, to
establish base levels of water quality,
and to check the possibility that the
observed conditions actually originated
at some point up.tream from Point A.
b A site or sites mUst be located down-
stream from Point A. Such a site
should be downstream a sufficient
distance to permit adequate mixing in
the receivinl water.
c Sampling would be necessary at Point
B in order to demonstrate that the
water quality is in fact impaired, and
that the impairment is due to infiuE'nl'E'1I
traced from Point A.

B Hydraulic Factor.
1 Flow rate and direction
a In Ii survey ot an extended body or
water it i. necessary to determine
the rate and direction or water move-
ment inflllence. selection or sam-
pling sites. Many workers plan
sampll.n8 stations representing not
less than the distance water flowe
in a 24-hour period. Thue, in
Figure 1. interven1ni samplin.
stations would be eelected at points
representing the di.tance water
would flow in about 24 houre.
b In a lake or impoundment direction
or flow 11 the major problem influenc-
ing selection or eampling stations.
Frequently it is neceuary to eBtab-
Ueh eome SOk.t or grid network ot
statione in the vicinity ot the sus-
pected sources or pollution.
c In a tidalutuary, the oecillating
nature ot water movement will re-
quire establishment or sampling
etations in both direction. rrom
euspected sourcee of pollution.
2 Introduction or other water
a In situation. in which a .tream being
.tudied is joilled by another stream
or significant Bize and character,
nmplin, stations will be located
immediately above the extraneous
stream, in the extraneous stream
above its point of juncture with the
main stream, and in the main
stream below the point or juncture.
b Similar .tations will be needed with
respect to other water discharres,
8uch as trom industrial outfalle,
other communities, or other inetal-
lations in whi~h water is introduced
into the main .tream.
3 Mixing
a Wherever pl>uible, one sampling
point at a nmple collection 8ite is
Sampling in Water Quality Studies -
u8ed in st~'eam surveys. This
ulually il near the surface of the
water, in the main channel or flow.
b In some streams mixing does not
occur quickly, and introduced water
moves dowr.!ltream for considerable
distance. below the point ot con-
fluence with the main streams.
Example: SUillquehanna River at
Harrisburg, where 3 such streams
are recogl'\izable in the main river.
Preliminary survey operations
should identify such situations.
When necessary, collect separate
samples at two or more points
acro.s the body or water.
c Similarly, ~'f!rtical mixing may not
be rapid. This is noted particularly
in tidal estuaries, where it may be
necessary to make collections both
trom near the bottom and near the
surlace or the water.
d Collection of multiple samples from
a station requires close coordination
with the laboratory, in terms of the
number at samples that can be
examined. Some types of sample s
may be composited. The decision
mu.t be ruched separately tor each
type dr sample.
C Types or Analytical Procedure
1 Samples collected ror physical, chemi-
cal, an:! bacteriological tests and
measurements may be collected from
tl\e same series or sampling stations.
2 Sampling stations selected for biological
(ecolol1ca1) investigation require
.election ot a series of similar aquatic
habitat, (a series of riffle areas, or a
series ot pool areas, or both), The
81tes used by the aquatic biologist may
or may not be compatible with those
used tor the rest of the survey. Accord-
ingly, in a given stream survey, the
stations used by the aquatic biologist
"uall,v are somewhat different from thp
stations used f;)r other eXamina~i"ns.

S~inl...!!J WateE...9uality Studies-
D Acce8S to Sampling Stations
For practical reasons, the sampling site
should be easily reached by automobile 11
a stream survey, or by boat 11 ~he survey
is an a large body of water. Hi8hway
bridges are particularly useful, 11 the
sample collector can operate in safety.
A Survey Objectiv811
B Time of Year
1 In short-term water qJ.ality investiga-
tions, particularly in pollution
investigations, there often is need to
demonstrate the extremes of pollution
effects on the aquatic environment.
For this reason, many short-term
surveys are conducteu during the
warmer season 01 the year, at such
times as the water flow rate and
vdlume is at a minimum and there is
minimum likelihood of f'xtensive
ra i»,fal1.
2 In a long-term investi;lation, sampling
typically is conducted at a11 seasons
01 the year.
C Daily Schedules
As shown in an introductory parall'apb,
water quality is subject to numerous
cyclic or intermittent vari,tiona. Sched-
uling of sample collections should be de-
signed to reveal such variations.
1 lit short-term surveys it is common
practice to collect samples Irom each
sampling site at stated intervals tbroulh
tbe 24-hour day, cont1nuine tbe program
for 1 - 3 weeks. Sampling at 3-hour
intervals is preferred by many workers.
tKough practical considerations may re-
quire extension to 4- ur even '-hour
~ In an exten:ied survey there is a ten-
dency to collect samples from each
site at not more than dai~y intervals,
or even lonler. In such caseB the
hour of the day should be varied through
the en'l!"e Prolram, In order- that the
final s\.!rvey show cyclic or irttermittent
variations 11 they exl.t.
3 In addition, samplinlln tidal waters
requires consideration of tidal flows.
If samples are collected but OI1ce daily,
many workers prefer to malee the col-
lections at low slack tide.
4 In lonl-term or any other survey in
which only once- daily samples are
collected, it is desirable to have an
occ8.iOl1al period of around-the-clock
A River Mile System
The FWPCA method of identifying points
on a water courle il by counting river
miles from the mouth (or junction with a
larger .tream) back to the lource. This
should not be confused with other systems,
such as tbolle in which the river mile is
started at the source of the stream and
proeeeds to the mouth of the .tream or
confluence with another b:xiy of water.
B STORET 5y.tem
The STORET 5y.tem is a computer-oriented
data procening system used by FWPCA for
storage, retrieval, and analysis of water
quaUty data collected by federal. state,
loeal, and private agendes.
The s)llltem includes a complex system -
ba.ed on the river mUe .yetem - for
identifying sampling location. on all rivers
and streams in the United State.. A recent
addition to the .ystem introdllCes a location
procedure baaed on geolraphic coordinates;
this procedure is especir.lly ad8Pted to
location of sampling sta..tbns in large bodies
of water such as laIcel and impoundments.

Not all location. have been coded at thil
Ume, althoulh tbe :'3odinl .y.tem. have
been e.tabl1.hed, The inter..ted worker
.houJd oanlUlt PUb!1c Health Service Publi-
cation No. 1263, "The Sto..... and Retrieval
of Data for Water Quality Control." 1963.

A Type. of Sample.
1 "Grab" sample - a grab .ample is u.ually
a manually co~ected .illlie portion of the
wastewater or .tream water. An analyd.
of a grab sample show. the concentration
of the constituents in the water at the time
the .ample was taken.
2 "Contlnuou." .ample - when .everal point.
are to be sampled at frequent interval. or
when a contl.mlOll8 record of qualit, at a
liven lamplinl .tatiOn 18 reqllired, an
automatic or continuou. .ampler may be
a Some automatic samplers collect a
given volume of lample at definite time
intervals; thi. i. .ati.factory when the
volume of flow i. constant.
b Other automatic aamplen take .ampl"
at variable rates in prOportion to chang-
ing rates of flow. This type of .ampler
requires .ome type of now mea.uri.lg
3 "Composite" .ample - a compo.ite
I&mple is the collection and m1xilll
together of various individual .amples
based upon the ratio of the volume of
now at the Ume the, individual sample.
were taken to Ihe total cumulative
volume of flow. The de. ired compo.ite
period will dictate the magnitude of the
cumulative volume of now. The more
frequently the !lamples are collected,
the more representative will be the
composite sample to the actual .1tua-
tion. Composite .ample. may be
obtained by:
a Manual sampling and volume of flow
determination made when each .am-
ple is taken.
Sampling in Water Quality Studies
b Con.tant &utomatic sampling (equal
volumu of nmple taken each time)
with flow determinations made as
each sample is taken.
c Automatic sampling which takes
aamples at pre-determined time
intervals an1 the volume of sample
taken is proportional to the volume
of flow at any given time.
B Type of Sampling Equipment
1 Manual sampling
a Equipment il1 specially designed
for collection of samples from the
bottom muds, at various depths.
or at water surfaces. Special
dedp. are related to protection of
.ample Intelrity in terms of the
water ch&racteristic or component
beina measured.
b For details of typical sampling equip-
ment u.ed in water quality surveys.
lIee outlinee dealing with biological,
bacteriological. and chemical tests
in this manual.

c Manual sampling equipment has
very broad application in field work.
as great mobility of opera tion is
pOllsible, at lQwer cost than may be
pouible with automatic sampling
2 Automatic samplinA: equipment
Automatic sampling equipment has
.everal important advantages over
manual methods. Probably the most
important consideration is the reduction
in perioMel requirements resulting
from the use of this equipment. It
a180 allows more frequent sampling
than i8 practical manually, and elimi-
nates many of the human errors in-
herent in manult! 8l1.mpling.
Automatic saI1'1i'ling equipment has
80me disadvantages. Probably the
most important or these is the tendency
of many automatic devices to become

~~~U1\1 in Water Qual~ Stuaif!s
cl0lled when liquids hiah in solids are
being sampled. Individ..\al portions of
composite I&Inple8 are uaually quite
small which may in 80me cases be
disadvant8aeous. In Ulin. automatic
sampler.. .ampling points are fixed.
which relults in a certain 1088 of
mobiliv as compared to manual
a Compositing liQ~lera
Automatic sampling equipment Ihould
not be used indiscriminately; some type:>
of samples - notably bac.teriolopcal.
biological. and 00 l&IqIlu - should
not be compolited. In Q~.el of doubt.
the appropriate anal1at &culd be
1) Jar and ~be8&mpler - tbia typl'
aamp!" e.£fectively wballow
is nearly constant. AI ...ter
drains from the upper carboy.
the vacuum created syph.1
waste into the lower one. The
rate-ot-flow 1s relUlated by the
pinch clamp to fill the lower
carboy dur ing the samplin,
period. (See Figure 2 )

Z) ScOOt tJpe
a) """1nI .coop
'tta18 ctevie e cOG.i8t. 01 .
pow'" driven .cOOp mOUlited
""'1',,11\ from a wetr. The
.coop 18 80 de8l.ped and
1D0000ted that the .amp18 yol-
\lime p'abbed on 8ch rotation
01 the .coop i. proportional to
tile flow, a. IOverned by tbe
head on the weir. The .coop
IMY be rotated at a coutIDt
epe.d or timed to Ample
at lilled time interval..
b) Revolvin, wheel with cupe
(1I'il\ll'e 3)
TU. device cmeiet8 ot a
power driven wbeel or dilc
mounted upltream trom a
weir. A number ot treely
.1I8p8:1ded bucket. are mount-
ed at varym, di.tlDc.. trom
the axie 80 that increaeed
now will caule more buclcet8
to be filled, thereby livin, a
.ample proportionate to now.
Both thie device and the
rotatin, ICOop .ampler can,
ot courle, be u.e4 tor non-
proportionate lamplin,.
c) Buc~"t elevatore
Thi. device may conailt at a
11nfle bucket altlrnately
lowered into and railed out of
the waite Itream, or it may
conl18t of . lerl81 ot buckets
on an endle.. main pa"ini
throup th8 w..te .tre.m. In
either c..e. it will include a
tripp1nf m.chanllm to caull
tile bucket 01' bucketl to .pill
into. sampl1nf funnel. Both
type. may be operated contin-
uOUI1)' or timed tor intermittent
operation. This method il not
well adapted to proportionate
Sampl1nrJ!!.~r Qua11ty- Stu~~
Willi GIll8\'
3 Pump.
a) Chemical feed pumps have
been found ueeful for sampling.
becDU8e of the ir ability to
met'!!r out .mall doses of
liquids. A timing mechanism
may be used to make the pump
run far longer periods during
heavy now, thereby allowing
collE1ction ot the sample in
proportion to now. These
pumpil are usually provided
with &djustable stroke and
variable speed 'feature s which
allo-.w variation ot the volume
of lample being pumped.
1I'igl,Jre 4 1llustratee a battery
operated pump.
b) Auiomatic shift sampler
en"..re 5 )
FtIJU'l S ,hows the automatic
shift sampler. It consists
fir8t of a Randolph or other
".queeiee-" type pump unit.

S~~i in Water Quality Studif!s
The 2-rpm gear motor drives
the pump at between 1 and 2
rpm through the spring-loaded
adjustable-pitch pulley and
adjustable motor-base arrange-
We use liS-in. (. 32-cm) ID
or 1/4-in. (. 64-em) ID
polyethylene tubing for sam-
ple intake from the waste
stream. The sample now is
delivered to the distributor
via a 3/16-in. ~.48-cm) ID
TYlon tube which is supported
100se1y by a wire attached to
the framework.
Operation of the distributor is
very simple. The 1-rpm clock
motor powers the chain-end-
sprocket drive which turns a
threaded bolt. Rotation of the
bolt moves the di.charge tube
down the pla.Uc trough at a
rate equal to one division
every eil{ht hours. With the
10 sample-jar receivers the
timer can bti: .et on Friday,
and the 9 week-end shift
.alBf"les can be picked up on
Figure 4
I~- 8
4) Solenoid-valve arrangements
A solenoid valve employed in
connection with a timing device
may be used for withdrawing
waste from a pipe under pressure.
Used in connection with a pump
such dev,lces may be employed ~
. sampl1n. free nowin, streams.
(See Figures 6 and 7. )
118IIU... -
5) Vacuum operated
In its simplest form, the vacuum
Is created by a suitably mounted
siphon. It 'collects the sample
at a uniform rate and is not
suitable for use when prCilportional
sampling is required.

,"It.. " ......
c:.et"n.. ""',
..... '\
III.... ...... ...,
-.- -.
-...., .....,.. .,
.. .....
Fl,ure 7
6) Air vent control
This type of device 18 illustrated
in Figure 8. The rate of sample
c collection i.. controlled by the
bubbling mechani8m. It is not
suitable for UJe when proportion-
ate samplin, 18 required.
L.. '--
Figure 8
Samplini in Water qua.l1ty Stlldies
7) Drip sampler
Two types of this device are
illustrated in Figures 9 and 10.
Both devi;:es are simple methods
01 obtaining a composite sample
at a fairly constant rate.
TO '''''''L AND IIMT
. 1/ WATlII ...,

Figure 9
.... -
Figure 10
I~- 9

Samplini in Water Quality Studies
Continuous recording equipment

Instruments have been developed
which provide dire( t measurement
of temperature, pH, conductivity,
color, and dis.olved oxygen. Such
instruments may be equipped for
continuous recording. Instruments
of this type are quite expensive and
their installation is otten difficult.
They are best adapted to permanent
tnatallations, although ,ood portable
non-recording instruments are
available for the measurement of
temperature, pH, and conductivity.
All procedure I in care and handlin, of sample.
between collection and thv performance of
observationll and testa are C:1rected toward
maintaining the reltabWty of the sample as an
indication of the characteristics of the sample
A Sample Quantity
Samples for a series of chemical
analY8es require d
that eaee, -::onvenlence, and reliability
of reeults are acceptable for the pur-
po8ee of the 8tudy.
Samples to be analyzed in the laboratory
require special protection to assure that
the quality measured In the .ample repre-
sents the condItion of the source. Many
8amples, IIIpeclaUy those 8Ubjected to
biological analy.is, require 8pecial pre-
servation, protection, and handling pro-
cedure8. In ca8e of doubt, the appropriate
analyst should be consulted. Most com-
mon procedur~s for 8ample protection
. Examination within brief time aner
b Temperature control.
c Protection from light.
d Addition of pre8ervatlve chemicals.
Applications of the8e .ample protective
procedures are along the fol1owin,
Early examinatlo:! of sample
Applicable to all types of samples.
Temperature ..:ontrol
a All biological materials for examina-
tion In a living state should be Iced
between collection and examination.
b Bacteriological samples should be
iced during a maximum tran8port
time of 6 hours. Such samples should
be refrigeratec:i upon receipt in the
laboratory and proceesed within 2 hours.
c Chemical samples often require
Preservation by refrigeration at 40C
II recommended for acidity, alkaUnity,
BOD, color, sulfate, threshold odor,
and other samples. Holding times vary.
~mp1inlr in Water Quality Studiel
Quick freez\ng will permit retention
of 1X1&IV' samp18s for up to several
months prior to laboratory examina-
5 Protection from llrht
a AIV' conetituent of water which may
be influenced by physiochemical
reactione involving light should be
protected. DO lamplee brought to
the iodine Itage, for example, should
be protected from light prior to
In addition. arIiY water oonstituent
(suoh ae dissolved oxypn) which
may be Influenced by aleal activity
ehould be protected frOlD light.
6 Addition of chemical preservatives
a Bacteriological eample. never
ehould be "protected" by "&dciition
of preservative agents. The only
permiseible chemical additive is
sodium thiosulfate, which is used
to neutralize free residual chlorine,
if present. '
Samples for biolqrical examination
should be protected by chemical
additives only under specific
direction of the prindpal biologist
in a water quality study.
For chemkal tests, preservatives
are useful for a number of water
components. The following examples
are cited:
Nitro~en and pholphorus analyses:
The addition of 40 mg HgC12 per
liter of sample and refrigeration at
40C will ret..rd biological activity
which might otherwise alte, the
concentration of these constituents

9I.mpllnl( in Water Qualitv Studi.s
Metals: The addition of 5 ml of
}{N03 acid per liter or .ample
prevent. preciptt..tion of the metal
in the container.
CO~ id O~'fc c::on: Addition
of m su r c ac per liter of
sampls i8 8U,...tid.
In ,.neral, sample. requ.irin. re-
be temporarU~ pre.erved by addi-
tion of chloroform; te.ts should be
run as soon a8 pOI.ible, however,
Cy~~ deterll\1nat1on. may be
de yed temporarily throu8h addition
of alkali to the sample. Addition of
sodium hydroxtde to pH 10 is recom.
Sulfide ua]y.is may be delayed up
to 7 days by addition of 2 ml/liter of
sample of 2N solution of zinc acetate.
Phenol analylis can be delayed
temporarily by acidification to pH 4. 0
with pholphoric acid and preaenation
with 1 rram CuI04' 5H20 per liter
of sample.
1 Stan~rd Method. for the Examination
Water, Sewa.e and Industrial Wastes.
13th Ed. A.P.H,A. 1871

2Plannin. and. Maldna lndII8trial Waste
Survey.. Ohio Rinr Vau.y.Water
Sanhat10n Commission.
Industry's 14.. Clinic. JOII1'Dalorthe
Water PoUution Control Federation.
AprU, 1185.
Thi8 0Iril1ne was prepared by H. L. Jeter,
Director, National Traim.nl Center, EP A,
WPO, Cincinnati, OH 45288 and P. F.
Atkinl, Jr., tormerly.Slnitar,y Engineer,
Training Actintiel.
Delcrt 01'1: Inltrumentation, Investigations,
atura reams, On- Sitl!lnvesti,ations,
Preservation, Sampler I, SsmpUne, Water
Analy8is, Water Sampliq

A Factors to Consider:
1 Locating sampling sites
2 Sampling equipment
3 Type of sample required
a grab
b . composite
4 Amount of sample required
5 Frequency of collection
6 Preservation measur.s, it any
B Decisive Criteria
1 Nature of the l!!Iample source
2 Stability of cons~ituent(s) to be measured
3 Ultimate use of data
If a .ample is to provide meaningful and
valid data about the parent population, it
must be repre.entative of the conditions
exriiIng In that parent .ource at the
sampling 10catlor..
A The container should be rln.ed two or
three times with the water to be collected.
B Compositj.ng Samples
1 For some sources, a composite of
samples is made which wiU represent
the average situation for stable
2 The nature ot the constituent to be
determined mLY require a .eries of
separate sample..
C The equipment uRed to collect the sample
is an important factor to consider.
ASTM(11 has a detailed section on various
sampling devices and techniques.
D Great care must be exercised when
collecting samples in sludge or mud areas
and near benthi~ deposits. No det1nite
procedure can bt; given, but careM
effort should bE: r.:lade to obtain a rep-
resentative sa~le.
A Each sample must be unmistakably
identified, preferably with a ta8 or label.
The required information should be planned
in advance.
B An information form preprinted on the
tRiS or labels pr::>vides uniformity of
sample recorde, assists the sampler, and
helps ensure that vital information will
not be omitted.
C Useful Identi11catiol'l Information includes:
1 sample identity code
2 signature of sampler
3 .ignature of witnen
4 description of sampling location de-
tailed enough to accommodate repro-
ducible 8ampling. (It may be more
convenient to record the details in the
field record book).
5 sampling equipment used
6 date of conection
7 time of conection
8 type of sample (grab or composite)
9 water temperature
10 sampling conditions such as weather,
water level, flow rate of source, etc.
11 any preservative additions or techniques
12 record of any determinations done in
the t1eld
13 type of analyscs to be done In laboratory

Sample Handlin" - Field Th~ou"h Labor.wry
A Available Materials
1 glass
2 plastic
3 hard rubber
B Considerations
- Or,anics
2 Nature of con8tituent(1I) to be determined
- Cations can adsorb readily on 80me
plastics and on certain ,..sware.
Metal or aluminum toil cap liners can
interfere with metal analY8es.

3 Preservatives to be used - Mineral
acids attack some pIasttc...
4 Matlina Requirement!, - Containers
should be large enou,~ to allow extra
volume for effects of temperature
changes during transit. AU caps
should be lecurely in place. Gla.s
containers must be protected apinst
breakale. Styrofoam lining. are
useful for protectlni I:la8sware.
C Preliminary Check
Any question of possible inte:rferences
related to the sample container Mould
be resolved before the atudy beiln8. A
preliminary check should be made usin,
correspondin, sample meterials, con-
tainers. preservative. and analysis.
D Cleaning
If new containers are to he used, prelim-
inary cleaning is usual11 not necessary.
If the sample contalnsrs have been used
previ6usly, they should be carefUlly
cleaned before use.
There are several clea~1 methods
available. Choosing the best method in-
volves careful consideration of the nature
of the sample and of the con.tituent(s) to
be determined.
1 Phosphate deterpnt8 should ,not be
used to clean containers tor pho8phOrus
samples .

2 TraMs of dichromate cleanJ1'1g solution
wID interfere with metal anaJyles;
E Stora,.
Sample container8 should be stored and
transported In a manner to as8ure their
readine.. for use.
Every eftort .hould be made to _,hieve'
the shortut po..lble Interval between
.ample coUection and analy.es. If there
must be a deJ&1 and it 18 long enough to
produce sign1f1c.nt chanees in the sample,
pr.servation measures are required.
At beat, however, preservation efforts
can only retard changes tha"t inevitably
continue after the sample is removed
from the parent population.

A Functions
Methods ot preservation are relatively
limited. The priR1617 functions of those
employed are:
1 to retard biolol1eal action
2 to retard pree1pitaUoa or the hydrol7sis
of chemical compounds and COIIlp1exes
3 to reduce yolatllity of constituents

B General Methoda
1 pH control - This aff.cts precipitation
of metals, salt formation ad can
inhibit bacterlal action.
2 ~~l1\!Jd1tiQl1 - '!be choice of
~ on the change tabe
Mercuric chloride 18 commODly used
a. a bacterial Inhibitor . D1-.osalof
the mercury-conta.irlini sample. is a
problem and effort. to find a 8ubstitUte
for this toxicant are underwII¥..

Sample HandUni: - Field Thro~gh Laboratory
To dispose of solutions of inorgamc
mercury salts, a recommended
procedure i8 to capture and retain the
mercury salts R8 the sulfide at a high
pH. Several firms have tentative~
agreed to accept the mercury sulfide for
re-processing after preliminary con-
ditions are met. (4)
3 ~efriierat1on and freezinJ - This is
the best presp,rvation technique avail-
able, but it il not applicable to all
types of samples. It is not always a
practical technique for field operations.

C Specific Methods
The EPA Methods Manual(2) includes a
table summarizin, the holding times and
preservation techniques for several
analytlcal.procedurl!s. This information
also can be found in the standard refer-
encss (1,2,3) as part of the discussion
of the determinlil.~lon of interest.
Standard reference books of analytical
procedures to df'termine the physical
and chemical characteristics of various
types of water s9.mples are available.
A EP A Methods ManUl\l
The Analytical Quality Control Laboratory,
National Environmental Research Center -
Cincinnati, Environmental Protection
Agency, has published a manual of analytical
procedures to be used in Water Programs
Operations laboratories for the ana~8is
of water and wastes. The title of this manu:u
is "Methods for Chemical Ana~8is of
Water and Waates" (1971).
For some procedures, the ana~st is
referred to Standard Method8 and/or to
ASTM Standards,
B Standard Method8
The American Public Health Auociation,
the American Water Works Association
and the Water PoUution Control Federation
prepare and publish a volume describing
methods of water ana~sis. Th.ae include
physical and chemical procedures. The
title of this book is "Standard Methods
for the Examina~!.on of Water and Waste-
water", 13th edUioJ1, 1971.
ASTM Standards
The American Society for Testing and
Materials publishes an annual "book"
of speclf1cations and methods for testing
materials. The "book" current~ con-
siats of 33 parts. The part applicable
to water is a book titled, "Annual Book of
ASTM Standards, Part 23, Water;
Atmospheric Analysis".
Other Reference3
Current literature and other books of
analytical procedures with related in-
formation are available to the analyst.
The ideal situation is to perform all
analyses shortly after sample collection.
In the practical order, this is rarely
possible. The allowable holding time
for preserved samples is the basis
for scheduling analyses.
The allowable holding time for samples
depend8 on the nature of the sample, the
stability of the const1tuent(s) to be de-
termined and the conditions of storage.
1 For some constituents and physical
values, immediate determination is
required, e. g. dissolved oxygen, pH.
2 Usine pre8enation techniques, the
holding timcs 10r other determinations
range from 6 hours (BOD) to 7 days
(COD). Metals may be held up to 6
months. (2)

3 The EPA Methods Manual(2) includes
a table summarizing holding times and
preservation techniques for several
analytical procedures. This information
can also be found in the standard

Sample Handlin. - Field ThnlWlIt lAboratory
(1' 2 3)
references" as part of the
discu.sion of the determinatim 01
4 If dissolved concentrationl are
Bought. filtration 8hould be done in
the field if at all po.sible. Other-
wise, the lample ilt filtered al 800n
as it is received in thO! laboratory.
A 0.45 micron membrane filter is
recommended lor reproducible
The time interval between collectlon
and analysi. is important and lhould be
recorded in the laboratory record book.
The importance of ml\fntainina .. bound,
lejible record of pertll\ent informauon
on samples cannot be over-emphalized.
Field Operations
A bound notebook should be used. Sample
information written in it would include:
1 Sample identification records (See
Part m)
2 Any information requested by the
analyst a8 sign1fi~ant .
3 Details of sample preservation
4 A complete record of data on any
determinations done in the field.
(See B, next)
5 Shipping detai18 and "cordll
Laboratory Operati0b8
Samples Ihould be lor,ed in as soon a.
received and the artalyl.S performed
as soon as possible.
A bound notebook should be uled.
Preprinted data forms provide uniformity
of records and help enlure that required
'information will be recorded. Such sheets
should be permanently bound.
Itema in tile :It!bor.tory notebook would
inoItaIIe :
1 sam.ple identifying code
2 date and time 01 coUection
3 date and time of analy8i8
" the 8I\alytic&l methpd u..d
5 any dev1atione from the anaJr'tie&1
method u.ed
6 data obtaiDBd durin, analysi8
7 quUty cotftrol checks used
8 any information wteful to those who
interpret and UI. the data
9 slptut"e of the an&1y81:
Valid data can be obtained 0114" ftoom a repre-
lentaUve sample, UlUni8tai'.abJ:r identified,
carefUlly coU.cted and stored. A sk1l1ed
analyst, u.~1 re11able method. of analyses
and perfornUnl the determinations within
the prescribed time limits, can produce data
for the sample. 'Ibis data win be of 'Y'&lue
only if a written record exists to verify sample
history from the field through the 1aboZ'atoi'y.

ASTM Standardl, Part 23. Water;
Atmospheric Analysis.
Metho~ for Qlemica~ AnaJ,y.i8 of Water
and Waites, EPA-AQCL, 19'71,
Cincinnati, OH 405268.
Standard Methods tor thf! Eilcamination of
Water and Wastewater. 1Mb edition,
Dean. R.. W'U118m., R. and Wise, R..
D11tJKJ.al 01 Mercury Wastes from
Wafer Laboratories, EnvtrohJnental
Science and Techno1.Gf1, Oetober. 1971.
~his outline -s prepa:t'el~ 'X. DOtwtue,
Chemist. National Trainin, Center. EPA,
WPO, Cincinnati, OH 45268.

~scr~r:t: Data CO)lectlons. InvesUp-
t ons, - it. Data Collections. on- Site InV'e8ti-
,aUon., P1aDninc, Preservation. Sampling.
SurveY8, Water AIIa1ysis. Water Sampling

The first step in the examination of a water
sl1pply for bacteriolopcal examination is
careful collection and handling of samples.
Information from bacteriolopcal tests is
useful in evaluating water purification,
bacteriological potability. wallte dilpoul,
and indUItria1 supply. Topics covered
include: represent:ltive site lelection,
frequency, number, size of samples,
satisfactory sample bottles, techniquee of
samplin" labeling, and transport.

The basis for locating sampling points is
collection of representative samples.
A Take samples for potability testing from
the distribution system through taps.
Choose representative points covering
the entire system. The tap iteel! should
be clean and con:1ected directly into the
system. Avoid leaky faucets because of
the danger of washing in extraneous
bacteria. Wells with pumps may be
considered similar to distribution systems.

B Grab sampln from streams are frequently
collected for control data or application of
regulatory requirements. A lI'ab sample
can be taken in the stream near the surface.
C For intensive etream studies on source
and extent of pollution, representative
sample. are taken by considering sUe,
method and time of sampling. The
sampling sites may be a compromise
between physical limitations of the
laboratory, detection of pollution peaks.
and frequency of sample collection in
certain types of surveys. First, decide
how many samples are needed to be
processed in a day. Second, decide
whether to measure cycles of immediate
pollution or more average pollution.

Sites for measuring cyclic pollution are
immediately below the pollution source.
Sampling is frequent, for example, every
three hours. .
A site designed to measure more average
conditions is far enough downstream for
a complete mixing of pollution and water.
W. BA. sa. ld.l. 73
Keep in mind that averaging does not
remove all varb.tion but only minimizes
sharp nuctuations. Downstream sites
sampling may nC't need to be so frequent.

Samples may be collected 1/4. 1/2 and
3/4 of the stream width at each site or
other distances, depending on survey
objectives. Often only one sample in the
channel of the stream is collected.
Samples are usually taken near the Burface.
D Samples from hIkes or rel!lervoirs are
frequently collected at the drawoff and
US\1a11y about the same depth and may be
colleoted over this entire surface.
E Collect samples of bathing beach water
at locations and times where the most
".thera 8wim.
A For determining lIampling frequency for
drinking water, consult the USPHS
1 The total numbel', frequency, and site
are established by agreement with
either state of PHS authorities.
2 The minimum number depends upon the
number of users. Figure 1 indicates
that the smal\er jJopulations call for
relatively more samples than larger
ones. The numbers on the left of the
graph refer tc actual users and not the
population shown by census.

3 In the event that coliform limits of the
standard are exceeded. daily samples
muet be taken at the same site.
Examinations ..hould continue until two
conlecutive samplshow coliform
level is utisfact . Such samples
are to be conside as special samples
and shan not be included in the total
number of 1!Ian-.ples examined.
4 Sampling programs described above
represent a minimum number which
may be increased by reviewing

CoUection and Handling of Samples for &acteriological Examination
B For sU'eam investigationl the type of
study governs frequency 
I-- --

~ ----- ..,~ -

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f-- --"
...---- -- ...

1---- --- >-
---- - - -- -
-- --

- ....
=~ _..
I--- ..-
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... _..- 1--.
..... j-.- _. ..
--- '-
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.. ~
-_.- -- ---- -
-- -
... ..
i-. e--
h --
.... .... --
- .... ~-
~ ---- ..

Conection ,nd Hand1irur C!Il Sample. for Bacteriological Examination
E Salt water or estuarine beaches are
sampled as needed with frequency
depending on u.e.

F Size of sample. dflpends upon examination
anticipated. Generally 100 mIls the
minimum size.
A The sample bothes should have c.apRcity
for at least 100 ml of sample, plus an
air .pace. The battle and cap must be of
bacteriololfical inert materials. Rellistant
glall or heat re.i8tant pla.tic are
acceptable. At the National Training
Center, wide mouth ground-gla8s
IItoppered bottlea (Figure 2) are used.

All bottl.. ml1.t be properly washed and
sterilized. Protect the top of the bottles
and cap from contamination by paper or
metal foU hood.. Both glull and heat
resi.tant plastic bottles may be
.terilized in sn 6.utoclave. Hold plastic
at 121°C for at but 10 minutes. Hot
air IIterilizaUon, 1 hour at 170oC, may
be used for glass bottles.

B Add lIodium thiosulfate to bottles intended
fo.r halogenated water samples. A quantity
of O. 1 ml of a 100}', solution provides 100
mg per liter concentration in a 100 ml
sample. This level shows no effect upon
viability or grov.th.
C Supply catalols list wide mouth ground
glall8 stoppered bottles of borosilicate
re.i.tance glass. specially for water
Follow aseptic technique as nearly as
pOllible. Nothing but sample water must
touch the inside of the bottle or cap. To
avoid loss of Bodium thiosulfate, fill the
bottle directly and do not rinse. Always
remember to leave sn air space.
A In sampling from a distribution system,
first run the faucet wide open until the
service line is cleared. A time of 3-5
minutes generally is sufficient. Reduce
the flow and fill the sample bottle without
splashing. Some authorities stress
flaming the tap before collection. A
chlorine determination is often made on
thll site.
B The bottle may be dipped into some waters
by hand. Avoid introduction of bacteria
from the human hand and from surface
debris. Some sl:.ggesttons follow:
Hold the bottle near the base with one
hand and with the other remove the hood
and cap. Push the bottle rapidly into the
water mouth down and tilt up towards the
current to fill. I. depth of about 6 inches
is satt.factory. When there Is no current
move the bottle through the water
horizontally and away from the hand. Lift
the bottle from the water. spill a small
amount of sample to provide an air space,
and return the uncontaminated cap.

CollecUon and Handlini: of Samples for BacterioloJ(i.cal Examination
C Samples ma,. be dipped from Bwimming
pools. Determine residl.:Bl chlorine on
the pool water at the site. Test the
sample at the laboratory to check chlorine
neutralization by the thiosulfate.

D Sample bathing beach water DY wadin, out
to the two foot depth and dipping the
sample up from about 8 inches below the
surtaoe. Use the procec1'lre described in
V. B.
E Wells with pumps are limilar to
distribution systems. With a hand pumped
well, waste water for about five minl1tes
before taking the sample. Sample a well
without a pump by loweri'1g a sterile
bottle attached to a weight. A device which
open I the bottle underneath the water
will avoid contamination by surface debris.

F Various types of sampling devices are
available where the sample point is
inaccessible or depth lamplel are desired.
The ,eneral problem il to put a sample
bottle'in place, open it, clole it, and
return it to the surface. No bacteria but
those in the sample must enter the bottle.
I The J - Z sampler described by Zobell
in 1941, was designed for deep sea
sampling but is useful elsewhere (Figure)
3). It has a metal frame, breaking
device for a glass tube, and sample
bottle. The heavy metal mel8enger
strtkes the lever arm which breaks
the glBSS tubing at a file mark. A
ben.t rubber tube straightens and the
water is drawn in several inches from
tM apparatus. Either glasl or collaplible
rubber bottles are sample containers.

Cammercial adaptations are ava1lable.
2 Note the VBne and lever mechanism on
the New York State Cc.nservaUon
Department's sampler in Figure 4.
. When the apparatus is at proper depth
the suspending line is given a sharp
pull. Water inerUa againlt the vane
raises the stopper and water pours
into the bottle. Sufficient sample is
collected prior to the detachment of
the stopper from the vane arm allowing
a closure of the lample bottle.

The New York State Conservation
Dt!partment's sampler is useful for
shallow depths and requires nothing
b8'llides Ila88 stoppered sample bottles.
Reproduced with permiuion of the Journal
of Marine Research 4:3, 173-188 (1941) by
the Department of Health, Educatioa and

CoUectiQn and HandUoa of Samples for Bacteoriolo,i1cal Examination
...._,... .... ~
fiGURE 4
3 A commercial sampler is available
which is an evacuated 8ealed tube with.
a capillary tip. When a lever on the
8upport rack break8 the tip, the tube
fills. Other samplers exist with a
lever for pulling the 8topper, while
another uses an eIectromagnet.
A Information generally includes: date, time
of collection, temperature of water, location
of sampling point, and name of the sample
collector. Codes are often used. The
location description must be exact enough
to guide another j)erson to the site.
Reference to bridges, road II , distance to
the nearest town may help. Use of the
surveyors' description and maps are
recommended. Mark identification on the
bottles or on securely fastened tags.
Gummed tags may soak off and are
B While a sanitary survey i8 an indispensable
part of the evaluat:on of It water supply, its
di8cussion is not within the scope of this
lec:ture. The samp'e collector oould supply
much information if 
Collection and Handling at Satnplea for Bacterioloaical EJ:8Jni~lon
5 Bathing Water Quality and Health m
Coastal Water. 134 pp. U. S .
Department of Health, EducaUon, and
Welfare, Public Health Service, Robert
A. T aft Sanitary Enpneering,
Cincinnati, Ohio. 1961.
8 Zobell, c. E. Apparatl,1S ffJr Col1.~tinr
Water Samplell from Ditferent Depths
for Bacteriological A'1aly.18. Journal
of Marine Reaearch, 4:3;173-88. 1941
Thill out1ine waa orlpnallJr prepared by
A. O. Joee, fOl'llMr MlcrobloJopat FWPCA
Traimng Activitl... SEC and,gpdated by
the Trainln, Staff. Nattcnal Tramlng Center,
~scl~tor\ . Equipment, Microbiology,
mp .. ateI' "mpllng

The determination of bacteriolo,ical quality
of water should include active participation
of the bacterial analyst in all phases of the
survey, from the planning stages through and
including the performance of the survey and
the final prepara~ton and presentation of the
finished report.
The principal bacteriologist may be 8 pro-
Ceasional bacteriologist serving 8S the prin-
cipal survey bacteriologist, 8 bacteriological
consultant, or, in practice, these duties
sometimes may be assumed by a qualified
eni:meer or sctentist nominally representing
some other professional discipline.
Chief responsibilities of the principal bacteri-
ologist in a survey are summarized in the
following paragra.phs.
A Through consultation with survey manage-
ment and review of survey objectives, he
determines what specific pollution problems
are related to bacteria.
B A determination is made of the kind of
bacteriological information which must be
developed in order to meet survey
C The related chemi{:al and physical tests
are identified in terms of their expected
need with r..pect to interpretation of
bacteriological data.
W. BA. 47a. 3. 74
In order to develop the kind of bacteriological
information required to meet survey objec-
tives, the following decisions must be made,
and pertinent information obtained:
A The specific indicator organisms used to
detect and evaluate pollution must be
B The methods used for their
quantitativt! measurement must be deter-
C Consultations must be included with re-
spect to bacteriological sampling.
1 The nmple points must be established.
2 The frequency of bacteriological testing
at each point must be determined.
3 The location of proposed sampling
stations with relerence to assumed
influences on bacterial levels must be
reviewed. These influences may be
alleged polh:.tion sources, entrance of
diluting waters, drainage areas, or,
in some cases, industrial drainage or
D Any existing data on bacterial densities,
as well 8S wat~r flow velocity and volume
should be obtained and studied.
E Based on the sampling plan am determin-
ation. to be made, a protocol of bacteri-
ological sample examination is developed.
Figure 1 i8 an C!xample of such a protocol,
used in a recem water pollution survey.

lIP an rlI ....,.....
-- 1-' (»
,.,...J III ..... at. )S'O

eo.t 1 fir p1DII: oolMlu,
~ 1181,. 10-20 _tor ~-
IIInllate -- .t )S'C Q...- T... ftoau1

+ ~ -::-..~.
::!:. - - at 44o~ + 0t020C ==::e~a :;;~tr.ptoeo_l
coU~ ,........

~~ IftUIIt (~~ 110 ... - IINtJ. co1U818 .....
~.... III ...... at. )5'0 _t (1I8IiIM")
I 188 - 18otJ. .oUt- --
110 'I- .. ...-, (...-...).
-.u.. - - "'-'1.. teat. -1'4 .. ... ..u.-
..lU.- .... eollform. preHnt MPN per 100 ,,,,1
ealeulate eonflrmed te.t MPN
per 100 ml.
. Inilio- ............. d88OI1.1I8d 88 ........ - 111 --...... - tor .. --... at
VaWr .. V_-, l3tl> ado (18T1)
{ll r.e- broth - be .._"'... .... la$ '"'"""'. broth.
I' .... 1latllrl8Da 111- cw"" ..... - -- ...baUt8- t... the ~ .... ..u. 118_"""'.
,: ft. IP Alar....... pla'" -- .., be """ 1.............,.1I1r 111- .. III' ..1- --iIII .........
Water Quality Surveys
La 1 t.r/,t- ..... (i)
( 1.. -I.
-- at. )SOC
"\8IIre 1
The bacteriolo,ical plan ill reviewed for
adequacy to accomplillh the objective.
of the lIurvey, based on a "paper review" and
on preliminary samples which should be
examined at the lIelected sampling lltattonll if
at all fe..!ble.
A Sufficient qualified peraoMel for the labora-
tory load shOl.lld be provided. EsUms tell of
work loads are .hown in Part 2 of tbill
B The detailed methods for all procedure.
are reviewed with all persoMel respon.i-
ble for collection and hand liD" tran.-
mi..ion to the laboratory, ud with the
laboratory per.annel re.pon.!ble ~r
sample tut1111 procedures.
C The duties all8ijp'1ed to all per.onnel muat
be reviewed with all concerned, work
scheduled, reviewed and understood by all.
Care must be taken to assl.lre that allsiJned
work is consistent with trainiq. ability,
and experience ot individuals concerned.
D Location and quality of the lahoratory
faciliUe. must be reviewe~ to ascertain
that sample testing procedure. will be in
accorctanee with established pIe, .tandard
proceclure., and .to assure that adequate
work space is available.

E Laboratory equipment and supplie8 must
be adequate for the expected work loads.
Glassware and other supplies which mu.t
be u..d 011 a rotational ba.:i.Ji shoukl, in
general, be 31 - . times the u:pected
daily need.
After the established daily work of the survey
has gotten well underway. the principal
bacteriolo,i.t IIhould review operations based
on the plan elltabUshed as the finsl plan for
survey operations. Typical cbeck points

Water Oualitv Surve~
A Review of sample collection and handlin,
procedures and assurance tl1at there I.
minimum delay between collection and
starting testing procedures on individual
with reference to matters not anticipated
when planning the survey.

4 Data are reviewed to determine validity
for use in the survey, in terms of
reliability of results.
B Evaluation of Laboratory Work
1 Determination of whether the methods
actually used are in conformance with
plans .
a When membrane filter or plate count
methods are used, check the correla-
tion between occasional duplicate or
triplicate plate count for
2 Elimination or justification of deviations
from planned procedures.
b When multiple dilution tube methods
are used, check the percentage of the
usual codes, according to the m,~thod
described by Woodward and Walton
(JAWWA. 1957; 49: 1060-1068, "How
Probable Is the Most Probable Number'?"
3 Problems which have arisen during the
operations are discussed, particularly
 Expectancy Positive tube combinations   Freq\.lency of occurrence
 group   constituting ,roup    By group  Cumulative'
          %  %
Group I (Frequent) ! 100 200 300 400 500 550 i 67.5  67.5
       510 551:   
       520 552    
       530 553    
       540 554    
Group 11 (Common) I 110 210 310 410 511     91. 1
   420 521   -L
  I     531  
  I     542  
  I  101 201      I 
Group IJT ,001  301 401 501 i 7. 9 I 99.0
  I      502 !  
(Uncommon) '010  120 211 311 411   
  i 020   220 320 421     
      330 430 522    
       431 532:   
       440 533    
Group IV (Rare)
i All other positive tube combinations
Ref.: Richard L. Woodward and Graham Walton. 1957.

Water Quality Survey.
 Positive tubf.          I       
 combination     Number of  I  II  m   IV I 
 (5 portions planted I      I     i 
 I   t1.mes found Frequent I C~cml  UncornmQn   Rare I 
 ill each of 3 decimal        i      I 
 dUut1on8)      i  i     I
 100 I    1  1      i   I
 110     1    1      
 200     2  2        
 220     1       1     
 300     6  6    :      
 301     1 I     I 1     
 310  I   3  i 3  I      
     I  i  r      I
 400  I   6 8   I      I
 401  !   1     I 1     
 410 i 12 i 12        
 411  I   2 j  !    2     I
  I           I
 412  I   1 j  :       1  
 420  I   4   4    i    ;
I 430  I   2 I      2    
 440     1 I      1     
 500     13 I 13          
 510   I  11 ! 11          
 511   I  2    -2        
I 512   I  1       1     
 520   !  9 I 9      I    
 521     2    2    !   
 530     9  9        
 531     3    3    I    
 540     3  3      :    :
 541     5 I   5    !    
 542    . 1 1  I 1        
 550     2  2    I     
 551    i 1  1         
 Total No.     108  811  33   9   1  
 Percent of total     100.0  60. 3 I 30.5   8.3   0.9  
 Cumulative percent     100 i 60.3 90.8   99.1   100.0  i
Ref.: Richard L. Woodward and Graham Walton. 1957.

c Data sometimes show apparent
anomalies which necessitate addi-
tional test procedures or special
tests on selected samples in order
to account tor seemingly unusual
results or to establish ~e validity
ot the ruults beln, obtained.
It is ellsential that periodic review ot data
be conducted during the survey. It is sUi-
gested that such reviews be conducted daily
auril1l the initiaIilltages ot the aurveyand
thereatter at weekly or biweekly intervals.
A Review can reveal the need tor changes in
procedures to m..et the Qbjectives ot the
B Changes needea In sampling plan can be
revealed. According to the type ot results
beln. obtained, it may be nece88ary to add
turther samplin( stations, to delete certain
Water Qual1t,r Surveys -
stations. or to modify the schedule of sam-
ple collection.

C Need for additional test procedures may be
D Interim data may show that certain test
procedures are producing data of no value
in meetin, surv"y objectives; such proce-
dur es can be drc:.pped.
Development of the survey plan. performance
ot the planned operations to meet survey
objectives. and the final data ILnaly.es and
preparation of repCJrt are interdependent
There are man,y ~echniques available for
summarizing andpresenting data for interpre-
tation ot ,findings. This topic is discussed at
greater length in the outline titled "Presenta-
tion and Interpretation of Bacteriological Data, "

Water Quality Survey.
lAboratory Operation.
Part 1 of thi. outline hu been concerned with
planning the bacteriological aapects of a .ur-
vey. With thie part of the outline, certain
epecific recommendatione are preeented with
re.pect to pereonnel qualiticlltion. and work
.chedules, desired location and feature8 of
laboratory facilitie8, and the care and handline
of eamplee prior to 8tarting te8t procedure8.
A SkUl Levels
1 The principal baoteriolo,i.t 8bould have
at leut 3 - 5 year. of profel8ional
experience in the .anitary bacteriology
of water. The duties at the principal
'bacteriolo,iet. who mayor may not
personally perform the laboratory pro-
cedures, have been identitied in Part l'
of thia ouUine.
2 Subordinate bacteriolo,iats preferably
should have at le.8t 1 - 2 years of
workine experience in water
The laboratory bacteriolo,ist is re-
, spon8ible for the oorrect t.sting of
eamples accordlne to predetermined
. plan. and for preparation of accurate
record. of resuUs, in an orderly
manner. Nonprotessionallaboratory
a8eistants, if employed, mU8t be
closely eupervised in r.l1 duties
. undertaken.
3 Nonprofes8ional1aboratory ..ai.tants
may be needed. In a short-term,
highly intensive survey, the writer ie
reluctant to employ any but experienced
laboratory helpers. Such experience
, can best be gained in a fixed laboratory
where close supervision and direction
are available for routine operations than
would be possible with the short-term
intensive survey operations.
%)uti.. of nonprofe.sional1i.boratory
...i.tants include washint and
sterUization of ,l..sware, preparation
of s.mple bottles and related '8Upplies,
preparation and maintenance of culture
media luppltes, and related duties.
J) Work Loa.s

I Bued on the assumption of
utm..Uon of birhl1 Ikilled,
fait, laborlotory1l'llt'kere, tlte
blocteriol"lt-~on.ultants of
many water quality eurv.y. bave
recommended a maximum ot 20
coliform determination. per.
For a short-term, hi,iIly intelUlive,
8urvey there usually 11 lUt1e difficulty
1n operating on this basil, provided
that perlonnel are motivated to the task
at hand. With long-term invesU,aUone,
personnel levels should be held at such
levels as t~ permit normal work-days
alid work-weeks with off-dilly daY8
provided .t periodic J.nlerval8.
A Location
The requirement for prompt examination
of samplea after collection demands
attention to the lucation of ~boratory
1 With suitable transportation arrange-
ments it may be possible to perform
examinations in eltabli8hed water
bacteriology laboratoriel.
Thill 18 to be preferred if at .U poi8ible.
Recently, ircre.sing attention hall been
given to air tran8port of 8amples as a
means of rellolving this problem.
2 With limitations on available trans-
portation it more commonly ill necell-
sary to e8tablish a temporary labora.
tory. Thi. rtlay be temporar:v
.pace in eltabUshed laboratories, as
in local or hospital laboratories, or it
may be nece8sary to use a mobile trailer
3 It ill not practiced to conduct the entire
bacteriolo,1cal examination of water in
stream surveYM under field condi-
tions, even with membrane filter
methods. In some case8, however, it
may be necessary to inoculate sample II
into primar;y tube media or on mem-
brane filters in the field. In lIuch sam-
ples, temporary incubation mUllt be
provided (no iCin, of inoculated sam-
plea) for transport of the nmple to the
laboratory where the remainder of the
incubation and 8ubsequent laboratory
procedures are performed.
Water QU!\ity Sprveys
B Space
The bacteriology laboratory requires
provision for .;everallunctions, each of
which requires a silldficant amount of
space. To give assurance that adequate
apace i8 provided, the following functions
mu.t be considered!
1 Bench IIpace
Bench space of the type used for chemi-
cal determinations 18 suitable for
bacteriologice.! work. Unless the num-
ber of bacterlolo,ical samples is quite
small, the bench 8pace should be re-
served for this function. About six
lineal fee~ of bench 8pace per worker
i. a minimum allow&Jl('e.
2 Preparation area
U the number of daily samples is low,
it may be feasible to make and dispense
culture media, prepare sample bottles,
etc., at the laboratory bench. If the
work load is Virge, one general area
should be reserved for media prepara-
tion, wa.hing and sterilization of glass-
ware, and other Bupportin, functions.
3 Incubation and sterilization
Space must be provided for certain
fixed laboratory equipment, such as
washing equipment, dry sterilizer and
autoclave, water bath, and incubator,
accordj,ng to the work load anticipated
for the survey.
A Source
In survey planning, consideration should
be given to preparing all culture media
and reu8able 8upplies in a centrallabora-
tory, and tran8porting luch supplies to the
field laboratol")' site. The practice fre-
quently is more economical than efforts to
provide such support at a field site.

~ QuaUty Surve~1
B Sample Bottles
1 All sample bottles mllst !:Ie clean,
sterile, and free of lubstances un-
favorable to bacterial .urvival.
Althouih a wide variety of containers
is acceptable, the preferred form is
a glaes-Itoppered. wtd,,-moutb boro-
eilicate alael bottle, having abollt 2&0
ml capacity.
2 Some samplel, such al efnuent trom
lorne wa.te treatment plante, may con-
tain relldual chlorine. In luch calel,
enough todium thiolu1fl,te iI inU'c>duced
intb the clean eampl. betore stlfrUiza-
tion to live 100 m./l tltio.ultate in the
lample. For exam.,!e, add O. 2 ml 01 a
10% solution of sodium thio.ultate to a
250 ml bottle luch as deecrlbed above.
This wUl dechlorinate u.mplee contain-
ini up to about 23 ml chlorine per UteI'.
The bottle il dry or moiet heat Iterilized
following introduction of the s04hun

Laboratory te8t!! have Ihown that thi.
amount of lodium thiosulfate haa no
adverle effect on the survival and
I(r6wth ot coliform biacteria or fecal
A Collection
I In many calel, samplinlil limited to
lurface ,ub lamplel. The opened
bottle i. plunled into the water to a
depth ot about one foot. The .ample
bottle never should be fUled more than
one-hall to three-fourthl full. This 18
to facilitate mixing by lhaitinl( when
laboratory tellts are itarted.
2 If but one eample i8 taken trom each
station at the selected ..'1tervall, It
u.l1ally I. collected from Ileal' the
center of the main channel 01 now.
preUm1nary te8ts may demon.trate
need for multiple 8ample. trom each
stAtion. In this ca.e, it may be nece8-
sary to collect samples from predeter-
mined point. acrose the body ot watlr
and' or at deelpated depth8. Such
8ampl.. are DOt comp08ited and
retail\. their id8ntity 88 .linP .~plell
ttirileihcNt the labotator, ta.Ung
3 The lam"le coUect1~ i8 made tn iluch
. manner.. to inlure ob~in' a lam-
pIe reprelentaUve of the itWree. Thus,
it ..mpliDg is from . boa t, the bottle
should be dipped into the upttream side
ot the boat, with the mOUth of the bbttle
al.o directed uplitream. If there is no
di.cernible current, thP.n taM battle I.
moved thtou,h the water in . dtfection
a.., from the hand hold in, ttie "ottle.

.. It depth sampiifti iB iridicaMd, it i. nec-
eBlary to u.e 'pectal deptH '-1'1411'8.
and open them at the dHired hyJth.
B Identification
1 AU samples muilt be immediately
and fully identified a:t the time
of collection, including at least
the 8ample location, arid the date
and time ot s~pl1nl' Maliy workers
ule . liupplemental she4!t to record
tlmperature. pH alid other data.

I T"e lample tag should be affixed to the
'ai:nple bottle. If the bttttle hat perma-
nelit identifyin, mark8. a 8ujJpietri8i1tal
sheet may be Gled to identify the lIample.
3 Wax pencils are not sati8factory. In-
delible peneU. are pr.rerrid. AiV
markinl( material that wUi run if wetted
w1U be unsaU'factor,y.

C Care in Tranlit.
1 Time between siunple cott.ctibn and
starting te8t8
The telt procedurea iibould
ldeaUy be run immed'-teq, or
If this 18 not fft.lIble, should
pl'lferabq be started ."Ithin 1
hour after sam.ple t:OUlection!
it never 8hould exceed 8 hours
(.be hOUt'B tranBit time and two
hour. laboratory).

With some samples, such as those
from heavily polluted streams, even
,reater limitation on this interval
is necessary.
Temporary storage
With temporary storage of samples,
"Standard Methods" now recommends
the practice of icing samples prior
to examination.
Water Quality Surveys
This outline was prepared by H. L. Jeter,
Director, National Training Center, WPO,
EPA, Cincinnati, OH 45268
Descriptors: Personnel, Planning, Labor-
atory Equipment, Laboratory Tests, Micro-
biology, Sampling, Surveys, Water Quality

Part I
Summarization of Data
The body of statiltical methods comprises
two groups generally (".ailed descriptive
statiltics and statiatical inference. Descrip-
tive statistics encompassea primarily graphical
techniques for the preaentation of data while
statistical inference is basically a mathemat-
ical approach to the problem of inferring
from a part to the whole.
In this section we con.ider the non-mathematical
techniques of summarizing a set of data in
order that the meanin!{ful information can be
extracted from it.
Cona1der the data in Table 1 which represent
aqueous fiuoride ion concentrations determined
by a colorimetric procedure.
 F-, mg/liter 
0.68 O. 'i7 0.85
0.71 0.79 0.85
0.72 0.80 0.87
0.74 0.81 0.90
0.75 0,82 0.91
0.77 0.83 0.95
A Frequency Table
As the first step in summarizing a set of
data we form a frequency table (See
Table 2). To const.ruct the table the data
are divided into a number of intervall of
equal length called class intervals and the
number of result~ falling into each interval
is recorded in the "frequency" column.
The number of class intervals chosen is
arbitrary. However, it is a rule of thumb
to choose the length of the c]ass interval
so that 7 to 15 intervals will include all
the data under consideration.
Class Interval
. 65 au*
.70 au
.75 au
.80 au
,85 au
.90 au
.95 au
* and under
B Frequency Polygon
As a further step we can graph the
frequencies recorded in the frequency
table. One way of doing this is to plot
the frequency along the ordinate (vertical
axis) and the midpoint of the class interval
along the abscilsa (horizontal axis), The
une connecting the plotted points in Figure 1
form a frequency polygon.
a I
i I
.." 1.11 7,11 ,.U '.7' '.2' t.I'

Iau'oduc.tion to Statistics
C H18tolP'am
Another method of graphm8 the frequency
information Is to cOlUltruct & h18tOfI'UD
&s shown in Fieure 2. The h18topoam i8
& two dimensional graph in which the
lencth of the cla.. intervali. tauA into
consideration. Bach class iftterva1
becomes the ba8e of a rectaftl\lW bar
WhOM hes,trt 18 equal to the corr.-pcmcUn,
frequncy .

..1 .n '.ra ... ... .11 .11 1088
'~U."'I e...........,....
D Histol(r&m of Relative Frequency

The hiatolram can be a very usetlll tool in
statistics espec1al111f we COIlvert the liven
frequency scale to II. relative scale 8\lob
that the sum of all the ordinate. equals one.
Thu, each ordinate value 18 derived by
divfdini the orilinal frequency by the
number of observatl0D8 in the sample.
For the data in Table 1 we wOll1d divide
each of the frequency value. Ift"TQ1e 2
by 1IL
The advanta,e of conatructiQla relative
freq1l8l1CY histOlr&m Uh the one in
F18Ure 3 il that we can intel'pl'- areas
under the histop-am aa prababi1iU.s pro.
vided we assume a scale on the absol..
such that each cla.. interval Is of unit
length. Then the probabiliv that a Ii"""
value wlU faU in any one interval ta the
area under the hlstolP'&m in that"mtenal.

For example, the probability that a value
win faU between. 70 and . '75 is tqual to
the area under the hiatolr&m in that
interval which is 3/18')( 1 . l/e.
i ,..
.N .'1 .11 ... .H ... .. I.'"
11M""""" ,.,... J --
PlOtTU 3.
!lC\aft~ IW.,,,l,'1 Dt.t~

From. rreq\t..... .b1e (s. Table :a}.we
ean ~ . ft1ndctift hCDI8RC1'
table a. .hDwn ill Tab1e 3.. Tbe CUll1U-


The cumulattwfttequncy ".... ia
T.li1e I .... ~.. -=1DbII tbe
frtqueaalft t8 ... :I .
p. Cone. Clam. J'nquelU!:y
  . .;..~
under .'70 1
II .'75 4
II .80 it
'II .85 12
" .110 1&
II .95 1'7
" 1.00 18
If we ccavert the cum.u1at1ve frequency
values to relative frequencies &s we dl.d.
to ooaatruct tIa8 re]att... fnquency hato.
IP"'m, We can p1Qt the rlt1&Uve values to
form. oumu.tlve fre..~ ourve ..
shown 1R Fipre". The pftIbabWty that
a reau1t wtU faU below . liven value can
be r_d from tb8 owaulaUve frequHcy
curve. "1'01' ....., to tbuS tile
~ ...,. renla will M leaathan

0.85 mg/Uter we rl!ad up to the curve at
the point x . . 85 and across to the value
.87 on the probability axis.

~ ;:- ..0
.. ..
... it .'0
~ S .60
... ~ .10
= ~ .40
: > .30
;::; .20
.n .'0 .,. ..0 ... ..0 .., 1.00
'" CONC.
Introduction to Statiatic8

Part n
Measures of Central Tendency and Dispersion
From a practical point of view, statistical
inference is the application of mathematical
tools to analyze and interpret the relults of
investigations in the physical and soclal
sciences with respect to the bypothesell under
investigation. The need for a statistical
approach rests on the well-founded assumption
that a~ such result c(.'ntains an inherent
component of error or random variation which
introduces uncertaint).. Ulto a~ conclusions
which may be drawn tr.om the observed
A Mathematical Distributions
It is further assumed that the error com-
ponents can be deecribed by a mathematical
function called a probability distribution.
Knowledge of the relevant type of theoret-
ical distribution provides us with the means
of auessing the un~ertainty (riek of being
incorrect) in conclusions drawn from
observed results.
B Population Parameters
The concept of distribution pertaine to the
totality of possible resulte which would be
observed in an infinite number of repetitions
of an experiment or investigation. This
theoretically infinite number of results is
caUed a population. A~ measurable
characteristic at a population distribution
Is called a parameter. For each type of
distribution there exists a characteristic
set Of parameters. Specific values for the
parameters of a given distributional form
define a particular distribution of that type.
In almost every case the entire population
can never be observed and therefore the
values for the parameters are unknown.
Popu1ation parameters are denoted by
lower case Greek letters.
C Sample Statistic Ii
The observed results from a particu1ar
investigation art! called a sample of the
relevant populatIon. In almost every case
the sample is a subset of the population.
A statistic is a tunctton of the sample
points (1. e., obs",rved results) which
usually elltimat'9s a population parameter.
Sample statistics are denoted by English
D Classification of Statistical Measures
It is implied above that the type of
popu1ation distribution determines the
particu:lar funcHon of the obeerved results
(1. e., sample s~t1stic) which will provide
the ''best'' estimate of the corresponding
population parameter. For this reason
it is advantageous to assume that any given
sample came from a population with a
specified form of distribution. The use of
graphical teclmiques to indicate the form
of distribution .hould now be apparent.
In most situation.. the distributions or
frequency functions of interest have a
single peak and characteristic dispersion
of area about thla peak. Parameters which
locate the distribution (by the peak, center
of gravity, midpoint, etc.) are called
location parameters and the corresponding
sample statistics are c1assified as measures
of central tendency. Parameters which
describe the dispersion of area are called
scale parameters and the corresponding
sample statistic.:s are c1assified as measures
of dispersion. Ideally, one would like to
encounter only d18trf.butions for which one
measure of central tendency and one
measure of dispersion summarize all of
the relevant information in the sample
about the population.
Prlcedlng page "ank

tfttroduction to Statistics
In this section and the ne"t we define and lay
out the computatianal format for the more
c:ommo~ used measures in statistics. We
let xl denote a typical obseM'ed re.ult .0 tbat
[xl' x2"'" xnl. representa a n.mp1e of n
A The Mean
The most commonly used mea.ure of
central tendency Is the m4'&n or arithmetic
!vera,e. We denote the sample "".s.by
x and t,he ~tlon m..n, oJ wbi- x i.
an estimate, b.r ~. The comp~1
.formula 1.:

- &xl
x. -
For the fluoride data In Tabie 1. we
calcu_te the ..mple m..n a. follow.:
tai 14.52. 0.807
x ."'i1818'
 F-, mg/liter 
0.68  0.'1T  0.8&
0.71  0.79  0.85
O. T2  0.80  0.87
0.74  0.81  O.tO
0.75  0.82  0.'1
0.17  0.83  0.95
B. The Median
Another common measure of central
tendency 1. the median. Th.' median 1.
the midpoint of an arrq 01 numben
(ordered according to ftl1l8). To ft.nd the
.amp1e median we need ~ arraqe the
data 1ft. ascendiq or de.candin, order and
pick the middle value. When there 1. an
even n\lmber 01 observat101\8 take the
avera,- of the two middle values. For
the data lis Table I the medt&n 18 0.805.

e The lItIo~
The macht 18 aaotber meaaure of central
tend-01. althoqh it 18 oI1ittle prac:tf.ca1
importan.ce. The-mode 18 the most
Irequ8ft&1;y occurrlDt ".1\08. ""'refDl'e.
the popWatloD mode i. the 'Y&lue oorrea-
poI\db1. 1:0 the peak 01 the .fio8q1leDCY
dintiblltUa curve. Fr8CJUeDCI1. distrtlnttiGls
with more tban one peak are ~ned .
mulUmoclal. In a aymmetriC!81 freqUilncy
curve the mean, median, ud J:IIII108 W.
all sq..
A The Staru~rd ~tion
A. w1th mea.ure. of central.1:eDdacy.
there an several me..ure. of dt8IPe!'IIian.
the most common of whlc~ i. the'-.ndard
de¥iatIG11, We deftote the ..mpk' by . and
the ~UOn va1Iae of wbioh a i_.
estimate bt (J . The CCIIftp\Ra1:SaIIIS
lormula it:'

1 -a
1; (xi - x,
. .
n - 1
However. compuaUon U8I88 th18 tormuJa
i. tedlou. and it 1. reJat1ve~ a1mp1e to
.how tI8e toUowtq reJatlGn8laip:

J -2 j 2 -2
. !:Cat -X). ntxl - U:x1)
. n-l 8,*-1}
The derived fonn1la Is pre'''''' ~U..
01 its ..pability to the deek cUa8ator.
W. calftiMe the amp1e .tMdu1t~ion
01 tbe I1IIortde dMa in T.w.. 1.....toU&Jowe:

. . JUt)(l1.~tft~.M\a:

8 J 213. 'H4 - 2111-;,.'-
:s6t .
8 Jl;:'.J 0.00fJ~. 0.073

B The Variance
The sample valUe s 18 referred to as the
sample variance wnd Is merely the square
of the standard devlt,t1on. Its formula 18:
8 .
~(.J - ';)2
n - 1
T~e popuJation variance 1s represented by
C1 . 1t8 formu1a 18:
2 t(XI - II)
a 8_-
This 1. the 8ams "s the fol'1ftw.. tor s2
except we use the true JIOj)uJat1on mean II
rather than It. estimate x &Del divide by n
Instead n - 1,
Obviously in calculating the sample
variance the true mean is not known and
we, therefore, use the estimate of the
mean from the data. In calculating the
sample mean and thun usin, It to calculate
the variance of the same data we lose what
is called a degree 01 freedom. It can be
shown that the esUmate of the variance
must be based upon the sum of independent
squared terms. We avera,e our values
over n - 1 because this is the number of
independent squared terms that we wID
have when using a mean that has been
estimated from the sample. In line with
above, we should draw the distinction
between the variance of the sample and
the sample variance. The variance of the
sample, we would cr..lculate for its own
sake and divide by n. However, we cal-
culate the sample vlu;iance as an estimate
of the population variance and divide by
n - 1. There Is'21o practical use of the
variance of the sample, therefore, we
always calculate the sample variance
dividing by n - 1 which we 8hall call the
number of degrees of freedom in our
sample. As a rule, in any calculation,
for every parameter that must be estimated,
one degree of freedom (d. f) i8 lost.
Introductso. to Statistics
C The Ranfe
The range is also used as a measure of
dispersion. It is the difference between
the high.st and low"lIt values in a 8et of
R . max(x!) - m!n(xi)
For the data in Tl&ble 1 the range is then:
R . 0.95 - 0.68 = 0.27
A rough estimate of s can be made by
diViding the ran.e of the sample by the
equare toot of n, the number of
obs.rvat10ft8, when n 1. 8mall.
s ~ 8-G~ 0,:7 ~ 0.067
The use of the range is limited to
in8tancss where ~he 1s.bor of computing
the standard deviation is impractical.
A The most important theoretical di8tribution
In statistics is the familiar bell-shaped
normal distribution which Is symmetric
about it. peak (Sel'! FI.lUre 1). The.
folJbw1n. a18umptlon. gtve ri8e to this
d18tributtbnal form:
1 Values above or below the mean are
equally likely to occur.
2 Small deviations from the mean are
extremely Ukely.
3 Large dev1atior.s from the mean are
extremely unlikely.

..OI.AL OIUle.uno.. CUIVI
18 -7

JmroduciiCID to StaU.ti08 I
The normal cStatribut1o" la oomp1e"q
def1ned by Ita m.an, II, and Ita a~rd
deviation a 1n the followiDI mumar:

1 The area uad8r the ~rmal curve
between II ndnua a aM.. plv.. tJ .. 88
perc.nt of the total....., to the near.at
I perC8Dt.
:I Th. area .er the Mrmal curve
betw..n '" minus :I a IIDd IA p1ua ,. 18
911 R'rceat.
3 The. area W\der the nor~l curv.
betw.en " m1nus 3 '1 8Dd '" pJu8 10 1.
99.' per~- of the t_l area. t. the
neareat 0.1 percent.
If a frequene)' curve ia . lood &pp1'Olt1maUon
to the normal curve, th.a~ c_raot...laUca
of the Dormal curve cao" ua,d to nad
SftIormation about the rreqQ8ftcy d1ltrib\ltion.
A Skewed Diatr1bution
In aome ar..a of 1nves'Aptt.Ga\ one oft-
encounters distribution. whie' al" not
.,mmetric. For eumpJ.., dl8tribut~
of ",ct. rial count a are oft. obaraot.r181tCi
by many mol" .xtremeq hiIb count.
r.Jalive to the median than .xtr.~ 10w
counta. The frequency curves of th.a,
dUtrlbutiona have a lon. 1'- taU .a .howa
in F1pr. :I. D1!1tr1buU., 0.- t11.. ~
dlapJay po.it1v. akewn....
'08CfIVI1Y IKlW.. 0111.
B Lo.rithadc Tre.zaBfonnati<*

For JUDy .....Oft., both pra~ ~d
theoretical, w. pref.r to ~".
.)'IDIft"1U cl&8trifttiolt. lik. .. .._1
CUI'VI. -naer.fo..., II i. ~18 to
tl'U8to1'm akn" data 1ft .... .1 that
a a~ric dl8t\rSbudao r""",
....em.bll.n. the .....1. Oae., of
d.rivf.nt an appl'08dma1:eq ~ diI...
tributiOft faooJn . PNltlve1.y --.~
di.tttbutiab 18 by "'1'''''' .. "18IMI
data 1ft terme oIlo.rltlnnl. -
artUlcial amp18 of coliform omm\8 ~d
th.1r lo8u'ithm8 a... a~ .~~ 2.
Compari.. of the frequ,,", -..-. lor ..
01'1...1 data and tite 10.. (~i-..cs
Table t, ....,.0U¥8J\Y) cl.~' u.t
the ]oprllltm. IDOH o1oce. ~~lrMw
a Q1D88hit cUlltJ'JInaUID.
W'''' .J LI.
1~ 041
2. U"

Imrodudion to Statistics
Class Interval
Frequency (MPN)
o au 400
400 au 800
800 au 1200
1200 au 1600

1600 au 2000
2000 au 2400
2400 au 2800
2800 au 3200
Class Interval
Frequency (log MPN)
1.000 au 1.300
1. 300 au 1. 600
1.600 au 1. 900
1. 900 au 2.200
2.200 au 2.500



2.500 au 2.800
2.800 au 3. 100
3.100 au 3.400
3.400 au 3.700
C Measures of Central Tendency of Skewed
If the logarithms of data from a positively
skewed distribution are approximately
normally distributed, we say that the
original data have a log-normal distribution.
The best estimate of central tendency of
log-normal data ill the geometric mean
defined as:
.J (XI) (x2)...(xn) 
Notice that
1;(108 xi)
10' Xg" n
110 that the geometric mean of the
original data is equal to the antilog
of the arithmetic :nean of the logarithms.
It is of interest that the population
,eometric mean is equal to the population
median. Thus. in the case of data from
a skewed distribvtlon. the sample median
i. a better estimate of central tendency
than the arithmetic mean of the original
data. For the coliform data in Table 2
we calculate:
1;(1og xi)
32.737 2.1825,
x . antilog (2. 1625) ~ 152,
. 130,
. - =
6~:2 " 442.
This outline was prepared by John H. Parker.
Former Statistician, Analytical Reference
Service, Training Program.
Delcriptors: Frequency Analysis, Histo-
grams, Statistical Methods, Statistical
Models, Statistics, Variability

The data collected during any water quality
survey must ultimately be reduced to some
simple, expressive form for presentation.
Ideally, the data presentation should be
easily understood by the least trained per-
sonnel as well as by.the trained profeuional.
The data presentation dOM ma)' determine
the response to the survey.
A Before one analyzes any information, the
data must be set into 10l1cal order and
the analyst must prel.'Oent. the information
to himself.
In developing this order, the analyst Ibould
keep in mind the obJectivel of the survey,
and the variables ~ncountered during the
The analyst's "private" presentation to
himself enables him to evaluate or inter-
pret the data in a logical and scientific

Presentation / f Lope demands
\ order

Logical' ,
I ' '.
Interpretation Know Audience
\ ... - -
Scientifically Accurate
This is one of the most important areas of
report writing an:! unfortunately is often
neglected or overlooked.

A Common Methods of Presenting Data
1 S1n61s valup.
2 Single value plus related information
3 Graphic methods
WP, SUR. in. 4a, 3.74
B Single Value Appi'oach
1 Methods commvnly used
a Arithmetic mean
b Geometric mean
c Median value
d Mode value
2 Advantaies of using ain,le value approach

a Econ~:nicat - engineering design
criteria Is oft.en baaed on average
values. In some instances minimum
and maximum values are more
b Inetrumentation - adwnced analytical
techniques and instrumentation are
accounting for the collection of tre-
mendO'J8 a.mOll:\t., of information.
The tendency is to compile this infor-
mation on computers and U'3e sophis-
ticated Itatist1cal methods to arrive
at representat1ve mean values.

c Audience - Arce some readers
Clocal. state or federal legislators)
do not have the teohnical bl1ckgrou:1d
or (especially). interest to read
through many pages of numbers, it
becomes cO!\'18r1lent to summarize
the numerous observations by using
sin,l. representative values. In
effect, the a.nalyst is a salesman,
hi. report be1n, his product.
C Single Value Plus Relat.ed Information
1 Methodtt commonly IJsed

a Sinale value plus maximum or mini-
mum value
b Sinale value plus percentllge range
(usuall,y BO"!e! of lurvey or laboratory
c Sinlle value p~u,~ standard deviation a
from the mean (depending upon de-
gree of confidence required)

d Distribu~ion and frequency distribution

Pre.entation of Data
2 Advanta,88 of u!Jin, .In,le val"e p1",
related Information.

a El1minltell 80me mi.lnterpl"8t&tion
cau!Jed by extremely hi,b or low
result. that occur rarel)'.
b Give. an Idea of wbat type of di.in-
bution I. occurin, (normal, .tewed,
etc. )
c Avera,. values are extremely u.e-
ful but it II U8Ually tte muimum
or minimum valu.. tbat po.. ..rioU8

D Graphic Metbod.
One of the .allelt, m08t attractive, aad
mOlt efficient ",aY8 to pre.ent numerical
Information 111 by ulle of. V.ph..

I Grapbl commonly uled
a Normalll"apb paper (Cartel1an
Coordinate paper) - Thil i. the
mOlt pnerally u.ed ,raph paper
and 1. mOlt u.etulln obt81nln, an
idea of the trend the numerical
obaervatlons are takinr. Thl. type
of paper ia mo.t helpfll11n prelimin-
ary plott1n, of Information ~ d.cide
it lemi-Io" 10,-10101' otber type.
ot functional p'aph paper would be
more d.lirable to ule.
b Functional rraph paper (..mi-lo"
10,-10') - Ulle at thi. paper aUOW'.
for the plottlnr at r..lI1t. In Itra1,ht
I1n. fa.hion that wC1ald appear on
normall1"aph paper (Cartea1an
Coordinate paper) a. a curved line.

c Arithmetic or pometrlc (I.,) pHba-
b1l1ty paper - thil t,ype cI ...apb Dlatl
the normal frequency cur.. (8e11
shaped curve) a. a Itl"td,ht 11ne.
d Nomofl'aph - Uaed wbere complex
equations or complicated matheJ:nati-
cal manipulation. and calculations
are involved. Nomo,raphl have
been developed tor numeroul

eSpecial p'aph8
1 3-dimensional cI1avaml
2 ple-dia,rams
3 hi.topanul, etc.
2 De.lnbl. cbaraeter1ltlc. cI ..apha
a I'alrly .mple
b Se able to Mand Uoe
c G...,h. .holl1d N --~cI 80 they
m.,. be ...Uy I'eprodlllled.

The pr.aentatlon of Information 18 ... of tbe
fir at &tepa in ..eport wr1UDc. It i. 0ftI of the
most importut part. of &n7 repDrt .....,. ~
properl)' hud1ed can produce a nlJOl't that
i8 brief, undaratandable and "ttful to the
reader. The manner In.which I'e.ult. are
pre.entecl ~ or oral) "&)' ..... aa a
ba.l. I... jwlfment cI the wbole .....y.
Thl. oUt~e... prepared by P. 1'. Atldii.,
Jr.. Sanitar,y EnPM.~ fonner~ with
Train1n. Actlv1"e., EPA.
~ D&~a CoUecti«m8, J;)ata

Bacteriological data analysis consists of an
orderly assembly and summary of the data
obtained in thf' investigation, in the simplest
available mannf'r, leading to demonstration
and explanation of the .,bserved results in
terms of the initial .survey objectives. The
topics of summary of data and interpretation
of data are discussed separately in this
presentation; however these are mutually
intE'I'dependent activities.
A There are many ways in which data can be
summarized and am.Jyzed. The most
commonly used procedures are described
in this outline.
1'1 A number of C'riteria apply to the selection
of procedures for data analysis:
ThE' analysis must be -
1 Understandable by readers of the report;
2 Consistent with survey objectives;
3 Accurate summary of data; and
4 Representative of technically sound
A Data on coliform bacteria, fecal coliforms,
and fecal streptococci must be presented
on a quantitative basis if uBeful interpreta-
tions are to be drewn. Partition
counting of col1forms (IMVIC typing) or
streptococci (biochemical characterization
or species identification) should be done on
a quantitative basis, through study of a
rE'presentative number of ( 100 for
example) pure cultures, with determination
of percentai{e of occurrence of each
identified variant.
WP. SUR. 30g. 3. 74
B Development of Expression of Central
1 Single central ..alue
a Median
The median is determined by assem-
bling all data in an array of ascend-
mil or descending order. The median
value is the central value, a posi-
tional value in the array.
b Arithmetic me~. or average. This
is much innuenced by individllal
c Geometric mean. (Logarithmic

Many workers prefer use of this
value, as It includes all determinate
values but minimizes the effects of
extremely high or extremely low
d In selecting the form of expression
of central vaille, it is necessary to
know the methods of calculation re-
quired in m~eting established stand-
ards. For eJlample, geometric
mean values often are lower than
arithmetic mean values on identical
data. If the standards of quality are
based on use of arithmetic mean
vaIlles, it wOllld be necessary to u'Be
the arithmE< tic mean value in the
data analysi!!.
2 Distributions of values
a Data may be calculated and arranged
on a distrib'.ltion table. The method
has particular usefulness when the
investigatio.1 is related to designated
standards of water quality.

W..ter Q\1Iility Surveys
b The central value ma,y be obtained
by one of the above method.. plus
showing the maximum value at the
designated sampling point.
c The central value may be obtained,
plus a percentage r&nlB for the data
from a .ingle location. Some
I.nve.t1ptors recommend U8e of the
geometric mean value, plus the
range of the middle 80 percent of all
value. from the data being con-
solidated. The method permits
expression of average water quality
(geometric mean) and the maximum
and minimum quality demonstrated.
Exclusion of the ext!"eme 10 percent
of high and low values reduce. the
danger of misinterpretation by
ascribing particular significance to
the sporadic extremes of values
which may not be t1'\l~ repre.entative
of quality of the water.
C Values V.ed to Describe Number. of
1 Number per 100 ml of water. This is
direct use of data obtained in laboratory
work, and may be a Most Probable
Number value; or it may be . value
obtained by membrane filter methods
or plate count methods in which a
silJlple calculation is made to relate the
number of colonies to the volume of
&le te.ted, comput1ne the number
of test organisms per 100 mi.
This form of data expreli.ion appear.
to be most applicable to inve.tigations
for compliance with established water
quality standards, or to trace pollution
indicators when there i8 a relatively
fbed volume of water.
2 In stream surveys where there may be
multiple sources of pollution with
chJnges in volume of the reoeiving
_tel' throuih waste outf.u... or
juncture with other streams, it may
be nece..ary to determine the total
number of bacteria and trace their
fate in the investiption.
Because the numbers of orpn1sma may
reach near~ astronomical proporUOftI.
and to simplity pre..ntaUQa,. COft8Ol1-
dated values often are ...d.
a Bacterial Qualtity Unit (QU) is the
number of bacteria pa..ing a given
point in one day if the CQnCentration
i8 1000 orHsma per millW.ter,
and the stream now is 1 cubic foot
per second. One QU ... equivaJ.nt
to 2.45 X 1011 bacteriA.
b The BacteriAl Population Equivalent
(BPE) i. another useful tool for
consolidating data and representing
the total number of organiems In
the water being stuaied. This value
does not represent the number of
coliform bacteria d1acharged by an
individual but. instead, represents
a value obtained by determining the
relationship between the number of
coliform bacteria p&ss1nt some down-
stream point and tbe nwnber of
individuals in the community die-
charpng wastes into the flowing
waterbody. The BPE (calculated
sewered popu1ations) are derived
by converting flows of ot. to flows
of 100 ml/day, muldp~ by
density of coliform. (MPN/lOO ml)
and d1v1d1ng the total number. by
400 billion/day (summer data) or
U5 bill1on/day (winter data).
Summer (15 C. or more):
BPE. Q (cts) XMPN/I00 mIX(6.1 X 10-5)
Winter (le.. than 15 c. )
BPE. Q (ct.) XMPN/I00 mlX(l,95 X 10-4)

D Methods of Presentation of Data
1 Distribution tables (Table 1)
Water Quality Surveys
2 Circles, hi.t\!~ams, or other geometric
figures superimposed on maps to show,
by relative dimensions, relative values
3 Graphs
4 Development of mathematical expreuions
to describe death rate of the organisms.
a The method involved determination of
a peak value 10r coliforms at some
point immediately downstream
(10 - 15 hours flow time) of the point
of discharge.
b The decreasing numberll of bacteria
often can be dellcribed by the

Y . A X 10.bt + e X10.dt
Y = the fraction of the peak number
of bacteria remaining after the
time, 1;
A . the fraction of the bacteria out
of the peak number which decrease
at a rapid rate;
b . the rate at which the "A" fraction
of bacteria decrease;
C a the fract1o:1 of the bacteria out
of the peak number which decrease
at a different (lesser) rate; and
d .. the I'!!.tl' at which the "e" fraction
of bacteria decrease.
The values of the rate coefficient b
and dare dete1:'m1ned on the basis of
the coliform dkta, using techniques
similar to those used for development
of the rate coefficient k in the BOD
equation. (80e Figure 1)
. ,
\ ...",
~ .:. \ '. "-
.\'.' "
!l101 \\ "-,
s, \ "\ ~
I \. \
:II '. "-
~ .....'...,
.... , ~
e I " "
t " ...
io ""',
~ --....


Water Quality Surveys
A Development of Ratios
Using the single central-tendency values
from each sampling station in a survey,
it often is ulleful to determine the ratio of
fecal coUforms to fecal btreptococci. Ir,
for example, at a given station the fecal
coliform value i5 given as 72,000 per 100
ml and the fecal streptococcus value i8
18,000, then the fecal coliform Ifecal
streptococcus ratio is 4. 5.
B Interpretation of Ratios

I When the fecal coUform/recal' strepto-
COCCUB ratio is greater than 4.0, thia
is regarded as overwhelming evidence
of pollution derived from human oripn;
or that if the pollutior. is of mixed
origin, the majority of such pollution
is of human origin.
2 Wtren the fecal coliform/fecal strepto-
coccus ratio i5 less than 0.7, this
suggests pollution derived predominantly
or entirely from liveetock or poultry
wa5tps. Feedlots, sto(:kyards, and even
storm water runoff usually produce such
3 Ra'tios falling between 4.0 - O. 7 are
not quite so certain. To be Bure, a
ratio of 3. 5, for example, would be
more suggestive of pollution reprellent-
inl predominantly human oripn; and Ii
ratio of 0.9 would be more succest1ve
of'animal Qrigin. A truly "gray-area"
of interprptation of these ratios is in
th~ range 2.0 to 1.0.
a When the ratio is in this range, it
frequently represents significant
mixtures of both human and animal
contribution, or
b The source of pollution may be some-
what remote, and due to differences
in the rates of disappearance of the
two bacterbal groups, the original
numerical relatior.sbips have been
4 Limitations on interpretation of fecal
coliform-fecal streptococcus ratios.
a The ratios have greatest reliability
for samples taker; not more than 24
hours flow time (or distance) from
the origin of the pollution, and
b The ratios must be baaed on'wate.rs
in pH range between 4.0 - 9. O.
c Total coliform c.:>unts cannot be.
ueed in determination or
interpretation of ratios with fecal.
A For Bome determinations, notably demon-
strations of pathogenic microorgaoisms,
quantitative methodolou either is lacking
or il so expensive and' time-conswning a8
to make lure tests completely impractical.
B In surveys involving pollution of intestiR&l
oripn. much attention i8 being given cur-
rently to the demonstration of pathogenic
bacteria~ notably bacteria -of the- genus
SalmoaU.. Such' orgaai8U)8. wheD found,
are interpreted to represent positive proof
of deleterious bacter101o8ical quality of the
water. since all members of the genus
are di8ea 8e- causing bacteria.
C The FWQA. survey report-of the Red
River Of the North presented data. on
occurrence of SabnoDella a8 shown in
Table 2 and Figure 2.

      Distance from     
     River waste source Flow time Total Fecal  
 Date Station mile (miles) (days) coli/lOOm! coli/100m! Salmonella i:;olated 
 Sept. 1964 plant 448 0  Not done Not done S. kentucky 
   RR 11 436 13 0.5 250,000 64,500 S. keDtucky 
   Rn 12 426 22 1.0 47,600 2,850 S. saint paul 
 New. 1964 RR 9 4&2 -14  314 49 Absent 
   RR 10 441 7 .3 432, 000 155, 000 s. typhimurium 
          S. braeDderup 
          S. readiDg 
   RR 11 436 12 .4 249,000 85,600 S. braellderup 
          S. heidelburg 
          S. reading 
   BR 12 426 22 .8 68,000 16, 800 s. bioclde1 
          8. braenderup 
   RR 16 386 62 3.0 6,630 1,610 S. beidelberg 
   RB 28 292 7 .3 39,800 2,970 S. reading 
   RR 29 286 13 .7 27,800 I, 030 S. reading 
          8., ialantis 
 Jan. 1965 RR 10 441 7 .2 182, 000 61,000 s. diester 
          S. tbompsOD 
          S. oranienburg 
          S. at. paul 
   RJtIl 436 12 .4 83,500 34,000 S. chester 
          S. beidelburg 
          S. st. paul ~
          S. enteritidis III
          S. typhimuriurn ...
   SH 13 42/1-1   650 218 Absent .,
   RR 14 416 32 1.2 18,600 9, 170 S. enteritidis f
          S. chester s:
          S. st. paul '<
          S. tbompson UJ
   RR 16 386 62 3.3 7,800 5,160 S. st. paul Ei
          S. enteritidis ;d
N          S. thompson I>
I   RR 18 375 73 4.0 6, 140 2,950 S. st. paul 
          S. thompsOft 

, Water Quality Surveys
- -- ------
RR-18(MLE 188)
RR-20(MlLE !4?)
Alt-18CMLE 557)

I' N. DAte.
I. .. DAK.
RR- i 5(MlLE 403)
SH-la(MlLE I)
RR-14(MILE 418)
.ltft-il(MlLE 4H)

"...... (MiLa 08)
-IO(MLI 440
RR-8 (MIL! 482)
Figure 2. Location of Sampling Stations and Station Numbers (River MUes) of Samples from tII..
Red River of the North. North Dakota and Minnesota

The following studie8 illu8trate the waY8 in
which certain pollution surveys became
needed, the examl.n&tionl made, and the ways
in which data were summarized and pre.ented,
leading to the final conclusion.
A Case Study U
1 Need
To determine whether the outfalls
discharging raw sewa,e from a
sewered population of 3820 persons
in VUlageville to River A between
miles 20 and 18, causes a deterioration
of water quality, thus con.tituting a
hazard to the city water plant of
Statesville with intake located at river
mile 15.2 and the County Water Plant
with the intake ~ocated at river mile
13. O.
2 Survey and procedures
A survey was eatablillhed for the IItUdy
of the reach of the river which included
the pollution in the river above the
alleged pollutiI1i city, pollution con-
tributed from a tributary creek, the
pollution contributed by Villageville
and the effect en water quality at the
water plant intakes. Samples were
collected and e1~amined every .ix hours
for seven con8ecutive day.. Bacteri-
ological procedures were the Standard
Methods multiple tube procedure by the
confirmed test for the coliform group
using three acceptable dilutions of 5/5/5;

the test for coliforms of fecal origin;
and the tentative plate count procedures
for fecal streptococcal group and their
confirmation by supporting biochemical
Water Quality Surveys
3 Results
These data are presented in Table 3
and Figure 3.
4 Interpretation of tha data
a The bacteriolo~cal data obtained at
eample points located at river miles
24 to 21 inclusive, established the
water in the river as being of
relatively good quality, with total
col1lorm density of approximately
600, 20 to 30 facal coliforms and 10 to
16 fecal streptococci per 100 mI. 'Th is
i. a re:Jatively high-quality raw-water
river supply in this area.

b The Clear Creek tributary was an
overflow from a lake with a coliform
den.ity of ap1'roximate1y 20 per
100 ml and both fecal coliforms and
fecal streptQ:=occi were absent. In
the absence of data on the cfe of
Clear Creek and of River A, it is not
pouible to partition the bacterial
densities at river mile 20 but it is
evident tha1 there is an apparent
improvement in water quality due
to the dilution factor.
c Between river mile 20 and 18, a
marked increase in pollution
occurred with 25,000, la, 000 and
4, 000, r~spectively, being the
den8ities of total coliform, fecal
coliform and the fecal streptococcal
groups. Periodic sampling of the
.ewer outfall" indicated the domestic
waite from VillagevWe was the sourCE;
of the bacterial densities.
d The bacterial data at river miles 16
to 10 incl\:sive demonstrated the poor
quality of the water due to the pollution
oripting from the untreated wastes
entering the river from Vil:Jageville.

Water QuAlity Surveys
e The presence 01 the fecal colUorm
group proves that th1s portion of the
total coliform group ora,:trated from
the gut of warm-blooded animale and
1s present in larp quantttWB.
I The presence of f.-eal pollution ma)'
be at al\)' time and frequently 1s
uaooiate" wi*h enter1c pathopDio'
btaoteria, ¥iral qents - pa,raait18 ,
or...s -8IId b7th8.. ......-ciatiaU.
1s . hazard to he&IIIh.
f The streptococcal group was proven
by a ser18s of b1ochen-..t_l reactions
to be identical with the '.c,l
streptococcal group lound in the eut
of warm-blooded ani;nals and there-
fore confirmed the interpretation of
the fecal coliform group.
h The presence of feall poUuUon, as
1nci1cated "7 the data . TtobIe 3.
cOR8tltutes I.JI. umacessar;y and
remedial !aUI'd and 1"1815 iD tile .
raw water supplies to the water
I River mUe Total Coliform Fecal Coliform  Remarks
i 24 610 30 38 
I 23 620 23 18 
 22 800 28 10 
 21 590 23 10 
 20 310 9 13 Clear Creek enters at
 river 1t~i1e 30. 4.
 19 2.000 600 180 Four 8f"wer ouUa1ls
 18 25,000 10, 000 4,000 between 1"1,,- lllUes
 19.7 alld 1...1i frQln
 17    ViJ:1ap"ille.
 16 20,000 10. 000 4,200 
 15    I StatesvUle water intake;
    . at mile 15. Z .
 14 17,000 9,000 3,900 
 13    COUnty water P»Dt
     , intake at mile 13. 0
 13 14,000 9.200 3,940 
 10 10,000 8, 900 3,700 
Bacterial densitlties caleuted a8 pometric mean value per 100 mI. Sample coUected and
examined every six hours for seven consecutive days.
Pppulation (sewered) of VWagevUle, 3, 830.
Consumers of water lrom city water plant, 70, 000.
Consumers from COUllty water plant, 205,000.
Velocity of river now, 0.5 mUes per hour.
Volume in cfs, information not avaUable.
Report of sanitary survey by engineers: no known sources of pollution observed
br.tween river mile 24 and 10 except &8 noted under remal'ks.
20- 8

in 1,000
Water Quality SUrveys
.j -
a:.j ILl
~ ~ ;'b!

, []
'..__..- '
 , 1 1
,  I II'
,  !
,  ~
  I- ~
  S ~
  iii a:
  i J
  )0 ~
  u z
18 II
Figure 3

Wa'er foNaUty ~rv~YI
B Cue ~udy 12
1 Need
A study was initiated at the request of
a south western state to prepare &
supplemental report on the baoterio1ol1cal
quality of & national wildlifl'! refute lake
based upon .tate gather.d data &s well
as an intenlive study by the FWQA
conducted -ring the same y...r. A
mailsive localized fish kill and observed
bacterial poUution indicator eft~la-
meftts on the southern half of the --
precipitated an urgent need for this
report. Filll!'e 4 depicte this lake,
Indian Hunt Lake, (here g1"l8n & fictious
name) and its major pollution tributary
source. A cloeer view cof thie souroe
in Figure 5 shows White Stone Creek
a8 the major influence with its tributary
Freehand Brook.
,.IIure ..

2 Survey and procedures
Indian Hunt LAke is a man made 2000
lI.cre wildlife refure and recreational'
area. Since there 1s a ]arg. population
of waterfowl (exceeding 1,025, 000 during
resting and wintering periods) and con-
tributing polluting sourcel from cattle
feed lots, it was apparent that data
would be required during dry periods
and during rainfall and subsequent Jand
runoff periods to de~ermine the extent
of this pollution effect. The necelsity
of this runoff data became even more
manditory when it was ascertained that
Freehand Brook was not a continual
free-running tributary and this effect
was only evident during periods of
rainfall and the dry periods produced
a dry bed and storage in Oxbow Lake.
Stream flow determinations wer made
and from 14 year averages peak flows
were determined and in this manner
maximum effects upon the Indian Hunt
Lake could be more easily determined
from current data.
Water ~lity Sur~s
Previous data for this study area was
compiled by the ltate agency and con-
.isted only of the total coliform
indicator taken by the MPN method in
monthly intervals. A four phase pJan
was estabUshed to accomplish the
survey objectives:
. Acquire bacbriological data for all
sample stations.
b Acquire additional bacteriological
data and note the effect of rec-
reational use on the bacteriological
quality of Indian Hunt Lake.
c Evaluate the effect of rainfall on the
receiving stream and Indian Hunt
d Data analysis and report preparation.
A mobile laboratorj' study was initiated
and samples we:re ualyzed by the
membrane fiher technique and the
indicator organisms included the total
coliforms, the fecal coliforms, and
the fecal streptococci.
...... Tr..'''.'''' ~I...t
~()- 'I

Wat~ Qual1.ty Surv~
3 Results
After the acquisition of backrrOWl.d data
from the study area Indian Hunt Lake
was aampled to ascertain the effect of
recreational activities on the bacteri-
olo8ical quality dur1na dry perlocl8.
Some of this data is reflected In
Fleure 6 which notes the fecal coliform
median values per 100 ml for th18
..Igur. «I
~Jo.. <>- . ~ .
period and the mrmber8 in t1£. . ... . ,wes
gives the median values per ~ :tM
duriDi periods of ra~ he. storm-
water runoff, aad SUbsequut .creach-
mant of runoff bacteria '."lab.
.2# - /2-
Arrow denot....mpUUe point. First ft1I1nber.
i8 median value of feaal ooUform per 100 ml
durbll dry per1od8 and eecond 1NIU8I' is
median vame of tecal coUform.per 1.oo.1D1
durlne pert0d8 of bea"" etormwatar .~.

4 Interpretation of data

Table 4 indicates the actual recom-
mendat10ns ,iven by the f1na1 report
and the column designated as Remark8
gives the interpretations derived from
the data.
Water Quality Surveys

Corrective Measures to More Adequately Control D1schargf!s from
Domest1c Sewage and Cattle Feedlot Drainage into IndilP.n Hunt Lake
1. Development of diversion dykes around
an cattle feedlots to channel drainage
into waste stabilization ponds.
Existing sewage treatment facilities
should be expanded to produce a better
quality effluent with a BOD reduction
goal of 85-900/0. Post chlorination is
desirable espec1ully during the recreat10n
season to further reduce pathogenic
hazards during thi, period.
3. Recreational restrictions will be
necessary whenever intense rainfall
occurs in a magnitude sufficient to
produce an inflow of more than 450
acre feet of water from White Stone
4. Restrictions to be placed upon the horse-
power and number of p:!.easure-boats in the
area of inflow frt'm Wh1te Stone Creek.
5. A buffer zone must be continually main-
tained and enforced between the wUdlife
refuge area and those areas designated
for swimming, wa
Water Quality ~rveY8
A similar swdy waa made of Whl,te Stone Creek and Freehand Brook and this Is .hown in FiFe '1
and FilUre 8. In both f1iure. (FiFe '1 - dry period. and F1l1U'e 8 - after heavy .tormwater
overflow) the circular areaa indicate the fecal coliform valU.. and al80 the FC/n ratio. (fecal'
coliform to fecal streptococc:1) are indicated for thia IIaDlpllAi potato .
Circular areas repr..ent fecal coliforma per
100 ml. FC/FS (hcal coliform tQ fecal
atreptococc1 ratio) ratios are indicated for
each station.

Ory P.rlo~
.act.r.ol..loal. Data
I'C/I'S .0..
8.&(1. Tr.atm.nt PI.,.t
.If} ,-I

Water Quality Surveys
FC/pre - 0."
Figure 13
H.avy Stormwater Overflow
Bacterlologlca, Date
070. ,~-

Water Quallty Survey.
,.I.ur. 8
Op.n.d to boatlnll .nd
'Ialllnll f,om
Marcil 1 . Oct 31
Wat.rfowl 8..aon
Nov 1 . ,..b 28
Indian KlInt lAha
Nation. W I lei," f. ...,"'..
KittraU. F. W. and Furtart.. S. A.
Ob..rvati0ft8 of Coitform Bacteria
in Str..mll, Jour, Water PoUutt.on
Control Federation 35:1381- 85. 1963.
In the preparation of thie CI1tllne the a\8hor
ha. m.de exteneive use of material made
avaiIable by Mr. Harold F. Clark (decea.ed)
formerly bacteriological con8Ultant to the'
Enforcement Branch. DWS & PC by Mr. F. W.
Kittrell (retired) formerly ehlef, Technical
Advisory and Inve.tigatlone Activt.ti88. FWQA
and Mr. Edwin Geldreich. OIief Bacteriolopat,
Bureau of Water Hygiene. Environmental
Protection Agency, Cincln."'/.att.. OH.
'1tht.e outb.n. wa. prepared by H. L. Jeter,
Director. and R. Ru..omanno. Micro-
biololf,8t. '*'lon81 Tr..in1nI Center. WPO.
EPA. C1nciftDati. OH 4&269.
~: Data Handlinl. Evaluation.
~, Survey., Water Pollution.
Water Qq&Uty
Outline, this manual, titled "Bacteriological
Indicators of Water Po1\ution" and
reIated references.
20- 16

The type of stream survey report to be pre-
pared depends on two basic factors. These
are the purpose and the audience for whom
A The Purpose of the Report
1 A report of findings or basic data
2 A report of existing causes and effects
together with an explanation of how and

3 An exposition of existing causes and
effects and a projf:lction of conditions
that reasonably may occur due to natural
variations in stream flow and temperature.
4 A purpose similE..r to 3 above plus a pre-
diotion of the effect8 of population growth
and industrial change.

5 The same purpose as 4 above plus an
estimate of the need to protect water
uses, and cause reduction in waste
B The Specific Audlen~e for Whom the Report
Is Prepared
1 For the record - no expository purp08e

2 Other technlcnl agencie8 with compe-
tencies in the 8ame field
3 Other technical agencies In other fields

4 Public officials 8upportlng or oppollng
the recommendations of the report
5 The general public

C Both in style and content the report should
be adequate to serve as a basis for action
to accomplish the recommended objective8.
WP. SUR.IBb. 3. H
A Title, Authors, Contents
B Acknowledgemetlt of Aid and Assistance
1 Can be included In a letter of trans-
mittal or 8ubmission
2 Can be incorporated in a preface or
3 Should include names of persons and
of corporationf-, public and private,
who auisted or aided the survey
C Authority
1 Source of A uthor1ty
2 Date of authorization
D Report Summary

1 A brief summary of the report and its
recommendations generally precedes
the report proper and should include
three topics:
a Summary of 8pecific findings
b ConclU8ions drawn from findings
c Recommendations in general terms.

2 Brevity is eS8ential but not at the
expense of clarity.
3 A very brief but lucid description of the
stream section involved should be
4 This will be the only part of the report
read by many ,)f its audience. Conse-
quently it 8hould be drafted with the
utmost care.
21- 1

Water Quality SlArveys
Figure -1
1\ FilWre 1 illustrates the principles of
written communication format. It 1s
important that technical reports be
presented in factual report arrange-
meltt and NOT in one similar tel that
of fiction.
6 Figure 2 shows how the various
portions of a survey report relate
to the generalized factual report.
7 Recommendations, although brieny
stalled in general terms, should be
couched in positive, unexa.gerated
8 Cost estimates of compliaAce with
recommendations is helrful but not
9 Botta tangible and intanJible benefits
may be listed brieny under cODdus1ons.
The body of the report should belin with a
statement of the problem and a discussion of
the reasons for and the location of the study.
..... ... ......_..n..
FillJre 2
1 Statement of the problltm
a Ade8cription of the area, empha.
siaing pertinent features, should
be included.

1) The inclusion of pertinent histor-
ical data is of value tor audience
2) The relationship of thi8 8tudy to
other current water resource
3) Water use and economic data may
be import:mt.
b An area map i8 an absolute necessity.
1) Feature8 included should be care-
fully selected and ..ot~on of the
stream involved should be
2) Do not include unnece8sary detail.
3) A general location map u8ually
orients the reader to the area
2 Objectives of the Survey
a A statement of the purpose by listing
the specific iLnswers sou,ht by the
survey to address various aspects of
the problem.
b The geo,raphical and tim-e sCO!," "f
the objectives ot the survey.
F Survey Methodology
A complete description of the methods of
study employed is an important part of
the record.
1 The time period of survey should be
2 All sampling and gauging station loca-
tions should be identified by river
3 Sampling and analytical method8
a Provide adequate de8cription of
all non-standard methods.
Water. Quality Surveys
.. An appendix for these descr1phoas
may be required if they are lengthy.
4 Frequency of sampling
5 Description of laboratory types and
6 Hydrological methods employed for:
a Times of WItter travel
b Stream flow data
c Any waste flow measurements
G Survey Results
1 Sources of wastes
a Computed waste loads based on
known contributing populations and
industrial waste strength
b Results of sampling and gauging

c Data summaries or displays suffice
for text (>f report.
2 Stream data
a Summari2:ed survey in the text
Complete tabulations including time
of collection and averages In appendix
3 Hydrological data
a Usually tabulated with analytical
results both in the text and in
b Time of water travel curve or curves
c Stream flow frequency charts
d Pertinent groundwater data
4 Aesthetic comdderations are of real

Water Quality Surveys
H Analysis and Interpretation of Date
 1 Three fUndamentel procedures are
  a COmparisoo of survey results and
   appropriate wa1.eJ' quality criteria
  b Projection of sunG)' data to provide
   for comparison of stream conditions
   with water quality criteria under
   more adverse c'3nditions
  c Eatimates of parma sible wastes
   loads under present and future
 2 Since this topic i. the aubject of other
  outlinea it will not be fuMber developed
 3 A description of the method. of analy.is
  and tnterpretation belong in the report.
  a Nece.sary auumpt1.ons should be
  b All statistical methods used ahould
   be identified. 
 4 Reaults of analysis and tnterpr~ation
  are belt presented in chart form insofar
  loa possible, to 8UPfOrt discus.ion and
  interpretation rationale. 
 1 Focua of attention, much of it c"itical,
  is on this aection of the report.
 2 Clearness. conaisenes., and positive-
  ness are essential. 
 3 This section indicates reuoning that
  leads from findin,s to conclusions.
J Recommendations 
  This is the crux of the report and
  should answer the question, "What
  needs to be dene to resolve problems
  'delineated in the report? "
Cost estimates are higbly desirable,
if polatble, and apfropnate.
K BibliolJ'aphy
Uaeful to the student and perhaps to
future workers
Entails additional time and effort in
a..embling references in proper form
A Prepare a Detailed O\atltne
1 This is an tmpol'tllRt step if the coverage
of the report t. to be completed and. its
arrangement Iollcal. It i. also a time-
laver if properly done.
2 Topics to be tnclucled in the outline
will become apparent from the fore-
going di.cus.ion of report content.
B Words are Your Tools
1 Keep dictionary, thesaurus, and
1J10...ry avaiJab1e.
2 Define any vague terms or abbreviations.
3 Control superJatives. and alang.
4 Avoid emphatic. luch a. "it il to be
noted" or "tti. a wen-known fact".
. 5 Limit intensive expreuions. such 8.
"extremely" or "undO\llDtedly."
6 Use active verbl! when poa8ible.

C Regu]arly review the report or gantzatton
and development with a coUealUe.
This outline was prepared by Staff Members,
National Trainlni Center, EPA, WPO,
Cincinnatt, OR 45268
~: Reporta, Surveys, Technical
~ter Quality

Part l: The Nature and Behavior of WateJ:'
The earth is phYlically divisible into the
llthosp!!!!:! or land malles, and the
hydrolphere which includes the oceans,
lakes, streams, and lubterranean waters.
A ]'pon the hYdrOI~ere are baled a number
of llciences whlc represent different
approaches. Hydrology is the general
science of water itself with its various
special fields such &II hydrography,
hydrauUcs, etc. These in turn merge
. into physical chemistry and chemistry.
B Limnology and oceanollI'aphy combine
aspects of all of theile, and deal not O:lly
with the physical Uquid water and its
various naturally occurring solutions and
forms, but also with livin, organisms
and the infinite interactionl that occur
between them and their environment.
C Water quality management, including
pollution control, thus looks to all
branches of aquatic. 8cience in efforts
to coordinate and improve man's
relationship with his aquatic: environment,
A Water i8 the only abundant Uquid on our
planet. It has many properties most
unusual for liquids, upon which depend
most of the familiar alpects of the world
about us as we know it. (See Table 1)
- PI'Gp.rty
HI.~..t hOlt .....elty (ipecllic hOlt) al Iny
lulld a,' liquid (.xc.pt NH,I

HI"'.lt latlnt h..l oIl..Ion (excepl NH,'

81....t h.., 01 evaporation of aft)' 8ub.tance
StabUi... hI......t....H of orpnl.m. .nd-
.oo....phlcal r..lonl
Thenn08tatic .tt.ct at freesme point

Imponant In h..t .nd ...ter tra",'er of
The only 8ub.taae. 1M' .... ltl Ift&xinu.m
denllty.. 8 liquid (40C)
P ...1Ia and b...ckWh water. have maximum
dlG.lty above Ir...In. paint. Thl. II
bnportant in .,.rttcal circ,.1ation pattern
in lake..
Hi,h..t .urlace '.n.- of any liquid
Dielo!"" more ,"""0.. in ar-t.r
quantity than IIIJ' other liquid
Controle lurrace and drop pIIenomena.
Importanl In cellular phy.lolalY

M8ku co...,l.. IIIttIop..1 1,.ltem pol.Ib1e.
Important lor !",,"portat"'" al mal.r1811
In _lion.
1..-d8 to hllh di8loc18t1on oIlaorpnlc
8U"tanO.' in .ollatlon
Pure w~ter hol Ihl """.11 cU-.llotrlc
-Itant of""" U'l"1d
Neut...l. yet coetllw both H+ and OH - tone
VOl')' littl. .1ealroJ,tta dl800al8t\on
".lallv," t....eparellt
Abeorb. much. _PlY III infra red and ul'ra
riollt ru,,". but Itttl. In vtllble ...n'8.
H8ftCe "color..."
BI, 21e,1. 74

The Aquatic Environment
B Physical Factors of Sitniftcance
1 Water sub8tanct'
Water is not simply "H20" but in
reality 18 a mixture of 80me 33
different substances involvinll three
isotopes each of hydroll~n and ox:y,en
(ordinary hydrogen HI, deuterium ~2,
and tr1t1um H3; ordinary oXYlLen 01 ,
oxygen 17. and oxypn 18) plU815
known types of Ions. The mol~cules
of a water mass tend to "8oc,iate
them8elv.. a8 polymer8 rather than
to remain as discrete units.
(See Fi8\11"e 1)
o 0
rip" I.
2 Density
Ii Temperature and dea81ty: ,Ice.
Water i8 the only, known substance
in which the 80lid state will float
on the liquid state. (See Table 2)

Temperature (OC)
- 8
- 6
- 4
- 2
1. 00000
. 98997
. 95838
. 939.7
* Tabular value8 for den.ity, 'etc., represent
estimates by variOU8 workers rather than
absolute value., due to the variability of
** RefUlar 108 i.known as -'ice I". Four or
more other "fot'ms" of ice are known to
exi8t (tee n, 108 nI. etc.), havin, densities
at 1 atm. pre.wre ranging from 1. 1595
to 1.87. These are of extremely restricted
occurrence and may be ipored in most
routine operations.
This ensures that ice usually
forms on top of a body of water
and tenc18 to insulate the remain-
in, water mass from further 10s8
at heat. Did ice iJ1nk, there
could be l1ttle or no carryover of
aquatic l1fe from aeason to 8eason
in the M,her latitudes. Frazil or
needle ice formscoUoid&lly at a
few thousandthfl of a degree
below 0:> C. It is ..dbesive and
may build up on submerpdobjects
as "anchor ice", but it i8 stlll
typical ice (ice I).

1) Seasonal increase in solar
radiation annually warms
surface waters In summer
whUe other factors result in
winter cooling. The density
differences resulting establish
two dassic layers: the epilimni.on
or surface layer, and the
hypolimnion or lower layer, and
in between is the thermocline
or shear-plane.
2) WhUe for certain theoretical
purposes a "thermocline" is
defined as a zone in which the
temperr.ture changes one
degree centigrade for each
meter of depth, in practice,
any transitional layer between
two relative1y stable masses
of water of different temper-
atures may be regarded as a
3) Obviously the greater the
temperature differences
between epilimnion and
hypolimnion and the sharper
the gradient in the thermocline,
the more stable will the
8 itua.tion be.
4) From information given above,
it Ihould be evident that while
the temperature of the
hypolimnion rarely drops
much below 40 C, the
epUimnion may range from
00 C upward.
5) When epilimnion and hypolimnion
achieve the same temperature,
stratification no lonier exists.
The entire body of water behaves
hydrologically as a unit, and
tends to as.ume uniform chemical
and physical characteristics.
Even a light breeze may then
cause the entire body of water
to circulate. Such events are called
overturns, and usually result in
water quality changes of consider-
able physical, chemical, and
biological significance.
The Aquatic Environment
Mineral-rich water from the
hypolimnion, for example,
is mixed with oxygenated
watE'r from the epilimnion.
This tlsually trigger s a
sucden growth or "bloom"
of plankton organisms.
6) When stratification is present,
ho'otever. each layer behaves
rell\tively independently, and
significant quality differences
may develop.
7) Thermal stratification as
described above has no
reference to the size of the
water mllss; it is found in
oceans and puddles.
b The relative densities of the
various isotopes of water
influence its molecular com-
position. For example, the
lighter 016 tends to go off
first in the process of evaporation,
leading t~ the relative enrichment
of air by 016 and the enrichment
of water by 017 and 018' This
can lead to a measurab];y higher
018 content in warmer climates.
Also, ~he temperature of water
in past geologic ages can be
close];y estimated from the ratio
of 018 in the carbonate of mollusc
c Dissolved and/or suspended solids
may also affect the density of
natural water masses (see Table 3)
Dissolved Solids
(Grams per Uter)



(at 40C)



35 (mean for sea water)

The Aquatic Environment
d ,Types of density .t~tification
1) Denl1ty difierence8 produce
ItriLtif1cation which may be
permanent, transient, or
2) Permanent stratification
exists for example where
there is a heavy mall of
brine in the deeper areas of
a buin which don not relpond,
to seasonal or other chanJin,
3) Tranlient stratification may
occur with the recurrent
lntlux of tidal water in an
estuary for example, or the
occasionallntlux of cold
mud~ water into a deep lake
or reservoir.
4) Seasonal stratification is
t)'pical~ thermal in nature,
and involve I the annual
establishment of the ep11imnion,
~11mnion, and thermocline
as described above.
5) Denlity stratification il not
limited to two-laqered .Yltems;
three. four, or even more
~rs may be encountered in
larler bodies of water.
e A "plunge line" (sometimes caned
"thermal line ") m-r develop at
the mouth of a .,tream. Heavier
water f10wing into a lake or
reBervoir plun,es below the
li,hter water mass of the epWminium
to flow alon, at a lower level. Such
a line 1a uBuaUy marked by an
accumulation of floating debril.
f Stratification m~ be modLfied
or entirely suppressed in lome
caaes when deemed expedient, by
means of a simple air lift.
3 The vi.colity of water is greater at
rc;;;rtemperatures(See Table 4).
Thi. ia important not onIJ in situation.
invol"l&n, the control of fiowing water
ae il1 . aand filter, but .110 since
overcomil), re.i8tu1ce to now ,en-
erate. heat, it is sipW.c.ant in the
heatln, of water by internal friction
from wave ad current ' action.
Llvil1, orlaniems mQN eal1~ support
theml.lve..in the more viscous
(and also dena.r) cold. waterapf the
arctic than in the lesa vilcous warm
water. of the tropic.. (See Table 4).
VISCOSITY 0.,. WA TER (lnmUlipoises at 1 atm)
 Dia801ved 8011. in fiTL 
TeInD. 0 C 0 5 10 30
-10 26.0 - --- ---- ---..
- 5 21.. ---- .. --- ----
o 17.94 18.1 18.24 18.7
I) 15.18 1&.3 15.5 16.0
10 13.10 13.2 13.4 13.8
30 8.00 8.1 8.2 8.6
100 2.84 .. -.- ---... ----
4 Surface ten810n haa biololical as well
as physical 8ipificance. Organisms
whoee bod,. 8urfaces cannot be wet by
water can either ride on the surface
film or in lOme m.tanee. may be
"trapped" on the ftrflice film, and be'
unable to re-eate%' the water.
5 Hea' or enern
Incident solar ra«aUon i8 the prime
source of enerQ for virtually all
orpllic and moat ir10rpntc processes
on earth. For the earth as a whole,
the total.mount lof enel'lY) received
annua1q mult exac~ balance that
loet by renection ad radiation into
space if climatic and related con-
ditions are to remain Z"elativ~
constant over ,eo1octo Jime.

The AqaMio ~vironment
a For a given body of water,
immediate sources of energy
include in addition to solar
irradiation: terrestrial heat,
transformat1:m. of kinetic energy
(wave and current action) to heat,
chemical and biochemical
reactions, convection from the
atmosphere, and condensation of
water vapor.
81gnt.ficL>1t chante in surface
level is de~ected. Shifts in
8ubmeried water masses oC
this type can have severe effects
on the bIota and also on human
water uses where withdrawals
are confined to a given depth.
Descriptions and analyses of
many other types and sub-types
of waves and wave-like movements
may be found in the literature.
b The proportion of li,ht reflected
depends on the an,le of incidence,
the temperature, color, and other
qualities of the water; and the
presence 01 absence of fl1ms
of lighter liquids such as 011.
In general, a. the depth increases
arithmetically, the light tends to
decrease geometrically. Blues,
greens, and yellows tend to
penetrate most deeply while ultra
violet, violets, and orange-reds
are most quickly absorbed. On
the order of 90% of the total
illumination which penetrates the
surface film is absorbed in the
first 10 meters of even the clearest
water, thus tending to warm the
uppe r layer s .
b Tides
1) Tides are the longest waves
known, and are responsible for
the once or twice a day rythmic
rise and fall of the ocean level
on most ahores around the world.
2) While part and parcel of the
same phenomenon, it is often
convel"ient to refer to the rise
and fall of the water level as
"tide, " and to the resulting
currents as "Udal currents. "
1) The beet !mown are traV'1~ni
wave. caused by wind. T ese are
err;ciive only against objects near
the surface. They have little
effect on the movement of large
masses of water.
3) Tides are basically caused by the
attraction of the sun and moon on
water masses, large and small;
however. it is only in the oceans
and possibly certain of the larger
lakes that true tidal action has
been demonstrated. The patterns
of tidal acUOD are enormously
complicated by local topography,
interaction with seiches, and other
factors. The Uterature on tides
is very large.
6 Water movements
a Waves or rhythmic movement
2), Seichee
c Currents (except tidal currents)
are steady arytl\mic water movements
which have had major study only in
oceanogra~hy although they are
most often observed in rivers and
streams. Tiley are primarily
concerned with the translocation of
water maseE's. They may be generated
internaUy by virtue of density changes,
or externally by wind or terrestrial
topo,raphy. Turbulence phenomena
or eddy currents are largely respon-
sible for lateral mixing in a current.
These are of far more importance
in the economy of a body of water than
mere lamina. flow.
Standing waves or seiches occur
in lakes, estuaries, and other
eneloseri bodies of water, but are
seldom large enough to be
observed. An "internal wave or
seich" is an oscillation in a
submersed mass of water such
as a hypolimnion, accompanied
by compenutm, oscillation in the
overlying water so that no

'tb, ACB1~tic El\v1ronment
d ~ force Is a result of inter-
actIOii'between the rotation of the
earth, and the movement 01 masses
or bodies on the earth. The net
renlt is a sl1pt ~endency for moving
objeQts to veer to therl,ht in the
northern hemisphere, and to the
left in the souther.l hem1aphere.
While the result in fruh waters Is
UluaU,y ne,l1Jible, it may be con-
si.fable in marine waters. For
example, other factors permitting,
there is a tendency in estuaries for
fresh waters to move toward the
ocean faster alone.the rlaht bank,
wbUe ..It tidal waters tend to
Intrude farther inJlmd aJon, the
left bt.nk. Efteetll are even more
dramatic in the open oceans.
To somewhat overaimpltfy the
COlicept. a series Clr'adjoinlng cells
mifh* be thoulht of .. chains of
biterlocJdDi paJ'8 in which at evet')'
cdher contact the ... are ris1nl
while at the ..1tenlate contact.. they
are siOk1n, (lI'tpr'e .).
e ~~ (or L. spirals)
1itIieTrite~ation of
somewhat cylindrical manes of
surface water under the influence
of wind action. The axes of the
cy11nders are p.ra1lel to the
direction of the wind.
~. r~ i8 ek1nptfed masses of
water tisinr or s'1ntiaDgtoptber.
This pI'Oducllifli the tIImillar "willd
rows" of foam, flotsam and jetsam,
or plankton otten"" strealcinf
w1nclt1own lake. or_ane. Certain
8GO-,Jaakton ~ to maintain
a potflntm near "~ce tend to
cOllect bi the down 4$rrent between
two ~1Ur c4tlU,' C&1I8b1g I'UCh
an are. to b. csne4""'. "red-daRce",
wbile the clear upWeUtn, watSt'
between i8 the "blue dan"".
This phenomenon may be important
in w.ter or pJankton 8&mpl1nR on
a windy day.
r/ b
~,. '1
_ai... ..:I

r..7~ WAt~.
.0' HIrAM
"',. 1:
Fllure 2. Langmuire Spirals
b. Blue dance, water rislal. r. Red
dance, water sinking, ftGatitlg or
swimminl! obiects concentrate4~

The pH of pure water has been deter-
mined between 5. 7 and 7. 01 by various
workers. The latter value is mOlt
widely accepted at the pre8ent time.
Natural waters of CO\lrse vary widely
according to circumstances.
C The elementl of nydroloiY mentioned
above represent a selection of some of
the more conspicuous physical factors
involved in working with water quality.
Other items no'~ specifically mentioned
include: molecular structure of waters.
interaction of water and radiation,
internal pressure, acoustical charac-
teriatics. pressure-volume-temperature
relationships, refractivity, luminescence,
color, dielectrical characteristics and
phenomena. solubi!ity. action and inter-
actions of gales, liquids and solids,
water vapor, phenomena of hydrostatics
and hydrodynamics in general.
The Aquatic Environment
1 Buswell, A. M. and Rodebush. W. H.
Water. 5ci, Am. April 1956.
2 Dorsey, N. Ernest. Properties of
Ordinary Water - Substance.
Reinhold Publ. Corp. New York.
pp. 1-673. 1940.
3 Fowle. Frederick E. Smithsonian
Phylical 'rabIes. Smithsonian
Miscellaneous Collection, 71(1),
7th revised ed., 1929.
4 Hutchelon, George E. A Treatise on
Limnology. John Wiley Company.
This outUne was prepared by H. W. Jackson,
Chief Biologist, National Training Center,
Water Programs Operations, EPA, Cincinnati.
OH 45268.
Aquatic 'EiiVironment, Estuarine Environment,
Lentic Environment, Lotic Environment.
Currents, Marshel>. Limnology, Water

Part 2: Tb8 'Aquatic Environment as an ECINI,..t!'m
Part 1 introduced the lithosphere and the
hydrosphere. Part 2 will .1 with certain
general aspects 01 the biolphere, or the
sphere of life on 'this earth, which photo-
graphs Irom space tmve ahowD is a rinite
globe in inrinite apace.
This i8 the habitat 01 man and the other
organisms. His relatkmships with the
aquatic biosphere are OUr common concern.
A We can only imagine what this world
must have been like before there wa. Ufe.
B The world a8 we know it i8 largely shaped
by the forces of life.
Primitive forms of life created organic
matter and eswbliahed soil.
Plants cover the lands and enormously
influence the forces of erosion.
The nature and rat. 01 .rosion affect
the redistribution of materials
(and mass) on the surface 01 the
earth (topographic changes).
4 Orlanisms tie up vast quantities of
certain chemica.ls, aueh as carbon
and oxygen.
R..piration of plants and animals
release!! carbon dioxide to the
atmosphere in influential quantities.
6 C02 affects the heat tranamission of
the atmosphere.
C Organisms respond to and in turn affect
their environment. Man is one of the
moet influential.
BI.;) 1..1.74
A The ecoeystem is the basic func~onal
unit of ecolOfD'. AfI¥ area of nature that
includes living organisms and nonliving
sub8tances interactlni to produce an
exchange of matezola11 between the living
and nonliving ps.rta, constitutes an
ecosystem. (~ 1959)
1 From a stnaotural standpoint, it is
convenient tc recoanize four
consUtu8l'1ts ae composing an
e..,stem (Figure I),

which are the physical stuff of
which living protoplasm will be
b Autotrophic (self-nourishing) or
PRODUCER orp.nisms. These
are 1arge~ the green plants
(holophytes), but other minor
groups must aiso be included
(See Firur8 2). They assimilate
the nutrient minerals, by the use
of cOl1side.rable energy, and combine
them into ]lYing orp.nic substance.

c Heterotrophic (other-nourishing)
CONSUMERS (holozoic), are chiefly
the animale. They ingest (or eat)
and digest organic matter, releasing
con.lderable energy in the process.
d Heterotrophic REDUCERS are chiefly
bacteria a,.old fungi that return
complex organic compounds back to
the orlpnal abiotic mineral condition,
thereby relea.ing the remaining
chemical energy.
2 From a functional standpoint, an
ecosystem has two parts (Figure 2)
Pr8C8~1a1 Plgi blilk

The Aa.uatle Env1.romnent
.1 I;' . I 118
'101 U CEI'
. E H,' E '~'If'
a The autQtt'ophl~ or pt'ocNCU'
orpnlaml, which oonltruct
orpnle eubatance,
b The heterotrophic or conlwner and
reduoer orpntsm. which destroy
orp.ntc subltanee.
3 Unlea. the autotrophlo and hetero-
trophic phaBea of the cyc]e approximate
a dYnamic equW.brtUll\. tha ecotty.tem
and the environment wUl otanf8,
B Each of these lP'0ups includ.. slrap1e,
.1aC1e-.ceUed representati..., perat.tin,
at knYer level8 on the evol~ional'1 eteml
ol the hi8her orpnilm., (Flp1'8 II)
1 These ,roup. span the gap. ~we.ft the
higher kln,doms with a multit1lde of
tran.ltional forml, They are.OO1MIctlvely
caUed the PROTISTA,
2 With1n the pl'Glti8ta, twe priDctpal sub-
IP'0UPI CaD be defined on the baaia of
Te1at1.e COfttp1e~t7 of atnature,

a The bacteria and blue-fr8en a1llM.
1&c- a nucl8..r m.1IIIIn'8ne m1fi3"
be conatd....ed aa tb8 .....~. .
(or ~).

b The am,le...oeUed a.... 8Dd
prow.oa ~. beat ref.".,.. to ..
the Rlpr ProUD.
C Dlatrlbuted thrOUfhcKrt the. po.,. wtU
be fOUftd mo8t of the tra~...,,!'pllar1a"
of c]&..10 btolol1.
A A,ood c:e. Is the transfer of food enerl)".
from.p Ida throup a ..rt"'.'OI'p1lttama
with repeated .attat and bt11th148tea.,
Food otaw are not iaolated Nquence. but
are interconnected.

The A9~tic E;nvirODm~t
Enerl)' Flow. 'rom Lett to RI"'t, Oeneral EvOlutionary Sequenc. I, Up_rei
Orpnlc Material PrGduct4
U8IIIlly by Pllow.;mtiiitI
Orpnic Mate..lalln,.eted or
D~.- Internally
Orpnlc Matertal lIe~.d
by 1!:xtrat:eUuw mjii1IOA
and IntraceUuw M.t8boUem
to Minerai CondtUon
J'low.rlnf PlInte IUId
C1\ab Mo...e, Ferne
Ltverwort., Mo...e
Multicellular Or.....
Red Aillae
Brown Aillae
Arachnid. Mammala
In..et. Bird.
Cru8taceanl R eptll..
Be.....ented Worms Amphibians
Mollu.c. F leh..
Bry.. 08 Prlm1tlYII
Roundworm. Echinoderm.
Funlli lIr.perfecti
Hl,her Phycomycete.
Unicellular Gre~n Allee
Pigmented Flallellate.
Blue Or..en AI,.e
Phototropic Bacteria
~ . ..

(or: Monera)
Chemoh'oplC Bacteria
B1. ECQ, pI. aa. I.. ..
(Chytrldialee, et. 81. )

Tn. Aavat1c Env1roMlem
:s ~ i. the interlock1nc pattern of
~ in an ecolyaem., (J.\'fcur.. 3, ,,)
In complex natural comm\1rl1fie.;' o~.m.
whoae food i8 obtained by i;he eame number
of .teps are .aid to be" ~o the .ame
trophic (r..d1QI) level.' .
c TroPhic Le.,.J"
1 First - Green plant. (producer.)
(Figure 5) fix biochemical energy and
.,Flthe.i"e basic or~c .~ce..
'lbi. I. primary produotion .
2 Second - Plant eatinC animal. (herbiwre.)
depend on the producer or.""m. for
3 Third - Primary carnivmoe., animal.
which teed on herbivore..
" Fourth - Secondary carnivor.. feed on
primary carn1vore8.
I) ast - Ultimate carnivore. are the last
or ultimate level of con.ume....
-" -.
,'~;-. ,
D Total A8l8imUa.tion

Tb.""" of .....,..h£lIh ftDwa Glroqh
a trophic level i8dt.trtl5uted Mttween tke
productton of biorna.. (UYiDlnllatlUlCe),
IIftC1 the demand. ot l''''ira~ (Internal
enet'l1l1le by Uftn.,or,.....) in a ratio
ot aJIIIII'8iJuC.~ 1:18. .
E T~o~n at" Eaof1lit.m
The interaction of thelood chain
phenome. fwtth enerl.1 :/Q..at Hch
tran.fer) Hault.in ftltou6clbm:lolUlUie.
hav1nlllleftD1te trophic .truClM'e orenerlY
leve18. ~c .tructure -'7"
meaeured_d de.cdbed either 111 terms
of the n+. crop per unit area or in
term. of_t'I7ftxed per.ua&t area ,per
unit time ., 1NOO8..ive tl'Op¥c levell,
Trophic IItrJI1ct1aaIe IIDd tuncUDa can be
shown ..,....., by mean. ., .co1opcal
P7Z'.m.~lrare 5),
, .

'.':"... ..
" "'.': .":" ,'. ',\:,",:.

Figure 3. Dlap8111 ., the poIICI..,-. lule uftit. ~ '';' iolJowal I. .bIock! ,""'.or. ..........-
........ ~UDdl; UA. proiI_1I-IIIiM8cI wptatlon; 118, prod--ph~1OIII 111-1"- pn.uy -
(IIIrW_)-bottom """'1111,11, JI'IIII8!Y - (h.rbi_)-~1IIIi 014. -- - c-
---II u"" 1IItIouJ- I"""" ......),IV, -"l'Il' r I II_ria ..,...,....,.

..-- ------_._.
-"--. .- -- ... -~---- The ~uatie E_vl.r~nrnent
.-."" L.t'~.., ...
1111"0\'1'1"'''' "".",
. """",,,..,.,,.,,...,,,.
"..' OlIO' 'II' '." ~"~' , '" 1;1
FiJUre 4. A MARINE ECOSYSTEM (After Clark. 11154 and Patten. ~966)
.2.)- 13

The Aquatic: Env1\"oronent
(a) Numbers of 1ndividual8, (b) Bioma.., and
(0) Energy (Shading Indicate. Bur., 1.0").
A PJankton are the macroscopic and
microscopic animals, pJam., bacterIA,
etc., floatm, free in the open water.
Many, cIoi filters, cau.e tastes, odor.,
and other troubles in water supplie..
Eiis and larvae of lar,er farms are
atten pre.ent.
1 Phytoplankton are plant-like. The.e
are the dominant producers of the
water., fresh and salt, "the lI'ass
of the Ie..".
2 Zooplankton are animal-like.
Includes many different animal types,
rahge in size fram minute protO'..
to gf.p.nt1c marine jelqfilbes.

B Periphyton (or Aufwuchs) - The communities
of micr08copic argani8n\8 lI..soctated with
submerged surfaces of any type 0'1' depth.
InclUde. bacteria, al...... pret.o,loa, and
ather rn1oro.co;k: anbnala. -.ad blten the
youn, or embl'fGOic -ees "f' a!lll' and
otber DrJUiIntS tut no~u,. jroW up
tobeeoiaea 1*" of tMbentliM-C"e below).
M.l\y plbktoJiic type. ~SU a* ~ete
to nl't'at:.. d,petip\111OD. ~ .,me
typi~ ,pllriphyttlft ma)' br~,otr and'
be collected is ptahMers.' . :

C Bent"- are tl1e pJantl"and aD~. livini
an, in. 0'1' eio..J:f as'Oclated ....14 the
-bottO'fn.. . They include .,-Jaats and'
D Nektan are the community af Itl'Clftg
a'lI'essive swimmers af the' ~ waters.
aften called pe11apc. Certain f~es,
w81.... &ad llwertebr.te. 1Ub.:..
shrimps and squids are 1r.Ic1v~'here.
E The marsb community is based on larger
"hlahwr" pl_.. f1oattit. and emergent.
Both m~ and fre.hWater mar8he. are
areas at enOrftUMl8 bidlcS~cal productian.
Cal1e.-,a..v kl'id\m a8 "wet1aad8". they
bridle .~. p, Ntwe'l\ .. ~er8 aDd the
dry 1andII. '

A The blo1oiical re8ultant of aU phy.ical
and chemical factors tn the quantity of
life that may acluaUy' be present. The
ability to produce this "blomas." is
otten,r8ferred to as the "prOdUct1Ytty"
of.a bady 01 water. This i. neither load
nar bad per 8e. A water af low pl'O-
ductiv1ty I.s a "poor" water bio1og1.:aUy,
and ,also a r.lative~ "pure" 0'1' "diatt"
water: _ee deairable as 11 water .upplf
ot' . ~thtn. b*,.ch. A praductive water
on the other hand may be a nu1'anoeto
man or hi&bly de.irable. It i8 a nutsance
if foul adars and/or weed.c:hocked
waterways resd, it i. desin.ble if
bumper crops ot bass. catfish, ar
oy'ters are produced. Open oceans have
a klw level of productivity u.. pnel"8l.

1 Clarke, G. L. Elements of Ecology.
John Wiley 8t Sons, New York. 1954.
2 Cooke, W. B. Trickling Filter Ecology.
Ecology 40(2):273-291. 1959.
3 Hanlon, E. D. Animal Divenlty.
Prentice-Hall, Inc., New Janey. 1964.
4 Hedgpeth, J. W.
Eco8)'stern .
Publ. No.3.
Aspects of the Estuarine
Arne!'. Fish. Soc., Spec.
The A Quatic Environment
5 Odum, E.P. Fundamentals of Ecology.
W. B. Saunders Company,
Philadelphia and London. 1959.
6 Patten, B. C. Systems Ecology.
Bio-Science. 16hl). 1966.
7 Whittaker, R. H. New Concepts of
Kinldoms. Science 163: 150-160. 1969.
This outline was prepared by H. W. Jackson,
Chief Biolog1.t, National Training Center,
Water ProgrllUns Operations, EP A,
Cincinnati, OH 45268.
Aquatic Environment.. Estuarine Environment,
Lentlc Environment, LoUc Environment,
Currents, Marshes, Limnology, Water Properties

Part 3. The Freshwater Environment
The freshwater environment as considered
herein refers to those inland waters not
detectably diluted by ocean waters, although
the lower portions of rivers are subject to
certain tidal flow effects.
Certain atypical inland waters such as saline
or a1kaline lakes, si>rin,s, etc., are not
treated, as the main objective here in typical
inland water.
All waters have certain basic biological cycles
and types of intera(:t.ions most of which have
already been presented, hence this outline
will concentrate on aspects essentially
peculiar to fresh inland waters.
A The history of a bodi)' of water determines
its present condition. Natural waters have
evolved in the cuurse of geolotic time
into what we know today.
B Streams
In the course of their evolution, streams
in general pass thr<>uih four stages of
development which may be called: birth,
youth, maturity, iU1d old age.
These terms or conditions may be
employed or considered in two contexts:
temporal, or spatial. In terms of geologic
~, a given poInt in a stream may pass
through each of the 8tages ducribed below
or: at any given time, these various stages
of development can be loosely identified
in successive reaches of a stream traveling
from its headwaters to base level in ocean
or major lake.
BI. 21e.l. 74
1 Establishment or birth. This
micht be a "dry r~" or headwater
stream-bed, before it had eroded
down to ~e level of ground water.
During periods of run-off after a
rain or sllow-melt, such a gulley
would have a f10w of water which
might range from torrential to a
mere trickle. Erosion may proceed
rapid1y as there is no permanent
aquatic flora or fauna to stabilize
streambed materials. On the other
hand, terrestrial grass or forest
growth may retard erosion. When
the run-off has passed, however,
the "streambed" is dry.
2 Youthful streams. When the
streambed is eroded below the
ground water level, spring or
seepage water enters, and the
stream becomes permanent. An
aquatic flora and fauna develops
and water flows the year round.
Yoll1 hful stl'eams typically have a
relatively steep gradient, rocky beds,
with rapids, falls, and small pools.
3 Mature streams. Mature streams
have wide valleys, a developed
flood plain, are deeper, more
turbid, and usual17 have warmer
water, sand, mud, silt, or clay
bottom materials which shift with
increase in flow. In their more
favorable reaches, streams in this
condition ~re at a peak of biological
productivity. Gradients are moderate,
riffles or rapidk are often separated
by long pools.
4 in old age, streams have approached
,eololic base level, usually the
ocean. During flood stage they scour
their beds and deposit materials on
the flood plain which may be very
broad and f:.at. During normal flON
the channel is refilled and many
shifting barR are developed.
Pr.c.ding page ~Int

The .\Qustic Environment
(Under the influence of man this
pattern lRay be broken UP. or
temporarily interrupted. Thul an
ellenti&Uy "youthfUl" Itream might
take on lome of the characteristici
of a "mature" stream following so11
er08ion, drganic enrichment, and
increaled surface runoff. Correction
ot the.. coodltionl miFt Hklwise be
followed by at least a partial reverlion
to the "original" condLtion).
C Lakes and Reservoirs
Oeolegtcal factors which 8ian111eut]y
affect the nature of either a .tream or
lake include the following:
1 The geolraphicallocation of the
drainap ba.in or watershed.
2 The 81&e and 8hape of the drabge
3 The ,eneral topography, i. e. ,
mountainou8 or plain8.
4 The character of the beciroc:kl and
80111. '
5 The character, amount. annual
diltribution, and rate of precipitation.
6 The natural vegetative cover of the
land 18, of courle. re.poolive to and
A Fresh waters in general and under
natural condition. by definition have a
lener supply of dissolved subltancell
than marine waters, and thul a lesser
basic potential for the growth of aquatic
organisms, By the same token, they
may be said to be more sensitive to the
addition of extrane(JUs materia18
(pollutants, nutrients, etc.) The
following notee are directed toward
natural geological and other environ.
mental factorl as they affect the
productivity of fresh waters.
B Factors Affectin5 Stream Productivity
(See Table 1)
(The productivity of sand bottoms is
taken as 1)
 Bottom Material ~elati~n,
 roduc vitv
Sand  1
M~rl  6
Fine Gravel 9
Gravel and sUt 14
Co.rse gravel 32
Moss on fine gravel 89
Fissidens (moss) on coarse 111
Ranunculus (water buttercup) 194
Watercress 301
Anachari! (waterweed) 452
"'Selected from Tanwell 1937
To De productive of aquatic life, a
stream must provide adequate nutrients,
light, a suitable temperature, and time
for growth to'take place.
1 Youthful streams, especially on rock
or sand substrates are low in essential
nutrients. Temp~ratures in moun-
tainous regions are usually low, and
due to the steep gradient, time for
lI'owth 18 short. Although ample
light is available, growth of true
p1anJcton i8 thus greatly limited.
The Aquatic En~ronment
2 As the stream flows toward a more
"mature" condition, nutrients tend to
accumulate, and gradient diminishes
and so time of flow increases, tem-
perature tends to increase, and
plankton flourish.

Should a heavy load of inert silt
develop on the other hand, the
turbidity would reduce the light
penetrati0n and consequently the
general plankton production would
~ As the stream approaches base level
(old age) and the time availab'!.e for
plankton growth increases, the
balance between turbidity, nutrient
levels, and temperature and other
seasonal conditions, determines the
o'\I'erall pl'oductivity.
C Factors Affecting the Productivity of
lakes (See Table :.!)
1 The size, shape, and depth of the
lake basin. Shallow water is more
productive than deeper water since
more ligt.t will reach the bottom to
stimulate rooted plant growth. As
a corollary, lakes with more shore-
line, having mQre shallow water,
are in ger-eral more productive.
Broad shdlow lakes and reservoirs
have the greatest production potential
(and. hence should be avoided for
water supplies).

(The productivity of sand bottoms is taken as 1)
Bottom Material Relative Productivity
Sand 1
Pebbles 4
Clay 8
Flat rubble 9
Block rubble 11
Shelving rock 77
. Selected from Tar~wel1 1937

'1'be A\1uatic Env1ronmel\t
2 Hard waters are generaDIY more
productive than soft wahl's IUI there
are more plant nutrient minerals
ava11able. This is often rreatly in-
fluenced by the character of the. soil
and rocks in the watershed Ind the
quality and quantity of ,round water
entering the lake, In pMra1. pH
range. of 8.8 to 8, a appear to be
most prOd\&ctive.
3 Turbidity reduces productivity as
light penetration is rc
C According to location, lakes and
reservoirs may be classified as polar,
temperate, or tropical. Differences in
climatic and geographic conditions
result in differ'!nces in their biology.
A A body of water such as a lake, stream,
or estuary represp-nts an intricately
balanced system in a state of dynamic
equilibrium. Modification imposed at
one point in the system automatically
results in compensatory adjustments at
alsociated points.

B 'The more thorough our knowledge of the
entire system, the better we can judge
where to impale control measures to
achieve a desired rtlsult.
1 Chamberlin, Thomas C. and Salisburg,
Rollin P. Geological Processes and
'Their Results. Geology 1: pp i-xix,
and 1-654. Hem')' Hoh and Company.
New York. 1904.
2 Frey, David G. Limnology in North
America. Univ. Wise. Press. 1963.
3 Hutcheson, George E. A Treatise on
Limnology Vc.1. I Geography, Physics
and Chemistry. 1957. Vol. II.
Introduction to Lake Biology and the
Limnoplankton. 1115pp. 1967.
John Wiley Co.

4 Hynes, H. B.N. The Ecology of Running
Waters. Unlv. Toronto Press.
555 pp. 1970.
5 Ruttner, Franz. Fu~damentals of
Limnology. University of Toronto
Press. pp. 1-242. 1953.
The Aquatic Environment
8 TarzweU, Clarence M. Experimental
Evidence on the Value of Trout 1937
Stream Improvement in Michigan.
American Fisheries Society Trans.
66:117-187. 1936.
7 U. S. Dept. of Health, Education, and
Welfare. Public Health Service.
Algae ar,d Mt:!tropolitan Wastes.
Transactions of a seminar held
April 27-29, 1960 at the Robert A.
Taft Sanitary Engineering Center.
Cincinnati, OH. No. SEC TR W61-3.
8 Ward and Whi[>ple. Fresh Water
Biology. (Introduction). John
Wiley Company. 1918.
This outUne was prepared by H. W. Jackson,
Chief Biologist, National Training Center,
Water Programs Operations, EPA, Cincinnati,
OH 45268.

Aquatic Environmeut, Estuarine .o!:nvironment,
Lent1c Environment, Lotic Environment,
Currents, Marshes, Limnology, Water

Part 4.
The Marine Environment and ite Role In the Total Aqu!.tic Environment
A The marine environment il arbitrarily
defined as the water mass e1ttend1ng
beyond the continental land masses,
including the plants and animals harbored
therein. This wate~ mass is larre and
deep, covering about 70 percent of the
earth'e eudace &I1d being al deep .1
7 mUe.. The .alt content avera,el
about 35 parts per U-.ousand. Ute extende
to aU depthe.
B The leneral nature of the water cycle on
earth il well known. Because the largest
portion of the sur:~ce area of the earth
is covered with water, rouply '70 percent
of the earth's ra1nfallis on the seas.
(Firure 1)
lrnl o.8ulc
.. $1"'ACI
nit"" 1.
""I VA"'. C1CLB
Since roughly one third of the
rain which fallB on tl1e land i8 again
recycled throup the .tmosph.re
(see Firure 1 again), the total amount
of water washln, over the earth's eurface
is 11gn1f1cantly lI'eater than one third of
the total world ralnfan. It 11 UN. not
lurpri.ing to note that the rivers which
flna1q empty Into the sea carry a
dilproportionate blU'den of di8.01ved and
luspended solids picked up from the land.
The chemical compe.ition of thi8 burden
depends on the composition of the rocks
and sou. throu,h which the river !lows,
the proximity of an ocean, the direction
of prevailing winds, end other factors.
This ie the subltarc. of geolopcal erosion.
(Table 1)
BI. 21e.1, 74
(Data from Clark. .,. W.. 1924. "The Compo.mon of River
and Lake Watera of \he UDI"'d !!\ate.". U. S. Gool. Surv..
Prof. Paper No. ISD; IlarYey. If. W.. 1857. "The Chemi.try
and Fertll11y of Seta Water.". Cuabrictce Unlver.ity Pre..,
 Delaware River Rio Granda 
Ion .t .t S.. Water
 Lamb.rtvUle, N. J. Laredo, Teu. 
II. '.70 14.78 30.'
K 1.'8 .85 1.1
Ca 17.'9 13.73 1.18
Nt '.81 3.03 3.7
C1 '.11 21.66 56.2
SO. 17.'9 30.10 7.7
CO, 31.86 11.55 It-HC03 0.35
C For thie presentatioD, the marine
environment will b", (1) described using
an ecolopcal approach, (2) characterized
.colopcalq bl comparing it with fresh-
water aDd eBtuaril1e environments, and
(8) conaider.d as a functional ecological
.;ylltem (.cosyetem),
Dtlltinct differences are found in phY8ical,
chem1oa1. and biotic fsetors in ,oing from
a freshwater to an o.::eanic environment.
In pneral' envirOl1ment&1 factors are more
conetant in frelhwater (rivere) and oceanic
environments than in th.' h1gb1y variable
and harsh environments of estuarine and
coutal waters. (Figure 2}
A PhY8ical and Ch.mica1 Factors
Rivers, .stuaries, and oceans are
compared in Figure a with reference to
the relative instability (or variation) of
several important parameters. In the
oceans, it will be noted. very little change
occurs in any parameter. In rivers, while
"salinity" (usually referred to as "dissolved
solids") and temperature (accepting normal
se..onal variations) change little, the ather
four parameter. vary coneiderably. In
e.tuaries, they all change.
Pr'C8~i.1 'III blalk

.!he AqUatic Environment
  De ree of ins tabU it   Avail-
Type of environment     
and ,enera! direction Salinity Temperature Water  ab\lity Tllrbi4Uy
of water m8hCire and offehore
oceanic re,icm8 tOfetber, are often
cla88Uied with ...terM- 'to 1iIht penetra-
~ and water depth. (Pi~e' ~)

1 ~. - RelatWely ehallow-water
~ch'ext~d8 &oltt'th_.-
tide mark to the edre or the
coidlnental ehelf.

The Aq\latic Environment
o D (; t .. N / c- I ,-'
I ~
, r./~""'1 ()
I ~
~ : l ..
0.; ""-..J--_----_._-_J "..
'... I \..
I //////////././/./;/.////C?J!-~:
. -
Nl.MIII /Wo...,
ItNTHIG el.It..,
Lit'.,., CIII''''h'."
"\ :/././././ .///./././ mQ" -

"'~ :,~.

..,,,,,.,.,/~ ....

.::. ///./,.3 r_..,.
.~ """.,.,.,;,
..,.... \"'. -

c :s :.M'
.",." ",., "" #""'"''
"K'''. '''''Hlt',01'",
"""'''''''.'''' .""".,,,..
FIGI1U 3-CltJ$S;fi("liolt 0/ m/j,ilt, ,nri,lIIIlIIlItll
a Stability of physical factors is
intermediatf! between uNar1ne
and oceanic environments.
1) Physical factors fluctuate
less than 1n the neritic zone.
b Phytop1ankters are the dominant
producers but in some locations
attached a1g...e are also important
as producers.
2) Producers are the phyto-
planktC'n and consumers are
the zoop1ankton and nekton.
c The animal consumers are
zooplankton. nekton, and benthic
b Bathyal zone - From the bottom
of the euphotic zone to about
2000 meters.
1) Physical factors relatively
constmt but light Is absent.
2 Oceanic - The repon 01 the ocean
beyond the cont!nental shelf. Divided
into three parts. aU relatively
poorly popu1&tfJd compared to the
neritic zone.
2) Producers are absent and
consumers are scarce.
c A~Sa1 z~e - All the sea below
the athya zone.
a Euphotic zone - Waters 1nto which
sunlight penetrates (often to the
bottom 1n the neritic zone). The
zone ot prlmary productivity often
extends to 500 feet below the 8urface.
1) Physical factors more con-
stant than in bathyal zone.
2) Producers absent and consumers
even 1es8 abundant than in the
bathyal zone.

~. Aquatic J!:nrll'Onment
A Sea water is a remarkabJ;y suitable
environment for living roen., .. it
contllins an of the chemical elements
essential to the growth and maJntenance
of plante and animals. The ratio and
often the concentration of the major
88lt8 of s.a water are stI-lldng4' similar
in the qtopla.m and body fluids of
marine or,ani.ms. Thi. similarity i.
al8o-evident, although modified 80mewhat
in the body fiuids of fruh water and
terrestrial animals. For example,
8terile aea water may be used in
emer,encle. a8 a subatitute for blood
plasma in man.
B S1nc~ marine orpnisml have an internal
salt content similar to that or their
surrounding medium (isotonic condition)
osmoregulation poses no problem. On the
other hand, fresh water organisms are
hypertonic (olmotic pressure of body
fiuidl Is higher than that of the 8urround-
ing water). Hence, freln water animal8
must con8tantJ;y expend more energy to
keep water out (1. e., hiih olmotic
pressure fiuids contain more salta, the
action being then to dilute thil concen-
tration with more water).
1 Generally, marine invertebrates are


wtththat of the external medium. This
has special lignificance in estuarine
situations where salt concentrationa
of the water often vary noft81derabJ;y
in short periods of time.

2 Marine bony fish (teleost.) have lower
-.1t content internalJ;y than the external
environment (hypotonic). In order to
prevent dehydration, water is in,..ted
apd salts are excreted throUJh special
cells 1n the gills.
.......---.-..::-..,~. .
A Salinity. Bal1nity is the sintle mo8t
conatU1t and cOl1tro1Un, fadOr in the
marifte 8DvirDlUn_t,p~Jiy followed
by temperature. It rangeI' around
311,000 l1li. per Wer, or "3& parts per
thousand" (Bfmbo1: 35".. ) in the 1anpage
of the oceana,rapher. While variations
in the open ocean are relativeJ;y small,
sa11nity decreases rapitily aa one
approaahes share and proceeds thrOU4fh
the en.r;y and up into frellh water wlih
a sal1a&ty ot "0 fa. (se~ Fipre 2)

B SaUnity and temperature as ,1imitinl
factora in eooliopcal diatribut1on.
1 Orpnisms difter in the ..linitie.
and temperature.. in which they
prefer to live, and in the variaWlitie8
of the8e parameters which they can
tolerate. These preferenoes and
tolerances often chanae with SUCce88f,ft
life history at&t.s, and I.D tum olten
dictate where the organisms live:
their "distribution. "
2 The.e requiremesats or preferences
otten lead to extensive migrations
of variO\18 .,.c1.. tor bree~,
feeding, and l1'owtng st.,e8. OII.e
vel')' 1mportut rHU1t of tais ill that
aD e8tuaril1e 8Dvironment 18 an
ab8011lte ne08..i1:7"for over half of
all coastal commero;:ial and aport
related species of fishes and invertebrate II,
for either aU or cerWJn pgrotWfa8 of their
I1fe Idstories. (Part V. fl8ure 8)
3 The Greek word root:! "eUl'y"
(meaninl wide) and "s1:erao" (meaning
narrow) are cuatomaru, combined
witb .uch words "8 "~" for aalt,
and iltbermal" tor tentpeJ.atu.e, to
live us "euryha1tne" .. an ad,1eotive
to characterbe an orpni8m able to
tolerate a wide rlDle of salinity, for
example; or "stel\otherlU.l" mean-Ing
one which cannot liand ImIch chan,e
in temperature. "Meso- ',' is a prefix
indicatinl an intermediate capadt)'.

C Marine, estua::ine. and fresh water
organisms. (See Figure 4)
o Salinity ca.35
F'igure 4. Salinity Tolerance of Organisms
1 Offshore marina organism. are, in
general, bo~h stenohaline and
stenothermal unless, as noted above,
they have certain life history require.
ments for estuarine conditions.
2 Fresh water organisms are also
stenohaline, and (except for seasonal
adaptation) meso. or stenothermal.
(Figure 2)
3 Indigenous or native estuarine species
that normally spend their entire lives
in the estuary are relatively few in
number. (See Figure 5). They are
generally meso- or euryha1ine and
meso. or eurythermal.


10 k~nft~ 25 30 3~

a Euryhaline, freshwater
b Indigenous, estuarine, (mesohaline)
c Euryhah"e, marine
Tt.e Aquatic Environment
Some well known and interesting
examples of migratory species which
ohange their environmental preferences
with the :tife history stage include the
shrimp hr..entioned above), striped bass,
many herrings and relatives, the salmons,
and many others. None are more
dramatic than the salmon hordes which
lay their eggs in freshwater streams,
migrate far out to sea to feed and grow,
then returt. to the stream where they
hatched to lay their own eggs before
5 Among euryhaline animals landlocked
(trapped), populations living in lowered
salinities often ~ve a smaller maximum
size than individuals of the same species
living in more saline waters. For
example, the lamprey (Petromyzon
marinus) attains a length of 30 - 36"
in the sea, while in the Great Lakes
the length is 18 . 24".
Usual1y the larvae of aquatic organisms
are more sensitive to changes in
saUnity than are the adults. This
characteristic both limits and dictates
the distribution and size of populations.
D The effects of tides on organisms.
1 Tidal fluctuations probably subject
the benthic or intertidal populations
to the most extreme and rapid variations
ot environmental stress encountered
in any aquatic habitat. Highly specialized
communities have developed in this
zone, some adapted to the rocky surf
zones of the op'!n coast, others to the
muddy inlets ~f protected estuaries.
Tidal reaches of fresh water rivers,
sandy beaches, coral reefs and
mangrove swamps i,n the tropics; all
have their own floras and faunas. All
must emerge and flourish when whatever
water there is rises and covers or
tears at them, all must collapse or
retract to endure drying, blazing
tropical sun, or freezing arctic ice
during the low dde interval. Such a
community is depicted in Figure 6.
25 - 27

The Aquat1c Env1I'Oftment
LUtorina nerl.toide.
L. rudill
I.. 011 tU.3.ta
L. littorea

. .~.....
.~",~,~".~o. ~


~~I~;;:'q;ili~~.l~f~[:~,t~ ...
~~1~)\- ,~~, ~'~'~~:/":'~'~~\~~I~:~
'SJ~d';,.,i+.rt>'ii.~..}'1'f)...~(t r"",- ..
.. ..~"b~~:J"'~~'~'',iI~!k~\)i)a
" ... ""'" .~..~~....:~,..." .
"'0. 0 G. ~ 1\(J~~ ~,:I ';"'~ ,:~
t! t> e ... (I  ,~,':: e(l:'''?~,,,. a~, 8
. - O"'~~..~~''t:"t!\.:: ,'....:,-e, "","'r".'!~.'"",. "::'..
,,.. ., ~d !t\.)O;.~,\I"I''IJ'-'''' "'f'-":,:v,. '~:''''
~ 8 "1M~t;Z~ l~;,;"'~Vf~:::":,, ':'(~i.'( "~:';..,.,::~;J:'r ..
~&ro~ ~ ;:~~~~{f1~~ :
~0~~#i~ ' ~q;J! ~,.~~,~~~~O~~/:~~d.
. ¥JftJI/1(l'ir;~\ I . : ,- ~\. ~~\,~~~~. It.:' J
fffiJjI4.~l1i!»~Jj}4),J!JIV)~:v.)~.;~. \*4'

Fiaure I

Zonation of planta, anaUe, and bamac~. on a ..~ .hon. While
thl.. diairam i. ba..d on the situation on the .outkw..t coast .f
England. the leneral idea of zonation may be applied to.ern)' temper-
al~ rocky ocean .~re. though the specie. wUl cUller. Tha ,ra)'
z~>ne con.1llota 1&1"61)' of lichen.. At the l.ft i. the zonation of rocu
with expo.ure too .treme to lupport al..; at the n.ht, .on a lwo
expo.ed .I.tuation. the animal. are mo.tq ob.CV8d b7 Ute alpa.
Fiaurn at the right hand marlin refer to the perce. of time tIW.t
lhe zone is ellpO'.eI, to the air. i. e.. the time that the tide I.. ftt.
Three major' IOI18a \188 be recolnized: the Uttorina zone (above the
il'a" zone); the BaJanoid zone (between the Iray zone and the
laminarias); and the Laminarl.a zone. a. pe~ve~ canaUc*J,a,-;
b. Ii'ucue Itpir&~1.; c. ~ nodo.urn; . ~ .er...'".;
e. Laminaria !!!J.U!!t. ~.ph.n.on) .
e Chthamalull .tellatu.
. Balanus balanoides
/fj A. pel'for.to

A The sea i8 in continuous circulation. With-
o'.1t circulation, nutrients of the ocean would
eventually become 1\ part of the bottom and
biological production would cease. Generally,
in all ocean8 there ex1.t. a warm sudace
layer which overJ.1es the colder water and
forms a two-layer syltem of per .istent
stability. Nutrient concentration i8 usually
greatest in the lower zone. Wherever a
mixing or disturbance of theBe two layers
. occurs biological production is greatest.
B The estuaries are &4S0 a mixing zone of
enormous importi.nce. Here the fertility
washed off the land i8 mingled with the
nutrient capacity of lIeawater, and many
of the would's most productive waters
C When man adds his cultural contributions
of sewage, fertilizer, sUt or toxic waste,
it is no wonder that the dynamic equilibrium
of the ages is rude~ upset, and the
environmentalist cries, "See what man
hath wrought" I
This outline contains selected material
from other outlines prepared by C. M.
TarzweU, Charles L. Brown, Jr.,
C. G. Gunnerson, W. Lee Trent, W. B.
Cooke, B. H. Ketchuro, J. K. McNulty,
J. L. Taylor, R. M. Sinclair, and others.
The Aquatic Environment
1 Harvey, H. W. The Chemistry and
Fertility of Sea Water (2nd Ed.).
Cambridge Univ. Press, New York.
234 pp. 1957.
2 Hedgpeth, J. W. (Ed.). Treatise on
Marine Ecology and Paleoecology.
Vol. I. Ecology Mero. 67 Geo1.
Soc. Aroer., New York. 1296 pp.
3 Hm, M. N. (Ed.). The Sea. Vol. n.
The Composition of Sea Water
Comparative and Descriptive
Oceanography. Intersc1ence PubIs.
John Wiley & Sons, New York.
554 pp. 1963.
4 Moore, H. B. Marine Ecology. John
Wiley & Sons, Inc., New York.
493 pp. 1958.
5 Reid, G. K. Ecology of Inland Waters
and Estuaries. Reinhold Publ.
Corp. New Yorl<. 375 pp. 1961.
6 Sverdrup, Johnson, and Fleming.
The Oceans. Prentice-HaU, Inc.,
New Yo.-k. 1087 pp. 1942.
This outline was prepared by H. W. Jackson,
Chief Biologist, National Training Center,
Water Programs Op.~rations, EP A, Cincinnati,
OH 45266.
Aquatic :Environment, Estuarine Environment,
Lenttc Environment, Lotic Environment,
Currents, Marsh.., Lironology, Water Properties

Part 5:
A Broadly defined, wetands are areas
which are "to W.1!t to plough but too
thick to flow." Th\! sol1 tends to be
saturated with water, salt or fresh,
and nl1merous channels or ponds 01
shallow or open water are common.
Due to ecological features too numerous
and variable to Ust here, they comprise
in general a rignrous (highly stressed)
habitat, occupied by a small relatively
.peciaUzed indigenous (native) flora
and fauna.
B They are prodigiously productive
however, and me.ny constitute an
absolutely esseni1al habitat for some
portion of the Ufe history of animal
forms general1y recognized as residents
of other habitats (Figure 8). This is
particularly true of tidal marshes as
mentioned below.
C Wetlands in toto comprise a remarkably
large proportion ()f the earth's surface,
and the total organic carbon bound in
their mass con.titutes an enormous
sink of energy.
D Since our main concern here is with
the "aquatic" envirt'nment, .primary
emphasis will be directed toward a
description of wetlands as the transitional
zone between the waters and the land, and
how their desecration by human culture
spreads degradation in both directions.
A "There is no other case in nature, save
in the coral reefs, where the adjustment
of organic relations to physical condition
is seen in such a beautiful way as the
balance between the growing marshes
and the tidal streams by which they are
at once nourished and worn away. "
(Shaler, 1886)

B Estuarine pollution studies are usually
devoted to the dynamics of the circulating
water, its chemical, physical, and
biological parameters, bottom deposits, etc.
C It is easy to overlook the intimate relation-
ships which enst between the bordering
marshland, th~ moving waters, the tidal
flats, subtidill deposition, and seston
whether of local, oceanic, or riverine
D The tidal marsh (some inland areas also
have ..It marshes) is generally considered
to be the marginal areas of estuaries and
coasts in the ir.tertidal zone, which are
dominated by emergent vegetation. They
general1y extend inland to the farthest
point reached by the spring tides, where
they merge into freshwater swamps and
marshes (Figure 1). They may range in
width from nonexistent on rocky coasts to
many kilometers.

.- -- '-=-ri-H
,~u-I---_m'~r~'~~ ~-~ (.~tl.tt
,..,. -
z. - - - -- - - ... - - - _.- - . - ~1..tJ\ I': - -....-- ~
......" ~. ......::' :;:: :- /: -
~r .::!I":::- P.., -.- - ~-O =-
..'. ....'" -- ~ ~ - - /' - -
, ---- - "'-
... Cia, -c5- Subltrat. -
- --~ -- -;;- 0::- -Q - "J
~ 10M'" 1ft. po8t""." !:naanll 811""'1')'. 1. SprLn, ttd. lev.l, 1. Mean hllIh 1I.dt.
......,... "* t ..,1IiNI . 1M d...... .-i.'o Ch1lnk Or ~ '.rf depwl.t." ~ ice.
~: :;::u-:-... ~w."""":' ~..tt,. .. ..Ip" (z.ot;:.~d _..1. ImodIoh8.-
_-'1---''''''-' '0. ...-.. ..
Preceding page blank

Th. Aquatic Environment
A In general, marsh sub.trnt.. are h18h in
organic content, relativeq 1Dw in minerals
and trace elements. The! upper layers
bound together with livtne roots called
turf, underlaid by more compacted peat
type mater1&l.
Such banks are l1keJy to be cliff"like,
and are otten undercut. Chunk.. of .
peat are otten found qUl.i about on
harder substrate belclw,hiah; tide, Une.
If face of cUff 18 well above higb'water,
overlyin, veptMima i..J1kttJ:r to be
typical1y terrestrf.&l. of the area.'.
Mar8h type vegetation I. probaDJr>\9
1 Rising or erodin, coa.tline. may
expo8e peat trom ancient marsh
growth to wave actior, which cuts
inte the 80ft peat rapidly (F~1'e 2).
T.r...trgl~. ~~~~~.W:il~
}.-- ... ---
.M ....-... follp. ;.<.:- ,..,,-;:;: ~ ~'-.

-:}. ~ ~_:::.':i'~?"~(;;~:~.:/~

c 1.... ", .' '.~.' .. -. oil ':
';'~:~";~.7':~~'..~',.::"J '" ~... ',' .", , .", ".
2 Low lying de1taJ.e. or Bin~ cout.
lines, or thoBe with low .QlrcyccW80Wl. ,
action are Wceq to have aattw-.rsh
formation in progre... Saftd' daIas .
are a180 common in BUch ar...
(FlI\Ire 3). General coa-.al.
conftpattoa. I. a fa~OI'.
.,..... DIa.,.,.....uc: 8(>('U"''' ......"Nt c:aut
.. '
J's,ur. 3

Dewlapat....t of & Ma...chu..tt. Marsh .lnce 1808 ac:, 1IMtIYtIII&a
18 foot rille In -tel' !.en1. Shaded &..- 1ndI.... .... cIa118.. N*
m8lll1d8..1q mal'8h Udal d..alDa... Ii.: 1300 Be, B: 11150 AD.

a RUlied or precipitous coasts or
.lowly ri8in, coast., typically
exhibit I\!irrC'W shelvea, sea clUfs,
fjar'ds, masslve beaches, and
relatively less marsh area (Figure 4).
An Alaskan fjord subject to recent
catastrophic I'ubsidence and rapid
deposition of ,lacialflour shows
evIdence of the recent encroachment
of saline waters in the presence of
recently buried trees and other
terrestrial ve,etation, exposure
01 layers of s.It marsh peat along
the edges of channels, and a poorly
compacted youn, marsh turf dsveloping
at the new high water level (1I'll\11"e 5).
ShINnI nat.
The Aquatic EJ1vironment
...,...... .. -.-,

n....... A IIIwP - ..... SIowJr lIi.ID, Co..l, Nol. ah.....
., -.. _~I ...... ..Jati""l1l1tU. mar ohland.
~ mWl1 Dati .Uppled ar. e.ten.tve.
I'1l1Un 5
lIome 1811....1 re1aU_hip. ID a nOMhern fjord with a M.lnl-t.r 1ev.1. 1. m..., low
water. I. maxlmwn hip tld., 3. Sedrocll... 01a"..1 tkNr to depth. In ",",cea. of
400 meten, I. Shlftlaa nat8 and channel., 8. 0IaDn.1 &pIn.t bedrock. 7. Burled
te~'r..tr..l ve,8IIr.tlon, .. OIatOropplnl' of _It marillt peal.
b Low lying cOB.Btal plains tend to be
fringed by barrier islands, broad
estuaries and deltas, and broad
associated marshlands (Fii\ll'e 3).
Deep tidal channels fan out through
innumerable branching and often
interconnecting rivulets. The
intervening grassy plains are
el!ll!lentiaUy at mean high tide level.

'J.'he Aquatic Environment
c Tropical and subtropical rel10ns
such as Florida, the Gulf cOast,
and Central America, are frequented
by man.rove swarllps. This unique
type of ,rowth is able to ..tablish
itself in shallow water and move out
into pro,ressive:b' deeper area.
(Figure 8), The stron, deeply
embedded root. enable the man,rove
to resiat considerable wave action
at time., and the tanlle of roots
quickly accumulatee a deep layer of
organic sediment, Mangroves
in the south may be considered to
be rouahly the equivr.1ent of the
Spartina marsh ,rass in the north
as a land builder. When ful:b'
developed, a mana-rove swamp 18 an
impenetrable thicket of root. over
the tidal flat affording shelter to an
assortment of .emi-aquatic organisms
such as various molluscs and
crustaceans, and providing access
from the nearby land to predaceous.
birds, reptiles, and mammals.
Mangroves are not r~stricted to
estuaries, but may develop out into
shallow oceanic lagoons, or upstream
into relative:b' fresh waters.
=~ ~ ...,=:...........
, 11'.
n,..... 6
Dtall'ammatic t..n..et 01 a 1IIAII,1'O'f8 -mp
.howtll. tran.1tion from martM to t..,...tri81
A Measuring the productivity of grasslands
is not easy, because today grass 18 seldom
used direct:b' as such bl man. It is thus
usually expressed as production of meat,
milk, or in the case of salt marshes, the
total crop Qf animals that obtain food p1!r
unit of area. The primary producer in a
tidal marsh is the marsh Jt&88, but very
little of it i.u8ed by mu .. 11'.'"
(Table 1)

The nutritional ana]ysis ct..veral
mar.h ,rallies as cotril*N~'to dry land
hay 18 shown in Table 2.
'TABU t. -..a Or-. of"""""" -......, -I)' ta-
.. - w.... '" a..... .tto. ..... A......-
..,.. .',.."
,......1_- ~
.--. ....-
-. -.........
-. -.............
s--. ..... - .......
-......... (......
TABLE a. Ana1f... of Som, Ttddllareh.aras-
TlA ,......,... ~iII'"
Dry w,. "_i" 'II fiber w-
B The actual utilizati.m of marsh grass is
accomplished prin,arU~ by its decom-
position and in,..Uon by micro organisms.
(Figure 7) A .man quantity of seeds and
solids is consumed directly by birds.
-~. .
1 -- _u- ~.
Fll'lre 7 Th. nutr1t1ve eompo.ltlon of
.uac...lv. .ta,.. of It.compo.ltlon of
~ mar.h ,ra.., .how1nl1ner.....
InPrOte1n and decr8&l. in c:arbol!Tdrat.
with Inereuln, .'" IUId deer.ulnf .Ize
ot d8trltu. parUel...
I The quantity of micro invertebrates
which thrive on this wealth of decaying
marsh has not been estimated, nor has
the actual production of smaU indigenous
fishes and invertebrates such as the
top minnows (Fundulus), or the mud
snails (Nassa), and others.
2 Many forms of oceanic life migrate
into the estuaries, espec1aUy the
mar.h areas, fC'r' important portions
of their life histories as is mentioned
elsewhere (Figure 8). It has been
estimated that in excess ot 600/. of the
marine commercial and sport fisheries
are estuarine or marsh dependent in
some way.
The Aquatic Environ~
FIcUt'e 8 DiaQam 01 th.. We cyele
ot whit. .hrlmp (att.r Ander.on and
Lufta 1"5),
3 An effort to make an indirect
estimate of productivity in a Rhode
Island marsh was made on a single
August day by recording the numbers
and kinds of birds that fed on a
relatively small area (Figure 9).
Between 700 and 1000 wild birds of
12 species, ranging from 100 least
sandpipers to ur.countable numbers
of seagulls were counted. One food
requirement estimate for three-
poun:! poultry in the confined inactivity
of a poultry )-ard i8 approximately one
ounce per pound of bird per day.
n".I" 9
Some Comrncn Mars" Birds

The Aquatic Environment
Gne-hundred black bellied pJovers
at approximately ten QWlce. each
would wei&h on the order 01 8ixty
pound.. At the same rate 011000
conwmption, this would tndicate
nearly lour pounds of f('od required
lor this .pecies alone. The much
,reater activity of the wUd "birds
would obvtously are.tly incre..e their
food requirements, a. would their
t'elatively smaller size.
Considering the raqe of food. con-
.umed, the sizes 01 the bird., and the
lact that at certain sealane, thousands
of miarating duck. and other. J».use
to feed here, the enormou. productivity
of such a marsh can be b"tter under-
A Much of what has been ;said of tidal
marshes a180 applies to inland wetlande.
As was mentioned earlier, not an inland
swamps are 8<-free, any more than all
marllhes aflected by t1dall')'thms are
B The specificity of specialized tlor.. to
particular types of wetlands Is perhaps
more spectacular in freshwater wetlands
than 'in the marine, where Juncus,
Spar-tina, and Mangroves tend to dominate.
1 ~ or peat r.108l, i.
proDa6Jy One of the mo"t wideepead
and abundant wetland pant. on lalib.
Deevey (1958) quote. an estbnaCe --
there Is probably upwu'd. 01.183
billion. (dry wellht) of tons 01 psat
in the world today, cMrived during
recent geologic time from ~num
bogs. Particularly in thu northern
r$glons, peat m088 tends to overgrow
pends and shallow depre..ion., eventually
farming the vast tundra plain. and
moore. of the north.
2 Long list. or other bog and marsh plant.
might be cited, each W1t" it. own
.~eclal requirement., topographical,
aM pographie distribution, I!Itc.
InclUded would be the lamW.ar oattan.,
lpike r1t8he., cotton gra...., Md,.I,
tr.,ot-18. aJder.,. and mlU1\Y, mMIT
e Types of ift}and wet~ds.
1 A. noted above (eI: Fipre 1)
tidal marshes otten :nerfe into
fresh",ater marlbe. &ItCi" t.qOU8.
Deltaic tidal swamps and marshes
are often sa1tAe tA.. ""ward
portion. and Iresh in the.landward
2 River bottom wetland8 differ from
those formed from lake., "since 'wide
flood plaJ.n8 subject to periodic
inundation are the f1na1 at.... 01
the .r08'1on of river V&1le)'s. . whereas
lake. in leneral tend to"be e1im1llated
by the pololic pro.::n... ot natOral
eutrophication otten tnw1*l
~ and peat formation.
~m marshes in the 8OU'thfl"n
United Stat.., with 14i,VOr.ble cs1Sma..,
have luxurhmt" fl'owUuI 8dI' as "the
canebrake of.the rawer MtUfa8tppl,
or a cb&ractert8Uc .~r ~
IUch .. cypr....
3 Althoqlt bird life ie the moat
conap!cuOQ animal element" ~ the
tauna (el: npr.8-), many mammal8,
such .. mu.lln.t.. beavers, otters.
and other. are al.o marah-ortented.
(Figure 12)
'1Iur. 12

A No .ingle statemel"t can .ummarize the
effect. of p,:>llut1on an marshlands as
di.ttnct from elfects noted elsewhere on
~her habitats.
B Reduction of Primary Productivity
The primary producers in most wetlands
are the grasses and peat mosses.
Production may be reduced or eliminated
1 Changes in the water level brought
about by f1o~ing or drainage.
a Marshland areas are sometimes
dLked and flooded to produce fresh-
water ponel.. This may be for
aesthetic rea~onB, to suppress the
growth of noxious marsh inhabitating
insects such as mosquitoes or biting
midges, to coostruct an industrial
waste holdiniC pond, a thermal or a
sewage stabilization pond, a
"convenier.t" result of highway
causeway construction. or other
reason. The result is the elim-
ination of an area of marsh. A
small compensating border of
marsh mayor may not develop.
b High tidal marshes were often
ditched and drained in former days
to stabilize the sad for .alt hay or
"thatch" harvesting which was highly
sought after In colonial days. This
inevitably changed the character
of the marRn, but it remained as
essentially marshland. Conversion
to outright agricultural land ha.
been less widespread because of the
necessity of diking to exclude the
periodic floods or t1dalincursions,
and carefully timed drainage to
eliminate excess precipitation.
Mechanical,pumping of tidal marshes
has not been economical in this
country, although the success of
the Dutch and others in this regard
is well known.
The ,J\q~tic ~v1ronment
2 Marsh grasves may also be eliminated
by smothering as, for example, by
deposition of dredge spoils, or the
.pWor dLscharge of sewage sludge.
3 Considerable marsh !Iorea has been
eliminated by indu.trial construction
activity such as wharf and dock con-
struction, oil well construction and
operation, ar.d the discharge of toxic
brines and other chemicals.
C Consumer prodUction (animal life) has
been drastical]"v reduced by the deliberate
distribution of pesticides. In some cases,
thil has been air.1ed at nearby agricultural
lands for economic crop pest control, in
other cases the marshes have been sprayed
or dusted directly to control noxious
1 The results have t>een universally
disastrous for the marshes, and the
benefits to the hl\man community often
2 Pesticides designed to kill nuisance
insects, are a1so toxic to other
arthropods so that in addition to the
target species, slich forage staples as
the various scuds (amphipods), fiddler
crabs, and (other macroinvertebrates
have either been drastically reduced
or entirely eliminated in many places.
For example, one familiar with fiddler
crabs can traverse miles of marsh
margins, still riddled with their burrows,
witho'lit seeing a single live crab.
3 DDT and related compounds have been
"eaten up the food chain" (biological
magnUication effect) until fish eating
and other predatory birds such as herons
and egrets (Figure 9), have been virtually
eliminated from vast areas, and the
accumulation of DDT in man himself
is only too well known.

.1b!..Aq~1c Environment
D Most eedaue of the marlh enemiee 1e
man himlelf. In hie quer. lor "l.heneraum"
n~ar the water, he haa aU but ld11ed the
water he IItrive8 to approach. Thus lip to
twenty percent 01 the n'i8.rab--e8tuartne
area in vartaul p~rtl ot the co_II')' haa
already been utterly de8tro)'1!d by cut and
fill !"eal e.ate development. (Fil11ree
10, 11).
E Swimmtn, bird8 .uch ae ducka, 10...
cormol'ant., pe11caa... aD4 muy othen
are s.evere1;y ~dl.ze. by Ooatint
po~ .uch.. 011.
F1IUre 10. Dia,rammat1c reprelentatiOft of cut-am1-fW for
real estate development. mlw. mean - .at..
OC ~If ~
FilUre 11. Tracing of portion of map of a eouthern
c1ty Ihowin, extent of cut-and-fill real
eltate development.

A Wetlands comprise the marshes. swamps,
bogs, and tundra areas of the world.
They are essential to the well-being of
our surface waters and ground waters.
They are essential ~o aquatic life of
all types living ~ the open waters. They
are essential as habitat for all forms of
B The tidal marsh Is the area of emergent
vegetation bordering the ocean or an
C Marshes are highly producUve areas,
euenUal to the maintenance of a well
rounded community of aquatic Ufe.
D Wetlands may be dPBtroyed by:
i Degradation of the Ufe forms of
which it is comp"sed in the name of
nuisance control.
2 Physical destruction by cut-and-fih
to create more land area.
1 Anderson, W. W. The Shrimp and the
Shrimp Fishery of the Southern
United States. USDI. FWS, BCF.
Fishery Leaflet 589. 1986.
2 Deevey, E. S.. ,Jr. Bogs. Sci. Am. Vol.
199(4): 115-122. October 1958.
3 Emery, K. O. and Stevenson. Estuaries
and Lagoons. Part D, Biological
Aspects by J. W. Hedgepeth, pp. 893-
728. in: Treatise on Marine Ecology
and PaleoeculolY. Geol. Soc. Am.
Mem. 87. Washington, DC. 1957.
.. Hesse, R., W. C. Allee, and K. P.
Schmidt. Ecological Animal
Geography. John Wiley & Sons. 1937.
The Aquatic Environment
5 Morgan. J, P. Ephemeral Estuaries of
the Deltaic Environment in: Estuaries,
pp. 115-120. Put>l. No. 83, Am.
Ass':lc. Adv. ScL Washington, DC. 1967.
6 Odum, E. P. and Dela Crug, A. A.
Particulate Organic Detritus in a
Georgia Salt Marsh - Estuarine
Ecosystem. in: Estuaries, pp. 383-
388, Publ. NO: 83, Am. Assoc. Adv.
Sd. Washington, DC. 1967.
7 Redfield, A. C. The Ontogeny of a Salt
Marsh Estuary. in: Estuaries, pp.
108-114. Publ. No. 83, Am. Assoc.
Adv, Se1. Washi'lgton, DC. 1967.
8 Stuckey, O. H. Measuring the Productivity
of Salt Marshes. Maritimes (Grad
School of Oenn., U.R. I.) Vol. 14(1):
9-11. February 1970.
9 Williams, R. B. Compartmental
Analysis of Production and Decay
of Juncus reomerianus. Prog.
Report, Radiobiol. Lab.. Beaufort, NC,
Fis'::al Year 1968, USDI, BCF, pp. 10-
This outline was prepared by H. W. Jackson,
Chief Biologist, National Training Center.
Water Programs Operations, EPA. Cincinnati,
OH 45288.
Aquatic Environment. Estuarine
LenUc Environment Lotic
Currents, Marshes, Limnology

A There are few major groups of organisms
that are either exclusively terrestrial or
aquatic. The follow!ng remarks will
therefore apply ir. large measure to both,
but primary attention will be directed to
aquatic types.
B One of the first ques"iions usual~ posed
about an organism is: "What is it, "
usually meaning ''What is it's name?"
The naming or class!fication of biological
organisms Is a science in itself (taxonomy).
Some of the principles involved need to be
understood by anyone working with
1 Names are the "key number, II "code
designation, " or "file references"
which we must have to find information
about an unknown organism.
2 Why are they so long and why must they
be in Latin and Greek? File references
in large systems have to be long in order
to designate the many divisions and
subdivisions. There are over a million
and a half items (or species) included
in the system of hlologlcal nomenclature
(very few libraries have'a million
3 The system of biological nomenclature
is regulated by international congresses.
a It is based on a system of groups and
super groups, of which the foundation
(which actually exists in nature) is
the species. Everything else has
been devised by man and Is subject
to change and revision as man's
knowledge and 'mderstanding increase.
b The categories employed are as follows:
Similar species are grouped into
genera (genus)
BI. AQ. 3f. 1. 74
Similar genera are grouped into
Similar r..:-nilies are grouped into

Similar orders are grouped into
Similar classes are grouped into
phyla (phylum)
Similar phyla are grouped into
4 The scientific name of an organism is
its genus name plus its species name.
This is analogous to our syStem of
surnames (family names) and given
names (Christian names).
a The generic (genus) name is always
capitalized and the species name
written with a smaU letter. They
should also be underlined or printed
in italics when used in a technical
sense, For exa'Ilple:
Homo sapiflns - modern man
Homo neanderthalis - neanderthal

Esox niger - chain pickerel
Esox lucius - northern pike
Esox masquinonltY - muskellunge
b Common names do not exist for
most of the smaller and less familiar
organisms. For" example, if we wish
to refer to members of the genus
Anabaena (~n alga), we must simply
use the generic name, and:

Anabaena planctonica,
Anabaena constricta, and
Anabaena fios-aquae

The Identification of Aquatic Organisms
are three distinct species which
have different significances to
water treatment plant operations.
5 A complete list of th~ various cate-
gories to which an orgllnism belonas
is known as its "cla8sifi;:at1on." This
may be written as follows for Phacus
pyrum a green flagellate, for example:
Kingdom Plantae
Phylum Thallophyta
Class Algae
Order Volvocales
Family Phacotaceae
Genus ~
Species pyrum
a It should be emphasized that since
all cateaories of above species are
essentially human concepts, there
is often divergence of opinion in
regard to how cerh&in organisms
should be grouped. Changes result.
b The most appropriate or correct
name for a given spedes ie also
sometimes disputed, and 110 species
names too are ohanged. The species
itself, as an entity in nature, how-
ever, is relatively tin.eless and 80
does not change to man's eye.
The following key is intended to provide an
introductory acquaintance with some of the
more common aquatic oraanisms. While
moet of the groups mentioned are animals,
a few oth.er groups often confused with them
are also included. Technical nomenclature
is kept to a minimum.
In the use of the key, each couplet should
be considered by itself, witho"t reference to
any other couplet. Select the alternative
that set!ms most reasonable and either proceed
to the couplet indicated, 0:' else accept the
name of the aroup as given if the selection
is a terminal statement. "Cf: --" means to
compare with the statement indicated. "POL"
means that members or the ,roup bAve been
reported to be tolerant or organic pollution.
la The bod,y of the oraanisms comprising a
sinpe microscopic independent cell, or
many similar and independwltly fllol1ctioning
cells BSIJociated in a colony. with little
or no difference between the cells
(i. e.. without forming ~issue8); or com-
prising mailses of multinLlCleate proto-
plasm; moa~1Y microscopic. Single celled
animals. . . . . . . . . . . . . . Phylum Protozoa
lb The body of the organism comprising
many cells of different kir.d8 (1. e. form -
ing ti8sue) may be microscopic or

macroscopic. . . . . . .. . . . . . . . . . . ... .. 2
2a Body of colony usually forming irregular
masses or layers sometimes cylindrical,
goblet-snaped, vase-shaped, e1onp.te.
serrate, or tree-like. Rante from
barely visible to large. . . . . . . . . . . . .. :3
2b Body or colony shows some type of
definite lIymmetry. . . . . . . . . . . . . . . . . 15
3a Colony rorntina irreaular masses, clumps,
or a,greptions of. material ,",wing
..round, over, or cllniirili to surface of
rocks. sticks. pipes, etc........... "
3b Irregular. or not obviously s~"Dlmetrical.
but ellsentially erect or plant-liJJe ind1vili-
uals or coloni.es............,..... 9
4a Freahwater....................... 8
4b Marine...........................
5a Surface of colony usually sJ1ghtly rough
or bristly in appearance \lnder the micro-
scope or band lens. . . . . . . . . . . . . ... .. 7
5b Surface smooth. . . . . . . . . . . . . . . . . . " &
5c Colony or oraaniam erect, plant-like,
branched or unbranched. ... . . . . . . . . . 12.
6a Mass very thin, u.ually piak or \¥bUe
(Coral al,ae). . . . . . . . .. . . . . . . . . . .. l3b

6b Ma.. gelatinous, either as relatively
thin sheet or clumps of tongue-like club
BMped projections. Transparent or
colored. Star shaped groups of individ-
ual organisms often evident. Colonial
tunicates or ascid~ans. Phylum Chordata,
Clus Tunicata (Cf: 12b)
7a Surface webbed or bristly in appearance,
stick-like crystals or "spicules" often
protruding from the general Burface.
Pores numerous, one or more usually
large and conspicuous. Sponges.
. . . . . . . . . . . . . . . . . . . .. Phylum Porifera
7b Colonies very thin, surface slighlly
roughened. Composed of minute individ-
ual cells or compr.rtments arranged in
various lacy or coral-like patterns. Each
compartment inhabited by a minute bilater-
ally symm('( rtcal animal which protrudes
a crown of delicate tentacles. Moss
animals, bryozoani!l.... Phylum Bryozoa
8a Surface of colony rough or bristly in
appearanc'e under the microscope or hand
lens. Grey, green, or brown. Sponges.
(Figure 1). . . . . . . . . . .. Phylum Porifera
Ob Surface colony relatively smooth, general
texture of mass gelatinous, transparent.
Clumps of minule individual organisms
variously distributed. Moss animals,
bryozoans. (Figure 2). . Phylum Bryozoa
9a Freshwater................ ......
Vb Marine.....
................... .
lOa Surface of structures rough or bristly
under the microscop'3.. . . . . . . . . . . . . .
lOb Individuals or branches tubular. Usually
covered with brownish cuticle to which
much sediment may adhere. Moss
animals, bryozoans.... Phylum Bryozoa
(Cf:8b) (Figure 2i
11a Surface of structure appears web-like or
br-istly under the mkroscope. . . . . . . .. 7a
llb Surface of structure an unbroken skin or
cuticle, except for a limited number of
functional apertures. . . . . . . . . . . . .. . 13
The Identification of Aquatic Organisms
12a Colonies minute, plant-like or coral-like.
(Figure 2)....... ....... ........13
12b Solitary, sac-like organism, either stalked
or growing directly on substrate. TranB-
parent, covered with mud or sand, or
variously colored. Bilateral symmetry
revealed on close investigation. Becomes
flaccid when exposed and relaxed at low
tide. Up to sevE-ral inches in length.
Seasquirts or tunicates. . Phylum Chordata,
Class Tunicata
13a Compartments o. apertures for separate
individual animals evident. . . . . . . . . 14
13b No evidence of l1.e presence of individual
animals. Structure profusely jointed.
Individual segments hard, smooth, usually
pil'\k or white. May a150 grow as hard
smooth thin sheet over surface of rocks.
Calcareous algae or coral algae. A
common Genus: . . . . . . . . . . . .. Corallina
14a Minute organisms protruding from holes
in colony are radially symmetrical. .. 17b
14b Organisms protruding are bilaterally
symmetrical. ('ihis may be assumed if
they are not obviously radial) Moss
animals, bryozml.l1s. (Figure 2) (Cf:7b)
. . . . . . . . . . . . . . . . . . . . .. Phylum Bryozoa
15a Radially symmetrical (body arranged around

an axis). . . . . . . . . . . . . . . . . . . . . . . . . .. 16
15b Fundamentally bilaterally symmetrical.
15c May be superficially spiral as Figure 13 35
16a Plan involves multiples of two, or else
rather large numbers of tentacles around
the margin of an umbrella; saucer-like,
or flower-like structure.... ... ....17
16b Body plan based on multiples of five.
Outer surface hard or rough to the touch.
Starfishes, brittle stars, sea urchins,
sand dollars, sea cucumbers.
. . . . . . . . .. .... Phylum Echinodermata
17a Jelly-like body, massive or ovoid, free
swimming nearly transparent in nature.
Eight rows comb-like paddle plates:
Comb Jellies. Phylum Ctenophora

The Identification of Aquatic Organisms
17b Body tube-like or umbrella -like, tentacles
around mouth, margin of disc, or both.
Jellyfishes, corals. hydroids. sea
anemones, etc... P~Lum S:0elenterata

18a Micro.cople. Action of two cUlated lobes
at anterior end in lite often gives appear-
ance of wheels. Body otten segmented.
accordion-like. Free 8wimminl or
attached. Rohfers or wheel animalcules
(Figure 3). Phylum Trochelmlnthes (Rottlera)
18b Often larger, worm -like or e18e havlRi
strong skeleton or s8le11. . . . . . . . . . . . . 19
19a Skeleton (or shell) present may be internal

or external. . . . . . . . . . . . , . . . . . . . . . .. 30
19b Body soft and/or worm-like. 8kin may
range from 80ft to parchment -like. ., 20
3) a Three or more pair. of well-formed
(though possibly small) jointed leg.
present. (Phylum Arthr?poda). . . . .. 36
20b Legs or appendages. if present, limited
to pairs of bumps or hooklf. Lobes or
tentacles 1f present loft .'td fie shy , not

jointed. . . . . . . . . . . . . . . . . . . . . . . . . .. 21
21a Batty round, oval or fiat in cross lIection

"""""""""""" -........ 22
21b Inverted U-shaped cr088 s"chon, with
flat slimy undersurface or foot tapering
off behind. Head with two pairs of
sensitive retractUe tentacles. Marine
or terrestrial. Marine forms have fancy
gill structures on back. Slugll.
Class Gastropoda (Cf: 35a)

(Phylum Mollusca).... ......... ... .35
22a 8t1"onlly depressed or fil.'.Uened. . . . ., 23
22b Oval or round in cross section. or, if
fiat, with sinile sucker disc at each end.

. .. ., .. . . .. .. . .. . . . . . . .. . . . .. . . . .. 25
23a Plirasillc in bodies of higher animals.
ExtremelylOng and fiat. divided into
sedions (like a Roman girdle). Life
history may involve intermediate host.
Tape worms. (Figure Ii) Clan ~
23b par.-Uic on bodies of higher animals,
very muscular. Itrong exte.-na1.uClker
discs (J'lCure 9). . . . . . . . . . . . , . . . . . . . 29b
23c Body a 8i.e unit. MoIIth "'.""".tive
system pre8ent {byt no anu.t., . . . . . .. 24
24a External or internal parasite on higher
animals. Sucking discs pr-..m.for attach-
ment. Life history m"y irIVcIIve two or
more iRtermeciiate hOllts or 8'.8.
Flukes..... .... .......C1a.. ~matoda

24b Free-!ivil\l- Entire body surt... covered
with locomotor cilia. Eyvd:arees in head
often appear "cro8sed." ptJt;. '-"eMiv1ng
fiatworlfts (Figure 6). Clul Tufllellaria
25a Lone, slender, with snake-1ikeatotion in
11fe. Covered with glistent,. eUMicle.
Par.sitic or free-living. MicrOllC'opic to
six feet in leAlth. ROl1lld worma
(Filu", T). . .. Phylum NesnatbekDinthes
25b Divided into sections or lIe,ment8
(Figures 8 and 9) . . . . . . . . . . . . . . . . .. 26
26a Head a more or less well-formed ~rd
capsule with jaws, eyes. and alttennae.
Pol. (Figure 8) Clals Inaecta.
(Cf:49a) . . . . . . . . . . . . . . . . . ~.I' D1ptera
26b Head structure soft (except ja-, if
present) (Filure 9) . . . . . . . . . . . . . . .. 27
27a Head conical or rounded. lateral appen~ .8
not con8picuou8 or numerous. .. . . . .. 28
27b Head 80mewhat broad and blunt. Retractile
jaws usually present. Soft fiesbt lobes or
tentacles often present in head region.. Tail
usually narrower. Lateral lobe. or fleshy
appendafes on each segment. (Pigure 8-A)
. . . . . . . . . . . . .. Phylum Anmtlida. . . . . 29
28a Minute dark colored retractile jaws present.
body taperin, somewhat at ~h 1h1d8. pairs
or rings of bumps or "tegs" often present,
even near tail. Class Irlsecta. (Cf:49a)
. . . . . . . . . . . . . . . . . . . . . . .. Order Dipter~

28b No jaws, sides of body ,ener~ .pal'al~l
except at endB. (simlla!" to Figure 9).

Tb1ekened area or ring usually present
part way back on 7>ody, Clumps of
minute bristles on most segments.
, . , . . . . . . . . . . . (Phylum Annelida)
29a Segments wj.th brhtles, and/or fleshy
lobes, or other extensions. Tube builder,
borer, or burt'ower. Often reddish or
greenish in color. Pol. Earthworms,
sludge worms, clam worms, etc.
. . . . . . . . . . . . . . . . . . Clan Chaetopoda
29b A sucker disc '3.t each end, the larler
one posterior. External bloodsucking
parasites on high6 r animals, often found
unattached to any host. Pol. Leeches.
(Figure 9 B). ...... . . . . Clas8 Hirudinea
30a Skeleton internal, of true bone
. , . . . . . . . . . , . . . " (Vertebrates)
30b Body covered with external skeleton or
shell. (Figure 4). . . . . . . . . . . . . , . . .. 31
31a li:xternal jointed skeleton or shell covers
legs and other appendages. often leathery
in nature. Jointed legged animals.
(Figures 11, 12, end 14 throuih 29)
. . . . . . . . . . . . . . . . (Phylum Arthropoda)36
31b Shells limey or calcar'eous. (Figures 10

and 13). . . . . . . . . . . . . . . . . . . . . . . . . .. 32
32a Marine, altached, shell conical, or purse-
shaped on fleshy stalk. Crowded individ-
uals may be more or less cylindrioal.
Shell composed of several parts, two of
which spread apart to permit extension
of "hand" for fet:ding. Barnacles.
Phylum Arthropoda, Class Cirripedia
32b Shells one or two, or several arranged
as transverse plat"s. . . . . . . . . . . , . . .. 33
33a Half inch or less in length. Two clam-
like shells, Soft parts inside inolude
delicate flattened jc.inted appendages.
Phyllopods or branchipods (Figures 11
and 12) Phylum Arthropoda, Class
Crustacea, Subcllrss Branchipoda
33b Soft parts covered with thin skin, often
slimy,. " . ...,... (Phylum Mollusca) 34
The I($entificat~on of Aquatic Org~isms
34a Marin- oval in shape, eight shells tranI!!-
versely jointed. Chitoos.
. .. . " . . . .. .. . .. Class Amphineura
34b Shells one or two in nwnber. Snails,
clama, etc. (Figures 10-13)........ 3&
35a Shell, single may bt" a spiral, a flattened
cone, dsgenerl1ote, or missing. Pol.
Snails and s1l11S. . . . . Class G..trope"

35b Shell, double, right and left halves, hinged
at one point. Mussels, clams, oysters.
. . . . . . . . . . . . . . . . .. Class Pelecypoda
36a Head, thorax, and abdominal body regions
usually distinct. Three pairs of regular
walking legs, or their rudiments. Wings
present in all adults and rudiments in
some larvae. I,eglike appendages or gills
present on abdomen!' of some larvae. No
marine forms. (Phylum Arthropoda,
, . . . . . . . . . . . . . . Class Ineecta) , . . . .. 47
36b More than three pairs of legs apparently
present. (Figures 14-19) . . . . . . . . .. 37
37a Body elongated, head broad and flat with
Itrong jaws. Appendages following first
three pairs of legs are rounded tapering
fUaments. Up to 3 inches long. Dobson
f]y and fish fly larvae. Class Insecta,
. . . . . . . . . . . . . . .. Order Megaloptera
37b Four or more Fairs of true legs. , . .. 38
38a Four pairs of legs. Body roullded, bulbous,
head minute. Often brown or red. Water
mites. (Figure 15) Phylum Arthropoda,
Class Arachnida, Order Acari
38b Five or more pairs of locomotor appendages
(walking or swimming legs) gills, two pairs
of atenna. (Figures 16-19, 11-12)
Crustaceans. Phylum Arthropoda,
Class Crustacea. . . . . . , . , . . . . . . . . .. 39
39a Ten or more pairs of flattened, leaf-like
swimming (and respiratory) appendages.
Many species swim constantly in life,
some normally upside down. (Figure 16)
Fairy Ihrimps, phyllopods, or branchipods.
... """" ... .... Subclass Branchipoda

The IdentificatioD of AqUl.U~ OriaJP,8m8
39b Less than ten pairs ollooomotor

append.,es. . . . . . ' . . . . . . . . . . . . .. .. 40
40a Body and le,. enclosed in bivalved
(3 halvel) lhell which may or may not
completely hide them. (Fi,urel U.12) 41

40b Appendagel more rounded than IMf..Like
in craIB leeU.Oft. or if appendaps are
nattened lor sw1mmi~, eyes are on
stalks (pelalic shrimpa). May be lar,.

or minute..... . . . . . . .. - .. .. .. . . ... 43
4la One pair of branched antennae enlarged
tor lQcomotion, extend outside of 8hell
(carapace) . Single eye WluaUy villtbJ.e.
Cyclops or claclocera. Subel... Cladocera
41b Locomotion accompl1shl!d b'o/ body 1..
(not antennae). (Figure IS). . . . . . .. . .42
42a Appe.e.s l.af-like, nattC!ned, more
tun ten pairs. (Branchipod8)....... 39a
42b Animal leu than three mm loR length.
Appendages more or 1..1 slender and
jointed, often used for walkinl. (Figure 11)
Shells opaque. Ostracodes.
. . . . . . . . . . . . . . . .. Subela.s OltnOO4a
43a Body a series of six or more es...1a1ly
limilar segments, differlnc mainly in

11ze. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 44
43b Front part of body enlarged into a 8ome-
what ..parate body unit (cephalothorax)
often covered with a single piece of shen
(carapace). Back part (aMomen) may be
relatively smaIl, even folded und8meath
front part. (Fi,ure 19). .. . . . . . . . . .. 45
44& Body compres8ed laterally. i. e., high
and thin. Scuds, amphipods. (Fipr8 IT)
. . . . . . . . . . . . . . . . . .. Subel.."s Amphipoda

44b Body deprel8ed, 1. e., low and broad.
Flat gille eontalned in chamber beneath
tail. (Fieure 18) Sowbu.s.
. . . . . . . . . . . . . . . . .. Subcla.. Isopoda
45a Minute in .i:l8. Often .or.~ewhat eloneated
&Ad cylindrical, with abdomen extending
Itrai,l:It out behind ending in two small
projection.. Two relatively enormoue
masses of 8,gS are otte!'1 aU.abed to
female. Locomotion b.r 1n.ans of two
en1.aa',ed, unbrlllUlbed ante_.; the CIIily
lar,- .PP""" on tIIe~. (Fi,uI'819)
CopepodIt............. Subc1... C~po",

45b La....r. Carepaee (shell of cephalothOru,
the eftlar..d front "etton) u.8IIally more
COft'pieucMi. IS pidrl at \1IUtttair 1...
incllad1ft, claws, if:prehnt. Appelllllllates
pre8ent on abdl:J.aaen. . . . . . . . . . . . . . .. 46
46a Eye. imDlilwabl., in broad shell s~ped
like a horll8lhoe, a mud dweller. Long
sharp poiote4 trianlQ1ar tail eKtend'8 out
behind. Flat "boo." gUll beneath abdbmen.
Hor...hoe crab. Su.bclue A,a~,

. . . . . . . . . . . . . . . . . . . . . Genus Limlliu.
46b Eyes 00 movable stalks. Abdomen tight1y
folded back under cephalothorax (crabl)
or contimaed out behind thorax to elHlin
an expanded "tUpper" or ewimmint paddle.
Crabe, Ihr1tnps, crayfishes, lobsters,
etc................. Subclass Deeapada
47a Two pairs of functional winls (one pail'
may be more 01' 1e.s harden84i as pI'Otection
for tt\e other ptlir). Adult insecta which
no!'mally live on or In th~ water (Flfuree 22,

2&,28,28) ....................... 57
47b No functional wings, though "pads" in
which they are developiq !!!!1 be visible.
Some resemble adults very closely, otherS
differ extremely (""ure. 8, 23, H, 26, 2'1)

........... Ii.................... .'.. 4-8
48a Extemal padl or e...s in wilich -.."
develop clearly vi8ib.1e... . .. . . . .. ... 53
48b More or le.s worm-like" no external
evidence of wine "slop.18nt........ 49
49a No jointed le,s (luckeI' .scs, hooks,
prole,s, breath1n& tube., and other 8uch
structure's me.y be pre.ent. 'Fi,uns 8, 91»
Larv.. of two wiftl'ld aiel, midges, etc.
Fl:>l. . . . . . . . . . . . . . . . . . . . Order D1~era

49b Three palr8 of Jointed thoracic 18... head
cap8i1le wen-formed. . . . . . . . . . . . . . .. 50
50a M1mate (2 - .4m~, ltvin, on surface Mm.
Tail a Itl'ORC or..n that can be hooked into-

a "catch" beneatb thorax. When released,
animal jumps 4ntQ the air. Not usually
classed as aquatic. These are adults.
no wings are ever grown. (Figure 20)
Springtails. . . . . . . . . . Order Collembola
50b Larger (rarely collected under 5 mm),
worm-like. living beneath the .urface.

... .. . . . . .. .. .. . . . . ... . . . . . .. . . . .. 51
51a Construct fixed or movable cases of
bottom materials. (Figure 21) Abdominal
segments often 1I\1th minute gill filaments.
no long lateral fUaments. Generally
cylindric in shape. Caddisfly larvae.
. . . . . . . . . . . . . . . . Order Trichoptera

5Ib Free living, i. e., not case-buUding. 52
52a Somewhat flattened and relatively massive
in appearance. First segment of thorax
about same size as head, following two
segments smaller. Each abdominal
segment with rather stout, tapering
lateral filament abcut as long as body is
wide. (Figure 14) Dobsonfly, alderfly,
and fishfly larvae. Order Megaloptera
52b Generally rounded in cross section.
Lateral filaments if present tend to be
longer and thinner. (Figure 23) (A few
forms extremely flattened, like a suction
cup: Psephenidae). Beetle larvae.
. . . . . . . . . . . . . . . . .. Order Coleoptera
53a Two or three fUaments or other structures
extending out from end of abdomen.
(Figures 24B, 2.m. 26. 27)........ 55
53b Abdomen ending abruptly, unless terminal
segment itself is extended as single
structure. (Figure 24C) . . . . . . . . . .. 54
541 Head broad, eyel:1 widely spaced. Front
of face covered by extensible folded mouth-
parts often called a "mask." Larvae of
dragonfiies or darning needles. Pol.
(Figure 24E). . . . . . . . . .. Order Odonata
. . . . . . . . . . . . . . . . . (Suborder Anisoptera)

54b Mouthparts for p~ercing and sucking.
Body form varies extremely. Water bugs,
walking sticks, wate.!' boatmen, back-
swimmers, electric 11ght bugs, water
strider_, and others. (Figure 25)
. . . . . . . . . . . . . . . . . . . Order Hemiptera
The Ic8a\ification Ct$ 4quatiQ Q!:aqUme
55a TaU extensions (caudal fUaments) two.
Stonefly larvae. (Fi.ure 26)
. .. . . . . . . . . . . . . . .. Order Plecoptera
55b TaU extensions usu:a.1ly three. (Figures 24B,

24D. 27)........................ .. 56
56a Tail extensions long and slender. Rows
of hairs may give them a feather-like
appearance. Larvae of mayflies.
. . . . . . . . . . . . . Order Ephemeroptera
5Gb Tail extension8 flat, elongated plates.
Head broad with widely spaced eyes,
abdomen relatively long and slender.
Larvae of damselflies. Order Odonata
. . . . . . . . . . . . . . . . . . (Suborder Zygoptera)
57 a External wings or wing covers form a
hard protective dome over the inner wings
folded beneath, and the abdomen. Beetles.
(Figure 28). . . . . . . . . Order Cole~~
57b External wings somewhat membranous.
usually relatively flat as compared to wing
covers of beetles. Mouthparts for piercing
and sucking. Body form various, (Cf:53b).
True bugs. (Figure 25) Order He~tera
58a Appendages present in pairs (fias, legs,

wings). . . . . . . . . . . . . . . . . . . . . . . . . . .. 60
58b Mouth a round sucHon disc. No paired
appendages. . . . . . . . . . . . . . . . . . . . . .. 59
59a Body long and sle~der. Several holes
alongside of head. Lampreys and hagfishes.
Sub-phylum Vert~brata. Class Cyclostomata
59b Body plump, oval, tail extending out abruptly.
Larvae of frogs and toads. Legs appear
one at a time during metamorphosis to
adult form. Tadpoles... Class Amphibia
60a Paired appendages are legs. . . . . . . . . . 62
60b Paired appendagelJ are fins. . . . . . . . . . 61
61a Gills covered with a flap or "operculum. "
True fishes. . . . . . . . . . . . . . Class Pisces
61b Gill chambers open directly to the outside.
Skin often rough and scratchy or penetrated
by clumps of shore sharp spines. Essentially
marine. Sharks, skates, and rays.
. . . . . . . . . . . . . Clus Elasmobranchi!

TM Ident1fic.tioq of AQ.uatic Ofa-..,..
82. lX,it. with claw., naUI; or hoof.. . .. 63

82b Skin .ed, no claw. 01\ dta1~.. "1."Of.,
toad8, and .a1amandel'8. C1&.. Am,pbibia
83. W.rm blooded. . . . . . . . . . . . . . . . . . . .. 84
83b B9dT coy.red with ho.,. Ileal.. or plat,..
Reptil... . . . . . . . . . . . . . . . . . <::1... Reptilia-

64. Body cov....d with featbu.. Sirda.

. " . . . . . . . .'. . . . .. . . . . . . . . . . Cla88 ~y-
64b Bod;y CCW1Jred with WI'. NI!IIIUn81..

. . . . . . . . ... *' . . . . . . . ... .. . cl88- ~....(,

The IdenWicat10n 01 ~uatie'Orlani'lDII
Pltyla or Cia. e.. or Cla.ees or Other
Subphyla Subpbyla Orden
Platyhelminth.. Turb.Uaria  
Annelida Chaetopoda  
Arthropoda Cruetacea Brancb1opoda 
 Arachnoidea Acari 
  Xiphoeura Limulu.
 Insecta Collembola 
MollusCa Gastropoda  
Chordata Tunicata  
Algae   Corallina

T. Jden.tific,.t~ of Aqli.U~ Ori&ni.zns
I'~'~ \
\~ , \ \. \
1. ~ 8pioule.
up to . a m.aa.loac.

3A. ~1f.r, ~.rthra
Up to . S mm.
3C. RoUfer, Ph1lodina
Up to.4 mm.

38. RoUf.l'; Xel'atel1a
Up to . I 11,\""
~. Joint.d 10,
TlU'lllellaria, 1\I..08t08a
'II to 1 em.
-', - 10
2B. Br:Joeoal man. Up to
..vera! fen d1-.
IA. Bryozoa, P1\IIDatella. IndiYidlla18 lip
to 2 mm. Iutert~d mas... mq be
vel')' extensive.
88. Turbellarla, Du,eeia
Up to 1.8 em.
4C. Jointed Ie.
5. Tapeworm heed,
Taenia.. Up to
25 yd.., 1001[
((f) 8 ,
7, Nematodes. Free livina
form.' comm~ lip ,to
1 mm.. aceutonally

The Identification of Aquatic Organisms
8A. Diptera. MC1.QIl1to larvae
Up to 15 mDt. Ion..
9A. A.neUd,
worm. \lp to
II. zn.tn
8B. Diptera. Moequito
pup.. Up to II mm.
8C. Diptera. Tendiped
larvae. Up to 3 cm.
9D. Diptera. Rattailed ma,fo1.
Up to 35 mm. without tube.
98. Annelid. leech up to 20 !!nt.
IDA. PeIe cyopod, Alumidonta
Side view, up to 18 em. long.
11A. Oat racoci, .£zPe ric U8
Side view. lip to 7 mm.
. "
llB. CypericU8. end view.
8E. Diptera. crane fly
lIupa. Up to 2.5 em.
lOB. Alaamidonta, end view.
12B. 3ranchiopod \
HOemina. Up
to 2 mm.

The Ident1r1cation of Aquatic Or.ani".
18. Gutroapad. V1viparu8
Up to S -iDebee.
14. J4eplopter..
A1derfly 1&1'\'ae
Up to 25 mm.
18.l'a1" Shrimp. J:ubrllDCb1~
Up tell em.
15. Water mite,
Up to S ma.
18. I80p0d, A8eUQ8
nn ~01lembala, Podlll'&
, to 3 mm. 10aI
19A. Calanoid eopepod,
Up to S mm.
~t - 13
198. CopepoKi eopepgd,
Up to J5 mm.

I ~ 
21A. UB. 2lC.
~ 6:
2lD. UE.
21. Trleopcera, Larvllll ea..., mo.tly 1-2 em.
The Identification of Aquatic Organisms
22. Mega10ptera, alderfly
Up to 2 em.
2M. s.ttt'L1t larvae.
D7U81c1a. ,
U.uatq about tern.
23B. Beetle larvae,
Uaually about 1 em.
2fA. Odonata. dragonfly
nYMph up to 3 or 4
24B. Odonata, tail of
damee lfl;y nymph
(aide view)
24D. Odonata, dam8e1fl.7
nymph (top vi.w)
24E. Odonata, front view
of dragonfly nymph
showing "maskl'
partially extended
Odonata, tail of
drqonflv nvmoh
(top v.

Tn. Identiflcation of Aquatic ':>t'aan1Im8
25A. Hemiptera.
Water Boatman
About I em.
27 .Eph..meropt.. 'a.
Mayfly nymph
Up to ! cm.
.:t(. - 1 4
~"". :Jiptera. ,-'ra~
Cy. Up 10 21 em.
26B. Hemiptera.
Water Scorpion
About 4 em.
28A. Coleopt..ra,
Water se.veneer
beetle. Up to 4 em.
26. Pleeopte ra.
Stonefly nymph
Up 10 i em.
28B. Coleoptera.
Dytiseld b..etle
Usually up to 2! em.
29B. Dipter". Mosquito
Up to 20 mm.

Keeler, C. R. Books on Aquatic BiololD'.
Freshwater and Marlne,Facile Press,
Tallahassee, Florida, 1965.
REFERENCES - Invertebrates
1 Eddy, S. and Hodson, A. C. Taxonomic
Keys to the Common Animals of the
North Central States. Burgess Publ.
Co" Minneapolis. 1955.
2 Edmond.on, W. '1'., Ed. Ward and
Whipple's Frerhwater Biology.
Wiley &. Sons. New York.
3 Hedgepeth. J., and Hinton, X. Common
Seuhore Ute ')f South~rn CalUornia,
Naturegraph Company, Heraldsburg,
CalUornia. 1981.
4 Jahn, T. L., and Jahn, F. F. How to
Know the Protozoa. Wm. C. Brown
Co., Dubuque, Ic;,wa. 1949.
5 Kudq, R. ProtozQlogy. Charles C.
TlKnnas, Publiahflr, Sprin8field, Illinois.
6 L18ht, S. F.. et aI. Intertidal Inverte-
brates of the ~ntral California Coast.
University 01 California Press,
Berkeley. 1981.
7 MacGin1tie, G. E., and N, Na tW"al
History of Marine Animals. MeGraw-
HiU. 1949.
8 Miner, R. W. Fielcl>ook of Seashore Life.
G, B. Putnam's Sons, New York. 1960.
9 Pennak, R. W. Freshwater Invertebrates
of the United States. The Ronald
Press Co., New York.
10 Pratt, H. W . A Manual of the Common
Invertebrate Animals Exclusive of
Insects. The B1a.k1ston Company,
Philadelphia. 1951.
The Identification of Aquatic Or£lU1isms
11 Ricketts, E., and Calvin, J. Between
Pacific Tides. .Revised by J.
Hedgepeth, Stanford Univ. Press,
Palo Alto, Ca)1fornia. 1952.
12 Smith, R. I. Keys to Marine Invertebrates
of the Woods Hole Region, Contribution
No. 11, MBL. Woods Hole, Mass.
1 American Fisheries Society. A List of
Common and Scientific Names of
Fishes from the United States and
Canada. Special Publication No.2.
Am. Fish Soc. Dr. E.A. Seaman,
Sec. - Treas. Box 483. McLean, Va.
1960. (Price $1. (10 paper, $2. 00 cloth)
2 Anon. Check List 01' the Florida Game
and Commerda1 Marine Fishes with
Common Names. Florida State Board
of Cons. Educational Bull. No. 12
3 Benton, A.H.. and Stewart. Keys to the
Vertebrates of the Northeastern States.
Burgen Pub. Co. 1964.
4 Breder, C. M. Fieldbook of Marine Fishes
of the Atlantic Coast. G. B. Putnam's
Sons, New York.
5 Clemens, W.A., and WUby, G. V.
Fishes of the Pacific Coast of Canada.
Fisheries Re'ilearch Board of Canada.
Bulletin No. 68, 2nd Ed. 1961.
6 Hubbs, C. L., and Lagler, K. F. Fishes
of the Great lAkes Region. Bull.
Cranbrook Inst. Science. Bloomfield
Hills. Michigan. 1949.
7 Perlmutter, A. Guide to Marine Fishes.
New York University, NY. 1961.
8 Schrenkeisen, R. Fieldbook of Fresh-
water Fishes of North America, north
of Mexico. Putnam's Sons. 1963.
26- 15

The IdenWioaU&m of Aquatic Or~
9 Ztm, H.S., and Shoemaker, H.H.
Flfh.I..A Fide to Fre8b 8Dd Salt
Water .,.... GoWen. 195'.
28- 18
This outline wa. prepared by H. W. JacUon,
011ef 8ioJo1S1t. Nat18Dal T~iIQI-cen...
Environmetltal PJ"Oteelton .....~. OWP,
Cincinnati, OH: 45211
~: Syltematlcs

Benthos is the noun.
Benthonic, benth41 and benthic are
A Composed ot a wide variety of life
forms that are related because they
occupy "common ground"--the water-
ways bottom substrates. Usually
they are attached or have relatively
weak powers of It'comotion. These
Hf. forms an:
1 Bacteria
A wide variety of decomposers work
on organic materials, breaking them
down to eIe:nental or simple com-
\ pounds (heterotrophic). Other forms
..- grow on basic nutrient compounds or
form more complex chemical com-
pounds (autotrophic).
2 Algae
Photosynthetic plants havin, no true
roots, stems, .,nd leaves. The basic
p:roducers of food that nurtures the
aftimal compol'1ents of the community.
3 Flowerin, Aquatic Plants (Pondweeds)
The largest flora, composed of
COmplex and differentiated tissues.
Many are rooted.
4 Microfauna
Animals th&:.t pus through a U. S.
Standard Series No. 30 sieve, but
are retained on a No. 100 sieve.
Examples are rottfers and micro-
crustaceans. Some forms have
organs for attachment to substrates,
HI. MET, fm. 8e.1. 74
while others burrow into soft
materials or occupy the interstices
between rocks. floral or faunal
5 Meiofauna
Animals, mostly metazoans, that
can pass a 1. 0 mm to O. 5 mm
screen. Exarnples are naiad
worms and flat\llorms.
6 Macrofauna
Animals that ar\! retained on a
No. 30 sieve. This group includes
the insects, worms. molluscs, and
occasionally fish. Fish are not
normally considered as benthos,
though there are bottom dwellers
8uch as scuJ:pins and darters.
It is a self-contained community. though
there is interchange with other commun-
ities. For example: Plankton settles
to it, nsh prey on 1\. and lay their eggs
there, terrestrial detritus leaves are
added to it, and mp.ny aquatic insects
migrate from ~.t to the terrestrial en-
vironment for their mating cycles.
It is a stationary water qual1ty monitor.
The low motility of the biotic compon-
ents requires that they "l1ve with" the
qual1ty changes of the over-passing
waters. Change!! imposed in the long-
lived components remain visible for
extended periods, even after the cause
has been eliminated. Only time will
allow a cure for the community by drift
and reproduction.
Ancient l1terature records the vermin
associated with fouled waters.
500-year-old fishing l1terature refers
to animal form.:! that are fish food and
used .s bait.

'U~. S,nthio Biota in Watef QuaUb' Eval_tionl
C The acientll1c Uteraturo a8l0c:lattna
biota to water pollution problema i.
over lOG year. old (MackentbuA and
Inll'am. laU).

DEarly thia c8fttury. appUect biolollcal
inve8tiption. were initiated.
1 The entrance of state board. of Health
into water pollution control actlvitiea.
2 Creat.ton of atate OOft8Eo!:'Vattm a,enciea.
3 Indu8triaUzation and ur1lanization.
4 Growth of Umnolopcal profram.
at univer8l.ties.
E A decided increaae in ~nth1o .tudies
occurred in the 1950 dec..de. and much
of today'. activitie8 are 8trOft'b' influenced
by developmental work conducted durin,
thi. period. Some of the rea.ona for thia
1 Movement of the un1venitiea from
lI..qade8n1o biolot1." to apJIUed
pollutiOn program,.
2 Entrance of the federal ,overnment
into 8fttorcement .apect. of water
pollution control.
3 A rl.ln. economy and the development
of federal.rant .ystema.
4 Enviromnental Protection Pl'Ofrarnll
are. current stimulua.
A It 111 a natural monitor
B The community contalnt all of the
component a of an ecow.r8tem.
1 Reducer.
2 Producers
3 Consumera
a Detritivore. and bacterial feeders
b Herbivores
c: Prldatora
C ~ of SUrvey
1 Manpower
2 TiJae
3 Equipment
D Extenalve Supporting Uterature
E Advafttap. of the Macrobetit'b08
1 ReWi..q .e.aile
2 We ht8tory length
3 Fi.h food organisms
4 ReUability of Sampling
5 DDUars/lnlormatian
6 Pl'ed1ctabWty

7 Univ......Uty

A Dllltruction 01 Orp.ni8m Type.
1 Beginninj with the most sen.iti;,e forms.
pollutanta ldU in order ~ li88itivttt
until the most tolel'8itt ftnom is the Jaat
aurvivor. This re.u1t. tD .. reductioa
of varl~ or dlvertity of orpnilm..

2 The u8Ual order of macrof.trftrtebrate
eIi.appearalu:e on a 8.88tvUy Bcale
below pollution source's 1.8 shown In
F1pe 2.
Water I

1 Water
As water qW6l1ty 1mprove8, these
reappear in the ..me order.
B The'lfUmber of Survivon Increa8e
1 Competition and predation are reduced
between foros.
2 When the pollutant is a food (plants,
fertilizers, animals, organic materials)

C The Number of Survivor8 Decrease
1 The materia~ added 1s toxic or has no
foOd value.
2 The material added produces toxic
conditions a8 a byproduct of decom-
p08ition (e. ,., large or,anic loadings
produce an anaerobic environment
relu1ting in tne production of toxic
.u1tl.des, methanes, etc.)
D The Effects May be ManUelt in Com-
1 'Of pollutants W1d their effects.
2 Vary with longitudinal distrihution
in a stream. (Figure 1)
E Tolerance to Enrichment Grouping
(Fi,ure 2)
Flexibility must be maintained in the
eetablishment of tolerance lilts based
on the response of organisms to the
environment because of complex relation-
lhips among varying environmental
conditions. Soree general tolerance
patterns can b" established. Stonel1y
nymphs, mayf]y /18.iads, helll(I'ammite8,
and caddisf1y larne represent a arouping
(8enlitive or into]erant) that is leneral-
bed quite senlit1ve to environmental
changes. Blackf1y larvae, scuds, 10W-
bu,s, snails, fiuaernaU clam8, dr-lOn-
ny D1JnPhs, damse1f1y nymphs, and most
kind. of midge !arve are intermediate
(facultative or intermediate) in tolerance.
Sludge-worms, some kinds of midge
~rvae (bloodworms), and some leeches
Usinlf Benthic Biota in Water Quality Evaluationll
. ,~...\
III' ,
to-' .
. I .
~, \
.. \

~ I \
, "
, "
--' "'.....

.. -- -. NUM8ER or ORGANISMS
FoUl ba81c 188pO""'. of bottom animo to ponutlon.
A. 0Iqcmk: wa- eliminate the "Mllive bottom animaIo
and proYide food In the form of 01l1li988 !or the .uwvlvIDq toler.
ant ..... .. Lanp quanlitiOle 01 decompoelnq orqanic waal88
eUmiDale ..naIU.,e bottom anima" and the .xcea.lve quanti-
Iiee of byproducll 01 orqanlc dtoCODlpoeltion inhibit the tolerant
form8: In lime, with natural lllream puriBcation. water qualltr
I8IpIO'I'W 80 that the tolerant forma can f1ourl8h. utilizlnq the
oludq" aa food. C. Toxic material. .limlnat. th. ..II8iUve
bottom anlmala: oludqe Is abeent and food 18 reatrlcled to !hat
lIatuIa1ly occ:wrIDq In the meam. which Umill tbe number of
tolerant own'l'illq form.. V tIl'f toxic matertala may eliminate
an 0I'IJCIII!8m8 below a wa.te IOUlce. D. Orqanlc .ludqe. with
to1IIc materialo reduce th.. number of kind. by eUminatlDq
-.III.,. form.. Tolerant euwvtVonl do nat utilize tbe orqcmlc
:oIud9W becauH the toxicity JHIrtcI8 their qrowth.

Figure 1
are tolerant to comparatively heavy loads
of organic pollutants. Sewage mosquitoes
and rat-tailed maggots are tolerant of
anaerobic environments.

U~~1l Benthic Biota in Wat.r QuaUty Evaluation.
F Structural Limitation.
1 The morpholoaica1 structure of a
.pedu limits the typIJ of environment
it,may occupy.

a Speci.. with cOll\plex appenda,e.
and exposed complicated re.piratory
structures, such a. atonefV
nymphs, mayf~ ~mphs, and
cadd18fly larvae, that are .ubjected
to a constant del'.1Ie.of sette able
particulate matter 1l00ft abandon
the polluted area betJause of the
con.tant preening required to main-
tain mobility or respirotory func-
tion.; otherwise, t'.1eyare soon
b Benthic animahi in depo.iting :tones
may also be burden'ld by "sewaae
fungu." growths including stalked
prO\OlOan8. MarlY or thue stalked
protozoans are hoat lpecific.
2 Spedel without conlplieated external
..tru~rel, such &1 b108clworma and
.lud88worms, are not so limited in
a A .ludieworm, for example, can
burrow in a deludp of particulate
organic matter and flouri.h on the
ab\lJl,dance of "manna. "
b Morphology also determines the
spedes that are fO'md in rHfies, on
vepta1:ion, on the bottom of. pool.,
or 11:1 bottom deposit..
1 Qualitative aampl1ng determines the
variety of species occupyinJ an area.
Samples may be taken by any method
that wiU capture representatives of the
lpecie. pre.ent. Collections from such
samp11Ags indicate changes iwthe
envirorunent, but pneralq do not
acc\U'ately rettect the del1'ee of
chan,e. MayfUes, for e~ple. may
be reduced from 100 to 1 per square
foot. Qualitative data wou1d mdicate
the presence of both specie8, but,migJrt
not neces8&rily delineate the change in
predominance from mayflies to sludge-.
worms. The stop net or kick sampUDg
technique i. often used.
2 Quantitative .ampling 18 performed to
observe changes in predominance. The
most common quantitative sampling
tools are the Petersen and Elkman

Drawings from Geck1er. J.. K, M. Mackenthun and W. M. Incram. 1963,
Gla..ary of Commonly Ueed Biological and Related Terms in Water and'
Wute Water Control,DHEW, PHS, Cincinnati, Oha.o, Pub. No. 999-WP-:a.
Stoneny nymph (P1ecoptera)
Mayfly nymph (Ephemeroptera)
Hellgrammite or
Dobaonfly ]a rvae (M".loptera)
Cadd1sfly larvae (Trlchoptera)
Black fly larvae ~Simuliidae)
Scud (A mphipoda)
-A q\atic 80wbug UeQPoda)
'SnaU 1G&.tropoda)
I Fingernail clam (Sphaeriidae)
J Dam8elfly nymph (Zygo~tera)
K Drago~ nymph (AniIOJ)tera)
L BloodworM or midge
fly larvae (Chironomidae)
M Leech (Hirudinea)
N Sludgeworm (Tubifi<:idae)
o ~wale fly larvae (Psychodidae)
P Rat-taU.d mallot (Tubifera-Eli8talia)

U.ini Benthic Biota in Water Quality Evaluations
t - . "
.- --\
~' E
F-r-.---.-,--"->-.. ,:
:~v-V-. r

11.... Be1fttUc WDta in WaHr Qualitv ~hlatiou
,rab. and the Surber stream bottom
01' sq\l&re-foot lamplt:r. Of these,
the Petersen ,rab samples the widelt
variety of substrate.. The Ekman
Itrab is 1I.mited to fine-textured and
80tt sub8tratel, such as sUt and Ilu..
The !harber sampler il dell.8ned tor
sampUn, rifne area.; Ii requires
movinl ~ter to tr4lnspert d1slodpd
drpDl8ms Into its ntt and is limited
to depths of two feet (II' lei..
3 The oolleoted sampll'! il screened with
. standard sieve td cOncentrate the
organisms; these are sorted from
the retained material, and the number
ot each kind determined. Data are then
adjusted to number per unit area,
usua~ to number of bottom per
.quare meter.
4 JndependentJ;y. neither qualitative not
quaDtitative data suff:l.oe for thoroulh
anaJrses of environmental conditions.
A C1lrllOry examination to detect dama,e
may h made with either method. but
a co~b1nation of the two pve8 a more
preoile determinatior.. It a choice mU8t
be made. quantitative Mnaplinc would
be belt, because it incorporate I a
p.1'Ual quaUtative sample.

S FlOra
1 Direct quantitative eampUn, of natu-
rally ,rowing bottom alpe ie d1mcult.
It is baaically one of colleetinl al,ae
from a standardor uniform area 01 the
bottom lubstratesW1thO\1t dl11turbln.
the deUcate growthl and thereby di8-
tort the sample. Indirect quantitative
sampUn, is the best Available m-*hod.
Artificial subatratel, luch as wood
blocks, ,]ass or plc.xil1aBl IUdes,
brick., etc., are placlld in a .tream.
Bottom-attached alpe will grow on
these artificial sl:lbstrates. Atter two
or more weeks. the artificial sub-
strat.. are removed for analysis.
Algallrowths are .craped from the
substrates and the qunatity measured.
Since the exposed substrate area and
exposure periods are equal at all of
the sampUng sites. differences in the
quantity of alpe can be related to
cllan.s in the quality of water tklwin,
OVlI' the sub8trat.s.
2 The quantity of alga. on artil1clal sub-
strate. can be measured 1D several
wa)'f. Mloroscop1<:: oount. of alpl
cel1. and dry well(ht of alpl material
are 10ftg established ~eth0d8.

M1c~!,.CQpic counu iIIJrolV1!ttboi'wah
.cr&ping, mixing, and !Nlpension of
the alial oeUa. From thI. aUxture
an aJ1quot of ceUs is witbdrawn for
enumeration under a micro.cope. Dry
weJ.altt 18 determineci by dryin, and
we1tblng the alpl 8amplB., then 11~
nidn, the ample to bum off the algal
mater1a18. leavinl inert inorpD1c
matenala that are apia _i&bed..
The difference between initial weiprt
and welaht atter ignition 18 attributed
to alpe.
Any orpnlc 8edimel).ts, hqwever, that
settle on the artificial substrate along
with the alpe are processed also.
Thus. if oraanie wa.e. are preant
appreciable erron IDa,. --I' in80
this method.
3 Durin, the paet decade, cbloroplaJJl
analysi8 haa become a popu]ar method
tor eetlmatiD, alp.l growth. Chloro-
p~llls extracted Irem the aliae and
Is used a8, an indeJ.; of tlae quantity of
alpe present. The a~ps of
ch1orCJPh7U analysis are npWity.
sl.mpJicity, and vivid platorlal results.

The alpe are sorubbed from the
artificial substrate _mp1n~ ground.
then eaoh Ample is IItqped hi equal
volume.. 90"'" aqueoua acetone, wh1<:h
extracts the chlorop~l1 from the alp].
cells. The chlorophyll ~racts may
be compared vi.ua~.

Ueina Bf!nthi~ Biota in Water Quality Evaluations
S.cauae the chlorophyll extracts fade
with time, Qolorhnentry should be used
for perman.~ recorda. For routine
, rlClordl, limple colorimeters will
lumce. At ve~y high chlorophyll
densities, interference with colori-
, $8try occurs, which must be corrected
throuih serial dtlution of the sample
or with a nomofl'aph.
4 Autotrophic Indu
The chlorophyll content of the periphyton
il \lsed to e8timate the alpl biomass and
&s an indicator of the nutrient content
(or trophic status) or toxicity of the
water and the taxonomic composition
of the community. Periphyton growing
in surface water relatively free of
organic pollution consists largeq of
&1...., which cor.tain approximately
1 to 2 percent chlorophyll & by dry
weight. .If cUssolved or particulate
organic matter 1s present in high con-
centrations, large populations of
filamentous bacteria, IIt&lked protozoa,
and,other non~hlorophyll bearing micro-
'organisms develop and the percentage
of chlorophyll a is then reduced. If the
biomass- chlorophyll a relationship is
expressed as a ratio (the autotrophic
index), values greater than 100 may
relult from org""1ic pollution (Weber
and McFarland. 1969; Weber, 1973).
~h-free Wjlt (ma~m2)
Autotrophic Index II Chlorophyll a (mg m2)
Two very important factors in data evalua-
tion are a thorouah kn"wledge of conditions
under which the data were collected and a
critical.llessment of the reliability of the
data's r6presentati:m of the situation.
A Maximum-Minimum Values
The evaluation of physical and chemical
data to determine their effects on aquatic
organisms is primarily dependent on
maximum and minimum observed values.
The mean is useful only when the data are
relatively uniform. The minimum or
maximum valnes usually create acute
conditions in the environment.
Precise identification of organisms to
species requires a lIpecialist in limited
taxonomic groups. Many immature
aquatic forms have not been associated
with the adult species. Therefore, one
who is certain of tbe genus but not the
species should utilize the generic name,
not a potentially incorrect species name.
The method of ~terpreting biological
data on the basis of numbers of kinds
and numbers of organisms will typicalq
C Lake and Stream Influence
Physical characteristics of a body of
water also affect animal populations.
Lakes or impounded bodies of water
support different fz.unal associations
from rivers. The number of kinds
present in a lake may be less than that
found in a stream because of a more
unifrom habitat. A lake is all pool,
but a river is composed of both pools
and riffles. The nonflowing water of
lake exhibits a more complete set-
tling of particulate organic matter that
naturalq SUPPOl'ts a higher population
of detritus consumers. For these
reasons, the bottom fauna of a lake
or impoundment cannot be directq
compared with that of a flowing stream.
D Extrapolation
How can bottom-dwelling macrofauna
data be extrapc,lated to other environ-
mental components? It must be borne
in mind that a component of the total
environment 1s being sampled. If the
sampled component exhibits changes,
then so must the other interdependent
components of the environment. For
example, a clean stream with a wide
variety of desirable bottom organisms

WIn BeIathk.Biota in Wau ~Ub' .Evalu8t1ou
would be expected to '-vo a wide varl-
ety of de.lrable bottom neh..; when
poUjtlon reduces the number of bottom
orp.n1am.. a comparable ~eduot1on
wou1d be expected in the number 01
fishes. Moreover, It would be JoatofJ.
to conclude that any factof that eJitn.
inate. aU bottom orplll"" wCJU1d
eliminate most other aqultKc iorme
of life,
A The Chemical Environment
1 D1..o1ved oxypn
2 Nutrtents
3 Toxic materials
4 Acidity and alkalinity
5 Etc.
B The Ph3flc&l Env1rClh&lient
1 Sua.d solid.
2 temperature
3 Ulht penetration
4 Sediment compo.ition
5 Etc.
A Damage A..essment
If ta stream 18 .uffering from pollutant.,
the biota wUI so indicate. A b101opat
can determl:ne dama.es by looldng at the
"critter" aa.emblap I:n a matter of hours.
Usually, if damage. are not found, it will
ndlt be nece...ary to alert the remainder
of the a",My's staff, Slack all the eqUip-
ment, pay travel and per die, a'l\d then
wait five dl.ys before enou,h data can be
a~semb1ed to begin eW.luatlon.
B B)' detft'lDintnC what dama,n have been
done, ... pot'''lal cause ''DM'' can be
reducedto a few items.for emptiatis and
the enUre "wendel'ful worlds" of Ictence
and ""'1'.., need not. ",..pra;et!ced with
the r.- that much data are discarded
)ateI' bt,8u.eCbty were --'llPPKi!:&b1e to
the ~tn b., in"lo~.
C Good benthic data associated withc:!hemi"eal,
phy.lca1. and .entmeeribr can b. data
U8ed to predict the direction of future
cban,.. and to dtlmate the a1'no\1l1t of
poUu--. that need to be ~moved fl'otn
1 Hyi1e.. H. B. N. The EcoJD&y of Running
Water'. t1mv. Toronto Proes'.. 19'70.
2 Keup, L. E., l81ratn, W. M. and
Mack..thun. It. M. The Role of
Bottom DweUtng MacrofaUtla in Water
PoUutlon Iftvelltigatlonll. USPHS
En\l'irGbment81 Health Serie8 Publ.
No. .....WP".'. 23 pp. 1986.
3 Keup, L.E.. _am, W.M. and
Mackenthun. K. M. Biology of Water
Population: ACollectioll of s.,lected
Paper. Oft Stream PoUutlO'n, Waste
Water, and Water Treatntent.
Federal Wa-t.r PoUution Control
Admiadeti'at18 PUb. No. CWA"3,
290 pp. lilt.
4 Mack1tnthun. K. M. Thtl Practice of
Water PouuUcm Bfo1OJy. FWQA.
281 pp. 1989.
I 8te_rt, R.K., In,ra-. W. M. atid
MaGIIeMhun, K. M. Water PoUutibb.
Control, Wafte Treatmet\t.and Water
Treatment: Selected Di.JjiC81. Ref-
erence. on Fresh and Marine Wat~r..
FWPCA Pub. No, wp..U. 136 Pp. 1986.

6 Weber, Cornel1n. I., Bioloaical Field
and Laboratory Methods for Mea8uring
the Quality of Surlace Waters and
Effluents. U. S. Environmental Pro-
tection Agency, NERC, Cincinnati,
08. Environmental Monitoring Series
6"10/4.73.001 July 1973
Usin" Benthic Biota in Water Quality Evaluations
This outline was prepared by Lowell E.
Keup, Chief, Technical Studies Branch.
Div. of Technici'.l Support. EPA, Wash-
ington, D. C. 20242, and revised by
Ralph M. Sinclair. Aquatic Biologist,
National Training Center. EP A. WPO,
Cincinnati, OH t5268.
Aquatic Life, Benthos, Water Quality.
Dear.dation, Environmental Effects,
Trophic Level, Biological Communities,
Ecological Distributor

A Due to the nature of ecolorical inter-
re1ation.hlpe. method. for the collection
of different "/pe. of aquatic orpnl.me
durer. In pfteral we can recornize
tho.e that ..1m 01' neat and thole that
crawl. tho.. that are big and thole that
are I1ttle. Each comprise. a part of
"the life" at a~ riven .urvey etatlon
and con.equentJ.r a "complete" collection
would include all type..
B Field method. in the following outline
are grouped under four ,eneral
cate,oriee. the collection of:
Bentho. (or bottom dwelling
organism.). Theee may be
attached. crawling. or burrowing
Plankton (p:.ancton). These are all
of the micro.coplc planb and
animall r.ormally .wimmlng or
luspendt!d in the open water.
Periphyton or "autwuchl". Thil is
the commurii1y of organllms
associated with the lurfaces of
'object.. Some are attached. .ome
crawl. Th. lJI'oup i. int.rmedlate
between the benthos and the plankton.
Nekton. Nekton are the larger.
free .wimmin, active animal. such
as .hrimp or n.bel.
C Aq18tic mam.nal. and bird.. in mo.t
oa.... require stUI other approaches
and are not included.
D Thsre i8 I1ttle ba.ic difference between
biolo,ical methodl for oceanic.
'Ituarine. or frelhwater .ituations
eXoept thole dictated by the phy.ical
nature of the environment8 and the
relative .ize. of the orpni.m..
Fi8h. bentho!i. and plankton collection
i8 e8sentially the ..me whether con-
ducted in Lake Michi..n, Jones'
Beach. or the Sargasso Sea.
Marine "rguisms range to larger
Itr.e.. and the corrosive nature of
s_water dic1atel Ipeclal care in
the delign and maintenance of
marine equipment. Site lelection
and collection schedules are
influenced by such factors as tidal
currents and periodicity, and
saUnity distribution, rather than
(river) currents. riffles, and pools,
, 2
FreshwlI.ter organisms are in
,eneral smaller. and the water is
seldom chemically corrosive on
equipment. mte selection in
.treams involves riffles. falls,
pools. etc.. and a unidirectional
flow pat~ern. Lake collection may
involve 'less predictable strati-
fication or flow patterns.
Definite objectivel should be eltablished
in advance as to the size range of
organilms to be collected and counted.
i. e.: micr03copic only. microscopic
and macroscopic. those retained by
"30 me.h" 'C1'e.".. invertebrates and I
or vertebratel, etc.
Certain standard supplementa ry
pro~edure8 aI'e a part of all field
techniques. In order to be interpreted
and u.ed. every collection must be
associated with a record of environ-
mental condi1ion. at the time of
Data recorded should include the
following 8S fAr as practicable.
Location (name of river, lake. etc,)
28- 1

JDoJoilcal Field Methodl
Station number (particular locIU:on
of which a full d.,crlptlon Ihould
be on record)
Date and hour
Air te.,eraturl
Water temperature (at varioul
depth, if appUc8h1e I
Salinity (at varlOUl depthl. if

Tidal t10w (ebb ~ flood)
Turbicltty (orU- pe8ltraUon. etc.)
Wind direction and veJoc1ty
Sky or cloud cover
Water oolor
r,pe of bottom
T"e oi coUeot&Dc device and
Method of colll«inJ
Type of 8ample «I-.aUtatlve or
Number of lample. at each .tatton
Chemical and pt!y.lcal data. e.,..
dillolved oXYfIII1, nutrlent.. pH.
etc. '
Collector'. ..me
Mi.ceUaneoul ob."rvatioh.- Cotten
very important)
All ool1ecUnl containers 8hould be
identified at 1nat with location,
station number, lample nmober.
and date. Sparel are very handy.
- Mlach tran.crlpUon of data can be
el1m1n8ted by ullnglheet. or carda
with a uniform arranlement fOr
includinl the above dlta. The
.me field data 8heei~incI1lde
field or JaboratQI'Y'analY'8i8.
Compact kitl of field-oo~ctinc.equip-
meAt and mater1&11 ir"~ in-c"...e
ooU8d1a& .mol.ncf. ..,.c1a»r if
coUeoUOn site 1. remote_..om.
Direct or indirect oolhlM/8.tionof Uilder-
water condition8 hubecome relatiVely
DtYiI14J spherel, pioneered by
William Beebe, eousteau, Honot.
Wi11m, and Manad at'e proving
very important for deep water
U.. of the aquaIUDfl,}lermits'dtrect
,.psanal .tudy do'wI'J >to Over
200 IMt.
Underwater te1e-..~ion (introduced
by the Bl'iti.h Admiralty for
mi1ttary'purpoe..)-tl now generally
available for biololical and other
Underwater 'phOtof~hy .1s
improvln8 in ftU&~,,''''d -factillty .
Underwater eW1rnmiag or use of
SCUBA ie quite valuable .for direct
ob.ervation and oollectlna.
Biological Fje14Methoda
tfoMd ..

Biolqlica~ Field MtU'lood8
8urber Sa...l...
AprOD Det
S,.ct_n or
.tJ ....... --
.If- 4

Biologi~ Fielp Methods
water i. a fundamental and much
u.ed ~bod for quickly "'''yin.
what i. pr..ent and what may be
expected an further uarch.
Patch.. ot ..weed and eelgra.s
and shallow weedy marlins any-
where are u....1J;y studied on a
qualitative ba.i. only.
The apron net is one of the best
toot. tor animals in weed bed.
or othe.' heavy vegetation. It
i. e...ntia1J;y a pointed wire
liev. on a lon, handle with
coar.e screening over the top
to keep out laavea and sticks.
Grapple hooks or a rake may
be u.ed to pull masses of
ve..tatton out on the bank
where the tauna may be
examined and collected a. they
crawl out.
Quantitdive e.timates of both
plants I4nd animal. can be made
with a "stove pipe" s.mpler
which i. forced down through
a weed mas. in shallow water
and embedded in the bottom.
Entire content. can then be
bailed out into a .ieve and
A frame of known dimenaions
may b. placed over an area to
be .ampled and the material
within cropped out. This is
especialq good for larger
plant. and lar,. bivalves.
This method yields quantitative
Sand and mud flat. in estuari.s and
.hallow lakes may be eampled
quantitatively by marking off a
desired are.. and el.ther di,ging
away .urrounding material or
excavattni the desired material
to a measured depth. Handle-
operated ...mpler. recently
developed b;r Jackson and
Larrimore. make for more
effective aampling of a variety
of bottom. clown to the depth of
the handles. Such samples are
then _shed through graded
acreens to r..trieve the organisms.
Ekman ,lrabs are most useful on
soft bottoms. This 1s a completely
closing clamshell type grab with
apr1n1 operated jaws. Size of grab
is usually 6" X6" or 9" X 9", the
12" X 12" size is impractical due
to its hellVY weight when filled with
bottom matel'ial.
For use in shallow water, it is
convenient to rig an Ekman with
a handle and a hand operated jaw-
release mt!chanism.
The Petersen type grab (described
below) without weights will take
satisfactory samples in firm muds,
but tend., to bury itself in very
soft bottoms. It is seldom used in
shallow water except as noted
below .
Collecting in i"reshwater Riffles or
The riffle is one of the most
saUsfactory habitats for comparing
stream conditions at different
The hand screen is the simplest
and easiest device to uBe in this
situation. Resulting collections
are qualitative only.
In use the screen is firmly
planted in the stream bed.
Upstream bottom is thoroughly
disturbed with the feet, or
worked over by hand by
another person. Organisms
dislodged are carried down
into the screen.
Screen is then lifted and
dumped into sorting tray or
collecting jar.

~lol1cal FM¥ KethoCl
The w.U-known ~re foot Surber
.amplel'l. one Jf the b..t quan-
maUve coUect1llC dflvlc.. for
It oon.I81:1 ()f a frame on. foot
.quare with a coa.tcal net
attached. It il uaDle onJ.y in
~o-rinl wat.r.
Its U8e it i. firmly pJ8nted on
the bottom. The bottom atonea
and iravel "lthin the Iquare
frame are thfln \3&r.tlally iOfte
ov.r by haM to enlur. that aU
or.nlame ha 7e be.n dillodfed
and carried b:y the current into
the net. A 8tW veietaDIe
bNlh 1a often uietulin thil
From three to five Iquare-foot
amplea Ihould be taken at elch
.tatlon to inlure that a realon-
able pereenta,e ()f the epeei..
p,.e.ent wiU be t8pr..ented.

Th. Petersen t)'P8 ...'ab may be uled
in dee, swift rUl1e. or where the
Surber la unlultable.
It il planted by hand on the
bottom. and worked down into
the bottom w~th the feet.
It il then c10led and lifted by
puUing on tbe rope in the \llual
1ft&Mer. '
A Itrolll medium w\tiaht dipnet il
the clo.est app1'elllch to a univeraal
eoUec:tlng tool.

a aw..pln. W..d beds and Stream
Thll ls u.ed with a Bweepln, motlon,
thto\aah we.4I, OWl' the bottoml or
in open wat8l'. A triaftJUlar IIbape
11 preferred by some.

b Stop net or KIcking Tec:hnl.que

Thil may be uaed a8 a roUP17 quan-
tit.tive deviCtl tn rifflel b)' boldine the
end fiat acab>et the bottom and
backina .1owq .,..wtream
diaturbln. the "atrate with
one'l teet. A ..ndard period
of time 1. u....
'lbe haadl. uboldd be from 4
- W 6 f.et lcftc. Ulltab08t the
weiabt of a ....eMf rake
handle. :
'lbe riq .ht-uldoe made of
-ateel or Iprift. brass. and
"CW'e~ futeDed to the
handle. It Ihoald be strong
but not c1UDber80me; .be of
. riq 8tock will clitpend on
diameter of ..iq.
The ba, or net mould be the
8trOnp8t available. not oyer
1/8 inch me.h, preferably
about 1/18 IAch. Avoid 30 or
more melhel to the inch; thtl
il so fine that the net plugs too
eal117 and il Ilow and heavy
to handle.
There mould be a wlde can...
apron lewed around the rim
and prot8Ct1q the ba,. The
r1m 1ft&y be pl'otected wlth
leather U d..lred.
Deep Water Benthic Collecting Plate UI
When eampUDI from vellel.. a
cnne and winch. eitber hand or
powu operated, i. aed. The
'8D8l'&l ldeal deacr1bed for .hallow
wa'terl &PIIl)' a180 to deeper watel'8,
when practicable.
The Petereen type lrab. aeems to
b. the b.lt all around. _..pIer for
the lI'8&t..t variet)' of bottoms at
aU'depthl, fror4 .bareline down to
OV8t' 10, 000 meterl. (Plate I)
It coee&at. of two h_vil)'
Biolol1cal Field Methods
Slolo,leal dred,e
Otter trawl
021"- 7

...J J'~k! J4et~
To ~ble them to bU. Into
hard bottom., or to be \I,ed. in
'trong current., wei..,t. may
be attached to brift, the total
weiaht up to between 50 and
100 lb..
Area. lampled ranlel~m
1/5th to l/lOth .quare met.r.
(1/10 Iquare oet.r....18
approximately 1.1 .quare ft.)
A Peteraen ,rab to be hauled
by hand ehould b~ fitted wtth
5/8 or 3/4 inch diameter twi.ted
rope in order to provide
adequate hand grip. It i. be.t
handled by mean. of wire rope.
and a wipeb.:
Other bottom aamplere include the
VanV.., Lee, Holme, Smith-
Melnt,...., Knu~ Ponar. and
A .prin.loaded _mp18r hu
reC8Dt~ been developed by Shipek
for ule on all type. 1If bottom.. -
It tak.. a half-cyUlldel' "'mple,
1/25th Iquare meterain area aed
approximately 4 inch.. deep at the
center. The device i. .\ltomaticaUy
tr1l1ered on contact with tbe
bottom, and the .ample i. com-
pletely protected enrOllte to the
lurfaoe. (Plate I)
Drag dredge I or .crrpel are otten
uled in marine -tel'. and deeper
lake. and Itream., 81~ .FOmpr1.e
the bta.ic equipm8Pt ~.ral typ..
of commercial ftllheri... Some
type. have been developed for
lhallow .treaml. In ,.neral
however, they have been little u.ed
in fre.h water.
The above il only a ~azot1al lilting
of the many lampling device.
avaU..ltle. Othet'. that are otten
encountered are the oranp.p.el
bucket, plow dredg., ilCaUop type
dredge, hydraulic dreclte., and
var1ou. coring devicel. Each 11&.
it. own adftnta,e8 and di.-
a_ntage. and U il up to the
wornI' and hi. open.t1cin to decide
wbat i. belt for hi. part1cu]ar needl.
The Peter.en type and Ekman grabs .
are perhap. the mo.t cOftilatonly
Trep. of many types are u.ed tor
varioul benthic or.lti.m.,
..peclal~ crabl lilad lobeterl.
Artificial eubltrat.. (belOW) are ~tn
.e.ence a t)'pe of trap.
Since mOlt biological cbmmuniti..
are not w.nly di.trl&uted,it il
adv1.lable to routine1y"take' at lieaet
two and preferably morel&mp1e.
from anyone station.
Artificial lubltrates re~;on the
ecolo,ict.l predilection' OfO~pm.ltnl
to ,row wherever they' rind a .uitable
habita,t. When a small pOl'Utift of
artificial habhat il provided, It tEinds
'to become popllated by aU available
8P8~ie. .,.rtial to that type of .ituation.
. The coUectOr- Gan ther. at wUlremove
ttse hAbitat or trap tb hill labOratory'and
Itudy the population'a\ leisure.'
Thl. vel'_till rele8:rch techllt.e is
much \lBed for both r01Jtiner niOftttorinC
and ftploratory Itudies or pollUtion.
It i. a180 exploited c6m:mercia1ly,
e.pec:ially for i.hellfi.h prodUction.
Type. or materials ulied'1riclude:
Cement plates and pane18.
Wood (eilpeciany ror~~i
form.). .:
Gl... .u@. tex: Catfte't"w'Ood
Multiple plate 'trap (tNtliMlte).
BUkets(or other (iontainerli) hdlcliftg
natural bottom mateMal and eitb:er
imbedded in the't)oftom, or sus-
pended in the overlying""."er.

Unadoraed rope. 1U8~ded In the
water. 01" .Uck. thru" into the
F Sorting and Preservation c:t eollection.
Benthic collection. u.ually consist
of a 'I'.t ma.s 01 mud and other
debri. amoR,.hieh the organism.
are hiduen. VariCft1s procedures
may be lollowed to separate the
TIle orpnism. may be picked
out on the spot by hand or the
enUre mes. taken into the
laboratory .here it can be
examined mOre efficiently
(especially In roullh weather).
I\OUfh1y equivalent time will
probably be required In either
Specimens may be .imply
obseMl.ad and recorded or they
may boe prenrved u a
permanent record.
Organisms may be simply
counted. wei,hed. or measured
volumetricaU;y; or they may be
.eparated and recorded In
groups or speeles.
If separation is in the field, this is
usually 10ne by hand picking.
.creenin" or some type of flotation
Hand pickin, il beat done on a
white I_roeled tray usln,
11lht touch llmnolo,ical forceps.
Screenin, is one 01 the mOlt
practical methods to eeparate
or.ni.ms from dabril in the
I£eld. Some prefer to use a
single fine screen. others
prefer a lerle. of 2 or 3
Icreens of graded lizes. The
conect!.on may be dumped
directly on the Icreen and the
mud and debris washed throullh.
8101oakalFieid M.~ds
or it may be dumped into a
buck.t or small tub. Water
is then added. the mixture i.
weil stirred. and the super-
natant ~oured through the
ecreen. The residue is then
ex.amiJIed for heavy forms that
win not float up.
A variation of thia method In
situation8 where there is no
murl II to pour a strong sugar
or salt solution over the
collection in the bucket. stir
it well. and again pour the
supernatant through the screen.
Th!.s time, bowever, saving
the flotntion solution for
re-use. The heavier-than-
water solution accentuates the
separation 01 organisms from
the debris (except for the
heavy shelled molluscs, etc.).
A oolut1011 of 2-1/2 lbs. of
sugar per gallon of water Is
ocmsidered to be optimum.
Preservation or 8tabilization is
usually lIece.Bary in the field.
95% etMnol (ethyl alcohol) is
hi(lhly satisfactory. A final
8trength of 7O'Y. Is necessary
for prolonged storage. If the
C.o1l8cl1on Is drained of water
and flooded with 95% ethanol
in ~he field, a laboratory
flotation separa tion can usually
be made later. thus saving
much time. COIUJiderable
que.ntitl.e. of ethanol are
required lor thll1 procedure.
Formaldehyde is more widely
available and Is effective in
concentrations of 3 10% of
the commercial formulation.
However, it shrinks and
hardenl epecimens, collector,
and laboratory analyst without
favor! In order to minimi ze
bad effectll from formalin.
neutralized formalin is

1K0101f.ca1 J'181d Methocif
recommend.d. Mollusc .hens
wID eventuaUy dietat.,rate in
acid formaUn
Properly preserved benthos
Mmplel may be r.tained
iftdelinitely, thereby enhancing
thlir util1t7.
Aefri,eraUOI'I or tcine ts very
A Thi. i. a relatively new area which
promiae. to be of l1'.at importance.
Tbe microfauna of mud and .and
bottome may be studied to .ome extent
Irom collections made with the varlou.
d8v1ce. mentioned above. In moet
ca.ee however, there 11 c0n8iderable
1,.. of the .maner lorm..
8 Moet .pec1al microfauna Amplere lor
aoft bottom. are e..enUally modified
corti ..mpler. in which an elfort i.
made to brin, up an ,'n~urbed portion
01 the bottom alon, with the tmm.cUately
overly1ft, water. The b..t type currently
seem. to be the Enequt.t ..mpler which
w.tibs some 35 k.. IIIld take. a 100 eq.
om Ample 50 cm. deep.
C Microfauna from the eurface 01 hard
.and or gravel bottom. may be Ampled
by the Hunt vacuum .mfoler. Thi. has
a ben-.haped "sampl1n,' tube ...led
by ,lu. diaphrapa: On contact with
the bottom, the g1&.. i. automatically
broken and the n....1.1 bottom material
18 ....Ipt up into a trap.
D Periptlyton attached to or a..ociated
with hard 8\1rIIC88 nch ae rock or
wood may be .ampled by .crapln, or
otherwi.e removtnc an lurface
materiallrom a meaeured ar_. The
perlphytOft, however, is more effectively
quantitaUyely sampled by artificial
eub.trate technique. described above.
Phytoplankton: A Planned Program I.
A plamed pro,ram of plankton
-.ly.te "hould. Involve periodic-
amplin, at we.Jcq or even more
h'.quent ifttervau..

A wen-planned 5tUcly or analy.is
01 the growth pattern Of plsnkton
in one '_I' will provide a ba.t.
for preclietini conditions the
loUowini :r_r .ince le880nal
,rowth pattern. tend to repeat
them"el...,. lrom '.':1" to year.
S~e the ....ons and the Yi!!ar.
di(f.r, recorda accumulated
over the y_re become more
A. the time lor an anticipated
bloom of some troublesome
epecl... approach.e, the
frequency of analyse. may be
Detection of a bloom In it. _rly
eta,8I wW facilitate more
economical control.
Field A.pect. of the Analy.ie. Program

Two lenaral .apeOw 01 plsnlttOb
analy.t. are cOtnmonly -recognized:
quantitative and qualitative.
Qualitative examb.t1on teu.
!:!!!! i. preAnt.
Quantitative te11.. how muc".
Eitner approach 18 1.1."1.11, a
combination 18 bellt.
Equip.....t for collecting 8&mpifl
in the fi.ld I. varied.
A half-liter bottle Will "serve
for .urfaae sample. Of
phytoplankton, if oarefuHy

Biological Fiel~ Methods-
Wisconsin net
.:zJ - 11

Bioloiical Field Methoda
A Kemmere!, Nansen, or
other special sampler (amall
battery operated pumpa are
time !laving) is sugielted for
depth sample8.
Plankton netr concentrate the
sample in the ad of collecting
and also capt.,jre certain larger
forms which eac..pe from the
bottles. Only the more
elaborate types are quantitative
however. For phytoplankton,
'30 or U5 a:ze neta are
co.-monly ueed. Uaually a net
di.rneter of 5 - 10 inche. ia
autttcient. The smaller forma
however, are 108t through any
C Zooplankton CollectL'1g
Since zooplankton have the ability
to swim away from water bottles,
etc. nets towed at moderately faat
speed are used for their capture.
Number 12 nets (1perature
8ize 0.119 mm, 125 meshes 1 inch)
or Imaller numbered net alzes are
commonly used. A net diameter
great..r than 5" 18 preferred.
f'requently half meler nets or
larger are employee. These may
be equipped with flow measuring
devicel Cor mea.urinll the amount
of water enterin, the net.
Other instruments such &s the
Clark-Bumpus. Gull-Stream,
Hardy continuous p,-nkton recorder,
and high-speed In,,trument8 are
used for collecting zooplankton, alia.
The devices used for collecting
plankton capture both the plant and
animal type.. The muh .Ize
(net no. , is a method for .electing
which category of p~nkton III to be
D The Location of SampUng Points
Both shall;:;" and deep samples are
"Shallow" samples 8hould be
taken at a d~pth of 8 inches to
one foot. Tha lurface film is
often 8ign1li,=*nt.
"Daep" samples should be
taken at such intervals
between lurface and bottom
as circumstance. dictate.
In general, the entire water
column should be sampled as
completely "s practicable,
and the plankton from each
level recorded .eparately.
For estuarine planktoo, it II
necessary to sample different
period. in the stage of the tide,
otherwise samJ:les would be biased
to a given time, or type of water
carried by the tidal currel1ts.
Plankton is subjected to the force
of the winds and currents. As a
result, the plankton is often in
patches or "willd rows" (Langmuir
cellI). For this reason when using
a net, it Is often desirable to tow
the net at right angles to the wind or
Nearly all plankton are horizontalJiy
discontinuoua. Planktonic organisms
tend to be numeroUS rtear the bottom
in daylight, but distributed more
evenly throuah the water column at
nieht. Ther8f(lre, a aerie a of towa
or lamples at different depths 18
necessary to obtain a complete
lampling. One technique often em-
ployed il to take an oblique tow
from the bottom to the tap of the
water oolumn,
PUot ltudies to indicate .ampling
locationl and intervals are often
mandatory. Some studies requir.e
random 8ampling points.

Tha number of sampling station..
that should be estabilahed is limited
by the capability of the laboratoJ"Y.tp
analyze the samples, but should
approach the needs of the object~.
&8 c1o..ly as possible.

Field conditions greatly affect the
plankton. and a record thereof
should be carefully identified with
the collection al in II above.
Provilions Ihould be made for the
field ltl.b1l1~tion of the ample
until tbt .bONtel")' exe.mtnaUon
can be mad. it ftIOre1han an hour
or 80 1. to .lap...
Refri,.rati. or icbtC il vel")'
helpful, but to. 8hould Diver
be placed ~ the Itmple;
Preservation by 5"!o formalin 1s
widely u8ed but badly shrinks
animals and makee all forms
brlttle 0
LulOls solution 1s a good
Ultra.violet eterilization i.
sometimes used in the laboratory
to retard the decompo.ition 01
A highly utisfactory merthlolate
pre.ervative has been de.cribed
by Weber (1968).
A Fish and other nekton must be lought in
the ob8cure and unlikely area. a. well
as the obvioul locationl in order for the
collection to be complete. Several
techniquel Ihould be employed where-
ever pouible (thil i. appropriate for
'all biota). It if'l advllable to check with
local authorities to inform them of the
reasons for lllI.mplini. becaule many of
the techniques are not legal for the
layman. In this area. perhapi more
than any other, profellionally trained
workers are important. Al.o, there
mu.t be at least one helper, as a lingle
individual alway. has difficulty in pulling
both ends of a 20 foot seine limultaneoully!
Biolopcal Field Method.
The more comnlon techniques are
Ulted below.
Straipt .eine. range from 4-6 feet
and upward8 in length. "Common
sense" minrlow seines with approxi-
mate~ l/~ Inch mesh are w1dely
u.l'd along Ihore for collecting the
smaller Iishe..
Baa 8eine. have an extra trap or
bag tied in the middle which helps
trap and holo:! fish when seining in
difficult situations.
GUI net. are of use in offshore and/or
deep "Waters. They range in length
from apprOlldmately 30 yards upward.
A me.h Ilze 18 designed to catch a
splcit1ed lize of fish. The trammel
net" a variation of the g111 net.
Trt&~ raftge from small wire boxes or
cy Ir. with inverted cone entrances
to semi-permanent weirs a half mile or
more in lenjfth. A 11 tend to induce fish
to .Wim intI) an inner chamber pro-
tected by an inverted cone or V - shaped
notch to prevent escape. Current
operated rotating fish traps are also
very effective (and equally illegal) in
suitable sUlIation8.
Trawls are 8ubmarine nets, usually of
COiii'iC!erabJe size. towed by vessels at
speeds sufficient to overtake and scoop
bl fish, etc. The mouth of the net must
be held open by some device such as a
long beam (beam trawl) or two or more
vanel or "otter boards" (otter trawl).
Beam and otter trawls are usually
fished on the bottom, but otter
trawl8 when suitably rigged are
now being used to fish mid-depths.
The midwo.ter trawl resembles a
huge p16..nkton net many feet in
diameter. It is proving very effec-
tive for collecting at mid-depths.

1 Field Metr,o~___-- ---
Bioloi1c~ --
...~(,.._- ..~:J~
- -~.~\" '-f~~'
. .~::~ ~~SS:i{~:~~: .
.;;, - 1 4

controlled door8



"""1 PW.t~&I.@U
Num...... .,.eta1, ..apa haft Men
dev.~. ~. VI
'I' Electric ...e. all4 .er"l18 are wtdeJr
elllltloyed'"'ij"'H8MI'1 worur. in .mall
anti. difftCNlt .trlam.. They mal a1.lo
be u.ed fD 1ha:UOW water UIIe ar.. with
ce_in re88l'Yat1ODI.
Q ~ ill much uaed in ftahel'1 8t8diM
~'e1'hlnt. Mo,t wtd817 ...ect and
II...ralq .tt.f&ct01'1 il rot...one ill
varyiDi formu:Jat1an8, ahhoup ID&IIi;Y
otben have been employed from time to
time, and 80me appear to be ..~ pd.
Under IlUitahle circum.taAce., ""'"y
Iven be IdUed aelecttveq accorila,~
H P...~1 ob.ervat1on bf ~
p...aonne1, and .180 1nfo1'1ll&1 ~el
and di.cua.tona with lacal r."-'"
wW often yield information of ...ll1le.
Many laymen arl, ke'" ob8ervar.,
althoup tbey do Dot .bra,.. lIader.and
what they are .eelal. ',The or.-zat.ed
c,.l cenall. techot... ytelda data on
what and how many n... are b_.
I ~lMYl remaina in ita own r1lht a very
~hnique in the hand. 01 t~ 8IdUed
p(l&cUtioner, for deiermiJUDI what n.h
...e preMllt. Spear-ft.tdft8.18o t. now
b.inlua.d in aome Rudie..
J F,tah and other nlktcm al'l often taald
to tr'&ce their movementl dlariD.
..i,ration and at other tilfte., MIII&a-
tere rad10 tran.mltter. can now be
attached or fed to 111b (and otber
or,.ni.ml) which enable them to be
tracked over con.iden1:J1e dl8taftoe.'.
Phyaiolopcal informatiOft 181 oft...
~tained ill thia way. Thi. i. known ..
telemetry ,
Handling biological ool1ect1ona (a. COII-
trained to chemical and phyaical aamp11nc)
on board boats differ. with the lize of the
craft and the- magn1tudeof operatlona.
Some ,..,..ala 1\8m. are ll8ted beklw.
HoiItSq &Ad mIUS7 other type. ~ .81' are
1A8ed in COIIUIIGI\ with otIwr type. 01
"""011/' aad w01 not be :Listed.
Special IAboioatory R~(.)

ConlltUlt now of <;:l"!i 1II&t.r fOr
e'!IMlI1'i111 or...la,.....(Se1ectton of
IIJ8t....ta18 ..dde.ttn ()f a 1Iy8t-'m to
1ft...... non -toarf.e'Wtiter III8y be n1"Y
tro""" tNt Wry bnJ'OI'taftt, )
UN 80K bUilt taU! .hlp'at water lenl
R.fripnitlaa 9ptem(8)
~or , tem~"of
, 1 orlMd*m* In
Ia '
For ~ aftd".orap'of
"""~be examined ]atft'.

Storap, -- '(Unrelri'(8r1ited)
'heattie. Nr the...., .torag._dul.
of m101"Oloapel and other ]aboftitory
Facilltie. 101' the .'e lito....,. and ue
of deck ""Ift.m~

Adlft1Dtatntl,.. ace... to the Captam
and TeGIIhd.l Leadlr in order to
cool'dtlate i'eqUir'eI8eftt8,for bIeiOIItial
con.adem (neb .. a 81WlrJliidlt()ri\tdW)
wi'" thCM8 for other' OOl18ett....,
Sal8tJ oIperaormel 11'0"-. 1ft arreJ'
U'08d'troat8/ .. weU,".IJr,oMiet' t1etd
actlvitt.. 8hlNid be H1"i".,'aon-
.Iared and promoted'at '.II,tbRit~
'ProdaCtl'Yft'y St1Hliu oll\len,y. ~
Life c:Ydeand Maaa,.mem

Dtltribut10n of Sport OJ' (~tlitfal1rJ~
Commlrcial Specie.

Scattering Layers and Other Submarine
Sound Studies
Artificial Culture of Marine Food Crops
Radioactive Uptake
Growth of Surlace-Fouling Organisms
Marine Borers
Dangerous Marine Organisms
Red Tide s
Many specialized ite:ns of biological
collecting equipm~nt 'ire not available
from the usual laboratory supply houses.
Consequent~. the American Society of
lLimnology and Oceanography has compiled
I l18t of companies handling such items
and released it as "Special Publication
No.1. Sources of Limnoligical and
Oceano,raphic Apparatus and Supplies. "
Available from the Secretary of the Society.
The hazards associated with work on or near
water require special consideration. Personnel
should not be a..igned to duty alone in boats.
and should be competent in the use of boating
equipment (courses lare offered by the U. S.
Coast Guard). Field training should a180 include
inltructions on the proper rigging and handling
of biological sampling gear.
Lite preservers(jacket type work vests) should
be wron at all times when on or near deep water.
Boats ahould have air-tight or foam-filled com-
partments for flotation and be equipped with
fire extinguishers. running lights. oars. and
anchor. The use ')f inflatable plastic or rubber
boata ia discouraged.

All boat trailera ahould have two rear running
and atop lights and turn signala and a license
Biological Field Methods
plate illuminator, Trailers 80 inches (wheel
to wheel) or more '.vide should be equipped with
amber marker lights on the front and rear of
the frame on both sides.
Laboratories should be provided with fire
extinguishers. fume hoods. and eye fountains.
Safety glasses should be worn when mixing
dangerous chemicals and preservatives.
A copy of the EP A Safety Manual is available
from the Office of Administration. Washington.
D. C. (Reference: 10)
1 Arnold. E.L.. Jr. and Gehringer. J.W.
High Speed Plankton Samplers.
U. S. Fish and Wildlife Spec. Sci.
Rept. Fish No. 88:1-6.
2 Barnes. H. (ed.). Symposium on New
Advances in Underwater Observations.
Brit. Assoc. Adv. Scl.. Liverpool.
pp. 49-64. 1953.
3 Hedgepeth. Joel W. Obtaining
Ecological Data in the Sea Chapter 4
in "Treatise on Marine Ecology and
Paleoecology" Mf\mois 67. Geol.
Soc. Am. 1063.
Isaacs. John D. and Columbus. O. D.
Oceanographic Instrumentation NCR
Div. Phys. Scl. Publ. 309. 233 pp.
5 Jackson. H. W. A Controlled Depth
Volumetric Bottom Sampler. Prog.
Fish Cult.. April. 1970.
6 Lagler. Karl F. Freshwater Fishery.
Biology, Wm. C. Brown Company.
Dubuque. 1956.
7 Standard Methods for the Examination
of Water and Wilstewater. APHA,
AWWA, WPFC. Publ. by Am. Pub.
Health Assoc. New York.
28- 17

BioloFcal Field Method8
8 Sverdrup, H. U. et al. Ob8ervat1on8
and,collections at !lea. Chapter X
in: The Oceans, Their Physics,
Chemi8try. and Biology. Prentice-
Ha11, Inc.. New York. 1087 pp. 1942.
U.iaeer. R. L. Aquatic ln8ecte of
CaUfornia (Section on Field Method.).
University of California Pre...
Berkeley. 1956.

10 Weber, C.I. Biolopcal Fteld and Lab
Methods for Measurin, the Quality of
Surface Waters and Effluent.. U. S.
Environmental Prote~ion Aiency. Nat-
ional Environmental RfI!learch Ctr.,
Cincinnati, OH Environmental Monitoring
Series 670/4-73-001. Juq, 1975.
11 We.lch, Paw S. LimnoJ.olical Methods.
The Blaki.ton Compar~. Philadelphia.
Penn.ylvan1a. 1948.
12 FWPCA, Investtptlna Ft.h Mortalities.
USDI, No. CWT-6, 1970. U.S. Gov't.
Ptint. Off. 19700-380-257
This outline was prepaNc! by H. W. Jacuon,
Former Chief Btologt.t, Na~lonal Tra.ina
Center, OfficI:: of Water Pro,ram., EPA,
Cinctanati, OH 45268, and revt"d by
Ralph M. Sinclair, Aquatic 81010Jist,
NatioJl&l Training Center.

De!lcx:tptor~ :
AquaUc Environment, Analytical Techniques.
28- 18

A The report of the Council on Environ-
mental Quality (1970) repeated~ stresses
the need for the development of predictive,
simulative, and mmagerial capabilities
to combat air anel water pollution. The
lalt capa.bility depends on the first two.
8 The standard static jar fish bioassay,
which uses death ::\s a response, enables
one to predict the toxicity of a particular
waste to fish. One limitation of this
procedure is that it uses a grab sample
which represents the quality of the waste
at only one point in time. The water
u~ed to make the dilJ.t1ons is also taken
at one point in time. At the actual
industrial site, tht' quality oC the waste
and the river water vary through time.
A composite waste sample partially
overcomes this limitation. but may mask
variations t.hat are biologically important.
C One could p11t fish in a continuou'l flow oC
:waste diluted with river wate:-, but then
there is one further limita~ion oC the
standard bioassay: death is used as the
response. In order to prevent damage
to organisms, it illl r..p.cessary to have an
early warning of danferous ,~onditions,
so that corrective action can be taken.
In other words, sym}Jtoms of ill health,
which occur before death, must be detect-
ed if there is to be time for diagnosis and
,\ Fiah Movement Patt 3.. m. the next day to allow the fish
to recove, ove~J\ight from handlin~.
Toxic solutions are introduced at 10:00 a. m,
after the experimental fish hav'" been
exposed to water containing no addC'd
2fJ- 1

i>(!'i!cial ApiJUcations and P~t;~~!9!..I!!2'i'!I!!!t_---
to,dcant for periods of 0.1e to slx d.1ys.
Ea ~h experimen~al fish thus serves a;
its .)",n cOIl~rol. In addition, one or
two fish are n~ver e:ll'posed to the
toxicant and serve as controls through~
out each experiment. In one experiment.,
using zinc as the toxicant, reported
in Table VI, six control fisa were
e~posed to water containing no added
zl,nc for four d~ys.
a Prelimin.~ry l'xidence suggested that
the data could be analyzed by separa-
ting the experimental day into lour
periods; a period from 6:00 to 8:00 a. m.
when the breathing rates changed
marked1.y, a period from 9:00 a. m.
to :; :00 p. m. when the rates were
comparatively high, another period
o~ rapid~hange from 6:00 to 8:00 p. m.,
ani a night period from 9:00 p. m. to
5 ;00 a. m. when the rates were compar-
atively low (Sp.1.rks, e.! a!., 1970).
b B1u~gills increase their orea+hing
rates when .>xposed to zinc (Cairns,
et al., 1970). An individual fish was
£i1US considered ~o have shown a
r.'spanse each time Us breathing rate
during a time period exceeded the
nl.lxlml1ln ;neathtng rate observed
during the corresponding period 'Jf the
HI's'. day, befoce any zinc was added. A
respollse was scored tor each value
011 the second day that was higher than
the fir8t day maximu n tor the compar-
ahle period. The control periods
(b~fore any zinc was aided) and the
e,0: ..loJ.3 (aitel' zinc was added) do!ter-
mln..j .'lOW r;uickly the method of
a lalysls could jetect zinc concentrations
in water.
I.m.- concent.rations were determined
.Jaily by atomic ahso!'ption spectro-
A Fish Movement Patterns
1 Table 1 shows t.he rC8ultsIJf one
continuous flow experimcnt ca.~ried
out for 20 days. During this experiment
fish were exposed to zinc.on .y 7
from 1:00 p.m. until 7:00 p.m. at
which time &he flow was ret.urned to
normal dilutlon. water. The zinc
concentrations reached their maximum
at 7:00 p. m. and atomic ahsorpt.ion
analyses on effluent s::\mples \1Qlklcied
at this time showed the following
com:entraUons: ~ank One, 1.3.32.
tank two, less than 0.08; tank iju'eE\.
11.39; tank four, 12.72;.~ank.five. ++
13.32; and tank six. 12.59 mgil Zn .
The results show tt-at these cOlI..cu.tra.t1ons
of zinc developing over the six hour
interval of exposure.. were- inl~uffic.ient
to cause a detectable change inUle
movement patterns o~ the f!sh. ,By
8:30 a. m. 0: day 8 the efc.wellt zinc
concentrations were .les8 than :>..:N.in
a11 cases.
2 To determine the p~rcent survival a'1d
recovery patterns d the fish. oncestJ'ess
detection occurred, zinc flow.. was
reinitiated at 1 :00 p. m. on day 13 of
thls experiment. Between 8:0J,and
9:00 p. m. on day 13 the zinc concentration
in the effluent reached a ma.1C1m.umoI:
7.51 for taRk one; less than .0,,05. for
tSl'lk two; 7.49 for. tank, :thre.e; 7. 112 (91'
tar.k fOUl'; 7.49 for tan1t C1ve~,and 7.~4.mg/l
for tank six. The concc\1tz:at.1o,ns 1!e.me.1.ned
n ear the above values 'JIltil ~e, statis~ical
analyses showed "stress detect.iQn"
du~ing the first !'laIr nigl1t V'~lues .oll-d~y
14 (Tab~.~ 1). As soon a'l.,~ress dete~
o;:cuI'red fJIe flow was !etu::-ned. to. noriQlllil
dilution water. At 10:00,a.m..~m.:h.y.15
zinc a'1a~ses showed ;lU dnu~Jlt ~~a-
tions to be less tnan J. 70 m&/l' ~++.
S~ress d,~t.ectioncontinJ.1e.d. to, be I'egi.s~~d
for two consecutive time, int.erv.a18 "(QUowing
the initial detectlpp., . bu~ a. "tel' thl;l.tn::l, str~ss
detection wa; regist.ered Nad:.t,be.fr..e,q,uenr;y
of abnormal patterns re~rned t();p!'"tr~8
levels within .i8 hoa~8. In tt)i..eX~nt

- __~~!!l.&PJ:!!!cations and Pr0s.ed'.1res for Bioassay-
as 'Nith all ot~t"8 in which dilution
water contafn1n. zinc was replaced with
dilution water minus z1.nc at that time
01 ,tress detection !1-!!ish survived!
3 The results rrom the seriea of experiments
at progressively lower zinc concentrations
indicate that the lowest detectable con-
centration is between 3.65 (Table II) and
2.. 93 mgll zinc (Table III) for a ~6-hour
B Fish Breat.hing
1 Table IV shows the 'Jreathing rates of
five fish on days 1, 2, and 7 of experiment
8. The first four fish were e:
~c;.~-~{I!.LclfJ~a!l.!f.F!,c.dqr.. ror -~as".Y
s\1nIish can b." uied to detect sublethal
CO:1.,~p.l1trat1o'1s reathing rate or activity dllt'ing one
time pcdod.
B In chQosing a specific criterion tor
detection, the risk of no~ detecting stressful
condiiions Boon '~nnug~1 mUAt be weighed
against the risk of (alse d~tections, a.':ld
the ::holce would probably b." determined
"1 th? nature of th~ p()llutant. If a ponutant
1s easily detected by the bl,olOgical monitoring
system, 1s slow-ading, and If the toxic
effects are reversible, then the criterio':l
fOJ' de~ectioll migl1t b~ responses by 3/4
0: the le"t fish, t<> avoid th" false detections
that would necessitate expensive remedial
a~t1ol'1 or a tempo,~ary shut-down. On the
oth~r hanrl, an in j 18try that produced an
effluent containinJ a fast-acting toxicant
whose effects are irreversible would
probal:>ly use a criterion that lead. to rapid
detection (rupol1s'!s 1:>y 1/4 to 1/2 of the
test fish), and would have to go to th."
e:(p':m8e af installing holding p-:mdA 0:-
reeycUn~ facilitiea to 1ccommodate a
relatively high n 1mber of false detectio.1s.
Alte~n,],tively, a safety factor could be
introdu:ed ~y metering proportionally
mo:"e wast~ into ~hp. dUutiol' wat.er delivered
to the teat fish than is :ieU,-ered to the
stream. Th!! safety factor could he
ddermim,-:J by growth i\1'I.1 reproduction
exp~riment. with fish.
C fn a 1 'lctualin~Jqtrial situation water and
waste q1J.111t1es an apt to 'lary u:1predlctably,
al'ld it would ::ertainly be desirlLble to l'Bve
a redll'1 hnt detedion system. It is conceiv-
a1)ll' ~h~t sn;ne harmiul combination of
t-I1Vil'o.1ment11 c-o.1diUo\1" and waste qU.llity
w0\11'J he detected b:l mo:u'oring on~ biological
funcHo,I, but not by mO':lttorinl another.
It 1" also pos,sible that excesstve turbidity
wouit: ,1:,8'"U&,t th" light beams of the movement
m >"itn . and not affe::t th~ breathing monitor;
0: th'i! ai' ~xe",'sive c()n::ell~ratio':l 0: electro-
lytes "muld ;..1 :f,,:t the dectrodea of the breath-
in.., munitor, b,t' n"t affed the a<:tivity monitor.
There:Ol'e, the a:tivity mon~toL' an:! the
bn'4thing mC'nit<>r h:we been combined :n our
labn".,t"rv in' fu"~h"r e:
-------- .-- ..... ._--~~ta.!.~.£.aJ!2!!U:.'tlir~u!!!.!~1!!.<>~.sl'Y..
G It is likely that "fish sensorll" in
continuous monitorlng units at industrial
.ites can warn of d.!veloping toxic conditions
in time to forestall acute damage to the
nsh populations b s~reams. In conjunction
with stream ww.ter quality standards for
chronic exposure, s1Jch biological monitoring
systems should make it possible for healthy
nsh population.s to co-exist with ind11strial
water us~.
This research was s'Jpported by grants 18050 EDP
and 18050 EDQ front the Water Quality Office, 10
Environmental Protection Agen::y.
Literatul'e Ci.ted
1 Bru11gs, W. A. 19GO. Chronic toxicity of
zinc to the fathead minnow, .!1~e'phale~
R,,~q!!l&l~ Rannesque. Trans. ArneI'.
Fish. Soc. 98(2):272-279.
2 Cairns, J., K. L. Dick:'lon, R. E. Sparks,
and W. T. Waner. 1970. A preliminary
report 011 rapid b~ological information
aystems fOl' water pollution control. Jour.
Water Poll. Contr. Fed. 42(5):685-703.
3 Eaton, S. G. 1970. Chronic malathion
toxicity to the bluegill (~Y.~T~ ~~~~~'!
Rafinesque). Water Ressarch. 4:673-63.1.
4 McKim, ,T. M., and D. A. Benoit. 1971.
Effects of long-term exposures to cooper
on survival, growth, and ,'eproductlon
of brook trout (Salvel1nus fontinal1s)
J. Fish. Res. Bd7Cana-da 28:655-'662.
5 Moor'~, J. G., Jr., CommissIoner. 1968.
~!!!: ~ 9'!~~ Report of the
National Teclmi-:al Advisory Committee
to ~he S~cretar~' of the Interior. U. S.
Govt. Print1ng Office. 234 pp.
6 Mount, D. I. 1968. Chronic toxicity of
cooper to fathea::! minn.)ws (~ephales
fr~~ Rafim'iique). Water Research.
7 Mount, D. I. and C. E. S~ephan. 1967.
A method for establishing ao::ceptable
toxican~ limits for fish-- Malathion
and the butoxyetha.'101 ester of 2, 4-D.
Trans. Amer. Fish. Soc. 96(2):185-193.
8 80kal, R. R. and F. J. RO:1lf. 1969.
~~~~. W. H. Freeman and Co.
776 pp.
9 Sparks. R. E., W. T. Waller, J. Cairns, Jr.
and A. G. Heath. 1970. Diurnal variation
in the behavior and physiology of blueg1lls
(!:-~1!1}~ ~S!"~~~~ Raf1nesqueJ.
The ASB Bull. 17(3):90 (Abstract).
Sprague, J. B. 1969. Measurement of
pollutant toxicity to fish I. Bioa:n:lY
methods for a ~ute toxicity. Wate::-
Ref!earch. 3:793-821.
11 Cairns, John Jr.; Sparks, R. E.; and
Waller. W. T. "A tentative proposal
for a rapid in-plant biological moni-
toring system. " in Biological Methods
for the Assessment of Water Quality.
ASTMSTP 526. American Society
for Test. a.'1d MaL, 1973. pp. 127-147.
ThiBO~as-p7epar-ed by John -cairn.s-:-J~-.
and Richurd E. SparkEs, Center for En'/iron-
mental Studies a"li Departmen~ of Biology,
Virginia Polytechnic Institute ann State
University, Blacl(sburg, VA 24061
Environmental Control, Bioassay, Toxicity,
Fish Behavior, Fisll, Monitoring

Fi~e 1.
Test chambE!r .ror monitorin~ syste~, showing t.he electrodes for
. . " ,jlI . . ,," . -" ...,
recording fish breathing ahd the light-beam system .ror recording
fish movement.




n '


Figure 3.





Arrangement of fish monitoring units at an industrial site.

--.-. ~ -- ------------- ~~!!.!e£~!!1.2!1!'.~.2 .E!s:2~9~.l.o~ssay
Figure 4.
Detail of a single fish monitoring unit. showing how the
experimental fish are exposed to waste diluter! with upstream
water and the control fish are exposed to upstream water alone.


A AfI1 organism encoWltered in a survey is
of .ianiftcance. Our problem is thus not
to determine which are of signtricance
but rather to decide "what is the signifi-
cance of each? "
B The ftrst step in interpretation is
recoanition. "The ftrst exercise in
ecoloi)' is systematic.. "
C Reco8l1ition implies identtrication and an
understanding of general relationships
(systematicI), The following outline 11'111
thus review the genf!ral relationships of
liv1na (a. contrasted to to..il) organisms
and briefly describe the various types.
D The .pecies problem
1 Necessity of identifying species

Studies of the scology of any habitat
require the identification of the
organisms found in it. One cannot
come up with definitive evaluations of
stress on the biota of a system Wlless
we can say what species constitute the
biota. Species vary in their responses
to the impact of thp. environment.
2 Solutions to the problem

a Evasion

Treat the ecosystem as a "black
box"--a Wlit--while ilnoring the
constitution of the system. This
may produce some broad generalizations
and w1ll certainly yield more questions
than answers.
b Compromise

Work on~ witb ti10se taxonomic
categories with which one has the
competence to 1ea1. Describe the
biotic component as a taxocenosis
limited to one or two numerically
dominant taxonomic categories.
bearing in mind that numerically
taxa which are ignored may be very
important to the ecology of the ecosystem.
~..AQ.22c.l, 74
c Comprehensive description

Attempt a comprehensive description
of the biota. No one can claim
competence to deal with more than
one or two groups. The cooperation
of experts must be obtained. The
Smithsonian Institution has a clear-
inghouse for this sort of thing. 1
Lists of expert taxonomists can be
obtained. Th
A,Q\1t.t1c Or.l118ms of Si~1f1cBnce
IE D U~: .'EI:8
Figure 1. BASIC CYCLES O:f Li,F,f
..Jot. 2

A The va8cular plants are usually larger
and polsess roots, stems, and leaves.
1 Some types emerge above the surface

2 SUbmersed typed typically do not
extend to the surface.
3 Floating types may be rooted or free-
B Aleae generally smaller, more delicate.
le88 complex in structure, possess
chlorophyll like ott:.er green plants.
For convenience thE'! following artificial
grouping is used in sanitary science:

1 "Blue- green algae" are typically small
and lack an organized nucleus, pigments
are dissolved in cell sap. Structure
very simple.
2 "Pigmented flagellates" possess nuclei,
chloroplasts, flagellae and a red eye
spot. This is an artificial group con-
taining several remotely related organ-
isms, may be green, red, brown, etc.
3 "Diatoms" have "pillbox" structure of
8i02 - may move. Extremely common.
Many minute in size, but colonial forms
may produce hair-like filaments.
Golden brown in color.
4 "Non-motile green algae" have no loco-
motor structure or ability in mature
condition. Another art1f1cial group.
a Unicellular representatives may be
extremely small.
b Multicellular forms may produce
great floating mats of material.
Lack chlorophyll and consequently most are
dependent on other organisms. They secrete
extracellular enzymcs and ~ complex
or,anic material to simple compounds which
they can absorb directly through the cell wall.
Aauatic OrD'aniAma of SUmificance
Schizomycetes or bacteria are typically
very small and do not have an organized
I Autotrophic bacteria utilize basic food
materials from inorganic substrates.
They may be ,hoto- synthetic or
2 Heterotrophic bacteria are most
common. The require organic
material on which to feed.
"True fungi" usually exhibit hyphae as the
basis of structure.
Lack chlorophyll and consequently feed on
or consume other organisms. Typically
ingest and digest their food.
The Animal Phy~a
I PROTOZOA are single celled organisms;
many resemhling algae but lacking
chlorophyll (e!: illustration in "Oxygen"
2 PORIFERA are the sponges; both marine
and freshwater representatives.
include corals, marine and fresh-
water jelly fishes, marine and
freshwater hyd:coids.
4 PLATYHELMINTHES are the flat worms
such as tape worms, flukes and Planeria.
5 NEMATHELMINTHES are the round
worms and illc lude both free-living
forms and many dangerous parasites.
6 ROTIF ERS are multicellular micro-
scopic predators.
7 BRYOZOA are small colonial sessile
forms, marine or freshwater.

Aquatic Orlt.n1lms of S11P111'1cance
8 lIIOLLUSCA include snails and slugs,
clams, mussels and oysterl, squids,
II1d octopi.
9 BRACHIOPODS are bivalved marine
acp.nisms usually observed as foslils.
10 ANNELIDS are the segmented worms
such as earthworm", sludge worms and
many marine spec~es.
11 ECHINODERMS incluc!e starfish, sea
urchins and brittle stars. They are
exc1uaively marine.
12 CTENOPHORES, or comb jellies, are
delicate jelly-like marine organisms.
13 ARTHROPODA, the largest of all
animal phyla. They have jointed ap-
pendages and a chitinous exoskelton.
a CRUSTACEA are divided into a
cephaJothoraz and abdomen, and
have many pairs of appendages,
including paired antennae.
1) CLAOOCERA include Daph:\na
a common frt.shwater micro-
crustacean; swim by means of
branched antennae.
are the fairy shrimps, given
to eruptive appearances in
temporary pools.
3) COPEPODES are marine and
freshwater mlcrocrustacea--
swim by means of unbranced
4) OSTRACODS are like micro-
scopic "clams with legs. "
5) ISOPODS are dorsoventrally
compressed; called sowbugs.
Terrestrial and aquatic, marine
and freshwater.
6) AMPHIPODA - known as scuds
laterally compressed. Marin:
and freshwater.
7) DECAPODA. crabs, shrimp,
crayfish, lobsters, etc.
Marine and freshwater.
b INSECTA - body divided into head,
thorax: and abdomen; 3 paria of legs;
adults typically with 2 pairs of
wings and one pair of antennae.
No common marine specie-so Nine
of the twenty-odd orders include
species wi.th freshwater-inhabiting
stages in their liie history as follows:
1) DlPTERA - two-winged flies

2) COLEOPTERA - beetles

3) EPHEMEROPTERA - may flies

4) TRICHOPTERA - caddis mes

5) PLECOPTER.A - stone flies

8) OOONATA - Gragon mes and
damsel flies

7) NEUROPTERA - alder mes,-
Dobson flies and fish flies,

8) HEMIPTERA - true bugs,
lUcking ihsp..-."1s such a8 water
striders, electric light bugs
and water boatman
9) LEPIDOPTERA - butterflies
and moths, f.p.cJudes a few
freshwater moths

c ARAC:HINIDA - bodT divided into
cephalothorax and abdomen;. 4. pat'rs
of legs - spiders, scorpions, ticks
and mites. Few a.quatic repre-
sentatiYes except for the, freshwater
mites and tardigrade8.
1 PROTOCHORDATE~ - primitive marine
forms such as acorn worme, !tea
squirts and lancel'Ct..
2 VERTEBRATES - all animals whteh
have a backbone
a PlCES or fishes: inclutUhg. such'
forms as sharks and rays,
lampreys, and hither f1800E!; both
marine and freuhwater

b AMPHIDIA - frogs, toads, and
salamanders - marine species
c REPTILA - snakes, lizards and
d MAMMALS - whales and other
warm-blooded vertebrates with
eAVES - birds - warm-blooded
vertebrates with feathers.
Whittaker, R. H. New Concepts of
Kingdoms of Organisms. Science
163:150-160. 1969.
This outline was prepared by H. W. Jackson,
formerly Chief Biologist, National Training
Center, Water Program Operations, EPA,
Cincinnati, OH 4')2&8, and revised by
Ralph M. Sinclair, Aquatic Biologist,
National Training Center.
Aquatic Life; Systematics
Aquatic Organisms of Significance
30- 5

- ---------- ---
.~. -. --
B800ter1a, free 11"111& r.present.t1ft.
...- . .
( 1\-'
.t. ,"
,f ~
Ph1oemroete. - $a>'role~nl.; A,detall of 1mmature reproduotive
Jt8@;.o; B, lIIature o,":onium and IWther1d1a with ec- S !ll1d fert1li-
aation tube'i C, de.d tadpole with !!;rawth' ot s. '.

A@ ~ t t
'1 :I fl
Iob1 &Olll)'oet68 -
, .
, ,
J ~".
#, "
IW roll 811 ter
~=~ -
'1Iyoo~o..te lApt'Jlllltuo.
~b1. tlnu. In01u10" pollution
t-'lllerUlt .peo ie8.
, .' I
, .
" .
, .,.
Azetollao ter
A,lIOomyoete - Spcoharo""/oes. .
yoast lnoluding poll. tol!erant
.peoi..s. ',sin,,;l.. oe11; a,budding;
C, ascospor. formation.
. it. t.

~ianJ.~~B _o~ Significance______- -- --- - --
--- --- ---- -
--- "- - - .--- - ----
~ll.tori. ~" filamente (tr1oho~.s) range fro~ ,6 to over
~ in diameter. Ubiquitous, pollution toller ant.
~~" sim1lar to Q50ilbtorta but has a sheath.
A, ~ iwtorte.. reported to 00 gen$re.lh intollerant
0(' pollution. B. L. bir"e1.

~- B

ADhanholMnOD l.lo. .oaQ use
A, ~olOQJt B,fl1ament
Aaabsena floe-aquae
A, akinete; B,heterO'Jyst
H. '.f 0 Jaol
-- --------------_._-~---

'::: -,;~ -~-----..

\.""" .l
Peel i... ~:"Ua!
op..ciec; 01 t tie Ge ~us S.~ d
. ,_ene e8mus

'-,'ofi .JI
l\quatlC vrga.u_tJu~i:t Vj, ''''''''5u........,--_nce

S. 41morphu8
-=i7c r

A tic OI'<1anisIn.-; ,,! SigniticatlL'"
~~-_u . ~- .'" -. - ~- -
-~--~-- -------
-- ------ ----
J ,\
A. to... of o!t::,':'1':..U I.a lon...
,7<:' ,.~

~tlc Orpn18mS of~i!icQJlce
'al" n...
G1rdll .,1...,
.t711.14 ~o .~
b..lo 41a1:o8
A 41.0014 or o.ntral
41atom .uoh ...
Iteph8llOc111101J. .
- --- - --- ~ ---- ---
~il(!J! lIbiIMIIM'Ii/':ll~
" pollllate or U.,lIUiU'
41ato. noh u
... 091any of !E&11.\I£a
~,1r418 .,1...)
B L ~:: ':':::... ,] B
1.,.,111" .,1_, l,c1r4l1
"oolo~ u1' ~to~10D81~..
(,11'411 .,1p.
~Cj ~J
Dhgr.. .bowing pror;ro..1" 41111nll.t1on In th. .110 of o.rta1n
trll.'~1I.1.. tAroUlh 8800...1.,. 0.11 ,IDeration. of . d1.toa.

------,--"'-'--- --,
- ----~--_._----~-
I. n~81la"4 'I'Oto.oa, 01&1. IlalUc0pol"a
k.1.llIQIIAY 111
Poll~tion t~lerant
Poll~tton ole rant
n. _bold Proto.oa, Cla.. 8u.1041na
Poll\tt1o~ toh'rant
III. ~111a~ Protol08, Cl... Cl11opbora
C,: ~1
, .:;../
Pollv.t\..c. 1:0 prant
20h (,0 J4
to ~ intolerant of
pollutan. 35 JA

f-' LA TJi; VII
Aquatic orgarusms 01 ~lgnificance
Pollution tolerant
60-500 )I
.IR1.In.U.I.. polll:ltlon
torerai1t'" Colon1.. otMn
B... J8IIk.0I1
.--..~:jC' ,2-

~c Orranisms ot:. Sl&nificl\.nce
.1" '.3
----- --
P"ran"rna trichophorum
. .' ",'" .,1..' . ,. .
;. ". ';',' ,\',' ,'....,
Ar c"Ua
vullari .
A ctmo_" phae nurn

J\quatlC urgam.mll 01 i)ip&lilO4N1CI
VaTl0U8 Forms of t:t:'rateUa cochlearis
pe ~ITi\iia
R,'t_a-l'la sp
,,'.cO . "fI'

Aquatic Orp.n1Bms out Significan~_____--
---- - --~-------
- -- - ~---
~.!! d
~d, 's-
.M'. Tine
.1rCr8t017 pon
.81h8l'7 cha4
8ell1n81 T.dc18
_T". deieTen.
eJaculator1 gland
ectal g1ud
Ctpulatory ep1cule
j,chrolladora ~

--- --------~-----
Phylum Annelida
//, / /;
//// .~
/ / / ' /

/ ~ ,.\. '>~
;' " , / \i~ "
/ ~---- mouth
// / ,//
. //'/ -\
/' '/, \
/ '/' /
,,/ ////

/ / /./
'1 aU- :~~ ~., -,c.ha.ta. ~...rtb.\rIor.ci
£Xl ~!!.
I th. ~l.: i« ""... '-, -.~II
, .."'''~ L1.bllall)
F /II .~ ,y:'kICh
Pl[,'P; )-,1
C1A.. Hirud1nea, leech~s
(JJ' hr Hegner)
anter10r end
Cl... ~olychaeta , polychaet worm.
tr.: !4&11a,yunrh. a m1nute. rATe, ~ubi
ouUdint; "~rll.
;.J.~ter Le1dy)
]I? AI::

~Uc Onl-mama of~1f1canc!!___~~ -----
01.111 CeptW.opc'4a'.
1q1d.48. ootopll. I
- --- ---"-- n-_____--
- - ~ - --
Ixciul1veiy ~1ne.
orhe giant 8quid 8hown
wal captured tn t~e
.Hla:nic 10 the early
n10teenth century.
(Atter Kegner)
& 81....
'" . '"
. : '. /""'..
en air hreathing maU & watsr breathing
01&.s. Ga8tropoda. enaile and ling8. (After Buchsbaum)
01&881 Pelecypodai clam.. mn..elB. oysters.
I.ooomotion ot a freshwater clR.lli eho"'l!1£ 11.0'1" fooi 18 extended, the t\p
expanded. and. the 1llU1I'.!U pulled dang to its OWl! ancher. (After Eueh&-
baum) R.W.Jack8on
. 31. 'I
F' LA 7 E ~,11

--- ---------- ------
Crqfi8:'. or .r_JII4,
a..b&r~8, Ord.r o.oapoda

10-2(" ;8
Wat.r n...;
.~'"' j 1u.J.i1\£
QN" ~b!pod.
C 1&.. CRUS1' tCU.
Fdry Shr1IWp;
~br.nDh12YJ. Ord.r
, .
(.opoopod I £r.2..~, tr4n Co~pod&
?-, -
"U\TE ."(III
Aquatic Qrgani!,.m8 of SiD1ifl,*,ce
r18h Lou... JI:&iliII
& p&r&l1t10 Copep04

Muatic Organisms of Significrmce
Two WingC'd Fli",,,
Adul t lliace
--- -- -----

Al1ul t nw~e ny
lat.-tailed macgot
A B.I1u1t; Jj,larva,
Sewage fly lerTa
111~. pupa
8eWII£" ny pupa
PLATE )'.1\'
------ -----
After various antLvr.

---- ----- -----
._~ />-


. d1v1ne b.e\l. (D1t1.cu.)
\Ak1J\c ur at the 8UJ'tao..

.~ 1i1Tl:141: ....:.le \~:'.ter). !h. d1T-
lac ~..\ln. lD~l~,. .o..-;r-th. lazC."
A:114 :.."" "'o"""lOU8 of '~QUe LA..ch.
A, ;....'.'"I!;;." 1.. ;.~iu-'.'
Wh1rl~~ b.etle ;,r~~II) A. 81de Ti..
ot h.ad of adul t 80110 DC videoS. ..,e;
II, Lan'a; C, Adul\. CIII'II1Torou8.
P'..\TE Xii
rJ.,..) II~~ I
:. 1,111 - -
, '" ~ .- '1
. J C
!he riffl. b..tl. (~h~m");
.1. ad.ul t; 11. &oreal 114.. 0 arTa;
C. velitral 1148 of lana. Pndoa1Z18DU;y
11 "~
Craw11nf, water b~etle;
A;aduH; B.lana. PredollinenU:r
H.W.JacE8on. Atter
I..dham. P8!Ul8k. More;an,and other.,.

jg!!It!c Orpn18ms of Slil1iflcance
Phylum Coelenterata
Medusa of
Hydra with bud;
extended, anC: contracted
Phylum Bryozoa
Creeplng colony
on rock
Single zooid, young 8tatoblasts in tube

"- -- --~..
,~qu~~~(:_Q.!]~anis~~_~, ?~~~f1.(a~Cl~
--~-'-'C!. '
CIU8 Aenatha. Jawle.. fi.he. (lamprey. and Itagft.heoj Family
PETROMYZONTIDAE. the lamprey.. Lampetr. aepyptera. the
Brook Lo.mprey A: adult. B, larva (enlarrd)

~.)~~~ /
,...' /./
A' \;..-}. A'
-':'\;~ )"'''' ,:r
\ "
'. .."'
, ,
Cia.. Ch(\ndrlchthye8 - carhlag~n"u8 fisheR (shark., ~k;tte8, rays)
FamUy DASYATIDAE ... RtingrayfoJ. DasyatlB c€'ntroura. th~ Roughlail Stingr:iy
: "",~" -".\-'I
,..".~,.( f-~),' .. .' '.,,1.."i,......1'0'" "''''-.
fl' . V - . 'due..,.; .~~~-
Cla8B 08tt'~chthyeB bony fishcR Faro lly ACIPENSE RIDAE. sturgeolJ.
Acipenller fulvescens. tht> Lakf.' Sturg.'on
~~--~~ '::'[ -: " ::
Cla88 08telchthye8 bony nsh.,s Famdy POLYODONTIDAE. the

paddlefiahe8. Polyodon spathub., thp Paddlpf1sh. A.s~df> vif>w B top vbw

-- ~,~,---".-:,~JP
ClU.08teichthyes bony 1,.h"8 Famdy LEPISOSTEIDAE
Lepiaoeteu8 O!l8f>UB, th(" Longnu~c G~ r
Clu. 08telchthyes bony nshes
AnUa calva, the Bowfin
Famlly AMIIDAE. bowflnB
Reproduced with p€'rmifiSlon, Trautman, lQS7"
BI. M... pI. 91. 6. 6Q
l'IJ\Tl' ',nr
'-.."(:1 ;;; /-

:~:' /; "'
." ~:::-- ~~:- - ~'f'

,~7' "~'-~\.

.,:¥7# :t~~::::~7\::';r;-;

...... ,
"ar;,.dy CLl..PEIDA:o;; - herrtngs
i!o,'.)1'Hm,a cepedia;1U1n - tho'> .",,-,,(',:1 giZ7,:,\rd 8bad
-..r-' '_""._~~." '.:
~ ~-~, "-4.
-.... . ---- - ~ .
.---------- ; ,
--,'.- -- - ----';;>:'7i.~;tJ \
Family ANGtILLEDAE - freshwater e€'ls
Anguilla r08t.ata - the AnlPrican eel
-' -, - ~,t:. 4~ , ,; . ,.-:~ ",!;f

Family ESOCIDAE - pikes

Esox lucius - the northern pike
Reproduced with permission; Trautman, 1957.
BI. AQ. pI. 9m. 6.60
male ." , ,';'~~-'~ /' '

# (.#-..~f)J/.J" - ~~-----

~~ ~~J\h!:ij;:~~~:~~~_:: .

~ '_-'~"'V', -
, . # --....\ . -- ..::..:--
G",~"""''''''l7'C<''- . ,) "'..---, :..-
~{,L~Y"(f'£i,\>\~U, " /"'~~,.'J'-":' ", - - "
:;J!e"~. ~\.~~iV1t'" ,_,r " .'. -

f~: ~J}':;~;\;:L~'P~.'~L ~_.)

Family POECILIIDAE - livebearers
Gambusia affinis - the mosquitofish


Family GADDIDAE - codfishes. hakes, haddock, burbot
Lota Iota - the eastern burbot
Family SCLAENIDAE - drums
Aplodinotus grunniens - the freshwater drum

A Importance of Laboratory Data
1 Water quality standards
The eatabUshing, implementation, and
enforcement of we-tel' quality standards
requires very accurate Wormation on
existing water quality in all the major
surface waters 01 the United States.
Determination of compliance with the
standards, al'id the economic, leau,
and 80cial actions associated with
pollutton abatement, mu.t rest on
reliable data.
2 Waste treatment
The efficienc)' of waste treatment
sy.tems must be accurately determined
by laboratory data, in order to evaluate
treatment proC'eue. and wa.te dis-
3 aesearch
In most research investigation.,
laboratory data defines the information
sou,ht, the success or failure of
alternative path_ys, and often
describes the final answer obtained.
B ReUabiUty Requirements
1 Specificity
To be useful, data on chemical con-
stituents must clearly indicate what is
bein, mealured, including the phYlical
state and the chemtcailitructure.
2 Accuracy
The accuracy required il defined by
the final use of the data, and t. directly
related to the interpretation of the
values reported in terml of water
quality or was~e conltituent concentration.
CH.MET.con. 6. 3. 74
1'01' proper int..rpretation. the limits
o! accuracy must also be known.
3 Precision
The analyst mUAt know the precision
of his methods, in order to determine
when the procedure is in control.
The precision establishes the con-
f1dence one can have in a single
reported result.
A Avan..ble Sources of Methods
1 Standard Methods fvr the Examination
of Water and Waste"Nater
2 ASTM Standards, Part 23, Water;
Atmospheric Analy.ta
3 Current literature
4 Methode researCD in WPO
B Selection Proce..
The sources listed above are reviewed
regularly and preliminary selection is
made by the staff of the Methods Development
and Quality Auurance Laboratory. After
collaborative testing. the final sp.lectton
1. reviewed and approved by WPO
Reponal AQC Coordinators or by
A dvteory Committ0e8.
C Status of WPO M"thods
The analytical procedures adopted are
used in aU WPO laboratories, except
where the analyst has determined that
another method must be used because of
unusual interferences or special data
requirements. The methods are also
used by contractors providing services
to WPO

AnaJ.yttcal Quality Control 111 WPO
D PubUcaUolUI
WPO methode are published by the
Methods Development 8. Quality A.surance
Laboratory and distributed to Federal.
.at_, and printe 1abol'atorte.'hrc:Iqh
the R.~ AQC CoordiDator..
A Collaborativ. T..Un,
1 PUl"p08e
To verUy the specificity, lensit1vUy.
prec11ion, and accuracy of the adopted
methocl8, round-rotJin ts8t1l1. i. GUTied
out throu&h the cooperation of WPO
and other Jaboratories. MaUsUcal
evaluation of telt data estabUshee the
.ul1abUtty of the mdhod III our
laboratories and determine. the need
flDr improvement or turther re.earch.
2 Sample studi..
Reterence sampl.s for a majority of
the parametere used in determ1nin.
water quaUty have been prepared or
are planned. The studi.. are de.iped
to teet the method at the concentration.
found in -tel' and wallte ..mple8. tD .
the pr..ence of the in~.rf.r..ce.
.." aormaUy ..oounterod. .
it Performanoe Testin.
Tbe abillty of the individual anaqat to
obtain a valid result 1s an 1mport8nt part
of the quality control provam, The
reterence sample Itudies are ~1ped
to evaluate the performance of the
an~y.t. in eompari8on with other.
partic1patin. in the study. 1bul the
~It. aDd bie lupervi80l'. eM I4eDtUy
problem. .....irin, Upt.r GOntrol or
additional traUainlf.
A Element, of a Control Program
An adequate quality control pro,ram in a
laboratCK'J ..,..W iaclude checo on:
1 Methocl8
2 R",Ints
3 Iutrum.ntation
4 Gluaware
5 DaU, ,.rformance (control chart.)
6 Data haDdlift, and reporting
B ReapoulbWtte. tor Qaality A..""'ance

The b..ic respon.ibility for the cauaUty.~
of laboratory data re.te with ttte analyst
him.elf. A formal pro,ram in the
laboratory. however, provide. the
laboratory dt.rector with a meCbMiarn
for evaluatl.at tlte reliability of the data
and maUMinln, ..t18faetory qua:U.ty.
Th18 autUM wa. prepared by D. 0..
Bal11n,er, Dir.etor. Methods Dev.1oPment
. Quality A..urance Re..arch LaWatol')'
EP A, NERC" Cincinnati
n..prit)*".... Che~tcal Ana1,yIit., QUality
Control, Water AnaJy8i8

Sat1.1actor7 evaluation 01 the quaUty of water
4IptftCIa upon the aw1JabIUty of adequate data.
Suob data must be not Oftly a. aCOIU'&te a.
.eoaomtea~ praC!ttca! but mutt al80 be 01
.ufftot.eM quantity 'to cupport reUabJe con-
elu.ionl. In general, the more laformation
a~le, the mol'. reliable wUI be the
In the pa.t, information on the chemical,
phy.lcal, and baeterto~lJ1cal quaUty of most
.urtaee waterl bat been obtained by periodic
Itr.am wrveys or tntrequent "8pOt" anaJyle',
Althoulh raw water .uppUe. are .ampled
~, the data obtained are re.trlcted to
thole telts 01 importance in water treatment
and do not include other poUutional param-
eter. or water r..ouroces not at pre.ent be1Dg
u..d for water wppq.

It 11 apparent that in many l1tuations .ignUi-
CIoI1t change. in water quality may occur often
and.brupt1y. Sea.onal chl.np. in flow, the
oocul'I'eatce of unpredictable induetr1a1 dl.-
Char... or spill., and the ehanJe. in flow
from impoundment I may alter the concen-
tration of many of the ...tanee. 01 intere.t
to the water u.er. In eom. cas.. th...
chan..s may occur within a lew hours: lor
example, the diurnal flUctuationl in dillolved
oxygen and the saUnity chan,el in tidal
elNaries. Thus cONIldenble advanta,. Is
pined U continuoU8 monitoring 01 water
quaUty can be accompU'hed. The use 01
manual amplin, and .tandard laboratory
aIlaJy.e. would be far too expen.lve; therefore
a wide wriety 01 autamatlc Ift8trument. bave
been developed.
The c:Ua,...m below 1llultratee a typical
Iylttm tor mon1tortnc In.trum.nt. which
laW.. electrical .ental".
CH,MET,lSb. 3.74
The lensor produr.es an electrical slenal
repre.entat1ve of concentration, the slenal
il CODverled, and ampUfled, then passed to
the recorder to develop a permanent .record
of cOl1centratton levels. For a clear
under.tandin, of the operational charac-
terlltio., the part. of the system are
di.cua8ed Ieparate1y.


t ....0.
"'V,. I
A Sensors
The sensor is the part of the system in
contact with the sample. Sensing ele-
ments may be immersed directly in the
stream, or plar.ed in f10w cells through
which the lample 18 pumped. Both
methods have advantages and limitationl.
When the sen80r is placed in the stream
the determination Is made "in situ. "
Therefore the sample 11 not affected by
pumping, temperature changes, or time
o! travel through the inltrument. Such
an installation, however, presents
certain problems. The sensing elements
mUlt be protected from floating debris
and must be mounted so as to remain in
.. fixed position in spite of changes in
velocity or direction of current. In bodies
01 water which fluctuate in surface leve],
euch as impoundments, or estuaries, the
relative depth of the sensor may change.
In addition, frequent inspection of the
sensin, elements for attached growths or
physical damage is difficult and apt to be

Autolll&tic In8trum.ntl for Water Quality Me(Llurernente
The u.e of the ehore-baeed ')'8tem, where
the .tream Ample ie pumped throup now
ceU. within the In.trument bOIa.In" i.
free from eome of the difficulti.. men-
tioned previou8ly. In8pect1on of the
eenaora ia "8)', cleanf.DI 18 8imp1Uled.
and replacement of .euta, elemente i."
r.adftr accomp11ehed. It.bou1d be
recolNzed, however, that the prec&ut118n8
required tor AU8faatOl'1 moantq and
protection of 8811.01' UIdt* in the strlam
appJ,y eq\&11r will to the pump intake.
Further, it 11 I..ential that tile Ample
bem, tested In the flow cell.18 truq
repr..entattve of th. etream water. If
di..oilved oxypn ia Included In the
parameter., a eubmer8ible pump ia
requfred, to awid cavftation and prevent
auction removal of die.olved ...... The
Intake ecreen must be caretuJq de8iped.
alnce a ftne ecreen will quicJcqc1ol(, while
a coal''' ecreeD may permit Qaatln,
material to enter the eyet.m. ,
Electrical eeneore may be concl\lctimetric,
potemiom.tric. polarol1'aphic, 01' .
cou1f.metric. The een.or may .ur.ctJ,y
mealUZ'e a constituent or property of the
8UI1p1e or it may b. ue.d al an Indicatln,
mechanl.8m in autom.UctitNtfoa.

B Ana~zer-Amp1Uier
The tunction of the ana],yzer 18 to convert
the el.pa1 fl'Olll the lelllor to . lltandard
EMF. una~ nnainl h'om 0-00 mWivolte.
Otten bridle circuit. are 8mployed. The
"~r I'ftut be ruaed to awl.d .!Iock
damace and the electr8l.c ol.l'OUitr)r muet
b. .table ove~ . wide rusl oIlIrIiroftmental
oondWon., eoneider8ble acn.nta,. 18
pined by the uee of etudard. reacUq
avai1lble component.. Provi8l.on ehou1d
b. made for a atandard eipal to "l"IDit
Intel'8&1 oMoIdaa of the circuit..
In mest ~... the eillll81 mutt be .mp1Uled
to provide euakient volta", to drt"e a
rlcai'der. AmplUtcataoa 18 ~
butlttnto th &1aqzer Cll.rcndtr7.
C Recorder
Althoulh read-out metere can be ueed, a
permanent record 18 duirable. Strip-
chart 'Nool"dera are the mollt popular
eVIII thouah they require line voU.,e.
A .low chart epeed ie nec...r,y, .ince
ob..nattcIe per!ode Of "venJ dip
ar. eomeoa. DUftC1l1tie8 with"... ....
oft.. euooantered due to val')'llll COI1-
ditiorl. of humidity aDd t.mpet'8It\ll'e.
The ..... of pl'8H1P'e-8eD8W.,. ""1' '
m&1 01.181' di8tI.IIIct ad¥8Dt8p.

Circular cbart. have 10Dl beea Uted bllt
they have certain dieadvant.,... V.,.
thlchart i. inconveniently 1aJwJe the oIIart'
diTi"'o~ are ver,y clo.e toptlwr meldlll'
hourq fll1ctuati0n8 difficult te ..rpret.
In adtlltioA. the circular chart don ralt
lend iteelf to mechanical handUaI of 'data
for compllilr PfOc",,,, Cbart '..,..... by
clock mechani8m 18 poe.sw.., bo,......r, "
e1imifta&- the need lor power ,..-dUm..
A preflrable eyltem for the r8QOl'dlDc cd
data i. the uee of diaital output. Water
quality Ialtrllm.nt8 can be equipJledwith
Automatic Instruments for Water Qt.ality Measurements
D parameters Mealured Electr1cliUy

The foUowing water ~uality parameters
CIR,'be measured by the use of electrodes:



Residual chlorine

tion potential
A prominent instrument manufacturer is
now conductiDl rtl.earch into the develop-
ment of a numbel' o~ 8las8 electl'odes
specifically desilned fol' the mea8urement
of other ions. It appeal's that the list above
may be con8iderably expanded in the future.
In most cases precision and accuracy is
not as IJOOd as with Itandard labol'atory
test8. but the advantac.s of continuous
record11\1 outweiih the limitations in
Becaule of the impol'tance of colorimetric
analysis in the water laboratory. considel'able
..UenUon hal been liven to the development of
continuous monitorinr instruments employing
this principle. A typical system is shown
in Pi.un 2.
It should be noted that the sample must be
pumped through the instrument with the
attendant problems described above.
A Measurement of Turbidity and Color
In the simplest photometric instrument,
a property of the ...mple, suoh as
turbidity or color, is measured directly.
The.e relatively simple parameters,
however. are rather difficult to determine.
Turbidity measurements are affected by
particlle' size and by the true color of the
sample. Conversely. color determinations
are subject to errurs caused by turbidity
and by the fact that the wave length of the
color in the Ample may vary widely.
Instruments of this type currently
avaHable do not satisfactorUy compensate
for these interferences and the data
obtained from them does not correlate
well with standard methods values.
..3'1- 3

~mat1c Inlt1'\lD1enti for Water Quality Mea8urement8
B Colorimetric Analyzerl
A lecond type of photometric instrument
i8 designed to reproduct! laboratory
colorimetric procedures. That i8, re-
agentl are added to the lample to produce
a color chanJe proportional to the con-
centration of the material being determined.
Since the 8ample is fiowinc continuou81y
the reagent I must be metered accurately
w mix.d thoroughly before photometric
meaeur.ment. In a properly d.8igned
.ylt.m, almost any color1metrtc proc.dure
can be duplicated and therefore the potential
ran,e of determinations i8 much wider '
Ihan in the electrometr1c inltrumentl.
A modification of the colorimetric in8tru-
ment is the continuous titrator. In thil
Byltem an indicator i8 added to the nOMng
.ample and the reagent i8 added at a vari-
able rate to maintain a conltant color.
The amount of reagent required is pro-
portional to the concentration of the re-
actant in the sample, and the current used
by the me tering pump actl as a sill1al for
the analyzer.
In .pite of the apparent advantages of the
photometric system8, cer',ain special
problems are inherent. The color and
turbidity of the sample may interfere, the
accumulation of sl1me in the cell8 may
.eriously reduce the sensitivity, and the
llmUations of the filter photometer (wide
band pUI) mU8t be considered. Further,
in mOlt colorimetric procedure8 the
amount of reagent required i8 proportional
to the concentration rang" of the lample,
a factor which would limit the appl1cabU1ty
or the instrument.

C Parameters Measured Photometrically
Continuous analytical procedures have
been developed for:


Chemical Oxyaen
Residua[ Chlorine
14-4 '
To illustrate the quality of the data which may
be obtained from an integrated water quality
monitor, the following table shows the results
of a performance test contiucted on a proto-
type instrument npplied by one manufacturer.
The "Acceptable" toleranceo were selected as
representative of the usual requirements for
continuous data acquisition and may be too
b1gh or too low, depending UPOII the accuracy
deemed nece8sary.
A Calibration
In the parlance of the instrument manu-
facturer' calibration involves two steps:
(1) the .etting of the readout to zero value
in the absence of senso." signal, and
(2t lIetting of the readout to some standard
value, such as 5mv. when the sensor
signal i8 replaced by 3. standard signal.
It ill readily apparent that a calibration
of this type adjusts the meter and amplifier
to reproduce correctly the signal received
from the sensing elements. It does not,
however, "calibrate" the total instrument
in terms or the concentration of measured
substance in the sample.
For proper calibration, it is essential
that the final readout of the instrument be
adjusted to correspond to e. true value for
the meaaured me.terial. Thus the only
adequate calibration must involve the
measurement of a standard solution, such
as a buffer of known pH or a salt solution
of known conductance. Since instrument
systems may lack linear response to a
wide signal range, the instrument should
be calibrated at several points over the
range of values anticipated. In some
cases current instrument design has not
included adequate means of replacing the
sample with 8tandard solutions for calibra-
tion purposes.
The frequency of recalibrat10n depends
upon both the Itability of tjle analyzer and
the sensor. In general, analyzer-amplifiers
are more stable than the sensing systems
now in use. The Basic Data Program,

A utomatic Instrument, for Water Quality Measurement. <
Acceptable deviation
Mean deviation found
% Accep~ablAl
! 0. I unit
+ 10
:!: 5%
Dissolved Oxygen
FWPCA, in per~onnance specifications
for monitoring instruments, has established
a two-week period :or unattended per-
formance. While such instrument stability
Is desirable, ctrcumstances of site location
ar.a s&mpl" charactenst1cs, often
necessitate rnc.e frequent checking and
B M':l1.:1ter.aflt'f'
It IS un":lse to assume that any 8tream
monitor"\g IOtJtrllTT1ent, no maher how
Wf>U deslgn..:i ~dj b<"lt, can function
ad'!quatt'ly f,_'r "j.'~:L",'dE'rF: ma:, EOtOf-' ,)C fail to print. The
fr-=qu."-cy of maintenance dependli upon a
j,..r@e number of factors both controllable
and acc ,«1<>n\al "n(~ can be determmed
001;1 b;v io;cg-terr tl'Etlng and actual field
ex::,er..!1c,<, Ease of maintenance can be
d"'8iii!I1"'; :10'" thf :'1<"rument by the ulie of
:-~~';lb:('<2,"J'~E cjc<'~'~~Cln,lc components and
"cu;!J~,b';:t.. C<,
A StpSficance
Orpnic !!ubstances in surface water are
pre.ent due to runoff, the metaboUc pro.
duct. of microor,anisms in streams,
dom..Uc wastes, and industrial wutes.
OrlUlic pollutant. are silftificant because
01 their possible oxyren liemand, color,
and ta.te and odor potenUal. In addition,
certain compounds may be per.istent in
the .tream or exhibit chronic toxicity.

B Me..urement
1 BOD, COD and carbon analysis have
been used for ,.neral estimates of
orranic pollution. Results of each
one carries a different connotation
in terms of oxy,.n demand criteria:

a The BOD ....ult includes an estimate
of oxygen demand of biolotrically
available materials includ1n, carbon,
bydro,.n, nitrogen, and sulfur
components under favorable conditions.
b The COD includes an estimate of
chemically oxidizable carbon hydro,en
and su]fur components. It is useful to
estimate first stage oxygen demand but
the oxypn equivafent of unoxidized
nitro,en mu.t be added to estimate the
long term demand.

CI Total carbon tTC) or total organic
carbon (TOC) reflect the carbon con-
tent specifically. It is possible to
..tImate ox,ypn equivalents on the
basis of backfround information on
.ample characteristics.
The three criteria each renect diff-
erent approaches and sample char-
acteristic.. They may be used inter-
chanreably in certain situaUons where
relation.hips have been estabUlhed.
More import",tly. they may be used
to classify 1& sample on the basis of
the variou!! determinations. Recent
development. include an instrumental
methodolol1 of estimating oxygen
demand including the four major
contributing components - carbon.
hydrogen, nitrogen and sulfur.
2 The more specific analysis of organic
contaminants in -tel' is made difficult
by the large number of complex
orranic substances in the sample as
well ae the very minute concentrations
usually found. A s a result. more
enensive evaluation or organic pol-
lution requires concentration and
clean-up techniques followed by con-
firmatory identification.
A Carbon Filter
1 Procedure
The use of activated carbon for the
adsorption of organic compounds is
well known. The technique has been
appUed to the recovery and concen-
tration of organic8 from water by
meane of the carbon fUter. The fUter
consists of a glass cylinder 18 inches
long and 3 inche. in diameter packed
with activated carbon. In use the
water to be sampled is pumped through
the fUter until approximately 5000
gallons has been sampled. The car-
bon is then removed from the cylinder,
dried at low tem.perature. and extracted
8erially with chloroform and ethyl
alcohol. The organic compounds
which have been recovered from the
8tream are then concentrated to small
volume by evaporation of the solvent.
By weighing the residue a value for
the total chloroform extractables can
be calculated. In m08t relatively
clean streams the total organics
recovered by this technique average
l88s than 200 mgll (aOO ppb). Where
indu8trial pollution i8 present, however,
values as high a8 1000 mg/l have been

Oraanlc Analyses ln Pollution Surveys
2 Umltationa
It is difficult to obtain quantitative
information ueing the carbon filler
because 1ts performance is .ffected
by such Vlll'iable. as now rate, pH.
particle 11118 01 cal'boft. quantity of
particuJat8 matter pre..", and type
of orpn1c compound bein, adsorbed.
Recent r.s..rch has lIbown that the
.,stem 18 most efUclent wbeD the
flow rate 18 very s]ow and the total
volume passed thrOufh U. filter Is
..ry smaU. Thu the Water Pollution
Surveillance System 1s now -m,
modU1ed _its which pus 1000 liters
(1&0 111laD8) at a flow rate 01100 ml/min.
(1/31 pn).
B L1q1lid-Liquld Extraction
L1q11id-liqu1d extraction teclm1ques are
based on the principle that an orpnic
8ubstanee will d18trib.. its.U 1ft some
ratio between water and an orpnic
solvent. If the retio lavor.the orpme
80l.ent, that 80lvent can be used to
extract th. orpnic 8~b8taDce from water.
The technique has l1m1iaU0ft8 111 water
analysis, however, becaue the extraction
of very lar,. eamples with v81'1lar,e
amounts of solvent 18 r8qulr84in ordsr
to recover large enOl&8h qaaaUties 01
A So1ubUity Separation
1 The residue f"om the carbon filter
repr...ts a mixture of most of the
or88Dic compounds present in the
or1pl stream .ample. In order to
obtam more information as to the
nature of 'the orpn1c contamination,
it ls nec..sary to separate -the mixture
intostructuralgrwpa. Th18 separation
is accompli8hed by talc1D, advantage
of the d1flerences in solubility of the
groups in va rlous aquews solutions.
The residue ls first d18.o1ved in ether
and the undi880lved material, caUed
"ether inso1ubles" is removed by
filtration. Then the. ether solution
01 the orpmcs ls successively
extracted wlth water. dilute Hel.
dilute NaHC03' abd dilute NaOH. to
y1eld the water soluble. a~ine, strong
acid, weak acid, and neutral tractions
of the orill.nal material. The weight
of theee fractions lives the relative
proport1one of these orpnic JI'Oups
in the stream.
2 Throqh a know led,e of the r.JatlY~
amOUllte ot various ~ract100. Present
in the sample, the probabl. _1'0.
of the orpD1c con3minadon.can be
dedl1cted. For ~le. .& la....
portion of -tel' soluble ' compounds
mi8ht indicate natunl mat8l1a18 from
veptation; a large amount of baseB
would indicat. pyrid1n. or 8indlar
Ditropn compounds. .
B ChromatOfl'&phic SeJ&retiQD
1 Usm, column chromatolrAphy with
sU1ca lel the n.utral fraction can
further be .eparated. I.o-.octaDe
elutes aliphatics; benzene elutes
aromaticB; finally, a 50:&0 mlxtur.
of chloroform and methulol elutes
the oXYlenated fraction.
Z A hip ratio of aUpbaticl to total
extract would point to probable con-
tamination by refinery waeteB.
Aromatic fractions are often odor.oue;
such compounds aB phell¥l ether,
ortho -nitrochlorobenze,ae and
chklrlnated hydrocarbon meecticidee
have been i80lat.d from this fraction.
C Other Techniques
1 In recent years the appUcation of thin
layer and paper chromatopplUc
.techniques to the clean-up-ofthe
aromatic fraction has r..Ulted in
further separatjon and eventual
ident1f1cation of orlan1c~tants.
:a Employment of chemical D\od1fications
to form derivativee of selected com-
pounde in a mixture may be employed

to Improve .pecUicity and .en.itivity
of infrared. p, chromatography or
other determinative method..
A IDfrared spectra of cleaned-up extracts
provide qualitative information as to the
chemical nature of the compound. in the
ample. In some case. the .peetra have
id8l1tU1ed pure compound..
B Ga. chromatography applied to extracts
aUio provides quali1.&tive information.
However. it. ,r..te.t asset il the ability
01 cenain detectors to provide quantitative
Information in the nanogram range. The
technique is also ueed at timee a. a pre-
liminary clean-up procedure before
Infrared identification.
A Ph,nole
1 Blcau.e of the importance of phenolic
. compound. as organic pollutants.
analysis of wastee and stream sample.
for phenols is often nece ssary .
2 When interfering substances such as
lulfur or nitrogen compounds are
prllent. the sample must be distilled
to remove the phenole from the inter-
ference.. Phenol concentration.
above O. 1 mill. as in waite samples.
may be determined by the aqueoul
4 -amino antipyrill& method. For
concentration. below 100 /Iogll the
4AAP method 1. modified by a chloro-
form extraction of the colored complex.
Sen.itivity of the modified procedure is
approximately 2 /Iogll (2 ppb).
3 The 4AAP rea,ent producee color with
a wide ran,e of phenolic compounds.
but the sen.1tivity varies with different
phenols. Studies have ehown that phenol
itlelf produces al much or more color
than other phenolic compounds usually
encountered. Thus when phenol is used
Ol'pnic Analyses in Pollution Surveys
as a Itandard and the results are
reported al phenol. the reported
valuel represent the minimum amount
of phenolic materials present in the
8 Surfactants
1 Surfactants include LAS (linear
alky1ate sulfonate) and alkyl sulfates
(anionic) and ethoxy ether alkylates
(non-ionics) among the active ingre-
dients of synthetic detergents. The
anionics and some of the non -ionics
are determinable in sewage and river
water by the formation of a stable
colored complex with methylene blue.
The method is accurate to approxi-
mately t O. 1 mgll river water samples.
2 Strell on use of biodegradable
detergents has required adoption
of methods to eyq.luate biodegradability.
Improved tests are essential to
eltimate surfactants not included in
the' methylene blue active surfactants
(MBAS) talt.
C Taste and Odor
Certain organic pollutants can be
measured according to their taste and
odor potentials. An evaluation of the
threshold odor numbl!r can be obtained
by setting up a series of dilutions of
sample. The dilution ratio at which odor
i. just detectable by a representative
panel i. defined as the threshold odor
D 011. and Grease
The prelence of heavy oils and grease
in surface water 9.re indicative of
Industrial pollution. Methods for the
general evaluation of such pollution
consiet of liquid extraction techniques.
For a more specific analysis it is
neceesary to utilize chromatographic
separation and infrared analysis.

O~anic; Anabr... in PoUution Surveys
E P..tlcide.
Th. FWPCA M' pubU.h.ed official methods
tor analy.i. of ch.lor1Dated hydrocarbon.
(3) .,plicab18 for mea~em.nt of ~icro-
Iram and piCftlram (10- ud 10 - I)
per Uter qll&8t1t1es a1aft8 tbe foUowm,
1 Chlorinated hydrocarbon.
a Recove1'7 of pe&tic1de. from water
u.1nf the carbon ft]ter or liquid-
liquid atractiOD.
b Separation and identification of
1) Solubt11ty and chl'omato,raphic
..pal'&tion .erve. a. a cllan-up
proCldure. Pe&tlclde. are
round in the aromatic fraction.
2) Tb1r11ayer chromato.raphy
.ervee both.. a clean-up
technique and a. . mean. for

3) Ga. chromato,raphy (both
electron capture and micro-
coulomet.ric) can b. uHd for
identification and quantitative
"II&)' of pe&tieide..
4) Paper chromatoaraphy can alia
be u.ed for COftrtrmatory
c Infrared identification
Ab.oIute ident1f1caU0Q8 of pe.ticid..
can be made u.in, micro infrared
spectro.copy. How.ver, it i.
nec...ry to have rtcovered 1-10
micl'Op'Uns of pelticide when u.in.
the PuJdn Elm.1' Model 421
1.n8u,ua.nt with 8Xb~ COIIdell8er
and a t. & mm K Br dUe.
2 QrpDIc pbo.phoru.
It i. po.lible to mea.ure orp.n1c
pho.phoru. insecticide. uain, electrOD
capture p' chromato,raphy foUowInf
liquid-liquid extraction. However,
more sp.c1f1c1ty and senaitiv1ty can
be obtained u.in. phosphorus detectors
ba.ed on a thermionic principle.
Techniques nece...ry for the evaluatioa. of
orpDic polJation are pre.ented. Such
evaluation req\l1re. tl.me-conaumin, and
eJ:pen.ive analys...
Thi. outUne CODtain. certain portion. of a
previous ouWne by D. G. Ballinger aad
B.A. PUftabOr.t.
1 BibUo.raph.y on Synthetic Deterlents in
Water and Wastes. EDar. Section.
BASB, DWSPC. Robert A. Taft
Sanitary En,ineerina Center. June
2 Booth, Robert L. and Engli.h, John N.
Evaluation of Sampling Conditions in
the Cerbon Ad80rpt1on Method.
J. AWWA. 57:215. February 1985.
3 Braus, R., Middleton, F. M. and
Ruchoff, C. C. Systematic Analysl.s
of arpnie Indu.trial Wastes.
Anal. Chem. 24:412. 1952.
.. FWPCA Method for Chlorinated
H)'drOct.rbOD Pesticides in Water
and Wa.t_ater. Dept. of Interior.
AprU 1989.
5 EtUn,er, M. E., Ruchotf, C. C. and
U8hka, R. J. Sen.tUve .4-Amino
Antipyrine Method for Phenolic
Compound., Anal. c;:hem. 23:1783.
19&1. .
6 Ludzack, F. J. and Whitfield, C. E.
Determination of High BoUlnf Paraffin
H)'drocarbona in Polluted. Water.
Anal. Chsm. 28:157. February 1956,

7 Ta.k Group Report. Determination of
Synthetic Deterlent Contlll1t 01 Raw
Water Supplie.. Journal AWWA,
&0:1343. U5B,
8 Standard Methods for the Examination of
Water and Wa.tewater. APHA,
AWWA, WPCF: 13th Edition. 1965,
Orpnic Analyses in Pollution Surveys
9 Van Hall, C. E., et a1. Rapid Com-
bu.tion Method for the Determination
of Orpnic Substancelil in Aqueous
Solutions. Anal. Chern.. 35:315.
March 1963.
This outUne was preps.red by F, J.
Ludzack, Chemist, National Training
Center, Office of Water Programs, EPA,
Cincinnati, OH 45226.
Analyt cal TechnIques, Chemi.ca1 Analysis,
Water Analysis, Organic Compounds,
Water Pollution, Water Quality, Phenols,
Surfactants, Taste, Odor, Oil, Pesticidcs.
35- 5

I Analytical results are an "imperfect"
eltimate of some ....1 value .ought as a
meanl to interpret, predict, or control
behavior. In sanitary engineerin" this
refers to some aspect at the aquatic
environment; or tor the purposes of this
outline, a chemiCkI determination related
to water ..enovation.
A Interpretation of analytical results may
be meaningful or misleading dependin, on
the interpreter'e reco,nition of various
factors .
1 Sample validity in representation of a
situation, location, time, or occurrence
i. a prerequi8lte tor reliance on
,ub.equent operation..
2 The analytical relult may include a
direct or indirect estimate of the
information lought. It may be relatively
specific or include a composite of
.everal related item.. Interpretation
requires a careful evaluation of the
information obtained in relation to the
use of it.
3 It is probable that several related
analysis, chemical, biolo,ical, or
physical, will be used for interpretation
of behavior. All of these should be
con.istent in terms of the applicable
conditions. If not, it is e8lentlal to
seek an explanation of the divergence.
8 Analytical data commonly are defined in
operational terms. i. e. the term is
defined in relationship to the methodl used
to obtain a number estimate. For example,
it 18 not possible to di8tinguish preci.ely
that which 1s in soluble or suspended form
in a given sample. The e.timate of the
suspended particulates is made on the
balil of some filter media such as a
membrane fUter, glass fiber fUter,
asbestos mat fUter, paper or other. The
pore sizes vary from 0.45 microns in a
commonly available membrane fUter to
WP.SUR.in.21b. 3.71
significantly larger sizes such as for
paper fUters. A given sample may have
particulates ranging from atomic or ionic
sized materials dispersed in a soluble
form to agglomerates of colloidal or
settleable size. The pore size of the
filter used determines the fraction
retained in or on the filter media classified
as suspended Bolids.
1 Analysis based upon two different filter
media, theref"re, may give different
results for the estimate of sample
suspended solids. Interpretation of
results would require recognition of
operational dlfferences used. Similarly
a given colorimetric test may give a
high or low estimate based upon control
of interference, reagent proportions,
type of reagents and time or measure-
ment factors.
2 Conditions under which the analysis
was made such as personnel experience.
work schedule, facUities, and
incentive all contribute to validity of
the results. An inexperienced person
working in the field Is unlikely to
obtain as good an estimate as the
professional under more ideal conditions.
3 Care in sampling and interference
control has a real effect upon
analytical validity.
The interpret or must ask himself whether
the analytical results apply to the situation
and to the desired objectives. Are the
assembled data uoable, and do they con-
tain the needed items? Close coordination
is essential among the sampling, field,
laboratory and management personnel to
insure that the results are productive and
consistent. Preliminary surveys are
intended to define the problem but situ-
ations may change. Early recognition of
pOI81ble situation changes hopefully should
be followed by adjustment of the program
to develop a meanLngful package. This
means that interpretation of data should

Interpretation of Chemical Data
be started with the first daY'8 re.ults and
continue throughout the 8urvey to be sure
that the engineering, biological, chemical
and field or laboratory results are con-
sistent and that the questions asked do not
lead to a mas8 of unneeded information or
to a pop in required information. Certain
..l.cted examples illu.trato problems that
may arl8e.

A An activated sludge operation was checked
for phoephoru. removal with the' following
901, of the phosphorus was removed on the
ba.is of the determination or influent snd
.fnuent p. 530/, of removed ph08phorus
was found in eXcess activated slud,. solids.

1 Casual interpretation pruent8 no
probleme but what happened to the
difference between 90 and 53 or 370/,
01. the P involved
a Considerat1on of the analy.is involved
revealed that the teeted samples were
filtered before determinative pro-
ceuin,; therefore, the result was
not total P but filterable P, The
influent sample is likely to contain
a larger fraction of total P in filter-
able form than the efnuent sample
because of the tendency for P to
associate with biolorical growth.
This tends to increase the indicated
percentage removal of P.
b The 53% P in the exceu solids may
have been in error becau8e of the
adsorbed P washed into the filtrate,
as a result of incomplete direstion
of organic P or as a ruult of sampling
problems. More complete digestion
technlquf's were indicated to be
necessary in this situation. Incom-
plete Information is available for
decision on washout ano! sampling.
Total P on a whole sample basis
preferable for material balance
c The information furni8hed is
inlfufficient because total P was not
included In the re8ults. The fraction
0' P removed by filtration and that
not included in solids dire.tion limits

B A creek with about 0.7 cfs flow received
the discharge of an ammonia plant stabi-
lization pond discharging about I cfs.
Lees than 25 lb.. of nitrogen! day passed
a given point above the lagoon discharge.
Lagoon discharge was about 7200 Ibs. I day
of nitrogen (N) as ammor-ia. Within 3 mUes
of flow ths stream contained less than 800
Ibs. N in the dail,. flow. Organic N varied
but not enou,h to aceou.nt for 900/', N
disappearance. Oxidized N was negligible
in the sample data. Further, there were
occasional increases of NH3 -N in the
flow that could not be attributed to tribu-
taries or shoreline occur.nts.

1 Apparent lack of conversion of ammonia
to oxidized form is partially explained
in terms of the hydrolo8Y involved.
The strsam was a series of pools and
riffl... Substantial quanit1tes of
ammonia may have been converted to
nitrates by attached growth In the
riffles but converted to N gas by
denitrUication in the pools where little,
if any, 00 was detected even durIng
dayli,ht. Under thue conditions little
oxidized N would be found re,ardless
of rapid oxidation in the rUfles because
it's hard to sample riffled flow.
2 Volatility of the N as NH3 from the
now would be a possibility considering
7000 plus Ibs. of NHS -N in 1. 7 cfa or
about 1.1 mgd oC flow. 7000 in
9,200,000 represents about 750 mg/l
as NH3 -N. Volatility in the riffle
areas could account for reduction of
N to the vicinity of 200 mgll but it must
be recornized that the flow used was
that below the lagoon and did not include
downstream dilution of more than 5
tim.. within 2 mile. of flow. It does
not appear likely that volatility could
account for more than 2!3 oC the loss
in NH3 -N but may have been larger
or smaner.
3 Unexplained rises in NH3 -N are likely
to be the result of deamination in pooled
areas. Solids discharged' would settle
in pools, become anaerobic with
orpnie N hydrolyzing to amines and
subsequentl)' converted to acids and
C A Survey of the Upper Missouri River

Total carbon and total or,anie carbon
(TC and TOC) determinations indicated
that there was a general tendency for
gradual increases from upstream to
downstream sampling points above
Kansas City, Missouri. Certain Increases

were not accountable In relation to
clI.tomary Interpretation.

1 Tb1. Ulustrates ~ problem related to
the use of a l'elative1y ne" analytical
orUerion for which a hi.tory ot
cbll'acteri.tic. to guide interpretation
I. .cant.
a Conventional oxygen demand criteria
tend to show a sharp ri.e below a
'O\Irce 01 poUution followed by a
decrease with down.tream travel to
the next source of pollution. Carbon
data apparently need additional or
different correlation conliderations.

3 A gradual increa.e in alkalinity may be
expected with down.tream travel,
eftects of deposition and re.uspension
may be expected, the conversion of
0X1,en demanding materials into
hurnls may be expected. Experience
under a variety of situations is
required to reveal other expectancies
needed for Interpretation of these data.
Interpretation of Chemical Data
liI The examples illustrate three different
situations leading to some 108S of clarity:
i. e., an analytical yield problem, a situ-
ation in which the usual questions didn't quite
fit, and one in which a new criterion required
a ditterent approach.

It would be desirable to be in a position to
"follow through" and clarify each situation.
Survey economics, manpower and time
rarely permit an unUmited application of
etfort. Judicious use of past experience and
knowledge of the situation are useful to
direct resoarces Into productive channels;
prompt review ot collected data for apparent
trends helps to keep them on path or adjust
'1'01' outUne was prepared I>y 1". J. Ludzack-:
Chemil!lt, National Training Center, Office
o! Water Programs, EP A, Cincinnati, OH
AnalyUc.1 Techn:ques. Water Analysis,
Chemical Analysis, Data [nterpretation,
Balic Data Collections

Establilhed practice include II common use
otthe BOD test a8 a tool for estimatioa of
the bio-oxidizable fraction of surface waters
or wastewaters di8charged to them. Any
index including a quantity per unit time 8uch
a8 the BODs is a rate expre18ion, The
ultimate demand i. more important than any
one point on the progree8ion. The relu]tll
of a bottle test with minimum leeding and
quiescent storage are not likely to be &II
hlF all those on the same influent in a m1x-
'!ng situation and abundant seed of secondary
treatment or receiving waters. The BOD
ia"a" fraction of total oxygen requ1remenfs.
A The particular technique used for BOD
commoiU3r is specified by State .genoles
and/or 8uperv1lors. They are required
to interpret the results as obtained by
laboratory testing. It is essential that
the te.ter and the interpreters have a
common underltandine of what waa done
and how. It is highly advisable to main-
tain a liven routine until all concerned
agree upon a chan,e.
1 Each particular routine has many un-
definable factol's. The particu1ar
routine Is not as Important u the con-
siltency and capabtl1ty with which the
result was obtained.

2 This outline and Standard Methods
dilcusses several valid approaches for
obtaining BOD results. Selection of
"method" is not intended in this outline
or in the EPA Methods Manual(2).
B The common 5-day incubation period for
BOD testing is a result of tradition and
cost. Initial lags are likely to be over
and some unknown fraction of the total
oxidizable mus has been 8atilfied after
5 daYI.

C A series of observations over a period of
time makes it possible to estimate the
total oxidizable mass and the fraction
Oxidized or remaining to be oxidized at
any given time. The problem I. to define
the shape of the deoxygenation pattern and
its limits. A fair estimate of the shape of
the deoxygenation pattern is available by
observations at I, 2, or 3 days, 7 days
and 14 days, Increased observations are
desirable for more valid estimates of
curve. shape. rate of oxidation and total
oxidizable mass or ultimate BOD.
D Increasing impoundment of surface waters
and concurrent increases in complexity
and stability of wastewater components
emphasize the importance of long-term
observation of BOD, The S-day observation
includel most of the J.'eadily oxidizable
materials but a very small fraction of the
stable components that are the main factors
in impoundment behavior.
A With relatively clean surface waters. the
BOD may be determined by incubation of
the undiluted sample for the prescribed
time interval. This method is applicable
only to those waters whose BOD is less
than 8 mg/l and assumes the sample
contains lIuitable organisms and accessory
nutrients fot optimum biological
stabiliz aUon.
B Treated effluents, polluted surface waters,
household and Industrial wastewaters
commonly require dilution to provide the
excess oxygen required for the oxygen
demand determination. General guidelines
for dilution requir~ments for a given BOD
range in terms of the percent of sample in
BOD dilution water are:
For a 5-day BOD of
5-20 mgll, use 25 to 1000/, sample

For a BOD of
20-100 mg/l. use 5 to 250/, sample
For a BOD of
100-500 mgll, use 1 to 50/, sample

For a BOD of
500-5000 mg/l, use 0,1 to 1. 00/, sample
A Cylinder Dilution Technique

Biochemical Oxygen Demand Test Procedures
1 Using an anumed or estimated BOD
value as a ,uide, calculate the factors
for. a ranp 01 dilutions to cover the
desired depletions. Those dilutions
ranpng from a depletion of 2 m,/I
and a residual of 1 m,/I are most
reli8ble. At least three dilutions in
duplicate should be used for an
unknown sample.

2 Into a one-liter graduate cylinder (or
larger container if necessary) measare
accW'atel,y the required amount of mixed
sample to give one liter of dillrted waste,
Fill to the on, titer mark with dilation
water. Carefully mix. The initial DO by
calculation Includes IDOD (VIII) a
determined init.al does not. Both are
euential to estimate significance of
IDOD. Entrapment 01 air bubbles during
manipulation must be avoiaed.
3 Siphon the mixture from the cylinder Into
three 300 ml glass stoppered bottles,
filling the bottles to overflowl.ni.
" Determine the 00 concentration on One
of the bottles by the appropriate
Winkler modification and record as
"Initial DO".
5 Incubate the two remaining bottles at
200C in complete darknees. The
inclJbated bottles should be water-sealed
by hnmer.ion in a tray cr by using a
special water-seal bottle.

6 After 5 days of incubation, or other
duired interval, determine the DO on
the bottles. Average the DO concentration
of the dupUc:ates and report ae "Final 00".
B Direct Dilution Technique
1 It may be more convenient to make the
dilution directly in sample bottles of
known capacity A measured volume of
sample may be add",d (a., i.,,:Ucated in
A -1) above. and thE" bottle f11.led with dilution
water to make the desired sample
concentration for incubation. In this
caee, the saI:'!ple must be precisely
measured, the bottle carefully fUled,
but not overfUled, and tbe bottle volumes
comparable and kn,}wn. Precision is
likely tt) be poorer than for cylinder
2 Continue the procedure' aB in A-4. 5,
and 6 above.
C Seeded Cylinder Dilution Technique

1 Many wastewaters may be partially or
completely sterile as a result of
chlorination, effects of other toxic
chemicals, heat, unfavorable pH or
other factors detrimental to biological
activity. Validity of the BOD reaalt
depends .upon the presence of organisms
capable 01 prompt and effectivebio-
de,radation and favorable conditions
during the particular test. Correction
of the cause resulting in sterilization
mll8t be corrected by adjustment,
dilution. et,c., prior to reinoculation to
achieve meaningful BOD data. Receiving
water. biologically treated effluents, and
soil suspensions are a good source of
organisms likely to be adapted for
stabilizatiQn of wastewaters. Untreated
wastewaters provide numerous organisms
but are likely to contain nutrients
contributing to excessive seed corrections
and may require appreciable time for
adaptation before test waste oxidation
becomes significant.
2 The amount of added inocalant must be
determined by trial. The concentration
added should initiate biochemical
activity promptly but should not exert
enoulJh oxygen demand to unduly reduce
the oxygen available for sample
3 Estimate the sample concentration
desired in accordance with A -1 and
C-Z above and add the sample aliquot to the
dilution cylinder.

4 Add approximately half of the required
amount 01 dilution water to the sam.ple
and mix. This is necessary to assure
that the concentrated waste does not
exert a toxic effect on the seed organisms.
5 Measure a suitable aliquot of seed into
the bottle or cylinder and fill with
dilution water. Mix the combined sample.
Be-ed and dilution water without BJCcessive
air entrairunent.
II Continue a8 in III-A steps +. 5, and 6 above,

Standard Methods includes a calculation
section that il valid and concise. Preceding
it are detaUs of reagent preparation and

procedure. for the test. These will not be
1'eprinted here. Th~ sect10n conlliders
certain items that may cau.e concern about
the va11d1ty of re.ults un1e.. they are care-
~ oonsidered and controlled.

A The initial 00 of the BOD test obviously
should be hi8h. The method of attain1ng
a high DO can trap the analyst.
1 Aeration of dilution water is the most
commonly considered treatment.
Tbi. technique does produce a high DO
but it is a treacherous ally.
a Dirty air passing through clean
dilution water can produce clean
air and dirty water. This is a
slmple air-wa.hing operation.
Filtering the entering air stream
may remove brickbats and 2 x 4's,
but filters tend to pass organic
,ases, fine aero.olll, and partic-
b A stream of air passing through
water tends to co~,l the water by
evaporation 1 to 3 C below ambient
temperature. The cooled liquid
picks up more DO than it can hold
at ambient temperature. The
physical 10:3s of oxygen may produce
an erroneously high depletion value
for a determined initial DO, or a
low depletion on a calculated
initial DO. Erroneous blanks are
a particular concern. The dilution
water temperature/DO shift is
2 Raising DO by allowing the sample to
equilibrate in a cotton-plugged bottle
for 2 or 3 days permits oxygenation
with minimum air volume contact.
3 Shakinl a partially fWed bottle for a
few seconds also oxygenates with min-
imum opportunhy of gas washing con-
tamination, supersaturation. or
temperature changes.
B Seeding always is a precarious procedure
but a very necessa&')' one at tlme.. otten
the application of seed correctioDs Is a
Biochemical QxYlleR Demand Test Procedures
" if you do, if you don't"
situation. Hopefully, seed corrections
are small because each individual
biological situation is a "universe" of
its own.
1 Unstable seeding materials such as
fresb wastewater have "seed" organisms
characteristic of their origin and
,history. Saprophytes resulting in
surface water stabilization may be a
small fraction of the population. Re-
actable oxygen-demanding components
produce excessIve demands upon test
oxygen resourc~s.
2 A seed containing viable organisms at
a lower energy state because of limited
nutritional availability theoretically is
the best available seed source. An
organism population grown under
simUar conditions should be most
effective for initiating biochemical
activity as soon as the nutrient situation
favors more activity. The population
should not be stored too long because
organism redistribution and die-out
become limiting. This type of seed
would most likely be fOWld in a surface
water or a treatment plant effluent
with a history of receiving the particular
material under consideration.
3 Seed sources ar.d amounts can only be
eva}uated by trial. Different seed
sources and locations require checkout
to determine the best available material
from a standpoint of rapid initiation of
aotivity, low correction, and predictable
high oxygen depletion under test.
C Chlorination and BOD results fundamentally
are incompatible. C1llorination objectives
include disinfection as the number one
goal. Chlorine is notoriously non-specific
in organism effects. Chlorine acts like
an oxidant in the DO determination. Test
organisms are less suitable for activity
than they were before chlorination.
Nutrients may be less available after
chlorination. Certainly the conditions are
lees euitable for biological response after
chlorination. Dechlorination is feasible
with respect to the oxidizing power of

Biochemical O¥YIef1 Demand Telt Procedures
chlorine, but many organic chlorine com-
pOW1ds that do not show strong oxidizing
action still have toxic eftects on biologic
Numbers are obtainable arter dechlorination
and reseed1ng. The meaning of the.e
numbers is ob.cure. At least two Itates
(New York and New Jer8ey) specify BOD's
before chlorination only.

A The 00 test precision often has been used
to suggest precilsion of the BOD result.
00 ppecision is a relatively minor and
controllable factor contributing to BOD
results. Cther factors such as organism
suitability, members, adaptation and
conditional variables are much more
difficult to control or to evaluate.
B The Analytical Reference Service report
on Water Oxygen Demand, July, 1950
(Sample type VII) included the results of
seeded samplel of glucose- glutamic acid
BOD results from 34 agencie. on 2, 3, 5
and 7 day incubations.

The relative geometric standard deviation

Biochemical Oxygen Demand Test Procedures
2 Dilution wl.ter problem I are eliminated,
to the extent that the stream !lample may
be tested without dilution.
3 Incubator storap Ipac. becomes a real
problem for multiple sample routine.
VlI Dillolved oxygen electrodes, polarographic
and others, are feasible for ule in BOD
determinations, often makin, it pOllible to
make an elt1mate of DO or BOD when lample
interference prevent. a valid Winkler DO

Electronic probe DO makel it poslible to
determine many 8ucceutve DO's at different
time intervals on the same bottle with
ne.lipb1e lample 1088. Reaeration or
extended time seriel, therelore, are more
Anothe.. outUne in this series describes
relponle 01 reaerated BOD5 with electronic
00 probel.
A It is the responsibility of the analyst to
1 Applicability of the Ipecified technique
and lample.

2 To determine requirements for mixing
and possible thermal effects while
mixing in terms of instrument response
and biochemical reaction.
3 To evaluate long-term calibration or
standardization and their effects upon
precision and accuracy of the BOD
Immediate dissolved oxygen demand includes
dislolved oxygen utilization requirements of
subltancel 8uch 1.1 ferrous iron, sulfite
and sulfide which are susceptible to high
rl'lte r.hp.mical oxidation.
..ol.d bottl..
. .
..al.d jug U

cI.o. .ampl.,

~ioc:hemical Ox"y,en Demand Teet Procedures
A The rooD is an apparent re.pon.. as
indicated by a specilled technique. Since
00 titraUon is based upon iodine titration,
any factor that causes 12 rssponse different
from that produced by tHe reaction of KI
and molecular oxypn cOnfu8u the mOD

B IDOD Determination
1 The IOOD determination includes the
determination of 00 on a .&mule and
dilution water separately. A waste
likely to bav. a slgnifioant IDOD is
unlikely to show Ii 00.

2 According to mixing theory, it should
be poseible to calculate the 00 of any
definite mixture of the sample and
dilution water from the 00 of component
parts and their proportion.
3 The same relative proportions of sample
and dilution water should be mixed
without air entrainment and the 00
Mtvmt'l!dJUYe~~e arbitrarily selected

4 Any difference betweer. the caloulst..."
initial 00 as obtained in 2 above, and
the 00 determined in 3 above, may be
designated as looD.

5 Sample aeration, 00 interference, and
other factors at act results for mOD.
C Sample Calculation of IDOD

1 Sample 00 checked and shown to
be 0.0 mill
OOution water DO found to be 8. 2 mgll

Assume a mixture of 9 parts of dilution
water and 1 part (vi V) of sample.'
Calculated DO -
9 X 8.2 - '73.8
10 parts of the mixtw-e contain '73. 8/10
or '7. 4 m, 0011. Note that mixing has
reduced the 00 concentration because
the OriJinal amount is pre.ent in a
larpr paoka,e.

2 The mixture deBcribed abo1'e was held
for 15 minl,ite. and the DO determined
was 4.3 m,/l .

IOOD - 00 &1- OOd t X ~100 --
c c em e
-31 m, IDOD/I

1 Standard Methode, APHA-AWWA-WPCF
13th £d. 1971.
2 Methods for Chemical Analysis of Water
and Wastes, EPA-AQCL. 1971.

This outline was prepared by F. J. Ludzack,
Chemist, National Traininl Center, EPA,
WPO. Cincinnati, OH 45268.
Descriptors: Biochemical Oxygen Demand.
Chemical Analy.ie, Chemical Oxypn
Demand, Dissolved Oxygen, Water Analysis

A Winkler Di880lved Oxygen (Alaterberg
1 Manganous sulfate
2 Alkaline iodide-azide
3 SWf\&ric acid (concentrated)
4 O.03'1N IOdtum thiolulf&te
5 Starch solution
B BOD 'test
Standard Methods Dilution Water
A DO gottles (300 ml)
Three bottles for each dilution (9 bottles
for each telt); ane bottle for initial DO
and 2 bottles for incubation, each dilution.
B Siphon
C Alpirator Bu]))
D I-liter Graduated Cylinder
E Aerator Plunger
F 3 - 2 ml Transfer Pipettes
G 1 lOO-ml Graduated Cylinder
1 2~.ml Graduated Cylinder
H 2
500-ml Erlenmeyer Flaskl
[ 25-ml or 5--m! Burette, stand
J BOD Incubator (20°C)
CH.O,Bod.Jab.la. 3. '11
A Preparation of Dilution
1 Mix sample thoroughly
2 Prepare separately 10/0, 50/0, and 10'}'.
dilutions of the sample: ~dd 10 mI.
50 ml, and 100 ml of sample,
Z'e.pe~ively. to the I-liter graduated
cylinder, and fill to the I-liter mark
with BOD dilution water mix.
3 Siphon contents of graduated cylinder
into 3 DO bottles. Repeat for each
dilution (10/.. 50/. and 100/.).
B Winkler DO Determination (on one of the
three bottles for each dilution) maintain
a full siphon tube throughout transfer.

1 Add 2 ml of manganous sulfate solution,
introduced below the liquid surface.
2 Add 2 ml of alkaline iodide-azide
solution, introduced below the liquid
surface. and immediately stopper the
3 Mix the contents of the bottle by
inverting 10-15 times.
4 Permit floc to settle halfway down the
bottle. and a.gain invert the bottle and
mix the contents.
5 After settling. add 2 ml of sulfuric
add (concentra.ted). stopper, and mix.
6 Pour entire contents of the bottle into
a 500 ml Erlenmeyer flask.
'I Titrate with a. 03'1N sodium thiosulfate
solution to a pale straw color; then add
5 drops of starch solution, and continue
titration until the blue color disappears.
8 Record the vQlume of sodium thiosulfate
used; the volume in ml represents the

Laboratory for Biochemical OxYlen Demand Te.t
number of ~Wl.lram. of di..olved
oxy,en per Uter of .ample.
9 Incubate the other two bottle. for eaob
.ample dilution, I.nverted under water
or with .tOJlller-weU fiUld, 5 day. at
200 C in the dark.
10 Remove bottle. after incubation period
and perform Winkler DO determlnaUon.
a. de.crlbe4 in step. 1 thru 8.

BOD . (i.D1U.l 00 - final 00) X 100
" of ample in dilution Incubated
U the 00 initial wa. 8,0
DO final wae S.2
2'" ..mpJ. te.ted
BOD. (8.0 - 3.2)XIOO
. 4.8 X 100
.4.8 X50
. 240.' m. BOD/I
B If the ample was .ulpected of hav1ni an
immediate dillolved oxygen demand (IooD).
a calculated initial would be required.
The calcu]ated initial would be hither
than the determined initial if an looD
wal involveci.
Sample 00
. 0,0 m-'l
Dilution Water 00
. 8.2 mill
2 XO.O
. O. (j oxygen units
98 X8. 2
100 part.
804.oxy,gen unit.
. iiOi total oxygen unit.

. TOO. 8.04 mill
Unit 00 concentratl.an
for the calculated DOl' since the 001
wa. 8.0, any lOOD mUlt be allumed
Tht. outUne wal revi.ed by C.R. Feldmann,
Chemilt, EPA, WPO. National Training
Center, Cincinnati, OH 45288.
De.criptor. :
Biochemical oxygen demud, Chemical.
AnalYli., Otemical OXygen Demand,
Dluolved OXygen, Water Analyst.

The Chemical Oxygen Demand (COD) test
is a measure of the oxygen equivalent
that portion of the organic matter in a
sample that Is susceptible to oxidation
under speclfic conditions of oxidizing
agent, temperat"re and time.
B A variety of terms have been and are used
for the test described here as COD:
1 Oxygen absorbed (OA) primarily in
British practice.
2 Oxygen consumed (DC) preferred by
some, but unpopular.
3 Chemical oxygen demand (COD) current
4 Complete oxy,en demand (COD)
5 Dichromate oxygen demand (DOC)
earlier distinction of the current pre-
ference for COD by dichromate or a
specified analysis such as Standard
6 Others have been and are being used.
Since 1960, terms have been generally
agreed upon within most professional
groups as indicated in I-A and B-3 and
the explanation in B-5.
C The concept of the COD is, almost as old
as the BOD. Many oxidants and varia-
tions in procedure have been proposed,
bl,lt none have been completely
1 Ceric sulfate has been investigated,
bllt in general it is not a strong
CH.O~o~. 10e.3.74
2 Potassium permanganate was one of
the earliest oxidp.nts proposed and
until recently appeared in Standard
Methods (9th ed.) as a standard pro-
cedure. It is currently used in
British practice as a 4-hr. test at
room temperature.
a The results obtained with perman-
pnate were dependent upon concen-
tration of reagent. time of oxidation,
temperature. etc.. so that results
were not reproducible.
3 Potassium iodate or iodic acid is an
excellent oxidant but methods employing
this reaction are time-consuming and
require a very close control.
4 A number of investigators have used
potassium dichromate under a variety
of conditions. The method proposed
by Moore at SEC is the basis of the
standard procedure. (1,2) Statistical
comparisons with other methods are
described. (3)
5 Effective determination of elemental
carbon in wastewater was sought by
Buswell as a water quality criteria.

a Van Slyke(4) described a carbon
determination based on anhydrous
samples and mixed oxidizing agents
including sulfuric, chromic, iodic
and phosphoric acids to obtain a
yield comparable to the theoretical
on a wide spectrum of components.
b Van 8all, et aI., (5) used a heated
combustion tube with infrared
detection to determine' carbon quickly
and effectively by wet sample
6 Current development show", a trend to
instrumental methods autnmatin~

Chemical.Oxygen Demand and COD/BOD Relationships
conventional procedure. or to seek
elemental or more specific group
A Table 1
Te.t Te.t Reaction Oxidation Variables 
Temp. °c time .ystem  
BOD 20 days Bioi. prod. Compouad, environ-
   Enz. Oxidn. ment, biota, time,
     numbers. Metabolic
     acceptability, ,etC.
COD 146 2 hrs. 50"," H2SO4 Susceptibility of
   KZCr207 the test sample to
   May be cata- the specified
   lyzed  oxidation 
WOD 20 15' Di88. oxy,. Includes materials
     rapidly oxidized by
     dirlft action,
     Fe . SH. 
V*n Slyke 400+ 1 hr. Ht04 Excellent approach
Carbon detn.   H03  to theoretical oxi-
   H2S04  .dation for most
   K2Cr207 compound. (N -nil)
Carbon by 950 minutes OxYlen atm. Comparable to
combu.tion   catalyud theoretical for
+IR     carbon only.
Chlorine 20 20 min. HOCI soln. Good NH 3 oxidn.
Demand     Variable for other
B From Table 1 it is apparent that oxidation
Is the only common item of this series 01
.eparate tests.
a liven sample to oxidation under
specified conditions that are different
lor each test.
1 Any relationships among COD. BOD
or any other tests are fortuitous be-
cause the conditions or u,st tend to give
results indicating the 8usceptibility 01
2 If the sample is primarily composed
01 compounds that are oxidized by
both procedures (BOD and COD) a
relationship may be established.

Chemical Oxy~en Demand and COD/BOD Relations~
a The COD procedure may be sub-
!!tituted (with proper qualification!!)
tor BOD or the COD may be u!!ed
88 an indication of the dilution
required for !!etting up BOD
b If the sample is characterized by a
predominance of material that 'can
be chemically. but not biochemi-
cally oxidized. the COD will be
Ireater than the BOD. Textile
wastes, paper mill waste!!, and
other waste!! containing high con-
centrations of cellulose have a
high COD, low BOD.
c It the situation in item b is reversed
the BOD w1l1 t.e higher than the
COD. Distillery wastes or refinery
wastes may have a high BOD. low
COD. unien catalyzed by silver
sulfa te.
d Any relationship established as in
2a will change in response to
.ample hi. tory and environment.
The BOD tends to decrease more
rapidly than the COD. Biological
cell mass or detritus produced by
biological action has a low BOD
but a relatively high COD. The
COD/BOD ratio tends to increasE'
with time, treatment, or conditions
favoring stabilization.
A Advantages
1 Time, manipulation, and equipment
co.ts are lower for the COD test.
2 COD oxidation conditions are effective
for a wider spectrum of chemical
3 COD test conditions can be standardized
more readily to give more precise
4 COD results are available while the
waste is in the plant, not several
days laier, hence, plant control is
5 COD results are useful to indicate
downstream dama.ge potential in the
form of sludge deposition.
6 The COD result plus the oxygen equiva-
lent for ammonia and organic nitrogen
is a good estimate of the ultimate BOD
for many municipal wastewaters.
B Limitations
1 Results are not applicable for estimating
BOD except as a result of experimental
evidence by both methods on a given
sample type.
2 Certain compounds are not susceptible
to oxidation under COD conditions or
are too volatile to remain in the oxida-
tion nask long enough to be oxidized.
Ammonia, aromatic hydrocarbons,
saturated hydrocarbons, pyridine, and
toluene are examples of materials with
a low analytical response in the COD
3 Dichromate in hot 500/0 sulfuric acid
requires close control to maintain
safety during manipulation.
4 Oxidation of chloride to chlorine is not
closely relat~d to BOD but may affect
COD results.
5 It is not advisable to expect precise
COD results on saline water.

A The COD procedure (1) considered dichro-
mate oxidation in 33 and 50 percent sul-
Curic acid. Results indicated preferencE'
of the 50 percent aCId concentration for
oxidation of sample components. This ~s
the basis for the present standard

Chemical OXYlLen Demand and COD/BOD Relationships
B Muers(6) lugi8sted add1tlon of silver
lulfate to catal;,ze oxidation of certain
low molecular weight aliphatic acids and
alcohols. The catalyst alao improves
oxidation of most other orpmc components
to lome extent but does not make the COD
test univeraally applicable for all chemical

C The unmod1f1ed COD test relult (A) include!!
oxidation of chloride to chlorine. Each mg
of chloride will have a COD equivalent of
0.23 mg. Chlorides must be determiner1
in the sample and the COD relult corrected

1 For example, if a sample shows 300
mg of COD per Uter and 200 mg Cl-
per liter the corrected COD rewlt will
be 300 -(200 x 0.23)or 300 - 46 . 254
mg COD/Ion a chloride corrected baaia.
2 Silver suUate addition as a catalyet
tends to caule partial precipitation of
lilver chloride even in the hot acid. solu.
tlon. Chloride corrections are ques-
tionable unlesl the chloride is oxidized
before addition of silver sulfate, i. e.,
reflux for 111 minutes for chloride ox-
idation, add Ag2S0 4,'- and cont1nue the
reflux or use orHgBO.(D).

D Dobbs and Williams (7) proposed prior
complexation of chlorides with HgS04 to
prevent chloride oxidati~ during the test.
A ratio of about 10 of Hi to 1 of Cl- (wt.
balis) appears essential. The Cl- mU8t
be complexed in acid solution before addi-
tionof dichromate and silv1!r sulfate.
1 For unexplained reasonl the H,so4
complexation doel not completely
prevent chloride oxidation in the
presence of high chloride concentrations.

2 Factors have been developed to provide
some estimste of error in the re.ult
due to incomplete control of chloride
behavior. These tend to vary with the
sample and technique employed.
E It is not likely that COD resultl will be
precile for samples containing high
chloridu. Sea water cont.uns 18000 to
21000 mgCl-/1 normally. Equivalent
chloride correction for COD exceeds
4000 ml/l. The error in chloride
determination may give negative COD
results upon application of the correctiol'
Incomplete control of chloride oxidation
with H,SO" may give equaUy confusing
HgSO" appears to give predse results
for COD when chlondes do not exceed
about 2000 mil/I. Interference in-
creases with increasing chlorldes at
higher levels:

F The 12th edition of Standard Methods re-
duced the amount of sample and reagents
to 40'rr of amounts utilized in previous
editions. There has been no change in
the relative proportions in the telt. This
ateI' -s taken to reduce the cost of pro-
viding expensive mercury and silver sul-
fatel required. Results are comparable
as lon, as the proportion!! are Identical.
Smaller aliquota of sample and reagents
req..ure more care during man1J)111ation
to promote precision.
G The EP A Methode for COD
1 For routine level COD (eamplea having
an organic carbon concentration
greater than 15 mg/liter and a chloride
concentration le!!S than 2000 mg/liter),
the EPA apeeWes th~'procedures found
in Standard Methods (Z)and in ASTM(8).
2 For low level COD (samples with less
than 15 mg/liter organic carbon and
chloride concentration less than 2000
mg/liter). EPA provides an analytical
procedure (9). The difference from
the routine procedure pritnar~ in-
volves a greater sample volume and
more dilute solutions of dichromate
and ferrous ammonium sulfate.
3 For Baline samples (chloride level
exceeds 2000 mg/liter), ,fiPA provides
an analytical procedure( involving
preparation of a standard curve of COD
verSUI mgfliter chloride to correct
the calculations. Volumes and concen-
trations for the sample and reagents
are adjusted for thil type of determination.

Chemical OXYien Demand and COD/BC>D Relationships
The precision of the unmodified COD
r..ult shows.. stAndard deviation of +
4'. of the mean (3) on low chloride -
samples. SUver sulfate modified COD
results are likely to show a standard
deviation about twice that without
catalysis, due to qUeitionable chloride
behavior. The determination of chloride
frequently shows a coefficient of vari-
ation (s/x. ot 10 to 15%, hence high
chloride ..mples result U\ COD pre-
cision controlled more by chloride
behavior than organic oxidation.
Sample size and COD limits for 0.25 N
reagents are approximately as given.
For 0.025 N reagents multiply COD by
0.1. Use thE:! weak reagent for COD's
in the range of 5- 50 mgl1, (low level).
Sample Size

20 ml
10 ml
5 ml
mg COO/1

Most organic materials oxidize rela-
tively rapidly under COD test condi-
tions. A significant fraction of
ozidation occurs during the heating upon
addition of acid. The color change of
dichromate remaining. If th.. mixed
sample color changes from yellow to
green after acid addition the sample was
too large. Discard without reflux and
repeat with a smaller aliquot until the
color after mixing does not go beyond
a brownish hue. The dichromate color
change is less rapid with sample com-
ponents that are slowly oxidized under
COD reactlon conditions.
Chloride concentrations should be known
for all test samples and results inter-
preted according]y.
Special precautions advisable for the
regular COD procdure and essential
when using 0.025 N reagents include:
Keep the apparatus assembled
when not in use.
. 2
Plug the condenser breather tube
with glass wool to minimze dust
Wipe the upper part of the flask
and lower part of the condenser
with a wet towel before disassembly
to minimize sample contamination.
Steam out the condenser after use
for high concentration samples and
periodically for regular samples.
Use the regular blank reagent mix
and heat, without use of condenser
water, to clean the apparatus of
residual oxidizable components.
Distilled water and sulfurnie acid
must be of very high quality to
maintain low blanks on the refluxed
samples for the 0.025 N oxidant.
Certain portions of this outline contain
training material from prior outlines by
R. C. Kroner, R. J. Lishka, and
J. W. Mandia.
Moore, W. A.. Kroner, R. C. and
Ruchhoft, C. C. Anal. Chern.
21:953. 1949.
Standard Methods, 13th Edition, APHA-
AWWA-WPCF, 1971.
Moore, W. A., Ludzack. F. J. and
Ruchhoft. C. C. Anal. Chern.
23: 1297, 1951.
Van Slyke, D. O. and FOlch, J. J.
BioI. Chern. 136:509, 1940.
Van Hall, C. E., Safranko. J. and
Stenger. V. A.. Anal. Chern.
35:315. 1963.

CMInical Oxy.pn Demand and CoP/BOP Relation.hip
Muerl, M. M. J. Soc. Chem.
Ind. (Londoft) U:711, 19S8.
Dobb., R. A. and WilUam., R. T.,
Anal. Chem.. 35:1064, 1963.
ASTM Stan~rd8, Part 23, Water:
Atmolpher1c Analyli., 1972.
Method. for Chemical Analy.i.
of Water and Wa.te., EPA-
AQCL, Cincinnati, OH, July
Thi8 out Un. was prepared by PO. J.
Lud..ck, Chem!8t, EPA, WPO, National
Tra1ntn, Center, Cincinnati, OR 45268.
D..crtptp Biochemical Ox1pn Demand,
Chemica, Chemical Aaaly81., Chemical
Oxypn Demand, Chemical Reactions,
Chloride., rn..olved Ox)',en, Orpnic
Compound., Oxidation, Oxypn. Oxygen
Demand, Oixypn Requirement., Saline
Water, Water Analylis

A Standard Pota.sium Dichromate (0.250 N):

Dissolve 12.258 g of primary standard
grade K"Cr ° , which had been dried at
1030 C ftlr tt.o "hours, in distilled water and
dilute to one liter.
B Ferrous Ammonium Sulfate (0. IN):

Dissolve 39 g of Fe (NH4)2 (504)2' 6H20
in distilled water.
Carefully add 20 ml of concentrated H2SO4 .
Cool and dilute to one liter.
C Ferroin Indicator:

Diuolve 1. 485 it I, 10-phenanthroline
monohydrate and 0.895 g FeSO . 7H ° in
water and dilute to 100 ml. Th1s indicator
may be purchased already prepared.
D Concentrated SulfurIc Acid (36 N):
E Mercuric Sulfate: Analytical Grade
F Silver Sulfate: Analytical Grade
G Concentrated Sulfu!'ic Acid - Silver Sulfate:

Dissolve 22 g of .ilver sulfate in a 9 lb
bottle of concentrated sulfuric acid.
(1-2 days required for d188olut1on)
Before use, the Erlenmeyer flask (500 mI.
24/40 standard taper joint) and reflux con-
denser (Friedrichs, 24/40 standard taper
joint) should be steamed out to remove trace
organic contaminants. Add 10 ml of
0.250 N K2.Cr207' 50 ml of distilled water,
and severlD b\Iinping stones to the flask.
Carefully add 20 ml of concentrated H2S04
and mix thorough~. Connect the flaSK
and condenser, but do not turn on the water
to the condenser. Bol1 the mixture so that
CH.O.oc.lab.3b. 3. 74
steam emerges from the top of the con-
denser for several minutes. Cool the
mixture, carefully discard the acid. and
rinse the condenaer and flask with distilled
water. In order to prevent contamination
from air -borne particles, the top of the
condenser should be lightly plugged with
glass wool during storage and use.
A Dilute 10.0 ml of the standard
potassium dichromate to about 100 ml
with distilled water.
B Add 30 ml of concentrated H2SO4 and
allow to cool.
C Add 2 -3 drops of ferroin indicator and
titrate to a red:lish-brown end point
with the ferrous ammonium sulfate.
Calculate the normality. N, of the
ferrous ammonium sulfate.
D Calculation
N of Fe (NH4)2 (S04)2 .

ml K2Cr207 XN of K2Cr207
ml Fe (NH4)2 (804)2
A Place 0.4 g HgSO 4. in a 500 ml 24/40
standard taper Er1.enmeyer flask.
Add 20 ml of sample, or an aliquot
diluted to 20 ml with distilled water.
B Add 2 ml of concentrated H2S04 and
swirl to dissolve the HgS04'

Laboratory Proc:edlll'e for RQUUne Level Chemical Oxy,en Demand
C Add 10.0 ml of the 0.250 N ~Cr207
and mix.
D Carefully add 28 ml of the Bulturtc acid -
silver sulfate reageftt.
E Add several pumice eranule8 or ,lu,
beadB to prevent bumping, and theft
swirl the mixture to inlUre complete
F Refiux the mixture for two hoa1'l.
G A Uow the solution to cool, waBh down
the condenBer with di8tiUed water, and
add a))out 75 ml water to brill, the
volUme to about 150 mi.
H Add 2 -3 drop. of the ferroiA indicator
and titrate the !!olution to a reddi.h-
brown end point with the ferrous
ammonium .ulfate.
I A blank consisting of 20 ml of distilled
water and containing all rea,snt. ie
refluxed and titrated in the ..me manner
ae the eample.
J Calculation

mg COD/I. (A -B) N X 8 X 1000
ml of sample
COD. chemical oxygen demand
A . ml Fe (NH4)2 (50.)2 used lor blank
B . ml Fe {NH4)2 (504)2 used lor sample
N . N of Fe(NH4)2 (804)2
8 . equivalent weight' of oxygen
Portion. 01 this outline were taken from
an outline prepared by R. J. U8ka.
1. Methoda lor Chemical Analysis of
Water & Wastes, EPA-AQCL, 1971.
2. Standard Methods, APHA-AWWA-
WPCF, 13th Edition. 1971.
3. ASTM Standards. Part 23, 1970.
This outline was prepared by Charles R.
Feldmann, Chemist, EPA. WPO National
Train1nf Center, Cincinnati, OK 452811.
Descriptor~ : Chemical Analy8is, Chemical
Oxygen Demand, Organic Compounds,
Oxidation. OxYlen, Oxygen Demand, Oxygen
Requirement., Water Analyei.s

Determination By The Winkler lodometrie Titration and Azide Modification
The ba.ic Winkler procedu.re (1888) has been
modified many tim.. to improve its work-
ability in polluted waters. None of these
modincations have been completely
lucce88ful. The most useful modification
wa. proposed by Aillterberg and consists of
the addition of .odium azide to control
nitrite interference durin, the iodometric
titration. The A zide modification of the
iodometric titration is recommended as
official by the EP A - WPO Quality Control
Committee for re1atively clean waters.
The determination of DO involves
a complex series of interactions
that mUlt be quantitative to provide
a valid DO re.ult. The number of
sequential reactions also compli-
cates interference control. The
reaction. will be presented first
followed by di.cu.sion of the
functional aspects.
MnS04 + 2 KOH -Mn(OH)2 + K2S04 (a)
2 Mn(OH)2 + 02 -2 MnO(OH)2 (b)
Dissolved OKypn Determination
times. allowing the solids to
lettle half way and repeat the
procells. Reaction il rapid;
contact ill the principal
problem in the two phase
lf the alkaline floc il white,
no oxygen is present.
Acidification (reactions c and d)
chan,es the oxy,enated manpnic
hydroxide to rnanpnic lulfate
which in turn reacts with
potassium iodide to form elemental
iodine. Under acid condition.,
oxygen cannot react directly with
the excess manpnous sulfate
remaining in solution.
Iodine (reaction e) may be titrated
with sodium thiosulfate standard
solution to indicate the amount of
diesolved oxygen orilinally
pres.nt in the nmple.
The blue color of the starch-
iodine complex commonly is
used as an indicl.tor. This
blue color diAppears when
elemental iodine ha. been
reacted with an equivalent
amount of thiosulfate.

Phenylanine oxide .olutions are
more expensive to obtain but
have better keeping qua11tie.
than thiosulfate solutions.
Occasional USI, field operations
and situation. where it is not
tealible to calibrate thio
.olutlons reaularly, UluaU.v
encourage use gf purchaseel.
PAO rea..ot..
For practical purpo.es the 00
determination Icheme involve. the
following operation..
Fill a 300 ml bottle* un~
condition. minimizinl 00
changes. This mean. that the
sample bottle must be flushed
with test solution to diaplace
the air in the. bottle with water
chara.cteristic of the te.ted
sample. .
.00 teat bottle volu.m1l11 lOould
be checked - d1lcard those
ollt.ide of the limits gf 300 ml
+ or - 3 ml.
To the filled bo~tle:
Add .MnSO 4 reagent (2 ml)

Add KOH, KI. NaN3 reagent
(2 m!)
Stopper. mix by inver$ion,
allow to settle half way and
repeat the operation.
Hiahly saline t..t waters
commonly lettle very
slowly at this Itage and
may not settle to the. half
way point in the ,time,
To the alkaline mix (se.tUed
about half way) add 2 ml of
slllfllr1c acid. stopper and mix
unUl the precipitate dissolves.
Tranaf.,r the contents,oftha
bottle to a 500 ml Erlenmeyer
flask and titrate with 0.0375
Normal Thiosulfate"'. Each
ml of reagent used repre.ents
1 ma of oo/11ter of .ample.

The same thing applies for
other sample volumes when
using an appropriate t1trant
normality such as:
For a 200 ml sample, use
0.025 N Thio
For a 100 ml sample, use
0.0125 N Thlo
"'EP A- WPO Method
The addition of the Hut two 00
reagents, (MnS04 and the KOH. KI
and NaN3 solut1ons) displaces an
equal quantity of the sample. This
is not the cue when add is added
because the clear liquid above the
floc does not contain di880lved
oxygen a8 all of it should be con-
verted to the particulate MnO(OH}2'
Some error i8 introduced by this
displacement of sample during
dosa,e of the Hrst two reagents,
The error upon addition of 2 ml of
each reagent to a 300 ml sample
Is 3~ X 100 or 1.330/. 1088 in 00.

This may be corrected by an
appropriate factor or by adjust-
ment of reagent normality, It is
generally considered small in
relation to other errorB in sampling,
manipulation and interference.
hence thi8 error ma,)' be recognized
but not corrected,
Reagent preparation and pro-
cedural detail8 can be found in
reference 1.
IV The 8equent1al reactions for the
Chemtcal DO determination provides
several situations where 8ignificant Inter-
ference may occur tn application on
poUuted water, Buch as:
Sampling errors may not be strictly
d.stgnated a8 Interference but have the
same effect of changing sample DO.
Inadequate flushing of the bottle con-
tent8 or exposure to air may raiBe the
DO of low oxygen samples or lower the
DO of 8upersaturated samples.
Dissolved Oxygen Determination
B Entrained air may be trapped in a DO
bottle by:
Rapid fUling of vigorously mixed
samples without allowing the
entrained air to escape before
cloBing the bottle and adding DO
Filling a bottle with low temperature
water holding more DO than that in
equilibrium after the samples warm
to working temperature.
Aeration is likely to cool the sample
permitting more DO to be introduced
than can be held at the room or
incubator temperatures.
Samples warmer than working
incubator temperatures will be
only partially full at equilibrium
Addition of DO reagents res1.11ts in
reaction with dissolved or entrained
oxygen. Results for DO are invalid
if there is any evidence of gas
bubbles in the s~mple bottle.
C The 00 reagents respond to any oxidant
or reductant in the sample capable of
reacting within the time allotted. HOCI
or H202 may rai!,e the DO titration
while H2S, & SH may react with sample
oxy,en to lower the sample titration.
The items ment10ned react rapidly and
raise or lower the DO r.f-t'ult pr~mptly.
Other items such as Fe or SO may
or may not r~act completely wit5in the
t1me allotted for reaction. Many.
organic material8 or complexes from
benthic deposits may have an effect 1.1pon
00 result~ that are difficult to predict.
They may ha,'e one effect during the
alkaline stage tL' release iodine from
Kl while favoring lrreversible
absorption of iodine r!uring the acid
stage. Degree of effer:t may increase
with reaction time. It is generally
inadvisable to use the iodometric
titration on 8&.mples containing large
amounts of organic contaminants or

Di.solved Oxygen Determination - II
benthic residues. It would be expected
that benthic residuel would tend tOY(ard
low results because of the reduced iron
and sulfur content - tbeycommonly
favor high results due to other factors
that react more rapidly, often giving
the same effect as in uncontrolled
nitrite interference durinl titration.
Nitrite is present to some extent in
natural waters or partially oxidized
treatment plant sample.. Nitrite is
associated with a cyclic reaction during
the acid S1&le of the 00 determination
that may lead to erroneous hi,h result..
These reactions may be repre-
sented &s follows:
2HNO + 2 HI .....1:2 + 4H20 + N2.02
t 2 I
211tN02 - + 1/202 + H20 + N202
Tnese reactiona are time, mixing
and concentration dependent and
can be minimized by rapid
Sodium azide (NaN3) reacts with
nitrite undel" acid conditions to
form a combination of N2 + N20
which effectively blocks1he
cyclic reaction by converting the
HNO to nonintertering compounds
of nilrogen.
Sodium azide added to fresh
alkaline KI reagent is adequate to
control interference up to about
20 mg of NO - N/ltter of sample
The azide is utstable and grad-
ually decomposes. If reBuspended
benthic sedim.ents are not detectable
in a Ample showing a returnine
blue color. it il likely that the
a zide has decompo.ed in the
alkaline KI azide reagent.
Surfactants, color and Fe+++ may
confuse endpoint detection if present
in sihttlificant quantities.
F Polluted water commonly contains
sirnificant interferences such as C.
It is advisable to use a membrane
protected sensor of the electronic type
for DO determinations in the presence
of these type. of interference.
G The order of reagent addition and prompt
completion of the DO determination i8
critical. Stable waters may give valid
DO results after extended delay of
titration durina the acidified stage. For
unstable water, u.due delay at any stage
of proc...in. accentuates interference
This outline COII.t&1n8 sijnif1cant materials
from previous outlines by J. . W. Mandia.
Review and comments by C. R. Hirth and
R. L. Booth are 8I'satIy appreciated.
This outline was prepared by F. J. Ludzack.
Chemist, National Training Center, EPA. I
WPO, Cindnnati, OH 45268, and revised
by C. R. PeldlD8lllD. Chemist, National
Training Center.
Descriptors: Chemical Analysis, Dissolved
Oxygen, OXy,en. Water Analysis

A Electronic measurement of DO is attractive
for leveral rea80ns:
1 Electronic methods are more readily
adaptable for automated analysis, con-
tinuous recording, remote sen8ing or
2 Application of electronic method. with
membrane protection of lIensorl affords
.. 'h1,h de ire. 01 interference control.
3 Versatility of the electronic system
permits de8ign for a particular measure-
ment, situation or ule.
~ Many more determinations per man-
hour are po88ible with a minor expend-
iture of time for calibration.
B EIsctronic methods of analysis imp08e
certain restrictions upon the analY8t to
inlure that the re8ponse does, in fact,
Indicate the item sought.
1 The ease of reading the indicator tends
to produce a false lIense of security.
Frequent and careful calibration8 are
e..ential to establish workability of the
apparatus and validity of its re8ponse.
2 The use of electrGnic devices requires
a greater de,ree of oompetence on the
part of the ana~8t. Underltanding of
the behavior of oxygen mult be supple-
mented by an under.tandin. of the
particu1&r Inltrument and itl behavior
durin, u.e.
C Definitions
1 Electrochemistry - a branch of chemistry
dealing with re1&tionlhips between
electrical and chemical changes.
2 Electronic measurements or electro-
metric procedures - procedures using
the measurement of potential differences
as an indicator of reactions taking
place at an electrode or plate.
3 Reduction - any process in which one
or more electrons are added to an atom
or an i"n, such as 02 + 2e - 20.
The oxygen has been reduced.
4 Oxidation - any process in which one
or more electrons are removed from
an atom ar an ion, such as Zno - 2e
- Zn +2. The zinc has been oxidized.
5 Oxidation - reduction reactions - in a
8trictly chemical reaction, reduction
caDnOt occur unless an equivalent
amount af some oxidizable substance
has been oxidized. For example:
2H2 + 02
2HZ - 4e
02 + 4e
= 2H20
= 4H +1

= 20-2
hydrogen oxidized
oxygen reduced
ChellLical reduction of oxygen may alllo
be accomplished by electrons supplied
to .. 110ble metal electrode by a battery
or other energizer.
6 AnOde - an electrode at which oxidation
of .e reactable substance occurs.
7 CatbDde - an electrode at which
re.t1on of some reactable substallicc
OCCurl. For example in I. C. 3. the
reduction of oxygen occurs at the
8 Electrochemical reaction - a reaction
involving simultaneous conversion of
chemical energy into electrical energy
or the reverse. These conversions are
Note' Mention of Commeretal Products and Manufacturers Dof'S
. Not Imply Endorsement by the Environmental Protection
CH.O, do. 32a. 3,74

Ct..olved OxYien Determination
equivalent in termB of chemical Uld
electrical energy and ,enerally are'
9 Electrolyte a Bolution. ,el, or mixture
capable of conductin, electrical enerlD'
and Bervin. a. a reaatiq media for
chftnical chanleB. The electrolyte
commonly contain. an appropriate
concentration of selected mobile ions
to promote the desired reactions.
10 Electrochemical cell - a device con-
sLatinl of an electrolyte in which 2
electrodes are immersed and connected
via an external metaUic conductor.
The electrodes may be in separate
compartments connected by tube con-
tain1nl electrolyte to complete the
internal circuit,
a Galvanic (or voltaic) cell - an
electrochemical cell operated in
such a way as to produce electrical
enerlD' from & chemical chanp,
web as a battery (lee FtlUre 1).
II. --
,"'" 1
0(/.,2 - 2
b Polaroar.pltic (electr~c) cell-
an electrochemical cell operated in
such a way &8 to produce. chemical
chance from eleetrical ner"
(See Fipre 2).
'." "
D As indicated in I. C. 10 the sip of Ul
electrode may ehanae a8 a reault of the
operatiDImode. The cOlWeraion br the
reactant of primary 1aterest at a liven
electrode therefore d"ilaate.. teradnoJ.ory
for that electrode &Dd operaUnt mode.
In electl'Oldc QX7Pft "..... U-
electrode at which o~..n reducU- OCCur8
Is des,,-ted the cathode.

E Each cell type has ebara-ateristte ad_n1aps
and l1mitatloD8. Botb may be UNCI
1 The ..!vanie cell dep.d8 upon
me.surement of electrical ellel'&r
produced as a result of OIIiYpn

reduction. It the oxygen content of the
aample is negligible, the measured
current is very low and indicator driving
force is ne,l1g1ble, therefore response
time is longer.
2 The polarographic cell uses a standing
current to provide energy for oxygen
reduction. The indicator response
depends upon a chan,e in the standing
current as a result of electrons
released during oxygen reduction.
Indicator response time therefore is
not dependent upon oxygen concentration.
3 Choice may depend upon availability,
habit, accessories, or the situation.
In each case it is necessary to use
care and judgment both in selection
and use for the objectives desired.
A Reduction of oxygen takes place in two
steps as shown in the following equations:
1 02 + 2H20 + 2e - H202 + 20H-
2 ~02 + 2e - - 20H-
Both equations require electron input to
activate reduction of oxygen. The first
reaction is more important for electronic
00 measurement becaul!le it occurs at a
potential (voltage) which is below that
required to activate reduction of most
intertering component!! (0.3 to 0.8 volts
re]ative to the l!Iaturated calomel electrode -
SCE). Interferences that may be reduced
at or below that required for oxygen
Ulualq are present at lower concentrations
in water or may be minimized by the use
of a selective membrane or other means.
When reduction occun, a definite quantity
of electrical energy is produced that is
proportional to the quantity of reductant
e"tering the reaction. Resulting current
mealurementl thu. are more Ipecific for
Oxygen reduction.
B MOlt electronic meaaurementa of oxygen
are baaed upon one of two techniques for
evaluating oxygen reduction in line with
Dis801ved Oxygen Determination
equation n.A. 1. Both require activating
energy, both produce a current propor-
tional to the quantity of reacting reductant.
The techniques differ in the means of
.upplying the activating potential; one
employs a source of outside energy, the
other uses I!Ipontaneous energy produced
by the electrode pair.
1 The poJarographic oxygen sensor
relies upon an outside source of
potential to activate oxygen reduction.
Electron gain by oxygen changes the
reference voltage.
a Traditionally, the dropping mercury
electrode (DME) has been used for
polarographic measurements. Good
results have been obtained for 00
using the DME but the difficulty of
maintaining a constant mercury drop
rate, temIJerature control. and
freedom from turbulence makes it
impractical for field use.
b Solid electrodes are attractive
because greater surface area
improves sensitivity. Poisoning
of the solid surface electrodes is
a recurrent problem. The use of
selective membranes over noble
metal electrodes has minimized
but not eliminated electrode con-
tamination. Feasibility has been
improved liufficiently to make this
type popular for regular use.
2 Galvanic oxygen electrodes consist of
a decomposable anode and a noble
metal cathode in a suitable electrolyte
to produce activating energy for oxygen
reduction (an air cell or battery). Lead
is commonly used as the anode because
its decomposition potential favors
spontaneous reduction of oxygen. The
process is continuous as long as lead
and oxygen are in contact in the electrolyte
and the electrical energy released at
the cathode may be dissipated by an
outside circuit. The anode may be
conserved by limiting oxygen a va ilability.
Interrupting the outside circuit may
produce erratic behavior for a time
after reconnection. The rE'f'ulting

Dil80lved OxYJlen DetermillaUon - n
current produced by oxygen reduction
may be converted to oxnen conoen-
tration by ule of a sensitivity coefficient
obtained during calibration. Provision
of a pulsed or interrupted lignal makes
it pOBBible to amplify or control the
sianal and adjust it for direct readinr
in terms of oxygen concentration or to
compensate for temperature effects.
A Polarographic or galvanic DO instruments
operate as a result of oxygen partial
pressure at the sensor lurface to produce
a signal characteristic of oxYlen reduced
at the cathode of some electrode pair.
This lignal il conveyed to an indicating
device with or without modification for
sensitivity and temperature or other
infiuences dlpending upon the inltrument
capabilitie8 and intended ule.
1 Many approaches and refinements have
been used to improve workability,
applicability, validity, stabUlty and
control of variables. Developments
are continuing. It Is posllble to produce
a device capable of meetini any reasonable
situation, but situations cWfer.
2 Most commercial DO instruments are
designed for use under lpecUied con-
ditions. Some are more veratile than
others. Benefits are commo~ reflected
in the price. It is essential to deter-
mine the requirements of the measure-
ment situation and objectlv.. for use.
Evialuation of a given instrument in
tetms of sensitivity. response time,
portability, stability, service
chll.racteristics, degree of automation,
and conststency are used for judgment
on a cost/benefit basil to select the
most acceptable unit.

B Variables Affecting Electronic 00
1 Temperature affects the solubility of
oxygen, the magnitude of the resulting
signa 1 and the permeabWty of the
protective membrane. A curve of
oxygen solubility in water versus
increasing temperature may be concave
downward witHe a simOar curve of
sensor reaponse versus temperature
is concave upward. Increasing
temperature decreases ~"11 solubWty
and increase I probe sensitivi~ and
membrane permeability. Thermistor
actuated compenaatlon ot probe
responle based upon a linear relation-
ship or average of oxygea. .01ubl11ty
and electrode sensitivity 18 not precisely
correct al the maximum .,read in
curvature occurs at about noc with
lower deviations from linearity above
or below that temperature. U the
instrument il calibrated at a temperature
within + or - 50 C of wor~g temperature,
the compenl!l&ted readout is likely to be
within 20/0 of the real value. li)epending
upon probe geometry, the laboratory
sensor may require 4 to 6" carrection
of signal per 0 C change in liquid
2 Increasing pressure tends to increase
electrode response by compression
and contact effects upon the electrolyte,
dissolved gases and electrode surfaces.-
As long as entrained gases are not
contained in the electrolyte or under
the membrane, these effects are
Inclusion of entrained pses results in
erratic response that increases with
depth of immersion.
3 Electrode sensitivity changes occur as
a relUlt of the nature and concentration
of contaminants at the electrode sur-
facel and polBible physical chemical or
electronic side reactions produced.
1be.. may take the form of a physical
barrier, internal short, high residual
current, or chemical chan,es in the
metal surface. The membrane is
intended to anow dissolved gas pene-
tration but to exclude passage of ions
or particulates. ApP:1rently some ions
or materials producing extraneous ions
within the electrode vicinity are able
to pass in limited amount. which

become significant in time. Dissolved
pses include 1) oxygen, 2) n1trol~n,
3) carbon dioxide, 4) hydrogen sulfide,
and certain others. Item 4 is 11bly to
be a major problem. Item 3 may pro-
duce deposits in alkaline media; most
electrolytes are alkaline or tend to
become so In line with reaction II. A . 1.
The usable life of the sensor varies
with the type of electrode system,
surface area, amount of electrolyte
and type, membrane characteristics,
nature of the samples to which the
system is expoled and the length of
exposure. For example, galvanic
electrodes used in activated sludge
units showed that the time between
cleanup was 4 to 6 months for electrodes
used for Intermittent daily checks of
effluent 00; continuous use In the mixed
liquor required electrode cleanup In 2
to" weeks. Each electrometric cell
configuration and operating mode has
its own response 'characteristics.
Some are more stable than others.
It is necessary to check calibration
frequency required under conditions
of use as none of them will maintain
uniform response indefinitely. Cali-
bration before and after daily use is
4 Electrolytes may consist of solutions
or gels of ionizable materials such as
acids, alkalies or salts. Bicarbonates,
KC1 and KI are frequently u8ed. The
electrolyte i8 the transfer and reaction
media, hence, it necessarily becomes
contaminated before damage to the
electrode surface may occur. Electro-
lyte concentration, nature, amount and
quality affect response time, sensitivity,
stability, and specificity of the sensor
system. Generalq a small quantity of
electrolyte gives a shorter response
time and higher sensitivity but also may
be affected to a greater extent by a
given quantity 01. contaminating sub-
5 Membranes may consist of teflon,
polyethylene, rubber, and certain
other polymeric films. Thickness
may vary from 0.5 to 3 mils (inches X
1/1000). A thinner membrane will
Dissolved Oxygen Qetermination
decrease response time and increase
sensitivity but is less selective and
may be ruptured more easily. The
choice of material and its uniformity
affects response time. -selectivity and
durability. The area of the membrane
and its permeability are directly
related to the quantity of transported
materials that may produce a signal.
The permeability of the membrane
material is related to temperature and
to residues accummulated on the
membrane surface or interior. A
cloudy membrane usually indicates
deposition and more or less loss of
6 Test media characteristics control the
Interval of usable life between cleaning
and rejuvenation for any type of
electrode. More frequent cleanup is
euential in low quality waters than for
high quality waters. Reduced sulfur
compounds are among the more
troublesome contaminants. Salinity
affects the partial pressure of oxygen
at any given temperature. This effect
is small compared to' most other
variables but is significant if salinity
changes by more than 500 mgll.
7 Agitation of the sample in the vicinity
of the electrode is important because
00 is reduced at the cathode. Under
quiescent conditions a gradient in
dissolved oxygen content would be
established on the sample side of the
membrane as well as on the electrode
side. resulting in atypical response.
The sample should be agitated
sufficiently to deliver a representative
portion of the main body of the liquid
to the outer face of the membrane.
It i8 commonly observed that no
agitation will result in a very low or
negigible response after a short period
of time. Increasing agitation will cause
the response to rise gradually until
some minimum liquid velocity is reached
that will not cause a further increase
in response with increased mixing
energy. It is important to check
mixing velocity to reach a stable high
signal that is independent of a reasonable
change in sample mixing. Excessive

Dl..olved OxY.len Determinat10n
mix,"-. may create a vorte:.: and e:.:po.e
the .en.1nJ .urface to all' rather than
.ample liquid. Thi. .hould be a"lded.
A Unear liquid veloolty 01 about. 1 tt/.ec
at the .en.1nJ .urface 11 \l8uaU;r
. DO ..nlor re.ponse repre..m. a
potential or current 11analin the
mllU-volt or mllU-amp raftie 1D a
hl.h res18tance syetem. A h1lh quality
electronic in.trument 18 e..ential to
maintain a u.able eipl-to-no1.e ratio.
Some 01 the more common d1ft1.cu1tie.
a Variable Une volta,e or low batterl..
in amplifier power circuit..
b Subltandard or unat_d,y amp.\1fier
or reliltor componentl.
c Undependable contact. or junctionl
In the .Inlor, connectiq cablel, or
in.trumuat control circuit..
d Inadequately ehielded electroQic
e I!;xce..ive exposure to mollture,
fume. or chem1cala In the wrona
,lacel l8d to 8tray currents,
inteI'M1 .hort8 or other ma11\anctioo.
C De.irable Features in a Portable DO
1 The unit .hould include stead,y state
performance electronl.c and indicatin.
componlntl in a convenient but Murd)'
paclca.e that i. 8maU ellOqtI to carry.

:I Thlre .hould be provillDzUl lor addition
01 .pecial acoee.orlll 8\lch a. bottle
or field .enlOl'8, ailtator., recorders,
line extlnsion8, II needed lor IpecU1c
requirem6ntl. Such addition. lhould
be read~ attachable and detachable
and maintain lOod wor~, character18t1c8.
3 The instrument should include a
sensitivity adjustment which upon
calJbration will provide lor dirlct
readil1i in terms of m. 01 DO/UteI'.
. Temperature cOmpenllation and temp-
erature readout Ihould be incorporated.
s rlUC in cOl1tact. .hou!d be positive,
lturd)', readily cleanable and situated..
to miD1mhe contamination. Water
leail 8bould be provided wllere
6 The senIOr shcNld be lultaJaJ;y de81gned
lor the purpole intended in terms of
8enlltivity, relpon8e, stabWt;y. and
prctection durin, u.e. It 8bouJd be
ealY to clean. and reassemble for use
with a minimum 1088 of lervice time.
7 Switchel, connectifti plup, aDd con-
tacts prelerabq 8hould be located on
or 1n the 1DItrument bO:K ratheJ;' than
at the "wet" end of the liDe near the
lenlOr. Connectin, cables Ibould be
multiple .trand to minimize 8eparate
line.. Calibration controll 8tMNid be
convenient but dellaned 80 tlat It Is
not likeq that they wlU be inadYertently
8hitted durin. u.e.
8 Alitator acce..odel for bottle use
impole .pecial problems becaOe they
.hould be smaU, selt contained. and
readily detachable but sturd,y 8IIOUgh
to live polmve aptation and electrical
continuity in a wet zone.
9 Major load batteries lbou1d be
reclarpable or readi~ rep1aeeable.
Line operation lbou1d be leaslble
wherever pollible.
10 Service and repJacement parts avail-
abWt;y are a primary consideration.
Drawiftil, part8 identU1cat1Da and
troubll Ibootin, memos should be
1Dcorporated with appUc:able operat1n1
in.tructiona 1n the 1n8trum.t _nual
1n an1nlonnative orpnized fonn.
D SenIOr and Inltrument Calibration
The instrument box is like~ to have some
form of check to ver1l;y electronic.,
battery or other power supp~ conditions
lor u... The lensor commonq 18 not
included in this check. A known reference

tamp1e uled with the inltrument in an
operating mode 1. the bellt aval~le
method to campen. ate for lIenlor variablee
under use condition.. It il adv1..ble to
caUbrate before and after daily use under
teet condition.; Severe condition., changes
in conditions, or possible damale call for
calibrations durin, the use period. The
readout scale 18 likely to be labeled -
caUbraUon is the buis for thi. label.
The following procedure is recommended;
1 Turn the in.trument on and allow it to
reach a stable condition. Perform the
recommended instrument check as
outUned in the operating manusl.
2 The instrument check ullually includes
an electronic zero correction. Check
each instrument again.t the readout
scale with the sensor immerlled in an
a,itated solution of sodium sulfite
containing suff1cient cobalt chloride to
catalyze the reaction of sulfite and
oxygen. The indicator should stabilize
on the zero reading. If it does not, it
may be the re.ult of residual or stray
currents, internal shortin. in the
electrode, or membrane rupture.
Minor adjuetments may be made using
the indicator rather than the electronic
controls. Ser1ou8 imbalance requires
electrode recondLtion1ng if the electronic
check is O. K. Sulfite must be carefully
rineed from the sensor until the readout
etabilizes to prevent carryover to the
next sample.
3 Fill two 00 bottles with replicate
lamples of clarified water similar to
that to be tested. This water should
not contain lip1ftcabt test interferences.

4 Determine the DO in one by the azide
modification of the iodometrio titration.
5 Insert a maanetic stirrer in the other
bottle or uee a probe agitator. Start
..itation after in.erUon of the sensor
a8lembly and note the point of
Dissolved Oxygen Determination
a Adjust the instrument calibration
controlU necellary to compare
with the titrated 00.
b If sensitivity adjustment is not
po. sible, note the instrument
stabilization point and designate
it a8 ua. A sensitivity coefficient,

4> is equal to ~ where DO is the

titrated value for the sample on
which us was obtained. An unknown

DO then becomes 00 .. !!!.. This
factor is applicable as long as the
sensitivity does not change.
6 Objectives of the test program and the
type of instrument influence calibration
requirem~nts. Precise work may
require calibration at 3 points in the
00 range of interest instead of at zero
and hieh ran.e 00. One calibration
point frequently may be adequate.
Calibration of a DO sensor in air is a
quick test for possible changes in
sensor response. The dUference in
oxypn content of air and of water il
too large for air calibration to be
lattlfactory for precise calibration
for Ule in water.
IV This section reviews characteristics of
several sample laboratory instruments.
Mention of a soecUic instrument does not
imply EPA endorsement or recommendation.
No attempt has been made to include all the
available instruments; those described are
used to indicate the approach used at one
stage of development which may 0[' may not
reprellent the current avaUable model.
A The electrode described by Cardt and
Kanwisher (1) is illustrated in Figure 3.
This electrode was an early example of
those using a membrane. The anode was
a silver - silver oxide refereftce cell with
a platinum disc cathode (1-3 cm diameter).
The salt bridge consisted of N /2 KCI and

Diaaolved Oxypn Determination
KOH. The polyethylene membrane was
held in place by a retaining rine. An
applied current was used in a polaro8I'aphic
mode. Temperature effects were relatively
large. Thermi.tor correction was Itudied
but not integrated with early models.
B The Beckman oxygen electrode 1.8 aIIOther
iUultrat10n of a polarographic 00 .ensor
(Figure 4). It consists of a 101d cathode,
a sHver anode, an electrolytic ,el oon-
taWng KCl. covered by a teflon membr..ne.
The instrument has a temperature readout
and compensatin, thermistor, a source
polarizin.g current, amplifier with alanal
adjustment and a readout 00 .eale with
recorder contacts.


! .. "Ot --I
Pi.IIM.. 0&..
EI......,.. 1Afe.
Figure 3
\ , -(.,
.011'" ..,..,
QU'" .0"
"~ "01''' 01'
111ft. ...
-Vol - 8
C The YSI Model 51 (3) is iUustrated in
Figure 5. This is another form of
polaroaraphic DO analyzer. The cell
conai.ts of a silver anode coil, a lold
rini cathode and a KCl electro4'te with
a te£]on membrane. The instrument has
a .ensl.tiv1ty adjustment, temperature and
00 readout. The model 51 A has temp.-
erature compenaation via manual preset
dial. A field probe and bottle probe are
available .
TII M".I 51 DO I.n...
c.th.tI. .1"1
Fipre !t
D The Model 54 YSI 00 analyzer (4) is based
upon the same electrode configuration but
modified to include automatic temperature
compenaat1on, 00 readout. and recorder
jacks. A motorized agitator bottle probe
i. avaUab1e for the Model 54 (Fipre 6).
Tit "'.1 .. A.,..... ......
a......, ~. '.iII
-..k... .........

E The Galvanic Cell Oxygen Analyzer (7,8)
employ. an indicator for proportional DO
lignal but doee not include thermi.tor
compenlation or .ipl adju.tment.
Temperature readout ie provided. The
leneor includes a lead anode ring, and
a sUver cathode with KOH electrolyte
(4 moJar) covered by a membrane film
(FilUre 7).
"..1.1.. ..1....1. c.n 0.".. "....
........, ,....
~II..., ce.h.4.
'''.rMI..., c.,'e
'..4 A".tI. II...
...,.th,.... .,,,,'ren.
F The Weeton and Stack Model 300 00
Analyzer (8) has a galvanic type lensor
with a pulled current amplUier adjultment
to provide for sipl and temperature
compen.ation. DO and temperature
readout is provided. The main power
supply is a recharaeable battery. The
senior (Figure 8) consilts of a lead anode
coil rec8lsed in the electrolyte cavity
(50% 1<1) with a pJatinum cathode in the tip.
The lensor is covered with a teflon mem-
brane. Membrane retention by rubber
band or by a plastic retention ring may be
used for the bottle agitator or depth
simpler respectively. The thermistor
and al1tator are mounted in a Ileeve that
1180 provides protection for the membrane.
Diseolved Oxygen Determination
G The ElL Model 15 A sensor is illustrated
in Figure 9. Thil is a galvanic cell with
thermistor activated temperature com-
Di8801vpc; Cr.'.l'gen Dctermina.tion

Cabl. Sealin,
A 15017
'0' Ii...
'0' lin,
L.ad Anod.
M... b,a n.- S.cu,in,
'0' lin,
A U0140
(With 51..v. 5241
'0' Ii.,
Silv., C.thod.

t :::::::::::::::J
'0' Ii..
Ilack wire 0' cabl. conn.ct. to Anod. Contact
Not.: R.d wi.. 0' c.bl. conn.ct. to Anod. Cont.ct Holdo,
Momlt,.no not shown E. I. L. part numb., T22
Figure 9
M...b,... S.cu,in,
End C.p
Fill., Scr..
'0' lin,

Dl..olved OxYllen Determination
       DO Temp. 
       Ste. Compo Accessories for
  Anode Cathode Elec Type MeumI' Adj. Temp. Rd,. which designed
Carr1t . lilver- Pt KCI pol. poqeth no no Reeordq temp.
Kanwisher .ilver ox. disc KOH     . sipl adj. self
  rinll  ~  tet1011   as,e~led
Beckman Aq Au pol ye. ~ recording
  tinll dilc iel    yes 
Yellow Sprinll AI Au KCI pol tefloo y.. ~ field and'bottle
 51 coil rmj( loIn    18s probe
Yellow Sprtnls " II " " II Y.' I!!. recording field
 54       ,es bottle&. alitator
   silver KOH gal~"'Polyeth   probes
Precision Pb no is?. 
Sei  DAlE ~IC 4N    yeS 
We.ton & Pb Pt ia galv tel1Ol1 yes X!.! apt. probe
Stack  col1 d1.c 40"10    yel depth sampler
ElL  Ph AI KHC03 plv tenon yel l!! recording
Delta  Lead Silver KOH plv tef1011  ye. tield bottle &
 yes l!.!
75   d~N    no !lIitator ~
De"lta  Lead Silver KOH plv tef10n yes l!! field bottle&.
85   d1.c IN    yes allitator probe
*Pol - Polarocraphic (or amperom.lc)     
**Galv -)Galvanic (or voltametric)      
1 Carrit, D.E. and Kanwilher, J.W.
Anal. Chem. 31:5. 1951.
5 Technical Bulletin TS-68850 Precleton
Scientiftc Company, Olicago, IL 60647,
2 Beckman Inltrument Company. Bulletin
7015, A Die.olved Oxygen Primer,
FuUerton, CA. 1982.
" Maney, K. H., Okun, D. A. and Reilley,
C.N. J. Electroanal. Chem. 4:65.
3 In.truction. for the YSI Model 51 Oxypn
Meter, Yellow Sprtnp In.trument
Company, Yellow Spr1n,., OH 45387.
7 ln8truct1on Bulletin, Weston and Stack
Model 300 Oxygen Analyzer. Roy F.
Welton, We.t Chester, PA 19380.
4 Instructions for the YSI Model 54 Oxygen
Meter, YelJow Sprin,s In.trument
Company, Yellow Spring., OK 45387.
8 Bri"l, R. and Viney, M. Desi8l1 and
Performance of Temperature Com-
pensated Electrode. for Oxygen
Mealuremente. Jour. of Sd.
In.truments 41:78-83. 1984.

9 Eden, R. E. BOD Determination Using
a Di8solved OxYien Meter. Water
Ponution Control. pp. 537-539. 1987.
10. Sko0l, D. A. and West, D. M. Fundamentals
of Analytical Chemistry. Holt,
Rinehart & Winston, Inc, 1986.
Dissolved Oxygen Determination
11 Methods for Chemical Analysis of Water
and Wastes. EPA -AQCL, Cincinl'lati,
OH, Ju1y, 1971.
This outline was prepared by F. J. Ludzack
Chemist, EPA, WPO, National Training
Center, Cincinnati, OH 45268 and Nate
Malol, Chemist, EPA. WPO, National
Field Investigations Center, Cincinnati, OH
~iptors : Chemical Analysis, Dissolved
Oxygen, Dissolved Oxygen Analyzers,
Instrumentation, 00- Site Tests, Water Analysis

I Pholphorus is closely associated with
water quality because of (a) its role in
aqUitiC productivity such as algal blooms,
(b) Us sequestering action, which causes
interference in coagulation, (e) the difficulty
of removing phosphorus from water to some
desirable low concentration, and (d) its
characteristic of converting from one to
another of many possible forms.
A Phosphorus is one of the primary nutrients
such as hydrogen (H), carbon (C),
nitrogen (N), sulfur (S) and phosphorus (P).
1 Phosphorus is unique among nutrients
in that its oxidation does not contribute
sianificant energy because it commonly
exists in oxidized form.
2 Phosphorus is intimately involved in
oxidative energy release from and
synthesis of other nutrients into cell
mass via:
a Transport of nutrients across
membranes into cell protoplasm is
likely to include phOllphorylation.
The release of energy for meta-
bolic purposes is likely to
include a triphosphate exchange
B Most natural waters contain relatively low
levels of P (0.01 to 0.05 mgll) in the
soluble state during periods of significant
1 Metabolic activity tends to convert
soluble P into cell mass (organic P) as
a part of the protoplasm, intermediate
products, or sorbed material.
2 Degradation of cell mass and incidental
P compounds results in a feedback of
lysed P to the water at rates governed
by the type of P and the environment.
Aquatic metabolic kinetics show marked
influences of this feedback.
CR. PROS. 4c. 3.74
3 The concentrations of P in hydrosoils,
sludges, tr~atment plant samples and
soils may range from 102 to 106 times
that in stabilized surface water. Both
concentration and interfering compo-
nents affect applicability of analytical
II The primary source of phosphorus in the
aqueous system is of geological origin.
Indirect sources are the processed mineral
products for use in agriculture, household,
industry or other activities.
A Agricultural fertilizer run-off is related
to chemicals applied, farming practice
and soil exchange capacity.
B Wastewaters primarily of domestic
origin contain major amounts of P from:
1 Human. animal and plant residues
2 Surfactants (cleaning agent) discharge
3 Microbial and other cell masses
C Wastewaters primarily of industrial
origin contain P related to:
1 Corrosion control
2 Scale control additives
3 Surfactants or dispersants
4 Chemical processing of materials
including P
5 Liquors from clean-up operations of
cbats, fumes, stack gases, or other
III Phosphorus terminology is commonly
confused because of the interrelations among
biological, chemical, engineering, physical,
and analytical factors.
43- 1

Determination of Pho~h?ru8 in the Aqueous Environment
A Biologically, phosphorus may be avaUable
as a nutrient, synthelized into living mals.
storeo in living or dead ceUI. agilomerates.
or mineral complexes. or converted to
degraded materiall.

B Chemtcally, P exiltl in leveral mineral
and organic forml that may be converted
from one to another under favorable
conditions. Analytical eltimatee are
based upon physical or chemical techniques
necelsary to convert varioul forml of P
into ortho pholphatel which alone can be
quantitated in terms of the molybdenum
blue colorimetric test.
C Engineering interest in phosphorus is
related to the prediction, treatment. or
control of aqueous syetems to favor
acceptable water quality objectives.
PholphQrul removal il a.lociated with
D SolubUity and temperature are major
phYlical factors in pholphorul behavior.
Soluble P is much more available than
inloluble P for chemical or biological
transformations and the rate of converlion
from One to another II Itrongly influenced
by temperature.
E Table 1 includel a clau1f1cation of the
four main typee of chemical P and lome
of the relationlhips controlling lolubUity
of each group. It is apparent that no
clear-cut leparation can be made on a
lolub1l1ty buil as molecular weight.
subltituent and other factore affect
F Table 2 includel a scheme of analytlcal
differentiation of various forms of P
based upon:
1 The technique required to convert an
unknown variety of pholphorul into
ortho P which Is the onq one quanti-
tated by the colorimetric teet.
2 Solubility characteristics of the sample
P or more precisely the means required
to cJ.artty the sample.
a Any clarification method il subject
to incomplete separation. Therefore.
It il e.lential to specify the method
ueed to interpret the yl.ll1 factor of
the separation technique. The
dearee of leparation of lolubles
and inlolubles will be 11anificantly
different for:
1 Membrane filter separation
(0.5 micron pore size)
2 CentrifUgation (at some specified
rpm and time) .
3 Paper filtration (spec1C, paper
4 Subsidence (specify time and
G Analytical separationl (Table 2) like those
in Table I, do not give a precise separa-
tion of the various forms of P which may
be included quantitatively with ortho or
poly P. Conveuely some of the poly and
organic P will be included with ortho P if
they have been partially hydrolyzed
durinl Itorale or analysis. Inaolubles
may likewise be included as a result of
poor separation and analytical conditions.
1 Th.e separation methods provide an
operational type of definition adequate
in moat situations if the "operation"
is known. Table 2 indicates the nature
of incidental P that may appear along
with the type sought.

Determination of P\1osphorus in the Aqueovs Environment
Table 1
Water Soluble(l)
Insoluble ( 1)
1. Ortho phosphates
Combined with monovalent
cationl such.. H7 Na:K~H:
Combined with multi
valent cations such
+2 +3 +3
as Ca Al Fe
2. Poly phosphates
-4 -5 -3
(Pa07)' (P301O) (P309)

and others depending upon
the de,ree of dehydration.
as in 1 above

Increasing dehydration
decrealel 80lubility
(a) as in 1 above
(b) multi P polyphosphates
(high mol. wt.) in-
cluding the "glassy"
3. Organic phosphorus
R-P, R-P-R (2)
(unulually varied nature)
(a) certain chemicals
(b) degradation products
(c) enzyme P
(d) phosphorylated nutrients
(a) certain chemicals
(b) cell mass, may be
colloidal or agglom-
(c) soluble P sorbed by
insoluble residues
4. Mineral phosphorus
(a) as in 1 above
(a) as in 1 above
(b) as in 2 above
(c) geological P such as
phosphosilicate s
(d) certain mineral com-
(1) Uled in reference to predominance under common conditions.
(2) R represents an orianic radical, Prepresents P, PO" or its dl'rivatives.
2 Total P in Table 2 includes liquid and
separated residue P that may exi.t in .
the whole sample includin, sUt, orgamc
sludge. or hydro.oils. This recognizes
that the feedback of soluble P from
deposited or suspended material has a
real effect upon the kinetics of the
aqueous environment.

Determination of Phosphorus in the Aqueous Environment
Table 2
Duired P Components
Incidental P ~cluded(2)
1. Ortbo phosphates
No tr..tment on clear
Easily hydrolyzed
(a) poly phosphat.. -
(b) oraaoic -P, -
(c) Mineral -P, + or -
2 . Polypbosphates
(2)-(1) . poly P
acid hydrolysis on clear
samples, dilute
(a) H2S04

(b) HCI
(a) ortho-P ...
(b) organic -P + or -
(c) mineral -P +or -
3. Or..nic phosphorus
(3) - (2) + ori P
t. Soluble phosphorus
(preferably classified
by clarification method)
acid + oxidizini hydrolysis
on whole sample. dilute

(a) H2S04 + HN03

(b) H2SO4 + (NH4)2S208

clariUed Uquid followin8
filtration, centrifu8at1on
or subsidence
(a) oriho P +
(b) poly P +
(c) mineral P + or -
generally includes
(a) 1. 2, or 3

(b) particulates not
completely separated
5. Insoluble phosphorus
(residue from clari-
Retained residues separated
durin. clarlficaUon
See (6)
(a) ienerally includes
sorbed or complexed
6. Total phoephorus
Stron8 acid + oxidant

(a) H2S04 + HN03

(b) H2S04 + HN03 + HClO4o

(c) H202 + Mg(N03)2 fusion
all components in
1, 2, 3, 4, 5 in the
whole sample
(1) Determinative step by phospho molybdate colorimetric method.
(2) Codin8: + quantitative yield
- a amall fr"ctlol1 of the amount preaent
+ or - depends upon the individual chemical and sample history

Determination 01 Phosphorus in the Aqueous Environment
IV Polyphosphates are of major interut in
cleaning agent formulation, as dispersants,
and for corrosion control.
A They are prepared by dehydration of ortho
phosphates to form products having two or
more phosphate derivatives per molecule.
1 The simplest polyphosphate may be
prepared as follow s:
NaO Na<{

:7 . 0 ...t, H~p

~.o °
H01 '\p . °
NaO H01
. °
+ H20
mono sodium ortho
phosphate (2)
dlsodium dihydrogen
polyphospha te
2 The general form for producing
polyphosphates from mono substituted
orthophosphates is:

n (NaH2P04) 20;_e:~ooc~ (NaP03)n+ n H20
3 rn-substituted oMho phosphates or
mixtures of substituted ortho phosphates
lead to other polyph08phates:
dlloc:Hum hydrolen +
ortho phOlphllte
NIiH2PO" ~ Na&P]OlO
mono .odium -t penta. odium
di hydrolen trA-phOlpha"
ortho pho.phate
+ 3H,O
+ water
4 The polyphosphate series usually
consist of the polyphosphate anion
with a negative charge of 2 to 5.
Hydrogen or metals commonly occupy
these sites. The polyphosphate can be
further dehydrated by heat as long as
hydrogen remains. Di or trivalent
cations generally produce a more
Insoluble polyphosphate than the same
cation in the form of insoluble ortho
phosphate. Insolubility increases with
the number of P atoms in the
polyphosphate. The "glassy" poly-
phosphates are a special group with
limited solubility that are used to aid
corrosion resistance in pipe distribu-
tion systems and similar uses.
B Polyphosphates tend to hydrolyze or
"revert" to the ortho P form by addition
of water. This occurs whenever
polyphosphates are found In the aqueous
environment. '
The major factors affecting the rate of
reversion of poly to orthophosphates
a) Temperature, increased T increases
b) pH, lower pH increases rate
c) Enzymes, hydrolase enzymes
increase rate
d) Colloidal gels, increase rate
e) Complexing cations and ionic
concentration increase rate
f) Concentration of the polyphosphate
Increases rate
Items a, band c have a larg" effect
upon reversion rate compared with
other factors listed. The actual
reversion rate is a combination of
listed items and other condLtions or
3 The differences among ortho and ortho
+ polyphosphates commonly are close to
experimental error of the colorimetric
test in stabilized surface water samples.
A significant difference generally
indicates that the sample was obtained
relatively close to a source of poly-
phosphates and was promptly analyzed.
This implies that the reversion rate of
polyphosphates is much higher than
generally believed.

Determiaation of PhOliphorvll in the Aqueoua Environment
A Samp11ng
1 Great care Ihovld be exercliled to
exclude any benthic depolitl from
water lample..
2 Glau containere .hould be acid rinled
before v.e.
3 Certain plalUC containers m~ be
uled. POllible adlorptlon of low con-
centration. of pho.phorvi should be
" If a dilferenUatton of phoephorul forme
is to be made, filtration .hould be
carried out immediateq upon eample
collection. A membrane filter of
O. .5j.1 pore eize ill recommended for
reproducible separations.
B Pre.ervaUon
1 If at all pollible, eamplel should be
ana]yzed on the d~ of collection. At
be.t, pre..rvation meaBure. onq
~etard poslible chan,e. in the .ample.
a POllible physical change. include
solubilization, precipitation,
ablorption on or clelorption from
sU8pended matter.
b POllible chemical changes include
reversion of poly to ortho P and
decomposition of orpmc or min-
eral P.
POllible biolopcal chan,.s
include microbial d.compolitton
of orpnic P and al8al 01'
bacterial growth tonatn, orpnic
2 Refrigeration at .0 C il recommended
if sample I are to be atored. Thi.
decrea.el hydro]ysis and reaction
ratn and al.o 10.lel due to YOlatWty.
Addition of 40 m, HgC12/liter is
recommended for longer storage
periodl. Thi8 chernicall1rnits
biololic&1 changeB.
H.<: 11. il an interference in the
ana1)'Ucal procedure if the
chloride level ill low (See Part
VI, 83). .
This il a colorimetric determination
lpeciflc for orthopholphate. DepencUng
on the nature of the eample and on the
type of data lOUght, the procedure in-
volv.1 two ,eneral operations:
Convereion of phosphorue forms to
soluble orthOphosphate (See Fig. 1):
sulfuric acid-hydro]ysis for
poqpho8phates, and some
orpnic P compounds,
persu1fate digeeUon for organic
. P compounds.
2 The color determination involves
reacting dilute solutions of phosphorus
with ammonium moqbdate and
pota.sium antimonyl tartrate in an
acid medium to form an antimony-
pholphomoqbdate complex. This
complex is reduced to an intenseq
blue-colored complex by aecorbic
acid. The color is proportional to
the orthopholphate concentration.
Color abeorbance ie measured at
880 nm and a concentration value
obtained uliog a Itandard curve.
ae.,ent preparation and the detailed
procedure can be found in the EP A
The methode described there are
ulable in the 0.01 to 0.5 ma/liter
phoaphorus range.

Determination of Phosphorus 1n the Aqueous Environment
~ l:ol.a J'flt..ul_~
. C.lod..trJ
O. tIS Micron ahmbrane FULer
inorl..Lo +
7lDURJ: 1
1 Erroneous re8ults from contam1natea
,Jas8ware is avoided by cleaning it
with hot 1: 1 HC1, treating it with
procedure reapnts and rin.ings
with distilled water. Preferably
thi. ,lassware should be used only
for the determination of pho.phorus
and protected from dust during
lItorage. Commercial deterllents
,~oq],d never'be U8ed.

2 Hi8h iron concentrations in samples
can precipitate pho8phorus.
3 ",H,Cl is used as a preservative,
it Interferes if the chloride level of
the sample is less than 50 m,
Cl/liter. Sp1k1n, with NaCl is then
4 Others have reported interference
from chlorine, chromium, sulfides,
nitrite, tannins, Ugnin and other
minerals and organics at high con-
Precision and AcCuracy(6)
1 Organic phosphate 33 analysts in
19 laboratories analyzed natural
water samples containing exact in-
crements of organic phosphate of
0.110, O. 132, 0.772, and 0.882 mg
Standard deviations obtained were
0.033, 0.051, 0.130 and 0.128
Accuracy results as bias, mg P /liter
were: +0.003, +0.016, +0.023 and
- O. 008, respectively.
2 Orthophosphate was determined by
26 analysts in 16 laboratories using
samples containing orthophosphate
in amounts of 0.029, 0.038, 0.335
and 0.383 mg P /Uter.
Standard deviations obtained were
O. OlD, 0.008, 0.018 and 0.023
iI.J - 7

eeterm,nation o( Pho.pho.r.ue 111 the AqueOlUl Environment
Accuracy results a. b1a., mg P/Uter
were -0.001, -0.002, -0,009 and
- 0.007 reapectively,
Beveralimportant variable. affect
formation of the yellow heteropoly
acid and it. reduced form, molybdenum
blue, in the colorimetric teat for P.
Acid Concentration during color dew1op-
ment 1s critical. Fii\U'e 2 shows that
color will appear in a .ample conta1ninr
no phosphate 1f the acid concentration
is low. Interfering color 1s ner~1e
when the normality with respect to
H2SO4 approaches 0.4,
1 Acid normality during color develop-
ment of 0,3 to slightly more than
0.4 1. fea.1ble for U8e. It 1s prefer-
able to control ac1dity caretully and
to seek a normality closer to the
hiaher limits of the acceptable rance.

2 '.It is e"8I\t1&1 to add the acid and
. molybdate 8.8 one lOlution.
3 The aUquot of .ample mUtt be
neutralized prior to adding the
color r.a.ent.
H"O NO."ifFY

Figure 2

Choice of Reductant - Reagent stability,
effective reduction and freedom "'om
de18teriCN' .ide effects are the bases
for reductant selection. Several re-
ductant. have been used effectively.
A.corbic acid reduction is hirhly
effective in both marin. and fresh water.
It 18 the nductant spec1f1ed in the
EPA m.etaod.
Temperatur! affects the rate of color
formation. Blank, standards, and
sampl.. must be adjusted to the same
temperature (t 10C), (prefera~ room
temperature), before addition of the
acid molybdate reagent.
Time for Color Development mu.t be
spec1fied and consistent. After addition
of reductant, the blue color develops
rapidly for 10 minutes then fades grad-
ually after 12 minutes.
Determination of total phosphorus
content involves om1uion of any
filtration procedure and usinl the acid-
hydroly.i. and persuUate treatments
to convert all phosphorus forma to the
te.t-"Mitiv. orthophc.phate form.
Determ1ninl total pho.phorus content
y1eld8 the moat meaningful data since
the various forma of phosphQ1'U8 may
chante from ane form to amtber in a
.hort period of time. (See part V. Bl)
Pho.phol'Ul ~sis received. intensive
inv..t~at1on; coordination and validation of
method. is more difficult than changing

"Determination q:( ;Phosphorus in the Aqueous Environment
A part of the problem in method!J arose
because of changes in analytical objectives
.uch as: '
i Methods suitable to gather "survey"
information may not be adequate for
"standards" .
a Methods acceptable for water are not
necessarUy effective in the presence
of significant mineral and organic
Interference characteristic of hydro-
10l.1s, marine samples. organic
Iludges and benthic depo.it..
3 Interest has been centered on "fresh"
water. it was essential to extend them
for marine waters.
" Inltrumentation and automation have
required adaptation of methodology.
B Analysts have tended to work on their own
special problems. If the method
appareru.ly served their situations. it was
used. ! Each has a "favorite" scheme that
may be quite effective but progress
toward widespread application of "one"
method has been slow. Consequently,
many methods are available. Reagent
'addity, Mo content. reductant and
leparation techniques are the major

C At the present time there is not sufficient
data to warrant EPA endorsement of the
P procedure for sediment-type sample.,
sludges, algal blooms, etc. Following
i. a procedure (not included in the EPA
manual) which is useful when solids
are present in samples: ---:II
1 If sample contains large particles.
grind and emulsify solids in a blender.

2 Transfer 50 ml sample, or aliquot
diluted to 50 ml, into a 250 ml
Erlenmeyer flask.
3 Add 6.0 ml of 18N H2S04, 5 ml
concentrated HNC':i' 2 berl saddles
and digest on hot plate.
4 Digest until the disappearance of nitric
acid fumes and the appearance of white
S03 fumes. Con~inue digestion for
approximately 5 minutes. Cool before
proceeding with Step 5.
5 Add 2 ml of HN03-HCI04 mixture
and 5 ml concentrated HN03' Continue
digestion until all of the nitric acid is
driven off and dense fumes of perchloric
evolve. Perchloric acid requires
dilution with sulfuric acid and prior
destruction of moet organics for safety.

6 Cool. Add approximately 40 ml
distilled water and transfer to 100 ml
volumetric flask.
7 Add 2-3 drops of phenolphthalein and
concentrated ammonium hydroxide
until a pink color is seen. Then
discharge the pink color with the
strong sulfuric acid. It is advisable
to add an equivalent amount of salt
formed during neutralization of digested
l:Iamples to the calibration standards to
equalize salt content during color

Determine orthophosphate according
to the usual color proce dure.
Materials in this outline include significant
portions of previous outlines by J. M. Cohen,
L. J. Kamphake, and R. J. Lishka. Important
contributions and assistance were made by
R. C. Kroner, E. F. Barth. W. Allen Moore,
Lloyd Kahn,. Clifford R~sJey, Lee Scarce.
John Winter, Ferd Ludzack and Charles
Jenkins, David. A Study of Methods
Suitable for the Analysis and
Preservation of Phosphorus Forms
in the Estuarine Environment. DHEW,
Central Pacific River Basins Project.
SERL Report No. 65-18, University
of California, Berkeley. Calif.
November 1965.

I)etercmaUon of Pho.phoru. 1n~~.!A~I;/~g ~"'1Jm",,,,"
2 Oalee, Morri. E., Jr., JuUan, Elmo C.,
and Kroner, Robert C., Method for
Quantitativ. Determl~UOll of Total
Pbo.phoru in Water. JAWWA 158:
(10) 13113. October 1988.
3 Lee, G. Fred, Cle8ceri, Nichola. L. and
F1tz,uald, Geor,. P., Studi.. on the
Analy.i. of Pho.pUt.. in Alral Culture..
Int. J. Air" Water Poll. titUl. 18815.
Barth, E. F. and Salott0.L V. V.,
Procedure for Total .I"haphol'll'
in Sewa.e and Slud.., UftJlUbU.hed
Memo, Cincinnati Water R..earch
Laboratory, FWQA. Apr111966.
43- 10
15 MOil, H. V., (Chairman, AASGP
Committee) Determination of Ortho
Pho.pbat., H1drolyzabl. Phaphate
and T4Ma1 Ph08Phate in Surface Wa~er.
JAWWA 56:1583. Decem~1' 11'58.
6 Method. fo~ OIem1cal Anaq81. of Water
&. Wute.., EPA-AQCL, C1noinDati, OH
45288, b71.
Thi8 out11M WI., prepared by Aud-rei 'E.
Donahue, OIeinWt,. Natioaal TrabUq Center,
DTTB. MPi. WPQ EP A. Cinc!noat1.
OR 415288.
De8criptor. :
Chemical An-ly.i.. Nutrients, Phosphates,
Ph08phoru., Pho.phoru. CompounC\8,
Pollutant Identification, Samplin" Water
Analy.i., Water Pollution SourCe8.

(Range O. 1 to 2 mil nitrate nitrogen/I)
A Water

Di.tUIed water free of nitrite and nitrate
should be used in the preparation of all
rea,ents and .tandards.
B Stock Potassium Nitrate
(1.0 ml = O. 1 mg N03 - N):

Dtuolve 0.7218 g of anhydrous KNO
in d1stUIed water and dilute to 1 Ute;'
C Standard Potassium Nitrate
(1.0 ml = O. 001 mg N03- N):

Dilute 10.0 ml of the stock solution to
1 Ute.. with distilled water. This solution
should be prepared fresh weekly.

D Sulfuric Acid:
Carefully add 500 ml of concentrated
HiSC. (sp. Ill'. 1. 84) to 125 ml of distilled
w tel'. CooT and keep tightly stoppered.

E Sodium Chloride (30%):
Dissolve 30 g of NaClin distilled water
and dilute to 100 ml.
F Brucine - sulfanilic acid reagent:

D1810lve 1 g of brucine sulfate.
(C23H26N204)2 . H2SO4' 7 H20. and

0.1 g of sulfanilic acid (NH2C6H4S03H . H20)
in '70 ml of hot distilled warer.
Add 3 ml of concentrated HCI. cool, and
dilute to 100 mI. This solution is stable
for .everal months if stored in a dark bottle
at 50C. The pink color which develops
slowly does not effect the usefulness of the
solution. The bottle should be marked "toxic".
G Acetic Acid:
Dilute 1 vol olglacial HC2H302 with 3 vols
of distilled water.
CH. N.lab. 2e. 3. 74
A In the case ol highly alkaline waters, it,
i. necessary to adjust the pH to
approximately 7 with the acetic acid.
B Filter through a O. 45 micron pore
size filter lfnece8sary.
A Pipet 0.0, 2. 0, 5.0, 7.0, and 10.0 ml
of the standard KNO solution into
50 ml test tubes held in a suitable rack.
Add sufficient distilled water to bring
the volume to 10. a ml.
B Pipet 2.0, 3. O. and 5.0 ml. of the
sample into 50 ml test tubes. The
purpose of using three different volumes
of the sample is to ensure that when the
colors are developed. at least one of the
three samples will give an absorbance
which lies within the range of the
calibration curve. A dd sufficient dis-
tilled water to bring the volume to
10 mi.
C If the samples are saline, pipet 2.0 ml
of 3()~o NaCI into the standard and
sample tubes. Mix well. This addition
i8 W1necessary for fresh water samples.
D Chill all of the tubes to 0
cold water bath.
100C in a
E Pipet 10.0 ml of the H2801 solution into
each standard and sample ube. mix
well by swirling, and again chill to
o 100 C.

t-aboratory Procedure for Nitrate N1trofen
F Pipet 0.5 ml of the brucine - luUanilic
acid reagent into the standard .nd
sample tube.. mix well by .w1dt",.. and
place the tubel In a boilln. water bath
lor 25 minute..
Q Remove the tubel from the boUUa, water
bath and cool to 20-250C in a water bath.
H If color or turbidity developI In the
..mple while the tube. are in.the boilin.
water bath, a blank mUlt be prepared
u.in. all reagents except the brucine -
luUanll1c add.
I Meaeure the ab.orbance valuee of the
Itandard8 and ..mples at 410 nm.
on a suitable .pectrophotometer.
Before each 80lution i. r...d, rln8e the
.pectrophotometer ceU at lea8t twice
with the 80lution.
J Prepare a calibration curve 01 ab80rbance
valu!8 for the ltancardl VI. mg of
NO! N. For example: 11 2. 0 ml of the
ItaAdard!eNO 80lution are u8ed, and
Itl concentration i80. OOI,ma of NOS - N/ml,
then O. 002mg of NO - il t.be vawe
.~ted on the oaUb1.UOft curve VI. the
Oatr.lpond1n. ab80rbance vl1u..
K Determine the m. of N03 - N pre8ent
in the .amplel ulinl the calibration
curve an' then calcu:late the m. of
NO, - NIl ulin, the formula:

mg 01 NO, - NIl.
mlJ 01 N03 - N Irom curve X :1000 mlll
.ftl « 8ample
Method. for Chemical Analyei. of Water
and Waite.. EPA-AQCL, 1971.
Thi8 outline Wall prepared by Charles R.
Feldmann, Chemi8t, National Training
Center, MDS, WPO, EPA, Cincinnati,
OH 45268

Chemical Analy8ts. Nltt'8te8, Nitro.en
Compound., Water Analy.tl

A Laboratory determinations with approved
equipment and convenient facilities by
experienced specialists generally are
ealier, faster, and more reliable. It is
advioble to tranlport the samples to the
laboratory whenever it is feasible to do so.
B Field tests are e.sential because:
1 Certain sample components are
inherently unstable with respect to
biological, chemical, or physical
chanles. Any test result performed in
the laboratory may not represent true
conditions on site at the time because
of delay, displacement, or changed
conditions .
2 Subsequent operations generally may be
coordinated and made more meaningful
by preliminary on site neld investigation
to identify and evaluate problems, locate
critical areas, and minimize surprises.
A Moving the laboratory into the field means
improvisation and adaptation to more
"primitive" conditions.
1 Rugged construction of field equipment
il a first consideration. Sturdy and
convenient cases are required; the case
often may be the only available work-
bench. Polyethylene bottles, beakers,
burets, and pipets generally are
necessary to eliminate breakage of
fralne glass.
2 Portability is essential, particularly
when the site i. not accessible by boat
or car. Ideally, the field kit should
be .mall enouah and light enough to be
carried by one man for extended periods.
3 Procedures for field use are restricted
in equipment and manipulation. In
general, it is not .possible to use long,
tedious routines or highly precise
measurements. Numerous reagents
generally cannot be carried. Quick
positive reactions are essential. Small
visual comparators, test papers or spot
tests are popular. Titration assemblies
must be modified for field use.
4 Instrumentation increases objectivity
of the measurements but the instruments
must be adapted to the items outlined in
II. A. I, 2 and 3.
The instruments commonly used must
be simplified yet rugged enough to keep
working in spite of temperature changes,
moisture, bumps, etc. A meaningful
validation procedure must be worked out
for calibratior. on site with simple
adjustment or repair for emergencies.
5 Personnel engaged in field testing often
are unaccustomed to laboratory
procedures. Advance training is
essential in procedures and instrumentation
used. Field testing is a multi-
disciplinarian operation:
The individuals must be good
observers and recognize what may
be significant later.
They must be good interpreters
of a variety of observed and
derived test information.
They must be good technicians to
follow prescribed procedures
diligently and recognize anomalous
behavior when it occurs.
They must be good reporters to
describe what happened, where
it happened, and when. Failure
to report anomalous behavior

Chemical Tests Observations and Measurements in the Field
and related events in an understandable
and consistent fashion may render the
entire eflort mellninlless because of
some doubt or 1nconsiatena)'.
Subsequent operations also may be
mislead by the ear~ observer.
B Use of field data imposes certain con-
straints relatable to the item, the methods,
and to the personnel involved:
I How valid is it?
2 How consistent is it in 11ne with other
observed or recorded information?
3 What backup lnformation is required
to verif,y suggested information?

C Decisions must be made on all field
data applications:
I Where "in-place" and "now"
measurements are required how much
of the laboratory must be moved out
to the site to obtain data reliable
enough to meet objectives?
2 Which items are to be determined on
site? Which items in a central
3 What use is to be made of the
preliminary field e.timates?
4 What do you "need" to know?
5 What would be "nice" to know?
6 In line with facUities, time, cost,
manpower and objectives, what
can you determine?
A Any item that changes rapidly during a
holding period as a result of temperature
or pressure variations, or is highly
reactive from a biololical, chemical,
or physical standpoint, usually means
that {t must be determined on site and 1n
place with minimum time lapse and
1 Dissolved gases, such as 0 , C02'
"28, Cl2' are sensitive to Jressure
or temperature changes and may
react with other sample components
readily. These require in p1ac::e and
now readout.
2 The collective analysis of all forms
of a given item may be preserved
and analyzed later. Estimates of
different forms of the same substance
are not subject to delay unless they
can be separated prompt~. Examples
of this include the relative ratios of
oxidized or reduied s'fl8tance.'+:fuch +3
as eu+l and Cu+ ,Fe and Fe ,Cr
and Cr +6; hydrolyais Is the principal
factor in the ratios of organtc and
NH3-N and in changes among organic,
poly and ortho- P .
3 Reactive substances contributing to
"oxygen demand" tests, such as BOD
or other respiratory tests generally
require a minimum time delay before
testing and are not amenable to
preservation without altering results.
4 Soluble I insoluble ratios commonly
change with time and conditions due
to complexation, hydrolysis
precipitation, agglomeration, or
other factors. Particulai"e size may
increase or decrease. Solubles may
be sorbed on or desorbed from solid
surfaces. Settleability or turbidity
are transient phenomena .ubject to
change with time or conditions.
5 Biological progressions are dynamic
entities that shift among "critters"
in relation to predominence in
variety, numbers, growth, and decay.
Whatever is, will c1w1ge in response
to nutrition and conditions.
Preliminary information often is needed
to guide situation evaluation, problem
identification, on site variation with
respect to cross section, depth, or
time. These data are usefUl to determine
whether there is a problem or not and
for planning of subsequent operations.

'Vi1~.,dcal TeBts Observations and Measurements in the Field
may be necessary to locate suspected
Jtlows, channels, or sources of items
.ffecting water quality.
Definition of mixing zones often is
w Laboratory time may be reduced if
approximate concentrations of items
sought are known.
4 Field tests or observations may
sUiiest other tests that are more
critical in evaluation of the given
5 Stratification may be evaluated for
lUidance of subsequent operations.
6 Knowledge of the distribution of
components is useful for selecting
meaningful future sampling sites.
The field test may be used to check
compliance with regulations prior to
more rigorous backup teBting.
1 An elltabUshed operation may be in
control or out of control.
2 The field test may reveal which of
multiple discharges are in conformance.
3 Undisclosed discharges may become
4 It may be necessary to "track" some
hazardous dischar ge down stream
to guide subsequent users on the
choice of intake or storage water.
5 A complaint or inquiry may be
evaluat ed by field tests.

: In-plant field type tests are essential
to IUide operations toward the production
or continuous high quality effluents.
Record tests commonly are provided too
late to do anything about the situation;
quick test results are emphasized for
procell control because time, manpower,
and change are critical.
1 Surprises in the form of wastewater
changes in now. concentration, or
significant componentll are common
for treatment process operators. They
need quick number!! rather than
impressions to distinguish real from
apparent problems.
2 Process upsets are minimized or
prevented by regular and meaningful
te sts from which trends may be
established. Small imbalances may
be corrected before they become
major upsets.
3 Backup or supplementary treatment
such as coagulation, adsorption,
neutralization, may prevent serious
process disruption if tests indicate
the problem and its magnitude in
time to do something about it.
pH is one of the simplest and most
valuable field tests. If the result is due
to mineral acids or alkalies in stable
form, color indicators or comparators,
impregnated paper or other devices
will serve. When free C02 in solution
is a major variable such as in the
vicinity of benthic deposits or in active
biological systems, the application of
an electronic sensor in-place and
extension wire to an indicator instrument
is essential. Any delay, pressure change
or manipulation will change free C02
and alter pH response. Ele ctronic pH
instruments are produced for field use
by many apparatus supply firms. With
care, they will function effectively.
Conductivity is a very useful index of
ionic materials and usually may be
correlated with wet chemical data to
give a reasonable correlation with total
dissolved solids for a given mixture.
This test is very useful to detect saline
intrustions or discharges, springs,
hidden channels of different salinity
from the main body of water Several
reliable instruments are marketed.

asemical Te.tB,ObiervationB, and Measurements in the Field
DO anaqzers are available for field
use appUcation from Beveral manufacturers.
They differ in portabW.ty accessory
equipment and in ver.atWty so that it is
p08lible to obtain one or more fitting almost
any requirement. Theile units are balled
upon reduction of oxygen at the cathode
surface to conve rt chemical to electrical
energy. Direct measurement of electrical
energy produced or the change in some
carrier current or voltaae may be used
for readout. The signal may be amplUied
in liDe with sensitivity to ,ive direct
concentration readout. Temperature
compenBatlon is usefUl and available.
Membrane covered sensors eelective
for dissolved gases protect sensor
lurfaces from most interleriDg components.
The anaqlt must learn to use his
particular instrwnent effectlveq to enable
him to obtain valid relultl under conditions
in which a wet chemical procedure would
be misleading.
DO titrations for clean water samples
may be adapted for field use by shifting
'0 dry reagents encaaed in plastic
pillowB, substituting phenyl arsene
oxide for th10 and using plastic burets,
sample containers, etc. Powdered
Itarch substitutes also are available.
Sample lizes may be altered along
with reagent concentration to maintain
the 1 to 1 ratio of DO to titrant volume.
Hach, La Motte and probably others,
provide DO test kits packaced for field
use. "Home made" kits are common.
Alkalinity' and hardness titration. are
commonly lought under field conditions
using IItandard acid and EDT A solutions.
Methyl purple or mixed indicator II
commonly are used in place of methyl
orange indicator because of better
visib1l1ty'in the field. . Powder pillows
for buffer and indicator are available
for the hardness telt.
Chlorine tests u.ing ortho to11d1ne in a
comparator, such as ,those by HUger
or Taylor and others, frequently are
used for rapid on site measurements.
The comparators, reagents an4 comparator
tubes commonq are available from
apparatus, equipment, or chlorine
Simple and rapid field test kits are
available from several manufacturers,
suppliers and scientific apparatus
hou.es for individual tests, a. well
as combination test kits for the items
mentioned and others.
It is poslible for the anaqst to devise
one suited to his particular requirements
with availf,ble or improvised material..
Effectiveness of the field operation
dependl on the knowledge and care
exercised in using the equipment and
CAUTION: Field test results are
useful, but not always valid
enough for the record.
Thil outline contains signUicant
material fram a prevl.ous outline by
D. G. BalUn..r.
This outline was prepared by F. J. Ludzack,
Chemist, National Training Center, WPO,
CiPlclnnati, OH 45268.
Eescriptors: Chemical Analysis, On-Site
Tests, Water Analysis

Attached rind FiJure 1 of the kit. This kit is
capable or mealuring DO, temperature,
alkalinity (both phenolphthalein and total)
and hardness under neld conditions. An
approximation of pH can be obtained with
indicators (phenolphthalein and methyl
purple) and indioator papers contained in
the kit. It is presumed that the chemist will
also carry a portable pH meter, conductivity
prob., and DO sampler, it desired. The
t.lt. can be performed with the following
+ 1
- s
o - 10 mgll :
0.2 mgll
o -
10 mgll j
1 mgll
Ph.nolphthale in

TotalAlkalinity 100 - 300 mg/l t
7 mgll
200 - 400 mg/1 t 15 mgll
Th. kit i8 constructed of high grade outdoor
plywood, reinforced with waterproof joints,
and painted with epoxy paint. Styrofoam
packin, is used inside the case (See Figure 4);
a polyurethane foam rectangular block
(15 1/4" X 7 1/2") is placed inside the top
lid \0 lecure the reagents and equipment
during travel. The pipets are held in two
.ponge rubber blocks. (See Figure 3)
(See Figure 2)
A Distilled Water (250 rnl polyethylene
bottle with wash bottle attachment).
To be used for rinsing buret used in
B Manganous Sulfate (powder pillows
available from Hach O1emical Company,
Ames, Iowa).
C Alkaline-Iodide-Azide (available from
Hach Chemical Company, Amel, Iowa).
D Sulfamic Acid. This powdered reagent
can be used instead. of sulfuric acid in
the 00 test. It 18 available as packa.ed
powder pillows from Hach Chemical
E 0.0250 N Phenylarsene Oxide, PAO
(available from Hach Chemical Company)
contained in a 250 ml polyethylene
bottle with a wash bottle attachment.
Add 90 ml of distiUed water to 180 m1
of the 0.0250 N PAO. This gives 250 ml
of 0.016 N PAO.
F Starch (in a small 100 ml bottle -
polyethylene). Superlose 500 - a 801uble
starch in powder form - is used. It 18
available from Stein Hall and Company,
285 Madison Avenue, New York, N. Y.
$25/100 lb.
G Glass Stoppered 00 Bottle (125 ml
nominal size, 129 ml actual size)
This bottle is used to collect the 00
sample. Reagents are directly added
NOTE: Mention of commercial products and manufacturers does not imply endorsement by the
omce of Water Programs, Environmental Protection Agency.
CR, Dc. 3, 74

Chemical Field Kit Description
and then entire sample is transferred to
250 ml Erlenmeyer flask for titration.
The bottle also has calibration marks at
25, 50, 75, and 100 ml for use to obtain
samples for other determinaticma.
H 250 ml Centrifuge Bottle or Erlenmeyer
Fla.k (Po~carbonate). To be u.ed for
I Phenolphthalein and methyl purple ( in
100 ml po~ethylene dropper bottles).
Methyl purple can be obtained from
Flei.her Chemical Company, Benjamin
Franklin Station, Walhington, D. C. 20204.
J 0.02 N Sulfuric Acid (in 250 m1 po~-
ethy18lle bottle with wash bottle attachment).
K Univer In (Hardness Indicator + Buffer)
powder pl.11ows. The8e are avaUable
from Hach O1emical Company.
L pH Testinj Paper (Range 2. 0 - 12.0 and
6.6 - 8.1). TheBe papers are available
in Itrip form from Oxyphen Products
Company, 1>.0. Box 148, Forest HIDs,
New York.
M BiDTA (0.01 M) - in 250 ml polyethylene
bbttle with wash bottle attachment.
N 10 ml pipet, buret tubinr, microburet clamp,
right angle clamp, Bcissors and 14 1/2"
lon8 aluminum rod. (See Figure 5)
o Thermometer - a pocket type of Weston
aU metal type.
P Chemical Kits and Reagents are alao
avaUable from other companies such 1.8
LAMotte Chemical Company, Chestertown,
Q Acidity Rea8ents can be cOl'lvenient~ sub-
stituted in the kit.
R If it is desirable, the kit can be manipulated
80 that it is possible to do more DO
determinations. This is done by sub-
stituting DO bottleB (125 ml) for either
or both alkalinity or hardness reagents.
A Di..olved Oxygen
1 Collect sample in 125 ml bottle.
2 Add 1 pillow manganous sulfate and
I piUow alkaline iodide.

3 Stopper bottle. Mix. Allow pre-
cipitate to settle. Repeat..p 3.
4 Add 1 pillow lIullamic acid. Mix well.
5 Transfer entire contents of 125- ml
bottle to Erl_meyer fJask.
6 Titrate to pale yellow with 0.016 N
PAO. At this point, add a pinch at
7 Finish titration to colorlen endpoint.
8 Calculation:
ml PAO ulled. mg oxygenll
B Hardnelill
1 Measure 25 mlof sample into
Erlenmeyer flask.
2 Add 1 indicator-buffer pillow.
3 Add a pinch of hardness indicator.
4 Titrate to blue endpoint with EDTA
(1 ml . 1 m8 CaC03)' Note: The
last few drops of tile titration should
be added at 3 sec. interval..
5 Calculation:
m8/1 hardnus as CaC03 " EDTA used X40
C Phenolphthalein Alkalinity
1 MealUre 50 ml sample into
Erlenmeyer flask.


Chemical Field Kit De8cription


Figure 1.
ALUMINUM .00 14.1/2 lONG
10 "'L PI"'"
- "::;:1
.I (1.1/4"d
I[ (1.1/4""
m (1/1"')
m 11-8/1"41
][ (1/2""
pH UST ,.,,.
m 11/2"411
r1- 3/8' d~
]I (1/2"8'
Z (S/I"'4'
lull't CLAM'
H01l "01 AUJMINUM 11'00
F'lgure 2.

.I 10 m I PIPITTfS
5 3/e-
8 ~ I '2"
'''; 2'

Chemical Field Tnp DescnptLOn

I ,
~ -
Fi(UI"6 5. BURET

C~ioa1 Field Kit D.'Cl'iption
2 Add 2 drop. phenolphthalein indicator.
3 If eolution i8 pink, titrate to colorle.e
endpoint with 0.02 N H2SO4.
4 Calculation:
m,ll phenolphthalein alkalinity ae CaC03 .
ml 0.02 N H2SO4 X 20
D Total-Alkalinity
1 To' eample (on which phenolphthalein
alkalinity has juet been determined)
add 5 drop. methyl purple indicator.
2 Titrate from green color to purple
endpoint with 0.02 N H..SO .
NOTE: A blue-grey co1or ippears
ju.t before the endpoint.

m,II total alkalinity as CaC03 . mg/l

phenolphth.lein .lkalinity + mg/l methyl
purple alkalinity (ml O. 02 N H2SO" ueed in
methyl purple titration X 20).
1 Kroner, Robert C.. Lon.bottom, Jamee
E., and Gorman, Richard.
A Compari8on of Various R_gente
Propoeed for Use in the Winkler
ProcedVre for Dissolved Oxygen.
PHS Water Pollution SurveWance
SYRem Applications and Development
Report 112. July 1964.
2 Water Minerals 13. Analytiaal
Refereilce Service. Trail11ng
Pro,ram, R9bert A. Taft Sanitary
Enltlt..rm.-Center. November 1961.
Thi8 outline ""8 r;8pared by B. A. Pqhorst,
Chemist, forlll'" with National Training Center,
FWQA. Cincinnati. OH 45228.
Dee<:riptor8:' Alkalinity, Chemical Analyeis,
Dileolved OxyJeD, Hardnes8, On- Site Te.t.,
Testing, Water Analysi.

Part 1. General Concepts
A Bacterial Indication of Pollution
1 In the broadest sense, a bacterial
indicator of pollution is any organism
which, by its presence, would demon-
strate that pollution has occurred, and
otten suggest the source of the pollution.
2 In a more restrictive sense, bacterial
indicators of pollution are associated
primarily with demonstration of con-
tamination of water, originating from
excreta of warm -blooded animals
(including man, domestic and wild
animals, and birds).
B Implications of Pollution of Intestinal
1 Intestinal wastes from warm-blooded
animals regularly include a wide
variety of genera and species of
bacteria. Among these the coliform
group may be listed, and species of
the genera Streptococcus, Lactobacillus,
Staphylococcus, Proteus, Pseudomonas,
certain spore-forming bacteria, and
2 In addition, many kinds of pathogenic
bacteria and other microorganisms
may be released in wastes on an inter-
mittent basis, varying with the geo-
graphic area, state of community
health, nature and degree of waste
treatment, and other factors. These
may include the following:
a Bacteria: Species of Salmonella,
Shigella, Leptospira, Brucella,
Mycobacterium, and ~ ~.
b Viruses: A wide variety, including
that of infectious hepatitis, Polio-
viruses, Coxsackie virus, ECHO
viruses (enteric cytopathogenic
human orphan -- "viruses in search
of a disease"), and unspecified
viruses postulated to account for
outbreaks of diarrheal and upper
respiratory diseases of unknown
etiology, apparently infective by
the water-borne route.
c Protozoa: Endamoeba histolytica
3 As routinely practiced, bacterial
evidence of water pollution is a test
for the presence and numbers of
bacteria in wastes which, by their
presence, indicate that intestinal
pollution has occurred. In this con-
text, indicator groups discussed in
subsequent parts of this outline are
as follows:
a Coliform group and certain sub-
b Fecal streptococci and certain
sub groupings
c Miscellaneous indicators of pollution
4 Evidence of water contamination by
intestinal wastes of warm-blooded
animals is regarded as evidence of
health hazard in the water being tested.
A A n "ideal" bacterial indicator of pollution
I Be applicable in all types of water
51- 1

BacterioloJr1cal Indicator8 of Water Pollution
2 Alway. be pre8ent 1n water when
patltogenic bacterial con8tituents of
fecal contamination are present.
Ramification8 of thi8 include --
a Its den8ity 8hould lave some direct
relationship to the degree of fecal
b It Mould have It'eater lurvival time
in water than enteric pathogens,
throuahout itl course of natural
disappearance from the water body.
c It should disappear rapidly from
water foUowing the disappearance
of pathogens, eUher through natural
01' man-made procelses.
d It always 8hould be ab sent in a
bacteriologicaD,y safe water.
3 Lend it8elf to rout1ne quantitative
testinl procedure8 without 1nterference
or'confU8ion of results due to extra-
neOU8 bacteria
4 Be harmless to man and other animals
B In aU1probabWty. an "ideal" bacterial
indicator does not exUt. The diecu8ston
,of baCterial1ndicatore of pollution in the
following parts of thi8 outline 1nclude
consideration of the merU8 and Umitatlons
of each It'0UP, with their applications 1n
evaluating bacterial quality of water.
A Test. for Compliance with Bacterial
Water Quality Standards
I Potability test8 on drinking water to
meet Interstate Quarant1ne or other
standard8 of regulatory agencies.
2 Determination of bacterial quality of
environmental water for which quality
Sfandarc18 may exi.t, such as 8helU18h
water., recreational waters, water
re80urces for municipal or other
3 Tests for compliance with establi.hed
standard. in cases involving the pro-
tection or prosecution of m181ictpa1ttie..
industrie.. etc.
B Treatment Plant Process Control
1 Water treatment plants
2 Wa.tewater treatment plant.
C Water Quality Survey.
1 Determination of intestinal pollution
in surface water to determlM type and
extent of treatment required for com-
pUance with standards
2 Tracing sources of pollution
3 Determination of effects on bacterial
flora, due to addition of orpnic or
other wastes
D Special Studies, such as
1 Tracing sources of intest1nalj>&thogens
in eptdemiologicalinve.ti,.~n.
2 Inve8t1pttons of problem. due to the
Sphaerotilua IrouP
3 Inv.sttaations of bacterial interference
to certain industrial proo....., with
r..p.ct to .uch orpniam. a. P.eudo-
~, Achromobacter, or other;--
The laboratory bacteriologist il not alone in
evaluation of indication of water pollution of
1ntestinalorillin. On-site studi)' (SanUary
Survey) of the aquatic environment and
adjacent area8, by a qualified person. Is a
necessary collateral study with the laboratory
work and frequently wUI reveal1nformation
regard1ni potential bacteriolollcal hazard
which mayor may not be demonstrated
through laboratory findinjs from a single
sample or short series of sample..

Bacteriololl1cal Indicators of Water Pollution
Part 2. The Coliform Group and Its Conatituents
A Backll'0und

1 In 1885, Escherich, a pioneer bacteri-
ologist, recovered certain bacteria from
human feces, which he found in such
numbers and consistency as to lead him
to term these organisms "the charac.
teristic organism of human feces. "
He named these organisms Bacterium
£2Y-commune and]!. .!!£!!! aerogenes.
In 1895, another bacteriologist,
Migula, renamed B. coli commune as
!lheriChiA ~ wh1Chtoday is the
o cial name for the type species.
2 Later work has substantiated much of
the original concept of Escherich, but
has shown that the above species are
in tact a heterogeneous complex of
bacterial species and species variants.
a This heterogeneous group occurs not
only in human feces but representatives
also are to be found in many environ-
mental media, including sewage,
surface freshwaters of all categories,
in and on soils, vegetation, etc.

b The group may be !lubdivided into
various categories on the basis of
numerous biochemical and other
differential tests that may be applied.
B Composition of the Coliform Group
1 Current definition
As defined In "Standard Methods for the
Examination of Water and Wastewater"
(l3th ed): "The coliform group includes
all of the aerobic and facultative
anaerobic, Gram-negative, nonspore-
forming rod- shaped bacteria which
ferment lactose with gas formation
within 48 hours at 350 C. "
2 The term "coliforms" or "coliform
group" is an inclusive one, including
the following bacteria which may
meet the definition above:
a Escherichia coli, E. aurescens,
~. freundii, ~. intermed1a
b Aerobacter aerogenes, ~. cloacae
c Biochemical intermediates between
the genera Escherichia and Aero-
bacter ----

* The above terminology is in accordance
with the current edition. of Standard
Methods and Bergey's Manual of Deter-
minative Bacteriology and will be
consistent throughout this manual until
these sources are mocUtied.
3 There is no provision in the definition
of coliform bacteria for "atypical" or
"aberrant" coliform strains.
a An individual strain of any of the above
species may fail to meet one of the
criteria of the coliform group.
b Such an organism, by definition, is
not a member of the coliform group,
even though a taxonomic bacteriologist
may be perfectly correct in classifying
the strain in one of the above species.
A Need
Single-test differentiations between
coliforms of "fecal" origin and those of
"nonfecal" origin are based on the
assumption that typical E. coli and
closely related strains are Offecal
origin while ~. aerogenes and its close
relatives are not of direct fecal origin.
(The latter assumption is not fully borne
out by investigations at this Center.
See Table 1, IMViC Type - -++). A
number of single differential tests have
been proposed to differentiate between
"fecal" and "nonfecal" coliforms.
51- 3

d&c~IiCJlloi1callD41catorl of Water ~iOD
Without dilcu'l!Iion of their relative merits,
,everd may be cited here:
B Type, of Siqle-Te.t Differentiation
1 Determination of p' ratio
Fermentation of gluool!le by E, coll
ruult. in ..S production, wTth-
hydropn and carbOn diOiltide beine
Jlroduced in equal.mounts.
Fermentation of glueo.e by A.
aerogene. result. in generaUOn of
twice a. much carbon dioxide as
Further 8tudies su,.eated ab80lute
correlation betwe- H../C~2 ratioll
and the terminal pH rl8UltlDf from
,luco.e f.rmentat:fon. Thi. led to the
.ubstitutl.on of the methyl red tut.
2 Methyl r~ test
Gluco.e fermentation by E. coll
typically result, in a cultiire medium
having terminal pH m the range 4.2 -
4. 6 (red color a po.itive test with the
addition of methyl red 1ftdicator).
A. aerolenes typically relults in a
CiUlture medium havinl pH 5.8 or
great... (yellow color, a negative test).
3 Indole
When tryptophane, an amino acid, ill
mcorporated in a nutrient broth,
typical!: 0011 straW are capAble of
produdn, IiiCfole (po.ltlve test) among
the end products, whereas!,. a.roaenes
/loes not (negative test).
In revi.win, techntcalllterature, the
worker should be alert to the method
..ed to detect indole formation, as the
result8 may be grea~ tntluenced by
the ana~ical prooedure.
4 Vo,es-Proekauer t..t (aoetylmethyl
carbinol te.t)
The telt i. for detection of acetylmethyl
carbinol, a derivative of 2, 3, butylene-
117001. a. a re.ult of ,luco..
fermentation in the pre.ncI of
peflcne. !. aeropnes produces
thi8 end p1'*luct (p08it1ve ft.)
whereas !,. ~ gives a ne_ve te.t.
a EKperience with coliform eulture.
livinl a po.ittve te.t has shown a
1081 of this ability with .torage on
18It0ratory media for 8 months to
2tyear., m 20 - 25% of cultures
(105 out of 458 culture.).
b Some worker. consider that all
eolifora llaotert& prodl1oe acetyl-
methr1 C8S"bl8olin glueo.. metab-
oli8m. 1hese worker. rqard
acetylme_l caJtJInoI-..,.tl.ve
aulture. as those wh1cl1 _ve
enzyme .,..tem. capAble Of further
degradation ot acetylmethyl
carbinol to other end prqdl1cts
wi\ich do not give a po.ttl.ve test
with the analytical procedlZ'e.
Cultures pving a positive test tor
acety1m8tb;y1 carbinollaok this
enzyme 'Yl!ltem.
c This real!loning leads to a hypothesis
(not experimentally pro"...) that the
change of reaction noted in certain
cultures in 4.a above 18 due to the
activation of a latent enzyme system,
5 Citrate utWzation
Cultures of E. coll are UDable to use
the carbon 01 citrate. (nepUve te.t)
in their metabolism, whereas cultures
of.!,. aero,enes are capable of u.in.
the carbon of citrates in their metab-
olism (positive teat).
Some workers (ueing Simmon. Citrate
Apr) incorporate a pH 1nd1ator
(brom thymol blue) in the culture
medium in order to demm8U'ate the
typi_l alkaline reaction (pH t.4 - 9. 0)
reaultf.ng with citrate utiU:&atlon.

6 Elevated temperature (Eijkman) test

a The tellt is based on evidence that
!.:. ~ and other coliforms of fecal

Bacteriolollical Indicators of Water Pollution
origin are capable of growing and
fermenting carbohydrates (glucose
or lactose) at temperatures signif-
icant1y higher than the body tem-
perature of warm-blooded animals.
Organisms not associated with direct
fecal origin would give a negative
test result, through their inability
to grow at the elevated temperature.

b While many media and techniques
have been proposed, EC Broth, a
medium developed by Perry and
Hajna, used as a confirmatory
medium for 24 hours at 44.5 t
0.20 C are the current recommended
medium and method of choice.
While the "EC" terminology of the
medium suggests "E. coli" the
worker should not N,gard this as a
specific procedure for i8olation of

c A similar medium, Boric Acid
Lactose Broth, has been developed
by Levine and his associates. This
medium gives results virtual1y
identical with those obtained from
EC Broth, but requires 48 hours of
d Elevated temperature tests require
incubation in a water bath. Standard
Methods 13th Ed. requires this
temperature to be 44.5:t 0.2oC.
Various workers have urged use of
temperatures ranging between
43. ooC and 46. ooC. Most of these
recommendations have provided a
tolerance of t 0.50 C from the rec-
ommended levels. However, Bome
workers, notably in the Shellfish
Program of the Public Health Service,
stipulate a temperature of 44.5 t
0.20 C. This requires use of a water
bath with forced circulation to main-
tain this close tolerance. This
tolerance range has been instituted
in the 13th Edition of Standard Methods
: and the laboratory worker should
conform to these new limits.
e The reliability of elevated temper-
ature tests is influenced by the
time required for the newly-
inoculated cultures to reach the
designated incubation temperature.
Critical workers insist on place-
ment of the cultures in the water
bath within 30 minutes, at most,
atter inoculation.
7 Other tests
Numerous other tests for differentiation
between coliforms of fecal vs. nonfecal
origin have been proposed. Current
studies suggest little promise for the
following tests in this application:
uric acid test, cellobiose fermentation,
gelatin liquefaction, production of
hydrogen sulfide, sucrose fermentation,
and others.
C IMViC Classification
1 In 1938, Parr reported on a review of
a literature survey on biochemical tests
used to differentiate between coliforms
of fecal vs. nonfecalorigin. A summary
No. of times
used for dif-
Methyl red test
Citrate utilization
Indole test
Uric acid test
Cellobiose fermentation
Gelatin liquefaction

Eijkman test

Hydrogen sulfide
Sucrose fermentation
a-Methyl-d- glucoside

Hacteriololi:1cal Indicators of Water Pollution
Based on this summary and on his own
studies, Parr recommended utilization
of a combination of tests, the indole,
methyl red, Voges-Proskauer, and the
citrate utilization tests for this differ-
entiation. This series of reactions is
designated by the mnemonic "IMViC".
Using this scheme, any coliform culture
can be described by an "IMViC Code"
according to the reactions for ..ach
culture. Thus, a typical culture of
E. coli would have a code ++- -. and a
tjp~~. aeroj(enes culture would
have a code --++.
Gruupings of coliforms into fecal,
non-fecal, and intermediate groups,
as shown in "Standard Methods for the
Examination of Water and Wastewater"
are shown at the bottom of thi5 page.
D t'
Bacter~oloJ[icallDdicators of Water Pollution
c A wider variety of environmental and
biolopcal 80urces i8 beinlltudied
than in any previous serie8 of reports.

d All studiel are based on freshly
recovered pure culture ilolates
from the designated sources.
e All studiel!l are based on cultures
recovered from the widest feasible
leographic range, collected at aU
seasons of the year. It is believed
that no more representative series
of studies ha8 been made or is in
:I Di8tribution of coliform types
Table 1 shows the consolidated results
of coliform distributions from various
biological and environmental sources.
a The result8 of these studies show a
high order of correlation between
known or probable fecal origin and
the typical E. coli IMViC code
(++--). ori'ihiOiher hand,
human feces a180 includes
numbers of~. aerogenes and other
IMViC types, which some regard as
"nonfecal" segments of the coliform
group. (Figure 1)
b The majority of coliforms attributable
to excretal origin tend to be limited
to a relatively small number of the
possible IMViC codes; on the other
hand, coliform bacteria recovered
from undisturbed soil, vegetation,
and insect life represent a wider
range of IMViC codel!l than fecal
sources, without clear dominance of
anyone type. (Figure 2)
c The most prominant IMViC code
from nonfecal sources is the inter-
mediate type, -+-+, which accounts
for almost half the coliform cultures
recovered from soils, and a high
percentage of th08e recovered from
vegetation and from insects. It
would appear that if any coliform
segment could be termed a "soil
type" it would be IMViC code -+-+.
d It should not be surprising that
cultures of typical E. col1 are
recovered in relativelysmaller
numbers from sources judged,
on the basis of sanitary survey,
to be unpolluted. There is no
known way to exclude the influence
of limited fecal pollution from small
animals and birds in such environ-
e The distribution of coliform types
from human sources should be
regarded as a representative value
for large numberl!l of sources.
Invel!ltigations have shown that there
can be ]arie differences in the
distribution of IMViC types from
person to person, or even from an
3 Differentiation between coliforms of
fecal vs. nonfecal origin
Table 2 is a summary of findings
based on a number of different criteria
for differentiating between coliforms
of fecal origin and those from other
a IMViC type ++- - is a measurement
of~. coli, Variety I, and appears
to give reasonably good correlation
between known or highly probable
fecal origin and doubtful fecal origin.
b The combination of IMViC types,
++--, +---. and -+--, gives
improved identification of probable
fecal origin, and appears also to
exclude most of the coliforms not
found in excreta of warm-blooded
animals in ]arge numbers.
c While the indole, methyl red,
Voges Proskauer, and citrate
utilization tests, each used alone,
appear to give useful answers when
applied only to samples of known
pollution from fecal sources, the
interpretation is not as clear when
applied to coliforms from sources
believed to be remote from direct
fecal pollution.


I ++--
I ~:::

I -++-
No. EC +
%EC +
I T ~~
Vegetation Insectll r UTTDdisturbed
,- No. I ,. of 'i No. ! ,. of I No. % of
sVains I total strains' total' strains total

I ' I
134 12.4 131
i I
113 110.4 I 443 18.8
o ;<0. 1 !
o <0.1 :
128 110.6
237 19.7
23 1. 9
0.2 !
14.0 I
9. 6

7.2 i
0.4 I
*120 of these
were ++--.
15 --++.
11 -+--
2.6 !
<0. I
<0. 1
*129 of these
were ++-~,
27 -+-+.
5 ++-+
48. 1
159 I 6. 8
67 : 2.9

4 0.2
<0. 1
<0. 1
No. t .,. of
strains' total
536 ! 80. 6

13 2.0

1 ~ 0.2 I

o '<0.1


87 i 13.0
22 ! 3.3
5 0.7
<0. 1
<0. 1
<0. 1
<0. 1
Eecal s urces I PoaIU'y I
HlJn on l.iv.".tnrJL. I --r-:;;--:;i
No. ,. of No. T,. oi1 No. ,'" of !
straw total strains' total' strains I total I
! I I I
2237 195.6! 1857: 97.9 !

o i <0. 1 1 O. 1 .
, I
20 ' 1.1 '

o 1<0.1

5 0.3

11 0.6 i
87.2 I
99 2.2 I
106 I 2.4 :
50 1.1
35 ! 0.8
21 ; 0.5 :
6 ! 0.1 '
14 i 0.2 :
2 I <0.1 I
o <0.1


14 0.6
59 i 2.5 ,
1 i <0.1 ,
27 ' 1. 2 :
o i <0.1 i
, I
o i <0.1 !
o ; <0.1 I
<0. 1

Bacterioloi1cal Indicators of Water Pollution
,---------------1 ++--1
// , .
'" / I
'" / I
",,,,'" /1 I
liC ~ 96."
BAli ~ 9".7
..6__--1 -+-+ I
liC $ 9'.7
.Al8 ~ 98.6
Typo 'o,.on' po.i';vo
+- 91.8
+- 1.5
11--- 0.1
-+ 2.8
liC~ 96.3
BAL' 95.3
I -+--}--
liC ldrt'kh. t't. .11.)
....of - - + + .t-..
"",-- --...
,..- i t-...
"."",'- -- ++- - ... -......
r ........ ..........,
.. ..
s/- 9

1JacterioloJi/ical Indicator. of Wa~er Pollution
AT 44.50 C ( ! 0.50) (12th ed. 1965; Standard Methods for the Examination 01 Water
and Wastewater)
  - Warm-blooded Soil: Soil: Vege- 
  Test  animal feces Unpolluted pollaated tation Insects
++ -   91. 8% 5.6% 80.6% 10.1\% 12.4%
+ + - -,       
+ - - - and   93.3% 8.90/0 80.7% 12.50/0 13.2%
- + -       
Indole positive  94.0% 19.4% 82.7"- 52. 50/. 52. 40/.
Methyl red positive 96.90/0 75.6% 97.9'- 63.6% 79. 90/.
Voges-proskauer positive 5. 1% 40.7% 97.3% 56.30/0 40.6%
Citrate utilizers  3.6% 88.2% 19.2"!o 85.10/. 86.7%
Elevated temperature (EC). 96.4% 9.2% 82. 9fo 14.1% 14.90/0
Number of I'ultures 8,741 2,348 665 1,203 1,084
Total Pure Cultures Studied: 14,047
d The elevated temperature test gives
excellent correlation with samples
of known or highly probable fecal
origin. The presence of smaUer,
but demonstrable, percentages 01
such organisms in environmental
sources not interpreted as beina
poUuted could be attributed largely
to the warm-blooded wildlife in the
area, includ1na birds, rodents, and
other smaU mammals.
e The elevated temperature test yields
results equal to those obtained from
the total IMViC code. It has marked
advantages in speed, ease and
simplicity of performance, and yields
quantitative results for each water
sample. Therelore, it i8 regarded
as the method of choice for differ-
entiation between coliforms of
probable direct fecal origin and those
which may have become established
in the bacterial flora of the aquatic
or terrestrial habitat.
A The Coliform Group as a Whole
1 Merits
a The absence of coliform bacteria is
evidence of a bacteriologically sale
b The den8'ity of coliforms is rough~
proportional to the amount of
excretal pollution present.
c If pathogenic bacteria 01 intestinal
origin are present, coliform
bacteria also are present, in much
greater number..
d Coliforms are always present in the
intestines of tmmans and other warm'
blooded animals, and are eliminated
in larlJe numbers in fecal wastes.

Bacte~iolQldcalindicator8 of Water Pollu1ion
e Coliforms are more persistent in
the aquatic environment than are
pathorenic bacteria of inte8tinal
b These organisms are of reJatively
infrequent occurrence except in
a..ociation with fecal pollution.
r Coliforms are generally harmleu
to humans and can be determined
quantitatively by routine laboratory
c Survival of the fecal coliform group
i. shorter in environmental waters
than for the coliform group as
whole. It follows, then. that high
densitiee of fecal coliforms is
indicative of relatively recent
2 Limitations
a Some of the constituents of the
coliform group have a wide environ-
mental distribution in addition to
their occurrence in the intestines
of warm-blooded animals.
d Fecal coliforms generally do not
multiply outside the intestines of
warm- blooded animals. In certain
high-carbohydrate wastes, such as
from the sugar beet refineries,
exceptions have been noted.
b Some strains of the coliform group
may multiply in certain polluted
waters ("attergrowth"), of high
nutritive values thereby adding to
the difficulty of evaluating a pOllution
situation in the aquatic environment.
Members of the!. aerO.ienes section
of the coliform are commonly
involved in this kind of problem.
c Because of occasional aftergrowth
problems, the age of the pollution
may be difficult to evaluate under
.ome circumstances.
a Feces from warm-blooded animals
include some (though proportionately
low) numbers of coliforms which do
not yield a positive fecal coliform
test when the elevated temperature
test is used as the crite rion of
differentiation. These organisms
are E. coli varieties by present
d Tests for coliforms are subjllct to
interferences due to other kinds of
bacteria. False negative re!Iults
sometimes occur when speci1es of
Pseudomonas are present. 1;;'a18e
positive results sometimes occur
when two or more kinds of non-
coliforms produce gas from Jactose,
when neither can do so alonf!
b There is at present no established
and consistent correlation between
ratios of total coliforms/fecal
coliforms in interpreting sanitary
quality of environmental waters.
a The majority (over 950/0 of the coli-
form bacteria from intest1/~es of
warm-blooded animals grow at the
elevated temperature.
In domestic sewage, the fecal
coliform density commonly is
greater than 900/0 of the total
coliform density. In environmental
waters reJatively free from recent
pollution, the fecal coliform density
may range from 10-300/0 of the total
coliforms. There are, however,
too many variables relating to
water- borne wastes and surface
water runoff to permit sweeping
generalization on the numerical
relationships between fecal- and
total coliforms.
B The Fecal Coliform Component of' the
CoUform Group (as determined by elevated
temperature teet)
1 Merits
c At this time, evaluations are
underway regarding the survival

Bacteriolollicallndlcators of Water Pollution
of fecal colUorms In polluted waters
cf'mpared with that of enteric
pathogenic bacteria. In recent
pollution .tudies, species of
Salmonella have been found In the
presence of 220 fecal coliform. per
100 ml (Spino), and 110 fecal
coillorms per 100 ml (Brezenski,
Raritan Bay Project).
A Current Statu. In Official Tests
1 The coliform group is designated, In
"St~dard Methods for the Examination
of Water and Wa"tewater" (13th ed..
1971), through the Completed Test
MPN procedure all the official test
for bacteriological potability of water.
The Confirmed Test MPN procedure
is accepted where it has been demon-
strated, through comparative tests,
to yield results equivalent to the
Co~pleted Test. The membrane filter
method .lso is accepted for examination
of waters subject to Interstate regulation.
2 The 12th edition of Standard Methods
Introduced the standard test for fecal
coliform bacteria. It is emphasized
that this is to be used in pollution
studies, and does not apply to the
evaluation of water for potability;
This procedure has been carried to
the 13th Edition.
B Applications
1 Tests for the coliform group as a
whole are used In official tests to
comply with Interstate drinking water
standards, state standards for shell-
fish waters. and In most, if not all,
cases where bacterial standards of
water quality have been established
for such use as In recreational or
bathing waters, water supplieR, or
industrial supplie s. Laboratory
personnel should be aware of possible
implementation of the fecal coliform
group as the official test for recreational
and bathing waters.
'2 The fecal coliform test has application
in water quality survey's, as an adjunct
to deterblnation of total colifOrm
density. The fecal coliform test is
being used increasingly in all water
quality surveys.
3 It is emphasized that no responsible
worker advocates substitution of .a
fecal coliform test for total coliforms
in evaluating drinking water quality.

Bacterioloaicallndicators of Water Pollution
Part 3. The Fecal Streptococci
Inv..Uptions re.&rding streptococci
proFelsed from the streptococci of medical
concern to those which were distributed in
differiDI enviromnental conditions which,
apJD, related to the welfare of man. The
Itreptococci were originally reported by Laws
and Andrews (1894), and Houston (1899, 1900)
conlidered those streptococci, which we now
can "fecal streptococcI," as . .. "indicative
of dan,.rous poUution, Bince they are readi]y
demonltrable In waters recently polluted and
seemlng]y alto,.ther absent from waters above
luspicion of contamination.
From their discovery to the present time the
fecal Itreptococci appear characteristic of
fecal pollution, bein~ consistent]y present in
both the feces of all warm-blooded animals
and In the environment associated with animal
dilchar,es. As ear]y as 1910 fecal strepto-
cocci were proposed as indicators to the
Metropolitan Water Board of London.
However, little progress resulted in the
United States until improved methodB of
detection and enumeration appeared after
World War II.
Renewed interest in the group as indicators
bepn with the introduction of azide dextrose
broth in 1950, CMallmann &. S'!U8JDann, 1950).
The method which is in the current edition
of Standard Methods appeared soon after.
CUt'lef, et al. 1955).
With the advent of improved methods for
detection and enumeration of fecal strep-
tococci, significant body of technical
literature has appeared.
Th1. outline will consider the findings of
various investiptors regarding the fecal
Itreptococci and the significance of discharges
of thele organi.ms into the aquatic environment.
A Definition
The terms "enterococci, " "fecal
streptococci," "Group D streptococci, "
"Streptococcus fe .::alis, " and even
"streptococci" have been used in a loose
and Interchangeable manner to indicate
the streptococci present in the enteric
tract of warm-blooded animals or of the
fresh fecal material excreted therefrom.
Enterococci are characterized by specific
taxonomic biochemistry. Serological
procedures diffe:rentiate the Group D>
streptococci from the various groups.
Althou£h they over1ap, the three grO\lpS,
fecal Itreptococcus, enterococcus, and
Group D streptococcus, are not synonymous.
Because our emphasis is on indicators of
unsanitary origin, fecal streptococcus is
the more appropriate term and will include
the enterococcus as well as other groups.
A rigid definition of the fecal streptococcus
group is not possible with our present
knowledge. The British Ministry of Health
(1956) defines the organisms as "Gram-
positive" cocci, generally occurring in
pairs or short chains, growing in the
presence of bile salt, usual1y capable of
development at 450 C, producing acid but
not gas in mannitol and lactose, failing to
attack raffinose, failing to reduce nitrate
to nitrite, producing acid in litmus milk
and precipitating the casein in the form of
a loose, but solid curd, and exhibiting a
greater resistance to heat, to alkaline
conditions and to high concentrations of
salt than most vegetative bacteria. II
HoW'ever, it is pointed out that "streptococci
departing in one or more particulars from
the type species cannot be disregarded
in water. "
For the proposes of this outline, and in line
with the consensus of most water micro-
biologists in this country, the definition
of the fecal streptococci is:
. . . "The group composed of Group D
species consistently present in
significant numbers in fresh f~cal
excreta of warm- blooded animals,
which includes all of the enterococcus
group in addition to other groups of
streptococci. "

Bacterioloalcallndlcators of Water Pollution
B Species Isolated
1 Findinis
a Human feces
ExamJnation of human fecal specimens
yield8 a high percentage of tile
enterococcus group and usualJy
demonstration of the S. sal1varius
which is generally considered a
member of the human throat nora
and to be surviving in human fecal
materials rather than actively
multiplying in the enteric tract.
AI80 present would be a small
percentage of variants or biotypes
of the enterococcus group.
b Nonhuman Feces
1) Fecal material which are from
nonhuman or not from fowl will
yield high percentages of the
S, bov1s and/or S. eQuinus
organisms with a concomitantly
reduced percenta&e of the
enterococcus group.
2) Fowl excreta
Excrement from fowl characteris-
tically yields a large percentage
of t!nterococcal biotypes as well
as a significant percentage of
enterococcus group.
2 Significance
Specie. a..oclation. with particular
animal hosts is an estabUshed fact and
leads to the important laboratory
technique of partition counting of colonies
from the membrane filter or pour
agar plates in order to establish or
confirm the source of excretal
pollution In certain aquatic investi-
It is important to realize that a suitable
medium is necessary in "rder to
allow an of the streptoco"cci which
we consider to be fecal streptococci
to grow in order to give credence to
the derived opinions. Use of Uquid
growth media into which direct
inoculations from the sample are
made bave not proven to be 8UQcessful
for partition counting due to the differing
growth rates of the various .,.cles of
streptococci alter1ne the or""l
percentage relationships. Due to the
limited survival capabWties of some
of the fecal streptococci it is necessary
to sample fresh fecal materul or water
samples in close proximity to the
pollution source especially when
multiple sources are contributing to a
reach of water. Also the pH range
must be within the raage of 4.0-9. C.
A General
From the foregoing it is apparent that
the preponderant human fecal Itreptococci
is composed of the enterococcus group
and, as this is the case, several media
presently available which will detect only
the enterococcal aroup will be suitable
for use with aquatic samples which are
known to be contaminated or potentially
contaminated with purely domestic
(human)'waates. On the 'Jther hand,
when it is known or suspected that other-
than-human wastel have potential egress
to the aquatic environment under 1nveIU-
gation, it is necessary to utilize those
media which are capable of quantitatina
the whole of the fecal streptococci group.
B Stormwaters and Combined Sewers
1 General
Storm sewers are a series of pipes
and conduits which rer.eive surface
runoffs from the action of rainstorms
and do not include .ewai. which are
borne by a 8Ystem of sanitary sewers.
Combined sewers receive both the runoff
rains as well as the water borne wastes
of the sanitary system. Both of these

BacterlQ!Qadcal 4~f&tw8 of Wa~er Pollution
types of runoffs can be dischar ged to
the aquatic environment and the usual
instance wt\ere this occurs, with respect
to the combined sewers, i8 when the
amount of flow i. in excess of the
amounts capable of being treated during
the high flow conditions. Both of these
discharge form. have been found to
usually contain large quantities of
fecal streptococci and in numbers
which generally are larger than that
of the fecal coliform indicator organism.
Stormwaters can be concluded to
represent a typical stream environment
with respect to the presence of chemical
constituents and show a wide range of
electrolytes which at times simulate
that of irrigation waters.
2 Bacteriological Findings
Table 1 repre'ients, 1n a modified form,
some of the findings of Geldreich and
Kenner (1969) with respect to the
densities of fecal streptococci when
considering Domestic sewage in contrast
to Stormwaters:
Table I
Fecal Streptococci
per 100 ml
median values
- Water Source
Domestic Sewage  
Preston, :lD 6~, 000 5.3
Fargo, ND 290,000 4.5
Moorehead, MN 330,000 4.9
Cincinnati, OH 2,470,000 4.4
Lawrence, MA 1.500,000 4.0,
Monroe, MI 700,000 27.9
Denver. CO 2,900,000 16.9
Business District 51.000 0.26
Residential 150,000 0.04
Rural 58, 000 0.05
The Ratio FC! FS is that of the
Fecal coliform and Fecal streptococci
and it will be noted that in each case,
when considering the Domestic
Sewage, it is 4.0 or greater while
it 1s less than 0.7 for stormwaters.
The use of this ratio is useful to
identify the source of pollution as
   Average indicator Average contribution 
   dens ity per gram per capita per 24 hr 
   of feces    
 Avg wt of  Fecal Fecal Fecal Fecal 
Animals Feces!24 hr, coliform, streptococci, coliform, streptococci, Ratio
 wet wt, g  million million million million FC!FS
Man 150  13.0 3.0 2,000 450 4.4
Duck 336  33.0 54.0 11, 000 18, 000 0.6
Sheep 1, 130  16.0 38.0 18.000 43,000 0.4
Chicken 182  1.3 3.4 240 620 0.4
Cow 23,600  0.23 1.3 5,400 31, 000 0.2
Turkey 448  0.29 2.8 130 1,300 0.1
Pig 2,700  3.3 84.0 8,900 230,000 0.04
*Pub4cation WP-20-3, P. 102    
       51- 15

Bacteriolodcallnd1~atQr. of Water Polhl.tion
being human or nonhuman warm-
blooded animal polluted. When the ratio
i8 greater than 4. 0 it is considered to be
human waste contaminated while a ratio
of le.. than 0.7 is considered to be
nonhuman. It is evident that the storm-
w.ten have been primarily polluted by
eXcreta 01 rats and other rodents and
possibly domestic anMor farm animals.
Species differences are the main cause
of different fecal coliform-fecal
st'reptococci ratios. Table 2 compares
fecal .treptococCUB and fecal coliform
count. for different species. Even
ttroup individuals vary widely, masses
of individuals in a species have charac-
teristic proportion of indicators.
C Surface Waters
In general, the occurrence of fecal
streptococci1.ndicates fecal pollution and
its absence indicates that little or no
warm-blooded fecal contribution. in
studies of remote surface waters the fecal
streptococci are infrequently i.olated and
occurrences of small numbers can be
attributed to wUd life and/or anow melts
and resultant drainage flows.
Various examples of fecal streptococcal
occurrences are shown in Table 3 in
relation to surface water!! of widely varying
quality. (Geldreich and Kenner 1969)
A Genft'al
Serious studies concerning the streptococci
were instituted when it became apparent
that they were the agent. responsible or
suspected for a wide variety of human
diseases. Natural priority then focused
itself to the taxonomy of these organisms
and this study is still causing consternation
as more and more microbiolopcal techniques
have been brought to bear em these questions.
The sanitary microbiologist is concerned
with those streptococci which inhabit the
enteric tract of warm-blooded animals,
their detection, and utilization in develop-
ing a criterium for water quality standards.
Table 3
Water Source col1for~ strt.ococci

Prak'te Waterebeds
Cherry Creek. WY
Saline River. KS
Cub River, ID
Clear Creek, CO
Recreational Water.

Lake Mead
Lake Moovalaya
Colorado River
Whitman River
Merrimack River
Public Water Int,.kes

Missouri River (1959)
Mile 470.5 11,500
Mile 434.5 22,000
Mile 408.8 14,000
Kabler (1962) discu.sed the slow acceptance
of the fecal streptococci ac indicators of
poUution resulting from:
1 Multip11city and difficulty of laboratory
2 Poor agreement between methods of
quantitative enumeration
a sources
3 Lack of systematic studie. of. . . .
b 8urvival. and
c interpretations, and
4 Undue attention to the S. faecalis group.

Bacteriololllcalindicators of Water Pollution
Increased at:tenticn to the fecal streptococci.
especially during the last decade. have
clarified many of the earlier cloudy issues
anti have elevated the stature of these
organisms a8 indicators of pollution.
Court precedents establishing legal status
and recommendations of various technical
advisory board. have placed the fecal
coliform group in a position of primacy
in many water quality applications. The
fecal streptococci have evolved from a
pOllition of a theoretically useful indicator
to one which was ancillary to the coliforms
to one which was '.1seful when discrepancies
or questions evolved as to the validity of
the coliform data to one where an equality
status was achieved in ce rtain applications.
In the future it is anticipated that. for
certain applications, the fecal streptococci
will achieve a position of primacy for
u8eful data, and, as indicated by Litsky
(1955) "be taken out of the realm of step-
children and given their legitimate place
in the field of santiary bacteriology as
indicators of sewage pollution. "
Advantages and l.1mttations
1 Survival
In general, the fecal streptococci have
been observed to have a more limited
survival time in the aquatic environment
when compared to the coliform group.
They are rivaled in this respect only
by the fecal coli1'orms. Except for cases
of persistence in waters of high electro-
lytic content, as may be common to
irrigation waters, the fecal streptococci
have not been observed to multiply in
polluted waters as may sometimes be
observed for some of the collforms.
Fecal streptococci usually require a
greater abundance of nutrients for sur-
vival as compared to the coliforms and
the coliforms are more dependent upon
the oxy gen tension in the waterbody.
In a number of situations it was concluded
that the fecal streptococci reached an
extinction point more rapidly in warmer
waters while the reverse was true in the
colder situations as the coliforms now
were tota1~ eliminated sooner.
2 Resistance to Disinfection
In artificial pools the source of
contaminatio:1 by the bathers is
usually limited to throat and skin
flora and thus increasing attention
has been paid to indicators other
than those traditionally from the
enteric tract. Thus, one of the
organisms considered to be a fecal
streptococci, namely, S. sillvarius,
can be a more reliable indicator
when detected along with the other
fecal streptococci upecially since
studies have confirmed the greater
resistance of the fecal streptococci
to chlorination. This greater
resistance to chlorination, when
compared to the fecal coliforms, Is
important since the die off curve'
differences are insignificant when
the curves of the fecal coliforms
are compared to various Gram
negative pathogenic bactE'ria which
reduces their effectiveness as
3 Ubiquitous Strains
Among the fecal streptococcus are
two organisms. one a biotype and
the other a variety of the S. faecalis,
which, being ubiquitous (omnipresent)
have limited sanitary significance.
The biotype, or atypical, S. faecalia
is characterized by its ability to
hydrolyze star..:h while the varietal
form, liquefac1ens, is nonbeta
haemolytic and ~apable of liquefying
gelatin. Quantitation of these organisms
in anomalous conditions is due to their
capability of survival in soil or high
electrolytic' waters and in waters with
a temperature of less than 12 Degrees C.
Samples have been encountered which
have been devoid of fecal coliforms
and yet contain a substantial number of
"fecal streptococci" of which these
ubiquitous stralns constitute the majority
or all of the isolations when analyzed

Bacterie>lOl!icallndlcators of Water Pollution
Acceptance and utilizatlon of Total Coliform
criteria, which mU8t now be con.idered a
pioneering effort, has largely been 8upplanted
in concept and in fact by tne fecal coliform8
in e8tablishing standards lor recreational
The first significant approach to the utiliza-
tion of the fecal streptococci as a criterium
for recreatlonal water standard. occurred in
1986 when a technical committee recommended
the utilization of the fecal streptococci with the
total coliform. as criteria fo r .tandard8
pertaining to the Calumet River and lower
Lake,Michii&l\ water.. Several .et8 of
criteria were established to fit the intended
uses for thi. area. The u.e of the lecal
8treptococci a8 a criterium i8 indicated to
be tentatfve pending the accumulation of
exisUn. densities and could be modified in
futurl! standard8.
With the existing state-of-the-art knowledge
of the presence of the fecal streptococci in
waters containing low number8 of fecal
coliforms it is difficult to establi8h a 8pecific
fecal streptococcus den8ity limit of below
100 organism8/l00 ml when used alone or
in conjunctlon with the total coliform..
51- 111

Bacteriological Indicators of Water Pollution
Part 4. Other Bacterial Indicator. of PoUution
A Hi.torical
1 The early studies of Robert Koch led
him to develop tentative standards of
water quality based on a limitation of
not more than 100 bacterial colonies
per ml on a ,elatin plating medium
incubated 3 day. at 200 C.
2 lJiI.ter developments led to inoculation
of .amples on duplicate plating media,
with one set incubated at 370 C and the
other at 200 C.
a Results were used to develop a ratio
between the 370 C counts and the
200C count..

b Waters having a predominant
count at 370 C were regarded as
being of probable sanitary signifi-
cance, while those giving
predominant counts at 200 C were
considered to 1:>e of probable soil
origin, or natural inhabitants of
the water being examined.
B Group. Tested
There is no such thing as "total" bacterial
cOlU1t In terms of a laboratory determination.
1 Direct microscopic counts do not
differentiate between living and dead
2 P1ate counting methods enumerate only
the bll,cteria which are capable of using
the culture medium provided. under the
temperature and other growth conditions
u.ed as a standard procedure. No one
culture medium and set of growth
conditions can provide, .imultaneously,
an acceptable environment for all the
heterogeneous. often conflicting.
requirement. of the total range of
bacteria which may be recovered from
C Utilization of Total Counts
1 Total bacterial counts, using plating
methods. are useful for:
a Detection of changes in the bacterial
composition of a water source
b Process contr"l procedure. in
treatment p1ant operations
c Determination of sanitary conditions
in plant equipment or distributional
2 Serious limitations in total bacterial
counts exist because:

a No information is given regarding
possible cr probable fecal origin
of bacterial changes. Large numbers
of bacteria can sometimes be
cultivated from waters \mown to be
free of fecal pollution.
b No information of any kind is given
about the species of bacteria
c There is no differentiation between
harmless or potentially dangerous
3 Status of total counts
a There is no total bacterial count
standard for any of the following:
Interstate Quarantine Drinking
Water Standards
PHS regulations for water
potability (as shown in
"Standard Methods" Public'
Health Service Drinking
Water Standards of 1962, )
b The most widely used current
application of total bacterial counts
in water bacteriology today is in

Ba.cterlo1olZ1callndlcators of Water Pollution
water treatment plants, where some
workers use standard plate counts
for process control and for deter-
mInation of the bacterial quality of
distribution systems and equipment.
c Total bacterial counts are not used
in PHS water quality studies, though
extensively u.ed until the 19.0'8.
B Spore- Forming Bacteria (Clostridium
perfrinftns, or C. 1Jelchii)
1 Distribution
This is one of the most widely distributed
specie8 of bacteria. It is regularly
present in the intestinal tract of warm-
blooded animals,
2 Nature of organ1am
C. perfringens i8 a Gram-positive,
spore-forming rod. The spores cause
a distinct swellJ.ng of the cell when
formed. The orp.nism is extremely
active in fermentation of carbohydrates,
and produces the well-known "stormy
fermentation" of milk.
3 Status
The or..nism, when present, indicates
that pollution has occurred at some
time. However, becau.e of the ex-
tremely extended viabWty of the spores,
it is impossible to obtain even an
approximation of the recency of pollution
ba8ed only on the presence of
f. perfringens.
The presence of the organism does not
necesarily indicate an unafe water.
C T.sts tor Pathogenic Bacteria of Intestinal
1 Group8 considered include Salmonella
sp, Shifella sp, Vibrio comma,
Mycobacterium sp:-pasteiirefi'a sp.
Lepto8)l1ra sp, aDd others.
2 Merits of direct test.:
Demonstration of any pathogenic
specie. would demonstrate an
un.atisfactory water quality, hazardous
to per80ns COn811min, or coming into
contact with that water.
3 UJnltat1on8
a There 18 no avaUable routine pro-
cedure for detection of the tull
rante of pathorenic bacteria cited
above .
b Quantitative methods are not avail-
able lor routine application to any
of the above.
c The intermittent relea8e 01 these
patho,ens makes it impo..ible to
r.,.,.d water as safe, even in the
absence 01 pathogens.

d After detection, the public already
would have been exposed to the
organism; thus, there is no built-in
margin 01 s.'ety, all exists with
t..ta tor the cou,tnrm ,roup.
4 Applications
a In tracing the source of pathogenic
bacteria in epidemiolo,icalinve8tt-
b In special research projects
c In water quality studies concerned
with enforcement actions a..inst
pollution, increasina .ttention i.
bem, iiven to the demonstration 01
enteric pathogenic bacteria in the
pruence 01 the bacterial Sadicators
ot pollution.
D Miscellaneous Indicatou
It 18 beyond this discusstun to explore the
total rUle of microbiolopcal JDdicatore
01 poUution that have been propoeed and

investigated to Borne extent. Mention can
be made. however. of consideration of
tests for the following.

1 Bacteriophages specific for any of a
number of kinds of bacteria
2 Serological procedures for detection
of coliforms and other indicators; a
oertain amount of recent attention has
been given to applications of fluorescent
antibodies in such tests
3 Tests for Pseudomonas aeruginosa

4 Tests for viruses. which may persist
in waters even longer than members
of the coliform group.
1 Standard Methods for the Examination of
Water and Wastewater. 13th ed..
APHA. AWWA. WPCF. Published by
American Public Health Association
1790 Broadway, New York, N. Y. 19'71.
2 Prescott. S.C.. Winslow. C.E.A., and
McCrady. M. Water Bacteriology.
John Wiley &. Sons. Inc. 1946.
3 Parr. L. W. Coliform Intermediates in
Human Feces. Jour. Bact. 36:1.
4 Clark. H. F. and Kabler, P. W. The
Physiology of the Coliform Group.
Procl'edings of the Rudo1fs Research
Conference on Principles and Appli-
cations in Aquatic Microbiology. 1963.

5 Geldreich. E. E.. Bordner, R. H., Huff,
C.B., Clark. H.F.. and Kabler. P.W.
Type Distribution of Coliform Bacteria
in the Feces of Warm-Blooded Animals.
JWPCF. 34:295-301. 1962.
6 Geldre1ch et al. The Fecal Coli-Aerogenee
Flora of Solls from Various Geographic
Areas. Journal of Applied Bacteriology
25 :87 -93. 1962.
Bacteriological Indicators of Water Pollution
'7 Geldreich, E.E., Kenner. B.A.. and
Kabler. P. W . Occurrence of
Coliforms. Fecal Coliforms. and
Streptococci on Veptation and Inllecte.
Applied MicrobiololY. 12:63-69.1964.
8 Kabler, P.W., Clark. H.F.. and
Geldreich. E. E. 9anitary Significance
of Coliform and Fecal Coliform
Organisms in Surface Water. Public
Health Repor'.s. 79:58-60. 1964.
9 Clark, H.F. and Kabler, P.W.
Re-evaluation of the Significance of the
Coliform Bacteria. Journal AWWA.
56:931-936. 1964.
10 Kenner. B. S.. Clark. H. F.. and
Kabler. P. W. Fecal Streptococci.
II. Quantification in Feces. Am. J.
Public Health. 50:1553-59. 1960.
11 Lit sky, W., Mallman. W. L., and Fifield,
C. W. ComFoarison of MPN of
Escherichia coli and Enterococci in
River Water.Am. Jour. Public Health.
45:1949. 1955.
12 Medrek, T. F. and LUsky, W.
Comparative Incidence of Coliform
Bacteria and Enterococci in
Undisturbed Soil. Applied Micro-
biology. 8:60-63. 1960.
13 MaIlman, W. L., and Litsky, W.
Survival of Selected Enteric Organisms
in Various Types of SoiL Am. J.
Public Health. 41:38-44. 1950.
14 MaIlman, W.L., and Seligman, E.B.. Jr.
A Comparative Study of Media for
Detection of Streptococci in Water and
Sewage. Am. J. Public Health.
40:286-89. 1950.
15 Ministry of Health (London). The
Bacterial Examination of Water Supplies.
Reports on Public Health and Medical
Subjects. 71 :34.
51- 21

Bacteriol~cal Indicators of Water Pollution
Morris, W. and Weaver, R.H.
Streptococci as Indices of Pollution
in Well Water. Applied MicrobioloiY,
2:282-2811. 19114.
11 Mundt, J.O., COllin, J.H.. Jr., and
John.on, L. F. Growth of
~ fecaUt var. liquefaciens
~pp'1.'r.'c1R\crobioIO/D" .
10:1152-1115. 1982.
18 Geldreich. E. E. Sanitary Sitnif1.cance
of Fecal Coliform. in the Environment.
U. S. Department of the Interior.
FWPCA Publ. WP-20-3. 1966.
19 Geldreich, E. E. and Kenner. B. A.
Concepts of Fecal Streptococci in
Stream Pollution. J. WPCF. 41:R336.
20 Kabler, P. W. Purification and Sanitary
Control of Water (Potable and Waste)
Ann. Rev. of Microbial. 16: 127 . 1962.
Litsky, W.. MaUman. W. L.. and Fifield,
C. W. Comparison of the Most Probable
Numbers of Eschericbia coli and
Enterococci in River Waters. A. J. P. H.
45:1049. 1955.
22 Geldreicb. E. E. Applyin. Bacteriological
Parameters to Recreational Water
Quality. J. AWWA. 61:113. 1970.
Geldreicb. E. E.. Be., L. C., Kenner, B. A.
and Van Donael, D. J. Tb. Bacteriolar-
ieal Aspecte of Stormwater Pollution.
J. WPCF. 40:1HO. 19...
24 FWPCA Report of Water Quality Criteria
Calumet Area - Lower Lake Michipn.
Chicalo, IL. Jan. 1988.
This outline was prepared by H. L. Jeter,
Director, National Trainin, Center and
revi.ed b7 R. R\I8aomanno. MicrGbiolo(fl.8t.
NaUonal TrainiDi Center, WPO. EPA,
Cincinnati. OH 45268.
De_cr~m;: Coliform_, E8cherichia coli ,
Fecal iforms, Fecal Streptococci. Indicator
Bacteria, MicrobioloQ, Sewa.. Bacteria.
Water Pollution

(Multiple Dilution Tube (MPN) Method8)
The .ubJect matter of Ws outline i. contained
in three parts, a8 fOUoW8:
A Part 1
1 Fundamental aspecte of multiple dilution
tube ("most probable number.") tests,
both from a qualitative tmd . quantitative
2 Laboratory bench reoorde.
S U.eful techniquee in multiple dilution
tube methods.
4 Standard supplies, equipment, and
media in multlple dilution tube teats.
a Part 2
Detailed, day-by- day, procedures in tests
. for the collform group and subgroups
within the colUorro group.
C Part 3
Detailed, day-by. day, procedures in tests
tor members of the fecal streptococci.
D Application of Tests to Routine Examinations
The fol1owing considerations (Table 1) apply
to the selection of the Presumptive Test,
the Confirmed Test, and the Completed
Test. Termination of testing at the
Presumptive Test leveli. not practiced

by laboratories of this agency. It must
be realized that the Presumptive Test alone
has limited use when water quality is to
be determined.
Examination Terminated at -
Type of Receivinll Presumptlve Confirmed Teet Completed Test
w.+.... Te8t  
Sew.,. Receiving Applicable Applicable Important where results
   p.re to be used for contr
Treatment Plant. Raw Applicable A DDlicable of raw or finished water
   Application to a statis-
Chlorinated Not Done ADDlicable ticany valid number of
   samples from the
B&thirur Not Done ADDlic.ble Confirmed Test to estab
   lish its validity in
Dr1nldn. Not Done ADDlicable deter~ining the sanitary
Other Information  Applicable in all 
  casee where Pre- 
  sumptive Test alone 
  18 unreliable. 
NOTE: Mention of COJIlmercial productll and manufacturers does not imply endorsement by the
Environmental Protection Agency.
W. aA. n. 3.74

MPN Methods
A Qualf.tative Alpects
1 For purely qualitative aspects of telting
for indicator organisms, it is convenient
to conaider the tests applied to one
sample portion, inoculated into a tube
of culture medium, and the follow.up
exa.minations and te.ts on results of the
original inoculation. Results of testing
proceduru are deftnite: positive
(presence of the orpnilm-group is
demon.trated) or neaative (presence of
the organism-group il riot demonltrated.)
2 '1'elt procedures are based on certain
f\Uldamental auumption.:
a Fir.t. even if only one living cell of
the test organism i. present in the
sample, it will be able to grow when
introduced into the primary inoculation
b Second. growth of the test organism
in the culture medium will produce
a result which indicates presence of
the test organi.m; and,

c Third, extraneous organisms will
not grow, or if they;do grow, they
wiU not limit growth of the t..t
oraani.m; nor wW they produce
growth effects that wUl be contused
with tho.e of the bacterial group for
which the test is del1Jned.
3 Meeting these assumptions usually
makes it necessary to conduct the telts
in a serle. of stage. (for example, the
Presumptive, Confirmed, and Completed
'lelt lta,.S, respectively, of Itandard
tests for the coliform ,roup).
4 Features of a full, mUlti-stage test
a Fir.t stage: The culture medium
usually serves primarily a. an
enrichment medium for the group
tested. A good first.ltage growth
medium should lupport growth of all
the living cells of the group tester
and it should include provi.ion for
indicating the presence of the test
orpQi8ID being studied. A first-
Ita,. medium may include some
component which 1nhi~its growth
of extraneous bacteria, but this
feature never should be included
if it alsOiiiiiTbits growth of any
cel18 of the group for which the
t.lt Ie deligned. The Preaumptive
Test for the coliform group 1s a
aood example. The medium
lupports growth, presu~1y. of
an UvinI cells of the coliform
rrouP; the culture container has a
fermentation vial for delDOl1stration
of &a' production resulUq from
lactose fermentation bJ"~orm
bacteria, if present; and sodium
lauryl lulfate may be iDelucled in
one Of the approved media for
luppre.lion of growth of Hrtain
noncoliform bacteria. TbU
additive apparently hat no adverse
effect on growth of members of the
coliform group in the concentration
used. If the result of the first-stage
test 1s neptive, the study of the
culture is terminated, and the result
1s recorded as a negative test. No
further study is made of negative
telts. If the result of the first-
stage te8t is positive, the culture
mty be subjected to further study
to verify. the findings of the first

b S8cond Ita/(e: A tranller is made
from p<*itive culture. of the rtrst-
atace te.t to a second culture medium.
This test stage emphasizes prOvision
to reduce confusio~ of results due to
Irowth effects of extraneous bacteria,
commonly achieved by addition of
8elective inhibitory .,ents. (The
Confirmed Test for coliform. meets
these requirements. Lactose aDd
fermentation vials are provided for
demonstration of col1forms in the
medium. Brilliant green dye and
bile salts are included as inhibitory
.,ents which tentS to eupprsss growth
of practically all kinds of noncoliform
bacteria, but do not suppress growth
of coliform bacteTi. when used as
directed) .

MPN Methods
If result of the second- Itange telt is
ne,ative, the Itudy of the culture il
terminated, and the result is recorded
as a negatiVe t8lt. A negative test here
means that the poettive results of the
first-stage telt were "false positive, "
due to one or more kindl of extraneous
bacteria. A politive lecond-stage test
il partial confirmation of the positive
relult s obtained in the first - stage test;
the culture may be subjected to final
identification through application of still
further testing procedures. In rCAltine
practice, most sample examinations
are terminated at the end of the second
stage, on the assumption that the result
would be positive if carried to the third,
and final Itage. This practice should be
followed only if adequate testing is done
to demonstrate that the assumption i8
valid. Some workers recommend contin-
uing at least 50/0 of all sample examina-
tions to the third stage to demonstrate
the reliabUtty 0: the second- stage results.
8 Quantitative Asp.cts of Tests
I These methods for determining bacterial
numbers are based on the assumption
that the bacteria can be separated from
one another (by ahaldn, or other means)
resulting in a suspension of individual
bacterial ceUs, uniformly d18tributed
through the ori,inal sample when the
primary inoculation is made.
2 Multiple dilution tube tests for quantita-
tive determinations apply a Most Probable
Number (MPN) technique. In this pro-
cedure one or more measured portions
of aach of a stipulated series of de-
creuing sample volumes is inoculated
into the first-stage culture medium.
Through decreasing the sample incre-
ments, eventually a volume i8 reached
where only one cell is introduced into
some tubes, and no ce1l8 are introduced
into other tubes. Each of the several
tubes of sample-inoculated first-stage
medium is tested independently,
accordin, to the principles previously
described, in the qualitative aspects
of testing procedures.
3 The oombination of positive and
negative results ib used in an application
of probability mathematics to secure
a single MPN value for the sample.
4 To obtain MPN values, the following
conditions must be met:
a The testing procedure must result
in one or more tubes in which the
test organism is demonstrated to
be present; and
b The testing procedure must result
in one or more tubes in which the
test organism is not demonstrated
to be pre8ent. -
I) The MPN value for a given sample is
obtained through the use of MPN Tables.
It is emphasized that the precision of
an individual MPN value is not great
when compared with most physical or
chemical determinations.
6 Standard practice in water pollution
surveys conducted by this organization,
is to plant five tubes in each of a series
of sample increments, in sample
volumes decreasing at decimal intervals.
For example, in testing known polluted
waters, the initial sample inoculations
might consist 'of 5 tubes each in volumes
of 0.1, 0.01, O. 001, and 0.0001 ml,
respectively. This series of sample
volumes will yield determinate results
from a low of 200 to a high of 1,600,000
organisms per 100 mI.
52- 3

A Features of a Good Bench Record Sheet
1 Provides complete identWcat10n of the
2 Provides for full, day-by-day informa-
tion about all tests performed on the
3 Providee eaey step-by"l1.ep record
applicable to any portion of the eample.
4. Provides for recording of the quantitative
result which will be tranecribed to IUb-
sequent reports.
~ Minimizes the amount al writing by the
II Identifies the analyst(s).
B There 18 no such thing as "standard"
bench sheet for multiple tube test.: there
are many vereions of bench sheet.. Some
are prescribed by adm1ni8trative authority
(euch as the Office of a State sanitary
Engineer); others are devised by laboratory
or project peraonnel to meet specific needa.
It 18 not the purpose of th1e discus.ion to
recommend an "ideal" bench form; however,
the form used in this train1q course
manual is e..entially l1rnilar to that ueed
in certain re.earch laboratories of th1e
organization. The etudent enrolled in the
COUrBe for which this manual 1s written
should make himself thorou(lhly familiar
with the bench sheet and its proper use.
See Figure 1.
A Each bacteriological examination of water
by multiple dilution tube methods requires
a cOl1liderab1e amount 01 manipulation;
much i. quite repetitious. I..II.boratory
worun mWit deYeJop and maintain good
routine wol'klna babits, with conetant
a1ertn..e to euard against lapIel into
careles.. el1p- ehod laboratO¥')' procedures
and "ehon outs" which onl,y oan,~d to
lowered ~ of ]aboratorywork.
The student reader is ur,ed to review the
form for laboratory IUrvey. (PHS-a7S,
Rev. 1988) uled by Public Health Service
personnel charged with responstbt1ity for
accreditation of laboratorie.'for examination
of water under Inter8tate Quaftntine
B Specinc attention is brought to the following
by no means exhaustive, critical aspects of
laboratory procedures in multiple dilution
tube teets:
1 Original sample
a Follow pre8cribed care and handling
procedures before t.eUng.
b Maintain abeolute i<1eotWcation of
eample at allltaJU iP .te8Ung.

c VlIOrou~ lbake u..mp.. (and
Ample c:I1l<1ona) before panting
in culture media.
2 Sample meaeurement into primary
culture meclbun
a Sample portions must be measured
acourateJ;y into the culture medium
for reliable quantitative teste to be
made. StaQdard Methode prescribes
that calibration errors IhCNld not
exceed t ~. 5'..

BACTERIOLOGY BENCH SHEET Multiple Dil\lt1on Tube Testa


S~pl. ..atl~~ - ,Jlj.


Date .L~JI!.." Time ~'Sl) By~-
T.m~_oC pH~
Other aa..rvat1on8-
_Analytical Record

Bench Number of Sample~
AnalYllt~~- ~~ ~
Tellt It ted at ~. . 87 tf --
I I I I ,
~'r : ,- ~--r--- --T-- I --;-.-
. -t--+ i --+----~----t---+----+-- --+---, -----,
---~-+-~----L----J-~._~- --~~. -------~
I I I I I 1'1 'H I i

---+\-~--t----i--~--+-- -~-- :---++--------i
: ~: :: =--=--=1
+-+-+-: +-+-----t--t--t--+--+ -=11=+! j :-~-----i
...L--t----+-_-!- - I - ----t--------'
I : I I: ;1 :: I
1 :-+-; ---+- I ~-- --+-. , -----------1
, : '_: -L-J i J: ~ ~ 8=L-~~~- -~~-1
tJ 1 i=+--t---t--t- i - r+--H--t--------~
f: I : --=r--B--+ , =~===tj=F£l~~==~l
:: : 1 ==t=-- - I -J -+----,,-::~- ~-:- -J

.. ,. , " -t--

, 1-: -t--T-- :--1 -I, -.- ~ -:- J--------~
. +---=-~-J- -- I i t=+--t=:::-=--::_~:
~-=~==:t-t j--_i_-t =:t= ~ -{== ==~==--=J
~ . I +-' :1 I I
'--L-+-- --+---t--- ~-- ---t -------1
...-+-~---- ---~--~-----~-_--:--...L_----!.-----..JI ------ --...1
----L-J--~---i---l---- '_1-
., ,
" '
ColUorm MPN /100 ml
FecI! Co1Uorm MPN:
Fecal Streptoc:..~ MPN IIO~~
A - D EVA:
S;2. - 5

MPN Methodl
SUilelted sampJe me&8urln. practices
are as 101]ows: Mohr mea.urini
pipetl are recommended. 10 ml
samples are delivered at the top 01
the culture tube, using 10 ml pipetl.
1.0 ml lamples are delivered down
into the culture tube, near the sur-
face of the medium. and "touched
off" at the side of the tube when the
delired amount of sample has been
delivered. 1. 0 ml or 2.0 m1 pipetl
are used for measurement of thil
volume. 0.1 mll&mples are
delivered in the lame manner .1 1.0
ml samples, uslnl great care that
the sample actual~ getl Into the
culture medium. Only 1.0 ml pipets

are used for this lample volume.
After delivery of all lample incre-
ments into the culture tubel, the
entire rack of culture tubes may be
shaken gently to carry down any of
the sample adhertft' to the wall of
the tube above the medium.

Workerl should demonstrate by actual
teets that the pipets and the technique
in ule actually delivers the rated volumes
within the prelcribed limits of error.

b Volumel as small as O. 1 ml routinely
can be delivered directq from the
sample with suitable pipetl. Leiser
sample volumes first should be diluted,
with subsequent delivery of suitable
volumes of diluted sample into the
culture medium. A diagrammatic
scheme for making dilutions is shown
in FifUI'e 2.
3 Reading of culture tubes for I&s
a On removal from the incubator,
shake culture rack iently, to
encourage release 01 gas which
may be supersaturated in the culture
b ~ tD any quantity is a potaiUve telt.
Ii 1. necell&ry to work 1D cGllditlonl
of .uttable lightm, for ea.y recog-
nition of the extremely .mall amounts.
of ..S inside the tops of lome
fermentation vials.
" Reading of liquid culture tubes for
e:d!i as indication of a po.1ti~ test
requires lOod lighting. Growth'is
8hown by any amount of increaaed
turbidity or opalescence in the culture
medium. with or without deW.lt of
sedimeat at the bottom of the tube.
5 Transfer of culturss wW11noC1lJation
loop. and needles
a Always sterilize inoculation loops
and needles in flame immediately
before transfer of culture; do not
lay it down or touch it to a~ non-
sterile object before makina the
b After sterilization, allow sufficient
time for cooling. in the air. to avoid
heat-killing bacterial cells on the
hot wire.
c Loops .hould be 3 mm in inside
diameter, with a capability of holding
. drop of water or culture.
For routine standard transfers
requiring transfer of 3 100psfulof
culture. many workers form three
3-rnm ]oops on the same len(th of
6 AI an alternative to use of standard
inoculation loop., the use of
"applicator lUck." have now been
..nct1oned by the 13th Edition of
Standard Methods.

Figure 2.
MPN Methods
Dilution Ratios:
Sam ph,
Dellv8ry volume
Tubes 1
1: 10000
Petri Dishes or Culture Tubes
Actual volume
of sample in tubp.
0.1 rol
The applicator sticks are dry heat
sterU1zed (autocJave sterilization is
not acceptable because of pouible
release of phenols if the wood is
steamed) and are used on a single-
service basi.. Thus, for every culture
tube traneferred, a new applicator
lUck is used.
'fhil use of applicator sticks is
parUcular~ attractive in field
situations where it is inconvenient or
impossible to provide a gas burner
suitable for sterilization of the
inoculation loop. In addition, use of
applicator sticks is favored in
laboratories where room temperatures
are significantly elevated by use of
p.. burners.
O. 01 ml O. 001 ml
O. 0001 ml
O. 00001 ml
7 Strealdnf cultures on acar surfaces
a All streak-inocuJations should be
made without breaking the surface
of (he agar. Learn to use a light
touch with the needle; however,
many inoculation needles are 80
sharp that they are virtually u8eless
in this respect. When the needle is
pJatinum or platinum- iridium wire,
it sometimes iR beneficial to fuse
the working tip into a small sphere.
This can be done by momentary
insertion of a well-insulated (against
electricity) wirt. into a carbon arc,
or some other extremely hot environ-
ment. The sphere should not be more
than twice the diameter of the wire
from which it is formed, otherwise
it will be entire~ too heat-retentive
to be useful.

MPN Methods
When the needle is nichrome
resistance wire, it cannot be heat-
tused; the writer prefer. to bend
the term1nall/18 - 1/8" of the wire
at a sUIht angle to the overall axis
of the needle. The side of the
terminal bent portion of the needle
then is used for 1nocu1at1on of a..r
surface. .
b When streaking for colony i80lation,
avoid using too much inoculum. The
streak1nt pattern 18 somewhat
variable according to indiv1d\81
preference. The procedure favored
by the writer is shown in the
accompw.nying fieures. Note
particularly that when aoine from
anyone atage of the strealdn. to the
next, the inocu1atlon needle Is "...t-
8 Preparation of cultures for Gram
a The Gram at.in always should be
made from. culture grown on .
nutrient agar surface (nutrient .gar
slants are used here) or from nutrient
b The culture should be YOURI, and
should be actively growing. Many
workers dGubt the validity of the
Gram 8tain made on a culture more
tMn 2. hours old.
c Prepare a thin smear for the staining
procedure.'ost beginning workers
tend to use too much bacterial sus-
penaion in prepar1n. the dried smear
for atalllinl. The amount of bacteria
8IIIould be '0 small that the dried film
1. barely vi8ib1e to the naked eye.
Consolidated Usts of equipment, supplies,
and culture media required for all multiple
dilution tube test. described in th18 outline
are shown III Table 2.:. Quantitative infor-
mation Is not presented: this is variable-
accordin. to the extent of the testing pro-
cedure, the number of dilution.s u.ed, and
the number of repUcate tubes per dilution.
It la noted that requirements for alternate
procedure. are ~ Usted and chOices are
made in accordance to laboratory preference.

MP~ Methods
1 a Flame-sterilize an inoculation needle and air-cool.

b Dip the ~ of the inoculation needle into the bac-
terial culture being studied.

c Streak the inoculation needle tip lightly back and
forth over half the agar surface, as in (1), avoid-
ing scratching or breaking the aer.r surface.

d Flame-aterilize the inoculation needle and air-cool.
2 a Turn the Petri dish one-quarter-turn and atreak the
inoculation needle tip lightly back and forth over one-
half the agar surface, working from area (1) into one-
half the unstreaked area of the agar.

b Flame-aterilize the inoculation needle and air-cool.
3 a Turn the Petri dish one-quarter-turn and streak the
inoculation needle tip lightly back and forth over one-
half the agar surface, wOl'king from area (2) into
area (3), the remaining unatreaked araa.

b Flame-sterilize the inoculation needle and set it aside.

c Close the culture container and incubate aa prescribed.
IMode,ate afowthl
AREA 3 !Isolated cololllSl
S.2 - 9

MPN Method.
------   -  -~'".l
D.ler",,- .f Item   
   r..!~." ""'T;:- ~.. (IIA"" II!IC b,!,,"
lAut)'ltr,nM'" _""II or Lat:to.. X     
broth. 10 mJ Iftll4NM. 0' 1.~~      
CClftc""raUOIII IDetI'um, in n A UO mm      
cult",,.. ... ..... "'vertd ,..........      
,,'1- mi., ftb1lilt capt.      
IAUr)'1 'r"JJUI1 -...... or Laeto.. X  .   
b"th. )....1 'IMImIt of tUI,..      
l'r8ltf\b m.c8... ,. 10 ~ 110 n\ftII      
eubure ..... wltb ''''WI'''''. ,.rllt...      
,,\Lon ,,"II, '."'18 capI.      
Bl'UUam I"'''' "cto.. bU. b",h. ft  X 11   
'ft 10 ta) ..na&8. .~ntle atr........      
in 10)( 110 ..... calt.... tub.. .....      
Inverted II"'''''U- rial_,      
1".1.1." CI8pII.       
Eo.... m,...,'en. bl",. ..." pou."  " 11   
in 100 J( U "'In Petri di,"..      
11ft" '." pwred'" Loa ~ III......  X    
Nut"..., ...,. ..... lera. cap""   X   
"riel acid ........ '",h, 10 1D1    "  
&IM\Ift1. ., ... "rent'" ,.......      
1ft r.........,... ''''0       
EC 8t'G4h, 10..1 amoWtl1 01.....     X 
4,r....." "',.... .. I.",....,...      
Pormlt8 rtc"""'h broth   I X   
Cllttllr. t_1 nck8. 10)( OJ opeftintli X X X X X 
etch 0,"'.1 10 aCl\:ept U "'"' Ifta.      
Pipeu... 10 ..~. Mohr ')'p', ..rU., "     
Pip.ne., I ..1 C."'-U, Mol'''''''. "     
.t...I1.. 1ft ..Koill C8ft.       
!!tip.UU, I .1. ""'r tfp8. ..erih "     
in n....I,1 lukM" CM8       
5t8nd8I'U ....Un" .hltlon ...t.t, X     I
8'erUe, "..-1 .....ounl. tn .0.....     
ea".d bottl...      
au burnu. hu... t)1H'   " X X "
I8oc...!aUOII ..... ..., ) mm dJa..  " " 11 " 
m.' u. 0' ..iaht-o..... or p18U....-      
Irten.... _Lft. .1 8 .. S "-.0, In      
euJtab18 "'1..1"', (01'" ."1'118 ..,u.'.r      
tAoC:III.U.. ...lIIU.e, DLchroas.. or  X 11   
,18UftlMla-lrldkUh wire, II.. 8    
"III'~, ill 1",l1abl. hold.r.      
1tk''''b8lcu', Id,u'ed ID 35 ~ O.IIC " " "   
Wa,.tDa", UIc1Ib8tor, adJulled"    X  
tI : o..'C      
w'.'erilath u..:1te.0I'. IdJu.ted"     X 
H.e! D,' C.      
OJ.... mlcro.c.<, .1I.e.. 1"'(1"   "   
Illd. r.ck. IDpt...1)    X   
Gram-.t,1n 8CllutiGl'l'. complete."   X   
CompcKlnd 8It1C'I'.eope. 011 ~rn......   X   
.~an II!II'. "bD,' eon.e"'.e,.     
"..e' reI'" dl.e8rded ('ul'III'" X " k "  
C.nL8~~.r '01' dl.cerd'" plpM'" X     
 ---.---- --    
$".z - 10

Part 2

A Te.t. Described
1 Presumptive Test
2 Confirmed Teet
3 Completed Test
4 Fecal Coliform Test
B Form of Presentation
The Presumptive, Confirmed, and
Completed Tests are presented as total,
independent procedures. It is recognized
that this form of presentation 18 somewhat
repetitious, lnasmuch as the Presumptive
Tilt is preliminary to the Confirmed
Test, and both the Presumptive Test and
the Confirmed Test are preliminary to the
Completed Test for total coliforms. .
In using these procedures, the worker
must know at the outset what 18 to be the
ii&ie at which the test 18 to be ended, and
the details of the procedures throughout,
in order to prevent the possib1l1ty of
di,cardp1i aas-po.1t1v~ tub~s before
~er transfer procedures hAve been
fo wed.
Thu., if the worker know8 that the test will
b. ended at the Confirmed Test, he will
turn at once to Section nI, TESTING TO
ignore Sections II and IV.
The Fecal Coliform Test i. described
separately, in Section V, as an
adjunct to the Confirmed Test and to the
Completed Te8t.
A First-Day Procedures
1 Prepare a laboratory data sheet for
the sample. Record the following
information: assigned laboratory
number, source of sample, date and
time of collection, temperature of the
source, name of sample collector,
date and time of receipt of sample in
the laboratory. Also show the date
and time of starting tests in the
laboratory, name(s) of worker(s) per-
forming the laboratory tests. and the
sample volumes planted.
2 Label the tubes i)f lauryl tryptose broth
required for th~ initial planting of the
sample (Table :j). The label should
bear three identifying marks. The
upper number is the identification of
the worker(s) performing the test
(applicable to personnel in training
courses), the number immediately
below is the assigned laboratory num-
ber, corre.ponding with the laboratory
record sheet. The lower number is the
code to designate the sample volume
and which tube of a replicate series is
NOTE: Be sure to use tubell containing
the correct concentrations of culture medium
for the inoculum/tube volumes. (See the
chapter on media and solutions for multiple
dilution tube methods or refer to the current
edition of Standard Methods for Water and

MPN Methods
 Tube Tube Tube Tube Tube Sample volume
 1 2 3 fo 5 repre.ented
Bench number 313 313 312 313 312 Tube. with 10 ml
Volume & tube Ii B C D E of nmple
8e""h numbe r 313 .313 313 313 313 Tube. with 1 ml
Volume'" tube a b c It e of .ample
Bench number 312 313 312 313 313 Tube. with O. 1 ml
Volume" tube ! ~ .£ ~ ! of .ample
Bench number 312 312 312 312 312 Tube. with 0.01 ml
Volume" tube la Ib lc Id Ie of .ample
Bench number 313 312 312 313 313 Tubee with 0.001 ml
Volume" tube 3a 2b 3c 3d 2e of .ample
Tbe labelln, of cultures can be redueed b)' IabeU", on1)" the tint tube 01
each eerie. of Identical eample volum... in the initIal plantinl of the.ample.
1111 .ubcuUure. from initial planUnp .hould be labeled complete I,..
3 Place the labeled culture tube. in an
ord81'ly arrangement In a culture tube
rack, with the tubel intended lor the
lar,..t .ample volume. In the front
row, and tho.. intended lor .maUer
volume. in the IUcceecling row..

4 Shake the ample vigoroueJ,y, .approxi-
mately 2& t11h8., in an arc of one foot
within ..ven .econd. and withdraw the
.ample portion at onc..
II Mea.ure the predetermined .ample
voluU1es into the labeled tub.. of Jauryl
trypto.e broth, usinr care to avoid
introduction of any bacteria into the
culture medium except tho.e in the
a U.e a 10 ml pipet lor 10 ml .ample
portion., and 1 ml pipete lor portiOD.
of 1 ml or les.. Handle sterUe pipet.
only near the mouthpiece, and protect
the delivery end Irom external con-
tamination. Do not remove the cotton
plurin the mouthpiece as this i.
intended to protect the user from
ingesting any sample.
Typical Example
l£b. Worker

Bench NlImber
Tube of Culture M2dium
b When u.in, the pipet to withdraw
ample portione, do not cUp the
pipet more than 1/2 inch Into the
eample; otherwiee eamp1e l'QDDing
down the outeide of the pipet wID
make mea8U1'ement. macCUl"8te.
6 After meaeuriftlan portione of 'he
Ample into their re8peCti'19 tube. 01
medium. ~ ebake the raok of
1nocuatedt\ibee to ineure IOod mixing
01 Ample with the culture medium.
Avoid vi.oroUi .baklng, as air bubbles
may be .baken into the lerm,ntat1on
vial. and thereb11.nvallclate the teet.

7 Pace the rack of inocw.ted tub.. in the
incubator at 3110 + O. ~oC lor 24 +
2 houre. - -
B 24-hour Proceduree
1 Remove the rack 01 Jauryl tryptoee
broth cultures Irom the incubator, and
shake gently. If pe i. about to appear
in the fermentation viall, the.baIdng
wID .peed the proce.s.

Examine each tube carefully. Record,
in the column "24" under LST on the
laboratory data sheet. each tube showing
gas in the fermentation vial as a positive
(+) test and each tube not showing gas
as a neptlve (-) test. GAS IN ANY
Discard aU 88s-positive tubes of lauryl
tryptose broth. and return all the gas-
negative tubes to the 3SoC incubator for
an additional 24 t 2 hours.
48-hour Procedures
Remove the rack of culture tubes from
the incubator. read and record gas
production for each tube.
Be sure to record all results under the
48-hour LTB column on the data sheet.
Discard all tubes, The Presumptive
Test is concluded at this point. and
Presumptive coliforms per 100 ml can
be computed according to the methods
described slllewhere in this manual.
Not. that the description starts with the
sample inoculation and includes the
Presumptive Teet stage. The Confirmed
Telt'preferred in Laboratoriel of thil agency
il accomplished by means of the brilliant
green lactose bile broth (BGLB) and the
acceptable alternate testl are mentioned in
UI F. In addition. the Fecal Coliform Test Is
included as an optional adjunct to the procedure.
A First- Day Procedures
Prepare a laboratory data sheet for the
sample. Record the following infor-
mation: assigned laboratory number.
source of sample. date and time of
collection. temperature of the source.
name 01 sample collector. date and
time of receipt of sample in the
laboratory. Also show the date and
MPN Methods
time of starting tests in the laboratory,
name(s) of worker(s) performing the
laboratory tests. and the sample
volumes planted.
Label the tubes of lauryl tryptose broth
required for the initial planting of the
sample. The label should bear three
identifying marks. The upper number
is the identiIicatbn of the worker( 9)
performing the test (applicable to
personnel in training courses), the
number immediately below is the
assigned laboratory number. corres-
ponding with the laboratory record
sheet. The lower number is the code
to designate the sample volume and
which tube of a replicate series is indicated.
NOTE: If 10-ml samples are being
planted. it is necessary to use tubes
containing the correct concentration
of culture mediumr This has previously
been noted in II A- 2,
Place the labeled culture tubes in an
orderly arrangement in a culture tube
rack. with the tube"! intended for the
largest sample volumes in the front
row. and those intended for smaller
volumes in the succeeding rowl!I.
Shake the sample vigorously, approxi-
mately 25 timel!l. in an up-and-down
motion .
Measure the predetermined sample
volumes Into the labeled tubes of lauryl
tryptose broth. using care to avoid
introduction of any bacteria into the
culture medium except those in the sample.
Use a 10- ml pipet for 10 ml sample
portions, and I-ml pipets for portions
of 1 ml or less. Handle sterUe pipets
only near the mouthpiece, and protect
the delivery end from external con-
tamination. Do not remove the cotton
plug in the mouthpiece as this is intended
to protect the user from ingesting any
52 - 13

MPN Methodll
b When using the pipet to withdraw
sample portions, do not dip the
pipet mOl'e than 1/2 inch into the
sample; otherwise sample runnirll
down the outside of the pipet w1ll
make measurements inaccurate.
c When delivering the sample into the
culture medium, deliver lIample
portions of 1 ml or less down into
the culture tube near the surface of
the medium. Do not deliver small
sample volumes aTIhe top of the tube
and allow them to run down inside
the tube; too much of the eample
will fall to r~ach the culture medium.
d Prepare preliminary dilutione of
samples tor portions 01 0.01 ml or
less before delivery into the culture
medium. See Table 1 for preparation
of dilutions. NOTE: Always deliver
diluted sample portionll into the
culture medium as s;~n a~ posiible
after preparation. e interva
between preparation of dilution and
introduction of lIample into the
medium never should be as much
as 30 minutes.
6 After me&8ur1ng all portions of the
sample into their respective tubes of
medium, .1!!.!!!!1 shake the rack of
inoculated tubes to insure good mixing
of sample with the culture medium.
Avoid vigorous shaking, as air bubbles
may be shaken into the fermentation
vials and thereby invalidate the test.
7 Place the rack of inoculated tubes in
the incubator at 350 : 0.50 C lor 24 :!
2 'houre,
B 24-hour Prooedures
1 Remove the rack oC lauryl tryptose
broth culturee from the incubator, and
shake gently. IC gas ie about to appear
in the fermentation vials, the shakinr
will speed the procen.
62 - 14
2 Examine each tube carefully. Record,
in the column "24" under LST on the
laboratory data sheet, each tube showing
iRs in the fermentation vial aa a
positive (+) test and'each tube not
showing gas as a neptive (-) test.
3 Retain all gas-positive tubes of lauryl
tryptose broth culture in their place
in the rack, and proceed.
4 Select the gas-positive tube. of Jauryl
tryptose broth culture for COIIf1rmed
Test procedures. Confirmed Test
procedures may not be required tor all
gas-positive cultures. It, after 24-hours
01 incubation, all five replicate cultures
are gas-positive for two or more con-
secutive eample volumee, then select
the eet of five cultures representing
the smallest volume of sample in which
all tubes were gas-positive. Apply
Confirmed Test procedures'to all these
cultures and to any other ga.-positive
cultures representing smaller volumes
01 sample, in which lIome tubee were
gas-positive and some were gas-negative.
5 l.abel one tube of brilliant green lactose
bile broth (BGLB) to corresponeS with
each tube of Jauryl tryptose broth
selected for Confirmed Test prOCedures.
6 Gently ehake the rack of Prewmptive
Teet cultures. With a flame-sterilized
inoculation loop transCer one loopfulof
culture from each iRs-positive tube to
the corresponding tube of BGLB. Place
each newly inoculated culture into BGLB
in the poeit1on of the ori.illal ps-positive
7 After making the transfers, the rack
should contain some 24-hour ps-
neptive tubes 01 Jauryl trypt08e broth
and the newly inoculated BGLB.
6 It the Fecal ColUorm Test is included
in the testq procedures, consult
Section V of this part of the outline ,of
testing procedures.

MPCi Methods
9 Incubate the 24-hour gas-negative
BGLB tubes and any new]y-inoculated
tubes of BG~ an additional 24 0\- 2
hours at 35 !: O.50C. -
C 48-hour Procedure I!
1 Remove the rack of culture tubes from
the incubator, read and record gas
production for each tube.
2 Some tubes will be lauryl tryptose broth
and some will be brilliant green lactose
bile broth (BGLB). Be sure to record
relults from L TB under the 48- hour
LTB column and the BGLB result. under
the 24-hour column of the data sheet.
3 Label tubes of BOLB to-correspond with
aU (If any) 48-hour gas-positive cultures
in lauryl tryptose broth. Transfer one
l00pfulof culture from each gas-positive
LTB culture to the correspondingly.
labeled tube of BGLB. NOTE: All
tub.. of LTB culture which wer;--
nepUve at 24 hours and became
positive at 48 hours are to be transferred.
The option described above tor 24-hour
cultures does not app1y at 48 hours.
4 If the Fecal Coliform Test Is included
in the testing procedure, consult
Section V of the part of the outline
of testing procedures.
5 Incubate the 24-hour gas-negative
BOLB tubes and any new]y-inoculated
tubes of BOLB 24 + 2 hours at 350 +
O. &0 C. - -
6 Discard aU tubes of LTB ..nd aU 24-hour
gal-positive BGLB cultures.
D 72-hour Procedures
1 If a~ cultures remain to be examined,
aU will be BOLB. Some may be 24
hours old and some may be 48 hours
old. Remove such cultures from the
incubator, examine each tube for gas
production, and record results on the
data sheet.
2 Be sure to record the results of 24-hour
BGLB cultures in the "24" column under
BGLB and the 48-hour results under the
"48" column of the data sheet.
3 Return any 24-h0ur gas-negative cultures
for incubation 24 + 2 hours at 35 +
0.50C. - -
4 Discard all gas-pvsitive BGLB cultures
and all 48-hour gas-negative cultures
from BGLB.
5 It Is possible that aU cultural work and
results for the Confirmed Test have
been finished at this point. If so, codify
result' and determine Confirmed Test
coliforms per 100 ml as described in
the outline on use of MPN Tables.
E Be-hour Procedures
At most only a few 4£-hcur cultures in
BOLB may be present. Read and record
ps production of such cultures in the "48"
column under BGLB on the data sheet.
Codify results and determine Confirmed
Test colUorms per 100 mI.
F Streak-plate methods for the Confirmed
Test, using eosin methylene blue agar or
Endo agar plates, are accepted procedures
in Standard Methods. The worker who
prefers to use one 01 these media in
preterence to BOLB (alao approved in
Standard Methode) is 3.dvieed to refer to
the current edition of "Standard Methods
for the Examination of Water and Waste-
water" tor procedures.

MPN Methods
(Note that this description starts with the
sample inocu1ation and proceeds through the
Presumptive and the Confirmed Test stages.
In addition, the Fecal Coliform Test is
reCerred to as an optional adjunct to the
procedure. )
A First-Day Procedures

1 Prepare a 1aboratory data sheet for the
sample. Record the following information:
assi,ned laboratory number, source of
sample, date and time of collection,
temperature of the source, name of
sample collector, date and time of
receipt of sample in the laboratory.
Alia show the date and time of starting
tests in the laboratory, name(s) of
worker(s) performing thE" laboratory
tests, and the sample volumes planted.
2 abel the tubes of lauryl tryptose broth
required Cor the initial planting of the
sample. The label should bear three
identifying marks. The upper number
is the identification of the worker(s)

performing the test (applicable to
persom,el in training courses),
the number immediately below is the
assigned laboratory number, corre.-
ponding with the laboratory record
sheet. The lower numbf:r is the cod..
to duirnate the sample volume and
which tube of a repUcate series is
indicated. Guidance on labeling for
laboratory data number and identification
of individual tubes is described els8-
where in this outline.
NOTE: If 10-ml samples are being
plated, it i. necessary to use tubes
containing the correct concentration
of culture medium. This has previously
been noted elsewhere in thil outline
and referrali. made to table..
3 P1ace the labeled culture tube. in an
orderly arrangement in a culture tube
rack, with the tubes intended for the
largelt sample volumes in the front
raw, and those intended for smaller
volumes in the succeeding rowe.
4 Shak. the sample vigorously, approxi-
mately 25 times, in an up-and-down
5 Measure the predetermined sample
volumes into the labeled tube. of lauryl
tryptose broth, using care to avoid
introduction of any bacteria into the
culture medium except those in the
a Use a 10-ml pipet for 10 ml sample
portions, and l-ml pipets for portions
of 1 ml or less. Handle sterile
pipets only near the mouthpiece,
and protect the delivery end from
external contamination. Do not move
the cotton plug in the mouthpiece
as this is intended to protect the
user from ingesting an;y sample.
b When using the pipet to withdraw
sample portions, do not dip the
p1pet more than 1/2 inch into the
sample; otherwise sample running
down the outside of the pipet will
make mea.urements inaccurate.
c When delivering the sample 1nto the
culture medium, deliver sample
portions of 1 ml or less down into

MPN Methods
the culture tube near the surface of
the medium. Do not del1ver small
lample volumes aThe top of the
tube and anow them to run down
inside the tube; too much of the
sample will faU to reach the culture
d Prepare preliminary dilutions of
samples for portions of 0.01 ml or
less before delivery into the culture
medium. See'Table 2 for preparation
of dilutions.. NOTE: Always del1ver
diluted .ample portions into the
culture medium as soon a. possible
after preparation. The Giterval
between preparation of dilution and
introduction of sample into the
medium never .hould be as much as
30 minutes.
6 After measuring all portion. of the
.ample into their respective tubes of
medium, ~ .bake the rack of
inocula tec:i"'tti'6es to insure good mixing
of .ample with the culture medium.
Avoid vigorous shaking, as air bubbles
may be shaken into the fermentation
viall and thereby invalidate the test.
7 Place the rack of inoculated tubes in
the incubator at 350 + 0.50 C for 24 +
2 hours. -
B 24-hour Procedures
1 Remove the rack of 1auryl tryptose broth
cultures from the incubator, and shake
gently. If..s is about to appear in the
fermentation vials, the shaking will
speed the process.
2 Examine each tube carefully. Record,
in the column "24" under LST on the
laboratory data sheet, each tube showing
pi in the fermentation vial as a positive
(+) test and each tube not showing gas
as a negative (-) test. GAS IN ANY'
3 Retain all gas-positive tubes of 1auryl
tryptose broth culture in their place in
the rack, and proceed.
4 Select the gas-positive tubes of lauryl
tryptose broth culture for the Confirmed
Test procedures. . Confirmed Test
procedures may not be required for
an gal-positive cultures. If, after
24-hours of incubation, all five
replicate cultures are gas-positive for
two or more consecutive sample
volume I, then select the set of five
cultures representmg the smallest
volume of sample in which all tubes
were gas-positive. Apply Confirmed
Telt procedures to all these cultures
and to any oth~r gas-positive cultures
representing smaller volumes of
sample, in which some tubes were
gal-politive and some were gas.
6 Label one tube of brilliant green lactose
bile broth (BGLB) to correspond with
each tube of 1auryl tryptose broth
lelected for Confh'med Test procedures.
6 Gent~ shake the rack of Presumptive
Test cultures. With a flame- sterilized
inoculation l~p transfer one loopful of
culture from each gas-positive tube to
the corresponding tube of BGLB. Place
each newly inoculated culture into
BGLB in the position of the original
ga.-positive tube.
7 If the Fecal Coliform Test is included
in the testing procedure, consult
Section V of this outline for details of
the testing procedure.
8 After making the transfer, the rack
should contain Borne 24- hour gas-
negative tubea of lauryl tryptose borth
and the newly inoculated BGLB.
Incubate the rack of cultures at 350 C
~ 0.50 C for 24 ~ 2 hours.
C 48-hour Procedures
1 Remove the rack of culture tubes from
the incubator, re..d and record gas
production for each tube.
2 Some tubes will be lauryl tryptose broth
and some will be brilliant green lactose

MPN Methods
bile broth (BGLB). Be sure to record
results from LTB under the 4B-hour
LTB column and the BGLB results
under the 24-hour column of the data
3 label tubes of BGLB to correspond with
all (if any) 48-hour gas-positive cultures
in lauryl tryptose broth. Transfer one
loopful of culture from each gas-positive
LTB.culture to the correspondingly-
labeled tube of BGLB. NOTE: All tubes
of LTB culture which were negative at
24 hours and became p6sitive at 4B hours
are to be transferred. The Option
described above for 24-hour LTB
cultures does not apply at 48 hours.
4 Incubate the 24-hour gas-negative BGLB
tubes and any newly-inoculated tubes of
BGLB 24 + 2 hours at 350 + O.50C.
Retain aU"24-hour gas-positive cultures
in BGLB for further test procedures.
Label a Petri dish preparation of eosin
methylene blue agar (EMB agar) to
correspond with each gas-positive
cult\lre in BGLB.
6 Prepare a streak plate for colony
isolation from each gas-positive culture
in BGLB on the correspondingly-labeled
EMS agar plate.
Incubate the EMB agar plates 24 + 2
hou.. at 350:!, 0.50 C. -
D 72-hour Procedures
Remove the cultures from the incubator.
So~e may be on BGLB; several EMB
agar plates also can be expected.
Examine and record gas production
results for any cultures in BGLB.
3 Retain any gas-positive BGLB cultures
and prepare streak plate inoculations
for colony isolation in EMB agar.
Incubate the EMB apr plates 24 +
2 hours at 35 + 0.50 C. Discard the
gas-positive BGLB cultures after
4 Reincubate any gas-negative BGLB
cultures 24 :!' 2 hours at 350 't 0.50 C.
5 Discard all 4B-hour gas-negative BGLB
6 Examine the EMB agar plates for the
type of colonies developed thereon.
Well-iso1ated colonies having a dark
center (when viewed from the lower
side, held toward a light) are termed
"nucleated or fisheye" colonies, and
are regarded as "typical" coliform
colonies. A surface sheen mayor may
not be present on "typical" colonies.
Colonies which are pink or opaque but
are not nucleated are regarded as
"atypical colonies." Other colony
types are considered "noncoliform. "
Read and record results as + for
"typical" (nucleated) colonies + for
"atypical" (non- nucleated pink -or
opaque colonies), and - for other types
of colonies which might develop.
7 With plates bearing "typical" colonies,
select at least one well-isolated colony
and transfer it to a correspondingly-
1abeled tube of lactose broth and to an
agar slant. As a second choice, select
at least two "atypical" colonies (if
typical colonies are not present) and
transfer them to labeled tubes of
lactose broth and to agar slants. As a
third choice, in the absence of typical
or atypical coliform-like colonies,
select at lea8t two well-isolated
colonies representative of those
appearing on the EMB plate, and trans-
fer them to lactose broth and to agar
B Incubate all cultures transfered from
EMB agar plates 24 + 2 hours at 35 +
O.50C. - -
E 96-hour Procedures
1 Subculture I from the samples being
studied may include: 4B-hour tubes
of BGLB, EMB agar plates, ]actose
broth tubes, and agar slant cultures.

MPN Methods
2 If any 48-hour tubes of BGLB are
present, read and record gas production
in the "48" column under BGLB. From
any gas-positive BGLB cultures pre-
pare streak plate inoculations for colony
isolation on EMB agar. Discard all
tubes of BGLB, and incubate EMB agar
plates 24 ~ 2 hours at 35 ~ 0.50 C.
3 If any EMB plates are present, examine
and record results in the "EMB" column
of the data sheet. Make transfers to
agar slants and to lactose broth from
all EMB agar plate cultures. In
decreasing order of preference, transfer
at least one typical colony, or at least
two atypical colonies, or at least two
colonies representative of those on the
4 Examine and record results from the
lactose broth cultures.
5 Prepare a Gram- stained smear from
each of the agar slant cultures, as
follow s :
NOTE: Always prepare Gram stain
from an actively growing culture,
preferab~ about 18 hours old, and
never more than 24 hours old. Failure
to observe this precaution often results
in irregular staining reactions.
a Thoroughly clean a glass slide to
free it of any trace of olly film,
b Place one drop of distilled water on
the slide.
c Use the inoculation needle to suspend
a ~ amount of growth from the
nutrient agar slant culture in the
drop of water.
d Mix the thin suspension of cells with
the tip o'Ti'he inoculation needle, and
anow the water to e'4porate.
e "Fix" the smear by gently warming
the slide over a flame.
f Stain the smear by flooding it for 1
minute with crystal violet solution.
g Flush the excess crystal violet
solution off in gently running water,
and gently blot dry with filter
paper or with other clean absorbent
h Flood the smear with Lugol's
iodine for 1 minute.
Wash the slide in gently running
water and blot dry with filter paper.
Decolorize the smear with 950/.
alcohol solution with gentle
agitation for 10- 30 seconds,
depending upon extent of removal
of crystal violet dye, then blot dry.
k Counterstain for 10 seconds with
safranin solution, then wash in
running water and blot dry.
Examine the slide under the
microscope, using the oil
immersion lens. Coliform
bacteria are Gram-negative,
nonspore-forming, rod- shaped
cells, occurl'ing singly, in pairs,
or rarely in short chains.
m If typical coliform staining reaction
and morphology are observed,
record + in the appropriate space
under the "Gram Stain" column of
the data sheet. If typical morphology
and staining reaction are not
observed, then mark it ~ or ,and
make suitable comment in the
"remarks" column at the right-hand
side of the data sheet.
n If spore-forming bacteria are
observed, it will be necessary to
repurify the culture from which
the observations were made.
Consult the instructor, or refer
to Standard Methods, for procedures.
At this point, it is possible that all
cultural work for the Completed Test
has been finished If so, codify results
and determine Completed Test coliforms
per 100 m!.

MPN Methods
F 120-hour Procedures and fol1owinll:
1 Any procedures to be undertaken from
this point are "strallgler" cultures on
media already described, and requLrinll
step- by- step methodology already liven
in detail. Such cultures may be on:
EMB plates, agar slama, or lactose
broth. The same time-And-temperature
of incubation required for earlier Itudies
appliel to the "stralillers" a8 do the
observations, staining reactions, and
interpretation of relults. On con-
clusion of all cultural procedures,
codify results and detennine Completed
Test coll.forms per 100 mI.
A General Information
1 The procedure described il an elevated
temperature test for fecal coillorm
2 Equipment required for the tests are
those required for the Presumptive
Test of Standard Methods, a water-bath
incubator, and the appr,;,priate culture
B Fecal Coliform Test with EC Broth
1 Sample: The test is applied to gas-
pOlitive tubes from the Standard
Methods Presumptive Test (lauryl
tryptole broth), in parallel with
Confirmed Test procedures.
2 24-hour ~erations. Initial procedures
are the p nting procedures described
for the Standard Metho~1 Prelumptive
Coliform teat.
a After reading and recordini gas-
production on lauryl tryptose broth,
temporarily retain all gas-politive
tubes. -
b Label a tube of EC broth to corre-
spond with each ps-positive tube
of lauryl tryptose broth. The option
regarding transfer of only a limited
numb41r of tubes to the Confirmed
Te.t sometimes can be applied here.
However, the worker is urged to
avoid exercise of this option until
he has assured the applicability of
the option by preliminary tests on
the sample source.
c Transfer one loopful of culture from
each gas-positive culture in lauryl
tryptose broth to the corresponding~
labeled tube of EC broth.
d Incubate EC broth tubes 24 i 2 hours
at 44.5.t O. ;l°C in a waterbath
with water depth sufficient to come
up at least as high as the top of the
culture medium in the tubes. Place
in waterbath as soon as p08Sible
after inocuJat1on and always within
30 minute. after ino~'.1lation.
3 48-hour operations
a Remove the rack of EC cultures
from the waterbath, s~ke gently,
and record ps production for each
tube. Gas in any quantity is a
positive test.
b AI soon a8 re8ultIJ are recorded,
discard all tubes. (This is a 24-
hour te8t for EC broth inoculations
and not a "8-hour test. )
c Transfer any additional 48- hour
gal-polUive tubes of lauryl tryptose
broth to correspondingly labeled
tubes of EC broth. Incubate 24 +
2 hours at 44.5.:!: 0.20C. -
4 72-hour operations
a Read and record gas production for
each tube. Discard all cultures.
b Codl.fy results and determine fecal
coll.form count per 100 ml of sample.

MPN Methods

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IIOD. A../08

Part 3
(Day- By- Day Procedures)
A The lame sampling and holdirli procedure.
apply a. for the coliform test.
B The number ot tecal Itreptococci in water
generally is lower than the number of
coliform bacteria. It is good practice
in multiple dilution tube tests to start the
sample planting series with one sample
increment larger than for the coliform
teat. For example: If a sample planting
series of 1.0, O. I, 0.01, and 0.001 ml
Is planned for the coliform test, it is
sUile~ed that a series of 10, 1.0, 0.1,
and 0.01 ml be planted for the fecal
streptococcus test.
C Equipment required for the test is the same
as required for the Standard Methods
Prelumpt1ve and Confirmed Telts, except
for the differences in culture media.
A Firlt-Day Operations
1 Prepare the sample data sheet and
labeled tubes of azide dextrose broth
in the same manner as for the
Presumptive Test. NOTE: If 10-ml
samples are included in the series, be
sure to use a special concentration
(ordinarily double-strength) of azide
dextrose broth for these sample
2 Shake the sample vigorouBly, approxi-
mately 25 times, in an up-and- down
3 Measure the predetermined sample
volumes into the labeled tubes of azide
dextrose broth, using the sample
measurement and delivery techniques
u8ed for the Presumptive Test.
Preceding page blank
4 Shake the rack o! tubee of inoculated
culture media, to insure good mixing
of sample with mp.dlum.
5 Place the rack of inoculated tubes in
the incubator at 350 + 0.50 C for 24 +
2 hours. - -
B 24-hour Operations
1 Remove the rack of tubes from the
incubator. Read and record the results
from each tube. Growth is a positive
test with this test-:-EViaence of growth
consists either of turbidity of the
medium, a "button" of sediment at the
bottom of the culture tube, or both.
2 abel a tube of ethyl violet azide broth
to correspond with each positive culture
of azide dextrose broth. It may be
permissible to use the same confirmatory
option as described for the coliform
Confirmed Test, in this outline.
3 Shake the rack of cultures gently, to
resuspend any living cells which have
settled out to the bottom of the culture
4 Transfer three loopfuls of culture from
each growth-positive tube of azide
dextrose broth to the correspondingly
labeled tube of ethyl violet azide broth.
5 As transfers are made, place the newly
inoculated tubes of ethyl violet azide
broth in the positions in the rack
formerly occupied by the growth-
positive tubes of azide dextrose broth.
Discard the tubes of azide dextrose
broth culture.
6 Return the rack, containing 24-hour
growth-negative azide dextrose broth
tubes and newly-inoculated tubes of
ethyl violet azide broth, to the incubator.
Incubate 24 ~ 2 hourB at 350 ~ 0.50 C.

MPN Methods
C 48-hOlSr Operations
1 Remove the rack of tubes from the
incubator. Read and report results.
Growth, either in azide dextrose broth
or in ethyl violet azide broth, 18 a
po.1t1ve te8t. Be sure to report the
re.ult8 of the azide dextrose broth
medium under the "48" column for that
medium and the results of the ethyl
v10let azide broth cultures under the
"24" column for that. medium.
2 Any 48-hour growth-p08itive cultures
of azide dextrose broth are to be
transferred (three loopfulls) to ethyl
violet azide broth. Discard all 48-hour
growth-neptive tubes of azide dextrose
broth and all 24-hour growth-positive
tubes of ethyl violet azide broth.
3 Incubate the 24-hour Irowth-negative
and the newly-inoculated tubes of ethyl
violet azide broth 24 + 2 hours at 350
~ 0.50C. -
D '72-hour Operations
1 Read and report growth result8 of all
tubes of ethyl violet azide broth.
2 Di.card all Irowth-positive cultures
and all 4S-hour growth-negative
culture 8 .
3 Reincubate any 24-hour growth-negative
cultures in ethyl violet azide broth 24
t 2 hours at 350 t O. SoC.

E 96-hour Operations
1 Read and report growth results of any
remainil\j tubes of ethyl violet azide
2 Codify results and determine fecal
.treptococd per 100 mI.
1 Standard Methods for th'3 Examf,nation of
Water and Wastewater (13th J!:d).
Prepared and published jointly by
American Public Health A8soclation,
American Water Works A88OCiation,
and Water Pollution Control
Federation:. 1971.
2 Geldreich, E.E.. Clark, H.F.. Kabler,
P. W., Huff. C. B. and Bordner, R. H.
The Coliform Group. II. Reactions
in EC Medium at 450C. Appl.
Microbiol. 8:347-348. 1958.
3 Geldreich, E. E., Bordner, R. H., Huff,
C. B.. Clark, H. F. and Kabler. P. W.
Type Distribution of Coliform Bacteria
in the Feces of Warm-Blooded Animals.
J. Water Pollution Control Federation.
34:295-301. 1962.
4 Recommend Proc. for th~ Bacteriological
Examination of Sea Water and Shellfish.
3rd Edition. American Public Health
Al8ocl&t1on. 1962.
This outUne was prepared by H. L. Jeter,
Director, National Training Center, Water'
Programs Operations. Environmental
Protection Agency, Cincl.nnad. OH 45268.
De8criptor8: Coliforms, Fecal Coliforms,
Fecal Streptococci, Indicator Bacteria,
Laboratory Equipment, Laboratory Tests,
Microbiol00', Most Probable Number. MPN,
Sewag. Bacteria, Water Analysi8

Multiple Dilution Tube Methode
The baeic procedure I! of mgltlple dilution tube
methods, often called the Most Probable
Number (MPN) Method, are described here,
.. applied in tests for the coliform group,
fecal coliform group, and for the fecal
IItreptococci. The method. are d8lcribed in
greater detail elsewhere in thi. course manual.
A Basic Assumptions of the Method
1 Bacteria in a s"mple can be freed from
one another by shaking, '0 that IIlngle-
cell unih are uniformly di.perlled in
the !lample.
2 Through inoculation of progressively
.maller volumes of sample into a series
of tubes of culture medium, some tubes
will receive single-cell units of the tellt
bacteria and some will not.
3 Cglture media being used in the proce-
dure will permit ,rowth of all cells of
the organi.ms for which the test is
4 The testin, procedure will give a simple
"Y8S" (positive test) or "no" (negative
test) answer for each inoculated tube,
with respect to presence of the orp.nism
for which the teet has been devised.
5 Probability mathematics can be applied
to the results of the testin, procedure
to give quantitative information (Most
Probable Number) on the density of the
test organisms in the sample source.
B Application
I Quantitative application
Most probable number methods can be
applied in almost any conceivable com-
bination of sample inoculation volumes
and number of replicate tubes per
volume. Standard practice of this
or,an1zation in water pollution surveys
is to inoculate 5 replicate tubes in a
series of sample volumes in successively
decreasing decimal increments. At one
time it was common to use 3 tubes per
decimal sample increment, but due to
the relative Jack of precision of results,
the practice has been largely abandoned.
Qualitative aspects
To assure arowth of the test organ-
isml, the tellts are performed in
stages. The stages of the Standard
Coliform Test are the well-known
Presumptive, Confirmed, and Com-
pleted Tests, respectively.
The fecal coliform test also is per-
formed in stages, using the standard
Presumptive Test a. the first stage,
and a special confirmatory procedure
to differentiate between total coli1'orms
and fecal colifc.rme.
Similarly, fecal streptococcus tests
are conducted in two stages, w1.th the
first stage serving primarily as an
enrichment and limited differential
test which is further verified by
results from the second, more selec-
tive, culture medium.
Presumptive Test
The Presumptive Test is the primary
stage of the procedures leading to the
Confirmed Test and the Completed Test.

Determination of Bacterial Pollution
Tne test alone is not applicable in
testing potability of drinking water,
but it may be used for intermediate
procels control in treatment plants,
or on occasion, in the study of waters
known to be heavUy polluted.
2 Culture medium
The bacteriologist has a choice between
standard lactose broth and lauryl tryp-
tose lactose broth, in fermentation tubes.
Lauryl tryptose lactose broth is preferred.
This medium is more selective for
coliform bacteria, and it exerts no
apparent suppressive effect on any
strains of the coliform group.
3 Incubation
In this and in all thE. Standard Methods
test procedures for coliforms, incuba-
tion is at 350C + a.lloc. This tempera-
ture is fixed arbitrarily. Cultures
may be incubated up to 48 ~ 3 hours.
Continued incubation of positive tubes
is unfavorable to coliform bacteria.
Cultures are examined for the develop-
ment of positive tests at the 24-hour
interval and negative cultures are re-
turned to the incubator for the remainder
of the Incubation period. Tubes givinll
positive results are lubject to the
Confirmed Test.
4 Procedure
Predetermined volumes of sample are
measured into the culture medium and
incubated up to 48 hours. Fermentation
tubes showing any amount of visible
gas are reported as positive tests.
Oas formation is another criterion for
B Confirmed Test
1 Applicafion
The Confirmed Test is the procedure
applied to tubes giving positive results
in the Presumptive Test. To lighten
- ----- -
the work-load, it may be permissible
to subject to the Confirmed Test only
certain positive Presumptive Test
tubes. Our usual practice is to apply
the Confirmed Test to all tubes repre-
lenting the smallest sample volume
in which all replicate sample incre-
ments gave positive results, and to all
positive tubes representing lesser
sample volumes. A preponderance of
coliform organisms is assumed.
All tubes giving positive results at 48
hours but not at 24 hourt' are subjected
to the Confirmed Test. Slow fermenters
are more likely to be noncoliforms.
The test may be used for routine
potability tests on drinking waters,
on bathing waters, and on
chlorinated effluents from
sewage treatment pJants.
When the MPN procedure is used for
coliform tests in stream surveys,
the Confirmed Test is the most-used
routine method.
It is strongly urged, however, that the
worker continue a limited number of tests
to the Completed Test stage in order to
demonstrate the applicabUtty of the
Confirmed Test. This is especially
important with watere known to receive
appreciable amounts of land drainage.
If there is unacceptable discrepancy
between the results obtained by the two
methods, high quality work will require
use of the more cumberRome Completed
Test for the entire survey.
2 Culture media
The bacteriologist has a choice among
three different media for the Confirmed
Test. These are, brilliant green lac-
tose bile broth, eosin methylene blue
agar plates, and Endo agar.
Brilliant green lactose bile broth in
fermentation tubes is the Confirmed
Test medium of choice in most

DeterminatIOn of. Bacterial pollution
3 Incubation
In order to show distinctive colony
form, the agar :>late media are incu-
bated up to 24 hours.
4 Procedure notes
If brilliant green lactose bUe
broth is used, one loopful of the
culture from each positive
Presumptive Test tube selected
for confirmation is adequate
inoculum. Within 48 hours,
coUforms should form gas in
.ome amount.
Streaking is done with an inoculating
needle, so manipulated as to ensure
development of several separated
colonies. After incubaUon the colony
morphology of separated colonies is
studied. Colonies having nucleated
centers, with or Without surface sheen,
are reported as positive tests. Opaque
or mucoid colonies lacking nuclei are
considered atypical coliforms. Other
types of colonies are reported as
negative tests.
C Completed Test
I Application
The Completed Test is the official coli-
form test of Standard Methods, and is
the test to be used on all water pota-
bility tests, subject to the exceptions
noted in the Confirmed Test.
The test is applied to all positive tests
from the Confirmed Test and to all
atypical results noted when eosin
methylene blue agar or Endo agar
plates are used. The test determines
whether bacteria in question are or are
not coliform!! by definition.
2 Culture media
If brilliant green lactose bile broth is
used for the Confirmed Test, then eosin
methylene blue agar plates or Endo
agar plates are n.!eded. In
addition. nutrient agar slants
and lactose broth fermenta-
tion tubes are needed.
3 Procedure notes
If brilliant green lactose bile broth
was used in the Confirmed Test, then
pure cultures must be obtained by the
streaking proceciures.
Typical coliform ::olonies. or atypical
colonies if typical colonies are not
present, are fished from one or more
plates. Tubes of lactose broth and
nutrient agar slants are then inoculated
for gas production and Gram stain
Smears for Gram stains should be pre-
pared from all nutrient agar slant cultures
within an incubation period of 24-hours
because Gram ri!action may become
variable. Incubation of the lactose broth
cultures is up to 48 hours. If gas is pro-
duced in any quantity, this portion of the
Completed Test is positive, and Gram
stains are made of the corresponding
A positive test requires compliance
with the coliform definition in all details.
A Application
The significance of this test is discussed
in the outline on bacterial indicators of
pollution. It should be recalled that
this test once was regarded by many as
a specific te!:t for E. coli.
2 The test is applied to the same positive
Presumptive Test tubes of the standard
coliform test that are subjected to the
standard Confirmed Test procedures.
The Presumptive tube enrichment
elevates test sen~itivity. With some
samples experiel'ce will demonstrate
need for applying the test to all positive
Presumptive Test cultures. -

Determination of Bacterial Pollutlon
B Procedure
One standard looptul from each po.itive
Presumptive Test culture to be tested
is transferred to EC Broth in fermen-
tation tubes, and incubated for 24::2
hours at 44. 50C.:!: O,20C in a forced
circulation water bath.
Gas in any quantity is a positive test.
Experimentally fecal coliforms give
gas; Other bacteria do not,
A The status of this group as pollution in-
dicators is discussed in the consideration
of bll'Cte rial indicators of pollution.
B Method of Lit.ky, Mallman, and Fifield
Pre8umptive Test
Predetermined sample increments are
delivered into azide-dextrose broth.
Turbidity in the medium indicates
growth and constitutes a posit{ve test.
Azide is a strong inhibitor and few
bacteria grow besides streptococci.
Azide also slows streptococcus growth
so that 48 hours incubatiC\n is necessary,
2 Confirmatory Test
Three looptuls of culture from positive
presumptive test tubes are transferred
to tubes of ethyl violet azide broth, and
incubated up to 48 hours at 35°C. Three
loopfuls are to overcome medium
Development of turbidity in the medium
indicates growth and is a positive test.
The original research in development of
this method required microscopic
examination of Gram- stained smears of
cultures. Standard Methods, 13th edition,
doel!l not require staining,

Determinatl?n of Bacterial pollution
Membrane Filter Procedures
A Scope
1 The membrane filter, as currently
used in our laboratories. consists of
a porous disk of cellulose esters
47 mm in diameter and approximately
0.15 mm thick. Filters used in water
bacteriology have pores of 0.45 microns
in diameter. The basic principles of
use in water bacteriology are as follows:
a A sterile me.mbrane filter is
fastened into a suitable filter holding
device and a predetermined volume
of a water sample is filtered through
the membrane filter.
b The filter is placed in a culture con-
tainer either on a paper pad impregnated
with moist culture medium or upon an
agar medium.

c The inoculated filter is incubated
under prescribed cooditions of
temperature and humidity for a
designated time.
d After incubation, the resulting
culture is examined, and the neces-
sary interpretations and/or additional
tests are made.
2 Through variations in such factors as
the composition of the culture medium,
incubation time and temperature. and
combinations with other cultural and
biochemical tests, several kinds of
bacterial determinations are possible.
B Basic Principles and Origins of Membrane
Filter Techniques
The purpose of this discussion is to pro-
vide orientation in the types and amount of
equipment and supplies needed in mem-
brane filter analyses in water quality sur-
veys. the basic methodology, limitations
of membrane filter procedures, and the
status of membrane filter methods in water
quality testing. Finally, it considers the
level of technical skill and the workloads
of personnel assigned to perform tests by
this method,
A Laboratory Supplies and Equipment
1 Membrane filters
.a Membrane filters are available
from at least 3 different commercial
sources, representing the United
States. Britain, and Germany. Each
source applies a distinctive trade-
name to its MF produc t:
"Millipore Filters" Millipore
Filter Corp., U. S. A.
"Bac-T- Flex Filters" Schleicher
and Schuell, U. S. A. representatives
for Sartorius - Werke. Germany.
"Oxoid Filters" - Oxo Limited,
b Membrane filters are available in
a wide variety of controlled pore
sizes. Each commercial source
will designate the filter most suitable
for water bacteriological tests upon
2 Absorbent pads for nutrients
Absorbent paper disks for mechanical
support of the filters during incubation
and for maintaining an adequate supply
of culture medium in contact with the
filtere. ordinarily are sold with the
membrane filters. Additional supplies
are obtainable from filter

Determination of Bacterial Pollution
3 FUter-holding units
a The filter-holdirli unit is a device
for supportini the membrane filter
and for holdil1i the sample until it
panel through the filter. During
the filtration, the sample passes
through a circular area, usually
about 35 mm in. diameter, in the
center of the fUter. The outer part
of the filter disk is clamped between
the two essential components of the
filter-holding unit.
1) The lower element, called the
filter base, or receptacle,
supports the membrane filter on
a plate about 50 mm in diameter.
The central part of this plate is
porous, to aHow free passage of
liquid. The outer part of the
plate is a smooth nonporous sur-
face. The lower element includes
fittings for mounting the unit in
a suction flask or other container
suitable for filtration with vacuum.
2) The upper element, usually called
the funnel, holds the sample until
it is drawn through the filter. Its
lower portion is a nat ring that
rests directly over the nonporoua
part of the filter support plate.
3) When assembled, the two
element. of the filtel'-holding
unit are joined by a locking ring
or by one or more clampa or
b Filter-holding units are either
metal or gla8s. Metal units are
avaUable from at least six commer-
cial sources, and 11ass unit. are
avaUable from at least two sources.
Most workers prefer to use metal
tllter-holdint units, due to their
Ireater durability. Most of the use
ot 11ass units seems to be dictated
by economic considerationa, as
the.e are much lower in initial
4 Culture media
a Culture media are commercially
available for use with membrane
tilters in the determination at
bacterial plate counts, for coliform
counts, fecal coliform counts, fecal
streptococcus counts and, in addition,
tor several special applications.
b These media can be obtained in
several torm., including:
1) Dehydrated media, requiring
weighing, addition ot a suitable
amount of distilled water, and
some form of sterilization prior
to use. This form ot media is
preterred as it appears to give
the most satisfactory combina-
tion of performance quality,
stability and economy.
2) Ready-to-use culture media,
sealed in glass ampoules, and
3) Dehydrated cultu~'e media,
impregnated in absorbent pads.
Media in this tOl'm require only
the addition of a suitable amount
of I!Iterile distilled water to the
paper pad to be ready for use.
These media have limited shelf
lite, and are not widely used at
thil!l time.
5 Culture containerl!l
Several forms of culture containers are
suitable for membrane filter work.
These include small glass Petri dishes,
and plastic Petri dishes. The plastic
Petri dil!lhes may be preterred, as they
are sufficiently cheap to encourage
single-service use.
8 Vacuum sources
In the fixed laborat.:>ry, central vacuum
service, laboratory-scale electric
vacuum pumps, or water pumps
("a8pirators") are suitable tor vacuum
t1Itration of samples.

7 Gla8sware
Conventional laboratory glassWare is
used in fixed laboratories faf'measure-
ment and delivery of samples for filtra-
tion, sample dilutions, and the like.
Under field condition8 it sometimes is
nece88ary to use a limited amount of
laboratory Ilassware, with some form
of field application of sterilization
8 General laboratory facilities
Such facilities a8 autoclave, incubators,
weighing equipment, and the like are
required in conventional bacteriology
laboratorie8, and are needed equally
when membrane filter methods are used.
B Field Equipment
Field operati0l'18 cannot be carried on
indefinitely without support from a cen-
tra1laboratory. Functions 8uch as pre-
paration of di8tilled water, pre -weighing
of culture media, sterilization of mem-
brane filters and culture containers, and
other general preparations are best per-
formed in a central laboratory facility.
1 With the introduction of membrane
filters to the bacteriological analysis
of water, a number of investigators
have given attention to the development
of membrane filter equipment which
can be used in the field in easily
portable, self-contained water labora-
tories. Such portable laboratory kits.
would have three major fields of use-
a They would be useful in certain
routine water quality control
operations. Examples include such
places as on board ships, in some
national parks, on airlines, etc.
Determination of .Bacterial Pollution
b They could be used in certain types
of stream surveys, where there is
an excessive delay between sample
collection and the time when
examination can be started, due to
time required for transportation of
c Finally, such units would be invalu-
able in emergencies, when existing
laboratories are overburdened or
inoperative. Portable kits already
have proven extremely useful in
testing many small water supplies
in a short perIod of time. Further,
there is a predictable need for such
equipment in the event of natural or
wartime civil defense disaster.
2 Availability of field equipment
Commercially development equipment
is available from several sources.
a Equipment of this type includes
provision for sample filtration,
field sterilization (where necessary)
of filtration equipment. limited
capacity for reserves of culture
media and other supplies, and
provision for incubation of cultures.
Such equ~pment does not include
satisfactory visual aid equipment
(a low powered microscope is needed
for streptococcus counts. and at
least a simple lens is needed for
coliform work), and provision for
many of the supplies is not included.
b Performance of the various forms
of commerciaily available field
equipment is not equal. Buyers of
field equipment are urged to study
the equipment specifications quite
carefully, and if necessary. per-
form comparative tests with labora-
tory type equipment to determine
the type of field equipment most
suited to their own needs.

Determination of Bacterial Pollution
A Standard Coltrorm Tut
I Basic pl'Ocedures
a Sample filtration volumes are
selected (usually 3 volumes) to give
at l.et one volume which will con-
tatn 20-80 typical coliform colonies.
b The sample volume. are filtered
throu,h membrane fUten, and
incubated at 350C for 20-22 hours
in an atmosphere at or near
c Following incubation, the typical
colUorm colonie. are counted and
reported as coliform. per 100 ml
of ...rnple.
2 Applications
The total coliform count has been used
moat widely of all procedures when
bacteriological data is collected in a
survey. Applicationa of membrane
fUter methods in eurveys necessarily
have a ehort history. The method has
been used in certain studies of the
Potomac River System, in the Illinois-
Great Lakes studies, and in certain
surveye in the West.
The Standard MF coliform test is
applicable when it can be initiated
within the recommended period between
sample collection and receipt at the
laboratory. The procedure is applicable
with MF field units.
B "Fecal Coliform" Teet with Membrane
I Procedure
a Sample filtration procedures are the
same as for the Standard Membrane
Filter coliform test. SamJ?le fil-
tration volumes should be selected
so that at least one filter will pro-
duce 20-60 fecal coliform colonies.
The culture medium i8 M-FC
Broth. The medium requires
addition of a ro80lic acid solution
prior to use.
Within 20 minutes after _mple
filtration, the membrane filters
are placed in emall pllUltic bags,
which are sealed, and immersed.in
a forced circulation water bath at
44. SOC Z O. 20C for a a. hour
incubation time.
Fecal foliform colonies are blue or
blue-hues, genera1]y 1 - 3 mm in
diameter. The fecalcol1form
deneity is reported.. fecal colliorms
per 100 ml of .ample.
The fecal foliform test ie being used
increasingly in water quality studies
for improved evidence of recent di.rect
oriiln of intestinal pollution in the
water .amples being tested.
C Fecal StreptococcU8 Tests
1 Baeic procedures
a The filtration procedures are the
lIame a. with the coliform tests.
The sample volumes ..bould be such
that at least one membrane filter
wUl produce 20 100 fecal strepto-
coccus colonies.
b The culture medium can be the
M-Enterococcu8 Allar developed by
Slanetz and hie aS80ciates, or it can
be KF-Broth. developed by Kenner
and his associates. In eitl].er case,
incubation is 48 hours at 35°C.
c With both media, fecal streptococcus
colonies are pale pink to deep red,
up to 2 mm in diameter.

d Because the density of fecal
streptococci in most waters is
lower than coliform density, it is
necessary to filter greater volumes
of water. Further, the number of
colonies that can be counted effec-
tively Is greater than the number of
coliform colonies. Sample volumes
often can be 10 to 100 -fold greater
for fecal streptococcus counts than
for coliform counts.
2 Application
Fecal streptococcus counts are
especially useful when supplementary
data will support information from the
coliform counts. Fecal streptococcus
counts are used in many states in
addition to coliform tests in examina-
tion of bathing waters as well as in
stream surveys.
Most of the recognized limitations of the
membrane filter m~thod have been related
to tests for members of the coliform group.
Extension of these recognized limitations to
tests for other pollution indicators requires
special interpretation.
A Need for Selection of Suitable Filtration
1 In tests for coliform bacteria, the
sample filtration volume s should be so
selected that one of volumes yields
20- 80 coliform colonies. With fewer
than 20 colonies, the random variation
of colony counts becomes great enough
to cause some difficulty in statistical
analysis; with more than 80 coliform
colonies, the proportionality between
number of coliform colonies differen-
tiated and sample volume begins to fail.
This is especially troublesome in
waters containing large numbers of
noncoliform bacteria capable of growing
on the filters.
Determination of Bacterial Pollutipn
2 In te.ts for the fecal streptococci,
fewer nonstreptococcus interference
colonies are capable of growing on
the medium. For this reason and
because fecal streptococcus colonies
are small, the sample filtration
volumes are so selected as to
yield 20-100 colonies on a
membrane filter.
B Influence of Turbidity
When the water being studied contains
large amounts of suspended matter, such
a8 clay, and has relatively low counts of
the indicator organisms being studied,
cultural difficulties often result. Thl'
particulate matter suspended in the water
is deposited on the surface of the membrane
fUter in sample filtrRtion, with any
bacteria contained in the sampll'. Culture
medium diffusing through the membrane
fUter forms a capillary layer around all
the particles of matter deposited on the
filter. This results in dl'velopment of
spreading, poorly defined bacterial
The solutions to problems of this type are:
1) to filter the sample in several smaller
increments so that the combined sample
fUtration volume is spread over several
membrane filters, e..ch of which has a
lesser amount of particulate matter
deposited on the surface; or, 2) perform
the analysis by means of the dilution tube
(MPN) procedure.
C Influence of large numbers of bacteria
representing forms other than the indicator
group being studied.
In coliform testing, this can be a
serious problem. The difflculty arises
when appreciable numbers of non-
coliform bacteria :ue pre se nt and are
capable of growing on the Endo- type
culture medium used. When lar ge
numbers of noncoliform bacteria grow
on the medium, and few coliform

Determination of Bacterial Pollution
bacteria are present, the sheen-
producing capacity of many of the
coliform colonies ill impaired, and
the result indicates the presence of
fewer coliform bacteria than actually
are prellent.
A solution for this type of problem
is to filter several smaller sample
increments, or else to use the MPN
2 In tests for fecal streptococci, the
pre6ence of large numbers of bacteria
of other groups has not proven trouble-
some. Thl' medium is extremely
selective, and the few forms that do
seem to grow on the medium produce
extremely small colonies which have
not.been shown to have any adverse
effect on the qualitative results obtained
in tests for fecal streptococci.
A American Public Health Association,
American Water Works Auociat1on,
and Water Pollution Control Federation,
in "St8roard Methods for the Examination
of Water and Wastewater." (l3th Edition,
1 A single-stage procedure, using
M-Endo BrQ-th MF for tellts for
members of the coliform group, is de-
8cribed as an official testing procedure.
2 A new two-stage procedure has been
3 A delayed incubation procedure, using
benzoated M-Endo Broth MF, is
de8ignated a Tentative Method.
4 A te8t for enterococci (fecal strepto-
cocci). U8ing M Enterococcus Agar or KF
broth, is designated a Tentative Method.
B The Technical Committee on Methods for
the Bacteriological Examination of
Boundary Water8 recommended use of
membrane filter methods for the examina-
tion of boundary waters. The Advisory
Committee adopted the recommendation in
January, 1957.
C The U. S. Government, in Interstate
Quarantine Drinking Water Standards
1 (Federal Register, Octob~r 23, 1956,
pp. 8110-11 and March 1, 1957, Vol.
22, p. 1271), Preliminary and flnal
authorization was granted to use mem-
brane filter coliform tests for the
examination of potable water8at inter-
8tate carrier water points and other
waters under water quality control
regulations of the Public Health
Service or other agencies of the U. S.

Supplemental Tests
A Need for Supplemental Tests
I To demonstrate applicability of non-
standard procedures to the particular
survey situation.
2 To clarify significance of results
obta 1ned through appl1ca tion of
standard procedures.
3 To investigate anomalous results
obtained by ~ou tine survey procedures.
such as evidehce of toxic substances in
the water. unexplained high densities of
pollution indicators. etc.
4 To develop information not otherwise
shown by the routine testing program.
B Scope
This discussion is concerned with further
differentiation of members of the coliform
group, fecal streptococcus group. enteric
pathogens, and with a bioassay test in
which bacteria are used as the test
A Nature of Tests
Pure cultures for IMViC classification
are obtained from agar plate or from
membrane filter isolations from the
sample source. For development of
relative numbers of the various IMViC
types. it never is acceptable to use
cultur£' isolates from the dilution tube
"IMViC" is a mnemonic expression,
referring to a series of four biochemical
tests. The tests and basic principles of
each are as follows:
Determination of Bactenal Poll,,,_:_, ,
a Indole test (I):
This is a test to determine if indol.
is produced as an end-product of tlw
utilization of the amino acid trypt"
pbane. The calture is transf£'rret! tf
tryptophane broth and incubated 21
hours. after which 4 ml of the cultup'
is tested with Kovac's reagent.
Development of a cherry- red color
in the reagent indicates presence of
Methyl red test (M):
This is a test of the terminal pH of
the culture after utilization of a
fermentable carbohydrate, glucose.
The culture is incubated 5 days in
a standard peptone- glucose medium.
after which a few drops of methyl red
indicator are added. DevE'lopment
of a red color constitutes a positive
test, demonstrating that the organism
being tested has brought the medium
to a more strongly acid condition
than is obtained with a negative result
(development of a yellow color).
c Voges-ProBkauer test (V):
This is to determine whE'ther acetyl-
methylcarbinol iB produced as one of
the end-products of the medjum used
for the methyl red test. After 48
hours of incubation. 5 ml of thf' medium
is removed from the culture and tested
with a-naphthol solution. Th', pre sence
of acetylmethylcarbinol is mdicated by
development of a red color.
d Citrate test (C):
This test is to determine whethpr
the strain of coliform organism s
being tested is capable of using
citrate as the Bole source of "arbon.
A small inoculum of <:'ulture is trans-
ferred to Koser's citrate medium or

Determination of Bacterial Pollution
Simmons' citrate apr. Growth in
or on either medium is evidence that
the organism can use citrate and
constitutes a potitive test.

B Application of IMViC Tests

1 Field survey workers make little routine
use .of IMViC testinl procedures in water
pollution surveys. EssentiaUy the eame
information relative to intestinal orilin
of coliforms is obtainable on a quan-
Utative basis by the fecal coliform test.

2 In the event that appreciable amounts of
land drainage are to be considered in a
survey, IMViC classification may be
use'ul, as a means of recoanizinl
IMViC types of coliform. which may
become predominant in soils (-+-+).
3 IMViC cla..1f1cation is a useful re-
search laboratory tool as a means of
segreptin, pure culturell of coliform
bacteria into groups havin. a similar
biochemical and behavioral character-
istics. It is noted that in theory 16
IMViC categories are ident1f1able.

A Nature ot Tests
1 Tests are applied to pure cultures
obtained from apr plates or mem-
brane filter isolations from the sample
source. As with IMViC tests. partition
counts are not acceptable if made from
dilution tube isolate..

2 A sertea of biochemical teats Is made
which lead to species or species-group
identification of each culture. A
simplified protocol for these teste is
shown in the following table:
KF apr
(pink-red colonies)
Growth in Brain-Heart Infusion Broth Within 2 Days at 450C and 5 Days at lOOC
~ ~
Growth at 450 and IOoC Growth at 450C Only
. I
Confirm with growth in S. bovis-S. equinu8
6; 5"/0 NaCI (brain -heart ...............
infusion broth. ) Starch hydrolysis
Lactose Fermentation Test
Positive N.egative
(Add) (No change)
S. bovis group S. equinus
Group D Streptococci
Starch hydrolysis
Positive Negative
I \
(Atypical Peptonization of
S. fecaUs) Litrnus Milk
Positive Negative
S. fecaUs enterococci
(var. liquetaclens)
NOTE, A small percentage of unclassified strains will be found since fecal str"'ptococci vary
greatly in their biochemical reactions.

B Applications
As described in the d18culision of indicator
organisms, certain streptococcal specie8
are normal inhabitants of the intestines of
domestic animals; others are not. The
tests are made to account for probable
human VS. probable animal origin of
pOllution-indicating bacteria found in
water .amples.
A Nature of Tests
1 The tests are performed in multiple stages,
and involve a combination of cultural
biochemical teste and serolo,icalidenti-
tication procedures. The e..ential steps
a Application of special sample collection
procedures such as filtration or gauze
pad concentration

b Enrichment of sample and primary
isolation of cultures.
c Tentative identification by cultural
determination of biochemical
d Confirmation and completion of
identification by serological
2 The details of these tests are far beyond
the scope of this discu8sion. The
interested wcrker should conault a
8tandard reference on the 8ubject.
"Identification of Enterobacteriaceae"
by P. R. Edward8 and W. H. Ewing
(Burgess Publishing Co.. Minneapolis
15, Minnesota) i8 particularly
B Application of Tests
In certain water pollution studies, survey
workers currently are making tests to
show presence of species of Salmonella
as positive evidence of microbiological
Determination o! Bacterial Pollutl')"
hazard in the waters being tested. In
this connection the following points are
I Demonatratio;'\ of presence of these
organisms is a qualitative, not a
quantitative, procedure.
2 Salmonella species, when demonstrated,
mayor may not be of human origin; hr.",-
ever, any Salmonella species dpmon-
strated is regarded as a potential hazard
to human health lince all are pathogenic.
3 Tests to determine the presence of
enteric pathogens are supplemental,
not alternatives, to tests for the estab-
lished bacterial indicators of pollution,
4 The tests require a high order of skill
and training, as well as special supplies,
beyond the pres~nt resources of most
water laboratories.
A Nature of Test
1 Contrary to all the other tests described,
this test does not necessarily involve
laboratory work on organisms recovered
Crom the water source, but is a test of
the water itself. The water is used as
solvent for a synthetic culture medium,
and used to cultivate a pure culture oC a
strain of Aerobacter aerogenes. A
parallel synthetic culture medium is
tested in a similar manner, but distilled
water of the highest purity is used in
media preparation. After 24 hours of
incubation, bacter ial dens iUes from
both media are compared.
2 Through various combinations of the
constituents of the culture medium, the
worker can determine whether substances
unfavorable to tbe test organism are
present, or he can demonstrate the pos-
sible presence of nutritive materials in
the water source, which might help to
account Cor unexpected elevations oC
denllity of the pollution indicator.
53 - 13

Determination of Bacterial Pollution
3 The details of the test correspond quite
clollely to the test for distilled water
quality, described in the current (13th)
edition of "Standard Methods for the Ex-
amination of Water and Wastewater. "
B Applications: The Teet Has potential
Use In
1 Studies in which pollution-indicatin,
bacteria seem to disappear more rapidly
thah expected. on the baais of experi.
encl' with environmental waters in
general, through demonstration of bac-
tericidal or bacteriostatic properties
of the test water; or
2 In studiel!l in which elevations of the
denl!lity of pollution indicators occur and
there is au,gestion of prolon,ed after-
growth of the organisms due to hiah
levels of available nutrient. This type
of tE'st must be undertaken with ,reat
caution, al!l the testing procedure re-
moves many of the environmental
factors which tend to cause disappear-
ance of the pollution indicators,
notably predatory organisms, other
microorpnisms which compete more
favorably for the available food supply,
arid certain chemical substances.

1 APHA, AWWA, WPCF, Standard Methods
for the Examination of Water and
Wastewater. 13th ed. Am. Pub.
Health Assn. New York, N. Y. 1965.
2 Geldreil'h. E. E., Clark, H. F., Kabler,
P. W., Bordner, R. H., and Huff, C. B.
The Coliform Group. II. Reactions of
Coliform IMViC Types in a Modified
EC Test at 45. ooC. Inc'ubation.
3 Kenner, Bernai-d A., Clark, H. F., and
Kabler, P. W. Fecal Streptococci. I.
Cultivation and EnUmeration of
S\reptococci in Surface Waters.
Applied Microbiology. 9: 15-20. 1981.
S3 -14
4 Kenner, Bernard A., Clark, H. F., and
Kabler, P. W. Fecal Streptococci. II.
Quantification of Streptococci in Feces.
Am. JCNI'. Pub. Health. 50: 1653-59.
5 Litaky, W., MaIlman, W. L., and
Fifield, C. W. A New Medium for the
Detection of Ent8rococci in Water.
Am. Jour. Pub. Health. 43:873-879.
6 Litslcy, W., Mallman, W. L., and Fifield,
C. W. Comparison of the Most probable
Numbers of Escherichia £2li and
Enterococci in River Waters. Am.
Jour. Pub. Health. 45: 1049-1053. 1955.
7 Fifield, C. W., and Schaufus, C. P.
Improved Membrane Filter Medium for
the Detection of Coliform Orpnisms.
Jour. Am. Water Works AssD. 50: 193.
8 Slanetz, L. W., Bent and Bartley. Use of
the Membrane Filter Technique to
Enumerate Enterococci in Water.
Public Health Reports. 70:87. 1955.
9 Slanetz, L. W., Bartley, C. H., and Ray,
V. A. Further Studies on Membrane
Filter Procedures for the Determina-
tion of Numbers of Enterococci in
Water and Sewage. Proc. Soc. Am.
BacterioIOfi.ts. 56th Meetiq. 1956.
10 Clark, H. F.. Kabler, P. W., .00
Geldreich, E. E. Advantages and
Limitatione of the Membrane Filter
Procedures. Water and Sewa,e Works.
September 1857.
11 Geldreich, Edwin E., Clark, H. F., Huff,
C. B., and Beat, Lois C. Fecal-
Coliform-Organism Medium for the
Membrane Filter Technique. Jour.
AWWA. 57:208-214. 1965.

Geldreich. E. E., Clark, H. F., and
Huff, C. B. A study of Pollution
Indicaton in a Wa.te Stabilization
Pond. Jour. Water Pollution Control
Federation. 36:1372-79. 1964.
Edwards, P,R., and Ewinl, W.H.
Identification of Enterobacteriaceae.
Burle.. PubU.hinl Company. 1962.
Determination of Bacterial pollul_'2!!
This outline was prepared by Harold L. Jf""r.
Director, National TrBining Center, EPA,
WPO, Cincinnati, OH 45268.
Descriptors; Colifocms, Fecal Coliforms, .
Fecal Streptococci, Filters. Indicator BaderJR.
Laboratory Equipment, Laboratory Tests,
Membranes, Microbiology, MPN, Most P,' .,b.
able Number, Sewage Bacteria, Water AnalY8,

A Introduction
Successful application of membrane filter
methods requires development of good
routine operational practices. The
detailed basic procedures described in
this Section are applicable to all mem-
brane filter methods in water bacteriology
for filtration, incubation, colony counting,
and reporting of results. In addition,
equipment and supplies used in all mem-
brane filter procedures are described here
and not repeated elsewhere in such detail.
Workers using membrane filter methods
for the first time are urged to become
thoroughly familiar with these basic
procedures and precautions.
B General Supplies and Equipment List
Table 1 is a check list of materials.
<;: "Sterilizing" Media
Set tubes in a boiling waterbath for 10
minutes. This mE'thod suffices for
medium in tubes up to 25 X 150 mm.
Frequent agitation improves dissolving
of the medium.
Alternately, coliform media can be
directly heated on a hotplate to the first
bubble of boiling. Stir the medium
frequent~ if direct heat is used, to avoid
charring the medium.
Do not autoclave.
D General Laboratory Procedures with
Membrane Filters
1 PreR/ire data sheet
Minimum data required are: sample
identification, test performed including
media and methods, sample filtration
volumes, and the bench numbers
assi8l'led to individual membrane filters.
2 Disinfect the laboratory bench surface.
Use a suitable disinfectant solution and
allow the surface to dry before
3 Set out sterile culture containers in an
orderLy arrangement.
4 Label the culture containers.
Numbers correspond with the filter
numbers shown on the data sheet.
5 Place one sterilE' absorbent pad" in
each culture container, unless an agar
medium is being used.
Use sterile forceps for all manipulations
of absorbent pads and membrane filters.
Forcep~.sterillty is maintained by
storing the working tips in about 1 inch
of methanol or ethanol. Because the
alcohol deteriorates the filter, dissipate
it by burning before using the forceps.
Avoid heating the forceps in the burner
as hot metal cha:-s the filter.
*When an agar medium is used, absorbent pads are !!21 used. The amount of medium should be
sufficient to make a layer approximately 1/8" deep in the culture container. In the 50 mm
plastic culture containers this corresponds to approximately 6-8 ml of culture medium.
NOTE: Mention of commercial products and manufacturers does not imply endorsement by the
Office Of Water Programs, Environmental Protection Agency.
W. BA.mem. 811. 3. 74

Membran(' Filter Laboratory and Field Procedures
     ~"n,," TeMe   '1'--" 
     M-Endo L.E.S. Delayed Fecal Feckl Varltled
  item  Broth ('-"lifoI'm Colllol'ln O>ltl'orm StrC"To......e"8 T-
Half-roW1d glass paper weight. for X X X X X 
('oloay counling. \\ Ith lower half of a      
2- o. metal ointment box       
Hanrl tall:--, single. unit acceptable, X X X X X 
hand or df'sk typp       
Stereo.copk (dissection) ml('ro8cope,. X X X X X X
maplflca lion of lOX or l5X. prefer-      "
able binocular wide field type     
Bacteriological inoculating needle      X
Wire racks for culture tubee,      X
10 openings Ly five openlnge pre-      
fe,'rpu, dlmensloTUI overall approxl-      
mately 6" )( 12"       
Phenol Red Lactooe Broth in 16 X      X
150 mm fermentation tubes with      
tnctal caps, 10 m1 per tube       
~:o.1n M..thyl.ne Blue AgoI'       X
~ Levine) 111 petri plates, prepared      
ready tor u".-         
\lutrirnt "Rar R1ant8, in 8CT@W      X
cappf"d tubE's, 16 < 126 mm       
ffram stain ~oluUon., 4 solutions      X
pt'lr t (lmpli'f. ad       
\tl~T'jb(':Opt', compound, binocular.      X
,\ Ith oil immt'l"sion lens, miC'ro...      
51'Ore i Imp and 1mmerelon 011      
\11lro!';l'op~' ~lide., new, cl~an.      X
! . I . ,:" ~-,!., I'         
Water proof pl.lstic b"is     X  
rOt" 1'('1.:1; ("colif(IJ'm C\llture       
dish incubation       
~1-EAdo 111('(II1J:n. MF dehydrated X  X   
Jl1l"riiUrtl In 2!) . ~5 n.m fJat bottomed      
t'(..'IT\\-".,ppt',i glaS8 vials, I. 44 I      
pf'r tutl', ..uncl"nt for 30 ml of      
f-~t~nol. «150/0 in .mall bntt1t'8 or X X X   
H( ~- capped tube_, about 20 ml      
II~~ l' tuhe         
SoeJium t",nzoat,(' solution. 12%   X   
aqueous, tn.25 x 150 mm 8cr~w"      
"a.pped tubes, about 10 ml per tube      
L.E.S. ~.:ndo Agar MF, dehydratad  X    
11.1 - Endo medium, 0.36 1/ per 25 X      
~~ mm fJa l hottomed .crew- capped      
gla... vIal, plus 0.45 II agar, for 30 ml      
'",,'to.o Lauryl Sulfate Tryptose Broth  X    
in 2~ X 150 mm teat tube without      
lIIcluded gas tube, about 25 mi. for      
enr!rhment in I.. E. S. method     I
.s-y - 2

Membrane Filter Laboratory and Field Procedures
     Standard Te.t.  Nonatandard Teste 
     M. Endo L.E.S. DeJa)'ed Fecal Fecal VerUIed
Item    Broth CoUlorm CoUlDrm CoUlorm Str.otococCU8 Teet
Funnel unit a..embHe.    X X X X X 
JUne .tand, with about.. 3" .pHt rln&, to X X X X X 
.uppart the filtration fllMel        
Forceps, curved-.nd round tipped,  X X X X X 
.peclal t)'pe for MF work         
Methanol, In .mall wide-mouthed bottle. X X X X X 
about 20 ml for .terU1Zlna forcep. '
SuCtion flask., .18s., 1 Ht.r, mouth to X X X X X 
fit No.8 stopper         
Rubber tubing, 2 - 3 feet, to connect  X X X X X 
.uctlon fJask to vacuum .emc.., Jatn      
rubber 3/16" I. D, by 3/32" wan        
Pinch clamps strona .nou", for tl.1It X X X X X 
compression of rubber tubln. abOYe       
Plp.ttes, 10 ml, .raduated, Mohr !)'pe, X X X X X 
.terU., dlspen.e 10 per can per workin.      
.pace per day. (Re.terl1ize dalq to      
meet need),          
Pipettes, 1 ml, II'duated, Mohr t)'pe, X X X X X 
.terlle, dl.penee 2' per can per workin.      
.pac. pe I' day, (1\88terl11u dalq to      
meet need),          
Pipette boxes, .t.rUe, for 1 ml and X X X X X 
10 ml pipettes (sterUize above plpett..      
In thue boxes).          
Cylinders, 100 ml.raduated, .terU.., X X X X X 
(re.terIHze daily to meet need).        
Jan, to receive u.ed pipette.   X X X X X 
Gas burner, Bun.en or aimllar   X X X X X X
laboratory type          
Wax pencils, red, .uitable for wrltln. X X X X X 
on .Ja.s          
Spon.e In dilute Iodine, to wipe down the X X X X X 
desk tops          
Membrane filtera (whUe, .rld marloed, X X X X X 
.terUe,and sultabls pore size for       
microbiological analysis of water)       
Absorbent pads for nutrient, (47 mm In X X X X X 
diameter!, sterUe, In unit. of 10 pad.      
per package. Not required if medium      
contain. apr.  plastic,  X X X X X 
Petri dlsh~s, disposable,  
50 X 12 mm, sterile         
Waterbath Incubator U.5 : O. 20(;     X
v e.etable crlspsrs, or cake boxes,  X X X  X 
pJastlc, with tiiht fitting covere, for      
membrane fUter incubations        
Fluorncent 18mp, with extension cord X X X X X X
equipped with.. .lmple lens of about      
'X mallRlficat10n          
th lam. utilit t e X X X X X 
Rlne .tand, wi
p ,
$"'7". 3

Membrane Filter Laboratory and Field Procedure.
      Standard TestB  NonBtandB dTestB 
      M-Endo L.E.S. Delayed Fecal Fecal Verified
  Item  Broth ColUorm Colitorm  .8t.......tOCOCCIIB Test
M - FC Broth for fecal coliform,    X  
dehydrat..d medium in 25 <: 9S mm      
flat bottomE'd 8crew-capped ,1&8S      
vials. 1. 11 g per tube, sufficient      
for ~o ml of culture medium      
ROBolic acid, 1 '';'" solution, in    X  
O. 2N NaOn, in 25 X 150 mm {]at      
bottomed .screw-capped tubes,      
about 5 ml per tube, freshly      
M-F;ntE"'ocuccus Agar, dehydrated     X 
n1edimn in 2~ ,,( 150 mm screw.      
capped tubes, sufficient for 30 ml,      
1.26 g pt'r tube         
Dilution bottles, 6-oz, preferable X X X X X 
boro- aUlea Ie gla.B, with screw-      
l'ap (or ruhbe,' stopper protected      
by paper) , each containing 99 ml      
of sterUe phosphate huffered      
di.tilled )Vater         
Electric hot plate surfacE' X X X X X 
AeahrB, 400 - 600 ml (for water- X X X X X 
bath in pl'l'p~ration of membrane      
filter \ uit",." media)       
Crucihlc hmgs, to b~ u8ed at X X X X X 
~lt'l tde.; hot plate8, for removal      
of hol lu\,.." of culture media for      
boiling walerbath         

Membt'ane Filter Laboratory and Field Procedu~
Deliver enoullh culture medium to
saturate each absorbent pad. '" using
a 8terile pipette.
Exact quantities cannot be stated
because pad a and culture containers vary.
SUfficient medium should be applied so
that when the culture container is tipped,
a rood-sized drop of culture medium
freely drains a1 t of the absorbent pad.
7 Oraanize I:fupplies and equipment for
convenient sample filtration. In
training courses, laboratory instructors
will suggest useful arrangements;
eventually the individual will select a
sy.tem of bench-top organization most
suited to his own needs. The important
point in any arrangement is to have all
needed equipment and supplies con-
veniently at hand, in such a pattern as
to minimi ze lost time in useless motions.
8 Lay a sterile membrane filter on the
filter holder, grid-side up, centered
over the porous part of the filter
support plate.
Membrane filters are extremely
delicate and easily damaged. For
manipulation, the sterile forceps
should always grasp the ~ part
of the filter disk, outside the part
of the filter through which the sample
9 Attach the funnel element to the base
of the filtration unit.
To aboid damage to the membrane
filter, loc'king forces should only be
applied at the locking arrangement.
The funnel element never should be
turned or twisted wtiile'being seated
and locked to the lower element of the
filter holding unit. Filter holding units
featuring a bayonet joint and locking
ring to join the upper element to the
lower element require special care on
the part of the operator. The locking
ring should be turned sufficiently to
give a snug fit, but should not be
tightened excessively.
10 Shake the sample thoroughly.
11 Measure sample into the funnel with
vacuum turned off.
The primary objectives here are:
1) accurate measurement of sample;
and 2) optimum distribution of colonie!>
on the filter after incubation. To
meet these objectives, methods of
measurement and dispensation to the
filtration assembly are varied with
different sample filtration volumes.
a With samples greater than 20 ml,
measure the sample with a sterile
graduated cylinder and pour it into
the funnel. It is important to rinse
this graduate with sterile buffered
distilled water to preclude the loss
of excessive sample volume. This
should be poured into the funnel.
b With samples of 10 ml to 20 mI.
measure the sample with a sterile
10 ml or 20 ml pipette. and pipette
on a dry membrane in the filtration
c With samples of 2 ml to 10 ml, pour
about 20 ml of sterile dilution water
into the fi Uration assembly. then
measure the sample into the sterile
buffered dilution water with a 10 ml
sterile pipette.
d With sample,; of O. & to 2 mI. pour
about 20 ml of sterile dilution water
into the funnel assembly, then
measure the sample into the sterile
dilution water in the funnel with a
1 ml or a 2 ml pipette.
e If a sample of less than O. & ml is to
be filtered, prepare appropriate
dilutions in sterile dilution water,
and proceed as applicable in item c
or d above.
When dilutior.s of samples are needed,
always make the filtrations as soon
as possible after dilution of the
sample; this never should exceed
NOTE: Mention of commercial products and manufacturers does not imply endorsement
by the Office of Water Programs. Environmental Protection Agency.

Membrane Filter Laboratory and Field Procedures
30 minutes. Always shake 8ample
tiilutions thoroughly before delivering
measured volumes.
12 Turn on the vacuum.
Open the appropriate 8pring clamp or
valve, and filter the sample.
After sample filtration a few droplets
of sample usually remain adhered to
the funnel walls. Unless these droplets
are removed. the bacteria contained in
them will be a 80urce of contamination
of later samples. (In laboratory
pr~ctice the funnel unit is not routinely
sterilized between successive filtrations
of a series). The purpose of the funnel
rinse is to fiush all droplets of a sample
from the fUJU1el walls to the membrane
filter. Extensive tests have 8hown that
with proper rinsing technique, bacterial
retention on the funnel walls is negligible.
13 Rinse the sample throulth the filter.

After all the sample has passed through
the membrane filter. rinse down the
.des of the fuMel walls with at least
20 ml of sterile dilution water. Repeat
the rinse twicl' after all the first rinse
has paued through the filter. Cut off
suction on the filtration a8sembly.
14 Remove the fUMel element of the filter
ho}dina unit.

It' a ring stand with spUt ring is u8ed,
hang the fuMel element on the ring;
otherwise. place the inverted funnel
element on the inner surface of the
wrapping material. This requires
care in open1ng the sterilized package,
but it i8 effective as a protection of the
funnel ring from contamination.
15 :!.,\ke the membrane £il1,:.r from the
tTIi er holder and carefu~ place it.
Ilrid-8ide \Q) on the medium.

Check that no air bubbles have been
trapped between the membrane lilter
and the under¥ni ab80rbent pad or
agar. Relay the membrane if necessary.
16 Pace in incubator alter ftni8hin,
~on series.

Invert the containers. The immedia~e
atmosphere 01 the irlcubat1ng membrane
tUter must be at or very near 1000/.
relative humidity.
Count colonies which have appeared
after 1ncubatinll tor the pre8cribed

A stereoscopic micro8cope magnifying
10-15 time8 and careful illumination
give best counts.
For reporting results. the computation
bacteria/100 ml .
:"0. C:Olonte8 c0;futed ~ 100
mple va urne ltere in ml
Example :
A total of 38 colonies grew after
filtering a 10 ml sample. The
number reported is:
36 colonie8
10 ml X 100 . 360 per 100 ml

Membra~e Fllter Laboratory and Field Procedures
A Standard Coliform Test (Based on M-Endo
Broth MF)
1 Culture medium
a, M-Endo Broth MF Dileo 0749-02
or the equivalent BBL M-Coliform
Broth 01-494
Preparation of Culture Medium
(M-Endo Broth) for Standard MF
Coliform Test
Yeast extract
Casitone or equivalent
Thiopeptone or equivalent
Sodium desoxycholate
Dipotassium phosphate
Monopotassium phosphate
Sodium chloride
Sodium lauryl sulfate
Basic fuchsin (bacteriological)
Sodium sulfite

Distilled water (containing
20,0 m! E'thanol)
1. 5 g
5.0 g
5.0 g
10.0 g
12.5 g
0.1 g
4.375 g
1.375 g
5.0 g
0.05 g
1.05 g
2.1 g

1000 ml
This medium is available in
dehydrated form and it is rec-
ommended that the commercially
available medium be used in
preference to compounding the
medium of its individual constituents.
To prepare the medium for use,
suspend the dehydrated medium at
the rate of 48 grams per liter of
water containing ethyl alcohol at
the rate of 20 ml per liter.
As a time-saving convenience, it is
recommended ~hat the laboratory
worker preweigh the dehydrated
medium in closed tubes for several
days, or even weeks, at one operation.
With this system, a large number
of increments of dehydrated mediun
(e. g., 1,44 grams), sufficient for
some convenient (e. g.. 30 ml)
volume of finished culture medium
are weighed and dispensed into
screw-capped culture tubes, and
stored until needed. Storage should
preferably be in a darkened disiccator.
A supply of distilled water containing
20 ml stock ethanol per liter is
When the medium is to be used, it
is reconstituted by adding 30 ml of
the distilled water-ethanol mixture
per tube of pre-weighed dehydrated
culture medium.
Medium is "sterilized" as directed
in I, C.
Finished medium can be retained
up to 96 hours if kept in a cool,
dark place. Many workers prefer
to reconstitute fresh medium daily.
2 Filtration and incubation procedures
are as given in I, D.
Special instructions:

a For counting, use the wide field
binocular dissecting microscope, or
simple lens. For illumination, use
a I1ght source perpendicular to the
plane of the membrane filter. A
small fluorescent lamp is ideal for
the purpose.
Coliform colonies have a "metallic"
surface sheen under reflected light
which may cover the entire colony, or
it may appear only in the center. Non.
coliform colonies range from
colorless to pink, but do not have
the characteristic sheen.
Record the colony counts on the
data sheet, and compute the coliform
count per 100 ml of sample.

Membrane Filter Laboratory and Field Procedures
B Standard Coliform Tests (Based on L. E. S.
Endo Agar.)
The dillt inction of the L. E. S. count is a
two hour enrichment incubation on LST
broth. M-Endo L.E.S. medmm is used
as agar rather than the broth.
1 Preparation of culture medium
(L.E.S. Endo Agar) for L.E.S.
coliform test
a Formula from McCarthy, Delaney,
and Grasso (2)
Bacto. Yeast Extract
Bacto- Caeitone
Bacto- Thiopeptone
Bacto- Tryptose
Bacto- Lactolle
Dipotassium phosphate
MonopotBssium phosphate
Sodium chloride
Sodium desoxycholate
Sodium lauryl sulfate
Sodium sulfite
Bacto-Basic fuchsin
1.2 g
3.7 g
3.7 g
7.5 i
9.4 i
3.3 i
1.0 i
3.7 g
0.1 g
0.05 g
1.6 g
0.8 g
15 g
DIstilled water (containing
20 m1 ethyl alcohol)
1000 ml
b To rehydrate the medium, SUI pend
51 grams in the water-ethyl alcohol
c Medium is "sterilized" &s directed
in I, C.
d Pour 4-6 m1 of freshly prepared Agar
into the smaller half of the container.
Allow the medium ~o cool and s.olidify.
2 Procedures for filtration and incubation

a Layout the culture dishes in a row
or series of rows al U8ual. Place
these with the upper (lid) or top
side down.
b Place one sterile absorbent pad in
the larger half of each container
(lid). Use sterile forceps for aU
manipulations of the pads.
(Agar occupies ,smaller half or
c Using a sterile pipette, deliver
enough Bingle strength lauryl
sulfate tryptoBe broth to saturate
the pad only. Excels interferes.
d Follow leneral procedures for
filtering in I, D. P1ace filters on
pad with lauryl sulfate tryptose
e Upon completion of the filtrations,
invert the culture containers and
incubate at 3S0 C for 1 1/2 to 2
3 2-hour procedures
a Transfer the membrane filter from
the enrichment pad in the upper half
to the ap.r medium in the lower
ha,lf of the container. Carefully
roll the membrane onto the agar
surface to avoid trapping air
bubbles beneath the membrane.
b Removal of the used absorbent pad
i8 optional.
c The container is inverted and
incubated 22 hours': 2 hours: O. soC.
4 Counting procedur.e8 are as in I, D.
S L. E. S. Endo Apr may be 'used as a
single-lRage medium (no enrichment
step) in the,eame roaMer a8 M-Endo
Broth, MF.
C Delayed Incubation Coliform Test
This technique is applicable in situations
where there is an excessive delay between
sample collection and plating. The procedure
i8 unnecessary when the interval be-
tween samp1e collectlcn and plating is
within acceptable limits.
1 Preparation of C'oJt.".,~ media for
delayed incubation coliform test
a Preservative r:lpr'ia M- Endo Broth

Membrane Filter Laboratorv and Field Procedl.P'~
To 30 ml of M-Endo Broth MF
prepared in accordance with
directions in n, A, 1 of thia
outline, add 1. 0 ml of a lIterile
120/0 aqueous solution of sodium
L. E. S. MF Holding Medium-
CoUform: Dissolve 12. 7 grams in
1 UteI' of distiUed water. No
heating is necessB.l1'. Final pH
7.1 + 0.1. This medium contains
sodium benzoate.
b Growth med1a
M-Endo Broth MF is used, prepared
as described in n, A, 1 earlier in
this outline. Alternately. L. E. S.
Endo Medium may be used.
2 General filtration followed is in I, D.
Special procedures are:
a Transfer the membrane filter from
the filtration apparatus to a pad
saturated with benzoated M-Endo
b Close the culture dishes and hold
in a container at ambient temperature.
This may be mailed or transported
to a central laboratory . The mailing
or transporting tube should contain
accurate transmittal data sheets which
correspond to properly labeled dishes.
Transportation time, in the case of
mailed containers, should not exceed
three days to the time of reception
by the testing laboratory.
c On receipt in the central laboratory,
unpack mailing carton, and layout
the culture containers on the labora-
tory bench.
d Remove the tops from the oulture
containers. Using sterile forceps,
remove each membrane and its
absorbent pad to the other half of
the culture container.
e With a sterile pipette or sterile
absorbent pad, remove preservative
medium from the c~ure container.
f Place a sterile absorbent pad in
each culture container, and deliver
enough freshly prepared M-Endo
Broth to saturate each pad.
g UII1ng sterile forceps, transfer the
membrane to the new absorbent pad
containing M-Endo Broth. Place
the membrane carefully to avoid
entrapment of air between the
membrane and the \U1derlying
absorbent pad. Discard the
absorbent pad containing pre-
servative medium.
h After incubation of 20 + 2 hours
at 350 C, count colonies as in the
above section A, 2.
i If L. E. S. Endo Agar i8 used. the
steps beginning with (e) above are
omitted; and the membrane filter is
removed from the preservative
medium and transferred to a fresh
culture container with L. E. S. Endo
Agar, Incubated, and colonies
counted in the usual way.
D Verified Membrane Filter Coliform Test
This procedure applieo to identification
of colonies growing on Endo-type media
used for determination of total coliform
CO\U1ts. Isolates from these colonies are
studied for gas production trom lactose
and typical coliform morphology. In
effect, the procedure corresponds with
the Completed Test stage of the multiple
fermentation tube test for coliforms.
1 Select a membrane filter bearing
several well-isolated coliform-type
2 Using sterile technique, pick all
colonies in a selected area with the
inoculation needle. making transfers
into tubes of phenol red lactose broth
(or lauryl sulfate tryptose lactose

Membrane Filter Laboratory and Field Procedures
broth). Using an appropriate data
sheet record the interpretation of
each colony, using, for instance,
"c" for colonies having the typical
color and sheen of colUorms; "NC"
for colonies not conforming to
colUurm colony appearance on
Endotype media.
3 Incubate the broth tubes at 350 C!: 0.5 C.
4 At 24 hours:
a Read and record the rellu1ts from
the lactose broth fermentation tubes.
The following code ill sug,ested:
No indication of acid or gas
production, either with or
without evidence of growth.

Evidence of acid but not gas
(applies on~ when a pH indicator
is included in the broth medium)

Growth with production of gas.
If pH indicator is used, USe
symbol AG to show evidence of
acid. Gas in any quantity is a
positive test.
b Tubes not showing gas production are
returned to the 350 C incubator.
c Gas-positive tubes are transferred
as follows;
1) Prepare a streak inocu]ation on
EMB agar for colony. isolation, and
using the same culture.
2) Inoculate a nutrient agar slant.
3) Incubate the EMB agar plates and
slants at 350 C !: o. SoC.
5 At 48 hours:
a Read and record results of ]actose
broth tubes which were negative at
24 hours and were returned for
further incubation.
b Gas-positive- cultures are subjected
to further transfers as. in 4c.
Gas-neptive cultul'es are discarded
without further study; they are
coU1orm- neptive.
c Examine the cultures transferred
to EMB agar plates and to nutrient
apr slants, as fol]ows:
1) Exam1De the EMB ap.r plate for
evidence of purity of culture; if
the culture represents more than
one colony type, discard the
nutrient agar culture and reisolate
each of the representative colonial
types on the EMB plate and resume
as with 4c for each isolation.
If purity of culture appears evident,
continue with c (2) below.
2) Prepare a smear and Gram stain
from each nutrient agar slant
culture. The Gram stam should
be made on a culture not more
than 24 hours o~d. Examine under
oll immersion for typical coliform
morphololY, and record reBults.
6 At 73 hours:
Perform procedures described in 5c
above, and record results.
7 Coliform colonies are considered
verified if the procedures demonstrate
a pure culture of bacteria which,are
gram negative nonspore-forming rods
and produce. gas from lar;:tose at 350 C
within 48 hours..
E Fecal Coliform Count (Based on M-FC
Broth Base)
The count depends upon growth on a
special medium at 44.5 :!' O.2oC.

I Preparation of Culture Medium
(M-FC Broth BI;'e' for Fecal
Coliform Count

Membrane Filter J ,aboratorv and Field Procedures
a Composition
Proteose Peptone No.3
Yeast extract
Sodium chloride
811e salts No.3
Rosolic acid* (Allied
Aniline blue (Allied Chemical)
3.0 g
5.0 g
12.5 g
1.5 g
10.0 ml
0.1 g
Distilled water
1000 m1
b To prepare the medium dissolve
37. 1 grams in a liter of distilled
water which contains 10 m1 of 10/0
rosolic acid (prepared in 0.2 N
Fresh solutions of rosolic acid give
best results. Discard solutions
which have changed from dark red
to orange.
c To sterilize, heat to boiling as
directed in I, C.
d Prepared medium may be retained
up to 4 days in the dark at 2- 80 C.
2 Special supplies
Small water proof p]astic sacks capable
of being sealed against water with
capacity of 3 to 6 culture containers.
3 Filtration procedures are as given in
I, D.
4 Elevated temperature incubation
a Place fecal coliform count mem-
branes at 44.5 :- O. 20C as rapidly
as possible.
*Prepare 1% solution of rosolic acid in 0.2 N Na0H.
Filter me.mbranes for fecal coliform
counts consecutively and immediately
place them in their culture containers.
Insert as many as six c:ulture containers
all oriented in the same way (1. e., all
grid sides facing the same direction)
into the sacks and seal. Tear off the

perforated top, grasp the side wires,
and twirl the sack to roll the open end
inside the folds of sack. Then submerj;\t:
the sacks with culture containers in-
verteg beneath the surface of a 44.5
.:: 0.2 C waterbath.
b Incubate for 22 :- 2 hours.
5 Counting procedures
Examine and count colonies as follows:
a Use a wide field binocular dissecting
microscope with 5 - lOX magnification.
b Low angle lighting from the side is
c Fecal coliform colonies are blue,
generally 1-3 mm in diameter.
d Record the colony counts on the
data sheet, and report the fecal
coliform count per 100 ml of sample.
(I, D, 17 illustrates method)
A 48 hour incubation period on a choice of
two different media. giving high selectivity
for fecal streptococci, are the distinctive
features of the tests.
This dye is practically insoluble in water
54- 11

Membrane Filter Laboratory and Field Procedures'
A Test for Members of Fecal Streptococcal
(Tentative, Standard Method8) M-
Enterococcus Ap.r Medium
I Preparation of the culture medium
a Formula (The DUco formula is shown,
but equivalent con8tituents from
other sources are equaUy acceptable).
Bactcl' yeast extract
Bacto dextrose
Dipotkssium phosphate
Sodium Azide
Bacto agar
2, 3, 5, Triphenyl
tetrazol1um chloride
20.0 g
5.0 g
2.0 g
4.0 g
0.4 g
10.0 g
0.1 g
b The medium is prepared by
rehydration at the rate of 42 grams
per 1000 ml of distilled water. It
is recommended that the medium in
dehydrated form be preweighed and
dispensed into culture container8
(about 25 X 150 mm) in quantities
sufficient for preparation of 30 m1
of culture medium (1.26 g per tube).
c Follow I, C, for "sterUizing" medium
and dispense whlle hot into culture
containers. Allow plates to harden
before use.
2 List of apparatus, materials, a8 given
in Table 1.
3 Procedure; in general, as given in I.
SpeCial iristru ctions
a Inc!ubale for 48 hours, inverted,
with 1000/. relative humidity, after
filtrations are completed. If the
eritire incubator does not have
saturated humidity, acceptable
conditions can be secured by p1acing
the cultUres in a tightJ;y closed
container with wet paper, towels,
or other moist material.
b After incubation; remove the
cultures from the incuba.tor, and
count all colonies under wide field
binocular dissecting microscope
with magnification set at lOX or
2OX. Fecal streptococcus colonies
are 0.5 - 2 mm in diameter, and
flat to raised smooth, and vary
from pale pink to dark red in color.
Report enterococcus count per
100 ml of sample. This is con-
venientJ;y computed:
No. fecal streptococci per 100 ml =
No. fecal streJ!!2s2.ccus colonies counted X 100
Sample filtration volume in ml
B Test for Members of Fecal Streptococcal
Group based on KF-Agar
1 Preparation of the culture medium
a Formula: (The dehydrated formula
of Bacto 0496 is shown, but
equivalent constituents from other
sources are acceptable). Formula
is in grams per Uter of reconstituted
Bado proteose peptone *3 10.0 g
Bac:to yeast extract 10.0 g
Sodium chloride (reagent grade) 5. a g
Sodium glycerphosphate 10. a g
Maltose (CP) 20. a g
Lactose (CP) 1.0 g
Sodium azide (Eastman) 0.4 g
Sodium carbonate 0.636 g
(Na2C03 reagent grade)
Brom cresol purple
(waier soluble)
Bacto agar
0.015 g
b Reagent
2, 3, 5-Triphenyl tetra.zolium
chloride reagent iTPTC)
This reagent. '" prepared by making
a 10/. aqueous ",'L'ion of the above
chemical passing it through a Seitz
filter or membrane filter. It can

Membrane Filter Laboratorv and Field PrDcedures
be kept in the refrigerator in a
screw- capped tube W1tU used.
c The dehydrated medium described
above is prepared for laboratory
use as follows:
Suspend 7. 64 grams of the dehydrated
medium in 100 ml of dist1lled water
in a f1ask with an aluminum foil
P1ace the flask in a boiling water-
bath, melt the dehydrated medium,
and leave in the boiling waterbath
an addiona.l 5 minutes.
Cool th.. medium to 500 - 6 00 C, add
1. 0 ml of the TPTC reagent, and
For mt'mbrane filter studies, pour
5-8 ml in each 50 mm glass or
plastic culture dish or enough to
mak" a layer ,appruximately 1/8"
thick. Re sure to pour plates before
agar cools and solidifies.
For plate counts, pour as for standard
agar plate counts.
NOTE: P1al3tic dishes containing
media may be stored in a dark, cool
place up to, 30 days without change
in producUvity of the medium, pro-
vided that no dehydration occurs.
P1astic dl.shes may be incubated in
an ordina'ry air incubator. Glass
dishes must be incubated in an
atmosph,~re with saturated humidity.
2 Apparatus, and materials as given in
Table 1.
3 General procedure is as given in I.
Spec,\.al instructions
a J,ncubate 4'8 hours, inverted with
100'70 relative humidity after
b After incubation, remove the
cultures from the incubator, and
count colon1es o.Illder wide field
binocular dissecting microscope,
with magnification set at lOX or
2OX. Fecal streptococcus colonies
are pale pink to dark wine- color.
In sile they range from barely
visible to approximately 2mm in
diameter. Colorless colonies are
not counted.
c Report fecal streptococcus count
per 100 ml of sample. This is
computed as follows:
No. fecal streptococci per 100 rol
No. fecal streptococcus ;;olon1es ;.; 100
Sample filtration volume in ml
C Verification of Streptococcus Colonies
1 Verification of .:oolony identification
may be required in waters containing
large numbers of Micrococclls orga-
nisms. This has been noted
particularly with bathing waters, but
the problem is by no means limited to
such waters.
2 A verification procedure is described
in "Standard Methods for the Examination
of Water and Wastewater;' 13th ed.
(1971), The worker should ust:
this reference for the step-by-
step procedure.
A Culture Media
1 The standard coliform media used with
laboratory tests are used.
2 To simplify field operations, it is
suggested that the medium Le sent to
the field, preweighed, in vials or
capped culture tubes. The medium
then requires only the addition of a
suitable volume of distilled water-
ethanol prior to sterilization.
54 - 13

Membrane Filter Laboraton and Field Proc.edur..
3 Ster1l1zation procedures in the field
are the _me a8 for laboratory methods.

4 LIfboratory preparation of the media,
reltdy for use, would be permi8sthle
prb'Vlded that the required limitation8
on time &iid conditione of stora,e are
B Oper~tion of the Sabro Field Unit
1 Equipment and materials
Sabro field unit
Membrane filters
Absorbent pads for nutrient
Culture containers
M-Endo Broth MF or L.E.S. Endo
Provision for heating water (optional)
Source of electricity
a Procedure (Based on M-Endo Broth MF)

a Connect the electric cord to the
power source and to Sabro field
unit. After about 15 minutes check
the teMperature in the incubator
drawer. The required temperature
Is 350 C (950 F). If the temperature
is too low It can be increased by
turning the thermostat adjustment
screw counterclockwise. This
screw is located at the front on the
recessled divider between the two
incub.:tor drawers. To lower
temperature, turn the adjustment
!lcrew clockwise.
b Review the suppq of expendable
materials to be ulled with the unit
and secure replacements as needed
(culture containers, medlwn,
rtiembrane filters, absorbent pads
for l\Utrlent, fuel, etc.).
c Sterilize the funnel unit by one of
the folloWing procedures.

1) lmmerlre the equipment 2 minutes
In boiling water. The temperature
should be at least 780C (l700F).
2) Flame-sterWae membrane filter
holder inside and both ends of
funnel (suggested by manufacturer).
d lay in a row all the culture con-
tainers to be used in the filtration
s'erle's, and number the containers
to correspond with numbers of the
data sheet.
e Place one sterile absorbent pad in
each culture container. Use sterile
forceps for all manipulation of
absorbent pads and filtera.
f Uaing sterile pipette deliver enough
culture mediwn to saturate each
absorbent pad. The amount of
culture mediwn required Is approXi-
mate~ 2 ml, but cannot be precisely
stated. Sufficient medium should be
applied, that when the culture con-
tainer Is tipped, a good-sized drop
of culture medium free~ drains out
of the absorbent pad.

I Ueing sterile forceps, place a mem-
brane filter. grid-side up, on the
MF receptacle of the funnel unit.
P1ace the funnel port,ion over the
membrane, and eJamp the unit with
the IIPring cJamp prO'Vided With the
portable kit.
h Pour the water SlUn\>le into the funnel
using a sterile pipette or graduate.
COru1ect the tubing of the vacuum
pump to the receptable on the base
of the fUterunit and 
Membrane Filter lAboratory and Field Procedures
filter. rinse down the sides of the funnel
walls with at least 20 ml of sterile dilu-
tion water. Repeat the rinse twice after
all the first rinse bas passed through
the filter.
Disassemble the funnel unit and with
sterile forceps transfer the membrane
filter grid-side up to the appropriate
culture container. The membrane
should be "rolled on" the absorbent
pad containing culture medium, to
prevent entrapment of air between
the pad and the membrane filter.
k Repeat steps g . j for additional
filtrations of the same or different
sampling volumes for the water
being tested.
I After completion of filtration, place
the culture container in an inverted
position (with membrane position
grid-side down) in the incubator

m After completion of the last filtration
from anyone sample, restf'rilize the
funnel unit by one of the procedures
descrtbed in instruction 2c.
n Allow the cultures to incubate
20 - 24 hours.
o Remove the cultures from the
incubator and count coliform colonies.
C Operation of Ml1lipore Water Testing Kit,
1 Supporting supplies and equipment are
the same as for the laboratory
2 Set the incubator voltage selector
switch to the voltage of the available
supply, turn on the unit and adjust as
necessary to establish operating
incubator temperature at 35:t 0.50C.
3 Sterilize the funnel unit assembly by
exposure to formaldehyde or by
immersion in bo1ling water. If a
laboratory autoclave is available, this
is preferred.
Formaldehyde is produced by soakin:;,
an asbestos ring (in the funnel base)
with meUaDcN. ig'1itin~, and after a
Cew seconds of burnin" closing the
unit by placing the stainless steel
flask over the funnel and base. This
results in incomplete combustion of
the methanol, whereby formaldehyde
ill produced. Leave the unit closed
for 16 minutes to allow adequate
exposure to formaldel:\)'de.
4 Filtration and incubation procedures
correspond with laboratory methods.
5 The unit is supplied with a booklet
containing detailed step-by. step
operational procedures. The worker
using the equipment should become
completely versed in its contents and
D Counting of Colonies on Membrane Filters
I Equipment and materials
Membrane filter cultures to be
Illumination source
Simple lens, 2X to 6X magnification
Hand tally (optional)
2 Procedure
a Remove the cultures from the
incubator and arrange them in
numerical sequence.
b Set up illumination source as that
light will originate from an area
perpendicular to the plane of
membrane filters being examined.
A small fluorescent lamp is ideal
for the purpose. It is highly
desirable that a simple lens be
attached to the light source.
c Examine results. Count all colliorm
and noncoliform colonies. Coliform
colonies have a "metallic" surface
sheen under reflected light, which
~over the entire colony or may
appear only on the center.

Me...-- alte.. ~rUor1 and new PI'ocedure.
NoncolUorm colonSe. ru,e from
cCJ1arJ88e to plftk or r.d. but do not
. have tbe char.oteri8tic "..eta~
d hwr..~c~.m~e~~
e _tel' ~e coliform count per 100 ml
ofaanple for each membrue bavinl
. COUI1table number OIl coUform
coJonie.. Computation 18 .. fOUoW8:

No. coUtoI'm per 100 ml .
1 Standard Method. for the 'Ex.mination of
W.ter and Wa.tewater. APHA.
AWWA. WPCF. 13th Edition. 1965.
McCartbf, J.A.. De~. J,E. and
G.....o. &.J. ~m, Colltorm.
ta ."1'. Water 8ad ....... W~k8.
1'81: &.428-31. 1881.
Thi. outUM ".. prepared"" H. L. Jeter,
Director. lfaUanal '1'niaI8I c.t.r,
EPA, WPO, CiDoiMaU, OR .'268.
~: B101o1ic:&l Membr8De8,
~"'C81 Co1ItcmDa, I'8cal
Streptooood, 1'U88r.. 1-,,,,,,,, "cteria,
Laborato17 Bq1dpID8llt. x.IIorll8or)' Teat.,
Membrua.. MicraIdoIiDl)'. WIHr ~a18

A Oxygen demand ~lys.. are basic water
pollution control tool.. Treatment plant
des1p i8 largely based on empirical
evidence expreased in terms at S-day
biochemical oxy,en demand (BOD). Fint-
at... BOD, sec:;ond-st..,e BOD, 20-day
BOD, and ultimate BOD are familiar
terme t~ ~xygen resource ana1ysts.
Modern research in treatment technology
employs more exotic analyses such as
chemical oxygen demand (COD) and various
methods of estimating theoretical oxygen
demand (TOD), It is perhaps fair to say
that more pollution control decisions are
baaed upon oKY,en demand data than upon
other analytical re8ults.

B Vet 'Interpretation of these data often seems
an Insurmountably difficult task. There
should be no mystery about such Inter-
pretation; oxygen demand analyses are
powerful tools which must be used with
informed 8kUl, Those problems which
arise are more related to complexity of the
environmental situation than to inherent
analytical errors.
A Positive oxyg.n demand results imply one
or more of the fonowing:
1 The presence of . compound which wUl
exert an oxygen demand in the natural
2 The presence of organic matter
B These statements are not necessartly both
true at the same time, i. e. . there are
compounds (e". NH3)whlch will exert
oxygen demand In nature but are not
chemically orpmc. Likewise there are
organic compounds which, under favorable
laboratory or environmental conditione,
will not exert a measurable oxygen demand.
C Nor i8 the converse of either statement
nece88ari~ true. The presence of a
suitable organic or Inorganic compound
doee not guarantee a positive oxygen
demand, eepec!ally under laboratory
D It Is. perhaps, better to. say that positive
oxygen demand values indicate the presence
of.. compound which may potentially
exert a natural 02 demand or may
potentially be orpmc in nature.
Generally one or both implications proves
true, but the except~ns are often crucial.
Oxygen demand terminology tends to present
a communications barrier. To a chemist
COD may represent the result of a pre-
scribed analytical procedure; to an engineer
it may represent a rather abstract cOl/.cept
of the amount of oxygen utilized in a purely
chemical reaction. Of course this sort of
confUct can never be decided; it is analagous
to the philosophical trap ot deciding whether
there is noise when a tree falls in the forest
in the absence of an observer. The practical
solution is that each practitioner develof his
knowledge of COD, BOD, etc. to include
several concepts. Durillf discussion or
writing. the sense in which an oxygen
demand term is used must be clearly defined.
of a waste is the aIrount of oxygen required
for oxidation of the component8 of that
waste to their highest attainable oxidation
states. Theoretical mr:rgen demand
would be attained only by perfect com-
bustion at elevated temperature in the
presence of excess oXYlen.
For example, if a waste were composed
of C+4. N02 -. ar.d S. the theoretical
oxygen demand of that waste would he the

O~'y.'1) Dezpand A~'eB
amount 9f oxy,en required to convert these
components to C02' NOg -, and SO 4. .

TOD repreaents the standard to which all
other oxy,en demand method. may be
conaparlid. It i. a unique, ab.tract value
whkh hi.ll never been actually meaallred.
defined.. the amount of oxy,en reqllired
for bioc:.hemical oxidation of or,anic and/or
inorpnic matter. The ULTIMATE
biochemical oxy..n demand would be exerted
U the bl.ochemical reaction. were allowed to
proFess to completion. the ULTIMATE
FIRST STAGE demand would be exerted
upon completion of oxidation 01 carbonaceous
compoupd.; 5-day biochemical ox"en
demand is the amount of o.,..n utilized
dllring Uve day. of biochemical oxidation.
Depending upon sample histor, theBe five
days may repre.ent any 5-d., .e,ment of
the BOD curve. The relation.hlp of the
various c1aues of BOD i. .hown in Firure 1.
Tbe sel;,ond step of the BOD cllrve may be
call.eel.by nitrUication and/or low-rate
oxidation of re.i.tant compoundl. The
.hape Pfthe.c,llrve is not a valid indicator
of the t.,ype of oxidation occllrring.
STAqt BOD are unique, ab.tract valueB
which can never be reached. If one pre-
suppo~es an infinite variety of organism.,
infinite biolo,ieji1 adapt&bU1ty, and infinite
reactiljln time. ULTIMATE BOD mll.t equal
4-D - 2
oxygen eqllivalent of the amount of active
a,ent consllmed during oxidation of .

;~~t~~~c=~~.~~~~a:; ~~~~~

compollnd. the rea,ents, and the proced-
ures emPI~d dllriDf analysis. Standard
MethodlilpecUI.. a dichromate oxid-
ation in 5~ 'IittW'l.e acid at approximately
145°C; an older method - called the OXYGEN
CONSUMED t8St - employ. potan:htm
permanpnate as an oxidinng arent. If
performed on the .ame 1I'&ete, th. COD
and OXYGEN CONSUMED analysu would
produee' ditterent ftumeri~al r8.1I1te.

oxygen de.and exerted lIpontaneou81y upon
contact of a waste with oxygen. It may
include oxypn utilized in reaction with
ferrous iron. sulfite., sulfides, aulfhydryl
FOUp8, or other reducing agents which
will react within 15 minate. of contact.
A Dissolved O.,.,.n (DO) Determinations

Quantitative measurements of d18801ved
oXYfen level. are made by chemical or
electrometric methods. Various ionll and
compound. interfere with the chemical
determination of DO; when u.lo, chemical
methods. one .hould be alert to their
preeence. The Azide Mfdification of the
basic Winkler' Metho1 i. the chemical
procedure most commonly used; other
modUications have been developed. to
correct for ..."tOIi. interference..
Electrometric methods have recently
become more popular; such method. may
produce a continuous oxygen record, are
easily adapted to remote telemetr:r. and
tend to overcome certain Interf..-ces with
the Winkler method.
Dis.olved oxy,en data may:

1 Serve a. an indirect me8Sl1r8ment of
photoayDtftet1c aetivtty;
2 1ndi~t1y indicate the progr ... of
biological stabl1ization;

3 Form the chemical bases f06' tbe BOD
and IOD t.st..

B Biochemical Oxygen Demand Test (BOD)

I Indicates oxygen demand potential of a
wute when exposed to a biological
2 Indirectly used as a measure of avail-
able material (specific analyses for a
myriad of organic compounds found in
domestic and industrial wastes would
be time-conluming and pointless for
most cases).
3 Gives an idea of the rate at which bio-
detradable organic material 11 being
4 Long-term BOD is invariably lees than
the theoretical oxygen demand because:

a Chemical compoundl differ with
respect to ava1lablUty as a biological
b Biological action tends to convert
nutrients to other lubstances of
decreasinr ava1lablUty.
c Adapted or,anisms may not be
d The environment is not always
tavorable for optimum biological
5 At the diAcretion of the analyat reported
BOD test results mayor may not include
immediate oxygen demand (IOD). When
relults are reported or analyzed,
inclusion or non-inclusion Ihould be

C Chemical Oxygen Demand Test (COD)

I COD indicates the presence of organic
and/or inorganic material sUlcepUble
to chemical oxidation. Thil material
may also be biologically oxidizable.

2 For certain wastes, a definite BOD! COD
relationship may exist; this is true with
domestic sewage, for which COD ~
approximate ultimate first-Itage .
OXYllen Demand Analyse' --
a High COD/ BOD ratio on an organic
waste which, from all known char-
acterisUcs should be biodegradable,
may indicate the presence of toxic
agent I in the waste.
b A high COD/BOD ratio may also
indicate that the waste is resistant
to biodegradati:)n.

S Although COD usually exceeds BOD, the
reverse may be true with certain wastes,
notably cellulose. In such cases, COD
is not a reUable mealure of deoxygen-
ation potential. Ideally, neither COD
nor BOD should be ulled as the sale
mealure of deoxygenation potential.
Oxygen demand analyses are key pollution
control tools. Poeitive relults indicate
~otential oxygen demanc! in nature and/ or
lie ~al presence of organic matter.
GeniriIIY1J1e implications are true, but
there may be important exceptions. Valid
ulle of euch data requires careful and
knowledgeable interpretatio:'!.
1 Standard Methodl for the Examination of
Water and Wastewater, AWWA, APHA,
WPCF, 12th Edition. (1965)
2 Sawyer, C. N. Chemiatry for Sanitary
Enpneers, McGrkw-HiU, New York.
Thil outline was pr.epared by F. P. Nixon,
former Acting Regional Training Officer,
Northeaat Regional Training Center, OWP,
Hudlon-Delaware Basins Office, Edison,
NJ 08817.
Delcriptors: Biochemical Oxygen Demand,
Chemical Oxygen Demand, Dissolved Oxygen,
Oxygen Demand

A The common equation Yt - L (1- 10.kt) for
BOD relationship indicates time as a
variable. The rate coefficient (kl)1ndicates
that a specific percentage of material
initially present (oxygen) will be used
during a given time unit. Each successive
unit of time has less reactant present
initially than the preceding interval, hence
a definite precentage decrease results in
successively smaller amounts of reactant
use per unit of lapsed time. Increasing
kl results in a 1arger percentage oxygen
use per unit of time and also increases the
chanae in reactant mass among successive
time intervals.
B Adney's work for the British Royal Com-
mission cited 5 days passage time from
source to the ocean as maximum for
English streams. The 8th Report (1909)
largely established BOD philosophy in-
cludina the 5-day interval. At 5 days,
initial lags generally have terminated and
a substantial fraction of the long-term
oxygen demand has been exerted. If only
one time intf'rval can be used, 7 days
permits better scheduling. Anyone time
interval is "a" fraction of the total oxygen
requirement; this is a poor reference
point if we do not know how it arrived.
For example, the percentage of
oxidizable material stabilized in terms
of oxygen use at various rate factors
0/0 oxidized
kb (loglO) in 5 days
O. 5 420/0
O. 10 67%
O. 15 84%
O. 25 94%
0.50 99+%
K 1 (log)
0.11 e
1. 15
This range (K = 2.3 k ) is commonly en-
1 1 b'l' t' 'th
countered in wastewa.ter sta 1 lza ion Wi
the hiJ{her rates characteristic of frest. oxi-
dizable material that is readily converted.
CH. O. bod, 56d, 3. 74
The lower coefficients are charaderistH'
of cell mass at latE'r stages of oxidation
and of low-rate reactants in gpneral.
C The oxygen utilization at specified inter-
vals of time are required to estimate kl'
and L, the estimate of oxygen use at
infinite time, It is common to observe
results at equal intervals of time but
this is not essential as long as
the time intervals are accurately known.
The initial time periods arE' critical as
an error of a few hours in time represent:..
a relatively large change in reactant mass
in a system at maximum instability. Un-
equal time periods can be plotted to define
the curve from whic~ any givpn intervals
can be selected as desirpd.
D Increasing impoundment of surfa<." watpr
provides more time for stabilization of
relatively inert soluble or suspended
pollutants and for organism adaptation
to the situation or pollutants. Long term
BOD's are essential to indicate changes
in the pattern of oxygen demand vs. time.
It may be expected that one or more
plateaus will be evident in the BOD curve
followed by a temporary rise in rate
during second stage oxidation or thereafter.
Anaerobiosis may cause a rise in rate
coefficient after aerobic conditions are
re-estabUshed. Eventually kl stabilizes
at very low values.
1 Rate coefficients tend to be difficult to
interpret during long term 80D's
because of progressive changes and
other factors.
a The relative error of the 00 test may
be a large fracEon of the incremental
DO change during low rate periods.
b Cell mass may agglomerate under
quiescent test conditions and decrease
nutrient availability.

Effect of Some Variables on the BOD Test
c It is not likely that recycled nutrients
under aerobic test conditions will
have as much effect a. recycle from
anaerobic benthic deposits in a
2 The BOD result tends to underestimate
deoxygenation relative to surface water
behavior because of interchanges,
turbulence, biota. and boundary effects.
Reseeding doe!! not occur in a sealed
bottle but reseeding is inevitable in a
stream or treatment unit.
A Effect on Oxidation Rate
Temperature is one of the import,nt con-
trolling factors in any biological system.
In the BOD rl'&ction. changes in tempera-
ture produce acceleration or depression
of the rate of oxidation. Figure 1 shows
the changes in the value of k at tempera-
tures from 0 - 25°C on a common
B Test Temperature
In the BOD test procedure an arbitrary
temperature is usually selected for
convenience even though a wide temperature
range exists under natural conditions.
Incubation of the test containers at 200C
for the whole period is now accepted
practice in the U.S.; 18. 50C is preferred
in Etlgland. Camp (ASCE, SA5 91: 1, Oct.
65) recommended light and dark bottle
immersion In the stream.
C Tt'mperature Correction
When it is necessary to calculate the rate
of oxidation at a temperature other than
20°, the following relationlhip may be
.2: e (Tl - T2)
O. Z
t ~~~

o 0.0
~ 0.0
,.J 0.0
> 0.0
zo ZI
o..,,1It C ;
k =
rate coefficient at temperature T 1
kZ = rate at'coefficient at temperature T 2
9 . temperature coefficient. for which
Streeter and Phelps obtained the value
1. 047. 9 changes with temperature; it
appears to be higher in the range of
5-150C than in the range of 30 to 400C.
The value given refers to 15-300.
The cited temperature coefficient appears
reasonable for household wastes. It may
not apply for other wastes where developing
or seed organisms may not tolerate tem-
perature changes as readily. A given
temperature coefficient should be checked
for applicability under specified conditions.
In pH
A The organisms involved in biochemical
conversiON! apparently have an optimum
response near a pH of 7.0 providing other
environmental factors are favorable; a pH
range of about 6.5 to 8. 3 apparently is
acceptable (Figure 2). Reactivity is likely
to be s1&nificantly lower on both sides of the
acceptable pH ran,e but microbial adapta-
tion may extend th~ "mite appreciably.
For example, trick lin!, ;iIters have operated
with better than 50'1/. treatment efficiency
at}lH 3 and 10 after adaptation.


~... 80
~ .,
/ .' \
4 pH Ie

B A djuBtment of Concentrated Samples

When wastes are more acid than pH 6.5 or
more alkaline than pH 8.3, adjustment to
pH 7.2 is advisable before reliable BOD
value. can be obtained.
C Dilution Samples

Standard dilution water is buffered at pH
7.2. Sample-dilution water mixtures should
be checked to make sure that the sample
buffer capacity does not exceed the capacity
of the dilution water for pH adjustment.
A Importancl.'

In 1932 Butterfield reported on the role of
certain minerals in the biochemical oxidation
of sewage and concluded that deficient
minerals ofteil upset metabolic response.
In addition, he found that inadequate nitrogen
and/or phosphorus was a common cause of
low BOD results in industrial wastewaters.
(Fii\lre 3)
Fi.u", 3
~ no
Time in Day.
F.tre"t ... Minerai Nutrient. an 80D
Effect of Some Variables on the BOD Te>ot
B Standard Methods Dilution Water

The dilution water specified for the BOD
test approximates USGS estimates for an
average U. S. mineral content of Burface
water except for added phosphate buffer.
It Is assumed to provide essential mineral
nutrients for most wastewaters but cannot
be expected to meet requirements for
grossly deficient wastewater nutrients both
mineral and organic. Ruchhoft (5. W. J.
13:669, 1941) summarized committee action
leading to the present dilution water.
C Other Dilution Considerations
There is a trend toward the use of recf'iving
water, storage-stabilized if necessary, to
evaluate waste behavior. It is advisable
to minimize dilution and consider the
nutrient level likely in the receiving wate!'
as most valid. Any change in the environ-
ment, such as dilution, upsets the
microbial balance and requires adaptive
A Need for Complex Flora and Fauna

Butterfield, Purdy, and Theriault (Pub.
Health Rep. 393, 193 1) demonstrated that
an isolated species of organisms was not
as effective in biological stabilization as
a variety of species. Figure 4 summarizes
some of their data. ghatta and Gaudy
(ASCE, SA3, 91:63, June 19(5) reinvestigated
this factor. Many studies haveemphasizf'd
the need for a mixed biota in the BOD test.
It appears that bacteria are capable of
varied activities. but all species are not
capable of synthesizing all required nutrients.
Certain bacterial species may be capable
of producing enzymes, amino acids, or
growth factors needed for their Use and by
other species for optimum performance.
It has been shown that oxygen demand
becomes minimal when sam£> limit of
bacterial population tas been reached.
Predation prevents such an approach to
maximum numbers and maintains a con-
tinuing bacterial growth and recycle of
nutrients among a mixeo population. The
net effect is a symbiotic relation among
mixed organisms tending to enhance the
rate of stabilization or utili za tion of
oxygen as in the BOD test.
61 - :3

Effect of Some Variables on the BOD Test
B Ori2-nism Adaptation
1 Early inveaUgations in relation to the
BOD test considered domestic wastewaters
primarily. The saprop~tic organisms
involved in atabilization either were
present in adequate numbers or quickly
multiplied to attain effective populaUons.
2 The period of adjuatment required to
shift enzyme production needed to utilize
an energy source different from that
previously utilized or to ahift population
variety from that favored by one food to
that favored by another food is con-
sidered an adaptation period. Dilution,
temperature, oxygen tenaion, pH,
nutrient type, inhibitory substance a,
light and other changes all are common
inducements for microbial adaptation.
Mutation of organisms may be encoW1tered
during adaptation but usually is not a
3 The developments in induatry and
technology have resulted in discharge
of new and more varied wastewater
conatituents. Microori2-nisms may
adapt themselves to the use of a new
substance as an energy source providing
the energy and environment are favor-
able. The receiving stream usually shows
development of an adapted microbiota
for a new or different dilcharge con-
stituent within hours, days or weeks
after fairly rei'llar di.charge. The
tirpe for adaptation depends on the nature
of the conltituent, available energy,
tolerance of the organisms, and environ-
mt:ntal conditions.
C S.eding
The amount of eeed and its selection muat
be determine~ experimentally. The most
effec;tive inoculant would be that which
would produce the maximum BOD response
with minimum lag period and neglipble
seed demand, This would mean some
maximum population adapted to feed and
conditions at a minimum equilibrium energy
nutrient supply.
1 Figure 5 indicates corrected BOD
progression on a synthetic feed with
river water and stale sewage inoculants
at several concentrations. The river
water resulted in higher BOD with
negligible lag and seed correction. The
seed correction at 200/. concentration
of inoculant was less than 0.3 mg.
00/1 at 5 days. It would be pos8ible to
use this river water a8 a diluent without
excessive oxygen 108s to produce more
valid BOD progression for that receiving
water. The lower waetewater inoculant
concentration resulted in a definite BOD
lag. Higher wastewater concentrations
produced comparable BOD progression
earlier but resulted in high seed
corrections and lowered availability of
dililllolved oxygen for the sample.
2 A good secondary treated effluent
produced results similar to river water
inoculation with higher seed corrections
per increment of applied inoculant.
Soil suspensions also are very effective
sources of seed organisms with minor
seed corrections if they are reasonably
stabilized surface soils.
3 It appears that the BOD progression
most nearq indicating receiving water
oxidation would be one based upon
receiving water dilution or inoculated
with organisms from it.
4 A new or unusual wastewater may
require adapted organisms not present
in sufficient numbers in the receiving
water. Development of an adapted seed
from soil sU8pensions, plant effluents
or receiving water may be necessary to
evab..te oxidation potential in a plant
or receivin, water at some future time.
Enrichment culture technique i8 bene-
ficial whers smaU concentrations of the
test wastewater are applied regularly
with increases in wastewater concen-
tration8 8S BOD or respiration activity
indicates increasing tolerance and
oxidation of the test waste. Both time
and concentration limits are useful to
characterbe the stewater and its
acceptability for :.i, ',J~ical stabilization.

-- ---_-Efect 0LSome~'ariab!!.~ on .the BOD Test
I .
g .
AU rorm. in rIVf'r .....r
-...-.. Mt.... .Icwr,. .. ptank'CM
- PuN ""'''r. B. A.ropn.. . ,18NIIOft
Mt..d c-.Ill\lr. b8('''rla
....-... run cu""", B. A.ro"MI
s , 7 .
ThM' in 0.,.
t>:rr.t t 01 H lololtcal Vorm. on O.,..n o.pl.tlOft
..,..,.. 4
10 II 11 IJ 14
. ............. IIYEI WAtEi
4 ~
J 0
1-20" liVER WAIII 
2 3 4 6 ' DAYS
HgUI'" "
" It mUst be recognized that HOLJ
progressions are most likel)' to err
on the low side. A meaningful BOD
test should seek the highest consistent
oxygen demand feasibl(' for sample and
D Algae
"hen laqcc numbers of algae are present
in surface v.aters, they produce significant
chang..,; in the oxy!!en content. Under the
influence of sunlight excess oxygen is
produced while a net deficit occurs in the
dark. The result is a wide variation in
surface water 00 depending on sample
When stream samples containing algae are
incubated in the laboratory the aljlae
survive fOI' a hme, then die beL'ause 01 th,.
lack of light. Short-term ROn df'termina-
tions may show the influenct' of ox)'!!en
production by the algae. When the algae
die, they release tht' ston'd organic load
for recycle and increaRe the ROD. There-
fore. samples incubated," the dark may
not be representalivt' of the dE'oxygenation
process in the strt'am, since the h..nefits
of photosynthesis art' Lacking. Converst'ly,
samples incubated in the lillht. under
,'onditions of continuai photosynthesis,
110111 yield 10110 BOD values.
The influence of algae on ROD is one of
the most difficult variables to evaluate.
\Iore research is needed to devE'lop
satisfactory methods for the accurate
determination of BOD in the pres,'nce 01
lar~e numbers of algae. Light and dark
holtle incubations suggest the magnitude
of effects.
-iinu' satisfactIon of the BOD is accom-
pished through the action of mkroorgan-
i!lms, the presence of toxic ~uhstance>;
\l ill result in depression of th.. oxidation
rate. In many caSt's. toxiL'it) will
pl'oduce a lag period, until tolerant
organism activity hecomes signifIcant.
Figure 6 shov. s '.hp effed of cyanide on 'he
ROn curve. .-\ prominent lag perio<.i IS
exhibited in the 2 ppm curv~, while at
10 ppm the lag ext...n
Effect of Some Var1ables on the BOD Test
~ I.
I I.
Tim!' in DaJ8
Frted of c,....... on BOD of Dome.Uc !Jew...
C."" ...... t.a Formull C Dilution W.ter)
Figure 6
Heavy metals have similar effects depending
on history and environment. The effects of
copper and chromium are illustrated in
Figure 7.
~ (oj,
(Hft(l"Il"\I '\'11
::. 41'
- '>1'
f. J"F"[( T I.' Hf \n ,1t:T.1jl" Q'\ BVI1
Figure 7
B Detection
In laboratory determinations of BOD the
abseljlce of toxic substances including
chlorine must be established before the
resuits can be accepted as valid.
Comparison of BOD values for several
dilutions of the waste w1llindicate the
presence or absence of toxicity. In Table 1
the calculated BOD for the dilutions 8how
higher values in the more dilute concen-
trations. It is apparent that toxicity was
present and that the toxic effect was diluted
out at a waate concentration of 2% or les8.
 Table I 
Waste Depletion 5 ~ BOD
1 oeJ. 3.51 35
5'1. 4.53 91
2"/. 2;80 140
1'/. 1.52 152
O. 5", 9.74 148
A Mechanism
The oxidation proces8, as exemplified by
the equation:
Y" L (l-10.~
prelumab1y involves th~ oxidation of
carbonaceous matter or 18t stage oxygen
C H ° -
C02 + H20
The rate coefficient is normally high, givin@
nearly complete oxidation in a few days.
When nitroienou8 material is present its
oxidation can be shown as:
02 - 02
NH3 - N02 - N03
Nitroien oxidation may be delayed for
several days during BOD tests unless
suitable micro-biota are initially available.
Under some circumstances these two
oxidation. can proceed simultaneously and
the reaultant BOD CUrve will be a com-
poaite of the two reactions.

y .. [L (l-IO.kct) + L (1-10.kn1]
t c n
wbere Yt .. the simultaneous BOD of the car-
,?onaceOUtLand nitroge:nQU8 phases or 1st and
2nd stap deJnU1ds.

kc and kn .. the rate coefficients appli-
cable to the carbonaceous and nitrogenous
materials respecti"ely.

Lc and L . the ultimate oxygen demands
cnaracteAstic of the two phases respectively.
This is the general formula for any system
characterized by two simultaneous reactions.
Principal conditions governing simultaneous
carbon and nitrogen oxidation:
1 Presence of an effective nitrifying
culture at the beginning of the test
Interval (nitriflers grow relatively
2 Maintenance of adequate DO, believed
to be a minimum of O. 5 to 1. 0 mgll,
for nitrifier activity.
3 Available nitrogen - In excess of that
required for synthesis. This is believed
to require a minimum of about 7 mgll
to support active nitrification on a
continuous basis.
4 Nitrifiers appear to be more sensitive
to toxicity than most saprophytic
organisms, hence are likely to be
Inhibited more readily. This is
particularly evident during nitrite to
'nitrate conversion.
B It may require 5 to 10 days to establish
nitrification if the population was not
nitrifying Initially. This is the basis for
the sequential carbonaceous and nitrogenous
oxidation of sewage oxidation.
1 Effects on the BOD curve Indicate a
typical pattern such as In Figure 8.
The Influence of nitrification In the
production of a secondary rille In the
BOD curve is so well known that any
secondary rise may be erroneously
attributed to nitrification whether or
not nitrification was Involved. Actually,
a secondary rise In the curve may be
due to any oxidation system assuming
dominance alter the Initial oxidation
system has been completed.
2 The nitrification phenomena occurs
simultaneously In many streams,
treated effluents or partially stabilized
samples. The designation of a secondary
Effect of Some Variables on the BOD Test
BOD rise to nitrification should be
based on analysis, not curve shape.
C The extent of nitrific'ition is conclusively
shown only by periodic analysis of
ammonia, organic, nitrite and nitrate
nitrogen. The conversion of ammonia
and organic nitrogen to oxidized nitrogen
is a definite indication of nitrification.
D Nitrification Inhibition
Plant efficiencies from a BOD standpoint
can be erroneous because nitrification
generally is not established during the
usual Incubation of influent samples but
may be a major factor in effluent
incubations. It requires about 2 times
the oxygen to convert NH3 -N to N03 -N
as to convert C to C02 hence this is a
major fraction of stream oxygen use.
Most secondary treated effluents are
characterized by a larger fraction of
carbon than nitrogen removal which
accentuates the problem.
Pasteurization of samples, methylene
blue, chromium, and acid treatment
followed by neutralization have been used
to inhibit nitrification for estimation of
carbonaceous BOD only. Any inhibition
of nitrification also produces a change in
the sample or its behavior and may
partially innibit carbonaceous oxidation.
Nitrification is a factor In stream self-
purification and treatment. It does not
appear realistic to alter it for convenience.
The most realistic approach to carbon-
aceous oxidation i8 the measurement of
C02 or C~D.
. .
. .
: .1
. , .
~,.., ... 0&'\
["£C.T Of' ,-,UtFI(lIr,olol ON 800
Figure 8

Effect of Some Variables on the BOD Test
When a series 01 dilution I are made on a
BOD sample usually the result s vary to the
extent that only an approximate BOD value
i., obtained.
Table 2
Sample cane.  DO; Depletion BOD
Inihal : 8,2 - -
1%  5.5 2,7 270
2'" ' 3.3 4,9 245
4'10 1 0.0 i complete .
A For example, in Table 2, I",., 2.,. and 4'10
concentrations ot sample were used. The
4'10 concentration became ana'erobic before
the end of 5 days. The 5-day BOD of the
1% concentration was 270 and that of the
2". concentration was 245.
B Statistically one value 18 more reliable
than the other.
00 depletion

5.S mill

3.3 mill
2.2 mill
The d1ffer~nce In depleUon betw.en 1 and
n'. dilutions II 2.2 mlh. This difference
may be attributed to an additional 1'" of
sample added to the orl,lnal 1%. If the
difference is multiplied by the dilution
factor.of 100 to obtain the BOD, the result
18 220 mg/l..
1 We now have three estimates of the
BOD on a one percent concentration
basil from the two dilutions:
a the actual 1% depletion gives 270
b 2%/2 depletion gives 245
c (2%- 1%) depletion gives 220
Statistically the probabilities of being
nearer the actual value goes with the
-nearest two of three. The 41. value
of 8. 2 depletlon/4 as a minimum
po8l1ble BOD 1". concentration gives
a BOD of at least 200.
2 There Is the pos8ibl~ty that higher
concentrations may reflect significant
toxicity while lower concentrBtlons
tend to reflect a greater proportion of
dilution water. The toxicity problem
does not appear to be significant since
the 4'" sample concentration indicated
a BOD of at least 200. The higher
BOD at 1% sample concentration may
be due to a contaminated dilution water
or to the fact that a similar number of
seed oraanlsms had less food and
utlUzed certain fractions that they had
pasled by when they had more choice
with the 2'10 sample concentration.
Data i8 Insufficient to resolve this one.
3 Incubations having a depletion of at
l.ast 2 mg DO/liter and a residual of
at least 1 mg DOlliter are indicated
to be most valid(1) . Both the 1 and
2.,.. concentrations fit this requirement
In Table 2. An average error of
+ or -0.1 ml on the DO titration would
have a smaUer relative error upon
the 2% depletion. '
4 We have a reasonable presumption
that the sample BOD of about 230 was
a good estimate. . We do not have an
unequivocal basis for so stating,
Possible variations in results with
different dllutions of a giveft sample
are subject to many uncertabities in
the te st routine.
U lIome cause is known - such as a
titration eror, the inclusion of ex.
traneous substances producln, high
or law response, or a definite procedural
error that rules out a valid 'elltimate of
the lIample BOD- that result 8hould be
labeled as a lost cause or unreported.
Otherwi!!e, report what was obtained
to the best of your .b1ltty with the
provision of uncer~~ ','ty for uncon-

Certain portions of this outline contain
training material from prior outlines by
D, G. Ba11inger and J, W, Mandla.
Standard Methods, APHA-AWWA-WPCF.
13th edition, 1971.
Effect of Some Variables on the BOD Tc:'.:-
This outline was prepared by F. J. Ludza",
Chemist, National Training Center, EPA,
WPO, Cincinnati, OH 45268.
Descriptors: Algae, Ammonia, Bacteria,
Biochemical Oxygen Demand, Essential.
Nutrients, Microorganisms, Nitrates, Nitri-
fication, Nitrites, Nutrients, Oxygen Demand,
Rates, Time, Toxicity, Waste Dilution

Factors Affecting 00 Concentration in Water
I The Dissolved Oxygen determination is
a very important water quality criteria for
many reaeons:
1 The rate of bIochemical activity in
terms of oxygen demand for a
given sample and conditions.
A OxYien is an essential nutrient for all
livini organbms. Dissolved oxygen ill
es.ential for survival of aerobic
orlanisms and permita facultative
organisms to metabolize more effectively.
Many desirable varieties of macro or
micro organisms cannot survive at
dissolved oxygen concentrations below
certain minimum valuee. These values
vary with the type of orranisms, stage
in their life history. activity, and other
2 The degree of acceptability
(a bioassay technique) for bio-
chemical stabilization of a given
microbiota in response to food,
inhibitory agents or test conditions
3 The degree of instability of a
water mass on the basis of test
sample DO changes over an
extended interval of time
B Dissolved oXYien levels may be used as
an indicator of pollution by oxygen
demanding wastes. Low DO concen-
trations are likely to be ...ociated with
low quality waters.
4 Permissible load variations in
surface water or treatment units
in terms of DO depletion versus
time, concentration, or ratio of
food to organism mass, solids, or
volume ratios.
C The presence of dissolved oxygen
prevents or minimizes the onset of
putrefactive decompoait1on and the
production of objectionable amounts of
malodorous sulfides. mercaptans,
amines. etc.
5 OxYienation requirements
necessary to meet the oxygen
demand in treatment units or
surface water situations.
G The 00 tellt is the only chemical test
included in all Water Quality Criteria,
Federal, State, Regional or local
D Dissolved oxygen is euential for
terminal stabilization wastewatenl.
Hiih DO concentrations are normally
associated with good quality water.
A Physical Factors:
E Dissolved oxygen changes with respect
to time. depth or section of a water'
mass are usefUl to indicate the degree
of stability or mixing characteristics
of that situation.
1 00 solubility in water for an
air/water system is limited to
about 9 mg DO/liter of water at
20oC. This amounts to about
0.00090/0 as compared to 210/0 by
weight of oxygen in air.
F The BOD or other respirometric test
methods for water quality are comm,:mly
based upon the difference between an
inUial and final 00 determination for a
given sample time interval and con-
dition. These measurements are
usefUl to indicate:
2 Transfer of oxygen from air to
water is limited by the interface
area, the oxygen deficit, partial
pressure, the conditions at the
WP.NAP. 25. 3. 74

Di..olved OxYlI:en Determination
interface area, m1xinll: phenomena
and' other items.
Certain factors tend to confuse
reeixy.enat1on mechanisms of
water aeration:
a The transfer of oxygen in air
to di..olved molecular oxygen
in water has two principal
Area of the air-water
Dispersion of the oxygen-
saturated water at the
interface Into the body liquid.
The first depend. upon the surface
area of the air bubble s in the water
or water drop. in the air; the
second depend. upon the mixine
eneriY in the liquid. U dfffusors
are placed in a line along the wall,
dead .pots may develop in the core.
Different diffusor placement or
mixin, enerllY may improve oXYlen
tran.fer to the liquid two or threefold.
b . Other variables in oxygen tran.fer
Oxygen deficit In th" liquid.
Oxygen content of the gas phase.
If the first four variables are
favorable. the process of water
oxyeenation is rapid until the liquid
approaches saturation. Much more
enerc and time are required to
increase oxygen .aturat1on from
about 95 to 100'" than to Increase
oxy,en saturation from 0 to about
911.,.. For example: 
solubility. A cold water often has
much more 00 than the solubility
limits at laboratory temperature,
Standing during warmup commonly
results in a loss of DO due to
oxygen diffusion from the super-
saturated sample. Samples
warmer than laboratory tempera-
ture may decrease in volume due
to the contraction of liquid as
temperature is lowered. The full
bottle at higher temperature will
be partial~ full after shrinkage
with air entrance around the stopper
to replace the void. Oxygen in the
air may be transferred to raise the
sample DO. For example, a
volumetric flask filled to the 1000 ml
mark at 300 C will show a water
level about 1/2 inch below the mark
when the water temperature is
reduced to 200 C. BOD dilutions
should be adjusted to 200 C + or
1 1/20 before filling and testing.
Water density varies with tem-
perature with maximum water
density at 40 C. Colder or warmer
waters tend to promote stratification
of water that interferes with
distribution of 00 because the
higher density waters tend to seek
the lower levels.
Oxygen diffusion in a water mass is
relatively slow, hence vertical and
lateral mixing are essential to
maintain relatively uniform oxygen
concentrations in a water mass.
Increasing salt concentration
decreales oxygen solubility
slightly but has a larger effect
upon density stratification in a
water mass.
The partial pressure of the oxygen
in the gas above the water interface
control. the oxygen solubility
limits in the water. For example,
the equilibrium concentration of
oxygen in water is about 9 mg DO/I
under one atmospheric pressure of
Dissolved Oxygen Determination
air, about 42 mg DO/liter in
contaot with ;:mre oxygen and 0 mg
DO/liter in contact with pure
nitrogen (@ 200 C).
Biological or Bio-Chemical Factors
Aquatic life requires oxygen for
relpiration to meet energ:
requirements for growth, repro-
duction, and motion. The net
effect is to deplete oxygen resources
in the water at a rate controlled
by the type, activity, and mass of
living materials present, the
availabUity of food and favor-
ability of conditions.
A 19ae, autotrophic bacteria, plants
or other organisms capable of
photosynthesis may use light
energy to synthesize cell materials
from mineralized nutrients with
oxygen released in process.
Photosynthesis occurs only
under the influence of adequate
light intensity.
Respiration of alga is
The dominant effect in terms
of oxygen assets or
liabilities of alga depends upon
algal activity, numbers and
light inteneity. Gross algal
productivity contributes to
significant diurnal DO
High rate deoxygenation commonly
accompanies assimilation of
readily available nutrients and
conversion into cell mass or
storage products, Deoxygenation
due to cell mass respiration
commonly occurs at some lower
rate dependent upon the nature of
the organisms present, the stage
of decomposition and the degree
of predation, lysis, mixing and
regrowth. Relatively high

Di8lolved Oxyaen Determination
deoxy,enation ratel commonly are
a..ociated with lilJ\if1cant ,rowth
or re,rowth of orpnilms.
Micro-organism. tend to flocculate
or ai,lomerate to form settleable
maues particularly at limiting
nutrient levels (after available
nutrient. have been a.similated or
the number of orianisms are lar,e
in proportion to available food).
Relu1ting benthic depositl
continue to respire a. bed
OXygen availability ill limited
beClulle the deposit is physically
removed from the source of
surface oxy,enation and alpl
activity usually il more
favorable near the surface.
Stratification is like~ to limit
oXYien tranlfer tothe bed load
The bed load commonly ill
oxy,en deficient and decomposes
by anaerobic action.
Anaerobic action commonly is
characterized by a dominant
hydrolytic or lolubUlzin, action
with relative~ low rate growth
of organam..
The net effect is to produce low
molecular wei,ht productll
from cell mas. with a corre-
.pondingly lar,e fraction of
feedback of nutrients to the
overlayin, waters. These
~.is product I have the effect
of a. hi,h rate or immediate
oxy,en demand upon mixture
with oxygen containin, waters.
Turbulence favarin, mixin, of
lurface waterl and benthic
lediments commonly are
aSlodated with extremely
rapid depletion of 00.
Recurrent resuspension of
thin benthic deposits may
contribute to hiahly erratic
00 patternl.
Lon, term depoaition areas
commonly act like point
sources of new pollution as
a result of the feedback of
nutrients from the depolit.
Rate of reaction may be low
for old materials but a low
percenia,e of a Jarge mass of
unltable material may produce
exceslive oxygen demands.
Tremendous 00 variations are likely
in a polluted water in reference to
depth, crosl section or time of day.
More stabilized waterll tend to show
decrealed 00 variations althouih it is
like~ that natural deposits such as leaf
mold will produce differences related
to depth in It ratified deep waters.
This outline contains ligntficant materials
from previous outlines by J. W. Mandia.
Review and comments by C. R. Hirth and,
R. L. Booth are greatly appreciated.
1 Method. for Chemical Analysis of
Water and Waltel, EPA-AQCL.
Cinci..-ti, OH, July 1971,
This outline w.. prepared by F. J. Ludzack.
Chemist, National Training' Center, MDS,
WPO, EPA, CincinnaU, OH 45268 and
revised by Charlel R. Feldmann. Chemist,
National Tratnin. Center.
Delcripiorl : Aeration, Aerobic Condi tions,
Air-Water Interfaces, Anaerobic Conditions,
Benthol, Biolqr ical Oxygen Demand, Dissolved
OxYien, Water Pollution, Water Quality

A Di..olved oxy,en is so economically,
biologically, and technologically important
that its signiftcance is nearly \Uliveually
recognized. All water quality standards
employ dissolved oxygen (DO) concen-
tration as a parameter of water quality.
B Effective water quality management is
largely dependent upon control of the
natural oxygen balance. Unless pollution
control decisions are based upon sO\Uld
technical knowledge, achievement of desired
water quality will be purely fortuitous.
C Full comprehenllon of the natural oxygen
balance Is required to:
1 Predict the relponse of the oxygen
resource to any stimulul (including
pollution) before it occurs,
2 Aiseis the effectiveness of exlltlng
pollution control measures, and
3 Determine the degree of treatment
required to maintain specified oxygen
To efficiently control the oxygen balance one
must: (1) aCCO\Ult for all proceslu which
innuence DO resources, and (2) quantitatively
estimate their effects.
A Two methods meet these requirements:
1 Oxygen budget method

Thil method requires measurement CJf
all oxygen resources and Uaqllities under
a given set of condition.. Should the
same conditions reoccur at lome future
time the observed oxygen values would
again prevail.
WP.NAP, 20b. 3. 74
The budget method has been severely
criticized because it tends to be
laborious. evaluates the balance only
under one set of environmental conditions.
and prelupposes that methods for
independent measurement of the various
assetl and liabilities exist. Nevertheless,
this method may be practical in
uncomplicated situations.
2 Mathematical modp.l
The mechanism of oxygen exchange in
natural waters could theoretically be
described by a mathematical equation
or model. A perfect mathematical
model would account for all the myriad
biological, chemical, and physical
factors influencing the oxygen balance
and could be used to predict oxygen
levels under various conditions.
The mathematical model might be
envisioned as a single equation con-
taining numerous terms, each
reprelenting the influence of a chemical,
phyllical, or biological variable. In
practice, portions of this hypothetical
equation have already been developed,
but the elusive master equation itself
is yet to be derived. At their present
state of development, such mathematical
models are not fully comprehensive but
may be used provided the limitations of
the analysis be known.
The values of coefficients and terms of
the model may be estimated in the
laboratory or derived from field
B The "proof of the pudding" of a budget or
model is its ability to accurately reproduce
what actually occurs in nature. [f the
predicted value of DO based on laboratory
results or field measurements equals the
ob8erved value the budget or model is
usually considered to be an accurate
reproduction of the natural process.
This practice ignores real or intentional

Oxygen Dynamics in Streams
compensating errors which do not affect
the predict'ed value but incor.rectly
indicate the relaUve effects of individual
processes. To be completely accurate
a model must not only predict the oxygen
sag curve but also accurately compare
C Generally it i8 ea81er to e8timate the
a verage oxygen exchange for a 8tream
reach than to precisely define the oxy,en
sag curve. If the former approach may
be taken technical constraint8 may b.
ea8ed and difficulties much aUevlated.
Natural oxygen exchange Is a complex
phenomenon involving several proce8se8.
The potential effect8 of these proce88es upon
the biochemical oxy,en demand (BOD) and/or
dls80lved oxygen (DO) resources of a
hypothetical reach of stream are indicated in
Table 1. BOD and DO resources are a88umed
to exist 801ely within the liquid pha8e.
Although it would be rare to find less than
two proces8es sljl1iticant, several of the
processes of Table 1 may be inoperative
throughout a given reach of 8tream. One
of the major difficulties of oxygen balance
analysis III independent estimation of the
effects of the Individual processes. If
acceptable, there are techniques available
to elltimate the combined effect of several
The following processes increase oxygen
liabUities. Effects are normally exerted
as oxygen depletion or increased BOD.
A Bacterial Re8plration

Bacterial re8plration has long been
recognized .s an oxygen-demanding pro-
ceSl!l and i8 generally re8ponsible for the
majority of natural demand. Although
most literature references a8cribe thl8
Addition Removal A dcKtlon . Removal BOD and DO,
Scour Sed1menaUOft PIlDt0878tb..i8 Im_cHate d_..d 
Df.ftII8loft (rom A deorpUoa A tmo""'"" Dlftlaelon to tbe 
bentbal layer  nae..Uoa ben1lla1leyer 
Local runoff VoleUU..Uon Local MIBOff PurliDl acUca yertl_. hDri_tal.
   of ...e Ii 1oa""",1 tIIa-
Pbotoeynthul. Re.plratlon of  Pleat reeptntioll 
 bacteria a..d   
   Re.piratlon of 
   beeter" ....d 
,,,- 2

demand to "bacteria" it may also Include
requirements of zoomicrobes of higher
tropic levels, such as protozoa and rotifers.
Bacterial and zoomicrobe respiration is
primarily dependent upon three variables:

1 Nature of compound

a Some organic material, such as
domestic sewage, is quite easily
degraded by bacteria, whereas other
compounds, e. g. ,lignlns and poly-
saccharides, are relatively resistant.
In the presence of a given organism
or association of organisms, an
organic compound will exhibit a
characteristic BOD rate coefficient
under standard conditions,

b As the BOD reaction proceeds, the
original compounds are converted to
materials of lower energy content
and availability, shifts occur In
microscopic population predominance,
and the apparent BOD rate coefficient
progressively changes. The BOD
reaction may be mathematically
described as a combination of several
logarithmically linear reactions acting
concurrently andlor In sequence. 1,2
Accurate prediction of 00 levels at
all points within a stream reach may
require frequent evaluation of the
apparent rate coefficient.
c There has been much controversy as
to whether nitrification can occur in
the presence of organic matter'3 In
the original work of Therriault
nitrification followed carbonaceous
oxidation. recent studies by 5
Courchaine and by Downing et al
indicate that nitrification may also
occur In the presence of organic
matter. Carbonaceous matter ~ se
appears to have no intrinsic
antagonistic effect upon nitrification
but may foster low DO levels, Increased
cell synthesis, or high levels of
predation. As long as DO concentrations
exceed 1 mgll and NH concentration
exceeds that required ~or cell synthesis
ammonia appears to function like an
Oxygen Dynamics in Strean'~-
individual organic compound during
the BOD reaction. Oxygen demand
for nitrification may be included in
apparently linear BOD reactions.
2 Seed
Some wastes contain bacteria capable
of degrading that particular waste.
Other wastes are essentially sterile
(due to toxic substances, low DO levels,
temperature, etc.). In streams
receiving waste discharges over a long
period of time naturally occurring
bacteria are usually present which are
capable of degradir..g ~he waste material.
Many compounds have been classified
as "non-degradable" in a biological
environment. The variety of organisms
existing in nature, their adaptive powers,
and the processes controlling effective
exposure time lire so complex that. In
the absence of contrary evidence, it is
perhaps conservatively advisable to
assume that any organic compound may
be degraded within a stream reach.
Many "non-degradable" compounds are
bioloi1cally utilize:i at such low rates
that conventional analytical techniques
do not detect reaction progress within
practical observation periods. Also,
many of the processes which af~
oxidation in nature cannot be accurately
duplicated in the laboratory.
Bacterial respiration is normally
evaluated by BOD bottle tests or
Warburg or large-volume respirometer
B Immediate Demand
Immediate oxygen demand is exerted
spontaneously upon contact of waste
components with oxygen. The demand
reaction is normally complete within 15
minutes and is exerted solely in the
liquid phase.
Immediate demand is especially harmful
because its effect'! are exerted at the
instant or location of discharge. rather

<>XYaen Dynamics in Streams
than being distributed over a reach of
stream as would be the case with BOD.
Fortunateq, this type of demand is not
common. .
C Benthal Demand
A large portion of stream oxygen demand
may be derived, not from bacterial-
zoomicrobe respiration in the soluble
phase, but from anaerobic-aerobic reactions
in organic bottom sludges. Such deposits
affect the oxygen balance in two ways.

1 They exert an oxygen demand through
aerobic respiration within the benthal
layer. Oxygen for this process must
be supplied by diffusion from theUquid
2 They release to the overlying liquid
simple organic compounds which exert
additional BOD.
Although the rate of aerobic oxidation in the
sludge ma88 Is generalq low the total
amount of unoxld1zed material in the deposit
may be so large that the oxygen resources
of the soluble phase become severe~ taxed.
The significance of organic sludge deposits
is dependent upon the aerobic-anaerobic
respiration rates and the amount of
oltidizable material in the sludge mass.

Benthal demands are determined by
measurement ~f oxygen depletions in
dark bell jara ~r special benthal
respirometers, or by deduction from
observed field data.
D Purging Action of Gases
Extrerheq active benthal deposits may
liberate gases, such as H2S. which
cause immediate oxygen demand. This
demand is difficult to evaluate separately,
and is UBualq included in determinations
of benthal demand.
64- 4
E Plant Respiration
Oxypn requirsments for plant respiration
may exceed all other demands U growths
are ai8D1ficant. Unfortunateq, thi,8 is
commonly the case. Whether plant
respiration rates are affected by OIItygen
concentration remains a controversial
que.tion. A..umption of constant
respiration rates has proven acceptable in
numerous field studies but controlled
laboratory research increasm,ly indicates
that re.piratton rates are affected.
Respiratory requirements are measured
in dark BOD bottle I, 8 dark large-volume
respirometers, dark alpl chambers, 9-10
or by field observation during Jarlcneu. 11
F Scour
During periods of high now or turbulence
organic matter which had previou.ly deposited
on the .tream bottom may be re-8uspended
and exert BOD at an increased rate. The
combination of potentially large sludge
depo8its and an increased rate of exertion
may cauee exceptlcmalq severe oxygen
Scour is usually evaluated by comparison
of the observed BOD, COD, or orpnic
carbon prolUe with the predicted profile
based on BOD bottle or respirometers
G Land Runoff
Surface runoff may contribute cORsiderable
BOD to a stream. For modeling purposes
it i8 assumed that the load is contributed
in a uniform manner along the stream
Normally this load is only a short-term
liability during periods of rainfall.

The following procellel enhance oxygen aSleta.
Effect. are normally exhibited IU increased
DO ot decrealed BOD.
A Natural Reaeratlon
Thl. .ource il ulually the most important
factor in supplying di.solved oxYllen to
receiYing water.. Some of the 'acton
affecting reaeratlon:
1 Oxy,en deficit
When oxygen II removed from solutton,
an Imbalance il created and the
deficiency il made up by atmolpheric
oxygen passing Into the solution. The
rate of reaeration 11 proportional to the
d18lo1ved oxygen defldt and may be
expressed a. follows:
where: KZ . Reaeratlon coeffldent (base e)
. OxYien deficit. oxygen at
saturation - actual oxygen
content (mll/l)
Z Phy.lcal and chemical factors of the
recelYing stream
a Roughness of the stream bed -
Roughne81 of a Itream bed impartl
turbulence to the stream flow and
enhances all,al tran.fer operationl
Uncludini reaeratlon).
b Wldth or lurtace area - Allows for
more surface contact between water
lurface and atmosphere.
c Depth - In ieneral deep streams have
lower reaeratton coefficlents than do
shallow streams.
d Velocity - Swift moving water will
tend to be more turbulent and will
enhance more efficient reaeration.
Oxygen Dynamic8 in Streams
e Solubility - Various wastes may
affect the solubility of oxygen In the
receiving water. This aspect may
readily be determlned in the
laboratory. An example of this is
the effect chloride concentration has
on 0XYllen lolubility In water.
f Pollution - OUy wastes may form a
blanket or surface fthn on the water
surface and reduce transfer of oxygen
from all' to the water.

3 O'Connor and DObbins12 have postulated
formulas for approximation of the
reaeration coefUdent of natural riverB
and Itreams. Two caseB were con-
a Non-isotropic turbulence where a
vertical velocity gradient and shear
stre88 extst (characteristic of
shallow stream8).
b I80tropic turbulence where neither
a signtUcant velodty gradlent nor
shearinll 8tress extst (characteristic
of deep streaml).
The appropriate formulas for
calculation of K (at ZOOC) under
nonisotrop1c an~ ilotrop1c conditione.
relpectively. are

48.6 (e) 1/4
(K2) - H 5/4
13.0 V
and (K2) - ~T2
200 C H
- reaeratton coefUdent
(base e) under stated
. velocity of Itream flow
. stream depth (ft)
- slope of stream bed ((t/ ft)

OxY.ien Dynamics in Streams
Thel!le formulas are based upon a
molecular diffusion coefficient, DV
for oxygen in water of 0.00195.
c Determination of the type of flow
existing in the river or stream Is
made on the basis of the magnitude
of the Chezy, or roughness.
coefficient of the channel, defined
as fOllows:
C .
If the value of C is le88 than 17 the
flow is considered to be non-Isotropic,
and if C is greater than 17, isotropic.
d The K2 derived for a temperature of
200C may be corrected to other
temperatures as follows:
(K2)ToC . (K2}200C X 1.0241 (5)
where T . temperature, 0 C
4 O'Connor and Dobbins' Isotropic formula
has recently been substantiated by
Tsivoglou et al, 13 Based on measurements
of air-water transfer of radioactive
argon, an experimentally confirmed ratio
between the gas transfer rates of argon
and oxygen, and determination of travel
time" with fluorescent tracers~ these
workers have estimated K2 Independently
of all other natural processes.
B Photosynthesis
Oxygen derived from photosynthesis may
constitute a lsrge fraction of the DO
resource. Whether this production should
be conilidered an asset i. problematical.
Since photosynthesis il not operative during
darknesl, supply of excels DO during the
daytime may be of little advantage. On
the other hand, it may be beneficial if
nighttime depletions are considered
1 If the oxygen budget method is used,
photolynthet1c production Is usually
measured as gross photosynthesis, P.
The respiratory demand of plant
metaboU.m is usually includediD
measurements of community re.piration.
2 If a mathematical model is used,
photolynthetic production Is assumed
to equal the difference between plant
reapiration and photosynthesis, (P-R).
Th1a value 18 uS\l8.lly derived from
analylis of fle:J,-l data.
Gross photosynthesis may be mTsured
by the light-dark bottle method. Net
photosynthesis, if-W' must be derived
from field data.' P1ant respirati0rs
has not It_. measured independently.
C Sedimentation
Sedimentation removes oxidizable matter
from solution and reduces the BOD of the
liquid phale. This effect is usually
evaluated by comparbon of the field BOD
profile with the predicted profil, based 00
laboratory determinations. 16, 1
D Biological Extraction
Growths on the stream bottom may remove
BOD from solution by adlorption. This
effect is evaluatled in the 8ame manner as
ledimentation.. 17 If ledimentatiOft and
biolortcal extraction occur in the same
reach, It may be difficult to determine
their effects IndependeDtly.
The cumulative effects of deoxygeratlon and
reoxygenatlon produce a characterlsUc
variation in Itreams called the "dlesolved
oxygen lag curve." Thil curve has been
described by leveral equationll.
A Streeter-Phelpl

1 In 1925 Streeter and PhelpllS propo.ed
the followinl equatton:

D . ~ -~ ~ -K,' -. -~

+ DA e -K2t
D . di8lolved oXYlen deficit (mg/l)
. time of flow (daYI)
LA . ultimate carbonaceou. BOD
at the upltream end of the
reach (mg/l)
o A . dillolved oxygen deficit at the
upstream end of the reach (mg/l)
K. . BOD rate coefficient deter-
I mined in the laboratory (per day)
K . reaeration coefficient (per day)
2 Thil equation aelumes that
a The stream flow 111 Iteady and uniform.
b The procell for the .tretch a. a whole
is a steady-ltate proceel. The
conditionl at every croll lection
beinl unchanled with timl.
c The BOD removal proce.. may be
delcribed by a linlle rate coefficient
throughout the entire stream reach.

d The removal of oxy,en by the benthal
demand and by plant r..piration, the
addition of oXYl8n by photolynthesi.,
aDd the addition of BOD from the benthal
layer or the local runoff are all
uniform alon, the stretch.
e The BOD and oxygen are uniformly
d1ltributed over each cro81 section,
thus permitting the equations to be
written in the usual one-dimensional
Oxylen D,ynam1cs in Streamp
f The ultimate carbonaceous BOD
represent. all oxYlen liab1l1t1es
which .",.111 be exerted within the
.tream reach.
Since these conditions are unlikely enr
gr_t lengths the stream is usually
modeled in successive, short reaches.
3 BOD removal coefficients may be
determiAed by bottle tests, respirometer
experimentl, or derivation from the
actual BOD profile.
a If BOD removal Ie solely a function
of bacterial respiration, the rate
coefficient derived from bottle or
re.pirometer experlmente may be
b U other facton are known to affect
the removal process the rate should
be derived from the field profile.
Dt:rlvation from the field profile
conlUtutes a preferred procedure.
c Either bottle tests, respirometer
experiments, or the field profile
may reveal a rate coefficient
proll"...lon which may be approxi-
mated by a sequence of linear
r8cUons .llf such a progression
IxlltS. prediction of the actual
profile requires application of different
coefficients and ultimate BOD values
to the several sub-reaches.
4 Values of ultimate BOD for substitution
into the equaUon may be estlmated by
three methods:
a Extension of a semi-logarithmic plot
of .hort term BOD bottle reeults'g
Combinations of 3-~Y and 7-day
or 2-day and 5-day 9 results have
been employed.
b A ddlt10n of the oxygen equivalent of
the organic and ~wmonia nitrogen
to COD results.

OxYI.n Dynamic. in Stream.
c Addition of the oxygen equivalent. of
ammonia and organic nitrogen and
orp.nic carbon.
5 If nitrification.1s known to occur within
the reach (or lIub-reach), its potential
demand .hould be either who~ included
in the estimate of L or who~ included
in ano'her term.
6 The overall change in BOD within the
reach.may be estimated by addition of
carbonaceous demand based on observed
rates and estimatell of ultimate first-
stage demand to stoichiometric oxYlen
reqUiremeni\ for ob.erved nitrate
7 The Streeter-Phelps eq18Uon i. quite
adequate for profile or overall chanee
prediction in simple environmsnts, and
may be utilized to predict overall
chan,es in complex environments. It
i. generally Inadequate for prediction
of profile. in complex situation..
B Thomall' Approach

Thomas hall proposed that the rate
coefUcient expressing natural BOD removal
be split Into two fractions a. follow.:
Kr . Kl + Ka
K1 . reaction coefficient determined
in the laboratory
K . ooelUcient expreslling the effect.
., of other variables
The cor.re8pondinll form of the Streeter-
PhE'lps would be
K - K
2 r
(_Kr' -r -~ (8)
-K t
+ eA e 2
When organic matter (BOD) is removed by
ancillary factors. Ka Is positive; when it
Ie returned to the nowing water, to be
measured at a downstream station, K3 may
be n.pUve.
C Dobbinll' equation

1 Dobbins has recently proposed an
equation which accounts for several
oXY,eD exchange processes:

D . K,( LA ~: kKr' -r -~

~ - Kr
+ DA e -K2t
. (DB
+ K1 ~( -K ~
K2 9~ - e 2 )
D . dis.olved oxyge~ deficit (mg/l)
. time of flow (day.)
LA 8 ultimate carbonaceoull BOD at the
uplltr..m end of the reach (mgll)
DA. dissolved oxygen deficit at the
upstree.m end of the reach (mg/l)
Kl 8 BOD removal rate coefficient as
determined by bottle or
rnpirometer tests
K2. reaeration rate coeffic.lent (pe.r day)
Kr 8 overall coefficient of BOD
removal derived from the field
profile (per day)
La 8 rate of addition of BOD along the
reach (mg/l javl
DB- the net rate of removal of oxy"en
by the benthal 1emand and the
effect of plan' mg/1/day)

2 Thi. equation i. ba.ed on the following
a. .umpt1on..
a The stream now i. steady and uniform.
b The proce.s for the .tretch as a
whole i. a .t.acty-.tate proce...
c The removal. of BOD by both bacterial
oxidation and other procenes are
first order reactions.
d The removal of oxygen by the benthal
demand and by plant respiration, the
addition of oxygen by photosynthesis,
and the addition of BOD from the
benthal layer or the local runoff are
all uniform along the stretch.
e The BOD and oxygen are uniformly
distributed over each cro.s .ection,
thu. permitting the equations to be
written in the u.ual one-dimen.ional
f The ultimate carbonaceous BOD
represent. all oxygen liabU1tie.
which will be exerted within the
.tream reach.
3 Ultimate BOD values and rate coefficients
may be estimated by method. suitable for
the Streeter-Phelps equation.
4 Nitrogenous demand may be evaluated
by inclusion in the ultimate BOD value
or calculation from ob.erved nitrate
5 Although thi. equation represents a
.ignificant improvement over other
forms, determination of the values of
the .everal constant. and terms may
prove difficult. The effect. of diffu8ion
of oxidizable matter from the benthal
layer, for example, may be included in
the value of either La or DB' In either
case, this effect would be combined with
that of other processes. This equation
represents the most rational expression
of the DO balance yet presented.
Oxygen Dynamics in Str_eam~
A The equations previously presented assume
that all incremental volumes of liquid
which originated at a !liven cross .ectiol1
reach a down.tream section at the samf'
time, i. e. that the flow time of every
incremental volume exactly equals the
di8tance divided by the mean stream
velocity. However, an unknown fraction
of the incremental volumes may reside in
the reach longer than the average flow-
through value, be exposed to the BOD
r..ction. longer, and cause greater 00
depletion at a downstream point than
would be expected durinrr the average
travel time, t.

B Dobbin.21 has presented an equation which
accountl for longitudinal dispersion:
D .
~ La~Gmx r~
KI LA - ~ e - e
K - K
2 r
+ D erx

+~: +

KI L~ ( - er~
K2 K-;J '( ,
D, t, LA' DA . terms previously
KI' K2' Kr' La (Section VI Cl)
m .
U2 + 4 Kr DL
2 DL
r .
U2 + 4 K2 DL (12)
2 DL
U . mear. stream velocity (ft/day)
DL . coefficient of longitudinal
disperaion (sq. ft./day)

OxYien Dynamics in Streams
C Based on the ratio of mx (which exprell8es
the rate of BOD removal in a dispersion-
affected system) to Krt (which expresses
BOD removal in a non-turbulent system),
Dobbins has presented rather convincing
evidence that elfects of 10n,1tudinal dispersion
will rarely be signUicant in flowing streams.
In slow-movin.. well-mixed swams
diapersion may be significant.
Sound analysis of the natural oxygen balance
is necessary to accurately predict the oXYlen
profile in sh'eams or to aaaess the influence
of various factors upon oXYlen resources.
Two methods of analysis are available: the
budtet method and mathematical models.
Several examples of the latter have been
presented. In addition, the proceues which
affect oxygen exchange have been identified
and methods for evaluation of their effects have
been indicated.
1 Tsivoglol,l, E, C.. Discl,lssion of Application
of Stream Data to Waste Treatment
Plant Dellign, Oxygen Relationships in
Streams, Technical Report W58-2,
U. S. Pl,Iblic Health Service. (1958)
2 BhatIa. M.N. and Gaudy, A.F., Jr..
R()le of Protozoa in the Diphasic Exertion
of BOD, Proceedings of the American
Society of Civil Engineers, 91, No. SA3,
June 1965.
3 Tn..rwu1t, E. J., Oxygen Demand of Polluted
Waters, Public Health Bulletin 173, 145.
(1927 )
4 Courchaine. R. J., The Significance of
Nitrification in Stream Analysis -
Effects on the Oxygen Balance,
Proceedings, 18th Industrial Waste
C:mference, Purdue University. (1965)
5 Dowmng, A. L. d a1, Nitrification in the
A eU vated-S~udge Process. Journal and
Proceeding. J[ the Institute of Sewage
Purification, Part 2, 130. (1964)
6 Fair, G. M. et al, The Natural Purification
of River Muda and Pollutional Sediments,
Sewage Works Journal, 13, 27. (1941)
7 Hanes, N. B. and Irvine, R. L., New
Teclmiques for Measuring Oxygen
Uptake of Benthal Systems, JOQl'nal
01 the Water Pollution Control Federation,
40,223, February 1968.
8 Standard Methods for the Examination of
Water and Wastewater, 12th Edition,
A merican Public Health Association,
American Water Works AS80ciation,
and the Water Pollution Control
Feden.Uon. (19115)
9 Camp, T. R., Field Estimate8 of Oxygen
, Ba1anee Parametera, Proceedings of
tbe American Society of Civil Engineers,
91, No. SA5, October 19115.
10 Hull, C.H.J., Photosynthetic Oxygenation
01 a Polluted Estuary, Journal of the
Water Pollution Control Federation,
35,587. (1963)
11 Odum, H. T., Primary Production in
Flowing Waters, Journal of Limnology
and Oceanolraphy, I, 102. (1956)
12 O'Connor, D.J. and Dobbins, W.E..
Mechanism of Reaeration in Natural
Streams, Tranaactlol\s, Amer~can
Society 01 Civil EDgineers, 123, 641.
( 1956)
13 Taivollou, E.C. et .1, Tracer Measure-
ment 01 Stream Reaeration. 11. Field
Studies, Journal 01 the Water Pollution
Control Federation, 40, 285, February
14 O'COIU1ell, R. L. and Thomaa, N.A..
Effect of Benthic Alpe on Stream
Di.solved Oxy,en, ProceedJ.nas 01' tht'
American Society of Civil Engineers,
91, No. SA3, June 1965.
15 Hull, C. H. J., DiScu9"'10n of Effect 01
Benthic Algae on S~ream Dissolved
Oxygen by O'Com '. p. L. and Thomas.
N.A., Proceedin!!>! v. the American
Society of Civil Engineers, 92, No. SA 1,
Proceedings Paper'4R37, Febrm'ry 19611.

Thomas, H. A., Jr., Pollution Load
Capacity of Streams, Water and Sewage
Works, 95, 409. (1948)
Gannon, J. J., River and Laboratory BOD
Rate Considerations, Proceeding. of
the American Society of Civil Ervineers,
92, No. SA1, February 1986.
Streeter, H. W. and Phelps. E. B., A Study
of the Pollution and Natural Purification
of the Ohio River, Public Health Bulletin
146, U. S. Public Heahh Service,
Washington, D. C. (1925)
19 Purdy, R. W., What Do We Know About
Natural Purification, Proceedings of the
American Society of Civil Engineere,
94, No. SA1, February 1968.
20 Wastewater Reclamation at Whittier
Narrows, State of California, The
Re.ources Agency, Publication No. 33,
Page ix. (1966)
Oxygen Dynamics in Streams
Dobbins, W. E., BOD and Oxygen
Relationships in Streams, Proceedings
of the Am= rican Society of Civil
Engineers, 90, No. SA3, June 1964.
O'Connor, D. J., Oxygen Balance of an
Estuary, Transactions, American
Society of Civil Engineers, 126.
Part m. (1961)
This Q1 tUne was prepared by F. P. Nixon,
formerly Acting Regional Training Officer,
Northeast Regional Training Center, Hudson-
Delaware Basins Offtce, Edison, NJ 08817.
Descriptors: Biochemical Oxygen Demand,
Dl.solved Oxygen, Equation., Mathematical
Models, Oxygen Demand, Oxygen Sag,
Photosynthetic Oxygen, Reaeration, Water
Quality Control

A Objectionable sludge deposits are now
commonly prohibited by regulatory
authorities. The final report of the
National Technical Tasf Committees on
Water Quality Criteria recommends that
sludge deposits be prohibited in receiving
waters rated for all major water uses.

B Such extensive control is largely based on
widespread recognition of the potential
oxygen demand of benthal deposits.
Although large benthal demands have
frequently been observed, prediction of
their magnitude and incidence is difficult.
A Benthal deposits affect oxygen resources
in two ways:
1 They exert an oxygen demand through
aerobic respiration within the benthal
layer. Oxygen for this procel!s must
be I!upplied by diffusion from or mixing
with liquid phase.
2 They release to the overlying liquid
simple organic compounds which exert
additional BOD. The composite rate
of biological degradation in the over-
lying liquid may increase or decrease
downstream of the area of deposition.

S Anomalies in the dissolved oxygen (DO)
sag curve may be caused by deposition.
The apparent effect, depicted in Figure I,
is similar to that of an unsuspected outfall.
TIJo\E 0 flOW
The magnitude and incidence of benthal
loads depend upon the amount and nature
of putrescible matter in the waste and the
frequency and duration of periods of
deposi tion.

C During periods of high flow or turbulence
biodegradable sludges may become re-
suspended and deplete overlying waters
of oxygen. Thus a body of oxygen deficient
water may pass downstream and harm
fish and other aquatic life for a consider-
able distance. Many feel that this effect
is more dangerous than exertion of a 3
continuous, albeit unsuspected demand.
A The potential for deposition may be
evaluated by examination of a plot of
cumulative time of passage versus distance
along the stream reach. as shown in
Figure 2. Deposition may be expected in
those sectIOns where v:flocity Is leBS than
a cntical value. Velz recommends a
critical velocity for sedimentation of 0.6
ft/ see and a critical velocity for sour of
1. 0-1. 5 ft/sec. The magnitudes of these
velocities would depend upon the nature of
the waste and may need to be determined
B Deposition potential IS usually evaluated
at critical flow.
Although the locatIOn of potential benthal
demand is rather easily determined,
WP. NAP. 2la. 3. 74

E;ffect of Benthic Deposits on Oxygen Dynamics

evaluation of itll magnitude is more difficult.
Benthal demand is dependent upon the nature
of the was~e and the amount of \ccumulation
in the area of deposition. Velz hall proposed
a procedure by which the effects of the
colloidal and dissolved portion of a waste may
be evaluated separately from those of the
settleable portion.

A The ultimate biochemical oXygen demand
(BOD) of the settleable fraction is assumed
to be tI~e difference between the ultimate
BOD of the raw waste and the ultimate
BOD o~ a sample allowed to settle for one
B The aerobic removals of BOD of the
colloidal and dissolved and the settleable
portions are plotted separately, as shown
in Figure 3. Rates of aerobic satisfaction
are either assumed or determined experi-
mentally. Rate coefficient progressions
may occur in either phase.
C At the point of deposition the remaining
settleable matter becomes wholly
incorporated in the benthal mass. If the
retention time of the sludge mass is large,
the demand exerted may nearly equal the
ultimate BOD of the daily addition of
settleable matter.
I The removal of BOD from the deposit
will equal the amount satisfied by
aerobic oxidation plus the amount
released to the overlying liquid.
Carbonaceous matter released to the
overlying liquid as methane (CH4) may
escape to the atmosphere or be utilized
in further bacterial metabolism.

2 The factors which influence the relative
importance of these processes are
extremely complex and not well
understood. Management decisions are
commonly based on the assumption that
the oxygen demand exerted on the over-
lying waters equals the ultimate BOD of
the daily addition to the deposit.
3 Nitrification becomes more established
as the age of the deposit increases.
Moore indicates that a large portion of
the total organic nitrogen content of
"young" deposits is liberated as ammonia
whereas NH3 evolution from "old" deposits
is minimal.
D Downstream of the benthal deposit the
composite BOD rate coefficient may increase
or decreass depending upon the amount of
elution, the nature of the compounds re-
leased, and the presence of acclimated
organisms. Several environmental variables
especially temperature and stream flow. .
influence this rellponlle.
Benth.al deposits affect oxygen resources by
exertmg an aerobic demand within the benthal
layer or by releasing to the overlying water
compounds which exert additional BOD. Tpe
location of areas of potential d
2 Velz, C. J. Significance of Organic
Sludge Deposits, Oxygen Relationships
in Streams, Technical Report W58-2,
U. S. Public Health Service, 1958.
3 Tarzwell. C. M. Informal Discussion.
Oxygen Relationships in Streams, p. 60.
Effect of Benthic Deposits on Oxygen Resourc< s
This outline was prepared by F. P. Nixon.
formerly Acting Regional Training Officer,
Northeast Regional Training Center, Hudson-
De1aware Basins Offi(.e, Edison, NJ 08817.
Descriptors: Benthos, Biochemical Oxygen
Demand, Oxygen Demand, Sedimentation,
Sewage Sludge, Sludge

A Various workers have emphasized the
effects of aquatic p'lants on oxygen
resources. CampI reported that two-
thirds of the di8solved oxygen (00)
resource of the Merrimack River ~s
derived from photosynthesis; Hull' 3
reported similar results for Baltimore
Harbor and the Delaware River estuary.
B Other investigators have found aquat1c
plants detrimental to the dissolved 4
oxygen resource. Gunnerson and Bailey

- .


benthic alma.
o .
(602) BIOL . P benthic
and O'Connell and Thomas5 have
reported extreme depletions and rapid
00 fluctuations in waters containing
large benthos and phytoplankton
A The oxygen concentration of a body of
water is the composite effect of add~tion
and depletion of oxygen from solutIOn
by biological and physical processes,
shown in Figure I.


zoop lank ton

.J..:~~ pended

(A02)BlOL 1 OF
- Rbenthic
. Pphytoplankton
- .phytoplankton
- R.u.pen4ed
. P perlphyton
- Rperlphyton
- R benthic
anima I.
BI. ECO. 21.3.74

Effects of Aquatic Plants on Oxygen Resources
EquatIon (1) indicates that the change
in value of thO' oxygen resource equals
the net effeel of all !;Jiological proc-
,'sses, (~02)BlOL' plus or minus gas
transfer at !lie water surface, DF.
Equation (2) indicates that the net
effect of all biological processes
equals the Rum of oxygen production
by photosynthesis, P . .. and
oxygen depletion by i~s'P'tration,
R . .. for the several habitats or
cft.'ldes of organism present, X, Y, . ..
Some organisms have no photosynthetic
capability and exert only a depleting
Terms of equations (1) and (2) may be
expressed as time rates of change
(g/sq mlhr, mg/l/hr) or as increments
of amount or concentration (lb, mg/I).
B In the presence of significant plant growth
the several photosynthetic and respiratory
processes combine to produce the elas-
sica} dissolved oxygen variation shown
in Figure 2. The classical curve reaches
a maXImum during mid-afternoon and a
minimum Just before sunrise. 00
concentration may exceed the 24-hr.
nwan fO!' a large portion of the day.
DU!'lng mid-afternoon 00 may exceed
saturation con(','ntration and oxygen will
he vI'nled 10 the atmosphere.
A Engineers are commonly asked to
estimate two quantities:
The fraction of the total DO resource
which is attributable to aquatic plants,
2 The contribution of aquatic p]ants to
community respiration.
3 In a complex environment it is very
difficult to independently estimate
the effects of each class of organism.
Effects are usual]y estimated
collectively for identifiable groups,
such as plankton, benthos, or the
entire community. Several methods
have been developed to estimate the
plant contributions; none exists for
independent measurement of plant
4 If a single organism'is predominant
within a reach the measured overall
effect is usually attributed to that
class. The degree of predominance
must be independently demonstrated.
B Photosynthetic Production
The amount of oxygen added to a body
of water by aquatic p]ants equals the
gross production of oxygen by photo-
synthesis less the oxygen requirement
for plant respiration and the amount of
oxygen vented to the atmosphere.
(P -R) is often called net production for
the habitat or c]ass of or.ganism involved.
Values of P, R, and (P-R) ma.y be
determined for planktonic communities
by light-dark bottle techniques. 1,2,3
a Gross photosynthesis, P, equals
the DO change in a bottle incubated
under light minus the change in a
bottle incubated in darkness.

b Planktonic respiration, H. equals
the DO depletion in a bottle
incubated in darkness, If both
bacteria and suspended algae are
present the depletion will be the
result of the activities of both
types of organisms.
c Net production equals the DO
change in a bottle exposed to light
and darkness for the same periods
ohserved in the natural environment.
Estimation of net production by
li!l,ht-dark bottle techniques may
I'equire submergence of several
sets of bottles in the stream itself,
In this manner natural conditions
are closely duplicated,
e The light -dark bottle technique is
mOl'e representative if DO concen-
tration does not exceed saturation,
The technique may be affected by
nutrient supply, lack of turbulencE',
or oxygen concentration,

2 Odum1s6 method
Net production may also be determined
by correction of the observed 00
variation for atmospheric diffusion.
The diurnal variation of oxygen is
plotted for a point or a stream
reach as shown in Figure 3A.
For each incremental sampling
period the rate of change of oxygen
concentration is calculated and
plotted as in Figure 3B.
Based on an assumed or determined
exchange coefficient the rate of
atmospheric 02 transfer is plotted
as in Figure 3 C.
Effects of Aquatic Plants on Oxygen Resoul'ct'';'
d Summation of curves 3B and 3<'
will produce a curve of the rat<.
of net production. Figure 3D.
This curve will indicate when
respiration exceeds production.
If the average value exceeds zero,
oxygen is added to the flow ing
water by aquatic plants.
e This technique includes the effec.s
of all influences and groups of
organisms. The effect of the
benthic community may be
estimated by subtraction of light-
dark bottle results from the overall
production values estimated by
Odum's method.
C Plant Respiration
Techniques for independent estimation of
plant respiration have not yet been
developed. Composite respiration
values may be estimated by dark bottle
or dark algal chamber techniques or b)
field measurement of oxygen exchange
during darkness.
The effect of aquatic plants on oxygen
resources is widely recognized. Such
plants have been called both beneficial
and detrimental. This classification i8
usually based upon the rated use of the
stream and the intensity of the diurnal
oxygen variation. Techniques are avail-
able for estimation of net production. (P -R),
for plankton. benthos. or the entire
community. Respiration can be determined
only collectively for several groups.
The basic principles of two major estimating
techniques have been presented.

Effects of Aquatic Plants on Oxygen Resources
 10. A
 8 -     
(../1) It -     
 NOCI4 3 6 9 MID-
 2 .     
.1.0 .
~ "
I "
,. ....
r ~,
~ "
~ '-
, ~
-1.0 ,

o --------.............
(ftI~/l/hr) ~ .'"

-5.0- -----..
(p - r)
" ~.
...... wi-

Camp, T. It. Field Estimates of Oxygen
Balance Parameters. Proceedings
of The American Society of Civil
Engineers, 91, No. SA5 1
October 1965. ' .
Hull, C. H. J. Photosynthetic Oxygenation
of a Polluted Estuary. Journal of the
Water Pollution Control Federation
34,275. March 1962. '
3 Hull, C. H. J. Oxygenation of Baltimore
Harbor by Planktonic Algae. Journal
of the Water Pollution Control
Federation, 3", 587. May 1963.
4 GW1nersnn, C. G. and Ba !ley, T. E.
Oxyg..n Relationships in the Sacramento
River. Proceedings of the American
Society of Civil Engineers, 91, SA3, 1.
.June IUr,,,.
Eft<:dS of Aquatic Plants on OXygen Resourc'"s
O'Connell, R. L. and Thomas, N. A.
Effect of Benthic Algae on Stream
Dissolved Oxygen. Proceeriings
of the American Society of Civil
Engineers, 91, SA3, 1. Jt'l1e 1965.
Odum, H. T. Primary Production in
Flowing Waters. Journal of
Limnology and Oceanography, I, 102.
This outline was prepared by F. p, Nixon.
former Acting Regiona.l Training Officer,
Hudson- Delaware Basins Office, Wate r
Quality Office, Edison, NJ 08817.
Descriptors: Aquatic Plants, Aquatic
Populations, Aquatic Productivity, Dissolved
Oxygen, Dirunal Distribution, Oxygen
Demand, Photosynthetic Oxygen, Productivity,

A Oxygen normally composes approximately
20% of the atmosphere. but from only 0.5
to 1% of unpolluted water by volume
(9 - 14 milligrams per liter. or parts per
B Physically, oxygen is a colorless gas,
responsive to the laws of solubility,
temperature, etc.
C The amount of oxygen present can be
readily determined by various methods,
both chemical and physical.
D Biologically, oxygen is one of the key
elements to allUfe, all living organisms
contain it along with hydrogen and carbon.
It is the physiological "complement" of
ca rbon dioxide.
E When properly interpreted, oxygen con-
centration can serve as an excellent index
to water quality.
1 It is widely present.
2 It is intimately involved in many
processes, chemical, physical, and
RELATIONSffiPS (Figure 1)
A Photosynthesis is the biological starting
point for synthesizing li-.rtng substance out
of non-living carbon dioxide, water, and
other materials. In the process, radiant
energy is adsorbed from the sun (hence it
is an endothermic reaction) and free
molecular oxygen is released to the environ-
ment as long as sulficient light iE; available,
i. e., during day light only.
1 The conventional empirical formula for
photosynthesis is a very grefJ.t sim-
pllfication, as many as twenty steps
BI. ECO. 23.3. 74
may actually be involved (like
respiration, controlled by systems
of enzymes).
6 C02 +6H20+ 673 Cal
(Energy sunlight)

j chlorophyll
(enzyme system)
CSH1206 + 602
(Simple sugar)
2 The end product 16 a basic organic
material known as a simph' sugar
(carbohydrate, monosaccl1aride, of
which "glucose" is an example,
see Figures 1, A) which serves as a
starting point for the elaboration of
higher organic substances, and wtlldl
may also be oxidized or "n~spired" by
the cell to release the stored chc'mical
energy .
Synthesis of organic materials (living
protoplasm) from simple sugars and
other substances in bacteria, algae,
and man.
a Glucose and other simple sugar
molecules can be combined by
living organisms such as algae into
other carbohydrl!-tes such as starch.
b After modification into fatty aCIds
and glycerol, fats can be made.
c Combination with nitrogen and
other minerals taken in from the
surroundin,? water produces aminn
acids. These can then b.. combined
into protein~, the most complicl' ':(,
of all life substances. PJants <11"
the onl~" organisms that can synth""'i,,
amino acids complet~ly "Irom scratch.
f,7 -1

Or.me Enriohment and D18l101ved Oxygen Relationships in Water
H. W. Jock.o"


c~A:1 .~~ I .... .. of Iooto. ....I. cll1ol'O II I!

=-.. -~ 1873.01.) ...... r.. Ii

, { - C,H1IO, + 0a
~ SArNJIyuo,,,: poly.occbarl"'- .u.occurt.o.- "l,.....euri...

g3 F.,,: fat. ond 1I,1...fotty od.. a". .Iye..ol~ ~

j ~ ;::.18.: ,rot.ta.-I'.""''''' 0".1-10"-1-1 c_Waad wl'l\ If
A. 2 -- ~ pro~.o... - oe'" \0'" 0'"''
~ R , '~ ...iural.

~ ur and om8r _ia~l -
:J .0....tal8. .xc rota 81. ECO. I. 11. !!
',",THUli Alfl:) I:)EOa.ADATIOIf OF norc.LA'w
Figure 1

Organic Ennd~:ll'nl "llU Di<:lsolved OXy£..i. :,.elationships in \\,,1'1'

HIE ~.t~tt>I!.ijneEil C'CLE 'n IA~~!!- '/T:-\\

C..RtIOH DIQXQ;M "r.R >U! - --
'. ,. '-_/"'"
.', ' 111 "
/ / E:NEFtJy
, / :
REU' '.r U '
(r.AY.~f;N tn~:"'E'A~"~)1'
800.. '..0' - ~ 0."0.'.0.
Figure 2
4 All of thesl' synthetic or grow1h
processes are endothermic and require
energy to consummate. This energy
is obtained by respiration.
B Respiration. Life processes such as those
mentioned pn'viously (II ,\ 3, also physical
movement) are energized by the controlled
oxidation or "respiration" of single sugars.
This is a cellular or physiological process
and continues constantly in all living things,
independently of light, as long as life
Types of respiration. Respiration is
an energy releasing or exothermic
biochemical process and is to be
distinguished from the external bodily
BI. ECO. 2&. 3.59
act of "breathing", which is a proc'~ss
involving a lung, gill, or oth,." organ,
whereby molecular oxygen is brought
inside the body of the organism and
carbon dioxide is released. Breathing
is transportation; respiration is
burning or biochemicalutilization.
a Aerobic respiration. The most
common form of respiration ib
illustrated by the oxidation of
glucose, using free dissolved
oxygen from the environm",nt.
This is also the most efficient
type since virtually all of the
chemical energy contaim:d in the
glucose molecule is reh,ased
(f)73 Cal. ) and the residue is com-
pletely mineralized or stabilized to
carbon dioxide and water"
(. 'I~ 3

Orpnic Enr1ctunent and Di..olved OItYlen Relationships in Water
Although actua~ taking place in
more than twenty steps involving
as many different enzymes, the
overaU process can be empirical1y
represented by the following
C6H1206 + 602
(a simple sugar such al gluoole)
6 C02 + 6 H20 + 673 Cal
(enzymes) (energy)
b Anaerobic respiration. If free
dissolved oxygen (DO) is not available
from the environment, certain types
of bacteria, flUlgi, and other orp.
nisms known ae "facultative aerobes"
can switch to another, less efficient
type of respiration and suB obtain
energy by breaking down simple
sugars. This is known as anaerobic
respiration. Some types of micro-
organisms, the true anaerobes, can
thrive 5 under anaerobic conditions.

The anaerobic respiration of glucose
involves splitting the molecule with-
out the use of outside oxygen so as
to release some of the contained
,-,nergy, but not aU, The remainder
is still available In the end products.
A common empirical relationship is
as follows:
3 CaH1206- 2 CH3(COOH)2
(.glucose) (enzymes) (lactic acid)
+ 2 CH3CH20H + 2 CH3 COOH
(ethyl alcohol) (acetic acid)
59 1/2 Cal
+ (energy)
c Anaerobic respintion of proteins
and their derivations by micro-
organisms (known as putrefaction)
often leads ~ the production of foul
smelling end 'products. Aerobic
respiration of proteine (known as
dry) seldom pro4uces offensive
o liS. In addition to carbohydrates,
proteins, fats, and various other
nat~l and unnatursl hydrocarbons,
and Other substances can also be
broken down or respired to rel-
atively stable end products by
2 The adaptability of life is remarkable.
By the same mechanisms of genetics
and natural selection which have
resulted in the evolution of present
day life on earth, microorganisms
have evolved which are capable of
obtaining energy from, or in other
words, "respiring" a great variety
of new .,mhetic materials, the products
of modern industry. This is the basis
for the biological treatment of wastes.
a The types of biological mechanisms
involved. in general, are !mown
and to a degree, IUlderstood.
b Many species or kinds of orga-
nisms may be involved in the
stabilization of a single type of
3 Significance of respiration in sanitary
engineering. The process of respi-
ration uses or "demands" oxygen from
the environment. ~ material, such
as sewage. an organic industrial waste.
motor 011, etc., which will support
life of any kind will thus eJCert (through
the organisms living on it) a "BOD".
a Respiration has two end "products":
energy, by which the organisms
live; and degraded. mineralized.
or stabilized material such as
digested sewage sludge which
retains little " no energy available
to organism... :ilat is, they have
no biochemical oxygen demand or
BOD remaining.

Organic Enrichment and Dissolved Oxygen Relationships in \\ at<'.
b Since it i8 independent of light. it
goes on night and day.
A Solubility of oxygen in pure water varies
with temperature and Pressure according
to well known laws and values.
I We expect to find less oxygen in warmer
waters and more in colder. This has
both seasonal and geographical
2 Dissolved substances such as salts tend
to reduce the solubility of oxygen at a
given temperature (See Figure :J -
Oxygen Solubility at Selected Salinities).

. I() .
OIllOLVlO C*Vltw , J,'"
.,~ - _Of, ..--.. --- ,-
Figure 3
The concentration of oxygen often is
expressed as percent of saturation.
a Organisms, however. are affected
by the real quantities present. and
actually demand the most oxygen at
the highest temperature. when the
solubility is lowest.
b It is therefore more significant,
where organisms and stream con-
ditions are concerned. to express
concentration as milligrams per
~ (or parts per million).
B The movement or distribution of oxygen
throughout a water mass is not entirely
dependent on molecular diffusion. but
also involves various types of gross
water movements. Reaeration (or aerlltl".l'
is the transfer of oxygen from the air intn
the water mass.
A Chemical toxicity may delay the exertion
of biological oxygen demands. or the
release of oxygen by biological mechanism",
B Chemical substances may themselv('~
demand oxygen from the water.
1 If oxygen is available. the chemical
oxygen demands tend to be satisfied
relatively quickly and h,~ncc are likely
to be a more local problem.
2 They are indeper.dent of any biological
3 They are in addition to biological
A Diurnal interactions of photosynthesis
and respiration. (Figure 6)
Life, growth. and hence the need for
oxygen for resp:L.ation continue
twenty-four hours a day. as long as
life persists.
2 Since photosynthesis in nature is
activated by radiant energy from the
sun, it is operative only during the
daytime. It releases approxima1 ely
twenty times as much oxygen as is
consumed in cellular respiration.
however. so there is a great excess
left over which diffuses out into the
surrounding water. Here it is availab],.
to biochemical oJ:idative demands of
other organisms with the result that
the ecological system becomes "ac:rfJIJlC;. .

Organic Enrichment and Dissolved Oxygen Relationships in Water
3 DO's affected by photosynthesis thus
tead to be hi hest in the da ime
(particularly mid-afternoon and lowest
at night (3 - 4 A.M.). -
B Orgaaic materials from domestic sewage
or similar wastes are readily available
to microorganisms. Assimilation of food,
and jtrowth and multiplication of the pop-
ulati"n can thus begin relatively quickly,
thus -establishing a demand for oxygen for
1 It takes a significant period of time for
a population of bacteria, fungi, and
other microorganisms such as protozoa
to become established, after the
initiation of growth of the few original
organisms, even under the best of
2 Dissolved oxygen does not thus di8appear
instantly from the water upon the
admixture of sewage, but rather begins
to diminish slowly. As the population of
Qxygen consuming organisms builds up,
the delicit in the water increases. As
this occurs, any equilibrium which may
have existed with the atmosphere is
destroyed, and the rate of aeration
through the BUrf'acefiim increases.
a As the population of microorganisms
grows Btilllarger, its rate of increase
begins to increase (logarithmic
growth). Very soon a size of pop-
ulation is achieved where the demand
for oxygen exceeds the amount
which can be supplied through the
surface film. The concentration of
free 00 now begins to drop sharply
and may go to zero.
b If sufficient food for the micro-
organisms remains, anaerobic
conditions will now prevail.
Oxygen, of course, continues to
enter at a maximum rate through
the surface film, but is immediately
used up. The overall rate of
oxidation of thF waste or1OOd
material however, is now very low
(although the total amount may be
"7- 6
large), as surface aeration can
supply but a small portion of tht'
oxygen needed.
c If there is no replenishment of the
food supply by repollution, the big
population of fungi and bacteria
eventua~ uses up most of the
available substrate or food, and
begins to starve. Growth now
begins to slow down, and the pop-
ulation eventua~ drops back to
its original low level.
d Meanwhile time has passed. An
abundance of fundamental plant
nutrients such as nitrate, phosphate,
potash, etc., have been released
to the water as a result of micro.
bial respiration. Algae begin to
grow on this substrate, and increase
rapi~ to tremendous numbers.
e Algal photosynthesis thus suddenly
begins to release great quantities
of oxygen in the daytime. At first
this is exhausted each night by
respiration of the algae and other
microorganisms, but soon persists
around the clock until the algae in
turn have exhausted their food
supply and return to numbers
normal for an unpolluted stream.
f With the above mentioned advent
of free DO from the algae, the more
efficient aerobic type of respiration
can again be employed by the
microorganisms. This hastens
the oxidation or stabilization process,
and leaves behind but a minimum
residue of well mineralized matt'rial
to accumulate on the bottom.
g It should be mentioned that turbidity
from suspended microorganisms
and other organic solids frequently
inhibit the establishment of an algal
population until the biochemical
oxidation or stabilization process
has been W" U started. Turbidity
inhtbits a1j.,~) :;:'owth primarily by
shading and suppression of photo-

Organic Enrichment and Dissolved Oxygen Relationships in Water
C When organic material is clumped into a
body of water faster than it can be oxidized
by the mechanism described above, it
tends to accumulate on the bottom in a
partially digested condition known as
1 Great quantities of nutrient energy are
still present, as well as a huge popu-
lation of microorganislT'/!. There is a
very great need for oxygen which, if
available, is avid~ taken up by the
organisms in the surface layers,
a Aerobic conditions in the overlying
waters thus tend to hasten the
stabilization of sludge banks by
providing oxygen for aerobic type
b Anaerobic waters, on the other hand,
tend to preserve the sludge by
restricting all organisms to anaer-
obic types of respiration. It has
been reported that the speed of
decomposition begins to be restricted
at concentrations below 1 ppm.
Figure 4

Non-Plimented, Non-OxYien-Produclni,
Protozoan Fla,ellalea

2 Anaerobic conditions are present
throughout the sludg<;! mass below the
surface. Stabilization is still
proceeding, but at a much slower
rate than if oxygen were present.
D In the deeper reservoirs, lakes, rivers,
and estuaries thermal stratification may
develop in the summer.
1 The epilimnion or upper layer, being
in contact with the atmosphere,
receives a continual replenishment of
oxygen by aeration processes.
2 If turbidity is not excessive, photo-
synthesis by algae (Figure 5) will
also provide oxygen.
The hypolimnion or lower layer, being
separated from the atmosphere and
frequently from the light, tends to
acquire an excess of carbon dioxide
and a deficiency of oxygen due to the
respiratory activities of micro-
organisms (such as protozoa, fungi,
and bacteria). (Figure 4)
This effect is heightened if the bottom
material is high in organic content
as has been shown below.
Figure 5

Pl8mented, OxYien-Produclni. Alial

,'lJI.'.~ ~
:;",\.1 h~~

.. ~
(~~.....>- ,
. '.''''::''~''!';;h
(, i

Or_nic Enrichment and Dissolved Oxygen Relationships in Water
(Figures 4. 5, 6)
A Problems to man may result when the total
"primary production" by algae leads to an
increase in the total organic content of the
water that interferes with a desired use.
I This may consist of a high algal
popUlation that produces a water with
high turbidity, taste and odor, or other
undesirable effect. High respiratory
needs may lead to nocturnal oxygen
2 Certain algae may cause tastes and
odors, clog filters; or otherwise inter-
fere with potable water processing.

(GCTGID I, .117)
'~ 0.&
3 Death of large algal populations may
lead to obnoxious odors through
bacterial decomposition. Oxygen
deficits may result at any time of day
in this process. Deposition of masses
of organic sediment or sludge in
estuaries and back waters may be
B The primary production of a:i.gae can also
serve a8 a supp1;y of food to consumer
organisms (animals), resulting in
increased production at several (trophic)
levels: (Figure 7)
zo'dmicrobe8, microinvertebrates.
macroinvertebrates, fishes.
19. CII. 111II1II
OCT. " 187

Figure 6

Organic Enrichment and Dissolved Oxygen Relationships in Water
Mean Surface Water Productivity Rates of th~ CreRt lakes
and Other Freshwater Lakes and Ocean Areas.
Lake or Ocean ProductiVit) Rate  Investigator
and Location In mg.CI m Iday 
Lake Superior  16.62 Parkos (1967-68) 
Lake "l'ron  23.04 Parkos (1968) 
Lake ~Iichilan 37.62 Parkos (1967) 
Lake Erie  175.20 Parkos (1968) 
Grand Traverse B.y, 0.34 Saunrle r s .!! a 1. (1962)
N.E. Lake ~1ichhan
Douglas Lake, Mich. 0.23 Saunders .!! a1. (1962)
Bras d'Or Lake, 23.5 Geen and Hargrave (19&&)
Nova Scotia, Ca~ada
Brooks Lake, Alaska 3.2 GoldMan (19 (0) 
Maknek Lake, Alaska 8.8 Goldman (19&0) 
Torne Trllsk, Sweden &.4 Rodhe (1958b) 
Ransaren, Sweden 8.8 Rodile (19 ~8bj 
Lake Er.ken, Sweden 221.0 Rodhe (1958b) 
     Jonasson and 
Lake Esrom, Denmark 1600 Mathiesen (1959) 
     Jonasson and 
Lake Fures4, Denmark 2100 Mathiesen (1959) 
N.E. Atlantic - 21 Steemann-~lelsen (1960)
npar Denmark    
North' Atlantic, same 7.5 Steemann-Nielsen (1958b)
lat. as Great Lakes    
Southeast PacIfic 3.3 Holmes (19&1) 
Southwest Pac if ic 7.2 Angot (1961) 
North Pacific, 6.4 Koblen tz-M1shke (1961)
Sub-Arctic ReRion    
Figure 7
0- a

Organic Enrichment and Dissolved Oxygen Relationships in Water
Earlier notation cited the release of
oxygen during utilization of C02 during
algal photosynthesis. This encourages
fuagal or bacterial breakdown of
2 Photosynthesis occurs in the presence
of adequate Ught and favorable conditions.
In darkness, the cells continue to
respire and may consume more oxygen
than they produced because photo-
synthesis increases the organic :load.
3 Photosynthesis tends to occur at the
surface where light intensity is greatest
(Figure 6). Poor vertical mixing
would result in stratification of water
super'saturated with oxygen over oxygen
deficient water. Depending upon con-
ditions, a significant fraction of the
oxygen could be lost to the atmosphere.
This outline contains certain material sub-
mitted by F. J. Ludzack and M. E. Bender.
1 Anonymous. Aquatic Life Water Quality
Criteria. Aquatic Life Advisory
Committee on the Ohio River Valley
Water Sanitation Commission. Second
Progress Report. Sewage and Ind.
Wa-st"s. 28(5):678-690. 1956.
2 Anonymous. Oxygen Relationships in
Strr'arns. Proc. of Sem. at R. A.
Taft Sanitary I~ngineering Center.
O..tol",r 30 - November 1, 1957.
Bartsch, A. F. Algae in Relation to
Oxidation Processes in Natural Waters.
Special Publ. No.2. Ecology of Algae.
Pymatuning Lab. of Field Biology.
U. of Pittsburgh. Pittsburgh, Pa.
pp. ,,6-71.
4 Carp,'nter, J. H., Pritchard, D. W., and
Whaley, R. C. Observations of
Eutrophication and Nutrient Cycles in
Some Coastal Plain Estuaries. in:
r;utroohication; Causes, Consequences,
Correctives; pp. 210-221. Proc. of
Symposium. June 11 ~ 15, 1967.
Pub I. Nat. Acad. Scl. Washington,
DC. 1969.

5 Custer, S. W. and Krutchkoff, R. G.
Stochastic Model for BOD and DO
in Estuaries. ASCE. Jour. San.
Eng. Div. Vol. 95. pp. 865-886.
6 Ketchum, B. H. Eutrophication of
Estuaries. in Eutrophication:
Causes, Consequences, Correctives;
pp. 197-209. Proc. of Symposium.
June 11-15, 1967. Publ. Nat. Acad.
ScL Washington, DC. 1969.
7 Olsen, Theodore A. Some Observations
on the Interrelationship of Sunlight,
Aquatic P1ant Life, and Fishes.
62nd Arumal Meeting of the Am. Fish
Soc. Baltimore. 1932.
8 R,ichards, F. A., and Corwin, N. Some
Oceanographic Applications of Recent
Determinations of the Solubility of
Oxygen in Sea Water. Limnology and
Oceanography. 1(4):263-267.
October 1956.
9 Ruttner, Franz. . (Translated by D. G.
Frey and F. E. J. Fry.) Fundamentals
of Limnology. Univ. of Toronto Press.
pp. 1-242. 1951.
I j
10 Truesdale, G.A.,Downing, AI, and
Lowden, G. F. The Solubility of
Oxygen in Pure Wlioter and Sea Water.
J. Appl. Chem. 5(2):53-62. 1955.
This outline was prepared by H. W. Jackson,
Chief Bilogist, National Training Center,
Office of Water ProgFams, EPA. Cincinnati,
Ohio 45268
Descriptors: Algae, Biochemical Oxygen
Demand, Chemical Oxygen Demand, Dissolved
Oxygen, Eutrophication. Microorganisms,
Oxygen, Oxygen Dema. " nxygenation, Photo-
synthesis, Photosythehc uxygen, Respiration

A Definition
The word "r'econnaissance" is derived from
the word "reconnoiter" which means to
conduct a preli~inary examination or survey.
Its earlier apphcations to engineering and
military requirements has been expanded
to include photomapping and interpretations
of natural resources. Aerial reconnaissance
can be defined as "airborne examination or
survey procedures performed by heavier-
than-air craft, lighter-than-air craft, or
ea rth orbiting satellites. "
B Types of Aerial Reconnaissance
1 Visual
Examination of the flight path by a human
observer with no provision for permanent
recording for later study. This form
of aerial observation has limited use but
in many cases can complement the other
2 Image forming sensor
Image recording of the covered flight
path where maximum advantage can be
made of image interpretation techniques.
Image forming sensors includes such
instruments as cameras, infrared
scanners, and radar.
3 Nonimage forming sensors
Nonimage forming sensors include such
devices as oscilloscopes, strip charts.
and dial indicators which directly indicate
parameter differentials as received by
the sensing elements from the target scan.
4 Combination image forming and nonimage
It is advantageous at times to combine
both of these techniques in order to
rapidly interpret flight path imagery.
WP. SUR.fm. 6a. 3. 74
This advantage Is shown in Fq{ure 1
where a heated effluent discharge is
traced to a canal by infrared imager'y
and indicated by the "lighter" plumf:
which parallels the shoreline. The
oscilloscope phototracing indicates the
temperature differential of the discharge
point as compared to the offshore bay
C Detected images can be identified by one
or more of the following criteria.
1 Size of image
2 Shape of image
3 Tonal qualities of image
4 Image profile shadowing
5 Location of image
6 Texture patterns of grouped images
7 Spatial relationship of image to
surrounding bodies
D Selection of Remote Sensing Method
I A knowledge of the electromagnetic
spectrum (Figur'e 2) is important in
order to select the most advantageous
method of remote sensing. It will be
noted that there are transmissibility
and sensitivity limits as well as existing
film limits with respect to equipment
wave length patterns.
2 Remote sensing methods can be
categorized into two areas.
a Source active: Utilization of an
instrument which is capable of
emitting a source of energy which
is transmitted to the target area and
emanations are received whose
characteristics are dependent upon
the nature of the specific target.
70- I

Aerial Reconnaissance in Pollution Surveillance
~ ~
~;~:- _L~"':
~:_= ~~---:_1' ..-: ~ .
... ~.-
~~-~-~ r"
flGUlf 1
An example of such a device is the
Racial' set (Figure 3) or special
Infrared Emitters both of which are
capable of being utilized in total
eneri)' "Beatterine" and yet not long
enough 10 that photographic recording
is imponible.
b Source panive: Utilization of
instrumentation which is only eapable
of receivinl target area emanations.
Such remote sensinlinltruments,
therefore, do not transmit artUicial
energy sources but depend upon the
sun as a Bource of energy which is
selectively received and individually
reflected by each object in the target
area. Aerial photography and most
of the remote sensing is by this
mejjhod and the included regions of
the electromagnetic spectrum are the
upper portions of the UV band to the
near infrared region.
Utilization of camera systems (Multi-
band) exposing discrete bands of the
photographic spectrum simultaneously
have allowed the photo-inte1'preter
to make discriminations which are not
possible with single bands if b1ack-
and-white photography. The in-
terpreter may use various pro;lect1on
devices which anow the viewing of
one, two, or more images super-
imposed for a more sophisticated
anaqsis ca~il1ty.
3 Selection of desired wavelength is
depen!ient upon the optimum emission
capability of the instrument which has
the shortest wavelength possible to
sharply differentiate small objects while
being long enough to preclude excessive
A Raciar(RA dio ~etection ~nd ,!!angine)

Radar is particularly u ..ful for discerning
certain type I of vegets- jr , which may not
appear in the best visual quality in
Panchromatic Color Film (Iensitive to the

Aerial K~cunnaissance in Pollution Surveillance
10-3 C.ntim.t.n,
AVE ANGSTROM UNITS 11 Antl,tram Unit= 0.1 Milt;micran,
TO 11,000 A
TO 12,000 A
;?~ - 3

Aerial Reconnaissance in Pollution surveiiiance
entire visible spectrum) and also has all~
weather and around~the-clock capabilities.
Radar in the lonier wavelength ranges is
capable of penetrating dense vegetative
cover a'hd this advantage has been found
to be useful in detecting drainage networks
and geological features.
B Conventional Photography
Conventional photography covers the entire
range df the visible spectrum and employs
both black-and-white film and color film.
Some 01 the special films are capable of
photographing a portion of the infrared band
(the "nt!!ar" infrared region) and this
capability is useful in special situations.
1 Direct photomapping and panoramic scan
Figure 4 illustrated the manner in which
direct photomapping differs from the
panoramic scanning technique and it can
be seen that continual attached mapping
scans can be made of a11 or portions of
the flight path to be analyzed.
2 Multiband photography
Multiband photography is, as its name
implies, the synchronized photography
of up to nine separate photographs of the
same target area each of which is
photographed in a ditfere\1t wavelength.
A distinct advantage of this method is
that, during photo analysis, distinct
anomalies may be evident between
objects appearing in the different bands
and further investigation may be
warranted. An example of this is shown
in Figure 5 where it was observed that
an anomaly in vegetative growth was
apparent in the Infrared wavelength
while not bein, apparent in the other
visual spectral ranges. Further
investigations proved that &ravel washings
in slug intervals had affected normal
growth of the streamside vegetation.
3 Infrared photography
Infrared photograpt 1 f,:'J been found
useful in depicting certain hydrological
features. Within the photographic

J, r
,'I' ,,).. ,'.-.
rl v,~'
J~ ;/
'\ ,/
( ,X
f-/(~'\\ ''-I,~\J
<-- ./

'""\.:iJ.;-,:--.~nLY ~~1_~~:,~1~~1()~~ Sl1!~r~~~~
? / /
(" .

f' J'1 'r,- rnlt S --/ I'

[\," --l< ,----/ ,/"

,,~~-;- -- '---,
t~ ~ - ---~/-
, '\. '.!
, I(~': p f: 4
'r J :},
" ' r'
. II
\ (.'
--. ImpOlred or
dead 'w'8getot1on
1/'1 t, cl8d
fIGU~E 5

Aerial Reconnaissance in Pollution S'.1rveillance
ranges it is not a heat-measuring tool
and therefore cannot detect thermal
anomalies within bodies of water but
instead sharply delineates shoreline and
tributary features and has haze
penetrating qualities. The blues appear
much darker by infrared photography
and the reds. greens, and yellows will
show up much lighter when compared to
a panchromatic color photograph.
4 Gamma Ray Spectrometer
This device functions in the very short
wavelengths and has been found to have
excellent detecting capabilities for
radioactivity. Such a device would serve
as an excellent means of searching for
radioactive waste spills which have
occurred either accidentally or
C Infrared Radiometry
Infrared imagery is different than infrared
photography since the tone of the imagery
is directly related to the infrared radiation
emitted from the target area. This type
of scanner converts the normally
unphotographic IR band to a thermal
imagery. An optical-mechanical device
is necessary since the normal thermal
energy emitted by the camera itself will
influence radiations received from the
target area and this elimination of detector
internal radiation is accomplished by
miniturization and application of extremely
low temperatures. One example of the
usefulness of infrared imagery is shown in
Figure 6 wherE' the dark areas along the
shoreline were found to be caused by cold
water infiltration by a previously unknown
source. Such a finding could be indicative,
for example, of a new untapped freshwater
source or a polluting influence from a
remote source. This cold water infiltration
was not evident by normal visual spectrum
photography and the IR photograph would
only indicate a mO\'e distinct shoreline.
D Automatic Analysis
In thf analysis of large numbers of prints
use can be made of photoelectric scanners
wherein automatic recording of degree of
brightness can be recorded on a tape
printout. An example of this procedure is
shown in Figure 7 where eac:t symbol has
a significance relating to "tone signature. "
The "D" zones are the darker areas while
the other symbols relate to lighter zones.
In the center the clear area is a rIver
tributary. Future predictions are that
automatic encoding can be channeled to
computers wherein decisions can be made,
for inBtance, in water management policies.
E Stereoscopic Analysis
The principleB and methods developed
during World War II for the stereoscopic
analysis of aerial photographs has been a
continuing and expanding &cience. ItB
principles are based upon the fact that the
human eyes are normally spaced about
65 millimeters apart and are capable of
compositing, via the optic nerve, the two
separate and distinct images viewed by
each orbit. Aerial stereoscopic analysis
depends upon the placing of two photographs
precise distances apart with precise
, overlapping as required by normal human
visual acuity. With the aid of special
stereoscopic viewing lenses the viewer iB
able, in mOBt caBeB, to compoBite both
of the photographs in the Bame m9.J1ner aB
with normal vision and thus a sense of
depth can be imparted materially enhancing
the analyzing of the terrain.
Aerial reconnaissance in pollution Bur-
veilJance can be accomplished in a variety
of way. and -the method chosen usually is
dictated by costs involved and what is
desired with reprd to immediate or
potential information.
A Visual Observations
The simplest and least expensive method
is to visually obse~"e the study area and
this can be useful .., ~c .ermining sampling
sites, locating previously undetermined
tributaries, pinpointing visual gross

v e 1l1an "::
. Pollution Sur
. Bance ill
1 K.eCOnnalB
ODD 0 1JJ. D [) D D D [',
XXX "'" t~H' C D D X ~ D D D D 0 ~
\ ») '" . .'r" DO DD DJ D .
. » cc e "", r "CCe DO xee 0 D D .'
( ( eee e "~'-~." ~ ~ ODD CcC, ~g D D i' ;l l
( ) ) 'C:' , CD 0 D .J f1 Cj D 0
( ( .".., .~:' 0 [) D D rF DDD D , D' J D D
») ) « X 0 D 0 0 CCDOO 0 JXJl
"') « e" '0 D e DD '" D D D
,,"hf , «) Jtrt. ro CDD D D D -
ceT (» ({) )CeO " CO"';C DO' DDO 0 " : ~"
(,( ..' ~DDD (; '1rr( D DgDg D ~ ~J
«X P* .«l (m'.« JD D D ~.,L
D D ~DDO) ( ( ( ( " " -'w
C"" D D D D "DD 0) ) 'i ( « ( ( I ( ,
':: : D D DDH, cc (( ) ) « ( » ,
"DDDDO cee() (I «
D'::" ~'x (c ( ;,'< \ \ ) ) (~ ( ) ( I
"'** " c e ,,..::. ( » ( »» )
"-~ ., ec XX ,. CC ( ) ( )
."" ex. () ( ( ) ) ) ) )
< C ( »
exxx"":D( ) (w
X ,-
- 7

Aerial Reconnaiuance in Pollution Surveillance
pollutant effluents. etc. To augment
these observations it is usually better
to have a permanent recording of the
survey area in the form of photographic
or imagery recordings.
B Photographic Techniques
Photographic evidence. either color.
black-and-white. or special f11m. can be
recotded in various attitudes or positions
of the aircraft in order to make best use
of interpretation techniques such &s
stereoscopic analysis.

1 Current patterns and now velocity can
be ascertained by a method developed
by the U. S. Coast and Geodetic Survey
whereby powdered aluminum is surface
distributed to a wide area and subsequent
photographic patterns can be analyzed.
Flowing bodies can either appear as
"d'epressions" or "elevations" depending
upon direction of flow with relation to
the photographing aircraft and from these
"parallax" anomalies the velocity can
be determined.
2 Photographic IR has found applications
in the areas of detecting sharp
delineations of shorelines and tributaries
especially when haze is prevalent.

3 The work of Strandbera: con,cludes that
"In aerial photographs. oxygen-deficient
water frequently appears black. or at
leil.st darker in tone and this appearance
may be caused by the incomplete reduction
of wastes by anaerobic bacteria." Thus
his black-and-white photographs of
waterbodies with low values of dissolv,ed
oxygen (DO) following a source of waste
discharge are characterized by a plume
discharge followed downstream by dark
water tones.
Further values ascribed to photography
in this manual include color photographs
dealing with fish kills where the extent
of damage can be better ascessed and
the us£' of "false" color infrared
70- 8
photography where algal masses can be
differentiated and. again. the low DO
concentration is manifested by a color
C Imagery Techniques
1 Ut11ization of the "mid" and "far"
infrared wavelengths in the form of
"imagery" has been found useful in
analyzing for thermal pollution and its
concomitant circulation and diffusion
patterns for which "thermographs" can
be described for large water bodies. 2
It is also poesible to establish naturally
occurring patterns of seasonal and
diurnal thermal ranges for surface waters.
2 It has been ascertained, in principle.
that it is possible to identify pollutants
by study of spectral emissivity
characteristics for particular wave-
lengths and this can he of value. for
instance. when one is searching for a
particular pollutant and the emissivity
characteristics for the waterbody can
be compared with known patterns for
the specific pollutant. 2
A hypothetical situation is plotted in
Figure 8 where spectrometric values
of pollu~ed water discharge are
compared to a "normal" pattern of the
water body.
Polluted Water Discharge
Normal 'Nater Body
o ,
400 500 600 700 800 900
~Wavelength in millicrons
Figure 8

Utilization of radar and orbiting
satellitl' platforms can produce imagery
of great usefulness. The radar tech-
nique can precisely delineate shoreline
characteristics without vegetative inter-
ferences and thus thoroughly image
drainage basins. Satelllte imagery and
photography can obt ain the same infor-
mation but on a much broader scale and
with great rapidity. Recent lines of
thought have postulated that this orbiting
scanner can be fitted with computing
devices to render decisions upon analysis
of its constant data collection. 3 Such
decisions can be of immeasurable aid
to the water conservationist who may
require long range predictions based
upon analysis of whole river basin!!.
Development of aerial reconnaissance in
pollution surveillance has proceeded in the
areas of technique and interpretation. With
regard to the techniques available most of
the earlier problems have been overcome to
a degree where available material is
amenable to analysis. Such early problems
as pitch and yaw of the aircraft, cloud cover,
imagery instrumentation, etc., has been
overcome to a large degree by available
engineering skills and the need for improve-
ment due to military needs. In the area of
interpretation, however, much has yet to
be accomplished to develop this tool as an
extension of laboratory analysis which it will
augment rather than replace. It is highly
possible that remote sensing will take the
Aerial Reconnaissance in Pollution Surveillance
final evolutionary form of fully automatic
computerized operations. A variety of
remote sensing devices can be envisioned
to collectively contribute to a final printout
of management decisions.
Strandberg, C. H. "Aerioal Discovery
Manual." John Wiley & Sons, Inc.,
New York.
Van Lopik, J. R., Rambie, G. S. and
Pressman, A. E. "pollution
Surveillance by Noncontact Infrar~d
Techniques." Jour. Water Poll.
Control Fed., 40, 3, 425, March 1968.
Colwell, R. N. "Rp.mote Sensing of
Natural Resources." Scientific
American. January 196's.--
This outline was prepared by R. Russomanno,
Microbiologist, National Training Center,
WPO, EPA, Cincinnati, OH 45268.
Descriptors: Aerial Photography, Cameras,
Mapping, Photogrammetry, Pollutants,
Radar, Remote Sensing, Water pollution

A Administrative Proceedings
1 Rule making
a Setting up of regulations having
general application, e. g., stream
c1assification!, and implementation
plan target dates
b Factors of sa,fety and absolute
prohibitions may be appropriate
2 Adjudications
a Determinations by agency having
expertise with respect to particular
discharge or discharger, e. g.,
approval of plans and specs and
time schedule of a particular
B Court Actions
1 Civil in behalf of state or federal
a Actions to compel action or sus-
pension of action - nuisance, health
hazard. etc.. - -including court
action following federal conference
--hearing procedure
b Violations of Water Quality Standards
c Violations of Effluent Standards or
discharge permits
d Tort or contract actions relating to
design and/ or operation of treatment
2 Criminal (dependent on content of
applicable statutes>.

a Discharge of specific materials
W .Q.le.la. 3. 74
b Discharges from specific industries
c Littering
d Discharges harmful to fish and/or
e Discharges harmful to specific
types of receiving waters
Discharges of poisons
NOTE--In some of these situations
doing the act may constitute
the violation; in others
proof of intent or knowledge
of effects may also have to
be proved.
3 Private actions for damages or
to compel action
a Alleged harm to plaintiff, e. g..
pollution of stream killing animals
C Procedural Matters
1 See Attached sheet "Administrative
and Court Proceedings" on Burden
of proof, fact finding, and methods of
presentation of evidence.
D Classes of Evidence - General Rules
1 Facts
a The material was floating from
the outfaJ1.
2 Derived values expert testimony
- test results and/or opinion as to
a The D. O. was zero; the waterway
was polluted; the plant can be built
in 6 months.
3 Hearsay
a Joe told me

Case Preparation and Courtroom Procedure
4 Relevancy
5 Admissibility vs. weight
a Even if admissible, the weight to be
given is up to fact !inder--credi-
E Admissibility of Results of Sampling
and Testing (Numbers)
1 Sampling
a Chain of custody
b Tags, etc.
c Containers
d Place and time
e Retention of samples (Proving that
the sample represents what is at
issue in the action (relevancy), that
there has been no opportunity for
tampering; and availability of
portions for analysis by other side
(non-transitory criteria) ).
2 Ana~sis
a Who performed (Can identity of
each participant be shown?)
b Admiuion through supervisor -
c Scientific acceptance of method.
Is there a particular method required
to be used by the agency?
d Propriety of conduct
e Retention of bench cards and other
indicia of results. (Your attorney
can make arrangements to substitute
copies for ori8inals).
3 Tests
a Comparison with actual conditions
b Mathematical models - how can a
computer be cross-examined?
Admissibility of Expert Opinion on
Causes and Effects
1 Who has special knowledge - and of
what particular areas?
2 Indicators
3 Significance of numerical determin-
ations or observations
4 Consistency with own prior publications
and testimony
5 Have under]y1ng facts been or need to
be proved- -first hand information of
this and/or comparable situations.
6 Use of treatises
G Conduct on the Witness Stand
1 General
a On direct - know what counsel will
ask and let him know generally
what you will answer, but don't
make it sound rehearsed.
b Use layman's language to extent
c Listen to question and answer it
to best of your ability.
d Speak so that court reporter, judge,
jury, and counsel can hear you.
e Speak in language that will be
understood; don't talk down.
Answer only what you are asked
--don't volunteer; however, answer
with precision.
g Tqere is nothing wrong with asking
to have a question repeated or
h There is nothing wrong with saying
that you conf :ted with your
attorney befr r ~'ou testified, but
beware of the question "Did Mr. X
tell you what to say? "

There is nothing wrong with thinking
out your answer before respand1ng.

You are not expected to know all
the answers--if you do not know
admit it. '
k Don't attempt to answer questions
outside your area of personal
knowledge (hearsay) or beyond your
expertise. (Your may be an expert
on conduding laboratory tests, but
not on epidimeo10gica1 inferences
from results).
Don't try to answer before the judge
rules on objection.
m Show that you are an impartial
dispense r of information and I or
opinion, not a protagonist.
n Don't be afraid to admit what may
appear to be damaging.
2 If you are testifying as an expert:
a Establish qualifications -- give
information relevant to your area
of expertise - - educational (in-
eluding this course?), work,
publications, number of times you
have testified previously.
b Differentiate between physical facts
(measurements and observations)
and opinion (derived values).
c Be prepared to discuss theory (in-
cluding assumptions) instruments
used, techniques(inc1uding choice
of a particular technique),physica1
limitations and errors, inter-
d If experiments were conducted,
be able to justify both as to theory
and relevancy to this litigation.
e If you're being paid to testify,
admit it.
3 Scientific personnel as advisers to
Case Preparation and Courtroom Procedure
a Review and refami1iarize self with
materials before you discuss with
your attorney.
b Be in a position to present all facts
known to you simply and concisely:
Who, What, When, Where, and
Why, How.
c Don't overlook fads and/or test
results because you don't think
they're important. Let attorney
decide what he needs.
d Use of standard report forms
e Ability to recommend additional
witnesses with needed specialized
f Ability to aid in cross - examination
of other side's experts and reconcile
opinions and/or results
g Be candid - sometimes better not
to start a lawsuit or accept a
settlement than lose in the end.
H Non-Verbal Presentation of Evidence
1 Exhibits - including photographs
2 Summaries
3 Business and/or government records
a Prepared contemporaneously and
in usual course of activities
4 Pre-prepared direct examination
a U suallv limit" j to actions before
ICC, FPC, and other federal
Criminal Procedure
1 Privilege Against Self Incrimination
(available only to persons)
a Warning and suspects
b Effect of duty to report spills

ea.e Preparation and Courtroom Procedure
c Effecrt of duty to obtain license or
permit and/or furnish operating
d ImmW1ity from pro.ecution
2 Double Jeopardy
Unreasonable search and seizure
Available to per80na and
4 Procedure. and need for arrest and
search warrants --po..ible cause
Thil ootUlae wa. prepared by David I.
Shedroff, Enforcement Aaaqllt, Office of
Enforcement and General COWUlel,
Cincinnati FielcllRveetip.ti0l'18 Clftter,
5555 Ridp AV81N8, Cl.nciImMl, OR 45268.
Descriptor.: Courtroom Procedure,
Law Enforcement, Le,u A.pect., SampUng,
Water AnaJ,y.18, Water PoJlution
Control, Water Quality Standards
Admin18t~tive & Court Proceedings,
and Excerpts from Revi.ed Draft of
Propo.ed Rules of Evidence for the
United States Court. can be found on the
foUowing pages.

Court or Agency
State Pollution Control
Rule making-adjudi-
Federal Water PoThrtion
Control Act
Civil Case --
- for money only
- inj\8lction

preliminary or
pe rmanent
- administrative
Criminal case
includes penalties
Fact Finder
Head of agency
Hearing B08.I'd
Judge or jury
Judge - whether
"arbitrary and
capricious" or sub-
stantial evidence.
Jury unless waived.
Burden of Proof
As per statute -
usually weight of
Weight of evidence
Must show immediate
harm or danger.
Usually clear and convincing.
Beyond reasonable
Hearing may be conducted by hearing
examiner. agency member. or full
agency. Appeal may be on facts and
law or law alone. depending on statute.
Reports acceptable.
Specific testimony.
Uses prior material, and may take
additional testimony.
Must also show likelihood of success at
final hearing - bond required for non-
government plaintiff.

"Balance Equities"
Sometimes have complete new trial.
Proof of intent may be required.

Cafe Preparation and Courtroom Procedure
Excerpts from Revised Draft of Proposed
Rule 102.

These rules shall be construed to secure fairness in administration, elimination
dt unjustifiable expense and delay, and promotion of growth and develo~ent of
the law of evidence to the end that the truth may be ascertained and proceedings
jllstly determined.
Rule 101.


Case Preparation and Courtroom Procedure
Rule 613.


(a) Examining Witness Concerning Prior Statement. In examining a witness
concerning a prior statement made by him, whether written or not, the state-
ment need not be shown or lts contents disclosed to him at that time but on
request the same shall be shown or disclosed to opposing couneel. '
Rule 201.
(b) Kinds of Facts. A judicially noticed fact must be one not subject to reasonable
dispute in that it is elther (1) generally known within the territorial jurisdiction of
the trial court or (2) capable of accurate and ready determination by resort to
sources whose accuracy cannot reasonably be questioned.

(g) Instructing Jury. The judge shall instruct the jury to accept as established
any facts judicially noticed.
Rule 401.

"Relevant evidence" means evidence having any tendency to make the existence
of any fact that is of consequence to the determination of the action more probable
or less probable than it would be without the evidence.
Rule 402.

All relevant evidence is admissible, except as otherwise provided by these rules,
by other rules adopted by the Supreme Court, by Act of Congress, or by the
Constitution of the United States. Evidence which is not relevant is not admissible.
Rule 601.
Every person is competent to be a wltne8B except as otherwise provided in these

Cue Preparation and Courtroom Procedure
Rule 602.
A witness may not testify to a matter unleu evidence is- introduced sumc1ent
to support a findini that he has personal knowledge of the matter. Evtcklnce to
prove personal knowledge may, but need not, consist of the testimony of the
witness himself. This rule is subject to the provisions of Rule 703, relating to
opinion testimony by expert witne..es.
Rule 702.
If scientific, technical, or other specialized knowledge will assist the trier of
fact to understand the evidence or to determine a fact in issue, a witness qualified
as an expert by knowledge, skill, experience; training, or education, may teatify
thereto in the form of an opinion or otherwise.
Rule 703.
The facts or data in the particular case upon which an expert bases an opinion or
infl!rence may be those perceived by or made known to him at or before the hearing.
If of a type reasonably relied upon by experts in the particular field in forming
opinions or inferences upon the subject, the facta or data need not be admissible
in evidence.
Rule 705.
The expert may teatify in terms at opinion or inference and give his reasons
therefore without prior diaclosure of the under:IiYing facts or data, unless the
juclge requires otherwise. The expert may in any event be required to disclose
the under:IiYing facts or data on cro.a-examination.
ij.ule 706.
(a) Appointment. The judge may on his own motion or on the motion of any
party enter an order to show cauae why expert witnesse. should not be appointed,
and may request the parties to aubmit nominations. The judge may appoint any
expert witneues agreed upon by the parties, and may appoint witnesse. of hill
own aelection. An expert witneas shall not be appointed by the judge unleas he
con.ent. to act. A witness ao appointed shall be informed of his dut- :3 by the
judge in writing, a copy of which shall be filed with the clerk, or at 1 '!lference
in which the parties shall have opportunity to participate. A witness so appointed
shall advise the parties of his findings, if any; his deposition may be taken by any
party; and he may be called to testify by the judge or any party. He .\.~ll be subject
to cross-examination by each party, including a party calling him as ""'1ess.

Case Preparation and Courtroom Procedure
Rule 801.
The following definitions apply under this Article:

(a) Statement. A "statement" is (1) an oral or written assertion or
(2) nonverbal conduct of a person, if it is intended by him as an assertion.

(b) Declarant. A "declarant" is a person who makes a statement.
(c) Hearsay. "Hearsay" is a statement, other than one made by the declarant
while testifying at the trial or hearing, offered in evidence to prove the truth
of the matter asserted.
Rule 802.
Hearsay iB not admissible except as provided by these rules or by other rules
adopted by the Supreme Court or by A ct of Congress.
Rule 803.
The following are not excluded by the hearsay rule, even though the declarant is
available as a witness:
(5) Recorded Recollection. A memorandum or record cuncerning a matter about
which a witness once had knowledge but now has insufficient recollection to enable
him to testify fully and accurately, shown to have been made when the matter was
fresh in his memory and to reflect that knowledge correctly. U admitted, the
memorandum or record may be read into evidence but may not itself be received
as an exhibit unless offered by an adverse party.
(6) Records of Regularly Conducted Activity. A memorandum, report, record,
or data compilation, in any form, of acts, events, conditions, opinions, or
diagnoses, made at or near the time by, or from information transmitted by,
a person with Imowledge, all in the course of a regularly conducted activity, as
shown by the testimony of the custodian or other qualified witness, unless the
sources of informaticm or other circumstances indicate lack of trustworthiness.
(18) Learned Treatises. To the extent called to the attention of an t!xpert witness
upon croBB-examination or relied upon by him in direct examination, statements
contained in published treatises, periodicals, or pamphlets on a subject of
history, medicine, or other science or art, established as a reliable authority
by the testimony or admission of the witness or by other expert testimony or by
judicial notice. If admitted, the statements may be read into evidence but may
not be received as exhibits.

£ase Preparation and Courtroom Procedure
Rule 901.
(a) Gen~ral Provision. The requirement of authentication or identification as a
condition precedent to admissibility is satisfied by evidence sufficient to support
a finding that the matter in question is what its proponent claims.
(b) Illustrations. By way of illustration only, and not by way of limitation, the
following are examples of authentication or identification conformin, with the
requirements of this rule:
(1) Testimony of Witness with Knowledge. Testimony that a matter is what it il
claimed to be.
(3) Comparison by Trier or Expert Witness. Comparison by the trier of fact or
by expert witnesses with specimens which have been authenticated.
(9) Process or System. Evidence describing a process or system used to produce
a result and showing that the process or system produces accurate result.
Rule 406.
(a) Admissibility. Evidence of the habit of a person or of the routine practice of an
organization, whether corroborated or not and regardless of the presence of eye-
witnesses, is relevant to prove that the conduct of the person or organization on a
particular occasion was in conformity with the habit or routine practice.
(b) Method of Proof. Habit or routine practice may be proved by testimony in the
for.m of an opinion or by spec1fic instances of conduct sufficient in number to warrant
a finding that the habit existed or that the practice was routine.
Rule 612
[f a witness uses a writing to refresh his memory, either before or while testifying,
an adverse party is entitled to have it produced at the hearing, to inspect it, to
cross-examine the witness thereon, and to introduce in evidence tho.e portion. which
relate to the testimony of the witness.
Rule 1006.
The contents of voluminous writings, recordings, or photographs which cannot
conveniently be examined in court may be presented in thE; form of a chart, summary,
or calculation. The originals, or duplicates, shall be made available for examination
or copying, or both, by other parties at a reasonable time and place The judge may
order that they be produced in court.


b~C'1r /Jog"
{,"/JrorJ of u,,, is re
"ff" OC'ti rei> '!>rOrJ
r rJ"tq 0" l>J"orf b tJC'erJ
11. U,OrJ'Y q eJ' of tJ,

fo p~::'~l)t
The standard eq\)ation for discharge
through a Cipolletti weir is

Q = 3.367 LH3/2
discharge, cfs
L = length of the level crest edge, feet
weir head, feet
The discharge of a Cipolletti
weir exceeds that of a suppressed
rectangular weir of eqllal crest
length by approximately 1 percent.
Triangular weirs
The crest of a triangular weir is
shaped like a V-notch with sides
equally inclined from the vertical.
The central angle of the notch is
normally 60 or 90 degrees. Since
the triangular weir develops more
head at a given discharge than does
a rectangular shape, it is especial1y
useful for measurement 01 smal1
or varying flow. It is preferred for
discharges less than 1 cfs, is as
accurate as other shapes up to 10
cfs, and is commonly used in plant
Flow Measurement
The st'tndard equation for dlsch.~ t ,.,
of a 90 triangular weir (Cone
formula) is
2. 4C,H2. 48
dischar ge, cfs
weir head, feet
Crest height and hf'ad
are measured to and from the pOint
of the notch. respectively.
I' Accuracy and installation
Quotations of weir accuracy express
the difference in performance hetween
two purportedly identical weirs and
do not include the effects of randon,
error in measurement of head. Weirs
installed according to the following
speclflcatlons should measure dis-
charge within:!: 50/, of the values
observed when the previously cited
standard equations were developed.

1) The upstream face of the bulkhead
and! or weir plate shall be smooth
and in a vertical plane perpendicular
to the axis of the channel.
2) The crest edge shall be level, shall
have a square upstream corner,
and shall not exceed 0.08 in (2 mm)
in thickness. If the weir plate is
thicker than the prescrIbed crest
thickness the downstream corner
of the crest shall be relieved by a
450 champfer. .
3) The pressure under the nappe
shall be atmospheric. The maXI-
mum water surface in the down-
stream channel shall be at least
0.2 ft. below the weir crest.
Vents shall be provided at the
ends of standard suppressed wel rs
to admit air to the space ben..ath
the nappe.

'i~1pling ill_Water Quality Studies
The 2-rpm gear motor drives
the pump at between 1 and 2
rpm thr~)Ugh the spring-loaded
adjustable-pitch pulley and
adjustable motor-base arrange-
We use 1/ 8- in. (. 32-cm) ID
or 1/4-in. (. 64-cm) ID
polyethylene tubing for sam-
ple intake from the waste
stream. The sample flow is
delivered to the distributor
via a 3/16-in. (.48-cm) ID
Tygon tube which is supported
loosely by a wire attached to
the framework.
Operation of the distributor is
very simple. The 1-rpm clock
motor powers the chain-and-
sprocket drive which turns a
threaded bolt. Rotation of the
bolt moves the discharge tube
down the prastic trough at a
rate equat'to one division
every eight hours. With the
10 sample-jar receivers the
timer can be set on Friday,
and the 9 week-end Shlft
samples can be picked up on
Figure 4
.t- \."6
C. (\\. t\\
o~ce ,\\\e~~ \6
1"I~0 \\ <.\ ,,\l
".- . ~e~ \~1 \1~0
:..-' e \s "o~\. \.0
1"1\\~ ~e~ ~o
....,,\S ~ ~ ~e \i\e
" \r- 0 \.\ot\
\:J(\C o,\C \\.
,,~o \e\.\\
~e~ {l

4) Solenoid-valv/? arrangl'n1ents
A solenoid valv,' .'mrlo}l'U ill
l'onnectiun with a timing d,'vi"
may bf' used for withuI'C1wing
waste from 3. pip" und,'r pr" ";SIII'"
Used in connection with a punlf'
such devices may be pmploy"cI ]11
. sampling frpe flowing strf'<1l1h.
(See Figures 6 and 7. )
!OLfN080 VAa[
/-,.,.. llle"
~,,"~lAT"'. ....
"""' -\ u..
- ""'101 '0
~CT ...... '-- .111M. c"l
Figure 6
5) Vacuum operated
In Its simplest form, th.' '.',J< )1'11'
is creatf'd hy a suitably 1'1 JIllt
siphOIl. It (ollect- th.. ",C'i ,pi,
at a uniform ratf and i ~ 11(>'
suitahll' fur U::i"-' ,';hl n r:onfJ!'tlr,' ~
,'ampling is required.